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Purpose: To determine the ability of RAD51 foci to predict platinum chemotherapy response in high-grade serous ovarian cancer (HGSOC) patient-derived samples. Experimental Design: RAD51 and γH2AX nuclear foci were evaluated by immunofluorescence in HGSOC patient-derived cell lines (n = 5), organoids (n = 11), and formalin-fixed, paraffin-embedded tumor samples (discovery n = 31, validation n = 148). Samples were defined as RAD51-High if >10% of geminin-positive cells had ≥5 RAD51 foci. Associations between RAD51 scores, platinum chemotherapy response, and survival were evaluated. Results: RAD51 scores correlated with in vitro response to platinum chemotherapy in established and primary ovarian cancer cell lines (Pearson r = 0.96, P = 0.01). Organoids from platinum-nonresponsive tumors had significantly higher RAD51 scores than those from platinum-responsive tumors (P < 0.001). In a discovery cohort, RAD51-Low tumors were more likely to have a pathologic complete response (RR, 5.28; P < 0.001) and to be platinum-sensitive (RR, ∞; P = 0.05). The RAD51 score was predictive of chemotherapy response score [AUC, 0.90; 95% confidence interval (CI), 0.78–1.0; P < 0.001). A novel automatic quantification system accurately reflected the manual assay (92%). In a validation cohort, RAD51-Low tumors were more likely to be platinum-sensitive (RR, ∞; P < 0.001) than RAD51-High tumors. Moreover, RAD51-Low status predicted platinum sensitivity with 100% positive predictive value and was associated with better progression-free (HR, 0.53; 95% CI, 0.33–0.85; P < 0.001) and overall survival (HR, 0.43; 95% CI, 0.25–0.75; P = 0.003) than RAD51-High status. Conclusions: RAD51 foci are a robust marker of platinum chemotherapy response and survival in ovarian cancer. The utility of RAD51 foci as a predictive biomarker for HGSOC should be tested in clinical trials.

Background The OlympiAD trial evaluated the PARP inhibitor (PARPi) olaparib versus a non-platinum standard chemotherapy in HER2-negative metastatic breast cancer (MBC) patients with a germline BRCA1/2 (gBRCA) mutation. Olaparib resulted in improved progression-free survival (PFS) and doubled the response rate vs chemotherapy. Nevertheless, the response rate to PARPi is 60% in the gBRCA1/2 population with MBC (current approval), suggesting a limited positive predictive value of gBRCA1/2 status. Moreover, patients with other relevant Homologous Recombination Repair defects (HRD) such as PALB2 or RAD51C/D mutation carriers, or HRD epigenetic silencing, are not captured with a gBRCA analysis. We have previoulsy shown that the functional HRD biomarker RAD51, tested in FFPE tumor samples using an optimized immunofluorescence-based assay, is associated with platinum response in early TNBC and PARPi response in preclinical BC models. We hypothesize that the RAD51 test would help to expand the clinical benefit of PARPi by predicting response to olaparib in MBC with germline/somatic BRCA1/2, PALB2 or RAD51C/D mutation and beyond. Study design RADIOLA is an open-label, single-arm, multicentre phase II study evaluating treatment with olaparib in male or female ≥18 years patients with HER2-negative MBC with ≤ two prior chemotherapy lines in two cohorts: cohort 1 (N=41) with known germline/somatic BRCA1/2, PALB2 or RAD51C/D mutation; cohort 2 (N=25) with functional HRD, namely RAD51-low score (≤10%), in wild-type/unknown mutation status at study entry. All patients will receive olaparib 300mg po BID until progression or unacceptable toxicity. Primary objective will assess, in terms of overall response rate (ORR), the capacity of the RAD51 score to predict olaparib efficacy in cohort 1. Secondary objectives include PFS, clinical benefit rate, duration of response, safety in both cohorts and ORR in cohort 2. Recruitment Recruitment (11 sites) started in March 2022. As of July 2022, 7 patients have been enrolled in Spain. Funding This study is financially supported by AstraZeneca. Citation Format: Judith Balmaña, Tomás Pascual, Alba Llop-Guevara, Pablo Tolosa, Isabel Blancas, Maria-Eva Perez-Lopez, Barbara Adamo, Iris Teruel, Jose Ponce, Marta Gonzalez, Gemma Viñas, Laura Lema, Francisco Javier Salvador, Mª Teresa Martinez, Alejandra Espinosa, Aleix Prat, Violeta Serra. SOLTI-1910: Predicting olaparib sensitivity in patients with unresectable locally advanced/metastatic HER2-negative breast cancer with BRCA1/2, PALB2, RAD51C/D mutations or HRD by the RAD51 test: RADIOLA TRIAL [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-11-02.

