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Comparison of Fluorescence In Situ Hybridization and Immunohistochemistry for the Evaluation of HER-2/neu in Breast Cancer

From the Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA; and PhenoPath Laboratories and IRIS, Seattle, WA.

Address reprint requests to Stuart J. Schnitt, MD, Department of Pathology, Beth Israel Deaconess Medical Center–East Campus, 330 Brookline Ave, Boston, MA 02215; email sschnitt{at}caregroup harvard.edu.

ABSTRACT

PURPOSE: To compare fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in the determination of HER-2/neu status of breast cancers.

MATERIALS AND METHODS: FISH and IHC for HER-2/neu were performed on formalin-fixed paraffin sections of 100 consecutive invasive breast cancers. FISH was performed at Beth Israel Deaconess Medical Center, Boston, MA, using the Oncor/Ventana INFORM kit (Ventana Medical Systems, Tucson, AZ; formerly sold by Oncor, Inc, Gaithersburg, MD) in a laboratory certified as proficient in this procedure. IHC was performed at PhenoPath Laboratories, Seattle, WA, using a polyclonal antibody to the HER-2/neu protein. FISH and IHC were analyzed in a blinded fashion, and the results were then compared. Procedure and interpretation times and reagent costs for FISH and IHC were also compared.

RESULTS: HER-2/neu was amplified by FISH in 26% of cases, and 23% were HER-2/neu–positive by IHC. FISHand IHC were both assessable in 90 cases. Concordance between FISH and IHC results was seen in 82 of these cases (91%, P < .001). The FISH procedure required more technologist time and more interpretation time per case for the pathologist than IHC. Reagent costs were substantially higher for FISH than for IHC.

CONCLUSION: There is a high level of correlation between FISH and IHC in the evaluation of HER-2/neu status of breast cancers using formalin-fixed paraffin-embedded specimens. Although the choice of which assay to use should be left for individual laboratories to make based on technical and economic considerations, our results may make it difficult to justify the routine use of FISH for determination of HER-2/neu status in breast cancer.

IN 1987, SLAMON et al¹ first reported a significant relationship between amplification of the HER-2/neu (c-erbB-2) oncogene and adverse clinical outcome in patients with breast cancer. Although subsequent studies have largely confirmed this association in patients with node-positive disease,^2-22 whether or not HER-2/neu gene amplification or overexpression is an independent prognostic factor in patients with node-negative breast cancer remains a matter of controversy.^{6-11,14-16,19,22-37} More recently, there has been considerable interest in the potential role of HER-2/neu gene amplification and overexpression as a predictor of response or resistance to various therapeutic modalities in patients with breast cancer. In particular, the results of recent clinical trials have indicated that treatment with a monoclonal antibody to the HER-2/neu protein (trastuzumab [Herceptin, Genentech, Inc, South San Francisco, CA]) may be useful in prolonging the survival of patients with metastatic disease.^38-40 Furthermore, some studies have indicated that tumors that overexpress HER-2/neu may show resistance to certain forms of cytotoxic therapy^{14,21,28,41-43} and sensitivity to others.^44-47 Finally, some recent experimental and clinical studies have suggested that HER-2/neu overexpression is associated with resistance to tamoxifen.^43,48-51 As a result of this information, there is a growing clinical demand for HER-2/neu analysis of current and archived breast cancer specimens.

There are a variety of methods available to determine the HER-2/neu status of breast cancers. These include assays to evaluate gene amplification, including Southern blot,^1,2 slot blot,^52,53 and dot blot analyses,^19,54 polymerase chain reaction,^55,56 in situ hybridization,⁵⁷ and fluorescent in situ hybridization (FISH)^53,58,59; assays to determine mRNA overexpression, such as Northern blot analysis,² slot blot analysis,⁵² and in situ hybridization⁵²; and methods to assess protein overexpression, including Western blot analysis,² immunoassays,⁶⁰ and immunohistochemistry (IHC).^2,61-64 Many of these methods are beyond the scope of most pathology laboratories for technical reasons. Furthermore, most of these assays require prospective collection of fresh tissue and are not applicable to archival material. Therefore, the most viable choices for HER-2/neu analysis in both routine clinical practice and in clinical research studies are FISH or IHC, performed on sections cut from formalin-fixed, paraffin-embedded specimens. Although each of these methods has its advantages and disadvantages, direct comparisons of these two assays have been few and are limited by small numbers of cases.^65-69 The purpose of this study, therefore, was to compare FISH and IHC in a single cohort of invasive breast cancers.

