Applications. Eppendorf Thermomixer: A low-cost hybridization station for high quality FISH analysis. Note 156 July Abstract.

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1 Applications Note 156 July 2007 Eppendorf Thermomixer: A low-cost hybridization station for high quality FISH analysis. Sascha Eghtessadi, Maria Christina Tsourlakis, Annastasia Tsaklakidis, Silvia Schnöger, and Ronald Simon Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Abstract Fluorescence in situ hybridization (FISH) is the gold standard methodology for gene copy number determination in cultured cells, metaphase spreads, tissue samples or cytologic specimens. While it has primarily been used as a research tool during the last decade, there is a growing demand for the use of FISH as a diagnostic tool. In such a setting, analysis speed, reliability and cost efficiency become important parameters. Perhaps the most critical step in the FISH procedure is the proper adjustment and maintenance of temperatures for DNA denaturation and probe hybridization. We have compared different devices for slide heating to estimate their suitability for FISH analysis. Our study demonstrates that programmable slide incubators are profoundly superior to the classical waterbath for FISH analysis. The Eppendorf add-on hybridization station is an attractive alternative for owners of the Thermomixer comfort device that allows for high-quality FISH analysis at low cost for machinery. Introduction Fluorescence in situ hybridization (FISH) technology is a means to directly visualize defined DNA sequences in cell nuclei of cell preparations, metaphase spreads, or histological tissue sections. FISH is a frequently used technique particularly in biological and medical research, for example to localize genes on chromosomes, map translocation breakpoints, or to determine gene copy numbers [1]. During the last decade FISH has massively gained importance also for medical diagnostics. Primary genetic events like chromosomal translocations, amplifications, or DNA mutations highlight genetic loci of genes with particular importance for cancer development, diagnosis, or therapy. In particular, genes that undergo amplification have proven to be suitable targets for a new class of gene specific anticancer drugs. Probably the bestknown example for a gene that frequently undergoes amplification is the ERBB2 (HER2/neu) transmembrane receptor. Located on chromosome 17q12, ERBB2 encodes a growth factor receptor tyrosine kinase that connects an extracellular growth trigger to an intracellular signal transduction pathway that finally results in cell growth [2]. Although ERBB2 is (except from heart) not expressed in normal human tissues, it is of major importance in breast cancer. About % of advanced breast cancers are characterized by amplification of the genomic region harboring the ERBB2 gene [3]. The amplification usually results in an elongated chromosome with dozens of adjacent ERBB2 gene copies. Since all ERBB2 gene copies are readily translated into protein, ERBB2 amplified cells are characterized by a massive overexpression of the receptor molecule at the cell surface. Because all these ERBB2 molecules are functional and can activate the growth cascade, ERBB2 amplification and overexpression obligatory leads to breast cancer. In 1998, the FDA approved trastuzumab (Herceptin), a monoclonal antibody that binds to the ERBB2 receptor. Clinical studies demonstrated that Herceptin treatment in combination with cytotoxic drugs provides a significant survival benefit for patients with ERBB2 positive breast cancer, making ERBB2 a paradigm for the new class of targeted therapies [4]. These findings have raised the issue of what is the best method for ERBB2 diagnosis in clinical praxis. Early studies used immunohistochemistry to directly detect ERBB2 protein overexpression in tissue sections from breast tumors. Later it was found that an indirect approach analyzing the ERBB2 gene copy number by means of fluorescence in situ hybridization (FISH) is more accurate and better predicts response to Herceptin treatment [5].

