Priti Lal, MD, 1 Paulo A. Salazar, 1 Clifford A. Hudis, MD, 2 Marc Ladanyi, MD, 1 and Beiyun Chen, MD, PhD 1. Abstract

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1 Anatomic Pathology / DUAL- VS SINGLE-COLOR SCORING IN IMMUNOHISTOCHEMICAL AND FISH HER-2 TESTING HER-2 Testing in Breast Cancer Using Immunohistochemical Analysis and Fluorescence In Situ Hybridization A Single-Institution Experience of 2,279 Cases and Comparison of Dual-Color and Single-Color Scoring Priti Lal, MD, 1 Paulo A. Salazar, 1 Clifford A. Hudis, MD, 2 Marc Ladanyi, MD, 1 and Beiyun Chen, MD, PhD 1 Key Words: HER-2/neu; ERBB2; Immunohistochemistry; Fluorescence in situ hybridization; FISH; Breast cancer DOI: /VE7862V2646BR6EX Abstract We analyzed concordance between immunohistochemical analysis and fluorescence in situ hybridization (FISH) in HER-2 status and studied the effect of dualcolor (D-FISH) vs single-color FISH (S-FISH) scoring on the assignment of tumors to amplified or nonamplified categories. The assays were performed on formalin-fixed, paraffin-embedded sections of 2,279 invasive breast carcinomas. Immunohistochemical results were interpreted as negative (0, 1+) or positive (2+, 3+). For FISH analyses, a ratio for HER-2/chromosome 17 of 2.0 or more (D-FISH) or an absolute HER-2 copy number per nucleus of more than 4.0 (S-FISH) were interpreted as positive gene amplification. We found 547 (24.0%) cases positive immunohistochemically, 326 (14.3%) by D-FISH, and 351 (15.4%) by S-FISH. Overall concordance in HER-2 status with immunohistochemical analysis was 87% for D-FISH and 86% for S-FISH. Excellent concordance was found among groups scored immunohistochemically as 0, 1+, and 3+ (with D-FISH, 97%; with S-FISH, 96%). The most discordant category was the group scored 2+ immunohistochemically, in which only a quarter of the 2+ tumors were FISH(+). D-FISH and S-FISH scoring results were discordant in 89 tumors (4%), of which 8 (9%) had 3+ immunohistochemical staining and none showed high-level HER-2 amplification. Among all FISH(+) tumors, 10% were negative by immunohistochemical analysis, and notably almost half (47%) showed borderline to low HER-2 amplification (D-FISH score, ); the clinical significance of these findings warrants further investigation. Breast cancer is a disease with highly variable biologic and clinical behavior. Besides the classic prognostic factors used in clinical practice, HER-2 (HER-2/neu, ERBB2), a proto-oncogene located on chromosome 17, has become an important prognostic indicator. Amplification and/or overexpression of HER-2 occur in 15% to 25% of human breast cancers and are associated with a poor clinical outcome. 1-4 With the introduction of trastuzumab (Herceptin, Genentech, South San Francisco, CA), a recombinant humanized monoclonal antibody against HER-2 for the treatment of metastatic breast cancer, the accurate assessment of HER-2 status has become essential in determining the clinical management of patients with breast cancer. Immunohistochemical analysis and fluorescence in situ hybridization (FISH) are the 2 most widely used methods to evaluate HER-2 status in breast cancer. Studies have shown that overexpression of the HER-2 protein is closely correlated with amplification of the HER-2 gene in tumors scored immunohistochemically as 3+, but not in tumors scored as Studies also have indicated that HER-2 gene amplification determined by FISH seems to be a better predictor of response to trastuzumab-based therapy than HER-2 overexpression determined by immunohistochemical scores of 2+ to 3+. 8,9 The accurate assessment of HER-2 gene amplification, therefore, has become critical in the management of these tumors. Currently, there are 2 FISH-based assays for the assessment of HER-2 gene amplification. One is the US Food and Drug Administration approved PathVysion HER-2 probe kit (Vysis, Downers Grove, IL). It uses probes for chromosome 17 centromere and HER-2 gene simultaneously, and HER-2 gene amplification is defined as a ratio of HER-2 gene copies Am J Clin Pathol 2004;121: DOI: /VE7862V2646BR6EX 631

