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Supplementary Fig. 1: Mutation rate and ploidy for each individual patient's primary and metastatic sample (cohort 1);Supplementary Fig. 2: Validation of genomic heterogeneity within primary tumor and between primary tumor and lymph node (LN) metastases detected by next generation sequencing (NGS) with fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC);Supplementary Fig. 3: PANGEA: Platform clinical trial of molecularly driven therapies in patients with previously untreated GEA;Supplementary Fig. 4: Biomarker profiling of PANGEA where the biomarker profiling of the metastatic tumor did not lead to treatment reassignment relative to treatments assigned based upon the primary tumor

Gastroesophageal adenocarcinoma (GEA) is a lethal disease where targeted therapies, even when guided by genomic biomarkers, have had limited efficacy. A potential reason for the failure of such therapies is that genomic profiling results could commonly differ between the primary and metastatic tumors. To evaluate genomic heterogeneity, we sequenced paired primary GEA and synchronous metastatic lesions across multiple cohorts, finding extensive differences in genomic alterations, including discrepancies in potentially clinically relevant alterations. Multiregion sequencing showed significant discrepancy within the primary tumor (PT) and between the PT and disseminated disease, with oncogene amplification profiles commonly discordant. In addition, a pilot analysis of cell-free DNA (cfDNA) sequencing demonstrated the feasibility of detecting genomic amplifications not detected in PT sampling. Lastly, we profiled paired primary tumors, metastatic tumors, and cfDNA from patients enrolled in the personalized antibodies for GEA (PANGEA) trial of targeted therapies in GEA and found that genomic biomarkers were recurrently discrepant between the PT and untreated metastases. Divergent primary and metastatic tissue profiling led to treatment reassignment in 32% (9/28) of patients. In discordant primary and metastatic lesions, we found 87.5% concordance for targetable alterations in metastatic tissue and cfDNA, suggesting the potential for cfDNA profiling to enhance selection of therapy.Significance: We demonstrate frequent baseline heterogeneity in targetable genomic alterations in GEA, indicating that current tissue sampling practices for biomarker testing do not effectively guide precision medicine in this disease and that routine profiling of metastatic lesions and/or cfDNA should be systematically evaluated. Cancer Discov; 8(1); 37–48. ©2017 AACR.See related commentary by Sundar and Tan, p. 14.See related article by Janjigian et al., p. 49.This article is highlighted in the In This Issue feature, p. 1

Supplementary Figure Legends

Supplementary Methods

Supplementary Table 1. Tumor and sequencing information for Cohort 1, paired primary and metastatic gastric adenocarcinoma samples; Supplementary Table 2. 243-gene targeted panel used for sequencing of Cohort 2 samples; Supplementary Table 3: Wild type and mutant read counts for paired primary and metastatic samples in cohort 1; Supplementary Table 4. Clinical and pathologic characteristics of Cohort 2; Supplementary Table 5. Complete sequencing results of Cohort 2; Supplementary Table 6. Clinical characteristics of Cohort 3; Supplementary Table 7. Treatment assignment algorithm of PANGEA clinical trial; Supplementary Table 8. Clinical Characteristics and Response Data of evaluable patients in the PANGEA cohort (N = 21)

While most resection specimens from patients with neoadjuvantly treated esophageal squamous cell carcinoma show therapy-related changes in the form of inflammation and fibrosis, others harbor a florid foreign body-type giant cell response to keratin debris. The purpose of our study was to perform a detailed clinicopathologic analysis of these histologic types of treatment responses and correlate these findings with patient outcome. Clinical and pathologic parameters from 110 esophagogastrectomies were recorded and analyzed. Two main types of histologic responses were observed: inflammatory-predominant response (59%) and florid foreign body-type giant cell response to keratin (41%). Irrespective of cG, cTNM, and amount of residual cancer, florid foreign body-type giant cell reaction was predominantly noted deep within the esophageal wall, while the inflammatory response was restricted to the mucosa, submucosa, and inner half of muscularis propria. Patients with foreign body-type giant cell response showed significantly better overall survival compared with the inflammatory response group (log-rank test P=0.015). Florid foreign body-type giant cell response was the only factor associated with improved survival in a multivariable analysis for overall survival (hazard ratio=0.5; 95% confidence interval=0.3-1.0; P=0.038), but not in the model for disease-specific survival, whereas ypTNM stage II was the only significant risk factor for disease-specific survival in multivariable analysis (hazard ratio=3.4; 95% confidence interval=1.0-11.2; P=0.047). Our results suggest that in addition to the College of American Pathologists Tumor Regression Score and ypTNM stage, subtype of histologic response to therapy may represent another prognostic marker for neoadjuvantly treated esophageal squamous cell carcinoma.

There is a growing interest in minimally invasive endoscopic gallbladder drainage therapies. Unlike stenting, a technology based on magnetic compression could theoretically produce a durable drainage anastomosis without residual foreign material. This study aimed to evaluate the safety and technical feasibility of a cholecysto-duodenal magnetic compression anastomosis.We performed a survival study of two Yorkshire pigs. Duodenal magnets were deployed endoscopically; reciprocal gallbladder magnets were placed laparoscopically, and the magnets were coupled. Pigs underwent serial endoscopy documenting magnet expulsion and evolution of cholecysto-duodenal anastomosis creation. Necropsies and histological evaluation were performed. Primary endpoints were technical success and safety. Secondary endpoints included anastomosis integrity, patency, and histologic characteristics.Magnets were successfully delivered and coupled. Patent, leak-free anastomoses formed by day 4. Magnets were expelled by day 10. All anastomoses were widely patent at one month (mean diameter 15 mm). Necropsy showed the absence of adhesions affecting the anastomosis. Histology showed complete re-epithelialization without inflammation or foreign body reaction.Magnetic cholecysto-duodenal anastomosis for gallbladder drainage appears safe and feasible in the animal model. The anastomoses are patent, leak-free, and without inflammation from the presence of foreign material. Technical modifications for magnet delivery under endoscopic ultrasound (EUS) guidance are currently underway.

AIMS SMAD4 (DPC4) is a tumour suppressor gene that is dysregulated in various tumour types, particularly pancreaticobiliary and gastrointestinal carcinomas. Corresponding loss of protein expression has been reported in approximately 50% of pancreatic and 25% of colonic adenocarcinomas. In the evaluation of carcinoma of unknown primary site, immunohistochemical loss of SMAD4 expression is often used to suggest pancreaticobiliary origin, but there are limited data on the spectrum of SMAD4 expression in carcinomas of other sites. This study evaluates the frequency of SMAD4 loss in a large cohort of carcinomas from diverse anatomical sites. METHODS AND RESULTS Immunohistochemistry for SMAD4 was performed on tissue microarrays or whole tissue sections of 1210 carcinomas from various organs: gastrointestinal tract, liver, pancreas/biliary tract, lung, breast, thyroid, kidney, ovary and uterus. Expression was considered lost when there was complete absence of staining in tumour cell nuclei, in the presence of intact staining in non-neoplastic cells. SMAD4 loss was seen in 58% of pancreatic adenocarcinomas, 27% of appendiceal adenocarcinomas, 19% of colorectal adenocarcinomas, 16% of cholangiocarcinomas, 10% of lung adenocarcinomas and

