Press MF, et al. - Given that, for HER2 testing by fluorescence in situ hybridization (FISH), an “equivocal” category (average HER2 copies per tumor cell ≥4-6 with HER2/CEP17 ratio <2.0) to be resolved as negative or positive by assessments with alternative control probes has been designated by the 2013/2014 American Society of Clinical Oncology and College of American Pathologists (ASCO-CAP) guidelines, researchers investigated whether genetic loci used as alternative controls are heterozygously deleted in a substantial proportion of breast cancers and whether false-positive assessments could result from using these loci for HER2 evaluation by FISH. They also investigated if there was any difference in outcomes between these HER2 false-positive breast cancer patients and patients with HER2-negative breast cancer. Findings revealed that, false-positive interpretations of HER2 status may happen with the indiscriminate use of alternative control probes to calculate HER2 FISH ratios in HER2-equivocal breast cancers. This was attributed to unrecognized heterozygous deletions in 1 or more of these alternative control genomic sites and incorrect HER2 ratio determinations.

Methods

• Included subjects were patients whose data were available via Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and whose tissues were available via the Breast Cancer International Research Group clinical trials.
• Retrospective assessment was carried out for the use of chromosome 17 p-arm and q-arm alternative control genomic sites (TP53, D17S122, SMS, RARA, TOP2A), as recommended by the 2013/2014 ASCO-CAP guidelines for HER2 testing.
• Data anlaysis was carried out using data from an international cohort database of invasive breast cancers (1980 participants) and international clinical trial of adjuvant chemotherapy in invasive, node-positive breast cancer patients.
• They evaluated frequency of heterozygous deletions in chromosome 17 genomic sites used as FISH internal controls for assessment of HER2 status among HER2-equivocal cancers.
• They also characterized influence of using deleted sites for determination of HER2-to-internal-control-gene ratios.
• Furthermore, evaluations were carried out for HER2 protein expression in each subgroup and for clinical outcome comparisons for each subgroup.

Results

• Completely assessed patients were 1,915 of 1,980 patients in METABRIC.
• Analysis included 100 HER2-equivocal breast cancers by FISH and 100 comparator FISH-negative breast cancers from the BCIRG-005 trial.
• In both HER2-amplified and HER2-not-amplified breast cancers, they commonly found heterozygous deletions, particularly in specific p-arm sites.
• Findings revealed high rates of false-positive ratios (HER2-to-alternative control ratio ≥2.0) resulting from use of alternative control probes from these regions to assess HER2 by FISH in HER2-equivocal as well as HER2-not-amplified breast cancers, this was attributed to heterozygous deletions of control p-arm genomic sites used in ratio denominators.
• In breast cancers with ASCO-CAP equivocal status and in breast cancers with an average of fewer than 4.0 HER2 copies per tumor cell, misclassification of HER2 status was observed when using alternative control probes.