Increased Expression and Altered Cellular Localization of Fibroblast Growth Factor Receptor-Like 1 (FGFRL1) Are Associated with Prostate Cancer Progression.

Fibroblast growth factor receptors (FGFRs) 1-4 are involved in prostate cancer (PCa) regulation, but the role of FGFR-like 1 (FGFRL1) in PCa is unclear. FGFRL1 expression was studied by qRT-PCR and immunohistochemistry of patient tissue microarrays (TMAs) and correlated with clinical patient data. The effects of FGFRL1 knockdown (KD) in PC3M were studied in in vitro culture models and in mouse xenograft tumors. Our results showed that FGFRL1 was significantly upregulated in PCa. The level of membranous FGFRL1 was negatively associated with high Gleason scores (GSs) and Ki67, while increased cytoplasmic and nuclear FGFRL1 showed a positive correlation. Cox regression analysis indicated that nuclear FGFRL1 was an independent prognostic marker for biochemical recurrence after radical prostatectomy. Functional studies indicated that FGFRL1-KD in PC3M cells increases FGFR signaling, whereas FGFRL1 overexpression attenuates it, supporting decoy receptor actions of membrane-localized FGFRL1. In accordance with clinical data, FGFRL1-KD markedly suppressed PC3M xenograft growth. Transcriptomics of FGFRL1-KD cells and xenografts revealed major changes in genes regulating differentiation, ECM turnover, and tumor-stromal interactions associated with decreased growth in FGFRL1-KD xenografts. Our results suggest that FGFRL1 upregulation and altered cellular compartmentalization contribute to PCa progression. The nuclear FGFRL1 could serve as a prognostic marker for PCa patients.

Benefit of Adjuvant Chemotherapy and Pelvic Lymph Node Dissection in pT3 and Node Positive Bladder Cancer Patients Treated with Radical Cystectomy.

Background: Benefits of adjuvant chemotherapy (AC) and extent of pelvic lymph node dissection (PLND) in radical cystectomy (RC) are debated. Results from randomized trials are still expected. Objective: To analyze the effects of AC and PLND in two academic centers with opposite policies regarding their use. Methods: 581 bladder cancer patients who underwent RC without neoadjuvant chemotherapy, from Toronto (University Health Network), Canada, and Turku University Hospital, Finland were included. Disease specific survival (DSS) and failure patterns were assessed. Results: Centers differed in PLND rate (93% and 36% in Toronto and Turku respectively, p <  0.001), PLND extent (≥10 removed nodes, 58% vs. 8%, p <  0.001) and AC rate (21% vs. 2%, p <  0.001). Survival between centers among pT≤1 or pT4 patients was similar. pT3 patients in Toronto had an improved 10 year DSS (43% vs. 22%, p = 0.025). Distant failures were less common after AC (HR 0.56, 95%  CI 0.33-0.98, p <  0.042). In node positive (N+) patients, mortality was significantly higher in Turku (HR 2.19, 95%  CI 1.44-3.34, p <  0.001) and lower in patients receiving AC (HR 0.60, 95%  CI 0.37-0.99, p = 0.044). 41% DSS at 10 years was observed in N+ Toronto patients. Limitations included the non-randomized retrospective design and absence of propensity score analysis. Conclusion: Combining AC and PLND to RC is associated with improved survival in pT3 and N+ patients. PLND did not affect survival independently but helps in selecting patients for AC. Our data adds to the growing body of evidence supporting the usefulness of AC in addition to PLND in high risk patients operated by cystectomy.

Increased expression of fibroblast growth factor 13 in prostate cancer is associated with shortened time to biochemical recurrence after radical prostatectomy.

