Modification of pancreatic carcinogenesis in the hamster model. 6. The effect of ductal ligation and excision

The effect of ligation and excision of the pancreatic duct in pancreatic carcinogenesis was examined in the hamster model. Animals were treated with a single dose (20 mg/kg body weight) of N-nitrosobis(2-oxopropyl)amine (BOP) either immediately (Group 1) or on Days 1 (Group 2), 3 (Group 3) or 7 (Group 4) after ligation and excision of the duct of the splenic lobe. Group 5 received BOP shortly after laparoscopy, and Group 6 consisted of BOP-treated controls. All hamsters were killed 46 weeks after BOP treatment. The results showed that despite advanced atrophy of the splenic lobe distal to the excised duct in Groups 1-4, some hamsters in Groups 2, 3, and 4 showed hyperplasia, dysplasia, and increased mitotic activities of ductal and ductular cells. However, carcinomas in the duct-excised atrophic lobe were found only in Groups 1-3. These data indicate that BOP carcinogenesis is mediated through blood circulation, and that cancer development is not inhibited in the duct-excised lobe for up to 3 days after surgery. However, in the entire pancreas, a significant reduction in tumor incidence was seen when the carcinogen was given immediately, or to a lesser extent, 1 day after surgery, regardless of whether or not excision was made. On the contrary, BOP, when given 3 and 7 days after duct excision, enhanced tumor development in the nonexcised (intact) pancreas, compared with other test groups and with BOP controls. Both inhibition and enhancement seemed due to a proportional decrease and increase, respectively, of BOP-responsive cells throughout the intact pancreas.     

The role of pancreatic islets in experimental pancreatic carcinogenicity.

Our previous studies have suggested that the presence of intact islets is essential for the induction of pancreatic exocrine tumors in the Syrian hamster model. To validate this, we investigated the effect of the carcinogen, N-nitrosobis(2-oxo-propyl)amine (BOP) in hamsters, in which homologous isolated intact islets were transplanted into the submandibular gland (SMG). Freshly isolated pure islets from hamster donors were transplanted into the left SMG of 20 female host hamsters. Ten of these hamsters (group 1) received BOP (40 mg/kg) weekly for 3 weeks. Another 10 hamsters (group 2) were kept untreated. In groups 3 and 4 (10 hamsters each) the salt solution or isolated pancreatic ductal cells, respectively, was injected into the gland. In other groups (10 hamsters each) islets were transplanted into the peri-SMG connective tissue (group 5) or into the renal subcapsular space (group 6). Hamsters of group 1 (40 mg/kg, weekly for 3 weeks) as were group 7 hamsters, which served as BOP-treated controls. All BOP-treated hamsters developed pancreatic lesions. Similar hyperplastic and atypical ductal/ductular proliferation and in situ carcinoma were found in the SMG of many group 1 hamsters. No such lesions were found in the SMG, peri-SMG, or renal subcapsular space of the other groups. Islets appear to be involved in carcinogenicity of BOP. The mechanism is obscure.     

Modification of pancreatic carcinogenesis in the hamster model. 3. Inhibitory effect of alloxan.

Alloxan, when given intravenously at a dose of 60 mg/kg body weight 2 hours prior to subcutaneous injection of the potent pancreatic carcinogen N-nitrosobis (2-oxopropyl) amine (BOP), inhibited the induction of hyperplastic and neoplastic pancreatic lesions in a statistically significant fashion (P less than 0.01). The number of lesions per animal affected was markedly less in these animals, compared with BOP-treated control animals. BOP administration 2 weeks after alloxan treatment, at which time pancreatic islet cell regeneration is considered completed, did not alter either the incidence or number of lesions. The results support our view that the pancreatic islet cells are the primary source of BOP metabolism. The concomitant inhibition of gallbladder tumors, but not of common duct neoplasms, in hamsters receiving BOP 2 hours after alloxan could indicate that alloxan's inhibitory effects on BOP carcinogenesis are not restricted to the pancreas.     

Stimulation of islet cell proliferation enhances pancreatic ductal carcinogenesis in the hamster model.

