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Importance: Intraoperative consultation (IOC) on surgical margins during curative intent resection of gastric and gastroesophageal adenocarcinoma presents sampling and interpretive challenges. A false-negative (FN) IOC result can affect clinical care. Many factors may be associated with higher risk for an FN result of IOC on surgical margins., Objective: To assess the prevalence and clinical implications of FN results of IOC on surgical margins during resection of gastric and gastroesophageal adenocarcinoma., Design, Setting, and Participants: This retrospective study assessed the results of IOC on surgical margins to determine the prevalence of FN results and the accuracy and clinical implications of the results for patients undergoing curative intent resection for gastric or gastroesophageal adenocarcinoma. The study examined patients with gastric or gastroesophageal adenocarcinoma who underwent resection with curative intent at a single-institution referral center from January 1, 1992, to December 31, 2015., Interventions: Curative intent gastric and/or esophageal resection., Main Outcomes and Measures: False-negative results of IOC on surgical margins, accuracy of the results, factors associated with decreased accuracy of the results, and clinical implications of FN results., Results: This study included 2002 patients (median age, 65 years; 1343 [67.1%] male; 1638 [81.8%] white) who received 3171 IOCs on surgical margins. Of the 3171 IOCs, the prevalence of FN results was 1.7%, with an accuracy of 98.1%. The prevalence of an FN IOC result was 1.2% for esophageal margins, 2.0% for gastric margins, and 2.5% for duodenal margins (P = .04). The prevalence of an FN IOC result was higher for patients with diffuse or signet ring disease compared with those without (2.6% vs 1.2%, P = .002) and for those not receiving neoadjuvant radiotherapy compared with those receiving neoadjuvant radiotherapy (1.4% vs 0.7%, P < .001). The prevalence of FN results of IOCs performed by nongastrointestinal pathologists was similar to that of IOCs performed by gastrointestinal pathologists (2.3% vs 1.9%, P = .60). The disease-specific survival was 34 months (95% CI, 20.7-47.2 months) for those with an FN result and 26.9 months (95% CI, 18.3-35.4; P = .72) for those with a true-positive result. Half of the patients with FN IOC results received further margin-directed therapy, including subsequent resection or radiotherapy., Conclusions and Relevance: This study found that IOC on surgical margins was accurate at a specialty center. Signet ring or diffuse disease, duodenal margins, and not receiving neoadjuvant radiotherapy were challenging scenarios for IOC on surgical margins. The use of IOC on surgical margins may be optimal when it will affect intraoperative decision making framed by the stage of disease, tumor location, and surgical fitness of the patient.

Purpose: Microsatellite instability (MSI) and/or mismatch repair deficiency (MMR-D) testing has traditionally been performed in patients with colorectal (CRC) and endometrial cancer (EC) to screen for Lynch syndrome (LS)-associated cancer predisposition. The recent success of immunotherapy in high-frequency MSI (MSI-H) and/or MMR-D tumors now supports testing for MSI in all advanced solid tumors. The extent to which LS accounts for MSI-H across heterogeneous tumor types is unknown. Here, we establish the prevalence of LS across solid tumors according to MSI status., Methods: MSI status was determined using targeted next-generation sequencing, with tumors classified as MSI-H, MSI-indeterminate, or microsatellite-stable. Matched germline DNA was analyzed for mutations in LS-associated mismatch repair genes ( MLH1, MSH2, MSH6, PMS2, EPCAM). In patients with LS with MSI-H/I tumors, immunohistochemical staining for MMR-D was assessed., Results: Among 15,045 unique patients (more than 50 cancer types), LS was identified in 16.3% (53 of 326), 1.9% (13 of 699), and 0.3% (37 of 14,020) of patients with MSI-H, MSI-indeterminate, and microsatellite-stable tumors, respectively ( P < .001). Among patients with LS with MSI-H/I tumors, 50% (33 of 66) had tumors other than CRC/EC, including urothelial, prostate, pancreas, adrenocortical, small bowel, sarcoma, mesothelioma, melanoma, gastric, and germ cell tumors. In these patients with non-CRC/EC tumors, 45% (15 of 33) did not meet LS genetic testing criteria on the basis of personal/family history. Immunohistochemical staining of LS-positive MSI-H/I tumors demonstrated MMR-D in 98.2% (56 of 57) of available cases., Conclusion: MSI-H/MMR-D is predictive of LS across a much broader tumor spectrum than currently appreciated. Given implications for cancer surveillance and prevention measures in affected families, these data support germline genetic assessment for LS for patients with an MSI-H/MMR-D tumor, regardless of cancer type or family cancer history.

Mutations of Hepatocyte-Nuclear-Factor-1-Homeobox-A (HNF1A) gene and loss of Liver-Fatty-Acid-Binding-Protein (LFABP) are well documented in hepatocellular adenoma. However, the role of HNF1A mutations in hepatocellular carcinoma remains to be determined. In this study, all hepatocellular neoplasms evaluated by our institutional Memorial Sloan Kettering-Integrated Mutational Profiling of Actionable Clinical Targets assay or the Cancer Genome Atlas sequencing, and cases reported in the literature, were queried for HNF1A mutations. Together, 11 of 672 (1.6%) hepatocellular carcinomas harbored HNF1A mutations. The single case from our institution (n = 153) was extremely well differentiated, arising in a background of adenomatosis. Both the adenoma and carcinoma component contained the same 2 somatic HNF1A mutations (p. E32* and L214Q), with loss of LFABP. From the literature, 2 of 146 (1.4%) hepatocellular carcinomas had HNF1A mutations, and both arose in a background of adenomatosis. Information on pre-existing adenoma for the remaining cases (8/373, from The Cancer Genome Atlas) was not available. HNF1A mutations in carcinomas were associated with negative viral hepatitis status (p = .004), mutually exclusive with catenin beta-1 (CTNNB1) hotspot mutations, and trended to occur more in females (p = .06) and without cirrhosis (p = .03). Grade was not associated with HNF1A status (p = .28). Somatic HNF1A mutations occur in approximately 1% to 2% of hepatocellular carcinoma, often in a background of adenomatosis. Our findings suggest that malignant transformation of HNF1A-mutated hepatocellular adenoma occurs, albeit infrequently. Hepatocellular adenomas with HNF1A mutation or adenomatosis with loss of LFABP warrant thorough sampling and examination., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Blockade of the interaction between PD-1 and its ligands PD-L1 has shown clinical efficacy across several tumor types, especially in mismatch-repair-deficient colorectal carcinoma. The aim of this study was to examine the pattern and cellular localization of PD-L1 expression in the different molecular subtypes of mismatch-repair-deficient colorectal cancers vs. their mismatch-repair-proficient counterparts. PD-L1/SATB2 double-antibody-immunohistochemistry was utilized to distinguish tumor cell from immune cell staining. We observed in our series of 129 colorectal adenocarcinomas that PD-L1 expression occurred primarily in tumor-associated-immune cells and most prominently at the tumor-stroma-interface of the invasive front. The level of invasive front immune cell staining was significantly higher in mismatch-repair-deficient tumors compared to mismatch-repair-proficient tumors (p < 0.001), but no difference was observed among the different subtypes of mismatch-repair-deficient tumors: Lynch syndrome-associated vs. MLH1-methylated vs. unexplained. While selected mismatch-repair-proficient tumors exhibited unusually high tumor-infiltrating-lymphocytes and had high level immune cell PD-L1 expression, a positive correlation between PD-L1 expression and high lymphocyte count was detected only in mismatch-repair-deficient tumors (r = 0.39, p < 0.001) and not in mismatch-repair-proficient tumors. Notably, true tumor cell PD-L1 expression in colorectal carcinoma was rare, present in only 3 of 129 tumors (2.3%): 2 MLH1-methylated and 1 mismatch-repair-proficient with high tumor-infiltrating-lymphocytes; and the staining in the tumor cells in all 3 was diffuse (>=50% of the tumor). These findings may serve to inform further efforts aiming to evaluate PD-L1 immunohistochemistry vis-à-vis molecular sub-classification as predictive biomarkers in the treatment of colorectal carcinoma.

The classification of neuroendocrine neoplasms (NENs) differs between organ systems and currently causes considerable confusion. A uniform classification framework for NENs at any anatomical location may reduce inconsistencies and contradictions among the various systems currently in use. The classification suggested here is intended to allow pathologists and clinicians to manage their patients with NENs consistently, while acknowledging organ-specific differences in classification criteria, tumor biology, and prognostic factors. The classification suggested is based on a consensus conference held at the International Agency for Research on Cancer (IARC) in November 2017 and subsequent discussion with additional experts. The key feature of the new classification is a distinction between differentiated neuroendocrine tumors (NETs), also designated carcinoid tumors in some systems, and poorly differentiated NECs, as they both share common expression of neuroendocrine markers. This dichotomous morphological subdivision into NETs and NECs is supported by genetic evidence at specific anatomic sites as well as clinical, epidemiologic, histologic, and prognostic differences. In many organ systems, NETs are graded as G1, G2, or G3 based on mitotic count and/or Ki-67 labeling index, and/or the presence of necrosis; NECs are considered high grade by definition. We believe this conceptual approach can form the basis for the next generation of NEN classifications and will allow more consistent taxonomy to understand how neoplasms from different organ systems inter-relate clinically and genetically.

