The Use of TMA for Interlaboratory Validation of FISH Testing for Detection of HER2 Gene Amplification in Breast Cancer
Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois (LKD,RG,NK,ELW), and Department of Pathology, University of Texas M.D. Anderson Cancer Center, Houston, Texas (NS)
Correspondence to: Leslie K. Diaz, MD, Northwestern Memorial Hospital, 251 E. Huron St., Feinberg 7-325, Chicago, IL 60611. E-mail: l-diaz{at}northwestern.edu
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: breast cancer HER2 quality assurance in situ hybridization
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Most laboratories use IHC as the preliminary test for the detection of HER2 protein expression in breast cancer specimens (Allred and Swanson 2000; Hanna 2001
). FISH testing is now the most widely accepted method for confirming the presence of HER2 gene amplification in breast cancer, and in many respects is considered superior to IHC (Pauletti et al. 2000
; Bartlett et al. 2001
). The FDA-approved HercepTest is a popular IHC-based test for HER2 protein detection, but many laboratories use other antibodies and methods. Laboratories using "home brew" methods for HER2 testing by IHC do so largely because of the unjustifiably high price and proprietary restrictions of the HercepTest. It has been recommended that HER2 FISH analysis should reserved for cases scored as "equivocal or borderline" for overexpression of HER2 protein (i.e., HercepTest scores of 2+ or 1+) (Ellis et al. 2000
; Hoang et al. 2000
; Jimenez et al. 2000
; Field et al. 2001
; Hanna 2001
; Thomson et al. 2001
; Tubbs et al. 2001
; Kobayashi et al. 2002
).
Many academic centers and commercial laboratories offer FISH testing, but limited numbers of community hospital-based laboratories perform these assays. FISH technology requires specialized training and the purchase of specific equipment. The introduction of FISH technology by a laboratory into a clinical setting requires technical validation and the establishment of quality assurance and quality control mechanisms (Allred and Swanson 2000; Mascarello et al. 2002
). For smaller laboratories this can be a daunting task, even in the face of ever-increasing demands for FISH testing by oncologists and their patients.
In light of recent reports questioning the accuracy of HER2 FISH testing results from "non-reference" laboratories, an urgent need has developed for methods to monitor the quality of these assays (Paik et al. 2002; Roche et al. 2002
). Laboratories introducing HER2 FISH as a clinical test should voluntarily subject themselves to a period of technical validation. Allred and colleagues consider an assay to have achieved technical validation when it is "sensitive, specific, reproducible, and interpreted in a uniform manner from laboratory to laboratory" (Allred and Swanson 2000
).
Considering the complexities associated with molecular techniques as well as the expense and potential morbidity associated with Herceptin therapy, testing for predictive markers such as HER2 deserves rigorous validation. The nature of HER2 testing adds an additional challenge because a sample must be determined to be either "positive" or "negative." TMA is a high-throughput method for biomarker evaluation that is gaining widespread acceptance. TMAs are composed of cores from "donor" blocks derived from many specimens and are ideal for use as control tissues and for quality assessment.
The use of TMA sections for the measurement of breast cancer prognostic and predictive markers using both IHC and in situ hybridization has recently been validated (Skacel et al. 2002; Zhang et al. 2003
). We believe that a practical extension of these findings would be the use of TMA for the technical validation of HER2 testing by FISH. Similarly, TMA would be useful for evaluating CISH, another technique for HER2 analysis. Recent data have suggested that CISH is comparable to FISH for HER2 analysis (Tubbs et al. 2002
; Gupta et al. 2003
). Here we evaluate the usefulness of TMA technology for technical validation of HER2 FISH testing.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FISH Analysis
FISH analysis was performed separately by two laboratories using similar protocols. Northwestern Memorial Hospital (Chicago, IL) was the "testing" laboratory conducting the validation. The clinical FISH laboratory of The M.D. Anderson Cancer Center (Houston, TX) served as the "reference" laboratory. The TMA slides were preheated at 60C overnight, then deparaffinized in xylene and ethanol. The slides were submerged in Vysis Paraffin Pretreatment Reagent (Vysis; Downers Grove, IL) for 13 min at 80C in a waterbath. After the sections were rinsed with deionized water, the slides were treated with protease solution (250 mg pepsin + 62.5 ml 0.2 N HCl, pH 1.0) for 13 min at 37C in a waterbath. After rinsing and and air-drying, 20 µl of probe was applied to each TMA slide. The Vysis PathVysion HER2 DNA probe kit was used, which contains the LSI-HER2 (spectrum orange) and CEP-17 (spectrum green) probes. The