Previous studies have shown that some N-nitrosobis (2-oxopropyl)amine (BOP)-induced ductal/ductular pancreatic cancers in the hamster model develop within islets and that streptozotocin (SZ) pretreatment that caused islet degeneration and atrophy inhibits pancreatic cancer induction. Hence, it appears that in this model islets play a significant role in exocrine pancreatic carcinogenesis. To examine whether stimulation of islet cell proliferation (nesidioblastosis) enhances pancreatic exocrine cancer development, we tested the effect of the pancreatic carcinogen BOP in hamsters after induction of nesidioblastosis by cellophane wrapping. Before wrapping, hamsters were treated with SZ to inhibit pancreatic tumor induction in the unwrapped pancreatic tissues. Control groups with a wrapped pancreas did not receive SZ. Six weeks after SZ treatment, all hamsters were treated with BOP (10 mg/kg body weight) weekly for 10 weeks and the experiment was terminated 38 weeks after the last BOP treatment. Many animals recovered from their diabetes at the time when BOP was injected and many more after BOP treatment. Only nine hamsters remained diabetic until the end of the experiment. Both SZ-treated and control groups developed proliferative and malignant pancreatic ductal-type lesions primarily in the wrapped area (47%) but less frequently in the larger segments of the pancreas, including the splenic lobe (34%), gastric lobe (13%), and duodenal lobe (6%). Only a few lesions developed in the unwrapped pancreatic region of nine diabetic hamsters with atrophic islets, whereas seven of these hamsters had tumors in the wrapped area. Histologically, most tumors appeared to originate from islets, many invasive carcinomas had foci of islets, and some tumor cells showed reactivity with anti-insulin. The results show that, in the BOP hamster model, islets are the site of formation of the major fraction of exocrine pancreatic cancer and that induction of nesidioblastosis enhances pancreatic carcinogenesis.     

Modification of pancreatic carcinogenesis in the hamster model. 7. Inhibitory effect of bethanechol chloride

Experiments were designed to investigate in the hamster model the effect on pancreatic carcinogenesis of bethanechol chloride (BC), which is known to increase pancreatic protein synthesis in rats. Hamsters received a single (15 mg/kg body weight) dose of BC either before, simultaneously with, or after a single dose of N-nitrosobis(2-oxopropyl)amine (BOP; 20 mg/kg body weight). A second group was treated daily with BC (7.5 mg/kg body weight) for 24 weeks, following BOP. The control groups consisted of animals treated with BOP only, with BC only, and with solvent only. The surviving hamsters were killed 46 weeks after BOP treatment. BC, whether given before, simultaneously with, or after BOP, significantly reduced the incidence of pancreatic ductal/ductular carcinomas. The multiplicity, size, and latency of carcinomas were also affected by BC. A more pronounced inhibition of cancer induction occurred in the group treated daily with BC after BOP. The possible mechanisms involved in the inhibitory action of BC on pancreatic carcinogenesis are discussed.     

Inhibitory effect of 1α-hydroxyvitamin D3 on N-nitrosobis(2-oxopropyl)amine-induced cholangiocarcinogenesis in Syrian hamsters

Sixty-three male 5-week-old Syrian hamsters received the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) s.c. in 5 weekly injections (the first, 70 mg/kg body, and the remaining, 20mg/kg each). The hamsters that received BOP were given intragastric administration of 0.2 ml of medium chain triglyceride (MCT) with or without 0.04 μg of 1α-hydroxyvitamin D3 [1α(OH)D3] through a feeding tube for 12 weeks. Thus, 3 groups were assigned:Group 1;BOP alone (n＝20), Group 2;BOP＋MCT (n＝18) and Group 3;BOP＋1α(OH)D3 (n＝25). The mean body weight of Group 3 was lower than those of Groups 1 and 2 at the end of the experiment (p＜0.001,Tukey-Kramer HSD test). At the end of week 12, all surviving hamsters were put to sleep. The incidences of liver tumors were 80%, 72% and 32% in Groups 1, 2 and 3, respectively. The incidence of tumors in Group 3 was significantly lower than in Group 1 and Group 2 (p＜0.05, χ2-test). All tumors were cholangiocarcinoma. These results indicated that BOP-induced cholangiocarcinogenesis was suppressed by the supplemental administration of 1α(OH)D3.     

