Satish Sankaran

Detection of high frequency of mutations in a breast and/or ovarian cancer cohort: implications of embracing a multi-gene panel in molecular diagnosis in India

Breast and/or ovarian cancer (BOC) are among the most frequently diagnosed forms of hereditary cancers and leading cause of death in India. This emphasizes on the need for a cost-effective method for early detection of these cancers. We sequenced 141 unrelated patients and families with BOC using the TruSight Cancer panel, which includes 13 genes strongly associated with risk of inherited BOC. Multi-gene sequencing was done on the Illumina MiSeq platform. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. We were able to detect pathogenic mutations in 51 (36.2%) cases, out of which 19 were novel mutations. When we considered familial breast cancer cases only, the detection rate increased to 52%. When cases were stratified based on age of diagnosis into three categories, ⩽40 years, 40–50 years and >50 years, the detection rates were higher in the first two categories (44.4% and 53.4%, respectively) as compared with the third category, in which it was 26.9%. Our study suggests that next-generation sequencing-based multi-gene panels increase the sensitivity of mutation detection and help in identifying patients with a high risk of developing cancer as compared with sequential tests of individual genes.

StrandAdvantage test for early-line and advanced-stage treatment decisions in solid tumors

Comprehensive genetic profiling of tumors using next‐generation sequencing (NGS) is gaining acceptance for guiding treatment decisions in cancer care. We designed a cancer profiling test combining both deep sequencing and immunohistochemistry (IHC) of relevant cancer targets to aid therapy choices in both standard‐of‐care (SOC) and advanced‐stage treatments for solid tumors. The SOC report is provided in a short turnaround time for four tumors, namely lung, breast, colon, and melanoma, followed by an investigational report. For other tumor types, an investigational report is provided. The NGS assay reports single‐nucleotide variants (SNVs), copy number variations (CNVs), and translocations in 152 cancer‐related genes. The tissue‐specific IHC tests include routine and less common markers associated with drugs used in SOC settings. We describe the standardization, validation, and clinical utility of the StrandAdvantage test (SA test) using more than 250 solid tumor formalin‐fixed paraffin‐embedded (FFPE) samples and control cell line samples. The NGS test showed high reproducibility and accuracy of >99%. The test provided relevant clinical information for SOC treatment as well as more information related to investigational options and clinical trials for >95% of advanced‐stage patients. In conclusion, the SA test comprising a robust and accurate NGS assay combined with clinically relevant IHC tests can detect somatic changes of clinical significance for strategic cancer management in all the stages.

Genetic variants in post myocardial infarction patients presenting with electrical storm of unstable ventricular tachycardia

Electrical storm (ES) is a life threatening clinical situation. Though a few clinical pointers exist, the occurrence of ES in a patient with remote myocardial infarction (MI) is generally unpredictable. Genetic markers for this entity have not been studied. In the present study, we carried out genetic screening in patients with remote myocardial infarction presenting with ES by next generation sequencing and identified 25 rare variants in 19 genes predominantly in RYR2, SCN5A, KCNJ11, KCNE1 and KCNH2, CACNA1B, CACNA1C, CACNA1D and desmosomal genes - DSP and DSG2 that could potentially be implicated in electrical storm. These genes have been previously reported to be associated with inherited syndromes of Sudden Cardiac Death. The present study suggests that the genetic architecture in patients with remote MI and ES of unstable ventricular tachycardia may be similar to that of Ion channelopathies. Identification of these variants may identify post MI patients who are predisposed to develop electrical storm and help in risk stratification.

Identification of prognostic and susceptibility markers in chronic myeloid leukemia using next generation sequencing

