Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Masafumi Nakamura Last modified date:2019.09.18

Professor / Dept. Surgery and Oncology / Department of Clinical Medicine / Faculty of Medical Sciences


Papers
1. Katano M, Morisaki T, Matsuo T, Nakamura M, Kubota E, Hisatsugu T, Interleuikin-2 (IL-2) production by human B-cell line , Cellular Immuno., 159, 2, 262-270, 1994.04, A human B-cell line (Hairy-BM) constitutively secreting interleukin-2 (IL-2) was established from tumor tissue resected surgically from a patient with breast cancer. Hairy-BM was found to be 100% CD20+, 98% surface immunoglobulin (sIg) G+, 98% sIg kappa chain+, 100% HLA-DR+, 94% IL-2 receptor (IL-2R alpha), 98% IL-2R beta, and devoid of T-cell, monocyte, and natural killer cell surface antigens. The B-cell origin of Hairy-BM was also confirmed by clonally rearranged Ig heavy- and Ig light-chain genes. The growth of Hairy-BM expressing IL-2R was promoted by recombinant IL-2 (rIL-2) and anti-CD25 antibody significantly blocked the growth enhancement. IL-2 secretion from Hairy-BM was confirmed by radioimmunoassay. By using a sensitive polymerase chain reaction technique, we demonstrated that Hairy-BM expressed IL-2 mRNA, IL-2R alpha mRNA, and IL-2R beta mRNA. These findings indicate that certain B-cells not only produce, but also respond to IL-2 in an autocrine fashion with increased proliferation.".
2. Katano M, Matsuo T, Morisaki T, Naito K, Nagumo F, Kubota E, Nakamura M, Hisatsugu T, Tadano J, Increased proliferation of a human breast carcinoma cell line by recombinant interleukin-2, Cancer Immunol. Immunother., 39, 3, 161-166, 1994.04, Two adenocarcinoma cell lines, Breast M25-SF and Breast M, were established from tumor tissue resected surgically from a patient with breast cancer. One, Breast M25-SF, expresses interleukin-2 receptor (IL-2R) on the cell surface and the other, Breast M does not. The effects of recombinant interleukin-2 (rIL-2) on the proliferation of these cell lines were investigated. The growth of Breast M25-SF was significantly promoted by rIL-2 ranging from 1.25 U/ml to 640 U/ml. Anti-CD25 (Tac) antibody significantly blocked the growth enhancement of Breast M25-SF by rIL-2. Breast M, however, did not respond to rIL-2. To confirm more directly the promotion of Breast M25-SF growth by rIL-2, cloning of IL-2 responders from parent Breast M25-SF cells was carried out by limiting dilution without feeder cells in 96-well microplates. No colony formation was found in 24 wells without rIL-2. Eleven, 13 and 6 clones were established from groups of 24 wells containing rIL-2 at 200, 20 and 2 U/ml respectively. All of the clones expressed IL-2R and respond to rIL-2. By using a sensitive polymerase chain reaction technique, we demonstrated that Breast M25-SF but not Breast M expressed IL-2 mRNA, and IL-2 secretion from Breast M25-SF but not Breast M was also confirmed by radioimmunoassay. These findings suggest a role for IL-2 in autocrine support of Breast M25-SF growth. IL-2 may play an important role in the growth control of breast carcinoma cells. ".
3. Morisaki T, Katano M, Ikubo A, Anan K, Nakamura M, Nakamura Kenjiro, Sato H, Tanaka M, Torisu M, Immunosuppressive cytokines (IL-10, TGF-beta) genes expression in human gastric carcinoma tissues., J. Surg. Oncol., 63, 4, 234-239, 1996.04, BACKGROUND: Contribution of immunosuppressive cytokines to tumor progression in many types of cancers has been suggested. To characterize the in vivo expression of immunosuppressive cytokines in gastric cancer, we analyzed the messenger RNA (mRNA) expression of transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) in human gastric carcinoma tissues. METHODS: Both tumor tissues and nontumor tissues from each resected specimen of 29 primary gastric carcinomas were tested for IL-10 and TGF-beta mRNA expression by the reverse transcriptase-polymerase chain reaction (RT-PCR), and the mRNA expression was correlated with various pathological parameters of the tumors. RESULTS: Among the 29 tumors, mRNAs of TGF-beta and IL-10 were detected in 79% and 62% of tumor samples, respectively. These cytokines were detected only in 31% for TGF-beta and 17% for IL-10 in nontumor samples. Both mRNAs were frequently expressed in the poorly differentiated adenocarcinomas and the tumor tissues with high degree of stage or lymph node metastasis. CONCLUSIONS: Local expression of immunosuppressive cytokines may contribute to the progression of primary gastric carcinomas possibly through immunosuppression.".
4. Noguchi E, Hayashi N, Azuma Y, Seki T, Nakamura M, Nakashima N, Yanagida M, He X, Mueller U, Sazer S, Nishimoto T, Dis3, implicated in mitotic control, binds directory to Ran and enhances the GEF activity of RCC1., EMBO J., 15, 20, 5595-5605, 1996.04, Using the two-hybrid method, we isolated a Saccharomyces cerevisiae cDNA encoding a protein homologous to Schizosaccharomyces pombe protein Dis3sp, using as bait, human GTPase Ran. The DIS3 gene is essential for viability and complements S.pombe mutant dis3-54 which is defective in mitosis. Although Dis3sc has no homology to RanBP1, it bound directly to Ran and the S.cerevisiae Ran homologue Cnr1, but not to the S.cerevisiae RCC1 homologue Srm1. Upon binding to Ran with a 1:1 molar ratio, Dis3sc enhanced a nucleotide-releasing activity of RCC1 on Ran. In the presence of Dis3sc, the K(m) of RCC1 on Ran decreased by half, while the kcat was unchanged. In vivo, Dis3sp was present as oligomers of M(r) 670-200 kDa as previously reported, and the 200 kDa oligomer of Dis3sp was found to include Spi1 and Pim1, the S.pombe homologues of Ran and RCC1, respectively. Although the biological function of the heterotrimeric oligomer consisting of Dis3, Spi1 and Pim1 is unknown, our results indicate that Dis3 is a component of the RCC1-Ran pathway. ".
5. Nakamura M, Katano M, Fujimoto K, Morisaki T, A new prognostic strategy for gastric carcinoma mRNA expression of tumor growth-related factors in endoscopic biopsy specimens., Ann. Surg. , 226, 1, 35-42, 1997.04, OBJECTIVE: The study analyzed the prognostic value of the transcription of several tumor growth-related genes in gastric carcinoma biopsy specimens. SUMMARY BACKGROUND DATA: The nodal status is one of the most significant prognostic factors in gastric carcinoma. There are, however, no satisfactory parameters for the preoperative assessment of nodal status. METHODS: A reverse transcriptase-polymerase chain reaction analysis was used to analyze the transcription of several tumor growth-related genes in endoscopic biopsy specimens from 78 gastric carcinomas. The factors examined were cyclin D1, cyclin E, urokinase-type plasminogen activator, 72-kd type IV collagenase, vascular endothelial growth factor, platelet-derived growth factor-A (PDGF-A), transforming growth factor-beta, and interleukin-10. The relation between the mRNA expression and the clinical pathologic parameters was analyzed statistically. RESULTS: The incidence of PDGF-A (p = 0.010) and transforming growth factor-beta (p = 0.009) mRNA expression increased as the pathologic stage advanced. Nodal metastasis correlated with cyclin D1 (p = 0.045), cyclin E (p = 0.037), urokinase-type plasminogen activator (p = 0.047), and PDGF-A (p = 0.003) mRNA. Interestingly, the expression of PDGF-A mRNA showed a positive correlation (p = 0.004) with the early presence of lymph node metastases. CONCLUSIONS: Tumor growth-related factor mRNA in biopsy specimens may be a new prognostic tool. ".
6. Nakamura M, Masuda H, Horii J, Kuma K, Yokoyama N, Ohba T, Nishitani H, Miyata T, Tanaka M, Nishimoto T, When overexpressed, a novel centrosomal protein, ranBPM, causes ectopic microtubule nucleation similar to γ-tubulin., J. Cell. Biol. , 143, 4, 1041-1052, 1998.04, A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM. ".
7. Nakamura M, Katano M, Fujimoto K, Miyazaki K, Morisaki T, Mori M, Transforming growth factor β1(TGF-β1)is a preoperative prognostic indicator in advanced gastric carcinoma., Br. J. Cancer., 78, 10, 1373-1378, 1998.04, It has been generally accepted that transforming growth factor beta1 (TGF-beta1) has both negative and positive effects on tumour growth and progression. This study analysed the prognostic value of TGF-beta1 mRNA in advanced gastric carcinoma. A reverse transcriptase-polymerase chain reaction analysis (RT-PCR) was used for TGF-beta1 in endoscopic biopsy specimens from 42 advanced gastric carcinomas. Thirty specimens expressed TGF-beta1 mRNA while 12 specimens did not. The follow-up duration ranged from 4 to 37 months (mean 22.8 months). TGF-beta1-positive group demonstrated a shorter overall survival compared with the TGF-beta1 -negative group (P=0.0014). A significant correlation was also found in the 32 patients who underwent curative resection (P=0.0048). Significant correlations were found between TGF-beta1 mRNA expression and both stage (P=0.0015) and nodal involvement (P=0.0060). Multivariate analysis demonstrated that only TGF-beta1 mRNA expression (P=0.0306) was an independent prognostic factor. All of ten patients who underwent non-curative resection expressed TGF-beta1 mRNA. Expression of TGF-beta1 mRNA in gastric biopsy specimens may be an important preoperative prognostic variable for advanced gastric carcinoma. .
8. Katano M, Nakamura M, Fujimoto K, Miyazaki K, Morisaki T, Prognostic value of platelet-derived growth factor-A(PDGF-A) in gastric carcinoma., Ann. Surg., 227, 3, 365-371, 1998.04, OBJECTIVE: Because our previous study indicated that PDGF-A mRNA expression in biopsy specimens might identify a subgroup of high-risk patients with gastric carcinoma, in this study we analyzed the prognostic value of platelet-derived growth factor-A (PDGF-A) gene expression in gastric carcinoma biopsy specimens. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analyze the PDGF-A gene expression in 65 gastric carcinoma endoscopic biopsy specimens. The 65 patients were divided into a PDGF-A-positive group (29 patients) and a PDGF-A-negative group (36 patients). RESULTS: On the basis of 2-year follow-up data, the PDGF-A-positive group demonstrated a shorter overall survival rate compared with the PDGF-A-negative group (p < 0.0001). A similar correlation was found in 34 advanced-stage patients (p = 0.003) and in 24 advanced-stage patients who underwent a curative resection (p = 0.003). Multivariance analysis indicated that the transcription of PDGF-A gene is a potent prognostic factor that is independent of the traditional pathologic parameters. CONCLUSIONS: Expression of PDGF-A mRNA in gastric biopsy specimens may be a new preoperative prognostic parameter in gastric carcinoma. ".
9. Katano M, Nakamura M, Kuwahara A, Fujimoto K, Matsunaga H, Miyazaki K, Morisaki T, Expression of interleukin(IL)-12 mRNA in gastric carcinoma specimens : cellular antitumor immune responses, J. Surg. Oncol., 67, 1, 11-17, 1998.04, BACKGROUND AND OBJECTIVES: Several tumor-related antigen peptides that are recognized by autologous cytolytic T cells (CTL) have been reported. However, most human solid tumors, including gastric carcinoma, are only weakly immunogenic. In this study, we focused on interleukin (IL)-12 and interferon-gamma (IFN-gamma) as key cytokines for estimating positive cellular immune responses. METHODS: To estimate the immunogenicity of gastric carcinomas, we examined IL-12 and IFN-gamma at mRNA levels by reverse transcription-polymerase chain reaction assay (RT-PCR) in tumor specimens and adjacent nontumor specimens from 36 gastric carcinoma patients. RESULTS: IL-12 expression was detected in 12 tumor specimens and in only two adjacent nontumor specimens (P = .003). The frequency of IFN-gamma gene expression was higher in the IL-12 mRNA-positive tumor specimens than in the IL-12 mRNA-negative tumor specimens (P = .015). In the IL-12 mRNA-positive tumors, IFN-gamma expression was higher in the tumor specimens than in the adjacent nontumor specimens (P = .007). Conversely, in the IL-12 mRNA-negative tumors, IFN-gamma expression was lower in the tumor specimens than in the nontumor specimens (P = .03). Many tumor-infiltrating mononuclear cells, predominantly T cells, were found in four of the 12 IL-12-mRNA-positive tumor specimens and in none of the 24 IL-12-mRNA-negative tumor specimens (P = .008). CONCLUSIONS: These data suggest that possible immune responses against a tumor may occur at the mRNA level in approximately one-third of gastric carcinomas. ".
10. Ohba T, Nakamura M, Nishitani H, Nishimoto T, Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran, Science, 284, 5418, 1356- 1358, 1999.04.
11. Kuwahara A, Katano M, Nakamura M, Fujimoto K, Miyazaki K, Mori M, Morisaki T, New therapeutic strategy for gastric carcinoma:a two-step evaluation of malignant potential from its molecular biologic and pathologic chartacteristics., J. Surg. Oncol., 72, 3, 142-149, 1999.04, BACKGROUND AND OBJECTIVES: A previous study of ours indicated that platelet-derived growth factor-A (PDGF-A) mRNA expression in biopsy specimens can identify a subgroup of high-risk gastric carcinoma patients, while clinicopathologic studies have shown that lymph node involvement is an important risk factor for predicting overall survival. To identify gastric carcinoma patients at high risk for recurrence, we assessed a two-step evaluation consisting of mRNA expression of tumor growth-related factors and the histopathologic findings. METHODS: The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assay the gene expression of PDGF-A and transforming growth factor-beta1 (TGF-beta1) in 69 gastric carcinoma endoscopic biopsy specimens (prospective cohort). The corresponding gastric carcinoma surgical specimens were classified histologically. Finally, the patients' survival curves were calculated. The relationships among the mRNA expression, histopathologic findings, and survival period were analyzed statistically. RESULTS: Nodal involvement correlated with PDGF-A and TGF-beta1 mRNA expression in early and advanced carcinomas, respectively. Both PDGF-A mRNA and TGF-beta1 mRNA expression were independent preoperative prognostic indicators in advanced cases. The ratio of involved nodes (n1) to total perigastric lymph nodes dissected (percentage of involved nodes) was the most independent postoperative prognostic indicator in advanced cases. Early carcinomas were divided preoperatively into two types. Advanced carcinomas were divided preoperatively into three. These were divided again postoperatively according to the percentage of involved nodes into high- and low-malignacy groups. CONCLUSIONS: A two-step evaluation of the malignant potential of gastric carcinoma by a combination of preoperative evaluation for PDGF-A and TGF-beta1 expression and postoperative pathologic examination would yield a more accurate prognosis for patients with gastric carcinoma. Copyright 1999 Wiley-Liss, Inc. ".
12. Sato N, Mizumoto K, Nakamura M, Nakamura Kenjiro, Kusumoto M, Niiyama H, Ogawa T, Tanaka M, Centrosome abnormalities in pancreatic ductal carcinoma, Clin. Cancer Res., 5, 5, 963-970, 1999.04, The centrosome plays an important role in microtubule nucleation and organization, ensuring the establishment of cell polarity and balanced chromosome segregation. Recent studies have suggested that the loss of cell polarity and/or chromosome missegregation (aneuploidy) in human malignant tumors could result from defects in centrosome function. Using immunofluorescence analysis with an antibody to gamma-tubulin (a well-characterized centrosomal component), we examined surgically resected human pancreatic tissues for centrosome abnormalities. The tissues included ductal carcinomas (n = 13), adenomas (n = 3), endocrine tumors (n = 3), chronic pancreatitis (n = 5), and normal pancreatic tissues (n = 12). We found that most (85%) carcinomas and some adenomas displayed abnormal centrosome profiles, characterized by an increase in size and number of centrosomes, and by their irregular distribution. In contrast, none of normal ductal and stromal tissues showed these abnormalities. These findings suggest that centrosome abnormalities may develop at a relatively early stage of pancreatic ductal carcinogenesis.".
13. Kusumoto M, Ogawa T, Mizumoto K, Ueno H, Niiyama H, Sato N, Nakamura M, Tanaka M, Adenovirus-mediated p53 gene transduction inhibits telomerase activity independent of its effects on cell cycle arrest and apoptosis in human pancreatic cancer cells., Clin. Cancer Res., 5, 8, 2140-2147, 1999.04, Evidence for a relationship between overexpression of wild-type p53 and telomerase activity remains controversial. We investigated whether p53 gene transduction could cause telomerase inhibition in pancreatic cancer cell lines, focusing on the relation of transduction to growth arrest, cell cycle arrest, and apoptotic cell death. The cells were infected with recombinant adenovirus expressing wild-type p53 or p21WAF1 at a multiplicity of infection of 100 or were continuously exposed to 10 microM VP-16, which is well known to induce apoptosis. Adenovirus-mediated p53 gene transduction caused G1 cell cycle arrest, apoptosis, and resultant growth inhibition in MIA PaCa-2 cells; the cell number 2 days after infection was 50% of preinfection value, and 13% of the cells were dead. Moreover, the transduction resulted in complete depression of telomerase activity through down-regulation of hTERT mRNA expression. In contrast, p21WAF1 gene transduction only arrested cell growth and cell cycle at G1 phase, and VP-16 treatment inhibited cell growth with G2-M arrest and apoptosis; after treatment, the cell number was 73% of pretreatment, and 12% of the cells were dead. Neither p21WAF1 gene transduction nor VP-16 treatment caused telomerase inhibition. Similar results were obtained in two other pancreatic cancer cell lines, SUIT-2 and AsPC-1. Thus, our results demonstrate that the p53 gene transduction directly inhibits telomerase activity, independent of its effects on cell growth arrest, cell cycle arrest, and apoptosis.".
14. Sato N, Mizumoto K, Nakamura M, Tanaka M, Radiation-induced centrosome overduplication and multiple mitotic spindles in human tumor cells, Exp. Cell Res. , 255, 2, 321-326, 2000.04, The centrosome is a highly regulated organelle and its proper duplication is indispensable for the formation of bipolar mitotic spindles and balanced chromosome segregation. To elucidate a possible linkage between centrosome duplication and radiation-induced nuclear damage, we examined centrosome dynamics in U2-OS osteosarcoma cells following gamma-irradiation. Nearly all control cells contained one or two centrosomes, and at mitosis more than 97% of the cells displayed typical bipolar spindles. In contrast, over 20% of cells at 48 h after 10 Gy gamma-irradiation contained more than two centrosomes, and 60% of the mitotic cells showed aberrant spindles organized by multiple poles. Remarkably, the cells with multiple centrosomes frequently exhibited changes in size and/or morphology of the nucleus, including micronuclei formation. We conclude that abnormal centrosome duplication could be one of the key events involved in nuclear fragmentation and perhaps even cell death following irradiation. Copyright 2000 Academic Press. .
15. Sato N, Mizumoto K, Nakamura M, Ueno H, Minamishima YA, Farber JL, Tanaka M, A possible role for centrosome overduplication in radiation-induced cell death, Oncogene, 19, 46, 5281-5290, 2000.04, Radiotherapy plays a key role in the treatment of many tumors; however, the precise mechanisms responsible for radiation-induced cell death remain uncertain. We have reported previously that ionizing radiation induces centrosome overduplication in human tumor cells. The present study was designed to elucidate a possible link between centrosome dysregulation and radiation-induced cell death. Exposure to 10 Gy gamma-radiation resulted in a substantial increase in cells containing an abnormally high number of centrosomes in a variety of cell lines derived from different types of human solid tumors. These aberrant centrosomes contribute to the assembly of multipolar spindles, thereby causing an unbalanced division of chromosomes and mitotic cell death characterized by the appearance of multi- or micronucleated cells. An extensive analysis of a panel of 10 tumor cell lines revealed a positive correlation between the fraction of cells with multiple centrosomes and the fraction with these nuclear abnormalities after irradiation. When the centrosome overduplication was blocked by enforced expression of p21Waf1/Cip1, the radiation-induced lethality was drastically rescued. Taken together, these results indicate that centrosome overduplication may be a critical event leading to mitotic failure and subsequent cell death following exposure to ionizing radiation. .
16. Kishi S, Wulf G, Nakamura M, Lu KP, Telomeric protein Pin2/TRF1 induces mitotic entry and apoptosis in cells with short telomeres and is down-regulated in human breast tumors., Oncogene, 20, 12, 1497-1508, 2001.04, Telomeres are essential for cell survival and have been implicated in the mitotic control. The telomeric protein Pin2/TRF1 controls telomere elongation and its expression is tightly regulated during cell cycle. We previously reported that overexpression of Pin2/TRF1 affects mitotic progression. However, the role of Pin2/TRF1 at the interface between cell division and cell survival remains to be determined. Here we show that overexpression of Pin2 induced apoptosis in cells containing short telomeres, but not in cells with long telomeres. Furthermore, before entering apoptosis, Pin2-expressing cells first accumulated in mitosis and strongly stained with the mitosis-specific MPM2 antibody. Moreover, Pin2-induced apoptosis is potentiated by arresting cells in mitosis, but suppressed by accumulating cells in G1. In addition, overexpression of Pin2 also resulted in activation of caspase-3, and its proapoptotic activity was significantly reduced by inhibition of caspase-3. These results indicate that up-regulation of Pin2/TRF1 can specifically induce entry into mitosis and apoptosis, likely via a mechanism related to activation of caspase-3. Significantly, we also found that, out of 51 human breast cancer tissues and 10 normal controls examined, protein levels of Pin2/TRF1 in tumors were significantly lower than in normal tissues, as detected by immunoblotting analysis and immunocytochemistry. Since down-regulation of Pin2/TRF1 allows cells to maintain long telomeres, these results suggest that down-regulation of Pin2/TRF1 may be important for cancer cells to extend their proliferative potential. ".
17. Shono M, Sato N, MizumotoK, Maehara N, Nakamura M, Nagai E, Tanaka M, Stepwise progression of centrosome defects associated with local tumor growth and metastatic process of human pancreatic carcinoma cells transplanted orthotopically into nude mice, Lab. Invest., 81, 7, 945-952, 2001.04, Recent evidence indicates that loss of centrosome integrity may be a major cause of genetic instability underlying various human cancers. The aim of this study was to define the role of centrosome defects during the in vivo tumor progression of pancreatic carcinoma using an orthotopic implantation model. Injection of Suit-2 human pancreatic cancer cells into the pancreata of nude mice reproduced the pattern of local tumor growth and distant metastasis observed in humans. Pancreatic xenografts, peritoneal disseminations, and hepatic metastases were harvested, and tumor cells were examined for centrosomes by immunofluorescence microscopy. Centrosome abnormalities, characterized by increased numbers of centrosomes, were detected in only a small fraction of parental Suit-2 cells in culture, whereas the frequency was markedly increased in cells isolated from the pancreatic xenografts. Abnormal centrosome numbers were found at higher frequencies in metastatic foci than in pancreatic xenografts. A significant positive correlation existed between the fraction of cells with multiple centrosomes and that with multipolar mitotic spindles, suggesting a functional involvement of aberrant centrosomes in spindle disorganization and chromosome missegregation. In addition, the increased frequency of abnormal centrosomes was associated with an enhanced degree of chromosomal instability. These findings suggest a novel model of pancreatic tumor progression whereby a stepwise increase in the magnitude of centrosomal abnormalities confers an increased chance for aberrant mitotic events, thus accelerating genetic instability and causing the tumor to progress to a more advanced stage. ".
18. Ryo A, Nakamura M, Wulf G, Liou YC, Lu KP, Pin1 regulates turnover and subcellular localization of betacatenin by inhibiting its interaction with APC., Nat. Cell Biol., 3, 9, 793-801, 2001.04, Phosphorylation on a serine or threonine residue preceding proline (Ser/Thr-Pro) is a key regulatory mechanism, and the conformation of certain phosphorylated Ser/Thr-Pro bonds is regulated specifically by the prolyl isomerase Pin1. Whereas the inhibition of Pin1 induces apoptosis, Pin1 is strikingly overexpressed in a subset of human tumours. Here we show that Pin1 regulates beta-catenin turnover and subcellular localization by interfering with its interaction with adenomatous polyposis coli protein (APC). A differential-display screen reveals that Pin1 increases the transcription of several beta-catenin target genes, including those encoding cyclin D1 and c-Myc. Manipulation of Pin1 levels affects the stability of beta-catenin in vitro. Furthermore, beta-catenin levels are decreased in Pin1-deficient mice but are increased and correlated with Pin1 overexpression in human breast cancer. Pin1 directly binds a phosphorylated Ser-Pro motif next to the APC-binding site in beta-catenin, inhibits its interaction with APC and increases its translocation into the nucleus. Thus, Pin1 is a novel regulator of beta-catenin signalling and its overexpression might contribute to the upregulation of beta-catenin in tumours such as breast cancer, in which APC or beta-catenin mutations are not common. ".
19. Nishitani H, Hirose E, Uchimura Y, Nakamura M, Umeda M, Nishii K, Mori N, Nishimoto T, Full-sized RanBMP cDNA encodes a protein possessing a long stretch of proline and glutamine within the N-terminal region, comprising a large protein complex, Gene, 272, 1-2, 25-33, 2001.04, Previously isolated RanBPM, a Ran-binding protein in the microtubule-organizing center, which had been thought to play a role in Ran-stimulated microtubule assembly, turned out to be a truncated protein. To clarify the function of RanBPM, we cloned the full-sized RanBPM cDNA that encodes a 90 kDa protein, compared to the previously isolated cDNA that encoded a 55 kDa protein. The newly cloned 5' coding region contains a great number of cytidine and guanidine nucleotides, like the CpG island. Thus, full-sized RanBPM cDNA encodes a long stretch of proline and glutamine residues in the N-terminal region. It comprises a protein complex of more than 670 kDa. Ran was detected in this complex when RanBPM and Ran were both ectopically expressed. New antibodies to RanBPM were prepared against three different regions of RanBPM. All of them detected a 90 kDa protein that is predominantly localized both in the nucleus and in the cytoplasmic region surrounding the centrosome, but none of them stained the centrosome. In this context, our previous notion that RanBPM is a centrosomal protein should be discarded. RanBPM is well conserved in the animal kingdom. It may play an important role in uncovering Ran-dependent nuclear events. ".
20. Shono M, Sato N, Mizumoto K, Minamishima Y, Nakamura M, Maehara N, Urashima T, Saimura M, Qian L, Nishio S, Nagai E, Tanaka M, Effect of serum depletion on centrosome overduplication and death of human pancreatic cancer cells after exposure to radiation., Cancer Lett., 170, 1, 81-89, 2001.04, The tumor microenvironment is one of the key factors affecting the cellular response to radiation; however, the influence of serum concentration on tumor radiosensitivity remains poorly understood. We recently discovered that gamma-irradiation of tumor cells causes centrosome overduplication, which may lead to lethal nuclear fragmentation through the establishment of multipolar mitotic spindles. In the present study, we investigated the effect of serum depletion on radiation-induced cell death in relation to the centrosome dynamics in human pancreatic cancer cells. Exposure of Capan-1 cells to gamma-irradiation resulted in a time-dependent increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death. Treatment of irradiated cells with serum depletion drastically accelerated centrosome overduplication and the formation of multipolar spindles, resulting in increased nuclear fragmentation and cell death. Cell cycle analysis of irradiated cultures revealed that the reduced serum level increased the population of cells arrested in the G2/M phase, which might be responsible for the abnormal centrosome accumulation. These findings suggest that serum concentration can influence radiation-induced cell killing through modulating cell cycle progression and possibly centrosome overduplication. ".
21. Nakamura M, Zhou XZ, Lu KP, Critical role for the EB1 and APC interaction in the regulation of microtubule polymerization., Curr. Biol., 11, 13, 1062-1067, 2001.04, Human EB1 was originally cloned as a protein that interacts with the COOH terminus of adenomatous polyposis coli (APC). Interestingly, this interaction is often disrupted in colon cancer, due to mutations in APC. EB1 also interacts with the plus-ends of microtubules and targets APC to microtubule tips. Since APC is detected on the kinetochores of chromosomes, it has been hypothesized that the EB1-APC interaction connects microtubule spindles to the kinetochores and regulates microtubule stability. In yeast, EB1 regulates microtubule dynamics, and its binding domain in APC may be conserved in Kar9, an EB1 binding protein involved in the microtubule-capturing mechanism. These results suggest that the interaction of EB1 and APC is important and may be conserved. However, it is largely unknown whether the EB1-APC interaction affects microtubule dynamics. Here, we show that EB1 potently promotes microtubule polymerization in vitro and in permeabilized cells, but, surprisingly, only in the presence of the COOH-terminal EB1 binding domain of APC (C-APC). Significantly, this C-APC activity is abolished by phosphorylation, which also disrupts its ability to bind to EB1. Furthermore, yeast EB1 protein effectively substitutes for the human protein but also requires C-APC in promoting microtubule polymerization. Finally, C-APC is able to promote microtubule polymerization when stably expressed in APC mutant cells, demonstrating the ability of C-APC to promote microtubule assembly in vivo. Thus, the interaction between EB1 and APC plays an essential role in the regulation of microtubule polymerization, and a similar mechanism may be conserved in yeast. ".
22. Sato N, Mizumoto K, Nakamura M, Maehara N, Minamishima Y, Nishio S, Nagai E, Tanaka M, Correlation between centrosome abnormalities and chromosomal instability in human pancreatic cancer cells, Cancer Genet. Cytogen., 126, 1, 13-19, 2001.04, Chromosomal instability, characterized by abnormal numbers or structures of chromosomes, is a common feature of human cancers, but the mechanisms behind these changes are still unclear. Since centrosomes play a pivotal role in balanced chromosomal segregation during mitosis, we attempted to investigate the association between centrosome abnormalities and chromosomal instability in a large number of human pancreatic cancer cell lines. Immunofluorescence microscopy revealed centrosomes that were highly atypical with respect to their size, shape, and number in most cell lines. These abnormal centrosomes contributed to the assembly of multipolar spindles, resulting in defective mitosis and chromosome mis-segregation. Interestingly, a high frequency of centrosome defects inversely correlated with the growth rate of cells in culture. Fluorescence in situ hybridization revealed a dramatic variation of chromosome numbers in cell lines with the defective centrosome phenotype. Furthermore, a significant positive correlation existed between the level of centrosome defects and the level of chromosomal imbalances. These results indicate that centrosome abnormalities can lead to spindle disorganization and chromosome segregation errors, which may drive the accumulation of chromosomal alterations. Thus, defects in centrosome function may be an underlying cause of genetic instability in human pancreatic cancers. ".
23. Nakamura M, Zhou XZ, Kishi S, Kosugi I, Tsutsui Y, Lu KP, A specific interaction between the telomeric protein Pin2/TRF1 and the mitotic spindle., Curr. Biol., 11, 19, 1512-1516, 2001.04, Pin2/TRF1 was independently identified as a telomeric DNA binding protein (TRF1) [1] and as a protein (Pin2) that can bind the mitotic kinase NIMA and suppress its ability to induce mitotic catastrophe [2, 3]. Pin2/TRF1 has been shown to bind telomeric DNA as a dimer [3-7] and to negatively regulate telomere length [8-11]. Interestingly, Pin2/TRF1 levels are regulated during the cell cycle, being increased in late G2 and mitosis and degraded as cells exit from mitosis [3]. Furthermore, overexpression of Pin2/TRF1 induces mitotic entry and then apoptosis [12]. This Pin2/TRF1 activity can be significantly potentiated by the microtubule-disrupting agent nocodazole [12] but is suppressed by phosphorylation of Pin2/TRF1 by ATM; this negative regulation is important for preventing apoptosis upon DNA damage [13]. These results suggest a role for Pin2/TRF1 in mitosis. However, nothing is known about how Pin2/TRF1 is involved in mitotic progression. Here, we describe a surprising physical interaction between Pin2/TRF1 and microtubules in a cell cycle-specific manner. Both expressed and endogenous Pin2/TRF1 proteins were localized to the mitotic spindle during mitosis. Furthermore, Pin2/TRF1 directly bound microtubules via its C-terminal domain. Moreover, Pin2/TRF1 also promoted microtubule polymerization in vitro. These results demonstrate for the first time a specific interaction between Pin2/TRF1 and microtubules in a mitosis-specific manner, and they suggest a new role for Pin2/TRF1 in modulating the function of microtubules during mitosis..
24. Ryo A, Liou Y. C, Wulf G, Nakamura M, Lee S. W, Lu K. P, PIN1 is an E2F target gene essential for Neu/Ras-induced transformation of mammary epithelial cells, Mol Cell Biol, 22, 15, 5281-5295, 2002.04.
25. Nakamura M, Zhou XZ, Kishi K, Lu KP , Involvement of the telomeric protein Pin2/TRF1 in the regulation of the mitotic spindle


, FEBS Lett., 514, 2-3, 193-198, 2002.04, Pin2/TRF1 was independently identified as a telomeric DNA-binding protein (TRF1) that regulates telomere length, and as a protein (Pin2) that can bind the mitotic kinase NIMA and suppress its lethal phenotype. We have previously demonstrated that Pin2/TRF1 levels are cell cycle-regulated and its overexpression induces mitotic arrest and then apoptosis. This Pin2/TRF1 activity can be potentiated by microtubule-disrupting agents, but suppressed by phosphorylation of Pin2/TRF1 by ATM; this negative regulation is critical in mediating for many, but not all, ATM-dependent phenotypes. Interestingly, Pin2/TRF1 specifically localizes to mitotic spindles in mitotic cells and affects the microtubule polymerization in vitro. These results suggest a role of Pin2/TRF1 in mitosis. However, nothing is known about whether Pin2/TRF1 affects the spindle function in mitotic progression. Here we characterized a new Pin2/TRF1-interacting protein, EB1, that was originally identified in our yeast two-hybrid screen. Pin2/TRF1 bound EB1 both in vitro and in vivo and they also co-localize at the mitotic spindle in cells. Furthermore, EB1 inhibits the ability of Pin2/TRF1 to promote microtubule polymerization in vitro. Given that EB1 is a microtubule plus end-binding protein, these results further confirm a specific interaction between Pin2/TRF1 and the mitotic spindle. More importantly, we have shown that inhibition of Pin2/TRF1 in ataxia-telangiectasia cells is able to fully restore their mitotic spindle defect in response to microtubule disruption, demonstrating for the first time a functional involvement of Pin2/TRF1 in mitotic spindle regulation.

