Carcinoma of the prostate is the most common form of cancer in men, accounting for 29% of cancer in the USA in 2007. Prostate cancer was responsible for 9% of cancer deaths in the USA in 2007; in Egypt, prostatic carcinoma represents 49.4% of malignant tumors of the male reproductive system (Mokhtar et al., 2007). There is a one in six lifetime probability of being diagnosed with prostate cancer. It is one of the most known tumors, showing a wide range of clinical behaviors from very aggressive lethal cancers to incidentally discovered clinically insignificant cancers (Cotran et al., 2004).
Prostatic intraepithelial neoplasia (PIN) consists of architecturally benign prostatic acini or ducts lined by cytologically atypical cells, and are subclassified into low-grade and high-grade PIN. The difference between low-grade and high-grade PIN is that prominent nucleoli may be found in high-grade PIN. Uncommonly, the diagnosis of high-grade PIN is made in the absence of prominent nucleoli if there is significant nuclear pleomorphism. There are also cases in which the degree of nucleolar prominence is borderline; some authorities may consider these as low-grade PIN and others as high-grade PIN. These features contribute toward the varying incidences of high-grade PIN reported on needle biopsy, ranging from 1 to 24%, with most studies in the 4 to 5% range. High-grade PIN is considered a premalignant condition. It precedes carcinoma by 10 years. Currently, high-grade PIN is associated with adenocarcinoma on rebiopsy in 25% of patients (Haber et al., 2010).
During metastasis, cancer cells commonly develop the ability to migrate and invade tissues by regulating their interaction with other cells and their extracellular environment. Many studies have shown that alterations in cell–cell adhesion correlate with epithelial tumor progression and metastasis (Liu et al., 2009). Cancer cells can invade either as single cells or collectively as groups of cells. Both types of invasion involve disruption of the epithelium, which usually requires a weakening of cell–cell contacts and a change in cell shape. Cadherins and catenins form adherens junctions, which are central mediators of cell–cell adhesion. The expression of adherens junction proteins is often decreased in tumors, and reconstitution of functional adherens junctions can revert the invasive phenotype of cancer cells (Nelson, 2008).
P-cadherin is a member of the cadherin family of cell surface glycoproteins that mediate Ca2+-dependent cell–cell adhesion and is expressed in a differential manner in normal epithelial tissues. The expression of P-cadherin in human prostate cancer has not been examined previously (Walker et al., 2008; Sarrió et al., 2009).
Periostin (POSTN) is a 93 kDa N-glycoprotein. It shows homology with the cell adhesion molecule fasciculin 1 (drosophila) and β-IgH3 (human), sharing features that may explain some of its functional characteristics, such as involvement in cell adhesion and osteoblast recruitment. Periostin has been found in several, mainly collagen-rich and fetal tissues as an extracellular matrix protein and is upregulated by mechanical stress during tissue repair and regeneration. Periostin expression can be induced by vascular injury, which in turn induces vascular endothelial growth factor receptor 2, with consequent promotion of angiogenesis (Tischler et al., 2010).
As a ligand to αvβ and αβ integrin, periostin appears to activate the Akt/PKB pathway, known to facilitate cell survival and tumorogenesis. High expression of periostin protein or mRNA may be detected in most solid tumors including breast, colon, head and neck, pancreatic, papillary thyroid, ovarian, lung, gastric, and liver carcinomas, as well as neuroblastoma in prostatic carcinoma (Tischler et al., 2010).
Aim of the work
This study aimed to evaluate the role of P-cadherin, periostin expression, and nuclear morphometry in PIN lesions and to compare them with cancer of the prostate in terms of the histopathological variables studied.
