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Contains fulltext : 244306.pdf (Publisher’s version ) (Open Access) Patients are the stewards of their own care and hence their voice is important when designing and implementing research. Patients should be involved not only as participants in research that impacts their care, as the recipients of that care and any associated harms, but also as research collaborators in prioritising important questions from the patient perspective and designing the research and the ways in which is it most appropriate to involve patients. The PIONEER Consortium, an international multistakeholder collaboration lead by the European Association of Urology, has developed a core outcome set (COS) for localised and metastatic prostate cancer relevant to all stakeholders in particular patients. Throughout the work of PIONEER, patient representatives were involved as collaborators in setting the research agenda, and a wider group of patients was involved as participants in developing COSs, for instance in consensus meetings on choosing important outcomes and appropriate definitions. This publication showcases the process for COS development and highlights the most important recommendations to ultimately inform future research projects co-created between patients and other stakeholders. PATIENT SUMMARY: An important step in involving patients in the selection of outcomes for clinical trials, clinical audits, and real-world evidence is the development of a core outcome set (COS) that is relevant to all stakeholders. This report highlights the patient participation throughout our PIONEER COS development. TAKE HOME MESSAGE: An important step in involving patients in the selection of outcomes for clinical trials, clinical audits, and real-world evidence is to develop a core outcome set (COS) that is relevant to all stakeholders. As part of the work of the PIONEER Consortium, we aim to highlight the patient participation throughout our PIONEER COS development.

There is an urgent need for new treatment options in metastatic drug-resistant prostate cancer. Combining immunotherapy with other targeted therapies may be an effective strategy for advanced prostate cancer. In the present study, we sought to investigate to enhance the efficacy of anti-CTLA-4 therapy against prostate cancer by the combination with STAT3 inhibition. Male C57BL6 mice were subcutaneously inoculated with the murine prostate cancer cell line RM-1. Tumor progression was monitored following treatment with vehicle, the small molecule STAT3 inhibitor GPB730, anti-CTLA-4 or GPB730 + anti-CTLA-4. Treatment with anti-CTLA-4 or anti-CTLA-4 + GPB730 significantly inhibited tumor growth and enhanced survival compared to vehicle. Combining anti-CTLA-4 treatment with GPB730 resulted in a significantly prolonged survival compared to anti-CTLA-4 alone. GPB730 significantly increased infiltration of CD45 + cells in tumors of anti-CTLA-4-treated mice compared to anti-CTLA-4 alone. The levels of tumor-infiltrating Tregs were significantly decreased and the CD8:Treg ratio significantly increased by GPB730 treatment in combination with anti-CTLA-4 compared to anti-CTLA-4 alone. Immunohistochemical analysis showed a significant increase in CD45-positive cells in anti-CTLA-4 and anti-CTLA-4 + GPB730-treated tumors compared to vehicle or GPB730 monotherapy. Plasma levels of IL10 were significantly increased by anti-CTLA-4 compared to vehicle but no increase was observed when combining anti-CTLA-4 with GPB730. In conclusion, STAT3 inhibition by GPB730 enhances the antitumoral activity of anti-CTLA-4 and decreases the intratumoral Treg frequency in a prostate cancer mouse model. These results support the combination of STAT3 inhibition with anti-CTLA-4 therapy to increase clinical responses in patients with prostate cancer. Supplementary Information The online version contains supplementary material available at 10.1007/s00262-021-02915-6.

Contains fulltext : 291547.pdf (Publisher’s version ) (Open Access) OBJECTIVES: Genome-wide association studies have revealed over 200 genetic susceptibility loci for prostate cancer (PCa). By combining them, polygenic risk scores (PRS) can be generated to predict risk of PCa. We summarize the published evidence and conduct meta-analyses of PRS as a predictor of PCa risk in Caucasian men. PATIENTS AND METHODS: Data were extracted from 59 studies, with 16 studies including 17 separate analyses used in the main meta-analysis with a total of 20,786 cases and 69,106 controls identified through a systematic search of ten databases. Random effects meta-analysis was used to obtain pooled estimates of area under the receiver-operating characteristic curve (AUC). Meta-regression was used to assess the impact of number of single-nucleotide polymorphisms (SNPs) incorporated in PRS on AUC. Heterogeneity is expressed as I(2) scores. Publication bias was evaluated using funnel plots and Egger tests. RESULTS: The ability of PRS to identify men with PCa was modest (pooled AUC 0.63, 95% CI 0.62-0.64) with moderate consistency (I(2) 64%). Combining PRS with clinical variables increased the pooled AUC to 0.74 (0.68-0.81). Meta-regression showed only negligible increase in AUC for adding incremental SNPs. Despite moderate heterogeneity, publication bias was not evident. CONCLUSION: Typically, PRS accuracy is comparable to PSA or family history with a pooled AUC value 0.63 indicating mediocre performance for PRS alone. 01 april 2023

Watchful waiting (WW) represents a deferred treatment option for prostate cancer (PCa) patients when curative treatment seems overtreatment right from the outset. Patients are ‘watched’ for the development of local or systemic progression with disease-related symptoms, at which stage they are then treated palliatively according to their symptoms, in order to maintain quality of life (QoL). When choosing WW, it is important to adequately assess life expectancy of patients. Although previous studies reported the outcomes of PCa patients managed with WW, their results might not be generalizable to contemporary patients. Moreover, which is the impact of individual patient characteristics and comorbidities on long-term outcomes is still largely unknown. In the face of such a paucity of data, the PIONEER, which is a novel project of the Innovative Medicine Initiative’s (IMI’s) “Big Data for Better Outcomes” program with the mission to transform PCa care with particular focus on improving cancer-related outcomes, health system efficiency and the quality of health and social care across Europe, aims at assessing which are the long-term outcomes of PCa patients undergoing WW overall and after stratification according to disease characteristics, comorbidities and life expectancy. Of note, this question emerged as the second one with the highest agreement score among different stakeholders after an international consensus to identify and prioritize the most important questions in the field of PCa performed within the PIONEER project.

