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Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. It is one of the most lethal human cancers and leads to an estimated number of 227,000 deaths per year worldwide (Raimondi et al., 2009). It has a high metastatic potential and the majority of diseases present themselves at a very late stage. It is estimated that 80%-90% of patients already in metastatic stage at the time of initial presentation, which results in a dismal prognosis.
As other cancers, The TNM system has been used to stage PDAC according to the American Joint Commission on Cancer (AJCC) and International Union against Cancer (UICC). 'TNM' stands for Tumor, Node, and Metastasis. This system can describe the size of a primary tumor, lymph nodes involved by cancer cells or not, cancer has spread or not. There are 5 stages in the current TNM classification (Table 1).
The median survival of PDAC is less than 6 months (Xu et al., 2011a). Surgical intervention is possible in only 10% of cases. Despite recent progress in chemotherapy, radiation therapy, and surgical resections, the overall survival rate of pancreatic cancer is still less than 5%. Around 95% of patients diagnosed with PDAC will die of this disease within 5 years, 3/4 within a year (Raimondi et al., 2009).
Pancreatic intraepithelial neoplasia (PanIN) is the presumed precursor lesion to infiltrating PDAC. PanINs are defined as neoplastic epithelial proliferations in the smaller caliber pancreatic ducts, and are divided into 3 grades based on the degree of architectural and nuclear atypical present (Hruban et al., 2001). The importance of these lesions as precursors to invasive PDAC has been paid great attention during these recent years because it should be possible to detect and treat these non-invasive precursor lesions before an incurable invasive cancer develops. Therefore, study of novel biomarkers with high sensitivity and specificity for early detection of either precursor or adenocarcinoma is giving hope for improving patient survival.
PanIN is associated with multiple genetic alterations, such as activating point mutations in K-ras oncogene and over-expression of human epidermal growth factor receptor 2 (HER-2)/neu during early stage, and inactivation of the p16 tumor suppressor gene at a later stage followed by the loss of p53, SMAD4, and breast cancer type 2 susceptibility protein (BRCA2) tumor suppressor genes (Harada et al., 2007; Legoffic et al., 2009). However, despite a wealth of molecular studies (Koliopanos et al., 2008), none of the proposed biomarkers are currently recommended for clinical use.
Comparing with other potential biomarkers, microRNA (miRNA) can be stabilized in body fluid and tissue samples, which makes it one of the most promising methods for earlier detection of cancer. Recent studies have focused on the impact of miRNA expression in PDAC, many of which have shown the diagnostic, predictive, and prognostic utility of miRNA profiling in PDAC identifying numerous potential targets including miR-21, miR-10b, miR-196a, and miR-221 (Basu et al., 2011; Bloomston et al., 2007; Giovannetti et al., 2010; Jamieson et al., 2012; Nakata et al., 2011; Szafranska et al., 2007). Most recently, the miRNA alterations that arise during the development of PanINs has also been identified (Yu et al., 2012). Indeed, miRNAs may serve as a long-awaited screening tool for PDAC progression in the future. Furthermore, miRNA expression pattern in PDAC maybe incorporated into modern treatment algorithms to enhance therapeutic management. Equally as exciting is the potential for novel therapies directed against these important disease mediators.
1.2 Pancreatic head cancer and body/tail cancer
1.2.1 Difference in anatomy and embryology
PDAC can be divided into head and body/tail cancers according to the anatomy. Distinctions in anatomical conditions are well known from embryology (In't Veld et al., 2010), histopathology (Atri et al., 1994; Zyromski et al., 2009), and imaging findings (Kawamoto et al., 2009; Schoennagel et al.; Wiersema et al., 1995; Yoshikawa et al., 2006).
Pancreatic development begins with the formation of ventral and dorsal buds, which becomes a ventral head (lower head and uncinate process) and dorsal pancreas (upper head, body and tail), respectively (Figure 1).
Figure 1. The development of the pancreas from dorsal and ventral buds. During maturation, The dorsal pancreatic bud gives rise to the accessory pancreatic duct, while the ventral pancreatic bud gives rise to the major pancreatic duct. 1: Head of pancreas; 2: Uncinate process of pancreas; 3: Pancreatic notch; 4: Body of pancreas; 5: Anterior surface of pancreas; 6: Inferior surface of pancreas; 7: Superior margin of pancreas; 8: Anterior margin of pancreas; 9: Inferior margin of pancreas;
This difference in ontogeny leads to significant differences in cell composition, blood supply and innervations between the head and body/tail of pancreas (In't Veld et al., 2010). The superior pancreaticoduodenal artery (from gastroduodenal artery) and the inferior pancreaticoduodenal artery (from superior mesenteric artery) run in the groove between pancreas and duodenum. They supply the head of pancreas. While the pancreatic branches of splenic artery supply the neck, body and tail of pancreas. The largest of those branches is called the arteria pancreatica magna. The head of pancreas drains into the superior mesenteric and portal veins, while the body and neck drain into splenic vein (Figure 2).
