Transcriptionally Active Human Papillomavirus Infection in a Minority of Esophageal Squamous Cell Carcinomas in North America.

The role of Human papillomavirus (HPV) infection in esophageal squamous cell carcinoma (ESCC) is a topic of ongoing debate. This study used two screening approaches to look for evidence of HPV infection in esophageal squamous cell carcinoma. We initially checked for HPV infection in a randomly selected group of 53 ESCC cases. We did not detect any tumors positive for high-risk HPV. However, during clinical practice, we identified an HPV-positive ESCC in the distal esophagus, which tested positive for HPV16. This index case was TP53 wild-type, as determined by next-generation DNA sequencing (NGS). Since TP53 mutations are rare in other HPV-driven cancers, we improved our screening method by limiting our screen to a subset of ESCC cases without TP53 mutations. A second screen of 95 ESCCs (from 93 patients) sequenced by NGS revealed an additional 7 ESCCs with TP53 wild-type status (7.3% of the total). Of the 7 cases, 2 cases were found to be high-risk HPV positive. Both patients also tested positive for circulating cell-free HPV DNA and had a complete response to neoadjuvant chemoradiation. The index patient had microscopic residual tumor following neoadjuvant therapy. The patient underwent adjuvant immunotherapy and remained disease free after 22 months of surveillance. This study affirms the transcriptionally active status of high-risk HPV in a minority of ESCC patients in North America.

Hypoxia-inducible factor-1α mediates reflux-induced epithelial-mesenchymal plasticity in Barrett's oesophagus patients.

INTRODUCTION: Epithelial-mesenchymal plasticity (EMP), the process through which epithelial cells acquire mesenchymal features, is needed for wound repair but also might contribute to cancer initiation. Earlier, in vitro studies showed that Barrett's cells exposed to acidic bile salt solutions (ABS) develop EMP. Now, we have (1) induced reflux oesophagitis in Barrett's oesophagus (BO) patients by stopping proton pump inhibitors (PPIs), (2) assessed their biopsies for EMP and (3) explored molecular pathways underlying reflux-induced EMP in BO cells and spheroids. METHODS: 15 BO patients had endoscopy with biopsies of Barrett's metaplasia while on PPIs, and 1 and 2 weeks after stopping PPIs; RNA-seq data were assessed for enrichments in hypoxia-inducible factors (HIFs), angiogenesis and EMP pathways. In BO biopsies, cell lines and spheroids, EMP features (motility) and markers (vascular endothelial growth factor (VEGF), ZEB1, miR-200a&b) were evaluated by morphology, migration assays, immunostaining and qPCR; HIF-1α was knocked down with siRNA or shRNA. RESULTS: At 1 and/or 2 weeks off PPIs, BO biopsies exhibited EMP features and markers, with significant enrichment for HIF-1α, angiogenesis and EMP pathways. In BO cells, ABS induced HIF-1α activation, which decreased miR-200a&b while increasing VEGF, ZEB1 and motility; HIF-1α knockdown blocked these effects. After ABS treatment, BO spheroids exhibited migratory protrusions showing nuclear HIF-1α, increased VEGF and decreased miR-200a&b. CONCLUSIONS: In BO patients, reflux oesophagitis induces EMP changes associated with increased HIF-1α signalling in Barrett's metaplasia. In Barrett's cells, ABS trigger EMP via HIF-1α signalling. Thus, HIF-1α appears to play a key role in mediating reflux-induced EMP that might contribute to cancer in BO. TRIAL REGISTRATION NUMBER: NCT02579460.

Genomic signatures of past and present chromosomal instability in Barrett's esophagus and early esophageal adenocarcinoma.

The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations but how these alterations arise is poorly understood. Here we perform haplotype-specific analysis of chromosomal copy-number evolution in the progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) on multiregional whole-genome sequencing data of BE with dysplasia and microscopic EAC foci. We identify distinct patterns of copy-number evolution indicating multigenerational chromosomal instability that is initiated by cell division errors but propagated only after p53 loss. While abnormal mitosis, including whole-genome duplication, underlies chromosomal copy-number changes, segmental alterations display signatures of successive breakage-fusion-bridge cycles and chromothripsis of unstable dicentric chromosomes. Our analysis elucidates how multigenerational chromosomal instability generates copy-number variation in BE cells, precipitates complex alterations including DNA amplifications, and promotes their independent clonal expansion and transformation. In particular, we suggest sloping copy-number variation as a signature of ongoing chromosomal instability that precedes copy-number complexity. These findings suggest copy-number heterogeneity in advanced cancers originates from chromosomal instability in precancerous cells and such instability may be identified from the presence of sloping copy-number variation in bulk sequencing data.

Pathological features associated with metastasis in patients with early invasive (pT1) colorectal carcinoma in colorectal polyps.

AIMS: Colorectal carcinoma (CRC) arising in a colorectal polyp with invasion limited to the submucosa is sufficiently treated by complete endoscopic resection alone in many cases. Histological features of the carcinoma including tumour size, vascular invasion and poor tumour differentiation or evidence of de-differentiation, such as tumour budding, are associated with a higher risk for metastasis such that oncological resection is recommended. However, most malignant polyps with these features do not have lymph node metastases at the time of resection, so there is a need for better refinement of the histological risk features. METHODS AND RESULTS: A total of 437 consecutive colorectal polyps with submucosal invasive carcinoma from a single centre, 57 of which had metastatic disease, were supplemented by 30 cases with known metastatic disease from two additional centres. Clinical and histological features of the polyp cancers were reviewed looking for differences between the 87 cancers with metastatic disease and the remaining cases without metastasis. A subgroup of 204 polyps removed intact was also analysed to ensure maximum histological accuracy. CONCLUSIONS: This study confirmed larger invasive tumour size, vascular invasion and poor tumour differentiation as adverse predictive features. Prominent peritumoral desmoplasia and high cytological grade were additional adverse features. A predictive logistic regression model comprised of (i) presence of any form of vascular invasion; (ii) presence of high tumour budding (BD3); (iii) width of invasive tumour component > 8 mm; (iv) depth of invasive tumour > 1.5 mm; and (v) the finding of prominent expansile desmoplasia located within and beyond the deep invasive edge of the carcinoma, showed excellent performance in predicting metastatic disease.

Esophageal squamous cell carcinoma with basaloid features are genetically and prognostically similar to conventional squamous cell carcinoma.

We compared clinicopathologic and molecular features of esophageal squamous cell carcinoma (SCC) with basaloid features to conventional SCC using surgical resections of treatment naïve esophageal carcinomas and cases available from the TCGA database. Twenty-two cases of SCC with basaloid features were identified in the Mass General Brigham pathology archives, including 9 cases with pure basaloid morphology and 13 cases with mixed other features such as conventional well- or poorly differentiated areas or sarcomatoid areas. Thirty-eight cases of conventional SCC matched by tumor stage were used as controls. HPV infection status was tested by p16 immunohistochemistry and HPV mRNA ISH. Digital slides for 94 cases of esophageal SCC from TCGA found in the Genomic Data Commons (GDC) Data Portal were reviewed. Five cases of SCC with basaloid features were identified. Genomic profiles of SCC with basaloid features were compared to the rest of 89 SCCs without basaloid features. In addition, eight tumor sections from six patients selected from our cohort underwent in-house molecular profiling. Compared to conventional SCC, SCC with basaloid features were more frequently associated with diffuse or multifocal squamous dysplasia (p < 0.001). P16 IHC was positive in 2/13 cases, whereas HPV mRNA ISH was negative in 17/17 cases (including both p16-positive cases). SCC with basaloid features and conventional SCC from TCGA showed similar rates of TP53 mutations, CDKN2A/B deletions, and CCDN1 amplifications. TP53 variants were identified in all in-house samples that had sufficient coverage. Survival analyses between SCC with basaloid features versus conventional SCC (matched for tumor stage) did not reveal any statistically significant differences. In conclusion, esophageal SCC with basaloid features has similar survival and genomic alterations to those of conventional SCC, are more frequently associated with diffuse or multifocal dysplasia, and are not associated with HPV (high-risk strains) infection.

Use of Real Time Specimen Radiography to Evaluate the Number of Stereotactic Core Biopsy Specimens Containing Calcifications Required for Diagnosis.

