Serum PSA detection is used for disease monitoring among men diagnosed with PCa[8-11]. Later, the tissue specificity for the prostate epithelium defined, and the high level of the serum PSA or remote expression of PSA in another tissue were found compatible with the diagnosis of the prostatic origin[12]. In combination with histopathological evidences, serum and tissue PAP levels, Gleason score and clinical stage of the disease, PSA levels can be predictive for the outcome of disease as well as the treatment, but still with some limitations[13,14]. Serum PSA level increases with age, and therefore the PCa cases of young males remain unnoticed until the disease progress and becomes clinically detectable, whereas the older males with PCa develop less aggressive disease. Recently, the increased serum PSA level and the low mortality rate for elderly were associated and this may explain the slow growth in prostate cancer with aging[15]. However, a recent review on “the use of classical and novel biomarkers” revealed the heterogeneity of the studies resulting in the inconclusiveness to use a single classical marker or the novel biochemical and histological markers on disease prognosis. Some of these new markers, including modified Gleason grade, STAT5, PAP, p53, Bcl-2, Androgen receptor (AR) with CAG (Cysteine-Adenine-Guanine) repeats, were inconsistent with disease prognosis[16]. Another classical tissue marker of PCa is AR which is also not accepted as a single marker[16-19]. Although the basal secretion of PSA level is unclear for disease prediction, for some cases, it is found reliable to combine the tissue AR, PAP and PSA levels for diagnosis[17,19-21]. AR is the marker observed in primary PCa varying through the disease progression in both hormone-sensitive and hormone refractory PCa[10,13,16-18].

Considering the fact that, the disease aggressiveness is directly related with its metastatic potential it is important to determine the levels of tumor suppressor gene expression in PCa. In this respect, a tumor suppressor gene product, NM23-H1, received a great attention and, in turn, studies focused to dissect the mechanism underlying its anti metastatic function[22-25]. However, the heterogeneity of PCa and the clinical studies were resulted in inconsistency to explain the role of NM23-H1 in disease prognosis. The expression level in healthy prostate tissue was reported with the same intensity in PCa, where healthy prostate gland showed more basal activity than the secretory. Since an increasing expression of NM23- H1 was seen with dormant cells and the decreasing levels in metastasized cancer cells, NM23-H1 appears to be a possible prognostic marker combined with other classical markers for disease aggressiveness[22-27]. Considering again that younger males are susceptible to develop more aggressive and highly metastatic prostate cancer, and elderly may be at risk for undergoing any up-to-date treatment modalities, new approaches to combine the novel and conventional parameters for the diagnosis and the prognosis of disease should be studied to show whether the patient may benefit from therapy in a short term, or the disease has a potential to become aggressive during the life time. On the other hand, based on recent studies with the classical markers, such as serum PSA and PAP levels, as well as tissue AR expression in combination with novel markers, such as nm23-H1, one can easily conclude that the more extensive clinical studies are necessary to reach more reliable results[2,4-39].

In this study, we investigated the expression levels of NM23-H1, androgen receptor, PAP and PSA in both prostate cancer and benign prostate hyperplasia (BPH) to understand and correlate their roles in disease progress, metastatic propensity and prognosis (9,37,40,41).

Statistical evaluation
Statistically, Kruskal-Wallis variants analysis, Mann- Whitney U test, Chi-Square test, Kendall's tau-c test, Spearman correlation test were applied to compare the expressions of NM23-H1, AR, PSA and PAP, their correlations with each other, and with the tumor grading, staging, serum PAP and PSA levels. For the statistical significance, alpha level was assigned as p=0.01 for Mann-Whitney U test and Kruskal-Wallis analysis, and as p=0.05 for other analyses.

