Inside the heterogeneous tumor tissue samples sent to clinical pathology laboratories for HER2 gene testing are cancer genomes with their own inherent complexity. The biopsy (or surgical excision) tissue represents a mixture of genomes from the tumor and normal stroma. Further compounding the heterogeneity problem, tumor genomes harboring HER2 gene amplifications may have amplified HER2 in as low as 10% of the tumor genome. Sound complicated? Yes it is! Yet in the era of precision medicine, pathologists are expected to identify all HER2 gene amplified breast and gastroesphageal (GEA) cancers in order to predict whether or not the patient will respond to specific gene-targeted therapies.
Traditionally an IHC score combined with FFPE tissue FISH has been the accepted method for determining the patient’s definitive HER2 status. However, heterogeneous tumors (for which a definitive positive or negative status cannot be assigned) have confused technologists and frustrated physicians for the past decade.
In light of the growing number of equivocal (neither negative nor positive) results, our laboratory has validated an approach that promises to resolve HER2 status in single tumor cell populations rather than as an aggregate of 50 cells randomly selected and representing bias of the technologist evaluating the case.
A total of 80 previously characterized FFPE tissue samples of Breast and GEA metastases origin were used for the study. Prior characterization was either through traditional FISH testing and/or HER2 status by IHC. The samples were dissociated into single cells, immunofluorescently stained and then sorted using DEPArray™ NxT technology to recover pure populations of stromal and tumor cells.
Fluorescence in situ hybridization (FISH) is performed only on the recovered tumor cell population in order to reveal the true status of the biopsy. Every cell on the slide is evaluated individually and an aggregate cut off for single cell amplification is defined on the basis of the counts. The scoring is in line with the CAP/ ASCO guidelines for both percent positive and the number of amplified gene copies per cell.
The method showed > 95% concordance with gold standard IHC and FISH results. Using single cells minimizes truncation or overlapping artifacts observed in traditional tissue FISH and is an effective tool to resolve heterogeneity.
The DEPArray HER2 assay can direct pathologists where to find the HER2 positive needles in a heterogenous tumor haystack and opens the door for a precision medicine approach to HER2 testing!