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.

Deep sequencing reveals recurrent somatic mutations and distinct molecular subgroups in gastric cancer in Mizo population, North East India.

In India, Mizoram has the highest incidence of gastric cancer (GC) which might be associated with environmental factors such as diet, Helicobacter pylori (H.pylori) and Epstein-Barr virus (EBV) infections, and somatic genomic alterations. We performed PCR cum sequencing and fragment analysis for detection of H. pylori/EBV infection and microsatellite Instability (MSI) in GC patients (N = 68). Somatic mutations were identified by targeted and exome sequencing. We found 87% of GC patients infected with H. pylori and or EBV. Pathogenic infections were mostly mutually exclusive with only 16% of coinfection. TP53, MUC6, and ARID1A were significantly mutated. Two molecular subgroups with distinctive mutational profiles were identified: (1) patients harboring mutations in TP53 and (2) patients harboring mutations in RTK/RAS/PI3-K signaling pathway and chromatin-remodeling genes. Therefore, EBV and H. pylori infections and somatic mutations in the genes involved in RTK/RAS/PI3K signaling pathway, chromatin-remodeling, and TP53 might drive GC development and progression in Mizo patients.

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.

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.

Association of exosomal miR-96-5p and miR-146a-5p with the disease severity in dengue virus infection.

Exosomes are small extracellular vesicles secreted by cells and have a major role in cell-to-cell signaling. As dengue infection progresses from a mild to a severe form of infection, the exosome's microRNA (miRNA) composition might change, which may contribute to pathogenesis. In this study, a comprehensive analysis of serum exosomal miRNAs was performed and their involvement in dengue virus-induced disease progression in an Indian cohort was assessed. Small RNA-seq showed 50 differentially expressed exosomal miRNAs that were significantly dysregulated during dengue infection. After extensive validation, miR-96-5p was found to be significantly upregulated, whereas miR-146a-5p was significantly downregulated with the progression of disease to severe form. Interestingly, a strong positive correlation was found between the expression levels of miR-96-5p and miR-146a-5p and the platelet levels of the patients. Further, study of miR-146a-5p showed that it regulates the expression of the proteins which are involved in the immune responses. These results suggest that miR-96-5p and miR-146a-5p could be used as diagnostic and prognostic markers for dengue disease progression, in addition to the already available biochemical and pathological parameters.

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.

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.

Application of Random Forest and data integration identifies three dysregulated genes and enrichment of Central Carbon Metabolism pathway in Oral Cancer.

BACKGROUND: Studies of epigenomic alterations associated with diseases primarily focus on methylation profiles of promoter regions of genes, but not of other genomic regions. In our past work (Das et al. 2019) on patients suffering from gingivo-buccal oral cancer - the most prevalent form of cancer among males in India - we have also focused on promoter methylation changes and resultant impact on transcription profiles. Here, we have investigated alterations in non-promoter (gene-body) methylation profiles and have carried out an integrative analysis of gene-body methylation and transcriptomic data of oral cancer patients. METHODS: Tumor and adjacent normal tissue samples were collected from 40 patients. Data on methylation in the non-promoter (gene-body) regions of genes and transcriptome profiles were generated and analyzed. Because of high dimensionality and highly correlated nature of these data, we have used Random Forest (RF) and other data-analytical methods. RESULTS: Integrative analysis of non-promoter methylation and transcriptome data revealed significant methylation-driven alterations in some genes that also significantly impact on their transcription levels. These changes result in enrichment of the Central Carbon Metabolism (CCM) pathway, primarily by dysregulation of (a) NTRK3, which plays a dual role as an oncogene and a tumor suppressor; (b) SLC7A5 (LAT1) which is a transporter dedicated to essential amino acids, and is overexpressed in cancer cells to meet the increased demand for nutrients that include glucose and essential amino acids; and, (c) EGFR which has been earlier implicated in progression, recurrence, and stemness of oral cancer, but we provide evidence of epigenetic impact on overexpression of this gene for the first time. CONCLUSIONS: In rapidly dividing cancer cells, metabolic reprogramming from normal cells takes place to enable enhanced proliferation. Here, we have identified that among oral cancer patients, genes in the CCM pathway - that plays a fundamental role in metabolic reprogramming - are significantly dysregulated because of perturbation of methylation in non-promoter regions of the genome. This result compliments our previous result that perturbation of promoter methylation results in significant changes in key genes that regulate the feedback process of DNA methylation for the maintenance of normal cell division.

Lymph node metastasis in oral cancer is strongly associated with chromosomal instability and DNA repair defects.

