IL-4 and IL-17A Cooperatively Promote Hydrogen Peroxide Production, Oxidative DNA Damage, and Upregulation of Dual Oxidase 2 in Human Colon and Pancreatic Cancer Cells.

Dual oxidase 2 (DUOX2) generates H2O2 that plays a critical role in both host defense and chronic inflammation. Previously, we demonstrated that the proinflammatory mediators IFN-γ and LPS enhance expression of DUOX2 and its maturation factor DUOXA2 through STAT1- and NF-κB‒mediated signaling in human pancreatic cancer cells. Using a panel of colon and pancreatic cancer cell lines, we now report the induction of DUOX2/DUOXA2 mRNA and protein expression by the TH2 cytokine IL-4. IL-4 activated STAT6 signaling that, when silenced, significantly decreased induction of DUOX2. Furthermore, the TH17 cytokine IL-17A combined synergistically with IL-4 to increase DUOX2 expression in both colon and pancreatic cancer cells mediated, at least in part, by signaling through NF-κB. The upregulation of DUOX2 was associated with a significant increase in the production of extracellular H2O2 and DNA damage-as indicated by the accumulation of 8-oxo-dG and γH2AX-which was suppressed by the NADPH oxidase inhibitor diphenylene iodonium and a DUOX2-specific small interfering RNA. The clinical relevance of these experiments is suggested by immunohistochemical, microarray, and quantitative RT-PCR studies of human colon and pancreatic tumors demonstrating significantly higher DUOX2, IL-4R, and IL-17RA expression in tumors than in adjacent normal tissues; in pancreatic adenocarcinoma, increased DUOX2 expression is adversely associated with overall patient survival. These data suggest a functional association between DUOX2-mediated H2O2 production and induced DNA damage in gastrointestinal malignancies.

Residual tissue repositories as a resource for population-based cancer proteomic studies.

BACKGROUND: Mass spectrometry-based proteomics has become a powerful tool for the identification and quantification of proteins from a wide variety of biological specimens. To date, the majority of studies utilizing tissue samples have been carried out on prospectively collected fresh frozen or optimal cutting temperature (OCT) embedded specimens. However, such specimens are often difficult to obtain, in limited in supply, and clinical information and outcomes on patients are inherently delayed as compared to banked samples. Annotated formalin fixed, paraffin embedded (FFPE) tumor tissue specimens are available for research use from a variety of tissue banks, such as from the surveillance, epidemiology and end results (SEER) registries' residual tissue repositories. Given the wealth of outcomes information associated with such samples, the reuse of archived FFPE blocks for deep proteomic characterization with mass spectrometry technologies would provide a valuable resource for population-based cancer studies. Further, due to the widespread availability of FFPE specimens, validation of specimen integrity opens the possibility for thousands of studies that can be conducted worldwide. METHODS: To examine the suitability of the SEER repository tissues for proteomic and phosphoproteomic analysis, we analyzed 60 SEER patient samples, with time in storage ranging from 7 to 32 years; 60 samples with expression proteomics and 18 with phosphoproteomics, using isobaric labeling. Linear modeling and gene set enrichment analysis was used to evaluate the impacts of collection site and storage time. RESULTS: All samples, regardless of age, yielded suitable protein mass after extraction for expression analysis and 18 samples yielded sufficient mass for phosphopeptide analysis. Although peptide, protein, and phosphopeptide identifications were reduced by 50, 20 and 76% respectively, from comparable OCT specimens, we found no statistically significant differences in protein quantitation correlating with collection site or specimen age. GSEA analysis of GO-term level measurements of protein abundance differences between FFPE and OCT embedded specimens suggest that the formalin fixation process may alter representation of protein categories in the resulting dataset. CONCLUSIONS: These studies demonstrate that residual FFPE tissue specimens, of varying age and collection site, are a promising source of protein for proteomic investigations if paired with rigorously verified mass spectrometry workflows.

NADPH oxidase 5 (NOX5)-induced reactive oxygen signaling modulates normoxic HIF-1α and p27Kip1 expression in malignant melanoma and other human tumors.

