Abstract 4878: Analytical and technical validation of a cost-effective diagnostic test for BRCA1, BRCA2 and TP53
Authors: Sen Manimala , Agrawal Pooja , Vittal P Vikram , Ghosh Mithua , Sheela M.L , Vishwanath Divya , Kumari Kiran , N.S.N Swetha , Pathak Vaibhavi , Deshpande Gouri , Mannan Ashraf , Gadkari Rupali , Kapoor Suman , Yadhav Jamuna , Yousuff Mohammed , Sankaran Satish , Hariharan Ramesh , Ramamoorthy Preveen , Subramanian Kalyanasundaram , Gupta Vaijayanti 
Published: August 2015

Abstract
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 (&lt;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