**3.2.1 CA19-9**

The best-established marker is CA19-9, which is a sialylated Lewis antigen of the MUC1 protein with an overall sensitivity ranging from 41 to 86% and specificity from 33 to 100%

Biomarkers in Gastrointestinal Cancer: Focus on Colon, Pancreatic and Gastric Cancer 59

their development and progression (see table 1, modified from (Wang & Sen, 2011)). MiR signatures specific for normal pancreas, chronic pancreatitis and cancer tissues have been identified and have been proposed to represent helpful markers for differential diagnosis of pancreatic cancer from chronic inflammatory disease of the pancreas and even other tumors (Hamada & Shimosegawa, 2011)(Vincent et al., 2011)(Wang & Sen, 2011). In addition, these differential-expressing miRs can also be profiled in blood as a minimally invasive biomarker assay for pancreatic cancer. This finding is extremely promising since there is no reliable biomarker assay, much less of minimally invasive nature, currently available for early

miR-10 Up (Bloomston et al., 2007)(Zhang et al., 2009b) miR-15b Up (Lee et al., 2007)(Zhang et al., 2009b)

2007)(Zhang et al., 2009b)(Mees et al., 2010)

(Szafranska et al., 2008)

(Szafranska et al., 2008)

2007)(Zhang et al., 2009b)

2007)(Szafranska et al., 2008)

2007)(Szafranska et al., 2008)

(Zhang et al., 2009b)

(Bloomston et al., 2007)

(Szafranska et al., 2007) (Bloomston et al., 2007) (Szafranska et al., 2008)

(Bloomston et al., 2007)

(Szafranska et al., 2007)(Bloomston et al.,

miR-21 Up (Lee et al., 2007)(Bloomston et al.,

detection, diagnosis and predicting prognosis of pancreatic cancer patients.

microRNA Expression profile References Let-7f-1 Up (Lee et al., 2007) Let-7d Up (Lee et al., 2007)

miR-16-1 Up (Lee et al., 2007)

miR-92 Up (Lee et al., 2007) miR-95 Up (Zhang et al., 2009b) miR-96 Down (Szafranska et al., 2007)

miR-139 Down (Lee et al., 2007) miR-142-P Down (Lee et al., 2007)

miR-146 Up (Szafranska et al., 2007)

miR-148b Down (Szafranska et al., 2007)

miR-143 Up

miR-148a Down

miR-23 Up (Bloomston et al., 2007) miR-24 Up (Lee et al., 2007)

miR-31 Up (Szafranska et al., 2007)

miR-99 Up (Bloomston et al., 2007)

miR-100 Up (Lee et al., 2007)(Bloomston et al., 2007) miR-103 Up (Bloomston et al., 2007)(Zhang et al., 2009b)

miR-125 Up (Lee et al., 2007)(Bloomston et al., 2007) miR-130b Down (Szafranska et al., 2007)(Bloomston et al.,

miR-145 Up (Szafranska et al., 2008)(Zhang et al., 2009b)

miR-107 Up (Lee et al., 2007)(Bloomston et al.,

(Bünger et al., 2011)(Buxbaum & Eloubeidi, 2010). As a marker for early pancreatic cancer, there are some important weaknesses. Approximately 10% of the population with the Lewis-negative genotype is not able to produce CA19-9, secondary to a lack of the enzyme involved in its synthesis, even if they have advanced pancreatic cancer. Recently it has been reported that patients with undetectable CA19-9 have a better prognosis than those with elevated levels. Patients with small pancreatic cancers often show false negative CA19-9 values, thus eliminating its value in early diagnosis. In addition, patients with certain blood types are incapable of expressing the antigen recognized by CA19-9. Moreover, CA19-9 elevation is common in patients with obstructive jaundice even without malignancy because of the reduction in clearance by the cholestatic liver. Furthermore, false positive CA19-9 elevation is also frequently observed in patients with cancers of the upper gastrointestinal tract, ovarian cancer, hepatocellular cancer, benign conditions of the hepatobiliary system and chronic pancreatitis (Xu et al., 2011). Nevertheless, continuous evaluation of this marker strongly suggests progressive disease during chemotherapy or recurrence after operation (Hamada & Shimosegawa, 2011). Thus CA19-9 is considered the standard for monitoring response to chemotherapy and recurrence following surgical resection in patients with pancreatic cancer but not for the initial diagnosis of the disease in the asymptomatic population (Xu et al., 2011)(Buxbaum & Eloubeidi, 2010)(Vincent et al., 2011).