AIMS Oesophageal verrucous carcinoma (VSCC) is a rare and morphologically distinct type of oesophageal squamous cell carcinoma (SCC). Diagnosing VSCC on biopsy material is challenging, given the lack of significant atypia and the presence of keratinising epithelium and exophytic growth. The molecular pathogenesis of VSCC remains unclear. The aim of this study was to characterise the genomic landscape of VSCC in comparison to conventional oesophageal SCC. METHODS AND RESULTS Three cases of VSCC from the Brigham and Women's Hospital pathology archive were identified. Formalin-fixed, paraffin-embedded (FFPE) tumour tissue was used for p16 immunohistochemistry (IHC), high-risk human papillomavirus (HPV) in-situ mRNA hybridisation (ISH) and DNA isolation. Tumour DNA was sequenced using a targeted massively parallel sequencing assay enriched for cancer-associated genes. Three additional cases of VSCC were identified by image review of The Cancer Genome Atlas (TCGA) oesophageal SCC cohort. VSCC cases were negative for p16 IHC and high-risk HPV ISH. TP53 mutations (P

Complex chromosomal alterations are a hallmark of advanced cancers but rarely seen in normal tissue. The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations that can drive their transformation through focal oncogenic amplifications. However, the etiology and evolution dynamics of these alterations are poorly understood. Here we study chromosomal copy-number evolution in the progression of Barrett’s esophagus (BE) to esophageal adenocarcinoma (EAC) by multi-regional whole-genome sequencing analysis of BE samples with dysplasia and microscopic EAC foci. Through haplotype-specific copy-number analysis of BE genome evolution, we identified distinct patterns of episodic copy-number evolution consistent with the outcomes of abnormal mitosis and dicentric chromosome breakage. While abnormal mitosis, including whole-genome duplication, accounts for most chromosome or arm-level copy-number changes, segmental copy-number alterations display signatures of multi-generational evolution of unstable dicentric chromosomes. Continuous evolution of dicentric chromosomes through breakage-fusion-bridge cycles and chromothripsis rapidly increases genomic complexity and diversity among BE cells, culminating in the generation of distinct focal amplifications. These mutational processes enable multiple subclones within small dysplastic areas to undergo parallel transformation to cancer following acquisition of distinct oncogenic amplifications. Our results demonstrate how chromosomal instability drives clonal diversification in precancer evolution and promotes tumorigenesis in primary human samples.

Outcomes for pancreatic cancer (PC) patients remain strikingly poor with a 5‐year survival of less than 8% due to the lack of effective treatment modalities. Here, a novel precision medicine approach for PC treatment is developed, which is composed of a rationally designed tumor‐targeting ICAM1 antibody‐drug conjugate (ADC) with optimized chemical linker and cytotoxic payload, complemented with a magnetic resonance imaging (MRI)‐based molecular imaging approach to noninvasively evaluate the efficiency of ICAM1 ADC therapy. It is shown that ICAM1 is differentially overexpressed on the surface of human PC cells with restricted expression in normal tissues, enabling ICAM1 antibody to selectively recognize and target PC tumors in vivo. It is further demonstrated that the developed ICAM1 ADC induces potent and durable tumor regression in an orthotopic PC mouse model. To build a precision medicine, an MRI‐based molecular imaging approach is developed that noninvasively maps the tumoral ICAM1 expression that can be potentially used to identify ICAM1‐overexpressing PC patients. Collectively, this study establishes a strong foundation for the development of a promising ADC to address the critical need in the PC patient care., A novel precision medicine approach for pancreatic cancer (PC) treatment is developed. It is composed of a rationally designed tumor‐targeting ICAM1 antibody‐drug conjugate (ADC) with an optimized chemical linker and cytotoxic payload, complemented by a magnetic resonance imaging (MRI)‐based molecular imaging approach to noninvasively evaluate the efficiency of ICAM1‐targeting immunotherapy.

Summary Paraneoplastic thrombocytosis has been associated with adverse outcomes in several cancers, but has not been described in oesophageal adenocarcinoma. The aim of our study was to examine the prognostic value of platelet counts in patients with oesophageal adenocarcinoma. A cohort of 584 patients who underwent oesophagectomy for oesophageal adenocarcinoma was identified. Platelet counts, history of neoadjuvant chemoradiation, and clinicopathological factors such as T and N stage, and overall survival were recorded. Patients with elevated platelet count (>450,000/μL) had a higher mortality rate than patients with normal platelet count (150,000–450,000/μL) (hazard ratio = 2.60, p = 0.0013). This effect was seen in patients with and without neoadjuvant chemoradiation therapy. Paraneoplastic thrombocytosis was also associated with increased likelihood of lymph node metastasis compared to normal platelet count (69% versus 31%, p Paraneoplastic thrombocytosis is associated with increased rate of lymph node metastasis and mortality in oesophageal adenocarcinoma. Further studies are needed to examine the mechanisms behind this phenomenon.

Oesophageal submucosal glands secrete mucins and other chemicals that are believed to serve as protectants of the mucosal surface from luminal noxious agents, either ingested or refluxed. Changes in the type, distribution or number of submucosal glands may contribute to, or be associated with, the development of Barrett's oesophagus and progression to cancer. The aim of this study was to investigate the anatomical, morphological and immunohistochemical characteristics of submucosal glands in Barrett's oesophagus-associated neoplasia in 64 oesophageal resections for Barrett's oesophagus-associated adenocarcinoma and 32 squamous cell carcinomas (as a control group). Gland density was not significantly different between the oesophageal adenocarcinoma (0.91/cm) and squamous cell carcinoma (0.81/cm) groups (p=0.7). In the oesophageal adenocarcinoma group, glands underlying Barrett's oesophagus-associated neoplastic epithelium showed a significant decrease in the percentage of mucinous acini and a significant increase in the percentage of atrophic acini compared to glands underlying epithelium without dysplasia or carcinoma (74% vs 83%, p=0.03; and 24% vs 14%, p=0.01). There was also an increase in the percentage of glands with moderate to severe inflammation underlying neoplastic epithelium compared to glands underlying epithelium without dysplasia or carcinoma (53% vs 33%, p=0.001). None of these differences was seen in the squamous cell carcinoma group. The immunohistochemical characteristics of the different histological subtypes were also distinct. Atrophic and oncocytic acini were diffusely and strongly positive for CK7, SOX2, SOX9 and CK5/6 (a progenitor cell phenotype) while mucinous acini showed weak or moderate staining for those markers. Our results suggest that submucosal glands play a role in the progression of neoplasia, possibly by offering less protection to the mucosal surface of the oesophageal epithelium from chemical injury.

Mismatch repair protein deficiency (MMR-D) and high microsatellite instability (MSI-H) are features of Lynch syndrome–associated colorectal carcinomas and have implications in clinical management. We evaluate the ability of a targeted next-generation sequencing panel to detect MMR-D and MSI-H based on mutational phenotype. Using a criterion of >40 total mutations per megabase or >5 single-base insertion or deletion mutations in repeats per megabase, sequencing achieves 92% sensitivity and 100% specificity for MMR-D by immunohistochemistry in a training cohort of 149 colorectal carcinomas and 91% sensitivity and 98% specificity for MMR-D in a validation cohort of 94 additional colorectal carcinomas. False-negative samples are attributable to tumor heterogeneity, and next-generation sequencing results are concordant with analysis of microsatellite loci by PCR. In a subset of 95 carcinomas with microsatellite analysis, sequencing achieves 100% sensitivity and 99% specificity for MSI-H in the combined training and validation set. False-positive results for MMR-D and MSI-H are attributable to ultramutated cancers with POLE mutations, which are confirmed by direct sequencing of the POLE gene and are detected by mutational signature analysis. These findings provide a framework for a targeted tumor sequencing panel to accurately detect MMR-D and MSI-H in colorectal carcinomas.