IntroductionAs part of the PIONEER (Prostate Cancer Diagnosis and Treatment Enhancement Through the Power of Big Data in Europe) Consortium, we will explore which diagnostic and prognostic factors (DPFs) are currently being researched to previously defined clinical and patient-reported outcomes for prostate cancer (PCa).Methods and analysisThis research project will follow the following four steps: (1) a broad systematic literature review of DPFs for all stages of PCa, covering evidence from 2014 onwards; (2) discussion of systematic review findings by a multidisciplinary expert panel; (3) risk of bias assessment and applicability with Prediction model Risk Of Bias Assessment Tool criteria, Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) and the Quality In Prognosis Studies tool (QUIPS) and (4) additional quantitative assessments if required.Ethics and disseminationWe aim to develop an online tool to present the DPFs identified in this research and make them available across all stakeholders. There are no ethical implications.

This is a study protocol for an observational health data analysis, submitted as a preprint to facilitate transparency and open science. Watchful waiting (WW) represents a deferred treatment option for prostate cancer (PCa) patients when curative treatment seems overtreatment right from the outset. Patients are ‘watched’ for the development of local or systemic progression with disease-related symptoms, at which stage they are then treated palliatively according to their symptoms, in order to maintain quality of life. When choosing WW, it is important to adequately assess life expectancy of patients. Although previous studies reported the outcomes of PCa patients managed with WW, which is the impact of individual patient characteristics and comorbidities on long-term outcomes is still largely unknown. The PIONEER, which is a novel project of the Innovative Medicine Initiative’s (IMI’s) “Big Data for Better Outcomes” program with the mission to transform PCa care with particular focus on improving cancer-related outcomes, health system efficiency and the quality of health and social care across Europe, aims at assessing which are the long-term outcomes of PCa patients undergoing WW overall and after stratification according to disease characteristics, comorbidities and life expectancy. Of note, this topic emerged as the second one with the highest agreement score among different stakeholders after an international consensus to identify and prioritize the most important questions in the field of PCa. This study aims to describe demographics, clinical characteristics and estimate outcomes of PCa patients under delayed treatment (WW) across a network of databases in the overall population and subgroups of patients identified by individual disease characteristics, demographics and comorbidities. The study will rely on large observational data, namely population-based registries, electronic health records and insurance claims data. The study will be an observational cohort study based on routinely collected health care data which has been mapped to the Observational Medical Outcomes Partnership (OMOP) Common Data Model (CDM).

Prostate Cancer Diagnosis and Treatment Enhancement Through the Power of Big Data in Europe (PIONEER) is a European network of excellence for big data in prostate cancer, consisting of 32 private and public stakeholders from 9 countries across Europe. Launched by the Innovative Medicines Initiative 2 and part of the Big Data for Better Outcomes Programme (BD4BO), the overarching goal of PIONEER is to provide high-quality evidence on prostate cancer management by unlocking the potential of big data. The project has identified critical evidence gaps in prostate cancer care, via a detailed prioritization exercise including all key stakeholders. By standardizing and integrating existing high-quality and multidisciplinary data sources from patients with prostate cancer across different stages of the disease, the resulting big data will be assembled into a single innovative data platform for research. Based on a unique set of methodologies, PIONEER aims to advance the field of prostate cancer care with a particular focus on improving prostate-cancer-related outcomes, health system efficiency by streamlining patient management, and the quality of health and social care delivered to all men with prostate cancer and their families worldwide.

²²⁵Ac-PSMA-617 targeted-therapy has demonstrated efficacy in 75-85% of patients; however, responses are not durable. We aimed to establish translatable mouse models of disseminated prostate cancer (PCa) to evaluate effectiveness of ²²⁵Ac-PSMA-617 at various disease stages. Methods: C4-2, C4-2B, or 22Rv1 cells were injected into the left ventricle of male NSG mice. Disease progression was monitored using bioluminescence imaging (BLI). For treatment, mice were injected with 40 kBq ²²⁵Ac-PSMA-617 at one (early treatment cohort) or three weeks (late treatment cohort) post-inoculation. Treatment efficacy was monitored by BLI of whole-body tumor burden. Mice were sacrificed based on body conditioning score. Results: C4-2 cells yielded metastases in liver, lungs, spleen, stomach, bones, and brain - achieving a clinically relevant model of widespread metastatic disease. The disease burden in the early treatment cohort was stable over 27 weeks in 5/9 mice and progressive in 4/9 mice. These mice were sacrificed due to brain metastases. Median survival of the late treatment cohort was superior to controls (13 vs. 7 weeks; p

The transcription factor STAT3 is a potential target for the treatment of castration-resistant prostate cancer. Galiellalactone (1), a direct inhibitor of STAT3, prevents the transcription of STAT3 regulated genes. In this study we characterized 6 (GPA512, Johansson , M. ; Sterner , O. Patent WO 2015/132396 A1, 2015 ), a prodrug of 1. In vitro studies showed that 6 is rapidly converted to 1 in plasma and is stable in a buffer solution. The pharmacokinetics of 6 following a single oral dose indicated that the prodrug was rapidly absorbed and converted to 1 with a tmax of 15 min. Oral administration of 6 in mice increased the plasma exposure of the active parent compound 20-fold compared to when 1 was dosed orally. 6 treated mice bearing DU145 xenograft tumors had significantly reduced tumor growth compared to untreated mice. The favorable druglike properties and safety profile of 6 warrant further studies of 6 for the treatment of castration-resistant prostate cancer.

