Acoustic Enrichment of Heterogeneous Circulating Tumor Cells and Clusters from Metastatic Prostate Cancer Patients.

BACKGROUND: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on the microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. METHODS: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility), resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. RESULTS: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogeneous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding a higher number of CTCs using acoustophoresis. CONCLUSION: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables the sensitive label-free enrichment of cells with epithelial phenotypes in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

[Molecular subtypes provide possibilities for precision medicine in a advanced prostate cancer]. / Molekylära subtyper och avancerad prostatacancer.

Increased molecular knowledge makes it possible to consider not only genetic defects but also expression profiles for precision medicine in advanced prostate cancer. Several prognostic and treatment-predictive classifiers for prostate cancer have been described, such as Prolaris, OncotypeDx, Decipher, Prostatype, PAM50, PCS1-2, and MetA-C, which all build upon transcript profiles. In research studies, the MetA-C classifier has shown clear prognostic information for patients with metastatic disease, in relation to outcome after androgen receptor targeting therapies, and so has immunohistochemical evaluation of tumor cell proliferation (Ki67) and PSA expression. Unfortunately, methods within clinical routine today do not allow molecular subclassification of prostate cancer. To enable comparison of the most promising treatment-predictive biomarkers and to evaluate the health economic value of implementing such precision medicine for prostate cancer, a prospective study is being planned as a joint initiative in Sweden that aims to evaluate and validate biomarkers and to establish a study platform for adaptive biomarker-driven clinical trials (sprintr.se).

Acoustic enrichment of heterogenous circulating tumor cells and clusters from patients with metastatic prostate cancer.

Background: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. Methods: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility) resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. Results: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogenous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC-clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding higher number of CTCs using acoustophoresis. Conclusion: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC-clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables sensitive label-free enrichment of cells with epithelial phenotype in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Magnetic Resonance Imaging as a Tool for Monitoring Intratibial Growth of Experimental Prostate Cancer Metastases in Mice.

Bone metastases cause morbidity and mortality in several human cancer forms. Experimental models are used to unravel the mechanisms and identify possible treatment targets. The location inside the skeleton complicates accurate assessment. This study evaluates the performance of magnetic resonance imaging (MRI) of prostate cancer tumors growing intratibially in mice. MRI detected intratibial tumor lesions with a sensitivity and specificity of 100% and 89%, respectively, compared to histological evaluation. Location and some phenotypical features could also be readily detected with MRI. Regarding volume estimation, the correlation between MRI and histological assessment was high (p < 0.001, r = 0.936). In conclusion, this study finds MRI to be a reliable tool for in vivo, non-invasive, non-ionizing, real-time monitoring of intratibial tumor growth.

Ki67 and prostate specific antigen are prognostic in metastatic hormone naïve prostate cancer.

BACKGROUND: For metastatic hormone naïve prostate cancer patients, androgen deprivation therapy (ADT) with escalation therapy including docetaxel and/or androgen targeting drugs is the standard therapy. However, de-escalation is preferable to avoid unnecessary side effects, especially from docetaxel, but markers to identify these patients are lacking. The purpose of the present study was to investigate the potential of PSA and Ki67 immunoreactive scores as prognostic and treatment-predictive markers. MATERIAL AND METHODS: Prostate biopsies from 92 patients with metastatic hormone naïve PC (PSA > 80 ng/mL or clinical metastases) were immunohistochemically evaluated for PSA and Ki67. Gene expression analysis was performed with Clariom D microarrays to identify the phenotypic profile associated with the immunohistochemistry scores of biopsies. Cox regression analysis for progression free survival after ADT adjustment for age, ISUP, and serum PSA and Kaplan-Meier analyses were performed to assess prognostic values of Ki67, PSA, and the Ki67/PSA ratio. RESULTS: The immunohistochemical score for PSA was the strongest prognostic factor for progression-free and overall survival after ADT. Consequently, the ratio between Ki67 and PSA displayed a stronger prognostic value than Ki67 itself. Further, mRNA expression data analysis showed an association between high Ki67/PSA ratio, cell-cycle regulation, and DNA damage repair. In an exploratory sub-analysis of 12 patients treated with early docetaxel as addition to ADT and matched controls, a high Ki67/PSA ratio showed potential to identify those who benefit from docetaxel. CONCLUSION: PSA and Ki67 immunoreactive scores are prognostic in the metastatic hormone-sensitive setting, with PSA being superior. The combination of Ki67 and PSA did not give additional prognostic value. The results suggest immunohistochemical scoring of PSA to have potential to improve identification of patients responding well to ADT alone.

