Gross tumor size using the AJCC 8th ed. T staging criteria does not provide prognostic stratification for neoadjuvant treated pancreatic ductal adenocarcinoma

The 8th edition AJCC T stage criteria for pancreatic ductal adenocarcinoma (PDAC) are now size based. These criteria provide better prognostic stratification in patients without neoadjuvant therapy. Our aim was to determine if gross tumor size is prognostically significant using the 8th ed. staging criteria for neoadjuvant treated PDAC. The study included 289 patients who underwent resection for PDAC following neoadjuvant therapy. By AJCC 7th ed., there were 12 (4.2%) ypT0, 32 (11.1%) ypT1, 64 (22.1%) ypT2, and 181 (62.6%) ypT3 patients. By AJCC 8th ed., there were 12 (4.2%) ypT0, 74 (25.6%) ypT1 (6 ypT1a, 1 ypT1b, 67 ypT1c), 161 (55.7%) ypT2, and 42 (14.5%) ypT3 patients. 182 patients had negative lymph nodes and 107 had positive lymph nodes. 77 patients were ypN1 and 30 were ypN2 by 8th ed. criteria. 7th ed. T stage significantly correlated with OS (p = 0.048), while 8th ed. T stage did not correlate with OS (p = 0.13). In ypN0 patients, neither the 7th ed. or 8th ed. T stages significantly correlated with patient OS (p = 0.065 and 0.26, respectively). Higher 7th ed. T stage correlated with lymph node status (p ≤ 0.001) more strongly than 8th ed. T stage (p = 0.04). 7th ed. and 8th ed. N stage correlated with OS (p = 0.004 and p = 0.0002, respectively). By 8th ed. AJCC staging criteria, gross tumor size does not provide good prognostic stratification in neoadjuvant therapy PDAC. Mapped grossing techniques combining gross and microscopic examination to determine tumor size may provide more accurate staging of neoadjuvant treated tumors.     

Pancreatic intraepithelial neoplasia – can we detect early pancreatic cancer?

Haugk B
(2010) Histopathology 57, 503–514
Pancreatic intraepithelial neoplasia – can we detect early pancreatic cancer? Pancreatic cancer is one of the most lethal cancers, with an incidence equalling mortality. Pancreatic cancer is a heterogeneous group in which pancreatic ductal adenocarcinoma (PDAC) is the most common. It is now established that PDAC develops through stepwise progression from precursor lesions. Detection and treatment of these precursor lesions would allow curative treatment. Three precursor lesions for PDAC have been identified. Two of these – mucinous cystic neoplasms (MCNs) and intraductal papillary mucinous neoplasms (IPMNs) – are rare, radiologically detectable, cystic precursor lesions which can be cured if treated at the preinvasive stage. The third and most common precursor lesion has recently been defined as pancreatic intraepithelial neoplasia (PanIN). PanINs are microscopic lesions with no clinical correlate. They display a spectrum of cyto‐architectural changes (PanIN‐1, PanIN‐2 and PanIN‐3) mirrored in an increasing accumulation of molecular genetic changes, with PanIN‐3 sharing many of the alterations with PDAC. Great advances in the understanding of pancreatic carcinogenesis have opened avenues for diagnosis and chemoprevention. However, access to the pancreas is limited, molecular tests are at the early stages and too little is known about the natural history of early PanINs to justify resection. Currently, screening focuses upon high‐risk individuals only.     

The epigenetic regulators Bmi1 and Ring1B are differentially regulated in pancreatitis and pancreatic ductal adenocarcinoma

Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are associated with major changes in cell differentiation. These changes may be at the basis of the increased risk for PDAC among patients with chronic pancreatitis. Polycomb proteins are epigenetic silencers expressed in adult stem cells; up‐regulation of Polycomb proteins has been reported to occur in a variety of solid tumours such as colon and breast cancer. We hypothesized that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development/progression. To test these ideas, we determined the expression of PRC1 complex proteins (Bmi1 and Ring1b) during pancreatic development and in pancreatic tissue from mouse models of disease: acute and chronic pancreatic injury, duct ligation, and in K‐Ras^G12V conditional knock‐in and caerulein‐treated K‐Ras^G12V mice. The study was extended to human pancreatic tissue samples. To obtain mechanistic insights, Bmi1 expression in cells undergoing in vitro exocrine cell metaplasia and the effects of Bmi1 depletion in an acinar cancer cell line were studied. We found that Bmi1 and Ring1B are expressed in pancreatic exocrine precursor cells during early development and in ductal and islet cells—but not acinar cells—in the adult pancreas. Bmi1 expression was induced in acinar cells during acute injury, in acinar–ductal metaplastic lesions, as well as in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B expression was only significantly and persistently up‐regulated in high‐grade PanINs and in PDAC. Bmi1 knockdown in cultured acinar tumour cells led to changes in the expression of various digestive enzymes. Our results suggest that Bmi1 and Ring1B are modulated in pancreatic diseases and could contribute differently to tumour development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Genetic analyses of isolated high‐grade pancreatic intraepithelial neoplasia (HG‐PanIN) reveal paucity of alterations in TP53 and SMAD4

