Integrated mutational landscape analysis of poorly differentiated high-grade neuroendocrine carcinoma of the uterine cervix.

High-grade neuroendocrine cervical cancers (NETc) are exceedingly rare, highly aggressive tumors. We analyzed 64 NETc tumor samples by whole-exome sequencing (WES). Human papillomavirus DNA was detected in 65.6% (42/64) of the tumors. Recurrent mutations were identified in PIK3CA, KMT2D/MLL2, K-RAS, ARID1A, NOTCH2, and RPL10. The top mutated genes included RB1, ARID1A, PTEN, KMT2D/MLL2, and WDFY3, a gene not yet implicated in NETc. Somatic CNV analysis identified two copy number gains (3q27.1 and 19q13.12) and five copy number losses (1p36.21/5q31.3/6p22.2/9q21.11/11p15.5). Also, gene fusions affecting the ACLY-CRHR1 and PVT1-MYC genes were identified in one of the eight samples subjected to RNA sequencing. To resolve evolutionary history, multiregion WES in NETc admixed with adenocarcinoma cells was performed (i.e., mixed-NETc). Phylogenetic analysis of mixed-NETc demonstrated that adenocarcinoma and neuroendocrine elements derive from a common precursor with mutations typical of adenocarcinomas. Over one-third (22/64) of NETc demonstrated a mutator phenotype of C > T at CpG consistent with deficiencies in MBD4, a member of the base excision repair (BER) pathway. Mutations in the PI3K/AMPK pathways were identified in 49/64 samples. We used two patient-derived-xenografts (PDX) (i.e., NET19 and NET21) to evaluate the activity of pan-HER (afatinib), PIK3CA (copanlisib), and ATR (elimusertib) inhibitors, alone and in combination. PDXs harboring alterations in the ERBB2/PI3K/AKT/mTOR/ATR pathway were sensitive to afatinib, copanlisib, and elimusertib (P < 0.001 vs. controls). However, combinations of copanlisib/afatinib and copanlisib/elimusertib were significantly more effective in controlling NETc tumor growth. These findings define the genetic landscape of NETc and suggest that a large subset of these highly lethal malignancies might benefit from existing targeted therapies.

Arsenic and UVR co-exposure results in unique gene expression profile identifying key co-carcinogenic mechanisms.

Changes in gene expression underlie many pathogenic endpoints including carcinogenesis. Metals, like arsenic, alter gene expression; however, the consequences of co-exposures of metals with other stressors are less understood. Although arsenic acts as a co-carcinogen by enhancing the development of UVR skin cancers, changes in gene expression in arsenic UVR co-carcinogenesis have not been investigated. We performed RNA-sequencing analysis to profile changes in gene expression distinct from arsenic or UVR exposures alone. A large number of differentially expressed genes (DEGs) were identified after arsenic exposure alone, while after UVR exposure alone fewer genes were changed. A distinct increase in the number of DEGs was identified after exposure to combined arsenic and UVR exposure that was synergistic rather than additive. In addition, a majority of these DEGs were unique from arsenic or UVR alone suggesting a distinct response to combined arsenic-UVR exposure. Globally, arsenic alone and arsenic plus UVR exposure caused a global downregulation of genes while fewer genes were upregulated. Gene Ontology analysis using the DEGs revealed cellular processes related to chromosome instability, cell cycle, cellular transformation, and signaling were targeted by combined arsenic and UVR exposure, distinct from UVR alone and arsenic alone, while others were related to epigenetic mechanisms such as the modification of histones. This result suggests the cellular functions we identified in this study may be key in understanding how arsenic enhances UVR carcinogenesis and that arsenic-enhanced gene expression changes may drive co-carcinogenesis of UVR exposure.

Nucleosome remodeling at origins of global genome-nucleotide excision repair occurs at the boundaries of higher-order chromatin structure.

Repair of UV-induced DNA damage requires chromatin remodeling. How repair is initiated in chromatin remains largely unknown. We recently demonstrated that global genome-nucleotide excision repair (GG-NER) in chromatin is organized into domains in relation to open reading frames. Here, we define these domains, identifying the genomic locations from which repair is initiated. By examining DNA damage-induced changes in the linear structure of nucleosomes at these sites, we demonstrate how chromatin remodeling is initiated during GG-NER. In undamaged cells, we show that the GG-NER complex occupies chromatin, establishing the nucleosome structure at these genomic locations, which we refer to as GG-NER complex binding sites (GCBSs). We demonstrate that these sites are frequently located at genomic boundaries that delineate chromosomally interacting domains (CIDs). These boundaries define domains of higher-order nucleosome-nucleosome interaction. We demonstrate that initiation of GG-NER in chromatin is accompanied by the disruption of dynamic nucleosomes that flank GCBSs by the GG-NER complex.

