Papouchado, Bettina G. MD*; Myles, Jonathan MD*; Lloyd, Ricardo V. MD, PhD†; Stoler, Mark MD‡; Oliveira, Andre M. MD†; Downs-Kelly, Erinn DO*; Morey, Adrienne FRCPA, Dphil§; Bilous, Michael MA, MB, ChB, FRCPA∥; Nagle, Ray MD¶; Prescott, Nichole BS*; Wang, Lin MD*; Dragovich, Lidija PhD♯; McElhinny, Abigail PhD♯; Garcia, Carole Ferrell BSN♯; Ranger-Moore, Jim PhD♯; Free, Heather MPH♯; Powell, William PhD♯; Loftus, Margaret BS*; Pettay, James BS, MT (A.S.C.P)*; Gaire, Fabien PhD♯; Roberts, Christopher♯; Dietel, Manfred MD**; Roche, Patrick PhD♯; Grogan, Thomas MD♯; Tubbs, Raymond DO*
Breast cancer is the second leading cause of cancer death in women, with approximately 200,000 new cases of invasive breast cancer diagnosed each year, and 40,000 women dying annually from the disease.10 The ERBB2 (HER2) oncogene, located on CHR17q21, is a member of the human epidermal growth factor receptor family.49 The gene product is a 185 kd protein that has an intracellular and extracellular domain with tyrosine kinase activity involved in the signal transduction of cell proliferation and development.4,17 Amplification of the HER2 oncogene is found in approximately 15% to 20% of invasive breast cancers presenting in women, and it is the primary mechanism for HER2 protein overexpression.40 Genomic amplification of HER2 is associated with more aggressive behavior in tumors and poor patient outcomes,9,44 including a higher rate of metastasis,23,43 higher mortality,36 more rapid progression, and increased likelihood of recurrence.39 HER2 amplification status is also of great importance as it predicts the likelihood of response to anthracycline-based regimens, and is being used as a treatment predictor for the humanized monoclonal antibody, trastuzumab (Herceptin), a target-specific therapy that acts by inhibiting tyrosine kinase activity.3,35,41 Therefore, it is critical that precise and reproducible assays be used in the clinical laboratory setting for determination of the HER2 status in patients with breast cancer.
The 2 methodologies in current clinical use to assess HER2 status in breast cancer are fluorescence in situ hybridization (FISH) to evaluate HER2 gene amplification and immunohistochemistry (IHC) to detect protein overexpression. IHC has been used widely in the diagnostic laboratory. It is easy to perform and relatively inexpensive. However, protein quantification by IHC analysis may be affected considerably by a number of factors, such as fixation time and processing, antigen retrieval, and antibody specificity and sensitivity. FISH has become universally accepted as a reference standard in the assessment of HER2 status and is often used to confirm equivocal results by IHC (corresponding to “2+” IHC scores). Despite many attempts by the international pathology community to improve accuracy of HER2 testing in routine practice, several peer-reviewed publications have addressed discordances between FISH and IHC assays.14,18,19,21,31,33,45
The Food Drug Administration (FDA)-approved Abbott Molecular Vysis FISH test (PathVysion) uses a direct dual-probe system to assess HER2 gene amplification as a ratio of the total HER2 signals to those of CEP17 (CHR17 centromere). The inclusion of CEP17 probe permits the recognition of the tumor cells that are aneusomic for CHR17, thereby correcting for HER2 pseudoamplification, enabling an accurate assessment of true HER2 amplification. However, this test has significant limitations, including expense, length of turnaround time attributable to requisite overnight incubation, and the necessity for specialized equipment and technical expertise to perform and interpret the assay; for these reasons, it is not readily available in many routine laboratories.