slides were coverslipped and sealed with rubber cement, denatured for 5 min at 73C, and then hybridized overnight at 37C. Denaturization and hybridization were performed on separate Boekel Slide Moats (Boekel Scientific; Feasterville, PA). For post-hybridization washing, the slides were immersed in 2 x SSC with 0.3% NP-40 at 73C in a waterbath for 1 min. The TMA slides were air-dried in the dark, then counterstained with 20 µl of DAPI (4,6-diamidino-2-phenylindole). The FISH signals were visualized using Vysis filter sets and an Olympus BX41 (Olympus America; Melville, NY) fluorescent microscope and a Zeiss (Carl Zeiss; Thornwood, NY) microscope attached to a QUIPS Image Capture system (Applied Imaging; Santa Clara, CA). The relative ratios of LSI-HER2 to CEP-17 signals were counted in 60 tumor cells for each case by two individuals (LKD and cytotechnologist K. Sikka) under oil at x1000 magnification. Two 1.5-mm cores were present in all but three cases, and 30 cells from each core were analyzed and the counts combined for a single score. HER2 to chromosome 17 ratios 2.0 were considered positive for HER2 gene amplification. The results were entered into a spreadsheet. Pairwise kappa scores were determined for each case assayed by the two laboratories (Cohen 1960
). The kappa statistic calculates the level of agreement between two observers. The range and strength of agreement for kappa values are as follows: <0.00 = poor; 0.000.20 = slight; 0.210.40 = fair; 0.410.60 = moderate; 0.610.80 = substantial; 0.810.99 = almost perfect; and 1.00 = perfect agreement.
CISH Analysis
CISH analysis was performed using the Zymed Spot-Light CISH polymer detection kit (Zymed; South San Francisco, CA) according to the suggested procedure (Tanner et al. 2000). This kit uses a diaminobenzidine (DAB) detection method and a digoxigenin-labeled probe to assay for the HER2 gene (Zymed Spot-Light HER2 DNA Probe). Two separate tissue sections from each TMA were deparaffinized and washed in absolute alcohol before steam heat pretreatment in Heat Pretreatment Solution. After two washes with PBS, enzymatic digestion with Pretreatment Enzyme was performed at 37C for 8 min. Three additional PBS washes and dehydration of the slides in graded alcohols were performed. After air-drying, 15 µl of probe was applied to each section and coverslips were applied. The slides were denatured at 95C for 5 min and the hybridization was performed overnight at 37C. After the hybridization, the slides were placed in pre-warmed 0.5 x SSC solution for 5 min and were washed with PBS/Tween-20 three times for 2 min each. According to the Spot-Light kit instructions, the signal was visualized by sequential incubations at room temperature with CAS-block (10 min), anti-digoxigenin fluorescein (30 min), anti-fluorescein peroxidase (30 min), and DAB (20 min). After rinsing with distilled water, the slides were lightly counterstained with hematoxylin and coverslipped. Scoring was performed by one pathologist (ELW) as previously described (Tanner et al. 2000
; Gupta et al. 2003
). Tumors with more than 10 gene copies or with large gene copy clusters were considered amplified and those with one to five gene copies were considered non-amplified. Low-level amplification was defined as six to ten gene copies in tumor cell nuclei.
IHC Analysis
Immunohistochemical analysis for HER2 protein expression was performed using a polyclonal rabbit anti-human antibody to the c-erbB-2 oncoprotein (A0485, dilution 1:1000). The antibody used for this study was purchased from DAKO (Carpinteria, CA) and is the antibody included as part of the DAKO HercepTest kit. We performed a standard (ABC) IHC procedure under conditions modified for our laboratory (Northwestern Memorial Hospital, Chicago, IL). IHC was performed using the Ventana NexES automated staining system and the Ventana ES DAB detection kit (Ventana Medical Systems; Tucson, AZ). Microwave antigen retrieval was performed for 12 min in citrated buffer, allowing 20 min for cooling. The staining procedure was performed according to the detection kit instructions. The TMA sections were scored for HER2 protein expression independently by two pathologists (LKD and ELW) and a consensus score was obtained for discrepant cases. Scoring was performed using the HercepTest system (score range 0 to 3+).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The concordance rate was 100% with the reference laboratory for positive and negative HER2 FISH results for the 36 cases that could be compared. Strong agreement for HER2 gene to chromosome 17 gene copy ratios were also observed (kappa = 0.85). These results indicate that the HER2 FISH assay, as performed by the testing laboratory, compares favorably with the assay performed by the reference laboratory. In addition to the TMA sections, standard histological sections from 30 additional clinical breast cancer cases were included in our formal validation of HER2 FISH (data not provided), largely to include "real life" testing conditions in the validation.
