**Coagulation Disorders in Pancreatic Cancer**

A. Albu, D. Gheban, C. Grad and D.L. Dumitrascu *University of Medicine and Pharmacy "Iuliu Hatieganu", Cluj-Napoca, Romania* 

#### **1. Introduction**

254 Pancreatic Cancer – Clinical Management

Jennings PE, Donald JJ, Coral A, et al (1989) Ultrasound –guided core biopsy. Lancet 1:1369-1371 Karlson BM, Forsman CA, Wilander E, et al (1996) Efficiency of percutaneous core biopsy in

Klimet M, Urban O, CEgan M et al, (2010) Endoscopic ultrasound-guided fine-needle

Levy MJ. (2006)Know when to biopsy 'em, know when to walk away. Gastrointest Endosc;

Livraghi T. (1984) A simple no-cost technique for real-time biopsy. J Clin Utrasound; 12 (1):

Mallery JS, Centeno BA, Hahn PF, et al (2002) Pancreatic tissue sampling guided by EUS, CT/US,

Matsubara J, Okusaka T, Morizane C, et al (2008) Ultrasound-guided percutaneous pancreatic

including sensitivity, specificity, and comlications. J Gastroenterol 43: 225-232 Micames C, Jowell PS, White R, et al (2003) Lower frequency of peritoneal carcinomatosis in

Paulsen SD, Nghiem HV, Negussie E, et al (2006) Evaluation of imaging-guided core bipsy

Siddiqui AA, Brown JL, Hong SSK, et al (2011) Relashionship of pancreatic mass size and

Sohn TA, Yeo CJ, Cameron JL, et al. (2000) Resected adenocarcinoma of the pancreas -616 patients: results, outcomes, and prognostic indicators. J Gastrointest Surg; 4: 576. Surveillance, Epidemiology, and End Results (SEER)Program (www.seer.cancer.gov) ,

Tillou A, Schwartz MR, Jordan PH Jr. (1996) Percutaneous needle biopsy of the pancreas: when should it be performed? World J Surg; 20: 283-286, discussion 287 Touchefeu Y, Le Rhun M, Coron E, et al (2009) Endoscopic ultrasound-guided fine-needle

Varadarajulu S, Wallace MB. (2004) Applications of endoscopic ultrasonography in

Volmar K, Vollmer R, Jowell P; et al (2005) Pancreatic FNA in 1000 cases: a comparison of

Yoshinaga S, Suzuki H, Oda I, et al (2011) Role of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) for diagnosis of solid pancreatic masses. Dig Endosc 1:29-33 Zamboni GA, D'Onofrio M, Principe F, et al. (2010) Focal pancreatic lesions: accuracy and

compliactions of US-guided fine-needle aspiration cytology. Abdom Imaging; 35:

FNA: a report of accuracy. Gastrointestinal endoscopy; 71: 1.

Yeo TP, Hruban RH, Leach SD et al (2002) Pancreatic Cancer. Curr Probl Cancer; 26: 176

Moossa AR, Altorki N. (1983) Pancreatic biopsy. Surg Clin North Am; 63: 1205-1214.

of pancreatic masses. AJR Am J Roentgenol 187:769-772

aspiration of pancreatic masses: the utility and impact of management of patients.

and surgery: a comparison of sensitivity and specificity. Gastrointest Endosc 56: 218-224

tumor biopsy in pancreatic cancer: a comparison with metastatic liver tumor biopsy,

patients with pancreatic cancer diagnosed by EUS-guided FNA vs percutaneous

diagnostic yield of endoscopic ultrasound-guided fine needle aspiration. Dig Dis

National Cancer Institute, DCCPS, Surveillance Research Program, Cancer Statistics Branch, released April 2008, based on the November 2007 submission Tamm E, Charnsangavej C. (2001) Pancreatic cancer: current concepts in imaging for

aspiration for the diagnosis of solid pancreatic masses: the impact of patientmanagement strategy. Alimentary Pharmacology and Therapeutics 30: 1070-1077 Turner BG, Cizginer S, Agarwal D, (2010) Diagnosis of pancreatic neoplasia with EUS and

pancreatic tumor diagnosis. Surgery 120: 75-79

Scandinavian Journal of Gatroenter; 45: 1372-1379

63: 630-634.

FNA. Gastrointest Endosc 58: 5

Sci; DOI 10. 1007s 10620-011-1782-z

diagnosis and staging. CancerJ; 7: 298-311.

pancreatic cancer. Cancer Control; 11:15-22.

imaging madalities. Gastrointest Endosc 61:854-861

60 – 62.

362-366

The association between cancers and thrombosis is well known for a long period of time. In 1865 Armand Trousseau noted for the first time that unexpected or migratory thrombophlebitis could be a sign of an undiagnosed visceral malignancy (Trousseau, 1865). Some years later it is said that he observed this complication on himself in the context of an occult gastric cancer that cased his death (Khorana, 2003).

The risk of developing thrombosis in cancer patients is considered to be increased 2- 7 fold compared with persons without cancer (Bloom et al, 2005; Heit et al, 2004). This risk is dependent on many factors. According to the type of tumor, the risk is thought to be the highest in tumors of the ovary, pancreas and central nervous system. Also the extent of the tumor, the presence of metastasis, age, immobility and the type of therapy increase this risk. Surgery for cancers (Rahr & Sørensen, 1992) and chemotherapy (Levine, 1997) are both associated with an important risk of venous thrombosis and embolism. In a large casecontrol study that included 3220 patients with cancer, it was reported an overall 7 times increased risk for venous thrombosis that depend on type of cancer and time since the cancer diagnosis. A very high relative risk was found for gastrointestinal, lung and hematological malignancies. Advanced stage of disease was associated with a further increase in risk (Blom et al, 2005).

Patients with cancer who develop venous thromboembolism have a poor prognosis than those without this vascular complication. The risk of recurrent thromboembolism and death from any cause is greater than three fold in patients with cancer compared to those without malignancy (Levitan et al, 1999).

Epidemiological studies looking for the incidence of cancer in patients with thromboembolic events found out that in 15-20% of patients, thromboses were associated with malignancy (Er & Zacharsky, 2006).

The association of cancer and thrombosis raises two distinct problems. On one hand, the diagnosis of thrombosis in one patient may represent, in some situations, a sign of an occult malignancy. On the other hand, a patient with cancer may develop some time, in the evolution of his malignant disease, a thromboembolic event, which may worsen his

Coagulation Disorders in Pancreatic Cancer 257

coagulation systems and increased angiogenesis. It is considered that activation of hemostasis in pancreatic cancer causes thrombosis but also tumor angiogenesis (Browder et

The key molecule in this process seems to be *tissue factor* (TF), the main physiologic initiator of the extrinsic pathway of coagulation (Gouaulthelimann & Josso, 1979; Nemerson, 1988). TF plays also an important role in angiogenesis (Mechtcheriakova et al, 1999; Zhang

TF, also called platelet tissue factor, factor III, or CD142 is a protein present in subendothelial tissue, platelets, and leukocytes. TF consists of three domains: extracellular that binds factor VIIa, transmembrane and intracellular involved in the signaling function (Nemerson, 1988). In healthy individuals there are little circulating amounts of active TF. In response to specific stimuli such as inflammation, malignant

As an initiator of coagulation, TF binds and activates factor VIIa, resulting in TF-VIIa complex which activates factor X leading to the synthesis of thrombin essential in clot formation (Gilbert & Arena, 1995). The activity of TF is regulated by several factors. The most important is TF pathway inhibitor which is composed of three different domains: the first inhibits FVII, the second inhibits FX and the function of the last one is still unknown (Broze, 1995; Girard et al, 1989; Echrish et al, 2011). TF pathway inhibitor is secreted by

The expression of TF can be controlled by epidermal growth factor receptor (Milsom et al, 2008) and by FX activated. Increased concentrations of FX activated inhibit the synthesis of

In cancers, TF is present on malignant cells and also on endothelial cells (Rickles et al, 2003). Some previous data indicate that TF is expressed in pancreatic malignant cells. It correlated with advanced histological stages and with a poor prognosis (Kakkar et al, 1995; Nitori et al, 2005). In a retrospective study, Khorana and colab. investigated the expression of TF in non invasive and invasive pancreatic cancers. They found an increased expression of TF in 77% of patients with pancreatic intraepithelial neoplasia and in 91% of patients with intraductal papillary mucinous neoplasms, two non invasive precursors of invasive pancreatic cancer. They concluded that TF expression is an early event in pancreatic cancer. In patients with pancreas resection, TF expression correlated with expression of vascular endothelial growth factor (VEGF) and increased neovascularization, suggesting an implication of TF in angiogenesis. They found an incidence of thromboembolism of 26.3% in patients with high TF expression levels compared to 4.5% in those with low expression of TF, suggesting an important role of TF in cancer associated thrombotic complications. (Khorana et al, 2007).

**Angiogenesis** has been documented in pancreatic cancer and it was associated with a rapid

The process of angiogenesis represents the formation of new blood vessels from the preexisting vascular bed. In cancers angiogenesis contributes to tumor growth (Folkman, 1995). Pancreatic cancer seems to be accompanied by an important increase in angiogenesis that is linked to the activation of coagulation. Proteins of coagulation are involved in angiogenesis

tumor growth and a poor prognosis (Lomberk, 2010).

processes, its expression increases (Ruf et al, 2000; Wada et al, 1995).

al, 2000).

et al, 1994).

endothelial cells.

TF (Ettelaie et al, 2007).

prognostic. That is why, for the clinical practice, the diagnosis of these associated diseases is very important.

#### **2. Epidemiology of thrombosis in pancreatic cancer**

Pancreatic cancer (PC) is known to be associated with a higher incidence of venous thromboembolism than other cancers. The first publication that noted the high incidence of thrombosis in PC was a postmortem study done in 1938 (Sproul, 1938). Since that, several other studies have been conducted and the incidence found ranges from 5% to 60% (Sack et al, 1977; Khorana & Fine, 2005).

In a cohort study of 202 patients with a first diagnosis of pancreas carcinoma the authors found that the risk of venous thrombosis is 6-fold increased compared with the general population, at a cumulative risk of 10% (Blom et al, 2006). In this study, tumors of the corpus and cauda of the pancreas had a 2-3-fold increase risk of venous thrombosis than tumors of the caput of the pancreas (Blom et al, 2006). Similar results showing a higher incidence of thrombotic events for tumors located in the corpus and cauda of the pancreas were reported by other authors (Sproul, 1938; Sack et al, 1977; Bick, 1992; Pinzon et al, 1986).

In a retrospective single institute study 6,870 patients with pancreatic cancer were evaluated for venous and arterial thrombosis. The incidence of all thrombotic events was 19%with venous thrombosis accounting for 17%, arterial thrombotic events for 2% and associated venous and arterial events in 0.9% of cases. Pulmonary embolism was found in 25% of patients with venous thrombosis (Epstein et al, 2010)

The risk of venous thrombosis increases in the presence of metastases. Blom and colab. found a 2-fold increase risk of venous thrombosis in patients with distant metastases, after adjusting for age, sex, surgery and chemo- or radiotherapy (Blom et al, 2006).

The risk of developing thrombosis is further increased with chemotherapy (Heit, 2002; Wall, 1989) and also with surgical treatment. Patients with PC treated with chemotherapy had a 4.8-fold increased risk of thrombosis compared to those without chemotherapy. The same study showed no significant increase in thrombotic risk patients treated with radiotherapy (Blom et al, 2006).

Patients with malignancies submitted to surgery have at least twice the risk of postoperative venous thrombosis and more than 3 times the risk of fatal PE compared with non-cancer patients undergoing a similar procedure (Geerts et al, 2004).

In patients with PC submitted to surgery there was a 4.5-folf increase in the risk of venous thrombosis during the postoperative period of 30 days (Blom et al, 2006).

The incidence of fatal pulmonary embolism was also evaluated. In one study 4 out of 541 (0.7%) died from pulmonary embolism (Neoptolemos et al, 2001). Concordant results were reported in another study that found 2 of 202 patients (1%) with fatal pulmonary embolism (Blom et al, 2006).

#### **3. Pathogenesis of thrombosis in pancreatic cancer**

The mechanisms underlying the association of venous thromboembolism with pancreatic cancer are not completely understood. Large and relevant data suggest an implication of

prognostic. That is why, for the clinical practice, the diagnosis of these associated diseases is

Pancreatic cancer (PC) is known to be associated with a higher incidence of venous thromboembolism than other cancers. The first publication that noted the high incidence of thrombosis in PC was a postmortem study done in 1938 (Sproul, 1938). Since that, several other studies have been conducted and the incidence found ranges from 5% to 60% (Sack et

In a cohort study of 202 patients with a first diagnosis of pancreas carcinoma the authors found that the risk of venous thrombosis is 6-fold increased compared with the general population, at a cumulative risk of 10% (Blom et al, 2006). In this study, tumors of the corpus and cauda of the pancreas had a 2-3-fold increase risk of venous thrombosis than tumors of the caput of the pancreas (Blom et al, 2006). Similar results showing a higher incidence of thrombotic events for tumors located in the corpus and cauda of the pancreas were reported

In a retrospective single institute study 6,870 patients with pancreatic cancer were evaluated for venous and arterial thrombosis. The incidence of all thrombotic events was 19%with venous thrombosis accounting for 17%, arterial thrombotic events for 2% and associated venous and arterial events in 0.9% of cases. Pulmonary embolism was found in 25% of

The risk of venous thrombosis increases in the presence of metastases. Blom and colab. found a 2-fold increase risk of venous thrombosis in patients with distant metastases, after

The risk of developing thrombosis is further increased with chemotherapy (Heit, 2002; Wall, 1989) and also with surgical treatment. Patients with PC treated with chemotherapy had a 4.8-fold increased risk of thrombosis compared to those without chemotherapy. The same study showed no significant increase in thrombotic risk patients treated with radiotherapy

Patients with malignancies submitted to surgery have at least twice the risk of postoperative venous thrombosis and more than 3 times the risk of fatal PE compared with non-cancer

In patients with PC submitted to surgery there was a 4.5-folf increase in the risk of venous

The incidence of fatal pulmonary embolism was also evaluated. In one study 4 out of 541 (0.7%) died from pulmonary embolism (Neoptolemos et al, 2001). Concordant results were reported in another study that found 2 of 202 patients (1%) with fatal pulmonary embolism

The mechanisms underlying the association of venous thromboembolism with pancreatic cancer are not completely understood. Large and relevant data suggest an implication of

by other authors (Sproul, 1938; Sack et al, 1977; Bick, 1992; Pinzon et al, 1986).

adjusting for age, sex, surgery and chemo- or radiotherapy (Blom et al, 2006).

**2. Epidemiology of thrombosis in pancreatic cancer**

patients with venous thrombosis (Epstein et al, 2010)

patients undergoing a similar procedure (Geerts et al, 2004).

**3. Pathogenesis of thrombosis in pancreatic cancer** 

thrombosis during the postoperative period of 30 days (Blom et al, 2006).

very important.

(Blom et al, 2006).

(Blom et al, 2006).

al, 1977; Khorana & Fine, 2005).

coagulation systems and increased angiogenesis. It is considered that activation of hemostasis in pancreatic cancer causes thrombosis but also tumor angiogenesis (Browder et al, 2000).

The key molecule in this process seems to be *tissue factor* (TF), the main physiologic initiator of the extrinsic pathway of coagulation (Gouaulthelimann & Josso, 1979; Nemerson, 1988). TF plays also an important role in angiogenesis (Mechtcheriakova et al, 1999; Zhang et al, 1994).

TF, also called platelet tissue factor, factor III, or CD142 is a protein present in subendothelial tissue, platelets, and leukocytes. TF consists of three domains: extracellular that binds factor VIIa, transmembrane and intracellular involved in the signaling function (Nemerson, 1988). In healthy individuals there are little circulating amounts of active TF. In response to specific stimuli such as inflammation, malignant processes, its expression increases (Ruf et al, 2000; Wada et al, 1995).

As an initiator of coagulation, TF binds and activates factor VIIa, resulting in TF-VIIa complex which activates factor X leading to the synthesis of thrombin essential in clot formation (Gilbert & Arena, 1995). The activity of TF is regulated by several factors. The most important is TF pathway inhibitor which is composed of three different domains: the first inhibits FVII, the second inhibits FX and the function of the last one is still unknown (Broze, 1995; Girard et al, 1989; Echrish et al, 2011). TF pathway inhibitor is secreted by endothelial cells.

The expression of TF can be controlled by epidermal growth factor receptor (Milsom et al, 2008) and by FX activated. Increased concentrations of FX activated inhibit the synthesis of TF (Ettelaie et al, 2007).

In cancers, TF is present on malignant cells and also on endothelial cells (Rickles et al, 2003). Some previous data indicate that TF is expressed in pancreatic malignant cells. It correlated with advanced histological stages and with a poor prognosis (Kakkar et al, 1995; Nitori et al, 2005). In a retrospective study, Khorana and colab. investigated the expression of TF in non invasive and invasive pancreatic cancers. They found an increased expression of TF in 77% of patients with pancreatic intraepithelial neoplasia and in 91% of patients with intraductal papillary mucinous neoplasms, two non invasive precursors of invasive pancreatic cancer. They concluded that TF expression is an early event in pancreatic cancer. In patients with pancreas resection, TF expression correlated with expression of vascular endothelial growth factor (VEGF) and increased neovascularization, suggesting an implication of TF in angiogenesis. They found an incidence of thromboembolism of 26.3% in patients with high TF expression levels compared to 4.5% in those with low expression of TF, suggesting an important role of TF in cancer associated thrombotic complications. (Khorana et al, 2007).

**Angiogenesis** has been documented in pancreatic cancer and it was associated with a rapid tumor growth and a poor prognosis (Lomberk, 2010).

The process of angiogenesis represents the formation of new blood vessels from the preexisting vascular bed. In cancers angiogenesis contributes to tumor growth (Folkman, 1995).

Pancreatic cancer seems to be accompanied by an important increase in angiogenesis that is linked to the activation of coagulation. Proteins of coagulation are involved in angiogenesis

Coagulation Disorders in Pancreatic Cancer 259

Central vein catheterization used for the administration of cancer therapy represents a risk factor for thrombosis in these patients. Patients with distant metastases have more increased risk for thrombosis in absence of antithrombotic prophylaxis. The incidence of clinically overt venous thrombosis in cancer patients with central venous catheter ranges from 0.3% to 28%, and rises to 27% - 66% when the diagnosis was assessed by venography (Verso &

Chemotherapy has been shown to be an independent risk factor for thrombosis in cancer patients. In a large population based study, the risk of thrombosis was increased 6.5 fold in patients receiving chemotherapy and 4,1 fold in patients with cancer not receiving this kind of therapy, compared to patients without malignancies (Heit et al, 2000, Kirwan et al, 2011). The risk is additionally increased if chemotherapy is combined with steroids (Shen et al, 2011) or erythropoietin (Bennet et al, 2008). The inhibitors of angiogenesis (thalidomide, lenalidomide, bevacizumab, sunitinib, sorafenib, and sirolimus) used as novel antineoplasic therapy are associated with an increase in arterial and venous thromboembolism and hemorrhage (Zangari et al, 2009). Gemcitabine is a deoxycytidine analogue related to cytarabine, that has been shown to improve evolution in patients with advanced PC. Deep venous thrombosis was found in one study in 3.2% of patients treated with gemcitabine

**4. Clinical outcome in pancreatic cancer patients with thrombosis** 

prognosis (Price et al, 2010). Perpancreatic veins may also be involved (Fig.2).

thrombotic events (13.7 month) (Epstein et al, 2010; Shah& Saif, 2010).

Patients with PC may present with signs of venous or arterial thrombosis. Venous thrombosis is more frequent and it can affect peripheral or visceral veins (Blom et al, 2006). Migratory superficial thrombophlebitis is highly suggestive for a malignancy (Fig. 1). Of the visceral vein thrombosis portal thrombosis has a very high incidence. In one study portal vein thrombosis was found in 32 of 108 patients (30%) and it was associated with a poor

The diagnosis is suggested by clinical signs and is usually confirmed ultrasonographically

Disseminated intravascular coagulation is another coagulation disturbance described in PC. It was associated with an increase in circulating TF (Ueda, 2001). This complication was also observed in patients suffering from metastatic pancreatic cancer treated with a recombinant adenoviral vector containing the cloned human wild type p53 suppressor

It is generally reported that patients with cancer and thrombotic complications have a poor prognosis (Levitan et al, 1999; Sorensen et al, 2000). In the retrospective study by Epstein and colab., 24% of patients with PC and thromboembolism, experienced pulmonary embolism. The authors found a reduced overall survival for patients with a thromboembolic event (12.9 month) if compared to those without (13.4 month). Treatment consisted of low molecular weight heparin, in 95% of patients and inferior vena cava filter was necessary in 19%. Patients with occult thrombotic events or with thrombosis diagnosed at the time of cancer diagnosis, had a poorer survival (6.2 month) compared with those with secondary

Agnelli, 2003; Verso et al, 2008).

(Kaye,1994).

(Fig. 3 and 4).

gene (Haag, 2000).

in two different ways, one clotting dependent and the other one clotting independent (Echrish et al, 2011). The clotting dependent mechanism is initiated by the activation of TF receptors. TF activates then the coagulation cascade that leading to fibrin formation and platelet activation (Falanga & Rickles, 1999). Activated platelets release mediators that promote angiogenesis such as VEGF, beta fibroblast growth factor (β-FGF) and platelet grows factor (PGF) (Palumbo et al, 2000; Echrish et al, 2011). In clotting independent mechanism thrombin plays a very important role by inducing the proteolytic cleavage of proteaseactivated receptors (PAR) (Traynelis & Trejo, 2007). The activation of PAR stimulates the synthesis of factors implicated in angiogenesis such as VEGF (Liu & Mueller, 2006).

Another mediator that involved in thrombosis and angiogenesis of pancreatic cancer is epithelial growth factor receptor (EGFR). An increased expression EGFR was noted in pancreatic cancer and correlated with enhanced angiogenesis, tumor growth and unfavorable evolution (Yamanaka et al, 1993).

**Microparticles** have also been studied in relation with thromboembolism in cancer. Microparticles are membrane vesicles released from stimulated or apoptotic cells in normal persons but they are also implicated in the activation of coagulation (Diamant et al, 2004).

Many recent data support the role of microparticles (MP) and of TF-MP complex in thrombotic complications of patients with malignancies (Tilley et al, 2008).

The level of TF activity associated with TF/MP seems to be higher in PC compared with other types of cancer. From the group of patients with cancer and thrombosis those with pancreatic malignancies have the highest TF activity (Tesselaar et al, 2009).

The role of *P-selectin* in thrombosis in these patients was also studied during the last two decades. P-selectin is released from platelets and endothelial cells and contributes to the adhesion of leucocytes on activated platelets and thrombus formation and to adhesion of cancer cells to stimulated endothelial cells. Experimental studies that have been done on primates suggest that P-selectin inhibition is as effective as low molecular weight heparin in promoting thrombus resolution and in preventing re-occlusion (Chen & Geng, 2006). In humans elevated levels of P-selectin may be predictive of thromboembolism in patients with cancers (Ay et al, 2008).

A large case-control study of venous thrombosis in patients with cancer found that the presence of factor V Leiden or prothrombin 20210A mutation increases by 12 to 17-fold the risk of developing thrombosis compared to those with out these modifications (Blom et al,2005).

Activation of endothelium by tumor-derived inflammatory cytokines, which could induce expression of various adhesive molecules such as V-CAM and E-selectin may promote the thrombotic process in cancer patients (Varki, 2007).

Thromboembolic events in PC patients are also influenced by particular conditions that generally increase the risk of thrombosis such as immobilization, advanced age, comorbidities (infections, cardiac or respiratory failure, obesity, etc.), history of venous thrombosis (Offord et al, 2004; Echrish et al, 2011). Also, the local effects of a great tumour, such as venous compression, that can predispose to an increased risk of thromboembolism (Dumitrascu et al, 2010) .

in two different ways, one clotting dependent and the other one clotting independent (Echrish et al, 2011). The clotting dependent mechanism is initiated by the activation of TF receptors. TF activates then the coagulation cascade that leading to fibrin formation and platelet activation (Falanga & Rickles, 1999). Activated platelets release mediators that promote angiogenesis such as VEGF, beta fibroblast growth factor (β-FGF) and platelet grows factor (PGF) (Palumbo et al, 2000; Echrish et al, 2011). In clotting independent mechanism thrombin plays a very important role by inducing the proteolytic cleavage of proteaseactivated receptors (PAR) (Traynelis & Trejo, 2007). The activation of PAR stimulates the

Another mediator that involved in thrombosis and angiogenesis of pancreatic cancer is epithelial growth factor receptor (EGFR). An increased expression EGFR was noted in pancreatic cancer and correlated with enhanced angiogenesis, tumor growth and

**Microparticles** have also been studied in relation with thromboembolism in cancer. Microparticles are membrane vesicles released from stimulated or apoptotic cells in normal persons but they are also implicated in the activation of coagulation (Diamant et al, 2004). Many recent data support the role of microparticles (MP) and of TF-MP complex in

The level of TF activity associated with TF/MP seems to be higher in PC compared with other types of cancer. From the group of patients with cancer and thrombosis those with

The role of *P-selectin* in thrombosis in these patients was also studied during the last two decades. P-selectin is released from platelets and endothelial cells and contributes to the adhesion of leucocytes on activated platelets and thrombus formation and to adhesion of cancer cells to stimulated endothelial cells. Experimental studies that have been done on primates suggest that P-selectin inhibition is as effective as low molecular weight heparin in promoting thrombus resolution and in preventing re-occlusion (Chen & Geng, 2006). In humans elevated levels of P-selectin may be predictive of thromboembolism in patients with

A large case-control study of venous thrombosis in patients with cancer found that the presence of factor V Leiden or prothrombin 20210A mutation increases by 12 to 17-fold the risk of developing thrombosis compared to those with out these modifications (Blom et

Activation of endothelium by tumor-derived inflammatory cytokines, which could induce expression of various adhesive molecules such as V-CAM and E-selectin may promote the

Thromboembolic events in PC patients are also influenced by particular conditions that generally increase the risk of thrombosis such as immobilization, advanced age, comorbidities (infections, cardiac or respiratory failure, obesity, etc.), history of venous thrombosis (Offord et al, 2004; Echrish et al, 2011). Also, the local effects of a great tumour, such as venous compression, that can predispose to an increased risk of thromboembolism

synthesis of factors implicated in angiogenesis such as VEGF (Liu & Mueller, 2006).

thrombotic complications of patients with malignancies (Tilley et al, 2008).

pancreatic malignancies have the highest TF activity (Tesselaar et al, 2009).

unfavorable evolution (Yamanaka et al, 1993).

thrombotic process in cancer patients (Varki, 2007).

cancers (Ay et al, 2008).

(Dumitrascu et al, 2010) .

al,2005).

Central vein catheterization used for the administration of cancer therapy represents a risk factor for thrombosis in these patients. Patients with distant metastases have more increased risk for thrombosis in absence of antithrombotic prophylaxis. The incidence of clinically overt venous thrombosis in cancer patients with central venous catheter ranges from 0.3% to 28%, and rises to 27% - 66% when the diagnosis was assessed by venography (Verso & Agnelli, 2003; Verso et al, 2008).

Chemotherapy has been shown to be an independent risk factor for thrombosis in cancer patients. In a large population based study, the risk of thrombosis was increased 6.5 fold in patients receiving chemotherapy and 4,1 fold in patients with cancer not receiving this kind of therapy, compared to patients without malignancies (Heit et al, 2000, Kirwan et al, 2011). The risk is additionally increased if chemotherapy is combined with steroids (Shen et al, 2011) or erythropoietin (Bennet et al, 2008). The inhibitors of angiogenesis (thalidomide, lenalidomide, bevacizumab, sunitinib, sorafenib, and sirolimus) used as novel antineoplasic therapy are associated with an increase in arterial and venous thromboembolism and hemorrhage (Zangari et al, 2009). Gemcitabine is a deoxycytidine analogue related to cytarabine, that has been shown to improve evolution in patients with advanced PC. Deep venous thrombosis was found in one study in 3.2% of patients treated with gemcitabine (Kaye,1994).

#### **4. Clinical outcome in pancreatic cancer patients with thrombosis**

Patients with PC may present with signs of venous or arterial thrombosis. Venous thrombosis is more frequent and it can affect peripheral or visceral veins (Blom et al, 2006). Migratory superficial thrombophlebitis is highly suggestive for a malignancy (Fig. 1). Of the visceral vein thrombosis portal thrombosis has a very high incidence. In one study portal vein thrombosis was found in 32 of 108 patients (30%) and it was associated with a poor prognosis (Price et al, 2010). Perpancreatic veins may also be involved (Fig.2).

The diagnosis is suggested by clinical signs and is usually confirmed ultrasonographically (Fig. 3 and 4).

Disseminated intravascular coagulation is another coagulation disturbance described in PC. It was associated with an increase in circulating TF (Ueda, 2001). This complication was also observed in patients suffering from metastatic pancreatic cancer treated with a recombinant adenoviral vector containing the cloned human wild type p53 suppressor gene (Haag, 2000).

It is generally reported that patients with cancer and thrombotic complications have a poor prognosis (Levitan et al, 1999; Sorensen et al, 2000). In the retrospective study by Epstein and colab., 24% of patients with PC and thromboembolism, experienced pulmonary embolism. The authors found a reduced overall survival for patients with a thromboembolic event (12.9 month) if compared to those without (13.4 month). Treatment consisted of low molecular weight heparin, in 95% of patients and inferior vena cava filter was necessary in 19%. Patients with occult thrombotic events or with thrombosis diagnosed at the time of cancer diagnosis, had a poorer survival (6.2 month) compared with those with secondary thrombotic events (13.7 month) (Epstein et al, 2010; Shah& Saif, 2010).

Coagulation Disorders in Pancreatic Cancer 261

Fig. 3. Thrombosis of peroneal vein in a patient with PC (2D and colour Doppler

Fig. 4. Thombosis of femoral vein in a patient with PC (2D echographic examination)

echographic examination)

Fig. 1. Migratory superficial thrombophlebitis in a case of PC (hematoxylin-eosin staining of superficial veins)

Fig. 2. Vascular invasion of PC with local vein thrombosis (hematoxylin-eosin staining)

Fig. 1. Migratory superficial thrombophlebitis in a case of PC (hematoxylin-eosin staining of

Fig. 2. Vascular invasion of PC with local vein thrombosis (hematoxylin-eosin staining)

superficial veins)

Fig. 3. Thrombosis of peroneal vein in a patient with PC (2D and colour Doppler echographic examination)

Fig. 4. Thombosis of femoral vein in a patient with PC (2D echographic examination)

Coagulation Disorders in Pancreatic Cancer 263

symptomatic thromboembolism in treated patients after 3 month (1.25% compared to 9.87% in non treated patients). This significant difference was also found after 12 months with an incidence of 5% in treated patients compared to 15.13% in non treated arm of the trial. There were no significant major hemorrhagic complications in both groups. The median overall survival was not different between the two groups (9.92 month in treated patients versus 8.15 month in no treatment group; p=0.054), for a median follow up period of 45.44 months

In the FRAGEM (Chemotherapy With or Without Dalteparin) trial, 123 patients were randomised to receive dalteparin. This study showed also a significant reduction in

The third trial aimed to assess the effects of the addition of low molecular weight heparin (nadoparin) to gemcitabine plus cisplatinum combination in 42 patients with advanced PC. The results showed a better mean time to progression in the group receiving prophylaxis (6.0+/-0.9 months) when compared to control group (3.0+/-1.5 months) (p=0.0001). Also median overall survival time for the nadoparin group was 9.0+/-1.9 months compared to

The results of these trials showed that the association of a low molecular weight heparin to chemotherapy in advanced pancreatic cancer patients reduces the risk of thromboembolic events. However, the CONKO-004 did not found any improvement in the overall survival

In patients with central vein catheter used commonly for the administration of chemotherapeutic agents and parenteral nutrition, anticoagulation is not recommended for routine prophylaxis of catheter related thrombosis in cancer patients (Geerts et al, 2004). Even if early studies showed risk of venous thrombosis related to central vein catheters (Montreal et al, 1996), a large multinational trial that investigated the efficacy of dalteparin in preventing catheter related thrombosis, found that the risk of thrombosis was not significantly different in the group treated with dalteparin compared to placebo-treated patients (Karthaus et al, 2006). Prophylaxis in patients with central vein catheters may be

Treatment of venous thombotic complications in patients with cancer is usually difficult due to the risk of recurrences and at the same time of bleeding with severe consequences. The aims of treatment are reduction of clinical manifestations of thrombosis and of the risks

The treatment of choice is the administration of a low molecular weight heparin for one week followed by an oral anticoagulant (vitamin K antagonist). Low molecular weight heparins have been shown to be as effective and save as unfractionated heparin. They are preferred like first line treatment because usually no laboratory monitoring is necessary, and the risks of developing heparin induced thrombocytopenia and osteoporosis is reduced. Also the administration of this type of heparin is convenient using once or twice daily doses as subcutaneous injection (Dolovich et al, 2000; van den Belt et al, 2000, Er &Zacharski,

thromboembolic events in patients receiving prophylaxis (Maraveyas et al, 2007).

and time to progression. This needs to be verified in future prospective trials.

