• MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé MeSH
• reziduální nádor * diagnóza   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH

We compared minimal/measurable residual disease (MRD) levels evaluated by routinely used real-time quantitative polymerase chain reaction (qPCR) patient-specific assays and by next-generation sequencing (NGS) approach in 780 immunoglobulin (IG) and T-cell receptor (TR) markers in 432 children with B-cell precursor acute lymphoblastic leukemia treated on the AIEOP-BFM ALL 2009 protocol. Our aim was to compare the MRD-based risk stratification at the end of induction. The results were concordant in 639 of 780 (81.9%) of these markers; 37 of 780 (4.7%) markers were detected only by NGS. In 104 of 780 (13.3%) markers positive only by qPCR, a large fraction (23/104; 22.1%) was detected also by NGS, however, owing to the presence of identical IG/TR rearrangements in unrelated samples, we classified those as nonspecific/false-positive. Risk group stratification based on the MRD results by qPCR and NGS at the end of induction was concordant in 76% of the patients; 19% of the patients would be assigned to a lower risk group by NGS, largely owing to the elimination of false-positive qPCR results, and 5% of patients would be assigned to a higher risk group by NGS. NGS MRD is highly concordant with qPCR while providing more specific results and can be an alternative in the front line of MRD evaluation in forthcoming MRD-based protocols.

• MeSH
• akutní lymfatická leukemie * diagnóza   genetika   terapie   MeSH
• dítě MeSH
• genová přestavba MeSH
• hodnocení rizik MeSH
• imunoglobuliny genetika   MeSH
• lidé MeSH
• pre-B-buněčná leukemie * diagnóza   genetika   terapie   MeSH
• receptory antigenů T-buněk genetika   MeSH
• reziduální nádor diagnóza   genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imunoglobuliny MeSH
• receptory antigenů T-buněk MeSH

Detection of patient- and tumor-specific clonally rearranged immune receptor genes using real-time quantitative (RQ)-PCR is an accepted method in the field of precision medicine for hematologic malignancies. As individual primers are needed for each patient and leukemic clone, establishing performance specifications for the method faces unique challenges. Results for series of diagnostic assays for CLL and ALL patients demonstrate that the analytic performance of the method is not dependent on patients' disease characteristics. The calibration range is linear between 10-1 and 10-5 for 90% of all assays. The detection limit of the current standardized approach is between 1.8 and 4.8 cells among 100,000 leukocytes. RQ-PCR has about 90% overall agreement to flow cytometry and next generation sequencing as orthogonal methods. Accuracy and precision across different labs, and above and below the clinically applied cutoffs for minimal/measurable residual disease (MRD) demonstrate the robustness of the technique. The here reported comprehensive, IVD-guided analytical validation provides evidence that the personalized diagnostic methodology generates robust, reproducible and specific MRD data when standardized protocols for data generation and evaluation are used. Our approach may also serve as a guiding example of how to accomplish analytical validation of personalized in-house diagnostics under the European IVD Regulation.

• Klíčová slova
• EuroMRD, IG rearrangement, IVDR, MRD, RQ-PCR, TR rearrangement, method validation, personalized diagnostics,
• Publikační typ
• časopisecké články MeSH

Cytomegalovirus (CMV) infection is associated with allograft rejection but the mechanisms behind are poorly defined yet. Although cross-reactivity of T cells to alloantigen and CMV has been hypothesized, direct evidence in patients is lacking. In this observational cohort study, we tested the pre-transplant effector/memory T cell response to CMV peptide pools and alloantigen in 78 living donor/recipient pairs using the interferon-gamma Enzyme-Linked ImmunoSpot (ELISPOT) assay. To prove the hypothesis of cross-reactivity, we analyzed by applying next-generation sequencing the T cell receptor ß (TCR- ß) repertoire of CMV- and alloantigen-reactive T cells enriched from peripheral pre-transplant blood of 11 CMV-seropositive and HLA class I mismatched patients. Moreover, the TCR-repertoire was also analyzed in the allograft biopsies of those patients. There was a significant association between the presence of pre-transplant CMV immediate-early protein 1 (IE-1)-specific effector/memory T cells and acute renal allograft rejection and function (p = 0.01). Most importantly, we revealed shared TCR-ß sequences between CMV-IE1 and donor alloantigen-reactive T cells in all pre-transplant peripheral blood samples analyzed in CMV-seropositive patients who received HLA class I mismatched grafts. Identical TCR sequences were also found in particular in post-transplant allograft biopsies of patients with concomitant CMV infection and rejection. Our data show the presence of functional, cross-reactive T cells and their clonotypes in peripheral blood and in kidney allograft tissue. It is therefore likely that CMV-donor cross-reactivity as well as CMV specific T cell elicited inflammation is involved in the processes that affect allograft outcomes.

• Klíčová slova
• ELISPOT, TCR repertoire, cross-reactivity, cytomegalovirus, heterologous immunity, kidney transplantation, rejection,
• MeSH
• alografty MeSH
• biopsie MeSH
• cytomegalovirové infekce * etiologie   genetika   imunologie   patologie   MeSH
• Cytomegalovirus imunologie   MeSH
• dospělí MeSH
• imunologická paměť * MeSH
• isoantigeny genetika   imunologie   MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• receptory antigenů T-buněk alfa-beta * imunologie   MeSH
• T-lymfocyty * mikrobiologie   patologie   MeSH
• transplantace ledvin * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky MeSH
• pozorovací studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• isoantigeny MeSH
• receptory antigenů T-buněk alfa-beta * MeSH

Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) was spiked into each sample as a library-specific quality control and calibrator. NGS identified 259 (average 5.2/sample, range 0-14) clonal sequences vs. Sanger-sequencing 248 (average 5.0/sample, range 0-14). NGS primers covered possible IG/TR rearrangement types more completely compared with local multiplex PCR sets and enabled sequencing of bi-allelic rearrangements and weak PCR products. The cPT-QC showed high reproducibility across all laboratories. These validated and reproducible quality-controlled EuroClonality-NGS assays can be used for standardized NGS-based identification of IG/TR markers in lymphoid malignancies.