2 Application Note 156 Page 2 Introduction The importance of FISH analysis for the emerging field of predictive molecular pathology is steadily growing. Additional genes have been identified that undergo amplification, and that represent targets for existing gene specific therapies (e.g. EGFR) [6], or targets for drugs under development (e.g. AKT) [7]. As a consequence, a growing need for high quality FISH analysis as a tool for medical routine diagnostic can be expected. FISH analysis is a technically challenging procedure. Besides the quality of the probe (which is generally excellent for commercial probes), highly standardized experimental conditions, particularly with respect to denaturation and hybridization temperatures, are crucial for analysis success. This study was designed to address the suitability of a low-cost add-on slide tray to the Eppendorf Thermomixer comfort device for the FISH based detection of ERBB2 amplification in breast cancer tissue samples. Materials and Methods Tissues. For evaluation of ERBB2 amplification, a total of 229 tissue samples were selected from the archive of the Department of Pathology of the University Medical Center Hamburg Eppendorf (Hamburg, Germany). A tissue array (TMA) was constructed from these samples according to the procedure described in Kononen et al [8]. Selected tissues included 171 samples of invasively growing high grade (BRE grade 3) ductal breast cancers that have the highest likelyhood to contain gene amplifications. In addition to the breast cancer samples, 58 control tissues including normal tissue samples of heart (n=2), kidney (n=2), lung (n=2), colon (n=2), endometrium (n=2), prostate (n=2), lymph node (n=2), sceletal muscle (n=2), skin (n=2), breast (n=10), as well as several different neoplastic tissues including lung cancers (n=10), colon cancers (n=10), and prostate cancers were included in the TMA (Figure 1). Fluorescence in situ hybridization. The TMA sections were treated according to the Paraffin Pretreatment Reagent Kit protocol (Vysis, Downers Grove, IL) before hybridization. To study the potential influence of different methods for slide heating and incubation, three experimental setups were tested. The different approaches are listed in Table 1. FISH was performed with a Spectrum Orange-labeled Fig. 1: Hematoxilin-eosin stained section of the tissue microarray. The diameter of each tissue spot is 0.6 mm. A magnification of a single tissue spot is shown in the insert. ERBB2 probe and a Spectrum Green-labeled chromosome 17 centromeric probe as reference (Vysis). Hybridization and posthybridization washes were according to the LSI procedure (Vysis). Slides were then counterstained with 1000 ng/ml 4,6-diamino-2-phenylindole in an antifade solution (Abbott, USA). Tab. 1: Experimental setup Device Vendor Features Capacity Denaturation Incubation ThermoBrite Thermomixer comfort Conventional water bath Abbott Molecular Eppendorf Various vendors Programmable slide incubator Device with heating, sooling and mixing function Can be operated with different exchangeable thermoblocks for various tube formats and slides 2 programmable incubation steps 12 slides in separate trays 4 slides in separate trays Almost unlimited; slides in coplin jars 72 C, 5 min; 10 µl denaturation solution per slide 72 C, 5 min; 10 µl denaturation solution per slide (no mixing) 72 C, 5 min; denaturation solution in coplin jar 37 C, overnight 37 C, overnight (no mixing) 37 C, overnight

3 Application Note 156 Page 3 Materials and Methods FISH scoring. ERBB2 and centromere 7 (Cen7) copy numbers were determined in 20 non-overlapping cell nuclei in each tissue spot using an epifluorescence microscope equipped with filtersets suitable for detection of the spectrum orange and spectrum green labeled FISH probes. Average signal numbers from the 20 cell nuclei were calculated for Cen7 and ERBB2. Amplification was defined as presence of at least two times more average ERBB2 gene signals than average centromere 7 signals. All samples not meeting this criterion were categorized as normal. Results Not all tissue spots were interpretable by FISH analysis (Table 2). Reasons for non-analyzable tissues were first of all related to the TMA technology, i.e. missing spots in the tissue section. The number of available tissue spots was n = 209 for the slides incubated in the ThermoBrite and the Theromixer comfort, and n = 207 in the slide incubated in the waterbath. Another fraction of tissue spots was not analyzable because of lacking FISH signals. Such failure occurred only rarely if the slides were incubated in the ThermoBrite (10/209 analyses failed, 5 %) or in the Thermomixer comfort device (13/209, 6 %). In contrast, more than one third of tissues (63/207, 36 %) did not show FISH signals in the water bath-incubated slide. Accordingly, ERBB2 status was interpretable in 184 tissues (including 153 cancers) pretreated in the ThermoBrite, in 186 tissues (142 cancers) pretreated in the Thermomixer comfort, and in only 134 tissues (89 cancers) that had been incubated using the waterbath. In addition, fluorescence intensity was overall slightly weaker for the slide incubated in the waterbath as compared to ThermoBrite or Theromixer comfort (Figure 2). b c a Fig. 2: Examples of ERBB2 amplification. A) ThermoBrite, B) Thermomixer comfort, C) waterbath However, cases with ERBB2 amplification were readily identified irrespective of the type of slide incubation. Small differences in the overall frequency of ERBB2 amplification in breast cancer tissues (18.3 %, 18.3 %, 22.5 %, respectively) only resulted from the different number of interpretable cases. All results are summarized in Table 2.

