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Cancer Immunol ImmunotherDOI 10.1007/s00262-014-1594-z

ORIGINAL ARTICLE

High placenta-specific 1/low prostate-specific antigen expression pattern in high-grade prostate adenocarcinoma

Roya Ghods · Mohammad-Hossein Ghahremani · Zahra Madjd · Mojgan Asgari · Maryam Abolhasani · Sanaz Tavasoli · Ahmad-Reza Mahmoudi · Maryam Darzi · Parvin Pasalar · Mahmood Jeddi-Tehrani · Amir-Hassan Zarnani

Received: 12 April 2014 / Accepted: 5 August 2014 © Springer-Verlag Berlin Heidelberg 2014

nonneoplastic/nonhyperplastic prostate tissues using micro-array-based immunohistochemistry (n = 227). The correla-tion of PLAC1 expression with certain clinicopathologi-cal parameters and expression of prostate-specific antigen (PSA), as a prostate epithelial cell differentiation marker, were investigated.Results Placenta-specific 1 (PLAC1) expression was increased in a stepwise manner from BPH to PCa, which expressed highest levels of this molecule, while in a major-ity of normal tissues, PLAC1 expression was not detected. Moreover, PLAC1 expression was positively associated with Gleason score (p ≤ 0.001). Interestingly, there was a negative correlation between PLAC1 and PSA expression in patients with PCa and HPIN (p ≤ 0.01). Increment of

Abstract Background The scarcity of effective therapeutic approaches for prostate cancer (PCa) has encouraged stead-ily growing interest for the identification of novel antigenic targets. Placenta-specific 1 (PLAC1) is a novel cancer–testis antigen with reported ectopic expression in a variety of tumors and cancer cell lines. The purpose of the present study was to investigate for the first time the differential expression of PLAC1 in PCa tissues.Methods We investigated the differential expression of PLAC1 in PCa, high-grade prostatic intraepithelial neo-plasia (HPIN), benign prostatic hyperplasia (BPH), and

Electronic supplementary material The online version of this article (doi:10.1007/s00262-014-1594-z) contains supplementary material, which is available to authorized users.

R. Ghods · M.-H. Ghahremani · P. Pasalar Department of Molecular Medicine, School of Advanced Medical Technologies, Tehran University of Medical Sciences, TUMS, Tehran, Iran

R. Ghods · A.-R. Mahmoudi · M. Darzi · M. Jeddi-Tehrani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

M.-H. Ghahremani Department of Pharmacology-Toxicology, Faculty of Medicine, Tehran University of Medical Sciences, TUMS, Tehran, Iran

M.-H. Ghahremani (*) School of Advanced Technologies in Medicine, Eastern side of Tehran University, 88, Italia St, P.O. box: 1417755469, Tehran, Irane-mail: mhghahremani@tums.ac.ir

Z. Madjd · M. Asgari · M. Abolhasani Oncopathology Research Center, Iran University of Medical Sciences, IUMS, Tehran, Iran

Z. Madjd Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, IUMS, Tehran, Iran

M. Asgari · M. Abolhasani Department of Pathology, Hasheminejad Kidney Center, Iran University of Medical Sciences, IUMS, Tehran, Iran

S. Tavasoli Department of Nutrition, Science and Research Branch, Azad University, Tehran, Iran

A.-H. Zarnani (*) Immunology Research Center, Iran University of Medical Sciences, IUMS, Hemmat Highway, P.O. box: 1449614535, Tehran, Irane-mail: zarnani@avicenna.ac.ir; zarnania@gmail.com

A.-H. Zarnani Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

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PLAC1 expression increased the odds of PCa and HPIN diagnosis (OR 49.45, 95 % CI for OR 16.17–151.25).Conclusion Our findings on differential expression of PLAC1 in PCa plus its positive association with Gleason score and negative correlation with PSA expression high-light the potential usefulness of PLAC1 for targeted PC therapy especially for patients with advanced disease.