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26. Morisaki T, Matsumoto K, Onishi H, Kuroki H, Baba E, Tasaki A, Kubo M, Nakamura M, Inaba S, Yamaguchi K, Tanaka M, Katano M, Dendritic cell-based combined immunotherapy with autologous tumor-pulsed dendritic cell vaccine and activated T cells for cancer patients: rationale, current progress, and perspectives, Hum Cell, 16, 4, 175-182, 2003.04, Effective adoptive cancer immunotherapy depends on an ability to generate tumor-antigen-presenting cells and tumor-reactive effector lymphocytes and to deliver these effector cells to the tumor. Dendritic cells (DCs) are the most potent antigen-presenting cells, capable of sensitizing T cells to new and recall antigens. Many studies have shown that tumors express unique proteins that can be loaded on DCs to trigger an immune response. The current experimental and clinical statuses of adoptive transfer of tumor antigen-pulsed DCs and vaccine-primed activated T cells are summarized herein. Clinical trials of antigen-pulsed DCs have been conducted in patients with various types of cancer, including non-Hodgkin lymphoma, multiple myeloma, prostate cancer, renal cell carcinoma, malignant melanoma, colorectal cancer, and non-small cell lung cancer. These studies have shown that antigen-loaded DC vaccination is safe and promising for the treatment of cancer. In addition, tumor vaccine-primed T cells have been shown to induce antitumor activity in vivo. Several clinical studies are being conducted on the use of vaccine-primed T cells such as tumor-drainage lymph node. It is reasonable to consider using both tumor antigen-pulsed DCs and vaccine-primed lymphocytes as adjuvants. We are now investigating the use of autologous whole tumor antigen-pulsed DCs and the DC vaccine-primed activated lymphocytes in patients with multiple metastasis of solid tumors.
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27. Morisaki T, Matsumoto K, Kuroki H, Kubo M, Baba E, Onishi H, Tasaki A, Nakamura M, Inaba S, Katano M, Combined immunotherapy with intracavital injection of activated lymphocytes, monocyte-derived dendritic cells and low-dose OK-432 in patients with malignant effusion.
, Anticancer Res., 23, 6a, 4459-4465, 2003.04, We have conducted a pilot study with combined immunotherapy using autologous lymphocytes activated ex vivo and monocyte-derived dendritic cells in combination with low-dose OK-432, a streptococcal preparation, in five patients with peritoneal or pleural carcinomatosis who were resistant to standard chemotherapy. All patients were given 3 to 10 courses of the combined immunotherapy. No severe adverse reactions occurred. Effusion production was decreased in all of the patients. Significant decreases in tumor markers of both effusions and sera as well as effusion volume occurred in all of the patients. Cytological examinations revealed a marked decrease or disappearance of cancer cells in those effusions. Three patients showed increase in IFN-gamma levels in the effusions. The overall prognosis of the patients was acceptable and the mean survival time was more than 9 months. The locoregional immunotherapy seems to be encouraging in view of therapeutic modality in patients who are resistant to standard chemotherapy. Our study provides a new protocol for immunotherapy and warrants further phase I/II clinical study for chemo-resistant patients with malignant effusion.
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28. Kubo M, Nakamura M, Tasaki A, Yamanaka N, Nakashima H, Nomura M, Kuroki S, Katano M, Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer
, Cancer Res. , 64, 17, 6071-6074, 2004.04, The Hedgehog (Hh) signaling pathway functions as an organizer in embryonic development. Genetic analysis has demonstrated a critical role for the Hh pathway in mammary gland morphogenesis. Disruption of Patched1, a component of the Hh pathway, results in abnormal growth of mammary duct. Recent studies have shown constitutive activation of the Hh pathway in various types of malignancies. However, it remains unclear whether this pathway is activated in human breast cancer. Here, we determined the expression of the components, including Sonic Hh, Patched1, and Gli1, of the Hh pathway by immunohistochemical staining in a series of 52 human breast carcinomas. All of 52 tumors display staining of high intensity for Gli1 when compared with adjacent normal tissue. The nuclear staining ratio of Gli1 correlates with expression of estrogen receptor and histologic type. Exposure to cyclopamine, a steroidal alkaloid that blocks the Hh pathway, suppresses expression of Gli1 and the growth of the Hh pathway-activated breast carcinoma cells. These data indicate that the Hh pathway is a new candidate for therapeutic target of breast cancer.
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29. Kim K. T, Ongusaha P. P, Hong Y. K, Kurdistani S. K, Nakamura M, Lu K. P, Lee S. W, Function of Drg1/Rit42 in p53-dependent mitotic spindle checkpoint, J Biol Chem, 279, 37, 38597-38602, 2004.04.
30. Matsumoto K, Morisaki T, Kuroki H, Kubo M, Onishi H, Nakamura K, Nakahara C, Kuga H, Baba E, Nakamura M, Hirata K, Tanaka M, Katano M, Exosomes secreted from monocyte-derived dendritic cells support in vitro naive CD4+ T cell survival through NF-(kappa)B activation, Cell Immunol, 231, 1-2, 20-29, 2004.04, Abstract

We investigated the effect of exosomes secreted from human monocyte-derived dendritic cells (Mo-DCs), which are generated from PBMCs in response to treatment with GM-CSF and IL-4, on naive CD4+ T cell survival in vitro. Exosomes isolated from culture supernatants of Mo-DCs (>90% purity) were purified with anti-HLA-DP, -DQ, -DR-coated paramagnetic beads. Purified exosomes prolonged the survival of naive CD4+ T cells (>98% purity) in vitro. Treatment with neutralizing mAb against HLA-DR significantly decreased the supportive effect of purified exosomes on CD4+ T cell survival. Exosomes increased nuclear translocation of NF-(kappa)B in naive CD4+ T cells, and NF-(kappa)B activation was significantly suppressed by anti-HLA-DR mAb or NF-(kappa)B inhibitor pyrrolidine dithiocarbamate (PDTC). In addition, PDTC inhibited the effect of exosomes on naive CD4+ T cell survival. Thus, exosomes secreted by Mo-DCs appear to support naive CD4+ T cell survival via NF-(kappa)B activation induced by interaction of HLA-DR and TCRs.
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31. Yamaguchi K, Nakamura M, Shirahane K, Konomi H, Torata N, Hamasaki N, Kawakita M, Tanaka M, Urine diacetylspermine as a novel tumour maker for pancreatobiliary carcinomas, Digestive and Liver disease , 37, 3, 190-194, 2005.04.
32. Koga K, Matsumoto K, Akiyoshi T, Kubo M, Yamanaka N, Tasaki A, Nakashima H, Nakamura M, Kuroki S, Tanaka M, Katano M, Purification, characterization and biological significance of tumor-derived exosomes., Anticancer Res, 25, 6A, 3703-3707, 2005.04, Abstract

Exosomes are nanovesicles that are released into the extracellular environment during the fusion of multivesicular bodies with the plasma membrane. Exosomes released from dendritic cells, dexosomes, have several biological functions, for example as immunostimulants. Some tumor cells also secrete exosomes (Tu-exosomes). Although experimental data obtained with the use of dexosomes suggest a biological function of Tu-exosomes, this still remains poorly understood. To examine the function of Tu-exosomes, we established a method for collecting highly purified Tu-exosomes, using paramagnetic beads coated with antibodies against tumor-specific proteins such as HER2/neu. With these antibody-coated beads (Ab-beads), it was possible to collect HER2-expressing Tu-exosomes of high purity. Tu-exosomes were also collected from malignant ascites, which contain exosomes secreted from various types of cells such as tumor cells, lymphoid cells and mesothelial cells. The isolation of Tu-exosomes was confirmed by FACS analysis. With regard to their biological functions, Tu-exosomes cultured with a human breast cancer cell line bound to the cell surface and increased tumor cell proliferation. These data indicate that Tu-exosomes may have physiological functions.
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33. Yamaguchi K, Nakamura M, Shirahane K, Kawamoto M, Konomi H, Ohta M, Tanaka M, Pancreatic juice cytology in IPMN of the pancreas.
, Pancreatology, 5, 4-5, 416-421, 2005.04, Background: Intraductal papillary-mucinous neoplasm (IPMN) of the pancreas is a disease ranging from adenoma to borderline (with moderate dysplasia) and further to carcinoma (noninvasive and invasive) and surgical strategy is different by the grades of dysplasia. Methods: Preoperativepancreatic juice cytology in IPMN was reviewed in 71 patients with IPMN who underwent surgical resection. Results: The IPMN was adenoma in 48 patients, borderline in 13 and carcinoma (invasive) in 10. The sensitivity of pancreatic juice cytology in malignant IPMN was 40% (4/10). In 4 patients with the 48 IPM adenomas, diagnosis of pancreatic juice cytology was class IV or V. One of the 4 cases was considered to be an overdiagnosis of cytology, but the other 3 cases were considered to be a consequence of accompanying carcinoma in situ (or PanIN-3) (2 patients) or invasive ductal adenocarcinoma (1 patient) apart from IPMN. Sensitivity of pancreatic juice cytology was higher in IPMN of the main duct type with mucin hypersecretion and with mural nodules. Conclusions: These findings suggest that pancreatic juice cytology in IPMN is useful especially in the main duct type with mucin hypersecretion and mural nodules. When the diagnosis of pancreatic juice cytology is malignant in otherwise benign-looking IPMNs, coexistence of pancreatic carcinoma should be suspected. Copyright (c) 2005 S. Karger AG, Basel and IAP.
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34. Kubo M, Morisaki T, Matsumoto K, Tasaki A, Yamanaka N, Nakashima H, Kuroki H, Nakamura K, Nakamura M, Katano M, Paclitaxel probably enhances cytotoxicity of natural killer cells against breast carcinoma cells by increasing perforin production, Cancer Immunol Immunother, 54, 5, 468-476, 2005.04.
35. Tasaki A, Akiyoshi T, Koga K, Nakashima h, Yamanaka N, Kubo M, Matsumoto K, Kojima M, Tanaka M, Nakamura M, Katano M, Immunohistochemical staining of hedgehog pathway-related proteins in human thymomas, Anticancer research, 25, 6A, 3697-3701, 2005.04, The thymus plays an essential role in the maturing of progenitor cells to functional T cells. Recent studies suggest that the Hedgehog (Hh) signaling pathway contributes to this differentiation process. However, there is limited information concerning the expression of Hh pathway-related proteins (Hh proteins) in the human thymus. The staining of Hh proteins in the thymic epithelium of 26 surgically resected thymoma tissues was examined by immunohistochemistry. The staining of sonic Hh (Shh) correlated relatively well with the World Health Organization histological classification of thymoma. The higher the grade, the fainter the staining. However, no significant difference in Shh staining was found between normal and neoplastic epithelia. Interestingly, Gli1 staining in thymomas was significantly greater than that in normal thymus (p < 0.0001). Thus, some members of the Hh signaling pathway may contribute to the development of thymoma..
36. Yamaguchi K, Shirahane K, Nakamura M, Su D, Konomi H, Motoyama K Sugitani A, Mizumoto K, Tanaka M, Frozen section and permanent diagnoses of the bile duct margin in gallbladder and bile duct cancer., HPB (the official journal of the International Hepato Pancreato Biliary Association) , 7, 2, 135-138, 2005.04.
37. Nakashima H, Tasaki A, Kubo M, Kuroki H, Matsumoto K, Tanaka M, Nakamura M, Morisaki T, Katano M, Effects of docetaxel on antigen presentation-related functions of human monocyte-derived dendritic cells.
, Cancer Chemother Pharmacol. , 55, 5, 479-487, 2005.04, PURPOSE: Docetaxel (TXT) is a unique chemotherapeutic agent that has been approved for treating various types of malignancies. TXT stabilizes microtubule assembly in cells and causes various dysfunctions of microtubule-dependent cellular events. Patients with advanced malignancies are beginning to receive TXT in combination with immunotherapy; however, the influence of TXT at clinically achievable serum concentrations (less than 10(-6) M) on antigen presentation-related functions of human monocyte-derived dendritic cells (Mo-DCs) remains unclear. METHODS: Immature Mo-DCs (imMo-DCs) were generated from peripheral blood monocytes with interleukin-4 and granulocyte-macrophage colony-stimulating factor in vitro. Mature Mo-DCs (mMo-DCs) were induced from imMo-DCs with tumor necrosis factor-alpha and prostaglandin E(2). RESULTS: TXT at concentrations lower than 10(-7) M did not significantly affect cellular viability, phagocytosis, or expression of antigen presentation-related molecules of Mo-DCs. In contrast, TXT at concentrations lower than 10(-9) M significantly suppressed directional motility of imMo-DCs toward MIP-1alpha and of mMo-DCs toward MIP-3beta. However, TXT had no effect on either CCR1 expression by imMo-DCs or CCR7 expression by mMo-DCs. No gross changes in the microtubule skeleton were evident by immunofluorescence microscopy after treatment with TXT at less than 10(-8) M. However, reduced numbers of imMo-DCs with podosomes localized primarily in one cell region were observed. CONCLUSIONS: The present results indicate that different concentrations of TXT influence antigen presentation-related functions differently. In particular, TXT at relatively low therapeutic doses disrupts chemotactic motility of Mo-DCs.
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38. Katano M, Morisaki T, Koga K, Nakamura M, Onishi H, Matsumoto K, Tasaki A, Nakashima H, Akiyoshi T, Nakamura M, Combination therapy with tumor cell-pulsed dendritic cells and activated lymphocytes for patients with disseminated carcinomas., Anticancer Res, 25, 6A, 3771-3776, 2005.04.
39. Nakashima H, Nakamura M, Yamaguchi H, Yamanaka N, Akiyoshi T, Koga K, Yamaguchi K, Tsuneyoshi M, Tanaka M, Katano M, Nuclear factor-kappaB contributes to hedgehog signaling pathway activation through sonic hedgehog induction in pancreatic cancer., Cancer Res., 66, 14, 7041-7049, 2006.04, Abstract

The hedgehog (Hh) signaling pathway, which functions as an organizer in embryonic development, is implicated in the development of various tumors. In pancreatic cancer, pathway activation is reported to result from aberrant expression of the ligand, sonic Hh (Shh). However, the details of the mechanisms regulating Shh expression are not yet known. We hypothesized that nuclear factor-kappaB (NF-kappaB), a hallmark transcription factor in inflammatory responses, contributes to the overexpression of Shh in pancreatic cancer. In the present study, we found a close positive correlation between NF-kappaB p65 and Shh expression in surgically resected pancreas specimens, including specimens of chronic pancreatitis and pancreatic adenocarcinoma. We showed that blockade of NF-kappaB suppressed constitutive expression of Shh mRNA in pancreatic cancer cells. Further activation of NF-kappaB by inflammatory stimuli, including interleukin-1beta, tumor necrosis factor-alpha, and lipopolysaccharide, induced overexpression of Shh, resulting in activation of the Hh pathway. Overexpression of Shh induced by these stimuli was also suppressed by blockade of NF-kappaB. NF-kappaB-induced Shh expression actually activated the Hh pathway in a ligand-dependent manner and enhanced cell proliferation in pancreatic cancer cells. In addition, inhibition of the Hh pathway as well as NF-kappaB suppressed the enhanced cell proliferation. Our data suggest that NF-kappaB activation is one of the mechanisms underlying Shh overexpression in pancreatic cancer and that proliferation of pancreatic cancer cells is accelerated by NF-kappaB activation in part through Shh overexpression.
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40. Akiyoshi T, Nakamura M, Nakashima H, Yao T, Tsuneyoshi M, Tanaka M, Katano M, Gli1, downregulated in colorectal cancers, inhibits proliferation of colon cancer cells involving Wnt signalling activation., Gut, 55, 7, 991-999, 2006.04, BACKGROUND: Early events in the progression of 90% of sporadic colorectal cancers depend on constitutive activation of Wnt signalling. Recent data also indicate a close association between the Hedgehog (Hh) and Wnt pathways in colonic epithelial cell differentiation. AIMS: To investigate whether expression of Gli1, a transactivator of Hh signalling, can suppress Wnt signalling and inhibit proliferation of human colorectal cancer cells. METHODS: Gli1 and nuclear beta-catenin expression were examined in a series of 40 human colorectal cancers by immunohistochemistry. We quantified Gli1 and nuclear beta-catenin staining as markers of Hh and Wnt pathway activation, respectively. Two human colon cancer cell lines, SW480 and HCT116, with mutations in APC and beta-catenin, respectively, were used. The effects of Gli1 overexpression on Wnt transcriptional activity, beta-catenin subcellular distribution, and proliferation in these cells were analysed. RESULTS: Nuclear accumulation of beta-catenin and the Gli1 staining level were inversely associated in the 40 human colorectal cancers. Wnt transcriptional activity was reduced in Gli1 transfected cells. These effects were observed even in Gli1 transfected cells cotransfected with mutated beta-catenin. Furthermore, nuclear accumulation of beta-catenin was diminished compared with that in empty vector transfected cells, and downregulated transcription of c-Myc was observed in Gli1 transfected cells. Proliferation of Gli1 transfected cells was also significantly suppressed compared with that in empty vector transfected cells. CONCLUSIONS: Our data suggest that Gli1 plays an inhibitory role in the development of colorectal cancer involving Wnt signalling, even in cases with the stabilising mutation of beta-catenin.

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41. Yanai K, Nagai S, Wada J, Yamanaka N, Nakamura M, Torata N, Noshiro H, Tsuneyoshi M, Tanaka M, Katano M, Hedgehog signaling pathway is a possible therapeutic target for gastric cancer., J Surg Oncol. , 95, 1, 55-62, 2007.04, BACKGROUND AND OBJECTIVES: It has been shown that the hedgehog (Hh) signaling pathway is activated in gastric cancer. To investigate the viability of the Hh pathway as a therapeutic target, we analyzed activation of the Hh pathway in gastric cancer. METHODS: Surgically resected gastric carcinoma specimens and lymph nodes were analyzed immunohistochemically. We used the percentage of cancer cells with nuclear translocation of Gli1 as a marker of Hh pathway activation. RESULTS: Nuclear localization of Gli1 was higher in 28 undifferentiated-type tumors than in 30 differentiated-type tumors. Eighteen of the fifty-eight cancer specimens consisted of a mixture of a histologically predominant part and a small area with different histology. In these 18 tumors, the percentage of cells showing nuclear staining of Gli1 was higher in the undifferentiated-type part than in the differentiated-type part. Nuclear staining of Gli1 in primary tumors was positively correlated with lymph node metastasis. The Gli1 nuclear staining percentage of metastatic lymph nodes correlated closely with that of each primary carcinoma. Cyclopamine, a Hh pathway inhibitor, suppressed the growth of gastric cancer cells in vitro. CONCLUSIONS: The Hh pathway may be a useful therapeutic target for such as undifferentiated-type gastric cancer with lymph node metastasis.

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42. Nakamura M, Kubo M, Yanai K, Mikami Y, Ikebe M, Nagai S, Yamaguchi K, Tanaka M, Katano M , Anti-patched-1 antibodies suppress hedgehog signaling pathway and pancreatic cancer proliferation., Anticancer Res., 27, 6A, 3743-3748, 2007.04, BACKGROUND: The hedgehog (Hh) signaling pathway is aberrantly activated in many human carcinomas including pancreatic cancer and regulates tumor cell growth. Overproduction of sonic hedgehog (Shh), a ligand of the Hh signaling pathway, increases the Hh signaling activity through transmitting the signal to patched-1 (Ptch1), the receptor of the Hh signaling pathway. MATERIALS AND METHODS: a-Ptch1 antibodies were raised against an oligo-peptide, designed according to the Ptch1 aminoacid sequence. The specificity of a-Ptch1 was examined by immunoblotting and immuno-fluorescence, and biological effects were detected by RT-PCR and cell proliferation assay using two pancreatic cancer cell lines, Panc1 and SUIT-2. RESULTS: a-Ptch1 recognized a 160 kDa protein as shown by immunoblotting and cell surface staining of pancreatic cancer cells. Incubation with a-Ptch1 suppressed Hh signaling activity and proliferation of pancreatic cancer cells. CONCLUSION: These results provide a new strategy for controling Hh dependent development of pancreatic cancer and other Hh related carcinomas.

PMID: 17970037 [PubMed - in process]
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43. Wada J, Yamasaki A, Nagai S, Yanai K, Fuchino K, Kameda C, Tanaka Haruo, Koga K, Nakashima H, Nakamura M, Tanaka M, Katano M, Morisaki T, Regulatory T-cells are possible effect prediction markers of immunotherapy for cancer patients., Anticancer Res., 28, 4C, 2401-2408, 2008.04, We previously showed that a combination therapy with tumor cell-pulsed monocyte-derived dendritic cells (DCs) and activated lymphocytes was well tolerated in patients with disseminated carcinomas. Recently, accumulating evidence has indicated that regulatory T-cells (Tregs), a unique population of CD4+ T-cells, are increased in patients with several advanced malignancies and prevent cell-mediated immune responses against tumors. However, reports analyzing the relationship between the Tregs population and the effects of immunotherapy are extremely rare. In the present study, 22 patients received an intravenous injection of DC-activated lymphocytes (DAK) and/or a subcutaneous injection of tumor-pulsed DCs (DC vaccine) every 2 to 4 weeks. The Tregs were defined based on their expression of CD4, CD25 and FOXP3, a transcription factor. Most CD4+CD25high T-cells expressed FOXP3. Therefore, CD4+CD25high T-cells were evaluated as Tregs in the present study. As reported previously, the percentage of Tregs (% Tregs) among total CD4+ T-cells in peripheral blood mononuclear cells (PBMCs) was significantly higher for advanced cancer patients than for healthy volunteers. When the patients were divided into three groups according to their survival time, i.e. 12 short-survival patients, 4 medium-survival patients and 6 long-survival patients, the % Tregs of the long-survival patients before the therapy was significantly lower than that of the short-survival patients (p=0.026). The % Tregs decreased after the therapy, although the difference did not reach statistical significance. When the patients were divided into a high group (>4.99%: 7 patients) and a low group (<4.99%: 15 patients) according to their % Tregs before the therapy, the survival times of the two groups differed significantly (p=0.0034). These data suggest that the % Tregs among the PBMCs might be used as an effect prediction factor of immunotherapy for patients with advanced cancer.

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44. Koga K, Nakamura M, Nakashima H, Akiyoshi T, Kubo M, Sato N, Kuroki S, Nomura M, Tanaka M, Katano M, Novel link between estrogen receptor alpha and hedgehog pathway in breast cancer., Anticancer Res. , 28, 2A, 731-740, 2008.04, Ligand-dependent constitutive activation of the hedgehog (Hh) pathway is important in the development of various carcinomas including breast cancer. A link between estrogen receptor alpha (ERalpha) and the Hh pathway in human breast cancer is shown here for the first time. In ERalpha-positive cells, estrogen depletion decreased the expression of sonic hedgehog (Shh), a ligand of the Hh pathway, while estrogen supplementation triggered Shh up-regulation. This estrogen-induced Shh expression activated the Hh pathway in a ligand-dependent manner, and increased cell proliferation. These effects were suppressed by ERalpha inhibitors, including ICI 182,780 (ICI), the dominant negative form of ERalpha and small interfering RNA (siRNA) against ERalpha. Consistent with the in vitro data, a positive correlation between ERalpha and Shh expression was found in breast cancer tissues. These data suggest that ERalpha regulates the Hh pathway through Shh induction, and promotes breast cancer development.

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45. Nagai S, Nakamura M, Yanai K, Wada J, Akiyoshi T, Nakashima H, Ohuchida K, Sato N, Tanaka M, Katano M, Gli1 contributes to the invasiveness of pancreatic cancer through matrix metalloproteinase-9 activation., Cancer Sci., 99 , 7, 1377-1384, 2008.04, The hedgehog (Hh) signaling pathway has been reported to be associated with the growth of pancreatic cancer, but its role in the invasive phenotype is poorly understood. Therefore, we investigated the role of the Hh pathway in pancreatic cancer cell invasiveness using a Matrigel invasion assay. Blockade of the Hh pathway by cyclopamine inhibited pancreatic cancer cell invasion in association with a decreased expression of matrix metalloproteinase (MMP)-9. By contrast, activation of the Hh pathway by the addition of exogenous Sonic hedgehog increased cell invasion and MMP-9 expression. Stable transfection of pancreatic cancer cells with Gli1 increased their invasiveness, which was associated with activation of MMP-9. We also showed that inhibition of MMP-9 by small interfering RNA blocked the increased invasiveness of Gli1-transfected cells. Furthermore, inhibition of Gli1 by small interfering RNA suppressed the invasiveness and MMP-9 expression of pancreatic cancer cells. Taken together, these findings suggest that members of the Hh pathway, especially Gli1, play an important role in the invasiveness of pancreatic cancer cells through the regulation of MMP-9 expression.

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46. Akiyoshi T, Nakamura M, Yanai K, Nagai S, Wada J, Koga K, Nakashima H, Sato N, Tanaka M, Katano M, Gamma-secretase inhibitors enhance taxane-induced mitotic arrest and apoptosis in colon cancer cells., Gastroenterology., 134, 1, 131-144, 2008.04, BACKGROUND & AIMS: Colorectal cancers are resistant to conventional chemotherapeutic treatments, including taxanes. gamma-Secretase is a multimeric membrane protein complex responsible for the intramembrane proteolysis of various type I transmembrane proteins, including amyloid beta-precursor protein and Notch. gamma-Secretase inhibitors have attracted increasing interest as anticancer drugs because of their ability to inhibit Notch signaling. However, the therapeutic usefulness of gamma-secretase inhibitors against colorectal cancers remains unclear. METHODS: The effects of gamma-secretase inhibitors on growth and apoptosis induced by various chemotherapeutic agents in colon cancer cells were evaluated using Hoechst 33342 staining, colony formation assay, and cell cycle analysis. The effect of gamma-secretase inhibitors on taxane-induced mitotic arrest was evaluated using the cyclin B1-associated histone H1 kinase assay and MPM-2 reactivity. The involvement of Notch signaling was evaluated by the silencing of Notch/CBF1 signaling by RNA interference. RESULTS: gamma-Secretase inhibitors enhanced taxane-induced mitotic arrest and apoptosis of colon cancer cells both in vitro and in vivo, although gamma-secretase inhibitors alone did not affect growth and apoptosis of colon cancer cells. We also showed that this effect by gamma-secretase inhibitors was restricted to taxanes and colon cancer cells. Silencing of Notch/CBF1 signaling failed to affect paclitaxel-induced mitotic arrest and apoptosis. CONCLUSIONS: These data suggest that gamma-secretase inhibitors could be a new therapeutic modality for overcoming resistance to taxanes in colorectal cancers..
47. Yanai K, Nakamura M, Akiyoshi T, Nagai S, Wada J, Koga K, Noshiro H, Nagai E, Tsuneyoshi M, Tanaka M, Katano M. , Crosstalk of hedgehog and Wnt pathways in gastric cancer., Cancer Lett, 263, 1, 145-156, 2008.04, Morphogenic signals like Hedgehog (Hh) and Wnt are reported to play critical roles in the progression of gastric cancer. We aimed to assess the relationship between Hh and Wnt signaling pathways. In 58 gastric cancer specimens, Wnt pathway activation was inversely correlated with Hh pathway activation. When AGS gastric cancer cells, in which Wnt signaling was constitutively active, were used as a target cell line, Gli1 overexpression suppressed Wnt transcriptional activity, nuclear beta-catenin accumulation and proliferation of AGS cells. Knock-down of beta-catenin by siRNA suppressed Wnt pathway activity and proliferation of AGS cells. Our data may provide some clues for the treatment of gastric cancer associated with Wnt signaling activation.

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48. Tanaka Haruo, Nakamura M, Kameda C, Kubo M, Sato N, Kuroki S, Tanaka M, Katano M, The Hedgehog signaling pathway plays an essential role in maintaining the CD44+CD24-/low subpopulation and the side population of breast cancer cells.
, Anticancer Res. , 29, 6, 2147-2157, 2009.04, The side population (SP) and the CD44(+)/CD24(-/low) population have been reported in separate studies to include more tumorigenic cells than other populations, and to have the ability to form new tumors and undergo heterogeneous differentiation in breast cancer tissue. However, the relationship between these two populations has not yet been explored in breast cancer cells. Here it is shown that the SP and the CD44(+)/CD24(-/low) populations are overlapping. Both populations were resistant to paclitaxel. Components of the Hedgehog (Hh) signaling pathway were more highly expressed in these cell populations at both the mRNA and protein levels compared with other populations. Furthermore, inhibition of Hh signaling activity suppressed the proliferation of both populations. The significance of Hh signaling activity in the proliferation of both populations was confirmed by the effect of an si-RNA against Gli1, a trans-activator of the Hh signaling pathway, on the proliferation of both populations. These data suggest that the Hh signaling pathway is essential for the proliferation of the tumorigenic population of breast cancer cells, and that this pathway might represent a new candidate for breast cancer therapy targeting cancer stem cells.

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49. Kameda C, Tanaka Haruo, Yamasaki A, Nakamura M, Koga K, Sato N, Kubo M, Kuroki S, Tanaka M, Katano M , The Hedgehog pathway is a possible therapeutic target for patients with estrogen receptor-negative breast cancer, Anticancer Res, 29, 3, 871-879, 2009.04, Understanding the expression patterns of estrogen receptor-alpha (ERalpha) is essential for determining therapeutic strategies for patients with breast cancer. The prognosis of patients with ERalpha-negative breast cancer is still poor. We have previously shown that Hedgehog (Hh) signaling is constitutively activated in breast cancer and that Hh signaling could be a new therapeutic target. Therefore, in this study, whether or not Hh signaling could be utilized as a therapeutic target for patients with ERalpha-negative breast cancer was examined. For this purpose, three ERalpha-negative breast cancer cell lines were used in which Hh pathway-related molecules such as the ligand Patched1 and the transcriptional factor Gli1 as target cells are expressed. Cyclopamine, an inhibitor of the Hh pathway, significantly suppressed both the cell proliferation and invasion ability of these cancer cells. In addition, the knockdown of Gli1 by RNA interference in these cells also significantly reduced both cell proliferation and invasion ability. Since our previous data have shown a constitutive activation of the Hh pathway in surgically-resected ERalpha-negative breast cancer specimens, the Hh pathway, especially Gli1, may be a useful therapeutic target for patients with ERalpha-negative breast cancer.