Fibroblast growth factor homologous factors (FHFs) belong to the fibroblast growth factor (FGF) superfamily, which plays an important role in prostate cancer (PCa). Mining of public database suggests that FGF13 (FHF2) mRNA expression is altered in over 30% of PCa cases. This study examined the FGF13 expression pattern in human PCa specimens and evaluated its potential as a biomarker for patient outcome after radical prostatectomy (RP). Immunohistochemistry (IHC) showed that FGF13 was detectable in the majority of human PCa samples, and FGF13 IHC scores were higher in high-grade prostatic intraepithelial neoplasia, in primary PCa and in metastatic PCa than in benign prostatic tissue. There was a significant association between high cytoplasmic FGF13 staining and a risk of biochemical recurrence (BCR) after RP. This was also evident in the intermediate to high-risk patient groups. In contrast, positive nuclear FGF13 staining along with low cytoplasmic FGF13 group showed a decreased BCR risk. Multivariate regression analysis indicated that high cytoplasmic FGF13 staining was associated with BCR and that this could serve as an independent prognostic marker in PCa. Several PCa cell lines showed increased FGF13 expression at the mRNA and protein levels compared to the immortalized prostate epithelial cell line PNT1a. Analysis of co-labeled cells suggested a possible interaction of FGF13 with α-tubulin and the voltage-gated sodium channel proteins (Na(V)s/VGSCs). Our data indicate that, for PCa patients after RP, FGF13 serves as a potential novel prognostic marker that improves prediction of BCR-free survival, in particular if combined with other clinical parameters.

Cancer-associated changes in the expression of TMPRSS2-ERG, PCA3, and SPINK1 in histologically benign tissue from cancerous vs noncancerous prostatectomy specimens.

OBJECTIVE: To investigate whether messenger ribonucleic acid (mRNA) expression of TMPRSS2-ERG fusion gene, a suggested prostate cancer (PCa) biomarker, was specific to cancerous lesions alone and to study the expression of SPINK1 and PCA3 mRNAs in the same cohort to also explore the proposed mutual exclusivity of TMPRSS2-ERG and SPINK1 expression. METHODS: Levels of 2 TMPRSS2-ERG transcripts, PCA3, and SPINK1 mRNAs were measured with highly standardized reverse transcription quantitative polymerase chain reaction assays in cystoprostatectomy specimens from 19 patients with invasive bladder cancer and 174 radical prostatectomy (RP) samples (88 histologically benign prostate [HBP] tissues and 86 from cancerous lesions) from 87 patients with clinically localized PCa. RESULTS: Expression of TMPRSS2-ERG transcripts was detected in 45 of 88 (51%) HBP tissues from RP specimens and more frequently (57 of 86, 66%) found in cancerous lesions. In contrast, TMPRSS2-ERG expression was detected in only 2 of 19 (11%) cystoprostatectomy specimens, both with incidental PCa foci elsewhere in the gland. Similar trends of changes in the expression of PCA3 and SPINK1 were present in HBP tissue from RP compared with cystoprostatectomy specimens. CONCLUSION: Although the expression of TMPRSS2-ERG, SPINK1, and PCA3 mRNA is higher or more frequently found in cancerous lesions, HBP tissues from patients with clinically localized PCa manifest molecular, mRNA level changes that are absent in cystoprostatectomy specimens lacking incidental PCa foci or infrequent in cystoprostatectomy specimens containing incidental PCa. If this finding is replicated, these molecular assays could be used to inform men with negative biopsy results about the likelihood of cancerous lesions in unsampled regions and hence the need for repeat biopsy.

Association of transcript levels of 10 established or candidate-biomarker gene targets with cancerous versus non-cancerous prostate tissue from radical prostatectomy specimens.

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 10(5)-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.

[Update on current care guidelines: benign prostatic hyperplasia]. / Eturauhasen hyvänlaatuinen liikakasvu.

Most benign prostatic hyperplasia (BPH) patients can be diagnosed and treated in primary care. Evaluation requires a medical history and a symptom score (DAN-PSS or IPSS) and a careful physical examination including a digital rectal examination. Careful follow-up and medical therapy are the usual first-line management options in uncomplicated BPH. Combination therapy with both alpha-blocker and 5alpha-reductase inhibitor is the most effective medical treatment modality. Transurethral resection (TURP) is a safe and effective way of treating BPH. The green laser is the most promising new endoscopic treatment modality.

Does patient age affect survival after radical cystectomy?

