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Background: There can be a diagnostic challenge in differentiating giant cell tumor of bone (GCTB) from its mimics. Lately, histone H3F3A (Histone 3.3) G34W has been identified as a promising immunohistochemical marker. Aims: This study was aimed at evaluating H3.3 G34W immunostaining in 100 GCTBs, including its value in resolving diagnostic dilemmas. Materials and Methods: Immunohistochemical staining for H3.3 G34W was graded in terms of staining intensity (1+ to 3+) and the percentage of tumor cells showing crisp nuclear staining. Results: One hundred GCTBs occurred in 58 males and 42 females (M: F ratio = 1.3), of 7-66 years age (average = 31.3, median = 28), commonly in distal femur (26), followed by proximal tibia (17), distal radius (12), proximal humerus (7), metacarpals (7), sacrum (6), proximal fibula (6), and relatively unusual sites (19), including a single multicentric case. Out of 92 GCTBs, wherein H3.3 G34W immunostaining worked, 81 (88.1%) showed positive staining in the mononuclear cells, including tumors with fibrous histiocytoma-like areas, sparing osteoclast-like giant cells, with 3+ staining intensity in 65/81 (80%) tumors. All 7/7 (100%) malignant GCTBs showed positive staining, including the pleomorphic/sarcomatous cells. All 7/7 (100%) metastatic GCTBs showed positive immunostaining. Seven out of 10 post-denosumab treated GCTBs showed positive H3.3 G34W immunostaining in the residual mononuclear cells. None of the other 37 “giant cell-rich” lesions displayed H3.3 G34W immunostaining. Four of 9 GCTBs tested for H3.3 G34W mutation showed positive results. Conclusions: The diagnostic sensitivity and specificity of H3.3 G34W for GCTB were 88.1% and 100%, respectively. This constitutes one of the first reports from our country, further validating the diagnostic value of H3.3 G34W in differentiating GCTB, including metastatic and malignant forms from its mimics, including small biopsy samples. Its value in various diagnostic dilemmas is presented and utility in identifying residual tumor cells in post-denosumab treated GCTBs is worth exploring.

Abstract Head and neck cancer is a major cause of morbidity and mortality worldwide. The identification of genetic alterations in head and neck cancer may improve diagnosis and treatment outcomes. In this study, we report the identification and functional characterization of UBE3C-LRP5 translocation in head and neck cancer. Our whole transcriptome sequencing and RT-PCR analysis of 151 head and neck cancer tumor samples identified the LRP5-UBE3C and UBE3C-LRP5 fusion transcripts in 5.3% of patients of Indian origin (n = 151), and UBE3C-LRP5 fusion transcripts in 1.2% of TCGA-HNSC patients (n = 502). Further, whole genome sequencing identified the breakpoint of UBE3C-LRP5 translocation. We demonstrate that UBE3C-LRP5 fusion is activating in vitro and in vivo, and promotes the proliferation, migration, and invasion of head and neck cancer cells. In contrast, depletion of UBE3C-LRP5 fusion suppresses the clonogenic, migratory, and invasive potential of the cells. The UBE3C-LRP5 fusion activates the Wnt/β-catenin signaling by promoting nuclear accumulation of β-catenin, leading to upregulation of Wnt/β-catenin target genes, MYC, CCND1, TCF4, and LEF1. Consistently, treatment with the FDA-approved drug, pyrvinium pamoate, significantly reduced the transforming ability of cells expressing the fusion protein and improved survival in mice bearing tumors of fusion-overexpressing cells. Interestingly, fusion-expressing cells upon knockdown of CTNNB1, or LEF1 show reduced proliferation, clonogenic abilities, and reduced sensitivity to pyrvinium pamoate. Overall, our study suggests that the UBE3C-LRP5 fusion is a promising therapeutic target for head and neck cancer and that pyrvinium pamoate may be a potential drug candidate for treating head and neck cancer harboring this translocation.