Keywords Immunohistochemistry · Gleason · PLAC1 · Prostate cancer · PSA

AbbreviationsBPH Benign prostatic hyperplasiaCI Confidence intervalDAB DiaminobenzidineHPIN High-grade prostatic intraepithelial neoplasiaIHC ImmunohistochemistryOR Odds ratioPAP Prostate acid phosphatasePCa Prostate cancerPLAC1 Placenta-specific 1PSA Prostate-specific antigenTMA Tissue microarrayTRUS Transrectal ultrasound

Introduction

Due to the existence of few effective therapeutic strategies and the associated morbidity, new therapeutic approaches such as immunotherapy for the treatment for prostate cancer (PCa) are highly desired for several reasons. First, several overexpressed and/or altered antigens exist in PC, which could be potential candidates for immunotherapy [1, 2]. Second, it takes a long time for localized PCa to metastasize to other tissues provid-ing enough time for an immunotherapeutic approach to take effect [1, 3]. Third, biomarkers such as prostate-specific anti-gen (PSA) [1, 3] and prostate acid phosphatase (PAP) [3] can be used for monitoring tumor progression.

The aim of the new therapeutic approaches was to target tumor-initiating rather than more differentiated cells [4]. CD133+/α2β1 integrinhigh/CD44+ phenotype has been pro-posed for selecting cancer-initiating cells in human prostate tumors [5]. Similarly, in prostate xenografts and cell lines, tumorigenic cells with coexpression of CD44/α2β1 integrin or CD44 expression alone have been isolated [6, 7]. In this regard, the value of markers such as CD133 in partitioning stem cell-like cells from PCa cell lines has been questioned by recent investigation [8]. Collectively, the prognostic value of these markers in prostate tumors is still a matter of debate. Moreover, many normal cell types also express substantial levels of these markers reflecting the ambiguity regarding their usefulness as an immunotherapy target. Several studies

have demonstrated a positive correlation between the PSA protein expression and overall degree of differentiation in PCa [9–11]. Interestingly, it has been recently shown that PCa cells that do not express or express low levels of PSA (PSA−/lo), which have high clonogenic potential, are refrac-tory to androgen deprivation and reveal a long-term tumor-propagating capacity. In contrast, PSA+ PCa cells possess more limited tumor-propagating capacity [12].

Although, based on the aforementioned features, the PSA−/lo cell population could be regarded as an appropri-ate target of immunotherapy, its targeting needs a specific marker, which is overexpressed instead of being downregu-lated. Some critical factors for the selection of a candidate biomarker suitable for cancer immunotherapy include lack of expression in normal tissues, a tumor-promoting func-tion, and expression in most cancerous tissues of the same type [13]. Placenta-specific 1 (PLAC1) is a novel X-linked gene [14] and a new member of the cancer–testis antigens [15, 16] with 212 amino acids [14]. PLAC1 expression is restricted to placenta [15, 17, 18]. Much lower levels of this molecule are detected in testis [15, 16], while other normal human tissues have no detectable expression of PLAC1 [16]. Predicted topology shows PLAC1 is a trans-membrane protein, suggesting that it is localized to a mem-branous compartment [15, 19]. The physiological role of PLAC1 remains to be elucidated. In a murine model, it has been shown that PLAC1 is essential for normal placental development [20]. Recently, ectopic expression of PLAC1 transcript, and in few instances PLAC1 protein, has been reported in a wide variety of tumor types such as lung [19], gastric [21], colorectal, hepatocellular [22], breast [19], and epithelial ovarian cancers [23] as well as in numerous can-cer cell lines [16, 19, 22]. It has been shown that knock-ing down PLAC1 by siRNA in MCF-7 and BT-549 breast cancer cell lines induces G1-S cell cycle arrest with nearly complete abrogation of cell proliferation and impaired motility, migration, and invasion [19].

The purpose of the present study was to investigate for the first time the differential expression of PLAC1 in a series of prostate tissues including PCa, high-grade prostatic intraepithelial neoplasia (HPIN), benign prostatic hyperpla-sia (BPH), and nonneoplastic/nonhyperplastic prostate tis-sue, to correlate PLAC1 expression with clinicopathological parameters in PCa, and to examine a potential link between PLAC1 and PSA expression.

Materials and methods

Production of anti-PLAC1-specific polyclonal antibody

A New Zealand White Rabbit was immunized against KLH-conjugated PLAC1-specific epitope corresponding

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to the published amino acid sequence (CVFSEEEHTQVP) [24]. Specific polyclonal antibodies were purified using peptide-coupled affinity column. The purity and reactivity of purified antibodies were assessed by SDS–PAGE and ELISA, respectively.

