**Meet the editor**

Dr. Sanjay K. Srivastava is a Professor of Biomedical Sciences at Texas Tech University Health Sciences Center (TTUHSC), Amarillo, Texas, specializing in cancer biology, cell signaling and nutritional chemoprevention. Dr. Srivastava served as an Assistant Professor in the Department of Pharmacology, University of Pittsburgh School of Medicine, and did his post-doc from Uni-

versity of Texas Medical Branch at Galveston, Texas. He received a M.S. in Biochemistry from Lucknow University and a Ph.D. in Biochemical Toxicology from Industrial Toxicology Research Center, India. Dr. Srivastava is funded by grants from the National Cancer Institute, NIH. He has authored/co-authored more than 100 research papers and book chapters and is in the editorial board of several journals. Dr. Srivastava has been the recipient of several awards including TTUHSC "President's Excellence in Research Award". His research has been featured by news agencies including BBC, MSNBC, CBS, ABC, Science News etc.

Contents

**Preface IX** 

Chapter 1 **The Genetics of Pancreatic Cancer 1** 

Dagan Efrat and Gershoni-Baruch Ruth

Chapter 2 **Systems and Network-Centric Understanding of** 

Irfana Muqbil, Ramzi M. Mohammad, Fazlul H. Sarkar and Asfar S. Azmi

Chapter 3 **Novel Biomarkers in Pancreatic Cancer 31** 

Anca Botezatu and Irinel Popescu

Chapter 5 **Temporal Trends in Pancreatic Cancer 77**  Tadeusz Popiela and Marek Sierzega

**Current Status and Future Targets 55**  Edward Livshin and Michael Michael

Chapter 6 **Current Perspectives and Future Trends of Systemic** 

Chapter 7 **Immunotherapy of the Pancreatic Cancer 109** 

Chapter 9 **Bacterial Immunotherapy-Antitumoral Potential** 

Joerg Emmrich and Michael Linnebacher

Yang Bo

**Therapy in Advanced Pancreatic Carcinoma 89**  Purificacion Estevez-Garcia and Rocio Garcia-Carbonero

Chapter 8 **An Overview on Immunotherapy of Pancreatic Cancer 137**  Fabrizio Romano, Luca Degrate, Mattia Garancini, Fabio Uggeri, Gianmaria Mauri and Franco Uggeri

> **of the Streptococcal Toxin Streptolysin S- 163**  Claudia Maletzki, Bernd Kreikemeyer, Peggy Bodammer,

Chapter 4 **Medical Therapy of Pancreatic Cancer:** 

**Pancreatic Ductal Adenocarcinoma Signalling 15** 

Simona O. Dima, Cristiana Tanase, Radu Albulescu,

### Contents

#### **Preface XI**


X Contents



### Preface

*Dedicated to my mother Vidya Srivastava and father Dr. Balramji Srivastava,* 

*who provided me constant love and support.*

Pancreatic cancer is one of the most fatal human malignancies with extremely poor prognosis making it the fourth leading cause of cancer-related deaths in the United States. The molecular mechanisms of pancreatic carcinogenesis are not well understood. The major focus of these two books is towards the understanding of the basic biology of pancreatic carcinogenesis, identification of newer molecular targets and the development of adjuvant and neoadjuvant therapies.

Book 1 on pancreatic cancer provides the reader with an overall understanding of the biology of pancreatic cancer, hereditary, complex signaling pathways and alternative therapies. The book explains nutrigenomics and epigenetics mechanisms such as DNA methylation, which may explain the etiology or progression of pancreatic cancer. Apart from epigenetics, book summarizes the molecular control of oncogenic pathways such as K-Ras and KLF4. Since pancreatic cancer metastasizes to vital organs resulting in poor prognosis, special emphasis is given to the mechanism of tumor cell invasion and metastasis. Role of nitric oxide and Syk kinase in tumor metastasis is discussed in detail. Prevention strategies for pancreatic cancer are also described. The molecular mechanisms of the anti-cancer effects of curcumin, benzyl isothiocyante and vitamin D are discussed in detail. Furthermore, this book covers the basic mechanisms of resistance of pancreatic cancer to chemotherapy drugs such as gemcitabine and 5 flourouracil. The involvement of various survival pathways in chemo-drug resistance is discussed in depth. Major emphasis is given to the identification of newer therapeutic targets such as mesothalin, glycosylphosphatidylinositol, cell cycle regulatory proteins, glycans, galectins, p53, toll-like receptors, Grb7 and telomerase in pancreatic cancer for drug development.

Book 2 covers pancreatic cancer risk factors, treatment and clinical procedures. It provides an outline of pancreatic cancer genetic risk factors, signaling mechanisms, biomarkers and disorders and systems biology for the better understanding of disease. As pancreatic cancer suffers from lack of early diagnosis or prognosis markers, this book encompasses stem cell and genetic makers to identify the disease in early stages. The book uncovers the rationale and effectiveness of monotherapy and combination therapy in combating the devastating disease. As immunotherapy is emerging as an attractive approach to cease pancreatic cancer progression, the present book covers various aspects of immunotherapy including innate, adaptive, active, passive and

#### X Preface

bacterial approaches. The book also focuses on the disease management and clinical procedures. Book explains the role of pre-existing conditions such as diabetes and smoking in pancreatic cancer. Management of anesthesia during surgery and pain after surgery has been discussed. Book also takes the reader through the role of endoscopy and fine needle guided biopsies in diagnosing and observing the disease progression. As pancreatic cancer is recognized as a major risk factor for vein thromboembolism, this book reviews the basics of coagulation disorders and implication of expandable metallic stents in the management of portal vein stenosis of recurrent and resected pancreatic cancer. Emphasis is given to neuronal invasion of pancreatic tumors along with management of pancreatic neuroendocrine tumors.

We hope that this book will be helpful to the researchers, scientists and patients providing invaluable information of the basic, translational and clinical aspects of pancreatic cancer.

> **Sanjay K. Srivastava, Ph.D.**  Department of Biomedical Sciences Texas Tech University Health Sciences Center Amarillo, Texas USA

X Preface

pancreatic cancer.

bacterial approaches. The book also focuses on the disease management and clinical procedures. Book explains the role of pre-existing conditions such as diabetes and smoking in pancreatic cancer. Management of anesthesia during surgery and pain after surgery has been discussed. Book also takes the reader through the role of endoscopy and fine needle guided biopsies in diagnosing and observing the disease progression. As pancreatic cancer is recognized as a major risk factor for vein thromboembolism, this book reviews the basics of coagulation disorders and implication of expandable metallic stents in the management of portal vein stenosis of recurrent and resected pancreatic cancer. Emphasis is given to neuronal invasion of pancreatic tumors along with management of pancreatic neuroendocrine tumors.

We hope that this book will be helpful to the researchers, scientists and patients providing invaluable information of the basic, translational and clinical aspects of

**Sanjay K. Srivastava, Ph.D.** 

Amarillo, Texas

USA

Department of Biomedical Sciences

Texas Tech University Health Sciences Center

**1**

**The Genetics of**

*University of Haifa,* 

*Israel* 

**Pancreatic Cancer** 

Dagan Efrat1,2 and Gershoni-Baruch Ruth1,3

*3The Ruth and Bruce Rapoport Faculty of Medicine,* 

*Technion-Institute of Technology, Haifa,* 

*1Institute of Human Genetics, Rambam Health Care Campus, Haifa,* 

*2Department of Nursing, the Faculty of Social Welfare and Health Sciences,* 

Globally, pancreatic cancer is considered a rare cause of cancer. More than 250,000 new cases, equivalent to 2.5% of all forms of cancer, were diagnosed in 2008 worldwide (Ferlay et al., 2008, 2010). Pancreatic adenocarcinoma currently represents the fourth most common cancer causing death in the United States and in most developed countries (Jemal et al., 2009, 2011). Despite advances in medical science, the overall prognosis of pancreatic cancer remains poor and five years survival is only 4% (Jemal et al., 2006). Those diagnosed early, with tumor limited to the pancreas, display a 25-30% five years survival following surgery

It has been suggested that it takes at least 10 years from tumor initiation to the development of the parental clone and another five years to the development of metastatic subclones, with patients dying within two years thereafter, on average (Costello & Neoptolemos, 2011). Given the limited treatment options there has been considerable focus on clinical and molecular harbingers of early disease. A mechanism for early detection and for early intervention remains to be elaborated. Current research is focused on the discovery and the development of diagnostic bio markers that can unveil pancreatic cancer in its early stages. Deciphering and understanding the genetics of sporadic and hereditary pancreatic cancer

Based on family aggregation and family history of pancreatic disease, it is estimated that around 10% of cases diagnosed with pancreatic cancer host a hereditary germ line mutation (Lynch et al., 1996; Hruban et al., 1998). Furthermore, it has been observed that pancreatic cancer occurs in excess of expected frequencies, in several familial cancer syndromes, which are associated with specific germ-line mutations. The best characterized include hereditary breast-ovarian cancer syndrome ascribed to mutations in BRCA1/2 genes, especially BRCA2; familial pancreatic and breast cancer syndrome due to mutations in PALB2 gene; familial isolated pancreatic cancer caused by mutations in PALLD encoding palladin; and familial multiple mole melanoma with pancreatic cancer (FAMMM-PC) attributed to

**1. Introduction**

(Ryu et al., 2010).

remains a fundamental milestone.

### **The Genetics of Pancreatic Cancer**

Dagan Efrat1,2 and Gershoni-Baruch Ruth1,3

*1Institute of Human Genetics, Rambam Health Care Campus, Haifa, 2Department of Nursing, the Faculty of Social Welfare and Health Sciences, University of Haifa, 3The Ruth and Bruce Rapoport Faculty of Medicine, Technion-Institute of Technology, Haifa, Israel* 

#### **1. Introduction**

Globally, pancreatic cancer is considered a rare cause of cancer. More than 250,000 new cases, equivalent to 2.5% of all forms of cancer, were diagnosed in 2008 worldwide (Ferlay et al., 2008, 2010). Pancreatic adenocarcinoma currently represents the fourth most common cancer causing death in the United States and in most developed countries (Jemal et al., 2009, 2011). Despite advances in medical science, the overall prognosis of pancreatic cancer remains poor and five years survival is only 4% (Jemal et al., 2006). Those diagnosed early, with tumor limited to the pancreas, display a 25-30% five years survival following surgery (Ryu et al., 2010).

It has been suggested that it takes at least 10 years from tumor initiation to the development of the parental clone and another five years to the development of metastatic subclones, with patients dying within two years thereafter, on average (Costello & Neoptolemos, 2011). Given the limited treatment options there has been considerable focus on clinical and molecular harbingers of early disease. A mechanism for early detection and for early intervention remains to be elaborated. Current research is focused on the discovery and the development of diagnostic bio markers that can unveil pancreatic cancer in its early stages. Deciphering and understanding the genetics of sporadic and hereditary pancreatic cancer remains a fundamental milestone.

Based on family aggregation and family history of pancreatic disease, it is estimated that around 10% of cases diagnosed with pancreatic cancer host a hereditary germ line mutation (Lynch et al., 1996; Hruban et al., 1998). Furthermore, it has been observed that pancreatic cancer occurs in excess of expected frequencies, in several familial cancer syndromes, which are associated with specific germ-line mutations. The best characterized include hereditary breast-ovarian cancer syndrome ascribed to mutations in BRCA1/2 genes, especially BRCA2; familial pancreatic and breast cancer syndrome due to mutations in PALB2 gene; familial isolated pancreatic cancer caused by mutations in PALLD encoding palladin; and familial multiple mole melanoma with pancreatic cancer (FAMMM-PC) attributed to

The Genetics of Pancreatic Cancer 3

Meckler et al., 2001). Genomewide linkage screen of a family, noted as 'family X', has shown significant linkage to chromosome 4q32-34 (Eberle et al., 2002). Pogue-Geile et al. (2006) later found a mutation, inducing a proline (hydrophobic) to serine (hydrophilic) amino acid change (P239S), in a highly conserved region of the gene encoding palladin (PALLD), segregating in all affected family members and absent in unaffected family members. Zogopoulous et al. (2007) identified this same mutation (P239S) in one of 84 (1.2%) patients with familial and early-onset pancreatic cancer and in one of 555 controls (0.002%). No evidence for palladin mutations in 48 individuals with familial pancreatic cancer was recorded by Klein et al. (2009). Further investigation is warranted in order to confirm the

*AKT2* (MIM 164731) - It has been suggested that the AKT2 oncogene, on chromosome 19q, contributes to the malignant phenotype of a subset of human ductal pancreatic cancers. Cheng et al., (1996) demonstrated that the AKT2 oncogene is over expressed in approximately 10-15% of pancreatic carcinomas. AKT2 encodes a protein belonging to a

*AIB1* (MIM 601937) - AIB1 gene, on chromosome 20q, is amplified in as many as 60% of pancreatic cancers (Anzick et al., 1997; Calhoun et al., 2003; Aguirre et al., 2004). Altered AIB1 expression may contribute to the development of steroid-dependent cancers. It has also been reported that amplification of a localized region on the long arm of chromosome 8 is commonly seen in pancreatic cancers, and this amplification corresponds to the oncogenic

In addition to these genes, numbers of amplicons, amplified from DNA fragments, have been identified in pancreatic cancers by using gene chip technologies (Aguirre et al., 2004). Employing array comparative genomic hybridization (CGH) technology, a high resolution analysis of genome-wide copy number aberrations, permits to identify over expression of DNA fragments in tumor transformed pancreatic cells. Understanding the mechanisms underlying the development of pancreatic cancer may aid target early detection, gene-

In pancreatic invasive adenocarcinoma, CDKN2A/INK4A, TP53, and DPC4/SMAD4/

The CDKN2A gene on chromosome 9p21 encodes proteins that control two critical cell cycle regulatory pathways, the p53 (TP53) pathway and the retinoblastoma (RB1) pathway. Through the use of shared coding regions and alternative reading frames, the CDKN2A gene produces 2 major proteins: p16(INK4), which is a cyclin-dependent kinase inhibitor checkpoint, and p14(ARF), which binds the p53-stabilizing protein MDM2 (Robertson and Jones, 1999). P16 inhibits cyclin D1 by binding to the cyclin-dependent kinases Cdk4 and Cdk6 thereby causing G1-S cell-cycle arrest (Schutte et al., 1997). Loss of p16 function,

pathogenecity of mutations in PALLD.

subfamily of serine/threonine kinases.

transcription factor CMYC (MIM 190080) (Aguirre et al., 2004).

specific therapies and thereby improve prognosis.

**3. Tumor suppressor genes** 

MADH4 are commonly inactivated.

**3.1 CDKN2A/INK4A gene (MIM 600160)** 

**2.4 Other oncogenes** 

mutations in CDKN2A. Other hereditary cancer syndromes demonstrating increased hereditary risk for pancreatic cancer, yet with less significance, include hereditary nonpolyposis colorectal syndrome - Lynch syndrome and Li-Fraumeni syndrome which is caused by mutations in p53 gene.

The identification of individuals at risk for pancreatic cancer would aid in targeting those who might benefit most from cancer surveillance strategies and early detection (Brentnall et al., 1999). This chapter describes the cutting edge data related to the genetics of sporadic and hereditary pancreatic cancer subdivided according to 'genes' function.

### **2. Oncogenes**

#### **2.1 KRAS gene (MIM 190070)**

Recent studies have shown that the KRAS oncogene on chromosome 12p is activated by point mutations in approximately 90% of pancreatic cancers tumors, and these mutations involve codon 12 most commonly, and codons 13 and 61 thereafter (Caldas & Kern, 1995). The RAS protein produced by wild-type KRAS binds GTPase-activating protein and regulates cell-cycle progression. Mutations in KRAS constitute the earliest genetic abnormalities underlying the development of pancreatic neoplasms (Maitra et al., 2006; Feldmann et al., 2007). KRAS may thus be a promising bio marker for early detection of curable non-invasive pancreatic neoplasia (Maitra et al., 2006).

#### **2.2 BRAF gene (MIM 164757)**

The BRAF gene maps to chromosome 7q and takes part in the RAF–MAP signaling pathway, critical in mediating cancer causing signals in the RAS corridor (Calhoun et al., 2003). BRAF mutations have been described in about 15% of all human cancers, including pancreatic cancer (Davies et al., 2002). The BRAF gene is activated by oncogenic RAS, leading to cooperative mutual effects in cells responding to growth factor signals. BRAF and KRAS appear to be alternately mutated in pancreatic cancers; thus, pancreatic cancers with KRAS gene mutations do not harbor BRAF gene mutations and vice versa (Maitra et al., 2006).

#### **2.3 PALLD gene (MIM 608092)**

Palladin RNA is over-expressed in tissues from both precancerous dysplasia and pancreatic adenocarcinoma in familial and sporadic pancreatic disease. The mutated gene is assumingly, best detected in very early precancerous dysplastic tissue, heralding neoplastic transformation before the overarching of genetic instability, underlying cancer, has occurred. Palladin is a component of actin-containing microfilaments that control cell shape, adhesion and contraction and is associated with myocardial infarction and pancreatic cancer. Palladin is most probably a proto-oncogene (Pogue-Geile et al., 2006).

#### **2.3.1 Familial pancreatic cancer associated PALLD gene (MIM 164757)**

Few families with isolated pancreatic cancer of early onset and high penetrance have been identified (Lynch et al., 1990; Brentnall et al., 1999; Banke et al., 2000; Hruban et al., 2001;

mutations in CDKN2A. Other hereditary cancer syndromes demonstrating increased hereditary risk for pancreatic cancer, yet with less significance, include hereditary nonpolyposis colorectal syndrome - Lynch syndrome and Li-Fraumeni syndrome which is

The identification of individuals at risk for pancreatic cancer would aid in targeting those who might benefit most from cancer surveillance strategies and early detection (Brentnall et al., 1999). This chapter describes the cutting edge data related to the genetics of sporadic and

Recent studies have shown that the KRAS oncogene on chromosome 12p is activated by point mutations in approximately 90% of pancreatic cancers tumors, and these mutations involve codon 12 most commonly, and codons 13 and 61 thereafter (Caldas & Kern, 1995). The RAS protein produced by wild-type KRAS binds GTPase-activating protein and regulates cell-cycle progression. Mutations in KRAS constitute the earliest genetic abnormalities underlying the development of pancreatic neoplasms (Maitra et al., 2006; Feldmann et al., 2007). KRAS may thus be a promising bio marker for early detection of

The BRAF gene maps to chromosome 7q and takes part in the RAF–MAP signaling pathway, critical in mediating cancer causing signals in the RAS corridor (Calhoun et al., 2003). BRAF mutations have been described in about 15% of all human cancers, including pancreatic cancer (Davies et al., 2002). The BRAF gene is activated by oncogenic RAS, leading to cooperative mutual effects in cells responding to growth factor signals. BRAF and KRAS appear to be alternately mutated in pancreatic cancers; thus, pancreatic cancers with KRAS gene mutations do not harbor BRAF gene mutations and vice versa (Maitra et al.,

Palladin RNA is over-expressed in tissues from both precancerous dysplasia and pancreatic adenocarcinoma in familial and sporadic pancreatic disease. The mutated gene is assumingly, best detected in very early precancerous dysplastic tissue, heralding neoplastic transformation before the overarching of genetic instability, underlying cancer, has occurred. Palladin is a component of actin-containing microfilaments that control cell shape, adhesion and contraction and is associated with myocardial infarction and pancreatic

Few families with isolated pancreatic cancer of early onset and high penetrance have been identified (Lynch et al., 1990; Brentnall et al., 1999; Banke et al., 2000; Hruban et al., 2001;

cancer. Palladin is most probably a proto-oncogene (Pogue-Geile et al., 2006).

**2.3.1 Familial pancreatic cancer associated PALLD gene (MIM 164757)** 

hereditary pancreatic cancer subdivided according to 'genes' function.

curable non-invasive pancreatic neoplasia (Maitra et al., 2006).

caused by mutations in p53 gene.

**2.1 KRAS gene (MIM 190070)** 

**2.2 BRAF gene (MIM 164757)** 

**2.3 PALLD gene (MIM 608092)** 

2006).

**2. Oncogenes** 

Meckler et al., 2001). Genomewide linkage screen of a family, noted as 'family X', has shown significant linkage to chromosome 4q32-34 (Eberle et al., 2002). Pogue-Geile et al. (2006) later found a mutation, inducing a proline (hydrophobic) to serine (hydrophilic) amino acid change (P239S), in a highly conserved region of the gene encoding palladin (PALLD), segregating in all affected family members and absent in unaffected family members. Zogopoulous et al. (2007) identified this same mutation (P239S) in one of 84 (1.2%) patients with familial and early-onset pancreatic cancer and in one of 555 controls (0.002%). No evidence for palladin mutations in 48 individuals with familial pancreatic cancer was recorded by Klein et al. (2009). Further investigation is warranted in order to confirm the pathogenecity of mutations in PALLD.

#### **2.4 Other oncogenes**

*AKT2* (MIM 164731) - It has been suggested that the AKT2 oncogene, on chromosome 19q, contributes to the malignant phenotype of a subset of human ductal pancreatic cancers. Cheng et al., (1996) demonstrated that the AKT2 oncogene is over expressed in approximately 10-15% of pancreatic carcinomas. AKT2 encodes a protein belonging to a subfamily of serine/threonine kinases.

*AIB1* (MIM 601937) - AIB1 gene, on chromosome 20q, is amplified in as many as 60% of pancreatic cancers (Anzick et al., 1997; Calhoun et al., 2003; Aguirre et al., 2004). Altered AIB1 expression may contribute to the development of steroid-dependent cancers. It has also been reported that amplification of a localized region on the long arm of chromosome 8 is commonly seen in pancreatic cancers, and this amplification corresponds to the oncogenic transcription factor CMYC (MIM 190080) (Aguirre et al., 2004).

In addition to these genes, numbers of amplicons, amplified from DNA fragments, have been identified in pancreatic cancers by using gene chip technologies (Aguirre et al., 2004). Employing array comparative genomic hybridization (CGH) technology, a high resolution analysis of genome-wide copy number aberrations, permits to identify over expression of DNA fragments in tumor transformed pancreatic cells. Understanding the mechanisms underlying the development of pancreatic cancer may aid target early detection, genespecific therapies and thereby improve prognosis.

#### **3. Tumor suppressor genes**

In pancreatic invasive adenocarcinoma, CDKN2A/INK4A, TP53, and DPC4/SMAD4/ MADH4 are commonly inactivated.

#### **3.1 CDKN2A/INK4A gene (MIM 600160)**

The CDKN2A gene on chromosome 9p21 encodes proteins that control two critical cell cycle regulatory pathways, the p53 (TP53) pathway and the retinoblastoma (RB1) pathway. Through the use of shared coding regions and alternative reading frames, the CDKN2A gene produces 2 major proteins: p16(INK4), which is a cyclin-dependent kinase inhibitor checkpoint, and p14(ARF), which binds the p53-stabilizing protein MDM2 (Robertson and Jones, 1999). P16 inhibits cyclin D1 by binding to the cyclin-dependent kinases Cdk4 and Cdk6 thereby causing G1-S cell-cycle arrest (Schutte et al., 1997). Loss of p16 function,

The Genetics of Pancreatic Cancer 5

carcinoma (Li et al., 1988). Several families with Li-Fraumeni syndrome presenting with

About 90% of human somatic pancreatic carcinomas show allelic loss at 18q. Hahn et al. (1996) reported the identification of a putative tumor suppressor gene, namely, Deleted in Pancreatic Carcinoma 4 or DPC4 (also known as SMAD4/MADH4) on chromosome 18q21.1. Loss of Dpc4 protein function interferes with intracellular signaling cascades leading to decreased growth inhibition and uncontrolled proliferation. SMAD4 plays a pivotal role in signal transduction of the transforming growth factor beta superfamily cytokines by mediating transcriptional activation of target genes. Immunohistochemical labeling for Dpc4 protein expression mirrors DPC4/SMAD4/MADH4 gene status with rare exceptions, and like TP53, loss of Dpc4 expression is a late genetic event in pancreatic carcinoma and is observed in about 30% of progression lesions (Feldmann et al., 2007).

Genome-wide association studies (GWAS) have provided evidence that a person's risk of developing pancreatic cancer is influenced by multiple common disease alleles with small effects (Low et al., 2010; Petersen et al., 2010). Further research is required to evaluate the epidemiological input of these markers to the development of pancreatic cancer and their availability for early detection (Costello & Neoptolemos, 2011). Other tumor-suppressor genes are targeted at low frequency in pancreatic cancer. These genes provide a significant insight unto the molecular mechanism that underlines pancreatic cancers, and may serve as

Several gene ensembles, that play a role in caring for genome stability, were found to be mutated in pancreatic cancer, more so, in familial rather than sporadic cancer, including familial pancreatic cancer. BRCA2 is with no doubt the prominent gene in this category.

*BRCA1* - The gene product of BRCA1, functions in a number of cellular pathways that maintain genomic stability, including DNA damage-induced cell cycle checkpoint activation and arrest, DNA damage repair, protein ubiquitination, chromatin remodeling, as well as transcriptional regulation and apoptosis (see for example review by Wu et al., 2010). BRCA1 forms several distinct complexes through association with different adaptor proteins, and

*BRCA2 –* BRCA2 plays a key role in recombinational DNA repair, maintenance of genomic integrity and resistance to agents that damage DNA or collapse replication forks. The role of BRCA2 is best understood during DNA double-strand break repair (see for example Schlacher et al., 2011) as it co-localizes with PALB2 gene in nuclear foci, thereby promoting its stability in nuclear structures and enabling its recombinational repair and checkpoint

Both BRCA1 and BRCA2 have transcriptional activation and seem to be mutually

each complex assemble in a mutually exclusive manner (Wang et al., 2009).

pancreatic cancer were occasionally described (Lynch et al., 1985; Casey et al., 1993).

**3.3 Deleted in pancreatic carcinoma 4 (DPC4) gene (MIM 600993)** 

therapeutic targets in the early stages of pancreatic cancer.

**4. Genome-maintenance genes** 

**4.1 BRCA1/2 genes (MIM 113705/600185)** 

functions (Xia et al., 2006).

interrelated.

consequent to several different mechanisms, including homozygous deletion, intragenic mutation and epigenetic silencing by gene promoter methylation, is seen in approximately 90% of pancreatic cancers (Caldas et al., 1994; Schutte et al., 1997; Ueki et al., 2000). As a bystander effect, homozygous deletions of the CDKN2A/INK4A gene can also delete both copies of the methylthio-adenosine phosphorylase (MTAP) gene, whose product is essential for the salvage pathway of purine synthesis. In about a third of pancreatic cancers codeletion of the MTAP and CDKN2A/INK4A genes is observed (Hustinx et al., 2005).

This observation has a potential therapeutic significance, since chemotherapeutic regimes selectively targeted to cells demonstrating loss of Mtap function are currently available.

#### **3.1.1 Familial Atypical Multiple Mole Melanoma – Pancreatic Cancer (FAMMM-PC) syndrome (MIM 606719)**

The association between mutations in p16 (CDKN2A) and familial pancreatic cancer was previously noted by Caldas et al. (1994) and others (Liu et al., 1995; Whelan et al., 1995; Schutte et al., 1997). Further evidence for a plausible role of CDKN2A in pancreatic cancer was provided by Whelan et al. (1995) who described a kindred at risk for pancreatic cancers, melanomas, and additional types of tumors, co-segregating with a CDKN2A mutation. CDKN2A mutations were detected individuals with pancreatic cancer from melanoma families (Goldstein et al., 1995). Later, Lynch et al., 2002, coined the term hereditary FAMMM-PC syndrome to describe families with both melanoma and pancreatic cancers. Although rare, the life time risk of CDKN2A carriers, to develop pancreatic cancer and melanoma was calculated to be 58% and 39%, respectively (McWilliams et al., 2010). Basically, CDKN2A is a small gene, containing 3 coding exons. However, lack of founder mutations impedes the screening of families at risk in the clinical setting.

#### **3.2 TP53 gene (MIM 191170)**

The TP53 gene on chromosome 17p undergoes bi-allelic inactivation in approximately 50– 75% of pancreatic cancers, almost always subject to the combination of an intragenic mutation and the loss of the second wild-type allele (Redston et al., 1994). The transcription factor p53 responds to diverse cellular stresses formulated to regulate target genes participating in G1-S cell cycle checkpoint, maintenance of G2-M arrest, cell cycle arrest, apoptosis, senescence and DNA repair (Redston et al., 1994). There is emerging evidence to suggest that loss of p53 function may contribute to the genomic instability observed in pancreatic cancers (Hingorani et al., 2005); and that TP53 gene mutations constitute late events in pancreatic cancer progression (Maitra et al., 2003).

#### **3.2.1 Li- Fraumeni syndrome (MIM 151623)**

Li-Fraumeni syndrome is a rare, clinically and genetically heterogeneous, inherited cancer syndrome caused by germline mutations in TP53. Li-Fraumeni syndrome is characterized by autosomal dominant inheritance and early onset of tumors, rather multiple tumors in one individual and multiple affected family members. In contrast to other inherited cancer syndromes, which are predominantly characterized by site-specific cancers, Li-Fraumeni syndrome presents with a variety of tumor types. The most common types are soft tissue sarcomas and osteosarcomas, breast cancer, brain tumors, leukemia, and adrenocortical

consequent to several different mechanisms, including homozygous deletion, intragenic mutation and epigenetic silencing by gene promoter methylation, is seen in approximately 90% of pancreatic cancers (Caldas et al., 1994; Schutte et al., 1997; Ueki et al., 2000). As a bystander effect, homozygous deletions of the CDKN2A/INK4A gene can also delete both copies of the methylthio-adenosine phosphorylase (MTAP) gene, whose product is essential for the salvage pathway of purine synthesis. In about a third of pancreatic cancers codeletion of the MTAP and CDKN2A/INK4A genes is observed (Hustinx et al., 2005).

This observation has a potential therapeutic significance, since chemotherapeutic regimes selectively targeted to cells demonstrating loss of Mtap function are currently available.

The association between mutations in p16 (CDKN2A) and familial pancreatic cancer was previously noted by Caldas et al. (1994) and others (Liu et al., 1995; Whelan et al., 1995; Schutte et al., 1997). Further evidence for a plausible role of CDKN2A in pancreatic cancer was provided by Whelan et al. (1995) who described a kindred at risk for pancreatic cancers, melanomas, and additional types of tumors, co-segregating with a CDKN2A mutation. CDKN2A mutations were detected individuals with pancreatic cancer from melanoma families (Goldstein et al., 1995). Later, Lynch et al., 2002, coined the term hereditary FAMMM-PC syndrome to describe families with both melanoma and pancreatic cancers. Although rare, the life time risk of CDKN2A carriers, to develop pancreatic cancer and melanoma was calculated to be 58% and 39%, respectively (McWilliams et al., 2010). Basically, CDKN2A is a small gene, containing 3 coding exons. However, lack of founder

The TP53 gene on chromosome 17p undergoes bi-allelic inactivation in approximately 50– 75% of pancreatic cancers, almost always subject to the combination of an intragenic mutation and the loss of the second wild-type allele (Redston et al., 1994). The transcription factor p53 responds to diverse cellular stresses formulated to regulate target genes participating in G1-S cell cycle checkpoint, maintenance of G2-M arrest, cell cycle arrest, apoptosis, senescence and DNA repair (Redston et al., 1994). There is emerging evidence to suggest that loss of p53 function may contribute to the genomic instability observed in pancreatic cancers (Hingorani et al., 2005); and that TP53 gene mutations constitute late

Li-Fraumeni syndrome is a rare, clinically and genetically heterogeneous, inherited cancer syndrome caused by germline mutations in TP53. Li-Fraumeni syndrome is characterized by autosomal dominant inheritance and early onset of tumors, rather multiple tumors in one individual and multiple affected family members. In contrast to other inherited cancer syndromes, which are predominantly characterized by site-specific cancers, Li-Fraumeni syndrome presents with a variety of tumor types. The most common types are soft tissue sarcomas and osteosarcomas, breast cancer, brain tumors, leukemia, and adrenocortical

**3.1.1 Familial Atypical Multiple Mole Melanoma – Pancreatic Cancer (FAMMM-PC)** 

mutations impedes the screening of families at risk in the clinical setting.

events in pancreatic cancer progression (Maitra et al., 2003).

**3.2.1 Li- Fraumeni syndrome (MIM 151623)** 

**syndrome (MIM 606719)** 

**3.2 TP53 gene (MIM 191170)** 

carcinoma (Li et al., 1988). Several families with Li-Fraumeni syndrome presenting with pancreatic cancer were occasionally described (Lynch et al., 1985; Casey et al., 1993).

#### **3.3 Deleted in pancreatic carcinoma 4 (DPC4) gene (MIM 600993)**

About 90% of human somatic pancreatic carcinomas show allelic loss at 18q. Hahn et al. (1996) reported the identification of a putative tumor suppressor gene, namely, Deleted in Pancreatic Carcinoma 4 or DPC4 (also known as SMAD4/MADH4) on chromosome 18q21.1. Loss of Dpc4 protein function interferes with intracellular signaling cascades leading to decreased growth inhibition and uncontrolled proliferation. SMAD4 plays a pivotal role in signal transduction of the transforming growth factor beta superfamily cytokines by mediating transcriptional activation of target genes. Immunohistochemical labeling for Dpc4 protein expression mirrors DPC4/SMAD4/MADH4 gene status with rare exceptions, and like TP53, loss of Dpc4 expression is a late genetic event in pancreatic carcinoma and is observed in about 30% of progression lesions (Feldmann et al., 2007).

Genome-wide association studies (GWAS) have provided evidence that a person's risk of developing pancreatic cancer is influenced by multiple common disease alleles with small effects (Low et al., 2010; Petersen et al., 2010). Further research is required to evaluate the epidemiological input of these markers to the development of pancreatic cancer and their availability for early detection (Costello & Neoptolemos, 2011). Other tumor-suppressor genes are targeted at low frequency in pancreatic cancer. These genes provide a significant insight unto the molecular mechanism that underlines pancreatic cancers, and may serve as therapeutic targets in the early stages of pancreatic cancer.

#### **4. Genome-maintenance genes**

Several gene ensembles, that play a role in caring for genome stability, were found to be mutated in pancreatic cancer, more so, in familial rather than sporadic cancer, including familial pancreatic cancer. BRCA2 is with no doubt the prominent gene in this category.

#### **4.1 BRCA1/2 genes (MIM 113705/600185)**

*BRCA1* - The gene product of BRCA1, functions in a number of cellular pathways that maintain genomic stability, including DNA damage-induced cell cycle checkpoint activation and arrest, DNA damage repair, protein ubiquitination, chromatin remodeling, as well as transcriptional regulation and apoptosis (see for example review by Wu et al., 2010). BRCA1 forms several distinct complexes through association with different adaptor proteins, and each complex assemble in a mutually exclusive manner (Wang et al., 2009).

*BRCA2 –* BRCA2 plays a key role in recombinational DNA repair, maintenance of genomic integrity and resistance to agents that damage DNA or collapse replication forks. The role of BRCA2 is best understood during DNA double-strand break repair (see for example Schlacher et al., 2011) as it co-localizes with PALB2 gene in nuclear foci, thereby promoting its stability in nuclear structures and enabling its recombinational repair and checkpoint functions (Xia et al., 2006).

Both BRCA1 and BRCA2 have transcriptional activation and seem to be mutually interrelated.

The Genetics of Pancreatic Cancer 7

regards DNA repair, it should be considered, in principle, as a caretaker gene. Like BRCA2, PALB2 participates in DNA damage response and both genes collectively cooperate allowing BRCA2 to escape the effects of proteasome-mediated degradation (Reid et al., 2007;

Germline mutations in PALB2 have been identified in approximately 1-2% of familial breast cancer and 3-4% of familial pancreatic cancer cases (Slater et al., 2010; Casadei et al., 2011; Hofstatter et al., 2011). Three pancreatic cancer patients out of 96, with a positive family history of pancreatic cancer were found to harbor a PALB2 germline deletion of 4 basepairs, that was absent in 1084 control samples (Jones et al., 2009; Rahman et al., 2007). PALB2 appears to be the second most commonly mutated gene implicated in hereditary pancreatic

Pancreatic cancer was infrequently described in families with hereditary non-polyposis colon cancer (Lynch et al., 1985; Miyaki et al., 1997). HNPCC subdivided into Lynch I, primarily affecting the colon, Lynch II mainly targeting extra colonic organs including the pancreas and Muir-Torre syndrome. HNPCC is a genetically heterogeneous disease, with

*MSH2 (MIM 609309)* - The microsatellite DNA instability that is associated with alteration in the MSH2 gene in hereditary nonpolyposis colon cancer and several forms of sporadic cancer is thought to arise from defective repair of DNA replication errors. MSH2 has a direct role in mutation avoidance and microsatellite stability in human cells (Fishel et al., 1994).

*MLH1 (MIM 609310) –* Similarly to MSH2, MLH1 gene encodes a protein involved in the identification and repair of DNA mismatch errors. The identification of germline mutations in *MLH1* and *MSH2* was rapidly followed by the discovery of other human genes that encode proteins involved in the mismatch repair (MMR) complex (see review by Lynch et

Pancreatic cancer is of the most lethal of all human malignancies caused by inherited and acquired (somatic) mutations. The poor prognosis of pancreatic cancer (Jemal et al., 2006) warrants early detection of asymptomatic individuals, at high risk, using imaging methods and molecular analyses and thereby providing them with a chance for better survival (Goggins et al., 2000). Understanding the complex genetic mechanisms underlying the development of pancreatic cancer, as depicted in this chapter, may conduit medical science in the path that will ultimately lead to early detection, tailored treatment and consequently

Although, novel mechanisms, sprout on the horizon, could be exploited for early detection, as depicted by the KRAS detection technology, it seems that most pancreatic neoplasms in the general population will remain undetectable before invasive cancer develops. However, the recognition of early genetic somatic changes can advocate for presymptomatic chemo or

**4.3 Hereditary non-polyposis colon syndrome – HNPCC (MIM 120435)** 

Xia et al., 2007).

al., 2009).

**5. Synopsis** 

**4.2.1 Familial pancreatic cancer associated PALB2** 

most mutations detected in MSH2 and MLH1 genes.

better prognosis for this incurable disease.

cancer after BRCA2 (Jones et al., 2009).

Traditionally BRCA1 and BRCA2 were classified as tumor suppressor genes. Nowadays, BRCA1 and BRCA2 are rather cataloged as 'caretaker' genes that act, amongst other, as nucleotide-excision-repair (NER) genes (Kinzler and Vogelstein, 1997). While, inactivated 'gatekeepers', namely, tumor suppressor genes, promote tumor initiation directly, the inactivation of caretaker genes leads to genetic instability resulting in increased mutations in other genes, including gatekeepers. Once a tumor is initiated by inactivation of a caretaker gene, it may progress rapidly due to an accelerated rate of mutations in other genes that directly control cell birth or death. Consistent with this hypothesis, mutations in BRCA1 and BRCA2 are rarely found in sporadic cancers, and the risk of cancer arising in people with BRCA somatic mutations is relatively low.

#### **4.1.1 Hereditary breast-ovarian cancer syndrome**

Since the late nineties of the 20th century, excess of pancreatic cancer cases was documented in families with hereditary breast-ovarian cancer syndrome, traditionally linked to BRCA1/2 genes. Several studies have shown high BRCA2 mutation carrier frequencies in pancreatic cancer patients, reaching 10-20%, more so in Jewish Ashkenazi compared to non-Jewish pancreatic cancer patients (Teng et al., 1996; Ozcelik et al., 1997; Slater et al., 2010), with greater penetrance for males over females (Risch et al., 2001; Murphy et al., 2002; McWilliams et al., 2005; Dagan, 2008; Dagan et al., 2010; Ferrone et al., 2009). BRCA1 mutations are less often associated with pancreatic cancer compared to BRCA2 mutations (Al-Sukhni et al., 2008; Dagan et al., 2010). Mutations within the OCCR-ovarian cancercluster region of the BRCA2 gene in exon 11 frequently cause either/or pancreatic cancer, ovarian cancer and other type of cancers (Risch et al., 2001; Thompson et al., 2001).

The distinction between gatekeepers and caretakers genes has important practical and theoretical ramifications. Tumors that have defective caretaker genes are expected to respond favorably to therapeutic agents that induce the type of genomic damage that is normally detected or repaired by the particular caretaker gene involved.

Poly (ADP-ribose) polymerase (PARP) inhibitors have raised recent excitement as to their deleterious effect on BRCA1 or BRCA2 associated ovarian, breast or pancreatic cancer cells. If either PARP or BRCA function remains intact, a cell will continue to survive. Thus, inhibiting PARP should not affect the non-cancerous cells that contain one functional copy of BRCA. Loss of both functions, however, is incompatible with life (Bryant et al., 2005; Helleday et al., 2005; Drew et al., 2011). With this in mind, this class of agents has the potential to potentiate cytotoxic therapy without increased side effects. Acting as sole agents, they are able to exterminate cancer cells with DNA repair defects. The genomic instability of tumor cells allows PARP inhibitors to selectively target tumor cells rather than normal cells. PARP proteins inhibitors have gained supremacy as ideal anticancer agents (Weil & Chen, 2011) and may promise better prognosis in pancreatic, ovarian and breast cancer due to hereditary mutations in BRCA1/2.

#### **4.2 Partner and localizer of BRCA2 (PALB2) gene (MIM 610355)**

PALB2 maps to chromosome 16p12 (Xia et al., 2006; Reid et al., 2007; Xia et al., 2007). Differential extraction showed that BRCA2 and PALB2 colocalize in S-phase foci and are associated with stable nuclear structures. As PALB2 is critical for the function of BRCA2 as

Traditionally BRCA1 and BRCA2 were classified as tumor suppressor genes. Nowadays, BRCA1 and BRCA2 are rather cataloged as 'caretaker' genes that act, amongst other, as nucleotide-excision-repair (NER) genes (Kinzler and Vogelstein, 1997). While, inactivated 'gatekeepers', namely, tumor suppressor genes, promote tumor initiation directly, the inactivation of caretaker genes leads to genetic instability resulting in increased mutations in other genes, including gatekeepers. Once a tumor is initiated by inactivation of a caretaker gene, it may progress rapidly due to an accelerated rate of mutations in other genes that directly control cell birth or death. Consistent with this hypothesis, mutations in BRCA1 and BRCA2 are rarely found in sporadic cancers, and the risk of cancer arising in people with

Since the late nineties of the 20th century, excess of pancreatic cancer cases was documented in families with hereditary breast-ovarian cancer syndrome, traditionally linked to BRCA1/2 genes. Several studies have shown high BRCA2 mutation carrier frequencies in pancreatic cancer patients, reaching 10-20%, more so in Jewish Ashkenazi compared to non-Jewish pancreatic cancer patients (Teng et al., 1996; Ozcelik et al., 1997; Slater et al., 2010), with greater penetrance for males over females (Risch et al., 2001; Murphy et al., 2002; McWilliams et al., 2005; Dagan, 2008; Dagan et al., 2010; Ferrone et al., 2009). BRCA1 mutations are less often associated with pancreatic cancer compared to BRCA2 mutations (Al-Sukhni et al., 2008; Dagan et al., 2010). Mutations within the OCCR-ovarian cancercluster region of the BRCA2 gene in exon 11 frequently cause either/or pancreatic cancer,

ovarian cancer and other type of cancers (Risch et al., 2001; Thompson et al., 2001).

normally detected or repaired by the particular caretaker gene involved.

The distinction between gatekeepers and caretakers genes has important practical and theoretical ramifications. Tumors that have defective caretaker genes are expected to respond favorably to therapeutic agents that induce the type of genomic damage that is

Poly (ADP-ribose) polymerase (PARP) inhibitors have raised recent excitement as to their deleterious effect on BRCA1 or BRCA2 associated ovarian, breast or pancreatic cancer cells. If either PARP or BRCA function remains intact, a cell will continue to survive. Thus, inhibiting PARP should not affect the non-cancerous cells that contain one functional copy of BRCA. Loss of both functions, however, is incompatible with life (Bryant et al., 2005; Helleday et al., 2005; Drew et al., 2011). With this in mind, this class of agents has the potential to potentiate cytotoxic therapy without increased side effects. Acting as sole agents, they are able to exterminate cancer cells with DNA repair defects. The genomic instability of tumor cells allows PARP inhibitors to selectively target tumor cells rather than normal cells. PARP proteins inhibitors have gained supremacy as ideal anticancer agents (Weil & Chen, 2011) and may promise better prognosis in pancreatic, ovarian and breast

PALB2 maps to chromosome 16p12 (Xia et al., 2006; Reid et al., 2007; Xia et al., 2007). Differential extraction showed that BRCA2 and PALB2 colocalize in S-phase foci and are associated with stable nuclear structures. As PALB2 is critical for the function of BRCA2 as

BRCA somatic mutations is relatively low.

**4.1.1 Hereditary breast-ovarian cancer syndrome** 

cancer due to hereditary mutations in BRCA1/2.

**4.2 Partner and localizer of BRCA2 (PALB2) gene (MIM 610355)** 

regards DNA repair, it should be considered, in principle, as a caretaker gene. Like BRCA2, PALB2 participates in DNA damage response and both genes collectively cooperate allowing BRCA2 to escape the effects of proteasome-mediated degradation (Reid et al., 2007; Xia et al., 2007).

#### **4.2.1 Familial pancreatic cancer associated PALB2**

Germline mutations in PALB2 have been identified in approximately 1-2% of familial breast cancer and 3-4% of familial pancreatic cancer cases (Slater et al., 2010; Casadei et al., 2011; Hofstatter et al., 2011). Three pancreatic cancer patients out of 96, with a positive family history of pancreatic cancer were found to harbor a PALB2 germline deletion of 4 basepairs, that was absent in 1084 control samples (Jones et al., 2009; Rahman et al., 2007). PALB2 appears to be the second most commonly mutated gene implicated in hereditary pancreatic cancer after BRCA2 (Jones et al., 2009).

#### **4.3 Hereditary non-polyposis colon syndrome – HNPCC (MIM 120435)**

Pancreatic cancer was infrequently described in families with hereditary non-polyposis colon cancer (Lynch et al., 1985; Miyaki et al., 1997). HNPCC subdivided into Lynch I, primarily affecting the colon, Lynch II mainly targeting extra colonic organs including the pancreas and Muir-Torre syndrome. HNPCC is a genetically heterogeneous disease, with most mutations detected in MSH2 and MLH1 genes.

*MSH2 (MIM 609309)* - The microsatellite DNA instability that is associated with alteration in the MSH2 gene in hereditary nonpolyposis colon cancer and several forms of sporadic cancer is thought to arise from defective repair of DNA replication errors. MSH2 has a direct role in mutation avoidance and microsatellite stability in human cells (Fishel et al., 1994).

*MLH1 (MIM 609310) –* Similarly to MSH2, MLH1 gene encodes a protein involved in the identification and repair of DNA mismatch errors. The identification of germline mutations in *MLH1* and *MSH2* was rapidly followed by the discovery of other human genes that encode proteins involved in the mismatch repair (MMR) complex (see review by Lynch et al., 2009).

#### **5. Synopsis**

Pancreatic cancer is of the most lethal of all human malignancies caused by inherited and acquired (somatic) mutations. The poor prognosis of pancreatic cancer (Jemal et al., 2006) warrants early detection of asymptomatic individuals, at high risk, using imaging methods and molecular analyses and thereby providing them with a chance for better survival (Goggins et al., 2000). Understanding the complex genetic mechanisms underlying the development of pancreatic cancer, as depicted in this chapter, may conduit medical science in the path that will ultimately lead to early detection, tailored treatment and consequently better prognosis for this incurable disease.

Although, novel mechanisms, sprout on the horizon, could be exploited for early detection, as depicted by the KRAS detection technology, it seems that most pancreatic neoplasms in the general population will remain undetectable before invasive cancer develops. However, the recognition of early genetic somatic changes can advocate for presymptomatic chemo or

The Genetics of Pancreatic Cancer 9

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surgical prevention schemes that may alleviate those with pre cancerous neoplasms before an invasive cancer had a chance to develop. This farfetched undertaking is already underway.

Although, pancreatic cancer is basically sporadic, about 10% of the patients harbor a germline mutation. It seems that BRCA2 is the major susceptibility gene contributing to hereditary pancreatic cancer, especially in populations segregating founder mutations, namely, Ashkenazi Jews, Icelandic (Thorlacius et al., 1996; Dagan, 2008; Dagan et al., 2010) and others. Beyond this, pancreatic cancer patients and family members at risk should follow the standard recommendations, as regards genetic counseling and diagnosis that befits hereditary breast-ovarian cancer. Thus, the follow-up surveillance schemes for BRCA1/2 mutation carriers have to focus, in addition to the standard recommendations, on early detection of pancreatic cancer.

Deciphering the precise functional role of genes, involved in the development of pancreatic cancer, may open new and exciting targets for chemotherapy. The recognition that BRCA1/2 and PARP proteins combine forces in maintaining genomic stability and DNA damage repair, as well as transcriptional regulation and apoptosis, has prompted the clinical development of PARP inhibitors. It has been recently shown that PARP inhibitors are selectively toxic to human cancer cell lines with BRCA1/2 mutations. Furthermore, these agents may have a therapeutic potential in tumors with defects in homologous recombinant DNA repair (HRR) system (Drew et al., 2010). Clinical trials of PARP inhibitors, especially with olaparib, in BRCA1/2 mutated cancer patients confirm their potential therapeutic effect. Further studies are required to address the many questions regarding safety and efficacy in the clinical setting (Fong et al., 2009).

#### **6. References**


surgical prevention schemes that may alleviate those with pre cancerous neoplasms before an invasive cancer had a chance to develop. This farfetched undertaking is already

Although, pancreatic cancer is basically sporadic, about 10% of the patients harbor a germline mutation. It seems that BRCA2 is the major susceptibility gene contributing to hereditary pancreatic cancer, especially in populations segregating founder mutations, namely, Ashkenazi Jews, Icelandic (Thorlacius et al., 1996; Dagan, 2008; Dagan et al., 2010) and others. Beyond this, pancreatic cancer patients and family members at risk should follow the standard recommendations, as regards genetic counseling and diagnosis that befits hereditary breast-ovarian cancer. Thus, the follow-up surveillance schemes for BRCA1/2 mutation carriers have to focus, in addition to the standard recommendations, on

Deciphering the precise functional role of genes, involved in the development of pancreatic cancer, may open new and exciting targets for chemotherapy. The recognition that BRCA1/2 and PARP proteins combine forces in maintaining genomic stability and DNA damage repair, as well as transcriptional regulation and apoptosis, has prompted the clinical development of PARP inhibitors. It has been recently shown that PARP inhibitors are selectively toxic to human cancer cell lines with BRCA1/2 mutations. Furthermore, these agents may have a therapeutic potential in tumors with defects in homologous recombinant DNA repair (HRR) system (Drew et al., 2010). Clinical trials of PARP inhibitors, especially with olaparib, in BRCA1/2 mutated cancer patients confirm their potential therapeutic effect. Further studies are required to address the many questions regarding safety and

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**2**

*USA* 

**Systems and Network-Centric Understanding of** 

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is intractable to currently available treatment modalities (Vincent et al. 2011). Failure of standard chemo-, radio- and neoadjuvant single pathway targeted therapies indicate that before newer treatment regimens are designed, one has to re-visit the basic understanding of the origins and complexity of PDAC. As such, PDAC is now appreciated to have not only a highly heterogeneous pathology but is also a disease characterized by dysregulation of multiple pathways governing fundamental cell processes (Kim and Simeone 2011). Such complexity has been suggested to be governed by molecular networks that execute metabolic or cytoskeletal processes, or their regulation by complex signal transduction originating from diverse genetic mutations (Figure 1). A major challenge, therefore, is to understand how to develop actionable modulation of this multivariate dysregulation, with respect to both how it arises from diverse genetic mutations and to how it may be ameliorated by prospective treatments in PDAC. Lack of understanding in both these areas is certainly a major underlying reason for failure of most of the available and clinically used drugs (Stathis and Moore 2010). The pharmaceutical industry handpicked drugs have been generally based on their specificity towards a particular protein and the subsequent targeted pathway (K-Ras, PI3K, MEK, EGFR, p53 etc) without considering the effect of modulating secondary and interacting pathways (Almhanna and Philip 2011; Philip 2011). However, as results from integrated network modeling and systems biology studies indicate, targeting one protein is not straightforward as each protein in a cellular system works in a complex interacting network comprised of a myriad interconnected pathways (Wist et al. 2009a). Silencing one protein/pathway can have multiple effects on different secondary pathways leading to secondary effects. For example, activation of salvage pathways (commonly observed in PDAC) can result in diminished drug response or in some cases acquired resistance. Therefore, in order to decode this complexity and to understand both the PDAC disease and identify drug targets, it requires a departure from a protein-centric to a more advanced network-centric view. This chapter deals with recent advancements on deciphering PDAC disease networks and drug response networks based on integrated systems and network biology-driven science. It is believed that such integrated and holistic approach will help in not only delineating the mechanism of resistance of this complex disease, it will also aid in the future design of targeted drug

**1. Introduction** 

combinations that will improve the dismal cure rate.

**Pancreatic Ductal Adenocarcinoma Signalling** 

Irfana Muqbil, Ramzi M. Mohammad, Fazlul H. Sarkar and Asfar S. Azmi

*Wayne State University,* 

Fanconi anemia is associated with a defect in the BRCA2 partner PALB2. Nature Genet. 39: 159-161.

Zogopoulous G, Rothenmund H, Eppel A, Ash C, Akbari MR, Hedley D, Narod SA, Gallinger S (2007) The P239S palladin variant does not account for a significant fraction of hereditary or early onset pancreatic cancer. Hum Genet. 121: 635-637.

### **Systems and Network-Centric Understanding of Pancreatic Ductal Adenocarcinoma Signalling**

Irfana Muqbil, Ramzi M. Mohammad, Fazlul H. Sarkar and Asfar S. Azmi *Wayne State University, USA* 

#### **1. Introduction**

14 Pancreatic Cancer – Clinical Management

Zogopoulous G, Rothenmund H, Eppel A, Ash C, Akbari MR, Hedley D, Narod SA,

Genet. 39: 159-161.

Fanconi anemia is associated with a defect in the BRCA2 partner PALB2. Nature

Gallinger S (2007) The P239S palladin variant does not account for a significant fraction of hereditary or early onset pancreatic cancer. Hum Genet. 121: 635-637.

> Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is intractable to currently available treatment modalities (Vincent et al. 2011). Failure of standard chemo-, radio- and neoadjuvant single pathway targeted therapies indicate that before newer treatment regimens are designed, one has to re-visit the basic understanding of the origins and complexity of PDAC. As such, PDAC is now appreciated to have not only a highly heterogeneous pathology but is also a disease characterized by dysregulation of multiple pathways governing fundamental cell processes (Kim and Simeone 2011). Such complexity has been suggested to be governed by molecular networks that execute metabolic or cytoskeletal processes, or their regulation by complex signal transduction originating from diverse genetic mutations (Figure 1). A major challenge, therefore, is to understand how to develop actionable modulation of this multivariate dysregulation, with respect to both how it arises from diverse genetic mutations and to how it may be ameliorated by prospective treatments in PDAC. Lack of understanding in both these areas is certainly a major underlying reason for failure of most of the available and clinically used drugs (Stathis and Moore 2010). The pharmaceutical industry handpicked drugs have been generally based on their specificity towards a particular protein and the subsequent targeted pathway (K-Ras, PI3K, MEK, EGFR, p53 etc) without considering the effect of modulating secondary and interacting pathways (Almhanna and Philip 2011; Philip 2011). However, as results from integrated network modeling and systems biology studies indicate, targeting one protein is not straightforward as each protein in a cellular system works in a complex interacting network comprised of a myriad interconnected pathways (Wist et al. 2009a). Silencing one protein/pathway can have multiple effects on different secondary pathways leading to secondary effects. For example, activation of salvage pathways (commonly observed in PDAC) can result in diminished drug response or in some cases acquired resistance. Therefore, in order to decode this complexity and to understand both the PDAC disease and identify drug targets, it requires a departure from a protein-centric to a more advanced network-centric view. This chapter deals with recent advancements on deciphering PDAC disease networks and drug response networks based on integrated systems and network biology-driven science. It is believed that such integrated and holistic approach will help in not only delineating the mechanism of resistance of this complex disease, it will also aid in the future design of targeted drug combinations that will improve the dismal cure rate.

Systems and Network-Centric Understanding of Pancreatic Ductal Adenocarcinoma Signalling 17

These early and late genetic alterations have fundamental roles affecting key guardians of cellular signaling, which induces instability of entire molecular systems such as cell growth, division, apoptosis and migration. Mutation in proto-oncogenes gives rise to oncogenes that are often present in PDAC. These mutations cause the protein products of oncogenes to be permanently activated, resulting in uncontrolled cell proliferation. Oncogenic mutations exhibit a dominant characteristic and deficiency of one allele (i.e. heterozygous mutation) is sufficient for a lethal outcome. There are several key proto-oncogenes involved in PDAC, including KRAS, Her2/Neu, CTNNB1 (β-catenin), PIK3CA or AKT1. The most common oncogenic mutation types are point mutations, deletions, gene amplifications, and gene re-

On the other hand, tumor suppressor genes code for proteins that act against cell proliferation. As a result of late event genetic alterations, their normal function may be reduced or even completely eliminated. Mutations in tumor suppressor genes have recessive characteristics and hence, the cell looses its function only when both alleles are affected. Commonly, described as a double hit model, one allele is initially mutated while the other is subsequently mutated or lost completely (Serra et al. 1997). In addition, there are numerous epigenetic controls of tumor suppressors that involve deactivation by hypermethylation (Herman et al. 1996). In PDAC, the frequently affected tumor suppressors include the guardian regulator TP53 (Barton et al. 1991), APC (Horii et al. 1992); SMAD4

Intense research over the last three decades have revealed that PDAC has a highly intricate web of de-regulatory signaling. In pancreatic duct cells, molecular biologist have identified some of the core signaling pathways that are aberrantly expressed that consequently leads to development of PDAC. Major cell surface receptor de-regulatory mechanisms include the c-MET/HGF (hepatocyte growth factor) signaling pathway which is a key factor in early progression of PDAC. This pathway is responsible for invasive growth of PDAC through activation of key oncogenes, angiogenesis and scattering (cell dissociation and metastasis). c-MET is a proto-oncogene that encodes an HGF receptor that has a primary function in embryonic development and wound healing (Chmielowiec et al. 2007). Even though c-MET mRNA is present at very small amounts in normal human exocrine pancreas, it is upregulated in a majority of PDAC. Interestingly overexpression of c-MET has been observed in regenerative tissue affected by acute pancreatitis (Otte et al. 2000), and has been linked to early events in PDAC carcinogenesis. HGF is a primary ligand of c-MET. Upon c-MET/HGF interaction, several different signaling pathways are activated, including the Ras, phosphoinositide 3-kinase (PI3K), JAK signal transducer and activator of transcription

The second major cell surface signaling found altered in PDAC is the Ras/Raf/MAPK pathway. The Ras/Raf/mitogen-activated protein kinase (MAPK) pathway is one of the most elaborately studied signaling pathways in PDAC and other cancers (Molina and Adjei 2006). The role of Ras/Raf/MAPK signaling is critical for many carcinogeneic processes, including cell growth, division, cell differentiation, invasion and migration, wound healing repair, and angiogenic processes. The central regulator of this multivariate signal transduction from extracellular to intracellular environment is the Ras protein, which is

arrangements.

(Bartsch et al. 1999) and TP16 (Caldas et al. 1994).

(STAT) and β-catenin (Wnt) pathways.

**2.1.1 Complex de-regulatory signaling mechanisms in PDAC** 

Fig. 1. Genetic alterations in PDAC are categorized into early state (oncogenes, K-Ras, Her2/Neu); Late Stage (tumor suppressors, p16, Smad4, BRCA2) and chromosomal instability pathways that accelerate progression from PanIN-1A lesions to metastatic PDAC.

#### **2. Complex PDAC genetic network**

PDAC is highly complex malignancy with myriad set of de-regulated mechanisms involved and affecting the tissue at different stages of the disease. Detailed molecular mechanisms of initiation, development and progression of PDAC have been thoroughly studied since the basic principles of the disease were revealed in the 1970s (Pour et al. 2003; Morosco et al. 1981; Morosco and Goeringer 1980). The most acceptable model is the classical one that describes morphological as well as molecular transformation from precursor lesions into invasive carcinoma (Hruban et al. 2000a; Hruban et al. 2000b). While the standard nomenclature and diagnostic criteria for classification of PDAC has primarily been based on grades of pancreatic intraepithelial neoplasia (PanIN) (Hruban et al. 2001), cumulatively it has been accepted that PDAC is a genetically and epigenetically complex disease that arises through a combination of events. It is increasingly being accepted that these complexities cannot be fully understood by traditional molecular biology techniques and integrated approaches may play pivotal role in the better understanding of PDAC as are discussed below.

#### **2.1 Interaction of oncogenes and tumor suppressor genes in PDAC**

PDAC origin and progression is broadly classified to be result of three major events (a) early stage genetic alterations in the proto-oncogenes mainly K-ras and Her-2/Neu; (b) late stage alterations in tumor suppressor genes such as p53, p16, Smad4 and BRCA2 and (c) chromosomal instability/precursor lesion in the normal duct (i.e. formation of PanIN-1a and PanIN-1B to Pan-IN-2 and Pan-IN3 (summarized in Figure 1).

Fig. 1. Genetic alterations in PDAC are categorized into early state (oncogenes, K-Ras, Her2/Neu); Late Stage (tumor suppressors, p16, Smad4, BRCA2) and chromosomal

**2.1 Interaction of oncogenes and tumor suppressor genes in PDAC** 

PanIN-1B to Pan-IN-2 and Pan-IN3 (summarized in Figure 1).

**2. Complex PDAC genetic network** 

below.

instability pathways that accelerate progression from PanIN-1A lesions to metastatic PDAC.

PDAC is highly complex malignancy with myriad set of de-regulated mechanisms involved and affecting the tissue at different stages of the disease. Detailed molecular mechanisms of initiation, development and progression of PDAC have been thoroughly studied since the basic principles of the disease were revealed in the 1970s (Pour et al. 2003; Morosco et al. 1981; Morosco and Goeringer 1980). The most acceptable model is the classical one that describes morphological as well as molecular transformation from precursor lesions into invasive carcinoma (Hruban et al. 2000a; Hruban et al. 2000b). While the standard nomenclature and diagnostic criteria for classification of PDAC has primarily been based on grades of pancreatic intraepithelial neoplasia (PanIN) (Hruban et al. 2001), cumulatively it has been accepted that PDAC is a genetically and epigenetically complex disease that arises through a combination of events. It is increasingly being accepted that these complexities cannot be fully understood by traditional molecular biology techniques and integrated approaches may play pivotal role in the better understanding of PDAC as are discussed

PDAC origin and progression is broadly classified to be result of three major events (a) early stage genetic alterations in the proto-oncogenes mainly K-ras and Her-2/Neu; (b) late stage alterations in tumor suppressor genes such as p53, p16, Smad4 and BRCA2 and (c) chromosomal instability/precursor lesion in the normal duct (i.e. formation of PanIN-1a and

These early and late genetic alterations have fundamental roles affecting key guardians of cellular signaling, which induces instability of entire molecular systems such as cell growth, division, apoptosis and migration. Mutation in proto-oncogenes gives rise to oncogenes that are often present in PDAC. These mutations cause the protein products of oncogenes to be permanently activated, resulting in uncontrolled cell proliferation. Oncogenic mutations exhibit a dominant characteristic and deficiency of one allele (i.e. heterozygous mutation) is sufficient for a lethal outcome. There are several key proto-oncogenes involved in PDAC, including KRAS, Her2/Neu, CTNNB1 (β-catenin), PIK3CA or AKT1. The most common oncogenic mutation types are point mutations, deletions, gene amplifications, and gene rearrangements.

On the other hand, tumor suppressor genes code for proteins that act against cell proliferation. As a result of late event genetic alterations, their normal function may be reduced or even completely eliminated. Mutations in tumor suppressor genes have recessive characteristics and hence, the cell looses its function only when both alleles are affected. Commonly, described as a double hit model, one allele is initially mutated while the other is subsequently mutated or lost completely (Serra et al. 1997). In addition, there are numerous epigenetic controls of tumor suppressors that involve deactivation by hypermethylation (Herman et al. 1996). In PDAC, the frequently affected tumor suppressors include the guardian regulator TP53 (Barton et al. 1991), APC (Horii et al. 1992); SMAD4 (Bartsch et al. 1999) and TP16 (Caldas et al. 1994).

#### **2.1.1 Complex de-regulatory signaling mechanisms in PDAC**

Intense research over the last three decades have revealed that PDAC has a highly intricate web of de-regulatory signaling. In pancreatic duct cells, molecular biologist have identified some of the core signaling pathways that are aberrantly expressed that consequently leads to development of PDAC. Major cell surface receptor de-regulatory mechanisms include the c-MET/HGF (hepatocyte growth factor) signaling pathway which is a key factor in early progression of PDAC. This pathway is responsible for invasive growth of PDAC through activation of key oncogenes, angiogenesis and scattering (cell dissociation and metastasis). c-MET is a proto-oncogene that encodes an HGF receptor that has a primary function in embryonic development and wound healing (Chmielowiec et al. 2007). Even though c-MET mRNA is present at very small amounts in normal human exocrine pancreas, it is upregulated in a majority of PDAC. Interestingly overexpression of c-MET has been observed in regenerative tissue affected by acute pancreatitis (Otte et al. 2000), and has been linked to early events in PDAC carcinogenesis. HGF is a primary ligand of c-MET. Upon c-MET/HGF interaction, several different signaling pathways are activated, including the Ras, phosphoinositide 3-kinase (PI3K), JAK signal transducer and activator of transcription (STAT) and β-catenin (Wnt) pathways.

The second major cell surface signaling found altered in PDAC is the Ras/Raf/MAPK pathway. The Ras/Raf/mitogen-activated protein kinase (MAPK) pathway is one of the most elaborately studied signaling pathways in PDAC and other cancers (Molina and Adjei 2006). The role of Ras/Raf/MAPK signaling is critical for many carcinogeneic processes, including cell growth, division, cell differentiation, invasion and migration, wound healing repair, and angiogenic processes. The central regulator of this multivariate signal transduction from extracellular to intracellular environment is the Ras protein, which is

Systems and Network-Centric Understanding of Pancreatic Ductal Adenocarcinoma Signalling 19

SMADs form a complex with SMAD4, which localizes it in the nucleus and where it interacts with other factors to stimulate transcription of genes that are important for cell cycle arrest and migration. SMAD4 is therefore a key mediator for TGF-β signals. Due to its frequent absence in proliferating PDAC tissue, it is also known as DPC or "deleted in pancreatic cancer" (Schutte et al. 1995). Relatively high frequency of SMAD4 mutations and loss of heterozygosity at the DPC4 locus (18q21.1) strongly suggest that the protein is a primary tumor suppressor involved in PDAC carcinogenesis process. However, it should be noted that reinstating SMAD4 expression results in tumor growth suppression only *in vivo* and not *in vitro*. It has also been found that a SMAD4-independent pathways may be

Wnt signaling is crucial to formation and maintenance of pancreas (Dessimoz and Grapin-Botton 2006; Dessimoz et al. 2005). During PDAC development, hyper-activation of Wnt triggers transcription of a number of genes that have a direct impact on cell proliferation, differentiation and migration (Cano et al. 2008; Rulifson et al. 2007). Activation of Wnt signaling is through interaction of a family of membrane-bound receptors known as Frizzleds with Wnt ligands. Once activated, the downstream signals may proceed through independent pathways. In a canonical pathway, signal transduction is mediated through stabilization and translocation of β-catenin from the cytosol into the nucleus followed by its interaction with T-cell factor that in turn activates transcription of target genes. The localization of high expression levels of β-catenin in the nucleus has been experimentally confirmed in various high grade PanIN lesions, as well as in advanced PDAC (Al-Aynati et al. 2004). In non-canonical, β-catenin-independent pathways, other signaling mediators are involved, that block the β-catenin assisted transcription. The nuclear localization of βcatenin and high expression levels of WNT5a, a gene involved in non-canonical Wnt

The cell cycle control genes have profound importance in PDAC and CDKN2A is one of key factors in its negative control. The CDKN2A has two promoters and alternative splicing sites that give rise to two alternative protein products: cyclin-dependent kinase inhibitor p16INK4a and p53-activator p14ARF. Although both proteins are active in negative control of the cell cycle, only the function of p16INK4a is frequently lost in PDAC due to point mutations, deletions or hypermethylation . p16INK4a protein (also known as p16) inhibits key elements of cell cycle progression at the G1 checkpoint. p16 inactivation is an early event in pancreatic carcinogenesis, and low levels of p16 expression are associated with larger tumors, risk of early metastases and poor survival. The network interactions of de-

In summary, the above comprehensive set of studies accumulated over the years clearly show that PDAC is a highly complex disease. Traditional molecular biology focuses on studying these alterations in a single protein-centric manner honing on individual pathways. There are unanswered questions regarding the interaction between these deregulatory signaling mechanisms that may be related to the cause of such dismal outcomes in PDAC. This is indeed the case as pharmaceutical companies handpick drugs to target individual protein and not multiple pathways. Even if a drug blocks one signaling molecule in the tumor, another salvage pathway becomes activated leading to diminished efficacy of the drugs. Therefore, we are of the view that an integrated holistic approach is needed to to

responsible for tumorigenic effect of TGF-β signaling (Levy and Hill 2005).

pathways, suggests involvement of both pathways in PDAC progression.

regulatory signaling pathways in PDAC are depicted in Figure 2.

localized at the inner side of the cellular membrane. Under normal physiological conditions, the hydrophobic Ras protein is in its inactive GDP-bound form. In the event of an extracellular signal coming through growth factor receptors, their is removal of GDP from Ras protein and its subsequent activation upon binding to GTP. Activated Ras complex triggers kinase activity of Raf kinase, which ultimately results in activation of an MAPK. MAPK kinase (MAPKK) in turn is an important regulator of DNA transcription and mRNA translation. Mutations that affect any of the Ras/Raf/MAPK members produce an increase in tumorigenicity through hyper-activation of DNA machinery and mRNA translation. Besides Raf and MAPK, there are other downstream effectors of Ras protein, including PI3K, thus providing crosstalk between multiple pathways.

Aside from Ras pathway, the PTEN/PI3K/AKT signaling axis is found altered in PDAC. This pathway is fundamentally based on regulated activation of AKT through its localization at the cell membrane (Carnero et al. 2008). PI3K and PTEN phosphatases are two important protein families involved in the membrane localization of AKT. PI3K phosphorylates certain membrane-bound lipids known as phosphoinositides producing three different phosphatidylinositol 3-phosphate (PIP), phosphatidylinositol (3,4) bisphosphate (PIP2), and phosphatidylinositol (3,4,5)-trisphosphate (PIP3). The phosphorylated forms, PIP3 and, to a lesser extent, PIP2, attract important protein kinases to the cell membrane. The most prominent is AKT, a family of serine/threonine protein kinases that trigger a number of key cellular processes, including glucose metabolism, cell proliferation, and apoptosis, transcription, and cell migration (Maitra and Hruban 2005). AKT activity is strongly dependent on its proper localization on the cell membrane. The positioning of AKT at the membrane is achieved through its strong binding to PIP3. In pancreatic carcinogenesis, AKT1 acts as an oncogene that upholds cell survival by overcoming cell cycle arrest, blocking apoptosis, and promoting angiogenesis. PTEN is a phosphatase that acts in opposition to PI3K. It has tumor suppression ability by converting PIP3 back to PIP2 and to PIP, hence disrupting membrane localization and reducing activity of AKT. In most cancers, expression levels of PI3Ks and AKT are high, while PTEN is often deactivated by mutation, or deleted completely. Through its key role in pancreatic carcinogenesis, PI3K/AKT/PTEN signaling is an important target for anticancer therapy.

The JAK/STAT signaling pathway also has an important role in regulation of DNA transcription by inducing chemical signals from cytokine receptors into the cell nucleus. The signal is phosphorylation dependent prompting activation and dimerization in a family of STAT proteins. Activated STAT dimers initiate DNA transcription inside the nucleus. It is known that inhibition of JAK/STAT signaling induces apoptosis in various human cancers, and is therefore, a primary focus for potential new drug candidates (Buettner et al. 2002). A recent study has reported reduced growth of pancreatic cancer cells *in vitro* when exposed to benzyl isothiocyanate, through suppression of STAT3 signaling and subsequent induction of apoptosis. This is suggested as a possible explanation of the anti-carcinogenic effect of cruciferous vegetables (such as broccoli, cauliflower, cabbage or horseradish) that are rich in isothiocyanates.

TGF-β is a ligand that binds to type II cytokine receptor dimer, which then interacts and activates type I cytokine receptor dimer, triggering phosphorylation of receptor-regulated SMADs (R-SMADs), mainly SMAD2 and SMAD3. In the phosphorylated form, the R-

localized at the inner side of the cellular membrane. Under normal physiological conditions, the hydrophobic Ras protein is in its inactive GDP-bound form. In the event of an extracellular signal coming through growth factor receptors, their is removal of GDP from Ras protein and its subsequent activation upon binding to GTP. Activated Ras complex triggers kinase activity of Raf kinase, which ultimately results in activation of an MAPK. MAPK kinase (MAPKK) in turn is an important regulator of DNA transcription and mRNA translation. Mutations that affect any of the Ras/Raf/MAPK members produce an increase in tumorigenicity through hyper-activation of DNA machinery and mRNA translation. Besides Raf and MAPK, there are other downstream effectors of Ras protein, including

Aside from Ras pathway, the PTEN/PI3K/AKT signaling axis is found altered in PDAC. This pathway is fundamentally based on regulated activation of AKT through its localization at the cell membrane (Carnero et al. 2008). PI3K and PTEN phosphatases are two important protein families involved in the membrane localization of AKT. PI3K phosphorylates certain membrane-bound lipids known as phosphoinositides producing three different phosphatidylinositol 3-phosphate (PIP), phosphatidylinositol (3,4) bisphosphate (PIP2), and phosphatidylinositol (3,4,5)-trisphosphate (PIP3). The phosphorylated forms, PIP3 and, to a lesser extent, PIP2, attract important protein kinases to the cell membrane. The most prominent is AKT, a family of serine/threonine protein kinases that trigger a number of key cellular processes, including glucose metabolism, cell proliferation, and apoptosis, transcription, and cell migration (Maitra and Hruban 2005). AKT activity is strongly dependent on its proper localization on the cell membrane. The positioning of AKT at the membrane is achieved through its strong binding to PIP3. In pancreatic carcinogenesis, AKT1 acts as an oncogene that upholds cell survival by overcoming cell cycle arrest, blocking apoptosis, and promoting angiogenesis. PTEN is a phosphatase that acts in opposition to PI3K. It has tumor suppression ability by converting PIP3 back to PIP2 and to PIP, hence disrupting membrane localization and reducing activity of AKT. In most cancers, expression levels of PI3Ks and AKT are high, while PTEN is often deactivated by mutation, or deleted completely. Through its key role in pancreatic carcinogenesis, PI3K/AKT/PTEN signaling is an important target for

The JAK/STAT signaling pathway also has an important role in regulation of DNA transcription by inducing chemical signals from cytokine receptors into the cell nucleus. The signal is phosphorylation dependent prompting activation and dimerization in a family of STAT proteins. Activated STAT dimers initiate DNA transcription inside the nucleus. It is known that inhibition of JAK/STAT signaling induces apoptosis in various human cancers, and is therefore, a primary focus for potential new drug candidates (Buettner et al. 2002). A recent study has reported reduced growth of pancreatic cancer cells *in vitro* when exposed to benzyl isothiocyanate, through suppression of STAT3 signaling and subsequent induction of apoptosis. This is suggested as a possible explanation of the anti-carcinogenic effect of cruciferous vegetables (such as broccoli, cauliflower, cabbage or horseradish) that are rich in

TGF-β is a ligand that binds to type II cytokine receptor dimer, which then interacts and activates type I cytokine receptor dimer, triggering phosphorylation of receptor-regulated SMADs (R-SMADs), mainly SMAD2 and SMAD3. In the phosphorylated form, the R-

PI3K, thus providing crosstalk between multiple pathways.

anticancer therapy.

isothiocyanates.

SMADs form a complex with SMAD4, which localizes it in the nucleus and where it interacts with other factors to stimulate transcription of genes that are important for cell cycle arrest and migration. SMAD4 is therefore a key mediator for TGF-β signals. Due to its frequent absence in proliferating PDAC tissue, it is also known as DPC or "deleted in pancreatic cancer" (Schutte et al. 1995). Relatively high frequency of SMAD4 mutations and loss of heterozygosity at the DPC4 locus (18q21.1) strongly suggest that the protein is a primary tumor suppressor involved in PDAC carcinogenesis process. However, it should be noted that reinstating SMAD4 expression results in tumor growth suppression only *in vivo* and not *in vitro*. It has also been found that a SMAD4-independent pathways may be responsible for tumorigenic effect of TGF-β signaling (Levy and Hill 2005).

