Abstract 6110: Multi-site evaluation of FFPE homologous recombination deficiency reference materials
Authors: Lowe Dana Ruminski , Whiting Robert M. , Butler Matthew G. , Konigshofer Yves , Huang Catherine , Chivukula Indira , Nahlik Krystyna , Xia Dianren , Garlick Russell , Anekella Bharathi 
Published: April 2023

Homologous recombination deficiency (HRD) arises when defects in DNA repair pathways occur, leading to genomic instability. HRD status is an emerging therapeutic biomarker; NGS assays that measure it can be used to stratify ovarian and breast cancer patients and determine eligibility for clinical trials, and PARP inhibitor- and platinum-based therapies. Seraseq FFPE HRD reference materials were developed to cover a range of genomic instability scores (GIS) to help NGS HRD assay validation and development. Here we discuss a multi-site evaluation across various platforms of our high-positive, low-positive, and negative HRD reference materials.

Tumor cell lines were characterized by sequencing and evaluated in collaboration with several IVD partners. Three breast cancer cell lines (along with their SNP-matched normal cell lines) were selected based on their GIS. Tumor cells were blended with their SNP-matched normal cells to achieve ~65% tumor content. Biosynthetic DNA containing mutations in homologous recombination repair (HRR) genes (ATM, BRIP1, RAD51C, RAD51D) were added to the high-positive and negative reference materials targeted at &gt;5% variant allele frequency (VAF) and measured by digital PCR. Formalin fixed paraffin embedded (FFPE) blocks were made and each block was tested for yield per curl using both the Qiagen QIAamp DNA FFPE Tissue Kit and the Maxwell RSC DNA FFPE Kit for extraction and Qubit dsDNA HS kit for concentration analysis. DNA quality was assessed using an Agilent gDNA ScreenTape Assay for the TapeStation. Whole genome shotgun sequencing was performed on extracted DNA using LGC NxSeq AmpFREE Low DNA Library Kit and Roche KAPA UDI adapters and Illumina NextSeq 2000 P1 flow cell. HRD status was evaluated by external collaborators with multiple NGS and microarray assays, including the Illumina TSO500 HRD RUO assay, the SOPHiA DDM HRD Solution and the OncoScan CNV Array

A breast cancer cell line with a GIS of ~75 was selected as the HRD high-positive reference material, a second breast cancer cell line with a GIS of ~60 was selected as HRD low-positive, and a third breast cancer cell line with a GIS of ~30 was selected as HRD negative. Representative DNA yields per 10-micron section (determined using the HRD positive FFPE curls extracted by Qiagen QIAamp method) were 165 ± 28 ng/curl. Digital PCR confirmed the presence of the 8 HRR mutations (in 4 genes) at levels &gt;5% VAF. GIS varied for each material across assays, but HRD status was consistent, confirming the wide applicability of the new reference materials

We have developed the Seraseq HRD reference materials to meet the needs of laboratories looking to analyze HRD in cancer patient samples. These reference materials facilitate standardization and quality control in HRD testing by clinical labs for current and new PARP inhibitor treatment stratification in expanded patient populations that may include those with WT BRCA1/2 genes

Citation Format: Dana Ruminski Lowe, Robert M. Whiting, Matthew G. Butler, Yves Konigshofer, Catherine Huang, Indira Chivukula, Krystyna Nahlik, Dianren Xia, Russell Garlick, Bharathi Anekella. Multi-site evaluation of FFPE homologous recombination deficiency reference materials. [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 6110.