Prostate-specific membrane antigen (PSMA)–targeted radioligand therapy (RLT) is effective against prostate cancer (PCa), but all patients relapse eventually. Poor understanding of the underlying resistance mechanisms represents a key barrier to development of more effective RLT. We investigate the proteome and phosphoproteome in a mouse model of PCa to identify signaling adaptations triggered by PSMA RLT. Methods: Therapeutic efficacy of PSMA RLT was assessed by tumor volume measurements, time to progression, and survival in C4-2 or C4-2 TP53(−/−) tumor-bearing nonobese diabetic scid γ-mice. Two days after RLT, the proteome and phosphoproteome were analyzed by mass spectrometry. Results: PSMA RLT significantly improved disease control in a dose-dependent manner. Proteome and phosphoproteome datasets revealed activation of genotoxic stress response pathways, including deregulation of DNA damage/replication stress response, TP53, androgen receptor, phosphatidylinositol-3-kinase/AKT, and MYC signaling. C4-2 TP53(−/−) tumors were less sensitive to PSMA RLT than were parental counterparts, supporting a role for TP53 in mediating RLT responsiveness. Conclusion: We identified signaling alterations that may mediate resistance to PSMA RLT in a PCa mouse model. Our data enable the development of rational synergistic RLT-combination therapies to improve outcomes for PCa patients.

BackgroundProstate-specific membrane antigen (PSMA)-directed radioligand therapy (RLT) is a promising yet not curative approach in castration-resistant (CR) prostate cancer (PC). Rational combination therapies may improve treatment efficacy. Here, we explored the effect of androgen receptor blockade (ARB) on PSMA expression visualized by PET and its potential additive effect when combined with 177Lu-PSMA RLT in a mouse model of prostate cancer.MethodsMice bearing human CRPC (C4-2 cells) xenografts were treated with 10mg/kg enzalutamide (ENZ), with 50mg/kg bicalutamide (BIC), or vehicle (control) for 21days. PSMA expression was evaluated by 68Ga-PSMA11 PET/CT and quantified by flow cytometry of tumor fine needle aspirations before treatment and on days 23, 29, 34, and 39 post-therapy induction. For the RLT combination approach, mice bearing C4-2 tumors were treated with 10mg/kg ENZ or vehicle for 21days before receiving either 15MBq (84GBq/μmol) 177Lu-PSMA617 or vehicle. DNA damage was assessed as phospho-γH2A.X foci in tumor biopsies. Reduction of tumor volume on CT and survival were used as study endpoints.ResultsTumor growth was delayed by ARB while 68Ga-PSMA11 uptake increased up to 2.3-fold over time when compared to controls. ABR-induced upregulation of PSMA expression was confirmed by flow cytometry. Phospho-γH2A.X levels increased 1.8- and 3.4-fold at 48h in response to single treatment ENZ or RLT and ENZ+RLT, respectively. Despite significantly greater DNA damage and persistent increase of PSMA expression at the time of RLT, no additional tumor growth retardation was observed in the ENZ+RLT group (vs. RLT only, p = 0.372 at day 81). Median survival did not improve significantly when ENZ was combined with RLT.ConclusionARB-mediated increases in PSMA expression in PC xenografts were evident by 68Ga-PSMA11 PET imaging and flow cytometry. 177Lu-PSMA617 effectively decreased C4-2 tumor size. However, while pre-treatment with ARB increased DNA damage significantly, it did not result in synergistic effects when combined with RLT.

Background Peptides labeled with positron-emitting isotopes are emerging as a versatile class of compounds for the development of highly specific, targeted imaging agents for diagnostic imaging via positron-emission tomography (PET) and for precision medicine via theranostic applications. Despite the success of peptides labeled with gallium-68 (for imaging) or lutetium-177 (for therapy) in the clinical management of patients with neuroendocrine tumors or prostate cancer, there are significant advantages of using fluorine-18 for imaging. Recent developments have greatly simplified such labeling: in particular, labeling of organotrifluoroborates via isotopic exchange can readily be performed in a single-step under aqueous conditions and without the need for HPLC purification. Though an automated synthesis has not yet been explored, microfluidic approaches have emerged for 18F-labeling with high speed, minimal reagents, and high molar activity compared to conventional approaches. As a proof-of-concept, we performed microfluidic labeling of an octreotate analog ([18F]AMBF3-TATE), a promising 18F-labeled analog that could compete with [68Ga]Ga-DOTATATE with the advantage of providing a greater number of patient doses per batch produced. Methods Both [18F]AMBF3-TATE and [68Ga]Ga-DOTATATE were labeled, the former by microscale methods adapted from manual labeling, and were imaged in mice bearing human SSTR2-overexpressing, rat SSTR2 wildtype, and SSTR2-negative xenografts. Furthermore, a dosimetry analysis was performed for [18F]AMBF3-TATE. Results The micro-synthesis exhibited highly-repeatable performance with radiochemical conversion of 50 ± 6% (n = 15), overall decay-corrected radiochemical yield of 16 ± 1% (n = 5) in ~40 min, radiochemical purity >99%, and high molar activity. Preclinical imaging with [18F]AMBF3-TATE in SSTR2 tumor models correlated well with [68Ga]Ga-DOTATATE. The favorable biodistribution, with the highest tracer accumulation in the bladder followed distantly by gastrointestinal tissues, resulted in 1.26 × 10−2 mSv/MBq maximal estimated effective dose in human, a value lower than that reported for current clinical 18F- and 68Ga-labeled compounds. Conclusions The combination of novel chemical approaches to 18F-labeling and microdroplet radiochemistry have the potential to serve as a platform for greatly simplified development and production of 18F-labeled peptide tracers. Favorable preclinical imaging and dosimetry of [18F]AMBF3-TATE, combined with a convenient synthesis, validate this assertion and suggest strong potential for clinical translation.

