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JMD 2000, Vol. 2, No. 2
Copyright © 2000 American Society for Investigative Pathology & Association for Molecular Pathology

Concomitant Oncoprotein Detection with Fluorescence in Situ Hybridization (CODFISH)

A Fluorescence-Based Assay Enabling Simultaneous Visualization of Gene Amplification and Encoded Protein Expression

Raymond R. Tubbs^*, James Pettay^*, Pat Roche, Mark H. Stoler, Robert Jenkins, Jon Myles^* and Thomas Grogan

From the Departments of Clinical and Anatomic Pathology, * the Cleveland Clinic Foundation, Cleveland, Ohio; the Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota; the Department of Pathology, University of Arizona School of Medicine, Tucson, Arizona; and the Robert E. Fechner Laboratory of Surgical Pathology, University of Virginia School of Medicine, Charlottesville, Virginia

	Abstract

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We sought the validation of a three-color fluorescence-based system that simultaneously profiles Her-2/neu oncogene copy by fluorescence in situ hybridization (FISH) and Her-2/neu encoded protein by the use of a versatile alkaline phosphatase chromogen fast red K in either fluorescence or bright-field mode. Nuclei were counterstained with DAPI. Nineteen infiltrating ductal carcinomas of breast were comprehensively evaluated for Her-2/neu amplification/overexpression by direct and indirect FISH using digoxigenin (DigFISH) and direct fluorescently labeled probes, autoradiographic RNA:RNA in situ hybridization, and immunohistochemistry using monoclonal antibody CB11. CODFISH results correlated well with DigFISH, direct-label FISH, mRNA expression, and oncoprotein expression as assessed with CB11, and enabled simultaneous visualization of gene copy and protein. In addition, qualitative immunohistochemistry may be followed by CODFISH gene copy enumeration to clarify ambiguous cases.

	Introduction

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Currently available assays for defining gene amplification and overexpression of encoded protein focus on only one aspect of the process. That is, either the gene copy is profiled directly, or the expression of the encoded protein is identified with immunofluorescence or immunohistochemistry, or (much less frequently) the correspondingmRNA is profiled by in situ hybridization. Such assays can be performed from extracted materials,^{1, 2} in situ methods visualizing DNA amplification and RNA or protein expression provide the important advantage of simultaneous assessment of morphology.^{3, 4, 5}

For most genes, gene copy number, message, and encoded protein measurements are concordant in that increased gene copy leads to enhanced message production and overexpression of protein. For other genes, external modulating influences on transcription and translation may be reflected in enhanced expression of mRNA and overproduction of encoded protein in the presence of two apparently normal gene copies measured by fluorescence in situ hybridization (FISH). A technique that could allow simultaneous assessment of gene copy number and production of its encoded protein could facilitate visualization of the process on a cell-by-cell level, enhancing understanding of both physiological and pathophysiological mechanisms.

A three-color fluorescence-based assay is described herein that combines conventional DNA:DNA FISH with immunohistochemical detection of the Her-2/neu cell membrane-associated oncoprotein using the bifunctional chromogen of alkaline phosphatase fast red K that has both chromogenic and fluorescent properties. In sections of formalin-fixed paraffin tissue counterstained with DAPI, the three-color fluorescence assay permitted precise quantitation of gene copy by FISH and simultaneous visualization of the encoded protein by immunohistochemistry (IHC).

Concomitant oncoprotein detection with FISH (CODFISH) represents a versatile assay for simultaneous assessment of Her-2/neu or other genomic amplification and oncoprotein expression. For Her-2/neu, the method is readily adaptable to a 2-stage algorithm whereby bright field IHC with CB11 can be followed by CODFISH to detect gene copy for clarification of ambiguous cases, significantly decreasing possible false negative or false positive interpretations now linked to important therapeutic decisions.

	Materials and Methods

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Patient samples from 19 primary infiltrating mammary carcinomas of ductal type were selected for study based on preliminary immunohistochemistry results using the monoclonal antibody CB11 (Ventana Medical Systems, Tucson, AZ)^{6, 7} and a semiquantitative scoring system approved by the Food and Drug Administration for interpretation of HercepTest (Dako) results.^{8, 9} Paraffin blocks of formalin-fixed cell lines SiHa and BT-474 containing normal and eightfold increased Her-2/neu gene copy were used also in preliminary experiments and as in-run controls. Each paraffin block from the 19 cases included sufficient redundant paraffin-embedded archived tissue to do all of the components of the study. All tissues were first evaluated with a presectioning hematoxylin and eosin (H&E)-stained section, as well as a section following preparation of the last unstained section, to confirm the presence of infiltrating carcinoma in material to be studied.

CODFISH
Unstained paraffin sections on electrostatically charged slides were deparaffinized and rehydrated in 3 changes each of xylene, absolute alcohol, and 95% and 80% alcohol, and then placed in 1x phosphate-buffered saline (PBS) for 5 minutes. Cell conditioning was achieved through the use of microwaving in citrate buffer for 15 minutes, followed by cooling of the slides in the citrate solution at room temperature for 20 minutes. The monoclonal antibody CB11 at manufacturer’s concentration was dispensed onto the sections and allowed to incubate for 1 hour at room temperature. Sections were then washed in 1x PBS, 2 changes of 3 minutes each, and biotinylated anti-mouse IgG (Ventana) applied at a concentration of 15 µg/ml for 30 minutes. Two changes of 1x PBS wash at room temperature were followed by the addition of avidin-alkaline phosphatase (Ventana) at a concentration of 3000 µg/ml for 30 minutes. Sections were then washed twice for 3 minutes each, in 1x PBS at room temperature, and then the chromogen reaction product developed from the fast red K proprietary solution (Ventana), 10 minutes at room temperature. Sections were then washed briefly in distilled water and counter stained with hemotoxylin for approximately 30 sections. Sections were then dehydrated in graded alcohols.

Ten microliters of digoxigenin-labeled Her-2/neu probe in proprietary probe solution (Oncor/Ventana) were applied to the section and coverslipped, then the probe solution and target tissue were codenatured at 90^o centigrade for 6 minutes followed by overnight hybridization at 37^o in a humidified chamber. The coverslip was removed by soaking the slides in 2x standard saline citrate (SSC) for 5 minutes at room temperature. Washes of 0.5x SSC for 5 minutes at 72^o centigrade followed. Slides were prewashed in 1x PBS containing 0.5% Tween 20 for 3 minutes at room temperature. Fluorescein-isothiocyanate-antidigoxigenin 1:50 in 1x bovine calf solution containing 0.5% bovine serum albumin was applied to the section, per slide, and allowed to incubate for 1 hour. The sections were then washed 3 times, 5 minutes each, in 1x PBS containing 0.5% Tween 20 (Sigma, St. Louis, MO). The sections were then counterstained with 20 µl DAPI in antifade solution (Oncor, Gaithersburg, MD) and mounted for FISH analysis.

The fast red K bright red chromogenic reaction product of alkaline phosphatase also displays brilliant pink-red fluorescence on excitation at 530 nm. The sections were screened by immunofluorescence without knowledge of the immunohistochemistry results, and scored in the same manner as described above.^{8, 9} The CODFISH Her-2/neu copy number was acquired through FISH analysis without knowledge of the DigFISH results. Signals were visualized on an Axioskop (Zeiss, Oberkochen, Germany) photomicroscope equipped with a triple bandpass filter. Images were captured through a photometric digital cooled CCD camera using a SmartCapture system (Vysis).¹⁰ Two representative fields were identified in the area preselected for probe application, and gene copy enumerated for a total of 40 nuclei, 20 in each of the two representative fields averaged.

DNA:DNA FISH (DigFISH), Site I
The same procedure sans the CB11 immunohistochemistry portion of the CODFISH protocol was used for the DigFISH assay.

Direct Two-Color Her/2-neu/Chromosome 17 Fluorescence Hybridization, Site II
FISH was performed using fluorescent-labeled DNA probes obtained from Vysis, Inc. (Downers Grover, IL). Five-micron paraffin sections were deparaffinized, dehydrated, incubated in 2x SSC at 75°C for 15 minutes, digested in pepsin solution (4 mg/ml in 0.9% NaCl, pH 1.5) for 15 minutes at 37°C, rinsed in 2x SSC at room temperature for 5 minutes, and air-dried. Dual-probe hybridization was performed using a Spectrum Green-labeled probe for the centromere region of chromosome 17 (CEP17) and a Spectrum Orange-labeled Her-2/neu probe. The probe mixture was hybridized to an area containing invasive carcinoma as determined on adjacent H&E-stained section. Probes and target DNA were denatured simultaneously in an 80°C oven for 5 minutes, and hybridization performed by incubation at 37°C overnight. Posthybridization washes were performed using 1.5 mol/L urea/0.1x SSC at 45°C for 30 minutes and 2x SSC at room temperature for 2 minutes. Nuclei were counterstained with 4,6-diamindino-2-phenylindole and antifade compound p-phenyldiamine. The number of HER2 signals and CEP17 signals were counted in 60 nonoverlapping nuclei, and an overall mean HER2/CEP17 ratio was calculated. Lesions with ratios 2.0 were defined as having HER2 amplification. Site I used the CEP17 probe in combination with the DigFISH method described. All other procedural steps were identical for both Sites I and II.

