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Rationale: Surgery results in rapid and progressively severe exposure of tumor tissue to hypoxia, up to the point of complete removal, but its effect on tumor gene expression is not well characterized. We document the molecular effects of surgery on primary breast cancer tumor with a serial tissue sampling strategy, including an intra-operative sample. Methods: We included treatment naïve, non-metastatic breast cancer patients and sampled tumor and tumour adjacent normal tissue during surgery at three time points: beginning of surgery (Sample A), after half the tumor circumference had been devascularized (Sample B) and from completely resected tumor (Sample C). Patients were divided into two groups: discovery and validation. Tumor or adjacent normal samples from the discovery group underwent whole transcriptome paired-end sequencing (RNA-Seq) generating at least 50 million reads using next generation sequencing. Findings from discovery group were evaluated in a validation group using qRT-PCR and Nanostring nCounter gene expression profiling. Results: 81 breast cancer patients were eligible for this study of whom 46 with at least 1 quality passed sample at time-point A, B or C comprised the discovery group whose samples underwent RNA-seq. Validation group comprised two subsets: 35 independent patients (8 patients’ samples qRT-PCR, data included here; 27 patients’ samples nCounter gene expression, data will be presented) and 17 patients from discovery group whose samples also underwent nCounter gene expression profiling (data will be presented). Individual patient based analyses for A vs B vs C in discovery group revealed 249 significantly de-regulated genes in at least 20% of patients, in at least one comparison (AvB or BvC or AvC). Genes involved in stress response (FOSB, FOS, JUN, JUNB, DUSP1, CYR61, EGR1-3, ATF3, RGS1, RGS2), inflammation (IL20, IL8, SOCS3, GABRP, PIGR, NR4A1-2, CCL2- 3, CCL21, CCL14, CCL18-19, CXCL2, CXCL9-10, CXCL14), invasion & migration (PTGS2, MMP9-13), lipid metabolism (CD36, LIPE, TAT, FABP4, PLIN1, PLIN4, LPL, LEP), epithelial markers (KRT5, KRT7, KRT14-17, KRT23, KRT6A-6B) and cellular differentiation(SOX10, LRP2, S100A2, S100A7-A9, S100B, S100P) were significantly deregulated at time-point B versus A and many of these genes were also significantly deregulated in C versus A comparison, suggesting sustained deregulation through surgical resection. Replicative analysis involving comparison of tumors grouped by time points (A vs B vs C) identified 192 genes uniquely de-regulated in any one of the 3 comparisons, of which 42 overlapped with patient-wise analysis. These 42 genes included all the AP-1 transcription factor network signaling genes, epithelial markers (KRT14, KRT6A), inflammation related genes (SOCS3, PIGR, GABRP, NR4A1, NR4A2), lipid metabolism related genes (PLIN1) and cellular differentiation & cell fate related genes (SOX10, LRP2). Pathway analyses will be presented. qRT-PCR on paired samples in 8 independent patients validated the differential expression of eight genes in either AvB or AvC or both comparisons: FOS, FOSB, JUNB, DUSP1, RGS1, NR4A2, ZFP36, andMMP13. Comparison of biopsy (corresponding to A) with surgical samples (corresponding to C) in 6 cancer types (breast, cervical, lymphoma, mesothelioma, esophageal and sarcoma) in TCGA studies showed statistically significant de-regulation of 11 AP-1 transcription factor network signaling related genes in at least 4 cancers, further validating our experimental results. Conclusions: Our experiment, uniquely including an intra-operative tumor sample, shows that surgical removal induces a conserved stress response in the primary tumor that is potentially capable of bestowing metastatic capability on the tumor cells. Citation Format: Sudeep Gupta, Rohan Chaubal, Nilesh Gardi, Sunil Pachakar, Dimple Bhatia, Poonam Gera, Nita Nair, Shalaka Joshi, Vani Parmar, Purvi Thakkar, Garvit Chitkara, Rasika Kadam, Sejal Gujarathi, Harsh Oza, Rohini Hawaldar, Vaibhav Vanmali, Kenneth Buetow, Amit Dutt, Rajendra Badwe. Molecular effects of surgical resection on primary breast tumor [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-05-01.