In addition to serum it has been shown that pancreatic juice might also be a source of pancreatic cancer tumor markers. Several groups evaluated the diagnostic value of CA19-9 in pancreatic juice. Some groups found that CA19-9 concentrations were significantly higher in patients with cancer than in patients with chronic pancreatitis and other non-neoplastic patients (Malesci et al., 1987) showing a diagnostic value approximately similar to that of serum CA19-9 (Nishida et al., 1988)(Chen et al., 1989). Other studies could not confirm the diagnostic value of CA19-9 in pancreatic juice (Matsumoto et al., 1994). Further investigation into the exact role of CA19-9 in pancreatic juice is required. However, other potentially interesting biomarkers, like *KRAS* mutations, 90K, CEA were identified in this pancreatic juice and need further investigation (Nakaizumi et al., 1999)(Gentiloni et al., 1995).

#### **3.2.2 Micro-RNA**

Small non-coding RNAs are now attracting increased attention as robust regulators of various biological processes, including cancer progression. The micro-RNAs (miRs) are a class of conserved small non-coding RNA's of 17-25 nucleotides in length that regulate gene expression by either repressing the translation or causing degradation of multiple target mRNAs. *MiR* genes represent about 1% of the genome in different species and it is estimated that about 30% of the protein-coding genes in the human genome are regulated by miRs (Wang & Sen, 2011). These miRs play a central role in the regulation of cellular functions, such as migration, invasion and stem cell functions (Vincent et al., 2011). Extensive mapping of the known *miR* genes revealed that these are often located in the genomic intervals rearranged in cancers including those displaying amplification, loss of heterozygosity, common breakpoints and fragile sites. Furthermore, functional analyses suggest that miRs play roles in cancer initiation, invasion and progression processes and, therefore, may prove to be informative biomarkers of detection, diagnosis and prognosis besides being potential targets of therapy (Wang & Sen, 2011).

Over 300 miRs have been identified, and widespread alterations in these miRs have been recognized in various types of cancer, including pancreatic cancer, and seem to contribute to

(Bünger et al., 2011)(Buxbaum & Eloubeidi, 2010). As a marker for early pancreatic cancer, there are some important weaknesses. Approximately 10% of the population with the Lewis-negative genotype is not able to produce CA19-9, secondary to a lack of the enzyme involved in its synthesis, even if they have advanced pancreatic cancer. Recently it has been reported that patients with undetectable CA19-9 have a better prognosis than those with elevated levels. Patients with small pancreatic cancers often show false negative CA19-9 values, thus eliminating its value in early diagnosis. In addition, patients with certain blood types are incapable of expressing the antigen recognized by CA19-9. Moreover, CA19-9 elevation is common in patients with obstructive jaundice even without malignancy because of the reduction in clearance by the cholestatic liver. Furthermore, false positive CA19-9 elevation is also frequently observed in patients with cancers of the upper gastrointestinal tract, ovarian cancer, hepatocellular cancer, benign conditions of the hepatobiliary system and chronic pancreatitis (Xu et al., 2011). Nevertheless, continuous evaluation of this marker strongly suggests progressive disease during chemotherapy or recurrence after operation (Hamada & Shimosegawa, 2011). Thus CA19-9 is considered the standard for monitoring response to chemotherapy and recurrence following surgical resection in patients with pancreatic cancer but not for the initial diagnosis of the disease in the asymptomatic

population (Xu et al., 2011)(Buxbaum & Eloubeidi, 2010)(Vincent et al., 2011).

juice and need further investigation (Nakaizumi et al., 1999)(Gentiloni et al., 1995).

besides being potential targets of therapy (Wang & Sen, 2011).

