Pawan Upadhyay, Ph.D.

Summary
With the emerging advances made in genomics and functional genomics approaches, there is a critical and growing unmet need to integrate plural datasets in order to identify driver genes in cancer. An integrative approach, with the convergence of multiple types of genetic evidence, can limit false positives through a posterior filtering strategy and reduce the need for multiple hypothesis testing to identify true cancer vulnerabilities. We performed a pooled shRNA screen against 906… Show more

Summary
With the emerging advances made in genomics and functional genomics approaches, there is a critical and growing unmet need to integrate plural datasets in order to identify driver genes in cancer. An integrative approach, with the convergence of multiple types of genetic evidence, can limit false positives through a posterior filtering strategy and reduce the need for multiple hypothesis testing to identify true cancer vulnerabilities. We performed a pooled shRNA screen against 906 human genes in the oral cancer cell line AW13516 in triplicate. The genes that were depleted in the screen were integrated with copy number alteration and gene expression data and ranked based on ROAST analysis, using an integrative scoring system, DepRanker, to compute a Rank Impact Score (RIS) for each gene. The RIS-based ranking of candidate driver genes was used to identify the putative oncogenes AURKB and TK1 as essential for oral cancer cell proliferation. We validated the findings, showing that shRNA mediated genetic knockdown of TK1 or pharmacological inhibition of AURKB by AZD-1152 HQPA in AW13516 cells could significantly impede their proliferation. Next we analysed alterations in AURKB and TK1 genes in head and neck cancer and their association with prognosis using data on 528 patients obtained from TCGA. Patients harbouring alterations in AURKB and TK1 genes were associated with poor survival. To summarise, we present DepRanker as a simple yet robust package with no third-party dependencies for the identification of potential driver genes from a pooled shRNA functional genomic screen by integrating results from RNAi screens with gene expression and copy number data. Using DepRanker, we identify AURKB and TK1 as potential therapeutic targets in oral cancer. DepRanker is in the public domain and available for download at http://www.actrec.gov.in/pi-webpages/AmitDutt/DepRanker/DepRanker.html. Show less

Abstract
TP53 is the most frequently mutated tumor suppressor gene in many cancers, yet biochemical characterization of several of its reported mutations with probable biological significance have not been accomplished enough. Specifically, missense mutations in TP53 can contribute to tumorigenesis through gain-of-function of biochemical and biological properties that stimulate tumor growth. Here, we identified a relatively rare mutation leading to a proline to leucine substitution (P152L)… Show more

Abstract
TP53 is the most frequently mutated tumor suppressor gene in many cancers, yet biochemical characterization of several of its reported mutations with probable biological significance have not been accomplished enough. Specifically, missense mutations in TP53 can contribute to tumorigenesis through gain-of-function of biochemical and biological properties that stimulate tumor growth. Here, we identified a relatively rare mutation leading to a proline to leucine substitution (P152L) in TP53 at the very end of its DNA-binding domain (DBD) in a sample from an Indian oral cancer patient. Although the P152Lp53 DBD alone bound to DNA, the full-length protein completely lacked binding ability at its cognate DNA motifs. Interestingly, P152Lp53 could efficiently tetramerize, and the mutation had only a limited impact on the structure and stability of full-length p53. Significantly, when we expressed this variant in a TP53-null cell line, it induced cell motility, proliferation, and invasion compared with a vector-only control. Also, enhanced tumorigenic potential was observed when P152Lp53-expressing cells were xenografted into nude mice. Investigating the effects of P152Lp53 expression on cellular pathways, we found that it is associated with up-regulation of several pathways, including cell-cell and cell-extracellular matrix signaling, epidermal growth factor receptor signaling, and Rho-GTPase signaling, commonly active in tumorigenesis and metastasis. Taken together, our findings provide a detailed account of the biochemical and cellular alterations associated with the cancer-associated P152Lp53 variant and establish it as a gain-of-function TP53 variant. Show less

Abstract
Objectives
Nodal metastases status among early stage tongue squamous cell cancer patients plays a decisive role in the choice of treatment, wherein about 70% patients can be spared from surgery with an accurate prediction of negative pathological lymph node status. This underscores an unmet need for prognostic biomarkers to stratify the patients who are likely to develop metastases.

