Genetic signature of prostate cancer mouse models resistant to optimized hK2 targeted α-particle therapy.

Hu11B6 is a monoclonal antibody that internalizes in cells expressing androgen receptor (AR)-regulated prostate-specific enzyme human kallikrein-related peptidase 2 (hK2; KLK2). In multiple rodent models, Actinium-225-labeled hu11B6-IgG1 ([225Ac]hu11B6-IgG1) has shown promising treatment efficacy. In the present study, we investigated options to enhance and optimize [225Ac]hu11B6 treatment. First, we evaluated the possibility of exploiting IgG3, the IgG subclass with superior activation of complement and ability to mediate FC-γ-receptor binding, for immunotherapeutically enhanced hK2 targeted α-radioimmunotherapy. Second, we compared the therapeutic efficacy of a single high activity vs. fractionated activity. Finally, we used RNA sequencing to analyze the genomic signatures of prostate cancer that progressed after targeted α-therapy. [225Ac]hu11B6-IgG3 was a functionally enhanced alternative to [225Ac]hu11B6-IgG1 but offered no improvement of therapeutic efficacy. Progression-free survival was slightly increased with a single high activity compared to fractionated activity. Tumor-free animals succumbing after treatment revealed no evidence of treatment-associated toxicity. In addition to up-regulation of canonical aggressive prostate cancer genes, such as MMP7, ETV1, NTS, and SCHLAP1, we also noted a significant decrease in both KLK3 (prostate-specific antigen ) and FOLH1 (prostate-specific membrane antigen) but not in AR and KLK2, demonstrating efficacy of sequential [225Ac]hu11B6 in a mouse model.

Improved cancer specificity in PSA assay using Aleuria aurantia lectin coated Eu-nanoparticles for detection.

OBJECTIVES: The objective was to study the differences in PSA fucosylation obtained from LNCaP and PC-3 prostate cancer cell lines, seminal plasma PSA and recombinant precursor form of PSA expressed in baculovirus, using Aleuria aurantia lectin (AAL). The aim was to assess whether differences in fucosylation (Fucα1-6/3GlcNAc carbohydrates) of PSA either in urine, blood or tissue enable the discrimination of patients with prostate cancer (PCa) from benign prostatic hyperplasia (BPH) and young males. DESIGN AND METHODS: Two novel lectin-immunoassays were developed for the analysis of fucosylation of PSA by measuring the time-resolved fluorescence of europium chelate. The lectin-immunoassays utilize free PSA-specific Fab-fragments for capturing the analyte and either europium-labeled AAL or AAL coupled to Eu(III)-chelate-dyed nanoparticles for the detection of Fucα1-6/3GlcNAc carbohydrates on PSA. RESULTS: Using the novel lectin-immunoassays, we showed higher levels of Fucα1-6/3GlcNAc on PSA derived from LNCaP and PC-3 cells compared to seminal plasma PSA. With the more sensitive nanoparticle-based lectin-immunoassay we detected a statistically significant increase in the PSA fucosylation in PCa tissue compared to benign tissue (p=0.001) and in urine from PCa patients compared to BPH patients (p=0.030), and an even greater discrimination (p=0.010) when comparing BPH patients to PCa patients with Gleason score≥7. CONCLUSIONS: AAL coupled to Eu(III)-chelate-dyed nanoparticles improved the sensitivity of immunoassay for the detection of Fucα1-6/3GlcNAc structures on PSA. The preliminary findings show an increased fucosylation in PCa compared to benign conditions. Further validation is required to assess the true clinical utility of AAL in PCa diagnosis.

Quantitative Time-Resolved Fluorescence Imaging of Androgen Receptor and Prostate-Specific Antigen in Prostate Tissue Sections.

