RESUMO
BACKGROUND: Positive HER2 status identifies breast carcinomas that might respond to trastuzumab treatment. Manual HER2 fluorescent in situ hybridisation (FISH) is the most readily used method to detect HER2 gene amplification which defines positive HER2 status in addition to HER2 protein overexpression. Automation of HER2 FISH may improve HER2 gene testing. The aim of our study was to evaluate an automated HER2 FISH assay for assessing the HER2 genomic status. METHODS: Core biopsies of 100 invasive breast carcinomas were analysed in parallel using the PathVysion™ HER-2 DNA Probe Kit and the Leica HER2 FISH System for BOND™. To assess inter-method agreement, concordance analysis was performed for various numerical and categorical HER2/CEP17 FISH parameters. RESULTS: Carcinomas with all HER2 immunohistochemical scores were included (0+: 20; 1+: 20; 2+: 30; 3+: 30). Using either HER2/CEP17 ratio >2.2 or ≥2.0 as criterion for HER2 amplification, high levels of concordance were observed between automated and manual FISH (concordance rate 96%, κ coefficient 0.92). High levels of inter-method agreement were also found for HER2 copy number, CEP17 copy number, HER2/CEP17 ratio, the percentage of carcinoma cells with HER2/CEP17 ratio >2.2, and the presence of HER2 genetic heterogeneity, HER2 clusters and CEP17 polyploidy. CONCLUSIONS: HER2 testing using automated FISH is feasible on breast carcinoma core biopsies. Automated HER2 FISH using the Leica HER2 FISH System for BOND is a practical and efficient alternative to manual HER2 FISH in evaluating the HER2 status of primary invasive breast carcinomas.
RESUMO
Co-amplification of the centromere on chromosome 17 (CEP17) and HER2 can occur in breast cancer. Such aberrant patterns (clusters) on CEP17 can be misleading to calculate the HER2/CEP17 ratio, and thus underreporting of HER2 amplification. We identified 14 breast cancers retrospectively with HER2/CEP17 co-amplification and performed FISH (fluorescence in situ hybridization) with additional chromosome 17 probes (17p11.1-q11.1, 17p11.2-p12, TP53 on 17p13.1, RARA on 17q21.1-3 and TOP2 on 17q21.3-22) to characterize the spanning of the amplicon in these cases. Furthermore, the HER2 status was analyzed by means of HER2 silver in situ hybridization (SISH) and immunohistochemistry (IHC). The co-amplification of HER2/CEP17 was compared between the three institutions. TP53 was eusomic in all cases, 17p11.2-p12 in 79% (11/14), whereas 17p11.1-q11.1 showed chromosomal gain in all cases. RARA was amplified in 10/14 cases (71%) and TOP2 in 3/14 cases (21%). HER2 was amplified with FISH/SISH in all 14 cases. 9/14 tumors were 3+ IHC positive (64%) and 3 cases were 2+ IHC positive. In our cohort the CEP17 amplicon almost always involves the HER2 but not the TOP2 locus. Overall agreement on HER2/CEP17 ratio (when applying ASCO/CAP guidelines) was only 64% (9/14 cases) between the institutions. Discrepant ratios varied from 1.1 to 14.3. The HER2/CEP17 co-amplification is not defined in the ASCO/CAP guidelines, and may result in inaccurate HER2-FISH/SISH status, particularly if only the calculated HER2/CEP17 ratio is reported. It is recommended to report separate CEP17 and HER2 signals in complex HER2/CEP17 patterns.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Centrômero/genética , Cromossomos Humanos Par 17/genética , Amplificação de Genes , Receptor ErbB-2/genética , Adulto , Idoso , Antígenos de Neoplasias/genética , Neoplasias da Mama/diagnóstico , Carcinoma Ductal de Mama/diagnóstico , Variações do Número de Cópias de DNA , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Erros de Diagnóstico , Feminino , Humanos , Hibridização in Situ Fluorescente , Pessoa de Meia-Idade , Proteínas de Ligação a Poli-ADP-Ribose , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Estudos Retrospectivos , Proteína Supressora de Tumor p53/genéticaRESUMO
AIMS: To determine the frequency of HER2 genetic heterogeneity according to the recent American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) definition (2009) in invasive breast carcinoma, and to identify clinicopathological features that characterise breast carcinomas with HER2 genetic heterogeneity. METHODS: 530 invasive breast carcinomas were retrospectively analysed for HER2 genetic heterogeneity, and investigated for a potential association of HER2 genetic heterogeneity with other HER2 FISH findings, clinicopathological parameters, oestrogen/progesterone receptor expression and DNA cytometric parameters in breast carcinomas with an equivocal (2+) HER2 immunohistochemical score. RESULTS: The overall frequency of HER2 genetic heterogeneity was 14.7% in a cohort of 218 consecutive breast carcinomas. HER2 genetic heterogeneity was most frequent in invasive breast carcinomas with an equivocal (2+) HER2 immunohistochemical score. Among the 151 carcinomas lacking HER2 amplification, 16.1% showed HER2 genetic heterogeneity. In an extended cohort of 345 carcinomas with a (2+) HER2 score, the frequency of HER2 genetic heterogeneity was 41%, and was associated with the absence of HER2 gene clusters, chromosome 17 polysomy, histological tumour grade, DNA ploidy category and 5c exceeding rate. CONCLUSION: HER2 genetic heterogeneity according to the ASCO/CAP definition is frequent in breast carcinoma, and is most often present in carcinomas with an equivocal (2+) HER2 score. Many carcinomas with HER2 genetic heterogeneity have a negative HER2 amplification status, although they contain a significant number of tumour cells with HER2 gene amplification. Single cell scoring of the HER2/17 centromeric probe (CEP17) ratio is necessary to identify carcinomas with HER2 genetic heterogeneity, because they lack specific clinicopathological characteristics.
