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- W135272007 abstract "Fluorescence in situ hybridization (FISH) represents an excellent method for profiling gene amplification in situ, but correlation with tissue morphology is difficult because of dark-field visualization. Validation of a bright-field assay for assessment of HER-2/neu gene amplification was investigated. Streptavidin-Nanogold was used to generate bright-field gene copy signals using GoldEnhance gold-based autometallography, catalyzed reported deposition, and a biotin-labeled probe. One hundred cases of invasive breast carcinoma were evaluated for which FISH gene copy results, and mRNA and oncoprotein gene expression, were known. Autometallographic signals were qualitatively evaluable without the use of oil immersion microscopy. Results correlated well with indirect and direct label FISH. Autometallographic gold-based in situ hybridization represents a promising bright-field assay for the assessment of HER-2/neu gene amplification. Fluorescence in situ hybridization (FISH) represents an excellent method for profiling gene amplification in situ, but correlation with tissue morphology is difficult because of dark-field visualization. Validation of a bright-field assay for assessment of HER-2/neu gene amplification was investigated. Streptavidin-Nanogold was used to generate bright-field gene copy signals using GoldEnhance gold-based autometallography, catalyzed reported deposition, and a biotin-labeled probe. One hundred cases of invasive breast carcinoma were evaluated for which FISH gene copy results, and mRNA and oncoprotein gene expression, were known. Autometallographic signals were qualitatively evaluable without the use of oil immersion microscopy. Results correlated well with indirect and direct label FISH. Autometallographic gold-based in situ hybridization represents a promising bright-field assay for the assessment of HER-2/neu gene amplification. Pharmacogenomics as a clinical and laboratory discipline has emerged as an important rational approach to the treatment of malignancy. Perhaps one of the best examples of pharmacogenomics is the relationship between oncogene genotype and responses to specific available therapy, such as the link between HER-2/neu oncogene genotyping (as reflected in overamplification of the gene) and response to the humanized monoclonal antibody Herceptin alone or in combination with adriamycin and other drugs.1Slamon DJ Leyland-Jones B Shak S Fuchs H Paton V Pharm D Bajamonde A Fleming T Eiermann W Wolter J Pegram M Baselga J Norton L Use of chemotherapy plus a monoclonal antibody against Her-2 for metastatic breast cancer that overexpresses Her-2.N Engl J Med. 2001; 344: 783-792Crossref PubMed Scopus (9169) Google Scholar, 2Seidman AD Fornier MN Esteva FJ Tan L Kaptain S Bach A Panageas KS Arroyo C Valero V Currie V Gilewski T Theodoulou M Moynahan ME Moasser M Sklarin N Dickler M D'Andrea G Cristofanilli M Rivera E Hortobagyi GN Norton L Hudis C Weekly Trastuzumab and Paclitaxel therapy for metastatic breast cancer with analysis of efficacy by Her-2 immunophenotype and gene amplification.J Clin Oncol. 2001; 19: 2587-2595Crossref PubMed Scopus (537) Google Scholar, 3Baselga J Tripathy D Mendelsohn J Baughman S Benz CC Dantis L Sklarin NT Seidman AD Hudis CA Moore J Rosen PP Twaddell T Henderson IC Norton L Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer.J Clin Oncol. 