Prognostic impact of kallikrein-related peptidase transcript levels in prostate cancer.

We aimed to study mRNA levels and prognostic impact of all 15 human kallikrein-related peptidases (KLKs) and their targets, proteinase-activated receptors (PARs), in surgically treated prostate cancer (PCa). Seventy-nine patients with localized grade group 2-4 PCas represented aggressive cases, based on metastatic progression during median follow-up of 11 years. Eighty-six patients with similar baseline characteristics, but no metastasis during follow-up, were assigned as controls. Transcript counts were detected with nCounter technology. KLK12 protein expression was investigated with immunohistochemistry. The effects of KLK12 and KLK15 were studied in LNCaP cells using RNA interference. KLK3, -2, -4, -11, -15, -10 and -12 mRNA, in decreasing order, were expressed over limit of detection (LOD). The expression of KLK2, -3, -4 and -15 was decreased and KLK12 increased in aggressive cancers, compared to controls (P < .05). Low KLK2, -3 and -15 expression was associated with short metastasis-free survival (P < .05) in Kaplan-Meier analysis. PAR1 and -2 were expressed over LOD, and PAR1 expression was higher, and PAR2 lower, in aggressive cases than controls. Together, KLKs and PARs improved classification of metastatic and lethal disease over grade, pathological stage and prostate-specific antigen combined, in random forest analyses. Strong KLK12 immunohistochemical staining was associated with short metastasis-free and PCa-specific survival in Kaplan-Meier analysis (P < .05). Knock-down of KLK15 reduced colony formation of LNCaP cells grown on Matrigel basement membrane preparation. These results support the involvement of several KLKs in PCa progression, highlighting, that they may serve as prognostic PCa biomarkers.

The roles of proteases in prostate cancer.

Since the proposition of the pro-invasive activity of proteolytic enzymes over 70 years ago, several roles for proteases in cancer progression have been established. About half of the 473 active human proteases are expressed in the prostate and many of the most well-characterized members of this enzyme family are regulated by androgens, hormones essential for development of prostate cancer. Most notably, several kallikrein-related peptidases, including KLK3 (prostate-specific antigen, PSA), the most well-known prostate cancer marker, and type II transmembrane serine proteases, such as TMPRSS2 and matriptase, have been extensively studied and found to promote prostate cancer progression. Recent findings also suggest a critical role for proteases in the development of advanced and aggressive castration-resistant prostate cancer (CRPC). Perhaps the most intriguing evidence for this role comes from studies showing that the protease-activated transmembrane proteins, Notch and CDCP1, are associated with the development of CRPC. Here, we review the roles of proteases in prostate cancer, with a special focus on their regulation by androgens.

Persistently elevated serum concentrations of human chorionic gonadotropin (hCG).

OBJECTIVES: We describe a woman with constantly elevated hCG levels in serum. Since assay interference, pregnancy or cancer did not explain the elevated levels, we measured the concentrations of hCG, its ß subunit (hCGß) and its core fragment (hCGßcf) in serum and urine using specific assays, to understand the nature of the elevated hCG levels. METHODS: We used 3 assays for total hCG (these assays also recognize hCGß and to various degrees hCGßcf), 3 for intact hCG heterodimer, 3 for free hCGß and one for hCGßcf. RESULTS: With an hCG assay detecting total hCG the serum concentrations were in the range of 150-260 IU/L for the whole study period of almost 5 years, except for a peak of 1,200 IU/L, coinciding with a spontaneous abortion. Quantitation of different forms of hCG with specific immunoassays showed that the immunoreactivity in serum consisted of hCGß. Urine contained hCGß and hCGßcf. CONCLUSIONS: The laboratory findings are in keeping with familial hCG syndrome. However, so far the condition remains to be determined in any family members. Elevated hCG levels without any explanation are problematic as they cause suspicion of cancer or ectopic pregnancy and may lead to harmful therapy. Specific assays, as used here, will aid in diagnosis of such cases.

Pancreatic cancer survival prediction via inflammatory serum markers.

