Obinutuzumab Pretreatment as a Novel Approach to Mitigate Formation of Anti-Drug Antibodies Against Cergutuzumab Amunaleukin in Patients with Solid Tumors.

PURPOSE: The immunocytokine cergutuzumab amunaleukin (CEA-IL2v) showed manageable safety and favorable pharmacodynamics in phase I/Ib trials in patients with advanced/metastatic carcinoembryonic antigen-positive (CEA+) solid tumors, but this was accompanied by a high incidence of anti-drug antibodies (ADA). We examined B-cell depletion with obinutuzumab as a potential mitigation strategy. EXPERIMENTAL DESIGN: Preclinical data comparing B-cell depletion with rituximab versus obinutuzumab are summarized. Substudies of phase I/Ib trials investigated the effect of obinutuzumab pretreatment on ADA development, safety, pharmacodynamics, and antitumor activity of CEA-IL2v ± atezolizumab in patients with advanced/metastatic or unresectable CEA+ solid tumors who had progressed on standard of care. RESULTS: Preclinical data showed superior B-cell depletion with obinutuzumab versus rituximab. In clinical studies, patients received CEA-IL2v monotherapy with (n = 16) or without (n = 6) obinutuzumab pretreatment (monotherapy study), or CEA-IL2v + atezolizumab + obinutuzumab pretreatment (n = 5; combination study). In the monotherapy study, after four cycles (every 2 weeks treatment), 0/15 evaluable patients administered obinutuzumab pretreatment had ADAs versus 4/6 patients without obinutuzumab. Obinutuzumab pretreatment with CEA-IL2v monotherapy showed no new safety signals and pharmacodynamic data suggested minimal impact on T cells and natural killer cells. Conversely, increased liver toxicity was observed in the combination study (CEA-IL2v + atezolizumab + obinutuzumab pretreatment). CONCLUSIONS: These preliminary findings suggest that obinutuzumab pretreatment before CEA-IL2v administration in patients with CEA+ solid tumors may be a feasible and potent ADA mitigation strategy, with an acceptable safety profile, supporting broader investigation of obinutuzumab pretreatment for ADA mitigation in other settings.

A Phase I Study of the Pan-Notch Inhibitor CB-103 for Patients with Advanced Adenoid Cystic Carcinoma and Other Tumors.

PURPOSE: CB-103 selectively inhibits the CSL-NICD (Notch intracellular domain) interaction leading to transcriptional downregulation of oncogenic Notch pathway activation. This dose-escalation/expansion study aimed to determine safety, pharmacokinetics, and preliminary antitumor activity. EXPERIMENTAL DESIGN: Patients ≥18 years of age with selected advanced solid tumors [namely, adenoid cystic carcinoma (ACC)] and hematologic malignancies were eligible. CB-103 was dosed orally in cycles of 28 days at escalating doses until disease progression. Notch-activating mutations were required in a dose confirmatory cohort. Endpoints included dose-limiting toxicities (DLT), safety, tumor response, pharmacokinetics, and pharmacodynamics. Exploratory analyses focused on correlates of Notch and target gene expression. RESULTS: Seventy-nine patients (64, 12 dose-escalation cohorts; 15, confirmatory cohort) enrolled with 54% receiving two or more lines of prior therapy. ACC was the dominant tumor type (40, 51%). Two DLTs were observed [elevated gamma-glutamyl transferase (GGT), visual change]; recommended phase II dose was declared as 500 mg twice daily (5 days on, 2 days off weekly). Grade 3-4 treatment-related adverse events occurred in 15 patients (19%), including elevated liver function tests (LFTs), anemia, and visual changes. Five (6%) discontinued drug for toxicity; with no drug-related deaths. There were no objective responses, but 37 (49%) had stable disease; including 23 of 40 (58%) patients with ACC. In the ACC cohort, median progression-free survival was 2.5 months [95% confidence interval (CI), 1.5-3.7] and median overall survival was 18.4 months (95% CI, 6.3-not reached). CONCLUSIONS: CB-103 had a manageable safety profile and biological activity but limited clinical antitumor activity as monotherapy in this first-in-human study. SIGNIFICANCE: CB-103 is a novel oral pan-Notch inhibitor that selectively blocks the CSL-NICD interaction leading to transcriptional downregulation of oncogenic Notch pathway activation. This first-in-human dose-escalation and -confirmation study aimed to determine the safety, pharmacokinetics, and preliminary antitumor efficacy of CB-103. We observed a favorable safety profile with good tolerability and biological activity but limited clinical single-agent antitumor activity. Some disease stabilization was observed among an aggressive NOTCH-mutant ACC type-I subgroup where prognosis is poor and therapies are critically needed. Peripheral downregulation of select Notch target gene levels was observed with escalating doses. Future studies exploring CB-103 should enrich for patients with NOTCH-mutant ACC and investigate rational combinatorial approaches in tumors where there is limited success with investigational or approved drugs.

