Monitoring of minimal residual disease (MRD) after hematopoietic stem cell transplantation (HSCT) in patients with acute lymphoblastic leukemia (ALL) is vital for timely therapeutic intervention planning. However, interpreting low-positive results from the current standard method, quantitative PCR (qPCR) of immunoglobulin and T-cell receptor gene rearrangements (IG/TR), poses challenges due to the risk of false positivity caused by non-specific amplification. We aimed to improve MRD detection specificity using the next-generation amplicon sequencing (NGS) of IG/TR rearrangements for better relapse prediction. In pediatric and young adult ALL patients undergoing sequential post-HSCT MRD monitoring, we prospectively re-tested positive non-quantifiable qPCR results with NGS-MRD using the EuroClonality-NGS approach. We were able to confirm 13 out of 47 (27.7%) qPCR positive results using the more specific NGS-MRD method. Out of 10 patients with at least one MRD positivity confirmed by NGS, six relapsed (60%) 1-3.7 months after testing. Among 25 patients with all NGS-MRD results negative, two relapses occurred (8%) after 5.1 and 12.1 months. One-year RFS was 40% versus 96% and 3-year OS was 33.3% versus 94.4% for the NGS-positive and NGS-negative groups, respectively. The difference was not attributable to a varying rate of therapeutic interventions. Six patients out of 14 who had immunosuppressive treatment tapered or received donor lymphocyte infusion in response to MRD positivity developed significant graft versus host disease, leading to one fatality. This underscores the importance of enhancing the post-HSCT relapse risk prediction accuracy through NGS-MRD testing to avoid unnecessary interventions.

• Publication type
• Journal Article MeSH

Proteomics is nowadays increasingly becoming part of the routine clinical practice of diagnostic laboratories, especially due to the advent of advanced mass spectrometry techniques. This review focuses on the application of proteomic analysis in the identification of pathological conditions in a hospital setting, with a particular focus on the analysis of protein biomarkers. In particular, the main purpose of the review is to highlight the challenges associated with the identification of specific disease-causing proteins, given their complex nature and the variety of posttranslational modifications (PTMs) they can undergo. PTMs, such as phosphorylation and glycosylation, play critical roles in protein function but can also lead to diseases if dysregulated. Proteomics plays an important role especially in various medical fields ranging from cardiology, internal medicine to hemato-oncology emphasizing the interdisciplinary nature of this field. Traditional methods such as electrophoretic or immunochemical methods have been mainstay in protein detection; however, these techniques are limited in terms of specificity and sensitivity. Examples include the diagnosis of multiple myeloma and the detection of its specific protein or amyloidosis, which relies heavily on these conventional methods, which sometimes lead to false positives or inadequate disease monitoring. Mass spectrometry in this respect emerges as a superior alternative, providing high sensitivity and specificity in the detection and quantification of specific protein sequences. This technique is particularly beneficial for monitoring minimal residual disease (MRD) in the diagnosis of multiple myeloma where traditional methods fall short. Furthermore mass spectrometry can provide precise typing of amyloid proteins, which is crucial for the appropriate treatment of amyloidosis. This review summarizes the opportunities for proteomic determination using mass spectrometry between 2012 and 2024, highlighting the transformative potential of mass spectrometry in clinical proteomics and encouraging its wider use in diagnostic laboratories.

• Keywords
• amyloidosis, liquid chromatography, mass spectrometry, multiple myeloma, proteomics,
• MeSH
• Amyloidosis * diagnosis   metabolism   MeSH
• Biomarkers analysis   MeSH
• Mass Spectrometry * methods   MeSH
• Humans MeSH
• Multiple Myeloma * diagnosis   metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Proteomics * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Biomarkers MeSH