Early changes in islet hormone secretion in the hamster pancreatic cancer model

The diabetic state that is seen at a high frequency in association with pancreatic cancer is characterized by elevated plasma levels of several islet hormones and by marked insulin resistance. Both the diabetic state and insulin sensitivity improve after tumor removal by sub-total pancreatectomy. Impaired glucose tolerance has also been found in the hamster pancreatic cancer model, but conflicting data regarding islet function have been reported. In order to further investigate islet function and secretion during early development of pancreatic cancer, we measured the concentrations of insulin, glucagon, somatostatin, and islet amyloid polypeptide (IAPP) in plasma, pancreatic tissue, and secretin-stimulated pancreatic juice at 12 and 27 weeks after the ductal-cell-specific carcinogen, BOP had been used to induce tumors in Syrian golden hamsters. At 12 weeks after BOP, plasma glucagon levels were significantly increased. An exaggerated plasma-glucose response and concomitant hyperinsulinemia were observed at 27 but not 12 weeks after BOP. Plasma IAPP concentrations, but not glucagon or somatostatin, were elevated at 27 weeks. Tissue concentrations of IAPP were substantially reduced in BOP-treated hamsters at 27 weeks. No differences in hormone concentrations were seen in pancreatic juice from the two groups at either of the two time points investigated. The study showed that islet hormone changes accompany the early development of pancreatic tumors in the hamster pancreatic model. The hormone changes and apparent insulin resistance resemble the metabolic changes found in humans with pancreatic cancer.     

In vitro induction of giant cell tumors from cultured hamster islets treated with N-Nitrosobis(2-Oxopropyl)amine

Giant cell carcinoma of the pancreas is a rare tumor. Its histogenesis is still controversial. In a Syrian hamster pancreatic cancer model, tumors similar to human giant cell carcinomas have been induced at an extremely low rate of incidence and after the use of high doses of pancreatic carcinogens. Thus far no tumors of giant cell type have been induced by the in vitro treatment of hamster pancreatic ductal cells with the potent pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine (BOP). In the present study we report the induction of giant cell carcinoma from hamster islets treated with BOP in vitro. The results suggest that in hamsters some component of islet cells, probably stem cells, are the origin of giant cell carcinoma.     

Experimental evidence for the origin of ductal-type adenocarcinoma from the islets of Langerhans.

To investigate the role of the islets of Langerhans in pancreatic carcinogenesis, freshly isolated islets from male Syrian hamsters were transplanted into the right submandibular glands of 50 female hamsters that were or were not pre-treated with streptozotocin. Thyroid gland fragments, cellulose powder, and immortal hamster pancreatic ductal cells were injected into the left submandibular gland of the same hamsters. All recipient hamsters were then treated with the potent pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine weekly at a dose of 40 mg/kg of body weight for 3 weeks. Between 3 and 8 weeks later, 18 of 75 (24%) hamsters developed large ductal-type adenocarcinomas in the submandibular gland region, where islets were transplanted, but none developed tumors in the left submandibular gland. In 9 of 18 hamsters, tumors were multiple so that a total of 31 cancers were found. Eleven of these carcinomas were in the vicinity of transplanted islets, eight of which showed intra-insular ductular or cyst formation as seen in the pancreas of hamsters during pancreatic carcinogenesis. The formation of ductular structures within islets was also demonstrated in vitro. Some tumor cells in the vicinity of these islets were reactive with anti-insulin. Y chromosome message was found by polymerase chain reaction analysis in one of the three tumors examined. Also, like the induced pancreatic tumors, all three submandibular gland tumors that were examined had the mutation of the c-Ki-ras oncogene at codon 12 and all tumors expressed blood group A antigen. These and other findings strongly suggest that some components of islets, most probably stem cells, are the origin of ductal-type adenocarcinomas in this model.     

Early lesions of pancreatic ductal carcinoma in the hamster model.

Syrian golden hamsters were treated weekly with 10 mg/kg body weight N-nitrosobis (2-oxopropyl) amine for life (Group 1) or 6 weeks and were sacrificed at biweekly intervals from 2 weeks (Group 1) and 8 weeks (Group 2) after initiation of the experiment. The pancreas was examined in step sections, and the sequential alterations noted for each interval were recorded. Lesions were found in intrapancreatic and extrapancreatic ducts. Equivalent alterations consisting of hyperplasia, metaplasia, atypia, and lesions characteristic of carcinoma in situ developed ubiquitously and simultaneously in pancreatic ducts of different sizes and in ductules, but not in acinar cells. Among the most significant findings were intrainsular ductular formations, their proliferation, and sequential malignant alteration comparable to the involved preexisting ductules. Differences between the two experimental groups were of a quantitative rather than qualitative nature. The incidence and multiplicity of neoplastic lesions at each interval according to group, sex, and anatomic locations of adenocarcinomas are outlined. Predilected areas for some lesions were found. Results indicate a common origin of all induced tumors from a pluripotent cell populating the pancreatic ductal system.     