HER2 silver-enhanced in situ hybridization (SISH), a newly introduced method, may overcome many of the disadvantages mentioned above as it allows the quantification of the number of centromeric CHR17 and HER2-specific signals by conventional bright field light microscopy. In addition, HER2 SISH is the only fully automated bright field in situ hybridization assay in which HER2 gene status in breast carcinoma can be determined in only 6 hours and provides stable and permanent archival slides that are evaluated with conventional bright field light microscopy. Thus, this study was undertaken with 2 aims: (a) to evaluate the concordance between SISH and FISH assay in determining the status of HER2 amplification and (b) to assess the variables that may affect interobserver interpretative reproducibility among pathologists.
MATERIALS AND METHODS
Tissue Samples and SISH
The HER2 gene amplification status of an initial cohort of 228 invasive breast carcinomas was determined by SISH as compared with historical FISH results to determine the concordance of the 2 methods using a single reviewing pathologist. In a second cohort of 305 invasive breast carcinomas for which FISH results were available, we determined the concordance of consensus SISH among 10 pathologists in a variety of clinical practice settings, and assessed the variables contributing to the interobserver interpretative variability and reproducibility. Consecutive unstained sections on electrostatically charged slides were prepared from the same paraffin blocks used for hematoxylin and eosin and were stained according to the manufacturer's protocols with the INFORM HER2 DNA and CHR17 probes. Both probes were labeled with dinitrophenol (DNP) and optimally formulated for use with the Ventana ultraView SISH Detection Kit on the Ventana BenchMark® XT automated slide stainer. All assay components including deparaffinization and counterstaining were performed on the instrument. The repeat sequence depleted HER2 DNA probe and an oligo probe for centromeric CHR17 sequences were codenatured with the HER2 and CHR17 DNA targets on separate slides, the HER2 probe at 95°C for 12 minutes followed by hybridization at 52°C for 2 hours and the CHR17 probe denatured at 95°C for 12 minutes followed by hybridization at 44°C for 2 hours. After hybridization, appropriate stringency washes (3 times at 72°C) were performed. The HER2 and CHR17 DNP-labeled probes were visualized using the rabbit anti-DNP primary antibody and the ultraView SISH Detection Kit. The Detection Kit contains goat anti-rabbit IgG antibody conjugated to horseradish peroxidase. The enabling chemistry of the SISH reaction, enzyme metallography, is driven by the sequential addition of Silver A (silver acetate), Silver B (hydroquinone), and Silver C (hydrogen peroxide). Here, the silver ions are reduced by the hydroquinone to metallic silver ions. This reaction is fueled by the substrate for horseradish peroxidase, hydrogen peroxide (Silver C). The silver precipitation is deposited in the nucleus and a single copy of the HER2 gene locus is visualized as a black dot. The specimen is then counterstained with hematoxylin II and Bluing reagent for interpretation under conventional light microscopy.
Comparison Between SISH and FISH
Two hundred twenty-eight (228) primary invasive breast cancer cases with known dual-color direct FISH (HER2/CEP17 PathVysion) as previously described21 were selected for the first study cohort. Tissues were obtained from archival needle core biopsies or excisional biopsies tissue blocks from the Department of Anatomic Pathology at the Cleveland Clinic Foundation, after obtaining Institutional Review Board approval. Each case was given a unique, serial identifier so patients could not be identified directly or indirectly by the study investigator. All tissues were formalin fixed and paraffin embedded. Whole tissue sections stained with hematoxylin and eosin were reviewed to ensure the presence of primary invasive carcinoma in the sections to be stained with the SISH assay. Discrepant cases between SISH and FISH were analyzed by IHC using the PATHWAY HER2/neu (4B5) rabbit monoclonal antibody (Ventana Medical Systems Inc.) as previously described.45
Interobserver Interpretive Reproducibility of SISH
Interobserver interpretive reproducibility of HER2 SISH was evaluated among 10 pathologists in a separate cohort of 305 sequential primary invasive breast carcinomas for which historical FISH results were known. Tissues were deidentified and unlinked from patient identifiers after procedures approved by the Cleveland Clinic Foundation Institutional Review Board. Before scoring, a tutorial study set was assembled and reviewed by all pathologists interpreting the SISH slides. The teaching set contained specimens that were negative, equivocal, and positive [as defined by American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) Guidelines] for HER2 gene amplification. Examples of aneusomy and inadequate staining were included. For the cases included in this study, each reviewer was provided with a hematoxylin and eosin slide along with the SISH slides for CHR17 and HER2. The reviewers were blinded to the scores of other reviewers, the FISH data and the IHC scores. Three pathologists (R.T., B.P., and J.M.) from the Cleveland Clinic, 2 pathologists (R.L. and A.O.) from Mayo Clinic, 2 pathologists (A.M. and M.B.) from St Vincent's Hospital and ICPMR Westmead Hospital, Sydney, respectively, 1 pathologist (M.S.) from University of Virginia, 1 pathologist (M.D.) from Charite University Hospital, and 1 pathologist (R.N.) from Arizona Cancer Center performed the SISH scoring.
