Carsten Stephan

Comparison of 10 serum bone turnover markers in prostate carcinoma patients with bone metastatic spread: Diagnostic and prognostic implications

Our aim was to assess the diagnostic accuracy of bone markers in serum of patients with prostate cancer (PCa) for early detection of bone metastases and their usefulness as predictors of PCa‐caused mortality. In sera of 117 PCa patients (pN0M0, n = 39; pN1M0, n = 34; M1, n = 44), 35 healthy men and 35 patients with benign prostatic hyperplasia, bone formation markers [total and bone‐specific alkaline phosphatase (tALP, bALP), amino‐terminal procollagen propeptides of type I collagen (P1NP), osteocalcin (OC)], bone resorption markers [bone sialoprotein (BSP), cross‐linked C‐terminal (CTX) and cross‐linked N‐terminal (NTX) telopeptides of type I collagen, tartrate‐resistant acid phosphatase isoenzyme 5b (TRAP)] and osteoclastogenesis markers [osteoprotegerin (OPG), receptor activator of nuclear factor κB ligand (RANKL)] were measured. tALP, bALP, BSP, P1NP, TRAP, NTX and OPG were significantly increased in PCa patients with bone metastases compared to patients without metastases. OPG showed the best discriminatory power to differentiate between these patients. Logistic regression analysis resulted in a model with OPG and TRAP as variables that predicted bone metastasis with an overall correct classification of 93%. Patients with concentrations of OPG, P1NP, tALP, bALP, BSP, NTX, TRAP and CTX above cut‐off levels showed significantly shorter survival than patients with low marker concentrations. Multivariate Cox proportional hazards regression revealed that only OPG and BSP were independent prognostic factors for PCa‐related death. Thus, the importance of serum OPG in detecting bone metastatic spread, alone or in combination with other bone markers, and predicting survival in PCa patients has been clearly demonstrated.

DECREASED CONCENTRATIONS OF PROSTATE-SPECIFIC ANTIGEN AND HUMAN GLANDULAR KALLIKREIN 2 IN MALIGNANT VERSUS NONMALIGNANT PROSTATIC TISSUE

OBJECTIVESnTo study quantitatively the relative expression of human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) in paired (from the same patient) cancerous and noncancerous prostatic tissue to evaluate whether these proteins are overexpressed or underexpressed in cancer.nnnMETHODSnWe studied 14 patients who underwent radical retropubic prostatectomy for prostate cancer. Cancerous and adjacent normal tissues were excised and then extracted to prepare cytosolic extracts. The extracts were analyzed for total protein, and for hK2 and PSA using sensitive and specific immunofluorometric procedures.nnnRESULTSnPSA was present in the prostatic extracts at about 50 to 100 times higher amounts than hK2. The correlation between PSA and hK2 values was good. Both prostate kallikreins were expressed more in noncancerous than in cancerous prostatic tissue.nnnCONCLUSIONSnOur results demonstrated that both PSA and hK2 are down-regulated in prostate cancer compared with noncancerous tissue. The degree of down-regulation was higher for PSA than for hK2. The mechanism and physiologic consequences of this down-regulation are unknown.

GOLPH2 protein expression as a novel tissue biomarker for prostate cancer: implications for tissue-based diagnostics

GOLPH2 is coding the 73-kDa type II Golgi membrane antigen GOLPH2/GP73. Upregulation of GOLPH2 mRNA has been recently reported in expression array analyses of prostate cancer. As GOLPH2 protein expression in prostate tissues is currently unknown, this study aimed at a comprehensive analysis of GOLPH2 protein in benign and malignant prostate lesions. Immunohistochemically detected GOLPH2 protein expression was compared with the basal cell marker p63 and the prostate cancer marker α-methylacyl-CoA racemase (AMACR) in 614 radical prostatectomy specimens. GOLPH2 exhibited a perinuclear Golgi-type staining pattern and was preferentially seen in prostatic gland epithelia. Using a semiquantitative staining intensity score, GOLPH2 expression was significantly higher in prostate cancer glands compared with normal glands (P<0.001). GOLPH2 protein was upregulated in 567 of 614 tumours (92.3%) and AMACR in 583 of 614 tumours (95%) (correlation coefficient 0.113, P=0.005). Importantly, GOLPH2 immunohistochemistry exhibited a lower level of intratumoral heterogeneity (25 vs 45%). Further, GOLPH2 upregulation was detected in 26 of 31 (84%) AMACR-negative prostate cancer cases. These data clearly suggest GOLPH2 as an additional ancillary positive marker for tissue-based diagnosis of prostate cancer.

The influence of prostate volume on the ratio of free to total prostate specific antigen in serum of patients with prostate carcinoma and benign prostate hyperplasia

Determining the ratio of free to total prostate specific antigen (f‐PSA to t‐PSA, calculated as the percentage of f‐PSA [f‐PSA%]) in serum allows for a clearer distinction between patients with prostate carcinoma (PCa) and patients with benign prostate hyperplasia (BPH) than determining the level of t‐PSA alone. To find influencing factors on f‐PSA%, the authors investigated prostate volume, TNM classification, and tumor stage.

