Cancer/testis antigens: novel tools for discerning aggressive and non-aggressive prostate cancer

The introduction of serum prostate-specific antigen (PSA) in the 1980s has dramatically altered and benefited the initial diagnosis of prostate cancer. However, the widespread use of PSA testing has resulted in overdetection and overtreatment of potentially indolent disease. Thus, a clinical dilemma today in the management of prostate cancer is to discern men with aggressive disease who need definitive treatment from men whose disease are not lethal. Although several serum and tissue biomarkers have been evaluated during the past decade, improved markers are still needed to enhance the accuracy, with which patients at risk can be discerned and treated more aggressively. The cancer/testis antigens (CTAs) are a group of proteins that are restricted to the testis in the normal adult, but are aberrantly expressed in several types of cancers. Because of their restricted expression pattern, the CTAs represent attractive biomarker candidates for cancer diagnosis/prognosis. Furthermore, several studies to date have reported the differential expression of CTAs in prostate cancer. Here, we review recent developments that demonstrate the potential of the CTAs as biomarkers to discern the aggressive phenotype of prostate cancer.     

Expression of the tumour antigen T21 is up-regulated in prostate cancer and is associated with tumour stage

What's known on the subject? and What does the study add? T21 is an immunogenic prostate‐associated tumor antigen identified by SEREX expression cloning and shown to have a highly restricted mRNA expression profile. This study shows the expression of T21 at the protein level in a panel of tissues and cell lines and demonstrates increasing levels of T21 protein expression in patients with more advanced stage prostate cancer.

OBJECTIVES

• ? To define the expression pattern of the tumour antigen T21 at the protein level in prostate tissues, prostate cell lines and a panel of normal tissues.
• ? To correlate the expression pattern of T21 in prostate cancer with clinical parameters.

PATIENTS AND METHODS

• ? Tissue samples were collected from 79 patients presenting at clinic with either prostate cancer (63 patients) or benign prostatic hyperplasia (BPH, 16 patients).
• ? A tissue microarray (TMA) was constructed from 44 of the prostate cancer tissues and areas of benign disease (43 patients) from these tissues were also included on the TMA. The remaining tissues (prostate cancer 19 patients and BPH 16 patients) were mounted fresh frozen onto cork boards and sectioned.
• ? Full ethical approval was granted for all aspects of the study and informed patient consent was taken before tissue collection.
• ? Immunohistochemistry was used on the prostate tumour TMA, the normal tissue TMA and the fresh‐frozen prostate tissues. Fluorescent microscopy and flow cytometry was performed on prostate cell lines.

RESULTS

• ? Expression of T21 was highly restricted within normal tissues with only the stomach, ovary, breast and prostate having detectable T21 expression.
• ? T21 was significantly over‐expressed in prostate cancer glands compared with benign tissue and was present in >80% of the malignant specimens analysed.
• ? Increased expression was positively correlated to pathological stage of prostate tumours.
• ? Additionally, T21 was associated with Gleason grade and prostate‐specific antigen recurrence, although statistical significance was not reached in this restricted cohort of patients.

CONCLUSION

• ? Taken together these results show that T21 is a potential new biomarker for advanced disease and that elevated levels of T21 appear relevant to prostate cancer development.

     

NY-ESO-1 protein expression and humoral immune responses in prostate cancer

BACKGROUND: Due to restricted expression in normal tissues cancer/testis (C/T) antigens represent candidate molecules for immunotherapy of cancer. NY-ESO-1 is a well-studied C/T antigen with unknown expression and immunogenicity in prostate cancer (PC) patients. METHODS: NY-ESO-1 expression was determined by immunohistochemistry and humoral immune responses against NY-ESO-1 assessed by enzyme-linked immuno-sorbent assay (ELISA) and Western blotting. Protein expression and serological responses were correlated with clinical findings and survival. RESULTS: NY-ESO-1 expression was found in biopsies from 2 of 66 localized PC and 7/48 hormone refractory prostate cancer (HRPC) patients, respectively. Anti-NY-ESO-1 antibodies were detected in sera from 1 of 112 localized PC and 18 of 95 HRPC patients. Two of four HRPC patients with NY-ESO-1 positive biopsies had mounted a serological response. Positive anti-NY-ESO-1 titers were correlated with poor survival in HRPC patients. CONCLUSIONS: NY-ESO-1 is expressed in a subset of HRPC patients and, together with other C/T antigens, may serve as a target antigen for development of immunotherapy of PC. Spontaneous serological responses against NY-ESO-1 may be associated with poor survival.     