The SISH slides were evaluated with conventional bright field microscopy using an ordinary light microscope and dry ×20,×40, and/or×60 objectives. The use of oil immersion was prohibited. The SISH interpretation was restricted to the invasive carcinoma. SISH signals were visualized as single copies, multiple copies, and clusters.
Normal HER2 or CHR17 signals (1 to 2 copies/cell) served as the internal positive control and were required to be visible in the sample using×20,×40, or×60 objectives. These distinct nuclear signals were located in various non-neoplastic cells including stromal fibroblasts, endothelial cells, lymphocytes, and benign breast epithelial cells.
Not every nucleus demonstrated 1 or 2 discrete small dense black signals because a 4 μ paraffin section contains both intact and truncated nuclei. In some cases, the SISH component was visualized as confluent black nuclear signal where individual signal enumeration was not possible. When clusters of dots representing many copies of HER2 gene were present, small clusters were scored as 6 copies and large clusters were scored as 12 copies.
Two methods for enumerating HER2 gene status were used: a semiquantitative method based upon pattern recognition (method 1) and a quantitative method (method 2/2A). In method 1, SISH slides were reviewed and scored semiquantitatively by estimating the average number of HER2 and CHR17 signals in the target area and recording the average HER2 signal, and the average CHR17 signal to derive a HER2/CHR17 ratio. Cases with a ratio <1.4 were deemed negative and cases with a ratio greater than 4.0 were deemed amplified. In cases with a HER2/CHR17 ratio greater than or equal to 1.4 but less than or equal to 4.0, additional enumeration using method 2 was employed. In method 2, the reviewer recorded the quantitative enumeration of HER2 and CHR17 signals in 20 nuclei within a target area and calculated the HER2/CHR17 ratio. For cases that were enumerated using method 2 and with a HER2/CHR17 ratio equal to or between 1.8 and 2.2, method 2A was used. In method 2A the reviewer selected a second target area for each HER2 and CHR17 and counted the number of signals in 20 additional nuclei. The HER2/CHR17 ratio was then calculated by dividing the total number of HER2 signals from both target areas by the total number of CHR17 signals from both target areas. The results of HER2 gene amplification status was reported using the conventional FDA scoring criteria (amplified if ratio≥2, nonamplified if ratio<2) and also reported using the ASCO/CAP scoring guidelines (positive if ratio≥2.2, negative if ratio<1.8, and equivocal if 1.8≤HER2/CHR17 ratio≤2.2).
Discrepant cases between SISH and FISH were analyzed by IHC using the PATHWAY anti-HER2 (4B5) rabbit monoclonal antibody (Ventana Medical Systems International) and scored as negative (0 or 1+: no staining or weak, incomplete membrane staining in any proportion of tumor cells), positive (3+: uniform intense membrane staining of >30% of invasive tumor cells), or equivocal (2+: complete membrane staining, ie, either nonuniform or weak in intensity but with circumferential distribution in at least 10% of cells).