Patients and tissue samples

Paraffin-embedded prostate tissue blocks were collected from Hasheminejad Kidney Center hospital, a major uni-versity-based referral urology hospital in Tehran, Iran. All patients were diagnosed between 2006 and 2011 with either prostate adenocarcinoma (PCa), high-grade prostatic intraepithelial neoplasia (HPIN), or benign prostate hyper-plasia (BPH). Prostate tissues were collected for pathologi-cal examination after radical prostatectomy or performing transrectal ultrasound (TRUS)-guided needle biopsy. The patients had no history of preoperation systemic treat-ment. Pathologic parameters were extracted from pathol-ogy report such as Gleason grade, perineurial and vascular invasion, and adjacent tissue involvement including semi-nal vesicles and bladder neck, lymph node involvement, and pathologic tumor stage. Medical records of all patients were also reviewed to obtain patients’ age and serum PSA level. Gleason scoring was performed based on the guide-lines released in 2005 by the International Society of Urological Pathology [25]. Tumor stage was determined based on the AJCC/UICC TNM staging system [26]. As the negative control group, nonneoplastic/nonhyperplastic prostate tissues adjacent to the tumor site were collected and processed similarly. Specimens from patients with benign prostate hyperplasia (BPH) were collected upon simple prostatectomy. Patient demographic data were col-lected from medical reports. A total of 273 prostate sam-ples were collected at the initial step, and after excluding 46 samples due to technical problems in tissue processing, 227 samples comprising 154 PCa (103 radical prostatec-tomy and 51 needle biopsy), 23 HPIN, 27 BPH, and 23 nonneoplastic/nonhyperplastic prostate tissues were stud-ied. This research was approved by the ethical committees of Avicenna Research Institute and Iran University of Med-ical Sciences, and all patients provided written consent. Patient data are fully anonymous.

Tissue microarray (TMA) preparation

Hematoxylin and Eosin (H&E)-stained slides for each case were reviewed by a pathologist to find the best area of benign and cancerous tissue and also foci of high-grade PIN for preparing TMA. Tissue microarray blocks were constructed as published previously [27, 28]. In brief, three cores of 0.6 mm diameter were punched from the cancer-ous regions of the donor blocks and precisely arrayed

into a new recipient paraffin block using Tissue Arrayer Minicore (ALPHELYS, Plaisir, France). Each TMA had nonneoplastic/nonhyperplastic tissue specimens for com-paring the IHC signal intensity. Tissue microarray blocks were constructed in three copies for each specimen, and the mean scoring of three cores was then calculated as the final score. Needle biopsies were not included in TMA slides, but stained simultaneously with TMA sections.

Immunohistochemistry (IHC)

Immunohistochemistry was performed on 3 micron paraf-fin sections of TMA as we published elsewhere with some modifications [29]. Human term placenta tissue and normal human endometrium served as positive and negative control tissues for PLAC1, respectively. Briefly, deparaffinized sec-tions were subjected to heat-activated antigen retrieval in cit-rate buffer (10 mM, pH 6) at 95 °C for 30 min in water bath.

Endogenous peroxidase activity and nonspecific bind-ing sites were blocked by 1 % H2O2 and 5 % normal sheep serum diluted in protein block (Dako, CA, USA), respectively.

Anti-PLAC1 (10 µg/ml) or Rabbit anti-PSA (Dako, CA, USA) antibody was added to the slides followed by Envision secondary antibody (Dako, Denmark) (for PLAC1) or per-oxidase-conjugated sheep anti-rabbit Ig (for PSA)(Avicenna Research Institute, Tehran, Iran). Signals were visualized by 3,3′-Diaminobenzidine (DAB) (Roche, USA). In nega-tive reagent control slides, primary antibodies were blocked with saturating concentrations of immunizing peptide or pro-tein (1:100 molar ratio) prior to being applied. Slides were counterstained with Harris hematoxylin, dehydrated, and mounted. Digital images were captured by a BX51 micro-scope and a DP70 CCD camera (Olympus, Japan).

Immunostaining evaluation and scoring

The immunostained tissue arrays were observed on a multi-headed microscope simultaneously by three expert observ-ers in a blind manner without having previous knowledge of pathological diagnosis. Each sample was scored inde-pendently by observers, and a consensus was achieved based on the similarity of the scores. All IHC signals were scored according to the semiquantitative scoring sys-tem [30]. Scoring was classified as 0 (no expression), 1+ (weak), 2+ (moderate), and 3+ (strong). After 2 months, re-scoring was blindly repeated to confirm the reproduc-ibility of our initial scoring system.

