Abstract 4404: PAThway based RNA and DNA Integration with tumor Organoid Testing for clinical therapeutics (PATRIOT)
Authors: Drenner Kevin , Srivastava Shreyesi , Halder Kuntal , Basu Gargi , Halder Tithi Ghosh , Prabhu Abishta , Ampanattu Sherin Daniel , de Villar Ryan Rodriguez , Weston Alexis , Thode Trason , Royce Tom , Kaadige Mohan , Moser Justin , Guarnieri Carol , Steinbach Margaux , Richards Jacqueline , Tsai Frank , Sachdev Jasgit , Borazanci Erkut , Gordon Michael , Trent Jeffery , Soldi Raffaella , Sharma Sunil 
Published: September 2020

Purpose: Whole genome sequencing is utilized in the clinic with limited benefit. Only a fraction of patients sequenced will have a driver mutation that can be targeted by a drug. Treating with a targeted therapy often demonstrates a response but ultimately, resistance results in progression. Many of the common mutations, like KRAS, are currently not targetable and are only used to predict poor prognosis. The narrow clinical therapeutic potential of DNA sequencing shows a need for integration of other analyses like RNA and proteomics. RNA sequencing is currently used by some CLIA certified clinical reports to identify novel fusions in patient data but has not been completely integrated into the clinic. The primary roadblocks for complete RNA sequencing integration are due to batch effect, proper control tissue selection, tumor purity, and false positives or negatives. In this study we investigated the utility of using a cohort of normal control samples for Differential Expression (DE) analysis and the ability of RNA and DNA integration to identify new therapeutic targets (PATRIOT). This study also implements the use of patient-derived tumor organoids to test and validate the therapeutic targets using multi-omic analysis.

Experimental Design: PATRIOT utilizes sequencing from 22 different normal tissue controls. The cut off for significant gene expression is a fold change greater than 1.5 in 50 percent of the controls and a standard deviation less than the fold change. Top 1000 overexpressed and top 1000 underexpressed genes were input into Qiagen's Ingenuity Pathway Analysis (IPA) to identify canonical pathways, upstream regulators, and causal networks. Literature searches identified potential single and combination drugs to use on the tumor organoids. Organoids were generated by dissociating the patient tumor sample and immortalizing the primary cells using hTERT. Drug dosing on organoids was performed using a 6 point 4 fold dilution. IC50 was calculated using Graphpad PRISM and synergy was calculated using Calcusyn.

Results: PATRIOT analysis identified potential therapeutic targets, aberrant pathways, and ranked targetable mutations. These drugable targets were validated on the tumor organoids. Organoids were successfully generated with limited tissue in a therapeutic window. In a melanoma patient with an IDH1(R132C) and NRAS(Q61L) mutation, PATRIOT analysis predicted inactivation of NRAS and also predicted IDH1 mutation induced reduction of expression of DNA damage repair genes. The tumor organoids showed limited response to Trametinib and Everolimus while Olaparib exhibited a large response.

Conclusion: Our data suggests that PATRIOT analysis using a cohort of normal control samples can generate reliable DE data. PATRIOT methodology could expand the therapeutic potential for patients with direct in vitro results.

This study was supported by funding from Flinn Foundation grant #2193.

Citation Format: Kevin Drenner, Shreyesi Srivastava, Kuntal Halder, Gargi Basu, Tithi Ghosh Halder, Abishta Prabhu, Sherin Daniel Ampanattu, Ryan Rodriguez de Villar, Alexis Weston, Trason Thode, Tom Royce, Mohan Kaadige, Justin Moser, Carol Guarnieri, Margaux Steinbach, Jacqueline Richards, Frank Tsai, Jasgit Sachdev, Erkut Borazanci, Michael Gordon, Jeffery Trent, Raffaella Soldi, Sunil Sharma. PAThway based RNA and DNA Integration with tumor Organoid Testing for clinical therapeutics (PATRIOT) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4404.