4.0+/-0.4 months (p=0.0034) in the control group (Icli et al, 2007).

imposed sometimes when additional risk factors are detected.

pulmonary embolism and postthrombotic syndrome.

**5.2 Treatment of venous thrombosis** 

2006).

(Reiss et al, 2010).

Looking for possible predictors of thromboembolism in pancreatic cancer, one previous study showed that higher levels of TF expression in tumor cells were associated with nearly 4 fold increase in venous thrombosis (Khorana et al, 2007). In a recent retrospective study that included patients diagnosed with pancreaticobiliary cancers between January 2005 and December 2008, looked for the association of TF with thromboembolism and survival. This study included 117 patients with a median age of 65 years of which 68% had pancreatic cancer and 29% biliary cancers. Thrombotic complications were found in 52 (44.4%) patients. Elevated levels of TF (greater than 2.5pg/ml) were associated with thromboembolic events (odds ratio=1.22;p=0.04). Also, TF levels were predictive for a worse overall survival (hazard ratio=1.05; p=0.01) (Barthuar et al, 2010). These results if confirmed in prospective studies suggest that TF expressed by neoplastic cells or plasma levels of TF could be used as independent predictive biomarkers for thromboembolic events in PC patients and also in other cancers (Khorana et al,2007; Barthuar et al, 2010).

#### **5. Prevention and treatment of thromboembolism in PC**

#### **5.1 Prophylaxis of venous trombosis**

Epidemiologic and pathogenic data clearly indicate that patients with malignancy had an important risk of thormboembolic events. In practice, risk stratification can be used to classify patients according to their thrombotic risk. The ACCP guidelines consider the patient with cancer in the very high risk category particularly when surgery is recommended. Other factors that may increase patient's risk are age, immobilization, prior history of venous thrombosis, obesity and central venous catheter (Geerts et al, 2004; Caprini et al, 2001).

Prophylaxis in cancer is indicated mainly in two distinct situations: in patients undergoing surgery and in medical patients receiving chemotherapy.

Patients undergoing abdominal surgery are a particularly high-risk population who may benefit for extended thromboprophylaxis. Low molecular weight heparins are preferred as they showed to be as effective and safe as unfractionated heparin. Several studies showed a reduction in thromboembolic complications in patients receiving prolonged prophylaxis for 3 or 4 weeks compared to those with 1 week of treatment in postoperative period. This beneficial effect was not accompaneied by an increase in hemorrhagic complications (Bergqvist et al, 2002; Rasmussen et al, 2003).

In patients treated with chemotherapy antithrombotic prophylaxis showed also a reduction in thromboembolic risk. There are 2 trials in patients with PC treated with gemcitabine and a low molecular weight heparin and another one in wich a low molecular weight heparin was associated to a combined chemotherapy gemcitabine and cisplatinum.

The results of the Charité Onkologie (CONKO)-004 trial were recently published. The principal objective of this trail was the evaluation of the reduction in symptomatic thromboembolic events in patients with advanced PC. The second end point was the overall survival. Between April 2004 and January 2009, 312 patients with histological confirmed advanced PC were randomized into two groups as follows: 160 patients received treatment with enoxaparin 1 mg/kg once a day for 3 month, followed by 40 mg daily and 152 did not receive antithrombotic prophylaxis. The results indicated a significant reduction of

Looking for possible predictors of thromboembolism in pancreatic cancer, one previous study showed that higher levels of TF expression in tumor cells were associated with nearly 4 fold increase in venous thrombosis (Khorana et al, 2007). In a recent retrospective study that included patients diagnosed with pancreaticobiliary cancers between January 2005 and December 2008, looked for the association of TF with thromboembolism and survival. This study included 117 patients with a median age of 65 years of which 68% had pancreatic cancer and 29% biliary cancers. Thrombotic complications were found in 52 (44.4%) patients. Elevated levels of TF (greater than 2.5pg/ml) were associated with thromboembolic events (odds ratio=1.22;p=0.04). Also, TF levels were predictive for a worse overall survival (hazard ratio=1.05; p=0.01) (Barthuar et al, 2010). These results if confirmed in prospective studies suggest that TF expressed by neoplastic cells or plasma levels of TF could be used as independent predictive biomarkers for thromboembolic events in PC patients and also in

Epidemiologic and pathogenic data clearly indicate that patients with malignancy had an important risk of thormboembolic events. In practice, risk stratification can be used to classify patients according to their thrombotic risk. The ACCP guidelines consider the patient with cancer in the very high risk category particularly when surgery is recommended. Other factors that may increase patient's risk are age, immobilization, prior history of venous thrombosis, obesity and central venous catheter (Geerts et al, 2004; Caprini

Prophylaxis in cancer is indicated mainly in two distinct situations: in patients undergoing

Patients undergoing abdominal surgery are a particularly high-risk population who may benefit for extended thromboprophylaxis. Low molecular weight heparins are preferred as they showed to be as effective and safe as unfractionated heparin. Several studies showed a reduction in thromboembolic complications in patients receiving prolonged prophylaxis for 3 or 4 weeks compared to those with 1 week of treatment in postoperative period. This beneficial effect was not accompaneied by an increase in hemorrhagic complications

In patients treated with chemotherapy antithrombotic prophylaxis showed also a reduction in thromboembolic risk. There are 2 trials in patients with PC treated with gemcitabine and a low molecular weight heparin and another one in wich a low molecular weight heparin

The results of the Charité Onkologie (CONKO)-004 trial were recently published. The principal objective of this trail was the evaluation of the reduction in symptomatic thromboembolic events in patients with advanced PC. The second end point was the overall survival. Between April 2004 and January 2009, 312 patients with histological confirmed advanced PC were randomized into two groups as follows: 160 patients received treatment with enoxaparin 1 mg/kg once a day for 3 month, followed by 40 mg daily and 152 did not receive antithrombotic prophylaxis. The results indicated a significant reduction of

was associated to a combined chemotherapy gemcitabine and cisplatinum.

other cancers (Khorana et al,2007; Barthuar et al, 2010).

surgery and in medical patients receiving chemotherapy.

(Bergqvist et al, 2002; Rasmussen et al, 2003).

**5.1 Prophylaxis of venous trombosis** 

et al, 2001).

**5. Prevention and treatment of thromboembolism in PC** 

symptomatic thromboembolism in treated patients after 3 month (1.25% compared to 9.87% in non treated patients). This significant difference was also found after 12 months with an incidence of 5% in treated patients compared to 15.13% in non treated arm of the trial. There were no significant major hemorrhagic complications in both groups. The median overall survival was not different between the two groups (9.92 month in treated patients versus 8.15 month in no treatment group; p=0.054), for a median follow up period of 45.44 months (Reiss et al, 2010).

In the FRAGEM (Chemotherapy With or Without Dalteparin) trial, 123 patients were randomised to receive dalteparin. This study showed also a significant reduction in thromboembolic events in patients receiving prophylaxis (Maraveyas et al, 2007).

The third trial aimed to assess the effects of the addition of low molecular weight heparin (nadoparin) to gemcitabine plus cisplatinum combination in 42 patients with advanced PC. The results showed a better mean time to progression in the group receiving prophylaxis (6.0+/-0.9 months) when compared to control group (3.0+/-1.5 months) (p=0.0001). Also median overall survival time for the nadoparin group was 9.0+/-1.9 months compared to 4.0+/-0.4 months (p=0.0034) in the control group (Icli et al, 2007).

The results of these trials showed that the association of a low molecular weight heparin to chemotherapy in advanced pancreatic cancer patients reduces the risk of thromboembolic events. However, the CONKO-004 did not found any improvement in the overall survival and time to progression. This needs to be verified in future prospective trials.

In patients with central vein catheter used commonly for the administration of chemotherapeutic agents and parenteral nutrition, anticoagulation is not recommended for routine prophylaxis of catheter related thrombosis in cancer patients (Geerts et al, 2004). Even if early studies showed risk of venous thrombosis related to central vein catheters (Montreal et al, 1996), a large multinational trial that investigated the efficacy of dalteparin in preventing catheter related thrombosis, found that the risk of thrombosis was not significantly different in the group treated with dalteparin compared to placebo-treated patients (Karthaus et al, 2006). Prophylaxis in patients with central vein catheters may be imposed sometimes when additional risk factors are detected.

#### **5.2 Treatment of venous thrombosis**

Treatment of venous thombotic complications in patients with cancer is usually difficult due to the risk of recurrences and at the same time of bleeding with severe consequences. The aims of treatment are reduction of clinical manifestations of thrombosis and of the risks pulmonary embolism and postthrombotic syndrome.

The treatment of choice is the administration of a low molecular weight heparin for one week followed by an oral anticoagulant (vitamin K antagonist). Low molecular weight heparins have been shown to be as effective and save as unfractionated heparin. They are preferred like first line treatment because usually no laboratory monitoring is necessary, and the risks of developing heparin induced thrombocytopenia and osteoporosis is reduced. Also the administration of this type of heparin is convenient using once or twice daily doses as subcutaneous injection (Dolovich et al, 2000; van den Belt et al, 2000, Er &Zacharski, 2006).

Coagulation Disorders in Pancreatic Cancer 265

Pancreatic cancer is associated with a very increased risk of thromboembolic events. The mechanisms underlying this association are complex and multifactorial but are not yet clearly understood. Thromboembolic complications in patients with PC indicate a poor prognosis and a reduction of life expectancy. Antithrombotic prophylaxis in advanced PC treated with chemotherapy reduces the risk of embolic complications and it may also improve survival and time to progression of cancer in these patients. The medication of choice in preventing and treating thrombosis are low molecular weight heparins. Anticoagulant therapy may help cancer patients due also to a possible antitumor effect.

This work was partly funded by the research grant INFORAD of the Romanian Ministry of

Alban, S. (2001). Molecular weight-dependent influence of heparin on the form of tissue

Altinbas, M.; Coskun, H.S.; Er, O.; Ozkan, M.; Eser, B.; Unal, A., et al. (2004). A randomized

Ay, C.; Simonek, R.; Vormittag, R., et al. (2008). High plasma levels of soluble P-selectin are

Bennett, C.L.; Silver, S.M.; Djulbegovic, B. et al. *(*2008) . Venous thromboembolism and

Bergqvist, D.; Agneli, G.; Cohen A., et al. (2002). Duration of prophylaxis against venous

Bick, R.L. (1992). Coagulation abnormalities in malignancy; a review. *Semin Thromb Hemost*,

Blom, J.W.; Doggen, C.J.M.; Osanto, S. & Rosendaal, F.R. (2005) Malignancies, prothrombotic

Blom, J.W.; Osanto, S. & Rosendaal, F.R. (2006). High risk of venous thrombosis in patients

*J Clin Oncol,* vol. 28 (15 suppl),pp. 4062, ISSN 2218-4333.

factor pathway inhibitor circulating in plasma. *Semin Thromb Hemost*. Vol.27, pp.

clinical trial of combination chemotherapy with and without low-molecular-weight heparin in small cell lung cancer. *J Thromb Haemost,* vol.2, pp. 1266-1271, ISSN 1538-

predictive of venous thromboembolism in cancer patients. Results from the Vienna cancer and thrombosis study. *Blood*, vol. 112, no.7, pp. 2703-2708, ISSN 0268-960X. Barthuar, A.; Khorana, A.A.; Hutson, A., et al (2010). Association of elevated tissue factor

(TF) with survival and thromboembolism (TE) in pancreaticobiliary cancers (PBC).

mortality associated with recombinant erythropoietin and darbepoetin administration for the treatment of cancer-associated anemia. *JAMA*, vol. *299*, pp.

thromboembolism with enoxaparin after surgery for cancer. *N Engl J Med* vol. 346,

mutations, and the risk of venous thrombosis. *JAMA*, vol. *293*, pp. 715-722, ISSN

with pancreatic cancer: A cohort study of 202 patients. *Eur J Cancer*, vol.42, pp. 410-

**6. Conclusions** 

**7. Acknowledgements** 

Education and Research.

7933.

503-511. ISSN 0094-6176.

914-924, ISSN 00987484.

00987484.

414, ISSN 1359-6349.

pp. 975-980, ISSN 0028-4793.

vol.18, no.4, pp.353-372, ISSN 0094-6176.

**8. References** 

Recurrent thrombosis needs long-term management. In cancer patients prolonged anti thrombotic, particularly with oral antivitamin K medication, is associated with increased risk of hemorrhagic complications that may be linked to malnutrition, liver dysfunction and metastases, reduced alimentary intake or vomiting. The risk of bleeding appears to correlate with the extent of the disease. In a study that investigated the risk of bleeding in patients with different extent of the disease, patients with moderately extensive cancer had a 2-3-fold increase in risk of major bleeding; patients with extensive cancer had a 5-fold increase in this risk (Prandoni et al, 2002).

Low molecular weight heparins are now preferred as long term secondary prevention treatment in these patients. This indication is based on the results of several randomized trials that showed a superior efficacy and safety of low molecular weight heparins compared with oral anticoagulants in long term administration. In a multicenter randomized trial patients were treated for 3 month with enoxaparin or with warfarin. Of the group receiving warfarin 15 (21%) of 71 patients had a major bleeding or a thrombotic recurrence, compared to 7 (10.5%) of 67 patients treated with enoxaparin (Meyer et al, 2002). A large multicenter trail, "The Randomized Comparison of Low–Molecular-Weight Heparin versus Oral Anticoagulant Therapy for the Prevention of Recurrent Venous Thromboembolism in Patients with Cancer (CLOT) compared treatment with dalteparin with oral anticoagulant therapy. After 6 months of treatment the probability of recurrent venous thrombosis was 17% in patients receiving oral anticoagulation compared to 9% in those treated with dalteparin. There were no significant differences between groups for the hemorrhagic complications (Lee et al, 2003).

#### **5.3 Antineoplastic effects of anticoagulants**

There are evidences that anticoagulant therapy may have also anticancer effects. Heparins in addition to activation of antithrombin, may promote the release of the tissue factor pathway inhibitor from the endothelium that blocks tissue factor expressed by tumor cells (Alban, 2001; Sandset et al, 2001). Heparin is also able to bind to and to inhibit some inflammatory cytokines that can activate endothelial cells and increase expression of adhesion molecules (Elsayed & Becker, 2003; Varki ,2007). Heparin may interfere with formation of the platelet "cloak" around tumor cells suggesting a possible effect in metastasis prevention (Borsig et al, 2001).

Several clinical studies support the efficacy of heparins in improuving tumor response and survival. The administration of nadroparin in patients with advanced solid cancers increased median survival to 8 month compared to 6.6 months in patients receiving placebo, after 6 weeks of treatment (Klerk et al, 2005). Dalteparin associated in the treatemnt of patients with small cell lung cancer, for 18 weeks improved tumor response and median overall survival from 8 to 13 months (Altinbas et al, 2004).

Beneficial effects have also been reported for warfarin in cancer patients. A prospective randomized trial showed that survival of patients with small-cell lung carcinoma had a significant prolonged survival if warfarin was added to standard therapy. The median survival and the time to first evidence of disease progression were increased in patients receiving warfarin (Zacharski et al, 1981).

Data suggesting the participation of coagulation mechanisms in tumour growth are important arguments for researchers to explore this novel therapeutic strategy in cancer patients.

#### **6. Conclusions**

264 Pancreatic Cancer – Clinical Management

Recurrent thrombosis needs long-term management. In cancer patients prolonged anti thrombotic, particularly with oral antivitamin K medication, is associated with increased risk of hemorrhagic complications that may be linked to malnutrition, liver dysfunction and metastases, reduced alimentary intake or vomiting. The risk of bleeding appears to correlate with the extent of the disease. In a study that investigated the risk of bleeding in patients with different extent of the disease, patients with moderately extensive cancer had a 2-3-fold increase in risk of major bleeding; patients with extensive cancer had a 5-fold increase in this

Low molecular weight heparins are now preferred as long term secondary prevention treatment in these patients. This indication is based on the results of several randomized trials that showed a superior efficacy and safety of low molecular weight heparins compared with oral anticoagulants in long term administration. In a multicenter randomized trial patients were treated for 3 month with enoxaparin or with warfarin. Of the group receiving warfarin 15 (21%) of 71 patients had a major bleeding or a thrombotic recurrence, compared to 7 (10.5%) of 67 patients treated with enoxaparin (Meyer et al, 2002). A large multicenter trail, "The Randomized Comparison of Low–Molecular-Weight Heparin versus Oral Anticoagulant Therapy for the Prevention of Recurrent Venous Thromboembolism in Patients with Cancer (CLOT) compared treatment with dalteparin with oral anticoagulant therapy. After 6 months of treatment the probability of recurrent venous thrombosis was 17% in patients receiving oral anticoagulation compared to 9% in those treated with dalteparin. There were no significant differences between groups for the

There are evidences that anticoagulant therapy may have also anticancer effects. Heparins in addition to activation of antithrombin, may promote the release of the tissue factor pathway inhibitor from the endothelium that blocks tissue factor expressed by tumor cells (Alban, 2001; Sandset et al, 2001). Heparin is also able to bind to and to inhibit some inflammatory cytokines that can activate endothelial cells and increase expression of adhesion molecules (Elsayed & Becker, 2003; Varki ,2007). Heparin may interfere with formation of the platelet "cloak" around

Several clinical studies support the efficacy of heparins in improuving tumor response and survival. The administration of nadroparin in patients with advanced solid cancers increased median survival to 8 month compared to 6.6 months in patients receiving placebo, after 6 weeks of treatment (Klerk et al, 2005). Dalteparin associated in the treatemnt of patients with small cell lung cancer, for 18 weeks improved tumor response and median

Beneficial effects have also been reported for warfarin in cancer patients. A prospective randomized trial showed that survival of patients with small-cell lung carcinoma had a significant prolonged survival if warfarin was added to standard therapy. The median survival and the time to first evidence of disease progression were increased in patients

Data suggesting the participation of coagulation mechanisms in tumour growth are important

arguments for researchers to explore this novel therapeutic strategy in cancer patients.

tumor cells suggesting a possible effect in metastasis prevention (Borsig et al, 2001).

risk (Prandoni et al, 2002).

hemorrhagic complications (Lee et al, 2003).

**5.3 Antineoplastic effects of anticoagulants** 

overall survival from 8 to 13 months (Altinbas et al, 2004).

receiving warfarin (Zacharski et al, 1981).

Pancreatic cancer is associated with a very increased risk of thromboembolic events. The mechanisms underlying this association are complex and multifactorial but are not yet clearly understood. Thromboembolic complications in patients with PC indicate a poor prognosis and a reduction of life expectancy. Antithrombotic prophylaxis in advanced PC treated with chemotherapy reduces the risk of embolic complications and it may also improve survival and time to progression of cancer in these patients. The medication of choice in preventing and treating thrombosis are low molecular weight heparins. Anticoagulant therapy may help cancer patients due also to a possible antitumor effect.

#### **7. Acknowledgements**

This work was partly funded by the research grant INFORAD of the Romanian Ministry of Education and Research.

#### **8. References**


Coagulation Disorders in Pancreatic Cancer 267

Gilbert, G.E. & Arena, A.A. (1995). Phosphatidylethanolamine induces high-affinity binding

Girard, T.J.; Warren, L.A.; Novotny, W.F.; Likert, K.M.; Brown, S.G.; Miletich, J.P.& Broze,

Gouaulthelimann, M. & Josso, F. (1979). Initiation invivo of blood coagulation-role of white

Haag, C.; Thiede C.; Hanig V. & Ehninger G. (2000). Disseminated Intravascular

Heit, J.A.; Silverstein, M.D.; Mohr, D.N.; Petterson, T.M.; O'Fallon, W.M. & Melton L.J. 3rd.

Heit, J.A.; O'Fallon, W.M.; Petterson, T.M.; Lohse, C.M.; Silverstein, M.D.; Mohr, D.N. &

Icli, F.; Akbulut, H.; Utkan, G., et al. (2007). Low molecular weight heparin (LMWH)

Kaye, S.B. (1994). Gemcitabine: current status of phase I and II trials. *J Clin Oncol*, vol. 12, pp.

Karthaus, M.; Kretzschmar, A.; Kroning, H., et al. (2006). Dalteparin for prevention of

Kirwan, C.C.; McDowell, G.; McCollum, C.N. & Byrne, G.J. (2011). Incidence of Venous

advanced malignancy. *J Clin Oncol*, vol. 23, pp. 2130-2135, ISSN 2218-4333. Khorana, A.A. (2003). Malignancy, thrombosis and Trousseau: the case for an eponym. *J* 

Khorana, A.A, & Fine, R.L. (2004). Pancreatic cancer and thromboembolic disease. *Lancet* 

outcome. *Anticancer Res*, vol. 31, no. 6, pp. 2383-2388, ISSN 0250-7005. Klerk, C.P.; Smorenburg, S.M.; Otten, H.M.; Lensing, A.W.; Prins, M.H.; Piovella, F., et al.

*Thromb Haemost*, vol. 1, pp. 2463-2465, ISSN: 1538-7933.

*Oncol*, vol. 5, pp. 655-663, ISSN 1470-2045.

pancreatic cancer. *J Surg Oncol*, vol. 95, pp. 507-512. ISSN:0022-4790. Kakkar, A.K.; Lemoine N.R.; Scully, M.F. et al. (1995). Tissue factor expression correlates

0012-3692.

0028-0836.

23, 2000.

0003-9926.

1245-1248, ISSN 0003-9926.

1527–1531, ISSN 2218-4333.

pp. 289-296, ISSN 0923-7534.

ISSN: 1365-2168.

vol. *270*, pp. 18500-18505, ISSN 0021-9258.

Antithrombotic and Thrombolytic Therapy. *Chest*, vol. 126, pp. 338-400S, ISSN

sites for factor-VIII on membranes containing phosphatidyl-L-serin. *J Biol Chem*,

G.J. (1989). Functional significance of the kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. *Nature*, vol.*338*, pp. 518-520, ISSN

blood-cells and tissue factor. *Nouv Presse Med,* vol. *8*, pp. 3249-3253, ISSN 0755-4982.

Coagulation (DIC) After Intraarterial Injection of Adenoviral Vector Containing P53 in Patients with Pancreatic Cancer in a Phase I/II Study. Proc Am Soc Clin Oncol vol. 19, (abstr 1813). 2000 ASCO Annual Meeting, New Orleans, LA, May 20-

(2000). Risk factors for deep vein thrombosis and pulmonary embolism: a population-based casecontrol study. *Arch Intern Med*, vol. 160, pp. 809-815, ISSN

Melton, L.J. (2002). Relative impact of risk factors for deep vein thrombosis and pulmonary embolism – A population-based study. *Arch Intern Med*, vol. *162*, pp.

increases the efficacy of cisplatinum plus gemcitabine combination in advanced

with histological grade in human pancreatic cancer. *Br J Surg* vol.82, pp. 1101-1104,

catheter-related complications in cancer patients with central venous catheters: final results of a double-blind, placebo controlled phase III trial. *Ann Oncol*, vol. 17,

Thromboembolism during Chemotherapy for Breast cancer Impact on cancer

(2005). The effect of low molecular weight heparin on survival in patients with


Borsig, L.; Wong, R.; Feramisco, J.; Nadeau, D.R.; Varki, N.M. & Varki, A. (2001). Heparin

Browder, T.; Folkmen, J. & Pirie-Shepherd, S. (2000). The hemostatic system as a regulator of

Broze, G.J.(1995). Tissue factor pathway inhibitor and the revised theory of coagulation.

Caprini, J.A.; Arcelus, J.I. & Reyna, J.J. (2001). Effective risk stratification of surgical and

Chen, M. & Geng, J.G. (2006). P-selectin mediates adhesion of leukocytes, platelets, and

Diamant, M.; Tushuizen, M.E.; Sturk, A. & Nieuwland R. (2004). Cellular microparticles:

Dolovich, L.R.; Ginsberg, J.S.; Douketis, J.D., et al. (2000). A meta-analysiscomparing low-

Dumitrascu, D.L.; Suciu, O.; Grad, C. & Gheban, D. (2010). Thrombotic complications of pancreatic cancer: classical knowledge revised. Dig Dis, vol. 28, pp. 350-354. Echrish, H.; Madden, L.A.; Greenman, J. & Maraveyas, A. (2011). The hemostasis apparatus

Elsayed, E. & Becker, R.C. (2003). The impact of heparin compounds on cellular

Ettelaie, C.; Li, C.; Collier, M.E.W.; Pradier, A.; Frentzou, G.A.; Wood, C.G.; Chetter, I.C.;

Falanga, A. & Rickles, F.R. (1999). Pathophysiology of the thrombophilic state in the cancer

Folkman, J. (1995). Angiogenesis inhibitors generated by tumors. *Mol Med*, vol.*1*, pp. 120-

Geerts, W.H.; Pineo, G.F.; Heit, J.A.; Bergqvist, D.; Lassen, M.R.; Colwell, C.W., et al. (2004).

patient. *Sem Throm Hemost*, vol. *25*, pp. 173-182, ISSN 0094-6176.

development. *J Thromb Thrombolysis*, vol. 15, pp. 11-18, ISSN 0929-5305. Epstein, A.S.; Crosbie, C.; Gardos, S. at al. (2010). A single institution, (MSKCC) analysis of

*Health Risk Manag*, vol. 2, pp. 351-356, ISSN 1176-6344.

angiogenesis. *J Biol Chem*; vol. 275, pp.1521-1524, ISSN 0021-9258.

*Immunol Ther Exp* (Warsz). Vol. 54, pp. 75-84, ISSN 0004-069X.

*Annu. Rev.Med.*, vol. *46*, pp. 103-112, ISSN 0066-4219.

38(2Suppl 5), pp. 12-19, ISSN 0037-1963.

3357, ISSN 0027-8424.

ISSN 0014-2972.

188, ISSN 0003-9926.

284, ISSN 2072-6694.

*194*, pp. 88-101, ISSN: 0021-9150.

122, ISSN 1226-3613.

and cancer revisited: mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis. *Proc Natl Acad Sci USA*, vol. 98, pp. 3352-

nonsurgical patients for venous thromboembolic disease. *Semin Hematol*, vol.

cancer cells in inflammation, thrombosis, and cancer growth and metastasis. *Arch* 

new players in the field of vascular disease? *Eur J Clin Invest*, vol. 34, pp. 392–401,

molecular-weight heparins with unfractionated heparin in the treatment of venous thromboembolism:examining some unanswered questions regarding location of treatment, product type, and dosing frequency. *Arch Intern Med*,vol. 160, pp.181-

in pancreatic cancer and its importance beyond thrombosis. *Cancers*, vol. *3*, pp. 267-

inflammatory responses: a construct for future investigation and pharmaceutical

incidence and clinical outcomes in patients with thromboembolic events and exocrine pancreas cancer. *J Clin Oncol*, vol. 28(15 suppl), pp.4126, ISSN 2218-4333. Er, O. & Zacharsky, L. (2006). Management of cancer associated venous thrombosis. *Vasc* 

McCollum, P.T.; Bruckdorfer, K.R. & James, N.J. (2007). Differential functions of tissue factor in the trans-activation of Cell Signall pathways. *Atherosclerosis*, vol.

Prevention of venous thromboembolism: the Seventh ACCP Conference on

Antithrombotic and Thrombolytic Therapy. *Chest*, vol. 126, pp. 338-400S, ISSN 0012-3692.


Coagulation Disorders in Pancreatic Cancer 269

Pinzon, R.; Drewinko, B.; Trujillo, J.M. , et al. (1986). Pancreatic carcinoma and Trousseau's

Prandoni, P.; Lensing, A.W.; Piccioli, A., et al (2002). Recurrent venous thromboembolism

Rahr, H.B. & Sørensen, J.V. (1992). Venous thromboembolism and cancer. *Blood Coagul*

Rasmussen, M.S.; Wille-Jorgensen, P.; Jorgensen, L.N., et al. (2003).

Reiss, H.; Pelzer, U.; Opitz, B., et al. (2010). A prospective, randomized trial of simultaneous

Ruf, W.; Fischer, E.G.; Huang, H.Y.; Miyagi, Y.; Ott, I.; Riewald, M. & Mueller, B.M. (2000).

Sack, Jr G.H.; Levine, J. & Bel, W.R. (1977). Trousseau's syndrome and other manifestations

Sandset, P.R.; Bendz, B. & Hansen, J.B. (2000). Physiological function of tissue factor

Shah, M.M. & Saif, M.W. (2010). Pancreatic cancer and thrombosis. Highlights from the

Shen, Y.; Zhou, X.; Wang, Z.; Yang, G.; Jiang, Y.; Sun, C.; Wang, J.; Tong, Y. & Guo, H.

Sørensen, H.T.; Mellemkjaer, L.; Olsen, J.H., et al. (2000). Prognosis of cancers associated with venous thromboembolism. *N Engl J Med*, vol. 343, pp. 1846-1850, ISSN 0028-4793. Sproul, E.E. (1938).Carcinoma and venous thrombosis: the frequency of association of

Tesselaar, M.E.T.; Romijn, F.; van der Linden, I.K.; Bertina, R.M. & Osanto, S. (2009).

thrombosis. *J Thromb Haematol*, vol. *7*, pp. 1421-1423, ISSN 1740 3340.

and venous thrombosis. *Blood*, vol. 100, pp. 3484–3488, ISSN 0268-960X. Price, L.H.; Nguyen M. B.; Picozzi V. J. & Kozarek R. A. (2010). Portal vein thrombosis in

Orlando, Florida, USA, 22-24, January, 2010.

*Fibrinolysis*, vol. 3, pp. 451-460, ISSN 09575235.

*Chest*, vol. 124, pp. 58-68S, ISSN 0012-3692.

*Immunol Res*, vol. *21*, pp. 289-292, ISSN: 0923-2494 .

*Leuk Res*, vol. 35, no. 2, pp. 147-151, ISSN:0145-2126.

*J Cancer*, vol. 34, pp. 566-573, ISSN: 1175-6357.

ISSN 2218-4333.

186, ISSN 0268-960X.

1-37, ISSN: 0025-7974.

48-56, ISSN: 0340-6245.

no. 4, pp. 331-333, ISSN 1590-8577.

syndrome: experience at a large cancer center. *J Clin Oncol*, vol. 4, no. 4, pp. 509-514,

and bleeding complications during anticoagulant treatment in patients with cancer

pancreatic cancer: Natural history, risk factors, and implications for patient management. Poster session Meeting: *2010 Gastrointestinal Cancers Symposium*,

Prolongedthromboprophylaxis with low molecular weight heparin (dalteparin) following major abdominal surgery for malignancy [abstract]. *Blood*, vol. 102pp.

pancreatic cancer treatemnet with enoxaparin and chemotherapy: Final results of the CONKO-004 trial. *J Clin Oncol,* vol. 28(15Suppl), pp. 4033, ISSN 2218-4333. Rickles, F.R.; Patierno, S. & Fernandez, P.M. (2003).Tissue factor, thrombin, and cancer.

Diverse functions of protease receptor tissue factor in inflammation and metastasis.

of chronic disseminated coagulopathy in patients with neoplasms : clinical, pathophysiologic, and therapeutic features. *Medicine* (Baltimore), vol. 56, no. 1, pp.

pathway inhibitor and interaction with heparins. *Haemostasis*, vol.30(suppl 2), pp.

"2010 ASCO Annual Meeting". Chicago, IL, USA. June 4-8, 2010. *JOP*; jul 5, vol.11,

(2011). Coagulation profiles and thromboembolic events of bortezomib plus thalidomide and dexamethasone therapy in newly diagnosed multiple myeloma.

carcinoma in the body or tail of the pancreas with multiple venous thromboses. *Am* 

Microparticleassociated tissue factor activity in cancer patients with and without


Khorana, A.A.; Ahrendt S.A. & Ryan C.K. (2007). Tissue factor expression, angiogenesis and

Lee, A.Y.; Levine, M.N.; Baker, R.I., et al. (2003). Low molecular weight heparin versus a

Levine, M.N. (1997). Prevention of thrombotic disorders in cancer patients undergoing chemotherapy. *Thromb Haemost*, vol. 78, pp. 133-136, ISSN: 0340-6245. Levitan, N.; Dowlati, A.; Remick, S.C., et al. (1999). Rates of initial and recurrent

Liu, Y. & Mueller, B.M. (2006). Protease-activated receptor-2 regulates vascular endothelial

Mechtcheriakova, D.; Wlachos, A.; Holzmuller, H.; Binder, B.R. & Hofer, E. (1999). Vascular

Milsom, C.C.; Yu, J.L.; Mackman, N.; Micallef, J.; Anderson, G.M.; Guha, A. & Rak, J.W.

