A phase I prospective, non-randomized trial of autologous dendritic cell-based cryoimmunotherapy in patients with metastatic castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is an immunologically cold disease with dismal outcomes. Cryoablation destroys cancer tissue, releases tumor-associated antigens and creates a pro-inflammatory microenvironment, while dendritic cells (DCs) activate immune responses through processing of antigens. Immunotherapy combinations could enhance the anti-tumor efficacy. This open-label, single-arm, single-center phase I trial determined the safety and tolerability of combining cryoablation and autologous immature DC, without and with checkpoint inhibitors. Immune responses and clinical outcomes were evaluated. Patients with mCRPC, confirmed metastases and intact prostate gland were included. The first participants underwent prostate cryoablation with intratumoral injection of autologous DCs in a 3 + 3 design. In the second part, patients received cryoablation, the highest acceptable DC dose, and checkpoint inhibition with either ipilimumab or pembrolizumab. Sequentially collected information on adverse events, quality of life, blood values and images were analyzed by standard descriptive statistics. Neither dose-limiting toxicities nor adverse events > grade 3 were observed in the 18 participants. Results indicate antitumor activity through altered T cell receptor repertoires, and 33% durable (> 46 weeks) clinical benefit with median 40.7 months overall survival. Post-treatment pain and fatigue were associated with circulating tumor cell (CTC) presence at inclusion, while CTC responses correlated with clinical outcomes. This trial demonstrates that cryoimmunotherapy in mCRPC is safe and well tolerated, also for the highest DC dose (2.0 × 108) combined with checkpoint inhibitors. Further studies focusing on the biologic indications of antitumor activity and immune system activation could be considered through a phase II trial focusing on treatment responses and immunologic biomarkers.

PRSS2 remodels the tumor microenvironment via repression of Tsp1 to stimulate tumor growth and progression.

The progression of cancer from localized to metastatic disease is the primary cause of morbidity and mortality. The interplay between the tumor and its microenvironment is the key driver in this process of tumor progression. In order for tumors to progress and metastasize they must reprogram the cells that make up the microenvironment to promote tumor growth and suppress endogenous defense systems, such as the immune and inflammatory response. We have previously demonstrated that stimulation of Tsp-1 in the tumor microenvironment (TME) potently inhibits tumor growth and progression. Here, we identify a novel tumor-mediated mechanism that represses the expression of Tsp-1 in the TME via secretion of the serine protease PRSS2. We demonstrate that PRSS2 represses Tsp-1, not via its enzymatic activity, but by binding to low-density lipoprotein receptor-related protein 1 (LRP1). These findings describe a hitherto undescribed activity for PRSS2 through binding to LRP1 and represent a potential therapeutic strategy to treat cancer by blocking the PRSS2-mediated repression of Tsp-1. Based on the ability of PRSS2 to reprogram the tumor microenvironment, this discovery could lead to the development of therapeutic agents that are indication agnostic.

MicroRNAs in Differentiation of Embryoid Bodies and the Teratoma Subtype of Testicular Cancer.

BACKGROUND: Testicular germ cell tumours (TGCTs) are the most frequent tumour type among young, adult men. TGCTs can be efficiently treated, but metastases of the teratoma subtype, for which there are no circulating biomarkers, represent a challenge. MATERIALS AND METHODS: Global microRNA expression in teratoma tissue and embryoid bodies was assessed using next-generation sequencing. Levels of microRNAs identified as potential biomarkers were obtained from serum of patients with teratoma and matched healthy men. RESULTS: We identified miR-222-5p, miR-200a-5p, miR-196b-3p and miR-454-5p as biomarker candidates from the tumour tissue and embryoid body screening but the expression of these microRNAs was very low in serum and not statistically different between patients and controls. miR-375-3p was highly expressed, being highest in patients with teratoma (p=0.012) but the levels of expression in serum from these patients and healthy controls overlapped. miR-371a-3p was not expressed in serum from patients with pure teratoma, only in patients with mixed tumours. CONCLUSION: The microRNA profiles of the teratoma subtype of TGCT and embryoid bodies were obtained and assessed for candidate circulating biomarkers, but none with high sensitivity and specificity for teratoma were identified in our study. We conclude that neither the proposed teratoma marker miR-375-3p nor miR-371a-3p are suitable as circulating teratoma markers.

Ten-Year Results From a Phase II Study on Image Guided, Intensity Modulated Radiation Therapy With Simultaneous Integrated Boost in High-Risk Prostate Cancer.

