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NGS aligned with FISH for detecting HER2 status in patients with breast cancer, in addition to revealing ERBB2 mutations and nuanced alterations.
In an abstract presented at the 2024 San Antonio Breast Cancer Symposium, researchers evaluated the correlation between fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) for assessing HER2 status in patients with breast cancer.
When HER2 status is equivocal by immunohistochemistry (IHC), FISH serves as the gold standard, per ASCO guidelines, in approximately 15% to 20% of cases. However, NGS has emerged as a promising tool to reduce equivocal cases by providing comprehensive genomic and transcriptomic insights.
Detecting ERBB2 Expression & HER2 Positivity
“NGS can confirm the HER2 status of breast cancer samples and, in turn, help reduce the incidence of equivocal samples,” wrote Nikita Kotlov and colleagues. “Here, we examined the correlation between FISH and NGS in annotating HER2 status of breast cancer samples and evaluated the clinical utility of NGS as a support tool for improving analysis outcomes.”
The investigators analyzed data from two cohorts: the BostonGene internal breast cancer cohort (n=358, FISH annotated=124) and The Cancer Genome Atlas Breast Invasive Carcinoma cohort (n=972, FISH annotated=285). Clinicians used RNA sequencing to quantify ERBB2 expression and DNA sequencing to assess ERBB2 amplification and mutation.
High ERBB2 expression was defined as at least 8.5 log2(TPM+1). NGS categorized ERBB2 status as “gain” (one gene copy increase relative to ploidy) or “amplification” (two or more gene copies above ploidy). HER2 positivity by FISH followed ASCO/CAP guidelines.
NGS Offers Additional Insights
The researchers found substantial agreement between FISH and NGS, with a Cohen’s kappa of 0.57. Positive FISH samples exhibited significantly higher ERBB2 expression (median, 8.8) compared with negative FISH samples (median, 6.6; P<0.001). Among positive FISH cases, 53% (n=37) showed high ERBB2 expression.
NGS-detected amplified samples had notably higher ERBB2 expression (median, 9.6) than non-amplified samples (median, 6.6; P<0.001). Additionally, in samples with ERBB2 gain or amplification, 31% (23 of 74) displayed high ERBB2 expression. Positive FISH samples with ERBB2 gain exhibited significantly higher ERBB2 expression (median, 10.5) compared with negative FISH samples with gain (median, 7.9; P<0.001).
NGS also identified 23 unique ERBB2 mutations, including 18 missense, 4 frameshift, and 9 gain-of-function mutations. Non-amplified samples with ERBB2 mutations showed higher ERBB2 expression (median, 7.4) than non-amplified samples with wild-type ERBB2 (median, 6.9; P=0.004).
The researchers concluded that NGS reliably detected increased ERBB2 expression in positive FISH samples, in addition to identifying mutations not covered by FISH.
“Since these mutations may be associated with increased ERBB2 expression, they are clinically relevant and warrant further study,” the authors wrote. “Last but not least, NGS detected focal ERBB2 amplifications and gains at a higher resolution than FISH.”
The authors added that their findings underscore the heterogeneous nature of HER2 amplification in breast cancer.
“The ability of NGS to identify these nuanced genomic alterations yields valuable insights into the tumor biology and potential resistance mechanisms of breast cancer that are crucial for designing personalized treatment strategies and improving patient outcomes,” Kotlov and colleagues wrote.
