Search

The neuroepithelial stem cell marker nestin is a cytoskeletal protein that regulates cell proliferation, invasion, and stemness in various tumors, including pancreatic tumors. In the present study, we examined the expression and roles of phosphorylated nestin in pancreatic cancer cells. Nestin phosphorylation at threonines 315 (Thr315) and 1299 (Thr1299) was observed during mitosis in human pancreatic cancer cells. Nestin phosphorylation was positively correlated with a cell proliferation marker, MIB‐1 expression in human pancreatic cancer samples. Transfection of MIA PaCa‐2 cells with nestin mutated at Thr315 and/or Thr1299 (to suppress phosphorylation) resulted in lower proliferation rates than those in control groups. Transfecting MIA PaCa‐2 cells with wild‐type nestin or with nestin mutated at Thr315 increased migration and invasion. In contrast, transfection with nestin mutated at both phosphorylation sites (Thr315 and Thr1299) did not enhance cell migration or invasion. In an intra‐splenic xenograft experiment using MIA PaCa‐2 cells, tumors expressing the nestin double mutant formed fewer liver metastases than tumors expressing wild‐type nestin. Nestin phosphorylation at these two sites was decreased upon treatment with inhibitors for cyclin dependent kinases, AKT, and Aurora in PANC‐1 cells, which express a high baseline level of phosphorylated nestin. These findings suggest that phosphorylation of nestin at Thr315 and/or Thr1299 affects cell proliferation, and inhibition of both phosphorylation sites suppresses invasion and metastasis of human pancreatic cancer. Inhibiting nestin phosphorylation at these two sites may represent a novel therapeutic strategy for pancreatic cancer.

After undergoing an upper gastrointestinal endoscopy, a 74-year-old woman with anemia was diagnosed with advanced lower gastric cancer. We performed laparotomy and identified the tumor as unresectable because of the direct invasion to the pancreas. S-1 was administered at 60mg/day for 2 weeks followed by 1-week discontinuation. After 6 weeks, we changed the schedule to the same dosage of S-1 for 1 week followed by 2-week discontinuation. CT and endoscopic findings showed complete response after 64weeks of S-1 administration. Since then, S-1 has been maintained at 60mg/day intermittently for 14 days in 7 weeks accordingto the patient's condition. The patient is currently doingwell with a complete response for more than 5 years.

The short-term safety and efficacy of insertion of a self-expandable metallic colonic stent (SEMS) followed by elective surgery, “bridge to surgery (BTS)”, for malignant large bowel obstruction (MLBO) have been well described; however, the influence on long-term oncological outcomes is unclear. The aim of this study was to evaluate changes in oncological characteristics in colorectal cancer (CRC) tissues after SEMS insertion, focusing on growth factors, cell cycle and apoptosis. From January 2013 to September 2014, a total of 25 patients with MLBO who underwent BTS at our single institution were retrospectively included. Paired CRC tissue samples before (endoscopic biopsy) and after SEMS insertion (surgically resected) were collected from each patient. EGFR, VEGF, Ki-67, p27kip1 and TUNEL expression were determined by immunohistochemistry. No clinical or subclinical perforations evaluated by mechanical ulceration pathologically were observed. Epithelial exfoliation, tumour necrosis, infiltration of inflammatory cells and fibrosis were observed in SEMS-inserted surgically-resected specimens. Overall, 84% (21/25) and 60% (15/25) of patients exhibited no change or a decrease in staining category, respectively, for EGFR and VEGF expression after SEMS insertion. A significant decrease in Ki-67 expression was observed in surgically-resected specimens compared with endoscopic biopsy specimens (P

Introduction The postoperative results of laparoscopic distal pancreatectomy for solid pseudopapillary neoplasm of the pancreas (SPN), including the effects of spleen-preserving resection, are still to be elucidated. Methods Of the 139 patients who underwent laparoscopic pancreatectomy for non-cancerous tumors, 14 consecutive patients (average age, 29.6 years; 1 man, 13 women) with solitary SPN who underwent laparoscopic distal pancreatectomy between March 2004 and June 2015 were enrolled. The tumors had a mean diameter of 4.8 cm. Laparoscopic spleen-preserving distal pancreatectomy was performed in eight patients (spleen-preserving group), including two cases involving pancreatic tail preservation, and laparoscopic spleno-distal pancreatectomy was performed in six patients (standard resection group). Results The median operating time was 317 min, and the median blood loss was 50 mL. Postoperatively, grade B pancreatic fistulas appeared in two patients (14.3%) but resolved with conservative treatment. No patients had postoperative complications, other than pancreatic fistulas, or required reoperation. The median postoperative hospital stay was 11 days, and the postoperative mortality was zero.None of the patients had positive surgical margins or lymph nodes with metastasis. The median follow-up period did not significantly differ between the two groups (20 vs 39 months, P = 0.1368). All of the patients are alive and free from recurrent tumors without major late-phase complications. Conclusion Laparoscopic distal pancreatectomy might be a suitable treatment for patients with SPN. A spleen-preserving operation is preferable for younger patients with SPN, and this study demonstrated the non-inferiority of the procedure compared to spleno-distal pancreatectomy.