Nitori, N.; Ino Y.; Nakanishi Y, et al. (2005). Prognostic significance of tissue factor in

Offord, R.; Lloyd, A.C.; Anderson, P. & Bearne, A. (2004). Economic evaluation of

Palumbo, J.S.; Kombrinck, K.W.; Drew, A.F.; Grimes, T.S.; Kiser, J.H.; Degen, J.L. &

patients. *Pharmacy World Sci*, vol. *26*, pp. 214-220, ISSN 0928-1231.

is mediated by EGR-1. *Blood*, vol. 93,pp. 3811–3823, ISSN 0268-960X. Meyer, G.; Marjanovic, Z.; Valcke, J., et al. (2002). Comparison of lowmolecular-weight

Angiogenesis. *Cancer Res*, vol. *68*, pp. 10068-10076, ISSN: 0008-5472. Nemerson, Y. (1988). Tissue factor and hemostasis. *Blood*, vol. *71*, pp. 1-8, ISSN 0268-960X. Neoptolemos, J.P.; Dunn, J.A.; Stocken, D.D., et al. (2001). Adjuvant chemoradiotherapy and

*Lancet*, vol. 358, no. 9293, pp. 1576-1585, ISSN: 0140-6736.

Lomberk, G. (2010). Angiogenesis. *Pancreatology*, vol. *10*, pp. 112-113, ISSN: 1424-3903. Maraveyas, A.; Holmes, M.; Lofts, F., et al. (2007). Chemoanticoagulation versus

with cancer. *N Engl J Med*, vol. 349, pp. 146-153, ISSN 0028-4793.

2875, ISSN: 1078-0432.

pp. 285-291, ISSN: 0025-7974.

1729-1735, ISSN 0003-9926.

1078-0432.

*Res*, vol. *344*, pp. 1263-1270, ISSN 0006-291X.

*Oncol*, vol. 25, pp. 4583, ISSN 2218-4333.

thrombosis in pancreatic cancer. *Clin Cancer Res* vol.13,no. 10, may 15, pp. 2870-

coumarin for the prevention of recurrent venous thromboembolism in patients

thromboembolic disease among patients with malignancy versus those without malignancy. Risk analysis using Medicare claims data. *Medicine (Baltimore),* vol. 78,

growth factor expression in MDA-MB-231 cells via MAPK pathways. *Biochem Bioph* 

chemotherapy in advanced pancreatic cancer: results of the interim analysis of the FRAGEM trial. Program and abstracts of the 43rd Annual Meeting of the American Society of Clinical Oncology; June 1-5, 2007; Chicago, Illinois. Abstract 4583. *J Clin*

endothelial cell growth factor-induced tissue factor expression in endothelial cells

heparin and warfarin for the secondary prevention of venous thromboembolism in patients with cancer: a randomized controlled study. *Arch Intern Med*, vol. 162, pp.

(2008). Tissue Factor Regulation by Epidermal Growth Factor Receptor and Epithelial-to-Mesenchymal Transitions: Effect on Tumor Initiation and

chemotherapy in respectable pancreatic cancer: a randomized controlled trial.

pancreatic ductal adenocarcinoma. *Clin Cancer Res*, vol. 11, pp. 2531-2539, ISSN:

enoxaparin for the prevention of venous thromboembolism in acutely ill medical

Bugge,T.H. (2000). Fibrinogen is an important determinant of the metastatic potential of circulating tumor cells. *Blood*, vol. *96*, pp. 3302-3309, ISSN 0268-960X.


**16**

Yoshinori Nio *Nio Surgery Clinic,* 

*Japan* 

**Clinical Implications of an Expandable Metallic Mesh Stent for Malignant Portal Vein Stenosis**

Pancreatic cancer (PC) remains one of the most lethal common malignancies. More than 80% of patients with PC cannot be cured by surgical resection (Li D et al., 2004); the actuarial 5 year survival rate after curative resection is approximately 20% (Crist et al., 1987), and the median survival time (MST) after surgical resection ranges between 11 and 24 months (Nitecki et al., 1995). In other words, most patients develop recurrent disease in the near

Advanced or recurrent PC frequently invades the surrounding organs or tissues, and the patients require substantial palliative interventions, especially against biliary obstruction, gastric or duodenal outlet obstruction, and severe abdominal or back pain. In addition, when the portal vein (PV) is invaded and occluded, the patient suffers from various portal hypertension (PH)-associated symptoms and liver dysfunction, including jaundice, ascites,

PC-associated portal obstruction is classified into two categories, intrahepatic obstruction and extrahepatic obstruction. In the case of intrahepatic or hilar PV stenosis, a wall-stent is usually applied (Tsukamoto et al., 2003); however, a wall-stent cannot be used for the extrahepatic PV stenosis, because it may occlude the splenic vein, which joins the extrahepatic PV, leading to serious complications. In patients with extrahepatic PV obstruction, we placed an expandable metallic mesh (EMM) stent into the PV *via* the ileocecal vein following a mini-laparotomy. A total of 14 patients with inoperable or recurrent PC were given an EMM-PV-stent and received subsequent ChT and/or RT, and the treatment results were retrospectively compared with

We treated a total of 97 patients with inoperable or recurrent PC. Of 97 patients, 68 received ChT, 28 received RT using LINAC at 40 - 60Gy (2Gy 20 - 30 times) and 14 were given an

and bleeding tendencies, which disturb chemotherapy (ChT) or radiotherapy (RT).

**1. Introduction** 

future even after curative resection.

patients without an EMM-PV-stent.

**2. Patients and methods** 

**2.1 Patients** 

**in Management of Unresectable**

**or Recurrent Pancreatic Cancer** 


### **Clinical Implications of an Expandable Metallic Mesh Stent for Malignant Portal Vein Stenosis in Management of Unresectable or Recurrent Pancreatic Cancer**

Yoshinori Nio *Nio Surgery Clinic, Japan* 

#### **1. Introduction**

270 Pancreatic Cancer – Clinical Management

Tilley, R.E.; Holscher, T.; Belani, R.; Nieva, J. & Mackman, N. (2008).Tissue factor activity is

Traynelis, S.F.&Trejo, J. (2007). Protease-activated receptor signaling: New roles and regulatorymechanisms. *Cur Opin Hematol*, vol. *14*, pp. 230-235, ISSN: 1065-6251. Trousseau, A. (1865). Plegmasia alba dolens. Lectures on clinical medicine, delivered at the

van den Belt, A.G.; Prins, M.H.; Lensing, A.W., et al. (2000). Fixed dose subcutaneous low

thromboembolism. *Cochrane Database Syst Rev*, CD001100, **ISSN**:1469-493X. Varki, A. (2007). Trousseau's syndrome: multiple definitions and multiple mechanisms.

Verso, M. & Agnelli, G. (2003). Venous thromboembolism associated with long-term use of central venous catheters in cancer patients. *J Clin Oncol*, vol. 21, pp. 3665-3675. Verso, M.; Agnelli, G.; Kamphuisen, P.W., et al. (2008). Risk factors for upper limb deep vein

Ueda, C.; Hirohata, Y.; Kihara, Y.; Nakamura, H.; Abe, B.; Akahane, K.; Okamoto, K.; Itoh,

Wada, H.; Wakita, Y. & Shiku, H. (1995). Tissue factor expression in endothelial-cells in

Wall, J.G.; Weiss, R.B.; Norton, L.; Perloff, M.; Rice, M.A.; Korzun, A.H. & Wood, W.C.

Yamanaka, Y.; Friess, H.; Kobrin, M.S.; Buchler, M.; Beger, H.G. & Korc, M. (1993).

Zacharski, L.R.; Henderson, W.G.; Rickles, F.R., et al. (1981). Effect of warfarin on survival in small cell carcinoma of the lung. *JAMA*, vol. 245, pp. 831-835, ISSN 00987484. Zangari, M.; Fink, L.M.; Elice, F., et al (2009). Thrombotic Events in Patients with Cancer

Zhang, Y.M.; Deng, Y.H.; Luther, T.; Muller, M.; Ziegler, R.; Waldherr, R.; Stern, D.M. &

Zwicker, J.I.; Kos, C.A.; Johnston, K.A.; Liebman, H.A.; Furie, B.C. & Furie, B. (2007). Tissue

health anddisease. *Blood Coagul Fibrin*, *6*, S26-S31, ISSN 09575235.

*Thromb Res*, vol. *122*, pp. 604-609, ISSN: 0049-3848.

Hotel-Dieu, Paris, vol. 5, pp. 281-332.

*Blood*, vol.110, pp. 1723-1729, ISSN 0268-960X.

Gastroenterol, vol. 36, no.12, pp. 848-850.

0002-9343.

565-569, ISSN: 0250-7005.

ISSN 0732-183X.

0049-3848.

1327, ISSN 0021-9738.

*Emerg Med*, vol. 3, no.2, pp. 117-122, ISSN: 1828-0447.

increased in a combined platelet and microparticle sample from cancer patients.

molecular weight heparins versus adjusted dose unfractionated heparin for venous

thrombosis associated with the use of central vein catheter in cancer patients. *Intern* 

H. & Otsuki, M. (2001). Pancreatic cancer complicated bydisseminated intravascular coagulation associated with production of tissue factor. J

(1989). Arterial thrombosis associated with adjuvant chemotherapy for breast carcinoma-a cancer and leukemia group study. *Am J Med*, vol.*87*, pp. 501-504, ISSN:

Coexpression of epidermal growth factor receptor and ligands in human pancreatic cancer is associated with enhanced tumor aggressiveness. *Anticancer Res*, vol.*13*, pp.

Receiving Antiangiogenesis Agents. *JCO* (October 10), vol. 27, no. 29, pp. 4865-4873,

Nawroth, P.P. (1994). Tissue factor controls the balance of angiogenic and antiangiogenic properties of tumour-cells in mice. *J Clin Invest*, vol. *94*, pp. 1320-

factor bearing microparticles is associated with an increased risk of venous thromboembolic events in cancer patients. *Thromb Res*, vol. *120*, pp. S143-S143, ISSN Pancreatic cancer (PC) remains one of the most lethal common malignancies. More than 80% of patients with PC cannot be cured by surgical resection (Li D et al., 2004); the actuarial 5 year survival rate after curative resection is approximately 20% (Crist et al., 1987), and the median survival time (MST) after surgical resection ranges between 11 and 24 months (Nitecki et al., 1995). In other words, most patients develop recurrent disease in the near future even after curative resection.

Advanced or recurrent PC frequently invades the surrounding organs or tissues, and the patients require substantial palliative interventions, especially against biliary obstruction, gastric or duodenal outlet obstruction, and severe abdominal or back pain. In addition, when the portal vein (PV) is invaded and occluded, the patient suffers from various portal hypertension (PH)-associated symptoms and liver dysfunction, including jaundice, ascites, and bleeding tendencies, which disturb chemotherapy (ChT) or radiotherapy (RT).

PC-associated portal obstruction is classified into two categories, intrahepatic obstruction and extrahepatic obstruction. In the case of intrahepatic or hilar PV stenosis, a wall-stent is usually applied (Tsukamoto et al., 2003); however, a wall-stent cannot be used for the extrahepatic PV stenosis, because it may occlude the splenic vein, which joins the extrahepatic PV, leading to serious complications. In patients with extrahepatic PV obstruction, we placed an expandable metallic mesh (EMM) stent into the PV *via* the ileocecal vein following a mini-laparotomy. A total of 14 patients with inoperable or recurrent PC were given an EMM-PV-stent and received subsequent ChT and/or RT, and the treatment results were retrospectively compared with patients without an EMM-PV-stent.

#### **2. Patients and methods**

#### **2.1 Patients**

We treated a total of 97 patients with inoperable or recurrent PC. Of 97 patients, 68 received ChT, 28 received RT using LINAC at 40 - 60Gy (2Gy 20 - 30 times) and 14 were given an

Clinical Implications of an Expandable Metallic Mesh Stent for

evaluate side-effects.

**2.6 Statistics** 

significant.

metastasis.

**3. Treatment results** 

I. Objective response

II. Other clinical benefits

Table 1. Objective response and clinical benefits

occlusion were observed (**Figure 1A,1B and 1C**).

Malignant Portal Vein Stenosis in Management of Unresectable or Recurrent Pancreatic Cancer 273

routine hematological and biochemical examinations, and serum tumor marker assays to

The effects of the therapies were evaluated with respect to the response rate (RR) of the tumor and the survival rate after therapy. The overall survival (OS) was calculated by the Kaplan-Meier method. Multivariate analysis of the maximum likelihood estimates using Cox's proportional hazard model was used to obtain the conditional risk of carcinoma-related death. All analyses were performed using StatView software (SAS Institute Inc., Cary, NC, USA) and a *p*-value less than 0.05 was considered statistically

The effects of the EMM-PV-stent are summarized in **Table 1**. In 4 cases, the EMM-PV-stent was very effective, and the ascites and/or hemorrhagic tendency were improved. Furthermore, ChT and RT were also effective and 3 CRs and 3 PRs were observed: the overall RR (CR + PR) was 42.9%, and SDs were observed in 3 patients. However, in the 2 remaining cases, the EMM-PV-stent was not effective: one patient died of gastrointestinal bleeding and the other died of liver dysfunction and cachexia due to increased liver

 Complete response (CR) 3 Partial response (PR) 3 Stable disease (SD) 3 Progressive disease (PD) 5

Overall response rate (CR+PR) 42.9%(6/14)

The procedure for an EMM-PV-stent is shown in the treatment course of one representative case in **Figure 1 - 4.** The patient had a pancreatic head carcinoma causing obstructive jaundice, and the PC was diagnosed as inoperable because splenic metastasis and PV

 Pain relief 2 Decrease or disappearance of ascites 2 Improvement in hyperglycemia 1 Improvement in thrombocytopenia 1

EMM-PV-stent. All patients were treated in the Department of Surgery, Shimane University School of Medicine.

#### **2.2 Methods**

A Bird Luminex EMM-stent (6 - 12 mm in diameter and 4 - 8 cm in length) was used. The patients received a mini-laparotomy at the ileocecal region and the ileocecal vein was cutdown. Under guidance with image roentgenography, the stenotic portion of the PV was dilated by a balloon catheter and the EMM-stent was placed. In one case, 3 stents were placed, and in the other 13 cases, a single stent was placed. All patients were given heparin continuously at 5,000 U/day for 7 days, and then biaspirin or warfarin for 1 - 3 months.

#### **2.3 Chemotherapy (ChT) and radiotherapy (RT)**

The ChT included oral UFT (uracil and tegafur) at 300 - 400 mg/day daily, oral cyclophosphamide (CPA) at 50 mg/day every other day, and/or gemcitabine (GEM) at 200 - 400 mg/body weekly or biweekly in combination or singly. The regimens administered were decided according to the performance status with fully informed consent of the patients and/or their families. Six patients were given a UC (UFT and CPA) regimen orally in combination with GEM, and the other 7 patients received other regimens: 2 UC, 2 GEM alone, 1 UC + cisplatin + epirubicin, 1 UFT alone, and one GEM + TS-1. However, 1 patient died without receiving any ChT.

RT was performed using LINAC at 40 - 60Gy (2Gy 20 - 30 fractions).

#### **2.4 Evaluation of the objective response (OR) to the therapies**

The OR of the tumor was assessed using roentgenography, computed tomography (CT), or ultrasonography (US) using the following standard criteria: i) a complete response (CR) indicated total disappearance of the tumor for at least 4 weeks, during which time the patient was free of all symptoms related to pancreatic cancer; ii) a partial response (PR) was defined as a 50% or greater reduction in the sum of the products of the two perpendicular diameters of all measurable tumor lesions as compared to their original size for at least 4 weeks. During this time, there must have been no increase of >25% in the size of any single lesion or the appearance of any new lesion; and iii) progressive disease (PD) was defined as a greater than a 25% increase in the sum of the products of the diameters of all measurable lesions, the appearance of any new lesion, or a deterioration in the clinical status that was consistent with disease progression; and iv) stable disease (SD) was indicated for those patients who failed to meet the criteria for a CR, PR or PD, and who remained in the study for at least 8 weeks. The duration of the response was measured from the first day of injection of the agents to the day of the increase in tumor size.

#### **2.5 Evaluation of side-effects**

The National Cancer Institute - Common Toxicity Criteria were used for evaluation of sideeffects (NCI-CTC version 2.0). All of the patients were followed by physical examination, routine hematological and biochemical examinations, and serum tumor marker assays to evaluate side-effects.

#### **2.6 Statistics**

272 Pancreatic Cancer – Clinical Management

EMM-PV-stent. All patients were treated in the Department of Surgery, Shimane University

A Bird Luminex EMM-stent (6 - 12 mm in diameter and 4 - 8 cm in length) was used. The patients received a mini-laparotomy at the ileocecal region and the ileocecal vein was cutdown. Under guidance with image roentgenography, the stenotic portion of the PV was dilated by a balloon catheter and the EMM-stent was placed. In one case, 3 stents were placed, and in the other 13 cases, a single stent was placed. All patients were given heparin continuously at 5,000 U/day for 7 days, and then biaspirin or warfarin for 1 - 3

The ChT included oral UFT (uracil and tegafur) at 300 - 400 mg/day daily, oral cyclophosphamide (CPA) at 50 mg/day every other day, and/or gemcitabine (GEM) at 200 - 400 mg/body weekly or biweekly in combination or singly. The regimens administered were decided according to the performance status with fully informed consent of the patients and/or their families. Six patients were given a UC (UFT and CPA) regimen orally in combination with GEM, and the other 7 patients received other regimens: 2 UC, 2 GEM alone, 1 UC + cisplatin + epirubicin, 1 UFT alone, and one GEM + TS-1. However, 1 patient

The OR of the tumor was assessed using roentgenography, computed tomography (CT), or ultrasonography (US) using the following standard criteria: i) a complete response (CR) indicated total disappearance of the tumor for at least 4 weeks, during which time the patient was free of all symptoms related to pancreatic cancer; ii) a partial response (PR) was defined as a 50% or greater reduction in the sum of the products of the two perpendicular diameters of all measurable tumor lesions as compared to their original size for at least 4 weeks. During this time, there must have been no increase of >25% in the size of any single lesion or the appearance of any new lesion; and iii) progressive disease (PD) was defined as a greater than a 25% increase in the sum of the products of the diameters of all measurable lesions, the appearance of any new lesion, or a deterioration in the clinical status that was consistent with disease progression; and iv) stable disease (SD) was indicated for those patients who failed to meet the criteria for a CR, PR or PD, and who remained in the study for at least 8 weeks. The duration of the response was measured from the first day of

The National Cancer Institute - Common Toxicity Criteria were used for evaluation of sideeffects (NCI-CTC version 2.0). All of the patients were followed by physical examination,

School of Medicine.

**2.2 Methods** 

months.

**2.3 Chemotherapy (ChT) and radiotherapy (RT)** 

RT was performed using LINAC at 40 - 60Gy (2Gy 20 - 30 fractions).

**2.4 Evaluation of the objective response (OR) to the therapies** 

injection of the agents to the day of the increase in tumor size.

died without receiving any ChT.

**2.5 Evaluation of side-effects** 

The effects of the therapies were evaluated with respect to the response rate (RR) of the tumor and the survival rate after therapy. The overall survival (OS) was calculated by the Kaplan-Meier method. Multivariate analysis of the maximum likelihood estimates using Cox's proportional hazard model was used to obtain the conditional risk of carcinoma-related death. All analyses were performed using StatView software (SAS Institute Inc., Cary, NC, USA) and a *p*-value less than 0.05 was considered statistically significant.

#### **3. Treatment results**

The effects of the EMM-PV-stent are summarized in **Table 1**. In 4 cases, the EMM-PV-stent was very effective, and the ascites and/or hemorrhagic tendency were improved. Furthermore, ChT and RT were also effective and 3 CRs and 3 PRs were observed: the overall RR (CR + PR) was 42.9%, and SDs were observed in 3 patients. However, in the 2 remaining cases, the EMM-PV-stent was not effective: one patient died of gastrointestinal bleeding and the other died of liver dysfunction and cachexia due to increased liver metastasis.


Table 1. Objective response and clinical benefits

The procedure for an EMM-PV-stent is shown in the treatment course of one representative case in **Figure 1 - 4.** The patient had a pancreatic head carcinoma causing obstructive jaundice, and the PC was diagnosed as inoperable because splenic metastasis and PV occlusion were observed (**Figure 1A,1B and 1C**).

Clinical Implications of an Expandable Metallic Mesh Stent for

A. An expandable metallic mesh stent (Bird Luminex)

Fig. 2. Procedure of portal stent

B. Arrow indicates portal stenosis

C. Balloon dilatation D. Insertion of three stents E. Portography after portal stent

Malignant Portal Vein Stenosis in Management of Unresectable or Recurrent Pancreatic Cancer 275


The patient underwent a laparotomy, but peritoneal dissemination and malignant ascites were also seen. In order to release the obstruction of the bile duct and duodenum, the patient received bypass surgeries with a cholecysto-jejunostomy and a gastro-jejunostomy. In addition, she received placement of an EMM-PV-stent with three metallic stents, as shown in **Figure 2A,2B,2C,2D and 2E**.

After surgery, she was treated with ChT consisting of oral UFT plus CPA with intravenous GEM, and RT to a total of 50 Gy. The tumor responded well to the therapies, and the splenic metastasis and primary lesion disappeared completely 4 months after the surgery (**Figure 3**). Finally, she died of malignant ascites 21 months after the initiation of treatment. **Figure 4**  summarizes the treatment course.

Fig. 2. Procedure of portal stent


274 Pancreatic Cancer – Clinical Management

Fig. 1. A representative case with portal stenosis

B. CT. Circle indicates a pancreatic head cancer

shown in **Figure 2A,2B,2C,2D and 2E**.

summarizes the treatment course.

C. Portography. Arrow indicates extrahepatic portal stenosis

A. Percutaneous transhepatic cholangiography. Arrows indicate stenosis.

The patient underwent a laparotomy, but peritoneal dissemination and malignant ascites were also seen. In order to release the obstruction of the bile duct and duodenum, the patient received bypass surgeries with a cholecysto-jejunostomy and a gastro-jejunostomy. In addition, she received placement of an EMM-PV-stent with three metallic stents, as

After surgery, she was treated with ChT consisting of oral UFT plus CPA with intravenous GEM, and RT to a total of 50 Gy. The tumor responded well to the therapies, and the splenic metastasis and primary lesion disappeared completely 4 months after the surgery (**Figure 3**). Finally, she died of malignant ascites 21 months after the initiation of treatment. **Figure 4** 


Clinical Implications of an Expandable Metallic Mesh Stent for

Circles indicate pancreas head and portal vein.

Fig. 4. Treatment course

Fig. 5. Comparative survival curves.

shown in **Figure 5**.

Malignant Portal Vein Stenosis in Management of Unresectable or Recurrent Pancreatic Cancer 277

The survival curves after the initiation of treatment and placement of the EMM-PV-stent are

Fig. 3. Comparative CT before and after PV-stent A. Before PV-stent B. Two months after PV-stent C. Six months after PV-stent

Circles indicate pancreas head and portal vein.

276 Pancreatic Cancer – Clinical Management

Fig. 3. Comparative CT before and after PV-stent

A. Before PV-stent

B. Two months after PV-stent C. Six months after PV-stent

Fig. 4. Treatment course

The survival curves after the initiation of treatment and placement of the EMM-PV-stent are shown in **Figure 5**.

Fig. 5. Comparative survival curves.

Clinical Implications of an Expandable Metallic Mesh Stent for

too small to draw any conclusive interpretations.

Malignant Portal Vein Stenosis in Management of Unresectable or Recurrent Pancreatic Cancer 279

stent group vs. 16.2% for the control group, and the MSTs were 13.0 vs. 4.0 months, respectively (p=0.0006). These RR and survival rates are high and long for PC, as compared with previous reports, in which the RR of a combination regimen with 5-FU, GEM and their combinations ranged between 5% and 25%, while the MST ranged between 4 and 10 months (Van Cutsem et al., 2004; Okusaka & Kosuge, 2004; Pasetto et al., 2004; Heinemann, 2002; Novarino et al., 2004; Berlin et al., 2002), although the sample size of the present study was

The present study also demonstrated that an EMM-PV-stent was not a significant prognostic factor, although the survival rate was significantly higher in the EMM-PV-stent group than the control group. However, ChT and RT were significant prognostic factors by multivariate analysis (p<0.001 and 0.0120, respectively). These results indicate that the EMM-PV-stent itself does not improve prognosis, but that ChT and RT may play important roles in regressing the tumor, and that an EMM-PV-stent helps to improve the efficacy of ChT and RT in patients with PH-associated complications that cause liver dysfunction and pancytopenia, especially thrombocytopenia and leucocytopenia (due to hypersplenism), and gastrointestinal bleeding. However, in order to achieve clinically beneficial treatment results, ChT and RT at a sufficient dose to regress the tumor are very important in patients with PH, as a dose of ChT or RT sufficient to regress the tumor cannot be administered. Since liver dysfunction and pancytopenia can easily be exacerbated by ChT and RT, there are major difficulties for the administration of a dose of ChT or RT sufficient to induce regression of PC. Therefore, placement of a PV-stent improves the efficacy of these adjuvant therapies by removing any PH-associated co-morbidities. Furthermore, in the present study, pain and other PH-

associated symptoms such as ascites and hyperglycemia were also improved.

clinical benefit response (CBR), and a 10.7 month MST (Nio et al., 2005.).

(Haga et al., 1999; Endo et al., 1999; Nio et al., 2007).

We administered UFT, CPA, and GEM as the ChT regimen in most patients. These regimens were unique to our team. GEM now plays a core role in ChT for advanced PC, and various combination regimens have been attempted. The present study used a low dose of GEM at 200 - 400 mg (almost equivalent to 150 - 300 mg/m2), although most studies used standard doses of GEM at 800 - 1000 mg/m2. However, this low dose was used in order to reduce the side-effects in combination with RT because our previous preliminary study on RT in combination with GEM at standard doses for inoperable PC resulted in serious myelosuppression, especially thrombocytopenia. Our previous study using this combination regimen with UFT, CPA and GEM at low doses resulted in a 27% RR and 23%

Here, we oral UFT instead of *iv* 5-fluorouracil (5-FU). In Japan, UFT has been used as a substitute for *iv* 5-FU for various malignancies such as gastric, colorectal, lung and breast cancer, and several studies in other countries have demonstrated that UFT was as effective as *iv* 5-FU, with a better toxicity profile (Sulkes et al., 1998; Van Cutsem & Peeters, 2000). Furthermore, the present ChT combined CPA in addition to GEM and UFT because previous reports including ours demonstrated that CPA augments the antitumor activity of fluoropyrimidines by modulating the activity of various enzymes, which are associated with pyrimidine metabolism, such as augmenting ribonucleotide reductase, inducing thymidine phosphorylase and inhibiting intratumoral activity of dihydropyrimidine dehydrogenase

As discussed above, the treatment results of advanced or recurrent PC are not satisfactory, and the EMM-stent itself has no effect to regress the tumor; it only improves the PH-

The survival curve of the EMM-PV-stent group was significantly higher than that of the remaining patients (control group, n=83) (p=0.0006 by Cox-Mantel): the 6 months and 1-year survival rates were 85.7% and 54.5% for the EMM-PV-stent group vs. 32.0% and 16.2% for the control group, respectively, while the MSTs were 13.0 vs. 4.0 months, respectively (**Table 2**).


Table 2. Comparative survival between the control and EMM-PV-stent groups

The implications of EMM-PV-stenting in the treatment results were analyzed by multivariate analysis (**Table 3**), but this demonstrated that an EMM-PV-stent was not a significant factor, while RT and ChT were significant prognostic factors. This suggests that an EMM-PV-stent itself does not improve the patients' survival, but it is beneficial for improving the efficacy of ChT or RT by reducing the risk of liver failure or hemorrhagic tendency.


Table 3. Multivariate analysis by Cox's proportional hazard risk model

#### **4. Discussion**

In the present study, we used an EMM-stent as the PV-stent, although in general, for a vascular stent, a wall stent is used. The reason for using an EMM-stent is that a wall stent occludes the splenic vein, which is joined to the PV, and may lead to serious complications. In intrahepatic PV stenosis cases, a wall stent can be used, but pancreatic cancer usually causes extrahepatic PV stenosis. Furthermore, in intrahepatic PV stenosis, a percutaneous transhepatic procedure is usually applied to place the wall stent into the PV. However, we placed an EMM-stent into the PV via the ileocecal vein using laparotomy because it is very difficult to define the occlusive site from the distal PV under image roentgenography, and a percutaneous transhepatic procedure carries various risks such as intra-abdominal bleeding and perforation, which can be more easily managed by laparotomy.

One of the disadvantages of placing an EMM-stent is that the tumor frequently invades through the mesh into the lumen, resulting in re-obstruction. Accordingly, RT and/or ChT are essential to inhibit tumor invasion into the lumen.

The present study included 14 patients who received placement of an EMM-PV-stent and adjuvant ChT or RT, and the RR was 43%: the 1-year survival rate was 54.5% for the EMPV-

The survival curve of the EMM-PV-stent group was significantly higher than that of the remaining patients (control group, n=83) (p=0.0006 by Cox-Mantel): the 6 months and 1-year survival rates were 85.7% and 54.5% for the EMM-PV-stent group vs. 32.0% and 16.2% for the control group, respectively, while the MSTs were 13.0 vs. 4.0 months, respectively

Control 32.0 16.2 5.9 0.0006 EMM-PV-stent 85.7 54.5 12.7

The implications of EMM-PV-stenting in the treatment results were analyzed by multivariate analysis (**Table 3**), but this demonstrated that an EMM-PV-stent was not a significant factor, while RT and ChT were significant prognostic factors. This suggests that an EMM-PV-stent itself does not improve the patients' survival, but it is beneficial for improving the efficacy of ChT or RT by reducing the risk of liver failure or hemorrhagic

Table 2. Comparative survival between the control and EMM-PV-stent groups

Variables Conditional risk ratio

Table 3. Multivariate analysis by Cox's proportional hazard risk model

and perforation, which can be more easily managed by laparotomy.

are essential to inhibit tumor invasion into the lumen.

Survival rate (%) Median survival Group 6-month 1-year (months) *p*-value

Age 1.000 (0.978 – 1.022) 0.9718 Palliative surgery 0.830 (0.485 – 1.423) 0.4986 PV-stent 0.537 (0.195 – 1.481) 0.2298 Chemotherapy 0.349 (0.206 – 0.590) <0.001 Radiotherapy 0.427 (0.220 – 0.830) 0.012

In the present study, we used an EMM-stent as the PV-stent, although in general, for a vascular stent, a wall stent is used. The reason for using an EMM-stent is that a wall stent occludes the splenic vein, which is joined to the PV, and may lead to serious complications. In intrahepatic PV stenosis cases, a wall stent can be used, but pancreatic cancer usually causes extrahepatic PV stenosis. Furthermore, in intrahepatic PV stenosis, a percutaneous transhepatic procedure is usually applied to place the wall stent into the PV. However, we placed an EMM-stent into the PV via the ileocecal vein using laparotomy because it is very difficult to define the occlusive site from the distal PV under image roentgenography, and a percutaneous transhepatic procedure carries various risks such as intra-abdominal bleeding

One of the disadvantages of placing an EMM-stent is that the tumor frequently invades through the mesh into the lumen, resulting in re-obstruction. Accordingly, RT and/or ChT

The present study included 14 patients who received placement of an EMM-PV-stent and adjuvant ChT or RT, and the RR was 43%: the 1-year survival rate was 54.5% for the EMPV-

*<sup>p</sup>*-value (95% confidence limit)

(**Table 2**).

tendency.

**4. Discussion** 

stent group vs. 16.2% for the control group, and the MSTs were 13.0 vs. 4.0 months, respectively (p=0.0006). These RR and survival rates are high and long for PC, as compared with previous reports, in which the RR of a combination regimen with 5-FU, GEM and their combinations ranged between 5% and 25%, while the MST ranged between 4 and 10 months (Van Cutsem et al., 2004; Okusaka & Kosuge, 2004; Pasetto et al., 2004; Heinemann, 2002; Novarino et al., 2004; Berlin et al., 2002), although the sample size of the present study was too small to draw any conclusive interpretations.