Oral squamous cell carcinoma (OSCC) is highly prevalent in south and southeast Asia. Many (30-50%) OSCC patients develop lymph node metastasis (LNM), which is the most important prognostic factor in OSCC. To identify genomic correlates of LNM, we compared exome sequences and copy number variation data of blood and tumor DNA from highly contrasting subgroups of patients to reduce false inferences-(i) patients with LNM and (ii) patients with late stage disease but without LNM. We found that LNM is associated with (i) specific hotspot somatic mutations in TP53 and CASP8; (ii) rare nonsilent germline mutations in BRCA2 and FAT1; (iii) mutations in mito-G2/M and nonhomologous end joining (NHEJ) pathways; (iv) recurrent deletion of genes for DNA repair by homologous recombination; and (v) chromosomal instability. LN+ patients with NHEJ pathway mutations have longer disease-free survival. Five genomic features have a high predictive value of LNM.

Whole genome DNA methylation profiling of oral cancer in ethnic population of Meghalaya, North East India reveals novel genes.

Oral Squamous Cell Carcinoma (OSCC) is a serious and one of the most common and highly aggressive malignancies. Epigenetic factors such as DNA methylation have been known to be implicated in a number of cancer etiologies. The main objective of this study was to investigate physiognomies of Promoter DNA methylation patterns associated with oral cancer epigenome with special reference to the ethnic population of Meghalaya, North East India. The present study identifies 27,205 CpG sites and 3811 regions that are differentially methylated in oral cancer when compared to matched normal. 45 genes were found to be differentially methylated within the promoter region, of which 38 were hypermethylated and 7 hypomethylated. 14 of the hypermethylated genes were found to be similar to that of the TCGA-HNSCC study some of which are TSGs and few novel genes which may serve as candidate methylation biomarkers for OSCC in this poorly characterized ethnic group.

Harnessing genomics to improve outcomes for women with cancer in India: key priorities for research.

Cumulatively, breast, cervical, ovarian, and uterine cancer account for more than 70% of cancers in women in India. Distinct differences in the clinical presentation of women with cancer suggest underlying differences in cancer biology and genetics. The peak age of onset of breast and ovarian cancer appears to be a decade earlier in India (age 45-50 years) than in high-income countries (age >60 years). Understanding these differences through research to develop diagnosis, screening, prevention, and treatment frameworks that ar e specific to the Indian population are critical and essential to improving women's health in India. Since the sequencing of the human genome in 2001, applications of advanced technologies, such as massively parallel sequencing, have transformed the understanding of the genetic and environmental drivers of cancer. How can advanced technologies be harnessed to provide health-care solutions at a scale and to a budget suitable for a country of 1·2 billion people? What research programmes are necessary to answer questions specific to India, and to build capacity for innovative solutions using these technologies? In order to answer these questions, we convened a workshop with key stakeholders to address these issues. In this Series paper, we highlight challenges in tackling the growing cancer burden in India, discuss ongoing genomics research and developments in infrastructure, and suggest key priorities for future research in cancer in India.

Pre-operative progesterone benefits operable breast cancer patients by modulating surgical stress.

PURPOSE: We have reported a survival benefit of single injection of hydroxyprogesterone prior to surgery for primary tumour in patients with node-positive operable breast cancer. Hydroxyprogesterone was meant to recapitulate the luteal phase of menstrual cycle in these women. We wanted to understand the molecular basis of action of hydroxyprogesterone on primary breast tumours in a peri-operative setting. METHODS: We performed whole transcriptome sequencing (RNA-Seq) of primary breast tumour samples collected from patients before and after hydroxyprogesterone exposure and controls. Paired breast cancer samples were obtained from patients who were given hydroxyprogesterone before surgery and a group of patients who were subjected to only surgery. RESULTS: A test of significance between the two groups revealed 207 significantly altered genes, after correction for multiple hypothesis testing. We found significantly contrasting gene expression patterns in exposed versus unexposed groups; 142 genes were up-regulated post-surgery among exposed patients, and down-regulated post-surgery among unexposed patients. Significantly enriched pathways included genes that respond to progesterone, cellular stress, nonsense-mediated decay of proteins and negative regulation of inflammatory response. These results suggest that cellular stress is modulated by hydroxyprogesterone. Network analysis revealed that UBC, a mediator of stress response, to be a major node to which many of the significantly altered genes connect. CONCLUSIONS: Our study suggests that pre-operative exposure to progesterone favourably modulates the effect of surgical stress, and this might underlie its beneficial effect when administered prior to surgery.

Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington's Disease.

Altered expression levels of protein-coding genes and microRNAs have been implicated in the pathogenesis of Huntington's disease (HD). The involvement of other ncRNAs, especially long ncRNAs (lncRNA), is being realized recently and the related knowledge is still rudimentary. Using small RNA sequencing and PCR arrays we observed perturbations in the levels of 12 ncRNAs in HD mouse brain, eight of which had human homologs. Of these, Meg3, Neat1, and Xist showed a consistent and significant increase in HD cell and animal models. Transient knock-down of Meg3 and Neat1 in cell models of HD led to a significant decrease of aggregates formed by mutant huntingtin and downregulation of the endogenous Tp53 expression. Understanding Meg3 and Neat1 functions in the context of HD pathogenesis is likely to open up new strategies to control the disease.

