Uracil as a biomarker for spatial pyrimidine metabolism in the development of gingivobuccal oral squamous cell carcinoma.

No biomarker has yet been identified that allows accurate diagnosis and prognosis of oral cancers. In this study, we investigated the presence of key metabolites in oral cancer using proton nuclear magnetic resonance (NMR) spectroscopy to identify metabolic biomarkers of gingivobuccal oral squamous cell carcinoma (GB-OSCC). NMR spectroscopy revealed that uracil was expressed in 83.09% of tumor tissues and pyrimidine metabolism was active in GB-OSCC; these results correlated well with immunohistochemistry (IHC) and RNA sequencing data. Based on further gene and protein analyses, we proposed a pathway for the production of uracil in GB-OSCC tissues. Uridinetriphosphate (UTP) is hydrolyzed to uridine diphosphate (UDP) by CD39 in the tumor microenvironment (TME). We hypothesized that UDP enters the cell with the help of the UDP-specific P2Y6 receptor for further processing by ENTPD4/5 to produce uracil. As the ATP reserves diminish, the weakened immune cells in the TME utilize pyrimidine metabolism as fuel for antitumor activity, and the same mechanism is hijacked by the tumor cells to promote their survival. Correspondingly, the differential expression of ENTPD4 and ENTPD5 in immune and tumor cells, respectively, indicatedtheir involvement in disease progression. Furthermore, higher uracil levels were detected in patients with lymph node metastasis, indicating that metastatic potential is increased in the presence of uracil. The presence of uracil and/or expression patterns of intermediate molecules in purine and pyrimidine pathways, such asCD39, CD73, and P2Y6 receptors together with ENTPD4 and ENTPD5, hold promise as biomarker(s) for oral cancer diagnosis and prognosis.

MAL expression downregulation through suppressive H3K27me3 marks at the promoter in HPV16-related cervical cancers is prognostically relevant and manifested by the interplay of novel MAL antisense long noncoding RNA AC103563.8, E7 oncoprotein and EZH2.

BACKGROUND: MAL (T-lymphocyte maturation-associated protein) is highly downregulated in most cancers, including cervical cancer (CaCx), attributable to promoter hypermethylation. Long noncoding RNA genes (lncGs) play pivotal roles in CaCx pathogenesis, by interacting with human papillomavirus (HPV)-encoded oncoproteins, and epigenetically regulating coding gene expression. Hence, we attempted to decipher the impact and underlying mechanisms of MAL downregulation in HPV16-related CaCx pathogenesis, by interrogating the interactive roles of MAL antisense lncRNA AC103563.8, E7 oncoprotein and PRC2 complex protein, EZH2. RESULTS: Employing strand-specific RNA-sequencing, we confirmed the downregulated expression of MAL in association with poor overall survival of CaCx patients bearing HPV16, along with its antisense long noncoding RNA (lncRNA) AC103563.8. The strength of positive correlation between MAL and AC103563.8 was significantly high among patients compared to normal individuals. While downregulated expression of MAL was significantly associated with poor overall survival of CaCx patients bearing HPV16, AC103563.8 did not reveal any such association. We confirmed the enrichment of chromatin suppressive mark, H3K27me3 at MAL promoter, using ChIP-qPCR in HPV16-positive SiHa cells. Subsequent E7 knockdown in such cells significantly increased MAL expression, concomitant with decreased EZH2 expression and H3K27me3 marks at MAL promoter. In silico analysis revealed that both E7 and EZH2 bear the potential of interacting with AC103563.8, at the same binding domain. RNA immunoprecipitation with anti-EZH2 and anti-E7 antibodies, respectively, and subsequent quantitative PCR analysis in E7-silenced and unperturbed SiHa cells confirmed the interaction of AC103563.8 with EZH2 and E7, respectively. Apparently, AC103563.8 seems to preclude EZH2 and bind with E7, failing to block EZH2 function in patients. Thereby, enhanced EZH2 expression in the presence of E7 could potentially inactivate the MAL promoter through H3K27me3 marks, corroborating our previous results of MAL expression downregulation in patients. CONCLUSION: AC103563.8-E7-EZH2 axis, therefore, appears to crucially regulate the expression of MAL, through chromatin inactivation in HPV16-CaCx pathogenesis, warranting therapeutic strategy development.