1996; 14: 697-699PubMed Google Scholar, 4Baselga J Norton L Albanell J Kim YM Mendelsohn J Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of Paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts.Cancer Res. 1998; 58: 2825-2831PubMed Google Scholar, 5Cobleigh MA Vogle CL Tripath D Robert NJ Scholl S Fehrenbacher L Paton V Shak S Lieberman G Slamon D Efficacy and safety of Herceptin (humanized anti-HER2 antibody) as a single agent in 222 women with HER2 overexpression who relapsed following chemotherapy for metastatic breast cancer.Proc Am Soc Clin Oncol. 1998; 17: 97aGoogle Scholar, 6Slamon D Leyland-Jones B Shak S Addition of Herceptin (humanized anti-HER2 antibody) to first line chemotherapy for HER2 overexpressing metastatic breast cancer (HER2+/MBC) markedly increases anticancer activity: a randomized, multinational controlled phase III trial.Proc Am Soc Clin Oncol. 1998; 17: 98aGoogle Scholar HER-2/neu amplification/overexpression has been linked to progression of breast carcinoma and responsiveness to tamoxifen, taxanes, and adriamycin.7Press MF Bernstein L Thomas PA Meisner LF Zhou JY Ma Y Hung G Robinson RA Harris C El-Naggar A Slamon DJ Phillips RN Ross JS Wolman SR Flom DJ HER-2/neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas.J Clin Oncol. 1997; 15: 2894-2904Crossref PubMed Scopus (715) Google Scholar, 8Ro J El-Naggar A Ro J C-erb B-2 amplification in node-negative human breast cancer.Cancer Res. 1989; 49: 6941-6944PubMed Google Scholar, 9Seshadri R Firgaria FA Horsfall DJ McCaul K Setlur V Kitchen P Clinical significance of Her-2/neu oncogene amplification in primary breast cancer.J Clin Oncol. 1993; 11: 1936-1942Crossref PubMed Scopus (474) Google Scholar, 10Slamon DJ Godolphin W Jones L Holt J Wong S Keith D Levin W Stuart S Udove J Ulrich A Press M Studies of the Her-2/neu proto-oncogene in human breast and ovarian cancer.Science. 1989; 244: 707-712Crossref PubMed Scopus (6172) Google Scholar, 11Tsuda H Hirohashi S Shimosato Y Hirota T Tsugane S Yamamoto H Miyajima N Toyoshima K Yamamoto T Yokota J Yoshda T Skamoto H Terada M Sugimura T Correlation between long-term survival in breast cancer patients and amplification of two putative oncogene coamplification units: hst-1 1/int-2 and c-erb B-2/er-1.Cancer Res. 1989; 49: 3104-3108PubMed Google Scholar, 12Kallioniemi OP Holli K Visakorpi T Koivula T Helin HH Isola JJ Association of c-erbB-2 protein over-expression with high rate of cell proliferation, increased risk of visceral metastasis and poor long-term survival in breast cancer.Int J Cancer. 1991; 49: 650-655Crossref PubMed Scopus (342) Google Scholar, 13Babiak J Hugh J Poppema S Significance of c-erbB-2 amplification and DNA aneuploidy. Analysis in 78 patients with node-negative breast cancer.Cancer. 1992; 70: 770-776Crossref PubMed Scopus (33) Google Scholar, 14Toikkanen S Helin H Isola J Joensuu H Prognostic significance of HER-2 oncoprotein expression in breast cancer: a 30-year follow-up.J Clin Oncol. 1992; 10: 1044-1048Crossref PubMed Scopus (280) Google Scholar, 15Thor AD Berry DA Budman DR Muss HB Kute T Henderson IC Barcos M Cirrincione C Edgerton S Allred C Norton L Liu ET erbB2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer.J Natl Cancer Inst. 1998; 90: 1346-1360Crossref PubMed Scopus (544) Google Scholar, 16Paik S Bryant J Park C Fisher B Tan-Chiu E Hyams D Fisher ER Lippman ME Wickerham DL Wolmark N erbB-2 and response to doxorubicin in patients with axillary lymph node-positive, hormone receptor-negative breast cancer.J Natl Cancer Inst. 1998; 90: 1361-1370Crossref PubMed Scopus (604) Google Scholar, 17Bianco AR De Laurentiis M Carlomagno C 20 year update of the Naples GUN trial of adjuvant breast cancer therapy: evidence of interaction between c-erb-B2 expression and tamoxifen efficacy.Proc Am Soc Clin Oncol. 