An Official Periodical of The International Academy of Cytology and the Italian Society of Urologic Pathology

Transcription

1 ANALYTICAL AND QUANTITATIVE CYTOLOGY AND HISTOLOGY ARTICLES An Official Periodical of The International Academy of Cytology and the Italian Society of Urologic Pathology Cell Proliferation and Apoptosis in Prostate Needle Biopsies with Adenocarcinoma Gleason Score 6 or 7 Antonio Lopez-Beltran, M.D., Ph.D., Liang Cheng, M.D., Ana Blanca, Ph.D., and Rodolfo Montironi, M.D., F.R.C.Path. OBJECTIVE: To analyze whether cell proliferation and apoptotic indexes in needle biopsies with prostate cancer Gleason 6 or 7 can identify more objectively Gleason score 6 or 7 in needle biopsy samples. STUDY DESIGN: Seventy patients diagnosed by needle biopsy and treated by radical prostatectomy were included. Fifty cases were Gleason score 6 and 20 were Gleason 7. Twenty-two cases were organ-confined and 48 non organ-confined. Histologic sections from needle biopsies were stained for cell proliferation using the MIB 1 (Ki- 67) antibody and in situ end-labeling technique for apoptosis and recorded as the percentage of positive cells. Statistical analysis included Student s t-test, Pearson s test, and logistic regression analysis. RESULTS: We found increased apoptotic and proliferation indexes from Gleason 6 to 7, and from organconfined to non organ-confined. Apoptotic index was not associated to stage or Gleason score. We identified an association between cell proliferation and Gleason score indicating that higher proliferation index is associated with a higher probability of presenting a Gleason score 7. There was no association between pathologic stage and cell proliferation. CONCLUSION: Cell proliferation and apoptosis can From the Unit of Anatomic Pathology, Department of Surgery, Faculty of Medicine, Cordoba University Medical School, and Urooncology Laboratory, Biomedical Research Unit, Cordoba University Hospital, Cordoba, Spain; Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana, U.S.A.; and Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, Ancona, Italy. Dr. Lopez-Beltran is Professor of Pathology, Unit of Anatomic Pathology, Department of Surgery, Faculty of Medicine, Cordoba University Medical School. Dr. Cheng is Professor of Pathology and Urology, Department of Pathology and Laboratory Medicine, Indiana University School of Medicine. Dr. Blanca is Professor of Pathology, Uro-oncology Laboratory, Biomedical Research Unit, Cordoba University Hospital. Dr. Montironi is Professor of Pathology, Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine. Funded in part by the Grant FIS 00/0898 (Ministry of Science, Madrid, Spain). Address correspondence to: Antonio Lopez-Beltran, M.D., Ph.D., Unit of Anatomic Pathology, Department of Surgery, Faculty of Medicine, Cordoba University Medical School, Avenida Menendez Pidal s/n, E Cordoba, Spain (em1lobea@uco.es). Financial Disclosure: The authors have no connection to any companies or products mentioned in this article /12/ /$18.00/0 Science Printers and Publishers, Inc. 61

2 62 Lopez-Beltran et al distinguish a subset of aggressive lesions in needle biopsies with Gleason 6 or 7 prostate adenocarcinoma. (Anal Quant Cytol Histol 2012;34:61 65) Keywords: apoptosis, cell proliferation, Ki-67, needle biopsy, prostate cancer. Gleason score is a known predictor of pathologic stage, and therefore it is considered a powerful tool to guide therapy. Gleason score 6 or 7 tumors are the most common rendered diagnosis in needle biopsy specimens, 1 thus establishing two pathologic groups with different therapeutic approaches. Currently, predictors of pathologic stage in patients with prostate cancer Gleason score 6 or 7 are needed. 2 If found, these predictors would have the potential to be applied to prostate needle biopsy and thus help guide therapy more objectively. 3,4 The aim of this study was to determine whether cell proliferation and apoptosis indexes measured by MIB1-Ki67 (marker of cell proliferation) and apoptosis detected by the in situ end-labeling technique could identify more objectively prostate cancer Gleason score 6 or 7 in needle biopsy samples. The expression of these markers was also correlated to pathologic stage (organ-confined vs. non organconfined disease). Materials and Methods All of the 70 tumors were diagnosed on needle biopsies, and the patients were subsequently treated by radical prostatectomy. Pathologic stage was established according to 1997 TNM revision. 