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Objectives: Heparin-induced thrombocytopenia can occur in obese subjects. The medical comorbidities associated with obesity may contribute to the pathogenesis of this disease. It is unknown, however, which specific medical comorbidities and if higher odds of thrombosis are present in obese heparin-induced thrombocytopenia patients. We sought to determine whether obese heparin-induced thrombocytopenia subjects had higher odds of both comorbidities and thrombosis, hypothesizing that this patient population would have higher odds of both these conditions. Methods: This was a multi-center retrospective study utilizing TriNetX © , an electronic health record database, in subjects aged 18–99 years diagnosed with heparin-induced thrombocytopenia. The cohort was divided into two groups (1) non-obese (body mass index

Background and objectiveAcute kidney injury (AKI) is a common complication in hospitalized pediatric patients. Previous studies focused on adults found that proteinuria detected during an admission urinalysis is fit to serve as an indicator for AKI and associated clinical outcomes. The objective of this study is to evaluate if proteinuria on the first day of hospital services in hospitalized children is associated with AKI, need for renal replacement therapy, shock and/or antibiotic use, critical care services, and all-cause mortality at 30 days, hypothesizing that it is associated with these outcomes.MethodsThis is a retrospective cohort study using TriNetX electronic health record data of patients 2 to 18 years of age who underwent urinalysis laboratory testing on hospital admission, had three subsequent days of hospital or critical care services billing codes and creatinine laboratory values, and no pre-existing renal-related complex chronic condition. This study evaluated for the frequency, odds, and severity of AKI as defined by Kidney Disease: Improving Global Outcomes modified criteria and assessed for associated clinical outcomes.ResultsThis study included 971 pediatric subjects [435 (44.7%) with proteinuria]. Proteinuria on the first day of hospital services was associated with an increased odds for higher severity AKI on any day of hospitalization (odds ratio [OR] 2.41, CI 1.8-3.23, pConclusionChildren with proteinuria on the first day of hospital care services may have an increased odds of higher severity AKI, need for renal replacement therapy, shock and/or antibiotic use, and all-cause mortality at 30 days post-admission, with no significant association found for critical care services, mechanical intubation, or inotrope or vasopressor use.

Triple-negative breast cancer (TNBC) is the most aggressive subtype with high metastasis and mortality rates. Given the lack of actionable targets such as ER and HER2, TNBC still remains an unmet therapeutic challenge. Despite harboring high CDK4/6 expression levels, the efficacy of CDK4/6 inhibition in TNBC has been limited due to the emergence of resistance. The resistance to CDK4/6 inhibition is mainly mediated by RB1 inactivation. Since our aim is to overcome resistance to CDK4/6 inhibition, in this study, we primarily used the cell lines that do not express RB1. Following a screening for activated receptor tyrosine kinases (RTKs) upon CDK4/6 inhibition, we identified the TAM (Tyro3, Axl, and MerTK) RTKs as a crucial therapeutic vulnerability in TNBC. We show that targeting the TAM receptors with a novel inhibitor, sitravatinib, significantly sensitizes TNBC to CDK4/6 inhibitors. Upon prolonged HER2 inhibitor treatment, HER2+ breast cancers suppress HER2 expression, physiologically transforming into TNBC-like cells. We further show that the combined treatment is highly effective against drug-resistant HER2+ breast cancer as well. Following quantitative proteomics and RNA-seq data analysis, we extended our study into the immunophenotyping of TNBC. Given the roles of the TAM receptors in promoting the creation of an immunosuppressive tumor microenvironment (TME), we further demonstrate that the combination of CDK4/6 inhibitor abemaciclib and sitravatinib modifies the immune landscape of TNBC to favor immune checkpoint blockade. Overall, our study offers a novel and highly effective combination therapy against TNBC and potentially treatment-resistant HER2+ breast cancer that can be rapidly moved to the clinic.