Summary: Lung cancer varies between Caucasians and Asians. There have been differences recorded in the epidemiology, genomics, standard therapies and outcomes, with variations according to the geography and ethnicity which affect the decision for optimal treatment of the patients. To better understand the profile of lung cancer in Southeast Asia, with a focus on India, we have comprehensively reviewed the available data, and discuss the challenges and the way forward. A substantial proportion of patients with lung cancer in Southeast Asia are neversmokers, and adenocarcinoma is the common histopathologic subtype, found in approximately a third of the patients. EGFR mutations are noted in 23–30% of patients, and ALK rearrangements are noted in 5–7%. Therapies are similar to global standards, although access to newer modalities and molecules is a challenge. Collaborative research, political will with various policy changes and patient advocacy are urgently needed.

Abstract Introduction The emergence of resistance to the highly successful BCL2-directed therapy is a major unmet need in acute myeloid leukemia (AML), an aggressive malignancy with poor survival rates. Towards identifying therapeutic options for AML patients who progress on BCL2-directed therapy, we studied a clinical-stage CDK7 inhibitor XL102, which is being evaluated in solid tumors (NCT04726332). Materials and methods To determine the anti-proliferative effects of XL102, we performed experiments including time-resolved fluorescence resonance energy transfer, target occupancy, cell cycle and apoptosis-based assays. We also included genetically characterized primary myeloid blasts from de novo and relapsed/refractory AML patients. For mechanistic studies, CRISPR/Cas9 mediated knockout of CDK7 and c-Myc and immunoblotting were performed. NOD/SCID orthotropic and subcutaneous AML xenografts were used to determine anti-leukemic effects. To assess the synergistic effects of XL102 with Venetoclax, we performed RNA sequencing and gene set enrichment analysis using Venetoclax sensitive and resistant model systems. Results XL102, a highly specific, orally bioavailable covalent inhibitor of CDK7. Inhibitory effect on CDK7 by XL102 in primary myeloid blasts (n = 54) was in nanomolar range (mean = 300 nM; range = 4.0-952 nM). XL102 treated AML cells showed a reduction in phosphorylation levels of Serine 2/5/7 at carboxy-terminal domain of RNA polymerase II. T-loop phosphorylation of CDK1(Thr161) and CDK2(Thr160) was inhibited by XL102 in dose-dependent manner leading to cell-cycle arrest. c-Myc downregulation and enhanced levels of p53 and p21 in XL102 treated cells were observed. Increased levels of p21 and activation of p53 by XL102 were mimicked by genetic ablation of CDK7, which supports that the observed effects of XL102 are due to CDK7 inhibition. XL102 treated AML xenografts showed remarkable reduction in hCD45 + marrow cells (mean = 0.60%; range = 0.04%-3.53%) compared to vehicle control (mean = 38.2%; range = 10.1%-78%), with corresponding increase in p53, p21 and decrease in c-Myc levels. The data suggests XL102 induces apoptosis in AML cells via CDK7/c-Myc/p53 axis. RNA-sequencing from paired Venetoclax-sensitive and Venetoclax-resistant cells treated with XL102 showed downregulation of genes involved in proliferation and apoptosis. Conclusion Taken together, XL102 with Venetoclax led to synergistic effects in overcoming resistance and provided a strong rationale for clinical evaluation of XL102 as a single agent and in combination with Venetoclax.

Inflammatory leiomyosarcoma (LMS) is a newly included rare tumor entity in the group of smooth muscle tumors in the recent WHO classification. Recent studies have shown skeletal muscle expression within this tumor and its proximity with histiocyte-rich rhabdomyoblastic tumor (HRRT). A 17-year-old male presented with a soft tissue lump over the back of his neck of one-year duration. Radiologically, a lesion measuring 5.9 cm in the largest dimension was seen, extending from the skull base up to the C2 vertebral level, abutting the occipital bone. The initial biopsy was reported as a fibrohistiocytic tumor at the referring laboratory. A microscopic review of the sections from the initial biopsy and subsequent resection revealed a well-circumscribed, cellular tumor composed of plump spindle and polygonal-shaped tumor cells with relatively bland nuclei, moderate to abundant eosinophilic cytoplasm and numerous interspersed histiocytes, including foam cells and lymphocytes. Immunohistochemically, the tumor cells were positive for desmin, MYOD1 and SMA, focally positive for myogenin, while negative for h-caldesmon, SOX10 and S100P. A diagnosis of inflammatory leiomyosarcoma/HRRT was offered. Subsequently, the tumor was tested for MYOD1 (L122R) mutation and was found to be negative. The patient underwent adjuvant radiation therapy and is free-of-disease at 12 months post-treatment. This case constitutes an extremely rare case of an inflammatory LMS/HRRT, identified in the neck region. This tumor should be differentiated from its close mimics, such as a spindle cell/sclerosing rhabdomyosarcoma, as the latter is treated more aggressively, including with chemotherapy, given its relatively poor prognosis.

