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Comparing Fluorescence in Situ Hybridization and Chromogenic In Comparing Fluorescence In Situ Hybridization and Chromogenic In Situ Hybridization Methods to Determine the HER2/neu Status in Primary Breast Carcinoma using Tissue Microarray Kyeongmee Park, M.D., Ph.D., Jungyeon Kim, M.D., Ph.D., Sungjig Lim, M.D., Ph.D., Sehwan Han, M.D., Ph.D., Jung Young Lee, M.D., Ph.D. Departments of Pathology (PK, KJ, LS) and Surgery (HS), Inje University Sanggye Paik Hospital; and the Department of Pathology (LJ), College of Medicine, The Catholic University, Seoul, Korea prognostic implication because its amplification Identification of HER2/neu status is important for was associated with aggressive biologic features of predicting response to specific chemotherapy in the breast carcinoma. Integration of tissue microar- breast carcinoma. Chromogenic in situ hybridiza- ray technology enabled high-throughput determi- tion was performed using tissue microarray tech- nation of HER2/neu amplification profile with ra- nology on 188 primary breast carcinomas. To vali- pidity and accuracy in large cohorts of the breast date the reliability of novel chromogenic in situ carcinoma. hybridization technology, the results of chromo- genic in situ hybridization were correlated with the KEY WORDS: Breast carcinoma, Chromogenic in results of two-color fluorescence in situ hybridiza- situ hybridization, Fluorescence in situ hybridiza- tion done with the same tumors. On tissue microar- tion, HER2/neu, Tissue microarray. ray panels containing 188 breast carcinoma tissues, Mod Pathol 2003;16(9):937–943 fluorescence in situ hybridization and chromogenic in situ hybridization were conducted simulta- Among the numerous oncogenes and their prod- neously. HER2/neu amplification was detected in 46 ucts, HER2/neu is the most widely exploited one in tumors (24.5%) by fluorescence in situ hybridiza- clinical oncology. HER2/neu has moved from a tion and in 43 tumors (22.9%) by chromogenic in laboratory-based prognostic factor to a target for situ hybridization. Results of each method agreed the specific therapy, trastuzumab (Herceptin; Ge- with each other in 177 tumors (concordance: nentech, Inc., South San Francisco, CA), which 94.1%). HER2/neu amplification by fluorescence in binds to HER2/neu protein. The HER2/neu is a 185- situ hybridization was associated with nuclear ple- kDa transmembrane tyrosine kinase, and overex- and HER2/neu amplification pression of HER2/neu protein arises from HER2/neu ,(021. ؍ omorphism (P by chromogenic in situ hybridization was associ- gene amplification, resulting in increased gene High con- number (1). HER2/neu overexpression has been .(037. ؍ ated with poor nuclear grade (P cordance between fluorescence in situ hybridiza- shown in 20–40% of human breast carcinomas and tion and chromogenic in situ hybridization is associated with poor clinical outcome, even with indicated that chromogenic in situ hybridization systemic chemotherapy (2–4). Recent studies sug- can be a tempting alternative to fluorescence in situ gest that HER2/neu is a useful determinant of re- hybridization for the detection of HER2/neu ampli- sponse to hormonal or cytotoxic chemotherapy. fication in breast carcinoma because of its accuracy Data from Cancer and Leukemia Group B 8869 and and relative low cost. HER2/neu appeared to have a the National Surgical Adjuvant Breast and Bowel Project protocol B-11 suggest that patients whose Copyright © 2003 by The United States and Canadian Academy of tumors overexpress HER2/neu may derive a prefer- Pathology, Inc. VOL. 16, NO. 9, P. 937, 2003 Printed in the U.S.A. ential benefit from treatment with doxorubicin (5, Date of acceptance: May 16, 2003. 6). With recent introduction of trastuzumab ther- Supported by Grant R04-2001-00036 from the Korea Science & Engineer- ing Foundation. apy, it is now reasonable to test all new cases of Address reprint requests to: Kyeongmee Park, Department of Pathology, breast carcinomas, both early and late stage, for Inje University Sanggye Paik Hospital, 761-1, Sanggye-dong, Nowon-gu, Seoul 139-707, Korea; fax: 82-2-950-1266; e-mail: kmpark@sanggyepaik. HER2/neu overexpression. However, there is no ac.kr. consensus about which is the most optimal and DOI: 10.1097/01.MP.0000086487.78558.7D accurate testing strategy. 937 A vast majority of HER2/neu studies has been MATERIALS AND METHODS performed using immunohistochemistry that de- tects the HER2/neu protein overexpression. Mea- Materials surement of HER2/neu gene amplification is more One hundred eighty-eight primary breast carci- accurate because protein overexpression is the re- nomas were collected at Inje University Sanggye sult of gene amplification. Introduction of fluores- Paik Hospital, Seoul, Korea. Histopathologic classi- cence in situ hybridization (FISH) allows assess- fication and determination of tumor collecting re- ment of the level of gene amplification with gions were done on hematoxylin and eosin–stained information about distribution of gene copies in slides. The invasive ductal carcinoma was graded I, histologic