BACKGROUND: For prognostic evaluation of pancreatic ductal adenocarcinoma (PDAC), the only well-established serum marker is carbohydrate antigen CA19-9. To improve the accuracy of survival prediction, we tested the efficacy of inflammatory serum markers. METHODS: A preoperative serum panel comprising 48 cytokines plus high-sensitivity CRP (hs-CRP) was analyzed in 173 stage I-III PDAC patients. Analysis of the effect of serum markers on survival utilized the Cox regression model, with the most promising cytokines chosen with the aid of the lasso method. We formed a reference model comprising age, gender, tumor stage, adjuvant chemotherapy status, and CA19-9 level. Our prognostic study model incorporated these data plus hs-CRP and the cytokines. We constructed time-dependent ROC curves and calculated an integrated time-averaged area under the curve (iAUC) for both models from 1 to 10 years after surgery. RESULTS: Hs-CRP and the cytokines CTACK, MIF, IL-1ß, IL-3, GRO-α, M-CSF, and SCF, were our choices for the prognostic study model, in which the iAUC was 0.837 (95% CI 0.796-0.902), compared to the reference model's 0.759 (95% CI 0.691-0.836, NS). These models divided the patients into two groups based on the maximum value of Youden's index at 7.5 years. In our study model, 60th percentile survival times were 4.5 (95% CI 3.7-NA) years (predicted high-survival group, n = 34) and 1.3 (95% CI 1.0-1.7) years (predicted low-survival group, n = 128), log rank p < 0.001. By the reference model, the 60th percentile survival times were 2.8 (95% CI 2.1-4.4) years (predicted high-survival group, n = 44) and 1.3 (95% CI 1.0-1.7) years (predicted low-survival group, n = 118), log rank p < 0.001. CONCLUSION: Hs-CRP and the seven cytokines added to the reference model including CA19-9 are potential prognostic factors for improved survival prediction for PDAC patients.

TATI, TAT-2, and CRP as Prognostic Factors in Colorectal Cancer.

OBJECTIVES: Colorectal cancer is the third most common cancer worldwide, with an obvious need for more accurate prognostics. Previous studies identified C-reactive protein (CRP) as a prognostic serum biomarker for colorectal cancer, whereas the biomarkers tumor-associated trypsin inhibitor (TATI) and tumor-associated trypsin-2 (TAT-2) are less well-known prognostic factors. Therefore, in this study, we aimed to compare the prognostic role of these biomarkers. MATERIALS AND METHODS: Our cohort consisted of 219 women and 274 men who underwent colorectal cancer surgery at Helsinki University Central Hospital from 1998 through 2005. Serum and plasma samples were collected before surgery, aliquoted, stored at -80°C, and then analyzed using high-sensitivity methods with commercially available time-resolved immunofluorometric assay kits. RESULTS: In univariate analysis, CRP (HR 1.67; 95% confidence interval [CI]: 1.25-2.23; p = 0.001), TATI (HR 1.87; 95% CI: 1.13-3.08; p = 0.014), and TAT-2 (HR 1.52; 95% CI: 1.13-2.06; p = 0.006) were significant prognostic biomarkers across the entire cohort. In subgroup analyses, TATI and TAT-2 represented significant negative prognostic factors among patients older than 66, while patients with left-sided disease, a high serum TAT-2, or a high plasma CRP experienced worse prognosis. None of the biomarkers emerged as important in the disease stage subgroup analysis nor did they serve as independent factors in the multivariate analysis. CONCLUSIONS: TATI and TAT-2 as well as CRP significantly, but not independently, served as prognostic factors in our cohort of colorectal cancer patients. Further research is needed to fully understand their clinical role in colorectal cancer.

Immunoassay for trypsinogen-4.

Trypsin has been identified as a pancreatic protease comprising three isoenzymes, trypsin-1, -2, and -3. However, the gene for trypsinogen-3, PRSS3, also gives rise to additional variants, trypsinogen-4A and B, which differ from trypsinogen-3 only with respect to the leader-peptide part, and when activated are identical to trypsin-3. The unique overlapping leader peptides of trypsinogen-4A and B allowed us to develop a specific sandwich-type immunofluorometric assay that detects both these isoforms, but not trypsinogen-3 or activated trypsinogen-4. We measured the concentrations of trypsinogen-4 in various cell line lysates and bile of primary sclerosing cholangitis patients. Lysates of cell lines MDA-MB-231 and PC-3, and astrocytes contained trypsinogen-4, while the conditioned media from these cells did not, suggesting that trypsinogen-4, lacking a classical signal sequence, is not secreted from the cells. Interestingly, 5.7% of the 212 bile samples analyzed contained measurable (>2.4 µg/l) trypsinogen-4. In conclusion, we have established a specific assay for trypsinogen-4 and demonstrated that trypsinogen-4 can be found in biological samples. However, the clinical utility of the assay remains to be established.