The significance of EGFR targeted therapy in the lung adenocarcinoma is paramount. Several controlled clinical trials have reported considerable survival of EGFR mutation positive patients on receiving the EGFR tyrosine kinase inhibitor (TKI). However, the real-world evidence of benefits of EGFR TKI would be further useful to understand how the designated therapeutic regimen benefits the patients. In this study, we report a decade long real-world evidence of EGFR molecular testing in lung cancer at Tata Memorial Hospital (Mumbai, India). Laboratory and hospital records containing basic demographic details, clinical characteristics, treatment regimen, survival outcome were collected retrospectively. Statistical association and survival analysis were performed using the R programming. The cohort includes 9,053 lung cancer patients tested for EGFR mutations during 2011 to 2019. Baseline T790M and compound mutations were the only mutations observed co-occurring while all other EGFR mutations were mutually exclusive. Furthermore, the baseline T790M were also observed to be associated with TTF1 positivity, smoking and local metastasis. Overall survival of the patients harboring co-occurring compound mutations was significantly lesser than the other EGFR positive patients. Overall, our study suggests that EGFR TKI may provide real-world benefit to the lung cancer patients harboring mutually exclusive EGFR mutations. On the other hand, further systematic study is essential to develop better therapeutic regimen for co-occurring baseline EGFR T790M and other compound mutations.

IntroductionThe diverse subtypes of thyroid carcinoma have distinct clinical outcomes despite a comparable spectrum of underlying genetic alterations. Beyond genetic alterations, sparse efforts have been made to characterize the microbes associated with thyroid cancer. In this study, we examine the microbial profile of thyroid cancer.MethodsWe sequenced the whole transcriptome of 70 thyroid cancers (40 papillary and 30 anaplastic). Using Infectious Pathogen Detector IPD 2.0, we analysed the relative abundance of 1060 microbes across 70 tumours from patients with thyroid cancer against 118 tumour samples from patients with breast, cervical, colorectal, and tongue cancer.ResultsOur analysis reveals a significant prevalence of Cutibacterium acnes in 58.6% thyroid cancer samples compared to other cancer types (p=0.00038). Immune cell fraction analysis between thyroid cancer samples with high and low Cutibacterium loads identify enrichment of immunosuppressive cells, including Tregs (p=0.015), and other anti-inflammatory cytokines in the tumour microenvironment, suggesting an immune evasion/immunosuppression milieu is associated with the infection. A higher burden of Cutibacterium acnes was also found to be associated with poor survival defining a distinct sub-group of thyroid cancer.ConclusionCutibacterium acnes is associated with immune suppression and poor prognosis in a subpopulation of thyroid cancer. This study may help design novel therapeutic measures involving appropriate antibiotics to manage the disease better.

MMP10, which is upregulated in 86% of primary tongue tumors with lymph node metastases, is negatively regulated by miR-944 and promotes nodal metastasis in an orthotopic tongue cancer mouse model through the AXL signaling pathway.

