Measurable residual disease (MRD) monitoring in childhood acute myeloid leukemia (AML) is used to assess response to treatment and for early detection of imminent relapse. In childhood AML, MRD is typically evaluated using flow cytometry, or by quantitative detection of leukemia-specific aberrations at the mRNA level. Both methods, however, have significant limitations. Recently, we demonstrated the feasibility of MRD monitoring in selected subgroups of AML at the genomic DNA (gDNA) level. To evaluate the potential of gDNA-based MRD monitoring across all AML subtypes, we conducted a comprehensive analysis involving 133 consecutively diagnosed children. Integrating next-generation sequencing into the diagnostic process, we identified (presumed) primary genetic aberrations suitable as MRD targets in 97% of patients. We developed patient-specific quantification assays and monitored MRD in 122 children. The gDNA-based MRD monitoring via quantification of primary aberrations with a sensitivity of at least 10-4 was possible in 86% of patients; via quantification with sensitivity of 5 × 10-4, of secondary aberrations, or at the mRNA level in an additional 8%. Importantly, gDNA-based MRD exhibited independent prognostic value at early time-points in patients stratified to intermediate-/high-risk treatment arms. Our study demonstrates the broad applicability, feasibility, and clinical significance of gDNA-based MRD monitoring in childhood AML.
- MeSH
- akutní myeloidní leukemie * diagnóza genetika terapie MeSH
- dítě MeSH
- genomika MeSH
- kohortové studie MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- prognóza MeSH
- průtoková cytometrie MeSH
- recidiva MeSH
- reziduální nádor diagnóza genetika MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Minimal/measurable residual disease (MRD) diagnostics using real-time quantitative PCR analysis of rearranged immunoglobulin and T-cell receptor gene rearrangements are nowadays implemented in most treatment protocols for patients with acute lymphoblastic leukemia (ALL). Within the EuroMRD Consortium, we aim to provide comparable, high-quality MRD diagnostics, allowing appropriate risk-group classification for patients and inter-protocol comparisons. To this end, we set up a quality assessment scheme, that was gradually optimized and updated over the last 20 years, and that now includes participants from around 70 laboratories worldwide. We here describe the design and analysis of our quality assessment scheme. In addition, we here report revised data interpretation guidelines, based on our newly generated data and extensive discussions between experts. The main novelty is the partial re-definition of the "positive below quantitative range" category by two new categories, "MRD low positive, below quantitative range" and "MRD of uncertain significance". The quality assessment program and revised guidelines will ensure reproducible and accurate MRD data for ALL patients. Within the Consortium, similar programs and guidelines have been introduced for other lymphoid diseases (e.g., B-cell lymphoma), for new technological platforms (e.g., digital droplet PCR or Next-Generation Sequencing), and for other patient-specific MRD PCR-based targets (e.g., fusion genes).
- MeSH
- akutní lymfatická leukemie genetika diagnóza MeSH
- genová přestavba MeSH
- geny pro imunoglobuliny MeSH
- kvantitativní polymerázová řetězová reakce metody normy MeSH
- lidé MeSH
- reziduální nádor * genetika diagnóza MeSH
- směrnice pro lékařskou praxi jako téma normy MeSH
- zajištění kvality zdravotní péče MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: We aimed to study prognostic factors and efficacy of allogeneic hematopoietic stem-cell transplantation (allo-HSCT) in first remission of patients with noninfant childhood acute lymphoblastic leukemia (ALL) with 11q23/KMT2A rearrangements treated with chemotherapy regimens between 1995 and 2010. PATIENTS AND METHODS: Data were retrospectively retrieved from 629 patients with 11q23/KMT2A-rearranged ALL from 17 members of the Ponte-di-Legno Childhood ALL Working Group. Clinical and biologic characteristics, early response assessed by minimal residual disease at the end of induction (EOI) therapy, and allo-HSCT were analyzed for their impact on outcomes. RESULTS: A specific 11q23/KMT2A translocation partner gene was identified in 84.3% of patients, with the most frequent translocations being