Loss of HER2 and decreased T-DM1 efficacy in HER2 positive advanced breast cancer treated with dual HER2 blockade: the SePHER Study

Abstract

Background: HER2-targeting agents have dramatically changed the therapeutic landscape of HER2+ advanced breast cancer (ABC). Within a short time frame, the rapid introduction of new therapeutics has led to the approval of pertuzumab combined with trastuzumab and a taxane in first-line, and trastuzumab emtansine (T-DM1) in second-line. Thereby, evidence of T-DM1 efficacy following trastuzumab/pertuzumab combination is limited, with data from some retrospective reports suggesting lower activity. The purpose of the present study is to investigate T-DM1 efficacy in pertuzumab-pretreated and pertuzumab naïve HER2 positive ABC patients. We also aimed to provide evidence on the exposure to different drugs sequences including pertuzumab and T-DM1 in HER2 positive cell lines.

Methods: The biology of HER2 was investigated in vitro through sequential exposure of resistant HER2 + breast cancer cell lines to trastuzumab, pertuzumab, and their combination. In vitro experiments were paralleled by the analysis of data from 555 HER2 + ABC patients treated with T-DM1 and evaluation of T-DM1 efficacy in the 371 patients who received it in second line. Survival estimates were graphically displayed in Kaplan Meier curves, compared by log rank test and, when possibile, confirmed in multivariate models.

Results: We herein show evidence of lower activity of T-DM1 in two HER2+ breast cancer cell lines resistant to trastuzumab+pertuzumab, as compared to trastuzumab-resistant cells. Lower T-DM1 efficacy was associated with a marked reduction of HER2 expression on the cell membrane and its nuclear translocation. HER2 downregulation at the membrane level was confirmed in biopsies of four trastuzumab/pertuzumab-pretreated patients. Among the 371 patients treated with second-line T-DM1, median overall survival (mOS) from diagnosis of advanced disease and median progression-free survival to second-line treatment (mPFS2) were 52 and 6 months in 177 patients who received trastuzumab/pertuzumab in first-line, and 74 and 10 months in 194 pertuzumab-naïve patients (p = 0.0006 and 0.03 for OS and PFS2, respectively).

Conclusions: Our data support the hypothesis that the addition of pertuzumab to trastuzumab reduces the amount of available plasma membrane HER2 receptor, limiting the binding of T-DM1 in cancer cells. This may help interpret the less favorable outcomes of second-line T-DM1 in trastuzumab/pertuzumab pre-treated patients compared to their pertuzumab-naïve counterpart.

Keywords: HER2+ breast cancer; T-DM1 efficacy; Trastuzumab/pertuzumab blockade.

Fig. 1

Characterization of resistant cell lines. Cell viability of control (CTR), Trastuzumab (T), Pertuzumab (P), and T + P cell lines treated with 10 and 100 μg/ml trastuzumab, pertuzumab and trastuzumab+pertuzumab for 5 days was evaluated by MTT assay (a). Western Blot (WB) analysis of phosphorylated and total HER2, HER3, EGFR, AKT and ERKs was performed on total cell lysates from untreated/treated parental cell lines for 7 days (b, leftmost panel). The basal levels of phosphorylated and total HER2, HER3, EGFR, AKT and ERKs were evaluated by WB in CTR, T, P, and T + P resistant cell lines (b, rightmost panel). HER2 total and phosphorylated levels evaluation by WB was repeated in CTR, T, P, and T + P cell lines following resistant cell lines establishment (c). The anti-HSP70 antibody was used to validate equivalent amount of loaded proteins in each lane. Proliferation curves (d) and Invasion assay (e, upper panel) of CTR, T, P, and T + P cell lines. Representative images of invaded cells are reported for each cell line (e, lower panel)

Fig. 2

Trastuzumab+pertuzumab combination is associated with HER2 downregulation. Cell viability of CTR, T, P, and T + P SkBr3 and BT474 cells treated with 0.1 μg/ml and 1 μg/ml T-DM1, respectively, for 48 h was evaluated by Crystal Violet Assay (a). The results are expressed as percentage of T-DM1-responsive cells relative to control cells, as mean +/− standard deviation. T-DM1 dose-response curves of T and T + P cells are reported for both SkBr3 and BT474 (b). The expression of phosphorylated and total HER2, HER3, EGFR, AKT and ERKs was evaluated by Western Blot (WB) following exposure to 1 μg/ml T-DM1 for 48 h (c). Representative pre- and post-therapy sections from 2 ABC patients, stained by immunohistochemistry with eosin-hematossilin and anti-HER2 antibody (d)

Fig. 3

Prolonged trastuzumab+pertuzumab induces HER2 nuclear translocation. Control, T, P, and T + P cell lines were plated on poly-l lysine coated slides, and stained 24 hous later with anti-HER2 (green signal) (a). These cells were counterstained with Hoechst to highlight nuclei. Red arrows indicate HER2 localization on cellular protrusions. Cytoplasmic and nuclear fractions extracted from control, T, P, and T + P cells were analysed by Western Blot (WB) for the expression of phosphorylated and total HER2. Lamin A and α-tubulin were used to validate purity of nuclear and cytoplasmic extracts respectively (b). Following pre-treatment with 5 μg/ml trastuzumab + 5 μg/ml pertuzumab for 72 h, cell viability of control cells, pre-treated and T + P BT474 exposed to1 μg/ml T-DM1 for 72 h was evaluated by Crystal Violet Assay (c). Cytoplasmicand nuclear fractions of control, pre-treated and T + P BT474 cells were analysed by WB for the expression of total HER2 (d)

Fig. 4

Overall survival (OS) from diagnosis of metastatic disease (a), OS from T-DM1 start (b) and progression free survival to the second-line of treatment (PFS2) (c), in patients treated with trastuzumab-based first-line and T-DM1 in second-line (T - > T-DM1) and in patients treated with pertuzumab-trastuzumab-based first-line and T-DM1 in second-line (P + T - > T-DM1)