1998; 17: 97aGoogle Scholar, 18Gusterson BA Gelbert RD Goldhirsch A Price KN Save-Soderborgh J Anbazhagan R Styles J Rudenstam CM Golouh R Reed R Prognostic importance of c-erbB-2 expression in breast cancer.J Clin Oncol. 1992; 10: 1049-1056Crossref PubMed Scopus (836) Google Scholar With some 180,000 new breast cancer cases diagnosed in women in the United States annually (http://www.cancer.org/statisticis/statistics.html), this new approach to therapy, which may be a paradigm for successful cancer treatment in the future, depends heavily on reliable clinical laboratory results assessing the suitability of a tumor for Herceptin therapy. There are several ways to measure endogenous gene amplification, including extraction methods based on Southern, Northern, or Western blots, immunohistochemistry, and in situ hybridization.10Slamon DJ Godolphin W Jones L Holt J Wong S Keith D Levin W Stuart S Udove J Ulrich A Press M Studies of the Her-2/neu proto-oncogene in human breast and ovarian cancer.Science. 1989; 244: 707-712Crossref PubMed Scopus (6172) Google Scholar, 11Tsuda H Hirohashi S Shimosato Y Hirota T Tsugane S Yamamoto H Miyajima N Toyoshima K Yamamoto T Yokota J Yoshda T Skamoto H Terada M Sugimura T Correlation between long-term survival in breast cancer patients and amplification of two putative oncogene coamplification units: hst-1 1/int-2 and c-erb B-2/er-1.Cancer Res. 1989; 49: 3104-3108PubMed Google Scholar, 12Kallioniemi OP Holli K Visakorpi T Koivula T Helin HH Isola JJ Association of c-erbB-2 protein over-expression with high rate of cell proliferation, increased risk of visceral metastasis and poor long-term survival in breast cancer.Int J Cancer. 1991; 49: 650-655Crossref PubMed Scopus (342) Google Scholar, 13Babiak J Hugh J Poppema S Significance of c-erbB-2 amplification and DNA aneuploidy. Analysis in 78 patients with node-negative breast cancer.Cancer. 1992; 70: 770-776Crossref PubMed Scopus (33) Google Scholar, 14Toikkanen S Helin H Isola J Joensuu H Prognostic significance of HER-2 oncoprotein expression in breast cancer: a 30-year follow-up.J Clin Oncol. 1992; 10: 1044-1048Crossref PubMed Scopus (280) Google Scholar, 15Thor AD Berry DA Budman DR Muss HB Kute T Henderson IC Barcos M Cirrincione C Edgerton S Allred C Norton L Liu ET erbB2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer.J Natl Cancer Inst. 1998; 90: 1346-1360Crossref PubMed Scopus (544) Google Scholar, 16Paik S Bryant J Park C Fisher B Tan-Chiu E Hyams D Fisher ER Lippman ME Wickerham DL Wolmark N erbB-2 and response to doxorubicin in patients with axillary lymph node-positive, hormone receptor-negative breast cancer.J Natl Cancer Inst. 1998; 90: 1361-1370Crossref PubMed Scopus (604) Google Scholar, 17Bianco AR De Laurentiis M Carlomagno C 20 year update of the Naples GUN trial of adjuvant breast cancer therapy: evidence of interaction between c-erb-B2 expression and tamoxifen efficacy.Proc Am Soc Clin Oncol. 1998; 17: 97aGoogle Scholar, 18Gusterson BA Gelbert RD Goldhirsch A Price KN Save-Soderborgh J Anbazhagan R Styles J Rudenstam CM Golouh R Reed R Prognostic importance of c-erbB-2 expression in breast cancer.J Clin Oncol. 1992; 10: 1049-1056Crossref PubMed Scopus (836) Google Scholar, 19Pauletti G Godolpin W Press MF Slamon DJ Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization.Oncogene. 1996; 13: 63-72PubMed Google Scholar, 20Jimenez RE Wallis T Tabasczka P Visscher DW Determination of Her-2/neu status in breast carcinoma: comparative analysis of immunohistochemistry and fluorescent in situ hybridization (FISH).Mod Pathol. 