The commonly mutated genes in pancreatic neuroendocrine tumors (PanNETs) are ATRX, DAXX, and MEN1. We genotyped 64 PanNETs and found 58% carry ATRX, DAXX, and MEN1 mutations (A-D-M mutant PanNETs) and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing and DNA-methylation analysis to reveal two distinct subgroups with one consisting entirely of A-D-M mutant PanNETs. Two genes differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature related to that of alpha-cells (FDR q-value < 0.009) of pancreatic islets including increased expression of HNF1A and its transcriptional target genes. This gene expression profile suggests that A-D-M mutant PanNETs originate from or transdifferentiate into a distinct cell type similar to alpha cells.

Background: Identification of pathogenic germline alterations (PGAs) has important clinical and therapeutic implications in pancreas cancer. We performed comprehensive germline testing (GT) in an unselected prospective cohort of patients with exocrine pancreatic neoplasms with genotype and phenotype association to facilitate identification of prognostic and/or predictive biomarkers and examine potential therapeutic implications., Methods: Six hundred fifteen unselected patients with exocrine pancreatic neoplasms were prospectively consented for somatic tumor and matched sample profiling for 410-468 genes. GT for PGAs in 76 genes associated with cancer susceptibility was performed in an "identified" manner in 356 (57.9%) patients and in an "anonymized" manner in 259 (42.1%) patients, using an institutional review board-approved protocol. Detailed clinical and pathological features, response to platinum, and overall survival (OS) were collected for the identified cohort. OS was analyzed with Kaplan-Meier curves., Results: PGAs were present in 122 (19.8%) of 615 patients involving 24 different genes, including BRCA1/2, ATM, PALB2, and multiple additional genes associated with the DNA damage response pathway. Of 122 patients with germline alterations, 41.8% did not meet current guidelines for GT. The difference in median OS was not statistically significant between patients with and without PGA (50.8 months, 95% confidence interval = 34.5 to not reached, two-sided P = .94). Loss of heterozygosity was found in 60.0% of BRCA1/2., Conclusions: PGAs frequently occur in pancreas exocrine neoplasms and involve multiple genes beyond those previously associated with hereditary pancreatic cancer. These PGAs are therapeutically actionable in about 5% to 10% of patients. These data support routinely offering GT in all pancreatic ductal adenocarcimona patients with a broad panel of known hereditary cancer predisposition genes.

Intrahepatic cholangiocarcinomas are histologically heterogenous. Using a cohort of 184 clinically defined, resected intrahepatic cholangiocarcinomas, we retrospectively classified the histology into 4 subtypes: large duct (LD), small duct (SD) (predominantly tubular [SD1] or predominantly anastomosing/cholangiolar, [SD2]), or indeterminate. Then, we tested the 4 subtypes for associations with risk factors, patient outcomes, histology, and immunophenotypic characteristics. SD was the most common (84%; 24% SD1 and 60% SD2) with lower proportions of LD (8%), and indeterminate (8%). Primary sclerosing cholangitis was rare (2%), but correlated with LD (P=0.005). Chronic hepatitis, frequent alcohol use, smoking, and steatosis had no histologic association. LD was associated with mucin production (P<0.001), perineural invasion (P=0.002), CA19-9 staining (P<0.001), CK7, CK19, CD56 immunophenotype (P=0.005), and negative albumin RNA in situ hybridization (P<0.001). SD was histologically nodular (P=0.019), sclerotic (P<0.001), hepatoid (P=0.042), and infiltrative at the interface with hepatocytes (P<0.001). Albumin was positive in 71% of SD and 18% of LD (P=0.0021). Most albumin positive tumors (85%) lacked extracellular mucin (P<0.001). S100P expression did not associate with subtype (P>0.05). There was no difference in disease-specific or recurrence-free survival among the subtypes. Periductal infiltration and American Joint Committee on Cancer eighth edition pT stage predicted survival by multivariable analysis accounting for gross configuration, pT stage, and histologic type. pT2 had worse outcome relative to other pT stages. Significant differences in histology and albumin expression distinguish LD from SD, but there is insufficient evidence to support further subclassification of SD.

Most adult carcinomas develop from noninvasive precursor lesions, a progression that is supported by genetic analysis. However, the evolutionary and genetic relationships among co-existing lesions are unclear. Here we analysed the somatic variants of pancreatic cancers and precursor lesions sampled from distinct regions of the same pancreas. After inferring evolutionary relationships, we found that the ancestral cell had initiated and clonally expanded to form one or more lesions, and that subsequent driver gene mutations eventually led to invasive pancreatic cancer. We estimate that this multi-step progression generally spans many years. These new data reframe the step-wise progression model of pancreatic cancer by illustrating that independent, high-grade pancreatic precursor lesions observed in a single pancreas often represent a single neoplasm that has colonized the ductal system, accumulating spatial and genetic divergence over time.

Purpose: Various genetic driver aberrations have been identified among distinct anatomic and clinical subtypes of intrahepatic and extrahepatic cholangiocarcinoma, and these molecular alterations may be prognostic biomarkers and/or predictive of drug response. Experimental Design: Tumor samples from patients with cholangiocarcinoma who consented prospectively were analyzed using the MSK-IMPACT platform, a targeted next-generation sequencing assay that analyzes all exons and selected introns of 410 cancer-associated genes. Fisher exact tests were performed to identify associations between clinical characteristics and genetic alterations. Results: A total of 195 patients were studied: 78% intrahepatic and 22% extrahepatic cholangiocarcinoma. The most commonly altered genes in intrahepatic cholangiocarcinoma were IDH1 (30%), ARID1A (23%), BAP1 (20%), TP53 (20%), and FGFR2 gene fusions (14%). A tendency toward mutual exclusivity was seen between multiple genes in intrahepatic cholangiocarcinoma including TP53:IDH1, IDH1:KRAS, TP53:BAP1 , and IDH1:FGFR2 Alterations in CDKN2A/B and ERBB2 were associated with reduced survival and time to progression on chemotherapy in patients with locally advanced or metastatic disease. Genetic alterations with potential therapeutic implications were identified in 47% of patients, leading to biomarker-directed therapy or clinical trial enrollment in 16% of patients. Conclusions: Cholangiocarcinoma is a genetically diverse cancer. Alterations in CDKN2A/B and ERBB2 are associated with negative prognostic implications in patients with advanced disease. Somatic alterations with therapeutic implications were identified in almost half of patients. These prospective data provide a contemporary benchmark for guiding the development of targeted therapies in molecularly profiled cholangiocarcinoma, and support to the use of molecular profiling to guide therapy selection in patients with advanced biliary cancers. Clin Cancer Res; 24(17); 4154-61. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: Hepatocellular adenomas (HCA) are benign liver tumors that may transform into hepatocellular carcinoma (HCC), but the molecular drivers of this transformation remain ill-defined. This study evaluates the molecular changes in HCA and HCC and in comparison to their adjacent non-neoplastic liver., Methods: 11 patients with HCA and 10 patients with HCC without underlying hepatitis or cirrhosis were included in this pilot study. Tumor and non-tumor liver tissues were selected for immunohistochemical staining, small RNA sequencing, and targeted gene sequencing. We compared microRNA expressions and mutations between HCA and HCC and non-neoplastic liver., Results: HCA were classified as inflammatory (n = 6), steatotic (n = 4), or β-catenin activated (n = 1) subtypes. MicroRNA profile of all 3 HCA subtypes clustered between that of normal liver and HCC in principal component analysis. In both HCA and HCC, miR-200a, miR-429, and miR-490-3p were significantly downregulated compared to normal liver, whereas miR-452, miR-766, and miR-1180 were significantly upregulated. In addition, compared to HCA, HCC had significantly higher expression of members of the chromosome 19 miRNA cluster (C19MC), including miR-515-5p, miR-517a, miR-518b, and miR-520c-3p., Conclusions: This study indicates that while there are significant differences in the molecular profile between HCA and HCC, several miRNAs are similarly deregulated in HCA and HCC compared to adjacent normal liver. These results may provide insights into the drivers of hepatocarcinogenesis and warrant further investigations., Competing Interests: The authors have declared that no competing interests exist.

Background: Pancreatic neuroendocrine neoplasms with a Ki-67 labeling index greater than 20% were reclassified in 2017 by the World Health Organization into well differentiated (WD) and poorly differentiated grade 3 neuroendocrine carcinoma (NEC). The authors describe the cytologic features of grade 3 WD pancreatic neuroendocrine neoplasms compared with grade 2 neoplasms and NEC., Methods: Fine-needle aspirates from 65 pancreatic neuroendocrine neoplasms were reviewed, and their cytomorphologic features were compared across grade 2, WD grade 3, and PD small cell type (PD-S), large cell type (PD-L), and type not otherwise specified (PD-NOS) neoplasms., Results: The 65 aspirates consisted of 19 grade 2 neoplasms, 32 WD grade 3 neoplasms, and 14 NECs (6 PD-S, 5 PD-L, and 3 PD-NOS). The medians Ki-67 proliferation index was 11% (range, 3.2%-17%) in grade 2 neoplasms, 40% (range, 21%-89%) in WD grade 3 neoplasms, 80% (range, 63%-95%) in PD-S neoplasms, 39% (range, 25%-61%) in PD-L neoplasms, and 70% (range, 30%-80%) in PD-NOS neoplasms. Both grade 2 and WD grade 3 neoplasms were associated with plasmacytoid morphology and smooth nuclear contours, but WD grade 3 neoplasms had significant increases in abundant cytoplasm (72% vs 17%; P = .007), nuclear tangles (75% vs 42%; P = .006), and apoptosis (86% vs 58%; P = .005). Compared with NECs, WD grade 3 neoplasms had increased plasmacytoid morphology (75% vs 7%; P < .001), smooth nuclear contours (94% vs 64%; P = .02), round nuclei (59% vs 21%; P = .01), and less pleomorphism (13% vs 50%; P = .004), molding (9% vs 79%; P < .001), and necrosis (13% vs 43%; P = .003). WD grade 3 neoplasms had less pleomorphism (13% vs 50%; P = .04), less necrosis (13% vs 60%; P = .04), and more plasmacytoid morphology (75% vs 20%; P = .03) than PD-L., Conclusions: The prevalence of cytologic features differs in WD grade 3 pancreatic neuroendocrine neoplasms compared with grade 2 neoplasms and NECs, and these differences assist in the recognition of this newly classified entity. Cancer Cytopathol 2018;126:326-35. © 2018 American Cancer Society., (© 2018 American Cancer Society.)

