Background Subclonal evolution during primary breast cancer treatment is largely unexplored. We aimed to assess the dynamic changes in subclonal composition of treatment-naïve breast cancers during neoadjuvant chemotherapy. Methods We performed whole exome sequencing of tumor biopsies collected before, at therapy switch, and after treatment with sequential epirubicin and docetaxel monotherapy in 51 out of 109 patients with primary breast cancer, who were included in a prospectively registered, neoadjuvant single-arm phase II trial. Results There was a profound and differential redistribution of subclones during epirubicin and docetaxel treatment, regardless of therapy response. While truncal mutations and main subclones persisted, smaller subclones frequently appeared or disappeared. Reassessment of raw data, beyond formal mutation calling, indicated that the majority of subclones seemingly appearing during treatment were in fact present in pretreatment breast cancers, below conventional detection limits. Likewise, subclones which seemingly disappeared were still present, below detection limits, in most cases where tumor tissue remained. Tumor mutational burden (TMB) dropped during neoadjuvant therapy, and copy number analysis demonstrated specific genomic regions to be systematically lost or gained for each of the two chemotherapeutics. Conclusions Sequential epirubicin and docetaxel monotherapy caused profound redistribution of smaller subclones in primary breast cancer, while early truncal mutations and major subclones generally persisted through treatment. Trial registration ClinicalTrials.gov, NCT00496795, registered on July 4, 2007.

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.