Nidhan K. Biswas

Mutational landscape of gingivo-buccal oral squamous cell carcinoma reveals new recurrently-mutated genes and molecular subgroups

Gingivo-buccal oral squamous cell carcinoma (OSCC-GB), an anatomical and clinical subtype of head and neck squamous cell carcinoma (HNSCC), is prevalent in regions where tobacco-chewing is common. Exome sequencing (n=50) and recurrence testing (n=60) reveals that some significantly and frequently altered genes are specific to OSCC-GB (USP9X, MLL4, ARID2, UNC13C and TRPM3), while some others are shared with HNSCC (for example, TP53, FAT1, CASP8, HRAS and NOTCH1). We also find new genes with recurrent amplifications (for example, DROSHA, YAP1) or homozygous deletions (for example, DDX3X) in OSCC-GB. We find a high proportion of C>G transversions among tobacco users with high numbers of mutations. Many pathways that are enriched for genomic alterations are specific to OSCC-GB. Our work reveals molecular subtypes with distinctive mutational profiles such as patients predominantly harbouring mutations in CASP8 with or without mutations in FAT1. Mean duration of disease-free survival is significantly elevated in some molecular subgroups. These findings open new avenues for biological characterization and exploration of therapies.

Increased risk of oral cancer in relation to common Indian mitochondrial polymorphisms and Autosomal GSTP1 locus

Polymorphisms at mitochondrial (mt) loci could modulate the risk of diseases including cancers. Here the mtDNA polymorphisms at 12,308 nucleotide pairs (np), 11,467 np, 10,400 np, and 10,398 np were studied to examine the association with the risk of oral cancer and leukoplakia, alone and in combination with polymorphisms at the GST loci.

A large, systematic molecular-genetic study of G6PD in Indian populations identifies a new non-synonymous variant and supports recent positive selection

Malaria has been endemic in India. G6PD deficiency is known to confer resistance to malaria. Many G6PD deficiency variants, some of which are India-specific, are known to occur in high frequencies in India. This is the first systematic molecular-genetic study in multiple populations from India drawn from diverse ethnic, socio-cultural and geographical backgrounds. Resequencing of the G6PD gene was carried out in 80 males and then the polymorphic variants were genotyped in 400 individuals of both genders, drawn from 10 ethnic groups of India. Our study has identified one new exonic variant (M159I; exon-5), occurring in multiple populations, that is predicted to result in G6PD deficiency. A strong geographical sub-structuring of known G6PD variants has also been established. We have compared all available data from public-domain resources with those generated in this study to identify the nature and extent of natural selection. Our results (a) provide indication of weak negative selection, and (b) reveal signals of recent positive selection for the G6PD Orissa and G6PD Coimbra mutation bearing haplotypes. These inferences have been interpreted in the light of malarial protection to the populations that have been long exposed to plasmodium infection.

Hedgehog signaling pathway is active in GBM with GLI1 mRNA expression showing a single continuous distribution rather than discrete high/low clusters.

Hedgehog (Hh) signaling pathway is a valid therapeutic target in a wide range of malignancies. We focus here on glioblastoma multiforme (GBM), a lethal malignancy of the central nervous system (CNS). By analyzing RNA-sequencing based transcriptomics data on 149 clinical cases of TCGA-GBM database we show here a strong correlation (r = 0.7) between GLI1 and PTCH1 mRNA expression—as a hallmark of the canonical Hh-pathway activity in this malignancy. GLI1 mRNA expression varied in 3 orders of magnitude among the GBM patients of the same cohort showing a single continuous distribution—unlike the discrete high/low-GLI1 mRNA expressing clusters of medulloblastoma (MB). When compared with MB as a reference, the median GLI1 mRNA expression in GBM appeared 14.8 fold lower than that of the “high-Hh” cluster of MB but 5.6 fold higher than that of the “low-Hh” cluster of MB. Next, we demonstrated statistically significant up- and down-regulation of GLI1 mRNA expressions in GBM patient-derived low-passage neurospheres in vitro by sonic hedgehog ligand-enriched conditioned media (shh-CM) and by Hh-inhibitor drug vismodegib respectively. We also showed clinically achievable dose (50 μM) of vismodegib alone to be sufficient to induce apoptosis and cell cycle arrest in these low-passage GBM neurospheres in vitro. Vismodegib showed an effect on the neurospheres, both by down-regulating GLI1 mRNA expression and by inducing apoptosis/cell cycle arrest, irrespective of their relative endogenous levels of GLI1 mRNA expression. We conclude from our study that this single continuous distribution pattern of GLI1 mRNA expression technically puts almost all GBM patients in a single group rather than discrete high- or low-clusters in terms of Hh-pathway activity. That is suggestive of therapies with Hh-pathway inhibitor drugs in this malignancy without a need for further stratification of patients on the basis of relative levels of Hh-pathway activity among them.

