Is Fluorescence In Situ Hybridization Really Superior to HercepTest?
Authors: Peter Birner, Georg Oberhuber, Michael F. Press, Leslie Bernstein, Kerry Flom, Dennis Slamon
Journal: Journal of Clinical Oncology (2002)

Article Tools SPECIAL DEPARTMENTS Article Tools OPTIONS & TOOLS Export Citation Track Citation Add To Favorites Rights & Permissions COMPANION ARTICLES No companion articles ARTICLE CITATION DOI: 10.1200/JCO.2002.99.178 Journal of Clinical Oncology - published online before print September 21, 2016 PMID: 12454125 Is Fluorescence In Situ Hybridization Really Superior to HercepTest? Peter BirnerxPeter BirnerSearch for articles by this author , Georg OberhuberxGeorg OberhuberSearch for articles by this author Michael F. PressxMichael F. PressSearch for articles by this author , Leslie BernsteinxLeslie BernsteinSearch for articles by this author , Kerry FlomxKerry FlomSearch for articles by this author , Dennis SlamonxDennis SlamonSearch for articles by this author Show More Clinical Institute of Pathology, University of Vienna, Vienna, AustriaUniversity of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CAUniversity of Southern California, Los Angeles, CAAbbott-Vysis, Inc, Downers Grove, ILUniversity of California Los Angeles School of Medicine, Los Angeles, CA https://doi.org/10.1200/JCO.2002.99.178 First Page Full Text PDF Figures and Tables © 2002 by American Society of Clinical OncologyjcoJ Clin OncolJournal of Clinical OncologyJCO0732-183X1527-7755American Society of Clinical OncologyResponse01122002In Reply:Birner et al1 compared immunohistochemistry (IHC) staining for HER-2/neu protein with fluorescence in situ hybridization (FISH) for gene amplification in a group of 207 node-positive breast cancers. None of these 207 breast cancers had been previously molecularly characterized using any other technique. Therefore, FISH served as the gold standard for comparison of four different IHC assay methods. It is important to note that Birner et al used an unconventional approach to assessing the final HER-2/neu gene amplification status. Instead of determining the amplification status based on the overall ratio of average HER-2/neu gene signals in tumor cells divided by average chromosome 17 centromere signals, with a ratio of ≥ 2.0 scored as amplified, as required by the United States Food and Drug Administration’s approval of this method of assessment, the investigators scored breast cancer specimens as “negative when less than 10% of tumor cells showed amplification of HER-2/neu, paralleling the situation in IHC.” This is not the accepted standard method of assessment. We are not aware of any other investigator who has used this approach to FISH enumeration. Although widely used in IHC, the scientific basis for selecting 10% as a threshold for scoring is not well established. On the contrary, we2 have shown remarkable homogeneity in HER-2/neu protein immunostaining throughout the tumor cell population when frozen tissue sections are used for analysis. Heterogeneous HER-2/neu immunostaining is observed almost exclusively in fixed, paraffin-embedded tissue sections, strongly suggesting that the variability in immunostaining intensity observed from area to area within these tumor specimens is related to tissue fixation and processing for paraffin embedding, not cell-to-cell differences in protein expression. The recognized heterogeneity that does occur in FISH analysis is a methodologic issue related to sectioning. It is well recognized that a 4- to 5-μm tissue section does not account for an entire nucleus and that hybridization signals might be outside the plane of section for a 10- to 20-μm tumor cell nucleus. This problem is easily overcome by counting an adequate number of nuclei, as previously published.3-5 We believe that applying a 10% cutoff to FISH without first rigorously validating this approach has no methodologic merit.In our study,6 we compared results obtained with two FISH assays and with four IHC assays in paraffin-embedded tissue sections from 117 breast cancers to the results obtained in the same breast cancers analyzed as frozen specimens using molecular methods for characterization of the HER-2/neu gene other than IHC or FISH. These molecular methods used to first determine gene amplification and gene expression were solid matrix DNA hybridization for gene amplification and Northern and Western blot analyses for gene product expression. In addition, the cohort was analyzed by IHC in frozen sections, where there is little possibility of fixation artifacts. Material from these comprehensively evaluated and fully molecularly characterized tissues were then used to perform the current analysis in paraffin-embedded tissue samples, as published.6 Our gold standard was, therefore, HER-2/neu status determined by comprehensive molecular characterization in frozen tissues, not FISH. As a result, our sensitivity, specificity, and accuracy assessments are quite different from those of Birner et al. We found FISH to be 96% to 97% accurate and IHC with the HercepTest (Dako, Glostrup, Denmark) and Pathway assay (Ventana Medical Systems, Inc, Tucson, AZ) to be 89% and 90% accurate, respectively. On review of Birner et al’s publication, we do not find analyses for sensitivity or specificity or accuracy rates presented. Agreement rates between IHC assay scores and FISH are included in Table 3. The kappa statistic is not a measure of correlation. It is a measure of agreement corrected for the possibility that some agreement will occur simply by chance alone. Given the data reported in the article by Birner et al, we calculate their kappa statistic to be 0.50 (95% confidence interval, 0.39 to 0.62) for FISH (negative v positive) compared with the Dako IHC assay, according to cutoffs recommended in the package insert as “positive” by the United States Food and Drug Administration (0, 1+ v 2+, 3+). In other words, following the “United States Food and Drug Administration–approved scoring and test system of HER-2 protein expression in breast cancer,” as stated in the title of the Birner et al’s article,1 leads to a HercepTest kappa statistic that is lower than the kappa statistic reported in Drs Birner and Oberhuber’s letter to the editor for IHC 3+ alone. Since we used a gold standard of breast cancers that were fully molecularly characterized for HER-2/neu as the basis for comparison of both FISH and IHC, we have determined kappa statistics for each of these assays. Birner and Oberhuber have no such independent gold standard but compare IHC to FISH. Therefore, their kappa statistic is not comparable to those published in our article.As pointed out in their letter, “Press et al . . . considered 2+ and 3+ cases at immunohistochemical analysis as positive, [Birner et al] considered only 3+ cases as positive.” It is not clear why none of the IHC 2+ cases in the Birner et al1 article show gene amplification by FISH. Other investigators find from 12% to 88% of IHC 2+ breast cancers to have HER-2/neu gene amplification.7-14 With the HercepTest, we found the highest accuracy relative to the known HER-2/neu status (determined by solid matrix blotting) when both 2+ and 3+ were considered to be “positive” for overexpression as recommended by the HercepTest package insert.6 In contrast to the results of Birner et al, we6 and other investigators7-14 do find at least some proportion of the 2+ IHC group to show gene amplification.In addition, we do not agree with Drs Birner and Oberhuber’s view that “if a [HER-2/neu] gene is amplified, it does not necessarily mean that the protein is also overexpressed, and vice versa.” Comparison of solid matrix blotting results in frozen tissue specimens has demonstrated an extremely close correlation between HER-2/neu gene amplification and HER-2/neu overexpression.2,4 In our studies of frozen tissue, we have no example of a breast cancer with HER-2/neu gene amplification by Southern hybridization that did not also have overexpression by at least two of three methods for evaluating expression (Northern hybridization, Western immunoblot analysis, and frozen section IHC). However, some of the known amplified–overexpressed breast cancers from this cohort have shown a lack of immunostaining in paraffin-embedded tissue blocks recovered from the surgical pathology laboratory.2,6,15 We consider this lack of immunostaining to be an artifact of tissue processing. Among our original series,2 we reported that 18 (10%) of 187 frozen breast cancer cases were “single-copy overexpressors,” ie, breast cancers lacking gene amplification but nevertheless having HER-2/neu overexpression. Subsequently, we have shown that these single-copy overexpressor breast cancers are predominantly stroma-rich breast cancers with relatively few carcinoma cells in the tumor specimen.4 Although they failed to demonstrate amplification by Southern hybridization, probably due to dilutional artifacts, the vast majority of these single-copy overexpressor breast cancers do have gene amplification by FISH.2,4 Ultimately, only 2% of the breast cancers in our series failed to show a close relationship between the amplification status and the expression status.4 We consider 2% to be within the range of experimental error for the methods used.4 Therefore, we do find a close correlation between HER-2/neu gene