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50. Wada J, Suzuki H, Fuchino R, Yamasaki A, Nagai S, Yanai K, Koga K, Nakamura M, Tanaka M, Morisaki T, Katano M, The contribution of vascular endothelial growth factor to the induction of regulatory T-cells in malignant effusions, Anticancer Res. , 29, 3, 881-888, 2009.04, AbstractIt has been suggested that immunosuppressive cytokines such as transforming growth factor beta (TGF-beta) and interleukin 10 play an important role in the induction and/or maintenance of regulatory T-cells (Tregs) in patients with cancer. In the present study, whether or not vascular endothelial growth factor (VEGF) contributes to the induction and/or maintenance of Tregs was examined, because of experience with a patient in whom a positive correlation between VEGF concentration and the percentage of Tregs (% Tregs) among the total CD4(+) T-cells in the pleural effusion was found during dendritic cell activated lymphocyte therapy. CD4(+)CD25(high) T-cells were estimated as Tregs in the present study. In an in vitro experimental system, VEGF-containing malignant effusions increased the % Tregs in autologous peripheral blood mononuclear cells (PBMCs), which could be suppressed by the addition of a humanized monoclonal anti-VEGF antibody (bevaciz
umab [Avastin]). When VEGF-producing hepatic carcinoma cells were mix-cultured with PBMCs, the % Tregs increased and this increase was also suppressed by the addition of bevacizumab. Whether or not bevacizumab can affect the % Tregs of PBMCs in patients with colon cancer was also examined. Three out of four patients showed a significant decrease of the % Tregs after intravenous injection of bevacizumab. Interestingly, the expression of VEGF receptor-2 (VEGFR-2) was higher in Tregs than in other CD4(+) T-cells. Taken together, the data presented here indicate a contribution of VEGF to induction and/or maintenance of Tregs in patients with cancer..
51. Nakamura M, Wada J, Suzuki H, Tanaka M, Katano M, Morisaki T, Long-term Outcome of Immunotherapy for Patients with Refractory Pancreatic Cancer, Anticancer Res, 29, 3, 831-836, 2009.04, BACKGROUND: Pancreatic cancer is one of the most fatal human cancers, with a 5-year survival rate of <5% . Although new chemotherapies have been used for pancreatic cancer, the outcome is still poor. Here, we retrospectively analyzed the outcome of immunotherapy in pancreatic cancer patients and revealed the potential of immunotherapy in advanced pancreatic cancer treatment. PATIENTS AND METHODS: Seventeen pancreatic cancer patients underwent immunotherapy in the Kyushu University and the Yakuin CA Clinic. Six patients had postoperative recurrence, 11 were diagnosed as inoperable because of metastasis, 16 had prior chemotherapy and developed chemotherapy-resistant cancers, while 1 patient had no prior chemotherapy for recurrent cancer after surgical resection because of leukopenia. Immunotherapy was combined with chemotherapy in 11 patients and without chemotherapy in 6 patients. Immunotherapy was classified into two groups; combined dendritic cell (DC) vaccination and intravenous or peritoneal injection of activated lymphocytes (DC vaccine therapy), or injection of lymphokine-activated killer lymphocytes (LAK) alone (LAK therapy). RESULTS: Immunotherapy of refractory pancreatic cancer resulted in a median survival of 9 months. Peritoneal metastasis tended to shorten the survival period. Combination immunotherapy and chemotherapy showed no obvious difference as compared to immunotherapy alone. DC vaccine therapy conferred a significantly better survival period than LAK alone. CONCLUSION: Our results suggest that immunotherapy utilizing DC vaccination may prolong the survival of refractory pancreatic cancer patients.

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52. Ikebe M, Kitaura Y, Nakamura M, Tanaka Haruo, Yamasaki A, Nagai S, Wada J, Yanai K, Koga K, Sato N, Kubo M, Tanaka M, Onishi H, Katano M, Lipopolysaccharide (LPS) increases the invasive ability of pancreatic cancer cells through the TLR4/MyD88 signaling pathway
, J Surg Oncol. , 100, 8, 725-731, 2009.04, BACKGROUND: Inflammation plays a multifaceted role in cancer progression, and NF-kappaB is one of the key factors connecting inflammation with cancer progression. We have shown that lipopolysaccharide (LPS) promotes NF-kappaB activation in colon cancer cells and pancreatic cancer cells. However, it is unclear why inflammatory stimuli can induce NF-kappaB activation in cancer cells. METHODS: We used two human pancreatic cancer cells, Panc-1 and AsPC-1, as target cells. LPS was used as an inflammatory stimulus. To confirm the participation of TLR4/NF-kappaB signaling pathway, we used three different NF-kappaB inhibitors (PDTC, IkappaBalpha mutant, and NF-kappaB decoy ODN) and siRNAs (against TLR4, MyD88, and MMP-9). Effect of LPS on pancreatic cancer cell invasive ability was determined by Matrigel invasion assay. RESULTS: LPS increased the invasive ability of pancreatic cancer cells, while blockade of NF-kappaB pathway decreased the LPS-dependent increased invasive ability. Blockade of TLR4 or MyD88 by siRNA also decreased the LPS-dependent increased invasive ability. CONCLUSION: These results suggest that TLR/MyD88/NF-kappaB signaling pathway plays a significant role in connecting inflammation and cancer invasion and progression. Copyright 2009 Wiley-Liss, Inc.

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53. Xu R, Sato N, Yanai K, Akiyoshi T, Nagai S, Wada J, Koga K, Mibu R, Nakamura M, Katano M, Enhancement of paclitaxel-induced apoptosis by inhibition of mitogen-activated protein kinase pathway in colon cancer cells, Anticancer Res. 2009 Jan;29(1):261-70., 29, 1, 261-270, 2009.04, Resistance to chemotherapy represents a major obstacle to improving the survival of patients with colorectal cancer. In this study, the inhibition of the mitogen-activated protein kinase (MAPK) pathway was demonstrated to markedly enhance the apoptosis of colon cancer cells induced by paclitaxel, one of the key chemotherapeutic drugs widely used to treat various types of cancer. The treatment of the colon cancer cell lines SW480 and DLD-1 with paclitaxel resulted in increased activation of the MAPK pathway, which was blocked by PD98059, a MEK inhibitor. In both cell lines, MAPK inhibition by PD98059 led to a dramatic enhancement of the paclitaxel-induced apoptosis, as determined by cell cycle analysis and Hoechst 33342 staining, although the inhibitor alone did not affect apoptosis. This effect was restricted to paclitaxel since PD98059 did not alter the sensitivity to other drugs, including 5-fluorouracil (5-FU) and camptothecin (CPT). Importantly, selective blockage of the MAPK pathway by small interfering RNA (siRNA) also increased the apoptotic cell death induced by paclitaxel. These findings highlight the importance of the MAPK pathway in paclitaxel-induced apoptosis and suggest that a combined treatment with paclitaxel and MEK inhibitors could be an attractive therapeutic strategy against colon cancer.
54. Yamasaki A, Shoda M, Iijima H, Nagai S, Wada J, Suzuki H, Chikazawa N, Tasaka T, Kameda C, Tanaka Haruo, Ikebe M, Jo E, Sato N, Nakamura M, Sekine F, Morisaki T, Katano M, A protein-bound polysaccharide, PSK, enhances tumor suppression induced by docetaxel in a gastric cancer xenograft model
, Anticancer Res., 29, 3, 843-850, 2009.04, BACKGROUND: We have reported previously that docetaxel (TXT) induces apoptosis and nuclear factor-kappaB (NF-kappaB) activation, and that blockade of NF-kappaB activation augments TXT-induced apoptosis in human gastric cancer cells. In addition, we have also shown that a protein-bound polysaccharide PSK enhances TXT-induced apoptosis through NF-kappaB inhibition in human pancreatic cancer cells. Based on these observations, in the present study the possibility that PSK could enhance TXT-mediated tumor suppression was examined in vivo and in vitro. MATERIALS AND METHODS: A gastric cancer xenograft model was used to examine the enhanced TXT-mediated tumor suppression by PSK in vivo. The effects of PSK on proliferation and apoptosis induced by TXT in gastric cancer cells were evaluated in vitro using a human gastric cancer cell line, MK-1. The effect of PSK on increased TXT-induced invasion was also measured. RESULTS: PSK enhanced TXT-mediated tumor suppression in vivo. Immunohistochemical analyses of the tumors revealed that TXT increased NF-kappaB activation in the tumors and this was suppressed by PSK. In the ex vivo experimental system, PSK enhanced the growth inhibition and apoptosis induced by TXT in the MK-1 cells and reduced the increased invasive ability induced by TXT. CONCLUSION: PSK enhanced TXT-induced tumor suppression in a gastric cancer xenograft model.

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55. Kameda C, Nakamura M, Tanaka Haruo, Yamasaki A, Kubo M, Tanaka M, Onishi H, Katano M, Oestrogen receptor-alpha contributes to the regulation of the hedgehog signalling pathway in ERalpha-positive gastric cancer
, Br J Cancer, 102, 4, 738-747, 2010.04, BACKGROUND: Oestrogen receptor-alpha (ERalpha) is highly expressed in diffuse-type gastric cancer and oestrogen increases the proliferation of ERalpha-positive gastric cancer. However, a detailed mechanism by which oestrogen increases the proliferation of these cells is still unclear. METHODS: We used 17-beta-oestradiol (E2) as a stimulator against the ERalpha pathway. Pure anti-oestrogen drug ICI 182 780 (ICI) and small interfering RNA against ERalpha (ERalpha siRNA) were used as inhibitors. Cyclopamine (Cyc) was used as the hedgehog (Hh) pathway inhibitor. Two human ERalpha-positive gastric cancer cells were used as target cells. Effects of the stimulator and inhibitor on E2-induced cell proliferation were also examined. RESULTS: In ERalpha-positive cells, E2 increased not only cell proliferation but also one of the ligands of the Hh pathway, Shh expression. 17-beta-Oestradiol-induced cell proliferation was suppressed by ICI, ERalpha siRNA or Cyc. The increased expression of Shh induced by E2 was suppressed by ICI and ERalpha siRNA but not by Cyc. Furthermore, recombinant Shh activated the Hh pathway and increased cell proliferation, whereas anti-Shh antibody suppressed E2-induced cell proliferation. When a relationship between ERalpha and Shh expressions was analysed using surgically resected gastric cancer specimens, a positive correlation was found, suggesting a linkage between the ERalpha and Hh pathways. CONCLUSION: Our data indicate that activation of the ERalpha pathway promotes cell proliferation by activating the Hh pathway in a ligand-dependent manner through Shh induction of ERalpha-positive gastric cancer.

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56. Sadakari Y, Otsuka T, Ohuchida K, Tsutsumi K, Takahata S, Nakamura M, Mizumoto K, Tanaka M, MicroRNA expression analyses in preoperative pancreatic juice samples of pancreatic ductal adenocarcinoma.
, JOP. , 11, 6, 587-592, 2010.04, Abstract
CONTEXT: Cytological assessment of pancreatic juice is commonly used to diagnose pancreatic ductal adenocarcinoma; however, the sensitivity of cytological assessment has been reported to be low. MicroRNAs are small RNAs regulating various cellular processes and have recently been identified as possible markers of malignant diseases including pancreatic ductal adenocarcinoma.

OBJECTIVE: The purposes of this study were to prove the existence of microRNAs in pancreatic juice and to determine whether specific microRNAs in pancreatic juice could be used for detecting pancreatic ductal adenocarcinoma.

METHODS: Relative expression levels of microRNA-21 and microRNA-155 in formalin-fixed paraffin-embedded tissues of resected specimens (no. 13) and pancreatic juice samples collected using preoperative endoscopic retrograde cholangiopancreatography (no. 21) were quantified and their expression levels were then compared to pancreatic ductal adenocarcinoma and chronic pancreatitis.

RESULTS: Relative expression levels of microRNA-21 in tissue and pancreatic juice samples were significantly higher in pancreatic ductal adenocarcinoma than those in chronic pancreatitis (P=0.009 and P=0.021, respectively). The same results were obtained in the expression levels of microRNA-155 in tissue and pancreatic juice between pancreatic ductal adenocarcinoma and chronic pancreatitis (P=0.014 and P=0.021, respectively). Expression levels of microRNA-21 and microRNA-155 did not correlate with the preoperative cytological results of pancreatic juice.

CONCLUSION: MicroRNA-21 and microRNA-155 in pancreatic juice have the potential of becoming biomarkers for diagnosing pancreatic ductal adenocarcinoma.

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57. Sadakari Y, Ohuchida K, Nakata K, Ohtsuka T, Aishima S, Takahata S, Nakamura M, Mizumoto K, Tanaka M, Invasive carcinoma derived from the nonintestinal type intraductal papillary mucinous neoplasm of the pancreas has a poorer prognosis than that derived from the intestinal type, Surgery , 147, 6, 812-817, 2010.04, BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is divided into 4 subtypes: an intestinal type, a gastric type, a pancreatobiliary type, and an oncocytic type. The purposes of this study were to clarify the outcomes and the characteristics of invasive carcinoma derived from IPMN (invasive IPMC) by focusing on these subtypes with a comparison to conventional invasive ductal carcinoma (IDC) of the pancreas. METHODS: A total of 30 patients with invasive IPMC were reviewed, and the tumors were divided into 2 pathologic subtypes, intestinal and nonintestinal type. The prognosis and characteristics of the 2 subtypes were evaluated. Furthermore, the prognosis of 119 patients with conventional IDC was compared with that of patients with invasive carcinoma derived from the intestinal or nonintestinal type IPMN. RESULTS: The 5-year survival rate of patients with the nonintestinal type (0.0%) was as poor as that of patients with conventional IDC (19.9%; P = .67). The patients with the intestinal type (66.7%) had a more favorable prognosis than patients with conventional IDC (P < .001). The nonintestinal type was characterized by positive lymphatic invasion and tubular invasive pattern. CONCLUSION: Invasive carcinoma derived from the nonintestinal type IPMN characterized by lymphatic invasion and tubular invasive pattern is associated with a poor prognosis. Copyright 2010 Mosby, Inc. All rights reserved.

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58. Chikazawa N, Tanaka Haruo, Tasaka T, Nakamura M, Tanaka M, Onishi H, Katano M, Inhibition of Wnt signaling pathway decreases chemotherapy-resistant side-population colon cancer cells
, Anticancer Res, 30, 6, 2041-2048, 2010.04, BACKGROUND: The prognosis of advanced or recurrent colorectal cancer is still poor. Dye-effluxing side population (SP) colon cancer cells are reportedly resistant to chemotherapeutic agents. Most sporadic colorectal cancers involve constitutive activation of the Wnt signaling pathway. In this study, we examined the effect of the Wnt signaling on SP cells and the possibility that inhibition of Wnt signaling may decrease the resistance to chemotherapeutic drugs in the human colon cancer cells.

MATERIALS AND METHODS: Drug resistance of SP cells to 5-fluorouracil (5-FU) and irinotecan, decrease of SP cells by the Wnt signaling inhibition and activation of the Wnt signaling of the sorted SP cells were examined using the SW480 colon cancer cell line. mRNA expressions of ATP-binding cassette (ABC) transporters when Wnt signaling was inhibited were evaluated with real-time PCR using colon cancer cell lines (SW480, DLD-1, HCT116, HT29 and LOVO). The sensitivity to irinotecan and paclitaxel when the Wnt signaling was inhibited was investigated using SW480. Inhibition of Wnt signaling was performed by siRNA of beta-catenin.

RESULTS: SP cells showed more resistance to 5-FU and irinotecan, and higher activation of the Wnt signaling pathway, than non-SP cells. Silencing of beta-catenin decreased significantly more SP cells than non-SP cells. Expression of ABC transporter genes, such as ABCB1 and ABCG2, was significantly higher in SP cells than non-SP cells. Silencing of beta-catenin decreased transcription of these ABC transporter genes; beta-catenin-silenced cells became relatively sensitive to paclitaxel and irinotecan.

CONCLUSION: These results indicate that inhibiting the Wnt signaling pathway may be a fruitful strategy for targeting chemotherapy-resistant colon cancer cells, including SP cells.

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59. Kobayashi K, Sadakari Y, Ohtsuka T, Takahata S, Nakamura M, Mizumoto K, Tanaka M , Factors in Intraductal Papillary Mucinous Neoplasms of the Pancreas Predictive of Lymph Node Metastasis
, Pancreatology, 10, 6, 720-725, 2010.04, Background: Little is known about the frequency of lymph node metastasis (LNM) in intraductal papillary mucinous neoplasms (IPMNs), and we have not been able to determine how much lymph node dissection is necessary in individual cases. The aim of this study was to investigate the predictive factors for the LNM in IPMNs. Methods: Medical records of 120 patients pathologically diagnosed as having IPMN were reviewed, and 16 possible predictive factors regarding the LNM were analyzed. Results: LNM was observed in 7 patients (6%), all of whom were diagnosed as having mural nodules preoperatively. Sensitivity, specificity, and accuracy of preoperative imaging for detecting mural nodules of IPMNs in this study were 84, 97, and 90%, respectively. Univariate analysis using 61 patients having mural nodules preoperatively revealed that the size of mural nodules ≥10 mm and positive imaging findings for invasive tumor and possible LNM were significant predictive factors for the LNM. Multivariate analysis demonstrated that only an imaging finding for invasive tumor was an independent significant predictive factor. Positive and negative predictive values of the imaging finding of invasive IPMNs for LNM were 50 and 98%, respectively. Conclusions: Standard lymph node dissection would be recommended in patients with IPMNs with mural nodules demonstrating preoperative imaging findings for invasive carcinomas. and IAP.

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60. Sadakari Y, Ienaga J, Kobayashi K, Miyasaka Y, Takahata S, Nakamura M, Mizumoto K, Tanaka M, Cyst Size Indicates Malignant Transformation in Branch Duct Intraductal Papillary Mucinous Neoplasm of the Pancreas Without Mural Nodules, Pancreas, 39, 2, 232-236, 2010.04, In branch duct intraductal papillary mucinous neoplasm (IPMN) of the pancreas, the importance of the cyst size to predict malignancy is still controversial. Our aim was to elucidate the malignant potential of branch duct IPMN without mural nodules (flat branch duct IPMN). METHODS:: Seventy-three patients with flat branch duct IPMNs were studied in our institution. RESULTS:: There were 6 malignant IPMNs in this series, all of which were 30 mm or more in size, whereas there was no malignancy in IPMNs of less than 30 mm. Statistically significant predictors of malignancy were atypical cytological condition and main pancreatic duct (MPD) diameter of 5 mm or more. The cyst size of 30 mm or more tended to be associated with malignancy. The frequency of malignancy in flat branch duct IPMNs with the size of 30 mm or more and MPD diameter of less than 5 mm was 3.6%, whereas there were 5 malignant cases (26.3%) in flat branch duct IPMNs with the size of 30 mm or more and MPD diameter of 5 mm or more. CONCLUSIONS:: We conclude that the size criteria (>/=30 mm) to predict malignancy proposed in the international consensus guidelines is appropriate and resection or meticulous follow-up using cytological examination and MPD dilatation is needed in patients with flat branch duct IPMNs.
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61. Aly MY, Tsutsumi K, Nakamura M, Sato N, Takahata S, Ueda J, Shimizu S, Redwan AA, Tanaka M, Comparative study of laparoscopic and open distal pancreatectomy
, J Laparoendosc Adv Surg Tech A , 20, 5, 435-440, 2010.04, BACKGROUND: Laparoscopic distal pancreatectomy (LDP) has been shown to be an effective surgical option for benign lesions in the body and tail of the pancreas. However, its advantages and disadvantages have not been well characterized. In this study, we compared the outcomes of LDP and open pancreatectomy performed in our clinic.

MATERIALS AND METHODS: Peri- and postoperative outcomes were retrospectively compared between patients with benign pancreatic disorders who underwent open distal pancreatectomy (ODP) (n = 35) and those who underwent LDP (n = 40). The peri- and postoperative factors analyzed included operative time, blood loss, hospital stay, postoperative recovery, biochemical findings, and complications.

RESULTS: LDP was associated with significantly less operative blood loss (363 versus 606 mL; P = 0.001) and shorter hospital stay (22 versus 27 day; P = 0.009), but longer operative time (342 versus 250 min; P = 0.000), compared with ODP. There were no significant differences between the two groups in complication rates or postoperative recovery, except for the significantly shorter duration of postoperative pain-killer intake and earlier improvement of the biochemical analysis in LDP than in ODP.

CONCLUSIONS: LDP appears to be a safe, desirable procedure for the management of benign pancreatic diseases, with outcomes similar to ODP
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62. Tsutsumi K, Ohtsuka T, Oda Y, Sadakari Y, Mori Y, Aishima S, Takahata S, Nakamura M, Mizumoto K, Tanaka M, A History of Acute Pancreatitis in Intraductal Papillary Mucinous Neoplasms of the Pancreas Is a Potential Predictive Factor for Malignant Papillary Subtype
, Pancreatology, 10, 6, 707-712, 2010.04, Background/Aims: There are several reports regarding intraductal papillary mucinous neoplasms (IPMNs) detected after the occurrence of acute pancreatitis. Although the presence of symptoms is regarded as a factor for predicting malignant IPMNs, there have been few reports demonstrating whether a history of acute pancreatitis is a predictor of malignancy. The aim of this study was to evaluate the relationship between a history of acute pancreatitis and clinicopathological features of IPMNs including the papillary subtype. Methods: The data of 150 IPMNs resected between 1990 and 2009 were retrospectively reviewed. They were classified into IPMNs with or without history of acute pancreatitis, and then the clinicopathological features were compared between the 2 groups. Results: Nineteen (13%) of the 150 patients had a history of acute pancreatitis. Nine of them had repeated episodes of pancreatitis; however, severe pancreatitis was uncommon. The diameter of the main pancreatic duct of the pancreatitis group was significantly larger than that of the nonpancreatitis group (p = 0.04). The pancreatitis group had a significantly higher frequency of carcinoma derived from IPMNs than the nonpancreatitis group (p = 0.03). The incidence of intestinal-type IPMNs in the pancreatitis group was significantly higher than that in the nonpancreatitis group (p < 0.001). Conclusion: Acute pancreatitis associated with IPMNs could predict malignant intestinal-type tumor. and IAP.

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63. Nakamura M, Ueda J, Kono H, Aly MY, Takahata S, Shimizu S, Tanaka M, Prolonged peri-firing compression with a linear stapler prevents pancreatic fistula in laparoscopic distal pancreatectomy.
, Surg Endosc. , 25, 3, 867-871, 2011.04, BACKGROUND: Laparoscopic distal pancreatectomy (Lap-DP) is one of the most accepted laparoscopic procedures in the field of pancreatic surgery. However, pancreatic fistula remains a major and frequent complication in Lap-DP, as in open surgery. The aim of this retrospective study is to clarify the advantages of prolonged peri-firing compression (PFC) with a linear stapler for prevention of pancreatic fistula after laparoscopic distal pancreatectomy.

PATIENTS AND METHODS: Incidence of pancreatic fistula in clinical levels (equivalent to grades B and C defined by the International Study Group of Pancreatic Fistula (ISGPF)) was retrospectively compared between patients who underwent Lap-DP with PFC (PFC group, n = 17) and those who underwent Lap-DP without PFC (no-PFC group, n = 25).

RESULTS: Incidence of clinical pancreatic fistula was significantly lower in the PFC group than in the no-PFC group. Consistent with the results for pancreatic fistula, peritoneal drainage period and postoperative hospital stay were shorter in the PFC group than in the no-PFC group.

CONCLUSIONS: Our data show that PFC effectively prevents pancreatic fistula and shortens postoperative hospital stay after Lap-DP.

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64. Nakamura M, Nagayoshi Y, Kono H, Mori Y, Otsuka T, Takahata S, Shimizu S, Tanaka M, Lateral approach for laparoscopic splenic vessel-preserving distal pancreatectomy.
, Surgery, 150, 2, 326-331, 2011.04, AIM: We sought to evaluate the feasibility of the lateral approach for laparoscopic splenic vessel-preserving distal pancreatectomy (LA-SVPDP).

BACKGROUND: Complete preservation of the splenic vessels is an ideal outcome in spleen-preserving distal pancreatectomy (SPDP). However, the preservation of the vessels is challenging in laparoscopic surgery because the splenic vein is often embedded in the pancreatic parenchyma. Herein we have described LA-SVPDP, the most feasible method for laparoscopic SPDP, and the outcome of our initial experience.

PATIENTS: Twenty-three patients underwent laparoscopic SPDP. Before we adopted LA-SVPDP, 8 patients underwent the Warshaw method and 6 underwent SVPDP. After the adoption of LA-SVPDP, 8 patients underwent LA-SVPDP and 1 donor underwent the Warshaw method.

RESULTS: None of patients undergoing LA-SVPDP required conversion to an open operation, whereas 2 patients undergoing the other procedures were converted to open operations. Five out of 8 patients who underwent the Warshaw method showed engorgement of the gastric veins, revealed by computed tomography. However, only 1 of the 5 patients showed mild gastric varices on endoscopy.

CONCLUSION: Although the Warshaw method is acceptable with a low incidence of gastric varices in our analysis, SVPDP is a feasible approach for SPDP. Our LA-SVPDP technique may contribute to safer and easier SVPDP in laparoscopic surgery.

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65. Chen G, Goto Y, Sakamoto R, Tanaka K, Matsubara E, Nakamura M, Zheng H. Lu J, Takayanagi R, . Nomura M , GLI1, a crucial mediator of sonic hedgehog signaling in prostate cancer, functions as a negative modulator for androgen receptor.
, Biochem Biophys Res Commun. , 404, 3, 809-815, 2011.04, Sonic hedgehog (SHH) signaling, acting in a combinatorial manner with androgen signaling, is essential for prostate patterning and development. Recently, elevated activation of SHH signaling has been shown to play important roles in proliferation, progression and metastasis of prostate cancer. In this report, we demonstrate for the first time, that GLI1, which has been shown to play a central role in SHH signaling in prostate cancer, can act as a co-repressor to substantially block androgen receptor (AR)-mediated transactivation, at least in part, by directly interacting with AR. Our observations suggest that the SHH-GLI pathway might be one of determinants governing the transition of prostate cancer from anandrogen-dependent to an androgen-independent state by compensating, or even superseding androgen signaling.

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66. Tsutsumi K, Sato N, Tanabe R, Mizumoto K, Morimatsu K, Kayashima T, Fujita H, Ohuchida K, Ohtsuka T, Takahata S, Nakamura M, Tanaka M, Claudin-4 Expression Predicts Survival in Pancreatic Ductal Adenocarcinoma
, Ann Surg Oncol. , 19, Suppl3, S491-499, 2011.04, Abstract
BACKGROUND: Identification of prognostic markers would be useful in the clinical management of patients with pancreatic ductal adenocarcinoma (PDAC). The clinical relevance of claudin-4 (CLDN4), recently identified as overexpressed in PDAC, is unknown.

METHODS: Using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), we analyzed CLDN4 mRNA expression in a panel of 9 pancreatic cancer cell lines and formalin-fixed paraffin-embedded (FFPE) tissues from 100 patients with PDAC. The CLDN4 expression levels were then correlated with clinicopathological variables and patient outcome. We also performed immunohistochemical analysis in 20 FFPE samples of PDAC to investigate the expression of CLDN4 protein.

RESULTS: Increased expression of CLDN4 was confirmed in all the pancreatic cancer cell lines tested compared with normal ductal epithelial cells and fibroblasts. We found that low expression of CLDN4 was significantly associated with shorter survival in patients with PDAC (hazard ratio; 1.362, 95% confidence interval; 1.011-1.873, P = 0.0419). Patients with high CLDN4 expression survived longer for a median of 63.0 months, compared with 14.7 months in patients with low CLDN4 expression (P = 0.0067). In immunohistochemical analysis, the level of CLDN4 mRNA expression was significantly correlated with the expression of CLDN4 protein (P = 0.0168).

CONCLUSION: Increased expression of CLDN4 mRNA predicts better prognosis in PDAC.

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67. Nakamura M, Tanaka Haruo, Nagayoshi Y, Nakashima H, Tsutsumi K, Otsuka T, Takahata S, Tanaka M, Okada H, Targeting the hedgehog signaling pathway with interacting peptides to Patched-1, J Gastroenterol. , 47, 4, 452-460, 2012.04, BACKGROUND:

The hedgehog (Hh) signaling pathway is aberrantly activated in many cancers. Overproduction of sonic hedgehog (Shh), a ligand in the Hh pathway, increases Hh signaling activity by inhibiting Patched-1 (Ptch1), a suppressive receptor in the Hh pathway. The purpose of this study was to establish a novel strategy for treating pancreatic cancer and other Hh-dependent cancers through control of the tumor-suppressive function of Ptch1.

METHODS:

We synthesized seven interacting peptides to the amino-acid sequence of the Ptch1 docking site for Shh. Human pancreatic cancer cell lines (AsPC-1, SUIT2) were cultured in the presence or absence of the peptides. Cell proliferation was assessed by cell counting and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The activity of the Hh pathway was estimated by real-time polymerase chain reaction of the target gene product Gli1. To confirm their anti-tumor activity in vivo, the effect of the peptides in a mouse model of pancreatic cancer was determined. Finally, the Hh signaling activity of the xenograft was examined.

RESULTS:

Three of the interacting peptides to Ptch1 suppressed the proliferation of the two pancreatic cancer cell lines and decreased the expression of Gli1, both in vitro and in vivo.

CONCLUSIONS:

This study suggests that interacting peptides to Ptch1 may be a new tool for controlling the Hh-dependent growth of pancreatic cancer.
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68. Ohuchida K, Mizumoto K, Lin C, Yamaguchi H, Ohtsuka T, Sato N, Toma H, Nakamura M, Nagai E, Hashizume M, Tanaka M, MicroRNA-10a is overexpressed in human pancreatic cancer and involved in its invasiveness partially via suppression of the HOXA1 gene, Ann Surg Oncol., 19, 7, 2394-2402, 2012.04, Abstract
BACKGROUND:
There is increasing evidence that microRNAs are differentially expressed in many types of cancers. Despite progress in analyses of microRNAs in several types of cancers, the functional contributions of microRNAs to pancreatic cancer remain unclear.
METHODS:
In the present study, the expression levels of specific microRNAs identified by microarray analyses were examined in a panel of 15 pancreatic cancer cell lines. We then investigated the functional roles of these microRNAs in the proliferation and invasion of pancreatic cancer cells.
RESULTS:
Based on the microarray data, we found frequent and marked overexpression of miR-10a, miR-92, and miR-17-5p in pancreatic cancer cell lines. Microdissection analyses revealed that miR-10a was overexpressed in pancreatic cancer cells isolated from a subset of primary tumors (12 of 20, 60%) compared with precursor lesions and normal ducts (P<.01). In vitro experiments revealed that miR-10a inhibitors decreased the invasiveness of pancreatic cancer cells (P<.01), but had no effect on their proliferation. Inhibition of HOXA1, a target of miR-10a, promoted the invasiveness of pancreatic cancer cells (P<.01).
CONCLUSIONS:
The present data suggest that miR-10a is overexpressed in a subset of pancreatic cancers and is involved in the invasive potential of pancreatic cancer cells partially via suppression of HOXA1..
69. Tanabe R, Otsuka T, Miyatake E, Kawamoto M, Nakamura M, Takahata S, Tanaka M, Manometric Evidence of Earlier Recovery of Fasting Gastric Motility after Antecolic Duodenojejunostomy than after Retrocolic Duodenojejunostomy following PPPD., Hepatogastroenterology, 59, 118, 1981-1985, 2012.04, Backgrounds/Aims: Gastric stasis is a unique complication of pylorus-preserving pancreatoduodenectomy (PPPD). Although some studies reported less prevalence of gastric stasis after antecolic duodenojejunostomy, there have been no reports on detailed comparison of gastric motility after antecolic vs. retrocolic duodenojejunostomy after PPPD. Methodology: Thirty-six patients underwent PPPD with the modified Child reconstruction. Retrocolic duodenojejunostomy was utilized in initial 13 patients (retrocolic group). For comparison, antecolic duodenojejunostomy was employed in subsequent 23 patients (antecolic group). A manometric tube assembly was inserted into the gastric antrum and jejunum during PPPD. Gastrointestinal motility was recorded for 3 hours a day, starting on 6 to 14 days after surgery and repeated at a weekly interval until the first appearance of phase 3 gastric motility. Various clinical parameters were also assessed. Results: Recovery of gastric phase 3 was identified in 19 of 36 patients. Recovery of phase 3 was faster in antecolic group than in retrocolic group (p<0.01). The amount of the gastric juice output during 14 postoperative days was larger in retrocolic group than in antecolic group (p<0.01). Resumption of water intake and food intake was earlier and the length of intravenous hyperalimentation and hospital stay was shorter in antecolic group than in retrocolic group (p<0.05). Conclusions: Antecolic duodenojejunostomy contributes to early recovery of gastric phase 3 motility in patients after PPPD, leading to prevention of early gastric stasis..
70. Mori Y, Otsuka T, Kono H, Ideno N, Aso T, Nagayoshi Y, Takahata S, Nakamura M, Ishigami K, Aishima S, Oda Y, Tanaka M, Management strategy for multifocal branch duct intraductal papillary mucinous neoplasms of the pancreas., Pancreas, 41, 7, 1008-1012, 2012.04, OBJECTIVES:

Branch duct intraductal papillary mucinous neoplasms of the pancreas (BD-IPMNs) often are composed of multifocal lesions. We aimed to clarify the clinicopathologic features of multifocal BD-IPMNs.

METHODS:

Medical records of 211 patients with BD-IPMNs (169 solitary and 42 multifocal) were retrospectively analyzed. We compared the pathological grade of resected IPMNs and the resulting clinical course between solitary and multifocal BD-IPMNs.

RESULTS:

Sixty-nine patients (54 with solitary and 15 with multifocal BD-IPMNs) underwent pancreatectomy, and of these patients, 62 exhibited at least 1 malignant predictor. There was no significant difference in the prevalence of malignancy in the resected BD-IPMNs between the 2 groups. In the remaining 142 patients who exhibited no malignant predictors, both groups demonstrated no differences in morphologic changes of BD-IPMNs. Seventeen distinct ductal carcinomas were identified in both groups, and there was no difference in the prevalence of ductal carcinoma between the 2 groups. Moreover, there was no significant difference in the disease-specific survival rate between the 2 groups.