MATERIALS AND METHODS

Study Design
The study population consisted of 100 consecutive cases of invasive breast cancer accessioned at Beth Israel Deaconess Medical Center, Boston, MA, between July 24, 1997, and February 18, 1998. These specimens had been fixed initially in alcoholic formalin (Anatech, Ltd, Battle Creek, MI) followed by fixation in 10% neutral-buffered formalin. Only cases with sufficient invasive carcinoma for multiple assays were included in the study. For each case, 4-µm thick tissue sections were cut from a representative paraffin block and applied to positively charged slides. FISH for HER-2/neu gene amplification was performed using the Oncor/Ventana INFORM Gene Detection System (Ventana Medical Systems, Tucson, AZ; formerly sold by Oncor, Inc, Gaithersburg, MD) at Beth Israel Deaconess Medical Center in a laboratory certified by Oncor as proficient in the procedure. IHC for HER-2/neu protein expression was performed at PhenoPath Laboratories, Seattle, WA, using a polyclonal antibody to the HER-2/neu protein. Interpretation of FISH and IHC were each performed by investigators blinded to the results of the other assay. The procedure (technologist) and interpretation (pathologist) time and reagent costs were determined for FISH and IHC.

FISH for HER-2/neu Gene Amplification
FISH for HER-2/neu was performed using the Oncor/Ventana INFORM HER-2/neu Gene Detection System as described in the guide accompanying the kit.⁷⁰ Tissue sections (on slides) were baked overnight at 65°C, deparaffinized in three 5-minute changes of xylene, transferred through two 2-minute changes of 100% ethanol, and allowed to air-dry. Slides were immersed for 15 minutes in 30% Oncor Pretreatment Solution (Oncor, Inc, Gaithersburg, MD) at 43°C, briefly washed in 2x sodium chloride/sodium citrate (SSC) at room temperature, dehydrated through 100% ethanol, and air-dried. Slides were then incubated for 60 minutes in Oncor Protein Digesting Enzyme Working Solution at 37°C. Slides were briefly washed in 2x SSC at room temperature, dehydrated through 100% ethanol, and allowed to air dry. Tissue sections were denatured by immersing slides in Oncor Denaturation Solution for 8 minutes at 75°C, followed by rinsing through 100 ethanol at -20°C, and air-drying. Oncor biotinylated HER-2/neu DNA probe was prewarmed for 5 minutes at 37°C before application. Slides were incubated with probe overnight (12 to 16 hours) at 37°C in a humidified chamber. The amount of probe used (range, 10 to 20 µL) was adjusted according to the size of the tissue section to be covered. After hybridization, slides were immersed in Oncor Post-Hybridization Wash Solution at 43°C for 15 minutes, which was followed by washing in 2x SSC at 37°C for 10 minutes with agitation. The wash was repeated with fresh 2x SSC. Slides were then immersed in 1x phosphate-buffered detergent (PBD) at 18° to 25^oC. After application of Oncor Blocking Reagent One for 5 minutes at room temperature, Oncor Fluorescein-Labeled Avidin Detection Reagent was applied to the slides for a 20-minute incubation in a humidified chamber at room temperature. Slides were then subjected to three 2-minute washes in 1x PBD at room temperature. After incubation with Oncor Blocking Reagent Two for 5 minutes at room temperature, Oncor Anti-Avidin Antibody was applied to the slides (for signal amplification) and incubated for 20 minutes at room temperature in a humidified chamber. Slides were subjected to three 2-minute washes in 1x PBD at room temperature and incubated with Oncor Blocking Reagent One for 5 minutes in a humidified chamber at room temperature. Oncor Fluorescein-Labeled Avidin Detection Reagent was then applied to the slides, with incubation for 20 minutes at room temperature followed by three 2-minute washes in 1x PBD at room temperature. Nuclei were counterstained with 4'-6'-diamidino-2'-phenylindole (DAPI)/Antifade. Slides were stored in the dark at -70°C for up to 5 days before analysis. Positive controls were included in each staining run and consisted of freshly cut paraffin sections of cases known to be amplified for the HER-2/neu gene by FISH.