The decision was made that a consensus read could only be determined if at least half the readers scored a case, that is, if there were fewer than 5 readers for a case, it would be treated as missing data. For those cases having 5 or more readers, consensus would be determined by majority vote, with ties being considered as missing data. The original data set contained 305 cases. Figure 1 depicts the case characteristics that determined which cases could be used in statistical analysis. Of the original cases, 4 were problematic for the SISH ratio as determined by package insert. Two of these cases were tied, and 2 had fewer than 5 readers. These same cases were also problematic for the SISH ratio as determined by ASCO/CAP guidelines for the same reasons. Finally, for cases assessed solely by the HER2 count, 4 cases resulted in ties. Three of these cases were unique, the fourth being tied by the other 2 methods as well. This resulted in 7 unique cases for which a consensus read could not be determined (depicted in bold in Fig. 1), so that 298 cases were available for the statistical analysis. The low rate of case loss owing to insufficient readers (2 cases or 0.7%) indicates strong adherence to the study design. The low rate of loss owing to ties (5 cases or 1.6%) suggests that the ability of readers to identify amplification status is relatively robust.
All 10 readers received training before reading slides. As part of the training, it was specified that areas should be read such that no CHR17 counts would be less than one. However, 5 out of the 10 readers assigned values <1 to CHR17 in one or more cases. With 4 readers, this occurred in very few cases (1, 2, 4, and 6). One reader assigned CHR17 values <1 in 33 cases. For the analyses that follow, CHR17 counts <1 were set as equal to one. This typically did not result in a change in amplification status. However, there were 10 instances (occurring in 7 cases) where amplification status changed. Seven amplified cases changed (4 to nonamplified and 3 to equivocal). One equivocal case became nonamplified and 2 cases changed from missing (owing to the inability to calculate the ratio when the CHR17 count equaled 0) to amplified. Sensitivity analyses were conducted to assess whether the recordings made a difference. In one set of sensitivity analyses, all 39 cases where at least one reader called the CHR17 value <1 were excluded. In a second set of analyses, only the 7 cases were excluded where recoding CHR17 values <1 to a value one resulted in a changed amplification status. There were essentially no difference to overall and negative percent agreements presented below, and a very modest effect on the positive percent agreements, typically less than a 1% difference, and seldom reaching even a 2% difference.
Analyses were conducted using both package insert and ASCO/CAP guidelines. For both FISH and SISH, the package insert condition scored a case as positive for HER2/CHR17 ratio ≥2 and as negative for HER2/CHR17 ratio <2. Under ASCO/CAP guidelines, cases were scored as positive for HER2/CHR17 ratio greater than 2.2 and as negative for HER2/CHR17 ratio <1.8. Cases in the range 1.8 to 2.2 were considered equivocal and excluded from analysis. In addition to the HER2/CHR17 ratio, some authors have suggested using the HER2 copy number rather than the HER2/CHR17 ratio. In this approach, cases were scores as positive for HER2 ≥6 and as negative for HER2 <6. In the results section, unless otherwise noted, the term SISH denotes use of the HER2/CHR17 ratio, whereas the term HER2 designates the HER2 copy number alone.
All comparisons of agreement rates were made between SISH and FISH using FISH as the reference category. For the following Table 1.
Agreement rates are defined as follows:
Percent overall agreement=(a+d)/(a+b+c+d)×100
Percent positive agreement=a/(a+c)×100
Percent negative agreement=d/(b+d)×100
All confidence intervals reported are 2-sided Wilson 95% confidence intervals.
Testing for statistically significant differences between tables relied on 2-sided Fisher exact tests.
Comparison Between HER2 SISH and Conventional HER2 FISH
We analyzed the paired results of 228 invasive breast cancer specimens. The data from the paired analysis were compared in 3 different ways: using the FDA approved reporting criteria, using the ASCO/CAP guidelines, and using the ASCO/CAP scheme with equivocal samples removed.
