Histo-molecular characterization of pancreatic cancer with microsatellite instability: intra-tumor heterogeneity, B2M inactivation, and the importance of metastatic sites.

Pancreatic ductal adenocarcinoma (PDAC) with microsatellite instability (MSI)/defective mismatch repair (dMMR) is the only subtype of pancreatic cancer with potential response to immunotherapy. Here, we report the histo-molecular characterization of MSI/dMMR PDAC with immunohistochemistry, MSI-based PCR, and next-generation sequencing. Five paradigmatic cases have been identified. The main results include the first report in pancreatic cancer of MSI/dMMR intra-tumor heterogeneity, the presence of microsatellite-stable metastases from MSI/dMMR primary and recurrent B2M gene inactivation, which may confer resistance to immunotherapy. In addition to the classic PDAC drivers, ARID1A was the most common mutated gene in the cohort. Intra-tumor heterogeneity, B2M inactivation, and metastatic sites should be carefully considered in MSI/dMMR PDAC, which should also be investigated in routine diagnostic practice with specific molecular analysis. The chromatin remodeler ARID1A represents another potential driver gene in this context.

Genomic characterization of hepatoid tumors: context matters.

Hepatoid tumors (HT) are rare neoplasms morphologically resembling hepatocellular carcinoma, which arise in several organs other than the liver. A comprehensive molecular profile of this group of neoplasms is still lacking. Genomic characterization of 19 HTs from different organs (three colon HTs, four esophagogastric HTs, four biliary HTs, six genitourinary HTs, two lung HTs) was performed using a multigene next-generation sequencing panel. NGS unraveled a composite molecular profile of HT. Their genetic alterations were clearly clustered by tumor site: (i) colorectal HT displayed microsatellite instability, high tumor mutational burden, mutations in ARID1A/B genes and NCOA4-RET gene fusion (2/3 cases); (ii) gastric HT had TP53 mutations (2/4); (iii) biliary HT displayed loss of CDKN2A (3/4) and loss of chromosome 18 (2/4); (iv) genital HT showed gain of chromosome 12 (3/6); (v) lung HT had STK11 somatic mutations (2/2). The only commonly mutated gene occurring in HT of different sites was TP53 (8/19 cases: colon 2, esophagogastric 2, biliary 2, genital 1, lungs 1). This study shows that most genetic alterations of HT were clustered by site, indicating that context matters. The novel potential targets for HT precision oncology are also clustered based on the anatomic origin. This study shed light on the biology of these rare cancers and may have important consequences for treatment decisions and clinical trial selection for HT patients.

Regulation of pancreas plasticity and malignant transformation by Akt signaling.

BACKGROUND & AIMS: Extensive evidence suggests that Akt signaling plays an important role in beta-cell mass and function, although its function in the regulation of the different pancreatic fates has not been adequately investigated. The goal of these studies was to assess the role of Akt signaling in the pancreatic differentiation programs. METHODS: For these experiments, we have generated a double reporter mouse model that provides activation of Akt signaling in a cell type-specific manner. This mouse model conditionally overexpresses a constitutively active form of Akt upon Cre-mediated recombination. Activation of Akt signaling in pancreatic progenitors and acinar and beta-cells was achieved by crossing this animal model to specific Cre-lines. RESULTS: We showed that overexpression of a constitutively active Akt in pancreatic and duodenal homeobox 1 (Pdx1) progenitors induced expansion of ductal structures expressing progenitor markers. This expansion resulted in part from increased proliferation of the ductal epithelium. Lineage-tracing experiments in mice with activation of Akt signaling in mature acinar and beta-cells suggested that acinar-to-ductal and beta-cell-to-acinar/ductal transdifferentiation also contributed to the expansion of the ductal compartment. In addition to the changes in cell plasticity, these studies demonstrated that chronic activation of Akt signaling in Pdx1 progenitors induced the development of premalignant lesions and malignant transformation in old mice. CONCLUSIONS: The current work unravels some of the molecular mechanisms of cellular plasticity and reprogramming, and demonstrates for the first time that activation of Akt signaling regulates the fate of differentiated pancreatic cells in vivo.

Post-obstructive cyst formation in pancreas and cystic acinar transformation: Are they same?