Background Insulinoma is a very serious functional tumor. Surgeons should confirm complete resection of insulinomas before completing the operation, even in laparoscopic surgery. Methods Between August 2007 and September 2014, 15 consecutive patients with biochemical evidence of an insulinoma underwent laparoscopic pancreatectomy. Intraoperatively, a peripheral arterial blood sample was taken, and insulin was measured by quick insulin assay. Insulin levels were determined before anesthesia induction, every 30 min thereafter, and every 30 min for at least 1 h after tumor resection to confirm insulin levels did not increase before surgery was completed. Results All 15 patients (3 men and 12 women, average age 57.2 years) successfully underwent laparoscopic resection. One patient had two tumors, and the remaining 14 patients had one tumor each (three in the head, five in the body, and eight in the tail of the pancreas). Preoperative localization and regionalization studies identified the tumor correctly through CT (12/15 [80.0%]), MRI (9/12 [75.0%]), angiography (11/13 [84.6%]), endoscopic ultrasonography (7/10 [70.0%]), and selective arterial calcium injection (14/14 [100%]). Intraoperative ultrasonography detected 13 of 15 tumors (86.7%), and intraoperative blood insulin monitoring confirmed the complete resection of 16 of 16 tumors (100%). All patients were discharged with normal insulin levels and have been followed up for 3–88 months. There has been no recurrence of symptoms in any patients and none has died. Conclusion Complete removal of an insulinoma can be reliably predicted by intraoperative blood insulin monitoring even in laparoscopic pancreatectomies.

Epithelial splicing regulatory protein 1 (ESRP1) binds the FGFR-2 auxiliary cis-element ISE/ISS-3, located in the intron between exon IIIb and IIIc, and primarily promotes FGFR-2 IIIb expression. Here we assessed the role of ESRP1 in pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analysis was performed using anti-ESRP1, FGFR-2 IIIb and FGFR-2 IIIc antibodies in 123 PDAC cases. ESRP1-expression vector and small interference RNA (siRNA) targeting ESRP1 were transfected into human PDAC cells, and cell growth, migration and invasion were analyzed. In vivo heterotopic and orthotopic implantations using ESRP1 overexpression clones were performed and effects on pancreatic tumor volumes and hepatic and pulmonary metastases determined. ESRP1 immunoreactivity was strong in the nuclei of cancer cells in well-to-moderately differentiated PDACs, but weak in poorly-differentiated cancers. Well-to-moderately differentiated cancers also exhibited high FGFR-2 IIIb and low FGFR-2 IIIc expression, whereas this ratio was reversed in the poorly-differentiated cancers. Increased ESRP1 expression was associated with longer survival by comparison with low-ESRP1 expression, and PANC-1 cells engineered to express ESRP1 exhibited increased FGFR-2 IIIb expression and decreased migration and invasion in vitro, whereas ESRP1 siRNA-transfected KLM-1 cells exhibited increased FGFR-2 IIIc expression and increased cell growth, migration and invasion. In vivo, ESRP1-overexpressing clones formed significantly fewer liver metastases as compared with control clones. ESRP1 regulates the expression pattern of FGFR-2 isoforms, attenuates cell growth, migration, invasion, and metastasis, and is a favorable prognostic factor in PDAC. Therefore, devising mechanisms to up-regulate ESRP1 may exert a beneficial therapeutic effect in PDAC.

A 77-year-old man presenting with diarrhea and upper abdominal pain was referred to our hospital owing to multiple liver nodules and bulky gastric lymph node noted on abdominal contrast computed tomography (CT). His serum alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) levels were 187,800 ng/mL and 135,000 mAU/mL, respectively. The liver tumors displayed an enhanced hepatocellular pattern on the contrast CT scan; however, there was no hepatitis viral infection or chronic liver cirrhosis. On contrast magnetic resonance imaging, the liver nodules showed high intensity on the T2-weighted image, and the differential diagnosis by CT was liver metastasis. Upper gastrointestinal endoscopy revealed an advanced gastric carcinoma (type 2) in the cardia, which was histopathologically diagnosed as gastric adenocarcinoma. The tumor cell expressed AFP, and histopathological findings of the liver tumor due to core needle biopsy were similar to those of the gastric lesion; however, the liver tumor was positive for AFP and DCP. Thus, the tumor was clinically diagnosed as a gastric carcinoma producing both AFP and DCP.

Introduction We introduce a technique for pancreaticojejunostomy with closure of the pancreatic stump by endoscopic linear stapler as a reliable intervention with benefits for pancreatic resection in laparoscopic pancreaticoduodenectomy (Lap-PD). Materials and Surgical Technique Following laparoscopic resection, we perform pancreaticojejunostomy under direct visualization. We employ the same method as in open surgery and enter via a 4–5-cm incision, the minimum size feasible for easy removal of resected material from the body, positioned directly above the stump of the distal pancreas. In January 2011, we began using endoscopic linear stapler when cutting the pancreas during Lap-PD in order to reduce the leakage of pancreatic juice, which may contain tumor cells from the neoplastic lesion. Since then, we have used this procedure in 12 subjects undergoing Lap-PD and 5 subjects undergoing laparoscopic central pancreatectomy. We have observed postoperative complication in only one of the laparoscopic central pancreatectomy cases, involving grade B/C pancreatic fistula, and in none of the Lap-PD cases. Discussion Our pancreaticojejunostomy with closure of the pancreatic stump by endoscopic linear stapler is a feasible procedure in Lap-PD and has produced positive results over a short time frame.