Genes encoding subunits of SWI/SNF (BAF) chromatin-remodeling complexes are collectively mutated in ∼20% of all human cancers. Although ARID1A is the most frequent target of mutations, the mechanism by which its inactivation promotes tumorigenesis is unclear. Here we demonstrate that Arid1a functions as a tumor suppressor in the mouse colon, but not the small intestine, and that invasive ARID1A-deficient adenocarcinomas resemble human colorectal cancer (CRC). These tumors lack deregulation of APC/β-catenin signaling components, which are crucial gatekeepers in common forms of intestinal cancer. We find that ARID1A normally targets SWI/SNF complexes to enhancers, where they function in coordination with transcription factors to facilitate gene activation. ARID1B preserves SWI/SNF function in ARID1A-deficient cells, but defects in SWI/SNF targeting and control of enhancer activity cause extensive dysregulation of gene expression. These findings represent an advance in colon cancer modeling and implicate enhancer-mediated gene regulation as a principal tumor-suppressor function of ARID1A.

Objective In an earlier study wherein we induced acute reflux by interrupting proton pump inhibitor (PPI) therapy in patients with reflux oesophagitis (RO) healed by PPIs, we refuted the traditional concept that RO develops as an acid burn. The present study explored our alternative hypothesis that RO results from reflux-stimulated production of pro-inflammatory molecules mediated by hypoxia-inducible factors (HIFs). Design Using oesophageal biopsies taken from patients in our earlier study at baseline and at 1 and 2 weeks off PPIs, we immunostained for HIF-1α, HIF-2α and phospho-p65, and measured pro-inflammatory molecule mRNAs. We exposed human oesophageal squamous cell lines to acidic bile salts, and evaluated effects on HIF activation, p65 function, pro-inflammatory molecule production and immune cell migration. Results In patient biopsies, increased immunostaining for HIF-2α and phospho-p65, and increased pro-inflammatory molecule mRNA levels were seen when RO redeveloped 1 or 2 weeks after stopping PPIs. In oesophageal cells, exposure to acidic bile salts increased intracellular reactive oxygen species, which decreased prolyl hydroxylase function and stabilised HIF-2α, causing a p65-dependent increase in pro-inflammatory molecules; conditioned media from these cells increased T cell migration rates. HIF-2α inhibition by small hairpin RNA or selective small molecule antagonist blocked the increases in pro-inflammatory molecule expression and T cell migration induced by acidic bile salts. Conclusions In patients developing RO, increases in oesophageal HIF-2α correlate with increased pro-inflammatory molecule expression. In oesophageal epithelial cells, acidic bile salts stabilise HIF-2α, which mediates expression of pro-inflammatory molecules. HIF-2α appears to have a role in RO pathogenesis. Trial registration number NCT01733810; Results.

The overall survival of gastric carcinoma patients remains poor despite improved control over known risk factors and surveillance. This highlights the need for new classifications, driven towards identification of potential therapeutic targets. Using sophisticated molecular technologies and analysis, three groups recently provided genetic and epigenetic molecular classifications of gastric cancer (The Cancer Genome Atlas, 'Singapore-Duke' study, and Asian Cancer Research Group). Suggested by these classifications, here, we examined the expression of 14 biomarkers in a cohort of 146 gastric adenocarcinomas and performed unsupervised hierarchical clustering analysis using less expensive and widely available immunohistochemistry and in situ hybridization. Ultimately, we identified five groups of gastric cancers based on Epstein-Barr virus (EBV) positivity, microsatellite instability, aberrant E-cadherin, and p53 expression; the remaining cases constituted a group characterized by normal p53 expression. In addition, the five categories correspond to the reported molecular subgroups by virtue of clinicopathologic features. Furthermore, evaluation between these clusters and survival using the Cox proportional hazards model showed a trend for superior survival in the EBV and microsatellite-instable related adenocarcinomas. In conclusion, we offer as a proposal a simplified algorithm that is able to reproduce the recently proposed molecular subgroups of gastric adenocarcinoma, using immunohistochemical and in situ hybridization techniques.

Background: No consensus guideline has been established for microsatellite instability testing in upper gastrointestinal tract cancers. This study aims to determine whether targeted cancer next-generation sequencing can accurately detect microsatellite instability in upper gastrointestinal tract cancers and screen for patients with Lynch syndrome. Methods: In a cohort of 645 upper gastrointestinal tract cancers, targeted next-generation sequencing assessed microsatellite instability by identifying characteristic insertion and deletion mutations. Sequencing classification was compared with mismatch repair protein IHC. Cancers with microsatellite instability by sequencing were analyzed using a testing protocol to identify patients with Lynch syndrome. Results: Sequencing identified microsatellite instability in 3.6% (23/645) of upper gastrointestinal tract cancers, including 28% (8/29) of small intestinal and 9% (9/97) of gastric carcinomas. In 20 cancers classified as having microsatellite instability, 19 demonstrated loss of expression of MLH1, PMS2, MSH2, or MSH6, and one cancer was indeterminate by IHC. In contrast, 52 control cancers demonstrated retained expression of all mismatch repair proteins. Using targeted sequencing as the initial screening test, 1.1% (7/645) of patients were identified to have pathogenic germline variants confirming a diagnosis of Lynch syndrome. Conclusions: Targeted cancer next-generation sequencing is an accurate first-line test to detect microsatellite instability in upper gastrointestinal tract cancers. Impact: This study provides a proof of concept for the use of targeted next-generation sequencing to detect microsatellite instability and screen for Lynch syndrome.

Complete response to neoadjuvant therapy, determined by pathologic examination of the resection specimen (pCR), is associated with a favorable outcome in esophageal adenocarcinomas (EAC), but there is significant heterogeneity in survival reported within this group. Our aim was to determine predictors of disease recurrence (DR) and survival in EAC patients with pCR to neoadjuvant therapy. A total of 93 EAC patients with pCR to neoadjuvant therapy were identified, and a predetermined set of clinicopathologic variables was examined, including patient age, sex, tumor location, pretreatment tumor size, endoscopic ultrasound T and N stage, histologic tumor type, and grade in pretreatment biopsies. The esophagectomy specimens were evaluated for the extent of sampling of the tumor bed, depth of treatment-related changes, presence of treatment effect in lymph nodes, and the total number of lymph nodes examined. Complete histologic examination of the tumor bed was the most significant predictor of favorable outcome for both DR (hazard ratio [HR]=0.42; 95% confidence interval [CI]: 0.21-0.82; P=0.011) and disease-specific mortality (HR=0.40; 95% CI: 0.22-0.70; P

