BRIEF COMMUNICATION

Tissue Microarray Assessment of Prostate Cancer Tumor Proliferation in African- American and White Men

Erin E. Perrone, Constatine Theoharis, Neil R. Mucci, Satoru Hayasaka, Jeremy M. G. Taylor, Kathleen A. Cooney, Mark A. Rubin

Affiliations of authors: E. E. Perrone, N. R. Mucci (Department of Internal Medicine), C. Theoharis (Department of Pathology), S. Hayasaka, J. M. G. Taylor (Department of Biostatistics), K. A. Cooney (Department of Internal Medicine and Section of Urology, Department of Surgery), M. A. Rubin (Department of Pathology and Section of Urology, Department of Surgery), University of Michigan, Ann Arbor.

Correspondence to: Mark A. Rubin, M.D., Department of Pathology, University of Michigan, 1500 E. Medical Center Dr., Rm. 2G332/Box 0054, Ann Arbor, MI 48109-0054 (e-mail: marubin{at}umich.edu).

Prostate cancer is a major health-care problem for African-American men. The age-adjusted incidence of prostate carcinoma in African-American men is approximately 50% greater than in white men. Furthermore, studies (13) have consistently demonstrated that the mortality rate from prostate cancer is significantly greater in African-American men than in white men. This fact remains true, even after adjustment for stage at presentation (2). Some researchers have speculated that variable access to health care may substantively contribute to the disparate prostate cancer survival between racial groups. Robbins et al. (3) recently reported on survival from prostate carcinoma in participants in a large health-care maintenance organization in which one would assume that prostate carcinoma was detected and treated similarly in all races. In that study, the death rate from prostate cancer was higher in African-American men than in white men after adjustment for age and stage. These epidemiologic observations have led to the hypothesis that prostate carcinoma in African-American men is more biologically aggressive than in white men.

To investigate whether this difference is due to tumor biology or epigenetic factors, we have used high-density tissue microarray technology, developed by Kononen et al. (4). Tissue microarrays can contain up to 1000 tissue samples and can be used for multiple studies, thereby conserving tissue and assuring the uniformity of test conditions.

Our aim was to create a collection of clinically matched tumor samples from African-American and white men with prostate carcinoma and to investigate potential differences in tumor proliferation between the two groups. We matched African-American and white men before surgery by serum prostate-specific antigen (PSA) levels and fine-needle biopsy Gleason scores to identify men who presented with similar clinical features, so that any biologic differences identified could be attributed to race.

The biomarker selected for this study was the proliferation marker Ki-67. Ki-67 is a nuclear protein that is expressed in G1, S, G2, and M phases of the cell cycle but is not detected in cells in G0 phase (5). We defined the Ki-67 labeling index as the percent nuclear area stained with Ki-67 (6). Previous studies (79) of prostate cancer have described consistent associations between the Ki-67 labeling index and the Gleason grade (i.e., tumor grade).

Thirty matched clusters consisting of one African-American man and two white men were identified from 632 patients who underwent radical prostatectomy at our institution for clinically localized prostate carcinoma from 1994 through 1998. An institutional review board-approved consent for the molecular analysis of prostate cancer was obtained from all participants in this study. Matching was based on the Gleason score determined from a biopsy specimen and serum PSA levels. Tumors were staged by the tumor–node–metastasis system (10), and multiple samples of normal, high-grade prostatic intraepithelial neoplasia (PIN) and prostate carcinoma were identified from each tumor for transfer into a tissue microarray block. A tissue microarray was assembled by use of a manual tissue arrayer (Beecher Instruments, Silver Spring, MD). Three tissue samples from normal areas, three from high-grade PIN areas, and six from prostate cancer areas were taken from each patient. If a patient had multiple tumor foci, each tumor was sampled. This approach was taken because a priori we did not know which tumor would have the highest proliferation rate; previous studies using standard slides have assumed that the dominant lesion would best predict outcome.

