BRCA1/2 genes play a critical role in multiple cellular processes governing genome stability including DNA repair. In addition to suppressing tumor growth, BRCA1/2 genes may play a role in the maintenance of cardiomyocyte survival and function (1). In animal models, loss of cardiomyocyte-specific BRCA1/2 is associated with DNA damage, apoptosis, cardiac dysfunction, and cardiac mortality following doxorubicin exposure (1, 2). BRCA1/2 genes may potentially mitigate against anthracycline-induced genotoxic stress and cardiomyocyte apoptosis and thus serve a cardioprotective role. However, whether these preclinical findings translate to humans is unclear (3–5).

In a single-center, cross-sectional study, we investigated differences in cardiac function and cardiopulmonary fitness through comprehensive phenotyping of breast cancer survivors with and without BRCA1/2 mutations. Furthermore, we performed an in vitro study using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to assess the impact of BRCA1 loss on cardiomyocyte survival following doxorubicin exposure.

The mean (SD) age of the 67 breast cancer survivors in the study cohort was 50 (11) years, 87% were White and 64% had stage II/III disease. The median (Q1, Q3) time from diagnosis at enrollment was 6 (3, 7) years. Table 1 summarizes baseline characteristics according to BRCA1/2 status and doxorubicin exposure.

Participants were assessed at a median (Q1, Q3) of 4 (2, 6) years after completion of chemotherapy. The age-adjusted left ventricular ejection fraction (LVEF) was significantly lower in participants treated with doxorubicin, regardless of BRCA1/2 mutation status (p = 0.03). In doxorubicin-treated BRCA1/2 mutation carriers and non-carriers, estimated differences were lower by 5.4% (95% CI; −9.3, −1.5) and 4.8% (95% CI; −9.1, −0.5), respectively, compared to carriers without doxorubicin exposure. These findings were consistent across additional cardiac function measures including circumferential and longitudinal strain, although less pronounced for the latter. There were no differences in diastolic function measures E/A, e', and E/e' (Figure 1, Supplementary Table 1). These findings remained consistent in a sensitivity analysis excluding 6 participants who had received HER2-targeted therapy (Supplementary Table 2).

Among CPET measures, the age-adjusted resting heart rate was significantly higher in the doxorubicin-treated groups regardless of BRCA1/2 status. However, we did not find significant differences across the three groups in VO_2max, peak heart rate or peak respiratory exchange ratio (Figure 1, Supplementary Table 1). Similar findings were observed in a sensitivity analysis excluding participants who received HER2-targeted therapy (Supplementary Table 2). We also performed additional sensitivity analysis comparing echocardiography and CPET measures across the groups using a non-parametric test (i.e., Kruskal-Wallis test) and the findings were largely similar.

In vitro, doxorubicin caused a dose-dependent reduction in cell viability with no differences between BRCA1 mutant and wild type iPSC-CMs (p>0.05). Estimates of cell viability (doxorubicin concentration) in BRCA1 mutant compared with wild type iPSC-CMs were 97.3 vs. 92.4% (1 nM), 91.9 vs. 96.7% (10 nM), 36.0 vs. 34.0% (50 nM), 4.4 vs. 4.1% (100 nM), and 4.1 vs. 4.1% (500 nM) (Figure 2).

Overall, our results suggest that women with breast cancer who have BRCA1/2 mutations are not at increased risk of anthracycline-induced cardiotoxicity relative to those with sporadic breast cancer. This is based on several lines of evidence. First, although we observed significantly lower left ventricular systolic function in breast cancer survivors treated with doxorubicin compared to those without doxorubicin exposure, we did not find differences in age-adjusted estimates of echocardiography-derived measures of systolic or diastolic dysfunction according to germline BRCA1/2 mutation status. Second, there were no significant differences in cardiopulmonary fitness measures as determined by CPET based on BRCA1/2 status. Third, complementary in vitro experiments showed a comparable dose-dependent reduction in cell viability in both loss of function BRCA1 mutant and wild type iPSC-CMs receiving doxorubicin.

