Integrated approach to functional analysis of an ERBB2 variant of unknown significance detected by a cancer gene panel test

Abstract

Purpose Dealing with variants of unknown significance (VUS) is an important issue in the clinical application of NGS-based cancer gene panel tests. We detected a novel ERBB2 extracellular domain VUS, c.1157A > G p.(E401G), in a cancer gene panel test. Since the mechanisms of activation by ERBB2 extracellular domain (ECD) variants are not fully understood, we aimed to clarify those mechanisms and the biological functions of ERBB2 E401G. Methods ERBB2 E401G was selected as VUS for analysis because multiple software tools predicted its pathogenicity. We prepared ERBB2 expression vectors with the E401G variant as well as vectors with S310F and E321G, which are known to be activating mutations. On the basis of wild-type ERBB2 or mutant ERBB2 expression in cell lines without ERBB2 amplification or variants, we evaluated the phosphorylation of human epidermal growth factor receptor 2 and related proteins, and investigated with molecular dynamics (MD) simulation the mechanisms conferred by the variants. The biological effects of ERBB2 E401G were also investigated, both in vitro and in vivo. Results We found that ERBB2 E401G enhances C-terminal phosphorylation in a way similar to S310F. MD simulation analysis revealed that these variants maintain the stability of the EGFR-HER2 heterodimer in a ligand-independent manner. Moreover, ERBB2 E401G-transduced cells showed an increased invasive capacity in vitro and an increased tumor growth capacity in vivo. Conclusion Our results provide important information on the activating mechanisms of ERBB2 extracellular domain (ECD) variants and illustrate a model workflow integrating wet and dry bench processes for the analysis of VUS detected with cancer gene panel tests.

Anti-cancer effect of afatinib, dual inhibitor of HER2 and EGFR, on novel mutation HER2 E401G in models of patient-derived cancer

“…Droplet digital PCR (ddPCR ™ ) for analyzing the ratio of E401G and wild type HER2 was performed as described previously [10]. Copy number assay using ddPCR was performed with a HER2 probe set (dHsaCP1000116, Bio-Rad Laboratories, Inc., USA) and the reference gene, RPP30, probe set (dHsaCP2500350, Bio-Rad Laboratories, Inc., USA), according to the manufacturer's protocol.…”

“…The clinical course of a 67-year-old Japanese woman with carcinoma of unknown primary up to enrollment in the JUPITER trial (jRCT2031180150) [20], a basket trial of trastuzumab and pertuzumab combination therapy, was described in detail in our previous report [10]. After initial treatment with carboplatin and gemcitabine, the patient's cancer gradually became refractory to the therapy.…”

“…We recently experienced a case of carcinoma of unknown primary with HER2 E401G, a variant of unknown significance (VUS) in the extracellular domain concomitant with HER2 amplification. Comprehensive analysis using cell line and animal models, as well as molecular dynamic simulation in silico, showed that HER2 E401G has an epidermal growth factor receptor (EGFR)-mediated activation mechanism, the same as HER2 S310F [10]. Determining therapeutic strategies in accordance with the activation mechanism of variants is an important challenge for precision medicine based on the cancer genome, which we aimed to put into practice in this case.…”