RNA:RNA in Situ Hybridization
The formalin-fixed, paraffin-embedded breast biopsy blocks were serially sectioned at 4 to 5 µm, and the sections were mounted on 3-aminopropyltriethoxysilane-coated slides. These sections served as the substrate for in situ hybridization. A final serial section was stained with H&E to verify preservation of the morphology.

Her2/neu in Situ Hybridization, Site III
In situ hybridization for Her-2/neu mRNA was carried out using well-characterized methods.^{11, 12, 13} The Her-2/neu DNA construct, pHER2–436-1 (originally in pUC12), was obtained from the American Type Culture Collection (Manassas, VA; stock number 59297). The 1.45-kb construct was recloned into pCDNA1, which was grown up in bulk, and template plasmids were prepared by cesium banding. Templates for riboprobe transcription were prepared by restriction digestion of the DNA with HindIII for antisense transcription using SP6 polymerase and XbaI for T7 directed sense orientation probes. In vitro transcription to generate ³H-labeled riboprobes was carried out in the presence of ³H-UTP and ³H-CTP (approximately 30 or 60 Ci/mmol/L, respectively), yielding probes with a specific activity of 1.1 x 10⁸ dpm/µg. The in situ hybridization mixture contained 50% formamide, 0.3 mol/L NaCl, 20 mmol/L Tris-Cl, 1 mmol/L EDTA, pH 8.0, 1x Denhardt’s solution, 500 ng/ml yeast tRNA, and 10% dextran sulfate. Hybridization was carried out at Tm -25°C = 45°C. The final probe concentrations were adjusted to just fully saturate the target messages (0.2 µg/ml/kb complexity). Following hybridization, the specimens were washed at high stringency, estimated to be Tm -5°C = (0.1x SSC at 65°C)(1x SSC = 0.15 mol/L NaCl, 0.0015 mol/L Na-citrate). Parallel hybridizations were simultaneously performed using a 1.8-kb actin antisense probe. This was used to assess RNA preservation and for normalization as a means to judge overexpression (see below). The slides were then overlaid with Kodak NTB-2 autoradiography emulsion, exposed for 2 weeks at 4°C, developed photographically, and lightly stained with H&E.

All in situ hybridization results were evaluated independently (M. H. S.) without any knowledge of the parallel results of the multiple FISH or IHC analyses. Under the conditions of these experiments, a semiquantitative scale was developed and normalized to the level of actin expression in the adjacent section as follows: Neg/0 = <5 grains/cell, trace+ = 5–10 grains/cell, 1+ = 10–15 grains/cell, 2+ = 15–20 grains/cell, and 3+ = 20–25 grains/cell. The typical breast biopsy had an actin signal of at least 1+. Cases with trace or negative actin were considered RNA degraded. Cases were considered to have strong overexpression of Her-2/neu if the level was two or more steps higher than the actin level on the same day/experiment. A one-step difference was graded as weak overexpression. Normal tissues within the sections typically had no overexpression and served as internal controls. Positive controls included an identically processed breast cancer cell line, BT-474, that has known eightfold amplification of Her-2/neu and a known high-grade ductal carcinoma that immunohistochemically was consistently 3+. These controls reproducibly were weak and strong Her-2/neu mRNA overexpressors, respectively. These controls were performed in each run, as were sense orientation controls to assess for background binding of probe.