Abstract Background A preoperative-progesterone intervention increases disease-free survival in patients with breast cancer, with an unknown underlying mechanism. We elucidated the role of non-coding RNAs in response to progesterone in human breast cancer. Methods Whole transcriptome sequencing dataset of 30 breast primary tumors (10 tumors exposed to hydroxyprogesterone and 20 tumors as control) were re-analyzed to identify differentially expressed non-coding RNAs followed by real-time PCR analyses to validate the expression of candidates. Functional analyses were performed by genetic knockdown, biochemical, and cell-based assays. Results We identified a significant downregulation in the expression of a long non-coding RNA, Down syndrome cell adhesion molecule antisense DSCAM-AS1, in response to progesterone treatment in breast cancer. The progesterone-induced expression of DSCAM-AS1 could be effectively blocked by the knockdown of progesterone receptor (PR) or treatment of cells with mifepristone (PR-antagonist). We further show that knockdown of DSCAM-AS1 mimics the effect of progesterone in impeding cell migration and invasion in PR-positive breast cancer cells, while its overexpression shows an opposite effect. Additionally, DSCAM-AS1 sponges the activity of miR-130a that regulates the expression of ESR1 by binding to its 3’-UTR to mediate the effect of progesterone in breast cancer cells. Consistent with our findings, TCGA analysis suggests that high levels of miR-130a correlate with a tendency toward better overall survival in patients with breast cancer. Conclusion This study presents a mechanism involving the DSCAM-AS1/miR-130a/ESR1 genomic axis through which progesterone impedes breast cancer cell invasion and migration. The findings highlight the utility of progesterone treatment in impeding metastasis and improving survival outcomes in patients with breast cancer.

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor, shows significant benefit among patients with EGFR T790M mutation at disease progression. We analyzed the whole exome sequence of 48 samples obtained from 16 lung cancer patients with a longitudinal follow-up: treatment-naïve-baseline primary tumors positive for EGFR activating-mutations, paired re-biopsies upon disease progression but negative for EGFR T790M mutation based on qPCR, and their matched normal blood samples. Our Next generation sequencing (NGS) analysis identified an additional set of 25% re-biopsy samples to harbor EGFR T790M mutation occurring at a low-allele frequency of 5% or less, undetectable by conventional qPCR-based assays. Notably, the clinical utility of osimertinib among patients harboring low-allele frequency of EGFR T790M in tissue biopsy upon disease progression remains less explored. We established erlotinib-resistant PC-9R cells and twenty single-cell sub-clones from erlotinib-sensitive lung cancer PC-9 cells using in vitro drug-escalation protocol. NGS and allele-specific PCR confirmed the low-allele frequency of EGFR T790M present at 5% with a 100-fold higher resistance to erlotinib in the PC-9R cells and its sub-clones. Additionally, luciferase tagged PC-9, and PC-9R cells were orthotopically injected through the intercostal muscle into NOD-SCID mice. The orthotopic lung tumors formed were observed by non-invasive bioluminescence imaging. Consistent with in vitro data, osimertinib, but not erlotinib, caused tumor regression in mice injected with PC-9R cells, while both osimertinib and erlotinib inhibited tumors in mice injected with PC-9 cells. Taken together, our findings could extend the benefit of osimertinib treatment to patients with low EGFR T790M mutation allele frequency on disease progression.

PURPOSEMultidisciplinary molecular tumor boards (MTBs) help in interpreting complex genomic data generated by molecular tumor profiling and improve patients' access to targeted therapies. The purpose of this study was to assess the impact of our institution's MTB on the clinical management of patients with cancer.METHODSThis study was conducted at a tertiary cancer center in India. Cases to be discussed in the MTB were identified by molecular pathologists, scientists, or oncologists. On the basis of the clinical data and molecular test reports, a course of clinical management was recommended and made available to the treating oncologist. We determined the proportion of patients who were recommended a change in the clinical management. We also assessed compliance of the treating oncologists with MTB recommendations.RESULTSThere were 339 discussions for 328 unique patients. The median age of the cohort was 54 years (range 17-87), and the majority of the patients were men (65.1%). Of 339 cases, 133 (39.2%) were recommended continuation of ongoing therapy while the remaining 206 (60.7%) were recommended a change in clinical management. Compliance with MTB recommendations for a change in clinical management was 58.5% (79 of 138 evaluable cases). Compliance and implementation for MTB's recommendation to start a new therapy in 104 evaluable cases were 60.5% and 44.2%, respectively. A total of 248 biopsies had at least one actionable mutation. A total of 646 mutations were identified in the cohort, with EGFR being the most frequently altered gene.CONCLUSIONMTBs help in interpreting results of molecular tests, understanding the significance of molecular abnormalities, and assessing the benefits of available targeted therapies and clinical trials in the management of patients with targetable genetic alterations.