Pancreatic "acinar cell cystadenoma" (PACA) is a rare benign pancreatic cystic lesion showing acinar cell differentiation. The neoplastic nature of PACA has been questioned and its exact pathogenesis remains unclear. To investigate that acinar cell differentiation is a non-specific metaplastic phenomenon that can occur in pancreatic ductal system, especially when chronically inflamed and dilated, and doesn't necessarily imply an acinar cell neoplasm, we retrospectively analyzed cases diagnosed as PACA and cases with post-obstructive cystic dilatation of pancreatic ducts for acinar cell differentiation using immunohistochemistry for trypsin. The etiology of obstruction was microscopic periductal pancreatic neuroendocrine tumors (PanNET) and pancreatic ductal adenocarcinomas (PDAC). All cases of post-obstructive cystic dilatation showed multiple varying sized cysts distal to the obstruction with histologic findings virtually identical to PACAs. The cysts in both conditions were lined by a single layer of non-dysplastic flattened to columnar ductal-type epithelium with areas of acinar cell differentiation. Trypsin immunohistochemistry confirmed presence of acinar cell differentiation in all cases of post-obstructive cysts and PACAs. Our results suggest that acinar cell differentiation is common in post-obstructive cyst formation, and changes are identical to PACAs. These findings further support the notion that PACA is a benign non-neoplastic cystic lesion with acinar cell differentiation. The findings also suggest that in cases with histology resembling "PACA" or showing diffuse ductal cystic dilation, careful gross examination should be carried out for a proximal obstructive lesion, which can be subtle and easily be missed on initial examination.

Clinicopathologic Features and Outcome of Young Adults With Stage IV Colorectal Cancer.

PURPOSE: Colorectal cancer has a distinct clinicopathologic presentation in younger patients. The aim of this paper was to evaluate the outcome of younger (age below 50 y) and older patients with stage IV (advanced) colorectal cancer in the modern era of combination chemotherapy. METHODS: Cases of metastatic colorectal cancer reported in Surveillance, Epidemiology, and End Results registry (1973 to 2008) were reviewed. Demographics, tumor characteristics, and overall and cancer-specific survivals in patients below 50 and above 50 years of age were compared by Cox proportional hazard analyses. Joinpoint regression analysis was used to evaluate secular trends in 2-year survival. RESULTS: Younger patients had a greater proportion of negative clinicopathologic features (male sex, African American ethnicity, and signet ring or mucinous histology). In multivariate analysis, older age, male sex, African American ethnicity, right-sided tumors, and signet ring histology were associated with higher mortality risk. Younger patients had improved survival (hazard ratio 0.72; 95% confidence interval: 0.70-0.75) compared with older patients, whereas all patients experienced increased 2-year survival by joinpoint analysis beginning in 1999-2000. CONCLUSIONS: The results confirm decreased mortality from advanced colorectal cancer in the era of modern combination chemotherapy in younger and older patients. Younger age, non-right-sided tumors, and absence of signet ring histology significantly associate with better survival.

Molecular mechanism(s) of actinomycin-D induced sensitization of pancreatic cancer cells to CD95 mediated apoptosis.

The death receptor CD95 transduces apoptotic death signaling in many cell types. However, in pancreatic tumor cells CD95 mediated apoptotic machinery is blocked by unknown protein(s). We and others have recently demonstrated that actinomycin-D (ActD) treatment induces sensitization of pancreatic cancer cells as well as other cell types to CD95 mediated apoptosis. In addition, NF-kappaB/Akt system has been implicated in the processes of CD95 mediated apoptosis, however the precise mechanism by which ActD sensitizes pancreatic tumor cells to CD95 mediated apoptosis is still unknown. In the present study, we demonstrated that HPAC and PANC1 pancreatic cancer cells constitutively express high levels of NF-kappaB and phosphorylated form of Akt. ActD at a dose that is known to sensitize pancreatic cancer cells to CD95 mediated apoptosis abrogated the DNA binding activity of NF-kappaB but did not affect expression of phosphorylated Akt. Co-treatment of pancreatic cancer cells with ActD and agonist anti-CD95 antibodies showed no effect on the abrogation on the DNA binding activity of NF-kappaB, but decreased the expression of phosphorylated Akt. Moreover, treatment with PI3-kinase inhibitor LY294002 did not show any sensitization of pancreatic cancer cells to CD95 mediated apoptosis. Our data suggest that ActD sensitizes pancreatic cancer cells to CD95 mediated apoptosis through the abrogation of DNA binding activity of NF-kappaB, rather than PI3-kinase/Akt system. Over-expression of phosphorylated Akt in pancreatic cancer cells is controlled by effective apoptotic signaling from CD95 receptors, but do not protect tumor cells from CD95 induced apoptosis. Thus, our results indicate that modulation of NF-kappaB activity rather then Akt may provide a useful tool to sensitize pancreatic cancer cells to CD95 mediated apoptosis.

Having pancreatic cancer with tumoral loss of ATM and normal TP53 protein expression is associated with a poorer prognosis.