OBJECTIVES: Minimal residual disease (MRD) status in multiple myeloma (MM) is an important prognostic biomarker. Personalized blood-based targeted mass spectrometry detecting M-proteins (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to MRD-assessment in bone marrow. However, MS-MRD still comprises of manual steps that hamper upscaling of MS-MRD testing. Here, we introduce a proof-of-concept for a novel workflow using data independent acquisition-parallel accumulation and serial fragmentation (dia-PASEF) and automated data processing. METHODS: Using automated data processing of dia-PASEF measurements, we developed a workflow that identified unique targets from MM patient sera and personalized protein sequence databases. We generated patient-specific libraries linked to dia-PASEF methods and subsequently quantitated and reported M-protein concentrations in MM patient follow-up samples. Assay performance of parallel reaction monitoring (prm)-PASEF and dia-PASEF workflows were compared and we tested mixing patient intake sera for multiplexed target selection. RESULTS: No significant differences were observed in lowest detectable concentration, linearity, and slope coefficient when comparing prm-PASEF and dia-PASEF measurements of serial dilutions of patient sera. To improve assay development times, we tested multiplexing patient intake sera for target selection which resulted in the selection of identical clonotypic peptides for both simplex and multiplex dia-PASEF. Furthermore, assay development times improved up to 25× when measuring multiplexed samples for peptide selection compared to simplex. CONCLUSIONS: Dia-PASEF technology combined with automated data processing and multiplexed target selection facilitated the development of a faster MS-MRD workflow which benefits upscaling and is an important step towards the clinical implementation of MS-MRD.

• Keywords
• M-protein, data independent acquisition, mass spectrometry, multiple myeloma, quantitative biomarker,
• MeSH
• Automation MeSH
• Precision Medicine methods   MeSH
• Humans MeSH
• Multiple Myeloma * diagnosis   blood   MeSH
• Workflow * MeSH
• Neoplasm, Residual * diagnosis   MeSH
• High-Throughput Screening Assays methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

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
• Leukemia, Myeloid, Acute * diagnosis   genetics   therapy   MeSH
• Child MeSH
• Genomics MeSH
• Cohort Studies MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• Prognosis MeSH
• Flow Cytometry MeSH
• Recurrence MeSH
• Neoplasm, Residual diagnosis   genetics   MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA, Messenger 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.

• Keywords
• CD48, CD99, T-lineage acute lymphoblastic leukemia, feature importance, flow cytometry, minimal residual disease, support vector machine,
• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma * diagnosis   MeSH
• Child MeSH
• Adult MeSH
• Humans MeSH
• Precursor T-Cell Lymphoblastic Leukemia-Lymphoma * diagnosis   MeSH
• Flow Cytometry MeSH
• Neoplasm, Residual diagnosis   MeSH
• T-Lymphocytes MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Pediatric-inspired protocols with prospective monitoring of minimal residual disease (MRD) are considered the standard of intensive treatment for adults with acute lymphoblastic leukemia (ALL). They have been used in the Czech Republic since 2007. PATIENTS AND METHODS: Two hundred and ninety-seven patients aged 18-65 years were treated at five hematology centers between 2007-2020 according to the GMALL 07/2003 protocol. This is a retrospective analysis of their treatment outcomes. RESULTS: In the Ph-negative cohort, 189 (93.1%) patients achieved complete remission, 5 (2.4%) patients were refractory, and early mortality was 3.0%. Seventy (34.5%) patients experienced relapse in a median of 10.6 months. Overall survival (OS) at 3 and 5 years was 63.5% and 55.9%, disease-free survival (DFS) at 3 and 5 years was 54.5% and 49.7%, respectively. Young adults under 35 years of age (P = 0.015), patients without initial CNS infiltration (P = 0.016), with MRD negativity before consolidation treatment (P < 0.001), transplanted in the 1st complete remission (P < 0.001), and subjects treated after 2012 (P = 0.05) had significantly better overall survival. In a multivariate analysis, MRD at week 11 was the only independent factor affecting OS (HR 3.06; P = 0.006). For DFS, baseline CNS infiltration (HR 2.08; P = 0.038) and MRD at week 11 (HR 2.15; P = 0.020) were significant. In the Ph-positive cohort, 84 (89.4%) patients achieved complete remission, 1 (1.0%) patient was refractory, early mortality was 4.3%. Twenty-six (27.7%) patients relapsed in a median of 8.6 months. Survival at 3 and 5 years was 57.2% and 52.4% for OS and 50.2% and 44.9% for DFS, respectively. Transplanted patients and patients diagnosed after 2012 had statistically better overall survival (P < 0.001). CONCLUSION: The introduction of pediatric-inspired protocols with treatment intensification according to MRD levels resulted in a significant improvement in the survival outcomes of adult patients with ALL.

