Neratinib plus trastuzumab is superior to pertuzumab plus trastuzumab in HER2-positive breast cancer xenograft models

Abstract

Lapatinib (L) plus trastuzumab (T), with endocrine therapy for estrogen receptor (ER)+ tumors, but without chemotherapy, yielded meaningful response in HER2+ breast cancer (BC) neoadjuvant trials. The irreversible/pan-HER inhibitor neratinib (N) has proven more potent than L. However, the efficacy of N+T in comparison to pertuzumab (P) + T or L + T (without chemotherapy) remains less studied. To address this, mice bearing HER2+ BT474-AZ (ER+) cell and BCM-3963 patient-derived BC xenografts were randomized to vehicle, N, T, P, N+T, or P+T, with simultaneous estrogen deprivation for BT474-AZ. Time to tumor regression/progression and incidence/time to complete response (CR) were determined. Changes in key HER pathway and proliferative markers were assessed by immunohistochemistry and western blot of short-term-treated tumors. In the BT474-AZ model, while all N, P, T, N + T, and P + T treated tumors regressed, N + T-treated tumors regressed faster than P, T, and P + T. Further, N + T was superior to N and T alone in accelerating CR. In the BCM-3963 model, which was refractory to T, P, and P + T, while N and N + T yielded 100% CR, N + T accelerated the CR compared to N. Ki67, phosphorylated (p) AKT, pS6, and pERK levels were largely inhibited by N and N + T, but not by T, P, or P + T. Phosphorylated HER receptor levels were also markedly inhibited by N and N + T, but not by P + T or L + T. Our findings establish the efficacy of combining N with T and support clinical testing to investigate the efficacy of N + T with or without chemotherapy in the neoadjuvant setting for HER2+ BC.

Fig. 1. Neratinib (N) containing anti-HER2 regimen shows superior anti-tumor efficacy than trastuzumab (T) and pertuzumab (P) in BT474 cell-derived xenograft model.

a Average growth curves of tumors treated with Vehicle, N, T, P, N + T, or P + T (n = 9–14). Results are presented as mean tumor volume ± SEM. Kaplan–Meier analysis of b time to tumor progression in mice treated with different anti-HER2 therapies, c time to complete response in mice treated with N, T, or N + T, d time to complete response in mice treated with T, P, or T + P, and e time to tumor regression and f time to complete response in mice treated with N + T or P + T. Shaded areas represent point-wise 95% confidence regions, calculated using the loglog transform. P values are indicated on plots, Wilcoxon test; Veh Vehicle, ED estrogen deprivation.

Fig. 2. Neratinib (N) containing anti-HER2 regimens effectively achieve tumor regression and eradication in a patient-derived xenograft model that is refractory to trastuzumab (T) and pertuzumab (P).

a Average growth curves of tumors treated with Vehicle, N, T, P, N + T, or P + T (n = 13–19). Results are presented as mean tumor volume ± SEM. Kaplan–Meier analysis of b time to tumor progression in mice treated with different anti-HER2 therapies, and c time to complete response and d time to tumor regression in mice treated with N or N + T. Shaded areas represent point-wise 95% confidence regions, calculated using the loglog transform. P values are indicated on plots, Wilcoxon test. Veh Vehicle.

Fig. 3. Neratinib-containing regimens significantly inhibit tumor cell proliferation and HER2 signaling in short-term treated BT474-AZ xenograft tumors.

a Western blot analyses of alterations in level and activation of proteins along the HER signaling axis. Each of the seven short-term treatment arms had six tumors, which were run in two separate gels with each gel containing three tumors per arm. Colored bars below the treatment arms in gray boxes denote duplicate samples loaded as comparison control between the left and right blots. b Representative HER2, pHER2, Ki67, pAKT, and pP44/42 ERK immunohistochemical staining. c Box plots showing average Ki67 (%), pAKT (H-Score), and pERK (H-Score) protein levels by immunohistochemistry. Box plots indicate median and interquartile range (IQR). The lower and upper hinges correspond to the first (25th percentile) and third (75th percentile) quartiles, respectively. The whiskers extend to about 1.5 × IQR from the hinge, and data points beyond the whiskers are suspected outliers. E2 estrogen, ED estrogen deprivation, Veh/V vehicle, N neratinib, T trastuzumab, P pertuzumab, IHC images in b are of ×40 magnification, Scale bar: 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4. Neratinib-containing regimens suppress cell proliferation and HER2 signaling in short-term treated BCM-3963 patient-derived xenografts.

a Western blot analyses of alterations in level and activation of proteins along the HER signaling axis. Each of the six short-term treatment arms had six tumors, which were run in two separate gels with each gel containing three tumors per arm. Colored bars below the treatment arms in gray boxes denote duplicate samples loaded as comparison control between left and right blots. b Representative HER2, pHER2, Ki67, pAKT, and pP44/42 ERK immunohistochemical staining. c Box plots showing average Ki67 (%), pAKT (H-Score), and pERK (H-Score) protein levels by immunohistochemistry. Box plots indicate median and interquartile range (IQR). The lower and upper hinges correspond to the first (25th percentile) and third (75th percentile) quartiles, respectively. The whiskers extend to about 1.5 × IQR from the hinge, and data points beyond the whiskers are suspected outliers. V vehicle, N neratinib, T trastuzumab, P pertuzumab. IHC images in b are of ×40 magnification, Scale bar: 50 μm. **P < 0.01, ***P < 0.001.