amplification and overexpression as well as a close correlation between lack of HER-2/neu amplification and lack of overexpression.Birner et al compared their FISH and IHC staining results to clinical outcome among women with node-positive breast cancer pooled from three different treatment modalities: combined adjuvant chemotherapy (44.6% of the women), combined adjuvant chemotherapy with tamoxifen (42.5% of the women), and tamoxifen alone (12.9% of women). They also found that by “univariate analysis, staining with the HercepTest revealed a significantly worse prognosis in 3+ cases.” In a multivariate analysis of overall survival, Birner et al1 found predictors to be tumor stage and tumor grade. However, their survival curves do not correct for stage or grade. In addition, Birner et al used 303 subjects for their IHC comparison with survival, whereas they used 207 subjects for their FISH comparison with survival. Given the different sample sizes, it is not appropriate to claim IHC analysis of HER-2/neu provides a significant survival difference while FISH does not. At least one of these comparisons seems to be statistically underpowered for this comparison.Our previously published results using one of the FISH assays to analyze HER-2/neu gene amplification in 326 women with node-negative breast cancers demonstrated statistically significant correlations with disease-free survival and overall survival both by univariate and multivariate analyses.5 These women were treated with surgery alone until there was a recurrence of disease. Hence, treatment effects did not complicate clinical outcome in our previous study. In addition, using the largest cohort published to date in which IHC and FISH were directly compared, Pauletti et al3 found a total of 189 (21%) of 900 patients to be positive by FISH and 147 (17.2%) of 856 to be positive by IHC. The HercepTest did not improve sensitivity and introduced false-positive results. Comparison of IHC with FISH demonstrated that patient survival was inversely associated progressively with gene amplification level as determined by FISH, whereas for IHC an association was found only in the highest (3+) immunostaining group. Among women with FISH-negative, IHC-positive tumors, the survival probability was similar to that of FISH-negative, IHC-negative patients, whereas FISH-positive, IHC-negative patients had a survival probability similar to that of FISH-positive, IHC-positive patients.Finally, and perhaps most importantly, in studies of HER-2/neu as a predictive marker of treatment response to trastuzumab, conducted in collaboration with Genentech, Inc (South San Francisco, CA) and described elsewhere,16-18 we have found FISH to be more strongly correlated with trastuzumab treatment response than the clinical trials assay, the prototype immunohistochemical assay used for both the HercepTest and Pathway assays. In fact, Food and Drug Administration approval of the HercepTest was contingent on a direct comparison of the clinical trials assay with the HercepTest to confirm that there was a minimum of a 75% correlation between the two assay methods (a 79% correlation was observed). We believe the primary goal in development of an effective molecular assay is to obtain one with a high level of sensitivity and specificity in detecting the molecular alteration in question. The most accurate assay methods should then be used to define clinical correlations.The issue of differences in assay costs raised by Drs Birner and Oberhuber, in our opinion, pale by comparison to the financial cost differential of the involved therapeutics, such as trastuzumab, used to treat women with HER-2/neu–amplified breast cancer. Misclassification of women by less expensive, less accurate assay methods also leads to substantial personal costs. Given these considerations, we believe the most accurate assay method should be used to evaluate molecular alterations in breast or other cancers without regard to minor differences in assay cost.1. Birner P, Oberhuber G, Stani J, et al: Evaluation of the United States Food and Drug Administration-approved scoring and test system of HER-2 protein expression in breast cancer. Clin Cancer Res 7:: 1669,2001-1675, Medline, Google Scholar2. Slamon D, Godolphin W, Jones L, et al: Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:: 707,1989-712, Crossref, Medline, Google Scholar3. Pauletti G, Dandekar S, Rong H, et al: Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: A direct comparison of fluorescence in situ hybridization and immunohistochemistry. J Clin Oncol 18:: 3651,2000-3664, Link, Google Scholar4. 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