The present study also demonstrated that an EMM-PV-stent was not a significant prognostic factor, although the survival rate was significantly higher in the EMM-PV-stent group than the control group. However, ChT and RT were significant prognostic factors by multivariate analysis (p<0.001 and 0.0120, respectively). These results indicate that the EMM-PV-stent itself does not improve prognosis, but that ChT and RT may play important roles in regressing the tumor, and that an EMM-PV-stent helps to improve the efficacy of ChT and RT in patients with PH-associated complications that cause liver dysfunction and pancytopenia, especially thrombocytopenia and leucocytopenia (due to hypersplenism), and gastrointestinal bleeding. However, in order to achieve clinically beneficial treatment results, ChT and RT at a sufficient dose to regress the tumor are very important in patients with PH, as a dose of ChT or RT sufficient to regress the tumor cannot be administered. Since liver dysfunction and pancytopenia can easily be exacerbated by ChT and RT, there are major difficulties for the administration of a dose of ChT or RT sufficient to induce regression of PC. Therefore, placement of a PV-stent improves the efficacy of these adjuvant therapies by removing any PH-associated co-morbidities. Furthermore, in the present study, pain and other PHassociated symptoms such as ascites and hyperglycemia were also improved.

We administered UFT, CPA, and GEM as the ChT regimen in most patients. These regimens were unique to our team. GEM now plays a core role in ChT for advanced PC, and various combination regimens have been attempted. The present study used a low dose of GEM at 200 - 400 mg (almost equivalent to 150 - 300 mg/m2), although most studies used standard doses of GEM at 800 - 1000 mg/m2. However, this low dose was used in order to reduce the side-effects in combination with RT because our previous preliminary study on RT in combination with GEM at standard doses for inoperable PC resulted in serious myelosuppression, especially thrombocytopenia. Our previous study using this combination regimen with UFT, CPA and GEM at low doses resulted in a 27% RR and 23% clinical benefit response (CBR), and a 10.7 month MST (Nio et al., 2005.).

Here, we oral UFT instead of *iv* 5-fluorouracil (5-FU). In Japan, UFT has been used as a substitute for *iv* 5-FU for various malignancies such as gastric, colorectal, lung and breast cancer, and several studies in other countries have demonstrated that UFT was as effective as *iv* 5-FU, with a better toxicity profile (Sulkes et al., 1998; Van Cutsem & Peeters, 2000). Furthermore, the present ChT combined CPA in addition to GEM and UFT because previous reports including ours demonstrated that CPA augments the antitumor activity of fluoropyrimidines by modulating the activity of various enzymes, which are associated with pyrimidine metabolism, such as augmenting ribonucleotide reductase, inducing thymidine phosphorylase and inhibiting intratumoral activity of dihydropyrimidine dehydrogenase (Haga et al., 1999; Endo et al., 1999; Nio et al., 2007).

As discussed above, the treatment results of advanced or recurrent PC are not satisfactory, and the EMM-stent itself has no effect to regress the tumor; it only improves the PH-

**17**

*USA* 

**Pancreatic Neuroendocrine Tumors:**

Neuroendocrine tumors comprise a spectrum of slow growing neoplasm, characterized by storage and secretion of variable peptides and neuroamines (Massironi et al., 2008). Pancreatic neuroendocrine tumors (PNET) are relatively rare, with an estimated incidence of less than 1 per 1000,000 individuals (Metz and Jensen, 2008). A recent review of surveillance epidemiology and end results (SEER) (1950-2007) database reported the frequency of PNET to be around 7% among all identified neuroendocrine tumors (Lawrence et al., 2011a). Furthermore, they comprise 1-2% of all pancreatic neoplasms (Metz and Jensen, 2008). However, the incidence is considered to be increasing , perhaps in part due to improved diagnostic capabilities. Median overall survival in PNET ranges from more than 10 years in localized disease to approximately 2 years in metastatic disease (Yao et al., 2008a). Recently, considerable headway has been made in the realm of therapeutics. Therefore, it is imperative that oncologists today have a heightened awareness of this disease entity in

PNETs have also been referred to as pancreatic endocrine or islet cell tumors. It is important to note that carcinoid and PNETs, although exhibiting identical characteristics histologically, should be considered separately. It is increasingly clear that these two tumor types are different in their biology and response to therapy. The clinical presentation of PNET is extremely variable which depends on the originating cell type and whether there is secretion of active hormones. Majority of patients remain asymptomatic, but a significant proportion present with clinical symptoms and hepatic metastases at the time of diagnosis

Most cases of PNET occur sporadically, however, approximately 10% of cases may be associated with multiple endocrine neoplasia type 1 (MEN1). MEN1 is an autosomal dominant syndrome associated with mutations in the tumor suppressor gene *menin* and characterized by multiple neuroendocrine tumors in the pancreas, parathyroid and pituitary glands (Agarwal et al., 2004). PNETs have also been associated with MEN2, Von Hippel-Lindau disease, Tuberous sclerosis and Neurofibromatosis (Kulke et al., 2011). Although the incidence of these inherited syndromes is low, it may be important to consider these

**1. Introduction** 

order to provide effective care.

(Modlin et al., 2008).

**2. Diagnosis, staging and classification** 

syndromes in the diagnostic work up of patients with PNETs.

**Emerging Management Paradigm**

Syed F. Zafar and Bassel El-Rayes

*Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta,* 

associated symptoms. Recently, various new agents have been introduced to the ChT for advanced PC, including TS-1, capecitabine, oxaliplatin, irinotecan, erlotinib, and taxanes, and these should help to improve the poor outcomes for patients with PC.

#### **5. Conclusion**

The placement of an EMM-PV-stent is very beneficial for managing PH-associated symptoms, as well as improving the efficacy of ChT and RT in pancreatic cancer with malignant PV stenosis or obstruction.

#### **6. References**


### **Pancreatic Neuroendocrine Tumors: Emerging Management Paradigm**

Syed F. Zafar and Bassel El-Rayes *Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, USA* 

#### **1. Introduction**

280 Pancreatic Cancer – Clinical Management

associated symptoms. Recently, various new agents have been introduced to the ChT for advanced PC, including TS-1, capecitabine, oxaliplatin, irinotecan, erlotinib, and taxanes,

The placement of an EMM-PV-stent is very beneficial for managing PH-associated symptoms, as well as improving the efficacy of ChT and RT in pancreatic cancer with

Berlin JD, et al.(2002). Phase III study of gemcitabine in combination with fluorouracil

Endo M, et al.(1999). Induction of thymidine phosphorylase expression and enhancement of

Haga S, et al.(1999). Antitumor efficacy of combination chemotherapy with UFT and

Heinemann V.(2002). Gemcitabine-based combination treatment of pancreatic cancer. Semin

NCI-CTC version 2.0. Common toxicity criteria, Notice of modifications. (http://ctep.info.

Nio Y, et al.(2005). Phase II study on low-dose gemcitabine plus oral chemotherapy with

Nio Y, et al.(2007). Cyclophosphamide augments the anti-tumor efficacy of uracil and

Nitecki SS, et al.(1995). Long-term survival after resection for ductal adenocarcinoma of the

Novarino A, et al.(2004). Phase II study of cisplatin. gemcitabine and 5-fluorouracil in

Okusaka T and Kosuge T.(2004). Systemic chemotherapy for pancreatic cancer. Pancreas *28*:

Pasetto LM, et al.(2004). Old and new drugs in systemic therapy of pancreatic cancer. Crit

Sulkes A, et al.(1998). Uracil-tegafur: An oral fluoropyrimidine active in colorectal cancer. J

Tsukamoto T, et al.(2003). Percutaneous transhepatic metallic stent placement for malignant

Van Cutsem E and Peeters M.(2000). Oral fluoropyrimidines in colorectal cancer. Semin

Van Cutsem E, et al.(2004). Systemic treatment of pancreatic cancer. Eur J Gastroenterol

recurrent and advanced pancreatic cancer. Oncol Rep *14*: 401-408.

pancreas. Is it really improving? Ann Surg *221*: 59-66.

advanced pancreatic cancer. Ann Oncol *15*: 474-477.

portal vein stenosis. Hepatogastroenterol *50*: 453-455.

Cooperative Oncology Group trial E2297. J Clin Oncol *20*: 3270-3275. Crist DW, et al.(1987). Improved hospital morbidity, mortality, and survival after the

Whipple procedure. Ann Surg 206: 358-365.

Anticancer Res *19* (*3A*): 1791-1796.

Rev Oncol Hematol *49*: 135-151.

Clin Oncol *16*: 3461-3475.

Oncol *27* (*Suppl 10*): 91-95.

Hepatol *16*: 265-27.

Li D, et al.(2004). Pancreatic Cancer. Lancet 363: 1049-1057.

Oncol *29*(*1 Suppl 3*): 25-35.

nih.gov/).

301-304.

mammary tumor models. Int J Cancer *83*: 127-134.

versus gemcitabine alone in patients with advanced pancreatic carcinoma: Eastern

efficacy of capecitabine or 5'-deoxy-5-fluorouridine by cyclophosphamide in

cyclophosphamide against human breast cancer xenografts in nude mice.

uracil-tegafur and cyclophosphamide in combination with radiotherapy against

tegafur by inhibiting dihydropyrimidine dehydrogenase. Oncol Rep 17(1): 153-159.

and these should help to improve the poor outcomes for patients with PC.

**5. Conclusion** 

**6. References** 

malignant PV stenosis or obstruction.

Neuroendocrine tumors comprise a spectrum of slow growing neoplasm, characterized by storage and secretion of variable peptides and neuroamines (Massironi et al., 2008). Pancreatic neuroendocrine tumors (PNET) are relatively rare, with an estimated incidence of less than 1 per 1000,000 individuals (Metz and Jensen, 2008). A recent review of surveillance epidemiology and end results (SEER) (1950-2007) database reported the frequency of PNET to be around 7% among all identified neuroendocrine tumors (Lawrence et al., 2011a). Furthermore, they comprise 1-2% of all pancreatic neoplasms (Metz and Jensen, 2008). However, the incidence is considered to be increasing , perhaps in part due to improved diagnostic capabilities. Median overall survival in PNET ranges from more than 10 years in localized disease to approximately 2 years in metastatic disease (Yao et al., 2008a). Recently, considerable headway has been made in the realm of therapeutics. Therefore, it is imperative that oncologists today have a heightened awareness of this disease entity in order to provide effective care.

#### **2. Diagnosis, staging and classification**

PNETs have also been referred to as pancreatic endocrine or islet cell tumors. It is important to note that carcinoid and PNETs, although exhibiting identical characteristics histologically, should be considered separately. It is increasingly clear that these two tumor types are different in their biology and response to therapy. The clinical presentation of PNET is extremely variable which depends on the originating cell type and whether there is secretion of active hormones. Majority of patients remain asymptomatic, but a significant proportion present with clinical symptoms and hepatic metastases at the time of diagnosis (Modlin et al., 2008).

Most cases of PNET occur sporadically, however, approximately 10% of cases may be associated with multiple endocrine neoplasia type 1 (MEN1). MEN1 is an autosomal dominant syndrome associated with mutations in the tumor suppressor gene *menin* and characterized by multiple neuroendocrine tumors in the pancreas, parathyroid and pituitary glands (Agarwal et al., 2004). PNETs have also been associated with MEN2, Von Hippel-Lindau disease, Tuberous sclerosis and Neurofibromatosis (Kulke et al., 2011). Although the incidence of these inherited syndromes is low, it may be important to consider these syndromes in the diagnostic work up of patients with PNETs.

Pancreatic Neuroendocrine Tumors: Emerging Management Paradigm 283

Several organizations, including the European Neuroendocrine Tumor Society (ENETS), and the American Joint Committee on Cancer (AJCC), have proposed staging systems for neuroendocrine tumors using the TNM notation (Edge and Compton, 2010). Although these two staging systems are similar for tumor arising in the luminal gut, they differ for earlier stage PNETs. The ENETS system incorporates tumor diameter in its assessment for T stage, whereas the AJCC incorporates factors determining resectability. However, both systems are nearly identical in defining stage IV disease. Because the AJCC system has been widely accepted and adopted in North America, this is preferably and more commonly used for

When functional, PNETs can be characterized by the type of hormone secreted leading to a specific clinical manifestation (Table 2). Specific details about some common tumors, based

Insulinomas are the most common PNET, comprising 30-40% of these tumors. Overall, they remain a rare entity with an incidence of approximately 0.4/100,000 patient years (Mathur et al., 2009).Classically, they present with "Whipple's Triad": a combination of symptoms of hypoglycemia, inappropriately high insulin level and associated blood glucose levels of <50 mg/dl with relief of symptoms on administration of glucose (Whipple and Frantz, 1935). In a 25-year Massachusetts General Hospital experience with insulinoma, the most common clinical symptoms in this series of 61 patients were confusion, visual disturbances and diaphoresis (Nikfarjam et al., 2008). Biochemical diagnosis requires confirmation of inappropriately elevated insulin, C-peptide and proinsulin levels in the presence of low serum glucose. Biochemical diagnosis is usually followed by radiological (CT or MRI) or endoscopic diagnosis. At early stages, the hypoglycemia can be managed with diazoxide and somatostatin analogues should be used cautiously as it can worsen hypoglycemia (Goode et al., 1986). Everolimus, an mTOR inhibitor, has been reported to be efficacious in

Gastrinoma and Zollinger-Ellison syndrome are suspected in a patient with recurrent or refractory peptic ulcer disease and unexplained secretory diarrhea. In such patients, fasting gastrin level >100 pg/ml is highly suspicious of this diagnosis (Jensen, 1996). Other common causes of gastric hypersecretion should be excluded, which includes treatment with proton pump inhibitors (PPI), atrophic gastritis and pernicious anemia. Approximately 25% of patients will present with diarrhea as primary manifestation without peptic ulcer disease (Perry and Vinik, 1995). Gastrinomas have a strong predilection for a "gastrinoma triangle" that includes the pancreatic head, first two-thirds of the duodenum and the porta hepatis (Howard et al., 1990). A significant proportion of gastrinomas are malignant, with up to onethird of patients presenting with liver metastases (Mittendorf et al., 2006). PPI therapy is highly effective for initial symptom management and somatostatin analogues have also shown effectiveness in controlling symptoms and concomitantly offering tumor stabilization

classification by tumor stage.

**3. Clinical manifestation** 

**3.1 Insulinoma** 

**3.2 Gastrinoma** 

on the presentation are discussed below.

cases of refractory hypoglycemia (Kulke et al., 2009a).

(Lambers et al., 1984; Shojamanesh et al., 2002).

It is important to discern the diagnosis of PNET from the more common pancreatic adenocarcinoma. Grossly, PNETs are solitary well demarcated, tan soft tumors which can have a nodular appearance, when they exhibit fibrosis. The histological criteria for diagnosis are well established. These tumors can range from well differentiated ,low grade tumors to more poorly differentiated high grade types. Well differentiated tumors can exhibit various histological patterns, ranging from a common solid nesting, trabecular to tubular-acinar and mixed patterns .The cells are characterized by round to ovoid shape, with eosinophilic granular cytoplasm and prominent nucleoli. Unusual types can exhibit a spindle cell morphology which is referred to as the "rhabdoid" type. High grade malignancies with high mitotic rate usually encompass large cell and small cell carcinomas (Asa, 2011).

The usually employed classification schemes, although inconsistent in their criteria, reflect a basic separation between more indolent, well differentiated and aggressive poorly differentiated ones. While a number of histologic classification systems have been proposed for PNET, tumors with high mitotic count (>20/10 high power field) or a Ki-67 proliferation rate of >20%, generally represent highly aggressive malignancies and should be evaluated apart from the more classic well differentiated tumors such as classic carcinoid or islet cell type. These high grade malignancies are generally treated according to small cell carcinoma guidelines (Asa, 2011; Kloppel et al., 2004; Rindi and Kloppel, 2004). Table 1 outlines the histologic classification of neuroendocrine tumors.


ENETS: European Neuroendocrine Tumor Society; WHO: World Health Organization.

Table 1. Histologic classification of Neuroendocrine Tumors.

Several organizations, including the European Neuroendocrine Tumor Society (ENETS), and the American Joint Committee on Cancer (AJCC), have proposed staging systems for neuroendocrine tumors using the TNM notation (Edge and Compton, 2010). Although these two staging systems are similar for tumor arising in the luminal gut, they differ for earlier stage PNETs. The ENETS system incorporates tumor diameter in its assessment for T stage, whereas the AJCC incorporates factors determining resectability. However, both systems are nearly identical in defining stage IV disease. Because the AJCC system has been widely accepted and adopted in North America, this is preferably and more commonly used for classification by tumor stage.

#### **3. Clinical manifestation**

When functional, PNETs can be characterized by the type of hormone secreted leading to a specific clinical manifestation (Table 2). Specific details about some common tumors, based on the presentation are discussed below.

#### **3.1 Insulinoma**

282 Pancreatic Cancer – Clinical Management

It is important to discern the diagnosis of PNET from the more common pancreatic adenocarcinoma. Grossly, PNETs are solitary well demarcated, tan soft tumors which can have a nodular appearance, when they exhibit fibrosis. The histological criteria for diagnosis are well established. These tumors can range from well differentiated ,low grade tumors to more poorly differentiated high grade types. Well differentiated tumors can exhibit various histological patterns, ranging from a common solid nesting, trabecular to tubular-acinar and mixed patterns .The cells are characterized by round to ovoid shape, with eosinophilic granular cytoplasm and prominent nucleoli. Unusual types can exhibit a spindle cell morphology which is referred to as the "rhabdoid" type. High grade malignancies with high

The usually employed classification schemes, although inconsistent in their criteria, reflect a basic separation between more indolent, well differentiated and aggressive poorly differentiated ones. While a number of histologic classification systems have been proposed for PNET, tumors with high mitotic count (>20/10 high power field) or a Ki-67 proliferation rate of >20%, generally represent highly aggressive malignancies and should be evaluated apart from the more classic well differentiated tumors such as classic carcinoid or islet cell type. These high grade malignancies are generally treated according to small cell carcinoma guidelines (Asa, 2011; Kloppel et al., 2004; Rindi and Kloppel, 2004). Table 1 outlines the

index (%)

<2 Without local invasion

3-20 With or without

>20 % Small cell or large

cell carcinoma, often widely invasive or metastatic.

(angioinvasion or perineural invasion). Traditionally include carcinoid and PNETs

gross local invasion or metastases. Traditionally include carcinoid, atypical carcinoid and some PNET

General features ENETS, WHO

classification

Neuroendocrine tumor grade 1, WHO type 1.1 (pancreatic)

Neuroendocrine tumor, Grade 2, WHO type 1.2

Neuroendocrine Carcinoma grade 3 (small cell or large cell), WHO type 3 (pancreatic)

and 2 (pancreatic)

mitotic rate usually encompass large cell and small cell carcinomas (Asa, 2011).

Grade Ki-67

Low Grade (G1)

Intermediate grade (G2)

High Grade (G3)

Table 1. Histologic classification of Neuroendocrine Tumors.

ENETS: European Neuroendocrine Tumor Society; WHO: World Health Organization.

histologic classification of neuroendocrine tumors.

count

<2 per 10 HPF

2-20 per 10 HPF

>20 per 10 HPF

Differentiation Mitotic

Well

Poorly

Differentiated

differentiated

Insulinomas are the most common PNET, comprising 30-40% of these tumors. Overall, they remain a rare entity with an incidence of approximately 0.4/100,000 patient years (Mathur et al., 2009).Classically, they present with "Whipple's Triad": a combination of symptoms of hypoglycemia, inappropriately high insulin level and associated blood glucose levels of <50 mg/dl with relief of symptoms on administration of glucose (Whipple and Frantz, 1935). In a 25-year Massachusetts General Hospital experience with insulinoma, the most common clinical symptoms in this series of 61 patients were confusion, visual disturbances and diaphoresis (Nikfarjam et al., 2008). Biochemical diagnosis requires confirmation of inappropriately elevated insulin, C-peptide and proinsulin levels in the presence of low serum glucose. Biochemical diagnosis is usually followed by radiological (CT or MRI) or endoscopic diagnosis. At early stages, the hypoglycemia can be managed with diazoxide and somatostatin analogues should be used cautiously as it can worsen hypoglycemia (Goode et al., 1986). Everolimus, an mTOR inhibitor, has been reported to be efficacious in cases of refractory hypoglycemia (Kulke et al., 2009a).

#### **3.2 Gastrinoma**

Gastrinoma and Zollinger-Ellison syndrome are suspected in a patient with recurrent or refractory peptic ulcer disease and unexplained secretory diarrhea. In such patients, fasting gastrin level >100 pg/ml is highly suspicious of this diagnosis (Jensen, 1996). Other common causes of gastric hypersecretion should be excluded, which includes treatment with proton pump inhibitors (PPI), atrophic gastritis and pernicious anemia. Approximately 25% of patients will present with diarrhea as primary manifestation without peptic ulcer disease (Perry and Vinik, 1995). Gastrinomas have a strong predilection for a "gastrinoma triangle" that includes the pancreatic head, first two-thirds of the duodenum and the porta hepatis (Howard et al., 1990). A significant proportion of gastrinomas are malignant, with up to onethird of patients presenting with liver metastases (Mittendorf et al., 2006). PPI therapy is highly effective for initial symptom management and somatostatin analogues have also shown effectiveness in controlling symptoms and concomitantly offering tumor stabilization (Lambers et al., 1984; Shojamanesh et al., 2002).

Pancreatic Neuroendocrine Tumors: Emerging Management Paradigm 285

As majority of PNETs are non-functional, hormonal assays cannot be used for clinical assessment. Hence, serum chromogranin A (CgA) has come to represent a common denominator peptide with the putative ability to serve as a marker of disease activity, in both functional and non functional tumors. Granins are found as major components of the soluble core of dense secretory granules in neuroendocrine cells and are secreted in a physiologically regulated manner (Kim et al., 2001). Eight members have been identified including CgA, chromogranin B, chromogranin C, SgIII, SgIV, SgV, SgVI and VGF nerve growth factorinducible. Granins have been proposed as playing important roles in secretory granule formation and development. CgA was the initial member identified, and originally detected in the chromaffin granules of the adrenal medulla (Blaschko et al., 1967). Although the definitive function of CgA remains unclear, CgA derived peptides mediate a number of biologic functions including regulation of parathyroid hormone secretion, carbohydrate metabolism,

Serum concentrations of CgA may decrease in patients responding to somatostatin analogs or other therapies. CgA should be used with caution as a marker of disease activity in patients treated with somatostatin analogs, because these agents significantly reduce plasma CgA levels which may falsely reflect any change in tumor size. Increased CgA concentrations assist in the clinical evaluation of PNETs but they are not specific for this kind of malignancy. Benign causes of CgA elevation should also be taken into consideration which include renal insufficiency, liver diseases and in patient taking proton pump inhibitors. Therefore, use of CgA as a diagnostic or screening test for PNET is discouraged.

**5. Conventional imaging and Somatostatin Receptor Scintigraphy (SRS)** 

intrapancreatic, and frequently missed by conventional imaging (Kulke et al., 2010a).

PNETs frequently overexpress somatostatin receptors and bind synthetic somatostatin analogues with high affinity. A number of radiolabeled analogues have been developed, with the most widely used worldwide and the only one available in the United States being 111In-DTPA-octreotide (Octreoscan). SRS usually utilizes both planar imaging with either whole body scanning or multiple static acquisitions and single-photon computed tomography (SPECT). The latter modality can potentially improve the accuracy of SRS. This can allow SRS to detect up to 50% to 70% of primary PNETs and more than 90% of patients with metastatic disease. False-positive localizations can occur in up to 12% of patients, so it is important to interpret the results cautiously (Dabizzi et al., 2010; Kulke et al., 2010a) .

Although conventional imaging which include CT or MRI scans are usually employed in the initial diagnostic workup, they detect less than 50% of most PNETs that are less than 1 cm, therefore frequently missing small tumors (especially insulinomas, duodenal gastrinomas) and small liver metastases (Noone et al., 2005; Rockall and Reznek, 2007). Although, CT imaging with contrast is perhaps the most common initial imaging obtained, in certain clinical scenarios endoscopic ultrasound (EUS) paired with fine needle aspiration, remains the main endoscopic diagnostic technique. Several small studies reveal impressive diagnostic capability of this modality with reported sensitivity between 80% and 90% (De Angelis et al., 2011). EUS is much more effective for localizing intrapancreatic PNETs than extrapancreatic PNETs such as duodenal gastrinomas or somatostatinomas. Moreover, EUS is particularly helpful in localizing insulinomas, which are small, almost always

lipid metabolism and catecholamine secretion etc (Lawrence et al., 2011b).

**4. Biochemical testing in PNET** 

#### **3.3 Glucagonoma**

Majority of the patients with glucagonomas present with a dermatitis called necrolytic migratory erythema, causing pruritis and often becoming secondarily infected (Perry and Vinik, 1995). The clinical manifestation may also include diabetes, depression and deep vein thrombosis. Glucagonomas are frequently found in the pancreatic tail and have a malignant potential with a predilection for metastases. A serum glucagon level >500 pg/ml is highly suspicious of the diagnosis, whereas, a concentration of >10,000 pg/ml is virtually diagnostic (Chastain, 2001). However, a normal level does not exclude the diagnosis as secretion of glucagon may be episodic and a high concentration may be seen in other clinical syndromes such as sepsis, renal and hepatic failure. Initial management with somatostatin analogues are usually very effective in controlling symptoms however, such treatment may not have an effect on tumor growth. (Jockenhovel et al., 1994)

#### **3.4 Somatostatinoma**

Pancreatic somatostatinoma are usually malignant, and can present clinically with a syndrome of diabetes, steatorrhea and cholelithiasis (Warner, 2005). The diagnosis can be confirmed biochemically with marked elevation of serum somatostatin followed by imaging and endoscopic ultrasound, as with other pancreatic neuroendocrine tumors. Management with somatostatin analogues may be effective in symptomatic patients.

#### **3.5 VIPoma**

Verner and Morrison first described pancreatic endocrine tumors with a clinical syndrome of watery diarrhea, hypokalemia and achlorohydria (Verner and Morrison, 1958). This syndrome was subsequently found to be due to ectopic vasoactive intestinal peptide (VIP) secretion. Biochemical analysis assists in establishing a diagnosis when a marked elevation (>200 pg/dl) in the serum level of VIP is found (Smith et al., 1998). Symptomatic control of the diarrhea can be achieved with somatostatin analogues (Kraenzlin et al., 1985).


WDHA: Watery diarrhea, hypokalemia and achlorohydria

Table 2. Clinical manifestation of Pancreatic Neuroendocrine Tumors.

#### **4. Biochemical testing in PNET**

284 Pancreatic Cancer – Clinical Management

Majority of the patients with glucagonomas present with a dermatitis called necrolytic migratory erythema, causing pruritis and often becoming secondarily infected (Perry and Vinik, 1995). The clinical manifestation may also include diabetes, depression and deep vein thrombosis. Glucagonomas are frequently found in the pancreatic tail and have a malignant potential with a predilection for metastases. A serum glucagon level >500 pg/ml is highly suspicious of the diagnosis, whereas, a concentration of >10,000 pg/ml is virtually diagnostic (Chastain, 2001). However, a normal level does not exclude the diagnosis as secretion of glucagon may be episodic and a high concentration may be seen in other clinical syndromes such as sepsis, renal and hepatic failure. Initial management with somatostatin analogues are usually very effective in controlling symptoms however, such treatment may

Pancreatic somatostatinoma are usually malignant, and can present clinically with a syndrome of diabetes, steatorrhea and cholelithiasis (Warner, 2005). The diagnosis can be confirmed biochemically with marked elevation of serum somatostatin followed by imaging and endoscopic ultrasound, as with other pancreatic neuroendocrine tumors. Management

Verner and Morrison first described pancreatic endocrine tumors with a clinical syndrome of watery diarrhea, hypokalemia and achlorohydria (Verner and Morrison, 1958). This syndrome was subsequently found to be due to ectopic vasoactive intestinal peptide (VIP) secretion. Biochemical analysis assists in establishing a diagnosis when a marked elevation (>200 pg/dl) in the serum level of VIP is found (Smith et al., 1998). Symptomatic control of

Tumor Type Symptoms or signs Incidence of metastases

confusion, visual disturbance,

(secretory), recurrent peptic ulcer

erythema, cachexia, depression,

Watery diarrhea (secretory),

Abdominal pain, diarrhea

deep vein thrombosis

<15%

2011)

2011;

75%

(Vinik and Gonzales,

50-85% (Batcher et al.,

Mittendorf et al., 2006)

(Batcher et al., 2011)

70-80% (Vinik and Gonzales, 2011)

60-85% (Vinik and Gonzales, 2011)

80% (Vinik and Gonzales, 2011)

the diarrhea can be achieved with somatostatin analogues (Kraenzlin et al., 1985).

diaphoresis.

hypokalemia

diabetes

jaundice

Table 2. Clinical manifestation of Pancreatic Neuroendocrine Tumors.

disease

Glucagonoma Diabetes, necrotizing migratory

Somatostatinoma Cholelithiasis, steatorrhea,

WDHA: Watery diarrhea, hypokalemia and achlorohydria

Non-functioning Abdominal pain, weight loss,

not have an effect on tumor growth. (Jockenhovel et al., 1994)

Insulinoma Hypoglycemia leading to

with somatostatin analogues may be effective in symptomatic patients.

**3.3 Glucagonoma** 

**3.4 Somatostatinoma** 

**3.5 VIPoma** 

Gastrinoma (Zollinger-Ellison Syndrome)

VIPoma, Verner-Morrison syndrome,WDHA syndrome As majority of PNETs are non-functional, hormonal assays cannot be used for clinical assessment. Hence, serum chromogranin A (CgA) has come to represent a common denominator peptide with the putative ability to serve as a marker of disease activity, in both functional and non functional tumors. Granins are found as major components of the soluble core of dense secretory granules in neuroendocrine cells and are secreted in a physiologically regulated manner (Kim et al., 2001). Eight members have been identified including CgA, chromogranin B, chromogranin C, SgIII, SgIV, SgV, SgVI and VGF nerve growth factorinducible. Granins have been proposed as playing important roles in secretory granule formation and development. CgA was the initial member identified, and originally detected in the chromaffin granules of the adrenal medulla (Blaschko et al., 1967). Although the definitive function of CgA remains unclear, CgA derived peptides mediate a number of biologic functions including regulation of parathyroid hormone secretion, carbohydrate metabolism, lipid metabolism and catecholamine secretion etc (Lawrence et al., 2011b).

Serum concentrations of CgA may decrease in patients responding to somatostatin analogs or other therapies. CgA should be used with caution as a marker of disease activity in patients treated with somatostatin analogs, because these agents significantly reduce plasma CgA levels which may falsely reflect any change in tumor size. Increased CgA concentrations assist in the clinical evaluation of PNETs but they are not specific for this kind of malignancy. Benign causes of CgA elevation should also be taken into consideration which include renal insufficiency, liver diseases and in patient taking proton pump inhibitors. Therefore, use of CgA as a diagnostic or screening test for PNET is discouraged.

#### **5. Conventional imaging and Somatostatin Receptor Scintigraphy (SRS)**

Although conventional imaging which include CT or MRI scans are usually employed in the initial diagnostic workup, they detect less than 50% of most PNETs that are less than 1 cm, therefore frequently missing small tumors (especially insulinomas, duodenal gastrinomas) and small liver metastases (Noone et al., 2005; Rockall and Reznek, 2007). Although, CT imaging with contrast is perhaps the most common initial imaging obtained, in certain clinical scenarios endoscopic ultrasound (EUS) paired with fine needle aspiration, remains the main endoscopic diagnostic technique. Several small studies reveal impressive diagnostic capability of this modality with reported sensitivity between 80% and 90% (De Angelis et al., 2011). EUS is much more effective for localizing intrapancreatic PNETs than extrapancreatic PNETs such as duodenal gastrinomas or somatostatinomas. Moreover, EUS is particularly helpful in localizing insulinomas, which are small, almost always intrapancreatic, and frequently missed by conventional imaging (Kulke et al., 2010a).

PNETs frequently overexpress somatostatin receptors and bind synthetic somatostatin analogues with high affinity. A number of radiolabeled analogues have been developed, with the most widely used worldwide and the only one available in the United States being 111In-DTPA-octreotide (Octreoscan). SRS usually utilizes both planar imaging with either whole body scanning or multiple static acquisitions and single-photon computed tomography (SPECT). The latter modality can potentially improve the accuracy of SRS. This can allow SRS to detect up to 50% to 70% of primary PNETs and more than 90% of patients with metastatic disease. False-positive localizations can occur in up to 12% of patients, so it is important to interpret the results cautiously (Dabizzi et al., 2010; Kulke et al., 2010a) .

