IMMUNOHISTOCHEMICAL EXPRESSION OF CYCLIN D1 AND KI 67 IN PREMALIGNANT AND MALIGNANT PROSTATIC LESIONS

IMMUNOHISTOCHEMICAL EXPRESSION OF CYCLIN D1 AND KI 67 IN PREMALIGNANT AND MALIGNANT PROSTATIC LESIONS. Samia M. Gabal MD1, Samar A. El Sheikh M.D1, Hala M. El-hanbuli MD2, Maram M. Ayoub, M.B. B.Ch2. Department of Pathology, Faculty of Medicine, Cairo University1 and Fayoum University2, Egypt. ......................................................................................................................


Introduction:-
Prostatic diseases are responsible for significant morbidity and mortality in men all over the world (1). In men older than 50 years, prostatic carcinoma represents the most common malignant tumor and is characterized by a highly variable clinical outcome (2). It accounts for about 50% of male genital tract malignancies in Egypt (3) , and prostatic intraepithelial neoplasia (PIN), is probably the most important precursor lesion of invasive prostatic cancer (4). Cyclin D1 has been identified as a short-lived nuclear protein involved in cell cycle transition from growth (G1) to synthesis (S) phase in both normal and neoplastic cells (5) .The relationship between cyclin D1 expression and prostate cancer remains controversial. Some researchers have found occasional cyclin D1 expression in prostate cancer, whereas others reported that prostatic tumors with high cyclin D1 expression were associated with a more aggressive disease (6). Ki-67 is a protein involved in cell cycle regulation and cell proliferation. It is expressed in proliferating cells during all active phases of the cell cycle (apart from G0). It is confirmed to be the most promising biomarker for routine practice applications (7), which may reflect the biologically malignant potential of prostatic carcinoma and justify its use in selecting candidates for active surveillance However, its significance remains controversial (8) Aim of the work:-Because the role of cyclin D1 and Ki-67 in prostate cancer is unclear, we studied the expression of cyclin D1 and Ki-67 in PIN, prostatic adenocarcinoma and benign prostatic hyperplasia (BPH) (as a control group) to evaluate the relationship of these proteins with some clinicopathological features as age, preoperative serum PSA and Gleason score and to declare a possible correlation between Cyclin D1 and Ki-67.

Material and Methods:-
Specimens:-Paraffin blocks prepared from 50 specimens of prostatic tissue were collected in this study, including: 7 radical prostatectomy specimens, 18 transurethral specimens (TUR), 22 transrectal needle biopsies (TRUS) and 3 cases of open prostatectomy. The specimens were obtained from the Department of Pathology, Faculty of Medicine, Cairo University, in the period from January 2013 to December 2014. The specimens included 25 cases of previously diagnosed prostatic carcinoma, 10 cases of high grade PIN and 15 cases of BPH (as a control group). For all cases, available clinical data such as age of the patients and preoperative serum Prostate-specific antigen (PSA) were evaluated.
Histopathological examination:-Each paraffin block was recut into serial sections and stained by Hematoxylin and Eosin (H&E). In histopathological evaluation of the prostatic adenocarcinoma, Gleason grading system was used modified by Amin et al. Immunohistochemical staining:-Immunohistochemical analysis was performed on routinely processed, formalin-fixed, paraffin-embedded tissue. Tissue sections were cut at 5 microns thickness, mounted on charged slides and stained by Cyclin D1 using Monoclonal Mouse anti-human Cyclin D1 (SPM587-NBP2-32840,Novus Biological, USA) and by Ki-67 using polyclonal Rabbit anti-human Ki-67(NB500-170, Novus Biological, USA).
After routine deparafinization in xylene, the sections were hydrated through a series of graded alcohols, distilled water, and phosphate-buffered saline (PBS) at ph.7.2-7.4.Antigen retrieval was performed using Tris-EDTA (PH.9) at the microwave for Cyclin D1 and using citrate buffer (PH=6) for Ki67 immunostaining.
Blocking reagent (Ultra V Block) was placed on each slide and incubated for 10 minutes at room temperature in the humidity chamber. Excess serum was shacked off without washing. Each primary antibody was placed on the slides. The slides were incubated overnight at room temperature in humid chamber. The slides were rinsed for 5 minutes. 2-3 drops of secondary antibody (biotinylated polyvalent) diluted in this buffer solution TBS, were placed on each slide. The slides were incubated in a humidity chamber at room temperature for 15 minutes. After tapping off excess buffer the slides were rinsed for 5 minutes, twice, in PBS and were incubated in Streptavidin enzyme for 15 minutes at room temperature. The slides then rinsed for 5 minutes, twice, in PBS. Working Diaminoben zidine tetrhychochloride (DAB) reagent was placed on each slide (approximately 500 ml per section). The slides were incubated for 4 minutes at room temperature in the humidity chamber and then washed thoroughly in distilled water. Counter stain with Mayer's hematoxyline for 1 minute was done then the slides were washed. The slides were placed in two changes of 95% ethyl alcohol, then two changes of absolute alcohol, each for two minutes. Finally, the slides were cleared in xylene and cover slips were fixed using mounting reagent. Each slide was evaluated by the authors.

