Solanidine is a sensitive and specific dietary biomarker for CYP2D6 activity.

BACKGROUND: Individual assessment of CYP enzyme activities can be challenging. Recently, the potato alkaloid solanidine was suggested as a biomarker for CYP2D6 activity. Here, we aimed to characterize the sensitivity and specificity of solanidine as a CYP2D6 biomarker among Finnish volunteers with known CYP2D6 genotypes. RESULTS: Using non-targeted metabolomics analysis, we identified 9152 metabolite features in the fasting plasma samples of 356 healthy volunteers. Machine learning models suggested strong association between CYP2D6 genotype-based phenotype classes with a metabolite feature identified as solanidine. Plasma solanidine concentration was 1887% higher in genetically poor CYP2D6 metabolizers (gPM) (n = 9; 95% confidence interval 755%, 4515%; P = 1.88 × 10-11), 74% higher in intermediate CYP2D6 metabolizers (gIM) (n = 89; 27%, 138%; P = 6.40 × 10-4), and 35% lower in ultrarapid CYP2D6 metabolizers (gUM) (n = 20; 64%, - 17%; P = 0.151) than in genetically normal CYP2D6 metabolizers (gNM; n = 196). The solanidine metabolites m/z 444 and 430 to solanidine concentration ratios showed even stronger associations with CYP2D6 phenotypes. Furthermore, the areas under the receiver operating characteristic and precision-recall curves for these metabolic ratios showed equal or better performances for identifying the gPM, gIM, and gUM phenotype groups than the other metabolites, their ratios to solanidine, or solanidine alone. In vitro studies with human recombinant CYP enzymes showed that solanidine was metabolized mainly by CYP2D6, with a minor contribution from CYP3A4/5. In human liver microsomes, the CYP2D6 inhibitor paroxetine nearly completely (95%) inhibited the metabolism of solanidine. In a genome-wide association study, several variants near the CYP2D6 gene associated with plasma solanidine metabolite ratios. CONCLUSIONS: These results are in line with earlier studies and further indicate that solanidine and its metabolites are sensitive and specific biomarkers for measuring CYP2D6 activity. Since potato consumption is common worldwide, this biomarker could be useful for evaluating CYP2D6-mediated drug-drug interactions and to improve prediction of CYP2D6 activity in addition to genotyping.

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.

Warfarin dose requirement in patients having severe thrombosis or thrombophilia.

AIMS: Warfarin dose requirement varies significantly. We compared the clinically established doses based on international normalized ratio (INR) among patients with severe thrombosis and/or thrombophilia with estimates from genetic dosing algorithms. METHODS: Fifty patients with severe thrombosis and/or thrombophilia requiring permanent anticoagulation, referred to the Helsinki University Hospital Coagulation Center, were screened for thrombophilias and genotyped for CYP2C9*2 (c.430C>T, rs1799853), CYP2C9*3 (c.1075A>C, rs1057910) and VKORC1 c.-1639G>A (rs9923231) variants. The warfarin maintenance doses (target INR 2.0-3.0 in 94%, 2.5-3.5 in 6%) were estimated by the Gage and the International Warfarin Pharmacogenetics Consortium (IWPC) algorithms. The individual warfarin maintenance dose was tailored, supplementing estimates with comprehensive clinical evaluation and INR data. RESULTS: Mean patient age was 47 years (range 20-76), and BMI 27 (SD 6), 68% being women. Forty-six (92%) had previous venous or arterial thrombosis, and 26 (52%) had a thrombophilia, with 22% having concurrent aspirin. A total of 40% carried the CYP2C9*2 or *3 allele and 54% carried the VKORC1-1639A allele. The daily mean maintenance dose of warfarin estimated by the Gage algorithm was 5.4 mg (95% CI 4.9-5.9 mg), and by the IWPC algorithm was 5.2 mg (95% CI 4.7-5.7 mg). The daily warfarin maintenance dose after clinical visits and follow-up was higher than the estimates, mean 6.9 mg (95% CI 5.6-8.2 mg, P < 0.006), with highest dose in patients having multiple thrombophilic factors (P < 0.03). CONCLUSIONS: In severe thrombosis and/or thrombophilia, variation in thrombin generation and pharmacodynamics influences warfarin response. Pharmacogenetic dosing algorithms seem to underestimate dose requirement.

No Tissue Expression of KRAS or BRAF Mutations in 61 Adult Patients Treated for Esophageal Atresia in Early Childhood.

BACKGROUND: Previous studies have reported an association among esophageal atresia (EA), Barrett's esophagus, and esophageal adenocarcinoma later in life. OBJECTIVE: The objective of the article is to evaluate KRAS and BRAF mutations as potential genetic markers for early detection of malignant transformation, we used an ultrasensitive technique to detect tissue expression of KRAS and BRAF mutations in endoscopic biopsies from 61 adult patients under follow-up after treatment for EA. MATERIALS AND METHODS: RNA was extracted from 112 fresh-frozen endoscopic tissue biopsies from 61 adult patients treated for EA in early childhood. RNA was reverse transcribed using the extendable blocking probe reverse transcription method. KRAS codons 12 and 13, as well as BRAF mutations were detected by quantitative polymerase chain reaction. RESULTS: No mutations of KRAS codon 12, KRAS codon 13, or BRAF were found in 112 endoscopic biopsy samples from 61 patients. CONCLUSION: Despite the presence of histological findings indicating long-standing gastroesophageal reflux in 25%, as well as symptomatic gastroesophageal reflux in more than 40%, there was no detectable tissue expression of KRAS or BRAF mutations in this cohort of patients.

EGFR gene copy number predicts response to anti-EGFR treatment in RAS wild type and RAS/BRAF/PIK3CA wild type metastatic colorectal cancer.

Anti-EGFR antibodies are used for the treatment of RAS wild type metastatic colorectal cancer. We previously showed that EGFR gene copy number (GCN) predicts response to anti-EGFR therapy in KRAS exon 2 wild type metastatic colorectal cancer. The aim of our study was to analyse the predictive role of EGFR GCN in RAS/BRAF/PIK3CA wild type metastatic colorectal cancer. The material included 102 patients with KRAS exon 2 wild type metastatic colorectal cancer treated with anti-EGFR ± cytotoxic therapy. Next generation sequencing was used for KRAS, NRAS, BRAF and PIK3CA gene mutation analyses. EGFR GCN was analysed by EGFR immunohistochemistry guided automated silver in situ hybridisation. Increased EGFR GCN (≥4.0) predicted a better response and prolonged progression free survival in anti-EGFR treated RAS/BRAF/PIK3CA wild type patients (Log-rank test, p = 0.0004). In contrast, survival of RAS/BRAF/PIK3CA wild type, EGFR GCN below 4.0 patients did not differ from patients with mutant RAS, BRAF or PIK3CA. Our study indicates that EGFR GCN predicts anti-EGFR treatment efficacy in patients with RAS/BRAF/PIK3CA wt metastatic CRC. Tumours with EGFR GCN below 4.0 appear to be as refractory to anti-EGFR treatment as tumours with mutation in any of the RAS/RAF/PIK3CA pathway genes.

Prospective immunohistochemical analysis of BRAF V600E mutation in melanoma.

The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E mutation is the most common activating genetic alteration of this oncogene and a predictive marker for the therapeutic use of BRAF inhibitors in melanoma. Our aim was to evaluate the performance of BRAF V600E mutation-specific monoclonal antibody (VE1) in a prospective diagnostic setting of melanoma patients (n = 102). All 41 cases (40.2%) that showed a V600E mutation in the cyclic minisequencing analysis of the DNA were also initially scored immunopositive. Two cases that were scored as BRAF V600E mutation positive by immunohistochemistry were negative in the DNA-based mutation analysis and determined to be immunonegative in a repeated staining with more representative specimens. Thus, BRAF V600E mutation detection using immunohistochemistry was 100% sensitive and 96.8% specific, when compared with the analysis of the DNA. None of the BRAF V600K mutations was detected by the VE1 antibody (n = 7). However, the VE1 antibody detected a rare V600E2 mutation. We also studied the role of BRAF V600E mutation in a set of melanoma patients who had been investigated for sentinel node metastasis. Melanoma lymph node metastases were diagnosed in 21.8% (12/55) of the sentinel nodes, and BRAF V600E immunopositivity was detected in 34.5% (19/55) of the cases. BRAF V600E mutation status did not correlate with any clinicopathological parameters. In conclusion, analysis of BRAF V600E mutation in melanoma by immunohistochemistry is a sensitive and specific method, which can be used to identify BRAF inhibitor-sensitive melanoma patients as a first-line method due to its rapid and affordable nature.

Automation of diagnostic genetic testing: mutation detection by cyclic minisequencing.

BACKGROUND: The rising role of nucleic acid testing in clinical decision making is creating a need for efficient and automated diagnostic nucleic acid test platforms. Clinical use of nucleic acid testing sets demands for shorter turnaround times (TATs), lower production costs and robust, reliable methods that can easily adopt new test panels and is able to run rare tests in random access principle. Here we present a novel home-brew laboratory automation platform for diagnostic mutation testing. METHOD: This platform is based on the cyclic minisequecing (cMS) and two color near-infrared (NIR) detection. Pipetting is automated using Tecan Freedom EVO pipetting robots and all assays are performed in 384-well micro plate format. The automation platform includes a data processing system, controlling all procedures, and automated patient result reporting to the hospital information system. CONCLUSIONS: We have found automated cMS a reliable, inexpensive and robust method for nucleic acid testing for a wide variety of diagnostic tests. The platform is currently in clinical use for over 80 mutations or polymorphisms. Additionally to tests performed from blood samples, the system performs also epigenetic test for the methylation of the MGMT gene promoter, and companion diagnostic tests for analysis of KRAS and BRAF gene mutations from formalin fixed and paraffin embedded tumor samples. Automation of genetic test reporting is found reliable and efficient decreasing the work load of academic personnel.

BRAF mutation in sporadic colorectal cancer and Lynch syndrome.

The aim of the study was to detect mutations of BRAF oncogene in colorectal cancer and to use this information to identify Lynch syndrome patients. Consecutive cases of primary colorectal cancer (n = 137) were analyzed for MLH1 protein expression using immunohistochemistry (IHC). BRAF V600E mutation was detected by IHC using a specific monoclonal antibody (VE1) and by qPCR. All MLH1 protein-negative cases were subjected to microsatellite instability analysis and MLH1 promoter methylation assay. MLH1 protein expression deficiency and high microsatellite instability (MSI-H) were detected in 18 of the 137 (13.1%) consecutive colorectal cancer specimens. Detection of the BRAF V600E mutation by IHC was 100% sensitive and specific as compared to qPCR, and this mutation was frequently present in the MSI-H group (77.8%; 14/18) and less frequently in the microsatellite-stable group (7.6%; 9/118). All BRAF V600E mutated cases of the MSI-H group presented with a MLH1 promoter methylation (14/14) as detected by methylation-specific multiplex ligation-dependent probe amplification. When BRAF was wild type in the MSI-H group, only one MLH1 promoter methylation was detected (1/4), and of the remaining three cases without MLH1 methylation, two were identified to harbor an MLH1 mutation consistent with Lynch syndrome. Finally, 11 previously confirmed Lynch syndrome cases were analyzed for BRAF V600E mutation, and all of them were wild type. In conclusion, detection of BRAF V600E in colorectal cancer specimens by IHC is sensitive and specific and may help to identify Lynch syndrome patients.

Novel heat pulse extension-PCR-based method for detection of large CTG-repeat expansions in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is an autosomal-dominant disease caused by an expansion of CTG repeats in the 3' untranslated region of the Dystrophia Myotonica Protein Kinase (DMPK) gene. Detection and accurate sizing of the CTG-repeat expansions is clinically important, because the number of CTG repeats correlates with the disease severity. Because difficulties in PCR amplification over large expansions, molecular diagnosis of DM1 is still primarily based on Southern blotting, which is technically demanding and time consuming and requires large amounts of genomic DNA samples. We have recently discovered that the use of multiple heat pulses during Heat Pulse Extension PCR (HPE-PCR) enables efficient amplification over repetitive and GC-rich sequences. Based on this principle, we have developed an assay for efficient amplification of large CTG-repeat expansions seen in DM1 patients. The HPE-PCR method was able to amplify different DMPK1 repeat expansions of up to 1750 CTG repeats in 78 clinical samples with a varying degree of tissue heterogeneity, even in the presence of the short wild-type allele. The CTG-repeat lengths and fragmentation patterns obtained with HPE-PCR were fully concordant with the original diagnostic Southern blotting results. This novel technique provides a PCR-based platform for molecular diagnosis of DM1, and it has been adopted for routine diagnostic use.

Technique for strand-specific gene-expression analysis and monitoring of primer-independent cDNA synthesis in reverse transcription.

Primer-independent cDNA synthesis during reverse transcription hinders quantitative analysis of bidirectional mRNA synthesis in eukaryotes as well as in cells infected with RNA viruses. We report a simple RT-PCR-based assay for strand-specific gene-expression analysis. By modifying the cDNA sequence during reverse transcription, the opposite strands of target sequences can be simultaneously detected by postamplification melting curve analysis and primer-initiated transcripts are readily distinguished from nonspecifically primed cDNA. We have utilized this technique to optimize the specificity of reverse transcription on a panel of 15 target genes. Primer-independent reverse transcription occurred for all target sequences when reverse transcription was performed at 42°C and accounted for 11%-57% of the final PCR amplification products. By raising the reaction temperature to 55°C, the specificity of reverse transcription could be increased without significant loss of sensitivity. We have also demonstrated the utility of this technique for analysis of (+) and (-) RNA synthesis of influenza A virus in infected cells. Thus, this technique represents a powerful tool for analysis of bidirectional RNA synthesis.

Multiple heat pulses during PCR extension enabling amplification of GC-rich sequences and reducing amplification bias.

PCR amplification over GC-rich and/or long repetitive sequences is challenging because of thermo-stable structures resulting from incomplete denaturation, reannealing, and self-annealing of target sequences. These structures block the DNA polymerase during the extension step, leading to formation of incomplete extension products and favoring amplification of nonspecific products rather than specific ones. We have introduced multiple heat pulses in the extension step of a PCR cycling protocol to temporarily destabilize such blocking structures, in order to enhance DNA polymerase extension over GC-rich sequences. With this novel type of protocol, we were able to amplify all expansions of CGG repeats in five Fragile X cell lines, as well as extremely GC-rich nonrepetitive segments of the GNAQ and GP1BB genes. The longest Fragile X expansion contained 940 CGG repeats, corresponding to about 2.8 kilo bases of 100% GC content. For the GNAQ and GP1BB genes, different length PCR products in the range of 700 bases to 2 kilobases could be amplified without addition of cosolvents. As this technique improves the balance of amplification efficiencies between GC-rich target sequences of different length, we were able to amplify all of the allelic expansions even in the presence of the unexpanded allele.

High serum angiogenin at diagnosis predicts for failure on long-term treatment response and for poor overall survival in non-Hodgkin lymphoma.

BACKGROUND: Angiogenin is a potent inducer of angiogenesis. We prospectively evaluated the prognostic significance of serum angiogenin from 204 consecutive non-Hodgkin lymphoma (NHL) patients diagnosed and treated in a single institution. METHODS: Serum angiogenin, VEGF, and bFGF concentrations at diagnosis were determined using a quantitative sandwich enzyme immunoassay technique. Kaplan-Meier survival curves were compared by the log-rank test. Multivariate survival analyses were performed using the parametric model of Weibull and the non-parametric proportional hazards model of Cox. RESULTS: Patients with a high serum angiogenin at diagnosis (>median; 401 ng/ml) had significantly lower 5-year survival rate than those with a low (≤ median) angiogenin (42% versus 63%, respectively; P = 0.0073). Serum angiogenin provided additional information to the International Prognostic Index (IPI) identifying a subgroup (serum angiogenin >median and IPI>1) with very poor prognosis (5-year survival 19%, P < 0.0001). In receiver operating characteristic (ROC) analyses the accuracy of the IPI to correctly classify patients with favourable or poor survival was improved from fair to good by complementing the IPI with serum angiogenin concentration. With patients who initially achieved complete response (CR) after chemotherapy, a high angiogenin at diagnosis (>median; relative risk (RR) 2.38; P = 0.0077) and an advanced tumour stage (III-IV; RR 2.41; P = 0.0087) were the only independent predictors for patients with unfavourable outcome although first responding well to therapy. CONCLUSIONS: We conclude that elevated serum angiogenin surfaced as an independent predictor for failure in long-term treatment response and for poor overall survival in a series of 204 NHL patients, and might thus also complement the IPI in identifying the patients with particularly aggressive and/or treatment resistant disease.

Use of cancer-specific genomic rearrangements to quantify disease burden in plasma from patients with solid tumors.

Detection of recurrent somatic rearrangements routinely allows monitoring of residual disease burden in leukemias, but is not used for most solid tumors. However, next-generation sequencing now allows rapid identification of patient-specific rearrangements in solid tumors. We mapped genomic rearrangements in three cancers and showed that PCR assays for rearrangements could detect a single copy of the tumor genome in plasma without false positives. Disease status, drug responsiveness, and incipient relapse could be serially assessed. In future, this strategy could be readily established in diagnostic laboratories, with major impact on monitoring of disease status and personalizing treatment of solid tumors.

Patients referred to an indoor air health clinic: exposure to water-damaged buildings causes an increase of lymphocytes in bronchoalveolar lavage and a decrease of CD19 leucocytes in peripheral blood.

BACKGROUND: Respiratory and other symptoms are often associated with exposure to microbes present in water-damaged buildings. MATERIAL AND METHODS: We examined 82 consecutive patients referred to the Indoor Air Clinic, Helsinki University Hospital, due to symptoms suspected of having been caused by long-term exposure to water damage in the home or workplace. Exposure to water damage was assessed by building inspections and microbial analyses as needed. Bronchoalveolar lavage, lung function measurements, skin prick tests to inhalant allergens and radiological examinations were performed in all patients. Leucocyte subsets in peripheral blood were analysed in 35 patients. RESULTS: Marked water damage was detected in the homes or workplaces of 47 (59%) patients; the remaining 34 patients formed the control group. The exposed group expressed more symptoms in total than the control group: fatigue, conjunctival symptoms, rhinitis with sinusitis, recurrent bronchitis and asthma were more common in the exposed group, but a significant difference was seen only for headache. In BAL (bronchoalveolar lavage) samples, lymphocytes represented 25% of the total cell population in non-smoking-exposed patients compared with 12% in control patients (p=0.004). In peripheral blood, CD19 leucocytes were significantly decreased in the exposed group (7.5% versus 12.3%; p<0.01). CONCLUSIONS: Confirmed exposure to water damage was associated with an increase in symptoms. Exposure to water damage caused a significant change in the cellular composition in BAL fluid (lymphocytosis) and blood (decrease of CD19 cells). The depletion of CD19 leucocytes in peripheral blood may indicate an active immune response in the lungs.

Hemostatic gene polymorphisms in severe acute pancreatitis.

OBJECTIVE: Systemic inflammatory reaction in acute pancreatitis (AP) is associated with activation of the coagulation system. The prothrombotic component of the coagulation system, which may promote microvascular thrombosis and vital organ injury, is strengthened by genetic factors such as polymorphism of plasminogen activator inhibitor type 1 (PAI-1) and factor V Leiden (FVL) mutation. This prompted us to study the occurrence of FVL and PAI-1 4G/5G polymorphisms in patients with AP. METHODS: This case control association study included 397 patients with AP and 310 controls. Severe AP was determined according to the Atlanta Classification. Genotyping was performed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry-assisted genotyping method. RESULTS: Factor V Leiden was identified in 5 (3.3%) of 152 cases of severe AP and in 8 (3.3%) of 245 cases of mild AP. The prothrombotic PAI-1 4G allele frequency was 0.49 for patients with severe AP and 0.57 for patients with mild AP (P < 0.05). Patients with septic infectious complications (n = 47) and patients with organ failure (n = 55) had genotype distribution not different from those with mild, uncomplicated disease (n = 245). CONCLUSIONS: The results do not support the hypothesis that prothrombotic polymorphisms such as FVL mutation and PAI-1 4G/5G are associated with AP severity.

Polymorphisms of the TNF, CD14, and HSPA1B genes in patients with acute alcohol-induced pancreatitis.

OBJECTIVES: Genotype assessment has been suggested to be a tool for predicting disease severity in acute pancreatitis (AP). To study this hypothesis, we performed genotype analysis of tumor necrosis factor (TNF) -308 A/G, CD14 -159C/T, and HSPA1B +1267 A/G polymorphisms. METHODS: This is a case-control association study of 397 patients with AP (214 of whom had an alcohol-induced AP) and 300 controls. The control group comprised 218 subjects with detailed data of alcohol consumption, 70 of whom were heavy drinkers (daily alcohol intake >40 g), and 92 blood donors. The severity of AP was determined according to the Atlanta classification. Genotyping was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-assisted genotyping method. RESULTS: Major allele frequency in TNF gene was 0.87 for patients with AP and 0.86 for controls. For CD14, the gene major allele frequency was 0.60 for patients and 0.63 for controls. For HSPA1B, the major allele frequencies were 0.52 for patients and 0.49 for controls, respectively. The allele frequencies did not differ significantly between AP patients with organ failure and those with mild disease, patients with alcohol-induced AP, or those with biliary AP. The patients with septic infectious complications (n = 47) had genotype distribution no different from those with mild, uncomplicated disease (n = 245). CONCLUSIONS: The TNF, CD14, and HSPA1B polymorphisms studied seem not to play a role in determining the severity of AP or the risk of alcohol-induced AP and thus do not serve as a tool for predicting disease severity.

Targeted gene-expression analysis by genome-controlled reverse transcription-PCR.

BACKGROUND: For gene-expression analysis, which is anticipated to play an important role in classification of tumors and premalignant conditions, PCR-based quantitative assays must have increased diagnostic quantitative accuracy and reproducibility and enable analysis of gene expression in formalin-fixed paraffin-embedded (FFPE) tissue samples. METHODS: We developed a reverse transcription-PCR-based quantitative assay that modifies the cDNA sequence to increase the melting temperature of short (56-64 bp) PCR amplicons, enabling their quantification in-tube by homogeneous melting-curve analysis. We used this method to analyze the expression of 8 genes, 7 potential colon cancer markers, and 1 control in samples obtained from 3 colon carcinoma cell lines, endoscopic biopsy from 8 patients undergoing gastroscopy for Barrett esophagus, and archival FFPE and frozen tissue from 20 patients who underwent surgery for colon carcinoma. RESULTS: The detection limit of the assay, when optimized for FFPE samples, was 100 copies of cDNA, and the dynamic range was 3 orders of magnitude. A prototype assay containing a panel of 8 genes displayed good reproducibility compared with the commercially available TaqMan assay (interassay CVs, 5%-20% vs 7%-43%, respectively). Gene-expression analysis was performed successfully in 26 (96%) of 27 endoscopic biopsy specimens, 30 (86%) of 35 archival FFPE samples, and 20 (100%) of 20 archival frozen samples. CONCLUSIONS: This new technology combines the reproducibility of competitive PCR with accurate quantitative detection by in-tube melting-curve analysis, enabling efficient analysis of mRNA profiles in samples with small numbers of cells or small amounts of tissue, as well as in archival FFPE tissues.

Fluorescence resonance energy transfer (FRET) using ssDNA binding fluorescent dye.

There is a need for simple and inexpensive methods for genotyping single nucleotide polymorphisms (SNPs) and short insertion/deletion variations (InDels). In this work, I demonstrate that a single-stranded DNA (ssDNA) binding dye can be used as a donor fluorophore for fluorescence resonance energy transfer (FRET). The method presented is a homogenous assay in which detection is based on the FRET from the fluorescence of the ssDNA dye bound to the unmodified detection primer to the fluorescent nucleotide analog incorporated into this detection primer during cyclic template directed primer extension reaction. Collection of the FRET emission spectrum with a scanning fluorescence spectrophotometer allows powerful data analysis. The fluorescence emission signal is modified by the optical properties of the assay vessel. This seems to be a completely neglected parameter. By proper selection of the optical properties of the assay plate one can improve the detection of the fluorescence emission signal.

Serum levels of mast cell tryptase, vascular endothelial growth factor and basic fibroblast growth factor in patients with acute pancreatitis.

PURPOSE: Mast cell tryptase, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) possibly play a role in the pathogenesis of acute pancreatitis (AP). The aim is to describe their serum levels in relation to severity of AP. METHODS: Seventy patients with AP were studied. Thirty-one had mild acute pancreatitis and 39 severe AP of whom 21 developed organ dysfunction. Serum concentration of tryptase was determined with fluoroimmunoassay (UniCAP), and VEGF and bFGF with ELISA at admission and on days 1, 2, and 7 post-hospitalization. RESULTS: The peak tryptase levels and tryptase levels at 2nd day after symptom onset, although mostly within normal range, were significantly higher in patients with organ dysfunction than in patients without organ dysfunction (6.6 microg/l (inter quartile range 4.8 to 12.6) versus 4.0 microg/l (2.7 to 6.2); P = 0.018 and 6.0 microg/l (4.4 to 7.6) versus 3.4 microg/l (2.3 to 4.8); P = 0.006, respectively). Median serum VEGF and bFGF concentrations increased during follow-up, were significantly higher on day 7 than on days 0, 1, and 2, but were not related to development of organ dysfunction. CONCLUSIONS: Mast cell activation, as defined by serum tryptase levels, may play a role in the development of remote organ dysfunction in patients with AP. However, neither tryptase nor the factors VEGF and bFGF serve as predictors of organ dysfunction in clinical AP.

CD14 and TNfa promoter polymorphisms in patients with acute arthritis. Special reference to development of chronic spondyloarthropathy.

OBJECTIVE: To examine CD14 and TNFalpha gene polymorphisms in early arthritis in relation to clinical outcome. METHODS: We studied 141 Caucasians who had had early arthritis 10 to 38 years earlier. We analysed CD14 (-159) and TNFalpha (-238, -308, -376) polymorphisms using a novel cycle minisequencing method. DNA pools from 370 Caucasian blood donors served as controls. RESULTS: CD14 (-159)C-->T allele frequencies were comparable among patients and controls (39% vs 40%). Fifty men and 42 women had recovered while 24 men and six women had chronic spondyloarthropathy (SpA). Mutant T allele frequency was higher in the chronic SpA group than in the recovered group in women (75% vs 32%, relative risk 1.3, 95% confidence limit 1.1 to 1.6, P = 0.011), but not in men (38% vs 44%). All female patients with chronic SpA had CD14 (-159)T allele and none had a possibly protective TNFalpha (-308)G-->A allele. CONCLUSIONS: Possession of CD14 (-159)T allele does not increase risk of ReA but may increase susceptibility of female patients to development of chronic SpA.