CONCLUSIONS:

In patients with multifocal BD-IPMNs, resection is only warranted for lesions that exhibit malignancy predictors; moreover, closer attention to the potential presence or development of distinct ductal carcinoma in patients with multifocal and solitary BD-IPMNs is warranted.
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71. Yasui T, Takahata S, Kono H, Nagayoshi Y, Mori Y, Tsutsumi K, Sadakari Y, Otsuka T, Nakamura M, Tanaka M, Is cholecystectomy necessary after endoscopic treatment of bile duct stones in patients older than 80 years of age?
, J Gastroenterol. , 47, 1, 65-70, 2012.04, Abstract
BACKGROUND AND AIMS: Although patients with cholecystocholedocholithiasis are generally referred to cholecystectomy after endoscopic sphincterotomy (ES) and common bile duct clearance, we often have a conflict whether cholecystectomy is necessary in very elderly patients with comorbid diseases. The aim of this study is to assess whether cholecystectomy in very elderly patients is justified after ES.

PATIENTS AND METHODS: Patients with cholecystocholedocholithiasis who underwent ES and stone extraction and were followed-up for more than 10 years were retrospectively reviewed. We divided these patients into two groups: the elderly group (equal to or more than 80 years old) and young group (less than 80 years old) and compared late biliary complications and mortality.

RESULTS: The 10-year cumulative incidence of overall biliary complications was significantly lower in cholecystectomized patients than in patients with gallbladder in situ in the young group (7.5 vs. 21.7%, p = 0.0037), but not different in the elderly group (8.3 vs. 7.4%, p = 0.92). When each complication was evaluated separately, the rate of recurrent common bile duct stones (CBDS) was not different, but that of acute cholecystitis was significantly lower in the elderly group than in the young group (4.1 vs. 22.6%, p = 0.011).

CONCLUSIONS: In very elderly patients the incidence of acute cholecystitis is low even when the gallbladder is preserved after endoscopic treatment of CBDS, with a similar risk of CBDS recurrence. Thus, it may not be necessary to recommend cholecystectomy after ES for CBDS in very elderly patients.

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72. Otsuka T, Kono H, Tanabe R, Nagayoshi Y, Mori Y, Sadakari Y, Takahata S, Oda Y, Aishima S, Igarashi H, Ito T, Ishigami K, Nakamura M, MizumotoK, Tanaka M, Follow-up study after resection of intraductal papillary mucinous neoplasm of the pancreas; special references to the multifocal lesions and development of ductal carcinoma in the remnant pancreas.
, Am J Surg, 204, 1, 44-48, 2012.04, Abstract
BACKGROUND: Frequency and characteristics of metachronous occurrence of multifocal intraductal papillary mucinous neoplasms (IPMNs) or distinct pancreatic ductal adenocarcinomas (PDACs) in the remnant pancreas during follow-up evaluation after pancreatectomy for IPMNs have not been well known. The aim of this study was to investigate the outcomes after resection of IPMNs, especially focusing on the metachronous occurrence of multifocal IPMNs and distinct PDACs.

METHODS: Medical records of 172 patients who underwent resection of IPMNs were reviewed retrospectively, and the data regarding the occurrence of metachronous IPMNs or PDACs in the remnant pancreas during a mean postoperative follow-up period of 64 months were collected.

RESULTS: The incidence including synchronous and metachronous multifocal occurrence of IPMNs was 20% (34 of 172), and that of distinct PDACs was 9.9% (17 of 172). Ten metachronous IPMNs developed in the remnant pancreas after a mean time of 23 postoperative months (range, 12-84 mo), and 2 with main duct IPMNs (both were carcinoma in situ) required remnant pancreatectomy. Six distinct PDACs developed in the remnant pancreas after a mean time of 84 postoperative months (range, 12-150 mo). Four of them were found to have a tumor with a size of less than 2 cm, whereas the remaining 2 PDACs were found to be unresectable more than 10 years after resection of IPMNs.

CONCLUSIONS: Intense long-term follow-up evaluation is necessary for the early detection of metachronous occurrence of distinct PDACs as well as malignant IPMNs after resection of IPMNs.

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73. Higashida M, Matsumoto H, Kubota H, Murakami H, Kawabe Y, Nakashima H, Oka Y, Okumura H, Nakamura M, Hirai T, Evaluation of 5-FU plasma concentration by 13C breath test in patients treated with oral 5-FU analogs, Anticancer Res, 32, 12, 5407-5414, 2012.04.
74. Mori Y, Otsuka T, Tsutsumi K, Yasui T, Ueda J, Takahata S, Nakamura M, Tanaka M, Different incretin responses after pancreatoduodenectomy and distal pancreatectomy, Pancreas, 41, 3, 455-460, 2012.04,


OBJECTIVES:

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are known as incretins to stimulate insulin secretion. The aims of this study were to investigate the postoperative β-cell function and hormonal responses of GLP-1 and GIP after pancreatoduodenectomy (PD) and distal pancreatectomy (DP).

METHODS:

Oral glucose tolerance tests were performed in 34 patients (20 PD and 14 DP) before and 1 month after operation. The changes in the serum glucose and insulin concentrations, homeostasis model assessment of insulin resistance, and pancreatic β-cell function (BCF) were analyzed. GLP-1 and GIP were also measured.

RESULTS:

There was no patient with postoperative deterioration of glucose tolerance after PD, whereas impairment of glucose metabolism was observed after DP. Homeostasis model assessment of insulin resistance decreased after PD, whereas those after DP showed no change. The postoperative BCF were lower than preoperative values in both groups. GLP-1 increased after DP but not after PD, whereas GIP decreased after PD but not after DP.

CONCLUSIONS:

The changes in glucose metabolism and incretin responses were different between PD and DP. The increased level of GLP-1 after DP might reflect the relatively insufficient BCF; and thus, perioperative administration of GLP-1 might improve the diabetic condition after DP.
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75. Tsutsumi K, Otsuka T, Mori Y, Fujino M, Yasui T, Aishima S, Takahata S, Nakamura M, Ito T, Tanaka M, Analysis of lymph node metastasis in pancreatic neuroendocrine tumors (PNETs) based on the tumor size and hormonal production, J Gastroenterol, 47, 6, 678-685, 2012.04, BACKGROUND:Because of the rarity and variety of pancreatic neuroendocrine tumors (PNETs), there have been few reports regarding the indication for lymph node dissection in patients with these tumors. This study aimed to evaluate the risk of lymph node metastasis of PNETs based on the tumor size and hormonal production.METHODS:Data for a total of 66 patients who had PNETs resected at our department between 1987 and 2010 were retrospectively studied. The clinicopathological features, including the disease-specific survival rate, were assessed based on the status of lymph node metastasis at the time of initial surgical resection. Then the cut-off point of tumor size to predict lymph node metastasis was estimated.RESULTS:There were 12 patients (18%) with lymph node metastasis. The frequency of lymph node metastasis tended to be higher in gastrinomas than that in other tumors (43 vs. 15%; P = 0.08). The size of PNETs with lymph node metastasis was signific
antly larger than that of the PNETs without metastasis (P = 0.04). The postoperative survival rate in the PNET patients with lymph node metastasis was significantly lower than that in the patients without metastasis (P < 0.0001). Only 2 (8%) of 26 PNETs with a tumor size of <15 mm had lymph node metastasis, and both of these were gastrinomas. On the other hand, 10 (25%) of the remaining 40 PNETs with a tumor size of ≥15 mm had lymph node metastasis. Notably, there were no PNETs with lymph node metastasis in 22 non-gastrinomas with a tumor size of <15 mm.CONCLUSIONS:Non-gastrinomas with a tumor size of ≥15 mm and all gastrinomas would be an indication for pancreatectomy with lymph node dissection..
76. Otsuka T, Kono H, Nagayoshi Y, Mori Y, Tsutsumi K, Sadakari Y, Takahata S, Morimatsu K, Aishima S, Igarashi H, Ito T, Ishigami K, Nakamura M, Mizumoto K, Tanaka M, An increase in the number of predictive factors augments the likelihood of malignancy in branch duct intraductal papillary mucinous neoplasm of the pancreas.
, Surgery. , 151, 1, 76-83, 2012.04, Abstract
BACKGROUND: International consensus guidelines for the management of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas provide several factors that can be used to predict which IPMNs will become malignant.The sensitivity of each factor's predictive accuracy, however, is relatively low, making it difficult to determine the appropriate treatment in individual cases. The aim of this study was to investigate whether increasing the number of predictive factors might augment the sensitivity of the established guidelines to detect malignant IPMNs.

METHODS: The medical records of 138 patients with IPMNs resected at our institution were reviewed. Possible malignant predictors were analyzed by univariate and multivariate analysis, and the effects of the number of factors and the predictive score of the pathologic results were examined. The cutoff points for the number of predictors to discriminate between malignant and nonmalignant IPMNs were established by constructing receiver operating characteristic curves.

RESULTS: A predictive analysis could not be carried out for the main duct IPMNs because of the high prevalence of malignancy and the small number of significant predictors associated with them. For malignant branch duct IPMNs, however, we identified 4 predictive factors that helped determine the correct diagnosis as follows: (1) the presence of a cyst ≥30 mm in diameter; (2) the presence of mural nodules; (3) a history of acute pancreatitis; and (4) atypical results of pancreatic juice cytology. An increase in the number of these factors significantly affected the sensitivity to predict malignancy. The area under the curve for the number of predictors for malignant branch duct IPMNs was 0.856, and the sensitivity and specificity were 96% and 71%, respectively, when the cutoff point was set at 2. The predictive scoring system also showed the same values of sensitivity and specificity for the number of factors.

CONCLUSION: Patients with branch duct IPMNs who have 2 or more of the 4 predictive factors described above should undergo standard pancreatectomy with lymph node dissection, whereas patients who present with 0 or 1 predictive factor can be treated by minimal pancreatectomy without nodal dissection or by careful observation without resection. All patients with main duct IPMNs, therefore, should be treated with resection as suspected malignancies.

Copyright © 2011 Mosby, Inc. All rights reserved.

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77. Matsumoto H, Higashida M, Kubota H, Murakami H, Tsutsumi K, Nakashima H, Oka Y, Okumura H, Nakamura M, Hirai T, An immunoassay method for the pharmacokinetics of 5-fluorouracil in patients with gastric cancer administered adjuvant chemotherapy, Anticancer Res, 32, 11, 5111-5114, 2012.04.
78. Matsumoto H, Hirabayashi Y, Kubota H, Murakami H, Higashida M, Haruma K, Hiratsuka J, Nakamura M, Hirai T, A combined therapy with docetaxel and nedaplatin for relapsed and metastatic esophageal carcinoma, Anticancer Res, 32, 5, 1827-1831, 2012.04.
79. Otsuka T, Ideno N, Aso T, Nagayoshi Y, Kono H, Mori Y, Takahata S, Oda Y, Aishima S, Igarashi H, Ito T, Ishigami K, Nakamura M, Mizumoto K, Tanaka M, Role of endoscopic retrograde pancreatography for early detection of pancreatic ductal adenocarcinoma concomitant with intraductal papillary mucinous neoplasm of the pancreas., J Hepatobiliary Pancreat Sci. , 20, 3, 356-361, 2013.04, BACKGROUND:

Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is often found with distinct pancreatic ductal adenocarcinoma (PDAC) in the same pancreas. The aim of this study was to clarify whether endoscopic retrograde pancreatography (ERP) would be useful for the early detection of concomitant PDACs in patients with IPMNs.

METHODS:

Medical records of 179 patients who were histologically confirmed to have IPMNs after resection between 1987 and 2011 were reviewed. The patients having concomitant PDACs were selected, and the diagnostic abilities to detect concomitant PDACs of computed tomography (CT), magnetic resonance imaging (MRI), endoscopic ultrasonography (EUS), and ERP were compared between early (stages 0-I according to Japanese General Rules for Pancreatic Cancer) and advanced (stages II-IV) PDACs.

RESULTS:

A total of 23 PDACs developed synchronously or metachronously in 20 patients, and the prevalence of PDACs concomitant with IPMNs was 11.2 % (20/179). Sensitivities of CT (16 vs. 87 %), MRI (29 vs. 93 %), and EUS (29 vs. 92 %) in the early group were significantly lower than those in the advanced group (p < 0.01). On the other hand, the sensitivity of ERP in the early group was as high as that in the advanced group (86 vs. 82 %, respectively, p > 0.99). Among 7 early PDACs, 3 were diagnosed only by ERP.

CONCLUSIONS:

ERP has an important role in the early diagnosis of distinct PDACs in patients with IPMNs. Further investigation is necessary to clarify the indication and the timing of ERP during management of IPMNs in term of early detection of concomitant PDACs.
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80. Murakami H, Matsumoto H, Nakamura M, Hirai T, Yamaguchi Y, Octreotide acetate-steroid combination therapy for malignant gastrointestinal obstruction, Anticancer Res, 33, 12, 5557-5560, 2013.04.
81. Nakamura M, Nakashima H, Laparoscopic distal pancreatectomy and pancreatoduodenectomy is it worthwhile? A meta-analysis of laparoscopic pancreatectomy, J Hepatobiliary Pancreat Sci, 20, 4, 421-428, 2013.04.
82. Ideno N, Ohtsuka T, Kono H, Fujiwara K, Oda Y, Aishima S, Ito T, Tokunaga S, Ohuchida K, Takahata S, Nakamura M, Mizumoto K, Tanaka M, Intraductal Papillary Mucinous Neoplasms of the Pancreas with Distinct Pancreatic Ductal Adenocarcinoma Are Frequently of Gastric Subtype, Ann Surg., 258, 1, 141-151, 2013.04, OBJECTIVE:: To identify a high-risk group of patients with pancreatic ductal adenocarcinoma (PDAC), independently arising in the pancreas with intraductal papillary mucinous neoplasm (IPMN), using histopathologic subtypes. BACKGROUND:: Pathologic features of IPMN with distinct PDAC, including histopathologic subtypes of IPMN and PDAC phenotypes, have not been well characterized. Mucin expression patterns and the mutational status of GNAS and KRAS are useful to explore the relationship between these 2 lesion types. METHODS:: Clinicopathologic data of 179 resected IPMNs and 180 resected PDACs without IPMNs as a control group were reviewed. IPMNs were classified into 4 grades (low-grade, intermediate-grade, high-grade dysplasia, and an associated invasive carcinoma) and 4 subtypes (gastric, intestinal, pancreatobiliary, and oncocytic). The expression of MUC1, MUC2, MUC5AC, MUC6, and CDX2 was investigated by immunohistochemistry in IPMNs and PDACs with an
d without IPMNs. The mutational status of GNAS and KRAS was evaluated by cycle sequencing in PDACs and pre-/coexisting IPMNs. RESULTS:: Twenty-six synchronous or metachronous PDACs were identified in 20 patients (11.2%) with IPMNs. Occurrence of concomitant PDACs was more frequently observed in gastric-type IPMNs (18/110, 16.4%) compared with intestinal (1/49, 2.0%), pancreatobiliary (1/17, 5.9%), or oncocytic-type (0/3, 0%) (P = 0.047). Both PDACs with and without IPMNs were frequently positive for MUC1, MUC5AC, and MUC6 expression, as assessed by immunohistochemistry, but were negative for MUC2 and CDX2. The mucin-staining patterns were similar to those of invasive tubular adenocarcinoma arising from gastric-type IPMNs. Mutation of GNAS within codon 201 was not detected in PDACs and gastric-type IPMNs, whereas most of these exhibited KRAS mutations. However, the R201H GNAS mutation was detected in 1 intestinal-type IPMN with distinct PDAC. CONCLUSIONS:: Mucin expression pa
tterns demonstrate that PDAC without GNAS mutations of an aggressive phenotype frequently arise in the pancreas with benign gastric-type IPMN in the absence of GNAS mutations..
83. Kono H, Nakamura M, Ohtsuka T, Nagayoshi Y, Mori Y, Takahata S, Aishima S, Tanaka M, High expression of microRNA-155 is associated with the aggressive malignant behavior of gallbladder carcinoma., Oncol Rep., 30, 1, 17-24, 2013.04, The prognosis of gallbladder cancer (GBC) remains poor despite recent advances in diagnostics and therapeutic strategies. Although the role of microRNAs (miRs) in GBC have not been well documented, miR-155 is known to be associated with inflammation-associated carcinogenesis in various types of cancers. The aim of this study was to investigate the clinical significance of miR-155 expression and the biological functions of miR-155 in GBC. The expression levels of miR-155 in surgically resected GBCs and gallbladders with pancreaticobiliary maljunction (PBM) were assessed by quantitative reverse transcription-polymerase chain reaction. The relationship between the expression levels of miR-155 and clinicopathological features of GBCs was analyzed. Human GBC cell lines were transfected with miR-155 inhibitors or mimics, and the effects on proliferation and invasion were assessed. miR-155 was significantly overexpressed in GBCs when compared with that in gallbladders with PBM (p=0.007) and normal gallbladders (p=0.04). The high expression level of miR-155 in GBCs was significantly associated with the presence of lymph node metastasis (p=0.01) and a poor prognosis (p=0.02). In vitro assays showed that aberrant expression of miR-155 significantly enhanced GBC cell proliferation and invasion. In conclusion, high miR-155 expression correlates with the aggressive behavior of GBCs, and miR-155 may become a prognostic marker and therapeutic target for GBC..
84. Nakamura M, Nakashima H, Tsutsumi K, Matsumoto H, Muta Y, Ueno D, Yoshida K, Hino K, Urakami A, Tanaka M, First jejunal vein oriented mesenteric excision for pancreatoduodenectomy, J Gastroenterol., 48, 8, 989-985, 2013.04, BACKGROUND:

Dissection of the pancreatic head from the superior mesenteric vein (SMV) and artery (SMA) are major points of bleeding in pancreaticoduodenectomy (PD) because of congestion of the pancreatic head. The "SMA-first" approach, which involves ligating the artery from the SMA first, can be used to solve this problem. However, the SMA-first approach has problematic anatomical issues. We applied a new surgical approach, first jejunal vein oriented mesenteric excision (FME), for PD. This study aimed to clarify the effect of FME on reduction of bleeding during PD.

METHODS:

The jejunal vein, the most frequent source of bleeding during dissection of the mesoduodenum, was identified at the beginning of dissection of the pancreatic head from SMV and SMA. The mesoduodenum, including plural IPDAs, was completely divided before dissection of the pancreatic head from the SMV. The perioperative outcomes of two groups, patients who underwent FME-based PD and patients who underwent standard PD, were compared. Additionally, the spatial characteristics of the first jejunal vein (FJV) were analyzed using computed tomography.

RESULTS:

FME-based PD significantly reduced intraoperative blood loss compared with conventional PD (569 vs. 1094 ml, P = 0.0315). The median distance of the FJV was 0 mm from the middle colic artery and 0 mm from the third portion of the duodenum. The FJV was posterior to the SMA in the majority of the patients but was anterior to the SMA in 16.7 % of patients.

CONCLUSIONS:

FME is useful for reducing intraoperative bleeding.
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85. Nakamura M, Ohtsuka T, Nakashima H, Nagayoshi Y, Kono H, Tsutsumi K, Takahata S, Tanaka M, Extensive distal pancreatectomy for pancreatic tumor., Anticancer Res, 33, 1, 267-270, 2013.04, Aim: The purpose of this study was to evaluate the feasibility and advantages of extensive distal pancreatectomy (ExDP).

PATIENTS AND METHODS:

We retrospectively analyzed our experience in 24 patients, who underwent ExDP or total pancreatectomy (TP) for the treatment of pancreatic cancer (22 patients) or benign tumor (two patients).

RESULTS:

ExDP was associated with less blood loss (p=0.0189), shorter operative times (p=0.024), lower rates of worsening of diabetes mellitus (p<0.0001), and shorter hospital stays (p=0.0009) than TP. ExDP also had a lower complication rate than TP (1/11 cases versus 4/13 cases), but this was not a significant difference. There was no difference in the curative resection rate for pancreatic cancer between the two procedures (p=0.685).

CONCLUSION:

ExDP is a feasible and function-preserving operation for the treatment of pancreatic tumors in the body of the pancreas near the portal vein.
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86. Murakami H, Matsumoto H, Kubota H, Higashida M, Nakamura M, Hirai T, Evaluation of electrical activity after vagus nerve-preserving distal gastrectomy using multichannel electrogastrography, J Smooth Muscle Res, 49, 1-14, 2013.04.
87. Kubota H, Matsumoto H, Higashida M, Murakami H, Nakashima H, Oka Y, Okumura H, Yamamura M, Nakamura M, Hirai T, Eicosapentaenoic acid modifies cytokine activity and inhibits cell proliferation in an oesophageal cancer cell line, Anticancer Res, 33, 10, 4319-4324, 2013.04.
88. Nakamura M, Kayashima T, Fujiwara K, Nagayoshi Y, Kono H, Ohtsuka T, Takahata S, Mizumoto K, Tanaka M, Combination therapy of portal vein resection and adjuvant gemcitabine improved prognosis of advanced pancreatic cancer, Hepato-Gastroenterology, 60, 122, 354-357, 2013.04, Abstract


Background/Aims: Although adjuvant chemotherapy (AC) using gemcitabine improves the prognosis of patients with resectable pancreatic cancer, the effect of gemcitabine AC on the prognosis of patients with borderline resectable pancreatic cancer is not clear. Methodology: We analyzed the prognosis of patients with pancreatic cancer who underwent curative pancreatoduodenectomy or total pancreatectomy in combination with portal/superior mesenteric vein resection (PVR) [PVR (+) group] or without PVR [PVR(-) group]. Results: MST of the PVR (+) group was significantly shorter than that of the PVR(-) group (p=0.017). In contrast, when we focused on the patients with gemcitabine AC, there was no significant difference in MST between the PVR (+) and the PVR (-) groups (p=0.247). Furthermore, we compared MST of two subgroups in the PVR (+) group depending on gemcitabine AC status. In the PVR (+) group, MST of the patients with gemcitabine AC was significantly longer than that without gemcitabine AC (p=0.003). This was also true for the patients with pancreatic cancer which had histologically proven invasion to portal/superior mesenteric vein (PV/SMV) (p=0.001). Conclusions: The prognosis of patients with pancreatic cancer invading PV/SMV can be improved by combination therapy with PVR and gemcitabine adjuvant chemotherapy.
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89. Matsumoto H, Higashida M, Kubota H, Murakami H, Shiotani A, Haruma K, Nakamura M, Hirai T, Clinical Benefit of Non-Curative Resection for Stage IV Gastric Cancer. , Abdominal Oncology, 1, 1, 1-5, 2013.04.
90. Mori Y, Otsuka T, Kono H, Nagayoshi Y, Ideno N, Aso T, Kozono S, Ohuchida K, Takahata S, Nakamura M, Mizumoto K, Tanaka M, A minimally invasive and simple screening test for detection of pancreatic ductal adenocarcinoma using biomarkers in duodenal juice., Pancreas., 42, 2, 187-192, 2013.04, OBJECTIVES:

The aim of this study was to establish a minimally invasive and simple screening test for detection of pancreatic ductal adenocarcinoma (PDAC) using duodenal juice (DJ).

METHODS:

Duodenal juice was collected prospectively before endoscopic retrograde cholangiopancreatography in 46 patients. A protease inhibitor was not added to the samples collected during the initial 2.5 minutes but was added in the latter 2.5 minutes. Thereafter, secretin was administered intravenously, and DJ was subsequently collected for additional 10 minutes. The sensitivities of carcinoembryonic antigen (CEA), S100 calcium-binding protein P (S100P), and interleukin 8 in DJ and pancreatic juice were assessed.

RESULTS:

There were 30 patients with PDAC and 16 with benign lesions. It was possible to collect an adequate amount of DJ without secretin administration. In the PDAC group, CEA concentrations in DJ were significantly higher than those in the benign group, even without the use of a protease inhibitor. S100P levels in DJ in the PDAC group were significantly higher than those in the benign group in the presence of the protease inhibitor.

CONCLUSIONS:

Duodenal juice collection during routine upper endoscopy and assessments of CEA and S100P in DJ might become a useful screening test for detection of PDAC.
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91. Tsuruta A, Hirai T, Nakamura M, Retrospective comparison of open versus laparoscopic ventral and incisional hernia repair, Asian J Endosc Surg,, 7, 3, 246-250, 2014.04, Abstract
INTRODUCTION:
We performed a retrospective study to determine the mid-term recurrence and complication rates of patients following laparoscopic ventral and incisional hernia repair (LVHR) with DualMesh, an expanded polytetrafluoroethylene (ePTFE) mesh. Additionally, a study of the mesh contraction rate was performed postoperatively.
METHODS:
We compared open mesh repair of ventral and incisional hernias (OR) and LVHR. We also analyzed the shrinkage rate of ePTFE mesh. We included 45 patients (21 OR, 24 LVHR) who underwent mesh repair for primary ventral and incisional hernias between January 2008 and December 2012. Patients' characteristics did not significantly differ between the two groups.
RESULTS:
Mean operating time was 152.7 min for the OR group and 143.1 min for the LVHR group (P = 0.25). Mean postoperative hospital stay was 13.4 days for the OR group and 6.8 days for the LVHR groups (P = 0.01). The postoperative complication rate was 28.6% for the OR group and 12.5% for the LVHR group (P = 0.03). Among OR patients, causes of morbidity were variable: two recurrent cases, one surgical-site infection, one re-recurrence, one case of enteritis, and one case of heart failure. Among the LVHR patients, there was one surgical-site infection and two cases of seroma. No patients in the LVHR group experienced recurrence, while 14.3% of OR patients had a recurrence. In the LVHR group, the mean ePTFE mesh contraction rate was 10.6%.
CONCLUSION:
LVHR has advantages compared with OR, and the post-insertion contraction rate of ePTFE mesh was 10.6%.
© 2014 Japan Society for Endoscopic Surgery, Asia Endosurgery Task Force and Wiley Publishing Asia Pty Ltd.
KEYWORDS:
DualMesh; ePTFE; laparoscopic ventral and incisional hernia repair (LVHR).
92. Nagayoshi Y, Nakamura M, Matsuoka K, Ohtsuka T, Mori Y, Kono H, Aso T, Takahata S, Ryo A, Takeda H, Ito T, Oda Y, Endo Y, Sawasaki T, Tanaka M, Profiling of autoantibodies in sera of pancreatic cancer patients, Ann Surg Oncol, 10.1245/s10434-014-3574-0, 21, Suppl3, S459-S465, 2014.04, BACKGROUND:

Although autoantibodies to cancer antigens are candidates for biomarkers, no comprehensive studies to detect cancer-specific antibodies have been performed. This study identified autoantibodies in the sera of pancreatic cancer (PC) patients using proteomics based on a wheat germ cell-free protein production system.

METHODS:

We constructed a biotinylated protein library of 2,183 genes. Interactions between biotinylated proteins and serum antibodies were detected by AlphaScreen® assay. Relative luminescence signals of each protein in 37 PC patients and 20 healthy controls were measured, and their sensitivity and specificity for PC were calculated.

RESULTS:

Luminescence signals of nine proteins were significantly higher than those of healthy controls, with calcium and integrin binding 1 (CIB1) protein showing the greatest significance (p = 0.002). Sensitivity, specificity, positive predictive value and negative predictive value of CIB1 autoantibody alone for PC were 76, 70, 82, and 61 %, respectively, and 97, 35, 74, and 88 %, respectively, when the four most significant proteins were combined. Presence of these autoantibodies did not vary significantly with other clinicopathological characteristics.

CONCLUSION:

Several autoantibodies, including CIB1, are potential biomarkers for PC.
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93. Nakamura M, Shindo K, Ideno N, Ueda J, Takahata S, Nakashima H, Ohtsuka T, Shimizu S, Oda Y, Tanaka M, Prediction of Pancreatic Fistula by Preoperatively Assessable Factors; Retrospective Review of Unified Operations by Single Surgeon, Hepato-Gastroenterology, 2014, 61, 834-837, 2014.04, Abstract
BACKGROUND/AIMS: This retrospective study was conducted to find preoperatively assessable risk factors for postoperative pancreatic fistula (POPF) in patients undergoing laparoscopic distal pancreatectomy (LDP) using a slow compression method with a stapler, which we call pen-firing compression (PFC).
METHODOLOGY: Fifty-two patients underwent LDP, of whom 42 underwent PFC for pancreatic division using a stapler. The relationship between preoperatively assessable factors and the incidence of clinical POPF was statistically analyzed.
RESULTS: Overall rate of POPF was 7.1% in 42 patients. Univariate analysis showed that greater BMI (p = 0.004) and thicker pancreatic stump (0.0022) were significant risk factors for POPF. BMI and stump thickness remained significant (P < 0.0001, P < 0.0001) by multivariate analysis. Cutoff points estimated by ROC curve were 27 kg/m2 for BMI and 27 mm for stump thickness.
CONCLUSIONS: High BMI value and thick pancreatic stump are significant risk factors for POPF after LDP. Alternative treatment of the pancreatic stump may prevent POPF in high-risk patients.
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94. Nakamura M, Ogo A, Yamura M, Yamaguchi Y, Nakashima H, Metformin suppresses sonic hedgehog expression in pancreatic cancer cells, Anticancer Res, 34, 4, 1765-1769, 2014.04, Abstract
BACKGROUND/AIM:
Metformin use has previously been associated with decreased cancer risk. The Hedgehog signaling pathway is a well-characterized early and late mediator of pancreatic cancer oncogenesis. The aim of the present study was to clarify the effect of metformin on factors involved in Hedgehog signaling.
MATERIALS AND METHODS:
BxPC3 human pancreatic cancer cells were treated with metformin, and Sonic hedgehog (Shh) mRNA and protein levels were examined by real time reverse transcription-polymerase chain reaction, immunohistochemistry and immunoblotting, respectively. The effect of metformin on Shh levels was also examined in three other cancer cell lines.
RESULTS:
Shh protein and mRNA expression was suppressed by metformin in BxPC3 cells. This phenomenon was further confirmed in three other cancer cell lines. Shh mRNA expression was inhibited by metformin in a concentration-dependent manner in two cancer cell lines.
CONCLUSION:
Metformin reduces the expression of Shh in several cancer cell lines including pancreatic cancer cell.
KEYWORDS:
Hedgehog signaling pathway; Pancreatic cancer; Sonic Hedgehog; metformin.
95. Ueda J, Kayashima T, Mori Y, Ohtsuka T, Takahata S, Nakamura M, Tanaka M, Hepaticocholecystojejunostomy as effective palliative biliary bypass for unresectable pancreatic cancer, Hepatogastroenterology, 61, 129, 197-202, 2014.04, Abstract
BACKGROUND/AIMS:
The majority of patients with pancreatic cancer present with far advanced disease and jaundice. With the advancement of endoscopic interventional techniques, the role of surgical bypass has declined. However, surgical bypass is still considered to be appropriate in patients who are able to tolerate surgery. We performed hepaticocholecystojejunostomy consecutively as a palliative surgical biliary bypass for the purpose of long-term palliation. The aim of this study was to analyze the results of our palliative surgical biliary bypass, hepaticocholecystojejunostomy.
METHODOLOGY:
Between January 2001 through December 2009, 69 patients received palliative surgical biliary bypass (bypass group) and 33 patients received endoscopic biliary stenting (stent group) for unresectable pancreatic cancers. Mortality, morbidity and survival between the two groups were compared.
RESULTS:
There was no in-hospital death in the bypass group, but 2 patients (6%) in the stent group died in the hospital (p = 0.04). The surgical morbidity rate was 15% in the bypass group, while 20 patients (61%) in the stent group developed complications, mainly due to stent blockage. There was no significant difference in overall survival between the two groups. Among patients who underwent systemic chemotherapy but did not present with jaundice at the time of diagnosis, those who underwent prophylactic surgical biliary bypass before chemotherapy showed better survival than those who underwent systemic chemotherapy preceding biliary bypass or biliary stenting after occurrence of jaundice (p = 0.01).
CONCLUSIONS:
Hepaticocholecystojejunostomy resulted in negligible mortality, low morbidity and effective long-term palliation. Prophylactic surgical biliary bypass with gastrointestinal bypass might be a good treatment option for non-jaundiced patients undergoing chemotherapy for unresectable pancreatic cancer..
96. Nakamura M, Nakashima H, Abe T, Ensako T, Yoshida K, Hino K, Gemcitabine-based adjuvant chemotherapy for patients with advanced gallbladder cancer, Anticancer Res., 34, 6, 3125-3129, 2014.04, AAbstract
AIM:
We investigated effects of gemcitabine-based adjuvant chemotherapy (GEM) on prognosis of patients with gallbladder cancer.
PATIENTS AND METHODS:
We retrospectively analyzed outcomes of 36 patients who underwent radical resection for gallbladder cancer from 2001 through to 2012, using χ(2) for prognostic factors and Kaplan-Meier estimator and log-rank tests for survival data.
RESULTS:
The GEM group had higher rates of lymph node positivity and distant metastasis, higher UICC stage and fewer R0 resections; their 5-year survival rate (60%) did not significantly differ from that of the controls (70.0%), nor was GEM a significant prognostic factor in univariate analysis. However, among patients who underwent R1 and R2 resections, GEM significantly improved prognosis in both univariate and multivariate analyses. Median survival of the R1/2 GEM group (66.4 months) was significantly better than that of controls (5.4 months) (p=0.002).
CONCLUSION:
GEM improved prognosis of patients with gallbladder cancer after R1/R2 resections.
Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
KEYWORDS:
Gallbladder cancer; adjuvant chemotherapy; curability; gemcitabine (GEM).
97. Matsumoto H, Kubota T, Higashida M, Yoden E, Hiratsuka J, Haruma K, Nakamura M, Hirai H, Docetaxel/ TS-1 with Radiation for Unresectable Squamous Cell Carcinoma of the Esophagus - A Phase II Trial, Anticancer Res, 34, 7, 3759-3763, 2014.04, Abstract
BACKGROUND:
We tried a new regimen of docetaxel / TS-1 (tegafur-gimestat-otastat potassium) combined with radiation for squamous cell carcinoma of the esophagus in a phase II trial.
PATIENTS AND METHODS:
The patients, whose tumor invaded other organs without other organ metastasis, were given TS-1 (60 mg/m2/day) from days 1 to 14, and docetaxel (20-30 mg/m2) on days 1 and 8. They received radiation in 2.0 Gy from days 1 to 21. Patients were given a seven-day rest after the first course, and then were treated with the same regimen from days 28 to 49.
RESULTS:
Seventeen cases were enrolled in the study. The response rate was 76.4% (13/17). The overall 5-year survival rate was 29.6% (5/17) and median survival time was 15.2 months. Adverse events more than grade 3 occurred in 10 cases.
CONCLUSION:
This combination therapy may be one of the most effective treatments because of its lower rate of non-hematological adverse events and higher response rate. Three cases also underwent salvage surgery when the tumor recurred, and in one case, chemoradiation to a metastatic nodule on the thoracic wall was added.
Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
KEYWORDS:
Docetaxel; TS-1; chemoradiation; unresectable squamous cell carcinoma of esophagus.
98. Tsutsumi K, Ohtsuka T, Fujino M, Nakashima H, Aishima S, Ueda J, Takahata S, Nakamura M, Oda Y, Tanaka M, Analysis of risk factors for recurrence after curative resection of well-differentiated pancreatic neuroendocrine tumors based on the new grading classification, J Hepatobiliary Pancreat Sci., 21, 6, 418-425, 2014.04,

BACKGROUND:

It is difficult to predict the malignant potential of pancreatic neuroendocrine tumors (PNETs) precisely. This study investigated the validity of a new grading system adopted by the World Health Organization 2010 classification to determine risk factors for recurrence of PNETs.

