Multi-target NGS
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BACKGROUND: Carriers of mutations in hereditary cancer predisposition genes represent a small but clinically important subgroup of oncology patients. The identification of causal germline mutations determines follow-up management, treatment options and genetic counselling in patients' families. Targeted next-generation sequencing-based analyses using cancer-specific panels in high-risk individuals have been rapidly adopted by diagnostic laboratories. While the use of diagnosis-specific panels is straightforward in typical cases, individuals with unusual phenotypes from families with overlapping criteria require multiple panel testing. Moreover, narrow gene panels are limited by our currently incomplete knowledge about possible genetic dispositions. METHODS: We have designed a multi-gene panel called CZECANCA (CZEch CAncer paNel for Clinical Application) for a sequencing analysis of 219 cancer-susceptibility and candidate predisposition genes associated with frequent hereditary cancers. RESULTS: The bioanalytical and bioinformatics pipeline was validated on a set of internal and commercially available DNA controls showing high coverage uniformity, sensitivity, specificity and accuracy. The panel demonstrates a reliable detection of both single nucleotide and copy number variants. Inter-laboratory, intra- and inter-run replicates confirmed the robustness of our approach. CONCLUSION: The objective of CZECANCA is a nationwide consolidation of cancer-predisposition genetic testing across various clinical indications with savings in costs, human labor and turnaround time. Moreover, the unified diagnostics will enable the integration and analysis of genotypes with associated phenotypes in a national database improving the clinical interpretation of variants.
- MeSH
- dědičné nádorové syndromy genetika MeSH
- genetická predispozice k nemoci MeSH
- genetické asociační studie MeSH
- genetické testování MeSH
- lidé MeSH
- mutace INDEL MeSH
- mutace MeSH
- nádorové biomarkery * MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- variabilita počtu kopií segmentů DNA MeSH
- výpočetní biologie metody MeSH
- vysoce účinné nukleotidové sekvenování * metody normy MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2- 99.8%. In order to rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e., pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a variant allele frequency (VAF) >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107 of 115 (93% concordance) mutations were detected by all six centers, while the remaining eight variants (7%) were undetected by a single center. Notably, 6 of 8 of these variants concerned minor subclonal mutations (VAF <5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAF >5%, after rigorous validation, the use of unique molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.
De novo sequence variants, including truncating and splicing variants, in the additional sex‑combs like 3 gene (ASXL3) have been described as the cause of Bainbridge‑Ropers syndrome (BRS). This pathology is characterized by delayed psychomotor development, severe intellectual disability, growth delay, hypotonia and facial dimorphism. The present study reports a case of a girl (born in 2013) with severe global developmental delay, central hypotonia, microcephaly and poor speech. The proband was examined using a multi‑step molecular diagnostics algorithm, including karyotype and array‑comparative genomic hybridization analysis, with negative results. Therefore, the proband and her unaffected parents were enrolled for a pilot study using targeted next‑generation sequencing technology (NGS) with gene panel ClearSeq Inherited DiseaseXT and subsequent validation by Sanger sequencing. A novel de novo heterozygous frameshift variant in the ASXL3 gene (c.3006delT, p.R1004Efs*21), predicted to result in a premature termination codon, was identified. In conclusion, the present study demonstrated that targeted NGS using a suitable, gene‑rich panel may provide a conclusive molecular genetics diagnosis in children with severe global developmental delays.