Fibroblast growth factor receptor 2 (FGFR2) is considered a novel therapeutic target for various cancer. We used a silencing strategy to clarify the effect of reduced FGFR2 expression in human colorectal cancer (CRC) cells. The invasive front of cancer cells exhibited stronger FGFR2 expression than the surface area of the cancers. FGFR2 shRNA-transfected LoVo cells inhibited cell migration, invasion and tumor growth in vitro and in vivo. Thus, FGFR2 plays important roles in CRC progression in association with tumor cell migration, invasion and growth, and FGFR2 might be a novel therapeutic target for CRC.

Chronic pancreatitis (CP) is a painful, yet benign inflammatory process of the pancreas. Surgical management should be individualized because the pain is multifactorial and its mechanisms vary from patient to patient. Two main pathogenetic theories for the mechanisms of pain in CP have been proposed: the neurogenic theory and the theory of increased intraductal/intraparenchymal pressures. The latter theory is strongly supported by the good results of drainage procedures in the surgical management of CP. Other possible contributing factors include pancreatic ischemia; a centrally sensitized pain state; and the development of complications, such as pseudocysts and stenosis of the duodenum or common bile duct. Common indications for surgery include intractable pain, suspicion of neoplasm, and complications that cannot be resolved with radiological or endoscopic treatments. Operative procedures have been historically classified into 4 categories: decompression procedures for diseased and obstructed pancreatic ducts; resection procedures for the proximal, distal, or total pancreas; denervation procedures of the pancreas; and hybrid procedures. Pancreaticoduodenectomy and pylorus-preserving pancreaticoduodenectomy, once the standard operations for patients with CP, have been replaced by hybrid procedures, such as duodenum-preserving pancreatic head resection, the Frey procedure, and their variants. These procedures are safe and effective in providing long-term pain relief and in treating CP-related complications. Hybrid procedures should be the operations of choice for patients with CP.

We describe a 43-year-old woman who underwent laparoscopic distal pancreatectomy preserving the spleen and splenic vessels for the treatment of insulinoma in the pancreatic body. The patient experienced cold sweats on fasting, received diagnosis of insulinoma, and was referred to our hospital for laparoscopic surgery. Blood biochemistry studies showed low fasting blood glucose of 42 mg/dL, serial immunoreactive insulin of 15.2 microU/mL, and a Fajans index (immunoreactive insulin/blood glucose) of 0.36 (normal

Torsion of the gallbladder is a rare condition that most commonly affects the elderly. This condition is rarely diagnosed preoperatively despite advances in diagnostic imaging. We report a case of torsion of the gall bladder diagnosed preoperatively. An 81-year-old woman presented with right upper quadrant pain. Initial laboratory tests revealed elevation of the white blood cell count to 15,900/μL(normal, 4,000 to 8,000/μL) and mild liver dysfunction. Ultrasonography and computed tomography revealed swelling of the gall bladder with increased wall thickness, but neither examination showed any gallstones. Percutaneous transhepatic gall bladder drainage was performed, and bloody bile juice was obtained. Cholangiography via the drainage catheter of the gall bladder and endoscopic retrograde cholangiography revealed smooth tapering of the neck of the gall bladder. We diagnosed acute torsion of gallbladder and brought the patient to the operating theatre for laparoscopic cholecystectomy. Intraoperatively, we observed that the gallbladder had undergone complete torsion and appeared gangrenous. Routine cholecystectomy was then performed, and the patient recovered without incident.

A 70-year-old man was admitted to our hospital for evaluation of multiple pancreatic tumors. Twelve years earlier he had undergone left radical nephrectomy for renal cell carcinoma (RCC). Computed tomography revealed two well-defined mass lesions in the head and tail of the pancreas, with strong contrast enhancement in the arterial phase. Fluorine-18 fluorodeoxyglucose positron emission tomography detected an elevated uptake within the lesions but no extrapancreatic uptake. The preoperative diagnosis was isolated multifocal metastatic pancreatic tumors from RCC. The patient underwent total pancreatectomy with splenectomy. Both of the tumors were well-demarcated, gray-white, and firm on gross observation. Microscopic examination, meanwhile, revealed solid tumors consisting of clear oval cells with severe nuclear atypia. These pathologic findings were consistent with the preoperative diagnosis of pancreatic metastasis from RCC. Radical resection improves the long-term survival of patients, and total pancreatectomy may be an appropriate procedure.

The postoperative results of laparoscopic distal pancreatectomy for solid pseudopapillary neoplasm of the pancreas (SPN), including the effects of spleen-preserving resection, are still to be elucidated.Of the 139 patients who underwent laparoscopic pancreatectomy for non-cancerous tumors, 14 consecutive patients (average age, 29.6 years; 1 man, 13 women) with solitary SPN who underwent laparoscopic distal pancreatectomy between March 2004 and June 2015 were enrolled. The tumors had a mean diameter of 4.8 cm. Laparoscopic spleen-preserving distal pancreatectomy was performed in eight patients (spleen-preserving group), including two cases involving pancreatic tail preservation, and laparoscopic spleno-distal pancreatectomy was performed in six patients (standard resection group).The median operating time was 317 min, and the median blood loss was 50 mL. Postoperatively, grade B pancreatic fistulas appeared in two patients (14.3%) but resolved with conservative treatment. No patients had postoperative complications, other than pancreatic fistulas, or required reoperation. The median postoperative hospital stay was 11 days, and the postoperative mortality was zero.None of the patients had positive surgical margins or lymph nodes with metastasis. The median follow-up period did not significantly differ between the two groups (20 vs 39 months, P = 0.1368). All of the patients are alive and free from recurrent tumors without major late-phase complications.Laparoscopic distal pancreatectomy might be a suitable treatment for patients with SPN. A spleen-preserving operation is preferable for younger patients with SPN, and this study demonstrated the non-inferiority of the procedure compared to spleno-distal pancreatectomy.

