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

Recently, we described B-cell precursor acute lymphoblastic leukemia (BCP-ALL) subtype with early switch to the monocytic lineage and loss of the B-cell immunophenotype, including CD19 expression. Thus far, the genetic background has remained unknown. Among 726 children consecutively diagnosed with BCP-ALL, 8% patients experienced switch detectable by flow cytometry (FC). Using exome and RNA sequencing, switch was found to positively correlate with three different genetic subtypes: PAX5-P80R mutation (5 cases with switch out of 5), rearranged DUX4 (DUX4r; 30 cases of 41) and rearranged ZNF384 (ZNF384r; 4 cases of 10). Expression profiles or phenotypic patterns correlated with genotypes, but within each genotype they could not identify cases who subsequently switched. If switching was not taken into account, the B-cell-oriented FC assessment underestimated the minimal residual disease level. For patients with PAX5-P80R, a discordance between FC-determined and PCR-determined MRD was found on day 15, resulting from a rapid loss of the B-cell phenotype. Discordance on day 33 was observed in all the DUX4r, PAX5-P80R and ZNF384r subtypes. Importantly, despite the substantial phenotypic changes, possibly even challenging the appropriateness of BCP-ALL therapy, the monocytic switch was not associated with a higher incidence of relapse and poorer prognosis in patients undergoing standard ALL treatment.

• MeSH
• PAX5 Transcription Factor genetics   MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma * MeSH
• B-Lymphocytes MeSH
• Immunophenotyping MeSH
• Humans MeSH
• Mutation MeSH
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma * diagnosis   genetics   MeSH
• Neoplasm, Residual MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• PAX5 Transcription Factor MeSH
• PAX5 protein, human MeSH Browser

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma diagnosis   genetics   metabolism   MeSH
• Antigens, Differentiation genetics   metabolism   MeSH
• Gene Expression MeSH
• Gene Rearrangement * MeSH
• Homeodomain Proteins genetics   MeSH
• Humans MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Biomarkers, Tumor * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Differentiation MeSH
• DUX4L1 protein, human MeSH Browser
• Homeodomain Proteins MeSH
• Membrane Proteins MeSH
• Biomarkers, Tumor * MeSH

ERG-deletions occur recurrently in acute lymphoblastic leukemia, especially in the DUX4-rearranged subtype. The ERG-deletion was shown to positively impact prognosis of patients with IKZF1-deletion and its presence precludes assignment into IKZF1 plus group, a novel high-risk category on AIEOP-BFM ALL trials. We analyzed the impact of different methods on ERG-deletion detection rate, evaluated ERG-deletion as a potential marker for DUX4-rearranged leukemia, studied its associations with molecular and clinical characteristics within this leukemia subtype, and analyzed its clonality. Using single-nucleotide-polymorphism array, genomic polymerase chain reaction (PCR) and amplicon-sequencing we found ERG-deletion in 34% (16 of 47), 66% (33 of 50) and 78% (39 of 50) of DUX4-rearranged leukemia, respectively. False negativity of ERG-deletion by single-nucleotide-polymorphism array caused IKZF1 plus misclassification in 5 patients. No ERG-deletion was found outside the DUX4-rearranged cases. Within DUX4-rearranged leukemia, the ERG-deletion was associated with higher total number of copy-number aberrations, and, importantly, the ERG-deletion positivity by PCR was associated with better outcome [5-year event-free survival (EFS), ERG-deletion-positive 93% vs. ERG-deletion-negative 68%, P=0.022; 5-year overall survival (OS), ERG-deletion-positive 97% vs. ERG-deletion-negative 75%, P=0.029]. Ultra-deep amplicon-sequencing revealed distinct co-existing ERG-deletions in 22 of 24 patients. In conclusion, our data demonstrate inadequate sensitivity of single-nucleotide-polymorphism array for ERG-deletion detection, unacceptable for proper IKZF1 plus classification. Even using more sensitive methods (PCR/amplicon-sequencing) for its detection, ERG-deletion is absent in 22-34% of DUX4-rearranged leukemia and does not represent an adequately sensitive marker of this leukemia subtype. Importantly, the ERG-deletion potentially stratifies the DUX4-rearranged leukemia into biologically/clinically distinct subsets. Frequent polyclonal pattern of ERG-deletions shows that late origin of this lesion is more common than has been previously described.

