Prostate cancer (PC) represents
Dysregulation of cell cycle regulating genes is believed to play a major part in cancers. Cyclin D1 is the key regulator during the G1 phase and
DJ-1, also known as Parkinson disease protein 7, is encoded by the PARK7 gene. It acts
Accurate diagnosis is necessary to ensure the best and effective management. However, in some circumstances, for example, small foci and minimal (< 1 mm) needle tissue biopsy, the diagnosis is challengeable, and the IHC may be helpful.[4,5]
Moreover, there are data suggesting the relation between overexpression of DJ-1, cyclin D1 and androgen receptor (AR) status.[6,7]
The aim of this study is to evaluate the expression level of cyclin D1 and DJ-1 in BPH and PC, correlation with clinicopathological features and assess the sensitivity and specificity of both as immune-markers in discerning some embarrassing cases.
The current retrospective study involved 89 prostate needle biopsy specimens that were suspicious of cancer on abnormal rectal examination and/or elevated PSA during the period from January 2010 to March 2015 from the archives of the Department of Pathology, Medical Oncology Department and Clinical Oncology Department, Faculty of Medicine, Zagazig University. 9 samples were excluded due to insufficient data. They were diagnosed histopathologically with 40 cases of PC and 40 cases of BPH. The clinic-pathological and demographic features including age, pathological features, TNM stage, serum prostatic specific antigen (PSA) level, Gleason score, and followed up period were collected from the files of patients. The immune-histochemical analysis was done using cyclin D1 and DJ-1 antibody.
Four-five-micron sections from the blocks were cut into positive-charged slides; air dried overnight, deparaffinized in xylene, hydrated through a series of graded alcohol and washed in distilled water and 0.01 PBS. The avidin–biotin-complex (ABC) method was used for the immunohistochemistry staining. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 minutes. The sections were then treated with microwave radiation for 10 min for antigen retrieval, and, to block intrinsic antibody binding, they were reacted with normal serum (mouse IgG) for 10 min at room temperature. The sections were incubated overnight with a solution of primary antibodies to: Rabbit monoclonal anti-Cyclin D1 antibody (Cat. from Thermo Scientific/Lab Vision Corporation, Fermont, USA, and clone: EPR2764. 0.09% sodium azide. Dilution 1:100) and Rabbit monoclonal anti DJ1 antibody (Cat. from Thermo Scientific/Lab Vision Corporation, Fermont, USA, and clone: EPR2359. 0.09% sodium azide. Dilution 1:100) with appropriate negative and positive controls, they were reacted with biotinylated anti-mouse antibody (secondary antibody) for 10 min and with ABC for another 10 min, with intervening washes. Diaminobenzidine tetrahydrochloride was used as the final chromogen, and sections were counterstained with Mayer’s hematoxylin before mounting. Positive controls were cancer breast and kidney tissue for cyclin D1 and DJ1 respectively. Negative control was employed by substituting primary antibody. Cyclin D1 scored as:
Categorical variables were expressed as a number (percentage). Percent of categorical variables were compared using Pearson’s Chi-square test or Fisher’s exact test when appropriate.
A total of 80 eligible patients were included, 40 cases of BPH and the other 40 cases had PC. In BPH group, the median age was 65.5 years (range, 53–65 years) and median serum PSA level was 11.6 ng/ml (range 3.1–20 ng/ml).
The median age was 65.5 years (range, 50–81 years) and median serum PSA level was 50.5 ng/ml (range, 10–91 ng/ml). Table 1 shows the main clinicopathological features and outcome of 40 patients with PC.
Clinicopathological features and outcome of 40 patients with prostatic carcinoma.
Gleason score | Relapse | ||||
< 7 | 7 | 17.5% | Absent | 16 | 40% |
= 7 | 11 | 27.5% | Present | 24 | 60% |
➢7 | 22 | 55% | |||
T | Distant metastasis | ||||
T1 | 5 | 12.5% | Absent | 16 | 40% |
T2 | 14 | 35% | Present | 24 | 60% |
T3 | 21 | 52.5% | |||
Stage | Hormone Refractory Relapse | (N=24) | |||
Stage I | 3 | 7.5% | Absent | 7 | 29.2% |
Stage IIa | 6 | 15% | Present | 17 | 70.8% |
Stage IIb | 10 | 25% | |||
Stage III | 21 | 25% |
Categorical variables were expressed as a number (percentage).