Immunohistochemistry Using Monoclonal Antibody CB11
Unstained paraffin sections on electrostatically charged slides were deparaffinized and rehydrated in three 1-minute changes each of xylene, absolute alcohol, and 95% and 80% alcohol, and then placed in 1x PBS for 5 minutes. The modified labeled avidin biotin method was used for detection. Cell conditioning was achieved through the use of microwaving in citrate buffer for 15 minutes, followed by cooling of the slides in the citrate solution at room temperature for 20 minutes. The monoclonal antibody c-erb-B2 at manufacturer’s proprietary concentration (Ventana) was dispensed onto the sections and allowed to incubate for 1 hour at room temperature. Sections were then washed in 1x PBS, two changes of 3 minutes each, and biotinylated goat anti-mouse IgG (Ventana) applied at a concentration of 15 µg/ml in PBS containing 0.05 proclin 300 for 30 minutes. Two changes of 1x PBS wash at room temperature were followed by the addition of avidin-alkaline phosphatase (Ventana) at a concentration of 3000 µg/L PBS with 0.05 proclin 300 for 30 minutes at room temperature. Sections were then washed twice for 3 minutes each, in 1x PBS at room temperature, and then the chromogen reaction product developed from fast red K proprietary solution (Ventana), 10 minutes at room temperature. Sections were then washed briefly in distilled water and counterstained with hematoxylin for approximately 30 sections. After dehydration in graded alcohols and xylene, coverslips were mounted with Permount and examined by conventional optical microscopy.

Statistics
Statistical comparisons were made using Statview (SAS Institute, Cary, NC).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The data comparing the various in situ hybridization and immunohistochemical methods are summarized in Table 1and illustrated in Figures 1 2 3 4 . There was overall excellent agreement among the different modalities evaluated. All cases were reviewed by standard histopathological evaluation and graded by the Scarff-Bloom-Richardson system before the submission for special ancillary studies. Material evaluated by the various techniques were consecutive sections.

View larger version (61K): [in this window] [in a new window]   Figure 1. Fluorescence confocal microscopy photomicrograph, cell line known to have normal Her-2/neu gene copy and absence of oncoprotein expression. One, three, or four Her-2/neu gene copies are identified in each nucleus (green). No surface oncoprotein is present.

View larger version (172K): [in this window] [in a new window]   Figure 2. Fluorescence confocal microscopy photomicrograph, BT-474 cell line. Numerous Her-2/neu gene copies (green) are simultaneously visualized along with abundant surface membrane and some cytoplasmic oncoprotein expression (red).

View larger version (161K): [in this window] [in a new window]   Figure 3. Fluorescence confocal microscopy photomicrograph. Infiltrating ductal carcinoma of breast demonstrating normal Her-2/neu gene copy and absence of oncoprotein expression.

View larger version (160K): [in this window] [in a new window]   Figure 4. Fluorescence confocal microscopy photomicrograph. Abundant Her-2/neu gene copy (green) and oncoprotein (red) overexpression are simultaneously visualized. Infiltrating ductal carcinoma of breast.

Comparison of Standard Immunohistochemistry Results and Oncoprotein Detection by CODFISH
Five breast carcinomas and one positive cell line showed a strong circumferential thick layer of cytoplasmic membrane staining with both a monoclonal and polyclonal antibodies, and with the CB11 component of CODFISH. Identical patterns and intensity of staining were noted. However, HercepTest did also stain (grade 2 intensity) two cases negative by all FISH methods, and by CB11 and the Zymed polyclonal antibody sans epitope retrieval.

Gene Copy Enumeration by in Situ Hybridization
Three methods were used to determine copy number. A direct two-color system, an indirect digoxigenin-labeled FISH system (DigFISH) and the DigFISH component of CODFISH were used for this purpose. There was excellent concordance among the three methods used (Table 2) . The presence of the intense fluorescent emission from fast red K did not interfere with either capture or enumeration of the digoxigenin-based gene enumeration by CODFISH.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Approaches to the identification of gene expression are legion, ranging from techniques that focus on amplification at the genomic level, identification of increase in Her-2/neu RNA, and profiles of protein production encoded by the gene. Such approaches may use extracted DNA, RNA, or protein, or may focus on detection in situ by either DNA:DNA in situ hybridization, RNA:RNA in situ hybridization, or IHC. But even for real time measurements of such events in living cells, such approaches focus on only one of the determinants along the gene-message-protein pathway. Here, we present a method whereby gene amplification and encoded protein production are simultaneously visualized.