Pancreatic Neuroendocrine Tumors: Emerging Management Paradigm 287

The high expression of somatostatin receptors in PNETs also provides a rationale for utilizing somatostatin analogs for therapeutic purposes. In the PROMID study, which was a randomized, placebo-controlled, prospective trial in patients with midgut carcinoid, treatment with somatostatin analog octreotide was associated with improved time to progression over placebo (Rinke et al., 2009). Whether this hold true for PNETs, remains to be seen and is currently being explored in a number of ongoing studies. According to the National Comprehensive Cancer Network guidelines, somatostatin analogs should be considered in patients with hormone hypersecretion, although the authors do state that no randomized studies to date have demonstrated anti-tumor effect of somatostatin analogs in PNETs (Kulke, 2011). Octreotide 150-250 mcg subcutaneously three times a day or octreotide LAR 20-30 mg intramuscularly every 4 weeks can be considered for symptom control. Short acting octreotide can be added to octreotide LAR for treatment of

A number of chemotherapeutic agents have been tested in advanced metastatic PNETs, with encouraging results showing antitumor activity. Streptozocin was approved by the FDA in July, 1982 as a treatment for advanced PNET after initial studies showed sufficient antitumor effect and response rates. A number of studies by Moertel et al in the 1970's were crucial in this area. One trial randomized 84 patients to either streptozocin alone or streptozocin and fluorouracil. Based on non-standard criteria, 63% of patients were reported to have a response to therapy, with 33% complete responses in the combination arm (Moertel et al., 1980). Other combinations that have been evaluated are streptozocin/doxorubicin or streptozocin/doxorubicin/fluorouracil (Kulke et al., 2010a; Moertel et al., 1992). Treatment with streptozocin and doxorubicin was associated in a combined radiological and biochemical response rate of 69% with a median survival approaching 2 years (Moertel et al., 1992). Based on retrospective data, the 3-drug regimen of streptozocin, 5-fluorouracil, and doxorubicin is associated with an overall response rate of 39% and a median survival duration of 37 months (Kouvaraki et al., 2004) .The combination of 5-fluorouracil, Cisplatin and streptozocin was tested in a series of 82 patients with advanced neuroendocrine tumor, prospectively identified from a database .Sixty percent of patients in this series were identified to have a pancreatic primary. Although, limited by a number of weaknesses in the study, the investigators reported a response rate of 38% in PNETs (Turner et al., 2010). Patients with advanced poorly differentiated PNETs should be treated along the small cell carcinoma guidelines with therapy based on platinum regimens. This approach has been shown to result in a response rate of 40 to 70% (Kulke et al., 2010a). Although, these data support the antitumor activity of streptozocin based regimens, the acceptability of this approach has been limited because of a cumbersome administration

Temozolomide has been combined with other biological agents such as thalidomide, bevacizumab and everolimus is phase II studies, yielding a response rate from 24-45% (Kulke et al., 2010b; Kulke et al., 2006a; Kulke et al., 2006b). Moreover, the combination of temozolomide and capecitabine has been reported to have an objective response rate of 70% (Strosberg et al., 2011) .There is also evidence to suggest that 06-methylguanine DNA

**8. Role of somatostatin analogs** 

breakthrough symptoms.

**9. Cytotoxic chemotherapy** 

schedule and toxicity profile.

#### **6. Role of surgery and liver directed therapy**

The therapeutic plan of PNETs is based on the histologic classification and tumor stage. Surgery remains the cornerstone of treatment of early stage PNETs. Surgical resection of localized PNETs offers excellent prognosis and curative potential. Depending on the site and size, in the absence of distant metastases enucleation may be sufficient. This approach can easily be employed for many PNETs specially insulinomas, small non functioning PNETs (<2 cm) and small gastrinomas (Kulke et al., 2010a) . The long term survival in certain cases may exceed 90% (Service et al., 1991). Whipple pancreatoduodenectomy, left pancreatectomy or total pancreatectomy can offer a 5-year survival rates of 61%-79% even in some advanced cases (Dabizzi et al., 2010). The role of surgery in patients with MEN1 syndrome is complicated and remains controversial because the risk of additional neoplasms within the remaining pancreas and other sites (Demeure et al., 1991).

In patients with limited hepatic metastases, surgical hepatic resection may be feasible to debulk the tumor burden and help alleviate symptoms. Surgical resection of majority of the tumor is possible in only 5-15% of PNETs with hepatic metastases (Norton, 2005; Que et al., 2006). This approach can offer improvement in symptoms in over 90% of patients (Sarmiento and Que, 2003).Even though most of the evidence in this area is derived from uncontrolled studies, many agree that surgical resection should be attempted in malignant PNET with limited hepatic metastases if it is deemed possible that >90% of viable tumor can be removed (Kulke et al., 2010a).

In patients who are not candidates for surgical hepatic resection, hepatic arterial embolization remains a viable palliative approach. Important characteristics that are important for patient consideration is a preserved performance status , liver confined disease and a patent portal vein. Response rates are generally encouraging ( >50% ) as measured by either radiographic regression or hormonal secretion (Gupta et al., 2003; O'Toole and Ruszniewski, 2005; Toumpanakis et al., 2007). Although a number of techniques exist, including bland embolization, chemo-embolization or radioisotopeembolization, no data exist determining the superiority of one approach over another.

Other radiological approaches that can be employed in treating the hepatic metastases in malignant PNET, are radiofrequency ablation and cryoablation (Toumpanakis et al., 2007). These approaches may not be a feasible option in bulky hepatic disease and the benefit derived in small volume disease is also not clear. The advantage may be that these techniques seem to cause less morbidity. Therefore, careful patient selection is crucial to consider ablative techniques in order to avoid any unwarranted adverse effects.

#### **7. Peptide Reception Radiation Technique (PRRT)**

Majority of PNETs express somatostatin receptors, which provides a rationale for PRRT in selected cases. The most frequently used radionucleotides for PRRT are yttrium (90Y) and lutetium (177Lu), which have different physical and biological characteristics. One study reported encouraging results with 129 patients with malignant NETs treated with [177Lu-DOTA-Tyr3]octreotate and resulted in a complete response in 2%, partial in 32%, and stabilization in 34% (Kwekkeboom et al., 2008). This form of treatment is generating widespread interest and more randomized studies are warranted in order to better explain its efficacy, role and toxicity.

#### **8. Role of somatostatin analogs**

286 Pancreatic Cancer – Clinical Management

The therapeutic plan of PNETs is based on the histologic classification and tumor stage. Surgery remains the cornerstone of treatment of early stage PNETs. Surgical resection of localized PNETs offers excellent prognosis and curative potential. Depending on the site and size, in the absence of distant metastases enucleation may be sufficient. This approach can easily be employed for many PNETs specially insulinomas, small non functioning PNETs (<2 cm) and small gastrinomas (Kulke et al., 2010a) . The long term survival in certain cases may exceed 90% (Service et al., 1991). Whipple pancreatoduodenectomy, left pancreatectomy or total pancreatectomy can offer a 5-year survival rates of 61%-79% even in some advanced cases (Dabizzi et al., 2010). The role of surgery in patients with MEN1 syndrome is complicated and remains controversial because the risk of additional

In patients with limited hepatic metastases, surgical hepatic resection may be feasible to debulk the tumor burden and help alleviate symptoms. Surgical resection of majority of the tumor is possible in only 5-15% of PNETs with hepatic metastases (Norton, 2005; Que et al., 2006). This approach can offer improvement in symptoms in over 90% of patients (Sarmiento and Que, 2003).Even though most of the evidence in this area is derived from uncontrolled studies, many agree that surgical resection should be attempted in malignant PNET with limited hepatic metastases if it is deemed possible that >90% of viable tumor can

In patients who are not candidates for surgical hepatic resection, hepatic arterial embolization remains a viable palliative approach. Important characteristics that are important for patient consideration is a preserved performance status , liver confined disease and a patent portal vein. Response rates are generally encouraging ( >50% ) as measured by either radiographic regression or hormonal secretion (Gupta et al., 2003; O'Toole and Ruszniewski, 2005; Toumpanakis et al., 2007). Although a number of techniques exist, including bland embolization, chemo-embolization or radioisotopeembolization, no data exist determining the superiority of one approach over another.

Other radiological approaches that can be employed in treating the hepatic metastases in malignant PNET, are radiofrequency ablation and cryoablation (Toumpanakis et al., 2007). These approaches may not be a feasible option in bulky hepatic disease and the benefit derived in small volume disease is also not clear. The advantage may be that these techniques seem to cause less morbidity. Therefore, careful patient selection is crucial to

Majority of PNETs express somatostatin receptors, which provides a rationale for PRRT in selected cases. The most frequently used radionucleotides for PRRT are yttrium (90Y) and lutetium (177Lu), which have different physical and biological characteristics. One study reported encouraging results with 129 patients with malignant NETs treated with [177Lu-DOTA-Tyr3]octreotate and resulted in a complete response in 2%, partial in 32%, and stabilization in 34% (Kwekkeboom et al., 2008). This form of treatment is generating widespread interest and more randomized studies are warranted in order to better explain

consider ablative techniques in order to avoid any unwarranted adverse effects.

**7. Peptide Reception Radiation Technique (PRRT)** 

neoplasms within the remaining pancreas and other sites (Demeure et al., 1991).

**6. Role of surgery and liver directed therapy** 

be removed (Kulke et al., 2010a).

its efficacy, role and toxicity.

The high expression of somatostatin receptors in PNETs also provides a rationale for utilizing somatostatin analogs for therapeutic purposes. In the PROMID study, which was a randomized, placebo-controlled, prospective trial in patients with midgut carcinoid, treatment with somatostatin analog octreotide was associated with improved time to progression over placebo (Rinke et al., 2009). Whether this hold true for PNETs, remains to be seen and is currently being explored in a number of ongoing studies. According to the National Comprehensive Cancer Network guidelines, somatostatin analogs should be considered in patients with hormone hypersecretion, although the authors do state that no randomized studies to date have demonstrated anti-tumor effect of somatostatin analogs in PNETs (Kulke, 2011). Octreotide 150-250 mcg subcutaneously three times a day or octreotide LAR 20-30 mg intramuscularly every 4 weeks can be considered for symptom control. Short acting octreotide can be added to octreotide LAR for treatment of breakthrough symptoms.

#### **9. Cytotoxic chemotherapy**

A number of chemotherapeutic agents have been tested in advanced metastatic PNETs, with encouraging results showing antitumor activity. Streptozocin was approved by the FDA in July, 1982 as a treatment for advanced PNET after initial studies showed sufficient antitumor effect and response rates. A number of studies by Moertel et al in the 1970's were crucial in this area. One trial randomized 84 patients to either streptozocin alone or streptozocin and fluorouracil. Based on non-standard criteria, 63% of patients were reported to have a response to therapy, with 33% complete responses in the combination arm (Moertel et al., 1980). Other combinations that have been evaluated are streptozocin/doxorubicin or streptozocin/doxorubicin/fluorouracil (Kulke et al., 2010a; Moertel et al., 1992). Treatment with streptozocin and doxorubicin was associated in a combined radiological and biochemical response rate of 69% with a median survival approaching 2 years (Moertel et al., 1992). Based on retrospective data, the 3-drug regimen of streptozocin, 5-fluorouracil, and doxorubicin is associated with an overall response rate of 39% and a median survival duration of 37 months (Kouvaraki et al., 2004) .The combination of 5-fluorouracil, Cisplatin and streptozocin was tested in a series of 82 patients with advanced neuroendocrine tumor, prospectively identified from a database .Sixty percent of patients in this series were identified to have a pancreatic primary. Although, limited by a number of weaknesses in the study, the investigators reported a response rate of 38% in PNETs (Turner et al., 2010). Patients with advanced poorly differentiated PNETs should be treated along the small cell carcinoma guidelines with therapy based on platinum regimens. This approach has been shown to result in a response rate of 40 to 70% (Kulke et al., 2010a). Although, these data support the antitumor activity of streptozocin based regimens, the acceptability of this approach has been limited because of a cumbersome administration schedule and toxicity profile.

Temozolomide has been combined with other biological agents such as thalidomide, bevacizumab and everolimus is phase II studies, yielding a response rate from 24-45% (Kulke et al., 2010b; Kulke et al., 2006a; Kulke et al., 2006b). Moreover, the combination of temozolomide and capecitabine has been reported to have an objective response rate of 70% (Strosberg et al., 2011) .There is also evidence to suggest that 06-methylguanine DNA

Pancreatic Neuroendocrine Tumors: Emerging Management Paradigm 289

group. At the data cut off point, the hazard ratio of death was 0.41 (95% CI, 0.19-0.89; P=0.02), with 10% of deaths reported in the sunitinib arm compared with 25% of deaths reported in the placebo arm (Raymond et al., 2011a). Grade 3/4 adverse events were uncommon in the treatment arm with the most common being neutropenia (12%) and hypertension (10%). Updated results, however, showed continued favorable trend for overall survival in the sunitinib arm but without statistical significance, with a hazard ratio of 0.737 (95% CI 0.465- 1.168; p=0.1926) (Raymond et al., 2011b). Based on this trial, FDA

mTOR is an intracellular protein kinase which regulates cellular response to nutrients and energy in addition to mediating signaling through downstream growth factors such as insulin-like growth factor (IGF-1).Sporadic neuroendocrine tumors are known to co-express both IGF-1 and its receptor. There is in vitro evidence suggesting stimulation of mTOR pathway and inhibition of this pathway has demonstrated tumor regression in preclinical models (von Wichert et al., 2000; Yao, 2007). Temsirolimus and everolimus are rapamycin derivatives that have been tested in PNET. Temsirolimus was evaluated in a phase II clinical trial in advanced neuroendocrine tumors which included 15 patients with PNET. Partial response rate of 6.7% was observed in the PNET patient population (Duran et al., 2006). In an initial phase 2 study the combination of everolimus and octerotide was evaluated, reporting a partial response of 27% in patients with PNET (Yao et al., 2008b). The activity of everoliumus was subsequently evaluated in an international phase II multicenter trial (RADIANT-1). A total of 160 patients with advanced PNET were enrolled into the study. In this non randomized study, treatment with everolimus was associated with an overall response rate of 4.4% and progression free survival duration of 16.7 months in patients receiving concomitant octerotide. In patients not receiving octerotide, the response rate was 9.6% and progression free survival duration was 9.7 months (Yao et al., 2010a). This was followed by an international phase III randomized clinical trial (RADIANT 3) assigning 410 patients to receive treatment with everolimus or placebo. Everolimus was administered as 10 mg once daily, in conjunction with best supportive care. Octreotide was given at the discretion of the investigator. More than 80% of patients had well differentiated disease and more than 90% had metastases to liver. The median progression free survival as assessed by the local investigator was 11 months in the everolimus group as compared to 4.6 months in the placebo arm (hazard ratio 0.35; 95% CI 0.27-0.45; p<0.001). Grade 3/4 adverse events were rare in the treatment group which included anemia (6%) and hyperglycemia (5%). The overall tumor response rate associated with everolimus in this study was 5% (Yao et al., 2011). Based on this trial, the FDA approved everolimus for advanced PNET in May, 2011.

Strategies to combine biological agents have begun in patients with advanced PNET. In a phase II trial, the combination of everolimus and bevacizumab was recently shown to be well tolerated and associated with an overall response rate of 26% in low to intermediate grade neuroendocrine tumors (Yao et al., 2010b). CALGB 80701 is currently randomizing patients with advanced PNET to receive either treatment with everolimus or everolimus + bevacizumab, to asses efficacy and toxicity. This trial will hopefully shed more light on the

role for combination strategy in the treatment armamentarium for PNET.

approved sunitinib for advanced PNET in May, 2011.

**10.2 Targeting mTOR pathway** 

**11. Combination strategies** 

methyltransferase (MGMT) deficiency can predict treatment responses to temozolomide in PNETs (Kulke et al., 2009b). Considering the available data, temozolomide based treatment has comparable efficacy to streptozocin based therapies with favorable toxicity profile. Further larger trials are warranted to further elaborate the role of temozolomide in the context of modern treatment paradigm in PNET.

#### **10. Biologically targeted therapies**

Recently, a number of studies have demonstrated activity in PNETs, targeting the vascular endothelial growth factor (VEGF) signaling and the mammalian target of rapamycin (mTOR) pathways. Although, obejective responses have been persistently low across studies, improvements in progression free survival have been encouraging.

#### **10.1 Targeting VEGF pathway**

PNETs are characterized by upregulation of VEGF and VEGF receptor (VEGFR). This correlates with increased angiogenesis, metastases and can potentially lead to decreased progression free survival (Zhang et al., 2007). Tyrosine kinase inhibitors with activity against VEGFR, such as pazopanib, sorafenib and sunitinib, have been evaluated in advanced PNET demonstrating encouraging results. Pazopanib was evaluated in a multiinstituion phase II study treating a total of 51 patients , 29 of which had PNET. Patients received pazopanib 800 mg daily, in addition to octerotide LAR. The response rate among patients with PNETs was reported to be 17%. Median PFS was reported to be 11.7 months. Grade 3/4 toxicities were relatively rare and included anemia, neutropenia, hypertriglyceridemia and liver function derangement (Phan et al., 2010). Another phase II trial is evaluating the role of pazopanib in patients with neuroendocrine tumors who may have had treatment with antiangiogenic and mTOR inhibitors. The trial is currently accruing and is expected to complete accrual in September 2011 (Capdevila et al., 2011). Sorafenib, another small molecule tyrosine kinase inhibitor, was evaluated in a phase II study that included 43 patients with PNET. Patients received sorafenib 400 mg twice daily. In a preliminary analysis, 10% of patients with PNET were observed to have a partial response (Hobday et al., 2007).

Sunitinib was evaluated in a multi-institutional phase II study that included 66 patients with PNET. Patients were treated with repeated 6 week cycles of oral sunitinib (50 mg/d) for 4 weeks followed by 2 weeks off treatment. Overall, objective response rate in PNET was observed to be 16.7% . One-year survival rate was reported to be 81% in the PNET group (Kulke et al., 2008). Based on encouraging results from this study, a phase III trial to confirm the activity of sunitinib was undertaken. Patients were randomized to receive once daily oral sunitinib at a dose of 37.5 mg or matching placebo. After enrollment of 171 patients, the data safety monitoring committee recommended the discontinuation of study and accrual was stopped before the preplanned efficacy analysis. The discontinuation of the study precluded definitive hypothesis testing for progression free survival difference between the two arms. An analysis of the enrolled patients, 86 of whom received sunitinib and 85 of whom received placebo, showed that the median progression free survival was significantly longer with sunitinib compared to placebo (11.4 months vs. 5.5 months ; hazard ratio= 0.42; p < 0.001). The objective response rate was 9.3% in the sunitinib group vs. 0% in the placebo group. At the data cut off point, the hazard ratio of death was 0.41 (95% CI, 0.19-0.89; P=0.02), with 10% of deaths reported in the sunitinib arm compared with 25% of deaths reported in the placebo arm (Raymond et al., 2011a). Grade 3/4 adverse events were uncommon in the treatment arm with the most common being neutropenia (12%) and hypertension (10%). Updated results, however, showed continued favorable trend for overall survival in the sunitinib arm but without statistical significance, with a hazard ratio of 0.737 (95% CI 0.465- 1.168; p=0.1926) (Raymond et al., 2011b). Based on this trial, FDA approved sunitinib for advanced PNET in May, 2011.

#### **10.2 Targeting mTOR pathway**

288 Pancreatic Cancer – Clinical Management

methyltransferase (MGMT) deficiency can predict treatment responses to temozolomide in PNETs (Kulke et al., 2009b). Considering the available data, temozolomide based treatment has comparable efficacy to streptozocin based therapies with favorable toxicity profile. Further larger trials are warranted to further elaborate the role of temozolomide in the

Recently, a number of studies have demonstrated activity in PNETs, targeting the vascular endothelial growth factor (VEGF) signaling and the mammalian target of rapamycin (mTOR) pathways. Although, obejective responses have been persistently low across

PNETs are characterized by upregulation of VEGF and VEGF receptor (VEGFR). This correlates with increased angiogenesis, metastases and can potentially lead to decreased progression free survival (Zhang et al., 2007). Tyrosine kinase inhibitors with activity against VEGFR, such as pazopanib, sorafenib and sunitinib, have been evaluated in advanced PNET demonstrating encouraging results. Pazopanib was evaluated in a multiinstituion phase II study treating a total of 51 patients , 29 of which had PNET. Patients received pazopanib 800 mg daily, in addition to octerotide LAR. The response rate among patients with PNETs was reported to be 17%. Median PFS was reported to be 11.7 months. Grade 3/4 toxicities were relatively rare and included anemia, neutropenia, hypertriglyceridemia and liver function derangement (Phan et al., 2010). Another phase II trial is evaluating the role of pazopanib in patients with neuroendocrine tumors who may have had treatment with antiangiogenic and mTOR inhibitors. The trial is currently accruing and is expected to complete accrual in September 2011 (Capdevila et al., 2011). Sorafenib, another small molecule tyrosine kinase inhibitor, was evaluated in a phase II study that included 43 patients with PNET. Patients received sorafenib 400 mg twice daily. In a preliminary analysis, 10% of patients with PNET were observed to have a partial response

Sunitinib was evaluated in a multi-institutional phase II study that included 66 patients with PNET. Patients were treated with repeated 6 week cycles of oral sunitinib (50 mg/d) for 4 weeks followed by 2 weeks off treatment. Overall, objective response rate in PNET was observed to be 16.7% . One-year survival rate was reported to be 81% in the PNET group (Kulke et al., 2008). Based on encouraging results from this study, a phase III trial to confirm the activity of sunitinib was undertaken. Patients were randomized to receive once daily oral sunitinib at a dose of 37.5 mg or matching placebo. After enrollment of 171 patients, the data safety monitoring committee recommended the discontinuation of study and accrual was stopped before the preplanned efficacy analysis. The discontinuation of the study precluded definitive hypothesis testing for progression free survival difference between the two arms. An analysis of the enrolled patients, 86 of whom received sunitinib and 85 of whom received placebo, showed that the median progression free survival was significantly longer with sunitinib compared to placebo (11.4 months vs. 5.5 months ; hazard ratio= 0.42; p < 0.001). The objective response rate was 9.3% in the sunitinib group vs. 0% in the placebo

studies, improvements in progression free survival have been encouraging.

context of modern treatment paradigm in PNET.

**10. Biologically targeted therapies** 

**10.1 Targeting VEGF pathway** 

(Hobday et al., 2007).

mTOR is an intracellular protein kinase which regulates cellular response to nutrients and energy in addition to mediating signaling through downstream growth factors such as insulin-like growth factor (IGF-1).Sporadic neuroendocrine tumors are known to co-express both IGF-1 and its receptor. There is in vitro evidence suggesting stimulation of mTOR pathway and inhibition of this pathway has demonstrated tumor regression in preclinical models (von Wichert et al., 2000; Yao, 2007). Temsirolimus and everolimus are rapamycin derivatives that have been tested in PNET. Temsirolimus was evaluated in a phase II clinical trial in advanced neuroendocrine tumors which included 15 patients with PNET. Partial response rate of 6.7% was observed in the PNET patient population (Duran et al., 2006). In an initial phase 2 study the combination of everolimus and octerotide was evaluated, reporting a partial response of 27% in patients with PNET (Yao et al., 2008b). The activity of everoliumus was subsequently evaluated in an international phase II multicenter trial (RADIANT-1). A total of 160 patients with advanced PNET were enrolled into the study. In this non randomized study, treatment with everolimus was associated with an overall response rate of 4.4% and progression free survival duration of 16.7 months in patients receiving concomitant octerotide. In patients not receiving octerotide, the response rate was 9.6% and progression free survival duration was 9.7 months (Yao et al., 2010a). This was followed by an international phase III randomized clinical trial (RADIANT 3) assigning 410 patients to receive treatment with everolimus or placebo. Everolimus was administered as 10 mg once daily, in conjunction with best supportive care. Octreotide was given at the discretion of the investigator. More than 80% of patients had well differentiated disease and more than 90% had metastases to liver. The median progression free survival as assessed by the local investigator was 11 months in the everolimus group as compared to 4.6 months in the placebo arm (hazard ratio 0.35; 95% CI 0.27-0.45; p<0.001). Grade 3/4 adverse events were rare in the treatment group which included anemia (6%) and hyperglycemia (5%). The overall tumor response rate associated with everolimus in this study was 5% (Yao et al., 2011). Based on this trial, the FDA approved everolimus for advanced PNET in May, 2011.

#### **11. Combination strategies**

Strategies to combine biological agents have begun in patients with advanced PNET. In a phase II trial, the combination of everolimus and bevacizumab was recently shown to be well tolerated and associated with an overall response rate of 26% in low to intermediate grade neuroendocrine tumors (Yao et al., 2010b). CALGB 80701 is currently randomizing patients with advanced PNET to receive either treatment with everolimus or everolimus + bevacizumab, to asses efficacy and toxicity. This trial will hopefully shed more light on the role for combination strategy in the treatment armamentarium for PNET.

Pancreatic Neuroendocrine Tumors: Emerging Management Paradigm 291

Goode, P.N., Farndon, J.R., Anderson, J., Johnston, I.D., and Morte, J.A. (1986). Diazoxide in the management of patients with insulinoma. World J Surg *10*, 586-592. Gupta, S., Yao, J.C., Ahrar, K., Wallace, M.J., Morello, F.A., Madoff, D.C., Murthy, R., Hicks,

Hobday, T.J., Rubin, J., Holen, K., Picus, J., Donehower, R., Marschke, R., Maples, W., Lloyd,

Howard, T.J., Stabile, B.E., Zinner, M.J., Chang, S., Bhagavan, B.S., and Passaro, E., Jr. (1990). Anatomic distribution of pancreatic endocrine tumors. Am J Surg *159*, 258-264. Jensen, R.T. (1996). Gastrointestinal endocrine tumours. Gastrinoma. Baillieres Clin

Jockenhovel, F., Lederbogen, S., Olbricht, T., Schmidt-Gayk, H., Krenning, E.P., Lamberts,

Kim, T., Tao-Cheng, J.H., Eiden, L.E., and Loh, Y.P. (2001). Chromogranin A, an "on/off" switch controlling dense-core secretory granule biogenesis. Cell *106*, 499-509. Kloppel, G., Perren, A., and Heitz, P.U. (2004). The gastroenteropancreatic neuroendocrine cell system and its tumors: the WHO classification. Ann N Y Acad Sci *1014*, 13-27. Kouvaraki, M.A., Ajani, J.A., Hoff, P., Wolff, R., Evans, D.B., Lozano, R., and Yao, J.C. (2004).

Kraenzlin, M.E., Ch'ng, J.L., Wood, S.M., Carr, D.H., and Bloom, S.R. (1985). Long-term

Kulke, M.H., Anthony, L.B., Bushnell, D.L., de Herder, W.W., Goldsmith, S.J., Klimstra, D.S.,

Kulke, M.H., Bendell, J., Kvols, L., Picus, J., Pommier, R., and Yao, J. (2011). Evolving

Kulke, M.H., Bergsland, E.K., and Yao, J.C. (2009a). Glycemic control in patients with

Kulke, M.H., Blaszkowsky, L., Zhu, A., and al., e. (2010b). Phase I/II study of everolimus

insulinoma treated with everolimus. N Engl J Med *360*, 195-197.

S.W., and Reinwein, D. (1994). The long-acting somatostatin analogue octreotide alleviates symptoms by reducing posttranslational conversion of prepro-glucagon to glucagon in a patient with malignant glucagonoma, but does not prevent tumor

Fluorouracil, doxorubicin, and streptozocin in the treatment of patients with locally advanced and metastatic pancreatic endocrine carcinomas. J Clin Oncol *22*, 4762-4771.

treatment of a VIPoma with somatostatin analogue resulting in remission of symptoms and possible shrinkage of metastases. Gastroenterology *88*, 185-187. Kulke, M.H. (2011). National Comprehensive Cancer Network guidelines: Neuroendocrine

Marx, S.J., Pasieka, J.L., Pommier, R.F., Yao, J.C.*, et al.* (2010a). NANETS treatment guidelines: well-differentiated neuroendocrine tumors of the stomach and

diagnostic and treatment strategies for pancreatic neuroendocrine tumors. J

(RAD001) in combination with temozolomide (TMZ) in patients with advanced pancreatic neuroendocrine tumors. 2010 Gastrointestinal Cancer Symposium. Kulke, M.H., Hornick, J.L., Frauenhoffer, C., Hooshmand, S., Ryan, D.P., Enzinger, P.C.,

Meyerhardt, J.A., Clark, J.W., Stuart, K., Fuchs, C.S.*, et al.* (2009b). O6-methylguanine

experience. Cancer J *9*, 261-267.

Gastroenterol *10*, 603-643.

Tumors.

growth. Clin Investig *72*, 127-133.

pancreas. Pancreas *39*, 735-752.

Hematol Oncol *4*, 29.

Vol 25, No 18S (June 20 Supplement), 2007: 4504.

M.E., and Ajani, J.A. (2003). Hepatic artery embolization and chemoembolization for treatment of patients with metastatic carcinoid tumors: the M.D. Anderson

R., Mahoney, M., and C., E. (2007). MC044h, a phase II trial of sorafenib in patients (pts) with metastatic neuroendocrine tumors (NET): A Phase II Consortium (P2C) study. Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings Part I

#### **12. Conclusions**

PNETs are a heterogeneous group of rare tumors with a wide range of biological activity, manifestation and variable prognosis. Accurate clinical, pathologic and histologic diagnosis is an important first step in developing an appropriate management plan. PNETs should be considered separately from carcinoid tumors as they are dissimilar in clinical behavior, response to treatment and prognosis. Surgical resection remains the mainstay of treatment for early stage disease. Advanced PNET often requires a multidisciplinary approach. Options for advanced stage include liver directed therapies including surgery and radioembolization techniques. Systemic treatment option include somatostatin analogs for symptom control, cytotoxic chemotherapy (temozolomide or streptozocin based regimens) and molecularly targeted agents (sunitinib and everolimus). No specific treatment sequence currently exists. Future studies will provide more insight into combination strategies and expand our treatment options for patients with this disease.

#### **13. References**


PNETs are a heterogeneous group of rare tumors with a wide range of biological activity, manifestation and variable prognosis. Accurate clinical, pathologic and histologic diagnosis is an important first step in developing an appropriate management plan. PNETs should be considered separately from carcinoid tumors as they are dissimilar in clinical behavior, response to treatment and prognosis. Surgical resection remains the mainstay of treatment for early stage disease. Advanced PNET often requires a multidisciplinary approach. Options for advanced stage include liver directed therapies including surgery and radioembolization techniques. Systemic treatment option include somatostatin analogs for symptom control, cytotoxic chemotherapy (temozolomide or streptozocin based regimens) and molecularly targeted agents (sunitinib and everolimus). No specific treatment sequence currently exists. Future studies will provide more insight into combination strategies and

Agarwal, S.K., Lee Burns, A., Sukhodolets, K.E., Kennedy, P.A., Obungu, V.H., Hickman,

Molecular pathology of the MEN1 gene. Ann N Y Acad Sci *1014*, 189-198.

Batcher, E., Madaj, P., and Gianoukakis, A.G. (2011). Pancreatic neuroendocrine tumors.

Blaschko, H., Comline, R.S., Schneider, F.H., Silver, M., and Smith, A.D. (1967). Secretion of

Capdevila, J., Teule, A., Castellano, D.E., Sastre, J., Garcia-Carbonero, R., Sevilla, I., Duran,

Chastain, M.A. (2001). The glucagonoma syndrome: a review of its features and discussion

Dabizzi, E., Panossian, A., and Raimondo, M. (2010). Management of pancreatic

De Angelis, C., Pellicano, R., Rizzetto, M., and Repici, A. (2011). Role of endoscopy in the

Demeure, M.J., Klonoff, D.C., Karam, J.H., Duh, Q.Y., and Clark, O.H. (1991). Insulinomas

Duran, I., Kortmansky, J., Singh, D., Hirte, H., Kocha, W., Goss, G., Le, L., Oza, A., Nicklee,

management of gastroenteropancreatic neuroendocrine tumours. Minerva

associated with multiple endocrine neoplasia type I: the need for a different

T., Ho, J.*, et al.* (2006). A phase II clinical and pharmacodynamic study of temsirolimus in advanced neuroendocrine carcinomas. Br J Cancer *95*, 1148-1154. Edge, S.B., and Compton, C.C. (2010). The American Joint Committee on Cancer: the 7th

edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol

neuroendocrine tumors. Minerva Gastroenterol Dietol *56*, 467-479.

surgical approach. Surgery *110*, 998-1004; discussion 1004-1005.