METHODS:

Data of 70 patients with PNETs who underwent curative resection were retrospectively examined by uni- and multivariate analyses. Histopathological findings were re-reviewed by experienced pathologists. NET G1 was defined as mitotic count <2 per 10 high power fields (HPF) and/or ≤2% Ki67 index, and NET G2 as 2-20 mitosis per 10 HPF and/or 3-20% Ki67 index.

RESULTS:

There were 58 patients with NET G1 and 12 with NET G2. Incidence of recurrence was 11.4%. Univariate analysis demonstrated significant risk factors for recurrence including NET G2 of histological grade (P = 0.0089), male gender (P = 0.0333), tumor size ≥ 20 mm (P = 0.0117), lymph node metastasis (P = 0.0004), liver metastasis (P < 0.0001), lymphatic invasion (P = 0.046), and neural invasion (P = 0.0002). By multivariate analysis, histological grade (hazard ratio; 59.76, P = 0.0022) and neural invasion (hazard ratio; 147.49, P = 0.0016) were significantly associated with recurrence of PNETs.

CONCLUSIONS:

This study confirmed the prognostic relevance of the new grading classification and that evaluation of perineural invasion and histological grade should be considered as prognostic predictors in well-differentiated PNETs (NET G1 and G2).

© 2013 Japanese Society of Hepato-Biliary-Pancreatic Surgery.


KEYWORDS:

Grading classification; Neural invasion; Pancreatic neuroendocrine tumor; Predictors of recurrence; WHO 2010 classification
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99. Cases AI, Ohtsuka T, Kimura H, Zheng B, Shindo K, Oda Y, Mizumoto K, Nakamura M, Tanaka M, Significance of expression of glucagon-like peptide 1 receptor in pancreatic cancer, Oncol Rep, 10.3892/or.2015.4138, 34, 4, 1717-1725, 2015.04, Abstract
Glucagon-like peptide 1 (GLP-1) induces insulin secretion and proliferation of pancreatic β-cells, and inhibits their apoptosis through the GLP-1 receptor (GLP-1R), thus providing a foundation for using GLP-1-based therapies for the treatment of type 2 diabetes. However, doubts have emerged regarding the drug safety of these therapies. We investigated the potential role of GLP-1R in pancreatic ductal adenocarcinoma (PDAC). GLP-1R expression was semi-quantitatively evaluated by immunohistochemistry in 48 PDAC samples, and its correlations with clinicopathological features were investigated. CFPAC-1 cells were used for GLP-1R knockdown to evaluate its effects on cell proliferation, migration and invasion. GLP-1R expression was positive in 23 tumors and negative in 25 tumors. No correlations were found between GLP-1R expression status and clinicopathological characteristics. Furthermore, GLP-1R expression status did not affect the patient prognosis (P=0.74). The majority of lymph node metastases (11 of 15 samples examined; 73%) were positive for GLP-1R expression. Immunoreactivity for GLP-1R was also noted in sites of perineural and lymphovascular invasion. GLP-1R knockdown significantly reduced the proliferation, migration and invasion of CFPAC-1 cells (P<0.05). In conclusion, although GLP-1R is not an independent prognostic factor in PDAC patients, it appears to have some implications for PDAC metastatic ability.
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100. Tsuruta A, Itoh T, Hirai T, Nakamura M, Multi-layered intra-abdominal adhesion prophylaxis following laparoscopic colorectal surgery, Surg Endosc. , 10.1007/s00464-014-3813-2 , 29, 6, 1400-1405, 2015.04, BACKGROUND:

Small bowel obstruction secondary to intra-abdominal adhesions is a frequent postoperative complication. Less invasive surgery carries a lower risk of postoperative adhesions, but adhesions may still occur after laparoscopic colorectal surgery. We present here some of our methods of adhesion prophylaxis for laparoscopic colorectal surgery.

METHODS:

The 167 patients who underwent laparoscopic colorectal surgery at our center from 2007 to 2012 were retrospectively reviewed. To prevent postoperative intra-abdominal adhesions, anti-adhesion barriers were placed using the half-overlap method. The rate of postoperative small bowel obstruction was compared among three groups: patients who received no adhesion prophylaxis (Group NP), patients who received single-layered adhesion prophylaxis adjacent to the incision (Group SP), and patients who received three layers of adhesion prophylaxis at different depths (Group MLP).

RESULTS:

The rate of postoperative ileus was significantly different among the three groups, at 9.7 % (6/62) in Group NP, 5.0 % (1/19) in Group SP, and 0 % (0/86) in Group MLP).

CONCLUSIONS:

This retrospective analysis found that placement of multi-layered anti-adhesion barriers using the half-overlap method provided the most effective prophylaxis. Prospective clinical trials are needed to further evaluate these methods of anti-adhesion prophylaxis.
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101. Matsumoto H, Murakami H, Kubota T, Higashida M, Nakamura M, Hirai H, Clinical Outcome of Lower Esophageal Sphincter- and Vagus Nerve-Preserving Partial Cardiectomy for Early Gastric Cancer of Subcardia, Gastric Cancer, 18, 3, 669-674, 2015.04, Abstract
BACKGROUND:
No definitive operative method has been established for the treatment of early subcardial gastric cancer. Our newly developed technique involves local resection of the subcardia while preserving the lower esophageal sphincter and vagus nerve. A new fornix is constructed to accept the transposed esophagus.
METHODS:
Thirty patients underwent this procedure between July 2003 and December 2010. Continuous gastric pH monitoring was performed immediately after surgery, and esophageal manometry was undertaken 1 month later. Serum total protein, albumin, total cholesterol, cholinesterase, and body mass index (BMI) were recorded every 3 months. Pre- and postoperative oral intake were compared, reflux symptoms were recorded, and reflux esophagitis was assessed by endoscopy after 1 year.
RESULTS:
Twenty-five patients (86 %) reported no symptoms of reflux, and 27 (92.8 %) patients could eat 70 % or more of what they had eaten before surgery. Lower esophageal pressures were found to be >10 mmHg in 66.7 % of patients, and the fraction of time that pH <4 was <5 % of the 24-h monitoring period in 70 %. Serum parameters and BMI were unchanged.
CONCLUSIONS:
This surgical technique is a useful means of preserving postoperative quality of life after local gastrectomy by preventing reflux and maintaining nutritional status..
102. Horioka K, Ohuchida K, Sada M, Zheng B, Moriyama T, Fujita H, Manabe T, Ohtsuka T, Shimamoto M, Miyazaki T, Mizumoto K, Oda Y, Nakamura M, Suppression of CD51 in pancreatic stellate cells inhibits tumor growth by reducing stroma and altering tumor-stromal interaction in pancreatic cancer, Int J Oncol, 48, 4, 1499-1508, 2016.04, Pancreatic stellate cells (PSCs) enhance the malignant behavior of pancreatic cancer by interacting with cancer cells and producing extracellular matrix (ECM). To date, several stroma-targeted therapies for pancreatic cancer have been attempted, but these therapies are still not in practical use. Integrins expressed in stromal cells are involved in fibrosis of several organs, as well as promoting tumor malignancy. We investigated whether CD51, also known as integrin αV, expressed in PSCs was associated with stromal formation of pancreatic cancer and enhancement of tumor malignancy. We also assessed the effects of suppression of CD51 in PSCs on pancreatic cancer. Immunohistochemistry for CD51 in resected pancreatic cancer tissues showed that high expression of CD51 in the tumor stroma was associated with lymph node metastasis (P=0.025), positive pathologic margin (P=0.025), and shorter patient survival times (P=0.043). Lentivirus-mediated short hairpin RNA knockdown of CD51 decreased the proliferation and migration of PSCs. Quantitative real-time polymerase chain reaction showed that expression levels of genes related with ECM and tumor-stromal interactions were decreased by CD51 knockdown in PSCs. In a co-implantation model of pancreatic cancer cells and PSCs, tumor growth in vivo was inhibited by CD51 knockdown in PSCs (P<0.05). We also found reduced tumor stroma and decreased proliferation of cancer cells in implanted cancer tissues with CD51-silenced PSCs (P<0.05). Our results showed that CD51 expression in pancreatic cancer stroma is associated with enhanced tumor malignancy and that CD51 may be a potential therapeutic target for pancreatic cancer. .
103. Kohno Y, Yamamoto H, Hirahashi M, Kumagae Y, Nakamura M, Oki E, Oda Y, Reduced MUTYH, MTH1, and OGG1 expression and TP53 mutation in diffuse-type adenocarcinoma of gastric cardia, Human Pathology, 52, 145-152, 2016.04, The effects of oxidative stress in adenocarcinomas of gastric cardia (AGCs) have not been fully elucidated. With a strict definition of AGC, we examined the immunohistochemical expressions of inducible nitric oxide synthase; 8-hydroxy-deoxyguanosine; and the base excision repair enzymes such as MUTYH, MTH1, and OGG1, and TP53 mutational status. Sixty-three cases of AGC were characterized by younger patient age (P = .0227) and more frequent venous invasion (P = .0106) compared with the adenocarcinomas of pylorus (APs). 8-hydroxy-deoxyguanosine was accumulated (P = .0011), whereas MUTYH (P = .0325) and OGG1 (P = .0007) were decreased, in the AGCs compared with the adjacent mucosa, but these differences were not detected in the APs. Among the AGCs, lower expressions of MUTYH (P = .0013) and MTH1 (P = .0059) were each significantly associated with diffuse-type histology. A lower expression of OGG1 was correlated with higher T-stage (P = .0011), lymphatic invasion (P = .004), and lymph node metastasis (P = .0094). In addition, the presence of TP53 mutation was associated with diffuse-type histology (P = .0153) and a lower level of MUTYH (P = .0221). The AGCs also showed a relatively high rate of a transversion-type mutation of TP53 (50%), whereas all TP53 mutations in the APs were transition type. Age 62years or older (P = .0073), diffuse-type histology (P = .0020), and TP53 mutation (P = .0066) were each associated with worse survival in the AGC patients. Our results indicate that oxidative stress accumulation and a downregulation of base excision repair enzymes may play an important role in the pathogenesis of AGC, in particular diffuse-type AGCs. Diffuse-type AGC might involve molecular pathways different from those of other subsets of gastric cancer. .
104. Miyasaka Y, Ohtsuka T, Tamura K, Mori Y, Shindo K, Yamada D, Takahata S, Ishigami K, Ito T, Tokunaga S, Oda Y, Mizumoto K, Nakamura M, Tanaka M, Predictive factors for the metachronous development of high-risk lesions in the remnant pancreas after partial pancreatectomy for intraductal papillary mucinous neoplasm, Ann Surg, 10.1097/SLA.0000000000001368 , 263, 6, 1180-1187, 2016.04, Abstract
OBJECTIVE:
To identify factors predicting the development of high-risk lesions in the remnant pancreas after surgery for intraductal papillary mucinous neoplasm (IPMN).
BACKGROUND:
IPMN has unique features, including multifocality, adenoma-carcinoma sequence, and the development of distinct pancreatic ductal adenocarcinoma (PDAC) in the same pancreas. Careful attention should, therefore, be paid to the metachronous occurrence of high-risk lesions, including high-grade dysplasia or invasive carcinoma (HGD/INV) of IPMN and concomitant PDAC in the remnant pancreas after partial pancreatectomy for IPMN.
METHODS:
Clinicopathologic and surveillance data for 195 patients who underwent partial pancreatectomy for IPMN were reviewed retrospectively.
RESULTS:
Thirteen patients exhibited metachronous development of high-risk lesions including 6 HGD/INV and 7 concomitant PDACs in the remnant pancreas. The 5- and 10-year cumulative incidences of metachronous high-risk lesions in the remnant pancreas were 7.8% and 11.8%, respectively. Twelve of 13 patients had high-risk lesions at the time of initial surgery, and 10 of the 13 IPMNs were located in the distal pancreas. The IPMN subtypes initially resected were gastric in 6 patients, intestinal in 5, and pancreatobililary in the remaining 2. Univariate and multiple regression analyses identified pathologic results of HGD/INV and IPMN located in the distal pancreas as independent predictive factors for metachronous HGD/INV of IPMN, and the pancreatobiliary subtype of IPMN and presence of concomitant PDAC for metachronous PDAC.
CONCLUSIONS:
Patients undergoing partial pancreatectomy for IPMN are at high risk of developing lesions requiring surgery in the remnant pancreas, and close, long-term surveillance should be considered in these patients..
105. Chijiiwa Y, Moriyama T, Ohuchida K, Nabae T, Ohtsuka T, Miyasaka Y, Fujita H, Maeyama R, Manabe T, Abe A, Mizuuchi Y, Oda Y, Mizumoto K, Nakamura M, Overexpression of microRNA-5100 decreases the aggressive phenotype of pancreatic cancer cells by targeting PODXL, Int J Oncol, 10.3892/ijo.2016.3389 , 48, 4, 1688-1700, 2016.04, Abstract

Metastasis is the main cause of cancer-associated death, and metastasis of pancreatic cancer remains difficult to treat because of its aggressiveness. MicroRNAs (miRNAs) play crucial roles in the regulation of various human transcripts, and many miRNAs have been reported to correlate with cancer metastasis. We identified an anti-metastatic miRNA, miR-5100, by investigating differences in miRNA profiling between highly metastatic pancreatic cancer cells and their parental cells. Overexpression of miR-5100 inhibited colony formation (P<0.05), cell migration (P<0.0001) and invasion (P<0.0001) of pancreatic cancer cells. In addition, we identified a possible target of miR-5100, podocalyxin-like 1 (PODXL), and demonstrated miR-5100 directly binds to the 3' untranslated region of PODXL and post-transcriptionally regulates its expression in pancreatic cancer cells. Silencing PODXL resulted in diminished cell migration (P<0.0001) and invasion (P<0.05). We also clarified the close relationship between expression of PODXL in human pancreatic cancer specimens and liver metastasis (P=0.0003), and determined that post-operative survival was longer in the low-PODXL expression group than in the high-PODXL expression group (P<0.05). These results indicate that miR-5100 and PODXL have considerable therapeutic potential for anti-metastatic therapy and could be potential indicators for cancer metastases in patients with pancreatic cancer.
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106. Nakamura M, Matsumoto H, Nakashima H, Ando Y, Hirai T, Yoshida K, Hino K, L-Carnitine Supplementation Improved Hepatic Steatosis After Pancreatectomy, Pancreas, 10.1097/MPA.0000000000000508 , 45, 3, e7-e9, 2016.04.
107. Takizawa K, Yamamoto H, Taguchi K, Ohno S, Tokunaga E, Yamashita N, Kubo M, Nakamura M, Oda Y, Insulin-like growth factor II messenger RNA-binding protein-3 is an indicator of malignant phyllodes tumor of the breast, Hum Pathol, 55, 9, 30-38, 2016.04, Abstract
The aim of this study was to elucidate the clinicopathological and prognostic significance of the expressions of insulin-like growth factor II mRNA-binding protein-3 (IMP3) and epidermal growth factor receptor (EGFR) in phyllodes tumors (PTs). Immunohistochemical staining for IMP3 and EGFR was performed in 130 cases of primary PTs (83 benign, 28 borderline, 19 malignant), 34 recurrent/metastatic PTs, and 26 fibroadenomas (FAs). Among the primary tumors, a high expression of IMP3 was significantly more frequently present in malignant PTs (17/19, 89%) than in the FAs (0/26, 0%), benign PTs (0/83, 0%) and borderline PTs (3/28, 11%). The recurrent and metastatic lesions of malignant PTs also showed high IMP3 expression (3/5 [60%] and 6/6 [100%], respectively). Most malignant PTs showed strong IMP3 expression at the interductal area or more diffusely, whereas weak and focal (low) expression of IMP3 was limited to the periductal area in FAs and benign PTs. EGFR overexpression was significantly correlated with tumor grade and high IMP3 expression. Overexpressions of IMP3 and EGFR were significantly associated with shorter periods of metastasis-free and disease-free survival. The results suggest that high expressions of IMP3 and EGFR with a characteristic staining pattern may be helpful for both identifying malignant PT and predicting the prognosis of these tumors..
108. Sada M, Ohuchida K, Horioka K, Okumura T, Moriyama T, Miyasaka Y, Ohtsuka T, Mizumoto K, Oda Y, Nakamura M, Hypoxic stellate cells of pancreatic cancer stroma regulate extracellular matrix fiber organization and cancer cell motility, Cancer Letters, 10.1016/j.canlet.2016.01., 372, 2, 210-218, 2016.04, Desmoplasia and hypoxia in pancreatic cancer mutually affect each other and create a tumor-supportive microenvironment. Here, we show that microenvironment remodeling by hypoxic pancreatic stellate cells (PSCs) promotes cancer cell motility through alteration of extracellular matrix (ECM) fiber architecture. Three-dimensional (3-D) matrices derived from PSCs under hypoxia exhibited highly organized parallel-patterned matrix fibers compared with 3-D matrices derived from PSCs under normoxia, and promoted cancer cell motility by inducing directional migration of cancer cells due to the parallel fiber architecture. Microarray analysis revealed that procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) in PSCs was the gene that potentially regulates ECM fiber architecture under hypoxia. Stromal PLOD2 expression in surgical specimens of pancreatic cancer was confirmed by immunohistochemistry. RNA interference-mediated knockdown of PLOD2 in PSCs abrogated para
llel fiber architecture of 3-D matrices, leading to decreased directional migration of cancer cells within the matrices. In conclusion, these findings indicate that hypoxia-induced PLOD2 in PSCs creates a permissive microenvironment for migration of cancer cells through architectural regulation of stromal ECM in pancreatic cancer..
109. Manabe T, Ueki T, Nagayoshi K, Moriyama T, Yanai K, Nagai S, Esaki M, Nakamura K, Nakamura M, Feasibility of laparoscopic surgery for complex Crohn's disease of the small intestine., Asian J Endosc Surg, 10.1111/ases.12287, 9, 4, 265-269, 2016.04, Abstract
BACKGROUND:
The laparoscopic approach for complex Crohn's disease (CD), which involves abscess formation, fistula formation, and recurrent CD, is controversial. The aim of this study was to investigate the feasibility and safety of the laparoscopic approach for complex CD.

METHODS:
Fifty-six patients who had undergone surgery for CD of the small bowel from January 2007 to August 2014 were divided into two groups: the laparoscopic approach for complex CD group (LC group, n = 31) and the laparoscopic approach for simple CD group (LS group, n = 25). The preoperative data and surgical outcomes of the LC group were compared with those of the LS groups.

RESULTS:
There were no significant differences in preoperative data and operating time between the two groups. Blood loss was not significantly different between the LC and LS groups. The incision length was longer in the LC group than the LS group (P = 0.004). The incidence of severe postoperative complications in the LC group was higher than in the LS group (P = 0.026). The length of postoperative stay was similar in the LC and LS groups.

CONCLUSIONS:
The laparoscopic approach for complex CD is feasible and provides good cosmesis that is comparable to that offered by simple CD..
110. Fujimoto T, Ohtsuka T, Date K, Kimura H, Matsunaga T, Mori Y, Miyasaka Y, Mochidome N, Oda Y, Nakamura M, Expression of Bcl-2 19-kDa interacting protein 3 predicts prognosis after ampullary carcinoma resection, J Hepatobiliary Pancreat Sci, 10.1002/jhbp.367 , 23, 8, 489-496, 2016.04, Abstract
BACKGROUND:
An adequate management strategy for ampullary carcinoma (AC), a rare neoplasm, has yet to be determined. The aim of this study was to identify specific molecular markers allowing for the adequate management of AC.
METHODS:
The clinicopathological data of 41 patients who underwent curative resection of AC were reviewed retrospectively. The expression of thymidylate synthase (TS) and Bcl-2 19-kDa interacting protein 3 (BNIP3), two sensitive markers for S-1 and gemcitabine, respectively, was evaluated immunohistochemically. The relationship between the expression levels of these markers and the clinicopathological data were then investigated.
RESULTS:
The 5-year overall survival rate in the study population was 62%. In univariate and multivariate analyses, lymph node metastasis, neural invasion, lymphatic invasion, and the high-level BNIP3 expression were significant predictive factors for a poor postoperative prognosis. Neither TS nor BNIP3 expression were able to predict survival or the disease recurrence rate in patients who received postoperative adjuvant chemotherapy for AC.
CONCLUSIONS:
BNIP3 expression may serve as a prognostic marker for patients with AC, but neither TS nor BNIP3 contributes to the selection criteria for adjuvant chemotherapy for AC, at least with respect to current drug regimens.
© 2016 Japanese Society of Hepato-Biliary-Pancreatic Surgery.
KEYWORDS:
Ampulla of Vater; BNIP3; Carcinoma; Chemotherapy; Thymidylate synthase.
111. Ueno D, Nakashima H, Higashida M, Yoshida K, Hino K, Irei I, Moriya T, Matsumoto H, Hirai T, Nakamura M, Emergent laparoscopic cholecystectomy for acute acalculous cholecystitis revisited, Surg Today, 10.1007/s00595-015-1173-8 , 46, 3, 309-312, 2016.04, Abstract
PURPOSE:
To compare the safety of emergent laparoscopic cholecystectomy for acute acalculous cholecystitis (AAC) with surgery for acute calculous cholecystitis (ACC).
METHODS:
We retrospectively reviewed the perioperative records of 111 patients who underwent emergent laparoscopic cholecystectomy for acute cholecystitis under the care of the Department of Digestive Surgery, Kawasaki Medical School, Kurashiki, between January 2010 and April 2014. Patients were divided into the AAC group (27 patients) and the ACC group (84 patients), and their perioperative outcomes were compared.
RESULTS:
Patients in the AAC group had significantly higher disease severity and American Society of Anesthesiologists physical status scores (p = 0.001 and 0.037, respectively), lower blood hemoglobin and albumin concentrations (p = 0.0005 and 0.017, respectively), and lower hematocrit and platelet count (p < 0.0001 and 0.040, respectively) than those in the ACC group. When we compared perioperative outcomes, we also found that patients in the AAC group were more likely to have received a blood transfusion (p = 0.002) and to have required conversion to open surgery (p = 0.008). There were no significant differences in morbidity, mortality or length of hospital stay.
CONCLUSIONS:
Early laparoscopic cholecystectomy is safe in acute acalculous as well as acute calculous cholecystitis.
KEYWORDS:
Acute acalculous cholecystitis; Early laparoscopic cholecystectomy; Emergent operation; Short-term clinical outcomes; Ultrasound scissors.
112. Tamura K, Ohtsuka T, Date K, Fujimoto T, Matsunaga T, Kimura H, Watanabe Y, Miyazaki T, Ohuchida K, Takahata S, Ishigami K, Oda Y, Mizumoto K, Nakamura M, Tanaka M, Distinction of Invasive Carcinoma Derived From Intraductal Papillary Mucinous Neoplasms From Concomitant Ductal Adenocarcinoma of the Pancreas Using Molecular Biomarkers, Pancreas, 10.1097/MPA.0000000000000563 , 45, 6, 826-835, 2016.04, OBJECTIVES:To clarify the usefulness of molecular biomarkers for distinguishing invasive carcinoma derived from intraductal papillary mucinous neoplasms (IPMNs [Inv-IPMN]) from concomitant pancreatic ductal adenocarcinoma (PDAC).METHODS:Data from 19 patients with resected concomitant PDAC were retrospectively reviewed. KRAS/GNAS mutations and immunohistochemical (IHC) expression of p53 and p16/CDKN2A were assessed in both IPMN and distinct PDAC. As controls, KRAS/GNAS mutations and IHC labeling were assessed between invasive and noninvasive components in 1 lesion of 22 independent patients.RESULTS:KRAS/GNAS mutation status of invasive and noninvasive components in Inv-IPMN was consistent in 18 (86%) of 21 patients. Conversely, mutational patterns in IPMN and distinct PDAC in the same pancreas differed from each other in 17 (89%) of 19. There were 10 (53%) and 8 (42%) of 19 patients who showed the same p53 and p16/CDKN2A staining between concomitant PDAC and d
istinct IPMN. In the Inv-IPMN cohort, 19 (86%) of 22 patients showed the same IHC expression pattern between the noninvasive and invasive components.CONCLUSIONS:It may be possible to distinguish Inv-IPMN from concomitant PDAC by assessing these molecular biomarkers. More precise distinction of Inv-IPMN and concomitant PDAC will lead to adequate recognition of the natural history of IPMNs and hence optimal management..
113. Kimura H, Ohtsuka T, Fujimoto T, Date K, Matsunaga T, Cases AI, Abe A, Mizuuchi Y, Miyasaka Y, Ito T, Oda Y, Nakamura M, Tanaka M, Different hormonal expression patterns between primary pancreatic neuroendocrine tumors and metastatic sites, Pancreas, 45, 7, 947-952, 2016.04, OBJECTIVES: Pancreatic neuroendocrine tumors (PNETs) are known to have heterogeneity in terms of their ability to produce multiple hormones. The aim of this study was to evaluate the heterogeneity of PNETs from the viewpoint of hormonal expression. METHODS: The expressions of 4 representative hormones, gastrin, insulin, glucagon, and somatostatin, in both primary and metastatic lesions, were analyzed by immunohistochemical staining in 20 patients with metastatic PNETs (6 gastrinomas, 1 insulinoma, 1 glucagonoma, and 12 nonfunctioning PNETs [NF-PNETs]). Metastatic sites included lymph nodes in all 20 patients and liver metastasis in 7 patients (2 gastrinomas and 5 NF-PNETs). RESULTS: There were 6 PNETs with multiple hormone secretion (30%), and positive expression of 1 or more hormones was found in 9 of 12 patients whose primary tumors were diagnosed as NF-PNETs. The positive concordance rate of the hormonal expression pattern between primary tumors and metast
atic lymph nodes and between primary tumors and hepatic metastasis were 50% and 11%, respectively. Three patients had metastatic lesions with positive hormonal expression, whereas their primary tumors were negative. CONCLUSIONS: Hormonal expressions are often different between the primary tumors and metastatic sites of PNETs..
114. Yawen Y, Nakamura M, Nakashima N, Designing Health Data Management Systems:Learning From Prominent Worldwide Applications, J Health Med Inform, 7, 216, 1-3, 2016.04.
115. Abe T, Ohuchida K, Miyasaka Y, Ohtsuka T, Oda Y, Nakamura M, Comparison of Surgical Outcomes Between Radical Antegrade Modular Pancreatosplenectomy (RAMPS) and Standard Retrograde Pancreatosplenectomy (SPRS) for Left-Sided Pancreatic Cancer, World J Surg, 10.1007/s00268-016-3526-x , 40, 9, 2267-75, 2016.04, BACKGROUND: Radical antegrade modular pancreatosplenectomy (RAMPS) is a modification of standard retrograde pancreatosplenectomy (SRPS) used to achieve the dissection of N1 lymph nodes, early vascular control, and negative posterior margins. However, there have been few comparative studies regarding the clinical outcomes of the RAMPS and SRPS procedures.METHODS: Ninety-three patients underwent distal pancreatectomy for the treatment of pancreas body and tail adenocarcinoma between 2000 and 2014. Clinicopathologic data were retrospectively analyzed in this study. We compared short- and long-term outcomes between RAMPS and SRPS. In addition, we investigated the significance of clinicopathologic factors in left-sided pancreatic cancers.RESULTS: Fifty-three patients underwent RAMPS and 40 patients underwent SRPS. RAMPS revealed a larger number of retrieved lymph nodes [28.4 ± 11.6 vs 20.7 ± 10.1; P = 0.0016], more frequent R0 resection [90.5 vs 67.5
%; P = 0.0053], less intraoperative bleeding than SRPS [485.4 ± 63.3 vs 682.3 ± 72.8 ml; P = 0.0444], and shorter operating time (267.3 ± 11.5 vs 339.4 ± 13.2 min; P < 0.0001) as compared with SRPS. In comparing RAMPS and SRPS, RAMPS showed a tendency for improvement of the median survival times than SRPS (47 vs 34 months) (P = 0.1920). In the multivariate analysis, R1 resection, histologic grade, and vascular invasion for overall survival (OS) were found to be independent factors.CONCLUSIONS: There were a decrease of intraoperative bleeding and an increase in the number of retrieved lymph nodes and the R0 resection rate using RAMPS as compared with SRPS..
116. Suyama K, Onishi H, Imaizumi A, Shinkai K, Umebayashi M, Kubo M, Mizuuchi Y, Oda Y, Tanaka M, Nakamura M, Katano, M, CD24 suppresses malignant phenotype by downregulation of SHH transcription through STAT1 inhibition in breast cancer cells, Cancer Letters, 374, 1, 44-53, 2016.04, Hedgehog (Hh) signaling has been found to be activated in breast cancer stem cells (BCSCs). However, the precise role of the BCSCs marker, CD24, remains unclear. Here, we describe a relationship between CD24 and Sonic Hedgehog (SHH), and reveal a role for this relationship in the induction of a malignant phenotype of breast cancer. CD24 siRNA-transfected breast cancer cells (BCCs) demonstrated higher expression of SHH and GLI1, increased anchorage-independent proliferation, and enhanced invasiveness and superior tumorigenicity compared with control. Conversely, CD24 forced-expressing BCCs possessed decreased SHH and GLI1 expression, anchorage-independent proliferation, and invasiveness. Suppression of SHH decreased invasiveness through inhibition of matrix metalloproteinase (MMP)-2 expression, GLI1 expression, anchorage-independent proliferation, tumorigenicity, and tumor volume in vivo in CD24 siRNA transfected BCCs. DNA microarray analysis identified STAT1 as a relationship between CD24 and SHH. CD24 siRNA-transfected BCCs with concurrent STAT1 inhibition exhibited decreased SHH expression, invasiveness, anchorage-independent proliferation, tumorigenicity, and tumor volume in vivo. These results suggest that CD24 suppresses development of a malignant phenotype by down-regulating SHH transcription through STAT1 inhibition. CD24 gene transfer or STAT1 inhibition may represent new effective therapeutic strategies to target refractory breast cancer.

Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
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117. Zheng B, Ohuchida K, Chijiiwa Y, Zhao M, Mizuuchi Y, Cui L, Horioka K, Ohtsuka T, Mizumoto K, Oda Y, Hashizume M, Nakamura M, Tanaka M, CD146 Attenuation in Cancer-Associated Fibroblasts Promotes Pancreatic Cancer Progression, Molecular Carcinogenesis, 55, 1560-1572, 2016.04.
118. Yoshida M, Miyasaka Y, Ohuchida K, Okumura T, Zheng B, Torata N, Fujita H, Nabae T, Manabe T, Shimamoto M, Ohtsuka T, Mizumoto K, Nakamura M, Calpain inhibitor calpeptin suppresses pancreatic cancer by disrupting cancer-stromal interactions in a mouse xenograft model., Cancer Sci, 10.1111/cas.13024 , 107, 10, 1443-1452, 2016.04, Desmoplasia contributes to the aggressive behavior of pancreatic cancer. However, recent clinical trials testing several antifibrotic agents on pancreatic cancer have not shown clear efficacy. Therefore, further investigation of desmoplasia-targeting antifibrotic agents by another mechanism is needed. Calpeptin, an inhibitor of calpains, suppressed fibroblast function and inhibited fibrosis. In this study, we investigated the anticancer effects of calpeptin on pancreatic cancer. We investigated whether calpeptin inhibited tumor progression using a mouse xenograft model. We used quantitative RT-PCR to evaluate the expression of calpain-1 and calpain-2 mRNA in pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs). We also undertook functional assays, including proliferation, migration, and invasion, to evaluate the inhibitory effects of calpeptin on PCCs and PSCs. Quantitative RT-PCR indicated that PCCs and PSCs expressed calpain-2 mRNA. C
alpeptin reduced tumor volume (P = 0.0473) and tumor weight (P = 0.0471) and inhibited the tumor desmoplastic reaction (P < 0.001) in xenograft tumors in nude mice. Calpeptin also inhibited the biologic functions of PCCs and PSCs including proliferation (P = 0.017), migration (P = 0.027), and invasion (P = 0.035) in vitro. Furthermore, calpeptin reduced the migration of PCCs and PSCs by disrupting the cancer-stromal interaction (P = 0.0002). Our findings indicate that calpeptin is a promising antitumor agent for pancreatic cancer, due not only to its suppressive effect on PCCs and PSCs but also its disruption of the cancer-stromal interaction..
119. Mori H, Kubo M, Nishimura R, Osako T, Arima N, Okumura Y, Okido M, Yamada M, Kai M, Kishimoto J, Miyazaki T, Oda Y, Otsuka T, Nakamura M, BRCAness as a Biomarker for Predicting Prognosis and Response to Anthracycline-Based Adjuvant Chemotherapy for Patients with Triple-Negative Breast Cancer, Plos One, 10.1371/journal.pone.0167016 , 11, 12, e0167016, 2016.04, BackgroundTriple-negative breast cancer (TNBC) is a heterogeneous tumor that encompasses many different subclasses of the disease. In this study, we assessed BRCAness, defined as the shared characteristics between sporadic and BRCA1-mutated tumors, in a large cohort of TNBC cases. MethodsThe BRCAness of 262 patients with primary TNBCs resected between January 2004 and December 2014 was determined through the isolation of DNA from tumor tissue. Classification of BRCAness was performed using multiple ligation-dependent probe amplification (MLPA). The tumor subtypes were determined immunohistochemically using resected specimens. ResultsOf the 262 TNBCs, the results of the MLPA assays showed that 174 (66.4%) tumors had BRCAness. Patients with BRCAness tumors were younger than patients with non-BRCAness tumors (P = 0.003). There was no significant difference between the two groups regarding their pathological stages. The BRCAness group had a significantly shorter
recurrence-free survival (RFS) compared with the non-BRCAness group (P = 0.04) and had a shorter overall survival (OS) although this did not reach statistical significance. Adjuvant treatments with anthracycline-based regimens provided significantly greater benefits to the BRCAness group (P = 0.003 for RFS, and P = 0.03 for OS). Multivariate Cox proportional hazard model analysis showed that BRCAness was an independent negative prognostic factor, and the anthracycline-based adjuvant chemotherapy was an independent positive prognostic factor for both RFS and OS in TNBC.ConclusionsThe 66.4% patients of TNBCs showed BRCAness. BRCAness is essential as a biomarker in the subclassification of TNBCs and might be of use for predicting their prognosis. Furthermore, this biomarker might be a predictive factor for the effectiveness of anthracycline-based adjuvant chemotherapy for patients with TNBCs..
120. Kawamoto M, Onishi H, Ozono K, Yamasaki A, Imaizumi A1, Kamakura S, Nakano K, Oda Y, Sumimoto H, Nakamura M, Tropomyosin-related kinase B mediated signaling contributes to the induction of malignant phenotype of gallbladder cancer, Oncotarget, 10.18632/oncotarget.16063, 8, 22, 36211-36224, 2017.04, Abstract
This study aims to demonstrate the clinical and biological significance of Brain derived neurotrophic factor (BDNF)/Tropomyosin-related kinase B (TrkB) signaling in gallbladder cancer (GBC) through a series of in vitro and in vivo experiments. TrkB expression was detected in 63 (91.3%) out of 69 surgically resected primary GBC specimens by immunohistochemistry. TrkB expression in the invasive front correlated with T factor (p=0.0391) and clinical staging (p=0.0391). Overall survival was lower in patients with high TrkB expression in the invasive front than in those with low TrkB expression (p=0.0363). In vitro experiment, we used five TrkB-expressing GBC cell lines with or without K-ras mutation. TrkB-mediated signaling increased proliferation and the invasiveness by inducing epithelial mesenchymal transition, and activating matrix metalloproteinases-2 (MMP-2) and MMP-9. Inhibition of TrkB-mediated signaling also decreased hypoxia-inducible factor-1α, vascular endothelial growth factor A (VEGF-A), VEGF-C, and VEGF-D expression. In vivo experiment, inhibition of TrkB-mediated signaling suppressed tumorigenicity and tumor growth in GBC. These findings demonstrate that TrkB-mediated signaling contributes to the induction of malignant phenotypes (proliferation, invasiveness, angiogenesis, lymphangiogenesis, and tumorigenesis) in GBC, and could be a promising therapeutic target regardless of K-ras mutation status..
121. Mori H, Kubo M, Yamaguchi R, Nishimura R, Osako T, Arima N, Okumura Y, Okido M, Yamada M, Kai M, Kishimoto J, Oda Y, Nakamura M, The combination of PD-L1 expression and decreased tumor- infiltrating lymphocytes is associated with a poor prognosis in triple-negative breast cancer, Oncotarget, 8, 9, 15584-15592, 2017.04, This study included patients with primary triple-negative breast cancer (TNBC) who underwent resection without neoadjuvant chemotherapy between January 2004 and December 2014. Among the 248 TNBCs studied, programmed cell death ligand-1 (PD-L1) expression was detected in 103 (41.5%) tumors, and high levels of tumor-infiltrating lymphocytes (TILs) were present in 118 (47.6%) tumors. PD- L1 expression correlated with high levels of TILs, but was not a prognostic factor. Patients with TILs-high tumors had better overall survival than those with TILs- low tumors (P = 0.016). There was a strong interaction between PD-L1 expression and TILs that was associated with both recurrence-free survival (P = 0.0018) and overall survival (P = 0.015). Multivariate Cox proportional hazards model analysis showed that PD-L1-positive/TILs-low was an independent negative prognostic factor for both recurrence-free survival and overall survival. Our findings suggest that PD-
L1-positive/TILs-low tumors are associated with a poor prognosis in patients with TNBC, and that it is important to focus on the combination of PD-L1 expression on tumor cells and TILs present in the tumor microenvironment. These biomarkers may be useful for stratification of TNBCs and for predicting prognosis and developing novel cancer immunotherapies..
122. Matsunaga T, Ohtsuka T, Asano K, Kimura H, Ohuchida K, Kitada H, Ideno N, Mori Y, Tokunaga S, Oda Y, Guha S, Raimondo M, Nakamura M, Tanaka M, S100P in Duodenal Fluid Is a Useful Diagnostic Marker for Pancreatic Ductal Adenocarcinoma, Pancreas, 10.1097/MPA.0000000000000940, 46, 10, 1288-1295, 2017.04, Abstract
OBJECTIVES:
The development of an effective screening method for pancreatic ductal adenocarcinoma (PDAC) is of paramount importance. This study assessed the diagnostic utility in pancreatic diseases of duodenal markers during upper gastrointestinal endoscopy (GIE) or endoscopic ultrasonography.

METHODS:
This study prospectively enrolled 299 consecutive participants, including 94 patients with PDACs, 144 patients with other pancreatic diseases, and 61 normal individuals as control subjects. All subjects underwent upper GIE or endoscopic ultrasonography either at Kyushu University Hospital (Fukuoka, Japan) or the Mayo Clinic (Jacksonville, Fla) from October 2011 to July 2014. Duodenal fluid (DF) was collected without secretin stimulation and of carcinoembryonic antigen and S100 calcium-binding protein P (S100P) concentrations were measured.

RESULTS:
Concentrations of S100P in DF were significantly higher in patients with PDAC and chronic pancreatitis than in control subjects (P < 0.01). A logistic regression model that included age found that the sensitivity and specificity of S100P concentration in diagnosing stages 0/IA/IB/IIA PDAC were 85% and 77%, respectively, with an area under the receiver operating characteristic curve of 0.82. Carcinoembryonic antigen concentrations in DF of patients with pancreatic disease did not differ significantly from control subjects.

CONCLUSIONS:
Analysis of S100P concentration in DF, in combination with routine screening upper GIE, may facilitate the detection of PDAC..
123. Miyasaka Y, Mori Y, Nakata K, Ohtsuka T, Nakamura M, Prophylactic biliary and gastrointestinal bypass for unresectable pancreatic head cancer: A retrospective case series, Journal of the Pancreas, 18, 6, 470-474, 2017.04.
124. Date K, Ohtsuka, T Fujimoto, T Tamura K, Kimura H, Matsunaga T, Mochidome N, Miyazaki T, Mori Y, Oda Y, Nakamura M Tanaka M, Molecular Evidence for Monoclonal Skip Progression in Main Duct Intraductal Papillary Mucinous Neoplasms of the Pancreas, Annals of Surgery, 10.1097/SLA.0000000000001755, 265, 5, 969-977, 2017.04, Objective:
To clarify clonality of distinct multisegmental main duct (MD)-intraductal papillary mucinous neoplasms (IPMNs) using microarray analysis.
Background:
IPMNs represent a pancreatic ductal cell field defect, which causes multiple occurrences of lesions. In addtion, it has been speculated that MD-IPMNs display features of monoclonal skip progression.
Methods:
Total RNA was extracted from fresh-frozen tissue samples of metachronous MD-IPMNs and nonneoplastic pancreas tissue from the same pancreas from two individuals, and whole human genome microarray analysis was performed. Formalin-fixed paraffin-embedded tissue specimens from 28 distinct IPMNs were then collected from 12 patients, genomic DNA was extracted, and GNAS/KRAS mutational status was investigated. Immunohistochemical analysis was performed to validate the expression pattern of the indicated proteins.
Results:
Microarray analysis revealed that metachronous MD-IPMNs from the same individual displayed pair-wise correlation coefficients of 0.9523 and 0.9512. In contrast, MD-IPMNs of the same histological grade from different individuals displayed coefficients of 0.8092 and 0.8211. Scatter plot analysis revealed that metachronous MD-IPMNs from the same individual displayed a closer linear relationship. Furthermore, heat map and hierarchical cluster analyses revealed that metachronous MD-IPMNs from the same individual were classified in the same branch, and the gene expression patterns were similar. The GNAS/KRAS mutational statuses of distinct MD-IPMNs were consistent with each other. Immunohistochemical assessment of five specific proteins demonstrated that the same expression pattern between two lesions was observed in 95% of the samples.
Conclusions:
These findings using molecular analyses indicate that MD-IPMNs might display features of monoclonal skip progression.
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125. Miyazaki T, Ohishi Y, Miyasaka Y, Oda Y, Aishima S, Ozono K, Abe A, Nagai E, Nakamura M, Oda Y, Molecular Characteristics of Pancreatic Ductal Adenocarcinomas with High-Grade Pancreatic Intraepithelial Neoplasia (PanIN) Are Different from Those without High-Grade PanIN, Pathobiology, 10.1159//000455194, 84, 4, 192-201, 2017.04, We reported that pancreatic ductal adenocarcinomas (PDACs) without high-grade pancreatic intraepithelial neoplasia (PanIN) in the vicinity had worse prognoses than PDACs with high-grade PanIN. However, the molecular characteristics of PDACs with and without high-grade PanIN have not been compared. The aim of this study is to clarify the molecular characteristics of PDACs with and without high-grade PanIN..
126. Date S, Noguchi H, Kaku K, Kurihara K, Miyasaka Y, Okabe Y, Nakamura U, Ohtsuka T, Nakamura M, Laparoscopy-Assisted Spleen-Preserving Distal Pancreatectomy for Living-Donor Pancreas Transplantation, Transplant Proc. , 10.1016/j.transproceed.2017.03.037, 49, 5, 1133-1137, 2017.04, BACKGROUND:Living pancreas transplantation plays an important role in the treatment of patients with severe type 1 diabetes. However, pancreatectomy is very invasive for the donor, and less-invasive surgical procedures are needed. Although some reports have described hand-assisted laparoscopic surgery for distal pancreatectomy in living-donor operations, less-invasive laparoscopy-assisted (LA) procedures are expected to increase the donor pool. We herein report the outcomes of four cases of LA spleen-preserving distal pancreatectomy (Warshaw technique [WT]) in living pancreas donors.PATIENTS AND METHODS:Four living pancreas donors underwent LA-WT at our institution from September 2010 to January 2013. All donors fulfilled the donor criteria established by the Japan Society for Pancreas and Islet Transplantation.RESULTS:The median donor age was 54 years. Two donors underwent left nephrectomy in addition to LA-WT for simultaneous pancreas-kidney transpl
antation. The median donor operation time for pancreatectomy was 340.5 minutes. The median pancreas warm ischemic time was 3 minutes. The median donor blood loss was 246 g. All recipients immediately achieved insulin independence. One donor required reoperation because of obstructive ileus resulting from a port-site hernia. Another donor developed a pancreatic fistula (International Study Group of Pancreatic Fistula grade B), which was controlled with conservative management. After a maximum follow-up of 73 months, no clinically relevant adverse events had occurred. These results were comparable with those of previous studies concerning living-donor pancreas transplantation.CONCLUSION:The LA-WT is a safe and acceptable operation for living-donor pancreas transplantation..
127. Manabe T, Koba R, Nagayoshi K, Sadakari Y, Fujita H, Nagai S, Ueki T, Nagai E, Nakamura M, Laparoscopic excision of neurogenic retrorectal tumors, Asian J Endosc Surg, 10.1111/ases.12337., 10, 2, 223-226, 2017.04, Abstract
Retrorectal tumors (RT) are uncommon and usually managed by open surgical excision. Laparoscopic excision for RT has been reported in only a small number of papers. We aimed to assess the laparoscopic approach for RT and to discuss the factors that made this procedure difficult. We performed laparoscopic excision using a five-trocar technique for neurogenic RT in three patients. Tumors were successfully excised laparoscopically in two patients. However, the third patient required open conversion because the tumor was strongly adhered to the sacrum and could not be mobilized by dissection, resulting in poor visualization of the dissected site. Laparoscopic excision for RT provides excellent intraoperative visualization and good cosmesis in selected patients, but firm adherence to the sacrum may cause difficulty with this procedure..
128. Ohuchida K, Nagai E, Moriyama T, Shindo K, Manabe T, Ohtsuka T, Shimizu S, Nakamura M, Feasibility and safety of modified inverted T-shaped method using linear stapler with movable cartridge fork for esophagojejunostomy following laparoscopic total gastrectomy, Transl Gastroenterol Hepatol., 23, 2, 50, 2017.04, Abstract
BACKGROUND:
We previously reported the use of an inverted T-shaped method to obtain a suitable view for hand sewing to close the common entry hole when the linear stapler was fired for esophagojejunostomy after laparoscopic total gastrectomy (LTG). This conventional method involved insertion of the fixed cartridge fork to the Roux limb and the fine movable anvil fork to the esophagus to avoid perforation of the jejunum. However, insertion of the movable anvil fork to the esophagus during this procedure often requires us to strongly push down the main body of the stapler with the fixed cartridge fork to bring the direction of the anvil fork in line with the direction of the long axis of the esophagus while controlling the opening of the movable anvil fork. We therefore modified this complicated inverted T-shaped method using a linear stapler with a movable cartridge fork. This modified method involved insertion of the movable cartridge fork into the Roux limb followed by natural, easy insertion of the fixed anvil fork into the esophagus without controlling the opening of the movable cartridge fork.
METHODS:
We performed LTG in a total of 155 consecutive patients with gastric cancer from November 2007 to December 2015 in Kyushu University Hospital. After LTG, we performed the conventional inverted T-shaped method using a linear stapler with a fixed cartridge fork in 61 patients from November 2007 to July 2011 (fixed cartridge group). From August 2011, we used a linear stapler with a movable cartridge fork and performed the modified inverted T-shaped method in 94 patients (movable cartridge group). We herein compare the short-term outcomes in 94 cases of LTG using the modified method (movable cartridge fork) with those in 61 cases using the conventional method (fixed cartridge fork).
RESULTS:
We found no significant differences in the perioperative or postoperative events between the movable and fixed cartridge groups. One case of anastomotic leakage occurred in the fixed cartridge group, but no anastomotic leakage occurred in the movable cartridge group.
CONCLUSIONS:
Although there were no remarkable differences in the short-term outcomes between the movable and fixed cartridge groups, we believe that the modified inverted T-shaped method is technically more feasible and reliable than the conventional method and will contribute to the improved safety of LTG..
129. Okumura T, Ohuchida K, Sada M, Abe T, Endo S, Koikawa K, Iwamoto C, Miura D, Mizuuchi Y, Moriyama T, Nakata K, Miyasaka Y, Manabe T, Ohtsuka T, Nagai E, Mizumoto K, Oda Y, Hashizume M, Nakamura M, Extra-pancreatic invasion induces lipolytic and fibrotic changes in the adipose microenvironment, with released fatty acids enhancing the invasiveness of pancreatic cancer cells, Oncotarget, 10.18632/oncotarget.15430., 8, 11, 18280-18295, 2017.04, Abstract
Pancreatic cancer progression involves components of the tumor microenvironment, including stellate cells, immune cells, endothelial cells, and the extracellular matrix. Although peripancreatic fat is the main stromal component involved in extra-pancreatic invasion, its roles in local invasion and metastasis of pancreatic cancer remain unclear. This study investigated the role of adipose tissue in pancreatic cancer progression using genetically engineered mice (Pdx1-Cre; LSL-KrasG12D; Trp53R172H/+) and an in vitro model of organotypic fat invasion. Mice fed a high fat diet had significantly larger primary pancreatic tumors and a significantly higher rate of distant organ metastasis than mice fed a standard diet. In the organotypic fat invasion model, pancreatic cancer cell clusters were smaller and more elongated in shape and showed increased fibrosis. Adipose tissue-derived conditioned medium enhanced pancreatic cancer cell invasiveness and gemcitabine resistance, as well as inducing morphologic changes in cancer cells and increasing the numbers of lipid droplets in their cytoplasm. The concentrations of oleic, palmitoleic, and linoleic acids were higher in adipose tissue-derived conditioned medium than in normal medium, with these fatty acids significantly enhancing the migration of cancer cells. Mature adipocytes were smaller and the concentration of fatty acids in the medium higher when these cells were co-cultured with cancer cells. These findings indicate that lipolytic and fibrotic changes in peripancreatic adipose tissue enhance local invasiveness and metastasis via adipocyte-released fatty acids. Inhibition of fatty acid uptake by cancer cells may be a novel therapy targeting interactions between cancer and stromal cells.
KEYWORDS:
adipose microenvironment; extra-pancreatic invasion; fatty acids; lipolysis; pancreatic cancer.
130. Yamauchi A, Yamamura M, Katase N, Itadani M, Okada N, Kobiki K, Nakamura M, Yamaguchi Y, Kuribayashi F, Evaluation of pancreatic cancer cell migration with multiple parameters in vitro by using an optical real-time cell mobility assay device, BMC cancer, 10.1186/s12885-017-3218-4., 17, 1, 234-234, 2017.04, Abstract
BACKGROUND:
Migration of cancer cell correlates with distant metastasis and local invasion, which are good targets for cancer treatment. An optically accessible device "TAXIScan" was developed, which provides considerably more information regarding the cellular dynamics and less quantity of samples than do the existing methods. Here, we report the establishment of a system to analyze the nature of pancreatic cancer cells using TAXIScan and we evaluated lysophosphatidic acid (LPA)-elicited pancreatic cell migration.
METHODS:
Pancreatic cancer cell lines, BxPC3, PANC-1, AsPC1, and MIAPaCa-2, were analyzed for adhesion as well as migration towards LPA by TAXIScan using parameters such as velocity and directionality or for the number of migrated cells by the Boyden chamber methods. To confirm that the migration was initiated by LPA, the expression of LPA receptors and activation of intracellular signal transductions were examined by quantitative reverse transcriptase polymerase reaction and western blotting.
RESULTS:
Scaffold coating was necessary for the adhesion of pancreatic cancer cells, and collagen I and Matrigel were found to be good scaffolds. BxPC3 and PANC-1 cells clearly migrated towards the concentration gradient formed by injecting 1 μL LPA, which was abrogated by pre-treatment with LPA inhibitor, Ki16425 (IC50 for the directionality ≈ 1.86 μM). The LPA dependent migration was further confirmed by mRNA and protein expression of LPA receptors as well as phosphorylation of signaling molecules. LPA1 mRNA was highest among the 6 receptors, and LPA1, LPA2 and LPA3 proteins were detected in BxPC3 and PANC-1 cells. Phosphorylation of Akt (Thr308 and Ser473) and p42/44MAPK in BxPC3 and PANC-1 cells was observed after LPA stimulation, which was clearly inhibited by pre-treatment with a compound Ki16425.
CONCLUSIONS:
We established a novel pancreatic cancer cell migration assay system using TAXIScan. This assay device provides multiple information on migrating cells simultaneously, such as their morphology, directionality, and velocity, with a small volume of sample and can be a powerful tool for analyzing the nature of cancer cells and for identifying new factors that affect cell functions.
KEYWORDS:
Chemotaxis; Inhibitor; Lipid mediator; Metastasis; Migration; TAXIScan.
131. Fujimoto T, Ohtsuka T, Nakashima Y, Gotoh Y, Date K, Mori Y, Sadakari Y, Takahata S, Oda Y, Nakamura M, Elevated bile amylase level without pancreaticobiliary maljunction is a risk factor for gallbladder carcinoma, J Hepatobiliary Pancreat Sci., 10.1002/jhbp.421., 24, 2, 103-108, 2017.04, Background: Elevated bile amylase level in patients with pancreaticobiliary maljunction (PBM) or high confluence of pancreaticobiliary ducts (HCPBD) is well known as a risk factor for gallbladder carcinoma (GBC) development. However, the effects of occult pancreaticobiliary reflux (OPR), a condition characterized by high bile amylase level in the presence of an anatomically normal pancreaticobiliary junction, on GBC development remain unclear. The aim of this study was to assess the relationship between OPR and GBC.Methods: Clinicopathological data of 52 patients who were preoperatively diagnosed with gallbladder (GB) tumor (22 malignant, 30 benign) were retrospectively reviewed. All of the patients underwent preoperative endoscopic retrograde cholangiopancreatography to evaluate pancreaticobiliary junction morphology and bile amylase level. The relationship between the histological diagnosis of GB lesions, and pancreaticobiliary junction morphology a
nd bile amylase level were investigated.Results: PBM, HCPBD, and normal pancreaticobiliary junction (NPJ) were identified in 12, 9, and 31 patients, respectively. The rates of GBC in patients with PBM, HCPBD, and NPJ were 58% (7/12), 67% (6/9), and 29% (9/31), respectively. Of the 31 patients with NPJ, 22 had OPR and 9 of these had GBC. None of the patients with NPJ and normal bile amylase level had GBC. Additionally, among patients with NPJ, bile amylase level was significantly higher in patients with GBC than in patients with benign tumors.Conclusions: OPR, like PBM and HCPBD, is a risk factor for GBC development..
132. Nakayama H, Ohuchida K, Yoshida M, Miyazaki T, Takesue S, Abe T, Endo S, Koikawa K, Okumura T, Moriyama T, Nakata K, Miyasaka Y, Shirahane K, Manabe T, Ohtsuka T, Toma H, Tominaga Y, Nagai E, Mizumoto K, Oda Y, Nakamura M, Degree of desmoplasia in metastatic lymph node lesions is associated with lesion size and poor prognosis in pancreatic cancer patients, Oncol Lett, 10.3892/ol.2017.6549, 14, 3, 3141-3147, 2017.04, Abstract
Pancreatic cancer is characterized by increased hyperplasia of fibrotic tissue, termed desmoplasia, and lymph node metastasis is an independent prognostic factor in this disease. However, there are no reports focused on desmoplasia in pancreatic cancer lymph node metastases. The present study evaluated a range of factors and investigated their association with poor prognosis in pancreatic cancer cases with lymph node metastasis, including the degree of desmoplasia in lesions. To identify the poor prognostic factors associated with lymph node metastasis, the present study retrospectively reviewed the clinical data of 65 patients with lymph node metastases that underwent surgical pancreatic cancer resection between 2007 and 2012 at a single institution. The investigation focused on the degree of fibrosis in metastatic lesions in 216 lymph nodes, and investigated associations with prognosis or clinicopathological findings. The ratios of the fibrotic area in metastatic lymph node lesions were evaluated and classified into three categories, high (≥70%), moderate (10-70%) and low (<10%). Desmoplasia was not observed in cancer-free lymph nodes. The size of metastatic lymph node lesions was additionally measured, and a significant association between metastatic lesion size and the degree of desmoplasia was observed (P<0.001). The degree of desmoplasia was additionally associated with local extranodal invasion. In the analysis of 65 pancreatic cancer patients with metastatic lymph nodes, the presence of multiple metastatic lymph nodes with moderate or high desmoplasia was significantly associated with poor survival (high, P=0.0048; moderate/high, P=0.0075). Of several clinicopathological factors, the presence of multiple metastatic lymph nodes with high or moderate desmoplasia was associated with overall survival in univariate (P=0.0098) and multivariate (P=0.0466) analyses. The degree of desmoplasia in metastatic lymph nodes is associated with lesion size, and the presence of multiple metastatic lymph nodes with desmoplasia is an independent poor prognostic factor, suggesting that the desmoplasia may have an important role in the malignant progression of lymph node metastases.
KEYWORDS:
desmoplasia; locally extranodal invasion; lymph node metastasis; pancreatic cancer; prognostic factor.
133. Ohtsuka T, Mori Y, Ishigami K, Fujimoto T, Miyasaka Y, Nakata K, Ohuchida K, Nagai E, Oda Y, Shimizu S, Nakamura M, Clinical significance of circumportal pancreas, a rare congenital anomaly, in pancreatectomy, Am J Surg, 214, 2, 267-272, 2017.04, Abstract
BACKGROUND:
Circumportal pancreas is a rare congenital pancreatic anomaly. The aim of this study was to clarify the clinical characteristics of patients with circumportal pancreases undergoing pancreatectomy.
METHODS:
The medical records of 508 patients who underwent pancreatectomy were retrospectively reviewed. The prevalence of circumportal pancreas and related anatomical variations were assessed. Surgical procedures and postoperative outcomes were compared in patients with and without circumportal pancreas.
RESULTS:
Circumportal pancreas was observed in 9 of the 508 patients (1.7%). In all nine patients, the portal vein was completely encircled by the pancreatic parenchyma above the level of the splenoportal junction, and the main pancreatic duct ran dorsal to the portal vein. The rate of variant hepatic artery did not differ significantly in patients with and without circumportal pancreas. Pancreatic fistula developed more frequently in patients with than without circumportal pancreas (44% vs. 14%, p = 0.03), but other clinical parameters did not differ significantly in these two groups.
CONCLUSIONS:
Despite being rare, circumportal pancreas may increase the risk of postoperative pancreatic fistula in patients undergoing pancreatectomy. However, a prospective, large-cohort study is necessary to determine the real incidence of relevant anatomical variations and the definitive clinical significance of this rare anomaly..
134. Abe T, Ohuchida K, Endo S, Ookubo F, Mori Y, Nakata K, Miyasaka Y, Manabe T, Ohtsuka T, Nagai E, Oda Y, Nakamura M, Clinical importance of intraoperative peritoneal cytology in patients with pancreatic cancer, Surgery, 161, 4, 951-958, 2017.04, BACKGROUND:The clinical importance of intraoperative peritoneal cytology in patients with pancreatic cancer remains incompletely understood.METHODS:Peritoneal washing samples were collected from 411 consecutive patients with pancreatic ductal adenocarcinoma from 1996 to 2014. Of the 411 patients, 335 underwent macroscopically curative resection and 76 with noncurative factors did not undergo resection. We compared long-term outcomes between patients with positive cytology (cytology+) and those with negative cytology (cytology-) and investigated the importance of clinicopathologic factors.RESULTS:Of 335 patients with curative resection, 300 (89.6%) were cytology- and 35 (10.4%) were cytology+. The median overall survival of cytology+ patients was less than that of cytology- patients (16 vs 31 months, respectively; P < .0001). The median overall survival of cytology+ patients with noncurative factors was significantly worse than that of cytology+ pat
ients with curative resection (6.9 vs 16.0 months, respectively; P = .0023). The median disease-free survival of cytology+ patients was less than that of cytology- patients (6.5 vs 16 months, respectively; P < .0001). In the multivariate analysis, cytology+ was an independent prognostic factor for overall survival and disease-free survival.CONCLUSION:Cytology+ without noncurative factors was a predictive factor for a poor prognosis. Therefore, it is important to regard patients with pancreatic cancer characterized by cytology+ as a special group that may warrant more aggressive adjuvant therapy..
135. Abe T, Ohuchida K, Koikawa K, Endo S, Okumura T, Sada M, Horioka K, Zheng B, Moriyama T, Nakata K, Miyasaka Y, Manabe T, Ohtsuka T, Nagai E, Mizumoto K, Hashizume M, Nakamura M, Cancer-associated peritoneal mesothelial cells lead the formation of pancreatic cancer peritoneal dissemination, Int J Oncol, 50, 2, 457-467, 2017.04, The interaction between the cancer cells and the peritoneal mesothelial cells (PMCs) plays an important role in the peritoneal dissemination in several types of cancer. However, the role of PMCs in the peritoneal dissemination of pancreatic cancer remains unclear. In the present study, we investigated the interaction between the pancreatic cancer cells (PCCs) and the PMCs in the formation of peritoneal dissemination in vitro and in vivo. The tumor-stromal interaction of PCCs and PMCs significantly enhanced their mobility and invasiveness and enhanced the proliferation and anoikis resistance of PCCs. In a 3D organotypic culture model of peritoneal dissemination, co-culture of PCCs and PMCs significantly increased the cells invading into the collagen gel layer compared with mono-culture of PCCs. PMCs pre-invaded into the collagen gel, remodeled collagen fibers, and increased parallel fiber orientation along the direction of cell invasion. In the tissues
of peritoneal dissemination of the KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+;Pdx-1-Cre) transgenic mouse, the monolayer of PMCs was preserved in tumor-free areas, whereas PMCs around the invasive front of peritoneal dissemination proliferated and invaded into the muscle layer. In vivo, intraperitoneal injection of PCCs with PMCs significantly promoted peritoneal dissemination compared with PCCs alone. The present data suggest that the cancer-associated PMCs have important promoting roles in the peritoneal dissemination of PCCs. Therapy targeting cancer-associated PMCs may improve the prognosis of patients with pancreatic cancer..
136. Ozono K, Ohishi Y, Onishi H, Nakamura K, Motoshita J, Kato M, Nakanishi R, Nakamura M, Oda Y, Brain-derived neurotrophic factor/tropomyosin-related kinase B signaling pathway contributes to the aggressive behavior of lung squamous cell carcinoma, Lab Invest, 10.1038/labinvest.2017.45 [Indexed for MEDLINE], 97, 11, 1332-1342, 2017.04, Abstract
The tropomyosin-related kinase (Trk) family consists of TrkA, TrkB, and TrkC, which play essential roles in tumor progression and/or suppression in various cancers. Little is known about the biological significance of the Trk family in human lung squamous cell carcinoma (SCC). Here we investigated the clinical significance of the protein expression of Trk family members in samples from 99 SCC patients, and we explored the relationship between invasion/proliferation activities and Trk expression using lung SCC cell lines to clarify the biological significance of the Trk family in lung SCC. Immunohistochemical high expression of TrkB was significantly correlated with vascular invasion (P=0.004), lymph node metastasis (P<0.001), and advanced stage (P=0.0015). The overall survival of the patients with TrkB-high expression was significantly shorter than those with TrkB-low expression (P=0.0110). TrkA/TrkC expressions were not predictors of poor prognosis. An in vitro assay demonstrated that the inhibition of brain-derived neurotrophic factor (BDNF) (a TrkB ligand) and TrkB by K252a (a Trk inhibitor) or siRNA (BDNF-siRNA, TrkB-siRNA) suppressed the invasion, migration, and proliferative activities of lung SCC cells. The administration of recombinant human BDNF (rhBDNF) enhanced the invasion, migration, and proliferation activities, which were abrogated by K252a. TrkB-siRNA transfection increased the protein expression of E-cadherin and decreased vimentin expressions in lung SCC cells. Matrix metalloproteinase-2 (MMP-2)-mediated gelatin degradations were decreased in lung SCC cells transfected with TrkB-siRNA. Thus, TrkB-high expression is an indicator of poor prognosis in lung SCC, probably due to invasion/proliferation activities promoted by the BDNF/TrkB signaling pathway, which could become a therapeutic target for lung SCC.
137. Endo S, Nakata K, Ohuchida K, Takesue S, Nakayama H, Abe T, Koikawa K, Okumura T, Sada M, Horioka K, Zheng B, Mizuuchi Y, Iwamoto C, Murata M, Moriyama T, Miyasaka Y, Ohtsuka T, Mizumoto K, Oda Y, Hashizume M, Nakamura M, Autophagy Is Required for Activation of Pancreatic Stellate Cells, Associated With Pancreatic Cancer Progression and Promotes Growth of Pancreatic Tumors in Mice, Gastroenterology, 10.1053/j.gastro.2017.01.010, 152, 6, 1492-1506, 2017.04, BACKGROUND & AIMS: Pancreatic stellate cells (PSCs) changefrom a quiescent to activated state in the tumor environmentand secrete extracellular matrix (ECM) molecules and cytokinesto increase the aggressiveness of tumors. However, it is notclear how PSCs are activated to produce these factors, orwhether this process can be inhibited. PSCs have morphologicand functional similarities to hepatic stellate cells, whichundergo autophagy to promote fibrosis and tumor growth. Weinvestigated whether autophagy activates PSCs, which promotesdevelopment of the tumor stroma and growth ofpancreatic tumors in mice. METHODS: We used immunofluorescencemicroscopy and immunohistochemistry to analyzepancreatic tumor specimens from 133 patients who underwentpancreatectomy in Japan from 2000 to 2009. PSCs werecultured from pancreatic tumor tissues or tissues of patientswith chronic pancreatitis; these were analyzed by immunofluorescencemicroscopy, immunoblots, quantitative
reversetranscription polymerase chain reaction, and in assays forinvasiveness, proliferation, and lipid droplets. Autophagy wasinhibited in PSCs by administration of chloroquine or transfectionwith small interfering RNAs. Proteins were knockeddown in immortalized PSCs by expression of small hairpinRNAs. Cells were transplanted into pancreatic tails of nudemice, and tumor growth and metastasis were quantified. RESULTS:Based on immunohistochemical analyses, autophagywas significantly associated with tumor T category (P シ .018),histologic grade (P シ .001), lymph node metastases (P < .001),stage (P シ .009), perilymphatic invasion (P シ .001), and perivascularinvasion (P シ .003). Autophagy of PSCs was associatedwith shorter survival times of patients with pancreatic cancer.PSC expression of microtubule-associated protein 1 lightchain 3, a marker of autophagosomes, was associated with pooroutcomes (shorter survival time, disease recurrence) forpati
ents with pancreatic cancer (relative risk of shorter survivaltime, 1.56). Immunoblots showed that PSCs from pancreatictumor samples expressed higher levels of markers of autophagythan PSCs from chronic pancreatitis samples. Inhibitorsof autophagy increased the number of lipid droplets of PSCs,indicating a quiescent state of PSCs, and reduced their productionof ECM molecules and interleukin 6, as well as theirproliferation and invasiveness in culture. PSCs exposedto autophagy inhibitors formed smaller tumors in nude mice(P シ .001) and fewer liver metastases (P シ .018) with lessperitoneal dissemination (P シ .018) compared to PSCs notexposed to autophagy inhibitors. CONCLUSIONS: AutophagicPSCs produce ECM molecules and interleukin 6 and areassociated with shorter survival times and disease recurrencein patients with pancreatic cancer. Inhibitors of PSC autophagymight reduce pancreatic tumor invasiveness by altering thetumor stroma..
138. Endo S, Nakata K, Sagara A, Koikawa K, Ando Y, Kibe S, Takesue S, Nakayama H, Abe T, Okumura T, Moriyama T, Miyasaka Y, Ohuchida K, Ohtsuka T, Mizumoto K, Nakamura M, Autophagy inhibition enhances antiproliferative effect of salinomycin in pancreatic cancer cells, Pancreatology, 10.1016/j.pan.2017.08.009, 17, 6, 990-996, 2017.04, Background: Salinomycin has cytotoxic effects on various types of malignancy and induces autophagy.However, it has not been clarified whether autophagy induced by salinomycin treatment has a protectiveor cytotoxic role. We investigated whether salinomycin affects autophagy in pancreatic cancer cells andwhether autophagy induced by salinomycin treatment has a protective or cytotoxic role in these cells.Methods: We investigated the effect of salinomycin using three pancreatic cancer cell lines. We investigatedeffect on proliferation and the CD133 positive fraction using flow cytometry. In addition, wemonitored the change in autophagic activity after salinomycin treatment using fluorescent immunostaining,western blotting, and flow cytometry. Finally, knockdown of ATG5 or ATG7 by siRNA was usedto investigate the impact of autophagy inhibition on sensitivity to salinomycin.Results: Salinomycin suppressed the proliferation of pancreatic cancer cells in a co
ncentration dependentmanner, and reduced the CD133 positive fraction. Salinomycin enhanced autophagy activity in these cellsin a concentration dependent manner. Autophagy inhibition made pancreatic cancer cells more sensitiveto salinomycin.Conclusions: Our data provide the first evidence indicating that autophagy induced by salinomycin havea protective role in pancreatic cancer cells. A new therapeutic strategy of combining salinomycin,autophagy inhibitors, and anticancer drugs could hold promise for pancreatic cancer treatment..
139. Nakamura M, Nakata K, Matsumoto H, Ohtsuka T, Yoshida K, Tokunaga S, Hino K, Acyl/free carnitine ratio is a risk factor for hepatic steatosis after pancreatoduodenectomy and total pancreatectomy, Pancreatology, 17, 1, 135-138, 2017.04, Abstract
OBJECTIVES:
Hepatic steatosis, one of the most frequent long-term complications of pancreatectomy, influences not only hepatic function but also survival rate. However, its risk factors and pathogenesis have not been established. The purpose of this study was to clarify the risk factors for hepatic steatosis after pancreatectomy.
METHODS:
In this retrospective study of 21 patients who had undergone pancreatectomy (19 cases of pancreatoduodenectomy and 2 cases of total pancreatectomy), serum carnitine concentrations, fractions of carnitine, and hepatic attenuation on computed tomography images were analyzed with the aim of identifying risk factors for hepatic steatosis.
RESULTS:
Thirteen (61.9%) of the 21 patients were diagnosed as having hypocarnitinemia after pancreatectomy. Average hepatic attenuation was as low as 42.2HU (±21.3 SD). A high ratio of acyl/free carnitine was associated with less pronounced hepatic attenuation according to both univariate (P < 0.001) and multivariate (P = 0.020) regression analyses.
CONCLUSIONS:
The serum carnitine concentrations were low after pancreatectomy in some patients. The statistical analyses suggest that a high ratio of acyl/free carnitine is an independent risk factor for hepatic steatosis after pancreatectomy..
140. Nobuhiro Torata, Makoto Kubo, Daisuke Miura, Kenoki Ohuchida, Yusuke Mizuuchi, Yoshinori Fujimura, Eisuke Hayakawa, Masaya Kai, Yoshinao Oda, Kazuhiro Mizumoto, Makoto Hashizume, Masafumi Nakamura, Visualizing Energy Charge in Breast Carcinoma Tissues by MALDI Mass-spectrometry Imaging Profiles of Low-molecular-weight Metabolites, Anticancer Res, doi:10.21873/anticanres.12723, 38, 7, 4267-4272, 2018.04, Abstract
BACKGROUND/AIM:
Metabolomics is widely used for biomarker discovery, but conventional mass-spectrometry extraction procedures lose the spatial localization of metabolites. In this study, we directly analyzed breast carcinoma tissues embedded in frozen tissue microarrays (fTMAs) using MALDI mass-spectrometry imaging (MALDI-MSI).
MATERIALS AND METHODS:
A total of 119 breast tissues (84 carcinoma and 35 normal) were used. MSI data were extracted from each tissue.
RESULTS:
Overall, 185 of 1,915 peaks which were commonly detected in 60% of target areas were subjected to further analysis. One hundred and fifty-two peaks of carcinoma showed significantly higher intensity than normal. Comparing metabolite profiles from carcinoma and normal tissues, energy charge (EC) and the sum of adenosine phosphate compound (AXP) indicated significantly higher intensities in cancerous tissues than normal. But comparisons of EC and AXP among lymph node metastasis, tumor size and tumor subtypes indicated no significant differences.
CONCLUSION:
Breast carcinoma tissues had higher EC and AXP values than normal. MALDI-MSI could be a tool for characterizing breast carcinoma.
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141. Date K, Ohtsuka T, Nakamura S, Mochidome N, Mori Y, Miyasaka Y, Oda Y, Nakamura M, Surveillance of patients with intraductal papillary mucinous neoplasm with and without pancreatectomy with special reference to the incidence of concomitant pancreatic ductal adenocarcinoma, Surgery, 10.1016/j.surg.2017.09.040, 163, 2, 291-299, 2018.04, Abstract
BACKGROUND:
The presence of an intraductal papillary mucinous neoplasm is important in the detection of concomitant pancreatic ductal adenocarcinoma. The aim of this study was to elucidate the incidence and timing of development of concomitant pancreatic ductal adenocarcinoma in patients with and without pancreatectomy for intraductal papillary mucinous neoplasm.

