Precision oncology, utilizing multi-gene panel testing via next-generation sequencing (NGS), is increasingly prevalent. HER2, encoding the human epidermal growth factor receptor 2, is a crucial driver gene; however, therapeutic strategies for HER2 extracellular domain mutations remain unclear. This study focuses on the HER2 extracellular domain mutation E401G, previously shown to have an EGFR-mediated activation mechanism. The researchers hypothesized that co-targeting EGFR and HER2 would be beneficial for cancers harboring this mutation. This hypothesis is based on the understanding that the E401G mutation activates HER2 through an EGFR-mediated pathway. The study aimed to evaluate the efficacy of afatinib, a dual inhibitor of HER2 and EGFR, and compare it to lapatinib (a weaker EGFR inhibitor) and trastuzumab plus pertuzumab (effective against wild-type HER2 amplification) in patient-derived models. The use of patient-derived xenografts (PDX) and cancer tissue-originated spheroids (CTOS) aimed to better reflect clinical treatment responses compared to traditional cell lines. The choice of these models is significant because they are considered to more accurately reflect the complexity and heterogeneity of patient tumors compared to traditional cell lines, providing a more realistic assessment of drug efficacy.

The literature review highlights the increasing use of NGS-based multi-gene panel testing in precision oncology, with basket trials investigating the efficacy of targeted agents across various cancer types sharing similar driver gene abnormalities. While HER2 mutations, particularly in the intracellular kinase domain, are well-studied targets, the therapeutic implications of extracellular domain mutations beyond S310F are less understood. Previous work by the authors showed that the HER2 E401G mutation, a variant of unknown significance (VUS), exhibits EGFR-mediated activation. This finding supports the rationale for exploring a dual-inhibitor approach targeting both HER2 and EGFR in treating cancers with this mutation. Existing literature on the efficacy of afatinib, lapatinib, and the combination of trastuzumab and pertuzumab in HER2-positive cancers informs the choice of these drugs for comparison in the study.

The study employed a multifaceted approach using both in vivo and in vitro models. A PDX model and a CTOS model were established from a patient with carcinoma of unknown primary and coexisting HER2 E401G mutation and amplification. The models were rigorously characterized using morphology, immunohistochemistry (IHC), and droplet digital PCR (ddPCR) to ensure retention of the original tumor characteristics. The efficacy of afatinib, lapatinib, and trastuzumab plus pertuzumab were compared using these models. In vivo experiments were conducted in Balb/c Rag-2 −/− Jak3 −/− (BRJ) mice. Drug administration was performed using oral administration for afatinib and lapatinib and intraperitoneal injection for trastuzumab and pertuzumab. Tumor size and HER2 copy number were monitored. In vitro experiments using CTOS and the H2170 cell line (wild-type HER2 amplification) were also conducted to assess drug sensitivity. ddPCR was employed to assess HER2 copy number and the ratio of mutant to wild-type HER2. Statistical analysis was performed using the Wilcoxon rank-sum test and Kruskal-Wallis test.

The PDX and CTOS models successfully preserved the morphological, immunohistochemical, and genetic characteristics of the original patient tumor. Afatinib showed superior anti-cancer efficacy in both PDX and CTOS models, significantly reducing tumor size compared to lapatinib and trastuzumab plus pertuzumab. Afatinib treatment also resulted in a significant reduction in HER2 copy number in the PDX model. In contrast, in the H2170 xenograft model with wild-type HER2 amplification, trastuzumab plus pertuzumab demonstrated the greatest anti-tumor effect, highlighting the difference in response based on the presence of the E401G mutation. The IC50 values from the CTOS assay further supported the superior efficacy of afatinib (0.35 μM) compared to lapatinib (1.8 μM) and the weak effect of trastuzumab plus pertuzumab.

The findings support the hypothesis that simultaneously suppressing EGFR and HER2 signaling is effective against cancers harboring the HER2 E401G mutation and amplification. The superior efficacy of afatinib over lapatinib is attributed to afatinib's potent and prolonged inhibition of both EGFR and HER2, which is further supported by previous research on other HER2 extracellular domain mutations such as S310F. The results from the H2170 cell line (with wild-type HER2 amplification) highlights the importance of considering the specific HER2 mutation context when selecting treatment strategies, underscoring the need for precision medicine approaches. The difference in efficacy between afatinib and trastuzumab plus pertuzumab is likely due to the distinct binding sites of these agents and the EGFR-mediated activation mechanism of the E401G mutation.

This study demonstrates that targeting both EGFR and HER2 signaling pathways using afatinib is a promising therapeutic strategy for cancers with the HER2 E401G mutation. The findings underscore the importance of understanding the activation mechanisms of HER2 mutations for developing effective precision medicine approaches. Future research could explore combination therapies involving afatinib and other agents, potentially to further enhance efficacy while carefully managing side effects.

The study is limited by the use of a single patient-derived model. While the PDX and CTOS models were well-characterized, additional models and larger sample sizes would strengthen the generalizability of the results. Further mechanistic studies are warranted to definitively establish the role of EGFR inhibition in afatinib's efficacy against HER2 E401G. The lack of molecular evidence linking EGFR inhibition and afatinib efficacy due to tissue limitations represents a key limitation. The study also didn't explore combination therapies involving afatinib and trastuzumab plus pertuzumab.