Abstract Background Rapid analysis of SARS-CoV-2 genomic data plays a crucial role in surveillance and adoption of measures in controlling spread of Covid-19. Fast, inclusive and adaptive methods are required for the heterogenous SARS-CoV-2 sequence data generated at an unprecedented rate. Results We present an updated version of the SARS-CoV-2 analysis module of our automated computational pipeline, Infectious Pathogen Detector (IPD) 2.0, to perform genomic analysis to understand the variability and dynamics of the virus. It adopts the recent clade nomenclature and demonstrates the clade prediction accuracy of 92.8%. IPD 2.0 also contains a SARS-CoV-2 updater module, allowing automatic upgrading of the variant database using genome sequences from GISAID. As a proof of principle, analyzing 208,911 SARS-CoV-2 genome sequences, we generate an extensive database of 2.58 million sample-wise variants. A comparative account of lineage-specific mutations in the newer SARS-CoV-2 strains emerging in the UK, South Africa and Brazil and data reported from India identify overlapping and lineages specific acquired mutations suggesting a repetitive convergent and adaptive evolution. Conclusions A novel and dynamic feature of the SARS-CoV-2 module of IPD 2.0 makes it a contemporary tool to analyze the diverse and growing genomic strains of the virus and serve as a vital tool to help facilitate rapid genomic surveillance in a population to identify variants involved in breakthrough infections. IPD 2.0 is freely available from http://www.actrec.gov.in/pi-webpages/AmitDutt/IPD/IPD.html and the web-application is available at http://ipd.actrec.gov.in/ipdweb/ .

The recently conducted ADAURA trial concludes daily dosing of adjuvant osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), improves disease-free survival with stage IB/II/IIIA EGFR -mutated non-small cell lung cancer patients in comparison to placebo. We have developed a preclinical orthotopic mouse model, using luciferase tagged lung adenocarcinoma cells harboring EGFR TKI sensitive exon 19 deletion to model and extend trial implications comparing a weekly vs daily dosing outcome of osimertinib to a first-generation TKI- erlotinib. We find that 100% of mice in both the groups receiving osimertinib daily or weekly before injection of cells show a complete absence of homing of cells in mice's lungs from day three until day 18 post-injection of cells. On the other hand, 25% and 75% of mice receiving erlotinib daily and weekly before injecting cells show homing of cells to the lungs. The tumors observed in the lungs, when dissected at day 30, confirmed the colonization of the injected cells homing to the organ. Thus, our study establishes the efficacy of pretreatment with osimertinib in reducing tumor cells' homing to mouse lungs in an in vivo mouse model.

Early diagnosis of SARS-CoV-2 infected patients is essential to control the dynamics of the COVID-19 pandemic. We develop a rapid and accurate one-step multiplex TaqMan probe-based real-time RT-PCR assay, along with a computational tool to systematically analyse the data. Our assay could detect to a limit of 15 copies of SARS-CoV-2 transcripts—based on experiments performed by spiking total human RNA with in vitro synthesized viral transcripts. The assay was evaluated by performing 184 validations for the SARS-CoV-2 Nucleocapsid gene and human RNase P as an internal control reference gene with dilutions ranging from 1-100 ng for human RNA on a cohort of 26 clinical samples. 5 of 26 patients were confirmed to be infected with SARS-CoV-2, while 21 tested negative, consistent with the standards. The accuracy of the assay was found to be 100% sensitive and 100% specific based on the 26 clinical samples that need to be further verified using a large number of clinical samples. In summary, we present a rapid, easy to implement real-time PCR based assay with automated analysis using a novel COVID qPCR Analyzer tool with graphical user interface (GUI) to analyze the raw qRT-PCR data in an unbiased manner at a cost of under $3 per reaction and turnaround time of less than 2h, to enable in-house SARS-CoV-2 testing across laboratories.

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.

