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Acute B lymphoblastic leukemia (B-ALL) is one of the most common types of childhood cancer worldwide and chemotherapy is the main treatment approach. Despite good response rates to chemotherapy regiments, many patients eventually relapse and minimal residual disease (MRD) is the leading risk factor for relapse. The evolution of leukemic clones duri...
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... studied 51 childhood B-ALL patients diagnosed in Shenzhen Children's Hospital. BM specimens and (or) PB were obtained at diagnosis and during treatment, and in total 169 specimen were collected (Table S1 in Supplementary Material). The study was carried out in accordance with the recommendations of Declaration of Helsinki and was approved by BGI-IRB. ...
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... randomly, without any prior knowledge of the clonal IGH rearrangement existence, collected 51 pediatric B-ALL patients in total. According to the disease risk stratification criteria, 19 of them were classified as SR, 18 as IR, and 14 as HR (Table S1 in Supplementary Material, details in the section "Materials and Methods"). The characteristics and clinical information of the patients, including diagnostic age, gender, and cytogenetics were collected and listed in Table S1 in Supplementary Material. ...
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... to the disease risk stratification criteria, 19 of them were classified as SR, 18 as IR, and 14 as HR (Table S1 in Supplementary Material, details in the section "Materials and Methods"). The characteristics and clinical information of the patients, including diagnostic age, gender, and cytogenetics were collected and listed in Table S1 in Supplementary Material. Ninety-six percent of patients (49/51) were aged between 1 and 10, and 2 patients were more than 10 years old. ...
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... to the definition of leukemic clones (see Materials and Methods), we identified leukemic IGH CDR3s with com- plete V-D-J rearrangement in a frequency of above 10% in the diagnostic BM samples in 47 of 51 patients (92.2%). Of the 47 patients with leukemic IGH clones, 24 had two or more leukemic clones (Table 1; Table S2 in Supplementary Material). Therefore, we identified 77 leukemic clones from the 47 patients in total (Table S2 in Supplementary Material). ...
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... BM aspiration can be painful, using PB instead of BM was evaluated for MRD detection. For 15 patients, we collected both diagnostic BM and PB samples (Table S1 in Supplementary Material). We investigated if the leukemic clones identified in BM also existed in PB with high frequencies. ...
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... test MRD detection in PB specimens and to compare it with BM, post-treatment BM and PB samples were collected simultaneously in 15 pairs from 11 patients (Table S1 in Supplementary Material). We determined the frequencies of leukemic clones using the same method. ...
Citations
... Therefore, it does not require patient-specific customisation and can detect clonal evolution during treatment. Many studies confirmed its high sensitivity, reliability in clinical settings, and universal applicability [11][12][13][14][15][16]. In patients with B-ALL who achieved MRD negativity after induction chemotherapy, NGS detected positive MRD conversion earlier than MFC, with conversion being detected a median of 4.7 months before clinical relapse [10]. ...
... NGS methodology has several notable advantages, including high sensitivity, broad applicability across diverse patient populations, elimination of the need for patientspecific oligonucleotide primers, and the ability to detect both oligoclonality and clonal evolution dynamics over time. It also offers the practical benefit of obviating the need for freshly acquired patient samples, in contrast to conventional methods such as flow cytometry [11][12][13][14][15][16]. In addition, NGS MRD has shown improvement in measurement precision, and prediction of relapse and survival outcomes of post-treatment MRD in pediatric B-ALL compared to MFC and PCR [11,18,19]. ...
Background
Assessment of measurable residual disease (MRD) is an essential prognostic tool for B-lymphoblastic leukaemia (B-ALL). In this study, we evaluated the utility of next-generation sequencing (NGS)–based MRD assessment in real-world clinical practice.
Method
The study included 93 paediatric patients with B-ALL treated at our institution between January 2017 and June 2022. Clonality for IGH or IGK rearrangements was identified in most bone marrow samples (91/93, 97.8%) obtained at diagnosis.
