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. 2018 May 1;29(5):1203-1210.
doi: 10.1093/annonc/mdy099.

RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer

C Cruz  1 , M Castroviejo-Bermejo  2 , S Gutiérrez-Enríquez  3 , A Llop-Guevara  2 , Y H Ibrahim  2 , A Gris-Oliver  2 , S Bonache  3 , B Morancho  4 , A Bruna  5 , O M Rueda  5 , Z Lai  6 , U M Polanska  7 , G N Jones  7 , P Kristel  8 , L de Bustos  2 , M Guzman  2 , O Rodríguez  2 , J Grueso  2 , G Montalban  3 , G Caratú  9 , F Mancuso  9 , R Fasani  10 , J Jiménez  10 , W J Howat  7 , B Dougherty  6 , A Vivancos  9 , P Nuciforo  10 , X Serres-Créixams  11 , I T Rubio  12 , A Oaknin  13 , E Cadogan  7 , J C Barrett  6 , C Caldas  14 , J Baselga  15 , C Saura  16 , J Cortés  17 , J Arribas  18 , J Jonkers  9 , O Díez  19 , M J O'Connor  20 , J Balmaña  21 , V Serra  22
Affiliations

RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer

C Cruz et al. Ann Oncol. .

Abstract

Background: BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity.

Patients and methods: We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi.

Results: RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor.

Conclusion: Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.

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Figures

Figure 1.
Figure 1.
Homologous recombination repair markers and PARPi response. (A) Antitumor activity of olaparib in gBRCA patient-derived tumor xenografts (PDXs). Best response to olaparib is plotted as the percentage of tumor volume change after at least 21 days of treatment. +20%, –30% and –95% are marked by dashed lines to indicate the range of CR (complete response), PR (partial response), SD (stable disease) and PD (progressive disease). Mut, mutation; B1, mutation in BRCA1; B2, mutation in BRCA2; Metastatic, PDX derived from a metastatic lesion (otherwise, derived from a primary tumor); TNBC, triple negative BC; ER+, estrogen receptor positive BC; OvCa, ovarian cancer. (B) Immunofluorescence staining of BRCA1, 53BP1 and RAD51 across the PARPi-sensitive and PARPi-resistant gBRCA PDX models. Detection of BRCA1 [with an antibody towards the N-terminus of BRCA1 (B1-NT) or C-terminus (B1-CT)], 53BP1 and RAD51 nuclear foci in olaparib-treated PDX models. CR, PD and SD are indicated. For BRCA1, the location of the mutation within the gene is indicated. DAPI staining is shown in blue. Green nuclei indicate geminin-positive cells (S/G2 phase of the cell cycle). (C) RAD51 nuclear foci formation discriminates PARPi-resistant tumors. Quantification of geminin positive cells with RAD51 nuclear foci detected by immunofluorescence in FFPE samples from tumors treated with vehicle (black bars) or olaparib (green bars). The graph displays mean ± SEM from three independent tumors. The association with PARPi-response is shown in the supplementary Table S1, available at Annals of Oncology online. Dark grey, CR; light gray, SD; white, PD. For RAD51, dark gray means high RAD51 score; light gray, intermediate RAD51; white, low RAD51. Expression of hypomorphic BRCA1 isoforms and loss of 53BP1 is depicted in dark gray. (D) RAD51 in patients’ tumors is associated with PARPi clinical response. IF of RAD51 and geminin in the pretreatment setting using a pretreatment tumor sample (or the most recent metastatic sample). Samples from three PARPi-resistant patients (Pt179, skin metastasis of TNBC; Pt183, dermal lymphatic carcinomatosis of ovarian cancer; Pt034, lymph node metastasis of ER+ BC) and four PARPi-sensitive patients (Pt310pre, liver metastasis of ER+ BC; Pt124pre, primary TNBC; Pt280, peritoneal implant of ovarian cancer; Pt04, lymph node metastasis TNBC) are shown. For acquired resistance, samples obtained from three patients at PARPi progression (Pt310post, liver metastasis; Pt124post, skin metastasis; Pt201, skin metastasis of ER+ BC) are shown. Empty arrowheads show geminin-positive cells devoid of RAD51 nuclear foci. Solid arrowheads indicate RAD51/geminin-positive cells. DAPI staining is shown in blue. (E) Quantification of RAD51/geminin-positive cells from tumor samples shown in panel D. Pt183 was not scored as the tumor did not contain 100 geminin-positive cells. Unpaired t test: *P < 0.05; **P < 0.01.
Figure 2.
Figure 2.
Cisplatin or ATM inhibition overcomes PARPi resistance. (A) Relative tumor volume (RTV) of vehicle, cisplatin or olaparib in PDX196, PDX280 and PDX252. Cisplatin was administrated 6 mg/kg weekly unless RTV < 0.5. Olaparib was administrated daily at 50 mg/kg (5 doses/week). Number of tumors per arm is indicated. (B) RTV of vehicle, cisplatin, olaparib or its thereof combination in PDX236 and PDX274. Cisplatin and olaparib were administrated as in panel A. (C) RTV of vehicle, olaparib, AZD0156 or the combination of treatments in PDX127, STG316 and PDX280. Olaparib was administrated daily at 50 mg/kg (5 doses/week) and AZD0156 was administered three times per week at 2 or 2.5 mg/kg. (D) Quantification of pan-nuclear γH2AX-positive cells in PDX127, STG316 and PDX280 treated with vehicle, olaparib, AZD0156 or the combination of drugs at the end point of experiments shown in panel C. All figures show mean and SEM. Statistical P-values are shown when relevant: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-way ANOVA).
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