Introduction This is an investigation of the capability of FAIMS (Field Asymmetric Ion Mobility Spectrometry) technology as a tool for non-invasive detection of Colorectal Cancer (CRC) through urinary volatile organic compound analysis. It expands on "Detection of Colorectal Cancer (CRC) by Urinary Volatile Organic Compound Analysis" (Ramesh P. Arasaradnam et al, 2014) with an increased sample population and local to Japan, also comparing in-house data of CA19-9 and carcinoembryonic antigen (CEA) markers. Experiment Conducted at Nippon Medical School Chiba Hokusoh Hospital, urine samples were collected and frozen at -80°C from 139 patients at various stages of CRC and 78 healthy control samples. The samples were thawed in batches and placed in ice hours prior to testing. 2 ml of each urine sample was aliquoted into 10 ml vials for processing with the commercial FAIMS device (Lonestar, Owlstone, UK). Each vial was heated in the device to 40°C to create headspace with sufficient VOCs then a carrier gas (clean dry air) delivered the headspace (0.5 L/min) diluted with a make-up flow (2 L/min). The FAIMS device was set to scan at 0 to 100% electric dispersion field in 51 steps and compensation voltage between -6 V and +6 V in 512 steps, producing data matrices for each sample's analysis. Principal Component Analysis (PCA) followed by Partial Least Squares Discriminant Analysis (PLS-DA) of each sample was conducted using SIMCA 13 (Umetrics, Sweden). See Table Conclusions FAIMS technology achieved a high rate of separation between the CRC and healthy control urine samples with 64.7% sensitivity and 82.1% specificity overall. As the CRC stage advances the sensitivity increased from 27.3% to 100%. Results show excellent potential to use FAIMS technology as an early screening tool for CRC, particularly impressive compared to in-house sensitivity data of CA19-9 and CEA markers. Further research into FAIMS screening of other cancer types through VOC biomarker analysis of urine, breath, and feces is recommended. >Detection ResultsCRC STAGEFAIMSOTHER MARKERSCA19-9CEATrue PositiveFalse NegativeSensitivityFalse PositiveTrue NegativeSpecificityNo. of SamplesSensitivitySensitivityI123227.3%146482.1%434.7%18.6%II181260.0%146482.1%3116.1%32.3%III45590.0%146482.1%5219.2%36.5%IV150100%146482.1%1735.3%64.7%ALL904964.7%146482.1%14316.1%33.6% Citation Format: Christopher Psutka, Marina Yamada, Akihisa Matsuda, Kazuya Yamahatsu, Satoshi Matsumoto, Toshihiko Kitayama, Nobuo Nakano, Jyunichi Koyano, Tohru Mikoshiba, Masao Miyashita. FAIMS technology in urinary volatile organic compound analysis to detect colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5303. doi:10.1158/1538-7445.AM2017-5303

We report a case of locally advanced pancreatic tail adenosquamous carcinoma that was treated by performing R0 resection after neoadjuvant chemotherapy with S-1 and gemcitabine. A 75-year-old man visited our hospital because of left lateral abdominal pain. On the basis of computed tomography and endoscopic biopsy findings, an 80-mm locally advanced pancreatic tail carcinoma with direct invasion to the gastric upper body, splenic flexure of the colon, and left kidney was diagnosed. Combined chemotherapy with S-1 and gemcitabine was initiated for reduction in the tumor size. After 11 courses of treatment, computed tomography revealed a partial response in tumor size reduction. Grade 3 neutropenia was observed as an adverse event. Distal pancreatectomy, proximal gastrectomy, partial resection of the descending colon, resection of the left kidney and left adrenal gland, and D2 lymph node dissection were performed. The pathological diagnosis was adenosquamous carcinoma in the pancreatic tail, and an R0 resection was achieved. However, a month after surgery, multiple distant liver metastases were observed. Neoadjuvant chemotherapy with S-1 and gemcitabine may reduce the tumor size in locally advanced pancreatic tail adenosquamous carcinoma and increase the R0 resection rate. However, treatment for distant metastasis is warranted in cases of pancreatic adenosquamous carcinoma.

Introduction: The recent advances of surgical techniques and technology allow minimally invasive surgery to be applied in patients with benign and malignant diseases of the pancreas. About malignancy, we still have concerns regarding the oncologic adequacy of laparoscopic pancreatectomy, with fewer studies reporting oncologic outcomes. We describe the surgical technique to improve the curability of laparoscopic pancreatectomy in the treatment of Pancreatic Adenocarcinoma (PDAC) and focuse on the oncologic outcomes and long-term outcomes of laparoscopic surgery for PDAC patients. Methods: From January 2004, patients who had been diagnosed with the tumor in the pancreas without suspicion for vascular involvement were eligible for laparoscopic pancreatectomy at Nippon Medical School. In Lap- PD for PDAC patients, we apply laparoscopic left mesenteric approach, which enables both accurate laparoscopic lymph node retrieval and complete pancreatic nerve plexuses dissection to achieve R0 resection. In Lap-DP for PDAC patients, we perform retroperitoneal tissue dissection, which often includes adrenalectomy. Results: We have experienced laparoscopic pancreatectomies in 148 patients including 25 PDAC patients. In the 25 patients with PDAC, the mean number of lymph nodes dissected was 22.4 ± 12.6 (6–57). Metastasis to the lymph nodes was observed in 8 of the 25 patients (32%). R0 resection was performed in 22 patients (88%). The median follow-up period for the PDAC patients undergoing laparoscopic pancreatectomy was 16 months (1–71 months). Six of the 25 patients died, at 2.5 months (stage IV), 15 months (stage IA), 29 months (stage IIB), 33 months (stage IIB), 24 months (stage IIA), and 18 months (stage IIB). Surviving 19 patients had no recurrence. Conclusion: Laparoscopic pancreatectomy for pancreatic cancer seems to achieve similar oncologic and longterm outcomes to open approach.