Abstract Liquid biopsy is increasingly gaining traction as an alternative to invasive solid tumor biopsies for prognosis, treatment decisions, and disease monitoring. Matched tumor‐plasma samples were collected from 180 patients across different cancers with >90% of the samples below Stage IIIB. Tumors were profiled using next‐generation sequencing (NGS) or quantitative PCR (qPCR), and the mutation status was queried in the matched plasma using digital platforms such as droplet digital PCR (ddCPR) or NGS for concordance. Tumor‐plasma concordance of 82% and 32% was observed in advanced (Stage IIB and above) and early (Stage I to Stage IIA) stage samples, respectively. Interestingly, the overall survival outcomes correlated to presurgical/at‐biopsy ctDNA levels. Baseline ctDNA stratified patients into three categories: (a) high ctDNA correlated with poor survival outcome, (b) undetectable ctDNA with good outcome, and (c) low ctDNA whose outcome was ambiguous. ctDNA could be a powerful tool for therapy decisions and patient management in a large number of cancers across a variety of stages.

Context: Trends in epidermal growth factor receptor (EGFR) mutation based on ethnicity assist the initial selection of targeted therapy regimen. Reported incidence of EGFR mutation in Indian NSCLC patients is variable, ranging from 22% to 51.8%. Aim and Settings and Design: This multicenter, noninterventional study evaluated the prevalence of EGFR mutation in Indian NSCLC patients, its association with patients’ demographics, and for the first time used a central laboratory for molecular testing. Subjects and Methods: Tissue samples from 252 NSCLC patients were tested at a Central Laboratory at Tata Memorial Hospital. Statistical Analysis Used: Patient demographics, baseline characteristics including smoking status from routine examination were recorded in a single visit. Chi-square or Fisher's exact test was used for association of EGFR mutation status with gender, age, smoking status, and histological subtypes. Results: The prevalence of EGFR mutation in Indian NSCLC patients was 23.4%. Among these, 55.9% patients had mutations in exon 19, 39% in exon 21, and 1.7% in exon 18. The incidence of EGFR mutation was higher in females than males (32.5% vs. 18.9%, respectively), and in 30.6% patients that had never smoked, 26.3% smokers, and 5.8% former smokers. The mean duration of transportation of tissue samples to the central laboratory was 48 h with an average turnaround time of 5 days for molecular testing. Conclusions: Molecular testing at a central laboratory is a feasible option in India. Prevalence of EGFR mutation in Indian NSCLC patients was similar across western and southern centers in India. A statistically significant association between EGFR mutation and gender as well as the smoking status of the patients was observed. Majority of the patients had in-frame deletions in exon 19.

Molecular diagnostics has changed the way lung cancer patients are treated worldwide. Of several different testing methods available, PCR followed by directed sequencing and amplification refractory mutation system (ARMS) are the two most commonly used diagnostic methods worldwide to detect mutations at KRAS exon 2 and EGFR kinase domain exons 18-21 in lung cancer. Compared to ARMS, the PCR followed by directed sequencing approach is relatively inexpensive but more cumbersome to perform. Moreover, with a limiting amount of genomic DNA from clinical formalin-fixed, paraffin-embedded (FFPE) specimens or fine biopsies of lung tumors, multiple rounds of PCR and sequencing reactions often get challenging. Here, we report a cost-effective single multiplex-PCR based method, CRE (for Co-amplification of five KRAS and EGFR exons), followed by concatenation of the PCR product as a single linear fragment for direct sequencing. CRE is a robust protocol that can be adapted for routine use in clinical diagnostics with reduced variability, cost and turnaround time requiring a minimal amount of template DNA extracted from FFPE or fresh frozen tumor samples. As a proof of principle, CRE is able to detect the activating EGFR L858R and T790M EGFR mutations in lung cancer cell line and primary tumors.