Regarding the staining, cyclin D1 and DJ-1 were positive in 38 patients (95%) and 34 patients (85%) in PC, respectively. While no nuclear staining was detected for cyclin D1, the DJ-1 was positive in only 1 sample (2.5%) in
If we use cyclin D1/DJ-1 extent, both positive (+/+) was detected in 34 patients (85%) in
Comparison between PC and BPH as regard cyclin D1 and DJ-1 (staining and overexpression)
Cyclin D1 extent | |||||
Negative | 2 | (5%) | 39 | (97.5%) | < 0.001 |
Positive | 38 | (95%) | 1 | (2.5%) | |
DJ1 extent | |||||
Negative | 6 | (15%) | 39 | (97.5%) | < 0.001 |
Positive | 34 | (85%) | 1 | (2.5%) | |
Cyclin D1 extent /DJ1 extent | |||||
Negative/Negative | 2 | (5%) | 38 | (95%) | < 0.001 |
Negative/Positive | 0 | (0%) | 1 | (2.5%) | |
Positive/Negative | 4 | (10%) | 1 | (2.5%) | |
Positive/Positive | 34 | (85%) | 0 | (0%) | |
Cyclin D1 intensity | |||||
0 | 2 | (5%) | 39 | (97.5%) | < 0.001 |
1 | 5 | (12.5%) | 1 | (2.5%) | |
2 | 16 | (40%) | 0 | (0%) | |
3 | 17 | (42.5%) | 0 | (0%) | |
DJ1 intensity | |||||
0 | 6 | (15%) | 39 | (97.5%) | < 0.001 |
1 | 4 | (10%) | 0 | (0%) | |
2 | 14 | (35%) | 1 | (2.5%) | |
3 | 16 | (40%) | 0 | (0%) | |
Cyclin D1 intensity/DJ1 intensity | |||||
0/0 | 2 | (5%) | 38 | (95%) | < 0.001 |
0/1 | 0 | (0%) | 0 | (0%) | |
0/2 | 0 | (0%) | 1 | (2.5%) | |
1/0 | 1 | (2.5%) | 1 | (2.5%) | |
1/1 | 4 | (10%) | 0 | (0%) | |
1/2 | 0 | (0%) | 0 | (0%) | |
2/0 | 3 | (7.5%) | 0 | (0%) | |
2/1 | 0 | (0%) | 0 | (0%) | |
2/2 | 10 | (25%) | 0 | (0%) | |
2/3 | 3 | (7.5%) | 0 | (0%) | |
3/2 | 4 | (10%) | 0 | (0%) | |
3/3 | 13 | (32.5%) | 0 | (0%) | |
Cyclin D1 overexpression | |||||
Negative | 23 | (57.5%) | |||
Positive | 17 | (42.5%) | |||
DJ-1 overexpression | |||||
Negative | 21 | (52.5%) | |||
Positive | 19 | (47.5%) |
Categorical variables were expressed as number (percentage);
Chi-square test;
p< 0.05 is significant.
Relapse and hormone refractory were statistically significantly correlated with
Relation between cyclin D1 and DJ-1 (staining and overexpression), clinicopathological parameters and relapse in 40 PC patients
Gleason score | |||||||
< 7 | 7 | (17.5%) | 7 | (100%) | 0 | (0%) | < 0.001§ |
= 7 | 11 | (27.5%) | 9 | (81.8%) | 2 | (18.2%) | |
➢7 | 22 | (55%) | 0 | (0%) | 22 | (100%) | |
T | |||||||
T1 | 5 | (12.5%) | 5 | (100%) | 0 | (0%) | < 0.001§ |
T2 | 14 | (35%) | 11 | (78.6%) | 3 | (21.4%) | |
T3 | 21 | (52.5%) | 0 | (0%) | 21 | (100%) | |
Stage | |||||||
Stage I | 3 | (7.5%) | 3 | (100%) | 0 | (0%) | < 0.001§ |
Stage IIa | 6 | (15%) | 6 | (100%) | 0 | (0%) | |
Stage IIb | 10 | (25%) | 7 | (70%) | 3 | (30%) | |
Stage III | 21 | (25%) | 0 | (0%) | 21 | (100%) | |
Cyclin D1 extent | |||||||
Negative | 2 | (5%) | 2 | (100%) | 0 | (0%) | 0.154‡ |
Positive | 38 | (95%) | 14 | (36.8%) | 24 | (63.2%) | |
DJ1 extent | |||||||
Negative | 6 | (15%) | 6 | (100%) | 0 | (0%) | 0.002‡ |
Positive | 34 | (85%) | 10 | (29.4%) | 24 | (70.6%) | |
Cyclin D1 extent /DJ1 extent | |||||||
−ve/−ve | 2 | (5%) | 2 | (100%) | 0 | (0%) | 0.003§ |
