Quantitative amplification ratio for established and emerging biomarkers in gastroesophageal (GE) cancer.
Authors: Strickland Matthew , Lee Jessica , Fendler Bernard , Schrock Alexa Betzig , Klempner Samuel J. 
Published: January 2025

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Background:
Gene copy number (CN) amplifications (amps) and/or protein overexpression in
ERBB2
,
EGFR
,
FGFR2
, and
MET
represent established and emerging clinical targets in GE. Various methodologies are used to report gene amp or overexpression with some data suggesting FISH ratio may predict therapy response. Quantitative methods for NGS CN and relationship to ISH/IHC remains largely unresolved. We studied the landscape of targetable CN amps in GE to understand correlations of quantitative CN amp ratio and outcomes.
Methods:
GE tissue (N = 20,468) and liquid (N = 1,760) samples underwent NGS-based comprehensive genomic profiling (CGP). A genome-wide CN model for each sample was generated, segmented, and combined with variant allele frequencies of heterozygous SNPs to estimate the sample purity, ploidy, and CN for each segment. Genes were considered amplified at CN ≥ ploidy + 3 for
ERBB2
and + 4 for other genes. Sub-analysis using amp ratio (ratio of the gene CN to the sample ploidy) ≥ 3 was performed. ctDNA tumor fraction (TF) was quantified using a composite algorithm (PMID: 38990098). The Flatiron Health-Foundation Medicine clinico genomic database, a nationwide (US-based) de-identified EHR-derived database linked to CGP data, was queried to assess external HER2 FISH or IHC and describe real-world (rw) progression-free survival (rwPFS) and overall survival (rwOS) of GE patients (pts) with
ERBB2
amp on trastuzumab-based regimens.
Results:
≥1 gene amps were detected in 15,341 (75%) GE tissue cases, and in 67% of cases using an amp ratio ≥3 threshold. The rates of amps in current clinical targets using each threshold were
ERBB2
: 17%/12%,
KRAS
: 13%/12%,
EGFR:
8.7%/7.2%,
MET:
5.2%/4.0%
, FGFR2:
3.1%/2.7% of tissue samples. The median amp ratio and percent of amps with ratio ≥ 3 were
ERBB2
: 7.1/73%,
KRAS
: 14/92%,
EGFR
: 13/82%,
MET
: 6.3/76%, and
FGFR2
: 29/88%. Amp ratio ≥ 3 was associated with focal amplicons vs amp ratio < 3 (median amplicon size 0.95 vs. 3.8 Mbp, p < 0.001). Of 1,382 pts with HER2 NGS and IHC results, concordance with IHC3+ was 68% and increasing
ERBB2
amp ratio was associated with IHC 3+ status. Of 604 pts with HER2 NGS and FISH, concordance was 80% (118 positive, 366 negative). Higher
ERBB2
amp ratio was also associated with longer rwOS and rwPFS as a continuous variable on first-line trastuzumab-based regimens. ≥1 gene amps were detected in 597 (34%) liquid samples (median TF 18%) and 32% with the amp ratio ≥3 threshold, increasing to 79% and 73% with TF ≥ 20% (n=355).
Conclusions:
Gene amps are common established or emerging GE targets and
ERBB2
amp ratio is associated with outcomes for pts on trastuzumab-based regimens. Amp detection in liquid correlates with higher TF and amp ratio. As the number of therapies targeting gene amps and overexpression grows, quantification of the degree of amplification and harmonization across methodologies may be an important tool to select pts for optimal outcomes.