PURPOSE: There is no consensus on how to treat high-risk prostate cancer, and long-term results from hypofractionated radiation therapy are lacking. We report 10-year results after image guided, intensity modulated radiation therapy with hypofractionated simultaneous integrated boost and elective pelvic field. METHODS AND MATERIALS: Between 2007 and 2009, 97 consecutive patients with high-risk prostate cancer were included, treated with 2.7 to 2.0 Gy × 25 Gy to the prostate, seminal vesicles, and elective pelvic field. Toxicity was scored according to Radiation Therapy Oncology Group criteria and biochemical disease-free survival (BFS) defined by the Phoenix definition. Patients were subsequently divided into 3 groups: high risk (HR; n = 32), very high risk (VHR; n = 50), and N+/s-prostate-specific antigen (PSA) ≥100 (n = 15). Differences in outcomes were examined using Kaplan-Meier analyses. RESULTS: BFS in the patients at HR and VHR was 64%, metastasis-free survival 80%, prostate cancer-specific survival 90%, and overall survival (OS) 72%. VHR versus HR subgroups demonstrated significantly different BFS, 54% versus 79% (P = .01). Metastasis-free survival and prostate cancer-specific survival in the VHR group versus HR group were 76% versus 87% (P = .108) and 74% versus 100% (P = .157). Patients reaching nadir PSA <0.1 (n = 80) had significantly better outcomes than the rest (n = 17), with BFS 70% versus 7% (P < .001). Acute grade 2 gastrointestinal tract (GI) and genitourinary tract (GU) toxicity occurred in 27% and 40%, grade 3 GI and GU toxicity in 1% and 3%. Late GI and GU grade 2 toxicity occurred in 1% and 8%. CONCLUSIONS: High-risk prostate cancer patients obtained favorable 10-year outcomes with low toxicity. There were significantly better results in the HR versus the VHR group, both better than the N+/PSA ≥100 group. A nadir PSA value < 0.1 predicted good prognosis.

Novel protein signatures suggest progression to muscular invasiveness in bladder cancer.

Patients with bladder cancer need frequent controls over long follow-up time due to high recurrence rate and risk of conversion to muscle invasive cancer with poor prognosis. We identified cancer-related molecular signatures in apparently healthy bladder in patients with subsequent muscular invasiveness during follow-up. Global proteomics of the normal tissue biopsies revealed specific proteome fingerprints in these patients prior to subsequent muscular invasiveness. In these presumed normal samples, we detected modulations of proteins previously associated with different cancer types. This study indicates that analyzing apparently healthy tissue of a cancer-invaded organ may suggest disease progression.

Outcome in intermediate or high risk prostate cancer patients receiving radiation dose and hormone therapy.

BACKGROUND: To analyse the impact of radiation dose escalation and hormone treatment in prostate cancer patients according to risk groups. MATERIAL AND METHODS: Totally 494 prostate cancer patients received external beam radiation therapy, with or without androgen deprivation, between January 1990 and December 1999. The patients were divided into three risk groups, where the low risk group (stage T(1c), pretreatment prostate-specific antigen (PSA) level < or =10 ng/ml and WHO Grade 1) included 26 patients, the intermediate risk group (either stage T(2), PSA 10.1-20 ng/ml or WHO Grade 2) comprised 149 patients whereas the high-risk group (either stage T(3), PSA >20 ng/ml or WHO Grade 3) included 319 patients. RESULTS: In the intermediate risk group, the 5-years bNED rate was 92%, 69% and 61% after a radiation dose of 70 Gy, 66 Gy or 64 Gy, respectively (p < 0.001). In the high-risk group, the 5-year bNED rate was 79%, 69% and 34% for the same dose levels (p < 0.001). The 5-years CSS rates were not significantly different between the dose levels in the intermediate risk group while for the high-risk group it was 93%, 92% and 80% for the three dose levels (p < 0.001). Risk group and radiation doses were independent predictors of bNED, CSS and overall survival, for bNED also hormone treatment was independent predictors. CONCLUSION: Radiation dose is important for the outcome in intermediate and high risk prostate cancer patients. A dose of 70 Gy should be considered the minimal dose for these patients.

Genome-wide profiling of histone h3 lysine 4 and lysine 27 trimethylation reveals an epigenetic signature in prostate carcinogenesis.

BACKGROUND: Increasing evidence implicates the critical roles of epigenetic regulation in cancer. Very recent reports indicate that global gene silencing in cancer is associated with specific epigenetic modifications. However, the relationship between epigenetic switches and more dynamic patterns of gene activation and repression has remained largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Genome-wide profiling of the trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) was performed using chromatin immunoprecipitation coupled with whole genome promoter microarray (ChIP-chip) techniques. Comparison of the ChIP-chip data and microarray gene expression data revealed that loss and/or gain of H3K4me3 and/or H3K27me3 were strongly associated with differential gene expression, including microRNA expression, between prostate cancer and primary cells. The most common switches were gain or loss of H3K27me3 coupled with low effect on gene expression. The least prevalent switches were between H3K4me3 and H3K27me3 coupled with much higher fractions of activated and silenced genes. Promoter patterns of H3K4me3 and H3K27me3 corresponded strongly with coordinated expression changes of regulatory gene modules, such as HOX and microRNA genes, and structural gene modules, such as desmosome and gap junction genes. A number of epigenetically switched oncogenes and tumor suppressor genes were found overexpressed and underexpressed accordingly in prostate cancer cells. CONCLUSIONS/SIGNIFICANCE: This work offers a dynamic picture of epigenetic switches in carcinogenesis and contributes to an overall understanding of coordinated regulation of gene expression in cancer. Our data indicate an H3K4me3/H3K27me3 epigenetic signature of prostate carcinogenesis.

Comparison of nucleic acid targets prepared from total RNA or poly(A) RNA for DNA oligonucleotide microarray hybridization.

The aim of this work was to compare DNA microarray results using either total RNA or affinity-purified poly(A) RNA from the same biological sample for target preparation. The high-density oligonucleotide microarrays of both Agilent Technologies (based on two-color detection) and Applied Biosystems (based on single-color detection) were evaluated. Real-time quantitative PCR was used to quantify messenger RNA (mRNA) and ribosomal RNA (rRNA) at different stages of target preparations. Poly(A) RNA versus total RNA target hybridizations exhibited slightly lower correlation coefficients than did self versus self hybridizations (i.e., poly(A) RNA targets vs. poly(A) RNA targets or total RNA targets vs. total RNA targets). Only a small fraction of all transcripts appeared to be significantly over- or underrepresented when total RNA targets or poly(A) RNA targets from the same biological sample were compared. Therefore, the conclusion is that poly(A) affinity purification from total RNA can be omitted during target preparation for routine mRNA expression analysis using high-density oligonucleotide microarrays. Among consistently overrepresented transcripts in total RNA targets were histone mRNAs known to lack poly(A) tails. Therefore, structurally exceptional RNA species can be identified by comparing targets derived from either poly(A) RNA or total RNA using microarray hybridization.

Increased expression of SIM2-s protein is a novel marker of aggressive prostate cancer.

PURPOSE: The human SIM2 gene is located within the Down's syndrome critical region of chromosome 21 and encodes transcription factors involved in brain development and neuronal differentiation. SIM2 has been assigned a possible role in the pathogenesis of solid tumors, and the SIM2-short isoform (SIM2-s) was recently proposed as a molecular target for cancer therapy. We previously reported SIM2 among the highly up-regulated genes in 29 prostate cancers, and the purpose of our present study was to examine the expression status of SIM2 at the transcriptional and protein level as related to outcome in prostate cancer. EXPERIMENTAL DESIGN: By quantitative PCR, mRNA in situ hybridization, and immunohistochemistry, we evaluated the expression and significance of SIM2 isoforms in 39 patients with clinically localized prostate cancer and validated the expression of SIM2-s protein in an independent cohort of 103 radical prostatectomies from patients with long and complete follow-up. RESULTS: The SIM2 isoforms (SIM2-s and SIM2-l) were significantly coexpressed and increased in prostate cancer. Tumor cell expression of SIM2-s protein was associated with adverse clinicopathologic factors like increased preoperative serum prostate-specific antigen, high histologic grade, invasive tumor growth with extra-prostatic extension, and increased tumor cell proliferation by Ki-67 expression. SIM2-s protein expression was significantly associated with reduced cancer-specific survival in multivariate analyses. CONCLUSIONS: These novel findings indicate for the first time that SIM2 expression might be important for clinical progress of human cancer and support the recent proposal of SIM2-s as a candidate for targeted therapy in prostate cancer.