2000; 13: 37-45Crossref PubMed Scopus (189) Google Scholar, 21Press MF Hung G Godolphin W Slamon DJ Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression.Cancer Res. 1994; 54: 2771-2777PubMed Google Scholar, 22Roche PC Ingle JN Increased Her-2 with US Food and Drug Administration approved antibody.J Clin Oncol. 1999; 17: 434-435PubMed Google Scholar, 23Roche PC Letter to editor.CAP Today. June 1999; : 16Google Scholar, 24Tubbs RR Breast cancer tests: her-2/neu versus FISH.Lab Med. 2001; 32: 518Crossref Scopus (1) Google Scholar, 25Persons DL Bui MM Lowery MC Mark HFL Yung JF Birkmeier JM Wong EY Yang SJ Masood S Fluorescence in situ hybridization (FISH) for detection of Her-2/neu amplification in breast cancer: a multicenter portability study.Ann Clin Lab Sci. 2000; 30: 41-48PubMed Google Scholar, 26Bankfalvi A Simon Brandt B Burger H Vollmer I Dockhorn-Dworniczak B Lelle R-J Boecker W Comparative methodological analysis of erbB-2/Her-2 gene dosage, chromosomal copy number and protein over-expression in breast cancer tissues for diagnostic use.Histopathology. 2000; 37: 411-419Crossref PubMed Scopus (64) Google Scholar Controversy persists as to which of these methods provides the most accurate, precise, and reproducible results as a basis for therapy.20Jimenez RE Wallis T Tabasczka P Visscher DW Determination of Her-2/neu status in breast carcinoma: comparative analysis of immunohistochemistry and fluorescent in situ hybridization (FISH).Mod Pathol. 2000; 13: 37-45Crossref PubMed Scopus (189) Google Scholar, 21Press MF Hung G Godolphin W Slamon DJ Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression.Cancer Res. 1994; 54: 2771-2777PubMed Google Scholar, 22Roche PC Ingle JN Increased Her-2 with US Food and Drug Administration approved antibody.J Clin Oncol. 1999; 17: 434-435PubMed Google Scholar, 23Roche PC Letter to editor.CAP Today. June 1999; : 16Google Scholar, 24Tubbs RR Breast cancer tests: her-2/neu versus FISH.Lab Med. 2001; 32: 518Crossref Scopus (1) Google Scholar, 25Persons DL Bui MM Lowery MC Mark HFL Yung JF Birkmeier JM Wong EY Yang SJ Masood S Fluorescence in situ hybridization (FISH) for detection of Her-2/neu amplification in breast cancer: a multicenter portability study.Ann Clin Lab Sci. 2000; 30: 41-48PubMed Google Scholar, 26Bankfalvi A Simon Brandt B Burger H Vollmer I Dockhorn-Dworniczak B Lelle R-J Boecker W Comparative methodological analysis of erbB-2/Her-2 gene dosage, chromosomal copy number and protein over-expression in breast cancer tissues for diagnostic use.Histopathology. 2000; 37: 411-419Crossref PubMed Scopus (64) Google Scholar, 27Gancberg D Lespagnard L Rouzs G Paesmans M Piccart M DiLeo A Nogaret JM Hertens D Verhest A Larsimont D Sensitivity of Her-2/neu antibodies in archival tissue samples of invasive breast carcinomas.Am J Clin Pathol. 2000; 113: 675-682Crossref PubMed Scopus (86) Google Scholar, 28Lebeau A Deimling D Kaltz C Sendelhofert A Iff A Luthardt B Untch M Löhrs U Her-2/neu analysis in archival tissue samples of human breast cancer: comparison of immunohistochemistry and fluorescence in situ hybridization.J Clin Oncol. 2001; 19: 354-363Crossref PubMed Scopus (301) Google Scholar, 29Couturier J Vincent-Salomon A Nicolas A Beuzeboc P Mouret E Zafrani B Sastre-Garau X Strong correlation between results of fluorescent in situ hybridization and immunohistochemistry for the assessment of the ERBB2 (Her-2/neu) gene status in breast carcinoma.Mod Pathol. 2000; 13: 1238-1243Crossref PubMed Scopus (100) Google Scholar, 30Tubbs RR Stoler MH The quality of Her-2/neu predictive immunohistochemistry: something FISHy?.Mod Pathol. 