t(4;11)(q21;q23) (n = 273; 51.5%), t(11;19)(q23;p13.3) (n = 106; 20.0%), t(9;11)(p21_22;q23) (n = 76; 14.3%), t(6;11)(q27;q23) (n = 20; 3.8%), and t(10;11)(p12;q23) (n = 14; 2.6%); 41 patients (7.7%) had less frequently identified translocation partner genes. Patient characteristics and early response varied among subgroups, indicating large biologic heterogeneity and diversity in therapy sensitivity among 11q23/KMT2A-rearranged ALL. The EOI remission rate was 93.2%, and the 5-year event-free survival (EFS) for the entire cohort was 69.1% ± 1.9%, with a range from 41.7% ± 17.3% for patients with t(9;11)-positive T-ALL (n = 9) and 64.8% ± 3.0% for patients with t(4;11)-positive B-ALL (n = 266) to 91.2% ± 4.9% for patients with t(11;19)-positive T-ALL (n = 34). Low EOI minimal residual disease was associated with favorable EFS, and induction failure was particularly predictive of nonresponse to further therapy and relapse and poor EFS. In addition, EFS was not improved by allo-HSCT compared with chemotherapy only in patients with both t(4;11)-positive B-ALL (n = 64 v 51; P = .10) and 11q23/KMT2A-rearranged T-ALL (n = 16 v 10; P = .69). CONCLUSION: Compared with historical data, prognosis of patients with noninfant 11q23/KMT2A-rearranged ALL has improved, but allo-HSCT failed to affect outcome. Targeted therapies are needed to reduce relapse and treatment-related mortality rates.
- MeSH
- akutní lymfatická leukemie * terapie MeSH
- biologické přípravky * MeSH
- lidé MeSH
- lidské chromozomy, pár 11 MeSH
- lymfoblastická leukemie-lymfom z prekurzorových T-buněk * MeSH
- prognóza MeSH
- recidiva MeSH
- retrospektivní studie MeSH
- reziduální nádor genetika MeSH
- translokace genetická MeSH
- transplantace hematopoetických kmenových buněk * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural 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
Východiska: Maligní lymfomy představují vysoce heterogenní skupinu nádorů s rozmanitým klinickým chováním – od indolentních až po velmi agresivní formy s přežitím v rámci měsíců. Tato onemocnění jsou od počátku považována za systémová, často se vyskytující na několika místech současně. Nicméně diagnóza a přesná klasifikace obvykle probíhá z biopsie jediné patologické uzliny či infiltrátu. Klinické zkušenosti přitom ukazují, že biologické chování lymfomu nemusí být v různých jeho lokalizacích zcela shodné. Ve snaze vyřešit tento problém, ale také zlepšit diagnostiku z obtížně dostupných kompartmentů, je poslední dobou intenzivně zkoumána tzv. volná cirkulující DNA (cfDNA), jejíž součástí je také DNA uvolněná z nádorových buněk – cirkulující nádorová DNA (ctDNA). Tuto DNA lze snadno získat z tekutých biopsií, jako je krev, případně jiné tělní tekutiny. Cíl: Tento článek shrnuje dosavadní poznatky o cfDNA a ctDNA, zejména pak právě v kontextu maligních lymfomů, a nastiňuje potenciální směry jejího budoucího praktického využití. Závěr: Detekce a analýza ctDNA představuje novou modalitu, která může v budoucnu vést ke zkvalitnění všech fází léčby maligních lymfomů od diagnostiky až po sledování tzv. minimální zbytkové choroby.
Background: Malignant lymphomas represent a highly heterogeneous group of tumors with varied clinical behavior – from indolent to very aggressive forms with survival in the order of months. From the very beginning, these diseases are considered systemic, often occurring in several anatomical locations simultaneously. However, diagnosis and exact classification are usually inferred from a biopsy of a single pathological lymph node or infiltrate, even though clinical experience shows that the biological behavior of lymphoma is not necessarily identical across anatomical locations. In an effort to address this issue as well as the problem of biopsy of not easily accessible compartments, circulating free DNA (cfDNA), which contains circulating tumor DNA (ctDNA) released from dead tumor cells, has been extensively studied in recent years. This DNA is easily accessible from liquid biopsies such as blood or other patient‘s bodily fluids. Purpose: This article summarizes current scientific knowledge on cfDNA and ctDNA, particularly in the context of malignant lymphoma, and foreshadows its potential future uses. Conclusion: Detection and analysis of cfDNA represents a new approach that can lead to future improvements in all phases of lymphoma treatment from diagnostics to minimal residual disease monitoring.
