Assessment ofERBB2/HER2Status inHER2-Equivocal Breast Cancers by FISH and 2013/2014 ASCO-CAP Guidelines

Importance The 2013/2014 American Society of Clinical Oncology and College of American Pathologists (ASCO-CAP) guidelines forHER2testing by fluorescence in situ hybridization (FISH) designated an “equivocal” category (averageHER2copies per tumor cell ≥4-6 withHER2/CEP17 ratio <2.0) to be resolved as negative or positive by assessments with alternative control probes. Approximately 4% to 12% of all invasive breast cancers are characterized asHER2-equivocal based on FISH. Objective To evaluate the following hypotheses: (1) genetic loci used as alternative controls are heterozygously deleted in a substantial proportion of breast cancers; (2) use of these loci for assessment ofHER2by FISH leads to false-positive assessments; and (3) theseHER2false-positive breast cancer patients have outcomes that do not differ from clinical outcomes for patients withHER2-negative breast cancer. Design, Setting, and Participants We retrospectively assessed 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 forHER2testing, in patients whose data were available through Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and whose tissues were available through the Breast Cancer International Research Group clinical trials. We used 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. Main Outcomes and Measures The primary objectives were to (1) assess frequency of heterozygous deletions in chromosome 17 genomic sites used as FISH internal controls for evaluation ofHER2status amongHER2-equivocal cancers; (2) characterize impact of using deleted sites for determination ofHER2-to-internal-control-gene ratios; (3) assessHER2protein expression in each subgroup; and (4) compare clinical outcomes for each subgroup. Results Of the 1980 patients in METABRIC,1915 patients were fully evaluated. In addition, 100HER2-equivocal breast cancers by FISH and 100 comparator FISH-negative breast cancers from the BCIRG-005 trial were analyzed. Heterozygous deletions, particularly in specific p-arm sites, were common in bothHER2-amplified andHER2-not-amplified breast cancers. Use of alternative control probes from these regions to assessHER2by FISH inHER2-equivocal as well asHER2-not-amplified breast cancers resulted in high rates of false-positive ratios (HER2-to-alternative control ratio ≥2.0) owing to heterozygous deletions of control p-arm genomic sites used in ratio denominators. Misclassification of HER2 status was observed not only in breast cancers with ASCO-CAP equivocal status but also in breast cancers with an average of fewer than 4.0HER2copies per tumor cell when using alternative control probes. Conclusions and Relevance The indiscriminate use of alternative control probes to calculateHER2FISH ratios inHER2-equivocal breast cancers may lead to false-positive interpretations of HER2 status resulting from unrecognized heterozygous deletions in 1 or more of these alternative control genomic sites and incorrectHER2ratio determinations.