BACKGROUND: Peptides labeled with positron-emitting isotopes are emerging as a versatile class of compounds for the development of highly specific, targeted imaging agents for diagnostic imaging via positron-emission tomography (PET) and for precision medicine via theranostic applications. Despite the success of peptides labeled with gallium-68 (for imaging) or lutetium-177 (for therapy) in the clinical management of patients with neuroendocrine tumors or prostate cancer, there are significant advantages of using fluorine-18 for imaging. Recent developments have greatly simplified such labeling: in particular, labeling of organotrifluoroborates via isotopic exchange can readily be performed in a single-step under aqueous conditions and without the need for HPLC purification. Though an automated synthesis has not yet been explored, microfluidic approaches have emerged for (18)F-labeling with high speed, minimal reagents, and high molar activity compared to conventional approaches. As a proof-of-concept, we performed microfluidic labeling of an octreotate analog ([(18)F]AMBF(3)-TATE), a promising (18)F-labeled analog that could compete with [(68)Ga]Ga-DOTATATE with the advantage of providing a greater number of patient doses per batch produced. METHODS: Both [(18)F]AMBF(3)-TATE and [(68)Ga]Ga-DOTATATE were labeled, the former by microscale methods adapted from manual labeling, and were imaged in mice bearing human SSTR2-overexpressing, rat SSTR2 wildtype, and SSTR2-negative xenografts. Furthermore, a dosimetry analysis was performed for [(18)F]AMBF(3)-TATE. RESULTS: The micro-synthesis exhibited highly-repeatable performance with radiochemical conversion of 50±6% (n=15), overall decay-corrected radiochemical yield of 16±1% (n=5) in ~40 min, radiochemical purity >99%, and high molar activity. Preclinical imaging with [(18)F]AMBF(3)-TATE in SSTR2 tumor models correlated well with [(68)Ga]Ga-DOTATATE. The favorable biodistribution, with highest tracer accumulation in the bladder followed distantly by gastrointestinal tissues, resulted in 1.26×10(−2) mSv/MBq maximal estimated effective dose in human, a value lower than that reported for current clinical (18)F- and (68)Ga-labeled compounds. CONCLUSIONS: The combination of novel chemical approaches to (18)F-labeling and microdroplet radiochemistry have the potential to serve as a platform for greatly simplified development and production of (18)F-labeled peptide tracers. Favorable preclinical imaging and dosimetry of [(18)F]AMBF(3)-TATE, combined with a convenient synthesis, validate this assertion and suggest strong potential for clinical translation.

Human tumors xenografted in immunodeficient mice are crucial models in nuclear medicine to evaluate the effectiveness of candidate diagnostic and therapeutic compounds. However, little attention has been focused on the biological profile of the host model and its potential effects on the bio-distribution and tumor targeting of the tracer compound under study. We specifically investigated the dissimilarity in bio-distribution of (111)In-DTPA-5A10, which targets free prostate specific antigen (fPSA), in two animal models.In vivo bio-distribution studies of (111)In-DTPA-5A10 were performed in immunodeficient BALB/c-nu or NMRI-nu mice with subcutaneous (s.c.) LNCaP tumors. Targeting-specificity of the tracer was assessed by quantifying the uptake in (a) mice with s.c. xenografts of PSA-negative DU145 cells as well as (b) BALB/c-nu or NMRI-nu mice co-injected with an excess of non-labeled 5A10. Finally, the effect of neonatal Fc-receptor (FcRn) inhibition on the bio-distribution of the conjugate was studied by saturating FcRn-binding capacity with nonspecific IgG1.The inherent biological attributes of the mouse model substantially influenced the bio-distribution and pharmacokinetics of (111)In-DTPA-5A10. With LNCaP xenografts in BALB/c-nu mice (with intact B and NK cells but with deficient T cells) versus NMRI-nu mice (with intact B cells, increased NK cells and absent T cells), we observed a significantly higher hepatic accumulation (26 ± 3.9 versus 3.5 ± 0.4%IA/g respectively), and concomitantly lower tumor uptake (25 ± 11 versus 52 ± 10%IA/g respectively) in BALB/c-nu mice. Inhibiting FcRn by administration of nonspecific IgG1 just prior to (111)In-DTPA-5A10 did not change tumor accumulation significantly.We demonstrated that the choice of immunodeficient mouse model importantly influence the bio-distribution of (111)In-DTPA-5A10. This study further highlighted important considerations in the evaluation of preclinical tracers, with respect to gaining information on their performance in the translational setting. Investigators utilizing xenograft models need to assess not only radiolabeling strategies, but also the host immunological status.