The Global Cancer Genomics Consortium (GCGC) is an international collaborative platform that amalgamates cancer biologists, cutting-edge genomics, and high-throughput expertise with medical oncologists and surgical oncologists; they address the most important translational questions that are central to cancer research and treatment. The annual GCGC symposium was held at the Advanced Centre for Treatment Research and Education in Cancer, Mumbai, India, from November 9 to 11, 2011. The symposium showcased international next-generation sequencing efforts that explore cancer-specific transcriptomic changes, single-nucleotide polymorphism, and copy number variations in various types of cancers, as well as the structural genomics approach to develop new therapeutic targets and chemical probes. From the spectrum of studies presented at the symposium, it is evident that the translation of emerging cancer genomics knowledge into clinical applications can only be achieved through the integration of multidisciplinary expertise. In summary, the GCGC symposium provided practical knowledge on structural and cancer genomics approaches, as well as an exclusive platform for focused cancer genomics endeavors. Cancer Res; 72(15); 3720–4. ©2012 AACR.

Genomic alterations of the epidermal growth factor receptor (EGFR) gene play a crucial role in pathogenesis of glioblastoma multiforme (GBM). By systematic analysis of GBM genomic data, we have identified and characterized a novel exon 27 deletion mutation occurring within the EGFR carboxyl-terminus domain (CTD), in addition to identifying additional examples of previously reported deletion mutations in this region. We show that the GBM-derived EGFR CTD deletion mutants are able to induce cellular transformation in vitro and in vivo in the absence of ligand and receptor autophosphorylation. Treatment with the EGFR-targeted monoclonal antibody, cetuximab, or the small molecule EGFR inhibitor, erlotinib, effectively impaired tumorigenicity of oncogenic EGFR CTD deletion mutants. Cetuximab in particular prolonged the survival of intracranially xenografted mice with oncogenic EGFR CTD deletion mutants, compared with untreated control mice. Therefore, we propose that erlotinib and, especially, cetuximab treatment may be a promising therapeutic strategy in GBM patients harboring EGFR CTD deletion mutants. Cancer Res; 71(24); 7587–96. ©2011 AACR.

Background: Hypoxia is defined as oxygen levels in tumor microenvironment of less than that in blood (90-100 mm Hg) and influences many aspects of tumour biology. During surgery tumour vasculature is cut off gradually leading to induction of acute hypoxia.The present study aims to experimentally test the genotypic and phenotypic effects of surgically induced acute hypoxia in breast cancer tumor samples and cell lines. Methodology: Core biopsy samples were collected from breast tumors (N=8 patients) at three time points during their curative surgery: prior (pre), mid-way (intra) and at the end (post). The samples were subjected to RNA-Seq and a list of differentially expressed genes (DEG) was prepared. A set of 26 DEG (‘pre’ Vs ‘intra’ Vs ‘post’) obtained from RNA-Seq analysis and additional 17 genes involved in inflammation, EMT and hypoxia pathways were chosen for validation in tumor samples. These genes were validated using a customized qPCR Array (Qiagen). A gene was considered validated if it was significantly deregulated in at least 4 out of 8 patients. In another experiment, MCF-7 cells were exposed to varying levels of oxygen concentrations (0.1-20%) for varying time periods ranging from 30 minutes to 72 hours, to study time and dose dependent effects of hypoxia on following functional characteristics: proliferation, invasion and cell cycle changes. Results: Concordant, statistically significant up-regulation of FOS, DUSP1, JUNB, FOSB, ZFP36, RGS1, S100A4, CXCL8 and CCL2 were observed in RNA-Seq and qPCR experiments while MMP13, HIF1A and VEGFA were up-regulated only in qPCR. However, 7 protein markers of inflammation, EMT and hypoxia did not show any significant change between pre, intra and post-operative samples. In MCF-7 cells, a dose and time dependent decrease in cell viability was observed with increasing severity of hypoxia as well as decrease in invasiveness, but there was no significant impact on cell cycle phases. When hypoxic cells were re-incubated under normoxic conditions an increase in cell proliferation and accumulation of cells in S phase (with a reduction in G2-M fraction) were observed, compared to cells grown under only normoxic conditions. Conclusion: Acute intra-operative hypoxia up-regulates expression of genes related to cell survival, chemoresistance, invasiveness, inflammation and angiogenesis in breast tumors. Breast cancer cells exposed to acute severe hypoxia followed by normoxia show increased proliferation. These effects may have implications for tumor cells that disseminate during surgery. Citation Format: Dimple R. Bhatia, Shalaka Joshi, Rohan Chaubal, Poonam Gera, Prajakta Kalkar, Farheen Naeem, Nisanth Mathew Raju, Nilesh Gardi, Nita Nair, Vaibhav Vanmali, Rohini W. Hawaldar, Amit Dutt, Rajendra A. Badwe, Sudeep Gupta. The effect of acute intraoperative hypoxia in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4524. doi:10.1158/1538-7445.AM2017-4524