Wnt signaling is crucial to formation and maintenance of pancreas (Dessimoz and Grapin-Botton 2006; Dessimoz et al. 2005). During PDAC development, hyper-activation of Wnt triggers transcription of a number of genes that have a direct impact on cell proliferation, differentiation and migration (Cano et al. 2008; Rulifson et al. 2007). Activation of Wnt signaling is through interaction of a family of membrane-bound receptors known as Frizzleds with Wnt ligands. Once activated, the downstream signals may proceed through independent pathways. In a canonical pathway, signal transduction is mediated through stabilization and translocation of β-catenin from the cytosol into the nucleus followed by its interaction with T-cell factor that in turn activates transcription of target genes. The localization of high expression levels of β-catenin in the nucleus has been experimentally confirmed in various high grade PanIN lesions, as well as in advanced PDAC (Al-Aynati et al. 2004). In non-canonical, β-catenin-independent pathways, other signaling mediators are involved, that block the β-catenin assisted transcription. The nuclear localization of βcatenin and high expression levels of WNT5a, a gene involved in non-canonical Wnt pathways, suggests involvement of both pathways in PDAC progression.

The cell cycle control genes have profound importance in PDAC and CDKN2A is one of key factors in its negative control. The CDKN2A has two promoters and alternative splicing sites that give rise to two alternative protein products: cyclin-dependent kinase inhibitor p16INK4a and p53-activator p14ARF. Although both proteins are active in negative control of the cell cycle, only the function of p16INK4a is frequently lost in PDAC due to point mutations, deletions or hypermethylation . p16INK4a protein (also known as p16) inhibits key elements of cell cycle progression at the G1 checkpoint. p16 inactivation is an early event in pancreatic carcinogenesis, and low levels of p16 expression are associated with larger tumors, risk of early metastases and poor survival. The network interactions of deregulatory signaling pathways in PDAC are depicted in Figure 2.

In summary, the above comprehensive set of studies accumulated over the years clearly show that PDAC is a highly complex disease. Traditional molecular biology focuses on studying these alterations in a single protein-centric manner honing on individual pathways. There are unanswered questions regarding the interaction between these deregulatory signaling mechanisms that may be related to the cause of such dismal outcomes in PDAC. This is indeed the case as pharmaceutical companies handpick drugs to target individual protein and not multiple pathways. Even if a drug blocks one signaling molecule in the tumor, another salvage pathway becomes activated leading to diminished efficacy of the drugs. Therefore, we are of the view that an integrated holistic approach is needed to to

Systems and Network-Centric Understanding of Pancreatic Ductal Adenocarcinoma Signalling 21

targets and that would ultimately benefit in designing personalized medicine (Figure 3 depicting integration of multiple high-throughput technologies for better approach and

> **Targets Biomarkers Personalized Medicine Mechanistic Understanding**

> > **Metabolomics Protein Networks**

**Tailored Drug Design**

**Interaction Networks**

Fig. 3. Systems Biology is a potent tool for designing personalized medicine, predicting

This type of association study can be applied to both affected and healthy cohorts, or in relation to particular phenotypes, such as disease susceptibility (for example, diabetes) (Saxena et al. 2007), or to study individual responses to drugs. As a result, genetic variations have been identified through comprehensive re-sequencing studies of cancer-related mutations in colon and breast tumors, leading to the identification of around 80 DNA alterations in a typical cancer (Wood et al. 2007). This technology has been applied to understand PDAC genetics, pathway interactions and in identifying PDAC stem cells and

As a proof of concept, the first study on the use of proteomic profiling was published by Lohr and group and they showed how integrated technologies could be utilized in obtaining PDAC biomarkers (Lohr et al. 2006). In this study, it was postulated that this type of proteomic approach was extremely necessary in the rationale for the design of drugs for this deadly malignancy. Later, a number of investigations have demonstrated that indeed this technology can be applied to unwind the complex web of interacting pathways in PDAC. For example, in an elegant study, Chelala and colleagues provided pancreatic expression database that was a generic model for organization, integration and mining of

biomarkers and targets and mechanistic understanding of complex diseases.

**3.1 Systems understanding of PDAC expression datasets** 

**MOLECULAR NETWORKS PDAC THERAPY**

treatment to a disease).

**Genotyping Sequencing Proteomic**

**High-throughput** 

**Bioinformatics**

are discussed below.

**Transcriptomics Comparative genomic hybridization**

first understand the interactions between individual pathways that will aid in the design of single or combination regimens for the elimination of PDAC.

Fig. 2. Complex de-regulatory network of PDAC obtained from Ingenuity Pathway Analysis Database.

#### **3. Systems biology and its use in understanding the complexity of PDAC**

Applicability of systems biology is slowly being realized in the clinic (Faratian et al. 2009). Currently, combining information on patient history with high throughput bioinformatics such as genotyping, transcriptomics and comparative genomic hybridization, sequencing, and proteomics, followed by molecular network analysis, one can predict biomarkers and

first understand the interactions between individual pathways that will aid in the design of

Fig. 2. Complex de-regulatory network of PDAC obtained from Ingenuity Pathway Analysis

Applicability of systems biology is slowly being realized in the clinic (Faratian et al. 2009). Currently, combining information on patient history with high throughput bioinformatics such as genotyping, transcriptomics and comparative genomic hybridization, sequencing, and proteomics, followed by molecular network analysis, one can predict biomarkers and

**3. Systems biology and its use in understanding the complexity of PDAC** 

Database.

single or combination regimens for the elimination of PDAC.

targets and that would ultimately benefit in designing personalized medicine (Figure 3 depicting integration of multiple high-throughput technologies for better approach and treatment to a disease).

Fig. 3. Systems Biology is a potent tool for designing personalized medicine, predicting biomarkers and targets and mechanistic understanding of complex diseases.

This type of association study can be applied to both affected and healthy cohorts, or in relation to particular phenotypes, such as disease susceptibility (for example, diabetes) (Saxena et al. 2007), or to study individual responses to drugs. As a result, genetic variations have been identified through comprehensive re-sequencing studies of cancer-related mutations in colon and breast tumors, leading to the identification of around 80 DNA alterations in a typical cancer (Wood et al. 2007). This technology has been applied to understand PDAC genetics, pathway interactions and in identifying PDAC stem cells and are discussed below.

#### **3.1 Systems understanding of PDAC expression datasets**

As a proof of concept, the first study on the use of proteomic profiling was published by Lohr and group and they showed how integrated technologies could be utilized in obtaining PDAC biomarkers (Lohr et al. 2006). In this study, it was postulated that this type of proteomic approach was extremely necessary in the rationale for the design of drugs for this deadly malignancy. Later, a number of investigations have demonstrated that indeed this technology can be applied to unwind the complex web of interacting pathways in PDAC. For example, in an elegant study, Chelala and colleagues provided pancreatic expression database that was a generic model for organization, integration and mining of

Systems and Network-Centric Understanding of Pancreatic Ductal Adenocarcinoma Signalling 23

serum biomarker identification the profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media was performed (Xiao et al. 2010). In this study the effect of oxythiamine chloride on PDAC cell secreteome was studied. The authors further improved on the existing biomarker discovery technology (i.e. coupling of proteomics and in vitro labeling of proteins in cells (SILAC) to enhance the efficacy of biomarker discovery. The authors concluded that labeling protein with 15N amino acids in conjunction with depleted serum allows the identification of actively secreted proteins from pancreatic cancer cells, and the rate of production of a secreted protein may be used as an independent

**3.1.2 Integrated analysis of pathways collectively targeted by co-expressed** 

**3.1.3 Proteomic profiling in identification of PDAC stems cells** 

PDAC tumors are heterogenous in nature and harbor many different types of cells. In recent years it has been realized that PDAC and other tumors carry a sub-population of cells with stem cell characteristics that are resistant to chemotherapeutic treatment modalities. However, this concept is still controversial since these cells have yet to be comprehensively identified and characterized. PDAC stem cells (CSCs) are such a group of cells that only constitute 0.2-0.8% of the total tumor cells but have been found to be the origin of carcinogenesis and metastasis. However, the extremely low availability of pancreatic tissue

Apart from investigations on signaling pathway de-regulation, multiple recent studies have found aberrant expression profiles of small non-coding RNAs (microRNAs) in PDAC. While several target genes have been experimentally identified for some microRNAs in various tumors, the global pattern of cellular functions and pathways affected by co-expressed microRNAs in PDAC remained elusive. Here too systems biology has found application in identification through computational approach and global analysis of the major biological processes and signaling pathways that are most likely to be affected collectively by coexpressed microRNAs in cancer cells. In a recent study, using five datasets of aberrantly expressed microRNAs in pancreatic and other cancers (breast cancer, colon cancer, lung cancer and lymphoma) and combinatorial target prediction algorithm miRgate and a twostep data reduction procedure Gene Ontology categories were determined (Gusev 2008; Gusev et al. 2007). These studies demonstrated biological functions, disease categories, toxicological categories and signaling pathways that are: targeted by multiple microRNAs; statistically significantly enriched with target genes; and known to be affected in PDAC. The analysis of predicted miRNA targets suggests that co-expressed miRNAs collectively provide systemic compensatory response to the abnormal phenotypic changes in cancer cells by targeting a broad range of functional categories and signaling pathways known to be affected in PDAC. The analysis revealed that E2F1 is a predicted microRNA target as well as caspase3 that were also validated experimentally as a target of multiple miRNAs in PDAC. Such a systems biology based approach provides new avenues for biological interpretation of miRNA profiling data and generation of experimentally testable hypotheses regarding collective regulatory functions of miRNA in PDAC for the design of

biomarker of the presence of tumor.

**microRNAs in PDAC** 

effective therapies.

complex pancreatic cancer datasets (Chelala et al. 2007). The database holds 32 datasets comprising 7636 gene expression measurements extracted from 20 different published gene or protein expression studies from various PDAC types, pancreatic precursor lesions (PanINs) and chronic pancreatitis. The pancreatic data are stored in a data management system based on the BioMart technology alongside the human genome gene and protein annotations, sequence, homologue, SNP and antibody data. Interrogation of the database can be achieved through both a web-based query interface and through web services using combined criteria from pancreatic (disease stages, regulation, differential expression, expression, platform technology, publication) and/or public data (antibodies, genomic region, gene-related accessions, ontology, expression patterns, multi-species comparisons, protein data, SNPs). This database enables connections between otherwise disparate data sources and allows relatively simple navigation between all data types and annotations. The database structure and content provides a powerful and high-speed data-mining tool for cancer research. It can be used for target discovery i.e. of biomarkers from body fluids, identification and analysis of genes associated with the progression of cancer, cross-platform meta-analysis, SNP selection for pancreatic cancer association studies, cancer gene promoter analysis as well as mining cancer ontology information. The data model is generic and can be easily extended and applied to other types of cancer and is available online with no restrictions for the scientific community at http://www.pancreasexpression.org/. Building on this database, the same group has updated their PDAC expression studies combining newly discovered and emerging molecules in 2011 (Cutts et al. 2011). These studies were not possible through traditional molecular biology approach which has its own limitations. In addition to the 32 datasets discovery, the group has added newer, more sophisticated query types that serve as a prototype for possible questions of interest that might be addressed towards greater understanding of PDAC (Chelala et al. 2009).

#### **3.1.1 Integrated systems biology in identification of PDAC biomarkers**

Comprehensive progress has been made on the use of systems biology in identification of biomarkers for PDAC. In a recent study, PDAC cell line related conditioned media and pancreatic juice were both mined for identification of putative diagnostic leads (Makawita et al. 2011). The proteome of the condition media were identified using strong cation exchange chromatography, followed by LC-MS/MS on an LTQ-Orbitrap mass spectrometer from six pancreatic cancer cell lines (BxPc3, MIA-PaCa2, PANC1, CAPAN1, CFPAC1 and SU.86.86), one normal human pancreatic ductal epithelial cell line, HPDE, and two pools of six pancreatic juice samples from ductal adenocarcinoma patients. These studies identified 1261 and 2171 proteins with two or more peptides, in each of the cell lines, while an average of 521 proteins were identified in the pancreatic juice pools. In total, 3,479 non-redundant proteins were identified with high confidence, of which ~40% were extracellular or cell membrane-bound based on genome ontology classifications. Three strategies were employed for identification of candidate biomarkers (1) examination of differential protein expression between the cancer and normal cell lines using label-free protein quantification, (2) integrative analysis, focusing on the overlap of proteins between the multiple biological fluids, and (3) tissue specificity analysis through mining of publically available databases. However, further validation of these proteins is warranted, as is the investigation of the remaining group of candidate biomarkers in PDAC. In another study on PDAC, secreted

complex pancreatic cancer datasets (Chelala et al. 2007). The database holds 32 datasets comprising 7636 gene expression measurements extracted from 20 different published gene or protein expression studies from various PDAC types, pancreatic precursor lesions (PanINs) and chronic pancreatitis. The pancreatic data are stored in a data management system based on the BioMart technology alongside the human genome gene and protein annotations, sequence, homologue, SNP and antibody data. Interrogation of the database can be achieved through both a web-based query interface and through web services using combined criteria from pancreatic (disease stages, regulation, differential expression, expression, platform technology, publication) and/or public data (antibodies, genomic region, gene-related accessions, ontology, expression patterns, multi-species comparisons, protein data, SNPs). This database enables connections between otherwise disparate data sources and allows relatively simple navigation between all data types and annotations. The database structure and content provides a powerful and high-speed data-mining tool for cancer research. It can be used for target discovery i.e. of biomarkers from body fluids, identification and analysis of genes associated with the progression of cancer, cross-platform meta-analysis, SNP selection for pancreatic cancer association studies, cancer gene promoter analysis as well as mining cancer ontology information. The data model is generic and can be easily extended and applied to other types of cancer and is available online with no restrictions for the scientific community at http://www.pancreasexpression.org/. Building on this database, the same group has updated their PDAC expression studies combining newly discovered and emerging molecules in 2011 (Cutts et al. 2011). These studies were not possible through traditional molecular biology approach which has its own limitations. In addition to the 32 datasets discovery, the group has added newer, more sophisticated query types that serve as a prototype for possible questions of interest that might be addressed towards greater

understanding of PDAC (Chelala et al. 2009).

**3.1.1 Integrated systems biology in identification of PDAC biomarkers** 

Comprehensive progress has been made on the use of systems biology in identification of biomarkers for PDAC. In a recent study, PDAC cell line related conditioned media and pancreatic juice were both mined for identification of putative diagnostic leads (Makawita et al. 2011). The proteome of the condition media were identified using strong cation exchange chromatography, followed by LC-MS/MS on an LTQ-Orbitrap mass spectrometer from six pancreatic cancer cell lines (BxPc3, MIA-PaCa2, PANC1, CAPAN1, CFPAC1 and SU.86.86), one normal human pancreatic ductal epithelial cell line, HPDE, and two pools of six pancreatic juice samples from ductal adenocarcinoma patients. These studies identified 1261 and 2171 proteins with two or more peptides, in each of the cell lines, while an average of 521 proteins were identified in the pancreatic juice pools. In total, 3,479 non-redundant proteins were identified with high confidence, of which ~40% were extracellular or cell membrane-bound based on genome ontology classifications. Three strategies were employed for identification of candidate biomarkers (1) examination of differential protein expression between the cancer and normal cell lines using label-free protein quantification, (2) integrative analysis, focusing on the overlap of proteins between the multiple biological fluids, and (3) tissue specificity analysis through mining of publically available databases. However, further validation of these proteins is warranted, as is the investigation of the remaining group of candidate biomarkers in PDAC. In another study on PDAC, secreted serum biomarker identification the profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media was performed (Xiao et al. 2010). In this study the effect of oxythiamine chloride on PDAC cell secreteome was studied. The authors further improved on the existing biomarker discovery technology (i.e. coupling of proteomics and in vitro labeling of proteins in cells (SILAC) to enhance the efficacy of biomarker discovery. The authors concluded that labeling protein with 15N amino acids in conjunction with depleted serum allows the identification of actively secreted proteins from pancreatic cancer cells, and the rate of production of a secreted protein may be used as an independent biomarker of the presence of tumor.

#### **3.1.2 Integrated analysis of pathways collectively targeted by co-expressed microRNAs in PDAC**

Apart from investigations on signaling pathway de-regulation, multiple recent studies have found aberrant expression profiles of small non-coding RNAs (microRNAs) in PDAC. While several target genes have been experimentally identified for some microRNAs in various tumors, the global pattern of cellular functions and pathways affected by co-expressed microRNAs in PDAC remained elusive. Here too systems biology has found application in identification through computational approach and global analysis of the major biological processes and signaling pathways that are most likely to be affected collectively by coexpressed microRNAs in cancer cells. In a recent study, using five datasets of aberrantly expressed microRNAs in pancreatic and other cancers (breast cancer, colon cancer, lung cancer and lymphoma) and combinatorial target prediction algorithm miRgate and a twostep data reduction procedure Gene Ontology categories were determined (Gusev 2008; Gusev et al. 2007). These studies demonstrated biological functions, disease categories, toxicological categories and signaling pathways that are: targeted by multiple microRNAs; statistically significantly enriched with target genes; and known to be affected in PDAC. The analysis of predicted miRNA targets suggests that co-expressed miRNAs collectively provide systemic compensatory response to the abnormal phenotypic changes in cancer cells by targeting a broad range of functional categories and signaling pathways known to be affected in PDAC. The analysis revealed that E2F1 is a predicted microRNA target as well as caspase3 that were also validated experimentally as a target of multiple miRNAs in PDAC. Such a systems biology based approach provides new avenues for biological interpretation of miRNA profiling data and generation of experimentally testable hypotheses regarding collective regulatory functions of miRNA in PDAC for the design of effective therapies.

#### **3.1.3 Proteomic profiling in identification of PDAC stems cells**

PDAC tumors are heterogenous in nature and harbor many different types of cells. In recent years it has been realized that PDAC and other tumors carry a sub-population of cells with stem cell characteristics that are resistant to chemotherapeutic treatment modalities. However, this concept is still controversial since these cells have yet to be comprehensively identified and characterized. PDAC stem cells (CSCs) are such a group of cells that only constitute 0.2-0.8% of the total tumor cells but have been found to be the origin of carcinogenesis and metastasis. However, the extremely low availability of pancreatic tissue

Systems and Network-Centric Understanding of Pancreatic Ductal Adenocarcinoma Signalling 25

can mediate the response to the drugs leading to both therapeutic and adverse effect**s.**  Understanding these beneficial secondary targets specially observed in potent synergistic combinations will provide fundamental information for the design of the most potent drug

Fig. 4. Traditional vs Network view of drug mechanism of action. Network view differs in understanding the mechanism of action of drugs. Classic view pools all secondary effects as off targets that are considered to cause side effects and toxicity. Network pharmacology categorizes secondary targets into off targets and interacting secondary targets which can mediate the response to the drugs to both the therapeutic or adverse effects. Adopted from

Azmi et al., 2011b

combination for individualized/personalized treatments.

CSCs (around 10 000 cells per xenograft tumor or patient sample) has limited the utilization of currently available molecular biology techniques. Global proteome profiling of pancreatic CSCs from xenograft tumors in mice using integrated systems biology is a promising way to unveil the molecular machinery underlying the signaling pathways in these CSCs. Using a capillary scale shotgun technique by coupling offline capillary isoelectric focusing (cIEF) with nano reversed phase liquid chromatography (RPLC) followed by spectral counting peptide quantification, Lubman and group investigated the proteomic profile of PDAC stems cells (Dai et al. 2010). In comparison with a non-tumorigenic tumor cell sample, among 1159 distinct proteins identified with FDR and less than 0.2%, 169 differentially expressed proteins are identified after multiple testing corrections where 24% of the proteins are up-regulated in the CSCs group. Ingenuity Pathway analysis of these differential expression signatures further indicated that a significant involvement of signaling pathways related to cell proliferation, inflammation, and metastasis were indentified. This was the first study to represents the proteome profiling study on PDAC stem cells from xenografted tumors in mice.

#### **4. Systems biology can aid understanding of the drug mechanism of action in PDAC**

Although partially successful in PDAC, new adjuvant targeted therapies (k-ras, EGFR, VEGF, src etc) have been met with more failure than success. The major reason for the low response is related to incomplete understanding and validation of the specific molecular targets at the gene level. The complexities of genetic and epigenetic changes in PDAC, coupled with redundancies and cross-talk in signaling pathways may explain the failure of single-pathway targeted therapies. This can be envisioned from the fact that of the 25,000 genes representing the human genome, about 1,800 are involved in the etiology of numerous diseases including cancer (Wist et al. 2009b). Currently available FDA approved drugs (~ 1200 in the market) have been designed to target approximately 400 genes (**Drugome)**. However, targeting this drugome by individually analyzing each gene is an impossible task because the functional product of each gene or (Proteome) is under multiple control, including splice variants and post translational modifications, giving rise to >40,000 functionally distinct proteins. In addition, such studies, thus far have been hindered by lack of suitable rapid technology. Therefore, novel and high-throughput data acquisition technologies coupled with integrated systems network modeling are urgently required to identify target genes in a tumor-specific manner. Such technologies are crucial for identifying and understanding the mechanisms of potential target candidates in complex diseases like PDAC.

#### **4.1 Systems pharmacology view of drug action**

Most of the known targeted drugs currently being used in the clinic were initially designed to affect a single gene. Unfortunately, contrary to the original idea, even the most specific drugs eventually target more than one gene (in most cases, >10 secondary targets). The use of systems pharmacology categorizes these off-targets into two types i) off-targets (resulting in side effects [often toxic]) and ii) secondary targets resulting in partial synergy] (Figure 4) (Berger and Iyengar 2009). These secondary targets exist within a complex network which

CSCs (around 10 000 cells per xenograft tumor or patient sample) has limited the utilization of currently available molecular biology techniques. Global proteome profiling of pancreatic CSCs from xenograft tumors in mice using integrated systems biology is a promising way to unveil the molecular machinery underlying the signaling pathways in these CSCs. Using a capillary scale shotgun technique by coupling offline capillary isoelectric focusing (cIEF) with nano reversed phase liquid chromatography (RPLC) followed by spectral counting peptide quantification, Lubman and group investigated the proteomic profile of PDAC stems cells (Dai et al. 2010). In comparison with a non-tumorigenic tumor cell sample, among 1159 distinct proteins identified with FDR and less than 0.2%, 169 differentially expressed proteins are identified after multiple testing corrections where 24% of the proteins are up-regulated in the CSCs group. Ingenuity Pathway analysis of these differential expression signatures further indicated that a significant involvement of signaling pathways related to cell proliferation, inflammation, and metastasis were indentified. This was the first study to represents the proteome profiling study on PDAC stem cells from xenografted

**4. Systems biology can aid understanding of the drug mechanism of action** 

Although partially successful in PDAC, new adjuvant targeted therapies (k-ras, EGFR, VEGF, src etc) have been met with more failure than success. The major reason for the low response is related to incomplete understanding and validation of the specific molecular targets at the gene level. The complexities of genetic and epigenetic changes in PDAC, coupled with redundancies and cross-talk in signaling pathways may explain the failure of single-pathway targeted therapies. This can be envisioned from the fact that of the 25,000 genes representing the human genome, about 1,800 are involved in the etiology of numerous diseases including cancer (Wist et al. 2009b). Currently available FDA approved drugs (~ 1200 in the market) have been designed to target approximately 400 genes (**Drugome)**. However, targeting this drugome by individually analyzing each gene is an impossible task because the functional product of each gene or (Proteome) is under multiple control, including splice variants and post translational modifications, giving rise to >40,000 functionally distinct proteins. In addition, such studies, thus far have been hindered by lack of suitable rapid technology. Therefore, novel and high-throughput data acquisition technologies coupled with integrated systems network modeling are urgently required to identify target genes in a tumor-specific manner. Such technologies are crucial for identifying and understanding the mechanisms of potential target candidates in complex

Most of the known targeted drugs currently being used in the clinic were initially designed to affect a single gene. Unfortunately, contrary to the original idea, even the most specific drugs eventually target more than one gene (in most cases, >10 secondary targets). The use of systems pharmacology categorizes these off-targets into two types i) off-targets (resulting in side effects [often toxic]) and ii) secondary targets resulting in partial synergy] (Figure 4) (Berger and Iyengar 2009). These secondary targets exist within a complex network which

tumors in mice.

diseases like PDAC.

**4.1 Systems pharmacology view of drug action** 

**in PDAC** 

can mediate the response to the drugs leading to both therapeutic and adverse effect**s.**  Understanding these beneficial secondary targets specially observed in potent synergistic combinations will provide fundamental information for the design of the most potent drug combination for individualized/personalized treatments.

Fig. 4. Traditional vs Network view of drug mechanism of action. Network view differs in understanding the mechanism of action of drugs. Classic view pools all secondary effects as off targets that are considered to cause side effects and toxicity. Network pharmacology categorizes secondary targets into off targets and interacting secondary targets which can mediate the response to the drugs to both the therapeutic or adverse effects. Adopted from Azmi et al., 2011b

Systems and Network-Centric Understanding of Pancreatic Ductal Adenocarcinoma Signalling 27

kB, cadherin anti-tumor module, the tumor suppressor EGR1 and MDM2 negative regulator CREBBP. Our more in-depth analysis using these integrated approach, revealed the prominent role of HNF4A (hepatocyte nuclear factor 4 alpha) that modulates a totally distinct yet p53-linked set of proteins driving apoptosis (Azmi et al. 2010c). The identification of HNF4A as a key player was certainly revealing since it has not been well defined in PDAC cells used in this study (Capan-2 (wt-p53)). However, a search of the literature indicated that this gene is highly expresses in pancreatic tumors compared to their normal counterpart. HNF4A is known to interact with the p53 positive regulator CREBBP (Yoshida et al. 1997) and thus, confirmed its role in augmenting apoptotic effects in this synergic combination. Therefore, not only does systems biology provide information on the networks involved in drug efficacy, it can also provide information on biomarkers of therapeutic response that can be utilized for evaluation of drug response during actual

PDAC is a complex disease that arises from a complex set of genetic mutations and pathway alterations. Traditional sciences have not been very successful in clearly delineating the interaction between these multiple pathways and this could be the primary reason for the observed failure of chemo- and targeted therapies. All of these genetic alterations can now be "re-discovered" using next-generation integrated systems technology. As described above, integrated sciences have revealed that these signaling pathways cross talk with one another and can regulate cell growth, proliferation, survival, angiogenesis and metastasis in PDAC. In addition, these high-throughput technologies can achieve many different goals such as cataloging the driver mutations, exploring functional role of cancer genes, proteins and interaction networks, identifying microRNAs, understanding protein–DNA interactions, and comprehensive analyses of transcriptomes and interactomes. Furthermore, these technologies can be utilized to identify, understand and differentiate sub population of CSCs in PDAC heterogeneous tumor mass. Systems biology has the power to catalog complex events leading to origin, progression, recurrence and resistance of PDAC and can greatly assist in understanding how cancer genomes operate as part of the whole biological system. Now, high-quality clinical treatment and outcomes (death or survival) data from biobanks, and extensive genetics and genomics data for some PDAC and other tumors, including breast, colorectal, and lung are available. How all these clinical and genetics data could be integrated into reverse engineering-based network modeling to approach the extremely complex genotype–phenotype map of different tumors is currently being explored. These studies will pave way for the discovery of new molecular innovations, both predictive markers and therapies, towards personalized treatment of PDAC. Therefore systems

We thank Dr. Frances W.J. Beck for critically evaluating this manuscript. All members of Dr.

Fazlul H. Sarkar's team who could not be added in this chapter are acknowledged.

clinical trials in PDAC patients.

biology can aid in the overall understanding of PDAC.

**6. Acknowledgment** 

**5. Conclusion** 

Such an understanding requires mechanistic studies in the laboratory to be coupled with robust, state of the art computational tools to obtain irrevocably strong proof for the integration of pathways involved in the observed synergy. One such approach involves the use of network modeling which provides mathematically and statistically robust information regarding the involvement of effector genes in the efficacy or synergy between two drugs. These network models can also predict key secondary targets of such interaction, thus, also providing information on novel previously unrecognized targets and pathways which could be useful for future therapeutic interventions in the treatment of different cancers where, at present, information is gravely lacking, such as in PDAC.

#### **4.1.1 Validation of the systems approach for predicting potent drug combination in PDAC**

Our laboratory has been working on a specific small molecule inhibitor of MDM2 (MI-219) and indentifying, in greater detail, its mechanism of action in PDAC (Azmi et al. 2010b). MI-219 is currently in Phase I clinical trial (Brown et al. 2009). Our initial studies were restricted to evaluating its efficacy against wt-p53 tumors. However, we have recently found that MDM2 inhibitor, when combined with chemotherapy such as oxaliplatin, synergistically enhanced apoptosis in wt-p53 cancers and most importantly, 50% of tumor bearing mice treated with this combination remained tumor free without recurrence for 120 days (Azmi et al. 2010a). We used this model to validate a systems approach in predicting potent drug combinations in PDAC and to obtain critical information into understanding the mechanism for this synergy. Therefore, our study included integrated microarray gene expression profiling (IGEMP) and pathway network modeling (PNM) (Azmi et al. 2011a). The systems analysis data for MI-219-oxaliplatin combination treated wt-p53 capan-2 cells revealed that indeed synergy is at the gene level. Principle component analysis showed that one can differentiate the gene signatures between single treatment versus combination. The emergence of certain unique synergyrelated genes indicated their potential as key players supporting the overall response of MI-219-oxaliplatin in positively regulating the p53 re-activation (Azmi et al. 2010c; Azmi et al. 2011b). Presented with this vast amount of information regarding the mechanism involved in the response to MI-219-oxaliplatin synergy, we believe it validates the applicability of this technology for use in identifying the relevant pathways involved in both cure and resistance. Ultimately, results of these studies will significantly aid in the design of clinically successful drug combinations for PDAC, which will benefit the overall survival of patients.

#### **4.1.2 Systems identification of biomarker of response with implications for PDAC therapy**

Our intended goal in using IGEMP and PNM analysis was to demonstrate the synergy between MI-219-oxaliplatin at the gene level and to demonstrate the local network of p53 and crucial neighboring network that augment p53 re-activation mediated events. Systems network modeling, although a powerful technological tool has not yet been fully explored for use in PDAC (the most genetically complex cancer). We had previously identified several genes responsible for cross-talk within the local network of p53 which included NF-

kB, cadherin anti-tumor module, the tumor suppressor EGR1 and MDM2 negative regulator CREBBP. Our more in-depth analysis using these integrated approach, revealed the prominent role of HNF4A (hepatocyte nuclear factor 4 alpha) that modulates a totally distinct yet p53-linked set of proteins driving apoptosis (Azmi et al. 2010c). The identification of HNF4A as a key player was certainly revealing since it has not been well defined in PDAC cells used in this study (Capan-2 (wt-p53)). However, a search of the literature indicated that this gene is highly expresses in pancreatic tumors compared to their normal counterpart. HNF4A is known to interact with the p53 positive regulator CREBBP (Yoshida et al. 1997) and thus, confirmed its role in augmenting apoptotic effects in this synergic combination. Therefore, not only does systems biology provide information on the networks involved in drug efficacy, it can also provide information on biomarkers of therapeutic response that can be utilized for evaluation of drug response during actual clinical trials in PDAC patients.

#### **5. Conclusion**

26 Pancreatic Cancer – Clinical Management

Such an understanding requires mechanistic studies in the laboratory to be coupled with robust, state of the art computational tools to obtain irrevocably strong proof for the integration of pathways involved in the observed synergy. One such approach involves the use of network modeling which provides mathematically and statistically robust information regarding the involvement of effector genes in the efficacy or synergy between two drugs. These network models can also predict key secondary targets of such interaction, thus, also providing information on novel previously unrecognized targets and pathways which could be useful for future therapeutic interventions in the treatment of different

**4.1.1 Validation of the systems approach for predicting potent drug combination in** 

**4.1.2 Systems identification of biomarker of response with implications for PDAC** 

Our intended goal in using IGEMP and PNM analysis was to demonstrate the synergy between MI-219-oxaliplatin at the gene level and to demonstrate the local network of p53 and crucial neighboring network that augment p53 re-activation mediated events. Systems network modeling, although a powerful technological tool has not yet been fully explored for use in PDAC (the most genetically complex cancer). We had previously identified several genes responsible for cross-talk within the local network of p53 which included NF-

Our laboratory has been working on a specific small molecule inhibitor of MDM2 (MI-219) and indentifying, in greater detail, its mechanism of action in PDAC (Azmi et al. 2010b). MI-219 is currently in Phase I clinical trial (Brown et al. 2009). Our initial studies were restricted to evaluating its efficacy against wt-p53 tumors. However, we have recently found that MDM2 inhibitor, when combined with chemotherapy such as oxaliplatin, synergistically enhanced apoptosis in wt-p53 cancers and most importantly, 50% of tumor bearing mice treated with this combination remained tumor free without recurrence for 120 days (Azmi et al. 2010a). We used this model to validate a systems approach in predicting potent drug combinations in PDAC and to obtain critical information into understanding the mechanism for this synergy. Therefore, our study included integrated microarray gene expression profiling (IGEMP) and pathway network modeling (PNM) (Azmi et al. 2011a). The systems analysis data for MI-219-oxaliplatin combination treated wt-p53 capan-2 cells revealed that indeed synergy is at the gene level. Principle component analysis showed that one can differentiate the gene signatures between single treatment versus combination. The emergence of certain unique synergyrelated genes indicated their potential as key players supporting the overall response of MI-219-oxaliplatin in positively regulating the p53 re-activation (Azmi et al. 2010c; Azmi et al. 2011b). Presented with this vast amount of information regarding the mechanism involved in the response to MI-219-oxaliplatin synergy, we believe it validates the applicability of this technology for use in identifying the relevant pathways involved in both cure and resistance. Ultimately, results of these studies will significantly aid in the design of clinically successful drug combinations for PDAC, which will benefit the overall

cancers where, at present, information is gravely lacking, such as in PDAC.

**PDAC** 

survival of patients.

**therapy** 

PDAC is a complex disease that arises from a complex set of genetic mutations and pathway alterations. Traditional sciences have not been very successful in clearly delineating the interaction between these multiple pathways and this could be the primary reason for the observed failure of chemo- and targeted therapies. All of these genetic alterations can now be "re-discovered" using next-generation integrated systems technology. As described above, integrated sciences have revealed that these signaling pathways cross talk with one another and can regulate cell growth, proliferation, survival, angiogenesis and metastasis in PDAC. In addition, these high-throughput technologies can achieve many different goals such as cataloging the driver mutations, exploring functional role of cancer genes, proteins and interaction networks, identifying microRNAs, understanding protein–DNA interactions, and comprehensive analyses of transcriptomes and interactomes. Furthermore, these technologies can be utilized to identify, understand and differentiate sub population of CSCs in PDAC heterogeneous tumor mass. Systems biology has the power to catalog complex events leading to origin, progression, recurrence and resistance of PDAC and can greatly assist in understanding how cancer genomes operate as part of the whole biological system. Now, high-quality clinical treatment and outcomes (death or survival) data from biobanks, and extensive genetics and genomics data for some PDAC and other tumors, including breast, colorectal, and lung are available. How all these clinical and genetics data could be integrated into reverse engineering-based network modeling to approach the extremely complex genotype–phenotype map of different tumors is currently being explored. These studies will pave way for the discovery of new molecular innovations, both predictive markers and therapies, towards personalized treatment of PDAC. Therefore systems biology can aid in the overall understanding of PDAC.

#### **6. Acknowledgment**

We thank Dr. Frances W.J. Beck for critically evaluating this manuscript. All members of Dr. Fazlul H. Sarkar's team who could not be added in this chapter are acknowledged.

Systems and Network-Centric Understanding of Pancreatic Ductal Adenocarcinoma Signalling 29

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**3**

*Romania* 

**Novel Biomarkers in**

Anca Botezatu3 and Irinel Popescu1

*2Biochemistry and Proteomics Department,* 

*3Viral Genetic Engineering Laboratory,* 

Simona O. Dima1, Cristiana Tanase2, Radu Albulescu2,

*Romanian Academy 'Stefan S. Nicolau' Virology Institute, Bucharest,* 

*Digestive Disease and Liver Transplantation, Bucharest,* 

*"Victor Babes" National Institute of Pathology, Bucharest,* 

*1Center of General Surgery and Liver Transplantation, Fundeni Clinical Institute of* 

Pancreatic ductal adenocarcinoma( PDAC) cancer is one of the most aggressive human cancers, and the fifth most frequent cause of cancer-related mortality in Western society. Pancreatic cancer is well known for high metastatic potential, early local invasion and poor outcome. The overall 5-year survival rate is less than 5%, respectively 10-30% for R0 resection (Huang et al., 2010). Less than 10% of newly diagnosed pancreatic cancers could be

Clinical research in the field of cancer biomarkers is essential in understanding the biology and the heterogeneity of cancer disease. The factors involved in early PDAC development remain unknown. The detection of pancreatic cancer at early stages, the prediction of the potential resectability, or response to therapy are the current major challenges in improving the clinical outcome of PDAC. Therefore, predictive markers of responsiveness to adjuvant therapy would allow patients selection to appropriate treatment (Duffy et al., 2007). The aim of the postoperative surveillance following curative surgery for PDAC is to detect

Currently, there are only few studies that have identified cancer biomarkers with clinical

Molecular biological factors are important tools for early diagnosis, prognosis, not only for therapeutic strategy but also for novel and more efficient therapeutic agents' identification. On the other hand a biomarker must be easily quantified, in order to minimize the invasiveness of therapeutically interventions. Recent finding in the molecular biology field of pancreatic cancer have assisted in translational research, giving hope for individualized

**1. Introduction** 

significance.

detected in early-stage (Takayama et al., 2010).

recurrences or metastases as early as possible.

therapy and better disease management.

**Pancreatic Cancer** 

Markowitz SD, Parmigiani G, Kinzler KW, Velculescu VE and Vogelstein B. (2007). *Science,* 318, 1108-1113.


### **Novel Biomarkers in Pancreatic Cancer**

Simona O. Dima1, Cristiana Tanase2, Radu Albulescu2, Anca Botezatu3 and Irinel Popescu1 *1Center of General Surgery and Liver Transplantation, Fundeni Clinical Institute of Digestive Disease and Liver Transplantation, Bucharest, 2Biochemistry and Proteomics Department, "Victor Babes" National Institute of Pathology, Bucharest, 3Viral Genetic Engineering Laboratory, Romanian Academy 'Stefan S. Nicolau' Virology Institute, Bucharest, Romania* 

#### **1. Introduction**

30 Pancreatic Cancer – Clinical Management

Xiao J, Lee WN, Zhao Y, Cao R, Go VL, Recker RR, Wang Q and Xiao GG. (2010). *Pancreas,*

Yoshida E, Aratani S, Itou H, Miyagishi M, Takiguchi M, Osumu T, Murakami K and

Fukamizu A. (1997). *Biochem Biophys Res Commun,* 241, 664-669.

*Science,* 318, 1108-1113.

39, e17-e23.

Markowitz SD, Parmigiani G, Kinzler KW, Velculescu VE and Vogelstein B. (2007).

Pancreatic ductal adenocarcinoma( PDAC) cancer is one of the most aggressive human cancers, and the fifth most frequent cause of cancer-related mortality in Western society. Pancreatic cancer is well known for high metastatic potential, early local invasion and poor outcome. The overall 5-year survival rate is less than 5%, respectively 10-30% for R0 resection (Huang et al., 2010). Less than 10% of newly diagnosed pancreatic cancers could be detected in early-stage (Takayama et al., 2010).

Clinical research in the field of cancer biomarkers is essential in understanding the biology and the heterogeneity of cancer disease. The factors involved in early PDAC development remain unknown. The detection of pancreatic cancer at early stages, the prediction of the potential resectability, or response to therapy are the current major challenges in improving the clinical outcome of PDAC. Therefore, predictive markers of responsiveness to adjuvant therapy would allow patients selection to appropriate treatment (Duffy et al., 2007). The aim of the postoperative surveillance following curative surgery for PDAC is to detect recurrences or metastases as early as possible.

Currently, there are only few studies that have identified cancer biomarkers with clinical significance.

Molecular biological factors are important tools for early diagnosis, prognosis, not only for therapeutic strategy but also for novel and more efficient therapeutic agents' identification. On the other hand a biomarker must be easily quantified, in order to minimize the invasiveness of therapeutically interventions. Recent finding in the molecular biology field of pancreatic cancer have assisted in translational research, giving hope for individualized therapy and better disease management.

Novel Biomarkers in Pancreatic Cancer 33

not so specific. The analysis of pancreatic cyst fluids obtained from various cystic lesions showed that specific histological lesions are associated with distinct protein patterns. Two important factors, olfactomedin-4 (antiapoptotic protein that promotes tumor growth) and mucin-18 (melanoma cell adhesion molecule) were proposed as biomarkers of pancreatic

CD99 (cell surface glycoprotein involved in leukocyte migration), a useful diagnostic marker for Ewing sarcoma/primitive neuroectodermal tumor (Rocchi et al., 2010) was proposed for differentiation of solid-pseudopapillary neoplasm from other pancreatic tumor. The tissues positivity for CD99 was investigated in a recent study (Guo et al., 2011) using immunohistochemical staining technique. The solid-pseudopapillary neoplasm cells tumors exhibited paranuclear dot-like immunoreactivity for CD99. In contrast, in pancreatic neuroendocrine tumors a small percent of PDAC stained positive for CD99 at cytoplasmatic and membrane level. Pancreatic solid-pseudopapillary neoplasm exhibits a unique dot-like staining pattern for CD99 and could provide a definitive diagnosis of solid-pseudopapillary

Immunohistochemical analysis of pancreatic cancer tissue provided also several candidate biomarkers for survival estimation. CDCP1 (CUB domain containing protein 1) determines anchorage- independent growth and migration of cancer cells. A higher expression level of this factor is correlated with the overall survival of pancreatic cancer patients (Miyazava et al., 2010). L1-CAM (L1-cell adhesion molecule) expression was also correlated with perineural invasion of pancreatic cancer cells and poor survival (Ben at al., 2010). Higher expression of B7-H3, a co-stimulatory immune molecule, plays a critical role in the T cellmediated immune response and presents a positive correlation with pancreatic cancer

KOC (K homology domain containing protein) gene encodes a protein that contains several KH domains, which are important in RNA binding and are known to be involved in RNA synthesis and metabolism. This protein was found to be overexpressed in pancreatic cancer. Immunohystochemical analysis showed strong staining for KOC protein in invasive pancreatic tissue carcinomas versus normal pancreatic tissue. It was proposed to be a molecular marker with a high sensitivity and specificity in discriminating PDAC from

Most of the adenocarcinomas develop gradually from precursor lesions PanINs (Pancreatic intraepithelial neoplasias). These events are accompanied by genetic modifications. Most genetic abnormalities reported in pancreatic cancer are represented by deletions and duplications of specific chromosomal loci, mutations/deletions of oncogenes and tumorsuppresor genes (KRAS, CDKN1A/p16, TP53, MADH4/SMAD4/DPC4 and BRCA2)

**3.1 Gene expression studies and potential factors involved in pancreas oncogenesis**  Quantification of target mRNA gene levels represents a new tool for genome function analysis. High-throughput technologies like gene expression profiling using microarray and sequencing become important investigation methods for normal physiological and

neoplasm and differentiation from other pancreatic tumors.

benign ductal epithelium (Toll et al., 2009).

**3. Genome candidate biomarkers** 

[www.cancer-genetics.org].

cancer (Cuoghi et al., 2011).

prognosis.

#### **2. Identification of new potential tumor tissue biomarkers**

Much effort goes into finding new accurate prognostic, diagnostic single or combined tumor biomarkers. Nowadays, the research in this field is focused not only on finding biomarkers that could discriminate between pathological pancreas conditions (disease related biomarkers), but also to evaluate the aggressiveness grade of PDAC and to determine the therapy response (drug related biomarkers).

Presently, screening for pancreatic cancer is based on state-of-the-art imaging or even invasive diagnostic methods (Balasenthil et al., 2011). Serum and other body fluids, such as urine, pancreatic juice represents sources available by less invasive methods for biomarkers screening.

Several techniques like immunohistochemistry, fluorescence and chromogenic *in situ* hybridization, expression profiling- performed by microarray or quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), and mutation analysis are used in identification of new biomarkers. Among these techniques, immunohistochemical tests remain the most widely used in routine practice and, importantly, in the assessment of biomarkers in translational research.

In a recent study, *Wacher et. al* investigated the expression level of an oncofetal protein, insulin-like growth factor II messenger ribonucleic acid-binding protein 3 (IMP3), which represent a marker for tumor aggressiveness in many different tumors (Wachter et al., 2011; Ozdemir et al., 2011).

The investigation was conducted on tissue biopsies from PDAC, chronic sclerosing pancreatitis, PDAC metastases cases in order to determine IMP3 expression. For IMP3 expression evaluation large tissue sections were used in the immunohistochemical analysis. The results obtained showed that PDAC were positive for IMP3 expression in a high percentage (88.4%) of cases, whereas normal or inflammatory pancreatic tissue was weakly positive (23.1%). A strong IMP3 expression was found in PDAC metastases (94.4%). The sensitivity and specificity of IMP3 expression test to discriminate between PDAC and chronic sclerosing pancreatitis using core needle biopsies were found to be 88.4% and 94.6%, respectively. The authors consider that IMP3 might be an easy to use and potentially new immunohistochemical marker for the diagnosis of PDAC in core needle biopsies (Asioli et al., 2010; Walter et al., 2009).

Another prognostic biomarker in PDAC which characterize the tumor aggressiveness recently studied was vimentin. Vimentin protein is a marker of mesenchymal differentiation, being correlated especially with aggressive carcinomas. In a high percentage primary pancreatic adenocarcinomas contain neoplastic cells that express vimentin, and the presence of this protein was correlated with poor histological differentiation and predicts a shorter postsurgical survival (Li et al., 2009; Handra-Luca et al., 2011).

Several technical strategies (SDS-PAGE, mass spectrometry, immunoblot) are used to find candidate biomarkers for the presurgical management of malignant and premalignant pancreatic conditions. Pancreatic cystic neoplasms represent 10−15% of primary cystic masses of the pancreas and are detected with an increasing frequency due to the use of advanced imaging modalities in clinical practice. On the other hand, the diagnosis of pancreatic cystic neoplasms remains a challenge because available diagnostic techniques are

Much effort goes into finding new accurate prognostic, diagnostic single or combined tumor biomarkers. Nowadays, the research in this field is focused not only on finding biomarkers that could discriminate between pathological pancreas conditions (disease related biomarkers), but also to evaluate the aggressiveness grade of PDAC and to determine the

Presently, screening for pancreatic cancer is based on state-of-the-art imaging or even invasive diagnostic methods (Balasenthil et al., 2011). Serum and other body fluids, such as urine, pancreatic juice represents sources available by less invasive methods for biomarkers

Several techniques like immunohistochemistry, fluorescence and chromogenic *in situ* hybridization, expression profiling- performed by microarray or quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), and mutation analysis are used in identification of new biomarkers. Among these techniques, immunohistochemical tests remain the most widely used in routine practice and, importantly, in the assessment of

In a recent study, *Wacher et. al* investigated the expression level of an oncofetal protein, insulin-like growth factor II messenger ribonucleic acid-binding protein 3 (IMP3), which represent a marker for tumor aggressiveness in many different tumors (Wachter et al., 2011;

The investigation was conducted on tissue biopsies from PDAC, chronic sclerosing pancreatitis, PDAC metastases cases in order to determine IMP3 expression. For IMP3 expression evaluation large tissue sections were used in the immunohistochemical analysis. The results obtained showed that PDAC were positive for IMP3 expression in a high percentage (88.4%) of cases, whereas normal or inflammatory pancreatic tissue was weakly positive (23.1%). A strong IMP3 expression was found in PDAC metastases (94.4%). The sensitivity and specificity of IMP3 expression test to discriminate between PDAC and chronic sclerosing pancreatitis using core needle biopsies were found to be 88.4% and 94.6%, respectively. The authors consider that IMP3 might be an easy to use and potentially new immunohistochemical marker for the diagnosis of PDAC in core needle biopsies (Asioli et

Another prognostic biomarker in PDAC which characterize the tumor aggressiveness recently studied was vimentin. Vimentin protein is a marker of mesenchymal differentiation, being correlated especially with aggressive carcinomas. In a high percentage primary pancreatic adenocarcinomas contain neoplastic cells that express vimentin, and the presence of this protein was correlated with poor histological differentiation and predicts a

Several technical strategies (SDS-PAGE, mass spectrometry, immunoblot) are used to find candidate biomarkers for the presurgical management of malignant and premalignant pancreatic conditions. Pancreatic cystic neoplasms represent 10−15% of primary cystic masses of the pancreas and are detected with an increasing frequency due to the use of advanced imaging modalities in clinical practice. On the other hand, the diagnosis of pancreatic cystic neoplasms remains a challenge because available diagnostic techniques are

shorter postsurgical survival (Li et al., 2009; Handra-Luca et al., 2011).

**2. Identification of new potential tumor tissue biomarkers** 

therapy response (drug related biomarkers).

biomarkers in translational research.

Ozdemir et al., 2011).

al., 2010; Walter et al., 2009).

screening.

not so specific. The analysis of pancreatic cyst fluids obtained from various cystic lesions showed that specific histological lesions are associated with distinct protein patterns. Two important factors, olfactomedin-4 (antiapoptotic protein that promotes tumor growth) and mucin-18 (melanoma cell adhesion molecule) were proposed as biomarkers of pancreatic cancer (Cuoghi et al., 2011).

CD99 (cell surface glycoprotein involved in leukocyte migration), a useful diagnostic marker for Ewing sarcoma/primitive neuroectodermal tumor (Rocchi et al., 2010) was proposed for differentiation of solid-pseudopapillary neoplasm from other pancreatic tumor. The tissues positivity for CD99 was investigated in a recent study (Guo et al., 2011) using immunohistochemical staining technique. The solid-pseudopapillary neoplasm cells tumors exhibited paranuclear dot-like immunoreactivity for CD99. In contrast, in pancreatic neuroendocrine tumors a small percent of PDAC stained positive for CD99 at cytoplasmatic and membrane level. Pancreatic solid-pseudopapillary neoplasm exhibits a unique dot-like staining pattern for CD99 and could provide a definitive diagnosis of solid-pseudopapillary neoplasm and differentiation from other pancreatic tumors.

Immunohistochemical analysis of pancreatic cancer tissue provided also several candidate biomarkers for survival estimation. CDCP1 (CUB domain containing protein 1) determines anchorage- independent growth and migration of cancer cells. A higher expression level of this factor is correlated with the overall survival of pancreatic cancer patients (Miyazava et al., 2010). L1-CAM (L1-cell adhesion molecule) expression was also correlated with perineural invasion of pancreatic cancer cells and poor survival (Ben at al., 2010). Higher expression of B7-H3, a co-stimulatory immune molecule, plays a critical role in the T cellmediated immune response and presents a positive correlation with pancreatic cancer prognosis.

KOC (K homology domain containing protein) gene encodes a protein that contains several KH domains, which are important in RNA binding and are known to be involved in RNA synthesis and metabolism. This protein was found to be overexpressed in pancreatic cancer. Immunohystochemical analysis showed strong staining for KOC protein in invasive pancreatic tissue carcinomas versus normal pancreatic tissue. It was proposed to be a molecular marker with a high sensitivity and specificity in discriminating PDAC from benign ductal epithelium (Toll et al., 2009).

#### **3. Genome candidate biomarkers**

Most of the adenocarcinomas develop gradually from precursor lesions PanINs (Pancreatic intraepithelial neoplasias). These events are accompanied by genetic modifications. Most genetic abnormalities reported in pancreatic cancer are represented by deletions and duplications of specific chromosomal loci, mutations/deletions of oncogenes and tumorsuppresor genes (KRAS, CDKN1A/p16, TP53, MADH4/SMAD4/DPC4 and BRCA2) [www.cancer-genetics.org].

#### **3.1 Gene expression studies and potential factors involved in pancreas oncogenesis**

Quantification of target mRNA gene levels represents a new tool for genome function analysis. High-throughput technologies like gene expression profiling using microarray and sequencing become important investigation methods for normal physiological and

Novel Biomarkers in Pancreatic Cancer 35

Heparanases are endoglycosidases that cleave the heparan sulfate side chain of heparan sulfate proteoglycans (major membrane components) inducing extracellular matrix remodelling. On the other hand, heparanase increased growth factor bFGF (fibroblast

Thrombospondin (TSP-1) expression level was linked to a good prognostic for PDAC development. TSP-1 protein was found abundantly in stroma surrounding tumor cells and

The role of cathepsins in pancreatic cancer development and progression is still controversed. Cathepsin b (CTSB) and cathepsin l CTSL was found to be overexpressd in PDAC. A correlation between CTSB expression level and perineural invasion concludes the

Zinc is the most abundant trace element in cells. For example, about 25% of the total zinc present is found within the cell nuclei, being a component of chromatin. Zinc is an important factor in cellular processes, including cell division and proliferation, immune function, and defense against free radicals; zinc deficiency may be associated with an increased risk of cancer (Christudoss et al.,2011; Prasad et al., 2002). Zinc is found in over 300 enzymes, including copper/zinc superoxide dismutase, which is an important antioxidant enzyme, involved in cellular protection components from oxidation and

In a current study, *Costello et al*. found a major loss of zinc in ductal and acinar epithelium in adenocarcinoma compared to the normal epithelium (Costello et al., 2011). The decrease in zinc quantity is a characteristic not only for pancreatic cancer, but also for precursor lesions. The mentioned group showed that the gene expression of ZIP3 (basilar membrane zinc uptake transporter) is present in normal ductal/acinar epithelium and absent in adenocarcinoma. The decreased expression of ZIP3 determines the loss of zinc in early and progressing malignancy. RREB1 transcription factor was found to be down regulated along with ZIP3 and might be the silencing cause of ZIP3 gene. Zinc treatment exhibited cytotoxic effect on Panc1 cell line. ZIP3 and RREB-1 expression level changes represent early events in the development of

FAP (fibroblast activation protein) is involved in the control of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis. It is highly expressed in PDAC and is considered to be related to a poor prognostic. Targeting FAP factors are considered to be a promising and a new

Maspin (SerpinB5- mammary serine protease inhibitor) is a tumor suppressor gene. Maspin induces apoptosis in neoplastic cells and expression of maspin are suppressed as the carcinoma progresses in breast and prostatic carcinoma (Jiang et al., 2002). The pattern of maspin gene expression is dependent of the disease stage. For example, highly expression

growth factor) release, stimulating the angiogenesis process (Rohloff et al., 2002)

its expression is inversely correlated with microvessel density(Tobita et al., 2002).

**3.3 ZIP3 (Zinc/iron regulated transporter-related protein 3) a possible tumor** 

role of cathepsins in local tumor invasion (Niedergethmann et al., 2000).

adenocarcinoma suggesting that ZIP3 might be a tumor suppressor gene.

**suppressor**

damage.

**3.4 Other important factors** 

therapeuthical road (Cohen et al., 2008).

pathological processes. Therefore, studies based on quantification of gene expression levels revealed new potential factors involved in pancreas oncogenesis. Using Affymetrix and cDNA microarrays, nearly 1,100 molecules have been reported to be overexpressed in PanIN and IPMN lesions. A large majority of these molecules showed elevated mRNA levels expression in PDAC and in precursor lesions. Molecules such as S100P, MMP7, MUC4, FSCN1, and MUC5AC are found to be overexpressed in all type of lesions PanIN, IPMN and PDAC (Harsha et al., 2009).

Badea et al. using microarray study identificated a number of genes whose over-expression appears to be inversely correlated with patient survival: keratin 7, laminin gamma 2, stratifin, platelet phosphofructokinase, annexin A2, MAP4K4 and OACT2 (MBOAT2), which are specifically upregulated in the neoplastic epithelium (Badea et al., 2008).

The attention was focused especially on the calcium-binding protein- S100P. This protein was found to be expressed in pancreatic precursor lesions PanIN 2 or PanIN 3 and in pancreatic tumor creating the possibility to use the quantification of its expression level for earlier detection (Dowen et al., 2005). Expression levels of *S100P* mRNA were found to be higher in pancreatic juice from patients with pancreatic cancer and IPMN (Ohucida et al., 2006). On the other hand, three members of S100A family (S100A2, S100A4, and S100A6) were found to be associated with poor prognostic. S100A family members were involved in cell cycle regulation and cell invasion (Tanase et al., 2010).

The STAT3 transcription factor was found to be constitutively activated in PDAC. This is an important factor in stem cell self-renewal process, cancer cell survival, and inflammation. A close correlation between the levels of tyrosine-phosphorylated STAT3 and of the gp130 receptor was found. An upregulation of the IL6/LIF-gp130 pathway was also showed to be involved in STAT3 activation in pancreatic cancer (Corcoran et al., 2011). The same study asserts that STAT3 is required for the development of the precursor pancreatic lesions, acinar-to-ductal metaplasia (ADM) and PanIN, therefore evaluation of gp130 and phospho-STAT3 expression may be an effective biomarker.

#### **3.2 Angiogenesis factors**

With the increasing use of antiangiogenic agents for the treatment of cancers, is necessary the identification of candidate biomarkers for evaluation of the response and resistance. Is also, important to identify new biomarkers and to eliminate the risk for antiangiogenic therapies failure (Duda et al., 2010).

EGF (Epidermal Growth Factor), VEGF (Vascular Endothelial, Growth Factor) , heparanase, thrombospondin, cathepsins were the most important angiogenic factor specific for pancreatic cancer involved in cell growth promoting. The overexpression of EGF and his receptor EGFR were linked to tumor staging, but no clear data regarding the overall survival are available at this moment (Heidemann et al.,2006).

Another important factor that acts on angiogenesis process is VEGF. His effects in PDAC are increased by interaction with MMP-9, a cellular matrix remodeling factor. The therapy against both MMP-9 and VEGF in pancreatic cancer resulted in a significant decrease in PDAC growth and microvessel density versus a single target treatment (Tanase-Pistol et al.,2008).

pathological processes. Therefore, studies based on quantification of gene expression levels revealed new potential factors involved in pancreas oncogenesis. Using Affymetrix and cDNA microarrays, nearly 1,100 molecules have been reported to be overexpressed in PanIN and IPMN lesions. A large majority of these molecules showed elevated mRNA levels expression in PDAC and in precursor lesions. Molecules such as S100P, MMP7, MUC4, FSCN1, and MUC5AC are found to be overexpressed in all type of lesions PanIN,

Badea et al. using microarray study identificated a number of genes whose over-expression appears to be inversely correlated with patient survival: keratin 7, laminin gamma 2, stratifin, platelet phosphofructokinase, annexin A2, MAP4K4 and OACT2 (MBOAT2), which

The attention was focused especially on the calcium-binding protein- S100P. This protein was found to be expressed in pancreatic precursor lesions PanIN 2 or PanIN 3 and in pancreatic tumor creating the possibility to use the quantification of its expression level for earlier detection (Dowen et al., 2005). Expression levels of *S100P* mRNA were found to be higher in pancreatic juice from patients with pancreatic cancer and IPMN (Ohucida et al., 2006). On the other hand, three members of S100A family (S100A2, S100A4, and S100A6) were found to be associated with poor prognostic. S100A family members were involved in

The STAT3 transcription factor was found to be constitutively activated in PDAC. This is an important factor in stem cell self-renewal process, cancer cell survival, and inflammation. A close correlation between the levels of tyrosine-phosphorylated STAT3 and of the gp130 receptor was found. An upregulation of the IL6/LIF-gp130 pathway was also showed to be involved in STAT3 activation in pancreatic cancer (Corcoran et al., 2011). The same study asserts that STAT3 is required for the development of the precursor pancreatic lesions, acinar-to-ductal metaplasia (ADM) and PanIN, therefore evaluation of gp130 and phospho-

With the increasing use of antiangiogenic agents for the treatment of cancers, is necessary the identification of candidate biomarkers for evaluation of the response and resistance. Is also, important to identify new biomarkers and to eliminate the risk for antiangiogenic

EGF (Epidermal Growth Factor), VEGF (Vascular Endothelial, Growth Factor) , heparanase, thrombospondin, cathepsins were the most important angiogenic factor specific for pancreatic cancer involved in cell growth promoting. The overexpression of EGF and his receptor EGFR were linked to tumor staging, but no clear data regarding the overall

Another important factor that acts on angiogenesis process is VEGF. His effects in PDAC are increased by interaction with MMP-9, a cellular matrix remodeling factor. The therapy against both MMP-9 and VEGF in pancreatic cancer resulted in a significant decrease in PDAC growth and microvessel density versus a single target treatment (Tanase-Pistol et

are specifically upregulated in the neoplastic epithelium (Badea et al., 2008).

cell cycle regulation and cell invasion (Tanase et al., 2010).

STAT3 expression may be an effective biomarker.

survival are available at this moment (Heidemann et al.,2006).

**3.2 Angiogenesis factors** 

al.,2008).

therapies failure (Duda et al., 2010).

IPMN and PDAC (Harsha et al., 2009).

Heparanases are endoglycosidases that cleave the heparan sulfate side chain of heparan sulfate proteoglycans (major membrane components) inducing extracellular matrix remodelling. On the other hand, heparanase increased growth factor bFGF (fibroblast growth factor) release, stimulating the angiogenesis process (Rohloff et al., 2002)

Thrombospondin (TSP-1) expression level was linked to a good prognostic for PDAC development. TSP-1 protein was found abundantly in stroma surrounding tumor cells and its expression is inversely correlated with microvessel density(Tobita et al., 2002).

The role of cathepsins in pancreatic cancer development and progression is still controversed. Cathepsin b (CTSB) and cathepsin l CTSL was found to be overexpressd in PDAC. A correlation between CTSB expression level and perineural invasion concludes the role of cathepsins in local tumor invasion (Niedergethmann et al., 2000).

#### **3.3 ZIP3 (Zinc/iron regulated transporter-related protein 3) a possible tumor suppressor**

Zinc is the most abundant trace element in cells. For example, about 25% of the total zinc present is found within the cell nuclei, being a component of chromatin. Zinc is an important factor in cellular processes, including cell division and proliferation, immune function, and defense against free radicals; zinc deficiency may be associated with an increased risk of cancer (Christudoss et al.,2011; Prasad et al., 2002). Zinc is found in over 300 enzymes, including copper/zinc superoxide dismutase, which is an important antioxidant enzyme, involved in cellular protection components from oxidation and damage.

In a current study, *Costello et al*. found a major loss of zinc in ductal and acinar epithelium in adenocarcinoma compared to the normal epithelium (Costello et al., 2011). The decrease in zinc quantity is a characteristic not only for pancreatic cancer, but also for precursor lesions. The mentioned group showed that the gene expression of ZIP3 (basilar membrane zinc uptake transporter) is present in normal ductal/acinar epithelium and absent in adenocarcinoma. The decreased expression of ZIP3 determines the loss of zinc in early and progressing malignancy. RREB1 transcription factor was found to be down regulated along with ZIP3 and might be the silencing cause of ZIP3 gene. Zinc treatment exhibited cytotoxic effect on Panc1 cell line. ZIP3 and RREB-1 expression level changes represent early events in the development of adenocarcinoma suggesting that ZIP3 might be a tumor suppressor gene.

#### **3.4 Other important factors**

FAP (fibroblast activation protein) is involved in the control of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis. It is highly expressed in PDAC and is considered to be related to a poor prognostic. Targeting FAP factors are considered to be a promising and a new therapeuthical road (Cohen et al., 2008).

Maspin (SerpinB5- mammary serine protease inhibitor) is a tumor suppressor gene. Maspin induces apoptosis in neoplastic cells and expression of maspin are suppressed as the carcinoma progresses in breast and prostatic carcinoma (Jiang et al., 2002). The pattern of maspin gene expression is dependent of the disease stage. For example, highly expression

Novel Biomarkers in Pancreatic Cancer 37

ALDH1 in ovarian cancer correlates with a favourable prognostic. In contrast, pancreatic cancer increased expression of ALDH1A1 was correlated with poor survival (Rasheed et al.,

ALDH1+ breast tumors are negative for estrogen receptor and progesterone receptor overexpression, but present a high level of Ki67 expression (a nuclear protein that is associated with cellular proliferation) (Morimoto et al., 2009). Therefore, *Kahlert et al*. showed that a high expression of ALDH1A1 is significantly correlated with the proliferation

Most of the pancreatic cancer patients have inoperable disease due to distant metastases or locally advanced tumor and the main therapeutic decision in this case is systemic chemotherapy or chemoradiation. Therefore, understanding the mechanisms that govern drug-related resistance and the factors involved in this process are a mandatory condition

Patients with advanced or metastatic pancreatic cancer are treated with gemcitabine as a first line chemotherapeutic agent. Gemcitabine is a 2′,2′-difluoro-2-deoxycytidine analogue that inhibits DNA replication and repair. Gemcitabine possesses radiosensitizing properties and its administration must be combined with radiotherapy. The sensitivity to gemcitabine and its efficiency were correlated with several factors. One of the factors that correlate with gemcitabine sensitivity is hENT1 (human equilibrative nucleoside transporter 1) (Nakano et

Pancreatic cancer cells that highly express hENT1 are sensitive to gemcitabine by uptaking this therapeutic agent in cancer cells. The absence hENT1 expression in metastatic pancreatic adenocarcinoma pacients treated with gemcitabine was associated with a poor prognostic compared with patients whom tumor cancer cells presented hENT1 expression (Hamada &

At the cellular level, gemcitabine is phosphorylated to its active metabolites by dCK (deoxycytidine kinase). Another group reported that not only hENT1 higher expression is a possible prognostic factor, but also dCK expression level and its activity correlates with gemcitabine activity (Ashida et al., 2009). High dCK enzyme activity was linked with gemcitabine sensitivity in experimental models (Kroep et al., 2002) and biopsy samples

Gemcitabine inactivation is performed through an enzymatic deamination process. There are three important enzymes involved in gemcitabine deamination (deoxycytidylate deaminase-DCD, cytidine deaminase-CDA and 5'-nucleotidase -5'-NT). The increased activity of such enzymes could induce the resistance to gemcitabine (Kroep et al., 2002; Giovannetti et al., 2006; Nakano et al., 2007) Gemcitabine resistance mechanism is also realized by high expression of RRM1 and RRM2, which are the two subunits (large and small) of ribonucleotide reductase. This enzyme regulates the rate of DNA synthesis, and is also known to convert ribonucleotides to deoxyribonucleotides. Gemcitabine exerts its

rate of pancreatic tumour cells (Kahlert et al., 2011).

**5.1 Metabolic prognosis factors for gemcitabine resistance** 

**5. Drug-related biomarkers** 

for therapeutic management success.

2010).

al., 2007).

Shimosegawa, 2011).

analysis (Sebastiani et al., 2006).

was observed in intraductal papillary mucinous neoplasms, mucinous cystic neoplasms. and in carcinomas, whereas in adenomas is lower.It was also observed a significant decrease in maspin expression when intraductal papillary mucinous neoplasms progresses to invasion. However, the group with high maspin expression presented microinvasion, whereas low expression maspin group showed extensive invasion (Kashima et al., 2008). These results conclude that maspine could be a good prognostic factor for invasion.

#### **4. Cancer stem cell biomarkers**

Stem cell markers are a promising group of new biomarkers. Solid tumors contain small proportions of cells that are capable of proliferation, self-renewal, and differentiation into various cell types. These types of cells (cancer stem cells) are characterized by treatment resistance, especially to ionizing radiation. Therefore, it is a very challenging situation in order to identify these cells, to understand the mechanisms of resistance, and to evaluate the patient therapy outcome regarding the response to treatment. Identifying the markers that characterize cancer stem cells is the main research and a specific pattern regarding cell surface markers is emerging. In breast cancer, stem cells presented a characteristic antigenic pattern, whereas in high-grade gliomas, expression of CD133 on the cell surface selects a population of treatment resistant cells (Woodward & Sulman, 2008).

In pancreatic cancer, several surface markers have been identified for a subpopulation of the tumor cells with stem cell characteristics. These cancer stem cells were identified by expression of the cell surface markers CD44, CD24, and ESA (Li et al., 2007). When injected in the pancreas of immunocompromised mice these human cells are able to self-renew and generate differentiated progeny, to recapitulate the phenotype of the tumor from which they were derived.

Another subpopulation of cancer stem cells highly tumorigenic was isolated from patients with PDAC. These cell types were CD133+ and were able to induce tumor formation in athymic mice. This subpopulation of migrating cancer stem cells are characterized also by expression of the CXCR4 receptor and are involved in tumor metastasis Hermann et al., 2007.

Another candidate cell marker investigated was aldehyde dehydrogenase 1A1 (ALDH1A1), which has been identified to label cancer stem cells in breast cancer (Ginestier et al., 2007), colon cancer (Huang et al., 2009), lung cancer (Jiang et al., 2009) and head and neck squamous cancer (Chen et al., 2009). ALDH1 is a member of the aldehyde dehydrogenase gene (ALDH) superfamily playing an important role in the metabolism of endogenous and exogenous aldehydes. This NAD(P)(+)-dependent enzyme is also involved in the formation of molecules that are important in cellular processes, like retinoic acid (acting like modulator of gene regulation and cell differentiation), betaine and gamma-aminobutyric acid and exhibits additional, non-enzymatic functions, being able to bind to some hormones (Yoshida et al., 1998). The ALDH1 gene expression is ubiquitous in many human tissues; the protein is found localized mainly in the cellular cytoplasm.

It was also shown that ALDH1 has the capacity to detoxify aldophosphamide, conferring chemoresistance against cyclophosphamide to overexpressing cell (Hilton, 1984). Data regarding the effect of ALDH1A1 overexpression are controversial. Increased expression of

was observed in intraductal papillary mucinous neoplasms, mucinous cystic neoplasms. and in carcinomas, whereas in adenomas is lower.It was also observed a significant decrease in maspin expression when intraductal papillary mucinous neoplasms progresses to invasion. However, the group with high maspin expression presented microinvasion, whereas low expression maspin group showed extensive invasion (Kashima et al., 2008).

Stem cell markers are a promising group of new biomarkers. Solid tumors contain small proportions of cells that are capable of proliferation, self-renewal, and differentiation into various cell types. These types of cells (cancer stem cells) are characterized by treatment resistance, especially to ionizing radiation. Therefore, it is a very challenging situation in order to identify these cells, to understand the mechanisms of resistance, and to evaluate the patient therapy outcome regarding the response to treatment. Identifying the markers that characterize cancer stem cells is the main research and a specific pattern regarding cell surface markers is emerging. In breast cancer, stem cells presented a characteristic antigenic pattern, whereas in high-grade gliomas, expression of CD133 on the cell surface selects a

In pancreatic cancer, several surface markers have been identified for a subpopulation of the tumor cells with stem cell characteristics. These cancer stem cells were identified by expression of the cell surface markers CD44, CD24, and ESA (Li et al., 2007). When injected in the pancreas of immunocompromised mice these human cells are able to self-renew and generate differentiated progeny, to recapitulate the phenotype of the tumor from which they

Another subpopulation of cancer stem cells highly tumorigenic was isolated from patients with PDAC. These cell types were CD133+ and were able to induce tumor formation in athymic mice. This subpopulation of migrating cancer stem cells are characterized also by expression of the CXCR4 receptor and are involved in tumor metastasis Hermann et al.,

Another candidate cell marker investigated was aldehyde dehydrogenase 1A1 (ALDH1A1), which has been identified to label cancer stem cells in breast cancer (Ginestier et al., 2007), colon cancer (Huang et al., 2009), lung cancer (Jiang et al., 2009) and head and neck squamous cancer (Chen et al., 2009). ALDH1 is a member of the aldehyde dehydrogenase gene (ALDH) superfamily playing an important role in the metabolism of endogenous and exogenous aldehydes. This NAD(P)(+)-dependent enzyme is also involved in the formation of molecules that are important in cellular processes, like retinoic acid (acting like modulator of gene regulation and cell differentiation), betaine and gamma-aminobutyric acid and exhibits additional, non-enzymatic functions, being able to bind to some hormones (Yoshida et al., 1998). The ALDH1 gene expression is ubiquitous in many human tissues; the protein

It was also shown that ALDH1 has the capacity to detoxify aldophosphamide, conferring chemoresistance against cyclophosphamide to overexpressing cell (Hilton, 1984). Data regarding the effect of ALDH1A1 overexpression are controversial. Increased expression of

These results conclude that maspine could be a good prognostic factor for invasion.

population of treatment resistant cells (Woodward & Sulman, 2008).

is found localized mainly in the cellular cytoplasm.

**4. Cancer stem cell biomarkers** 

were derived.

2007.

ALDH1 in ovarian cancer correlates with a favourable prognostic. In contrast, pancreatic cancer increased expression of ALDH1A1 was correlated with poor survival (Rasheed et al., 2010).

ALDH1+ breast tumors are negative for estrogen receptor and progesterone receptor overexpression, but present a high level of Ki67 expression (a nuclear protein that is associated with cellular proliferation) (Morimoto et al., 2009). Therefore, *Kahlert et al*. showed that a high expression of ALDH1A1 is significantly correlated with the proliferation rate of pancreatic tumour cells (Kahlert et al., 2011).

#### **5. Drug-related biomarkers**

Most of the pancreatic cancer patients have inoperable disease due to distant metastases or locally advanced tumor and the main therapeutic decision in this case is systemic chemotherapy or chemoradiation. Therefore, understanding the mechanisms that govern drug-related resistance and the factors involved in this process are a mandatory condition for therapeutic management success.

#### **5.1 Metabolic prognosis factors for gemcitabine resistance**

Patients with advanced or metastatic pancreatic cancer are treated with gemcitabine as a first line chemotherapeutic agent. Gemcitabine is a 2′,2′-difluoro-2-deoxycytidine analogue that inhibits DNA replication and repair. Gemcitabine possesses radiosensitizing properties and its administration must be combined with radiotherapy. The sensitivity to gemcitabine and its efficiency were correlated with several factors. One of the factors that correlate with gemcitabine sensitivity is hENT1 (human equilibrative nucleoside transporter 1) (Nakano et al., 2007).

Pancreatic cancer cells that highly express hENT1 are sensitive to gemcitabine by uptaking this therapeutic agent in cancer cells. The absence hENT1 expression in metastatic pancreatic adenocarcinoma pacients treated with gemcitabine was associated with a poor prognostic compared with patients whom tumor cancer cells presented hENT1 expression (Hamada & Shimosegawa, 2011).

At the cellular level, gemcitabine is phosphorylated to its active metabolites by dCK (deoxycytidine kinase). Another group reported that not only hENT1 higher expression is a possible prognostic factor, but also dCK expression level and its activity correlates with gemcitabine activity (Ashida et al., 2009). High dCK enzyme activity was linked with gemcitabine sensitivity in experimental models (Kroep et al., 2002) and biopsy samples analysis (Sebastiani et al., 2006).

Gemcitabine inactivation is performed through an enzymatic deamination process. There are three important enzymes involved in gemcitabine deamination (deoxycytidylate deaminase-DCD, cytidine deaminase-CDA and 5'-nucleotidase -5'-NT). The increased activity of such enzymes could induce the resistance to gemcitabine (Kroep et al., 2002; Giovannetti et al., 2006; Nakano et al., 2007) Gemcitabine resistance mechanism is also realized by high expression of RRM1 and RRM2, which are the two subunits (large and small) of ribonucleotide reductase. This enzyme regulates the rate of DNA synthesis, and is also known to convert ribonucleotides to deoxyribonucleotides. Gemcitabine exerts its

Novel Biomarkers in Pancreatic Cancer 39

was found in pancreatic cancer xenografts and primary pancreatic adenocarcinomas. Reexpression of the *TFPI-2* gene led in the proliferation, migration and invasion of cancer cells

 GATA gene family members were also epigenetic silenced in pancreatic cancer. For example, *GATA-5* was frequently methylated in pancreatic cancers, whereas *GATA-4* was

Another gene commonly silenced epigenetically in pancreatic cancer is BNIP3. The protein encoded by this gene contains a BH3 domain and a transmembrane domain associated with pro-apoptotic function. BNIP3 gene silencing induced a drug resistance mechanism in

*Shimizu et al.* using a novel method called "microarray coupled with methyl-CpG targeted transcriptional activation" (MeTA-array for short), identified 16 genes hypermethylated in three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2 and PANC-1. Among these 16 genes several presented higher methylation level (CSMD2, SLC32A1, TMEM204 and TRH). CSMD2, SLC32A1 and TRH genes presented also a hypermethylated pattern in

In contrast, a great number of genes are overexpressed in pancreatic cancer. These genes presented hypomethylation of the promoter in pancreatic cancer versus normal pancreatic

DNA hypomethylation of promoter CpGs were identified in genes with overexpression pattern including claudin4, lipocalin2, 14-3-3sigma/ stratifin, trefoil factor 2, S100A4,

Covalent histone modifications are important regulatory elements in many biological processes. These modifications control the chromatin status by electrostatic interaction changes and non-histonic protein recruitement. Histones suffer specific N-terminal end post-translational changes represented by acethylation, methylation, phosphorylation, sumoylation, ubiquitination and ADP-ribosylation. These modifications alter DNA-histones

Certain histone modifications influence gene transcription level. The interplay between histone modifications led to 'histone code hypothesis'. For example, lysine acetylation neutralizes the charge between DNA and histone tails and correlates with a more

Hypermethylated CpG islands of silenced tumor-suppressor genes are correlated with deacetylation of histones H3 and H4, methylation of H3 lysine 9 (H3K9), H3 lysine 27 (H3K27), and H4 lysine 20 (H4K20), and demethylation of H3 lysine 4 (Rosenfeld et al., 2009; Barski et al., 2007). Methylation of histone H3 lysine 4 (H3K4) and H3 lysine 36 is associated with relaxed chromatin status (Benevolenskaya et al., 2007). Histone modifications recruit effector proteins. Acetylated lysines are recognized by bromodomains within nucleosome remodeling complexes, methylated H3K4 and the helicase Chd1 chromodomain recruits activating complexes of chromatin (Daniel et al., 2005). In contrast, methylated H3K9 and

pancreatic cancer cells, as a potential drug resistance mechanism (Okami et al., 2004)

(Sato et al., 2005).

tissue.

hypomethylated (Fu et al., 2007).

primary pancreatic cancers (Shimizu et al., 2011).

mesothelin, PSCA, S100P and maspin (Sato et al., 2003).