Background Previous studies have demonstrated epidemiological trends in individual metastatic cancer subtypes; however, research forecasting long-term incidence trends and projected survivorship of metastatic cancers is lacking. We assess the burden of metastatic cancer to 2040 by (1) characterizing past, current, and forecasted incidence trends, and (2) estimating odds of long-term (5-year) survivorship. Methods This retrospective, serial cross-sectional, population-based study used registry data from the Surveillance, Epidemiology, and End Results (SEER 9) database. Average annual percentage change (AAPC) was calculated to describe cancer incidence trends from 1988 to 2018. Autoregressive integrating moving average (ARIMA) models were used to forecast the distribution of primary metastatic cancer and metastatic cancer to specific sites from 2019 to 2040 and JoinPoint models were fitted to estimate mean projected annual percentage change (APC). Results The average annual percent change (AAPC) in incidence of metastatic cancer decreased by 0.80 per 100,000 individuals (1988–2018) and we forecast an APC decrease by 0.70 per 100,000 individuals (2018–2040). Analyses predict a decrease in metastases to liver (APC = −3.40, 95% CI [−3.50, −3.30]), lung (APC (2019–2030) = −1.90, 95% CI [−2.90, −1.00]); (2030–2040) = −3.70, 95% CI [−4.60, −2.80]), bone (APC = −4.00, 95% CI [−4.30, −3.70]), and brain (APC = −2.30, 95% CI [−2.60, −2.00]). By 2040, patients with metastatic cancer are predicted to have 46.7% greater odds of long-term survivorship, driven by increasing plurality of patients with more indolent forms of metastatic disease. Conclusions By 2040, the distribution of metastatic cancer patients is predicted to shift in predominance from invariably fatal to indolent cancers subtypes. Continued research on metastatic cancers is important to guide health policy and clinical intervention efforts, and direct allocations of healthcare resources.

Introduction: Hypoalgesic, or “silent”, inflammatory bowel disease (IBD) is a poorly understood condition that has been associated with poor clinical outcomes. There is evidence that lifestyle factors, including diet, exercise and substance use can influence inflammatory activity and symptoms in IBD. It is unclear, though, whether these issues impact pain experience in IBD. We performed this study to evaluate the potential relationship between several key lifestyle factors and silent IBD. Methods: We performed a retrospective analysis using an IBD natural history registry based in a single tertiary care referral center. We compared demographic and clinical features in two patient cohorts defined using data from simultaneous pain surveys and ileocolonoscopy: a) active IBD without pain (silent IBD), and b) active IBD with pain. We also evaluated the relative incidence of characteristics related to diet, exercise, sexual activity and substance abuse. Results: One hundred eighty IBD patients had active disease and 69 (38.3%) exhibited silent IBD. Silent IBD patients exhibited incidences of disease type, location, and severity as pain-perceiving IBD patients. Silent IBD patients were more likely to be male and less likely to exhibit anxiety and/or depression or to use cannabis, analgesic medication or corticosteroids. There were no significant differences in dietary, exercise-related or sexual activities between silent and pain-perceiving IBD patients. Conclusions: Silent IBD was associated with reduced incidence of substance and analgesic medication use. No relationships were found between silent IBD and diet, exercise or sexual activity, though specific elements of each require further dedicated study.

Introduction: Wnt/β-catenin signaling controls cell division and lineage specification during embryonic development, and is crucial for stem cells maintenance and gut tissue regeneration in adults. Aberrant activation of Wnt/β-catenin signaling is also essential for the pathogenesis of a variety of malignancies. The RNA-binding protein IGF2BP1 is a transcriptional target of Wnt/β-catenin signaling, normally expressed during development and often reactivated in cancer cells, where it regulates the stability of oncogenic mRNA.Methods: In this study, we employed iCLIP and RNA sequencing techniques to investigate the role of IGF2BP1 in the post-transcriptional regulation of Wnt/β-catenin-induced genes at a global level within colorectal cancer (CRC) cells characterized by constitutively active Wnt/β-catenin signaling.Results and Discussion: In our study, we show that, in contrast to normal cells, CRC cells exhibit a much stronger dependency on IGF2BP1 expression for Wnt/β-catenin-regulated genes. We show that both untransformed and CRC cells have their unique subsets of Wnt/β-catenin-regulated genes that IGF2BP1 directly controls through binding to their mRNA. Our iCLIP analysis revealed a significant change in the IGF2BP1-binding sites throughout the target transcriptomes and a significant change in the enrichment of 6-mer motifs associated with IGF2BP1 binding in response to Wnt/β-catenin signaling. Our study also revealed a signature of IGF2BP1-regulated genes that are significantly associated with colon cancer-free survival in humans, as well as potential targets for CRC treatment. Overall, this study highlights the complex and context-dependent regulation of Wnt/β-catenin signaling target genes by IGF2BP1 in non-transformed and CRC cells and identifies potential targets for colon cancer treatment.