Slides were evaluated for HER-2/neu gene copy number using a Zeiss Axioscope fluorescence microscope (Zeiss, Inc, Thornwood, NY) by one observer (T.W.J.). Each slide was examined using DAPI, dual-band pass, and triple-band pass filters. Slides were scanned at low power (10x objective) using the DAPI filter to identify areas with optimal tissue digestion and nonoverlapping nuclei. Twenty randomly selected invasive tumor nuclei in each of two separate, distinct microscopic areas were evaluated (ie, a total of 40 nuclei per case). Cases were scored as amplified by FISH when the mean number of fluorescent signals per nucleus was greater than four. Cases with 20 mean signals per nucleus were scored as 20. Borderline cases (ie, 3.5 to 4.5 mean signals per nucleus) were scored by a second observer (S.J.S.). For each case, the corresponding hematoxylin and eosin slide was reviewed before FISH interpretation. In cases where invasive carcinoma could not be reliably identified by DAPI during the interpretation of FISH, the corresponding hematoxylin- and eosin-stained slide was re-reviewed concurrently. For each case, the following factors were also noted: adequacy of digestion, intensity of specific fluorescent signal, nonspecific fluorescent background, and tissue histology and quality (including degree of overlapping of tumor nuclei). If any of these factors precluded reliable interpretation of a case (as outlined in the Oncor Procedure and Interpretation Guide⁷⁰), then the case was evaluated by a second observer (S.J.S.) and the assay was repeated. A case was categorized as uninformative (uninterpretable) if FISH could not be reliably interpreted after repeating the assay a second time.

IHC for HER-2/neu Protein Expression
Four-µm sections from each case were deparaffinized and rehydrated in graded alcohols. The slides were subjected to heat-induced epitope retrieval by immersing them in 0.01 mol/L citrate buffer (pH 6.0) preheated to greater than 90°C and then heated in a Black & Decker vegetable steamer (Black & Decker Corp, Towson, MD) for 20 minutes, followed by a 20-minute cooldown period at room temperature. Slides were then incubated with an anti–HER-2/neu polyclonal antibody (Dako Corp, Carpinteria, CA; 1:1,000 dilution) on a Dako Autostainer (Dako Corp) for 30 minutes at room temperature. The primary antibody used in this study is the same anti–HER-2/neu antibody currently available in the Dako HercepTest kit (Dako Corp). However, in the HercepTest kit, the antibody is provided in a prediluted form. For each case, one slide was incubated with phosphate-buffered saline (PBS) instead of the primary antibody as a negative control. For positive controls, a composite slide composed of formalin-fixed cell pellets of the following four cell lines (obtained from Dr. Nora Disis, Fred Hutchinson Cancer Research Center, Seattle, WA) was used: MCF-7 (a cell line negative for HER-2/neu overexpression) and three human carcinoma cell lines showing increasing levels of overexpression of HER-2/neu (BT-20 [low overexpressor], SKBR-3 [intermediate overexpressor], and SKOV-3 [high overexpressor]). Antibody was localized using the LSAB+ Detection System (labeled streptavidin biotin immunoperoxidase; Dako Corp) according to the manufacturer's instructions using the Dako Autostainer (Dako Corp) and counterstained with hematoxylin.

All slides were scored by one observer (A.M.G.). Only membrane staining intensity and pattern were evaluated using a 0 to 4+ scale (0, completely negative; 1+, faint membranous positivity; 2+, moderate membranous positivity; 3+, strong, circumferential membranous positivity; and 4+, extremely strong, circumferential membranous positivity). Cytoplasmic immunostaining was noted but not incorporated into the final scoring. For each case, infiltrating carcinoma and adjacent normal epithelium (if available) were separately scored. A final subtracted score of the tumor minus normal epithelium was used to correct for variability in background staining of normal epithelium (which should not overexpress the HER-2/neu protein). Either a final subtracted score of 2+ or tumor cell staining of 3+ or greater was required for the case to be considered positive.

Procedure Time, Interpretation Time, and Reagent Costs
The procedure time for each run of the FISH procedure was recorded by the technologist who performed the assay, and the average time per run was calculated. Similar data were recorded for the IHC procedure. The time required for interpretation of each FISH and IHC slide was recorded by the interpreting pathologist, and the average interpretation time per case was calculated for each procedure. The reagent cost per case was calculated by determining the actual retail cost of the FISH reagents and IHC reagents required to stain the study cases and dividing these total costs by the number of study cases evaluated by each assay.