Clinical (177)Lu-PSMA-617 radioligand therapy (RLT) is applied in advanced-stage prostate cancer. However, to the best of our knowledge murine models to study the biologic effects of various activity levels have not been established. The aim of this study was to optimize specific and total activity for (177)Lu-PSMA-617 RLT in a syngeneic model of murine prostate cancer. Methods: Murine-reconstituted, oncogene-driven prostate cancer cells (0.1 × 10(6)) (RM1), transduced to express human prostate-specific membrane antigen (PSMA), were injected into the left flank of C57Bl6 immunocompetent mice. RLT was performed by administering a single tail vein injection of (177)Lu-PSMA-617 at different formulations for specific (60 MBq at high, 62 MBq/nmol; intermediate, 31 MBq/nmol; or low 15 MBq/nmol specific activity) or total activity (30, 60, or 120 MBq). Organ distribution was determined by ex vivo γ-counter measurement. DNA double-strand breaks were measured using anti–gamma-H2A.X (phospho S139) immunohistochemistry. Efficacy was assessed by serial CT tumor volumetry and (18)F-FDG PET metabolic volume. Toxicity was evaluated 4 wk after the start of RLT. Results: Mean tumor-to-kidney ratios ± SEM were 19 ± 5, 10 ± 5, and 2 ± 0 for high, intermediate, and low (each n = 3) specific activity, respectively. Four of 6 (67%) mice treated with intermediate or high specific activity and none of 6 (0%) mice treated with low specific activity or formulation demonstrated significant DNA double-strand breaks (≥5% γ-H2A.X–positive cells). High when compared with intermediate or low specific activity resulted in a lower mean ± SEM tumor load by histopathology (vital tissue, 4 ± 2 vs. 8 ± 3 mm(2); n = 3 vs. 6), day-4 (18)F-FDG PET (metabolic volume, 87 ± 23 vs. 118 ± 14 mm(3); n = 6 vs. 12), and day-7 CT (volume, 323 ± 122 vs. 590 ± 46 mm(3); n = 3 vs. 6; P = 0.039). (177)Lu-PSMA-617 (120 MBq) with high specific activity induced superior tumor growth inhibition (P = 0.021, n = 5/group) without subacute hematologic toxicity (n = 3/group). Conclusion: (177)Lu-PSMA-617 (120 MBq) and high specific activity resulted in the highest efficacy in a syngeneic model of murine prostate cancer. The model will be useful for studying the effects of PSMA-directed RLT combined with potentially synergistic pharmacologic approaches.

Clinical 177Lu-PSMA-617 radioligand therapy (RLT) is applied in advanced-stage prostate cancer. However, to the best of our knowledge murine models to study the biologic effects of various activity levels have not been established. The aim of this study was to optimize specific and total activity for 177Lu-PSMA-617 RLT in a syngeneic model of murine prostate cancer. Methods: Murine-reconstituted, oncogene-driven prostate cancer cells (0.1 · 106) (RM1), transduced to express human prostate-specific membrane antigen (PSMA), were injected into the left flank of C57Bl6 immunocompetent mice. RLT was performed by administering a single tail vein injection of 177Lu-PSMA-617 at different formulations for specific (60 MBq at high, 62 MBq/nmol; intermediate, 31 MBq/nmol; or low 15 MBq/nmol specific activity) or total activity (30, 60, or 120 MBq). Organ distribution was determined by ex vivo g-counter measurement. DNA double-strand breaks were measured using anti–gamma-H2A.X (phospho S139) immunohistochemistry. Efficacy was assessed by serial CT tumor volumetry and 18F-FDG PET metabolic volume. Toxicity was evaluated 4 wk after the start of RLT. Results: Mean tumor-to-kidney ratios 6 SEM were 19 6 5, 10 6 5, and 2 6 0 for high, intermediate, and low (each n 5 3) specific activity, respectively. Four of 6 (67%) mice treated with intermediate or high specific activity and none of 6 (0%) mice treated with low specific activity or formulation demonstrated significant DNA double-strand breaks ($5% g-H2A.X–positive cells). High when compared with intermediate or low specific activity resulted in a lower mean 6 SEM tumor load by histopathology (vital tissue, 4 6 2 vs. 8 6 3 mm2; n 5 3 vs. 6), day-4 18F-FDG PET (metabolic volume, 87 6 23 vs. 118 6 14 mm3; n 5 6 vs. 12), and day-7 CT (volume, 323 6 122 vs. 590 6 46 mm3; n 5 3 vs. 6; P 5 0.039). 177Lu-PSMA-617 (120 MBq) with high specific activity induced superior tumor growth inhibition (P 5 0.021, n 5 5/group) without subacute hematologic toxicity (n 5 3/group). Conclusion: 177Lu-PSMA-617 (120 MBq) and high specific activity resulted in the highest efficacy in a syngeneic model of murine prostate cancer. The model will be useful for studying the effects of PSMA-directed RLT combined with potentially synergistic pharmacologic approaches.

In pre-clinical imaging, tumor response to radionuclide therapy can be monitored with PET imaging. Radionuclides used for therapy such as 177Lu emit a significant amount of low energy photons. These photons may have an energy high enough to penetrate the imaged object and are the prone to be detected. Although these phtons are likely to be rejected electronically, they add dead-time to the system since they need to be processed by the electronics. This is a problem in high-sensitivity pre-clinical PET system with a low number of readout channels, such as the Genisys G8 investigated in this work. The low energy gammas may also affect image quality due to increased probability of pulse pile-up. The use of high-attenuating shields designed to absorb most of the low energy photons emitted from the therapeutic radionuclide were investigated. Cylindrical led shields were constructed with thicknesses between 1 and 3 mm. A 3mm thick cylindrical shield was also constructed out of Rose metal (50% Bi, 28% Pb, and 22% Sn). The diameter of the shield was wide enough to accommodate a NEMA IQ phantom and a mouse. The attenuation of the shields for annihilation radiation was measured with a 22Na point source placed at the center of the FOV. Measurements of the coincidence rate were performed with the lead shields in place. At a of thicknesses of 1 and 3mm, the coincidence rate was reduced by a factor or 0.70 and 0.40, respectively. To study the effect of the presence of a background of low energy gammas on the coincidence count rate and the efficacy of the lead shields, 177Lu was added to a 1 cm diameter hollow sphere. In the presence of 100 MBq of 177Lu, the coincidence count rate was reduced by a factor 0.20 due to the detector dead-time. Although the count rate was reduced by a factor of 0.40 with the 3mm shield around the source, the dead-time effects due to the 177Lu background were less than 7%. 18F imaging of a NEMA phantom and a tumor bearing mouse showed dramatic image distortions in the presence of the 177Lu background. When imaging of with the 3mm shield in place, the image distortions were eliminated and were comparable in to the images acquired without the background activity.

