Abstract PD3-11:HER2/ERBB2status in “HER2equivocal” breast cancers by FISH and ASCO-CAP guidelines: False-positives due to heterozygous deletions of alternative control loci

Background. The ASCO-CAP guidelines for HER2 testing by fluorescence in situ hybridization (FISH) have a category, referred to as “equivocal” (average HER2 copies per tumor cell >4-6 with HER2/CEP17 ratio <2·0), which is neither “HER2-positive” nor “HER2-negative”. Approximately 4% - 12% of invasive breast cancers are “HER2-equivocal” based on FISH. Cancers in this category may be resolved as “negative” or “positive” by FISH alternative control probes (2013/2014 guidelines) or HER2 immunohistochemistry (IHC) (2018 update). Our objectives were to evaluate the following hypotheses: 1.) Genetic loci used as alternative controls show heterozygous deletion in a substantial proportion of breast cancers; 2.) Use of these loci for assessment of HER2 by FISH leads to false-positives; 3.) HER2 FISH false-positive breast cancer patients have outcomes that do not differ from clinical outcomes for HER2-negative breast cancer patients; and 4.) HER2-equivocal breast cancers seldom show HER2 protein overexpression (IHC 3+). Methods. 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, in patients whose data were available through the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC)(N=1980) or whose tissues were available from the BCIRG-005 clinical trial (N=3298). We used either FDA-approved HER2 IHC (HercepTest) or laboratory-developed HER2 (10H8) IHC assays to assess HER2 protein expression. Results. Using METABRIC we found heterozygous deletions, particularly in specific p-arm sites, were common in both HER2-amplified and HER2-not-amplified breast cancers. Use of alternative control probes from these regions to assess HER2 by FISH in “HER2 equivocal” as well as HER2-not-amplified breast cancers resulted in high rates of false-positive ratios (HER2-to-alternative control ratio >2·0) due to heterozygous deletions of control p-arm genomic sites used as ratio denominators. Misclassifications of HER2 status was observed not only in breast cancers with ASCO-CAP “equivocal” status but also in breast cancers with an average of <4·0 HER2 copies per tumor cell. These deletions were also identified by FISH. IHC demonstrated <1% of FISH “HER2-equivocal” breast cancers in BCIRG-005 had IHC3+ immunostaining, consistent with HER2-not-amplified status. Clinical outcomes of “HER2-equivocal” breast cancer patients with HER2-to-alternative control ratio >2·0 did not differ significantly from clinical outcomes of those with HER2-to-alternative control ratio<2·0. Conclusion. Using chromosome 17 p-arm alternative controls, as recommended by 2013/2014 ASCO-CAP guidelines, instead of CEP17 for resolution of “HER2 equivocal” cases, is problematic due to frequent heterozygous deletions of these loci in breast cancers. The indiscriminate use of alternative control probes to calculate a HER2 FISH ratio in “HER2-equivocal” breast cancers leads to false-positive interpretations of HER2 status resulting from unrecognized heterozygous deletions in one or more of these alternative control genomic sites and incorrect HER2 ratio determinations. Citation Format: Press MF, Seoane JA, Curtis C, Quinaux E, Guzman R, Sauter G, Eiermann W, Mackey JR, Robert N, Pienkowski T, Crown J, Martin M, Valero V, Bee V, Ma Y, Villalobos I, Slamon DJ. HER2/ERBB2 status in “HER2 equivocal” breast cancers by FISH and ASCO-CAP guidelines: False-positives due to heterozygous deletions of alternative control loci [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD3-11.