In-house testing for homologous recombination repair deficiency (HRD) testing in ovarian carcinoma: a feasibility study comparing AmoyDx HRD Focus panel with Myriad myChoiceCDx assay

Summary Background Homologous recombination repair (HRR) is the main mechanism of repair of DNA double-strand breaks. Its deficiency (HRD) is a common feature of epithelial ovarian cancers (EOCs). BRCA1/2 mutations and/or other aberrations in genes of HRR are well known causes of HRD and genomic instability. Poly ADP-ribose polymerase inhibitors (PARPi) have revolutionized the management of BRCA mutant EOCs and demonstrated activity in HRD tumor cells. Determining HRD status can provide informations on the magnitude of benefit for PARPi therapy. Myriad MyChoice CDx is a next generation sequencing- based in vitro diagnostic test that assesses the Genomic Instability Score (GIS) which is an algorithmic measurement of loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions using DNA isolated from formalin-fixed paraffin embedded tumor tissue specimens. However Myriad MyChoice CDx, is a centrally performed and costly assay, with no reimbursement scheduled, at least in Italy. Methods In this report, we described our experience in performing the HRD Focus AmoyDx (Amoy Diagnostics Ltd, Xiamen, Fujian, China) on the same samples of EOCs evaluated with Myriad MyChoiceCDx assay. Results The overall percent agreement between AmoyDx and Myriad was 87.8% (65 of 74 tumors tested). All the 36 AmoyDx negative cases were confirmed to be negative by Myriad (negative predictive value, 100%). Conclusions The concordance of the results with the gold standard Myriad MyChoice CDx assay suggest the feasibility and reliability of HRD testing in diagnostic laboratories with high-throughput NGS platforms and qualified personnel.


Introduction
Ovarian cancer represents the 3% of all cancers occurring in female and is the sixth cause of cancer-related death in women worldwide, globally accounting for 294,000 new cases and 198,000 deaths per year 1 and 5000 new cases and more than 3000 deaths per year in Italy 2 . The high grade serous ovarian carcinoma (HGSOC) is the most frequent (about 70%) and lethal ovarian cancer histotype. The large majority of HGSOC are diagnosed in advanced stage (FIGO stage III-IV) and despite cytoreductive surgery associated with platinum-based chemotherapy, will relapse within two years 3 . The introduction of PARP inhibitors (PARPi) in first-line therapeutic regimens of women with platinum-sensitive ovarian cancers has dramatically changed clinical outcomes, both in terms of progression free and overall survival [4][5][6][7] . Except for BRCA1/2 mutated cancers, which present the higher magnitude of clinical benefit for PARPi, this class of drugs show great efficacy also in BRCA wild type tumors with homologous recombination repair deficiency (HRD) [6][7][8] . Clinically meaningful improvements reported in recent trials 6,7 have lead to the approval of PARPis alone or in combination with antiangiogenetic therapy for the maintenance treatment of patients with HRD-positive advanced ovarian cancer (Food and Drug Administration 9 and European Medicines Agency 10 in 2020, and Agenzia Italiana del Farmaco 11 in 2022). As recently reported in European expert consensus recommendations 12 , BRCA1/2 tumor assessment should be associated with the evaluation of homologous recombination repair (HRR) status, as a pivotal step to extend effective PARPi treatment to the largest number of patients, considering that about 20-25% of HGSOCs harbor BRCA1/2 alterations and more than 50% are characterized by HRD 13 . The challenging topic is how to evaluate HRD in routine clinical practice. In the clinical trials PAOLA1 7 , PRIMA 6 and VELIA 8 , HRD assessment was performed with the FDA approved myChoiceCDx (Myriad) assay, which considered BRCA1 and BRCA2 status and HRD-induced genomic scar. However, this assay is centrally performed, costly and not reimbursed by the National Healthcare System. Next generation sequencing panels evaluating HRR genes, beyond BRCA1/2, may improve the detection rate of tumour with HRD by only 5-6% 13 . Commercial assays applicable in diagnostic laboratories that screen for HRR genes along with genomic scar have been recently developed 14 .
In this study, we report our first experience with inhouse HRD testing, using the HRD Focus panel (AmoyDx), which evaluates both BRCA1/2 status and genomic instability. We performed a double-blind evaluation of HRD status in a consecutive series of high grade epithelial ovarian cancers that were analyzed in our laboratory with HRD Focus panel and sent to Myriad for MyChoiceCDx testing. We aimed: i) to evaluate the feasibility of HRD testing with an assay applicable in a diagnostic clinical setting; ii) to compare HRD assessment obtained with the HRD Focus panel and the reference assay myChoiceCDx.