METHODS:
We reviewed retrospectively the surveillance data for 22 patients who underwent pancreatectomy for pancreatic ductal adenocarcinoma concomitant with intraductal papillary mucinous neoplasm (pancreatic ductal adenocarcinoma-resection group), 180 who underwent pancreatectomy for intraductal papillary mucinous neoplasm (intraductal papillary mucinous neoplasm-resection group), and 263 whose intraductal papillary mucinous neoplasms were left untreated (nonresection group). The incidence and timing of the development of a concomitant pancreatic ductal adenocarcinoma during the surveillance of patients with and without partial pancreatectomy for intraductal papillary mucinous neoplasm were investigated using the Kaplan-Meier method.

RESULTS:
During a median surveillance period of 40 months (range 6-262 months), 5 patients in the pancreatic ductal adenocarcinoma-resection group, 6 in the intraductal papillary mucinous neoplasm-resection group, and 8 in the nonresection group developed concomitant pancreatic ductal adenocarcinoma. The estimated 5-year (17%) and 10-year (56%) cumulative incidences of secondary pancreatic ductal adenocarcinoma in the pancreatic ductal adenocarcinoma-resection group were significantly greater than those in the other two groups (P < .01). Conversely, the difference in the estimated cumulative incidence of concomitant pancreatic ductal adenocarcinoma between the intraductal papillary mucinous neoplasm-resection and nonresection groups was not significant (5-year, 5.0% vs 2.2%; 10-year, 5.0% vs 8.7%; P = .87).

CONCLUSION:
Long-term (≥5-year) surveillance in patients with intraductal papillary mucinous neoplasm is necessary and important because of the potential for development of concomitant pancreatic ductal adenocarcinoma. Those with a history of resection of concomitant pancreatic ductal adenocarcinoma at the time of the initial operation are at quite high risk for the development of secondary pancreatic ductal adenocarcinoma..
142. Yamada S, Fujii T, Kawai M, Shimokawa T, Nakamura M, Murakami Y, Satoi S, Eguchi H, Nagakawa Y, Kodera Y, Yamaue H, Splenic vein resection together with the pancreatic parenchyma versus separated resection after isolation of the parenchyma during distal pancreatectomy (COSMOS-DP trial): study protocol for a randomised controlled trial., Trials, 10.1186/s13063-018-2756-7 , 19, 1, 369, 2018.04, Abstract
BACKGROUND:
In distal pancreatectomy (DP), it is customary to ligate and divide the splenic vein after isolating it from the pancreatic parenchyma. This is considered essential to prevent disruption of the stump of the splenic vein and consequent intra-abdominal haemorrhage in the event of pancreatic fistula (PF). However, this procedure can be technically demanding, especially when the vein is firmly embedded in the pancreatic parenchyma. The objective of the COSMOS-DP trial is to confirm the non-inferiority of resection of the splenic vein embedded in the pancreatic parenchyma compared with the conventional technique of isolating the splenic vein before resection during DP using a mechanical stapler.
METHODS:
Patients with diseases of the pancreatic body and tail whose pancreatic parenchyma and splenic vein can be divided concurrently during open or laparoscopic DP are considered eligible for inclusion. This study is designed as a multicentre prospective randomised phase III trial. Eligible patients will be centrally randomised to either Arm A (resection of the splenic vein after isolation from the pancreatic parenchyma) or Arm B (co-resection of the vein together with the pancreas). This study aims to establish the non-inferiority of the safety of Arm B compared with that of Arm A; the primary endpoint is the incidence of PF (ISGPF grade B/C).
DISCUSSION:
The COSMOS-DP trial will establish the safety of this procedure, such that it can be recommended with more confidence. The use of this procedure will likely result in significant reductions in operative time and blood loss during DP.
TRIAL REGISTRATION:
ClinicalTrials.gov, NCT02871804 . Registered on 27 July 2016.
.
143. Kameda C, Watanabe M, Suehara N, Watanabe Y, Nishihara K, Nakano T, Nakamura M, Safety of laparoscopic distal gastrectomy for gastric cancer when performed by trainee surgeons with little experience in performing open gastrectomy, Surg Today, 10.1007/s00595-017-1569-8, 48, 2, 211-216, 2018.04, Purpose: This study aimed to evaluate the surgical outcomes and clinical safety of laparoscopic distal gastrectomy (LDG) when performed by trainee surgeons with little prior experience in performing open gastrectomy, under the guidance of trainer surgeons.Methods: From January 2008 until March 2015, 17 trainee surgeons and 5 trainer surgeons performed LDGs to treat 371 patients with clinical stage T1-T3 gastric cancer. Of these patients, 140 and 231 underwent LDG performed by trainee surgeons and trainer surgeons, respectively. We retrospectively analyzed the surgical outcomes of the two groups.Results: Trainee surgeons required significantly longer operation times than the trainer surgeons, with respective mean operation times of 262 and 223 minutes (p < 0.001). However, the mean blood loss volumes, average numbers of retrieved lymph nodes, postoperative complications, and postoperative hospital stay lengths did not differ significantly between LD
Gs performed by trainee surgeons and trainer surgeons. Conclusions: The study findings suggest that, under the guidance of trainer surgeons, trainee surgeons with little experience with open gastrectomy and even without prior experience with LDG can perform radical surgeries safely..
144. Ohtsuka T, Gotoh Y, Nakashima Y, Okayama Y, Nakamura S, Morita M, Aly MYF, Velasquez VVDM, Mori Y, Sadakari Y, Nakata K, Miyasaka Y, Ishigami K, Fujimori N, Mochidome N, Oda Y, Shimizu S, Nakamura M, Role of SpyGlass-DStm in the preoperative assessment of pancreatic intraductal papillary mucinous neoplasm involving the main pancreatic duct, Pancreatology, 10.1016/j.pan.2018.04.012, 18, 5, 566-571, 2018.04, Abstract
BACKGROUND/OBJECTIVES:
It is often difficult to determine an adequate resection line during pancreatectomy for intraductal papillary mucinous neoplasm involving the main pancreatic duct during partial pancreatectomy. The aim of this study was to evaluate the usefulness of improved peroral pancreatoscopy using SpyGlass-DStm in the preoperative assessment of intraductal papillary mucinous neoplasm involving the main pancreatic duct.
METHODS:
We collected and retrospectively analyzed clinicopathological data from seven consecutive patients who underwent preoperative assessment of intraductal papillary mucinous neoplasm involving the main duct using SpyGlass-DStm.
RESULTS:
Good imaging quality of the intraductal protruding lesion was obtained in all seven patients, and only one adverse event was noted wherein a patient had mild pancreatitis. Six patients underwent pancreatectomy. In one patient, masked-type concomitant pancreatic ductal adenocarcinoma and low-length dysplastic lesion was found near the surgical margin, which was not detected by preoperative imaging modalities including SpyGlass-DStm. The sensitivity of targeting biopsy during SpyGlass-DStm to diagnose high-grade dysplasia was 0%.
CONCLUSIONS:
SpyGlass-DStm can be safely performed in patients with intraductal papillary mucinous neoplasm involving the main duct, and has excellent visualization of the target lesion. However, challenges include poor diagnostic ability of targeting biopsy, and, therefore, intraoperative frozen section is still needed to obtain negative surgical margins
.
145. Abe T, Nakata K, Kibe S, Mori Y, Miyasaka Y, Ohuchida K, Ohtsuka T, Oda Y, Nakamura M, Prognostic Value of Preoperative Nutritional and Immunological Factors in Patients with Pancreatic Ductal Adenocarcinoma, Ann Surg Oncol., 10.1245/s10434-018-6761-6, 25, 13, 3996-4003, 2018.04.
146. Noguchi H, Miyasaka Y, Kaku K, Nakamura U, Okabe Y, Ohtsuka T, Ishigami K, Nakamura M, Preoperative Muscle Volume Predicts Graft Survival after Pancreas Transplantation: A Retrospective Observational Cohort Study, Transplantation Proceedings, 10.1016/j.transproceed.2018.03.018, 50, 5, 1482-1488, 2018.04, Abstract
Background
Several studies have suggested that decreased muscle volume is associated with attenuation of immune function. The recipient’s immune system is responsible for rejection of transplanted organs, which is a major cause of graft loss after transplantation. We aimed to determine whether muscle volume is correlated with graft survival after pancreas transplantation (PT).

Methods
Forty-three patients underwent PT for type 1 diabetes mellitus at our institution from August 2001 to May 2016. The quantity of skeletal muscle was evaluated using the psoas muscle mass index (PMI). The correlation between the PMI and outcome after PT was assessed.

Results
A total of 32 and 11 recipients underwent simultaneous pancreas–kidney transplantation (SPK) and PT alone/pancreas after kidney transplantation, respectively. Patients with a surviving graft showed a significantly lower PMI than those with graft loss (P = 0.0451). We divided the recipients into two groups according to the PMI cutoff values which were established using receiver operating characteristic curves. The cumulative graft survival rate was significantly higher in patients with a low than normal PMI (P = 0.0206). A multivariate Cox regression analysis revealed that a low PMI (P= 0.0075) is an independent predictive factor for better graft survival. A low PMI was not a significant predictive factor for acute rejection, but was an independent predictive factor for graft survival after the first acute rejection (P= 0.0025).

Conclusions

Our data suggest that muscle volume could be a predictor of graft survival after PT.

Key words
pancreas transplantation; sarcopenia; graft rejection; graft survival

Abbreviations
AR, acute rejection; ATG, rabbit antithymocyte globulin; BMI, body mass index; CI, confidence interval; CT, computed tomography; DM, diabetes mellitus; HR, hazard ratio; IL, interleukin; ND, not done; PAK, pancreas-after-kidney transplantation; PMI, psoas muscle mass index; PT, pancreas transplantation; PTA, pancreas transplantation alone; SPK, simultaneous pancreas–kidney transplantation; TNF-α, tumor necrosis factor-alpha; Tregs, regulatory T cells.
147. Koikawa K, Ohuchida K, Takesue S, Ando Y, Kibe S, Nakayama H, Endo S, Abe T, Okumura T, Horioka H, Sada M, Iwamoto C, Moriyama T, Nakata K, Miyasaka Y, Ohuchida R, Manabe T, Ohtsuka T, Nagai E, Mizumoto K, Hashizume M, Nakamura M, Pancreatic stellate cells reorganize matrix components and lead pancreatic cancer invasion via the function of Endo180, Cancer letters, 10.1016/j.canlet.2017.10.010, 1, 412, 143-154, 2018.04, Specific cell populations leading the local invasion of cancer are called “leading cells”. However, the underlying mechanisms are unclear. Here, we identified leading cells in pancreatic cancer and deter- mined how these cells lead and promote cancer cell invasion in the extracellular matrix (ECM). Using three-dimensional matrix remodeling assay, we found that pancreatic stellate cells (PSCs) frequently invaded the collagen matrix with pancreatic cancer cells (PCCs), which invaded behind the invading PSCs. In addition, invading PSCs changed the alignment of collagen fibers, resulting in ECM remodeling and an increase in the parallel fibers along the direction of invading PSCs. Endo180 expression was higher in PSCs than in PCCs, Endo180 knockdown in PSCs attenuated the invasive abilities of PSCs and co- cultured PCCs, and decreased the expression level of phosphorylated myosin light chain 2 (MLC2). In mouse models, Endo180-knockdown PSCs
suppressed tumor growth and changes in collagen fiber orientation in co-transplantation with PCCs. Our findings suggest that PSCs lead the local invasion of PCCs by physically remodeling the ECM, possibly via the function of Endo180, which reconstructs the actin cell skeleton by phosphorylation of MLC2..
148. Kanno A, Masamune A, Hanada K, Maguchi H, Shimizu Y, Ueki T, Hasebe O, Ohtsuka T, Nakamura M, Takenaka M, Kitano M, Kikuyama M, Gabata T, Yoshida K, Sasaki T, Serikawa M, Furukawa T, Yanagisawa A, Shimosegawa T, Multicenter study of early pancreatic cancer in Japan, Pancreatology, 10.1016/j.pan.2017.11.007, 18, 1, 61-67, 2018.04, Abstract
BACKGROUND/OBJECTIVES:
The diagnosis of early-stage pancreatic ductal adenocarcinoma (PDAC) is still challenging. We conducted a multicenter study to clarify the clinical features of early-stage PDAC in Japan.

METHODS:
We collected patients with stage 0 and stage I PDAC according to the sixth edition of the Japanese Classification of Pancreatic Carcinoma. We retrospectively analyzed the clinical profiles including opportunities for medical examination, imaging modalities and findings, methods of cytological diagnosis, and prognosis according to the stages at diagnosis.

RESULTS:
Two hundred cases with Stage 0 and stage I PDAC were reported from 14 institutions, which accounted for approximately 0.7% and 3% of all PDAC cases, respectively. Overall, 20% of the early-stage PDAC cases were symptomatic. Indirect imaging findings such as dilatation of the main pancreatic duct were useful to detect early-stage PDAC. In particular, local fatty changes may be specific to early-stage PDAC. For preoperative pathologic diagnosis, cytology during endoscopic retrograde cholangiopancreatography was more commonly applied than endoscopic ultrasound fine-needle aspiration. Although the overall prognosis was favorable, new PDAC lesions developed in the remnant pancreas in 11.5% cases.

CONCLUSIONS:
This multicenter study revealed several key points concerning the diagnosis and management of early-stage PDAC, including screening of asymptomatic cases, importance of indirect imaging findings, application of cytology during endoscopic retrograde cholangiopancreatography, and the risk of carcinogenesis in the remnant pancreas..
149. Nakata K, Shikata S, Ohtsuka T, Ukai T, Miyasaka Y, Mori Y, Velasquez VVDM, Gotoh Y, Ban D, Nakamura Y, Nagakawa Y, Tanabe M, Sahara Y, Takaori K, Honda G, Misawa T, Kawai M, Yamaue H, Morikawa T, Kuroki T, Mou Y, Lee WJ, Shrikhande SV, Tang CN, Conrad C, Han HS, Chinnusamy P, Asbun HJ, Kooby DA, Wakabayashi G, Takada T, Yamamoto M, Nakamura M, Minimally invasive preservation versus splenectomy during distal pancreatectomy: a systematic review and meta-analysis, J HepatoBiliary Pancreat Sci., 10.1002/jhbp.569 , 25, 11, 476-488, 2018.04, Abstract
BACKGROUND:
Minimally invasive distal pancreatectomy (MIDP) has gained in popularity recently. However, there is no consensus on whether to preserve the spleen or not. In this study, we compared MIDP outcomes between spleen-preserving distal pancreatectomy (SPDP) and distal pancreatectomy with splenectomy (DPS); as well as outcomes between splenic vessel preservation (SVP) and Warshaw's technique (WT).
METHODS:
A systematic search of PubMed (MEDLINE) and Cochrane Library was conducted and the reference lists of review articles were hand-searched.
RESULTS:
Fifteen relevant studies with 769 patients were selected for meta-analyses of DPS and SPDP, while another 15 studies with 841 patients were used for the analysis between SVP and WT. Compared with the DPS group, SPDP patients had significantly lower incidences of infectious complications (P = 0.006) and pancreatic fistula (P = 0.002), shorter operative time (P < 0.001), and less blood loss (P = 0.01). Compared with WT, SVP patients had significantly lower incidences of splenic infarction (P < 0.001) and secondary splenectomy (P = 0.003). Subanalysis for laparoscopic surgery alone had similar results.
CONCLUSIONS:
Based on this study, SPDP has significantly superior outcomes compared to DPS. When a spleen is preserved, SVP has better outcomes over WT for reducing splenic complications.
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150. Inui K, Masamune A, Igarashi Y, Ohara H, Tazuma S, Sugiyama M, Suzuki Y, Miyoshi H, Yamamoto S, Takeyama Y, Nakano E, Takuma K, Sakagami J, Hayashi K, Kogure A, Ito T, Mukai T, Maetani I, Nagahama M, Serikawa M, Ueki T, Furuya K, Isayama H, Moriyama I, Shigeno M, Mizukami K, Nanashima 1, Oana S, Ikehata A, Watanabe N, Hirooka Y, Ogoshi K, Sasaki Y, Iwata Y, Kudo Y, Nakayama A, Nakamura M., Management of Pancreatolithiasis: A Nationwide Survey in Japan, Pancreas, 10.1097/MPA.0000000000001071., 47, 6, 708-714, 2018.04, Abstract
OBJECTIVES:
The aim of this study was to assess prevailing treatment of pancreatolithiasis in Japan.
METHODS:
We surveyed clinical data from 1834 patients (1479 men and 355 women) at 125 hospitals.
RESULTS:
Extracorporeal shock-wave lithotripsy (ESWL) was performed alone in 103 patients (5.6%), ESWL plus an endoscopic procedure in 446 (24.3%), endoscopic treatment alone in 261 (14.2%), and surgery in 167 (9.1%). Other treatments were given to 358 (19.5%), whereas 499 (27.2%) received no treatment. Symptoms were relieved in 85.7% after ESWL, 80.8% after endoscopic treatment alone, and 92.8% after surgery. Early complication rates within 3 months after ESWL, endoscopic treatment alone, and surgery were 8%, 4.5%, and 27.1%, respectively. Late complications after ESWL, endoscopic procedures alone, and surgery were 1.7%, 2.5%, and 8.2%, respectively. Symptom relief but also early and late complications were greater after surgery than after ESWL and endoscopic treatment. Among 417 patients undergoing ESWL, 61 (14.6%) required surgery, as did 32 (16%) of 200 patients treated endoscopically. Surgery was required less frequently following initial operative treatment (11/164 patients [6.7%]). Nonsurgical initial treatments were chosen more frequently.
CONCLUSIONS:
First-line treatment of pancreatolithiasis should be ESWL with or without endoscopy because of minimal invasiveness and fewer complications.
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151. Nagakawa Y, Nakamura Y, Honda G, Gotoh Y, Ohtsuka T, Ban D, Nakata K, Sahara Y, Velasquez VVDM, Takaori K, Misawa T, Kuroki T, Kawai M, Morikawa T, Yamaue H, Tanabe M, Mou Y, Lee WJ, Shrikhande SV, Conrad C, Han HS, Tang CN, Palanivelu C, Kooby DA, Asbun HJ, Wakabayashi G, Tsuchida A, Takada T, Yamamoto M, Nakamura M, Learning curve and surgical factors influencing the surgical outcomes during the initial experience with laparoscopic pancreaticoduodenectomy, J HepatoBiliary Pancreat Sci., 10.1002/jhbp.586 , 25, 11, 498-507, 2018.04, Abstract
BACKGROUND:
Laparoscopic pancreaticoduodenectomy (LPD) requires sufficient laparoscopic training for optimal outcomes. Our aim is to determine the learning curve and investigate the factors influencing surgical outcomes during the learning curve.
METHODS:
We analyzed surgical results of 150 consecutive cases of LPD performed by three hepatopancreatobiliary surgeons during their 50 first cases. Learning curves were constructed by cumulative sum (CUSUM) analysis. Preoperative factors influencing resection time and blood loss were investigated in the introductory and stable periods. RESULTS : The learning curve could be divided into three phases: initial (1-20 cases), plateau (21-30), and stable (31-50). Resection time with lymph node dissection was significantly longer during the introductory period (initial and plateau periods) (P < 0.01) but not the stable phase (P = 0.51). Multivariate analysis revealed that patients with pancreatitis had longer resection times and massive blood loss in both the introductory and stable periods (stable phase). High visceral fat area was also significantly related to massive blood loss in the introductory period (P = 0.04).
CONCLUSIONS:
Hepatopancreatobiliary surgeons need more than 30 cases until LPD becomes stable. Lymph node dissection and patients with high visceral fat area and concomitant pancreatitis should be avoided during the introductory period of the learning curve..
152. Aly MYF, Mori Y, Miyasaka Y, Ohtsuka T, Sadakari Y, Nakata K, Oda Y, Shimizu S, Nakamura M, Laparoscopic surgery for congenital biliary dilatation: a single-institution experience, Surg Today, 10.1007/s00595-017-1545-3, 48, 1, 44-50, 2018.04, Abstract
PURPOSE:
Laparoscopic surgery as a treatment for congenital biliary dilatation is uncommon. We herein present a series of laparoscopic surgeries for congenital biliary dilatation performed in our institution and review our experience with this approach over a long period of time.

METHODS:
Medical records of 36 consecutive patients who underwent laparoscopic surgery for congenital biliary dilatation from 1996 to 2015 were retrospectively reviewed. Data on patient demographics, operative time, blood loss, hospital stay, and complications were evaluated. A comparison between the former period (Group A, 1996-2005) and the latter period (Group B, 2006-2015) was performed.

RESULTS:
The patients comprised 23 females and 13 males with a median age of 34 years. The median operative time, blood loss, and hospital stay was 493 min, 154 g, and 11 days, respectively. Total early and late complications occurred in 7 (19%) and 2 (5%) patients, respectively. A comparison between Groups A and B revealed no significant difference in operative time or complications, but operative blood loss, open conversion, and hospital stay were significantly lower in Group B than in Group A (P < 0.05).