Background Incidence of Chronic Myeloid Leukemia (CML) is continuously increasing and expected to reach 100,000 patients every year by 2030. Though the discovery of Imatinib Mesylate (IM) has brought a paradigm shift in CML treatment, 20% patients show resistance to this tyrosine kinase inhibiter (TKI). Therefore, it is important to identify markers, which can predict the occurrence and prognosis of CML. Clinical Exome Sequencing, panel of more than 4800 genes, was performed in CML patients to identify prognostic and susceptibility markers in CML. Methods Enrolled CML patients (n=18) were segregated as IM responders (n=10) and IM failures (n=8) as per European Leukemia Net (ELN), 2013 guidelines. Healthy controls (n=5) were also enrolled. DNA from blood of subjects was subjected to Next Generation Sequencing. Rare mutations present in one patient group and absent in another group were considered as prognostic markers, whereas mutations present in more than 50% patients were considered as susceptibility markers. Result Mutations in genes associated with cancer related functions were found in different patient groups. Four variants: rs116201358, rs4014596, rs52897880 and rs2274329 in C8A, UNC93B1, APOH and CA6 genes, respectively, were present in IM responders; whereas rs4945 in MFGE8 was present in IM failures. Mutations in HLA-DRB1 (rs17878951), HLA-DRB5 (rs137863146), RPHN2 (rs193179333), CYP2F1 (rs116958555), KCNJ12 (rs76684759) and FUT3 (rs151218854) were present as susceptibility markers. Conclusion The potential genetic markers discovered in this study can help in predicting response to IM as frontline therapy. Susceptibility markers may also be used as panel for individuals prone to have CML.

Analysis of solid tumor mutation profiles in liquid biopsy

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.

Incidental Findings in Male Breast Carcinoma: A Genetic Counseling Approach

Abstract Male breast carcinoma (MBC) is a rare cancer type that accounts 1 percent of the total breast cancer cases. This substantially invites diagnosis challenges and social burdens where the individual needs more personalized therapies and genetic counseling to cope with the condition. This study aims to analyse a total of four male breast cancer cases with or without secondary recurrence. Tissue or saliva samples were analysed with informed and written consent for each individual subjecting to next generation sequencing aiming high throughput investigations. The results revealed two cancer syndromes in an individual with breast and thyroid carcinoma and mutations in PI3KCA, PTEN, NBN, RB1 genes. The rest three cases were identified with alterations in NBN, BRIP1 and BRCA2 mutations. Genetic counseling was provided to each participant and the responses were noted upon post-test targeted therapies.

Abstract P6-07-01: Multigene profiling to identify clinically relevant actionable mutations in breast cancer: An Indian study

Background: Numerous chemotherapeutic agents are available against breast cancer (BC), but a vast majority of patients diagnosed with this disease still develop treatment resistance and eventually succumb to disease. It remains an unmet need to identify specific molecular defects against which targeted therapy are available for improving clinical outcomes in BC. Our study aims to identify frequent hotspot mutations in BCs and determine their clinical impact. Methods: 200 women with BC(early diagnosed and/or metastatic) aged 26-75 yrs (median age 50.5yrs) diagnosed at HCG from April 2013-15 were consented to be profiled by targeted deep sequencing for hotspot mutations in 48 cancer-related genes using Illumina9s TSCAP panel and MiSeq technology in an IRB-approved prospective study in a CLIA compliant laboratory. All the cases had pathology review for stage, histological type, hormonal status and Ki67. The average coverage across 220 hot spots was greater than 1000X. Data was processed using Strand Avadis NGS™. Mutations identified in the tumor were assessed for 9actionability9 i.e. response to therapy and impact on prognosis. Results: Somatic variants were detected in 75% of cases with direct impact on therapy or prognosis. Genetic aberrations were identified in PI3K/AKT/ mTOR signalling pathway in substantial fraction (27%) of breast cancer cases, out of which 17% had PIK3CA activating mutations,13 and 5 cases had PTEN and AKT deletions or truncating mutations respectively. Aberration in this pathway was more prevalent in HR+ve (53%) and HER2-ve including TNBC (61%) than in HR+/HER2+ve tumors (10.6%) of IDC histology. However, no correlation was found with stage and Ki67 index of the tumor. Notably 80% of BC cases presented with liver metastasis at the time of diagnosis were detected with PIK3CA mutation indicating its role as a surrogate marker of organ specific metastasis. PIK3CA was found to be co mutated with p53 in 16 cases (9%) of which 4 cases showed npCR post NACT. Also disruptive and non-disruptive mutations in TP53 alone were found in 25% of BC, varying widely among different histologies. A follow up of few cases showed shorter PFS and poor outcome in resected BC treated with NACT indicating its robust prognostic value in NACT setting. Furthermore, two patients were detected with cKIT mutations indicating sensitivity to imatinib and therefore enrolled on a clinical trial. The other variants were found in RB1(n=8),Her2 (n=2),FGFR amplification(n=1), KRAS(n=2),NRAS(n=3)CDH1(n=1),FBXW7(n=2) and EGFR(n=1).All these variants detected indicated resistance to conventional therapy and suggested sensitivity to available targeted therapy, either approved or in clinical trials. The response and outcome are being monitored in about 20 (10%) patients who have been enrolled in clinical trials and receiving mutation specific targeted therapy. Conclusions: This study confirms the utility of multigene profiling in early diagnosed and advanced BC patients, to stratify them on their molecular profile who could potentially benefit from targeted therapy. Prospective studies and randomized clinical trials are ongoing to confirm the independent prognostic and therapeutic value of the mutations in a larger cohort of Indian population. Citation Format: Ghosh M, Sheela ML, Choudhury S, Bahadur U, Patil S, Satheesh CT, Murugan K, Nayak R, Sridhar PS, Rao N, Mahesh B, Shashidhara HP, Krishnamoorthy N, Gupta V, Sankaran S, Subramanian K, Ajaikumar BS. Multigene profiling to identify clinically relevant actionable mutations in breast cancer: An Indian study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-01.