2 Lal et al / DUAL- VS SINGLE-COLOR SCORING IN IMMUNOHISTOCHEMICAL AND FISH HER-2 TESTING per chromosome 17 copy equal to or greater than ,10 The other FISH assay is the Ventana INFORM HER-2 test (Ventana, Tucson, AZ). It uses the HER-2 probe alone, and defines HER-2 gene amplification as the absolute HER-2 gene copy number per tumor nucleus of greater than ,12 Some investigators 7,13 have found correction for chromosome 17 critical for determination of true gene amplification as opposed to increased HER-2 gene copy number due to polysomy 17, while others 14,15 believed it unnecessary. We assessed concordance between 2 Food and Drug Administration approved methods for evaluating HER-2 status, ie, the immunohistochemically based HercepTest (DAKO, Carpinteria, CA) and the FISH-based PathVysion assay. We also examined how the use of dual-color (D- FISH) vs single-color FISH (S-FISH) scoring might affect the assignment of tumors to amplified or nonamplified categories. Materials and Methods Samples Among tumors received for HER-2 testing during a 20- month period, all tumors of invasive breast carcinoma including primary and metastatic breast carcinoma were included in this study. There were a total of 2,279 tumors successfully analyzed. All immunohistochemical and FISH tests were performed as part of routine diagnostic workup in the Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY. Immunohistochemical Analysis Serial sections 4- to 5-µm thick were used for immunohistochemical and FISH tests. Immunohistochemical analysis was performed by using the HercepTest kit, and the results were interpreted as follows: 0, no membrane staining; 1+, faint, partial membrane staining; 2+, weak complete membrane staining in greater than 10% of invasive cancer cells; 3+, intense complete membrane staining in greater than 10% of invasive cancer cells. Fluorescence In Situ Hybridization The PathVysion HER-2 probe kit was used for the FISH analysis. 7,10 In brief, the sections were baked overnight at 56 C, and the invasive carcinoma components were marked based on a corresponding H&E-stained section. Unstained sections were deparaffinized in CitriSolv (Vysis), dehydrated in 100% ethanol, and air dried. Slides then were subjected to protease digestion for 45 to 60 minutes, denatured, and hybridized with prewarmed probes for the HER-2 gene and chromosome 17 centromere (Her2/neu/CEP17 SG probe, Vysis) overnight at 37 C. They then were washed with posthybridization wash buffer at 72 C and counterstained with 4,6-diamidino-2-phenylindole (DAPI) in antifade solution, mounted, and scored. Slides first were scanned at low magnification ( 100) using a DAPI filter to identify areas of optimal tissue digestion and nonoverlapping nuclei within the area of invasive carcinoma. The signal enumeration was performed under high magnification ( 1,000). The number of chromosome 17 signals, HER-2 signals, and tumor nuclei scored were recorded for each tumor. Tumors were interpreted as amplified when the ratio of HER-2 signals divided by chromosome 17 centromere signals was equal to or greater than 2.0. This method of interpretation is designated as D-FISH. The results also were analyzed using the absolute HER- 2 copy number: the tumor was interpreted as positive for HER-2 gene amplification when the mean number of HER-2 signals per tumor nucleus was greater than 4.0. This method of interpretation is designated as S-FISH. This is equivalent to the interpretation guidelines for the Ventana INFORM HER-2 FISH assay. 