Dedifferentiated liposarcoma of the mesentery is an extremely rare tumor. A 71-year-old man with a 2-month history of abdominal distention was admitted to our department for evaluation and treatment of an abdominal mass. Computed tomography and magnetic resonance imaging revealed an 11 × 9 cm mass lesion with fat density in the upper right abdominal cavity, displacing the ascending and transverse colon ventrally. Abdominal angiography showed small feeding vessels of the tumor from the ileocolic artery and the middle colic artery. On basis of these findings, liposarcoma arising from the mesocolon ascendens was diagnosed, and complete removal of the tumor and central pancreatectomy (partial resection of the body of the pancreas) were performed. The histopathological diagnosis was dedifferentiated liposarcoma, and the patient is free from recurrence 6 months after surgery. The treatment strategy for abdominal dedifferentiated liposarcoma is surgical resection with a wide surgical margin.

We introduce a technique for pancreaticojejunostomy with closure of the pancreatic stump by endoscopic linear stapler as a reliable intervention with benefits for pancreatic resection in laparoscopic pancreaticoduodenectomy (Lap-PD).Following laparoscopic resection, we perform pancreaticojejunostomy under direct visualization. We employ the same method as in open surgery and enter via a 4-5-cm incision, the minimum size feasible for easy removal of resected material from the body, positioned directly above the stump of the distal pancreas. In January 2011, we began using endoscopic linear stapler when cutting the pancreas during Lap-PD in order to reduce the leakage of pancreatic juice, which may contain tumor cells from the neoplastic lesion. Since then, we have used this procedure in 12 subjects undergoing Lap-PD and 5 subjects undergoing laparoscopic central pancreatectomy. We have observed postoperative complication in only one of the laparoscopic central pancreatectomy cases, involving grade B/C pancreatic fistula, and in none of the Lap-PD cases.Our pancreaticojejunostomy with closure of the pancreatic stump by endoscopic linear stapler is a feasible procedure in Lap-PD and has produced positive results over a short time frame.

The class VI intermediate filament protein, nestin is reported to be a progenitor cell marker in various tissues. In the present study, we analyzed the expression and roles of nestin in angiogenesis of pancreatic ductal adenocarcinomas, and determined whether nestin is a potential target for inhibiting tumor angiogenesis using a gene silencing strategy. Nestin expression was detected only in small vessels, whereas CD34, CD31 and factor VIII were also expressed in large-sized blood vessels in PDAC. The number of nestin-positive vessels was approximately 20% the number of CD34-positive vessels, and the average dimension of nestin-positive vessels was approximately 75% that of CD34-positive vessels. The PCNA labeling indices of nestin-positive vessels were higher than those of CD34-positive vessels and nestin-negative vessels. Reducing nestin expression by use of siRNA targeting nestin transcripts inhibited growth of the vascular endothelial cell lines, but there was no difference in cell motility. In xenograft models, administration of siRNA targeting mouse-nestin suppressed subcutaneous human pancreatic cancer cell growth in nude mice. In conclusion, nestin was expressed in small proliferating blood vessels in pancreatic cancer tissues and may be a useful marker of angiogenesis in pancreatic ductal adenocarcinoma tissues. Furthermore, nestin is a potential novel therapeutic target in pancreatic cancers to inhibit tumor angiogenesis.

A 73-year-old man was admitted with bloody stool. Duodenoscopy showed a hemorrhagic ulceration in the duodenum on the side opposite to the papilla of Vater. Abdominal CT demonstrated a well-defined hypervascular mass, adjacent to the lesion of the duodenum. Although as duodenal GIST was diagnosed, histologic examination for frozen sections during the procedure revealed tubular adenocarcinoma of the duodenum and pancreaticoduodenal lymph node metastasis of neuroendocrine carcinoma. He underwent a subtotal stomach-preserving pancreaticoduodenectomy. Clinicopathologically, the neuroendocrine carcinoma of the pancreaticoduodenal lymph node was considered to be metastasis from an unknown primary lesion.

Xenograft transplantation of human tumor cells into immunodeficient mice is an important method to clarify the roles of specific molecules or chemicals in vivo. Recently, this method has been reported as a definitive examination to identify tumor stem cells. In this study, the authors compared the morphology and the quality and quantity of ribonucleic acid (RNA) and protein in paraffin-embedded tissues of nude mice implanted with human uterine cervical cancer cells, followed by fixation with commonly used fixatives, including 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF), 20% NBF, and 99% ethanol (EtOH). The quality of the isolated RNA from PFA- and NBF-fixed paraffin-embedded tissues was high, while EtOH-fixed tissues showed degradation of RNA. NBF-fixed tissues showed excellent quality of morphology, but EtOH-fixed tissues showed contraction of cells. Immunohistochemical results showed differences depending on fixations. The 99% EtOH-fixed samples showed decreases of Ki-67 and VEGF-A immunoreactivities, but improved cytokeratin immunoreactivity. This study indicated that formalin fixation is better than alcohol fixation for RNA preservation in paraffin-embedded cancer cell implantation models. Immunohistochemical results differed markedly depending on fixation materials and antibodies; therefore, suitable fixations are needed to quantify and compare the results of immunohistochemical staining on cancer cell implanted nude mice tissues.

