MRD monitoring
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The DNA methyl-transferase 3A gene (DNMT3A) is the third most frequently mutated gene in cytogenetically normal acute myeloid leukemia (CN-AML) patients (20-30 %), who belong to a group of patients with intermediate risk. About 60 % of mutations in this gene have been identified in the arginine codon R882. To date, there is no consensus on whether these mutations can be used as biomarkers for monitoring of minimal residual disease and management of preemptive AML therapy. We studied the occurrence of mutations in the DNMT3A gene in our cohort of patients and their persistence during AML treatment. Using next-generation sequencing, we identified four mutations in 11/25 of our analyzed patients--frequent R882C and R882H mutations, rare Y735S mutation, and a novel L347P mutation. Mutation R882C was detected in 5/11, R882H in 4/11 patients, and Y735S and L347P in one patient each. In 4/7 patients initially carrying mutations in the R882 codon, we found the persistence of mutations also during complete remission with, however, no correlation to AML kinetics. Our findings suggest that mutations in the DNMT3A gene can only be used as a biomarker for those AML patients in whom DNMT3A mutation is lost after therapy.
- MeSH
- akutní myeloidní leukemie genetika MeSH
- DNA-(cytosin-5-)methyltransferasa genetika MeSH
- dospělí MeSH
- kodon genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- missense mutace * MeSH
- monitorování fyziologických funkcí * MeSH
- nádorové biomarkery genetika MeSH
- nádorové proteiny genetika MeSH
- reziduální nádor MeSH
- senioři MeSH
- substituce aminokyselin MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: Monitoring of residual disease (RD) by flow cytometry in childhood acute myeloid leukemia (AML) may predict outcome. However, the optimal time points for investigation, the best antibody combinations, and most importantly, the clinical impact of RD analysis remain unclear. PATIENTS AND METHODS: Five hundred forty-two specimens of 150 children enrolled in the AML-Berlin-Frankfurt-Muenster (BFM) 98 study were analyzed by four-color immunophenotyping at up to four predefined time points during treatment. For each of the 12 leukemia-associated immunophenotypes and time points, a threshold level based on a previous retrospective analysis of another cohort of children with AML and on control bone marrows was determined. RESULTS: Regarding all four time points, there is a statistically significant difference in the 3-year event-free survival (EFS) in those children presenting with immunologically detectable blasts at 3 or more time points. The levels at bone marrow puncture (BMP) 1 and BMP2 turned out to have the most significant predictive value for 3-year-EFS: 71% +/- 6% versus 48% +/- 9%, P(Log-Rank) = .029 and 70% +/- 6% versus 50% +/- 7%, P(Log-Rank) = .033), resulting in a more than two-fold risk of relapse. In a multivariate analysis, using a combined risk classification based on morphologically determined blasts at BMP1 and BMP2, French-American-British classification, and cytogenetics, the influence of immunologically determined RD was no longer statistically significant. CONCLUSION: RD monitoring before second induction has the same predictive value as examining levels at four different time points during intensive chemotherapy. Compared with commonly defined risk factors in the AML-BFM studies, flow cytometry does not provide additional information for outcome prediction, but may be helpful to evaluate the remission status at day 28.
- MeSH
- akutní myeloidní leukemie * diagnóza farmakoterapie MeSH
- analýza přežití MeSH
- analýza rozptylu MeSH
- dítě MeSH
- dospělí MeSH
- kojenec MeSH
- kostní dřeň patologie MeSH
- lidé MeSH
- mladiství MeSH
- prediktivní hodnota testů MeSH
- předškolní dítě MeSH
- přežití bez známek nemoci MeSH
- prognóza MeSH
- proporcionální rizikové modely MeSH
- protokoly protinádorové kombinované chemoterapie terapeutické užití MeSH
- průtoková cytometrie * MeSH
- retrospektivní studie MeSH
- reziduální nádor * diagnóza MeSH
- rizikové faktory MeSH
- výsledek terapie MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- multicentrická studie MeSH
- práce podpořená grantem MeSH
Selection of the proper target is crucial for clinically relevant monitoring of minimal residual disease (MRD) in patients with acute lymphoblastic leukemia using the quantitation of clonal-specific immunoreceptor (immunoglobulin/T cell receptor) gene rearrangements. Consequently, correct interpretation of the results of the entire analysis is of utmost importance. Here we present an overview of the quality control measures that need to be implemented into the process of marker identification, selection, and subsequent quantitation of the MRD level.