Unfurling the functional association between long intergenic noncoding RNAs (lincRNAs) and HPV16-related cervical cancer pathogenesis through weighted gene co-expression network analysis of differentially expressed lincRNAs and coding genes.

Long intergenic noncoding RNAs (lincRNAs) do not overlap annotated coding genes and are located in intergenic regions, as opposed to antisense and sense-intronic lncRNAs, located in genic regions. LincRNAs influence gene expression profiles and are thereby key to disease pathogenesis. In this study, we assessed the association between lincRNAs and HPV16-positive cervical cancer (CaCx) pathogenesis using weighted gene co-expression network analysis (WGCNA) with coding genes, comparing differentially expressed lincRNA and coding genes (DElincGs and DEcGs, respectively) in HPV16-positive patients with CaCx (n = 44) with those in HPV-negative healthy individuals (n = 34). Our analysis revealed five DElincG modules, co-expressing and correlating with DEcGs. We validated a substantial number of such module-specific correlations in the HPV16-positive cancer TCGA-CESC dataset. Four such modules, displayed significant correlations with patient traits, such as HPV16 physical status, lymph node involvement and overall survival (OS), highlighting a collaborative effect of all genes within specific modules on traits. Using the DAVID bioinformatics knowledgebase, we identified the underlying biological processes associated with these modules as cancer development and progression-associated pathways. Next, we identified the top 10 DElincGs with the highest connectivity within each functional module. Focusing on the prognostic module hub genes, downregulated CTD-2619J13.13 expression was associated with poor patient OS. This lincRNA gene interacted with 25 coding genes of its module and was associated with such biological processes as keratinization loss and keratinocyte differentiation, reflecting severe disease phenotypes. This study has translational relevance in fighting various cancers with high mortality rates in underdeveloped countries.

Correlation of severity & clinical outcomes of COVID-19 with virus variants: A prospective, multicentre hospital network study.

BACKGROUND OBJECTIVES: The clinical course of COVID-19 and its prognosis are influenced by both viral and host factors. The objectives of this study were to develop a nationwide platform to investigate the molecular epidemiology of SARS-CoV-2 (Severe acute respiratory syndrome Corona virus 2) and correlate the severity and clinical outcomes of COVID-19 with virus variants. METHODS: A nationwide, longitudinal, prospective cohort study was conducted from September 2021 to December 2022 at 14 hospitals across the country that were linked to a viral sequencing laboratory under the Indian SARS-CoV-2 Genomics Consortium. All participants (18 yr and above) who attended the hospital with a suspicion of SARS-CoV-2 infection and tested positive by the reverse transcription-PCR method were included. The participant population consisted of both hospitalized as well as outpatients. Their clinical course and outcomes were studied prospectively. Nasopharyngeal samples collected were subjected to whole genome sequencing to detect SARS-CoV-2 variants. RESULTS: Of the 4972 participants enrolled, 3397 provided samples for viral sequencing and 2723 samples were successfully sequenced. From this, the evolution of virus variants of concern including Omicron subvariants which emerged over time was observed and the same reported here. The mean age of the study participants was 41 yr and overall 49.3 per cent were female. The common symptoms were fever and cough and 32.5 per cent had comorbidities. Infection with the Delta variant evidently increased the risk of severe COVID-19 (adjusted odds ratio: 2.53, 95% confidence interval: 1.52, 4.2), while Omicron was milder independent of vaccination status. The independent risk factors for mortality were age >65 yr, presence of comorbidities and no vaccination. INTERPRETATION CONCLUSIONS: The authors believe that this is a first-of-its-kind study in the country that provides real-time data of virus evolution from a pan-India network of hospitals closely linked to the genome sequencing laboratories. The severity of COVID-19 could be correlated with virus variants with Omicron being the milder variant.

Single-cell transcriptomic analysis of gingivo-buccal oral cancer reveals two dominant cellular programs.