Introduction: Tongue cancer is the most predominant form of oral cancer in developed countries with varying incidence in developing countries. In India, tongue cancer accounts for 21% of head and neck squamous cell carcinoma (HNSCC) and known to display occult node metastasis during early stages of disease. The major etiological factors associated with tongue cancer includes tobacco related products, alcohol and human papilloma virus (HPV) infections. Objective: Portrait of genomic aberrations underlying the genome of tobacco/nut chewing HPV-negative early stage tongue tumors. Materials and Methods: Whole transcriptome sequencing of 17 HPV-negative early stage tongue squamous cell carcinoma (TSCC) tumors and 4 HNSCC cell lines. Validation of findings in an additional set of 44 paired HPV-negative early stage TSCC tumor samples. Results: Using bioinformatics approaches, we present the first glance of a portrait of 242 tumor specific transcript fusions, followed by exhaustive validation of 12 candidate fusion transcripts across 44 paired HPV-negative early TSCC tumor samples and 4 HNSCC cell lines. Comparative analysis of our data with various fusion databases revealed 48 previously described transcript fusions in various cancer types. We have identified and validated novel somatic recurrent fusion transcripts in tumor samples. Here, we present a comprehensive landscape of transcript fusions underlying the genome of HPV-negative early stage tongue tumors. Conclusions: Characterization of the recurrent transcript fusions described here could serve as attractive candidates to facilitate in diagnosis of HPV-negative early stage TSCC patients.

Introduction: Gallbladder cancer is relatively a rare lethal malignancy with dismal prognosis. While in India there is high incidence (3.9-8.6/1, 00,000) with majority of patients having advanced disease. Recent developments in next generation sequencing technologies have enabled the discovery of new molecular therapeutic targets in many human cancers. Objectives: Interrogate the landscape of somatic alterations in Indian gall bladder cancer using whole exome sequencing technology. Material and Methods: We interrogated the coding region of 27(10 paired and 7 unpaired) Indian gall bladder cancer samples using whole exome sequencing at an average coverage of 100X and above. We further validated the findings using an additional set of 27 FFPE samples. Results: Using a bioinformatics filtering approach, we identify a total of 5060 somatic variants found across 17 tumors consisted of 3239 missense, 1449 silent, 131 nonsense, 135 indels and 106 splice site mutations The average mutation rate considering the paired tumors is about 7.7 mutations/mb. We found TP53 (35.2%), ERBB2 (17.6%), SF3B1 (17.6%), ATM (17.6%) and AKAP11 (17.6%) mutations in more than two samples by exome sequencing analysis. Furthermore, we examined our exome sequencing data for identifying Salmonella sequences as well as presence of 143 HPV types using computation subtraction based on HPVDetector. Based on our evaluation we found association of typhoidal Salmonella strains in 11 of 26 gall bladder cancer samples and non-typhoidal Salmonella species in 12 of 26 samples, 6 samples were co-infected with both. Conclusions: The profiling of somatic alterations and identification of non typhoidal Salmonella traces may aid in changing the current treatment paradigm of gall bladder cancer.

Analysis of mutational spectra across various cancer types has given valuable insights into tumorigenesis. Different approaches have been used to identify novel drivers from the set of somatic mutations, including the methods which use sequence conservation, geometric localization and pathway information. Recent computational methods suggest use of protein domain information for analysis and understanding of the functional consequence of non-synonymous mutations. Similarly, evidence suggests recurrence at specific position in proteins is robust indicators of its functional impact. Building on this, we performed a systematic analysis of TCGA exome derived somatic mutations across 6089 PFAM domains and significantly mutated domains were identified using randomization approach. Multiple alignment of individual domain allowed us to prioritize for conserved residues mutated at analogous positions across different proteins in a statistically disciplined manner. In addition to the known frequently mutated genes, this analysis independently identifies low frequency Meprin and TRAF-Homology (MATH) domain in Speckle Type BTB/POZ (SPOP) protein, in prostate adenocarcinoma. Results from this analysis will help generate hypotheses about the downstream molecular mechanism resulting in cancer phenotypes.