Asa, S.L. (2011). Pancreatic endocrine tumors. Mod Pathol *24 Suppl 2*, S66-77.

A.B., Mullendore, M.E., Whitten, I., Skarulis, M.C., Simonds, W.F.*, et al.* (2004).

a chromaffin granule protein, chromogranin, from the adrenal gland after

I., Escudero, P., Fuster, J., and Grande Pulido, E. (2011). PAZONET: A phase II trial of pazopanib in patients with metastatic neuroendocrine tumors (NETs) who may have previously received antiangiogenic or mTOR treatment. J Clin Oncol 29: 2011

expand our treatment options for patients with this disease.

splanchnic stimulation. Nature *215*, 58-59.

of new perspectives. Am J Med Sci *321*, 306-320.

**12. Conclusions** 

**13. References** 

Endocr Res *36*, 35-43.

(suppl; abstr TPS171).

*17*, 1471-1474.

Gastroenterol Dietol *57*, 129-137.


Pancreatic Neuroendocrine Tumors: Emerging Management Paradigm 293

Noone, T.C., Hosey, J., Firat, Z., and Semelka, R.C. (2005). Imaging and localization of islet-

Norton, J.A. (2005). Endocrine tumours of the gastrointestinal tract. Surgical treatment of neuroendocrine metastases. Best Pract Res Clin Gastroenterol *19*, 577-583. O'Toole, D., and Ruszniewski, P. (2005). Chemoembolization and other ablative therapies

Perry, R.R., and Vinik, A.I. (1995). Clinical review 72: diagnosis and management of functioning islet cell tumors. J Clin Endocrinol Metab *80*, 2273-2278. Phan, A.T., Yao, J.C., Fogelman, D.R., Hess, K.R., Ng, C.S., Bullock, S.A., Malinowski, P., Regan,

*19*, 195-211.

Gastroenterol *19*, 585-594.

cell tumours of the pancreas on CT and MRI. Best Pract Res Clin Endocrinol Metab

for liver metastases of gastrointestinal endocrine tumours. Best Pract Res Clin

E., and M.H., K. (2010). A prospective, multi-institutional phase II study of GW786034 (pazopanib) and depot octreotide (sandostatin LAR) in advanced low-grade neuroendocrine carcinoma (LGNEC). J Clin Oncol 28:15s, 2010 (suppl; abstr 4001). Que, F.G., Sarmiento, J.M., and Nagorney, D.M. (2006). Hepatic surgery for metastatic gastrointestinal neuroendocrine tumors. Adv Exp Med Biol *574*, 43-56. Raymond, E., Dahan, L., Raoul, J.L., Bang, Y.J., Borbath, I., Lombard-Bohas, C., Valle, J.,

Metrakos, P., Smith, D., Vinik, A.*, et al.* (2011a). Sunitinib malate for the treatment

Metrakos, P., Smith, D., Vinik, A.*, et al.* (2011b). Updated overall survival (OS) and progression-free survival (PFS) by blinded independent central review (BICR) of sunitinib (SU) versus placebo (PBO) for patients (Pts) with advanced unresectable pancreatic neuroendocrine tumors (NET). J Clin Oncol 29: 2011 (suppl; abstr 4008).

Aminossadati, B., Pape, U.F., Blaker, M.*, et al.* (2009). Placebo-controlled, doubleblind, prospective, randomized study on the effect of octreotide LAR in the control of tumor growth in patients with metastatic neuroendocrine midgut tumors: a

of pancreatic neuroendocrine tumors. N Engl J Med *364*, 501-513.

and classification. Neuroendocrinology *80 Suppl 1*, 12-15.

Best Pract Res Clin Endocrinol Metab *21*, 43-68.

tumors. Surg Oncol Clin N Am *12*, 231-242.

study. Mayo Clin Proc *66*, 711-719.

Raymond, E., Niccoli, P., Raoul, J., Bang, Y., Borbath, I., Lombard-Bohas, C., Valle, J.W.,

Rindi, G., and Kloppel, G. (2004). Endocrine tumors of the gut and pancreas tumor biology

Rinke, A., Muller, H.H., Schade-Brittinger, C., Klose, K.J., Barth, P., Wied, M., Mayer, C.,

Rockall, A.G., and Reznek, R.H. (2007). Imaging of neuroendocrine tumours (CT/MR/US).

Sarmiento, J.M., and Que, F.G. (2003). Hepatic surgery for metastases from neuroendocrine

Service, F.J., McMahon, M.M., O'Brien, P.C., and Ballard, D.J. (1991). Functioning

Shojamanesh, H., Gibril, F., Louie, A., Ojeaburu, J.V., Bashir, S., Abou-Saif, A., and Jensen,

with metastatic pancreatic endocrine carcinomas. Cancer *117*, 268-275.

insulinoma--incidence, recurrence, and long-term survival of patients: a 60-year

R.T. (2002). Prospective study of the antitumor efficacy of long-term octreotide treatment in patients with progressive metastatic gastrinoma. Cancer *94*, 331-343. Smith, S.L., Branton, S.A., Avino, A.J., Martin, J.K., Klingler, P.J., Thompson, G.B., Grant, C.S.,

and van Heerden, J.A. (1998). Vasoactive intestinal polypeptide secreting islet cell tumors: a 15-year experience and review of the literature. Surgery *124*, 1050-1055. Strosberg, J.R., Fine, R.L., Choi, J., Nasir, A., Coppola, D., Chen, D.T., Helm, J., and Kvols, L.

(2011). First-line chemotherapy with capecitabine and temozolomide in patients

report from the PROMID Study Group. J Clin Oncol *27*, 4656-4663.

DNA methyltransferase deficiency and response to temozolomide-based therapy in patients with neuroendocrine tumors. Clin Cancer Res *15*, 338-345.


Kulke, M.H., Lenz, H.J., Meropol, N.J., Posey, J., Ryan, D.P., Picus, J., Bergsland, E., Stuart,

Kulke, M.H., Stuart, K., Earle, C., and al., e. (2006a). A phase II study of temozolomide and

Kulke, M.H., Stuart, K., Enzinger, P.C., Ryan, D.P., Clark, J.W., Muzikansky, A., Vincitore,

Kwekkeboom, D.J., de Herder, W.W., Kam, B.L., van Eijck, C.H., van Essen, M., Kooij, P.P.,

Lambers, C.B., Lind, T., Moberg, S., Jansen, J.B., and Olbe, L. (1984). Omeprazole in

Lawrence, B., Gustafsson, B.I., Chan, A., Svejda, B., Kidd, M., and Modlin, I.M. (2011a). The

Lawrence, B., Gustafsson, B.I., Kidd, M., Pavel, M., Svejda, B., and Modlin, I.M. (2011b). The

neuroendocrine tumors. Endocrinol Metab Clin North Am *40*, 111-134, viii. Massironi, S., Sciola, V., Peracchi, M., Ciafardini, C., Spampatti, M.P., and Conte, D. (2008).

Mathur, A., Gorden, P., and Libutti, S.K. (2009). Insulinoma. Surg Clin North Am *89*, 1105-

Metz, D.C., and Jensen, R.T. (2008). Gastrointestinal neuroendocrine tumors: pancreatic

Mittendorf, E.A., Shifrin, A.L., Inabnet, W.B., Libutti, S.K., McHenry, C.R., and Demeure,

Modlin, I.M., Oberg, K., Chung, D.C., Jensen, R.T., de Herder, W.W., Thakker, R.V., Caplin,

Gastroenteropancreatic neuroendocrine tumours. Lancet Oncol *9*, 61-72. Moertel, C.G., Hanley, J.A., and Johnson, L.A. (1980). Streptozocin alone compared with

Moertel, C.G., Lefkopoulo, M., Lipsitz, S., Hahn, R.G., and Klaassen, D. (1992). Streptozocin-

Nikfarjam, M., Warshaw, A.L., Axelrod, L., Deshpande, V., Thayer, S.P., Ferrone, C.R., and

M., Delle Fave, G., Kaltsas, G.A., Krenning, E.P.*, et al.* (2008).

streptozocin plus fluorouracil in the treatment of advanced islet-cell carcinoma. N

doxorubicin, streptozocin-fluorouracil or chlorozotocin in the treatment of

Fernandez-del Castillo, C. (2008). Improved contemporary surgical management of insulinomas: a 25-year experience at the Massachusetts General Hospital. Ann Surg

patients with neuroendocrine tumors. Clin Cancer Res *15*, 338-345.

neuroendocrine tumors. J Clin Oncol *26*, 3403-3410.

efficacy, and survival. J Clin Oncol *26*, 2124-2130.

endocrine tumors. Gastroenterology *135*, 1469-1492.

M.J. (2006). Islet cell tumors. Curr Probl Surg *43*, 685-765.

advanced islet-cell carcinoma. N Engl J Med *326*, 519-523.

suppl; abstr 4044.

Clin North Am *40*, 1-18, vii.

Gastroenterol *14*, 5377-5384.

Engl J Med *303*, 1189-1194.

*247*, 165-172.

401-406.

1121.

DNA methyltransferase deficiency and response to temozolomide-based therapy in

K., Tye, L., Huang, X.*, et al.* (2008). Activity of sunitinib in patients with advanced

bevacizumab in patients with advanced neuroendocrine tumors. J Clin Oncol *24*,

M., Michelini, A., and Fuchs, C.S. (2006b). Phase II study of temozolomide and thalidomide in patients with metastatic neuroendocrine tumors. J Clin Oncol *24*,

Feelders, R.A., van Aken, M.O., and Krenning, E.P. (2008). Treatment with the radiolabeled somatostatin analog [177 Lu-DOTA 0,Tyr3]octreotate: toxicity,

Zollinger-Ellison syndrome. Effects of a single dose and of long-term treatment in patients resistant to histamine H2-receptor antagonists. N Engl J Med *310*, 758-761.

epidemiology of gastroenteropancreatic neuroendocrine tumors. Endocrinol Metab

clinical relevance of chromogranin A as a biomarker for gastroenteropancreatic

Neuroendocrine tumors of the gastro-entero-pancreatic system. World J


**18**

*Germany* 

**Generation and Impact of Neural**

Ihsan Ekin Demir, Helmut Friess and Güralp O. Ceyhan

Pancreatic cancer (PCa) as one of the most aggressive malignancies of mankind has an unparallelled propensity to invade intrapancreatic nerves. This "neural invasion" is therefore one of the most frequent routes of spread in PCa in addition to lymphatic and vascular paths. The major clinical relevance of neural invasion (NI) has triggered intense research efforts to understand its pathomechanims, and the findings derived from all these studies show how multi-faceted this peculiar route of cancer invasion in PCa is. This chapter is devoted to a thorough description of the characteristics, the pathomechanism and the clinical impact of NI in PCa, with the discussion of the most important pathways which may

NI is a relatively new and more comprehensive term for the traditionally used description "perineural invasion". In several malignancies, e.g. in the prostate, head and neck, but also several gastrointestinal malignancies, cancer cells are commonly encountered around nerves (Liebig et al., 2009). The frequent presence of cancer cells along the perineurium, the protective sheet around neural fascicles, has hence made pathologists adopt the term "perineural invasion". Classically, cancer cells which penetrate through the epineurium come to lie between the epineurium and the underlying perineurium and "push" on the nerve fascicles within the constrained intraneural area. The earliest report on perineural invasion stems from Cruveilheir in 1835 where he noticed that cancer cells can actually extend along the invaded nerves (Demir et al., 2010). Interestingly, although pathologists frequently observed perineural invasion in several types of tumors, its role except for serving as an additional path of cancer spread has not been genuinely investigated and understood until 1990s. In his pioneering article on the ultrastructural features of perineural invasion in PCa, Dale Bockman from Augusta, Georgia, USA performed an electron microscopic analysis of invaded nerves in PCa (Bockman et al., 1994). There, he noted that, in contrast with the traditional assumption, PCa cells penetrate through the perineurium and become intimately associated with the interior of nerve fascicles, i.e. axons and Schwann cells (Bockman et al., 1994). These observations made by Bockman during mid-1990s laid the foundation for our understanding and the subsequent

**1. Introduction** 

be future targets for therapeutic intervention.

research on NI in PCa into the present time.

**2. What is "neural invasion" in pancreatic cancer?** 

**Invasion in Pancreatic Cancer** 

*Department of Surgery, Klinikum rechts der Isar, Technische Universität München, Munich,* 


### **Generation and Impact of Neural Invasion in Pancreatic Cancer**

Ihsan Ekin Demir, Helmut Friess and Güralp O. Ceyhan *Department of Surgery, Klinikum rechts der Isar, Technische Universität München, Munich, Germany* 

#### **1. Introduction**

294 Pancreatic Cancer – Clinical Management

Toumpanakis, C., Meyer, T., and Caplin, M.E. (2007). Cytotoxic treatment including

Turner, N.C., Strauss, S.J., Sarker, D., Gillmore, R., Kirkwood, A., Hackshaw, A.,

Verner, J.V., and Morrison, A.B. (1958). Islet cell tumor and a syndrome of refractory watery

Vinik, A.I., and Gonzales, M.R. (2011). New and emerging syndromes due to

Warner, R.R. (2005). Enteroendocrine tumors other than carcinoid: a review of clinically

Whipple, A.O., and Frantz, V.K. (1935). Adenoma of Islet Cells with Hyperinsulinism: A

Yao, J.C. (2007). Neuroendocrine tumors. Molecular targeted therapy for carcinoid and islet-

Yao, J.C., Hassan, M., Phan, A., Dagohoy, C., Leary, C., Mares, J.E., Abdalla, E.K., Fleming,

Yao, J.C., Lombard-Bohas, C., Baudin, E., Kvols, L.K., Rougier, P., Ruszniewski, P., Hoosen,

Yao, J.C., Phan, A.T., Chang, D.Z., Wolff, R.A., Hess, K., Gupta, S., Jacobs, C., Mares, J.E.,

Yao, J.C., Phan, A.T., Fogleman, D., Ng, C.S., Jacobs, C.B., C.D., D., Leary, C., and Hess, K.R.

Yao, J.C., Shah, M.H., Ito, T., Bohas, C.L., Wolin, E.M., Van Cutsem, E., Hobday, T.J.,

Zhang, J., Jia, Z., Li, Q., Wang, L., Rashid, A., Zhu, Z., Evans, D.B., Vauthey, J.N., Xie, K., and

functional biomarker. J Clin Oncol 28:15s, 2010 (suppl; abstr 4002).

pancreatic neuroendocrine tumors. N Engl J Med *364*, 514-523.

J.B., Vauthey, J.N., Rashid, A.*, et al.* (2008a). One hundred years after "carcinoid": epidemiology of and prognostic factors for neuroendocrine tumors in 35,825 cases

S., St Peter, J., Haas, T., Lebwohl, D.*, et al.* (2010a). Daily oral everolimus activity in patients with metastatic pancreatic neuroendocrine tumors after failure of cytotoxic

Landgraf, A.N., Rashid, A.*, et al.* (2008b). Efficacy of RAD001 (everolimus) and octreotide LAR in advanced low- to intermediate-grade neuroendocrine tumors:

(2010b). Randomized run-in study of bevacizumab (B) and everolimus (E) in lowto intermediate-grade neuroendocrine tumors (LGNETs) using perfusion CT as

Okusaka, T., Capdevila, J., de Vries, E.G.*, et al.* (2011). Everolimus for advanced

Yao, J.C. (2007). Elevated expression of vascular endothelial growth factor correlates with increased angiogenesis and decreased progression-free survival among patients with low-grade neuroendocrine tumors. Cancer *109*, 1478-1486.

in human neuroendocrine tumor cells. Cancer Res *60*, 4573-4581.

cell carcinoma. Best Pract Res Clin Endocrinol Metab *21*, 163-172.

significant advances. Gastroenterology *128*, 1668-1684.

in the United States. J Clin Oncol *26*, 3063-3072.

chemotherapy: a phase II trial. J Clin Oncol *28*, 69-76.

results of a phase II study. J Clin Oncol *26*, 4311-4318.

diarrhea and hypokalemia. Am J Med *25*, 374-380.

Endocrinol Metab *21*, 131-144.

Review. Ann Surg *101*, 1299-1335.

Cancer *102*, 1106-1112.

embolization/chemoembolization for neuroendocrine tumours. Best Pract Res Clin

Papadopoulou, A., Bell, J., Kayani, I., Toumpanakis, C.*, et al.* (2010). Chemotherapy with 5-fluorouracil, cisplatin and streptozocin for neuroendocrine tumours. Br J

neuroendocrine tumors. Endocrinol Metab Clin North Am *40*, 19-63, vii.von Wichert, G., Jehle, P.M., Hoeflich, A., Koschnick, S., Dralle, H., Wolf, E., Wiedenmann, B., Boehm, B.O., Adler, G., and Seufferlein, T. (2000). Insulin-like growth factor-I is an autocrine regulator of chromogranin A secretion and growth

> Pancreatic cancer (PCa) as one of the most aggressive malignancies of mankind has an unparallelled propensity to invade intrapancreatic nerves. This "neural invasion" is therefore one of the most frequent routes of spread in PCa in addition to lymphatic and vascular paths. The major clinical relevance of neural invasion (NI) has triggered intense research efforts to understand its pathomechanims, and the findings derived from all these studies show how multi-faceted this peculiar route of cancer invasion in PCa is. This chapter is devoted to a thorough description of the characteristics, the pathomechanism and the clinical impact of NI in PCa, with the discussion of the most important pathways which may be future targets for therapeutic intervention.

#### **2. What is "neural invasion" in pancreatic cancer?**

NI is a relatively new and more comprehensive term for the traditionally used description "perineural invasion". In several malignancies, e.g. in the prostate, head and neck, but also several gastrointestinal malignancies, cancer cells are commonly encountered around nerves (Liebig et al., 2009). The frequent presence of cancer cells along the perineurium, the protective sheet around neural fascicles, has hence made pathologists adopt the term "perineural invasion". Classically, cancer cells which penetrate through the epineurium come to lie between the epineurium and the underlying perineurium and "push" on the nerve fascicles within the constrained intraneural area. The earliest report on perineural invasion stems from Cruveilheir in 1835 where he noticed that cancer cells can actually extend along the invaded nerves (Demir et al., 2010). Interestingly, although pathologists frequently observed perineural invasion in several types of tumors, its role except for serving as an additional path of cancer spread has not been genuinely investigated and understood until 1990s. In his pioneering article on the ultrastructural features of perineural invasion in PCa, Dale Bockman from Augusta, Georgia, USA performed an electron microscopic analysis of invaded nerves in PCa (Bockman et al., 1994). There, he noted that, in contrast with the traditional assumption, PCa cells penetrate through the perineurium and become intimately associated with the interior of nerve fascicles, i.e. axons and Schwann cells (Bockman et al., 1994). These observations made by Bockman during mid-1990s laid the foundation for our understanding and the subsequent research on NI in PCa into the present time.

Generation and Impact of Neural Invasion in Pancreatic Cancer 297

2007), the investigators analyzed consecutive sections of surgically resected PCa specimens in order to elucidate the main patterns of cancer cell growth along nerves: (1) direct invasion of the nerves, (2) continuous tumor cells growth in the perineural space, (3) branching of the growing tumor mass along neural branches, (4) formation of a foremost growth cone of tumor cells, and (5) direct invasion of contiguous lymph nodes (Kayahara et al., 2007). Hence, the authors could provide an anatomical mechanism for the manifestation of NI and particularly extrapancreatic neural plexus invasion. Importantly, their study proved the continuous growth of PCa cells along intrapancreatic nerves towards the extrapancreatic

There are several factors which make NI a crucial aspect of PCa and an attractive field of research. First, NI has an utmost high prevalence in PCa, varying between 88% to 100% (Takahashi et al., 2001; Liu&Lu, 2002). Interestingly, according to a study by Kayahara et al., NI in PCa is significantly more common than in cancers which originate from direct anatomical neighbours of the pancreas, e.g. cancers of the distal bile duct or carcinoma of the papilla of Vater (Kayahara et al., 1991; Kayahara et al., 1993; Kayahara et al., 1994; Kayahara et al., 1995; Kayahara et al., 1996). Unfortunately, intrapancreatic NI is nearly always accompanied by invasion to the extrapancreatic neural plexus: In their series, Nakao et al detected intrapancreatic NI in 116 out of 129 (90%) patients, of whom 80 (69%) showed extrapancreatic nerve plexus involvement (Nakao et al., 1996). Based on these findings, it seems that clinicians should assume the presence of NI in every patient with PCa even if the pathology report does not include a statement regarding this histopathological feature.

In the face of such a high prevalence, NI is at the same time one of the foremost reasons for local tumor recurrence after curative tumor resection (Kayahara et al., 1991; Nagakawa et al., 1991; Kayahara et al., 1995; Kayahara et al., 1996; Nagakawa et al., 1996; Ozaki et al., 1999). In a study by Kayahara et al., the investigators analyzed the mode of recurrence in 30 patients who had originally undergone macroscopically curative resection (Kayahara et al., 1993; Liu&Lu, 2002). They showed that the rate of local retroperitoneal recurrence, i.e. the prevalence of extrapancreatic NI was 80%, of hepatic metastasis 66%, of peritoneal dissemination 53%, and of lymph node recurrence 47%, an observation which was confirmed by further antemortem studies (Kayahara et al., 1993; Kayahara et al., 1995; Liu&Lu, 2002). Among the several parts of the extrapancreatic neural plexus which demonstrate local tumor recurrence, pancreatic head plexus and splenic plexus are the most common sites of tumor recurrence (Liu&Lu, 2002). Based on the frequency of NI towards retropancreatic neural plexus, several surgeons advocated routine extended resections (including celiac plexus) or at least more aggressive surgery in the surgical treatment of PCa (Hiraoka et al., 1986; Nagakawa et al., 1991; Nagakawa et al., 1996; Imamura et al., 1999). However, subsequent clinical studies confirmed that, while extended resection - even in combination with radiotherapy- can contribute to local tumor control, there is no survival benefit for patients due to the early spread pattern of PCa (Bachmann et al., 2006; Takamori et al., 2008; Yokoyama&Nagino, 2011). However, these studies mostly concentrated on more extensive lymphadenectomy rather than plexus resection as the actual measure to reduce NI. In further newer studies (Hirano et al., ; Sperti et al., ; Kondo et al., 2001; Hirano et al., 2007; Chakravarty et al., 2011), the feasibility and safety of an en bloc resection including celiac artery, plexus and ganglia was demonstrated, but the actual survival beneift from this

**4. Why is neural invasion so deciding in the course of PCa?** 

neural plexus (Kayahara et al., 2007).

It should be noted that NI is often used to denote invasion of intrapancreatic nerves (Liu&Lu, 2002). However, it is at the same time a more comprehensive term for intra- as well as extrapancreatic nerve invasion (Bockman et al., 1994; Takahashi et al., 1997; Takahashi et al., 2001; Mitsunaga et al., 2007). In contrast with their European counterparts, several studies from the Far East were devoted to the study of extrapancreatic nerve invasion through collection of specimens from retro- and peripancreatic nerves during autopsy (Nakao et al., 1996; Takahashi et al., 1997; Hirai et al., 2002; Liu&Lu, 2002; Mitsunaga et al., 2007). In those studies, "extrapancreatic nerve invasion" is often used synonymously with NI (Nakao et al., 1996; Takahashi et al., 1997; Hirai et al., 2002; Liu&Lu, 2002; Mitsunaga et al., 2007).

#### **3. What are the specific histological characteristics of NI in PCa?**

A review of scientific literature on NI in PCa reveals that NI has mostly been perceived as the presence of PCa on and along the perineurium (Kayahara et al., 2007; Liebig et al., 2009). Recent studies, however, revealed that PCa cells are readily encountered in the endoneural area, i.e. between nerve fascicles, as originally observed by Bockman (Ceyhan et al., 2009). Consequently, subsequent studies applied a scoring system to describe the degree of penetration of PCa cells into intrapancreatic nerves, classifying NI into "no invasion" (score of 0/zero), "perineural invasion" (score of I/one) and "endoneural invasion" (score of II/two) (Ceyhan et al., 2006; Ceyhan et al., 2009; Ceyhan et al., 2011). Importantly, the presence of PCa cells in the interior of nerves should not necessarily be perceived as the invasion of the "endoneurium" which is the connective tissue within nerve fascicles of a nerve. Similarly, invasion around nerves does not directly imply invasion of the "perineurium" as the connective tissue layer encircling nerve fascicles. Rather, "endo-" and "perineural" stand for "between" or "around" nerve fascicles (Figure 1) (Ceyhan et al., 2009; Ceyhan et al., 2011).

Careful examination of PCa tissue specimens also reveals that the presence of NI is not independent of the localization of the nerves within the pancreatic tissue. Particularly, we could demonstrate that NI in ductal adenocarcinoma of the pancreas is detected more frequently in areas with severe desmoplasia (Ceyhan et al., 2009). The reasons for this association between desmoplasia and NI are so far not known. However, it is assumed that the extracellular matrix is a rich source of growth factors which may be trophic upon nerves (Zhu et al., 1999; Demir et al., 2010). Still, NI should not be assumed to be limited to desmoplastic areas. In a histopathological study on the normal pancreatic regions of PCa patients who underwent pancreatic resection, NI was also encountered in normal pancreatic areas which are distant from the actual tumor (Takahashi et al., 1997). In the original study, this type of NI termed "nex" was found in more than 50% of resected pancreatic specimens and correlated to the grade of intrapancreatic neural invasion or the presence of extrapancreatic neural plexus invasion (Takahashi et al., 1997). Moreover, its presence was also found to correlate to worse survival after removal of the tumor (Takahashi et al., 1997). Therefore, the presence of NI in the supposedly normal regions of the pancreas implies that NI is a rapidly progressive process where PCa cells grow very early along intrapancreatic nerves.

The different histological appearances of NI in PCa have also been employed to understand its pathomechanism and spread pattern. In a study by Kayahara et al. (Kayahara et al.,

It should be noted that NI is often used to denote invasion of intrapancreatic nerves (Liu&Lu, 2002). However, it is at the same time a more comprehensive term for intra- as well as extrapancreatic nerve invasion (Bockman et al., 1994; Takahashi et al., 1997; Takahashi et al., 2001; Mitsunaga et al., 2007). In contrast with their European counterparts, several studies from the Far East were devoted to the study of extrapancreatic nerve invasion through collection of specimens from retro- and peripancreatic nerves during autopsy (Nakao et al., 1996; Takahashi et al., 1997; Hirai et al., 2002; Liu&Lu, 2002; Mitsunaga et al., 2007). In those studies, "extrapancreatic nerve invasion" is often used synonymously with NI (Nakao et al., 1996; Takahashi et al., 1997; Hirai et al., 2002; Liu&Lu,

A review of scientific literature on NI in PCa reveals that NI has mostly been perceived as the presence of PCa on and along the perineurium (Kayahara et al., 2007; Liebig et al., 2009). Recent studies, however, revealed that PCa cells are readily encountered in the endoneural area, i.e. between nerve fascicles, as originally observed by Bockman (Ceyhan et al., 2009). Consequently, subsequent studies applied a scoring system to describe the degree of penetration of PCa cells into intrapancreatic nerves, classifying NI into "no invasion" (score of 0/zero), "perineural invasion" (score of I/one) and "endoneural invasion" (score of II/two) (Ceyhan et al., 2006; Ceyhan et al., 2009; Ceyhan et al., 2011). Importantly, the presence of PCa cells in the interior of nerves should not necessarily be perceived as the invasion of the "endoneurium" which is the connective tissue within nerve fascicles of a nerve. Similarly, invasion around nerves does not directly imply invasion of the "perineurium" as the connective tissue layer encircling nerve fascicles. Rather, "endo-" and "perineural" stand for "between" or "around" nerve fascicles (Figure 1) (Ceyhan et al., 2009;

Careful examination of PCa tissue specimens also reveals that the presence of NI is not independent of the localization of the nerves within the pancreatic tissue. Particularly, we could demonstrate that NI in ductal adenocarcinoma of the pancreas is detected more frequently in areas with severe desmoplasia (Ceyhan et al., 2009). The reasons for this association between desmoplasia and NI are so far not known. However, it is assumed that the extracellular matrix is a rich source of growth factors which may be trophic upon nerves (Zhu et al., 1999; Demir et al., 2010). Still, NI should not be assumed to be limited to desmoplastic areas. In a histopathological study on the normal pancreatic regions of PCa patients who underwent pancreatic resection, NI was also encountered in normal pancreatic areas which are distant from the actual tumor (Takahashi et al., 1997). In the original study, this type of NI termed "nex" was found in more than 50% of resected pancreatic specimens and correlated to the grade of intrapancreatic neural invasion or the presence of extrapancreatic neural plexus invasion (Takahashi et al., 1997). Moreover, its presence was also found to correlate to worse survival after removal of the tumor (Takahashi et al., 1997). Therefore, the presence of NI in the supposedly normal regions of the pancreas implies that NI is a rapidly progressive process where PCa cells grow very early along intrapancreatic

The different histological appearances of NI in PCa have also been employed to understand its pathomechanism and spread pattern. In a study by Kayahara et al. (Kayahara et al.,

**3. What are the specific histological characteristics of NI in PCa?** 

2002; Mitsunaga et al., 2007).

Ceyhan et al., 2011).

nerves.

2007), the investigators analyzed consecutive sections of surgically resected PCa specimens in order to elucidate the main patterns of cancer cell growth along nerves: (1) direct invasion of the nerves, (2) continuous tumor cells growth in the perineural space, (3) branching of the growing tumor mass along neural branches, (4) formation of a foremost growth cone of tumor cells, and (5) direct invasion of contiguous lymph nodes (Kayahara et al., 2007). Hence, the authors could provide an anatomical mechanism for the manifestation of NI and particularly extrapancreatic neural plexus invasion. Importantly, their study proved the continuous growth of PCa cells along intrapancreatic nerves towards the extrapancreatic neural plexus (Kayahara et al., 2007).

#### **4. Why is neural invasion so deciding in the course of PCa?**

There are several factors which make NI a crucial aspect of PCa and an attractive field of research. First, NI has an utmost high prevalence in PCa, varying between 88% to 100% (Takahashi et al., 2001; Liu&Lu, 2002). Interestingly, according to a study by Kayahara et al., NI in PCa is significantly more common than in cancers which originate from direct anatomical neighbours of the pancreas, e.g. cancers of the distal bile duct or carcinoma of the papilla of Vater (Kayahara et al., 1991; Kayahara et al., 1993; Kayahara et al., 1994; Kayahara et al., 1995; Kayahara et al., 1996). Unfortunately, intrapancreatic NI is nearly always accompanied by invasion to the extrapancreatic neural plexus: In their series, Nakao et al detected intrapancreatic NI in 116 out of 129 (90%) patients, of whom 80 (69%) showed extrapancreatic nerve plexus involvement (Nakao et al., 1996). Based on these findings, it seems that clinicians should assume the presence of NI in every patient with PCa even if the pathology report does not include a statement regarding this histopathological feature.