• Keywords
• acute lymphoblastic leukemia, hematopoietic stem cell transplantation, minimal residual disease, pediatric-inspired protocol, treatment, type 2 diabetes,
• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma * therapy   MeSH
• Adult MeSH
• Humans MeSH
• Young Adult MeSH
• Disease-Free Survival MeSH
• Prospective Studies MeSH
• Retrospective Studies MeSH
• Neoplasm, Residual diagnosis   MeSH
• Hematopoietic Stem Cell Transplantation * MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Young Adult MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic epidemiology   MeSH

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5'-KMT2A, two patients had a 5'-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma * genetics   MeSH
• Leukemia, Myeloid, Acute * genetics   MeSH
• Child MeSH
• Adult MeSH
• Gene Fusion MeSH
• Histone-Lysine N-Methyltransferase * genetics   MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Child, Preschool MeSH
• Myeloid-Lymphoid Leukemia Protein * genetics   MeSH
• Aged MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Child, Preschool MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Histone-Lysine N-Methyltransferase * MeSH
• KMT2A protein, human MeSH Browser
• Myeloid-Lymphoid Leukemia Protein * MeSH

INTRODUCTION: The malignant transformation leading to a maturation arrest in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occurs early in B-cell development, in a pro-B or pre-B cell, when somatic recombination of variable (V), diversity (D), and joining (J) segment immunoglobulin (IG) genes and the B-cell rescue mechanism of VH replacement might be ongoing or fully active, driving clonal evolution. In this study of newly diagnosed BCP-ALL, we sought to understand the mechanistic details of oligoclonal composition of the leukemia at diagnosis, clonal evolution during follow-up, and clonal distribution in different hematopoietic compartments. METHODS: Utilizing high-throughput sequencing assays and bespoke bioinformatics we identified BCP-ALL-derived clonally-related IGH sequences by their shared 'DNJ-stem'. RESULTS: We introduce the concept of 'marker DNJ-stem' to cover the entirety of, even lowly abundant, clonally-related family members. In a cohort of 280 adult patients with BCP-ALL, IGH clonal evolution at diagnosis was identified in one-third of patients. The phenomenon was linked to contemporaneous recombinant and editing activity driven by aberrant ongoing DH/VH-DJH recombination and VH replacement, and we share insights and examples for both. Furthermore, in a subset of 167 patients with molecular subtype allocation, high prevalence and high degree of clonal evolution driven by ongoing DH/VH-DJH recombination were associated with the presence of KMT2A gene rearrangements, while VH replacements occurred more frequently in Ph-like and DUX4 BCP-ALL. Analysis of 46 matched diagnostic bone marrow and peripheral blood samples showed a comparable clonal and clonotypic distribution in both hematopoietic compartments, but the clonotypic composition markedly changed in longitudinal follow-up analysis in select cases. Thus, finally, we present cases where the specific dynamics of clonal evolution have implications for both the initial marker identification and the MRD monitoring in follow-up samples. DISCUSSION: Consequently, we suggest to follow the marker DNJ-stem (capturing all family members) rather than specific clonotypes as the MRD target, as well as to follow both VDJH and DJH family members since their respective kinetics are not always parallel. Our study further highlights the intricacy, importance, and present and future challenges of IGH clonal evolution in BCP-ALL.

• Keywords
• DNJ-stem, IGH rearrangements, VH replacement, acute lymphoblastic leukemia, clonal evolution, high-throughput sequencing, minimal residual disease,
• MeSH
• Burkitt Lymphoma * genetics   MeSH
• Adult MeSH
• Genes, Immunoglobulin MeSH
• Bone Marrow pathology   MeSH
• Humans MeSH
• Polymerase Chain Reaction MeSH
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma * diagnosis   genetics   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• Publication type
• Journal Article 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.

• Keywords
• Acute lymphoblastic leukemia, Minimal residual disease, Next-generation sequencing, PCR, Quality control,
• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma * diagnosis   genetics   MeSH
• Biomarkers MeSH
• Immunoglobulins genetics   MeSH
• Humans MeSH
• Neoplasm, Residual diagnosis   genetics   MeSH
• Quality Control MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Biomarkers MeSH
• Immunoglobulins MeSH