Oral squamous cell carcinoma of the gingivo-buccal region (OSCC-GB) is the most common cancer among men in India, and is associated with poor prognosis and frequent recurrence. Cellular heterogeneity in OSCC-GB was investigated by single-cell RNA sequencing of tumors derived from the oral cavity of 12 OSCC-GB patients, 3 of whom had concomitant presence of a precancerous lesion (oral submucous fibrosis [OSMF]). Unique malignant cell types, features, and phenotypic shifts in the stromal cell population were identified in oral tumors with associated submucous fibrosis. Expression levels of FOS, ATP1A, and DUSP1 provided robust discrimination between tumors with or without the concomitant presence of OSMF. Malignant cell populations shared between tumors with and without OSMF were enriched with the expression of partial epithelial-mesenchymal transition (pEMT) or fetal cell type signatures indicative of two dominant cellular programs in OSCC-GB-pEMT and fetal cellular reprogramming. Malignant cells exhibiting fetal cellular and pEMT programs were enriched with the expression of immune-related pathway genes known to be involved in antitumor immune response. In the tumor microenvironment, higher infiltration of immune cells than the stromal cells was observed. The T cell population was large in tumors and diverse subtypes of T cells with varying levels of infiltration were found. We also detected double-negative PLCG2+ T cells and cells with intermediate M1-M2 macrophage polarization. Our findings shed light on unique aspects of cellular heterogeneity and cell states in OSCC-GB.

Upper respiratory tract microbiome profiles in SARS-CoV-2 Delta and Omicron infected patients exhibit variant specific patterns and robust prediction of disease groups.

IMPORTANCE: The role of the upper respiratory tract (URT) microbiome in predicting lung health has been documented in several studies. The dysbiosis in COVID patients has been associated with disease outcomes by modulating the host immune system. However, although it has been known that different SARS-CoV-2 variants manifest distinct transmissibility and mortality rates in human populations, their effect on the composition and diversity of the URT microbiome has not been studied to date. Unlike the older variant (Delta), the newer variant (Omicron) have become more transmissible with lesser mortality and the symptoms have also changed significantly. Hence, in the present study, we have investigated the change in the URT microbiome associated with Delta and Omicron variants and identified variant-specific signatures that will be useful in the assessment of lung health and can be utilized for nasal probiotic therapy in the future.

Identification of HPV16 positive cervical cancer subsets characterized by divergent immune and oncogenic phenotypes with potential implications for immunotherapy.

BACKGROUND: Cervical cancers (CaCx), like many other cancer types, portray high molecular heterogeneity that affects response to therapy, including immunotherapy. In India and other developing countries, CaCx mortality rates are very high because women report to the clinics with advanced cancers in absence of organized screening programs. This calls for implementation of newer therapeutic regimens for CaCx, like immunotherapy, which is again not used commonly in such countries. OBJECTIVE: Therefore, we focused on dissecting tumour immune heterogeneity, if any, identify immune gene-based biomarkers of heterogeneity and subsets of such cancers with the potential for immunotherapy. We also attempted to characterize the cancer-associated phenotypes of such subsets, including viral load, to decipher the relationship of tumour immunogenicity with oncogenicity. METHODS: Employing RNA-seq analysis of 44 HPV16 positive CaCx patients, immune subtypes were identified by unsupervised hierarchical clustering of global immune-gene expression profiles. Proportions of tumor infiltrating immune cells in the tumor milieu were estimated, employing Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT), using gene expression data from RNA-seq. The oncogenic phenotypes of the immune subtypes of CaCx were deciphered through differential gene expression (DEGs) and pathway enrichment analysis. Viral load was estimated through TaqMan-based qRT-PCR analysis. RESULTS: Analysis revealed the presence of two immune subtypes of CaCx, A (26/44; 59.09%) and B (18/44; 40.90%). Compared to Subtype-A, Subtype-B portrayed overexpression of immune genes and high infiltration of immune cells, specifically CD8+ T cells (p < 0.0001). Besides, a significant correlation between PD-1 and PD-L1 co-expression among Subtype-B, as opposed to Subtype-A, confirmed the interactive roles of these immune checkpoint molecules in Subtype B. Stepwise discriminant analysis pin-pointed ten immune-genes that could classify 100% of the patients significantly (p < 0.0001) into the two immune subtypes and serve as potential biomarkers of CaCx immunity. Differential gene expression analysis between the subtypes unveiled that Subtype-B was more biologically aggressive than Subtype-A, reflecting loss of structural integrity and promotion of cancer progression. The viral load was significantly lower in Subtype-B (average viral load = 10.74/100 ng of genomic DNA) compared to Subtype-A (average viral load = 14.29/100 ng of genomic DNA). Thus viral load and the ten-gene panel underscore their association with immunogenicity and oncogenicity. CONCLUSION: Our study provides strong evidence that only a subset, about 41% of HPV16 positive CaCx patients in India, portray immune enrichment of the tumor milieu coupled with aggressive phenotypes. Such subtypes are therefore likely to benefit through checkpoint molecule-based or tumor infiltrating lymphocyte-based immunotherapy, which could be a leap forward in tackling aggressive forms of such CaCx in India and other developing countries.