Abstract Background When central or peripheral intravenous access cannot be achieved in a timely manner, intraosseous (IO) access is recommended as a safe and equally effective alternative for pediatric resuscitation. IO usage and its complications in the pediatric population have been primarily studied in the setting of cardiac arrest. However, population-based studies identifying noncardiac indications and complications associated with different age groups are sparse. Results This was a retrospective observational cohort study utilizing the TriNetX® electronic health record data. Thirty-seven hospitals were included in the data set with 1012 patients where an IO procedure code was reported in the emergency department or inpatient setting. The cohort was split into two groups, pediatric subjects < 1 year of age and those ≥ 1 year of age. A total incidence of IO line placement of 18 per 100,000 pediatric encounters was reported. Total mortality was 31.8%, with a higher rate of mortality seen in subjects < 1 year of age (39.2% vs 29.0%; p = 0.0028). A diagnosis of cardiac arrest was more frequent in subjects < 1 year of age (51.5% vs 38.0%; p = 0.002), and a diagnosis of convulsions was more frequent in those ≥ 1 of age (28.0% vs 13.8%; p

Bladder cancer can often exhibit genomic and morphological heterogeneity. Here, the authors use genomics analysis to show lineage plasticity of bladder cancers with squamous differentiation, and identify key transcription factors related to this morphological and immune heterogeneity.

Abstract Human cancers display a restricted set of expression profiles, despite diverse mutational drivers. This has led to the hypothesis that select sets of transcription factors act on similar target genes as an integrated network, buffering a tumor’s transcriptional state. Noninvasive papillary urothelial carcinoma (NIPUC) with higher cell cycle activity has higher risk of recurrence and progression. In this paper, we describe a transcriptional network of cell cycle dysregulation in NIPUC, which was delineated using the ARACNe algorithm applied to expression data from a new cohort (n = 81, RNA sequencing), and two previously published cohorts. The transcriptional network comprised 121 transcription factors, including the pluripotency factors SOX2 and SALL4, the sex hormone binding receptors ESR1 and PGR, and multiple homeobox factors. Of these 121 transcription factors, 65 and 56 were more active in tumors with greater and less cell cycle activity, respectively. When clustered by activity of these transcription factors, tumors divided into High Cell Cycle versus Low Cell Cycle groups. Tumors in the High Cell Cycle group demonstrated greater mutational burden and copy number instability. A putative mutational driver of cell cycle dysregulation, such as homozygous loss of CDKN2A, was found in only 50% of High Cell Cycle NIPUC, suggesting a prominent role of transcription factor activity in driving cell cycle dysregulation. Activity of the 121 transcription factors strongly associated with expression of EZH2 and other members of the PRC2 complex, suggesting regulation by this complex influences expression of the transcription factors in this network. Activity of transcription factors in this network also associated with signatures of pluripotency and epithelial-to-mesenchymal transition (EMT), suggesting they play a role in driving evolution to invasive carcinoma. Consistent with this, these transcription factors differed in activity between NIPUC and invasive urothelial carcinoma.

Human papillomavirus (HPV)-induced oropharyngeal cancer now exceeds HPV-induced cervical cancer, with a noticeable sex bias. Although it is well established that women have a more proficient immune system, it remains unclear whether immune control of oral papillomavirus infections differs between sexes. In the current study, we use genetically modified mice to target CCR2 and Stat1 pathways, with the aim of investigating the role of both innate and adaptive immune responses in clearing oral papillomavirus, using our established papillomavirus (MmuPV1) infection model. Persistent oral MmuPV1 infection was detected in Rag1ko mice with T and B cell deficiencies. Meanwhile, other tested mice were susceptible to MmuPV1 infections but were able to clear the virus. We found sex differences in key myeloid cells, including macrophages, neutrophils, and dendritic cells in the infected tongues of wild type and Stat1ko mice but these differences were not observed in CCR2ko mice. Intriguingly, we also observed a sex difference in anti-MmuPV1 E4 antibody levels, especially for two IgG isotypes: IgG2b and IgG3. However, we found comparable numbers of interferon-gamma-producing CD8 T cells stimulated by E6 and E7 in both sexes. These findings suggest that males and females may use different components of innate and adaptive immune responses to control papillomavirus infections in the MmuPV1 mouse model. The observed sex difference in immune responses, especially in myeloid cells including dendritic cell (DC) subsets, may have potential diagnostic and prognostic values for HPV-associated oropharyngeal cancer.