4 Application Note 156 Page 4 Results Tab. 2: Influence of different slide incubation devices on the success of HER2-FISH analysis in breast cancer. ThermoBrite Thermomixer comfort Waterbath TMA contents Tissue spots on TMA slide Tumors Controls Studied tissues Spots available for study Successfully analyzed spots Successfully analyzed cancer spots ERBB2 amplification (n) ERBB2 amplification (%) Discussion The results of our study demonstrate that the laboratory equipment used for adjusting and maintaining proper slide temperature has profound influence on the success of FISH analysis. Computer-controlled devices with special slots for microscopic slides are clearly superior over conventional waterbaths. We used a tissue microarray approach to compare analysis results in more than 200 breast tissue samples. TMAs are perfectly suited for such kind of protocol comparisons, because a high number of samples can be simultaneously analyzed under highly standardized conditions. In particular, enzymatic tissue pretreatment, which is a critical step for optimal penetration of the FISH probe into the cell nucleus, was performed simultaneously for the three slides, therefore strictly avoiding any potential experimental bias. In addition, a commercially available, FDA approved ERBB2 FISH probe was used that is optimized for analysis of paraffin-embedded, formalin fixed breast cancer tissues. As expected, FISH results were highly comparable if the ThermoBrite or the Thermomixer comfort device were used for slide heating. Both devices are specifically made for this purpose, and differ mainly only in the capacity. Whereas the Abbott TermoBrite (12 slots) is perfectly suited for medium to large scale FISH analysis (e.g. in routine diagnostic labs), the Eppendorf Thermomixer comfort (4 slots) represents a low cost add-on device that may be wanted by customers already owning a Thermomixer comfort device and whishing to expand the abilities of their labs for occasional high-quality FISH analysis. General availability in labs, as well as a great slide capacity, is an advantage of the waterbath-approach. However, a critical step in FISH analysis is maintenance of the proper temperature of DNA denaturation [9]. Insufficient denaturation usually results in low hybridization efficiency and loss of signal strength, whereas overdenaturation because of too high temperature or excessive denaturation time causes damage to the chromosomes and blurred FISH signals that cannot be properly scored. For denaturation, coplin jars with denaturation solution are put into the water and preheated to 75 C, before the slides are added. According to our own experience, the temperature might drop significantly (2 5 C, depending on the number of slides) in the moment the cold slides are put in. Even in our present study where only one single slide was denatured at a time, this effect resulted in a particularly high fraction (36 %) of non-analyzable tissue spots. In comparison, only 5 6 % nonanalyzable tissue spots where found using the ThermoBrite or Thermomixer comfort. The obvious difficulties in adjusting and maintaining proper temperature, and the risk of considerable additional costs in case of failed analyses that must be repeated using expensive commercial FISH probes, clearly argument against a waterbath as first choice for FISH analysis. Remarkably, presence of ERBB2 amplification was properly identified independently from the method or device used for slide incubation, provided that FISH analysis was successful. Even the use of a suboptimal procedure (i.e. waterbath incubation) merely led to a poor overall success rate of FISH analysis, but did not produce any false positive or false negative results with respect to ERBB2 amplification. This finding illustrates an important advantage of FISH over immunohistochemistry (IHC) for diagnostic ERBB2 analysis. In IHC analysis, non reactive samples inevitably produce a seemingly negative result. This is typically caused by improper tissue fixation or antigen retrieval procedures that prevent antibody binding,