Using HapMap data: a cautionary note

The HapMap data are being widely used in human genetic studies. We show by direct resequencing of a ∼6-kb region of chromosome 1 that the HapMap data are unreliable for this region. This region contains a recent mitochondrial (mt) DNA insertion. The HapMap data report the corresponding mtDNA variation and not the nuclear DNA variation. In view of mtDNA insertions of varying lengths throughout the human genome and considerable segmental duplications, it is necessary to use the HapMap data cautiously.

Somatic mutations in arachidonic acid metabolism pathway genes enhance oral cancer post-treatment disease-free survival.

The arachidonic acid metabolism (AAM) pathway promotes tumour progression. Chemical inhibitors of AAM pathway prolong post-treatment survival of cancer patients. Here we test whether non-synonymous somatic mutations in genes of this pathway, acting as natural inhibitors, increase post-treatment survival. We identify loss-of-function somatic mutations in 15 (18%) of 84 treatment-naïve oral cancer patients by whole-exome sequencing, which we map to genes of AAM pathway. Patients (n = 53) who survived ≥ 12 months after surgery without recurrence have significantly (P = 0.007) higher proportion (26% versus 3%) of mutations than those who did not (n = 31). Patients with mutations have a significantly (P = 0.003) longer median disease-free survival (24 months) than those without (13 months). Compared with the presence of a mutation, absence of any mutation increases the hazard ratio for death (11.3) significantly (P = 0.018). The inferences are strengthened when we pool our data with The Cancer Genome Atlas (TCGA) data. In patients with AAM pathway mutations, some downstream pathways, such as the PI3K-Akt pathway, are downregulated.

Variant allele frequency enrichment analysis in vitro reveals sonic hedgehog pathway to impede sustained temozolomide response in GBM

Neoplastic cells of Glioblastoma multiforme (GBM) may or may not show sustained response to temozolomide (TMZ) chemotherapy. We hypothesize that TMZ chemotherapy response in GBM is predetermined in its neoplastic clones via a specific set of mutations that alter relevant pathways. We describe exome-wide enrichment of variant allele frequencies (VAFs) in neurospheres displaying contrasting phenotypes of sustained versus reversible TMZ-responses in vitro. Enrichment of VAFs was found on genes ST5, RP6KA1 and PRKDC in cells showing sustained TMZ-effect whereas on genes FREM2, AASDH and STK36, in cells showing reversible TMZ-effect. Ingenuity pathway analysis (IPA) revealed that these genes alter cell-cycle, G2/M-checkpoint-regulation and NHEJ pathways in sustained TMZ-effect cells whereas the lysine-II&V/phenylalanine degradation and sonic hedgehog (Hh) pathways in reversible TMZ-effect cells. Next, we validated the likely involvement of the Hh-pathway in TMZ-response on additional GBM neurospheres as well as on GBM patients, by extracting RNA-sequencing-based gene expression data from the TCGA-GBM database. Finally, we demonstrated TMZ-sensitization of a TMZ non-responder neurosphere in vitro by treating them with the FDA-approved pharmacological Hh-pathway inhibitor vismodegib. Altogether, our results indicate that the Hh-pathway impedes sustained TMZ-response in GBM and could be a potential therapeutic target to enhance TMZ-response in this malignancy.

Profiling of genomic alterations of mitochondrial DNA in gingivobuccal oral squamous cell carcinoma: Implications for disease progress

We have identified 164 somatic mutations in mitochondrial DNA in gingivobuccal oral cancer by deep sequencing the mitochondrial genome from paired tumor and blood DNA samples from 89 patients. We have found evidence of positive selection of somatic nonsynonymous mutations. Non-synonymous mutations in mitochondrial respiratory genes were found to increase the risk of lymph node metastasis (P = 0.0028). We have observed a significant reduction in mitochondrial DNA copy number in tumor DNA of these patients compared to the DNA from adjacent normal tissue samples (P < 1 × 10-6). Analysis of transcriptome data of tumor and adjacent normal tissue revealed patients harboring mutations in mitochondrial protein-coding genes exhibited reduced expression of mitochondrial transcripts.