Nivolumab for Patients With High-Risk Oral Leukoplakia: A Nonrandomized Controlled Trial.

Importance: Proliferative verrucous leukoplakia (PVL) is an aggressive oral precancerous disease characterized by a high risk of transformation to invasive oral squamous cell carcinoma (OSCC), and no therapies have been shown to affect its natural history. A recent study of the PVL immune landscape revealed a cytotoxic T-cell-rich microenvironment, providing strong rationale to investigate immune checkpoint therapy. Objective: To determine the safety and clinical activity of anti-programmed cell death 1 protein (PD-1) therapy to treat high-risk PVL. Design, Setting, and Participants: This nonrandomized, open-label, phase 2 clinical trial was conducted from January 2019 to December 2021 at a single academic medical center; median (range) follow-up was 21.1 (5.4-43.6) months. Participants were a population-based sample of patients with PVL (multifocal, contiguous, or a single lesion ≥4 cm with any degree of dysplasia). Intervention: Patients underwent pretreatment biopsy (1-3 sites) and then received 4 doses of nivolumab (480 mg intravenously) every 28 days, followed by rebiopsy and intraoral photographs at each visit. Main Outcomes and Measures: The primary end point was the change in composite score (size and degree of dysplasia) from before to after treatment (major response [MR]: >80% decrease in score; partial response: 40%-80% decrease). Secondary analyses included immune-related adverse events, cancer-free survival (CFS), PD-1 ligand 1 (PD-L1) expression, 9p21.3 deletion, and other exploratory immunologic and genomic associations of response. Results: A total of 33 patients were enrolled (median [range] age, 63 [32-80] years; 18 [55%] were female), including 8 (24%) with previously resected early-stage OSCC. Twelve patients (36%) (95% CI, 20.4%-54.8%) had a response by composite score (3 MRs [9%]), 4 had progressive disease (>10% composite score increase, or cancer). Nine patients (27%) developed OSCC during the trial, with a 2-year CFS of 73% (95% CI, 53%-86%). Two patients (6%) discontinued because of toxic effects; 7 (21%) experienced grade 3 to 4 immune-related adverse events. PD-L1 combined positive scores were not associated with response or CFS. Of 20 whole-exome sequenced patients, all 6 patients who had progression to OSCC after nivolumab treatment exhibited 9p21.3 somatic copy-number loss on pretreatment biopsy, while only 4 of the 14 patients (29%) who did not develop OSCC had 9p21.3 loss. Conclusions and Relevance: This immune checkpoint therapy precancer nonrandomized clinical trial met its prespecified response end point, suggesting potential clinical activity for nivolumab in high-risk PVL. Findings identified immunogenomic associations to inform future trials in this precancerous disease with unmet medical need that has been difficult to study. Trial Registration: ClinicalTrials.gov Identifier: NCT03692325.

Human cancer genomes harbor the mutational signature of tobacco-specific nitrosamines NNN and NNK.

Tobacco usage is linked to multiple cancer types and accounts for a quarter of all cancer-related deaths. Tobacco smoke contains various carcinogenic compounds, including polycyclic aromatic hydrocarbons (PAH), though the mutagenic potential of many tobacco-related chemicals remains largely unexplored. In particular, the highly carcinogenic tobacco-specific nitrosamines NNN and NNK form pre-mutagenic pyridyloxobutyl (POB) DNA adducts. In the study presented here, we identified genome-scale POB-induced mutational signatures in cell lines and rat tumors, while also investigating their role in human cancer. These signatures are characterized by T>N and C>T mutations forming from specific POB adducts damaging dT and dC residues. Analysis of 2,780 cancer genomes uncovered POB signatures in ∼180 tumors; from cancer types distinct from the ones linked to smoking-related signatures SBS4 and SBS92. This suggests that, unlike PAH compounds, the POB pathway may contribute uniquely to the mutational landscapes of certain hematological malignancies and cancers of the kidney, breast, prostate and pancreas.

The mutagenic forces shaping the genomic landscape of lung cancer in never smokers.