Prediction of the Optimal Dosing Regimen Using a Mathematical Model of Tumor Uptake for Immunocytokine-Based Cancer Immunotherapy.

Purpose: Optimal dosing is critical for immunocytokine-based cancer immunotherapy to maximize efficacy and minimize toxicity. Cergutuzumab amunaleukin (CEA-IL2v) is a novel CEA-targeted immunocytokine. We set out to develop a mathematical model to predict intratumoral CEA-IL2v concentrations following various systemic dosing intensities.Experimental Design: Sequential measurements of CEA-IL2v plasma concentrations in 74 patients with solid tumors were applied in a series of differential equations to devise a model that also incorporates the peripheral concentrations of IL2 receptor-positive cell populations (i.e., CD8+, CD4+, NK, and B cells), which affect tumor bioavailability of CEA-IL2v. Imaging data from a subset of 14 patients were subsequently utilized to additionally predict antibody uptake in tumor tissues.Results: We created a pharmacokinetic/pharmacodynamic mathematical model that incorporates the expansion of IL2R-positive target cells at multiple dose levels and different schedules of CEA-IL2v. Model-based prediction of drug levels correlated with the concentration of IL2R-positive cells in the peripheral blood of patients. The pharmacokinetic model was further refined and extended by adding a model of antibody uptake, which is based on drug dose and the biological properties of the tumor. In silico predictions of our model correlated with imaging data and demonstrated that a dose-dense schedule comprising escalating doses and shortened intervals of drug administration can improve intratumoral drug uptake and overcome consumption of CEA-IL2v by the expanding population of IL2R-positive cells.Conclusions: The model presented here allows simulation of individualized treatment plans for optimal dosing and scheduling of immunocytokines for anticancer immunotherapy. Clin Cancer Res; 24(14); 3325-33. ©2018 AACRSee related commentary by Ruiz-Cerdá et al., p. 3236.

Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor.

T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1hi TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1hi TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1int and PD-1neg T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1hi expressing cells; in contrast, patients with abundance of PD-1hi expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1hi T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction.

Progression of Lung Cancer Is Associated with Increased Dysfunction of T Cells Defined by Coexpression of Multiple Inhibitory Receptors.

Dysfunctional T cells present in malignant lesions are characterized by a sustained and highly diverse expression of inhibitory receptors, also referred to as immune checkpoints. Yet, their relative functional significance in different cancer types remains incompletely understood. In this study, we provide a comprehensive characterization of the diversity and expression patterns of inhibitory receptors on tumor-infiltrating T cells from patients with non-small cell lung cancer. In spite of the large heterogeneity observed in the amount of PD-1, Tim-3, CTLA-4, LAG-3, and BTLA expressed on intratumoral CD8(+) T cells from 32 patients, a clear correlation was established between increased expression of these inhibitory coreceptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Coexpression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T-cell function only in a subset of patients. A high percentage of PD-1(hi) T cells was correlated with poor restoration of T-cell function upon PD-1 blockade. Of note, PD-1(hi) expression marked a particularly dysfunctional T-cell subset characterized by coexpression of multiple inhibitory receptors and thus may assist in identifying patients likely to respond to inhibitory receptor-specific antibodies. Overall, these data may provide a framework for future personalized T-cell-based therapies aiming at restoration of tumor-infiltrating lymphocyte effector functions.

