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Objective interpretation of FC results may still be hampered by limited technical stan-dardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients’ samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA-vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B-and T-cells (but not on leukemic blasts, clonal B-and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein.

Introduction: One-quarter of the relapses in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occur very early (within 18 months, before completion of treatment), and prognosis in these patients is worse compared to cases that relapse after treatment has ended. Methods: In this study, we performed a genomic analysis of diagnosis–relapse pairs of 12 children who relapsed very early, followed by a deep-sequencing validation of all identified mutations. In addition, we included one case with a good initial treatment response and on-treatment relapse at the end of upfront therapy. Results: We observed a dynamic clonal evolution in all cases, with relapse almost exclusively originating from a subclone at diagnosis. We identified several driver mutations that may have influenced the outgrowth of a minor clone at diagnosis to become the major clone at relapse. For example, a minimal residual disease (MRD)-based standard-risk patient with ETV6-RUNX1-positive leukemia developed a relapse from a TP53-mutated subclone after loss of the wildtype allele. Furthermore, two patients with TCF3-PBX1-positive leukemia that developed a very early relapse carried E1099K WHSC1 mutations at diagnosis, a hotspot mutation that was recurrently encountered in other very early TCF3-PBX1-positive leukemia relapses as well. In addition to alterations in known relapse drivers, we found two cases with truncating mutations in the cohesin gene RAD21. Conclusion: Comprehensive genomic characterization of diagnosis–relapse pairs shows that very early relapses in BCP-ALL frequently arise from minor subclones at diagnosis. A detailed understanding of the therapeutic pressure driving these events may aid the development of improved therapies.

01 september 2023, Item does not contain fulltext

Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukemia (BCP-ALL) carries an immunoglobulin-MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukemia and use of individualized treatment schedules of unproven efficacy. Here we compare the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered in a national BCP-ALL clinical trial/registry. When present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analyses. The outcomes analyzed were 3-year event-free survival and overall survival. IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either established BCP-ALL-specific abnormalities (high hyperdiploidy, n=3; KMT2A-rearrangement, n=6; iAMP21, n=1; BCR-ABL1, n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J-rearranged cases, clearly distinct from Burkitt leukemia/lymphoma. Children with IG-MYC-r within that subgroup had a 3-year event-free survival of 47% and overall survival of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this group of patients must be allowed access to contemporary, minimal residual disease-adapted, prospective clinical trial protocols.

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.

Item does not contain fulltext, INTRODUCTION: One-quarter of the relapses in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occur very early (within 18 months, before completion of treatment), and prognosis in these patients is worse compared to cases that relapse after treatment has ended. METHODS: In this study, we performed a genomic analysis of diagnosis-relapse pairs of 12 children who relapsed very early, followed by a deep-sequencing validation of all identified mutations. In addition, we included one case with a good initial treatment response and on-treatment relapse at the end of upfront therapy. RESULTS: We observed a dynamic clonal evolution in all cases, with relapse almost exclusively originating from a subclone at diagnosis. We identified several driver mutations that may have influenced the outgrowth of a minor clone at diagnosis to become the major clone at relapse. For example, a minimal residual disease (MRD)-based standard-risk patient with ETV6-RUNX1-positive leukemia developed a relapse from a TP53-mutated subclone after loss of the wildtype allele. Furthermore, two patients with TCF3-PBX1-positive leukemia that developed a very early relapse carried E1099K WHSC1 mutations at diagnosis, a hotspot mutation that was recurrently encountered in other very early TCF3-PBX1-positive leukemia relapses as well. In addition to alterations in known relapse drivers, we found two cases with truncating mutations in the cohesin gene RAD21. CONCLUSION: Comprehensive genomic characterization of diagnosis-relapse pairs shows that very early relapses in BCP-ALL frequently arise from minor subclones at diagnosis. A detailed understanding of the therapeutic pressure driving these events may aid the development of improved therapies.

Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) carries an immunoglobulin-MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukaemia and use of unproven individualised treatment schedules. Here we contrast the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered on a national BCP-ALL clinical trial/registry. Where present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analysis. Outcome was analysed to define 3-year event free survival (EFS) and overall survival (OS). IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either: established BCP-ALL specific abnormalities (high hyperdiploidy n=3, KMT2A-rearrangement n=6, iAMP21 n=1, BCR-ABL n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J rearranged cases, clearly distinct from Burkitt leukaemia/lymphoma. Children with IG-MYC-r within that subgroup had 3-year EFS of 47% and OS of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this patient group must be allowed access to contemporary, minimal residual disease adapted, prospective clinical trial protocols.