Microsatellite instability, a well-established driver pathway in colorectal carcinogenesis, can develop in both sporadic and hereditary conditions via different molecular alterations in the DNA mismatch repair (MMR) genes. MMR protein immunohistochemistry (IHC) is currently widely used for the detection of MMR deficiency in solid tumors. The IHC test, however, can show varied staining patterns, posing challenges in the interpretation of the staining results in some cases. Here we report a case of an 80-year-old female with a colonic adenocarcinoma that exhibited an unusual "null" IHC staining pattern with complete loss of all four MMR proteins (MLH1, MSH2, MSH6, and PMS2). This led to subsequent MLH1 methylation testing and next generation sequencing which demonstrated that the loss of all MMR proteins was associated with concurrent promoter hypermethylation of MLH1 and double somatic truncating mutations in MSH2. These molecular findings, in conjunction with the patient's age being 80 years and the fact that the patient had no personal or family cancer history, indicated that the MMR deficiency was highly likely sporadic in nature. Thus, the stringent Lynch syndrome type surveillance programs were not recommended to the patient and her family members. This case illustrates a rare but important scenario where a null IHC phenotype signifies complex underlying molecular alternations that bear clinical management implications, highlighting the need for recognition and awareness of such unusual IHC staining patterns.

Gastric carcinoma with lymphoid stroma is an uncommon variant enriched for mutually exclusive Epstein-Barr virus (EBV) positivity and mismatch repair (MMR) deficiency. We performed this study to evaluate molecular alterations in this morphologically homogeneous subtype and compare them with 295 conventional gastric cancers analyzed in The Cancer Genome Atlas study. We identified 31 study cases and subjected them to in situ hybridization for EBV-encoded RNAs and assessment for MMR status. Immunostains for PD-L1, β-catenin, and HER2 were performed; extracted DNA was sequenced with a Comprehensive Cancer Panel. Most study patients were older adult men with stage I or II disease (76%). Tumors were classified as EBV/MMR-proficient (MMR-P) (n=7), EBV/MMR deficient (n=12), and EBV/MMR-P (n=12). EBV/MMR-P tumors were usually located in the proximal stomach (83%) and showed heterogenous growth patterns with glandular differentiation (83%). Tumors in all groups showed numerous tumor infiltrating lymphocytes and PD-L1 expression, infrequent nuclear β-catenin accumulation (10%), and lacked both membranous HER2 staining and HER2 amplification. EBV/MMR-deficient tumors showed significantly higher tumor mutation burden (P=0.001) and KRAS alterations (56%) compared with EBV/MMR-P tumors (9%, P=0.05). TP53 variants were more common among EBV/MMR-P tumors (82%) compared with EBV/MMR proficient (0%, P=0.01) and EBV/MMR-deficient (11%, P<0.01) tumors. Alterations in KRAS, ARID1A, PIK3CA, and TP53 followed similar patterns of distribution compared with The Cancer Genome Atlas dataset. We conclude that gastric carcinomas with lymphoid stroma show a spectrum of molecular changes and frequent PD-L1 expression, raising the possibility that this subgroup of tumors may be susceptible to checkpoint inhibitors and/or agents that target receptor tyrosine kinase-mediated signaling.

Purpose: Immune checkpoint inhibition has been shown to generate profound and durable responses in mismatch repair deficient (MMR-D) solid tumors and has elicited interest in detection tools and strategies to guide therapeutic decision-making. Herein we address questions on the appropriate screening, detection methods, patient selection, and initiation of therapy for MMR-D pancreatic ductal adenocarcinoma (PDAC) and assess the utility of next-generation sequencing (NGS) in providing additional prognostic and predictive information for MMR-D PDAC. Experimental Design: Archival and prospectively acquired samples and matched normal DNA from N = 833 PDAC cases were analyzed using a hybridization capture-based, NGS assay designed to perform targeted deep sequencing of all exons and selected introns of 341 to 468 cancer-associated genes. A computational program using NGS data derived the MSI status from the tumor-normal paired genome sequencing data. Available germline testing, IHC, and microsatellite instability (MSI) PCR results were reviewed to assess and confirm MMR-D and MSI status. Results: MMR-D in PDAC is a rare event among PDAC patients (7/833), occurring at a frequency of 0.8%. Loss of MMR protein expression by IHC, high mutational load, and elevated MSIsensor scores were correlated with MMR-D PDAC. All 7 MMR-D PDAC patients in the study were found to have Lynch syndrome. Four (57%) of the MMR-D patients treated with immune checkpoint blockade had treatment benefit (1 complete response, 2 partial responses, 1 stable disease). Conclusions: An integrated approach of germline testing and somatic analyses of tumor tissues in advanced PDAC using NGS may help guide future development of immune and molecularly directed therapies in PDAC patients. Clin Cancer Res; 24(6); 1326-36. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: Well-differentiated (WD) and poorly differentiated (PD) pancreatic neuroendocrine neoplasms are biologically distinct entities with different therapies and prognoses. WD neoplasms with elevated proliferation (Ki-67 > 20%) have been shown to have an overlapping histology with PD neuroendocrine carcinomas. This study compared expert cytomorphologic assessments of differentiation in pancreatic neuroendocrine neoplasms in a multi-institutional study., Methods: Fine-needle aspiration specimens from pancreatic neuroendocrine neoplasms (grade 2 [G2] and grade 3 [G3] according to the 2017 World Health Organization classification; n = 72) were diagnosed independently by 3 cytopathologists as WD or PD (poorly differentiated large cell type [PD-L] or poorly differentiated small cell type [PD-S]) purely on the basis of cytomorphology. Their diagnoses were compared with a final classification supported by immunohistochemistry (retinoblastoma (RB), death domain- associated protein (DAXX), and α thalassemia/mental retardation syndrome X-linked (ATRX) protein expression), targeted mutation analysis (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets), prior history of G1/G2 histology, and consensus., Results: The rate of agreement on differentiation was 38% (15 WD cases and 12 PD cases) for the 70 cases included (55 WD cases [n = 19 G2, n = 31 G3, and n = 5 could not be graded] and 15 PD cases [n = 6 PD-S, n = 6 PD-L, and n = 3 PD, not otherwise specified). Two cases could not be classified by the employed methods. PD carcinomas had a higher rate of agreement (10 of 15 [67%]) than WD neoplasms (15 of 55 [27%]). Round nuclei and plasmacytoid cells were associated with agreement for WD cases, whereas apoptosis and angulated nuclei were associated with disagreement. Necrosis was associated with agreement for PD cases., Conclusions: A purely morphologic approach to the distinction between G2 and G3 pancreatic neuroendocrine neoplasms based on cytology can be challenging, with disagreement found among experienced cytopathologists. Cancer Cytopathol 2018;126:44-53. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

With increasing accessibility and advancements in abdominal imaging modalities, the incidence of pancreatic neuroendocrine neoplasms has increased steadily during the past few decades. By definition, neuroendocrine neoplasms of the pancreas show neuroendocrine differentiation, but they represent a broad and heterogeneous group of neoplasms with diverse clinical and pathological characteristics. The majority of pancreatic neuroendocrine neoplasms can be classified as well-differentiated pancreatic neuroendocrine tumours (PanNETs) or poorly differentiated pancreatic neuroendocrine carcinomas (PanNECs). While PanNETs and PanNECs are distinct entities with respect to clinical presentation, outcome and therapeutic approach, they may exhibit overlapping histopathological features. Moreover, the frequent modifications in nomenclature and prognostic grading systems over the years of not only pancreatic neuroendocrine neoplasms, but neuroendocrine neoplasms from other organ sites, has created confusion for both pathologists and clinicians as to the appropriate use of terminology and grading when evaluating these neoplasms. This review examines the current concepts and issues of nomenclature and grading of PanNETs and PanNECs. In addition, considering the morphological overlap between high-grade (G3) PanNETs and PanNECs, we discuss an integrative and practical diagnostic approach to aid in discriminating challenging cases., (© 2017 John Wiley & Sons Ltd.)

Purpose: We assessed the usefulness of real-time molecular profiling through next-generation sequencing (NGS) in predicting the tumor biology of advanced pancreatic neuroendocrine tumors (panNETs) and in characterizing genomic evolution., Methods: Patients with metastatic panNETs were recruited in the routine clinical practice setting (between May 2014 and March 2017) for prospective NGS of their tumors as well as for germline analysis using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) sequencing platform. When possible, NGS was performed at multiple time points., Results: NGS was performed in 96 tumor samples from 80 patients. Somatic alterations were identified in 76 of 80 patients (95%). The most commonly altered genes were MEN1 (56%), DAXX (40%), ATRX (25%), and TSC2 (25%). Alterations could be defined in pathways that included chromatin remodeling factors, histone methyltransferases, and mammalian target of rapamycin pathway genes. Somatic loss of heterozygosity was particularly prevalent (50 of 96 samples [52%]), and the presence of loss of heterozygosity resulted in inferior overall survival ( P < .01). Sequencing of pre- and post-treatment samples revealed tumor-grade progression; clonal evolution patterns were also seen (molecular resistance mechanisms and chemotherapy-associated mutagenesis). Germline genetic analysis identified clinically actionable pathogenic or likely pathogenic variants in 14 of 88 patients (16%), including mutations in high-penetrance cancer susceptibility genes ( MEN1 , TSC2 , and VHL )., Conclusion: A clinical NGS platform reveals pertubations of biologic pathways in metastatic panNETs that may inform prognosis and direct therapies. Repeat sequencing at disease progression reveals increasing tumor grade and genetic evolution, demonstrating that panNETs adopt a more aggressive behavior through time and therapies. In addition to frequent somatic mutations in MEN1 and TSC2 , germline mutations in these same genes underlie susceptibility to panNETs and highlight the need to re-evaluate whether germline genetic analysis should be performed for all patients with panNETs.