Prostate cancer (PCa) is one of the most common cancers in men and is the second leading cause of cancer-related deaths in the USA. In the United States, it is the second most frequently diagnosed cancer after skin cancer, and in Europe it is number one. According to the American Cancer Society, approximately 221,000 men in the United States would be diagnosed with PCa during 2015, and approximately 28,000 would die of the disease. According to the International Agency for Research on Cancer, approximately 345,000 men were diagnosed with PCa in Europe during 2012, and despite more emphasis placed on early detection through routine screening, 72,000 men died of the disease. Hence, the need for improved therapy modalities is of utmost importance. And targeted therapies based on radiolabeled specific antibodies or peptides are a very interesting and promising alternative to increase the therapeutic efficacy and overall chance of survival of these patients. There are currently several preclinical and some clinical studies that have been conducted, or are ongoing, to investigate the therapeutic efficacy and toxicity of radioimmunotherapy (RIT) against PCa. One thing that is lacking in a lot of these published studies is the dosimetry data, which are needed to compare results between the studies and the study locations. Given the complicated tumor microenvironment and overall complexity of RIT to PCa, old and new targets and targeting strategies like combination RIT and pretargeting RIT are being improved and assessed along with various therapeutic radionuclides candidates. Given alone or in combination with other therapies, these new and improved strategies and RIT tools further enhance the clinical response to RIT drugs in PCa, making RIT for PCa an increasingly practical clinical tool.

Signal transducer and activator of transcription 3 (STAT3) is known to be involved in the progression of prostate cancer (PCa) and is a key factor in drug resistance and tumor immunoescape. As a result, it represents a promising target for PCa therapy. We studied the effects of the STAT3 inhibitor galiellalactone (GL) on tumor growth and metastatic spread in vitro and in vivo. The effect of GL on cell viability, apoptosis, and invasion was studied in vitro using androgen-independent DU145 and DU145-Luc cell lines. For in vivo studies, mice were injected orthotopically with DU145-Luc cells and treated with daily intraperitoneal injections of GL for 6 wk. GL significantly reduced the growth of the primary tumor and the metastatic spread of PCa cells to regional and distal lymph nodes in vivo. Treatment with GL also resulted in decreased cell proliferation and increased apoptosis compared with controls. In vitro, GL reduces the viability and invasive abilities of DU145-Luc cells and induces apoptosis. Our results showed that tumor growth and early metastatic dissemination of PCa can be significantly reduced by GL, indicating its potential use as a therapeutic compound in advanced metastatic PCa. Patient summary In this study, we tested the STAT3 inhibitor galiellalactone (GL) in an animal model of PCa. We found that mice treated with GL had smaller primary tumors and decreased lymph node metastases compared with mice treated with vehicle. GL has potential for treating advanced metastatic PCa.

Prostate cancer patients with high WNT5A expression in their tumors have been shown to have more favorable prognosis than those with low WNT5A expression. This suggests that reconstitution of Wnt5a in low WNT5A-expressing tumors might be an attractive therapeutic approach. To explore this idea, we have in the present study used Foxy-5, a WNT5A mimicking peptide, to investigate its impact on primary tumor and metastasis in vivo and on prostate cancer cell viability, apoptosis and invasion in vitro. We used an in vivo orthotopic xenograft mouse model with metastatic luciferase-labeled WNT5A-low DU145 cells and metastatic luciferase-labeled WNT5A-high PC3prostate cancer cells. We provide here the first evidence that Foxy-5 significantly inhibits the initial metastatic dissemination of tumor cells to regional and distal lymph nodes by 90% and 75%, respectively. Importantly, this effect was seen only with the WNT5A-low DU145 cells and not with the WNT5A-high PC3 cells. The inhibiting effect in the DU145-based model occurred despite the fact that no effects were observed on primary tumor growth, apoptosis or proliferation. These findings are consistent with and supported by the in vitro data, where Foxy-5 specifically targets invasion without affecting apoptosis or viability of WNT5A-low prostate cancer cells. To conclude, our data indicate that the WNT5A-mimicking peptide Foxy-5, which has been recently used in a phase 1 clinical trial, is an attractive candidate for complimentary anti-metastatic treatment of prostate cancer patients with tumors exhibiting absent or low WNT5A expression.

The aim of this study was to use multi-radionuclide autoradiography to compare the different distributions of three radiolabelled tracers in an atherosclerotic mouse model. This method, along with immunohistochemistry, was applied to investigate the intra-aortic distribution of 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)F-FDG), (131)I/(125)I labeled anti-oxidized Low Density Lipoprotein (oxLDL), and non-binding control antibodies. Aortas were isolated from a total of 12 apoB-100/LDL receptor deficient mice 73 h post injection of radioiodine-labeled anti-oxLDL and control antibody and 1 h post injection of (18)F-FDG. A solid-state real-time digital autoradiography system was used to image the slide mounted aortas. Contributions from each radionuclide were separated by half-life and emission energy and the aortas were subsequently stained with Oil Red O for plaque to aorta contrast ratios. Immunohistochemical staining was performed to detect anti-oxLDL and control antibody localization. Radiolabeled anti-oxLDL showed increased total activity uptake in the aorta over control antibody and immunohistochemical analysis of plaques indicated increased binding of the specific antibody compared to control. The intra-aortic activity distribution of the anti-oxLDL antibody was however very similar to that of the control antibody although both had higher atherosclerotic plaques to aorta wall ratios than (18)F-FDG. Given the right choice of radionuclides, multi-radionuclide digital autoradiography can be employed to compare several tracers ex vivo in the same animal. The distribution of anti-oxLDL antibodies did not significantly differ from the control antibody but it did appear to have a better plaque to aorta contrast at 73 h post injection than (18)F-FDG at 1 h post injection.