Background: Recent studies have shown that epithelial splicing regulatory protein 1 (ESRP1) plays important roles in the epithelial mesenchymal transition of cancer cells via regulating the splicing of several RNAs, including fibroblast growth factor receptor 2 (FGFR2). Previously we reported that the expression of FGFR2IIIb correlates with vascular invasion and liver metastasis of pancreatic ductal adenocarcinoma (PDAC) (Cho, et al. Am J Pathol. 2008), while the expression of FGFR2IIIc correlates with the growth and invasion of PDAC cells (Ishiwata, et al. AACR. 2010). However, the expression and roles of ESRP1 in PDAC have not been well-defined. Methods: We performed immunohistochemical analysis of ESRP1, FGFR2IIIb, and FGFR2IIIc using pancreatic tissue specimens of PDAC (N=52) and non-tumorous pancreatic tissue (N=8). The ESRP1 expression vector and small interference RNA (siRNA) targeting ESRP1 were used to transfect human PDAC cells, and cell growth, morphology, migration, and invasion were analyzed. To clarify the ESRP1-regulating molecules, we performed proteomic analysis of ESRP1-gene-transfected PDAC cells using mass spectrometry. Results: In immunohistochemical analysis, ESRP1 was strongly expressed in the nuclei of PDAC cells in well- and moderately differentiated adenocarcinoma, while weakly expressed in poorly differentiated adenocarcinoma and normal pancreatic ducts. Well-moderately differentiated adenocarcinomas showed high-FGFR2IIIb/low-FGFR2IIIc pattern, with opposite for poorly differentiated adenocarcinoma. The overall and disease-free survival rates of the high-ESRP1 group were significantly than those of the low-ESRP1 group. In vitro, transient transfection of PDAC cells with ESRP1 expression vector led to an increase in FGFR2IIIb expression and decrease in migration and invasion. Stable transfection of PDAC cells with the ESRP1 expression vector resulted in an increase of FGFR2IIIb expression and decrease in migration and invasion. In contrast, ESRP1 siRNA-transfected PDAC cells induced FGFR2IIIc expression and increased cell growth, migration, and invasion. In proteomic analysis, transiently ESRP1 transfected PDAC cells showed a decreased expression of vimentin, 14-3-3 protein epsilon, heat shock protein 70, and the IQ motif containing GTPase activating protein 1, but an increased expression of filamin alpha. Several proteins as determined by proteomic analysis are involved in cell growth, migration, and invasion. Conclusion: The expression of ESRP1 correlates with the differentiation and prognosis of PDAC, and it modulates cell growth, migration, and invasion in PDAC cells via the alternation of several proteins including FGFR2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2412. doi:1538-7445.AM2012-2412

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a high incidence of distant metastasis. Recent studies have shown that cancer stem cells (CSCs) are important in cancer cell growth, invasion, metastasis, and recurrence. Several studies have revealed some CSC-specific markers for PDAC, such as CD133, CD24, CD44, CXCR4, ESA, and nestin. Some evidence also suggests that PDAC progresses through a multistep process comprised of noninvasive precursor lesions known as pancreatic intraepithelial neoplasias (PanINs). The reports of CSCs in PanINs are limited. We performed a comprehensive analysis of the expression of CSC markers in PDACs, PanINs, and normal pancreatic tissues. Materials & Methods: CD24, CD44, ESA, CD133, CXCR4, and nestin were used as CSC markers. Human PDACs (n=67), PanIN-1 (n=35), PanIN-2 (n=52), PanIN-3 (n=18), and normal pancreatic tissues (n=54), which were obtained from patients who underwent surgical operations in the surgical department of Nippon Medical School, were used for immunohistochemical analysis. Human PDAC cell lines, PANC-1, KLM-1, and MIA PaCa-2, were used for flow cytometric assays and quantitative RT-PCR analysis. Results: Regarding the clinicopathological features, the positivity of CD44 and CD133 showed significant correlations for UICC classifications and histological features In the survival analysis, CD133-positive PDACs led to a significantly poorer prognosis. In the immunohistochemical analysis, CD24, CD44, ESA, CXCR4, and nestin showed a gradual increase of positivity along with the grades of PanINs. Flow cytometric assays and RT-PCR analysis confirmed the expression profile in PDAC. Conclusion: CSC markers were expressed to various extents in PDAC, and the expression of CD133 and CD44 was correlated with the prognosis and stage. Furthermore, CSC markers showed a gradual increase along with the grades of PanINs; therefore, CSC may be involved in the carcinogenesis of PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 412. doi:1538-7445.AM2012-412

