HER2 represents a complex and emerging biomarker in NSCLC, requiring sophisticated testing approaches to differentiate among various alteration types. The complexity stems from the relatively recent development of targeted medications that can specifically address HER2 alterations with reduced off-target side effects. Unlike EGFR mutations, HER2 exon 20 insertion mutations are relatively rare, with approximately 80% to 90% of HER2 intracellular kinase domain mutations occurring within exon 20. Additional mutations can occur in extracellular and transmembrane domains, adding to the testing complexity.

Testing becomes more challenging when distinguishing between amplification and overexpression events. HER2 amplification occurs in approximately 3% to 5% of cases depending on definition criteria, though effective targeting strategies remain limited. HER2 overexpression is measured through protein expression levels using IHC with a 3+ expression cutoff for strong expression in NSCLC. While amplification can lead to overexpression, epigenetic changes more commonly drive HER2 overexpression, making these alterations attractive targets for antibody-drug conjugates like trastuzumab deruxtecan, which is currently guideline approved.

The multilayered nature of HER2 biomarker testing requires comprehensive approaches combining multiple methodologies. Mutational and amplification testing can be performed through next-generation sequencing (NGS) on a DNA basis, while epigenetic expression requires IHC assessment. This necessitates incorporating multiple IHC tests alongside NGS testing, including PD-L1 and 3 additional IHC markers, to comprehensively cover the spectrum of HER2 exon 20 insertion mutations and other alterations in clinical practice.