**3.2.2 Micro-RNA** 

In addition to serum it has been shown that pancreatic juice might also be a source of pancreatic cancer tumor markers. Several groups evaluated the diagnostic value of CA19-9 in pancreatic juice. Some groups found that CA19-9 concentrations were significantly higher in patients with cancer than in patients with chronic pancreatitis and other non-neoplastic patients (Malesci et al., 1987) showing a diagnostic value approximately similar to that of serum CA19-9 (Nishida et al., 1988)(Chen et al., 1989). Other studies could not confirm the diagnostic value of CA19-9 in pancreatic juice (Matsumoto et al., 1994). Further investigation into the exact role of CA19-9 in pancreatic juice is required. However, other potentially interesting biomarkers, like *KRAS* mutations, 90K, CEA were identified in this pancreatic

Small non-coding RNAs are now attracting increased attention as robust regulators of various biological processes, including cancer progression. The micro-RNAs (miRs) are a class of conserved small non-coding RNA's of 17-25 nucleotides in length that regulate gene expression by either repressing the translation or causing degradation of multiple target mRNAs. *MiR* genes represent about 1% of the genome in different species and it is estimated that about 30% of the protein-coding genes in the human genome are regulated by miRs (Wang & Sen, 2011). These miRs play a central role in the regulation of cellular functions, such as migration, invasion and stem cell functions (Vincent et al., 2011). Extensive mapping of the known *miR* genes revealed that these are often located in the genomic intervals rearranged in cancers including those displaying amplification, loss of heterozygosity, common breakpoints and fragile sites. Furthermore, functional analyses suggest that miRs play roles in cancer initiation, invasion and progression processes and, therefore, may prove to be informative biomarkers of detection, diagnosis and prognosis

Over 300 miRs have been identified, and widespread alterations in these miRs have been recognized in various types of cancer, including pancreatic cancer, and seem to contribute to their development and progression (see table 1, modified from (Wang & Sen, 2011)). MiR signatures specific for normal pancreas, chronic pancreatitis and cancer tissues have been identified and have been proposed to represent helpful markers for differential diagnosis of pancreatic cancer from chronic inflammatory disease of the pancreas and even other tumors (Hamada & Shimosegawa, 2011)(Vincent et al., 2011)(Wang & Sen, 2011). In addition, these differential-expressing miRs can also be profiled in blood as a minimally invasive biomarker assay for pancreatic cancer. This finding is extremely promising since there is no reliable biomarker assay, much less of minimally invasive nature, currently available for early detection, diagnosis and predicting prognosis of pancreatic cancer patients.


Biomarkers in Gastrointestinal Cancer: Focus on Colon, Pancreatic and Gastric Cancer 61

In addition, successful therapeutic targeting of miRs (silencing, antisense blocking and miR modification of oncogenic miRs) also holds significant promise towards improved clinical management of patients with cancer, especially those with pancreatic carcinomas, since these patients have very limited treatment options available at this time (Wang & Sen,

Malignant cells have high constitutive glucose uptake and metabolism compared with normal cells (Pizzi et al., 2009). A family of glucose transporter isoforms (GLUT), which is currently composed of 13 members, facilitates the entry of glucose into cells. These are passive carriers and function as an energy-independent system that transports glucose down a concentration gradient. GLUT-1, a member of this family, is considered to be the predominantly upregulated glucose transporter in malignant epithelial tissue and mesothelium, and has been found to correlate with biological behavior in various