Statistical Methods
Statistical significance was set at P < .05. Data were analyzed by the Mann-Whitney rank sum test, ² test, and Fisher's exact test, where appropriate. Computations were performed with the SigmaStat for Windows software (Version 2.03, SPSS, Inc, Chicago, IL).

RESULTS

Patient Data and Histologic Features of Carcinomas
The median age of the patients was 53.5 years (range, 27 to 89 years). Seventy-two carcinomas were of infiltrating ductal type, 11 were infiltrating lobular, nine were invasive cancers with both ductal and lobular features, three were mucinous (colloid) carcinomas, three were tubular carcinomas, one was an invasive micropapillary carcinoma, and one was a metaplastic carcinoma. The median size of the tumors was 15 mm (range, 6 to 102 mm). Histologic grading was performed using the Elston and Ellis⁷¹ modification of the Bloom-Richardson grading system. Twenty-seven carcinomas were grade 1, 37 were grade 2, and 35 were grade 3. The metaplastic carcinoma was not graded, because there are no universally accepted criteria for the grading of such lesions.

FISH for HER-2/neu
Ninety-three of the 100 cases were interpretable for HER-2/neu gene amplification by FISH. In 24 of these 93 cases (25.8%), HER-2/neu was interpreted as amplified (ie, mean fluorescent signals per nucleus > four). The average number of signals per nucleus in the amplified cases ranged from 4.2 to 20.0 (median, 12.0). In nonamplified cases, the mean number of fluorescent signals per nucleus ranged from 1.3 to 3.6 (median, 1.8). Two cases were considered borderline, demonstrating 4.2 and 3.6 mean signals each per nucleus. However, in the subsequent analyses, the former case was considered amplified and the latter nonamplified. For eight of the 100 cases, the FISH assay was repeated a second time because the initial slides were unassessable. Only one of these cases was interpretable after the second assay. Of the seven cases considered uninterpretable after two assays, all had low-level specific fluorescent signal. Five also had high nonspecific fluorescent background staining, five were underdigested, and four had overlapping tumor nuclei. Re-review of hematoxylin- and eosin-stained slides concurrent with the FISH evaluation was necessary in 41 cases to reliably identify invasive tumor cells. There were several different reasons that such re-review was necessary. In 11 of these 41 cases (27%), invasive carcinoma could not be reliably distinguished from carcinoma-in-situ on the DAPI stain alone. Similarly, well differentiated (grade 1) invasive carcinoma could not be accurately distinguished from benign glands in seven of 41 cases (17%) on the DAPI stain. Seven of 41 cases (17%) were infiltrating lobular carcinomas, six had small tumor cell groups or glands, and one each was mucinous carcinoma, metaplastic carcinoma, or partially overdigested. All seven cases determined to be uninterpretable after a second assay required concurrent review of the hematoxylin- and eosin-stained sections. A second observer was required for the evaluation of the FISH slides in 28 cases, including all eight cases that were reassayed as well as the two borderline cases. Of the seven amplified cases that required a second observer, five had fewer than seven mean signals per nucleus. Eight of 24 cases scored as amplified had fewer than 10 mean signals per nucleus (range, 4.2 to 8.7 signals). In six of these cases, the fluorescent signal was dispersed (as opposed to clustered).

IHC for HER-2/neu
Ninety-six of the 100 cases were assessable for HER-2/neu, with 22 of these 96 cases (22.9%) interpreted as positive. All four unassessable cases had tumor cell membrane staining of 1+ or 2+ but no normal breast ducts or lobules on the slide. The IHC assay was repeated a second time in three cases. These cases had insufficient tumor present on the slides in the initial assay.