In the face of such a high prevalence, NI is at the same time one of the foremost reasons for local tumor recurrence after curative tumor resection (Kayahara et al., 1991; Nagakawa et al., 1991; Kayahara et al., 1995; Kayahara et al., 1996; Nagakawa et al., 1996; Ozaki et al., 1999). In a study by Kayahara et al., the investigators analyzed the mode of recurrence in 30 patients who had originally undergone macroscopically curative resection (Kayahara et al., 1993; Liu&Lu, 2002). They showed that the rate of local retroperitoneal recurrence, i.e. the prevalence of extrapancreatic NI was 80%, of hepatic metastasis 66%, of peritoneal dissemination 53%, and of lymph node recurrence 47%, an observation which was confirmed by further antemortem studies (Kayahara et al., 1993; Kayahara et al., 1995; Liu&Lu, 2002). Among the several parts of the extrapancreatic neural plexus which demonstrate local tumor recurrence, pancreatic head plexus and splenic plexus are the most common sites of tumor recurrence (Liu&Lu, 2002). Based on the frequency of NI towards retropancreatic neural plexus, several surgeons advocated routine extended resections (including celiac plexus) or at least more aggressive surgery in the surgical treatment of PCa (Hiraoka et al., 1986; Nagakawa et al., 1991; Nagakawa et al., 1996; Imamura et al., 1999). However, subsequent clinical studies confirmed that, while extended resection - even in combination with radiotherapy- can contribute to local tumor control, there is no survival benefit for patients due to the early spread pattern of PCa (Bachmann et al., 2006; Takamori et al., 2008; Yokoyama&Nagino, 2011). However, these studies mostly concentrated on more extensive lymphadenectomy rather than plexus resection as the actual measure to reduce NI. In further newer studies (Hirano et al., ; Sperti et al., ; Kondo et al., 2001; Hirano et al., 2007; Chakravarty et al., 2011), the feasibility and safety of an en bloc resection including celiac artery, plexus and ganglia was demonstrated, but the actual survival beneift from this

Generation and Impact of Neural Invasion in Pancreatic Cancer 299

radical operation remains to be demonstrated. Importantly, a common denominator of these studies on "en bloc" resection of retropancreatic neural plexus is the pronounced pain relief as a result of the resection of celiac plexus(Kondo et al., 2001; Hirano et al., 2007). This deciding association between extrapancreatic neural plexus and pain sensation builds up the link to the concept of "pancreatic neuropathy" in PCa which encompasses NI and several other neural alterations in PCa(Ceyhan et al., 2009), as explained in the

The pancreas is one of the most densely innervated visceral organs(Bradley&Bem, 2003). The extrinsic component of its innervation is composed of nerve fibers running within the vagal and splanchnic nerves which originate from vagal nuclei or DRGs, respectively. Like the intestine, it also has an intrinsic innervation which is represented by intrapancreatic neurons. Importantly, enteric and intrapancreatic neurons are embryologically closely related: intrapancreatic neurons develop from a subgroup of neural crest-derived enteric nervous system (ENS) precursors and thus belong to the ENS (Kirchgessner&Gershon, 1990; Kirchgessner&Gershon, 1991). Moreover, there exists a direct innervation of the pancreas from the duodenum termed "entero-pancreatic innervations", as evidenced by the entrance of nerve fibers directly from the duodenal ENS into intrapancreatic ganglia

In the currently most comprehensive systematic analysis of NI in PCa, our group aimed at the study of nerve morphology in 546 patients with different pancreatic tumors, including ductal adenocarcinoma, neuroendocrine tumors, intraductal papillary mucinous neoplasms (IPMN), serous and mucinous cystadenoma and other neoplasms of the pancreas (Ceyhan et al., 2009). In the mentioned study, we could demonstrate that ductal adenocarcinoma of the pancreas exhibits the highest degree of NI in comparison to all other pancreatic tumors (Ceyhan et al., 2009). Interestingly, ductal adenocarcinoma (PCa) also harbored an unparalleled degree of nerve alterations among all these tumors (Ceyhan et al., 2009). In particular, PCa was characterized by a prominently increased neural density, a pronounced neural hypertrophy and neural inflammatory cell infiltration ("pancreatic neuritis") (Ceyhan et al., 2006; Ceyhan et al., 2009). Moreover, we could also detect a key link between the severity of NI in PCa and the extent of intrapancreatic neuroplastic alterations: The more nerves and neural hypertrophy were present, the higher was the extent/severity of NI in

This association between pancreatic neuroplasticity and NI gained a further dimension in a subsequent study where the pancreatic "innervation quality" in PCa was studied and compared to normal human pancreas (NP) (Ceyhan et al., 2009). Interestingly, not only had nerves in PCa tissue fewer sympathetic nerve fibers than in NP, but nerves with NI had at the same time reduced amounts of both sympathetic and cholinergic nerve fibers (Ceyhan et al., 2009). This "neural remodeling" in PCa implies that PCa cells may not be arbitrarily invading intrapancreatic nerves but also aiming at specific fiber qualities for so far unknown reasons. Overall, these neural alterations which seem to be specific for PCa, i.e. pancreatic neuroplasticity, neural remodeling and the high degree of NI, are the three hallmarks of so-

**5. Neural invasion as part of "pancreatic neuropathy" in PCa** 

(Kirchgessner&Gershon, 1990; Kirchgessner&Gershon, 1991).

called "pancreatic neuropathy" in PCa (Ceyhan et al., 2009).

following section.

PCa (Ceyhan et al., 2009).

Fig. 1. Severity of neural invasion (NI) in pancreatic cancer (PCa). Examination of intrapancreatic nerves with NI reveals that PCa cells demonstrate varying degrees of interaction with the nerves. In many cases, PCa cells surround intrapancreatic nerves without breaching the epineural barrier (A, also termed epineural association). On the other hand, several nerves demonstrate a lack of the epineural barrier where pancreatic cancer cells surround the fascicles along their perineurium (B, perineural invasion). In most severe cases, PCa cells are encountered between nerve fascicles, along their endoneurium (C, endoneural invasion). There is a significant association between the severity of NI and the degree of pain sensation among PCa patients (please refer to the main text for the respective references). All images at 200x magnification.

Fig. 1. Severity of neural invasion (NI) in pancreatic cancer (PCa). Examination of intrapancreatic nerves with NI reveals that PCa cells demonstrate varying degrees of interaction with the nerves. In many cases, PCa cells surround intrapancreatic nerves without breaching the epineural barrier (A, also termed epineural association). On the other hand, several nerves demonstrate a lack of the epineural barrier where pancreatic cancer cells surround the fascicles along their perineurium (B, perineural invasion). In most severe cases, PCa cells are encountered between nerve fascicles, along their endoneurium (C, endoneural invasion). There is a significant association between the severity of NI and the degree of pain sensation among PCa patients (please refer to the main text for the respective

references). All images at 200x magnification.

radical operation remains to be demonstrated. Importantly, a common denominator of these studies on "en bloc" resection of retropancreatic neural plexus is the pronounced pain relief as a result of the resection of celiac plexus(Kondo et al., 2001; Hirano et al., 2007). This deciding association between extrapancreatic neural plexus and pain sensation builds up the link to the concept of "pancreatic neuropathy" in PCa which encompasses NI and several other neural alterations in PCa(Ceyhan et al., 2009), as explained in the following section.

#### **5. Neural invasion as part of "pancreatic neuropathy" in PCa**

The pancreas is one of the most densely innervated visceral organs(Bradley&Bem, 2003). The extrinsic component of its innervation is composed of nerve fibers running within the vagal and splanchnic nerves which originate from vagal nuclei or DRGs, respectively. Like the intestine, it also has an intrinsic innervation which is represented by intrapancreatic neurons. Importantly, enteric and intrapancreatic neurons are embryologically closely related: intrapancreatic neurons develop from a subgroup of neural crest-derived enteric nervous system (ENS) precursors and thus belong to the ENS (Kirchgessner&Gershon, 1990; Kirchgessner&Gershon, 1991). Moreover, there exists a direct innervation of the pancreas from the duodenum termed "entero-pancreatic innervations", as evidenced by the entrance of nerve fibers directly from the duodenal ENS into intrapancreatic ganglia (Kirchgessner&Gershon, 1990; Kirchgessner&Gershon, 1991).

In the currently most comprehensive systematic analysis of NI in PCa, our group aimed at the study of nerve morphology in 546 patients with different pancreatic tumors, including ductal adenocarcinoma, neuroendocrine tumors, intraductal papillary mucinous neoplasms (IPMN), serous and mucinous cystadenoma and other neoplasms of the pancreas (Ceyhan et al., 2009). In the mentioned study, we could demonstrate that ductal adenocarcinoma of the pancreas exhibits the highest degree of NI in comparison to all other pancreatic tumors (Ceyhan et al., 2009). Interestingly, ductal adenocarcinoma (PCa) also harbored an unparalleled degree of nerve alterations among all these tumors (Ceyhan et al., 2009). In particular, PCa was characterized by a prominently increased neural density, a pronounced neural hypertrophy and neural inflammatory cell infiltration ("pancreatic neuritis") (Ceyhan et al., 2006; Ceyhan et al., 2009). Moreover, we could also detect a key link between the severity of NI in PCa and the extent of intrapancreatic neuroplastic alterations: The more nerves and neural hypertrophy were present, the higher was the extent/severity of NI in PCa (Ceyhan et al., 2009).

This association between pancreatic neuroplasticity and NI gained a further dimension in a subsequent study where the pancreatic "innervation quality" in PCa was studied and compared to normal human pancreas (NP) (Ceyhan et al., 2009). Interestingly, not only had nerves in PCa tissue fewer sympathetic nerve fibers than in NP, but nerves with NI had at the same time reduced amounts of both sympathetic and cholinergic nerve fibers (Ceyhan et al., 2009). This "neural remodeling" in PCa implies that PCa cells may not be arbitrarily invading intrapancreatic nerves but also aiming at specific fiber qualities for so far unknown reasons. Overall, these neural alterations which seem to be specific for PCa, i.e. pancreatic neuroplasticity, neural remodeling and the high degree of NI, are the three hallmarks of socalled "pancreatic neuropathy" in PCa (Ceyhan et al., 2009).

Generation and Impact of Neural Invasion in Pancreatic Cancer 301

technique does not necessarily prove the neuropathic character of pain in PCa, it underlines the deciding contribution of nerves and the transmitted signals in the generation of the pain syndrome in PCa (Ceyhan et al., 2008) Considering the neuropathic character of pain in PCa, one can assume that neuropathic analgesics may be of benefit to treat PCa-associated pain. As of today, the impact of neuropathic analgesic regimens to treat of patients with advanced

Researchers and clinicians have long puzzled about why PCa cells are frequently encountered around intrapancreatic nerves. Early reports had claimed that PCa cells enter nerves through the perineurium at its weakest points, i.e. along neural lymph vessels(di Mola&di Sebastiano, 2008), which, however, could not be confirmed in later studies. In later studies, investigators suggested that PCa cells grow along the path of least resistance after entering nerves, which was thought to be the perineural space (Rodin et al., 1967; Bockman et al., 1994; di Mola&di Sebastiano, 2008). Indeed, a higher proliferative index and decreased apoptosis in the perineural space could previously be shown for prostate cancer cells invading nerves (Ayala et al., 2004). However, newer studies could demonstrate that limiting PCa cells' presence around nerves to the local physical circumstances may be an oversimplification of the utmost frequent NI in Pca (Demir et al., 2010). In particular, the development of novel in vitro research tools to study NI in PCa has enabled the discovery of a true cancer-nerve affinity as an important biological mechanism in Pca (Zhu et al., 1999; Zhu et al., 2002). Especially, we know today that peripheral nerves in the tumor microenvironment can serve a source of tumor-trophic factors and cancer-attracting molecules (Zhu et al., 1999; Ceyhan et al., 2008; Gil et al., 2010). This "biological cancer-nerve affinity" in PCa is today one of the cardinal pathomechanistic concepts in our understanding of NI in Pca (Zhu et al., 1999; Ceyhan et al., 2008; Demir et al., 2010; Gil et al.,

This increased appreciation of cancer-nerve affinity in PCa was largely possible owing to increased efforts to develop novel advanced in vitro models of NI in PCa. These models generally employ heterotypic co-cultures of neurons and PCa cells as in of the earliest models by Dai et al (Dai et al., 2007). In their study, the investigators co-cultivated the human PCa cell line MiaPaCa-2 with neurons from mouse dorsal root ganglia (DRG). In accordance with the hypothesized trophic effect of nerves, PCa cells which were co-cultured with DRG exhibited stronger growth than non-co-cultured control PCa cells and overexpressed prosurvival genes like MALT1 and TRAF (Dai et al., 2007). As a frequent observation also made by other current models, also PCa cells supported the growth of the

This mutual trophic effect gained a further dimension in a recent study by our group where we presented another in vitro model which allows a precise spatiotemporal monitoring of NI by PCa cells (Ceyhan et al., 2008). As shown in the original article, different PCa cell lines were co-cultured together with rat DRG or myenteric plexus (MP) cells in a threedimensional (3D) extracellular matrix (ECM)-based migration assay (Ceyhan et al., 2008).

neurons, as evidenced by their increased neurite growth (Dai et al., 2007).

**7. Why are pancreatic cancer cells attracted to nerves? Molecular** 

PCa has not yet been systematically investigated.

**mechanisms of neural invasion in PCa** 

**7.1 In vitro models** 

2010).

While the mechanisms of these neuroplastic alterations are not completely understood, there is increasing evidence that these neuropathic alterations in PCa can in part be attributed to the neurotrophic character of the tumor microenvironment in PCa (Demir et al., 2010). In a novel *in vitro* neuroplasticity assay, we could demonstrate that tissue extracts of PCa, PCa cell supernatants and supernatants of human pancreatic stellate cells as main generators of desmoplasia can all induce axonal sprouting, increased neurite density and perikaryonal hypertrophy of neurons isolated from dorsal root ganglia or myenteric plecus under *in vitro* conditions (Demir et al., 2010). In a very recent study, Li et al. added a novel dimension to our understanding of neural alterations in PCa: In accordance with their former hypothesis (Li&Ma, 2008), patients with hyperglycemia demonstrate more pronounced neural hypertrophy and increased neural density than normoglycemic patients (Li et al., 2011). Hence, it is to be expected that research on pancreatic neuroplasticity and especially NI in PCa may take a direction towards increased investigation of the impact of impaired glucose metabolism upon pancreatic neuropathy in PCa.

#### **6. The role of neural invasion in the pain due to PCa**

Decreased survival and local tumor recurrence are undoubtedly among the leading factors which make NI into a highly relevant clinical subject. However, within the true clinical impact of NI, its role in *pain* sensation occupies a special place. It has long been accepted that the extension of PCa along the intrapancreatic nerves towards extrapancreatic neural plexus may be a causal factor in the generation of pain in advanced Pca (Kayahara et al., 1991; Nakao et al., 1996; Kayahara et al., 2007). Bockman also postulated a significant role for NI in pain generation in PCa(Bockman et al., 1994), but the actual pioneering study in this context came from Zhu et al. who for the first time demonstrated the correlation between the intrapancreatic expression of the nerve growth factor (NGF)(Zhu et al., 1999), the frequency of perineural invasion and the degree of pain sensation in PCa patients, an observation which was later also discovered for the expression of NGF receptor TrkA (Zhang et al., 2005; Dang et al., 2006). Owing to this study and its successors, it became increasingly clear that the extent of NI in the pancreas affects pain sensation, where nervederived molecules like NGF play a key role in both pain sensation and potentially in the attraction of PCa cell to nerves (Demir et al., 2010). The resulting interest in such nervederived mediators, especially in neurotrophic factors, have inaugurated the era of research on the molecular biological mechanisms of NI which last until the present time (Demir et al., 2010). Moreover, the identified cross-link between NI, pancreatic neuroplastic alterations and pain sensation by PCa patients revealed the potential involvement of "neuropathic" pain mechanisms in PCa (Ceyhan et al., 2009; Demir et al., 2010). Hence, researchers and clinicians have recently and increasingly understood that damage to nerves within the pancreas may be the actual pain-triggering mechanism in PCa (Ceyhan et al., 2009; Demir et al., 2010).

One can assume that the blockade of pain transmission via the damaged nerves from the pancreas may be of major benefit to treat pain due to PCa. As the celiac plexus contains a large portion of the afferent nerve fibers from the pancreas, several studies have tested the efficieny of celiac plexus blockade/neurolysis in the treatment of pain due to PCa. In all these studies, patients had significant pain relief (Wong et al., 2004; Stefaniak et al., 2005; Yan&Myers, 2007) following this intervention. While the efficiency of this "denervation" technique does not necessarily prove the neuropathic character of pain in PCa, it underlines the deciding contribution of nerves and the transmitted signals in the generation of the pain syndrome in PCa (Ceyhan et al., 2008) Considering the neuropathic character of pain in PCa, one can assume that neuropathic analgesics may be of benefit to treat PCa-associated pain. As of today, the impact of neuropathic analgesic regimens to treat of patients with advanced PCa has not yet been systematically investigated.

#### **7. Why are pancreatic cancer cells attracted to nerves? Molecular mechanisms of neural invasion in PCa**

#### **7.1 In vitro models**

300 Pancreatic Cancer – Clinical Management

While the mechanisms of these neuroplastic alterations are not completely understood, there is increasing evidence that these neuropathic alterations in PCa can in part be attributed to the neurotrophic character of the tumor microenvironment in PCa (Demir et al., 2010). In a novel *in vitro* neuroplasticity assay, we could demonstrate that tissue extracts of PCa, PCa cell supernatants and supernatants of human pancreatic stellate cells as main generators of desmoplasia can all induce axonal sprouting, increased neurite density and perikaryonal hypertrophy of neurons isolated from dorsal root ganglia or myenteric plecus under *in vitro* conditions (Demir et al., 2010). In a very recent study, Li et al. added a novel dimension to our understanding of neural alterations in PCa: In accordance with their former hypothesis (Li&Ma, 2008), patients with hyperglycemia demonstrate more pronounced neural hypertrophy and increased neural density than normoglycemic patients (Li et al., 2011). Hence, it is to be expected that research on pancreatic neuroplasticity and especially NI in PCa may take a direction towards increased investigation of the impact of impaired glucose

Decreased survival and local tumor recurrence are undoubtedly among the leading factors which make NI into a highly relevant clinical subject. However, within the true clinical impact of NI, its role in *pain* sensation occupies a special place. It has long been accepted that the extension of PCa along the intrapancreatic nerves towards extrapancreatic neural plexus may be a causal factor in the generation of pain in advanced Pca (Kayahara et al., 1991; Nakao et al., 1996; Kayahara et al., 2007). Bockman also postulated a significant role for NI in pain generation in PCa(Bockman et al., 1994), but the actual pioneering study in this context came from Zhu et al. who for the first time demonstrated the correlation between the intrapancreatic expression of the nerve growth factor (NGF)(Zhu et al., 1999), the frequency of perineural invasion and the degree of pain sensation in PCa patients, an observation which was later also discovered for the expression of NGF receptor TrkA (Zhang et al., 2005; Dang et al., 2006). Owing to this study and its successors, it became increasingly clear that the extent of NI in the pancreas affects pain sensation, where nervederived molecules like NGF play a key role in both pain sensation and potentially in the attraction of PCa cell to nerves (Demir et al., 2010). The resulting interest in such nervederived mediators, especially in neurotrophic factors, have inaugurated the era of research on the molecular biological mechanisms of NI which last until the present time (Demir et al., 2010). Moreover, the identified cross-link between NI, pancreatic neuroplastic alterations and pain sensation by PCa patients revealed the potential involvement of "neuropathic" pain mechanisms in PCa (Ceyhan et al., 2009; Demir et al., 2010). Hence, researchers and clinicians have recently and increasingly understood that damage to nerves within the pancreas may be the actual pain-triggering mechanism in PCa (Ceyhan et al., 2009; Demir et

One can assume that the blockade of pain transmission via the damaged nerves from the pancreas may be of major benefit to treat pain due to PCa. As the celiac plexus contains a large portion of the afferent nerve fibers from the pancreas, several studies have tested the efficieny of celiac plexus blockade/neurolysis in the treatment of pain due to PCa. In all these studies, patients had significant pain relief (Wong et al., 2004; Stefaniak et al., 2005; Yan&Myers, 2007) following this intervention. While the efficiency of this "denervation"

metabolism upon pancreatic neuropathy in PCa.

al., 2010).

**6. The role of neural invasion in the pain due to PCa** 

Researchers and clinicians have long puzzled about why PCa cells are frequently encountered around intrapancreatic nerves. Early reports had claimed that PCa cells enter nerves through the perineurium at its weakest points, i.e. along neural lymph vessels(di Mola&di Sebastiano, 2008), which, however, could not be confirmed in later studies. In later studies, investigators suggested that PCa cells grow along the path of least resistance after entering nerves, which was thought to be the perineural space (Rodin et al., 1967; Bockman et al., 1994; di Mola&di Sebastiano, 2008). Indeed, a higher proliferative index and decreased apoptosis in the perineural space could previously be shown for prostate cancer cells invading nerves (Ayala et al., 2004). However, newer studies could demonstrate that limiting PCa cells' presence around nerves to the local physical circumstances may be an oversimplification of the utmost frequent NI in Pca (Demir et al., 2010). In particular, the development of novel in vitro research tools to study NI in PCa has enabled the discovery of a true cancer-nerve affinity as an important biological mechanism in Pca (Zhu et al., 1999; Zhu et al., 2002). Especially, we know today that peripheral nerves in the tumor microenvironment can serve a source of tumor-trophic factors and cancer-attracting molecules (Zhu et al., 1999; Ceyhan et al., 2008; Gil et al., 2010). This "biological cancer-nerve affinity" in PCa is today one of the cardinal pathomechanistic concepts in our understanding of NI in Pca (Zhu et al., 1999; Ceyhan et al., 2008; Demir et al., 2010; Gil et al., 2010).

This increased appreciation of cancer-nerve affinity in PCa was largely possible owing to increased efforts to develop novel advanced in vitro models of NI in PCa. These models generally employ heterotypic co-cultures of neurons and PCa cells as in of the earliest models by Dai et al (Dai et al., 2007). In their study, the investigators co-cultivated the human PCa cell line MiaPaCa-2 with neurons from mouse dorsal root ganglia (DRG). In accordance with the hypothesized trophic effect of nerves, PCa cells which were co-cultured with DRG exhibited stronger growth than non-co-cultured control PCa cells and overexpressed prosurvival genes like MALT1 and TRAF (Dai et al., 2007). As a frequent observation also made by other current models, also PCa cells supported the growth of the neurons, as evidenced by their increased neurite growth (Dai et al., 2007).

This mutual trophic effect gained a further dimension in a recent study by our group where we presented another in vitro model which allows a precise spatiotemporal monitoring of NI by PCa cells (Ceyhan et al., 2008). As shown in the original article, different PCa cell lines were co-cultured together with rat DRG or myenteric plexus (MP) cells in a threedimensional (3D) extracellular matrix (ECM)-based migration assay (Ceyhan et al., 2008).

Generation and Impact of Neural Invasion in Pancreatic Cancer 303

et al. showed that Schwann cells of peripheral nerves express myelin-associated glycoprotein (MAG) which can serve as a receptor for the transmembrane mucin MUC1 on PCa cells (Swanson et al., 2007). Hence, based on this study, it seems that nerves in PCa tissue not only chemo-attract PCa cells but also can undergo direct physical contact, as

It is conceivable that the presented *in vitro* models in the literature would enable the identification of a gene set which would reflect the differentially upregulated genes in NI in PCa. Accordingly, in a recent study, Abiatari et al. aimed at obtaining a transcriptome signature of NI in PCa by means of an *in vitro / ex vivo* model (Abiatari et al., 2009). Specifically, they confronted human PCa cell lines with explanted rat vagus nerves and quantified the differentially regulated genes in highly versus less nerve-invasive PCa cells (Abiatari et al., 2009). Interestingly, the differentially regulated genes which were identified by this study were primarily related to cell motility, including kinesin family member 14 (KIF14) and Rho-GDP dissociation inhibitor beta (ARHGDIbeta), a gene set which they could expand in a subsequent by two molecules, i.e., the microtubule-associated protein MAPRE2 and the nuclear protein YPEL2 (Abiatari et al., 2009; Abiatari et al., 2009; Abiatari et al., 2009). Based on these important observations, the investigators underlined the importance of increased PCa cell motility in the generation of NI in PCa as additional

The increasing number of efforts to elucidate the pathomechanism of NI in PCa necessitated the creation of *in vivo* models which better mimic NI in human PCa. The common characteristic of these models is that they involve implantation of PCa cells as xenograft tumors in one out of several locations, e.g. into the pancreas, under the skin or in the proximity of large-diameter peripheral nerves to allow NI by PCa cells. In the first one of these models, Eibl et al. aimed at creating a model to simulate the high rates of local recurrence and NI after curative resection (Eibl&Reber, 2005). For this purpose, they performed complete surgical resection of the tumor at 4, 6, and 8 weeks after orthotopic implantation of the PCa cell lines MiaPaCa-2 (undifferentiated) and Capan-2 (welldifferentiated) in nude mice pancreas. Six weeks after tumor implantation, local tumor recurrence with extensive retroperitoneal nerve invasion and distant organ metastasis were observed in nude mice who had received MiaPaCa-2 cells (Eibl&Reber, 2005). Astonishingly, although the investigators achieved a successful simulation of the local recurrence and NI associated with PCa in this murine model, there has since been no application of this model to identify further pathomechanistic features of NI in PCa. Certainly, the model is probably not suitable to study the initial, early events leading to PCa, however, it can possibly be employed to examine the therapeutic potential of different agents on NI in PCa in an animal model of PCa. In another model, Koide et al. subcutaneously (s.c.) implanted different PCa cell lines with or without human peripheral nerves in nonobese diabetes/severe combined immunodeficient mice and analyzed the frequency of NI by these different cell lines (Koide et al., 2006). Furthermore, they performed an oligonucleotide microarray to obtain the expression profiles of high and low perineurally invasive cell lines. Interestingly, only two well-differentiated cell lines (Capan-1 and Capan-2) demonstrated invasion of mouse s.c. nerves. In these invasive cell lines, they

initially observed by Bockman (Swanson et al., 2007; Demir et al., 2010).

molecular biological mechanism (Abiatari et al., 2009; Abiatari et al., 2009).

**7.2 In vivo models** 

The presented assay offers several advantages: First, it initiates from a clear-cut physical separation of PCa cells and neurons, as it is the case under *in vivo* conditions. Therefore, the model allows exact monitoring of cell behavior from the very beginning. Second, the model includes a pre-defined migration path for PCa cells, i.e. defined ECM-bridges, which allows the generation of a chemical gradient for any chemotactic factor (Ceyhan et al., 2008). Using this novel assay, we could demonstrate that PCa cells react to the presence of neurons with a characteristic morphological alteration including cell flattening, grouping, colony formation and spike-like cellular polarization directed towards neurites. Following their targeted migration towards neurons, PCa cells established physical contact with neurites along which they were guided in their migration (Ceyhan et al., 2008). Similar to the findings by Dai et al., we also observed increased neurite growth from DRG neurons towards PCa cells when compared to non-co-cultured DRG neurons (Ceyhan et al., 2008). Moreover, the presented 3D-migration assay for the first time included a neuronal subtype which represents the intrinsic pancreatic neurons, i.e. neurons of the enteric nervous system (ENS). The key role of neurotrophins which was initially demonstrated by Zhu et al. for NGF could be confirmed by means of this novel assay where we monitored the quantitative alterations in the expression of neurotrophins, their receptors and the members of the glial-cell-derived neurotrophic factor (GDNF) family (Zhu et al., 1999; Ceyhan et al., 2008). As opposed to several members of the GDNF family, the nerve growth factor (NGF) increased continuously throughout the migration process of 120 hours (Ceyhan et al., 2008). Certainly, this novel 3D migration assays is at the same time a novel tool to investigate the contribution of numerous molecular factors from different cellular sources to NI in PCa. A very similar model, though without pre-defined paths for chemical gradient generation, has been recently reported by Gil et al. where the investigators could demonstrate a potent chemotactic effect of GDNF from DRG neurons upon PCa cells (Gil et al., 2010). In a further study, our group showed the potent enhancer effect of the neurotrophic factor artemin, a member of the GDNF family of neurotrophic factors, upon invasiveness of PCa cells (Ceyhan et al., 2006).

In the presence of an increasing number of studies on the role of neurotrophic factors in NI in PCa, there is only a limited of reports on the role of chemokines in NI in PCa (Marchesi et al., 2008; Marchesi et al., 2010; Marchesi et al., 2010). In one of the first studies where the potential role of chemokines was recognized, Marchesi et al could show that the neural immunoreactivity for the receptor of the chemokine fractalkine, i.e. CX3CR1, was significantly higher in perineural invasive lesions of PCa (Marchesi et al., 2008). By using CX3CR1-overexpressing PCa cells for an *in vivo* implantation model, they could also demonstrate that CX3CR1-overexpressing PCa cells exhibited a more pronounced infiltration of peripheral nerves (Marchesi et al., 2008). This study stands out in the literature due to the seminal investigation of chemokines in the generation of NI in PCa and pointed to the CX3CR1-CX3CL1 as a potential therapeutic target.

Interestingly, beyond molecules with chemoattractive potential, other nerve-derived molecules have also been the focus of recent research on NI in PCa. From these, in a mouse perineural invasion and orthotopic transplantation model, the stable knockdown of synuclein gamma (synuclein-γ) via by short hairpin RNA significantly reduced the incidence of perineural invasion and liver and lymph node metastasis (Hibi et al., 2009). In another study where the investigators provided a novel perspective on NI in PCa, Swanson et al. showed that Schwann cells of peripheral nerves express myelin-associated glycoprotein (MAG) which can serve as a receptor for the transmembrane mucin MUC1 on PCa cells (Swanson et al., 2007). Hence, based on this study, it seems that nerves in PCa tissue not only chemo-attract PCa cells but also can undergo direct physical contact, as initially observed by Bockman (Swanson et al., 2007; Demir et al., 2010).

It is conceivable that the presented *in vitro* models in the literature would enable the identification of a gene set which would reflect the differentially upregulated genes in NI in PCa. Accordingly, in a recent study, Abiatari et al. aimed at obtaining a transcriptome signature of NI in PCa by means of an *in vitro / ex vivo* model (Abiatari et al., 2009). Specifically, they confronted human PCa cell lines with explanted rat vagus nerves and quantified the differentially regulated genes in highly versus less nerve-invasive PCa cells (Abiatari et al., 2009). Interestingly, the differentially regulated genes which were identified by this study were primarily related to cell motility, including kinesin family member 14 (KIF14) and Rho-GDP dissociation inhibitor beta (ARHGDIbeta), a gene set which they could expand in a subsequent by two molecules, i.e., the microtubule-associated protein MAPRE2 and the nuclear protein YPEL2 (Abiatari et al., 2009; Abiatari et al., 2009; Abiatari et al., 2009). Based on these important observations, the investigators underlined the importance of increased PCa cell motility in the generation of NI in PCa as additional molecular biological mechanism (Abiatari et al., 2009; Abiatari et al., 2009).

#### **7.2 In vivo models**

302 Pancreatic Cancer – Clinical Management

The presented assay offers several advantages: First, it initiates from a clear-cut physical separation of PCa cells and neurons, as it is the case under *in vivo* conditions. Therefore, the model allows exact monitoring of cell behavior from the very beginning. Second, the model includes a pre-defined migration path for PCa cells, i.e. defined ECM-bridges, which allows the generation of a chemical gradient for any chemotactic factor (Ceyhan et al., 2008). Using this novel assay, we could demonstrate that PCa cells react to the presence of neurons with a characteristic morphological alteration including cell flattening, grouping, colony formation and spike-like cellular polarization directed towards neurites. Following their targeted migration towards neurons, PCa cells established physical contact with neurites along which they were guided in their migration (Ceyhan et al., 2008). Similar to the findings by Dai et al., we also observed increased neurite growth from DRG neurons towards PCa cells when compared to non-co-cultured DRG neurons (Ceyhan et al., 2008). Moreover, the presented 3D-migration assay for the first time included a neuronal subtype which represents the intrinsic pancreatic neurons, i.e. neurons of the enteric nervous system (ENS). The key role of neurotrophins which was initially demonstrated by Zhu et al. for NGF could be confirmed by means of this novel assay where we monitored the quantitative alterations in the expression of neurotrophins, their receptors and the members of the glial-cell-derived neurotrophic factor (GDNF) family (Zhu et al., 1999; Ceyhan et al., 2008). As opposed to several members of the GDNF family, the nerve growth factor (NGF) increased continuously throughout the migration process of 120 hours (Ceyhan et al., 2008). Certainly, this novel 3D migration assays is at the same time a novel tool to investigate the contribution of numerous molecular factors from different cellular sources to NI in PCa. A very similar model, though without pre-defined paths for chemical gradient generation, has been recently reported by Gil et al. where the investigators could demonstrate a potent chemotactic effect of GDNF from DRG neurons upon PCa cells (Gil et al., 2010). In a further study, our group showed the potent enhancer effect of the neurotrophic factor artemin, a member of the GDNF family of neurotrophic factors, upon invasiveness of PCa cells

In the presence of an increasing number of studies on the role of neurotrophic factors in NI in PCa, there is only a limited of reports on the role of chemokines in NI in PCa (Marchesi et al., 2008; Marchesi et al., 2010; Marchesi et al., 2010). In one of the first studies where the potential role of chemokines was recognized, Marchesi et al could show that the neural immunoreactivity for the receptor of the chemokine fractalkine, i.e. CX3CR1, was significantly higher in perineural invasive lesions of PCa (Marchesi et al., 2008). By using CX3CR1-overexpressing PCa cells for an *in vivo* implantation model, they could also demonstrate that CX3CR1-overexpressing PCa cells exhibited a more pronounced infiltration of peripheral nerves (Marchesi et al., 2008). This study stands out in the literature due to the seminal investigation of chemokines in the generation of NI in PCa and pointed

Interestingly, beyond molecules with chemoattractive potential, other nerve-derived molecules have also been the focus of recent research on NI in PCa. From these, in a mouse perineural invasion and orthotopic transplantation model, the stable knockdown of synuclein gamma (synuclein-γ) via by short hairpin RNA significantly reduced the incidence of perineural invasion and liver and lymph node metastasis (Hibi et al., 2009). In another study where the investigators provided a novel perspective on NI in PCa, Swanson

to the CX3CR1-CX3CL1 as a potential therapeutic target.