Approximately 1-2% of pancreatic neoplasms are acinar cell carcinomas. Recently, BRAF gene rearrangements were identified in over 20% of acinar-type neoplasms, which included both pure acinar cell carcinomas and mixed carcinomas with acinar differentiation, using next-generation sequencing-based platforms, providing a potential therapeutic target for patients with these neoplasms. Thus, it is clinically important to develop a rapid, cost- and material-efficient assay to screen for BRAF gene fusions in pancreatic acinar-type neoplasms. We developed a dual color, break-apart FISH assay to detect BRAF gene rearrangements in these neoplasms and evaluated its performance in comparison to next-generation sequencing-based studies. A blinded BRAF rearrangement FISH investigation was performed on 31 acinar-type neoplasms that had been studied previously by next-generation sequencing-based analysis as well as on 18 additional acinar-type neoplasms that were accrued over the past 2 years. In total, BRAF fusions were identified in 12/49 (24%) acinar-type neoplasms by FISH. BRAF fusion partners were uncovered by using targeted next-generation sequencing studies in 11 FISH-positive cases that had sufficient material for next-generation sequencing studies. SND1 was the most frequent fusion partner involved in BRAF-fusion acinar-type neoplasms (50%), followed by HERPUD1 (18%). No BRAF fusions were identified by next-generation sequencing in any of the FISH-negative cases investigated. FISH analysis showed that BRAF rearrangements were diffusely present across tumor-rich areas in BRAF-fusion acinar-type neoplasms, which is consistent with an oncogenic driver alteration pattern. Thus, we demonstrated that, in comparison to targeted next-generation sequencing-based technologies, the FISH assay is highly sensitive and specific as well as time- and cost-efficient in the detection of BRAF fusions in acinar-type neoplasms. The FISH assay can be easily implemented in diagnostic settings to identify acinar-type neoplasms patients potentially suitable for targeted therapy to inhibit MAPK pathway activity.

Background: On the basis of historical data, patients with cancer of unknown primary (CUP) are generally assumed to have a dismal prognosis with overall survival of less than 1 year. Treatment is typically cytotoxic chemotherapy guided by histologic features and the pattern of metastatic spread. The purpose of this study was to provide a clinical and pathologic description of patients with CUP in the modern era, to define the frequency of clinically actionable molecular alterations in this population, to determine how molecular testing can alter therapeutic decisions, and to investigate novel uses of next-generation sequencing in the evaluation and treatment of patients with CUP., Patients and Methods: Under Institutional Review Board approval, we identified all CUP patients evaluated at our institution over a recent 2-year period. We documented demographic information, clinical outcomes, pathologic evaluations, next-generation sequencing of available tumor tissue, use of targeted therapies, and clinical trial enrollment., Results: We identified 333 patients with a diagnosis of CUP evaluated at our institution from 1 January 2014 through 30 June 2016. Of these patients, 150 had targeted next-generation sequencing carried out on available tissue. Median overall survival in this cohort was 13 months. Forty-five of 150 (30%) patients had potentially targetable genomic alterations identified by tumor molecular profiling, and 15 of 150 (10%) received targeted therapies. Dominant mutation signatures were identified in 21 of 150 (14%), largely implicating exogenous mutagen exposures such as ultraviolet radiation and tobacco., Conclusions: Patients with CUP represent a heterogeneous population, harboring a variety of potentially targetable alterations. Next-generation sequencing may provide an opportunity for CUP patients to benefit from novel personalized therapies., (© The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Intraductal tubulopapillary neoplasm is a relatively recently described member of the pancreatic intraductal neoplasm family. The more common member of this family, intraductal papillary mucinous neoplasm, often carries genetic alterations typical of pancreatic infiltrating ductal adenocarcinoma (KRAS, TP53, and CDKN2A) but additionally has mutations in GNAS and RNF43 genes. However, the genetic characteristics of intraductal tubulopapillary neoplasm have not been well characterized. Twenty-two intraductal tubulopapillary neoplasms were analyzed by either targeted next-generation sequencing, which enabled the identification of sequence mutations, copy number alterations, and selected structural rearrangements involving all targeted (≥300) genes, or whole-exome sequencing. Three of these intraductal tubulopapillary neoplasms were also subjected to whole-genome sequencing. All intraductal tubulopapillary neoplasms revealed the characteristic histologic (cellular intraductal nodules of back-to-back tubular glands lined by predominantly cuboidal cells with atypical nuclei and no obvious intracellular mucin) and immunohistochemical (immunolabeled with MUC1 and MUC6 but were negative for MUC2 and MUC5AC) features. By genomic analyses, there was loss of CDKN2A in 5/20 (25%) of these cases. However, the majority of the previously reported intraductal papillary mucinous neoplasm-related alterations were absent. Moreover, in contrast to most ductal neoplasms of the pancreas, MAP-kinase pathway was not involved. In fact, 2/22 (9%) of intraductal tubulopapillary neoplasms did not reveal any mutations in the tested genes. However, certain chromatin remodeling genes (MLL1, MLL2, MLL3, BAP1, PBRM1, EED, and ATRX) were found to be mutated in 7/22 (32%) of intraductal tubulopapillary neoplasms and 27% harbored phosphatidylinositol 3-kinase (PI3K) pathway (PIK3CA, PIK3CB, INPP4A, and PTEN) mutations. In addition, 4/18 (18%) of intraductal tubulopapillary neoplasms had FGFR2 fusions (FGFR2-CEP55, FGFR2-SASS6, DISP1-FGFR2, FGFR2-TXLNA, and FGFR2-VCL) and 1/18 (5.5%) had STRN-ALK fusion. Intraductal tubulopapillary neoplasm is a distinct clinicopathologic entity in the pancreas. Although its intraductal nature and some clinicopathologic features resemble those of intraductal papillary mucinous neoplasm, our results suggest that intraductal tubulopapillary neoplasm has distinguishing genetic characteristics. Some of these mutated genes are potentially targetable. Future functional studies will be needed to determine the consequences of these gene alterations.

Activating neurotrophic tyrosine receptor kinase (NTRK) fusions, typically detected using nucleic-acid based assays, are highly targetable and define certain tumors. Here, we explore the utility of pan-TRK immunohistochemistry (IHC) to detect NTRK fusions. NTRK rearrangements were detected prospectively using MSK-IMPACT, a DNA-based next-generation sequencing assay. Transcription of novel NTRK rearrangements into potentially functional fusion transcripts was assessed via Archer Dx fusion assay. Pan-Trk IHC testing with mAb EPR17341 was performed on all NTRK rearranged cases and 20 cases negative for NTRK fusions on Archer. Of 23 cases with NTRK rearrangements, 15 had known activating fusions. Archer detected fusion transcripts in 6 of 8 novel NTRK rearrangements of uncertain functional significance. Pan-Trk IHC was positive in 20 of 21 cases with NTRK fusion transcripts confirmed by Archer. The discordant negative case was a mismatch repair- deficient colorectal carcinoma with an ETV6-NTRK3 fusion. All 20 additional Archer-negative cases had concordant pan-TRK IHC results. Pan-Trk IHC sensitivity and specificity for transcribed NTRK fusions was 95.2% and 100%, respectively. All positive IHC cases had cytoplasmic staining while the following fusion partner-specific patterns were discovered: all 5 LMNA-NTRK1 fusions displayed nuclear membrane accentuation, all 4 TPM3/4 fusions displayed cellular membrane accentuation, and half (3/6) of ETV6-NTRK3 fusions displayed nuclear staining. Pan-Trk IHC is a time-efficient and tissue-efficient screen for NTRK fusions, particularly in driver-negative advanced malignancies and potential cases of secretory carcinoma and congenital fibrosarcoma. Pan-Trk IHC can help determine whether translation occurs for novel NTRK rearrangements.