High‐grade pancreatic intraepithelial neoplasia (HG‐PanIN) is the major precursor of pancreatic ductal adenocarcinoma (PDAC) and is an ideal target for early detection. To characterize pure HG‐PanIN, we analysed 23 isolated HG‐PanIN lesions occurring in the absence of PDAC. Whole‐exome sequencing of five of these HG‐PanIN lesions revealed a median of 33 somatic mutations per lesion, with a total of 318 mutated genes. Targeted next‐generation sequencing of 17 HG‐PanIN lesions identified KRAS mutations in 94% of the lesions. CDKN2A alterations occurred in six HG‐PanIN lesions, and RNF43 alterations in five. Mutations in TP53, GNAS, ARID1A, PIK3CA, and TGFBR2 were limited to one or two HG‐PanINs. No non‐synonymous mutations in SMAD4 were detected. Immunohistochemistry for p53 and SMAD4 proteins in 18 HG‐PanINs confirmed the paucity of alterations in these genes, with aberrant p53 labelling noted only in three lesions, two of which were found to be wild type in sequencing analyses. Sixteen adjacent LG‐PanIN lesions from ten patients were also sequenced using targeted sequencing. LG‐PanIN harboured KRAS mutations in 94% of the lesions; mutations in CDKN2A, TP53, and SMAD4 were not identified. These results suggest that inactivation of TP53 and SMAD4 are late genetic alterations, predominantly occurring in invasive PDAC. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Comparative analysis of tumorbiology and CD133 positivity in primary and recurrent pancreatic ductal adenocarcinoma

In over 70% of the cases, patients with curative surgery and adjuvant chemotherapy for pancreatic ductal adenocarcinoma (PDAC) develop recurrent tumors. The cancer stem cell (CSC) hypothesis suggests that CSCs are chemoresistant and enriched in recurrent tumors. This study analyzes tumorbiology, expression of the metastasis-promoting CXCR4 and actinin-4, and of the CSC marker CD133 in primary and recurrent PDAC. Twenty-six patients underwent resection for primary and recurrent PDAC and most developed tumor recurrence within 2 years. In 81% the histologic tumor grade was unchanged. Immunohistochemistry could be performed with 15 pairs of primary and recurrent PDAC. The mean Ki-67 proliferation index increased (P = 0.06). About 30% of tumor cells were positive for CXCR4 and almost all tumor cells expressed actinin-4, but there were neither significant changes in the expression levels in recurrent PDAC, nor specifically enhanced levels in metastases. The prominent CD133 pattern was an apical membrane staining of inflammatorily altered, non-neoplastic ductal structures equally observed in primary and recurrent PDAC. The membrane CD133 positivity was consistently absent in neoplastic PDAC cells. Cytoplasmic CD133 positivity was extremely rare (0.85 and 0.34 cells/cm² in primary and recurrent PDAC, respectively; P = 0.07). Tumor grade is mainly unchanged and the expression of CXCR4, actinin-4 and CD133 are not enhanced in recurrent PDAC. The apical membrane CD133 positivity of normal and inflammatorily altered ductal structures and its lack in tumor cells bring the role of CD133 as a specific CSC marker in PDAC into question.     

Tumor expression of Integrin-linked kinase (ILK) correlates with the expression of the E-cadherin repressor Snail: an immunohistochemical study in ductal pancreatic adenocarcinoma