INTRODUCTION Glioblastoma resistance and recurrence is attributed to the presence of innately Radiation Resistant (RR) cells present in the heterogeneous parent tumour. However, targeting these cells has been impossible due to inaccessibility of these cells. METHODOLOGY We therefore recapitulated clinical scenario of resistance in a cellular model developed from fresh primary GBM patient samples and cell lines. The model allowed us to capture 1) Parent cells 2) innately Radiation Resistant cells - less than 10% of the parent population and 3) Relapse (R) cells. To identify the targetable proteins governing the survival of RR cells, we performed iTRAQ based quantitative proteomic analysis on all the three populations from GBM cell line (SF 268). RESULTS The proteomic data analysis identified 34 proteins as differentially present in RR population of which 22 were upregulated and 12 were downregulated. A GENE STRING analysis of all the differential proteins in RR population revealed putative interaction of a novel serine threonine kinase DCLK3 with 14-3-3 zeta. The increased expression of DCLK3 and 14-3-3 zeta was confirmed by western blot in RR cells of two GBM cell lines and 8 patient samples. Meta-analysis of 242 tumor samples from COSMIC database showed DCLK3 overexpression in 232 tumors. Furthermore, it harbours 8 missense deleterious mutations, 6 of which were in the kinase domain, indicating towards an important kinase function of this protein. We hypothesized that DCLK3 mediated interaction and phosphorylation of 14-3-3 zeta modulated 14-3-3 zeta functions facilitating RR cell survival. For this, first we wanted to see if DCLK3 and 14-3-3 zeta can interact together. In silico docking of DCLK3 with 14-3-3 zeta did show interaction of 14-3-3 zeta and the kinase domain of DCLK3 kinase. This interaction was confirmed by in vitro immunoprecipitation studies. Further studies are ongoing to understand the importance of these proteins and their interaction in GBM recurrence. Additionally, pathway analysis of differentially upregulated proteins in RR cells revealed deregulation of the proteins involved in Ubiquitin-Proteasome System. Indeed, proteasome activity assay showed increased proteosome activity in the RR population of GBM cell lines and Patient samples. Accordingly, Bortezomib, a proteosome inhibitor induced significant apoptosis in the RR population at a concentration significantly lower than that required for inducing apoptosis in the parent cells. SIGNIFICANCE In conclusion this is the first study to identify a proteome signature of innately radiation resistant cells of GBM and identify proteosome pathway and a novel serine-threonine kinase DCLK3 in RR cells as a potential therapeutic target to inhibit GBM radioresistance and recurrence. Citation Format: Jacinth Rajendra, Keshava Datta, , Sheikh Burhan Ud Din Farooqee, Raja Reddy, Nilesh Gardi, Ekjot Kaur, Ketaki Patkar, Aliasgar Moiyadi, Prasanna Venkataraman, Kakoli Bose, Amit Dutt, Harsha Gowda, Shilpee Dutt. Identification of proteosome pathway and a novel serine threonine kinase DCLK3: Potential therapeutic targets for innately radiation resistant glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5845. doi:10.1158/1538-7445.AM2017-5845