Target-specific molecular imaging with radiolabeled antibodies has experienced a rapid growth over the past 20 years, and is now an essential tool to provide information on disease presence and extent. Despite all efforts to detect and control prostate cancer at an early stage, a number of men are still progressing to develop incurable metastatic disease. Because of this, development of new methods based on target-specific imaging is becoming increasingly important in the assessment and management of prostate cancer. The main purpose of this work was to investigate three target antigens: Ku70/Ku80, free prostate specific antigen (fPSA) and ICAM-1, as potential candidates for imaging of prostate cancer using radiolabeled antibodies. The studies were designed to assess the in vivo and ex vivo tumor targeting potential using animal models of prostate cancer. Preclinical SPECT/CT and PET/CT in vivo imaging modalities, ex vivo multi-radionuclide digital autoradiography, ex vivo activity measurements and immunohistochemistry were used to obtain biokinetics and specific activity uptake. In these studies, we found that radiolabeled INCA-X mAb could efficiently target the Ku70/Ku80 antigen in prostate cancer xenografts in mice that first receive a predose of non-labeled antibody. We also demonstrate that two antibodies specific for fPSA (PSA30 and 5A10) can efficiently target PSA positive prostate cancer xenografts. In that study, we also show that the animal models immune deficiency status can affect the antibody performance. Lastly, we show that 111In-R6.5 mAb outperformed the 177Lu-R6.5 or control IgG mAb and can be used as a complement to clinically used metabolic and proliferative probes. Our ex vivo and in vivo investigations presented in this dissertation should act as support for further studies of the Ku70/Ku80, fPSA and ICAM-1 antigens. Future studies should include therapeutic applications based on dosimetry calculation to evaluate possible therapeutic efficacy.

This study investigated the feasibility of targeting the free, unbound forms of prostate-specific antigen (fPSA) for in vivo imaging of prostate adenocarcinomas (PCa), as PSA is produced and secreted at abundance during every clinical stage and grade of PCa, including castration-resistant disease. We injected (125)I-labeled monoclonal antibody PSA30 (specific for an epitope uniquely accessible on fPSA alone) intravenously in male nude mice carrying subcutaneous xenografts of LNCaP tumors (n=36). Mice were sacrificed over a time course from 4 hours to 13 days after injecting (125)I-labeled PSA30. Tissue uptake of (125)I-PSA30 at 48 and 168 hours after intravenous injection was compared with two clinically used positron emission tomography radiopharmaceuticals, (18)F-fluoro-deoxy-glucose ((18)F-FDG) or (18)F-choline, in cryosections using Digital AutoRadiography (DAR) and also compared with immunohistochemical staining of PSA and histopathology. On DAR, the areas with high (125)I-PSA30 uptake corresponded mainly to morphologically intact and PSA-producing LNCaP cells, but did not associate with the areas of high uptake of either (18)F-FDG or (18)F-choline. Biodistribution of (125)I-PSA30 measured in dissected organs ex vivo during 4 to 312 hours after intravenous injection demonstrated maximum selective tumor uptake 24-48 hours after antibody injection. Our data showed selective uptake in vivo of a monoclonal antibody highly specific for fPSA in LNCaP cells. Hence, in vivo imaging of fPSA may be feasible with putative usefulness in disseminated PCa.

Traditional Botanical Supplement-101 (TBS-101) is a newly developed proprietary botanical agent containing seven standardized botanical extracts, including: Panax ginseng, cranberry, green tea, grape skin, grape seed, Ganoderma lucidum and chamomile. Each of the components has been consumed either in the regular diet or as natural supplement. When used as a single agent, each of these seven botanicals has been implicated in chemoprevention and therapy in various types of cancer. The anticancer effect of TBS-101, with the specific combination of these anti-cancer botanicals for the treatment of prostate cancer (PCa), has not been tested.The IC(50) and the effect of TBS-101 on the proliferation and apoptosis of PC-3 cells were determined. Tumor xenograft mice were generated by subcutaneously implanting PC-3 cells into mice and tumors were allowed to grow to different sizes before starting the treatment. The effects of TBS-101 on tumor growth were assessed by measuring tumor size and by histological, pathological and immunohistochemical analyses. A basic toxicity study was performed to test the tolerance of the mice to high doses of TBS-101.Treatment of the PC-3 cells with TBS-101 resulted in a dose-dependent inhibition of cell growth, with an IC(50) of 1.4 microg/ml. A concomitant induction of apoptosis in PC-3 cells treated with TBS-101 was also observed. Upon the treatment with TBS-101, all three groups of mice bearing moderate or large tumors showed significant inhibition of tumor growth and invasion. In contrast, control mice treated with vehicle alone had significant tumor growth and lymph node metastasis. In the basic toxicity studies, high doses of TBS-101 exerted no toxicity in healthy or tumor-bearing mice.The natural botanical agent TBS-101 has a good safety profile and significant anticancer activities in hormone-refractory PC-3 cells and large aggressive PC-3 tumors in a xenograft mouse model and has great potential for the treatment of aggressive prostate cancer.