**6.2 Histone modifications hallmarks for oncogenesis process** 

transcription permissive status of chromatin (Jenuwein et al., 2001).

interaction having a major impact on chromatin structure (Strahl et al., 2000).

cytotoxicity by inhibiting ribonucleotide reductase (Davidson et al., 2004; Bergman et al., 2005; Nakahira et al., 2006)

#### **6. Epigenetic biomarkers**

Cancer initiation and progression is traditionally characterized as a genetic alteration, but recent years crystallized in a new direction regarding epigenetic mechanisms involvement in oncogenesis. Epigenetic mechanisms are essential for normal development and for maintaining of a tissue specific pattern of gene expression. Epigenetic modifications lead to an abnormal genetic expression and further to malign transformation. The research in epigenetic field has demonstrated the involvement of an intensive reprogramming of epigenetic machinery (DNA methylation, histone modifications, microRNA expression). The reverse nature of epigenetic abnormalities permitted the development of epigenetic therapy field.

#### **6.1 Gene promoter methylation status and pancreatic oncogenesis**

The most known epigenetic modification in oncogenesis is DNA hypermethylation. This event is accompanied by genetic silencing. Identification and characterization of epigenetically silenced genes is important in order to understand the roles of such epigenetic modification in oncogenesis and to discover new tumor markers. It was reported that in pancreas cancer some tumor suppressor genes presented aberrant CpG islands hypermethylation at gene promoter level.

The first tumor suppressor gene identified to be specific for pancreatic cancer was p16/CDKN1A (Schutte et al., 1997). Subsequently, new hypermethylated genes were associated with pancreatic cancer (hMLH1, E-cadherin, ppENK, CDKN1C, SPARC, TFPI-2, GATA4,5, BNIP3, TSLC1, HHIP, MUC2, reprimo, CXCR4 si SOCS1) (Omura et al., 2009).

Several important epigenetically silenced factors have been identified (hsa-miR-9-1, ZNF415, CNTNAP2 si ELOVL4) (Omura et al 2009; Grady et al.,2008; Lehmann et al,.2008; Fabbri et al., 2007; Moriss et al., 2004).

Using methylated CpG island amplification (MCA) and representational difference analysis (RDA), Ueki *et al* group identified that gene preproenkephalin (*ppENK*) presented a hypermethylated promoter in most pancreatic cancers (Ueki et al., 2001). *ppENK* encodes an opioid peptide which presents growth-suppressor properties.

It was showed that CDKN1C/p57KIP2 gene presented a decrease expression in intraductal papillary mucinous neoplasm. The gene encodes for cyclin-dependent kinase inhibitor, and is a negative regulator of cell proliferation (Sato et al., 2005). Partial methylation of the *CDKN1C* promoter was found in pancreatic cancer cell lines and pancreas cancer.

SPARC (secreted protein acidic and rich in cysteine, or osteonectin/BM40) is a matrixassociated protein; calcium binding, that inhibits cell-cycle progression, and influences the synthesis of extracellular matrix. The gene codifying this protein was found aberrantly methylated in pancreatic cancer by. SPARC is a factor involved in many processes like cell migration, proliferation, matrix cell adhesion (Sato et al., 2003, Gao et al., 2010).

Tissue factor pathway inhibitor 2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor, which has been identified as a putative tumor-suppressor gene. Aberrant methylation of *TFPI*-2

cytotoxicity by inhibiting ribonucleotide reductase (Davidson et al., 2004; Bergman et al.,

Cancer initiation and progression is traditionally characterized as a genetic alteration, but recent years crystallized in a new direction regarding epigenetic mechanisms involvement in oncogenesis. Epigenetic mechanisms are essential for normal development and for maintaining of a tissue specific pattern of gene expression. Epigenetic modifications lead to an abnormal genetic expression and further to malign transformation. The research in epigenetic field has demonstrated the involvement of an intensive reprogramming of epigenetic machinery (DNA methylation, histone modifications, microRNA expression). The reverse nature of epigenetic abnormalities permitted the development of epigenetic therapy field.

The most known epigenetic modification in oncogenesis is DNA hypermethylation. This event is accompanied by genetic silencing. Identification and characterization of epigenetically silenced genes is important in order to understand the roles of such epigenetic modification in oncogenesis and to discover new tumor markers. It was reported that in pancreas cancer some tumor suppressor genes presented aberrant CpG islands

The first tumor suppressor gene identified to be specific for pancreatic cancer was p16/CDKN1A (Schutte et al., 1997). Subsequently, new hypermethylated genes were associated with pancreatic cancer (hMLH1, E-cadherin, ppENK, CDKN1C, SPARC, TFPI-2, GATA4,5, BNIP3, TSLC1, HHIP, MUC2, reprimo, CXCR4 si SOCS1) (Omura et al., 2009).

Several important epigenetically silenced factors have been identified (hsa-miR-9-1, ZNF415, CNTNAP2 si ELOVL4) (Omura et al 2009; Grady et al.,2008; Lehmann et al,.2008; Fabbri et

Using methylated CpG island amplification (MCA) and representational difference analysis (RDA), Ueki *et al* group identified that gene preproenkephalin (*ppENK*) presented a hypermethylated promoter in most pancreatic cancers (Ueki et al., 2001). *ppENK* encodes an

It was showed that CDKN1C/p57KIP2 gene presented a decrease expression in intraductal papillary mucinous neoplasm. The gene encodes for cyclin-dependent kinase inhibitor, and is a negative regulator of cell proliferation (Sato et al., 2005). Partial methylation of the

SPARC (secreted protein acidic and rich in cysteine, or osteonectin/BM40) is a matrixassociated protein; calcium binding, that inhibits cell-cycle progression, and influences the synthesis of extracellular matrix. The gene codifying this protein was found aberrantly methylated in pancreatic cancer by. SPARC is a factor involved in many processes like cell

Tissue factor pathway inhibitor 2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor, which has been identified as a putative tumor-suppressor gene. Aberrant methylation of *TFPI*-2

*CDKN1C* promoter was found in pancreatic cancer cell lines and pancreas cancer.

migration, proliferation, matrix cell adhesion (Sato et al., 2003, Gao et al., 2010).

**6.1 Gene promoter methylation status and pancreatic oncogenesis** 

opioid peptide which presents growth-suppressor properties.

2005; Nakahira et al., 2006)

**6. Epigenetic biomarkers** 

hypermethylation at gene promoter level.

al., 2007; Moriss et al., 2004).

was found in pancreatic cancer xenografts and primary pancreatic adenocarcinomas. Reexpression of the *TFPI-2* gene led in the proliferation, migration and invasion of cancer cells (Sato et al., 2005).

 GATA gene family members were also epigenetic silenced in pancreatic cancer. For example, *GATA-5* was frequently methylated in pancreatic cancers, whereas *GATA-4* was hypomethylated (Fu et al., 2007).

Another gene commonly silenced epigenetically in pancreatic cancer is BNIP3. The protein encoded by this gene contains a BH3 domain and a transmembrane domain associated with pro-apoptotic function. BNIP3 gene silencing induced a drug resistance mechanism in pancreatic cancer cells, as a potential drug resistance mechanism (Okami et al., 2004)

*Shimizu et al.* using a novel method called "microarray coupled with methyl-CpG targeted transcriptional activation" (MeTA-array for short), identified 16 genes hypermethylated in three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2 and PANC-1. Among these 16 genes several presented higher methylation level (CSMD2, SLC32A1, TMEM204 and TRH). CSMD2, SLC32A1 and TRH genes presented also a hypermethylated pattern in primary pancreatic cancers (Shimizu et al., 2011).

In contrast, a great number of genes are overexpressed in pancreatic cancer. These genes presented hypomethylation of the promoter in pancreatic cancer versus normal pancreatic tissue.

DNA hypomethylation of promoter CpGs were identified in genes with overexpression pattern including claudin4, lipocalin2, 14-3-3sigma/ stratifin, trefoil factor 2, S100A4, mesothelin, PSCA, S100P and maspin (Sato et al., 2003).

#### **6.2 Histone modifications hallmarks for oncogenesis process**

Covalent histone modifications are important regulatory elements in many biological processes. These modifications control the chromatin status by electrostatic interaction changes and non-histonic protein recruitement. Histones suffer specific N-terminal end post-translational changes represented by acethylation, methylation, phosphorylation, sumoylation, ubiquitination and ADP-ribosylation. These modifications alter DNA-histones interaction having a major impact on chromatin structure (Strahl et al., 2000).

Certain histone modifications influence gene transcription level. The interplay between histone modifications led to 'histone code hypothesis'. For example, lysine acetylation neutralizes the charge between DNA and histone tails and correlates with a more transcription permissive status of chromatin (Jenuwein et al., 2001).

Hypermethylated CpG islands of silenced tumor-suppressor genes are correlated with deacetylation of histones H3 and H4, methylation of H3 lysine 9 (H3K9), H3 lysine 27 (H3K27), and H4 lysine 20 (H4K20), and demethylation of H3 lysine 4 (Rosenfeld et al., 2009; Barski et al., 2007). Methylation of histone H3 lysine 4 (H3K4) and H3 lysine 36 is associated with relaxed chromatin status (Benevolenskaya et al., 2007). Histone modifications recruit effector proteins. Acetylated lysines are recognized by bromodomains within nucleosome remodeling complexes, methylated H3K4 and the helicase Chd1 chromodomain recruits activating complexes of chromatin (Daniel et al., 2005). In contrast, methylated H3K9 and

Novel Biomarkers in Pancreatic Cancer 41

The hnRNP A2/B1 protein plays an important role in the biogenesis and transport of mRNA. Abnormal expression of this protein leads to alteration of normal transcription. In concordance with this study a previous work found high levels of hnRNP A2/B1 expression in a limited number of human pancreatic adenocarcinomas from smokers and two pancreatic tumor cell lines, HPAF-11 and SU 86 (Shen et al., 2004). In contrast, carboxyl ester lipase (CEL pancreatic exocrine enzyme) expression level progressively decreased with

DMBA implantation into the rat pancreas is an effective method to induce PanINs and pancreas cancer in order to determine which the first change in proteins expression pattern

Actually, diagnostic methods for pancreatic cancer include invasive procedures (tissue sampling by endoscopy), involving risks and causing complications. The necessity for less invasive diagnostic methods is increasing; therefore the development of non-invasive

The major directions of proteomic range from basic research to discovery, validation and use of clinical applications. Protein profiling methods include high resolution twodimensional gels, two-dimensional differential in-gel electrophoresis, LC-MS and LC-MS/MS using accurate mass tags, and protein identifications using mass spectrometry methods. These methods were used in many studies for identification of prognostic and/or

The only clinically available serum biomarker for PDAC is CA 19-9, which is useful for the follow-up of pancreatic cancer patients receiving treatment, but has not been recommended for cancer screening (Goggins et al., 2000; Locker et al., 2006). The American Society of Clinical Oncology (ASCO) 2006 guidelines for the use of tumor markers do not recommend CA19-9 as a screening test for pancreatic cancer (Rosty et al., 2002; Liang et al., 2009). Several

Recent papers published new promising biomarkers, which can potentially detect early

Roberts *et al.* analyzed serum samples from patients with locally advanced or metastatic adenocarcinoma of the pancreas. Patient group was selected based on length of survival and type of therapy, and serum was subjected to liquid chromatography coupled to tandem mass spectrometry analysis (LC-MS-MS) (Roberts et al., 2011). The proteins presenting important changes in expression levels were validated by enzyme-linked immunosorbent assay (ELISA). After the data were analyzed, the authors selected 1 putative prognostic protein, alpha 1 antichymotrypsin (AACT), and 2 putative predictive proteins, histidine-rich glycoprotein (HRG) and complement factor H (CFH). AACT was found to be negatively correlated with overall survival, whereas CFH was found to have no predictive value as prognostic factor for overall survival. AACT may be a useful prognostic marker in patients with advanced stage

**8. Novel biomarkers for the non-invasive diagnosis of pancreatic cancer** 

DMBA-induced disease severity.

**8.1 Plasma biomarkers** 

is, and to identify markers for pancreas lesions progression.

biomarkers in pancreatic cancer is mandatory.

predictive biomarkers that may help stratify patients.

stage pancreatic cancer (Chen et al., 2011).

other serum markers have been proposed for pancreatic cancer.

pancreatic carcinoma, although additional validation studies are needed.

H3K27 interacts with heterochromatin protein 1 (HP 1) and Polycomb-group (PcG) proteins leading to chromatin compaction (Fischle et al., 2003). PcG proteins function as transcriptional repressors, but the molecular mechanisms of Polycomb repressive complex (PRC)-mediated repression is not clear (Sparmann et al., 2006). PcG proteins recruits DNMTs (DNA methyl transferase) involved in the hypermethylation of tumor suppressor genes (Vire et al., 2006).

Only few studies have examined genes that are regulated by histone modifications in pancreatic cancers. For example, mucin family gene underwent histone alterations in pancreatic cancers in association with gene overexpression. The 5' region of MUC1 gene transcriptional start site is enriched in tri/dimethylated H3K9 and methylated DNA in nontumor cells (Yamada et al., 2008). Transcriptional start site of MUC2 is highly enriched in diand tri-methylated H3K4, acetylated H3K9, and acetylated H3K27 in pancreatic cancer cells. Vincent *et al* demonstrated that *MUC4* transcription activity is affected by many factors (DNMT3A, DNMT3B, HDAC1 and HDAC3, DNA methylation, histone modification (Vincent et al., 2008).

#### **7. Model organisms studies provide potential biomarkers in pancreas oncogenesis**

Model organisms are widely used to explore potential causes and treatments for human disease. This strategy is made possible by the conservation of metabolic and developmental pathways and genetic material over the course of evolution.

It is known that Enolase 1 (α-enolase or non-neuronal enolase –NNE), is an isoenzyme of enolase, which catalyze the conversion of 2-phosphoglycerate into phosphoenolpyruvate.

Several studies have shown that enolase 1 plays an important role in in tumorigenesis, cancer invasion and metastasis. Proteomic studies reported that expression of enolase 1 is increased in cancers, such as hepatocellular carcinoma (Takashima et al. 2008, Hamaguchi et al., 2008) , non-small lung cancer (He et al., 2007), esophageal adenocarcinoma (Zhao et al., 2007), prostate cancer (van den Bemd et al., 2006), colon cancer (Katayama et al., 2006), oral epithelial and squamous cell carcinoma (Ito et al., 2007).

In pancreatic cancer Mikuriya et al using two-dimensional electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry showed that the expression levels of glycolytic enzymes, including enolase 1, increased in the cancerous pancreatic tissues patients compared with the paired non-cancerous tissues (Mikuriya et al., 2007).

In order to evaluate Enolase 1 expression changes, Lei et al. used chemical induced carcinogenesis in rats. Implantation of 7,12-dimethylbenzanthracene in rat pancreas leads to pancreatic cancer and PanINs. Alpha-enolase was specifically overexpressed in tumors compared with normal and pancreatic tissues (Lei et al., 2011).

The group found several proteins overexpressed in this carcinogenesis model, along with enolase 1 (Tumor protein translationally controlled 1, Expressed in non-metastatic cells 2, Pancreatic elastase 3B , Necdin, Hbp23, Chromodomain helicase DNA-binding protein, Albumin+retinoid X receptor-interacting protein, Heterogeneous nuclear ribonucleoprotein A2/B1-hnRNP A2/B1).

H3K27 interacts with heterochromatin protein 1 (HP 1) and Polycomb-group (PcG) proteins leading to chromatin compaction (Fischle et al., 2003). PcG proteins function as transcriptional repressors, but the molecular mechanisms of Polycomb repressive complex (PRC)-mediated repression is not clear (Sparmann et al., 2006). PcG proteins recruits DNMTs (DNA methyl transferase) involved in the hypermethylation of tumor suppressor

Only few studies have examined genes that are regulated by histone modifications in pancreatic cancers. For example, mucin family gene underwent histone alterations in pancreatic cancers in association with gene overexpression. The 5' region of MUC1 gene transcriptional start site is enriched in tri/dimethylated H3K9 and methylated DNA in nontumor cells (Yamada et al., 2008). Transcriptional start site of MUC2 is highly enriched in diand tri-methylated H3K4, acetylated H3K9, and acetylated H3K27 in pancreatic cancer cells. Vincent *et al* demonstrated that *MUC4* transcription activity is affected by many factors (DNMT3A, DNMT3B, HDAC1 and HDAC3, DNA methylation, histone modification

**7. Model organisms studies provide potential biomarkers in pancreas** 

pathways and genetic material over the course of evolution.

epithelial and squamous cell carcinoma (Ito et al., 2007).

compared with normal and pancreatic tissues (Lei et al., 2011).

Model organisms are widely used to explore potential causes and treatments for human disease. This strategy is made possible by the conservation of metabolic and developmental

It is known that Enolase 1 (α-enolase or non-neuronal enolase –NNE), is an isoenzyme of enolase, which catalyze the conversion of 2-phosphoglycerate into phosphoenolpyruvate.

Several studies have shown that enolase 1 plays an important role in in tumorigenesis, cancer invasion and metastasis. Proteomic studies reported that expression of enolase 1 is increased in cancers, such as hepatocellular carcinoma (Takashima et al. 2008, Hamaguchi et al., 2008) , non-small lung cancer (He et al., 2007), esophageal adenocarcinoma (Zhao et al., 2007), prostate cancer (van den Bemd et al., 2006), colon cancer (Katayama et al., 2006), oral

In pancreatic cancer Mikuriya et al using two-dimensional electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry showed that the expression levels of glycolytic enzymes, including enolase 1, increased in the cancerous pancreatic tissues

In order to evaluate Enolase 1 expression changes, Lei et al. used chemical induced carcinogenesis in rats. Implantation of 7,12-dimethylbenzanthracene in rat pancreas leads to pancreatic cancer and PanINs. Alpha-enolase was specifically overexpressed in tumors

The group found several proteins overexpressed in this carcinogenesis model, along with enolase 1 (Tumor protein translationally controlled 1, Expressed in non-metastatic cells 2, Pancreatic elastase 3B , Necdin, Hbp23, Chromodomain helicase DNA-binding protein, Albumin+retinoid X receptor-interacting protein, Heterogeneous nuclear ribonucleoprotein

patients compared with the paired non-cancerous tissues (Mikuriya et al., 2007).

genes (Vire et al., 2006).

(Vincent et al., 2008).

A2/B1-hnRNP A2/B1).

**oncogenesis** 

The hnRNP A2/B1 protein plays an important role in the biogenesis and transport of mRNA. Abnormal expression of this protein leads to alteration of normal transcription. In concordance with this study a previous work found high levels of hnRNP A2/B1 expression in a limited number of human pancreatic adenocarcinomas from smokers and two pancreatic tumor cell lines, HPAF-11 and SU 86 (Shen et al., 2004). In contrast, carboxyl ester lipase (CEL pancreatic exocrine enzyme) expression level progressively decreased with DMBA-induced disease severity.

DMBA implantation into the rat pancreas is an effective method to induce PanINs and pancreas cancer in order to determine which the first change in proteins expression pattern is, and to identify markers for pancreas lesions progression.

#### **8. Novel biomarkers for the non-invasive diagnosis of pancreatic cancer**

Actually, diagnostic methods for pancreatic cancer include invasive procedures (tissue sampling by endoscopy), involving risks and causing complications. The necessity for less invasive diagnostic methods is increasing; therefore the development of non-invasive biomarkers in pancreatic cancer is mandatory.

#### **8.1 Plasma biomarkers**

The major directions of proteomic range from basic research to discovery, validation and use of clinical applications. Protein profiling methods include high resolution twodimensional gels, two-dimensional differential in-gel electrophoresis, LC-MS and LC-MS/MS using accurate mass tags, and protein identifications using mass spectrometry methods. These methods were used in many studies for identification of prognostic and/or predictive biomarkers that may help stratify patients.

The only clinically available serum biomarker for PDAC is CA 19-9, which is useful for the follow-up of pancreatic cancer patients receiving treatment, but has not been recommended for cancer screening (Goggins et al., 2000; Locker et al., 2006). The American Society of Clinical Oncology (ASCO) 2006 guidelines for the use of tumor markers do not recommend CA19-9 as a screening test for pancreatic cancer (Rosty et al., 2002; Liang et al., 2009). Several other serum markers have been proposed for pancreatic cancer.

Recent papers published new promising biomarkers, which can potentially detect early stage pancreatic cancer (Chen et al., 2011).

Roberts *et al.* analyzed serum samples from patients with locally advanced or metastatic adenocarcinoma of the pancreas. Patient group was selected based on length of survival and type of therapy, and serum was subjected to liquid chromatography coupled to tandem mass spectrometry analysis (LC-MS-MS) (Roberts et al., 2011). The proteins presenting important changes in expression levels were validated by enzyme-linked immunosorbent assay (ELISA). After the data were analyzed, the authors selected 1 putative prognostic protein, alpha 1 antichymotrypsin (AACT), and 2 putative predictive proteins, histidine-rich glycoprotein (HRG) and complement factor H (CFH). AACT was found to be negatively correlated with overall survival, whereas CFH was found to have no predictive value as prognostic factor for overall survival. AACT may be a useful prognostic marker in patients with advanced stage pancreatic carcinoma, although additional validation studies are needed.

Novel Biomarkers in Pancreatic Cancer 43

This strong association between NP with inflammation determined to evaluate the expression and activity of this protein in PDAC, specifically in patients with antecedent inflammatory conditions like chronic pancreatitis, and pancreatic intraepithelial neoplasia (PanIN) (Rebours et al., 2010). Therefore, this correlation makes NP levels quantification to

Saliva is a body fluid that can be easily obtained without using a special technique. Recent reports suggested the possible utility of saliva in quantification of specific factors to discriminate between pancreatic cancer patients and patients with normal or chronic

Using transcriptome profiles, Zhang *et al*. group could differentiate pancreatic cancer patients from healthy subject with a sensitivity of 90.0% and a specificity of 95.0%. The group found 12 mRNA biomarkers specific for pancreatic cancer patients. Seven genes were found to be up-regulated (*MBD3L2*, *KRAS*, *STIM2*, *DMXL2ACRV1*, *DMD*, *CABLES1*), whereas five genes presented a decreased expression *TK2*, *GLTSCR2*, *CDKL3, DPM1, TPT1* 

A similar approach has been made by another group, which evaluated metabolites in saliva using mass spectrometry. Pancreatic cancer cases were successfully detected based on the pancreatic cancer-specific signature (Sugimoto et al., 2010). The levels of ornithine and putrescine were higher in patients with breast or pancreatic cancer, and were markedly higher in patients with oral cancer. The level of tryptophan is also increased in oral and pancreatic cancer, in contrast to arginine level which is decreased several cancers including breast, colonic and pancreatic cancer, which might be due to increased uptake of arginine by

A new and important atractive tool is represented by small non-coding RNA. These are regulators of various biological processes like gene expression and are involved in cancer progression. One of the most important players is microRNAs which control many cellular functions, such as migration, invasion and stem cell functions. Abnormal microRNA gene expression was found in many cancers, including pancreatic cancer (Rachangani et al., 2010,

MicroRNA molecules are present in various body fluids (blood, urine, cerebrospinal fluid, pancreatic juice, billiary secretion). Moreover, several microRNAs from sera seemed to be identical for many cancer types. Very important is the fact that miRNAs from sera are stable and exhibit resistance to RN-ase activity, intermittent frosting and defrosting, high

Using microarray technology it was established that several microRNAs are highly expressed in pancreatic cancer (miR-21, miR-17-5p,miR-191,miR-29b-2,miR-223 miR-128b, miR-199a-1, miR-24-1, miR-24-2,miR-146, miR-181b-1, miR-20a,miR-107,miR-32 , miR-92-2, miR-214,miR-30c, miR-25, miR-221, miR-106) (Volinia et al., 2006). More recently, was established that microRNAs, miR-200a and miR-200b are highly expressed in pancreatic

temperature values and extreme pH ( Mitchell et al., 2008; Albulescu et al., 2011).

be a useful marker for surveillance progression from inflammation to PDAC.

**8.3 Saliva biomarkers** 

pancreatitis.

(Zhang et al., 2010)*.*

tumor tissues with high arginase activity.

**8.4 Non- coding RNA as new era biomarkers** 

Eis et al., 2005, Calin et al., 2005, Yanaihara et al., 2006).

Another study involving quantification in patient serum of tumor cell metabolites, or secreted factor was conducted by He *et al.* . This study focused on DJ-1 oncoprotein secreted by cancer cells (He et al., 2011). The study group involved patients diagnosed with pancreatic cancer and chronic pancreatitis, along with healthy subjects. DJ-1 serum level and the conventional tumor marker carbohydrate antigen 19-9 (CA 19-9) were measured in order to establish the diagnostic and prognostic value of DJ-1. Serum DJ-1 level was increased in patients with pancreas cancer compared with chronic pancreatitis and healthy individuals. Serum DJ-1 levels were higher than CA 19-9, and combined the two biomarkers provided a sensitivity of 87.5%. After resection DJ-1 levels decreased and patient with lower value of this factor had a better prognosis. This study provides a potential clinical biomarker, easy to quantify from serum, in order to establish a rapid diagnosis and to evaluate prognosis in patients with pancreas cancer.

#### **8.2 Pancreatic juice biomarkers**

Analysis of protein expression profiles of pancreatic juice samples harvested from the pancreatic duct has the potential to identify markers that could serve for diagnosis triage of benign from malignant pancreatic lesions and to discriminate between different stages of PDAC.

The research performed by *Vareed et al*. has shown that 56 proteins were found to be elevated in pancreatic juice of PDAC patients compared to benign controls (Vareed et al., 2011).

Protein profiles studies revealed an unique presence of proteins associated with Parkinson's disease namely: aSyn and PARK7 (Bonifati et al., 2003; Singleton et al., 2003).

Increased expression of aSyn has been also identified in melanoma (Matsuo et al., 2010), while its isoform gamma-synuclein (cSyn) has been shown to be elevated in tumors of breast, uterine, colorectal and pancreas (Ye et al., 2009; Hibi et al., 2009; Ahmad et al., 2007; Li et al., 2004; Gupta et al., 2003; Jia et al., 1999; Morgan et al., 2009). Moreover, due to the structural omology between cSyn and aSyn, these factors potentiate invasion in these tumors.

Interestingly, tissue arrays demonstrated a strong staining for aSyn in tumors, and this protein expressed in a subset of PDAC patients was identified in its aggregated form similar to Lewy Bodies seen in Parkinson's disease (Polymeropoulus et al., 1997). Another interesting coincidence is the presence of increased levels of Parkinson's disease associated protein PARK7 (DJ1) (van Duijin et al., 2001) in pancreatic ductal juice of adenocarcinomas. The mechanism by which the protein PARK-7 exerts its oncogenic effect remains unclear. It is presumed the involvement of p38 mitogen activated-protein kinase signaling (Mo et al., 2010).

On the other hand PDAC-associated secretory proteome analysis also revealed increased levels of several metabolic enzymes. The most important metabolic factor was Purine NucleosidePhosphorylase (NP), enzyme involved in salvage pathway of purines, which is operational during inflammation and neoplastic progression, (Bantia et al., 2010). NP activity has been reported to be high in cancer sera (Roberts et al., 2004) and NP expression has also been used to determine the clinical severity of various types of cancers, in combination with another factor adenosine deaminase (ADA) (Mesarosova et al., 1993).

This strong association between NP with inflammation determined to evaluate the expression and activity of this protein in PDAC, specifically in patients with antecedent inflammatory conditions like chronic pancreatitis, and pancreatic intraepithelial neoplasia (PanIN) (Rebours et al., 2010). Therefore, this correlation makes NP levels quantification to be a useful marker for surveillance progression from inflammation to PDAC.

#### **8.3 Saliva biomarkers**

42 Pancreatic Cancer – Clinical Management

Another study involving quantification in patient serum of tumor cell metabolites, or secreted factor was conducted by He *et al.* . This study focused on DJ-1 oncoprotein secreted by cancer cells (He et al., 2011). The study group involved patients diagnosed with pancreatic cancer and chronic pancreatitis, along with healthy subjects. DJ-1 serum level and the conventional tumor marker carbohydrate antigen 19-9 (CA 19-9) were measured in order to establish the diagnostic and prognostic value of DJ-1. Serum DJ-1 level was increased in patients with pancreas cancer compared with chronic pancreatitis and healthy individuals. Serum DJ-1 levels were higher than CA 19-9, and combined the two biomarkers provided a sensitivity of 87.5%. After resection DJ-1 levels decreased and patient with lower value of this factor had a better prognosis. This study provides a potential clinical biomarker, easy to quantify from serum, in order to establish a rapid diagnosis and to

Analysis of protein expression profiles of pancreatic juice samples harvested from the pancreatic duct has the potential to identify markers that could serve for diagnosis triage of benign from malignant pancreatic lesions and to discriminate between different stages of

The research performed by *Vareed et al*. has shown that 56 proteins were found to be elevated in pancreatic juice of PDAC patients compared to benign controls (Vareed et al.,

Protein profiles studies revealed an unique presence of proteins associated with Parkinson's

Increased expression of aSyn has been also identified in melanoma (Matsuo et al., 2010), while its isoform gamma-synuclein (cSyn) has been shown to be elevated in tumors of breast, uterine, colorectal and pancreas (Ye et al., 2009; Hibi et al., 2009; Ahmad et al., 2007; Li et al., 2004; Gupta et al., 2003; Jia et al., 1999; Morgan et al., 2009). Moreover, due to the structural omology between cSyn and aSyn, these factors potentiate invasion in these

Interestingly, tissue arrays demonstrated a strong staining for aSyn in tumors, and this protein expressed in a subset of PDAC patients was identified in its aggregated form similar to Lewy Bodies seen in Parkinson's disease (Polymeropoulus et al., 1997). Another interesting coincidence is the presence of increased levels of Parkinson's disease associated protein PARK7 (DJ1) (van Duijin et al., 2001) in pancreatic ductal juice of adenocarcinomas. The mechanism by which the protein PARK-7 exerts its oncogenic effect remains unclear. It is presumed the involvement of p38 mitogen activated-protein kinase signaling (Mo et al., 2010). On the other hand PDAC-associated secretory proteome analysis also revealed increased levels of several metabolic enzymes. The most important metabolic factor was Purine NucleosidePhosphorylase (NP), enzyme involved in salvage pathway of purines, which is operational during inflammation and neoplastic progression, (Bantia et al., 2010). NP activity has been reported to be high in cancer sera (Roberts et al., 2004) and NP expression has also been used to determine the clinical severity of various types of cancers, in combination with another factor adenosine deaminase (ADA) (Mesarosova et al., 1993).

disease namely: aSyn and PARK7 (Bonifati et al., 2003; Singleton et al., 2003).

evaluate prognosis in patients with pancreas cancer.

**8.2 Pancreatic juice biomarkers** 

PDAC.

2011).

tumors.

Saliva is a body fluid that can be easily obtained without using a special technique. Recent reports suggested the possible utility of saliva in quantification of specific factors to discriminate between pancreatic cancer patients and patients with normal or chronic pancreatitis.

Using transcriptome profiles, Zhang *et al*. group could differentiate pancreatic cancer patients from healthy subject with a sensitivity of 90.0% and a specificity of 95.0%. The group found 12 mRNA biomarkers specific for pancreatic cancer patients. Seven genes were found to be up-regulated (*MBD3L2*, *KRAS*, *STIM2*, *DMXL2ACRV1*, *DMD*, *CABLES1*), whereas five genes presented a decreased expression *TK2*, *GLTSCR2*, *CDKL3, DPM1, TPT1*  (Zhang et al., 2010)*.*

A similar approach has been made by another group, which evaluated metabolites in saliva using mass spectrometry. Pancreatic cancer cases were successfully detected based on the pancreatic cancer-specific signature (Sugimoto et al., 2010). The levels of ornithine and putrescine were higher in patients with breast or pancreatic cancer, and were markedly higher in patients with oral cancer. The level of tryptophan is also increased in oral and pancreatic cancer, in contrast to arginine level which is decreased several cancers including breast, colonic and pancreatic cancer, which might be due to increased uptake of arginine by tumor tissues with high arginase activity.

#### **8.4 Non- coding RNA as new era biomarkers**

A new and important atractive tool is represented by small non-coding RNA. These are regulators of various biological processes like gene expression and are involved in cancer progression. One of the most important players is microRNAs which control many cellular functions, such as migration, invasion and stem cell functions. Abnormal microRNA gene expression was found in many cancers, including pancreatic cancer (Rachangani et al., 2010, Eis et al., 2005, Calin et al., 2005, Yanaihara et al., 2006).

MicroRNA molecules are present in various body fluids (blood, urine, cerebrospinal fluid, pancreatic juice, billiary secretion). Moreover, several microRNAs from sera seemed to be identical for many cancer types. Very important is the fact that miRNAs from sera are stable and exhibit resistance to RN-ase activity, intermittent frosting and defrosting, high temperature values and extreme pH ( Mitchell et al., 2008; Albulescu et al., 2011).

Using microarray technology it was established that several microRNAs are highly expressed in pancreatic cancer (miR-21, miR-17-5p,miR-191,miR-29b-2,miR-223 miR-128b, miR-199a-1, miR-24-1, miR-24-2,miR-146, miR-181b-1, miR-20a,miR-107,miR-32 , miR-92-2, miR-214,miR-30c, miR-25, miR-221, miR-106) (Volinia et al., 2006). More recently, was established that microRNAs, miR-200a and miR-200b are highly expressed in pancreatic

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Settleman, J., Engelman, J.A. & Bardeesy, N. (2011) STAT3 Plays a Critical Role in

cancer cell lines, and their expression levels were significantly increased in the sera from pancreatic cancer patients, suggesting that microRNA itself could be a biomarker for pancreatic cancer (Li et al., 2010). The attention was also focused on two pancreas specific miRNAs miR-216 and miR-217. The expression of those miRNAs is decreased or even absent in PADC and in cell lines (Sood et al., 2006). Only miR-217 and miR-196a are able to discriminate between normal pancreas, chronic pancreatitis and tumor PDAC (Szafranska et al., 2008). Furthermore, miR-196a expression is likely specific to PDAC cells and is positively associated with the progression of PDAC.

miR-21 and miR-155 are overexpressed in pancreatic tumor, as compared to tissues from normal pancreas and chronic pancreatitis. Both miR-21 and miR-155 have been suggested to have a proto-oncogene role being overexpressed in several cancers (breast cancer, lung cancer, Burkitt lymphoma, B-cell lymphoma) (Metzler et al., 2004;Yin et al., 2008)

Several studies demonstrated that miR-155 transcription is regulated by transforming growthfactor β -TGF β/Smad, nuclear factor-κB and activator protein-1 family transcription factors through direct interaction with the miR-155/BIC bidirectional promoter. These studies suggests that overexpression of miR-155 in cancer is due to transcriptional activation, involving other cellular deregulated mechanisms (Kong et al., 2008).

#### **9. Conclusions**

Asymptomatic pancreatic cancer is hard to detect, but possibly curable. Recent research identified novel biomarkers of pancreatic cancer, but screening for early pancreatic cancer is still challenging. Future work should be addressed to the development of diagnostic techniques with a higher sensitivity to detect even asymptomatic cases. Currently no clinically useful interventions to screen for patients with PDAC are available.

#### **10. Acknowledgements**

The authors are grateful for the funding support offered by POSDRU/89/1.5/S/60746 Grant.

#### **11. References**


cancer cell lines, and their expression levels were significantly increased in the sera from pancreatic cancer patients, suggesting that microRNA itself could be a biomarker for pancreatic cancer (Li et al., 2010). The attention was also focused on two pancreas specific miRNAs miR-216 and miR-217. The expression of those miRNAs is decreased or even absent in PADC and in cell lines (Sood et al., 2006). Only miR-217 and miR-196a are able to discriminate between normal pancreas, chronic pancreatitis and tumor PDAC (Szafranska et al., 2008). Furthermore, miR-196a expression is likely specific to PDAC cells and is positively

miR-21 and miR-155 are overexpressed in pancreatic tumor, as compared to tissues from normal pancreas and chronic pancreatitis. Both miR-21 and miR-155 have been suggested to have a proto-oncogene role being overexpressed in several cancers (breast cancer, lung

Several studies demonstrated that miR-155 transcription is regulated by transforming growthfactor β -TGF β/Smad, nuclear factor-κB and activator protein-1 family transcription factors through direct interaction with the miR-155/BIC bidirectional promoter. These studies suggests that overexpression of miR-155 in cancer is due to transcriptional

Asymptomatic pancreatic cancer is hard to detect, but possibly curable. Recent research identified novel biomarkers of pancreatic cancer, but screening for early pancreatic cancer is still challenging. Future work should be addressed to the development of diagnostic techniques with a higher sensitivity to detect even asymptomatic cases. Currently no

The authors are grateful for the funding support offered by POSDRU/89/1.5/S/60746

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**4** 

*Australia* 

**Medical Therapy of Pancreatic Cancer:** 

Pancreatic cancer is a major cause of cancer-related mortality relative to its incidence. In the US alone, it is estimated that there were 43,140 new cases in 2010 with 36,800 deaths making

Typically patients come to clinical attention at an advanced stage of their disease with only 10-15% having potentially operable disease. Surgery is the only established method shown to cure pancreatic adenocarcinoma, yet the rate of cure amongst patients with resectable disease still remains low. Improvements in survival with the addition of chemotherapy or

The medical management of pancreatic cancer in the adjuvant and advanced settings will be reviewed. The current standard of care in both settings is gemcitabine, with modest improvements in survival provided by the addition of erlotinib in the advanced setting. Despite arguably poor evidence for added survival benefit from combination cytotoxic regimens or other biological agents in the advanced setting, recent evidence for considering this in select patients will be discussed, along with a recent non-gemcitabine containing combination cytotoxic approach (FOLFIRINOX), that has challenged the traditional

Some of the important molecular signaling pathways involved in pancreatic cancer growth, invasion, angiogenesis, metastasis, and drug resistance will also be summarised. It is hoped that in future, survival outcomes may be improved by better targeting of these pathways in

There is an established survival advantage with adjuvant systemic therapy in pancreatic cancer. Adjuvant systemic therapy can be delivered either solely, or in combination with

the individual patient, aided by appropriate predictive and prognostic biomarkers.

**2. Chemotherapy and chemoradiotherapy in resected pancreatic cancer** 

it the fourth leading cause of cancer-related mortality.[1]

radiotherapy have only been relatively modest.

**1. Introduction** 

paradigm.

**Current Status and Future Targets** 

Edward Livshin1 and Michael Michael2

*Peter MacCallum Cancer Centre,Victoria,* 

*1Division of Cancer Medicine,* 

*2Division of Cancer Medicine, Upper GI Oncology Service, Peter MacCallum Cancer Centre, University of Melbourne, Victoria,* 


## **Medical Therapy of Pancreatic Cancer: Current Status and Future Targets**

Edward Livshin1 and Michael Michael2

*1Division of Cancer Medicine, Peter MacCallum Cancer Centre,Victoria, 2Division of Cancer Medicine, Upper GI Oncology Service, Peter MacCallum Cancer Centre, University of Melbourne, Victoria, Australia* 

### **1. Introduction**

54 Pancreatic Cancer – Clinical Management

Yin, Q., Wang, X., McBride, J., Fewell, C. & Flemington, E. (2008). B-cell receptor activation

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pancreatic cancer. *Gastroenterology,* 138:949–957.

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D.T.(2010). Salivary transcriptomic biomarkers for detection of resectable

Giordano, T.J., Beer, D.G., Lubman, D.M. (2007). Comparative proteomics analysis of Barrett metaplasia and esophageal adenocarcinoma using two-dimensional

> Pancreatic cancer is a major cause of cancer-related mortality relative to its incidence. In the US alone, it is estimated that there were 43,140 new cases in 2010 with 36,800 deaths making it the fourth leading cause of cancer-related mortality.[1]

> Typically patients come to clinical attention at an advanced stage of their disease with only 10-15% having potentially operable disease. Surgery is the only established method shown to cure pancreatic adenocarcinoma, yet the rate of cure amongst patients with resectable disease still remains low. Improvements in survival with the addition of chemotherapy or radiotherapy have only been relatively modest.

> The medical management of pancreatic cancer in the adjuvant and advanced settings will be reviewed. The current standard of care in both settings is gemcitabine, with modest improvements in survival provided by the addition of erlotinib in the advanced setting. Despite arguably poor evidence for added survival benefit from combination cytotoxic regimens or other biological agents in the advanced setting, recent evidence for considering this in select patients will be discussed, along with a recent non-gemcitabine containing combination cytotoxic approach (FOLFIRINOX), that has challenged the traditional paradigm.

> Some of the important molecular signaling pathways involved in pancreatic cancer growth, invasion, angiogenesis, metastasis, and drug resistance will also be summarised. It is hoped that in future, survival outcomes may be improved by better targeting of these pathways in the individual patient, aided by appropriate predictive and prognostic biomarkers.

#### **2. Chemotherapy and chemoradiotherapy in resected pancreatic cancer**

There is an established survival advantage with adjuvant systemic therapy in pancreatic cancer. Adjuvant systemic therapy can be delivered either solely, or in combination with

Medical Therapy of Pancreatic Cancer: Current Status and Future Targets 57

of patients who received chemotherapy alone improved from 30 to 40% and 8 to 12% when compared to those who did not receive chemotherapy. Therefore in this analysis, patients did not benefit from a combined modality approach, and in fact their outcome appeared to be worse. Based on the results of ESPAC-1 it was difficult to justify the role of adjuvant

The question of incorporating infusional 5-FU and gemcitabine into adjuvant radiotherapy has been addressed in the Radiation Therapy Oncology Group (RTOG) 9704 trial.[6] This was a phase III trial of 442 patients with pathological T1-4 and nodal stage N0-1 pancreatic cancer. Participants were randomised to either adjuvant chemotherapy with either weekly 5-FU or gemcitabine three weeks prior and for 12 weeks post chemoradiotherapy sandwiched in between. Radiotherapy was delivered at a dose of 50.4Gy (at 1.8 Gy/fraction/day) concurrent with continuous infusional 5-FU at 250mg/m2/day. Most patients (n= 381) had tumours confined to the pancreatic head. More patients with stage T3 and 4 disease received gemcitabine and more grade 4 haematologic toxicity was experienced in the gemcitabine arm (14% vs. 2%). Rates of treatment completion were comparable. Although no overall survival advantage of gemcitabine over 5-FU was seen if all pancreatic lesions were included, the subgroup of patients with pancreatic head tumours assigned to the gemcitabine group had a trend toward a more favourable survival (20.5 months vs. 16.9 months with a hazard ratio (HR) for death of 0.82; 95% CI, 0.65-1.03; p = 0.09). The 3-year

rate of survival was also higher (31 vs. 21%) also favouring the gemcitabine group.

survival advantage favouring chemotherapy over surgery alone.[7]

Older adjuvant cytotoxic regimes such as the triplet of doxorubicin, mitomycin and 5 fluorouracil (AMF) for six cycles to treat pancreatic and papillary cancers showed no overall survival advantage beyond two years, although there was a 1 and 2 year relapse-free

In addition to the survival advantage shown ESPAC-1, the Charité Onkologie (CONKO-001) study[8] published in 2007 demonstrated a survival benefit with adjuvant gemcitabine over surgery alone. Patients with R0 or R1 resections were assigned to observation alone or gemcitabine delivered at 1000mg/m2/week (days 1, 8 and 15 of a 28 day cycle) for a total of six cycles. There was a trend toward an improved median overall survival (22.8 vs. 20.2 months p=0.06) as well as a statistically significant improvement in disease-free survival (13.4 vs. 6.9 months p <0.001) over surgery alone. Importantly the rate of 5-year survival was significantly better in those patients receiving adjuvant gemcitabine over observation

In the largest adjuvant pancreatic trial to date, ESPAC-3[9-10] involved 1088 patients with R0 or R1 resected pancreatic adenocarcinoma, randomising patients into either observation alone, 5-FU/LV, or gemcitabine. Notably the 5-FU was delivered as five bolus doses (425mg/m2 with leucovorin 20mg/m2 days 1-5 of a 28 day cycle) rather than as an infusion. 551 patients received 5-FU and 537 received gemcitabine with treatment for a total of six months. The observational arm was discontinued after the outcome of the CONKO-001 trial was made available. At a median follow up of 34.2 months after 753 deaths, there was no advantage seen between the intervention arms (23.0 vs. 23.6 months p=0.39). 12 and 24 month survival was 78.5% and 48.1% respectively in those who received 5-FU with 80.1%

chemoradiotherapy over chemotherapy with bolus 5-FU alone.

**2.2 Adjuvant chemotherapy strategies** 

alone (21% vs. 9%).

radiotherapy following pancreatic resection, however the role of the latter is more controversial, and will be briefly summarised. *A further discussion of radiotherapy and its role in the neoadjuvant and adjuvant setting is discussed elsewhere.* 

#### **2.1 Adjuvant chemoradiotherapy compared with surgery alone**

A Gastrointestinal Study Group (GITSG) trial assessed the role of concurrent post-operative radiotherapy and radiosensitising bolus 5-fluorouracil (5-FU) compared with surgery alone.[2]

Patients were randomised to a split-course of radiotherapy in combination with bolus 5-FU compared with post-operative observation alone. Chemotherapy was given at 500mg/m2 per day over the first three days of each course of radiotherapy. Patients were given 20 Gray (Gy) in 10 fractions followed by a 14 day break, then a further course of radiotherapy up to a dose of 40Gy. Although demonstrating a median overall survival of 21 months vs. 11 months (p=0.035) favouring the chemoradiotherapy group, criticisms include small patient numbers (43 patients), a slow patient accrual of 8 years, and selection bias where only a more prognostically favourable group of patients with microscopically clear (R0 resection) margins, were included in the study. A later GITSG analysis[3] of an additional 30 patients all treated with adjuvant combined therapy - showed a median overall survival of 18 months.

The larger European Organization of Research and Treatment of Cancer (EORTC) 40891 study[4] however only showed a non-statistically significant trend towards an improved overall survival with chemoradiotherapy in a subgroup of 114 out of 218 patients with carcinoma of the pancreatic head. The median overall survival was 17.1 months vs. 12.6 months in the observation alone arm (p = 0.099). 5-FU delivery here was given as bolus daily doses at 25mg/kg up to 1,500mg/day, days 1-7 of each course of radiotherapy. There were two courses of radiotherapy given up to a total of 40Gy. EORTC 40891 included patients with T1 or T2 disease, and allowed patients with node-positive (N1) disease. 45% however had T1-3 periampullary disease. Shortcomings included the lack of maintenance chemotherapy, a significant (20%) of patients not proceeding with combination therapy and the large percentage of patients with periampullary cancers affecting the interpretation of outcome in pancreatic cancer.

The largest body of evidence has come from The European Study Group for Pancreatic Cancer (ESPAC) publishing the results the ESPAC-1 trial in 2004.[5] This study employing a 2x2 factorial design allowed a comparison between adjuvant radiotherapy or no radiotherapy, chemotherapy or no chemotherapy, and chemoradiotherapy vs. chemotherapy alone. Chemoradiotherapy was given as two courses of 20Gy separated by 14 days, combined with bolus 5-FU (500mg/m2) given for three days during each course. Following this, patients continued with a maintenance course of chemotherapy with 5FU/leucovorin (LV). Chemotherapy was given as bolus 5-FU (425mg/m2) with LV (20mg/m2) days 1-5 every 28 days, for a total of six cycles. 53% of patients had nodal involvement and 19% had involved margins. Patients who received chemotherapy compared with those who did not, survived a median of 20.6 months vs. 15.5 months (HR 0.71; 95% CI, 0.55-0.92 p=0.009). Patients who received chemoradiotherapy survived a median of only 15.9 months vs. 17.9 months in those who did not receive chemoradiotherapy (HR 1.28 ; 95% CI, 0.99-1.66 p=0.05). Notably, 2 and 5 year survival rates

radiotherapy following pancreatic resection, however the role of the latter is more controversial, and will be briefly summarised. *A further discussion of radiotherapy and its role* 

A Gastrointestinal Study Group (GITSG) trial assessed the role of concurrent post-operative radiotherapy and radiosensitising bolus 5-fluorouracil (5-FU) compared with surgery alone.[2] Patients were randomised to a split-course of radiotherapy in combination with bolus 5-FU compared with post-operative observation alone. Chemotherapy was given at 500mg/m2 per day over the first three days of each course of radiotherapy. Patients were given 20 Gray (Gy) in 10 fractions followed by a 14 day break, then a further course of radiotherapy up to a dose of 40Gy. Although demonstrating a median overall survival of 21 months vs. 11 months (p=0.035) favouring the chemoradiotherapy group, criticisms include small patient numbers (43 patients), a slow patient accrual of 8 years, and selection bias where only a more prognostically favourable group of patients with microscopically clear (R0 resection) margins, were included in the study. A later GITSG analysis[3] of an additional 30 patients all treated with adjuvant combined therapy - showed a median overall survival of 18

The larger European Organization of Research and Treatment of Cancer (EORTC) 40891 study[4] however only showed a non-statistically significant trend towards an improved overall survival with chemoradiotherapy in a subgroup of 114 out of 218 patients with carcinoma of the pancreatic head. The median overall survival was 17.1 months vs. 12.6 months in the observation alone arm (p = 0.099). 5-FU delivery here was given as bolus daily doses at 25mg/kg up to 1,500mg/day, days 1-7 of each course of radiotherapy. There were two courses of radiotherapy given up to a total of 40Gy. EORTC 40891 included patients with T1 or T2 disease, and allowed patients with node-positive (N1) disease. 45% however had T1-3 periampullary disease. Shortcomings included the lack of maintenance chemotherapy, a significant (20%) of patients not proceeding with combination therapy and the large percentage of patients with periampullary cancers affecting the interpretation of

The largest body of evidence has come from The European Study Group for Pancreatic Cancer (ESPAC) publishing the results the ESPAC-1 trial in 2004.[5] This study employing a 2x2 factorial design allowed a comparison between adjuvant radiotherapy or no radiotherapy, chemotherapy or no chemotherapy, and chemoradiotherapy vs. chemotherapy alone. Chemoradiotherapy was given as two courses of 20Gy separated by 14 days, combined with bolus 5-FU (500mg/m2) given for three days during each course. Following this, patients continued with a maintenance course of chemotherapy with 5FU/leucovorin (LV). Chemotherapy was given as bolus 5-FU (425mg/m2) with LV (20mg/m2) days 1-5 every 28 days, for a total of six cycles. 53% of patients had nodal involvement and 19% had involved margins. Patients who received chemotherapy compared with those who did not, survived a median of 20.6 months vs. 15.5 months (HR 0.71; 95% CI, 0.55-0.92 p=0.009). Patients who received chemoradiotherapy survived a median of only 15.9 months vs. 17.9 months in those who did not receive chemoradiotherapy (HR 1.28 ; 95% CI, 0.99-1.66 p=0.05). Notably, 2 and 5 year survival rates

*in the neoadjuvant and adjuvant setting is discussed elsewhere.* 

months.

outcome in pancreatic cancer.

**2.1 Adjuvant chemoradiotherapy compared with surgery alone** 

of patients who received chemotherapy alone improved from 30 to 40% and 8 to 12% when compared to those who did not receive chemotherapy. Therefore in this analysis, patients did not benefit from a combined modality approach, and in fact their outcome appeared to be worse. Based on the results of ESPAC-1 it was difficult to justify the role of adjuvant chemoradiotherapy over chemotherapy with bolus 5-FU alone.

The question of incorporating infusional 5-FU and gemcitabine into adjuvant radiotherapy has been addressed in the Radiation Therapy Oncology Group (RTOG) 9704 trial.[6] This was a phase III trial of 442 patients with pathological T1-4 and nodal stage N0-1 pancreatic cancer. Participants were randomised to either adjuvant chemotherapy with either weekly 5-FU or gemcitabine three weeks prior and for 12 weeks post chemoradiotherapy sandwiched in between. Radiotherapy was delivered at a dose of 50.4Gy (at 1.8 Gy/fraction/day) concurrent with continuous infusional 5-FU at 250mg/m2/day. Most patients (n= 381) had tumours confined to the pancreatic head. More patients with stage T3 and 4 disease received gemcitabine and more grade 4 haematologic toxicity was experienced in the gemcitabine arm (14% vs. 2%). Rates of treatment completion were comparable. Although no overall survival advantage of gemcitabine over 5-FU was seen if all pancreatic lesions were included, the subgroup of patients with pancreatic head tumours assigned to the gemcitabine group had a trend toward a more favourable survival (20.5 months vs. 16.9 months with a hazard ratio (HR) for death of 0.82; 95% CI, 0.65-1.03; p = 0.09). The 3-year rate of survival was also higher (31 vs. 21%) also favouring the gemcitabine group.

#### **2.2 Adjuvant chemotherapy strategies**

Older adjuvant cytotoxic regimes such as the triplet of doxorubicin, mitomycin and 5 fluorouracil (AMF) for six cycles to treat pancreatic and papillary cancers showed no overall survival advantage beyond two years, although there was a 1 and 2 year relapse-free survival advantage favouring chemotherapy over surgery alone.[7]

In addition to the survival advantage shown ESPAC-1, the Charité Onkologie (CONKO-001) study[8] published in 2007 demonstrated a survival benefit with adjuvant gemcitabine over surgery alone. Patients with R0 or R1 resections were assigned to observation alone or gemcitabine delivered at 1000mg/m2/week (days 1, 8 and 15 of a 28 day cycle) for a total of six cycles. There was a trend toward an improved median overall survival (22.8 vs. 20.2 months p=0.06) as well as a statistically significant improvement in disease-free survival (13.4 vs. 6.9 months p <0.001) over surgery alone. Importantly the rate of 5-year survival was significantly better in those patients receiving adjuvant gemcitabine over observation alone (21% vs. 9%).

In the largest adjuvant pancreatic trial to date, ESPAC-3[9-10] involved 1088 patients with R0 or R1 resected pancreatic adenocarcinoma, randomising patients into either observation alone, 5-FU/LV, or gemcitabine. Notably the 5-FU was delivered as five bolus doses (425mg/m2 with leucovorin 20mg/m2 days 1-5 of a 28 day cycle) rather than as an infusion. 551 patients received 5-FU and 537 received gemcitabine with treatment for a total of six months. The observational arm was discontinued after the outcome of the CONKO-001 trial was made available. At a median follow up of 34.2 months after 753 deaths, there was no advantage seen between the intervention arms (23.0 vs. 23.6 months p=0.39). 12 and 24 month survival was 78.5% and 48.1% respectively in those who received 5-FU with 80.1%

Medical Therapy of Pancreatic Cancer: Current Status and Future Targets 59

Erlotinib, an oral tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR), has to date been the only EGFR inhibitor combined with a cytotoxic to show, an albeit modest, survival advantage in a phase III study. [14] It was evaluated with gemcitabine in patients with locally advanced or metastatic disease. Patients received either gemcitabine at 1000mg/m2 (weekly for 7 out of 8 weeks) then continued with weekly treatment (in 3 out of 4 weeks), or the equivalent strategy combined with erlotinib at 100mg or 150mg per day. The latter dose was provided to a cohort of Canadian patients. Median overall survival improved with the combination approach of erlotinib with gemcitabine, compared with gemcitabine alone [(6.24 months versus 5.91 months; HR 0.82 (95% CI, 0.69 to 0.99; P = 0.038)]. The superiority of 150mg erlotinib over 100mg was not proven. One could argue that the benefit on overall survival is not economically justified, however with a 1-year survival rate improvement from 17 to 23% (95% CI, 18% to 28%, 95% CI, 12% to 21%, P =

Gefitinib, another EGFR inhibitor TKI has less evidence, but was evaluated in combination with gemcitabine in a phase II study[15] which reported either disease stability or response in 18/53 patients. The median progression-free survival was 4.1 months and median overall

Phase II and III studies combining other EGFR inhibitors such as cetuximab[16] or lapatinib (a dual HER2/EGFR inhibitor)[17] in combination with gemcitabine have not provided an additional survival advantage. Similarly a trial adding cetuximab to a gemcitabine/ cisplatin doublet did not progress beyond a phase II trial, as time to progression was equivalent at 5 months, despite a higher disease control rate.[18] Dual EGFR inhibition with erlotinib and panitumumab has recently been examined in a randomised phase II study, with a modest 3.3 vs. 2.0 month PFS advantage, though mature survival data and statistical

The role of erlotinib incorporated into the management of patients with locally advanced disease is being evaluated in the Groupe Cooperateur Multidisciplinaire en Oncologie (GERCOR) LAP07 phase III trial.[20] Patients are randomised initially to either induction gemcitabine or gemcitabine/erlotinib. In those patients who do not progress after four months, there is a secondary randomisation into a chemotherapy (with either gemcitabine or gemcitabine/erlotinib), or a chemoradiotherapy arm (with concurrent capecitabine) until

A number of gemcitabine-containing combinations with either fluoropyrimidines or platinum agents have been attempted. Individually these trials have not provided

**3.2 Combination chemotherapy and epidermal growth factor receptor (EGFR)** 

0.023), this has become an acceptable standard of care in many centres.

survival was 7.3 months. The reported 1-year survival rate was 27%.

**3.3 Combination chemotherapy: Gemcitabine-containing regimens** 

significance has not been published to date[19].

**Gemcitabine/fluoropyrimidine doublets** 

**Gemcitabine/platinum doublets** 

tumour progression.

**inhibition** 

**Gemcitabine/erlotinib** 

**Gemcitabine/other EGFR inhibitor combinations** 

and 49.1% respectively in the gemcitabine arm. The side effect profile however favoured gemcitabine in terms of grade 3-4 toxicity and hospitalisation. Grade 3 and 4 mucositis was seen in 10% of patients who received 5-FU (compared with no patients on gemcitabine). Grade 3-4 diarrhoea was also significantly higher in the 5-FU group. The gemcitabine treated group did however experience higher rates of grade 3 and 4 thrombocytopenia, although the absolute risk of this remained small (1.5 vs. 0%) (p=0.003). Quality-of-life was also comparable.

Thus, survival outcomes were not significantly improved by gemcitabine over 5-FU group in ESPAC-3. This outcome differs to that seen in the advanced setting.[13] One reason could be that the 5-FU intensity was greater in ESPAC-3 than that seen in the Burris *et al*. trial.

#### **2.3 Recommendations**

Adjuvant chemotherapy in resected pancreatic cancer is the standard of care, yet the role of chemoradiotherapy remains controversial. Gemcitabine for six cycles is preferable over 5-FU based treatment due to its more favourable toxicity profile. Although modest improvements in median survival have been shown, progression-free and 5-year survival rates are improved.

#### **3. Medical therapy of locally advanced and metastatic disease: First-line strategies**

#### **3.1 Single-agent chemotherapy**

#### **5-fluorouracil (5-FU), capecitabine, and gemcitabine**

5-fluorouracil (5-FU) has been used for half a century in advanced pancreatic cancer.[11] As a single agent, objective responses rates have typically been less than 10% with some historical data reporting higher response rates probably based on cruder estimations of disease burden such as physical examination and ultrasound. Typically responses were usually for less than six months.

Capecitabine is an oral fluoropyrimidine prodrug which is metabolised to 5-FU. A small phase II study[12] in patients with locally advanced or metastatic pancreatic cancer was performed in 42 patients at a dose of 1,250mg/m2 given twice a day in 3-week cycles, with 2 weeks of treatment followed by a 1-week break. Disease response evaluation was based on either computerised tomography (CT) or physical examination. Of the 41 patients with evaluable disease the objective response rate (ORR) was 7.3% (3 patients), with 41% having stable disease. 38% had progressive disease within the first 7 weeks. Median survival was quoted at 182 days (95% CI, 85-274 days). 52% of patients developed hand-foot syndrome (HFS) (41% Grade 2-3) and 48 % had nausea (24% Grade 2-3). 12% had grades 2-3 mucositis.

The randomised trial leading to the acceptance of gemcitabine as standard therapy in advanced pancreatic cancer was published in 1997.[13] This study compared gemcitabine with bolus weekly 5-FU. Gemcitabine was favoured over 5-FU with a modest improvement in median survival (5.7 vs. 4.4 months, p=0.0025). More significantly, the rate of 1-yr survival was improved (18% vs. 2%), and importantly the rate of clinical symptom improvement (measured by at least four weeks of improvement in either pain, reduced analgesic use, improved weight loss or performance status) favoured the gemcitabine arm (24% vs. 5%).

#### **3.2 Combination chemotherapy and epidermal growth factor receptor (EGFR) inhibition**

#### **Gemcitabine/erlotinib**

58 Pancreatic Cancer – Clinical Management

and 49.1% respectively in the gemcitabine arm. The side effect profile however favoured gemcitabine in terms of grade 3-4 toxicity and hospitalisation. Grade 3 and 4 mucositis was seen in 10% of patients who received 5-FU (compared with no patients on gemcitabine). Grade 3-4 diarrhoea was also significantly higher in the 5-FU group. The gemcitabine treated group did however experience higher rates of grade 3 and 4 thrombocytopenia, although the absolute risk of this remained small (1.5 vs. 0%) (p=0.003). Quality-of-life was

Thus, survival outcomes were not significantly improved by gemcitabine over 5-FU group in ESPAC-3. This outcome differs to that seen in the advanced setting.[13] One reason could be that the 5-FU intensity was greater in ESPAC-3 than that seen in the Burris *et al*. trial.

Adjuvant chemotherapy in resected pancreatic cancer is the standard of care, yet the role of chemoradiotherapy remains controversial. Gemcitabine for six cycles is preferable over 5-FU based treatment due to its more favourable toxicity profile. Although modest improvements in median survival have been shown, progression-free and 5-year survival rates are improved.

5-fluorouracil (5-FU) has been used for half a century in advanced pancreatic cancer.[11] As a single agent, objective responses rates have typically been less than 10% with some historical data reporting higher response rates probably based on cruder estimations of disease burden such as physical examination and ultrasound. Typically responses were usually for

Capecitabine is an oral fluoropyrimidine prodrug which is metabolised to 5-FU. A small phase II study[12] in patients with locally advanced or metastatic pancreatic cancer was performed in 42 patients at a dose of 1,250mg/m2 given twice a day in 3-week cycles, with 2 weeks of treatment followed by a 1-week break. Disease response evaluation was based on either computerised tomography (CT) or physical examination. Of the 41 patients with evaluable disease the objective response rate (ORR) was 7.3% (3 patients), with 41% having stable disease. 38% had progressive disease within the first 7 weeks. Median survival was quoted at 182 days (95% CI, 85-274 days). 52% of patients developed hand-foot syndrome (HFS) (41% Grade 2-3) and 48 % had nausea (24% Grade 2-3). 12% had grades 2-3 mucositis. The randomised trial leading to the acceptance of gemcitabine as standard therapy in advanced pancreatic cancer was published in 1997.[13] This study compared gemcitabine with bolus weekly 5-FU. Gemcitabine was favoured over 5-FU with a modest improvement in median survival (5.7 vs. 4.4 months, p=0.0025). More significantly, the rate of 1-yr survival was improved (18% vs. 2%), and importantly the rate of clinical symptom improvement (measured by at least four weeks of improvement in either pain, reduced analgesic use, improved weight loss or performance status) favoured the gemcitabine arm

**3. Medical therapy of locally advanced and metastatic disease: First-line** 

also comparable.

**strategies** 

less than six months.

(24% vs. 5%).

**2.3 Recommendations** 

**3.1 Single-agent chemotherapy** 

**5-fluorouracil (5-FU), capecitabine, and gemcitabine** 

#### **Gemcitabine/other EGFR inhibitor combinations**

Erlotinib, an oral tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR), has to date been the only EGFR inhibitor combined with a cytotoxic to show, an albeit modest, survival advantage in a phase III study. [14] It was evaluated with gemcitabine in patients with locally advanced or metastatic disease. Patients received either gemcitabine at 1000mg/m2 (weekly for 7 out of 8 weeks) then continued with weekly treatment (in 3 out of 4 weeks), or the equivalent strategy combined with erlotinib at 100mg or 150mg per day. The latter dose was provided to a cohort of Canadian patients. Median overall survival improved with the combination approach of erlotinib with gemcitabine, compared with gemcitabine alone [(6.24 months versus 5.91 months; HR 0.82 (95% CI, 0.69 to 0.99; P = 0.038)]. The superiority of 150mg erlotinib over 100mg was not proven. One could argue that the benefit on overall survival is not economically justified, however with a 1-year survival rate improvement from 17 to 23% (95% CI, 18% to 28%, 95% CI, 12% to 21%, P = 0.023), this has become an acceptable standard of care in many centres.

Gefitinib, another EGFR inhibitor TKI has less evidence, but was evaluated in combination with gemcitabine in a phase II study[15] which reported either disease stability or response in 18/53 patients. The median progression-free survival was 4.1 months and median overall survival was 7.3 months. The reported 1-year survival rate was 27%.

Phase II and III studies combining other EGFR inhibitors such as cetuximab[16] or lapatinib (a dual HER2/EGFR inhibitor)[17] in combination with gemcitabine have not provided an additional survival advantage. Similarly a trial adding cetuximab to a gemcitabine/ cisplatin doublet did not progress beyond a phase II trial, as time to progression was equivalent at 5 months, despite a higher disease control rate.[18] Dual EGFR inhibition with erlotinib and panitumumab has recently been examined in a randomised phase II study, with a modest 3.3 vs. 2.0 month PFS advantage, though mature survival data and statistical significance has not been published to date[19].

The role of erlotinib incorporated into the management of patients with locally advanced disease is being evaluated in the Groupe Cooperateur Multidisciplinaire en Oncologie (GERCOR) LAP07 phase III trial.[20] Patients are randomised initially to either induction gemcitabine or gemcitabine/erlotinib. In those patients who do not progress after four months, there is a secondary randomisation into a chemotherapy (with either gemcitabine or gemcitabine/erlotinib), or a chemoradiotherapy arm (with concurrent capecitabine) until tumour progression.

#### **3.3 Combination chemotherapy: Gemcitabine-containing regimens**

#### **Gemcitabine/fluoropyrimidine doublets**

#### **Gemcitabine/platinum doublets**

A number of gemcitabine-containing combinations with either fluoropyrimidines or platinum agents have been attempted. Individually these trials have not provided

Medical Therapy of Pancreatic Cancer: Current Status and Future Targets 61

An earlier non-gemcitabine containing regimen of irinotecan and docetaxel was examined in a phase II study randomising patients into two arms with or without cetuximab but response rates were 7 and 4.5% respectively. This did not meet a pre-determined goal to

The recent French PRODIGE 4 (ACCORD 11) study[29] randomised 342 patients with metastatic pancreatic carcinoma, who had an Eastern Cooperative Oncology Group performance status score of 0 or 1, to either a regimen of gemcitabine (1000mg/m2 weekly for 7 of 8 weeks followed by weekly treatment for 3 out of four weeks) or FOLFIRINOX. FOLFIRINOX patients received oxaliplatin (85mg/m2), irinotecan (180mg/m2), leucovorin (400mg/m2), with bolus (400mg/m2) then infusional (2400mg/m2 over 46 hours) 5-FU. Treatment was delivered every two weeks. It is important to note that more patients in the FOLFIRINOX arm (42.5%) received granulocyte colony stimulating factor (G-CSF) support

Using overall survival as its primary end point, and with an intended treatment period of six months, FOLFIRINOX treated patients had an impressive median 11.1 month overall survival, compared with only 6.8 months in those treated with gemcitabine alone (HR for death, 0.57; 95% CI, 0.45 - 0.73; p<0.001). Progression-free survival was also superior (6.4 vs. 3.4 months (HR, 0.47; P <0.0001). Objective response rates were significantly higher in the FOLFIRINOX group (31.6%) compared with gemcitabine (9.4%) (p<0.001). This advantage was at the expense of higher rates of grade 3 or 4 neutropenia (febrile neutropenia of 5.4% vs. 0.6% P=0.0001), thrombocytopenia (9.1% vs. 2.4% p=0.008), neuropathy, diarrhoea and grade 2 alopecia. There was one toxicity-related death in each arm of the trial. Despite the increased toxicity, quality of life scores were more preserved at six months in the FOLFIRINOX-treated patients. This regimen is therefore being considered a suitable option for some patients, particularly those with a good performance status. A survey of US Oncologists recently revealed that 18% would now adopt FOLFIRINOX over a gemcitabine-erlotinib doublet in the first-line setting for

The standard of care in the first-line setting of advanced pancreatic cancer remains gemcitabine or gemcitabine with erlotinib for most patients. The alternative of 5-FU remains if gemcitabine is poorly tolerated. Those who are particularly fit with a performance status of ECOG 0-1, might be considered for a gemcitabine-platinum or a gemcitabine-capecitabine doublet (based on subset- and recent meta-analyses), or the non-gemcitabine regimen of FOLFIRINOX. Recent phase III evidence for the latter challenges the traditional paradigm of a gemcitabine-containing backbone, but it must be balanced with the higher risks of toxicity when recommending treatment. Enrolment in clinical trials should always be considered if

**3.4 Combination chemotherapy: Non-gemcitabine containing regimens** 

**Irinotecan-docetaxel** 

proceed to a phase III study.[28]

than those in the gemcitabine arm (5.3%).

patients with a performance status of ECOG 1.[30]

**3.5 Recommendations** 

possible.

**FOLFIRINOX** 

significant improvements in survival over gemcitabine alone. However subset-analyses of some of these trials, as well as a meta-analysis suggest that doublets may confer a meaningful survival improvement in the fittest patients with Karnofsky Performance Status (KPS) scores of 90% or above.[21]

Gemcitabine and 5-FU was examined in a phase III trial[22] which randomised 322 patients to a schedule of gemcitabine 1000mg/m2 (three weeks out of four), with or without bolus 5FU 600mg/m2/week), however did not produce a statistically significant improvement in overall survival compared with gemcitabine alone (6.7 vs. 5.4 months respectively p=0.09).

Gemcitabine and capecitabine was also examined in a phase III trial comparing a gemcitabine/capecitabine doublet with gemcitabine with previously untreated locally advanced or metastatic disease.[23] It suggested a significantly higher objective response rate (ORR) of 19.1% vs. 12.4%; (P = 0.034), as well as an improvement in progression-free survival (HR 0.78 95% CI, 0.66 to 0.93; P=0.004) favouring the doublet. However it only demonstrated a trend toward an improved overall survival (HR 0.86; 95% CI, 0.72 to 1.02; P=0.08). Another study of this combination also showed no significant difference in the primary end-point of overall survival [(8.4 months with the combination arm vs. 7.2 months with gemcitabine alone (p= 0.234)]. However a post-hoc subgroup analysis did reveal evidence for more favourable survival in the combination arm if performance status was better. Patients with KPS of 90-100% receiving combination therapy had a median overall survival of 10.1 vs. 7.4 months compared with gemcitabine alone (p= 0.014).[24]

Combination gemcitabine and cisplatin was assessed in 195 patients enrolled in a phase III trial comparing gemcitabine 1000mg/m2 (days 1, 8 and 15 of a 28 day cycle) with gemcitabine 1000mg/m2 and cisplatin 50mg/m2 (days 1 and 15). Tumour responses were similar in the combination (10.2%) vs. standard treatment arms (8.2%), with an improved progression-free survival and equivalent toxicity. However, despite a trend toward an improvement in overall survival (the primary endpoint of this study) within the combination arm (7.5 vs. 6.0 months), this did not reach statistical significance (p=0.15).[25]

Louvet e*t al*[26] compared a combination gemcitabine/oxaliplatin doublet (GEMOX) with gemcitabine. Patients received either treatment with gemcitabine 1000mg/m2 and oxaliplatin 100mg/m2 every 2 weeks compared with weekly gemcitabine 1000mg/m2. The combination was shown to improve response rates (26.8 vs. 17.3% respectively, P=0.04), as well as progression-free survival (5.8 vs. 3.7 months P=0.04). However differences in median overall survival were not statistically significant (9.0 vs. 7.1 months P= 0.13). The combination arm was associated with greater rates of grade 3-4 thrombocytopenia, vomiting and sensory neuropathy. Some patients received radiotherapy for local control at the oncologists' discretion after they had completed 3 months of systemic therapy. The overall survival data may have been influenced by a proportion of gemcitabine patients receiving platinum-containing second-line therapy, once they had progressed and were off study.

Gemcitabine in combination with irinotecan was assessed in a trial that randomised 360 patients to gemcitabine 1000mg/m2 and irinotecan 100mg/m2 on days 1 and 8 every 21 days or gemcitabine alone.[27] Rates of diarrhoea, nausea and vomiting were higher in the combination arm with no improvement in the overall survival.

#### **3.4 Combination chemotherapy: Non-gemcitabine containing regimens**

#### **Irinotecan-docetaxel**

#### **FOLFIRINOX**

60 Pancreatic Cancer – Clinical Management

significant improvements in survival over gemcitabine alone. However subset-analyses of some of these trials, as well as a meta-analysis suggest that doublets may confer a meaningful survival improvement in the fittest patients with Karnofsky Performance Status

Gemcitabine and 5-FU was examined in a phase III trial[22] which randomised 322 patients to a schedule of gemcitabine 1000mg/m2 (three weeks out of four), with or without bolus 5FU 600mg/m2/week), however did not produce a statistically significant improvement in overall survival compared with gemcitabine alone (6.7 vs. 5.4 months respectively p=0.09). Gemcitabine and capecitabine was also examined in a phase III trial comparing a gemcitabine/capecitabine doublet with gemcitabine with previously untreated locally advanced or metastatic disease.[23] It suggested a significantly higher objective response rate (ORR) of 19.1% vs. 12.4%; (P = 0.034), as well as an improvement in progression-free survival (HR 0.78 95% CI, 0.66 to 0.93; P=0.004) favouring the doublet. However it only demonstrated a trend toward an improved overall survival (HR 0.86; 95% CI, 0.72 to 1.02; P=0.08). Another study of this combination also showed no significant difference in the primary end-point of overall survival [(8.4 months with the combination arm vs. 7.2 months with gemcitabine alone (p= 0.234)]. However a post-hoc subgroup analysis did reveal evidence for more favourable survival in the combination arm if performance status was better. Patients with KPS of 90-100% receiving combination therapy had a median overall

survival of 10.1 vs. 7.4 months compared with gemcitabine alone (p= 0.014).[24]

Combination gemcitabine and cisplatin was assessed in 195 patients enrolled in a phase III trial comparing gemcitabine 1000mg/m2 (days 1, 8 and 15 of a 28 day cycle) with gemcitabine 1000mg/m2 and cisplatin 50mg/m2 (days 1 and 15). Tumour responses were similar in the combination (10.2%) vs. standard treatment arms (8.2%), with an improved progression-free survival and equivalent toxicity. However, despite a trend toward an improvement in overall survival (the primary endpoint of this study) within the combination arm (7.5 vs. 6.0 months), this did not reach statistical significance (p=0.15).[25]

Louvet e*t al*[26] compared a combination gemcitabine/oxaliplatin doublet (GEMOX) with gemcitabine. Patients received either treatment with gemcitabine 1000mg/m2 and oxaliplatin 100mg/m2 every 2 weeks compared with weekly gemcitabine 1000mg/m2. The combination was shown to improve response rates (26.8 vs. 17.3% respectively, P=0.04), as well as progression-free survival (5.8 vs. 3.7 months P=0.04). However differences in median overall survival were not statistically significant (9.0 vs. 7.1 months P= 0.13). The combination arm was associated with greater rates of grade 3-4 thrombocytopenia, vomiting and sensory neuropathy. Some patients received radiotherapy for local control at the oncologists' discretion after they had completed 3 months of systemic therapy. The overall survival data may have been influenced by a proportion of gemcitabine patients receiving platinum-containing second-line therapy, once they had progressed and were

Gemcitabine in combination with irinotecan was assessed in a trial that randomised 360 patients to gemcitabine 1000mg/m2 and irinotecan 100mg/m2 on days 1 and 8 every 21 days or gemcitabine alone.[27] Rates of diarrhoea, nausea and vomiting were higher in the

combination arm with no improvement in the overall survival.