Hematopoietic stem cell transplantation (HSCT) is a curative post-remission treatment in patients with acute myeloid leukemia (AML), but relapse after transplant is still a challenging event. In recent year, several studies have investigated the molecular minimal residual disease (qPCR-MRD) as a predictor of relapse, but the lack of standardized protocols, cut-offs, and timepoints, especially in the pediatric setting, has prevented its use in several settings, including before HSCT. Here, we propose the first collaborative retrospective I-BFM-AML study assessing qPCR-MRD values in pretransplant bone marrow samples of 112 patients with a diagnosis of AML harboring t(8;21)(q22; q22)RUNX1::RUNX1T1, or inv(16)(p13q22)CBFB::MYH11, or t(9;11)(p21;q23)KMT2A::MLLT3, or FLT3-ITD genetic markers. We calculated an ROC cut-off of 2.1 × 10−4 that revealed significantly increased OS (83.7% versus 57.1%) and EFS (80.2% versus 52.9%) for those patients with lower qPCR-MRD values. Then, we partitioned patients into three qPCR-MRD groups by combining two different thresholds, 2.1 × 10−4 and one lower cut-off of 1 × 10−2, and stratified patients into low-, intermediate-, and high-risk groups. We found that the 5-year OS (83.7%, 68.6%, and 39.2%, respectively) and relapse-free survival (89.2%, 73.9%, and 67.9%, respectively) were significantly different independent of the genetic lesion, conditioning regimen, donor, and stem cell source. These data support the PCR-based approach playing a clinical relevance in AML transplant management.

For the last two decades, measurable residual disease (MRD) has become one of the most powerful independent prognostic factors in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the effect of therapy on the bone marrow (BM) microenvironment and its potential relationship with the MRD status and disease free survival (DFS) still remain to be investigated. Here we analyzed the distribution of mesenchymal stem cells (MSC) and endothelial cells (EC) in the BM of treated BCP-ALL patients, and its relationship with the BM MRD status and patient outcome. For this purpose, the BM MRD status and EC/MSC regeneration profile were analyzed by multiparameter flow cytometry (MFC) in 16 control BM (10 children; 6 adults) and 1204 BM samples from 347 children and 100 adult BCP-ALL patients studied at diagnosis (129 children; 100 adults) and follow-up (824 childhood samples; 151 adult samples). Patients were grouped into a discovery cohort (116 pediatric BCP-ALL patients; 338 samples) and two validation cohorts (74 pediatric BCP-ALL, 211 samples; and 74 adult BCP-ALL patients; 134 samples). Stromal cells (i.e., EC and MSC) were detected at relatively low frequencies in all control BM (16/16; 100%) and in most BCP-ALL follow-up samples (874/975; 90%), while they were undetected in BCP-ALL BM at diagnosis. In control BM samples, the overall percentage of EC plus MSC was higher in children than adults (p = 0.011), but with a similar EC/MSC ratio in both groups. According to the MRD status similar frequencies of both types of BM stromal cells were detected in BCP-ALL BM studied at different time points during the follow-up. Univariate analysis (including all relevant prognostic factors together with the percentage of stromal cells) performed in the discovery cohort was used to select covariates for a multivariate Cox regression model for predicting patient DFS. Of note, an increased percentage of EC (>32%) within the BCP-ALL BM stromal cell compartment at day +78 of

Contains fulltext : 220784.pdf (publisher's version ) (Open Access), Glucocorticoid (GC) resistance is a crucial determinant of inferior response to chemotherapy in pediatric acute lymphoblastic leukemia (ALL); however, molecular mechanisms underlying this phenomenon are poorly understood. Deregulated splicing is a common feature of many cancers, which impacts drug response and constitutes an attractive therapeutic target. Therefore, the aim of the current study was to characterize global splicing profiles associated with GC resistance and determine whether splicing modulation could serve as a novel therapeutic option for GC-resistant patients. To this end, 38 primary ALL samples were profiled using RNA-seq-based differential splicing analysis. The impact of splicing modulators was investigated in GC-resistant leukemia cell lines and primary leukemic specimens. Our findings revealed, for the first time, markedly distinct splicing landscapes in ALL samples of B-cell precursor (BCP)-ALL and T-ALL lineages. Differential splicing events associated with GC resistance were involved in RNA processing, a direct response to GCs, survival signaling, apoptosis, cell cycle regulation and energy metabolism. Furthermore, our analyses showed that GC-resistant ALL cell lines and primary samples are sensitive to splicing modulation, alone and in combination with GC. Together, these findings suggest that aberrant splicing is associated with GC resistance and splicing modulators deserve further interest as a novel treatment option for GC-resistant patients.

Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection.

Glucocorticoid (GC) resistance is a crucial determinant of inferior response to chemotherapy in pediatric acute lymphoblastic leukemia (ALL); however, molecular mechanisms underlying this phenomenon are poorly understood. Deregulated splicing is a common feature of many cancers, which impacts drug response and constitutes an attractive therapeutic target. Therefore, the aim of the current study was to characterize global splicing profiles associated with GC resistance and determine whether splicing modulation could serve as a novel therapeutic option for GC-resistant patients. To this end, 38 primary ALL samples were profiled using RNA-seq-based differential splicing analysis. The impact of splicing modulators was investigated in GC-resistant leukemia cell lines and primary leukemic specimens. Our findings revealed, for the first time, markedly distinct splicing landscapes in ALL samples of B-cell precursor (BCP)-ALL and T-ALL lineages. Differential splicing events associated with GC resistance were involved in RNA processing, a direct response to GCs, survival signaling, apoptosis, cell cycle regulation and energy metabolism. Furthermore, our analyses showed that GC-resistant ALL cell lines and primary samples are sensitive to splicing modulation, alone and in combination with GC. Together, these findings suggest that aberrant splicing is associated with GC resistance and splicing modulators deserve further interest as a novel treatment option for GC-resistant patients.

Contains fulltext : 218617.pdf (publisher's version ) (Open Access), Genomic alterations in relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) may provide insight into the role of specific genomic events in relapse development. Along this line, comparisons between the spectrum of alterations in relapses that arise in different upfront treatment protocols may provide valuable information on the association between the tumor genome, protocol components and outcome. Here, we performed a comprehensive characterization of relapsed BCP-ALL cases that developed in the context of 3 completed Dutch upfront studies, ALL8, ALL9, and ALL10. In total, 123 pediatric BCP-ALL relapses and 77 paired samples from primary diagnosis were analyzed for alterations in 22 recurrently affected genes. We found pronounced differences in relapse alterations between the 3 studies. Specifically, CREBBP mutations were observed predominantly in relapses after treatment with ALL8 and ALL10 which, in the latter group, were all detected in medium risk-treated patients. IKZF1 alterations were enriched 2.2-fold (p = 0.01) and 2.9-fold (p < 0.001) in ALL8 and ALL9 relapses compared to diagnosis, respectively, whereas no significant enrichment was found for relapses that were observed after treatment with ALL10. Furthermore, IKZF1 deletions were more frequently preserved from a major clone at diagnosis in relapses after ALL9 compared to relapses after ALL8 and ALL10 (p = 0.03). These data are in line with previous studies showing that the prognostic value of IKZF1 deletions differs between upfront protocols and is particularly strong in the ALL9 regimen. In conclusion, our data reveal a correlation between upfront treatment and the genetic composition of relapsed BCP-ALL.

A fully-standardized EuroFlow 8–color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of £1025, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)–based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR–based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (£1025), if sufficient cells (>4 3 106, preferably more) are evaluated.