11,12 Statistical Analysis Statistical analyses were performed using the Mann- Whitney test and the χ 2 test. All reported P values are 2-tailed. Results The correlation between HER-2 immunohistochemical and D-FISH results is summarized in Table 1. HER-2 gene amplification was detected in 14.3% of all tumors. The number of tumors showing HER-2 gene amplification in the immunohistochemically negative groups was relatively small, representing 1.1% and 3.1% of all tumors scored immunohistochemically as 0 and 1+, respectively. These tumors constitute 10.1% (33/326) of all FISH-positive tumors. Approximately one quarter of tumors scored immunohistochemically as 2+ and almost 90% of those scored as 3+ showed HER-2 gene amplification. Within the groups scored immunohistochemically as 0, 1+, or 3+, the overall concordance between immunohistochemical analysis and FISH was 97%. Among FISH-positive tumors, the mean ratios of HER-2/chromosome 17 within the groups scored immunohistochemically as 0, 1+, and 2+ were similar (3.4, 3.5, 3.6, respectively), but they were significantly lower than the ratio in the 3+ group (5.1; P <.0001). This was because most tumors with high-level amplification were scored immunohistochemically as 3+ with a median ratio of 4.7 vs 2.4, 2.9, and 2.8 in the groups scored immunohistochemically as 0, 1+, and 2+, respectively (Table 1).When the FISH results were interpreted using the absolute HER-2 copy number (S-FISH scoring 632 Am J Clin Pathol 2004;121: DOI: /VE7862V2646BR6EX

3 Anatomic Pathology / ORIGINAL ARTICLE Table 1 HER-2 Immunohistochemical Analysis and Dual-Color Fluorescence In Situ Hybridization Correlation * Ratio <2.0 Ratio 2.0 Immunohisto- Total No. (%) chemical Score of Cases No. (%) Mean (Range) No. (%) Mean (Range) Median 0 1,017 (44.6) 1,006 (98.9) 1.1 (1-1.9) 11 (1.1) 3.4 ( ) (31.4) 693 (96.9) 1.2 (1-1.9) 22 (3.1) 3.5 ( ) (13.7) 230 (73.5) 1.2 (1-1.9) 83 (26.5) 3.6 ( ) (10.3) 24 (10.3) 1.2 (1-1.7) 210 (89.7) 5.1 ( ) 4.7 Total 2,279 (100.0) 1,953 (85.7) 326 (14.3) * χ 2 = 905; P <.0001 for overall correlation between immunohistochemical results (grouped as 0-1+ and 2+-3+) and dual-color fluorescence in situ hybridization. P <.0001 (significantly higher than mean ratio in the groups with immunohistochemical scores of 0 to 2+). criteria), a similar correlation was seen with immunohistochemical results Table 2. The main differences were as follows: (1) more immunohistochemically negative (0 or 1+) tumors were FISH-positive (HER-2, >4.0); and (2) among all FISH-positive tumors, the mean HER-2 copy number in the group scored immunohistochemically as 2+ was intermediate between that in the immunohistochemically negative groups (8.1 vs ; P <.0001) and that in the group scored immunohistochemically as 3+ (8.1 vs 10.7; P <.0001). Based on the distribution of the HER-2 FISH results (dual- or single-color), the tumors could be classified into 4 categories of amplification: (1) none, (2) borderline, (3) low, and (4) high Table 3. An interesting finding is that two thirds of the FISH-positive tumors in each of the groups scored immunohistochemically as 0, 1+, or 2+ showed borderline to low-level amplification. In contrast, only one third of FISH-positive tumors in the group scored immunohistochemically as 3+ showed borderline to low-level amplification (data not shown). The D-FISH and the S-FISH results were concordant in 2,190 (96%) of 2,279 tumors Table 4 (χ 2 = 1,633; P <.0001). Results for 89 tumors were discordant: 57 tumors were negative by D-FISH (Table 4) Table 5 but positive by S-FISH, and 32 were positive by D-FISH but negative by S- FISH (Tables 4 and 5). Of the 89 tumors with discrepant D- FISH and S-FISH results, 81 (91%) were in the groups scored immunohistochemically as 0 to 2+ (Table 5). In all 57 tumors with negative D-FISH and positive S-FISH scores, the absolute HER-2 copy numbers per tumor cell (range, ) were within the borderline to low-level amplification categories; and the average chromosome 17 copy Table 2 HER-2 Immunohistochemical Analysis and Single-Color Fluorescence In Situ Hybridization Correlation * HER-2 Copy No. 4.0 HER-2 Copy No. >4.0 Immunohisto- Total No. (%) chemical Score of Cases No. (%) Mean (Range) No. (%) Mean (Range) 0 1,017 (44.6) 996 (97.9) 2.0 ( ) 21 (2.1) 6.1 ( ) (31.4) 674 (94.3) 2.3 ( ) 41 (5.7) 6.2 ( ) (13.7) 236 (75.4) 2.5 ( ) 77 (24.6) 8.1 ( ) (10.3) 22 (9.4) 2.7 ( ) 212 (90.6) 10.7 ( ) Total 2,279 (100.0) 1,928 (84.6) 351 (15.4) * χ 2 = 774; P <.0001 for overall correlation between immunohistochemical results (grouped as 0-1+ and 2+-3+) and single-color fluorescence in situ hybridization. P <.0001 (significantly higher than mean HER-2 copy number in the groups with immunohistochemical scores of 0 to 1+). Table 3 Distribution of Tumors With Nonamplified, Borderline-Amplified, Low-Amplified, and High-Amplified HER-2 by FISH in 2,279 Cases * HER-2 Gene Amplification Status D-FISH Score No. (%) of Cases S-FISH Score No. (%) of Cases Nonamplification <2.0 1,953 (85.7) 4.0 1,928 (84.6) Borderline (2.7) (2.8) Low (4.0) (4.3) High (7.6) (8.2) D, dual-color; FISH, fluorescence in situ hybridization; S, single-color. * D-FISH score, ratio of HER-2/chromosome 17; S-FISH score, absolute HER-2 copy numbers per nucleus. Am J Clin Pathol 2004;121: DOI: /VE7862V2646BR6EX 633

4 Lal et al / DUAL- VS SINGLE-COLOR SCORING IN IMMUNOHISTOCHEMICAL AND FISH HER-2 TESTING Table 4 Correlation of Dual-Color vs Single-Color FISH Dual-Color FISH Single-Color Total No. (%) FISH Negative Positive of Cases Negative 1, ,928 (84.6) Positive (15.4) Total No.(%) 1,953 (85.7) 326 (14.3) 2,279 (100.0) of cases FISH, fluorescence in situ hybridization. number was 3.31 (range, ) (Table 5). While in all 32 tumors with positive D-FISH and negative S-FISH scores, the ratios of HER-2/chromosome 17 (range, ) were within the borderline to low-level amplification categories, the average chromosome 17 copy number was only 1.25 (range, ) (Table 5). Overall, the correlation with immunohistochemical results (grouped into 0-1+ and 2+-3+) was similar for D-FISH (87% concordance) and for S-FISH (86% concordance) (Tables 1 and 2). Discussion Consistent with the existing data in the literature, 5-7,14,16-19 we found excellent concordance of HER-2 status between immunohistochemical analysis and FISH in groups scored immunohistochemically as 0, 1+, and 3+ but not in the 2+ group. The 2+ category has been well recognized as the main source of discrepancy between immunohistochemical and FISH results. The present study and other studies 5-7 have documented that the majority of 2+ tumors do not show HER- 2 amplification by FISH. Significant interobserver variability among pathologists in the interpretation of the 2+ staining pattern has been well recognized. 20 In the College of American Pathologists 2000 HER-2 immunohistochemical analysis survey, 20 a substantial number of observers scored the same 2+ tumor as 1+ or 3+. In the present study, the HER-2 immunohistochemical results were interpreted by more than 20 pathologists and were obtained directly from the pathology reports in an attempt to reflect a real-world situation. We recommend that, in clinical practice, tumors scored immunohistochemically as 2+ should be considered negative for HER-2 until proven to show gene amplification by FISH. The response rate to trastuzumab-based therapy in tumors showing 2+ HER-2 immunohistochemical overexpression is lower than that of 3+ tumors. 9,21 One of the widely acknowledged reasons is that the majority of 2+ tumors do not show HER-2 amplification by FISH. 14,16-19 The present study revealed, in addition, that even among the FISH-positive tumors scored immunohistochemically as 2+, the degree of HER-2 amplification was significantly lower than that of 3+ tumors, but rather similar to that of FISHpositive tumors scored immunohistochemically as negative for HER-2. The relatively low level of HER-2 amplification observed in the 2+ tumors also might contribute to the lower response rate. Our data also revealed that approximately 10% of all FISH-positive tumors were classified as negative for HER-2 by immunohistochemical analysis. The clinical significance of this finding is unclear because patients with such tumors were not included