(KPS) scores of 90% or above.[21]

off study.

An earlier non-gemcitabine containing regimen of irinotecan and docetaxel was examined in a phase II study randomising patients into two arms with or without cetuximab but response rates were 7 and 4.5% respectively. This did not meet a pre-determined goal to proceed to a phase III study.[28]

The recent French PRODIGE 4 (ACCORD 11) study[29] randomised 342 patients with metastatic pancreatic carcinoma, who had an Eastern Cooperative Oncology Group performance status score of 0 or 1, to either a regimen of gemcitabine (1000mg/m2 weekly for 7 of 8 weeks followed by weekly treatment for 3 out of four weeks) or FOLFIRINOX. FOLFIRINOX patients received oxaliplatin (85mg/m2), irinotecan (180mg/m2), leucovorin (400mg/m2), with bolus (400mg/m2) then infusional (2400mg/m2 over 46 hours) 5-FU. Treatment was delivered every two weeks. It is important to note that more patients in the FOLFIRINOX arm (42.5%) received granulocyte colony stimulating factor (G-CSF) support than those in the gemcitabine arm (5.3%).

Using overall survival as its primary end point, and with an intended treatment period of six months, FOLFIRINOX treated patients had an impressive median 11.1 month overall survival, compared with only 6.8 months in those treated with gemcitabine alone (HR for death, 0.57; 95% CI, 0.45 - 0.73; p<0.001). Progression-free survival was also superior (6.4 vs. 3.4 months (HR, 0.47; P <0.0001). Objective response rates were significantly higher in the FOLFIRINOX group (31.6%) compared with gemcitabine (9.4%) (p<0.001). This advantage was at the expense of higher rates of grade 3 or 4 neutropenia (febrile neutropenia of 5.4% vs. 0.6% P=0.0001), thrombocytopenia (9.1% vs. 2.4% p=0.008), neuropathy, diarrhoea and grade 2 alopecia. There was one toxicity-related death in each arm of the trial. Despite the increased toxicity, quality of life scores were more preserved at six months in the FOLFIRINOX-treated patients. This regimen is therefore being considered a suitable option for some patients, particularly those with a good performance status. A survey of US Oncologists recently revealed that 18% would now adopt FOLFIRINOX over a gemcitabine-erlotinib doublet in the first-line setting for patients with a performance status of ECOG 1.[30]

#### **3.5 Recommendations**

The standard of care in the first-line setting of advanced pancreatic cancer remains gemcitabine or gemcitabine with erlotinib for most patients. The alternative of 5-FU remains if gemcitabine is poorly tolerated. Those who are particularly fit with a performance status of ECOG 0-1, might be considered for a gemcitabine-platinum or a gemcitabine-capecitabine doublet (based on subset- and recent meta-analyses), or the non-gemcitabine regimen of FOLFIRINOX. Recent phase III evidence for the latter challenges the traditional paradigm of a gemcitabine-containing backbone, but it must be balanced with the higher risks of toxicity when recommending treatment. Enrolment in clinical trials should always be considered if possible.

Medical Therapy of Pancreatic Cancer: Current Status and Future Targets 63

Recently a nanoparticle liposomal encapsulated form of irinotecan (PEP02) was evaluated as a single agent in a phase II trial at 120mg/m2 given every 2 weeks in 37 patients who had progressed on gemcitabine.[39] A 74% 3-month overall survival endpoint was reached with initial reports of a 52% disease control rate. However 31% and 25% of patients had grade 3 or more fatigue and neutropenia respectively. Further prospective randomised evidence is

There is phase II evidence of 18 patients utilising weekly paclitaxel monotherapy with good tolerability.[40] Five patients had stable disease with one patient who achieved a complete response lasting beyond one year. The reported median overall survival was 17.5 weeks. Treatment was well tolerated with only one patient developing grade 3 myelotoxicity. A further report described evidence for activity using single agent docetaxel, combination docetaxel-gemcitabine or capecitabine regimes, however this was a small heterogeneous

SPARC (Secreted protein acidic and rich in cysteine) is frequently expressed by stromal fibroblasts adjacent to pancreatic adenocarcinoma cells, and immunohistochemical expression within the peritumoral stroma is an independent predictor for poorer survival, whereas expression by cancer cells is not. An analysis of 299 pancreaticoduodenectomy specimens showed that patients who expressed SPARC had a median survival of 15 months whereas patients who did not, had double the median survival of 30 months (p <0.001).[42] Nanoparticle albumin-bound (nab) paclitaxel (Abraxane®; Abraxis BioScience) is believed to allow better paclitaxel delivery by allowing albumin to bind to SPARC. It also has the advantages of avoiding the Cremophor® - related hypersensitivity reactions associated with

In a phase I/II study, patients with metastatic pancreatic adenocarcinoma were given firstline nab-paclitaxel (100-150mg/m2) in combination with gemcitabine 1000mg/m2 (days 1,8 and 15 of a 28 day cycle).[43] Of the 63 patients in the study, 35 had tissue available for immunohistochemical analysis. 29% of patients were SPARC positive. If SPARC positive, this predicted a metabolic response on positron emission tomography (PET) in 75% of those patients as well as a progression-free survival advantage of 6.2 vs. 4.8 months. A further phase II study of single agent nab-paclitaxel in patients who had progressed on gemcitabine however was less impressive with 63% of patients progressing by RECIST criteria at their first response assessment.[44] These patients were not preselected based on SPARC status.

The question of whether incorporating nanoparticle bound-paclitaxel into first-line chemotherapy with gemcitabine leads to a clinically meaningful improvement in survival is yet to be answered by a prospective randomised clinical trial currently awaiting completion.[45] Although tissue analysis for SPARC is included in this trial, the

To date there is no established standard of care in the second-line setting or beyond. Treatment must therefore be tailored to each patient but may include oxaliplatin,

awaited.

**4.4 Taxanes/nanoparticle – bound paclitaxel** 

group of patients and assessment was retrospective.[41]

standard paclitaxel, as well as delivery with a shorter infusion time.

interventional arm will not be enriched with SPARC positive patients.

**4.5 Recommendations** 

#### **4. Medical therapy of locally advanced and metastatic disease: Second-line strategies in gemcitabine-refractory disease**

#### **4.1 Oxaliplatin-based doublets**

The strategy of continuing gemcitabine with the addition of oxaliplatin (GEMOX) was evaluated in patients who have progressed on gemcitabine alone in a phase II trial of 33 patients with locally advanced and metastatic disease.[31] A partial response was seen in 7 of the 31 patients with evaluable disease and stable disease for 2 months or more was seen in 11 patients. The median survival was 6 months.

Second-line combination oxaliplatin/5-FU was examined in the Charité Onkologie trial (CONKO-003).[32] This began as a phase III trial with the intention to compare a 5-FUoxaliplatin doublet (the OFF regimen) with best supportive care (BSC).[33] The OFF regimen differs from FOLFOX being a 42-day cycle where infusional 5-FU (2000mg/m2 over 24 hours) with bolus LV (200mg/m2) is given days 1,8,15, and 22. Oxaliplatin (85mg/m2) is given on days 8 and 22. The protocol was revised due to poor acceptance of the best supportive care arm and later altered to include a 5-FU/LV arm as the control. Despite this methodological alteration, the study when presented as an abstract, did show an improvement in overall survival from 13 to 26 weeks favouring the doublet arm.[33]

There is phase II evidence showing activity with a doublet of oxaliplatin and capecitabine in the gemcitabine-refractory setting.[34] In a study of 41 patients, capecitabine was given at 1000mg/m2 BD days 1-14 with oxaliplatin 130mg/m2 every 3 weeks (doses of 850mg/m2 and 110mg/m2 respectively were used in patients greater than 65). Reported median overall survival was 23 weeks (95% CI, 17.0-31.0) with a progression-free survival of 9.9 weeks (95% CI, 9.6-14.5 weeks). Six month and 1 year survival rates were 44% and 21% respectively (95% CI 31-62% and 11-38%). Another recent phase II study has also confirmed activity in a mixed cohort of patients with pancreatic and biliary tract carcinomas.[35]

#### **4.2 Capecitabine/erlotinib**

A phase II study of capecitabine (1000mg/m2 BD days 1-14 of 21 day cycles) combined with erlotinib 150mg daily enrolled 32 patients.[36] The objective radiological response (ORR) was only 10% and median survival duration was 6.5months. 17% had CA 19-9 reductions of more than 50% of baseline. Diarrhoea, fatigue, rash and hand-foot syndrome were common toxicities. This has been suggested as an active first or second-line option, especially if gemcitabine is not tolerated.

#### **4.3 Irinotecan – based therapy**

Single agent irinotecan (150mg/m2) given every 2 weeks has demonstrated activity in the second-line setting.[37] 33 patients were evaluated in a phase II study where 48% had either stable disease or a partial response. The median time to progression was 4 months. With combination 5-FU and irinotecan regimens, disease control rates of 44.3-50% with overall survivals of 6 months or more have been reported.[38] Some patients received this in the third-line setting. However, patients were highly selected and much of the data is retrospective.

Recently a nanoparticle liposomal encapsulated form of irinotecan (PEP02) was evaluated as a single agent in a phase II trial at 120mg/m2 given every 2 weeks in 37 patients who had progressed on gemcitabine.[39] A 74% 3-month overall survival endpoint was reached with initial reports of a 52% disease control rate. However 31% and 25% of patients had grade 3 or more fatigue and neutropenia respectively. Further prospective randomised evidence is awaited.

#### **4.4 Taxanes/nanoparticle – bound paclitaxel**

62 Pancreatic Cancer – Clinical Management

**4. Medical therapy of locally advanced and metastatic disease: Second-line** 

The strategy of continuing gemcitabine with the addition of oxaliplatin (GEMOX) was evaluated in patients who have progressed on gemcitabine alone in a phase II trial of 33 patients with locally advanced and metastatic disease.[31] A partial response was seen in 7 of the 31 patients with evaluable disease and stable disease for 2 months or more was seen in

Second-line combination oxaliplatin/5-FU was examined in the Charité Onkologie trial (CONKO-003).[32] This began as a phase III trial with the intention to compare a 5-FUoxaliplatin doublet (the OFF regimen) with best supportive care (BSC).[33] The OFF regimen differs from FOLFOX being a 42-day cycle where infusional 5-FU (2000mg/m2 over 24 hours) with bolus LV (200mg/m2) is given days 1,8,15, and 22. Oxaliplatin (85mg/m2) is given on days 8 and 22. The protocol was revised due to poor acceptance of the best supportive care arm and later altered to include a 5-FU/LV arm as the control. Despite this methodological alteration, the study when presented as an abstract, did show an

There is phase II evidence showing activity with a doublet of oxaliplatin and capecitabine in the gemcitabine-refractory setting.[34] In a study of 41 patients, capecitabine was given at 1000mg/m2 BD days 1-14 with oxaliplatin 130mg/m2 every 3 weeks (doses of 850mg/m2 and 110mg/m2 respectively were used in patients greater than 65). Reported median overall survival was 23 weeks (95% CI, 17.0-31.0) with a progression-free survival of 9.9 weeks (95% CI, 9.6-14.5 weeks). Six month and 1 year survival rates were 44% and 21% respectively (95% CI 31-62% and 11-38%). Another recent phase II study has also confirmed activity in a mixed

A phase II study of capecitabine (1000mg/m2 BD days 1-14 of 21 day cycles) combined with erlotinib 150mg daily enrolled 32 patients.[36] The objective radiological response (ORR) was only 10% and median survival duration was 6.5months. 17% had CA 19-9 reductions of more than 50% of baseline. Diarrhoea, fatigue, rash and hand-foot syndrome were common toxicities. This has been suggested as an active first or second-line option, especially if

Single agent irinotecan (150mg/m2) given every 2 weeks has demonstrated activity in the second-line setting.[37] 33 patients were evaluated in a phase II study where 48% had either stable disease or a partial response. The median time to progression was 4 months. With combination 5-FU and irinotecan regimens, disease control rates of 44.3-50% with overall survivals of 6 months or more have been reported.[38] Some patients received this in the third-line setting. However, patients were highly selected and much of the data is

improvement in overall survival from 13 to 26 weeks favouring the doublet arm.[33]

cohort of patients with pancreatic and biliary tract carcinomas.[35]

**strategies in gemcitabine-refractory disease** 

11 patients. The median survival was 6 months.

**4.1 Oxaliplatin-based doublets** 

**4.2 Capecitabine/erlotinib** 

gemcitabine is not tolerated.

retrospective.

**4.3 Irinotecan – based therapy** 

There is phase II evidence of 18 patients utilising weekly paclitaxel monotherapy with good tolerability.[40] Five patients had stable disease with one patient who achieved a complete response lasting beyond one year. The reported median overall survival was 17.5 weeks. Treatment was well tolerated with only one patient developing grade 3 myelotoxicity. A further report described evidence for activity using single agent docetaxel, combination docetaxel-gemcitabine or capecitabine regimes, however this was a small heterogeneous group of patients and assessment was retrospective.[41]

SPARC (Secreted protein acidic and rich in cysteine) is frequently expressed by stromal fibroblasts adjacent to pancreatic adenocarcinoma cells, and immunohistochemical expression within the peritumoral stroma is an independent predictor for poorer survival, whereas expression by cancer cells is not. An analysis of 299 pancreaticoduodenectomy specimens showed that patients who expressed SPARC had a median survival of 15 months whereas patients who did not, had double the median survival of 30 months (p <0.001).[42] Nanoparticle albumin-bound (nab) paclitaxel (Abraxane®; Abraxis BioScience) is believed to allow better paclitaxel delivery by allowing albumin to bind to SPARC. It also has the advantages of avoiding the Cremophor® - related hypersensitivity reactions associated with standard paclitaxel, as well as delivery with a shorter infusion time.

In a phase I/II study, patients with metastatic pancreatic adenocarcinoma were given firstline nab-paclitaxel (100-150mg/m2) in combination with gemcitabine 1000mg/m2 (days 1,8 and 15 of a 28 day cycle).[43] Of the 63 patients in the study, 35 had tissue available for immunohistochemical analysis. 29% of patients were SPARC positive. If SPARC positive, this predicted a metabolic response on positron emission tomography (PET) in 75% of those patients as well as a progression-free survival advantage of 6.2 vs. 4.8 months. A further phase II study of single agent nab-paclitaxel in patients who had progressed on gemcitabine however was less impressive with 63% of patients progressing by RECIST criteria at their first response assessment.[44] These patients were not preselected based on SPARC status.

The question of whether incorporating nanoparticle bound-paclitaxel into first-line chemotherapy with gemcitabine leads to a clinically meaningful improvement in survival is yet to be answered by a prospective randomised clinical trial currently awaiting completion.[45] Although tissue analysis for SPARC is included in this trial, the interventional arm will not be enriched with SPARC positive patients.

#### **4.5 Recommendations**

To date there is no established standard of care in the second-line setting or beyond. Treatment must therefore be tailored to each patient but may include oxaliplatin,

Medical Therapy of Pancreatic Cancer: Current Status and Future Targets 65

and the regulation of apoptosis. When activated by the associated EGFR, Ras leads to further downstream activation of Raf, MAP2K, MAPK and PI3K-Akt cascades. K-ras mutations lead to cell-cycle progression, and promote tumour cell survival. Mutated K-ras, seen in over 90% of pancreatic cancer is mostly identified in codon 12 but may also be seen

There has been an attempt in an adjuvant phase II study to vaccinate against k-ras, in patients who harbour codon 12 k-ras mutations.[64] In 24 patients, this was felt to be safe, however less than half of patients had a detected immune response and the protective value

Another approach has been to inhibit the KRAS protein itself. This has been attempted through targeting its attachment to the cell membrane by inhibiting farnesyltransferase with tipifarnib - a farnysyltransferase inhibitor (FTI).[66] Inhibiting Ras-driven signal transduction and interfering with Ras-membrane binding with other small molecule drugs such a salirasib, or antisense/RNA inhibitors are early in clinical development.[65] Unfortunately to date the only strategy reaching a phase III study, combining tipifarnib with gemcitabine in advanced pancreatic carcinoma, did not provide any significant difference in either the clinical benefit rate, median progression-free, or overall survival.[66] This is likely due to

Downstream Ras pathway inhibition of mitogen-activated protein kinase (MAPK) with a MEK inhibitor has not shown any phase II activity.[67] This was despite preclinical evidence showing synergistic activity by dual inhibition with the EGFR TKI inhibitor gefitinib and the

Activation of this pathway leads to downstream signaling events through MAPK, PI3K-Akt and the STAT family of proteins. STAT proteins also have roles in cell proliferation, survival, motility, invasion and adhesion. Over-expression of this pathway and its ligands (EGF and TGF-) are common in pancreatic cancer.[69-70] The clinical evidence for targeting the EGFR is outlined above. As previously mentioned, the addition of erlotinib or cetuximab to gemcitabine has resulted in only modest and no additional overall survival benefit

VEGF overexpression is common in pancreatic adenocarcinoma and is associated with a poorer prognosis.[71] Despite this being an attractive target, multiple anti-angiogenic strategies added to a backbone of gemcitabine have been disappointing. Two phase III trials in advanced pancreatic adenocarcinoma, have shown no overall survival benefit with the addition of the VEGF monoclonal antibody bevacizumab[72] to either single-agent gemcitabine, or a doublet of gemcitabine with erlotinib.[73] The latter study did however demonstrate a difference in progression-free survival (HR, 0.73; 95% CI, 0.61 to 0.86; P =

Sorafenib is an oral multitargeted kinase inhibitor which inhibits the VEGF-receptor tyrosine kinase as well as Raf-1, the platelet-derived growth factor receptor (PDGFR), c-kit and FLT-

in codons 13 and 61.[47]

of this strategy is unknown.

alternate pathways that still allow the prenylation of Ras.

**5.1.2 The epidermal growth factor receptor (EGFR) pathway** 

**5.1.3 Angiogenesis, matrix metalloproteinases (MMPs) and integrins** 

MAPK inhibitor CI-1040 (PD184532).[68]

respectively.

0.0002).

fluoropyrimidine or taxane-based regimens, as outlined above. There is very limited evidence for irinotecan-based treatment. A 5-FU/oxaliplatin or capecitabine-erlotinib doublet is an option. Consideration for enrolment in a clinical trial should be given if available.

#### **5. Future targets in pancreatic cancer**

Because attempts at improving survival in pancreatic cancer with cytotoxic and biologic therapy have been modest at the most thus far, newer strategies of targeting the core signaling pathways implicated in pancreatic cancer are needed.

Previously, genetic mutations affecting genes such as TP53, KRAS, CDKN2A and SMAD4 were known to be associated, but a more recent genome-wide analysis has identified a broader range of aberrant pathways implicated in pancreatic cancer growth.[46] In most of the 24 cancers examined in this series, the majority of the genetic mutations were felt to be disrupting one or more of 12 core signaling pathways.

In pancreatic cancer, aberrations can occur in signal transduction and other pathways that promote cell survival and allow proliferation. These include KRAS,[47] PI3K/Akt/mTOR,[49- 50] EGFR,[52] insulin-like growth factor (IGF-1) (which is co-expressed with Src),[52] hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF).[53] There are embryonic developmental signaling pathways that also lead to progression such as the Hedgehog, Notch, and Wnt pathways.[54-57] Matrix metalloproteinases (MMPs) also play a part in promoting neovascularisation and tumour invasion, and abnormalities in core pathways involved in DNA repair as well as apoptosis control such as p53, SMAD/TGF and p14 AFR/p16 are also seen.[58-59]

Finally there is also documented activity or upregulation of other factors such as cyclooxygenase,[60] focal adhesion kinase (FAK) (which in turn interacts with the IGF-1 receptor),[61] telomerase,[62] as well as cholecystokinin, gastrin and gastrin receptors.[63]

#### **5.1 Current evidence and future strategies targeting specific pathways in pancreatic cancer**


#### **5.1.1 The ras pathway**

K-ras is part of the Ras group of genes, which code for GTP-binding proteins in the cellular membrane. Ras is important in cellular differentiation and proliferation, as well as adhesion

fluoropyrimidine or taxane-based regimens, as outlined above. There is very limited evidence for irinotecan-based treatment. A 5-FU/oxaliplatin or capecitabine-erlotinib doublet is an option. Consideration for enrolment in a clinical trial should be given if

Because attempts at improving survival in pancreatic cancer with cytotoxic and biologic therapy have been modest at the most thus far, newer strategies of targeting the core

Previously, genetic mutations affecting genes such as TP53, KRAS, CDKN2A and SMAD4 were known to be associated, but a more recent genome-wide analysis has identified a broader range of aberrant pathways implicated in pancreatic cancer growth.[46] In most of the 24 cancers examined in this series, the majority of the genetic mutations were felt to be

In pancreatic cancer, aberrations can occur in signal transduction and other pathways that promote cell survival and allow proliferation. These include KRAS,[47] PI3K/Akt/mTOR,[49- 50] EGFR,[52] insulin-like growth factor (IGF-1) (which is co-expressed with Src),[52] hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF).[53] There are embryonic developmental signaling pathways that also lead to progression such as the Hedgehog, Notch, and Wnt pathways.[54-57] Matrix metalloproteinases (MMPs) also play a part in promoting neovascularisation and tumour invasion, and abnormalities in core pathways involved in DNA repair as well as apoptosis control such as p53, SMAD/TGF-

Finally there is also documented activity or upregulation of other factors such as cyclooxygenase,[60] focal adhesion kinase (FAK) (which in turn interacts with the IGF-1 receptor),[61] telomerase,[62] as well as cholecystokinin, gastrin and gastrin receptors.[63]

**5.1 Current evidence and future strategies targeting specific pathways in pancreatic** 

K-ras is part of the Ras group of genes, which code for GTP-binding proteins in the cellular membrane. Ras is important in cellular differentiation and proliferation, as well as adhesion

available.

**cancer**  - K-ras





**5.1.1 The ras pathway** 

**5. Future targets in pancreatic cancer** 

signaling pathways implicated in pancreatic cancer are needed.

disrupting one or more of 12 core signaling pathways.

and p14 AFR/p16 are also seen.[58-59]





and the regulation of apoptosis. When activated by the associated EGFR, Ras leads to further downstream activation of Raf, MAP2K, MAPK and PI3K-Akt cascades. K-ras mutations lead to cell-cycle progression, and promote tumour cell survival. Mutated K-ras, seen in over 90% of pancreatic cancer is mostly identified in codon 12 but may also be seen in codons 13 and 61.[47]

There has been an attempt in an adjuvant phase II study to vaccinate against k-ras, in patients who harbour codon 12 k-ras mutations.[64] In 24 patients, this was felt to be safe, however less than half of patients had a detected immune response and the protective value of this strategy is unknown.

Another approach has been to inhibit the KRAS protein itself. This has been attempted through targeting its attachment to the cell membrane by inhibiting farnesyltransferase with tipifarnib - a farnysyltransferase inhibitor (FTI).[66] Inhibiting Ras-driven signal transduction and interfering with Ras-membrane binding with other small molecule drugs such a salirasib, or antisense/RNA inhibitors are early in clinical development.[65] Unfortunately to date the only strategy reaching a phase III study, combining tipifarnib with gemcitabine in advanced pancreatic carcinoma, did not provide any significant difference in either the clinical benefit rate, median progression-free, or overall survival.[66] This is likely due to alternate pathways that still allow the prenylation of Ras.

Downstream Ras pathway inhibition of mitogen-activated protein kinase (MAPK) with a MEK inhibitor has not shown any phase II activity.[67] This was despite preclinical evidence showing synergistic activity by dual inhibition with the EGFR TKI inhibitor gefitinib and the MAPK inhibitor CI-1040 (PD184532).[68]

#### **5.1.2 The epidermal growth factor receptor (EGFR) pathway**

Activation of this pathway leads to downstream signaling events through MAPK, PI3K-Akt and the STAT family of proteins. STAT proteins also have roles in cell proliferation, survival, motility, invasion and adhesion. Over-expression of this pathway and its ligands (EGF and TGF-) are common in pancreatic cancer.[69-70] The clinical evidence for targeting the EGFR is outlined above. As previously mentioned, the addition of erlotinib or cetuximab to gemcitabine has resulted in only modest and no additional overall survival benefit respectively.

#### **5.1.3 Angiogenesis, matrix metalloproteinases (MMPs) and integrins**

VEGF overexpression is common in pancreatic adenocarcinoma and is associated with a poorer prognosis.[71] Despite this being an attractive target, multiple anti-angiogenic strategies added to a backbone of gemcitabine have been disappointing. Two phase III trials in advanced pancreatic adenocarcinoma, have shown no overall survival benefit with the addition of the VEGF monoclonal antibody bevacizumab[72] to either single-agent gemcitabine, or a doublet of gemcitabine with erlotinib.[73] The latter study did however demonstrate a difference in progression-free survival (HR, 0.73; 95% CI, 0.61 to 0.86; P = 0.0002).

Sorafenib is an oral multitargeted kinase inhibitor which inhibits the VEGF-receptor tyrosine kinase as well as Raf-1, the platelet-derived growth factor receptor (PDGFR), c-kit and FLT-

Medical Therapy of Pancreatic Cancer: Current Status and Future Targets 67

and had to discontinue therapy.[86] A phase III trial of gemcitabine with or without a combination of curcumin and celecoxib (a cyclo-oxygenase-2 (COX-2) inhibitor) is currently

The cyclo-oxygenase (COX) pathway is also important. Inhibition with celecoxib has been proven to suppress tumour proliferation as well as VEGF expression in pancreatic cancer.[88] However phase II trial responses in combination with gemcitabine have been mixed. The most favourable phase II study was performed in 42 patients (most with metastatic rather than locally advanced disease) who received gemcitabine 1000mg/m2 (on days 1 and 8 only of a 3-week cycle) in combination with celecoxib 400mg BD. The clinical benefit rate in 30 patients was reported as 71% [95% CI, 58-84%]), and the median overall survival was 9.1 months (95% CI, 7.5-10.6 months).[89] However another phase II study showed that despite a clinical benefit rate of 52% in 25 patients, the 12 month survival rate was 15%, which did not

TGF- binds to cell receptors that lead to downstream activation of SMAD4 which in turn moves into the cell nucleus to activate gene transcription. TGF-is also involved with activating other pathways including Ras, PI3K and MAPK. Although tumour suppressive in epithelial cells, it is also involved in mediating invasion and metastasis. In pancreatic cancer, mutations in SMAD4 are seen in 50% and up to 4% of TGF receptors.[91] Mutations of the former can lead to reduced TGF- tumour suppression as well as increased tumour cell invasiveness. Exploitation of this pathway with inhibitors such as antisense oligonucleotides specific to the TGF receptor are in early phase clinical development in several solid

Overexpression of the c-MET proto-oncogene which codes for MET (mesenchymalepithelial transition factor) is common in a number of solid malignancies such as colon, gastric, lung, breast, ovarian, bladder and pancreatic cancer.[93] The resultant protein hepatocyte transcription factor receptor (HGFR) is stimulated by HGF which is produced by fibroblasts in the stromal microenvironent. This in turn, leads to further tumour growth, angiogenesis, invasion, and metastasis formation. Similarly, the insulin-like growth factor (IGF-1) and focal adhesion kinase (FAK) pathways which are implicated in tumour growth and survival are overexpressed in pancreatic cancer. Inhibitors such as the selective cMET inhibitor tivatanib (ARQ 197) and anti IGF-1 receptor antibody cixutumumab are also early

Src is a proto-oncogene which codes for a non-receptor tyrosine kinase (RTK). Src proteins are a family of kinases involved in cell adhesion, and fibroblast division. Expression has been documented in a variety of cancers including pancreatic cancer, where overexpression is seen in 70%.[94] Overexpressed Src can lead to upregulation of the IGF-1 receptor.[52] Phase I trials of the BCR/Abl, c-kit and Src family inhibitor dasatinib have been performed in patients in a variety of solid tumours but at present another dual Src and Abl tyrosine

reach predetermined efficacy in order to proceed to a phase III trial.[90]

malignancies including pancreatic cancer.[92]

in clinical development.

**5.1.8 Src** 

**5.1.7 Transforming growth factor- (TGF-), SMAD4, MET, and IGF-1** 

in progress.[87]

**5.1.6 Cyclo-oxygenase** 

3. It has not shown any significant additive activity in a phase II study. [74] Similarly axitinib (a selective oral inhibitor of multiple VEGF receptors), has also failed to show improved efficacy when combined with gemcitabine in the phase III setting despite promise in an earlier phase II trial.[75-76] A phase III study randomised 546 patients with metastatic pancreatic cancer to gemcitabine with aflibercept (the VEGF 'trap') vs. gemcitabine with placebo (clinicaltrials.gov identifier NCT00574275). This was also terminated early due to no significant improvement in the primary or secondary end points of overall and progressionfree survival.

Matrix metalloproteinases (MMPs) are enzymes that break down the extracellular matrix and are required for tumour spread and neovascularisation. However, randomised trials utilising the MMP inhibitor marimastat in metastatic disease did not show any added survival benefit over gemcitabine alone.[77-79] Whether there might be a role in the adjuvant setting remains unknown.

Volociximab is a monoclonal antibody that inhibits fibronectin binding to α5β1-integrin, which promotes apoptosis in tumour endothelial cells. A small, phase II study combining this agent with gemcitabine in 20 patients showed activity with stable disease in half of patients and a partial response in one patient. The median time to progression (TTP) was 4.3 months with 37% of patients alive at 12 months. However there is no prospective randomised evidence to date.[80] Cilengitide is another agent that interferes with integrin binding leading to proliferative endothelial cell apoptosis, but it was not shown to be of added benefit when combined with gemcitabine.[81] There are other integrin inhibitors in preclinical and early clinical stages of evaluation.

#### **5.1.4 The PI3k/Akt/mTOR pathway**

The phosphoinositide 3'-kinase (PI3k)/Akt/mammalian target of rapamycin (mTOR) pathway which is regulated upstream by KRAS is important in pancreatic tumorigenesis and angiogenesis. Activation in pancreatic cancer is common, and is associated with loss of the tumour suppressor PTEN, and with poorer outcomes as well as gemcitabine resistance.[82] Despite this, the mTOR inhibitors everolimus and temsirolimus have shown no objective responses in phase II studies, and when the former was combined with erlotinib, also no objective responses were seen.[83-84] It is felt that they are unlikely to have a role - at least as a single agent strategy - in this disease. The PI3K and Akt inhibitors (BKM-120 and MK-2206) are in phase I development. RX-0201 (an Akt-1 mRNA antisense oligonucleotide) is being evaluated in a phase II trial in combination with gemcitabine.[82]

#### **5.1.5 NFk**

Nuclear factor kappa light-chain enhancer of activated cells (NFk) is also activated by the PI3k/Akt/mTOR pathway. Curcumin (diferuloyl methane) - a component of the common Indian spice turmeric - has been shown to inhibit NFk. A phase II study using 8g of curcumin as a single agent daily for two months found that this agent was tolerable in 25 patients, two of which received prolonged (up to 12 month) periods of stable disease. One patient achieved a partial response.[85] A further phase II study of 17 patients with curcumin in combination with gemcitabine showed that 5 patients either had stable or partial responses but another 5 patients could not tolerate treatment due to abdominal discomfort, and had to discontinue therapy.[86] A phase III trial of gemcitabine with or without a combination of curcumin and celecoxib (a cyclo-oxygenase-2 (COX-2) inhibitor) is currently in progress.[87]

#### **5.1.6 Cyclo-oxygenase**

66 Pancreatic Cancer – Clinical Management

3. It has not shown any significant additive activity in a phase II study. [74] Similarly axitinib (a selective oral inhibitor of multiple VEGF receptors), has also failed to show improved efficacy when combined with gemcitabine in the phase III setting despite promise in an earlier phase II trial.[75-76] A phase III study randomised 546 patients with metastatic pancreatic cancer to gemcitabine with aflibercept (the VEGF 'trap') vs. gemcitabine with placebo (clinicaltrials.gov identifier NCT00574275). This was also terminated early due to no significant improvement in the primary or secondary end points of overall and progression-

Matrix metalloproteinases (MMPs) are enzymes that break down the extracellular matrix and are required for tumour spread and neovascularisation. However, randomised trials utilising the MMP inhibitor marimastat in metastatic disease did not show any added survival benefit over gemcitabine alone.[77-79] Whether there might be a role in the adjuvant

Volociximab is a monoclonal antibody that inhibits fibronectin binding to α5β1-integrin, which promotes apoptosis in tumour endothelial cells. A small, phase II study combining this agent with gemcitabine in 20 patients showed activity with stable disease in half of patients and a partial response in one patient. The median time to progression (TTP) was 4.3 months with 37% of patients alive at 12 months. However there is no prospective randomised evidence to date.[80] Cilengitide is another agent that interferes with integrin binding leading to proliferative endothelial cell apoptosis, but it was not shown to be of added benefit when combined with gemcitabine.[81] There are other integrin inhibitors in

The phosphoinositide 3'-kinase (PI3k)/Akt/mammalian target of rapamycin (mTOR) pathway which is regulated upstream by KRAS is important in pancreatic tumorigenesis and angiogenesis. Activation in pancreatic cancer is common, and is associated with loss of the tumour suppressor PTEN, and with poorer outcomes as well as gemcitabine resistance.[82] Despite this, the mTOR inhibitors everolimus and temsirolimus have shown no objective responses in phase II studies, and when the former was combined with erlotinib, also no objective responses were seen.[83-84] It is felt that they are unlikely to have a role - at least as a single agent strategy - in this disease. The PI3K and Akt inhibitors (BKM-120 and MK-2206) are in phase I development. RX-0201 (an Akt-1 mRNA antisense oligonucleotide) is being evaluated in a phase II trial in combination with gemcitabine.[82]

Nuclear factor kappa light-chain enhancer of activated cells (NFk) is also activated by the PI3k/Akt/mTOR pathway. Curcumin (diferuloyl methane) - a component of the common Indian spice turmeric - has been shown to inhibit NFk. A phase II study using 8g of curcumin as a single agent daily for two months found that this agent was tolerable in 25 patients, two of which received prolonged (up to 12 month) periods of stable disease. One patient achieved a partial response.[85] A further phase II study of 17 patients with curcumin in combination with gemcitabine showed that 5 patients either had stable or partial responses but another 5 patients could not tolerate treatment due to abdominal discomfort,

free survival.

**5.1.5 NFk**

setting remains unknown.

preclinical and early clinical stages of evaluation.

**5.1.4 The PI3k/Akt/mTOR pathway** 

The cyclo-oxygenase (COX) pathway is also important. Inhibition with celecoxib has been proven to suppress tumour proliferation as well as VEGF expression in pancreatic cancer.[88] However phase II trial responses in combination with gemcitabine have been mixed. The most favourable phase II study was performed in 42 patients (most with metastatic rather than locally advanced disease) who received gemcitabine 1000mg/m2 (on days 1 and 8 only of a 3-week cycle) in combination with celecoxib 400mg BD. The clinical benefit rate in 30 patients was reported as 71% [95% CI, 58-84%]), and the median overall survival was 9.1 months (95% CI, 7.5-10.6 months).[89] However another phase II study showed that despite a clinical benefit rate of 52% in 25 patients, the 12 month survival rate was 15%, which did not reach predetermined efficacy in order to proceed to a phase III trial.[90]

#### **5.1.7 Transforming growth factor- (TGF-), SMAD4, MET, and IGF-1**

TGF- binds to cell receptors that lead to downstream activation of SMAD4 which in turn moves into the cell nucleus to activate gene transcription. TGF-is also involved with activating other pathways including Ras, PI3K and MAPK. Although tumour suppressive in epithelial cells, it is also involved in mediating invasion and metastasis. In pancreatic cancer, mutations in SMAD4 are seen in 50% and up to 4% of TGF receptors.[91] Mutations of the former can lead to reduced TGF- tumour suppression as well as increased tumour cell invasiveness. Exploitation of this pathway with inhibitors such as antisense oligonucleotides specific to the TGF receptor are in early phase clinical development in several solid malignancies including pancreatic cancer.[92]

Overexpression of the c-MET proto-oncogene which codes for MET (mesenchymalepithelial transition factor) is common in a number of solid malignancies such as colon, gastric, lung, breast, ovarian, bladder and pancreatic cancer.[93] The resultant protein hepatocyte transcription factor receptor (HGFR) is stimulated by HGF which is produced by fibroblasts in the stromal microenvironent. This in turn, leads to further tumour growth, angiogenesis, invasion, and metastasis formation. Similarly, the insulin-like growth factor (IGF-1) and focal adhesion kinase (FAK) pathways which are implicated in tumour growth and survival are overexpressed in pancreatic cancer. Inhibitors such as the selective cMET inhibitor tivatanib (ARQ 197) and anti IGF-1 receptor antibody cixutumumab are also early in clinical development.

#### **5.1.8 Src**

Src is a proto-oncogene which codes for a non-receptor tyrosine kinase (RTK). Src proteins are a family of kinases involved in cell adhesion, and fibroblast division. Expression has been documented in a variety of cancers including pancreatic cancer, where overexpression is seen in 70%.[94] Overexpressed Src can lead to upregulation of the IGF-1 receptor.[52] Phase I trials of the BCR/Abl, c-kit and Src family inhibitor dasatinib have been performed in patients in a variety of solid tumours but at present another dual Src and Abl tyrosine

Medical Therapy of Pancreatic Cancer: Current Status and Future Targets 69

transport proteins to enter cells and to have a therapeutic effect. Both hENT1 and 2 allow this with hENT1 being more selective. A lack of hENT1 expression has been shown to interfere with gemcitabine influx, and is associated with reduced efficacy and decreased survival in patients.[102-105] However it is not yet clear whether immunohistochemical hENT1 expression or gene expression will be the most predictive measure, or whether there is a

Once gemcitabine is transported into the cell, it is phosphorylated by deoxycytidine kinase (dCK) to difluorodeoxycytidine. It is gemcitabine triphosphate's (dFdCTP) incorporation into DNA that leads to strand termination. DFdCTP is metabolised by cytidine deaminase (CDA). There is evidence of correlation between dCK and CDA levels, and also detected single nucleotide polymorphisms (SNPs) in genes that code for these and other proteins involved in gemcitabine transport and metabolism, and overall survival.[106] However, to date, attempts at increasing the effective intracellular concentration of gemcitabine and its metabolite dFdCTP have not translated into improved patient survival in the phase III trial setting. Fixed-dose rate (FDR) gemcitabine (1,500mg/m2/150mins) only modestly improved overall survival (6.2 vs. 4.9 months, HR 0.83 stratified log-rank p = 0.04) compared with standard gemcitabine, and did not meet predetermined efficacy. It was also associated with

A modified form of gemcitabine, CP-4126 (gemcitabine-5'-elaidic acid ester, Clavis Pharma) bypasses nucleoside transporters. It is undergoing phase II evaluation in patients with

Other promising predictive and prognostic biomarkers may include variations in cellular histone modification patterns.[110] Immunohistochemical analyses of histone H3 lysine 4 and 9, dimethylation and histone H3 lysine 18 acetylation were performed on tissue banks. Tissue was derived from patients with resected pancreatic tumours (including those in the RTOG 9704 study which compared adjuvant 5-FU and gemcitabine). Low levels of some histone modifications predicted a poorer disease-free survival if patients were treated with

DPC4 (SMAD4) gene expression has recently found to be prognostic and associated with local failure following adjuvant chemoradiotherapy, or with metastatic spread in locally advanced disease.[111-112] However prospective validation is still required, especially if therapeutic targeting of this pathway is a future therapeutic option. These, and other markers such as mismatch repair polymorphisms, are also yet to be prospectively validated.

Despite research into the medical management of pancreatic cancer, survival remains poor. Numerous agents and combinations have been attempted in early phase clinical trials, but to date, very modest improvements have been made in overall survival. Single agent gemcitabine still remains the standard of care for most patients in both the adjuvant and advanced settings with adjuvant chemoradiotherapy being more controversial. In the advanced settings, gemcitabine or gemcitabine with erlotinib are appropriate for most, but fit patients may benefit from gemcitabine-containing cytotoxic doublets. FOLFIRINOX is now considered an option in the fittest of patients, but its toxicity is significant. Although no

advanced pancreatic cancer, after a phase I study showed a good safety profile.[108-109]

concordance between hENT1 expression in primary and metastatic disease.

greater haematological toxicity.[107]

adjuvant 5-FU compared with gemcitabine.

**7. Conclusion** 

kinase inhibitor SKI-606 (bosutinib) is undergoing phase I/II evaluation with gemcitabine as adjuvant therapy in the postoperative setting.[95]

#### **5.1.9 Hedgehog/Wnt pathways/Notch**

The hedgehog signaling pathway plays an important part in embryonic development but when aberrant, may be implicated in tumorigenesis. [96] Two transmembrane proteins work in tandem. Ptch (patched), which is tumour suppressing, inhibits the Smo (smoothened) protein which when activated by a Ptch mutation, allows hedgehog proteins to bind. This leads to downstream activation of GLI-1 which promotes nuclear transcription. One of the hedgehog proteins (Sonic Hedgehog - SHH) is expressed in 70% of pancreatic adenocarcinoma. Preclinical evidence points to the drug cyclopamine inhibiting Smo, but further trial evidence for hedgehog pathway inhibitors in pancreatic cancer patients is awaited.

The Wnt pathways are also important in normal embryonic development and mutations are implicated in tumorigenesis. If the Wnt--catenin cascade pathway is aberrant (65% of pancreatic cancer), abnormal overactivation of -catenin occurs which promotes abnormal nuclear transcription.[97] There is preclinical evidence that blocking this pathway can lead to pancreatic cell death, which may be a future potential target for treatment. It is thought that chemokine receptor 4 (CXCR4) is key in tumour angiogenesis and metastasis. Specific blockade of this chemokine receptor or its ligand SDF-1 may be a further potential future clinical strategy. It is thought that inhibiting both these pathways may have anti cancer stem cell effects.[97]

Notch genes code for proteins also responsible for tumour differentiation, proliferation and apoptosis and the pathway requires the enzyme gamma-secretase to be activated. Notch 3 is also expressed in most pancreatic cancers with preclinical evidence of a potential role using siRNA and secretase inhibitors in therapy.[98]

#### **5.1.10 Gastrin and cholecystokinin receptors**

Targeting gastrin And the cholecystokinin receptor CCK-BR, with the intravenous agent JB95008 (gastrozole) has been attempted in advanced pretreated pancreatic cancer but was found to be no better than 5-FU in terms of survival.[99]

Another novel oral gastrin inhibitor named Z-360 has been was examined in a phase Ib/IIa study and found to be active when given in combination with gemcitabine, with a future randomised controlled trial planned.[100-101]

#### **6. Biomarkers in pancreatic cancer**

In contrast to other solid tumour malignancies, there have been relatively modest or poor responses achieved with molecularly targeted agents to date in unselected patients with pancreatic cancer. There is an urgent need for a personalised approach to better define biomarkers in order to predict patients that are more likely to benefit from a particular cytotoxic or molecular targeted therapy.

The biomarker with the most preclinical and clinical evidence is human equilibrative nucleoside transporter 1 (hENT1). Gemcitabine requires transmembrane nucleoside transport proteins to enter cells and to have a therapeutic effect. Both hENT1 and 2 allow this with hENT1 being more selective. A lack of hENT1 expression has been shown to interfere with gemcitabine influx, and is associated with reduced efficacy and decreased survival in patients.[102-105] However it is not yet clear whether immunohistochemical hENT1 expression or gene expression will be the most predictive measure, or whether there is a concordance between hENT1 expression in primary and metastatic disease.

Once gemcitabine is transported into the cell, it is phosphorylated by deoxycytidine kinase (dCK) to difluorodeoxycytidine. It is gemcitabine triphosphate's (dFdCTP) incorporation into DNA that leads to strand termination. DFdCTP is metabolised by cytidine deaminase (CDA). There is evidence of correlation between dCK and CDA levels, and also detected single nucleotide polymorphisms (SNPs) in genes that code for these and other proteins involved in gemcitabine transport and metabolism, and overall survival.[106] However, to date, attempts at increasing the effective intracellular concentration of gemcitabine and its metabolite dFdCTP have not translated into improved patient survival in the phase III trial setting. Fixed-dose rate (FDR) gemcitabine (1,500mg/m2/150mins) only modestly improved overall survival (6.2 vs. 4.9 months, HR 0.83 stratified log-rank p = 0.04) compared with standard gemcitabine, and did not meet predetermined efficacy. It was also associated with greater haematological toxicity.[107]

A modified form of gemcitabine, CP-4126 (gemcitabine-5'-elaidic acid ester, Clavis Pharma) bypasses nucleoside transporters. It is undergoing phase II evaluation in patients with advanced pancreatic cancer, after a phase I study showed a good safety profile.[108-109]

Other promising predictive and prognostic biomarkers may include variations in cellular histone modification patterns.[110] Immunohistochemical analyses of histone H3 lysine 4 and 9, dimethylation and histone H3 lysine 18 acetylation were performed on tissue banks. Tissue was derived from patients with resected pancreatic tumours (including those in the RTOG 9704 study which compared adjuvant 5-FU and gemcitabine). Low levels of some histone modifications predicted a poorer disease-free survival if patients were treated with adjuvant 5-FU compared with gemcitabine.

DPC4 (SMAD4) gene expression has recently found to be prognostic and associated with local failure following adjuvant chemoradiotherapy, or with metastatic spread in locally advanced disease.[111-112] However prospective validation is still required, especially if therapeutic targeting of this pathway is a future therapeutic option. These, and other markers such as mismatch repair polymorphisms, are also yet to be prospectively validated.

#### **7. Conclusion**

68 Pancreatic Cancer – Clinical Management

kinase inhibitor SKI-606 (bosutinib) is undergoing phase I/II evaluation with gemcitabine as

The hedgehog signaling pathway plays an important part in embryonic development but when aberrant, may be implicated in tumorigenesis. [96] Two transmembrane proteins work in tandem. Ptch (patched), which is tumour suppressing, inhibits the Smo (smoothened) protein which when activated by a Ptch mutation, allows hedgehog proteins to bind. This leads to downstream activation of GLI-1 which promotes nuclear transcription. One of the hedgehog proteins (Sonic Hedgehog - SHH) is expressed in 70% of pancreatic adenocarcinoma. Preclinical evidence points to the drug cyclopamine inhibiting Smo, but further trial evidence for hedgehog pathway inhibitors in pancreatic cancer patients is awaited. The Wnt pathways are also important in normal embryonic development and mutations are implicated in tumorigenesis. If the Wnt--catenin cascade pathway is aberrant (65% of pancreatic cancer), abnormal overactivation of -catenin occurs which promotes abnormal nuclear transcription.[97] There is preclinical evidence that blocking this pathway can lead to pancreatic cell death, which may be a future potential target for treatment. It is thought that chemokine receptor 4 (CXCR4) is key in tumour angiogenesis and metastasis. Specific blockade of this chemokine receptor or its ligand SDF-1 may be a further potential future clinical strategy. It is thought that inhibiting both these pathways may have anti cancer stem

Notch genes code for proteins also responsible for tumour differentiation, proliferation and apoptosis and the pathway requires the enzyme gamma-secretase to be activated. Notch 3 is also expressed in most pancreatic cancers with preclinical evidence of a potential role using

Targeting gastrin And the cholecystokinin receptor CCK-BR, with the intravenous agent JB95008 (gastrozole) has been attempted in advanced pretreated pancreatic cancer but was

Another novel oral gastrin inhibitor named Z-360 has been was examined in a phase Ib/IIa study and found to be active when given in combination with gemcitabine, with a future

In contrast to other solid tumour malignancies, there have been relatively modest or poor responses achieved with molecularly targeted agents to date in unselected patients with pancreatic cancer. There is an urgent need for a personalised approach to better define biomarkers in order to predict patients that are more likely to benefit from a particular

The biomarker with the most preclinical and clinical evidence is human equilibrative nucleoside transporter 1 (hENT1). Gemcitabine requires transmembrane nucleoside

adjuvant therapy in the postoperative setting.[95]

**5.1.9 Hedgehog/Wnt pathways/Notch** 

siRNA and secretase inhibitors in therapy.[98]

randomised controlled trial planned.[100-101]

**6. Biomarkers in pancreatic cancer** 

cytotoxic or molecular targeted therapy.

**5.1.10 Gastrin and cholecystokinin receptors** 

found to be no better than 5-FU in terms of survival.[99]

cell effects.[97]

Despite research into the medical management of pancreatic cancer, survival remains poor. Numerous agents and combinations have been attempted in early phase clinical trials, but to date, very modest improvements have been made in overall survival. Single agent gemcitabine still remains the standard of care for most patients in both the adjuvant and advanced settings with adjuvant chemoradiotherapy being more controversial. In the advanced settings, gemcitabine or gemcitabine with erlotinib are appropriate for most, but fit patients may benefit from gemcitabine-containing cytotoxic doublets. FOLFIRINOX is now considered an option in the fittest of patients, but its toxicity is significant. Although no

Medical Therapy of Pancreatic Cancer: Current Status and Future Targets 71

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Increased knowledge of the molecular pathogenesis of pancreatic cancer has allowed new targets and therapeutic strategies to emerge. However, true progress in the personalised management of this disease will only be likely with equally important research into the identification, and validation of appropriate predictive and prognostic biomarkers.

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**5**

*Poland* 

**Temporal Trends in Pancreatic Cancer** 

Pancreatic cancer is one of the most common malignancies of the digestive system and, depending on the geographic area, fourth or fifth leading cause of cancer deaths (Lillemoe *et al*, 2000; Simon & Printz, 2001). Between 1960 and 1980, the incidence rates had increased significantly in most industrialised countries, including Poland. Corresponding 5-year survival rates demonstrated only slight variations and remained stable at about 1-3%. Surprisingly, results obtained from some population databases suggest that only about 30% of patients registered as pancreatic cancer have been adequately verified by histopathology (Wood *et al*, 2006). Moreover, since many studies included only small groups of patients, previous reports did not properly reflect the actual changes, including long-term results of treatment (Gudjonsson, 1995). As incidence rates were relatively high and the efficacy of therapeutic methods was questionable, pancreatic cancer was subject to numerous clinical trials (Jafari & Abbruzzese, 2004). However, no clear conclusions could be drawn in terms of the best therapeutic approach due to marked differences between individual studies

Many epidemiological studies published over the last 50 years provided detailed data for some general trends related to incidence and mortality rates for pancreatic cancer. Changes in other areas of interest, such as variation in surgical and systemic therapy or long-term outcomes are much less examined. Taking into account these facts, an analysis of temporal trends for some surgical aspects of pancreatic disorders may provide significant information

A literature search was performed using two bibliographic databases, i.e. PubMed and Ovid. Databases were searched using combinations of the following keywords: (pancreatic neoplasm or pancreatic cancer) and (trends or time related changes). Additionally all patients diagnosed with pancreatic duct cell cancer (*adenocarcinoma ductale*) treated between 1972 and 2003 at the 1st Department of General and GI Surgery of Jagiellonian University Medical College in Kraków were reviewed. Other pancreatic tumours verified as non-duct cell cancers and periampullary neoplasms were excluded. Clinical and demographic data, including age, gender and type of therapeutic interventions, were collected from medical records. Tumours were staged according to the TNM classification of *Union Internationale Contre Le Cancer* (UICC) of 1997. The type and extent of surgical treatment was categorised

**1. Introduction** 

(Gudjonsson, 1995).

**2. Methods** 

supplementing the results of previous studies.

Tadeusz Popiela and Marek Sierzega *Jagiellonian University Medical College* 


### **Temporal Trends in Pancreatic Cancer**

Tadeusz Popiela and Marek Sierzega *Jagiellonian University Medical College Poland* 

#### **1. Introduction**

76 Pancreatic Cancer – Clinical Management

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[106] Spratlin J, Mackey, JR. Human equilibrative nucleoside transporter 1 (hENT1) in

[107] Poplin E, Feng Y, Berlin J, et al. Phase III, randomised study of gemcitabine and

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[111] Herman JM, Hsu CC, Fishman EK, et al. Correlation of DPC4 status with outcomes in

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Cooperative Oncology Group. *J Clin Oncol* 2009 10;27(23):Epub 2009 Jul 6 [108] Hendlisz A, Voest EE, Schellens A, et al. Phase I study if oral CP-4126, a gemcitabine

locally advanced pancreatic cancer. *Cancer* 2010, 116, 5325-5335.

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*Gastroenterology* 2009; 136:187-196.

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antagonist on tumor growth in human pancreatic adenocarcinoma cell lines in vivo and mode of action determinations in vitro. *Cancer Chemother. Pharmacol.* 

controlled study to evaluate the safety and pharmacokinetics of Z-360 in subjects with unresectable advanced pancreatic cancer in combination with gemcitabine

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nucleoside transporter 1 is associated with reduced survival in patients with gemcitabine-treated pancreas adenocarcinoma. *Clin Cancer Res* 2004; 10: 6956-6961.

levels predict response to gemcitabine in patients with pancreatic cancer.

pancreatic adenocarcinoma: Towards individualised treatment decisions. *Cancers* 

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analogue, in patients with advanced solid tumours. *J Clin Oncol* 28, 2010 (suppl;

prognosis and treatment response in resectable pancreatic adenocarcinoma: results

pancreatic adenocarcinoma patients receiving adjuvant chemoradiation. *J Clin* 

diagnostic cytology specimens to predict the pattern of tumour progression in locally advanced pancreatic cancer patients (LAPC). *J Clin Oncol* 29: 2011 (suppl 4; Pancreatic cancer is one of the most common malignancies of the digestive system and, depending on the geographic area, fourth or fifth leading cause of cancer deaths (Lillemoe *et al*, 2000; Simon & Printz, 2001). Between 1960 and 1980, the incidence rates had increased significantly in most industrialised countries, including Poland. Corresponding 5-year survival rates demonstrated only slight variations and remained stable at about 1-3%. Surprisingly, results obtained from some population databases suggest that only about 30% of patients registered as pancreatic cancer have been adequately verified by histopathology (Wood *et al*, 2006). Moreover, since many studies included only small groups of patients, previous reports did not properly reflect the actual changes, including long-term results of treatment (Gudjonsson, 1995). As incidence rates were relatively high and the efficacy of therapeutic methods was questionable, pancreatic cancer was subject to numerous clinical trials (Jafari & Abbruzzese, 2004). However, no clear conclusions could be drawn in terms of the best therapeutic approach due to marked differences between individual studies (Gudjonsson, 1995).

Many epidemiological studies published over the last 50 years provided detailed data for some general trends related to incidence and mortality rates for pancreatic cancer. Changes in other areas of interest, such as variation in surgical and systemic therapy or long-term outcomes are much less examined. Taking into account these facts, an analysis of temporal trends for some surgical aspects of pancreatic disorders may provide significant information supplementing the results of previous studies.

#### **2. Methods**

A literature search was performed using two bibliographic databases, i.e. PubMed and Ovid. Databases were searched using combinations of the following keywords: (pancreatic neoplasm or pancreatic cancer) and (trends or time related changes). Additionally all patients diagnosed with pancreatic duct cell cancer (*adenocarcinoma ductale*) treated between 1972 and 2003 at the 1st Department of General and GI Surgery of Jagiellonian University Medical College in Kraków were reviewed. Other pancreatic tumours verified as non-duct cell cancers and periampullary neoplasms were excluded. Clinical and demographic data, including age, gender and type of therapeutic interventions, were collected from medical records. Tumours were staged according to the TNM classification of *Union Internationale Contre Le Cancer* (UICC) of 1997. The type and extent of surgical treatment was categorised

Temporal Trends in Pancreatic Cancer 79

Most authors agree that this phenomenon is mainly related to the exposure to carcinogens, particularly smoking. The role of the latter factor has been confirmed by increasing incidence trends in populations with a high proportion of smoking individuals and lowering incidence in countries where smoking is decreasing, i.e. Sweden (Bobak, 2003;

Between 1972 and 2003, a total of 1708 patients with chronic pancreatic and periampullary disorders were hospitalised, including 947 patients with histopathologically verified pancreatic duct cell cancer (Popiela et al, 2007). Fifty-eight per cent of 947 patients with pancreatic cancer were males (n=549) and 42% (n=398) females. Although the proportion of males increased temporarily to 64% in period 2, it subsequently decreased to values observed in period 1 (tab. 1). The median age was 62 years (95% confidence interval [CI] 61 – 62) and demonstrated a significant increasing trend over time. Similarly to other authors, we have recorded a significant increase in the proportion of females diagnosed with pancreatic cancer over the last twenty years. The significant increasing trend for the median age shown in our series was similar to other reports where growing numbers of pancreatic resections were carried out in elderly patients (Delcore *et al*, 1991; DiCarlo *et al*, 1998; Sohn *et al*, 1998).

Numerous changes have been observed worldwide in the diagnostics of pancreatic cancer during the last five decades. The number of cases diagnosed at laparotomy carried out due to jaundice or epigastric complaints decreased sharply as abdominal ultrasound was introduced into routine clinical practice (Soreide *et al*, 2010). Subsequently, ultrasound was gradually replaced by less operator-dependent imaging techniques, such as computed tomography (CT) and magnetic resonance imaging (MRI). A gradual increase in the use of endoscopic ultrasonography (EUS) and positron emission tomography (PET) has been observed over the recent years, but their application is usually limited to some specific clinical situations. The proportion of patients diagnosed solely with US and CT nowadays varies between 75% and 85%, while other imaging techniques (MRI, PET, EUS) are used less

The majority of lesions treated in our centre was located in the head of the pancreas (n=629, 66%), whereas cancers of the body and tail were found in 28% (n=261) and 6% (n=57) of cases, respectively. There were no significant differences in the proportions of tumours located in the head, body and tail of the pancreas over time (Popiela *et al*, 2007). However, other authors suggested some variation over time. Based on 43,946 cases of pancreatic cancer recorded between 1973 and 2002 in the Surveillance, Epidemiology, and End Results (SEER), Lau et al. found a 46% increase in the incidence of body/tail cancers (Lau *et al*, 2010). Reports from other geographic areas mostly failed to demonstrate any significant

Technical improvements in imaging methods and their wider accessibility should theoretically allow for an earlier diagnosis of pancreatic cancer leading to reduced proportions of advanced tumours and increased resectability rates. Surprisingly, most cohort studies failed to demonstrate any marked increase in the proportion of cancers at a lower stage (Cress *et al*, 2006; Janes *et al*, 1996; Niederhuber *et al*, 1995; Riall *et al*, 2006; Sener *et al*, 1999). The percentage of patients with stage IV tumours in our series, similarly to other reports, showed only a slight lowering trend. Although this phenomenon was accompanied by increasing resectability rates,

Flook & van Zanten, 2009; Luo *et al,* 2008; Mulder *et al*, 2002; Simon & Printz, 2001).

**4. Changes in pathological findings** 

frequently (David *et al*, 2009; Ngamruengphong *et al*, 2010).

change in the prevalence of distally-located cancers.

based on commonly accepted criteria (Pedrazzoli *et al*, 1999). To analyse temporal trends for pancreatic cancer, the study interval was divided into three periods, i.e. period 1 (1972- 1983), period 2 (1984-1993) and period 3 (1994-2003).

#### **3. General epidemiological trends**

Worldwide epidemiological studies on pancreatic cancer have demonstrated slightly higher incidence rates in males irrespectively of the geographic area (Katanoda & Dongmei, 2008; Katanoda & Yako-Suketomo, 2010; Levi *et al*, 2003; Michaud, 2004; Sahmoun *et al*, 2003).


Table 1. Demographic and clinical data of patients with pancreatic cancer

(PD –pancreatoduodenectomy, PPPD – pylorus-preserving PD, † chi-square test;

‡ ANOVA analysis of variance, § log-rank test)

based on commonly accepted criteria (Pedrazzoli *et al*, 1999). To analyse temporal trends for pancreatic cancer, the study interval was divided into three periods, i.e. period 1 (1972-

Worldwide epidemiological studies on pancreatic cancer have demonstrated slightly higher incidence rates in males irrespectively of the geographic area (Katanoda & Dongmei, 2008; Katanoda & Yako-Suketomo, 2010; Levi *et al*, 2003; Michaud, 2004; Sahmoun *et al*, 2003).

> Period 2 1984-1993 (n=294)

> 106 (36%) 188 (64%)

> 199 (68%) 71 (24%) 24 (8%)

> 7 (2%) 2 (1%) 11 (4%) 274 (93%)

> 228 (78%) 66 (22%)

> 46 (20%) 182 (80%)

22 (48%) 4 (8%) 10 (22%) 10 (22%)

62 (34%) 64 (35%) 13 (7%) 43 (24%)

222 (76%) 72 (24%)

6.2 (5.2-7.2) 14.3 (11.2-17.4) 5.5 (4.6-6.5)

Period 3 1994-2003 (n=508)

222 (44%)

341 (67%) 140 (28%) 27 (5%)

5 (1%) 16 (3%) 32 (6%) 455 (90%)

376 (74%)

115 (31%) 261 (69%)

46 (40%) 14 (12%) 22 (19%) 33 (29%)

81 (31%) 19 (7%) 67 (26%) 94 (36%)

231 (45%) 277 (55%)

7.6 (6.7-8.5) 20.0 (13.7-26.3) 5.9 (5.1-6.6)

132 (26%) 0.192†

286 (56%) 0.027†

P

0.147†

0.047†

<0.001†

0.557†

<0.001†

<0.01†

<0.001§ 0.041§ <0.001§

Period 1 1972-1983 (n=145)

70 (48%) 75 (52%)

89 (61%) 50 (35%) 6 (4%)

0 (0%) 3 (2%) 8 (6%) 134 (92%)

117 (81%) 28 (19%)

11 (9%) 106 (91%)

> 8 (73%) 0 (0%) 2 (18%) 1 (9%)

38 (36%) 55 (52%) 4 (4%) 9 (8%)

138 (95%) 7 (5%)

5.2 (4.7-5.6) 26.6 (10.4-42.8) 5.0 (4.5-5.6)

Table 1. Demographic and clinical data of patients with pancreatic cancer (PD –pancreatoduodenectomy, PPPD – pylorus-preserving PD, † chi-square test;

Age, median (95%CI) 58 (57-62) 60 (58-62) 63 (62-65) <0.001‡

1983), period 2 (1984-1993) and period 3 (1994-2003).

**3. General epidemiological trends**

Gender female male

Location head body tail

Therapy surgical conservative

 resective non-resective

 PD PPD

Type of surgical procedures

Type of pancreatic resections

 distal pancreatectomy total pancreatectomy

Type of non-resective surgery

Median survival, months (95%CI)

‡ ANOVA analysis of variance, § log-rank test)

 pancreatic resections unresectable tumours

 laparotomy biliary bypass gastro-enteric bypass biliary and enteric bypass

Chemotherapy no yes

overall

Stage I II III IV Most authors agree that this phenomenon is mainly related to the exposure to carcinogens, particularly smoking. The role of the latter factor has been confirmed by increasing incidence trends in populations with a high proportion of smoking individuals and lowering incidence in countries where smoking is decreasing, i.e. Sweden (Bobak, 2003; Flook & van Zanten, 2009; Luo *et al,* 2008; Mulder *et al*, 2002; Simon & Printz, 2001).

Between 1972 and 2003, a total of 1708 patients with chronic pancreatic and periampullary disorders were hospitalised, including 947 patients with histopathologically verified pancreatic duct cell cancer (Popiela et al, 2007). Fifty-eight per cent of 947 patients with pancreatic cancer were males (n=549) and 42% (n=398) females. Although the proportion of males increased temporarily to 64% in period 2, it subsequently decreased to values observed in period 1 (tab. 1). The median age was 62 years (95% confidence interval [CI] 61 – 62) and demonstrated a significant increasing trend over time. Similarly to other authors, we have recorded a significant increase in the proportion of females diagnosed with pancreatic cancer over the last twenty years. The significant increasing trend for the median age shown in our series was similar to other reports where growing numbers of pancreatic resections were carried out in elderly patients (Delcore *et al*, 1991; DiCarlo *et al*, 1998; Sohn *et al*, 1998).

#### **4. Changes in pathological findings**

Numerous changes have been observed worldwide in the diagnostics of pancreatic cancer during the last five decades. The number of cases diagnosed at laparotomy carried out due to jaundice or epigastric complaints decreased sharply as abdominal ultrasound was introduced into routine clinical practice (Soreide *et al*, 2010). Subsequently, ultrasound was gradually replaced by less operator-dependent imaging techniques, such as computed tomography (CT) and magnetic resonance imaging (MRI). A gradual increase in the use of endoscopic ultrasonography (EUS) and positron emission tomography (PET) has been observed over the recent years, but their application is usually limited to some specific clinical situations. The proportion of patients diagnosed solely with US and CT nowadays varies between 75% and 85%, while other imaging techniques (MRI, PET, EUS) are used less frequently (David *et al*, 2009; Ngamruengphong *et al*, 2010).

The majority of lesions treated in our centre was located in the head of the pancreas (n=629, 66%), whereas cancers of the body and tail were found in 28% (n=261) and 6% (n=57) of cases, respectively. There were no significant differences in the proportions of tumours located in the head, body and tail of the pancreas over time (Popiela *et al*, 2007). However, other authors suggested some variation over time. Based on 43,946 cases of pancreatic cancer recorded between 1973 and 2002 in the Surveillance, Epidemiology, and End Results (SEER), Lau et al. found a 46% increase in the incidence of body/tail cancers (Lau *et al*, 2010). Reports from other geographic areas mostly failed to demonstrate any significant change in the prevalence of distally-located cancers.

Technical improvements in imaging methods and their wider accessibility should theoretically allow for an earlier diagnosis of pancreatic cancer leading to reduced proportions of advanced tumours and increased resectability rates. Surprisingly, most cohort studies failed to demonstrate any marked increase in the proportion of cancers at a lower stage (Cress *et al*, 2006; Janes *et al*, 1996; Niederhuber *et al*, 1995; Riall *et al*, 2006; Sener *et al*, 1999). The percentage of patients with stage IV tumours in our series, similarly to other reports, showed only a slight lowering trend. Although this phenomenon was accompanied by increasing resectability rates,

Temporal Trends in Pancreatic Cancer 81

The most important aspect of surgery-related trends in pancreatic cancer is associated with markedly decreasing postoperative mortality. We have observed similar changes in the early postoperative outcomes and long-term survival as those reported by other authors (Popiela *et al*, 2002b; Sierzega *et al*, 2006). In particular, a significant lowering trend of postoperative mortality rates was found from values exceeding 10% in the early eighties to

> 95% confidence interval Correlation coefficient r = -0,55

1970 1975 1980 1985 1990 1995 2000 2005 Year

Pancreatic surgery has reached a plateau in terms of long-term survival observed for patients with pancreatic cancer (Popiela *et al*, 2002a; Popiela *et al*, 2002c). Therefore, further improvements should only be expected from combined modality therapy. IN our series, various regimens of chemotherapy were used in 38% (n=356) of cases and the percentage of patients qualified of systemic therapy increased significantly from 5% in period 1 to 55% in period 3. Fluorouracil was the primary chemotherapeutic agent until 1997 and afterwards

Although there is no uniform consensus on adjuvant therapy of pancreatic cancer, a recent meta-analysis of clinical trials have supported the benefits of chemotherapy found in our study (Stocken *et al*, 2005). The most recent analysis of data from the SEER registry in 1910 patients who underwent resections for pancreatic adenocarcinoma performed between 1991 and 2002 reflects the overall change in the proportion of patients qualified for systemic treatment after surgical intervention (Simons *et al*, 2010). The proportion of subjects receiving adjuvant chemoradiotherapy in US increased from 26% in 1991–1993 to 37% in 2000-2002. The role of palliative chemotherapy is also increasing as demonstrated in a recent

Fig. 1. Postoperative mortality rates for pancreatic resections in consecutive years

was replaced by multidrug regimens based on gemcitabine, cisplatin and irinotecan.


**6. Trends in systemic therapy** 

Postoperative mortality

an average of 4.1% in the last decade (fig. 1) (Popiela, 1979; Popiela *et al*, 2004).

the proportion of stage groups in patients undergoing pancreatic resections remained stable. Similarly to our findings, other authors reported only slight variations in staging patients with pancreatic cancer. In a population of 2986 cases of pancreatic cancer from the Digestive Cancer Registry of Burgundy (France) over a 30-year period (1976–2005) the overall proportion of stage I, II and III tumours was 1.3%, 2.2% and 5.4%, respectively (David *et al*, 2009). The proportion of stage I–II cancers slightly increased from 2.8% in the 1976–1980 period to 8.8% in the 2001–2005 period, though these changes were highly significant (P<0.001). Nevertheless, metastatic and/or non-resected cases decreased only by about 10% from 95.2% to 85.5%. The increasing trend of resectability rates found in this study was also confirmed by authors from various geographic areas. In a group of 16,758 patients treated between 1980 and 2000 in Sweden, the proportion of resectable tumours observed by Linder et al. increased from 7.2% to 15.1% (Linder *et al*, 2006). A similar trend was reported for the US population in a recent study involving 24,016 patients (Riall *et al*, 2006).

#### **5. Surgical trends**

Improved diagnostic methods increased the percentage of patients diagnosed with metastatic disease before surgery. Simultaneous development of endoscopic methods allows to perform biliary or duodenal stenting, and along with better imaging tests, has contributed to the decreasing rates of open surgery in patients with disseminated disease (Lefebvre *et al*, 2009).

Two hundred and twenty-six of 947 analysed patients (24%) were disqualified from surgical intervention. The remaining 721 (76%) patients were subject to surgical therapy and this proportion decreased insignificantly from 81% to 74% in the last decade. Pancreatic resections were performed in 172 (24%) patients and the resectability rate increased significantly from 9% to 31% between period 1 and 3, respectively. No significant changes in the type of pancreatic resections could be demonstrated for the whole cohort. However, an increasing proportion of pylorus-preserving pancreaticoduodenectomy (Traverso procedure) from 0% to 23% in the last period was found for lesions located in the head of the pancreas. The percentage of patients undergoing only exploratory laparotomy was stable over time with a mean value of 33%. The proportion of patients with biliary bypass significantly decreased with a concomitant increase in the ratio of gastro-enteric and double bypass procedures.

A gradual increase in endoscopic procedures is commonly reported in most reports. Linder et al. reported a significant reduction in the proportion of patient subject to surgical biliary bypass from 45.9% between 1980 and 1986 to 18.1% between 1994 and 2000 (Linder *et al*, 2006). These changes were accompanied by a lowering percentage of gastro-enteric bypass from 33.8% to 22.8%. Lefebvre et al. reported a similar decreasing trend for palliative surgery from 55% in 1978–1982 to 32% in 1998–2002 due to the more common use of endoscopic stenting (Lefebvre *et al*, 2009). We have found analogical variations in biliary bypass from 52% to 7%, but as opposed to Linder et al. the percentage of gastro-enteric and simultaneous biliary and enteric bypasses increased from 4% to 26% and from 8% to 36%, respectively. This change in the therapeutic strategy was related mainly to our analysis of patients requiring open surgery for upper gastrointestinal ileus and results of randomized clinical trials supporting the idea of prophylactic gastro-enteric bypass (Lillemoe *et al*, 1999; Popiela *et al*, 2002b; Van Heek *et al*, 2003). The increasing proportion of pylorus-preserving pancreaticoduodenectomy observed in the last decade for patients undergoing pancreatic head resections reflects the current belief that the procedure does not impair oncological radicality and, as suggest some authors, reduces adverse metabolic consequences of pancreatic resections (Schafer *et al*, 2002).

the proportion of stage groups in patients undergoing pancreatic resections remained stable. Similarly to our findings, other authors reported only slight variations in staging patients with pancreatic cancer. In a population of 2986 cases of pancreatic cancer from the Digestive Cancer Registry of Burgundy (France) over a 30-year period (1976–2005) the overall proportion of stage I, II and III tumours was 1.3%, 2.2% and 5.4%, respectively (David *et al*, 2009). The proportion of stage I–II cancers slightly increased from 2.8% in the 1976–1980 period to 8.8% in the 2001–2005 period, though these changes were highly significant (P<0.001). Nevertheless, metastatic and/or non-resected cases decreased only by about 10% from 95.2% to 85.5%. The increasing trend of resectability rates found in this study was also confirmed by authors from various geographic areas. In a group of 16,758 patients treated between 1980 and 2000 in Sweden, the proportion of resectable tumours observed by Linder et al. increased from 7.2% to 15.1% (Linder *et al*, 2006). A similar trend was reported for the US population in a recent study

Improved diagnostic methods increased the percentage of patients diagnosed with metastatic disease before surgery. Simultaneous development of endoscopic methods allows to perform biliary or duodenal stenting, and along with better imaging tests, has contributed to the decreasing rates of open surgery in patients with disseminated disease (Lefebvre *et al*, 2009). Two hundred and twenty-six of 947 analysed patients (24%) were disqualified from surgical intervention. The remaining 721 (76%) patients were subject to surgical therapy and this proportion decreased insignificantly from 81% to 74% in the last decade. Pancreatic resections were performed in 172 (24%) patients and the resectability rate increased significantly from 9% to 31% between period 1 and 3, respectively. No significant changes in the type of pancreatic resections could be demonstrated for the whole cohort. However, an increasing proportion of pylorus-preserving pancreaticoduodenectomy (Traverso procedure) from 0% to 23% in the last period was found for lesions located in the head of the pancreas. The percentage of patients undergoing only exploratory laparotomy was stable over time with a mean value of 33%. The proportion of patients with biliary bypass significantly decreased with a concomitant increase

A gradual increase in endoscopic procedures is commonly reported in most reports. Linder et al. reported a significant reduction in the proportion of patient subject to surgical biliary bypass from 45.9% between 1980 and 1986 to 18.1% between 1994 and 2000 (Linder *et al*, 2006). These changes were accompanied by a lowering percentage of gastro-enteric bypass from 33.8% to 22.8%. Lefebvre et al. reported a similar decreasing trend for palliative surgery from 55% in 1978–1982 to 32% in 1998–2002 due to the more common use of endoscopic stenting (Lefebvre *et al*, 2009). We have found analogical variations in biliary bypass from 52% to 7%, but as opposed to Linder et al. the percentage of gastro-enteric and simultaneous biliary and enteric bypasses increased from 4% to 26% and from 8% to 36%, respectively. This change in the therapeutic strategy was related mainly to our analysis of patients requiring open surgery for upper gastrointestinal ileus and results of randomized clinical trials supporting the idea of prophylactic gastro-enteric bypass (Lillemoe *et al*, 1999; Popiela *et al*, 2002b; Van Heek *et al*, 2003). The increasing proportion of pylorus-preserving pancreaticoduodenectomy observed in the last decade for patients undergoing pancreatic head resections reflects the current belief that the procedure does not impair oncological radicality and, as suggest some authors,

reduces adverse metabolic consequences of pancreatic resections (Schafer *et al*, 2002).

involving 24,016 patients (Riall *et al*, 2006).

in the ratio of gastro-enteric and double bypass procedures.

**5. Surgical trends** 

The most important aspect of surgery-related trends in pancreatic cancer is associated with markedly decreasing postoperative mortality. We have observed similar changes in the early postoperative outcomes and long-term survival as those reported by other authors (Popiela *et al*, 2002b; Sierzega *et al*, 2006). In particular, a significant lowering trend of postoperative mortality rates was found from values exceeding 10% in the early eighties to an average of 4.1% in the last decade (fig. 1) (Popiela, 1979; Popiela *et al*, 2004).

Fig. 1. Postoperative mortality rates for pancreatic resections in consecutive years

#### **6. Trends in systemic therapy**

Pancreatic surgery has reached a plateau in terms of long-term survival observed for patients with pancreatic cancer (Popiela *et al*, 2002a; Popiela *et al*, 2002c). Therefore, further improvements should only be expected from combined modality therapy. IN our series, various regimens of chemotherapy were used in 38% (n=356) of cases and the percentage of patients qualified of systemic therapy increased significantly from 5% in period 1 to 55% in period 3. Fluorouracil was the primary chemotherapeutic agent until 1997 and afterwards was replaced by multidrug regimens based on gemcitabine, cisplatin and irinotecan.