2000; 12: 1-3Crossref Scopus (14) Google Scholar, 31Hoang MP Sahin AA Ordòñez NG Sneige N Her-2/neu gene amplification compared with Her-2/neu protein overexpression and interobserver reproducibility in invasive breast carcinoma.Am J Clin Pathol. 2000; 113: 852-859Crossref PubMed Scopus (190) Google Scholar, 32Ridolfi RL Jamehdor MR Arber JM Her-2/neu testing in breast carcinoma: a combined immunohistochemical and fluorescence in situ hybridization approach.Mod Pathol. 2000; 13: 866-873Crossref PubMed Scopus (181) Google Scholar, 33O'Malley FP Parkes R Latta E Tjan S Zadro T Mueller R Arneson N Blackstein M Andrulis I Comparison of Her-2/neu status assessed by quantitative polymerase chain reaction and immunohistochemistry.Am J Clin Pathol. 2001; 115: 504-511Crossref PubMed Scopus (70) Google Scholar, 34Kakar S Puangsuvan N Stevens JM Serenas R Mangan G Sahai S Mihalov ML HER-2/neu assessment in breast cancer by immunohistochemistry and fluorescence in situ hybridization: comparison of results and correlation with survival.Mol Diagn. 2000; 5: 199-207Crossref PubMed Google Scholar, 35Kakar S Puangsuvan N Stevens JM Serenas R Mangan G Sahai S Mihalov M Comparison of PathVysion and INFORM fluorescence in situ hybridization kits for assessment of Her-2/neu status in breast carcinoma.Mol Diagn. 2000; 5: 193-197PubMed Google Scholar, 36Tubbs RR Pettay JD Roche PC Stoler MH Jenkins RB Grogan TM Discrepancies in clinical laboratory testing of eligibility for Trastuzumab therapy: apparent immunohistochemical false-positives do not get the message.J Clin Oncol. 2001; 19: 2714-2721Crossref PubMed Scopus (335) Google Scholar Perhaps some of this angst reflects pathologists’ discomfort with “working in the dark,” ie, reliance on fluorescence in situ hybridization (FISH) in which morphology and gene amplification are primarily disconnected. Counting gene copy signals using oil immersion high-magnification microscopy, paying someone else to count signals, and/or purchasing special image analysis equipment is expensive and time consuming and not easily accommodated in the usual diagnostic surgical pathology practice. Other FISH limitations include fluorescent signal fading and bleaching limiting slide archiving. We tested the hypothesis that a bright-field assay for HER-2/neu gene amplification, qualitatively evaluable by conventional light microscopy without the need for oil immersion microscopy, yields results for gene amplification equal to fluorescence-based methods (FISH). HER-2/neu was used as the endogenous gene of interest. This work was based on a series of cases of invasive ductal carcinomas of breast, using direct label FISH as the reference standard. Results were also correlated with digoxigenin-based indirect FISH, RNA:RNA in situ hybridization, and immunohistochemistry using both monoclonal and polyclonal antibodies. Patient samples from 100 primary infiltrating mammary carcinomas were selected for study from a cohort recently published.36Tubbs RR Pettay JD Roche PC Stoler MH Jenkins RB Grogan TM Discrepancies in clinical laboratory testing of eligibility for Trastuzumab therapy: apparent immunohistochemical false-positives do not get the message.J Clin Oncol. 