Figure 2. The blood supply and venous drainage of pancreas.
Pancreas is both an endocrine gland producing important hormones (e.g. insulin, glucagon, somatostatin, and pancreatic polypeptide) and a digestive organ secreting pancreatic juice containing digestive enzymes, which assist the absorption of nutrients in the small intestine. The endocrine part of the pancreas is made up of approximately one million cell clusters called islets of Langerhans. The endocrine cells have different secret function: cells secrete glucagon (increase glucose in blood), cells secrete insulin (decrease glucose in blood), cells secrete somatostatin (regulates/stops and cells), and pancreatic polypeptide cells secrete pancreatic polypeptide. The endocrine cells (-, -, - and pancreatic polypeptide cells) are remarkably differentially distributed between head and body/tail of pancreas (In't Veld et al., 2010). The number of endocrine (Langerhans) islets is greater in the body and tail. Insulin-positive endocrine cells are highly refractory to malignant transformation under normal conditions, but recent study has found that insulin-positive endocrine cells could serve as a cell-of-origin of PDAC under oncogenic mutation inducement in combination with pancreatic injury (Gidekel Friedlander et al., 2009).
In addition, the distinction between pancreas head, body, and tail regions is also reflected by different appearances in ultrasonography (Wiersema et al., 1995), computer tomography (Kawamoto et al., 2009), and magnetic resonance imaging (Yoshikawa et al., 2006). Studies have shown that fatty infiltration is usually most prominent in the anterior aspect of the pancreas head and may simulate pancreatic neoplasm (Hori et al., 2011; Kawamoto et al., 2009; Zyromski et al., 2009). In this sense, pancreatic head and body/tail cancers may have different malignant potential.
1.2.2 Difference in clinical presentation
Although increasing evidence has shown difference on clinical presentation (incidence, symptoms, resectability) between pancreatic head and body/tail cancers (Bilimoria et al., 2007; Lau et al., 2010; Matsuno et al., 2004; van Oost et al., 2006), the diversity between the two subtypes of PDAC has not been clarified so far.
Data from Surveillance, Epidemiology, and End Results (SEER) registries of Unite States (1973-2002) have shown that about 77.5% (34,072/43,946) of pancreatic cancer originate at the head of the pancreas, on which consequently most discussion on pancreatic cancer has been focused (Lau et al., 2010). The overall annual incidence of pancreatic head cancer is 5.6 per 100,000, compared with 1.6 of pancreatic body/tail cancer (Lau et al., 2010). Data from National Pancreatic Cancer Registry of Japan (1981-2002, n = 9290) (Matsuno et al., 2004) and Eindhoven Cancer Registry of Netherland (1995-2000, n = 1128) (van Oost et al., 2006) also demonstrated much higher incidences of pancreatic head cancer (62.3% and 56.5%, respectively) than body/tail cancer (17.5% and 12.7%, respectively). For resectable tumors (Stage I: T1N0M0 and T2N0M0), about 70% (6676/9559) located in the head of pancreas as shown by another database in the Unite States (National Cancer Data Base, 1995-2004) (Bilimoria et al., 2007).
Symptoms often do not appear until the disease is in an advanced stage, thus making early detection difficult. Notably, a patient's symptoms will vary depending on the location of the cancer within the pancreas. Both pancreatic head and body/tail cancers can cause non-specific symptoms, such as abdominal pain, nausea, loss of appetite and weight loss. However, only tumor blocking the bile ducts, which pass through the head of pancreas, can cause jaundice (Figure 3). A study from China investigated the clinical-pathological characteristics between pancreatic head cancer (n = 541) and body/tail caner (n = 106) from 1980 to 2003. They found that patients primarily diagnosed with pancreatic body/tail cancer were associated with much less jaundice but more pain, higher serum albumin level, higher carcinoembryonic antigen but lower carbohydrate antigen 19-9 positive rates, and higher metastasis rate (Wu et al., 2007). Other studies also confirmed that patients with pancreatic body/tail cancer were more likely to be diagnosed at an advanced stage (Eyigor et al., 2010; Sener et al., 1999). SEER registries database reported that patients with pancreatic body/tail cancer had a higher proportion of the distant stage diseases (72.7% vs. 39.2%) compared with patients with pancreatic head cancer (Lau et al., 2010).
