Unraveling the clinicopathological and molecular changes induced by neoadjuvant chemotherapy and endocrine therapy in hormone receptor-positive/HER2-low and HER2-0 breast cancer

Background The characterization and comparison of gene expression (GE) and intrinsic subtypes (IS) changes induced by neoadjuvant chemotherapy (NACT) and endocrine therapy (NET) in hormone receptor-positive(HR+)/HER2-low vs. HR+/HER2-0 breast cancer (BC) has not been conducted so far. Most evidence on the association of HER2 status with pathologic responses and prognosis in HR+/HER2-negative BC is controversial and restricted to NACT-treated disease. Similarly, a temporal heterogeneity in HER2 status has been described only with NACT. Methods We retrospectively recruited a consecutive cohort of 186 patients with stage I-IIIB HR+/HER2-negative BC treated with neoadjuvant therapy (NAT). Available diagnostic biopsies and surgical samples were characterized for main pathological features, PAM50 intrinsic subtypes (IS) and risk-of-relapse (ROR)-P score, and GE. Associations with pathologic complete response (pCR), residual cancer burden (RCB)-0/I, event-free survival (EFS) and overall survival (OS) based on HER2 status were assessed. Pre/post pathologic/molecular changes were analyzed in matched samples. Results The HER2-low (62.9%) and HER2-0 (37.1%) cohorts did not differ significantly in main baseline features, treatments administered, breast conserving surgery (BCS), pCR and RCB-0/I rates, EFS and OS. NAT induced, regardless of HER2 status, a significant reduction of ER/PgR and Ki67, a downregulation of PAM50 proliferation- and luminal-related genes/signatures, an upregulation of selected immune genes and a shift towards less aggressive IS and lower ROR-P. Moreover, 25% of HER2-0 changed to HER2-low and 34% HER2-low became HER2-0. HER2 shifts were significant after NACT (p<0.001), not NET (p=0.063), with consistent ERBB2 mRNA level dynamics. HER2 changes were not associated to EFS/OS. Conclusions HER2 status changes after NAT in ∼1/4 of cases, mostly after NACT. Targeted adjuvant strategies should be investigated accordingly. Molecular downstaging with current chemo/endocrine agents and immunotherapy should not rely on HER2 immunohistochemical levels in HR+/HER2-negative BC. Instead, HER2-low-targeted approaches should be explored to pursue more effective and/or less toxic dimensional downstaging. Highlights 1. Hormone receptor-positive (HR+)/HER2-low and HER2-0 breast cancer (BC) showed similar post-neoadjuvant surgical outcomes. 2. Neoadjuvant therapy (NAT) induced a shift towards less aggressive subtypes and ROR-P classes regardless of HER2 status. 3. All NAT strategies induced a downregulation of proliferation- and luminal biology-related genes, regardless of HER2 status. 4. NAT induced changes in HER2 status, with a discordance rate of 34% and HER2-low showing higher instability than HER2-0. 5. HER2 status at baseline, after surgery and its dynamics were not significantly associated to long-term outcomes. ### Competing Interest Statement F.S. declares personal fees for educational events and/or materials from Gilead, Daiichy Sankyo and Novartis; travel expenses from Gilead, Daiichy Sankyo and Novartis; advisory fees from Pfizer. A.P. reports advisory and consulting fees from Roche, Daiichi-Sankyo, AstraZeneca, Pfizer, Novartis, Guardant Health, and Peptomyc, lecture fees from Roche, Novartis, AstraZeneca, and Daiichi Sankyo, institutional financial interests from Boehringer, Novartis, Roche, AstraZeneca, Daiichi-Sankyo, MedSIR, SL, Celgene, Astellas and Pfizer; stockholder and consultant of Reveal Genomics, SL; a patent PCT/EP2016/080056, the HER2DX patent filed (PCT/EP2022/086493), the DNADX patent filed (EP22382387.3) and the TNBCDX patent filed (EP23382703.9). F.B-M. has the HER2DX patent filed (PCT/EP2022/086493), the DNADX patent filed (EP22382387.3) and the TNBCDX patent filed (EP23382703.9). O.M.S. reports advisory and consulting fees from Roche and Reveal Genomic, lecture fees from Roche, Novartis, Eisai and Daiichi Sankyo, and travel expenses fees from Gilead, Pfizer and MSD. M.B. has a patent filed for the novel molecular subgroups for HR+/HER2+ early BC at the Institute of Cancer Research, and declares personal fees for educational events and/or materials from Astra Zeneca, Novartis and Eisai, and advisory fees from Pfizer. The other authors declare no conflicts of interest. ### Funding Statement This study was conducted in collaboration with AstraZeneca Farmaceutica Spain S.A. and Daiichy-Sankyo Spain S.A.U. (funding to A.P. laboratory). Any views, opinions, findings, conclusions, or recommendations expressed in this material are those solely of the authors and do not necessarily reflect those of funding entities. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Hospital Clinic of Barcelona gave ethical approval for this work (IRB n.HCB/2021/0007). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data generated in this study are not publicly available due to information that could compromise patient privacy or consent but are available upon reasonable request from the corresponding author.