Exome sequencing reveals the likely involvement of SOX10 in uveal melanoma.

PURPOSE: To identify the spectrum of somatic mutations in an Asian Indian patient with uveal melanoma (UM) without metastasis using exome sequencing. CASE REPORT: A 49-year-old man from India was diagnosed as having cilio-choroidal (uveal) melanoma (UM), without metastasis, in his right eye with the help of magnetic resonance imaging. This was later confirmed by histopathological evaluation. Two individuals from India with non-neoplastic blind eyes were recruited as controls. The affected eyes from the UM patient and the two control individuals were enucleated, and uveal tissues were collected. DNA was extracted from uveal tissue, and the matched blood sample from each of the three individuals was followed by exome sequencing. Statistical and bioinformatic analyses were done to identify somatic mutations and their putative associations with UM. Thirty-one somatic mutations (25 amino acid altering) in protein-coding (exonic) regions were detected in the UM patient. Of the amino acid-altering somatic mutations, 16 mutations were predicted to be candidate mutations relevant to UM. Somatic mutations, putatively causal for UM, were identified in GNAQ, SF3B1, and SOX10. CONCLUSIONS: Somatic mutations in GNAQ and SF3B1 genes were probable drivers of UM in the Indian patient; these were also reported earlier in some White patients. In addition, a frameshift deletion of 20 base pairs has been identified in SOX10 in the UM patient. Somatic mutations in SOX10, a transcription factor, which acts upstream of microphthalmia-associated transcription factor and synergizes with microphthalmia-associated transcription factor, was identified in some melanoma cell lines. The transcription factor SOX10 was found to have an essential role in melanocyte development and pigmentation. Our finding of the frameshift deletion (p.H387fs) in exon 4 of SOX10 in UM provides an important insight and complements earlier findings of mutations in GNAQ and SF3B1 on the genomic basis of UM.

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.

A FACS-based novel isolation technique identifies heterogeneous CTCs in oral squamous cell carcinoma.

Purpose: Isolating circulating tumour cells (CTCs) from the blood is challenging due to their low abundance and heterogeneity. Limitations of conventional CTC detection methods highlight the need for improved strategies to detect and isolate CTCs. Currently, the Food and Drug Administration (FDA)-approved CellSearch™ and other RUO techniques are not available in India. Therefore, we wanted to develop a flexible CTC detection/isolation technique that addresses the limitation(s) of currently available techniques and is suitable for various downstream applications. Methods: We developed a novel, efficient, user-friendly CTC isolation strategy combining density gradient centrifugation and immuno-magnetic hematogenous cell depletion with fluorescence-activated cell sorting (FACS)-based positive selection using multiple CTC-specific cell-surface markers. For FACS, a stringent gating strategy was optimised to exclude debris and doublets by side scatter/forward scatter (SSC/FSC) discriminator, remove dead cells by 4',6-diamidino-2-phenylindole (DAPI) staining, and eliminate non-specific fluorescence using a "dump" channel. APC-labelled anti-CD45mAB was used to gate remaining hematogenous cells, while multiple epithelial markers (EpCAM, EGFR, and Pan-Cytokeratin) and an epithelial-mesenchymal transition (EMT) marker (Vimentin) labelled with fluorescein isothiocyanate (FITC) were used to sort cancer cells. The technique was initially developed by spiking Cal 27 cancer cells into the blood of healthy donors and then validated in 95 biopsy-proven oral squamous cell carcinoma (OSCC) patients. CTCs isolated from patients were reconfirmed by Giemsa staining, immuno-staining, and whole transcriptome amplification (WTA), followed by qRT-PCR. In vitro culture and RNA sequencing (RNA-Seq) were also performed to confirm their suitability for various downstream applications. Results: The mean detection efficiency for the Cal 27 tongue cancer cells spiked in the whole blood of healthy donors was 32.82% ± 12.71%. While ~75% of our patients (71/95) had detectable CTCs, the CTC positivity was independent of the TNM staging. The isolated potential cancer cells from OSCC patients were heterogeneous in size. They expressed different CTC-specific markers in various combinations as identified by qRT-PCR after WTA in different patients. Isolated CTCs were also found to be suitable for downstream applications like short-term CTC culture and RNA-Seq. Conclusion: We developed a sensitive, specific, flexible, and affordable CTC detection/isolation technique, which is scalable to larger patient cohorts, provides a snapshot of CTC heterogeneity, isolates live CTCs ready for downstream molecular analysis, and, most importantly, is suitable for developing countries.