Involvement of inflammatory factors in pancreatic carcinogenesis and preventive effects of anti-inflammatory agents

Chronic inflammation is known to be a risk for many cancers, including pancreatic cancer. Heavy alcohol drinking and cigarette smoking are major causes of pancreatitis, and epidemiological studies have shown that smoking and chronic pancreatitis are risk factors for pancreatic cancer. Meanwhile, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are elevated in pancreatitis and pancreatic cancer tissues in humans and in animal models. Selective inhibitors of iNOS and COX-2 suppress pancreatic cancer development in a chemical carcinogenesis model of hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). In addition, hyperlipidemia, obesity, and type II diabetes are also suggested to be associated with chronic inflammation in the pancreas and involved in pancreatic cancer development. We have shown that a high-fat diet increased pancreatic cancer development in BOP-treated hamsters, along with aggravation of hyperlipidemia, severe fatty infiltration, and increased expression of adipokines and inflammatory factors in the pancreas. Of note, fatty pancreas has been observed in obese and/or diabetic cases in humans. Preventive effects of anti-hyperlipidemic/anti-diabetic agents on pancreatic cancer have also been shown in humans and animals. Taking this evidence into consideration, modulation of inflammatory factors by anti-inflammatory agents will provide useful data for prevention of pancreatic cancer.     

The impact of laparoscopic biopsy of pancreatic lymph nodes with helium and carbon dioxide on port site and liver metastasis in BOP-induced pancreatic cancer in hamster

The influence of pancreatic biopsy during laparoscopy with carbon dioxide (CO₂) and helium on the incidence of port site and liver metastasis in pancreatic carcinoma is still unknown. Ductal adenocarcinoma of the pancreas was induced in Syrian hamsters (n=30) by injection of N-nitrosobis-2-oxopropylamin (BOP, 10 mg/kg body weight/week) for 12 weeks. In week 13, hamsters were randomized in 3 groups (n=10): While in group 1 (gr. 1) a laparotomy and biopsy of pancreatic lymph nodes was performed, gr. 2 and gr. 3 underwent a laparoscopic biopsy either with CO₂ or helium. Therefore, one trocar was located in the left (biopsy) and the right abdominal wall (camera). In the 18th week all animals were sacrified and the incidence of abdominal wall, port site and liver metastases was histologically determined. While there were abdominal wall metastases after laparotomy in 10% (n=1), we observed trocar metastases in the CO₂ group in 20% (n=2). However, there were no trocar metastases in the helium group. The incidence of liver metastasis did not differ between the laparotomy and the helium group (20% vs 30%), but was increased in the CO₂ group (60%). Laparoscopic biopsy of pancreatic lymph nodes with CO₂ increased the incidence of port site and liver metastases in pancreatic cancer. The helium group was equal to the laparotomy group in this respect. Thus, staging laparoscopy with helium might become an alternative to explorative laparotomy in pancreatic cancer.     

Development of intrapancreatic transplantable model of pancreatic duct adenocarcinoma in Syrian golden hamsters.

Intrapancreatic and subcutaneous (SC) inoculation of cultured pancreatic cancer cells, derived from an induced primary pancreatic cancer in a Syrian hamster, resulted in tumor take in all recipient hamsters. The intrapancreatic allografts grew rapidly, were invasive, and metastasized into the lymph nodes and liver in 2 of 9 cases. In comparison, SC tumors grew relatively slower and formed a large encapsulated mass without invasion and metastases. Histologically, tumors of both sites showed fairly well-differentiated adenocarcinomas of ductal/ductular type resembling the induced primary cancer. Similar to the primary induced pancreatic cancers, tumor cells of both allografts expressed blood-group-related antigens, including A, B, H, Le(b), Le(y), Le(x), and tumor-associated antigen TAG-72. The tumor cells did not express Le(a), CA 19-9, 17-1A, or DU-PAN-2. The expression of these antigens was retained in the metastases and presented the same patterns of reactivity as the allografts. Thus intrapancreatic transplantation provides a rapid model for production of pancreatic cancer with morphologic similarities to human pancreatic cancer.     