Metaplastic epithelial cells of Barrett's esophagus transformed by the combination of p53-knockdown and oncogenic Ras expression are known to activate signal transducer and activator of transcription 3 (STAT3). When phosphorylated at tyrosine 705 (Tyr705), STAT3 functions as a nuclear transcription factor that can contribute to oncogenesis. STAT3 phosphorylated at serine 727 (Ser727) localizes in mitochondria, but little is known about mitochondrial STAT3's contribution to carcinogenesis in Barrett's esophagus, which is the focus of this study. We introduced a constitutively active variant of human STAT3 (STAT3CA) into the following: 1) non-neoplastic Barrett's (BAR-T) cells; 2) BAR-T cells with p53 knockdown; and 3) BAR-T cells that express oncogenic H-RasG12V. STAT3CA transformed only the H-RasG12V-expressing BAR-T cells (evidenced by loss of contact inhibition, formation of colonies in soft agar, and generation of tumors in immunodeficient mice), and did so in a p53-independent fashion. The transformed cells had elevated levels of both mitochondrial (Ser727) and nuclear (Tyr705) phospho-STAT3. Introduction of a STAT3CA construct with a mutated tyrosine phosphorylation site into H-RasG12V-expressing Barrett's cells resulted in high levels of mitochondrial phospho-STAT3 (Ser727) with little or no nuclear phospho-STAT3 (Tyr705), and the cells still formed tumors in immunodeficient mice. Thus tyrosine phosphorylation of STAT3 is not required for tumor formation in Ras-expressing Barrett's cells. We conclude that mitochondrial STAT3 (Ser727) can contribute to oncogenesis in Barrett's cells that express oncogenic Ras. These findings suggest that agents targeting STAT3 might be useful for chemoprevention in patients with Barrett's esophagus.

Gastroesophageal adenocarcinoma (GEA) is a lethal disease where targeted therapies, even when guided by genomic biomarkers, have had limited efficacy. A potential reason for the failure of such therapies is that genomic profiling results could commonly differ between the primary and metastatic tumors. To evaluate genomic heterogeneity, we sequenced paired primary GEA and synchronous metastatic lesions across multiple cohorts, finding extensive differences in genomic alterations, including discrepancies in potentially clinically relevant alterations. Multiregion sequencing showed significant discrepancy within the primary tumor (PT) and between the PT and disseminated disease, with oncogene amplification profiles commonly discordant. In addition, a pilot analysis of cell-free DNA (cfDNA) sequencing demonstrated the feasibility of detecting genomic amplifications not detected in PT sampling. Lastly, we profiled paired primary tumors, metastatic tumors, and cfDNA from patients enrolled in the personalized antibodies for GEA (PANGEA) trial of targeted therapies in GEA and found that genomic biomarkers were recurrently discrepant between the PT and untreated metastases. Divergent primary and metastatic tissue profiling led to treatment reassignment in 32% (9/28) of patients. In discordant primary and metastatic lesions, we found 87.5% concordance for targetable alterations in metastatic tissue and cfDNA, suggesting the potential for cfDNA profiling to enhance selection of therapy. Significance: We demonstrate frequent baseline heterogeneity in targetable genomic alterations in GEA, indicating that current tissue sampling practices for biomarker testing do not effectively guide precision medicine in this disease and that routine profiling of metastatic lesions and/or cfDNA should be systematically evaluated. Cancer Discov; 8(1); 37–48. ©2017 AACR. See related commentary by Sundar and Tan, p. 14. See related article by Janjigian et al., p. 49. This article is highlighted in the In This Issue feature, p. 1

Induction therapy followed by esophagectomy has become standard for treatment of intermediate-stage esophageal cancer in many centers. Herein we evaluate the feasibility and safety of the 3-hole minimally invasive esophagectomy (3HMIE) approach in patients who received induction radiation and chemotherapy. Between 2003 and 2012, the records of 119 consecutive patients with esophageal cancer who underwent 3HMIE were reviewed for perioperative complications and long-term outcomes. Comparison was made between procedures performed for patients receiving neoadjuvant chemoradiation and patients who were treated with only surgery. Of them, 78 patients received neoadjuvant chemoradiation and 41 patients were treated with only surgery. Tumor locations were upper (2), middle (16), distal (64), and gastroesophageal junction (37). In all, 76 patients were at clinical stage IIA or above at presentation. Increased requirement for blood replacement in the induction therapy group was significant compared with the surgery-only group. Operative time, estimated blood loss, proximal and distal margin lengths, and length of stay were not significantly different between the cohorts. There was a 30-day perioperative death (0.8%), and this patient was from the surgery-only group. No conduit necrosis or need for diversion was recorded. Overall, 5-year survival was 62% among the 107 patients with early-stage esophageal cancer. 3HMIE is feasible with low mortality and acceptable morbidity even in patients with locally advanced esophageal cancer who received neoadjuvant radiochemotherapy. Overall perioperative and survival outcomes are similar to or better than those reported in the published literature on esophagectomy after induction therapy.

Aims Esophageal adenocarcinoma (EAC) tumorigenesis has been primarily linked to loss-of-function mutations in tumor suppressor genes. Knowledge of specific oncogenes that drive tumor progression, and their relationship to outcomes, is limited. High Mobility Group AT-Hook 2 (HMGA2) has been reported to be amplified in a subset of EACs, but the clinicopathologic and prognostic implications of HMGA2 expression in EAC is unknown. Methods and Results We performed HMGA2 immunohistochemistry and fluorescence in-situ hybridization (FISH) in EAC to determine its clinicopathologic and prognostic significance. Ninety-one primary EAC resections without neoadjuvant treatment were identified and immunohistochemistry for HMGA2 was performed. The presence or absence of nuclear staining was evaluated and correlated with predetermined clinicopathologic parameters and patient outcomes. A selected subset of tumors was subjected to FISH to identify alterations at the HMGA2 locus. HMGA2 expression was present in 25/91 (27.4%) tumors. HMGA2 expressing cells were present in solid, poorly differentiated areas of the tumors at the invasive front, or as single infiltrating cells. FISH showed that 3-4 copies of HMGA2 are frequently present in EAC irrespective of HMGA2 protein expression and that high level HMGA2 amplification is a rare event. HMGA2 expression was associated with numerous adverse clinicopathologic parameters including higher T- and N-stage, the presence of lymphovascular invasion, and with a worse recurrence free and overall survival. Conclusion Our data suggests that HMGA2 is primarily regulated in EAC through non-chromosomal level alterations that lead to increased HMGA2 expression. HMGA2 positive EAC correlates with adverse pathologic features and worse patient outcomes. This article is protected by copyright. All rights reserved.

Biologic agents are effective treatments for rheumatoid arthritis but are associated with important risks, including severe infections. Tumor Necrosis Factor (TNF) α inhibitors are known to increase the risk of systemic fungal infections such as disseminated histoplasmosis. Abatacept is a biologic agent with a mechanism different from that of TNFα inhibitors: It suppresses cellular immunity by competing for the costimulatory signal on antigen-presenting cells. The risk of disseminated histoplasmosis for patients on abatacept is not known. We report a case of abatacept-associated disseminated histoplasmosis and review the known infectious complications of abatacept. While the safety of resuming biologic agents following treatment for disseminated histoplasmosis is also not known, abatacept is recommended over TNFα inhibitors for rheumatoid arthritis patients with a prior serious infection. We discuss the evidence supporting this recommendation and discuss alternative treatments for rheumatoid arthritis patients with a history of a serious infection.

