Determination of Her-2/Neu Status in Breast Carcinoma: Comparative Analysis of Immunohistochemistry and Fluorescent In Situ Hybridization

Abstract

Her-2/neu (H2N) status in breast carcinoma has been considered a prognostic factor that may have therapeutic implications; however, the correlation between H2N overexpression and gene amplification has not been completely defined. A consecutive series of ductal carcinomas (34 invasive and 7 in situ) were analyzed by fluorescent in situ hybridization for H2N gene and chromosome 17 copy number using touch preps of intact cells and by immunohistochemistry, using three different commercial antibodies to H2N protein (Zymed, clone 31G7; Ventana, clone CB11; and Dako, polyclonal) in corresponding formalinfixed, paraffin-embedded tissue sections. Gene amplification was classified as unequivocal if more than five signals were present in more than 80% of the counted nuclei and absent if more than 80% of the nuclei counted contained two or fewer gene copies. Cases that did not fulfill the above criteria were considered equivocal for amplification. Immunostaining was classified as follows: 0 ‫؍‬ no staining; 1؉ ‫؍‬ faint, incomplete membranous pattern; 2؉ ‫؍‬ moderate, complete membranous pattern; 3؉ ‫؍‬ strong membranous pattern. Of the 34 invasive tumors, 10 (29%) had unequivocal gene amplification. Furthermore, all had more than 10 copies of the gene in more than 60% of the counted nuclei. An additional nine cases (26%) had equivocal amplification, which was usually the result of chromosome 17 aneuploidy (seven of nine) or heterogeneity. With the Zymed and Dako antibodies, all tumors with 3؉ staining had unequivocal gene amplification and all cases with 2؉, 1؉, or 0 staining were negative or equivocal for gene amplification. Her-2/neu (H2N) status in breast carcinoma has long been implicated as a prognostic biomarker (1, 2), and recent studies suggest that it may be a determinant of response to adjuvant therapy (3, 4). Thus, determination of H2N status in breast cancer

Defining a Test for HER-2/neu Evaluation in Breast Cancer in the Diagnostic Setting

“…Press et al (26) found FISH to be 98% sensitive and 100% specific compared with PCR and that FISH results were an independent predictor of poor outcome. Similar data showing concordance between IH and FISH were obtained by P. Jimminez et al (27) and Jacobs et al (28). These studies suggest that very few cases (3-5%) show protein overexpression without gene amplification indicating that FISH or PCR could be false negative in about 5% of cases.…”

Strong Correlation between Results of Fluorescent In Situ Hybridization and Immunohistochemistry for the Assessment of the ERBB2 (HER-2/neu) Gene Status in Breast Carcinoma

“…Farabegoli et al (28), obtained a correlation of only 72% between FISH and IHC in a study of 79 primary invasive breast carcinoma samples, because of the observation of an excess of immunopositive cases without amplification. Similarly, Jimenez et al (32) observed, in a comparative study of 41 cases, a proportion of 3 to 15% of cases, according to the antibody used, showing moderate immunostaining without amplification. Jacobs et al (29), in a study of 100 consecutive paraffinembedded breast carcinomas samples, demonstrated a concordance of 92% between the results of FISH and IHC, using the Oncor Inform kit and the Dako A485 polyclonal antibody.…”

“…Slamon et al (2) have shown the existence of a direct correlation between amplification and overexpression of ERBB2. However, Press et al (32) have stressed the variability of the sensitivity of HER-2/neu antibodies as potential source of errors in the assessment of the expression of the gene. The present controversy about the reliability of a U. S. Food and Drug Administration-approved HER-2/ neu IHC test (33)(34) illustrates that this issue remains incompletely solved.…”

“…Indeed, results of molecular methods may be affected by the dilution of tumor material by normal cells and by the determination of the threshold defining the existence of an amplification or an overexpression (2,7,21). Recent technical developments have made possible the detection of the ERBB2 gene in tissue sections, touch preparations, and fine-needle aspirates using fluorescent in situ hybridization (FISH) (16,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). The most reliable method to routinely evaluate the status of ERBB2 in prospective and archival material remains to be determined.…”

Protein Overexpression and Gene Amplification of c-erbB-2 in Pulmonary Carcinomas: A Comparative Immunohistochemical and Fluorescence In Situ Hybridization Study

“…This score is basically similar to those used for breast cancers (17, 18) except 1ϩ. Many studies have chosen to ignore the cytoplasmic staining (18,19); however, we tentatively included this in 1ϩ, because it was virtually impossible to differentiate faint membranous staining from cytoplasmic staining in several cases and to compare previous findings using similar standards.…”