OBJECTIVE: To determine the minimum number of stereotactic biopsy specimens containing calcifications sufficient for pathologic diagnosis and the minimum number of specimens containing calcifications sufficient for immunohistochemistry (IHC) in cases of malignancy. METHODS: In this IRB-exempt quality assurance initiative, individual specimens from 126 patients with 129 calcified targets retrieved using a stereotactic system with real time specimen imaging were prospectively analyzed. Pathology was reported independently for each specimen containing calcifications. In every case, the pathologist reported which specimen containing calcifications was sufficient for diagnosis and, in cases of malignancy, which calcified specimen was sufficient for diagnosis and IHC. RESULTS: A diagnosis was made from the first calcified specimen in 74% of cases (95/129), from the first two calcified specimens in 92% (119/129) of cases, and from the first three calcified specimens in 100% of cases. Pathology was benign in 66% (85/129) of cases, with the diagnosis made from the first calcified specimen in 78% (66/85) of cases. High-risk lesions were the primary pathology in 8% (11/129) of cases, with 55% (6/11) diagnosed from the first calcified specimen. Pathology was malignant in 26% (33/129) of cases. The first calcified specimen was sufficient for diagnosis and IHC in 73% (24/33) of malignancies and the first three calcified specimens were sufficient for diagnosis and IHC in all cases of malignancy. CONCLUSION: Three cores verified to contain calcifications on real time specimen imaging were sufficient to make a diagnosis in all cases and to make a diagnosis and obtain IHC in nearly all cases of malignancy.

Florid Foreign Body-type Giant Cell Response to Keratin Is Associated With Improved Overall Survival in Patients Receiving Preoperative Therapy for Esophageal Squamous Cell Carcinoma.

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.

Verrucous carcinoma of the oesophagus is a genetically distinct subtype of oesophageal squamous cell carcinoma.

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 < 0.001) and copy number variants (CNVs) for CDKN2A (P < 0.001), CDKN2B (P < 0.01) and CCND1 (P < 0.01) were absent in VSCC and significantly less frequent in comparison to conventional SCC. Five VSCC cases featured SMARCA4 missense mutations or in-frame deletions compared to only four of 88 conventional SCC cases (P < 0.001). VSCC featured driver mutations in PIK3CA, HRAS and GNAS. Recurrent CNVs were rare in VSCC. CONCLUSIONS: VSCC is not only morphologically but also genetically distinct from conventional oesophageal SCC, featuring frequent SMARCA4 mutations and infrequent TP53 mutations or CDKN2A/B CNVs. Molecular findings may aid in establishing the challenging diagnosis of VSCC.

Magnetic cholecysto-duodenal anastomosis.

INTRODUCTION: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

A Rationally Designed ICAM1 Antibody Drug Conjugate for Pancreatic Cancer.

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.

Role of submucosal glands in the development and progression of carcinoma in Barrett's oesophagus.

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.

Loss of SMAD4 protein expression in gastrointestinal and extra-gastrointestinal carcinomas.

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 <5% of oesophageal, breast, gastric and mucinous ovarian adenocarcinomas. All papillary thyroid, hepatocellular, non-mucinous ovarian, endometrial and renal cell carcinomas showed intact SMAD4 nuclear expression. CONCLUSION: In addition to pancreaticobiliary, appendiceal and colonic tumours, SMAD4 loss is also seen in a small subset of other carcinomas, specifically breast, lung, oesophageal and gastric adenocarcinomas, all of which are typically CK7-positive, similar to pancreaticobiliary carcinoma. Awareness of SMAD4 loss in these other carcinoma types is helpful in the evaluation of carcinomas of unknown or uncertain primary site.

Targeted Cancer Next-Generation Sequencing as a Primary Screening Tool for Microsatellite Instability and Lynch Syndrome in Upper Gastrointestinal Tract Cancers.

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.

Acidic Bile Salts Induce Epithelial to Mesenchymal Transition via VEGF Signaling in Non-Neoplastic Barrett's Cells.

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.

Columnar-Lined Esophagus Develops via Wound Repair in a Surgical Model of Reflux Esophagitis.