Background: Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and predictive of response to PARP inhibition in the primary setting (Eikesdal et al, Ann Oncol, 2021). However, the prevalence of HRD across breast cancer subtypes has not been established. Methods: Pretreatment tumor biopsies from 201 patients (32 TNBC and 169 non-TNBC) with primary breast cancer in the phase II PETREMAC trial (ClinicalTrials #NCT02624973) were examined. These samples underwent targeted cancer gene panel sequencing and BRCA1 promoter methylation analysis to assess HRD status defined by homologous recombination repair (HRR) gene mutations and/or BRCA1 promoter methylation. HRR genes included BRCA1, BRCA2, BRIP1, BARD1, and PALB2 by strict definition (HRR-S), and additionally ABL1, ATM, ATR, ATRX, BLM, CDK12, CHEK1, EMSY, ERCC4, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, MEN1, MRE11, NBN, PTEN, and SETD2 by wider definition (HRR-W). HRD strict (HRD-S) was defined as biallelic gene inactivation by HRR-S mutations or BRCA1 methylation. Finally, tumors underwent PAM50 gene expression subtyping and evaluation of functional HRD by RAD51 nuclear foci analysis, for which a low score has been associated with HRD. Results: HRD-S was present in 13% of the breast cancers (total: n= 27/201; TNBC: 15/32; 47%; non-TNBC: 12/169; 7%), whereas HRD-W (HRR-W or BRCA1 methylation) was observed in 29% (total: n=58/201; TNBC: 19/32; 59%; non-TNBC: 39/169; 23%). Among 190 tumors analyzed for PAM50 intrinsic subtype, HRD-S was detected in 3/60 and 4/48 (5% and 8%) of tumors classified as luminal A and B, respectively, 1/35 (3%) of HER2-enriched, 4/21 (19%) of normal-like, and 12/26 (46%) of basal-like tumors. Out of 58 non-TNBC biopsies examined by RAD51 staining, four (7%) were classified as HRD-S and all these were scored as RAD51 low. The remaining 54 non-TNBC samples were homologous recombination proficient, and none of these exhibited functional HRD by RAD51 low scores. All four HRD-S/RAD51 low tumors were hormone receptor-positive, HER2 negative, and belonged to the luminal A (n=1), luminal B (n=2), and basal-like (n=1) subtypes, with HRD caused by germline BRCA1 (gBRCA1), gBRCA2, somatic BRCA1 mutations and BRCA1 methylation, respectively. Conclusion: The prevalence of HRD across all breast cancer subtypes suggests that HRD analysis and therapy targeting such DNA repair defects should be tested in future clinical trials. Citation Format: Christina Engebrethsen, Synnøve Yndestad, Andrea Herencia-Ropero, Oleksii Nikolaienko, Olav Karsten Vintermyr, Reidun K. Lillestøl, Laura Minsaas, Beryl Leirvaag, Gjertrud Iversen, Bjørnar Gilje, Egil Blix, Helge Espelid, Steinar Lundgren, Jürgen Geisler, Liv Jorunn Vassbotn, Hildegunn S. Aase, Turid Aas, Alba Llop-Guevara, Violeta Serra, Per Eystein Lønning, Stian Knappskog, Hans Petter Eikesdal. Homologous recombination deficiency across subtypes of primary breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-04.