NADPH oxidase 5 (NOX5) generated reactive oxygen species (ROS) have been implicated in signaling cascades that regulate cancer cell proliferation. To evaluate and validate NOX5 expression in human tumors, we screened a broad range of tissue microarrays (TMAs), and report substantial overexpression of NOX5 in malignant melanoma and cancers of the prostate, breast, and ovary. In human UACC-257 melanoma cells that possesses high levels of functional endogenous NOX5, overexpression of NOX5 resulted in enhanced cell growth, increased numbers of BrdU positive cells, and increased γ-H2AX levels. Additionally, NOX5-overexpressing (stable and inducible) UACC-257 cells demonstrated increased normoxic HIF-1α expression and decreased p27Kip1 expression. Similarly, increased normoxic HIF-1α expression and decreased p27Kip1 expression were observed in stable NOX5-overexpressing clones of KARPAS 299 human lymphoma cells and in the human prostate cancer cell line, PC-3. Conversely, knockdown of endogenous NOX5 in UACC-257 cells resulted in decreased cell growth, decreased HIF-1α expression, and increased p27Kip1 expression. Likewise, in an additional human melanoma cell line, WM852, and in PC-3 cells, transient knockdown of endogenous NOX5 resulted in increased p27Kip1 and decreased HIF-1α expression. Knockdown of endogenous NOX5 in UACC-257 cells resulted in decreased Akt and GSK3ß phosphorylation, signaling pathways known to modulate p27Kip1 levels. In summary, our findings suggest that NOX5 expression in human UACC-257 melanoma cells could contribute to cell proliferation due, in part, to the generation of high local concentrations of extracellular ROS that modulate multiple pathways that regulate HIF-1α and networks that signal through Akt/GSK3ß/p27Kip1 .

Predictors of all-cause mortality and liver-related mortality in patients with non-alcoholic fatty liver disease (NAFLD).

AIM: Non-alcoholic steatohepatitis (NASH) patients are at increased risk for progression to cirrhosis. The aim of this study was to assess all-cause and liver-specific mortality in a cohort of non-alcoholic fatty liver disease (NAFLD) patients. METHODS: Biopsy-proven NAFLD patients with and without NASH from two historic databases were included. Clinico-demographic information from the time of biopsy was available. Mortality data were obtained from National Death Index-Plus and used for estimating overall and cause-specific mortality. The non-parametric Kaplan-Meier method with log-rank test and multivariate analyses with Cox proportional hazard model were used to compare cohorts. RESULTS: Two hundred eighty-nine NAFLD patients were included (50.3 ± 14.5 years old, 39.4 % male, 78.6 % Caucasian, 46.0 % obese, 26.0 % diabetic, 5.9 % with family history of liver diseases). Of these, 59.2 % had NASH whereas 40.8 % had non-NASH NAFLD. NASH patients were predominantly female, had higher aspartate aminotranserase, alanine aminotransferase and fasting serum glucose. During follow-up (median 150 months, maximum 342 months), patients with NASH had higher probability of mortality from liver-related causes than non-NASH NAFLD patients (p value = 0.0026). In the entire NAFLD cohort, older age [aHR = 1.07 (95 % CI = 1.05-1.10)] and presence of type II diabetes [aHR = 2.09 (1.39-3.14)] were independent predictors of overall mortality. However, in addition to age [aHR = 1.06 (1.02-1.10)] having histologic NASH [aHR = 9.16 (2.10-9.88)] was found to be an independent predictor of liver-related mortality. Additionally, presence of type II diabetes was associated with liver-related mortality [aHR = 2.19 (1.00-4.81)]. CONCLUSIONS: This long-term follow-up of NAFLD patients confirms that NASH patients have higher risk of liver-related mortality than non-NASH. Additionally, patients with NAFLD and type II diabetes are at highest risk for overall and liver-related mortality.

Pathologic criteria for nonalcoholic steatohepatitis: interprotocol agreement and ability to predict liver-related mortality.