Materials and methods
We performed high throughput sequencing of fifty four samples… Show more

Abstract
Objectives
Nodal metastases status among early stage tongue squamous cell cancer patients plays a decisive role in the choice of treatment, wherein about 70% patients can be spared from surgery with an accurate prediction of negative pathological lymph node status. This underscores an unmet need for prognostic biomarkers to stratify the patients who are likely to develop metastases.

Materials and methods
We performed high throughput sequencing of fifty four samples derived from HPV negative early stage tongue cancer patients habitual of chewing betel nuts, areca nuts, lime or tobacco using whole exome (n = 47) and transcriptome (n = 17) sequencing that were analyzed using in-house computational tools. Additionally, gene expression meta-analyses were carried out for 253 tongue cancer samples. The candidate genes were validated using qPCR and immuno-histochemical analysis in an extended set of 50 early primary tongue cancer samples.

Results and conclusion
Somatic analysis revealed a classical tobacco mutational signature C:G > A:T transversion in 53% patients that were mutated in TP53, NOTCH1, CDKN2A, HRAS, USP6, PIK3CA, CASP8, FAT1, APC, and JAK1. Similarly, significant gains at genomic locus 11q13.3 (CCND1, FGF19, ORAOV1, FADD), 5p15.33 (SHANK2, MMP16, TERT), and 8q24.3 (BOP1); and, losses at 5q22.2 (APC), 6q25.3 (GTF2H2) and 5q13.2 (SMN1) were observed in these samples. Furthermore, an integrated gene-expression analysis of 253 tongue tumors suggested an upregulation of metastases-related pathways and over-expression of MMP10 in 48% tumors that may be crucial to predict nodal metastases in early tongue cancer patients. In overall, we present the first descriptive portrait of somatic alterations underlying the genome of tobacco/nut chewing HPV-negative early tongue cancer, and identify MMP10 as a potential prognostic biomarker to stratify those likely to develop metastases. Show less

Bystander cells of the tumor microenvironment show evidence of DNA damage and inflammation that can lead to their oncogenic transformation. Mediator(s) of cell–cell communication that brings about these pro-oncogenic pathologies has not been identified. We show here that cell-free chromatin (cfCh) released from dying cancer cells are the key mediators that trigger both DNA damage and inflammation in the surrounding healthy cells. When dying human cancer cells were cultured along with NIH3T3… Show more

Bystander cells of the tumor microenvironment show evidence of DNA damage and inflammation that can lead to their oncogenic transformation. Mediator(s) of cell–cell communication that brings about these pro-oncogenic pathologies has not been identified. We show here that cell-free chromatin (cfCh) released from dying cancer cells are the key mediators that trigger both DNA damage and inflammation in the surrounding healthy cells. When dying human cancer cells were cultured along with NIH3T3 mouse fibroblast cells, numerous cfCh emerged from them and rapidly entered into nuclei of bystander NIH3T3 cells to integrate into their genomes. This led to activation of H2AX and inflammatory cytokines NFκB, IL-6, TNFα and IFNγ. Genomic integration of cfCh triggered global deregulation of transcription and upregulation of pathways related to phagocytosis, DNA damage and inflammation. None of these activities were observed when living cancer cells were co-cultivated with NIH3T3 cells. However, upon intravenous injection into mice, both dead and live cells were found to be active. Living cancer cells are known to undergo extensive cell death when injected intravenously, and we observed that cfCh emerging from both types of cells integrated into genomes of cells of distant organs and induced DNA damage and inflammation. γH2AX and NFκB were frequently co-expressed in the same cells suggesting that DNA damage and inflammation are closely linked pathologies. As concurrent DNA damage and inflammation is a potent stimulus for oncogenic transformation, our results suggest that cfCh from dying cancer cells can transform cells of the microenvironment both locally and in distant organs providing a novel mechanism of tumor invasion and metastasis. The afore-described pro-oncogenic pathologies could be abrogated by concurrent treatment with chromatin neutralizing/degrading agents suggesting therapeutic possibilities. Show less

Background
Lung cancer is the leading cause of cancer-related deaths across the world. In this study, we present therapeutically relevant genetic alterations in lung adenocarcinoma of Indian origin.