Results
In 421 monitoring samples, concordance was 74.8% between NGS and multiparameter flow cytometry and 70.7% between NGS and reverse transcription-PCR. Elevated quantities of clones of IGH alone (P < 0.001; hazard ratio [HR], 22.2; 95% confidence interval [CI], 7.1–69.1), IGK alone (P = 0.011; HR, 5.8; 95% CI, 1.5–22.5), and IGH or IGK (P < 0.001; HR, 7.2; 95% CI, 2.6–20.0) were associated with an increased risk of relapse. Detection of new clone(s) in NGS was also associated with inferior relapse-free survival (P < 0.001; HR, 18.1; 95% CI, 3.0–108.6). Multivariable analysis confirmed age at diagnosis, BCR::ABL1-like mutation, TCF3::PBX1 mutation, and increased quantity of IGH or IGK clones during monitoring as unfavourable factors.
Conclusion
In conclusion, this study highlights the usefulness of NGS-based MRD as a routine assessment tool for prognostication of paediatric patients with B-ALL.
... Adverse circumstances for MRD assessment are clonal selection and clonal evolution, since the associated index might be missed. Potentially impacted are leukemia with initial oligoclonality as observed in approximately 15% of B-ALL, and up to 1000 subclones have been reported (Wu et al. 2016). Phenotypic plasticity under treatment and massive lymphocyte regeneration can cause false negativity or positivity, a solvable problem by applying mentioned high-throughput methodologies. ...
In ALL evaluation of molecular treatment response, assessment of minimal residual disease, nowadays named measurable residual disease (MRD), is a substantial independent predictor of outcome, as proven by randomized studies (Conter et al. 2010; Gökbuget et al. 2012; Bassan and Spinelli 2015). Consequently, MRD is implemented in virtually all clinical protocols in order to supplement or to redefine multifactorial risk stratification with optional customized treatment intensity. The detection of leukemic cells below the limit of classical cytomorphology is feasible by either disease-specific alterations of the immune phenotype or unique genetic features. Several competing and complementing MRD methods have been developed with preference application according to clinical protocols (Van der Velden et al. 2007; van Dongen et al. 2015).
... The clinical consequence to be avoided regarding clonal evolution is the absence of the diagnostic rearrangement at relapse. The use of HTS, by enabling the detection and tracking of multiple clones, has to potential to circumvent this problem.58 Several studies, most retrospective, have suggested that HTSbased assays have an increased sensitivity compared to MFC and qPCR and could potentially lead to improved risk stratification and better outcomes in ALL,51,53,59 what need to be confirmed in more prospective comparative studies. ...
... El término de enfermedad mínima residual (EMR) (MRD, por sus siglas en inglés) surgió en la década de 1980, cuando se usó por primera vez para su detección la microscopia de inmunof luorescencia, que permitió el monitoreo de disminución y recrecimiento de células malignas en pacientes diagnosticados con leucemia linfoblástica aguda (LLA), este reconocimiento de células emergentes hace referencia a la existencia de blastos leucémicos menores al 5 % de forma residual no detectables por técnicas rutinarias en médula ósea pero con una recuperación completa en sangre periférica (9, 15,16). El desarrollo de nuevas técnicas como la CMF, hace posible la identificación de esta población aberrante con una sensibilidad de 10-3 a 10-5 y una aplicabilidad >90% tanto en el linaje B (LLA-B) y linaje T (LLA-T), ayudando a establecer la etapa de diferenciación/madurez celular y dando lugar la clasificación o estratificación de riesgo de recaída como bajo (0 %), riesgo normal (47 %) y riesgo alto (94 %), donde la persistencia >10 -4 de linfoblastos neoplásicos hace referencia a enfermedad mínima residual (EMR) positivo (9, [16][17][18][19]. ...