Inhibition of pancreatic hepatocyte induction in hamsters treated sequentially with ethionine, a protein-free diet and then methionine by prior N-nitrosobis-(2-oxopropyl)amine administration

A 100% yield of pancreatic hepatocytes was induced in pancreas tissues of female hamsters treated with twice-repeated sequential administrations of DL-ethionine (ethionine) together with a protein-free diet and then L-methionine (methionine) for 10 weeks. The cells were also found in 40% of hamsters receiving 20 mg/kg body weight of the pancreatic carcinogen, N-nitrosobis(2-oxopropyl)amine (BOP) given twice at the peak of pancreatic regeneration stimulated by methionine after ethionine-induced cell damage. However, BOP at doses of 30, 70, and 100 mg/kg body weight administered before the occurrence of pancreatic regeneration dose-dependently inhibited their appearance, with reduction of the yield to 40%, 25%, and 8.3% respectively, and BOP per se did not induce any development of pancreatic hepatocytes. Stein iodine staining revealed bile pigments in the induced hamster pancreatic eosinophilic cell populations.     

Inhibition of Pancreatic Hepatocyte Induction in Hamsters Treated Sequentially with Ethionine, a Protein-free Diet and then Methionine by Prior N-nitrosobis-(2-oxopropyI)amine Administration

A 100% yield of pancreatic hepatocytes was induced in pancreas tissues of female hamsters treated with twice-repeated sequential administrations of DL-ethionine (ethionine) together with a protein-free diet and then L-methionine (methionine) for 10 weeks. The cells were also found in 40% of hamsters receiving 20mg/kg body weight of the pancreatic carcinogen, N nitrosobis(2-oxopropyl)amine (BOP) given twice at the peak of pancreatic regeneration stimulated by methionine after ethionine induced cell damage. However, BOP at doses of 30, 70, and 100 mg/kg body weight administered before the occurrence of pancreatic regeneration dose dependently inhibited their appearance, with reduction of the yield to 40%, 25%, and 8.3% respectively, and BOP per se did not induce any development of pancreatic hepatocytes. Stein iodine staining revealed bile pigments in the induced hamster pancreatic eosinophilic cell populations.     

Impact of Octreotide and SOM-230 on liver metastasis and hepatic lipidperoxidation in ductal pancreatic adenocarcinoma in Syrian Hamster

Octreotide is a somatostatin analogue binding on two receptor subtypes. In previous trials Octreotide showed inhibitory effects on tumour growth and liver metastasis in experimental pancreatic cancer. Thus we evaluated whether the new somatostatin analogue SOM-230 binding on 4 receptor subtypes has superior effects on carcinogenesis in pancreatic carcinoma. About 120 Syrian hamsters were randomised into six groups (n = 20): Gr.1: Aqua/Aqua, Gr.2: BOP/Aqua, Gr.3: Aqua/Octreotide, Gr.4: BOP/Octreotide, Gr.5: Aqua/SOM-230, Gr.6: BOP/SOM-230. Tumour groups 2,4,6 subcutaneously received 10 mg/kg body weight N-nitrosobis-2-oxopropylamin (BOP) weekly for 10 weeks, healthy control Gr.1,3,5 were given aqua. In the 17th week therapy started with Octreotide and SOM-230 for 16 weeks, after 32 weeks animals were sacrificed. Pancreas and liver were histopathologically analysed. Hepatic lipidperoxidation was determined by activities of antioxidative enzymes gluthation-peroxidase (GSH-Px) and superoxiddismutase (SOD) as well as concentration of thiobarbituric-acid reactive substances (TBARS). Incidence of liver metastases was 88.2% in Gr.2 (BOP/Aqua), it was decreased in Gr.4 (BOP/Octreo: 40%) and Gr.6 (BOP/SOM-230: 50%) (P < 0.05). Mean number/animal and mean-2-dimensional size of liver metastases did not differ between tumour groups. Comparing GSH-Px-activity in intrametastatic and extrametastatic hepatic tissue revealed a significant increase extrametastatically in Gr.2 (BOP/Aqua) and Gr.6 (BOP/SOM-230). SOD-activity in liver metastases was decreased in Gr.2 (1,801) (P < 0.05) versus Gr.4 (8,304) and Gr.6 (7,038). Intrametastatic TBARS concentration was increased in Gr.2 compared to Gr.4 (BOP/Octreotid) and Gr.6 (BOP/SOM-230) (P < 0.05). Octreotide and SOM-230 equally reduced liver metastasis in ductal pancreatic adenocarcinoma probably by a reduction of lipidperoxidation.     