Purpose: Molecular profiling in cancer has identified potential actionable drug targets that have prompted attempts to discover clinically validated biomarkers to guide therapeutic decision-making and enrollment to clinical trials. We evaluated whether comprehensive genetic analysis of patients with pancreatic adenocarcinoma is feasible within a clinically relevant timeframe and whether such analyses provide predictive and/or prognostic information along with identification of potential targets for therapy. Experimental Design: Archival or prospectively acquired FFPE samples and matched normal DNA from N = 336 patients with pancreatic cancer were analyzed using a hybridization capture-based, next-generation sequencing assay designed to perform targeted deep sequencing of all exons and selected introns of 410 key cancer-associated genes. Demographic and treatment data were prospectively collected with the goal of correlating treatment outcomes and drug response with molecular profiles. Results: The median time from protocol consent to reporting of the genomic results was 45 days with a median time from tissue delivery of 20 days. All genetic alterations identified were stratified based upon prior evidence that the mutation is a predictive biomarker of drug response using the MSKCC OncoKB classification. Three of 225 patients (1%) received a matched therapy based upon the sequencing results. Conclusions: The practical application of molecular results to guide individual patient treatment is currently limited in patients with pancreatic adenocarcinoma. Future prospective molecular profiling efforts should seek to incorporate routine germline genetic analysis and the identification of DNA profiles that predict for clinical benefit from agents that target DNA damage repair and or immunotherapy. Clin Cancer Res; 23(20); 6094-100. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Importance: Guidelines for cancer genetic testing based on family history may miss clinically actionable genetic changes with established implications for cancer screening or prevention., Objective: To determine the proportion and potential clinical implications of inherited variants detected using simultaneous sequencing of the tumor and normal tissue ("tumor-normal sequencing") compared with genetic test results based on current guidelines., Design, Setting, and Participants: From January 2014 until May 2016 at Memorial Sloan Kettering Cancer Center, 10 336 patients consented to tumor DNA sequencing. Since May 2015, 1040 of these patients with advanced cancer were referred by their oncologists for germline analysis of 76 cancer predisposition genes. Patients with clinically actionable inherited mutations whose genetic test results would not have been predicted by published decision rules were identified. Follow-up for potential clinical implications of mutation detection was through May 2017., Exposure: Tumor and germline sequencing compared with the predicted yield of targeted germline sequencing based on clinical guidelines., Main Outcomes and Measures: Proportion of clinically actionable germline mutations detected by universal tumor-normal sequencing that would not have been detected by guideline-directed testing., Results: Of 1040 patients, the median age was 58 years (interquartile range, 50.5-66 years), 65.3% were male, and 81.3% had stage IV disease at the time of genomic analysis, with prostate, renal, pancreatic, breast, and colon cancer as the most common diagnoses. Of the 1040 patients, 182 (17.5%; 95% CI, 15.3%-19.9%) had clinically actionable mutations conferring cancer susceptibility, including 149 with moderate- to high-penetrance mutations; 101 patients tested (9.7%; 95% CI, 8.1%-11.7%) would not have had these mutations detected using clinical guidelines, including 65 with moderate- to high-penetrance mutations. Frequency of inherited mutations was related to case mix, stage, and founder mutations. Germline findings led to discussion or initiation of change to targeted therapy in 38 patients tested (3.7%) and predictive testing in the families of 13 individuals (1.3%), including 6 for whom genetic evaluation would not have been initiated by guideline-based testing., Conclusions and Relevance: In this referral population with selected advanced cancers, universal sequencing of a broad panel of cancer-related genes in paired germline and tumor DNA samples was associated with increased detection of individuals with potentially clinically significant heritable mutations over the predicted yield of targeted germline testing based on current clinical guidelines. Knowledge of these additional mutations can help guide therapeutic and preventive interventions, but whether all of these interventions would improve outcomes for patients with cancer or their family members requires further study., Trial Registration: clinicaltrials.gov Identifier: NCT01775072.

Metastatic neoplasms of unknown primary site pose a major challenge to patient management. As targeted therapies are now being tailored to neuroendocrine tumors (NETs) of different primary sites, identifying the origin of metastatic NETs has become increasingly important. Compared with more extensive efforts on metastatic adenocarcinomas of unknown primary, the literature on metastatic NETs (often to the liver) is relatively sparse and most studies are based on primary tumors. We sought to study metastatic well-differentiated NETs to the liver to identify markers that predict the site of origin. Eighty-five metastatic NETs to the liver were retrieved from the pathology archive. The primary sites were determined based on either pathologic review of the primary tumors (in most cases) or radiologic/clinical findings. Immunohistochemical labeling for TTF1, CDX2, ISL1, NKX2.2, and PDX1 was performed on either tissue microarrays or whole sections. The primary sites of the NETs in the study cohort included: pancreas (35%), small intestine (32%), rectum (8%), stomach (2%), bile duct (1%), lung (9%), and unknown primary (12%). We found predominant expression of TTF1 in lung carcinoid (63%), CDX2 in small intestinal (89%) and ISL1 in pancreatic NETs (77%), respectively. NKX2.2 was mainly expressed in NETs of the digestive organs. PDX1 was detected in a small percentage of pancreatic, small intestinal and the single bile duct NET. There was no statistically significant association between tumor grade (World Health Organization G1 vs. G2) and the expression of any of the above markers. The 3-marker panel (TTF1, CDX2, and ISL1) had sensitivities of 81%, 89%, and 63%, specificities of 100%, 94%, and 100%, positive predictive values of 100%, 89%, and 100%, and negative predictive values of 84%, 94%, and 96% in separating metastatic NETs into 3 major primary sites: pancreas/rectum, small intestine, and lung, respectively, with an overall accuracy of 82%. Furthermore, this panel predicted a primary site for 6 of the 10 NETs of unknown primary, which reduced the NETs of unknown primary from 12% to 5%. Thus, through immunohistochemical study of a large series of metastatic NETs to the liver, we have demonstrated the utility of a 3-marker panel for the identification of one or more potential primary sites of most metastatic NETs, which could provide practical guidance in patient management.

Objectives: Alkylating agents have activity in well-differentiated pancreatic neuroendocrine tumors (WD panNETs). In glioblastoma multiforme, decreased activity of O-methylguanine DNA methyltransferase (MGMT) predicts response; in panNETs, MGMT relevance is unknown., Methods: We identified patients with WD panNETs treated with alkylating agents, determined best overall response by Response Evaluation Criteria In Solid Tumors (RECIST) 1.1, and performed MGMT activity testing., Results: Fifty-six patients were identified; 26 (46%) of the 56 patients experienced partial response, 24 (43%) of 56 experienced stable disease, and 6 (11%) of 56 experienced progression of disease. O-methylguanine DNA methyltransferase status was available for 36 tumors. For tumors with partial response, 10 (67%) of 15 were MGMT deficient, and 5 (33%) of 15 were MGMT intact. For tumors with stable disease, 7 (47%) of 15 were MGMT deficient, and 8 (53%) of 15 were MGMT intact. For tumors with progression of disease, 3 (50%) of 6 were MGMT deficient, and 3 (50%) of 6 were MGMT intact., Conclusions: We observed response and resistance to alkylating agents in MGMT-deficient and MGMT-intact tumors. O-methylguanine DNA methyltransferase status should not guide alkylating agent therapy in WD panNETs.

Here, comprehensive analysis was performed on the molecular and clinical features of colorectal carcinoma harboring chromosome 20q amplification. Tumor and normal DNA from patients with advanced colorectal carcinoma underwent next-generation sequencing via MSK-IMPACT, and a subset of case samples was subjected to high-resolution microarray (Oncoscan). Relationships between genomic copy number and transcript expression were assessed with The Cancer Genome Atlas (TCGA) colorectal carcinoma data. Of the colorectal carcinoma patients sequenced ( n = 401) with MSK-IMPACT, 148 (37%) had 20q gain, and 30 (7%) had 20q amplification. In both the MSK-IMPACT and TCGA datasets, BCL2L1 was the most frequently amplified 20q oncogene. However, SRC was the only recognized 20q oncogene with a significant inverse relationship between mRNA upregulation and RAS/RAF mutation (OR, -0.4 ± 0.2, P = 0.02). In comparison with 20q diploid colorectal carcinoma, 20q gain/amplification was associated with wild-type KRAS ( P < 0.001) and BRAF ( P = 0.01), microsatellite stability ( P < 0.001), distal primary tumors ( P < 0.001), and mutant TP53 ( P < 0.001), but not stage. On multivariate analysis, longer overall survival from the date of metastasis was observed with chromosome 20q gain ( P = 0.02) or amplification ( P = 0.04) compared with diploid 20q. Implications: 20q amplification defines a subset of colorectal cancer patients with better overall survival from the date of metastasis, and further studies are warranted to assess whether the inhibition of 20q oncogenes, such as SRC, may benefit this subset of patients. Mol Cancer Res; 15(6); 708-13. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically defined tumor types, coupled with an expanding portfolio of molecularly targeted therapies, demands flexible and comprehensive approaches to profile clinically relevant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative using a comprehensive assay, MSK-IMPACT, through which we have compiled tumor and matched normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel noncoding alterations, and mutational signatures that were shared by common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by resistance to currently employed chemotherapeutic approaches. Members of the mir-17~92 cluster of microRNAs (miRNAs) are upregulated in PDAC, but the precise roles of these miRNAs in PDAC are unknown. Using genetically engineered mouse models, we show that loss of mir-17~92 reduces ERK pathway activation downstream of mutant KRAS and promotes the regression of KRASG12D-driven precursor pancreatic intraepithelial neoplasias (PanINs) and their replacement by normal exocrine tissue. In a PDAC model driven by concomitant KRASG12D expression and Trp53 heterozygosity, mir-17~92 deficiency extended the survival of mice that lacked distant metastasis. Moreover, mir-17~92-deficient PDAC cell lines display reduced invasion activity in transwell assays, form fewer invadopodia rosettes than mir-17~92-competent cell lines and are less able to degrade extracellular matrix. Specific inhibition of miR-19 family miRNAs with antagomirs recapitulates these phenotypes, suggesting that miR-19 family miRNAs are important mediators of PDAC cell invasion. Together these data demonstrate an oncogenic role for mir-17~92 at multiple stages of pancreatic tumorigenesis and progression; specifically, they link this miRNA cluster to ERK pathway activation and precursor lesion maintenance in vivo and identify a novel role for miR-19 family miRNAs in promoting cancer cell invasion.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare variant of HCC that most frequently affects young adults. Because of its rarity and an absence of preclinical models, our understanding of FL-HCC is limited. Our objective was to analyze chromosomal alterations and dysregulated gene expression in tumor specimens collected at a single center during two decades of experience with FL-HCC. We analyzed 38 specimens from 26 patients by array comparative genomic hybridiziation (aCGH) and 35 specimens from 15 patients by transcriptome sequencing (RNA-seq). All tumor specimens exhibited genomic instability, with a higher frequency of genomic amplifications or deletions in metastatic tumors. The regions encoding 71 microRNAs (miRs) were deleted in at least 25% of tumor specimens. Five of these recurrently deleted miRs targeted the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) gene product, and a correlating 100-fold upregulation of IGF2BP1 mRNA was seen in tumor specimens. Transcriptome analysis demonstrated intrapatient tumor similarity, independent of recurrence site or time. The p53 tumor suppressor pathway was downregulated as demonstrated by both aCGH and RNA-seq analysis. Notch, EGFR, NRAS, and RB1 pathways were also significantly dysregulated in tumors compared with normal liver tissue. The findings illuminate the genomic and transcriptomic landscape of this rare disease and provide insight into dysregulated oncogenic pathways and potential therapeutic targets in FL-HCC.

