Other Histological Prognostic Factors in Prostate
reticulum also regulate autophagy, a survival pathway that limits metabolic stress, genomic instability and tumourigenesis5. Among the most important regulators of apoptosis and programmed cell death are that of B- cell lymphoma proteins( Bcl-2) and its related proteins. The protein encoded by this gene is a potent blocker of apoptosis. Complex interaction among the three subgroups within the Bcl-2 family controls the signaling events of apoptosis4. Over- expression of the Bcl-2 protein occurs in a wide variety of human cancers and presumably contributes to neoplastic expansion by prolonging cell survival. Bcl- 2 proteins at endoplasmic reticulum also regulate autophagy, a survival pathway that limits metabolic stress, genomic instability and tumourigenesis5.
In normal prostate Bcl-2 protein is present in the androgen independent basal cells that line the basement membrane of the prostate gland but not in the differentiated luminal secretory cells that are androgen dependent and undergo apoptosis when deprived of testosterone. In our study we evaluated the correlation of apoptotic index and Bcl-2 protein expression immuno-histocamically in prostate carcinoma with known histological prognostic factors like Gleason’s grading, scoring and angiogenic index.
performed by means of a modified labeled avidin- biotin technique, using monoclonal antibody and staining kit (DAKO LSAB2 System, HRP).The percentage of immunopositive tumour cells were scored as follows5: 1=0 to <1%;2=1 to 25%; 3=26 to 50%; 4=51 to 75% and 5=76 to 100%. Immuno-staining intensity was rated as follows: 0(none); 1(weak);
2(moderate) and 3(intense). Specimens were considered immunopositive when ³ 1% of the tumour cells had clear evidence of staining.Micro vessels marked with anti-CD31 in highly vascular tumour areas were counted in four adjacent high power fields.
Micro vessel density was calculated per square millimeter of tumour tissue.
Statistical analysis: We categorized our sample (total specimens=35) into two groups: intermediate (13) and high grade cases (22). There were no low grade adenocarcinomas (Gleason score _ 4) in any of our specimens. After calculating apoptotic index and angiogenic index and evaluating Bcl-2, the statistical analysis was performed to draw the significance, if any, amongst various grades of prostate adenocarcinoma.
Chi-sq. test was applied to find out any correlation between apoptotic index and Bcl-2. By using student’s‘t’ test correlation was observed between the angiogenic index and apoptotic index.
RESULTS
The study group consisted of 35 specimens of prostate adenocarcinoma which were categorized according to Gleason grading system. Apoptotic index was calculated using light microscopy on H&E stained sections. No statistically significant correlation was found between Gleason’s grade and apoptotic index (p>0.05): (Table-2): (Bar diagram). Bcl-2 immunostaining demonstrated highly variable expression in the malignant epithelium (Figure 1). On the average, 22.86% of cases were Bcl-2 positive. Bcl-2 positive percentage increased with increasing Gleason’s grade. Strong expression of Bcl-2 protein was related to high tumour category, metastatic disease, poor histological differentiation, weak infiltration of the tumour by lymphocytes. Statistically Bcl-2 and apoptotic index were not found to be significantly correlated (p=0.08).There were cases that showed apoptotic bodies and were Bcl-2 positive (Figure 2).
These cases were also subjected to CD-31 staining to find out angiogenic index . It was observed that angiogenic index increased with increasing grade(Figure 3). It showed statistically significant correlation between angiogenic index and Gleason’s grade (p<0.05).
Table 1. Case distribution of randomly sampled prostatic adenocarcinoma.
S. Case Number Percentage
No. Obtained
1 Trup 23 65.71
2 Radical Prostatectomy 11 31.43
3 Needle Biopsy 1 2.86
Total 35 100.00
MATERIAL AND METHODS
Thirty five cases of prostate adenocarcinoma were randomly selected among the Patients registered in Department of Surgery and Pathology of S.N. Medical College, Agra. Random sampling of 35 cases consisted of 23 cases of TURP, 11 cases of Radical prostatectomy and one needle biopsy specimen Table 1.
Haematoxylin - eosin stained slides from each specimen were reviewed and the Gleason’s grade was assigned. Tumours were classified as high grade when the combined Gleason’s score was _8 and intermediate when score was between 5 and 7 Apoptotic index was calculated in histological tumor material. Apoptotic index was calculated by measuring apoptotic cells per 1000 tumor cells per 10high power fields6. Immuno- histochemical staining for Bcl-2 and CD-31 was
DISCUSSION
Most prostate cancers, at the time of clinical diagnosis, present as mixture of androgen dependent and androgen independent cells. A vast majority of prostate cancers respond initially to androgen ablation since the population of androgen dependent cells undergoes rapid apoptosis upon androgen withdrawal. However, androgen ablation rarely cures patients, most of whom will experience recurrence due to takeover of the tumour mass by androgen independent tumour cells as well as emergence of apoptosis- resistant clones as a result of further genetic alterations such as Bcl-2 amplification. Proteins coded by the Bcl-2 family of genes are important regulators of programmed cell death and apoptosis8. Bcl-2 is a potent antiapototic protein which help in development of neoplasm, by maintaining the longevity of cells via suppressing their programmed death. Its expression is more common in tumour with invasive behavior, high proliferation rate and abnormal cell differentiation9.We observed a significant correlation in the Bcl-2 protein expression between the different tumour grades Table 2. The expression of Bcl-2 family in prostate cancer (gene bax, bcl-xl and mcl-1) positively correlates with Gleason’s grade7. Our results are in full agreement with those of Hering et al.10, who found positive correlation in frequency of Bcl-2 positive expression in prostate adenocarcinoma with low and high Gleason score. They concluded that over expression of Bcl-2 is significantly higher in patients with an initially elevated Gleason score (8, 9, and 10).