Materials and methods
Forty-five primary lung adenocarcinoma tumors were sequenced for 676 amplicons using RainDance cancer panel at an average coverage of 1500 × (reads per million mapped reads). To validate the findings, 49 mutations across 23 genes were genotyped in an additional set… Show more

Background
Lung cancer is the leading cause of cancer-related deaths across the world. In this study, we present therapeutically relevant genetic alterations in lung adenocarcinoma of Indian origin.

Materials and methods
Forty-five primary lung adenocarcinoma tumors were sequenced for 676 amplicons using RainDance cancer panel at an average coverage of 1500 × (reads per million mapped reads). To validate the findings, 49 mutations across 23 genes were genotyped in an additional set of 363 primary lung adenocarcinoma tumors using mass spectrometry. NIH/3T3 cells over expressing mutant and wild-type FGFR3 constructs were characterized for anchorage independent growth, constitutive activation, tumor formation and sensitivity to FGFR inhibitors using in vitro and xenograft mouse models.

Results
We present the first spectrum of actionable alterations in lung adenocarcinoma tumors of Indian origin, and shows that mutations of FGFR3 are present in 20 of 363 (5.5%) patients. These FGFR3 mutations are constitutively active and oncogenic when ectopically expressed in NIH/3T3 cells and using a xenograft model in NOD/SCID mice. Inhibition of FGFR3 kinase activity inhibits transformation of NIH/3T3 overexpressing FGFR3 constructs and growth of tumors driven by FGFR3 in the xenograft models. The reduction in tumor size in the mouse is paralleled by a reduction in the amounts of phospho-ERK, validating the in vitro findings. Interestingly, the FGFR3 mutations are significantly higher in a proportion of younger patients and show a trend toward better overall survival, compared with patients lacking actionable alterations or those harboring KRAS mutations.

Conclusion
We present the first actionable mutation spectrum in Indian lung cancer genome. These findings implicate FGFR3 as a novel therapeutic in lung adenocarcinoma. Show less

Recurrent mutations in the myogenic transcription factor MYOD1 and PIK3CA were initially described in a subset of embryonal rhabdomyosarcomas. We analyzed a much larger cohort of 49 primary rhabdomyosarcoma tumor samples of various subtypes, collected over a period of 9 years, for the presence of MYOD1 (L122R), PIK3CA (H1047), and PIK3CA (E542/E545) mutations, along with immunohistochemical analysis of desmin, myogenin, and MYOD1. Although activating PIK3CA mutations were absent across the… Show more

Recurrent mutations in the myogenic transcription factor MYOD1 and PIK3CA were initially described in a subset of embryonal rhabdomyosarcomas. We analyzed a much larger cohort of 49 primary rhabdomyosarcoma tumor samples of various subtypes, collected over a period of 9 years, for the presence of MYOD1 (L122R), PIK3CA (H1047), and PIK3CA (E542/E545) mutations, along with immunohistochemical analysis of desmin, myogenin, and MYOD1. Although activating PIK3CA mutations were absent across the sample set analyzed, we report 20% MYOD1 (L122R) mutation in rhabdomyosarcomas, found exclusively in 10 of 21 spindle cell and sclerosing rhabdomyosarcomas, occurring mostly in the head and neck region along with extremity sites (64%), than the paratesticular and intra-abdominal sites. Furthermore, while all 10 MYOD1 mutant spindle cell and sclerosing rhabdomyosarcoma samples showed diffuse and strong MYOD1 immunoexpression, 7 of 31 samples of rhabdomyosarcoma with wild-type MYOD1 were negative for MYOD1 expression. Clinically, a striking correlation was found between MYOD1 mutation and the clinical outcomes available for 15 of 21 cases: 5 of 7 patients with spindle cell and sclerosing rhabdomyosarcomas, harboring MYOD1 mutation, were alive-with-disease and 2 of 8 patients with spindle cell and sclerosing rhabdomyosarcomas, with mutant MYOD1, were free-of-disease. Taken together, we present the first report of MYOD1 (L122R) mutation in the largest cohort of 49 rhabdomyosarcomas reported so far, that are associated with a relatively aggressive clinical course. Moreover, consistent with the earlier two studies, this study further reinforces a relationship between spindle cell and the sclerosing rhabdomyosarcoma—now recognized as a single subtype, distinct from an embryonal rhabdomyosarcoma. Show less

Background
Notch pathway plays a complex role depending on cellular contexts: promotes stem cell maintenance or induces terminal differentiation in potential cancer-initiating cells; acts as an oncogene in lymphocytes and mammary tissue or plays a growth-suppressive role in leukemia, liver, skin, and head and neck cancer. Here, we present a novel clinical and functional significance of NOTCH1 alterations in early stage tongue squamous cell carcinoma (TSCC).

