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author:("lila, Hans")
1.  Mortality results from the Göteborg Randomised Prostate Cancer Screening Trial 
The lancet oncology  2010;11(8):725-732.
Summary
Background
Prostate cancer is one of the leading causes of death from malignant disease among men in the Western world. One strategy to decrease the risk of dying from this disease is screening with Prostate-Specific Antigen (PSA); however, the extent of benefit and harm with such screening is under continuous debate.
Methods
In December 1994, 20 000 men born 1930 to 1944, randomly sampled from the Population Register, were computer randomised in a 1:1 ratio to a screening group invited for biennial PSA testing or to a control group not invited. In each arm, 48 men were excluded from analysis due to either death or emigration before randomization date or prevalent prostate cancer. The primary endpoint was prostate cancer specific mortality analyzed according to the intention-to-screen principle. Men in the screening group were invited up to the upper age limit (median 69, range 67–71 years) and only men with elevated PSA were offered additional tests such as digital rectal examination and prostate biopsies. The study is still ongoing inviting men who have not yet reached the upper age limit. This is the first planned report on cumulative prostate cancer incidence and mortality calculated up to Dec 31 2008. This study is registered [as an International Standard Randomised Controlled Trial], number [ISRCTN49127736].
Findings
Among men randomised to screening 7578/9952 (76%) attended at least once (attendees). During a median follow-up of 14 years, 1138 men in the screening group and 718 in the control group were diagnosed with prostate cancer resulting in a cumulative incidence of prostate cancer of 12.7% in the screening arm and 8.2% in the control arm (hazard ratio 1.64; 95% confidence interval [CI] 1.50–1.80; p<0.0001). The absolute cumulative risk reduction of death from prostate cancer at 14 years was 0.40% (95% CI 0.17–0.64%), from 0.90% in the control group to 0.50% in the screening group. The incidence rate ratio for death from prostate cancer was 0.56 (95% CI 0.39–0.82; p=0.002) in the screening compared to the control group. The incidence rate ratio of attendees compared to the control group was 0.44 (95% CI 0.28–0.68; p=0.0002). Overall, 293 men needed to be invited for screening and 12 to be diagnosed to prevent one prostate cancer death.
Interpretation
The benefit of prostate cancer screening compares favourably to other cancer screening programs and in this study prostate cancer mortality was reduced almost by half over 14 years. However, the risk of over diagnosis is substantial and the number needed to treat is at least as high as in breast cancer screening.
Funding
The Swedish Cancer Society, the Swedish Research Council and the National Cancer Institute.
doi:10.1016/S1470-2045(10)70146-7
PMCID: PMC4089887  PMID: 20598634
2.  Association of transcript levels of 10 established or candidate-biomarker gene targets with cancerous versus non-cancerous prostate tissue from radical prostatectomy specimens 
Clinical biochemistry  2013;46(0):670-674.
OBJECTIVES
The benefits of PSA (prostate specific antigen)-testing in prostate cancer remain controversial with a consequential need for validation of additional biomarkers. We used highly standardized reverse-transcription (RT)-PCR assays to compare transcript levels of 10 candidate cancer marker genes – BMP6, FGF-8b, KLK2, KLK3, KLK4, KLK15, MSMB, PCA3, PSCA and Trpm8 – in carefully ascertained non-cancerous versus cancerous prostate tissue from patients with clinically localized prostate cancer treated by radical prostatectomy.
DESIGN AND METHODS
Total RNA was isolated from fresh frozen prostate tissue procured immediately after resection from two separate areas in each of 87 radical prostatectomy specimens. Subsequent histopathological assessment classified 86 samples as cancerous and 88 as histologically benign prostate tissue. Variation in total RNA recovery was accounted for by using external and internal standards and enabled us to measure transcript levels by RT-PCR in a highly quantitative manner.
RESULTS
Of the ten genes, there were significantly higher levels only of one of the less abundant transcripts, PCA3, in cancerous versus non-cancerous prostate tissue whereas PSCA mRNA levels were significantly lower in cancerous versus histologically benign tissue. Advanced pathologic stage was associated with significantly higher expression of KLK15 and PCA3 mRNAs. Median transcript levels of the most abundantly expressed genes (i.e. MSMB, KLK3, KLK4 and KLK2) in prostate tissue were up to 105-fold higher than those of other gene targets.
CONCLUSIONS
PCA3 expression was associated with advanced pathological stage but the magnitude of overexpression of PCA3 in cancerous versus non-cancerous prostate tissue was modest compared to previously reported data.
doi:10.1016/j.clinbiochem.2013.01.019
PMCID: PMC3628411  PMID: 23391636
Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction
3.  Influence of blood prostate specific antigen levels at age 60 on benefits and harms of prostate cancer screening: population based cohort study 
Objective To determine the relative risks of prostate cancer incidence, metastasis, and mortality associated with screening by serum prostate specific antigen (PSA) levels at age 60.
Design Population based cohort study.
Setting General male population of Sweden taking part in a screening trial in Gothenburg or participating in a cardiovascular study, the Malmö Preventive Project.
Participants The screened group consisted of 1756 men aged 57.5-62.5 participating in the screening arm of the Gothenburg randomized prostate cancer screening trial since 1995. The unscreened group consisted of 1162 men, born in 1921, participating in the Malmö Preventive Project, with PSA levels measured retrospectively in stored blood samples from 1981.
Intervention PSA screening versus no screening.