Alterations in pancreatic islet function produced by carcinogenic nitrosamines in the Syrian hamster.

Exposure of hamsters to 5 daily doses of 20 mg/kg N-nitrosobis(2-oxopropyl)amine (BOP) or 76 mg/kg N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), resulted in reduced insulin secretion in freshly isolated pancreatic islets. These treatments also reduced plasma insulin and glucose levels, and were hepatotoxic. The inhibition of insulin secretion, however, was transient. Islets isolated from treated hamsters that were then placed in culture secreted elevated levels of insulin for many months. When cultured islets were directly exposed to the nitrosamines for 3 days, there was also a transient reduction of insulin secretion that was subsequently normalized after removal of the nitrosamine from the medium. These results show that BOP and HPOP modify beta-cell function both directly, and possibly indirectly, via damage to the liver. Furthermore, the lack of immediate inhibition of insulin secretion when islets were incubated in the presence of BOP or HPOP as well as glucose, suggests that the nitrosamines do not bind to the glucose receptor.     

Expression of thrombospondin-1 in pancreatic carcinoma: correlation with microvessel density

Thrombospondin-1 (TSP-1) is a multifunctional platelet and extracellular matrix protein that is involved in angiogenesis. Under certain pathological conditions, e.g., malignant tumors, high concentrations of TSP-1 work as an angiogenic agonist. Here we examined 98 pancreatic carcinomas with respect to TSP-1 immunoreactivity and its correlation to intratumoral microvessel density (MVD), a representation of the overall degree of angiogenesis in carcinomas. Northern blot analysis for TSP-1 mRNA was performed in seven additional cases. Eighty-seven tumors showed strong TSP-1 immunoreactivity, nine carcinomas were only weakly positive, and two lesions were negative for TSP-1. TSP-1 immunoreactivity was detected in the extracellular matrix, mostly at the invasion front of the tumor. Using Northern blot analysis, we observed high levels of TSP-1 mRNA in three out of seven pancreatic carcinomas. The mean MVD in pancreatic carcinoma was 38.8 vessels per mm2. Tumors with a high expression of TSP-1 showed a higher MVD and the correlation between TSP-1 immunoreactivity and microvessel density was highly significant (P=0.003). As a modulator of angiogenesis, TSP-1 is strongly expressed in most pancreatic adenocarcinomas and is likely to contribute to the extensive neovascularization and spread of this highly aggressive tumor.     

Superficial vs deep pancreatic parenchymal invasion in the extrahepatic bile duct carcinomas: a significant prognostic factor.

Pancreatic invasion of the extrahepatic bile duct (EBD) carcinomas is known to have a poor outcome. We hypothesized that EBD carcinoma showing shallow invasion to the pancreas may have a better outcome than the usual deep pancreatic invasion. We divided 87 cases of the distal EBD carcinomas into superficial and deep pancreatic invasion groups according to degrees of the pancreatic invasion. The superficial pancreatic invasion group included cases with tumor abutting the pancreatic lobule or pancreatic parenchymal invasion equal to or less than 1 mm from the uppermost portion of the pancreatic lobule or tumors invading into the fibroadipose tissue between pancreatic lobules without parenchymal invasion. The deep invasion group consisted of tumors with more than 1 mm pancreatic parenchymal invasion. The cases with superficial pancreatic invasion showed significantly better survival rate than those with deep pancreatic invasion (P<0.001). Therefore, we recommend that a specific remark on the pathology report about the presence or absence of parenchymal invasion and the depth of invasion of the pancreas is required for managing patients and determining the prognosis. We also recommend that the current pT3 stage of distal EBD carcinomas be subdivided into superficial (pT3a) and deep pancreatic invasion (pT3b).