Colorectal medullary carcinoma, recognized by the World Health Organization as a distinct histologic subtype, is commonly regarded as a specific entity with an improved prognosis and unique molecular pathogenesis. A fundamental but as yet unaddressed question, however, is whether it can be diagnosed reproducibly. In this study, by analyzing 80 colorectal adenocarcinomas whose dominant growth pattern was solid (thus encompassing medullary carcinoma and its mimics), we provided a detailed description of the morphological spectrum from "classic medullary histology" to nonmedullary poorly differentiated histologies and demonstrated significant overlapping between categories. By assessing a selected subset (n=30) that represented the spectrum of histologies, we showed that the interobserver agreement for diagnosing medullary carcinoma by using 2010 World Health Organization criteria was poor; the κ value among 5 gastrointestinal pathologists was only 0.157 (95% confidence interval, 0.127-0.263; P=.001). When we arbitrarily classified the entire cohort into "classic" and "indeterminate" medullary tumors (group 1, n=19; group 2, n=26, respectively) and nonmedullary poorly differentiated tumors (group 3, n=35), groups 1 and 2 were more likely to exhibit mismatch repair protein deficiency than group 3 (P<.001); however, improved survival could not be detected in either group compared with group 3. Our findings suggest that the diagnosis of medullary carcinoma, as currently applied, may only serve as a morphological descriptor indicating an increased likelihood of mismatch-repair deficiency. Additional evidence including a more objective classification system is needed before medullary carcinoma can be regarded as a distinct entity with prognostic relevance. Until such evidence becomes available, caution should be exercised when making this diagnosis, as well as when comparing results across different studies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Cancer Genome Atlas data on colorectal carcinoma have provided a comprehensive view of the tumor's genomic alterations and their tumorigenic roles. Tumor morphology, however, has not been fully integrated into the analysis. The aim of this study was to explore relevant associations between tumor morphology and the newly characterized genomic alterations in colorectal carcinoma. Two hundred and seven colorectal carcinomas that had undergone whole-exome sequencing as part of The Cancer Genome Atlas project and had adequate virtual images in the cBioPortal for Cancer Genomics constituted our study population. Upon analysis, a tight association between 'microsatellite instability-high histology' and microsatellite instability-high (P<0.001) was readily detected and helped validate our image-based histology evaluation. Further, we showed, (1) among all histologies, the not otherwise specified type had the lowest overall mutation count (P<0.001 for entire cohort, P<0.03 for the microsatellite-instable group), and among the microsatellite-instable tumors, this type also correlated with fewer frameshift mutations in coding mononucleotide repeats of a defined set of relevant genes (P<0.01); (2) cytosine phosphate guanine island methylator phenotype-high colorectal cancers with or without microsatellite instability tended to have different histological patterns: the former more often mucinous and the latter more often not otherwise specified; (3) mucinous histology was associated with more frequent alterations in BRAF, PIK3CA, and the transforming growth factor-β pathway when compared with non-mucinous histologies (P<0.001, P=0.01, and P<0.001, respectively); and (4) few colorectal cancers (<9%) exhibited upregulation of immune-inhibitory genes including major immune checkpoints; these tumors were primarily microsatellite-instable (up to 43%, vs <3% in microsatellite-stable group) and had distinctly non-mucinous histologies with a solid growth. These morphology-molecular associations are interesting and propose important clinical implications. The morphological patterns associated with alterations of immune checkpoint genes bear the potential to guide patient selection for clinical trials that target immune checkpoints in colorectal cancer, and provide directions for future studies.

Intraductal tubulopapillary neoplasm (ITPN) is a relatively recently described member of the pancreatic intraductal neoplasm family. Thus, the literature on its histologic and immunohistochemical features, clinical behavior, and its similarities and differences from other pancreatic neoplasms is limited. Thirty-three cases of ITPN, the largest series to date, were identified. Immunohistochemical labeling for cytokeratins, glycoproteins, pancreatic enzymes, markers for intestinal and neuroendocrine differentiation, and antibodies associated with genetic alterations previously described in pancreatic neoplasms was performed. Clinicopathologic features and survival was assessed. Seventeen patients were female and 14 were male. Mean age was 55 years (range, 25 to 79 y). Median overall tumor size was 4.5 cm (range, 0.5 to 15 cm). Forty-five percent of the tumors occurred in the head, 32% in the body/tail, and 23% showed diffuse involvement. Microscopically, the tumors were characterized by intraductal nodules composed of tightly packed small tubular glands lined by cuboidal cells lacking apparent mucin. Although it was often challenging to determine its extent, invasion was present in 71%. Almost all tumors labeled for CAM5.2, CK7, and CK19; most expressed CA19.9, MUC1, and MUC6. CDX2, MUC2, trypsin, chymotrypsin, chromogranin, and synaptophysin were not expressed. SMAD4 expression was retained in 100%; p16 expression and p53 overexpression was seen in 33% and 27%, respectively. Follow-up information was available for 22 patients (median follow-up, 45 mo; range, 11 to 173 mo). Two patients with invasive carcinoma died of disease at 23 and 41 months, respectively. One patient died of unrelated causes at 49 months. Twelve patients were alive with disease. Seven patients were alive with no evidence of disease. The overall 1-, 3-, and 5-year survival rates were 100% in patients without an invasive component and 100%, 91%, and 71%, respectively, in patients with an invasive component (P=0.7). ITPN is a distinct clinicopathologic entity in the pancreas. Despite the difficulties of determining the extent of invasive carcinoma in many cases, the overall outcome seems to be relatively favorable and substantially better than that of conventional pancreatic ductal adenocarcinoma, even when only the cases with invasive carcinoma are considered.

Background: Histological features and Ki-67 index have known usefulness in predicting prognosis and guiding therapy among patients with metastatic pancreatic neuroendocrine neoplasms. Fine-needle aspiration may offer advantages for Ki-67 assessment because the technique obtains highly cellular, well-preserved specimens with the potential for broader tumor sampling. In the current study, the authors evaluated concordance for grade and differentiation between concurrent core needle biopsy and cytology preparations. Cytological features and grade then were correlated with survival., Methods: Differentiation, grade by Ki-67 index, and correlation of these features with survival were compared between concurrent core needle biopsy and cytology specimens from 44 patients with metastatic pancreatic neuroendocrine neoplasms., Results: Differentiation by cytology smear resulted in 38 cases of well (86%) and 6 cases of poor (14%) differentiation. Agreement for differentiation between smear and cell block, smear and core needle biopsy, and cell block and core needle biopsy was 88%, 94%, and 83%, respectively, and agreement for grade was 68%, 54%, and 22%, respectively. Cytology differentiation and cytology grade were found to be strong predictors of outcome with respective hazard ratios of 8.3 (95% confidence interval [95% CI], 3.1-22.1; P<.001) and 1.9 (95% CI, 1.2-2.9) for each ascending grade. The median disease-specific survival cytology projections were 121 months (95% CI, 57-185 months [estimated]) for grade 1, 45 months (95% CI, 29-87 months) for grade 2, and 19 months (95% CI, 1-44 months) for grade 3, with median survivals of 45 months and 3 months, respectively, for patients with well-differentiated and poorly differentiated neuroendocrine tumors (P<.001)., Conclusions: Grading of pancreatic neuroendocrine neoplasms on cytology may not correlate exactly with concurrent core needle biopsy, but cytology differentiation and grade are predictive of survival based on stage-adjusted analysis. Cancer Cytopathol 2017;125:188-196. © 2016 American Cancer Society., (© 2016 American Cancer Society.)

Objectives: Emerging data suggest that not all grade 3 (G3) pancreatic neuroendocrine neoplasms (panNENs) behave the same; tumor differentiation may predict outcome., Methods: Patients with G3 panNENs treated at our institution between 1999 and 2014 were identified. Demographics, response to therapy, and overall survival were determined., Results: Forty-five patients were identified, 16 with G3 well differentiated pancreatic neuroendocrine tumors (WD-panNETs) and 29 with poorly differentiated neuroendocrine carcinomas (PDNEC). Median overall survival in G3 WD-panNET patients was 52.2 months (95% confidence interval, 19.3-86.9 months) compared with 10.1 months (95% confidence interval, 6.9-12.4 months) in PDNEC patients (P = 0.0009). Response rate to platinum agents was 10% in G3 WD-panNETs and 37% in PDNEC. Response rate to alkylating agents was 50% in G3 WD-panNETs and 50% in PDNEC., Conclusions: Both G3 WD-panNETs and PDNEC responded to platinum and alkylating agents. Overall survival was significantly greater in G3 WD-panNETs compared with PDNEC. These findings challenge current classification and suggest that G3 panNENs should be classified by morphology.