Androgen receptor (AR) and prostate-specific antigen (PSA) are expressed in the prostate and are involved in prostate cancer (PCa). The aim of this study was to develop reliable protocols for reproducible quantification of AR and PSA in benign and malignant prostate tissue using time-resolved fluorescence (TRF) imaging techniques. AR and PSA were detected with TRF in tissue microarrays from 91 PCa patients. p63/ alpha-methylacyl-CoA racemase (AMACR) staining on consecutive sections was used to categorize tissue areas as benign or cancerous. Automated image analysis was used to quantify staining intensity. AR intensity was significantly higher in AMACR+ and lower in AMACR- cancer areas as compared with benign epithelium. The PSA intensity was significantly lower in cancer areas, particularly in AMACR- glands. The AR/PSA ratio varied significantly in the AMACR+ tumor cells as compared with benign glands. There was a trend of more rapid disease progression in patients with higher AR/PSA ratios in the AMACR- areas. This study demonstrates the feasibility of developing reproducible protocols for TRF imaging and automated image analysis to study the expression of AR and PSA in benign and malignant prostate. It also highlighted the differences in AR and PSA protein expression within AMACR- and AMACR+ cancer regions.

Immuno-based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer.

Extracellular vesicles (including the subclass exosomes) secreted by cells contain specific proteins and RNA that could be of interest in determining new markers. Isolation/characterization of PCa-derived exosomes from bodily fluids enables us to discover new markers for this disease. Unfortunately, isolation with current techniques (ultracentrifugation) is labor intensive and other techniques are still under development. The goal of our study was to develop a highly sensitive time-resolved fluorescence immunoassay (TR-FIA) for capture/detection of PCa-derived exosomes. In our assay, biotinylated capture antibodies against human CD9 or CD63 were incubated on streptavidin-coated wells. After application of exosomes, Europium-labeled detection antibodies (CD9 or CD63) were added. Cell medium from 37 cell lines was taken to validate this TR-FIA. Urine was collected (after digital rectal exam) from patients with PCa (n = 67), men without PCa (n = 76). As a control, urine was collected from men after radical prostatectomy (n = 13), women (n = 16) and patients with prostate cancer without digital rectal exam (n = 16). Signal intensities were corrected for urinary PSA and creatinine. This TR-FIA can measure purified exosomes with high sensitivity and minimal background signals. Exosomes can be measured in medium from 37 cell lines and in urine. DRE resulted in a pronounced increase in CD63 signals. After DRE and correction for urinary PSA, CD9 and CD63 were significantly higher in men with PCa. This TR-FIA enabled us to measure exosomes with high sensitivity directly from urine and cell medium. This TR-FIA forms the basis for testing different antibodies directed against exosome membrane markers to generate disease-specific detection assays.

Phage display aided improvement of a unique prostate-specific antigen (PSA) antibody unreactive with Lys(145)-Lys(146) internally cleaved forms.

Prostate specific antigen (PSA) is a commonly used marker of prostate cancer. A panel of four kallikrein immunoassays has been reported to improve the prediction of prostate biopsy outcome (cancer vs benign) in men with elevated PSA in the circulation. Assay of one of the kallikrein forms, intact free PSA (fPSA-I), is based on a unique monoclonal antibody (4D4), which is specific for PSA without the internal cleavage at Lys(145)-Lys(146). Due to high dissociation rate the 4D4 antibody is less than optimal for achieving a highly sensitive robust assay. In this study, we cloned the 4D4 Mab into a recombinant fragment (Fab) format and constructed three mutant libraries with the aim to increase its binding affinity. The libraries contained targeted mutations either in the CDR-H1, CDR-H2 or CDR-L3 region. PSA-I specific antibodies were enriched from the libraries by phage display technology. We identified fourteen unique clones with 1-5 mutated amino acids showing reduced dissociation of the PSA conjugate compared to the wt-4D4 Fab. Five of these mutant antibodies had 2-6 times higher binding affinity compared to the wt-4D4 Fab yet retaining the original specificity for PSA-I. The analytical sensitivity of fPSA-I assay with mutant L3-2 Fab was 0.12 µg/L compared to 4.46 µg/L with the original wt-4D4 Fab. In the method comparison study, the developed assay showed an excellent correlation to the existing fPSA-I assay. The high affinity and specificity of these mutant antibodies have potential to provide sensitive and robust detection of intact and nicked PSA from patient samples in different test formats.