Although there is no uniform consensus on adjuvant therapy of pancreatic cancer, a recent meta-analysis of clinical trials have supported the benefits of chemotherapy found in our study (Stocken *et al*, 2005). The most recent analysis of data from the SEER registry in 1910 patients who underwent resections for pancreatic adenocarcinoma performed between 1991 and 2002 reflects the overall change in the proportion of patients qualified for systemic treatment after surgical intervention (Simons *et al*, 2010). The proportion of subjects receiving adjuvant chemoradiotherapy in US increased from 26% in 1991–1993 to 37% in 2000-2002. The role of palliative chemotherapy is also increasing as demonstrated in a recent

Temporal Trends in Pancreatic Cancer 83

months and 29.4%, respectively. The corresponding duration of median survival for microscopically (R1) and macroscopically (R2) non-radical resections of 11.4 and 11 months was significantly shorter. No patient survived 5 years after either R1 or R2 resection. A significant increasing trend for overall survival was found between period 1 and 3 (fig. 3, tab. 1). The correlation coefficient for the median survival of patients treated in consecutive years was 0.59 and this increasing trend was statistically significant (fig. 4). The median survival of patients undergoing pancreatic resections during the last decade (20 months, 95%CI 13.7 to 26.3) was significantly longer than for the period 1984-1993 (14.3 months, 95%CI 11.2 to 17.4). However, the differences between median survival during 1972-1983 and 1984-1993 or 1972-1983 and 1994-2003 were statistically insignificant (fig. 5). Nevertheless, the median survival of patients with unresectable tumours increased

Long-term results in patients treated for pancreatic cancer demonstrate only slight variations over the last 30 years with 5-year survival rates of 1-3% (Gudjonsson, 1995; Lillemoe *et al*, 2000; Tsiotos *et al*, 1999). Nevertheless, the number of reports describing improving survival trends is growing (Riall *et al*, 2006; Wood *et al*, 2006). The 2.4-month increase in the overall median survival found in our patients was due to improvements observed in both resectable (from 14.3 months between 1984 and 1993 to 20 months between 1994 and 2003), and unresectable cases (from 5 months between 1972 and 1983 to 5.9 months in the last decade). The relatively high median survival (26.6 months) in resectable patients treated in period 1 was related to the small number of cases (11 patients). In a recent publication of 1423 patients undergoing pancreaticoduodenectomy for pancreatic cancer, the median survival increased significantly from 8 months in the eighties to 19 months in patients operated after 2000 with comparable proportions of stage groups (Winter *et al*, 2006). A similar trend was also reported by Riall et al. in a large study on unresectable

> 0 6 9 15 21 27 33 39 45 51 57 63 Time (months)

 1994-2003 1984-1993 1972-1983

P<0,001 P<0,001

significantly between consecutive periods (fig. 6).

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0

Fig. 3. Overall survival functions for individual time periods

Cumulative proportion surviving

meta-analysis of clinical trials, where chemotherapy significantly prolonged survival compared to symptomatic therapy (Yip *et al*, 2009). Another meta-analysis on gemcitabine combined with platinum agents provided additional proofs for combination therapy, as the concomitant use of both drugs significantly increased response rates and prolonged time to progression (Xie *et al*, 2006). Results of our studies showed that any gemcitabine based regimen of palliative chemotherapy produced better results than observed in control groups and the combination of gemcitabine and cisplatin was the most effective regimen (Popiela *et al*, 2001; Popiela *et al*, 2005). Increased rates of adjuvant and palliative chemotherapy have been reported by several authors (David *et al*, 2009; Lefebvre *et al*, 2009).

#### **7. Changes in prognosis**

The overall median survival in our series was 7.1 months (95%CI 6.6 to 7.6) and was significantly longer after pancreatic resections (median 14.8 months; 95%CI 11.5 to 16.9) than in the remaining cases (median 5.8 months; 95%CI 4.4 to 6.9). The overall 5-year survival rate was 4.5% and increased to 14.3% for resective cases. No patient with unresectable tumour survived 5 years from the time of diagnosis. Pairwise comparisons of survival functions demonstrated statistically significant differences between each stage according to UICC 1997 (fig. 2).

Fig. 2. Stage-specific survival for resectable pancreatic cancer

The 5-year survival rate for stage I was 50.3% with a median survival of 63.1 months (95%CI 7.6 to 118.6). Five-year survival rates of 16.5% and 5.9% for stage II and III, respectively, were significantly lower. Corresponding median survival times were 33.2 months (95%CI 21.0 to 45.5) and 20.1 months (95%CI 14.8 to 25.3). The median survival of patients with stage IV cancers was 6.2 months (95%CI 5.7-6.6) and no patient survived 5 years. The median and 5-year survival rates after curative resections (R0 according to UICC) were 27.9

meta-analysis of clinical trials, where chemotherapy significantly prolonged survival compared to symptomatic therapy (Yip *et al*, 2009). Another meta-analysis on gemcitabine combined with platinum agents provided additional proofs for combination therapy, as the concomitant use of both drugs significantly increased response rates and prolonged time to progression (Xie *et al*, 2006). Results of our studies showed that any gemcitabine based regimen of palliative chemotherapy produced better results than observed in control groups and the combination of gemcitabine and cisplatin was the most effective regimen (Popiela *et al*, 2001; Popiela *et al*, 2005). Increased rates of adjuvant and palliative chemotherapy have

The overall median survival in our series was 7.1 months (95%CI 6.6 to 7.6) and was significantly longer after pancreatic resections (median 14.8 months; 95%CI 11.5 to 16.9) than in the remaining cases (median 5.8 months; 95%CI 4.4 to 6.9). The overall 5-year survival rate was 4.5% and increased to 14.3% for resective cases. No patient with unresectable tumour survived 5 years from the time of diagnosis. Pairwise comparisons of survival functions demonstrated statistically significant differences between each stage according to

> I II III IV

P<0,05 P<0,05 P<0,05

0 6 9 15 21 27 33 39 45 51 57 63 Time (months)

The 5-year survival rate for stage I was 50.3% with a median survival of 63.1 months (95%CI 7.6 to 118.6). Five-year survival rates of 16.5% and 5.9% for stage II and III, respectively, were significantly lower. Corresponding median survival times were 33.2 months (95%CI 21.0 to 45.5) and 20.1 months (95%CI 14.8 to 25.3). The median survival of patients with stage IV cancers was 6.2 months (95%CI 5.7-6.6) and no patient survived 5 years. The median and 5-year survival rates after curative resections (R0 according to UICC) were 27.9

been reported by several authors (David *et al*, 2009; Lefebvre *et al*, 2009).

**7. Changes in prognosis** 

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0

Fig. 2. Stage-specific survival for resectable pancreatic cancer

Cumulative proportion surviving

UICC 1997 (fig. 2).

months and 29.4%, respectively. The corresponding duration of median survival for microscopically (R1) and macroscopically (R2) non-radical resections of 11.4 and 11 months was significantly shorter. No patient survived 5 years after either R1 or R2 resection. A significant increasing trend for overall survival was found between period 1 and 3 (fig. 3, tab. 1). The correlation coefficient for the median survival of patients treated in consecutive years was 0.59 and this increasing trend was statistically significant (fig. 4). The median survival of patients undergoing pancreatic resections during the last decade (20 months, 95%CI 13.7 to 26.3) was significantly longer than for the period 1984-1993 (14.3 months, 95%CI 11.2 to 17.4). However, the differences between median survival during 1972-1983 and 1984-1993 or 1972-1983 and 1994-2003 were statistically insignificant (fig. 5). Nevertheless, the median survival of patients with unresectable tumours increased significantly between consecutive periods (fig. 6).

Long-term results in patients treated for pancreatic cancer demonstrate only slight variations over the last 30 years with 5-year survival rates of 1-3% (Gudjonsson, 1995; Lillemoe *et al*, 2000; Tsiotos *et al*, 1999). Nevertheless, the number of reports describing improving survival trends is growing (Riall *et al*, 2006; Wood *et al*, 2006). The 2.4-month increase in the overall median survival found in our patients was due to improvements observed in both resectable (from 14.3 months between 1984 and 1993 to 20 months between 1994 and 2003), and unresectable cases (from 5 months between 1972 and 1983 to 5.9 months in the last decade). The relatively high median survival (26.6 months) in resectable patients treated in period 1 was related to the small number of cases (11 patients). In a recent publication of 1423 patients undergoing pancreaticoduodenectomy for pancreatic cancer, the median survival increased significantly from 8 months in the eighties to 19 months in patients operated after 2000 with comparable proportions of stage groups (Winter *et al*, 2006). A similar trend was also reported by Riall et al. in a large study on unresectable

Fig. 3. Overall survival functions for individual time periods

Temporal Trends in Pancreatic Cancer 85

 1994-2003 1984-1993 1972-1993 P<0,001

P<0,001

0 3 6 6 9 12 15 18 21 24 27 30 33 Time (months)

pancreatic cancer (Riall *et al*, 2006). In 12043 cases of disseminated disease, the proportion of patients who survived 2 years increased from 1.4% between 1988 and 1991 to 2.3% between 1996 and 1999. A recent analysis of the SEER database showed a similar improving trend for overall survival in patients with pancreatic cancer (Lau *et al*, 2010). The overall 3-year survival rate increased from 4.3% to 6.2% from 1973 to 1987 to 1988 to 2002 for tumours of the pancreatic head and from 2.8% to 3.9% in pancreatic body/tail cancers. Similar observations were reported from the South Australian Cancer Registry covering the period from 1977 to 2006 with 4,166 pancreatic cancers (Luke *et al*, 2009) and 21,663 patients from

The analysis of 947 patients with pancreatic cancer treated between 1972 and 2003 demonstrated the existence of significant trends mainly for early postoperative and long-term outcomes. Postoperative mortality rates significantly decreased from values exceeding 10% in the early eighties to an average of 4.1% in the last decade. The overall median survival increased from 5.2 to 7.6 months and this change was reflected by improving outcomes in both resectable and unresectable disease. The observed changes are attributed mainly to the increasing role of combined therapy and emphasise the importance of such an approach.

Even with easily accessible imaging tests, the majority of patients with either cancer is still diagnosed at an advanced stage. Therefore, improved diagnostic procedures at the level of prehospital care are the key for actual improvement of patients' survival. Primary care physicians and specialists diagnosing patients with obstructive jaundice are of particular importance since endoscopic procedures commonly performed by gastroenterologists cannot be regarded as

curative therapy and the need for surgical exploration should always be considered.

Fig. 6. Changes in median survival in consecutive years for unresectable tumours

the Cancer Registry of Norway for the period 1965–2007 (Soreide *et al*, 2010).

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0

**8. Conclusion** 

Cumulative proportion surviving

Fig. 4. Changes in median survival in consecutive years

Fig. 5. Changes in survival in consecutive years for resectable tumours

1970 1975 1980 1985 1990 1995 2000 2005 Year

> 1994-2003 1984-1993 1972-1983

P<0,04 NS

0 6 9 15 21 27 33 39 45 51 57 63 Time (months)

Fig. 5. Changes in survival in consecutive years for resectable tumours

3

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0

Cumulative proportion surviving

4

5

6

7

Median survival (months)

8

9

10

95% confidence interval Correlation coefficient r = 0.59

Fig. 4. Changes in median survival in consecutive years

11

Fig. 6. Changes in median survival in consecutive years for unresectable tumours

pancreatic cancer (Riall *et al*, 2006). In 12043 cases of disseminated disease, the proportion of patients who survived 2 years increased from 1.4% between 1988 and 1991 to 2.3% between 1996 and 1999. A recent analysis of the SEER database showed a similar improving trend for overall survival in patients with pancreatic cancer (Lau *et al*, 2010). The overall 3-year survival rate increased from 4.3% to 6.2% from 1973 to 1987 to 1988 to 2002 for tumours of the pancreatic head and from 2.8% to 3.9% in pancreatic body/tail cancers. Similar observations were reported from the South Australian Cancer Registry covering the period from 1977 to 2006 with 4,166 pancreatic cancers (Luke *et al*, 2009) and 21,663 patients from the Cancer Registry of Norway for the period 1965–2007 (Soreide *et al*, 2010).

#### **8. Conclusion**

The analysis of 947 patients with pancreatic cancer treated between 1972 and 2003 demonstrated the existence of significant trends mainly for early postoperative and long-term outcomes. Postoperative mortality rates significantly decreased from values exceeding 10% in the early eighties to an average of 4.1% in the last decade. The overall median survival increased from 5.2 to 7.6 months and this change was reflected by improving outcomes in both resectable and unresectable disease. The observed changes are attributed mainly to the increasing role of combined therapy and emphasise the importance of such an approach.

Even with easily accessible imaging tests, the majority of patients with either cancer is still diagnosed at an advanced stage. Therefore, improved diagnostic procedures at the level of prehospital care are the key for actual improvement of patients' survival. Primary care physicians and specialists diagnosing patients with obstructive jaundice are of particular importance since endoscopic procedures commonly performed by gastroenterologists cannot be regarded as curative therapy and the need for surgical exploration should always be considered.

Temporal Trends in Pancreatic Cancer 87

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Niederhuber JE, Brennan MF, Menck HR (1995) The National Cancer Data Base report on

Pedrazzoli S, Beger HG, Obertop H, Andren-Sandberg A, Fernandez-Cruz L, Henne-Bruns

Popiela T (1979) Early detection and surgical treatment of pancreatic cancer. *Pol Przeg Chir*

Popiela T, Kedra B, Sierzega M (2001) Efficacy of gemcitabine in patients wit non-resectable pancreatic cancer: prospective clinical study. *Nowotwory* 51: 117-121 Popiela T, Kedra B, Sierzega M (2002a) Does extended lymphadenectomy improve survival

Popiela T, Kedra B, Sierzega M, Gurda A (2004) Risk factors of pancreatic fistula following

Popiela T, Kedra B, Sierzega M, Kubisz A (2002b) Chirurgisch palliative Therapie beim

Popiela T, Kedra B, Sierzega M, Kubisz A (2002c) Patienten mit nicht-fortgeschrittenen

Popiela T, Kulig J, Sierzega M, Legutko J (2005) A prospective randomized trial on two

Popiela T, Kulig J, Sierzega M, Richter P, Legutko J (2007) Temporal trends of pancreatic

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Pankreaskarzinomen profitieren von der ausgedehnten Lymphadenektomie.

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cancer and cancer of the ampulla of Vater treated between 1972 and 2003.

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Michaud DS (2004) Epidemiology of pancreatic cancer. *Minerva Chir* 59(2): 99-111

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**9. References** 


**6**

*Spain* 

**Current Perspectives and Future**

**Advanced Pancreatic Carcinoma** 

Purificacion Estevez-Garcia and Rocio Garcia-Carbonero

Pancreatic carcinoma is one of the most lethal solid tumors, with particularly high mortalityto-incidence rates. Indeed, about 278,684 people were diagnosed worldwide of pancreatic cancer in 2008, of whom 266,669 dyed from the disease in the same year (Ferlay et al, 2010). The greatest impact is observed in developed countries were pancreatic cancer has become

Pancreatic ductal adenocarcinoma represents more than 90% of pancreatic malignancies. The majority arise in the head, neck or uncinate process (60-70%), being less commonly encountered in the body (5-10%) or tail (10-15%) of the gland (Solcia et al, 1997). Clinical presentation is often related to the location of the primary tumor within the gland, although many patients often undergo an initial period of nonspecific symptoms such as back pain or vague gastrointestinal distress. Jaundice may be a relatively early symptom for tumors located in the head or uncinate process of the pancreas. However, left-sided pancreatic tumors may remain asymptomatic for long periods of time. Other associated disorders include acute pancreatitis or diabetes mellitus, and when they develop in patients without risk factors or in conjunction with other associated symptoms such as pain, anorexia or weight loss, the possibility of an underlying malignancy should be considered. Thromboembolic complications are also very common and are associated with a poor prognosis, with an incidence ranging from 17% to 57% (Khorana & Fine, 2004). Anorexia, weight loss or gastric outlet obstruction generally occur late in the course of the disease. Nevertheless, even early symptoms in this tumor are usually indicative of

Clinical features of pancreatic adenocarcinoma translate its extremely high propensity for local invasion and distant spread, underscoring the great difficulty to obtain an early diagnosis. In fact, more than 70% of patients present with unresectable, locally advanced or metastatic disease at the time of diagnosis (Stathis & Moore, 2010), and 70-80% of resected tumors will eventually relapse following surgery. Once the tumor has progressed beyond

the fourth leading cause of cancer-related death (Jemal et al, 2010).

**1. Introduction** 

advanced disease.

**Trends of Systemic Therapy in** 

*GI Oncology Unit, Medical Oncology Department,* 

*Instituto de Biomedicina de Sevilla (IBIS), Seville,* 

*Virgen del Rocio University Hospital,* 


### **Current Perspectives and Future Trends of Systemic Therapy in Advanced Pancreatic Carcinoma**

Purificacion Estevez-Garcia and Rocio Garcia-Carbonero *GI Oncology Unit, Medical Oncology Department, Virgen del Rocio University Hospital, Instituto de Biomedicina de Sevilla (IBIS), Seville, Spain* 

#### **1. Introduction**

88 Pancreatic Cancer – Clinical Management

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Simon B, Printz H (2001) Epidemiological trends in pancreatic neoplasias. *Dig Dis* 19(1): 6-14 Simons JP, Ng SC, McDade TP, Zhou Z, Earle CC, Tseng JF (2010) Progress for resectable

Sohn TA, Yeo CJ, Cameron JL, Lillemoe KD, Talamini MA, Hruban RH, Sauter PK, Coleman

Soreide K, Aagnes B, Moller B, Westgaard A, Bray F (2010) Epidemiology of pancreatic

Stocken DD, Buchler MW, Dervenis C, Bassi C, Jeekel H, Klinkenbijl JH, Bakkevold KE,

Tsiotos GG, Farnell MB, Sarr MG (1999) Are the results of pancreatectomy for pancreatic

Van Heek NT, De Castro SM, van Eijck CH, van Geenen RC, Hesselink EJ, Breslau PJ, Tran

Winter JM, Cameron JL, Campbell KA, Arnold MA, Chang DC, Coleman J, Hodgin MB,

Wood HE, Gupta S, Kang JY, Quinn MJ, Maxwell JD, Mudan S, Majeed A (2006) Pancreatic

Xie DR, Liang HL, Wang Y, Guo SS, Yang Q (2006) Meta-analysis on inoperable pancreatic

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and mortality. *Aliment Pharmacol Ther* 23(8): 1205-14

gemcitabine alone. *World J Gastroenterol* 12(43): 6973-81

experience. *J Gastrointest Surg* 10(9): 1199-210; discussion 1210-1

treatment and survival trends for 100,313 patients diagnosed from 1985-1995, using

nodes is an independent prognostic factor in patients with node-positive pancreatic

pancreatic [corrected] cancer?: a population-based assessment of US practices.

J, Ord SE, Grochow LB, Abrams RA, Pitt HA (1998) Should pancreaticoduodenectomy be performed in octogenarians? *J Gastrointest Surg* 2(3):

cancer in Norway: trends in incidence, basis of diagnosis and survival 1965-2007.

Takada T, Amano H, Neoptolemos JP (2005) Meta-analysis of randomised adjuvant

TC, Kazemier G, Visser MR, Busch OR, Obertop H, Gouma DJ (2003) The need for a prophylactic gastrojejunostomy for unresectable periampullary cancer: a prospective randomized multicenter trial with special focus on assessment of

Sauter PK, Hruban RH, Riall TS, Schulick RD, Choti MA, Lillemoe KD, Yeo CJ (2006) 1423 pancreaticoduodenectomies for pancreatic cancer: A single-institution

cancer in England and Wales 1975-2000: patterns and trends in incidence, survival

cancer: a comparison between gemcitabine-based combination therapy and

radiotherapy for inoperable advanced pancreatic cancer. *Cochrane Database Syst* 

pancreatic cancer and chronic pancreatitis. *Ann Surg* 236(2): 137-48

the National Cancer Database. *J Am Coll Surg* 189(1): 1-7

head cancer. *Pancreas* 33(3): 240-5

*Scand J Gastroenterol* 45(1): 82-92

cancer improving? *World J Surg* 23(9): 913-9

*Cancer* 116(7): 1681-90

*Rev*(4): CD002093

207-16

Pancreatic carcinoma is one of the most lethal solid tumors, with particularly high mortalityto-incidence rates. Indeed, about 278,684 people were diagnosed worldwide of pancreatic cancer in 2008, of whom 266,669 dyed from the disease in the same year (Ferlay et al, 2010). The greatest impact is observed in developed countries were pancreatic cancer has become the fourth leading cause of cancer-related death (Jemal et al, 2010).

Pancreatic ductal adenocarcinoma represents more than 90% of pancreatic malignancies. The majority arise in the head, neck or uncinate process (60-70%), being less commonly encountered in the body (5-10%) or tail (10-15%) of the gland (Solcia et al, 1997). Clinical presentation is often related to the location of the primary tumor within the gland, although many patients often undergo an initial period of nonspecific symptoms such as back pain or vague gastrointestinal distress. Jaundice may be a relatively early symptom for tumors located in the head or uncinate process of the pancreas. However, left-sided pancreatic tumors may remain asymptomatic for long periods of time. Other associated disorders include acute pancreatitis or diabetes mellitus, and when they develop in patients without risk factors or in conjunction with other associated symptoms such as pain, anorexia or weight loss, the possibility of an underlying malignancy should be considered. Thromboembolic complications are also very common and are associated with a poor prognosis, with an incidence ranging from 17% to 57% (Khorana & Fine, 2004). Anorexia, weight loss or gastric outlet obstruction generally occur late in the course of the disease. Nevertheless, even early symptoms in this tumor are usually indicative of advanced disease.

Clinical features of pancreatic adenocarcinoma translate its extremely high propensity for local invasion and distant spread, underscoring the great difficulty to obtain an early diagnosis. In fact, more than 70% of patients present with unresectable, locally advanced or metastatic disease at the time of diagnosis (Stathis & Moore, 2010), and 70-80% of resected tumors will eventually relapse following surgery. Once the tumor has progressed beyond

Current Perspectives and Future Trends of

**2.2 Combination chemotherapy** 

(Table 1).

Systemic Therapy in Advanced Pancreatic Carcinoma 91

Conversely, preclinical data had suggested a dose-response relationship independent of infusion duration. In light of these data, a randomized phase II trial conducted in 92 pancreatic cancer patients was designed to assess the efficacy of two dose-intense schedules of gemcitabine: a dose-intense schedule administering gemcitabine as a standard 30-minute infusion (2200 mg/2/week) versus gemcitabine administered at a fixed dose rate (FDR) of 10 mg/m2/min (1500 mg/m2/week 150-minute infusion) (Gelibter et al, 2005; Tempero et al, 2003). Patients in the FDR infusion arm experienced increased survival rates (18% vs 2% at 2 years, p=.007), consistent with the higher intracellular gemcitabine triphosphate concentrations observed in these patients, although at the expense of increased hematologic toxicity. However, a confirmatory phase III trial failed to confirm a survival advantage for

Although the benefit of chemotherapy in patients with advanced pancreatic cancer is well established, the magnitude of the effect is rather small, with an absolute improvement of survival at 5 years of 3% to 6% (survival rates from 1975-77 to 1999-2005) (Oberstein & Saif, 2011). Over the past decade, multiple randomized trials have been performed to assess a number of gemcitabine-combination chemotherapy regimens in an effort to improve these modest results. These have included combinations with 5-FU (Berlin et al, 2002; Riess et al, 2005), capecitabine (Herrmann et al, 2007; Bernhard et al, 2008; Cunningham et al, 2009), cisplatin (Heinemann et al, 2006; Colucci et al, 2002, 2009), oxaliplatin (Louvet et al, 2005; Poplin et al, 2009), irinotecan (Rocha et al, 2004; Stathopoulos et al, 2006), exatecan (Abou Alfa et al, 2006) and pemetrexed (Oettle et al, 2005a). Individually, although many of these studies observed some improvement in terms of response rate and progression free survival favoring combination therapy, the great majority failed to demonstrate a survival benefit

The largest and most recent meta-analysis, however, confirm a modest although significant benefit in survival for gemcitabine combinations over gemcitabine alone (HR 0.91; 95%CI: 0.85 to 0.97; p=0.004) in patients with locally advanced or metastatic pancreatic cancer (Sultana et al, 2007; Heinemann et al, 2008b). The magnitude of this benefit was remarkably greater (HR 0.76; 95%CI: 0.67 to 0.87; p<0.0001) in patients with good performance status (representing 38% of all patients included in the meta-analysis). In subgroup analysis, platinum compounds (3 trials, 1077 patients; HR 0.85; 95%CI 0.74-0.96) and capecitabine (3 trials, 935 patients; HR 0.83; 95%CI 0.72-0.96) in combination with gemcitabine consistently showed improved survival over single-agent gemcitabine. Insufficient evidence was observed, nevertheless, to support combination of gemcitabine with 5FU or irinotecan.

The rationale for the combined use of gemcitabine and cisplatin is based on the preclinical evidence that gemcitabine not only increases cisplatin-induced DNA cross links, but also effectively inhibits their repair, and cisplatin, on the other hand, enhances the incorporation of gemcitabine triphosphate into DNA. In vitro studies show synergistic cytotoxicity and several non-controlled clinical studies suggested improved efficacy. Some early randomized studies observed increased response rates and progression free survival for patients treated with the cisplatin-gemcitabine combination as compared to those treated with gemcitabine alone (Colucci et al, 2002; Heinemann et al, 2006), with a non-significant trend towards a longer survival. However, more recent and larger trials have failed to confirm a significant

the FDR regimen over the standard administration (Poplin et al, 2009).

surgical resectability, prognosis is rather poor, with median survival ranging from 6 to 9 months and 5-year overall survival rates of less than 5% (National Cancer Institute, 2010; Jemal et al, 2008).

In recent years there has been only minimal progress in the systemic treatment of metastatic pancreatic cancer. Current standard therapies have a limited impact on the natural history of this disease and improvements in systemic therapy are desperately needed in order to improve the prognosis of these patients. However, intense translational and clinical research has lead to a better and deeper understanding of the complex molecular biology of this tumor and shall help improve the development of new more effective drugs in this disease.

#### **2. Conventional cytotoxic therapy**

#### **2.1 Monotherapy**

Early randomized trials demonstrated that several 5-fluorouracil (5FU)-based combination chemotherapy regimens improved survival (hazard ratio [HR] = 0.64; 95%CI, 0.42 to 0.98) and quality of life of patients with advanced pancreatic cancer over best supportive care (BSC) alone (Sultana et al, 2007). Subsequent studies showed, however, that 5FU-based combination therapy did not result in better overall survival compared with 5FU alone (HR = 0.94; 95% CI, 0.82 to 1.08). 5FU monotherapy became, consequently, the standard of care for pancreatic cancer. Reported response rates widely ranged from 0% to 19% (Evans et al, 1997), partly due to the lack of standardized criteria to assess response in these early trials, with median survival times of 4.2 to 5.5 months (Burris et al, 1997).

During the 1990s several non-controlled trials suggested some promising activity of a new drug in pancreatic cancer, gemcitabine. The pivotal study by Burris et al was responsible for the change in practice from 5FU to gemcitabine based on a marginal survival advantage and an improvement in clinical benefit response favoring gemcitabine-treated patients. This trial enrolled 126 patients with chemotherapy-naïve advanced symptomatic pancreatic cancer who were randomly allocated to receive gemcitabine (1000 mg/m2/week x 7 followed by 1 week of rest, and then weekly x 3 every 4 weeks) or 5-FU (600 mg/m2/week) until disease progression, clinical deterioration or unacceptable toxicity (Burris et al, 1997). The primary efficacy outcome was clinical benefit response (CBR), a term introduced for the first time in this trial, which was a composite of measurements of pain (analgesic consumption and pain intensity), Karnofsky performance status and weight. No statistically significant difference was found between study arms in terms of objective response (gemcitabine 5.4% vs 5-FU 0%), but patients in the gemcitabine arm experienced improved CBR (24% vs 5%) and overall survival (5.65 months vs 4.41 months, p=0.0025), with 1-year survival rates also favoring gemcitabine-treated patients (18% vs 2%).

Further trials aimed to optimize gemcitabine administration schedule. Gemcitabine (difluorodeoxycytidine) is a nucleoside analogue capable of inhibiting ribonucleotide reductase to deplete nucleoside pools, and its phosphorylated metabolite is incorporated into DNA causing chain termination and inhibition of DNA synthesis, function and repair. Phosphorylation of gemcitabine to the monophosphate by deoxycytidine kinase is the ratelimiting step in the accumulation of the active diphosphate and triphosphate metabolites. Some early clinical studies observed the rate of gemcitabine triphosphate accumulation by mononuclear cells and leukemia cells was optimized using dose rates of 10 mg/m2/min.

surgical resectability, prognosis is rather poor, with median survival ranging from 6 to 9 months and 5-year overall survival rates of less than 5% (National Cancer Institute, 2010;

In recent years there has been only minimal progress in the systemic treatment of metastatic pancreatic cancer. Current standard therapies have a limited impact on the natural history of this disease and improvements in systemic therapy are desperately needed in order to improve the prognosis of these patients. However, intense translational and clinical research has lead to a better and deeper understanding of the complex molecular biology of this tumor and shall help improve the development of new more effective drugs in this disease.

Early randomized trials demonstrated that several 5-fluorouracil (5FU)-based combination chemotherapy regimens improved survival (hazard ratio [HR] = 0.64; 95%CI, 0.42 to 0.98) and quality of life of patients with advanced pancreatic cancer over best supportive care (BSC) alone (Sultana et al, 2007). Subsequent studies showed, however, that 5FU-based combination therapy did not result in better overall survival compared with 5FU alone (HR = 0.94; 95% CI, 0.82 to 1.08). 5FU monotherapy became, consequently, the standard of care for pancreatic cancer. Reported response rates widely ranged from 0% to 19% (Evans et al, 1997), partly due to the lack of standardized criteria to assess response in these early trials,

During the 1990s several non-controlled trials suggested some promising activity of a new drug in pancreatic cancer, gemcitabine. The pivotal study by Burris et al was responsible for the change in practice from 5FU to gemcitabine based on a marginal survival advantage and an improvement in clinical benefit response favoring gemcitabine-treated patients. This trial enrolled 126 patients with chemotherapy-naïve advanced symptomatic pancreatic cancer who were randomly allocated to receive gemcitabine (1000 mg/m2/week x 7 followed by 1 week of rest, and then weekly x 3 every 4 weeks) or 5-FU (600 mg/m2/week) until disease progression, clinical deterioration or unacceptable toxicity (Burris et al, 1997). The primary efficacy outcome was clinical benefit response (CBR), a term introduced for the first time in this trial, which was a composite of measurements of pain (analgesic consumption and pain intensity), Karnofsky performance status and weight. No statistically significant difference was found between study arms in terms of objective response (gemcitabine 5.4% vs 5-FU 0%), but patients in the gemcitabine arm experienced improved CBR (24% vs 5%) and overall survival (5.65 months vs 4.41 months, p=0.0025), with 1-year survival rates also

Further trials aimed to optimize gemcitabine administration schedule. Gemcitabine (difluorodeoxycytidine) is a nucleoside analogue capable of inhibiting ribonucleotide reductase to deplete nucleoside pools, and its phosphorylated metabolite is incorporated into DNA causing chain termination and inhibition of DNA synthesis, function and repair. Phosphorylation of gemcitabine to the monophosphate by deoxycytidine kinase is the ratelimiting step in the accumulation of the active diphosphate and triphosphate metabolites. Some early clinical studies observed the rate of gemcitabine triphosphate accumulation by mononuclear cells and leukemia cells was optimized using dose rates of 10 mg/m2/min.

with median survival times of 4.2 to 5.5 months (Burris et al, 1997).

favoring gemcitabine-treated patients (18% vs 2%).

Jemal et al, 2008).

**2.1 Monotherapy** 

**2. Conventional cytotoxic therapy** 

Conversely, preclinical data had suggested a dose-response relationship independent of infusion duration. In light of these data, a randomized phase II trial conducted in 92 pancreatic cancer patients was designed to assess the efficacy of two dose-intense schedules of gemcitabine: a dose-intense schedule administering gemcitabine as a standard 30-minute infusion (2200 mg/2/week) versus gemcitabine administered at a fixed dose rate (FDR) of 10 mg/m2/min (1500 mg/m2/week 150-minute infusion) (Gelibter et al, 2005; Tempero et al, 2003). Patients in the FDR infusion arm experienced increased survival rates (18% vs 2% at 2 years, p=.007), consistent with the higher intracellular gemcitabine triphosphate concentrations observed in these patients, although at the expense of increased hematologic toxicity. However, a confirmatory phase III trial failed to confirm a survival advantage for the FDR regimen over the standard administration (Poplin et al, 2009).

#### **2.2 Combination chemotherapy**

Although the benefit of chemotherapy in patients with advanced pancreatic cancer is well established, the magnitude of the effect is rather small, with an absolute improvement of survival at 5 years of 3% to 6% (survival rates from 1975-77 to 1999-2005) (Oberstein & Saif, 2011). Over the past decade, multiple randomized trials have been performed to assess a number of gemcitabine-combination chemotherapy regimens in an effort to improve these modest results. These have included combinations with 5-FU (Berlin et al, 2002; Riess et al, 2005), capecitabine (Herrmann et al, 2007; Bernhard et al, 2008; Cunningham et al, 2009), cisplatin (Heinemann et al, 2006; Colucci et al, 2002, 2009), oxaliplatin (Louvet et al, 2005; Poplin et al, 2009), irinotecan (Rocha et al, 2004; Stathopoulos et al, 2006), exatecan (Abou Alfa et al, 2006) and pemetrexed (Oettle et al, 2005a). Individually, although many of these studies observed some improvement in terms of response rate and progression free survival favoring combination therapy, the great majority failed to demonstrate a survival benefit (Table 1).

The largest and most recent meta-analysis, however, confirm a modest although significant benefit in survival for gemcitabine combinations over gemcitabine alone (HR 0.91; 95%CI: 0.85 to 0.97; p=0.004) in patients with locally advanced or metastatic pancreatic cancer (Sultana et al, 2007; Heinemann et al, 2008b). The magnitude of this benefit was remarkably greater (HR 0.76; 95%CI: 0.67 to 0.87; p<0.0001) in patients with good performance status (representing 38% of all patients included in the meta-analysis). In subgroup analysis, platinum compounds (3 trials, 1077 patients; HR 0.85; 95%CI 0.74-0.96) and capecitabine (3 trials, 935 patients; HR 0.83; 95%CI 0.72-0.96) in combination with gemcitabine consistently showed improved survival over single-agent gemcitabine. Insufficient evidence was observed, nevertheless, to support combination of gemcitabine with 5FU or irinotecan.

The rationale for the combined use of gemcitabine and cisplatin is based on the preclinical evidence that gemcitabine not only increases cisplatin-induced DNA cross links, but also effectively inhibits their repair, and cisplatin, on the other hand, enhances the incorporation of gemcitabine triphosphate into DNA. In vitro studies show synergistic cytotoxicity and several non-controlled clinical studies suggested improved efficacy. Some early randomized studies observed increased response rates and progression free survival for patients treated with the cisplatin-gemcitabine combination as compared to those treated with gemcitabine alone (Colucci et al, 2002; Heinemann et al, 2006), with a non-significant trend towards a longer survival. However, more recent and larger trials have failed to confirm a significant

Current Perspectives and Future Trends of

benefits for the GEMOX regimen (Poplin et al, 2009).

Systemic Therapy in Advanced Pancreatic Carcinoma 93

however more commonly induced by the combination, particularly thrombocytopenia, emesis and neurotoxicity. More recently published trials, again, did not confirm these

Combination of gemcitabine plus capecitabine is the other cytotoxic chemotherapy doublet that has shown some advantage over gemcitabine alone. Two recent phase III studies consistently demonstrated a gain in terms of progression free survival (PFS) for the combination, although the benefit in overall survival (OS) only achieved statistical significance in the meta-analysis of these trials (Cunningham et al, 2009; Herrmann et al, 2007). Cunningham et al randomized 533 patients to receive gemcitabine (1000 mg/m2 in 30-min infusion weekly x 3 every 4 weeks) plus capecitabine (830 mg/m2/12 hours day 1-21 every 28 days) versus gemcitabine alone. Combination therapy obtained higher response rates (19.1% vs 12.4%, p=0.034) and PFS (5.3 vs 3.8 months; HR 0.78, 95% CI 0.66-0.93, p=0.004) and a trend toward better OS of borderline significance (7.1 vs 6.2 months; HR 0.86, 95% CI 0.72-1.02, p=0.08). Herrmann and colleagues randomized 319 patients to receive either gemcitabine (1000 mg/m2 days 1 and 8 every 21 days) plus capecitabine (650 mg/m2/12 hours days 1-14 every 21 days) or gemcitabine alone (1000 mg/m2 weekly for 7 weeks and one week off, and then weekly x 3 every 4 weeks). No significant differences were observed among study arms in terms of response rate, clinical benefit or quality of life (Bernhard et al, 2008), and the primary endpoint of the study, OS, was not reached (8.4 vs 7.2 months, p=0.234). However, post hoc analysis did show a significant survival advantage for the gemcitabine-capecitabine combination in patients with good performance status (10.1 vs 7.4 months, p=0.004). In both studies toxicity in the combination arm was tolerable, with a low incidence of grade 3-4 adverse events, being neutropenia and diarrhea the most commonly encountered toxicities. In light of these results, treatment with gemcitabine plus

capecitabine may be considered in fit patients with advanced pancreatic cancer.

Other multidrug combinations have also been investigated over the past years in several phase II-III trials, including PEFG (cisplatin, epirubicin, gemcitabine and 5-FU) (Reni et al, 2005), G-FLIP (irinotecan, gemcitabine, 5-FU, leucovorin and cisplatin) (Goel et al, 2007), and active schedules in other gastrointestinal cancers such as FOLFOX-6 (oxaliplatin, 5-FU and folinic acid) (Ghosn et al, 2007) or FOLFIRI.3 (irinotecan, 5-FU and folinic acid) (Taïeb et al, 2007). Increased tumor responses and progression free survival have been reported for some of these regimens (Reni et al, 2005), although at the expense of a worse toxicity profile with no impact on survival. However, the combination of Gemcitabine and *nab*-paclitaxel, an albumin-bound formulation of paclitaxel particles (Celgene, Summit, NJ), deserves special mention (Von Hoff et al, 2011). *nab*-Paclitaxel has shown antitumor activity in various advanced cancer types that overexpress the albumin-binding protein SPARC (secreted protein acidic and rich in cysteine), including breast, lung, and melanoma. Results of the phase I/II trial of this combination, with an overall response rate of 48%, a median survival of 12.2 months, and a 1-year survival rate of 48% at the MTD are among the highest ever reported for a phase II study in patients with advanced pancreatic cancer. Interestingly, SPARC expression in the stroma, but not in the tumor, was correlated with improved survival (median survival of 17.8 *v* 8.1 months for high- vs low- SPARC tumors, respectively; *P=* .0431), suggesting SPARC could be a potential new predictive biomarker of nab-paclitaxel activity. This promising results have prompted the conduction of a large international phase III study that is close to complete accrual. Also recently reported, results


5FU, 5-fluoruracil; GEM, gemcitabine; CAPE, capecitabine; CIS, cisplatin; OX, oxaliplatin; IRI, irinotecan; EXE, exatecan; PEM, pemetrexed; RR, response rate; PFS, progression free survival; OS, overall survival.

Table 1. Selected phase III trials of gemcitabine-based chemotherapy in advanced pancreatic cancer

impact on overall survival, whereas combination therapy was associated with greater hematological toxicity (Colucci et al, 2010). Similar findings have been observed with the combination of gemcitabine with oxaliplatin (GEMOX). GEMOX was superior to gemcitabine in terms of response rate (26.8% v 17.3%; p=0.04), progression-free survival (5.8 v 3.7 months; p=0.04), and clinical benefit (38.2% v 26.9%; p=0.03), with a trend for an improved survival (9.0 v 7.1 months, p=0.13) (Louvet et al, 2005). Severe toxicities were

533 12 vs 19

107 9.2 vs 26.4

400 10.1 vs 12.9 (p=0.37)

313 17.3 vs 26.8 (p=0.04)

832 6 vs 10 vs 9 (p=0.11)

145 10 vs 15

360 4.4 vs 16.1

565 7.1 vs 14.8

5FU, 5-fluoruracil; GEM, gemcitabine; CAPE, capecitabine; CIS, cisplatin; OX, oxaliplatin; IRI, irinotecan; EXE, exatecan; PEM, pemetrexed; RR, response rate; PFS, progression free survival; OS,

(p=0.39)

(p<0.001)

(p=0.004)

Table 1. Selected phase III trials of gemcitabine-based chemotherapy in advanced pancreatic

impact on overall survival, whereas combination therapy was associated with greater hematological toxicity (Colucci et al, 2010). Similar findings have been observed with the combination of gemcitabine with oxaliplatin (GEMOX). GEMOX was superior to gemcitabine in terms of response rate (26.8% v 17.3%; p=0.04), progression-free survival (5.8 v 3.7 months; p=0.04), and clinical benefit (38.2% v 26.9%; p=0.03), with a trend for an improved survival (9.0 v 7.1 months, p=0.13) (Louvet et al, 2005). Severe toxicities were

349 4.6 vs 6.3 3.8 vs 3.7

(p=0.034)

(p=0.02)

Response Rate (%)

327 5.6 vs 6.9 2.2 vs 3.4

319 7.8 vs 10 3.9 vs 4.3

195 8.2 vs 10.2 3.1 vs 5.3

PFS (months)

(p=0.022)

(p=0.103)

3.8 vs 5.3 (p=0.004)

1.8 vs 4.6 (p=0.048)

3.9 vs 3.8 (p=0.80)

(p=0.053)

3.7 vs 5.8 (p=0.04)

2.6 vs 3.5 (p=0.04) vs 2.7 (p=0.1)

2.8 vs 2.9 (p=0.79)

3 vs 3.5 (p=0.352)

3.3 vs 3.9 (p=0.11)

(p=0.22)

OS (months)

5.4 vs 6.7 (p=0.09)

7.2 vs 8.4 (p=0.234)

6.2 vs 7.1 (p=0.08)

5 vs 7.5 (p=0.43)

8.3 vs 7.2 (p=0.38)

6 vs 7.6 (p=0.15)

7.1 vs 9 (p=0.13)

4.9 vs 6.2 (p=0.04) vs 5.7 (p=0.22)

6.4 vs 6.5 (p=0.97)

6.6 vs 6.3 (p=0.789)

6.3 vs 6.2 (p=0.847)

6.2 vs 6.7 (p=0.52)

of patients

Reference Treatment Number

GEM vs GEM+5FU

GEM vs GEM+ CAP

GEM vs GEM+ CAP

GEM vs GEM+CIS

GEM vs GEM+CIS

GEM vs GEM+CIS

GEM vs GEM+OX

GEM vs GEM FDR GEM+OX

GEM vs GEM+IRI

GEM vs GEM+IRI

GEM vs GEM+ PEM

GEM vs GEM+EXA

Berlin *et al*  (2002)

Herrmann *et al* (2007)

Cunningh am *et al* (2009)

Colucci *et al* (2002)

Colucci et al (2010)

Heineman n *et al* (2006)

Louvet *et al* (2005)

Poplin *et al* (2009)

Stathopoul os *et al* (2006)

Rocha Lima *et al* (2004)

Oettle *et al* (2005a)

Abou Alfa *et al* (2006)

overall survival.

cancer

however more commonly induced by the combination, particularly thrombocytopenia, emesis and neurotoxicity. More recently published trials, again, did not confirm these benefits for the GEMOX regimen (Poplin et al, 2009).

Combination of gemcitabine plus capecitabine is the other cytotoxic chemotherapy doublet that has shown some advantage over gemcitabine alone. Two recent phase III studies consistently demonstrated a gain in terms of progression free survival (PFS) for the combination, although the benefit in overall survival (OS) only achieved statistical significance in the meta-analysis of these trials (Cunningham et al, 2009; Herrmann et al, 2007). Cunningham et al randomized 533 patients to receive gemcitabine (1000 mg/m2 in 30-min infusion weekly x 3 every 4 weeks) plus capecitabine (830 mg/m2/12 hours day 1-21 every 28 days) versus gemcitabine alone. Combination therapy obtained higher response rates (19.1% vs 12.4%, p=0.034) and PFS (5.3 vs 3.8 months; HR 0.78, 95% CI 0.66-0.93, p=0.004) and a trend toward better OS of borderline significance (7.1 vs 6.2 months; HR 0.86, 95% CI 0.72-1.02, p=0.08). Herrmann and colleagues randomized 319 patients to receive either gemcitabine (1000 mg/m2 days 1 and 8 every 21 days) plus capecitabine (650 mg/m2/12 hours days 1-14 every 21 days) or gemcitabine alone (1000 mg/m2 weekly for 7 weeks and one week off, and then weekly x 3 every 4 weeks). No significant differences were observed among study arms in terms of response rate, clinical benefit or quality of life (Bernhard et al, 2008), and the primary endpoint of the study, OS, was not reached (8.4 vs 7.2 months, p=0.234). However, post hoc analysis did show a significant survival advantage for the gemcitabine-capecitabine combination in patients with good performance status (10.1 vs 7.4 months, p=0.004). In both studies toxicity in the combination arm was tolerable, with a low incidence of grade 3-4 adverse events, being neutropenia and diarrhea the most commonly encountered toxicities. In light of these results, treatment with gemcitabine plus capecitabine may be considered in fit patients with advanced pancreatic cancer.

Other multidrug combinations have also been investigated over the past years in several phase II-III trials, including PEFG (cisplatin, epirubicin, gemcitabine and 5-FU) (Reni et al, 2005), G-FLIP (irinotecan, gemcitabine, 5-FU, leucovorin and cisplatin) (Goel et al, 2007), and active schedules in other gastrointestinal cancers such as FOLFOX-6 (oxaliplatin, 5-FU and folinic acid) (Ghosn et al, 2007) or FOLFIRI.3 (irinotecan, 5-FU and folinic acid) (Taïeb et al, 2007). Increased tumor responses and progression free survival have been reported for some of these regimens (Reni et al, 2005), although at the expense of a worse toxicity profile with no impact on survival. However, the combination of Gemcitabine and *nab*-paclitaxel, an albumin-bound formulation of paclitaxel particles (Celgene, Summit, NJ), deserves special mention (Von Hoff et al, 2011). *nab*-Paclitaxel has shown antitumor activity in various advanced cancer types that overexpress the albumin-binding protein SPARC (secreted protein acidic and rich in cysteine), including breast, lung, and melanoma. Results of the phase I/II trial of this combination, with an overall response rate of 48%, a median survival of 12.2 months, and a 1-year survival rate of 48% at the MTD are among the highest ever reported for a phase II study in patients with advanced pancreatic cancer. Interestingly, SPARC expression in the stroma, but not in the tumor, was correlated with improved survival (median survival of 17.8 *v* 8.1 months for high- vs low- SPARC tumors, respectively; *P=* .0431), suggesting SPARC could be a potential new predictive biomarker of nab-paclitaxel activity. This promising results have prompted the conduction of a large international phase III study that is close to complete accrual. Also recently reported, results

Current Perspectives and Future Trends of

OS (3.5 vs 3.1 months, respectively).

**3. Molecularly targeted therapies** 

(Poplin et al, 2009).

Systemic Therapy in Advanced Pancreatic Carcinoma 95

Combining gemcitabine and oxaliplatin (GEMOX) has been another commonly evaluated therapeutic schedule. Two small non-controlled trials investigated the efficacy of oxaliplatin plus fixed-dose rate gemcitabine in patients who had progressed on single agent gemcitabine. Although reported response rates were relevant (21-24% of partial responses), toxicity was not negligible, with up to half of the patients developing at least one grade 3 adverse event (Demols et al, 2006, as cited in Gounaris et al, 2010; Fortune el al, 2009, as cited in Gounaris et al, 2010). These results, together with the findings of the phase III E6201 conducted in chemotherapy-naïve patients failing to demonstrate a survival advantage for the combination, do not warrant further evaluation of this regimen in the second-line setting

Irinotecan has been tested both as single agent and in combination with oxaliplatin or fluoropyrimidines showing some activity and an acceptable toxicity profile (Yi et al, 2009; Cantore et al, 2004). A direct comparison between oxaliplatin- and irinotecan-based regimens was made by Hwang and colleagues in a small randomized phase II trial (Hwang et al, 2009). Sixty patients were enrolled and randomly allocated to receive FOLFOX (oxaliplatin, folinic acid and infusional 5FU) or FOLFIRI.3 (the same folinic acid and 5FU schedule combined with irinotecan) after gemcitabine failure. No significant differences were observed among study arms neither in PFS (1.4 vs 1.9 months, p>0.05) nor in OS (4 months both regimens). In light of these results, both regimens may be reasonable options for secondline therapy in appropriately selected patients with advanced pancreatic cancer. Other irinotecan-based regimens including combinations with raltitrexed (Ulrich-Pur, 2003, as cited in Gounaris, 2010), docetaxel (Ko et al, 2008), docetaxel and mitomycin C (Reni et al, 2004) or ifosfamide (Cereda et al, 2011) have not achieved positive results in small phase II trials.

Rubitecan, an orally bioavailable camptothecin derivative, was the subject of the largest study conducted in gemcitabine-resistant pancreatic cancer, despite results of an initial single arm study were not particularly encouraging (median TTP and OS of 1.9 and 3 months, respectively). Subsequently, a large phase III study was launched the results of which have only been reported in abstract form (Jacobs et al, 2004). Four-hundred and nine patients were randomized to receive treatment with rubitecan or physician's best choice (chemotherapy 89%, supportive care only 11%). There were more responses in the rubitecan arm (11% vs. 1%) and the difference in median PFS, although clinically modest, reached statistical significance (1.9 vs. 1.6 months). There was no significant difference however in

Other tested drugs in this setting, such as taxanes or pemetrexed, have not shown particularly promising results in small studies (Gounaris et al, 2010; Boeck et al, 2007b; Mazzer et al, 2009). Multidrug combinations such as PEFG (cisplatin, epirubicin, 5-FU and gemcitabine) (Reni et al, 2008, as cited in Gounaris et al, 2010) or G-FLIP (gemcitabine, irinotecan, folinic acid, 5-FU and cisplatin) (Kozuch et al, 2001, as cited in Gounaris, 2010) appear to show improved efficacy with impressive median survival of 8.3 and 10.3 months, respectively. Selection bias may at least partially explain these outstanding results as reported toxicity was rather high, which in any case would limit their use in the general population.

Pancreatic adenocarcinoma is a malignant disease that results from the successive accumulation of gene mutations (Vogelstein & Kinzsler, 2004) evolving from premalignant

of the PRODIGE 4/ACCORD 11 trial comparing gemcitabine alone (1000 mg/m2 weekly x 7 every 8 weeks and then weekly x 3 every 4 weeks) to FOLFIRINOX (oxaliplatin 85 mg/m2, irinotecan 180 mg/m2, 5-FU 400 mg/m2 given as a bolus followed by 2400 mg/m2 given as a 46-hour continuous infusion; and leucovorin 400 mg/m2; every 2 weeks) demonstrated remarkable and significant improvements in response, progression free and overall survival rates favoring patients treated with FOLFIRINOX (31% vs. 9%, 6.4 months *vs*. 3.3 months, and 11.1 months *vs*. 6.8 months, respectively) (Conroy, 2011). These results are somewhat surprising, given the known modest activity of each of the individual drugs included in the regimen, and shall be confirmed. In addition, the higher toxicity profile of this combination limits its widespread use as standard of care in patients with metastatic disease, often frail. However, it may be an excellent option for carefully selected patients, particularly those with locally advanced borderline resectable disease. Anyhow, this is the first phase III randomized trial that has demonstrated a benefit in overall survival of unquestionable clinical relevance for patients with advanced pancreatic cancer, and it may change the classical paradigm of gemcitabine as the keystone in the management of advanced pancreatic cancer.

#### **2.3 Gemcitabine-resistant disease**

Once the disease progresses to gemcinatine-based therapy there is no accepted standard of care and most patients will not be suitable candidates for further therapy due to clinical deterioration. Second-line chemotherapy may be considered, however, in patients who maintain good performance status, although efficacy in this setting is questionable. Overall, it is estimated that approximately 30% of patients are in good condition (including good performance status and adequate organ function) for consideration of second-line treatment (Gounaris et al, 2010). A number of trials have been performed assessing the efficacy of different antineoplastic agents in this context. Most of the published evidence, however, consists of small phase II studies testing a variety of drugs in a heterogeneous population.

Oxaliplatin-fluoropyrimidine doublets are probably the chemotherapy regimens most widely evaluated in gemcitabine-resistant disease. Several small phase II studies showed some promising activity with different combinations of oxaliplatin and 5FU or capecitabine (FOLFOX, OFF, XELOX,..), with median survival (6-7 months) that did not substantially differed from that observed in chemotherapy-naïve patients (Tsavaris et al, 2005; Xiong et al, 2008). These results prompted the development of a phase III study (Charité Onkologie; CONKO 003) that aimed to evaluate the efficacy of the OFF regimen (oxaliplatin, fluorouracil and folinic acid) compared with best supportive care in gemcitabine-pretreated patients. Unfortunately, the control arm was closed after 46 of the planned 165 patients were enrolled due to clinician reluctance to enroll in a no-treatment arm (Oettle et al, 2005b). The results of this initial cohort, however, showed a substantial improvement in overall survival for treated patients (22 vs 10 weeks, p=0.0077). The trial design was then modified to include an alternative comparator arm consisting of 5FU plus folinic acid (FF regimen) and 165 patients were subsequently enrolled. Toxicity was acceptable with few grade 3-4 adverse events. Median progression-free survival and overall survival were significantly better in the OFF arm (13 vs 9 weeks, p=0.012, and 26 vs 13 weeks, p=0.014, respectively) (Pelzer et al, 2008).

of the PRODIGE 4/ACCORD 11 trial comparing gemcitabine alone (1000 mg/m2 weekly x 7 every 8 weeks and then weekly x 3 every 4 weeks) to FOLFIRINOX (oxaliplatin 85 mg/m2, irinotecan 180 mg/m2, 5-FU 400 mg/m2 given as a bolus followed by 2400 mg/m2 given as a 46-hour continuous infusion; and leucovorin 400 mg/m2; every 2 weeks) demonstrated remarkable and significant improvements in response, progression free and overall survival rates favoring patients treated with FOLFIRINOX (31% vs. 9%, 6.4 months *vs*. 3.3 months, and 11.1 months *vs*. 6.8 months, respectively) (Conroy, 2011). These results are somewhat surprising, given the known modest activity of each of the individual drugs included in the regimen, and shall be confirmed. In addition, the higher toxicity profile of this combination limits its widespread use as standard of care in patients with metastatic disease, often frail. However, it may be an excellent option for carefully selected patients, particularly those with locally advanced borderline resectable disease. Anyhow, this is the first phase III randomized trial that has demonstrated a benefit in overall survival of unquestionable clinical relevance for patients with advanced pancreatic cancer, and it may change the classical paradigm of gemcitabine as the keystone in the management of advanced

Once the disease progresses to gemcinatine-based therapy there is no accepted standard of care and most patients will not be suitable candidates for further therapy due to clinical deterioration. Second-line chemotherapy may be considered, however, in patients who maintain good performance status, although efficacy in this setting is questionable. Overall, it is estimated that approximately 30% of patients are in good condition (including good performance status and adequate organ function) for consideration of second-line treatment (Gounaris et al, 2010). A number of trials have been performed assessing the efficacy of different antineoplastic agents in this context. Most of the published evidence, however, consists of small phase II studies testing a variety of drugs

Oxaliplatin-fluoropyrimidine doublets are probably the chemotherapy regimens most widely evaluated in gemcitabine-resistant disease. Several small phase II studies showed some promising activity with different combinations of oxaliplatin and 5FU or capecitabine (FOLFOX, OFF, XELOX,..), with median survival (6-7 months) that did not substantially differed from that observed in chemotherapy-naïve patients (Tsavaris et al, 2005; Xiong et al, 2008). These results prompted the development of a phase III study (Charité Onkologie; CONKO 003) that aimed to evaluate the efficacy of the OFF regimen (oxaliplatin, fluorouracil and folinic acid) compared with best supportive care in gemcitabine-pretreated patients. Unfortunately, the control arm was closed after 46 of the planned 165 patients were enrolled due to clinician reluctance to enroll in a no-treatment arm (Oettle et al, 2005b). The results of this initial cohort, however, showed a substantial improvement in overall survival for treated patients (22 vs 10 weeks, p=0.0077). The trial design was then modified to include an alternative comparator arm consisting of 5FU plus folinic acid (FF regimen) and 165 patients were subsequently enrolled. Toxicity was acceptable with few grade 3-4 adverse events. Median progression-free survival and overall survival were significantly better in the OFF arm (13 vs 9 weeks, p=0.012, and 26 vs 13 weeks, p=0.014, respectively) (Pelzer et al,

pancreatic cancer.

**2.3 Gemcitabine-resistant disease** 

in a heterogeneous population.

2008).

Combining gemcitabine and oxaliplatin (GEMOX) has been another commonly evaluated therapeutic schedule. Two small non-controlled trials investigated the efficacy of oxaliplatin plus fixed-dose rate gemcitabine in patients who had progressed on single agent gemcitabine. Although reported response rates were relevant (21-24% of partial responses), toxicity was not negligible, with up to half of the patients developing at least one grade 3 adverse event (Demols et al, 2006, as cited in Gounaris et al, 2010; Fortune el al, 2009, as cited in Gounaris et al, 2010). These results, together with the findings of the phase III E6201 conducted in chemotherapy-naïve patients failing to demonstrate a survival advantage for the combination, do not warrant further evaluation of this regimen in the second-line setting (Poplin et al, 2009).

Irinotecan has been tested both as single agent and in combination with oxaliplatin or fluoropyrimidines showing some activity and an acceptable toxicity profile (Yi et al, 2009; Cantore et al, 2004). A direct comparison between oxaliplatin- and irinotecan-based regimens was made by Hwang and colleagues in a small randomized phase II trial (Hwang et al, 2009). Sixty patients were enrolled and randomly allocated to receive FOLFOX (oxaliplatin, folinic acid and infusional 5FU) or FOLFIRI.3 (the same folinic acid and 5FU schedule combined with irinotecan) after gemcitabine failure. No significant differences were observed among study arms neither in PFS (1.4 vs 1.9 months, p>0.05) nor in OS (4 months both regimens). In light of these results, both regimens may be reasonable options for secondline therapy in appropriately selected patients with advanced pancreatic cancer. Other irinotecan-based regimens including combinations with raltitrexed (Ulrich-Pur, 2003, as cited in Gounaris, 2010), docetaxel (Ko et al, 2008), docetaxel and mitomycin C (Reni et al, 2004) or ifosfamide (Cereda et al, 2011) have not achieved positive results in small phase II trials.

Rubitecan, an orally bioavailable camptothecin derivative, was the subject of the largest study conducted in gemcitabine-resistant pancreatic cancer, despite results of an initial single arm study were not particularly encouraging (median TTP and OS of 1.9 and 3 months, respectively). Subsequently, a large phase III study was launched the results of which have only been reported in abstract form (Jacobs et al, 2004). Four-hundred and nine patients were randomized to receive treatment with rubitecan or physician's best choice (chemotherapy 89%, supportive care only 11%). There were more responses in the rubitecan arm (11% vs. 1%) and the difference in median PFS, although clinically modest, reached statistical significance (1.9 vs. 1.6 months). There was no significant difference however in OS (3.5 vs 3.1 months, respectively).

Other tested drugs in this setting, such as taxanes or pemetrexed, have not shown particularly promising results in small studies (Gounaris et al, 2010; Boeck et al, 2007b; Mazzer et al, 2009). Multidrug combinations such as PEFG (cisplatin, epirubicin, 5-FU and gemcitabine) (Reni et al, 2008, as cited in Gounaris et al, 2010) or G-FLIP (gemcitabine, irinotecan, folinic acid, 5-FU and cisplatin) (Kozuch et al, 2001, as cited in Gounaris, 2010) appear to show improved efficacy with impressive median survival of 8.3 and 10.3 months, respectively. Selection bias may at least partially explain these outstanding results as reported toxicity was rather high, which in any case would limit their use in the general population.

#### **3. Molecularly targeted therapies**

Pancreatic adenocarcinoma is a malignant disease that results from the successive accumulation of gene mutations (Vogelstein & Kinzsler, 2004) evolving from premalignant

Current Perspectives and Future Trends of

Moore et al (2007)

Philip et al (2007)

Van Cutsem et al (2009)

Kindler et al (2010)

Moore et al (2003)

Bramhall et al (2001)

Bramhall et al (2002)

Van Cutsem et al (2004)

Reference Treatment Number

GEM+ERLOT+PLA

GEM+ERLOT+BEV

GEM+PLA vs GEM+BEV

MARIMASTAT

GEM+MARIMASTAT

to try to improve patient selection and therapeutic benefit.

GEM+TIPIFARNIB

GEM vs

GEM vs

GEM vs

progression free survival; OS, overall survival

GEM + PLA vs GEM+ERLOT

GEM vs GEM+CETUX

vs

Systemic Therapy in Advanced Pancreatic Carcinoma 97

of patients

GEM vs BAY 12-9566 277 6.59 vs 3.74

GEM, gemcitabine; PLA, placebo; ERLOT, erlotinib; BEV, bevacizumab; RR, response rate; PFS,

functions downstream of the EGFR signaling pathway, and mutations in the KRAS protein lead to constitutive activation independent of extracellular stimuli. This is a well established mechanism of resistance to EGFR blockade in colorectal cancer, and, indeed, EGFR-targeted therapy is only to be used in KRAS wild-type tumors. The potential predictive value of KRAS mutation status and EGFR gene copy number in pancreatic cancer was evaluated in 26% of the patients included in the PA.3 trial who had tumor samples available for analysis. KRAS mutations were detected in 79% of tested samples. EGFR copy number was not correlated with treatment effect. However, the HR of death between gemcitabine/erlotinib and gemcitabine/placebo was 1.07 for patients with KRAS-mutated tumors versus 0.66 for those with KRAS wild-type tumors. Although this difference did not reach statistical significance probably due to small numbers, this plausible trend shall be further evaluated

Erlotinib has also been tested as second-line treatment of patients with advanced disease. Kulke et al evaluated the combination of erlotinib and capecitabine in 30 patients with gemcitabine-refractory pancreatic cancer. Objective radiologic responses were observed in 10% of patients and the median survival was 6.5 months. In addition, 17% of treated patients experienced decreases in tumor marker (CA 19-9) levels of more than 50% from baseline. However, common toxicities, particularly diarrhea and skin rash, were significant and required treatment dose reductions in 66% of patients (Kulke et al, 2007). More recently, this treatment regimen has been tested against erlotinib-gemcitabine in a phase III AIO trial. This trial included 279 chemotherapy naïve patients that were randomly allocated to receive

Table 2. Selected phase III trials of targeted agents in advanced pancreatic cancer

OS (months)

(p=0.038)

(p=0.14)

(p=0.95)

(p<0.01)

(p=0.75)

607 6 vs 7.1 3.6 vs 4.6

569 5.91 vs 6.24

766 6 vs 6.5

602 5.9 vs 5.8

688 6 vs 6.3

PFS (months)

3.55 vs 3,.75 (p=0.004)

3 vs 3.5 (p=0.058)

(p=0.0002)

2.9 vs 3.8 (p=0.07)

3.5 vs 1.68 (p<0.01)

3.6 vs 3.7 (p=0.72)

414 5.5 vs 4.1 3.8 vs 1.9 25.8 vs

239 5.4 vs 5.4 3.1 vs 3 16 vs

RR (%)

8 vs 8.6

14 vs 12

8.6 vs 13.5

10 vs 13


2.8

11

8 vs 6

lesions in the ductal epithelium to invasive cancer. These include activating mutations of KRAS2 oncogene (90% of pancreatic tumors), and inactivation of the tumor-suppresor genes CDKN2A (95%), TP53 (50-75%) or DPC4 (50%). More recent comprehensive genetic analysis have shown that molecular features in pancreatic cancer may be extremely complex and heterogeneous (Jones et al, 2008), although these genetic abnormalities may be classified in 12 core cancer signaling pathways involving not only pancreatic cancer cells but also other fundamental components of neoplasia such as cancer stem cells and tumor stroma (Hidalgo, 2010). As molecular pathways governing pancreatic cancer development are unraveled, novel targets emerge that may provide some promise to improve the dismal results obtained with conventional cytotoxic therapy.

#### **3.1 EGFR-RAS-MEK-ERK pathway**

EGFR (epidermal growth factor receptor), also known as HER-1 or ErbB-1, is activated by several ligands that include EGF (epidermal growth factor), TGF-α (transforming growth factor alpha), HB-EGF (heparin-binding EGF), amphiregulin, epiregulin, betacellulin and neuregulin. Activated EGFR forms homo- or heterodimeric complexes with other members of the ErbB family, triggering downstream signaling pathways such as Ras/MAP kinase, phosphatidylinositol 3'-kinase (PI3K)/Akt, Janus kinase (JAK)/Stat and phospholipase C/protein kinase C, that ultimately activate genes involved in cell proliferation, migration, adhesion, differentiation and apoptosis (Di Marco et al, 2010). Overexpression of EGFR and its ligands is very common in pancreatic cancer, and it is linked to increased tumor aggressiveness and poor prognosis. Preclinical studies have shown that blocking EGFR signaling inhibits growth and metastasis of pancreatic tumors in xenograft models and synergistic activity has been documented when combined with gemcitabine (Tempero et al, 2011).

Two strategies to antagonize EGFR signaling have been evaluated in the clinic to date: inhibition of the tyrosine kinase intracellular domain by small molecules and EGFR inhibition by monoclonal antibodies directed against the extracellular ligand binding domain. Erlotinib is an oral tyrosine kinase inhibitor [TKI] against EGFR, and the only targeted drug that has demonstrated some efficacy in pancreatic cancer thus far. The National Cancer Institute of Canada PA.3 trial was a phase III randomized study evaluating standard gemcitabine plus erlotinib (100 or 150 mg/day) versus gemcitabine plus placebo in 569 patients with chemo-naïve advanced pancreatic cancer (Table 2). Both PFS (PFS 3.75 vs 3.55 months, HR 0.77, p=0.004) and OS (6.24 vs 5.91 months, HR 0.82, p=0.038) were significantly improved in the experimental arm (Moore et al, 2007). Most common toxicity was, as expected, diarrhea and skin rash, which were of grade 1-2 in the majority of cases without negatively impacting patient´s quality of life. Interestingly, patients that developed grade 2 or higher skin rash had significantly longer survival compared to those who developed mild or no rash (10.5 vs 5.8 vs 5.3 months, respectively, HR 0.74, p=0.037). Levels of EGFR expression, however, were not correlated with survival. This was the pivotal study that granted erlotinib marketing authorization by regulatory authorities, although the small magnitude of benefit has precluded widespread acceptance by oncologists in Europe of the gemcitabine-erlotinib combination as the new standard of care for first line therapy of advanced pancreatic cancer.

One potential explanation for this modest effect of EGFR inhibition in pancreatic cancer is the fact that KRAS mutations occur in 70-90% of these tumors (Tempero et al, 2011). KRAS

lesions in the ductal epithelium to invasive cancer. These include activating mutations of KRAS2 oncogene (90% of pancreatic tumors), and inactivation of the tumor-suppresor genes CDKN2A (95%), TP53 (50-75%) or DPC4 (50%). More recent comprehensive genetic analysis have shown that molecular features in pancreatic cancer may be extremely complex and heterogeneous (Jones et al, 2008), although these genetic abnormalities may be classified in 12 core cancer signaling pathways involving not only pancreatic cancer cells but also other fundamental components of neoplasia such as cancer stem cells and tumor stroma (Hidalgo, 2010). As molecular pathways governing pancreatic cancer development are unraveled, novel targets emerge that may provide some promise to improve the dismal results

EGFR (epidermal growth factor receptor), also known as HER-1 or ErbB-1, is activated by several ligands that include EGF (epidermal growth factor), TGF-α (transforming growth factor alpha), HB-EGF (heparin-binding EGF), amphiregulin, epiregulin, betacellulin and neuregulin. Activated EGFR forms homo- or heterodimeric complexes with other members of the ErbB family, triggering downstream signaling pathways such as Ras/MAP kinase, phosphatidylinositol 3'-kinase (PI3K)/Akt, Janus kinase (JAK)/Stat and phospholipase C/protein kinase C, that ultimately activate genes involved in cell proliferation, migration, adhesion, differentiation and apoptosis (Di Marco et al, 2010). Overexpression of EGFR and its ligands is very common in pancreatic cancer, and it is linked to increased tumor aggressiveness and poor prognosis. Preclinical studies have shown that blocking EGFR signaling inhibits growth and metastasis of pancreatic tumors in xenograft models and synergistic activity has been documented when combined with gemcitabine (Tempero et al,

Two strategies to antagonize EGFR signaling have been evaluated in the clinic to date: inhibition of the tyrosine kinase intracellular domain by small molecules and EGFR inhibition by monoclonal antibodies directed against the extracellular ligand binding domain. Erlotinib is an oral tyrosine kinase inhibitor [TKI] against EGFR, and the only targeted drug that has demonstrated some efficacy in pancreatic cancer thus far. The National Cancer Institute of Canada PA.3 trial was a phase III randomized study evaluating standard gemcitabine plus erlotinib (100 or 150 mg/day) versus gemcitabine plus placebo in 569 patients with chemo-naïve advanced pancreatic cancer (Table 2). Both PFS (PFS 3.75 vs 3.55 months, HR 0.77, p=0.004) and OS (6.24 vs 5.91 months, HR 0.82, p=0.038) were significantly improved in the experimental arm (Moore et al, 2007). Most common toxicity was, as expected, diarrhea and skin rash, which were of grade 1-2 in the majority of cases without negatively impacting patient´s quality of life. Interestingly, patients that developed grade 2 or higher skin rash had significantly longer survival compared to those who developed mild or no rash (10.5 vs 5.8 vs 5.3 months, respectively, HR 0.74, p=0.037). Levels of EGFR expression, however, were not correlated with survival. This was the pivotal study that granted erlotinib marketing authorization by regulatory authorities, although the small magnitude of benefit has precluded widespread acceptance by oncologists in Europe of the gemcitabine-erlotinib combination as the new standard of care for first line therapy of

One potential explanation for this modest effect of EGFR inhibition in pancreatic cancer is the fact that KRAS mutations occur in 70-90% of these tumors (Tempero et al, 2011). KRAS

obtained with conventional cytotoxic therapy.

**3.1 EGFR-RAS-MEK-ERK pathway** 

2011).

advanced pancreatic cancer.


GEM, gemcitabine; PLA, placebo; ERLOT, erlotinib; BEV, bevacizumab; RR, response rate; PFS, progression free survival; OS, overall survival

Table 2. Selected phase III trials of targeted agents in advanced pancreatic cancer

functions downstream of the EGFR signaling pathway, and mutations in the KRAS protein lead to constitutive activation independent of extracellular stimuli. This is a well established mechanism of resistance to EGFR blockade in colorectal cancer, and, indeed, EGFR-targeted therapy is only to be used in KRAS wild-type tumors. The potential predictive value of KRAS mutation status and EGFR gene copy number in pancreatic cancer was evaluated in 26% of the patients included in the PA.3 trial who had tumor samples available for analysis. KRAS mutations were detected in 79% of tested samples. EGFR copy number was not correlated with treatment effect. However, the HR of death between gemcitabine/erlotinib and gemcitabine/placebo was 1.07 for patients with KRAS-mutated tumors versus 0.66 for those with KRAS wild-type tumors. Although this difference did not reach statistical significance probably due to small numbers, this plausible trend shall be further evaluated to try to improve patient selection and therapeutic benefit.

Erlotinib has also been tested as second-line treatment of patients with advanced disease. Kulke et al evaluated the combination of erlotinib and capecitabine in 30 patients with gemcitabine-refractory pancreatic cancer. Objective radiologic responses were observed in 10% of patients and the median survival was 6.5 months. In addition, 17% of treated patients experienced decreases in tumor marker (CA 19-9) levels of more than 50% from baseline. However, common toxicities, particularly diarrhea and skin rash, were significant and required treatment dose reductions in 66% of patients (Kulke et al, 2007). More recently, this treatment regimen has been tested against erlotinib-gemcitabine in a phase III AIO trial. This trial included 279 chemotherapy naïve patients that were randomly allocated to receive

Current Perspectives and Future Trends of

2001, as cited in Stathis & Moore, 2010).

improvement in survival was observed in this trial.

**3.2 Antiangiogenic agents** 

Systemic Therapy in Advanced Pancreatic Carcinoma 99

be tested. It is a farnesyl transferase inhibitor which demonstrated antiproliferative activity in a wide range of tumors in preclinical models. Farnesylation is an impotant posttraslational event required for Ras activation. A large phase III clinical trial, however, failed to demonstrate an improvement in survival of adding tipifarnib to gemcitabine over gemcitabine alone in patients with advanced pancreatic cancer (Table 2) (Van Cutsem et al, 2004). Some authors have postulated as a potential explanation for these negative results the fact that KRAS mutation could be an early event in the development of pancreatic cancer, becoming cancer cells less dependent on this pathway as the disease progresses. In addition, other mechanisms involved in the regulation of Ras activation (i.e. prenylation by other enzymes) may limit the therapeutic success of farnesyl transferase inhibition (Lobell R et al,

Other agents targeting downstream effectors of the EGFR pathway currently under evaluation include MEK inhibitors. Phase I trials have established the recommended dose for further clinical development and have documented rash, diarrhea and central serous retinopathy as dose limiting toxicities, all of them reversible (Messersmith et al, 2011). Several phase I and II trial combining MEK inhibitors with standard chemotherapy and other targeted agents are ongoing, the results of which are awaited with great interest.

Angiogenesis is a widely validated target for cancer therapy. Overexpression of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) has been described in pancreatic cancer and correlated with disease progression and poor prognosis. Bevacizumab is a recombinant humanized anti-VEGF monoclonal antibody and the most widely tested antiangiogenic agent. Promising data of several bevacizumab combination regimens in phase II clinical trials, with response rates of up to 24% and median survival of up to 11 months (Kindler et al, 2005; Walkins et al, 2010; Iyer et al, 2008, as cited in Di Marco et al, 2010), encouraged the development of two large phase III trials that unfortunately failed to yield positive results. The first one enrolled 602 patients that were randomized to receive gemcitabine plus bevacizumab or gemcitaine plus placebo. No significant differences were observed among study arms neither in PFS (PFS 3.8 vs 2.9 months) nor in OS (5.8 vs 5.9 months) (Kindler et al, 2010). The second one evaluated the addition of bevacizumab to the gemcitabine-erlotinib doublet (Table 2). Although PFS was better for the experimental arm (4.6 vs 3.6 months, HR 0.73, p=0.0002), the primary objective of the study was not met as the addition of bevacizumab did not improve overall survival (7.1 vs 6.0 months, HR 0.89, p=0.2) (Van Cutsem et al, 2009). A correlation between development of skin rash and

Other broadly tested agents that interfere with angiogenesis include small molecules targeting multiple kinases such as axitinib or sorafenib. Axitinib, an oral inhibitor of VEGFR-1, VEGFR-2 and VEGFR-3, was initially evaluated in a phase II randomized trial in combination with gemcitabine versus gemcitabine alone. This trial enrolled 103 patients and showed a small improvement in survival favoring the combination arm (6.9 vs 5.6 months), although this difference did not reach statistical significance (Spano et al, 2008). Nevertheless, a phase III trial was undertaken but was prematurely discontinued due to

capecitabine-erlotinib versus gemcitabine-erlotinib as the control arm. Crossover to gemcitabine or capecitabine alone was allowed at the time of progression. Neither time to treatment failure of second-line therapy (TTF2), which was the primary endpoint of the trial, nor OS were significantly different among study arms (TTF2 4.4 vs 4.2 months, HR 0.98, p=0.43; OS 6.9 vs 6.6 months, HR 0.96, p=0.78). Of note, overall survival was significantly correlated with KRAS mutation status (8.0 months vs 6.6 months for KRAS wild-type versus mutated tumors, respectively; HR 1.62; p=0.011). However, the study design, which included erlotinib in both treatment arms, does not allow to elucidate whether KRAS mutation status is predictive of efficacy of EGFR-targeted therapy or just a prognostic factor independent of therapy (Boeck et al, 2010). Anyhow, this regimen may represent an acceptable treatment option in patients who experience treatment failure with standard gemcitabine first-line therapy or for whom gemcitabine may not be an appropriate treatment option.

The other strategy to antagonize EGFR signaling consists of monoclonal antibodies directed against the extracellular domain of the receptor, such as cetuximab or panitumumab. They are currently approved for treatment of other advanced malignancies such as colorectal or head and neck cancer. Preclinical and early clinical trials suggested some efficay too in pancreatic cancer. Disappointingly, a large phase III trial comparing the combination of cetuximab plus gemcitabine vs gemcitabine alone (Table 2), which enrolled 366 patients, did not demonstrate a benefit in survival for the combination regimen (Philip et al, 2007). Other approaches explored include dual EGFR inhibition (TKI inhibitors plus monoclonal antibodies). Preliminary results of a phase II randomized study suggest a small benefit in terms of PFS (3.3 months vs 2.0 months) for the addition of panitumumab to gemcitabineerlotinib, although statistical significance was not reported and final data including overall survival are awaited for definitive conclusions (Kim et al, 2010).

