Adipocytes play a vital role in glucose metabolism. 3T3 L1 pre adipocytes after differentiation to adipocytes serve as excellent in vitro models and are useful tools in understanding the glucose metabolism. The traditional approaches adopted in pre adipocyte differentiation are lengthy exercises involving the usage of IBMX and Dexamethasone. Any effort to shorten the time of differentiation and quality expression of functional differentiation in 3T3 L1 cells in terms of enhanced Insulin sensitivity has an advantage in the drug discovery process. Thus, there is a need to develop a new effective method of differentiating the pre adipocytes to adipocytes and to use such methods for developing efficacious therapeutic molecules. We observed that a combination of Dexamethasone and Troglitazone generated differentiated adipocytes over fewer days as compared to the combination of IBMX and Dexamethasone which constitutes the standard protocol followed in our laboratory. The experiments conducted to compare the quality of differentiation yielded by various differentiating agents indicated that the lipid droplet accumulation increased by 112 % and the GLUT4 mediated glucose uptake by 137 % in cells differentiated with Troglitazone and Dexamethasone than in cells differentiated traditionally. The comparative studies conducted for evaluating efficient measurable glucose uptake by GOPOD assay, radioactive 3 H-2-deoxy-D-glucose assay and by non-radioactive 6-NBDG (fluorescent analog of glucose) indicated that the nonradioactive method using 6-NBDG showed a higher signal to noise ratio than the conventional indirect glucose uptake method (GOPOD assay) and the radioactive 3 H-2-deoxy-D-glucose uptake method. Differentiated 3T3 L1 cells when triggered with 2.5 ng/mL of Insulin showed 3.3 fold more glucose uptake in non-radioactive method over the radioactive 3 H-2-deoxy-Dglucose uptake method. The results of this study have suggested that a combination of Dexamethasone and Troglitazone for 3T3 L1 cell differentiation helps in better quality differentiation over a short period of time with increased sensitivity to Insulin. The application of these findings for developing new methods of screening novel Insulin mimetics and for evaluating the immunological responses has been discussed.

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.

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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