Giovanni Sais

MAGE-C2/CT10 Protein Expression Is an Independent Predictor of Recurrence in Prostate Cancer

The cancer-testis (CT) family of antigens is expressed in a variety of malignant neoplasms. In most cases, no CT antigen is found in normal tissues, except in testis, making them ideal targets for cancer immunotherapy. A comprehensive analysis of CT antigen expression has not yet been reported in prostate cancer. MAGE-C2/CT-10 is a novel CT antigen. The objective of this study was to analyze extent and prognostic significance of MAGE-C2/CT10 protein expression in prostate cancer. 348 prostate carcinomas from consecutive radical prostatectomies, 29 castration-refractory prostate cancer, 46 metastases, and 45 benign hyperplasias were immunohistochemically analyzed for MAGE-C2/CT10 expression using tissue microarrays. Nuclear MAGE-C2/CT10 expression was identified in only 3.3% primary prostate carcinomas. MAGE-C2/CT10 protein expression was significantly more frequent in metastatic (16.3% positivity) and castration-resistant prostate cancer (17% positivity; p<0.001). Nuclear MAGE-C2/CT10 expression was identified as predictor of biochemical recurrence after radical prostatectomy (p = 0.015), which was independent of preoperative PSA, Gleason score, tumor stage, and surgical margin status in multivariate analysis (p<0.05). MAGE-C2/CT10 expression in prostate cancer correlates with the degree of malignancy and indicates a higher risk for biochemical recurrence after radical prostatectomy. Further, the results suggest MAGE-C2/CT10 as a potential target for adjuvant and palliative immunotherapy in patients with prostate cancer.

Differential Patterns of Large Tumor Antigen-Specific Immune Responsiveness in Patients with BK Polyomavirus-Positive Prostate Cancer or Benign Prostatic Hyperplasia

ABSTRACT The role of the polyomavirus BK (BKV) large tumor antigen (L-Tag) as a target of immune response in patients with prostate cancer (PCa) has not been investigated thus far. In this study, we comparatively analyzed humoral and cellular L-Tag-specific responsiveness in age-matched patients bearing PCa or benign prostatic hyperplasia, expressing or not expressing BKV L-Tag-specific sequences in their tissue specimens, and in non-age-matched healthy individuals. Furthermore, results from patients with PCa were correlated to 5-year follow-up clinical data focusing on evidence of biochemical recurrence (BR) after surgery (prostate specific antigen level of ≥0.2 ng/ml). In peripheral blood mononuclear cells (PBMC) from patients with PCa with evidence of BR and BKV L-Tag-positive tumors, stimulation with peptides derived from the BKV L-Tag but not those derived from Epstein-Barr virus, influenza virus, or cytomegalovirus induced a peculiar cytokine gene expression profile, characterized by high expression of interleukin-10 (IL-10) and transforming growth factor β1 and low expression of gamma interferon genes. This pattern was confirmed by protein secretion data and correlated with high levels of anti-BKV L-Tag IgG. Furthermore, in PBMC from these PCa-bearing patients, L-Tag-derived peptides significantly expanded an IL-10-secreting CD4+ CD25+(high) CD127− FoxP3+ T cell population with an effector memory phenotype (CD103+) capable of inhibiting proliferation of autologous anti-CD3/CD28-triggered CD4+ CD25− T cells. Collectively, our findings indicate that potentially tolerogenic features of L-Tag-specific immune response are significantly associated with tumor progression in patients with BKV+ PCa.

A HCMV pp65 polypeptide promotes the expansion of CD4+ and CD8+ T cells across a wide range of HLA specificities

Human cytomegalovirus (HCMV) can cause life‐threatening disease in infected hosts. Immunization with human leukocyte antigen (HLA)‐restricted immunodominant synthetic peptides and adoptive transfer of epitope‐specific T cells have been envisaged to generate or boost HCMV‐specific cellular immunity, thereby preventing HCMV infection or reactivation. However, induction or expansion of T cells effective against HCMV are limited by the need of utilizing peptides with defined HLA restrictions. We took advantage of a combination of seven predictive algorithms to identify immunogenic peptides of potential use in the prevention or treatment of HCMV infection or reactivation. Here we describe a pp65‐derived peptide (pp65340–355, RQYDPVAALFFFDIDL: RQY16‐mer), characterized by peculiar features. First, RQY‐16mer is able to stimulate HCMV pp65 specific responses in both CD4+ and CD8+ T cells, restricted by a wide range of HLA class I and II determinants. Second, RQY‐16mer is able to induce an unusually wide range of effector functions in CD4+ T cells, including proliferation, killing of autologous HCMV‐infected target cells and cytokine production. Third, and most importantly, the RQY‐16mer is able to stimulate CD4+ and CD8+ T‐cell responses in pharmacologically immunosuppressed patients. These data suggest that a single reagent might qualify as synthetic immunogen for potentially large populations exposed to HCMV infection or reactivation.

