Evaluation of homologous recombination repair (HRR) status in metastatic prostate cancer by next-generation sequencing and functional tissue-based immunofluorescence assays.
Authors: Cresta Morgado Pablo , Arce-Gallego Sara , Delgado-Serrano Luisa , Simonetti Sara , González Macarena , Marmolejo Castañeda David Humberto , Morales-Barrera Rafael , Planas Jacques , Romero-Lozano Paula , Maldonado Xavier , Suárez Cristina , Figols Mariona , Cros Sara , Dienstmann Rodrigo , Nuciforo Paolo , Vivancos Ana , Llop-Guevara Alba , Carles Joan , Serra Violeta , Mateo Joaquin 
Published: June 2024

5024

Background: Metastatic prostate cancer (mPC) is enriched for HRR gene alterations; these biomarkers have prognostic and predictive value. Next-generation sequencing (NGS) allows for patient stratification, but widespread clinical implementation is still limited. Moreover, not all mutations in HRR genes result in functional HRR loss in the tumor. We investigated the correlation between genomic and functional loss of HRR, using NGS and an optimized RAD51 immunofluorescence (RAD51-IF) assay in mPC biopsies. Methods: Observational study including patients with mPC. Either primary tumor or metastatic biopsies underwent NGS (custom VHIO-300 targeted panel (Panel) and/or whole-exome sequencing (WES)), and RAD51-IF from FFPE tissue specimens. A previously defined threshold of 10% RAD51-IF positive cells was used to defined HRD based on RAD51-IF. Genomic scars (LOH, LST, NtAI, and HRD-sum) were obtained from Panel and WES data. Clinical data was extracted from electronic patient records. Results: 219 tumor tissues from 187 patients were acquired, including primary (151/219) and metastatic (68/219) cases collected either in the hormone-sensitive (HSPC) (169/219) or castration-resistant (CRPC) (50/219) setting. Genomic profiling was obtained for 181/219 samples (Panel n=139, WES n=80, both n=38). Gene alterations were common in TP53 (40%), PTEN (14%), AR (15%), MYC (10%), BRCA2 (9%), ATM (8%) and BRCA1 (2%). Tissue for RAD51-IF was available for 206 samples; of those, 140/206 (68%) were evaluable for RAD51-IF. The median RAD51-IF score was 28.5. 21% samples had RAD51-low results compatible with HRR deficiency (HRD). No RAD51-IF score differences were seen between primary/metastatic tumors (p=0.7) nor HSPC/CRPC (p=0.49). Sample matched RAD51-IF and genomics data were obtained for 128 biopsies (117 patients). BRCA1/2 alterations associated with lower RAD51-IF scores (median 3.5, IQR 9.8 – 8.5 for BRCA1/2 altered vs median 29.7, IQR 19.0 - 44.5 for BRCA1/2-WT), resulting in high sensitivity (71%) and specificity (85%) to identify cases with BRCA1/2 alterations (sensitivity 68% and specificity 87% when considering a larger set of HRR genes. RAD51-IF was able to classify as HRR proficient BRCA1/2 altered cases after secondary resistance to platinum or with retained BRCA1 expression by IF. Based on HRD-sum (HRD>=42) 27.5% and 20.1% cases were classified as HRD on WES and Panel, respectively. CRPC samples were more likely to be classified as HRD-sum “high” (OR 4.07 WES, OR 5.21 targeted panel) HRD-sum was significantly associated with BCRA1/2 (Panel, p= 0.004; WES, p=0.002), and with RAD-IF low for Panel ( p=0.021) and for WES once adjusted by castration-sensitivity status (p=0.03). Conclusions: RAD51-IF is feasible in clinical samples from mPC patients and associates strongly with clinically relevant HRR gene alterations.