Mutations; Gastric cancers; PARP inhibitors Mutacions; Càncers gàstrics; Inhibidors de PARP Mutaciones; Cánceres gástricos; Inhibidores de PARP Despite negative results of clinical trials conducted on the overall population of patients with gastric cancer, PARP inhibitor (PARPi) therapeutic strategy still might represent a window of opportunity for a subpopulation of patients with gastric cancer. An estimated 7% to 12% of gastric cancers exhibit a mutational signature associated with homologous recombination (HR) failure, suggesting that these patients could potentially benefit from PARPis. To analyze responsiveness of gastric cancer to PARPi, we exploited a gastroesophageal adenocarcinoma (GEA) platform of patient-derived xenografts (PDX) and PDX-derived primary cells and selected 10 PDXs with loss-of-function mutations in HR pathway genes. Cell viability assays and preclinical trials showed that olaparib treatment was effective in PDXs harboring BRCA2 germline mutations and somatic inactivation of the second allele. Olaparib responsive tumors were sensitive to oxaliplatin as well. Evaluation of HR deficiency (HRD) and mutational signatures efficiently stratified responder and nonresponder PDXs. A retrospective analysis on 57 patients with GEA showed that BRCA2 inactivating variants were associated with longer progression-free survival upon platinum-based regimens. Five of 7 patients with BRCA2 germline mutations carried the p.K3326* variant, classified as “benign.” However, familial history of cancer, the absence of RAD51 foci in tumor cells, and a high HRD score suggest a deleterious effect of this mutation in gastric cancer. In conclusion, PARPis could represent an effective therapeutic option for BRCA2-mutated and/or high HRD score patients with GEA, including patients with familial intestinal gastric cancer. This work was funded by the Italian Association for Cancer Research (AIRC), IG 20210 and IG 27531 to S. Giordano; IG 23624 to F. Pietrantonio; IG 21770 to S. Corso. FPRC 5×1000 2015 Min. Salute “Strategy” to SG; Fondazione Piemontese per la Ricerca sul Cancro (FPRC) 5×1000 MS2017 PTCRC-intra 2020 to S. Giordano; Ricerca Locale Dept. Oncology 2021 to S. Corso; Italian Ministry of Health-Ricerca Corrente 2022–23. B. Pellegrino was supported by ESMO with a Clinical Translational Fellowship aid supported by Roche and received research grants from GOIRC. Fondazione CR Firenze to M. Benelli.

Cholangiocarcinoma (CCA) is usually diagnosed at advanced stages, with limited therapeutic options. Preclinical models focused on unresectable metastatic CCA are necessary to develop rational treatments. Pathogenic mutations in IDH1/2, ARID1A/B, BAP1, and BRCA1/2 have been identified in 30\\%–50\\% of patients with CCA. Several types of tumor cells harboring these mutations exhibit homologous recombination deficiency (HRD) phenotype with enhanced sensitivity to PARP inhibitors (PARPi). However, PARPi treatment has not yet been tested for effectiveness in patient-derived models of advanced CCA.We have established a collection of patient-derived xenografts from patients with unresectable metastatic CCA (CCA\_PDX). The CCA\_PDXs were characterized at both histopathologic and genomic levels. We optimized a protocol to generate CCA tumoroids from CCA\_PDXs. We tested the effects of PARPis in both CCA tumoroids and CCA\_PDXs. Finally, we used the RAD51 assay to evaluate the HRD status of CCA tissues.This collection of CCA\_PDXs recapitulates the histopathologic and molecular features of their original tumors. PARPi treatments inhibited the growth of CCA tumoroids and CCA\_PDXs with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1. In line with these findings, only CCA\_PDX and CCA patient biopsy samples with mutations of BRCA2 showed RAD51 scores compatible with HRD.Our results suggest that patients with advanced CCA with pathogenic mutations of BRCA2, but not those with mutations of IDH1, ARID1A, or BAP1, are likely to benefit from PARPi therapy. This collection of CCA\_PDXs provides new opportunities for evaluating drug response and prioritizing clinical trials. The authors would like to thank the patients and their families for their support. This work was supported by grants from the Fundaci o Marat o TV3 awarded to T. Macarulla, M. Mel e, and S. Peir o; BeiGene research grant awarded toT. Macarulla and S. Peir o; AECC (INVES20036TIAN), Ram on y Cajal investigator program (RYC2020-029098-I), Proyecto de IþDþi (PID2019-108008RJ-I00), and FERO Foundation grant awarded to T.V. Tian; Proyecto de Investigaci on en Salud from the Instituto de Salud Carlos III (ISCIII) (PI20/00898) awarded to T. Macarulla; FIS/FEDER from the Instituto de Salud Carlos III (ISCIII) (PI12/01250; CP08/00223; PI16/00253 and CB16/12/00449) awarded to S. Peir o; and Ram on y Cajal investigator program (RYC-2017-22249) awarded to M. Mel e. Q. Serra-Camprubí is a recipient of the Ph.D. fellowship from La Caixa Foundation (LCF/PR/PR12/51070001). A. LlopGuevara was supported by the AECC (INVES20095LLOP) and V. Serra by the ISCIII (CPII19/00033). E.J. Arenas was funded by the AECC (POSTD211413AREN).J. Arribas is funded by the Instituto de Salud Carlos III (AC15/00062, CB16/12/00449, and PI22/00001). This publication is based upon the work of COST Action CA18122, European Cholangiocarcinoma Network, supported by the COST (European Cooperation in Science and Technology, www.cost.eu), a funding agency for research and innovation networks. The authors would like to thank Dr. V.A. Raker for manuscript editing and Drs. N. Herranz and J. Mateo for scientific discussions. The authors acknowledge the infrastructure and support of the FERO Foundation, La Caixa Foundation, and the Cellex Foundation. Peer Reviewed "Article signat per 31 autors/es: Queralt Serra-Camprubí; Helena Verdaguer; Winona Oliveros; Núria Lupión-Garcia; Núria Lupión-Garcia;Alba Llop-Guevara; Cristina Molina; Maria Vila-Casadesús; Anthony Turpin; Cindy Neuzillet; Joan Frigola; Jessica Querol; Mariana Yáñez-Bartolomé; Florian Castet; Carles Fabregat-Franco; Carmen Escudero-Iriarte; Marta Escorihuela; Enrique J. Arenas; Cristina Bernadó-Morales; Noemí Haro; Francis J. Giles; Óscar J. Pozo; Josep M. Miquel ; Paolo G. Nuciforo; Ana Vivancos; Marta Melé; Violeta Serra ; Joaquín Arribas; Josep Tabernero; Sandra Peiró; Teresa Macarulla; Tian V. Tian"

Molecular testing; Ovarian cancer Proves moleculars; Càncer d'ovaris Pruebas moleculares; Cáncer de ovarios Background The detection of homologous recombination deficiency (HRD) can identify patients who are more responsive to platinum and poly ADP ribose polymerase inhibitors (PARPi). MyChoice CDx (Myriad) is the most used HRD test in ovarian cancer (OC). However, some limitations of commercial tests exist, because of the high rate of inconclusive results, costs, and the impossibility of evaluating functional resistance mechanisms. Patients and methods Two academic genomic tests and a functional assay, the RAD51 foci, were evaluated to detect HRD. One hundred patients with high-grade OC enrolled in the MITO16A/MaNGO-OV2 trial and treated with first-line therapy with carboplatin, paclitaxel, and bevacizumab were analyzed. Results The failure rate of the two genomic assays was 2%. The sensitivity in detecting HRD when compared with Myriad was 98.1% and 90.6%, respectively. The agreement rate with Myriad was 0.92 and 0.87, with a Cohen’s κ coefficient corresponding to 0.84 and 0.74, respectively. For the RAD51 foci assay, the failure rate was 30%. When the test was successful, discordant results for deficient and proficient tumors were observed, and additional HRD patients were identified compared to Myriad; sensitivity was 82.9%, agreement rate was 0.65, and Cohen’s κ coefficient was 0.18. The HRD detected by genomic assays and residual tumor at primary surgery and stage was correlated with progression-free survival at multivariate analysis. Conclusions Results suggest the feasibility of academic tests for assessing HRD status that show robust concordance with Myriad and correlation with clinical outcome. The contribution of the functional information related to the RAD51 foci test to the genomic data needs further investigation. This work was supported by funding from the AIRC [grant numbers IG 2016 – ID. 18921 and IG 2021 – ID. 25932 projects – P.I. SP and CO-2018-12367051 (Ministero della Salute) P.I SP]; Ricerca Corrente grant M2/7 from Ministero della Salute to DC, Ricerca Corrente from Ministero della Salute to SP. SM is supported by the Italian Association for Cancer Research [grant number IG-2017 n: IG19997]. MITO16A/MaNGO-OV2 trial was partially supported by Roche. AL is a recipient of a grant from the Asociación Española contra el Cáncer (AECC) [grant number INVES20095LLOP]. VS is a recipient of a grant from the Instituto de Salud Carlos III [grant number CPII19/00033] and a European grant for personalized medicine [grant number ERAPERMED 2019-215]. BP is a recipient of a grant from GOIRC. BP was supported by ESMO with a Clinical Translational Fellowship aid supported by Roche. Any views, opinions, findings, conclusions, or recommendations expressed in this material are those solely of the authors and do not necessarily reflect those of ESMO or Roche. NC has received funding from AstraZeneca (to the institution). FP has received funding from Roche, AstraZeneca, Pfizer, Merck Sharp & Dome, Bayer, Incyte, Taiho Oncology, Janssen Cilag, Exelixis, Aileron, and Daiichi Sankyo (grants to the institution for clinical trial activities).