HER2 Gene Amplification Testing by Fluorescent In Situ Hybridization (FISH): Comparison of the ASCO-College of American Pathologists Guidelines With FISH Scores Used for Enrollment in Breast Cancer International Research Group Clinical Trials

Abstract

Purpose ASCO and the College of American Pathologists (ASCO-CAP) recently recommended further changes to the evaluation of human epidermal growth factor receptor 2 gene (HER2) amplification by fluorescent in situ hybridization (FISH). We retrospectively assessed the impact of these new guidelines by using annotated Breast Cancer International Research Group (BCIRG) -005, BCIRG-006, and BCIRG-007 clinical trials data for which we have detailed outcomes. Patients and Methods The HER2 FISH status of BCIRG-005/006/007 patients with breast cancers was re-evaluated according to current ASCO-CAP guidelines, which designates five different groups according to HER2 FISH ratio and average HER2 gene copy number per tumor cell: group 1 (in situ hybridization [ISH]-positive): HER2-to-chromosome 17 centromere ratio ≥ 2.0, average HER2 copies ≥ 4.0; group 2 (ISH-positive): ratio ≥ 2.0, copies < 4.0; group 3 (ISH-positive): ratio < 2.0, copies ≥ 6.0; group 4 (ISH-equivocal): ratio < 2.0, copies ≥ 4.0 and < 6.0; and group 5 (ISH-negative): ratio < 2.0, copies < 4.0. We assessed correlations with HER2 protein, clinical outcomes by disease-free survival (DFS) and overall survival (OS) and benefit from trastuzumab therapy (hazard ratio [HR]). Results Among 10,468 patients with breast cancers who were successfully screened for trial entry, 40.8% were in ASCO-CAP ISH group 1, 0.7% in group 2; 0.5% in group 3, 4.1% in group 4, and 53.9% in group 5. Distributions were similar in screened compared with accrued subpopulations. Among accrued patients, FISH group 1 breast cancers were strongly correlated with immunohistochemistry 3+ status (P < .0001), whereas groups 2, 3, 4, and 5 were not; however, groups 2, 4 and, 5 were strongly correlated with immunohistochemistry 0/1+ status (all P < .0001), whereas group 3 was not. Among patients accrued to BCIRG-005, group 4 was not associated with significantly worse DFS or OS compared with group 5. Among patients accrued to BCIRG-006, only group 1 showed a significant benefit from trastuzumab therapy (DFS HR, 0.71; 95% CI, 0.60 to 0.83; P < .0001; OS HR, 0.69; 95% CI, 0.55 to 0.85; P = .0006), whereas group 2 did not. Conclusion Our findings support the original categorizations of HER2 by FISH status in BCIRG/Translational Research in Oncology trials.

Trial registration: ClinicalTrials.gov NCT00021255 NCT00312208.

Conflict of interest statement

Authors’ disclosures of potential conflicts of interest are found in the article online at www.jco.org. Author contributions are found at the end of this article.

Figures

Fig 1.

Specimen accountability on the basis of the CONSORT statement. Breast cancers from patients were evaluated in one of two central laboratories as either human epidermal growth factor receptor 2 gene (HER2) not amplified or HER2 amplified for eligibility to one of three concurrently conducted clinical trials (BCIRG-005, BCIRG-006, AND BCIRG-007). One of the trials, BCIRG-005, required patients whose breast cancers wereHER2 not amplified and the other two trials, BCIRG-006 and BCIRG-007, required patients whose breast cancers were HER2gene 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, completeHER2 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 as a result of lack of probe hybridization. AC-T, anthracycline, cyclophosphamide, and docetaxel; ACTH, anthracycline, cyclophosphamide, docetaxel, and trastuzumab; TAC, taxotere, docetaxel, and cyclophosphamide. TCH, docetaxel, carboplatin, and trastuzumab.

Fig 2.