Osteoclasts directly influence castration-resistant prostate cancer cells.

Metastasis to bone is the leading cause of death from prostate cancer. Interaction between tumor cells and bone cells can promote progression and influence tumor phenotype. It is known that prostate cancer cells support osteoclast differentiation, and degradation of bone matrix by osteoclasts releases growth factors stimulating tumor cell proliferation and invasion. In the present study osteolytic (PC-3) and osteoblastic (LNCaP-19) castration-resistant prostate cancer (CRPC) cells were co-cultured with mature osteoclasts or their precursor cells (RAW 264.7) to characterize direct effects of mature osteoclasts on CRPC cells. Osteoclasts increased proliferation and decrease apoptosis of CRPC cells as assessed with flow cytometry. RNA sequencing revealed that osteolytic CRPC cells were more responsive to osteoclast stimulation regarding gene expression, but the overall induced expression patterns were similar between the prostate cancer cell lines. Genes related to DNA repair were upregulated by osteoclasts, while genes related to endoplasmic reticulum stress-induced apoptosis and cholesterol synthesis were downregulated. The results of this study shows that osteoclasts directly influence CRPC cells, increasing proliferation, decreasing apoptosis, and affecting gene expression pathways that can affect sensitivity to DNA damage and endoplasmic reticulum function. This suggests targeting of osteoclasts to be a possible way to affect efficacy of other drugs by combination regimens in treating prostate cancer metastases.

Androgens, aging, and prostate health.

Due to late onset hypogonadism (LOH), there is an increased usage of testosterone replacement therapy (TRT) in the aging male population. Since prostate is a target organ for androgens and anti-androgenic strategies are used to treat and palliate benign prostate hyperplasia (BPH) and prostate cancer (PC), the prevalence of both increases with age, the possible influence of TRT on prostate health becomes highly relevant. The present review summarizes existing data on the associations between endogenous hormone concentrations and prostate growth and concludes that circulating concentrations of androgens do not appear to be associated with the risks of development of BPH or initiation or progression of PC. The explanation for these findings relates to an apparent insensitivity of prostatic tissue to changes of testosterone concentrations within the physiological range.

Association between pharmaceutical modulation of oestrogen in postmenopausal women in Sweden and death due to COVID-19: a cohort study.

OBJECTIVE: Determine whether augmentation of oestrogen in postmenopausal women decreases the risk of death following COVID-19. DESIGN: Nationwide registry-based study in Sweden based on registries from the Swedish Public Health Agency (all individuals who tested positive for SARS-CoV-2); Statistics Sweden (socioeconomical variables) and the National Board of Health and Welfare (causes of death). PARTICIPANTS: Postmenopausal women between 50 and 80 years of age with verified COVID-19. INTERVENTIONS: Pharmaceutical modulation of oestrogen as defined by (1) women with previously diagnosed breast cancer and receiving endocrine therapy (decreased systemic oestrogen levels); (2) women receiving hormone replacement therapy (increased systemic oestrogen levels) and (3) a control group not fulfilling requirements for group 1 or 2 (postmenopausal oestrogen levels). Adjustments were made for potential confounders such as age, annual disposable income (richest group as the reference category), highest level of education (primary, secondary and tertiary (reference)) and the weighted Charlson Comorbidity Index (wCCI). PRIMARY OUTCOME MEASURE: Death following COVID-19. RESULTS: From a nationwide cohort consisting of 49 853 women diagnosed with COVID-19 between 4 February and 14 September 2020 in Sweden, 16 693 were between 50 and 80 years of age. We included 14 685 women in the study with 11 923 (81%) in the control group, 227 (2%) women in group 1 and 2535 (17%) women in group 2. The unadjusted ORs for death following COVID-19 were 2.35 (95% CI 1.51 to 3.65) for group 1 and 0.45 (0.34 to 0.6) for group 2. Only the adjusted OR for death remained significant for group 2 with OR 0.47 (0.34 to 0.63). Absolute risk of death was 4.6% for the control group vs 10.1% and 2.1%, for the decreased and increased oestrogen groups, respectively. The risk of death due to COVID-19 was significantly associated with: age, OR 1.15 (1.14 to 1.17); annual income, poorest 2.79 (1.96 to 3.97), poor 2.43 (91.71 to 3.46) and middle 1.64 (1.11 to 2.41); and education (primary 1.4 (1.07 to 1.81)) and wCCI 1.13 (1.1 to 1.16). CONCLUSIONS: Oestrogen supplementation in postmenopausal women is associated with a decreased risk of dying from COVID-19 in this nationwide cohort study. These findings are limited by the retrospective and non-randomised design. Further randomised intervention trials are warranted.