Study cohort
This single-institution study obtained specific Review Board approval (UID 2386). From the institutional electronic database, we selected all patients with high grade serous and endometrioid ovarian cancer treated at the European Institute of Oncology (IEO), Milan, Italy who underwent molecular analysis of HRR deficiency between April 2021 and April 2022. Demographic, clinicopathological and surgical characteristics were abstracted from clinical records. First line therapy indications were considered according with EMA criteria. Study data were collected and managed using REDCap (Research Electronic Data Capture) electronic data capture tools 15 .

hrd teSting
In-house HRD evaluation was performed with the HRD Focus Assay (CE-IVD) provided by AmoyDx (AmoyDx, Xiamen, China), following the manufacturer's instructions. Briefly, 100 ng of DNA (50 ng minimum yield request) extracted from representative formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks were used for library preparation, and then sequenced on Illumina NextSeq platform. This assay allowed the simultaneous analysis of SNVs and indels in the whole coding regions and exon-intron boundaries of BRCA1 and BRCA2, and estimated a genomic scar score (GSS) based on the analysis of 24,000 SNPs 16 . A GGS equal or higher than 50 was indicative of HRD positivity. The bioinformatic algorithm applied for the NGS data analysis was the version 1.1. The same FFPE tumor blocks used for AmoyDx evaluation were sent to Myriad, for performing myChoi-ceCDx assay. BRCA1 and BRCA2 status was evaluated along with HRD assessment, measured by a genomic instability score (GIS) score encompassing measured by loss of heterozygosity, telomeric allelic imbalance) and large-scale state transitions across the entire genome. A GIS equal or higher than 42 was indicative of HRD positivity.

StatiStical analySiS
Statistical analysis was carried out using SPSS Statistic 25 software. Chi-Square test with Yates correction and t-test calculators were used for data comparison of categorical variables. p-values < 0.05 were considered statistically significant.

Results
Among the 101 ovarian cancer patients referred to the Clinical Unit of Oncogenomics for HRD analysis, 6 were excluded from the current analysis as they had already undergone BRCA testing externally and were evaluated with Myriad myChoiceCDx only. The remaining 95 cases reached the minimum tumor cellular content required for AmoyDx (above than 30%) and Myriad (above than 20%) and were included in the present study. The most representative FFPE tumor block was sent to Myriad after section cutting (6 sections 5 µm-thick) for AmoyDxHRD testing (Fig. 1). The clinicopathological characteristics of this population are reported in Table I.

amoydx hrd focuS panel reSultS
95 cases underwent DNA extraction,obtaining a median concentration of 38.6 ng/µl (range 1.1-99.7 ng/ µl).In 84 cases (88.4%) DNA was extracted from surgical specimens, while in 11 cases (11.6%) DNA was extracted from biopsies.15 of 95 (15.8%) samples, including 5 biopsies and 10 surgical specimens, were not adequate for analysis with AmoyDx HRD Focus panel due to the low DNA yield and were addressed

Discussion
In the last years, the clinical management of women affected by ovarian cancers has been through a rapid evolution, prompted by progress in precision medicine. The recently introduced HRD assessment beyond BRCA1/2 status as a clinically relevant biomarker for therapeutic indications, poses a major challenge in hospital workflow as the gold standard assay for HRD evaluation. Myriad MyChoiceCDx is a centrally performed and costly assay, with no reimbursement scheduled, at least in Italy.
In this report, we describe our experience in with the HRD Focus AmoyDx (CE-IVD) in our diagnostic workflow, focusing on the feasibility and reliability of in-house HRD-testing. To our knowledge, this is one of the firsts report of feasibility of HRD-testing in a real-life diagnostic setting, evaluating a consecutive series of advanced carcinoma who may change their treatment indications. We observed a successful rate of 84.2%, with failures due to low extracted DNA yield, mainly related to small biopsies or DNA quality suboptimal, linked to formalin treatment and preanalytical condition that determined DNA deamination and fragmentation. Notably, the AmoyDx median TAT from the test request to the available report was 7 days, which was significantly shorter than the Myriad TAT of 18 days, the latter is also effected by logistic handling and transportation. Considering the clinical need to schedule the most effective therapy for the single patient in a short timeframe, both the successful rate and the TAT are crucial parameters to be taken under consideration to establish the clinical utility of an assay. Applying the HRD Focus panel, we identified 47.5% HRD positive tumors, in line with the incidence reported in the PAOLA1 (48%) 7 , PRIMA (50.9%) 6 and

funding
This work was partially supported by the Italian Ministry of Health with "RicercaCorrente" , "5 × 1000" .

ethical conSideration
All information regarding human material was managed using anonymous numerical codes, and all samples were handled in compliance with the Helsinki Declaration. The study received the European Institute Review Board approval: UID 2386. Informed consent was obtained from all subjects involved in the study.