Integrin-linked kinase (ILK) is a key molecule involved in mediating several biological functions including cell-matrix interactions, angiogenesis, and invasion, as well as playing a role in epithelial to mesenchymal transition (EMT) in cancer cells. In ductal pancreatic adenocarcinoma, increased expression of ILK has been linked to tumor prognosis and correlated with increased chemoresistance to drugs, such as gemcitabine. However, the precise relationship between ILK, Snail, E-cadherin, and N-cadherin expression on the stepwise development of pancreatic cancer is unknown. Hence, the purpose of this work was to investigate levels of expression of ILK, Snail, and the cadherins in pancreatic intraepithelial neoplasia (PanIN), and cancer. Resection specimens of 25 randomly selected patients, who underwent a pyloric preserving pancreatoduodenectomy for ductal pancreatic adenocarcinoma, were utilized for this study. Formalin-fixed paraffin embedded pancreatic tissue was immunostained for ILK, E-cadherin, N-cadherin, and Snail by standard techniques. The extent of staining positivity was scored and the results correlated with clinicopathological parameters. In 23 of 25 cases, ILK expression showed extensive positivity (>50%), while two cases did not demonstrate any ILK staining. PanIN grades 1 (n = 16), 2 (n = 11), and 3 (n = 19) lesions demonstrated only focal positivity (<10%) for ILK. E-cadherin showed a reciprocal staining pattern to ILK in 21 of 25 cases, with only focal expression of the marker in pancreatic adenocarcinoma. Interestingly, 15 of 19 PanIN-3 lesions expressed extensive E-cadherin staining. N-cadherin, however, was moderately expressed in the majority of cases (n = 18). Snail expression (n = 22) correlated with ILK expression in ductal pancreatic adenocarcinoma (ρ = 0.8168, p = 0.02), but only minimal Snail staining activity was detected in PanIN lesions. The increase in expression of the E-cadherin repressor Snail, as well as the related increase in the ILK expression, may point towards an ILK-mediated induction, opening possible avenues for targeted drug therapy.     

Mesenchymal–epithelial transition of pancreatic cancer cells at perineural invasion sites is induced by Schwann cells

Epithelial–mesenchymal transition (EMT) promotes invasion and metastasis of pancreatic ductal adenocarcinoma (PDAC). However, the importance of its reverse process, mesenchymal–epithelial transition (MET), for PDAC remains unclear. We aimed to characterize the histological finding “focal differentiation” in PDAC at perineural invasion sites in the context of MET and to investigate the role of Schwann cells in inducing tumor MET. Tumor differentiation and immunohistochemical expressions of E‐cadherin, SMAD3, and vimentin at perineural invasion sites were examined in 168 PDAC tissues. Four PDAC cell lines were co‐cultured with Schwann cells to investigate cell morphology, motility, or EMT‐related markers using immunocytochemistry and quantitative PCR. Of 168 tumors, 124 (74%) showed focal differentiation with enhanced E‐cadherin membrane expression (P < 0.001) and decreased nuclear accumulation of SMAD3 (P < 0.001). Among 115 PDACs harboring grade 1/2 tumor, tumors with focal differentiation showed worse survival compared to those without focal differentiation (P = 0.019). PDAC cells co‐cultured with Schwann cells demonstrated a sheet‐like appearance, increased E‐cadherin expression, decreased expressions of SMAD3 and vimentin, and reduced cell motility. In conclusion, MET‐like change is induced by Schwann cells, suggesting that Schwann cells contribute to PDAC colonization in pancreatic nerves through activating the MET machinery inside tumor cells in the pancreatic tumor microenvironment.     

Aberrant deleted in liver cancer‐1 expression is associated with tumor metastasis and poor prognosis in urothelial carcinoma

This study explored the potential role of deleted in liver cancer‐1 (DLC‐1) as a prognostic indicator of cancer metastasis and survival in urothelial carcinoma (UC). Tissue microarrays were constructed from paraffin‐embedded specimens from 88 UC patients, and immunohistochemical staining was performed to investigate the association of DLC‐1 with clinicopathologic characteristics and clinical outcome. The DLC‐1 expression showed a significant positive correlation with tumor location (p = 0.041) and a significant negative correlation with advanced histological grade (p = 0.013). In tumors with low DLC‐1 expression, Bcl‐2 positivity was observed in 24.4% of cases. The DLC‐1 expression had significant negative associations with Bcl‐2 expression (p = 0.032) and with highly metastatic UC (p = 0.032). Kaplan–Meier analysis showed that DLC‐1 protein expression was negatively associated with both overall survival (OS) (p = 0.035) and with distant metastasis‐free survival (DMFS) (p = 0.041), but not with disease‐free survival. Multivariate analyses indicated that tumor size was the significant independent predictors of OS (p = 0.048); however, only DLC‐1 expression was a significant independent predictor of DMFS (p = 0.019). In conclusion, reduced DLC‐1 protein expression may be an important factor in tumor progression and a useful prognostic molecular marker in UC.     