A Phase 2 Trial of the Effect of Antiandrogen Therapy on COVID-19 Outcome: No Evidence of Benefit, Supported by Epidemiology and In Vitro Data.

BACKGROUND: Men are more severely affected by COVID-19. Testosterone may influence SARS-CoV-2 infection and the immune response. OBJECTIVE: To clinically, epidemiologically, and experimentally evaluate the effect of antiandrogens on SARS-CoV-2 infection. DESIGNS, SETTINGS, AND PARTICIPANTS: A randomized phase 2 clinical trial (COVIDENZA) enrolled 42 hospitalized COVID-19 patients before safety evaluation. We also conducted a population-based retrospective study of 7894 SARS-CoV-2-positive prostate cancer patients and an experimental study using an air-liquid interface three-dimensional culture model of primary lung cells. INTERVENTION: In COVIDENZA, patients were randomized 2:1 to 5 d of enzalutamide or standard of care. OUTCOME MEASUREMENTS: The primary outcomes in COVIDENZA were the time to mechanical ventilation or discharge from hospital. The population-based study investigated risk of hospitalization, intensive care, and death from COVID-19 after androgen inhibition. RESULTS AND LIMITATIONS: Enzalutamide-treated patients required longer hospitalization (hazard ratio [HR] for discharge from hospital 0.43, 95% confidence interval [CI] 0.20-0.93) and the trial was terminated early. In the epidemiological study, no preventive effects were observed. The frail population of patients treated with androgen deprivation therapy (ADT) in combination with abiraterone acetate or enzalutamide had a higher risk of dying from COVID-19 (HR 2.51, 95% CI 1.52-4.16). In vitro data showed no effect of enzalutamide on virus replication. The epidemiological study has limitations that include residual confounders. CONCLUSIONS: The results do not support a therapeutic effect of enzalutamide or preventive effects of bicalutamide or ADT in COVID-19. Thus, these antiandrogens should not be used for hospitalized COVID-19 patients or as prevention for COVID-19. Further research on these therapeutics in this setting are not warranted. PATIENT SUMMARY: We studied whether inhibition of testosterone could diminish COVID-19 symptoms. We found no evidence of an effect in a clinical study or in epidemiological or experimental investigations. We conclude that androgen inhibition should not be used for prevention or treatment of COVID-19.

Clinical and biological relevance of the transcriptomic-based prostate cancer metastasis subtypes MetA-C.

To improve treatment of metastatic prostate cancer, the biology of metastases needs to be understood. We recently described three subtypes of prostate cancer bone metastases (MetA-C), based on differential gene expression. The aim of this study was to verify the clinical relevance of these subtypes and to explore their biology and relations to genetic drivers. Freshly-frozen metastasis samples were obtained as hormone-naive (n = 17), short-term castrated (n = 21), or castration-resistant (n = 65) from a total of 67 patients. Previously published sequencing data from 573 metastasis samples were also analyzed. Through transcriptome profiling and sample classification based on a set of predefined MetA-C-differentiating genes, we found that most metastases were heterogeneous for the MetA-C subtypes. Overall, MetA was the most common subtype, while MetB was significantly enriched in castration-resistant samples and in liver metastases, and consistently associated with poor prognosis. By gene set enrichment analysis, the phenotype of MetA was described by high androgen response, protein secretion and adipogenesis, MetB by high cell cycle activity and DNA repair, and MetC by epithelial-to-mesenchymal transition and inflammation. The MetB subtype demonstrated single nucleotide variants of RB transcriptional corepressor 1 (RB1) and loss of 21 genes at chromosome 13, including RB1, but provided independent prognostic value to those genetic aberrations. In conclusion, a distinct set of gene transcripts can be used to classify prostate cancer metastases into the subtypes MetA-C. The MetA-C subtypes show diverse biology, organ tropism, and prognosis. The MetA-C classification may be used independently, or in combination with genetic markers, primarily to identify MetB patients in need of complementary therapy to conventional androgen receptor-targeting treatments.