ERG upregulation and related ETS transcription factors in prostate cancer.

The aim of this study was to identify and validate differentially expressed genes in matched pairs of benign and malignant prostate tissue. Samples included 29 histologically verified primary tumors and 23 benign controls. Microarray analysis was initially performed using a sequence verified set of 40,000 human cDNA clones. Among the genes most consistently and highly upregulated in prostate cancer was the ETS family transcription factor ERG (ETS related gene). This finding was validated in an expanded patient series (37 tumors and 38 benign samples) using DNA oligonucleotide microarray and real-time quantitative PCR assays. ERG was 20- to more than 100-fold overexpressed in prostate cancer compared with benign prostate tissue in more than 50% of patients according to quantitative PCR. Surprisingly, ERG mRNA levels were found to be significantly higher in the endothelial cell line, HUVEC, than in the prostate cell lines PC3, DU145 and LNCaP. In situ hybridization of prostate cancer tissue revealed that ERG was abundantly expressed in both prostate cancer cells and associated endothelial cells. The consistency and magnitude of ERG overexpression in prostate cancer appeared unique, but several related ETS transcription factors were also overexpressed in matched pairs of tumor and benign samples, whereas ETS2 was significantly underexpressed. Our findings support the hypothesis that ERG overexpression and related ETS transcription factors are important for early prostate carcinogenesis.

A concomitant tumour boost in bladder irradiation: patient suitability and the potential of intensity-modulated radiotherapy.

BACKGROUND AND PURPOSE: In radiotherapy (RT) of bladder cancer, dose escalation without increased adverse effects could be achieved with a concomitant bladder tumour boost. In this study we quantified (1) the fraction of patients suitable for this approach, and (2) the potential of intensity-modulated RT (IMRT) to achieve this boost while also sparing normal tissues. MATERIALS AND METHODS: The fraction of patients suitable for this boost approach was quantified using both a series of 30 radical therapy candidates, and a series of 15 consecutive RT patients. IMRT plans with 3, 5, 7 and 9 equi-spaced beams were set up for the patients in the RT series found suitable for a boost. Two sets of targets were defined, with (i) wide and (ii) narrow margins around both the tumour (prescribed 120% dose) and the non-involved bladder (prescribed 100% dose). The inverse planning optimisation minimised the dose deviation across the targets whilst fulfilling dose-volume histogram (DVH) constraints--based on what could be achieved with conformal RT (CRT)--for both the normal tissues and the targets. RESULTS: Fourteen of the 30 radical therapy candidates (47%) and 10 of the 15 RT patients (67%) were suitable for a boost. The 20% boost could be obtained while maintaining target coverage with at least one IMRT plan in 9 of 10 cases with wide margins and for all 10 cases with narrow margins. Using wide margins, all 3-field plans were unacceptable, the 5-field plans were acceptable for 5 of 10, and the 7- and 9-field plans for 9 of the 10 patients. The normal tissue volumes receiving doses >100% were on average reduced by a factor of 3-4 compared with CRT. The normal tissue volumes receiving intermediate doses (73-88%) decreased slightly, whereas volumes receiving the lowest doses (30-48%) increased with the number of beams. The use of narrow margins resulted in markedly lower normal tissue irradiation. CONCLUSION: This study has shown bladder tumour boosting to be both clinically relevant and technically feasible using IMRT. This approach is ready for clinical implementation, although further improvement could be expected if integrated with target localisation techniques.

Radiation dose escalation combined with hormone therapy improves outcome in localised prostate cancer.

We present the impact of systematic radiation dose escalation from 64 Gy to 66 Gy to 70 Gy on the outcome after radiation therapy (RT) alone or combined with hormonal treatment (HT) in a series of 494 consecutive localised prostate cancer patients treated during 1990-1999. Prognostic factors for prostate-specific antigen (PSA) failure, overall survival (OS) and prostate cancer specific survival (CSS) were investigated using multivariate analysis. T stage, pre-treatment PSA, grade, radiation dose and HT were found to be independent predictors of PSA failure. T stage, grade and HT were also independent predictors of both OS and CSS, while radiation dose was a significant predictor for OS and indicated a trend (p = 0.07) for CSS. A dose of 70 Gy combined with hormonal treatment improves PSA failure free survival and survival in localised prostate cancer compared with doses of 64-66 Gy.

Predictors of prostate cancer evaluated by receiver operating characteristics partial area index: a prospective institutional study.

