Tumor-associated autoantibodies correlate with poor outcome in prostate cancer patients treated with androgen deprivation and external beam radiation therapy.

Standard cancer treatments trigger immune responses that may influence tumor control. The nature of these responses varies depending on the tumor and the treatment modality. We previously reported that radiation and androgen-deprivation therapy (ADT) induce tumor-associated autoantibody responses in prostate cancer patients. This follow-up analysis was conducted to assess the relationship between autoantibody responses and clinical outcome. Patients with non-metastatic prostate cancer received external beam radiation therapy (EBRT) plus neoadjuvant and concurrent androgen deprivation. Treatment-induced autoantibodies were detected in almost a third of patients receiving combinatorial ADT and EBRT. Unexpectedly, patients that developed autoantibody responses to tumor antigens had a significantly lower 5-year biochemical failure-free survival (BFFS) than patients that did not develop an autoantibody response. Thus, tumor-reactive autoantibodies may be associated with increased risk of biochemical failure and immunomodulation to prevent autoantibody development may improve BFFS for select, high-risk prostate cancer patients receiving both ADT and EBRT.

CD20+ tumor-infiltrating lymphocytes have an atypical CD27- memory phenotype and together with CD8+ T cells promote favorable prognosis in ovarian cancer.

PURPOSE: Tumor-infiltrating lymphocytes (TIL), in particular CD8(+) T cells and CD20(+) B cells, are strongly associated with survival in ovarian cancer and other carcinomas. Although CD8(+) TIL can mediate direct cytolytic activity against tumors, the role of CD20(+) TIL is poorly understood. Here, we investigate the possible contributions of CD20(+) TIL to humoral and cellular tumor immunity. EXPERIMENTAL DESIGN: Tumor and serum specimens were obtained from patients with high-grade serous ovarian cancer. CD8(+) and CD20(+) TIL were analyzed by immunohistochemistry and flow cytometry. Immunoglobulin molecules were evaluated by DNA sequencing. Serum autoantibody responses to the tumor antigens p53 and NY-ESO-1 were measured by ELISA. RESULTS: The vast majority of CD20(+) TIL were antigen experienced, as evidenced by class-switching, somatic hypermutation, and oligoclonality, yet they failed to express the canonical memory marker CD27. CD20(+) TIL showed no correlation with serum autoantibodies to p53 or NY-ESO-1. Instead, they colocalized with activated CD8(+) TIL and expressed markers of antigen presentation, including MHC class I, MHC class II, CD40, CD80, and CD86. The presence of both CD20(+) and CD8(+) TIL correlated with increased patient survival compared with CD8(+) TIL alone. CONCLUSIONS: In high-grade serous ovarian tumors, CD20(+) TIL have an antigen-experienced but atypical CD27(-) memory B-cell phenotype. They are uncoupled from serum autoantibodies, express markers of antigen-presenting cells, and colocalize with CD8(+) T cells. We propose that the association between CD20(+) TIL and patient survival may reflect a supportive role in cytolytic immune responses.

A viral vaccine encoding prostate-specific antigen induces antigen spreading to a common set of self-proteins in prostate cancer patients.

PURPOSE: We previously reported a randomized phase II clinical trial combining a poxvirus-based vaccine encoding prostate-specific antigen (PSA) with radiotherapy in patients with localized prostate cancer. Here, we investigate whether vaccination against PSA induced immune responses to additional tumor-associated antigens and how this influenced clinical outcome. EXPERIMENTAL DESIGN: Pretreatment and posttreatment serum samples from patients treated with vaccine + external beam radiation therapy (EBRT) versus EBRT alone were evaluated by Western blot and serologic screening of a prostate cancer cDNA expression library (SEREX) to assess the development of treatment-associated autoantibody responses. RESULTS: Western blotting revealed treatment-associated autoantibody responses in 15 of 33 (45.5%) patients treated with vaccine + EBRT versus 1 of 8 (12.5%) treated with EBRT alone. SEREX screening identified 18 antigens, which were assembled on an antigen array with 16 previously identified antigens. Antigen array screening revealed that 7 of 33 patients (21.2%) treated with vaccine + EBRT showed a vaccine-associated autoantibody response to four ubiquitously expressed self-antigens: DIRC2, NDUFS1, MRFAP1, and MATN2. These responses were not seen in patients treated with EBRT alone, or other control groups. Patients with autoantibody responses to this panel of antigens had a trend toward decreased biochemical-free survival. CONCLUSIONS: Vaccine + EBRT induced antigen spreading in a large proportion of patients. A subset of patients developed autoantibodies to a panel of four self-antigens and showed a trend toward inferior outcomes. Thus, cancer vaccines directed against tumor-specific antigens can trigger autoantibody responses to self-proteins, which may influence the efficacy of vaccination.

Polyfunctional T-cell responses are disrupted by the ovarian cancer ascites environment and only partially restored by clinically relevant cytokines.

BACKGROUND: Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment. METHODOLOGY/PRINCIPAL FINDINGS: Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1ß, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited. CONCLUSIONS/SIGNIFICANCE: The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.

