Anatomic Pathology / Visualization of FISH Probes by Dual-Color CISH

Visualization of FISH Probes by Dual-Color Chromogenic In Situ Hybridization

Kirsten Hoff, MSc, PhD,1 Jan T. Jrgensen, MScPharm, PhD,2 Sven Mller, MSc, PhD,3 Else Rngaard,4 Ole Rasmussen, MSc, PhD,5 and Andreas Schnau, MSc3
Key Words: Chromogenic in situ hybridization; CISH; Fluorescence in situ hybridization; FISH; HER2; Signal conversion; TOP2A
DOI: 10.1309/AJCP12MHRTFZJPKW

CME/SAM

Upon completion of this activity you will be able to: describe the principles for conversion of fluorescence in situ hybridization signals to chromogenic signals by the DAKO dual-color chromogenic in situ hybridization (CISH) assay. describe the application area for CISH. discuss the advantages of CISH.

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit per article. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 336. Exam is located at www.ascp.org/ajcpcme.

Abstract
The overall purpose of the study was to demonstrate applicability of the DAKO dual-color chromogenic in situ hybridization (CISH) assay (DAKO Denmark, Glostrup) with respect to 4 fluorescence in situ hybridization (FISH) probes: MYC (c-MYC), EGFR, ERBB2 (HER2), and TOP2A. The study showed that the dual-color CISH assay can convert Texas red and fluorescein isothiocyanate (FITC) signals into chromogenic signals with an almost complete 1:1 conversion ratio. Agreement studies between the FISH assays for HER2 and TOP2A and the corresponding CISH conversion assays showed 100% concordance ( values of 1.0) between the CISH and FISH methods for HER2 and TOP2A status. The correlations of the gene copy number to centromere-17 ratios were similarly high, with a correlation coefficient (r) for HER2 and TOP2A of more than 0.95. Owing to the relatively small number of specimens in this study, it is important that the data are confirmed in a larger study.

Fluorescence in situ hybridization (FISH) has achieved widespread use for a range of gene copy number detection probes and translocation probes. It is considered a very accurate and sensitive method, eg, the College of American Pathologists has published that FISH is to be regarded as a gold standard for HER2 testing.1 However, the FISH method is perceived as having some limitations. The evaluation of tumor morphologic features through FISH may be difficult, and the method requires a fluorescence microscope, which is costly and not readily available in all pathology laboratories. Furthermore, the fluorescence signals fade relatively quickly, which makes archiving of the slides difficult. These limitations can be overcome by chromogenic in situ hybridization (CISH), which converts the fluorescence signals into chromogenic precipitates, and can visualize FISH-labeled probes along with the morphologic features using a brightfield microscope. We have developed a dual-color CISH assay in which the green fluorescein isothiocyanate (FITC) FISH signals are converted into blue chromogenic precipitates and the Texas red FISH signals are converted into red chromogenic precipitates. In principle, the assay can be applied on top of all FISH probes labeled with Texas red and FITC fluorochromes, including probes designed for detection of amplification and deletion in formalin-fixed, paraffin-embedded tissue sections. In relation to copy number detection, a major advantage of the dual-color approach is that the gene signals and the corresponding reference signals can be visualized in the same nuclei on 1 slide, allowing direct comparison between signals. Hence, the dualcolor technique makes it easier to distinguish true gene amplification or deletion from chromosomal aneuploidy.2
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The purpose of this study was to demonstrate the general use of the DAKO dual-color CISH prototype assay (DAKO Denmark, Glostrup) by applying it to a range of gene copy number detection probes, such as c-MYC, EGFR, HER2, and TOP2A. Furthermore, the agreement between the CISH and the FISH assays was investigated.

blinded manner. The tumor specimens used in the study originated from Danish tissue banks and were fully anonymized with respect to the identity of the patients. Fluorescence In Situ Hybridization The FISH analyzes for EGFR and c-MYC were performed using FISH probes for EGFR and c-MYC, respectively (DAKO) in combination with the Histology FISH Accessory kit (DAKO). The FISH analyses for HER2 and TOP2A were performed using the HER2 FISH pharmDx (DAKO) and the TOP2A FISH pharmDx (DAKO) assays. All stainings were performed according to the respective package insert and subsequently evaluated using a fluorescence microscope. The slides that were analyzed using the dual-color CISH kit followed the same protocols but were not mounted. Instead, they continued as described in the following paragraph. Chromogenic In Situ Hybridization The dual-color CISH assay contains reagents required to complete a 2-step immunohistochemical staining procedure to detect DAKO Texas red and FITC-labeled FISH probes. First, the aforementioned FISH procedures were followed, except the last dehydration and mounting step was omitted and the tissue specimens for CISH analysis were immersed in the CISH Wash Buffer (DAKO). The next step in the CISH procedure is to block the tissue specimens for endogen peroxidase with a ready-to-use Peroxidase Blocker (DAKO). Peroxidase blocking is followed by incubation with a readyto-use CISH Antibody Mix (DAKO), which comprises a mixture of anti-FITC conjugated with horseradish peroxidase and antiTexas red conjugated with alkaline phosphatase. Tissue specimens were then incubated with a red chromogen followed by incubation with a blue chromogen.3 The enzymatic conversion of the added red and blue chromogens results in formation of visible red and blue dots at the antigen site (FISH probes) Figure 1. Finally, the specimens were counterstained and coverslipped (Tissue-Mount, Sakura Finetek, Zoeterwoude, the Netherlands), and the results were evaluated using a bright-field microscope. Evaluation of the HER2 and TOP2A CISH and FISH Slides For all tumor specimens in this pilot study, the HER2 and TOP2A signals from 20 nuclei were counted, and the gene/ centromere 17 (CEN-17) ratios were calculated. The ratios were subsequently translated to HER2 or TOP2A status using the cutoff values shown in Table 1. Statistical Analyses for HER2 and TOP2A The agreement between FISH and CISH assays was estimated by calculating the percentage of agreement and by statistics.4 The correlation between CISH and FISH ratios for
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Materials and Methods

The study was divided into two parts, one demonstrating the general applicability of the dual-color CISH assay, including dot-to-dot conversion, and another evaluating the agreement between CISH and FISH. For the latter part, agreement was investigated for HER2 and TOP2A probes. For the HER2 probe, 24 formalin-fixed, paraffin-embedded primary breast cancer specimens were included, and for the TOP2A comparison, 20 specimens were chosen. The CISH and FISH analyses were performed on serial sections of the same tumor specimens, and the evaluation was done in a

FISH
Target DNA (gene) Target DNA (reference) Target DNA (gene)

CISH
Target DNA (reference)

Target DNA (gene)

Target DNA (reference)

Target DNA (gene)

Target DNA (reference)

Mount and read slides

Red chromogenic detection AntiTexas red conjugated with alkaline phosphatase (AP) Texas redlabeled DNA probe Target DNA (gene) Target DNA (reference)

Blue chromogenic detection Anti-FITC conjugated with horseradish phosphatase (HRP) FITC-labeled FISH PNA probe

Target DNA (gene)

Target DNA (reference)

Mount and read slides

Figure 1 The use of dual-color chromogenic in situ hybridization (CISH) in combination with fluorescence in situ hybridization (FISH) probes. FITC, fluorescein isothiocyanate; PNA, peptide nucleic acid.

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HER2 or TOP2A gene copy number to CEN-17 were investigated, and the correlation coefficients (r) were calculated. Dot-to-Dot Conversion A number of already mounted FISH slides were converted into the CISH format by the following procedure. Following inspection and photographing of the mounted FISH slides, the coverslips were carefully removed and the slides were washed twice for 5 minutes each in 99.9% ethanol to remove the mounting medium. This procedure was followed by rehydration (2 minutes each in 96%, 85%, and 70% ethanol) and washing in FISH wash buffer twice for 3 minutes each. The washed FISH slides were then ready for conversion into the CISH format by standard handling according to the CISH protocol. A second photo was then taken of the converted slide.

Table 1 Cutoff Values for HER2 and TOP2A Status

Gene/Status HER2 Normal Amplified TOP2A Deleted Normal Amplified
CEN-17, centromere 17.

Definition

HER2/CEN-17 ratio <2.0 HER2/CEN-17 ratio 2.0 TOP2A/CEN-17 ratio <0.8 TOP2A/CEN-17 ratio 0.8 but <2.0 TOP2A/CEN-17 ratio 2.0