Patients and Methods
We… Show more

Background
Notch pathway plays a complex role depending on cellular contexts: promotes stem cell maintenance or induces terminal differentiation in potential cancer-initiating cells; acts as an oncogene in lymphocytes and mammary tissue or plays a growth-suppressive role in leukemia, liver, skin, and head and neck cancer. Here, we present a novel clinical and functional significance of NOTCH1 alterations in early stage tongue squamous cell carcinoma (TSCC).

Patients and Methods
We analyzed the Notch signaling pathway in 68 early stage TSCC primary tumor samples by whole exome and transcriptome sequencing, real-time PCR based copy number, expression, immuno-histochemical, followed by cell based biochemical and functional assays.

Results
We show, 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. HNSCC cell lines over expressing NOTCH1, when plated in the absence of attachment, are enriched in stem cell markers and form spheroids. Furthermore, we show that inhibition of NOTCH activation by gamma secretase inhibitor or shRNA mediated knockdown of NOTCH1 inhibits spheroid forming capacity, transformation, survival and migration of the HNSCC cells suggesting an oncogenic role of NOTCH1 in TSCC. Clinically, Notch pathway activation is higher in tumors of non-smokers compared to smokers (50% Vs 18%, respectively, P=0.026) and is also associated with greater nodal positivity compared to its non-activation (93% Vs 64%, respectively, P=0.029).

Conclusion
We anticipate that these results could form the basis for therapeutic targeting of NOTCH1 in tongue cancer. Show less

Abstract
Notch pathway play a context-dependent tumor-suppressive or pro-oncogenic role in solid tumors and hematological malignancies. In head and neck cancer, inactivating mutations in NOTCH1 are associated with poor prognosis. Here we perform meta-analysis of DNA copy number and gene expression profiling of NOTCH signaling pathway genes in 35 paired primary T1 and T2 stage tongue tumors, comprising of Notch ligands, receptors and target genes. We find 32% of primary tongue squamous cell… Show more

Abstract
Notch pathway play a context-dependent tumor-suppressive or pro-oncogenic role in solid tumors and hematological malignancies. In head and neck cancer, inactivating mutations in NOTCH1 are associated with poor prognosis. Here we perform meta-analysis of DNA copy number and gene expression profiling of NOTCH signaling pathway genes in 35 paired primary T1 and T2 stage tongue tumors, comprising of Notch ligands, receptors and target genes. We find 32% of primary tongue squamous cell carcinoma (TSCC) tumors amplify NOTCH signaling pathway components; and over express activated NOTCH1based on immuno-histochemical staining in an additional series of 50 surgically resected paired TSCC tumors. Interestingly, these alterations: amplification at Notch ligand DLL3 (χ2 = 7.575, P = 0.023); over expression of Notch pathway target gene HEY2 transcript (χ2 = 9.885, P = 0.007); and expression of activated NOTCH1 (χ2 = 7.575, P = 0.023) positively correlate with N+ve and non- smoking status of patients. In a parallel effort, we characterized four HNSCC cell lines– NT8E, OT9, AW13516, and AW8507– established from Indian patients using whole exome, whole transcriptome sequencing and SNP array assays. Significant amplification and over expression of NOTCH signaling pathway components were observed in AW13516 and NT8E cells. Inhibition of NOTCH activation by gamma secretase inhibitor or shRNA mediated knockdown of NOTCH1 could inhibit transformation, survival, migration and invasion of NT8E cells. Taken together, we present that activation of Notch signaling pathway in a subset of TSCC patients is associated with nodal positivity and non-smoking status among early tongue cancer patients. Show less

Oral cancer is the leading malignancy in Indian men. About 70% of patients present in clinical T1 and 30% in T4 stage that have clinically lymph node negative (N0) neck examination at the time of presentation. However, the current standard of care is to perform potentially mutilating and morbid neck dissection in all patients irrespective of their nodal status, because of a 27% chance of having pathologically involved lymph nodes. Therefore, its an imperative to find accurate predictors of… Show more

