ABSTRACT
OBJECTIVES: Guidelines suggest that secondary reflex testing may be useful for resolving HER2 status in breast cancers with equivocal results by both immunohistochemistry (IHC) and in situ hybridization (ISH). We compared two reflex ISH assays and a polymerase chain reaction (PCR) assay for this application. METHODS: Twenty-nine breast cancers with equivocal IHC and ISH results were retested two ways: (1) ISH using differentially labeled probes targeting ERBB2 ( HER2 , 17q12) and either RAI1 (17p11.2) or ORC4 (2q22.3-2q23.1) in two separate assays and (2) real-time quantitative PCR amplification of ERBB2 and a control locus ( EIF5B , 2q11.2). RESULTS: Results of the HER2 / RAI1 and HER2 / ORC4 ISH assays were concordant for 21 (72%) cases, and results of all three secondary reflex assays were concordant for only 18 (62%) cases. Result discrepancies between the two ISH readers were observed for cases close to the cutoff threshold. CONCLUSIONS: Use of different control loci for ISH is associated with discordant results, and PCR is more likely to classify cases as nonamplified, possibly due to contamination with nontumor cells. While resolution of HER2 -equivocal results is desirable from a clinical perspective, different secondary reflex assays yield different results, and the correlation of these results with clinical outcomes is unknown.
Subject(s)
Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Receptor, ErbB-2/metabolism , Breast Neoplasms/diagnosis , Breast Neoplasms/pathology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Female , Humans , Immunohistochemistry , In Situ Hybridization , Origin Recognition Complex/genetics , Origin Recognition Complex/metabolism , Real-Time Polymerase Chain Reaction , Receptor, ErbB-2/genetics , Reflex , Trans-Activators , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
OBJECTIVES: Guidelines for HER2 testing define an equivocal range for HER2 using two approved testing methods, immunohistochemistry (IHC) and in situ hybridization (ISH). We investigated genome-wide copy number alterations in this subgroup. METHODS: Ten breast cancers with equivocal HER2 status by both IHC and ISH were analyzed by single-nucleotide polymorphism cytogenomic microarray (SNP array). DNA ploidy analysis by flow cytometry was performed on nine cases with sufficient material remaining. RESULTS: SNP array analysis showed uniform gain of chromosome 17 (polysomy) in one case and segmental copy number gains encompassing HER2 and the centromere in five other cases. Flow cytometry revealed hyperdiploidy in six cases, all but one of which also had HER2 gains on SNP array. Although there was no evidence of HER2 amplification by SNP array, six cases showed amplification of other genomic regions, including known oncogenes in four cases. CONCLUSIONS: A combination of hyperdiploidy and segmental copy number gains contributes to HER2 ISH-equivocal results in most breast cancers. Cases in which HER2 copy number gain is not corroborated by genomic analysis suggest the presence of other contributing variables influencing ISH results. Genomic copy number analysis also implicates non-HER2 oncogenic drivers in many cases that are HER2 equivocal.
Subject(s)
Breast Neoplasms/genetics , Carcinoma, Ductal, Breast/genetics , Gene Amplification , Receptor, ErbB-2/genetics , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/pathology , Chromosomes, Human, Pair 17 , Comparative Genomic Hybridization , DNA Copy Number Variations , Female , Flow Cytometry , Humans , Polymorphism, Single NucleotideABSTRACT
UroVysion FISH detects chromosomal aberrations associated with urothelial carcinoma. In our laboratory, UroVysion FISH was initially evaluated manually with a change to image-aided interpretation using the BioView Duet imaging system. This retrospective study examined diagnostic findings over an 8.6 year period, with 1,869 manual interpretations over 4.8 years and 3,936 image-aided interpretations over 3.8 years. Although the initial goal was to evaluate possible impacts of the imaging system on diagnostic interpretations, the most important finding was that the demographics of the test population changed significantly. Female specimens increased incrementally from an average of 29% compared to 43% of the samples during periods of manual interpretation versus image-aided interpretation, respectively. The shift may reflect a gradual increase in the percentage of low-risk hematuria patients being evaluated for initial diagnosis of bladder cancer, rather than bladder cancer recurrence. Interpretation rates, evaluated separately for males and females, changed significantly over the test period. Male interpretation results were negative (75.1 vs. 67%), positive (18.6 vs. 14.6%), unsatisfactory (5.0 vs. 16.9%), and equivocal (1.4 vs. 1.5%) during periods of manual versus image-aided interpretation, respectively (Fisher Exact Test P-value = <0.0001). For females, results were negative (86.1 vs. 79.3%), positive (9.2 vs. 11.1%), unsatisfactory (2.8 vs. 8.9%), and equivocal (1.8 vs. 0.7%) over the same periods (Fisher Exact Test P-value = <0.0001). Logistic regression analysis identified the change in test population as the variable with the greatest impact on observed interpretation rate changes.