Main outcome measures Incidence rate ratios for the effect of screening on prostate cancer diagnosis, metastasis, and death by PSA levels at age 60.
Results The distribution of PSA levels was similar between the two cohorts. Differences in benefits by baseline PSA levels were large. Among men with baseline levels measured, 71.7% (1646/2295) had a PSA level <2 ng/mL. For men aged 60 with PSA level <2 ng/mL, there was an increase in incidence of 767 cases per 10 000 without a decrease in prostate cancer mortality. For men with PSA levels ≥2 ng/mL, the reduction in cancer mortality was large, with only 23 men needing to be screened and six diagnosed to avoid one prostate cancer death by 15 years.
Conclusions The ratio of benefits to harms of PSA screening varies noticeably with blood PSA levels at age 60. For men with a PSA level <1 ng/mL at age 60, no further screening is recommended. Continuing to screen men with PSA levels >2 ng/mL at age 60 is beneficial, with the number needed to screen and treat being extremely favourable. Screening men with a PSA level of 1-2 ng/mL is an individual decision to be based on a discussion between patient and doctor.
doi:10.1136/bmj.g2296
PMCID: PMC3968958  PMID: 24682399
4.  Prostate Cancer Mortality in Areas With High and Low Prostate Cancer Incidence 
Background
The effect of prostate-specific antigen (PSA) screening on prostate cancer mortality remains debated, despite evidence from randomized trials. We investigated the association between prostate cancer incidence, reflecting uptake of PSA testing, and prostate cancer mortality.
Methods
The study population consisted of all men aged 50 to 74 years residing in eight counties in Sweden with an early increase in prostate cancer incidence and six counties with a late increase during two time periods. Incidence of metastatic prostate cancer was investigated in the period from 2000 to 2009, and prostate cancer–specific mortality and excess mortality were investigated in the period from 1990 to 1999 and the period from 2000 to 2009 by calculating rate ratios for high- vs low-incidence counties and rate ratios for the period from 2000 to 2009 vs the period from 1990 to 1999 within these two groups. All statistical tests were two-sided.
Results
There were 4528134 person-years at risk, 1577 deaths from prostate cancer, and 1210 excess deaths in men with prostate cancer in high-incidence counties and 2471373 person-years at risk, 985 prostate cancer deaths, and 878 excess deaths in low-incidence counties in the period from 2000 to 2009. Rate ratios in counties with high vs low incidence adjusted for time period were 0.81 (95% confidence interval [CI] = 0.73 to 0.90) for prostate cancer– specific mortality and 0.74 (95% CI = 0.64 to 0.86) for excess mortality, and the rate ratio of metastatic prostate cancer was 0.85 (95% CI = 0.79 to 0.92).
Conclusions
The lower prostate cancer mortality in high-incidence counties reflecting a high PSA uptake suggests that more-intense as compared with less-intense opportunistic PSA screening reduces prostate cancer mortality.
doi:10.1093/jnci/dju007
PMCID: PMC3982781  PMID: 24610909
5.  Empirical estimates of prostate cancer overdiagnosis by age and prostate-specific antigen 
BMC Medicine  2014;12:26.
Background
Prostate cancer screening depends on a careful balance of benefits, in terms of reduced prostate cancer mortality, and harms, in terms of overdiagnosis and overtreatment. We aimed to estimate the effect on overdiagnosis of restricting prostate specific antigen (PSA) testing by age and baseline PSA.
Methods
Estimates of the effects of age on overdiagnosis were based on population based incidence data from the US Surveillance, Epidemiology and End Results database. To investigate the relationship between PSA and overdiagnosis, we used two separate cohorts subject to PSA testing in clinical trials (n = 1,577 and n = 1,197) and a population-based cohort of Swedish men not subject to PSA-screening followed for 25 years (n = 1,162).
Results
If PSA testing had been restricted to younger men, the number of excess cases associated with the introduction of PSA in the US would have been reduced by 85%, 68% and 42% for age cut-offs of 60, 65 and 70, respectively. The risk that a man with screen-detected cancer at age 60 would not subsequently lead to prostate cancer morbidity or mortality decreased exponentially as PSA approached conventional biopsy thresholds. For PSAs below 1 ng/ml, the risk of a positive biopsy is 65 (95% CI 18.2, 72.9) times greater than subsequent prostate cancer mortality.
Conclusions
Prostate cancer overdiagnosis has a strong relationship to age and PSA level. Restricting screening in men over 60 to those with PSA above median (>1 ng/ml) and screening men over 70 only in selected circumstances would importantly reduce overdiagnosis and change the ratio of benefits to harms of PSA-screening.
doi:10.1186/1741-7015-12-26
PMCID: PMC3922189  PMID: 24512643
Prostate cancer; Early detection; Overdiagnosis; PSA; Screening
6.  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 β-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.
doi:10.1093/jnci/djs486
PMCID: PMC3565627  PMID: 23213189
7.  EVALUATING THE PROSTATE CANCER PREVENTION TRIAL HIGH GRADE PROSTATE CANCER RISK CALCULATOR IN TEN INTERNATIONAL BIOSY COHORTS: RESULTS FROM THE PROSTATE BIOPSY COLLABORATIVE GROUP 
World journal of urology  2012;32(1):185-191.
OBJECTIVES
To assess the applicability of the Prostate Cancer Prevention Trial High Grade (Gleason grade ≥ 7) Risk Calculator (PCPTHG) in ten international cohorts, representing a range of populations.