Number of screening rounds attended and incidence of high-risk prostate cancer in the Finnish Randomized Study of Screening for Prostate Cancer (FinRSPC).

BACKGROUND: The European Randomized Study of Screening for Prostate Cancer has shown a 20% reduction in prostate cancer (PC) mortality by prostate-specific antigen-based screening. In addition, screening has been shown to reduce the risk of advanced PC. The objective of the current study was to analyze the impact of screening participation on the incidence of PC by risk group. METHODS: The participants in the screening arm of the Finnish trial (31,867 men) were classified according to screening attendance in a time-dependent fashion. Initially, all men in the screening arm were regarded as nonattenders until the first screening attendance; they then remained in the once-screened group until the second screen and similarly for the possible third round. The control arm formed the reference group. Follow-up started at randomization and ended at the time of diagnosis of PC, emigration, or the end of 2015. PC cases were divided into risk groups according to European Association of Urology definitions. RESULTS: The incidence of low-risk PC increased with the number of screens, whereas no clear relation with participation was noted in the intermediate-risk and high-risk cases. For patients with advanced PC, attending screening at least twice was associated with a lower risk. CONCLUSIONS: Screening reduces the risk of advanced PC after only 2 screening cycles. A single screen demonstrated no benefit in terms of PC incidence. Repeated screening is necessary to achieve screening advantages.

The Relationship between the Tissue Expression of TLR2, TLR4, TLR5, and TLR7 and Systemic Inflammatory Responses in Colorectal Cancer Patients.

BACKGROUND: Colorectal cancer (CRC) is the third most commonly diagnosed malignancy globally. CRC patients with elevated plasma C-reactive protein (CRP) levels exhibit compromised prognoses. Toll-like receptors (TLRs), activating the innate and adaptive immune systems, may contribute to pro- and antitumorigenic inflammatory responses. We aimed to identify a possible link between local and systemic inflammatory responses in CRC patients by investigating the association between tissue TLRs and plasma CRP. METHODS: Tissue expressions of TLR2, TLR4, TLR5, and TLR7 were assessed using immunohistochemistry of tissue microarray slides from 549 CRC patients surgically treated between 1998 and 2005. Blood samples were drawn preoperatively, centrifuged, aliquoted, and stored at -80°C until analysis. Plasma CRP was determined through high-sensitivity time-resolved immunofluorometric assay. We investigated the association of TLRs to clinicopathologic variables, plasma CRP, and survival. RESULTS: High TLR2 expression (hazard ratio [HR] 0.59; 95% confidence interval [CI] 0.41-0.85; p = 0.005), high TLR5 expression (HR 0.60; 95% CI 0.45-0.83; p = 0.002), positive TLR7 expression (HR 0.49; 95% CI 0.33-0.72; p < 0.001), and low CRP (HR 1.48; 95% CI 1.08-2.11; p = 0.017) were associated with a better prognosis. A high TLR2 immunoexpression was associated with a better prognosis among low-CRP patients (HR 0.53; 95% CI 0.35-0.80; p = 0.002), high TLR4 expression among high-CRP patients (HR 2.04; 95% CI 1.04-4.00; p = 0.038), high TLR5 expression among low-CRP patients (HR 0.059; 95% CI 0.37-0.92; p = 0.021), and positive TLR7 expression among low-CRP patients (HR 0.53; 95% CI 0.28-1.00; p = 0.049). In multivariate analyses, no biomarkers emerged as significant independent variables. CONCLUSIONS: High tissue TLR2, TLR5, and TLR7 levels were associated with a better prognosis. Among low-CRP patients, those with high TLR2, TLR5, and TLR7 immunoexpressions exhibited a better prognosis. Among high CRP patients, a high TLR4 immunoexpression was associated with a better prognosis.

Repeated Negative Urine Trypsinogen-2 Dipstick Test Rules Out Diagnosis of Post-ERCP Pancreatitis.