Allogeneic stem cell transplantation (alloSCT) is used for treating patients with T-prolymphocytic leukemia (T-PLL). However, direct evidence of GvL activity in T-PLL is lacking. We correlated minimal residual disease (MRD) kinetics with immune interventions and T-cell receptor (TCR) repertoire diversity alterations in patients after alloSCT for T-PLL. Longitudinal quantitative MRD monitoring was performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3) Although post-transplant immunomodulation (immunosuppression tapering or donor lymphocyte infusions) resulted in significant reduction (>1 log) of MRD levels in 7 of 10 occasions, durable MRD clearance was observed in only two patients. In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. Novel clonotypes that could explain a clonal GvL effect did not emerge. In conclusion, TCR-based MRD quantification appears to be a suitable tool for monitoring and guiding treatment interventions in T-PLL. The MRD responses to immune modulation observed here provide first molecular evidence for GvL activity in T-PLL which, however, may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.
- MeSH
- buněčné klony imunologie MeSH
- dospělí MeSH
- genová přestavba T-lymfocytů genetika MeSH
- homologní transplantace MeSH
- imunomodulace * MeSH
- kinetika MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- reakce štěpu proti leukémii * MeSH
- receptory antigenů T-buněk analýza genetika MeSH
- reziduální nádor diagnóza genetika MeSH
- senioři MeSH
- T-buněčná prolymfocytární leukemie diagnóza terapie MeSH
- transplantace kmenových buněk metody MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVE: To evaluate the prognostic power of minimal residual disease (MRD) monitored by polymerase chain reaction at defined time points during early treatment in adult patients with acute lymphoblastic leukemia (ALL). METHODS: Seventy-one patients were treated according to the GMALL 07/2003 protocol and evaluated for MRD in bone marrow by specific clonal rearrangements of Ig/TCR in BCR-ABL negative ALL or fusion gene transcript in BCR-ABL positive ALL. RESULTS: Three-year overall survival (OS) was 94% in patients with BCR-ABL negative ALL reaching complete molecular response (CMR) after the first course of chemotherapy (vs. 32% if MRD >10(-4) ; P = 0.001). Patients with CMR prior to the start of consolidation chemotherapy at week 11 had 3-yr OS 82% (vs. 18% if MRD >10(-4) ; P = 0.001). Patients with BCR-ABL positive ALL showed slower MRD dynamics. There was a trend to better OS in patients with ≥ 4 log reduction of BCR-ABL transcript prior to HSCT (92% vs. 50%; P = 0.065). None of the patients with detectable MRD (both BCR-ABL positive and negative) after HSCT survived 3 yr. CONCLUSION: Early MRD kinetics is an important tool for new prognostication models with direct clinical impact irrespective of standard prognostic factors in patients with BCR-ABL negative ALL.
- MeSH
- akutní lymfatická leukemie genetika mortalita patologie terapie MeSH
- analýza přežití MeSH
- bcr-abl fúzní proteiny genetika MeSH
- dospělí MeSH
- genová přestavba MeSH
- geny TcR MeSH
- imunoglobuliny genetika MeSH
- indukce remise MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- prognóza MeSH
- protokoly protinádorové kombinované chemoterapie terapeutické užití MeSH
- reziduální nádor diagnóza genetika MeSH
- senioři MeSH
- výsledek terapie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chromosomal translocation t(11;14)(q13;q32) is a characteristic molecular marker of mantle cell lymphoma (MCL) and leads to the fusion of the immunoglobulin heavy chain enhancer-promoter with the cyclin D1 gene. Both aberrant cyclin D1 expression and underlying chromosomal aberration may be used as molecular targets for monitoring minimal residual disease (MRD). The present study aims to assess the usefulness of quantitative cyclin D1 gene expression compared to the standardised but more technologically demanding DNA-based method for immunoglobulin heavy chain (IGH) or t(11;14) clone-specific gene rearrangement quantification in a cohort of bone marrow (BM) and peripheral blood (PB) samples from patients with MCL. We simultaneously evaluated DNA-MRD and cyclin D1 expression levels in 234 samples from 57 patients. We observed that both in DNA-MRD positive and negative BM/PB pairs from the same time points the expression levels of cyclin D1 are lower in PB than in BM (median 19×, BM/PB range 0.41-352). The correlation of cyclin D1 transcript levels with DNA-MRD or with flow cytometry was good only in samples with a very high infiltration. In DNA-MRD-negative BM samples, we observed a significant heterogeneity of cyclin D1 expression (in the range of more than three orders of magnitude). This is in contrast to previous reports demonstrating the usefulness of cyclin D1 for MRD monitoring that did not use DNA-based method as a reference. In PB, the specificity of cyclin D1 expression was better due to a lower physiological background. In conclusion, we show that cyclin D1 is unsuitable for MRD monitoring in BM.