2001; 19: 2714-2721Crossref PubMed Scopus (335) Google Scholar Each paraffin block from the 100 cases included sufficient redundant paraffin-embedded archived tissue for completion of all of the components of the study. All tissues were first evaluated with a presectioning hematoxylin- and eosin-stained section, as well as a section after preparation of the last unstained section, to confirm the presence of infiltrating carcinoma in material to be studied. Direct FISH, indirect FISH (DigFISH), RNA:RNA autoradiographic in situ hybridization, and immunohistochemistry using CB11 monoclonal antibody (Ventana Medical Systems, Tucson, AZ) and HercepTest (Dako, Carpinteria, CA), were performed as previously described.36Tubbs RR Pettay JD Roche PC Stoler MH Jenkins RB Grogan TM Discrepancies in clinical laboratory testing of eligibility for Trastuzumab therapy: apparent immunohistochemical false-positives do not get the message.J Clin Oncol. 2001; 19: 2714-2721Crossref PubMed Scopus (335) Google Scholar, 37Tubbs RR Pettay JD Roche PC Stoler MH Jenkins RB Myles J Grogan TM Concomitant oncoprotein detection with fluorescence in situ hybridization (CODFISH): a fluorescence-based assay enabling simultaneous visualization of gene amplification and encoded protein expression.J Mol Diagn. 2000; 2: 78-83Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 38Angerer LM Stoler MH Angerer RC In situ hybridization with RNAprobes—an annotated recipe.in: Valentino K Eberwine J Barchus J In-Situ Hybridization Applications to Neurobiology. Oxford University Press, New York1987: 42-70Google Scholar, 39Stoler MH Tissue in situ hybridization.in: Henry JB Clinical Diagnosis and Management by Laboratory Methods. W. B. Saunders, New York1996: 1400-1412Google Scholar Statistical comparisons were made using Statview (SAS Institute, Cary, NC). The method is summarized in Figure 1. Unstained 4-μm paraffin sections on electrostatically charged slides (Superfrost; Fisher, Pittsburgh, PA) were baked overnight at 60°C, and deparaffinized and rehydrated in two changes of xylene, 5 minutes each, two 1-minute washes in absolute alcohol, two 1-minute washes in 95% alcohol, and then soaked in distilled water for 5 minutes. Sections were then cell conditioned using target retrieval solution (DAKO, Carpinteria, CA) for 40 minutes at 95°C, then allowed to cool for 20 minutes at ambient temperature. Multiple changes of distilled water throughout a 5-minute period were followed by enzyme digestion with Proteinase K (DAKO) at a 1:5000 dilution in 50 mm of Tris-HCl, pH 7.6, for 4 minutes at room temperature. After 5 minutes of washing and several changes of distilled water, endogenous peroxidase was blocked for 20 minutes using 3% hydrogen peroxide (H2O2) in absolute methanol at room temperature. After a 20-minute distilled water wash at room temperature, sections were dehydrated with graded alcohols (absolute, 95%, 70%), and air-dried. Biotinylated cDNA probe (Ventana Medical Systems International, Tucson, AZ) preincubated with COT 1 DNA (1 mg/ml; Roche, Indianapolis, IN), and salmon sperm DNA (Sigma Chemical Co., St. Louis, MO) was applied as 10 μl, and a coverslip applied and sealed with rubber cement. The probe and target were co-denatured for 6 minutes at 90°C, and allowed to hybridize at 37°C overnight in a humidified chamber. Coverslips were then removed by soaking in 2× standard saline citrate (Sigma Chemical Co.), and subjected to a stringency wash of 0.5× standard saline citrate at 72°C for 5 minutes. Sections were then washed with 1× phosphate-buffered saline (PBS) containing 0.1% Tween 20 for 3 minutes at room temperature. Streptavidin-horseradish peroxidase (DAKO SA/HRP from Gen Point Kit at 1:800) 100 μl total volume, was applied for 15 minutes at room temperature. Sections were washed in 1× PBS with 0.1% Tween 20 three times, 5 minutes each wash, at room temperature. Prediluted tyramide signal amplification (TSA) biotin (DAKO Gen Point Kit) was then applied to the sections for 5 minutes at room temperature. Sections