Abstract Several symptoms have been connected to increased healthcare resource utilization (HRU) in the context of inflammatory bowel disease (IBD), including both Crohn’s disease (CD) and ulcerative colitis (UC). This study was designed to investigate the prevalence of IBD-associated symptoms and to determine whether any are independently associated with HRU. We undertook a retrospective analysis of data related to consecutive IBD patient encounters from a tertiary care referral center between 1/1/2015 and 8/31/2019. Demographics, clinical activity, endoscopic severity, IBD-related symptom scores, anxiety and depression scores, and other key clinical data were abstracted. Four hundred sixty-seven IBD patients [247f.: 220 m; 315 CD, 142 UC and 11 indeterminate colitis] were included in this study. The most common symptoms were fatigue (83.6%), fecal urgency (68.2%) and abdominal pain (63.5%). Fatigue, abdominal pain, anxiety or depression, corticosteroids, and opioids were each positively associated with HRU, while NSAID and mesalamine use were inversely associated on bivariate analysis. The only factor that demonstrated a statistically significant association with HRU in the whole cohort on multivariable analysis was abdominal pain. Abdominal pain is independently associated with HRU and should be specifically screened for in IBD patients to identify individuals at risk of undergoing expensive interventions. This study also reinforces the importance of optimizing diagnostic and therapeutic management of abdominal pain in IBD.

The essential role of store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels in T cells is well established. In contrast, the contribution of individual Orai isoforms to SOCE and their downstream signaling functions in B cells are poorly understood. Here, we demonstrate changes in the expression of Orai isoforms in response to B cell activation. We show that both Orai3 and Orai1 mediate native CRAC channels in B cells. The combined loss of Orai1 and Orai3, but not Orai3 alone, impairs SOCE, proliferation and survival, nuclear factor of activated T cells (NFAT) activation, mitochondrial respiration, glycolysis, and the metabolic reprogramming of primary B cells in response to antigenic stimulation. Nevertheless, the combined deletion of Orai1 and Orai3 in B cells did not compromise humoral immunity to influenza A virus infection in mice, suggesting that other in vivo co-stimulatory signals can overcome the requirement of BCR-mediated CRAC channel function in B cells. Our results shed important new light on the physiological roles of Orai1 and Orai3 proteins in SOCE and the effector functions of B lymphocytes.

Background Patients with bladder cancer (BC) who are cisplatin ineligible or have unresectable disease have limited treatment options. Previously, we showed targeting programmed death-ligand 1 (PD-L1) with durvalumab (durva) and radiation therapy (RT) combination was safe in BC. We now report results from a phase II study evaluating the toxicity and efficacy of durva and RT in localized BC.Methods This is a single-arm, multi-institutional phase II study; N=26. Enrolled patients had pure or mixed urothelial BC (T2-4 N0-2 M0) with unresectable tumors and were unfit for surgery or cisplatin ineligible. Patients received durva concurrently with RT ×7 weeks, followed by adjuvant durva × 1 year.Primary endpoints: (A) progression-free survival (PFS) at 1 year and (B) disease control rate (DCR) post adjuvant durva. Key secondary endpoints: (A) complete response (CR) post durvaRT (8 weeks), (B) overall survival (OS), (C) PFS and (D) toxicity. Correlative studies included evaluation of baseline tumor and blood (baseline, post durvaRT) for biomarkers.Results Median follow-up was 27 months. Evaluable patients: 24/26 post durvaRT, 22/26 for DCR post adjuvant durva, all patients for PFS and OS. Post adjuvant durva, DCR was seen in 72.7%, CR of 54.5%. 1-year PFS was 71.5%, median PFS was 21.8 months. 1-year OS was 83.8%, median OS was 30.8 months. CR at 8 weeks post durvaRT was 62.5%. Node positive (N+) patients had similar median PFS and OS. DurvaRT was well tolerated. Grade ≥3 treatment-related adverse events: anemia, high lipase/amylase, immune-nephritis, transaminitis, dyspnea (grade 4-COPD/immune), fatigue, rash, diarrhea and scleritis. No difference in outcome was observed with PD-L1 status of baseline tumor. Patients with CR/PR or SD had an increase in naïve CD4 T cells, a decrease in PD-1+CD4 T cells at baseline and an increase in cytokine-producing CD8 T cells, including interferon gamma (IFNγ) producing cells, in the peripheral blood.Conclusion Durva with RT followed by adjuvant durva was safe with promising efficacy in localized BC patients with comorbidities, including N+ patients. Larger randomized studies, like S1806 and EA8185, are needed to evaluate the efficacy of combining immunotherapy and RT in BC.Trial registration number NCT02891161.

Jeffrey Damrauer, Markia Smith et al. used existing datasets from cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) to characterize two subsets of HCC distinct from prototypical HCC tumors, based on comprehensive analysis of molecular data. The two classes differed from HCC by their copy number, gene expression and mutational signature and exhibited worse progression free survival, highlighting the need to identify class-specific biomarkers and develop targeted therapies for these forms of cancer.