Expression of NKX3.1 in normal and malignant tissues

BACKGROUND: NKX3.1, a member of the NK-class of homeodomain proteins, is expressed primarily in the adult prostate and has growth suppression and differentiating effects in prostate epithelial cells. METHODS: We performed immunohistochemical analysis for NKX3.1 and PSA expression in 4,061 samples included in a tissue microarray of a broad spectrum of human cancers and normal tissues. RESULTS: NKX3.1 expression was seen in prostate epithelial cells, prostate cancer, normal testis, 9% of primary and 5% of metastatic infiltrating ductal breast carcinoma, and 27% of primary and 26% of metastatic infiltrating lobular breast carcinoma. In a cohort of 474 primary breast cancers with median follow-up over 62.5 month survival, we found no effect of NKX3.1 expression on prognosis. NKX3.1 expression was more restricted than the spectrum of prostate specific antigen expression. CONCLUSIONS: Expression of NKX3.1 is highly restricted and is found primarily in benign and malignant prostatic epithelial cells and also in normal testis and lobular carcinoma of the breast.     

Preferential humoral immune response in prostate cancer to cellular proteins p90 and p62 in a panel of tumor-associated antigens

BACKGROUND: Cytoplasmic p90 autoantigen was recently cloned from a cDNA expression library using serum antibody from a cancer patient. The humoral immune response to p90 in prostate cancer and benign prostatic hyperplasia (BPH) was examined. METHODS: An antigenic fragment of recombinant p90 protein and several other tumor-associated antigens (TAAs) were used in ELISA and Western blotting to detect antibodies in sera from patients with prostate cancer, BPH, and other controls. RESULTS: Autoantibodies to p90 were detected in 30.8% of 133 prostate cancer patients versus 1.5% in 68 BPH patients. When a selected panel of six TAAs including p90 were used for immunoscreening, the cumulative positive reactions in prostate cancer sera reached 92.5%, significantly higher than in BPH and other control sera. Antibodies to p90 showed the highest frequency in prostate cancer (30.8%), followed by antibodies to p62 (22.6%). CONCLUSIONS: A panel of six selected TAAs was shown to have high sensitivity and specificity as immunodiagnostic markers in prostate cancer.     

Identification of an HLA-A*0201-restricted T-cell epitope derived from the prostate cancer-associated protein trp-p8

BACKGROUND: New concepts for the immunotherapy of prostate carcinoma (PCa) largely depend on the identification of suitable target antigens that are present in a high percentage of prostate tumors. Their expression in normal tissues should be restricted to the prostate and they should be immunogenic in vivo. The number of antigens displaying these properties is still limited. Here, we identify for the first time an immunogenic peptide derived from the prostate-specific protein transient receptor potential-p8 (trp-p8) that is recognized by cytotoxic T lymphocytes (CTLs) from PCa patients. METHODS: To determine the abundance of trp-p8 in prostate tumors, the expression level of trp-p8 mRNA was quantitatively analyzed in a panel of prostate cancer tissues. Trp-p8-derived human leukocyte antigen (HLA)-A*0201-restricted peptides were selected and tested for the in vitro activation of CTLs when loaded on autologous dendritic cells (DCs). RESULTS: Trp-p8 mRNA was found to be expressed in all prostate tumors and in the corresponding normal prostate tissue. Of five selected trp-p8-derived peptides, only peptide GLMKYIGEV was shown to activate specific CTLs, which effectively lysed PCa cells confirming the endogenous generation and presentation of this peptide by tumor cells. CONCLUSIONS: Our results suggest this antigen as a suitable target for the T-cell-based immunotherapy of PCa.     

Alternative splicing isoforms of hippostasin (PRSS20/KLK11) in prostate cancer cell lines

BACKGROUND: Hippostasin is a kallikrein-like protease (PRSS20/KLK11), which is expressed preferentially in the hippocampus and prostate. We have reported that alternative splicing variants of human hippostasin are regulated in a tissue-specific manner. Brain-type hippostasin consists of 250 amino acids including a typical signal sequence, and is expressed in the brain and prostate. The prostate-type hippostasin, which has 32 extra amino acids at the N-terminal end, is expressed only in the prostate. METHODS: We analyzed the expression and localization of hippostasin in normal prostate tissue, BPH tissue, and prostate cancer cell lines. We performed northern blotting, in situ hybridization, immunohistochemistry, and RT-PCR. RESULTS: Hippostasin mRNA is expressed preferentially in the normal prostate and weakly in the testis. It was detected in prostate secretory epithelium. Hippostasin protein was localized in the prostate secretory epithelium, and western blotting showed that hippostasin was present in semen. All tested prostate cancer cell lines, including PSA-negative cell lines, expressed hippostasin. Interestingly, all the prostate cancer cell lines expressed only brain-type but not prostate-type hippostasin, while normal prostate and BPH expressed both types of hippostasin CONCLUSIONS: Our results suggest the possibility that hippostasin may be a useful marker by which prostate cancer and BPH can be distinguished.     