Expression of indoleamine 2,3-dioxygenase induced by IFN-γ and TNF-α as potential biomarker of prostate cancer progression.

Inflammation has been suggested to play an important role in onset and progression of prostate cancer (PCa). Histological analysis of prostatectomy specimens has revealed focal inflammation in early stage lesions of this malignancy. We addressed the role of inflammatory stimuli in the release of PCa-specific, tumor-derived soluble factors (PCa-TDSFs) already reported to be mediators of PCa morbidity, such as indoleamine 2,3-dioxygenase (IDO) and interleukin (IL)-6. Inflammation-driven production and functions of PCa-TDFSs were tested “in vitro” by stimulating established cell lines (CA-HPV-10 and PC3) with IFN-γ or TNF-α. Expression of genes encoding IDO, IL-6, IFN-γ, TNF-α, and their receptors was investigated in tumor tissues of PCa patients undergoing radical prostatectomy, in comparison with benign prostatic hyperplasia (BPH) specimens. IFN-γ and TNF-α-treatment resulted in the induction of IDO and IL-6 gene expression and release in established cell lines, suggesting that the elicitation of PCa-TDSFs by these cytokines might contribute to progression of cancer into an untreatable phenotype. An analysis based on timing of biochemical recurrence revealed the prognostic value of IDO but not IL-6 gene expression in predicting recurrence-free survival in patients (RFS) with PCa. In addition, a urine-based mRNA biomarker study revealed the diagnostic potential of IDO gene expression in urines of men at risk of PCa development.

Implication of vascular endothelial growth factor A and C in revealing diagnostic lymphangiogenic markers in node-positive bladder cancer

Several lymphangiogenic factors, such as vascular endothelial growth factors (VEGFs), have been found to drive the development of lymphatic metastasis in bladder cancer (BCa). Here, we have analyzed the gene expression of lymphangiogenic factors in tissue specimens from 12 non-muscle invasive bladder cancers (NMIBC) and 11 muscle invasive bladder cancers (MIBC), considering tumor and tumor-adjacent normal bladder areas obtained from the same organs. We then compared the results observed in patients with those obtained after treating human primary bladder microvascular endothelial cells (MEC) with either direct stimulation with VEGF-A or VEGF-C or by co-culturing (trans-well assay) MEC with bladder cancer cell lines varying in VEGF-A and VEGF-C production based on tumor grade. The genes of three markers of lymphatic endothelial commitment and development (PDPN, LYVE-1 and SLP-76) were significantly overexpressed in tissues of MIBC patients showing positive lymphovascular invasion (LVI+), lymph node metastasis (Ln+) and tumor progression. Their expression was also significantly enhanced either after direct stimulation of MEC by VEGF-A and VEGF-C or in the trans-well assay with each bladder cancer cell line. SLP-76 showed the highest gene expression. Both VEGF-A and VEGF-C also enhanced the expression of SLP-76 protein in MEC. However, a correlation between increase of SLP-76 gene expression and the ability of MEC to migrate could only be seen after induction by VEGF-C. The significant expression of SLP-76 in LVI+/Ln+ progressive MIBC and its overexpression in MEC after VEGF-A and VEGF-C stimulation suggest the need to develop this regulator of developmental lymphangiogenesis as a diagnostic tool in BCa.