Overexpression of CD73 is associated with recurrence and poor prognosis of gingivobuccal oral cancer as revealed by transcriptome and deep immune profiling of paired tumor and margin tissues.

BACKGROUND: For various cancers, differences in response to treatment and subsequent survival period have been reported to be associated with variation in immune contextures. AIM: We sought to identify whether such association exists in respect of gingivobuccal oral cancer. MATERIALS AND METHODS: We performed deep immune profiling of tumor and margin tissues collected from 46 treatment naïve, Human Papillomavirus (HPV) negative, patients. Each patient was followed for 24 months and prognosis (recurrence/death) noted. Key findings were validated by comparing with TCGA-HNSC cohort data. RESULTS: About 28% of patients showed poor post-treatment prognosis. These patients exhibited a high probability of recurrence even within 1 year and death within 2 years. There was restricted immune cell infiltration in tumor, but not in margin, among these patients. Reduced expression of eight immune-related genes (IRGs) (NT5E, THRA, RBP1, TLR4, ITGA6, BMPR1B, ITGAV, SSTR1) in tumor strongly predicted better quality of prognosis, both in our patient cohort and in TCGA-HNSC cohort. Tumors of patients with better prognosis were associated with (a) lower CD73+ cells with concomitant lower expression level of NT5E/CD73, (b) higher proportions of CD4+ and CD8+ T cells, B cells, NK cells, M1 macrophages, (c) higher %Granzyme+ cells, (d) higher TCR and BCR repertoire diversities. CD73 expression in tumor was associated with low CD8+ and CD4+ T cells, low immune repertoire diversity, and advanced cancer stage. DISCUSSION AND CONCLUSION: High infiltration of anti-tumor immune cells in both tumors and margins results in good prognosis, while in patients with minimal infiltration in tumors in spite of high infiltration in margins results in poor prognosis. Targeted CD73 immune-checkpoint inhibition may improve clinical outcome.

Mapinsights: deep exploration of quality issues and error profiles in high-throughput sequence data.

High-throughput sequencing (HTS) has revolutionized science by enabling super-fast detection of genomic variants at base-pair resolution. Consequently, it poses the challenging problem of identification of technical artifacts, i.e. hidden non-random error patterns. Understanding the properties of sequencing artifacts holds the key in separating true variants from false positives. Here, we develop Mapinsights, a toolkit that performs quality control (QC) analysis of sequence alignment files, capable of detecting outliers based on sequencing artifacts of HTS data at a deeper resolution compared with existing methods. Mapinsights performs a cluster analysis based on novel and existing QC features derived from the sequence alignment for outlier detection. We applied Mapinsights on community standard open-source datasets and identified various quality issues including technical errors related to sequencing cycles, sequencing chemistry, sequencing libraries and across various orthogonal sequencing platforms. Mapinsights also enables identification of anomalies related to sequencing depth. A logistic regression-based model built on the features of Mapinsights shows high accuracy in detecting 'low-confidence' variant sites. Quantitative estimates and probabilistic arguments provided by Mapinsights can be utilized in identifying errors, bias and outlier samples, and also aid in improving the authenticity of variant calls.

NOTCH pathway inactivation reprograms stem-like oral cancer cells to JAK-STAT dependent state and provides the opportunity of synthetic lethality.