UNLABELLED: Since the initial description of nonalcoholic steatohepatitis (NASH), several sets of pathologic criteria for its diagnosis have been proposed. However, their interprotocol agreement and ability to predict long-term liver-related mortality (LRM) have not been demonstrated. In this study, we examined patients with biopsy-proven nonalcoholic fatty liver disease (NAFLD) for whom liver biopsy slides and clinical and mortality data were available. Liver biopsy samples were evaluated for a number of pathologic features and were classified according to the presence or absence of NASH by (1) the original criteria for NAFLD subtypes, (2) the nonalcoholic fatty liver disease activity score (NAS), (3) the Brunt criteria, and (4) the current study's criteria. All NASH diagnostic criteria and individual pathologic features were tested for agreement and for their independent associations with LRM, which were determined with a Cox proportional hazards model. Two hundred fifty-seven NAFLD patients with complete data were included. The diagnoses of NASH by the original NAFLD subtypes and by the current study's definition of NASH were in almost perfect agreement (κ = 0.896). However, their agreement was moderate with NAS (κ = 0.470 and κ = 0.511, respectively) and only fair to moderate with the Brunt criteria (κ = 0.365 and κ = 0.441, respectively). Furthermore, the agreement of the Brunt criteria with NAS was relatively poor (κ = 0.178). During the follow-up (median = 146 months), 31% of the patients died (9% were LRM). After we controlled for confounders, a diagnosis of NASH by the original criteria for NAFLD subtypes [adjusted hazard ratio = 9.94 (95% confidence interval = 1.28-77.08)] demonstrated the best independent association with LRM. Among the individual pathologic features, advanced fibrosis showed the best independent association with LRM [adjusted hazard ratio = 5.68 (95% confidence interval = 1.50-21.45)]. CONCLUSION: The original criteria for NAFLD subtypes and the current study's criteria for NASH were in almost perfect agreement, but their level of agreement with the NAS and Brunt criteria was lower. A diagnosis of NASH by the original criteria for NAFLD subtypes demonstrated the best predictability for LRM in NAFLD patients.

Efficacy and Safety of a Botanical Formula Fuzheng Huayu for Hepatic Fibrosis in Patients with CHC: Results of a Phase 2 Clinical Trial.

Background: Hepatitis C virus (HCV) is a common cause of progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma worldwide. Despite the availability of effective direct-acting antivirals, patients often have significant hepatic fibrosis at the time of diagnosis due to delay in diagnosis and comorbidities which promote fibrogenesis. Thus, antifibrotic agents represent an attractive adjunctive therapy. Fuzheng Huayu (FZHY), a traditional Chinese medicine botanical formulation, has been used as an antifibrotic agent in chronic HBV infection. Our aim was to assess FZHY in patients with HCV infection and active viremia. Method: We randomized 118 patients with active viremia from 8 liver centers in the U.S. to receive oral FZHY (n = 59) or placebo (n = 59) for 48 weeks. Efficacy was assessed by histopathologic changes at the end of therapy. A subset of biopsies was further analyzed using qFibrosis to detect subtle changes in fibrosis in different zones of the hepatic lobules. Results: FZHY was well tolerated and safe. Patients with baseline Ishak fibrosis stages F3 and F4 had better response rates to FZHY than patients with baseline F0-F2 (p=0.03). qFibrosis zonal analysis showed significant improvement in fibrosis in all zones in patients with regression of the fibrosis stage. Conclusions: FZHY produced antifibrotic effects in patients with baseline Ishak F3 and F4 fibrosis stages. Reduction in fibrosis severity was zonal and correlated with the severity of inflammation. Based on its tolerability, safety, and efficacy, FZHY should be further investigated as a therapy in chronic liver diseases because of its dual anti-inflammatory and antiibrotic properties. Lay Summary. This is the first US-based, multicenter and placebo-controlled clinical trial that shows statistically significant reduction in fibrosis in patients with active HCV using an antifibrotic botanical formula. This has important implications as there is an immediate need for effective antifibrotic agents in treating many chronic diseases including NASH that lead to scarring of the liver. With artificial intelligence-based methodology, qFibrosis, we may provide a more reliable way to assess the FZHY as a therapy in chronic liver diseases because of its dual anti-inflammatory and antifibrotic properties.

Farglitazar lacks antifibrotic activity in patients with chronic hepatitis C infection.