Background: 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. Material and Methods: 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. Differential expression of transcriptome was performed using tophat-cufflinks-cummeRbund pipeline and miRNA using standard array data analysis pipeline. Alterations identified were validated using orthologous technologies in an extended cohort of tongue squamous cell carcinoma patients. Finally, we 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. Linking expression profiles of whole transcriptome and miRNAs with their annotated functions, we 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. Conclusion: Knowledge from integrated expression analysis along with functional validation identify a novel matrix metalloproteinase as a clinically relevant biomarker to prognosticate metastases in patients with early stage oral tongue cancers. Citation Format: Pawan Upadhyay, Nilesh Laxman Gardi, Hemant Ramesh Dhamne, Kavitha Sonawane, Anil D'Cruz, Sudhir Nair, Amit Dutt. Discovery of a matrix metalloproteinase MMP10 as a clinically relevant biomarker to predict lymph node metastasis in tongue squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5161. doi:10.1158/1538-7445.AM2015-5161

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. Citation Format: Pawan Upadhyay, Jyotirmoi Aich, Vidyalakshmi Sethunath, Prachi Dani, Sadhana Kannan, Pratik Chandrani, Kavitha Sonawane, Beamon Agarwal, Shubhada Kane, Sudhir Nair, Amit Dutt. Pro-oncogenic role of NOTCH1 in early tongue squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4811. doi:10.1158/1538-7445.AM2015-4811

Inhibitors targeting specific genomic alterations have revolutionized cancer therapy and dramatically improved patient outcomes; nonetheless, after a period response, patient tumors almost universally develop resistance to these inhibitors and recur. In the case of squamous cell carcinoma of the lung, it has been demonstrated that focal amplifications of the FGFR1 gene occur in at least 10% of patients and that a subset of these events are sensitive to FGFR targeted therapies. Here, we describe the generation of resistant clones of the cell line NCI-H2077 to several FGFR inhibitors with clinical potential. This cell line is a squamous lung line with a focal amplification of FGFR1 that demonstrates dependency in the presence of both small molecule inhibitors and knockdown of the FGFR1 protein. After generation of resistant clones, we performed RTK phosphorylation arrays in the presence and absence of inhibitor to determine whether there were changes in the activity of other kinases, and the lines were sequenced to determine whether secondary mutations in FGFR1 had arisen. Follow-up experiments were performed to determine alternative sensitivities of the resistant clones. These experiments contribute to our growing understanding of the resistance mechanisms that arise in response to kinase inhibitors, and it is our hope that they will benefit patients undergoing treatment for FGFR1-amplified lung squamous cell carcinoma. Citation Format: Rachel G. Liao, Andrey Sivachenko, Gad Getz, Amit Dutt, Peter S. Hammerman, Matthew Meyerson. Characterizing acquired resistance mechanisms to targeted therapy in FGFR1-amplified lung squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 963. doi:10.1158/1538-7445.AM2013-963

The Cancer Genome Atlas (TCGA) Consortium recently completed a multidimensional analysis of glioblastoma, characterizing somatic alterations across a large sample set, and identifying genetic lesions that likely contribute to tumor fitness (TCGA Network, Nature, 2008). However, functional experiments are required to determine whether significantly altered genes are in fact oncogenes or tumor suppressor genes in glioblastoma. We experimentally analyzed mutations in one of the significantly mutated genes, ERBB2. Unlike ERBB2 mutations in lung adenocarcinoma, ERBB2 mutations in glioblastoma are primarily located in the extracellular region, clustering around the domain that mediates receptor dimerization. We have determined that a subset of the reported mutations are gain-of-function and oncogenic, require kinase activity for oncogenic transformation, and are sensitive to specific small molecule kinase inhibitors. This discovery has therapeutic implications for glioblastoma patients who harbor somatic ERBB2 mutations. Citation Information: Cancer Res 2009;69(23 Suppl):A2.