BACKGROUND: We have recently provided the evidence of interconvertible cellular states, driving non-genetic heterogeneity among stem-like oral cancer cells (oral-SLCCs). Here, NOTCH pathway-activity status is explored as one of the possible mechanisms behind this stochastic plasticity. METHODS: Oral-SLCCs were enriched in 3D-spheroids. Constitutively-active and inactive status of NOTCH pathway was achieved by genetic or pharmacological approaches. RNA sequencing and real-time PCR was performed for gene expression studies. in vitro cytotoxicity assessments were performed by AlamarBlue assay and in vivo effects were studied by xenograft growth in zebrafish embryo. RESULTS: We have observed stochastic plasticity in oral-SLCCs, spontaneously maintaining both NOTCH-active and inactive states. While cisplatin refraction was associated with post-treatment adaptation to the active-state of NOTCH pathway, oral-SLCCs with inactive NOTCH pathway status showed aggressive tumor growth and poor prognosis. RNAseq analysis clearly suggested the upregulation of JAK-STAT pathway in NOTCH pathway-inactive subset. The 3D-spheroids with lower NOTCH-activity status displayed significantly higher sensitivity to JAK-selective drugs, Ruxolitinib or Tofacitinib or siRNA mediated downregulation of tested partners STAT3/4. Oral-SLCCs were programmed to adapt the inactive status of NOTCH pathway by exposing to Î³-secretase inhibitors, LY411575 or RO4929097, followed by targeting with JAK-inhibitors, Ruxolitinib or Tofacitinib. This approach resulted in a very significant inhibition in viability of 3D-spheroids as well as xenograft initiation in Zebrafish embryos. CONCLUSION: Study revealed for the first time that NOTCH pathway-inactive state exhibit activation of JAK-STAT pathways, as synthetic lethal pair. Therefore, co-inhibition of these pathway may serve as novel therapeutic strategy against aggressive oral cancer.

Emergence of a unique SARS-CoV-2 Delta sub-cluster harboring a constellation of co-appearing non-Spike mutations.

Accumulation of diverse mutations across the structural and nonstructural genes is leading to rapid evolution of SARS-CoV-2, altering its pathogenicity. We performed whole genome sequencing of 239 SARS-CoV-2 RNA samples collected from both adult and pediatric patients across eastern India (West Bengal), during the second pandemic wave in India (April-May 2021). In addition to several common spike mutations within the Delta variant, a unique constellation of eight co-appearing non-Spike mutations was identified, which revealed a high degree of positive mutual correlation. Our results also demonstrated the dynamics of SARS-CoV-2 variants among unvaccinated pediatric patients. 41.4% of our studied Delta strains harbored this signature set of eight co-appearing non-Spike mutations and phylogenetically out-clustered other Delta sub-lineages like 21J, 21A, or 21I. This is the first report from eastern India that portrayed a landscape of co-appearing mutations in the non-Spike proteins, which might have led to the evolution of a distinct Delta subcluster. Accumulation of such mutations in SARS-CoV-2 may lead to the emergence of "vaccine-evading variants." Hence, monitoring of such non-Spike mutations will be significant in the formulation of any future vaccines against those SARS-CoV-2 variants that might evade the current vaccine-induced immunity, among both the pediatric and adult populations.

Autosomal recessive inheritance of a novel missense mutation of ITGB4 for Epidermolysis-Bullosa pyloric-atresia: a case report.

Epidermolysis-Bullosa (EB), a rare Mendelian disorder, exhibits complex phenotypic and locus-heterogeneity. We identified a nuclear family of clinically unaffected parents with two offsprings manifesting EB-Pyloric-Atresia (EB-PA), with a variable clinical severity. We generated whole exome sequence data on all four individuals to (1) identify the causal mutation behind EB-PA (2) understand the background genetic variation for phenotype variability of the siblings. We assumed an autosomal recessive mode of inheritance and used suites of bioinformatic and computational tools to collate information through global databases to identify the causal genetic variant for the disease. We also investigated variations in key genes that are likely to impact phenotype severity. We identified a novel missense mutation in the ITGB4 gene (p.Ala1227Asp), for which the parents were heterozygous and the children homozygous. The mutation in ITGB4 gene, predicted to reduce the stability of the primary alpha6beta4-plectin complex compared to all previously studied mutations on ITGB4 reported to cause EB.

Contrasting Distribution of SARS-CoV-2 Lineages across Multiple Rounds of Pandemic Waves in West Bengal, the Gateway of East and North-East States of India.