Analysis of the whole transcriptome from gingivo-buccal squamous cell carcinoma reveals deregulated immune landscape and suggests targets for immunotherapy

Background Gingivo-buccal squamous cell carcinoma (GBSCC) is one of the most common oral cavity cancers in India with less than 50% patients surviving past 5 years. Here, we report a whole transcriptome profile on a batch of GBSCC tumours with diverse tobacco usage habits. The study provides an entire landscape of altered expression with an emphasis on searching for targets with therapeutic potential. Methods Whole transcriptomes of 12 GBSCC tumours and adjacent normal tissues were sequenced and analysed to explore differential expression of genes. Expression changes were further compared with those in TCGA head and neck cohort (n = 263) data base and validated in an independent set of 10GBSCC samples. Results Differentially expressed genes (n = 2176) were used to cluster the patients based on their tobacco habits, resulting in 3 subgroups. Immune response was observed to be significantly aberrant, along with cell adhesion and lipid metabolism processes. Different modes of immune evasion were seen across 12 tumours with up-regulation or consistent expression of CD47, unlike other immune evasion genes such as PDL1, FUT4, CTLA4 and BTLA which were downregulated in a few samples. Variation in infiltrating immune cell signatures across tumours also indicates heterogeneity in immune evasion strategies. A few actionable genes such as ITGA4, TGFB1 and PTGS1/COX1 were over expressed in most samples. Conclusion This study found expression deregulation of key immune evasion genes, such as CD47 and PDL1, and reasserts their potential as effective immunotherapeutic targets for GBSCC, which requires further clinical studies. Present findings reiterate the idea of using transcriptome profiling to guide precision therapeutic strategies.

Mutational landscape of cytokine genes across major tumour types identifies new targets

Introduction: Components of immune system communicate extensively in tumour micro environment. Normally, immune system engages with tumours to inhibit its further progression. Simultaneously, cancer cells learn cues derived from immune system to its own growth advantage. Cytokines are cell signaling messengers that affect disease pathogenesis, non-specific response to infection, specific response to antigen, etc. A battery of cytokines are produced in the tumour microenvironment, when released in response to infections and inflammations, can function to inhibit tumour development and progression. Cancer cells also release cytokines that promote growth, extenuate apoptosis and perform invasion and metastasis. Hypothesis: Alterations in cytokine signaling genes might help tumour to misguide immune system. The aim of the study is to identify such genomic alterations in cytokine genes that may drive major human cancers. Methods: We did extensive literature survey to prepare a list of known cytokine genes (n=776) which were validated in multiple databases. To know the baseline DNA variation in cytokine genes, we analyzed DNA variations in healthy human population from the 1000 Genome project dataset. Somatic mutational landscape for cytokine genes were analyzed in 32 different human cancer types (TCGA data). Significantly mutated genes were detected using MutSig2CV and Oncodrive FM analysis. Genes found significant in both analysis were tabulated. Standard statistical and bioinformatics analysis were done further to identify putative driver events. Result: We detected 9 significantly mutated cytokine genes across major tumor types. EDN1 was found to be most significantly mutated, in multiple tumour types; apart from genes like CDH1, B2M, HLA-B, IL4, TRIM22, TGFB1, GDF1 and CRABP2. Discussion: Our systematic survey of somatic mutations in cytokine genes, in major tumour types, identified novel genes targets such as EDN1 gene. EDN1 is a chemokine, also a potent vasoconstrictor. EDN1 signaling modifies tumour microenvironment by regulating contribution of cells around tumor stroma through both autocrine and paracrine mechanisms, by promoting tumour cell proliferation, neovascularization, etc. Other significantly mutated genes are associated with antigen presentation, cell proliferation and chemoattraction. Rational combination therapy with current inhibitors to disrupt these signaling networks in tumor microenvironment, may improve clinical outcomes in patients. Citation: Bhattacharya, A., Roy, V.L., Das, S., Das, C., Mukherjee, T. and Biswas, N. Mutational landscape of cytokine genes across major tumour types identifies new targets [Abstract]. In: Abstracts of the NGBT conference; Oct 02-04, 2017; Bhubaneswar, Odisha, India: Can J biotech, Volume 1, Special Issue, Page 67. https://doi.org/10.24870/cjb.2017-a54