METHODS
25,512 biopsies from 10 cohorts (6 European, 1 UK, and 3 US) were included; 4 implemented 6-core biopsies and the remaining had 10- or higher schemes; 8 were screening cohorts and 2 were clinical. PCPTHG risks were calculated using prostate-specific antigen (PSA), digital rectal examination, age, African origin and history of prior biopsy and evaluated in terms of calibration plots, areas underneath the receiver operating characteristic curve (AUC), and net benefit curves.
RESULTS
The median AUC of the PCPTHG for high grade disease detection in the 10- and higher-core cohorts was 73.5% (range 63.9% to 76.7%) compared to a median of 78.1 (range = 72.0 to 87.6) among the four 6-core cohorts. Only the 10-core Cleveland Clinic cohort showed clear evidence of under-prediction by the PCPTHG, and this was restricted to risk ranges less than 15%. The PCPTHG demonstrated higher clinical net benefit in higher- compared to six-core biopsy cohorts, and among the former, there were no notable differences observed between clinical and screening cohorts, nor between European and US cohorts.
CONCLUSIONS
The PCPTHG requires minimal patient information and can be applied across a range of populations. PCPTHG risk thresholds ranging from 5 to 20%, depending on patient risk averseness, are recommended for clinical prostate biopsy decision-making.
doi:10.1007/s00345-012-0869-2
PMCID: PMC3702682  PMID: 22527674
Calibration; Discrimination; Net Benefit; High Grade Prostate Cancer; Risk; Prostate Cancer Prevention Trial
8.  Chromosome 19 Annotations with Disease Speciation – A First Report from the Global Research Consortium 
Journal of proteome research  2012;12(1):135-150.
A first research development progress report of the Chromosome 19 Consortium with members from Sweden, Norway, Spain, USA, China and India, a part of the Chromosome-Centric Human Proteome Project (C-HPP) global initiative is presented (http://www.c-hpp.org). From the chromosome 19 peptide-targeted library constituting 6159 peptides, a pilot study was conducted using a sub-set with 125 isotope-labeled peptides. We applied an annotation strategy with triple quadrupole, ESI-Qtrap, and MALDI mass spectrometry platforms, comparing the quality of data within, and in-between these instrumental set-ups. LC-MS conditions were outlined by multiplex assay developments, followed by MRM assay developments. SRM was applied to biobank samples, quantifying kallikrein 3 (prostate specific antigen) in plasma from prostate cancer patients. The antibody production has been initiated for more than 1200 genes from the entire chromosome 19, and the progress developments are presented. We developed a dedicated transcript microarray, to serve as the mRNA identifier by screening cancer cell lines. NAPPA protein arrays were built to align with the transcript data with the Chromosome 19 NAPPA chip, dedicated to 90 proteins, as the first development delivery. We have introduced an IT-infrastructure utilizing a LIMS system that serves as the key interface for the research teams in order to share and explore data generated within the project. The cross-site data repository will form the basis for sample processing, including biological samples, as well as patient samples from national Biobanks.
doi:10.1021/pr3008607
PMCID: PMC3539432  PMID: 23249167
Proteins; Genes; Antibodies; mRNA; Mass spectrometry; Bioinformatics; Protein microarray; Human Disease
9.  Rapid elimination kinetics of free PSA or human kallikrein-related peptidase 2 after initiation of gonadotropin-releasing hormone-antagonist treatment of prostate cancer: potential for rapid monitoring of treatment responses 
Background
The utility of conventional prostate-specific antigen (PSA) measurements in blood for monitoring rapid responses to treatment for prostate cancer is limited because of its slow elimination rate. Prior studies have shown that free PSA (fPSA), intact PSA (iPSA) and human kallikrein-related peptidase 2 (hK2) are eliminated more rapidly after radical prostatectomy. In contrast, all three markers have similarly slow elimination rates after castration induced by GnRH agonists, possibly due to the slow onset of castration. Therefore, we assessed elimination rates of tPSA, fPSA, iPSA and hK2 after rapid induction of castration with degarelix (Firmagon®), a novel GnRH antagonist.
Methods
This study included 24 patients treated with degarelix. Blood was taken at 1, 3, 7, 14, 21, and 28 days after injection of degarelix. Free and total PSA were measured with a commercial dual-label assay, and with in-house research assays of intact PSA and hK2.
Results
Median (interquartile range, IQR) tPSA at baseline was 23.4 (15.8, 59.8). Twenty-two patients (92%) reached castrate levels of testosterone within 24 hours of degarelix initiation, and all patients did so within 72 hours. All kallikrein forms declined in an exponential fashion after degarelix administration. The median time to 50% reduction in biomarker level was 8–9 days for tPSA or complexed PSA versus 2–4 days for hK2, iPSA and fPSA. The percentage eliminated at day 3 and day 7 was significantly higher for hK2, iPSA and fPSA than for tPSA (all p<0.02), while tPSA and complexed PSA were similar.
Conclusions
The rapid decline of fPSA, iPSA and hK2 after fast induction of castration with degarelix is similar to that reported after prostatectomy and offers a novel, informative method to monitor rapid onset of therapeutic action targeting signaling of the androgen receptor,
doi:10.1515/cclm-2011-0967
PMCID: PMC3474140  PMID: 22718641
androgen deprivation therapy; human kallikrein-related peptidase 2; prostate cancer; prostate-specific antigen; tumor markers
10.  Microfluidic, Label-Free Enrichment of Prostate Cancer Cells in Blood Based on Acoustophoresis 
Analytical chemistry  2012;84(18):7954-7962.
