Abstract
PurposeDealing 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.MethodsERBB2 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.ResultsWe 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.ConclusionOur 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.
Highlights
The use of comprehensive genomic panel tests based on next-generation sequencing (NGS) has increased in clinical practice for cancer patients [1]
We found that ERBB2 gene amplification (ERBB2 copy number of 107) was accompanied by a missense variant, E401G, with an allele fraction of 99.2% (Fig. 1c)
We found that cells expressing ERBB2 E401G or S310F exhibited increased phosphorylation levels of HER2 and EGFR, to a greater extent than cells expressing ERBB2 Wild type (WT) (Fig. 3)
Summary
The use of comprehensive genomic panel tests based on next-generation sequencing (NGS) has increased in clinical practice for cancer patients [1]. The number of cancer patients who can receive treatment based on the results of sequence data has been reported to be approximately 10% among those tested with NGS [2, 3]. VUS is defined as tier III when convincing evidence of cancer association is not found in a published database or when the variant has not been reported at a significant allele frequency in general or subpopulation-specific databases. Such a VUS is not regarded as a targetable variant, the possibility that it is amenable to treatment cannot be ruled out
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