The final tissue microarray consisted of 892 total arrayed samples in two blocks (Fig. 1Go, A–G). The identity of each 0.6-mm sample was tracked by their coordinate (X–Y) position and linked to a clinical database. Histologic review of the tissue microarray showed that, for each patient, the microarray contained, on average, 3.0, 0.9, and 4.6 samples of normal tissue, high-grade PIN, and prostate cancer, respectively. Immunostaining for Ki-67 (1:25 dilution; Coulter-Immunotech, Miami, FL) was performed, and results were quantified by use of an image analysis system (CAS2000 System; Bacus Labs, Lombard, IL). Examples of the prostate tissue samples from this study are shown in Fig. 1Go, E and F. Prostate xenografts with a high Ki-67 labeling index from the rapid autopsy program (11) were used as positive internal controls (Fig. 1Go, G and H). Six measurements of the Ki-67 labeling index were made for each tissue sample that contained 50–100 nuclei (total, 300–600 nuclei per tumor and 150–300 nuclei in normal tissues or high-grade PIN in the total tissue microarray).



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Fig. 1. Various steps of the tissue microarray assembly process. The 0.6-mm tissue core is shown on a standard slide stained with hematoxylin–eosin (A) and at higher magnification (B). An example of a paraffin tissue microarray block divided into four quadrants (C) and the slide stained with hematoxylin–eosin made from this block (D) are shown. Examples of a clinically localized prostate cancer (E and F), a prostate xenograft tumor (G and H) stained with hematoxylin–eosin (E and G), and the proliferation marker Ki-67 (F and H) are shown. The clinically localized tumor (F) has little Ki-67 expression (shown by arrows) in contrast to the high Ki-67 expression seen in the xenograft tumor (H).

 
Evaluation of preanalysis-matching criteria confirmed that biopsy Gleason scores within each cluster were identical and that there was no statistically significant difference in the mean preoperative PSA levels for African-American men (8.60 ng/mL) and for white men (8.70 ng/mL) (P = .682). However, the white men (mean age, 60.7 years) were older than the African-American men (mean age, 56.6 years) (P = .006). The preoperative matching produced two groups of patients with similar tumor grades and stage (data not shown).

For each person in the study, there was, on average, 3.0 normal samples (range, 2–4), 4.6 tumor samples (range, 2–6), and 0.9 high-grade PIN samples (range, 0–3). The average total number of Ki-67 measurements per person was 5.5 for high-grade PIN samples (range, 0–24), 18.3 for normal samples (range, 0–60), and 27.6 for tumor samples (range, 0–60). For each tissue type and each patient, a single summary Ki-67 was defined as the 90th percentile of these measurements. The 90th percentile was used because of its similarity to the largest value that is believed to have the greatest biologic importance but is less susceptible to outlying observations (our unpublished data).

Fig. 2Go shows a plot of the distribution of the Ki-67 labeling index for three tissue types and two racial groups. Conditional logistic regression adjusted for clusters found no statistically significant racial difference for the Ki-67 labeling index for any of the following tissue types examined: normal tissue (P = .537), high-grade PIN (P = .301), and prostate cancer (P = .260). This result is consistent with conditional logistic regression by use of the logarithmically transformed Ki-67 labeling index for the following tissue types: normal tissue (P = .443), high-grade PIN (P = .144), and prostate cancer (P = .364).



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Fig. 2. Distribution of Ki-67 expression for African-American and white men for the three tissue types (i.e., normal tissue, high-grade prostatic intraepithelial neoplasia [PIN], and prostate cancer). In the boxplot, the lines connect the medians, the boxes cover the 25th to 75th percentiles, and the minimum and maximum values are shown by the ends of the bars. AA = African-American; PCA = prostate cancer; LI = labeling index.

 
A mixed regression model that adjusted the matched clusters found no statistically significant differences between the Ki-67 labeling index for each tissue type and race (P = .195). However, statistically significant differences in the Ki-67 labeling index were seen between the three tissue types as follows: The Ki-67 labeling index of high-grade PIN was 0.621 (P<.001; 95% confidence interval [CI] = 0.4–0.84) higher than normal, and the Ki-67 labeling index of prostate cancer was 0.244 (P = .030; 95% CI = 0.03–0.46) higher than high-grade PIN on a logarithmically transformed scale. These differences corresponded to high-grade PIN having a Ki-67 labeling index that was 1.9 times higher than normal and prostate cancer having a Ki-67 labeling index that was 1.3 times higher than high-grade PIN. However, some overlap in the Ki-67 labeling indexes between tissue types was seen (Fig. 2Go). These findings are similar to a recently concluded biomarker study using biopsy specimens and standard slides (our unpublished data).