Characterization of human prostate and breast cancer cell lines for experimental T cell-based immunotherapy

BACKGROUND: In order to develop experimental immunotherapy for prostate and breast cancer it is of outmost importance to have representative target cell lines that through human leukocyte antigen (HLA) class I molecules present relevant levels of peptides from tumor-associated antigens for cytotoxic T lymphocyte (CTL) recognition. METHODS: We sequenced the HLA-A and HLA-B loci of eight commonly used prostate and breast cancer cell lines and analyzed the surface expression of HLA-ABC, HLA-DR, CD40, CD80, CD86, and CD54 by flow cytometry. We also analyzed the cell lines for mRNA expression from 25 genes reported to be specifically or preferentially expressed by prostate cells. RESULTS: Among the analyzed cell lines we found that LNCaP, PC-346C and MCF-7 are HLA-A*0201 positive. However, the HLA-A2 expression level is low and only MCF-7 upregulates HLA-A2 in response to IFN-gamma stimulation. MCF-7 also expresses high levels of CD54, which further improve its value as a CTL target cell line. On the other hand, LNCaP and PC-346C express 25 and 23 out of 25 prostate-related genes, respectively, while MCF-7 expresses 16 out of 25 genes. CONCLUSIONS: None of the analyzed prostate cancer cell lines are optimal CTL target cells. However, MCF-7 could in many cases be used as a complement to HLA-A*0201 positive prostate cancer cells. The LNCaP and PC-346C cell lines are rich sources of prostate-related antigens that may be valuable for cancer vaccine development.     

抗前列腺特异膜抗原单克隆抗体的前列腺癌免疫病理研究

目的 观察前列腺特异膜抗原（ＰＳＭ）在前列腺癌（ＰＣａ）中的表达情况,探讨其与前列腺癌病理组织学分级及与内分泌治疗效果的关系,方法 采用ＥｎＶｉｓｉｏｎ＾ＴＭ两步法免疫组织化学染色方法,用ＰＳＭ单克隆抗体Ｅｄ－５对３６例前列腺癌（Ｐｃａ）、１０例正常前列腺组织、１０例前列腺良性增生、６是性前列腺炎、３８例非前列腺肿瘤（包括膀胱癌、肾透明细胞癌、睾丸肿瘤等）进行染色。结果 ＰＳＭ在正常前列腺组织、慢     

Immunohistochemical expression of tumor antigens MAGE-A1, MAGE-A3/4, and NY-ESO-1 in cancerous and benign prostatic tissue

OBJECTIVE: To investigate immunohistochemical expression of MAGE-A and NY-ESO-1/LAGE-1, cancer testis antigens in prostate tissues showing evidence of malignant transformation or benign hyperplasia. METHODS: 112 prostate samples from patients undergoing surgery at the Urology Clinic at the Zagreb Clinical Hospital Center from 1995 to 2003 were investigated in this study. Of these, 92 carcinoma samples were obtained by radical prostatectomy, and 20 benign prostatic hyperplasia samples by transvesical prostatectomy. Three monoclonal antibodies were used for immunohistochemical staining: 77B for MAGE-A1, 57B for multi-MAGE-A and D8.38 for NY-ESO-1 expression. RESULTS: Expression of MAGE-A1 was observed in 10.8% of carcinoma samples, whereas multi-MAGE-A and NY-ESO-1/LAGE-1 stained 85.9% and 84.8% of samples. Immunohistochemical staining was only detectable in the cytoplasm. A significant heterogeneity could be observed within a same tissue sample where areas with strong positivities coexisted with cancer testis antigens negative areas. Interestingly, a majority of 57B positive cases were also found to be D8.38 positive (correlation coefficient r=0.727 (P<0.01)). Cancer testis antigens expression was neither significantly correlated with PSA values nor with Gleason score. In benign prostatic hyperplasia tissues MAGE-A1 expression was detected in 5%, while 57B and D8.38 staining was observed in 15% samples, and in all cases percentages of positive cells were always <10%. CONCLUSION: Our data underline the peculiar relevance of cancer testis antigens expression in prostate cancers, with potential implications regarding both diagnosis and therapy.     