Statistical analysis

Regarding PLAC1 and PSA expression score catego-ries, Kruskal–Wallis and Pearson’s χ2 tests were used to

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compare continuous and categorical variables, respectively. In the case of unbalanced values in table cells, exact tests were replaced. Spearman’s correlation test analysis was performed to find the correlation between categorical vari-ables. To investigate whether or not Gleason grades 3 + 4 and 4 + 3 show different patterns of PLAC1 or PSA expres-sion, χ2 test was used. Binary logistic regression analysis was performed to estimate the odds ratio (OR) and 95 % confidence intervals (CI) of PCa and HPIN versus BPH and nonneoplastic/nonhyperplastic diagnosis, using PLAC1 and PSA expressions as predictors. To exclude the effect of empty cells in regression analysis, PLAC1 and PSA expres-sions were also converted to dichotomous variables “no to weak expression” vs. “moderate to strong expression”. p < 0.05 was considered to be statistically significant.

Results

Study population

Overall mean age of the study population was 66.83 years (ranged 39–90). Mean age of different groups was as

follows: BPH 69.96 (SD 9.04), HPIN 65.70 (SD 7.08), needle biopsy adenocarcinoma 69.43 (SD 9.08), and radi-cal prostatectomy adenocarcinoma 65.58 (SD 7.83). PSA levels (range 0.6–89, mean 17.19 ng/ml) were grouped as <4, 4–10, and >10 ng/ml [31]. Of 99 cases for whom the PSA data were available, 7 (7.0 %) had a PSA of <4 ng/ml, 46 (46.5 %) had PSA of 4–10, and 46 (46.5 %) had PSA >10 ng/ml. The Gleason score of patients was categorized as 6–7 and ≥8. Of 154 PCa patients, 96 (62.3 %) had Glea-son score of 6–7 and 58 (37.7 %) had Gleason score of ≥8. Clinicopathological data of adenocarcinoma patients are summarized in Table 1.

Validation of anti-PLAC1 antibody

To determine the specificity of anti-PLAC1 antibody, IHC was carried out on human term placenta and normal human endometrium as negative tissue control. The results clearly showed the specific pattern of immunoreactivity on human term placenta as shown in Supplementary Figure 1. The anti-body recognized PLAC1 in differentiated trophoblasts, and localization was restricted mostly to the cytoplasmic com-partment and to some extent to microvillus plasma membrane

Table 1 Clinicopathological prognostic factors in prostate adenocarcinoma patients (n = 154)

PSA prostate-specific antigen† Number of cases with the available data for each parameter is presented in parenthesis‡ These variables were present only for radical prostatectomy PCa patients

Clinicopathological parameters† Value [n (%)] Clinicopathological parameters† Value [n (%)]

PSA (n = 89) Laterality (n = 95) ‡

<4 ng/mL 3 (3.4) Unilateral 19 (20.0)

4–10 ng/mL 41 (46.1) Bilateral 76 (80.0)

>10 ng/mL 45 (50.6)

Gleason score (n = 154) Involvement of margins (n = 88) ‡

6–7 96 (62.3) Not identified 54 (61.4)

≥8 58 (37.7) Present 34 (38.6)

Extraprostatic extension (n = 143) Involvement of bladder neck (n = 53) ‡

Not identified 95 (66.4) Not identified 46 (86.8)

present 48 (33.6) Present 7 (13.2)

Perineural invasion (n = 143)

Not identified 11 (7.7)

Present 132 (92.3)

Involvement of regional lymph nodes (n = 92) ‡

Free 87 (94.6)

Tumor stage (n = 97) ‡ Involved 5 (5.4)

T2 2 (2.1) Involvement of seminal vesicle (n = 93) ‡

T2a 6 (6.2) Free 73 (78.5)

T2b 10 (10.3) Involved 20 (21.5)

T2c 42 (43.3) Vascular invasion (n = 79) ‡

T3a 15 (15.5) Not identified 75 (94.9)

T3b 22 (22.6) Present 4 (5.1)

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of syncytiotrophoblasts. Moreover, very few cytotrophoblasts exhibited specific immunoreactivity. Endometrium, the tissue adjacent to placenta, was assessed for its reactivity with anti-PLAC1 and shown to be always negative (Supplementary Figure 1). No signal was detected in negative reagent control sections in which primary antibody was pre-adsorbed or sub-stituted by pre-immune-purified rabbit IgG.