• MeSH
• akutní lymfatická leukemie genetika   MeSH
• genetické markery genetika   MeSH
• genová přestavba T-lymfocytů genetika   MeSH
• geny pro imunoglobuliny genetika   MeSH
• geny TcR genetika   MeSH
• imunoglobuliny genetika   MeSH
• lidé MeSH
• receptory antigenů T-buněk genetika   MeSH
• referenční standardy MeSH
• rekombinace genetická genetika   MeSH
• reprodukovatelnost výsledků MeSH
• reziduální nádor genetika   MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genetické markery MeSH
• imunoglobuliny MeSH
• receptory antigenů T-buněk MeSH

Minimal residual disease (MRD) is the most important independent prognostic factor in acute lymphoblastic leukemia (ALL). Since it has been implemented into in treatment stratification strategies, cure rates have improved significantly for all age groups. Real time quantitative (RQ)-PCR of clonal immunoglobulin and T-cell receptor gene rearrangements using allele-specific primers is currently regarded as the gold standard for MRD analysis in ALL, as it is not only highly sensitive and specific but also provides accurate MRD quantification. Following recent advances in next-generation sequencing (NGS), much attention has been devoted to the development of NGS-based MRD assays. This new technique can enhance sensitivity provided that sufficient numbers of cells are analyzed. Recent reports have shown that NGS-MRD also tends to be more specific for relapse prediction than RQ-PCR. In addition, NGS provides information on the physiological B- and T-cell repertoire during and after treatment, which has been shown to be prognostically relevant. However, before implementation of NGS-MRD detection in clinical practice, several issues must be addressed and the whole workflow needs to be standardized, including not only the analytical phase (spike-in calibrators, quality controls) but also the pre-analytical (e.g. sample preparation) and the post-analytical phases (e.g. bioinformatics pipeline, guidelines for correct data interpretation). These topics are currently addressed by a European network, the EuroClonality-NGS Consortium. In conclusion, NGS is a promising tool for MRD detection with the potential to overcome most of the limitations of RQ-PCR and to become the new gold standard for MRD detection in ALL.

• Klíčová slova
• Acute Lymphoblastic Leukemia, Marker Identification, Minimal Residual Disease, Minimal Residual Disease Detection, Multicolor Flow Cytometry,
• MeSH
• akutní lymfatická leukemie genetika   MeSH
• lidé MeSH
• prognóza MeSH
• receptory antigenů T-buněk genetika   MeSH
• reziduální nádor MeSH
• sekvenční analýza DNA metody   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• receptory antigenů T-buněk MeSH

Minimal residual disease (MRD) monitoring via quantitative PCR (qPCR) detection of Ag receptor gene rearrangements has been the most sensitive method for predicting prognosis and making post-transplant treatment decisions for patients with ALL. Despite the broad clinical usefulness and standardization of this method, we and others have repeatedly reported the possibility of false-positive MRD results caused by massive B-lymphocyte regeneration after stem cell transplantation (SCT). Next-generation sequencing (NGS) enables precise and sensitive detection of multiple Ag receptor rearrangements, thus providing a more specific readout compared to qPCR. We investigated two cohorts of children with ALL who underwent SCT (30 patients and 228 samples). The first cohort consisted of 17 patients who remained in long-term CR after SCT despite having low MRD positivity (<0.01%) at least once during post-SCT monitoring using qPCR. Only one of 27 qPCR-positive samples was confirmed to be positive by NGS. Conversely, 10 of 15 samples with low qPCR-detected MRD positivity from 13 patients who subsequently relapsed were also confirmed to be positive by NGS (P=0.002). These data show that NGS has a better specificity in post-SCT ALL management and indicate that treatment interventions aimed at reverting impending relapse should not be based on qPCR only.

• MeSH
• akutní lymfatická leukemie * krev   diagnóza   genetika   terapie   MeSH
• dítě MeSH
• falešně pozitivní reakce MeSH
• lidé MeSH
• mladiství MeSH
• polymerázová řetězová reakce * MeSH
• předškolní dítě MeSH
• prognóza MeSH
• reziduální nádor MeSH
• transplantace hematopoetických kmenových buněk * MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky MeSH
• multicentrická studie MeSH

A fully-standardized EuroFlow 8-color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of ≤10-5, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)-based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR-based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (≤10-5), if sufficient cells (>4 × 106, preferably more) are evaluated.

• MeSH
• dítě MeSH
• dospělí MeSH
• genová přestavba MeSH
• kojenec MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• novorozenec MeSH
• pre-B-buněčná leukemie diagnóza   MeSH
• předškolní dítě MeSH
• průtoková cytometrie metody   normy   MeSH
• receptory antigenů B-buněk genetika   MeSH
• reziduální nádor diagnóza   MeSH
• senioři MeSH
• senzitivita a specificita MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• receptory antigenů B-buněk MeSH

• MeSH
• genetické markery genetika   MeSH
• genová přestavba * MeSH
• geny pro imunoglobuliny MeSH
• lidé MeSH
• receptory antigenů T-buněk genetika   MeSH
• reprodukovatelnost výsledků MeSH
• reziduální nádor diagnóza   MeSH
• teoretické modely MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genetické markery MeSH
• receptory antigenů T-buněk MeSH