Circulating tumor cells (CTC) are shed in peripheral blood at advanced metastatic stages of solid cancers. Surface-marker-based detection of CTC predicts recurrence and survival in colorectal, breast, and prostate cancer. However, scarcity and variation in size, morphology, expression profile, and antigen exposure impairs reliable detection and characterization of CTC. We have developed a non-contact, label-free microfluidic acoustophoresis method to separate prostate cancer cells from white blood cells (WBC) through forces generated by ultrasonic resonances in microfluidic channels. Implementation of cell pre-alignment in a temperature-stabilized (±0.5°C) acoustophoresis microchannel dramatically enhanced the discriminatory capacity and enabled the separation of 5-μm microspheres from 7-μm microspheres with 99% purity. Next, we determined the feasibility of employing label-free microfluidic acoustophoresis to discriminate and divert tumor cells from WBCs using erythrocyte-lysed blood from healthy volunteers spiked with tumor cells from three prostate cancer cell-lines (DU145, PC3, LNCaP). For cells fixed with paraformaldehyde, cancer cell recovery ranged from 93.6% to 97.9% with purity ranging from 97.4% to 98.4%. There was no detectable loss of cell viability or cell proliferation subsequent to the exposure of viable tumor cells to acoustophoresis. For non-fixed, viable cells, tumor cell recovery ranged from 72.5% to 93.9% with purity ranging from 79.6% to 99.7%. These data contribute proof-in-principle that label-free microfluidic acoustophoresis can be used to enrich both viable and fixed cancer cells from WBCs with very high recovery and purity.
doi:10.1021/ac301723s
PMCID: PMC3445767  PMID: 22897670
11.  Susceptibility Loci Associated with Prostate Cancer Progression and Mortality 
Purpose
Prostate cancer is a heterogenous disease with a variable natural history that is not accurately predicted by currently used prognostic tools.
Experimental Design
We genotyped 798 prostate cancer cases of Ashkenazi Jewish ancestry treated for localized prostate cancer between June 1988 and December 2007. Blood samples were prospectively collected and de-identified before being genotyped and matched to clinical data. The survival analysis was adjusted for Gleason score and PSA. We investigated associations between 29 single nucleotide polymorphisms (SNPs) and biochemical recurrence, castration-resistant metastasis, and prostate cancer-specific survival. Subsequently, we performed an independent analysis using a high resolution panel of 13 SNPs.
Results
On univariate analysis, 2 SNPs were associated (p<0.05) with biochemical recurrence; 3 SNPs were associated with clinical metastases; and 1 SNP was associated with prostate cancer-specific mortality. Applying a Bonferroni correction (p<0.0017), one association with biochemical recurrence (p=0.0007) was significant. Three SNPs showed associations on multivariable analysis, although not after correcting for multiple testing. The secondary analysis identified an additional association with prostate cancer-specific mortality in KLK3 (p<0.0005 by both univariate and multivariable analysis).
Conclusions
We identified associations between prostate cancer susceptibility SNPs and clinical endpoints. The rs61752561 in KLK3 and rs2735839 in the KLK2-KLK3 intergenic region associated strongly with prostate cancer-specific survival, and rs10486567 in 7JAZF1 gene associated with biochemical recurrence. A larger study will be required to independently validate these findings and determine the role of these SNPs in prognostic models.
doi:10.1158/1078-0432.CCR-10-0028
PMCID: PMC3732009  PMID: 20460480
Single nucleotide polymorphisms; Prostate cancer; Prognosis
12.  Copy Number Variants in the Kallikrein Gene Cluster 
PLoS ONE  2013;8(7):e69097.
The kallikrein gene family (KLK1-KLK15) is the largest contiguous group of protease genes within the human genome and is associated with both risk and outcome of cancer and other diseases. We searched for copy number variants in all KLK genes using quantitative PCR analysis and analysis of inheritance patterns of single nucleotide polymorphisms. Two deletions were identified: one 2235-bp deletion in KLK9 present in 1.2% of alleles, and one 3394-bp deletion in KLK15 present in 4.0% of alleles. Each deletion eliminated one complete exon and created out-of-frame coding that eliminated the catalytic triad of the resulting truncated gene product, which therefore likely is a non-functional protein. Deletion breakpoints identified by DNA sequencing located the KLK9 deletion breakpoint to a long interspersed element (LINE) repeated sequence, while the deletion in KLK15 is located in a single copy sequence. To search for an association between each deletion and risk of prostate cancer (PC), we analyzed a cohort of 667 biopsied men (266 PC cases and 401 men with no evidence of PC at biopsy) using short deletion-specific PCR assays. There was no association between evidence of PC in this cohort and the presence of either gene deletion. Haplotyping revealed a single origin of each deletion, with most recent common ancestor estimates of 3000-8000 and 6000-14 000 years for the deletions in KLK9 and KLK15, respectively. The presence of the deletions on the same haplotypes in 1000 Genomes data of both European and African populations indicate an early origin of both deletions. The old age in combination with homozygous presence of loss-of-function variants suggests that some kallikrein-related peptidases have non-essential functions.
doi:10.1371/journal.pone.0069097
PMCID: PMC3718828  PMID: 23894413
14.  A Novel Automated Platform for Quantifying the Extent of Skeletal Tumour Involvement in Prostate Cancer Patients Using the Bone Scan Index 
European Urology  2012;62(1):78-84.