Evaluation of Cyclin D1 expression:
Cyclin D1 expression was graded on the basis of the intensity of staining within the tumor cells. Only nuclear staining was considered positive, while isolated cytoplasmic staining whether in tumor tissue or in few foci of benign prostatic tissue found adjacent to tumor was ignored (10) . The scoring system used for evaluation of Cyclin D1 expression (11) was as follows: 1. Negative (no staining) 2. Mild (nuclear staining of <10% cells), 3. Moderate (staining of 10-49% cells) 4. Strong (staining of ≥50% cells).

Evaluation of Ki 67 expression:
The tumors were divided into five groups regarding the percentage of Ki-67 nuclear stained cells (12). 1. Cases in which the percentage of stained cells was ≤2% were considered negative. 2. Cases with Ki-67 index of<2% -≤25% were considered 1+ 3. 26-50% as 2+ 4. 51-75% as 3+ 5. 76-100% as 4+ Statistical analysis:-SPSS (statistical package for social sciences) version 22.0 was used for data management and data analysis. Mean ±standard deviation described quantitative variables and median with range when appropriate (distribution did not follow normality). Number and percentages described qualitative data and Chi-square or / Fisher exact tested proportion independence. For comparing mean values of 2 independent groups, parametric and non-parametric t test were used. For comparing means of more than two independent groups one way ANOVA (analysis of variance) and Kruskal Wallis ANOVA were used. Correlation analysis was used to show strength and significance of association between quantitative variables. P value is always 2 tailed and significant at 0.05 level.

Results:-
Among the fifty studied cases, twenty five (50%) were previously diagnosed as prostatic adenocarcinoma, fifteen cases (30%) were diagnosed as BPH and ten (20%) cases were diagnosed as PIN based on hematoxylin and eosinstained sections.
The age of the studied cases was between 50 -85 years old and the highest median was 67 which was detected in prostatic carcinoma group. The preoperative serum PSA in the studied cases ranged from 1 ng/dl up to 313 ng/dl with a highest PSA mean value seen in prostatic carcinoma group (Table 1). The Gleason scoring of the studied prostatic carcinoma cases ranged between 4 -10. The most prevalent score in the studied cases was Gleason score 8 (28%) ( Table 2). Regarding Cyclin D1 immunohistochemical staining (Figures 1, 2); there was a statistically significant difference in expression of cyclin D1 between BPH compared with PIN and carcinoma cases (P < 0.001) with the highest expression in carcinoma cases (Table 3).  No significant relationship was found between the intensity of Cyclin D1 expression in prostate cancer cases and other clinicopathological factors as age of the patient (P=0.687) and preoperative PSA (P=0.286).Also there is no significant association or correlation was found between the intensity of Cyclin D1 expression and Gleason scoring of cancer prostate cases (P=0.337) ( Table 4).  (Figures 3, 4); there was a statistically significant difference in expression of Ki-67 between BPH and prostate carcinoma is observed (P value of < 0.001) ( Table 5).   No significant relationship was found between the intensity of Ki-67 expression in prostate cancer cases and age of the patients (P=0.616), preoperative PSA (P=0.199), but there is statistically significant positive correlation between Ki-67 positivity and increased Gleason's score (P < 0.001) ( Table 6). A statistically significant positive correlation was observed between Cyclin D1 and Ki-67 expression in cancer group, PIN group and BPH group with P value 0.03, 0.001 and 0.04 respectively.

Discussion:-
In the present study, twenty three of the prostate cancer cases (92%) revealed positive nuclear staining for Cyclin D1. Nearly similar results were given by previous studies performed by Gupta  In the present study only one case (6.7%) of BPH cases showed positive Ki 67 expression. These results are close to a previous study by Verma et al (2015) (20) which reported that Ki-67 was expressed in only one out of 10 (10%) of BPH cases.
A statistically significant difference in expression of Ki-67 between BPH and prostate carcinoma was observed and this agrees with results of previous works (20, 22 ,23) .
There was no significant association or correlation between the intensity of Ki-67 expression in prostate cancer cases and age of the patients, preoperative PSA, but there is statistically significant positive correlation between Ki-67 positivity and increased Gleason's score and this is in concordance with other studies (12, 20, 23, 24) which concluded that Ki-67 can be used as a prognostic factor for prostate cancer. But in contrast to a single work performed by Muñoz and his colleagues (2003) (19) which detected no significant difference between the Gleason's score and Ki-67 immunostaining.
In this study a statistically significant positive correlation was observed between Cyclin D1 and Ki-67 expression in cancer group, PIN group and BPH group, and this was similar to a previous study by Aaltomaa and his colleagues (2006) (25) .