BACKGROUND: Pancreatic cancer is associated with extremely high mortality rates due to rapid progression and a high incidence of metastases. Liver and lung metastases in the early postoperative period are one of the causes for the poor prognosis of patients with resected pancreatic cancer. Information on the pathobiology of metastatic pancreatic cancer and elucidation of the underlying molecular mechanisms are urgently needed in order to develop novel and effective therapeutic approaches to treatment. Injection of human pancreatic cancer cells in the spleens of nude mice is one of the major liver metastasis models. However, this model yields few metastatic tumors in the livers and none in the lungs. NOG mice have severe immunodeficiency, including defects of T, B and NK cell functions, and dysfunction of dendritic cells. Human pancreatic cancer cells are reported to easily proliferate and metastasize to other organs in NOG mice. In this study, we established and characterized human pancreatic cancer cell lines from liver and lung metastases in NOG mice. METHODS: PANC-1 cells (1×105 cells) were injected into the spleens of NOG mice. The mice were euthanized 8 weeks after injection, and the livers and lungs were removed. Metastatic tumors of the liver and lung were cut into small pieces and dispersed into single cells in a medium containing antibiotics. Biological and morphological characteristics, and gene expression patterns of mesenchymal markers of the liver and lung metastatic cells, were compared with parental cells. RESULTS: These metastatic cells were confirmed as being of human-cell origin using PCR with primer pair of human mitochondrial DNA. There were no morphological changes between liver and lung metastatic cells and parental cells, but the metastatic cells proliferated more slowly than parental cells as determined by MTT assays and cell counts. By contrast, the metastatic cells exhibited greater migration than the parental cells, as determined in Boyden chamber assays and by time lapse analysis. Attachment assays revealed that adhesion of metastatic cells to the type I and IV collagen, laminin and fibronectin was stronger than that of parental cells. Nestin, a cancer stem cell marker, was highly expressed in the liver and lung metastatic cells. CONCLUSION: These findings suggest that human pancreatic cancer cell lines derived from liver and lung metastases in NOG mice have different characteristics from parental cells. These new cell lines may contribute to resolving the mechanisms of metastasis of pancreatic cancer and the roles of cancer stem cells in metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5240. doi:10.1158/1538-7445.AM2011-5240

Background: Tumor angiogenesis is an important factor in the proliferation and metastasis of pancreatic ductal adenocarcinoma (PDAC). CD34, CD31 and factor VIII-related antigen are commonly used as endothelial cell markers for tumor vessels. However, these markers identify not only newly formed small tumor vessels, but also pre-existing large blood vessels. Nestin, a class VI intermediate filament protein, has been reported to be up-regulated in endothelial cells accompanying the process of angiogenesis. In an acute pancreatitis murine model, we have previously reported that nestin is strongly expressed in proliferating endothelial cells. Furthermore, we have reported that nestin-positive blood vessels correlated with a poor prognosis in colorectal cancer. In this study, we examined the effectiveness of nestin as an angiogenic marker and potent target for anti-angiogenic therapy in PDAC. Methods: Tissues from 45 patients with PDAC were immunostained with nestin and other common vascular endothelial markers including CD34, CD31 and factor VIII-related antigen. We measured the number and dimension of the nestin- and CD34-positive blood vessels using image analyzing software. In addition, we compared proliferation activity between nestin- and CD34-positive vessels as determined by PCNA-labeling indices. To clarify the roles of nestin in endothelial cells, we transfected siRNA targeting nestin transcripts to mouse endothelial TKD2cells, and performed cell growth and migration assays. Results: Immunohistochemically, CD34, CD31 and factor VIII-related antigen were localized in blood vessels of all sizes, while nestin was localized only in the small blood vessels in PDAC tissues. Nestin was also expressed in myofibroblasts and nerve fibers, but not in lymphatic vessels. In image analyzing software, nestin-positive vessels were small in number, and they formed small lumen compare with CD34-positive blood vessels. Nestin-positive vessels showed higher PCNA-labeling indices than those of CD34-positive vessels. Nestin was expressed in small and proliferating blood vessels in PDAC tissues, suggesting that nestin plays important roles in tumor angiogenesis in cancer. Knock down of nestin in mouse endothelial cells using siRNA inhibited the cell growth, but not cell migration in vitro. Conclusion: Nestin was specifically expressed in small and proliferating blood vessels in pancreatic cancer tissues, indicating that nestin is a useful angiogenic marker in cancer. Furthermore, nestin may be a novel therapeutic target for inhibition of tumor angiogenesis in pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5145. doi:10.1158/1538-7445.AM2011-5145

Background: Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell/progenitor cell marker. Nestin expression has also been shown to be up-regulated in progenitor cells in muscle, testis, teeth and pancreas. In the pancreas, lineage-tracing experiments have indicated that exocrine cells are derived from Nestin-expressing progenitor cells. Moreover, activation of K-ras in the nestin cell lineage is sufficient for the initiation of premalignant pancreatic intraepithelial neoplasia lesions in mice. To examine cancer stem cell (CSC) functions, floating cultures in plates with a low-attachment surface have been widely employed. CSCs are reported to be able to grow in floating spheres in serum-free culture medium supplemented with epidermal growth factor (EGF) and/or fibroblast growth factor-2 (FGF-2), whereas non-CSCs, more differentiated non-CSCs cannot form spheres. In this study, we examined nestin expression in spheres and clarified whether nestin regulates sphere forming activity. Methods: Human pancreatic ductal adenocarcinoma (PDAC) cell lines, PANC-1, KLM-1, MIAPaCa-2 and PK-45H were used to perform sphere-forming assay in ultra-low attachment plates supplemented with EGF and FGF-2. Expression of CSC markers including CD133, CD44, CD24, epithelial specific antigen (ESA), and nestin in spheres were determined by real time RT-PCR and immunocytochemistry. Sphere-forming cells were dispersed with trypsin, and then cell growth and migration were assessed. Next, short hairpin RNA (shRNA) targeting nestin was stably transfected into PANC-1 and PK-45H, human PDAC cells, which express high levels of nestin, in order to assess the effects of decreased nestin expression on sphere-forming ability. Conversely, KLM-1 cells which express low level of nestin were stably transfected with a nestin expression vector and their sphere forming ability was then determined. Results: All the PDAC cell lines formed spheres, and all CSC markers examined were expressed in the spheres. The sphere cells showed low cell growth rates and increased cell migration as compared to non-sphere cells. Expression levels of nestin in the spheres of PDAC cells were higher than those of non-sphere cells. Nestin shRNA transfected PANC-1 and PK-45H cells showed a marked decrease in sphere number and diameter. By contrast, nestin-gene-transfected KLM-1 cells exhibited increased sphere formation. Conclusion: Nestin is a pancreatic CSC marker that promotes the sphere-forming ability of CSCs. Therefore, nestin may represent a novel therapeutic target for CSCs in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2457. doi:10.1158/1538-7445.AM2011-2457