All trans-retinoic acid abrogates the pro-tumorigenic phenotype of prostate cancer tumor-associated macrophages.

Tumor-associated macrophages (TAMs) are a prominent cell type of the tumor stroma and stimulate malignant cell growth, survival and metastasis. The present manuscript demonstrates that prostate cancer cell-derived factors induce a pro-tumoral TAM-like phenotype characterized by increased proliferation and increased expression of pro-angiogenic, immunosuppressive and pro-metastatic factors. These effects were abrogated by all trans-retinoic acid (ATRA), a clinically available molecule with known immune-modulating properties. Furthermore, ATRA inhibited the cancer cell-stimulated proliferation of the pro-tumoral macrophages and restored their cytotoxic capacity towards prostate cancer cells. These findings suggest the use of ATRA as an immunomodulating agent to block the activity of prostate cancer TAMs.

[18 F]FDG-PET imaging is an early non-invasive pharmacodynamic biomarker for a first-in-class dual MEK/Raf inhibitor, RO5126766 (CH5126766), in preclinical xenograft models.

BACKGROUND: Positron emission tomography (PET) with [2-18 F]-2-fluoro-2-deoxy-D-glucose ([18 F]FDG-PET) was acquired at multiple time-points a) to monitor the early response to RO5126766 (CH5126766) in xenograft models b) to evaluate non-invasive small animal [18 F]FDG-PET imaging as a biomarker for MEK inhibitors for translation into dose-finding studies in cancer patients and c) to explore the underlying mechanism related to FDG uptake in tumors treated with RO5126766. METHODS: [18 F]FDG uptake was studied in HCT116 (K-ras), COLO205 (B-raf) mutants and COLO320DM (wild type) xenografts from day 0 to 3 of RO5126766 treatment using a microPET Focus 120 and complemented with in vitro incubations, ex-vivo phosphor imaging and immunohistochemical (IHC) analyses. RESULTS: In the HCT116 (K-ras) and COLO205 (B-raf) mutant xenografts, significant decreases in [18 F]FDG uptake were detected in vivo on day 1 with 0.3 mg/kg and ex vivo on day 3 with 0.1 mg/kg RO5126766. [18 F]FDG changes correlated with decreases in tumor cells proliferation (Ki-67) and with changes in expression levels of GLUT1. No effects were observed in drug resistant COLO320DM cells. The cellular fractionation and Western blotting analyses suggested that the change of [18 F]FDG uptake associated with RO5126766 is due to translocation of GLUT1 from membrane to cytosol, similar to the results reported in the literature with EGFR tyrosine kinase inhibitors, which also target the MAPK pathway. CONCLUSIONS: RO5126766 inhibition resulted in a rapid time - and dose - dependent decline in [18 F]FDG uptake in both mutant xenografts. These results strongly resemble the clinical observations obtained with MEK/Raf inhibitors support the use of preclinical [18 F]FDG-PET as a translational tool for decision support in preclinical and early clinical development of MEK inhibitors.

DNA vaccine coding for the rhesus prostate specific antigen delivered by intradermal electroporation in patients with relapsed prostate cancer.

We tested safety, clinical efficacy and immunogenicity of a DNA vaccine coding for rhesus prostate specific antigen (PSA) delivered by intradermal injection and skin electroporation. Fifteen patients with biochemical relapse of prostate cancer without macroscopic disease participated in this phase I study. Patients were started on a 1 month course of androgen deprivation therapy (ADT) prior to treatment. Vaccine doses ranged from 50 to 1,600 µg. Study subjects received five vaccinations at four week intervals. All patients have had at least one year of follow-up. No systemic toxicity was observed. Discomfort from electroporation did not require analgesia or topical anesthetic. No clinically significant changes in PSA kinetics were observed as all patients required antiandrogen therapy shortly after completion of the 5 months of vaccination due to rising PSA. Immunogenicity, as measured by T-cell reactivity to the modified PSA peptide and to a mix of overlapping PSA peptides representing the full length protein, was observed in some patients. All but one patient had pre-study PSA specific T-cell reactivity. ADT alone resulted in increases in T-cell reactivity in most patients. Intradermal vaccination with skin electroporation is easily performed with only minor discomfort for the patient. Patients with biochemical relapse of prostate cancer are a good model for testing immune therapies.