Can one blood draw replace transrectal ultrasonography-estimated prostate volume to predict prostate cancer risk?

OBJECTIVE: To explore whether a panel of kallikrein markers in blood: total, free and intact prostate-specific antigen (PSA) and kallikrein-related peptidase 2, could be used as a non-invasive alternative for predicting prostate cancer on biopsy in a screening setting. SUBJECTS AND METHODS: The study cohort comprised previously unscreened men who underwent sextant biopsy owing to elevated PSA (≥3 ng/mL) in two different centres of the European Randomized Study of Screening for Prostate Cancer, Rotterdam (n = 2914) and Göteborg (n = 740). A statistical model, based on kallikrein markers, was compared with one based on established clinical factors for the prediction of biopsy outcome. RESULTS: The clinical tests were found to be no better than blood markers, with an area under the curve in favour of the blood measurements of 0.766 vs. 0.763 in Rotterdam and 0.809 vs. 0.774 in Göteborg. Adding digital rectal examination (DRE) or DRE plus transrectal ultrasonography (TRUS) volume to the markers improved discrimination, although the increases were small. Results were similar for predicting high-grade cancer. There was a strong correlation between the blood measurements and TRUS-estimated prostate volume (Spearman's correlation 0.60 in Rotterdam and 0.57 in Göteborg). CONCLUSIONS: In previously unscreened men, each with indication for biopsy, a statistical model based on kallikrein levels was similar to a clinical model in predicting prostate cancer in a screening setting, outside the day-to-day clinical practice. Whether a clinical approach can be replaced by laboratory analyses or used in combination with decision models (nomograms) is a clinical judgment that may vary from clinician to clinician depending on how they weigh the different advantages and disadvantages (harms, costs, time, invasiveness) of both approaches.

Levels of beta-microseminoprotein in blood and risk of prostate cancer in multiple populations.

BACKGROUND: A common genetic variant (rs10993994) in the 5' region of the gene encoding ß-microseminoprotein (MSP) is associated with circulating levels of MSP and prostate cancer risk. Whether MSP levels are predictive of prostate cancer risk has not been evaluated. METHODS: We investigated the prospective relationship between circulating plasma levels of MSP and prostate cancer risk in a nested case-control study of 1503 case subjects and 1503 control subjects among black, Latino, Japanese, Native Hawaiian, and white men from the Multiethnic Cohort study. We also examined the ability of MSP to serve as a biomarker for discriminating prostate cancer case subjects from control subjects. All statistical tests are two-sided. RESULTS: In all racial and ethnic groups, men with lower MSP levels were at greater risk of developing prostate cancer (odds ratio = 1.02 per one unit decrease in MSP, P < .001 in the prostate-specific antigen [PSA]-adjusted analysis). Compared with men in the highest decile of MSP, the multivariable PSA-adjusted odds ratio was 3.64 (95% confidence interval = 2.41 to 5.49) for men in the lowest decile. The positive association with lower MSP levels was observed consistently across racial and ethnic populations, by disease stage and Gleason score, for men with both high and low levels of PSA and across all genotype classes of rs10993994. However, we did not detect strong evidence of MSP levels in improving prostate cancer prediction beyond that of PSA. CONCLUSIONS: Regardless of race and ethnicity or rs10993994 genotype, men with low blood levels of MSP have increased risk of prostate cancer.

Immunoassay for the discrimination of free prostate-specific antigen (fPSA) forms with internal cleavages at Lys(145) or Lys(146) from fPSA without internal cleavages at Lys(145) or Lys(146).