Background: Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell/progenitor cell marker. Nestin expression has also been shown to be up-regulated in progenitor cells in muscle, testis, teeth and pancreas. In the pancreas, lineage-tracing experiments have indicated that exocrine cells are derived from Nestin-expressing progenitor cells. Moreover, activation of Kras in the Nestin cell lineage is sufficient for the initiation of premalignant pancreatic intraepithelial neoplasia lesions in mice. We have reported that Nestin was expressed in 30% of human pancreatic ductal adenocarcinoma (PDAC) cases, and Nestin expression in PDAC positively correlated with nerve involvement and invasion of the peripancreatic tissue margin. Therefore, we hypothesized that Nestin may play an important role in the migration and invasive potential of PDAC cells. In this study, we used a silencing strategy to clarify the roles of Nestin in human pancreatic cancer cells. Methods: An expression vector carrying a short hairpin RNA (shRNA) targeting Nestin was stably transfected into PANC-1 and PK-45H human pancreatic cancer cells, which express high levels of Nestin. Changes in the morphology and alignment of actin filaments were analyzed using phase-contrast images and immunocytochemistry. Effects of decreased expression of Nestin on cell growth, migration in scratch and Boyden chamber assays, invasion through matrigel, and cell adhesion on extracellular matrices were examined. Differences in mRNA levels of selected signaling molecules were examined by PCR arrays. Results: Targeting Nestin with shRNA caused a marked decrease in Nestin mRNA and protein levels. Nestin shRNA-transfected cells (Nes-sh cells) exhibited a sheet-like appearance with tight cell-cell adhesion, and increased expression of filamentous (F)-actin by comparison with sham-transfected cells, whereas anchorage-dependent and -independent cell growth and cell-extracellular matrix adhesion of Nes-sh cells did not differ from those of sham cells. By contrast, migration and invasion of Nes-sh cells were markedly attenuated. To clarify the mechanisms underlying this observation, we analyzed differences in selected signaling molecules by PCR arrays that could determine the expression of mRNA closely related to tumor metastasis. The gene expressing the greatest value in Nes-sh cells was E-cadherin. Real-time PCR and western blotting confirmed that E-cadherin expression was increased in Nes-sh cells by comparison with sham cells. Conclusion: Nestin participates in the regulation of pancreatic cancer cell migration and invasion, in part, by altering F-actin and E-cadherin expression. These observations suggest that Nestin may modulate the migration of Nestin-positive progenitor cells during pancreatic development, and may serve as a novel target for suppression of invasion and metastasis in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 408.

Background: Lumican is a member of a small leucine-rich proteoglycan family and its overexpression has been reported in breast, colorectal, neuroendocrine cell, uterine cervical and pancreatic cancers. The expression of lumican in stromal tissues in breast cancer is associated with a high tumor grade, a low estrogen receptor expression level, and young age. Lumican expression in the cytoplasm in advanced colorectal cancer is correlated with a poor prognosis. Lumican expression was previously reported in pancreatic cancer, but the role of lumican in pancreatic cancer is still not well understood. In this study, we clarified the role of lumican in pancreatic cancer. Methods: Immunohistochemical analysis using anti-lumican antibody was performed in 65 pancreatic cancer patients. Lumican mRNA and protein were examined in pancreatic cancer cell lines (i.e. PANC-1, MIA PaCa-2, KLM-1) by real-time PCR and western blot. Lumican stably transfected PANC-1 cells were prepared. Then, the growth rates in vitro and in vivo were compared with mock cells. The lumican expression of PANC-1 cell was knocked down using siRNA. Then, the growth rate in vitro was compared with the control cell. Results: Immunohistochemically, lumican was localized in the cytoplasm of cancer cells in 33 of 65 patients (51%). Lumican expression in cancer cells did not correlated with clinicopathological factors. However, the survival rate of patients with lumican-positive cancer cells was significantly lower than those with lumican-negative cancer cells in the surgery-only group. Lumican mRNA and the protein were detected in all pancreatic cancer cell lines and PANC-1 cells expressed median level of lumican in the cancer cell lines examined. The growth rate of lumican-transfected PANC-1 cells was significantly higher than that of mock cells both in vitro and in vivo. Moreover, the growth rate of lumican-knocked down PANC-1 cell was significantly lower than that of the control cell. Conclusion: Lumican might regulate pancreatic cancer cell growth and become a new target to develop anti-cancer drug for pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2264.