+ve/−ve | 4 | (10%) | 4 | (100%) | 0 | (0%) | |
+ve/+ve | 34 | (85%) | 10 | (29.4%) | 24 | (70.6%) | |
Cyclin D1 intensity | |||||||
0 | 2 | (5%) | 2 | (100%) | 0 | (0%) | 0.002§ |
1 | 5 | (12.5%) | 5 | (100%) | 0 | (0%) | |
2 | 16 | (40%) | 5 | (31.3%) | 11 | (68.8%) | |
3 | 17 | (42.5%) | 4 | (23.5%) | 13 | (76.5%) | |
DJ1 intensity | |||||||
0 | 6 | (15%) | 6 | (100%) | 0 | (0%) | < 0.001§ |
1 | 4 | (10%) | 4 | (100%) | 0 | (0%) | |
2 | 14 | (35%) | 6 | (42.9%) | 8 | (57.1%) | |
3 | 16 | (40%) | 0 | (0%) | 16 | (100%) | |
Cyclin D1 intensity/DJ1 intensity | |||||||
0/0 | 2 | (5%) | 2 | (100%) | 0 | (0%) | < 0.001§ |
1/0 | 1 | (2.5%) | 1 | (100%) | 0 | (0%) | |
1/1 | 4 | (10%) | 4 | (100%) | 0 | (0%) | |
2/0 | 3 | (7.5%) | 3 | (100%) | 0 | (0%) | |
2/2 | 10 | (25%) | 2 | (20%) | 8 | (80%) | |
2/3 | 3 | (7.5%) | 0 | (0%) | 3 | (100%) | |
3/2 | 4 | (10%) | 4 | (100%) | 0 | (0%) | |
3/3 | 13 | (32.5%) | 0 | (0%) | 13 | (100%) | |
Cyclin D1 overexpression | |||||||
Negative | 23 | (57.5%) | 16 | (69.6%) | 7 | (30.4%) | < 0.001‡ |
Positive | 17 | (42.5%) | 0 | (0%) | 17 | (100%) | |
DJ1 overexpression | |||||||
Negative | 21 | (52.5%) | 16 | (76.2%) | 5 | (23.8%) | < 0.001‡ |
Positive | 19 | (47.5%) | 0 | (0%) | 19 | (100%) |
Categorical variables were expressed as number (percentage);
Chi-square test;
Chi-square test for trend;
p < 0.05 is significant.
Relation between cyclin D1 and DJ-1 (staining and overexpression), clinicopathological parameters and hormone response in 40 PC patients
Gleason score | |||||||
= 7 | 2 | (8.3%) | 0 | (0%) | 2 | (100%) | 1.000‡ |
➢7 | 22 | (91.7%) | 7 | (31.8%) | 15 | (68.2%) | |
T | |||||||
T2 | 3 | (12.5%) | 1 | (33.3%) | 2 | (66.7%) | 1.000‡ |
T3 | 21 | (87.5%) | 6 | (28.6%) | 15 | (71.4%) | |
Stage | |||||||
Stage IIb | 3 | (12.5%) | 1 | (33.3%) | 2 | (66.7%) | 1.000‡ |
Stage III | 21 | (87.5%) | 6 | (28.6%) | 15 | (71.4%) | |
Cyclin D1 extent | |||||||
Positive | 24 | (100%) | 7 | (29.2%) | 17 | (70.8%) | --- |
DJ1 extent | |||||||
Positive | 24 | (100%) | 7 | (29.2%) | 17 | (70.8%) | --- |
Cyclin D1 extent /DJ1 extent | |||||||
+ve/+ve | 24 | (100%) | 7 | (29.2%) | 17 | (70.8%) | --- |
Cyclin D1 intensity | |||||||
2 | 11 | (45.8%) | 0 | (0%) | 11 | (100%) | 0.006‡ |
3 | 13 | (54.2%) | 7 | (53.8%) | 6 | (46.2%) | |
DJ1 intensity | |||||||
2 | 8 | (33.3%) | 0 | (0%) | 8 | (100%) | 0.054‡ |
3 | 16 | (66.7%) | 7 | (43.8%) | 9 | (56.3%) | |
Cyclin D1 intensity/DJ1 intensity | |||||||
2/2 | 8 | (33.3%) | 0 | (0%) | 8 | (100%) | 0.006§ |
2/3 | 3 | (12.5%) | 0 | (0%) | 3 | (100%) | |
3/3 | 13 | (54.2%) | 7 | (53.8%) | 6 | (46.2%) | |
Cyclin D1 overexpression | |||||||
Negative | 7 | (29.2%) | 7 | (100%) | 0 | (0%) | <0.001‡ |
Positive | 17 | (70.8%) | 0 | (0%) | 17 | (100%) | |
DJ1 overexpression | |||||||
Negative | 5 | (20.8%) | 5 | (100%) | 0 | (0%) | <0.001‡ |
Positive | 19 | (79.2%) | 2 | (10.5%) | 17 | (89.5%) |
Categorical variables were expressed as number (percentage);
Chi-square test;
p < 0.05 is significant.