Total levels of circulating prostate-specific antigen (tPSA) are strongly associated with prostate cancer (PCa) risk and outcome but benign prostate disease is the most frequent cause of a moderately elevated PSA level. Free PSA (fPSA) forms are independently associated with PCa risk and contribute modest diagnostic enhancements above and beyond tPSA alone. We developed an immunoassay for fPSA subfractions containing internal cleavages at Lys(145) or Lys(146) (fPSA-N). The assay was based on blocking intact single-chain fPSA (fPSA-I) with antibody 4D4 which does not detect PSA containing internal cleavages at Lys(145) or Lys(146). We also measured fPSA-N in blood from healthy volunteers and in anti-coagulated plasma from 76 men with or without evidence of PCa at biopsy. The analytical and functional detection limits of this assay were 0.016 ng/mL and 0.10 ng/mL, respectively. The median recovery of male fPSA-N from female plasma was 95.0%. All 12 female samples (average age 28 years) had fPSA-N concentrations at or below the analytical detection limit. The median fPSA-N concentration (0.050 ng/mL) in 9 healthy male volunteers (age<40 years) was below the functional detection limit, 0.420 ng/mL in 27 patients with benign prostate conditions and 0.239 ng/mL in 49 patients with PCa. Deming regression analysis of the patient samples showed that the measured fPSA-N concentrations were generally 23% lower than the previously calculated (fPSA minus fPSA-I) concentrations, likely due to differences in the antibody combinations used. In conclusion, we have developed a sensitive, specific and direct immunoassay for fPSA-N which can be used to study the clinical relevance of this PSA isoform.

Intact and internally cleaved free prostate-specific antigen in patients with prostate cancer with different pathologic stages and grades.

OBJECTIVES: To measure the concentration levels of free prostate-specific antigen (PSA) isoforms in patients with prostate cancer selected for curative treatment using radical prostatectomy and to study the association between the isoforms and the pathologic cancer stage and grade. METHODS: Preoperative plasma samples were obtained from 309 consecutive patients scheduled to undergo radical prostatectomy at Turku University Hospital. The pathologic TNM stage, Gleason score, and World Health Organization grade of the tumors were recorded. The total, free, and intact PSA (tPSA, fPSA, and fPSA-I, respectively) concentrations of the archived samples were measured with in-house immunoassays, and the nicked PSA (fPSA-N) concentrations (fPSA minus fPSA-I) and ratios of different PSA forms were calculated. These were compared with the prostate cancer stage, Gleason score, and World Health Organization grade. RESULTS: The median fPSA-I and fPSA-N concentrations in the patients with prostate cancer was 0.42 and 0.28 ng/mL, constituting an average of 60% and 40% of fPSA, respectively. The nicked/total PSA and free/total PSA ratios had the strongest negative correlations with a higher pathologic stage, Gleason score, and World Health Organization grade (Spearman rho -0.205 to -0.262, P < .05). Within a patient subgroup with tPSA <10 ng/mL, fPSA-N as a single marker had a negative correlation with a higher Gleason score (rho -0.160, P = .021). CONCLUSIONS: Lower proportions of fPSA-I and fPSA-N to total PSA were associated with a more advanced cancer stage and grade. A long-term follow-up study and a comparison with currently used clinical methods are needed to evaluate the usefulness of the analytes as prognostic markers for cancer aggressiveness in individual patients.

A common prostate cancer risk variant 5' of microseminoprotein-beta (MSMB) is a strong predictor of circulating beta-microseminoprotein (MSP) levels in multiple populations.

BACKGROUND: ß-Microseminoprotein (MSP) is one of the three most abundantly secreted proteins of the prostate and has been suggested as a biomarker for prostate cancer risk. A common variant, rs10993994, in the 5' region of the gene that encodes MSP (MSMB) has recently been identified as a risk factor for prostate cancer. METHODS: We examined the association between rs10993994 genotype and MSP levels in a sample of 500 prostate cancer-free men from four racial/ethnic populations in the Multiethnic Cohort (European Americans, African Americans, Latinos, and Japanese Americans). Generalized linear models were used to estimate the association between rs10993994 genotype and MSP levels. RESULTS: We observed robust associations between rs10994994 genotype and MSP levels in each racial/ethnic population (all P < 10(-8)), with carriers of the C allele having lower geometric mean MSP levels (ng/mL; CC/CT/TT genotypes: European Americans, 28.8/20.9/10.0; African Americans, 29.0/21.9/10.9; Latinos, 29.2/17.1/8.3; and Japanese Americans, 25.8/16.4/6.7). We estimated the variant accounts for 30% to 50% of the variation in MSP levels in each population. We also observed significant differences in MSP levels between populations (P = 3.5 × 10(-6)), with MSP levels observed to be highest in African Americans and lowest in Japanese Americans. CONCLUSIONS: Rs10993994 genotype is strongly associated with plasma MSP levels in multiple racial/ethnic populations. IMPACT: This supports the hypothesis that rs10993994 may be the biologically functional allele.