Lung cancer in never smokers (LCINS) accounts for up to 25% of all lung cancers and has been associated with exposure to secondhand tobacco smoke and air pollution in observational studies. Here, we evaluate the mutagenic exposures in LCINS by examining deep whole-genome sequencing data from a large international cohort of 871 treatment-naïve LCINS recruited from 28 geographical locations within the Sherlock-Lung study. KRAS mutations were 3.8-fold more common in adenocarcinomas of never smokers from North America and Europe, while a 1.6-fold higher prevalence of EGFR and TP53 mutations was observed in adenocarcinomas from East Asia. Signature SBS40a, with unknown cause, was found in most samples and accounted for the largest proportion of single base substitutions in adenocarcinomas, being enriched in EGFR-mutated cases. Conversely, the aristolochic acid signature SBS22a was almost exclusively observed in patients from Taipei. Even though LCINS exposed to secondhand smoke had an 8.3% higher mutational burden and 5.4% shorter telomeres, passive smoking was not associated with driver mutations in cancer driver genes or the activities of individual mutational signatures. In contrast, patients from regions with high levels of air pollution were more likely to have TP53 mutations while exhibiting shorter telomeres and an increase in most types of somatic mutations, including a 3.9-fold elevation of signature SBS4 (q-value=3.1 × 10-5), previously linked mainly to tobacco smoking, and a 76% increase of clock-like signature SBS5 (q-value=5.0 × 10-5). A positive dose-response effect was observed with air pollution levels, which correlated with both a decrease in telomere length and an elevation in somatic mutations, notably attributed to signatures SBS4 and SBS5. Our results elucidate the diversity of mutational processes shaping the genomic landscape of lung cancer in never smokers.

Prevalence and characterization of multidrug-resistant zoonotic Enterobacter spp. in poultry of Bangladesh.

Poultry and poultry products are major contributors of zoonotic pathogens. Limited data are available on Enterobacter spp. as a potent zoonotic pathogen in poultry. The present study is a first endeavor on the emergence of multidrug-resistant zoonotic Enterobacter spp. and its prevalence arising from poultry in Bangladesh. Cloacal swabs from poultry samples of five different farms at Savar, Dhaka, Bangladesh were collected and from 106 isolates, 18 presumptive Enterobacter spp. were obtained. Antibiogram using 19 used antibiotics belonging to 15 major groups revealed that all of the 18 isolates were completely resistant to penicillin and rifampicin, but differed in their drug resistance pattern against ampicillin (94.4%), clindamycin (94.4%), erythromycin (94.4%), vancomycin (88.9%), sulfonamides (72.2%), imipenem (66.6%), streptomycin (55.6%), nitrofurantoin (33.3%), doxycycline (33.3%), tetracyclines (33.3%), cefepime (11.1%), and gentamicin (5.6%). All Enterobacter spp. were found to be plasmid free, implying that multidrug-resistant properties are chromosomal borne. The vanA and sulI were detected by polymerase chain reaction assay in 17 and 13 isolates, respectively. Amplified ribosomal DNA restriction analysis and randomly amplified polymorphic DNA distributed the 18 multidrug-resistant Enterobacter spp. into three genotypes. Phylogenetic analysis of the representatives of the three genotypes using partial 16S rRNA gene sequence (approximately 900 bp) showed that the genotypically diverse groups belonged to Enterobacter hormaechei, E. cloacae, and E. cancerogenus, respectively. The clinical significance of the close relative Enterobacter spp. is indicative of their zoonotic potential. Therefore, urgent intervention is required to limit the emergence and spread of these bacteria in poultry feed as well as prudent use of antibiotics among poultry farmers in Bangladesh.

Arsenic is a potent co-mutagen of ultraviolet light.

Environmental co-exposures are widespread and are major contributors to carcinogenic mechanisms. Two well-established environmental agents causing skin cancer are ultraviolet radiation (UVR) and arsenic. Arsenic is a known co-carcinogen that enhances UVR's carcinogenicity. However, the mechanisms of arsenic co-carcinogenesis are not well understood. In this study, we utilized primary human keratinocytes and a hairless mouse model to investigate the carcinogenic and mutagenic properties of co-exposure to arsenic and UVR. In vitro and in vivo exposures revealed that, on its own, arsenic is neither mutagenic nor carcinogenic. However, in combination with UVR, arsenic exposure has a synergistic effect leading to an accelerated mouse skin carcinogenesis as well as to more than 2-fold enrichment of UVR mutational burden. Notably, mutational signature ID13, previously found only in UVR-associated human skin cancers, was observed exclusively in mouse skin tumors and cell lines jointly exposed to arsenic and UVR. This signature was not observed in any model system exposed purely to arsenic or purely to UVR, making ID13 the first co-exposure signature to be reported using controlled experimental conditions. Analysis of existing genomics data from basal cell carcinomas and melanomas revealed that only a subset of human skin cancers harbor ID13 and, consistent with our experimental observations, these cancers exhibited an elevated UVR mutagenesis. Our results provide the first report of a unique mutational signature caused by a co-exposure to two environmental carcinogens and the first comprehensive evidence that arsenic is a potent co-mutagen and co-carcinogen of UVR. Importantly, our findings suggest that a large proportion of human skin cancers are not formed purely due to UVR exposure but rather due to a co-exposure of UVR and other co-mutagens such as arsenic.