IMPAIRED IMMUNE PROINFLAMMATORY CYTOKINE PROFILING IN PROSTATE CANCER PATIENTS UPON INDUCTION USING PEPTIDES WITHIN POLYOMAVIRUS BK-P53 BINDING REGIONS

Prostate cancer (PCa) is a leading cause of cancer death in men. Nearly one third of annually new diagnosed cancers are prostate tumors. A contemporary model for prostate cancer induction and progression should include the potential contribution of inflammation to the development of preneoplastic or neoplastic lesions. Human Polyomavirus BK (BKV) has been associated to pre-early stages of cancer in the urinary tract and it is postulated to play an important role in the pathogenesis of prostate cancer. The BKV oncogenic effect appears to be highly associated to the activity of its main regulatory protein Large Tumor antigen (L-Tag) because of this antigen capability to bind and inactivate the products of tumor suppressor genes. The involvement of BKV L-Tag in the alteration of critical pathways of the human cell cycle together with the detection and expression of BKV L-Tag sequences in preneoplastic prostate tissues prompted us to investigate the role of this viral antigen as target of cellular immune surveillance in prostate cancer. Our previous results suggest that specific HLA-A*0201-associated BKV L-Tag candidate peptides nesting within regions responsible for L-Tag binding to p53 could efficiently be used to test the immune response in HLA-A*0201+ BKV seropositive donors. In this study we propose a comprehensive characterization of an epitope specific T cell response against BKV L-Tag in BKV-experienced patients bearing either PCa or benign prostate hyperplasia (BPH), as compared to gender-matched healthy donors. The specific aim of this study is to test the hypothesis whether an inefficient immune responsiveness to antigens specifically expressed by oncogenic viruses located in the urinary tract may define a role for BKV L-Tag immune surveillance in organ specific tumorigenesis and subsequent neoplastic progression. Furthermore, we want to test if a systemic boosting of T cells from prostate cancer patients using immunogenic peptides within BKV L-Tag regions prevalently binding products of tumor suppressor genes (i.e. p53) and highly expressed in cancer cells would implement a T cell immune response in favor to a pro-inflammatory immune activity. We thus want to fully characterize the functional features of BKV specific T cell immune response elicited by L-Tag in patients bearing PCa.

Abstract 4278: IFN-γ and TNF-α favor the progression of prostate cancer in distinct ways

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction Although screening methods for prostate cancer (PCa) have substantially improved, the outcome for patients with advanced PCa remains poor. The elucidation of the molecular mechanism that drives the progression from a slow-growing, organ-confined tumor to a highly invasive and castration-resistant cancer (CRPC) is therefore important. Although a causal correlation between inflammation and PCa has not been established, literature suggests that inflammation might play an important role in its onset and progression. The aim of this study is to investigate the effect of inflammation on progression of PCa. We have evaluated the effect of IFN-γ and TNF-α on proliferation, migration, and invasion of PCa cells, and potentially identified the molecular mechanisms underlying these effects. Materials and Methods The CA-HPV-10 and PC3 cell-lines were stimulated with 300U/ml of IFN-γ or TNF-α. Relative gene expressions were quantified by RT-PCR. Protein quantification was performed by HPLC or ELISA. Functionality of cells was tested using: acridine orange (viability), WST-1 (proliferation), Tscratch (migration) and the Chemicon Cell Invasion Assay (invasion). Results IFN-γ induced de-novo expression of both IDO1 and IDO2 genes in CA-HPV-10 and their over expression in PC3. TNF-α induced over-expression of MMP9, TNF- ≤ and IL-6 in CA-HPV-10, and of 7 genes in PC3 (MMP9, IDO1 and IDO2, TNF-α, IL-6, IL-1α and ROS1). IFN-γ and TNF- ≤ do neither induce apoptosis nor affect the proliferation of both cell-lines. TNF-α significantly increases migraton of metastatic cells (p<0.001) and markedly affects the invasiveness of both CA-HPV-10 and PC3 (increase of 32% and 51%, respectively), whereas IFN-γ has no or little effect. Discussion IFN-γ induces IDO1 and IDO2, whose roles in modulating immune tolerance have been extensively investigated. Conversely, IFN-γ does not affect other mechanism of cancer progression, such as cell migration and invasion. TNF-α, on the other hand, induces stable over-expression of MMP9 in localized cancer, and of 6 other genes in metastatic cells. MMP9 plays an important role in proliferation, migration and invasion and its elevated levels have been reported in many tumors. These notions could explain our findings showing that TNF-α enhances PCa cell migration and invasion. Furthermore TNF-α enhances the ability of cancer cells to actively maintain a pro-inflammatory microenvironment, due to the over-production of TNF-α, IL-6 and IL-1α. High levels of these cytokines in patients with advanced PCa have been hypothesized to play an important role in the onset of a CRPC. In conclusion, IFN-γ and TNF-α favor cancer progression by enhancing its malignant features in distinct ways. IFN-γ favors tumor immune-escape by inducing over-expression of IDO1 and IDO2, while TNF-α enhances PCa cell migration and invasion, sustains the active maintenance of a pro-inflammatory microenvironment and favors the onset of a CRPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4278. doi:1538-7445.AM2012-4278