Prostate-specific antigen, its molecular forms, and other kallikrein markers for detection of prostate cancer ☆

P antigen (PSA) is a serine protease produced by the prostate gland at very high concentrations. PSA is secreted into the seminal plasma in high concentrations (0.5 to 5 g/L), where it plays a role in semen liquefaction. The retrograde release of PSA into the bloodstream is a rare event in young, healthy men. It occurs with a frequency of less than one PSA molecule per million secreted PSA molecules, leading to a concentration of less than 4 ng/mL PSA in the serum. The perturbation of the prostate gland architecture often results in excessive escape of PSA into the circulation. Prostate cancer, benign prostatic diseases, and physical trauma to the prostate can result in significant increases in serum PSA. Thus, an elevated serum PSA level is a sensitive marker of prostate gland abnormalities, including prostate cancer and many other conditions affecting the integrity of the prostate gland. These noncancerous alterations have severely challenged the usefulness of PSA as a tumor marker for the early detection of prostate cancer. This disease is the most common malignancy in men, with an estimated 198,100 new cases and 31,500 deaths in 2001.1 Several calculated parameters, such as PSA density, PSA transition zone density, PSA velocity, and age and racespecific PSA ranges, have been only partially successful in enhancing the specificity of PSA and thus reducing somewhat the number of unnecessary prostate biopsies.2 The discovery of different PSA forms, such as free PSA (fPSA) and PSA bound to alpha1-antichymotrypsin (PSA-ACT) in the early 1990s renewed the clinical research on this biomarker.3,4 This review summarizes the current clinical use of fPSA and focuses on new developments regarding the molecular forms of PSA and the promising role of additional kallikreins (especially hK2) for prostate cancer detection.

Quantitative analysis of macrophage inhibitory cytokine-1 (MIC-1) gene expression in human prostatic tissues

Macrophage inhibitory cytokine-1 (MIC-1) gene is a member of transforming growth factor-β superfamily and was reported to be highly overexpressed in human prostate cancer using microarray technology. The aim of this study was to evaluate the quantitative expression of MIC-1 in malignant and benign prostate tissues and to associate expression levels with clinicopathological parameters of prostate cancer. Matched (paired) prostatic tissue samples from the cancerous and noncancerous parts of the same prostates were obtained from 66 patients who underwent radical prostatectomy. Quantitative RT–PCR was performed using SYBR Green I on the Roche LightCyclerTM system. Macrophage inhibitory cytokine-1 gene overexpression in cancerous tissues was observed in 88% of cases, compared to noncancerous tissues (P<0.001). The expression level of MIC-1 in cancerous tissues was significantly higher than in noncancerous tissue (P<0.001). Higher expression of MIC-1 gene was significantly associated with higher Gleason score (P=0.004). The expression of the MIC-1 gene in prostate cancer is significantly higher than in noncancerous tissues, especially in more aggressive forms of the disease (Gleason score>5). This is in contrast to prostate-specific antigen that is downregulated in higher-grade tumours. The upregulation of MIC-1 in prostate cancer and in advanced and more aggressive prostatic tumours suggests that MIC-1 protein should be evaluated as a potential diagnostic and prognostic biomarker.

An artificial neural network to predict the outcome of repeat prostate biopsies

OBJECTIVESnTo develop an advanced artificial neural network (ANN) to predict the presence of prostate cancer (PCa) and to predict the outcome of repeat prostate biopsies. The predictive accuracy was compared with the accuracy obtained using standard cutoffs for the free/total (f/t) prostate-specific antigen (PSA) ratio, PSA density (PSAD), PSA density of the transition zone (PSA-TZ), and the total and transition zone volumes. Clinical and biochemical diagnostic tests have been shown to improve PCa detection. When these tests are combined using an ANN, significant increases in specificity at high sensitivity are observed.nnnMETHODSnThe Vienna-based multicenter European referral database for early PCa detection of 820 men with a PSA level between 4 and 10 ng/mL was used. The presence of PCa was determined using transrectal ultrasound-guided octant needle repeat biopsy. Variables in the database consisted of age, PSA, f/t PSA ratio, digital rectal examination findings, PSA velocity, and the transrectal ultrasound-guided variables of prostate volume, transition zone volume, PSAD, and PSA-TZ. The ANN used in the analysis was an advanced multilayer perceptron selected for accuracy by a genetic algorithm.nnnRESULTSnThe repeat biopsy PCa detection rate was 10% (n = 83). At 95% sensitivity, the specificity for ANN was 68% compared with 54%, 33.5%, 21.4%, 14.7%, and 8.3% for multivariate logistic regression analysis, f/t PSA ratio, PSA-TZ, PSAD, and total PSA, respectively. The ANN reduced unnecessary repeat biopsies by 68% in this study. The area under the curve was 83% for the ANN versus 79%, 74.5%, 69.1%, 61.8%, and 60.5% for multivariate analysis, f/t PSA ratio, PSA-TZ, PSAD, and total PSA, respectively.nnnCONCLUSIONSnThe current ANN found a strong pattern predictive of PCa in patients with a negative initial biopsy. By combining the individual clinical and biochemical markers into the ANN, 68% specificity at 95% sensitivity was achieved. The ANN allows more accurate and individual counseling of patients with a negative initial biopsy.