Oral cancer is the leading malignancy in Indian men. About 70% of patients present in clinical T1 and 30% in T4 stage that have clinically lymph node negative (N0) neck examination at the time of presentation. However, the current standard of care is to perform potentially mutilating and morbid neck dissection in all patients irrespective of their nodal status, because of a 27% chance of having pathologically involved lymph nodes. Therefore, its an imperative to find accurate predictors of pathological lymph node status, whereby a considerable fraction can be spared unnecessary surgery.
We report an integrated analysis of whole transcriptome and miRNA expression profiling of primary tongue squamous cell carcinomas (TSCC) to identify clinically relevant molecular biomarkers to predict lymph node metastases. We also present functional validation of the candidate genes using biochemical analysis and cell-based assay.
Results: We investigated simultaneously the transcriptional changes of miRNA and mRNA expression levels of eleven N0 and N+ TSCC, and six paired control samples. To examine global miRNA and mRNA expression patterns, we perfomed an integrative analysis of whole transcriptome sequencing and miRNA expression profile of primary tumors. Integarting analysis find a negatively correlated network of 30 miRNA and 60 mRNA. We present here robust molecular validation of a novel and dynamic interplay of two miRNAs mir-944, mir-183-5p and a matrix metalloproteinase MMP10 expression, along with clinical correlation in an independent set of 40 primary N0 and N+ TSCC tumors to collectively predict nodal metastases in TSCC. Immunohistochemical validation of the candidate biomarker MPP10 among primary TSCC tumors to establish its utility in clinical settings will be discussed.
Knowledge from integrated expression analysis along with functional validation a clinically relevant biomarker to prognosticate metastases in patients with early stage oral tongue cancers. Show less

Background:
Human papilloma virus (HPV) accounts for the most common cause of all virus-associated human cancers. Here, we describe the first graphic user interface (GUI)-based automated tool ‘HPVDetector’, for non-computational biologists, exclusively for detection and annotation of the HPV genome based on next-generation sequencing data sets.

Methods:
We developed a custom-made reference genome that comprises of human chromosomes along with annotated genome of 143 HPV types as… Show more

Background:
Human papilloma virus (HPV) accounts for the most common cause of all virus-associated human cancers. Here, we describe the first graphic user interface (GUI)-based automated tool ‘HPVDetector’, for non-computational biologists, exclusively for detection and annotation of the HPV genome based on next-generation sequencing data sets.

Methods:
We developed a custom-made reference genome that comprises of human chromosomes along with annotated genome of 143 HPV types as pseudochromosomes. The tool runs on a dual mode as defined by the user: a ‘quick mode’ to identify presence of HPV types and an ‘integration mode’ to determine genomic location for the site of integration. The input data can be a paired-end whole-exome, whole-genome or whole-transcriptome data set. The HPVDetector is available in public domain for download: http://www.actrec.gov.in/pi-webpages/AmitDutt/HPVdetector/HPVDetector.html.

Results:
On the basis of our evaluation of 116 whole-exome, 23 whole-transcriptome and 2 whole-genome data, we were able to identify presence of HPV in 20 exomes and 4 transcriptomes of cervical and head and neck cancer tumour samples. Using the inbuilt annotation module of HPVDetector, we found predominant integration of viral gene E7, a known oncogene, at known 17q21, 3q27, 7q35, Xq28 and novel sites of integration in the human genome. Furthermore, co-infection with high-risk HPVs such as 16 and 31 were found to be mutually exclusive compared with low-risk HPV71.

Conclusions:
HPVDetector is a simple yet precise and robust tool for detecting HPV from tumour samples using variety of next-generation sequencing platforms including whole genome, whole exome and transcriptome. Two different modes (quick detection and integration mode) along with a GUI widen the usability of HPVDetector for biologists and clinicians with minimal computational knowledge. Show less

Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored. We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei… Show more

Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored. We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer. Show less

Abstract
Background
Several statistical tools have been developed to identify genes mutated at rates significantly higher than background, indicative of positive selection, involving large sample cohort studies. However, studies involving smaller sample sizes are inherently restrictive due to their limited statistical power to identify low frequency genetic variations.

