Assessment of ERBB2/HER2 Status in HER2-Equivocal Breast Cancers by FISH and 2013/2014 ASCO-CAP Guidelines

Abstract

Importance: The 2013/2014 American Society of Clinical Oncology and College of American Pathologists (ASCO-CAP) guidelines for HER2 testing by fluorescence in situ hybridization (FISH) designated 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. Approximately 4% to 12% of all invasive breast cancers are characterized as HER2-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 of HER2 by FISH leads to false-positive assessments; and (3) these HER2 false-positive breast cancer patients have outcomes that do not differ from clinical outcomes for patients with HER2-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 for HER2 testing, 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 of HER2 status among HER2-equivocal cancers; (2) characterize impact of using deleted sites for determination of HER2-to-internal-control-gene ratios; (3) assess HER2 protein 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, 100 HER2-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 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) 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 4.0 HER2 copies per tumor cell when using alternative control probes.

Conclusions and relevance: The indiscriminate use of alternative control probes to calculate HER2 FISH ratios in HER2-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 incorrect HER2 ratio determinations.

Figure 1.. Participant Flow Diagram and Specimen Accountability

Breast cancers from patients were evaluated in 1 of 2 central laboratories (laboratory) as either human epidermal growth factor receptor 2 gene HER2-not-amplified or HER2-amplified for eligibility to 1 of 3 concurrently conducted clinical trials (BCIRG-005, BCIRG-006, and BCIRG-007). One of the trials, BCIRG-005, required patients whose breast cancers were HER2-not-amplified and the other 2 trials, BCIRG-006 and BCIRG-007, required patients whose breast cancers were HER2-amplified, as determined with fluorescent in situ hybridization (FISH). Although 10 948 patients were screened in the Breast Cancer International Research Group central laboratories for trial accrual, complete HER2 FISH assay results were available from 10 468 patients for a variety of reasons, including lack of invasive carcinoma in samples submitted, tissue sections that detached from slides during processing, and FISH assay failure owing to lack of probe hybridization. BCIRG-005 randomized patients with HER2-not-amplified breast cancers to sequential (arm 1) or concurrent (arm 2) anthracycline, cyclophosphamide, and docetaxel chemotherapy. BCRIG-006 randomized patients with HER2-amplified breast cancers to standard anthracycline-containing chemotherapy (arm 1, AC-T) alone, AC-T with trastuzumab (arm 2, ACTH) or a nonanthracycline chemotherapy regimen with trastuzumab (arm 3, TCH). The breast cancers from these trials were subsequently pooled according to the ASCO-CAP guidelines for HER2 testing by FISH as recommended into 5 in situ hybridization (ISH) groups, identified in the lower portion of the figure (ASCO-CAP ISH group 5, ASCO-CAP ISH group 4, ASCO-CAP ISH group 3, ASCO-CAP ISH group 2, and ASCO-CAP ISH group 1) and reanalyzed for correlations with HER2 protein expression and clinical outcomes. Since ASCO-CAP ISH group 4 is composed exclusively of HER2-equivocal breast cancers by FISH, the focus of the current investigation, group 4 served as the source of all HER2-equivocal breast cancers (N = 100) characterized in this study by FISH with alternative control probes. As a comparator group, ASCO-CAP ISH group 5, breast cancers were selected for similar analyses by FISH using the same alternative control probes (N = 100). AC-T indicates anthracycline, cyclophosphamide, and docetaxel; ACTH, anthracycline, cyclophosphamide, docetaxel, and trastuzumab; TAC, taxotere, docetaxel, and cyclophosphamide; TCH, docetaxel, carboplatin, and trastuzumab.

Figure 2.. Relative Copy Number of ERBB2 and Genomic Sites Used as Alternate Controls for Assessment of HER2 Status by FISH (METABRIC Cohort, SNP Chip Data for 1980 Patients)

A, Schematic illustration of the positions of alternative control genomic sites (p-arm: LIS1, TP53, D17S122, RAI1, SMS; and q-arm: RARA, TOP2A) relative to ERBB2/HER2 on chromosome 17. The location of chromosome 17 centromere is highlighted in red. B, Relative copy number of ERBB2/HER2 and genomic sites used as alternative controls for assessment of HER2 Status by FISH (METABRIC SNP array data for 1980 patients). Samples were ordered by their HER2 CN value and plotted alongside the copy number profiles for alternative probes. A linear regression line was fit for each gene (probe). Top bar shows annotations for samples based on IHC, ASCAT, and GISTIC.

Figure 3.. Comparison of Clinical Outcomes for ASCO-CAP Group 4 (HER2-Equivocal) and ASCO-CAP Group 5 (HER2-Negative) Patients With Breast Cancer

A, Disease-free survival of ASCO-CAP FISH group 4 (HER2-equivocal) compared with ASCO-CAP FISH group 5 (HER2-not-amplified). There was no significant difference in disease-free survival for the 100 patients with ASCO-CAP FISH group 4 (HER2-equivocal) breast cancers compared with the 100 with ASCO-CAP FISH group 5 (HER2-not-amplified) breast cancer. B, Overall survival of ASCO-CAP FISH group 4 (HER2-equivocal) compared with ASCO-CAP FISH group 5 (HER2-not-amplified) patients with breast cancer. There was no significant difference in overall survival for the 100 patients with ASCO-CAP FISH group 4 (HER2-equivocal) breast cancers compared with the patients with 100 ASCO-CAP FISH group 5 (HER2-not-amplified) breast cancer. C, ASCO-CAP FISH group 4 (HER2-equivocal): OS for Alternative Control Probe D17S122 (HER2/D17S122) ratios ≥2.0 vs <2.0. Among women with HER2-equivocal breast cancers with an D17S122 alternative control probe ratio ≥2.0 does not identify a subgroup with a worse overall survival. D, ASCO-CAP FISH group 4 (HER2-equivocal): OS for alternative control probe SMS (HER2/SMS) ratios ≥2.0 vs <2.0. Among women with HER2-equivocal breast cancers, those who had a SMS alternative control probe ratio ≥2.0 appear to have a slightly better overall survival than those whose breast cancers had a SMS alternative control probe ratio <2.0; however, this difference was not significant.