The clinicopathological parameters in the form of high GS, large tumor size and higher stage were statistically significantly associated with disease relapse (p < 0.001). However, this association was lost in the case of cyclin D1 extent (p < 0.15). Moreover, the same findings were detected in the case of overexpression and intensity of both cyclin D1 and DJ-1 (p < 0.001) (Table 3).
In addition, there was no statistically significant association with GS, tumor size, stage and hormone refractory (p = 1.000). Meanwhile, those associations were regained with the overexpression and intensity of both cyclin D1 and DJ-1.
DJ-1 was more sensitive than cyclin D1 in predicting PC diagnosis (80%, 70%, respectively). However, cyclin D1 was more specific (100%) (p = 0.000) (Figure 4, 5).
Both environmental and genetic factors may be implicated in increasing the diagnosis of PC. The clinical course ranges from indolent behavior to highly aggressive that ultimately causes significant morbidity and even death.[10] To achieve
The cell cycle regulators have been involved in many types of cancer including PC and associated with tumor aggressiveness and poor prognosis.[11]
Cyclin D1 is a nuclear protein that is involved in shortening the G1 (growth) –S (synthesis) transition. Its
The relationships between PC and
In our study, cyclin D1 immunostaining was detected in 95% of PC group and was not detected in BPH group (0%), which is in agreement with a study done by Ueda et al.[16] In another retrospective study on 100 prostatic specimens, which divided into 50 cases were BPH and 50 cases were PC, Qahtani et al. demonstrated that cyclin D1
On the other hand, our results contradict some other studies that revealed cyclin D1
DJ-1 is a diverse signaling protein associated with multiple cellular processes, such as cellular transformation, response to oxidative stress, and androgen-receptor signaling.[20] It acts as
As regards DJ-1 immunostaining, our presenting results showed that 85% of PC was positive, compared to 2.5% was positive in BPH group; these
The findings of our study provided that involvement in cyclin D1 and DJ-1,
This is supported by
In addition, we found a significant association with cyclin D1/DJ-1 overexpression with tumor size, stage, and GS > 7. Moreover, similar results validated the same association and were reported by many studies.[17,26,27] On the other hand, this association was not proved in many earlier studies.[9,14,16,24,28]
Sensible explanations were suggested to prove that these differences are sample size, technical varieties of biopsy taking, staining type (cytoplasmic vs nuclear) and scoring system.
Our data showed that
In the PC group, the sensitivity and specificity of cyclin D1 expression were 70% and 100%, respectively, while in a retrospective study done by Atta et al., on 60 cases of PC were 93.3% and 86.6%, respectively. In addition, they were 80% and 95% respectively for DJ-1 expression, while by Osman et al., they were 93.33% and 86.67%, respectively.[23]
Can we use these results of clinical practice to help in diagnosis in special cases?
Based on
From a practical point of view and owing to their
Previous studies were focused mainly on the prognostic value of cyclin D1 and DJ-1 in PC. In our study, besides the prognostic value, there was statistically significant staining and overexpression of cyclin D1 and DJ-1 in PC in relation to BPH. Cyclin D1 had 70% sensitivity while DJ-1 had 80%, which made them markers for diagnosis in difficult cases such as limited tissue sample, small foci of carcinoma, or benign mimics of prostate cancer. We need to think out of