Figure 3. Pancreatic cancer located in the head (A) and body/tail (B) of pancreas. Bile duct passes through the head of pancreas. Therefore, pancreatic head cancer can easily block the bile ducts and cause jaundice.
188.8.131.52 Diagnostic characterization of genetic markers
Biomarkers are so important to the early diagnosis and prompt treatment of diseases. Carbohydrate antigen 19-9 is a clinically routinely used serum biomarker for PDAC with relative high specificity. Study from China found that pancreatic body/tail cancer were associated with lower serum carbohydrate antigen 19-9 but higher serum carcinoembryonic antigen levels (Wu et al., 2007). However, there is lack of a high sensitive biomarker for PDAC, which make the early diagnosis difficult and lead to a dismal prognosis.
Most recently, as a consequence of technical advances, whole sequencing of the cancer exome has been performed and leads to greater insight into the mutational spectrum of human cancers, including PDAC (Iacobuzio-Donahue, 2012; Jiao et al., 2011; Jones et al., 2004; Sjoblom et al., 2006). In 2008, a comprehensive genetic analysis containing 24 PDACs determined an average of 63 genetic alterations in the sequences of 23,219 transcripts, representing 20,661 protein-coding genes. These alterations defined a core set of 12 cellular signaling pathways and processes explaining the major features of pancreatic tumorigenesis (Jones et al., 2008). Fifteen out of 24 PDAC samples were obtained from the primary tumors. However, the precise locations were not provided, presenting a comparison between pancreatic head and body/tail cancers impossible in this context.
1.2.3 Difference in surgical treatment
In clinic, a pancreatectomy is the surgical removal of pancreas (part or all). Several types of pancreatectomy exist, such as pancreaticoduodenectomy (Whipple procedure), distal pancreatectomy, segmental pancreatectomy, and total pancreatectomy. Pancreatic head and body/tail cancers are usually treated with pancreaticoduodenectomy and distal pancreatectomy, respectively (Figure 4).
Figure 4 Pancreatic head and body/tail cancers are treated with pancreaticoduodenectomy (Whipple procedure) and distal pancreatectomy, respectively
The Whipple procedure consists of removal of the distal half of the stomach (antrectomy), the gall bladder and its cystic duct (cholecystectomy), the common bile duct (choledochectomy), the head of the pancreas, duodenum, proximal jejunum, and regional lymph nodes. Reconstruction (the original one is called Child's reconstruction) consists of attaching the pancreas to the jejunum (pancreaticojejunostomy), attaching the hepatic duct to the jejunum (hepaticojejunostomy), and attaching the stomach to the jejunum (gastrojejunostomy). So that digestive juices and bile can flow into the gastrointestinal tract and food can also pass through.
A distal pancreatectomy is the resection of pancreatic tissue to the left of the superior mesenteric vascular. It has been considered as the standard technique for management of benign and malignant pancreatic disorders in the body and tail. With the improvement of surgical technique, a surgical procedure is available where the spleen is preserved removing only the pancreas, which is also known as spleen preserving distal pancreatectomy.
1.2.4 Difference in prognosis
It is not surprising that pancreatic head cancer has a better overall patient survival than those with pancreatic body/tail cancer, because much more patients with pancreatic body/tail cancer are diagnosed at a relatively advanced stage. SEER registries database showed pancreatic head cancer had a 4% lower overall mortality risk compared with pancreatic body/tail cancer (Lau et al., 2010). However, within the same local-stage, pancreatic head cancer had a much lower 3-year survival rate than pancreatic body/tail (Lau et al., 2010). The National Cancer Data Base of Unite States (1985-1995) including information from 100,313 patients also presented a higher 5-year survival rate for local-stage pancreatic tail cancer (32.4%) compared with local-stage pancreatic head cancer (11.1%) (Sener et al., 1999). Consistent with the results from Western countries, the study from Japan, as mentioned before (Matsuno et al., 2004), showed a significantly higher median survival time (10.2 vs. 9.2 months) and 5-year survival rate (13.8% vs. 10.7%) for pancreatic body/tail cancer (n = 1629) than pancreatic head cancer (n = 5788) in invasive types.
As long as operation is possible, the pancreatic head cancer is surgically treated by pancreaticoduodenectomy, while pancreatic body/tail cancer is treated by a distal pancreatectomy. As mentioned previously, more tumors are diagnosed at early stage and the resectability is higher in pancreatic head cancer. Regarding the complexity of the surgical procedure, pancreatic body/tail cancer shows lower morbidity and mortality than pancreatic head cancer after surgery (Glanemann et al., 2008).