(Ceyhan et al., 2006).

The increasing number of efforts to elucidate the pathomechanism of NI in PCa necessitated the creation of *in vivo* models which better mimic NI in human PCa. The common characteristic of these models is that they involve implantation of PCa cells as xenograft tumors in one out of several locations, e.g. into the pancreas, under the skin or in the proximity of large-diameter peripheral nerves to allow NI by PCa cells. In the first one of these models, Eibl et al. aimed at creating a model to simulate the high rates of local recurrence and NI after curative resection (Eibl&Reber, 2005). For this purpose, they performed complete surgical resection of the tumor at 4, 6, and 8 weeks after orthotopic implantation of the PCa cell lines MiaPaCa-2 (undifferentiated) and Capan-2 (welldifferentiated) in nude mice pancreas. Six weeks after tumor implantation, local tumor recurrence with extensive retroperitoneal nerve invasion and distant organ metastasis were observed in nude mice who had received MiaPaCa-2 cells (Eibl&Reber, 2005). Astonishingly, although the investigators achieved a successful simulation of the local recurrence and NI associated with PCa in this murine model, there has since been no application of this model to identify further pathomechanistic features of NI in PCa. Certainly, the model is probably not suitable to study the initial, early events leading to PCa, however, it can possibly be employed to examine the therapeutic potential of different agents on NI in PCa in an animal model of PCa. In another model, Koide et al. subcutaneously (s.c.) implanted different PCa cell lines with or without human peripheral nerves in nonobese diabetes/severe combined immunodeficient mice and analyzed the frequency of NI by these different cell lines (Koide et al., 2006). Furthermore, they performed an oligonucleotide microarray to obtain the expression profiles of high and low perineurally invasive cell lines. Interestingly, only two well-differentiated cell lines (Capan-1 and Capan-2) demonstrated invasion of mouse s.c. nerves. In these invasive cell lines, they

Generation and Impact of Neural Invasion in Pancreatic Cancer 305

One of the initial models

Initial physical separation

of different cells Observation of initial cellular morphological

"Bridges" to enable chemical gradient generation

Usage of myenteric

reactions

neurons

Simulation of extrapancreatic NI

Easy to perform

Table 1. Experimental models of neural invasion (NI) in pancreatic cancer (PCa). The listed in vitro and in vivo models represent complicated heterotypic culture systems and possess several differences among each other. Their advantages, however, clearly overweigh their limitations. ECM: extracellular matrix. Please refer to the manuscript for the respective

paralysis

Therapeutic monitoring by observing degree of

Apt for studying expression changes

Advantages Limitations

No pre-defined chemical gradient for chemo-

No remark on the initial cellular reactions Different species of confronted cells (murine neurons vs. human PCa

Different species of confronted cells (murine neurons vs. human PCa

"large-caliber" vagal nerve

Different species of confronted cells (murine neurons vs. human PCa

Lacking monitoring of initial pathophysiological

Confrontation with the "large-caliber" sciatic

attractants

cells)

cells)

cells)

events

nerve

Easy to perform Confrontation with the

Investigator Model

Dai et al. Matrigel-based

Ceyhan et al. ECM-based three-

Abiatari et al. Ex vivo co-culture

Eibl et al. Orthotopic PCa cell

Gil et al. Tumor injection

(murine)

nerve

*In Vivo Models* 

references.

PCa cells

of rat nerves with

injection followed by tumor resection

onto murine sciatic

dimensional heterotypic migration assay

*In Vitro Models* 

characteristics

heterotypic coculture

identified over-expression of CD74 in the specifically perineural invasive cells, which they confirmed in human PCa tissue specimens (Koide et al., 2006). Despite the implantation of PCa cells under skin and not the actual organ of origin (i.e. the pancreas), this model sticks out owing to its uncomplicated performance. Still, it has to be underlined that none of these *in vivo* models has so far found widespread application.

Fig. 2. Different sources of molecular actors in neural invasion (NI) in pancreatic cancer (PCa). Research from the past 15 years revealed that NI results from a complicated interplay of numerous molecular agents derived from different sources, e.g. PCa cells, neurons, Schwann cells and stromal cells. Please refer to the main text for the respective references


identified over-expression of CD74 in the specifically perineural invasive cells, which they confirmed in human PCa tissue specimens (Koide et al., 2006). Despite the implantation of PCa cells under skin and not the actual organ of origin (i.e. the pancreas), this model sticks out owing to its uncomplicated performance. Still, it has to be underlined that none of these

Fig. 2. Different sources of molecular actors in neural invasion (NI) in pancreatic cancer (PCa). Research from the past 15 years revealed that NI results from a complicated interplay of numerous molecular agents derived from different sources, e.g. PCa cells, neurons, Schwann cells and stromal cells. Please refer to the main text for the respective references

*in vivo* models has so far found widespread application.


Table 1. Experimental models of neural invasion (NI) in pancreatic cancer (PCa). The listed in vitro and in vivo models represent complicated heterotypic culture systems and possess several differences among each other. Their advantages, however, clearly overweigh their limitations. ECM: extracellular matrix. Please refer to the manuscript for the respective references.

Generation and Impact of Neural Invasion in Pancreatic Cancer 307

Therefore, future *in vivo* models of NI in PCa should be superior to the current ones in the above-mentioned aspects, especially because they should increasingly be employed to

Neural invasion in PCa bears a unique importance in the biology of this disease due its impact on patient survival, local tumor reccurence and neuropathic pain sensation. Higher interest in NI has paved path for increased research on the biology of NI and accelarated the development of numerous experimental models. The discussed *in vitro* and *in vivo* models which shall help to eludicate the pathomechanisms of NI in PCa may provide novel tools to control and to reduce NI in this highly aggressive human malignancy. Considering the dismal average prognosis associated with PCa, one may wonder about the actual benefit of reducing the specific invasion of nerves in this tumor entity. Here, it should be underlined that reduction of NI can be regarded as one of several possibilities to control tumor growth, just as adjuvant therapy as an oncological therapy regimen aims at reaching microscopic tumor presence and reducing the systemic tumor burden. The control of NI, however, bears a further special importance since NI is not only the probably most common mode of spread for PCa, but also because nerves represent the most frequent site of local tumor recurrence in PCa. Moreover, limitation of NI is likely to have a considerable impact upon the neuropathic pain syndrome and thus quality of life of patients with PCa. Therefore, NI may find increased attention in the future as an additional therapeutic target for increased survival, enhanced postoperative outcome and improved quality of life among all patients

The authors are grateful to Dr. Matthias Maak for his assistance with the figures.

invasion of pancreatic cancer cells." *Int J Oncol* 35(5): 1111-6.

perineural invasion in prostate cancer." *Cancer Res* 64(17): 6082-90.

Abiatari, I., DeOliveira, T., Kerkadze, V., Schwager, C., Esposito, I., Giese, N. A., Huber, P.,

Abiatari, I., Gillen, S., DeOliveira, T., Klose, T., Bo, K., Giese, N. A., Friess, H. & Kleeff, J.

Abiatari, I., Kiladze, M., Kerkadze, V., Friess, H. & Kleeff, J. (2009). "Expression of YPEL1 in pancreatic cancer cell lines and tissues." *Georgian Med News*(175): 60-2. Ayala, G. E., Dai, H., Ittmann, M., Li, R., Powell, M., Frolov, A., Wheeler, T. M., Thompson,

Bachmann, J., Michalski, C. W., Martignoni, M. E., Buchler, M. W. & Friess, H. (2006). "Pancreatic resection for pancreatic cancer." *HPB (Oxford)* 8(5): 346-51. Bockman, D. E., Buchler, M. & Beger, H. G. (1994). "Interaction of pancreatic ductal carcinoma with nerves leads to nerve damage." *Gastroenterology* 107(1): 219-30.

Bergman, F., Abdollahi, A., Friess, H. & Kleeff, J. (2009). "Consensus transcriptome signature of perineural invasion in pancreatic carcinoma." *Mol Cancer Ther* 8(6):

(2009). "The microtubule-associated protein MAPRE2 is involved in perineural

T. C. & Rowley, D. (2004). "Growth and survival mechanisms associated with

deduce therapeutic targets and strategies.

**10. Summary and conclusion** 

with this dreadful malignancy.

**11. Acknowledgements** 

1494-504.

**12. References** 

#### **8. Efforts of controlling neural invasion in PCa**

Among all studies in the literature, there are so far two *in vivo* models of NI in PCa where a primarily therapeutic goal was pursued: In the first study, Gil et al. aimed at treating NI by means of an attenuated, replication-competent, oncolytic herpes simplex virus which inhabits nerves (Gil et al., 2007). After injection of PCa cell lines into the perineurium of the sciatic nerve of athymic mice, they monitored limb function for 9 days after injection. Excitingly, a single injection of the oncolytic herpes simplex virus 7 days after PCa cell injection effectively eradicated NI without compromising physiologic nerve function (Gil et al., 2007). The same group utilized this model in a subsequent study for intraoperative diagnosis of NI: By using enhanced green fluorescent protein (eGFP)-expressing oncolytic herpes virus, they could detect invaded nerves following intrasciatic implantation of PCa cell lines via intraoperative fluorescent stereoscopic imaging (Gil et al., 2008). Thereby, they proposed a novel tool for enhanced diagnosis and therapy of NI in PCa and for facilitated detection of invaded nerves in cases where an extended resection may be considered (Gil et al., 2008). In a third study, the group applied PCa cell injection onto mouse sciatic nerves and subsequently treated the mice with pyrazolopyrimidine-1, a tyrosine kinase inhibitor targeting the RET pathway (Gil et al., 2010). Strikingly, systemic therapy with this agent diminished nerve invasion toward the spinal cord and prevented limb paralysis (Gil et al., 2010). Still, the sensitivity and true effectiveness of their method remains to be confirmed in future studies.

#### **9. Future directions in research on NI in PCa**

Today, research on NI in PCa is in an era of "data collection" and "expansion of knowledge". The increasing number of *in vitro* and *in vivo* models, together with the rapidly growing scientific interest in NI, create the best possible conditions to learn and discover about this peculiar histopathological phenomenon. However, we are convinced that future studies should aim at the generation of models with an increasingly therapeutic intention, because there is urgent need to employ additional, novel tools to treat PCa. Furthermore, the main pathomechanistic hypothesis for the generation of NI, i.e. the neuro-affinity of PCa cells, has to be carefully reviewed. It should certainly be considered that PCa cells may not be responsible for every aspect of NI, but rather be reacting to the signals coming from the nerves. The increasing number of nerve-derived molecules like neurotrophic factors or neuronal chemokines which are continuously shown to contribute to NI should serve as a motivation to delve deeper into the involvement of neuronal molecules during NI.

While novel *in vitro* models of NI in PCa are being steadily developed, the currently available *in vivo* models still exhibit major deficits. In particular, these models lack:


Therefore, future *in vivo* models of NI in PCa should be superior to the current ones in the above-mentioned aspects, especially because they should increasingly be employed to deduce therapeutic targets and strategies.

#### **10. Summary and conclusion**

306 Pancreatic Cancer – Clinical Management

Among all studies in the literature, there are so far two *in vivo* models of NI in PCa where a primarily therapeutic goal was pursued: In the first study, Gil et al. aimed at treating NI by means of an attenuated, replication-competent, oncolytic herpes simplex virus which inhabits nerves (Gil et al., 2007). After injection of PCa cell lines into the perineurium of the sciatic nerve of athymic mice, they monitored limb function for 9 days after injection. Excitingly, a single injection of the oncolytic herpes simplex virus 7 days after PCa cell injection effectively eradicated NI without compromising physiologic nerve function (Gil et al., 2007). The same group utilized this model in a subsequent study for intraoperative diagnosis of NI: By using enhanced green fluorescent protein (eGFP)-expressing oncolytic herpes virus, they could detect invaded nerves following intrasciatic implantation of PCa cell lines via intraoperative fluorescent stereoscopic imaging (Gil et al., 2008). Thereby, they proposed a novel tool for enhanced diagnosis and therapy of NI in PCa and for facilitated detection of invaded nerves in cases where an extended resection may be considered (Gil et al., 2008). In a third study, the group applied PCa cell injection onto mouse sciatic nerves and subsequently treated the mice with pyrazolopyrimidine-1, a tyrosine kinase inhibitor targeting the RET pathway (Gil et al., 2010). Strikingly, systemic therapy with this agent diminished nerve invasion toward the spinal cord and prevented limb paralysis (Gil et al., 2010). Still, the sensitivity and true effectiveness of their method remains to be confirmed in

Today, research on NI in PCa is in an era of "data collection" and "expansion of knowledge". The increasing number of *in vitro* and *in vivo* models, together with the rapidly growing scientific interest in NI, create the best possible conditions to learn and discover about this peculiar histopathological phenomenon. However, we are convinced that future studies should aim at the generation of models with an increasingly therapeutic intention, because there is urgent need to employ additional, novel tools to treat PCa. Furthermore, the main pathomechanistic hypothesis for the generation of NI, i.e. the neuro-affinity of PCa cells, has to be carefully reviewed. It should certainly be considered that PCa cells may not be responsible for every aspect of NI, but rather be reacting to the signals coming from the nerves. The increasing number of nerve-derived molecules like neurotrophic factors or neuronal chemokines which are continuously shown to contribute to NI should serve as a

motivation to delve deeper into the involvement of neuronal molecules during NI.

available *in vivo* models still exhibit major deficits. In particular, these models lack:

4. The specific extension of NI towards the extrapancreatic neural plexus

5. Accompanying neuropathic and desmoplastic alterations

2. Histopathological confirmation of the tumor phenotype as ductal adenocarcinoma 3. The confirmation of the presence of NI even at early stages of tumor development and

While novel *in vitro* models of NI in PCa are being steadily developed, the currently

**8. Efforts of controlling neural invasion in PCa** 

**9. Future directions in research on NI in PCa** 

1. Tumors that directly originate from the pancreas

future studies.

progression

6. Neuropathic pain sensation

Neural invasion in PCa bears a unique importance in the biology of this disease due its impact on patient survival, local tumor reccurence and neuropathic pain sensation. Higher interest in NI has paved path for increased research on the biology of NI and accelarated the development of numerous experimental models. The discussed *in vitro* and *in vivo* models which shall help to eludicate the pathomechanisms of NI in PCa may provide novel tools to control and to reduce NI in this highly aggressive human malignancy. Considering the dismal average prognosis associated with PCa, one may wonder about the actual benefit of reducing the specific invasion of nerves in this tumor entity. Here, it should be underlined that reduction of NI can be regarded as one of several possibilities to control tumor growth, just as adjuvant therapy as an oncological therapy regimen aims at reaching microscopic tumor presence and reducing the systemic tumor burden. The control of NI, however, bears a further special importance since NI is not only the probably most common mode of spread for PCa, but also because nerves represent the most frequent site of local tumor recurrence in PCa. Moreover, limitation of NI is likely to have a considerable impact upon the neuropathic pain syndrome and thus quality of life of patients with PCa. Therefore, NI may find increased attention in the future as an additional therapeutic target for increased survival, enhanced postoperative outcome and improved quality of life among all patients with this dreadful malignancy.

#### **11. Acknowledgements**

The authors are grateful to Dr. Matthias Maak for his assistance with the figures.

#### **12. References**


Generation and Impact of Neural Invasion in Pancreatic Cancer 309

Gil, Z., Cavel, O., Kelly, K., Brader, P., Rein, A., Gao, S. P., Carlson, D. L., Shah, J. P., Fong, Y.

Gil, Z., Kelly, K. J., Brader, P., Shah, J. P., Fong, Y. & Wong, R. J. (2008). "Utility of a herpes

Gil, Z., Rein, A., Brader, P., Li, S., Shah, J. P., Fong, Y. & Wong, R. J. (2007). "Nerve-sparing

Hibi, T., Mori, T., Fukuma, M., Yamazaki, K., Hashiguchi, A., Yamada, T., Tanabe, M.,

Hirai, I., Kimura, W., Ozawa, K., Kudo, S., Suto, K., Kuzu, H. & Fuse, A. (2002). "Perineural

Hirano, S., Kondo, S., Hara, T., Ambo, Y., Tanaka, E., Shichinohe, T., Suzuki, O. & Hazama,

pancreatectomy with en-bloc celiac axis resection." *Dig Surg* 27(3): 212-6. Hiraoka, T., Watanabe, E., Katoh, T., Hayashida, N., Mizutani, J., Kanemitsu, K. & Miyauchi,

Imamura, M., Hosotani, R. & Kogire, M. (1999). "Rationale of the so-called extended resection for pancreatic invasive ductal carcinoma." *Digestion* 60 Suppl 1: 126-9. Kayahara, M., Nagakawa, T., Futagami, F., Kitagawa, H., Ohta, T. & Miyazaki, I. (1996).

Kayahara, M., Nagakawa, T., Konishi, I., Ueno, K., Ohta, T. & Miyazaki, I. (1991).

invasion of the extrapancreatic neural plexus." *Int J Pancreatol* 10(2): 105-11. Kayahara, M., Nagakawa, T., Tsukioka, Y., Ohta, T., Ueno, K. & Miyazaki, I. (1994). "Neural

Kayahara, M., Nagakawa, T., Ueno, K., Ohta, T., Takeda, T. & Miyazaki, I. (1993). "An

Kayahara, M., Nagakawa, T., Ueno, K., Ohta, T., Tsukioka, Y. & Miyazaki, I. (1995). "Surgical

analysis of nodal involvement and plexus invasion." *Surgery* 117(6): 616-23. Kayahara, M., Nakagawara, H., Kitagawa, H. & Ohta, T. (2007). "The nature of neural

complete denervation of the pancreas." *Am J Surg* 152(5): 549-51.

peripheral nerves." *J Natl Cancer Inst* 102(2): 107-18.

pancreatic cancer." *Clin Cancer Res* 15(8): 2864-71.

and tail of the pancreas." *Cancer* 78(12): 2485-91.

invasion by pancreatic cancer." *Pancreas* 35(3): 218-23.

invasion in pancreatic cancer." *Pancreas* 24(1): 15-25.

53.

*Res* 13(21): 6479-85.

41(2): 190-4.

23.

& Wong, R. J. (2010). "Paracrine regulation of pancreatic cancer cell invasion by

oncolytic virus for the detection of neural invasion by cancer." *Neoplasia* 10(4): 347-

therapy with oncolytic herpes virus for cancers with neural invasion." *Clin Cancer* 

Aiura, K., Kawakami, T., Ogiwara, A., Kosuge, T., Kitajima, M., Kitagawa, Y. & Sakamoto, M. (2009). "Synuclein-gamma is closely involved in perineural invasion and distant metastasis in mouse models and is a novel prognostic factor in

K. (2007). "Distal pancreatectomy with en bloc celiac axis resection for locally advanced pancreatic body cancer: long-term results." *Ann Surg* 246(1): 46-51. Hirano, S., Kondo, S., Tanaka, E., Shichinohe, T., Tsuchikawa, T., Kato, K. & Matsumoto, J.

"Postoperative bowel function and nutritional status following distal

Y. (1986). "A new surgical approach for control of pain in chronic pancreatitis:

"Lymphatic flow and neural plexus invasion associated with carcinoma of the body

"Clinicopathological study of pancreatic carcinoma with particular reference to the

invasion and nodal involvement in distal bile duct cancer." *Hepatogastroenterology*

evaluation of radical resection for pancreatic cancer based on the mode of recurrence as determined by autopsy and diagnostic imaging." *Cancer* 72(7): 2118-

strategy for carcinoma of the pancreas head area based on clinicopathologic


Bradley, E. L., 3rd & Bem, J. (2003). "Nerve blocks and neuroablative surgery for chronic

Ceyhan, G. O., Bergmann, F., Kadihasanoglu, M., Altintas, B., Demir, I. E., Hinz, U., Muller,

Ceyhan, G. O., Demir, I. E., Altintas, B., Rauch, U., Thiel, G., Muller, M. W., Giese, N. A.,

Ceyhan, G. O., Demir, I. E., Rauch, U., Bergmann, F., Muller, M. W., Buchler, M. W., Friess,

Ceyhan, G. O., Giese, N. A., Erkan, M., Kerscher, A. G., Wente, M. N., Giese, T., Buchler, M.

Ceyhan, G. O., Liebl, F., Maak, M., Schuster, T., Becker, K., Langer, R., Demir, I. E., Hartel,

Ceyhan, G. O., Michalski, C. W., Demir, I. E., Muller, M. W. & Friess, H. (2008). "Pancreatic

Chakravarty, K. D., Hsu, J. T., Liu, K. H., Yeh, C. N., Yeh, T. S., Hwang, T. L., Jan, Y. Y. &

Dai, H., Li, R., Wheeler, T., Ozen, M., Ittmann, M., Anderson, M., Wang, Y., Rowley, D.,

Dang, C., Zhang, Y., Ma, Q. & Shimahara, Y. (2006). "Expression of nerve growth factor

Demir, I. E., Ceyhan, G. O., Liebl, F., J.G., D. H., Maak, M. & Friess, H. (2010). "Neural

Demir, I. E., Ceyhan, G. O., Rauch, U., Altintas, B., Klotz, M., Muller, M. W., Buchler, M. W.,

di Mola, F. F. & di Sebastiano, P. (2008). "Pain and pain generation in pancreatic cancer."

Eibl, G. & Reber, H. A. (2005). "A xenograft nude mouse model for perineural invasion and

II pancreatic adenocarcinoma." *World J Gastroenterol* 16(8): 997-1002.

pancreatic cancer: an in vitro approach." *Hum Pathol* 38(2): 299-307.

M. W., Giese, T., Buchler, M. W., Giese, N. A. & Friess, H. (2009). "Pancreatic neuropathy and neuropathic pain--a comprehensive pathomorphological study of

Friess, H. & Schafer, K. H. (2008). "Neural invasion in pancreatic cancer: a mutual tropism between neurons and cancer cells." *Biochem Biophys Res Commun* 374(3):

H. & Schafer, K. H. (2009). "Pancreatic neuropathy results in "neural remodeling" and altered pancreatic innervation in chronic pancreatitis and pancreatic cancer."

W. & Friess, H. (2006). "The neurotrophic factor artemin promotes pancreatic

M., Friess, H. & Rosenberg, R. (2011). "The severity of neural invasion is a crucial prognostic factor in rectal cancer independent of neoadjuvant radiochemotherapy."

Chen, M. F. (2011). "Prognosis and feasibility of en-bloc vascular resection in stage

Younes, M. & Ayala, G. E. (2007). "Enhanced survival in perineural invasion of

receptors is correlated with progression and prognosis of human pancreatic

Invasion in Pancreatic Cancer: The Past, Present and Future." *Cancers* 2(3): 1513-

Friess, H. & Schafer, K. H. (2010). "The microenvironment in chronic pancreatitis and pancreatic cancer induces neuronal plasticity." *Neurogastroenterol Motil* 22(4):

pancreatitis." *World J Surg* 27(11): 1241-8.

*Am J Gastroenterol* 104(10): 2555-65.

*Ann Surg* 252(5): 797-804.

cancer invasion." *Ann Surg* 244(2): 274-81.

pain." *Best Pract Res Clin Gastroenterol* 22(1): 31-44.

cancer." *J Gastroenterol Hepatol* 21(5): 850-8.

*Langenbecks Arch Surg* 393(6): 919-22.

recurrence in pancreatic cancer." *Pancreas* 31(3): 258-62.

442-7.

1527.

480-90, e112-3.

546 cases." *Gastroenterology* 136(1): 177-186 e1.


Generation and Impact of Neural Invasion in Pancreatic Cancer 311

Nakao, A., Harada, A., Nonami, T., Kaneko, T. & Takagi, H. (1996). "Clinical significance of

Ozaki, H., Hiraoka, T., Mizumoto, R., Matsuno, S., Matsumoto, Y., Nakayama, T., Tsunoda,

Sperti, C., Berselli, M. & Pedrazzoli, S. "Distal pancreatectomy for body-tail pancreatic cancer: is there a role for celiac axis resection?" *Pancreatology* 10(4): 491-8. Stefaniak, T., Basinski, A., Vingerhoets, A., Makarewicz, W., Connor, S., Kaska, L., Stanek,

Swanson, B. J., McDermott, K. M., Singh, P. K., Eggers, J. P., Crocker, P. R. & Hollingsworth,

Takahashi, S., Hasebe, T., Oda, T., Sasaki, S., Kinoshita, T., Konishi, M., Ueda, T., Ochiai, T.

Takahashi, T., Ishikura, H., Motohara, T., Okushiba, S., Dohke, M. & Katoh, H. (1997).

Takamori, H., Hiraoka, T., Kanemitsu, K., Tsuji, T., Tanaka, H., Chikamoto, A., Horino, K.,

Wong, G. Y., Schroeder, D. R., Carns, P. E., Wilson, J. L., Martin, D. P., Kinney, M. O.,

Yan, B. M. & Myers, R. P. (2007). "Neurolytic celiac plexus block for pain control in

Yokoyama, Y. & Nagino, M. (2011). "Role of extended surgery for pancreatic cancer: critical

Zhang, Y., Dang, C., Ma, Q. & Shimahara, Y. (2005). "Expression of nerve growth factor

cancer: a randomized controlled trial." *Jama* 291(9): 1092-9.

unresectable pancreatic cancer." *Am J Gastroenterol* 102(2): 430-8.

*Pancreas* 12(4): 357-61.

by prostatic carcinoma." *Cancer* 20(10): 1772-9.

splanchnicectomy." *Eur J Surg Oncol* 31(7): 768-73.

perineural invasion." *Cancer Res* 67(21): 10222-9.

*Anticancer Res* 21(2B): 1407-12.

*Hepatobiliary Pancreat Sci*.

*Hepatobiliary Pancreat Surg* 15(6): 603-7.

164-70.

161-71.

carcinoma invasion of the extrapancreatic nerve plexus in pancreatic cancer."

T., Suzuki, T., Monden, M., Saitoh, Y., Yamauchi, H. & Ogata, Y. (1999). "The prognostic significance of lymph node metastasis and intrapancreatic perineural invasion in pancreatic cancer after curative resection." *Surg Today* 29(1): 16-22. Rodin, A. E., Larson, D. L. & Roberts, D. K. (1967). "Nature of the perineural space invaded

A., Kwiecinska, B., Lachinski, A. J. & Sledzinski, Z. (2005). "A comparison of two invasive techniques in the management of intractable pain due to inoperable pancreatic cancer: neurolytic celiac plexus block and videothoracoscopic

M. A. (2007). "MUC1 is a counter-receptor for myelin-associated glycoprotein (Siglec-4a) and their interaction contributes to adhesion in pancreatic cancer

& Ochiai, A. (2001). "Extra-tumor perineural invasion predicts postoperative development of peritoneal dissemination in pancreatic ductal adenocarcinoma."

"Perineural invasion by ductal adenocarcinoma of the pancreas." *J Surg Oncol* 65(3):

Beppu, T., Hirota, M. & Baba, H. (2008). "Long-term outcomes of extended radical resection combined with intraoperative radiation therapy for pancreatic cancer." *J* 

Mantilla, C. B. & Warner, D. O. (2004). "Effect of neurolytic celiac plexus block on pain relief, quality of life, and survival in patients with unresectable pancreatic

review of the four major RCTs comparing standard and extended surgery." *J* 

receptors and their prognostic value in human pancreatic cancer." *Oncol Rep* 14(1):


Kirchgessner, A. L. & Gershon, M. D. (1990). "Innervation of the pancreas by neurons in the

Kirchgessner, A. L. & Gershon, M. D. (1991). "Innervation and regulation of the pancreas by

Koide, N., Yamada, T., Shibata, R., Mori, T., Fukuma, M., Yamazaki, K., Aiura, K., Shimazu,

Kondo, S., Katoh, H., Omi, M., Hirano, S., Ambo, Y., Tanaka, E., Okushiba, S., Morikawa, T.,

Li, J. & Ma, Q. (2008). "Hyperglycemia promotes the perineural invasion in pancreatic

Li, J., Ma, Q., Liu, H., Guo, K., Li, F., Li, W., Han, L., Wang, F. & Wu, E. (2011). "Relationship

Liebig, C., Ayala, G., Wilks, J. A., Berger, D. H. & Albo, D. (2009). "Perineural invasion in

Liu, B. & Lu, K. Y. (2002). "Neural invasion in pancreatic carcinoma." *Hepatobiliary Pancreat* 

Marchesi, F., Locatelli, M., Solinas, G., Erreni, M., Allavena, P. & Mantovani, A. (2010). "Role

Marchesi, F., Piemonti, L., Fedele, G., Destro, A., Roncalli, M., Albarello, L., Doglioni, C.,

behavior of pancreatic ductal adenocarcinoma." *Cancer Res* 68(21): 9060-9. Marchesi, F., Piemonti, L., Mantovani, A. & Allavena, P. (2010). "Molecular mechanisms of

Mitsunaga, S., Hasebe, T., Kinoshita, T., Konishi, M., Takahashi, S., Gotohda, N., Nakagohri,

Nagakawa, T., Konishi, I., Ueno, K., Ohta, T., Akiyama, T., Kayahara, M. & Miyazaki, I.

Nagakawa, T., Nagamori, M., Futakami, F., Tsukioka, Y., Kayahara, M., Ohta, T., Ueno, K. &

body: a preliminary report on perfect pain relief." *Jop* 2(3): 93-7.

cancer: a review of the literature." *Cancer* 115(15): 3379-91.

system by cancer." *J Neuroimmunol* 224(1-2): 39-44.

M., Hirohashi, S., Nimura, Y. & Sakamoto, M. (2006). "Establishment of perineural invasion models and analysis of gene expression revealed an invariant chain (CD74) as a possible molecule involved in perineural invasion in pancreatic

Kanai, M. & Yano, T. (2001). "Radical distal pancreatectomy with en bloc resection of the celiac artery, plexus, and ganglions for advanced cancer of the pancreatic

between neural alteration and perineural invasion in pancreatic cancer patients

of CX3CR1/CX3CL1 axis in primary and secondary involvement of the nervous

Anselmo, A., Doni, A., Bianchi, P., Laghi, L., Malesci, A., Cervo, L., Malosio, M., Reni, M., Zerbi, A., Di Carlo, V., Mantovani, A. & Allavena, P. (2008). "The chemokine receptor CX3CR1 is involved in the neural tropism and malignant

perineural invasion, a forgotten pathway of dissemination and metastasis." *Cytokine* 

T. & Ochiai, A. (2007). "Detail histologic analysis of nerve plexus invasion in invasive ductal carcinoma of the pancreas and its prognostic impact." *Am J Surg* 

(1991). "Surgical treatment of pancreatic cancer. The Japanese experience." *Int J* 

Miyazaki, I. (1996). "Results of extensive surgery for pancreatic carcinoma." *Cancer*

gut." *J Neurosci* 10(5): 1626-42.

cancer." *Clin Cancer Res* 12(8): 2419-26.

cancer." *Med Hypotheses* 71(3): 386-9.

*Dis Int* 1(3): 469-76.

*Growth Factor Rev* 21(1): 77-82.

*Pathol* 31(11): 1636-44.

*Pancreatol* 9: 135-43.

77(4): 640-5.

with hyperglycemia." *PLoS One* 6(2): e17385.

neurons in the gut." *Z Gastroenterol Verh* 26: 230-3.


Zhu, Z., Friess, H., diMola, F. F., Zimmermann, A., Graber, H. U., Korc, M. & Buchler, M. W.

Zhu, Z., Kleeff, J., Kayed, H., Wang, L., Korc, M., Buchler, M. W. & Friess, H. (2002). "Nerve

pain in human pancreatic cancer." *J Clin Oncol* 17(8): 2419-28.

pancreatic cancer cells." *Mol Carcinog* 35(3): 138-47.

(1999). "Nerve growth factor expression correlates with perineural invasion and

growth factor and enhancement of proliferation, invasion, and tumorigenicity of

### *Edited by Sanjay K. Srivastava*

This book covers pancreatic cancer risk factors, treatment and clinical procedures. It provides an outline of pancreatic cancer genetic risk factors, biomarkers 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 bacterial approaches. 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.

Photo by royaltystockphoto / iStock

Pancreatic Cancer - Clinical Management

N

Pancreatic Cancer

Clinical Management

*Edited by Sanjay K. Srivastava*