Phase 1 study results of the type II glycoengineered humanized anti-CD20 monoclonal antibody obinutuzumab (GA101) in B-cell lymphoma patients.

Whereas the chimeric type I anti-CD20 Ab rituximab has improved outcomes for patients with B-cell malignancies significantly, many patients with non-Hodgkin lymphoma (NHL) remain incurable. Obinutuzumab (GA101) is a glycoengineered, humanized anti-CD20 type II Ab that has demonstrated superior activity against type I Abs in vitro and in preclinical studies. In the present study, we evaluated the safety, efficacy, and pharmacokinetics of GA101 in a phase 1 study of 21 patients with heavily pretreated, relapsed, or refractory CD20(+) indolent NHL. Patients received GA101 in a dose-escalating fashion (3 per cohort, range 50/100-1200/2000 mg) for 8 × 21-day cycles. The majority of adverse events (AEs) were grades 1 and 2 (114 of 132 total AEs). Seven patients reported a total of 18 grade 3 or 4 AEs. Infusion-related reactions were the most common AE, with most occurring during the first infusion and resolving with appropriate management. Three patients experienced grade 3 or 4 drug-related infusion-related reactions. The best overall response was 43%, with 5 complete responses and 4 partial responses. Data from this study suggest that GA101 was well tolerated and demonstrated encouraging activity in patients with previously treated NHL up to doses of 2000 mg. This trial is registered at www.clinicaltrials.gov as NCT00517530.

Distribution of Foxp3-, CD4- and CD8-positive lymphocytic cells in benign and malignant prostate tissue.

Foxp3 is a transcription factor that inhibits antitumor immune response and is expressed in regulatory T cells (Tregs). High levels of Tregs have been reported in several human cancers. This study investigates the distribution of cells positive for Foxp3, CD4 and CD8 in benign prostatic tissues and prostatic carcinoma. Tissue microarrays were constructed from radical prostatectomy specimens of 36 patients. From each patient, six cores were taken: two cores from cancer, one from benign tissue of each of the peripheral (PZ), transition (TZ) and central zones (CZ) and one from atrophy. Foxp3-, CD4- and CD8-positive cells were more common in cancer than in non-atrophic benign tissue (p < 0.01) and more common in atrophy than in non-atrophic PZ, but did not differ significantly between cancer and atrophy. Cells positive for Foxp3 and CD4 were less prevalent in CZ than in PZ and TZ. Tregs infiltrate more in prostate cancer (PC) than in benign tissue. Their presence in atrophy may have relevance for the hypothesis on atrophy as a potential precursor lesion of PC. CZ has the lowest Treg levels, and a possible role for the low rate of cancer in this zone remains to be investigated.

Skin electroporation: effects on transgene expression, DNA persistence and local tissue environment.

BACKGROUND: Electrical pulses have been used to enhance uptake of molecules into living cells for decades. This technique, often referred to as electroporation, has become an increasingly popular method to enhance in vivo DNA delivery for both gene therapy applications as well as for delivery of vaccines against both infectious diseases and cancer. In vivo electrovaccination (gene delivery followed by electroporation) is currently being investigated in several clinical trials, including DNA delivery to healthy volunteers. However, the mode of action at molecular level is not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: This study investigates intradermal DNA electrovaccination in detail and describes the effects on expression of the vaccine antigen, plasmid persistence and the local tissue environment. Gene profiling of the vaccination site showed that the combination of DNA and electroporation induced a significant up-regulation of pro-inflammatory genes. In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression. However, when the more immunogenic prostate specific antigen (PSA) was co-administered, PSA-specific T cells were induced and concurrently the luciferase expression became undetectable. Electroporation did not affect the long-term persistence of the PSA-expressing plasmid. CONCLUSIONS/SIGNIFICANCE: This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid. As the described vaccination approach is currently being evaluated in clinical trials, the data provided will be of high significance.