BACKGROUND & AIMS: Farglitazar (GI262570), an insulin-sensitizing agent, selectively binds and activates peroxisome proliferator-activated receptor gamma (PPARgamma) and inhibits stellate cell activation. We evaluated its antifibrotic effect in patients with chronic hepatitis C that did not respond to standard-of-care therapy. METHODS: Patients with fibrosis of Ishak stages 2-4 (n = 265), based on analysis of liver biopsy samples, were randomly assigned to groups given once-daily doses of 0.5 mg farglitazar, 1.0 mg farglitazar, or placebo for 52 weeks; repeat liver biopsy samples were then obtained. The primary end points were changes in levels of alpha-smooth muscle actin (SMA) expression and collagen, based on morphometry and ranked histologic assessments. RESULTS: Two hundred nine patients had paired biopsy specimens adequate for analysis (81.5% with pretreatment Ishak scores of stage 2 or 3). There was no overall difference in SMA (P = .58) or collagen (P = .99) levels at week 52. SMA levels increased by a median of 49% in samples from patients given placebo, 58% in patients given 0.5 mg farglitizar and 52% in patients given 1.0 mg farglitizar, respectively. Collagen increased by 27% in placebo samples and 31% in samples from patients given either dose of farglitizar. There were no significant differences between treatment groups in the ranked assessment of paired biopsy specimens or in the proportion of patients with a change in fibrosis score > or = Ishak stage. CONCLUSIONS: In patients with chronic hepatitis C and moderate fibrosis, 52 weeks of treatment with farglitazar does not affect stellate cell activation or fibrosis (measured by morphometry or comparison of paired biopsy specimens).

Fibrolamellar carcinomas are positive for CD68.

Fibrolamellar carcinomas are a unique type of liver carcinoma that arise in non-cirrhotic livers of young individuals. Despite their distinctive appearance, recent studies have demonstrated a lack of consistency in how fibrolamellar carcinomas are diagnosed by pathologists. As a potential aide in diagnosis, we investigated the staining properties of CD68. The CD68 gene encodes for a transmembrane glycoprotein located within lysosomes and endosomes. Macrophages as well as other cell types rich in lysosomes/endosomes are CD68 positive. Cases of fibrolamellar carcinoma were collected from four academic centers. Control groups included hepatocellular carcinomas arising in both non-cirrhotic livers and cirrhotic livers. A group of cholangiocarcinomas were also stained. CD68 immunostaining was scored for both intensity and distribution on a scale of 0 to 3+. Twenty-three primary fibrolamellar carcinomas and 9 metastases (total of 24 individuals) were immunostained and showed a distinctive granular, dot-like or stippled pattern of cytoplasmic staining in nearly all cases (31/32), with a median distribution and intensity score of 3+. In control hepatocellular carcinomas that arose in non-cirrhotic livers, 10/39 showed CD68 staining with a median distribution and intensity score of 2+. In hepatocellular carcinomas arising in cirrhotic livers, 3/27 cases showed CD68 positivity, all with stippled dot-like cytoplasmic staining similar to that of fibrolamellar carcinomas. All five cholangiocarcinomas were negative. Overall, CD68 positivity was strongly associated with fibrolamellar carcinomas, P<0.001 and had a sensitivity of 96%, a specificity of 80%, and a negative predictive value of 98%. In sum, tumor positivity for CD68 staining was highly sensitive for fibrolamellar carcinoma and a lack of CD68 staining should suggest caution in making a diagnosis of fibrolamellar carcinoma.

Biospecimen Collection During the COVID-19 Pandemic.

OBJECTIVES: Millions of biospecimens will be collected during the coronavirus disease 2019 (COVID-19) pandemic. As learned from severe acute respiratory syndrome (SARS), proper biospecimen handling is necessary to prevent laboratory-related infections. METHODS: Centers for Disease Control and Prevention and World Health Organization severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interim biosafety guidelines continue to be updated. Presented here are additional considerations intended to complement the interim guidance. These considerations draw on prior SARS recommendations and recent COVID-19 reports. RESULTS: SARS-CoV-2 viral RNA has been detected in various biospecimen types; however, studies are needed to determine whether viral load indicates viable virus. Throughout the pandemic, biospecimens will be collected for various purposes from COVID-19 known and suspected cases, as well as presymptomatic and asymptomatic individuals. Current data suggest the pandemic start may be as early as October 2019; thus, all biospecimens collected since could be considered potentially infectious. CONCLUSIONS: All entities handling these biospecimens should do risk assessments in accordance with institutional policies and adhere to any guidance provided. The scientific community has a responsibility to safely handle and maintain all biospecimens collected during the COVID-19 pandemic. Soon, it will be imperative to convene expert working groups to address the current and long-term storage and use of these biospecimens. Ideally, worldwide guidelines will be established to protect the personnel handling these biospecimens and communities at large.