Purpose: Microsatellite instability (MSI)/mismatch repair (MMR) status is increasingly important in the management of patients with cancer to predict response to immune checkpoint inhibitors. We determined MSI status from large-panel clinical targeted next-generation sequencing (NGS) data across various solid cancer types., Methods: The MSI statuses of 12,288 advanced solid cancers consecutively sequenced with Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets clinical NGS assay were inferred by using MSIsensor, a program that reports the percentage of unstable microsatellites as a score. Cutoff score determination and sensitivity/specificity were based on MSI polymerase chain reaction (PCR) and MMR immunohistochemistry., Results: By using an MSIsensor score ≥ 10 to define MSI high (MSI-H), 83 (8%) of 996 colorectal cancers (CRCs) and 42 (16%) of 260 uterine endometrioid cancers (UECs) were MSI-H. Validation against MSI PCR and/or MMR immunohistochemistry performed for 138 (24 MSI-H, 114 microsatellite stable [MSS]) CRCs, and 40 (15 MSI-H, 25 MSS) UECs showed a concordance of 99.4%. MSIsensor also identified 68 MSI-H/MMR-deficient (MMR-D) non-CRC/UECs. Of 9,591 non-CRC/UEC tumors with MSS MSIsensor status, 456 (4.8%) had slightly elevated scores(≥3 and <10) of which 96.6% with available material were confirmed to be MSS by MSI PCR. MSI-H was also detected and confirmed in three non-CRC/UECs with low exonic mutation burden (< 20). MSIsensor correctly scored all 15 polymerase ε ultra-mutated cancers as negative for MSI., Conclusion: MSI status can be reliably inferred by MSIsensor from large-panel targeted NGS data. Concurrent MSI testing by NGS is resource efficient, is potentially more sensitive for MMR-D than MSI PCR, and allows identification of MSI-H across various cancers not typically screened, as highlighted by the finding that 35% (68 of 193) of all MSI-H tumors were non-CRC/ UEC.

Objective: The aim of this study was to evaluate and validate the proposed 8th edition American Joint Committee on Cancer (AJCC) system for T and N staging of pancreatic adenocarcinoma., Summary of Background Data: Investigators have questioned the clinical relevance and reproducibility of previous AJCC staging for pancreatic adenocarcinoma., Methods: Prospective databases at Memorial Sloan Kettering (MSK), Massachusetts General Hospital (MGH), and Johns Hopkins Hospital (JHH) were queried for patients who had undergone resection for pancreatic adenocarcinoma. Patients who underwent a margin-negative (R0) resection, and who had previously undergone pathologic review, were included. Patients were staged according to 7th edition AJCC criteria, as well as the proposed 8th edition system that includes different definitions of tumor size (T) and nodal status (N). The dataset was randomly split into training and test sets., Results: Two thousand three hundred eighteen patients were identified who met inclusion criteria. Recursive partitioning on the training set (n = 1551) identified statistically appropriate cutoffs for tumor size (<2.2 cm, ≥4.8 cm,) and nodal status (no positive nodes, 1 to 3 positive nodes, ≥4 positive nodes) that supported the proposed 8th edition changes. Median survival in patients staged as T3, N0 by the 7th edition definitions was different between institutions (median Center 1, 24 mo; Center 2, 37 mo; Center 3, 29 mo; P = 0.054). This difference was not observed when patients were staged as T3, N0 by 8th edition criteria. Stage, and stage-specific outcome (7th edition), on the test set revealed a predominance of patients (68%) within the IIB subgroup, and a concordance probability estimate (CPE) of 0.57 for stage-specific survival. When assessed with 8th edition criteria, no stage subgroup had a majority of patients, and the CPE was 0.58., Conclusions: The proposed 8th edition changes for T and N classification were statistically valid and may allow a more reproducible system of T staging. This system also stratifies patients more evenly across stages without sacrificing prognostic accuracy.

Pancreatic ductal adenocarcinoma (PDAC), a poor prognostic cancer, commonly develops following activating mutations in the KRAS oncogene. Activation of WNT signaling is also commonly observed in PDAC. To ascertain the impact of postnatal activation of WNT-stimulated signaling pathways in PDAC development, we combined the Elastase-tva-based RCAS-TVA pancreatic cancer model with the established LSL-Kras G12D , Ptf1a-cre model. Delivery of RCAS viruses encoding β-catenin S37A and WNT1 stimulated the progression of premalignant pancreatic intraepithelial neoplasias (PanIN) and PDAC development. Moreover, mice injected with RCAS-β-catenin S37A or RCAS-Wnt1 had reduced survival relative to RCAS-GFP-injected controls (P<.05). Ectopic expression of active β-catenin, or its DNA-binding partner TCF4, enhanced transformation associated phenotypes in PDAC cells. In contrast, these phenotypes were significantly impaired by the introduction of ICAT, an inhibitor of the β-catenin/TCF4 interaction. By gene expression profiling, we identified Cyr61 as a target molecule of the WNT/β-catenin signaling pathway in pancreatic cancer cells. Nuclear β-catenin and CYR61 expression were predominantly detected in moderately to poorly differentiated murine and human PDAC. Indeed, nuclear β-catenin- and CYR61-positive PDAC patients demonstrated poor prognosis (P<.01). Knockdown of CYR61 in a β-catenin-activated pancreatic cancer cell line reduced soft agar, migration and invasion activity. Together, these data suggest that the WNT/β-catenin signaling pathway enhances pancreatic cancer development and malignancy in part via up-regulation of CYR61., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

mTOR signaling controls several critical cellular functions and is deregulated in many cancers, including pancreatic cancer. To date, most efforts have focused on inhibiting the mTORC1 complex. However, clinical trials of mTORC1 inhibitors in pancreatic cancer have failed, raising questions about this therapeutic approach. We employed a genetic approach to delete the obligate mTORC2 subunit Rictor and identified the critical times during which tumorigenesis requires mTORC2 signaling. Rictor deletion resulted in profoundly delayed tumorigenesis. Whereas previous studies showed most pancreatic tumors were insensitive to rapamycin, treatment with a dual mTORC1/2 inhibitor strongly suppressed tumorigenesis. In late-stage tumor-bearing mice, combined mTORC1/2 and PI3K inhibition significantly increased survival. Thus, targeting mTOR may be a potential therapeutic strategy in pancreatic cancer. Cancer Res; 76(23); 6911-23. ©2016 AACR., Competing Interests: The authors have no competing interests to disclose, (©2016 American Association for Cancer Research.)

Intraductal oncocytic papillary neoplasm (IOPN) of the pancreas is classified as a variant of intraductal papillary mucinous neoplasm (IPMN) in the WHO guidelines. However, the neoplastic cells of IOPNs are unique, with distinctive architecture/oncocytic cytoplasm. Although molecular/immunohistochemical features of other IPMN variants have been extensively studied, those of IOPNs have not been well characterized. Expression profile of antibodies associated with genetic alterations previously described for ductal adenocarcinomas (DAs) and IPMNs (SMAD4/β-catenin/p53/mesothelin/claudin-4) as well as antibodies to mucins and differentiation markers [MUC1/MUC2/MUC5AC/MUC6/CDX2/hepatocyte paraffin-1 (HepPar-1)] was investigated in 24 IOPNs and 22 IPMNs to assess the similarities/differences between these tumors. Expression of mesothelin and claudin-4 was dissimilar between these tumor types: A higher proportion of IOPNs labeled with mesothelin [21/24 (87.5 %) of IOPNs, 6/22 (27 %) of IPMNs, p < 0.001], while the reverse was true for claudin-4 [2/23 (9 %) of IOPNs, 9/22 (41 %) of IPMNs, p = 0.01]. The results of immunolabeling for SMAD4/β-catenin/p53 were similar in both: None of the cases showed SMAD4 loss in the intraductal components, and only 1/21 (5 %) of IOPNs and 2/22 (9 %) of IPMNs revealed abnormal β-catenin expression (p = 0.49). Nuclear p53 accumulation was seen mostly in architecturally complex/high-grade dysplasia areas in both. Immunolabeling for MUC proteins showed that almost all lesions expressed MUC5AC. Twelve of the 24 (50 %) IOPNs and 6/22 (27 %) of IPMNs (p = 0.11) labeled for MUC1, whereas 7/24 (29 %) of IOPNs and 10/22 (45 %) of IPMNs labeled for MUC2 (p = 0.25). MUC6 was expressed in 8/9 (89 %) of IOPNs (strong) and 6/21 (29 %) of IPMNs (weak) (p = 0.002). Fourteen of the 23 (61 %) IOPNs and 4/22 (18 %) of IPMNs labeled for HepPar-1 (p = 0.003). These results show that IOPNs have distinct immunoprofile and provide support for the proposition that IOPN is a distinct entity developing through a mechanism different from other pancreatic ductal neoplasms., Competing Interests: The authors declare no conflict of interest.