in the clinical trials using trastuzumabbased therapy reported to date. 8,,21,22 Although most of these tumors showed borderline or low-level HER-2 amplification, some of them, nevertheless, might respond to trastuzumab therapy and warrant further clinical investigation. The standard FISH method not only analyzes whether the HER-2 gene is amplified but also assesses the degree of amplification, thus providing a more quantitative measurement than Table 5 The Effect of Chromosome 17 Copy Number on Tumors With Discordant D-FISH and S-FISH Results Score (Range) Immunohisto- No. (%) Chromosome 17 Copy chemical Score of Cases D-FISH S-FISH No. per Nucleus (Range) Negative D-FISH/positive S-FISH results 0 14 (25) 1.5 ( ) 5.0 ( ) 3.35 ( ) (47) 1.5 ( ) 4.7 ( ) 3.20 ( ) (19) 1.6 ( ) 5.0 ( ) 3.08 ( ) 3+ 5 (9) 1.3 ( ) 5.3 ( ) 4.35 ( ) Total 57 (100) 1.5 ( ) 4.9 ( ) 3.31 ( ) Positive D-FISH/negative S-FISH results 0 4 (13) 2.2 ( ) 2.6 ( ) 1.16 ( ) 1+ 8 (25) 2.5 ( ) 3.0 ( ) 1.24 ( ) (53) 2.4 ( ) 3.0 ( ) 1.26 ( ) 3+ 3 (9) 2.6 ( ) 3.4 ( ) 1.39 ( ) Total 32 (100) 2.4 ( ) 3.0 ( ) 1.25 ( ) D, dual-color; FISH, fluorescence in situ hybridization; S, single-color. 634 Am J Clin Pathol 2004;121: DOI: /VE7862V2646BR6EX

5 Anatomic Pathology / ORIGINAL ARTICLE immunohistochemical analysis. An important point of this quantification is that, although borderline and low-level HER-2 amplification occurred infrequently among all invasive breast carcinomas (in 2.7% and 4.0%, respectively, of all tumors in the present study), in aggregate, borderline or lowlevel amplification was observed in almost half (152/326 [46.6%]) of all FISH-positive tumors. Previous clinical studies have shown that a significant proportion of tumors with even HER-2 immunohistochemical results of 3+ and gene amplification by FISH do not respond to trastuzumab therapy. 8,9,21 The gene dosage phenomenon could have a significant role in the spectrum of therapeutic responses and warrants further clinical investigation. Two commonly used FISH assays are the Vysis PathVysion assay and the Ventana INFORM assay. The key difference is the inclusion of a probe for chromosome 17 in the Vysis assay but not in the Ventana assay. We analyzed our data using the interpretation guidelines of both assays. Only 3.9% of tumors (89/2,279) showed discrepant results between the 2 methods. The discrepancy apparently was due to polysomy 17 in tumors that were negative by D-FISH and positive by S-FISH and monosomy 17 with concurrent duplication of the HER-2 gene on the remaining chromosome 17 in tumors that were positive by D-FISH and negative by S-FISH, as suggested by other studies as well. 10,23,24 The present study does not permit a determination of which FISH assay is better for clinical testing. The issue of polysomy 17 has been raised in previous studies, 7,10,13,24 and the clinical significance remains unclear. In our data, all 57 tumors with discrepant FISH results and increased chromosome 17 copy number showed borderline to low-level amplification based on the absolute HER-2 copy number. Again, whether this group of tumors will behave and respond to trastuzumab differently from tumors that are FISH-positive by both methods of interpretation is unknown. In conclusion, for assessment of HER-2 status, immunohistochemical and FISH results were highly concordant in groups scored immunohistochemically as 0, 1+, and 3+ but not in the 2+ group. In clinical practice, 2+ tumors should be considered negative for HER-2 until proven to have gene amplification by FISH. Borderline to low-level HER-2 amplification was observed in almost half of FISH-positive tumors, and its clinical significance warrants further investigation. Discordance between D-FISH and S-FISH was low and occurred only in tumors with borderline to low-level HER-2 amplification. From the Departments of 1 Pathology and 2 Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY. 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