Expression of PLAC1 in PCa, HPIN, BPH, and nonneoplastic/nonhyperplastic prostate tissues

Figure 1 depicts PLAC1 expression in nonneoplastic/nonhyperplastic, BPH, HPIN, and PCa tissues. PLAC1 was expressed with varying intensities in nearly all examined

tissues except for the nonneoplastic/nonhyperplastic ones. PLAC1 was localized to both cytoplasmic compartment and plasma membrane. In most cases, PLAC1 expression was localized predominantly to the apical surface of epithe-lial cells. Immunoreactivity was restricted to the epithelial cells of prostate tissues, and no expression was observed in nonepithelial cells including stromal and endothelial cells. Interestingly, expression of PLAC1 was increased in a step-wise manner from the nonneoplastic/nonhyperplastic group with the lowest PLAC1 expression to HPIN and PCa groups with the highest expression levels of this molecule (Fig. 2) (χ2 tests, p < 0.001). The results of Spearman’s correlation test also showed a significant positive correlation between PLAC1 expression and diagnosis groups (r = 0.650,

Fig. 1 PLAC1 and PSA expression pattern in nonneoplastic/nonhyperplastic prostate, BPH, HPIN, and PCa. A series of 227 samples including PCa (n = 154), HPIN (n = 23), BPH (n = 27), and nonneoplastic/nonhyperplastic prostate tissues (n = 23) were collected and assessed for the expression of PLAC1 and PSA

by immunohistochemistry using specific antibodies. In each panel (PLAC1 and PSA), photographs of low- and high-power fields are represented. In PCa, two series of photographs representing PLAC1 and PSA staining of samples with Gleason scores of 6 (m-p) and 9 (q-t) are shown

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p < 0.001). Notably, none of the PCa and HPIN samples showed “no expression” of PLAC1, while 69.6 % of HPIN and 86.4 % of PCa samples expressed moderate or high lev-els of PLAC1. Moreover, no moderate or strong staining was observed in the nonneoplastic/nonhyperplastic group. On average, 65 % of nonneoplastic/nonhyperplastic samples failed to express PLAC1 and 34.8 % of them showed weak immunoreactivity. In BPH group, all staining patterns were observed except strong staining with 22.2, 63.0, and 14.8 % of samples showing no expression (0), weak (1 +), and moderate (2 +) expression of PLAC1, respectively (Fig. 2).

Placenta-specific 1 (PLAC1) expression in relation to clinicopathological features of prostate cancer

The association between clinicopathological prognos-tic parameters and PLAC1 expression was also assessed in prostate adenocarcinoma patients (Table 2). There is a strong positive correlation between Gleason scores and PLAC1 expression in PCa patients (χ2 test: p < 0.001) (Fig. 3a). No significant correlation was observed between PLAC1 expression and other clinicopathological parame-ters. There was no significant difference in PLAC1 expres-sion between Gleason scores 3 + 4 and 4 + 3, but PCa tis-sues with 4 + 3 Gleason score showed significantly lower percent of moderate and high PSA expression compared to those with 3 + 4 Gleason score (p < 0.05).

PSA expression and its correlation with clinicopathologic parameters and PLAC1 expression

The expression of PSA in serial sections of all tissues was determined and scored. Results showed that the majority

of nonneoplastic/nonhyperplastic (76.2 %) and BPH (92.6 %) tissues exhibited intense staining (3+) for PSA. In HPIN group, the percentage of specimens with moder-ate and weak staining was increased, and this pattern of reactivity was further fortified in the cancer group in which most specimens (64.5 %) showed weak or moderate PSA expression.

Interestingly, in patients with higher Gleason scores, lower PSA expression was more frequent (p < 0.001) (Fig. 3b). Evaluation of the association between PSA expression and clinicopathological prognostic parameters showed that there was a significant negative association between PSA expression and extra-prostatic extension (p < 0.01). The correlation of PSA expression with PLAC1 in PCa and HPIN groups was also analyzed. As shown in Fig. 4, PSA expression showed a significant negative corre-lation with PLAC1 expression (p < 0.001). By performing binary logistic regression, we found a significant predic-tive value of PLAC1 and PSA expression in patients’ diag-nosis (PCa and HPIN vs. nonneoplastic/nonhyperplastic and BPH). The analysis showed that increment of PLAC1 expression increased the odds of PCa and HPIN diagnosis (OR 49.45, 95 % CI for OR 16.17–151.25), whereas incre-ment of PSA expression decreased the mentioned odds (OR 0.08, 95 % CI for OR 0.01–0.70).

