Integrated analysis of Head and Neck squamous cell carcinoma: A genomic approach
Researcher:
Dr. Pawan Kumar Upadhyay
Funding:
DBT-Wellcome Trust India Alliance
Collaborators:
Dr. Sudhir Nair, Tata Memorial Hospital, Mumbai

Tongue cancer is the most predominant form of oral cancer in developed countries with varying incidence in developing countries wherein etiological factors such as chewing betel-quid comprising betel leaf, areca nut and slaked lime is a part of the tradition. While several large-scale genome-sequencing efforts of advanced stage primary oral tumors have been described, systematic efforts to catalogue somatic alterations in tobacco/ nut chewing associated early stage (pT1/2) HPV-negative tongue tumors has been lacking.

In a comprehensive effort to characterize tongue tumors derived from Indian patients:

  1. First, we performed functional analysis of biological relevant alterations of 3 primary tumor-derived oral cancer cell lines (AW13516, AW8507 and NT8E) established from Indian head and neck cancer patients (by other groups at the Centre) using integrated genomic approach. The findings establish mutant nuclear receptor binding protein NRBP1 as a novel oncogene in oral cancer [2].
  2. Second, using in house developed HPVDetector [1], we established that unlike Caucasian population, tongue tumors of Indian origin display significantly low HPV infectivity (n=46), suggesting an alternative mode underlying the disease;
  3. Thirdly, we characterized the somatic alterations underlying the tongue cancer genome of 57 tobacco/nut chewing HPV-negative early stage tongue tumors by whole exome (n=47) and transcriptome (n=17) sequencing [5]. Post depleting variants against in-house developed TMC-SNPdb, the study revealed a classical tobacco mutational signature C:G>A:T transversion in 53% patients [3]. Of significance, unlike TCGA HNSCC data set, NOTCH1 harbors significantly lower frequency of inactivating mutations (4%), is somatically amplified and overexpressed in 31% and 37% of early stage TSCC patients, respectively;
  4. Fourth, to functionally validate the relevance of Notch pathway, oral-cancer cell lines with higher basal NOTCH1 expression are enriched in stem cell markers and form spheroids. Inhibition of NOTCH activation by gamma secretase inhibitor or shRNA mediated knockdown of NOTCH1 could inhibit spheroid forming capacity, transformation, survival and migration of the HNSCC cells suggesting an oncogenic role of NOTCH1 in TSCC, thus suggesting NOTCH1 as a therapeutic target [4]; and,
  5. Finally, an integrated gene-expression analysis of 253 tongue tumor identify upregulation of metastases related pathways and over-expression of MMP10 in 48% tumors along with concomitant downregulation of a miR-944 that is associated with poor prognosis [5].


    Figure: Identification of somatic mutations and DNA copy number changes in HPV-negative early stage TSCC tumors.
    (a) Mutational features of 25 early tongue squamous carcinoma samples (b) Somatic DNA copy number changes identified using Exome sequencing data. Somatic DNA copy number gains and losses were generated using Segments-of-Gain-Or-Loss (SGOL) scores across 23 TSCC patients. Representative amplified and deleted regions are annotated for HNSCC-associated genes and denoted by an arrow. (Oral Oncology. 2017).

Identification and characterization of potential biomarkers to predict metastasis of early stage oral cancer
Researcher:
Bhasker Dharavath, Ph.D-SRF
Funding:
DBT-Wellcome Trust India Alliance
Collaborators:
Dr. Sudhir Nair Tata Memorial Hospital, Mumbai
Dr. Anil D’Cruz Tata Memorial Hospital, Mumbai
Dr. Kumar Prabhash, Tata Memorial Hospital, Mumbai

Nodal metastases status among early stage tongue cancer patients (pT1-pT2) plays a decisive role for choice of treatment, wherein about 70% patients may be spared from surgery with accurate prediction of negative pathological lymph node status. Thus, there is an unmet need for identification of prognostic biomarkers to stratify the early stage tongue cancer patients who are likely to develop metastases. To identify genes and miRNAs implicated in early stage metastasis of tongue cancer, we performed whole transcriptome sequencing and microRNA profiling of early stage tongue tumor samples and metastatic lymph nodes. Further, the candidate genes and miRNAs are being validated in extended cohort of oral cancer patient samples, followed by functional characterization of target genes using in vitro and in vivo studies. Preliminary studies suggest that a matrix metalloproteinase gene, MMP10, and a set of 8 miRNAs could play an important role in invasion and metastasis of oral cancer and could act as potential candidate biomarkers to predict nodal metastasis in early tongue cancer patients.