The roles of RUNX2 and osteoclasts in regulating expression of steroidogenic enzymes in castration-resistant prostate cancer cells.

Intratumoral steroidogenesis is involved in development of castration-resistant prostate cancer (CRPC) as bone metastases. The osteoblast transcription factor RUNX2 influences steroidogenesis and is induced in CRPC cells by osteoblasts. This study investigates osteoclastic influence on RUNX2 in intratumoral steroidogenesis. Steroidogenic enzymes and steroid receptors were detected with immunohistochemistry in xenograft intratibial tumors from CRPC cells. In vitro, expression of RUNX2 was increased by osteoclasts in osteoblastic LNCaP-19 cells, but not in osteolytic PC-3. Silencing of RUNX2 downregulates expression of CYP11A1, CYP17A1 and HSD3B1 in LNCaP-19 cells co-cultured with osteoclasts, leading to inhibition of KLK3 expression. Osteoclasts promoted CYP11A1 and RUNX2 promoted AKR1C3, HSD17B3 and CYP19A1, but suppressed ESR2 in PC-3 cells. This study shows that osteoclasts promote RUNX2 regulated induction of key steroidogenic enzymes, influencing activation of androgen receptor in CRPC cells. The potential of RUNX2 as a target to inhibit progression of skeletal metastases of CRPC needs further investigation.

COVIDENZA - A prospective, multicenter, randomized PHASE II clinical trial of enzalutamide treatment to decrease the morbidity in patients with Corona virus disease 2019 (COVID-19): a structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: The main goal of the COVIDENZA trial is to evaluate if inhibition of testosterone signalling by enzalutamide can improve the outcome of patients hospitalised for COVID-19. The hypothesis is based on the observation that the majority of patients in need of intensive care are male, and the connection between androgen receptor signalling and expression of TMPRSS2, an enzyme important for SARS-CoV-2 host cell internalization. TRIAL DESIGN: Hospitalised COVID-19 patients will be randomised (2:1) to enzalutamide plus standard of care vs. standard of care designed to identify superiority. PARTICIPANTS: Included participants, men or women above 50 years of age, must be hospitalised for PCR confirmed COVID-19 symptoms and not in need of immediate mechanical ventilation. Major exclusion criteria are breast-feeding or pregnant women, hormonal treatment for prostate or breast cancer, treatment with immunosuppressive drugs, current symptomatic unstable cardiovascular disease (see Additional file 1 for further details). The trial is registered at Umeå University Hospital, Region Västerbotten, Sweden and 8 hospitals are approved for inclusion in Sweden. INTERVENTION AND COMPARATOR: Patients randomised to the treatment arm will be treated orally with 160 mg (4x40 mg) enzalutamide (Xtandi®) daily, for five consecutive days. The study is not placebo controlled. The comparator is standard of care treatment for patients hospitalised with COVID-19. MAIN OUTCOMES: The primary endpoints of the study are (time to) need of mechanical ventilation or discharge from hospital as assessed by a clinical 7-point ordinal scale (up to 30 days after inclusion). RANDOMISATION: Randomisation was stratified by center and sex. Each strata was randomized separately with block size six with a 2:1 allocation ratio (enzalutamide + "standard of care": "standard of care"). The randomisation list, with consecutive subject numbers, was generated by an independent statistician using the PROC PLAN procedure of SAS version 9.4 software (SAS Institute, Inc, Cary, North Carolina) BLINDING (MASKING): This is an open-label trial. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): The trial is designed to have three phases. The first, an exploration phase of 45 participants (30 treatment and 15 control) will focus on safety and includes a more extensive laboratory assessment as well as more frequent safety evaluation. The second prolongation phase, includes the first 100 participants followed by an interim analysis to define the power of the study. The third phase is the continuation of the study up to maximum 600 participants included in total. TRIAL STATUS: The current protocol version is COVIDENZA v2.0 as of September 10, 2020. Recruitment started July 29, 2020 and is presently in safety pause after the first exploration phase. Recruitment is anticipated to be complete by 31 December 2021. TRIAL REGISTRATION: Eudract number 2020-002027-10 ClinicalTrials.gov Identifier: NCT04475601 , registered June 8, 2020 FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

RGS2 is prognostic for development of castration resistance and cancer-specific survival in castration-resistant prostate cancer.