Expression of a human cell adhesion molecule, MUC18, in prostate cancer cell lines and tissues

BACKGROUND: Over expression of huMUC18, a cell adhesion molecule in the immunoglobulin gene superfamily, causes a non-metastatic human melanoma cell line to become metastatic in a nude mouse system. To determine if MUC18 expression correlates with the malignant progression of prostate cancer, we investigated differential expression of human MUC18 (huMUC18) in normal prostate epithelial cells, prostate cancer cell lines, and prostatic normal and cancer tissues. METHODS: RT-PCR and Western blot analyses were used to analyze the expression of MUC18 mRNA and protein in four human prostate cancer cell lines, cultured primary normal prostate epithelial cells, normal prostate and malignant prostate tissues. Immunohistochemistry was used to determine the expression of MUC18 antigen in prostatic tissues at different stages of malignancy. RESULTS: Human MUC18 mRNA and protein was expressed in three different prostate cancer cell lines (TSU-PR1, DU145, and PC-3), but not in one prostate cancer cell line (LNCaP.FGC). HuMUC18 protein was also expressed at high levels in extracts prepared from tissue sample sections containing high grade prostatic intraepithelial neoplasia (PIN), but weakly expressed in extracts prepared from either cultured primary normal prostatic epithelial cells or the normal prostate gland. Immunohistochemical analysis showed that huMUC18 was expressed at higher levels in the epithelial cells of high-grade PIN and prostatic carcinomas and in cells of a lymph node metastasis compared to that in normal or benign hyperplastic epithelium (BPH). CONCLUSIONS: We therefore conclude that MUC18 is expressed at higher levels in pre-malignant and malignant prostatic epithelium, including metastasis. We suggest that over-expression of MUC18 may be a new marker of human prostate cancer and also implicates its possible role in development and progression of prostate cancer.     

Kallikrein 10基因在前列腺癌组织中的表达及意义

目的：探讨Kallikrein10（KLK10）基因在不同前列腺组织中的表达情况。方法：采用荧光定量PCR方法检测3例正常前列腺组织、5例BPH组织、6例前列腺癌细胞株、35例前列腺癌组织中KLK10mRNA的表达水平。结果：正常前列腺组织、良性前列腺增生组织、前列腺癌细胞株、前列腺癌组织KLK10 mRNA表达相对值分别为0．521、0．487、0．021、0．018,组间比较差异有统计学意义（P〈0．01）。骨转移前列腺癌与未转移前列腺癌KLKIOmRNA表达相对值分别为0．003、0．023,两组比较差异有统计学意义（P〈0．05）。结论：KLK10在前列腺癌组织中表达下调,在转移性前列腺癌中表达更低。KLK10表达下调可促进肿瘤的发生与进展。     

组织激肽释放酶基因7在前列腺癌组织中的表达

目的 探讨组织激肽释放酶基因7(KLK7)在不同前列腺组织中的表达情况.方法运用逆转录聚合酶链反应法检测正常前列腺(5例)、良性前列腺增生(BPH)及BPH细胞株(BPH1,13例)、前列腺癌及前列腺癌细胞株(8例)的上皮细胞中KLK7mRNA表达水平;蛋白质印迹法检测不同前列腺组织上皮细胞中KLK7蛋白表达水平;免疫组化分析正常前列腺(20例)、BPH(50例)、前列腺癌(103例)组织中KLK表达水平.根据染色强度分为4个等级(-,+,++,+++)进行半定量分析,染色强度++及+++者判定为阳性.结果 正常组、BPH组和前列腺癌组KLK7 mRNA表达相对值分别为0.59、0.52、0.02,组间比较差异有统计学意义(F=13.03,P＜0.01),前列腺癌上皮中KLK7 mRNA表达下调(P＜0.01),正常前列腺和BPH上皮中KLK7 mRNA表达差异无统计学意义(P＞0.05).KLK7蛋白在正常前列腺、增生前列腺、DU145、LNCaP、PC3、22RV1、BPH细胞株中表达水平相对值分别为0.22、0.40、0.01、0.05、0、0.03、0.14.免疫组化染色结果 显示正常前列腺组织、BPH组织、前列腺癌中KLK7蛋白表达阳性率分别为65.0%(13/20)、76.0%(38/50)、17.5%(18/103),前列腺癌组与前2组比较差异均有统计学意义(P＜0.01),前2组间比较差异无统计学意义(P＞0.05).结论 KLK7在前列腺癌组织中表达下调,提示KLK7在前列癌的发生和进展中可能起一定作用.     

