Immunohistochemistry and alternative FISH testing in breast cancer with HER2 equivocal amplification

Abstract

PurposeWhile HER2 testing is well established in directing appropriate treatment for breast cancer, a small percentage of cases show equivocal results by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Alternative probes may be used in equivocal cases. We present a single community-based institution’s experience in further evaluating these cases.Patients and methodsBetween 2014 and 2016, 4255 samples were submitted for HER2 amplification testing by alternative probes, TP53, RAI1, and RARA. Of the patients tested by FISH, 505/3908 (12.9%) also had IHC data.ResultsMost (73.9%) FISH equivocal cases remained equivocal after IHC testing. However, 50.5% of equivocal cases were classified as HER2 amplified by alternative probes. Most cases were positive by more than one probe: 78% of positive cases by RAI1 and 73.9% by TP53. There was a significant difference between IHC and FISH alternative testing (p < 0.0001) among the equivocal cases by conventional FISH testing, 44% of IHC negative cases became positive while 36% of the positive IHC cases became negative by alternative FISH testing. Available data showed that 41% of patients were treated with palbociclib and were positive by alternative FISH.ConclusionThe prevalence of double HER2 equivocal cases and the discrepancy between IHC and alternative FISH testing suggest that FISH alternative testing using both RAI1 and TP53 probes is necessary for conclusive classification. Because almost half of FISH equivocal cases converted to HER2 amplified upon alternative testing, clinical studies to determine the benefit of anti-HER2 therapy in these patients are urgently needed.

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

“…Several studies reported the use of alternative controls among patients whose cancers had ISH-equivocal status. 19 , 20 , 21 , 22 , 26 In these studies, if any HER2 -to-alternative probe ratio was 2.0 or more, the breast cancer was upgraded to ISH-positive. 19 , 20 , 21 , 26 …”

“…The second part involved reassessment of HER2 status in breast cancers from women accrued to BCIRG-005 (ClinicalTrials.gov Identifier NCT00312208 ) whose cancers had HER2 -to-CEP17 FISH ratios of less than 2.0 and an average of 4.0 to 5.99 HER2 genes per tumor cell (ISH-equivocal) or cancers with FISH ratios of less than 2.0 and average HER2 gene copies less than 4.0 per tumor cell (ISH-negative) using alternative control probes to reassess HER2 status, according to the 2013/2014 ASCO-CAP guidelines. 19 , 20 , 22 , 26 Finally, we assessed clinical outcomes in these subgroups.…”

“…We included all patients whose breast cancers were both ISH-equivocal by standard FISH ( HER2 /CEP17) (183 participants) ( Figure 1 ) and whose breast cancers were successfully hybridized with all alternative controls used in this study to replace CEP17 ( TP53 , SMS, D17S122, RARA , and TOP2A ) (100 participants) ( Figure 1 ) to recalculate the HER2 FISH ratio, as recommended. 13 , 14 , 19 , 21 , 22 This portion of our study is based on 100 ISH-equivocal and 100 ISH-negative cases from BCIRG-005 successfully reanalyzed with 5 alternative controls by FISH. For comparison with ISH-equivocal, we selected 100 HER2 -not-amplified breast cancers that, according to the guidelines, are ISH-negative (our ASCO-CAP ISH group 5 breast cancers 17 , 18 ) beginning with those samples that had an average HER2 gene copy number just less than 4.0 and selected every case in sequential order beginning with 3.99 copies per tumor cell until we had a comparison group of 100 cases that also had successful hybridization with all alternative control probes used in the study (100 participants) ( Figure 1 ).…”

“…This approach was widely adopted by both commercial testing laboratories and academic centers. As described, 19 , 20 , 21 , 22 when any of these alternative control probes leads to a HER2 -to-control ratio of 2.0 or more, the breast cancer is designated “ISH-positive.”…”

Next‐generation assessment of human epidermal growth factor receptor 2 gene (ERBB2) amplification status in invasive breast carcinoma: a focus on Group 4 by use of the 2018 American Society of Clinical Oncology/College of American Pathologists HER2 testing guideline

“…also detected RARA amplification with FISH in 71% (10 of 14 samples) of ERBB2 ‐amplified cases. RARA has been used as an alternative chromosome 17 probe in cases with equivocal HER2 FISH results (although this is no longer recommended by the updated ASCO/CAP guidelines); however, given its proximity to ERBB2 and resultant frequent coamplification, as shown in this study, it often provides little discriminatory value in such cases 21 . RARA is more notable for its role in the characteristic chromosomal translocation t(15;17)(q24;q21) in acute promyelocytic leukaemia, which leads to the fusion of RARA to the promyelocytic leukaemia gene ( PML ) 22–24 …”