Discussion

One major drawback of PCa immunotherapy is the paucity of specific markers that could be potentially used for tar-geting. More importantly, most of the proposed markers for PC immunotherapy including PSA have not been shown so far to have a known role in cancer development and pro-gression. Indeed, as we showed here, there was an inverse correlation between PSA expression and tumor aggres-siveness and prognosis as judged by Gleason scoring. The elegant work done by Qin et al. [12] clearly demonstrated that PSA−/lo rather than PSA+ prostate cells are highly clo-nogenic and possess long-term tumor-propagating capacity. This implies that novel immunotherapeutic strategies for PCa should focus on PSA−/lo cells as a more suitable target. These observations raise the fundamental question of how PSA−/lo PCa cells could be targeted?

In this study, we investigated for the first time the dif-ferential expression profile of PLAC1 and PSA expression in a wide variety of prostate tissues with different diagnosis including nonneoplastic/nonhyperplastic, BPH, HPIN, and PCa.

Immunohistochemistry (IHC) results displayed a dis-criminating profile of PLAC1 expression in carcinoma tissues relative to the nonneoplastic/nonhyperplastic con-trols (p < 0.001). PLAC1 was mostly absent or weakly

Fig. 2 Distribution of PLAC1 expression score in nonneoplastic/nonhyperplastic, BPH, HPIN, and PCa groups. n = 227, χ2 test: p < 0.001

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Table 2 Association of clinicopathological prognostic factors with PLAC1 expression score in prostate adenocarcinoma patients (n = 154)

* Statistically significant † Represented values show mean (standard deviation)‡ Categorical variable values are presented as number (%)§ These variables were present only for radical prostatectomy specimens

Clinicopathological parameters PLAC1 expression‡ p value

Weak (n = 21) Moderate (n = 69) Strong (n = 64)

Age† (n = 154) 63.05 (9.29) 67.55 (7.76) 67.36 (8.65) 0.251

Serum PSA (n = 89)

<4 ng/mL 0 (0.0 %) 3 (7.0 %) 0 (0.0 %) 0.394

4–10 ng/mL 7 (46.7 %) 21 (48.8 %) 13 (41.9 %)

>10 ng/mL 8 (53.3 %) 19 (42.2 %) 18 (40.0 %)

Gleason score (n = 154)

6–7 20 (91 %) 49 (72.1 %) 27 (42.2 %) <0.001*

≥8 2 (9 %) 19 (27.9 %) 37 (57.8 %)

Extraprostatic extension (n = 143)

Not identified 11 (52.4 %) 44 (69.8 %) 40 (67.8 %) 0.340

Present 10 (47.6 %) 19 (30.2 %) 19 (32.2 %)

Perineural invasion (n = 143)

Not identified 0 (0.0 %) 5 (7.8 %) 6 (10.2 %) 0.364

Present 20 (100 %) 59 (92.2 %) 53 (89.8 %)

Laterality (n = 95)§

Unilateral 4 (20.0 %) 9 (18.8 %) 6 (22.2 %) 0.937

Bilateral 16 (80.0 %) 39 (81.3 %) 21 (77.8 %)

Tumor stage (n = 97) ‡

T2 0 (0.0 %) 2 (4.1 %) 0 (0.0 %)

T2a 0 (0.0 %) 3 (6.1 %) 3 (10.7 %)

T2b 2 (10 %) 5 (10.2 %) 3 (10.7 %)

T2c 6 (30 %) 22 (44.9 %) 14 (50 %) 0.423

T3a 6 (30 %) 5 (10.2 %) 4 (14.3 %)

T3b 6 (30 %) 12 (24.5 %) 4 (14.3 %)

Margins involvement (n = 88)§ 7 (36.8 %) 18 (40.0 %) 9 (37.5 %) 0.964

Bladder neck involvement (n = 53)§ 4 (28.6 %) 1 (4.2 %) 2 (13.3 %) 0.089

Lymph node metastasis (n = 92)§ 1 (5.0 %) 4 (8.5 %) 0 (0.0 %) 0.355

Seminal vesicle involvement (n = 93)§ 6 (30.0 %) 10 (20.8 %) 4 (16.0 %) 0.518

Vascular invasion (n = 79)§ 2 (12.5 %) 2 (5.0 %) 0 (0.0 %) 0.240

Fig. 3 Association of Gleason score with PLAC1 and PSA expression in PCa patients. Gleason score and PLAC1 a; n = 152, Gleason score and PSA b; n = 147, χ2 test: p < 0.001 for both analyses

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expressed in nonneoplastic/nonhyperplastic tissues, while its expression level was increased in a stepwise manner in BPH, HPIN, and PCa, suggesting PLAC1 expression as an early event in prostate carcinogenesis. These find-ings strongly suggest PLAC1 as a potential target for PCa immunotherapy. Expression of PLAC1 in healthy individu-als was reported to be restricted to placenta and testis with no detectable expression in other normal tissues [15–18]. Thus, one may not expect nonneoplastic/nonhyperplastic prostate tissues to express PLAC1; nonetheless, in our study, about 35 % of nonneoplastic/nonhyperplastic tissues expressed marginal levels (1+) of this antigen. We obtained nonneoplastic/nonhyperplastic tissues from areas adjacent to cancerous tissues; therefore, it is possible that these areas would be affected by molecular events responsible for carcinogenesis.

Placenta-specific 1 (PLAC1) may play a role in cancer cell proliferation [19]. Our results on relatively increased level of PLAC1 expression in BPH specimens compared to nonneoplastic/nonhyperplastic tissues are in line with this assumption. Nonetheless, the exact function of this cancer–testis antigen in prostate tumor biology remains to be eluci-dated. Expression of PLAC1 transcript in PCa cell line has also been reported recently [16]. Nonetheless, no functional activity of PLAC1 in these cells has been surveyed.

In this study, we did not find statistical associations between PLAC1 expression and most of the examined clin-icopathological parameters except Gleason score for which we observed a positive correlation. Prostate cancer is a het-erogeneous mixture of differentiated and undifferentiated tumor cells with different proliferation potentials. Whether PLAC1 expression is a function of the differentiation state of PCa cells is not clear; however, our finding on the posi-tive correlation between PLAC1 expression and Gleason

score suggests PLAC1 as a marker of poorly differenti-ated PC cells. In line with this assumption, we found that tumors with higher expression of PLAC1 showed lower PSA expression. According to a recent report, PSA expres-sion positively correlates with the degree of differentiation, and differentiated cells expressed high levels of PSA, while PSA−/lo cells exhibited high clonogenic and long-term tumor-propagating capacity [12].

Interestingly, lower tissue PSA transcript in PCa cor-relates with worse clinical outcomes, including high tumor grade, lymph node positivity, metastasis, recur-rence, and reduced patient survival [12]. Given that high-grade PCas are usually unresponsive to conven-tional therapeutic regimens, PLAC1 may be consid-ered as a proper candidate molecule to be targeted in such cases. Considering the inverse correlation between PSA and PLAC1 expression, this molecule may also be regarded as a potential therapeutic target in meta-static PCas. Importantly, recent work by Chen et al. [32] showed that Tp53 mutations in cancer cells trigger the derepression of a PLAC1 promoter. Interestingly, p53 mutation is reported in advanced stages of PCa, as well as in the recurrent and metastatic disease [33]. Our find-ing on the positive correlation between PLAC1 expres-sion and Gleason score support these data.

Conclusion

Here, we showed for the first time the differential expres-sion of PLAC1 in PCas and nonneoplastic/nonhyperplastic prostate tissues. The positive association between PLAC1 expression and Gleason score may highlight the potential usefulness of PLAC1 in targeted PC therapy especially for patients with advanced disease. Whether other epithelial solid tumors take advantage of PLAC1 expression is our ongoing research plan.

Acknowledgments This study was funded by grants from Iran Uni-versity of Medical Sciences (Grant No: 92-02-13-22395), and Tehran University of Medical Sciences (Grant No: 90-03-13-13530). The authors would like to thank Dr M. Bozorgmehr for carefully proof-reading the manuscript, Mr. E. Mirzadegan for technical assistance and preparation of figures, and Mrs. J. Taeb for arranging clinico-pathological data.

Conflict of interest All authors declare that there is no conflict of interest.

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Fig. 4 Association of PLAC1 and PSA expression in HPIN and PCa groups. n = 171, Spearman correlation test; r = −0.218, p < 0.01

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