PURPOSE: To our knowledge we introduce the ROC partial area under the curve (AUC) index as a method of evaluating the discriminative power of different prostate cancer predictors. Peripheral zone volume and peripheral zone prostate specific antigen (PSA) density are introduced as potential predictors and compared with other known predictors of prostate cancer. MATERIALS AND METHODS: During 1999, 220 consecutive patients with suspected early prostate cancer were examined using total PSA, free PSA, total prostate volume, transition zone volume and transrectal ultrasonography guided sextant biopsy of the prostate. The free-to-total PSA ratio, PSA density, transition zone PSA density, peripheral zone volume and peripheral zone PSA density were calculated. Usually total AUC is used to evaluate the discriminative power of different parameters. In this study parameters were evaluated by the ROC partial area index, which includes only the AUC in highly sensitivity parts of the ROC curve. Explorative analysis using logistic regression analysis was performed to investigate the ability of combinations of parameters to predict cancer. RESULTS: Of the 220 patients 75 were diagnosed with cancer. In the subgroup of 160 patients with PSA less than 10 microg/l 44 had cancer. Transition zone PSA density and PSA density had significant discriminative power in the total group, while none of the parameters were discriminative in the subgroup of patients. CONCLUSIONS: When high sensitivity is demanded, the ROC partial area index seems to be meaningful for evaluating the discriminative power of potential predictors. In our study none of the evaluated parameters had discriminative power in patients with PSA less than 10 microg/l, while transition zone PSA density and PSA density showed discriminative power in the total group of patients.

Gene expression profiles in prostate cancer: association with patient subgroups and tumour differentiation.

Prostate carcinoma is the most common cancer of western men and is a markedly heterogeneous disease. The aim of this study was to identify signatures of differentially expressed genes in prostate cancer using DNA microarray technology, evaluating expression profiles in matched pairs of benign and malignant tissue. Samples were collected from 33 radical prostatectomies, and 52 specimens were included, representing 29 histologically verified primary tumours, 19 paired samples of malignant and benign tissue, and 4 non-paired benign tissue samples. Microarray analysis was performed using an expanded sequence verified set of 40,000 human cDNA clones, revealing several genes with significant differences between malignant and benign tissue, including recently reported genes like alpha-methylacyl-CoA racemase (AMACR) and hepsin, as well as genes relevant for tumour development and progression. Leave out cross validation (LOCV) test correctly predicted tumour or benign tissue in 47 (90.3%) out of 52 cases, significantly better than cross validation tests using randomly permuted tissue labels. Unsupervised clustering analysis revealed 3 distinct patient clusters significantly associated with Gleason score, and high grade tumours (Gleason score >/=7) accumulated in cluster 1 (C1). Gene expression profiles correctly predicted 100% of tumour samples segregating to C1, as also validated by LOCV. Gene expression profiles were analysed in filtered and floored datasets with similar results, and a pair-wise design was also tested. Gene expression profiles provided tumour clusters linked to differentiation, and revealed novel markers relevant for molecular classification, grading and therapy of prostate cancer.

Benign growth of different prostate zones in aging men with slightly elevated PSA in whom prostate cancer has been excluded: a prospective study of 510 patients.

OBJECTIVES: To study, in a selected series of patients, whether the peripheral/central zone volumes also change with age. The reported normal total prostate volume in the third decade seems not to exceed 25 to 30 cm(3). Benign prostatic hyperplasia is generally accepted to originate in the transition zone and periurethral tissue, which accordingly show substantial growth with age. METHODS: From January 1997 through December 2000, we performed transrectal ultrasound volume measurements of the different prostate zones in patients admitted for suspected prostate cancer. The information was registered according to a prospectively designed protocol. A total of 872 patients were examined, of whom 360 shown to have prostate cancer were excluded. Two more patients were excluded because of missing volume data, leaving 510 patients with noncancerous prostates for inclusion in this study. RESULTS: The mean age was 62.8 years and the mean total prostate-specific antigen level was 9.8 microg/L. It was found that 64.9% (331 of 510) had a peripheral/central zone volume larger than 30 cm(3), indicating that some form of growth had occurred also in this zone. A weak but significant correlation was found between age and the peripheral/central zone volume. CONCLUSIONS: The results of our study indicate that the peripheral/central zone may contribute to the benign growth of the prostate gland in men younger than 70 years old with slightly elevated total prostate-specific antigen levels.