Neratinib is effective in breast tumors bearing both amplification and mutation of ERBB2 (HER2)

Abstract

Mutations in ERBB2, the gene encoding epidermal growth factor receptor (EGFR) family member HER2, are common in and drive the growth of "HER2-negative" (not ERBB2 amplified) tumors but are rare in "HER2-positive" (ERBB2 amplified) breast cancer. We analyzed DNA-sequencing data from HER2-positive patients and used cell lines and a patient-derived xenograft model to test the consequence of HER2 mutations on the efficacy of anti-HER2 agents such as trastuzumab, lapatinib, and neratinib, an irreversible pan-EGFR inhibitor. HER2 mutations were present in ~7% of HER2-positive tumors, all of which were metastatic but not all were previously treated. Compared to HER2 amplification alone, in both patients and cultured cell lines, the co-occurrence of HER2 mutation and amplification was associated with poor response to trastuzumab and lapatinib, the standard-of-care anti-HER2 agents. In mice, xenografts established from a patient whose HER2-positive tumor acquired a D769Y mutation in HER2 after progression on trastuzumab-based therapy were resistant to trastuzumab or lapatinib but were sensitive to neratinib. Clinical data revealed that six heavily pretreated patients with tumors bearing coincident HER2 amplification and mutation subsequently exhibited a statistically significant response to neratinib monotherapy. Thus, these findings indicate that coincident HER2 mutation reduces the efficacy of therapies commonly used to treat HER2-positive breast cancer, particularly in metastatic and previously HER2 inhibitor-treated patients, as well as potentially in patients scheduled for first-line treatment. Therefore, we propose that clinical studies testing the efficacy of neratinib are warranted selectively in breast cancer patients whose tumors carry both amplification and mutation of ERBB2/HER2.

Fig. 1:. Co-existence of ERBB2 amplification and mutations in metastatic breast cancer.

(A) Schematic representation of the detection of HER2 mutations in HER2-positive breast cancer patients treated with anti-HER2 therapy for the indicated time. “HER2 3⁺” refers to tumors that were highly positive for HER2 staining by IHC. (B) OncoPrint software-generated genomic alterations heatmap of the ERBB2-amplified patients available in the MSK database. (C) Graph indicating the proportion of primary or metastatic patients with tumors bearing concomitant mutation and amplification of ERBB2. (D) Kaplen-Meyer curves showing the progression-free survival of the MSK cohort of HER2-positive patients treated with the standard-of-care trastuzumab/pertuzumab/paclitaxel combination (THP). Displayed inset is the hazard ratio (HR) with 95% confidence interval (95%CI) of patients with ERBB2-mutant/amplified (coincident) tumors versus ERBB2-amplified–only tumors.

Fig. 2.. HER2 L755S mutation confers resistance to lapatinib.

(A and C) Western blot analyses of total HER2, AKT, ERK and S6 as well as phospho-Tyrosine, phospho AKT (Ser473), phospho ERK (Thr202/Tyr204 and phospho S6 (Ser235/236) in whole-cell extracts from Sk-Br-3 (A) and BT-474 (C) cells expressing empty-vector, wild-type (WT) HER2, or HER2 L755S and cultured in various concentrations of lapatinib for 4 hours. (p, phosphorylated). (B and D). Cell viability by CTG assay of the cells described in (A) and (C), respectively after incubation with increasing concentrations of lapatinib (ranging from 4 to 2000nM). Data are means ± SD from three independent experiments. P value obtained by two-tailed Student’s t-test.

Fig. 3.. HER2 L755S mutation induces resistance to HER2-targeted therapy.

Clonogenic growth assays performed by crystal violet staining on Sk-Br-3 (A) and BT-474 (B) cells expressing empty-vector, wild-type or mutant (L755S) HER2 constructs and cultured with trastuzumab (20 μg/ml), lapatinib (500 nM) or the combination, as indicated, for ten days. Results from treated cultures were quantified relative to non-treated cells. Data are means ± SEM from three independent experiments. *P<0.05 by two-tailed Student’s t-test.

Fig. 4.. Activity of neratinib against breast cancer cells carrying both the amplification and mutation of HER2.

(A and C) Western blot analyses of total HER2, AKT, ERK and S6 as well as phospho-Tyrosine, phospho AKT (Ser⁴⁷³), phospho ERK (Thr²⁰²/Tyr²⁰⁴) and phospho S6 (Ser^235/236) in whole-cell extracts from Sk-Br-3 (A) and BT-474 (C) cells expressing empty-vector, wild-type (WT) HER2, or HER2 L755S and cultured in various concentrations of neratinib for 4 hours. (p, phosphorylated). (B and D) Cell viability by CTG assay of the cells described in (A) and (C), respectively after incubation with increasing concentrations of lapatinib (ranging from 0.05 to 25nM). Data are means ± SD from three independent experiments. P value obtained by two-tailed Student’s t-test.

Fig. 5.. In vivo efficacy of neratinib against HER2-amplified and mutant PDXs.

Antitumor effects of neratinib in xenografts bearing in ERBB2. Growth of patient-derived xenografts containing coincident ERBB2 amplification and HER2 D769Y mutation, in mice treated with vehicle (control), trastuzumab (10 mg/kg; ip; 2xwkly), lapatinib (100mg/kg daily) or neratinib (40mg/kg daily). Data are k means ± SEM from 8 mice each condition. P<0.001 by two-tailed Student’s t-test