Materials and methods
Clinical investigation and tissue sample
The present study was carried out on 70 patients with prostatic lesions, including 40 patients with nonconsecutive, retrospective selected prostatic carcinoma, 20 patients with PIN (six patients with high-grade PIN and 14 with low-grade PIN), and 10 patients with non-neoplastic inflammatory prostatic lesions as controls. Cases were collected between 2004 and 2009. They were selected from the files of pathology and urology departments of Faculty of Medicine, Benha University, and the National Cancer Institute, Cairo University. They were selected according to the availability of clinical and follow-up data. Prostate glands were inked and subjected to routine histopathological examination. Staging was carried out according to the TNM classification (Sobin and Wittekind, 1997). There were 20 stage II cases, 15 stage III cases, and five stage IV cases. There were 25 lymph node-positive cases and 15 lymph node-negative cases. Tumors were graded according to the Gleason system (Gleason and Mellinger, 2002; Pier Paolo et al., 2002; Wu et al., 2007). There were 10 grade I cases, 20 grade II cases, and 10 grade III cases. Each specimen was assessed for extension of the tumor to an inked specimen margin as well as extra capsular extension into periprostatic tissue. Only patients with primary cancer prostate who had not undergone any previous irradiation or chemotherapeutic treatment were included in this study. Two experienced pathologists blindly and independently confirmed the histological diagnosis of each prostatic lesion and the Gleason grading. Normal prostatic tissue close to the tumor area in the specimens examined was used as a normal control. Formalin-fixed paraffin-embedded prostatic tissues were used in this study. Three sections of 4 μm thickness were obtained for each case. One section was H&E stained for diagnosis and review of Gleason’s sum scores. The other two sections were mounted on positively charged slides, and stained immunohistochemically for P-cadherin and periostin using the Ultra Vision Detection System (Anti-polyvalent, HRP/DAB, ready to use; Lab Vision Corporation, New York, USA).
Paraffin-embedded tissue sections, 3–4 μm thick, were mounted on positively charged slides and heated at 60° centigrade for 30 min, then deparaffinized and rehydrated through a series of xylene and alcohol before staining. After antigen retrieval with microwave treatment in 10 mmol/l citrate buffer (Neo-Markers, Cat. #AP-9003, New York, USA), pH 6, endogenous peroxidase was blocked with 3% hydrogen peroxide for 20 min. The sections were washed three times with cold 0.01 mol/l PBS. After blocking with 10% normal rabbit serum, the sections were incubated with antibody. Immunohistochemistry staining of tissues was carried out using the polyclonal antibody against human periostin [OSF-2/periostin (BioVendor Laboratory Medicine, Modrice, Czech Republic), RD181045050 RD-932, at a dilution of 1 : 500 (Lab Vision Corporation), and P-cadherin, ready to use (Lab Vision Corporation)].
The slides were incubated for 2 h with P-cadherin and overnight with periostin. The DAB-substrate-chromogen solution prepared was applied and incubated for 5–15 min until the color intensity was achieved. Finally, sections were counterstained with Mayer’s hematoxylin. Dako-positive control slides including sections of a human skin for positive expression of P-cadherin and breast carcinoma for positive expression of periostin were examined. Negative control was established by replacing the antibody by a normal nonimmune serum.
Interpretation and evaluation of immunohistochemical staining
Evaluation of epithelial and stromal periostin expression:
An immunoreactive score (IRS) including the intensity and number of cells stained was used. The staining intensity was scored negative (0), weak (+1), moderate (+2), or strong (+3). The number of stained cells was scored zero (0), <10% (+1), 10–50% (+2), 51–80% (+3), and>80% (+4). The intensity and number were multiplied (IRS, range 0–12) with low IRS (<6) and high IRS (>6) (Tischler et al., 2010).
Evaluation of P-cadherin expression:
Tumors were considered as negative if less than 10% of the tumor was stained and positive if 10% or more of the tumor cells were positively stained. Also, the pattern of staining of P-cadherin was recorded as membranous-only or membranous and cytoplasmic immunostaining (Paredes et al., 2005).
Evaluation of nuclear morphometry:
Nuclear morphometry was carried out using the Olympus soft imaging system (Tokyo, Japan), analysis life science program, to measure the nuclear area of the examined nuclei of prostatic lesions with an optical magnification ×400 on routine H&E-stained sections. For this, 60 tumor cell nuclei in randomly chosen fields within a well-preserved and highly cellular area of the tumor were selected for each case. The mean nuclear area (MNA) was determined in µm2 (Helmy, 2010).
All statistical analyses were carried out using the SPSS 8.0 statistical package (New York, USA) for Microsoft Windows. Associations between the periostin expression, P-cadherin expression, and different clinicopathological parameters were determined. P value of 0.05 or less was considered significant. P values between 0.06 and 0.1 were considered borderline significant (i.e. did not reach a significant value).
In the present study, 70 cases of prostatic biopsies were examined histologically and immunohistochemically for P-cadherin and periostin; the results are shown in Table 1.
All control cases of non-neoplastic inflammatory prostatic control cases showed positive P-cadherin expression in all 20 examined PIN cases (100%).