Comparison of HER-2/neu FISH and IHC
The results of both the FISH and IHC assays were assessable in 90 cases. Results of these two assays were concordant in 82 of these cases (91.1%; P < .001) (Table 1). Of 23 cases amplified by FISH, five were negative by IHC, and of 67 cases not amplified by FISH, three were positive by IHC (Table 1; Figs 1 and 2). Four of five cases that were negative by IHC and amplified by FISH had fewer than 10 mean signals per nucleus (range, 4.2 to 8.4 signals), with one case having 10.9 mean signals per nucleus (Fig 1). The three cases that were nonamplified by FISH and positive by IHC had between 1.7 and 2.1 mean fluorescent signals per nucleus. Of the four cases that were unassessable by IHC, one was also uninterpretable by FISH, one was amplified, and two were nonamplified by FISH. Of the seven cases that were uninterpretable by FISH, one was also unassessable by IHC, one was positive, and five were negative by IHC.

View larger version (15K): [in this window] [in a new window]   Fig 1. Average number of HER-2/neu signals per nucleus by FISH for invasive breast carcinoma cases interpreted as HER-2/neu–negative by IHC (n = 90). Cases with more than four signals per nucleus by FISH were considered amplified.

View larger version (16K): [in this window] [in a new window]   Fig 2. Average number of HER-2/neu signals per nucleus by FISH for invasive breast carcinoma cases interpreted as HER-2/neu–positive by IHC (n = 90). Cases with more than four signals per nucleus by FISH were considered amplified.

The FISH procedure required more technologist time than did IHC (10.4 hours over 2 days v 6 hours on 1 day). Pathologist interpretation time per case was longer for FISH than for IHC (mean 8.9 minutes v 2.5 minutes). Reagent costs were substantially higher for FISH than for IHC (Table 2). A total of nine Oncor/Ventana INFORM kits were required for 108 FISH assays (100 cases, eight of which were repeated a second time) at a cost of $2,000 per kit (total cost of $18,000). Therefore, the average cost per case for FISH was $166.67 ($18,000 divided by 108). In contrast, the total cost of the IHC reagents required for 103 assays (100 cases, three of which were repeated a second time) was $608 (primary antibody, $208; detection system, $350; diaminobenzidine [chromagen], $50). The average cost per case for IHC was, therefore, $5.90 ($608 divided by 103).

DISCUSSION

The results of this study indicate that there is a high level of correlation between FISH and IHC in the evaluation of HER-2/neu status of invasive breast cancers using formalin-fixed, paraffin-embedded tissue. The levels of HER-2/neu gene amplification (26%) and overexpression (23%) found in our study are in keeping with the published range of 20% to 30%.^1,72 In addition, the level of concordance of FISH and IHC (91%) is similar to that seen in prior studies comparing assays of HER-2/neu gene amplification with those of HER-2/neu protein expression.^{2,53,55,58,59,73}

When FISH and IHC results were compared, five cases interpreted as negative by IHC were scored as amplified by FISH. All but one of these cases had a mean of fewer than 10 fluorescent signals per nucleus (range, 4.2 to 8.4 signals per nucleus) by FISH. In a study by Ciocca et al,⁷³ the HER-2/neu gene was amplified (as measured by Southern blot analysis) without overexpression of protein (as measured by Western blot analysis) in 9% of cases. However, these authors did not distinguish invasive from in situ carcinoma. Of cases that showed HER-2/neu gene amplification by Southern blot, Slamon et al² found discordance (ie, no overexpression) on Western blot in 6%, Northern blot in 2%, and IHC in 1%. However, at least one assay of gene expression was concordant with the Southern blot results in these cases. At the cellular level, Szollosi et al⁷⁴ found good overall correlation between HER-2/neu oncoprotein expression and gene copy number. However, on a cell-by-cell basis, heterogeneity was found between these parameters. It is possible that in cases with low-level gene amplification, gene transcription and posttranscriptional and translational events could be abnormal or downregulated, leading to low HER-2/neu oncoprotein expression or abnormal protein or epitope production. Alternatively, the low gene copy number (< 10 mean signals/nucleus) could represent aneuploidy for chromosome 17 rather than gene amplification per se. In a study correlating HER-2/neu gene expression and DNA ploidy, cases that were negative for HER-2/neu oncoprotein by IHC had DNA indices that ranged from diploid to up to three times normal.⁷⁵ Use of a reference probe, such as to chromosome 17 alpha-satellite DNA or D21S16 on 17q,⁵³ could resolve the ploidy status of such cases. However, the Oncor/Ventana INFORM kit does not include such a probe, which precluded testing for this in our study.