UNLABELLED: What's known on the subject? and What does the study add? Elderly patients have more years to compound comorbidities and it has previously been shown that comorbidity is an important predictor of overall survival in patients with bladder cancer, including those treated with radical cystectomy (RC). Other studies have also demonstrated higher stage at diagnosis, higher rate of upstaging on final pathology and a longer delay to definitive therapy for older patients. Because of these findings, elderly patients are being offered RC less often than younger patients. Whether or not this practice is justified has come under recent scrutiny and there has been much conflicting data in the literature. While some studies have shown worse outcomes for elderly patients, others have shown similar results for both elderly and younger patients. Large population-based databases have recently been used to try to determine whether age effects outcome after RC but their conclusions may not be as generalizable as ours for several reasons: billing code data was used to build patient cohorts, patients were generally recipients of Medicare, lack of pathological review, and lack of available and accurate clinical data. Our series is unique in that it comprises a large group of patients from two major tertiary care academic institutions using a very robust dataset. Pathological specimens were reviewed by dedicated genitourinary pathologists, including those recovered from peripheral hospitals. Our sample size is one of the largest single- or multi-institutional studies. OBJECTIVE: • To analyse the impact of patient age on survival after radical cystectomy (RC). PATIENTS AND METHODS: • After ethics review board approval, two databases of patients with bladder cancer (BC) undergoing RC at the University Heath Network, Toronto, Canada (1992-2008) and the University of Turku, Turku, Finland (1986-2005) were retrospectively analysed. • A total of 605 patients who underwent this procedure between June 1985 and March 2010 were included. • Patients were divided into four age groups: ≤ 59, 60-69, 70-79 and ≥ 80 years. • Demographic, clinical and pathological data were compared, as well as recurrence-free survival (RFS), disease-specific survival (DSS) and overall survival (OAS) rates. RESULTS: • Compared with younger patients (age ≤ 79 years), elderly patients (age ≥ 80 years) had higher American Society of Anesthesiologists scores (P < 0.001), a greater number of lymph nodes removed during surgical dissection (P < 0.001), and underwent less adjuvant treatment (P < 0.001). • Choice of urinary diversion differed among the groups, with ileal conduit being used for all patients ≥ 80 years (P < 0.001). • No differences were noted between age groups with respect to RFS (P= 0.3), DSS (P= 0.4) or OAS (P= 0.4). CONCLUSION: • Although RC is an operation with significant morbidity, it is a viable treatment option for carefully selected elderly patients. Senior patients (≥ 80 years) should not be denied RC if they are deemed fit to undergo surgery. • Senior adults do not suffer from adverse histopathological features as compared with younger patients.

Improved detection of localized prostate cancer using co-registered MRI and 11C-acetate PET/CT.

OBJECTIVES: We aimed to study the ability of contrast enhanced MRI at 1.5 T and 11C-acetate PET/CT, both individually and using fused data, to detect localized prostate cancer. METHODS: Thirty-six men with untreated prostate cancer and negative for metastatic disease on pelvic CT and bone scan were prospectively enrolled. A pelvic 11C-acetate PET/CT scan was performed in all patients, and a contrast enhanced MRI scan in 33 patients (6 examinations using both endorectal coil and surface coils, and 27 examinations using surface coils only). After the imaging studies 10 patients underwent prostatectomy and 26 were treated by image guided external beam radiation treatment. Image fusion of co-registered PET and MRI data was performed based on anatomical landmarks visible on CT and MRI using an advanced in-house developed software package. PET/CT, MRI and fused PET/MRI data were evaluated visually and compared with biopsy findings on a lobar level, while a sextant approach was used for patients undergoing prostatectomy. RESULTS: When using biopsy samples as method of reference, the sensitivity, specificity and accuracy for visual detection of prostate cancer on a lobar level by contrast enhanced MRI was 85%, 37%, 73% and that of 11C-acetate PET/CT 88%, 41%, 74%, respectively. Fusion of PET with MRI data increased sensitivity, specificity and accuracy to 90%, 72% and 85%, respectively. CONCLUSIONS: Fusion of sequentially obtained PET/CT and MRI data for the localization of prostate cancer is feasible and superior to the performance of each individual modality alone.

[Treatment of benign prostatic hyperplasia]. / Eturauhasen hyvänlaatuisen liikakasvun hoito.