Background: The transcription factor STAT3 is a promising target for the treatment of castration-resistant prostate cancer (CRPC) as STAT3 is implicated in drug resistance and castration resistance as well as metastatic spread, tumor growth and immune escape. Galiellalactone (GL) is a direct inhibitor of STAT3 that prevents DNA binding, inhibits proliferation of prostate cancer cells expressing active STAT3 and induces apoptosis by down-regulation of STAT3 activated genes. In this study we aimed to characterize a prodrug of GL, GPA512, with improved drug-like properties and to demonstrate its effect on tumor growth in a xenograft model of prostate cancer following oral administration. Methods: Stability studies of prodrugs based on GL were performed in 0.1 M PBS buffer (pH 7.4) and in plasma at 37°C. In vitro efficacy of prodrugs was studied by WST-1 proliferation assay in DU145 prostate cancer cells expressing active STAT3. The systemic exposure of GL in mice was studied following a single oral dose of GPA512 or GL (both 10 mg/kg). The plasma concentrations of GL were determined by LC-MS/MS. For the xenograft study NMRI-nude male mice were inoculated subcutaneously with DU145 cells and once tumors were established the mice were divided in two groups with ten mice in each. Mice were treated orally with 40 mg/kg GPA512 daily five times/week for four weeks. Tumor growth was measured by caliper and at the end of the study tumors were harvested for subsequent analyses using immunohistochemistry and mRNA expression analysis. Results: In vitro studies showed that the prodrug GPA512 is rapidly converted to GL in plasma and that GPA512 is stable in buffer solution and has similar inhibitory effects on proliferation as GL on DU145 cells. The pharmacokinetics of GPA512 following a single oral dose indicated that the compound was rapidly absorbed and converted to GL with a tmax of 15 min. Oral administration of GPA512 in mice increased the plasma exposure (AUC) of the active parent compound 20-fold compared to when GL was dosed orally. GPA512 treated mice bearing subcutaneous DU145 tumors had significantly smaller tumors compared to mice treated with vehicle. No adverse effects or weight loss were observed. Analysis of tumors showed decreased cell proliferation and increased amount of apoptotic cells in GPA512 treated mice compared to control. The mRNA expression of STAT3 regulated anti-apoptotic gene MCl-1 was significantly reduced by GPA512 treatment. Conclusions: The drug-like properties and safety profile of the prodrug GPA512 and galiellalactone's direct inhibition of STAT3, warrant further studies of GPA512 as a drug candidate for treatment of patients with CRPC. Citation Format: Rebecka Hellsten, Zilma Escobar, Anders Bjartell, Giacomo Canesin, Susan Evans Axelsson, Olov Sterner, Martin Johansson. Preclinical characterization of GPA512: A prodrug of a direct STAT3 inhibitor for the treatment of prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C179.

Background and Aims Wnt5a protein levels are upregulated in prostate cancer (PCa) compared to benign tissue and patients with high Wnt5a protein levels have a better outcome after radical prostatectomy compared to patients with low Wnt5a levels (Syed Khaja AS, 2011; 2012). Thus, the reconstitution of the Wnt5a signaling pathway could be a promising therapeutic approach in PCa, as already shown in breast cancer (Säfholm A, 2008). Constitutively active STAT3 (pSTAT3) has been correlated to PCa progression and disruption of its signaling pathway could represent a promising strategy for the treatment of patients with advanced PCa (Hellsten R, 2008). We have analyzed the expression of Wnt5a and pSTAT3 in a cohort of patients with metastatic Castration Resistant Prostate Cancer (mCRPC). Using in vitro and in vivo techniques we have explored the effect of the Wnt5a-mimicking peptide FOXY5 for the reconstitution of the Wnt5a pathway in PCa, and we have studied the effect of the STAT3 inhibitor Galiellalactone (GL) on PCa cell viability, apoptosis, invasion, tumor growth and metastases. Methods Wnt5a and pSTAT3 protein expression was analyzed by IHC on a TMA construct with duplicate cores of malignant tissues from 38 patients with mCRPC. Statistical and correlation analyses were performed using the SPSS version 21. Cell viability was determined by MTT assay; apoptotic cells were visualized with M30 CytoDeath antibody 24h after treatment with FOXY5 or GL. Cell invasion was analyzed using matrigel pre-coated cell culture inserts in the presence or absence of FOXY5 or GL. For the animal study, NMRI nude mice were injected orthotopically with 1×106 DU145-Luc cells, and treated IP with GL every day or with FOXY5 every second day. Primary tumor growth and metastatic spread were evaluated weekly by in vivo luminescence using the IVIS Lumina II system. 9 weeks after injection animals were sacrificed and organs were analyzed for the presence of metastases. Results IHC analyses of TMA showed high pSTAT3 and low Wnt5a expression in CRPC patients, with a small but significant correlation (r = 0.4646; p = 0.0038). Our results show that GL inhibits viability and induces apoptosis of DU145-Luc cells in vitro, and that it reduces tumor growth and metastatic spread to lymph nodes in vivo. FOXY5 treatment has no effects on cell viability or on primary tumor growth, but it significantly reduces cell invasion in vitro and metastatic spread to lymph nodes in vivo. Conclusions Our results confirm that the reconstitution of the Wnt5a pathway and the inhibition of the STAT3 pathway are a promising therapeutic approach for the treatment of mCRPC. We found that GL and FOXY5 are good candidates for the treatment of mCRPC, as they inhibit tumor growth and metastatic spread to lymph nodes. Since these two compounds act through different mechanisms, our future studies will explore the possibility of a combination of GL and FOXY5 for the treatment of PCa. Citation Format: Giacomo Canesin, Susan Evans-Axelsson, Rebecka Hellsten, Nicholas Don-Doncow, Tommy Andersson, Anders Bjartell. Targeting Wnt5a and STAT3 pathways for the treatment of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4476. doi:10.1158/1538-7445.AM2015-4476

e22186 Background: The transcription factor STAT3 is a promising target for the treatment of castration-resistant prostate cancer (CRPC) as STAT3 is implicated in drug resistance, the progression o...