• MeSH
• Gene Deletion * MeSH
• Child MeSH
• Gene Rearrangement * MeSH
• Homeodomain Proteins genetics   MeSH
• Infant MeSH
• Humans MeSH
• Survival Rate MeSH
• Adolescent MeSH
• Biomarkers, Tumor genetics   MeSH
• Follow-Up Studies MeSH
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma classification   diagnosis   genetics   MeSH
• Child, Preschool MeSH
• Prognosis MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Retrospective Studies MeSH
• Transcriptional Regulator ERG genetics   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DUX4L1 protein, human MeSH Browser
• ERG protein, human MeSH Browser
• Homeodomain Proteins MeSH
• Biomarkers, Tumor MeSH
• Transcriptional Regulator ERG MeSH

Traditionally, genetic abnormalities detected by conventional karyotyping, fluorescence in situ hybridization, and polymerase chain reaction divided childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) into well-established genetic subtypes. This genetic classification has been prognostically relevant and thus used for the risk stratification of therapy. Recently, the introduction of genome-wide approaches, including massive parallel sequencing methods (whole-genome, -exome, and -transcriptome sequencing), enabled extensive genomic studies which, together with gene expression profiling, largely expanded our understanding of leukemia pathogenesis and its heterogeneity. Novel BCP-ALL subtypes have been described. Exact identification of recurrent genetic alterations and their combinations facilitates more precise risk stratification of patients. Discovery of targetable lesions in subsets of patients enables the introduction of new treatment modalities into clinical practice and stimulates the transfer of modern methods from research laboratories to routine practice.

• Keywords
• acute lymphoblastic leukemia, children, massive parallel sequencing, new BCP-ALL subtypes,
• MeSH
• Acute Disease MeSH
• Child MeSH
• Exome genetics   MeSH
• Genomics methods   MeSH
• Risk Assessment methods   MeSH
• Humans MeSH
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma classification   genetics   MeSH
• Gene Expression Profiling MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma pathology   MeSH
• Leukemia, Myeloid, Acute pathology   MeSH
• Acute Disease MeSH
• Child MeSH
• Adult MeSH
• Immunophenotyping methods   MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Antigens, CD19 immunology   MeSH
• Child MeSH
• Infant MeSH
• Humans MeSH
• Neoplasm Recurrence, Local immunology   pathology   MeSH
• Adolescent MeSH
• Biomarkers, Tumor immunology   MeSH
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma drug therapy   immunology   pathology   MeSH
• Child, Preschool MeSH
• Antibodies, Bispecific therapeutic use   MeSH
• Antineoplastic Agents therapeutic use   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, CD19 MeSH
• blinatumomab MeSH Browser
• Biomarkers, Tumor MeSH
• Antibodies, Bispecific MeSH
• Antineoplastic Agents MeSH

Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. Despite enormous improvement of prognosis during the last half century, ALL remains a major cause of childhood cancer-related mortality. During the past decade, whole genomic methods have enhanced our knowledge of disease biology. Stratification of therapy according to early treatment response measured by minimal residual disease allows risk group assignment into different treatment arms, ranging from reduction to intensification of treatment. Progress has been achieved in academic clinical trials by optimization of combined chemotherapy, which continues to be the mainstay of contemporary treatment. The availability of suitable volunteer main histocompatibility antigen-matched unrelated donors has increased the rates of hematopoietic stem cell transplantation (HSCT) over the past two decades. Allogeneic HSCT has become an alternative treatment for selected, very-high-risk patients. However, intensive treatment burdens children with severe acute toxic effects that can cause permanent organ damage and even toxic death. Immunotherapeutic approaches have recently come to the forefront in ALL therapy. Monoclonal antibodies blinatumomab and inotuzumab ozogamicin as well as gene-modified T cells directed to specific target antigens have shown efficacy against resistant/relapsed leukemia in phase I/II studies. Integration of these newer modalities into combined regimens with chemotherapy may rescue a subset of children not curable by contemporary therapy. Another major challenge will be to incorporate less toxic regimens into the therapy of patients with low-risk disease who have a nearly 100% chance of being cured, and the ultimate goal is to improve their quality of life while maintaining a high cure rate.

• Keywords
• ALL, HSCT, immunotherapy, leukaemia, monoclonal antibodies, paediatric,
• Publication type
• Journal Article MeSH
• Review MeSH

• MeSH
• B-Lymphocytes metabolism   MeSH
• Philadelphia Chromosome * MeSH
• Humans MeSH
• Macrophages cytology   MeSH
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Letter MeSH
• Comment MeSH