P-cadherin expression was found in all the six high-grade PIN cases (100%) examined, but was positive in eight of 14 (57.1%) cases of low-grade PIN, with a cut-off value of 10%, which was statistically highly significant (P<0.01) (Fig. 1).
In terms of the tumor nuclear grade, grade I showed no P-cadherin expression (0%), whereas P-cadherin expression was found in five of 20 grade II cases examined (25%) and in two of 10 grade III cases (20%). Thus, P-cadherin positivity was found to be inversely correlated with the tumor nuclear grade Figs 2 and 3. However, this relationship was statistically nonsignificant (P>0.05).
According to Gleason’s sum score, low-score cases (<7) showed positive P-cadherin expression in six of 30 cases (20%), whereas high-score cases (>7) showed high expression in only one of 10 (10%). This means that the higher the Gleason score, the lower the P-cadherin expression; this relationship was statistically significant (P<0.05).
Stage II cases showed positive P-cadherin expression in seven of 20 cases (35%); stage III and stage IV cases showed no P-cadherin expression in (0%). This indicated a highly significant inverse relationship between P-cadherin expression and the tumor stage (P<0.01).
In terms of lymph node status, five of the 15 lymph node-negative cases (33.3%) showed positive P-cadherin expression, whereas this was found in only two of 25 lymph node-positive cases (8%). This relationship was highly significant (P<0.01) (Table 2).
The control cases examined showed low-IRS periostin expression in all 10 cases examined (100%), but they did not show a high IRS periostin score.
High IRS periostin expression was found in all the high-grade PIN cases examined (100%) in relation to high periostin IRS in eight of 14 cases examined (57.8%) of low-grade PIN cases, which was statistically highly significant (P<0.01) (Fig. 4).
High-score IRS periostin expression was found in nine of 10 cases of grade III prostatic carcinoma examined (90%) in relation to the tumor nuclear grade, whereas grade I cases showed high IRS periostin expression in two of 10 cases (20%). This relationship was statistically significant (P<0.05).
In terms of Gleason’s sum score, high IRS periostin expression was found in five of 10 (50%) cases with Gleason’s score of 7 or more examined, whereas high IRS periostin expression was found in 12 of 30 (40%) in cases with Gleason’s score less than 7. This means that the higher the Gleason score, the higher the periostin expression. This relationship was statistically nonsignificant (P>0.05).
Stage II cases showed high IRS periostin expression in five of 20 (25%), stage III cases showed high periostin expression in seven of 15 (41%), whereas all stage IV cases (100%) showed high IRS periostin expression. There was a highly significant positive relationship between periostin expression and tumor stage (P<0.01).
In terms of lymph node status, five of the 15 lymph node-negative cases (33.3%) showed high IRS periostin expression, which was found in 12 of 25 lymph node-positive cases (48%). This direct relationship was nonsignificant (P>0.05) (Figs 4 and 5; Table 3).
PIN: the MNA ranges from 19.3 to 41.4 μm2, with a mean value of 29.8 μm2 (Fig. 6).
Prostatic carcinoma: the MNA ranges from 42.8 to 66.9 μm2. The mean value of MNA for all cases was 58.3 μm2.
Statistically, there was a significant positive correlation between MNA and nuclear grade in Gleason’s sum score cases (P value<0.05) (Fig. 7).
Carcinoma of the prostate is the most common form of cancer in men, accounting for 29% of cancer in the USA in 2007. The distinction between low-grade and high-grade PIN can be made with the finding of prominent nucleoli in high-grade PIN. There are also cases in which the degree of nucleolar prominence is borderline; some may consider these as low-grade PIN and others as high-grade PIN. High-grade PIN is considered as a premalignant condition. It precedes carcinoma by 10 years. Currently, high-grade PIN is associated with adenocarcinoma on rebiopsy in 25% of patients (Haber et al., 2010).
The prostate is a complex organ with distinguishable anatomical and functional areas and composed of cells of different embryonic origins. However, the current classification of prostate cancers is restricted to tumor grading and descriptions of unusual histological patterns. It has been difficult to associate histological patterns of prostatic cancers with distinctive cell types or tissue structures. This has hampered attempts to correlate histological types of prostatic tumors with prognostic factors or indicators of clinical relevance.