Quantitative Analysis of Kallikrein 15 Gene Expression in Prostate Tissue

PURPOSEnThe newly discovered human kallikrein 15 gene KLK15 has been shown in preliminary analysis to be associated with more aggressive types of prostate cancer. We quantitatively measured and compared gene expression of KLK15 in malignant and benign prostate tissues.nnnMATERIALS AND METHODSnMatched prostate tissue samples from the cancerous and noncancerous parts of the same prostates were obtained from 90 patients who underwent radical prostatectomy. Quantitative reverse transcriptase-polymerase chain reaction using SYBR Green I and the LightCycler system (Roche Applied Science, Mannheim, Germany) was performed. Associations of KLK15 expression with clinicopathological parameters were analyzed.nnnRESULTSnKLK15 over expression in cancerous versus noncancerous tissue was found in 76 of the 90 patient samples (84.4%, p <0.001). The ratio of cancerous-to-noncancerous KLK15 expression tended to be higher in patients with stage pT3/4 versus pT2 tumors (p = 0.1). KLK15 expression tended to be higher in grade 3 than in grade 2 tumors and in Gleason score 7 or greater than in Gleason score less than 7 tumors (p = 0.18 and 0.23, respectively). A 1.7 cutoff at the 40th percentile provided a significant difference in stages pT2 and pT3/4 tumors (p = 0.029).nnnCONCLUSIONSnOn quantitative real-time polymerase chain reaction KLK15 expression was significantly higher in cancerous than in noncancerous tissue. Up-regulation of the KLK15 gene in advanced and more aggressive tumors may indicate a possible role for KLK15 protein as future serum marker for prostate cancer and for distinguishing tumor aggressiveness.

Matrix‐metalloproteinases and their inhibitors in plasma and tumor tissue of patients with renal cell carcinoma

Matrix‐metalloproteinases (MMPs) are associated with invasive and metastatic behavior of several human malignant tumors. We have determined MMP‐2 and MMP‐9 and tissue inhibitors of MMPs (TIMP‐1 and TIMP‐2) in blood plasma and in renal tissue samples of patients with renal cell carcinoma (RCC) from cancerous and non‐cancerous parts of the same kidney. In tumor tissue, MMP‐9 and TIMP‐1 were significantly higher than in normal counterparts. MMP‐2 was not different between tumor tissue and normal counterparts. TIMP‐2 values could not be measured. In plasma, MMP‐9 concentrations were significantly higher in RCC patients than in healthy controls, MMP‐2 and TIMP‐2 concentrations were higher in healthy controls and TIMP‐1 concentrations were not different. Int. J. Cancer 85:801–804, 2000.

Differential expression of kallikrein gene 5 in cancerous and normal testicular tissues

OBJECTIVESnKallikrein gene 5 (KLK5; formerly designated as kallikrein-like gene 2, or human stratum corneum tryptic enzyme) is one of the new members of the human kallikrein gene family on chromosome 19q13.4. Although it is expressed at low levels in various tissues, KLK5 expression is highest in the human mammary gland and testis. Previous investigations have established that the expression of KLK5 is estrogen and progestin driven in the BT-474 breast cancer cell line. In this study, we focused on KLK5 expression in normal and cancerous testicular tissue.nnnMETHODSnFourteen matched testicular tumor and adjacent normal tissue samples were minced on dry ice and homogenized. Total RNA was extracted and mRNA was reverse transcribed. The cDNA samples were amplified by real-time quantitative polymerase chain reaction with KLK5-specific primers to compare the relative KLK5 levels in normal and cancerous testicular tissues.nnnRESULTSnIn 13 (93%) of the 14 patients, KLK5 expression in the cancerous area was significantly lower than in the adjacent, histologically confirmed, normal testicular tissue samples (P <0.001). The KLK5 level was 9.0 +/- 3.9 (mean +/- standard error, arbitrary units) in the noncancerous tissue and 4.5 +/- 2.9 in the cancerous tissue. We noted significantly lower KLK5 expression in seminomas than in nonseminomas (P = 0.009), as well as in late-stage (II/III) tumors versus early-stage (I) tumors (P = 0.026). KLK5 expression was also associated with the extent of primary tumor, with tumors with vascular/lymphatic invasion (T2/T3) expressing lower KLK5 message than did tumors limited to the testis and epididymis (T1) (P = 0.008).nnnCONCLUSIONSnWe found significantly lower expression of KLK5 in testicular tumors than in normal testicular tissue. More studies are necessary to investigate the mechanism behind this finding.