The analysis of FISH signals using the TMA sections was uncomplicated, and DAPI staining provided acceptable morphology for TMA navigation under the fluorescent microscope. Although biological heterogeneity is believed to be an issue with TMA use, it is not relevant when identical TMA sections are compared between laboratories. Although not the focus of this study, we observed markedly discrepant HER2 to chromosome 17 signal ratios in only a single case between the paired cores obtained from the center and the periphery of the 41 tumors evaluated.
Evaluation of the performance of IHC testing for HER2 by the testing laboratory was conducted as part of the validation. The IHC results (scored as 0,1+, 2+, and 3+) were compared to the results obtained by FISH in a fashion often published in the literature (Diaz 2001). Overall, concordance for HER2 testing by IHC and FISH was good. The concordance rates between IHC cases scored as 3+ and 0 were excellent for FISH-positive and -negative cases, respectively. For IHC cases scored as 1+ and 2+, one case from each group was later determined to be HER2 gene-amplified by FISH. One of these tumors (scored as 2+) displayed marked chromosome 17 polysomy when assayed by FISH. Non-correlative IHC results compared to FISH, such as these, are in keeping with findings of past reports (Jacobs et al. 1999
; Hoang et al. 2000
; Ridolfi et al. 2000
; Tubbs et al. 2001
).
In addition to comparing HER2 FISH testing between institutions, we also compared FISH testing results with those determined by CISH. Recently, Gupta et al. (2003) compared these two in situ hybridization techniques in 31 clinical breast cancer cases. They found that in four cases with low-level amplification for the HER2 oncogene by CISH, chromosome 17 polysomy was present in three of these cases as assayed by FISH (PathVysion). We found similar findings in our comparison, in which 4/32 cases contained chromosome 17 polysomy and all were low-level amplified by CISH. Gupta et al. (2003)
concluded that use of a newly marketed chromosome 17 CISH-based probe (Zymed) would be necessary when low-level HER2 gene amplification was detected initially using CISH to rule out aneusomy.
In summary, we utilized TMA sections for the validation of HER2 molecular testing by our laboratory. Although these sections enabled our laboratory to rapidly achieve technical validation for HER2 FISH testing, we will continue to monitor our clinical testing results on traditional histological sections. We evaluated both FISH- and CISH-based HER2 testing methods and compared our results to those of a reference laboratory with considerable FISH experience. We found TMA to be a robust and effective method for the technical validation of HER2 FISH testing, and we believe that its use should be considered for external quality assessment programs.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allred DC, Swanson PE (2000) Testing for erbB-2 by immunohistochemistry in breast cancer. Am J Clin Pathol 113:171175[Medline]
Bartlett JM, Going JJ, Mallon EA, Watters AD, Reeves JR, Stanton P, Richmond J, Donald B, et al. (2001). Evaluating HER2 amplification and overexpression in breast cancer. J Pathol 195:422428[Medline]
Battifora H (1986) The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing. Lab Invest 55:244248[Medline]
Camp RL, Charette LA, D. Rimm L (2000) Validation of tissue microarray technology in breast carcinoma. Lab Invest 80:19431949[Medline]
Cobleigh MA, Vogel CL, Tripathy D, Robert NJ, Scholl S, Fehrenbacher L, Wolter JM, et al. (1999) Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol 17:26392648
Cohen J (1960) A coefficient of agreement for nominal scales. Educ Psych Meas 20:3746
Diaz NM (2001) Laboratory testing for HER2/neu in breast carcinoma: an evolving strategy to predict response to targeted therapy. Cancer Control 8:415418[Medline]
Ellis IO, Dowsett M, Bartlett J, Walker R, Cooke T, Gullick W, Gusterson B, et al. (2000) Recommendations for HER2 testing in the UK. J Clin Pathol 53:890892
Field AS, Chamberlain NL, Tran D, Morey AL (2001) Suggestions for HER-2/neu testing in breast carcinoma, based on a comparison of immunohistochemistry and fluorescence in situ hybridisation. Pathology 33:278282[Medline]
Gancberg D, Di Leo A, Rouas G, Jarvinen T, Verhest A, Isola J, Piccart MJ, et al. (2002) Reliability of the tissue microarray based FISH for evaluation of the HER-2 oncogene in breast carcinoma. J Clin Pathol 55:315317
Gupta DL, Middleton LP, Whitaker MJ, Abrams J (2003) Comparison of fluorescence and chromogenic in situ hybridization for detection of HER-2/neu oncogene in breast cancer. Am J Clin Pathol 119:381387[Medline]
Hanna W (2001) Testing for HER2 status. Oncology 61(suppl 2):2230[Medline]
Hoang MP, Sahin AA, Ordonez NG, Sneige N (2000) HER-2/neu gene amplification compared with HER-2/neu protein overexpression and interobserver reproducibility in invasive breast carcinoma. Am J Clin Pathol 113:852859[Medline]
Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ (1999) Specificity of HercepTest in determining HER-2/neu status of breast cancers using the United States Food and Drug Administration-approved scoring system. J Clin Oncol 17:19831987
Jimenez RE, Wallis T, Tabasczka P, Visscher DW (2000) Determination of Her-2/Neu status in breast carcinoma: comparative analysis of immunohistochemistry and fluorescent in situ hybridization. Mod Pathol 13:3745[Medline]
Kobayashi M, Ooi A, Oda Y, Nakanishi I (2002) Protein overexpression and gene amplification of c-erbB-2 in breast carcinomas: a comparative study of immunohistochemistry and fluorescence in situ hybridization of formalin-fixed, paraffin-embedded tissues. Hum Pathol 33:2128[Medline]
Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, et al. (1998) Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nature Med 4:844847[Medline]
Mascarello JT, Brothman AR, Davison K, Dewald GW, Herrman M, McCandless D, Park JP, et al. (2002) Proficiency testing for laboratories performing fluorescence in situ hybridization with chromosome-specific DNA probes. Arch Pathol Lab Med 126: 14581462[Medline]
Menard S, Fortis S, Castiglioni F, Agresti R, Balsari A (2001) HER2 as a prognostic factor in breast cancer. Oncology 61(suppl 2):6772[Medline]
Paik S, Bryant J, TanChiu E, Romond E, Hiller W, Park K, Brown A, et al. (2002) Real-world performance of HER2 testingNational Surgical Adjuvant Breast and Bowel Project experience. J Natl Cancer Inst 94:852854
Pauletti G, Dandekar S, Rong H, Ramos L, Peng H, Seshadri R, Slamon DJ (2000) 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 18:36513664
Ridolfi RL, Jamehdor MR, Arber JM (2000) HER-2/neu testing in breast carcinoma: a combined immunohistochemical and fluorescence in situ hybridization approach. Mod Pathol 13:866873[Medline]
Rilke F, Colnaghi MI, Cascinelli N, Andreola S, Baldini MT, Bufalino R, Della Porta G, et al. (1991) Prognostic significance of HER-2/neu expression in breast cancer and its relationship to other prognostic factors. Int J Cancer 49:4449[Medline]
Roche PC, Suman VJ, Jenkins RB, Davidson NE, Martino S, Kaufman PA, Addo FK, et al. (2002) Concordance between local and central laboratory HER2 testing in the breast intergroup trial N9831. J Natl Cancer Inst 94:855857
Skacel M, Skilton B, Pettay JD, Tubbs RR (2002) Tissue microarrays: a powerful tool for high-throughput analysis of clinical specimens: a review of the method with validation data. Appl Immunohistochem Mol Morphol 10:16[Medline]
Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, et al. (1989) Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707712[Medline]
Slamon DJ, LeylandJones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, et al. (2001) Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344:783792
Tanner M, Gancberg D, Di Leo A, Larsimont D, Rouas G, Piccart MJ, Isola J (2000) Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples. Am J Pathol 157:14671472
Thomson TA, Hayes MM, Spinelli JJ, Hilland E, Sawrenko C, Phillips D, Dupuis B, et al. (2001) HER-2/neu in breast cancer: interobserver variability and performance of immunohistochemistry with 4 antibodies compared with fluorescent in situ hybridization. Mod Pathol 14:10791086[Medline]
Torhorst J, Bucher C, Kononen J, Haas P, Zuber M, Kochli OR, Mross F, et al. (2001) Tissue microarrays for rapid linking of molecular changes to clinical endpoints. Am J Pathol 159:22492256
Tubbs R, Pettay J, Skacel M, Powell R, Stoler M, Roche P, Hainfeld J (2002) Gold-facilitated in situ hybridization: a bright-field autometallographic alternative to fluorescence in situ hybridization for detection of Her-2/neu gene amplification. Am J Pathol 160:15891595
Tubbs RR, Pettay JD, Roche PC, Stoler MH, Jenkins RB, Grogan TM (2001) Discrepancies in clinical laboratory testing of eligibility for trastuzumab therapy: apparent immunohistochemical false-positives do not get the message. J Clin Oncol 19:27142721
Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, Slamon DJ, et al. (2001). First-line Herceptin monotherapy in metastatic breast cancer. Oncology 61(suppl 2):3742[Medline]
Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, Slamon DJ, et al. (2002) Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 20:719726
Zhang D, SaltoTellez M, Putti TC, Do E, Koay ES (2003) Reliability of tissue microarrays in detecting protein expression and gene amplification in breast cancer. Mod Pathol 16:7985[Medline]