Glycogen synthase kinase‐3β ablation limits pancreatitis‐induced acinar‐to‐ductal metaplasia

Acinar‐to‐ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre‐malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase‐3beta (GSK‐3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK‐3β promotes TGF‐α‐induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK‐3β attenuates caerulein‐induced ADM formation and PanIN progression in Kras^G12D transgenic mice. Furthermore, we demonstrate that GSK‐3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas‐driven cell proliferation. Mechanistically, we show that GSK‐3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK‐3β participates in early pancreatitis‐induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

EGFR expression in pancreatic intraepithelial neoplasia and ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignant tumor with poor prognosis. Epidermal growth factor receptor (EGFR) is an important cell adhesion and signaling pathway mediator. The aim of this study was to evaluate the expression of EGFR in both pancreatic intraepithelial neoplasia (PanIN) and PDA and their relationship to clinicopathologic characteristics. Formalin-fixed, paraffin-embedded tissues including 81 cases with pancreatic ductal adenocarcinoma, 27 with normal pancreas, 16 with PanIN-1A, 18 with PanIN-1B, 11 with PanIN-2, and 24 with PanIN-3 were used for construction of tissue microarrays. Imunohistochemistry for EGFR was performed. Normal pancreatic ducts, PanIN-1A, and PanIN-1B did not show EGFR overexpression. EGFR overexpression was observed in 18.2% (2/9) of PanIN-2, 41.7% (10/14) of PanIN-3, and 64.2% (52/81) of PDA, respectively. Significantly higher EGFR overexpression was observed in PDAs than in PanIN lesions (P<0.05). No statistically significant correlation was observed between EGFR overexpression and patient age, sex, tumor location, size, histological grade, vascular invasion, lymph node metastasis and stage at presentation, respectively. In conclusion, EGFR expression increased from PanIN to PDA. EGFR may be involved in early stage in development of PDA.     

Sineoculis homeobox homolog 1 protein overexpression as an independent biomarker for pancreatic ductal adenocarcinoma

Sineoculis homeobox homolog 1 (SIX1) is a member of the SIX gene family. It is highly expressed in cancers derived from tissues that play a fundamental role during embryogenesis. Recent studies suggest that inappropriate expression of SIX1 can both initiate tumorigenesis and promote metastasis. To investigate the clinicopathological significance of SIX1 expression in pancreatic ductal adenocarcinoma (PDAC), and to further identify its role as a potential biomarker and therapeutic target in PDAC, 103 PDAC tissue samples and 45 normal pancreatic tissue samples were immunohistochemically stained for SIX1 protein. The localization of SIX1 protein was detected in Panc-1 cancer cells using immunofluorescence staining. Correlations between SIX1 overexpression and the clinicopathological features of pancreatic cancer were evaluated using Chi-square (χ²) tests, differences in survival curves were analyzed using log-rank tests, and multivariate survival analysis was performed using the Cox proportional hazard regression model. In results, SIX1 protein showed mainly cytoplasmic/perinuclear staining pattern in PDAC with immunohistochemistry. The strongly positive rate of SIX1 protein was 60.2% (62/103) in PDAC, which was significantly higher than normal pancreatic tissue (6.7%, 3/45). SIX1 overexpression was positively correlated with tumor size, TNM stage, lymph node metastasis, and grade of PDAC (P < 0.001). SIX1 high expression levels influenced overall survival rates in G1, G2, stage I–II and stage III–IV groups of PDAC; and high expression levels had significantly lower overall survival rates than SIX1 low expression levels. In conclusion, SIX1 emerged as a significant independent prognostic factor in PDAC. SIX1 overexpression appears to be associated with PDAC, and may be a potential biomarker for early diagnosis and prognostic evaluation of PDAC.     

Loss of membranous E-cadherin expression in pancreatic cancer: Correlation with lymph node metastasis,high grade,and advanced stage

Epithelial cadherin (E-cadherin) is a Ca²⁺-dependent cell-cell adhesion molecule that connects cells via homotypic interactions. Its function is critical in the induction and maintenance of cell polarity and differentiation, and its loss of downregulation is associated with an invasive and poorly differentiated phenotype in colon and other tumours. We have used an avidin-biotin immunoperoxidase technique to localize E-cadherin in microwave-treated, paraffin-embedded sections from 36 patients with pancreatic adenocarcinomas. E-cadherin was expressed by normal ductal and acinar cells with typical membranous staining at the intercellular junctions. Loss of normal surface E-cadherin expression was found in 19/36 (53 per cent) tumours compared to the adjacent normal ductal cells. Abnormal E-cadherin expression was found more frequently in poorly differentiated (grade III) (6/7, 86 per cent) than in well-differentiated tumours (grade I) (4/14, 28 per cent) (P=0·012). Membranous E-cadherin expression was also lost more frequently in primary tumours with lymph node (stage III) (14/23, 61 per cent) and distant metastasis (stage IV) (2/2, 100 per cent) compared with 3/11 (27 per cent) lymph node-negative tumours (stage I) (P=0·043). In conclusions, our data indicate that loss of membranous E-cadherin expression is associated with high grade and advanced stage in pancreatic cancer.     