In the HER2 amplified cases, the SISH component was visualized as large confluent black signals when the copy number was generally greater than 12 signals per nucleus (each small cluster represents 6 signals and each large clusters represents 12 signals), whereas individual signal enumeration was possible in cases that were amplified with 6 to 12 signals per nucleus (Fig. 2). The cases identified by historical FISH as aneusomic (80% or more of tumor cells with three or more CEP17 signals by FISH) demonstrated 3 to 5 HER2 black SISH signals per nucleus rather than the expected 2 copies (Fig. 3). HER2 nonamplified cases had 1 or 2 discrete black signals per nucleus (Fig. 4). Endogenous HER2 signal was also seen as 1 or 2 nuclear signals in stromal cells in each case.
Overall agreement between the HER2 SISH assay and the historical PathVysion FISH results was found in 214 of 228 cases (94%) using conventional FDA-approved scoring criteria and 209 of 219 cases (95%) using the ASCO/CAP result reporting scheme (with equivocal cases by either assay removed). When using conventional criteria, discrepancies between SISH and FISH occurred in 14 cases. Among 13 cases categorized as HER2 amplified by FISH and nonamplified by SISH, 8 were scored negative by IHC and 5 were scored positive. The one case categorized as nonamplified by FISH and amplified by SISH was negative by IHC. When using the ASCO/CAP result reporting scheme, discrepancies between SISH and FISH occurred in 10 cases. Among 9 cases categorized as HER2 amplified by FISH and nonamplified by SISH, 4 were scored negative by IHC, 3 were scored positive, and 2 were equivocal. The tenth discrepant case categorized as HER2 amplified by SISH and nonamplified by FISH was negative by IHC.
Comparison Between HER2 SISH and Conventional HER2 FISH and HER2 SISH Interobserver Interpretative Reproducibility
Interobserver interpretative reproducibility of HER2 SISH was evaluated among 10 pathologists in a subset of cases. The cases consisted of 298 scorable cases in a consecutive series of 305 primary breast carcinomas for which historical FISH results were known.
Variability of Reader Ratings
Although HER2/CHR17 ratios varied from reader to reader (from a low mean ratio of 1.55 to a high mean ratio of 2.19 across the 10 readers), all of the perreader confidence intervals overlapped, suggesting that the interreader differences were not statistically significant.
A variance components analysis was conducted on all HER2 copy number and HER2/CHR17 ratios across all cases. A random effects model incorporating case and reader was conducted using SAS 9.2. Estimates of mean copy number (or ratio) and its variance were used to calculate the percentage coefficient of variation (% CV), defined as the SD divided by the mean×100. For HER2 copy number, the case-to-case % CV was 330% and the reader-to-reader % CV was 136%. For the HER2/CHR17 ratio, the case-to-case % CV was 122% and the reader-to-reader % CV was 10%. These results suggest higher variability among the cases and less variability owing to reader effects. The ratio demonstrated more stability than the HER2 copy number, with the low % CV for reader-to-reader variation suggesting that the ratio is likely to be more reproducible across readers.
Consensus Agreement Rates Versus FISH
Tables 2 and 3 show the comparison of consensus SISH by package insert with the historical FISH status, first by frequency, and then presenting the agreement rates.
The overall and negative agreement rates were high. The lower positive agreement rate was due to the systematic nature of discordances. All 10 discordant cases were amplified by FISH, but nonamplified by consensus SISH. However, Tables 4 and 5 demonstrate that the majority of these discordant cases were equivocal by ASCO/CAP guidelines. Use of the HER2 signal count alone was also assessed. Tables 6 to 9 show analyses parallel to those already presented for HER2 signal count.
Under package insert guidelines, whether achievement of a consensus SISH read used HER2/CHR17 ratio or HER2 copy count made no difference, with all agreement rates being exactly the same. Under the ASCO/CAP scoring guidelines, a single case that was discordant by HER2/CHR17 ratio was concordant by HER2 copy count, resulting in slightly higher overall and positive agreement rates. However, this difference was not statistically significant.
Reflex Rates to Methods 2/2A
Overall reflex rates to methods 2 and/or 2A were examined for the cases presented in Table 6. Of the total 298 cases, 110 (37%) were reflexed by at least one reader, whereas only 6 (2%) were reflexed by a majority of readers. Most reflexed cases (57/110=52%) were reflexed by only one reader. Perhaps the best estimate for the reflex rate to expect in clinical practice is the percentage of reads (rather than readers) resulting in reflex: out of 2936 reads in the study, 228 (7.7%) resulted in reflex to method 2 and/or 2A.