were then washed with PBS/Tween 20 three times for 5 minutes each at room temperature, treated with Lugol’s iodine (Fisher) by immersion for 5 minutes at room temperature, followed by three rinses in distilled water, and cleared by immersion for ∼5 seconds in sodium thiosulfate (2.5%). The iodine solution was used to remove contaminating heavy metals in the sections that would interfere with autometallography. Sections were then washed in double-distilled water for a total of 7 minutes using five rinses. Slides were then immersed for 5 minutes in PBS, pH 7.6, containing 0.1% cold water fish gelatin (Sigma). Streptavidin-Nanogold (Nanoprobes, Inc., Yaphank, NY) prediluted to 1:400 with PBS, pH 7.6, containing 1% bovine serum albumin was applied and then incubated for 30 minutes at room temperature. Sections were then washed with PBS, pH 7.6, for two 5-minute washes, immersed in PBS, pH 7.6, with 0.1% cold water fish gelatin for 5 minutes, rinsed in double-distilled water for using multiple changes throughout 10 minutes, and the autometallographic signal developed with GoldEnhance LM/Blot (Nanoprobes, Inc.); the autometallographic signal was developed for 4 minutes at room temperature, and the reaction terminated by adding 500 μl of 2.5% sodium thiosulfate, and then double-distilled water for 3 minutes at room temperature (Figure 2, Figure 3). Sections were counterstained with nuclear fast red (Newcomer, Middleton, WI) for 8 minutes, dehydrated in graded alcohols and xylene, and coverslipped and mounted with Cytoseal/60 (Stephens Scientific, Riverdale, NJ). Controls for each experiment included positive and negative controls, which consisted of tumors independently and previously evaluated in triplicate for the presence or absence of gene amplification by direct FISH and for which chromosome 17 enumeration had been established. An additional internal positive hybridization control was present on every slide, consisting of the identification of endogenous nonamplified HER-2/neu gene signals in nonneoplastic cells. Figure 2, Figure 3 illustrate the mechanism of signal development. Gold-facilitated fluorescence in situ hybridization (GOLDFISH) slides were interpreted as amplified, low-level amplified, or nonamplified using conventional microscopy at ×100 and ×400. GOLDFISH preparations were designated as “nonamplified” if only one or two small discreet black signals were identified within nuclei of the invasive carcinoma. Approximately 30 to 40% of tumor cell nuclei demonstrated one or two individual small discreet signals—a 4-μm section contains primarily truncated nuclei and the gene target may not be present in all of the cells in an individual 4-μm section. The designation “low-level amplification” was rarely made, and reserved for cases with four to eight (usually four or five) discreet black small nonconfluent signals within invasive tumor nuclei. The designation “HER-2/neu amplified” was reserved for invasive carcinomas demonstrating massive black confluent signals in invasive tumor cell nuclei that were usually located centrally in the nucleus and occupied most of the nuclear area.Figure 2Mechanism for metallic gold deposition via autometallography using GoldEnhance. Gold ions in solution are catalytically deposited onto the Nanogold particle as metallic gold (Au0). Particle grows in size with development time: shorter times are used for electron microscopy, longer times for light microscopy and blots.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Mechanism of signal diameter and intensity of growth.