Abstract 556: L-Tag-driven systemic immune regulatory profile as a potential signature for polyomavirus BK involvement in prostate cancer

Background: Polyomavirus BK (BKV) large tumor antigen (L-Tag) contributes to oncogenesis by regulating crucial pathways of human cell cycle when non-permissive cells are infected. Therefore, L-Tag has been identified as an important target of immune surveillance in L-Tag expressing tumors. The role of L-Tag as a target of tumor immune surveillance in prostate cancer (PCa) patients has not been investigated so far. In this study we thus analyzed the immunological profile exerted by L-Tag peptide-specific induction in newly diagnosed PCa patients by means of BKV L-Tag serology and local molecular testing, and compared it to age-matched benign prostatic hyperplasia (BPH) patients. Methods: We enrolled 60 newly diagnosed PCa patients and 50 age-matched BPH. Detection of BKV L-Tag DNA was carried out in random punches from tumor areas of surgically excised specimens. BKV L-Tag seroprevalence was determined by enzyme immunoassay (EIA). Systemic immune-regulatory activity was tested upon BKV L-Tag peptide-pool induction and analyzed using cytokine gene expressions (qrt-PCR), antigen-specific regulatory T cell (Treg) characterization (flow cytometry) and immune-regulatory protein release (multiplex fluorescent bead immunoassay). Data were correlated to 5-year follow-up clinical information. Five-year follow-up after surgery was completed on 48/60 PCa patients. Biochemical recurrence (BR) with early censoring was defined as the first PSA ≤ 0.2ng/ml. Results: In peripheral blood mononuclear cells (PBMCs) from PCa patients with evidence of biochemical recurrence (BR) and BKV L-Tag positive lesions, BKV L-Tag peptide-pool, but not the Epstein Barr/Influenza virus/Cytomegalovirus (CEF) peptide-pool, induced a particular gene profile, which was characterized by high expression of IL-10 and TGFβ-1 and a low expression of IFN-γ and TNF-α genes. This signature was confirmed by protein secretion data and correlated with levels of anti-BKV L-Tag IgG activity. Furthermore, in these PCa patients the L-Tag peptide pool significantly boosted IL-10 secreting CD4 + CD25 +(high) /FoxP3 +(high) /CD103 + T cells exerting suppressive functions on autologous effectors (CD4 + , CD25 − and CD8 + T cell fractions) by reducing cell proliferation and IFN-γ production. Conclusion: Significant associations between immune regulatory activity exerted by BKV L-Tag induction, BKV L-Tag DNA positive tumor lesions and IgG activity against L-Tag were observed in PCa patients. In detail, BR+ PCa patients shared features such as functional BKV L-Tag specific immune regulatory profile with marked BKV L-Tag positive lesion and strong L-Tag-specific IgG activity, thereby hinting at a potential BKV involvement in prostate cancer progression. Our findings are thus consistent with a systemic BKV L-Tag-related tolerogenic environment governing prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 556. doi:1538-7445.AM2012-556

Abstract 2538: The anti-proliferative effect of PUVA treatment on human bladder cancer cell lines