Lapatinib, an oral TKI which reversibly inhibits both EGFR/HER1 and HER2/neu, has also been evaluated. Preclinical assays suggested activity alone and in combination with other drugs such as capecitabine. Moreover, a phase I trial combining lapatinib with either gemcitabine or GEMOX showed encouraging results with median survival of 10 months (Safran et al, 2008, as cited in Di Marco et al, 2010). More recently, preliminary results of a single arm phase II trial evaluating the combination of capecitabine and lapatinib as firstline treatment in advanced pancreatic cancer have been presented. Survival of 6 months was not reached in 7 of the 9 enrolled patients, and none of them obtained objective responses (McDermott et al, 2011). This data led to the premature termination of the study.

HER2 may be also targeted by monoclonal antibodies such as trastuzumab. HER2 is overexpressed in some pancreatic cancers, with results widely varying from 0 to 82% in different studies. One early trial evaluated gemcitabine plus trastuzumab in 34 metastatic pancreatic cancer patients with 2+/3+ Her2-positive tumors determined by immunohistochemistry. Only 4 patients (12%) presented Her2 neu 3+ expression. Partial responses were observed in 6% of patients (2/32) (Safran et al, 2004). Further studies would be needed to appropriately assess the role of this agent in pancreatic cancer.

Other therapeutic strategies have aimed to target some of the downstream effectors of EGFR. The high incidence of KRAS mutations in pancreatic cancer provided a strong rationale for the evaluation of KRAS inhibition. Tipifarnib was the first agent of this class to

capecitabine-erlotinib versus gemcitabine-erlotinib as the control arm. Crossover to gemcitabine or capecitabine alone was allowed at the time of progression. Neither time to treatment failure of second-line therapy (TTF2), which was the primary endpoint of the trial, nor OS were significantly different among study arms (TTF2 4.4 vs 4.2 months, HR 0.98, p=0.43; OS 6.9 vs 6.6 months, HR 0.96, p=0.78). Of note, overall survival was significantly correlated with KRAS mutation status (8.0 months vs 6.6 months for KRAS wild-type versus mutated tumors, respectively; HR 1.62; p=0.011). However, the study design, which included erlotinib in both treatment arms, does not allow to elucidate whether KRAS mutation status is predictive of efficacy of EGFR-targeted therapy or just a prognostic factor independent of therapy (Boeck et al, 2010). Anyhow, this regimen may represent an acceptable treatment option in patients who experience treatment failure with standard gemcitabine first-line therapy or for whom gemcitabine may not be an appropriate

The other strategy to antagonize EGFR signaling consists of monoclonal antibodies directed against the extracellular domain of the receptor, such as cetuximab or panitumumab. They are currently approved for treatment of other advanced malignancies such as colorectal or head and neck cancer. Preclinical and early clinical trials suggested some efficay too in pancreatic cancer. Disappointingly, a large phase III trial comparing the combination of cetuximab plus gemcitabine vs gemcitabine alone (Table 2), which enrolled 366 patients, did not demonstrate a benefit in survival for the combination regimen (Philip et al, 2007). Other approaches explored include dual EGFR inhibition (TKI inhibitors plus monoclonal antibodies). Preliminary results of a phase II randomized study suggest a small benefit in terms of PFS (3.3 months vs 2.0 months) for the addition of panitumumab to gemcitabineerlotinib, although statistical significance was not reported and final data including overall

Lapatinib, an oral TKI which reversibly inhibits both EGFR/HER1 and HER2/neu, has also been evaluated. Preclinical assays suggested activity alone and in combination with other drugs such as capecitabine. Moreover, a phase I trial combining lapatinib with either gemcitabine or GEMOX showed encouraging results with median survival of 10 months (Safran et al, 2008, as cited in Di Marco et al, 2010). More recently, preliminary results of a single arm phase II trial evaluating the combination of capecitabine and lapatinib as firstline treatment in advanced pancreatic cancer have been presented. Survival of 6 months was not reached in 7 of the 9 enrolled patients, and none of them obtained objective responses

HER2 may be also targeted by monoclonal antibodies such as trastuzumab. HER2 is overexpressed in some pancreatic cancers, with results widely varying from 0 to 82% in different studies. One early trial evaluated gemcitabine plus trastuzumab in 34 metastatic pancreatic cancer patients with 2+/3+ Her2-positive tumors determined by immunohistochemistry. Only 4 patients (12%) presented Her2 neu 3+ expression. Partial responses were observed in 6% of patients (2/32) (Safran et al, 2004). Further studies would

Other therapeutic strategies have aimed to target some of the downstream effectors of EGFR. The high incidence of KRAS mutations in pancreatic cancer provided a strong rationale for the evaluation of KRAS inhibition. Tipifarnib was the first agent of this class to

(McDermott et al, 2011). This data led to the premature termination of the study.

be needed to appropriately assess the role of this agent in pancreatic cancer.

survival are awaited for definitive conclusions (Kim et al, 2010).

treatment option.

be tested. It is a farnesyl transferase inhibitor which demonstrated antiproliferative activity in a wide range of tumors in preclinical models. Farnesylation is an impotant posttraslational event required for Ras activation. A large phase III clinical trial, however, failed to demonstrate an improvement in survival of adding tipifarnib to gemcitabine over gemcitabine alone in patients with advanced pancreatic cancer (Table 2) (Van Cutsem et al, 2004). Some authors have postulated as a potential explanation for these negative results the fact that KRAS mutation could be an early event in the development of pancreatic cancer, becoming cancer cells less dependent on this pathway as the disease progresses. In addition, other mechanisms involved in the regulation of Ras activation (i.e. prenylation by other enzymes) may limit the therapeutic success of farnesyl transferase inhibition (Lobell R et al, 2001, as cited in Stathis & Moore, 2010).

Other agents targeting downstream effectors of the EGFR pathway currently under evaluation include MEK inhibitors. Phase I trials have established the recommended dose for further clinical development and have documented rash, diarrhea and central serous retinopathy as dose limiting toxicities, all of them reversible (Messersmith et al, 2011). Several phase I and II trial combining MEK inhibitors with standard chemotherapy and other targeted agents are ongoing, the results of which are awaited with great interest.

#### **3.2 Antiangiogenic agents**

Angiogenesis is a widely validated target for cancer therapy. Overexpression of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) has been described in pancreatic cancer and correlated with disease progression and poor prognosis. Bevacizumab is a recombinant humanized anti-VEGF monoclonal antibody and the most widely tested antiangiogenic agent. Promising data of several bevacizumab combination regimens in phase II clinical trials, with response rates of up to 24% and median survival of up to 11 months (Kindler et al, 2005; Walkins et al, 2010; Iyer et al, 2008, as cited in Di Marco et al, 2010), encouraged the development of two large phase III trials that unfortunately failed to yield positive results. The first one enrolled 602 patients that were randomized to receive gemcitabine plus bevacizumab or gemcitaine plus placebo. No significant differences were observed among study arms neither in PFS (PFS 3.8 vs 2.9 months) nor in OS (5.8 vs 5.9 months) (Kindler et al, 2010). The second one evaluated the addition of bevacizumab to the gemcitabine-erlotinib doublet (Table 2). Although PFS was better for the experimental arm (4.6 vs 3.6 months, HR 0.73, p=0.0002), the primary objective of the study was not met as the addition of bevacizumab did not improve overall survival (7.1 vs 6.0 months, HR 0.89, p=0.2) (Van Cutsem et al, 2009). A correlation between development of skin rash and improvement in survival was observed in this trial.

Other broadly tested agents that interfere with angiogenesis include small molecules targeting multiple kinases such as axitinib or sorafenib. Axitinib, an oral inhibitor of VEGFR-1, VEGFR-2 and VEGFR-3, was initially evaluated in a phase II randomized trial in combination with gemcitabine versus gemcitabine alone. This trial enrolled 103 patients and showed a small improvement in survival favoring the combination arm (6.9 vs 5.6 months), although this difference did not reach statistical significance (Spano et al, 2008). Nevertheless, a phase III trial was undertaken but was prematurely discontinued due to

Current Perspectives and Future Trends of

line metastatic pancreatic cancer (Hidalgo, 2010).

**IGF-1R** 

**TNF-α**

**Multikinase inhibitor** 

**Death receptors** 

**Other pathways** 

gemcitabine with or without masitinib.

safety profile of this combination.

pancreatic cancer (Hidalgo, 2010).

Systemic Therapy in Advanced Pancreatic Carcinoma 101

IGF-1R mediated signaling plays an important role in cell growth regulation and survival. Several monoclonal antibodies targeting IGF-IR have undergone clinical investigation (AMG479, MK0646, R1507). Based on promising preclinical and early clinical data, a phase III trial has been initiated to evaluate the combination of AMG479 plus gemcitabine in first-

TNF-α shows potent anticancer activity, but high systemic toxicity limits its use. AdEgr.TNF.11D (TNFerade) is a gene delivery strategy to increase local peritumoral TNF concentrations through intratumoral injections of an adenoviral vector expressing hTNF, in an attempt to improve local activity while minimizing systemic effects. Effectiveness in combination with gemcitabine has been demonstrated in human pancreatic xenografts (Murugesan et al, 2009). A phase III trial is currently evaluating the addition of TNFerade to

Masitinib is a multikinase inhibitor that has greater activity and selectivity against KIT than imatinib. Masitinib also potently inhibits PDGFR (platelet-derived growth factor receptor) and the intracellular kinase Lyn, and to a lesser extent, FGFR3 (fibroblast growth factor receptor 3). Synergistic activity with gemcitabine was demonstrated in preclinical assays. A phase II trial combining gemcitabine and masitinib in 22 patients reported median PFS of 6.4 months and OS of 7.1 months, with a 23% 18-months survival rate. Toxicity was acceptable, being cytopenia, diarrhea and rash the most common severe events (Hammel et al, 2009). A subsequent phase III trial is ongoing comparing

AMG655 is a monoclonal antibody against human death receptor 5 (DR5) that activates caspases and, as a result, induces apoptosis in tumor cells. It showed preclinical activity and synergy with gemcitabine. Early clinical data from a phase I trial that included 13 patients reported promising results for the combination of AMG655 with gemcitabine, with a response rate of 31%, median PFS of 5.3 months and a 6-month survival rate of 76.8%. Toxicity was however not negligible, with severe adverse events observed in 69% of patients (Kindler et al, 2009). A phase II is ongoing to assess efficacy and further characterize the

Other pathways highly implicated in pancreatic tumorigenesis are at earlier stages of investigation. Hedgehog, Notch and Wnt signaling are important developmental pathways related to pancreatic cancer stem cells, and new agents are being developed to target these pathways (GDC-0449, IPI-926,..). Other agents in development include monoclonal antibodies against cell-membrane proteins such as mesothelin (MORAb-009). Specific mechanisms of cell killing are still not well defined but preclinical research suggest a role in

5-FU plus radiotherapy in unresectable pancreatic cancer (Stathis & Moore, 2010).

the lack of benefit observed in an interim analysis for the addition of axitinib to the standard gemcitabine therapy. Sorafenib has also been evaluated in combination with both gemcitabine and gemcitabine-erlotinib in different non-controlled trials with disappointing results (Wallace et al, 2007; Cohen et al, 2011). The lack of success of antiangiogenic strategies in pancreatic cancer could be potentially related to the fact that most tumors display intense fibrosis and are of hypovascular nature (Stathis & Moore, 2010).

#### **3.3 Matrix metalloproteinases (MMP) inhibitors**

MMPs are a family of zinc-dependent proteolytic enzymes implicated in the degradation of extracellular matrix proteins both in physiological and pathological conditions. Aberrant MMP expression contributes to neovascularization, dissemination and metastasis of a variety of solid malignancies (Stathis & Moore, 2010). Several compounds developed to inhibit MMPs have been completely unsuccessful in clinical trials over the last decade. Marimastat was the first agent to be tested (Table 2). Two large phase III trials enrolling over 900 patients showed marimastat, either alone or in combination with gemcitabine, was not able to improve survival or disease control of patients with advanced pancreatic cancer (Bramhall et al, 2001, 2002). Similar negative results were obtained with other agents of this class. Standard gemcitabine monotherapy was compared to BAY 12-9566, in a design that allowed for crossover after disease progression. Interim analyses demonstrated a deleterious effect on survival of the MMP inhibitor as compared to the control arm (OS 3.74 vs 6.59 months, p<0.01), and led to early trial termination (Moore et al, 2003). In light of this data, this approach has been definitively abandoned.

#### **3.4 Other pathways**

#### **Phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR**

The PI3K/Akt/mTOR pathway is determinant for processes related to cell proliferation and inhibition of apoptosis, and constitutive activation of this pathway has been documented in pancreatic cancer (Royal et al, 2008). NVP-BEZ235 is a novel dual PI3K/mTOR inhibitor that has demonstrated activity in both human pancreatic cancer cell lines and mice models, and some synergy has been observed when combined with gemcitabine and antiangiogenic EMAP II (endothelial monocyte activating polypeptide II) (Awasthi et al, 2011). Further research will define the role of these new drugs in pancreatic cancer.

#### **Src kinase**

Src tyrosine kinase is a non-receptor protein implicated in tumor progression. It is overexpressed in more than two thirds of pancreatic adenocarcinomas. Src inhibitors (dasatinib, saracatinib) have been developed demonstrating antitumor activity in cancer cell lines and mice models (Royal et al, 2008). A recent phase II trial tested saracatinib (AZD0530) in 19 gemcitabine-refractory patients. No responses were seen and the minimum of 18% 6-month survival required for continuation of the trial was not achieved. A pharmacodiagnostic pre-selection strategy is planned to be implemented to better define patients most likely to respond (Nallapareddy et al, 2010).

#### **IGF-1R**

100 Pancreatic Cancer – Clinical Management

the lack of benefit observed in an interim analysis for the addition of axitinib to the standard gemcitabine therapy. Sorafenib has also been evaluated in combination with both gemcitabine and gemcitabine-erlotinib in different non-controlled trials with disappointing results (Wallace et al, 2007; Cohen et al, 2011). The lack of success of antiangiogenic strategies in pancreatic cancer could be potentially related to the fact that most tumors display intense fibrosis and are of hypovascular nature (Stathis & Moore,

MMPs are a family of zinc-dependent proteolytic enzymes implicated in the degradation of extracellular matrix proteins both in physiological and pathological conditions. Aberrant MMP expression contributes to neovascularization, dissemination and metastasis of a variety of solid malignancies (Stathis & Moore, 2010). Several compounds developed to inhibit MMPs have been completely unsuccessful in clinical trials over the last decade. Marimastat was the first agent to be tested (Table 2). Two large phase III trials enrolling over 900 patients showed marimastat, either alone or in combination with gemcitabine, was not able to improve survival or disease control of patients with advanced pancreatic cancer (Bramhall et al, 2001, 2002). Similar negative results were obtained with other agents of this class. Standard gemcitabine monotherapy was compared to BAY 12-9566, in a design that allowed for crossover after disease progression. Interim analyses demonstrated a deleterious effect on survival of the MMP inhibitor as compared to the control arm (OS 3.74 vs 6.59 months, p<0.01), and led to early trial termination (Moore et al, 2003). In light of this data,

The PI3K/Akt/mTOR pathway is determinant for processes related to cell proliferation and inhibition of apoptosis, and constitutive activation of this pathway has been documented in pancreatic cancer (Royal et al, 2008). NVP-BEZ235 is a novel dual PI3K/mTOR inhibitor that has demonstrated activity in both human pancreatic cancer cell lines and mice models, and some synergy has been observed when combined with gemcitabine and antiangiogenic EMAP II (endothelial monocyte activating polypeptide II) (Awasthi et al, 2011). Further

Src tyrosine kinase is a non-receptor protein implicated in tumor progression. It is overexpressed in more than two thirds of pancreatic adenocarcinomas. Src inhibitors (dasatinib, saracatinib) have been developed demonstrating antitumor activity in cancer cell lines and mice models (Royal et al, 2008). A recent phase II trial tested saracatinib (AZD0530) in 19 gemcitabine-refractory patients. No responses were seen and the minimum of 18% 6-month survival required for continuation of the trial was not achieved. A pharmacodiagnostic pre-selection strategy is planned to be implemented to better define

2010).

**3.3 Matrix metalloproteinases (MMP) inhibitors** 

this approach has been definitively abandoned.

**Phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR** 

research will define the role of these new drugs in pancreatic cancer.

patients most likely to respond (Nallapareddy et al, 2010).

**3.4 Other pathways** 

**Src kinase** 

IGF-1R mediated signaling plays an important role in cell growth regulation and survival. Several monoclonal antibodies targeting IGF-IR have undergone clinical investigation (AMG479, MK0646, R1507). Based on promising preclinical and early clinical data, a phase III trial has been initiated to evaluate the combination of AMG479 plus gemcitabine in firstline metastatic pancreatic cancer (Hidalgo, 2010).

#### **TNF-α**

TNF-α shows potent anticancer activity, but high systemic toxicity limits its use. AdEgr.TNF.11D (TNFerade) is a gene delivery strategy to increase local peritumoral TNF concentrations through intratumoral injections of an adenoviral vector expressing hTNF, in an attempt to improve local activity while minimizing systemic effects. Effectiveness in combination with gemcitabine has been demonstrated in human pancreatic xenografts (Murugesan et al, 2009). A phase III trial is currently evaluating the addition of TNFerade to 5-FU plus radiotherapy in unresectable pancreatic cancer (Stathis & Moore, 2010).

#### **Multikinase inhibitor**

Masitinib is a multikinase inhibitor that has greater activity and selectivity against KIT than imatinib. Masitinib also potently inhibits PDGFR (platelet-derived growth factor receptor) and the intracellular kinase Lyn, and to a lesser extent, FGFR3 (fibroblast growth factor receptor 3). Synergistic activity with gemcitabine was demonstrated in preclinical assays. A phase II trial combining gemcitabine and masitinib in 22 patients reported median PFS of 6.4 months and OS of 7.1 months, with a 23% 18-months survival rate. Toxicity was acceptable, being cytopenia, diarrhea and rash the most common severe events (Hammel et al, 2009). A subsequent phase III trial is ongoing comparing gemcitabine with or without masitinib.

#### **Death receptors**

AMG655 is a monoclonal antibody against human death receptor 5 (DR5) that activates caspases and, as a result, induces apoptosis in tumor cells. It showed preclinical activity and synergy with gemcitabine. Early clinical data from a phase I trial that included 13 patients reported promising results for the combination of AMG655 with gemcitabine, with a response rate of 31%, median PFS of 5.3 months and a 6-month survival rate of 76.8%. Toxicity was however not negligible, with severe adverse events observed in 69% of patients (Kindler et al, 2009). A phase II is ongoing to assess efficacy and further characterize the safety profile of this combination.

#### **Other pathways**

Other pathways highly implicated in pancreatic tumorigenesis are at earlier stages of investigation. Hedgehog, Notch and Wnt signaling are important developmental pathways related to pancreatic cancer stem cells, and new agents are being developed to target these pathways (GDC-0449, IPI-926,..). Other agents in development include monoclonal antibodies against cell-membrane proteins such as mesothelin (MORAb-009). Specific mechanisms of cell killing are still not well defined but preclinical research suggest a role in pancreatic cancer (Hidalgo, 2010).

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#### **4. Conclusions**

Pancreatic cancer continues to be a major challenge for oncologists as it is a highly chemoresistant malignancy carrying an extremely poor prognosis. Despite the intense research carried out over the last decades no major improvements have been achieved in patient's outcomes. Most patients present with locally advanced or metastatic disease and will therefore require systemic therapy. Conventional chemotherapy modestly improves survival and quality of life of patients with advanced disease. Gemcitabine has been the reference treatment for over a decade and little progress has been made since its introduction in clinical practice in 1997. Gemcitabine-combination therapy with capecitabine, platinum agents or erlotinib may be considered in patients with good performance status, although the small magnitude of benefit they confer shall be balanced against the increased toxicity they induce, particularly considering that prognosis is in any case rather poor and symptomatic relief shall be a major objective of disease management. FOLFIRINOX may be a preferred option for carefully selected fit patients, particularly those with locally advanced borderline resectable disease.

Nevertheless, there is much room for improvement, and more efforts in basic, translational and clinical research will be necessary in the following years for progress to be made. Indeed, a better understanding of the biology of pancreatic cancer shall enable the discovery of new targets of potential diagnostic or therapeutic interest. Meanwhile, as the molecular pathways governing pancreatic cancer are unraveled, efforts shall be made to improve selection of patients most likely to benefit from specific therapies (SPARC, kras,..). Small randomized phase II trials of both non-selected and enriched patient populations will help to adequately identify potentially active new agents. Phase III trials should only be initiated in appropriate patients based on strong clinical and biological grounds. In this context, the need for further collaborative research is highly warranted.

#### **5. References**


Pancreatic cancer continues to be a major challenge for oncologists as it is a highly chemoresistant malignancy carrying an extremely poor prognosis. Despite the intense research carried out over the last decades no major improvements have been achieved in patient's outcomes. Most patients present with locally advanced or metastatic disease and will therefore require systemic therapy. Conventional chemotherapy modestly improves survival and quality of life of patients with advanced disease. Gemcitabine has been the reference treatment for over a decade and little progress has been made since its introduction in clinical practice in 1997. Gemcitabine-combination therapy with capecitabine, platinum agents or erlotinib may be considered in patients with good performance status, although the small magnitude of benefit they confer shall be balanced against the increased toxicity they induce, particularly considering that prognosis is in any case rather poor and symptomatic relief shall be a major objective of disease management. FOLFIRINOX may be a preferred option for carefully selected fit patients, particularly those

Nevertheless, there is much room for improvement, and more efforts in basic, translational and clinical research will be necessary in the following years for progress to be made. Indeed, a better understanding of the biology of pancreatic cancer shall enable the discovery of new targets of potential diagnostic or therapeutic interest. Meanwhile, as the molecular pathways governing pancreatic cancer are unraveled, efforts shall be made to improve selection of patients most likely to benefit from specific therapies (SPARC, kras,..). Small randomized phase II trials of both non-selected and enriched patient populations will help to adequately identify potentially active new agents. Phase III trials should only be initiated in appropriate patients based on strong clinical and biological grounds. In this context, the

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**7** 

Yang Bo

*China* 

**Immunotherapy of** 

**the Pancreatic Cancer** 

*HepatoBiliary Department of Surgery* 

*3rd Affiliated Hospital of Soochow University, Changzhou, Jiangsu,* 

Pancreatic cancer, which we refer to pancreatic ductal adenocarcinoma, is the forth most common cause of cancer-realated-death disease. In 2010, there were 43,140 new cases and 36,800 patients died of pancreatic cancer in USA(1). Although surgical resection may be the only available treatment for this horrible disease, there are beyond 80% patients when dignosised cannot be cured by surgical treatment(2). In the past 50 years, despite of the progress in surgery skill, hospital morbidity and mortality rates were decreased from 59% and 24% to 36% and 2%, respectively(3) , in the patients received the most optimal surgical operation, the median survival ranged from 15 to 19 months, and 5-year survival rate was still approximately 20%(4). Even if Gemcitabine became the new standard of chemotherapy in pancreatic cancer in 1997(5). The outcome of the pancreatic cancer is still dismal, overall five-year survival rate is blow 5%. With an increasing incidence of pancreatic cancer in the world and conventional treatments often have limited effects and substantial toxicity, a strong need exists for novel therapies. Biological approaches, including gene therapy and immunotherapy, which are targeting pancreatic cancer at a molecular or protein level, are rapidly evolving and seem to be promising strategies for this devastating and virtually

**1. Introduction** 

unexceptionally lethal malignancy.

**2. Immune target and Immune response in pancreatic cancer** 

immunologically distinct and potential targets for the host immune system.

ras oncogene plays a very important role in tumor origination and progression(8).

Cancer is fundamentally a gene associated disease, it has become increasingly clear that some genomic instability and aberrant gene expression lead to biologic behaviour abnormality in tumor cells. In pancreatic cancer, Several genes have high mutation rate in different phase, so the tumor cell may express abnormal antigens that make them

**K-Ras**: The mutation of K-ras oncogene (homologous to the ras gene of Kirsten murine sarcoma virus) occurs in 75-100% of pancreatic cancer(6). With the progression from minimally dysplasia epithelium(PanIN 1A, 1B) to more severe dysplasia(PanIN 2, 3) and invasive cancer(7), the mutation rate of K-ras oncogene is increaseing successively, denotes k-


## **Immunotherapy of the Pancreatic Cancer**

Yang Bo

*HepatoBiliary Department of Surgery 3rd Affiliated Hospital of Soochow University, Changzhou, Jiangsu, China* 

#### **1. Introduction**

108 Pancreatic Cancer – Clinical Management

Tsavaris N, Kosmas C, Skopelitis H, et al. Secon-line treatment with oxaliplatin, leucovorin

Van Cutsem E, van de Velde H, Karasek P, Oettle H, Vervenne WL, Szawlowski A et al.

Vogelstein B & Kinszler KW. Cancer genes and the pathways they control. *Nat Med* 2004;

Von Hoff DD, Ramanathan R, Borad M, Laheru D, Smith L, Wood T et al. SPARC

Von Hoff DD, Ramanathan RK, Borad MJ, Laheru DA, Smith LS, Wood TE, Korn RL, Desai

Walkins DJ, Starling N, Chau I, Thomas J, Webb J, Oates JR et al. The combination of

adenocarcinoma: the TARGET study. J Clin Oncol 2010; 28 (15 Suppl): 4036. Xiong HQ, Varadhachary GR, Blais JC, Hess KR, Abbruzzese JL and Wolff RA. Phase II trial

Yi SY, Park YS, Kim HS, Jun HJ, Kim KH, Chang MH et al. Irinotecan monotherapy as

Zhang Y, Yang Q, Jiang Z, Ma W, Zhou S, Xie de R. Overall survival of patients with

placebo in advanced pancreatic cancer. J Clin Oncol 2004; 22:1430-38. Van Cutsem E, Vervenne WL, Bennouna J et al. Phase III trial of bevacizumab in

II study. Invest New Drugs 2005; 23:369-75.

cancer. J Clin Oncol 2009; 27:2231-37.

ahead of print] PubMed PMID: 21969517

Pharmacology 2009; 63: 1141-5.

2011; 12(2):131-7.

advanced pancreatic cancer. Cancer 2008; 113: 2046-52.

10:789-99.

27 (15 Suppl): 4525.

and 5-fluorouracil in gemcitabine-pretreated advanced pancreatic cancer: A phase

Phase III trial of gemcitabine plus tipifarnib compared with gemcitabine plus

combination with gemcitabine and erlotinib in patients with metastatic pancreatic

correlation with response to gemcitabine (G) plus nab-paclitaxel (nab-P) in patients with advanced metastatic pancreatic cancer: a phase I/II study. J Clin Oncol 2009;

N, Trieu V, Iglesias JL, Zhang H, Soon-Shiong P, Shi T, Rajeshkumar NV, Maitra A, Hidalgo M. Gemcitabine Plus nab-Paclitaxel Is an Active Regimen in Patients With Advanced Pancreatic Cancer: A Phase I/II Trial. J Clin Oncol. 2011 Oct 3. [Epub

chemotherapy doublet (gemcitabine plus capecitabine) with a biologic doublet (bevacizumab plus erlotinib) in patients with advanced pancreatic

of oxaliplatin plus capecitabine (XELOX) as second-line therapy for patients with

second-line treatment in advanced pancreatic cancer. Cancer Chemotherapy

advanced pancreatic cancer improved with an increase in second-line chemotherapy after gemcitabine-based therapy. Journal of pancreatic cancer (JOP) Pancreatic cancer, which we refer to pancreatic ductal adenocarcinoma, is the forth most common cause of cancer-realated-death disease. In 2010, there were 43,140 new cases and 36,800 patients died of pancreatic cancer in USA(1). Although surgical resection may be the only available treatment for this horrible disease, there are beyond 80% patients when dignosised cannot be cured by surgical treatment(2). In the past 50 years, despite of the progress in surgery skill, hospital morbidity and mortality rates were decreased from 59% and 24% to 36% and 2%, respectively(3) , in the patients received the most optimal surgical operation, the median survival ranged from 15 to 19 months, and 5-year survival rate was still approximately 20%(4). Even if Gemcitabine became the new standard of chemotherapy in pancreatic cancer in 1997(5). The outcome of the pancreatic cancer is still dismal, overall five-year survival rate is blow 5%. With an increasing incidence of pancreatic cancer in the world and conventional treatments often have limited effects and substantial toxicity, a strong need exists for novel therapies. Biological approaches, including gene therapy and immunotherapy, which are targeting pancreatic cancer at a molecular or protein level, are rapidly evolving and seem to be promising strategies for this devastating and virtually unexceptionally lethal malignancy.

#### **2. Immune target and Immune response in pancreatic cancer**

Cancer is fundamentally a gene associated disease, it has become increasingly clear that some genomic instability and aberrant gene expression lead to biologic behaviour abnormality in tumor cells. In pancreatic cancer, Several genes have high mutation rate in different phase, so the tumor cell may express abnormal antigens that make them immunologically distinct and potential targets for the host immune system.

**K-Ras**: The mutation of K-ras oncogene (homologous to the ras gene of Kirsten murine sarcoma virus) occurs in 75-100% of pancreatic cancer(6). With the progression from minimally dysplasia epithelium(PanIN 1A, 1B) to more severe dysplasia(PanIN 2, 3) and invasive cancer(7), the mutation rate of K-ras oncogene is increaseing successively, denotes kras oncogene plays a very important role in tumor origination and progression(8).

Immunotherapy of the Pancreatic Cancer 111

normal and tumor cells, there is a remarkable diversity in oligosaccharide moieties between

**Mesothelin:** Mesothelin is a 40-kDa glycosyl phosphatidylinositol anchored cell surface protein and is a c-terminus menbrane-bound form of a 69-kDa precursor protein encoded by the Mesothelin gene(MSLN). Normally, mesothelin is only expressed on mesothelial cells which lining peritoneal, pleural and pericardial cavities(18). The biologic functions are not clearly understood. Some early studies have shown mesothelin playing role in tumor adhesion and dissemination(19). In pancreaticobiliary adenocarcinomas, the expression rate of mesotheline is 100%, whereas none in normal pancreas and chronic pancreatitis(20).

In the past 50 years, with the advances in cellular, molecular biology of cancer and development of immunology, people comes to realizes the relationship between tumor and immune cells is just like a cat and mouse game. The human immune system assume the responsibility to get rid of the extrinsic and endogenic abnormal antigen, it can produce actived immunocyte or immune material such as antibody to react anomalous antigen and

finally eliminate the target, but the fact is not under our desire. (Picture 2)

Picture 2. Immune system:From Robert A. Weinberg, The Biology of Cancer. 2007

At the genesis of the cancer, under ideal condition, the innate immune system responds to "danger signals", macrophages and fibroblasts are enlisted to construct the microenvironment surrounding the cancer cell, just like inflmmation, many cytokines and growth factors are produced to activate innate effector cells with antitumor activity, stimulate professional antigen-presenting cells (APCs, mainly dendritic cell) to capture tumor-derived antigens and migrate to draining lymph nodes to priming an adaptive response by activating T and B lymphocytes. Unfortunately, the growth factors can also

normal and cancer cells(17).

**2.1 Immune escape and immunosuppression** 

Picture 1. Associated genes in pancreatic cancer progression. from Paula Ghaneh, et al. Biology and management of pancreatic cancer. Gut 2007;56:1134-1152.

K-ras gene encodes a 21 kDa membrane-bound guanosine triphosphate(GTP) –binding protein. Before localization at cell membrane, K-ras protein must be farnesylated or geranylgeranylated on the same cysteine residue, it is involved in the transduction of signal from growth factor receptors and other signal inputs, as an upstream activator, it will activat several signaling pathways including Raf/MEK/ERK, P13K/Akt and RalEGF/Ral(9)to regulate gene expression and prevent apoptosis. The mutation of the K-ras oncogene, which occers mostly at codon 12 but also occasionally at codon 13 and 61, will lead to impaired GTPhosphatase(GTPase) activity, resulting in lock the protein locked in GTP-bound state and thus activating downstream signalling cascades(10). According to the META Analyse, point mutation occurred in codon 12 mainly divided into several types, the wild type GGT is replaced by GAT(47%), GTT(28%), CGT(15%), TGT(7%), AGT(2%) and GCT(1%). so in the protein, the 12th amino acid Guanine is replaced by Aspartic acid, Valine, Arginine, Cysteine, Alanine, Serine(11). The K-RAS function changes due to the abnormality in protein structure. The mutation also provide the epitope which might be the target in immunotherapy.

**MUC1:** Mucins are large glycoproteins with carbohydrate content and marked diversity both in the apoprotein and in the oligosaccharide moieties(12). MUC1 is a heavily glycosylated typeⅠmembrane protein with several extracellular tandem repeat domains, which is expressed by nearly all human glandular epithelial and its expression is limited to the apical membrane of the cells. In pancreatic cancer, MUC1 expression is upregulated with an expression pattern over the entire cell surface(13). The core peptide of MUC1 not only serves as a counter-receptor for myelin-associated glycoprotein in pancreatic cancer and is related to perineural invasion(14), but also block death receptor-mediated apoptosis by binding to caspase 8 and FADD(15). MUC1 molecular has sialic acid-containing oligosaccharides in a highly O-glycosylated tandem-repeat domain, the structure has wide range and a large molecular weight(16). Although the core protein of MUC1 is similar in both

Picture 1. Associated genes in pancreatic cancer progression. from Paula Ghaneh, et al.

K-ras gene encodes a 21 kDa membrane-bound guanosine triphosphate(GTP) –binding protein. Before localization at cell membrane, K-ras protein must be farnesylated or geranylgeranylated on the same cysteine residue, it is involved in the transduction of signal from growth factor receptors and other signal inputs, as an upstream activator, it will activat several signaling pathways including Raf/MEK/ERK, P13K/Akt and RalEGF/Ral(9)to regulate gene expression and prevent apoptosis. The mutation of the K-ras oncogene, which occers mostly at codon 12 but also occasionally at codon 13 and 61, will lead to impaired GTPhosphatase(GTPase) activity, resulting in lock the protein locked in GTP-bound state and thus activating downstream signalling cascades(10). According to the META Analyse, point mutation occurred in codon 12 mainly divided into several types, the wild type GGT is replaced by GAT(47%), GTT(28%), CGT(15%), TGT(7%), AGT(2%) and GCT(1%). so in the protein, the 12th amino acid Guanine is replaced by Aspartic acid, Valine, Arginine, Cysteine, Alanine, Serine(11). The K-RAS function changes due to the abnormality in protein structure. The mutation also provide the epitope which might be the target in

**MUC1:** Mucins are large glycoproteins with carbohydrate content and marked diversity both in the apoprotein and in the oligosaccharide moieties(12). MUC1 is a heavily glycosylated typeⅠmembrane protein with several extracellular tandem repeat domains, which is expressed by nearly all human glandular epithelial and its expression is limited to the apical membrane of the cells. In pancreatic cancer, MUC1 expression is upregulated with an expression pattern over the entire cell surface(13). The core peptide of MUC1 not only serves as a counter-receptor for myelin-associated glycoprotein in pancreatic cancer and is related to perineural invasion(14), but also block death receptor-mediated apoptosis by binding to caspase 8 and FADD(15). MUC1 molecular has sialic acid-containing oligosaccharides in a highly O-glycosylated tandem-repeat domain, the structure has wide range and a large molecular weight(16). Although the core protein of MUC1 is similar in both

Biology and management of pancreatic cancer. Gut 2007;56:1134-1152.

immunotherapy.

normal and tumor cells, there is a remarkable diversity in oligosaccharide moieties between normal and cancer cells(17).

**Mesothelin:** Mesothelin is a 40-kDa glycosyl phosphatidylinositol anchored cell surface protein and is a c-terminus menbrane-bound form of a 69-kDa precursor protein encoded by the Mesothelin gene(MSLN). Normally, mesothelin is only expressed on mesothelial cells which lining peritoneal, pleural and pericardial cavities(18). The biologic functions are not clearly understood. Some early studies have shown mesothelin playing role in tumor adhesion and dissemination(19). In pancreaticobiliary adenocarcinomas, the expression rate of mesotheline is 100%, whereas none in normal pancreas and chronic pancreatitis(20).

#### **2.1 Immune escape and immunosuppression**

In the past 50 years, with the advances in cellular, molecular biology of cancer and development of immunology, people comes to realizes the relationship between tumor and immune cells is just like a cat and mouse game. The human immune system assume the responsibility to get rid of the extrinsic and endogenic abnormal antigen, it can produce actived immunocyte or immune material such as antibody to react anomalous antigen and finally eliminate the target, but the fact is not under our desire. (Picture 2)

Picture 2. Immune system:From Robert A. Weinberg, The Biology of Cancer. 2007

At the genesis of the cancer, under ideal condition, the innate immune system responds to "danger signals", macrophages and fibroblasts are enlisted to construct the microenvironment surrounding the cancer cell, just like inflmmation, many cytokines and growth factors are produced to activate innate effector cells with antitumor activity, stimulate professional antigen-presenting cells (APCs, mainly dendritic cell) to capture tumor-derived antigens and migrate to draining lymph nodes to priming an adaptive response by activating T and B lymphocytes. Unfortunately, the growth factors can also

Immunotherapy of the Pancreatic Cancer 113

beta positive) tumors but not Eso2 (TGF-beta negative). Naive CD4+25-Foxp3- T cells, when adoptively had transferred into Rag-/- mice, were converted into Foxp3+ Treg in the presence of Pan02 but not Eso2 tumors. Induction of Treg in Pan02 mice was blocked by systemic injection of an anti-TGF-beta antibody. If Rag-/- mice were instead reconstituted with naive CD4+25- T cells expressing a mutated TGF-beta receptor, induction of Foxp3+ Treg in Pan02 bearing mice was blocked. Collectively, The observations supported the role

Recent studies have shown that increased numbers of tumor-infiltrating Tregs were associated with poorer prognosis in pancreatic cancer(37)(38), so the presence of Tregs in pancreatic cancers highlighes the importance of targeting the suppressive function of these

In Yamamoto's study, a cytotoxicity assay, enzyme-linked immunosorbent spot (ELISPOT) assay and measuring cytokine secretion, were used to study the efficacy of Treg depletion by anti-CD25 antibody added to a dendritic cell/tumor cell (DC/TC) fusion hybrid vaccine in a murine pancreatic cancer model. All the mice treated with the combined therapy of fusion hybrid vaccine and Treg depletion rejected tumor growth in a challenging test, although the rejection rate was 20% both for mice that received the fusion hybrids alone or Treg depletion alone. In addition, combined therapy showed a significantly improved survival in comparison to other treatment or control groups. The NK cell activity for DC/TC fusion + Treg depletion was significantly higher than that for the other treatment groups. Cytotoxic T lymphocyte (CTL) activity for DC/TC could potentially be enhanced by the addition of Treg depletion therapy. The treatments including DC/TC fusion induced IFNgamma secreting effector cells in ELISPOT assays. Furthermore, a cytometric beads array assay used to measure cytokine secretion showed that DC/TC fusion + Treg depletion stimulated the highest levels of IFN-gamma Th1/Th2 ratios and Th17. The results demonstrate that Treg depletion combined with DC/TC fusion hybrid vaccine enhanced the

efficacy of immunotherapy in pancreatic cancer by activating CTLs and NK cells(39).

depleting therapies synergize to enhance vaccine efficacy(41).

In both human pancreatic adenocarcinoma and a murine pancreatic tumor model (Pan02), tumor cells produce increased levels of ligands for the CCR5 chemokine receptor and, reciprocally, CD4(+) Foxp3(+) Tregs, compared with CD4(+) Foxp3(-) effector T cells, preferentially express CCR5. When CCR5/CCL5 signaling is disrupted, either by reducing CCL5 production by tumor cells or by systemic administration of a CCR5 inhibitor , Treg migration to tumors is reduced and tumors are smaller than in control mice. Thus, the study demonstrates the importance of Tregs in immune evasion by tumors, how blockade of Treg migration might inhibit tumor growth, and, specifically in pancreatic adenocarcinoma, the role of CCR5 in the homing of tumor-associated Tregs. Selective targeting of CCR5/CCL5 signaling may represent a novel immunomodulatory strategy for the treatment of cancer(40). In murine mesothelin-expressing pancreatic tumor model (Panc02), vaccine with the immune-relevant mesothelin-derived peptides and in sequence with low-dose cyclophosphamide (CY) and an anti-CD25 IL-2Rα monoclonal antibody (PC61), which are known to deplete subpopulations of T regulatory cells (Tregs), showed that combined Treg-

**Myeloid-derived suppressor cells:** Myeloid-derived suppressor cells(MDSCs) are a heterogeneous population of cells that expand during cancer, inflammation and infection,

of TGF-beta in the induction of Treg in pancreas adenocarcinoma(36).

cells in future immunotherapy research.

stimulate cancer cells proliferation and progression. The cancer cells are so clever that can learn to avoid detection or to escape or overwhelm the immune response. Immunosuppressive tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg) reside in tumors, and their products along with tumor derived products (such as VEGF, TGFbeta and IL-10), create a microenvironment that resists immune activation and attack.

Many strategies are found to escape from immune surveillance(21). 1) Physical exclusion of immune cells from tumor site. It has been proved in epithelial cancer that basal-membranelike structures around the tumor can prevent lymphocytes from infiltrating and tumorspecific T cells expanding(22). 2) Poor immunogenicity by reducing expression of major histocompatability complex(MHC) or co-stimulatory proteins(23) and disruption of natural killer(NK) and natural killer T (NKT) cell recognition(24). The other ways are to change themselves by losing whole protein or TAA expression, which changes in immunodominant T-cell epitopes that alter T-cell recognition, antigen processing or binging to the MHC. 3) Secreting soluble immunosuppressive proteins such as interleukin (IL-10) to prevent inflammatory response from triggering, or vascular endothelial growth factor(VEGF) to interfere with dendritic cells(DC) activation and differentiation(25). 4) Increasing expression of STAT3 protein to block the production of pro-inflammatory molecules(26).

If the specific reaction had been established, being attacked by activated NK cells, antibodies or cytotoxic T lymphocytes, cancer cells can escape elimination according to downregulating targeted antigens, rendering tuomr-reative cell anergic(27), or inducing responding T cell apotosis specifically. The pro-apoptotic function of FasL on carcinoma cells has been demonstrated in both in virto and in vivo, FasL expressed cancer cells can induce apoptosis of lymphocytes in Fas-dependent manner(28), and in patient's biopsies, the present of FasL on cancer cells is in parallel with reduced number(29) and apoptosis (30)of tumor-infiltrating immune cells (TICs). In pancreatic ductal carcinoma, the expression of FasL is 82% in primary versus 100% in hepatic metastases and is associate with shorter survival(31). At last, the eventual developed tumor reflects immunoediting with selection of poorly immunogenic and/or immune-resistant malignant cells(32).

**Treg cells:** CD4+25+Foxp3+ regulatory T cells (Tregs) have been discovered in the 1960s, which can suppres T-cell response and compromise the development of effective tumor immunity(33). these cells are distinguished in high expressed CD4, CD25, CTLA-4, the glucocorticoid-induced TNF-related receptor (GITR) and the forkhead transcription Foxp3. they can arise in response to persistent antigen stimulation in the absence of inflammatory signals, especially in the presence of TGF-β(34). The tumor-induced expansion of regulatory T cells by conversion of CD4+CD25+ lymphocytes is thymus and proliferation independent(35).

Tregs play a critical role in the induction of tolerance to tumor-associated antigens and suppression of antitumor immunity. Additional evidence showed that Tregs were increased locally within the tumor microenvironment by a mechanism that seems dependent on TGFbeta receptor expression and the presence of tumor derived TGF-beta. The murine pancreas cancer cell line Pan02 produces high levels of TGF-beta both in vitro and in vivo. In contrast, the esophageal murine cancer cell line, Eso2, does not. Immunohistochemical staining of Foxp3 in explanted tumors showed an identifiable population of Treg in the Pan02 (TGF-

stimulate cancer cells proliferation and progression. The cancer cells are so clever that can learn to avoid detection or to escape or overwhelm the immune response. Immunosuppressive tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg) reside in tumors, and their products along with tumor derived products (such as VEGF, TGFbeta and IL-10), create a microenvironment that

Many strategies are found to escape from immune surveillance(21). 1) Physical exclusion of immune cells from tumor site. It has been proved in epithelial cancer that basal-membranelike structures around the tumor can prevent lymphocytes from infiltrating and tumorspecific T cells expanding(22). 2) Poor immunogenicity by reducing expression of major histocompatability complex(MHC) or co-stimulatory proteins(23) and disruption of natural killer(NK) and natural killer T (NKT) cell recognition(24). The other ways are to change themselves by losing whole protein or TAA expression, which changes in immunodominant T-cell epitopes that alter T-cell recognition, antigen processing or binging to the MHC. 3) Secreting soluble immunosuppressive proteins such as interleukin (IL-10) to prevent inflammatory response from triggering, or vascular endothelial growth factor(VEGF) to interfere with dendritic cells(DC) activation and differentiation(25). 4) Increasing expression

If the specific reaction had been established, being attacked by activated NK cells, antibodies or cytotoxic T lymphocytes, cancer cells can escape elimination according to downregulating targeted antigens, rendering tuomr-reative cell anergic(27), or inducing responding T cell apotosis specifically. The pro-apoptotic function of FasL on carcinoma cells has been demonstrated in both in virto and in vivo, FasL expressed cancer cells can induce apoptosis of lymphocytes in Fas-dependent manner(28), and in patient's biopsies, the present of FasL on cancer cells is in parallel with reduced number(29) and apoptosis (30)of tumor-infiltrating immune cells (TICs). In pancreatic ductal carcinoma, the expression of FasL is 82% in primary versus 100% in hepatic metastases and is associate with shorter survival(31). At last, the eventual developed tumor reflects immunoediting with selection of

**Treg cells:** CD4+25+Foxp3+ regulatory T cells (Tregs) have been discovered in the 1960s, which can suppres T-cell response and compromise the development of effective tumor immunity(33). these cells are distinguished in high expressed CD4, CD25, CTLA-4, the glucocorticoid-induced TNF-related receptor (GITR) and the forkhead transcription Foxp3. they can arise in response to persistent antigen stimulation in the absence of inflammatory signals, especially in the presence of TGF-β(34). The tumor-induced expansion of regulatory T cells by conversion of CD4+CD25+ lymphocytes is thymus and proliferation

Tregs play a critical role in the induction of tolerance to tumor-associated antigens and suppression of antitumor immunity. Additional evidence showed that Tregs were increased locally within the tumor microenvironment by a mechanism that seems dependent on TGFbeta receptor expression and the presence of tumor derived TGF-beta. The murine pancreas cancer cell line Pan02 produces high levels of TGF-beta both in vitro and in vivo. In contrast, the esophageal murine cancer cell line, Eso2, does not. Immunohistochemical staining of Foxp3 in explanted tumors showed an identifiable population of Treg in the Pan02 (TGF-

of STAT3 protein to block the production of pro-inflammatory molecules(26).

poorly immunogenic and/or immune-resistant malignant cells(32).

resists immune activation and attack.

independent(35).

beta positive) tumors but not Eso2 (TGF-beta negative). Naive CD4+25-Foxp3- T cells, when adoptively had transferred into Rag-/- mice, were converted into Foxp3+ Treg in the presence of Pan02 but not Eso2 tumors. Induction of Treg in Pan02 mice was blocked by systemic injection of an anti-TGF-beta antibody. If Rag-/- mice were instead reconstituted with naive CD4+25- T cells expressing a mutated TGF-beta receptor, induction of Foxp3+ Treg in Pan02 bearing mice was blocked. Collectively, The observations supported the role of TGF-beta in the induction of Treg in pancreas adenocarcinoma(36).

Recent studies have shown that increased numbers of tumor-infiltrating Tregs were associated with poorer prognosis in pancreatic cancer(37)(38), so the presence of Tregs in pancreatic cancers highlighes the importance of targeting the suppressive function of these cells in future immunotherapy research.

In Yamamoto's study, a cytotoxicity assay, enzyme-linked immunosorbent spot (ELISPOT) assay and measuring cytokine secretion, were used to study the efficacy of Treg depletion by anti-CD25 antibody added to a dendritic cell/tumor cell (DC/TC) fusion hybrid vaccine in a murine pancreatic cancer model. All the mice treated with the combined therapy of fusion hybrid vaccine and Treg depletion rejected tumor growth in a challenging test, although the rejection rate was 20% both for mice that received the fusion hybrids alone or Treg depletion alone. In addition, combined therapy showed a significantly improved survival in comparison to other treatment or control groups. The NK cell activity for DC/TC fusion + Treg depletion was significantly higher than that for the other treatment groups. Cytotoxic T lymphocyte (CTL) activity for DC/TC could potentially be enhanced by the addition of Treg depletion therapy. The treatments including DC/TC fusion induced IFNgamma secreting effector cells in ELISPOT assays. Furthermore, a cytometric beads array assay used to measure cytokine secretion showed that DC/TC fusion + Treg depletion stimulated the highest levels of IFN-gamma Th1/Th2 ratios and Th17. The results demonstrate that Treg depletion combined with DC/TC fusion hybrid vaccine enhanced the efficacy of immunotherapy in pancreatic cancer by activating CTLs and NK cells(39).

In both human pancreatic adenocarcinoma and a murine pancreatic tumor model (Pan02), tumor cells produce increased levels of ligands for the CCR5 chemokine receptor and, reciprocally, CD4(+) Foxp3(+) Tregs, compared with CD4(+) Foxp3(-) effector T cells, preferentially express CCR5. When CCR5/CCL5 signaling is disrupted, either by reducing CCL5 production by tumor cells or by systemic administration of a CCR5 inhibitor , Treg migration to tumors is reduced and tumors are smaller than in control mice. Thus, the study demonstrates the importance of Tregs in immune evasion by tumors, how blockade of Treg migration might inhibit tumor growth, and, specifically in pancreatic adenocarcinoma, the role of CCR5 in the homing of tumor-associated Tregs. Selective targeting of CCR5/CCL5 signaling may represent a novel immunomodulatory strategy for the treatment of cancer(40).

In murine mesothelin-expressing pancreatic tumor model (Panc02), vaccine with the immune-relevant mesothelin-derived peptides and in sequence with low-dose cyclophosphamide (CY) and an anti-CD25 IL-2Rα monoclonal antibody (PC61), which are known to deplete subpopulations of T regulatory cells (Tregs), showed that combined Tregdepleting therapies synergize to enhance vaccine efficacy(41).

**Myeloid-derived suppressor cells:** Myeloid-derived suppressor cells(MDSCs) are a heterogeneous population of cells that expand during cancer, inflammation and infection,

Immunotherapy of the Pancreatic Cancer 115

Treatment of tumor-bearing mice with CDDO-Me did not affect the proportion of MDSCs in the spleens but eliminated their suppressive activity. This effect was independent of antitumor activity. CDDO-Me treatment decreased tumor growth in mice. Experiments with severe combined immunodeficient-beige mice indicated that this effect was largely mediated by the immune system. CDDO-Me substantially enhanced the antitumor effect of a cancer vaccines. treatment of pancreatic cancer patients with the synthetic triterpenoid (CDDO-Me) didn't affect the number of MDSCs in peripheral blood but significantly improved the immune response. The research demonstrated MDSCs is the key of the

**3. Nonspecific immunotherapy - Innate Immune system and cytokine** 

Nature kill cells are the central component of the innate immunity and play an important role in cancer immunosurveilance. It has been reported that NK cells can recognize and control tumor growth by direct cellular cytotoxicity and secrete immunostimulatory cytokines such as IFN-γ. The further researches have demonstrated NK cells can eliminate tumor cell by inhibiting cellular proliferation, angiogenesis, promoting apoptosis and stimulate the adaptive immune system. In mouse experimental models, NK cell-mediated elimination of tumor cells induced the subsequent development of tumor-specific T cell responses to the parental tumor cells as a bridge between innate and adaptive immune

In 1984, K. Funa has found patients with pancreatic adenocarcinomas expressing deficiencies in the NK-IFN system at least three levels:(1)diminished basal NK activities, (2)decreased sensitivity of NK to IFN in virto, (3)decreased atypical IFN production by

In a recent clinical trial, a patient exhibited regression of several pancreatic cancer metastases following the administration of the immune modulator Ipilimumab (anti-CTLA-4 antibody). Tumor infiltrating lymphocytes (TIL-2742) and an autologous tumor line (TC-2742) were expanded from a regressing metastatic lesion excised from this patient. Natural killer (NK) cells predominated in the TIL (92% CD56(+)) with few T cells (12% CD3(+)). A majority (88%) of the NK cells were CD56(bright)CD16(-). TIL-2742 secreted IFN-γ and GM-CSF following co-culture with TC-2742 and major histocompatibility complex mismatched pancreatic tumor lines. After sorting TIL-2742, the purified CD56(+)CD16(-)CD3(-) subset showed reactivity similar to TIL-2742 while the CD56(-)CD16(-)CD3(+) cells exhibited no tumor recognition. In co-culture assays, TIL-2742 and the NK subset expressed high reactivity to several pancreatic cancer cell lines and could lyse the autologous tumor as well as pancreas cancer lines. Reactivity was partially abrogated by blockade of TRAIL. This represents the first report of CD56(+)CD16(-) NK cells with apparent specificity for pancreatic cancer cell lines and associated with tumor regression following the treatment

Clinical and experimental evidence demonstrate the extent of NK cell activity in peripheral blood is associated with cancer risk in adults(51). In recent years, novel studies have discovered the phenotypic status and functionality of NK cells in tumor site and also in peripheral blood of cancer patients. Research has shown that only a few infiltrating NK cells which are unlikely to greatly contribute to eliminate the tumoe cells(52). Due to NK's

immunotherapy(47).

responses(48).

*staphylococcus aureus* cowan Ⅰ(SACoI)(49).

with an immune modulating agent(50).

and have a remarkable ability to suppress T-cell responses(42). They contributes negative regulation of immune response and can be activated by factor produced by activated T cells and tumor stromal cells(43).

Myeloid derived suppressor cells (MDSC), which are observed with increased prevalence in the peripheral blood and tumor microenvironment of cancer patients, including pancreatic cancer. Accumulation of MDSC in the peripheral circulation has been related to extent of disease, and correlates with stage. MDSC have primarily been implicated in promoting tumor growth by suppressing antitumor immunity. There is also compelling evidence MDSC are also involved in angiogenesis and metastatic spread. Two main subsets of MDSC have been identified in cancer patients: a monocytic subset, characterized by expression of CD14, and a granulocytic subset characterized by expression of CD15. Both subsets of MDSC actively suppress host immunity through a variety of mechanisms including production of reactive oxygen species and arginase. Just as in humans, accumulation of monocytic and granulocytic MDSC has been noted in the bone marrow, spleen, peripheral circulation, and tumors of tumor bearing mice. Successful targeting of MDSC in mice is associated with improved immune responses, delayed tumor growth, improved survival, and increased efficacy of vaccine therapy. By further elucidating mechanisms of MDSC recruitment and maintenance in the tumor environment, strategies could be developed to reverse immune tolerance to tumor(44).

In a comprehensive analysis of circulating myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) in pancreatic, esophageal and gastric cancer Patients Peripheral blood was collected from 131 cancer patients (46 pancreatic, 60 esophageal and 25 gastric) and 54 healthy controls. PBMC were harvested with subsequent flow cytometric analysis of MDSC (HLADR(-) Lin1(low/-) CD33(+) CD11b(+)) and Treg (CD4(+) CD25(+) CD127 (low/-) FoxP3(+)) percentages. MDSCs and Tregs were statistically significantly elevated in pancreatic, esophageal and gastric cancer compared with controls, and MDSC numbers correlated with Treg levels. Increasing MDSC percentage was associated with increased risk of death, and in a multivariate analysis, MDSC level was an independent prognostic factor for survival. A unit increase in MDSC percentage was associated with a 22% increased risk of death (hazard ratio 1. 22,95% confidence interval 1.06-1.41). The result showed MDSCs are an independent prognostic factor for survival(45).

In mice with spontaneous pancreatic tumours, mice with premalignant lesions as well as wild-type mice, Myeloid-derived suppressor cells (MDSC)were analysed. An increase in the frequency of MDSC early in tumour development was detected in lymph nodes, blood and pancreas of mice with premalignant lesions and increased further upon tumour progression. The MDSC from mice with pancreatic tumours have arginase activity and suppress T-cell responses, which represent the hallmark functions of these cells. The study suggests that immune suppressor mechanisms generated by tumours exist as early as premalignant lesions and increase with tumour progression and highlight the importance of blocking these suppressor mechanisms early in the disease in developing immunotherapy protocols(46).

Nagaraj reported use of the synthetic triterpenoid(CDDO-Me) can completely abrogated immune suppressive activity of MDSC in vitro, CDDO-Me reduced reactive oxygen species in MDSCs but did not affect their viability or the levels of nitric oxide and arginase.

and have a remarkable ability to suppress T-cell responses(42). They contributes negative regulation of immune response and can be activated by factor produced by activated T cells

Myeloid derived suppressor cells (MDSC), which are observed with increased prevalence in the peripheral blood and tumor microenvironment of cancer patients, including pancreatic cancer. Accumulation of MDSC in the peripheral circulation has been related to extent of disease, and correlates with stage. MDSC have primarily been implicated in promoting tumor growth by suppressing antitumor immunity. There is also compelling evidence MDSC are also involved in angiogenesis and metastatic spread. Two main subsets of MDSC have been identified in cancer patients: a monocytic subset, characterized by expression of CD14, and a granulocytic subset characterized by expression of CD15. Both subsets of MDSC actively suppress host immunity through a variety of mechanisms including production of reactive oxygen species and arginase. Just as in humans, accumulation of monocytic and granulocytic MDSC has been noted in the bone marrow, spleen, peripheral circulation, and tumors of tumor bearing mice. Successful targeting of MDSC in mice is associated with improved immune responses, delayed tumor growth, improved survival, and increased efficacy of vaccine therapy. By further elucidating mechanisms of MDSC recruitment and maintenance in the tumor environment, strategies could be developed to

In a comprehensive analysis of circulating myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) in pancreatic, esophageal and gastric cancer Patients Peripheral blood was collected from 131 cancer patients (46 pancreatic, 60 esophageal and 25 gastric) and 54 healthy controls. PBMC were harvested with subsequent flow cytometric analysis of MDSC (HLADR(-) Lin1(low/-) CD33(+) CD11b(+)) and Treg (CD4(+) CD25(+) CD127 (low/-) FoxP3(+)) percentages. MDSCs and Tregs were statistically significantly elevated in pancreatic, esophageal and gastric cancer compared with controls, and MDSC numbers correlated with Treg levels. Increasing MDSC percentage was associated with increased risk of death, and in a multivariate analysis, MDSC level was an independent prognostic factor for survival. A unit increase in MDSC percentage was associated with a 22% increased risk of death (hazard ratio 1. 22,95% confidence interval 1.06-1.41). The result showed MDSCs

In mice with spontaneous pancreatic tumours, mice with premalignant lesions as well as wild-type mice, Myeloid-derived suppressor cells (MDSC)were analysed. An increase in the frequency of MDSC early in tumour development was detected in lymph nodes, blood and pancreas of mice with premalignant lesions and increased further upon tumour progression. The MDSC from mice with pancreatic tumours have arginase activity and suppress T-cell responses, which represent the hallmark functions of these cells. The study suggests that immune suppressor mechanisms generated by tumours exist as early as premalignant lesions and increase with tumour progression and highlight the importance of blocking these suppressor mechanisms early in the disease in developing immunotherapy

Nagaraj reported use of the synthetic triterpenoid(CDDO-Me) can completely abrogated immune suppressive activity of MDSC in vitro, CDDO-Me reduced reactive oxygen species in MDSCs but did not affect their viability or the levels of nitric oxide and arginase.

and tumor stromal cells(43).

reverse immune tolerance to tumor(44).

are an independent prognostic factor for survival(45).

protocols(46).

Treatment of tumor-bearing mice with CDDO-Me did not affect the proportion of MDSCs in the spleens but eliminated their suppressive activity. This effect was independent of antitumor activity. CDDO-Me treatment decreased tumor growth in mice. Experiments with severe combined immunodeficient-beige mice indicated that this effect was largely mediated by the immune system. CDDO-Me substantially enhanced the antitumor effect of a cancer vaccines. treatment of pancreatic cancer patients with the synthetic triterpenoid (CDDO-Me) didn't affect the number of MDSCs in peripheral blood but significantly improved the immune response. The research demonstrated MDSCs is the key of the immunotherapy(47).

#### **3. Nonspecific immunotherapy - Innate Immune system and cytokine**

Nature kill cells are the central component of the innate immunity and play an important role in cancer immunosurveilance. It has been reported that NK cells can recognize and control tumor growth by direct cellular cytotoxicity and secrete immunostimulatory cytokines such as IFN-γ. The further researches have demonstrated NK cells can eliminate tumor cell by inhibiting cellular proliferation, angiogenesis, promoting apoptosis and stimulate the adaptive immune system. In mouse experimental models, NK cell-mediated elimination of tumor cells induced the subsequent development of tumor-specific T cell responses to the parental tumor cells as a bridge between innate and adaptive immune responses(48).

In 1984, K. Funa has found patients with pancreatic adenocarcinomas expressing deficiencies in the NK-IFN system at least three levels:(1)diminished basal NK activities, (2)decreased sensitivity of NK to IFN in virto, (3)decreased atypical IFN production by *staphylococcus aureus* cowan Ⅰ(SACoI)(49).

In a recent clinical trial, a patient exhibited regression of several pancreatic cancer metastases following the administration of the immune modulator Ipilimumab (anti-CTLA-4 antibody). Tumor infiltrating lymphocytes (TIL-2742) and an autologous tumor line (TC-2742) were expanded from a regressing metastatic lesion excised from this patient. Natural killer (NK) cells predominated in the TIL (92% CD56(+)) with few T cells (12% CD3(+)). A majority (88%) of the NK cells were CD56(bright)CD16(-). TIL-2742 secreted IFN-γ and GM-CSF following co-culture with TC-2742 and major histocompatibility complex mismatched pancreatic tumor lines. After sorting TIL-2742, the purified CD56(+)CD16(-)CD3(-) subset showed reactivity similar to TIL-2742 while the CD56(-)CD16(-)CD3(+) cells exhibited no tumor recognition. In co-culture assays, TIL-2742 and the NK subset expressed high reactivity to several pancreatic cancer cell lines and could lyse the autologous tumor as well as pancreas cancer lines. Reactivity was partially abrogated by blockade of TRAIL. This represents the first report of CD56(+)CD16(-) NK cells with apparent specificity for pancreatic cancer cell lines and associated with tumor regression following the treatment with an immune modulating agent(50).

Clinical and experimental evidence demonstrate the extent of NK cell activity in peripheral blood is associated with cancer risk in adults(51). In recent years, novel studies have discovered the phenotypic status and functionality of NK cells in tumor site and also in peripheral blood of cancer patients. Research has shown that only a few infiltrating NK cells which are unlikely to greatly contribute to eliminate the tumoe cells(52). Due to NK's

Immunotherapy of the Pancreatic Cancer 117

pancreatic cancer. Also, mesothelin specific T cell responses are detected/enhanced in some patients treated with CG8020/CG2505 immunotherapy. In addition, Cy modulated immunotherapy resulted in median survival in a Gemzar resistant population similar to

Interlukin-2 (IL-2) is a growth factor that stimulates innate immunity cells. Different dose of IL-2 has been proved either enhance or decrease cellular and humoral immune functions. Rosenberg used it developing lymphokine-activated killer(LAK) therapy for cancer(62). In a randomized study, preoperative subcutaneously IL-2 immunotherapy at 12 million IU for 3 consecutive days before surgery is able to abrogate the effects of the surgical trauma and recover a normal immunofunction in pancreatic cancer patients(63). Recombinant interleukin-2 (rIL-2) was used in a study which aimed to evaluate the toxicity of pre- and postoperative rIL-2 treatment and the effects on innate immunity both in peripheral blood and in cancer tissue of patients with resectable pancreatic adenocarcinoma. Seventeen patients received high dose rIL-2 preoperative subcutaneous administration and two low dose postoperative cycles. NK cell and eosinophil count were evaluated in blood and in pancreatic surgical specimens. The result showed toxicity was moderate. In the early postoperative period, blood NK cells and eosinophils significantly increased compared to basal values (p < 0.02). Preoperative high dose rIL-2 administration is able to counteract surgery-induced deficiency of NK cells and eosinophils in peripheral blood in the early postoperative period, although it cannot overcome local mechanisms of immune tumor escape in cancer tissue. The amplification of innate immunity, induced by immunotherapy,

may improve the control of metastatic cells spreading in the perioperative period(64).

As a bridge between innate and adaptive immune response(65), IL-12 is independently identified as natural killer-stimulating factor (NKSF) and cytotoxic lymphocyte maturation factor(66), which induces proliferation of NK and T1 cells and production of cytokines, especially IFN-γ,and also enhances the generation and activity of CTLs, through activation

The combination of IL-12 and IL-27 can modify the polarization of Th2 effectors by both reduction of IL-5, GM-CSF and IL-13 and induction of IFN-gamma production, which lasted after cytokine removal. Besides, the combined treatment functionally modulated the Th2 polarization of CEA-specific CD4(+) T cells and enhanced pre-existing Th1 type

In recent study, IL-12 was coformulated with the biodegradable polysaccharide chitosan which could enhance the antitumor activity of IL-12 while limiting its systemic toxicity. Antitumor efficacy of IL-12 alone and IL-12 coformulated with chitosan (chitosan/IL-12) was assessed in mice bearing established pancreatic (Panc02) tumors. Additional studies involving depletion of immune cell subsets, tumor rechallenge, and CTL activity were designed to elucidate mechanisms of regression and tumor-specific immunity. Coformulation with chitosan increased local IL-12 retention from 1 to 2 days to 5 to 6 days. Weekly i. t. injections of IL-12 alone eradicated ≤10% of established Panc02 tumors, while i. t. chitosan/IL-12 immunotherapy caused complete tumor regression in 80% to 100% of mice. Depletion of CD4(+) or Gr-1(+) cells had no impact on chitosan/IL-12-mediated tumor regression. However, CD8(+) or NK cell depletion completely abrogated antitumor activity. I. t. chitosan/IL-12 immunotherapy generated systemic tumor-specific immunity, as >80%

chemotherapy alone(61).

of STAT4(67).

immunity(68).

inefficient homing into malignant tissues, the situation may be overcome by cytokinemediated activation in immunotherapeutical regimen(53). However, novel studies of tumorassociated NK cells demonstrated a striking phenotype, supporting the notion that tumorinduced alterations of activating NK cell receptor expression may hamper immune surveillance and promote tumor progression.

Bhat R reported the finding:besides its intrinsic oncolytic activity, parvovirus H-1PV is able to enhance NK cell-mediated killing of pancreatic adenocarcinoma cells. The experiment show that H-1PV infection of Panc-1 cells increases NK cell capacity to release IFN-γ, TNF-α and MIP-1α/β. Multiple activating receptors are involved in the NK cell-mediated killing of Panc-1 cells. Indeed, blocking of the natural cytotoxicity receptors-NKp30, 44 and 46 in combination, and NKG2D and DNAM1 alone inhibit the killing of Panc-1 cells. Interestingly, H-1PV infection of Panc-1 cells overcomes the part of inhibitory effects suggesting that parvovirus may induce additional NK cell ligands on Panc-1 cells. The enhanced sensitivity of H-1PV-infected pancreatic adenocarcinoma cells to NK celldependent killing could be traced back to the upregulation of the DNAM-1 ligand, CD155 and to the downregulation of MHC class I expression. The data suggests that NK cells display antitumor potential against PDAC and that H-1PV-based oncolytic immunotherapy could further boost NK cell-mediated immune responses and help to develop a combinatorial therapeutic approach against pancreatic cancer(54).

NK cells can eliminate tumor cells through their ability to mediate antibody-dependent cellular cytotoxicity(ADCC). Nk cell recognition of an antibody-coated target cell results in rapid NK cell activation and degranulation(55). NK-cell mediated ADCC play a part in mechanisms of tumor-targeted mAbs which targeting CD20, Her2/neu, epidermal growth factor receptor(EGFR)(56)(57). Because HLA class Ⅰis a ligand for inhibitory receptor family, killer cell immunoglobulin-like receptor of NK cells(58), loss of HLA class Ⅰexpression can lead to escape of antigen-dependent cytotoxicity of CD8+ CTL and increase the possibility as a target of NK cell cytotoxicity. In pancreatic cancer, total HLA class Ⅰ loss is 6% in primary versus 43% in metastastic tumors;0 in G1, 33% in G2 and 67% in G3(59).

In research of nonspecific immunotherapy, many cytokines were used to elevate the ability of the immune system. it is possible to activate tumor-specific antitumor immune responses by systemic injection of cytokine or introduction of cytokine gene into tumors through activating natural killer(NK) cells and tumor-specific CD4+ T cells and cytotoxic T lymphocytes(CTL). Different cytokines may stimulate antitumor immune responses by different mechanisms.

Granulocyte Marcophage Colony-Stimulating Factor(GM-CSF) and IL-2 are the most popular cytokines used in cancer immunotherapy. GM-CSF, which can stimulate bone marrows differentiating and maturing to neutrophils, monocytes and dendritic cells, is used to generate cancer immunotherapy called GAVX(60). In clinical trials using the GAVX, induction of systemic antitumor immune response and clinical activity was observed in pancreatic cancer, melanoma, and renal cell carcinoma. In a study of combination of chemotherapy and immunotherapy, two GM-CSF secreting pancreas cancer cell lines (CG8020/CG2505) as immunotherapy were administered alone or in sequence with Cy in patients with advanced pancreatic cancer. Results showed GM-CSF secreting pancreas cancer cell lines demonstrated minimal treatment-related toxicity in patients with advanced

inefficient homing into malignant tissues, the situation may be overcome by cytokinemediated activation in immunotherapeutical regimen(53). However, novel studies of tumorassociated NK cells demonstrated a striking phenotype, supporting the notion that tumorinduced alterations of activating NK cell receptor expression may hamper immune

Bhat R reported the finding:besides its intrinsic oncolytic activity, parvovirus H-1PV is able to enhance NK cell-mediated killing of pancreatic adenocarcinoma cells. The experiment show that H-1PV infection of Panc-1 cells increases NK cell capacity to release IFN-γ, TNF-α and MIP-1α/β. Multiple activating receptors are involved in the NK cell-mediated killing of Panc-1 cells. Indeed, blocking of the natural cytotoxicity receptors-NKp30, 44 and 46 in combination, and NKG2D and DNAM1 alone inhibit the killing of Panc-1 cells. Interestingly, H-1PV infection of Panc-1 cells overcomes the part of inhibitory effects suggesting that parvovirus may induce additional NK cell ligands on Panc-1 cells. The enhanced sensitivity of H-1PV-infected pancreatic adenocarcinoma cells to NK celldependent killing could be traced back to the upregulation of the DNAM-1 ligand, CD155 and to the downregulation of MHC class I expression. The data suggests that NK cells display antitumor potential against PDAC and that H-1PV-based oncolytic immunotherapy could further boost NK cell-mediated immune responses and help to develop a

NK cells can eliminate tumor cells through their ability to mediate antibody-dependent cellular cytotoxicity(ADCC). Nk cell recognition of an antibody-coated target cell results in rapid NK cell activation and degranulation(55). NK-cell mediated ADCC play a part in mechanisms of tumor-targeted mAbs which targeting CD20, Her2/neu, epidermal growth factor receptor(EGFR)(56)(57). Because HLA class Ⅰis a ligand for inhibitory receptor family, killer cell immunoglobulin-like receptor of NK cells(58), loss of HLA class Ⅰexpression can lead to escape of antigen-dependent cytotoxicity of CD8+ CTL and increase the possibility as a target of NK cell cytotoxicity. In pancreatic cancer, total HLA class Ⅰ loss is 6% in

In research of nonspecific immunotherapy, many cytokines were used to elevate the ability of the immune system. it is possible to activate tumor-specific antitumor immune responses by systemic injection of cytokine or introduction of cytokine gene into tumors through activating natural killer(NK) cells and tumor-specific CD4+ T cells and cytotoxic T lymphocytes(CTL). Different cytokines may stimulate antitumor immune responses by

Granulocyte Marcophage Colony-Stimulating Factor(GM-CSF) and IL-2 are the most popular cytokines used in cancer immunotherapy. GM-CSF, which can stimulate bone marrows differentiating and maturing to neutrophils, monocytes and dendritic cells, is used to generate cancer immunotherapy called GAVX(60). In clinical trials using the GAVX, induction of systemic antitumor immune response and clinical activity was observed in pancreatic cancer, melanoma, and renal cell carcinoma. In a study of combination of chemotherapy and immunotherapy, two GM-CSF secreting pancreas cancer cell lines (CG8020/CG2505) as immunotherapy were administered alone or in sequence with Cy in patients with advanced pancreatic cancer. Results showed GM-CSF secreting pancreas cancer cell lines demonstrated minimal treatment-related toxicity in patients with advanced

primary versus 43% in metastastic tumors;0 in G1, 33% in G2 and 67% in G3(59).

surveillance and promote tumor progression.

different mechanisms.

combinatorial therapeutic approach against pancreatic cancer(54).

pancreatic cancer. Also, mesothelin specific T cell responses are detected/enhanced in some patients treated with CG8020/CG2505 immunotherapy. In addition, Cy modulated immunotherapy resulted in median survival in a Gemzar resistant population similar to chemotherapy alone(61).

Interlukin-2 (IL-2) is a growth factor that stimulates innate immunity cells. Different dose of IL-2 has been proved either enhance or decrease cellular and humoral immune functions. Rosenberg used it developing lymphokine-activated killer(LAK) therapy for cancer(62). In a randomized study, preoperative subcutaneously IL-2 immunotherapy at 12 million IU for 3 consecutive days before surgery is able to abrogate the effects of the surgical trauma and recover a normal immunofunction in pancreatic cancer patients(63). Recombinant interleukin-2 (rIL-2) was used in a study which aimed to evaluate the toxicity of pre- and postoperative rIL-2 treatment and the effects on innate immunity both in peripheral blood and in cancer tissue of patients with resectable pancreatic adenocarcinoma. Seventeen patients received high dose rIL-2 preoperative subcutaneous administration and two low dose postoperative cycles. NK cell and eosinophil count were evaluated in blood and in pancreatic surgical specimens. The result showed toxicity was moderate. In the early postoperative period, blood NK cells and eosinophils significantly increased compared to basal values (p < 0.02). Preoperative high dose rIL-2 administration is able to counteract surgery-induced deficiency of NK cells and eosinophils in peripheral blood in the early postoperative period, although it cannot overcome local mechanisms of immune tumor escape in cancer tissue. The amplification of innate immunity, induced by immunotherapy, may improve the control of metastatic cells spreading in the perioperative period(64).

As a bridge between innate and adaptive immune response(65), IL-12 is independently identified as natural killer-stimulating factor (NKSF) and cytotoxic lymphocyte maturation factor(66), which induces proliferation of NK and T1 cells and production of cytokines, especially IFN-γ,and also enhances the generation and activity of CTLs, through activation of STAT4(67).

The combination of IL-12 and IL-27 can modify the polarization of Th2 effectors by both reduction of IL-5, GM-CSF and IL-13 and induction of IFN-gamma production, which lasted after cytokine removal. Besides, the combined treatment functionally modulated the Th2 polarization of CEA-specific CD4(+) T cells and enhanced pre-existing Th1 type immunity(68).