BACKGROUND: Regulator of G-protein signaling 2 (RGS2) is a multifaceted protein with a prognostic value in hormone-naïve prostate cancer (PC). It has previously been associated with the development of castration resistance. However, RGS2 expression in clinical specimens of castration-resistant prostate cancer (CRPC) and its clinical relevance has not been explored. In the present study, RGS2 was assessed in CRPC and in relation to the development of castration resistance. METHODS: In the present study, RGS2 expression was evaluated with immunohistochemistry in patient materials of hormone-naïve and castration-resistant primary tumors, also in matched specimens before and after 3 months of androgen deprivation therapy (ADT). Cox regression and Kaplan-Meier curves were used to evaluate the clinical significance of RGS2 expression. RGS2 expression in association to castration-resistant growth was assessed experimentally in an orthotopic xenograft mouse model of CRPC. In vitro, hormone depletion of LNCaP and enzalutamide treatment of LNCaP, 22Rv1, and VCaP was performed to evaluate the association between RGS2 and the androgen receptor (AR). Stable RGS2 knockdown was used to evaluate the impact of RGS2 in association to PC cell growth under hormone-reduced conditions. Gene and protein expression were evaluated with quantitative polymerase chain reaction and Western blot analysis, respectively. RESULTS: RGS2 expression is increased in CRPC and enriched under ADT. Furthermore, a high RGS2 level is prognostic for poor cancer-specific survival for CRPC patients and significantly reduced failure-free survival (FFS) after an initiated ADT. Additionally, the prognostic value of RGS2 outperforms prostate-specific antigen (PSA) in terms of FFS. The present study furthermore suggests that RGS2 expression is reflective of AR activity. Moreover, low RGS2-expressing cells display hampered growth under hormone-reduced conditions, in line with the poor prognosis associated with high RGS2 expression. CONCLUSIONS: High levels of RGS2 are associated with aggressive forms of castration-resistant PC. The results demonstrate that a high level of RGS2 is associated with poor prognosis in association with castration-resistant PC growth. RGS2 alone, or in association with PSA, has the potential to identify patients that require additional treatment at an early stage during ADT.

Targeted alpha therapy with astatine-211-labeled anti-PSCA A11 minibody shows antitumor efficacy in prostate cancer xenografts and bone microtumors.

PURPOSE: Targeted alpha therapy (TAT) is a promising treatment for micrometastatic and minimal residual cancer. We evaluated systemic α-radioimmunotherapy (α-RIT) of metastatic castration-resistant prostate cancer (mCRPC) using the α-particle emitter 211At-labeled to the anti-PSCA A11 minibody. A11 is specific for prostate stem cell antigen (PSCA), a cell surface glycoprotein which is overexpressed in more than 90% of both localized prostate cancer and bone metastases. METHODS: PC3-PSCA cells were implanted subcutaneously (s.c.) and intratibially (i.t) in nude mice. Efficacy of α-RIT (two fractions-14-day interval) was studied on s.c. macrotumors (0, 1.5 and 1.9 MBq) and on i.t. microtumors (~100-200 µm; 0, 0.8 or 1.5 MBq) by tumor-volume measurements. The injected activities for therapies were estimated from separate biodistribution and myelotoxicity studies. RESULTS: Tumor targeting of 211At-A11 was efficient and the effect on s.c. macrotumors was strong and dose-dependent. At 6 weeks, the mean tumor volumes for the treated groups, compared with controls, were reduced by approximately 85%. The separate myelotoxicity study following one single fraction showed reduced white blood cells (WBC) for all treated groups on day 6 after treatment. For the 0.8 and 1.5 MBq, the WBC reductions were transient and followed by recovery at day 13. For 2.4 MBq, a clear toxicity was observed and the mice were sacrificed on day 7. In the long-term follow-up of the 0.8 and 1.5 MBq-groups, blood counts on day 252 were normal and no signs of radiotoxicity observed. Efficacy on i.t. microtumors was evaluated in two experiments. In experiment 1, the tumor-free fraction (TFF) was 95% for both treated groups and significantly different (p < 0.05) from the controls at a TFF of 66%). In experiment 2, the difference in TFF was smaller, 32% for the treated group versus 20% for the controls. However, the difference in microtumor volume in experiment 2 was highly significant, 0.010 ± 0.003 mm3 versus 3.79 ± 1.24 mm3 (treated versus controls, respectively), i.e., a 99.7% reduction (p < 0.001). The different outcome in experiment 1 and 2 is most likely due to differences in microtumor sizes at therapy, or higher tumor-take in experiment 2 (where more cells were implanted). CONCLUSION: Evaluating fractionated α-RIT with 211At-labeled anti-PSCA A11 minibody, we found clear growth inhibition on both macrotumors and intratibial microtumors. For mice treated with multiple fractions, we also observed radiotoxicity manifested by progressive loss in body weight at 30 to 90 days after treatment. Our findings are conceptually promising for a systemic TAT of mCRPC and warrant further investigations of 211At-labeled PSCA-directed vectors. Such studies should include methods to improve the therapeutic window, e.g., by implementing a pretargeted regimen of α-RIT or by altering the size of the targeting vector.