Background
There is little consensus on a standard approach to analysing bone scan images. The Bone Scan Index (BSI) is predictive of survival in patients with progressive prostate cancer (PCa), but the popularity of this metric is hampered by the tedium of the manual calculation.
Objective
Develop a fully automated method of quantifying the BSI and determining the clinical value of automated BSI measurements beyond conventional clinical and pathologic features.
Design, setting, and participants
We conditioned a computer-assisted diagnosis system identifying metastatic lesions on a bone scan to automatically compute BSI measurements. A training group of 795 bone scans was used in the conditioning process. Independent validation of the method used bone scans obtained ≤3 mo from diagnosis of 384 PCa cases in two large population-based cohorts. An experienced analyser (blinded to case identity, prior BSI, and outcome) scored the BSI measurements twice. We measured prediction of outcome using pretreatment Gleason score, clinical stage, and prostate-specific antigen with models that also incorporated either manual or automated BSI measurements.
Measurements
The agreement between methods was evaluated using Pearson’s correlation coefficient. Discrimination between prognostic models was assessed using the concordance index (C-index).
Results and limitations
Manual and automated BSI measurements were strongly correlated (ρ = 0.80), correlated more closely (ρ = 0.93) when excluding cases with BSI scores ≥10 (1.8%), and were independently associated with PCa death (p < 0.0001 for each) when added to the prediction model. Predictive accuracy of the base model (C-index: 0.768; 95% confidence interval [CI], 0.702–0.837) increased to 0.794 (95% CI, 0.727–0.860) by adding manual BSI scoring, and increased to 0.825 (95% CI, 0.754–0.881) by adding automated BSI scoring to the base model.
Conclusions
Automated BSI scoring, with its 100% reproducibility, reduces turnaround time, eliminates operator-dependent subjectivity, and provides important clinical information comparable to that of manual BSI scoring.
doi:10.1016/j.eururo.2012.01.037
PMCID: PMC3402084  PMID: 22306323
15.  Microchannel Acoustophoresis does not Impact Survival or Function of Microglia, Leukocytes or Tumor Cells 
PLoS ONE  2013;8(5):e64233.
Background
The use of acoustic forces to manipulate particles or cells at the microfluidic scale (i.e. acoustophoresis), enables non-contact, label-free separation based on intrinsic cell properties such as size, density and compressibility. Acoustophoresis holds great promise as a cell separation technique in several research and clinical areas. However, it has been suggested that the force acting upon cells undergoing acoustophoresis may impact cell viability, proliferation or cell function via subtle phenotypic changes. If this were the case, it would suggest that the acoustophoresis method would be a less useful tool for many cell analysis applications as well as for cell therapy.
Methods
We investigate, for the first time, several key aspects of cellular changes following acoustophoretic processing. We used two settings of ultrasonic actuation, one that is used for cell sorting (10 Vpp operating voltage) and one that is close to the maximum of what the system can generate (20 Vpp). We used microglial cells and assessed cell viability and proliferation, as well as the inflammatory response that is indicative of more subtle changes in cellular phenotype. Furthermore, we adapted a similar methodology to monitor the response of human prostate cancer cells to acoustophoretic processing. Lastly, we analyzed the respiratory properties of human leukocytes and thrombocytes to explore if acoustophoretic processing has adverse effects.
Results
BV2 microglia were unaltered after acoustophoretic processing as measured by apoptosis and cell turnover assays as well as inflammatory cytokine response up to 48 h following acoustophoresis. Similarly, we found that acoustophoretic processing neither affected the cell viability of prostate cancer cells nor altered their prostate-specific antigen secretion following androgen receptor activation. Finally, human thrombocytes and leukocytes displayed unaltered mitochondrial respiratory function and integrity after acoustophoretic processing.
Conclusion
We conclude that microchannel acoustophoresis can be used for effective continuous flow-based cell separation without affecting cell viability, proliferation, mitochondrial respiration or inflammatory status.
doi:10.1371/journal.pone.0064233
PMCID: PMC3664584  PMID: 23724038
16.  Targeting Free Prostate-Specific Antigen for In Vivo Imaging of Prostate Cancer Using a Monoclonal Antibody Specific for Unique Epitopes Accessible on Free Prostate-Specific Antigen Alone 
Abstract
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 125I-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 125I-labeled PSA30. Tissue uptake of 125I-PSA30 at 48 and 168 hours after intravenous injection was compared with two clinically used positron emission tomography radiopharmaceuticals, 18F-fluoro-deoxy-glucose (18F-FDG) or 18F-choline, in cryosections using Digital AutoRadiography (DAR) and also compared with immunohistochemical staining of PSA and histopathology. On DAR, the areas with high 125I-PSA30 uptake corresponded mainly to morphologically intact and PSA-producing LNCaP cells, but did not associate with the areas of high uptake of either 18F-FDG or 18F-choline. Biodistribution of 125I-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.
doi:10.1089/cbr.2011.1088
PMCID: PMC3353767  PMID: 22489659
digital autoradiography; dual isotope imaging; free PSA; prostate cancer; prostate-specific antigen; radioimmunodetection
17.  Evaluating The PCPT Risk Calculator in Ten International Biopsy Cohorts: Results from the Prostate Biopsy Collaborative Group 
World journal of urology  2011;30(2):181-187.