Figure: Differential expression profile of tongue squamous cell carcinoma using mRNA sequencing and meta-analysis identifies MMP10 up regulation.
Differential expression analysis to identify the distinct gene expression profile of tongue tumors. (a) Volcano plot representation of differentially expressed in between early tongue tumors and adjacent normal tongue tissues. The red and green dots denote the up-regulated and down-regulated differentially expressed genes with P value < 0.05 and fold changes ≥ 2 or ≤ -2 for, respectively. (b) The tabular representation of a number of genes overlapped in tongue cancer across different studies. (c) Schematic representation of commonly up-regulated genes qRT-PCR validation in a cohort of 35 paired tongue tumor samples. The Red denotes up-regulation, blue as downregulation, black as basal expression and gray color; experiment could not be done or results could not be acquired. The ≥2 mean fold change is for up-regulation, ≤0.5 mean fold change for down-regulation and in between 1.99-0.501 mean fold change as a basal level expression compared to the adjacent normal tissue sample. (d) qRT-PCR analysis of MMP10 transcript expression in early tongue tumors. Dot plot representation of MMP10 transcript ΔCt value distribution and its significance between normal and tumors tongue tissue sample (n=35). Each dot represents the average normalized ΔCt value of MMP10 in a single sample. Median with interquartile range is shown for MMP10 for normal and tumor samples. (e) Dot plot representation of immunohistochemical score of MMP10 expression in tongue tumors and adjacent normal tissues (n=50). Each dot represents that final IHC score for each sample and median with interquartile range is shown. Median with interquartile range is shown for MMP10 protein expression in normal and tumor samples. (f) Representative IHC stained photomicrographs tongue tumors and paired normal samples are shown. The brown color indicates positive expression of MMP10 protein. The P-value was calculated by Mann- Whitney U test using GraphPad Prism 5 program and p value ≤0.05 was considered as a threshold for statistical significance. P value is denoted as ***; P < 0.0001[5].

Publications:

  1. Chandrani P, Kulkarni V, Iyer P, Upadhyay P, Chaubal R, Das P, Mulherkar R, Singh R, Dutt A. NGS-based approach to determine the presence of HPV and their sites of integration in human cancer genome. Br J Cancer PMID:  25973533
  2. Chandrani P, Upadhyay P, Iyer P, Tanna M, Shetty M, Raghuram GV, Oak N, Singh A, Chaubal R, Ramteke M, Gupta S, Dutt A. Integrated genomics approach to identify biologically relevant alterations in fewer samples. BMC Genomics. PMID: 26572163
  3. Upadhyay P, Gardi N, Desai S, Sahoo B, Singh A, Togar T, Iyer P, Prasad R, Chandrani P, Gupta S, Dutt A.TMC-SNPdb: an Indian germline variant database derived from whole exome sequences.Database (Oxford). . PMID: 27402678
  4. Upadhyay P, Nair S, Kaur E, Aich J, Dani P, Sethunath V, Gardi N, Chandrani P, Godbole M, Sonawane K, Prasad R, Kannan S, Agarwal B, Kane S, Gupta S, Dutt S, Dutt A. Notch pathway activation is essential for maintenance of stem-like cells in early tongue cancer. Oncotarget. PMID:  27391340
  5. Upadhyay P, Gardi N, Desai S, Chandrani P, Joshi A, Dharavath B, Arora P, Bal M, Nair S, Dutt A. Genomic characterization of tobacco/nut chewing HPV-negative early stage tongue tumors identify MMP10 as a candidate to predict metastases. Oral Oncology. PMID: 2893907