The results of nuclear morphometry showed that there was a significant positive correlation between MNA and histopathological type of prostatic lesions as we found that the mean value of MNA in PIN cases was 29.8 μm2, but for cases of prostatic carcinoma, it was 58.3 μm2.This is in agreement with the work of Mijovic and Mihailovic (2002), Li et al. (2003), and Helmy (2010) on melanocytic lesions, Özer et al. (2001) in renal cell carcinoma, Lim et al. (2006) in columnar cell lesions, and Wang et al. (2005) in differentiation between follicular adenoma and follicular carcinoma. In contrast to these results, Yan et al. (2007) reported that morphometric parameters (minimum nuclear diameter and maximum nuclear diameter) but not the MNA might be valuable to predict substantial development of prostatic cancer among patients with benign prostatic lesions.
Here, we found that the expression of P-cadherin and periostin in non-neoplastic and neoplastic tissues obtained from prostatic tissues is mutually exclusive. In this study, we provide evidence for periostin upregulation during prostate cancer progression. Periostin expression was found both in epithelial cancer cells and in peritumor stroma. Periostin is known to be a marker for epithelial mesenchymal transition (EMT) in lung cancer. EMT is correlated with tumor progression and represents an important form of tumor–stroma interaction facilitating stromal invasion by cancer cells. Periostin seems to play an important part in this prognostically adverse transdifferentiation process. However, the regulation mechanisms of periostin in tumor progression have not been elucidated as yet.
Our study found a significant association between periostin expression and pT stage, Gleason’s grade, indicating that EMT is very important for prostate cancer progression. Our results are in agreement with those of Tischler et al. (2010), who studied 418 patients with prostatic carcinoma, and found that increased epithelial periostin expression positively correlated to grade and stage and increased stromal periostin correlated positively to grade. Augmentation of both epithelial and stromal periostin is a characteristic of the advanced and more aggressive cases of prostate cancer. This observation is further supported by the finding that only two of 38 benign prostate tissues expressed stromal periostin. In contrast, Tsunada et al. (2004) studied a cohort of 77 patients with prostate cancer, and found increased periostin expression in early prostate cancer stages as well as in the stroma of patients with advanced prostate cancer. The difference may be attributed to the difference in number of cases and differences in the grade and stage.
The results of IHC of P-cadherin showed that there was a statistically significant difference in the extent of its expression among patients with PIN and prostatic carcinoma (P value<0.01). We found that P-cadherin positivity correlated inversely with the tumor nuclear grade. However, this relationship was statistically nonsignificant (P>0.05). In terms of Gleason’s sum score, the higher the Gleason score, the lower the P-cadherin expression; this relationship was statistically significant (P<0.05). These results are in agreement with those by Paredes et al. (2005), who found that in breast carcinoma, P-cadherin was expressed in (31.3%) of the cases and was absent in (68.7%) of the cases. Also, it was found that P-cadherin was found in myoepithelial cells from normal ducts, acini, and ducts containing in situ carcinoma, but not in acinar or ductal normal epithelial cells. In prostatic carcinomas, Gravdal et al. (2007) found that P-cadherin expression is restricted to the cell–cell border of basal epithelial cells in normal prostate samples. This staining is downregulated in prostatic intraepithelial neoplasia and is absent in the entire well-differentiated to poorly differentiated prostate cancer specimens analyzed. Also, Oue et al. (2004) reported that in gastric carcinomas, P-cadherin was expressed in 54% of the gastric carcinomas examined, and the expression was unstable in most of them. Similarly, Lorenzo et al. (2005) found that in esophageal squamous cell carcinoma, 55.2% of cases showed a positive expression, whereas 44.8% of cases showed a negative expression for P-cadherin.
In contrast to the current results, Bauer et al. (2006), Helmy (2010) reported that a significantly lower P-cadherin expression was shown by dermal nevi. Similarly, Taniuchi et al. (2005) reported that overexpression of P-cadherin in pancreatic cancer cells (94.1%) is associated with increased cell motility, a high degree of aggressiveness, and potential for invasion and metastasis. Also, Ingunn et al. (2004) reported a significant correlation between the extent of P-cadherin expression and other clinicopathological variables in endometrial carcinoma. In conclusion, P-cadherin and periostin immunoreactivity could be considered as important independent predictors for outcome in patients with prostatic cancer. Moreover, the nuclear area may be valuable in differentiation between cases with PIN and prostatic carcinoma cases, and it may be useful for a more in-depth evaluation and assessment of clinical outcome in those patients, which may facilitate progress during therapy. Further large-scale studies with a large number of patients and long-term follow-up may be recommended to confirm these results.
Conflicts of interest
There are no conflicts of interest.
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