Fig 1: The serum PSA expression levels correlated with the corresponding tumor stages with respect to the mean follow-up in months (Mann-Whitney U, P<001)

Fig 2: (A&B;). (A): IHC staining of BPH and (B): IHC staining of PCa samples against PAP with strong staining pattern shown here for benign glands of BPH (x25) and for atypical acinar glands and infiltrative tumor cells of high grade PCa (x50)

Fig 3: (A&B;). (A): IHC staining of BPH and (B): IHC staining of PCa samples against PSA shown here for BPH with strong staining pattern and for PCa with Gleason grade 3 with moderate to strong stain patterns (x25)

The strong expression pattern was observed for 70.7% for PAP and 46.6 % for PSA and their expression levels were correlated with the disease stages (Figure 4). Here, 13% of stage II and 24% of Stage III cases exhibited weaker PSA stain compared to PAP, whereas one patient with stage T1 was positive for PAP but negative for PSA. Except five PSA negative NM cases, all cases with metastasis revealed positive PAP and PSA staining (Figures 2 and 3). The mean serum PSA level was determined as 39.2 ng/ml in cases with the Gleason grade 0-7, and was 42.6 ng/ml in cases with the Gleason grade 8-9. No statistical difference between high or low Gleason grade with serum PSA level was determined (t test: p= 0.054).

Fig 4: IHC staining against PAP and PSA is presented as percent tissue expression and correlated with the disease stages

NM 23-H1 immunohistochemistry showed extensive density and intensity staining with red-brown and granular staining pattern located in cytoplasm (Figure 5). The strong intensity pattern was observed for the 28% of Stage I, 54% of stage II, 33% of stage III, and 55% of NM tumors (Figure 6-A). The staining density pattern for NM23-H1 was found dense (75-100%) for 78.2% of patients and weak to moderately dense (25-75%) for 16.4% of the patients. Five point four percent exhibited no stain pattern (Figure 6-B). However, no statistical association was determined between the decrease in NM23-H1 intensity or density and advanced tumor staging by chi-square analysis (p>0.01).

Fig 5: (A&B;). (A): IHC staining of BPH and (B): IHC staining of PCa samples against NM23-H1 with strong nuclear staining pattern (x25)

Fig 6: (A&B;). (A): NM23-H1 and AR expression distributions based on the degree of the staining intensity. (B): NM23-H1 and AR expression distributions based on the degree of the staining density. NM23-H1 and AR was not correlated in terms of density or intensity distribution (Kruskal-Wallis Analysis, p>0.05). The intensity pattern scaling is 0: negative; 1: weak; 2: fair; and 3: strong. The density pattern scaling is 0: negative (0-25%); 1: weak 25-50%); 2: moderate (50-75%), and 3: strong (75-100%)

NM23-H1 and AR was not associated with density or intensity distribution (Kruskal-Wallis Analysis, p>0.05). AR, was characterized with nuclear red-brown and granular staining pattern (Figure 7). The strong intensity pattern was observed for 23.1% of patients, and for 68.1% the intensity was scaled in moderate to weak (Figure 6-A). In terms of stain density for AR, 60.8% of patients displayed dense (75-100%) and 24% displayed moderately dense (25-75%) staining pattern (Figure 6-B). Evaluation of AR staining intensity with respect to stage revealed the strong intensity pattern for the 6% of T1 stage, 20% of T2, 21% of T3, 10% of NM and 40% of BPH patients, and the strong association (Table 3) between the elevating AR expression with BPH and early PCa was determined by Chi-Square analysis (p<0.01). With Kruskal-Wallis analysis, no significant association between NM23-H1 and AR was determined in terms of their expression intensity (p>0.05).

Fig 7: (A&B;). IHC staining of (A): moderate and (B): high grade PCa samples against AR with strong nuclear staining pattern (x25)