sections (7). A number of reports have II, or III with the Nottingham histologic grading verified its accuracy and apparent superiority over system (14) in ascending degree of malignancy and immunohistochemistry in a prediction of response was graded I, II, or III with the Black’s nuclear grading system (15) in descending degree of to trastuzumab in metastatic breast carcinoma pa- malignancy. tients (8, 9). The main difficulty for adopting FISH in a clinical setting is the need of additional equip- ment for analysis such as fluorescence microscopy Tissue Microarray Block and multiband fluorescence filters. Recently, novel Recipient blocks were made with purified agar in technology to detect DNA probe has been devel- 3.8 ϫ 2.2–cm frames. Holes with 2 mm in each size oped. Chromogenic in situ hybridization uses a were made on the recipient blocks by core needle, simple immunohistochemistry-like peroxidase re- and agar core was discarded. Donor blocks were action (10). Chromogenic in situ hybridization is a prepared after through evaluation of hematoxylin tempting technology to overcome the practical lim- and eosin slides. Representative cancer portions itations of FISH, although its standardization has caught from matching donor blocks were trans- not been validated. planted to the recipient blocks using a 2-mm core Completion of the human genome sequence has needle. Recipient blocks were framed in the mold provided the basic structural information on all that is used to frame conventional paraffin block, human genes. Functional technique such as cDNA and then paraffin was added to the frame. Consec- microarrays enables analysis of expression levels of utive sections in 3.5-␮m thickness were cut from thousands of genes and proteins at once (11). Com- the recipient blocks using an adhesive-coated slide pared with the high-throughput techniques of system (Instrumedics Inc., New Jersey) supporting genomics and proteomics, most tissue-based mo- the cohesion of the 2-mm array elements on the lecular analyses have been tedious and require ex- glass (Fig. 1). tensive manual interaction. Tissue microarray is a novel technology of harvesting small disks of tissue Fluorescence In Situ Hybridization from individual donor paraffin-embedded tissue ␮ blocks and placing them in a recipient block with Two-color FISH was done on a 3.5- m-thick con- defined array coordinates (12). Tissue microarray secutive microarray sections from the same paraffin technology allows high-throughput molecular pro- blocks with chromogenic in situ hybridization. Be- filing of cancer from DNA to protein level by en- fore hybridization, the sections were deparaf- finized, air dried, and dehydrated in 100% ethanol abling the simultaneous analysis of hundreds of after incubation at 56° C for 24 hours. Microarray tissue specimens (13). This technology provides slides were treated in wash buffer (Vysis Inc., maximal use of limited tissue resources and renders Downers Grove, IL) for 3 minutes after treatment the advantage of generating gene expression pro- with 0.2 N HCl for 20 minutes. Pretreatment solu- files of cells as they occur in actual neoplastic tis- tion (Vysis) at 80° C was applied for 30 minutes, and sues in vivo. Tissue microarray technology has the the slides were washed with purified water. Slides potential to significantly accelerate molecular stud- were treated with wash buffer twice for 5 minutes ies and has become one of the most promising tools serially. Immersed slides in protease solution (Vy- in cancer research fields. sis) at 37° C was applied for 10 minutes, and the In this report, we summarize the results of simul- slides were washed with wash buffer at 45–50° C taneous analyses for HER2/neu amplification in 188 and air dried. Slides were fixed in 10% buffered human breast carcinomas using tissue microarray formalin for 10 minutes and were washed with technology. Chromogenic in situ hybridization ap- wash buffer at 45–50° C. For denaturation, slides peared as a reasonable alternative to FISH in the were immersed in denaturation solution (Vysis) for current study, and genetic analyses on the archival 5 minutes at 72° C, followed by dehydration with cancer tissues were successful with novel 70%, 85%, 100% ethanol serially at 45–50° C. For technologies. hybridization, 20 ␮LofLSIHER-2/CEP17 probe 938 Modern Pathology FIGURE 1. A portion of tissue microarray section of breast carcinoma (H & E, 10ϫ). (PathVysionTM, Vysis) was applied, and a coverslip digoxigenin-labeled HER2/neu probe. The slides was applied over the probe. After overnight hybrid- were treated on a 95° C hot plate for 5–10 minutes ization at 37° C in a humidified chamber, the slides and were incubated at 37° C for 16–24 hours. After were washed with 72° C posthybridization wash incubation, microarray slides were treated in 0.5ϫ buffer (Vysis) for 2 minutes. Nuclei were counter- sodium chloride citrate for 5 minutes and were stained with 20 ␮L 4,6-diamino-2-phenylindole washed with phosphate-buffered saline/Tween so- (DAPI) (Vysis). The centromere 17 (CEP) and HER2/ lution.
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