Results
General Appearance and Dot-to-Dot Conversion The dual-color CISH assay was tested with the HER2/ CEN-17, TOP2A/CEN-17, EGFR/CEN-7, and c-MYC/CEN-8 probes. This part of the study was qualitative, evaluating the general appearance of CISH staining with respect to tissue morphologic features and the balance between red and blue signal size, sharpness, and intensity. For all 4 probes, the general appearance seemed almost identical between the 2 methods. An example of HER2 amplified breast carcinoma stained with dual-color CISH is shown in Image 1. Examples of the dot-to-dot conversions are shown in Image 2A and Image 2B (EGFR cluster amplification), Image 2C and Image 2D (EGFR polysomy), Image 2E and Image 2F (HER2 amplification), Image 2G and Image 2H (TOP2A deletion). HER2 All 24 specimens were stained and analyzed successfully by CISH and FISH. The HER2/CEN-17 ratios for CISH and FISH are shown in Figure 2. A 100% concordance was found between FISH and CISH when classifying the tumor specimens according to HER2 status. The corresponding coefficient was 1.0. The cross-tabulation of HER2 status for CISH and FISH is shown in Table 2. The correlation of the HER2/CEN-17 ratio for FISH and CISH is shown in Figure 3 (r = 1.0). TOP2A The 20 specimens available for the TOP2A part of the study were all stained and analyzed successfully by CISH and FISH. The TOP2A/CEN-17 ratios for CISH and FISH are shown in Figure 4. A 100% concordance was found between FISH and CISH when classifying the tumor
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Image 1 HER2-amplified breast carcinoma stained with dualcolor chromogenic in situ hybridization.

specimens according to TOP2A status. The corresponding coefficient was 1.0. The cross-tabulation of TOP2A status for CISH and FISH is shown in Table 3. The correlation of the TOP2A/CEN-17 ratio for FISH and CISH is shown in Figure 5 (r = 0.954).

Discussion
The present study showed that the dual-color CISH prototype assay can convert Texas red and FITC FISH signals into chromogenic signals with an almost complete 1:1 conversion ratio in context with preserved morphologic features. The agreement analyses with respect to HER2 and TOP2A status likewise showed 100% concordance, and the corresponding values were 1.0. The correlations of the gene copy number to CEN-17 ratios were similarly high,
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Image 2 Examples of dot-to-dot conversion of fluorescence in situ hybridization (FISH) (A, C, E, G) to chromogenic in situ hybridization (CISH) (B, D, F, H) signals. A and B, Lung carcinoma with epidermal growth factor receptor (EGFR) cluster amplification. C and D, Lung carcinoma with EGFR polysomy. E and F, Breast carcinoma with HER2 amplification.

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G and H, Breast carcinoma with TOP2A deletion.

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with correlation coefficients (r) for HER2 and TOP2A more than 0.95. More careful examination of the individual ratios seems to show a weak trend toward higher ratios for FISH than for CISH (Figures 2 and 4). This is especially the situation in the most amplified cases. This observation may partly be explained by looking at the gene copy number counts (data not shown). For TOP2A and HER2, these figures seem to be marginally higher for FISH than for CISH. However, this trend in differences of the gene copy number do not seem to influence the overall agreement between the 2 assays, which in this study was 100%. Owing to the relatively small number

Ratio

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Figure 2 HER2/centromere 17 (CEN-17) ratios for chromogenic in situ hybridization (white bars) and fluorescence in situ hybridization (black bars), case by case. Dotted line shows the cutoff ratio for amplification (2.0).

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Table 2 Cross-Tabulation of HER2 Status for CISH and FISH

CISH FISH Normal Amplified Total Normal 15 0 15 Amplified 0 9 9 Total 15 9 24

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CISH, chromogenic in situ hybridization; FISH, fluorescence in situ hybridization.

Figure 3 Correlation between HER2/centromere 17 (CEN-17) for fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH). r = 1.0.

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2.50 2.00 1.50 1.00 0.50 0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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Figure 4 TOP2A/centromere 17 ratios for chromogenic in situ hybridization (white bars) and fluorescence in situ hybridization (black bars), case by case. The upper dotted line shows the cutoff ratio for amplification (2.0), and the lower line shows the cutoff ratio for deletion (0.8). Table 3 Cross-Tabulation of TOP2A Status for CISH and FISH
CISH FISH Deleted Normal Amplified Total Deleted 3 0 0 3 Normal 0 13 0 13 Amplified 0 0 4 4 Total 3 13 4 20

CISH, chromogenic in situ hybridization; FISH, fluorescence in situ hybridization.

