SHORT REPORT Antibodies against the cancer-testis antigen CTSP-1 are frequently found in prostate cancer patients and are an independent prognostic factor for biochemical-recurrence Raphael B. Parmigiani 1 , Fabiana Bettoni 1 , Daniela M Grosso 1 , Ademar Lopes 2 , Isabela W. Cunha 2 , Fernando A. Soares 2 , Andr e L. Carvalho 2 , Francisco Fonseca 2 and Anamaria A. Camargo 1 * 1 Laboratory of Molecular Biology and Genomics, Ludwig Institute for Cancer Research, Hospital Alem~ ao Oswaldo Cruz, S ~ ao Paulo, SP, Brazil 2 Hospital A.C.Camargo, S ~ ao Paulo, SP, Brazil Cancer-testis (CT) antigens are immunogenic proteins expressed in normal gametogenic tissues and in different types of tumors. CTSP-1 is a CT antigen frequently expressed in prostate tumors, and capable of eliciting humoral response in prostate cancer patients. Here, we analyzed the presence of anti-CTSP-1 antibod- ies in 147 patients with localized prostate cancer and determined its prognostic value for predicting biochemical-recurrence after radical prostatectomy. Anti-CTSP-1 antibodies were detected in 25% of the patients and a significant correlation (p 5 0.017) between CTSP-1 protein expression and the presence of specific humoral response was observed. No association was found between the presence of antibodies and the pathological variables analyzed. On univariate analysis, patients without antibodies against CTSP-1 had a lower biochemical-recurrence free survival than did those with anti-CTSP-1 antibodies, although the differ- ence between the groups was not statistically significant (57 vs. 75%, p 5 0.075). However, the presence of antibodies against CTSP-1 was significantly associated with a better prognosis in patients with higher Gleason score (36 vs. 80%, p 5 0.028). On multivariate analysis, antibodies against CTSP-1 were associated with a better prognosis (Hazard ratio 5 0.41, 95% IC 0.18–0.90 p 5 0.039), being the third most powerful prognostic factor among Gleason score and preoperative PSA levels. CTSP-1 should be considered a promising candidate for prostate cancer immuno- therapy, since it is frequently expressed in prostate tumors and ca- pable of eliciting humoral immune response in prostate cancer patients. Our results also suggest that humoral response against CTSP-1 could be used as a prognostic marker, especially among patients with a high Gleason score. ' 2008 Wiley-Liss, Inc. Key words: cancer/testis antigen; CTSP-1; humoral response; immunotherapy; prostate cancer Prostate cancer is the most commonly diagnosed malignancy, and the second leading cause of cancer-related death in men in the United States. 1 Radical retropubic prostatectomy (RRP) is the most common and effective treatment for clinically localized pros- tate cancer. However, 35% of prostate cancer patients submit- ting to RRP will have biochemical-recurrence of the disease within 10 years after surgery, as defined by an increase in Prostate Specific Antigen (PSA) level to >0.2 ng/mL. 2 Subsequently, these patients will develop clinical relapse of the disease, typically with the development of bone metastases. Biochemical-recurrence after RRP has been associated with multiple factors including preoperative PSA level and velocity, clinical stage, Gleason score, level of extracapsular extension, seminal vesicle invasion, pelvic lymph node status and surgical margin status and at present there is no cure for metastatic prostate cancer. 3,4 However, due to the significant clinical heterogeneity of the disease, these factors are far from accurate in identifying patients at high risk for cancer progression and the characteriza- tion of novel biomarkers to predict biochemical-recurrence, as well as the development of targeted therapies for prostate cancer are, thus, of considerable interest. 5–8 Cancer/testis (CT) antigens are proteins expressed in normal gametogenic tissues and in different types of tumors. 9,10 Although it seems to exist an association between CT antigen expression and more aggressive tumors, this association varies according to antigen and tumor type and the potential use of CT antigens as tu- mor markers remains poorly explored. 11 However, most CT anti- gens are highly immunogenic, eliciting both humoral and cellular immune responses in cancer patients. These antigens are not im- munogenic in normal tissues, as their expression is restricted to MHC class I-negative germ cells. Thus, CT antigens are consid- ered ideal targets for cancer immunotherapy. 9,10,12 We have recently described a novel CT antigen, named CTSP- 1, which is aberrantly expressed in 58% of prostate tumors and ca- pable of eliciting a humoral immune response in 20% of prostate cancer patients. 13 In the present study, we analyzed CTSP-1 pro- tein expression and the presence of antibodies against CTSP-1 in a larger population of prostate cancer patients with clinically local- ized disease. Our results suggest that CTSP-1 can be considered a promising target for prostate cancer immunotherapy, since it is frequently expressed in localized prostate tumors and is capable of eliciting humoral immune response in a significant fraction of prostate cancer patients. We also demonstrated that the presence of anti-CTSP-1 antibodies in the plasma of prostate cancer patients is associated with a better prognosis and could be used as a prognostic marker for biochemical-recurrence in these patients. Patients and methods Patients The study included 147 consecutive prostate cancer patients who underwent RRP with simultaneous bilateral pelvic lymphade- nectomy at the Urology Division of the Pelvic Surgery Depart- ment of the Hospital do Cancer, S~ ao Paulo, Brazil, between No- vember 1998 and July 2005. All cases had clinically localized dis- ease as suggested by elevated PSA serum levels or palpable nodules on digital rectal examination, and then confirmed by transrectal ultrasound-guided needle biopsy of the prostate. Patients with a second nonprostate primary tumor, or who were submitted to radiotherapy before the surgery, were excluded from this study. Twenty-two patients received neoadjuvant androgen This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0020-7136/suppmat. Grant sponsors: The Ludwig Institute for Cancer Research (LICR), the Fundac ¸~ ao de Amparo a Pesquisa do Estado de S~ ao Paulo (FAPESP), Con- selho Nacional de Desenvolvimento Cient  ıfico e Tecnol ogico (CNPq). Raphael B. Parmigiani’s current address is: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. *Correspondence to: Laboratory of Molecular Biology and Genomics, Ludwig Institute for Cancer Research, Hospital Alem~ ao Oswaldo Cruz, Rua Jo~ ao Juli ~ ao 245, 01323-930 S~ ao Paulo, SP, Brazil. Fax: 155-11-32847821. E-mail: anamaria@compbio.ludwig.org.br Received 22 May 2007; Accepted after revision 23 November 2007 DOI 10.1002/ijc.23369 Published online 23 January 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 122, 2385–2390 (2008) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer