In the current report, we set out to evaluate serum antibody responses to a panel of prostate tissue- and prostate cancer-associated antigens in order to determine if a subset of these antigens were commonly recognized in patients with prostate cancer, and whether higher numbers of antigens were recognized in patients with more advanced disease. In an initial small screen we identified that immune responses to proteins originally identified as immunological targets in patients with chronic prostatitis were commonly recognized in patients with advanced prostate cancer relative to volunteer male blood donors. This suggested to us that many of these prostatitis antigens represent true immunologically recognized antigens of the prostate, many of which are recognized with the development of prostate cancer. Our subsequent studies specifically evaluated whether patients with prostate cancer of earlier stage, and patients with prostatitis, had IgG responses to these antigens, and whether responses to multiple antigens was more frequent with later stages of disease. Overall we found that immune responses to at least one member of a subset of 23 of these antigens were detectable as frequently in patients with early stage disease as in patients with advanced, metastatic disease. In addition, immune responses were as frequently detected in patients with clinical prostatitis, however were uncommon in sera from male subjects with other types of cancer. Responses to multiple members of this panel were not more frequent in patients with more advanced prostate cancer than patients with prostatitis. This latter observation may be related to the group of antigens prioritized, associated primarily with prostatitis, and not to gene products that might only be expressed in advanced tumors. These findings suggest an association between prostate tissue inflammation and cancer, and at least with this group of antigens, suggest that “antigen spread” does not necessarily occur with increased tumor burden.
Chronic inflammation is highly associated with the development of several solid tumors, notably lung and colorectal cancers. At present, much experimental and epidemiological evidence also suggests that chronic inflammation may similarly drive the development of prostate cancers (19
). Tumor-infiltrating lymphocytes are commonly seen in prostate tumor specimens, and chronic inflammatory cells are commonly seen adjacent to the earliest premalignant lesions of the prostate (23
). Moreover, in rodent models chronic prostatitis appears to drive the development of prostate tumors (24
). Viral and bacterial pathogens have been cited as possible etiologic agents for human prostate inflammation and cancer, however a definitive causal link has not been established (25
). Human prostate-infiltrating lymphocytes, obtained at the time of surgery, have been demonstrated to be oligoclonal, suggesting these lymphocytes may recognize tissue-specific antigens (26
). Our current findings, while not suggesting causality, suggest an association between chronic inflammatory conditions such as prostatitis and prostate cancer, or at least that common targets of a prostate-associated immune response can be recognized in these disorders. Our findings further suggest that there may be a subset of common antigens expressed by the prostate, recognition of which can be shared by multiple individuals. While the detection of these IgG responses itself does not appear to be highly sensitive or specific as a test to distinguish prostate cancer from patients with prostatitis, the evaluation of IgG responses to prostate-associated antigens could potentially identify patients in a premalignant inflammatory state at risk for developing prostate cancer. We plan to test this hypothesis using sera obtained from patients without a history of prostatitis but who developed prostate cancer within several years, suggesting this could be developed as a test to identify subjects in a premalignant stage at risk for developing prostate cancer.
Other groups have sought to identify antibody responses to prostate tissue antigens as a means of prostate cancer detection. In reports by Wang and Bradford, the investigators identified autoantibodies specific for peptides expressed by prostate cancer tissue using phage-encoded epitope screening (15
). They reported that the specificity and sensitivity for prostate cancer diagnosis exceeded that obtained with standard serum PSA screening using phage-encoded peptides derived from these screens. In separate reports, this same investigator group has evaluated antibody responses to individual proteins, including huntingtin-interacting protein 1 (HIP1) (29
) and alpha-methylacyl-CoA racemase (AMACR) (30
), and demonstrated that antibody responses detectable to these proteins are highly specific for prostate cancer. Our findings support the findings of these groups, and add further feasibility to the general field of antibody-based diagnostics. However, given that our current panel of antigens was chosen from prior studies, the prioritized antigens identified here are not entirely prostate-specific in terms of expression. Notwithstanding, their recognition appears to be biased towards patients with prostate disease, given that they were not similarly recognized in sera from patients with other malignancies. This is not entirely unexpected, as antibodies to ubiquitously expressed antigens are common in patients with autoimmune disorders such as systemic lupus erythematosis (31
). If this panel of antigens were to be developed as a diagnostic test for prostate cancer, future studies would explore the frequency of recognition of these antigens using larger numbers of sera from patients with other malignancies.
Our demonstration of antibody responses to antigens identified as prostatitis antigens, and other antigens similarly recognized by patients with prostatitis, suggests that IgG responses to many antigens might develop very early in the transformation process. In addition, most groups assessing immune responses to prostate-associated antigens have used control samples from normal male control blood donors or patients with benign prostatic hypertrophy. Our findings suggest that sera from patients with prostatitis should be considered in these studies as well, since antibody responses in this nonmalignant population are detectable, including responses to cancer-testis antigens that would be predicted to be tumor specific. In any case, the association of immune responses to defined antigens with a premalignant inflammatory state might be useful for identifying patients at risk for developing cancer, potentially well before an elevation of a serum protein such as PSA could be detected. If this is true, this concept of identifying antigens recognized in inflammatory tissue states could have application to the other malignancies associated with chronic inflammation for which serum-based tests are not available.
Advantages of the phage immunoblot approach we describe here are the ease of transfection and protein expression in bacteria, particularly for novel proteins for which there are no available reagents. In addition, the ability to simultaneously evaluate IgG responses to multiple proteins at once from a particular serum sample is an advantage over traditional ELISA. However, the sensitivity of this approach is lower than ELISA, and in fact we did not detect IgG responses to antigens such as PSA or AR LBD in individual sera samples in which we could identify low level IgG by ELISA (data not shown). In addition, background reactivity to E. coli produced regional variations on some membranes that made densitometry evaluation difficult. For defining individual antigen reactivity as strictly “positive” or “negative” we relied on visual interpretation of replicates which significantly adds to the time and potential subjectivity of the interpretation. Notwithstanding, we believe this method provides a robust means to prioritize antigens for which more sensitive methods could be developed.
Finally, the identification of antibody responses to multiple prostate-associated proteins provides us a potential tool for the evaluation of immune responses to the prostate. For example, in addition to antigen-specific immunotherapies, many new immune-based therapies have entered clinical testing for which there is not a defined target antigen. In the case of prostate cancer, agents such as cell-based vaccines and T-cell checkpoint inhibitors have shown evidence of anti-tumor effect in clinical trials (32
). A challenge in the development of these therapies, however, has been the absence of biomarkers for tumor-specific immunological effect that are associated with clinical responses. Studies with anti-CTLA-4 monoclonal antibodies, in particular, have sought to identify whether amplification of other T-cell co-stimulatory molecules (35
), or antibodies to defined antigenic tumor-associated proteins (33
), might be useful as biomarkers. For whole cell tumor vaccines where there is not a specific defined antigen being targeted, surrogate antigens known to be expressed by the tumor vaccine have been used as a means of monitoring immune responses from the vaccine (37
). The use of immunologically-recognized surrogate antigens, including HER-2/neu, MUC1 and p53, has been possible in the case of breast cancer where IgG responses to these antigens have been identified. In the case of prostate cancer, however, there has not been a defined panel of commonly recognized antigenic proteins. Most of the antigens identified in this report are not specifically expressed in prostate tissue, and the cancer-testis antigens identified here are recognized in sera from patients with different malignancies. Nonetheless, the antigens identified might still serve as a panel of gene products commonly associated with prostate inflammation. Future studies will explore whether IgG responses to other members of this panel are similarly recognized in other malignancies or other inflammatory conditions. As new immune-based and immune-modulating agents are developed, it will be important to establish tools able to define whether immune responses to specific tumors are elicited. The ability to rapidly evaluate antibody responses to a panel of commonly recognized antigens, such as we report here, before or after treatment could be useful in these determinations.