PURPOSE: To determine how often loss of ataxia-telangiectasia-mutated (ATM) protein expression occurs in primary pancreatic ductal adenocarcinomas and to determine its prognostic significance. EXPERIMENTAL DESIGN: The expression of ATM and TP53 was determined by immunohistochemistry in 397 surgically resected pancreatic ductal adenocarcinomas (Hopkins; Johns Hopkins Medical Institutions, Baltimore, MD), a second set of 159 cases (Emory; Emory University Hospital, Atlanta, GA), and 21 cancers after neoadjuvant chemoradiotherapy. Expression was correlated with the clinicopathologic parameters, including survival. RESULTS: Tumoral ATM loss was observed in one cancer known to have biallelic inactivation of ATM and 50 of the first 396 (12.8%) cases, significantly more often in patients with a family history of pancreatic cancer (12/49; 24.5%) than in those without (38/347; 11.0%; P = 0.019). In the Hopkins series, ATM loss was associated with a significantly decreased overall survival in patients whose cancers had normal TP53 expression (P = 0.019) and was a significant independent predictor of decreased overall survival (P = 0.014). Seventeen (10.7%) of 159 Emory cases had tumoral ATM loss and tumoral ATM loss/normal TP53 was associated with poorer overall survival (P = 0.1). Multivariate analysis of the combined Hopkins/Emory cases found that tumoral ATM loss/normal TP53 was an independent predictor of decreased overall survival [HR = 2.61; confidence interval (CI), 1.27-5.37; P = 0.009]. Of 21 cancers examined after neoadjuvant chemoradiotherapy, one had tumoral loss of ATM; it had no histologic evidence of tumor response. CONCLUSIONS: Tumoral loss of ATM protein was detected more often in patients with a family history of pancreatic cancer than in those without. Patients whose pancreatic cancers had loss of ATM but normal TP53 had worse overall survival after pancreatic resection.

Novel synthetic curcumin analogues EF31 and UBS109 are potent DNA hypomethylating agents in pancreatic cancer.

DNA methylation is a rational therapeutic target in pancreatic cancer. The activity of novel curcumin analogues EF31 and UBS109 as demethylating agents were investigated. MiaPaCa-2 and PANC-1 cells were treated with vehicle, curcumin, EF31 or UBS109. EF31 and UBS109 resulted in significantly higher inhibition of proliferation and cytosine methylation than curcumin. Demethylation was associated with re-expression of silenced p16, SPARC, and E-cadherin. EF31 and UBS109 inhibited HSP-90 and NF-κB leading to downregulation of DNA methyltransferase-1 (DNMT-1) expression. Transfection experiments confirmed this mechanism of action. Similar results were observed in vitro when subcutaneous tumors (MiaPaCa-2) were treated with EF31 and UBS109.

Vacuolated cell pattern of pancreatobiliary adenocarcinoma: a clinicopathological analysis of 24 cases of a poorly recognized distinctive morphologic variant important in the differential diagnosis.

Pancreatic ductal adenocarcinoma (PDCA) is characterized by well-defined tubular units in the vast majority of the cases; however, variations in this theme do occur. It is important to recognize the morphologic spectrum of PDCA to avoid misdiagnosis especially in small specimens and also in metastatic foci. Here, we document a morphologic variant of PDCA that is characterized by a distinctive pattern of infiltrating cribriform nests in a distinctive "microcystic" or "secretory" pattern. Twenty-four cases of PDCA have been identified in a review of 505 cases diagnosed with PDCA. Histologically, this pattern was characterized by infiltrating nests of tumor cells with large vacuoles and "signet-ring" like appearance imparting a cribriform growth pattern. The vacuoles were one to five cells in size, often merging to form multilocular spaces separated by a thin rim of cell membrane. Many of these spaces contained CA19.9 positive granular secretory material. The nuclei were often pushed to the periphery and compressed in a pattern resembling adipocytes, although the nuclei were often densely hyperchromatic and displayed significant atypia. Especially in biopsies from the peripancreatic fat and peritoneum, these neoplastic cells had been misdiagnosed as degenerating adipocytes, and in the lymph nodes, they had been misinterpreted as lipogranulomas. Clinical findings of the patients were similar to that of conventional PDCA, except higher incidence of history of smoking (83% vs. 60%; p=0.034). In conclusion, vacuolated cell adenocarcinoma is a distinct morphologic variant of PDCA, and the presence of this peculiar pattern in a metastatic site, although not specific, should raise the suspicion of a PDCA.

Clinicopathological correlates of pancreatic intraepithelial neoplasia: a comparative analysis of 82 cases with and 152 cases without pancreatic ductal adenocarcinoma.

Pancreatic intraepithelial neoplasia is often associated with pancreatic ductal adenocarcinoma and is presumed to be its precursor. It has been difficult to determine the frequency of these lesions because until recently, there was no consensus regarding the terminology and criteria for their grading. Here we compare the frequency and clinical correlates of pancreatic intraepithelial neoplasia in pancreata involved by ductal adenocarcinoma and in benign ones, using the criteria put forward recently. We evaluated pancreatectomy specimens from 82 patients with ductal adenocarcinoma and 152 patients who underwent pancreatectomy for reasons other than primary malignancy (trauma, pancreatitis, and metastatic tumor to pancreas) for the presence, grade, and number of foci of pancreatic intraepithelial neoplasia. Cases were graded by the highest grade of pancreatic intraepithelial neoplasia focus identified. An average of 5.3 sections of pancreas was available for evaluation (range, 1-28 sections). Overall, the frequency of pancreatic intraepithelial neoplasia lesions in ductal adenocarcinoma patients, including Grade 1A (mucinous duct lesions), was 82%, which was significantly higher than the one in benign pancreata -54%, P <.001. There was a progressive increase from normal pancreata to pancreatitis and to ductal adenocarcinoma in the frequency of overall pancreatic intraepithelial neoplasia lesions (16%, 60%, and 82%, respectively) and Grade 3 pancreatic intraepithelial neoplasia (0%, 4%, and 40%, respectively). In most instances, in any given case of higher-grade pancreatic intraepithelial neoplasia lesion, there were also several foci of lower grade lesions. The frequency of higher-grade pancreatic intraepithelial neoplasia lesions (2 and 3) in pancreata resected for ductal adenocarcinoma was 59%, significantly higher than in those without primary carcinoma (17%). This progressive increase in frequency of pancreatic intraepithelial neoplasia from incidental pancreatectomies (presumed to have a nonpathologic pancreas) to pancreatitis (considered a risk factor for carcinoma) and to ductal adenocarcinoma constitutes an indirect support for the precancerous role attributed to pancreatic intraepithelial neoplasia lesions. The relatively high absolute occurrence of pancreatic intraepithelial neoplasia Grade 1A (mucinous duct lesions) in benign conditions (43%) suggests that this group represents a combination of neoplastic and non-neoplastic lesions.

Epidermal growth factor receptor-related protein: a potential therapeutic agent for colorectal cancer.

BACKGROUND & AIMS: Epidermal growth factor receptor is frequently implicated in epithelial cancers and is, therefore, being considered as a potential target for therapy. Recently, we reported the isolation and characterization of epidermal growth factor receptor-related protein, a negative regulator of epidermal growth factor receptor. To discern whether epidermal growth factor receptor-related protein could be an effective therapeutic agent for colorectal cancer, we generated epidermal growth factor receptor-related protein fusion protein and studied its effect on the growth of colon cancer cells in vivo and in vitro. We also studied whether epidermal growth factor receptor-related protein expression is altered in colorectal cancer. METHODS: A 55-kilodalton epidermal growth factor receptor-related protein fusion protein with V5 and His tags was generated in a drosophila expression system and subsequently purified by a His antibody affinity column. Rabbit polyclonal antibodies against epidermal growth factor receptor-related protein were used to examine the expression of epidermal growth factor receptor-related protein. RESULTS: Epidermal growth factor receptor-related protein expression was found to be high in benign human colonic epithelium but low in adenocarcinoma. Exposure of the colon cancer cell lines HCT-116 and Caco-2 to purified recombinant epidermal growth factor receptor-related protein caused a marked inhibition of proliferation, as well as attenuation of basal and ligand-induced stimulation of epidermal growth factor receptor phosphorylation. Epidermal growth factor receptor-related protein-induced inhibition of proliferation of colon cancer cells was prevented by epidermal growth factor receptor-related protein antibodies. Reduced epidermal growth factor receptor phosphorylation was partly due to sequestration of epidermal growth factor receptor ligands by epidermal growth factor receptor-related protein, resulting in the formation of inactive heterodimers with epidermal growth factor receptor. Intratumoral or subcutaneous (away from the tumor site) injections of purified epidermal growth factor receptor-related protein caused regression of palpable colon cancer xenograft tumors in some severely compromised immunodeficient mice and arrested tumor growth in others. CONCLUSIONS: We propose that epidermal growth factor receptor-related protein inhibits cellular growth by attenuating epidermal growth factor receptor signaling processes and is an effective therapeutic agent for colorectal cancer.