Reflex rates were significantly higher in cases discordant between SISH and FISH. All discordant cases (10 of 10) were reflexed by at least one reader, a statistically significantly greater rate than the 35.4% (100 of 288) of concordant cases reflexed (P<0.001). Twenty percent of discordant cases (2 of 10) were reflexed by a majority of readers, again a significantly higher rate than the 1.4% (4 of 288) of concordant cases reflexed (P=0.01). Resort to reflex, then, was clearly correlated with difficulty in case classification.
Discordant Case Resolution
For all consensus agreement tables, a total of 11 distinct cases were found to be in disagreement for SISH and FISH either by HER2/CHR17 ratio or HER2 copy count. Nine of the 11 discordant cases were located and the SISH slides were reread by one principal investigator of the study. The results of this reread are presented in Table 10.
Of the 9 discordant cases that were reread, 6 cases had equivocal FISH ratios under ASCO/CAP guidelines. By rereading these cases and obtaining a “resolved” ratio, only one discrepant case was aligned with FISH (0779) under package insert criteria, and an additional 2 cases were aligned (0776 and 0935) under ASCO/CAP guidelines. The HER2 values were also investigated to determine if rereads allowed SISH values to align with FISH values. With the exception of a few cases, most SISH HER2 values were nearly identical to FISH HER2 values. This suggests that the discrepancy between SISH and FISH ratios stems from the CHR17 counts.
A rapid, accurate, reliable, and reproducible laboratory method for determining HER2 status is essential for identifying patients with cancer who are candidates for treatment with the anti-HER2 humanized monoclonal antibody trastuzumab, which has been shown to significantly prolong survival in HER2-positive breast cancer.41 Response to some types of chemotherapy and hormonal therapy also seems to be dependent on HER2 status.5,16,22,28 However, a consensus has not yet been reached as to the optimum method to evaluate HER2 status.
IHC to detect protein overexpression on the tumor cell membrane and FISH to detect HER2 gene amplification on CHR17 are the most frequently used methods in the clinical laboratory. IHC detection of HER2 protein can be affected by a number of factors including variations in antigen retrieval and tissue fixatives and fixation methods, varying sensitivities of reagents, false-positive and false-negative results, and subjectivity in evaluation of staining intensity.20,30,45 The FISH assay is relatively expensive compared to IHC and requires special instrumentation and technical expertise, making it somewhat impractical for widespread use as the primary testing modality in many laboratories. The 4',6-diamidino-2-phenylindole counterstain can be used as a surrogate hematoxylin counterstain to correlate with areas of tumor preselected by hematoxylin and eosin-stained consecutive sections, but may be challenging in cases of microinvasion. The disconnection between primary reporting pathologists and subsequent FISH testing in specialized laboratories may also introduce delays in reporting results. Moreover, despite several previously published HER2 gene/protein studies that have shown good to excellent concordance between FISH and IHC in the research setting, a clinically significant number of cases have demonstrated discordance between these 2 methodologies, particularly in the “real world” testing environment.25,27,33,34,45 When discordant cases were evaluated by profiling mRNA expression, FISH testing was found to be more accurate and reliable than IHC.21 The clinical importance of accurate determination of the HER2 status is considerable, with implications for the identification of patients who are the most likely to benefit from Herceptin therapy.31
For these reasons, bright field ISH methods were developed, using enzymatic labeling with conventional organic chromogens. This allows detection of gene copy status using a conventional peroxidase-base reaction and standard bright field light microscope.1,8,15,24,29,38,42,48 Previous studies have shown good correlation between chromogenic ISH and FISH assays.24,32,42,46 However, the characteristics of the staining are not ideal: diffuse signals can reduce ease of quantification, signals can be difficult to distinguish from counterstains, and a lack of sensitivity leading to increased numbers of nondiagnostic cases may also be observed.
Gold-facilitated ISH (GOLDFISH) was subsequently developed to detect amplification of the HER2 gene in paraffin-embedded breast carcinoma sections.47 GOLDFISH demonstrated good correlation with FISH and excellent interobserver interpretive reproducibility.46,47 The GOLDFISH assay was configured for simplified, rapid manual interpretation without the need for enumeration of individual signals as the autometallographic process was optimized to give large, confluent signals in amplified cases rather than discrete spots that could be counted. However, GOLDFISH proved to be somewhat problematic in breast cancers aneusomic for CHR17 producing false-positive gene amplification signals in some cases.
Subsequently, we developed a bright field assay to simultaneously visualize HER2 gene amplification and HER2 protein expression (EnzMet GenePro). This assay demonstrated excellent concordance with the HER2 status determined by FISH and allowed for a semiquantitative analysis of the HER2 gene status. In addition, most of the components of this assay have been automated using technology routinely available in clinical laboratories.12
In this study we adopted HER2 SISH, a newly introduced SISH method. This assay may overcome many of the disadvantages mentioned above as it allows for the quantification of the number of CHR17 and HER2 specific signals and correlation of results with morphologic features by the pathologist using conventional bright field light microscopy. It is the only fully automated bright field chromogenic in situ hybridization assay for HER2 and CHR17 detection which uses preformulated reagents and a run time of 6 hours. SISH also offers a permanent slide record. Without the need for a specialized microscope, the pathologist can integrate this test into the surgical pathology workflow as simply another slide in the tray of slides along with the hematoxylin and eosin stained and IHC slides.
The SISH assay format used in this study employed single color locus detection on 2 separate slides—a theoretical disadvantage with respect to 2 color FISH. However, using FISH as the reference standard, SISH demonstrated excellent concordance and reproducibility for detection of HER2 status in breast cancer. Of the 228 cases selected for the method comparison study (Cohort 1), overall agreement between the HER2 SISH assay and the historical Abbot Molecular Vysis FISH results was found in 214 (94%) cases using conventional FDA-approved criteria and 209 (95%) cases using ASCO/CAP result reporting scheme (with equivocal cases removed). Of the 298 evaluable cases comprising the interobserver reproducibility study (Cohort 2), overall agreement between the HER2 SISH assay and the historical Abbott Molecular Vysis FISH results was found in 288 of 298 (96.6%) cases using conventional FDA-approved criteria, and 282 of 285 (98.9%) cases using ASCO/CAP result reporting scheme (with equivocal cases removed). Our results are consistent with other reported SISH studies that have demonstrated excellent concordance between SISH and corresponding FISH assays.2,6,7,11,13,26,37 No test for HER2 status is perfect, and the precision of counting of FISH and the interobserver data for FISH are probably no better—if not worse—given the difficulty and time required to count in the dark. Indeed it is impossible to do such an interobserver study on the scale reported herein with FISH. As with IHC and FISH, cases will certainly be tested by SISH for which equivocal results are obtained (HER2/CHR17 ratio 1.8 to 2.2); in these instances, use of the ASCO/CAP guideline algorithm is recommended—use of another FDA-approved IHC or FISH assay to resolve the equivocal result.
In summary, the results of this study demonstrated that SISH is well suited for use as an alternative to FISH in determining the status of HER2 gene amplification. Overall, there was excellent concordance between SISH and corresponding FISH results. Furthermore, the high interobserver reproducibility validated the interpretative reliability of this method for assessing HER2 status. Results of the variance components analysis detailed above for HER2 copy number versus HER2/CHR17 ratios in Cohort 2 demonstrated more stability and lower % CV for reader-to-reader variation for the HER2/CHR17 ratio than for the HER2 copy number, suggesting that the ratio is likely to be more reproducible across readers. Compared with FISH, SISH may offer a convenient method for the assessment of HER2 gene amplification status in routinely processed breast cancer samples, using instrumentation widely available in diagnostic pathology laboratories.
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