5 Application Note 156 Page 5 Discussion and cannot be distinguished from true negative (lack of protein expression) results. Even the use of positive control tissues or cell lines cannot avoid such misinterpretation, because they merely control for proper processing of the IHC assay, but not for insufficient tissue quality of the test slide. Such false negative results cannot occur in FISH analysis, because tissues lacking any detectable FISH signals will be readily recognized as non-reactive and excluded from analysis. In summary, our study demonstrates that programmable slide incubators are profoundly superior to the classical waterbath for FISH analysis. The Eppendorf add-on hybridization station is an attractive alternative for owners of the Thermomixer comfort device that allows for high-quality FISH analysis at low cost for machinery. References [1] Trask BJ. DNA sequence localization in metaphase and interphase cells by fluorescence in situ hybridization. Methods Cell Biol 1991; 35:3 35. [2] Bargmann CI, Hung MC, Weinberg RA. The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature 1986; 319: [3] Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, and McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987; 235: [4] Pegram MD, Lipton A, Hayes DF, Weber BL, Baselga JM, Tripathy D, Baly D, Baughman SA, Twaddell T, Glaspy JA, and Slamon DJ. Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185her2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J Clin Oncol 1998; 16: [5] Mass, RD, Press MF, Anderson S, Cobleigh MA, Vogel CL, Dybdal N, Leiberman G, and Slamon DJ. Evaluation of clinical outcomes according to HER2 detection by fluorescence in situ hybridization in women with metastatic breast cancer treated with trastuzumab. Clin Breast Cancer 2005; 6: [6] Hirsch FR, Varella-Garcia M, McCoy J, West H, Xavier AC, Gumerlock P, Bunn PA Jr., Franklin WA, Crowley J, and Gandara DR. Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: a Southwest Oncology Group Study. J Clin Oncol 2005; 23: [7] Thomas SA, Li T, Woods KW, Song X, Packard G, Fischer JP, Diebold RB, Liu X, Shi Y, Klinghofer V, Johnson EF, Bouska JJ, Olson A, Guan R, Magnone SR, Marsh K, Luo Y, Rosenberg SH, Giranda VL, and Li Q. Identification of a novel 3,5-disubstituted pyridine as a potent, selective, and orally active inhibitor of Akt1 kinase. Bioorg Med Chem Lett 2006;16: [8] Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, and Kallioniemi OP. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998; 4: [9] Petersen BL, Sorensen MC, Pedersen S, and Rasmussen M. Fluorescence in situ hybridization on formalin-fixed and paraffin-embedded tissue: optimizing the method. Appl Immunohistochem Mol Morphol 2004; 12:

6 Application Note 156 Page 6 Ordering information Description Order no. international Order no. North America Thermomixer comfort (Thermomixer R), without thermoblock V, Hz V, Hz V, Hz Exchangeable thermoblock for 4 slides ThermoBrite is a trademark of Abbott Molecular, Des Plaines, USA Your local distributor: Eppendorf AG Hamburg Germany Tel Fax eppendorf@eppendorf.com Eppendorf North America, Inc. One Cantiague Road, P.O. Box 1019 Westbury, N.Y USA Tel Toll free phone Fax info@eppendorf.com Application Support Europe, International: Tel support@eppendorf.com North America: Tel ext support_na@eppendorf.com Asia, Pacific: Tel support_asiapacific@eppendorf.com Eppendorf is a registered trademark. Order No. AA15 6PW 020/GB1/WEB/0707/NEUH