Schematic diagram of the ASCO and College of American Pathologists (ASCO-CAP) algorithm for human epidermal growth factor receptor 2 (HER2) testing by fluorescent in situ hybridization (FISH) as published by the ASCO-CAP guidelines committee,, modified here by introduction of the numbers 1 to 5 to identify the various ASCO-CAP FISH groups categorized, followed by FISH and immunohistochemistry (IHC) photomicrographs of representative cases from each of the five groups. (A) Breast cancers withHER2-to-chromosome 17 centromere (CEP17) ratios ≥ 2.0 are divided in two groups, one with an average HER2 gene copy number per tumor cell ≥ 4.0 (in situ hybridization [ISH] positive; our group 1) and one with an average HER2 gene copy number per tumor cell < 4.0 (ISH positive; our group 2). Breast cancers withHER2-to-CEP17 ratios < 2.0 are separated into three additional groups: one with average HER2 gene copy number per tumor cell ≥ 6.0 (ISH positive; our group 3), another with averageHER2 gene copy number per tumor cell ≥ 4.0 but < 6.0 (ISH equivocal; our group 4), and one with breast cancers that contained an average HER2 gene copy number per tumor cell < 4.0 (ISH negative; our group 5). Therefore, according to the ASCO-CAP guidelines, breast cancers in groups 1, 2, and 3 are interpreted as ISH positive, group 4 as ISH equivocal, and group 5 as ISH negative. (B-M) ASCO-CAP guidelines algorithm ISH groups compared with observed HER2 gene amplification status by FISH and HER2 protein expression status by IHC staining using the DAKO HercepTest IHC assay. ASCO-CAP guidelines algorithm identification of subdivisions byHER2 FISH ratios and average HER2 gene copy number into group 1 is categorized as ISH positive, with results as illustrated in panels B (FISH) and C (IHC); group 2 is also categorized as ISH positive, but with our contradictory results as illustrated in panels D (FISH) and E (IHC); group 3 is categorized as ISH positive, but with mixed results as illustrated in panels F (FISH), G (IHC), H (FISH), and I (IHC); group 4 is categorized as ISH equivocal, but with contradictory results as illustrated in panels J (FISH) and K (IHC); and group 5 is categorized as ISH negative, with confirmatory results as illustrated in panels L (FISH) and M (IHC). (B) ASCO-CAP group 1 breast cancer with HER2 gene amplification by FISH, consistent with the ASCO-CAP guidelines designation of ISH positive (and Breast Cancer International Research Group [BCIRG] designation ofHER2 amplified). Average HER2 gene copy number for this case was 16.85 copies per tumor cell, and the CEP17 copy number per cell was 2.28 with a HER2-to-CEP17 FISH ratio of 7.38.HER2 signals are sufficiently numerous and are not captured in a single plain of focus in this photomicrograph so that some appear out of focus. Computer enhancement was not used for any image (BCIRG01661, original photomicrograph at 1,000×). (C) ASCO-CAP group 1 breast cancer case with HER2 protein overexpression, IHC3+ by the HercepTest IHC assay (BCIRG01661, original magnification, ×400). (D) ASCO-CAP group 2 breast cancer. Average HER2 gene copy number for this breast cancer was 3.75 copies per tumor cell, with a CEP17 copy number of 1.80 per cell and aHER2-to-CEP17 FISH ratio of 2.08. This breast cancer was evaluated in the BCIRG/Translational Research in Oncology (TRIO) central laboratory as HER2 not amplified by FISH, which contradicted the ASCO-CAP guidelines designation of ISH positive, and the patient was accrued to the BCIRG-005 trial. Of 52 patients whose breast cancers were in this group, three were accrued to BCIRG-005 and 46 were accrued to BCIRG-006 (BCIRG02899, original magnification, ×1,000). (E) ASCO-CAP group 2 breast cancer, corresponding to the breast cancer in panel D, with HER2 protein expression determined as IHC0 with HercepTest IHC assay, which contradicted the ASCO-CAP