In recent study, IL-12 was coformulated with the biodegradable polysaccharide chitosan which could enhance the antitumor activity of IL-12 while limiting its systemic toxicity. Antitumor efficacy of IL-12 alone and IL-12 coformulated with chitosan (chitosan/IL-12) was assessed in mice bearing established pancreatic (Panc02) tumors. Additional studies involving depletion of immune cell subsets, tumor rechallenge, and CTL activity were designed to elucidate mechanisms of regression and tumor-specific immunity. Coformulation with chitosan increased local IL-12 retention from 1 to 2 days to 5 to 6 days. Weekly i. t. injections of IL-12 alone eradicated ≤10% of established Panc02 tumors, while i. t. chitosan/IL-12 immunotherapy caused complete tumor regression in 80% to 100% of mice. Depletion of CD4(+) or Gr-1(+) cells had no impact on chitosan/IL-12-mediated tumor regression. However, CD8(+) or NK cell depletion completely abrogated antitumor activity. I. t. chitosan/IL-12 immunotherapy generated systemic tumor-specific immunity, as >80%

Immunotherapy of the Pancreatic Cancer 119

5-FU based chemoradiation, patient received 5 immunotherapy. The median disease-free survival was 17.3 months with median survival of 24.8 months. The administration of immunotherapy was well tolerated. Besides, the postimmunotherapy induction of mesothelin-specific CD8+ T cells in HLA-A1+ and HLA-A2+ patients correlates with disease-free survival. The research concluded that an immunotherapy approach intergated

**VEGF** Vascular endothelial growth factor receptor 2 (VEGFR2) is an essential factor in tumor angiogenesis and in the growth of pancreatic cancer. Immunotherapy using epitope peptide for VEGFR2 (VEGFR2-169) is expected to improve the clinical outcome. A phase I clinical trial combining of VEGFR2-169 with gemcitabine was conducted for patients with metastatic and unresectable pancreatic cancer. Gemcitabine was administered at a dose of 1000 mg/m(2) on days 1, 8, and 15 in a 28-day cycle. The VEGFR2-169 peptide was subcutaneously injected weekly in a dose-escalation manner (doses of 0.5, 1, and 2 mg/body, six patients/one cohort). No severe adverse effect of grade 4 or higher was observed. Of the 18 patients who completed at least one course of the treatment, 15 (83%) developed immunological reactions at the injection sites. Specific cytotoxic T lymphocytes (CTL) reacting to the VEGFR2-169 peptide were induced in 11 (61%) of the 18 patients. The disease control rate was 67%, and the median overall survival time was 8. 7 months. This combination therapy for pancreatic cancer patients was tolerable at all doses. Peptidespecific CTL could be induced by the VEGFR2-169 peptide vaccine at a high rate, even in combination with gemcitabine. From an immunological point of view, the optimal dose for

**Ras** peptide is the first agent tested in immunetherapy in pancreatic cancer. Gjertsen used an intradermal vaccine of APCs loaded ex vivo with synthetic ras peptide corresponding to the mutation found in patients. In this phase I/II trial , two of five patients with advanced pancreatic cancer showed induced immune response(75). In further phase I/II trial in 48 pancreatic cancer patients with different clinic stages, ras peptide in combination with GM-CSF could induce peptide-specific immunty in 58% patients. Compared to non-responders, survival time were prolonged in patients with advanced disease, the association between prolonged survival and an immune response against the vaccine suggests that a clinical

In 24 Patients with resected pancreatic cancer, with K-ras mutations at codon 12, were vaccinated once monthly for 3 months with a 21-mer peptide vaccine containing the corresponding K-ras mutation of the patient's tumor. Immune responses were evaluated by delayed-type hypersensitivity (DTH) tests and the enzyme-linked immunosorbent spot assays. Results showed there were no grade 3-5 vaccine-specific toxicities. The only National Cancer Institute grade 1 and 2 toxicity was erythema at the injection site (94%). Nine patients (25%) were evaluable for immunologic responses. One patient (11%) had a detectable immune response specific to the patient's K-ras mutation, as assessed by DTH. Three patients (13%) displayed a DTH response that was not specific. Median recurrence free survival time was 8. 6 months (95% confidence interval, 2.96-19.2) and median overall survival time was 20. 3 months (95% confidence interval, 11.6-45.3). It suggested K-ras vaccination for patients with resectable pancreatic adenocarcinoma proved to be safe and

benefit of ras peptide vaccination may be obtained for this group of patients(76).

tolerable with however no elicitable immunogenicity and unproven efficacy(77).

with chemoradiation is safe and helpful for resected pancreas cancer(73).

further clinical trials might be 2 mg/body or higher(74).

of mice cured with i. t. chitosan/IL-12 immunotherapy were at least partially protected from tumor rechallenge. Furthermore, CTLs from spleens of cured mice lysed MC32a and gp70 peptide-loaded targets. The reasearch has demonstrated Chitosan/IL-12 immunotherapy increased local retention of IL-12 in the tumor microenvironment, eradicated established, aggressive murine tumors, and generated systemic tumor-specific protective immunity(69).

#### **4. Specific immunotherapy**

Specific immunotherapy, which seems be more important in cancer treatment research, could be divide into 3 parts:monoclonal antibody, adoptive cellular therapy, and vaccine. Infusion of antibody or activated cells is called Passive Immunotherapy, on the other, vaccine can induce active immunotherapy. The simplest model of immune cell-mediated antigen-specific tumor rejection consists of three elements: appropriate antigen specific for the tumor, efficient antigen presentation and the generation of potent effector cells.

#### **4.1 Active immunotherapy**

**Vaccine:** The development of human therapeutic cancer vaccines has come a long way since the discovery of major histocompatability complex (MHC) restricted tumor antigens. As an new method to reconsituting immunity, cancer vaccionation can actively harness the intrinsic power of the immne system to recognize and destroy tumors. The ideal designed vaccine should actively generate antigen-specific immune response to abnomal protein expressed in tumor cells, including activating distinct components of the immune system:antigen presenting cells, B cells and T cells, producing the advantages of high specificity, minimal toxicity and permanently effective immunologic memory. Antigen could be delivered in the form of DNA or peptide, as well as tumor cells or antigen-pulsed DCs.

**GM-CSF** is an important growth factor for granulocytes and monocytes, and has a crucial role in the growth and differentiation of DCs. Kimura M has found in vivo growth of AsPC-1 cells, which retrovirally transduced with the GM-CSF gene, was inhabited and associated with increased survival of the nude mice(70).

A series of clinical trails have been reported by researchers in John Hopkins in recent 10 years. Jaffee et al conducted a phase Ⅰ study using allogeneic GM-CSF-secreting whole-cell tumor vaccine for pancreatic cancer. As vaccines, Two pancreatic cancer lines(PANC 10.05 and 6.03), which had been genetically modified to express GM-CSF, were given to patients who had undergone pancreaticoduodenectomy eight weeks prior. Three of the eight patients who received ≥10×107 vaccine cells developed post-vaccination delayed-type hypersensitivity (DTH) responses associated with increased disease free survival time, and remained disease-free for longer than 25 months after diagnosis. Side effects were mainly limited to local skin reactions at the site of vaccination(71). Further phase Ⅱ study of 60 patients with resected pancreatic adenocarcinoma, patients received five treatments of 2.5×108 vaccine cells, together with 5-Fu and radiotherapy. The reported median survival was 26 months, with a 1- and 2-year survival of 88% and 76% respectively(72). In latest report, a single institution phase Ⅱ study of 60 patients with resected pancreatic adenocarcinoma was performed, each treatment consisted of a total of 5×108 GM-CSFsecreting cells distributed equally among 3 lymph node regions. Subsequently, had received

of mice cured with i. t. chitosan/IL-12 immunotherapy were at least partially protected from tumor rechallenge. Furthermore, CTLs from spleens of cured mice lysed MC32a and gp70 peptide-loaded targets. The reasearch has demonstrated Chitosan/IL-12 immunotherapy increased local retention of IL-12 in the tumor microenvironment, eradicated established, aggressive murine tumors, and generated systemic tumor-specific protective immunity(69).

Specific immunotherapy, which seems be more important in cancer treatment research, could be divide into 3 parts:monoclonal antibody, adoptive cellular therapy, and vaccine. Infusion of antibody or activated cells is called Passive Immunotherapy, on the other, vaccine can induce active immunotherapy. The simplest model of immune cell-mediated antigen-specific tumor rejection consists of three elements: appropriate antigen specific for

**Vaccine:** The development of human therapeutic cancer vaccines has come a long way since the discovery of major histocompatability complex (MHC) restricted tumor antigens. As an new method to reconsituting immunity, cancer vaccionation can actively harness the intrinsic power of the immne system to recognize and destroy tumors. The ideal designed vaccine should actively generate antigen-specific immune response to abnomal protein expressed in tumor cells, including activating distinct components of the immune system:antigen presenting cells, B cells and T cells, producing the advantages of high specificity, minimal toxicity and permanently effective immunologic memory. Antigen could be delivered in the form of DNA or peptide, as well as tumor cells or antigen-pulsed

**GM-CSF** is an important growth factor for granulocytes and monocytes, and has a crucial role in the growth and differentiation of DCs. Kimura M has found in vivo growth of AsPC-1 cells, which retrovirally transduced with the GM-CSF gene, was inhabited and associated

A series of clinical trails have been reported by researchers in John Hopkins in recent 10 years. Jaffee et al conducted a phase Ⅰ study using allogeneic GM-CSF-secreting whole-cell tumor vaccine for pancreatic cancer. As vaccines, Two pancreatic cancer lines(PANC 10.05 and 6.03), which had been genetically modified to express GM-CSF, were given to patients who had undergone pancreaticoduodenectomy eight weeks prior. Three of the eight patients who received ≥10×107 vaccine cells developed post-vaccination delayed-type hypersensitivity (DTH) responses associated with increased disease free survival time, and remained disease-free for longer than 25 months after diagnosis. Side effects were mainly limited to local skin reactions at the site of vaccination(71). Further phase Ⅱ study of 60 patients with resected pancreatic adenocarcinoma, patients received five treatments of 2.5×108 vaccine cells, together with 5-Fu and radiotherapy. The reported median survival was 26 months, with a 1- and 2-year survival of 88% and 76% respectively(72). In latest report, a single institution phase Ⅱ study of 60 patients with resected pancreatic adenocarcinoma was performed, each treatment consisted of a total of 5×108 GM-CSFsecreting cells distributed equally among 3 lymph node regions. Subsequently, had received

the tumor, efficient antigen presentation and the generation of potent effector cells.

**4. Specific immunotherapy** 

**4.1 Active immunotherapy** 

with increased survival of the nude mice(70).

DCs.

5-FU based chemoradiation, patient received 5 immunotherapy. The median disease-free survival was 17.3 months with median survival of 24.8 months. The administration of immunotherapy was well tolerated. Besides, the postimmunotherapy induction of mesothelin-specific CD8+ T cells in HLA-A1+ and HLA-A2+ patients correlates with disease-free survival. The research concluded that an immunotherapy approach intergated with chemoradiation is safe and helpful for resected pancreas cancer(73).

**VEGF** Vascular endothelial growth factor receptor 2 (VEGFR2) is an essential factor in tumor angiogenesis and in the growth of pancreatic cancer. Immunotherapy using epitope peptide for VEGFR2 (VEGFR2-169) is expected to improve the clinical outcome. A phase I clinical trial combining of VEGFR2-169 with gemcitabine was conducted for patients with metastatic and unresectable pancreatic cancer. Gemcitabine was administered at a dose of 1000 mg/m(2) on days 1, 8, and 15 in a 28-day cycle. The VEGFR2-169 peptide was subcutaneously injected weekly in a dose-escalation manner (doses of 0.5, 1, and 2 mg/body, six patients/one cohort). No severe adverse effect of grade 4 or higher was observed. Of the 18 patients who completed at least one course of the treatment, 15 (83%) developed immunological reactions at the injection sites. Specific cytotoxic T lymphocytes (CTL) reacting to the VEGFR2-169 peptide were induced in 11 (61%) of the 18 patients. The disease control rate was 67%, and the median overall survival time was 8. 7 months. This combination therapy for pancreatic cancer patients was tolerable at all doses. Peptidespecific CTL could be induced by the VEGFR2-169 peptide vaccine at a high rate, even in combination with gemcitabine. From an immunological point of view, the optimal dose for further clinical trials might be 2 mg/body or higher(74).

**Ras** peptide is the first agent tested in immunetherapy in pancreatic cancer. Gjertsen used an intradermal vaccine of APCs loaded ex vivo with synthetic ras peptide corresponding to the mutation found in patients. In this phase I/II trial , two of five patients with advanced pancreatic cancer showed induced immune response(75). In further phase I/II trial in 48 pancreatic cancer patients with different clinic stages, ras peptide in combination with GM-CSF could induce peptide-specific immunty in 58% patients. Compared to non-responders, survival time were prolonged in patients with advanced disease, the association between prolonged survival and an immune response against the vaccine suggests that a clinical benefit of ras peptide vaccination may be obtained for this group of patients(76).

In 24 Patients with resected pancreatic cancer, with K-ras mutations at codon 12, were vaccinated once monthly for 3 months with a 21-mer peptide vaccine containing the corresponding K-ras mutation of the patient's tumor. Immune responses were evaluated by delayed-type hypersensitivity (DTH) tests and the enzyme-linked immunosorbent spot assays. Results showed there were no grade 3-5 vaccine-specific toxicities. The only National Cancer Institute grade 1 and 2 toxicity was erythema at the injection site (94%). Nine patients (25%) were evaluable for immunologic responses. One patient (11%) had a detectable immune response specific to the patient's K-ras mutation, as assessed by DTH. Three patients (13%) displayed a DTH response that was not specific. Median recurrence free survival time was 8. 6 months (95% confidence interval, 2.96-19.2) and median overall survival time was 20. 3 months (95% confidence interval, 11.6-45.3). It suggested K-ras vaccination for patients with resectable pancreatic adenocarcinoma proved to be safe and tolerable with however no elicitable immunogenicity and unproven efficacy(77).

Immunotherapy of the Pancreatic Cancer 121

intradermally and MUC1-CTLs were given intravenously. The result showed one patient with multiple lung metastases experienced a complete response. Five patients had stable disease. The mean survival time was 9.8 months. No grade II-IV toxicity was observed. The research suggested adoptive immunotherapy with MUC1-DC and MUC1-CTL may be

**Mesothelin** It is first reported by Thomas that specific CD8+ T-cell response which targeting mesothelin epitopes in pancreatic cancer can be induced via cross-presentation by an approach that recruits APCs to the vaccination site(85). Combinated with anti-glucocorticoidinduced TNF receptor antibody (anti-GITR), the mesotheline DNA vaccine can induce immune pretection in mice with sungeneic mesothelin-expressing pancreatic cancer. 50% of animals treated with mesothelin were tumor-free 25 days after tumor injection compared to

DNA vaccines employing single-chain trimers (SCT) have been shown to bypass antigen processing and presentation and result in significant enhancement of DNA vaccine potency. In a study, a DNA vaccine employing an SCT targeting human mesothelin and characterized the ensuing antigen-specific CD8+ T cell-mediated immune responses and anti-tumor effects against human mesothelin-expressing tumors in HLA-A2 transgenic mice. The results showed that vaccination with DNA employing an SCT of HLA-A2 linked to human mesothelin epitope aa540-549 (pcDNA3-Hmeso540-beta2m-A2) generated strong human mesothelin peptide (aa540-549)-specific CD8+ T cell immune responses in HLA-A2 transgenic mice. Vaccination with pcDNA3-Hmeso540-beta2m-A2 prevented the growth of HLA-A2 positive human mesothelin-expressing tumor cell lines in HLA-A2 transgenic mice in contrast to vaccination with DNA encoding SCT linked to OVA CTL epitope. Thus, the employment of SCT of HLA-A2 linked to the human mesothelin epitope aa540-549 represents a potential opportunity for the clinical translation of DNA vaccines against

Passive immunotherapy could be accomplished by infusion monoantibody and tumor specific T-cell which was actived in vitro. With advances in structural and functional genomics, recent work has focused on targeted molecular therapy using monoclonal antibodies. Many monoantibodies were used to target molecules on the tumor cell surface and normal tissue stroma, which are related to pancreatic cancer oncogenesis, tumor growth or resistance to chemotherapy, as well as molecules involved in regulating inflammation and host immunoresponses. Although progress made by monoantibody in pancreatic cancer treatment, especially in preclinical studies, its clinical application requires further investigation. Besides the function bind to target antigen to block the corresponding signal transduction pathways, antibody-dependent cellular cytotoxicity (ADCC) can also be

Monoclonal antibodies against human tumor targets were initially in rodents, which will induce immunologic responses from patient against mouse antibodies. With the

human mesothelin-expressing tumors, including pancreatic cancer(87).

feasible and effective for pancreatic cancer(84).

0 in untreated mice(86).

**4.2 Passive immunotherapy** 

**4.2.1 Antibody** 

observed in some pancreatic cancer cell lines.

In another phase Ⅱstudy, a specific mutant ras peptide vaccine was tested as an adjuvant immunotherapy in pancreatic and colorectal cancer patients. Five pancreatic and seven colorectal cancer patients were vaccinated subcutaneously with 13-mer mutant ras peptide, corresponding to their tumor's ras mutation. Vaccinations were given every 4 weeks, up to a total of six vaccines. The result showed no serious acute or delayed systemic side effects were seen. Five out of eleven patients showed a positive immune response. Furthermore, the five pancreatic cancer patients have shown a mean disease-free survival (DFS) of 35. 2+ months and a mean overall survival (OS) of 44.4+ months. The study suggested it is feasible to use mutant ras vaccine in the adjuvant setting. This vaccine is safe, can induce specific immune responses, and it appears to have a positive outcome in overall survival(78). In a follow-up study, Twenty-three patients who were vaccinated after surgical resection for pancreatic adenocarcinoma (22 pancreaticoduodenectomies, one distal resection). The vaccine was composed of long synthetic mutant ras peptides designed mainly to elicit Thelper responses. Seventeen of 20 evaluable patients (85%) responded immunologically to the vaccine. Median survival for all patients was 27.5 months and 28 months for immune responders. The 5-year survival was 22% and 29%, respectively. Strikingly, 10-year survival was 20% (four patients out of 20 evaluable) versus zero (0/87) in a cohort of nonvaccinated patient treated in the same period. Three patients mounted a memory response up to 9 years after vaccination. The observation indicates that K-ras vaccination may consolidate the effect of surgery and represent an adjuvant treatment option for the future(79).

**MUC1** In order to create MUC1-specific immune response, a vaccine composed of MUC1 peptide and SBAS2 adjuvant was tested in a phase Ⅰstudy, There was an increase in the percentage of CD8+ T cells and MUC1-specific antibody(80).

The other approach to induce MUC1-specific immune response is antigen-pulsed DCs. A Phase I/II clinical trial of a MUC1 peptide-loaded DC vaccine was carried out in 12 pancreatic and biliary cancer patients following resection of their primary tumors. The vaccine was well tolerated and no toxicity was observed. Prior to vaccination, patients entered onto this trial had a significantly higher percentage of FoxP3+CD4+T cells compared to age matched healthy controls. The percentage of these cells also increased transiently following each injection, returning to baseline or below before the next injection. Vaccinated patients have been followed for over four years and four of the twelve patients are alive, all without evidence of recurrence(81).

Another phase I/II trial used human autologous DCs transfected with MUC1 cDNA as vaccine, 4 of 10 patients showed a two- to ten-fold increase in the frequency of mucinspecific IFN-γ-secreting CD8+ T cells, suggesting an immune response(82). But in a phase Ⅲ trial of 255 patients using vaccine consisted of recombinant vaccinia and fowlpox viruses coexpressing CEA/MUC1/TRICOM, researchers failed to improve overall survival compared to palliative chemotherapy or best supportive care(83).

In Kondo H's clinical trial, Peripheral blood mononuclear cells (PBMCs) of twenty patients with unresectable or recurrent pancreatic cancer were separated into adherent cells for induction of MUC1-DCs and floating cells for MUC1-CTLs. MUC1-DCs were generated by culture with granulocyte monocyte colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) and then exposed to MUC1 peptide and TNF-alpha. MUC1-CTLs were induced by coculture with YPK-1 and then with interleukin-2 (IL-2). MUC1-DCs were injected

In another phase Ⅱstudy, a specific mutant ras peptide vaccine was tested as an adjuvant immunotherapy in pancreatic and colorectal cancer patients. Five pancreatic and seven colorectal cancer patients were vaccinated subcutaneously with 13-mer mutant ras peptide, corresponding to their tumor's ras mutation. Vaccinations were given every 4 weeks, up to a total of six vaccines. The result showed no serious acute or delayed systemic side effects were seen. Five out of eleven patients showed a positive immune response. Furthermore, the five pancreatic cancer patients have shown a mean disease-free survival (DFS) of 35. 2+ months and a mean overall survival (OS) of 44.4+ months. The study suggested it is feasible to use mutant ras vaccine in the adjuvant setting. This vaccine is safe, can induce specific immune responses, and it appears to have a positive outcome in overall survival(78). In a follow-up study, Twenty-three patients who were vaccinated after surgical resection for pancreatic adenocarcinoma (22 pancreaticoduodenectomies, one distal resection). The vaccine was composed of long synthetic mutant ras peptides designed mainly to elicit Thelper responses. Seventeen of 20 evaluable patients (85%) responded immunologically to the vaccine. Median survival for all patients was 27.5 months and 28 months for immune responders. The 5-year survival was 22% and 29%, respectively. Strikingly, 10-year survival was 20% (four patients out of 20 evaluable) versus zero (0/87) in a cohort of nonvaccinated patient treated in the same period. Three patients mounted a memory response up to 9 years after vaccination. The observation indicates that K-ras vaccination may consolidate the effect

of surgery and represent an adjuvant treatment option for the future(79).

percentage of CD8+ T cells and MUC1-specific antibody(80).

compared to palliative chemotherapy or best supportive care(83).

are alive, all without evidence of recurrence(81).

**MUC1** In order to create MUC1-specific immune response, a vaccine composed of MUC1 peptide and SBAS2 adjuvant was tested in a phase Ⅰstudy, There was an increase in the

The other approach to induce MUC1-specific immune response is antigen-pulsed DCs. A Phase I/II clinical trial of a MUC1 peptide-loaded DC vaccine was carried out in 12 pancreatic and biliary cancer patients following resection of their primary tumors. The vaccine was well tolerated and no toxicity was observed. Prior to vaccination, patients entered onto this trial had a significantly higher percentage of FoxP3+CD4+T cells compared to age matched healthy controls. The percentage of these cells also increased transiently following each injection, returning to baseline or below before the next injection. Vaccinated patients have been followed for over four years and four of the twelve patients

Another phase I/II trial used human autologous DCs transfected with MUC1 cDNA as vaccine, 4 of 10 patients showed a two- to ten-fold increase in the frequency of mucinspecific IFN-γ-secreting CD8+ T cells, suggesting an immune response(82). But in a phase Ⅲ trial of 255 patients using vaccine consisted of recombinant vaccinia and fowlpox viruses coexpressing CEA/MUC1/TRICOM, researchers failed to improve overall survival

In Kondo H's clinical trial, Peripheral blood mononuclear cells (PBMCs) of twenty patients with unresectable or recurrent pancreatic cancer were separated into adherent cells for induction of MUC1-DCs and floating cells for MUC1-CTLs. MUC1-DCs were generated by culture with granulocyte monocyte colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) and then exposed to MUC1 peptide and TNF-alpha. MUC1-CTLs were induced by coculture with YPK-1 and then with interleukin-2 (IL-2). MUC1-DCs were injected intradermally and MUC1-CTLs were given intravenously. The result showed one patient with multiple lung metastases experienced a complete response. Five patients had stable disease. The mean survival time was 9.8 months. No grade II-IV toxicity was observed. The research suggested adoptive immunotherapy with MUC1-DC and MUC1-CTL may be feasible and effective for pancreatic cancer(84).

**Mesothelin** It is first reported by Thomas that specific CD8+ T-cell response which targeting mesothelin epitopes in pancreatic cancer can be induced via cross-presentation by an approach that recruits APCs to the vaccination site(85). Combinated with anti-glucocorticoidinduced TNF receptor antibody (anti-GITR), the mesotheline DNA vaccine can induce immune pretection in mice with sungeneic mesothelin-expressing pancreatic cancer. 50% of animals treated with mesothelin were tumor-free 25 days after tumor injection compared to 0 in untreated mice(86).

DNA vaccines employing single-chain trimers (SCT) have been shown to bypass antigen processing and presentation and result in significant enhancement of DNA vaccine potency. In a study, a DNA vaccine employing an SCT targeting human mesothelin and characterized the ensuing antigen-specific CD8+ T cell-mediated immune responses and anti-tumor effects against human mesothelin-expressing tumors in HLA-A2 transgenic mice. The results showed that vaccination with DNA employing an SCT of HLA-A2 linked to human mesothelin epitope aa540-549 (pcDNA3-Hmeso540-beta2m-A2) generated strong human mesothelin peptide (aa540-549)-specific CD8+ T cell immune responses in HLA-A2 transgenic mice. Vaccination with pcDNA3-Hmeso540-beta2m-A2 prevented the growth of HLA-A2 positive human mesothelin-expressing tumor cell lines in HLA-A2 transgenic mice in contrast to vaccination with DNA encoding SCT linked to OVA CTL epitope. Thus, the employment of SCT of HLA-A2 linked to the human mesothelin epitope aa540-549 represents a potential opportunity for the clinical translation of DNA vaccines against human mesothelin-expressing tumors, including pancreatic cancer(87).

#### **4.2 Passive immunotherapy**

Passive immunotherapy could be accomplished by infusion monoantibody and tumor specific T-cell which was actived in vitro. With advances in structural and functional genomics, recent work has focused on targeted molecular therapy using monoclonal antibodies. Many monoantibodies were used to target molecules on the tumor cell surface and normal tissue stroma, which are related to pancreatic cancer oncogenesis, tumor growth or resistance to chemotherapy, as well as molecules involved in regulating inflammation and host immunoresponses. Although progress made by monoantibody in pancreatic cancer treatment, especially in preclinical studies, its clinical application requires further investigation. Besides the function bind to target antigen to block the corresponding signal transduction pathways, antibody-dependent cellular cytotoxicity (ADCC) can also be observed in some pancreatic cancer cell lines.

#### **4.2.1 Antibody**

Monoclonal antibodies against human tumor targets were initially in rodents, which will induce immunologic responses from patient against mouse antibodies. With the

Immunotherapy of the Pancreatic Cancer 123

patients (39%). Although the addition of cetuximab to the combination of gemcitabine and oxaliplatin is well tolerated, the reasearch failed to increase response or survival in patients

But the effect of the cetuximab is limited by the affinity of expressed EGFR in pancreatic cancer, other factors including mutation of K-ras, PTEN expression or host complement level. these may be the reasons of failure in some trails. In a phase Ⅱ trail, within the cetuximab group and noncetuximab group, no significant differences were found in objective response rate (17.5% Vs12.2%), median progression-free survival (3.4 months Vs 4.2 months), median overall survival (7.5 months Vs 7.8 months)(93), The result can't prove a synergistic effect in combination of cetuximab and gemcitabine/cisplatin treatment in

Another phase Ⅲ trail of Patients with unresectable locally advanced or metastatic pancreatic adenocarcinoma were randomly assigned to receive gemcitabine alone or gemcitabine plus cetuximab. A total of 745 eligible patients were accrued. No significant difference was seen between the two arms of the study with respect to the median survival time (6. 3 months for the gemcitabine plus cetuximab arm v5.9 months for the gemcitabine alone arm; hazard ratio = 1.06; 95% CI, 0.91 to 1.23; P = .23, one-sided). Objective responses and progression-free survival were similar in both arms of the study. Although time to treatment failure was longer in patients on gemcitabine plus cetuximab (P=.006), the difference in length of treatment was only 2 weeks longer in the combination arm. Among patients who were studied for tumoral EGFR expression, 90% were positive, with no treatment benefit detected in this patient subset. The author think in patients with advanced pancreas cancer, the anti-EGFR monoclonal antibody cetuximab did not improve the outcome compared with patients treated with gemcitabine alone. Alternate targets other

Matuzumab(EMD 72000) is a humanized IgG1 mAb against EGFR. Laboratory studies have shown promising inhibitory effects on tumor growth and angiogenesis, include L3. 6pl in an orthotopic rat model(95). In an phase Ⅰclinical trail, matuzumab was given at a dose of 400- 800 mg once weekly for 8 weeks, followed by gemcitabine 1000mg/m2 weekly for two cycles. The partial response or stable disease in 12 evaluated advanced pancreatic cancer

Trastuzumab(Herceptin) is a humanized mAb, which has shown significant growth inhibition of a pancreatic cancer cell line and xenografts established with the same line. In a study focusing on HER2 overexpressing pancreatic cancer, trastuzumab was combined with fluoropyrimidine S-1 to treat cancer cells in vivo and in vitro, pancreatic cell growth inhibition is observed not only by inhibition of the HER2 signal transduction pathway, but also by antibody-dependent cellular cytotoxicity(ADCC) induced by trastuzumab(97). In another research, although in four pancreatic cell lines, trastuzumab didn't express inhibitor effect and synergistic effect with gemcitabine, ADCC were observed in three cells which expressed HER2 in mice. In Capan-1 xenografted mice, trastuzumab inhibited tumor growth

than EGFR should be evaluated for new drug development(94).

with metastatic pancreatic cancer(92).

pancreatic cancer.

patients was 66.7%(96).

**4.2.1.2 Anti-ErbB2/HER2 antibodies** 

significantly and prolonged survival(98).

drvelopment of recombinant DNA technology, this problem was solved and chimeric antibodies, antibody fragments or intact fully human antibodies were produced and tested clinically. Base on moral principles, antibodies were used as adjunctive treement with chemotherapy agents, small molecule signal transduction inhibitors, or radiation in clinical trails, to study if can help patients lengthen the survival. The targets are generally classified into three major categories:cell surface proteins;antigen associated with the tumor stroma;antigen on tumor-associated vasculature and angiogenic ligands(88).

#### **4.2.1.1 Anti-EGFR antibodies**

Cetuximab is a chimeric mouse-human antibody against an epitope located in extra-celluar part of EGFR. In preclinical studies, cetuximab could decrease cell proliferation and phosphorylation of EGFR, and blocked the binding of the adaptor protein Grb2 to EGFR upon activation by EGF(89). Another preclinical study, the combination of cetuximab together with gemcitabine and radiation effectively prolonged the tumor xenograft volume doubling time (30.1±3.3days), compared with gemcitabine monotherapy (11.6±3.1days), radiation monotherapy (16.7±3.1days), cetuximab with gemcitabine (20.1±3.1days)and cetuximab with radiation (22.5±3.3days)(90).

In many clinical trails, synergistic effects were observed using combination of cetuximab therapy and chemotherapy agents. In a multicenter phase II trial, Patients with measurable locally advanced or metastatic pancreatic cancer who had never received chemotherapy for their advanced disease and had immunohistochemical evidence of EGFR expression were treated with cetuximab at an initial dose of 400 mg/m(2), followed by 250 mg/m(2) weekly for 7 weeks. Gemcitabine was administered at 1,000 mg/m(2) for 7 weeks, followed by 1 week of rest. In subsequent cycles, cetuximab was administered weekly, and gemcitabine was administered weekly for 3 weeks every 4 weeks. In sixty-one patients who were screened for EGFR expression, 58 patients (95%) had at least 1+ staining, and 41 were enrolled onto the trial, result showed Five patients (12.2%) achieved a partial response, and 26 (63.4%) had stable disease. The median time to disease progression was 3.8 months, and the median overall survival duration was 7.1 months. One-year progression-free survival and overall survival rates were 12% and 31.7%, respectively. The most frequently reported grade 3 or 4 adverse events were neutropenia (39.0%), asthenia (22.0%), abdominal pain (22.0%), and thrombocytopenia (17.1%). Cetuximab in combination with gemcitabine showed promising activity against advanced pancreatic cancer(91).

Another multicenter pahse Ⅱ study which is combination treatment with cetuximab and gemcitabine/oxaliplatin. Patients which had histological or cytological diagnosis of metastatic pancreatic adenocarcinoma received cetuximab 400 mg m(-2) at first infusion followed by weekly 250 mg m(-2) combined with gemcitabine 1000 mg m(-2) as a 100 min infusion on day 1 and oxaliplatin 100 mg m(-2) as a 2-h infusion on day 2 every 2 weeks. The intention-to-treat analysis of 61 evaluable patients showed an overall response rate of 33%, including 1 (2%) complete and 19 (31%) partial remissions. There were 31% patients with stable and 36% with progressive disease or discontinuation of the therapy before re-staging. The presence of a grade 2 or higher skin rash was associated with a higher likelihood of achieving objective response. Median time to progression was 118 days, with a median overall survival of 213 days. A clinical benefit response was noted in 24 of the evaluable 61

drvelopment of recombinant DNA technology, this problem was solved and chimeric antibodies, antibody fragments or intact fully human antibodies were produced and tested clinically. Base on moral principles, antibodies were used as adjunctive treement with chemotherapy agents, small molecule signal transduction inhibitors, or radiation in clinical trails, to study if can help patients lengthen the survival. The targets are generally classified into three major categories:cell surface proteins;antigen associated with the tumor

Cetuximab is a chimeric mouse-human antibody against an epitope located in extra-celluar part of EGFR. In preclinical studies, cetuximab could decrease cell proliferation and phosphorylation of EGFR, and blocked the binding of the adaptor protein Grb2 to EGFR upon activation by EGF(89). Another preclinical study, the combination of cetuximab together with gemcitabine and radiation effectively prolonged the tumor xenograft volume doubling time (30.1±3.3days), compared with gemcitabine monotherapy (11.6±3.1days), radiation monotherapy (16.7±3.1days), cetuximab with gemcitabine (20.1±3.1days)and

In many clinical trails, synergistic effects were observed using combination of cetuximab therapy and chemotherapy agents. In a multicenter phase II trial, Patients with measurable locally advanced or metastatic pancreatic cancer who had never received chemotherapy for their advanced disease and had immunohistochemical evidence of EGFR expression were treated with cetuximab at an initial dose of 400 mg/m(2), followed by 250 mg/m(2) weekly for 7 weeks. Gemcitabine was administered at 1,000 mg/m(2) for 7 weeks, followed by 1 week of rest. In subsequent cycles, cetuximab was administered weekly, and gemcitabine was administered weekly for 3 weeks every 4 weeks. In sixty-one patients who were screened for EGFR expression, 58 patients (95%) had at least 1+ staining, and 41 were enrolled onto the trial, result showed Five patients (12.2%) achieved a partial response, and 26 (63.4%) had stable disease. The median time to disease progression was 3.8 months, and the median overall survival duration was 7.1 months. One-year progression-free survival and overall survival rates were 12% and 31.7%, respectively. The most frequently reported grade 3 or 4 adverse events were neutropenia (39.0%), asthenia (22.0%), abdominal pain (22.0%), and thrombocytopenia (17.1%). Cetuximab in combination with gemcitabine

Another multicenter pahse Ⅱ study which is combination treatment with cetuximab and gemcitabine/oxaliplatin. Patients which had histological or cytological diagnosis of metastatic pancreatic adenocarcinoma received cetuximab 400 mg m(-2) at first infusion followed by weekly 250 mg m(-2) combined with gemcitabine 1000 mg m(-2) as a 100 min infusion on day 1 and oxaliplatin 100 mg m(-2) as a 2-h infusion on day 2 every 2 weeks. The intention-to-treat analysis of 61 evaluable patients showed an overall response rate of 33%, including 1 (2%) complete and 19 (31%) partial remissions. There were 31% patients with stable and 36% with progressive disease or discontinuation of the therapy before re-staging. The presence of a grade 2 or higher skin rash was associated with a higher likelihood of achieving objective response. Median time to progression was 118 days, with a median overall survival of 213 days. A clinical benefit response was noted in 24 of the evaluable 61

stroma;antigen on tumor-associated vasculature and angiogenic ligands(88).

showed promising activity against advanced pancreatic cancer(91).

**4.2.1.1 Anti-EGFR antibodies** 

cetuximab with radiation (22.5±3.3days)(90).

patients (39%). Although the addition of cetuximab to the combination of gemcitabine and oxaliplatin is well tolerated, the reasearch failed to increase response or survival in patients with metastatic pancreatic cancer(92).

But the effect of the cetuximab is limited by the affinity of expressed EGFR in pancreatic cancer, other factors including mutation of K-ras, PTEN expression or host complement level. these may be the reasons of failure in some trails. In a phase Ⅱ trail, within the cetuximab group and noncetuximab group, no significant differences were found in objective response rate (17.5% Vs12.2%), median progression-free survival (3.4 months Vs 4.2 months), median overall survival (7.5 months Vs 7.8 months)(93), The result can't prove a synergistic effect in combination of cetuximab and gemcitabine/cisplatin treatment in pancreatic cancer.

Another phase Ⅲ trail of Patients with unresectable locally advanced or metastatic pancreatic adenocarcinoma were randomly assigned to receive gemcitabine alone or gemcitabine plus cetuximab. A total of 745 eligible patients were accrued. No significant difference was seen between the two arms of the study with respect to the median survival time (6. 3 months for the gemcitabine plus cetuximab arm v5.9 months for the gemcitabine alone arm; hazard ratio = 1.06; 95% CI, 0.91 to 1.23; P = .23, one-sided). Objective responses and progression-free survival were similar in both arms of the study. Although time to treatment failure was longer in patients on gemcitabine plus cetuximab (P=.006), the difference in length of treatment was only 2 weeks longer in the combination arm. Among patients who were studied for tumoral EGFR expression, 90% were positive, with no treatment benefit detected in this patient subset. The author think in patients with advanced pancreas cancer, the anti-EGFR monoclonal antibody cetuximab did not improve the outcome compared with patients treated with gemcitabine alone. Alternate targets other than EGFR should be evaluated for new drug development(94).

Matuzumab(EMD 72000) is a humanized IgG1 mAb against EGFR. Laboratory studies have shown promising inhibitory effects on tumor growth and angiogenesis, include L3. 6pl in an orthotopic rat model(95). In an phase Ⅰclinical trail, matuzumab was given at a dose of 400- 800 mg once weekly for 8 weeks, followed by gemcitabine 1000mg/m2 weekly for two cycles. The partial response or stable disease in 12 evaluated advanced pancreatic cancer patients was 66.7%(96).

#### **4.2.1.2 Anti-ErbB2/HER2 antibodies**

Trastuzumab(Herceptin) is a humanized mAb, which has shown significant growth inhibition of a pancreatic cancer cell line and xenografts established with the same line. In a study focusing on HER2 overexpressing pancreatic cancer, trastuzumab was combined with fluoropyrimidine S-1 to treat cancer cells in vivo and in vitro, pancreatic cell growth inhibition is observed not only by inhibition of the HER2 signal transduction pathway, but also by antibody-dependent cellular cytotoxicity(ADCC) induced by trastuzumab(97). In another research, although in four pancreatic cell lines, trastuzumab didn't express inhibitor effect and synergistic effect with gemcitabine, ADCC were observed in three cells which expressed HER2 in mice. In Capan-1 xenografted mice, trastuzumab inhibited tumor growth significantly and prolonged survival(98).

Immunotherapy of the Pancreatic Cancer 125

213bismuch. In vitro study showed specific cytotoxic to MUC1-expressing pancreatic cancer

SS1P is a recombinant immunotoxin that consists of an anti-mesothelin scFv(ss1) fused to PE38, a 38Kda portion of Pseudomonas exotoxin. Sing-chain Fv(scFv)v was isolated from a phage display library obtained from the spleen of mice immunized with mesothelinexpression plasmid. After binding to mesothelin and subsequent internalisation into cells, it

In preclinical study, SS1P plus radition in treating mesothelin-expressing tumor xenografts, combination treatment significantly prolonged the doubling time of tumors(104);meanwhile, synergic result was observed when treat with gemcitabin, the tumors were induced

In the further phase Ⅰ clinical study, SS1P was administered by intravenous infusion in 34 patients with mesothelin-expressing tumor, including 2 pancreatic cancer patients, the results showed that it was well-tolerated with self-limiting pleuritis as dose-limiting toxicity, 12% tumor size decresed from 20-50% and lasted for more than 4 weeks, 56% patients

Another monoclonal antibody against mesothelin, MORAb-009, is a chimeric of a mouse and human mAb derived from a phage-display library and re-engineered(107). In a phase I clinical trial, treatment of MORAb-009 in patients with advanced mesothelin-expressing cancers has been determined if safety, dose-limiting toxicity (DLT), and maximum tolerated dose (MTD). A total of 24 subjects were treated including 13 mesothelioma, 7 pancreatic cancer, and 4 ovarian cancer patients. The median number of MORAb-009 infusions was 4 (range 1-24 infusions). At the 400 mg/m(2) dose level, 2 subjects experienced DLT (grade 4 transaminitis and a grade 3 serum sickness). Thus, although there were other contributing causes of these adverse events, 200 mg/m(2) was considered the MTD. Other adverse events at least possibly related to MORAb-009 included 7 drug hypersensitivity events (all grade 1 or 2) and a thromboembolic event (grade 4). Eleven subjects had stable disease. There was a dose-dependent increase in serum MORAb-009 concentration. The result suggested that MORAb-009 is well tolerated and the MTD when administered weekly is conservatively set at 200 mg/m(2). Phase II studies of MORAb-009 in different mesothelin-expressing cancers

In cellular antitumor immunity, T-cells must first be activated by bone marrow—derived APCs that present tumor antigens and provide essential co-stimulatory signals, migrate and gain access to the tumor microenvironment, and overcome obstacles to effective triggering posed by the tumor. Dendritic cells, which are the strongest antigen presenting cells in the body. Their generation for anti-tumor immunity has been the focus of a vast array of scientific and clinical studies. DC's specialized capacity to cross-present exogenous Ags onto major histocompatability (MHC) class I molecules for the generation of T-Ag-specific cytotoxic T lymphocytes (CTLs) has made it possible to produce actived T cell in vitro and

showed stable disease and 29% of the patients had progressive disease(106).

cells in a concentration-dependent manner compared to controls(103).

**4.2.1.4 Anti-mesothelin antibodies** 

regression completely(105).

are ongoing(108).

**4.2.2 Adoptive cell transfer** 

inhibits protein synthesis and results in apoptosis.

Larbouret reported combination treatment of matuzumab and trastuzumab could enhance the inhibitory effect on HER2 phosphorylation, lead to significantly decrease xenograft tumor sizes or induce more complete remissions when compared to antibody alone, then prolonged survival in BxPC-3 and MIA PaCa-2 pancreatic cancer cells xenograft mice(99). The further study which took placed in nude mice, bearing human pancreatic carcinoma xenografts, combined anti-EGFR (cetuximab) and anti-HER2 (trastuzumab) or gemcitabine were given as trement and tumor growth was observed. Result showed in first-line therapy, mice survival was significantly longer in the 2mAbs group compared with gemcitabine (P<0.0001 for BxPC-3, P=0.0679 for MiaPaCa-2 and P=0.0019 for Capan-1) and with controls (P<0.0001). In second-line therapy, tumor regressions were observed after replacing gemcitabine by 2mAbs treatment, resulting in significantly longer animal survival compared with mice receiving continuous gemcitabine injections (P=0.008 for BxPC-3, P=0.05 for MiaPaCa-2 and P<0.001 for Capan-1). Therapeutic benefit of 2mAbs was observed despite K-Ras mutation. Interestingly, concerning the mechanism of action, coinjection of F(ab')(2) fragments from 2mAbs induced significant tumor growth inhibition, compared with controls (P=0.001), indicating that the 2mAbs had an Fc fragment-independent direct action on tumor cells. This preclinical study demonstrated a significant improvement of survival and tumor regression in mice treated with anti-EGFR/anti-HER2 2mAbs in first- and second-line treatments, compared with gemcitabine, independently of the K-Ras status(100).

#### **4.2.1.3 Anti-MUC1 antibodies**

PAM4 is a murine antibody to MUC1 obtained from mice immunized with purified mucin from a human pancreatic cancer xenograft sample. In a preclinical study, 90Yttriumlabelled PAM4 monoclonal antibody was combined with gemcitabine in mice bearing Capan-1, the result showed increased inhibition of tumor growth and prolonged survival of the mice(101). The recent clinical trail took place in 21 patients with advanced pancreatic cancer. 111In-hPAM4 showed normal biodistribution with radiation dose estimates to red marrow and solid organs acceptable for radioimmunotherapy and with tumor targeting in 12 patients. One patient withdrew before (90)Y-hPAM4; otherwise, 20 patients received (90)Y doses of 15 (n=7), 20 (n=9), and 25 mCi/m(2) (n=4). Treatment was well tolerated; the only significant drug-related toxicities were (NCI CTC v.3) grade 3 to 4 neutropenia and thrombocytopenia increasing with (90)Y dose. There were no bleeding events or serious infections, and most cytopenias recovered to grade 1 within 12 weeks. Three patients at 25 mCi/m(2) encountered dose-limiting toxicity with grade 4 cytopenias more than 7 days, establishing 20 mCi/m(2) as the maximal tolerated (90)Y dose. Two patients developed HAHA of uncertain clinical significance. Most patients progressed rapidly and with CA19-9 levels increasing within 1 month of therapy, but 7 remained progression-free by CT for 1.5 to 5.6 months, including 3 achieving transient partial responses (32%-52% tumor diameter shrinkage). The study concluded (90)Y-Clivatuzumab tetraxetan was well tolerated with manageable hematologic toxicity at the maximal tolerated (90)Y dose, and is a potential new therapeutic for advanced pancreatic cancer(102).

In a Phase Ⅰtrial for patients with stage Ⅲ and Ⅳ pancreatic cancer, another antibody C595, which is targeting the protein core of MUC1, was conjugated with the α-particle-emitting

Larbouret reported combination treatment of matuzumab and trastuzumab could enhance the inhibitory effect on HER2 phosphorylation, lead to significantly decrease xenograft tumor sizes or induce more complete remissions when compared to antibody alone, then prolonged survival in BxPC-3 and MIA PaCa-2 pancreatic cancer cells xenograft mice(99). The further study which took placed in nude mice, bearing human pancreatic carcinoma xenografts, combined anti-EGFR (cetuximab) and anti-HER2 (trastuzumab) or gemcitabine were given as trement and tumor growth was observed. Result showed in first-line therapy, mice survival was significantly longer in the 2mAbs group compared with gemcitabine (P<0.0001 for BxPC-3, P=0.0679 for MiaPaCa-2 and P=0.0019 for Capan-1) and with controls (P<0.0001). In second-line therapy, tumor regressions were observed after replacing gemcitabine by 2mAbs treatment, resulting in significantly longer animal survival compared with mice receiving continuous gemcitabine injections (P=0.008 for BxPC-3, P=0.05 for MiaPaCa-2 and P<0.001 for Capan-1). Therapeutic benefit of 2mAbs was observed despite K-Ras mutation. Interestingly, concerning the mechanism of action, coinjection of F(ab')(2) fragments from 2mAbs induced significant tumor growth inhibition, compared with controls (P=0.001), indicating that the 2mAbs had an Fc fragment-independent direct action on tumor cells. This preclinical study demonstrated a significant improvement of survival and tumor regression in mice treated with anti-EGFR/anti-HER2 2mAbs in first- and second-line treatments, compared with

PAM4 is a murine antibody to MUC1 obtained from mice immunized with purified mucin from a human pancreatic cancer xenograft sample. In a preclinical study, 90Yttriumlabelled PAM4 monoclonal antibody was combined with gemcitabine in mice bearing Capan-1, the result showed increased inhibition of tumor growth and prolonged survival of the mice(101). The recent clinical trail took place in 21 patients with advanced pancreatic cancer. 111In-hPAM4 showed normal biodistribution with radiation dose estimates to red marrow and solid organs acceptable for radioimmunotherapy and with tumor targeting in 12 patients. One patient withdrew before (90)Y-hPAM4; otherwise, 20 patients received (90)Y doses of 15 (n=7), 20 (n=9), and 25 mCi/m(2) (n=4). Treatment was well tolerated; the only significant drug-related toxicities were (NCI CTC v.3) grade 3 to 4 neutropenia and thrombocytopenia increasing with (90)Y dose. There were no bleeding events or serious infections, and most cytopenias recovered to grade 1 within 12 weeks. Three patients at 25 mCi/m(2) encountered dose-limiting toxicity with grade 4 cytopenias more than 7 days, establishing 20 mCi/m(2) as the maximal tolerated (90)Y dose. Two patients developed HAHA of uncertain clinical significance. Most patients progressed rapidly and with CA19-9 levels increasing within 1 month of therapy, but 7 remained progression-free by CT for 1.5 to 5.6 months, including 3 achieving transient partial responses (32%-52% tumor diameter shrinkage). The study concluded (90)Y-Clivatuzumab tetraxetan was well tolerated with manageable hematologic toxicity at the maximal tolerated (90)Y dose, and

In a Phase Ⅰtrial for patients with stage Ⅲ and Ⅳ pancreatic cancer, another antibody C595, which is targeting the protein core of MUC1, was conjugated with the α-particle-emitting

gemcitabine, independently of the K-Ras status(100).

is a potential new therapeutic for advanced pancreatic cancer(102).

**4.2.1.3 Anti-MUC1 antibodies** 

213bismuch. In vitro study showed specific cytotoxic to MUC1-expressing pancreatic cancer cells in a concentration-dependent manner compared to controls(103).

#### **4.2.1.4 Anti-mesothelin antibodies**

SS1P is a recombinant immunotoxin that consists of an anti-mesothelin scFv(ss1) fused to PE38, a 38Kda portion of Pseudomonas exotoxin. Sing-chain Fv(scFv)v was isolated from a phage display library obtained from the spleen of mice immunized with mesothelinexpression plasmid. After binding to mesothelin and subsequent internalisation into cells, it inhibits protein synthesis and results in apoptosis.

In preclinical study, SS1P plus radition in treating mesothelin-expressing tumor xenografts, combination treatment significantly prolonged the doubling time of tumors(104);meanwhile, synergic result was observed when treat with gemcitabin, the tumors were induced regression completely(105).

In the further phase Ⅰ clinical study, SS1P was administered by intravenous infusion in 34 patients with mesothelin-expressing tumor, including 2 pancreatic cancer patients, the results showed that it was well-tolerated with self-limiting pleuritis as dose-limiting toxicity, 12% tumor size decresed from 20-50% and lasted for more than 4 weeks, 56% patients showed stable disease and 29% of the patients had progressive disease(106).

Another monoclonal antibody against mesothelin, MORAb-009, is a chimeric of a mouse and human mAb derived from a phage-display library and re-engineered(107). In a phase I clinical trial, treatment of MORAb-009 in patients with advanced mesothelin-expressing cancers has been determined if safety, dose-limiting toxicity (DLT), and maximum tolerated dose (MTD). A total of 24 subjects were treated including 13 mesothelioma, 7 pancreatic cancer, and 4 ovarian cancer patients. The median number of MORAb-009 infusions was 4 (range 1-24 infusions). At the 400 mg/m(2) dose level, 2 subjects experienced DLT (grade 4 transaminitis and a grade 3 serum sickness). Thus, although there were other contributing causes of these adverse events, 200 mg/m(2) was considered the MTD. Other adverse events at least possibly related to MORAb-009 included 7 drug hypersensitivity events (all grade 1 or 2) and a thromboembolic event (grade 4). Eleven subjects had stable disease. There was a dose-dependent increase in serum MORAb-009 concentration. The result suggested that MORAb-009 is well tolerated and the MTD when administered weekly is conservatively set at 200 mg/m(2). Phase II studies of MORAb-009 in different mesothelin-expressing cancers are ongoing(108).

#### **4.2.2 Adoptive cell transfer**

In cellular antitumor immunity, T-cells must first be activated by bone marrow—derived APCs that present tumor antigens and provide essential co-stimulatory signals, migrate and gain access to the tumor microenvironment, and overcome obstacles to effective triggering posed by the tumor. Dendritic cells, which are the strongest antigen presenting cells in the body. Their generation for anti-tumor immunity has been the focus of a vast array of scientific and clinical studies. DC's specialized capacity to cross-present exogenous Ags onto major histocompatability (MHC) class I molecules for the generation of T-Ag-specific cytotoxic T lymphocytes (CTLs) has made it possible to produce actived T cell in vitro and

Immunotherapy of the Pancreatic Cancer 127

of PDAK cells in this patient appeared to be substantially higher compared to those in PD patients. The results suggest that adoptive immunotherapy using PDAK cells for cancer

However, in another clinical study, data demonstrate that MUC1 peptide-based immunization elicits mature MUC1-specific CTLs in the peripheral lymphoid organs. The mature CTLs secrete IFN-gamma and are cytolytic against MUC1-expressing tumor cells in vitro. Unfortunately, active CTLs that infiltrate the pancreas tumor microenvironment become cytolytically anergic and are tolerized to MUC1 antigen, allowing the tumor to grow. The CTL tolerance could be reversed at least in vitro with the use of anti-CD40 costimulation. The pancreas tumor cells secrete immunosuppressive cytokines, including IL-10 and TGF-beta that are partly responsible for the down-regulation of CTL activity. In addition, they down-regulate their MHC class I molecules to avoid immune recognition. CD4+CD25+T regulatory cells, which secrete IL-10, were also found in the tumor environment. Together these data indicate the use of several immune evasion mechanisms by tumor cells to evade CTL killing. Thus altering the tumor microenvironment to make it more conducive to CTL killing may be key in developing a successful anti-cancer

In a syngeneic pancreatic tumor mouse model, T-cells were produced in vitro by coculturing human lymphocytes with telomerase peptide-pulsed dendritic cells(DCs) or in vivo by injection of peptide, animals treated with telomerase-specific T cells showed significantly

With the identification of novel mesothelin CTL epitopes, T-cell lines generated from one of these epitopes were shown to lyse pancreatic tumor cells. Several agonist epitopes were defined and were shown to (a) have higher affinity and avidity for HLA-A2, (b) activate mesothelin-specific T cells from normal individuals or cancer patients to a greater degree than the native epitope in terms of induction of higher levels of IFN-gamma and the chemokine lymphotactin, and (c) lyse several mesothelin-expressing tumor types in a MHCrestricted manner more effectively than T cells generated using the native peptide. External beam radiation of tumor cells at nontoxic levels was shown to enhance the expression of mesothelin and other accessory molecules, resulting in a modest but statistically significant increase in tumor cell lysis by mesothelin-specific T cells. The result supports and extends observations that mesothelin is a potential target for immunotherapy of pancreatic cancers, as well as mesotheliomas. Combination of immunotherapy and chemoradiotherapy may be

Although it is used as adjuvant treatment in preclinical or clinical trail, immunotherapy may be the next great hope for pancreatic cancer treatment. While monoclonal antibodies, cytokines, vaccines and CTL have individually shown some promise, it's hard to say which is better in nonspecific and specific immunotherapy. It seems to be the best strategy to

patients with antigen-positive lung metastasis is safe and feasible(111).

immunotherapy(112).

**4.2.2.3 Telomerase-specific CTLs** 

delayed disease progression(113). **4.2.2.4 Mesothelin-specific CTLs** 

a better choice for the patients(114).

**4.3 Future perspective** 

in vivo. Adoptive immunotherapy involves harvesting the patient's peripheral blood Tlymphocytes, stimulating and expanding the autologous tumor-reactive T-cells, finally transferring them back into the patient.

#### **4.2.2.1 K-ras-specific CTLs**

In vitro, Immature DCs had pulsed with synthesized mutant K-ras peptide (YKLVVVGAV). When the DCs were matured, Kras antigen epitope can express on the DC's surface effectively and Cytotoxic T lymphocytes (CTLs) can be induced when autogeneic and homologous T cells co-cultured with the mutant K-ras peptide-pulsed DCs. The reasearch demonstrated the induced CTLs can kill the pancreatic cancer cell line Patu8988 which expresss the same K-ras mutation type effectively in virto and in vivo. Without damage the normal tissue cells, the killing rate of activated K-ras specific CTLs to the tumor cell when the ratios of CTL: Patu8988 cells were 10:1, 20:1, and 50:1 were (21.2+/-1.9)%, (32.4 +/- 2.1)%, and (45.7+/-5.3)% respectively, all while the killing efficiency significantly superior to those of the non-specific activated T lymphocyte (all P < 0.05). Eight days after CTL injection into the nude mice the tumor size of the intratumor injection group was (68 +/- 13) mm3, significantly smaller than those of the control group and IL-2 activated nonspecific CTL intra-tumor injection group [(87+/-14) mm3 and (79 +/- 19) mm3, both P < 0.05]. The survival rates of the nude mice of the K-ras specific CTL intra-tumor injection group, CTL caudal vein injection group, and IL-2 activated non-specific CTL intra-tumor injection group were all significantly higher than that of the control group (all P < 0.05), and the survival rate of the K-ras specific CTL intra-tumor injection group was significantly higher than that of the IL-2 activated non-specific CTL intra-tumor injection group (P <0.05). Immunohistochemical staining confirmed that K-ras specific CTL had the ability to move toward tumor. The result showed antigen-specific-CTLs induced in virto and transferred into the patient can used be a effective treatment for pancreatic cancer(109).

#### **4.2.2.2 MUC1-specific CTLs**

In MUC1 expressing Tumor-bearing mice , there were low affinity MUC1-specific CTLs that have no effect on the spontaneously occurring pancreatic tumors in vivo. However, adoptive transfer of these CTLs was able to completely eradicate MUC1-expressing injectable tumors in MUC1 transgenic mice, and these mice developed long-term immunity. These CTLs were MHC class I restricted and recognized peptide epitopes in the immunodominant tandem repeat region of MUC1. The MET mice appropriately mimic the human condition and are an excellent model with which to elucidate the native immune responses that develop during tumor progression and to develop effective antitumor vaccine strategies(110).

In a study of 11 patients with lung metastases from different cancer, CTLs were generated in virto using cultured DCs, synthetic peptide, peripheral blood lymphocytes, IL-2 and anti-CD3 antibody. The patients received either Muc-1, CEA, gpl00, Her-2 or SART-3-PDAK cells generated in vitro, All transfers of peptide-pulsed dendritic cell-activated killer(PDAK) cells, which showed peptide/HLA-specific lysis, were well-tolerated in all patients, and adverse effects (elevation of transaminase, fever, and headache) were observed primarily at grade 1, but in no case greater than grade 2. One partial response (PR) of lung metastasis occurred in a pancreatic cancer patient who received 3x10(7) Muc-1-PDAK cells/kg. The cytolytic units

in vivo. Adoptive immunotherapy involves harvesting the patient's peripheral blood Tlymphocytes, stimulating and expanding the autologous tumor-reactive T-cells, finally

In vitro, Immature DCs had pulsed with synthesized mutant K-ras peptide (YKLVVVGAV). When the DCs were matured, Kras antigen epitope can express on the DC's surface effectively and Cytotoxic T lymphocytes (CTLs) can be induced when autogeneic and homologous T cells co-cultured with the mutant K-ras peptide-pulsed DCs. The reasearch demonstrated the induced CTLs can kill the pancreatic cancer cell line Patu8988 which expresss the same K-ras mutation type effectively in virto and in vivo. Without damage the normal tissue cells, the killing rate of activated K-ras specific CTLs to the tumor cell when the ratios of CTL: Patu8988 cells were 10:1, 20:1, and 50:1 were (21.2+/-1.9)%, (32.4 +/- 2.1)%, and (45.7+/-5.3)% respectively, all while the killing efficiency significantly superior to those of the non-specific activated T lymphocyte (all P < 0.05). Eight days after CTL injection into the nude mice the tumor size of the intratumor injection group was (68 +/- 13) mm3, significantly smaller than those of the control group and IL-2 activated nonspecific CTL intra-tumor injection group [(87+/-14) mm3 and (79 +/- 19) mm3, both P < 0.05]. The survival rates of the nude mice of the K-ras specific CTL intra-tumor injection group, CTL caudal vein injection group, and IL-2 activated non-specific CTL intra-tumor injection group were all significantly higher than that of the control group (all P < 0.05), and the survival rate of the K-ras specific CTL intra-tumor injection group was significantly higher than that of the IL-2 activated non-specific CTL intra-tumor injection group (P <0.05). Immunohistochemical staining confirmed that K-ras specific CTL had the ability to move toward tumor. The result showed antigen-specific-CTLs induced in virto and transferred into the patient can used be a effective treatment for pancreatic cancer(109).

In MUC1 expressing Tumor-bearing mice , there were low affinity MUC1-specific CTLs that have no effect on the spontaneously occurring pancreatic tumors in vivo. However, adoptive transfer of these CTLs was able to completely eradicate MUC1-expressing injectable tumors in MUC1 transgenic mice, and these mice developed long-term immunity. These CTLs were MHC class I restricted and recognized peptide epitopes in the immunodominant tandem repeat region of MUC1. The MET mice appropriately mimic the human condition and are an excellent model with which to elucidate the native immune responses that develop during tumor progression and to develop effective antitumor

In a study of 11 patients with lung metastases from different cancer, CTLs were generated in virto using cultured DCs, synthetic peptide, peripheral blood lymphocytes, IL-2 and anti-CD3 antibody. The patients received either Muc-1, CEA, gpl00, Her-2 or SART-3-PDAK cells generated in vitro, All transfers of peptide-pulsed dendritic cell-activated killer(PDAK) cells, which showed peptide/HLA-specific lysis, were well-tolerated in all patients, and adverse effects (elevation of transaminase, fever, and headache) were observed primarily at grade 1, but in no case greater than grade 2. One partial response (PR) of lung metastasis occurred in a pancreatic cancer patient who received 3x10(7) Muc-1-PDAK cells/kg. The cytolytic units

transferring them back into the patient.

**4.2.2.1 K-ras-specific CTLs** 

**4.2.2.2 MUC1-specific CTLs** 

vaccine strategies(110).

of PDAK cells in this patient appeared to be substantially higher compared to those in PD patients. The results suggest that adoptive immunotherapy using PDAK cells for cancer patients with antigen-positive lung metastasis is safe and feasible(111).

However, in another clinical study, data demonstrate that MUC1 peptide-based immunization elicits mature MUC1-specific CTLs in the peripheral lymphoid organs. The mature CTLs secrete IFN-gamma and are cytolytic against MUC1-expressing tumor cells in vitro. Unfortunately, active CTLs that infiltrate the pancreas tumor microenvironment become cytolytically anergic and are tolerized to MUC1 antigen, allowing the tumor to grow. The CTL tolerance could be reversed at least in vitro with the use of anti-CD40 costimulation. The pancreas tumor cells secrete immunosuppressive cytokines, including IL-10 and TGF-beta that are partly responsible for the down-regulation of CTL activity. In addition, they down-regulate their MHC class I molecules to avoid immune recognition. CD4+CD25+T regulatory cells, which secrete IL-10, were also found in the tumor environment. Together these data indicate the use of several immune evasion mechanisms by tumor cells to evade CTL killing. Thus altering the tumor microenvironment to make it more conducive to CTL killing may be key in developing a successful anti-cancer immunotherapy(112).

#### **4.2.2.3 Telomerase-specific CTLs**

In a syngeneic pancreatic tumor mouse model, T-cells were produced in vitro by coculturing human lymphocytes with telomerase peptide-pulsed dendritic cells(DCs) or in vivo by injection of peptide, animals treated with telomerase-specific T cells showed significantly delayed disease progression(113).

#### **4.2.2.4 Mesothelin-specific CTLs**

With the identification of novel mesothelin CTL epitopes, T-cell lines generated from one of these epitopes were shown to lyse pancreatic tumor cells. Several agonist epitopes were defined and were shown to (a) have higher affinity and avidity for HLA-A2, (b) activate mesothelin-specific T cells from normal individuals or cancer patients to a greater degree than the native epitope in terms of induction of higher levels of IFN-gamma and the chemokine lymphotactin, and (c) lyse several mesothelin-expressing tumor types in a MHCrestricted manner more effectively than T cells generated using the native peptide. External beam radiation of tumor cells at nontoxic levels was shown to enhance the expression of mesothelin and other accessory molecules, resulting in a modest but statistically significant increase in tumor cell lysis by mesothelin-specific T cells. The result supports and extends observations that mesothelin is a potential target for immunotherapy of pancreatic cancers, as well as mesotheliomas. Combination of immunotherapy and chemoradiotherapy may be a better choice for the patients(114).

#### **4.3 Future perspective**

Although it is used as adjuvant treatment in preclinical or clinical trail, immunotherapy may be the next great hope for pancreatic cancer treatment. While monoclonal antibodies, cytokines, vaccines and CTL have individually shown some promise, it's hard to say which is better in nonspecific and specific immunotherapy. It seems to be the best strategy to

Immunotherapy of the Pancreatic Cancer 129

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obtained more efficient results in combination with a variety of antigens, or vaccine and antibody combinations. A nonspecific and specific immunotherapy combination offers another potent strategy. With the combination, the ultimate achievable goal may be a durable anti-tumor immune response that can destory and prevent it from recurrence over the course of a patient's life.

According to the existed profiles, The key of the immunotherapy on pancreatic cancer is to break through cancer microenvironment's defence. Suppressing the function of immunosuppression cells, such as immunosuppressive tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) reside in tumors is as important as inducing the specific immune agent, such as antibody or CTL.

Combating with each other, tumor and immune system are like two warriors on the other side of balance. What we can do is to break the balance and help the immune system win the war. Traditional methods, surgical operation, chemoradiation, can decrease the number of the tumor cells to the minimum while do harmful to immune system in the same time. So as the followed treatment, the passive immunotherapy may be the best choice to supply enough actived immune agents in a short period to kill the metastatic cancer cells. When patient recovered, Cytokines and vaccine will help to establish long term specific immune response to keep watch on and get rid of residuary cancer cells. Owing to pancreatic cancer cells expressing different abnormal antigens, the combination of 2 or more epitopes vaccines will obstain better effect to prevent from recurrence. and metastasis.

#### **5. References**


obtained more efficient results in combination with a variety of antigens, or vaccine and antibody combinations. A nonspecific and specific immunotherapy combination offers another potent strategy. With the combination, the ultimate achievable goal may be a durable anti-tumor immune response that can destory and prevent it from recurrence over

According to the existed profiles, The key of the immunotherapy on pancreatic cancer is to break through cancer microenvironment's defence. Suppressing the function of immunosuppression cells, such as immunosuppressive tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) reside in tumors

Combating with each other, tumor and immune system are like two warriors on the other side of balance. What we can do is to break the balance and help the immune system win the war. Traditional methods, surgical operation, chemoradiation, can decrease the number of the tumor cells to the minimum while do harmful to immune system in the same time. So as the followed treatment, the passive immunotherapy may be the best choice to supply enough actived immune agents in a short period to kill the metastatic cancer cells. When patient recovered, Cytokines and vaccine will help to establish long term specific immune response to keep watch on and get rid of residuary cancer cells. Owing to pancreatic cancer cells expressing different abnormal antigens, the combination of 2 or more epitopes vaccines will obstain better effect to prevent from recurrence. and

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**8**

*Italy* 

**An Overview on Immunotherapy**

Fabrizio Romano, Luca Degrate, Mattia Garancini, Fabio Uggeri,

Pancreatic cancer is the fourth leading cause of cancer mortality in both men and women. Approximately 32,000 Americans each year will develop and also die from this disease . Despite aggressive surgical and medical management, the mean life expectancy is approximately 15–18 months for patients with local and regional disease, and 3–6 months for patients with metastatic disease 1-2. Even in case of radical surgery it is associated with a poor prognosis and a 5-year survival rate of less than 4%. Early detection methods are under development but do not yet exist in practice for pancreatic cancer. Therefore, most patients present with advanced disease that cannot be cured by surgery (pancreaticoduodenectomy). Clinically, pancreatic cancer is characterized by rapid tumor progression, early metastatization and unresponsiveness to most conventional treatment modalities. In a recent analysis using a database from 1973 to 2003 based on modeled period analysis, 5-year survival of pancreatic cancer patients was 7.1% and 10-year survival was below 5%3. The survival rate is apparently related to the disease stage with a low rate at 1.6–3.3% among patients with distant metastases. Curative resection remains the most important factor determining outcome for resectable tumors. However, the resection rate for pancreatic carcinoma is only 10% and the overall five-year survival rate after resection is still only 10 to 20%. Early diagnosis and effective treatment to control the advanced stages of disease may prolong the survival rate of pancreatic cancer. Otherwise pancreatic cancer remains a disease with high mortality despite numerous efforts that have been made to improve its

In developing cancer immunotherapy, the following aims must be considered: (1) detection of immune response to autologous tumor cells, (2) identification of tumor antigens and analysis of the immune responses in patients, (3) analysis of tumor escape mechanisms and development of methods to overcome them, and (4) development of a more efficient immune intervention system by way of animal model experiments and clinical trials. Identification of tumor antigens in the first objective is important because it subsequently allows their use not only as targets for immunotherapy in a more immunogenic form but also enables quantitative and qualitative monitoring of immune responses to tumor cells during immunotherapy. In many animal tumors and in human melanoma, T cells play an

**1. Introduction** 

survival rates.

**of Pancreatic Cancer** 

*University Of Milan Bicocca, Monza,* 

Gianmaria Mauri and Franco Uggeri *Department of Surgery, San Gerardo Hospital,* 


### **An Overview on Immunotherapy of Pancreatic Cancer**

Fabrizio Romano, Luca Degrate, Mattia Garancini, Fabio Uggeri, Gianmaria Mauri and Franco Uggeri *Department of Surgery, San Gerardo Hospital, University Of Milan Bicocca, Monza, Italy* 

#### **1. Introduction**

136 Pancreatic Cancer – Clinical Management

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[114] Yokokawa J, Palena C, Arlen P, et al. Identification of novel human CTL epitopes and their agonist epitopes of mesothelin. Clin Cancer Res. 2005;11(17):6342-51.

activated killer(PDAK) cells. Anticancer Res 2005;25(3c):2407-2415.

cells in virto and in vivo. Cancer 2006;106(4):759-764.

immunotherapy for metastatic lung tumors using peptide-pulsed dendritic cells-

Pancreatic cancer is the fourth leading cause of cancer mortality in both men and women. Approximately 32,000 Americans each year will develop and also die from this disease . Despite aggressive surgical and medical management, the mean life expectancy is approximately 15–18 months for patients with local and regional disease, and 3–6 months for patients with metastatic disease 1-2. Even in case of radical surgery it is associated with a poor prognosis and a 5-year survival rate of less than 4%. Early detection methods are under development but do not yet exist in practice for pancreatic cancer. Therefore, most patients present with advanced disease that cannot be cured by surgery (pancreaticoduodenectomy). Clinically, pancreatic cancer is characterized by rapid tumor progression, early metastatization and unresponsiveness to most conventional treatment modalities. In a recent analysis using a database from 1973 to 2003 based on modeled period analysis, 5-year survival of pancreatic cancer patients was 7.1% and 10-year survival was below 5%3. The survival rate is apparently related to the disease stage with a low rate at 1.6–3.3% among patients with distant metastases. Curative resection remains the most important factor determining outcome for resectable tumors. However, the resection rate for pancreatic carcinoma is only 10% and the overall five-year survival rate after resection is still only 10 to 20%. Early diagnosis and effective treatment to control the advanced stages of disease may prolong the survival rate of pancreatic cancer. Otherwise pancreatic cancer remains a disease with high mortality despite numerous efforts that have been made to improve its survival rates.

In developing cancer immunotherapy, the following aims must be considered: (1) detection of immune response to autologous tumor cells, (2) identification of tumor antigens and analysis of the immune responses in patients, (3) analysis of tumor escape mechanisms and development of methods to overcome them, and (4) development of a more efficient immune intervention system by way of animal model experiments and clinical trials. Identification of tumor antigens in the first objective is important because it subsequently allows their use not only as targets for immunotherapy in a more immunogenic form but also enables quantitative and qualitative monitoring of immune responses to tumor cells during immunotherapy. In many animal tumors and in human melanoma, T cells play an

An Overview on Immunotherapy of Pancreatic Cancer 139

serve two purposes. One is to help generate and maintain self-tolerance, by eliminating T cells that are specific for self-antigens. The other is to restrain the amplitude of normal T-cell responses so that they do not 'overshoot' in their natural response to foreign pathogens. The prototypical immunological checkpoint is mediated by the cytotoxic-T-lymphocyteassociated protein 4 (CTLA4) counter-regulatory receptor that is expressed by T cells when they become activated15, 23. CTLA4 binds two B7-FAMILY members on the surface APCs — B7.1 (also known as CD80) and B7.2 (also known as CD86) — with roughly 20-fold higher affinity than the T-cell surface protein CD28 binds these molecules. CD28 is a co-stimulatory receptor that is constitutively expressed on naive T cells. Because of its higher affinity, CTLA4 out-competes CD28 for B7.1/B7.2 binding, resulting in the downmodulation of T-

A range of B7-family members interact with co-stimulatory and counter-regulatory inhibitory receptors on T cells. Two recently discovered B7-family members, B7-H1 (also known as PD-L1) and B7-DC (also known as PD-L2) also seem to interact with T-cell costimulatory and counter-regulatory inhibitory receptors18, 29, 30. PD-L1, which is upregulated on T cells when they become activated, seems to control a counter-regulatory immunological checkpoint when it binds PD-1 26,28,29. Activating receptors for B7-DC and B7-H1 have not yet been definitively identified. B7-DC is expressed on DCs, and is likely to have a co-stimulatory role in increasing activation of naive or resting T cells. In contrast to B7.1, B7.2 and B7-DC, B7-H1 is also expressed on several peripheral tissues and on many

Another new B7-family member, B7-H4, seems to mediate a predominantly inhibitory function in the immune system14. Recent data indicate that pancreatic tumours also express B7-H4 (D.L. and E.M.J., manuscript in preparation), and both B7-H1 and B7-H4 probably protect tumours from immune-system attack. Preclinical studies have already demonstrated that it is possible to downregulate B7-H1 signalling in mice, improving the antitumour response to vaccination18. Monoclonal antibodies that downregulate B7-H1 and B7-H4 are currently in clinical development. These antibodies will probably begin clinical testing in

Clinical trials using various immunotherapies, active immunization with tumor antigens, or tumor cell–derived products, and adoptive immunotherapy using antitumor immune cells were conducted in various cancers, most extensively in melanoma, and tumor regression was observed in some patients. Active Immunization Immunizations with synthetic peptides, particularly MHC class I–binding epitopes, were performed in various trials. Since native epitopes have relatively low immunogenicity, various immunoaugmenting methods, including coadministration of adjuvants and cytokines [incomplete Freund adjuvant (IFA), IL-2, IL-12, or GM-CSF], were applied to achieve efficient immunization. Tumor regression in melanoma patients was observed in various clinical trials using melanocytespecific antigens such as MART-1 and gp100 and, in particular, the HLA high-binding modified peptide. Since CD4+ T cells appear to be directly and indirectly important in tumor rejection, combined immunization with both Th and CTL antigens is being attempted. Immunization with proteins containing multiple Th and CTL epitopes may be effective,

cell responses20.

tumours, including pancreatic tumours30.

patients with pancreatic cancer within 2 to 3 years.

**3. Cancer immunotherapy protocols** 

important role in in vivo tumor rejection. Because of their expression of MHC class I, CD8+ T cells are integral in the eradication of most solid tumors. However, CD4+ T cells are also important in the induction and maintenance of final effectors, such as CD8+ T cells and macrophages, as well as for the accumulation of CD8+ T cells in tumor tissues. Thus, we are applying various methods to identify human tumor antigens recognized by T cells.

Immunotherapy has an advantage over radiation therapy and chemotherapy because it can act specifically against the tumor without damaging normal tissue. Immunotherapeutic approaches to PC have included the use of monoclonal antibodies 47, cytokines 8, vaccine 9 and lymphokine activated killer (LAK) cells (10).

#### **2. Immune surveillance and tumour evasion**

The extraordinary features of the immune system make it possible to discern self from nonself. However, most human cancers, and pancreatic cancer in particular, are known to be poorly immunogenic, as crucial somatic genetic mutations can generate pancreatic cancer proteins that are essentially altered self proteins. Furthermore, promising immunotherapeutic approaches that have been used for relatively immunogenic cancers such as melanoma have met with variable success6. These observations have revealed that for tumours to form and progress, they must develop local and/or systemic mechanisms that subsequently allow them to escape the normal surveillance mechanisms of the intact immune system. Immune-based therapies must therefore incorporate at least one agent against a pancreatic cancer target as well as one or more agents that will modify both local and systemic mechanisms of pancreatic-cancer-induced IMMUNE TOLERANCE.

It is now clear that both local characteristics of the tumour microenvironment as well as systemic factors are important for the immune evasion of tumours. For example, T-cell recognition of pancreatic tumours might be inhibited or suppressed due to the downregulation of human leukocyte antigen (HLA) CLASS I tumour-antigen complexes on tumour cells by a range of intracellular mechanisms4, 7 — upregulation of immuneinhibition molecules11, 12, 13, 14, 15, 16, 17, loss of immune-regulation signals15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, defects in immune-cell tumour localization31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 and loss of co-stimulatory molecules52, 53, 54, 55, 56, 57. Such alterations within a tumour cell would not be unexpected, as they have unstable genomes. The local inflammatory reaction is also an important triggering event in the recruitment of professional ANTIGEN-PRESENTING CELLS (APCs) and effector cells, such as T cells and NATURAL KILLER (NK) CELLS, to the tumour site. However, pancreatic tumour cells express a range of proteins that inhibit pro-inflammatory cytokines and DENDRITIC CELL (DC) MATURATION58, 59, 60.

In addition, the numbers of CD4+CD25+ T regulatory (TReg) CELLS — a subset of T cells that are known to be important in the suppression of self-reactive T cells (peripheral tolerance) — accumulate in pancreatic tumours61, 62, 63. Although these cells are thought to be activated during the immunization process, TReg cells seem to localize to tumour sites. Tumour production of the chemokine CCL22 probably attracts the TReg cells by interacting with the CCR4 receptor that is expressed by these cells64.

