Carboplatin and paclitaxel induced-gonadotoxicity on the ovarian reserve of young breast cancer patients with BRCA1 mutation

Abstract

Study question: Are ovarian tissue fragments from patients with BReast CAncer gene 1 (BRCA1)-mutated breast cancer (BC) more sensitive to carboplatin and/or paclitaxel exposure compared to those from non-mutated patients with BC?

Summary answer: Carboplatin and paclitaxel treatment showed similar gonadotoxicity, irrespective of the genetic background.

What is known already: Studies have shown that mutations of BRCA1 gene negatively impact the ovarian reserve due to defects in DNA repair mechanisms. As a result, patients with BRCA germline mutations might be more vulnerable to chemotherapy-induced gonadotoxicity. Carboplatin and paclitaxel are known moderately gonadotoxic drugs, but the impact of their combination on fertility remains unclear, particularly in BRCA-mutated patients.

Study design, size, duration: Cryopreserved ovarian tissue fragments from patients with BC, either carrying a BRCA1 germline mutation (n = 4) or not (n = 4), were exposed to chemotherapy using two models: (i) in vitro culture or (ii) in vivo xenotransplantation model. First, thawed ovarian tissue fragments were cultured for 3 days with carboplatin (10 µg/ml), paclitaxel (1 µM), carboplatin, and paclitaxel or vehicle (dimethyl sulfoxide). Next, ovarian tissue fragments from the same patients were xenografted into the peritoneum of immunodeficient mice, followed by 3-week injections with either carboplatin (50 mg/kg/week) and paclitaxel (10 mg/kg/every 3 days) or saline solution as a control.

Participants/materials, setting, methods: Ovarian cortex was processed for histological analyses to assess follicle activation and survival in both experimental models. Follicle counting and morphological assessment were performed to evaluate the rates of follicles at different developmental stages, as well as the rate of atretic follicles. Immunostainings were performed for follicle activation (KL and p-RPS6), apoptosis (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay), and DNA repair mechanisms (γH2AX, RAD51, and DNA PKcs).

Main results and the role of chance: While chemotherapy exposure did not significantly affect the proportion of primordial follicles in vitro, an increase in the proportion of quiescent follicles was observed after xenografting in the treated conditions compared to their respective controls, regardless of the presence of a BRCA mutation (BRCA+: 79.6 ± 5.07% versus 35.4 ± 8.26%, P = 0.0003; BRCA-: 81.8 ± 10.50% versus 17.9 ± 21.93%, P = 0.0014), reflecting the massive destruction of the pool of growing follicles. No difference was observed in the rate of atretic follicles, but the TUNEL assay revealed that chemotherapy, alone or in combination, increased DNA fragmentation rates (BRCA+: 37-49%; BRCA-: 43-55%) compared to the control conditions (BRCA+: 13-19%; BRCA-: 17-23%) both in vitro and in vivo. DNA repair mechanisms were affected following chemotherapy exposure, as evidenced by a significant increase in γH2AX-stained follicles in vitro (both populations) and in vivo (BRCA-mutated tissue) compared to the respective controls. Finally, chemotherapy had a similar impact on follicular atresia and apoptosis in both populations. However, BRCA-mutated tissue had lower rates of apoptotic (41% in BRCA+ versus 56% in BRCA-; P = 0.0184) and γH2AX-positive follicles (20% in BRCA+ versus 42% in BRCA-; P = 0.0142) than non-mutated fragments when exposed to carboplatin alone in vitro.

Large scale data: N/A.

Limitations, reasons for caution: Experiments on human tissue have limitations, particularly with rare material such as ovarian tissue from young BRCA-mutated patients donated for research. The study is limited by the small sample size and high intra- and inter-patient variability.

Wider implications of the findings: Studies on BRCA-mutated ovarian tissue are essential to better understand how BRCA mutations impact the ovarian reserve and respond to gonadotoxic treatments. Unexpectedly, this study suggested that follicles from young BRCA-mutated patients below 35 are not more sensitive to chemotherapy than the one from non-mutated patients, although DNA repair mechanisms seems to be differentially affected. Altogether, the results can help to provide appropriate fertility preservation counseling in BRCA1-mutated patients.

Study funding/competing interest(s): This study is funded by Télévie, a grant of the Fonds National de la Recherche Scientifique-FNRS (Grant Nos 7.4531.22 and 7.6509.24), the Fondation contre le Cancer (Grant No. FAF-C/2018/1274), and supported by Fonds Erasme.

Trial registration number: N/A.

Keywords: BRCA mutations; DNA damage; chemotherapy; fertility preservation; ovarian tissue.