Expression of immunomodulating genes in prostate cancer and benign prostatic tissue.

OBJECTIVE: To investigate the expression of immunomodulating genes in prostate cancer and benign prostatic tissue. STUDY DESIGN: We investigated by quantitative real-time polymerase chain reaction the expression of indoleamine 2,3-dioxygenase, arginase 1, arginase 2, inducible form of nitric oxide synthase, cyclooxygenase 2 (COX-2), programmed death ligand 1 and interleukin 10 in 36 matched pairs of samples from prostate cancer and benign prostatic tissue. RESULTS: Among the genes analyzed, arginase 2 and COX-2 showed statistically significant up-regulation and down-regulation, respectively, in malignant compared to benign prostate tissue. In addition, arginase 1 was more often present in cancer than benign samples. No significant modulation was detected for the other genes under investigation. CONCLUSION: Our data suggest that arginine metabolism may be involved in prostate cancer immune response evasion, whereas COX-2 may play a role in pathogenesis. We provide a snapshot of immunosuppressive gene expression at the transcriptional level in the prostate tumor microenvironment.

Tumor-specific bacteriophages induce tumor destruction through activation of tumor-associated macrophages.

We recently reported that administration of tumor-specific bacteriophages initiates infiltration of neutrophilic granulocytes with subsequent regression of established B16 tumors. The aim of the current study was to investigate the mechanism of action of bacteriophage-induced tumor regression and to examine possible stimulatory effects of bacteriophages on macrophages. We observed that the mechanism of phage-induced tumor regression is TLR dependent as no signs of tumor destruction or neutrophil infiltration were observed in tumors in MyD88(-/-) mice in which TLR signaling is abolished. The microenvironment of bacteriophage-treated tumors was further analyzed by gene profiling through applying a low-density array preferentially designed to detect genes expressed by activated APCs, which demonstrated that the M2-polarized tumor microenvironment switched to a more M1-polarized milieu following phage treatment. Bacteriophage stimulation induced secretion of proinflammatory cytokines in both normal mouse macrophages and tumor-associated macrophages (TAMs) and increased expression of molecules involved in Ag presentation and costimulation. Furthermore, mouse neutrophils selectively migrated toward mediators secreted by bacteriophage-stimulated TAMs. Under these conditions, the neutrophils also exhibited increased cytotoxicity toward B16 mouse melanoma target cells. These results describe a close interplay of the innate immune system in which bacteriophages, located to the tumor microenvironment due to their specificity, stimulate TAMs to secrete factors that promote recruitment of neutrophils and potentiate neutrophil-mediated tumor destruction.

A modified epitope identified for generation and monitoring of PSA-specific T cells in patients on early phases of PSA-based immunotherapeutic protocols.

Efficacy of vaccination in cancer patients on immunotherapeutic protocols can be difficult to evaluate. The aim of this study was therefore to identify a single natural or modified epitope in prostate-specific antigen (PSA) with the ability to generate high levels of PSA-specific T cells to facilitate monitoring in patients after vaccination against prostate cancer. To the best of our knowledge, this study describes for the first time the peptide specificity of T cells stimulated by endogenously processed PSA antigen. The peptide specificity of HLA-A*0201-restricted CD8(+) T cells against human and rhesus PSA was investigated both in vivo after DNA vaccination in HLA-A*0201-transgenic mice and in vitro after repetitive stimulation of human T cells with DNA-transfected human dendritic cells (DCs). One of seven native PSA peptides, psa53-61, was able to activate high levels of PSA-specific CD8(+) T cells in HLA-A*0201-transgenic mice after PSA DNA vaccination. Psa53-61 was also the only peptide that induced human T cells to produce IFNgamma after stimulation with PSA transfected DCs, however not in all donors. Therefore, plasmids encoding modified epitopes in predicted HLA-A*0201 sequences were constructed. One of these modified PSA plasmids consistently induced IFNgamma producing CD8(+) T cells to the corresponding modified peptide as well as to the corresponding native peptide, in all murine and human T cell cultures. This study demonstrates a novel concept of introducing a modified epitope within a self-tumor antigen, with the purpose of eliciting a reliable T cell response from the non-tolerized immune repertoire, to facilitate monitoring of vaccine efficacy in cancer patients on immunotherapeutic protocols. The purpose of such a modified epitope is thus not to induce therapeutically relevant T cells but rather to, in case of weak or divergent T cell responses to self antigens/peptides, help answer questions about efficacy of vaccine delivery and about the possibility to induce immune responses in the selected and often immunosuppressed cancer patients.