Due to the changing population structure, the proportion of aged men has increased, whereby there is an increasing need for the treatment of prostatic hyperplasia. Most men can be treated with medication within the primary health care. Although the disease is mostly mild in nature and merely lowering the quality of life, it may cause severe complications in the urinary tract, if left untreated. In the treatment of severe cases, the common transurethral prostatic resection has maintained an important status, but has been accompanied by several mini-invasive treatment methods.

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.

Functional imaging of localized prostate cancer aggressiveness using 11C-acetate PET/CT and 1H-MR spectroscopy.

UNLABELLED: We assessed the ability of (11)C-acetate PET/CT, MRI, and proton MR spectroscopy ((1)H-MRS) to image localized prostate cancer and detect its aggressiveness, using qualitative and quantitative approaches. METHODS: Twenty-one patients with untreated localized prostate cancer, diagnosed using transrectal ultrasound-guided biopsy, were prospectively enrolled. Cancer laterality was based on the percentage of cancer and the highest Gleason score determined from biopsies. In addition to PET/CT, 3-dimensional (1)H-MRS of the entire prostate volume using a quantitative approach was performed. The imaging and histologic findings of 8 patients undergoing subsequent prostatectomy were compared on a sextant level. For each lobe and sextant, standardized uptake values (SUVs) and (choline + creatine + polyamines)-to-citrate (CCP/C) ratios were obtained from (11)C-acetate PET/CT and (1)H-MRS, respectively. The visual and quantitative findings on PET/CT and MRI data were compared with cancer laterality and aggressiveness based on the Gleason score and with prostate-specific antigen (PSA) velocity and international risk group classification. RESULTS: The sensitivity, specificity, and accuracy, on a lobar level using visual analysis, of (11)C-acetate PET/CT were 80%, 29%, 71%, respectively, and 89%, 29%, 79%, respectively, using contrast-enhanced MRI. The sensitivity and accuracy of (11)C-acetate PET/CT decreased to 64% and 63% and specificity increased to 62% when sextant analysis was performed. The agreement between prostate cancer laterality based on biopsy findings and visual interpretation of (11)C-acetate PET/CT and contrast-enhanced MRI was similar at 71%. The mean SUV maximum and CCP/C maximum for the dominant tumor lesion were 5.5 and 1.48, respectively, and did not differ significantly from values in the nondominant lobe. The dominant-lesion SUVs or CCP/C values were not associated with histologically determined prostate cancer aggressiveness, nor did PSA velocity correlate with the SUV or CCP/C values from the entire gland. CONCLUSION: (11)C-acetate PET/CT, MRI, and (1)H-MRS enable detection of localized prostate cancer with comparable and limited accuracy but fail to provide information on cancer aggressiveness.

N-terminal truncation of Stat5a/b circumvents PIAS3-mediated transcriptional inhibition of Stat5 in prostate cancer cells.

Transcription factor Stat5a/b is critical for prostate cancer cell survival and for prostate xenograft tumor growth. In addition, the Stat5a/b signaling pathway may contribute to progression of organ-confined prostate cancer to castration-resistant and/or metastatic disease. Expression of nuclear Stat5a/b is clustered to high grade human prostate cancers, and nuclear Stat5a/b in primary prostate cancer predicts early disease recurrence after initial treatment. Here, we show by Western blotting and electromobility shift assay that Stat5a/b protein in human prostate cancer is N-terminally truncated. This short form of Stat5a/b is generated post-translationally in vivo in prostate cancer cells and is the predominant form of Stat5a/b that binds to DNA. We further demonstrate by mutagenesis and co-immunoprecipitations that the N-domain of Stat5a/b is required for binding to PIAS3, and that PIAS3 inhibits transcriptional activity of Stat5a/b in breast cancer cells but not in prostate cancer cells. Thus, the proteolytic cleavage of the N-terminus of Stat5a/b may be a mechanism by which Stat5 evades the transcriptional repression by PIAS3 in prostate cancer cells, and results in increased Stat5-driven gene expression and prostate cancer progression.

Twenty-year experience of radical cystectomy for bladder cancer in a medium-volume centre.