Associations between the Ki-67 labeling index and pathology results for white patients (e.g., extraprostatic extension, seminal vesicle invasion, and Gleason score) revealed that the Ki-67 labeling index showed a statistically significant association for extraprostatic extension (P = .019) and seminal vesicle invasion (P = .011) but not Gleason score (P = .214). Associations between the Ki-67 labeling index and pathology results for African-American men revealed that the Ki-67 labeling index showed a trend toward association with worse pathology. However, these differences were not statistically significant (extraprostatic extension, P = .181; seminal vesicle invasion, P = .344; and Gleason score, P = .203). In a multivariate analysis adjusting for race, we found statistically significant associations between extraprostatic extension (P = .006) and seminal vesicle invasion (P = .012). There were no statistically significant differences in the multivariate analysis with respect to race and the four pathology variables.

In this brief communication, we have used a method involving high-throughput analyses of prostate cancer specimens by use of tissue microarray technology to investigate whether biologic differences in prostate cancer in African-American and white patients can be detected. The strategy of matching African-American and white patients resulted in two groups with final pathology results that were similar. Thus, this tissue microarray block should be useful in identifying biologic differences that may be associated with race.

We selected Ki-67 as the biomarker because its expression is associated with biochemical recurrence as measured by elevation in PSA levels after radical prostatectomy (8,1214). Although this analysis failed to detect racial differences in the Ki-67 labeling indexes, data from longitudinal follow-up may.

NOTES

Supported by Public Health Service grant CA69568 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.

REFERENCES

1 Parker SL, Davis KJ, Wingo PA, Ries LA, Heath CW Jr. Cancer statistics by race and ethnicity. CA Cancer J Clin 1998;48:31–48.[Abstract/Free Full Text]

2 Parker SL, Tong T, Bolden S, Wingo PA. Cancer statistics, 1997. CA Cancer J Clin 1997;47:5–27.[Free Full Text]

3 Robbins AS, Whittemore AS, Van Den Eeden SK. Race, prostate cancer survival, and membership in a large health maintenance organization. J Natl Cancer Inst 1998;90:986–90.[Abstract/Free Full Text]

4 Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998;4:844–7.[Medline]

5 Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984;133:1710–5.[Abstract/Free Full Text]

6 Bacus J, Grace J. Optical microscope system of standardized cell measurements and analysis. Appl Optics 1987;26:3280–93.

7 Bubendorf L, Tapia C, Gasser TC, Casella R, Grunder B, Moch H, et al. Ki67 labeling index in core needle biopsies independently predicts tumor-specific survival in prostate cancer. Hum Pathol 1998;29:949–54.[Medline]

8 Bettencourt MC, Bauer JJ, Sesterhenn IA, Mostofi FK, McLeod DG, Moul JW. Ki-67 expression is a prognostic marker of prostate cancer recurrence after radical prostatectomy. J Urol 1996;156:1064–8.[Medline]

9 Kallakury BV, Sheehan CE, Rhee SJ, Fisher HA, Kaufman RP Jr, Rifkin MD, et al. The prognostic significance of proliferation-associated nucleolar protein p120 expression in prostate adenocarcinoma: a comparison with cyclins A and B1, Ki-67, proliferating cell nuclear antigen, and p34cdc2. Cancer 1999;85:1569–76.[Medline]

10 Schroder FH, Hermanek P, Denis L, Fair WR, Gospodarowicz MK, Pavone-Macaluso M. The TNM classification of prostate cancer. Prostate Suppl 1992;4:129–38.[Medline]

11 Rubin M, Putzi M, Mucci N, Smith D, Wojno K, Korenchuk S, et al. Rapid ("warm") autopsy study for procurement of metastatic prostate cancer. Clin Cancer Res 2000;6:1038–45.[Abstract/Free Full Text]

12 Bubendorf L, Sauter G, Moch H, Schmid HP, Gasser TC, Jordan P, et al. Ki67 labelling index: an independent predictor of progression in prostate cancer treated by radical prostatectomy. J Pathol 1996;178:437–41.[Medline]

13 Keshgegian AA, Johnston E, Cnaan A. Bcl-2 oncoprotein positivity and high MIB-1 (Ki-67) proliferative rate are independent predictive markers for recurrence in prostate carcinoma. Am J Clin Pathol 1998;110:443–9.[Medline]

14 Stapleton AM, Zbell P, Kattan MW, Yang G, Wheeler TM, Scardino PT, et al. Assessment of the biologic markers p53, Ki-67, and apoptotic index as predictive indicators of prostate carcinoma recurrence after surgery. Cancer 1998;82:168–75.[Medline]

Manuscript received November 12, 1999; revised March 15, 2000; accepted March 23, 2000.


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