Nationwide databases have implicated an increased incidence of renal cell carcinoma (RCC). The Pennsylvania (PA) Cancer Registry was queried to better define incidence, geographic distribution, and statewide trends of new RCC cases over a 27-year period. JoinPoint Trend Analysis Software modeled average annual percent changes (APCs) in age-adjusted rates (AAR). Maps plotting county-level incidence rates and stage distribution of disease across the state in 5-year time intervals were created using R 4.0.2 software. Overall, 59,628 cases of RCC were recorded in PA from 1990 to 2017. Eighty six percent of patients were >50 years of age, 61% were males, and 89% were Caucasian. Stage distribution using the SEER staging system included 64% local, 17% regional, and 16% distant. Over the study interval, AAR of all RCC cases increased from 9.9 to 18.0 patients per 100,000 population with an APC of 2.3% (p < 0.01). AAR of local disease increased from 5.4 to 12.7 patients per 100,000 population with an APC of 3.2% (p < 0.01). AAR of regional disease also increased from 1.9 to 2.9 patients per 100,000 population with an APC of 1.0% (p = 0.01). Younger patients (

Breast cancer is the second leading cause of death for women in the United States, and early detection could offer patients the opportunity to receive early intervention. The current methods of diagnosis rely on mammograms and have relatively high rates of false positivity, causing anxiety in patients. We sought to identify protein markers in saliva and serum for early detection of breast cancer. A rigorous analysis was performed for individual saliva and serum samples from women without breast disease, and women diagnosed with benign or malignant breast disease, using isobaric tags for relative and absolute quantitation (iTRAQ) technique, and employing a random effects model. A total of 591 and 371 proteins were identified in saliva and serum samples from the same individuals, respectively. The differentially expressed proteins were mainly involved in exocytosis, secretion, immune response, neutrophil-mediated immunity and cytokine-mediated signaling pathway. Using a network biology approach, significantly expressed proteins in both biological fluids were evaluated for protein–protein interaction networks and further analyzed for these being potential biomarkers in breast cancer diagnosis and prognosis. Our systems approach illustrates a feasible platform for investigating the responsive proteomic profile in benign and malignant breast disease using saliva and serum from the same women.

The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, but the roles of ORAI2 and ORAI3 remained obscure. Here authors show that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation.

Contraceptives such as Depo-medroxyprogesterone (DMPA) are used by an estimated 34 million women worldwide. DMPA has been associated with increased risk of several viral infections including Herpes simplex virus-2 (HSV-2) and Human immunodeficiency virus (HIV). In the current study, we used the mouse papillomavirus (MmuPV1) anogenital infection model to test two hypotheses: (1) contraceptives such as DMPA increase the susceptibility of the anogenital tract to viral infection and (2) long-term contraceptive administration induces more advanced disease at the anogenital tract. DMPA treatments of both athymic nude mice and heterozygous NU/J (Foxn1nu/+) but ovariectomized mice led to a significantly increased viral load at the anogenital tract, suggesting that endogenous sex hormones were involved in increased viral susceptibility by DMPA treatment. Consistent with previous reports, DMPA treatment suppressed host anti-viral activities at the lower genital tract. To test the impact of long-term contraceptive treatment on the MmuPV1-infected lower genital tract, we included two other treatments in addition to DMPA: 17β-estradiol and a non-hormone based contraceptive Cilostazol (CLZ, Pletal). Viral infections were monitored monthly up to nine months post infection by qPCR. The infected vaginal and anal tissues were harvested and further examined by histological, virological, and immunological analyses. Surprisingly, we did not detect a significantly higher grade of histology in animals in the long-term DMPA and 17β-estradiol treated groups when compared to the control groups in the athymic mice we tested. Therefore, although DMPA promotes initial papillomavirus infections in the lower genital tract, the chronic administration of DMPA does not promote cancer development in the infected tissues in our mouse model.

Despite the established role of mitochondria in cancer, the mechanisms by which mitochondrial Ca2+ (mtCa2+) regulates tumorigenesis remain incompletely understood. The crucial role of mtCa2+ in tumorigenesis is highlighted by altered expression of proteins mediating mtCa2+ uptake and extrusion in cancer. Here, we demonstrate decreased expression of the mitochondrial Na+/Ca2+/Li+ exchanger NCLX (SLC8B1) in human colorectal tumors and its association with advanced-stage disease in patients. Downregulation of NCLX causes mtCa2+ overload, mitochondrial depolarization, decreased expression of cell-cycle genes and reduced tumor size in xenograft and spontaneous colorectal cancer mouse models. Concomitantly, NCLX downregulation drives metastatic spread, chemoresistance, and expression of epithelial-to-mesenchymal, hypoxia, and stem cell pathways. Mechanistically, mtCa2+ overload leads to increased mitochondrial reactive oxygen species, which activate HIF1α signaling supporting metastasis of NCLX-null tumor cells. Thus, loss of NCLX is a novel driver of metastasis, indicating that regulation of mtCa2+ is a novel therapeutic approach in metastatic colorectal cancer.

Purpose Summative evaluation forms assessing a student’s clinical performance are often completed by a faculty preceptor at the end of a clinical training experience. At our institution, despite the use of an electronic system, timeliness of completion has been suboptimal, potentially limiting our ability to monitor students’ progress. The aim of the present study was to determine whether a student-directed approach to summative evaluation form collection at the end of a pediatrics clerkship would enhance timeliness of completion for third-year medical students. Methods This was a pre- and post-intervention educational quality improvement project focused on 156 (82 pre-intervention, 74 post-intervention) third-year medical students at Penn State College of Medicine completing their 4-week pediatric clerkship. Utilizing REDCap (Research Electronic Data Capture) informatics support, student-directed evaluation form solicitation was encouraged. The Wilcoxon rank-sum test was applied to compare the pre-intervention (May 1, 2017 to March 2, 2018) and post-intervention (April 2, 2018 to December 21, 2018) percentages of forms completed before the rotation midpoint. Results In total, 740 evaluation forms were submitted during the pre-intervention phase and 517 during the post-intervention phase. The percentage of forms completed before the rotation midpoint increased after implementing student-directed solicitation (9.6% vs. 39.7%, P

The molecular nature of large-cell neuroendocrine lung carcinomas (LCNEC) has remained unclear. Here, the authors show LCNECs represent a distinct transcriptional subgroup among lung cancers and comprise two molecular subgroups, type I (TP53 and STK11/KEAP1 alterations) and type II (TP53 and RB1 inactivation).

Renal cell carcinoma (RCC) is a common and aggressive malignancy. Here, the authors generate two mouse models of the most common RCC subtypes: the human papillary RCC throughMYC activation and clear cell RCC through MYC activation combined with Vhl and Cdkn2adeletion.

Ovarian cancer remains the most lethal gynecologic malignancy, and is primarily diagnosed at late stage when considerable metastasis has occurred in the peritoneal cavity. At late stage abdominal cavity ascites accumulation provides a tumor-supporting medium in which cancer cells gain access to growth factors and cytokines that promote survival and metastasis. However, little is known about the redox status of ascites, or whether antioxidant enzymes are required to support ovarian cancer survival during transcoelomic metastasis in this medium. Gene expression cluster analysis of antioxidant enzymes identified two distinct populations of high-grade serous adenocarcinomas (HGSA), the most common ovarian cancer subtype, which specifically separated into clusters based on glutathione peroxidase 3 (GPx3) expression. High GPx3 expression was associated with poorer overall patient survival and increased tumor stage. GPx3 is an extracellular glutathione peroxidase with reported dichotomous roles in cancer. To further examine a potential pro-tumorigenic role of GPx3 in HGSA, stable OVCAR3 GPx3 knock-down cell lines were generated using lentiviral shRNA constructs. Decreased GPx3 expression inhibited clonogenicity and anchorage-independent cell survival. Moreover, GPx3 was necessary for protecting cells from exogenous oxidant insult, as demonstrated by treatment with high dose ascorbate. This cytoprotective effect was shown to be due to GPx3-dependent removal of extracellular H2O2. Importantly, GPx3 was necessary for clonogenic survival when cells were cultured in patient-derived ascites fluid. While oxidation reduction potential (ORP) of malignant ascites was heterogeneous in our patient cohort, and correlated positively with ascites iron content, GPx3 was required for optimal survival regardless of ORP or iron content. Collectively, our data suggest that HGSA ovarian cancers cluster into distinct groups of high and low GPx3 expression. GPx3 is necessary for HGSA ovarian cancer cellular survival in the ascites tumor environment and protects against extracellular sources of oxidative stress, implicating GPx3 as an important adaptation for transcoelomic metastasis. Keywords: Extracellular glutathione peroxidase, GPx3, Ovarian cancer, Ascites, Ascorbate