HER-2/neu mediated down-regulation of MHC class I antigen processing prevents CTL-mediated tumor recognition upon DNA vaccination in HLA-A2 transgenic mice.

To study DNA vaccination directed against human HER-2 in the HHD mouse Tg strain, we created a novel HER-2-expressing syngeneic tumor transplantation model. We found that a DNA vaccine encoding the full length HER-2 DNA protected HHD mice from HER-2(+) tumor challenge by a CTL independent mechanism. A more efficient approach to induce HLA-A2 restricted CTLs, through immunization with a multi-epitope DNA vaccine expressing the HLA-A2 restricted HER-2 369-377, 435-443 and 689-697 epitopes, resulted in high numbers of peptide specific T cells but failed to induce tumor protection. Subsequently we discovered that HER-2 transfected tumor cells down-regulated MHC class I antigen expression and exhibited a series of defects in the antigen processing pathway which impaired the capacity to produce and display MHC class I peptide-ligands to specific CTLs. Our data demonstrate that HER-2 transfection is associated with defects in the MHC class I presentation pathway, which may be the underlying mechanism behind the inability of CTLs to recognize tumors in this HLA-A2 transgenic model. As defective MHC class I presentation may be a common characteristic of HER-2 expressing tumors, vaccines targeting HER-2 should aim at inducing an integrated immune response where also CD4(+) T cells and antibodies are important components.

Bevacizumab: direct anti-VEGF therapy in renal cell carcinoma.

Bevacizumab, in combination with IFN, is approved in the EU as first-line therapy for advanced and/or metastatic renal cell carcinoma (mRCC). Data from Avastin and Roferon in Renal Cell Carcinoma [BO17705] (AVOREN), a Phase III trial, demonstrated that bevacizumab plus IFN significantly improves progression-free survival and response rate in patients with previously untreated mRCC compared with IFN plus placebo. Furthermore, bevacizumab plus IFN is well tolerated and has a predictable and well-established tolerability profile; reducing the dose of IFN, when necessary, can effectively manage IFN-related side effects without compromising efficacy. The rapid evolution of options for RCC therapy means that the optimal use of available agents to maximize patient benefit is not currently well defined. Combination regimens and sequencing of agents are both being investigated to maximize future outcomes, with bevacizumab playing a key role in first-line regimens. Trials over the next 5 years will guide clinical practice, but bevacizumab plus IFN is currently a standard first-line option for mRCC.

Expression of PDX-1 in prostate cancer, prostatic intraepithelial neoplasia and benign prostatic tissue.

Pancreatic duodenal homeobox 1 (PDX-1), a Hox type transcription factor, is necessary for differentiation of exocrine and endocrine pancreas, and regulates insulin gene transcription. PDX-1 expression was studied by immunohistochemistry on a tissue microarray (TMA) of 289 primary prostate cancers (PCa) from radical prostatectomy (RP) specimens with median follow-up of 48.9 months. We separately arrayed benign prostatic tissue, atrophy, high-grade prostatic intraepithelial neoplasia (HGPIN) and PCa from 40 men and also 17 lymph node metastases. Intensity and extent of immunoreactivity and their product (IRp) were evaluated by two independent observers. PDX-1 was overexpressed in cancer vs benign tissue (p<0.001), but also in atrophy and HGPIN vs cancer (p<0.001 and p=0.022, respectively). PDX-1 expression did not correlate with biochemical recurrence, but decreased with higher Gleason pattern (p<0.001) and in metastases vs primary PCa (p<0.001). Weighted kappa for interobserver agreement of intensity, extent and IRp was 0.65, 0.13 and 0.54, respectively. Presence of PDX-1 protein in benign and malignant prostatic tissue was confirmed by Western blot. In view of recent attention to the role of insulin systems in men with PCa, this protein is of interest in the pathogenesis of PCa.

