HER2 in situ hybridization test in breast cancer: quantifying margins of error and genetic heterogeneity.

The aim of the present study was to evaluate the effect of counting increasing number of invasive cancer cells in the result of the HER2 in situ hybridization (ISH) test in breast cancer as well as to compare two different approaches of measuring genomic heterogeneity (single cell and population based). A cohort of 100 consecutive breast cancer cases (primary and metastatic) were evaluated for HER2 gene amplification with bright-field ISH. The evaluation of the samples included scoring 20 nuclei, in five different areas, measuring the margins of error for each case. Genomic heterogeneity (GH) was defined by the 2018 ASCO/CAP guideline as a discrete population of tumor cells with HER2 amplification. We also evaluated GH as single tumor cells with HER2 amplification. The stabilization of the coefficient of variation of HER2/CEP17 ratio requires about 60 invasive cancer cells. The average margin of error of HER2/CEP17 ratio and of HER2 copy number was 0.40 and 0.53, respectively, when counting 20 cells, decreasing to 0.20 and 0.26 when counting 100 cells. Population GH was observed in 1% of the cases, while single cell GH was observed in 27% of the cases, reaching its maximum value in cases near the thresholds of positivity. Therefore, margins of error in HER2 ISH test are high, and the minimal cell number recommended in current guidelines should be raised to at least 60 cells. Population GH is a rare event and single cell GH is maximal in cases near the thresholds.