The homeostatic iron regulator protein HFE is involved in regulation of iron acquisition for cells. The prevalence of two common HFE gene variants (H63D, C282Y) has been studied in many cancer types; however, the impact of HFE variants, sex and HFE gene expression in lung cancer has not been studied. We determined the prevalence of HFE variants and their impact on cancer phenotypes in lung cancer cell lines, in lung cancer patient specimens, and using The Cancer Genome Atlas (TCGA) database. We found that seven out of ten human lung cancer cell lines carry the H63D or C282Y HFE variant. Analysis of lung cancer specimens from our institute (Penn State Hershey Medical Center) revealed a sex and genotype interaction risk for metastasis in lung adenocarcinoma (LUAD) patients; H63D HFE is associated with less metastasis in males compared to wild type (WT) HFE; however, females with the H63D HFE variant tend to develop more metastatic tumors than WT female patients. In the TCGA LUAD dataset, the H63D HFE variant was associated with poorer survival in females compared to females with WT HFE. The frequency of C282Y HFE is higher in female lung squamous cell carcinoma (LUSC) patients of TCGA than males, however the C282Y HFE variant did not impact the survival of LUSC patients. In the TCGA LUSC dataset, C282Y HFE patients (especially females) had poorer survival than WT HFE patients. HFE expression level was not affected by HFE genotype status and did not impact patient's survival, regardless of sex. In summary, these data suggest that there is a sexually dimorphic effect of HFE polymorphisms in the survival and metastatic disease in lung cancer.

Background: Hypoalgesic inflammatory bowel disease (IBD), a condition in which patients with active disease do not perceive and/or report abdominal pain, is associated with serious complications and there is a lack of cost-effective, reliable diagnostic methods to identify “at-risk” patients. The voltage-gated sodium channels (VGSC's), Nav1.7, Nav1.8, and Nav1.9, are preferentially expressed on nociceptive neurons, and have been implicated in visceral inflammatory pain. At least 29 VGSC single nucleotide polymorphisms (SNPs) have been implicated in chronic somatic pain syndromes, but little is known about their role in human visceral sensation. We hypothesized that disruptive VGSC polymorphisms result in anti-nociceptive behavior in IBD.Methods and Findings: We performed targeted exome sequencing and/or TaqMan genotyping to evaluate the Nav1.7, Nav1.8, and Nav1.9 genes (SCN9A, SCN10A and SCN11A) in 121 IBD patients (including 41 “hypoalgesic” IBD patients) and 86 healthy controls. Allelic and genotypic frequencies of polymorphisms were compared among study groups who had undergone characterization of intestinal inflammatory status and abdominal pain experience. Forty-nine total exonic SNPs were identified. The allelic frequency of only one non-synonymous SNP (rs6795970 [SCN10A]) approached significance in hypoalgesic IBD patients when compared to other IBD patients (p = 0.096, Fisher's exact test). Hypoalgesic IBD patients were more likely to be homozygous for this polymorphism (46 vs. 22%, p = 0.01, Fisher's exact test).Conclusions: This is the first human study to demonstrate a link between a genetic variant of SCN10A and abdominal pain perception in IBD. These findings provide key insights into visceral nociceptive physiology and new diagnostic and therapeutic targets to consider in IBD and other gastrointestinal conditions associated with chronic abdominal pain. Further studies are required to elucidate the precise pathophysiological impact of the rs6795970 polymorphism on human gastrointestinal nociception.

Summary: This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smoking and/or human papillomavirus (HPV). SCCs harbor 3q, 5p, and other recurrent chromosomal copy-number alterations (CNAs), DNA mutations, and/or aberrant methylation of genes and microRNAs, which are correlated with the expression of multi-gene programs linked to squamous cell stemness, epithelial-to-mesenchymal differentiation, growth, genomic integrity, oxidative damage, death, and inflammation. Low-CNA SCCs tended to be HPV(+) and display hypermethylation with repression of TET1 demethylase and FANCF, previously linked to predisposition to SCC, or harbor mutations affecting CASP8, RAS-MAPK pathways, chromatin modifiers, and immunoregulatory molecules. We uncovered hypomethylation of the alternative promoter that drives expression of the ΔNp63 oncogene and embedded miR944. Co-expression of immune checkpoint, T-regulatory, and Myeloid suppressor cells signatures may explain reduced efficacy of immune therapy. These findings support possibilities for molecular classification and therapeutic approaches. : Campbell et al. reveal that squamous cell cancers from different tissue sites may be distinguished from other cancers and subclassified molecularly by recurrent alterations in chromosomes, DNA methylation, messenger and microRNA expression, or by mutations. These affect squamous cell pathways and programs that provide candidates for therapy. Keywords: genomics, transcriptomics, proteomics, head and neck squamous cell carcinoma, lung squamous cell carcinoma, esophageal squamous cell carcinoma, cervical squamous cell carcinoma, bladder carcinoma with squamous differentiation, human papillomavirus

[This corrects the article DOI: 10.1371/journal.pone.0056823.].