Other important elements in regulating the T-cell recognition of pancreatic tumours are the inhibitory pathways, known as 'immunological checkpoints'. Immunological checkpoints

important role in in vivo tumor rejection. Because of their expression of MHC class I, CD8+ T cells are integral in the eradication of most solid tumors. However, CD4+ T cells are also important in the induction and maintenance of final effectors, such as CD8+ T cells and macrophages, as well as for the accumulation of CD8+ T cells in tumor tissues. Thus, we are

Immunotherapy has an advantage over radiation therapy and chemotherapy because it can act specifically against the tumor without damaging normal tissue. Immunotherapeutic approaches to PC have included the use of monoclonal antibodies 47, cytokines 8, vaccine 9

The extraordinary features of the immune system make it possible to discern self from nonself. However, most human cancers, and pancreatic cancer in particular, are known to be poorly immunogenic, as crucial somatic genetic mutations can generate pancreatic cancer proteins that are essentially altered self proteins. Furthermore, promising immunotherapeutic approaches that have been used for relatively immunogenic cancers such as melanoma have met with variable success6. These observations have revealed that for tumours to form and progress, they must develop local and/or systemic mechanisms that subsequently allow them to escape the normal surveillance mechanisms of the intact immune system. Immune-based therapies must therefore incorporate at least one agent against a pancreatic cancer target as well as one or more agents that will modify both local

and systemic mechanisms of pancreatic-cancer-induced IMMUNE TOLERANCE.

It is now clear that both local characteristics of the tumour microenvironment as well as systemic factors are important for the immune evasion of tumours. For example, T-cell recognition of pancreatic tumours might be inhibited or suppressed due to the downregulation of human leukocyte antigen (HLA) CLASS I tumour-antigen complexes on tumour cells by a range of intracellular mechanisms4, 7 — upregulation of immuneinhibition molecules11, 12, 13, 14, 15, 16, 17, loss of immune-regulation signals15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, defects in immune-cell tumour localization31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 and loss of co-stimulatory molecules52, 53, 54, 55, 56, 57. Such alterations within a tumour cell would not be unexpected, as they have unstable genomes. The local inflammatory reaction is also an important triggering event in the recruitment of professional ANTIGEN-PRESENTING CELLS (APCs) and effector cells, such as T cells and NATURAL KILLER (NK) CELLS, to the tumour site. However, pancreatic tumour cells express a range of proteins that inhibit pro-inflammatory cytokines and DENDRITIC CELL

In addition, the numbers of CD4+CD25+ T regulatory (TReg) CELLS — a subset of T cells that are known to be important in the suppression of self-reactive T cells (peripheral tolerance) — accumulate in pancreatic tumours61, 62, 63. Although these cells are thought to be activated during the immunization process, TReg cells seem to localize to tumour sites. Tumour production of the chemokine CCL22 probably attracts the TReg cells by interacting

Other important elements in regulating the T-cell recognition of pancreatic tumours are the inhibitory pathways, known as 'immunological checkpoints'. Immunological checkpoints

applying various methods to identify human tumor antigens recognized by T cells.

and lymphokine activated killer (LAK) cells (10).

(DC) MATURATION58, 59, 60.

with the CCR4 receptor that is expressed by these cells64.

**2. Immune surveillance and tumour evasion** 

serve two purposes. One is to help generate and maintain self-tolerance, by eliminating T cells that are specific for self-antigens. The other is to restrain the amplitude of normal T-cell responses so that they do not 'overshoot' in their natural response to foreign pathogens. The prototypical immunological checkpoint is mediated by the cytotoxic-T-lymphocyteassociated protein 4 (CTLA4) counter-regulatory receptor that is expressed by T cells when they become activated15, 23. CTLA4 binds two B7-FAMILY members on the surface APCs — B7.1 (also known as CD80) and B7.2 (also known as CD86) — with roughly 20-fold higher affinity than the T-cell surface protein CD28 binds these molecules. CD28 is a co-stimulatory receptor that is constitutively expressed on naive T cells. Because of its higher affinity, CTLA4 out-competes CD28 for B7.1/B7.2 binding, resulting in the downmodulation of Tcell responses20.

A range of B7-family members interact with co-stimulatory and counter-regulatory inhibitory receptors on T cells. Two recently discovered B7-family members, B7-H1 (also known as PD-L1) and B7-DC (also known as PD-L2) also seem to interact with T-cell costimulatory and counter-regulatory inhibitory receptors18, 29, 30. PD-L1, which is upregulated on T cells when they become activated, seems to control a counter-regulatory immunological checkpoint when it binds PD-1 26,28,29. Activating receptors for B7-DC and B7-H1 have not yet been definitively identified. B7-DC is expressed on DCs, and is likely to have a co-stimulatory role in increasing activation of naive or resting T cells. In contrast to B7.1, B7.2 and B7-DC, B7-H1 is also expressed on several peripheral tissues and on many tumours, including pancreatic tumours30.

Another new B7-family member, B7-H4, seems to mediate a predominantly inhibitory function in the immune system14. Recent data indicate that pancreatic tumours also express B7-H4 (D.L. and E.M.J., manuscript in preparation), and both B7-H1 and B7-H4 probably protect tumours from immune-system attack. Preclinical studies have already demonstrated that it is possible to downregulate B7-H1 signalling in mice, improving the antitumour response to vaccination18. Monoclonal antibodies that downregulate B7-H1 and B7-H4 are currently in clinical development. These antibodies will probably begin clinical testing in patients with pancreatic cancer within 2 to 3 years.

#### **3. Cancer immunotherapy protocols**

Clinical trials using various immunotherapies, active immunization with tumor antigens, or tumor cell–derived products, and adoptive immunotherapy using antitumor immune cells were conducted in various cancers, most extensively in melanoma, and tumor regression was observed in some patients. Active Immunization Immunizations with synthetic peptides, particularly MHC class I–binding epitopes, were performed in various trials. Since native epitopes have relatively low immunogenicity, various immunoaugmenting methods, including coadministration of adjuvants and cytokines [incomplete Freund adjuvant (IFA), IL-2, IL-12, or GM-CSF], were applied to achieve efficient immunization. Tumor regression in melanoma patients was observed in various clinical trials using melanocytespecific antigens such as MART-1 and gp100 and, in particular, the HLA high-binding modified peptide. Since CD4+ T cells appear to be directly and indirectly important in tumor rejection, combined immunization with both Th and CTL antigens is being attempted. Immunization with proteins containing multiple Th and CTL epitopes may be effective,

An Overview on Immunotherapy of Pancreatic Cancer 141

cancer. Strong antitumor effects, however, were not observed in the reported clinical trials. In pancreatic cancer, vaccination with GM-CSF transduced allogeneic pancreatic cancer cell lines along with adjuvant radiation and chemotherapy following surgical excision demonstrated possible benefit in disease-free survival, which appeared to be associated with

Passive immunotherapy with large doses of activated antitumor lymphocytes was also employed since there was a possibility that active immunization would be insufficient to induce enough of an immune response to cause tumor regression in the immunosuppressed patient with a large tumor burden. Adoptive transfer of tumor-reactive T cells cultured from tumor-infiltrating lymphocytes, along with IL-2, resulted in a clinical response in melanoma patients.65 Adoptive transfer of EBV-specific T cells resulted in regression of EBV-associated lymphoma. Intraportal infusion of in vitro MUC1-stimulated T cells was performed in pancreatic cancer, yielding preliminary results that indicate inhibition of liver metastasis. Although the clinical use of tumor-reactive T cells was previously limited due to the difficulty in generating tumor-reactive T cells for most cancers, it is now possible to generate these cells from the PBMC of cancer patients by in vitro stimulation, using the identified tumor antigens.66 Tumor-reactive T cells from patients preimmunized with tumor antigens were generated more efficiently, which suggests that combined use of active and passive immunotherapies is ideal. One of the problems that arises from adoptive transfer of cultured T cells is the low efficiency of administered T cells in in vivo maintenance and accumulation in tumor tissues. However, it was recently reported that nonmyeloablative, lymphodepletive pre-treatment with cyclophosphamide and fludarabine resulted in extended persistence of administered tumor-reactive T cells in peripheral blood and tumor tissues and increased tumor regression, which may be due to suppression of patient immune responses or the need to make room for homeostatic proliferation of transferred lymphocytes.67 Adoptive immunotherapy with IL- 2–activated PBMC, LAK (lymphokine activated killer) cells displayed some antitumor effects when locally administered (ie, by intrapleural or intraarterial infusion) for lung or liver cancer. Intraportal administration following intraoperative irradiation in pancreatic cancer patients is reported to result in

Adoptive immunotherapy involves harvesting the patient's peripheral blood Tlymphocytes, stimulating and expanding the autologous tumour-reactive T-cells using IL-2 and CD3-specific antibody, before subsequently transferring them back into the patient. Twelve patients with advanced pancreatic cancer who underwent resection, intraoperative radiotherapy and intraportal infusion of LAK cells with recombinant IL-2 had lower incidence of liver metastasis compared to controls (three of 12 vs ten of 15; p<0.05)69. There was no significant difference in overall survival, but more patients were alive three years

**Telomerase—**Telomerase is a reverse transcriptase that contains a RNA template used to synthesise telomeric repeats onto chromosomal ends. Activation of telomerase and its maintenance of telomeres play a role in immortalisation of human cancer cells, as telomeres

the increase of postvaccination DTH responses against autologous tumor cells.

**4. Adoptive Immunotherapy with antitumor** 

**4.1 Immune cells** 

possible prolongation of survival68.

later (36% vs none).

although production of recombinant GMP-grade proteins is costly, and modifications such as particle formation may be required for effective presentation of MHC class I–restricted epitopes. To facilitate peptide immunization in melanoma, coadministration of the anti-CTLA4 antibody, which blocks regulatory T cells and negative feedback regulation of T-cell activation, was carried out. Although tumor regression along with autoimmune reactions was observed, augmentation of the immune response to the administered peptides was not observed in peripheral blood.24 In pancreatic cancer, intradermal immunization with the mutated K-*ras* peptides and GM-CSF resulted in the induction of a memory CD4+ T-cell response and prolonged survival, compared with nonresponders.15 Immunization with the MUC1 peptide and BCG resulted in augmented immune responses without tumor regression.22 Immunization with recombinant viruses or plasmids containing tumor antigen cDNA (DNA immunization) rather than peptide/proteins may be applied. In melanoma clinical trials, a generation of neutralizing antibodies against viral proteins appeared to interfere with the induction of immune response to tumor antigens following immunization with recombinant adenovirus and vaccinia virus.25 However, recent protocols using a recombinant fowlpox virus containing the modified gp100 cDNA or the ER signal sequence– conjugated gp100-epitope minimal gene demonstrated frequent induction of tumor reactive T cells.26 Interestingly, tumor regression was observed in patients after subsequent administration of IL-2.

Intramuscular immunization with the recombinant gp100 plasmids appeared to be insufficient to induce an antitumor T-cell response.27 DC are the most potent professional APC that can process antigens for both MHC class I and II pathways and activate both naive CD4+ T cells and CD8+T cells in vivo. In murine studies, immunization with DC pulsed with tumor antigens resulted in better antitumor effects than direct peptide administration. In immunization trials using DC pulsed with tumor lysates or synthetic peptides, tumor regression was observed in patients with various cancers, including melanoma, prostate cancer, colon cancer, and B-cell lymphoma.28 Although most clinical trials have used monocyte-derived DC, peripheral blood DC as well as CD34+ cell–derived DC have been used in some protocols.29 Antigen loading on DC using various antigens including RNA, cDNA, recombinant virusand cell-penetrating peptide conjugated proteins has also been exploited. DC fused with tumor cells and leukemia clone– derived DC have also been used in clinical trials. K-ras– specific T cells were detected in pancreatic cancer patients following multiple intravenous infusions of peptide-pulsed antigen presenting mononuclear cells obtained by leukapheresis, although no therapeutic effect in patients was observed. In addition, no tumor regression was observed following immunization with DC transfected with MUC1 cDNA. A decrease in tumor marker was observed in a patient with a pancreatic neuroendocrine tumor, following immunization with DC pulsed with autologous tumor lysates. Intratumoral administration of immature DC following intraoperative irradiation is currently being conducted in Japan. Thus far, any antitumor effects observed in these DCbased clinical trials for pancreatic cancer are weak. Protocols for the optimal use of DC in immunotherapy, including the source of DC, kinds of tumor antigens, methods for maturation and antigen loading, site and schedule for administration, remain to be determined. Based on murine experiments, immunization with more immunogenic tumor cells that are modified using various techniques, including hapten conjugation, foreign antigen introduction, and transfection with various genes such as cytokines (eg, GM-CSF, IL-2, TNF-\_, IFN-\_, IL-4) have been employed in melanoma, prostate cancer, and lung cancer. Strong antitumor effects, however, were not observed in the reported clinical trials. In pancreatic cancer, vaccination with GM-CSF transduced allogeneic pancreatic cancer cell lines along with adjuvant radiation and chemotherapy following surgical excision demonstrated possible benefit in disease-free survival, which appeared to be associated with the increase of postvaccination DTH responses against autologous tumor cells.

#### **4. Adoptive Immunotherapy with antitumor**

#### **4.1 Immune cells**

140 Pancreatic Cancer – Clinical Management

although production of recombinant GMP-grade proteins is costly, and modifications such as particle formation may be required for effective presentation of MHC class I–restricted epitopes. To facilitate peptide immunization in melanoma, coadministration of the anti-CTLA4 antibody, which blocks regulatory T cells and negative feedback regulation of T-cell activation, was carried out. Although tumor regression along with autoimmune reactions was observed, augmentation of the immune response to the administered peptides was not observed in peripheral blood.24 In pancreatic cancer, intradermal immunization with the mutated K-*ras* peptides and GM-CSF resulted in the induction of a memory CD4+ T-cell response and prolonged survival, compared with nonresponders.15 Immunization with the MUC1 peptide and BCG resulted in augmented immune responses without tumor regression.22 Immunization with recombinant viruses or plasmids containing tumor antigen cDNA (DNA immunization) rather than peptide/proteins may be applied. In melanoma clinical trials, a generation of neutralizing antibodies against viral proteins appeared to interfere with the induction of immune response to tumor antigens following immunization with recombinant adenovirus and vaccinia virus.25 However, recent protocols using a recombinant fowlpox virus containing the modified gp100 cDNA or the ER signal sequence– conjugated gp100-epitope minimal gene demonstrated frequent induction of tumor reactive T cells.26 Interestingly, tumor regression was observed in patients after subsequent

Intramuscular immunization with the recombinant gp100 plasmids appeared to be insufficient to induce an antitumor T-cell response.27 DC are the most potent professional APC that can process antigens for both MHC class I and II pathways and activate both naive CD4+ T cells and CD8+T cells in vivo. In murine studies, immunization with DC pulsed with tumor antigens resulted in better antitumor effects than direct peptide administration. In immunization trials using DC pulsed with tumor lysates or synthetic peptides, tumor regression was observed in patients with various cancers, including melanoma, prostate cancer, colon cancer, and B-cell lymphoma.28 Although most clinical trials have used monocyte-derived DC, peripheral blood DC as well as CD34+ cell–derived DC have been used in some protocols.29 Antigen loading on DC using various antigens including RNA, cDNA, recombinant virusand cell-penetrating peptide conjugated proteins has also been exploited. DC fused with tumor cells and leukemia clone– derived DC have also been used in clinical trials. K-ras– specific T cells were detected in pancreatic cancer patients following multiple intravenous infusions of peptide-pulsed antigen presenting mononuclear cells obtained by leukapheresis, although no therapeutic effect in patients was observed. In addition, no tumor regression was observed following immunization with DC transfected with MUC1 cDNA. A decrease in tumor marker was observed in a patient with a pancreatic neuroendocrine tumor, following immunization with DC pulsed with autologous tumor lysates. Intratumoral administration of immature DC following intraoperative irradiation is currently being conducted in Japan. Thus far, any antitumor effects observed in these DCbased clinical trials for pancreatic cancer are weak. Protocols for the optimal use of DC in immunotherapy, including the source of DC, kinds of tumor antigens, methods for maturation and antigen loading, site and schedule for administration, remain to be determined. Based on murine experiments, immunization with more immunogenic tumor cells that are modified using various techniques, including hapten conjugation, foreign antigen introduction, and transfection with various genes such as cytokines (eg, GM-CSF, IL-2, TNF-\_, IFN-\_, IL-4) have been employed in melanoma, prostate cancer, and lung

administration of IL-2.

Passive immunotherapy with large doses of activated antitumor lymphocytes was also employed since there was a possibility that active immunization would be insufficient to induce enough of an immune response to cause tumor regression in the immunosuppressed patient with a large tumor burden. Adoptive transfer of tumor-reactive T cells cultured from tumor-infiltrating lymphocytes, along with IL-2, resulted in a clinical response in melanoma patients.65 Adoptive transfer of EBV-specific T cells resulted in regression of EBV-associated lymphoma. Intraportal infusion of in vitro MUC1-stimulated T cells was performed in pancreatic cancer, yielding preliminary results that indicate inhibition of liver metastasis. Although the clinical use of tumor-reactive T cells was previously limited due to the difficulty in generating tumor-reactive T cells for most cancers, it is now possible to generate these cells from the PBMC of cancer patients by in vitro stimulation, using the identified tumor antigens.66 Tumor-reactive T cells from patients preimmunized with tumor antigens were generated more efficiently, which suggests that combined use of active and passive immunotherapies is ideal. One of the problems that arises from adoptive transfer of cultured T cells is the low efficiency of administered T cells in in vivo maintenance and accumulation in tumor tissues. However, it was recently reported that nonmyeloablative, lymphodepletive pre-treatment with cyclophosphamide and fludarabine resulted in extended persistence of administered tumor-reactive T cells in peripheral blood and tumor tissues and increased tumor regression, which may be due to suppression of patient immune responses or the need to make room for homeostatic proliferation of transferred lymphocytes.67 Adoptive immunotherapy with IL- 2–activated PBMC, LAK (lymphokine activated killer) cells displayed some antitumor effects when locally administered (ie, by intrapleural or intraarterial infusion) for lung or liver cancer. Intraportal administration following intraoperative irradiation in pancreatic cancer patients is reported to result in possible prolongation of survival68.

Adoptive immunotherapy involves harvesting the patient's peripheral blood Tlymphocytes, stimulating and expanding the autologous tumour-reactive T-cells using IL-2 and CD3-specific antibody, before subsequently transferring them back into the patient. Twelve patients with advanced pancreatic cancer who underwent resection, intraoperative radiotherapy and intraportal infusion of LAK cells with recombinant IL-2 had lower incidence of liver metastasis compared to controls (three of 12 vs ten of 15; p<0.05)69. There was no significant difference in overall survival, but more patients were alive three years later (36% vs none).

**Telomerase—**Telomerase is a reverse transcriptase that contains a RNA template used to synthesise telomeric repeats onto chromosomal ends. Activation of telomerase and its maintenance of telomeres play a role in immortalisation of human cancer cells, as telomeres

An Overview on Immunotherapy of Pancreatic Cancer 143

effect of gemcitabine with or without Virulizin in 434 chemotherapy-naïve patients with advanced pancreatic cancer [341]. MS was not significantly better for the gemcitabine and Virulizin group compared to gemcitabine with placebo (6.3 vs 6 months). However for stage 3 patients who received Virulizin in a salvage setting, a significant difference in survival was

Pancreatic cancer could thus constitute a paradigmatic example of neoplasia where tumorrelated variables and host immunosuppressive status have the same importance in determining an unfavourable prognosis. The severe suppression of anticancer immunity, which characterizes patients suffering from pancreatic cancer, is further aggravated by surgical treatment 98. In fact, it is known that surgery may inhibit anticancer immunity by provoking a postoperative decline in the absolute number of circulating lymphocytes 99-101, which play a fundamental role in generating an effective anticancer immune reaction; this is

Surgery-induced immunosuppression could represent one of the main factors responsible for relapse in cancer patients treated by radical surgery, by possibly promoting the growth of micro-metastases, already existing at the time of the surgical removal of the tumor . Previous clinical studies have shown that the immunosuppressive status occurring during the postoperative period is particularly severe in patients with pancreatic cancer and this evidence could explain, at least in part, the high percentage of recurrences occurring in patients radically operated for cancer of the pancreas 103. At present, the only molecule which has been proven to correct the lymphocytopenia is IL-2, representing the main growth factor for lymphocytes, including T lymphocytes and natural killer (NK) cells 104 and the stimulation of lymphocyte proliferation would constitute the main mechanism responsible for the antitumor activity of IL-2 in the immunotherapy of cancer 105. Moreover, the preoperative administration of IL-2 for only few days prior to surgery was effective in preventing surgery-induced lymphocytopenia 106. In addition, the abrogation of surgeryinduced lymphocyte decline has been shown to improve the prognosis of patients with colorectal cancer in whether treated by radical or palliative surgery 107. The therapeutic impact of IL-2 presurgical administration remains to be better defined in gastric cancer 108, despite its efficacy in preventing the postoperative lymphocytopenia. Finally, the prevention of postoperative lymphocyte decline by IL-2 presurgical immunotherapy was associated with clear lymphocyte and eosinophil intratumoral infiltration in colorectal cancer patients, which, in contrast, was less evident in patients with gastric carcinoma. Preliminary clinical studies have suggested that preoperative injection of IL-2 may also prevent surgery-induced lymphocytopenia in patients with pancreatic cancer 109. According to previous investigations, IL-2 presurgical immunotherapy may also completely abrogate surgeryinduced lymphocytopenia also patients with pancreatic carcinoma, as well as previously described for both colorectal and gastric carcinomas. Moreover, in agreement with the clinical results previously reported for colorectal cancer patients and in contrast to those more controversially reported in gastric cancer, this study would suggest that a preoperative immunotherapy with IL-2 may improve the clinical course of the pancreatic cancer in terms of both FFPP and OS. Therefore, particularly because of its unfavourable prognosis, presurgical immunotherapy with IL-2 could represent a simple but effective

demonstrated (10.9 vs 7.4 months, p=0.017).

fundamentally an IL-2-dependent phenomenon 102.

**4.3 IL-2** 

shrink after each cell division 70. Telomerase activity is found in 92-95% of pancreatic cancers 71-72, and is associated with increased potential of invasion and metastasis and poor prognosis 73-74. Upregulation of telomerase may also be responsible for the development of chemotherapy resistance 75. Adenovirus-mediated transduction of p53 gene inhibited telomerase activity in MIAPaCa-2, SUIT-2 and AsPC-1 cells, independent of its effect on apoptosis, cell growth and cycle arrest 76. Antisense to the RNA component of telomerase seemed to increase susceptibility of Panc-1 cells to cisplatin 77. Telomerase reverse transcriptase antisense oligonucleotide (hTERT-ASO) was found to inhibit the proliferation of BxPC-3 cells *in vitro* by decreasing telomerase activity and increasing apoptosis 78. Adoptive transfer of telomerase-specific T-cells was studied in a syngeneic pancreatic tumour mouse model 79. T-cells were produced *in vitro* by coculturing human lymphocytes with telomerase peptide-pulsed dendritic cells (DCs) or *in vivo* by injection of peptide with adjuvant into C57BL/6 mice. Animals treated with these T-cells showed significantly delayed disease progression.

**MUC1—**Adoptive transfer of MUC1-specific cytotoxic T-lymphocytes (CTLs) was able to completely eradicate MUC1-expressing tumours in mice 80. Intraportal infusion of *In vitro*  MUC1-stimulated T-cells was performed in patients with pancreatic cancer, with subsequent inhibition of liver metastasis 81. In a study of eleven patients with lung metastases (from colorectal, pancreatic, breast, lung, or melanoma primaries), effector cells were generated *in vitro* using cultured DCs, synthetic peptide, peripheral blood lymphocytes, IL-2 and anti-CD3 antibody 82. A partial response of the lung metastases was observed in a patient with pancreatic cancer who received these cells stimulated with MUC1.

#### **4.2 Cytokines and immunomodulators**

**TNFerade—**TNF-α is a multifunctional cytokine that has shown antitumour potency 83-85. TNFerade Biologic (TNFerade) is a replication-deficient adenovirus carrying the gene for human TNF-α, regulated by a radiation-inducible promoter Early Growth Response (Egr-1). The latter would ensure maximal gene expression when infected tissue is irradiated 86. TNFerade was effective in combination with radiation in a number of human xenograft models, including glioma 87, prostate 88, oesophageal 89 and radiation-resistant laryngeal cancers 90. The multicentre phase II/III Pancreatic Cancer Clinical Trial with TNFerade (PACT) is currently ongoing and involved patients with locally advanced pancreatic cancer. Patients were given radiotherapy, 5-FU with or without CT-guided transabdominal injection of TNFerade. Preliminary data of 51 patients revealed that the one-year survival increased from 28% to 70.5% with the addition of TNFerade, with MS of 335 and 515 days respectively91.

**Virulizin—**Virulizin (Lorus Therapeutics Inc.) is a biological response modifier obtained from bovine bile 92. It stimulates the expression of TNF-α and activates macrophages, which subsequently activates natural killer cells via IL-12 93-94. Evidence exists to show that it also induces the production of IL-17E with resulting eosinophilia 95.

*In vivo* studies showed that Virulizin significantly inhibited the growth of human pancreatic cancer xenografts (BxPC-3, SU 86.86 and MIAPaCa-2) in nude mice, as well as potentiated the antitumour effect of gemcitabine and 5-FU 96-97. A phase III trial was conducted to study the effect of gemcitabine with or without Virulizin in 434 chemotherapy-naïve patients with advanced pancreatic cancer [341]. MS was not significantly better for the gemcitabine and Virulizin group compared to gemcitabine with placebo (6.3 vs 6 months). However for stage 3 patients who received Virulizin in a salvage setting, a significant difference in survival was demonstrated (10.9 vs 7.4 months, p=0.017).

#### **4.3 IL-2**

142 Pancreatic Cancer – Clinical Management

shrink after each cell division 70. Telomerase activity is found in 92-95% of pancreatic cancers 71-72, and is associated with increased potential of invasion and metastasis and poor prognosis 73-74. Upregulation of telomerase may also be responsible for the development of chemotherapy resistance 75. Adenovirus-mediated transduction of p53 gene inhibited telomerase activity in MIAPaCa-2, SUIT-2 and AsPC-1 cells, independent of its effect on apoptosis, cell growth and cycle arrest 76. Antisense to the RNA component of telomerase seemed to increase susceptibility of Panc-1 cells to cisplatin 77. Telomerase reverse transcriptase antisense oligonucleotide (hTERT-ASO) was found to inhibit the proliferation of BxPC-3 cells *in vitro* by decreasing telomerase activity and increasing apoptosis 78. Adoptive transfer of telomerase-specific T-cells was studied in a syngeneic pancreatic tumour mouse model 79. T-cells were produced *in vitro* by coculturing human lymphocytes with telomerase peptide-pulsed dendritic cells (DCs) or *in vivo* by injection of peptide with adjuvant into C57BL/6 mice. Animals treated with these T-cells showed significantly

**MUC1—**Adoptive transfer of MUC1-specific cytotoxic T-lymphocytes (CTLs) was able to completely eradicate MUC1-expressing tumours in mice 80. Intraportal infusion of *In vitro*  MUC1-stimulated T-cells was performed in patients with pancreatic cancer, with subsequent inhibition of liver metastasis 81. In a study of eleven patients with lung metastases (from colorectal, pancreatic, breast, lung, or melanoma primaries), effector cells were generated *in vitro* using cultured DCs, synthetic peptide, peripheral blood lymphocytes, IL-2 and anti-CD3 antibody 82. A partial response of the lung metastases was observed in a patient with pancreatic cancer who received these cells stimulated with

**TNFerade—**TNF-α is a multifunctional cytokine that has shown antitumour potency 83-85. TNFerade Biologic (TNFerade) is a replication-deficient adenovirus carrying the gene for human TNF-α, regulated by a radiation-inducible promoter Early Growth Response (Egr-1). The latter would ensure maximal gene expression when infected tissue is irradiated 86. TNFerade was effective in combination with radiation in a number of human xenograft models, including glioma 87, prostate 88, oesophageal 89 and radiation-resistant laryngeal cancers 90. The multicentre phase II/III Pancreatic Cancer Clinical Trial with TNFerade (PACT) is currently ongoing and involved patients with locally advanced pancreatic cancer. Patients were given radiotherapy, 5-FU with or without CT-guided transabdominal injection of TNFerade. Preliminary data of 51 patients revealed that the one-year survival increased from 28% to 70.5% with the addition of TNFerade, with MS of 335 and 515 days

**Virulizin—**Virulizin (Lorus Therapeutics Inc.) is a biological response modifier obtained from bovine bile 92. It stimulates the expression of TNF-α and activates macrophages, which subsequently activates natural killer cells via IL-12 93-94. Evidence exists to show that it also

*In vivo* studies showed that Virulizin significantly inhibited the growth of human pancreatic cancer xenografts (BxPC-3, SU 86.86 and MIAPaCa-2) in nude mice, as well as potentiated the antitumour effect of gemcitabine and 5-FU 96-97. A phase III trial was conducted to study the

induces the production of IL-17E with resulting eosinophilia 95.

delayed disease progression.

**4.2 Cytokines and immunomodulators** 

MUC1.

respectively91.

Pancreatic cancer could thus constitute a paradigmatic example of neoplasia where tumorrelated variables and host immunosuppressive status have the same importance in determining an unfavourable prognosis. The severe suppression of anticancer immunity, which characterizes patients suffering from pancreatic cancer, is further aggravated by surgical treatment 98. In fact, it is known that surgery may inhibit anticancer immunity by provoking a postoperative decline in the absolute number of circulating lymphocytes 99-101, which play a fundamental role in generating an effective anticancer immune reaction; this is fundamentally an IL-2-dependent phenomenon 102.

Surgery-induced immunosuppression could represent one of the main factors responsible for relapse in cancer patients treated by radical surgery, by possibly promoting the growth of micro-metastases, already existing at the time of the surgical removal of the tumor . Previous clinical studies have shown that the immunosuppressive status occurring during the postoperative period is particularly severe in patients with pancreatic cancer and this evidence could explain, at least in part, the high percentage of recurrences occurring in patients radically operated for cancer of the pancreas 103. At present, the only molecule which has been proven to correct the lymphocytopenia is IL-2, representing the main growth factor for lymphocytes, including T lymphocytes and natural killer (NK) cells 104 and the stimulation of lymphocyte proliferation would constitute the main mechanism responsible for the antitumor activity of IL-2 in the immunotherapy of cancer 105. Moreover, the preoperative administration of IL-2 for only few days prior to surgery was effective in preventing surgery-induced lymphocytopenia 106. In addition, the abrogation of surgeryinduced lymphocyte decline has been shown to improve the prognosis of patients with colorectal cancer in whether treated by radical or palliative surgery 107. The therapeutic impact of IL-2 presurgical administration remains to be better defined in gastric cancer 108, despite its efficacy in preventing the postoperative lymphocytopenia. Finally, the prevention of postoperative lymphocyte decline by IL-2 presurgical immunotherapy was associated with clear lymphocyte and eosinophil intratumoral infiltration in colorectal cancer patients, which, in contrast, was less evident in patients with gastric carcinoma. Preliminary clinical studies have suggested that preoperative injection of IL-2 may also prevent surgery-induced lymphocytopenia in patients with pancreatic cancer 109. According to previous investigations, IL-2 presurgical immunotherapy may also completely abrogate surgeryinduced lymphocytopenia also patients with pancreatic carcinoma, as well as previously described for both colorectal and gastric carcinomas. Moreover, in agreement with the clinical results previously reported for colorectal cancer patients and in contrast to those more controversially reported in gastric cancer, this study would suggest that a preoperative immunotherapy with IL-2 may improve the clinical course of the pancreatic cancer in terms of both FFPP and OS. Therefore, particularly because of its unfavourable prognosis, presurgical immunotherapy with IL-2 could represent a simple but effective

An Overview on Immunotherapy of Pancreatic Cancer 145

serve as T-cell and antibody targets. These advances now make it possible to exploit the

By the time that patients are diagnosed with pancreatic cancer, the tumour has typically progressed and invaded adjacent structures. Perineural invasion, metastasis to lymph nodes and liver, and an intense DESMOPLASTIC STROMAL REACTION are commonly observed. A range of signalling pathways, including epidermal growth factor receptor (EGFR) and the PI3K–AKT–mTOR–S6K cascades, are known to mediate pancreatic tumour growth and progression111n addition, new blood-vessel formation (angiogenesis) is required for the growth of primary pancreatic tumours and is essential for metastasis. In pancreatic tumours, this process is probably regulated by fibroblast growth factor, platelet-derived endothelialcell growth factor and VEGF family members. In fact, several pancreatic-cancer-associated genes have been linked to angiogenesis. DPC4 upregulates VEGF expression, and mutated

Monoclonal antibodies that target a range of these pathways have demonstrated efficacy in preclinical models113-115dition, monoclonal antibodies that target EGFR and VEGF receptor have been tested in patients with a range of cancers, including pancreatic cance115,117hough these antibodies have demonstrated only modest results as single agents, the pathways they

Preclinical evidence has also shown that specific inhibitors of these signalling pathways can also increase immune activation. For example, VEGF is a key inhibitor of pro-inflammatory cytokines as well as dendritic-cell maturation, and it can also directly inhibit T-cell development. So antibodies that block signalling by this growth factor can promote antitumour immune responses. Furthermore, downregulation of the ERBB-receptor-family members with drugs such as herceptin promotes tumour-antigen presentation by HLA class I molecules, improving the potential for T-cell recognition and lysis118onoclonal antibodies that target these signalling pathways are now being developed for clinical trials as agents

that potentially synergize with other immune-based approaches, including vaccines.

To develop the ideal vaccine for pancreatic cancer, the following wish list would probably need to be fulfilled. First, specific cell-surface proteins must be identified that are that are crucial in the cancer growth or progression pathway and are unique to pancreatic cancer tumours. Second, these tumour-exclusive proteins should be shown to elicit a vigorous tumour-protein-specific immune response. Third, the best carrier to deliver the appropriate immunogenic tumour proteins should be identified. Fourth, molecules that are immune stimulatory as well as molecules that can abrogate the natural immune-inhibition signalling that is seen in pancreatic cancer should be identified to enhance the immune response. Fifth, additional synergistic immune help should be identified (for example, antibodies or *ex vivo* tumour-reactive T cells). Several proteins, such as carcinoembryonic antigen (CEA), mutated KRAS, mucin-1 (MUC1) and gastrin, have in fact been identified to be specifically overexpressed in most pancreatic cancers119-125 antigens were identified over 10 years ago using various methods to analyse gene expression in cancer cells. Vaccines and antibodies

immune system in the fight against pancreatic cancer.

KRAS expression is associated with increased micro-vessel density112.

affect are also candidate targets for immune intervention.

**4.6 Vaccines against pancreatic tumour antigens** 

**4.5 Targeting signalling molecules** 

clinical strategy to improve the prognosis of pancreatic cancer patients undergoing macroscopical radical surgery.

#### **4.4 Allogeneic antigen-specific immunotherapy**

Allogeneic antigen-specific immunotherapies, nonmyeloablative SCT (minitransplant) and DLI (donor leukocyte infusion), are reported to have some antitumor effect [graft versus tumor (GVT)] on solid tumors, including RCC, breast cancer, and pancreatic cancer, in addition to haematological malignancies.110GVT effects were also observed in pancreatic cancer patients in minitransplant protocols conducted in Japan. Although the mechanisms of the antitumor effects, such as allogeneic responses to minor histocompatibility antigens (mHa), on hematological malignancies are well studied, they remain unclear with regard to solid tumors. One of the major problems in allogeneic treatment of the solid tumor is severe GVHD. Several strategies for the separation of GVT and GVHD have been developed for hematological malignancies. Whether this separation is possible for solid tumors, however, is unclear.

Was reported on the efficacy of adoptive immunotherapy (AIT) with cytotoxic T lymphocytes (CTLs), induced from autologous pancreatic tumors but not from AIT with LAK cells. Although these immunotherapies have a potential as alternative treatments for PC, the effects have been limited.

Pancreatic cancer cells present an enormous challenge, as they are naturally resistant to current chemotherapy and radiation therapy. In addition, known pancreatic cancer antigens have generated relatively weak immune responses. This is probably due to a combination of mutations in oncogenes such as *KRAS* and tumour-suppressor genes such as *TP53*, *CDKN2A*, *DPC4* (deleted in pancreas cancer 4), *BRCA2* and *ERBB2* (also known as HER2/neu), as well as overexpression of growth factors such as transforming growth factor- (TGF), interleukin-1 (IL-1), IL-6 and IL-8, tumour-necrosis factor-a (TNF), or vascular endothelial growth factor (VEGF), their receptors, or constitutive expression of multidrugresistant genes2, 3, 4, 5. Alternative therapeutic approaches are therefore urgently needed for this disease.

Immune-based therapies aim to recruit and activate T cells that recognize tumour-specific antigens. In addition, recombinant monoclonal antibodies are being designed to target tumour-specific antigens — these would kill tumour cells either by direct lysis or through delivery of a conjugated cytotoxic agent. Both approaches are attractive for the treatment of pancreatic cancer for several reasons. First, these immune-based therapies act through a mechanism that is distinct from chemotherapy or radiation therapy, and represent a noncross-resistant treatment with an entirely different spectrum of toxicities. Second, through the genetic recombination of their respective receptors, the B cells and T cells of the immune system are capable of recognizing a diverse array of potential tumour antigens. In addition, both T and B cells can distinguish small antigenic differences between normal and transformed cells, providing specificity while minimizing toxicity. New insights into the mechanisms by which T cells are successfully activated and by which tumours evade immune recognition are driving the development of new combinatorial immunotherapy approaches. In addition, recent advances in gene-expression analysis have allowed for the identification of new pancreatic targets, including candidate tumour antigens that might serve as T-cell and antibody targets. These advances now make it possible to exploit the immune system in the fight against pancreatic cancer.

#### **4.5 Targeting signalling molecules**

144 Pancreatic Cancer – Clinical Management

clinical strategy to improve the prognosis of pancreatic cancer patients undergoing

Allogeneic antigen-specific immunotherapies, nonmyeloablative SCT (minitransplant) and DLI (donor leukocyte infusion), are reported to have some antitumor effect [graft versus tumor (GVT)] on solid tumors, including RCC, breast cancer, and pancreatic cancer, in addition to haematological malignancies.110GVT effects were also observed in pancreatic cancer patients in minitransplant protocols conducted in Japan. Although the mechanisms of the antitumor effects, such as allogeneic responses to minor histocompatibility antigens (mHa), on hematological malignancies are well studied, they remain unclear with regard to solid tumors. One of the major problems in allogeneic treatment of the solid tumor is severe GVHD. Several strategies for the separation of GVT and GVHD have been developed for hematological malignancies. Whether this separation is possible for solid tumors, however,

Was reported on the efficacy of adoptive immunotherapy (AIT) with cytotoxic T lymphocytes (CTLs), induced from autologous pancreatic tumors but not from AIT with LAK cells. Although these immunotherapies have a potential as alternative treatments for

Pancreatic cancer cells present an enormous challenge, as they are naturally resistant to current chemotherapy and radiation therapy. In addition, known pancreatic cancer antigens have generated relatively weak immune responses. This is probably due to a combination of mutations in oncogenes such as *KRAS* and tumour-suppressor genes such as *TP53*, *CDKN2A*, *DPC4* (deleted in pancreas cancer 4), *BRCA2* and *ERBB2* (also known as HER2/neu), as well as overexpression of growth factors such as transforming growth factor- (TGF), interleukin-1 (IL-1), IL-6 and IL-8, tumour-necrosis factor-a (TNF), or vascular endothelial growth factor (VEGF), their receptors, or constitutive expression of multidrugresistant genes2, 3, 4, 5. Alternative therapeutic approaches are therefore urgently needed for

Immune-based therapies aim to recruit and activate T cells that recognize tumour-specific antigens. In addition, recombinant monoclonal antibodies are being designed to target tumour-specific antigens — these would kill tumour cells either by direct lysis or through delivery of a conjugated cytotoxic agent. Both approaches are attractive for the treatment of pancreatic cancer for several reasons. First, these immune-based therapies act through a mechanism that is distinct from chemotherapy or radiation therapy, and represent a noncross-resistant treatment with an entirely different spectrum of toxicities. Second, through the genetic recombination of their respective receptors, the B cells and T cells of the immune system are capable of recognizing a diverse array of potential tumour antigens. In addition, both T and B cells can distinguish small antigenic differences between normal and transformed cells, providing specificity while minimizing toxicity. New insights into the mechanisms by which T cells are successfully activated and by which tumours evade immune recognition are driving the development of new combinatorial immunotherapy approaches. In addition, recent advances in gene-expression analysis have allowed for the identification of new pancreatic targets, including candidate tumour antigens that might

macroscopical radical surgery.

PC, the effects have been limited.

is unclear.

this disease.

**4.4 Allogeneic antigen-specific immunotherapy** 

By the time that patients are diagnosed with pancreatic cancer, the tumour has typically progressed and invaded adjacent structures. Perineural invasion, metastasis to lymph nodes and liver, and an intense DESMOPLASTIC STROMAL REACTION are commonly observed. A range of signalling pathways, including epidermal growth factor receptor (EGFR) and the PI3K–AKT–mTOR–S6K cascades, are known to mediate pancreatic tumour growth and progression111n addition, new blood-vessel formation (angiogenesis) is required for the growth of primary pancreatic tumours and is essential for metastasis. In pancreatic tumours, this process is probably regulated by fibroblast growth factor, platelet-derived endothelialcell growth factor and VEGF family members. In fact, several pancreatic-cancer-associated genes have been linked to angiogenesis. DPC4 upregulates VEGF expression, and mutated KRAS expression is associated with increased micro-vessel density112.

Monoclonal antibodies that target a range of these pathways have demonstrated efficacy in preclinical models113-115dition, monoclonal antibodies that target EGFR and VEGF receptor have been tested in patients with a range of cancers, including pancreatic cance115,117hough these antibodies have demonstrated only modest results as single agents, the pathways they affect are also candidate targets for immune intervention.

Preclinical evidence has also shown that specific inhibitors of these signalling pathways can also increase immune activation. For example, VEGF is a key inhibitor of pro-inflammatory cytokines as well as dendritic-cell maturation, and it can also directly inhibit T-cell development. So antibodies that block signalling by this growth factor can promote antitumour immune responses. Furthermore, downregulation of the ERBB-receptor-family members with drugs such as herceptin promotes tumour-antigen presentation by HLA class I molecules, improving the potential for T-cell recognition and lysis118onoclonal antibodies that target these signalling pathways are now being developed for clinical trials as agents that potentially synergize with other immune-based approaches, including vaccines.

#### **4.6 Vaccines against pancreatic tumour antigens**

To develop the ideal vaccine for pancreatic cancer, the following wish list would probably need to be fulfilled. First, specific cell-surface proteins must be identified that are that are crucial in the cancer growth or progression pathway and are unique to pancreatic cancer tumours. Second, these tumour-exclusive proteins should be shown to elicit a vigorous tumour-protein-specific immune response. Third, the best carrier to deliver the appropriate immunogenic tumour proteins should be identified. Fourth, molecules that are immune stimulatory as well as molecules that can abrogate the natural immune-inhibition signalling that is seen in pancreatic cancer should be identified to enhance the immune response. Fifth, additional synergistic immune help should be identified (for example, antibodies or *ex vivo* tumour-reactive T cells). Several proteins, such as carcinoembryonic antigen (CEA), mutated KRAS, mucin-1 (MUC1) and gastrin, have in fact been identified to be specifically overexpressed in most pancreatic cancers119-125 antigens were identified over 10 years ago using various methods to analyse gene expression in cancer cells. Vaccines and antibodies

An Overview on Immunotherapy of Pancreatic Cancer 147

As IL-2 is involved in T-cell-mediated immune response, a vaccine consisting of mutant ras peptide in combination with GM-CSF and IL-2 was tested in a phase II trial of 17 patients with advanced cancers (14 colorectal, one non-small cell lung and two pancreatic cancers) 138. Of the six patients with positive immune response (by means of IFN-γ mRNA copies), the MS and the median PFS were 39.9 and 17.9 months compared to 18.5 and 15.6 months for nonresponders, respectively. Grade III toxicities led to IL-2 dose reduction in three of the

 **CEA and MUC1:** Carcinoembryonic antigen (CEA) glycoprotein is expressed at a low level in normal colonic epithelium but is overexpressed in many malignant diseases, including those of the colon, rectum, stomach and pancreas (85-90%) 139. Its serum level is sometimes used as a marker for the diagnosis of pancreatic cancer, with a

To boost MUC1-specific immune response, a vaccine composed of MUC1 peptide and SBAS2 adjuvant was tested in a phase I study 142. There was an increase in the percentage of CD8+ T-cells and MUC1-specific antibody (some developed IgG). Hope for the CEA or MUC1 vaccine was nevertheless crushed when a phase III trial of 255 patients using PANVAC-VF (vaccine consisted of recombinant vaccinia and fowlpox viruses coexpressing CEA, MUC-1 and TRICOM) failed to improve overall survival compared to palliative

 **Gastrin:** G17DT (Gastrimmune or Insegia) is an immunoconjugate of the aminoterminal sequence of gastrin-17 (G-17) linked by means of a spacer peptide to diphtheria toxoid. Given intramuscularly it induces the formation of antibodies that can neutralise both amidated-G-17 and the precursor glycine-extended G17 143. In a phase II study of 30 patients, 67% mounted an antibody response. A significantly higher response (82%) was achieved in those given the highest dose of 250μg compared to 46% in the 100μg group. MS was significantly higher (217 days) for the antibody responders

When used as a monotherapy for patients with advanced pancreatic cancer unwilling or unsuitable to take chemotherapy, MS was 151 compared to 82 days in the placebo group (p=0.03) [360]. G17DT was subsequently tested in a phase III trial with or without gemcitabine in 383 untreated patients with locally advanced, recurrent or metastatic pancreatic adenocarcinoma. This unfortunately showed that the addition of G17DT did not improve overall survival or secondary endpoints Increasing -17 antibody titre levels in a

 **Mesothelin:** Thomas and colleagues provided the first direct evidence, by using mesothelin epitopes, that pancreatic cancer-specific CD8+ T-cell response can be generated via crosspresentation by an approach that recruits APCs to the vaccination

compared to non-responders (148 and 61 days respectively; p=0.0002).

sensitivity of 25-40% and a specificity of 70-90% 140-141.

compared to non-responders (121 days; p=0.0023).

subset of patients, however, were associated with increased survival.

chemotherapy or best supportive care.

patients.

phase I/II study of five patients with advanced pancreatic cancer, two of them showed induced immune response. They also studied ras peptide in combination with GM-CSF in a phase I/II trial involving 48 patients with pancreatic adenocarcinoma of variable stage 137. Peptide-specific immunity was induced in 58% of patients. Of patients with advanced disease, those who responded to treatment showed increased survival

designed to target these antigens have been tested in early-phase clinical trials126-131hese antigens are known to have weak inherent immune potential, various immune-modulating agents were co-administered, including granulocyte–macrophage colony-stimulating factor (GM-CSF), and interleukin-2 (IL-2). So far, a few studies have demonstrated postvaccination immune responses to the relevant peptides or whole proteins. Significant clinical responses have not yet been observed. This might be due to the lack of pooling of the right antigens, to the existence of host mechanisms of immune tolerance, the inability of the relevant immune cells to effectively localize to the sites of disease, or a combination of these factors.

Vaccination involves administering an antigen that is unique for a particular type of tumour with the aim of stimulating tumour-specific immunity. Antigens could be delivered in the form of DNA or peptide, as well as tumour cells or antigen-pulsed DCs. Additional synergistic help is added to elicit a more vigorous and effective immune response, such as cytokines and immunostimulating adjuvants.

**Whole-Cell—**GM-CSF is one of a few cytokines that has shown significant antitumour effect *in vivo* [342]. It is an important growth factor for granulocytes and monocytes, and has a crucial role in the growth and differentiation of DCs, the most potent antigen-presenting cells (APCs) for triggering immune response. *In vivo* growth of AsPC-1 cells, retrovirally transduced with the GM-CSF gene, was inhibited and associated with increased survival of the nude mice, even in the mature T-cell-deficient condition 132. Jaffee et al. conducted a phase I study using allogeneic GM-CSF-secreting whole-cell tumour vaccine for pancreatic cancer 133. This is based on the concept that the localisation of GMCSF in the implanted tumour environment together with the shared tumour antigen expressed by the primary cancer would effectively induce an antitumour immune response. In this study two pancreatic cancer cell lines (PANC 10.05 and PANC 6.03) were used as the vaccine, both genetically modified to express GM-CSF. 14 pancreatic cancer patients who had undergone pancreaticoduodenectomy eight weeks prior were given variable doses of the vaccine intradermally. Three of the eight patients who received ≥10 × 107 vaccine cells developed postvaccination delayed-type hypersensitivity (DTH) responses associated with increased disease free survival time, and remained disease-free for longer than 25 months after diagnosis. Side effects were mainly limited to local skin reactions at the site of vaccination. In a recently completed phase II study of 60 patients with resected pancreatic adenocarcinoma, patients received five treatments of 2.5 × 108 vaccine cells, together with 5- FU and radiotherapy134. The reported MS was 26 months, with a one- and two-year survival of 88% and 76% respectively.

#### **4.7 Peptide and DNA**

 **Ras:** As described earlier, mutated ras is highly prevalent in pancreatic cancer. A phase II study was done using mutant ras peptide-based subcutaneous vaccine in 12 cancer patients (five with fully resected pancreatic and seven with colorectal cancers). Five out of 11 patients showed showed ≥1.5 fold increase in interferon-γ (IFN-γ) mRNA copies in peripheral blood mononuclear cells. The pancreatic cancer patients showed a diseasefree survival of >35.2 months and post-vaccination survival of >44.4 months 135. Gjertsen et al tested an intradermal vaccine of APCs loaded *ex vivo* with synthetic ras peptide corresponding to the ras mutation found in the patient's tumour 136. In this

designed to target these antigens have been tested in early-phase clinical trials126-131hese antigens are known to have weak inherent immune potential, various immune-modulating agents were co-administered, including granulocyte–macrophage colony-stimulating factor (GM-CSF), and interleukin-2 (IL-2). So far, a few studies have demonstrated postvaccination immune responses to the relevant peptides or whole proteins. Significant clinical responses have not yet been observed. This might be due to the lack of pooling of the right antigens, to the existence of host mechanisms of immune tolerance, the inability of the relevant immune cells to effectively localize to the sites of disease, or a combination of

Vaccination involves administering an antigen that is unique for a particular type of tumour with the aim of stimulating tumour-specific immunity. Antigens could be delivered in the form of DNA or peptide, as well as tumour cells or antigen-pulsed DCs. Additional synergistic help is added to elicit a more vigorous and effective immune response, such as

**Whole-Cell—**GM-CSF is one of a few cytokines that has shown significant antitumour effect *in vivo* [342]. It is an important growth factor for granulocytes and monocytes, and has a crucial role in the growth and differentiation of DCs, the most potent antigen-presenting cells (APCs) for triggering immune response. *In vivo* growth of AsPC-1 cells, retrovirally transduced with the GM-CSF gene, was inhibited and associated with increased survival of the nude mice, even in the mature T-cell-deficient condition 132. Jaffee et al. conducted a phase I study using allogeneic GM-CSF-secreting whole-cell tumour vaccine for pancreatic cancer 133. This is based on the concept that the localisation of GMCSF in the implanted tumour environment together with the shared tumour antigen expressed by the primary cancer would effectively induce an antitumour immune response. In this study two pancreatic cancer cell lines (PANC 10.05 and PANC 6.03) were used as the vaccine, both genetically modified to express GM-CSF. 14 pancreatic cancer patients who had undergone pancreaticoduodenectomy eight weeks prior were given variable doses of the vaccine intradermally. Three of the eight patients who received ≥10 × 107 vaccine cells developed postvaccination delayed-type hypersensitivity (DTH) responses associated with increased disease free survival time, and remained disease-free for longer than 25 months after diagnosis. Side effects were mainly limited to local skin reactions at the site of vaccination. In a recently completed phase II study of 60 patients with resected pancreatic adenocarcinoma, patients received five treatments of 2.5 × 108 vaccine cells, together with 5- FU and radiotherapy134. The reported MS was 26 months, with a one- and two-year

 **Ras:** As described earlier, mutated ras is highly prevalent in pancreatic cancer. A phase II study was done using mutant ras peptide-based subcutaneous vaccine in 12 cancer patients (five with fully resected pancreatic and seven with colorectal cancers). Five out of 11 patients showed showed ≥1.5 fold increase in interferon-γ (IFN-γ) mRNA copies in peripheral blood mononuclear cells. The pancreatic cancer patients showed a diseasefree survival of >35.2 months and post-vaccination survival of >44.4 months 135. Gjertsen et al tested an intradermal vaccine of APCs loaded *ex vivo* with synthetic ras peptide corresponding to the ras mutation found in the patient's tumour 136. In this

these factors.

cytokines and immunostimulating adjuvants.

survival of 88% and 76% respectively.

**4.7 Peptide and DNA** 

phase I/II study of five patients with advanced pancreatic cancer, two of them showed induced immune response. They also studied ras peptide in combination with GM-CSF in a phase I/II trial involving 48 patients with pancreatic adenocarcinoma of variable stage 137. Peptide-specific immunity was induced in 58% of patients. Of patients with advanced disease, those who responded to treatment showed increased survival compared to non-responders (148 and 61 days respectively; p=0.0002).

As IL-2 is involved in T-cell-mediated immune response, a vaccine consisting of mutant ras peptide in combination with GM-CSF and IL-2 was tested in a phase II trial of 17 patients with advanced cancers (14 colorectal, one non-small cell lung and two pancreatic cancers) 138. Of the six patients with positive immune response (by means of IFN-γ mRNA copies), the MS and the median PFS were 39.9 and 17.9 months compared to 18.5 and 15.6 months for nonresponders, respectively. Grade III toxicities led to IL-2 dose reduction in three of the patients.

 **CEA and MUC1:** Carcinoembryonic antigen (CEA) glycoprotein is expressed at a low level in normal colonic epithelium but is overexpressed in many malignant diseases, including those of the colon, rectum, stomach and pancreas (85-90%) 139. Its serum level is sometimes used as a marker for the diagnosis of pancreatic cancer, with a sensitivity of 25-40% and a specificity of 70-90% 140-141.

To boost MUC1-specific immune response, a vaccine composed of MUC1 peptide and SBAS2 adjuvant was tested in a phase I study 142. There was an increase in the percentage of CD8+ T-cells and MUC1-specific antibody (some developed IgG). Hope for the CEA or MUC1 vaccine was nevertheless crushed when a phase III trial of 255 patients using PANVAC-VF (vaccine consisted of recombinant vaccinia and fowlpox viruses coexpressing CEA, MUC-1 and TRICOM) failed to improve overall survival compared to palliative chemotherapy or best supportive care.

 **Gastrin:** G17DT (Gastrimmune or Insegia) is an immunoconjugate of the aminoterminal sequence of gastrin-17 (G-17) linked by means of a spacer peptide to diphtheria toxoid. Given intramuscularly it induces the formation of antibodies that can neutralise both amidated-G-17 and the precursor glycine-extended G17 143. In a phase II study of 30 patients, 67% mounted an antibody response. A significantly higher response (82%) was achieved in those given the highest dose of 250μg compared to 46% in the 100μg group. MS was significantly higher (217 days) for the antibody responders compared to non-responders (121 days; p=0.0023).

When used as a monotherapy for patients with advanced pancreatic cancer unwilling or unsuitable to take chemotherapy, MS was 151 compared to 82 days in the placebo group (p=0.03) [360]. G17DT was subsequently tested in a phase III trial with or without gemcitabine in 383 untreated patients with locally advanced, recurrent or metastatic pancreatic adenocarcinoma. This unfortunately showed that the addition of G17DT did not improve overall survival or secondary endpoints Increasing -17 antibody titre levels in a subset of patients, however, were associated with increased survival.

 **Mesothelin:** Thomas and colleagues provided the first direct evidence, by using mesothelin epitopes, that pancreatic cancer-specific CD8+ T-cell response can be generated via crosspresentation by an approach that recruits APCs to the vaccination

An Overview on Immunotherapy of Pancreatic Cancer 149

being targeted. Inhibitors to EGFR and to VEGF have been tested in combination with gemcitabine and are currently in Phase III trials either with other approaches have used dendritic cells as the carrier of the antigen of interest. To date, CEA and MUC1 antigens have been among the initial antigens tested, with mixed results153-154 se of adoptively transferred pancreatic-cancer-specific T cells has been proposed to be another opportunity to augment the immune response. Although this strategy has been promising preclinically, and has been used with some success in melanoma, there have not been any clinical trials in

A current limitation to the development of vaccines for pancreatic cancer has been the inability to correlate *in vitro* measures of antitumour immunity with *in vivo* responses. Post-vaccination DTH responses to autologous tumour are a potential useful surrogate, but this approach is not ideal. At present, it is technically challenging to produce sufficient quantity and purity of autologous tumour material for testing, as tumours vary in their composition of tumour cells versus other cell types between patients. Although other biological end points, such as an antibody response or *in vitro* CYTOLYTIC T LYMPHOCYTE (CTL) ASSAY against a vaccinedelivered tumour antigen (or antigens), have been measured and provide important 'proof of concept' data, these end points have also not been demonstrated to be predictors of traditional

It is difficult to assess whether the lack of improved survival after immunotherapy is due to inefficient antigen delivery, which could result in ineffective immunization, inappropriate selection of antigen targets, or both. As discussed above, there are formidable barriers to inducing an antitumour immune response, even when the vaccine itself is potent enough to reduce significant cancer burdens in more immunogenic tumour systems. Effective immunization will therefore require the targeting of relevant pancreatic tumour antigens using optimized antigen-delivery systems with immune-stimulating cytokines, in sequence with other therapeutic interventions that alter immune checkpoints in the tumour microenvironment, such as inhibitors to regulatory molecules on T cells (for example,

The inability of previously tested antigens (including CEA, KRAS, MUC1 and gastrin) to induce immune-specific responses underscores the challenge to identify more relevant immunogenic targets. Indeed, these antigens were chosen only because they were overexpressed or had altered expression in pancreatic tumours, and not because they had been shown to be immunogenic. Therefore, there might be additional as-yet-unidentified antigens that might be more immunogenic for inducing effective immunity against pancreatic cancers. How will such new candidate pancreatic cancer antigens be discovered? Two methods are routinely used in an attempt to identify new targets. The first method, serological analysis of recombinant tumour cDNA expression libraries (SEREX), uses serum to screen phage-display libraries prepared from tumour cells to identify candidate antigen targets that have elicited both humoral and cell-mediated immune responses in cancer patients. This method has identified coactosin-like protein (an actin-filament-binding protein that interacts directly with 5-liopoxygenase and has an important role in cellular leukotriene synthesis) as a potential pancreatic cancer target antigen. This protein seems to

be recognized by antibody and T-cell responses in patients with pancreatic cancer155.

clinical end points, including tumour response and survival benefit.

pancreatic cancer so far.

antibody to CD152/CTLA4).

**5. New immunotherapy targets** 

site 144. Gaffney et al studied the mesothelin DNA vaccine in combination with the anti-glucocorticoid-induced TNF receptor antibody (anti-GITR) in mice with syngeneic mesothelin-expressing pancreatic cancer 145. 50% of animals treated with mesothelin were tumour-free 25 days after tumour injection compared to 0% of non-treated mice. This increased to 94% with the addition of anti-GITR. The agonist anti-GITR served to enhance T-cell-mediated response of the vaccine 146-147.


**Antigen-pulsed DCs—**Antigen-specific T-cell responses are initiated by DCs. They capture antigens secreted or shed by tumour cells and present peptides in association with the MHC class I and II molecules. This results in the expression and upregulation of cytokines and costimulatory molecules which in turn stimulate CD4+ and CD8+ T-cells to mount an antitumour response. As such DCs that carry the tumour antigen of interest is an ideal adjuvant in cancer immunotherapy.

 **MUC1:** In a phase I/II trial, human autologous DCs transfected with MUC1 cDNA were used as a vaccine for ten patients with advanced breast, pancreatic or papillary cancer 151. Four patients showed a two- to ten-fold increase in the frequency of mucinspecific IFN-γ-secreting CD8+ T-cells, suggesting an immune response. In a phase 1b study, eight patients with pancreatic or biliary tumours were vaccinated with DCs pulsed with MUC1 152.

As discussed previously, monoclonal antibodies have so far been the most successful form of immunotherapy clinically. They are being used as diagnostic tools, prognostic indicators, and for the treatment of many cancers. Advantages include their specific targeting of tumour cells while sparing normal tissue, their relative ease of administration, and their low toxicity profile. The major disadvantages include the absence of T-cell activation, which therefore precludes T-cell-mediated cytotoxic killing and the generation of memory immune responses. In addition, a potential limiting factor in its use involves tumour heterogeneity. Specifically, all tumour cells within a proliferating mass might not express the antigen that is

 **Telomerase:** The telomerase peptide vaccine GV1001 was tested in a phase I/II study of 48 patients with unresectable pancreatic cancer 148. They received intradermal injection in combination with GM-CSF. Immune responses, as measured by DTH skin reaction and T-cell proliferation *in vitro*, were demonstrated in 24 of 38 evaluable patients, with the highest percentage (75%) in the intermediate dose group. MS for this group was significantly longer at 8.6 months, and one-year survival was 25%. GV1001 was given to patients in a phase I trial using imiquimod as an adjuvant149. Imiquimod acts by binding to Toll-like receptor 7 on immune cells, resulting in the production of cytokines such as IFN-α, IFN-β and IL-12. Immune response was found in up to six (46%) of 13

 **Survivin:** Survivin-specific CTLs were isolated from pancreatic cancer patients and these could lyse pancreatic carcinoma cell lines *in vitro* 150. Vaccination with survivin DNA prolonged survival in murine pancreatic and lymphoma tumour models, associated with slower tumour growth and increased lymphocyte infiltration . Survivin peptide was tested in a patient with gemcitabine refractory pancreatic cancer . Whilst on treatment he had complete remission of liver metastases after six months. However when he was weaned from the vaccination he developed recurrent disease. Vaccineinduced immune activity was detected by IFN-γ enzyme-linked immunospot

**Antigen-pulsed DCs—**Antigen-specific T-cell responses are initiated by DCs. They capture antigens secreted or shed by tumour cells and present peptides in association with the MHC class I and II molecules. This results in the expression and upregulation of cytokines and costimulatory molecules which in turn stimulate CD4+ and CD8+ T-cells to mount an antitumour response. As such DCs that carry the tumour antigen of interest is an ideal

 **MUC1:** In a phase I/II trial, human autologous DCs transfected with MUC1 cDNA were used as a vaccine for ten patients with advanced breast, pancreatic or papillary cancer 151. Four patients showed a two- to ten-fold increase in the frequency of mucinspecific IFN-γ-secreting CD8+ T-cells, suggesting an immune response. In a phase 1b study, eight patients with pancreatic or biliary tumours were vaccinated with DCs

As discussed previously, monoclonal antibodies have so far been the most successful form of immunotherapy clinically. They are being used as diagnostic tools, prognostic indicators, and for the treatment of many cancers. Advantages include their specific targeting of tumour cells while sparing normal tissue, their relative ease of administration, and their low toxicity profile. The major disadvantages include the absence of T-cell activation, which therefore precludes T-cell-mediated cytotoxic killing and the generation of memory immune responses. In addition, a potential limiting factor in its use involves tumour heterogeneity. Specifically, all tumour cells within a proliferating mass might not express the antigen that is

enhance T-cell-mediated response of the vaccine 146-147.

evaluable patients.

(ELISPOT) assay.

adjuvant in cancer immunotherapy.

pulsed with MUC1 152.

site 144. Gaffney et al studied the mesothelin DNA vaccine in combination with the anti-glucocorticoid-induced TNF receptor antibody (anti-GITR) in mice with syngeneic mesothelin-expressing pancreatic cancer 145. 50% of animals treated with mesothelin were tumour-free 25 days after tumour injection compared to 0% of non-treated mice. This increased to 94% with the addition of anti-GITR. The agonist anti-GITR served to being targeted. Inhibitors to EGFR and to VEGF have been tested in combination with gemcitabine and are currently in Phase III trials either with other approaches have used dendritic cells as the carrier of the antigen of interest. To date, CEA and MUC1 antigens have been among the initial antigens tested, with mixed results153-154 se of adoptively transferred pancreatic-cancer-specific T cells has been proposed to be another opportunity to augment the immune response. Although this strategy has been promising preclinically, and has been used with some success in melanoma, there have not been any clinical trials in pancreatic cancer so far.

A current limitation to the development of vaccines for pancreatic cancer has been the inability to correlate *in vitro* measures of antitumour immunity with *in vivo* responses. Post-vaccination DTH responses to autologous tumour are a potential useful surrogate, but this approach is not ideal. At present, it is technically challenging to produce sufficient quantity and purity of autologous tumour material for testing, as tumours vary in their composition of tumour cells versus other cell types between patients. Although other biological end points, such as an antibody response or *in vitro* CYTOLYTIC T LYMPHOCYTE (CTL) ASSAY against a vaccinedelivered tumour antigen (or antigens), have been measured and provide important 'proof of concept' data, these end points have also not been demonstrated to be predictors of traditional clinical end points, including tumour response and survival benefit.

It is difficult to assess whether the lack of improved survival after immunotherapy is due to inefficient antigen delivery, which could result in ineffective immunization, inappropriate selection of antigen targets, or both. As discussed above, there are formidable barriers to inducing an antitumour immune response, even when the vaccine itself is potent enough to reduce significant cancer burdens in more immunogenic tumour systems. Effective immunization will therefore require the targeting of relevant pancreatic tumour antigens using optimized antigen-delivery systems with immune-stimulating cytokines, in sequence with other therapeutic interventions that alter immune checkpoints in the tumour microenvironment, such as inhibitors to regulatory molecules on T cells (for example, antibody to CD152/CTLA4).

#### **5. New immunotherapy targets**

The inability of previously tested antigens (including CEA, KRAS, MUC1 and gastrin) to induce immune-specific responses underscores the challenge to identify more relevant immunogenic targets. Indeed, these antigens were chosen only because they were overexpressed or had altered expression in pancreatic tumours, and not because they had been shown to be immunogenic. Therefore, there might be additional as-yet-unidentified antigens that might be more immunogenic for inducing effective immunity against pancreatic cancers. How will such new candidate pancreatic cancer antigens be discovered? Two methods are routinely used in an attempt to identify new targets. The first method, serological analysis of recombinant tumour cDNA expression libraries (SEREX), uses serum to screen phage-display libraries prepared from tumour cells to identify candidate antigen targets that have elicited both humoral and cell-mediated immune responses in cancer patients. This method has identified coactosin-like protein (an actin-filament-binding protein that interacts directly with 5-liopoxygenase and has an important role in cellular leukotriene synthesis) as a potential pancreatic cancer target antigen. This protein seems to be recognized by antibody and T-cell responses in patients with pancreatic cancer155.

An Overview on Immunotherapy of Pancreatic Cancer 151

effective therapy will require a combined approach incorporating the best targeted interventions taken from each respective modality. Preclinical models have already revealed the synergy between immunotherapy and other targeted therapeutics, such as inhibitors of VEGF and EGF signalling. These combinations are about to be tested in patients with

Pancreatic cancer remains one of the most resistant cancers to traditional forms of therapy. Until techniques for early detection can be developed, most patients will continue to present with incurable disease. The pancreatic cancer research community is committed to developing new therapies for this disease. Pancreatic cancer patients and their families, through a number of national pancreatic cancer non-profit organizations such as Pancreas Cancer Action Network have organized to support this effort. It is crucial that we move forward with scientifically driven innovative therapies, as the empirical approaches have failed. Recent developments in the design of mouse models that recapitulate early preinvasive genetic changes in *KRAS* activation, inactivation of *CDKN2A*, *TP53* and *SMAD4* tumour-suppressor genes should provide the opportunity to test such approaches in a

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**7. References** 

The second method uses tumour-specific T cells that have been isolated from patients with pancreatic cancer to screen cDNA libraries prepared from autologous tumour cells. This method requires the isolation and culture of tumour-specific T cells, along with tumour cells, from patients with pancreatic cancer and is a technically challenging approach. This approach has been most successful in identifying melanoma-associated antigens156.

A relatively newer, more promising method of tumour-antigen identification is the use of the patient's lymphocytes to evaluate proteins that are found to be differentially expressed by pancreatic cancer157-158 approach has several advantages. First, it allows for a rapid screen of a large number of candidate antigens but requires the isolation from patients of only a few lymphocytes, which are limited in availability. Second, this approach is not dependent on the availability of autologous tumour cells, which are difficult to isolate in large enough numbers for generating cDNA libraries. Third, this approach can be used to identify tumour antigens that are expressed by any HLA type, allowing for the generalization of this approach to most patients. Finally, this approach has the potential to rapidly identify 'immune relevant' antigens, as it uses immunized lymphocytes from patients vaccinated with a whole-tumour-cell vaccine approach who ideally have demonstrated clinical evidence of immune activation following vaccination. So this method provides the best insurance that the antigens identified are ones that the patient's immune system is reacting to after immunization.

As additional 'immune relevant' pancreatic tumour antigens are identified, the next significant challenge lies in developing strategies to improve the *in vivo* delivery of these antigens to APCs and thereby allow effective antigen processing and presentation, and subsequent activation of a potent antitumour immune response. DCs are now accepted as the most efficient APCs in B- and T-cell activation. Several clinical trials have tested *ex vivo* expanded and primed DCs as a vaccine approach. However, these studies have revealed the difficulty in reliably producing phenotypically mature DCs for clinical testing, as only mature DCs are capable of efficiently presenting antigens to T cells. If an antigen is not presented in the proper context by mature DCs, immune downregulation or tolerance can occur. It has been shown in animal models that immature DCs induce T-cell tolerance. As an alternative to DC-based delivery, recombinant viral- and bacterial-vector delivery systems are currently under development or are already undergoing clinical testing. The use of modified viral particles or targeted bacteria to deliver tumour antigens to the immune system is based on the innate ability of the agent to efficiently infect APCs *in vivo*. Early approaches have included viruses such as vaccinia 159,160 er, the use of immunogenic vectors in cancer patients who have been previously exposed to a similar vector often induces vigorous immune responses against the vector before effective priming against the tumour antigen can occur. As such, other viral particles and bacterial delivery systems are currently nearing or are already undergoing clinical development for the treatment of pancreatic cancer.

#### **6. Future directions**

The limitations of currently available therapy for pancreatic cancer are more clearly exposed as we begin to appreciate the molecular changes behind the complex transformation of normal pancreatic ductal cells into frank pancreatic cancers, and the mechanisms of pancreatic cancer resistance to traditional anticancer modalities. It is clear that the most effective therapy will require a combined approach incorporating the best targeted interventions taken from each respective modality. Preclinical models have already revealed the synergy between immunotherapy and other targeted therapeutics, such as inhibitors of VEGF and EGF signalling. These combinations are about to be tested in patients with pancreatic cancer.

Pancreatic cancer remains one of the most resistant cancers to traditional forms of therapy. Until techniques for early detection can be developed, most patients will continue to present with incurable disease. The pancreatic cancer research community is committed to developing new therapies for this disease. Pancreatic cancer patients and their families, through a number of national pancreatic cancer non-profit organizations such as Pancreas Cancer Action Network have organized to support this effort. It is crucial that we move forward with scientifically driven innovative therapies, as the empirical approaches have failed. Recent developments in the design of mouse models that recapitulate early preinvasive genetic changes in *KRAS* activation, inactivation of *CDKN2A*, *TP53* and *SMAD4* tumour-suppressor genes should provide the opportunity to test such approaches in a timely manner161,162.

#### **7. References**

150 Pancreatic Cancer – Clinical Management

The second method uses tumour-specific T cells that have been isolated from patients with pancreatic cancer to screen cDNA libraries prepared from autologous tumour cells. This method requires the isolation and culture of tumour-specific T cells, along with tumour cells, from patients with pancreatic cancer and is a technically challenging approach. This

A relatively newer, more promising method of tumour-antigen identification is the use of the patient's lymphocytes to evaluate proteins that are found to be differentially expressed by pancreatic cancer157-158 approach has several advantages. First, it allows for a rapid screen of a large number of candidate antigens but requires the isolation from patients of only a few lymphocytes, which are limited in availability. Second, this approach is not dependent on the availability of autologous tumour cells, which are difficult to isolate in large enough numbers for generating cDNA libraries. Third, this approach can be used to identify tumour antigens that are expressed by any HLA type, allowing for the generalization of this approach to most patients. Finally, this approach has the potential to rapidly identify 'immune relevant' antigens, as it uses immunized lymphocytes from patients vaccinated with a whole-tumour-cell vaccine approach who ideally have demonstrated clinical evidence of immune activation following vaccination. So this method provides the best insurance that the antigens identified are ones that the patient's immune system is reacting

As additional 'immune relevant' pancreatic tumour antigens are identified, the next significant challenge lies in developing strategies to improve the *in vivo* delivery of these antigens to APCs and thereby allow effective antigen processing and presentation, and subsequent activation of a potent antitumour immune response. DCs are now accepted as the most efficient APCs in B- and T-cell activation. Several clinical trials have tested *ex vivo* expanded and primed DCs as a vaccine approach. However, these studies have revealed the difficulty in reliably producing phenotypically mature DCs for clinical testing, as only mature DCs are capable of efficiently presenting antigens to T cells. If an antigen is not presented in the proper context by mature DCs, immune downregulation or tolerance can occur. It has been shown in animal models that immature DCs induce T-cell tolerance. As an alternative to DC-based delivery, recombinant viral- and bacterial-vector delivery systems are currently under development or are already undergoing clinical testing. The use of modified viral particles or targeted bacteria to deliver tumour antigens to the immune system is based on the innate ability of the agent to efficiently infect APCs *in vivo*. Early approaches have included viruses such as vaccinia 159,160 er, the use of immunogenic vectors in cancer patients who have been previously exposed to a similar vector often induces vigorous immune responses against the vector before effective priming against the tumour antigen can occur. As such, other viral particles and bacterial delivery systems are currently nearing or are already undergoing clinical development for the treatment of pancreatic

The limitations of currently available therapy for pancreatic cancer are more clearly exposed as we begin to appreciate the molecular changes behind the complex transformation of normal pancreatic ductal cells into frank pancreatic cancers, and the mechanisms of pancreatic cancer resistance to traditional anticancer modalities. It is clear that the most

approach has been most successful in identifying melanoma-associated antigens156.

to after immunization.

cancer.

**6. Future directions** 


An Overview on Immunotherapy of Pancreatic Cancer 153

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**9** 

,

*Germany* 

**Bacterial Immunotherapy** 

, Peggy Bodammer³

**-Antitumoral Potential of the** 

Joerg Emmrich³ and Michael Linnebacher1

*Section of Molecular Oncology and Immunotherapy,* 

*Medicine University of Rostock, Rostock,* 

*²Institute of Medical Microbiology, Virology and Hygiene, ³ Division of Gastroenterology Department of Internal* 

**Streptococcal Toxin Streptolysin S-**

*1Department of General, Vascular, Thoracic and Transplantation Surgery,* 

Claudia Maletzki1, Bernd Kreikemeyer²

Chronic infections can lead to cancer. However, acute infection has beneficial effects often contributing to complete eradication of tumors. In the wake of this, bacteria and their related products were applied therapeutically for experimental immunotherapy. They exhibit direct antitumoral potential and are recognized by the host's immune system via Toll-like receptors (TLRs) finally promoting pro-inflammatory, often Th1-directed immune

Recently, we described that local injection of live as well as lysed gram-positive Group A Streptococci (GAS) eradicates established pancreatic tumors in mice (Linnebacher et al., 2008; Maletzki et al., 2008). This antitumoral effect could be attributed to activation of immune response mechanisms including both the innate and even more important, the adaptive arm of the immune system. In the face of the vigorous immune attack induced by *S. pyogenes*, the identification of factors responsible for tumor disintegration might provide the basis for development of therapeutic approaches. Amongst other virulence factors delivered by *S. pyogenes*, the cytolysins Streptolysin O (SLO) and S (SLS) represent the most obvious therapeutically active candidates (Fraser & Proft, 2008; Hobohm et al., 2008; Nizet et al., 2008). SLO is an oxygen-labile, pore-forming toxin mediating cytolysis by disturbing the balance between influxes and effluxes across the cell membrane. While SLS is nonimmunogenic in the natural course of infection and can clinically be identified by betahaemolysis surrounding GAS colonies grown on blood agar. Besides their capacity to lyse erythrocytes, SLS also exerts cytolytic effects towards tumor cells and is by weight one of the

To address the question of the SLS contribution to the antitumoral effects observed in our previous studies, we performed a series of *in vivo* experiments in our murine syngeneic

most potent cytotoxins known (Ginsberg, 1999; Taketo & Taketo, 1966).

**1. Introduction** 

responses.