- MeSH
- dítě MeSH
- lidé MeSH
- mikrocefalie genetika MeSH
- pilotní projekty MeSH
- poruchy řeči genetika MeSH
- posunová mutace MeSH
- rodokmen MeSH
- svalová hypotonie genetika MeSH
- transkripční faktory genetika MeSH
- vývojové poruchy u dětí genetika MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
BACKGROUND: Multi-parametric magnetic resonance imaging (mpMRI)-directed biopsy for prostate cancer (PC) diagnosis improves the detection of clinically significant prostate cancer (CSPC) and decreases the rate of over-diagnosis of insignificant disease. The aim of this study was to investigate the value of mpMRI combined with prostate specific antigen density (PSAD) in the decision making related to the biopsy. METHODS: mpMRI and mpMRI/transrectal ultrasound fusion targeted biopsies with subsequent systematic biopsies were performed in 397 patients (223 biopsy-naïve and 174 with a previous biopsy). Detection rates of (CSPC) and insignificant PC were stratified using the PIRADS score, and the number of avoidable biopsies and missed (CSPC) were plotted against PSAD values of 0.1-0.5 ng/mL2. RESULTS: PIRADS <3 and PSAD <0.2 ng/mL2 were the safest criteria for not performing a biopsy. When applied to the biopsy-naïve group, 21.5% (48/223) of the biopsies could have been avoided and 3.7% (3/82) of CSPC would have been missed. In the repeat biopsy group, 12.6% (22/174) of biopsies could have been avoided and 6.9% (4/58) of (CSPC) would have been missed. CONCLUSIONS: A combination of mpMRI and PSAD might reduce the number of biopsies performed with the cost of missing <4% of CSPC.
- MeSH
- biopsie * MeSH
- časná detekce nádoru normy MeSH
- lékařská onkologie normy MeSH
- lidé středního věku MeSH
- lidé MeSH
- multiparametrická magnetická rezonance * MeSH
- nádory prostaty diagnostické zobrazování patologie MeSH
- prostata patologie MeSH
- prostatický specifický antigen krev MeSH
- reprodukovatelnost výsledků MeSH
- rozhodování MeSH
- senioři MeSH
- ultrasonografie MeSH
- ultrazvukem navigovaná biopsie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: High-intensity focused ultrasound (HIFU) emerged as a novel approach for the treatment of localized prostate cancer (PCa). However, prospective studies on HIFU-related outcomes and predictors of treatment failure (TF) remain scarce. MATERIALS AND METHODS: We conducted a multinational prospective cohort study among patients undergoing HIFU therapy for localized, low- to intermediate-risk PCa. Follow-up data on serial prostate specific antigen (PSA), multi-parametric magnetic resonance imaging (mpMRI), targeted/systematic biopsies, adverse events and functional outcomes were collected. The primary endpoint was TF, defined as histologically confirmed PCa requiring whole-gland salvage treatment. Uni- and multi-variable adjusted hazard ratios (HRs) were calculated using Cox proportional hazard regression models. RESULTS: At baseline, mean (standard deviation) age was 64.14 (7.19) years, with the majority of patients showing T-stage 1 (73.9%) and International Society of Urological Pathology grading system Grade 2 (58.8%). PSA nadir (median, 1.70 ng/mL) was reached after 6 months. Of all patients recruited, 16% had clinically significant PCa, as confirmed by biopsy, of which 13.4% had TF. Notably, T-stage and number of positive cores at initial biopsy were independent predictors of TF during follow-up (HR [95% CI] 1.27 [1.02-1.59] and 5.02 [1.80-14.03], respectively). Adverse events were minimal (17% and 8% early and late adverse events, respectively), with stable or improved functional outcomes in the majority of patients. CONCLUSIONS: This interim analysis of a multinational study on HIFU therapy for the management of low-to-intermediate-risk PCa reveals good functional outcomes, minimal adverse events and low incidence of TF over the short-term. Data on long-term outcomes, specifically as it relates to oncological outcomes, are awaited eagerly.
- MeSH
- ablace intenzivním ultrazvukovým paprskem metody MeSH
- kohortové studie MeSH
- lidé středního věku MeSH
- lidé MeSH
- multiparametrická magnetická rezonance MeSH
- nádory prostaty * terapie patologie MeSH
- neúspěšná terapie MeSH
- proporcionální rizikové modely MeSH
- prospektivní studie MeSH
- prostatický specifický antigen * metabolismus krev MeSH
- senioři MeSH
- stupeň nádoru MeSH
- transrektální ultrazvuk o vysoké intenzitě MeSH
- záchranná terapie metody MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