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Figure 5 Correlation between TOP2A/centromere 17 for fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH). r = 0.954.

of specimens in the study, one should be careful to not overinterpret the results, and it is important that the data from the present study be confirmed in larger studies. A larger validation study is now initiated with the dual-color CISH assay, and data from this study will be available in 2010. A number of previously published studies with HER2 probes have likewise shown high concordance between FISH and CISH assays, in the range of 91% to 100%, which seems to confirm the reliability of CISH.2,5-8 However, most of these studies have been performed with a single-color probe and without simultaneous detection of the CEN-17 reference on the same slide. We have found that the use of a dual-color probe makes it easy to calculate the HER2/CEN-17 copy number ratio, thereby making it possible to distinguish true gene amplification or deletion from chromosomal aneuploidy.2 For a single-color probe, this may also be possible, but only if the CEN-17 control probe is applied on a consecutive tissue section, which is more labor-intensive, and still this approach prevents counting of gene and reference signals in the same nuclei. The dual-color CISH compatibility with Texas red and FITC-labeled probes opens for application of the assay on all Texas red and FITC-labeled FISH probes. Besides the HER2 and TOP2A probes, the dual-color CISH assay was tested in this study on the EGFR and c-MYC probes, evaluating dotto-dot conversion. The conversion of fluorescence signals to a chromogen precipitate requires that the same nuclei are sequentially inspected in the FISH and CISH formats. Consequently, tissues representing gene amplification, deletion, polysomy, and clustered amplification were stained in the FISH protocol, examined with a fluorescence microscope, and subsequently converted to CISH. When already mounted FISH slides were converted into the CISH format, the intensity of the blue reference signal appeared a little compromised as seen when a pure CISH slide (Image 1) is compared with a CISH slide converted from an already mounted FISH slide (Image 2F). However, this slight drop in quality does not seem to hinder evaluation of the copy number status, as the comparison of the FISH and CISH signals in identical nuclei revealed an almost complete dot-to-dot conversion. HER2 status is established as an important predictive test for treatment with trastuzumab (Herceptin, Genentech, South San Francisco, CA) and lapatinib (Tykerb, GlaxoSmithKline, Middlesex, England) in breast cancer.9 Likewise, data generated within the last 3 to 5 years have shown that TOP2A is an important predictive biomarker for treatment with anthracyclines in primary breast cancer.9,10 To our knowledge, the data from the present study are the first published showing agreement between the FISH and CISH assays with respect to aberrations of the TOP2A gene. It is expected that the CISH assay, within a relatively short period, will be an accepted method in the routine diagnostic
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evaluation of HER2 status in breast cancer.11,12 The use of a light microscope instead of a fluorescence microscope and the ability to readily observe background morphologic features will be viewed as advantages compared with the current FISH assays. Based on the data from this study, the DAKO dual-color CISH assay seems to be as sensitive and specific as FISH, and, owing to its familiarity with immunohistochemical analysis, it may have the potential of being the preferred HER2 gene test in pathology laboratories in the future.12
From 1Antibody Development, 3Molecular Pathology, 4Reagent Development, and 5System Development, Dako Denmark, Glostrup; and 2Research and Development, CMC Contrast, Copenhagen, Denmark. Address reprint requests to Mr Schnau: Dako Denmark, Produktionsvej 42, DK-2600 Glostrup, Denmark.

References
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6. Gong Y, Gilcrease M, Sneige N. Reliability of chromogenic in situ hybridization for detecting HER-2 gene status in breast cancer: comparison with fluorescence in situ hybridization and assessment of interobserver reproducibility. Mod Pathol. 2005;18:1015-1021. 7. Vocaturo A, Novelli F, Benevolo M, et al. Chromogenic in situ hybridization to detect HER2/neu gene amplification in histological and ThinPrep-processed breast cancer fineneedle aspirates: a sensitive and practical method in the trastuzumab era. Oncologist. 2006;11:878-886. 8. Dietel M, Ellis IO, Hfler H, et al. Comparison of automated silver enhanced in situ hybridization (SISH) and fluorescence ISH (FISH) for the validation of HER2 gene status in breast carcinoma according to the guidelines of the American Society of Clinical Oncology and the College of American Pathologists. Virchows Arch. 2007;451:19-25. 9. Jrgensen JT, Nielsen KV, Ejlertsen B. Pharmacodiagnostics and targeted therapies: a rational approach for individualizing medical anti-cancer therapy in breast cancer. Oncologist. 2007;12:397-405. 10. Nielsen KV, Ejlertsen B, Mller S, et al. The value of TOP2A gene copy number variation as a biomarker in breast cancer: update of DBCG trial 89D. Acta Oncol. 2008;47:725-743. 11. Palma SD, Collins N, Faulkes C. Chromogenic in situ hybridisation (CISH) should be an accepted method in the routine diagnostic evaluation of HER2 status in breast cancer. J Clin Pathol. 2007;60:1067-1068. 12. Ross JS, Symmans FW, Puszati L, et al. Personalized medicine for breast cancer: moving forward and going back. Personalized Med. 2006;3:363-370.

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