Immune checkpoint blockade targeting the programmed death-1 (PD-1) pathway has shown efficacy in several types of cancers including mismatch-repair-deficient colorectal carcinoma. In some tumor types, programmed death-ligand 1 (PD-L1) expression detected by immunohistochemistry has shown utility as a predictive marker for response to anti-PD-1 therapies. This utility, however, remains to be determined in colorectal carcinoma. In addition, although tumor-infiltrating lymphocytes have been associated with better prognosis in colorectal carcinoma, the prognostic value of PD-1 expression in these lymphocytes and its interaction with PD-L1 expression still await investigation. To address these questions, we performed a pilot study to evaluate the patterns of PD-L1 and PD-1 immunohistochemical expression on colorectal carcinoma cells and their tumor-infiltrating lymphocytes, respectively. Using tissue microarray, we found that 5&percnt; (19&sol;394) of colorectal carcinomas exhibited high tumor PD-L1 expression, and 19&percnt; (76&sol;392) had elevated numbers of PD-1-positive tumor-infiltrating lymphocytes. PD-L1 levels correlated with PD-1 levels (P&lt;0.001), and mismatch-repair-deficient tumors had significantly higher rates of high PD-L1 and PD-1 expression when compared with mismatch-repair-proficient tumors (18&percnt; vs 2&percnt; and 50&percnt; vs 13&percnt;, respectively; P&lt;0.001 for both). Staining intensity was also stronger for both markers in mismatch-repair-deficient tumors. Furthermore, we observed that among patients with mismatch-repair-deficient colorectal carcinoma, PD-1&sol;PD-L1 expression stratified recurrence-free survival in an inter-dependent manner: an association between high PD-1-positive tumor-infiltrating lymphocytes and improved recurrence-free survival (P&equals;0.041) was maintained only when the tumors had low-level PD-L1 expression (P&equals;0.006); patients whose tumors had both high PD-1-positive tumor-infiltrating lymphocytes and high PD-L1 expression had a significantly worse recurrence-free survival (P&lt;0.001). Thus, our results not only provide a foundation for further assessment of PD-L1 immunohistochemistry as a predictive marker for anti-PD-1 therapy in colorectal carcinoma, they also shed light on the prognostic impact of tumor-infiltrating lymphocytes in different subsets of mismatch-repair-deficient colorectal carcinomas., Competing Interests: Disclosure/conflict of interest The authors declare no conflict of interest.

Pancreatic intraductal papillary mucinous neoplasms (IPMN) are surgically treatable cancer precursors, if high-risk features can be identified. Analysis of proteins and genes in cyst fluid helps diagnostically, but prior biomarkers had limited ability to detect high-risk cases. Cyst fluid telomerase activity may be a useful biomarker for high-risk IPMNs. Clin Cancer Res; 22(20); 4966-7. ©2016 AACRSee related article by Hata et al., p. 5141., (©2016 American Association for Cancer Research.)

The pathology of biliary carcinomas is diverse with different gross and histological features in tumors arising in the different segments of the biliary system. Various epidemiological risk factors, varied genetic makeup, and tissue microenvironment are contributory factors. As biliary tumors have been shown to be a part of the Lynch syndrome tumor spectrum, it is plausible to speculate that DNA mismatch repair (MMR) deficiency plays a role in biliary tumors. Literature data suggest that DNA MMR deficiency indeed occurs in these tumors, albeit infrequently with the reported frequencies (weighted for sample size) of high level microsatellite instability (MSI) being 5% each for gallbladder carcinoma and carcinoma of extra-hepatic bile ducts, and 10% each for intrahepatic cholangiocarcinoma and ampullary carcinoma. Importantly, the presence of MMR deficiency in these tumors has been shown to have different implications with regard to its association with Lynch syndrome, tumor histological features, and other clinical characteristics, when compared with non-biliary tumors or among the biliary tumors from the different segments of the biliary system. Ongoing and future efforts that utilize large scale sequencing techniques and aim at detecting actionable molecular targets should emphasize a multidisciplinary approach that integrates genomic discoveries with not only functional studies but also studies of tumor pathology and the tumor's clinical and biological behavior.

In human studies and mouse models, the contributions of p53 and p16 Ink4a /p19 Arf loss are well established in pancreatic ductal adenocarcinoma (PDAC). Although loss of functional p53 pathway and loss of Ink4a/Arf in human pancreatic acinar cell carcinoma (PACC) and pancreatic neuroendocrine tumor (PanNET) are identified, their direct roles in tumorigenesis of PACC and PanNET remain to be determined. Using transgenic mouse models expressing the viral oncogene polyoma middle T antigen (PyMT), we demonstrate that p53 loss in pancreatic Pdx1+ progenitor cells results in aggressive PACC, whereas Ink4a/Arf loss results in PanNETs. Concurrent loss of p53 and Ink4a/Arf resembles loss of p53 alone, suggesting that Ink4a/Arf loss has no additive effect to PACC progression. Our results show that specific tumor suppressor genotypes provocatively influence the tumor biological phenotypes in pancreatic progenitor cells. Additionally, in a mouse model of β-cell hyperplasia, we demonstrate that p53 and Ink4a/Arf play cooperative roles in constraining the progression of PanNETs., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

ETV6-NTRK3 fusion was identified in several cancers including the recently described mammary analog secretory carcinoma (MASC) of the salivary glands and a minority of papillary thyroid carcinomas. We describe three cases of primary MASC of the thyroid gland and provide a detailed clinical and pathological characterization of the tumor morphology, immunoprofile, and genetic background. Immunohistochemistry for PAX8, TTF-1, thyroglobulin, mammaglobin, GCDFP-15, S-100 protein, and p63 was used to define the tumor immunophenotype. Fluorescence in situ hybridization for ETV6 rearrangement was performed in three, and the next-generation sequencing assay MSK-IMPACT™ (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) was performed in two cases. Primary MASC of the thyroid occurred in two women and one man, age 47-72 years. All patients presented with high T stage, infiltrative, locally aggressive tumors with extrathyroidal extension. Two cases were associated with well-differentiated papillary thyroid carcinoma. Histologically, they appeared as low-grade tumors, resembling MASC of the salivary glands and labeled positive for mammaglobin, GCDFP-15, S-100 protein, p63, weakly positive for PAX8, and negative for TTF-1 and thyroglobulin. Fluorescence in situ hybridization revealed ETV6 rearrangement in all cases. In two tested cases MSK-IMPACT™ confirmed the presence of ETV6-NTRK3 gene fusion. Two patients had at least two local recurrences, one was alive with disease, and one was alive and free of disease after 14 and 17 years, respectively. The third patient was alive and free of disease after 2 years. MASC of the thyroid is histologically, immunophenotypically, and genetically similar to its salivary gland counterpart. Thyroid MASC can be associated with a well-differentiated papillary thyroid carcinoma component, supporting follicular cell origin. Clinically, these carcinomas may show frequent recurrences but are associated with long-term survival. Patients with MASC of the thyroid may potentially benefit from Trk molecular-targeted therapy., Competing Interests: Disclosure/conflict of interest The authors declare no conflict of interest.

The presence of invasion into the extra-hepatic portion of the portal vein or the development of distant metastases renders hepatocellular carcinoma (HCC) patients ineligible for the only potential curative options for this malignancy-tumor resection or organ transplantation. Gene expression profiling of murine HCC cell lines identified KLF6 as a potential regulator of HCC cell migration. KLF6 knockdown increases cell migration, consistent with the correlation between decreased KLF6 mRNA levels and the presence of vascular invasion in human HCC. Concordantly, single-copy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs and decreased survival, indicating that KLF6 suppresses both HCC development and metastasis. By combining gene expression profiling and chromatin immunoprecipitation coupled to deep sequencing, we identified novel transcriptional targets of KLF6 in HCC cells including VAV3, a known activator of the RAC1 small GTPase. Indeed, RAC1 activity is increased in KLF6-knockdown cells in a VAV3-dependent manner, and knockdown of either RAC1 or VAV3 impairs HCC cell migration. Together, our data demonstrate a novel function for KLF6 in constraining HCC dissemination through the regulation of a VAV3-RAC1 signaling axis.

Purpose To establish the prognostic value of biopsy of the central and marginal ablation zones for time to local tumor progression (LTP) after radiofrequency (RF) ablation of colorectal cancer liver metastasis (CLM). Materials and Methods A total of 47 patients with 67 CLMs were enrolled in this prospective institutional review board-approved and HIPAA-compliant study between November 2009 and August 2012. Mean tumor size was 2.1 cm (range, 0.6-4.3 cm). Biopsy of the center and margin of the ablation zone was performed immediately after RF ablation (mean number of biopsy samples per ablation zone, 1.9) and was evaluated for the presence of viable tumor cells. Samples containing tumor cells at morphologic evaluation were further interrogated with immunohistochemistry and were classified as either positive, viable tumor (V) or negative, necrotic (N). Minimal ablation margin size was evaluated in the first postablation CT study performed 4-8 weeks after ablation. Variables were evaluated as predictors of time to LTP with the competing-risks model (uni- and multivariate analyses). Results Technical effectiveness was evident in 66 of 67 (98%) ablated lesions on the first contrast material-enhanced CT images at 4-8-week follow-up. The cumulative incidence of LTP at 12-month follow-up was 22% (95% confidence interval [CI]: 12, 32). Samples from 16 (24%) of 67 ablation zones were classified as viable tumor. At univariate analysis, tumor size, minimal margin size, and biopsy results were significant in predicting LTP. When these variables were subsequently entered in a multivariate model, margin size of less than 5 mm (P < .001; hazard ratio [HR], 6.7) and positive biopsy results (P = .008; HR, 3.4) were significant. LTP within 12 months after RF ablation was noted in 3% (95% CI: 0, 9) of necrotic CLMs with margins of at least 5 mm. Conclusion Biopsy proof of complete tumor ablation and minimal ablation margins of at least 5 mm are independent predictors of LTP and yield the best oncologic outcomes. (©) RSNA, 2016.