Abstract 231: Evaluation of a 293 gene oncology-focused exome sequencing panel for screening of ovarian cancer patients for BRCA mutations as a prerequisite for autologous cellular immunotherapy manufacturing
Authors: Willoughby David , Bognar Ernie , Shen Weiming , Nejedlik Laura , Nagel Casey , Pruthi Aman , Zhang Peter , Walter Adam , Zupanc Heidi , Jay Chris , Neumunaitis John 
Published: June 2023

Gemogenovatucel-T is a cellular immunotherapy manufactured from harvested tumor tissue transfected with a plasmid which encodes granulocyte macrophage colony stimulating factor (GM-CSF) and a short harpin RNA which specifically reduces the expression of furin and downstream targets TGF-β1, and TGF- β2. In the Phase IIB VITAL study involving stage 3/4 ovarian cancer patients, recurrence-free survival and overall survival in patients who were BRCA1/2 wild-type was improved in patients receiving Gemogenovatucel-T as compared to placebo. The same improvements in recurrence-free and overall survival were not observed in patients with pathogenic or likely pathogenic mutations in BRCA1/2 genes. Similar improvements were demonstrated in patients who were homologous recombination proficient.

For future clinical trials it is necessary to rapidly examine genomic DNA from blood of ovarian cancer patients to determine BRCA mutation status for eligibility to manufacture autologous Gemogenovatucel-T. An oligonucleotide hybridization-based capture panel targeting the coding exons and adjacent intronic regions of 293 oncology-related genes was developed for use in enriching DNA libraries molecules for sequencing on the Illumina platform. The purpose of the current study was to evaluate the utility of this panel for detection of likely pathogenic and pathogenic mutations in the BRCA1/2 genes.

Genomic DNA from Gemogenovatucel-T preparations from the Phase IIB trial including 10 patients harboring known mutations in the BRCA1/2 genes and 2 BRCA wild type patients was processed through library preparation, hybridization-based capture using the 293 gene probe set, and sequencing on the Illumina NextSeq 2000. All eleven previously identified pathogenic mutations in BRCA1/BRCA2 gene, including 8 short insertions/deletions (INDELs) and 3 single nucleotide polymorphisms (SNPs) were detected with the method. No false positive pathogenic BRCA mutations were detected in any of the 12 samples. The panel was evaluated further using a set of reference genomic DNAs from Horizon Discovery containing known polymorphisms in BRCA as well as 30 other oncology-related genes. 100% of the expected SNPs and INDELs with an allele frequency of &gt;3% were detected; no false positive likely pathogenic or pathogenic polymorphisms with an allele frequency of &gt;2.5% were detected in the genomic DNA of healthy control patients.

The 293 gene panel will be suitable for identification of BRCA mutations in patients to screen patients eligible for Gemogenovatucel-T manufacturing. The entire process starting with blood DNA extraction and ending with report generation can be completed in about 3.5 days. The results show that a comprehensive oncology-focused sequencing panel can be an effective tool for focused gene-specific variant analysis.

Citation Format: David Willoughby, Ernie Bognar, Weiming Shen, Laura Nejedlik, Casey Nagel, Aman Pruthi, Peter Zhang, Adam Walter, Heidi Zupanc, Chris Jay, John Neumunaitis. Evaluation of a 293 gene oncology-focused exome sequencing panel for screening of ovarian cancer patients for BRCA mutations as a prerequisite for autologous cellular immunotherapy manufacturing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 231.