DNA damage and somatic mutations in mammalian cells after irradiation with a nail polish dryer.

Ultraviolet A light is commonly emitted by UV-nail polish dryers with recent reports suggesting that long-term use may increase the risk for developing skin cancer. However, no experimental evaluation has been conducted to reveal the effect of radiation emitted by UV-nail polish dryers on mammalian cells. Here, we show that irradiation by a UV-nail polish dryer causes high levels of reactive oxygen species, consistent with 8-oxo-7,8-dihydroguanine damage and mitochondrial dysfunction. Analysis of somatic mutations reveals a dose-dependent increase of C:G>A:T substitutions in irradiated samples with mutagenic patterns similar to mutational signatures previously attributed to reactive oxygen species. In summary, this study demonstrates that radiation emitted by UV-nail polish dryers can both damage DNA and permanently engrave mutations on the genomes of primary mouse embryonic fibroblasts, human foreskin fibroblasts, and human epidermal keratinocytes.

Arsenic is a potent co-mutagen of ultraviolet light.

Arsenic enhances the carcinogenicity of ultraviolet radiation (UVR). However, the mechanisms of arsenic-driven oncogenesis are not well understood. Here, we utilize experimental systems to investigate the carcinogenic and mutagenic properties of co-exposure to arsenic and UVR. In vitro and in vivo exposures indicate that, by itself, arsenic is not mutagenic. However, in combination with UVR, arsenic exposure has a synergistic effect leading to an accelerated mouse skin carcinogenesis and to more than 2-fold enrichment of UVR mutational burden. Notably, mutational signature ID13, previously found only in UVR-associated human skin cancers, is observed exclusively in mouse skin tumors and cell lines jointly exposed to arsenic and UVR. This signature was not observed in any model system exposed purely to arsenic or purely to UVR, making ID13, to the best of our knowledge, the first co-exposure signature to be reported using controlled experimental conditions. Analysis of existing skin cancer genomics data reveals that only a subset of cancers harbor ID13 and these exhibit an elevated UVR mutagenesis. Our results report a unique mutational signature caused by a co-exposure to two environmental carcinogens and provide comprehensive evidence that arsenic is a potent co-mutagen and co-carcinogen of UVR.

Circulatory white spot syndrome virus in South-West region of Bangladesh from 2014 to 2017: molecular characterization and genetic variation.

White Spot Syndrome Virus (WSSV), the etiological agent of White Spot Disease (WSD) is a major impediment for shrimp aquaculture in the worldwide. A critical threshold level of WSSV load in infected shrimp is an important trait for disease manifestation and WSSV transmission in cultured shrimp and subsequently make outbreaks. The present study investigated 120 naturally infected cultured shrimp samples by SYBR Green based qPCR assay for WSD diagnosis and quantification of WSSV load. Among them, 94 samples resulted a variable count of WSSV load ranging from 2.1 × 108 to 2.64 × 1014 copies/g of shrimp tissue. The severity of WSSV infection was assessed based on the established critical threshold load of WSSV in shrimp tissue. Compared to the established critical threshold value of WSSV load in shrimp tissue, our findings showed the horrifying scenario of the severity of WSSV infection in cultured shrimps of Bangladesh that was found to be above the critical limit to initiate an outbreak in the Bangladeshi shrimp aquaculture industry. The latest phylogenetic pattern was altered from the former monophyletic history among WSSVs of Bangladesh due to a variation at 500th nucleotide of VP28 coding gene. Viruses characterized from recent outbreaks in 2015 and 2017 displayed amino acid substitution at position 167 (G→E) on the surface of VP28 protein which has demonstrated the probable replacement of indigenous virus pool. Therefore, it is imperative to take initiative for the management and prevention of WSSV outbreak to sustain shrimp aquaculture in South-West region of Bangladesh.

Complete Genome Sequence of Foot-and-Mouth Disease Virus Serotype O Isolated from Bangladesh.

Foot-and-mouth disease (FMD) is a highly infectious enzootic disease caused by FMD virus. The complete genome sequence of a circulatory FMD virus (FMDV) serotype O isolated from Natore, Bangladesh, is reported here. Genomic analysis revealed antigenic heterogeneity within the VP1 region, a fragment deletion, and insertions at the 5' untranslated region (UTR) and 3A region compared to the genome of the available vaccine strain.