Three cases interpreted as HER-2/neu–positive by IHC were scored as nonamplified by FISH. This could represent single-copy overexpression of the HER-2/neu gene at the mRNA transcription level and/or beyond, with resultant overexpression of protein. Alternatively, it may be due to gene amplification that is below the detection level of the FISH assay. These scenarios were alluded to in the study of Slamon et al,² in which low HER-2/neu DNA levels were found with overexpression of mRNA and protein levels in 10% of cases. Similar findings of absent HER-2/neu gene amplification with protein expression have been reported in 3% to 7% of cases in other studies.^53,58,59 Regardless of the molecular mechanisms involved, the findings of discordant gene and gene product results may have therapeutic implications, because the gene product (ie, HER-2/neu oncoprotein) is now being used as the target for treatment modalities, such as the anti-HER-2 monoclonal antibody, trastuzumab (Herceptin).^38-40,76 Therefore, IHC may well be the more biologically relevant assay on the basis of these considerations.

A number of previous studies have suggested that IHC for HER-2/neu lacks sensitivity and specificity and that this assay is subject to considerable interobserver variability in the interpretation of the results.^63,77-79 The largest and perhaps the most widely cited study in this regard is that of Press et al.⁶³ These investigators performed HER-2/neu IHC on formalin-fixed paraffin-embedded tissue, comparing the sensitivity and specificity of 28 different antibodies. The results were compared with known Southern blot, Northern blot, Western blot, and frozen section IHC for the cases. The sensitivity of the various antibodies studied ranged from 6% to 82%, and with one antibody, the rate of tumor positivity was as low as 2%. However, there are several important limitations to the IHC assays used in that study. First, epitope retrieval methods were not used for 27 of the 28 antibodies evaluated. It is now clear that epitope retrieval is important for obtaining optimal staining of formalin-fixed paraffin-embedded tissue with at least some of the commercially available HER-2/neu antibodies.⁸⁰ Second, the peroxidase/antiperoxidase method was used as the detection system. This method has substantially lower sensitivity than the avidin-biotin complex systems that are currently in widespread use. Finally, only a very small tissue sample was evaluated for each case, because the authors used multitumor tissue blocks. This could have resulted in cases that exhibited regional variation in HER-2/neu staining being erroneously categorized as negative. Therefore, it is difficult to extrapolate the results of the study of Press et al⁶³ to studies of HER-2/neu IHC that use current reagents and methodology.

In this study, the FISH assay required more technologist time and more interpretation time by the pathologist than did IHC. Moreover, FISH requires the use of a fluorescence microscope and often required re-review of the hematoxylin- and eosin-stained sections to definitely identify areas of invasive carcinoma. IHC interpretation is performed using a standard light microscope and permits simultaneous evaluation of immunostaining and morphology on the same slide. The FISH slides must be stored at -20°C or lower and are subject to quenching of the fluorescent signal, whereas IHC-stained slides can be stored in standard slide files and the reaction product is permanent. Finally, the cost of the FISH procedure is substantially greater than that of the IHC assay.

In summary, we have documented a high level of concordance between FISH and IHC in the evaluation of HER-2/neu status on the same cases of invasive breast carcinoma. The technical and interpretation times and reagent costs per case are substantially lower for IHC compared with FISH. However, there are a number of important caveats in the interpretation of our results. First, our findings are based on the analysis of consecutive breast cancer cases that were fixed and processed in a relatively uniform manner in a single pathology department. It remains to be determined if similar results can be obtained in laboratories that analyze specimens that are subject to more variability in tissue fixation and processing, such as reference laboratories that receive specimens from a variety of different institutions. It is also not known which of these procedures (IHC or FISH) is subject to greater interlaboratory concordance with regard to both performance of the assay and interpretation of the results, and studies are needed to address this important issue. Finally, it is imperative that these two assays be compared directly in the same study population to determine which, if either of these, has greater clinical relevance with regard to predicting prognosis and response to therapy. Although the choice of which assay to use for evaluating the HER-2/neu status of breast cancers should be left for individual institutions to make based on technical and economic considerations, our results may make it difficult to justify the routine use of FISH for the determination of HER-2/neu status in breast cancer.

ACKNOWLEDGMENTS

We thank Oncor Corp, Gaithersburg, MD, and Dako Corp, Carpinteria, CA, for generously supplying the reagents for the FISH and IHC assays, respectively.

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Submitted January 15, 1999; accepted April 7, 1999.

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