BACKGROUND: A dipstick test for urine trypsinogen-2 has been used in the diagnosis of acute pancreatitis, but there are only a few studies exploring the effectiveness of this test for early diagnose of post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP). GOALS: The authors explore if the rapid point-of-care urine trypsinogen-2 dipstick test can replace assay of amylase in diagnosing PEP. STUDY: For this prospective study, from Helsinki University Hospital 400 ERCP patients were enrolled in whom the authors analyzed plasma amylase or pancreas-specific amylase, bilirubin, and urine trypsinogen-2, and urine trypsinogen-2 with dipstick before, 4 and 24 hours after ERCP. RESULTS: PEP developed in 15 (3.8%) patients. Urine trypsinogen-2 concentrations were significantly higher in PEP than in non-PEP patients 24 hours after ERCP (P=0.001, Mann-Whitney U test) but not 4 hours after ERCP (P=0.094). When combined with abdominal pain symptoms at 4 hours the dipstick test had a sensitivity of 60%, a specificity of 99%, a positive predictive value of 64%, and a negative predictive value 98%. At 24 hours, sensitivity was 100%, specificity 98%, positive predictive value 71%, and negative predictive value 100%. CONCLUSIONS: A positive dipstick seems to identify PEP cases and a negative test excludes PEP with high accuracy.

Recommendations for validation testing of home pregnancy tests (HPTs) in Europe.

Home pregnancy tests (HPTs) available in Europe include accuracy and other performance claims listed on their packaging. Due to the lack of guidance on the standardisation of such products, it is often difficult to replicate these claims when tested on a clinical sample, whether in a laboratory setting or by lay users. The In Vitro Diagnostic Regulation is a set of requirements that mandate comprehensive validation data on human pregnancy tests and other in vitro devices. It is due to replace the current European Directive (98/79/EC) and fully implemented in Europe by 2022. In June 2019, a panel of seven experts convened to discuss the validation studies required to provide the information needed to meet the new regulation for HPTs in Europe and proposed 15 recommendations for best practice. Defining best practice at all stages of validation of these important tests may ensure that tests marketed in Europe are fit for purpose, enabling lay users to be confident of the high quality of the HPT results they obtain. The panelists believe that the recommendations proposed here for the validation of HPTs may constructively contribute to improved standardisation of validation procedures in Europe.

Proteolytic Cleavage of Bioactive Peptides and Protease-Activated Receptors in Acute and Post-Colitis.

The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been studied extensively using synthetic fluorogenic substrates targeting specific sets of proteases. We explored activities in colonic tissue from a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model by investigating the cleavage of bioactive peptides. Pure trypsin- and elastase-like proteases on the one hand and colonic tissue from rats with TNBS-induced colitis in the acute or post-inflammatory phase on the other, were incubated with relevant peptides to identify their cleavage pattern by mass spectrometry. An increased cleavage of several peptides was observed in the colon from acute colitis rats. The tethered ligand (TL) sequences of peptides mimicking the N-terminus of protease-activated receptors (PAR) 1 and 4 were significantly unmasked by acute colitis samples and these cleavages were positively correlated with thrombin activity. Increased cleavage of ß-endorphin and disarming of the TL-sequence of the PAR3-based peptide were observed in acute colitis and linked to chymotrypsin-like activity. Increased processing of the enkephalins points to the involvement of proteases with specificities different from trypsin- or chymotrypsin-like enzymes. In conclusion, our results suggest thrombin, chymotrypsin-like proteases and a set of proteases with different specificities as potential therapeutic targets in IBD.

Evaluation of the expression levels of BRAFV600E mRNA in primary tumors of thyroid cancer using an ultrasensitive mutation assay.

BACKGROUND: The BRAFV600E gene encodes for the mutant BRAFV600E protein, which triggers downstream oncogenic signaling in thyroid cancer. Since most currently available methods have focused on detecting BRAFV600E mutations in tumor DNA, there is limited information about the level of BRAFV600E mRNA in primary tumors of thyroid cancer, and the diagnostic relevance of these RNA mutations is not known. METHODS: Sixty-two patients with thyroid cancer and non-malignant thyroid disease were included in the study. Armed with an ultrasensitive technique for mRNA-based mutation analysis based on a two step RT-qPCR method, we analysed the expression levels of the mutated BRAFV600E mRNA in formalin-fixed paraffin-embedded samples of thyroid tissues. Sanger sequencing for detection of BRAFV600E DNA was performed in parallel for comparison and normalization of BRAFV600E mRNA expression levels. RESULTS: The mRNA-based mutation detection assay enables detection of the BRAFV600E mRNA transcripts in a 10,000-fold excess of wildtype BRAF counterparts. While BRAFV600E mutations could be detected by Sanger sequencing in 13 out of 32 malignant thyroid cancer FFPE tissue samples, the mRNA-based assay detected mutations in additionally 5 cases, improving the detection rate from 40.6 to 56.3%. Furthermore, we observed a surprisingly large, 3-log variability, in the expression level of the BRAFV600E mRNA in FFPE samples of thyroid cancer tissue. CONCLUSIONS: The expression levels of BRAFV600E mRNA was characterized in the primary tumors of thyroid cancer using an ultrasensitive mRNA-based mutation assay. Our data inspires further studies on the prognostic and diagnostic relevance of the BRAFV600E mRNA levels as a molecular biomarker for the diagnosis and monitoring of various genetic and malignant diseases.

Tumor-associated trypsin inhibitor (TATI) and tumor-associated trypsin-2 (TAT-2) predict outcomes in gastric cancer.

Introduction: Tumor-associated trypsin inhibitor (TATI) limits serine proteases, promotes carcinogenesis in several cancers and functions as an acute-phase reactant. Tumor-associated trypsin-2 (TAT-2), a proteolytic target enzyme for TATI, can enhance invasion by promoting extracellular matrix degradation. Here, we aimed to study serum TATI and TAT-2 levels, including the TAT-2/TATI ratio, as prognostic and diagnostic biomarkers in gastric cancer. We compared the results with the plasma level of C-reactive protein (CRP).Material and Methods: We selected 240 individuals operated on for gastric adenocarcinoma at the Helsinki University Hospital, Finland, between 2000 and 2009. We determined the preoperative serum TAT-2, TATI and plasma CRP levels using time-resolved immunofluorometric assays using monoclonal antibodies.Results: The medium serum TAT-2 level was higher among gastric cancer patients [8.68 ng/ml; interquartile range (IQR) 5.93-13.2] than among benign controls (median 5.41 ng/ml; IQR 4.12-11.8; p = .005). Five-year survival among patients with a high serum TAT-2 was 22.9% [95% confidence interval (CI) 11.7-34.1], compared to 52.2% (95% CI 44.6-59.8; p < .001) among those with a low level. The five-year survival among patients with a high serum TATI was 30.6% (95% CI 20.4-40.8), compared to 52.9% (95% CI 44.7-61.1; p < .001) among those with a low level. The serum TATI level remained significant in the multivariable survival analysis (hazard ratio 2.01; 95% CI 1.32-3.07). An elevated plasma CRP level associated with a high serum TATI level (p = .037).Conclusions: This study shows for the first time that a high serum TAT-2 may function as a prognostic biomarker in gastric cancer and that TAT-2 levels may be elevated compared to controls. Additionally, we show that the prognosis is worse among gastric cancer patients with a high serum TATI. These biomarkers serve as prognostic factors particularly among patients with a metastatic or a locally advanced disease.

Dramatic increase in serum trypsinogens, SPINK1 and hCGß in aortic surgery patients after hypothermic circulatory arrest.

The concentrations of several diagnostic markers have been found to increase dramatically in critically ill patients with a severe disturbance of normal physiological homeostasis, without indication of the diseases they are normally associated with. To prevent false diagnoses and inappropriate treatments of critically ill patients, it is important that the markers aiding the selection of second-line treatments are evaluated in such patients and not only in the healthy population and patients with diseases the markers are associated with. The levels of trypsinogen isoenzymes, the trypsin inhibitor serine peptidase inhibitor Kazal type 1 (SPINK1), hCG and hCGß, which are used as pancreatitis and cancer markers, were analyzed by immunoassays from serum samples of 17 adult patients who have undergone surgery of the ascending aorta during hypothermic circulatory arrest (HCA) with optional selective cerebral perfusion. Highly elevated levels of trypsinogen-1, -2 and -3, SPINK1 and hCGß were observed in patients after HCA. This was accompanied by increased concentrations of S100ß and NSE. In conclusion, this study highlights the importance of critically evaluating the markers used for aiding selection of second line of treatments in critically ill patients.

Bias-corrected estimates of effects of PSA screening decisions on the risk of prostate cancer diagnosis and death: Analysis of the Finnish randomized study of screening for prostate cancer.

More information is needed about effects of prostate-specific antigen (PSA) screening for informed decision making. The objective of our study is to evaluate the effects of an implemented screening decision on the risk of prostate cancer (PC) diagnosis and PC death. In a randomized trial, 31,867 Finnish men aged 55-67 years were allocated to the screening arm and 48,282 to the control arm during 1996-1999. Two to three screening rounds were offered to the screening arm with a PSA cut-off of 4.0 ng/ml. A counterfactual exclusion method was used to adjust for the effects of screening noncompliance and PSA contamination on risk of PC death and PC incidence by prognostic group at 15 years of follow up. After correcting for noncompliance and contamination, PSA screening led to 32.4 (95% CI 26.4, 38.6) more PC diagnoses per 1,000 men after 15 years and 1.4 (95% CI 0.0, 2.8) fewer PC deaths compared to the control arm. The corresponding results of an intention-to-screen analysis were 16.5 (95% CI 12.3, 20.7) and 0.8 (95% CI 0.5, 2.0), respectively. These results can be used for patient counseling in informed decision making about PC screening. A limitation of the study was the lack of comprehensive data on contamination.

Prostate Cancer Risk-Associated Single-Nucleotide Polymorphism Affects Prostate-Specific Antigen Glycosylation and Its Function.

BACKGROUND: Genetic association studies have reported single-nucleotide polymorphisms (SNPs) at chromosome 19q13.3 to be associated with prostate cancer (PCa) risk. Recently, the rs61752561 SNP (Asp84Asn substitution) in exon 3 of the kallikrein-related peptidase 3 (KLK3) gene encoding prostate-specific antigen (PSA) was reported to be strongly associated with PCa risk (P = 2.3 × 10-8). However, the biological contribution of the rs61752561 SNP to PCa risk has not been elucidated. METHODS: Recombinant PSA protein variants were generated to assess the SNP-mediated biochemical changes by stability and substrate activity assays. PC3 cell-PSA overexpression models were established to evaluate the effect of the SNP on PCa pathogenesis. Genotype-specific correlation of the SNP with total PSA (tPSA) concentrations and free/total (F/T) PSA ratio were determined from serum samples. RESULTS: Functional analysis showed that the rs61752561 SNP affects PSA stability and structural conformation and creates an extra glycosylation site. This PSA variant had reduced enzymatic activity and the ability to stimulate proliferation and migration of PCa cells. Interestingly, the minor allele is associated with lower tPSA concentrations and high F/T PSA ratio in serum samples, indicating that the amino acid substitution may affect PSA immunoreactivity to the antibodies used in the clinical immunoassays. CONCLUSIONS: The rs61752561 SNP appears to have a potential role in PCa pathogenesis by changing the glycosylation, protein stability, and PSA activity and may also affect the clinically measured F/T PSA ratio. Accounting for these effects on tPSA concentration and F/T PSA ratio may help to improve the accuracy of the current PSA test.

Risk Prediction of Prostate Cancer with Single Nucleotide Polymorphisms and Prostate Specific Antigen.

PURPOSE: Combined information on single nucleotide polymorphisms and prostate specific antigen offers opportunities to improve the performance of screening by risk stratification. We aimed to predict the risk of prostate cancer based on prostate specific antigen together with single nucleotide polymorphism information. MATERIALS AND METHODS: We performed a prospective study of 20,575 men with prostate specific antigen testing and 4,967 with a polygenic risk score for prostate cancer based on 66 single nucleotide polymorphisms from the Finnish population based screening trial of prostate cancer and 5,269 samples of 7 single nucleotide polymorphisms from the Finnish prostate cancer DNA study. A Bayesian predictive model was built to estimate the risk of prostate cancer by sequentially combining genetic information with prostate specific antigen compared with prostate specific antigen alone in study subjects limited to those with prostate specific antigen 4 ng/ml or above. RESULTS: The posterior odds of prostate cancer based on 7 single nucleotide polymorphisms together with the prostate specific antigen level ranged from 3.7 at 4 ng/ml, 14.2 at 6 and 40.7 at 8 to 98.2 at 10 ng/ml. The ROC AUC was elevated to 88.8% (95% CI 88.6-89.1) for prostate specific antigen combined with the risk score based on 7 single nucleotide polymorphisms compared with 70.1% (95% CI 69.6-70.7) for prostate specific antigen alone. It was further escalated to 96.7% (95% CI 96.5-96.9) when all prostate cancer susceptibility polygenes were combined. CONCLUSIONS: Expedient use of multiple genetic variants together with information on prostate specific antigen levels better predicts the risk of prostate cancer than prostate specific antigen alone and allows for higher prostate specific antigen cutoffs. Combined information also provides a basis for risk stratification which can be used to optimize the performance of prostate cancer screening.

Tumor expression of human chorionic gonadotropin beta mRNA and prognosis of prostate cancer treated by radical prostatectomy.

The beta subunit of human chorionic gonadotropin (hCGß) is encoded by six genes (CGB) classified as type I and type II. CGB mRNA is produced in large amounts by trophoblastic tissues and in small amounts by several cancerous tissues including prostate cancer and by a few benign tissues, including the prostate. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to study the expression levels of all CGB mRNAs together (total CGB mRNA) and the two types of CGB mRNA separately in non-cancerous (n = 74) and cancerous prostatic tissue obtained by radical prostatectomy (n = 193). RNA was isolated from formalin-fixed paraffin-embedded (FFPE) samples and mRNA levels of CGB were correlated with disease-specific survival. Total CGB mRNA concentrations were significantly lower (p < .0001) in cancerous than non-cancerous prostatic tissue. Separate analysis of type I CGB and type II CGB mRNA showed that both type I CGB (p < .0001) and type II CGB mRNA (p = .007) are lower in cancerous tissue than in non-cancerous tissue. Low type II CGB mRNA level in cancerous tissue was associated with shorter cancer-specific survival (p = .001) of prostate cancer patients treated by radical prostatectomy.

Specificity profiling of human trypsin-isoenzymes.

In humans, three different trypsin-isoenzymes have been described. Of these, trypsin-3 appears to be functionally different from the others. In order to systematically study the specificity of the trypsin-isoenzymes, we utilized proteome-derived peptide libraries and quantitative proteomics. We found similar specificity profiles dominated by the well-characterized preference for cleavage after lysine and arginine. Especially, trypsin-1 slightly favored lysine over arginine in this position, while trypsin-3 did not discriminate between them. In the P1' position, which is the residue C-terminal to the cleavage site, we noticed a subtle enrichment of alanine and glycine for all three trypsins and for trypsin-3 there were additional minor P1' and P2' preferences for threonine and aspartic acid, respectively. These findings were confirmed by FRET peptide substrates showing different susceptibility to cleavage by different trypsins. The preference of trypsin-3 for aspartic acid in P2' is explained by salt bridge formation with the unique Arg193. This salt bridge enables and stabilizes a canonical oxyanion conformation by the amides of Ser195 and Arg193, thus manifesting a selective substrate-assisted catalysis. As trypsin-3 has been proposed to be a therapeutic target and marker for cancers, our results may aid the development of specific inhibitors for cancer therapy and diagnostic probes.

Postoperative CEA is a better prognostic marker than CA19-9, hCGß or TATI after resection of colorectal liver metastases.

Liver metastases of colorectal cancer can be operated with a curative intent in selected cases. However, more than half of the patients have a recurrence. The aim of this study was to evaluate the prognostic and predictive value of carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), human chorionic gonadotropin ß (hCGß) and tumour-associated trypsin-inhibitor (TATI) in colorectal cancer patients before and 3 months after resection of liver metastases. Marker concentrations were determined in blood samples from 168 colorectal cancer patients, who underwent liver resection between the years 1998 and 2007 at Helsinki University Hospital, Finland. The samples were taken before and 3 months after curative resection. Increased concentrations of CEA (>5 µg/L) and hCGß (>1 pmol/L) 3 months after liver resection correlated with recurrence and impaired overall survival and increased CA19-9 (>26 kU/L) with impaired overall survival, but postoperative TATI was not prognostic. Preoperatively elevated CEA and CA19-9 correlated with impaired overall survival, but not with recurrence. Neither preoperative hCGß nor TATI was prognostic. In conclusion, CEA is a useful prognostic marker, when measured 3 months after resection of colorectal liver metastases. CA19-9 also has prognostic significance and may have additional value.