... La muestra de médula ósea (MO) es la más utilizada para el diagnóstico, clasificación y detección de la EMR en la LLA (10, 16, 17, 25-27, 31); sin embargo, estudios realizados usando sangre periférica, lo cual, demostraron que los niveles de EMR en sangre en pacientes diagnosticados con LLA-T eran compatibles o 1 log más bajo que en MO y en pacientes con LLA-B se detectaron en sangre 1 a 3 log más bajos que en MO (16,17,(31)(32)(33). En consecuencia, lo más conveniente para monitorear la enfer-medad mínima residual independiente del origen T o B, es la muestra de médula ósea o aspirado medular, donde se recomienda recolectar un volumen entre 2 a 5 mL para examinar >5x10 6 células por citometría de flujo y obtener un rango de sensibilidad 10 -4 células blásticas aberrantes (16,34). ...
Antecedentes. La citometría de flujo (CMF) es una técnica que permite el análisis multiparamétrico de poblaciones celulares, siendo esencial en la investigación biomédica y como herramienta diagnóstica. Esta técnica rápida tiene una alta sensibilidad, evaluandocaracterísticas en la población de interés como es el caso del tamaño, granularidad, complejidad del citoplasma celular y proteínas de que permiten la clasificación fenotípica y funcional de un gran número de células. Por estas razones, esta técnica ha adquiridoimportancia en el diagnóstico y seguimiento de enfermedades y anomalías hematológicas, como leucemias, síndromes mielodisplásicos y síndromes mieloproliferativos, entre otras. Objetivo. La presente revisión se enfoca en los avances en la implementación de la CMF en la Enfermedad Mínima Residual (EMR) presente en la Leucemia Linfoblástica Aguda (LLA), la cual es una población mínima leucémica que se detecta en un paciente después de suministrar un tratamiento oncológico, donde se evalúa su eficacia, el riesgo de una recaída y el proceso de remisión completa. Metodología. Se realiza una revisión no sistemática deliteratura en bases de datos, de los últimos 15 años, donde evalúen las implicaciones del uso de citometría de flujo en la EMR, de esta revisión se extraen aspectos relevantes al momento de emplear la CMF para el diagnóstico y seguimiento de pacientes con leucemias. Resultados. La CMF es una técnica muy versátil e importante para el diagnóstico y seguimiento de la EMR por su alta sensibilidad para la detección de bajos números de células resistentes a la terapia. Adicionalmente se muestra la importancia de la estandarización de protocolos como EUROFLOW para un adecuado procesamiento y análisis clínico de las muestras de pacientes.
... 45 Unlike ASO-qPCR, NGS-based IG/TR MRD is not clone-biased, meaning that new clones may be identified as they emerge in remission and relapse samples if the necessary clonal evolution and new clone identifica-tion tools are included in the assessment. 46 The sensitivity of NGS MRD approximates 0.0001% (10 -6 ) in the bone marrow; 47 however, the significance of MRD below the traditional threshold of 10 -4 has not been defined across different clinical scenarios. Similar to flow MRD, the sensitivity of NGS MRD depends on the amount of input, which is the amount of DNA in this method. ...
Measurable residual disease (MRD) is the most powerful independent predictor of risk of relapse and long-term survival in adults and children with acute lymphoblastic leukemia (ALL). For almost all patients with ALL there is a reliable method to evaluate MRD, which can be done using multi-color flow cytometry, quantitative polymerase chain reaction to detect specific fusion transcripts or immunoglobulin/T-cell receptor gene rearrangements, and high-throughput next-generation sequencing. While next-generation sequencing-based MRD detection has been increasingly utilized in clinical practice due to its high sensitivity, the clinical significance of very low MRD levels (<10-4) is not fully characterized. Several new immunotherapy approaches including blinatumomab, inotuzumab ozogamicin, and chimeric antigen receptor T-cell therapies have demonstrated efficacy in eradicating MRD in patients with B-ALL. However, new approaches to target MRD in patients with T-ALL remain an unmet need. As our MRD detection assays become more sensitive and expanding novel therapeutics enter clinical development, the future of ALL therapy will increasingly utilize MRD as a criterion to either intensify or modify therapy to prevent relapse or de-escalate therapy to reduce treatment-related morbidity and mortality.