A four-kallikrein panel predicts prostate cancer in men with recent screening: data from the European Randomized Study of Screening for Prostate Cancer, Rotterdam.

PURPOSE: We have developed a statistical prediction model for prostate cancer based on four kallikrein markers in blood: total, free, and intact prostate-specific antigen (PSA), and kallikrein-related peptidase 2 (hK2). Although this model accurately predicts the result of biopsy in unscreened men, its properties for men with a history of PSA screening have not been fully characterized. EXPERIMENTAL DESIGN: A total of 1,501 previously screened men with elevated PSA underwent initial biopsy during rounds 2 and 3 of the European Randomized Study of Screening for Prostate Cancer, Rotterdam, with 388 cancers diagnosed. Biomarker levels were measured in serum samples taken before biopsy. The prediction model developed on the unscreened cohort was then applied and predictions compared with biopsy outcome. RESULTS: The previously developed four-kallikrein prediction model had much higher predictive accuracy than PSA and age alone (area under the curve of 0.711 versus 0.585, and 0.713 versus 0.557 with and without digital rectal exam, respectively; both P < 0.001). Similar statistically significant enhancements were seen for high-grade cancer. Applying the model with a cutoff of 20% cancer risk as the criterion for biopsy would reduce the biopsy rate by 362 for every 1,000 men with elevated PSA. Although diagnosis would be delayed for 47 cancers, these would be predominately low-stage and low-grade (83% Gleason 6 T(1c)). CONCLUSIONS: A panel of four kallikreins can help predict the result of initial biopsy in previously screened men with elevated PSA. Use of a statistical model based on the panel would substantially decrease rates of unnecessary biopsy.

Reducing unnecessary biopsy during prostate cancer screening using a four-kallikrein panel: an independent replication.

PURPOSE: We previously reported that a panel of four kallikrein forms in blood-total, free, and intact prostate-specific antigen (PSA) and kallikrein-related peptidase 2 (hK2)-can reduce unnecessary biopsy in previously unscreened men with elevated total PSA. We aimed to replicate our findings in a large, independent, representative, population-based cohort. PATIENTS AND METHODS: The study cohort included 2,914 previously unscreened men undergoing biopsy as a result of elevated PSA (> or = 3 ng/mL) in the European Randomized Study of Screening for Prostate Cancer, Rotterdam, with 807 prostate cancers (28%) detected. The cohort was randomly divided 1:3 into a training and validation set. Levels of kallikrein markers were compared with biopsy outcome. RESULTS: Addition of free PSA, intact PSA, and hK2 to a model containing total PSA and age improved the area under the curve from 0.64 to 0.76 and 0.70 to 0.78 for models without and with digital rectal examination results, respectively (P < .001 for both). Application of the panel to 1,000 men with elevated PSA would reduce the number of biopsies by 513 and miss 54 of 177 low-grade cancers and 12 of 100 high-grade cancers. Findings were robust to sensitivity analysis. CONCLUSION: We have replicated our previously published finding that a panel of four kallikreins can predict the result of biopsy for prostate cancer in men with elevated PSA. Use of this panel would dramatically reduce biopsy rates. A small number of men with cancer would be advised against immediate biopsy, but these men would have predominately low-stage, low-grade disease.

Homogeneous dual-parameter assay for prostate-specific antigen based on fluorescence resonance energy transfer.