Context.— The clinicopathologic and prognostic significance of ARID1A mutation in esophageal adenocarcinoma (EAC) is unknown. Objective.— To determine the morphological correlates and prognostic significance of ARID1A-deficient EAC. Design.— One hundred twenty cases of primary EAC were evaluated for a predetermined set of histologic features and immunohistochemistry for ARID1A, p53, and MLH1 performed on EAC, as well as adjacent Barrett esophagus and Barrett esophagus–associated dysplasia, when feasible. Associations between categorical clinicopathologic variables were analyzed by Fisher exact test, and survival analysis was performed by a Cox proportional hazards analysis. Results.— The study group included 97 men and 23 women (mean age, 66 years). Loss of ARID1A expression was seen in 12 of 120 EACs (10%). ARID1A-deficient tumors showed a strong correlation with a medullary and mucinous phenotype, and 8 of 12 (67%) had at least one feature reminiscent of high microsatellite instability colon carcinomas (mucinous or medullary differentiation, marked intratumoral or peritumoral lymphoid infiltrate). A mutant p53 pattern was present in 52 of 120 EACs (43%) and showed no correlation with ARID1A deficiency (P > .05). MLH1 loss was present in only 2 of 120 EACs (2%); both of which were also deficient in ARID1A. ARID1A-deficient EACs showed a trend toward increased risk of nodal metastasis but had no effect on overall patient survival. Conclusions.— ARID1A-deficient EACs show a phenotype similar to colon cancer with high microsatellite instability but do not appear to have any prognostic significance. Concurrent MLH1 loss is not seen in most ARID1A-deficient tumors, suggesting that ARID1A may be a primary driver of carcinogenesis in a subset of EACs.

Importance The histologic changes associated with acute gastroesophageal reflux disease (GERD) have not been studied prospectively in humans. Recent studies in animals have challenged the traditional notion that reflux esophagitis develops when esophageal surface epithelial cells are exposed to lethal chemical injury from refluxed acid. Objective To evaluate histologic features of esophageal inflammation in acute GERD to study its pathogenesis. Design, Setting, and Participants Patients from the Dallas Veterans Affairs Medical Center who had reflux esophagitis successfully treated with proton pump inhibitors (PPIs) began 24-hour esophageal pH and impedance monitoring and esophagoscopy (including confocal laser endomicroscopy [CLE]) with biopsies from noneroded areas of distal esophagus at baseline (taking PPIs) and at 1 week and 2 weeks after stopping the PPI medication. Enrollment began May 2013 and follow-up ended July 2015. Interventions PPIs stopped for 2 weeks. Main Outcomes and Measures Twelve patients (men, 11; mean age, 57.6 year [SD, 13.1]) completed the study. Primary outcome was change in esophageal inflammation 2 weeks after stopping the PPI medication, determined by comparing lymphocyte, eosinophil, and neutrophil infiltrates (each scored on a 0-3 scale) in esophageal biopsies. Also evaluated were changes in epithelial basal cell and papillary hyperplasia, surface erosions, intercellular space width, endoscopic grade of esophagitis, esophageal acid exposure, and mucosal impedance (an index of mucosal integrity). Results At 1 week and 2 weeks after discontinuation of PPIs, biopsies showed significant increases in intraepithelial lymphocytes, which were predominantly T cells (median [range]: 0 (0-2) at baseline vs 1 (1-2) at both 1 week [ P = .005] and 2 weeks [ P = .002]); neutrophils and eosinophils were few or absent. Biopsies also showed widening of intercellular spaces (confirmed by CLE), and basal cell and papillary hyperplasia developed without surface erosions. Two weeks after stopping the PPI medication, esophageal acid exposure increased (median: 1.2% at baseline to 17.8% at 2 weeks; Δ, 16.2% [95% CI, 4.4%-26.5%], P = .005), mucosal impedance decreased (mean: 2671.3 Ω at baseline to 1508.4 Ω at 2 weeks; Δ, 1162.9 Ω [95% CI, 629.9-1695.9], P = .001), and all patients had evidence of esophagitis. Conclusions and Relevance In this preliminary study of 12 patients with severe reflux esophagitis successfully treated with PPI therapy, stopping PPI medication was associated with T lymphocyte–predominant esophageal inflammation and basal cell and papillary hyperplasia without loss of surface cells. If replicated, these findings suggest that the pathogenesis of reflux esophagitis may be cytokine-mediated rather than the result of chemical injury. Trial Registration clinicaltrials.gov Identifier:NCT01733810.

Huang Q, Fan X, Agoston A T, Feng A, Yu H, Lauwers G, Zhang L & Odze R D (2011) Histopathology59, 188–197 Comparison of gastro-oesophageal junction carcinomas in Chinese versus American patients Aims: To compare the clinical and pathological features of gastro-oesophageal junction (GEJ) carcinomas in Chinese and American patients. Methods and results: Eighty consecutive patients with a GEJ carcinoma (43 from mainland China, and 37 from the USA) were evaluated for association with Barrett oesophagus (BO), chronic Helicobacter pylori gastritis, intestinal metaplasia, and outcome. GEJ carcinomas were defined as tumours that were located within 20 mm of, and crossed, the GEJ. Overall, GEJ carcinomas from Chinese patients revealed significantly more frequent location in the proximal stomach, higher pathological stage, larger size, younger patient age, and association with chronic H. pylori gastritis. In contrast, GEJ cancers from American patients showed a strong association with distal oesophageal location, BO, and associated intestinal metaplasia and dysplasia. Pathologically, GEJ carcinomas from American patients were predominantly adenocarcinomas, whereas Chinese patients showed a higher proportion of mucinous, adenosquamous, acinar or neuroendocrine tumours. Overall, 3- and 5-year survival rates were statistically similar between both patient groups, but upon multivariate analysis, Chinese patients showed statistically better survival rates for stage III tumours. Conclusions: Most GEJ carcinomas in patients from China represent proximal gastric cancers associated with chronic H. pylori gastritis, and BO-associated carcinomas are rare among this patient population.

Background & Aims Metaplastic glands buried under squamous epithelium are frequently detected in patients with Barrett esophagus (BE). This subsquamous intestinal metaplasia might be responsible for cancers that develop despite endoscopic surveillance and for metaplasia recurrences after endoscopic ablation. To determine whether reflux induces BE cells to undergo an epithelial-to-mesenchymal transition (EMT) that produces subsquamous intestinal metaplasia, we assessed EMT in BE cells exposed to acidic bile salts and in rat and human esophageal tissues. Methods We compared markers of EMT and cell motility in trans-well and 3-dimensional organotypic culture systems among dysplastic BE epithelial cell lines, nondysplastic telomerase-immortalized BE cell lines (BAR-T), and BAR-T cells exposed acutely or for 20 weeks to acidic bile salts. Vascular endothelial growth factor (VEGF) A was inhibited with a neutralizing antibody or CRISPR-Cas9n and VEGF receptor 2 was inhibited with SU1498 or shRNA, and cells were analyzed by immunohistochemistry, quantitative polymerase chain reaction, or immunoblotting for markers of VEGF signaling and EMT; cell motility was assessed by trans-well assay. We used immunohistochemistry and quantitative polymerase chain reaction to assess EMT markers in the columnar-lined esophagus of rats with surgically induced reflux esophagitis and in esophagectomy specimens from patients with BE. Results We detected features of EMT (decreased cadherin 1 [CDH1]; increased fibronectin 1, vimentin, and matrix metalloproteinase 2; and increased motility) in dysplastic BE epithelial cell lines and in BAR-T cells exposed for 20 weeks, but not in unexposed BAR-T cells. Acute acidic bile salt exposure induced expression of zinc finger E-box binding homeobox 1 and 2 (ZEB1/2) in BAR-T cells, which decreased their expression of CDH1 and increased motility; inhibitors of VEGF signaling blocked these effects. Columnar-lined esophagus of rats with reflux esophagitis had increased expression of ZEB1/2 and decreased expression of CDH1 compared with controls. Dysplastic BE tissues also had significantly increased levels of ZEB1 and significantly decreased levels of CDH1 compared with nondysplastic BE tissues. Conclusions In BE cell lines, acidic bile salts induce EMT by VEGF signaling, which increases expression of ZEB1/2, repressors of CDH1. These observations suggest that reflux induces EMT in metaplastic BE tissues, which promotes development of subsquamous intestinal metaplasia.