Castration induces autoantibody and T cell responses that correlate with inferior outcomes in an androgen-dependent murine tumor model.

We recently reported that hormone therapy induces antigen-specific autoantibody responses in prostate cancer patients. However, the contribution of autoantibody responses to clinical outcomes is unknown. We used an animal model to test the hypothesis that hormone therapy-induced immune responses may be associated with delayed tumor recurrence. Male DD/S mice bearing established tumors from the androgen-dependent Shionogi carcinoma line were castrated to induce tumor regression. Tumor-specific autoantibody responses were measured by immunoblot, and the underlying antigen was identified by serological screening of a cDNA expression library. T cell responses were assessed by immunohistochemistry and IFN-gamma ELISPOT. Following castration, 97% of mice underwent complete tumor regression. Of these, 72% experienced tumor recurrence 18-79 days postcastration, whereas the remaining 28% remained tumor-free for the duration of the experiment. In 55% of mice, castration induced autoantibody responses to an antigen identified as poly(A) binding protein nuclear 1 (PABPN1). Castration also induced PABPN1-specific T cell responses, which were highly correlated to autoantibody responses, and this was accompanied by dense infiltration of tumors by CD3+ T cells 1-2 weeks after castration. Unexpectedly, mice that developed autoantibody and T cell responses to PABPN1 showed a higher rate and shorter latency of tumor recurrence. In mice with recurrent tumors, T cell responses to PABPN1 were still detectable; however, T cell infiltrates were restricted to the peripheral stroma of tumors. In conclusion, castration-induced immune responses are associated with inferior outcomes in the Shionogi carcinoma model, raising concerns about the influence of treatment-induced immune responses on clinical outcomes in humans.

Tumor-infiltrating T cells correlate with NY-ESO-1-specific autoantibodies in ovarian cancer.

BACKGROUND: Tumor-infiltrating CD8+ T cells are correlated with prolonged progression-free and overall survival in epithelial ovarian cancer (EOC). A significant fraction of EOC patients mount autoantibody responses to various tumor antigens, however the relationship between autoantibodies and tumor-infiltrating T cells has not been investigated in EOC or any other human cancer. We hypothesized that autoantibody and T cell responses may be correlated in EOC and directed toward the same antigens. METHODOLOGY AND PRINCIPAL FINDINGS: We obtained matched serum and tumor tissue from 35 patients with high-grade serous ovarian cancer. Serum samples were assessed by ELISA for autoantibodies to the common tumor antigen NY-ESO-1. Tumor tissue was examined by immunohistochemistry for expression of NY-ESO-1, various T cell markers (CD3, CD4, CD8, CD25, FoxP3, TIA-1 and Granzyme B) and other immunological markers (CD20, MHC class I and MHC class II). Lymphocytic infiltrates varied widely among tumors and included cells positive for CD3, CD8, TIA-1, CD25, FoxP3 and CD4. Twenty-six percent (9/35) of patients demonstrated serum IgG autoantibodies to NY-ESO-1, which were positively correlated with expression of NY-ESO-1 antigen by tumor cells (r = 0.57, p = 0.0004). Autoantibodies to NY-ESO-1 were associated with increased tumor-infiltrating CD8+, CD4+ and FoxP3+ cells. In an individual HLA-A2+ patient with autoantibodies to NY-ESO-1, CD8+ T cells isolated from solid tumor and ascites were reactive to NY-ESO-1 by IFN-gamma ELISPOT and MHC class I pentamer staining. CONCLUSION AND SIGNIFICANCE: We demonstrate that tumor-specific autoantibodies and tumor-infiltrating T cells are correlated in human cancer and can be directed against the same target antigen. This implies that autoantibodies may collaborate with tumor-infiltrating T cells to influence clinical outcomes in EOC. Furthermore, serological screening methods may prove useful for identifying clinically relevant T cell antigens for immunotherapy.

Standard treatments induce antigen-specific immune responses in prostate cancer.

PURPOSE: Prostate tumors express antigens that are recognized by the immune system in a significant proportion of patients; however, little is known about the effect of standard treatments on tumor-specific immunity. Radiation therapy induces expression of inflammatory and immune-stimulatory molecules, and neoadjuvant hormone therapy causes prominent T-cell infiltration of prostate tumors. We therefore hypothesized that radiation therapy and hormone therapy may initiate tumor-specific immune responses. EXPERIMENTAL DESIGN: Pretreatment and posttreatment serum samples from 73 men with nonmetastatic prostate cancer and 50 cancer-free controls were evaluated by Western blotting and SEREX (serological identification of antigens by recombinant cDNA expression cloning) antigen arrays to examine whether autoantibody responses to tumor proteins arose during the course of standard treatment. RESULTS: Western blotting revealed the development of treatment-associated autoantibody responses in patients undergoing neoadjuvant hormone therapy (7 of 24, 29.2%), external beam radiation therapy (4 of 29, 13.8%), and brachytherapy (5 of 20, 25%), compared with 0 of 14 patients undergoing radical prostatectomy and 2 of 36 (5.6%) controls. Responses were seen within 4 to 9 months of initiation of treatment and were equally prevalent across different disease risk groups. Similarly, in the murine Shionogi tumor model, hormone therapy induced tumor-associated autoantibody responses in 5 of 10 animals. In four patients, SEREX immunoscreening of a prostate cancer cDNA expression library identified several antigens recognized by treatment-associated autoantibodies, including PARP1, ZNF707 + PTMA, CEP78, SDCCAG1, and ODF2. CONCLUSION: We show for the first time that standard treatments induce antigen-specific immune responses in prostate cancer patients. Thus, immunologic mechanisms may contribute to clinical outcomes after hormone and radiation therapy, an effect that could potentially be exploited as a practical, personalized form of immunotherapy.