OBJECTIVE: To evaluate long-term survival after radical cystectomy (RC) for bladder cancer (BC) and to define risk factors for BC-specific death. MATERIAL AND METHODS: Patients having RC for BC with curative intent in Turku University Hospital 1986-2005 were assessed. Survival results were recorded and 10 risk factors for BC-specific death were analysed. RESULTS: In total, 248 patients with a median age of 64 years were included in the study. Sixty-four per cent of the tumours were intravesical and the lymph-node metastasis rate was 9%. Disease recurrence was observed in 90 patients (36%). Median time for local recurrence and distant metastasis after RC was 9 and 12 months, respectively. The mortality rate for both local recurrence and distant metastasis was 93%. Upper urinary tract and urethral recurrences were less common (3% and 5%, respectively) and occurred later (median time to recurrence 26 and 18 months, respectively). The 5-, 10-, and 15-year BC-specific survival was 69%, 67% and 66%, respectively. Extravesical tumour status, high tumour grade, positive node status and no history of intravesical therapy before RC were significant risk factors for BC-specific death. Other variables (neoadjuvant radiation, lymphadenectomy, age, time period, gender, smoking) did not affect the risk. CONCLUSIONS: The survival results are comparable with those of high-volume centres and demonstrate the possibility of excellent local control in all cases and a high rate of cure in tumours confined to the bladder. Extravesical tumour growth, high grade and lymph-node metastasis are the most important risk factors for BC-specific mortality.

Risk factors for mortality and morbidity related to radical cystectomy.

OBJECTIVES: To evaluate the risk factors for mortality and morbidity related to radical cystectomy (RC) in a medium-sized academic centre, and to analyse the rate and trends of perioperative morbidity and mortality, as although complications related RC to are lower in modern than historic series, RC is still associated with marked risks. PATIENTS AND METHODS: The study included 258 patients undergoing RC for bladder cancer in Turku University Hospital in 1986-2005. Basic patient characteristics and in-hospital, early (from hospital discharge to 3 months) and combined morbidity and mortality were analysed. Risk analysis included 16 risk factors for complications. Trends were analysed by comparing the two study decades (1986-1995 vs 1996-2005). RESULTS: The total complication rate was 34%, with minor and major complications in 26%, and 11% of patients, respectively. There were no significant changes in total morbidity, but the number of myocardial infarctions and atrial fibrillations decreased significantly (P = 0.045). Operative mortality was 2.7%, with an insignificant decrease (4.2% to 0.9%, P = 0.11) over time. Salvage RC, high American Society of Anesthesiologists (ASA) score (> or = 3), extensive blood loss (>3 L), a high number of transfusions (five or more), several comorbidities (two or more), age (> or = 65 vs <65 years), and extravesical tumours were significant risk factors for major complications. An ASA score of > or = 3 and five or more transfusions were the only factors associated with mortality. A high ASA score (odds ration 3.25, 95% confidence interval 1.08-9.74) and high number of transfusions (2.74, 1.05-7.15) were independent risk factors for major complications. CONCLUSION: Although RC is associated with acceptable morbidity, attention should be given to risk factors identified at the time of patient selection, and to meticulous haemostasis at the time of surgery. A predictable outcome comparable to that in high-volume centres is also possible in a medium-sized hospital.

Quantitative real-time RT-PCR assay for PCA3.

OBJECTIVES: To develop a quantitative, internally standardized real-time RT-PCR assay for prostate cancer antigen 3 (PCA3), a non-translated gene found to be prostate-specific and highly overexpressed in cancer, and examine the role of PCA3 in peripheral blood with a small sample cohort. DESIGN AND METHODS: The RT-PCR assay for PCA3 is based on target-specific lanthanide probes. Peripheral blood from 91 prostatic cancer/disorder patients and healthy controls was assayed for PCA3 and prostate-specific antigen (PSA) expression. RESULTS: The dynamic range of the assay reaches over eight orders of magnitude and the limit of quantification is 800 copies per milliliter blood. Peripheral blood from 2/9 patients with metastasized cancers were PCA3 positive, whereas all the other samples were negative. Eight samples were PSA positive. CONCLUSIONS: The degree of PCA3 positivity in circulating cells from prostate cancer patients is low compared to that of PSA. In contrast to some previous reports, we found no PCA3 expression in healthy individuals.