The evolution of viral variants and their impact on viral transmission have been an area of considerable importance in this pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We analyzed the viral variants in different phases of the pandemic in West Bengal, a state in India that is important geographically, and compared the variants with other states like Delhi, Maharashtra, and Karnataka, located in other regions of the country. We have identified 57 pango-lineages in 3,198 SARS-CoV-2 genomes, alteration in their distribution, as well as contrasting profiles of amino acid mutational dynamics across different waves in different states. The evolving characteristics of Delta (B.1.617.2) sublineages and alterations in hydrophobicity profiles of the viral proteins caused by these mutations were also studied. Additionally, implications of predictive host miRNA binding/unbinding to emerging spike or nucleocapsid mutations were highlighted. Our results throw considerable light on interesting aspects of the viral genomic variation and provide valuable information for improved understanding of wave-defining mutations in unfolding the pandemic. IMPORTANCE Multiple waves of infection were observed in many states in India during the coronavirus disease 2019 (COVID19) pandemic. Fine-scale evolution of major SARS-CoV-2 lineages and sublineages during four wave-window categories: Pre-Wave 1, Wave 1, Pre-Wave 2, and Wave 2 in four major states of India: Delhi (North), Maharashtra (West), Karnataka (South), and West Bengal (East) was studied using large-scale virus genome sequencing data. Our comprehensive analysis reveals contrasting molecular profiles of the wave-defining mutations and their implications in host miRNA binding/unbinding of the lineages in the major states of India.

Emergence of hybrid states of stem-like cancer cells correlates with poor prognosis in oral cancer.

Cancer cell state transitions emerged as powerful mechanisms responsible for drug tolerance and overall poor prognosis; however, evidences were largely missing in oral cancer. Here, by multiplexing phenotypic markers of stem-like cancer cells (SLCCs); CD44, CD24 and aldehyde dehydrogenase (ALDH), we characterized diversity among multiple oral tumor tissues and cell lines. Two distinct patterns of spontaneous transitions with stochastic bidirectional interconversions on 'ALDH-axis', and unidirectional non-interconvertible transitions on 'CD24-axis' were observed. Interestingly, plastic 'ALDH-axis' was harnessed by cells to adapt to a Cisplatin tolerant state. Furthermore, phenotype-specific RNA sequencing suggested the possible maintenance of intermediate hybrid cell states maintaining stemness within the differentiating subpopulations. Importantly, survival analysis with subpopulation-specific gene sets strongly suggested that cell-state transitions may drive non-genetic heterogeneity, resulting in poor prognosis. Therefore, we have described the phenotypic-composition of heterogeneous subpopulations critical for global tumor behavior in oral cancer; which may provide prerequisite knowledge for treatment strategies.

Computational prediction of the molecular mechanism of statin group of drugs against SARS-CoV-2 pathogenesis.

Recently published clinical data from COVID-19 patients indicated that statin therapy is associated with a better clinical outcome and a significant reduction in the risk of mortality. In this study by computational analysis, we have aimed to predict the possible mechanism of the statin group of drugs by which they can inhibit SARS-CoV-2 pathogenesis. Blind docking of the critical structural and functional proteins of SARS-CoV-2 like RNA-dependent RNA polymerase, M-protease of 3-CL-Pro, Helicase, and the Spike proteins ( wild type and mutants from different VOCs) were performed using the Schrodinger docking tool. We observed that fluvastatin and pitavastatin showed fair, binding affinities to RNA polymerase and 3-CL-Pro, whereas fluvastatin showed the strongest binding affinity to the helicase. Fluvastatin also showed the highest affinity for the SpikeDelta and a fair docking score for other spike variants. Additionally, molecular dynamics simulation confirmed the formation of a stable drug-protein complex between Fluvastatin and target proteins. Thus our study shows that of all the statins, fluvastatin can bind to multiple target proteins of SARS-CoV-2, including the spike-mutant proteins. This property might contribute to the potent antiviral efficacy of this drug.

Integrative analysis of genomic and transcriptomic data of normal, tumour, and co-occurring leukoplakia tissue triads drawn from patients with gingivobuccal oral cancer identifies signatures of tumour initiation and progression.