guidelines designation of ISH positive (BCIRG02899, original magnification, ×400). (F) ASCO-CAP group 3 breast cancer. One of our group 3N cases was reported to have a lack of HER2 gene amplification by FISH in the BCIRG/TRIO central laboratory, contrary to the current ASCO-CAP guidelines designation of ISH positive. AverageHER2 gene copy number for this breast cancer was 7.35 copies per tumor cell, average CEP17 copy number was 4.20 per cell, and, therefore, there was a HER2-to-CEP17 FISH ratio of 1.75 (BCIRG04086, original magnification, ×1,000). (G) ASCO-CAP group 3 breast cancer. Our Group3N, with low HER2 protein expression by IHC (IHC0/1+), reported previously as HER2 not amplified, contrary to the current ASCO-CAP guidelines designation of ISH positive (BCIRG04086, original magnification, ×400). (H) ASCO-CAP group 3 breast cancer, one of the BCIRG group 3A cases, with an average HER2 gene copy number of 27.50 per tumor cell, an average CEP17 copy number of 20.67 per tumor cell, and, therefore, a HER2 FISH ratio of only 1.33. Please note that the HER2 gene signals (orange) and CEP17 signals (green) are aggregated together in a limited geographic area of the nucleus, making assessment of individual signals challenging without the aid of single band-pass filters (Data Supplement Figure S1). This breast cancer was reported as HER2 amplified in the BCIRG/TRIO central laboratory, and the patient was accrued to BCIRG-006. This case is consistent with the ASCO-CAP guidelines designation of ISH positive (BCIRG00575, original magnification, ×1,000). (I) ASCO-CAP group 3 breast cancer, the same group3A in panel H, with HER2 protein overexpression by IHC (IHC3+ by HercepTest), consistent with the ASCO-CAP guidelines designation of ISH positive (BCIRG00575, original magnification, ×400; Data Supplement Figure S1E). (J) ASCO-CAP group 4 breast cancer, referred to by the current ASCO-CAP guidelines as ISH equivocal. BCIRG/TRIO central laboratory reported the case as HER2 not amplified by FISH, with an average HER2 gene copy number of 4.22 per tumor cell, an average CEP17 copy number of 2.23 per tumor cell, and, therefore, an HER2-to-CEP17 FISH ratio of 1.89. The patient was randomly assigned to BCIRG-005 (BCIRG01911, original magnification, ×1,000). (K) ASCO-CAP group 4 breast cancer, as in panel J, with low HER2 protein expression by HercepTest (IHC0; BCIRG01911, original magnification, ×400). (L) ASCO-CAP group 5 breast cancer, consistent with the guidelines designation of ISH negative, which was reported by the BCIRG/TRIO central laboratory as HER2 not amplified by FISH. The case had an average HER2 gene copy number of 1.35 per tumor cell, with 1.50 CEP17 copies per cell and anHER2-to-CEP17 ratio of 0.90 (BCIRG04095, original magnification, ×1,000). (M) ASCO-CAP group 5 breast cancer, see panel L, with low HER2 protein expression by IHC with HercepTest (IHC0), consistent the ASCO-CAP guidelines designation of ISH negative (BCIRG04095, original magnification, ×400). This figure has been modified with permission from Figure 3 of the previously published article by Wolff et al. Copyright 2013 American Society of Clinical Oncology.

Fig 3.

Distribution of average human epidermal growth factor receptor 2 gene (HER2) copy number and HER2 FISH ratios among breast cancers successfully screened for enrollment into Breast Cancer International Research Group trials from 2000 to 2004. (A) Plot of averageHER2 gene copy number per tumor cell nucleus from lowest to highest, with cases identified according to the ASCO and College of American Pathologists (ASCO-CAP) guidelines as groups 1 (blue), 2 (purple), 3 (green), 4 (orange), and 5 (yellow; N = 10,468. (B) Plot of HER2 FISH ratios from lowest to highest, as in panel A, with identification of ASCO-CAP groups 1 (blue), 2 (purple), 3 (green), 4 (red), and 5 (yellow; N = 10,468).