NADPH oxidase 1 is highly expressed in human large and small bowel cancers.

To facilitate functional investigation of the role of NADPH oxidase 1 (NOX1) and associated reactive oxygen species in cancer cell signaling, we report herein the development and characterization of a novel mouse monoclonal antibody that specifically recognizes the C-terminal region of the NOX1 protein. The antibody was validated in stable NOX1 overexpression and knockout systems, and demonstrates wide applicability for Western blot analysis, confocal microscopy, flow cytometry, and immunohistochemistry. We employed our NOX1 antibody to characterize NOX1 expression in a panel of 30 human colorectal cancer cell lines, and correlated protein expression with NOX1 mRNA expression and superoxide production in a subset of these cells. Although a significant correlation between oncogenic RAS status and NOX1 mRNA levels could not be demonstrated in colon cancer cell lines, RAS mutational status did correlate with NOX1 expression in human colon cancer surgical specimens. Immunohistochemical analysis of a comprehensive set of tissue microarrays comprising over 1,200 formalin-fixed, paraffin-embedded tissue cores from human epithelial tumors and inflammatory disease confirmed that NOX1 is overexpressed in human colon and small intestinal adenocarcinomas, as well as adenomatous polyps, compared to adjacent, uninvolved intestinal mucosae. In contradistinction to prior studies, we did not find evidence of NOX1 overexpression at the protein level in tumors versus histologically normal tissues in prostate, lung, ovarian, or breast carcinomas. This study constitutes the most comprehensive histopathological characterization of NOX1 to date in cellular models of colon cancer and in normal and malignant human tissues using a thoroughly evaluated monoclonal antibody. It also further establishes NOX1 as a clinically relevant therapeutic target in colorectal and small intestinal cancer.

Toward Improving Practices for Submission of Diagnostic Tissue Blocks for National Cancer Institute Clinical Trials.

OBJECTIVES: The National Cancer Institute (NCI) National Clinical Trials Network performs phase II and III clinical trials, which increasingly rely on the submission of diagnostic formalin-fixed, paraffin-embedded tissue blocks for biomarker assessment. Simultaneously, advances in precision oncology require that clinical centers maintain diagnostic specimens for ancillary, standard-of-care diagnostics. This has caused tissue blocks to become a limited resource for advancing the NCI clinical trial enterprise and the practice of modern molecular pathology. METHODS: The NCI convened a 1-day workshop of multidisciplined experts to discuss barriers and strategic solutions to facilitate diagnostic block submission for clinical trial science, from the perspective of patient advocates, legal experts, pathologists, and clinical oncologists. RESULTS: The expert views and opinions were carefully noted and reported. CONCLUSIONS: Recommendations were proposed to reduce institutional barriers and to assist organizations in developing clear policies regarding diagnostic block submission for clinical trials.

State of the Art: Toward Improving Outcomes of Lung and Liver Tumor Biopsies in Clinical Trials-A Multidisciplinary Approach.

PURPOSE: National Cancer Institute (NCI)-sponsored clinical trial network studies frequently require biopsy specimens for pharmacodynamic and molecular biomarker analyses, including paired pre- and post-treatment samples. The purpose of this meeting of NCI-sponsored investigators was to identify local institutional standard procedures found to ensure quantitative and qualitative specimen adequacy. METHODS: NCI convened a conference on best biopsy practices, focusing on the clinical research community. Topics discussed were (1) criteria for specimen adequacy in the personalized medicine era, (2) team-based approaches to ensure specimen adequacy and quality control, and (3) risk considerations relevant to academic and community practitioners and their patients. RESULTS AND RECOMMENDATIONS: Key recommendations from the convened consensus panel included (1) establishment of infrastructure for multidisciplinary biopsy teams with a formalized information capture process, (2) maintenance of standard operating procedures with regular team review, (3) optimization of tissue collection and yield methodology, (4) incorporation of needle aspiration and other newer techniques, and (5) commitment of stakeholders to use of guideline documents to increase awareness of best biopsy practices, with the goal of universally improving tumor biopsy practices.