CONCLUSION:
Laparoscopic surgery for congenital biliary dilatation is feasible and provides acceptable results. Further prospective studies of larger numbers of patients are needed..
153. Yamada S, Arase H, Tachibana S, Tomita K, Eriguchi M, Fujisaki K, Okabe Y, Nakamura M, Nakano T, Tsuruya K, Kitazono T, Immobilization-induced severe hypercalcaemia successfully treated with reduced dose of zoledronate in a maintenance haemodialysis patient., Nephrology, 10.1111/nep.13246 , 23, 10, 963-964, 2018.04.
154. Ideno N, Yamaguchi H, Ghosh B, Gupta S, Okumura T, Steffen DJ, Fisher CG, Wood LD, Singhi AD, Nakamura M, Gutkind JS, Maitra A, GNASR201C Induces Pancreatic Cystic Neoplasms in Mice That Express Activated KRAS by Inhibiting YAP1 Signaling, Gastroenterology, 10.1053/j.gastro.2018.08.006 , 155, 5, 1593-1607, 2018.04, Abstract
BACKGROUND & AIMS:
Mutations at hotspots in GNAS, which encodes stimulatory G-protein, α subunits, are detected in approximately 60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. We generated mice with KRAS-induced IPMNs that also express a constitutively active form of GNAS in pancreas and studied tumor development.
METHODS:
We generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras;Gnas mice); pancreatic tissues of these mice express activated KRAS and also express a mutant form of GNAS (GNASR201C) upon doxycycline administration. Mice that were not given doxycycline were used as controls, and survival times were compared by Kaplan-Meier analysis. Pancreata were collected at different time points after doxycycline administration and analyzed by histology. Pancreatic ductal adenocarcinomas (PDACs) were isolated from mice and used to generate cell lines, which were analyzed by reverse transcription polymerase chain reaction, immunoblotting, immunohistochemistry, and colony formation and invasion assays. Full-length and mutant forms of yes-associated protein (YAP) were expressed in PDAC cells. IPMN specimens were obtained from 13 patients with IPMN undergoing surgery and analyzed by immunohistochemistry.
RESULTS:
All Kras;Gnas mice developed pancreatic cystic lesions that resemble human IPMNs; the grade of epithelial dysplasia increased with time. None of the control mice developed cystic lesions. Approximately one third of Kras;Gnas mice developed PDACs at a median of 30 weeks after doxycycline administration, whereas 33% of control mice developed PDACs. Expression of GNASR201C did not accelerate the development of PDACs compared with control mice. However, the neoplasms observed in Kras;Gnas mice were more differentiated, and expressed more genes associated with ductal phenotypes, than in control mice. PDACs isolated from Kras;Gnas mice had activation of the Hippo pathway; in cells from these tumors, phosphorylated YAP1 was sequestered in the cytoplasm, and this was also observed in human IPMNs with GNAS mutations. Sequestration of YAP1 was not observed in PDAC cells from control mice.
CONCLUSIONS:
In mice that express activated KRAS in the pancreas, we found expression of GNASR201C to cause development of more differentiated tumors, with gene expression pattern associated with the ductal phenotype. Expression of mutant GNAS caused phosphorylated YAP1 to be sequestered in the cytoplasm, altering tumor progression.
Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.
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155. Gotoh Y, Ohtsuka T, Nakamura S, Shindo K, Ohuchida K, Miyasaka Y, Mori Y, Mochidome N, Oda Y, Nakamura M, Genetic assessment of recurrent pancreatic high-risk lesions in the remnant pancreas: Metachronous multifocal lesion or local recurrence?, Surgery, 10.1016/j.surg.2018.10.025 , 165, 4, 767-774, 2018.04, Abstract
BACKGROUND: It is difficult to determine whether a second high-risk lesion, including pancreatic ductal adenocarcinoma or high-grade pancreatic intraepithelial neoplasm, is a metachronous multifocal lesion or represents local recurrence after resection of the first high-risk lesion. This study attempts to clarify the characteristics of second high-risk lesions in the remnant pancreas using genetic analyses.
METHODS: Clinicopathologic data were collected from 12 patients who underwent pancreatectomy for a second high-risk lesion in the remnant pancreas. We performed mutational and immunohistochemical analyses of 4 major genes-KRAS, TP53, CDKN2A, and SMAD4-associated with pancreatic ductal adenocarcinoma progression, as well as targeted next-generation sequencing.
RESULTS: Mutations in the four genes in the second high-risk lesion were consistent with the first lesion in four patients but were inconsistent in the remaining eight patients, and thus we considered that the latter eight patients likely had metachronous multifocal high-risk lesions and the other four patients had local recurrence. The estimated cumulative recurrence rate after resection of the second high-risk lesion was greater in the local recurrence group compared with the metachronous multifocal group, and the estimated cumulative disease-specific survival rate was greater in the metachronous multifocal group. Targeted next-generation sequencing demonstrated that the second lesions in the metachronous multifocal high-risk lesion group showed differences in founder mutations compared with the first lesion. In the local recurrence group, the founder mutations in the second lesion were common with those in the first lesion.
CONCLUSION: Genetic assessment might help discriminate metachronous multifocal high-risk lesions from local recurrence.
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156. Ohtsuka T, Mori Y, Fujimoto T, Miyasaka Y, Nakata K, Ohuchida K, Nagai E, Oda Y, Shimizu S, Nakamura M, Feasibility of Prophylactic Pancreatojejunostomy in Possible High-Risk Patients for Prevention of Pancreatic Fistula during Enucleation or Limited Pancreatic Resection, Am Surg, 84, 1, 149-153, 2018.04, Abstract
The aim of this study was to assess the feasibility of prophylactic pancreatojejunostomy after enucleation or limited pancreatic resection regarding the risk of postoperative pancreatic fistula (PF). We retrospectively reviewed the medical records of 32 patients who underwent enucleation or limited pancreatic resection and compared the clinical parameters between patients with (n = 10) and without (n = 22) prophylactic pancreatojejunostomy. Prophylactic pancreatojejunostomy was performed in patients with a possible high risk ofPF. No operation-related mortality occurred. Operation time was significantly longer (P < 0.01) and blood loss significantly greater (P < 0.01) in patients with pancreatojejunostomy. Overall complications were more frequent (P = 0.02) and postoperative hospital stay was significantly longer (P = 0.02) in patients with pancreatojejunostomy. However, other assessed factors including the prevalence of postoperative PF did not differ between groups. In conclusion, prophylactic pancreatojejunostomy is feasible, and its efficacy in preventing PF after enucleation or limited pancreatic resection in high-risk patients will require further study..
157. Mori H, Kubo M, Kai M, Kurata K, Yamada M, Nakamura M, Efficacy and Safety of Bi-weekly Pegfilgrastim for Dose-dense Chemotherapy-induced Neutropenia in Breast Cancer Patients, Anticancer Research, 10.21873/anticanres.12740 , 38, 7, 4381-4386, 2018.04, Abstract
BACKGROUND/AIM:
The dose-dense doxorubicin and cyclophosphamide (ddAC) for patients with HER-2-negative breast cancer is recommended by the National Comprehensive Cancer Network guideline in US. However, there are little data on serum G-CSF concentrations in patients undergoing bi-weekly dose-dense therapy with pegfilgrastim. The objective of this study was to compare the serum G-CSF concentrations in patients receiving pegfilgrastim in bi- or tri-weekly regimens.
PATIENTS AND METHODS:
This study included 26 patients who received ddAC or docetaxel and cyclophosphamide (TC) for primary breast cancer. Serum G-CSF concentrations were measured by ELISA.
RESULTS:
Serum G-CSF concentrations peaked in the second week of ddAC cases and in the ninth week of TC cases. Neutrophils gradually increased until the sixth week in ddAC cases, while they were slightly decreased during the first three weeks in TC cases. Treatments were completed without febrile neutropenia or treatment delays.
CONCLUSION:
Primary prophylactic pegfilgrastim administrations increased serum G-CSF concentrations, helping to maintain the absolute neutrophil counts that are required to undergo chemotherapy. The treatment of ddAC with 3.6 mg pegfilgrastim is completely safe for female Japanese patients.
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158. Tamura S, Isobe T, Ariyama H, Nakano M, Kikushige Y, Takaishi S, Kusaba H, Takenaka K, Ueki T, Nakamura M , Akashi K, Baba E, E‑cadherin regulates proliferation of colorectal cancer stem cells through NANOG
, Oncol Rep., 10.3892/or.2018.6464 , 40, 2, 693-703, 2018.04.
159. Ohtsuka T, Ban D, Nakamura Y, Nagakawa Y, Tanabe M, Gotoh Y, Velasquez VVDM, Nakata K, Sahara Y, Takaori K, Honda G, Misawa T, Kawai M, Yamaue H, Morikawa T, Kuroki T, Mou Y, Lee WJ, Shrikhande SV, Tang CN, Conrad C, Han HS, Palanivelu C, Asbun HJ, Kooby DA, Wakabayashi G, Takada T, Yamamoto M, Nakamura M, Difficulty scoring system in laparoscopic distal pancreatectomy, J HepatoBiliary Pancreat Sci., 10.1002/jhbp.578, 25, 11, 489-497, 2018.04, AbstractBACKGROUND: Several factors affect the level of difficulty of laparoscopic distal pancreatectomy (LDP). The purpose of this study was to develop a difficulty scoring (DS) system to quantify the degree of difficulty in LDP.METHODS: We collected clinical data for 80 patients who underwent LDP. A 10-level difficulty index was developed and subcategorized into a three-level difficulty index; 1-3 as low, 4-6 as intermediate, and 7-10 as high index. The automatic linear modeling (LINEAR) statistical tool was used to identify factors that significantly increase level of difficulty in LDP.RESULTS: The operator's 10-level DS concordance between the 10-level DS by the reviewers, LINEAR index DS, and clinical index DS systems were analyzed, and the weighted Cohen's kappa statistic were at 0.869, 0.729, and 0.648, respectively, showing good to excellent inter-rater agreement. We identified five factors significantly affecting level of difficulty in LDP; type of operation, resection line, proximity of tumor to major vessel, tumor extension to peripancreatic tissue, and left-sided portal hypertension/splenomegaly..
160. Fujimoto H, Saito Y, Ohuchida K, Kawakami E, Fujiki S, Watanabe T, Ono R, Kaneko A, Takagi S, Najima Y, Hijikata A, Cui L, Ueki T, Oda Y, Hori S, Ohara O, Nakamura M, Saito T, Ishikawa F, Deregulated Mucosal Immune Surveillance through Gut-Associated Regulatory T Cells and PD-1+ T Cells in Human Colorectal Cancer, The Journal of Immunology, 10.4049/jimmunol.1701222 , 200, 9, 3291-3303, 2018.04, Abstract
Disturbed balance between immune surveillance and tolerance may lead to poor clinical outcomes in some malignancies. In paired analyses of adenocarcinoma and normal mucosa from 142 patients, we found a significant increase of the CD4/CD8 ratio and accumulation of regulatory T cells (Tregs) within the adenocarcinoma. The increased frequency of Tregs correlated with the local infiltration and extension of the tumor. There was concurrent maturation arrest, upregulation of programmed death-1 expression, and functional impairment in CD8+ T cells (CTLs) isolated from the adenocarcinoma. Adenocarcinoma-associated Tregs directly inhibit the function of normal human CTLs in vitro. With histopathological analysis, Foxp3+ Tregs were preferentially located in stroma. Concurrent transcriptome analysis of epithelial cells, stromal cells, and T cell subsets obtained from carcinomatous and normal intestinal samples from patients revealed a distinct gene expression signature in colorectal adenocarcinoma-associated Tregs, with overexpression of CCR1, CCR8, and TNFRSF9, whereas their ligands CCL4 and TNFSF9 were found upregulated in cancerous epithelium. Overexpression of WNT2 and CADM1, associated with carcinogenesis and metastasis, in cancer-associated stromal cells suggests that both cancer cells and stromal cells play important roles in the development and progression of colorectal cancer through the formation of a tumor microenvironment. The identification of CTL anergy by Tregs and the unique gene expression signature of human Tregs and stromal cells in colorectal cancer patients may facilitate the development of new therapeutics against malignancies.
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161. Nakano M, Kikushige Y, Miyawaki K, Kunisaki Y, Mizuno S, Takenaka K, Tamura S, Okumura Y, Ito M, Ariyama H, Kusaba H, Nakamura M, Maeda T, Baba E, Akashi K, Dedifferentiation process driven by TGF-beta signaling enhances stem cell properties in human colorectal cancer., Oncogene, 10.1038/s41388-018-0480-0 , 38, 6, 780-793, 2018.04, Abstract
Cancer stem cells (CSCs) possess the capacity for self-renewal and the potential to differentiate into non-CSCs. The recent discoveries of dynamic equilibrium between CSCs and non-CSCs revealed the significance of acquiring CSC-like properties in non-CSCs as an important process in progression of cancer. The mechanism underlying acquisition of CSC-like properties has mainly been investigated in the context of epithelial-mesenchymal transition. Here, we demonstrate the dedifferentiation process may be an alternative mechanism in acquisition of CSC-like properties in human colorectal cancer cells. By exploring the single-cell gene expression analysis of organoids developed from CD44+ CSCs, we identified TWIST1 as a key molecule for maintaining the undifferentiated state of cancer cells. Consistent with the finding, we found that TGF-beta signaling pathway, a regulator of TWIST1, was specifically activated in the undifferentiated CD44+ CSCs in human colorectal cancer using microarray-based gene expression analysis and quantitative pathology imaging system. Furthermore, we showed that external stimulation with TGF-beta and the induction of TWIST1 converted CD44- non-CSCs into the undifferentiated CD44+ CSCs, leading to the significant increment of CSCs in xenograft models. This study strongly suggests dedifferentiation driven by TGF-beta signaling enhances stem cell properties in human colorectal cancer.
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162. Matsuda R, Miyasaka Y, Ohishi Y, Yamamoto T, Saeki K, Mochidome N, Abe A, Ozono K, Shindo K, Ohtsuka T, Kikutake C, Nakamura M, Oda Y, Concomitant Intraductal Papillary Mucinous Neoplasm in Pancreatic Ductal Adenocarcinoma Is an Independent Predictive Factor for the Occurrence of New Cancer in the Remnant Pancreas, Ann Surg., 10.1097/SLA.0000000000003060, 2018.04, AbstractOBJECTIVE: To determine the factors predicting the subsequent development of pancreatic ductal adenocarcinoma in remnant pancreas (PDAC-RP) after partial pancreatectomy for PDAC.SUMMARY BACKGROUND DATA: PDAC-RP after partial pancreatectomy for PDAC is currently not so rare because of improved prognosis of PDAC patients due to recent advances in surgical techniques and adjuvant therapy. However, the predictive factors related to PDAC-RP remain unknown.METHODS: We retrospectively reviewed the clinicopathological data of a consecutive series of 379 patients with PDAC treated by partial pancreatectomy between 1992 and 2015; 14 patients (3.69%) had PDAC-RP. Clinicopathological variables were compared between PDAC-RP and non-PDAC-RP.RESULTS: In univariate analysis, concomitant intraductal papillary mucinous neoplasm (IPMN) (P = 0.0005), cancer location (body/tail) (P = 0.0060), and lower T factor in UICC (P = 0.0039) were correlated with PDAC-RP development. Multivariate analysis revealed concomitant IPMN (P = 0.0135) to be an independent predictive factor for PDAC-RP. PDAC concomitant with IPMN had higher cumulative incidence of PDAC-RP (47.5%/10 yrs) than PDAC without IPMN (9.96%/10 yrs) (P = 0.0071). Moreover, the density of pancreatic intraepithelial neoplasia lesions in the background pancreas of cases of PDAC concomitant with IPMN (1.86/cm) was higher than that of cases of PDAC without IPMN (0.91/cm) (P = 0.0007).CONCLUSIONS: Concomitant IPMN in PDAC is an independent predictive factor for the development of new PDAC in remnant pancreas. Cancer susceptibility of remnant pancreas after resection for PDAC concomitant with IPMN is probably due to an increased density of pancreatic intraepithelial neoplasia lesions..
163. Kawamoto M, Umebayashi M, Tanaka H, Koya N, Nakagawa S, Kawabe K, Onishi H, Nakamura M, Morisaki T, Combined Gemcitabine and Metronidazole Is a Promising Therapeutic Strategy for Cancer Stem-like Cholangiocarcinoma, Anticancer Res., 10.21873/anticanres.12516, 38, 5, 2739-2748, 2018.04, Abstract
BACKGROUND/AIM:
Metronidazole (MNZ) is a common antibiotic that exerts disulfiram-like effects when taken together with alcohol. However, the relationship between MNZ and aldehyde dehydrogenase (ALDH) activity remains unclear. This study investigated whether MNZ reduces cancer stemness by suppressing ALDH activity and accordingly reducing the malignancy of cholangiocarcinoma (CCA).

MATERIALS AND METHODS:
We developed gemcitabine (GEM)-resistant TFK-1 cells and originally established CCA cell line from a patient with GEM-resistant CCA. Using these cell lines, we analyzed the impacts of MNZ for cancer stem cell markers, invasiveness, and chemosensitivity.

RESULTS:
MNZ reduced ALDH activity in GEM-resistant CCA cells, leading to decreased invasiveness and enhanced chemosensitivity. MNZ diminished the invasiveness by inducing mesenchymal-epithelial transition and enhancing chemosensitivity by increasing ENT1 (equilibrative nucleoside transporter 1) and reducing RRM1 (ribonucleotide reductase M1).

CONCLUSION:
MNZ reduced cancer stemness in GEM-resistant CCA cells. Combined GEM and MNZ would be a promising therapeutic strategy for cancer stem-like CAA..
164. Nakano K, Yamamoto H, Fujiwara M, Koga Y, Tsuruta S, Ihara E, Oki E, Nakamura M, Ogawa Y, Oda Y, , Clinicopathologic and Molecular Characteristics of Synchronous Colorectal Carcinoma With Mismatch Repair Deficiency, Am J surg Pathol, 10.1097/PAS.0000000000000947, 42, 2, 172-182, 2018.04, Abstract
Synchronous colorectal carcinoma (CRC) is a unique disease associated with a high prevalence (∼35%) of microsatellite instability and occasionally with Lynch syndrome. The clinicopathologic and molecular features of synchronous CRC are poorly understood, particularly in Japanese patients. We examined 118 Japanese patients (236 tumors) with synchronous CRC and 117 Japanese patients (117 tumors) with solitary CRC with immunohistochemical staining for TP53 and mismatch repair (MMR) protein (MLH1, MSH2, PMS2, and MSH6) and mutation analyses of KRAS and BRAF genes. The results revealed no significant differences in clinicopathologic, histologic, and molecular findings between the synchronous and solitary CRC groups. Among the 118 synchronous CRC patients, 15 (12.7%) showed loss of MMR protein(s) expression in at least 1 tumor, whereas 103 (87.3%) showed intact expression of all 4 MMR proteins in both tumors. Of note, all patients with MMR deficiency had excellent prognoses. The 15 patients were further subdivided into 2 groups: the Concordant group, with concordant MMR loss (n=9, 7.6%) and the Discordant group, with discordant MMR loss (n=6, 5.1%). The Concordant patients showed concurrent MLH1/PMS2 loss (n=3), concurrent MSH2/MSH6 loss (n=4) and isolated MSH6 loss (n=2) in both tumors, whereas the Discordant patients showed concurrent MLH1/PMS2 loss (n=2), isolated PMS2 loss (n=2) and isolated MSH6 loss (n=2) in a single tumor. On the basis of the MMR expression pattern and BRAF mutation, the Concordant and Discordant groups were suspected to include Lynch syndrome, Lynch-like syndrome and sporadic MLH1 promoter hypermethylated CRC. In addition, KRAS mutation was present in only 1 tumor in a single patient in each group. In conclusion, the frequency of MMR protein deficiency in synchronous CRC in the Japanese population may be lower compared with the reported data from Western populations. MMR protein loss and KRAS and BRAF mutations in synchronous CRCs were heterogenous even in an individual patient.
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165. Mori H, Kubo M, Kai M, Velasquez VV, Kurata K, Yamada M, Okido M, Kuroki S, Oda Y, Nakamura M, BRCAness Combined With a Family History of Cancer Is Associated With a Poor Prognosis for Breast Cancer Patients With a High Risk of BRCA Mutations, Clin Breast Cancer., 10.1016/j.clbc.2018.05.008, 18, 5, e1217-e1227, 2018.04.
166. Koikawa K, Ohuchida K, Ando Y, Kibe S, Nakayama H, Takesue S, Endo S, Abe T, Okumura T, Iwamoto C, Moriyama T, Nakata K, Miyasaka Y, Ohtsuka T, Nagai E, Mizumoto K, Hashizume M, Nakamura M, Basement membrane destruction by pancreatic stellate cells leads to local invasion in pancreatic ductal adenocarcinoma, Cancer Letters, 10.1016/j.canlet.2018.03.031 , 1, 425, 65-77, 2018.04, Stroma invasion is an important step in pancreatic cancer progression. However, how pancreatic ductal adenocarcinoma (PDAC) with ductal structure invades the surrounding stroma has not been clear. Here, we elucidated the mechanism of stromal invasion of PDAC, using organoids. From resected PDAC specimens, we established human PDAC organoids, which developed ductal and basement membrane (BM) structures. When the organoids were co-cultured with pancreatic stellate cells (PSCs) in a collagen matrix, organoids lost their BM and ductal structures, and invaded collagen matrix more frequently than did mono-cultured organoids. Interestingly, direct contact by PSCs to PDAC organoids was observed before BM destruction. Matrix metalloproteinase (MMP) 2 or membrane type-1 MMP (MT1MMP) knockdown in PSCs significantly attenuated BM destruction by PSCs, and retained the ductal structures in organoids. Our results imply that direct contact by PSCs induces BM destruct
ion and stromal invasion of PDAC via MMP2 which binds to MT1MMP on PSCs..
167. Nagakawa Y, Hosokawa Y, Sahara Y, Takishita C, Hijikata Y, Osakabe H, Nakajima T, Shirota T, Katsumata K, Nakamura M, Tsuchida A, Approaching the superior mesenteric artery from the right side using the proximal-dorsal jejunal vein preisolation method during laparoscopic pancreaticoduodenectomy, Surgical Endoscopy, 10.1007/s00464-018-6118-z , 32, 9, 4044-4051, 2018.04, Abstract
BACKGROUND:
Although the artery-first approach is widely used in open pancreaticoduodenectomy, it is difficult to laparoscopically expose the origin of the inferior pancreaticoduodenal artery (IPDA) from the left side of the superior mesenteric artery (SMA). By contrast, damaging the inferior pancreaticoduodenal veins (IPDVs) is possible when approaching the IPDA from the right side of the SMA. To facilitate the artery-first approach in laparoscopic pancreaticoduodenectomy (LPD), we focused on the proximal-dorsal jejunal vein (PDJV) that branched from the superior mesenteric vein (SMV) dorsal side and drained the IPDVs. This study aimed to clarify the usefulness of the right SMA approach using the PDJV preisolation method.
METHODS:
The PDJV was first isolated, and the IPDVs were divided along the PDJV on the right side of the SMA. Then, the IPDA was divided at the root without first separating the pancreatic head from the portal vein and the SMV. Overall, 21 patients underwent this approach, and the results were retrospectively compared with those of 21 patients who underwent the artery-first approach, which was performed on the left side of the SMA. Anatomical characteristics of the PDJV were evaluated using multidetector computed tomography for the two groups.
RESULTS:
Operative times and resection times were significantly lower for the PDJV preisolation group than for the conventional LPD group (489.3 vs. 541.7 min, respectively; p = 0.002). During anatomical evaluation, 41 patients (97.6%) had a PDJV that drained from the SMV dorsally and was in contact with the anterior aspect of the uncinate process. The PDJV was confirmed as the first jejunal vein in 31 patients (73.8%) and as the second jejunal vein in 10 patients (23.8%).
CONCLUSIONS:
This approach facilitates dissection of the IPDA on the right side of the SMA, thereby reducing operative times.
168. Sadakari Y, Nagai S, Velasquez VV, Nagayoshi K, Fujita H, Ohuchida K, Manabe T, Ohtsuka T, Nakamura M, Application of ultrasonography to high-tie and low-tie vascular ligation of the inferior mesenteric artery in laparoscopic colorectal cancer surgery: technical notes., Surgical Endoscopy, 10.1007/s00464-018-6302-1, 33, 1, 309-314, 2018.04, Abstract
BACKGROUND:
Two ligation techniques can be applied in laparoscopy for left-sided colorectal cancer: (1) high-tie (HT), transection at the level of the inferior mesenteric artery (IMA); and (2) low-tie (LT), transection below the IMA, at the level of superior rectal artery (SRA), preserving the left colic artery (LCA). However, even with preoperative images, it can still be a challenge to identify these structures due to intraoperative individual conditions. In this study, we assess the use intraoperative ultrasonography (IOUS) to aid us in identifying the IMA and its branches to the SRA, LCA, and sigmoid artery.
METHODS:
We performed IOUS in 18 patients diagnosed with left-sided colorectal cancer. Preoperatively, a three-dimensional computed tomography (3D-CT) angiography was obtained in majority of the patients, to visualize the IMA and its branches. Two patients were contraindicated to receive a contrast study, hence, was unable to undergo 3D-CT angiography. The resected specimen was grossly examined for the study. The bifurcation types were identified and compared using different modalities: preoperative 3D-CT, IOUS, and gross examination of the resected specimen.
RESULTS:
The branching of the IMA revealed by IOUS was consistent to the findings preoperatively by the 3D-CT and postoperatively by the resected specimen. The IOUS result of the two patients without preoperative 3D-CT evaluation was also consistent with the post-operative bifurcation type.
CONCLUSIONS:
IOUS is an easy and feasible modality which aids in detecting the branching of the IMA during LT and HT ligation in laparoscopic left-sided colorectal surgery. It can serve as an adjunct modality for 3D-CT angiography and can also be considered a safe alternative option for cases wherein 3D-CT angiography is unavailable
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169. Okumura T, Ohuchida K, Kibe S, Iwamoto C, Ando Y, Takesue S, Nakayama H, Abe T, Endo S, Koikawa K, Sada M, Horioka K, Mochidome N, Arita M, Moriyama T, Nakata K, Miyasaka Y, Ohtsuka T, Mizumoto K, Oda Y, Hashizume M, Nakamura M, Adipose tissue-derived stromal cells are sources of cancer-associated fibroblasts and enhance tumor progression by dense collagen matrix, Int J Cancer., 10.1002/ijc.31775 , 144, 6, 1401-1413, 2018.04, Abstract
Although recent studies revealed that adipose tissue accelerates pancreatic tumor progression with excessive extracellular matrix, key players for desmoplasia in the adipose microenvironment remains unknown. Here, we investigated the roles of adipose tissue-derived stromal cells (ASCs) in desmoplastic lesions and tumor progression by in vitro and in vivo experiments. In a three-dimensional (3-D) organotypic fat invasion model using visceral fat from CAG-EGFP mice, GFP-positive fibroblastic cells infiltrated toward cancer cells. When tumor cells were inoculated into transplanted visceral fat pads in vivo, tumor weights and stromal components were enhanced compared to subcutaneous and orthotopic tumor cells inoculated without fat pads. Expression of αSMA in established human ASCs was lower compared to cancer associated fibroblasts, and the 3-D collagen matrices produced by ASCs cultured in cancer cell-conditioned medium changed from loose to dense structures that affected the motility of cancer cells. Microarray analyses revealed upregulation of S100A4 in ASCs, while S100A4-positive stromal cells were observed at extrapancreatic invasion sites of human pancreatic cancer. The present findings indicate that ASCs are recruited to extrapancreatic invasion sites and produce dense collagen matrices that lead to enhanced tumor progression. Both inhibition of ASCs recruitment and activation could lead to a novel antistromal therapy.
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170. Hiroshi Kurahara, Hiroyuki Shinchi, Takao Ohtsuka, Yoshihiro Miyasaka, Taketo Matsunaga, Hirokazu Noshiro, Tomohiko Adachi, Susumu Eguchi, Naoya Imamura, Atsushi Nanashima, Kazuhiko Sakamoto, Hiroaki Nagano, Masayuki Ohta, Masafumi Inomata, Akira Chikamoto, Hideo Baba, Yusuke Watanabe, Kazuyoshi Nishihara, Masafumi Yasunaga, Koji Okuda, Shoji Natsugoe, Masafumi Nakamura, Significance of neoadjuvant therapy for borderline resectable pancreatic cancer: a multicenter retrospective study, Langenbecks Arch Surg, 10.1007/s00423-019-01754-5, 404, 2, 167-174, 2019.04, Abstract
PURPOSE:
Neoadjuvant therapy (NAT) is increasingly used to improve the prognosis of patients with borderline resectable pancreatic cancer (BRPC) albeit with little evidence of its advantage over upfront surgical resection. We analyzed the prognostic impact of NAT on patients with BRPC in a multicenter retrospective study.
METHODS:
Medical data of 165 consecutive patients who underwent treatment for BRPC between January 2010 and December 2014 were collected from ten institutions. We defined BRPC according to the National Comprehensive Cancer Network guidelines, and subclassified patients according to venous invasion alone (BR-PV) and arterial invasion (BR-A).
RESULTS:
The rates of NAT administration and resection were 35% and 79%, respectively. There were no significant differences in resection rates and prognoses between patients in the BR-PV and BR-A subgroups. NAT did not have a significant impact on prognosis according to intention-to-treat analysis. However, in patients who underwent surgical resection, NAT was independently associated with longer overall survival (OS). The median OS of patients who underwent resection after NAT (53.7 months) was significantly longer than that of patients who underwent upfront (17.8 months) or no resection (14.9 months). The rates of superior mesenteric or portal vein invasion, lymphatic invasion, venous invasion, and lymph node metastasis were significantly lower in patients who underwent resection after NAT than in those who underwent upfront resection despite similar baseline clinical profiles.
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171. Hiroshi Noguchi, Yoichi Kakuta, Masayoshi Okumi, Kazuya Omoto, Yasuhiro Okabe, Hideki Ishida, Masafumi Nakamura, Kazunari Tanabe, Pure versus hand-assisted retroperitoneoscopic live donor nephrectomy: a retrospective cohort study of 1508 transplants from two centers., Surg Endosc., doi: 10.1007/s00464-019-06697-y, 2019.04.
172. Nakamura S, Sadakari Y, Ohtsuka T, Okayama T, Nakashima Y, Gotoh Y, Saeki K, Mori Y, Nakata K, Miyasaka Y, Onishi H, Oda Y, Goggins M, Nakamura M, Pancreatic Juice Exosomal MicroRNAs as Biomarkers for Detection of Pancreatic Ductal Adenocarcinoma, Annals Surgical Oncology, 10.1245/s10434-019-07269-z, 2019.04, Abstract
BACKGROUND:
Pancreatic ductal adenocarcinoma (PDAC) is a lethal neoplasm because of difficulties in early detection. Several studies have recently suggested that exosomes may have potential as novel biomarkers. This study aimed to isolate exosomes from pancreatic juice and to investigate whether exosomal microRNAs (ex-miRs) could be used as biomarkers for PDAC.
METHODS:
Pancreatic juice was collected from patients with PDAC and chronic pancreatitis (CP) by endoscopic retrograde pancreatography. Exosomes were extracted by ultracentrifugation. The presence of exosomes was confirmed by electron microscopy and Western blotting using anti-CD63, -CD81, and -TSG101 antibodies. Relative levels of ex-miR-21 and ex-miR-155 were quantified and compared between PDAC and CP patients.
RESULTS:
A total of 35 pancreatic juice samples (27 PDAC and 8 CP) were collected. Relative levels of both ex-miR-21 and ex-miR-155 were significantly higher in PDAC patients compared with CP patients (p < 0.001 and p = 0.008, respectively). By contrast, no significant difference was apparent in relative levels of miR-21 and miR-155 in whole pancreatic juice from PDAC patients compared with CP patients (p = 0.08 and p = 0.61, respectively). Ex-miR-21 and ex-miR-155 levels discriminated PDAC patients from CP patients with area under the curve values of 0.90 and 0.89, respectively. The accuracies of ex-miR-21 levels, ex-miR-155 levels, and pancreatic juice cytology were 83%, 89%, and 74%, respectively. When combining the results of ex-miR profiling with pancreatic juice cytology, the accuracy was improved to 91%.
CONCLUSIONS:
We successfully extracted exosomes from pancreatic juice. Ex-miRs, including ex-miR-21 and ex-miR-155, in pancreatic juice may be developed as biomarkers for PDAC
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173. Yoshihiro Miyasaka, Takao Ohtsuka, Ryuichiro Kimura, Ryota Matsuda, Yasuhisa Mori, Kohei Nakata, Daisuke Kakihara, Nao Fujimori, Takamasa Ohno, Yoshinao Oda, Masafumi Nakamura, Neoadjuvant Chemotherapy with Gemcitabine Plus Nab-Paclitaxel for Borderline Resectable Pancreatic Cancer Potentially Improves Survival and Facilitates Surgery, Ann Surg Oncol, 26, 5, 1528-1534, 2019.04.
174. Yoshifumi Miura, Hiroshi Nogichi, Yasuhiro Okabe, Kosuke Masutani, Shoji Tokunaga, Masafumi Nakamura, Effects of Telmisartan and Candesartan on the Metabolism of Lipids and Glucose in Kidney Transplantat Patients: A prospective, Randomized Crossover Study, Transplantation Direct, 10.1097/TXD.000000000000861, 5, 2, e423, 2019.04.
175. Kato H, Senoh M, Honda H, Fukuda T, Tagashira Y, Horiuchi H, Chiba H, Suzuki D, Hosokawa N, Kitazono H, Norisue Y, Kume H, Mori N, Morikawa H, Kashiwagura S, Higuchi A, Kato H, Nakamura M, Ishiguro S, Morita S, Ishikawa H, Watanabe T, Kojima K, Yokomaku I, Bando T, Toimoto K, Moriya K, Kasahara K, Kitada S, Ogawa J, Saito H, Tominaga H, Shimizu Y, Masumoto F, Tadera K, Yoshida J, Kikuchi T, Yoshikawa I, Watanabe T, Honda M, Yokote K, Toyokawa T, Miyazato H, Nakama M, Mahe C, Reske K, Olsen MA, Dubberke ER., Clostridioides (Clostridium) difficile infection burden in Japan: A multicenter prospective study., Anaerobe, 10.1016/j.anaerobe.2019.03.007, 2019.04.
176. Nakashima Y, Ohtsuka T, Nakamura S, Mori Y, Nakata K, Miyasaka Y, Ishigami K, Matsuda R, Oda Y, Nakamura M, Clinicopathological characteristics of non-functioning cystic pancreatic neuroendocrine tumors, Pancreatology, 10.1016/j.pan.2018.11.010, 19, 1, 50-56, 2019.04, Abstract
BACKGROUND/OBJECTIVES:
The biological features of cystic pancreatic neuroendocrine tumors (PNETs) remain unclear. The aim of this study was to clarify the clinicopathological characteristics of non-functioning PNETs (NF-PNETs) with a cystic component.
METHODS:
The medical records of 75 patients with NF-PNETs who had undergone resection in our institution were retrospectively reviewed. Clinicopathological factors were compared between PNETs with and without a cystic component. Expression of somatostatin 2 receptor (SSTR-2) was also analyzed.
RESULTS:
Cystic PNETs were diagnosed in 14 patients (19%). The proportion of men was significantly higher for cystic than solid PNETs (79% vs. 44%, P < 0.05) and cystic PNETs were significantly larger than solid PNETs (25 mm vs. 17 mm, P < 0.01). However, there were no significant differences in the prevalence of lymph node metastases (14% vs. 10%, P = 0.64), hepatic metastasis (7% vs. 3%, P = 0.54), or disease-free survival rate (both 86%, P = 0.29) between PNETs with and without a cystic component. SSTR-2 expression was more frequently observed in PNETs with a cystic component than in those without (100% vs. 70%, P < 0.01).
CONCLUSIONS:
Although cystic PNETs were larger upon diagnosis than solid PNETs in this study, prognosis after surgical resection did not differ significantly between these types of PNET. Somatostatin receptor scintigraphy and somatostatin analogues may be more useful for diagnosing and treating cystic PNETs, respectively.
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