Pilomatricoma, also known as 'calcifying epithelioma of Malherbe', is a common skin adnexal tumor that mimics hair growth. Its proliferating cells seem distinctly programmed to undergo terminal differentiation and death. We report the first cytogenetic investigations of pilomatricoma. Trisomy 18 was shown, in an index case, by G-banded karyotyping. This aberration was corroborated by interphase fluorescence in situ hybridization, using a chromosome 18 pericentromeric probe, in the basaloid epithelial component of 7 of 11 pilomatricomas, including the index case. Trisomy 18 was present in a small subset of cells, suggesting a role in pilomatricoma progression, rather than in tumor initiation. We conclude that trisomy 18 is a consistent feature in pilomatricoma, suggesting that genes carried on this chromosome, such as that for the antiapoptotic oncoprotein BCL2, may have a role in the growth and differentiation of this benign self-limited tumor.

We probed the clinicopathologic context of commercially available cell lines using sequencing data from the recently released Metastatic Breast Cancer (MBC) Project. Additionally, we identified cohorts in the MBC with similar profiles to the Wood cell model we recently derived and investigated alignment between clinicopathologic characteristics of these patients and the patient from which the model was derived. Using a validated cancer panel, we analyzed 592 genes in the Wood cell model detecting mutations and CNAs. We then accessed mutation and CNA data from 1) the MBC Project for 103 primary and metastatic samples from 78 patients and 2) the Cancer Cell Line Encyclopedia (CCLE) database for 56 breast cancer cell lines. We performed unsupervised clustering on the 159 total specimens, incorporating both mutation and copy number data in the clustering analysis by labeling genes either altered or unaltered. MBC patient specimens were grouped based on the clustering results and assessed for trends across multiple clinicopathologic data categories. Specimens representing metastatic sites and the primary site from the same patient were both included in the analysis. Clustering analysis revealed three major groups representing high, medium and low alteration levels (means of 237, 104 and 29 panel genes altered). The High mutation load group had 34 cell lines and 6 patient samples, the Medium group had 15 cell lines and 71 patent samples, and the Low group had 7 cell lines (including Wood) and 26 patient samples. The 6 patient specimens in the High group represented 5.8% of the MBC data, yet were represented by a majority (60.7%) of CCLE breast cancer lines. All patients in the High group self-reported extraordinary response to treatment; the other two groups had even distribution. None of the specimens in the High group had evidence of DCIS reported in the path report, whereas observed DCIS was distributed equally outside of the High group. Expectedly, markers of aggressiveness of disease were lower in the Low group compared to the rest of the MBC patients, including histologic grade (p=0.00072) and Ki67 percentage (p=0.026). The Low group MBC samples were more likely to be HER2(-) (p=0.02) and ER(+) (p=0.037), consistent the Wood patient. The eight patients clustering most closely with the Wood cell model had low N-stage disease when compared to the rest (p=0.036) which was consistent with the Wood patient N0 status. CCLE breast cancer cell lines over-represent a small MBC patient subset with a very high load of genomic alterations that also trends toward exceptionally good response to treatment- consistent with the notion that cancer cell lines over-predict drug efficacy. Complete lack of DCIS in the High group is an interesting observation that may describe a disease progression resulting in absent or minimal DCIS precursor. As more MBC patient data are released, these analyses will be repeated to increase statistical power. Citation Format: Rick Nicoletti, Pamela Shaw, Naghmeh Salimi, Agoston T. Agoston, Elin S. Agoston. Genomic representation of metastatic breast cancer patients by commercially available cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1054.

Histone deacetylases (HDAC) play a critical role in chromatin modification and gene expression. Recent evidence indicates that HDACs can also regulate functions of nonhistone proteins by catalyzing the removal of acetylated lysine residues. Here, we show that the HDAC inhibitor LBH589 down-regulates DNA methyltransferase 1 (DNMT1) protein expression in the nucleus of human breast cancer cells. Cotreatment with the proteasomal inhibitor MG-132 abolishes the ability of LBH589 to reduce DNMT1, suggesting that the proteasomal pathway mediates DNMT1 degradation on HDAC inhibition. Deletion of the NH2-terminal 120 amino acids of DNMT1 diminishes LBH589-induced ubiquitination, indicating that this domain is essential for its proteasomal degradation. DNMT1 recruits the molecular chaperone heat shock protein 90 (Hsp90) to form a chaperone complex. Treatment with LBH589 induces hyperacetylation of Hsp90, thereby inhibiting the association of DNMT1 with Hsp90 and promoting ubiquitination of DNMT1. In addition, inactivation of HDAC1 activity by small interfering RNA and MS-275 is associated with Hsp90 acetylation in conjunction with reduction of DNMT1 protein expression. We conclude that the stability of DNMT1 is maintained in part through its association with Hsp90. Disruption of Hsp90 function by HDAC inhibition is a unique mechanism that mediates the ubiquitin-proteasome pathway for DNMT1 degradation. Our studies suggest a new role for HDAC1 and identify a novel mechanism of action for the HDAC inhibitors as down-regulators of DNMT1. (Mol Cancer Res 2008;6(5):873–83)

We have examined the mechanism of normal DNA methyltransferase 1 (DNMT1) degradation as well as its mechanism of dysregulation in cancer. We have previously reported that DNMT1 protein levels were elevated and abnormally stabilized because of defective degradation through its N-terminal destruction domain. Here, we report that DNMT1 was abnormally stabilized in several cancer cell lines and that, in cells with normal DNMT1 destruction, depletion of CDC20 or FZR1 (two substrate recognition adaptor components of the anaphase-promoting complex) resulted in stabilization of DNMT1 that was partially dependent on the N-terminal destruction domain, thus implicating this cell cycle regulator in the destruction of DNMT1. MAD2, an inhibitor of CDC20, was shown to stabilize DNMT1 levels, and overexpression of MAD2, a consequence of retinoblastoma (RB) pathway dysregulation, was shown to correlate with impaired G1 phase DNMT1 destruction and RB inactivation by hyperphosphorylation in several normal and cancer cell lines. Furthermore, in a series of 85 cases of human breast cancer, a moderately strong, but highly significant, correlation between MAD2 and DNMT1 immunohistochemical staining was observed, yielding a Spearman rank order correlation coefficient of 0.37 (P < 0.001). This suggests that RB pathway inactivation, a common dysfunction in cancer cells, may be the underlying cause of DNMT1 dysregulation.