5 The biopsies were routinely fixed in 10% buffered formalin, embedded in paraffin, cut at 4 5 μm, and stained with hematoxylin-eosin (H-E). On each sample the primary and secondary patterns were evaluated, and the resulting Gleason sum 6 or 7 was recorded. Gleason score was assign by one dedicated pathologist (A.L.-B.), and cases with uncertain score were reviewed by two additional pathologists blind to clinical data, thus reaching a consensus diagnosis. Apotosis Detection To stain apoptotic cells by utilizing the in situ endlabeling (ISEL) technique (Apoptag Kit, Oncor Inc., Gaithersburg, Maryland, U.S.A.), formalin-fixed, paraffin-embedded tissue sections 5 μm thick, additional to those used for diagnostic purpose, were mounted on xyalinated glass slides. The sections were deparaffinized in xylene followed by sequential washes in graded ethanol to phosphatebuffered saline (PBS). Tissue sections were denatured by 10 minutes exposure to proteinase K (20 μg/ml) at 37 C. Endogenous peroxidase activity was blocked with 2% H 2 O 2 in PBS. The sections were counterstained with methyl green. Apoptotic cells were identified as positive to the immunostaining reaction. MIB1-Ki-67 Immunohistochemistry Staining for cell proliferation used MIB1-Ki67 antibody (Ki-67 is a monoclonal antibody that reacts with a nuclear antigen in the cell cycle G1, S, G2, and M, but not in G0) in formalin-fixed, paraffinembedded tissue mounted on xyalinated glass slides. The sections were deparaffinized, and endogenous peroxidase activity was blocked with 3% H 2 O 2 in methanol for 15 minutes. Then the sections were treated with sodium citrate buffer (ph 6.0) in the microwave for 5 minutes twice and allowed to cool for 20 minutes at room temperature. Afterward, preparations were incubated with primary antibody (Novocastra Biosystems, Wetzlar, Germany) overnight at 4 C, followed by a secondary biotinylated rabbit antimouse antibody, and the avidin-biotin-complex (Vectastain ABC kit, Vector Lab, Inc., Burlingame, California, U.S.A.). The color was developed with diaminobenzidine and the section counterstained with H-E. Qualitative and Quantitative Analysis of Apoptotic and Ki-67 Labeling Indexes The quantitation of apoptotic bodies and Ki-67 positive cells was performed as described by Aihara et al. 6 Random fields measuring μm 2 delineated by 1 cm 2 graded ocular grid attached to the eyepiece of a Nikon microscope (Tokyo, Japan) were chosen inside areas of tumor with primary Gleason pattern and with secondary Gleason pattern and were examined under high power ( 40) view, counting a mean of 1,600 cells per case. For Ki-67, all nuclear staining, regardless of intensity, was considered positive. For apoptosis, staining was considered positive if there were apoptotic bodies or intense nuclear staining. In each field, the apoptotic bodies that were shed into the gland s lumen or in the stroma around neoplastic glands were excluded. Chance of necrotic areas was avoided. The apoptotic and proliferation indexes were calculated as the percentage of positive tumor cells. Statistical Analysis All statistics were done on a PC computer using the

3 Volume 34, Number 2/April 2012 Cell Proliferation and Apoptosis in Prostate Cancer 63 Statistical Package for the Social Sciences software (SPSS, Inc., Chicago, Illinois, U.S.A). Cox s logistic regression analysis was used to study the associations among variables (age, Gleason score, stage, apoptotic index [AI], proliferation index [PI]).The Pearson s test (Pearson correlation coefficient) analyzed the correlation between quantitative data. Also a Student s t-test was included to analyze comparisons between AI and PI in Gleason score 6 vs. 7 or pt stage (organ-confined [ pt2] vs. non organconfined disease [> pt2], respectively). Results All 75 cases were available for study. Patients age (mean ± SE) was ± 1.25 (range, 55 69). Serum PSA ranged from 3.2 to Fifty cases were Gleason score 6 and 20 were Gleason 7. Twenty-two cases were organ-confined (pt2a, 10 cases; pt2b, 8 cases; pt2c, 4 cases) and 48 non organ-confined (pt3a, 18 cases; pt3b, 24 cases; pt4a, 6 cases). Main quantitative data regarding AI and PI are presented in Table I. The mean values of AI and