- MeSH
- cirkulující nádorová DNA analýza MeSH
- difúzní velkobuněčný B-lymfom diagnóza genetika MeSH
- lidé MeSH
- lymfom diagnóza genetika klasifikace MeSH
- progrese nemoci MeSH
- reziduální nádor diagnóza genetika MeSH
- tekutá biopsie metody MeSH
- volné cirkulující nukleové kyseliny * analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Recently, we defined "CML-like" subtype of BCR::ABL1-positive acute lymphoblastic leukemia (ALL), resembling lymphoid blast crisis of chronic myeloid leukemia (CML). Here we retrospectively analyzed prognostic relevance of minimal residual disease (MRD) and other features in 147 children with BCR::ABL1-positive ALL (diagnosed I/2000-IV/2021, treated according to EsPhALL (n = 133) or other (n = 14) protocols), using DNA-based monitoring of BCR::ABL1 genomic breakpoint and clonal immunoglobulin/T-cell receptor gene rearrangements. Although overall prognosis of CML-like (n = 48) and typical ALL (n = 99) was similar (5-year-EFS 60% and 49%, respectively; 5-year-OS 75% and 73%, respectively), typical ALL presented more relapses while CML-like patients more often died in the first remission. Prognostic role of MRD was significant in the typical ALL (p = 0.0005 in multivariate analysis for EFS). In contrast, in CML-like patients MRD was not significant (p values > 0.2) and inapplicable for therapy adjustment. Moreover, in the typical ALL, risk-prediction could be further improved by considering initial hyperleukocytosis. Early distinguishing typical BCR::ABL1-positive ALL and CML-like patients is essential to enable optimal treatment approach in upcoming protocols. For the typical ALL, tyrosine-kinase inhibitors and concurrent chemotherapy with risk-directed intensity should be recommended; in the CML-like disease, no relevant prognostic feature applicable for therapy tailoring was found so far.
- MeSH
- akutní lymfatická leukemie * genetika farmakoterapie MeSH
- akutní nemoc MeSH
- bcr-abl fúzové proteiny genetika MeSH
- chronická myeloidní leukemie * farmakoterapie genetika MeSH
- dítě MeSH
- lidé MeSH
- retrospektivní studie MeSH
- reziduální nádor genetika MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Publikační typ
- časopisecké články 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.
Článek předkládá vybraná sdělení z Druhé konference translačního výzkumu: Akutní lymfoblastické leukemie pořádané Evropskou hematologickou školou. Zaměřuje se přitom na roli nových technologií molekulární biologie a genetiky v dosažení nedávných výrazných pokroků v pochopení biologie akutní lymfoblastické leukemie a ve zlepšení diagnostiky, prognostiky a řízení léčby tohoto onemocnění.
The article presents selected communications from the 2nd Translational Research Conference: Acute Lymphoblastic Leukaemia organized by the European School of Haematology. It focuses on the role of new molecular biology and genetic technologies in the recent significant advances in our understanding of the biology of acute lymphoblastic leukaemia and in the improvement of disease diagnostics, prognostics and therapy management.
BACKGROUND: NPM1 plasmid standards are required for absolute quantification of minimal residual disease in acute myeloid leukemia patients. The standards are usually obtained, next to commercially constructed gene fragments, from transgenic bacteria colonies. However, this procedure is laborious and very time consuming. METHODS AND RESULTS: We have developed a PCR method that speeds up, simplifies, and streamlines the process of preparing NPM1 plasmid standards. The method is based on a combination of three primers, two surrounding the usual NPM1 mutation position and one over the mutation site. With this method, we were able to clearly distinguish plasmids with at least 15 different NPM1 mutations from the wild-type NPM1 plasmid. CONCLUSIONS: With the new approach, preparing NPM1 plasmid standards is easier, identifying NPM1-positive colonies is possible in less than a day and moreover, for a lower price than commercially constructed gene fragments.
- MeSH
- akutní myeloidní leukemie * genetika MeSH
- jaderné proteiny * genetika MeSH
- lidé MeSH
- mutace genetika MeSH
- nukleofosmin MeSH
- plazmidy genetika MeSH
- reziduální nádor genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
