Closing the gap in HER2 testing: CEP17-low FISH results and NGS discordance.

Abstract

e15192 Background: Discordance between HER2 amplification detected by fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) has been observed across tumor types, often in the context of chromosome 17 alterations. Low CEP17 control signal, due to monosomy 17, partial centromeric loss, truncation artifact or probe failure, can artifactually inflate the HER2/CEP17 ratio, resulting in FISH positivity in the absence of true ERBB2 gene amplification. The biologic underpinnings of FISH and NGS differ: FISH evaluates single cell nuclei, while NGS measures bulk tumor DNA and may be influenced by tumor purity, policy and heterogeneity. As HER2-targeted therapies expand across malignancies, accurate genomic classification is critical to avoid unnecessary toxicity in patients unlikely to benefit. Methods: We retrospectively identified patients with HER2 immunohistochemistry (IHC) 2+ tumors who underwent reflex HER2 FISH testing between 2020 and 2025 at a single institution. Cases with HER2 FISH-positive results were selected for analysis. NGS results were collected when available. Absolute HER2 copy number and CEP17 signal were analyzed. Anti-HER2 therapy use, disease progression, and cardiotoxicity were summarized using descriptive statistics. Results: Among 33 HER2 FISH-positive tumors, the majority were breast primaries (84.8%), with 15.1% gastroesophageal origin. NGS was available in 81.8% of cases (27/33), confirming HER2 amplification in only 11.1% (3/27), yielding a discordance rate of 88.9%. A low CEP17 signal was observed in 42.4% (14/33). Notably, absolute HER2 copy number was ≥6.0 in 42.4% of tumors, while 57.6% (19/33) met FISH positivity criteria despite HER2 copy number < 6.0, supporting CEP17-driven ratio inflation as a major contributor to discordance. Anti-HER2 therapy was administered in 75.8% (25/33) of patients; among treated patients, 36.0% experienced progression and 24.0% developed cardiotoxicity. Conclusions: This single-institution cohort reveals a high rate of discordance between HER2 FISH positivity and NGS-confirmed amplification in IHC 2+ tumors, often driven by artificially low CEP17 signals. A modest HER2 copy number represents a clinically relevant pitfall that may lead to HER2-directed treatment in patients without clear genomic amplification. These findings highlight the limitations of ratio-based FISH interpretation alone and support multimodal HER2 adjudication incorporating absolute HER2 copy number, low CEP17 signal, and NGS copy-number context. Prospective validation is warranted to refine HER2 testing algorithms and to guide HER2 targeted therapy decisions in discordant cases.