Various studies have shown a close relationship between GLUT-1 expression and tumor aggressiveness and poor prognosis in squamous cell carcinoma of the head and neck and in carcinomas of the lung, stomach, gallbladder, colorectum, kidney, bladder, ovary and cervix. An increased GLUT-1 expression has also proved to be associated with pancreatic cancer invasiveness both *in vitro* and *in vivo* (Basturk et al., 2011). However, literature data regarding the prognostic significance of immunohistochemical GLUT-1 expression in pancreatic ductal adenocarcinoma are limited and non consistent, as a prognostic significance of GLUT-1 expression has been found by some research groups (Sun et al., 2007)(Pizzi et al., 2009) and not by the other (Lyshchik et al., 2007). Differences can be ascribed to heterogeneity of histological types of pancreatic cancer and to the different scoring systems. Overall, GLUT-1 overexpression is regarded as a relative early event in pancreatic carcinogenesis and may be ascribed to local hypoxia. Furthermore, GLUT-1 expression seems to correlate to a higher glucose uptake in undifferentiated and highly proliferative pancreatic cancer cells (Pizzi et al., 2009). Moreover, GLUT-1 promotes cellular invasiveness in pancreatic cancer, which is matrix metalloproteinase 2 (MMP2)-dependent, with MMP-2 being transcriptionally activated by increased GLUT-1 levels (Ito et al., 2004). In addition, an increased expression of GLUT-1 molecules in pancreatic tumors has been suggested to contribute to the higher rate of fluorine 18 fluorodeoxyglucose (18F-FDG) uptake into tumor cells compared with normal pancreatic tissue, as determined by standardized uptake value (SUV). Also, SUV has been found to be a predictor of survival in patients with ductal adenocarcinomas. Therefore, in addition to being of diagnostic value imaging-wise, GLUT-1 may also be a potential therapeutic target to limit glucose uptake and metabolism, thereby limiting the proliferative potential of malignant cells (Basturk et al., 2011). Apigenin, a flavonoid with significant anti-proliferative properties that inhibit pancreatic cancer cell proliferation, has been shown to inhibit glucose uptake as well as both GLUT-1 mRNA and protein expression in human pancreatic cancer cell lines. In addition, the PI3K/Akt pathway may be involved in mediating apigenin's effects on downstream targets such as GLUT-1 (Melstrom et al., 2008). However, literature regarding the biological significance of GLUT-1 expression in pancreatic neoplasia has been limited and

2011)(Rachagani et al., 2010).

malignancies (Basturk et al., 2011).

controversial (Basturk et al., 2011).

**3.3 Glucose transporter isoforms (GLUT)** 


Table 1. Deregulated microRNAs in pancreatic ductal adenocarcinoma (modified from (Wang & Sen, 2011)).

In addition, successful therapeutic targeting of miRs (silencing, antisense blocking and miR modification of oncogenic miRs) also holds significant promise towards improved clinical management of patients with cancer, especially those with pancreatic carcinomas, since these patients have very limited treatment options available at this time (Wang & Sen, 2011)(Rachagani et al., 2010).

#### **3.3 Glucose transporter isoforms (GLUT)**

60 Biomarker

miR-181a Up (Lee et al., 2007)(Bloomston et al., 2007)

miR-181c Up (Lee et al., 2007)(Bloomston et al., 2007)

miR-181b Up (Bloomston et al., 2007)

miR-181d Up (Bloomston et al., 2007) miR-186 Up (Zhang et al., 2009b) miR-190 Up (Zhang et al., 2009b) miR-194 Up {Mees:2010fr}

miR-196a Up (Szafranska et al., 2007)

miR-196b Up (Szafranska et al., 2007) miR-199a Up (Bloomston et al., 2007)

miR-200c Up {Mees:2010fr} miR-203 Up (Ikenaga et al., 2010)

miR-212 Up (Lee et al., 2007) miR-213 Up (Bloomston et al., 2007) miR-217 Down (Szafranska et al., 2007)

miR-220 Up (Bloomston et al., 2007)

miR-222 Up (Szafranska et al., 2007)

miR-301 Up (Lee et al., 2007) miR-345 Down (Lee et al., 2007)

miR-376a Up (Lee et al., 2007) miR-424 Up (Lee et al., 2007) miR-429 Up {Mees:2010fr}