- MeSH
- cyklin D1 genetika metabolismus MeSH
- kostní dřeň metabolismus patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- lymfom z plášťových buněk diagnóza genetika patologie MeSH
- messenger RNA analýza MeSH
- monitorování fyziologických funkcí metody MeSH
- nádorové biomarkery analýza genetika MeSH
- regulace genové exprese u nádorů MeSH
- reziduální nádor MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články 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
T-lineage acute lymphoblastic leukemia (T-ALL) accounts for about 15% of pediatric and about 25% of adult ALL cases. Minimal/measurable residual disease (MRD) assessed by flow cytometry (FCM) is an important prognostic indicator for risk stratification. In order to assess the MRD a limited number of antibodies directed against the most discriminative antigens must be selected. We propose a pipeline for evaluating the influence of different markers for cell population classification in FCM data. We use linear support vector machine, fitted to each sample individually to avoid issues with patient and laboratory variations. The best separating hyperplane direction as well as the influence of omitting specific markers is considered. Ninety-one bone marrow samples of 43 pediatric T-ALL patients from five reference laboratories were analyzed by FCM regarding marker importance for blast cell identification using combinations of eight different markers. For all laboratories, CD48 and CD99 were among the top three markers with strongest contribution to the optimal hyperplane, measured by median separating hyperplane coefficient size for all samples per center and time point (diagnosis, Day 15, Day 33). Based on the available limited set tested (CD3, CD4, CD5, CD7, CD8, CD45, CD48, CD99), our findings prove that CD48 and CD99 are useful markers for MRD monitoring in T-ALL. The proposed pipeline can be applied for evaluation of other marker combinations in the future.
- MeSH
- akutní lymfatická leukemie * diagnóza MeSH
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- lymfoblastická leukemie-lymfom z prekurzorových T-buněk * diagnóza MeSH
- průtoková cytometrie MeSH
- reziduální nádor diagnóza MeSH
- T-lymfocyty MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Za standardní postup při sledování minimální reziduální nemoci u dětských akutních lymfoblastickýchleukemií se v současné době považuje kvantitativní detekce klonálně specifických přestaveb imunoreceptorovýchgenů (receptorů T buněk a imunoglobulinů). Optimalizace detekce dvou výše uvedenýchnezávislých přestaveb s citlivostí alespoň jedné maligní buňky mezi desetitisícem normálních buněkse však nezdaří u všech pacientů.Fúzní gen TEL/AML1 je nejčastější chromozomální aberací u dětskýchakutních lymfoblastických leukemií a nalézá se u více než 20 % pacientů. Srovnání hladin reziduálnínemoci paralelně vyšetřených pomocí kvantitativní polymerázové řetězové reakce na transkriptTEL/AML1 a na přestavby imunoreceptorových genů ve 41 vzorku ukázalo celkově velmi dobroukorelaci s pouhými dvěma výjimkami (R2 = 0,847). Kvantitativní detekce transkriptu TEL/AML1 tedymůže posloužit jako alternativní cíl pro sledování MRN u pacientů s TEL/AML1 pozitivní leukemiís nedostatkem citlivých standardních znaků – přestaveb imunoreceptorových genů. Mezi 52 pacientys vyšetřenou hladinou reziduální nemoci na konci indukce detekovatelné TEL/AML1 pozitivní buňkyjednoznačně oddělily pacienty se špatnou prognózou (přežití bez relapsu = 50 %; 7 relapsů ze 14 dětí)od dětí s výbornou šancí na přežití (přežití bez relapsu = 92 %; 3 děti z 38; p = 0,0007). DetekovatelnéTEL/AML1 pozitivní buňky na konci indukce tedy předpovídají s vysokou pravděpodobností relaps,i když se vyskytne až 57 měsíců od diagnózy.
Currently the quantitative detection of clonal-specific rearrangements of immunoreceptor genes (T-cellreceptors and immunoglobulins) is considered to be the standard approach in minimal residual disease(MRD) monitoring in childhood acute lymphoblastic leukaemia. However, optimisation of two independentrearrangements with sensitivity of at least one malignant cell among 10 000 normal cells is notsuccessful in all patients. TEL/AML1 fusion gene is the most common chromosomal aberration inchildhood acute lymphoblastic leukaemia and is present in more than 20% of patients. Comparison ofresidual disease levels examined simultaneously by quantitative polymerase chain reaction onTEL/AML1 transcript and on immunoreceptor gene rearrangements in 41 samples showed very goodoverall correlation with only two exceptions (R2=0,847). Thus, quantitative detection of TEL/AML1transcript can serve as an alternative target for MRD monitoring in patients with TEL/AML1-positiveleukaemia and a lack of sensitive standard markers – immunoreceptor gene rearrangements. Among52 patients with residual disease level tested at the end of induction therapy, the presence ofTEL/AML1-positive cells reliably separated patients with poor prognosis (relapse free survival = 50%, 7 relapses in14 patients) from children with excellent outcome (relapse free survival = 92%, 3 relapses in 38 patients,p=0,0007). Detectable TEL/AML1-positive cells at the end of induction therapy thus predict relapse witha high probability even though the relapse occurs 57 months from the original diagnosis.