... MN is an immune-related disease. If the treatment is effective, the immune status of the human body will be different from that before treatment, and the IGH repertoire will also be changed significantly, which has been reported in numerous studies (39)(40)(41). In this study, we found the frequency of IGHV3-66 was significantly lower in the post-therapy patients than in the pretherapy patients, and some IGHV genes skewed in MN patients were also significantly different between the CR and non-CR groups. ...
Membranous nephropathy (MN), an autoimmune glomerular disease, is one of the most common causes of nephrotic syndrome in adults. In current clinical practice, the diagnosis is dependent on renal tissue biopsy. A new method for diagnosis and prognosis surveillance is urgently needed for patients. In the present study, we recruited 66 MN patients before any treatment and 11 healthy control (HC) and analyzed multiple aspects of the immunoglobulin heavy chain (IGH) repertoire of these samples using high-throughput sequencing. We found that the abnormalities of CDR-H3 length, hydrophobicity, somatic hypermutation (SHM), and germ line index were progressively more prominent in patients with MN, and the frequency of IGHV3-66 in post-therapy patients was significantly lower than that in pre-therapy patients. Moreover, we found that the IGHV3-38 gene was significantly related to PLA2R, which is the most commonly used biomarker. The most important discovery was that several IGHV, IGHD transcripts, CDR-H3 length, and SHM rate in pre-therapy patients had the potential to predict the therapeutic effect. Our study further demonstrated that the IGH repertoire could be a potential biomarker for prognosis prediction of MN. The landscape of circulating B-lymphocyte repertoires sheds new light on the detection and surveillance of MN.
... Another study showed that while the leukemic clone could be identified in both DNA and RNA, the transcriptional clonal frequencies were generally lower [56]. Caution is warranted because not all rearrangements are transcribed, as shown by several studies [57][58][59]. ...
Malignant lymphoproliferative disorders collectively constitute a large fraction of the hematological cancers, ranging from indolent to highly aggressive neoplasms. Being a diagnostically important hallmark, clonal gene rearrangements of the immunoglobulins enable the detection of residual disease in the clinical course of the patients down to a minute fraction of malignant cells. The introduction of next-generation sequencing has provided unprecedented assay specificity, with a sensitivity matching that of PCR-based measurable residual disease (MRD) detection down to the 10⁻⁶ level. While reaching 10⁻⁶–10⁻⁷ is theoretically feasible, employing a sufficient amount of DNA and sequencing coverage is put in the perspective of the practical challenges when relying on clinical samples in contrast to controlled serial dilutions. As we discuss, the randomness of subsampling must be taken into account to accommodate the sensitivity threshold – both in terms of the required number of cells and sequencing coverage. As a substantial part of the reviewed studies do not state the depth of coverage or even amount of DNA in some cases, we call for increased transparency in order to enable critical assessment of the MRD assays for clinical implementation and feasibility.
... The clinical consequence to be avoided regarding clonal evolution is the absence of the diagnostic rearrangement at relapse. The use of HTS, by enabling the detection and tracking of multiple clones, has to potential to circumvent this problem.58 Several studies, most retrospective, have suggested that HTSbased assays have an increased sensitivity compared to MFC and qPCR and could potentially lead to improved risk stratification and better outcomes in ALL,51,53,59 what need to be confirmed in more prospective comparative studies. ...