Table 1. Deregulated microRNAs in pancreatic ductal adenocarcinoma (modified from

miR-200b Up (Zhang et al., 2009b){Mees:2010fr}

 (Lee et al., 2007)(Szafranska et al., 2007)(Szafranska et al., 2008)

(Bloomston et al., 2007)(Zhang et al., 2009b)

(Szafranska et al., 2008)(Zhang et al., 2009b)

(Bloomston et al., 2007)(Szafranska et al.,

(Szafranska et al., 2007)(Bloomston et al., 2007)(Zhang et al., 2009b){Mees:2010fr}

(Bloomston et al., 2007)(Zhang et al., 2009b)

(Szafranska et al., 2007)(Bloomston et al.,

2007)(Szafranska et al., 2008)

(Zhang et al., 2009b)

(Szafranska et al., 2007) (Bloomston et al., 2007) (Szafranska et al., 2008)

(Szafranska et al., 2007) (Bloomston et al., 2007) (Szafranska et al., 2008)

(Szafranska et al., 2007)

2008)(Zhang et al., 2009b)

(Szafranska et al., 2008)

(Lee et al., 2007)

miR-155 Up

miR-205 Up

miR-210 Up

miR-221 Up

miR-223 Up

miR-375 Down

(Wang & Sen, 2011)).

Malignant cells have high constitutive glucose uptake and metabolism compared with normal cells (Pizzi et al., 2009). A family of glucose transporter isoforms (GLUT), which is currently composed of 13 members, facilitates the entry of glucose into cells. These are passive carriers and function as an energy-independent system that transports glucose down a concentration gradient. GLUT-1, a member of this family, is considered to be the predominantly upregulated glucose transporter in malignant epithelial tissue and mesothelium, and has been found to correlate with biological behavior in various malignancies (Basturk et al., 2011).

Various studies have shown a close relationship between GLUT-1 expression and tumor aggressiveness and poor prognosis in squamous cell carcinoma of the head and neck and in carcinomas of the lung, stomach, gallbladder, colorectum, kidney, bladder, ovary and cervix. An increased GLUT-1 expression has also proved to be associated with pancreatic cancer invasiveness both *in vitro* and *in vivo* (Basturk et al., 2011). However, literature data regarding the prognostic significance of immunohistochemical GLUT-1 expression in pancreatic ductal adenocarcinoma are limited and non consistent, as a prognostic significance of GLUT-1 expression has been found by some research groups (Sun et al., 2007)(Pizzi et al., 2009) and not by the other (Lyshchik et al., 2007). Differences can be ascribed to heterogeneity of histological types of pancreatic cancer and to the different scoring systems. Overall, GLUT-1 overexpression is regarded as a relative early event in pancreatic carcinogenesis and may be ascribed to local hypoxia. Furthermore, GLUT-1 expression seems to correlate to a higher glucose uptake in undifferentiated and highly proliferative pancreatic cancer cells (Pizzi et al., 2009). Moreover, GLUT-1 promotes cellular invasiveness in pancreatic cancer, which is matrix metalloproteinase 2 (MMP2)-dependent, with MMP-2 being transcriptionally activated by increased GLUT-1 levels (Ito et al., 2004). In addition, an increased expression of GLUT-1 molecules in pancreatic tumors has been suggested to contribute to the higher rate of fluorine 18 fluorodeoxyglucose (18F-FDG) uptake into tumor cells compared with normal pancreatic tissue, as determined by standardized uptake value (SUV). Also, SUV has been found to be a predictor of survival in patients with ductal adenocarcinomas. Therefore, in addition to being of diagnostic value imaging-wise, GLUT-1 may also be a potential therapeutic target to limit glucose uptake and metabolism, thereby limiting the proliferative potential of malignant cells (Basturk et al., 2011). Apigenin, a flavonoid with significant anti-proliferative properties that inhibit pancreatic cancer cell proliferation, has been shown to inhibit glucose uptake as well as both GLUT-1 mRNA and protein expression in human pancreatic cancer cell lines. In addition, the PI3K/Akt pathway may be involved in mediating apigenin's effects on downstream targets such as GLUT-1 (Melstrom et al., 2008). However, literature regarding the biological significance of GLUT-1 expression in pancreatic neoplasia has been limited and controversial (Basturk et al., 2011).