The association of NM23-H1 expression with PAP and PSA, 22 patients (40% of cases) showed both strong NM23 and PAP expression and only 11 patients (20% of cases) showed both strong NM23 and PSA expressions (Tables 1 and 2). Mean Serum PSA and PAP values were also compared with the tissue expression levels of NM23- H1, PAP and PSA (Tables 1 and 2). Serum PSA was in the range of 36-38 ng/ml for patients showing both weak and strong tissue expression levels of NM23-H1 (p>0.01). For those patients, serum PAP levels were in the range of 29-39 ng/ml. The similar results correlating the serum PSA and PAP with the tissue expression of AR was also observed (p>0.05). Kruskal-Wallis one-way analysis showed no statistical association between serum PAP and PSA levels with the NM23-H1 and AR tissue expressions (Table 4). On the other hand, tissues from prostatic cancers showing moderate PSA staining demonstrated slightly more intense NM23-H1 staining and this correlation was statistically significant but weak with Kendall tau-c rank correlation test which showed the coefficient value of 0.018 (Table 5). When PAP and PSA expressions in tissues were compared with their serum levels, patients with weak PSA expressions showed higher serum PSA (37.3 ng/ml) and PAP (40.6 ng/ml) levels whereas, those with strong expressions showed the lower mean serum PSA (22 ng/ml) and PAP (22.3 ng/ml) levels. This reverse correlation for PAP and PSA was statistically significant when applying Kruskal-Wallis analysis (p<00.1).

Table 1: The comparison of NM23-H1 (NM23) and PAP in terms of percent distribution of expression intensity among the samplesevaluated

Table 2: The comparison of NM23-H1 (NM23) and PSA in terms of percent distribution of expression intensity among the samples evaluated

Table 3: The association of NM23-H1 and AR expression with tumor stages

Table 4: The association of serum PSA and PAP levels with tissue expressions of NM23-H1, AR, PSA, PAP

Table 5: The comparison of intensity of NM23-H1 tissue expression with the intensity of PSA expression (Kendall’s Tau-c rank correlation Analysis, coefficient value is 0.018)

In this study, all patients with metastasis showing a positive staining for PAP whereas, 5 out of those were negative for PSA staining. Patients with a weak PSA expressions showed high serum PAP and PSA levels and the cases with the strong PSA expression shared the lower serum PSA and PAP. This reverse correlation is statistically significant. We noted the different expressions of PAP and PSA from cell to cell even in adjacent cells which cannot be totally explained by the antibody dilutional variations in the same slides. We consider that each individual cells might have a different somatic and metabolic differentiation and maturation as well as the genetic alteration of the enzyme cascade.

The correlation of NM23-H1 and AR expression with the serum PAP and PSA, was not statistically significant. No correlation between Gleason Grading and serum PAP and PSA level were seen. PSA appears to be more specific than PAP. The patients with moderate PSA intensity showed a slightly more intense staining for NM23-H1 with no statistical correlation. No statistically significant relation between cancer stages and NM23-H1 expression was observed either. Only in the patients with moderately differentiated tumors, somewhat a weak correlation between the strong NM23-H1 staining and the moderate PSA expression was noted. The strong AR expression in patients with BPH and early PCa was observed, and for those with the weak or negative AR expressions were correlated with the worse prognosis.

According to previous studies, NM23-H1 level and tissue AR levels may be combined with the disease stage for patient specific prognosis[50-52]. These results found supporting the previous studies where the increase in NM23-H1 expression was found in dormant cells and the decrease was observed in metastasized cancer cells, revealing that it appears to be a possible prognostic marker combined with other classical markers for disease aggressiveness[22-27].

Our results based on the staining intensity and density show no statistical significant correlation between cancer stages and nm23-H1 expression. The moderately differentiated tumors with a moderate PSA expression have a statistically significant but a weak correlation with the increased NM23-H1 expression. The combination of the classical markers and histopathological analysis of NM23-H1, AR, PSA, PAP and tumor grading did not reveal a clear correlation for the disease prognosis, however, we observed a variable but steady NM23 staining in all different stages, we consider that tissue expression of NM23-H1 is less likely to be a reliable prognostic parameter for the patients. However, only the moderately differentiated tumors with moderate PSA expression levels did have a weak correlation with an increased expression of the NM23-H1, revealing that for the metastatic potential, NM23-H1 can be combined with classical tumor markers for the tumors where the moderate tissue PSA expressions are seen. This correlation cannot be generalized since NM23-H1 expression features could not be associated significantly with the other differentiation levels or the stages. The further studies are necessary to detail the mechanism of NM23 gene-enzyme pathway for predicting the prognosis for prostate cancer and the reasons of the variations should be clarified for the other crucial pathways of NM23 gene family which may also function as a regulatory gene as well as having the suppressor activity.

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