Background & Aims: After esophagojejunostomy, rodents develop reflux esophagitis and a columnar-lined esophagus with features of Barrett's metaplasia. This rodent columnar-lined esophagus has been proposed to develop from cellular reprogramming of progenitor cells, but studies on early columnar-lined esophagus development are lacking. We performed a systematic, histologic, and immunophenotypic analysis of columnar-lined esophagus development in rats after esophagojejunostomy. Methods: At various times after esophagojejunostomy in 52 rats, the esophagus was removed and tissue sections were evaluated for type, location, and length of columnar lining. Molecular characteristics were evaluated by immunohistochemistry and immunofluorescence. Results: At week 2, ulceration was seen in esophageal squamous epithelium, starting distally at the esophagojejunostomy anastomosis. Re-epithelialization of the distal ulcer segment occurred via proliferation and expansion of immature-appearing glands budding directly off jejunal crypts, characteristic of wound healing. The columnar-lined esophagus's immunoprofile was similar to jejunal crypt epithelium, and columnar-lined esophagus length increased significantly from 0.15 mm (±0.1 SEM) at 2 weeks to 5.22 mm (±0.37) at 32 weeks. Neoglands were found within esophageal ulcer beds, and spindle-shaped cells expressing epithelial-mesenchymal transition markers were found at the columnar-lined esophagus's leading edge. Only proliferative squamous epithelium was found at the proximal ulcer border. Conclusions: After esophagojejunostomy in rats, metaplastic columnar-lined esophagus develops via a wound healing process that does not appear to involve cellular reprogramming of progenitor cells. This process involves EMT-associated migration of jejunal cells into the esophagus, where they likely have a competitive advantage over squamous cells in the setting of ongoing gastroesophageal reflux disease.

Detection of Mutations in Barrett's Esophagus Before Progression to High-Grade Dysplasia or Adenocarcinoma.

BACKGROUND & AIMS: Barrett's esophagus (BE) is the greatest risk factor for esophageal adenocarcinoma (EAC), but only a small proportion of patients with BE develop cancer. Biomarkers might be able to identify patients at highest risk of progression. We investigated genomic differences in surveillance biopsies collected from patients whose BE subsequently progressed compared to patients whose disease did not progress. METHODS: We performed a retrospective case-control study of 24 patients with BE that progressed to high-grade dysplasia (HGD, n = 14) or EAC (n = 10). The control group (n = 73, called non-progressors) comprised patients with BE and at least 5 years of total endoscopic biopsy surveillance without progression to HGD or EAC. From each patient, we selected a single tissue sample obtained more than 1 year before progression (cases) or more than 2 years before the end of follow-up (controls). Pathogenic mutations, gene copy numbers, and ploidy were compared between samples from progressors and non-progressors. RESULTS: TP53 mutations were detected in 46% of samples from progressors and 5% of non-progressors. In this case-control sample set, TP53 mutations in BE tissues increased the adjusted risk of progression 13.8-fold (95% confidence interval, 3.2-61.0) (P < .001). We did not observe significant differences in ploidy or copy-number profile between groups. We identified 147 pathogenic mutations in 57 distinct genes-the average number of pathogenic mutations was higher in samples from progressors (n = 2.5) than non-progressors (n = 1.2) (P < .001). TP53 and other somatic mutations were recurrently detected in samples with limited copy-number changes (aneuploidy). CONCLUSIONS: In genomic analyses of BE tissues from patients with or without later progression to HGD or EAC, we found significantly higher numbers of TP53 mutations in BE from patients with subsequent progression. These mutations were frequently detected before the onset of dysplasia or substantial changes in copy number.

A Case of Disseminated Histoplasmosis in a Patient with Rheumatoid Arthritis on Abatacept.

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.

In vivo tissue regeneration with robotic implants.

Robots that reside inside the body to restore or enhance biological function have long been a staple of science fiction. Creating such robotic implants poses challenges both in signaling between the implant and the biological host, as well as in implant design. To investigate these challenges, we created a robotic implant to perform in vivo tissue regeneration via mechanostimulation. The robot is designed to induce lengthening of tubular organs, such as the esophagus and intestines, by computer-controlled application of traction forces. Esophageal testing in swine demonstrates that the applied forces can induce cell proliferation and lengthening of the organ without a reduction in diameter, while the animal is awake, mobile, and able to eat normally. Such robots can serve as research tools for studying mechanotransduction-based signaling and can also be used clinically for conditions such as long-gap esophageal atresia and short bowel syndrome.

Genomic Heterogeneity as a Barrier to Precision Medicine in Gastroesophageal Adenocarcinoma.

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. 14See related article by Janjigian et al., p. 49This article is highlighted in the In This Issue feature, p. 1.

Paraneoplastic thrombocytosis is associated with increased mortality and increased rate of lymph node metastasis in oesophageal adenocarcinoma.

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 < 0.01). 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.