Minimal Residual Disease (MRD) is the most important independent prognostic factor in acute lymphoblastic leukemia (ALL) and refers to the deep level of measurable disease in cases with complete remission by conventional pathologic analysis, especially by cytomorphology. MRD can be detected by multiparametric flow cytometry, molecular approaches such as quantitative polymerase chain reaction for immunoglobulin and T‐cell receptor (IG/TR) gene rearrangements or fusion genes transcript, and high‐throughput sequencing for IG/TR. Despite the proven clinical usefulness in detecting MRD, these methods have differences in sensitivity, specificity, applicability, turnaround time and cost. Knowing and understanding these differences, as well as the principles and limitations of each technology, is essential to laboratory standardization and correct interpretation of MRD results in line with treatment time points, therapeutic settings, and clinical trials. Here, we review the methodological approaches to measure MRD in ALL and discuss the advantages and limitations of the most commonly used techniques.
... 51106). The minimal residual disease (MRD) detection in T-ALL patients was conducted following the method introduced in our previous paper (40). All the TRB clonotypes in this paper refer to the CDR3 sequences. ...
... The feasibility of immune repertoire sequencing technologies in MRD detection for blood cancer has been demonstrated in many studies (17,40,43,44). Wood et al. (44) also reported that the patients with an MRD level of 0.01% by clonoSEQ, but negative by FC, had worse EFS compared with the patients who had a negative MRD by clonoSEQ (44). ...
Accurate T cell receptor repertoire profiling has provided novel biological and clinical insights in widespread immunological settings; however, there is a lack of reference materials in the community that can be used to calibrate and optimize the various experimental systems in different laboratories. In this study, we designed and synthesized 611 T cell receptor (TCR) beta chain (TRB) templates and used them as reference materials to optimize the multiplex PCR experimental system to enrich the TRB repertoire. We assessed the stability of the optimized system by repeating the experiments in different batches and by remixing the TRB templates in different ratios. These TRB reference materials could be used as independent positive controls to assess the accuracy of the experimental system, and they can also be used as spike-in materials to calibrate the residual biases of the experimental system. We then used the optimized system to detect the minimal residual disease of T cell acute lymphoblastic leukemia and showed a higher sensitivity compared with flow cytometry. We also interrogated how chemotherapy affected the TCR repertoire of patients with B-cell acute lymphoblastic leukemia. Our result shows that high-avidity T cells, such as those targeting known pathogens, are largely selected during chemotherapy, despite the global immunosuppression. These T cells were stimulated and emerged at the time of induction treatment and further expanded during consolidation treatment, possibly to fight against infections. These data demonstrate that accurate immune repertoire information can improve our understanding of the adaptive immunity in leukemia and lead to better treatment management of the patients.
... Several groups have shown the value of NGS technologies for MRD detection in precursor and mature B-cell tumors [13][14][15][16][17][18][56][57][58]. NGS can be used to detect clone-specific IG/TCR index sequences; clonal sequences detected at diagnosis can be re-detected and quantified in each follow-up sample. ...
Minimal residual disease (MRD) monitoring has proven to be one of the fundamental independent prognostic factors for patients with acute lymphoblastic leukemia (ALL). Sequential monitoring of MRD using sensitive and specific methods, such as real-time quantitative polymerase chain reaction (qPCR) or flow cytometry (FCM), has improved the assessment of treatment response and is currently used for therapeutic stratification and early detection. Although both FCM and qPCR yield highly consistent results with sensitivities of 10‒4, each method has several limitations. For example, qPCR is time-consuming and laborious: designing primers that correspond to the immunoglobulin (IG) and T-cell receptor (TCR) gene rearrangements at diagnosis can take 3‒4 weeks. In addition, the evolution of additional clones beyond the first or index clone during therapy cannot be detected, which might lead to false-negative results. FCM requires experienced technicians and sometimes does not achieve a sensitivity of 10‒4. Accordingly, a next generation sequencing (NGS)-based method has been developed in an attempt to overcome these limitations. With the advent of high-throughput NGS technologies, a more in-depth analysis of IG and/or TCR gene rearrangements is now within reach, which impacts all applications of IG/TR analysis. However, standardization, quality control, and validation of this new technology are warranted prior to its incorporation into routine practice.