Autocrine prolactin promotes prostate cancer cell growth via Janus kinase-2-signal transducer and activator of transcription-5a/b signaling pathway.

The molecular mechanisms that promote progression of localized prostate cancer to hormone-refractory and disseminated disease are poorly understood. Prolactin (Prl) is a local growth factor produced in high-grade prostate cancer, and exogenously added Prl in tissue or explant cultures of normal and malignant prostate is a strong mitogen and survival factor for prostate epithelium. The key signaling proteins that mediate the biological effects of Prl in prostate cancer are Signal Transducer and Activator of Transcription (Stat)-5a/5b via activation of Janus kinase-2. Importantly, inhibition of Stat5a/b in prostate cancer cells induces apoptotic death. Using a specific Prl receptor antagonist (Delta1-9G129R-hPRL), we demonstrate here for the first time that autocrine Prl in androgen-independent human prostate cancer cells promotes cell viability via Stat5 signaling pathway. Furthermore, we examined a unique clinical material of human hormone refractory prostate cancers and metastases and show that autocrine Prl is expressed in 54% of hormone-refractory clinical human prostate cancers and 62% prostate cancer metastases. Finally, we demonstrate that autocrine Prl is expressed from both the proximal and distal promoters of the Prl gene in clinical human prostate cancers and in vivo and in vitro human prostate cancer models, independently of pituitary transcription factor-1 (Pit-1). Collectively, the data provide novel evidence for the concept that autocrine Prl signaling pathway is involved in growth of hormone-refractory and metastatic prostate cancer. The study also provides support for the use of Prl receptor antagonists or other therapeutic strategies to block the Prl-Janus kinase-2-Stat5 signaling pathway in advanced prostate cancer.

Novel homogenous time-resolved fluorometric RT-PCR assays for quantification of PSA and hK2 mRNAs in blood.

OBJECTIVES: The purpose of this study was to design, validate, and optimize internally standardized real-time quantitative RT-PCR assays and to identify and avoid problems with assay reliability and examine the impact of an exogenous internal standard. DESIGN AND METHODS: The model system consisted of internally standardized quantitative real-time RT-PCR assays specific for PSA and hK2 mRNA based on time-resolved fluorometric detection of lanthanide chelates. RESULTS: Reproducibility was best when large copy numbers (>5000 per milliliter blood) were analyzed. Addition of an exogenous target-mimicking internal standard had no significant effect on the reproducibility of the method, but increased the calculated copy numbers by an average of 2-fold. CONCLUSIONS: We developed an internally standardized, specific and reproducible real-time RT-PCR analysis method for PSA and hK2 mRNA in circulating cells in the bloodstream. Both PSA and hK2 assays are sufficiently sensitive to detect two LNCaP cells per milliliter whole blood.

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.

Free and total human glandular kallikrein 2 in patients with prostate cancer.

OBJECTIVES: The use of prostate-specific antigen (PSA, hK3) results in the overdiagnosis and overtreatment of prostate cancer. Markers are needed that could identify aggressive, fast-growing tumors and help decide which patients would benefit most from aggressive treatment. Human glandular kallikrein 2 (hK2) could be such a marker. The aim of this study was to test how PSA and hK2 could predict the pathologic stage and grade in a set of patients with clinically organ-confined disease. METHODS: Heparin plasma was collected from 188 patients who had undergone radical prostatectomy at the Turku University Central Hospital. Total and free PSA, as well as total and free hK2, were measured and the results compared with the pathologic TNM stage, tumor World Health Organization grade, and Gleason score. RESULTS: Free and total hK2 performed similarly to PSA in their ability to separate groups of patients with different stages or grades. Concentrations of both kallikreins were significantly different in patients with World Health Organization grade 1 cancer compared with grade 2. Neither marker could separate patients with different Gleason scores. Although PSA concentrations increased most between patients with Stage pT2b and those with pT3a, the increase in hK2 was most pronounced between those with Stage pT3a and those with pT3b. CONCLUSIONS: Although hK2 could not predict the cancer stage or grade better than PSA, changes in the hK2 and PSA concentrations occurred at different points in cancer progression. hK2 may have a role in the prognosis of prostate cancer, but additional studies with longer follow-up are required to determine whether hK2 can help when selecting treatment options.