Gene Expression Alterations during Development of Castration-Resistant Prostate Cancer Are Detected in Circulating Tumor Cells.

Development of castration-resistant prostate cancer (CRPC) is associated with alterations in gene expression involved in steroidogenesis and androgen signaling. This study investigates whether gene expression changes related to CRPC development can be identified in circulating tumor cells (CTCs). Gene expression in paired CTC samples from 29 patients, before androgen deprivation therapy (ADT) and at CRPC relapse, was compared using a panel including 47 genes related to prostate cancer progression on a qPCR platform. Fourteen genes displayed significantly changed gene expression in CTCs at CRPC relapse compared to before start of ADT. The genes with increased expression at CRPC relapse were related to steroidogenesis, AR-signaling, and anti-apoptosis. In contrast, expression of prostate markers was downregulated at CRPC. We also show that midkine (MDK) expression in CTCs from metastatic hormone-sensitive prostate cancer (mHSPC) was associated to short cancer-specific survival (CSS). In conclusion, this study shows that gene expression patterns in CTCs reflect the development of CRPC, and that MDK expression levels in CTCs are prognostic for cancer-specific survival in mHSPC. This study emphasizes the role of CTCs in exploring mechanisms of therapy resistance, as well as a promising biomarker for prognostic and treatment-predictive purposes in advanced mHSPC.

Analysis of regulator of G-protein signalling 2 (RGS2) expression and function during prostate cancer progression.

Prostate cancer (PC) represents the second highest cancer-related mortality among men and the call for biomarkers for early discrimination between aggressive and indolent forms is essential. Downregulation of Regulator of G-protein signaling 2 (RGS2) has been shown in PC, however the underlying mechanism has not been described. Aberrant RGS2 expression has also been reported for other carcinomas in association to both positive and negative prognosis. In this study, we assessed RGS2 expression during PC progression in terms of regulation and impact on tumour phenotype and evaluated its prognostic value. Our experimental data suggest that the RGS2 downregulation seen in early PC is caused by hypoxia. In line with the common indolent phenotype of a primary PC, knockdown of RGS2 induced epithelial features and impaired metastatic properties. However, increased STAT3, TWIST1 and decreased E-cadherin expression suggest priming for EMT. Additionally, improved tumour cell survival and increased BCL-2 expression linked decreased RGS2 levels to fundamental tumour advantages. In contrast, high RGS2 levels in advanced PC were correlated to poor patient survival and a positive metastatic status. This study describes novel roles for RGS2 during PC progression and suggests a prognostic potential discriminating between indolent and metastatic forms of PC.

Circulating tumor cells mirror bone metastatic phenotype in prostate cancer.

Circulating tumor cells (CTCs) are promising biomarkers in prostate cancer (PC) because they derive from primary tumor and metastatic tissues. In this study, we used quantitative real-time PCR (qPCR) to compare the expression profiles of 41 PC-related genes between paired CTC and spinal column metastasis samples from 22 PC patients that underwent surgery for spinal cord compression. We observed good concordance between the gene expression profiles in the CTC and metastasis samples in most of the PC patients. Expression of nine genes (AGR2, AKR1C3, AR, CDH1, FOLH1, HER2, KRT19, MDK, and SPINK1) showed a significant correlation between the CTC and metastasis samples. Hierarchical clustering analysis showed a similar grouping of PC patients based on the expression of these nine genes in both CTC and metastasis samples. Our findings demonstrate that CTCs mirror gene expression patterns in tissue metastasis samples from PC patients. Although low detection frequency of certain genes is a limitation in CTCs, our results indicate the potential for CTC phenotyping as a tool to improve individualized therapy in metastatic prostate cancer.