We chose Her-2/neu as a model system for first developing the assay in previously characterized cell lines, then extending the technique to a series of human inva-sive breast carcinomas for which gene copy number, mRNA expression, and Her-2/neu-encoded protein production were well established. The current controversy surrounding the accuracy of an immunohistochemistryassay for profiling this oncogene’s overproduction as a means for screening women for serotherapy with a humanized monoclonal antibody highlights the potential benefits of an assay that simultaneously profiles both characteristics.^{3, 5, 8, 13}

We were concerned that differences in cell conditioning and the use of immunohistochemistry reagents might adversely effect the DNA in situ hybridization portion of the assay. In fact correlations between DigFISH and the digoxigenin-based: DNA in situ hybridization component of CODFISH were excellent. The leveraging of the fluorescent properties of fast red K may have numerous advantages over other applications whereby FISH and labeling of specific cellular populations may be of value. Figures 1 2 3 4 used confocal microscopy; in clinical practice, the expense and time required for confocal microscopy is not necessary, as gene copy counting is easily performed by examining multiple nuclear focus planes.

We have also concluded that the CODFISH assay could be part of a cost-effective algorithm in the clinical approach to profiling gene amplification and oncoprotein overexpression. Concern has been expressed in the relevant literature regarding the clinical import of a 2+ versus a 3+ IHC result in the management of patients with breast carcinoma; a significant portion of such cases appear to be false-positive IHC results. The cardiotoxicity of Herceptin, alone or in combination with Adriamycin, itself a cardiotoxic drug, requires the greatest possible accuracy in selection of patients for Herceptin serotherapy. The CODFISH assay could be used in an algorithm whereby qualitative IHC assessment is done with brightfield mode, and any cases demonstrating borderline or equivocal results by immunohistochemistry could be very easily subjected to the balance of the CODFISH assay (DigFISH), correlating the ambiguous IHC results with definitive gene copy enumeration.

	Footnotes

 
Address reprint requests to Raymond R. Tubbs, Cleveland Clinic Foundation, 9500 Euclid Avenue L11, Cleveland, OH 44195. E-mail: TubbsR{at}ccf.org

Accepted for publication February 18, 2000.

	References

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1. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL: Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987, 235:177-182[Abstract/Free Full Text]
2. Slamon DJ, Godolphin W, Jones LA, Holt JA, Keith DE, Wong SG, Levin WJ, Stuart SG, Udove J, Ullrich A, Press M: Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989, 244:707-712[Abstract/Free Full Text]
3. Pauletti G, Godolphin 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, 13:63-72[Medline]
4. Press MF, Jones LA, Godolphin W, Edwards CL, Slamon DJ: HER-2/neu oncogene amplification and expression in breast and ovarian cancers. Prog Clin Biol Res 1990, 209–221
5. Press 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, 54:2771-2777[Abstract/Free Full Text]
6. Bukowski RM, Olencki T, Gunn H, McLain D, Budd GT, Sandstrom K, Tuason L, Redovan C, Rayman P, Tubbs R, Resta D, Elson P, Finke J: Phase I trial of subcutaneous interleukin 3 in patients with refractory malignancy: hematological, immunological, and pharmacodynamic findings. Clin Cancer Res 1996, 2:347-357[Abstract/Free Full Text]
7. Segal GH, Stoler MH, Tubbs RR: The "CD43 only" phenotype: An aberrant, nonspecific immunophenotype requiring comprehensive analysis for lineage resolution. Am J Clin Pathol 1992, 97:861-865[Medline]
8. Roche PC, Ingle JN: Increased Her-2 with US Food and Drug Administration approved antibody. J Clin Oncol 1999, 17:434-435[Free Full Text]
9. Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ: 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 1999, 17:1983-1987[Abstract/Free Full Text]
10. Tbakhi A, Pettay J, Sreenan JJ, Abdel-Razeq H, Kaylacio M, Hoeltge G, Miller ML, Tubbs RR: Comparative analysis of interphase FISH and RT-PCR to detect bcr-abl translocation in chronic myelogenous leukemia and related disorders. Am J Clin Pathol 1998, 109:16-23[Medline]
11. Angerer LM SM, Angerer RC: In situ hybridization with RNA probes: an annotated recipe. In Situ Hybridization: Applications to Neurobiology. Edited by Valentino KEJ, Barchus J. New York, Oxford University Press, 1987, pp 42–70
12. Stoler MH: In situ hybridization. Clin Lab Med 1990, 10:215-236[Medline]
13. Segal GH, Shick HE, Tubbs RR, Fishleder AJ, Stoler MH: In situ hybridization analysis of lymphoproliferative disorders: assessment of clonality by immunoglobulin light-chain messenger RNA expression. Diagn Mol Pathol 1994, 3:170-177[Medline]

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tubbs, R. R. Articles by Grogan, T. Search for Related Content PubMed PubMed Citation Articles by Tubbs, R. R. Articles by Grogan, T.