Radiofrequency ablation (RFA), with or without endoscopic mucosal resection (EMR), is a safe, effective, and durable treatment option for Barrett esophagus (BE)-associated dysplasia (DYS), but few studies have identified predictors of treatment failure in BE-associated intramucosal adenocarcinoma (IMC). The aim of this study was to determine the rate of IMC eradication when using RFA±EMR and to identify clinical and pathologic predictors of treatment failure. A retrospective review of medical records and a central review of index histologic parameters were performed for 78 patients who underwent RFA±EMR as the primary treatment for biopsy-proven IMC at 4 academic tertiary medical centers. Complete eradication (CE) (absence of IMC/DYS on first follow-up endoscopy) was achieved in 86% of patients, and durable eradication (DE) (CE with no recurrence of IMC/DYS until last follow-up) was achieved in 78% of patients, with significant variation between the 4 study sites (P=0.03 and 0.09 by analysis of variance for DE and CE, respectively). Use of EMR before RFA significantly reduced the risk for treatment failure for IMC/DYS (hazard ratio, 0.15; 95% confidence interval, 0.05-0.48; P=0.001), whereas IMC involving ≥50% of the columnar metaplastic area on index examination significantly increased the risk for treatment failure (hazard ratio, 4.24; 95% confidence interval, 1.53-11.7; P=0.005). Endoscopic and pathologic factors associated with treatment failure in BE-associated IMC treated with RFA±EMR may help identify the subset of IMC patients for whom a more aggressive initial approach may be justified.

Barrett's esophagus is thought to progress to esophageal adenocarcinoma (EAC) through a stepwise progression with loss of CDKN2A followed by TP53 inactivation and aneuploidy. Here we present whole-exome sequencing from 25 pairs of EAC and Barrett's esophagus and from 5 patients whose Barrett's esophagus and tumor were extensively sampled. Our analysis showed that oncogene amplification typically occurred as a late event and that TP53 mutations often occurred early in Barrett's esophagus progression, including in non-dysplastic epithelium. Reanalysis of additional EAC exome data showed that the majority (62.5%) of EACs emerged following genome doubling and that tumors with genomic doubling had different patterns of genomic alterations, with more frequent oncogenic amplification and less frequent inactivation of tumor suppressors, including CDKN2A. These data suggest that many EACs emerge not through the gradual accumulation of tumor-suppressor alterations but rather through a more direct path whereby a TP53-mutant cell undergoes genome doubling, followed by the acquisition of oncogenic amplifications., National Human Genome Research Institute (U.S.) Large-Scale Sequencing Program (Grant U54 HG0003067)

Ipilimumab is a monoclonal antibody that inhibits the CTLA4 receptor on cytotoxic T lymphocytes, resulting in immune-mediated tumor cell death. Ipilimumab is most often used in the treatment of metastatic melanoma, and rarely liver toxicity necessitating cessation of treatment occurs. The aim of this study was to characterize the histologic features and clinical course of ipilimumab-associated hepatitis. Eleven patients with clinical suspicion of ipilimumab-induced hepatitis, due to the development of abnormal liver function tests (LFTs) while receiving treatment, and who underwent liver biopsy, were identified over a 6-year period. Ten patients were male and 1 female (median age 58 y), and all received 1 to 4 doses of ipilimumab. None had known preexisting liver disease. Two patients were obese, and another had a history of alcohol abuse. Viral and autoimmune serologies were negative in all patients except 1 who had a mildly elevated ANA titer. Nine biopsies showed active hepatitis with 2 distinct histologic patterns: panlobular hepatitis in 6 cases and zone 3 hepatitis in 3. The inflammatory infiltrate was similar in composition in both patterns, composed predominantly of CD8+ T lymphocytes, admixed histiocytes, scattered plasma cells, and eosinophils. Prominent histiocytic sinusoidal infiltrates were present in 7 cases and frequently formed loose histiocytic aggregates. Central vein endothelialitis was present in 8 cases. Patients in this group tended to have markedly elevated ALT, AST, and total bilirubin. Two cases did not fit into the above 2 histologic groups: 1 showed portal inflammation with cholangitis, and the other showed morphologic features indistinguishable from nonalcoholic steatohepatitis. Discontinuation of ipilimumab and administration of immunosuppressives resulted in resolution or marked improvement of LFTs in all patients within 3 months of presentation. Ipilimumab may potentially unmask previously subclinical liver disease, for example, fatty liver disease, and the diagnosis of ipilimumab-induced liver injury may only be recognized with certainty after cessation of the drug leads to normalization of LFTs. Overall, ipilimumab-associated hepatitis most often presents with a panlobular active hepatitis that resembles autoimmune hepatitis. Prominent sinusoidal histiocytic infiltrates and central vein damage with endothelialitis may be helpful histologic clues to the diagnosis of ipilimumab-associated hepatitis.

We report that DNA methyltransferase 1 (DNMT1) expression is dysregulated in breast cancer. The elevated protein levels are not a result of increased mRNA levels, but rather an increase in protein half-life. We found that DNMT1 protein levels were elevated in breast cancer tissues and in MCF-7 breast cancer cells relative to normal human mammary epithelial cells (HMECs) without a concomitant increase in DNMT1 mRNA or proliferative fraction. Although DNMT1 mRNA levels were properly S-phase-regulated in both cell types, DNMT1 protein levels did not follow S-phase fraction in MCF-7 cells. Rather, an increase in DNMT1 protein stability was found for MCF-7 cells relative to HMECs, and a destruction domain was mapped to the N-terminal 120 amino acids of DNMT1, which was required for its proper ubiquitination and degradation in HMECs. Furthermore, overexpression of DNMT1 with this deleted destruction domain in HMECs resulted in significantly increased genomic 5-methylcytosine levels relative to overexpression of the full-length protein. The regulation of DNMT1 destruction via this domain may be dysfunctional in cancer cells leading to subsequent cytosine hypermethylation in the genome.

Although relevant, the genomic and proteomic alterations between original tumor tissue and newly established cell lines are frequently uncharacterized. In developing a novel cell model (Powder) of high grade serous carcinoma (HGSC), we followed the status of clinically relevant biomarkers to identify changes taking place between the tissue and cultured cell population. Understanding these changes enables proper utilization of new models and continued model optimization. Using a validated panel, we analyzed 592 genes in the Powder cell model and tumor from which it was derived, detecting several classes of genomic alterations. This included point mutations, indels, copy number variations, fusions, and variant transcripts. p53 mutation status of Powder was matched to a panel of previously reported HGSC cell lines. We further subdivided the panel by p53 protein expression level by reanalyzing previously published reverse phase protein array (RPPA) data. Significance of differential expression between groups was determined with a Welch's t-test. A majority (14/21 or 67%) of the mutations detected in the HGSC tumor were conserved in the Powder cell model. Two additional mutations, undetected in the tumor, were found in the cell model. The tumor exhibited moderate to strong diffuse positive p53 immunostaining, and p53 mutations were measured in 79% of alleles. While the p53 mutation did not carry to the cell model, the cells were positive for CK7 and PAX8, consistent with ovarian cancer cells. In the panel of HGSC cell lines, we examined p53 mutation status and protein level, identifying 3 groups: (1) p53-mut/high protein, (2) p53-mut/low protein and (3) p53-WT/low protein. Powder identified with the p53-WT/low protein group. Previous work identifies the p53-WT/low protein group of cell models as part of a larger set of novel ovarian cell models with a stem-like molecular profile and higher drug resistance. Analysis of differential protein expression revealed upregulation in the p53 low protein group of both normal cell cycle genes and of notable cancer-related proteins. This included p21 and BAD, which are upregulated in cells with functional p53, eIF4E, a candidate cancer therapeutic target, and BOP1, which is disregulated in multiple solid cancers, including ovarian. These results underscore the need for further characterization of the Powder cell model to clarify its applications to specific research questions. Continued examination of Powder and other p53-WT cell models derived from HGSC tumors may uncover independent pathways driving disease. Additionally, understanding which mutations are underrepresented by cell models is necessary for ongoing methods development. Citation Format: Pamela S. Shaw, Rick Nicoletti, Naghmeh Salimi, Helen L. Yang, Abigail E. Witt, Agoston T. Agoston, Elin S. Agoston. Loss and retention of mutations in cell culture model systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 821. doi:10.1158/1538-7445.AM2017-821

Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are collectively mutated in ~20% of all human cancers, but the mechanism by which these mutations promote tumorigenesis is unclear. In this study, we investigate the tumor suppressor role of ARID1A, the subunit of SWI/SNF complexes that is the most frequent target of mutations. We develop an inducible Arid1a-knockout mouse model and find that mice develop invasive ARID1A-deficient colon adenocarcinoma. These cancers show prominent mucinous differentiation and tumor-infiltrating lymphocytes, features associated particularly with microsatellite-instable (MSI) human colorectal cancer - the subtype with the highest frequency of inactivating ARID1A mutations (37-39%). Although deregulation of Wnt signaling components (APC/β-catenin) is commonly utilized to model intestinal tumorigenesis, Arid1a-knockout mice reflect human colorectal cancer with greater accuracy. These mice do not show deregulation of Wnt signaling components, thus demonstrating a novel pathway to colon tumorigenesis that is independent of established models. Chromatin immunoprecipitation sequencing reveals that SWI/SNF complexes are targeted primarily to enhancers, where they function in coordination with transcription factors to activate gene expression. ARID1A loss impairs the targeting of SWI/SNF complexes to thousands of enhancers, which subsequently lose activity - showing reduced levels of H3K27ac and expression of nearest genes. Residual SWI/SNF complexes in ARID1A-deficient cells bind enhancers that remain active; these complexes contain ARID1B, the subunit that is mutually exclusive with ARID1A and has been identified as a therapeutic target in ARID1A-deficient cancers. Enhancers associated with developmental gene expression programs are most affected by ARID1A loss, both in the mouse colonic epithelium and in human colon cancer cells. Collectively, these findings represent an advance in colon cancer modeling and implicate enhancer-mediated gene regulation as the principal tumor suppressor function of ARID1A. Citation Format: Radhika Mathur, Burak H. Alver, Adrianna K. San Roman, Boris G. Wilson, Xiaofeng Wang, Agoston T. Agoston, Peter Park, Ramesh Shivdasani, Charles W. Roberts. ARID1A loss impairs enhancer-mediated gene regulation and drives colon cancer in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-286. doi:10.1158/1538-7445.AM2017-LB-286

Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy., Despite methodological improvements, it remains extremely difficult to routinely establish cell lines from human primary tumours. Here, the authors develop a culture medium that allows efficient cell line establishment and use this to generate 25 ovarian cancer cell lines.

The tumor suppressor gene MEN1 is frequently mutated in sporadic pancreatic neuroendocrine tumors (PanNET) and is responsible for the familial multiple endocrine neoplasia type 1 (MEN-1) cancer syndrome. Menin, the protein product of MEN1, associates with the histone methyltransferases (HMT) MLL1 (KMT2A) and MLL4 (KMT2B) to form menin–HMT complexes in both human and mouse model systems. To elucidate the role of methylation of histone H3 at lysine 4 (H3K4) mediated by menin–HMT complexes during PanNET formation, genome-wide histone H3 lysine 4 trimethylation (H3K4me3) signals were mapped in pancreatic islets using unbiased chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq). Integrative analysis of gene expression profiles and histone H3K4me3 levels identified a number of transcripts and target genes dependent on menin. In the absence of Men1, histone H3K27me3 levels are enriched, with a concomitant decrease in H3K4me3 within the promoters of these target genes. In particular, expression of the insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) gene is subject to dynamic epigenetic regulation by Men1-dependent histone modification in a time-dependent manner. Decreased expression of IGF2BP2 in Men1-deficient hyperplastic pancreatic islets is partially reversed by ablation of RBP2 (KDM5A), a histone H3K4-specific demethylase of the jumonji, AT-rich interactive domain 1 (JARID1) family. Taken together, these data demonstrate that loss of Men1 in pancreatic islet cells alters the epigenetic landscape of its target genes. Implications: Epigenetic profiling and gene expression analysis in Men1-deficient pancreatic islet cells reveals vital insight into the molecular events that occur during the progression of pancreatic islet tumorigenesis. Mol Cancer Res; 13(4); 689–98. ©2014 AACR.

Type 3 deiodinase (D3), the physiologic inactivator of thyroid hormones, is induced during tissue injury and regeneration. This has led to the hypotheses that D3 impacts injury tolerance by reducing local T3 signaling and contributes to the fall in serum triiodothyronine (T3) observed in up to 75% of sick patients (termed the low T3 syndrome). Here we show that a novel mutant mouse with hepatocyte-specific D3 deficiency has normal local responses to toxin-induced hepatonecrosis, including normal degrees of tissue necrosis and intact regeneration, but accelerated systemic recovery from illness-induced hypothyroxinemia and hypotriiodothyroninemia, demonstrating that peripheral D3 expression is a key modulator of the low T3 syndrome.

Most gastric cancers develop via an intestinal metaplasia (IM)-dysplasia-carcinogenic pathway. We have noted that some patients with chronic gastritis have dysplasia-like atypia (DLA) limited to the pit epithelium but without involvement of the surface epithelium. We performed this study to determine the clinical and biological characteristics and outcome of DLA, to gain insight into its role in the pathogenesis of gastric cancer. The study consisted of 102 consecutive patients with resected gastric cancer, a separate cohort of patients (n = 166) with chronic gastritis and IM in their index gastric biopsies, and 44 controls. All specimens were evaluated for clinical and pathologic features of the cancer (in the resection cohort) and background mucosa. Of 102 patients with gastric cancer, 50 (49%) had DLA in areas of mucosa adjacent to or near either conventional dysplasia or cancer. This value was significantly higher than controls (DLA 6.8%; P < .0001). Gastric cancer patients with DLA showed a significantly higher age at presentation; intestinal-type adenocarcinoma; low-grade differentiation; stage 1 tumors; and a higher rate of chronic gastritis, IM, atrophy, and conventional dysplasia in the background mucosa compared to patients without DLA. DLA showed intestinal-type differentiation, and a higher Ki-67 rate and MUC6 positivity compared with IM. Of the 166 patients with biopsies, DLA was identified in 24 (14%). Upon follow-up, 38% of positive cases showed persistent DLA, whereas 25% progressed to conventional low-grade dysplasia. Based on these results, we conclude that DLA represents an important precursor lesion in gastric carcinogenesis and supports its interpretation as a neoplastic lesion.