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To study the prognostic relevance of rare genetic aberrations in acute myeloid leukemia (AML), such as t(16;21), international collaboration is required. Two different types of t(16;21) translocations can be distinguished: t(16;21)(p11;q22), resulting in the FUS-ERG fusion gene; and t(16;21)(q24;q22), resulting in RUNX1-core binding factor (CBFA2T3). We collected data on clinical and biological characteristics of 54 pediatric AML cases with t(16;21) rearrangements from 14 international collaborative study groups participating in the international Berlin-Frankfurt-Münster (I-BFM) AML study group. The AML-BFM cohort diagnosed between 1997 and 2013 was used as a reference cohort. RUNX1-CBFA2T3 (n 5 23) had significantly lower median white blood cell count (12.5 3 109/L, P 5 .03) compared with the reference cohort. FUS-ERG rearranged AML (n 5 31) had no predominant French-American-British (FAB) type, whereas 76% of RUNX1-CBFA2T3 had an M1/M2 FAB type (M1, M2), significantly different from the reference cohort (P 5 .004). Four-year event-free survival (EFS) of patients with FUS-ERG was 7% (standard error [SE] 5 5%), significantly lower compared with the reference cohort (51%, SE 5 1%, P < .001). Four-year EFS of RUNX1-CBFA2T3 was 77% (SE 5 8%, P 5 .06), significantly higher compared with the reference cohort. Cumulative incidence of relapse was 74% (SE 5 8%) in FUS-ERG, 0% (SE 5 0%) in RUNX1-CBFA2T3, compared with 32% (SE 5 1%) in the reference cohort (P < .001). Multivariate analysis identified both FUS-ERG and RUNX1-CBFA2T3 as independent risk factors with hazard ratios of 1.9 (P < .0001) and 0.3 (P 5 .025), respectively. These results describe 2 clinically relevant distinct subtypes of pediatric AML. Similarly to other core-binding factor AMLs, patients with RUNX1-CBFA2T3 rearranged AML may benefit from stratification in the standard risk treatment, whereas patients with FUS-ERG rearranged AML should be considered high-risk.

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.

Contains fulltext : 169866.pdf (publisher's version ) (Open Access), Deletions and mutations affecting lymphoid transcription factor IKZF1 (IKAROS) are associated with an increased relapse risk and poor outcome in B-cell precursor acute lymphoblastic leukemia. However, additional genetic events may either enhance or negate the effects of IKZF1 deletions on prognosis. In a large discovery cohort of 533 childhood B-cell precursor acute lymphoblastic leukemia patients, we observed that single-copy losses of BTG1 were significantly enriched in IKZF1-deleted B-cell precursor acute lymphoblastic leukemia (P=0.007). While BTG1 deletions alone had no impact on prognosis, the combined presence of BTG1 and IKZF1 deletions was associated with a significantly lower 5-year event-free survival (P=0.0003) and a higher 5-year cumulative incidence of relapse (P=0.005), when compared with IKZF1-deleted cases without BTG1 aberrations. In contrast, other copy number losses commonly observed in B-cell precursor acute lymphoblastic leukemia, such as CDKN2A/B, PAX5, EBF1 or RB1, did not affect the outcome of IKZF1-deleted acute lymphoblastic leukemia patients. To establish whether the combined loss of IKZF1 and BTG1 function cooperate in leukemogenesis, Btg1-deficient mice were crossed onto an Ikzf1 heterozygous background. We observed that loss of Btg1 increased the tumor incidence of Ikzf1+/- mice in a dose-dependent manner. Moreover, murine B cells deficient for Btg1 and Ikzf1+/- displayed increased resistance to glucocorticoids, but not to other chemotherapeutic drugs. Together, our results identify BTG1 as a tumor suppressor in leukemia that, when deleted, strongly enhances the risk of relapse in IKZF1-deleted B-cell precursor acute lymphoblastic leukemia, and augments the glucocorticoid resistance phenotype mediated by the loss of IKZF1 function.

Contains fulltext : 170242.pdf (publisher's version ) (Closed access)

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The most important reason for therapy failure in pediatric acute myeloid leukemia (AML) is relapse. In order to identify miRNAs that contribute to the clonal evolution towards relapse in pediatric AML, miRNA expression profiling of 127 de novo pediatric AML cases were used. In the diagnostic phase, no miRNA signatures could be identified that were predictive for relapse occurrence, in a large pediatric cohort, nor in a nested mixed lineage leukemia (MLL)-rearranged pediatric cohort. AML with MLL- rearrangements are found in 15-20% of all pediatric AML samples, and reveal a relapse rate up to 50% for certain translocation partner subgroups. Therefore, microRNA expression profiling of six paired initial diagnosis-relapse MLL-rearranged pediatric AML samples (test cohort) and additional eight paired initial diagnosisrelapse samples with MLL-rearrangements (validation cohort) was performed. A list of 53 differentially expressed miRNAs was identified of which the miR-106b~25 cluster, located in intron 13 of MCM7, was the most prominent. These differentially expressed miRNAs however could not predict a relapse in de novo AML samples with MLLrearrangements at diagnosis. Furthermore, higher mRNA expression of both MCM7 and its upstream regulator E2F1 was found in relapse samples with MLL-rearrangements. In conclusion, we identified the miR-106b~25 cluster to be upregulated in relapse pediatric AML with MLL-rearrangements.