Introduction: Lung tumors with mutations in epidermal growth factor receptor (EGFR) gene represent a clinically distinct subtype of lung cancer and are observed at a frequency of 23% among Indian patients. The standard practice for treatment of EGFR mutated lung cancer patients includes tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib. Although initial clinical responses are observed, resistance to TKIs develops within year from the start of treatment. In about fifty percent of cases, the resistance is caused due to a secondary T790M mutation in the EGFR gene. Additionally, MET amplification and histological transformation of tumors are known to confer TKI resistance in a small subset of patients. Nonetheless, there is an unmet need to elucidate novel ways by which lung tumors acquire resistance to EGFR targeting TKIs. Objectives: To delineate novel mechanisms of acquired resistance to EGFR-TKIs by characterizing the differential profile of drug sensitive and resistant state among lung tumors using integrated genomics approaches. Material and Methods: A retrospective collection of FFPE DNA samples (n=45) from tumors at baseline and rebiopsy along with paired blood sample was done for a total of 15 EGFR mutated lung cancer patients. Only tumor samples which were negative for EGFR T790M (as confirmed by orthologous technologies) were selected in the study with an anticipation that such samples would be enriched novel resistance mechanisms. Whole exome sequencing at an average coverage of 100X was performed for these samples. Results: The whole exome data was analyzed using an in-house developed pipeline. Of all the known resistance mutations, we identified EGFR T790M mutation in five out of fifteen patients. Other than T790M we expect to identify novel resistance causing mutations from the analysis of ten patients with unknown resistance mechanisms. Functional validation of these resistance specific alterations would be performed in vitro using drug sensitive lung cancer cell lines.

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.

Screening for EGFR mutation is a key molecular test for management of lung cancer patients. Outcome of patients with mutation receiving EGFR tyrosine kinase inhibitor is known to be better across different ethnic populations. However, frequency of EGFR mutations and the clinical response in most other ethnic populations, including India, remains to be explored. We conducted a retrospective analysis of Indian lung cancer patients who were managed with oral tyrosine kinase inhibitors. Majority of the patients in the study had adenocarcinoma and were non-smokers. 39/111 patients tested positive for EGFR kinase domain mutations determined by Taqman based real time PCR. The overall response to oral TKI therapy was 30%. Patients with an activating mutation of EGFR had a response rate of 74%, while the response rate in patients with wild type EGFR was 5%, which was a statistically significant difference. Progression free survival of patients with EGFR mutations was 10 months compared to 2 months for EGFR mutation negative patients. Overall survival was 19 months for EGFR mutation patients and 13 months for mutation negative patients. This study emphasizes EGFR mutation as an important predictive marker for response to oral tyrosine kinase inhibitors in the Indian population.

Squamous cell lung carcinomas account for approximately 25% of new lung carcinoma cases and 40,000 deaths per year in the United States. Although there are multiple genomically targeted therapies for lung adenocarcinoma, none has yet been reported in squamous cell lung carcinoma.Using SNP array analysis, we found that a region of chromosome segment 8p11-12 containing three genes-WHSC1L1, LETM2, and FGFR1-is amplified in 3% of lung adenocarcinomas and 21% of squamous cell lung carcinomas. Furthermore, we demonstrated that a non-small cell lung carcinoma cell line harboring focal amplification of FGFR1 is dependent on FGFR1 activity for cell growth, as treatment of this cell line either with FGFR1-specific shRNAs or with FGFR small molecule enzymatic inhibitors leads to cell growth inhibition.These studies show that FGFR1 amplification is common in squamous cell lung cancer, and that FGFR1 may represent a promising therapeutic target in non-small cell lung cancer.

During Caenorhabditis elegans vulval development, the anchor cell (AC) in the somatic gonad secretes an epidermal growth factor (EGF) to activate the EGF receptor (EGFR) signaling pathway in the adjacent vulval precursor cells (VPCs). The inductive AC signal specifies the vulval fates of the three proximal VPCs P5.p, P6.p, and P7.p. The C. elegans Rhomboid homolog ROM-1 increases the range of EGF, allowing the inductive signal to reach the distal VPCs P3.p, P4.p and P8.p, which are further away from the AC. Surprisingly, ROM-1 functions in the signal-receiving VPCs rather than the signal-sending AC. This observation led to the discovery of an AC-independent activity of EGF in the VPCs that promotes vulval cell fate specification and depends on ROM-1. Of the two previously reported EGF splice variants, the longer one requires ROM-1 for its activity, while the shorter form acts independently of ROM-1. We present a model in which ROM-1 relays the inductive AC signal from the proximal to the distal VPCs by allowing the secretion of the LIN-3L splice variant. These results indicate that, in spite of their structural diversity, Rhomboid proteins play a conserved role in activating EGFR signaling in C. elegans, Drosophila, and possibly also in mammals.