The incidence of thyroid cancer has risen dramatically in the past few decades. The cause of this is unclear, but several lines of evidence indicate it is largely due to overdiagnosis, the diagnosis of tumors that would have never manifest clinically if untreated. Practices leading to overdiagnosis may relate to defensive medicine. In this study, we evaluated the association between malpractice climate and incidence of thyroid, breast, prostate, colon, and lung cancer in U.S. states from 1999-2012 using publicly available government data. State-level malpractice risk was quantified as malpractice payout rate, the number of malpractice payouts per 100,000 people per state per year. Associations between state-level cancer incidence, malpractice payout rate, and several cancer risk factors were evaluated. Risk factors included several social determinants of health, including factors predicting healthcare access. States with higher malpractice payout rate had higher thyroid cancer incidence, on both univariate analysis (r = 0.51, P = 0.009, Spearman) and multivariate analysis (P

Epigenetic aberrations are prominent in bladder cancer (BC) and contribute to disease pathogenesis. We characterized histone deacetylase (HDAC) expression, a family of deacetylation enzymes, in both in vitro and in vivo BC model systems and analyzed expression data from The Cancer Genome Atlas (TCGA). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analysis was used to determine the expression status of Class I and II HDACs in ten human BC cell lines, while qRT-PCR was used to determine HDAC expression in 24 human tumor specimens. The TCGA cohort consists of 408 muscle invasive BC (MIBC) clinical samples and analysis of this data set identified expression of HDAC4 and -9 as being associated with basal−squamous disease. These findings agree with qRT-PCR results identifying increased expression of HDAC4, -7, and -9 in basal BC cell lines (p < 0.05; Kruskal−Wallis test) and in clinical specimens with invasive bladder cancer (not statistically significant). We also observed increased expression in Hdac4, -7, and -9 in commonly used BC mouse models. Here, we identify suitable preclinical model systems for the study of HDACs, and show increased expression of Class IIa HDACs, specifically HDAC4 and HDAC9, in basal BC cell lines and in invasive clinical specimens. These results suggest this class of HDACs may be best suited for targeted inhibition in patients with basal BC.

The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07-0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11-1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal heterogeneous disease. Beyond the role of human papilloma virus (HPV), no validated molecular characterization of the disease has been established. Using an integrated genomic analysis and validation methodology we confirm four molecular classes of HNSCC (basal, mesenchymal, atypical, and classical) consistent with signatures established for squamous carcinoma of the lung, including deregulation of the KEAP1/NFE2L2 oxidative stress pathway, differential utilization of the lineage markers SOX2 and TP63, and preference for the oncogenes PIK3CA and EGFR. For potential clinical use the signatures are complimentary to classification by HPV infection status as well as the putative high risk marker CCND1 copy number gain. A molecular etiology for the subtypes is suggested by statistically significant chromosomal gains and losses and differential cell of origin expression patterns. Model systems representative of each of the four subtypes are also presented.

Lung adenocarcinoma (LAD) has extreme genetic variation among patients, which is currently not well understood, limiting progress in therapy development and research. LAD intrinsic molecular subtypes are a validated stratification of naturally-occurring gene expression patterns and encompass different functional pathways and patient outcomes. Patients may have incurred different mutations and alterations that led to the different subtypes. We hypothesized that the LAD molecular subtypes co-occur with distinct mutations and alterations in patient tumors.The LAD molecular subtypes (Bronchioid, Magnoid, and Squamoid) were tested for association with gene mutations and DNA copy number alterations using statistical methods and published cohorts (n = 504). A novel validation (n = 116) cohort was assayed and interrogated to confirm subtype-alteration associations. Gene mutation rates (EGFR, KRAS, STK11, TP53), chromosomal instability, regional copy number, and genomewide DNA methylation were significantly different among tumors of the molecular subtypes. Secondary analyses compared subtypes by integrated alterations and patient outcomes. Tumors having integrated alterations in the same gene associated with the subtypes, e.g. mutation, deletion and underexpression of STK11 with Magnoid, and mutation, amplification, and overexpression of EGFR with Bronchioid. The subtypes also associated with tumors having concurrent mutant genes, such as KRAS-STK11 with Magnoid. Patient overall survival, cisplatin plus vinorelbine therapy response and predicted gefitinib sensitivity were significantly different among the subtypes.The lung adenocarcinoma intrinsic molecular subtypes co-occur with grossly distinct genomic alterations and with patient therapy response. These results advance the understanding of lung adenocarcinoma etiology and nominate patient subgroups for future evaluation of treatment response.