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.

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.

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.

Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity.

Here we provide the first strategy to use a competitive Extendable Blocking Probe (ExBP) for allele-specific priming with superior selectivity at the stage of reverse transcription. In order to analyze highly similar RNA variants, a reverse-transcriptase primer whose sequence matches a specific variant selectively primes only that variant, whereas mismatch priming to the alternative variant is suppressed by virtue of hybridization and subsequent extension of the perfectly matched ExBP on that alternative variant template to form a cDNA-RNA hybrid. This hybrid will render the alternative RNA template unavailable for mismatch priming initiated by the specific primer in a hot-start protocol of reverse transcription when the temperature decreases to a level where such mismatch priming could occur. The ExBP-based reverse transcription assay detected BRAF and KRAS mutations in at least 1000-fold excess of wild-type RNA and detection was linear over a 4-log dynamic range. This novel strategy not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific expression.

Genomic data into everyday work of a medical practitioner - digital tools for decision-making.

Recent technological development has enabled fast and cost-effective simultaneous analyses of several gene variants or sequence of even the whole genome. For medical practitioners this has created challenges although genomic information may be clinically useful in new applications such as finding out individual risk for diseases influenced by as many as 50,000 variable DNA regions or in detecting pharmacogenetic risks prior to prescribing a medicine. New digital tools have paved the way for utilization of genomic data via easy access and clear clinical interpretation for both doctor and patient. In this review we describe some of these tools and applications for clinical use.

Somatic MED12 mutations in prostate cancer and uterine leiomyomas promote tumorigenesis through distinct mechanisms.

BACKGROUND: Mediator is a multiprotein interface between eukaryotic gene-specific transcription factors and RNA polymerase II. Mutations in exon 2 of the gene encoding MED12, a key subunit of the regulatory kinase module in Mediator, are extremely frequent in uterine leiomyomas, breast fibroadenomas, and phyllodes tumors. These mutations disrupt kinase module interactions and lead to diminished Mediator-associated kinase activity. MED12 mutations in exon 26, resulting in a substitution of leucine 1224 to phenylalanine (L1224F), have been recurrently observed in prostate cancer. METHODS: To elucidate the molecular mechanisms leading to tumorigenesis in prostate cancer, we analyzed global interaction profiles of wild-type and L1224F mutant MED12 with quantitative affinity purification-mass spectrometry (AP-MS). Immunoprecipitation and kinase activity assay were used to further assess the interactions between Mediator complex subunits and kinase activity. The presence of L1224F mutation was analyzed in altogether 877 samples representing prostate hyperplasia, prostate cancer, and various tumor types in which somatic MED12 mutations have previously been observed. RESULTS: In contrast to N-terminal MED12 mutations observed in uterine leiomyomas, the L1224F mutation compromises neither the interaction of MED12 with kinase module subunits Cyclin C and CDK8/19 nor Mediator-associated CDK activity. Instead, the L1224F mutation was shown to affect interactions between MED12 and other Mediator components (MED1, MED13, MED13L, MED14, MED15, MED17, and MED24). Mutation screening revealed one mutation in a Finnish (Caucasian) prostate cancer patient, whereas no mutations in any other tumor type were observed. CONCLUSIONS: Specific somatic MED12 mutations in prostate cancer and uterine leiomyomas accumulate in two separate regions of the gene and promote tumorigenesis through clearly distinct mechanisms.

Hyperglycosylated human chorionic gonadotropin in serum of testicular cancer patients.

BACKGROUND: Hyperglycosylated human chorionic gonadotropin (hCG-h) contains larger and more complex carbohydrate chains than regular human chorionic gonadotropin (hCG). hCG-h is thought to be the major form of hCG produced by testicular cancers and it has been suggested to play a key role in tumor invasion, but studies on hCG-h in testicular cancer are limited. We studied whether serum hCG is hyperglycosylated, and whether measurement of hCG-h in serum offers clinical value in the management of testicular cancer. METHODS: We determined the serum concentrations of hCG-h, hCG, and the free ß subunit of hCG (hCGß) by time-resolved immunofluorometric assays in 176 serum samples (preoperative n = 67, relapse n = 20, follow-up n = 89) obtained from 84 testicular cancer patients. We analyzed the association between preoperative serum concentrations of hCG, hCG-h, and hCGß with known prognostic factors and progression-free survival time. RESULTS: A major proportion of hCG was hyperglycosylated preoperatively, at relapse, and shortly after treatment. The serum concentrations of hCG-h and hCG correlated strongly with each other and had similar diagnostic value. The preoperative serum concentration of hCG-h correlated with prognostic factors and outcome in the same way as hCG. Increased preoperative hCGß concentration predicted shorter progression-free survival. CONCLUSIONS: Most of the hCG expressed by testicular cancers is hyperglycosylated and therefore it is important that hCG assays used for management of testicular cancer recognize hCG-h.

Association of tumor-associated trypsin inhibitor (TATI) expression with molecular markers, pathologic features and clinical outcomes of urothelial carcinoma of the urinary bladder.

PURPOSES: To describe the differential tissue expression of tumor-associated trypsin inhibitor (TATI) in normal bladder urothelium, primary urothelial carcinoma of the bladder (UCB) and metastatic UCB and to assess the association of TATI expression with molecular markers commonly altered in UCB and clinical outcomes after radical cystectomy. METHODS: Slides from eight cystectomy patients without cancer, 191 radical cystectomy patients, 20 lymph nodes without metastasis and 40 lymph nodes with UCB were stained. Tissue expression of TATI, cyclin E1, cyclin D1, p53, p21, p27, pRB, Ki-67, Bcl-2, Caspase-3, Survivin and Cyclooxigenase-2 was measured in a tissue microarray. Cancer-specific and recurrence-free survival after radical cystectomy was recorded. RESULTS: TATI was expressed in 100% of patients without cancer, while 71% of radical cystectomy specimens and 90% of lymph node metastases exhibited decreased or no TATI expression. In radical cystectomy specimens, TATI expression decreased with advancing pathologic stage (P < 0.001) and lymphovascular invasion (P = 0.055). In univariate analyses, but not in multivariable Cox proportional hazard regression analyses, decreased TATI expression was associated with increased probability of tumor recurrence and cancer-specific mortality. Decreased TATI expression was correlated with altered expression of Cyclooxigenase-2 (P = 0.005), p21 (P = 0.035) and Ki-67 (P = 0.004). CONCLUSIONS: We found that normal urothelium expresses TATI and that TATI expression decreases with advancing tumor stage. While there was no prognostic benefit to TATI when adjusted for standard clinicopathologic features, it seems to play an important biologic role in UCB pathogenesis and invasion. Its association with markers involved in the cell cycle, proliferation and inflammation serves as hypothesis for molecular interactions.

How Communicating Polygenic and Clinical Risk for Atherosclerotic Cardiovascular Disease Impacts Health Behavior: an Observational Follow-up Study.

BACKGROUND: Prediction tools that combine polygenic risk scores with clinical factors provide a new opportunity for improved prediction and prevention of atherosclerotic cardiovascular disease, but the clinical utility of polygenic risk score has remained unclear. METHODS: We collected a prospective cohort of 7342 individuals (64% women, mean age 56 years) and estimated their 10-year risk for atherosclerotic cardiovascular disease both by a traditional risk score and a composite score combining the effect of a polygenic risk score and clinical risk factors. We then tested how returning the personal risk information with an interactive web-tool impacted on the participants' health behavior. RESULTS: When reassessed after 1.5 years by a clinical visit and questionnaires, 20.8% of individuals at high (>10%) 10-year atherosclerotic cardiovascular disease risk had seen a doctor, 12.4% reported weight loss, 14.2% of smokers had quit smoking, and 15.4% had signed up for health coaching online. Altogether, 42.6% of persons at high risk had made one or more health behavioral changes versus 33.5% of persons at low/average risk such that higher baseline risk predicted a favorable change (OR [CI], 1.53 [1.37-1.72] for persons at high risk versus the rest, P<0.001), with both high clinical (P<0.001) and genomic risk (OR [CI], 1.10 [1.03-1.17], P=0.003) contributing independently. CONCLUSIONS: Web-based communication of personal atherosclerotic cardiovascular disease risk-data including polygenic risk to middle-aged persons motivates positive changes in health behavior and the propensity to seek care. It supports integration of genomic information into clinical risk calculators as a feasible approach to enhance disease prevention.

Specific immunoassay reveals increased serum trypsinogen 3 in acute pancreatitis.

BACKGROUND: Trypsinogen 3 is a minor trypsinogen isoform in the pancreas. In contrast with trypsin 1 and 2, trypsin 3 degrades pancreatic secretory trypsin inhibitor, which may lead to an excess of active trypsin and acute pancreatitis (AP). We developed an immunoassay for trypsinogen 3 and studied whether an assay of serum trypsinogen 3 is of clinical utility in the diagnosis of AP. METHODS: Monoclonal antibodies were generated using recombinant human trypsinogen 3 as the antigen and used to establish a sandwich-type immunoassay. We analyzed serum trypsinogen 3 concentrations in 82 patients with AP and 63 patients with upper abdominal pain (controls). The reference interval was determined using serum samples from 172 apparently healthy individuals. RESULTS: The measuring range of the trypsinogen 3 assay was 1.0-250 µg/L. Intra- and interassay CVs were <11%, and cross-reactivity with other trypsinogen isoenzymes was <0.1%. The median trypsinogen 3 concentration in serum from healthy individuals was <1.0 µg/L, and the upper reference limit was 4.4 µg/L. We observed increased trypsinogen 3 concentrations in patients with mild (median 9.5 µg/L) and severe (15.0 µg/L) AP; in both groups, the concentrations were significantly higher than in controls (median <1.0 µg/L) (P < 0.0001). In ROC analysis, the area under the curve of trypsinogen 3 for separation between AP and controls was 0.90 (P < 0.0001). CONCLUSIONS: We established for the first time a specific immunoassay for trypsinogen 3 using monoclonal antibodies. Patients with AP were found to have increased serum concentrations of trypsinogen 3. The availability of this assay will be useful for studies of the clinical utility of trypsinogen 3.

Clinical validation of automated and rapid mariPOC SARS-CoV-2 antigen test.

COVID-19 diagnostics was quickly ramped up worldwide early 2020 based on the detection of viral RNA. However, based on the scientific knowledge for pre-existing coronaviruses, it was expected that the SARS-CoV-2 RNA will be detected from symptomatic and at significant rates also from asymptomatic individuals due to persistence of non-infectious RNA. To increase the efficacy of diagnostics, surveillance, screening and pandemic control, rapid methods, such as antigen tests, are needed for decentralized testing and to assess infectiousness. A novel automated mariPOC SARS-CoV-2 test was developed for the detection of conserved structural viral nucleocapsid proteins. The test utilizes sophisticated optical laser technology for two-photon excitation and individual detection of immunoassay solid-phase particles. We validated the new method against qRT-PCR. Sensitivity of the test was 100.0% (13/13) directly from nasopharyngeal swab specimens and 84.4% (38/45) from swab specimens in undefined transport mediums. Specificity of the test was 100.0% (201/201). The test's limit of detection was 2.7 TCID50/test. It showed no cross-reactions. Our study shows that the new test can detect infectious individuals already in 20 min with clinical sensitivity close to qRT-PCR. The mariPOC is a versatile platform for syndromic testing and for high capacity infection control screening of infectious individuals.

Increased serum levels of tumour-associated trypsin inhibitor independently predict a poor prognosis in colorectal cancer patients.

BACKGROUND: There is an insufficient number of reliable prognostic and response predictive biomarkers in colorectal cancer (CRC) management. In a previous study, we found that high tumour tissue expression of tumour-associated trypsin inhibitor (TATI) correlated with liver metastasis and an impaired prognosis in CRC. The aim of this study was to investigate the prognostic validity of serum TATI (s-TATI) in CRC. We further assessed the prognostic value of carcino-embryonic antigen in serum (s-CEA) and the interrelationship between s-TATI and TATI in tissue (t-TATI). METHODS: Using an immunofluorometric assay, s-TATI levels were analysed in 334 preoperatively collected serum samples from patients with CRC. Spearman's Rho and Chi-square test were used for analysis of correlations between s-TATI and clinicopathological parameters, s-CEA and t-TATI. Kaplan-Meier analysis and Cox uni- and multivariate regression analysis were used to estimate disease free survival (DFS) and overall survival (OS) according to quartiles of s-TATI and cut-offs derived from ROC-analysis of s-TATI and s-CEA. RESULTS: Increased levels of s-TATI were associated with a reduced DFS (HR = 2.00; 95% CI 1.40-2.84, P < 0.001) and OS (HR = 2.40; 95% CI 1.74-3.33, P < 0.001). (HR = 2.89; 95% CI 1.96-4.25). This association remained significant in multivariate analysis. The association for OS remained significant in multivariate analysis (HR = 1.51; 95% CI 1.03-2.22, P = 0.034 for DFS and HR = 1.78; 95% CI 1.25-2.53, P = 0.001 for OS). There was no significant association between s-TATI and t-TATI. The prognostic value of s-CEA was also evident, but somewhat weaker than for s-TATI. CONCLUSIONS: High preoperative s-TATI levels predict a poor prognosis in patients with CRC, and the prognostic value is independent of established prognostic parameters and t-TATI expression. These data suggest that s-TATI might be a useful marker for prognostic stratification in CRC.

The proportion of free PSA and upgrading of biopsy Gleason score after radical prostatectomy.

INTRODUCTION: Upgrading of biopsy Gleason score (GS) after radical prostatectomy (RP) to GS >or=7 is common in patients with low-grade prostate cancer in biopsy. We evaluated whether a low proportion of free PSA (%fPSA) and total PSA (tPSA) predict significant upgrading after RP. PATIENTS AND METHODS: 122 patients with biopsy GS 5 or 6 prostate cancer and a tPSA <10 ng/ml who underwent RP in our academic center were included in the study. The utility of prebiopsy %fPSA and tPSA as predictors of significant upgrading was evaluated. RESULTS: Among patients undergoing RP, upgrading to GS >or=7 was found in 61 of 122 (50%) patients. A low %fPSA was a predictor of significant upgrading of tumors with biopsy GS 5 or 6 prostate cancer in patients with tPSA <10 ng/ml. CONCLUSIONS: Our results suggest that the selection of prostate cancer patients for active surveillance or re-biopsy may be improved by using %fPSA as a predictor of significant upgrading.

Free beta-subunit of human chorionic gonadotropin in serum is a diagnostically sensitive marker of seminomatous testicular cancer.

BACKGROUND: We studied whether measurement of the free beta subunit of human chorionic gonadotropin (hCGbeta) in serum offers additional diagnostic information compared to determination of intact hCG alone in testicular cancer. METHODS: We determined hCG and hCGbeta with ultrasensitive assays in 94 serum samples obtained preoperatively, 22 samples obtained during relapse, and 3687 samples obtained during routine follow-up of 351 patients with testicular tumors. RESULTS: In preoperative samples, isolated increases of hCGbeta were seen in 40% of the samples from seminoma patients (n = 42) and in 8% of those from patients with nonseminomatous testicular cancer (NSGCT) (n = 51). Both markers were increased in 12% of the seminoma and 71% of the NSGCT patients and were within reference intervals in 43% of the seminoma and 20% of the NSGCT patients. Specific determination of hCGbeta increased the frequency of marker-positive seminomas from 17% to 57% and of marker-positive relapses from 32% to 59% (n = 22). Theoretically, about 40% of marker-positive seminomas and relapses would have been missed with an assay measuring hCG and hCGbeta together. Preoperative hCG and hCGbeta concentrations correlated with stage, tumor histology, and disease-related mortality. Additionally, hCGbeta correlated with tumor size. CONCLUSIONS: hCGbeta is a diagnostically sensitive marker for testicular cancer. In patients with seminomatous testicular cancer, hCGbeta is superior to hCG, and in some NSGCT patients it provides additional information.

Expression of human chorionic gonadotropin beta-subunit type I genes predicts adverse outcome in renal cell carcinoma.

Expression of the free beta-subunit of human chorionic gonadotropin (hCGbeta) in malignant tumors is frequently associated with aggressive disease. The pretreatment serum concentration of hCGbeta is an independent prognostic variable in renal cell carcinoma (RCC). The three so-called type II genes (hCGbeta 3/9, 5, and 8) have been shown to be up-regulated in relation to type I genes (hCGbeta 6/7) in some malignant tumors. We developed a reverse transcription-polymerase chain reaction method for quantification of relative levels of the mRNAs for the two types of hCGbeta genes and studied the association between the expression in RCC tissue (n = 104) and clinical outcome. hCGbeta mRNA expression was detected in 40% (42 of 104) of the tumors, and in 40 of these (93%), this consisted of hCGbeta type I mRNA only, whereas type II hCGbeta mRNA was detected in two samples. hCGbeta mRNA expression was significantly associated with a shorter disease-specific (log-rank P = 0.023; median survival 1.4 versus 7.9 years) and overall survival (log-rank P = 0.011). In a Cox regression model, stage (P < 0.0001) and hCGbeta mRNA expression (P < 0.0001) were independent prognostic variables. We conclude that expression of type I hCGbeta genes indicates adverse prognosis in RCC.

Human chorionic gonadotropin in cancer.

Human chorionic gonadotropin (hCG) is mainly used for detection and monitoring of pregnancy and pregnancy-related disorders but it is also an extremely sensitive and specific marker for trophoblastic tumors of placental and germ cell origin. Thus treatment of relapsing choriocarcinomas and testicular germ cell tumors is often initiated on the basis of rising hCG levels even in the absence of clinical or histological evidence of a relapse. While these tumors mostly produce the intact heterodimeric hormone consisting of an alpha (hCGalpha), and a beta subunit (hCGbeta), many nontrophoblastic tumors produce only hCGbeta This is usually a sign of aggressive disease and elevated serum levels of hCGbeta are strongly associated with poor prognosis. Elevated serum levels are observed in 45-60% of patients with biliary and pancreatic cancer and in 10-30% of most other cancers. Methods that detect hCG and hCGbeta together are mainly used for measurement of hCG-like immunoreactivity in serum. However, the reference range for hCG is 5-8 fold higher than that for hCGbeta and thus moderately elevated levels can be identified only with a specific and sensitive hCGbeta assay.