Prognostic implications of the coexisting precursor lesion types in invasive gallbladder cancer.

Invasive gallbladder carcinoma (GBC) is preceded by two main types of precursor lesions: intracholecystic papillary-tubular neoplasms (ICPNs) and biliary intraepithelial neoplasias (BilINs). Invasive GBCs with an ICPN component have more favorable prognoses than those without an ICPN component. Some BilINs show a relatively exophytic papillary pattern but do not meet the ICPN criteria; at our institution, we call these papillary neoplasias. To clarify the clinical significance of papillary neoplasia, we herein examined 80 invasive GBCs and classified them into three groups based on the type of preinvasive lesions: those with ICPN (ICPN group, n = 35), those with papillary neoplasia (pap-neoplasia group, n = 13), and those without ICPN/papillary neoplasia (group without ICPN/pap-neoplasia, n = 32). We then compared the prognostic differences and characterized the tumors of each group by determining the immunohistochemical expressions of various biomarkers. The overall survival periods of the ICPN and pap-neoplasia groups were significantly longer than that of the group without ICPN/pap-neoplasia (P < 0.0001, P = 0.0036, respectively). Multivariate analysis revealed that lacking ICPN/papillary neoplasia was independently associated with poor prognosis (P = 0.0007), as were poor differentiation (P = 0.0395), presence of preoperative symptoms (P = 0.0488), and advanced stage (P = 0.0234). Invasive components of the ICPN and pap-neoplasia groups were characterized by higher expressions of p16 and p53 compared with those of the group without ICPN/pap-neoplasia. The prognoses of the invasive GBCs with either papillary neoplasia or ICPN were thus more favorable than those of the invasive GBCs without ICPN/pap-neoplasia. Invasive GBCs with exophytic papillary preinvasive lesions (ICPN and papillary neoplasia) may be biologically different from those without such lesions.

Neuroendocrine tumor of the pancreas with rhabdoid feature.

Imaging of a 53-year-old Japanese man revealed two tumors in the liver and a tumor in the head of the pancreas with a swelling lymph node. A needle biopsy for the liver tumors was performed, revealing a neuroendocrine tumor. Enucleation, lymphadenectomy, and partial hepatectomy were performed. The microscopic examination identified many tumor cells with intracytoplasmic inclusions arranged in a nested, cord, or tubular fashion. The intracytoplasmic inclusions displayed densely eosinophilic globules and displaced the nuclei toward the periphery, which constitutes "rhabdoid" features. The tumor cells were positive for synaptophysin and weakly positive for NCAM, but negative for chromogranin A. Epithelial markers (AE1/AE3 and CAM5.2) accentuated intracytoplasmic globules. Pancreatic neuroendocrine tumors with rhabdoid features are very rare. Generally, rhabdoid features are aggressive and dedifferentiated characteristics of various types of tumor. Pancreatic neuroendocrine tumors containing rhabdoid cells tend to display extrapancreatic spread at the time of presentation, although some of these tumors with rhabdoid features are not always associated with aggressive behavior.

'Microcystic pattern' should be recognised as part of the morphological spectrum of solid-pseudopapillary neoplasm of the pancreas.

AIM: Solid pseudopapillary neoplasm (SPN) is an uncommon pancreatic tumour characterised by solid and pseudopapillary growth patterns. We have observed SPNs can show a microcystic pattern (microcystic SPN), which has been poorly described and may be confused with microcystic neoplasms. We conducted the present study to clarify the clinicopathological and immunohistochemical features of microcystic SPNs. METHODS AND RESULTS: We examined a consecutive series of 44 SPNs and 10 serous cystadenomas (SCAs), and classified them into 13 microcystic SPNs (29.5%) and 31 conventional SPNs (70.5%). Clinicopathological analysis, immunohistochemical staining and mucin histochemistry were performed. Clear cell change, hyalinised stroma and haemorrhage were observed significantly more frequently in the microcystic SPNs compared to the conventional SPNs. Immunohistochemically, the microcystic SPNs showed significantly lower frequencies of CD10 (0%) and CD56 expression (62%) compared to the conventional SPNs (87%; P < 0.001, 90%; P < 0.0085, respectively). There were no significant differences in other clinicopathological and immunohistochemical features between the two groups (i.e. the nuclear expression of ß-catenin, E-cadherin, progesterone receptor (PgR), lack of forkhead box (Fox)L2 and occasional oestrogen receptor (ER), AE1/AE3 expression). Microcystic SCAs lack such a characteristic immunophenotype. The myxoid stroma of microcystic SPNs contained hyaluronan revealed by Alcian blue stain with hyaluronidase digestion. CONCLUSION: We thus conclude that the microcystic pattern should be recognised as a part of the morphological spectrum of SPNs. Our findings may contribute to the correct diagnosis of the pancreatic neoplasms with the microcystic pattern. In addition, we speculate that stromal change caused by an accumulation of hyaluronan may contribute to the microcystic pattern of SPN.

