Background: Although tremendous progress has been achieved with targeted therapy for HER2-positive (HER2+) metastatic breast cancer, most advanced tumors eventually develop resistance. Improving our understanding of mechanisms of resistance to anti-HER2 therapy is needed to develop new therapeutic approaches. The purpose of this study was to identify the mechanisms of resistance to treatment with ado-trastuzumab emtansine (T-DM1) and/or taxane/pertuzumab/trastuzumab (TPH).

Methods: In our preclinical analysis, HER2+ cell lines resistant to treatment with T-DM1 (n=5), and pertuzumab/trastuzumab (n=3) were generated. HER2 expression in the original and resistant cell lines was compared using Western blot, and HER2 gene amplification was compared in them using fluorescence in situ hybridization (FISH) and a Droplet Digital Polymerase Chain Reaction HER2 copy-number-validation assay. In our clinical analysis, nine patients with HER2+ metastatic breast cancer who had progressed on T-DM1 and/or TPH were enrolled. Patients underwent biopsies following treatment with T-DM1 and/or TPH. Targeted next-generation sequencing was performed using the FoundationOne® assay (Foundation Medicine, Inc.) to identify gene alterations. Also, the HER2 expression before and after the therapy was compared using immunohistochemistry and/or FISH.

Results: In preclinical analysis, HER2 expression/amplification by Western blot and gene copy-number analysis was significantly decreased in T-DM1–resistant cell lines (four of five cell lines; P < 0.01) but not in pertuzumab/trastuzumab-resistant cell lines (none of three cell lines). In clinical analysis, the patients' median age was 54 years (range, 45-77 years), and five patients (56%) were ER+. Five patients (56%) received first-line anti-HER2 therapy, and four patients (44%) received two lines of anti-HER2 therapy prior to enrollment. We observed loss of HER2 expression in four of nine patients (44%) after undergoing anti-HER2 therapy. After receiving TPH, one of eight patients (13%) lost HER2 positivity according to FISH. In contrast, after T-DM1, three of four tested patients (75%) lost HER2 amplification by FISH. As for next-generation sequencing, we analyzed seven samples: three after treatment with TPH and four after treatment with T-DM1. In four of these samples (57%), we observed loss of HER2 amplification: one after treatment with TPH and three after treatment with T-DM1. TP53 mutations were seen in all patients. Additionally, we observed TOP2A and MCL1 amplification in two patients with ERBB2 amplificationand AKT1 amplification in one patient with ERBB2 amplification loss.

Conclusions: We show for the first time that T-DM1–resistant breast cancer cells lose HER2 expression and amplification. Additionally, we observed loss of HER2 expression in patient samples following treatment with HER2 targeted therapy. Further study of resistant tumor samples is required to understand the impact of HER2 loss on outcomes. For the time being, repeating biopsy analysis of a metastatic site after treatment with T-DM1 to determine the HER2 expression status is reasonable, and it may increase the efficacy of future anti-HER2 therapy.

Citation Format: Kida K, Lee J, Liu H, Lim B, Murthy RK, Sahin AA, Tripathy D, Ueno NT. Changes in the expression of HER2 and other genes in HER2-positive metastatic breast cancer induced by treatment with ado-trastuzumab emtansine and/or pertuzumab/trastuzumab [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-23.