Clinical Evolution of Epithelial-Mesenchymal Transition in Human Carcinomas.

The significance of the phenotypic plasticity afforded by epithelial-mesenchymal transition (EMT) for cancer progression and drug resistance remains to be fully elucidated in the clinic. We evaluated epithelial-mesenchymal phenotypic characteristics across a range of tumor histologies using a validated, high-resolution digital microscopic immunofluorescence assay (IFA) that incorporates ß-catenin detection and cellular morphology to delineate carcinoma cells from stromal fibroblasts and that quantitates the individual and colocalized expression of the epithelial marker E-cadherin (E) and the mesenchymal marker vimentin (V) at subcellular resolution ("EMT-IFA"). We report the discovery of ß-catenin+ cancer cells that coexpress E-cadherin and vimentin in core-needle biopsies from patients with various advanced metastatic carcinomas, wherein these cells are transitioning between strongly epithelial and strongly mesenchymal-like phenotypes. Treatment of carcinoma models with anticancer drugs that differ in their mechanism of action (the tyrosine kinase inhibitor pazopanib in MKN45 gastric carcinoma xenografts and the combination of tubulin-targeting agent paclitaxel with the BCR-ABL inhibitor nilotinib in MDA-MB-468 breast cancer xenografts) caused changes in the tumor epithelial-mesenchymal character. Moreover, the appearance of partial EMT or mesenchymal-like carcinoma cells in MDA-MB-468 tumors treated with the paclitaxel-nilotinib combination resulted in upregulation of cancer stem cell (CSC) markers and susceptibility to FAK inhibitor. A metastatic prostate cancer patient treated with the PARP inhibitor talazoparib exhibited similar CSC marker upregulation. Therefore, the phenotypic plasticity conferred on carcinoma cells by EMT allows for rapid adaptation to cytotoxic or molecularly targeted therapy and could create a form of acquired drug resistance that is transient in nature. SIGNIFICANCE: Despite the role of EMT in metastasis and drug resistance, no standardized assessment of EMT phenotypic heterogeneity in human carcinomas exists; the EMT-IFA allows for clinical monitoring of tumor adaptation to therapy.

Pathology of chronic hepatitis C in children: liver biopsy findings in the Peds-C Trial.

There is relatively little information in the literature on the histopathology of chronic hepatitis C in children. The Peds-C Trial, designed to test the efficacy and safety of peginterferon alfa-2a and ribavirin in children, provided an opportunity to examine liver biopsies from 121 treatment-naïve children, ages 2 to 16 (mean, 9.8 years) infected with the hepatitis C virus (HCV) and with no other identifiable cause for liver disease, signs of hepatic decompensation, or another significant nonhepatic disease. Liver biopsies were scored for inflammation, fibrosis, steatosis, and other histological features. Inflammation in the biopsy was minimal in 42%, mild in 17%, moderate in 38%, and severe in only 3%. Five had bridging fibrosis, and 2 had cirrhosis. Steatosis was absent in 56%, minimal in 34%, and mild in 10%. Inflammation scores correlated with fibrosis scores, serum alanine aminotransferase levels, and duration of infection, but not with age, body mass index z score, or HCV genotype. Fibrosis scores correlated with inflammation but not with age, HCV genotype, body mass index z score, or steatosis parameters. Steatosis correlated with serum alanine aminotransferase levels and body mass index z scores; overweight children had more fibrosis than the non-overweight. In conclusion, in this cohort of HCV-infected children, inflammation, fibrosis, and steatosis were milder than reported for treatment-naïve adults with chronic hepatitis C, but there were several with bridging fibrosis or cirrhosis. The positive correlation of inflammation with duration of infection and fibrosis and of obesity with fibrosis suggest that children with chronic hepatitis C will be at risk for progressive liver disease as they age and possibly acquire other comorbid risk factors.

Synovial sarcoma of the stomach: a clinicopathologic, immunohistochemical, and molecular genetic study of 10 cases.

