The prognostic value of HER2 gene dosage and heterogeneity at a single-cell resolution in gastroesophageal adenocarcinoma.
Authors: Parashar Yashika , Culnane Leigh , Remland Joshua , Nambiar Chetan , Yuan Chen , Nasta Shagun , Bard Adam , Park Haeseong , Klempner Samuel J. , Giannakis Marios , Aguirre Andrew , Ng Kimmie , Meyerhardt Jeffrey A. , Wolpin Brian M. , Yaeger Rona , Cleary James M. , Wang Charlotte , Enzinger Peter C. , Sethi Nilay S. , Singh Harshabad 
Published: January 2025

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Background:
HER2 gene dosage (i.e. level of amplification) and heterogeneity are key prognostic variables for HER2+ gastroesophageal adenocarcinoma (GEA), yet no standardized approach to test these in routine clinical use exists. Here we modify the analytical pipeline for HER2 FISH to quantitatively estimate these prognostic variables and correlate them with clinical outcomes.
Methods:
Patients with metastatic HER2+ GEA who received trastuzumab-based therapy between 2011-2024 at Dana-Farber Cancer Institute and had available pre-treatment tissue for FISH analysis were included. HER2 FISH was conducted in a CLIA approved clinical pathology laboratory. Standard HER2/CEP17 ratio (an average of 50 cells) and individual cell HER2/CEP17 ratio were calculated. Kaplan-Meier method was used to estimate progression free (PFS) on trastuzumab-based therapy and overall survival (OS). Survival outcomes were adjusted for age, gender, tumor site, grade, prior resection, number of metastatic sites, and HER2 IHC using cox proportional hazard regression models.
Results:
A total of 77 patients were included. Median age was 60 years, 88% were male, and 92% had HER2 3+ tumors. Individual cells with HER2/CEP17 ratio ≥ 2.0 were defined as amplified (same cutoff as current bulk standard) and those with HER2/CEP17 ratio ≥ 4.95 (population median) were labeled highly HER2 amplified providing objective metrics to assess heterogeneity and gene dosage. Using these metrics we defined three subgroups: 1. Heterogenous (Het, n = 15): <75% cells HER2+; 2. Homogenous, low gene dosage (HL, n = 30): >75% cells HER2+, <50% cells with high HER2 amplification; 3. Homogenous, high gene dosage (HH, n = 32): >75% cells HER2+, >50% cells with high HER2 amplification. Cutoffs for both metrics were determined using data distribution patterns. The subgroups were prognostically relevant and PFS and OS were significantly lower for heterogenous tumors and numerically lower for homogenous tumors with low gene dosage after multivariable adjustment (Table).
Conclusions:
HER2 gene dosage and heterogeneity significantly impact outcomes in HER2+ GEA. More importantly by deconvoluting clinical HER2 FISH data at single cell level, we demonstrate an objective metric with prognostic value that could be adopted for routine clinical use and for patient stratification. Further insights into differing biology of these molecular subtypes are needed.

Outcomes of patients by HER2 heterogeneity and gene dosage. Hazards ratio (HR) and p values are adjusted using multivariable cox proportional hazard regression model.

PFS
OS

Patients, No.
Median, mo
HR (95%CI)

P
Value

Median, mo
HR (95%CI)

P
Value

HH
32
16.2
1

16.4
1

HL
30
12.1
1.70 (0.79-3.66)
0.173
15.7
1.71 (0.93-3.14)
0.085

Het
15
6.7
3.12 (1.35-7.2)
0.008
12.4
2.72 (1.28-5.75)
0.009