Objectives
To evaluate the discrimination, calibration and net benefit performance of the Prostate Cancer Prevention Trial Risk Calculator (PCPTRC) across five European Randomized study of Screening for Prostate Cancer (ERSPC), 1 United Kingdom, 1 Austrian and 3 US biopsy cohorts.
Methods
PCPTRC risks were calculated for 25,733 biopsies using prostate-specific antigen (PSA), digital rectal examination, family history and history of prior biopsy, and single imputation for missing covariates. Predictions were evaluated using the areas underneath the receiver operating characteristic curves (AUC), discrimination slopes, chi-square tests of goodness of fit, and net benefit decision curves.
Results
AUCs of the PCPTRC ranged from a low of 56% in the ERSPC Goeteborg Rounds 2-6 cohort to a high of 72% in the ERSPC Goeteborg Round 1 cohort, and were statistically significantly higher than that of PSA in 6 out of the 10 cohorts. The PCPTRC was well-calibrated in the SABOR, Tyrol and Durham cohorts. There was limited to no net benefit to using the PCPTRC for biopsy referral compared to biopsying all or no men in all five ERSPC cohorts and benefit within a limited range of risk thresholds in all other cohorts.
Conclusions
External validation of the PCPTRC across ten cohorts revealed varying degree of success highly dependent on the cohort, most likely due to different criteria for and work-up before biopsy. Future validation studies of new calculators for prostate cancer should acknowledge the potential impact of the specific cohort studied when reporting successful versus failed validation.
doi:10.1007/s00345-011-0818-5
PMCID: PMC3616370  PMID: 22210512
receiver operating characteristic curve; risk; prostate cancer; calibration; net benefit
18.  Imaging androgen receptor signaling with a radiotracer targeting free prostate specific antigen 
Cancer Discovery  2012;2(4):320-327.
Despite intense efforts to develop radiotracers to detect cancers or monitor treatment response, few are widely used due to challenges with demonstrating clear clinical utility. We reasoned that a radiotracer targeting a validated clinical biomarker could more clearly assess the advantages of imaging cancer. The virtues and shortcomings of measuring secreted prostate specific antigen (PSA), an androgen receptor (AR) target gene, in prostate cancer (PCa) patients are well documented, making it a logical candidate for assessing whether a radiotracer can reveal new (and useful) information beyond that conferred by serum PSA. Therefore, we developed 89Zr-5A10, a novel radiotracer that targets “free” PSA. 89Zr-5A10 localizes in an AR-dependent manner in vivo to models of castration resistant prostate cancer, a disease state where serum PSA may not reflect clinical outcomes. Finally, we demonstrate that 89Zr-5A10 can detect osseous PCa lesions, a context where bone scans fail to discriminate malignant and non-malignant signals.
doi:10.1158/2159-8290.CD-11-0316
PMCID: PMC3351274  PMID: 22576209
PET; prostate cancer; prostate specific antigen; androgen receptor; biomarker; PSA; PCa; AR; CRPC; 18F-FDG; fPSA; mAbs; PET; MRI; PSMA; ITLC
19.  Evaluation of Multiple Risk-Associated Single Nucleotide Polymorphisms Versus Prostate-Specific Antigen at Baseline to Predict Prostate Cancer in Unscreened Men 
European Urology  2011;61(3):471-477.
Background
Although case-control studies have identified numerous single nucleotide polymorphisms (SNPs) associated with prostate cancer, the clinical role of these SNPs remains unclear.
Objective
Evaluate previously identified SNPs for association with prostate cancer and accuracy in predicting prostate cancer in a large prospective population-based cohort of unscreened men.
Design, setting, and participants
This study used a nested case-control design based on the Malmö Diet and Cancer cohort with 943 men diagnosed with prostate cancer and 2829 matched controls. Blood samples were collected between 1991 and 1996, and follow-up lasted through 2005.
Measurements
We genotyped 50 SNPs, analyzed prostate-specific antigen (PSA) in blood from baseline, and tested for association with prostate cancer using the Cochran-Mantel-Haenszel test. We further developed a predictive model using SNPs nominally significant in univariate analysis and determined its accuracy to predict prostate cancer.
Results and limitations
Eighteen SNPs at 10 independent loci were associated with prostate cancer. Four independent SNPs at four independent loci remained significant after multiple test correction (p < 0.001). Seven SNPs at five independent loci were associated with advanced prostate cancer defined as clinical stage ≥T3 or evidence of metastasis at diagnosis. Four independent SNPs were associated with advanced or aggressive cancer defined as stage ≥T3, metastasis, Gleason score ≥8, or World Health Organization grade 3 at diagnosis. Prostate cancer risk prediction with SNPs alone was less accurate than with PSA at baseline (area under the curve of 0.57 vs 0.79), with no benefit from combining SNPs with PSA. This study is limited by our reliance on clinical diagnosis of prostate cancer; there are likely undiagnosed cases among our control group.
Conclusions
Only a few previously reported SNPs were associated with prostate cancer risk in the large prospective Diet and Cancer cohort in Malmö, Sweden. SNPs were less useful in predicting prostate cancer risk than PSA at baseline.
doi:10.1016/j.eururo.2011.10.047
PMCID: PMC3269546  PMID: 22101116
Prostate cancer; Biomarkers; SNPs; PSA; Sensitivity and specificity
20.  Molecular Microheterogeneity of Prostate Specific Antigen in Seminal Fluid by Mass Spectrometry 
Clinical Biochemistry  2011;45(4-5):331-338.