PI in Gleason score 7 tumors and non organ-confined prostatic carcinoma were higher than in Gleason 6 and in organ-confined prostate cancer; there is an increased mean AI (p = 0.013) and Ki-67 labeling index (p = 0.028) from Gleason 6 to 7 and from organ-confined (p = 0.012) to non organ-confined (p = 0.031) (Table I). We found an association between PI and Gleason score (OR = 1.28; 95% CI, ), suggesting that higher PI is associated with a high probability of presenting a Gleason score of 7 (p = 0.041). Likewise, no association was observed between AI and Gleason score (OR = 1.16; 95% CI, ; p = 0.184) (Figure 1). Also, we identified a significant correlation between both AI and PI (r = 0.386, p = 0.022). Discussion This is the first report, to our knowledge, measuring cell proliferation and apoptosis in needle biopsy specimens of patients with prostatic adenocarcinoma. Relatively few studies have been performed looking at either cell proliferation or apoptosis in prostate cancer In some studies, progression to invasive cancer from prostatic intraepithelial neoplasia was accompanied by no increase in proliferation, yet a decrease in cell death. 11,12 Metastatic prostate cancers had an increase in proliferation and decrease in cell death compared to localized prostate cancer. 11,12 These findings suggest that the relationship between cell proliferation and cell death in prostate cancer is complex and differs according to the extent of disease. 13 Consequently, prostate cancer prognosis using either cell proliferation or cell death by themselves may not be sufficient. Our study in needle biopsy samples addressed whether a correlation could be found in the group of Gleason score 6 vs. 7 cancers or pathologic stage (organ-confined vs. non organ-confined) and cell PI or AI. Although studies have been performed looking at either proliferation or apoptosis in prostate cancer, 3,14 few have restricted their studies to low and intermediate-grade carcinomas and tried to correlate pathologic stage with proliferation and apoptosis because most previous studies have focused on prostatectomy specimens. In needle biopsy specimens, 13 we could not find any association between apoptosis and Gleason score, which is in contradiction to the results found by Aihara et al. 6 A possible explanation for this is that Aihara et al 6 identified apoptotic bodies on H-E stained slides, whereas in our cases the staining for apoptotic bodies was by using the ISEL technique. 7 This method not only Table I Mean (± SE) Apoptotic and Cell Proliferation Indexes According to Gleason Score 6 vs. 7 and Organ-Confined vs. Non Organ- Confined Disease Apoptotic index Cell proliferation index Score N (%) (mean + SE) *p Value (mean + SE) *p Value Normal prostate 70 (100) Gleason score (71) (29) Pathologic stage Organ-confined ( pt2) 22 (31) Non organ-confined (> pt2) 48 (69) *Student s t-test.

4 64 Lopez-Beltran et al Figure 1 (A) Example of positively stained apoptotic cells. (B) Example of positively stained proliferating cells. Apoptotic and proliferation indexes according to (C) Gleason score 6 vs. 7 and (D) organ-confined vs. non organ-confined disease. (A, ISEL method, 200; B, MIB-Ki67, 200.) identifies apoptotic bodies but also cells that have initiated apoptosis before they have formed apoptotic bodies. 10 A more recent study by Aihara et al 15 suggested, similar to our study, that apoptosis shows no association with pathologic stage or Gleason score. Most studies have looked at proliferation and prostate cancer using both Ki-67 and proliferating cell nuclear antigen. Several studies have agreed that proliferation does correlate with histologic grade, 11,13,16-20 but in our study in needle biopsy specimens we identified an association between cell proliferation and Gleason score with higher PI associated with high probability of presenting a Gleason sum of 7. When looking at stage, however, investigations have found no correlation with proliferation, 11,13 similar to our observations. An interesting point from some reports is that certain