Results
We performed an integrated characterization of copy number, mutation and expression analyses of four… Show more

Abstract
Background
Several statistical tools have been developed to identify genes mutated at rates significantly higher than background, indicative of positive selection, involving large sample cohort studies. However, studies involving smaller sample sizes are inherently restrictive due to their limited statistical power to identify low frequency genetic variations.

Results
We performed an integrated characterization of copy number, mutation and expression analyses of four head and neck cancer cell lines - NT8e, OT9, AW13516 and AW8507-- by applying a filtering strategy to prioritize for genes affected by two or more alterations within or across the cell lines. Besides identifying TP53, PTEN, HRAS and MET as major altered HNSCC hallmark genes, this analysis uncovered 34 novel candidate genes altered. Of these, we find a heterozygous truncating mutation in Nuclear receptor binding protein, NRBP1 pseudokinase gene, identical to as reported in other cancers, is oncogenic when ectopically expressed in NIH-3 T3 cells. Knockdown of NRBP1 in an oral carcinoma cell line bearing NRBP1 mutation inhibit transformation and survival of the cells.

Conclusions
In overall, we present the first comprehensive genomic characterization of four head and neck cancer cell lines established from Indian patients. We also demonstrate the ability of integrated analysis to uncover biologically important genetic variation in studies involving fewer or rare clinical specimens. Show less

The completion of the Human Genome Project established a baseline for human genome reference
sequence allowing characterization of various alterations underlying several human diseases, including bcancer and has brought the field of genomics to this unprecedented moment of a great scientific ferment. It has also sparked a concomitant revolution in sequencing technologies that have become a fundamental tool for genome analysis with potential to transform medical practice. As an emerging… Show more

The completion of the Human Genome Project established a baseline for human genome reference
sequence allowing characterization of various alterations underlying several human diseases, including bcancer and has brought the field of genomics to this unprecedented moment of a great scientific ferment. It has also sparked a concomitant revolution in sequencing technologies that have become a fundamental tool for genome analysis with potential to transform medical practice. As an emerging field, the next-generation sequencing (NGS) technology has stimulated rapid cataloguing of all alterations in cancer genomes and has enabled researchers to look at large-scale genome events such as chromosomal lesions and copy-number variations as well as small-scale aberrations represented by point mutations, small insertions and deletions. Several advancements with smaller and faster versions of available technologies have recently been introduced enabling more democratic usage of the technology. Here, we review the application of NGS technology in understanding the underlying goal to catalogue human cancer-causing somatic mutations. Show less

Abstract
Background
During the past decade, the incidence of EGFR mutation has been shown to vary across different ethnicities. It occurs at the rate of 10–15% in North Americans and Europeans, 19% in African-Americans, 20–30% in various East Asian series including Chinese, Koreans, and Japanese. Frequency of EGFR mutations in India however remains sparsely explored.

Methodology/Principal Findings
We report 23% incidence of Epidermal growth factor receptor (EGFR) mutations in… Show more

Abstract
Background
During the past decade, the incidence of EGFR mutation has been shown to vary across different ethnicities. It occurs at the rate of 10–15% in North Americans and Europeans, 19% in African-Americans, 20–30% in various East Asian series including Chinese, Koreans, and Japanese. Frequency of EGFR mutations in India however remains sparsely explored.

Methodology/Principal Findings
We report 23% incidence of Epidermal growth factor receptor (EGFR) mutations in 907 Non small cell lung cancer (NSCLC) patients of Indian ethnicity, in contrast to 10–15% known in Caucasians and 27–62% among East Asians. In this study, EGFR mutations were found to be more common in never-smokers 29.4% as compared to smokers 15.3%. Consistent with other populations, mutation rates among adenocarcinoma-males were predominantly lower than females with 32% incidence. However unlike Caucasians, EGFR mutation rate among adenocarcinoma-never-smoker females were comparable to males suggesting lack of gender bias among never smokers likely to benefit from EGFR targeted therapy.

Conclusions/Significance
This study has an overall implication for establishing relevance for routine EGFR mutation diagnostics for NSCLC patients in clinics and emphasizes effectiveness for adoption of EGFR inhibitors as the first line treatment among Indian population. The intermediate frequency of EGFR mutation among Indian population compared to Caucasians and East Asians is reminiscent of an ancestral admixture of genetic influence from Middle Easterners, Central Asians, and Europeans on modern- Indian population that may confer differential susceptibility to somatic mutations in EGFR. Show less