Objectives
Prostate specific antigen (PSA) is a widely used and clinically valuable marker for prostate disease. In order to enable the development of new PSA assays and progress the understanding of the biology of PSA we have analyzed PSA in seminal plasma.
Design and Methods
PSA in seminal plasma from men attending a fertility clinic and healthy controls was analyzed using SDS-PAGE, Western blotting and mass spectrometry.
Results
Using mass spectrometry, different forms of PSA could be identified in 1–9 bands seen on SDS-PAGE analysis of the respective sample. However, a majority of these molecular forms of PSA were not observed on Western blots. Enzymatic activity of PSA isoforms was demonstrated by sequencing data in zymogram gels. Multivariate analysis of clinical data revealed well-separated patient groups.
Conclusions
We demonstrated that PSA in seminal plasma occurs in several isoforms, yet not all were detectable using an antibody based clinical routine method. The heterogeneity of PSA expression might be of clinical significance, by an improved patient phenotyping.
doi:10.1016/j.clinbiochem.2011.11.018
PMCID: PMC3292698  PMID: 22209970
prostate specific antigen; isoform; seminal plasma; infertility; MALDI LTQ Orbitrap XL; ESI-LTQ FT-ICR
21.  Intra-individual short-term variability of prostate-specific antigen and other kallikrein markers in a serial collection of blood from men under evaluation for prostate cancer 
BJU international  2010;107(11):1769-1774.
Objective
To assess variation of total prostate-specific antigen (tPSA), free PSA (fPSA), percent fPSA, human glandular kallikrein 2 (hK2), and intact PSA measured three times within two weeks. Knowledge of the variation in an individual’s PSA level is important for clinical decision-making.
Patients and Methods
Study participants were 149 patients referred for prostate biopsy, of which 97 had benign disease and 52 had prostate cancer. Three blood samples were drawn with a median of four hours between first and second samples and 12 days between first and third samples. Variability was described by absolute differences, ratios and intra-individual coefficients of variation. Total PSA, fPSA, hK2, and intact PSA were measured in anti-coagulated blood plasma.
Results
At baseline, the median tPSA was 6.8 (IQR 4.5, 9.6) ng/mL. The intra-individual variation was low for all biomarkers, and lowest for tPSA. For 80% of participants, the ratio between first and second time points for tPSA was between 0.91 and 1.09 and the ratio for percent fPSA was between 0.89 and 1.15. Total coefficients of variation between time 1 and 2 for tPSA, fPSA, percent fPSA, hK2 and intact PSA were 4.0%, 6.6%, 6.0%, 9.2%, and 9.5%, respectively. The measurements taken several days apart varied more than those taken on the same day, but the variation between both time points were not large.
Conclusion
The intra-individual variation for all the kallikrein-like markers studied was relatively small, especially for samples drawn the same day. Few cases are reclassified between the time points. This indicates high short-term biological and technical reproducibility of the tests in clinical use.
doi:10.1111/j.1464-410X.2010.09761.x
PMCID: PMC3572723  PMID: 20955263
Free PSA; Prostate cancer; PSA; Screening; Variation
22.  Tumor markers in prostate cancer I: blood-based markers 
Acta oncologica (Stockholm, Sweden)  2011;50(Suppl 1):61-75.
INTRODUCTION
The introduction of total prostate specific antigen (total PSA) testing in blood has revolutionized the detection and management of men with prostate cancer (PCa). The objective of this review was to discuss the challenges of PCa biomarker research, definition of the type of PCa biomarkers, the statistical considerations for biomarker discovery and validation, and to review the literature regarding total PSA velocity and novel blood-based biomarkers.
METHODS
An English-language literature review of the Medline database (1990 to August 2010) of published data on blood-based biomarkers and PCa was undertaken.
RESULTS
The inherent biological variability of total PSA levels affects the interpretation of any single result. Men who will eventually develop PCa have increased total PSA levels years or decades before the cancer is diagnosed. Total PSA velocity improves predictiveness of total PSA only marginally, limiting its value for PCa screening and prognostication. The combination of PSA molecular forms and other biomarkers improve PCa detection substantially. Several novel blood-based biomarkers such as human glandular kallikrein 2 (hK2), urokinase plasminogen activator (uPA) and its receptor (uPAR), transforming growth factor-beta 1 (TGF-β1); interleukin-6 (IL-6) and its receptor (IL-6R) may help PCa diagnosis, staging, prognostication, and monitoring. Panels of biomarkers that capture the biologic potential of PCa are in the process of being validated for PCa prognostication.
CONCLUSIONS
PSA is a strong prognostic marker for long-term risk of clinically relevant cancer. However, there is a need for novel biomarkers that aid clinical decision making about biopsy and initial treatment. There is no doubt that progress will continue based on the integrated collaboration of researchers, clinicians and biomedical firms.
doi:10.3109/0284186X.2010.542174
PMCID: PMC3571678  PMID: 21604943
Prostate neoplasms; molecular markers; prostate specific antigen
23.  Genome-wide Association Study Identifies Loci at ATF7IP and KLK2 Associated with Percentage of Circulating Free PSA1 2 
Neoplasia (New York, N.Y.)  2013;15(1):95-101.