Only a few synovial sarcomas arising in the gastrointestinal tract have been reported, most of them are from the esophagus. We report clinical, histopathologic, and immunohistochemical features of 10 gastric synovial sarcomas. These tumors occurred in 4 males and 6 females with mean and median age of 52 years (range, 29 to 68 y). None of the patients had evidence of synovial sarcoma elsewhere. The tumor sizes ranged from 0.8 to 15 cm (mean, 3 cm). Two tumors were large transmural masses of 8 and 15 cm, and 8 were 0.8 to 6 cm, ulcerated cuplike or plaquelike or oval lesions predominantly involving the luminal side. Histologically, 9 tumors were monophasic one also having a poorly differentiated round cell component, and one was biphasic. Microscopic calcifications were present in 2 tumors. At least focal keratin (AE1/AE3 cocktail, keratin 7) and/or epithelial membrane antigen-positivity were detected in all tumors, and there was no CD34 or KIT-immunoreactivity. SYT-SSX fusion transcripts were demonstrated in 7 cases studied by a polymerase chain reaction-based fusion transcript assay. Five patients had a partial gastrectomy, and 5 underwent wedge or segmental resections. Two patients had received chemotherapy after surgery, but none had postoperative radiation. Four patients with plaquelike or cuplike tumors < or =3 cm were alive and well 1, 2, 2, and 18.5 years after surgery. Two patients died of tumor 25 and 29 months after surgery. One of them had a large 8-cm tumor, and another had a 2-cm tumor with a poorly differentiated component. Two patients were alive with recurrences 6 and 48 months after diagnosis. Synovial sarcoma rarely occurs as a gastric primary tumor. It has a variable prognosis depending on tumor size and differentiation, and should be considered in the differential diagnosis of KIT-negative gastric spindle cell neoplasms.

Glypican-3 expression in benign liver tissue with active hepatitis C: implications for the diagnosis of hepatocellular carcinoma.

Glypican 3 is an oncofetal antigen that shows great promise as an adjunct to the diagnosis of hepatocellular carcinoma. To investigate whether glypican 3 might also appear in nonneoplastic liver cells, we performed immunostains on 60 biopsies of chronic hepatitis C, 30 with low-grade activity and 30 with high-grade activity. Glypican 3 immunoreactivity was detected in 25 (83.6%) of 30 in the high-grade but in none in the low-grade group. In 20% of the positive cases, glypican 3 produced strong cytoplasmic staining of more than 25% of hepatocytes and could potentially lead to a misdiagnosis of hepatocellular carcinoma. Pathologists should be aware of this phenomenon and exercise caution in interpreting biopsies or other specimens of suspected hepatocellular carcinoma when active necroinflammatory disease is present.

What Can Be Done to Improve Research Biopsy Quality in Oncology Clinical Trials?

PURPOSE:: Research biopsy specimens collected in clinical trials often present requirements beyond those of tumor biopsy specimens collected for diagnostic purposes. Research biopsies underpin hypothesis-driven drug development, pharmacodynamic assessment of molecularly targeted anticancer agents, and, increasingly, genomic assessment for precision medicine; insufficient biopsy specimen quality or quantity therefore compromises the scientific value of a study and the resources devoted to it, as well as each patient's contribution to and potential benefit from a clinical trial. METHODS:: To improve research biopsy specimen quality, we consulted with other translational oncology teams and reviewed current best practices. RESULTS:: Among the recommendations were improving communication between oncologists and interventional radiologists, providing feedback on specimen sufficiency, increasing academic recognition and financial support for the time investment required by radiologists to collect and preserve research biopsy specimens, and improving real-time assessment of tissue quality. CONCLUSION:: Implementing these recommendations at the National Cancer Institute's Developmental Therapeutics Clinic has demonstrably improved the quality of biopsy specimens collected; more widespread dissemination of these recommendations beyond large clinical cancer centers is possible and will be of value to the community in improving clinical research and, ultimately, patient care.

Globular hepatic amyloid: an early stage in the pathway of amyloid formation: a study of 20 new cases.

