Human epidermal growth factor receptor-2 (HER2) expression is an important biomarker for the management of RAS wild-type metastatic colorectal carcinoma (CRC). Immunohistochemistry (IHC) with reflex in situ hybridization (ISH) is accepted as a standard method of assessment, yet there are currently the following 2 sets of criteria used to interpret results: the HER2 Amplification for Colorectal Cancer Enhanced Stratification (HERACLES) criteria and the MyPathway criteria. The HER2 Amplification for Colorectal Cancer Enhanced Stratification criteria require ISH confirmation when IHC staining is 3+ in 10% to 49% of cells, whereas the MyPathway criteria mirror those for gastric HER2 assessment and do not recommend ISH confirmation in the previously referenced scenario. We aimed to assess the prevalence of HER2 3+ heterogeneity and its association with ERBB2 copy number amplification to evaluate the necessity of ISH testing when IHC staining is 3+ in <
50% of cells. Next-generation sequencing of DNA (592-gene panel or whole exome sequencing) was performed for 13,208 CRC tumors submitted to Caris Life Sciences. HER2 (4B5) expression was tested using IHC. A subset of tumors was tested for ERBB2 amplification via chromogenic ISH and/or via next-generation sequencing (copy number amplification). χ