Although surgical resection remains to be the only potential cure for PDAC, only 15-20% of patients newly diagnosed with PDAC are considered for surgical resection (Castellanos et al., 2011). Chemo and/or radiotherapy have emerged as a key factor to improve patient survival both in resectable and non-resectable tumors (Sata et al., 2009). Reports from both Western countries and Japan showed that tumor site (head vs. body/tail) did not relate to progression free-survival or overall survival in patients with advanced or metastatic pancreatic cancer treated with Chemo and/or radiotherapy (Chang et al., 2009; Marechal et al., 2007; Morganti et al., 2003; Tanaka et al., 2008). For resectable tumors, adjuvant chemotherapy was found to be a significant independent predictor of a favorable prognosis in a cohort of 34 Japanese patients with pancreatic body/tail cancer (Murakami et al., 2009), and also was recommend for patients with pancreatic head cancer (Katz et al., 2010). However, no large study has yet been conducted for comparing the different response to Chemo and/or radiotherapy such as toxicity and tumor progression between different tumor sites.
1.3 Difference between pancreatic head and body/tail cancers in vitro models
Cancer cell lines recapitulate the genomic events leading to tumor changes seen in clinical tissues and are valuable tools in studies of tumor cell biology. Different PDAC cell lines arising from primary tumors, liver metastasis, ascites, or lymph node metastasis, exhibit a great deal of diversity in structure and function. To compare the cell lines derived from pancreatic head and body/tail cancers, we reviewed current information characterizing the frequently used PDAC cell lines originating from the primary tumors (BxPC-3, Capan-2, MIA PaCa-2 and Panc-1). Other well-known cell lines such as Colo357, PSN-1 and Panc II-1, which were derived from primary tumor but the exact site of origin were not defined, were not included.
Panc-1 and MIA PaCa-2, which are matched by donor age (10 years), tumor stage, histological differentiation (Table 2) and ultrastructural features (Table 3) (Deer et al., 2010; Sipos et al., 2003), were selected as the one representing pancreatic head cancer and body/tail cancer, respectively.
a: Grading of ultrastructural features of monolayer cultures of PDAC cell lines (grade 1 score 5-8, grade 2 score 9-12, grade 3 score 13-15)
b: Grading of ultrastructural features of spheroid cultures of PDAC cell lines (grade 1 score 7-11, grade 2 score 12-16, grade 3 score 17-21)
c: Estimated score because no spheroid formation.
1.3.1 Cell migration and invasion
Tumor cell motility is the hallmark of invasion and is an initial step in metastasis, while ECM adhesion is a key mediator in cell motility. Compared to MIA PaCa-2, Panc-1 showed a higher binding affinity to collagens (type 1 and 4), the most abundant protein in the ECM, and also exhibited higher adhesion ability to endothelial cells (Deer et al., 2010; ten Kate et al., 2006). In addition, Panc-1 expressed greater levels of a series of cell adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1), sialyl Lewis a (sLea), sialyl Lewis x (sLex), lymphocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) (ten Kate et al., 2006). Moreover, Panc-1 exhibited much higher expression of invasion-associated molecules than MIA PaCa-2, such as EphA2 (Duxbury et al., 2004a).
Actually, invasion is a consequence of the cross-talk that occurs between cancer cells and the stroma cells. For better mimicking of the tumor environment, a study using co-culture system with PDAC cells and tumor-derived fibroblasts demonstrated that hepatocyte growth factor produced by the fibroblasts could initiate an apparent invasion-stimulating response in strong c-met expressing Panc-1 cells but not in weak expressing MIA PaCa-2 cells (Qian et al., 2003).
1.3.2 Pro-angiogenic potential
Angiogenesis is critical for tumor growth and metastasis. Vascular endothelial growth factor (VEGF), which principally mediates angiogenesis, can determine the tumor angiogenic switch in cancer. Compared to Panc-1, MIA PaCa-2 secreted lower levels of VEGF (Holloway et al., 2006). Both Panc-1 and MIA PaCa-2 do not express cyclooxygenase-2 (COX-2) protein, an important mediator of angiogenesis and tumor growth, which may indicate a relative low pro-angiogenic potential (Deer et al., 2010).
Tumorigenicity in vivo is an efficient way for evaluation of the tumor formation and metastatic potential of cancer cells, and is commonly measured by several parameters such as tumor mass, tumor size, rate of growth and metastasis. However, the tumor formation abilities varied among different methods, such as subcutaneous cancer cell injection, intra-peritoneal cancer cell injection, orthotopic tumor implantation and orthotopic cancer cell injection models (Deer et al., 2010). Compared to other methods, orthotopic injection of cancer cell could better reflect the clinical microenvironment and provide more convincing data. In this model, both Panc-1 and MIA PaCa-2 presented high intra-pancreatic tumorigenicity, local infiltration and distant metastasis potential (Hotz et al., 2003; Loukopoulos et al., 2004). However, data directly comparing the tumorigenicity of the two PDAC cell lines were not given in this model.