Background
Percentage of free-to-total prostate-specific antigen (%fPSA) is an independent predictor of risk for prostate cancer among men with modestly elevated level of total PSA (tPSA) in blood. Physiological and pathological factors have been shown to influence the %fPSA value and diagnostic accuracy.
Materials/Methods
To evaluate genetic determinants of %fPSA, we conducted a genome-wide association study of serum %fPSA by genotyping 642,584 single nucleotide polymorphisms (SNPs) in 3192 men of European ancestry, each with a tPSA level of 2.5 to 10 ng/ml, that were recruited in the REduction by DUtasteride of Prostate Cancer Events study. Single nucleotide polymorphisms (SNPs) with P < 10-5 were further evaluated among the controls of a population-based case-control study in Sweden (2899 prostate cancer cases and 1722 male controls), including 464 controls having tPSA levels of 2.5 to 10 ng/ml.
Results
We identified two loci that were associated with %fPSA at a genome-wide significance level (P <5 x 10-8). The first associated SNP was rs3213764 (P = 6.45 x 10-10), a nonsynonymous variant (K530R) in the ATF7IP gene at 12p13. This variant was also nominally associated with tPSA (P = .015). The second locus was rs1354774 (P = 1.25 x 10-12), near KLK2 at 19q13, which was not associated with tPSA levels, and is separate from the rs17632542 locus at KLK3 that was previously associated with tPSA levels and prostate cancer risk. Neither rs3213764 nor rs1354774 was associated with prostate cancer risk or aggressiveness.
Conclusions
These findings demonstrate that genetic variants at ATF7IP and KLK2 contribute to the variance of %fPSA.
PMCID: PMC3556942  PMID: 23359319
24.  Bioinformatic Strategies for Unambiguous Identification of Prostate Specific Antigen in Clinical Samples 
Journal of proteomics  2011;75(1):202-210.
Prostate specific antigen (PSA), as a widely used clinical biomarker in prostate cancer diagnostics, exists in multiple molecular forms. However, all of these forms might not be recognized in a given sample by the standard immunoassays. Therefore, we have investigated PSA isoforms separated by size using mass spectrometric analyses. The objective of these developments was to identify and specify the various forms of PSA. To optimize successful identification of different PSA forms, we have developed a bioinformatic strategy, consisting of high resolution MALDI-MS PMF and sequencing MS/MS data searches. To improve sequence-based identification, the recently introduced Proteios software environment was employed, allowing the combination of multiple database search engines in an automated manner.
We could unambiguously identify PSA in clinical samples by all detectable tryptic peptides, which were found to be common in several isoforms.
doi:10.1016/j.jprot.2011.06.008
PMCID: PMC3220806  PMID: 21723425
prostate specific antigen isoforms; MALDI LTQ Orbitrap XL; ESI-LTQ FT-ICR; Proteios software environment; kallikrein-2
25.  TMPRSS2-ERG Status in Circulating Tumor Cells as a Predictive Biomarker of Sensitivity in Castration-Resistant Prostate Cancer Patients Treated With Abiraterone Acetate 
European urology  2011;60(5):897-904.
Background
Abiraterone acetate (AA) is an androgen biosynthesis inhibitor shown to prolong life in patients with castration-resistant prostate cancer (CRPC) already treated with chemotherapy. AA treatment results in dramatic declines in prostate-specific antigen (PSA) in some patients and no declines in others, suggesting the presence of molecular determinants of sensitivity in tumors.
Objective
To study the role of transmembrane protease, serine 2 (TMPRSS2)–v-ets erythroblastosis virus E26 oncogene homolog (ERG) fusion, an androgen-dependent growth factor, in circulating tumor cells (CTCs) as a biomarker of sensitivity to AA.
Design, setting, and participants
The predictive value of TMPRSS2-ERG status was studied in 41 of 48 men with postchemotherapy-treated CRPC enrolled in sequential phase 2 AA trials.
Intervention
Patients received AA 1000 mg daily and continuously.
Measurements
TMPRSS2-ERG status was characterized by a sensitive, analytically valid reverse transcription polymerase chain reaction assay in CTCs enriched from ethylene-diaminetetraacetic acid anticoagulated blood obtained prior to AA treatment. Outcomes were measured by PSA Working Group 1 criteria.
Results and limitations
Standard procedures for specimen acquisition, processing, and testing using the validated TMPRSS2-ERG assay on a multiplex platform gave intra-assay and interassay coefficients of variation <7%. TMPRSS2-ERG fusion was present in 15 of 41 patients (37%), who had a median baseline CTC count of 17 (interquartile range: 7–103 cells per 7.5 ml). A PSA decline ≥50% was observed in 7 of 15 patients (47%) with the fusion and in 10 of 26 patients (38%) without the fusion. Although limited by the low number of patients, a posttherapy CTC count of less than five per 7.5 ml was prognostic for longer survival relative to a CTC count five or more. TMPRSS2-ERG status did not predict a decline in PSA or other clinical outcomes.
Conclusions
Molecular profiles of CTCs with an analytically valid assay identified the presence of the prostate cancer–specific TMPRSS2-ERG fusion but did not predict for response to AA treatment. This finding demonstrates the role of CTCs as surrogate tissue that can be obtained in a routine practice setting.
doi:10.1016/j.eururo.2011.07.011
PMCID: PMC3185163  PMID: 21802835
Abiraterone; Biomarker; Circulating tumor cells; Prostate cancer; Prostate-specific antigen; TMPRSS2-ERG fusion

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