Three monoclonal antibodies (Mab) specific for prostate-specific antigen (PSA) were used to design a homogeneous dual-parameter immunoassay based on fluorescence resonance energy transfer (FRET). One antibody was labeled with terbium(III) chelate, which acted as a donor, and the other two antibodies were labeled with fluorescent acceptor dyes (either Alexa Fluor (AF) 488 or Alexa Fluor 680). Due to the selection of the antibodies, sensitized emission of the AF488 could be measured only if uncomplexed, free PSA (PSA-F) was present in the sample. The amount of total PSA (PSA-T) was obtained by measuring the sensitized emission of AF680. Thus, the assay could simultaneously measure the amount of both PSA-F and PSA-T from a single sample. The lowest limits of detection with buffer calibrators were 0.74 and 0.60 ng/mL for PSA-F and PSA-T, respectively. Both of the assays were linear up to 100 ng/mL. The performance of the assay was also tested against heterogeneous single-parameter assays using spiked female plasma samples. The Pearson's correlations were 0.994 for PSA-F and 0.997 for PSA-T assays. However, due to the calibrator matrix being different from the sample matrix, the obtained concentrations with homogeneous assay, especially for PSA-F, were slightly less than with heterogeneous assays. In conclusion, it was shown that a homogeneous dual-parameter assay based on the measurement of the sensitized emission of two different acceptors in combination with a single donor can be performed. The assay was done in a single step using only one excitation wavelength and was functional within the clinically important analyte concentrations.

Intact free prostate-specific antigen and free and total human glandular kallikrein 2. Elimination of assay interference by enzymatic digestion of antibodies to F(ab')2 fragments.

Various blood constituents can interfere with immunoassays, usually by binding the Fc portion of antibodies. Our previously developed assays for intact free prostate-specific antigen (PSA), free human kallikrein 2 (hK2), and total hK2 frequently yielded falsely high results despite including an excess of scavenger antibodies. We investigated whether this interference could be eliminated by replacing monoclonal capture or tracer antibodies with F(ab')2 or recombinant Fab fragments. Female heparin plasma samples (n = 1092), which should have negligible PSA and hK2, and male samples (n = 957) were analyzed to identify samples manifesting interference, which then were used to optimize protocols for the immunoassays. We compared original assays (monoclonal antibodies) versus optimized assays (F(ab')2 fragments: denatured mouse IgG added as scavenger) using another set of EDTA plasma (n = 113), heparin plasma (n = 160), and serum samples (n = 171). With the original assays, the frequency of falsely elevated hK2 and intact free PSA was 15 and 13%, respectively. The optimized assays eliminated 70-85% of these falsely elevated results and importantly reduced the magnitude in the remainder. F(ab')2 fragmentation was the most important factor in reducing interference. The optimized intact free PSA, free hK2, and total hK2 assays manifested high accuracy close to the lower limit of detection.

TMPRSS2 fusions with oncogenic ETS factors in prostate cancer involve unbalanced genomic rearrangements and are associated with HDAC1 and epigenetic reprogramming.

Translocations fusing the strong androgen-responsive gene, TMPRSS2, with ERG or other oncogenic ETS factors may facilitate prostate cancer development. Here, we studied 18 advanced prostate cancers for ETS factor alterations, using reverse transcription-PCR and DNA and RNA array technologies, and identified putative ERG downstream gene targets from the microarray data of 410 prostate samples. Out of the 27 ETS factors, ERG was most frequently overexpressed. Seven cases showed TMPRSS2:ERG gene fusions, whereas the TMPRSS2:ETV4 fusion was seen in one case. In five out of six tumors with high ERG expression, array-CGH analysis revealed interstitial 2.8 Mb deletions between the TMPRSS2 and ERG loci, or smaller, unbalanced rearrangements. In silico analysis of the ERG gene coexpression patterns revealed an association with high expression of the histone deacetylase 1 gene, and low expression of its target genes. Furthermore, we observed increased expression of WNT-associated pathways and down-regulation of tumor necrosis factor and cell death pathways. In summary, our data indicate that the TMPRSS2:ERG translocation is common in advanced prostate cancer and occurs by virtue of unbalanced genomic rearrangements. Activation of ERG by fusion with TMPRSS2 may lead to epigenetic reprogramming, WNT signaling, and down-regulation of cell death pathways, implicating ERG in several hallmarks of cancer with potential therapeutic importance.