Biomarkers in Gastrointestinal Cancer: Focus on Colon, Pancreatic and Gastric Cancer 63

form is usually poorly differentiated, located most frequently in the proximal stomach and its incidence is rising at an alarming rate in overweight young men suffering from gastroesophageal reflux. Diffuse type cancers have usually a worse prognosis (Wagner &

Surgical resection remains the mainstay of treatment and cure in localized, non-metastatic gastric cancer while no globally accepted consensus exists on the best treatment regimen to be used in advanced gastric cancer (De Vita et al., 2010)(Lorenzen & Lordick, 2011). At present, the combination of a fluoropyrimidine and a platinum analogue either alone or in combination with a third drug such as an antracycline or taxanes are the most effective combinations resulting in a median survival of 8-10 months (Lorenzen & Lordick, 2011). These observations suggest the need for new therapeutic approaches, based on the implementation of predictive biomarkers, to further improve the outcome of patients with advanced gastric cancer (De Vita et al., 2010)(Wagner & Moehler, 2009)(Lorenzen & Lordick, 2011). A better understanding of the molecular basis of cancer has contributed to the development of rationally designed molecular targeted therapies, which interfere with the signaling cascades involved in cell differentiation, proliferation and survival (Gravalos & Jimeno, 2008). Recently, the evidence that upregulation of signaling pathways of EGFRfamily plays a central role in cell differentiation, proliferation, and survival has supported the development of antitumor strategies against these targets (De Vita et al., 2010). One of

the most considerable innovative targets in human cancer is the HER family.

The epidermal growth factor receptor (EGFR) family is composed of four members: HER1 also known as EGFR1, HER2, HER3 and HER4, amongst which the EGFR1 and HER2 represents targets for drugs currently under development for gastric cancer (Wagner & Moehler, 2009). The HER2 protein is a 185 kDa transmembrane tyrosine kinase (TK) receptor encoded by a gene located on chromosome 17q21, with an extracellular ligand-binding domain, a short transmembrane domain and an intracellular domain with TK activity. Up to now, no ligands have been identified for its extracellular domain, but it seems to be the preferred heterodimerization partner for other members of the HER family (De Vita et al., 2010). HER2 functions as an oncogene and its amplification or overexpression plays a central role in the initiation, progression and metastasis of some common cancers. Aberrant HER2 expression or function has been implicated in about 10-34% of invasive breast cancers. In addition, HER 2 also appears to be overexpressed in colon, bladder, ovarian, endometrium, lung, uterine cervix, head and neck, and esophageal carcinomas. The first description of HER2 overexpression in gastric cancer, using IHC, was reported in 1986. Since then, a number of studies have confirmed these findings, reporting a HER2 positivity rate in a wide range (6-35%) of gastric carcinomas. Moreover, HER2 expression varies depending on histology and on primary tumor location (Lorenzen & Lordick, 2011)(De Vita et al., 2010). The randomized open-label, multinational phase III ToGA (Trastuzumb for Gastric Cancer) trial, in which by now the largest population of 3807 gastric cancers were centrally screened for *HER2* gene amplification (Fluorescent in situ hybridization (FISH)) and HER2 protein overexpression (IHC 3+), reported a HER2 positivity of 22.1%, with a high degree of concordance between IHC and FISH (87,2%). Furthermore, HER2 positivity rates were found to be higher in esophagogastric junction cancer than in gastric cancer and

Moehler, 2009).

**4.2 HER2** 