Cyclin D1, p16INK4A and p27Kip1 in pancreatic adenocarcinoma: assessing prognostic implications through quantitative image analysis

The prognostic significance of cyclin D1, p16^INK4A and p27^Kip1 expression has been documented in several human malignancies; however, their prognostic potential in pancreatic adenocarcinoma is still unclear. This study aimed to assess the correlation of the aforementioned molecules with clinicopathological parameters and prognosis. Sixty patients with pancreatic ductal adenocarcinoma underwent surgical resection at a single institution; immunohistochemical staining of the studied markers was quantified by Ιmage analysis system. Cyclin D1 overexpression was positively associated with grade, neural infiltration and vascular invasion, whereas p27 positively correlated with age. Higher cyclin D1 expression indicated poorer survival (adjusted HR = 9.75, 95%CI: 1.48–64.31, p = 0.018, increment: one unit in H‐score), whereas a marginal trend toward an association between p16 positivity and improved survival was observed (adjusted HR = 0.58, 95%CI: 0.32–1.05, p = 0.072 regarding positive vs negative cases). No significant association with overall survival was noted regarding p27. In conclusion, cyclin D1 overexpression and possibly p16 loss of expression in pancreatic adenocarcinoma seem to be adverse prognostic factors, whereas p27 expression did not seem to possess such prognostic properties. Further validation of the present findings in studies encompassing larger samples seems to be needed.     

Relationship between pancreatic intraepithelial neoplasias,pancreatic ductal adenocarcinomas,and single nucleotide polymorphisms in autopsied elderly patients

We comparatively analyzed serially autopsied, elderly Japanese patients (n = 2205) with pancreatic intraepithelial neoplasias (PanINs) and pancreatic ductal adenocarcinomas (PDACs) on the basis of their pancreatic lesions, clinical information, and single nucleotide polymorphisms (SNPs). The incidence of PanIN‐1, ?2, ?3, and PDACs in these patients was 55%, 12%, 1.4%, and 2.4%, respectively. The occurrence of PanINs was associated with female sex, increasing age, and lower body mass index. We did not identify any common SNPs between PanINs and PDACs. There were no common SNPs associated with PanINs and PDACs between men and women. In previously reported pancreatic cancer‐associated SNPs, rs3790844 (NR5A2) showed a significant correlation with PDAC in our cohort. Six SNPs (rs7016880, rs10096633, rs10503669, rs12678919, rs17482753, rs328) that were correlated with blood lipid levels were associated with the risk for PDACs. Our data suggest that different clinicopathological characteristics and predispositions may affect pancreatic carcinogenesis in elderly Japanese patients.     

The vacuolar-ATPase modulates matrix metalloproteinase isoforms in human pancreatic cancer

The vacuolar-ATPase (v-ATPase) is a proton transporter found on many intracellular organelles and the plasma membrane (PM). The v-ATPase on PMs of cancer cells may contribute to their invasive properties in vitro. Its relevance to human cancer tissues remains unclear. We investigated whether the expression and cellular localization of v-ATPase corresponded to the stage of human pancreatic cancer, and its effect on matrix metalloproteinase (MMP) activation in vitro. The intensity of v-ATPase staining increased significantly across the range of pancreatic histology from normal ducts to pancreatic intraepithelial neoplasms (PanIN), and finally pancreatic ductal adenocarcinoma (PDAC). Low-grade PanIN lesions displayed polarized staining confined to the basal aspect of the cell in the majority (86%) of fields examined. High-grade PanIN lesions and PDAC showed intense and diffuse v-ATPase localization. In pancreatic cancer cells, PM-associated v-ATPase colocalized with cortactin, a component of the leading edge that helps direct MMP release. Blockade of the v-ATPase with concanamycin or short-hairpin RNA targeting the V?E subunit reduced MMP-9 activity; this effect was greatest in cells with prominent PM-associated v-ATPase. In cells with detectable MMP-2 activities, however, treatment with concanamycin markedly increased MMP-2's most activated forms. V-ATPase blockade inhibited functional migration and invasion in those cells with predominantly MMP-9 activity. These results indicate that human PDAC specimens show loss of v-ATPase polarity and increased expression that correlates with increasing invasive potential. Thus, v-ATPase selectively modulates specific MMPs that may be linked to an invasive cancer phenotype.