A thickened, white patch - leukoplakia - in the oral cavity is usually benign, but sometimes (in ~9% of individuals) it progresses to malignant tumour. Because the genomic basis of this progression is poorly understood, we undertook this study and collected samples of four tissues - leukoplakia, tumour, adjacent normal, and blood - from each of 28 patients suffering from gingivobuccal oral cancer. We performed multiomics analysis of the 112 collected tissues (four tissues per patient from 28 patients) and integrated information on progressive changes in the mutational and transcriptional profiles of each patient to create this genomic narrative. Additionally, we generated and analysed whole-exome sequence data from leukoplakia tissues collected from 11 individuals not suffering from oral cancer. Nonsynonymous somatic mutations in the CASP8 gene were identified as the likely events to initiate malignant transformation, since these were frequently shared between tumour and co-occurring leukoplakia. CASP8 alterations were also shown to enhance expressions of genes that favour lateral spread of mutant cells. During malignant transformation, additional pathogenic mutations are acquired in key genes (TP53, NOTCH1, HRAS) (41% of patients); chromosomal-instability (arm-level deletions of 19p and q, focal-deletion of DNA-repair pathway genes and NOTCH1, amplification of EGFR) (77%), and increased APOBEC-activity (23%) are also observed. These additional alterations were present singly (18% of patients) or in combination (68%). Some of these alterations likely impact immune-dynamics of the evolving transformed tissue; progression to malignancy is associated with immune suppression through infiltration of regulatory T-cells (56%), depletion of cytotoxic T-cells (68%), and antigen-presenting dendritic cells (72%), with a concomitant increase in inflammation (92%). Patients can be grouped into three clusters by the estimated time to development of cancer from precancer by acquiring additional mutations (range: 4-10 years). Our findings provide deep molecular insights into the evolutionary processes and trajectories of oral cancer initiation and progression. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Reciprocal interplay between asporin and decorin: Implications in gastric cancer prognosis.

Effective patient prognosis necessitates identification of novel tumor promoting drivers of gastric cancer (GC) which contribute to worsened conditions by analysing TCGA-gastric adenocarcinoma dataset. Small leucine-rich proteoglycans, asporin (ASPN) and decorin (DCN), play overlapping roles in development and diseases; however, the mechanisms underlying their interplay remain elusive. Here, we investigated the complex interplay of asporin, decorin and their interaction with TGFß in GC tumor and corresponding normal tissues. The mRNA levels, protein expressions and cellular localizations of ASPN and DCN were analyzed using real-time PCR, western blot and immunohistochemistry, respectively. The protein-protein interaction was predicted by in-silico interaction analysis and validated by co-immunoprecipitation assay. The correlations between ASPN and EMT proteins, VEGF and collagen were achieved using western blot analysis. A significant increase in expression of ASPN in tumor tissue vs. normal tissue was observed in both TCGA and our patient cohort. DCN, an effective inhibitor of the TGFß pathway, was negatively correlated with stages of GC. Co-immunoprecipitation demonstrated that DCN binds with TGFß, in normal gastric epithelium, whereas in GC, ASPN preferentially binds TGFß. Possible activation of the canonical TGFß pathway by phosphorylation of SMAD2 in tumor tissues suggests its role as an intracellular tumor promoter. Furthermore, tissues expressing ASPN showed unregulated EMT signalling. Our study uncovers ASPN as a GC-promoting gene and DCN as tumor suppressor, suggesting that ASPN can act as a prognostic marker in GC. For the first time, we describe the physical interaction of TGFß with ASPN in GC and DCN with TGFß in GC and normal gastric epithelium respectively. This study suggests that prevention of ASPN-TGFß interaction or overexpression of DCN could serve as promising therapeutic strategies for GC patients.

dbGENVOC: database of GENomic Variants of Oral Cancer, with special reference to India.

Oral cancer is highly prevalent in India and is the most frequent cancer type among Indian males. It is also very common in southeast Asia. India has participated in the International Cancer Genome Consortium (ICGC) and some national initiatives to generate large-scale genomic data on oral cancer patients and analyze to identify associations and systematically catalog the associated variants. We have now created an open, web-accessible database of these variants found significantly associated with Indian oral cancer patients, with a user-friendly interface to enable easy mining. We have value added to this database by including relevant data collated from various sources on other global populations, thereby providing opportunities of comparative geographical and/or ethnic analyses. Currently, no other database of similar nature is available on oral cancer. We have developed Database of GENomic Variants of Oral Cancer, a browsable online database framework for storage, retrieval and analysis of large-scale data on genomic variants and make it freely accessible to the scientific community. Presently, the web-accessible database allows potential users to mine data on ∼24 million clinically relevant somatic and germline variants derived from exomes (n = 100) and whole genomes (n = 5) of Indian oral cancer patients; all generated by us. Variant data from The Cancer Genome Atlas and data manually curated from peer-reviewed publications were also incorporated into the database for comparative analyses. It allows users to query the database by a single gene, multiple genes, multiple variant sites, genomic region, patient ID and pathway identities. Database URL: http://research.nibmg.ac.in/dbcares/dbgenvoc/.