Androgen-independent growth of LNCaP prostate cancer cells is mediated by gain-of-function mutant p53.

Mutations of p53 are common in hormone-refractory prostate cancer (CaP), suggesting the possibility that these mutations may be involved in the progression of CaP to androgen-independent (AI) growth. However, at present no direct evidence has been presented linking p53 mutations with AI growth of CaP. We established five stably transfected LNCaP cell lines: four containing gain-of-function (GOF) mutant p53 alleles (G245S, R248W, R273H, and R273C) and one containing a non-GOF p53 mutant allele (P151S). The four GOF p53 sublines were able to grow under androgen-depleted conditions, whereas the LNCaP parental line, vector-only line, and the non-GOF line were unable to grow. To investigate the mechanism of the AI growth displayed by the GOF p53 mutants, Western blotting or ELISA were used to examine the expression of the androgen receptor (AR), the AR-regulated prostate-specific antigen (PSA), as well as Akt and Bcl-2 under androgen-depleted conditions. On androgen ablation, the levels of AR decreased in the four GOF p53 sublines compared with the control lines. This decreased AR expression was accompanied by attenuated receptor activity, because a decrease in prostate-specific antigen levels compared with parental LNCaP cells was also observed. Levels of phosphorylated Akt increased in both the GOF p53 sublines and the control lines. Bcl-2 remains unchanged or showed reduced expression in all of the cell lines in the absence of androgen compared to the presence of androgen. These observations suggest that GOF p53 mutants mediate the AI growth of LNCaP cells in an AR-independent fashion, and that both Akt and Bcl-2 are not involved in this process.

Mutational analysis of patients with the diagnosis of choroideremia.

All reported mutations in the choroideremia (CHM) gene result in the truncation or complete absence of Rab escort protein 1 (REP1). Molecular analysis was carried out on 57 families diagnosed with CHM. Confirmation of the clinical diagnosis is important as end-stage CHM may be clinically similar to the end stages of other retinal degenerative diseases such as RP. The primary means of confirming the diagnosis of CHM is to sequence all 15 exons. An alternative method involves detection of the REP1 protein, as described in MacDonald et al. [1998]. A monoclonal antibody to REP1 does not detect truncated REP1 by immunoblot analysis, presumably due to instability and subsequent degradation of the truncated protein. This analysis provides relatively fast confirmation of the diagnosis, however, protein samples are not always available and are susceptible to degradation, affecting the accurate interpretation of results. CHM gene mutations were found in 54 of 57 families studied. The majority of mutations (>42%) were transitions and transversions. Complete deletions of the CHM gene and deletion/insertion mutations each accounted for almost 4% of the total, while over 9% had large intragenic and other partial deletions. Almost 28% of the mutations were deletions of fewer than 5 base pairs (bp) and almost 13% were splice site mutations. Despite the fact that mutations are found throughout the gene with no common mutation for the disorder, identical mutations have been characterized in unrelated individuals. The majority of these mutations are C to T transitions, changing an arginine residue (CGA) to a stop codon (TGA). Four of the five CGA codons in the CHM gene are sites of recurring mutations.

Complex functions of mutant p53 alleles from human prostate cancer.

BACKGROUND: Few studies have used multiple assays to examine the functionality of mutant p53 in prostate cancer (CaP). We employed seven functional assays to study 16 representative mutant p53 alleles, six from localized and ten from metastatic CaP. METHODS: Yeast assays were employed to determine loss of function (LOF), partial function (PF), and dominant-negative status. Assays using p53-null Saos2 cells were used to determine whether mammalian cells transfected with mutant p53 could up-regulate the MDR-1 or PCNA promoters, alter IL-6 expression or confer the ability to grow in soft agar. As a further test of gain of function (GOF), p53-null PC3 cells stably transfected with these mutant p53 alleles were examined for cell cycle distributions. RESULTS: All 16 mutant p53 alleles demonstrated either total or partial LOF. All but one allele also had at least one gain of function; however, the pattern of GOF was different for each mutant allele. Alleles derived from both localized and metastatic CaP had similar GOF characteristics; however, only alleles from metastatic disease had significantly increased S-phase fractions. CONCLUSIONS: Different mutant p53 alleles from CaP had different, complex functional profiles. The lack of predictable patterns for these alleles suggest that each mutation may uniquely affect p53 function.