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Results of the gold-based autometallographic assay were compared directly with two FISH assays (direct FISH and indirect DigFISH) using digoxigenin-labeled probe), mRNA autoradiographic in situ hybridization, and paraffin section immunohistology. Results are summarized in Table 1. The correlation of the autometallographic assay with both DigFISH and the reference direct FISH standard was excellent and identical (Table 1 and Figure 4, Figure 5, Figure 6, Figure 7). When the assay results were compared to mRNA and protein expression, correlation with mRNA was reasonable but correlation with immunohistochemistry quite variable (Table 1). Discrepancies between GOLDFISH and immunohistochemistry results were identified, consistent with the discordances well-documented in the literature when immunohistochemistry for HER-2/neu is compared with FISH.20Jimenez RE Wallis T Tabasczka P Visscher DW Determination of Her-2/neu status in breast carcinoma: comparative analysis of immunohistochemistry and fluorescent in situ hybridization (FISH).Mod Pathol. 2000; 13: 37-45Crossref PubMed Scopus (189) Google Scholar, 21Press MF Hung G Godolphin W Slamon DJ Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression.Cancer Res. 1994; 54: 2771-2777PubMed Google Scholar, 22Roche PC Ingle JN Increased Her-2 with US Food and Drug Administration approved antibody.J Clin Oncol. 1999; 17: 434-435PubMed Google Scholar, 23Roche PC Letter to editor.CAP Today. June 1999; : 16Google Scholar, 24Tubbs RR Breast cancer tests: her-2/neu versus FISH.Lab Med. 2001; 32: 518Crossref Scopus (1) Google Scholar, 25Persons DL Bui MM Lowery MC Mark HFL Yung JF Birkmeier JM Wong EY Yang SJ Masood S Fluorescence in situ hybridization (FISH) for detection of Her-2/neu amplification in breast cancer: a multicenter portability study.Ann Clin Lab Sci. 2000; 30: 41-48PubMed Google Scholar, 26Bankfalvi A Simon Brandt B Burger H Vollmer I Dockhorn-Dworniczak B Lelle R-J Boecker W Comparative methodological analysis of erbB-2/Her-2 gene dosage, chromosomal copy number and protein over-expression in breast cancer tissues for diagnostic use.Histopathology. 2000; 37: 411-419Crossref PubMed Scopus (64) Google Scholar, 27Gancberg D Lespagnard L Rouzs G Paesmans M Piccart M DiLeo A Nogaret JM Hertens D Verhest A Larsimont D Sensitivity of Her-2/neu antibodies in archival tissue samples of invasive breast carcinomas.Am J Clin Pathol. 2000; 113: 675-682Crossref PubMed Scopus (86) Google Scholar, 28Lebeau A Deimling D Kaltz C Sendelhofert A Iff A Luthardt B Untch M Löhrs U Her-2/neu analysis in archival tissue samples of human breast cancer: comparison of immunohistochemistry and fluorescence in situ hybridization.J Clin Oncol. 2001; 19: 354-363Crossref PubMed Scopus (301) Google Scholar, 29Couturier J Vincent-Salomon A Nicolas A Beuzeboc P Mouret E Zafrani B Sastre-Garau X Strong correlation between results of fluorescent in situ hybridization and immunohistochemistry for the assessment of the ERBB2 (Her-2/neu) gene status in breast carcinoma.Mod Pathol. 2000; 13: 1238-1243Crossref PubMed Scopus (100) Google Scholar, 30Tubbs RR Stoler MH The quality of Her-2/neu predictive immunohistochemistry: something FISHy?.Mod Pathol. 2000; 12: 1-3Crossref Scopus (14) Google Scholar, 31Hoang MP Sahin AA Ordòñez NG Sneige N Her-2/neu gene amplification compared with Her-2/neu protein overexpression and interobserver reproducibility in invasive breast carcinoma.Am J Clin Pathol. 2000; 113: 852-859Crossref PubMed Scopus (190) Google Scholar, 32Ridolfi RL Jamehdor MR Arber JM Her-2/neu t" @default.
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