5 Volume 34, Number 2/April 2012 Cell Proliferation and Apoptosis in Prostate Cancer 65 markers that do not predict stage seem to predict progression after radical prostatectomy, similar to the findings by Aihara et al 15 that showed a positive association between the AI and progression at 5 years, even though there was no association with pathologic stage. It appears that pathologic stage and disease progression are two different outcomes and a marker for one is not necessarily a marker for the other. In conclusion, cell proliferation and apoptosis do not correlate with pathologic stage when assessed in needle biopsies from patients with Gleason score 6 or 7 tumors, although higher PI is associated with a high probability of presenting a Gleason sum of 7; thus the cell PI can detect a subset of aggressive lesions in needle biopsy with moderately differentiated prostatic adenocarcinoma. The importance of these results warrants further investigation. References 1. Epstein JI, Pizov G, Walsh PC: Correlation of pathologic findings with progression after radical retropubic prostatectomy. Cancer 1993;71: Bostwick DG: Gleason grading of prostatic needle biopsies: Correlation with grade in 316 matched prostatectomies. Am J Surg Pathol 1994;18: Harris DR, Savill J: Apoptosis and the prostate. Br J Urol 1995;75: Montironi R, Pomante R, Diamanti L, Magi-Galluzzi C: Apoptosis in prostate adenocarcinoma following complete androgen ablation. Urol Int (Suppl 1) 1998;60: Edge SB, Byrd DR, Compton CC, Fritz AG, Green FL, Trotti A (editors): AJCC Cancer Staging Handbook. 7th edition. New York, Springer, Aihara M, Truong LD, Dunn JK, Wheeler TM, Scardino PT, Thompson TC: Frequency of apoptotic bodies positively correlates with Gleason grade in prostate cancer. Hum Pathol 1994;25: Gavrieli Y, Sherman Y, Bensasson SA: Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J Cell Biol 1992;119: Wyllie AH: Apoptosis and the regulation of cell numbers in normal and neoplastic tissues: An overview. Cancer Metast Rev 1992;11: Haussler O, Epstein JI, Amin MB, Heitz PU, Hailemariam S: Cell proliferation, apoptosis, oncogene, and tumor suppressor gene status in adenosis with respect to benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and cancer. Hum Pathol 1999;30: Koch M, de Miguel M, Höfler H, Diaz-Cano SJ: Kinetic profiles of intraepithelial and invasive prostatic neoplasia: The key role of down-regulated apoptosis in tumor progression. Virchows Arch 2000;436: Berges RR, Vukanovic J, Epstein JI, CarMichel M, Cisek L, Johnson DE, Veltri RW, Walsh PC, Isaacs JT: Implication of cell kinetic changes during the progression of human prostatic cancer. Clin Can Res 1995;1: Montironi R, Galluzzi CM, Diamanti L, Giannulis I, Pisani E, Scarpelli M: Prostatic intraepithelial neoplasia: Expression and location of proliferating cell nuclear antigen in epithelial, endothelial, and stromal nuclei. Virchows Arch A Pathol Anat 1993;422: Brown C, Sauvageot J, Kahane H, Epstein JI: Cell proliferation and apoptosis in prostate cancer: Correlation with pathologic stage? Mod Pathol 1996;9: Gaffney EF: The extent of apoptosis in different types of high grade prostatic carcinoma. Histopathology 1994;25: Aihara M, Scardino PT, Truong LD, Wheeler TM, Goad JR, Yang G, Thompson TC: The frequency of apoptosis correlates with prognosis of Gleason grade 3 adenocarcinoma of the prostate. Cancer 1995;75; Gonzalez-Campora R, Davalos-Casanova G, Beato-Moreno A, Luque RJ, Alvarez-Kindelan J, Requena MJ, Montironi R, Lopez-Beltrán A: Apoptotic and proliferation indexes in primary superficial bladder tumors. Cancer Lett 2006;242: Lopez-Beltran A, Maclennan GT, de la Haba-Rodriguez J, Montironi R, Cheng L: Research advances in apoptosismediated cancer therapy: A review. Anal Quant Cytol Histol 2007;29: Nagao K, Yamamoto Y, Hara T, Komatsu H, Inoue R, Matsuda K, Matsumoto H, Hara T, Sakano S, Baba Y, Matsuyama H: Ki67 and BUBR1 may discriminate clinically insignificant prostate cancer in the PSA range < 4 ng/ml. Jpn J Clin Oncol 2011;41: Zellweger T, Günther S, Zlobec I, Savic S, Sauter G, Moch H, Mattarelli G, Eichenberger T, Curschellas E, Rüfenacht H, Bachmann A, Gasser TC, Mihatsch MJ, Bubendorf L: Tumour growth fraction measured by immunohistochemical staining of Ki67 is an independent prognostic factor in preoperative prostate biopsies with small-volume or low-grade prostate cancer. Int J Cancer 2009;124: Mazzucchelli R, Barbisan F, Santinelli A, Lopez-Beltran A, Cheng L, Scarpelli M, Montironi R: Immunohistochemical expression of prostate stem cell antigen in cystoprostatectomies with incidental prostate cancer. Int J Immunopathol Pharmacol 2009;22: