Collaborative exercise: analysis of age estimation using a QIAGEN protocol and the PyroMark Q48 platform.

Human age estimation from trace samples may give important leads early in a police investigation by contributing to the description of the perpetrator. Several molecular biomarkers are available for the estimation of chronological age, and currently, DNA methylation patterns are the most promising. In this study, a QIAGEN age protocol for age estimation was tested by five forensic genetic laboratories. The assay comprised bisulfite treatment of the extracted DNA, amplification of five CpG loci (in the genes of ELOVL2, C1orf132, TRIM59, KLF14, and FHL2), and sequencing of the amplicons using the PyroMark Q48 platform. Blood samples from 49 individuals with ages ranging from 18 to 64 years as well as negative and methylation controls were analyzed. An existing age estimation model was applied to display a mean absolute deviation of 3.62 years within the reference data set. Key points: Age determination as an intelligence tool during investigations can be a powerful tool in forensic genetics.In this study, five laboratories ran 49 samples and obtained a mean absolute deviation of 3.62 years.Five markers were analyzed on a PyroMark Q48 platform.

Dysfunction of peripheral somatic and autonomic nervous system in patients with severe forms of Crohn's disease on biological therapy with TNFα inhibitors-A single center study.

OBJECTIVE: Crohn's disease (CD) can be associated with a wide range of extraintestinal manifestations (EIMs), including neurological ones. Published studies differ in their conclusions about the epidemiology and etiopathogenesis of neurological EIMs. The aims of this study were to demonstrate the presence and find risk factors of peripheral (somatic and autonomic) neuropathy patients with severe CD on anti-TNFα biological therapy. MATERIAL AND METHODS: A clinical examination focusing on detection of peripheral sensor-motor nervous dysfunction (including Sudoscan) and examination of autonomic nervous system dysfunction (using Ewing´s battery tests and spectral analysis) together with laboratory tests and collection of demographic data followed by administration of questionnaires were performed on a total of 30 neurologically asymptomatic outpatients with severe CD on anti-TNFα biological therapy. RESULTS: Peripheral sensor-motor nervous function via clinical neurological examination was pathological in 36.7% and Sudoscan in 33.3% of cases. Statistically significant associations between vibration perception test and age, CD and biological therapy duration, body mass index and Crohn's Disease Activity Index were proved while statistically significant associations between temperature perception test and age and BMI were proved as well. Additionally, a decrease of total protein in a patient´s serum below the physiological cut-off in the 6 months prior to measurement was associated with a pathological result of a Sudoscan. Cardiovascular autonomic neuropathy based on Ewing´s battery tests was present in 56.7% of patients, no statistically significant risk factors were found. Our peripheral neuropathy questionnaire correlated with the results of the Sudoscan test and some tests of the clinical examination of peripheral sensor-motor nervous function (discriminatory contact perception test, temperature perception test). CONCLUSIONS: This study demonstrated a relatively high prevalence of peripheral (especially autonomic) neuropathy and verified some risk factors for the development of peripheral somatic neuropathy in asymptomatic patients with severe form of CD on anti-TNFα biological therapy.

Age-related changes in sperm DNA methylation and their forensic and clinical implications.

As a link between a stable genome and a dynamic environment, epigenetics is a promising tool for mapping age-related changes in human DNA. Methylated cytosine changes at specific loci are generally less studied in sperm DNA than in somatic cell DNA. Age-related methylation changes can be connected to various reproductive health problems and multiple disorders in offspring. In addition, they can be helpful in forensic fields, where testing of specific loci in semen samples found at sexual assault crime scenes can predict a perpetrator's age and narrow down the police investigation. This review focuses on age-related methylation changes in sperm. It covers the biological role of methylation, methylation testing techniques and the implications of methylation changes in forensics and clinical practice.

DNA methylation is a biological process that can change the activity of a gene without changing its sequence. We do not know much about DNA methylation in sperm and what changes methylation undergoes during the lifespan. These changes can, however, be important both for health and solving crimes. Presperm cells renew themselves, which gives rise to new sperm cells, from youth to death, accumulating cell divisions prone to error. This is why sperm cells are affected by age more than nondividing eggs. Methylation is specific in different tissues of the body. The ratio between number of sperm cells, white blood cells, and other cell types is highly variable and hardly predicted, which may distort the results. Clinical studies have shown that older fathers have worse reproductive health. Their children can develop metabolic, neurological and behavioral disorders. This also applies to younger men whose DNA methylation pattern is similar to that of older men. Methylation changes allow us to build a model capable of predicting the age of an unknown person with a mean error of about 5 years. This can be helpful for police investigators in cases of sexual assault, when biological material is found but there is no match in the police database.

Clinical and prognostic significance of detecting CEA, EGFR, LunX, c-met and EpCAM mRNA-positive cells in the peripheral blood, tumor-draining blood and bone marrow of non-small cell lung cancer patients.

Background: Surgical treatment of early-stage non-small cell lung cancer (NSCLC) yields highest expectations for recovery. However, the frequency of further disease progression remains high since micro-metastatic disease may be undetected by conventional diagnostic methods. We test the presence and prognostic impact of circulating tumor cells (CTCs) in peripheral blood (PB), tumor-draining pulmonary blood (TDB) and bone marrow (BM) samples from NSCLC patients. Methods: The presence of circulating/disseminated tumor cells (CTCs/DTCs) was detected by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis in PB, TDB and BM samples before surgery in 119 stage IA-IIIA NSCLC patients (Clinical Trial NS10285). Results: NSCLC patients with the presence of carcinoembryonic antigen (CEA) mRNA-positive CTCs/DTCs in TDB and BM had significantly shorter cancer-specific survival (CSS) (P<0.013, resp. P<0.038). Patients with the presence of epithelial cellular adhesion molecule (EpCAM) mRNA-positive CTCs in TDB samples had significantly shorter CSS and disease-free survival (DFS) (P<0.031, resp. P<0.045). A multivariate analysis identified the presence of CEA mRNA-positive CTCs in the PB as an independent negative prognostic factor for DFS (P<0.005). No significant correlation of CTCs/DTCs presence and other prognostic factors was found. Conclusions: In NSCLC patients undergoing radical surgery, the presence of CEA and EpCAM mRNA-positive CTCs/DTCs is associated with poorer survival.

Factors affecting participation in the colorectal cancer screening program: a cross-sectional population study.

PURPOSE: Fecal immunochemical test (FIT) once a year or colonoscopy once in 10 years is the option approved for colorectal cancer (CRC) screening for asymptomatic individuals aged ≥ 50 years in the Czech Republic. We analyzed participation in the screening program to determine possible improvements. METHODS: In this observational cross-sectional study, data were collected from 4044 randomly chosen individuals from the Czech population (1866 men, 2178 women) aged ≥ 50 years by questionnaires. Individuals who underwent colonoscopy within the last 10 years or/and FIT within the last 2 years were classified as participants in the screening. RESULTS: 1050 individuals underwent FIT, 464 colonoscopy, and 558 underwent both. Adjusted for age, gender, and education, a higher chance of participation in the screening was observed in groups of non-smokers (OR = 1.25; CI 1.05-1.48), ex-smokers (OR = 1.51; CI 1.26-1.83), consuming smoked meat products less than once a week (OR = 1.26; CI 1.09-1.45), practicing physical activity at least once a week (OR = 1.25; CI 1.03-1.51), hospitalized in the past 12 months (OR = 1.73; CI 1.47-2.05), or consulting a general practitioner (GP) in the past 12 months (OR = 2.26; CI 1.87-2.74). The chance of participation of individuals having a risk factor for CRC (obesity, smoking, diabetes, low physical activity, alcohol drinking) was not higher compared to those without the risk factors. CONCLUSION: Individuals with a tendency to a healthy lifestyle or being in recent contact with the healthcare system by various means, mainly visiting a GP, had a higher participation in the screening for CRC. Among groups with an increased risk for CRC, higher participation was not shown.

Efficacy and safety of SARS-CoV-2 vaccination in patients with inflammatory bowel disease on immunosuppressive and biological therapy: Prospective observational study.

BACKGROUND AND AIMS: SARS-CoV-2 is a worldwide serious health problem and vaccination seems to have a crucial role in managing the COVID-19 pandemic. The aim of this prospective observational study was to monitor the trend of antibodies against SARS-CoV-2 after vaccination with BNT162b2 (COMIRNATY) in patients with inflammatory bowel disease treated by immunosuppressive and/or biological therapy, demonstrate whether any type of this therapy is associated with poorer production of antibodies against COVID-19 and evaluate the safety of vaccination against COVID-19 in these patients. METHODS: Eighty-seven eligible patients from one tertiary gastroenterological center with inflammatory bowel disease (60 with CD, 27 with UC) treated by immunosuppressive and/or biological therapy from the antiTNFα group were indicated to vaccination against SARS-CoV-2. Effectiveness of vaccination was evaluated by the values of antibodies before and 4 weeks after 2nd dose of vaccine. Additional goal was to evaluate adverse events of vaccination. RESULTS: Before the 2nd dose of vaccine, geometric mean of SARS-CoV-2 IgG antibodies were 40.7 U/ml in the biological therapy group, 34.8 U/ml in the azathioprine group and 44.8 U/ml in the combination therapy group of patients. The geometric means were 676.5.7 U/ml in the biological therapy group, 614.4 U/ml in the azathioprine group and 500.1 U/ml in the combination therapy group of patients four weeks after 2nd dose. Statistically significant differences between these groups were not proved. Several non-severe local and general adverse events were present in our patients with a majority of these events on the day of vaccine administration and the day after, no anaphylactic reactions were present. CONCLUSIONS: Our measurements proved the efficacy and safety of vaccination against SARS-CoV-2 in patients with inflammatory bowel disease treated by immunosuppressive and/or biological therapy. Statistically significant differences between our groups of patients were not proved.

Adalimumab biosimilars in the therapy of Crohn´s disease and ulcerative colitis: Prospective multicentric clinical monitoring.

OBJECTIVE: The adalimumab biosimilars FKB327 and GP2017 were approved for the therapy of patients with inflammatory bowel disease (IBD). Relatively few prospective studies with biosimilar adalimumab in patients with IBD have been published. The aim of this prospective observational study was to evaluate the effectiveness and safety of the biosimilar adalimumab. MATERIAL AND METHODS: Adalimumab biosimilars FKB327 (Hulio®) and GP2017 (Hyrimoz®) were indicated to 50 naive patients in terms of biological therapy with Crohn's disease (CD) or ulcerative colitis (UC). Effectiveness of therapy was evaluated via the Crohn's Disease Activity Index [CDAI] or the Mayo Scoring System [MSS] in patients with CD or UC, respectively, before and after 12 weeks. Additional goals were to evaluate weight changes, laboratory tests and complications or adverse events of this therapy. RESULTS: In CD patients, remission (CDAI <150) was achieved in 73.5% of cases, partial response (≥70-point decrease in CDAI score from baseline) in 11.8%, no response in 11.8% and 2.9% patients discontinued therapy. In UC patients, remission (total score on partial Mayo index ≤2 points) was achieved only in 18.8% of cases, partial response (≥2-point decrease in partial Mayo score from baseline) in 43.8%, no response in 25.0% and 12.5% patients discontinued therapy. There were statistically significant improvements in CDAI, MSS, haemoglobin, fecal calprotectin, albumin and CRP serum levels after 12 weeks of therapy. Seven adverse events were identified, three of which resulted in therapy being discontinued. CONCLUSIONS: This prospective observational study proved the effectiveness of the adalimumab biosimilars FKB327 and GP2017 in IBD.

Very late complications of oncotherapy in glioblastoma patients: A case series.

BACKGROUND: Stroke-like syndrome is defined as a rare, delayed complication of brain oncotherapy. Cases with more favorable brain cancer diagnoses and longer life expectancy have been previously reported, but here we present, for the first time, three long-term survivors of glioblastoma with stroke-like syndromes. METHODS AND RESULTS: Three young or middle-aged patients underwent tumor resection and chemoradiotherapy. They received regular clinical and imaging follow-up with stable neurological status and no signs of tumor recurrence. They exhibited varied signs and symptoms (motor and sensory deficits, aphasia, memory and cognitive disorders, seizures, and headache) accompanied by imaging abnormalities. Stroke-like syndromes developed within 2-5 days and resolved in 2-6 weeks. Diffusion-weighted MRI and T2 brain perfusion abnormalities were demonstrated in all patients. In addition, there was focal T1 MRI contrast enhancement due to blood-brain barrier disruption. In addition to tumor recurrence, classic stroke, encephalitis, metabolic and mitochondrial disorders, and post-seizure swelling should be excluded. The imaging indicated intensive MRI scanning and symptomatic medication (steroids supplemented by antiepileptics, vasoactive agents, etc.) for judicious management. With respect to the course, an invasive procedure was still considered an option. CONCLUSION: All stroke-like syndromes are diagnoses of exclusion. To avoid misinterpretation of imaging findings as glioblastoma recurrence and avert recall oncotherapy or redundant interventions, better understanding of delayed complications of brain tumor therapy is crucial.

Development and extensive analytical validation of deep amplicon sequencing for detecting KRAS and NRAS mutations in metastatic colorectal cancer samples.

The presence of wild-type RAS alleles, as determined by genotyping codons 12, 13, 59, 61, 117, and 146, is a prerequisite for personalized anti-EGFR treatment of metastatic colorectal cancer (mCRC) patients. Here we describe analytical validation of in-house developed massively parallel sequencing technology (MPS) in comparison to the in vitro diagnostics (IVD) certified qPCR method. DNA extracted from FFPE samples from CRC patients (n=703) and reference standards (n=33) were tested for KRAS and NRAS mutations in 6 codons of exons 2, 3, and 4 using deep amplicon sequencing (DAS) on a MiSeq benchtop sequencer (Illumina). Two different amplicon lengths and two different library preparation methods (long-RAS and short-RAS) were tested in order to evaluate their impact on DAS performance. In parallel, identical tumor DNA was tested by the following IVD assays: therascreen KRAS RGQ PCR Kit (Qiagen), cobas® KRAS Mutation Test (Roche Diagnostics), and SNaPshot assay (Thermo Fisher Scientific). Both DAS assays detected all the mutations present in reference standards and external quality control samples, except for the artificially generated KRAS codon 146 mutation. The DAS assays performed sufficient analytical specificity and sensitivity (≥0.95). The use of shorter amplicons prolonged the preparation steps but significantly improved the sequencing success rate of FFPE-derived DNA. RAS mutation frequencies in the Czech CRC patients were similar to previous reports, although rare mutations were also detected. DAS with short amplicons is a good strategy for routine assessment of somatic mutations in low-quality FFPE-derived DNA.

Whole genome and exome sequencing reference datasets from a multi-center and cross-platform benchmark study.

With the rapid advancement of sequencing technologies, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples and generated whole-genome (WGS) and whole-exome sequencing (WES) data using sixteen library protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.

Toward best practice in cancer mutation detection with whole-genome and whole-exome sequencing.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

Establishing community reference samples, data and call sets for benchmarking cancer mutation detection using whole-genome sequencing.

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.

Conjugated Hyperbilirubinemia in Infants: Is There Still a Role for ERCP?

Over a twenty-year period, we performed 255 ERCP procedures in infants aged up to 1 year. ERCP was indicated in cholestatic infants with suspicion of biliary obstruction. The most common diagnosis was biliary atresia (48%), choledochal cysts (13%), and choledocholithiasis (4%). The procedure complication rate was 13.7%. Hyperamylasemia occurred in 12.9%. More severe complications were rare-0.8% of ERCP procedure. There were no cases of postprocedural pancreatitis or death. Our study has proved that ERCP is a safe and reliable method in this age group. Its high specificity and negative predictive value for extrahepatic biliary atresia can prevent unnecessary surgeries in patients with normal bile ducts or endoscopically treatable pathologies.

Commonly available but highly effective protection against SARS-CoV-2 during gastrointestinal endoscopies.

BACKGROUND AND AIMS: SARS-CoV-2 is a worldwide serious health problem. The aim of this study was to demonstrate the number of potentially infectious particles present during endoscopic procedures and find effective tools to eliminate the risks of SARS-CoV-2 infection while performing them. METHODS: An experimental model which focused on aerosol problematics was made in a specialized laboratory. This model simulated conditions present during endoscopic procedures and monitored the formation of potentially infectious fluid particles from the patient's body, which pass through the endoscope and are then released into the environment. For this reason, we designed and tested a prototype of a protective cover for the endoscope's control body to prevent the release and spread of these fluid particles from its working channel. We performed measurements with and without the protective cover of the endoscope's control body. RESULTS: It was found that liquid coming through the working channel of the endoscope with forceps or other instruments inside generates droplets with a diameter in the range of 0.1-1.1 mm and an initial velocity of up to 0.9 m/s. The average number of particles per measurement per whole measured area without a protective cover on the endoscope control body was 51.1; with this protective cover on, the measurement was 0.0, p<0.0001. CONCLUSIONS: Our measurements proved that fluid particles are released from the working channel of an endoscope when forceps are inserted. A special protective cover for the endoscope control body, made out of breathable material (surgical cap) and designed by our team, was found to eliminate this release of potentially infectious fluid particles.

The Influence of Gene Aberrations on Survival in Resected IDH Wildtype Glioblastoma Patients: A Single-Institution Study.

This prospective population-based study on a group of 132 resected IDH-wildtype (IDH-wt) glioblastoma (GBM) patients assesses the prognostic and predictive value of selected genetic biomarkers and clinical factors for GBM as well as the dependence of these values on the applied therapeutic modalities. The patients were treated in our hospital between June 2006 and June 2015. Clinical data and tumor samples were analyzed to determine the frequencies of TP53, MDM2, EGFR, RB1, BCR, and CCND1 gene aberrations and the duplication/deletion statuses of the 9p21.3, 1p36.3, 19q13.32, and 10p11.1 chromosome regions. Cut-off values distinguishing low (LCN) and high (HCN) copy number status for each marker were defined. Additionally, MGMT promoter methylation and IDH1/2 mutation status were investigated retrospectively. Young age, female gender, Karnofsky scores (KS) above 80, chemoradiotherapy, TP53 HCN, and CCND1 HCN were identified as positive prognostic factors, and smoking was identified as a negative prognostic factor. Cox proportional regression models of the chemoradiotherapy patient group revealed TP53 HCN and CCND1 HCN to be positive prognostic factors for both progression-free survival and overall survival. These results confirmed the influence of key clinical factors (age, KS, adjuvant oncotherapy, and smoking) on survival in GBM IDH-wt patients and demonstrated the prognostic and/or predictive importance of CCND1, MDM2, and 22q12.2 aberrations.

Dabrafenib monotherapy in BRAF+ non-small cell lung cancer - our experience.

BACKGROUND: Activating BRAF mutations result in constitutive activation of the MAP kinase signaling cascade, stimulating cell proliferation. BRAF mutations are typical for malignant melanoma, but occur less frequently in other tumors, including in 1-2% cases of non-small cell lung cancer (NSCLC) [1,2]. CASE: We present two case reports of BRAF+ NSCLC patients, treated with 3rd line dabrafenib monotherapy on our department, and also brief review of available information about dabrafenib and its use in monotherapy of BRAF+ NSCLC. CONCLUSION: Monotherapy with BRAF inhibitors presents a viable alternative for BRAF+ NSCLC patients, incapable of combined therapy with trametinib. The lack of proper indication and reimbursement for NSCLC cases remains a problem, and individual treatment approval is required.

The absence of high-risk human papillomavirus in Czech non-small cell lung cancer cases.

AIMS: The purpose of our study was to examine the presence of human papillomavirus (HPV) DNA in Czech patients with non-small cell lung cancer (NSCLC). METHODS: A highly sensitive quantitative polymerase chain reaction (qPCR) detecting the E6 gene of HPV16, 18, 31, and 56 was designed. The limit of detection was assessed using serial dilutions of HPV-positive plasmids. The qPCR was validated on a set of 402 cervical swabs where the qPCR, Cobas, and PapilloCheck methods were tested in parallel. Finally, qPCR was used for HPV detection in a set of 80 patients with primary NSCLC, both from formalin-fixed paraffin-embedded (FFPE) and fresh frozen (FF) tissue samples. RESULTS: The qPCR method was able to reliably detect at least 4 copies of the E6 gene per reaction in HPV16, 18, and 31, and 40 copies per reaction in HPV56. The sensitivity and specificity of the qPCR were 75.6-99.3% and 63.9-100% respectively, depending on the HPV genotype and reference method used. HPV DNA was not detected in the FFPE and FF samples from the set of 80 NSCLC patients. CONCLUSION: No hrHPV DNA was found in primary NSCLC tumors from a Czech population.

Pitfalls of commercially available HPV tests in HPV68a detection.

BACKGROUND: Human papillomavirus 68 (HPV68) is a probable carcinogenic HPV genotype which is included in almost all HPV screening assays and exists as two genetically variable subtypes (HPV68a and HPV68b). Routine HPV sample testing has shown that the cobas 4800 HPV Test (Roche) provides higher false-negative rates for HPV68 status than PapilloCheck HPV-Screening (Greiner Bio-One). The aim of our study was to evaluate the efficacy of cobas 4800 in HPV68 detection. METHODS: A total of 2,145 cervical/cervicovaginal samples from women aged 17-88 were tested for HPV68 status using the cobas 4800 and PapilloCheck HPV tests. Viral load was assessed by quantitative PCR in all of the HPV68-positive cases. HPV68a/b subtyping was performed with real-time PCR followed by high resolution melting curve analysis, and was subsequently confirmed by Sanger sequencing. RESULTS: Cobas 4800 detected HPV positivity in only 13/33 HPV68 single-genotype infection cases. Viral load was comparable across both tested subgroups. HRM analysis and Sanger sequencing identified the HPV68a subtype in all of the 20 instances of cobas 4800 false negatives. HPV68a and HPV68b were detected in 3/13 and 10/13 cases identified as other HPV-positive by cobas 4800. CONCLUSION: The HPV68a subtype was missed by cobas 4800 in more than 85% of all HPV68a-positive cases. Therefore, commercially available assays may underestimate HPV68 prevalence.

ERCP in infants, children, and adolescents-Different roles of the methods in different age groups.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) is seldom used in children, and published series have limited numbers of pediatric patients. The aim of this retrospective observational study was to assess the efficacy and safety of pediatric ERCP in a large group of children. METHODS: Data were evaluated from 626 children with biliopancreatic disorders admitted to University Hospital Motol, Prague, between January 1999 and January 2018. Clinical data were obtained by retrospective evaluation of our database of pediatric ERCP procedures and from clinical records. RESULTS: We performed 856 ERCPs on 626 pediatric patients; of these procedures, 59% were therapeutic and 41% were diagnostic. We achieved 96% technical success. Indications for ERCP and pathological findings differed in different age groups. The main role of ERCP was in excluding biliary atresia in those aged less than one year. In children aged 1 to 6 years, the most frequent diagnoses were choledochal cyst followed by choledocholithiasis. In children aged 7 to 12 years and 13 to 19 years, the most frequent diagnoses were choledocholithiasis followed by pancreatic pathology. The overall complication rate found in this study was similar to rates observed in adult populations. CONCLUSIONS: Our study shows the efficacy and safety of diagnostic and therapeutic ERCP in a large series of infants and children with technical success and complication rates comparable to those in adults. Our data show that ERCP had different roles in different age groups of children.

IDH Mutation Analysis in Glioma Patients by CADMA Compared with SNaPshot Assay and two Immunohistochemical Methods.

Mutations in IDH1/2 genes are a marker of good prognosis for glioma patients, associated with low grade gliomas and secondary glioblastomas. Immunohistochemistry and Sanger sequencing are current standards for IDH1/2 genotyping while many other methods exist. The aim of this study was to validate Competitive amplification of differentially melting amplicons (CADMA) PCR for IDH genotyping by comparison with SNaPshot assay and two immunohistochemical methods. In our study, 87 glioma patients (46 from Olomouc and 41 from Ostrava) were analyzed. IDH1/2 mutations in native bioptical samples were analyzed at DNA level by CADMA and SNaPshot while IDH1 mutations in FFPE samples were analyzed at protein level by two IHC methods. CADMA PCR sensitivity for IDH1 was 96.4% and specificity 100% for 86 concluded samples. SNaPshot assay sensitivity was 92.9% and specificity of 100% for 85 concluded samples. IHC in the laboratory no. 2 reached sensitivity 85.7% and specificity 100% for 86 concluded samples. IHC in the laboratory no. 4 reached sensitivity of 96.4% and specificity of 79.7% in 74 concluded samples. Only one IDH2 mutation was found by SNaPshot while CADMA yielded false negative result. In conclusion, CADMA is a valid method for IDH1 p.(R132H) testing with higher sensitivity than SNaPshot assay. Also, molecular genetic methods of IDH1 testing from native samples were more robust than IHC from FFPE.