Abstract 4878: Analytical and technical validation of a cost-effective diagnostic test for BRCA1, BRCA2 and TP53

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA BRCA1, BRCA2 and TP53 encode tumor suppressor proteins in humans that help repair damaged DNA and play critical roles in ensuring genome stability. Several inherited mutations in any of the above 3 genes substantially increase the risk of cancer. Together, BRCA1 and BRCA2 mutations account for about 20 to 25 percent of hereditary breast and ovarian cancers. Germline mutations in TP53 are the most common cause of Li-Fraumeni syndrome, a rare disorder that increases the risk of developing multiple tumors such as breast, soft-tissue and leukemias, in children and young adults. In India, where the incidence of cancer has seen a steep rise in the last decade, there is a pressing need to develop cost-effective screening tests that can identify known and novel mutations in commonly associated genes. We have developed and offer a 3-Gene panel (Strand® - 3 gene) covering all known HGMD/ClinVar mutations and all coding exons of BRCA1, BRCA2 and TP53 genes. Current Sanger based methods query for restricted loci across these genes. Our test is based on an NGS enrichment protocol using xGen lockdown probes that allows parallel sequencing of upto 32 - 96 samples. The test would be offered at a tenth of the cost of current Sanger based tests anywhere in the world. In this study we present the technical and clinical validation data obtained from this assay. For technical validation, we included “gold standard” HAPMAP characterized as part of 1000 Genome Project, seven cell lines with known BRCA and TP53 mutations. For clinical validation, we enrolled thirty seven (37) patients who were consented on an IRB-approved study at HCG hospital for collecting saliva / blood. These patients were stratified / selected based on their family history, known risk of hereditary cancers and availability of previously characterized clinical samples. The overall sensitivity and specificity of this panel is 99.78% and 99.74% respectively with a reproducibility of 100%. On an average, 99.75% and 97% of the bases are covered at 0.2x and 0.5x mean coverage and the average gap (<20 reads per base) is 0.0056% in the validation study. We have identified 3 separate cases of Li-Fraumeni from the cohort of thirty seven patients. We further present clinical validation data from these 3 case studies in which we have identified both known and novel mutations. Further clinical validation of panel is ongoing. In summary this panel will provide a cost effective screening method for early detection of pathogenic variants in pre-symptomatic individuals and in families with known risk of hereditary cancer. Citation Format: Manimala Sen, Pooja Agrawal, Vikram Vittal P, Mithua Ghosh, M.L Sheela, Divya Vishwanath, Kiran Kumari, Swetha N.S.N, Vaibhavi Pathak, Gouri Deshpande, Ashraf Mannan, Rupali Gadkari, Suman Kapoor, Jamuna Yadhav, Mohammed Yousuff, Satish Sankaran, Ramesh Hariharan, Preveen Ramamoorthy, Kalyanasundaram Subramanian, Vaijayanti Gupta. Analytical and technical validation of a cost-effective diagnostic test for BRCA1, BRCA2 and TP53. [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 4878. doi:10.1158/1538-7445.AM2015-4878