By using immunohistochemical analysis in vivo, Panc-1 and MIA PaCa-2 tumors demonstrated quite similar major morphology, mucin accumulation, cytokeratin (CK) profile (CK7/CK19 and CK8/CK18), trans- (Vimentin, CK20, Chr-A and 1-chym) and dedifferentiation (pdx-1, shh and ptc) patterns (Neureiter et al., 2005).
1.3.4 Chemo and/or radiotherapy resistance
Chemo and/or radiotherapy promote cancer cell death primarily by the induction of apoptosis. A number of studies have proven abundant evidence that Panc-1 exhibits much higher half maximal inhibitory concentration (IC50) values for 5-fluorouracile (5-FU) and gemcitabine, and radiation than MIA PaCa-2 (Dai et al., 2010; Galloway et al., 2009; Pan et al., 2008). In addition, MIA PaCa-2 was more sensitive than Panc-1 to oncolytic therapy and presented a dramatic increase in apoptosis (Dai et al., 2010). The possible reason was that the expression of anti-apoptotic proteins were much higher in Panc-1 than those in MIA PaCa-2 (Galloway et al., 2009; Pan et al., 2008). Ribonucleotide reductase M2 subunit (RRM2), a gemcitabine-resistant enzyme was also found to have significantly higher expression in Panc-1 than MIA PaCa-2 (Duxbury et al., 2004b).
1.3.5 Genetic alterations and expression patterns
Genetic alterations in PDAC are common, both at the cell and tissue levels. Cancer progression through the accumulation of genetic alterations results in a gain of cell growth and proliferation, and subsequently in increased dissemination and metastatic potential. The genetic basis of PDAC is usually elucidated using a candidate gene approach, which has identified the four most frequent mutations. Mutation exists in KRAS, TP53 and CDKN2A/p16 genes but not in SMAD4/DPC4 in both cell lines (Deer et al., 2010; Loukopoulos et al., 2004; Sipos et al., 2003).
A meta-analysis including a consensus set of 2984 genes from four independent studies strictly compared the gene expression profiles between PDAC and normal pancreatic tissue, and found 62 differentially expressed genes already known to associate with tumorigenesis of PDAC (Grutzmann et al., 2005). Using this database (Grutzmann et al., 2005), we compared the PDAC-associated gene profiles (http://www.pancreasexpression.org/index.html) between Panc-1 and MIA PaCa-2 and found 52 differentially expressed genes which had at least a 2-fold change in expression. Fourteen out of the 52 genes have already been described in detail (related PDAC cells events) and are presented in Table 4.
1.4 Aim of the study
In pancreatic serous cystic neoplasms, tumor location in the head of pancreas were independently associated with local invasiveness (Khashab et al., 2011). In colon cancer, a number of studies have demonstrated that right- and left-sided tumors exhibit different genetic, biological and demographical characteristics and risk factors, suggesting that the carcinogenesis and tumor progression of colon cancer may differ with tumor location (Slattery et al., 2011; Weiss et al., 2011). These findings support the theory of different mechanisms in carcinogenesis of tumors at different locations and confirm the importance of subsite division. However, the genetic or molecular diversity between the pancreatic head and body/tail cancers has not been elucidated yet. Given the complex and heterogeneous nature of PDAC, it is reasonable to hypothesize that these two subtypes may be separated from each other and differ in phenotype and genotype.
So far, not enough attention has been paid to the diversity between pancreatic head and body/tail cancers and the overall information is limited. The current clinical data support higher incidence and easier detection of pancreatic head cancer compared with pancreatic body/tail cancer. For tumors at the local-stage, pancreatic head cancer has a lower survival than pancreatic body/tail cancer. Although the previous reports have large cohort of patients, the evidence is still not so convincing because there is a lack of strictly case-matched comparison between the two subtypes of PDAC. Further pioneering studies on patient tumor samples are needed.
Our aim was to evaluate the genetic diversity at molecular level between the pancreatic head cancer and pancreatic body/tail cancer. We performed a comparison of miRNA expression profile between pancreatic body/tail cancer and pancreatic head cancer, and found two miRNAs were significantly differentially expressed. We further aim to clarify the role of the differentially expressed miRNA in the phenotype varity between pancreatic body/tail cancer and pancreatic head cancer in vitro study.