Only 25 cases of globular hepatic amyloidosis have been reported, mostly from the early 1980s. We reviewed clinical, histopathologic, and immunohistochemical features of 20 cases of hepatic globular amyloid out of 208 cases of liver amyloidosis seen at the Armed Forces Institute of Pathology since 1970. Fourteen (70%) were men and 6 were women with a median age of 67 years (range, 40 to 92 y). More than half of the patients were Hispanic. Ten of 20 patients were diagnosed with systemic amyloidosis. Histologically, all cases revealed round to oval-shaped sometimes laminated globules, 1 to 40 mum in diameter, and 6 cases had evidence of transition from globular to the more usual linear form. In all 20 cases, Congo red and/or Sirius red stained the globules red and showed an apple green birefringence under polarized light. Portal tracts and parenchyma were involved in 15/20 the cases, and sinusoidal deposition alone in 5 cases. Vascular deposition was very common with more than 3/4 of the cases showing mainly perivenular amyloid with both the terminal hepatic venules and portal vein branches being equally involved. A few intrahepatocellular globules were present in half of the cases. In conclusion, hepatic amyloidosis can rarely occur as a globular form, and the finding of intracellular amyloid globules and transitional forms of globular to linear patterns of deposition suggest that this is an early form of hepatic involvement by systemic amyloidosis.

Expression of estrogen and progesterone receptors and inhibin-alpha in hepatobiliary cystadenoma: an immunohistochemical study.

To further characterize the immunohistochemical features of hepatobiliary cystadenoma with mesenchymal stroma, a battery of stains was performed on nine typical cases. All nine tumors had been resected from female patients who ranged in age from 30 to 59 years. Freshly cut sections were stained with antibodies to estrogen receptor (ER), progesterone receptor (PR), alpha-smooth muscle actin (SMA), inhibin-alpha, and cytokeratins (CK) 7, 8, 18, and 19. Nuclear staining of the mesenchymal stromal cells for ER and PR was present in all and seven out of nine cases, respectively. A strong cytoplasmic staining of the mesenchymal stromal cells for SMA was seen in all cases. A patchy pale cytoplasmic staining of the tumor epithelium for ER and PR was seen in five out of nine and four out of nine cases, respectively. Immunoreactivity of luteinized stromal cell for inhibin-alpha was documented in three out of nine cases. All tumors (nine out of nine) demonstrated strong cytoplasmic positivity of the epithelial lining of the cysts to CK7, CK8, CK18, and CK19, typical of biliary-type epithelium. The expression of ER, PR, and inhibin-alpha in the ovarian-like stroma supports the likely hormonal dependence of this tumor and probably explains its almost exclusive occurrence in women.

Nonalcoholic steatofibrosis independently predicts mortality in nonalcoholic fatty liver disease.

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease (NAFLD). The minimal pathologic criteria for NASH include hepatic steatosis, ballooning degeneration, and lobular inflammation. The resolution of NASH, which relies on the loss of ballooning degeneration, is subject to sampling and observer variability in pathologic interpretation. Ballooning is associated with advanced hepatic fibrosis in cross-sectional studies but is not a predictor of mortality in NAFLD. Fibrosis staging, while still subject to some sampling variability, has less observer variability and is a robust predictor of liver-related mortality in NAFLD. In this study, we hypothesize that, regardless of the diagnosis of NASH, the presence of steatofibrosis (steatosis accompanied by fibrosis) regardless of other pathologic features can also be a robust predictor of mortality in NAFLD. We used our previously reported cohort of patients with NAFLD with liver biopsies and long-term mortality follow-up. Cox proportional hazard models were used to determine the predictors of overall and liver-related mortality. Of 209 enrolled NAFLD subjects, 97 can be classified as having steatofibrosis. During follow-up (median 150 months), 64 (30.6%) patients died, with 18 (8.6%) from liver-related causes. Adjusted for age, both diagnostic categories of NASH and steatofibrosis were significantly and similarly associated with liver-related mortality (adjusted hazard ratio [aHR], 9.9; 95% confidence interval (CI), 1.3-74.9; P = 0.027; aHR, 6.7; 95% CI, 1.5-29.8; P = 0.013, respectively). However, only steatofibrosis showed independent association with overall mortality (aHR, 1.76; 95% CI, 1.02-3.05; P = 0.043). Conclusion: Steatofibrosis and NASH are similarly associated with liver-related mortality, but only steatofibrosis is associated with overall mortality in patients with NAFLD. Given the inherent observer variability in ballooning degeneration, a key diagnostic component of NASH, we suggest that steatofibrosis should be considered a viable diagnostic classification for NAFLD subjects at risk or adverse outcomes and provides a simpler endpoint for clinical trials of therapeutic agents. (Hepatology Communications 2017;1:421-428).