Introduction and Objectives: Although immunotherapy and chemotherapy are the most used intravesical treatments for non-muscle-invasive bladder cancer (NMIBC), progression and recurrence rates are high. So far, several attempts have been done to hire a safer and more efficient treatment. Psoralen (a photoreactive chemical substance) metabolites followed by Ultra Violet A (UVA) irradiation (PUVA therapy) have been previously used to treat a variety of diseases. UVA radiation forms covalent bonds between psoralen molecule and pyrimidine bases of DNA to create interstrand cross-links, which blocks DNA replication, thereby inhibiting cell proliferation. In addition, PUVA therapy leads to apoptosis and local immune reaction. The aim of this study was to evaluate the anti-proliferative effects of PUVA treatment on human bladder cancer cell lines as the first step of further investigations, in order to consider it as a new candidate for NMIBC treatment. Material and Methods: Human bladder cancer cell lines (T24, grade III and RT4, grade I) were cultured in vitro, treated with 500 ng/ml 8-methoxypsoralen (8-MOP, a natural psoralen metabolite) and incubated in the dark room for 45 minutes. Cells were then irradiated with UVA (365 nm) at different doses (0, 0.5, 2 and 5 J/cm2). After 24- or 48-hour PUVA treatment, cells were counted to measure cell growth. MTT assay was performed in order to analyze cell viability and BrdU incorporation assay to evaluate the anti-proliferative effect of PUVA treatment. Cells were also stained with Annexin V-FITC and observed under fluorescence microscope in order to measure the amount of apoptotic cells. Results: Both T24 and RT4 cells treated with 2 and 5 J/cm2 UVA showed a significant decrease (P In MTT assay, PUVA-treated RT4 cells significantly reduced viability with 0.5, 2 and 5 J/cm2 UVA-treatments, as compared to no UVA-treatment. Differently, T24 cells showed a significant reduction of viability with only 5 J/cm2 UVA treatment. BrdU incorporation assay in both cell lines revealed a significant reduction of cell proliferation after PUVA treatment with all the three different UVA doses in comparison to no UVA treatment. Fluorescent microscopic observation of cell lines stained using Annexin V-FITC showed an increased number of apoptotic cells among those particularly treated with 2 and 5 J/cm2 UVA doses, as compared to no UVA-treated cells. Conclusions: PUVA treatment, mostly at higher UVA doses, has a significant anti-proliferative effect on human bladder cancer cell lines by inhibiting cell growth, reducing cell viability and inducing apoptosis. These results suggest a possible relevant use of PUVA therapy for the treatment of NMIBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2538. doi:10.1158/1538-7445.AM2011-2538

Abstract 1360: The role of inflammatory stimuli in modulating tumor derived factor expression in metastatic prostate cancer microenvironment

Tumor microenvironment is the battlefield were not well defined relationships between oncogenesis and immune surveillance to cancer take place. Upon transformation due to accumulation of several genetic mutations and epigenetic alterations, tumor cells proceed to either their full elimination or progression to overt cancer depending on immunity fitness. In particular, tumor escape occurs through the secretion of different tumor derived factors (TDFs) with immune suppressive properties, such as indoleamine, 2-3 dioxygenase (IDO), interleukin (IL) 10, transforming growth factor beta (TGF-β), and/or cytokines relevant in modulating the TDFs network. Among them, our interest focused on those microenvironmental modifiers that have been reported as possible mediators of prostate cancer (PCa) morbidity (IDO, IL-6, TGF-β). For this study, two prostate cancer cell-lines with invasive (CA-HPV-10) and metastatic behavior (PC3) were used. After culturing upon stimulation over 24 hours with IFN-γ and TNF-α, cells were harvested, total RNA extracted and transcribed into cDNA in order to perform gene expression by using qrt-PCR. As housekeeping genes, either rRNA18S or β-actin was tested. A 2-ΔΔCt methods was used to compute fold changes among experimental conditions. IDO-1 was constitutively higher expressed in bone metastases (20fold) than in localized cancer (2fold) and the finding was inversely correlated to serine protease inhibitor maspin expression (8-10fold less in PC3) while c-myc, relevantly expressed in both cell lines, was not modulated over time. A significant higher expression of IL-10 and IL-6 (15fold and 20fold, respectively) was detected in PC3 while there were no variations for TNF-α and TGF-β, although a constitutive strongest expression was observed for IL-6 and TGF-β in both cell-lines, as compared to the other cytokines (2000fold). IL-2 and IL-4 gene expression was almost negligible. Upon 12 hours stimulation with IFN-γ, an inducible higher amount of IDO-1 was detected (450fold) over the constitutive level but at a lesser extent for IDO-2 (20fold). However, the fold change seen between both enzymes at a constitutive expression was proportionally maintained. Conversely, TNF-α treatment produced a lower increase of expression for both IDO (5-15fold), as compared to constitutive values but a significant modulation for IL-6 over 24 hours (8fold over the baseline). Factors involved in immune suppressions such as IDO, IL-10, TGF-β and most likely mediators for PCa morbidity, also including IL-6, appear to be expressed at higher levels during PCa progression. Furthermore, their expression is even more increased in a microenvironment characterized by inflammatory stimuli. An extended analysis on PCa patients might better inform on the role that inflammation plays in TDFs expression and activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1360.