RASSF1A promoter methylation is frequently detected in both pre-malignant and non-malignant microdissected prostatic epithelial tissues

BACKGROUND: The RASSF1A gene is a tumor suppressor gene inactivated by hypermethylation in a very wide variety of malignant tumors including prostate cancer. METHODS: In this study we have used laser capture microdissection to provide pure cell populations to investigate the methylation status of 16 CpG sites in the promoter region of this gene in prostatic intra-epithelial neoplasia, in histologically normal epithelial cells associated with these lesions and in epithelial cells from benign prostatic hyperplasia. RESULTS: Unexpectedly, frequent methylation, detected by sequence analysis following bisulphite treatment, was observed in benign epithelium as well as in the lesions associated with prostatic intra-epithelial neoplasia and at high risk of cancer formation. Fifty percent or more CpG sites were methylated in 7/14 prostatic intra-epithelial neoplasms, 8/11 histologically normal epithelial cells and 8/12 specimens of benign prostatic tissue. CONCLUSION: These observations suggest that methylation of the RASSF1A gene is present in both pre-malignant and benign epithelia and suggests quantitation is required for it to be an effective marker of early prostate cancer.     

Sharpin蛋白在前列腺癌中的表达及其与Gleason评分、t—PSA关系的研究

目的探讨Sharpin蛋白在人不同前列腺癌细胞株中与前列腺癌组织中的表达及其与Gleason评分、血清PSA的关系。方法采用实时荧光定量PCR法,检测Sharpin在DUl45、PC-3和LNCaP3种常见的前列腺癌细胞株和RWPE．1正常前列腺上皮细胞株中的表达。同时采用免疫组织化学方法检测Sharpin在前列腺增生及前列腺癌组织中的表达,并探讨与临床病理特征的关系。结果Sharpin在3种前列腺癌细胞株中的mRNA水平（1．62±0．31,1．36±0．23,2．1±0．1）要明显高于正常前列腺上皮细胞RWPE-1（0．6±0．11）。免疫组织化学结果示Sharpin在前列腺癌组织中高表达,前列腺癌中的阳性表达率远远高于前列腺增生组织,平均染色得分也要远远高于前列腺增生组织。另外,Sharpin在前列腺癌组织中的表达与患者的Gleason评分和术前血清的t-PSA密切相关,均呈正相关（P〈0．05）。结论Sharpin可能是前列腺癌的肿瘤相关抗原,sharpin的表达可能具有评估前列腺癌患者病情、指导临床治疗方案的指导及判断预后及复发的作用。     

Prostasin基因在前列腺细胞株中表达情况研究

目的 探讨Prostasin在前列腺癌中的功能。方法 运用RT-PCR方法检测人前列腺增生细胞株BPH-1，无转移能力前列腺癌细胞株LNCaP，及有转移能力的前列腺癌细胞株PC-3、DU-145中Prostasin基闪表达情况。结果 Prostasin基斟在BPH-l和LNCaP中正常表达，在PC-3、DU-145中低表达。BPH-l中Prostasin的表达与DU-145和PC-3比较差异有显著性，同样LNCaP中Prostasin的表达与DU-145和PC-3比较差异亦有显著性（P〈0．01）。BPH-1与LNCaP之间表达差异无显著性，DU-145和PC-3之间表达差异亦无显著性，（P〉0.05）。结论 ProStasin可能是前列腺癌的转移抑制剂。     

Inducible expression of a prostate cancer-testis antigen, SSX-2, following treatment with a DNA methylation inhibitor

BACKGROUND: Active immunotherapies are one approach being developed as novel treatments for prostate cancer. Critical to the success of these therapies is the identification of appropriate target antigens. We have been seeking to identify immunologically recognized proteins, cancer-testis antigens (CTA) in particular, in patients with prostate cancer that would be rational target antigens. METHODS: Using a previously reported panel of 29 different CTA, we used sera from 98 patients with prostate cancer and 50 healthy male blood donor controls to detect CTA-specific IgG. We then further evaluated the expression of one antigen, SSX-2, in prostate cancer cell lines and tissues. RESULTS: We identified IgG specific for NY-ESO-1, LAGE-1, NFX-2, and SSX-2 in at least 1/98 individuals with prostate cancer. We demonstrated that SSX-2 is a prostate CTA, and its expression is associated with metastatic prostate cancer. In addition, we report that the treatment of at least two human prostate cancer cell lines with the DNA methylation inhibitor 5-aza-2'-deoxycytidine induced the expression of SSX-2. In contrast, treatment of a normal prostate epithelial cell line (RWPE-1) with 5-aza-2'-deoxycytidine did not induce SSX-2 expression. CONCLUSIONS: Our findings suggest that SSX-2 could be further pursued as an immunotherapeutic target in prostate cancer, and that treatment with 5-aza-2'-deoxycytidine could be exploited to modulate antigen expression in combination with immunotherapeutic approaches.