High-Throughput Functional Evaluation of Variants of Unknown Significance in ERBB2

Abstract

The advent of next-generation sequencing technologies has enabled the identification of several activating mutations of Erb-B2 receptor tyrosine kinase 2 (ERBB2) among various cancers. However, the significance of infrequent mutations has not been fully investigated. Herein, we comprehensively assessed the functional significance of the mutations in a high-throughput manner. We evaluated the transforming activities and drug sensitivities of 55 nonsynonymous mutations using the mixed-all-nominated-in-one (MANO) method. G776V, G778_S779insG, and L841V were newly revealed to be activating mutations. Although afatinib, neratinib, and osimertinib were shown to be effective against most of the mutations, only osimertinib demonstrated good efficacy against L755P and L755S mutations, the most common mutations in breast cancer. In contrast, afatinib and neratinib were predicted to be more effective than other inhibitors for the A775_776insYVMA mutation, the most frequent mutation in lung cancer. We surveyed the prevalence of concurrent mutation with gene amplification and found that approximately 30% of ERBB2-amplified urothelial carcinomas simultaneously carried mutations, altering their sensitivity to trastuzumab, an mAb against ERBB2. Furthermore, the MANO method was applied to evaluate the functional significance of 17 compound mutations within reported in the COSMIC database, revealing that compound mutations involving L755S were sensitive to osimertinib but insensitive to afatinib and neratinib. Several mutations showed varying sensitivities to ERBB2-targeted inhibitors. Our comprehensive assessment of mutations offers a fundamental database to help customize therapy for ERBB2-driven cancers.We identified several mutations as activating mutations related to tumorigenesis. In addition, our comprehensive evaluation revealed that several mutations showed varying sensitivities to ERBB2-targeted inhibitors, and thus, the functional significance of each variant should be interpreted precisely to design the best treatment for each patient. .

Mutation Variants and Co-Mutations as Genomic Modifiers of Response to Afatinib in HER2-Mutant Lung Adenocarcinoma

“…But generally, compared with other variants, HER2 YVMA insertion correlated with significantly worse clinical outcomes on afatinib (ORR, 0%; median PFS, 1.2 months; 95% CI, 0–2.4). In accordance with our results, Nagano et al reported higher half maximal inhibitory concentration of afatinib against A775_G776insYVMA compared with G776delinsVC and V777L in preclinical models .…”

High-Throughput Functional Evaluation ofBRCA2Variants of Unknown Significance

“…It is important to note that wild-type BRCA2 -induced cells were significantly more sensitive than parental DLD1 cells; therefore, we should establish a sensitive method in which all comparisons could be made with wild-type-induced cells rather than parental cells. We thus modified the mixed-all-nominated-in-one (MANO) method 28,29 that was originally developed to enable high-throughput evaluation of VUSs in transforming genes and invented the MANO- BRCA (MANO-B) method for the functional evaluation of BRCA2 variants ( Fig. 1 ).…”

“…The original MANO method is a high-throughput functional assay previously reported by our laboratory 28,29 . Individually established DLD1 BRCA2 (-/-) cells with stable BRCA2 expression were mixed equally and cultured competitively.…”

Biomarkers to diagnose metastatic breast carcinoma to the pancreas: A case report and update

“…This mutation lies within the protein kinase domain of the HER2 protein, although the functional status of the protein alteration remains controversial. Two studies identified I767M as a gain‐of‐function mutation with transformative potential, resulting in increased HER2 kinase activity and sustained AKT phosphorylation . However, another study demonstrated no difference in cell proliferation or viability of HER2 I767M compared to wild‐type HER2 .…”

“…Two studies identified I767M as a gain-of-function mutation with transformative potential, resulting in increased HER2 kinase activity and sustained AKT phosphorylation. 19,20 However, another study demonstrated no difference in cell proliferation or viability of HER2 I767M compared to wild-type HER2. 21 HER2 mutations have been well studied in breast cancer, including HER2 nonamplified breast cancer.…”