SARS-CoV-2 mutation 614G creates an elastase cleavage site enhancing its spread in high AAT-deficient regions.

SARS-CoV-2 was first reported from China. Within three months, it evolved to 10 additional subtypes. Two evolved subtypes (A2 and A2a) carry a non-synonymous Spike protein mutation (D614G). We conducted phylodynamic analysis of over 70,000 SARS-CoV-2 coronaviruses worldwide, sequenced until July2020, and found that the mutant subtype (614G) outcompeted the pre-existing type (614D), significantly faster in Europe and North-America than in East Asia. Bioinformatically and computationally, we identified a novel neutrophil elastase (ELANE) cleavage site introduced in the G-mutant, near the S1-S2 junction of the Spike protein. We hypothesised that elevation of neutrophil elastase level at the site of infection will enhance the activation of Spike protein thus facilitating host cell entry for 614G, but not the 614D, subtype. The level of neutrophil elastase in the lung is modulated by its inhibitor α1-antitrypsin (AAT). AAT prevents lung tissue damage by elastase. However, many individuals exhibit genotype-dependent deficiency of AAT. AAT deficiency eases host-cell entry of the 614G virus, by retarding inhibition of neutrophil elastase and consequently enhancing activation of the Spike protein. AAT deficiency is highly prevalent in European and North-American populations, but much less so in East Asia. Therefore, the 614G subtype is able to infect and spread more easily in populations of the former regions than in the latter region. Our analyses provide a molecular biological and evolutionary model for the higher observed virulence of the 614G subtype, in terms of causing higher morbidity in the host (higher infectivity and higher viral load), than the non-mutant 614D subtype.

Two novel cell culture models of buccal mucosal oral cancer from patients with no risk-habits of tobacco smoking or chewing.

OBJECTIVE: Tobacco consumption is one of the major etiological factors for oral cancer, but it also develops in non-tobacco users, with unknown etiologies. Cellular models for tobacco associated oral cancer are available, however; reports of cellular models for studying non-tobacco associated oral cancer are limiting. We report here the establishment and characterization of two novel buccal mucosal cancer cell lines 'GBC02' and 'GBC035' derived from non-tobacco users. MATERIALS AND METHODS: Short tandem repeats (STR) profiling, Next-generation sequencing for whole-genome, exome and copy number alterations, immunofluorescence, flow-cytometry, proliferation, live-cell chemotaxis, 3D-spheroid formation, chemotherapy response, gene-expression microarray, gene-set enrichment analysis and xenograft development were performed. RESULTS: Sources of the established cultures were matched to their donors through STR profiling. Genome sequence analysis revealed somatic mutations in TP53, CASP8, CDKN2A for GBC02 with deletions and amplifications encompassing CDKN2A, FAT1 and CCND1, PIK3CA, SOX2, EGFR, MYC genes, respectively. GBC035 harbored mutations in FAT1, NOTCH1, HRAS, CDKN2A, HLA-B, HLA-A genes. While GBC035 cells showed higher E-Cadherin positive cell-cell junctions and collective cell migration in chemotaxis; GBC02 cells were vimentin-positive and demonstrated individual cell migration. Further, exhibiting their relevance to preclinical research, GBC02 3D-spheroids demonstrated enrichment of development-related gene-signatures in microarray transcriptome analysis and were resistant to Cisplatin, but showed sensitivity to cancer stem cells-targeting drug, Salinomycin. Additionally, tumorigenic ability of GBC02 was demonstrated. CONCLUSIONS: Altogether, we present here comprehensively characterized unique cell lines established from non-tobacco associated tumors, which may serve as models for preclinical investigations of oral cancers caused independent of tobacco usage.