Degree of desmoplasia in metastatic lymph node lesions is associated with lesion size and poor prognosis in pancreatic cancer patients.

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.

Molecular Evidence for Monoclonal Skip Progression in Main Duct Intraductal Papillary Mucinous Neoplasms of the Pancreas.

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.

SLC2A1/GLUT1 expression in mural nodules of intraductal papillary mucinous neoplasm of the pancreas.

In intraductal papillary mucinous neoplasms (IPMNs), the presence of a mural nodule showing a papillary or nodular proliferation of tumor cells in the dilated pancreatic duct is an indication for resection of IPMN. Solute carrier family 2, facilitated glucose transporter member 1, known as glucose transporter type 1 (SLC2A1/GLUT1) mediates cellular glucose uptake in many carcinomas and is correlated with increased 18F-fluorodeoxyglucose (18F-FDG) uptake. We examined SLC2A1/GLUT1 expression in the mural nodules of 180 IPMN specimens to distinguish malignant/benign tumors. A mural nodule was detected in 80 (44.4%) of the IPMNs, and was detected in 18.6% (13/70) of the IPMN-low (dysplasia) specimens, 36.1% (13/36) of the IPMN-int, 93.3% (28/30) of the IPMN-high, and 59.1% (26/44) of the IPMN-inv (with an associated invasive carcinoma) specimens. The sensitivity for detecting mural nodules was 81.7% by endoscopic ultrasonography, 70% by contrast-enhanced computed tomography and 54% by endoscopic retrograde cholangiopancreatography. SLC2A1/GLUT1 expression in the mural nodules was recognized in the basal and basolateral cytomembrane of tumor cells and was expressed in 15.4% (2/13) of the IPMN-low, 15.4% (2/13) of the IPMN-int, 71.4% (20/28) of the IPMN-high and 84.6% (22/26) of the IPMN-inv groups. The SLC2A1/GLUT1 expression was significantly higher in the IPMN-high and IPMN-inv mural nodules than in those of the IPMN-low and IPMN-int groups. Our findings suggest that SLC2A1/GLUT1 is expressed late in the adenoma-carcinoma sequence during carcinogenesis in IPMN, and SLC2A1/GLUT1 act as therapeutic target for malignant IPMN.

Molecular Characteristics of Pancreatic Ductal Adenocarcinomas with High-Grade Pancreatic Intraepithelial Neoplasia (PanIN) Are Different from Those without High-Grade PanIN.

AIMS: 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. METHOD AND RESULTS: We reviewed all of a consecutive series of 100 patients with PDACs and divided them into 2 groups: the PDACs with PanIN-2 or PanIN-3 in the background (the PanIN-high group, n = 60) and the PDACs without PanIN-2 or PanIN-3 in the background (the PanIN-low group, n = 40). We evaluated the p53, p16, and SMAD4 expressions in the invasive ductal carcinoma (IDC) components by immunohistochemical staining. KRAS mutation was also analyzed in 80 tumors. The PanIN-low group showed significantly more frequent "high p53 expression" and "loss of SMAD4 expression" than the PanIN-high group (p = 0.048 and p = 0.019, respectively). Loss of p16 expression was not significantly different between the groups. The rate of KRAS wild type was significantly higher in the PanIN-low group than the PanIN-high group (p = 0.024). CONCLUSIONS: Our results demonstrated that the molecular characteristics in the PDACs with high-grade PanIN were different from those in the PDACs without high-grade PanIN. PDACs without high-grade PanIN may develop via a pathway other than the PanIN-carcinoma sequence.