DNA vaccination for prostate cancer.

DNA-based cancer vaccines have been used successfully in mice to induce cytotoxic T lymphocytes (CTLs) specific for prostate antigens. Translation of a prostate-specific antigen (PSA) DNA vaccine into a phase I clinical trial demonstrated that PSA-specific immune responses could be induced but at a significantly lower level compared with those in mice. To enhance the efficacy of DNA vaccination against prostate cancer, we have explored and optimized intradermal electroporation as an effective way of delivering a PSA DNA vaccine. The results demonstrated that intradermal DNA vaccination using low amounts of DNA, followed by two sets of electrical pulses of different length and voltage, effectively induced PSA-specific T cells. Here we describe in detail how to perform intradermal DNA electroporation to induce high gene expression in skin and, more important, how to induce and analyze PSA-specific T cell responses.

Zoledronic acid modulates antitumoral responses of prostate cancer-tumor associated macrophages.

Macrophages are considered a key component of the immunosuppressive environment present in solid tumors, where they support tumor growth through the production of pro-angiogenic factors and active suppression of effector immune responses. Zoledronic acid (ZA), an aminobisphosphonate clinically approved for treatment of symptomatic skeletal events, has recently been shown to have immunomodulatory properties that can be exploited in cancer immunotherapy. Here, we utilize an in vitro model of prostate cancer cell-macrophage interaction to dissect the effect of ZA, on the function of prostate cancer tumor-associated macrophages (PC-TAM). We show that prostate cancer cells recruit macrophages, which in turn express a variety of proangiogenic and immunosuppressive mediators. ZA selectively suppressed the expression of MMP-9 by PC-TAM, whereas the expression of other mediators was not limited. PC-TAM treated with ZA, on the other hand, could effectively drive the proliferation of activated Tgammadelta lymphocytes, which lysed bisphosphonate-pulsed prostate cancer cells. Moreover, ZA boosted the production of type-1 cytokines by PC-TAM in response to immunomodulators such as IL-12 and polyI:C, which are known to polarize macrophages towards an anti-tumoral M1 phenotype. Overall, we provide evidence that ZA shifts the balance of PC-TAM from a tumor promoting to a tumor-eliminating phenotype and also suggest a potential use of this pharmacological agent as an immunotherapeutic adjuvant.

Current perspectives in the treatment of advanced prostate cancer.

Prostate cancer (PC) continues to be an important world health problem for men. Patients with locally confined PC are treated with either radiotherapy or surgery. However, treatment of more advanced stages of the disease is problematic. Initially, androgen deprivation offers a period of clinical stability, which is however invariably followed by progression to non-responsiveness to hormonal manipulation. Current management of patients with androgen-independent prostate cancer (AIPC) displays modest response rates and achieves only short-term benefit. Recently, knowledge in the complex pathophysiology of advanced PC has led to the identification of mechanisms and target molecules permitting the introduction of new therapies. Consequently, many investigational treatments are ongoing for AIPC in Phase-II and Phase-III trials aiming at the combination of chemotherapeutic regimens along with immunotherapy targeting PC-associated antigens. Other attractive options are gene therapy, as well as the targeting of survival signaling, differentiation, and apoptosis of the malignant PC cells. Further treatment modalities are directed against the tumor microenvironment, bone metastasis, or both. Collectively, the aforementioned efforts introduce a new era in the management of advanced PC. Novel pharmaceutical compounds and innovative approaches, integrated into the concept of individualized therapy will hopefully, during the next decade, improve the outcome and survival for hundreds of thousands of men worldwide.