The Bcl-2 expression probably exerts a role in the longevity of the cells submitted to hormonal therapy11. Khor et al(2007)12 observed that combination of negative Bcl-2 / normal Bax expression seemed more significantly related to reduced biochemical or any other treatment failure. In a previous analysis in prostate cancer it was suggested that the expression of Bcl-2 seems to be related to androgen independence, while in breast cancer expression of Bcl-2 is sex steroid dependent13. Accordingly, the results suggest that, along with increasing genetic instability (i.e. increased G-grade); the abnormal expression of Bcl-2 protein becomes more common in prostatic cancer.
We observed that Bcl-2 positive immunoreaction did not correlate with apoptotic index (p=0.08). The relationship between inhibited apoptosis and expression of Bcl-2 protein is not clear cut; since Bcl-2 positive tumour also showed apoptosis. However, in these tumours the apoptosis rate may be reduced in relation to mitosis rate and other regulatory mechanisms, such as androgen and other growth factors may be involved. Expression of Bcl-2 protein is not a critical factor that determines the degree of apoptosis14.
Table 2. Correlation of Gleason grade with apoptotic index (A.I.), Bcl2-immunopositive cases and
angiogenic index (Ag CD-31).
S. Gleason's Cases Bcl2 AgCD31
No. grade positive%
1 Intermediate
grade (5-7) 13 0-6(2.23±1.72) 1(7.69) 109.82 2 High grade (8-10) 22 0-3(1.00±0.85) 7(21.82) 247.35
TOTAL 35 8(22.86) 357.17
Fig. 1: Intense Bcl-2 positivity in Gleason Grade –III+IV ( score-7) Adenocarcinoma (10x40).
Fig. 3: Angiogenesis in Gleason Grade 3 Adenocarcinoma (CD 31 immunostain x 40)
Fig.2: Apoptosis in Gleason Grade –III Adenocarcinoma ( H& E 10 X 40)
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In our study, we quantified apoptosis by calculating apoptotic index (A.I.) but did not find any correlation of A.I. with Gleason’s grade (p >0.05). Findings of apoptotic index in prostate cancer were in accordance with those of Amirghofran et al.15, who did not observe any correlation between apoptosis and different grades of carcinoma. They found a significant correlation between bax expression and stage of carcinoma, but not with apoptotic index, suggesting the presence of non functional bax protein or the role of other proapoptotic molecules in oncogenesis. Similarly Brown et al (1996)16 found that the pathologic stage did not correlate with cell proliferation, apoptosis or overall daily growth. Their study concluded that cell proliferation and apoptosis do not correlate with pathological stage in clinically organ confined cancer.
Similarly Matushima et al. (1998)17 showed A.I. does not correlate with Gleason score nor was there any significant correlation between AI and pathologic stage. Tu et al. (1996)18 found that localized prostate cancer cells exhibited a relatively low rate of apoptosis, which was significantly lower than the apoptotic index of normal prostate glandular epithelial cells. Metastatic prostate tumor cells, however, exhibited a significantly higher apoptotic index compared with localized prostate cancer cells.
In our study we observed that angiogenic index increases with Gleason’s grade and was inversely related to apoptotic index (p<.05) Spontaneous apoptosis is related inversely to angiogenesis and positively related to proliferative activity. Quantitative micro vessel density has been shown to provide important staging and prognostic information in prostatic carcinoma. Correlation between angiogenic index and apoptotic index has been shown to be altered significantly by androgen ablation17. The increase in micro vessels from low to high grade was in accordance with the finding of Bettencourt et al.19. They found that micro vessel density count in the tumour are significantly increased with increasing Gleason’s
sum and nuclear grade but did not increase significantly in adjacent benign prostate.
Thus beside grading, scoring, lymphovascular invasion, perineural invasion; quantification of Bcl-2 antiapoptotic protein and angiogenic index can be of value to predict prognosis of prostate carcinoma. Bcl- 2 immunostain can be used on TURP specimens where often grading and scoring may not be what is given, as radical prostatectomy is not done. Similar large scale studies are required to derive a cut-off value of bcl-2 immuno positivity which may help in predicting progression of patients and their better management.
It is also seen that high
Bcl-2 positivity is present at the spreading edge of the tumour. The reason of this interesting finding could be decreased apoptosis at the spreading edge ensuring survival of cells to achieve invasiveness or it could be that malignant neoplasm has different cell kinetics in different areas according to survival/invasive need.
Of the many new molecular prognostic markers being evaluated in carcinoma prostate Bcl-2 and angiogenic index appear to have promise. There is an increase in Bcl-2 positive percentage with increasing Gleason’s grade and a significant inverse correlation (p<0.05) exists between angiogenic index and apoptotic index. These biomarkers appear useful in Radical Prostatectomy as well as TURP (Trans urethral resection of prostate) specimens.
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