BRCAness as a Biomarker for Predicting Prognosis and Response to Anthracycline-Based Adjuvant Chemotherapy for Patients with Triple-Negative Breast Cancer.

BACKGROUND: Triple-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. METHODS: The 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. RESULTS: Of 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. CONCLUSIONS: The 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.

Suppression of CD51 in pancreatic stellate cells inhibits tumor growth by reducing stroma and altering tumor-stromal interaction in pancreatic cancer.

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.

Distinction of Invasive Carcinoma Derived From Intraductal Papillary Mucinous Neoplasms From Concomitant Ductal Adenocarcinoma of the Pancreas Using Molecular Biomarkers.

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 distinct 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.

GNAS and KRAS mutational analyses of intraductal papillary neoplasms of the pancreas and bile duct developing in the same individual: A case report.

Intraductal papillary mucinous neoplasm (IPMN) of the pancreas and intraductal papillary neoplasm of the bile duct (IPNB) are considered as counterparts of each other, and it is suggested that these two entities have similar molecular alteration pathways. However, the occurrence of IPMN of the pancreas and IPNB in the same patient is rare. We report a surgical case of a 69-year-old woman who developed invasive IPMN of the pancreas and underwent pancreatectomy, 6 months after hepatic resection of invasive IPNB. Molecular analysis revealed GNAS/KRAS mutation in both invasive IPMN of the pancreas and IPNB. This is believed to be the first case report investigating GNAS/KRAS mutational status in both IPMN of the pancreas and IPNB developing in the same patient, and these two entities may show similar molecular alternations.

Predictors and Diagnostic Strategies for Early-Stage Pancreatic Ductal Adenocarcinoma: A Retrospective Study.

OBJECTIVES: As a strategy to diagnose early-stage pancreatic ductal adenocarcinoma (PDAC) is urgently needed, we aimed to clarify characteristics of early-stage PDAC. METHODS: We retrospectively reviewed medical records of 299 consecutive patients who underwent R0 or R1 surgical resection for PDAC between 1994 and 2013 and compared clinical characteristics between patients with early-stage (stages 0-I by Japanese General Rules for Pancreatic Cancer) and advanced-stage (stages II-IV) disease. Diagnostic processes were also analyzed. RESULTS: Twenty-four patients (8%) had early-stage PDAC (stage 0: 11; stage I: 13). Univariate and multivariate analyses showed that presence or history of intraductal papillary mucinous neoplasm (P < 0.01), history of pancreatitis (P < 0.01), and presence or history of extrapancreatic malignancies (P = 0.01) independently predicted detection of early-stage PDAC. Cytological examination during endoscopic retrograde pancreatography cytology was ∼65% sensitive in preoperative diagnosis of early-stage PDAC, whereas other imaging modalities were only 29% to 38% sensitive; 9 of 24 early-stage PDACs were diagnosed by endoscopic retrograde pancreatography cytology alone. CONCLUSIONS: Endoscopic retrograde pancreatography cytology for patients with intraductal papillary mucinous neoplasm or pancreatitis may help diagnose early-stage PDAC. Surveillance of extrapancreatic malignancies might also provide opportunities to detect early-stage PDAC as a second malignancy.

Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

Anterior gradient 2 (AGR2), a member of the protein disulfide isomerase family, has been implicated in various cancers including pancreatic ductal adenocarcinoma (PDAC) and is known to promote cancer progression. However, the prognostic value of AGR2 expression and the interaction with epithelial-mesenchymal transition (EMT) remain unclear. We investigated the clinical significance of AGR2 and EMT markers in PDAC patients by immunohistochemical analyses. Although AGR2 expression was not observed in normal pancreas, all pancreatic precursor neoplastic lesions were positive for AGR2, even at the earliest stages, including pancreatic intraepithelial neoplasia-1A, AGR2 expression was reduced in 27.7% (54/195 cases) of PDAC patients. AGR2 downregulation correlated with EMT markers (vimentin overexpression and reduced membranous E-cadherin expression), high Union for International Cancer Control stage (P<0.0001), high histological cellular grade (P<0.0001), and adverse outcome (P<0.0001). In vitro, targeted silencing of AGR2 in cancer cells using siRNA reduced cell proliferation, colony formation, cell invasiveness, and migration, but did not alter EMT markers. To confer a more aggressive phenotype and induce EMT in PDAC cells, we co-cultured PDAC cell lines with primary-cultured pancreatic stellate cells (PSCs) and found that AGR2 was downregulated in co-cultured PDAC cells compared with PDAC monocultures. Treatment with transforming growth factor beta-1 (TGF-ß), secreted from PSCs, decreased AGR2 expression, whereas inhibition of TGF-ß signaling using recombinant soluble human TGF-ß receptor type II and TGF-ß-neutralizing antibodies restored AGR2 expression. We conclude that AGR2 downregulation is a useful prognostic marker, induced by EMT, and that secreted TGF-ß from PSCs may partially contribute to AGR2 downregulation in PDAC patients. AGR2 downregulation does not induce EMT or a more aggressive phenotype, but is a secondary effect of these processes in advanced PDAC.

Assessment of clonality of multisegmental main duct intraductal papillary mucinous neoplasms of the pancreas based on GNAS mutation analysis.

BACKGROUND: Main duct intraductal papillary mucinous neoplasms (MD-IPMNs) may occur in 1 or multiple segments of the pancreatic duct. Unlike multifocal branch duct (BD)-IPMNs, the clonality of multisegmental MD-IPMNs remains unclear. GNAS mutations are common and specific for IPMNs, and mutational assessment might be useful to determine the clonality of IPMNs as well as to detect high-risk IPMN with distinct ductal adenocarcinoma (pancreatic ductal adenocarcinoma [PDAC]). Our aim was to clarify clonality using GNAS status in multisegmental MD-IPMNs. METHODS: We retrospectively reviewed the medical records of 70 patients with MD-IPMN. Histologic subtypes and KRAS/GNAS mutations were investigated, and the clonal relationships among multisegmental MD-IPMNs were assessed. Mutational analysis was performed using high-resolution melting analysis and subsequent Sanger/pyrosequencing. RESULTS: Thirteen patients had multiple synchronous and/or metachronous lesions. Seven of these 13 patients had multiple MD-IPMNs; 3 had multiple MD-IPMNs and distinct BD-IPMNs; 1 had multiple MD-IPMNs and a distinct PDAC; 1 had a solitary MD-IPMN, BD-IPMN, and PDAC; and 1 had a solitary MD-IPMN and PDAC. KRAS/GNAS mutations were consistent in 10 of 11 multisegmental MD-IPMNs, whereas MD-IPMNs, BD-IPMNs, and PDACs tended to show different mutational patterns. The frequency of malignant IPMNs was significantly higher in the multisegment cohort; malignant IPMNs constituted 90% (9/10) of the multiple cohort and 56% (32/57) of the solitary cohort (P = .04). Mutant GNAS was more frequently observed in the intestinal subtype (94%) than the others. CONCLUSION: MD-IPMNs can be characterized by monoclonal skip progression. Close attention should be paid to the possible presence of skip areas during or after partial pancreatectomy.

Pancreatic intraepithelial neoplasia in the background of invasive ductal carcinoma of the pancreas as a prognostic factor.

AIMS: Of the recognized precursor lesions of pancreatic adenocarcinoma, pancreatic intraepithelial neoplasia (PanIN) is the most common form. However, little is known about the relationship between the grade of PanIN and prognosis for patients with invasive ductal carcinoma. METHODS AND RESULTS: In 124 patients with invasive ductal carcinoma, we examined the grade and number of PanIN lesions in all slides of resected pancreas. The prevalence rates of PanIN-1A, PanIN-1B, PanIN-2 and PanIN-3 were 86%, 84%, 57% and 30%, respectively. We allocated PanIN-2 and PanIN-3 cases into a PanIN-high group, and cases showing PanIN-1A, PanIN-1B or absence of PanIN into a PanIN-low group. In clinicopathological analysis, PanIN-high status was significantly correlated with the number of PanIN lesions (P < 0.0001). Disease-free and overall survival were statistically better in the PanIN-high group than in the PanIN-low group (P = 0.0005 and P = 0.0003). Univariate and multivariate analyses revealed that tumour size and PanIN-low status were statistically significant factors for a poorer prognosis (P = 0.042 and P = 0.007). CONCLUSIONS: In a pathological examination, it is important to evaluate the grade and number of PanINs in assessing the prognosis of pancreatic cancer.