BRCA1/2 mutations may be associated with increased risk of doxorubicin cardiotoxicity, but human data are limited. One prior exploratory study of 401 patients, including 232 BRCA1 and 159 BRCA2 mutation carriers, showed an increased risk of heart failure based on self-reported symptoms elicited on an anonymous survey, relative to historical controls drawn from the general population (3). In this study, however, the authors were unable to verify reported symptoms using objective confirmatory data such as echocardiogram reports in most participants, and there was no direct comparator control group. In contrast, two other studies found no significant differences between rates of cardiomyopathy in BRCA1/2 mutation carriers vs. wild type controls receiving anthracyclines, though each had limitations (4, 5). One prospective study was underpowered to assess for differences in cardiac dysfunction between groups, excluded participants with hypertension or those who received trastuzumab, and did not demonstrate expected LVEF declines among BRCA1/2 mutation carriers receiving anthracyclines (4). A second retrospective study only evaluated the incidence of either asymptomatic decline in LVEF to <50% or heart failure and lacked detailed assessment of subclinical measures of cardiovascular function (5). Only a minority of participants included in the study underwent follow-up LVEF assessment after completion of anthracycline therapy limiting the ability to detect asymptomatic declines in cardiac function. Our study fills an important evidence gap by comprehensively characterizing cardiac function using both quantitative echocardiography and CPET and performing complementary in vitro experiments using iPSC-CMs.

Our human data contrast with the results of murine studies, where loss of BRCA1/2 in cardiomyocytes was associated with worse cardiac function and increased mortality following doxorubicin exposure (1, 2). There are several possible explanations for this. First, significant differences exist in the physiology of human and murine cardiomyocytes including calcium cycling, expression of ion channels, energetics, and myofilament composition (7). Second, cardiomyocyte specific BRCA1/2 knockouts in mice are biologically distinct from inherited germline BRCA1/2 mutations in humans. Third, mice used in preclinical studies were either exclusively male or the sex was not disclosed and administered relatively higher anthracycline doses compared to standard chemotherapy dosing regimens, potentially contributing to discrepancies in results (1, 2).

Our study has limitations. Though the study is one of the few studies to date to assess the impact of BRCA1/2 mutations on detailed measures of cardiac function in breast cancer patients receiving anthracyclines, statistical power was limited due to sample size. Our analyses were adjusted for age alone given the relatively small sample size, and confounding remains possible. Furthermore, limitations related to unequal group sizes should be considered. Our in vitro experiments do not incorporate hemodynamic or neurohormonal stressors inherent to in vivo studies, which may diminish observed differences, particularly with respect to BRCA1 status (8). In addition, we focused on cell viability in the in vitro study, and other measures related to iPSC-CM structure and function were not evaluated.

In conclusion, we present both detailed phenotypic characterization of cardiac function, including echocardiography and CPET, in breast cancer survivors with and without BRCA1/2 mutations treated with anthracyclines, and in vitro characterization using anthracycline-treated, wild type vs. gene-modified human iPSC-CMs with a loss of function mutation in BRCA1. Overall, we found no strong evidence to support associations between BRCA1/2 mutations and anthracycline-induced cardiac dysfunction based on echocardiography, CPET or in vitro data. Our study fills an important evidence gap and adds support to the lack of increased cardiotoxicity risk in breast cancer patients with BRCA1/2 mutations.

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the University of Pennsylvania Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.

BK, PS, and AS contributed to conception and design of the study. BK, AS, KS, and KMS contributed to clinical data collection. WL, CM-R, BK, and KM contributed to the design and execution of the in vitro study. BD and BK performed statistical analysis. BD, NW, and BK wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the NIH under Award Number UL1TR001878 and R01HL118018 (BK) and R35HL145203 (KM). KMS was supported by grant from the National Center for Advancing Translational Sciences (5UL1TR002014 and 5KL2TR002015).

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