Precursor Acute Lymphoblastic Leukemia Research Articles

Long Non-coding RNA RP11-252C15.1 is a Potential Biomarker of Prognosis and Hallmark for Leukemogenesis in Children with B-cell Precursor Acute Lymphoblastic Leukemia.

Improved understanding of the prognostic biomarkers associated with childhood acute lymphoblastic leukemia (ALL) is needed for accurate risk group stratification. This study aimed to identify potential long-non coding RNA (lncRNA) markers and evaluate their prognostic value in children with ALL. We selected 50 children with newly diagnosed ALL and 20 age-matched patients with idiopathic immune thrombocytopenia (controls). RNA sequencing was performed to identify differentially expressed lncRNAs between the ALL and control groups. Correlation analysis was performed to determine the relationships between candidate lncRNAs, clinical features, and the risk of leukemogenesis. A total of 1,019 differentially expressed lncRNAs were identified between the ALL and control groups. Reverse-transcriptase (RT-qPCR) revealed that lncRNA RP11-252C15.1 and lncRNA RP11-701P16.2 were significantly upregulated in patients with ALL. Furthermore correlation analysis showed that lncRNA RP11-252C15.1 and lncRNA RP11-701P16.2 represent potential predictors of leukemogenesis; however, only lncRNA RP11-252C15.1 was associated with clinical features and outcome in children with B-cell precursor ALL (BCP-ALL). In vitro experiments confirmed that lncRNA RP11-252C15.1 was significantly overexpressed in BCP-ALL cell lines and promoted proliferation, and repressed apoptosis in MHH-CALL-3 cells. LncRNA RP11-252C15.1 is a potential oncogene in BCP-ALL pathogenesis and a prognostic biomarker in children with BCP-ALL.

Raman spectroscopy can recognize the KMT2A rearrangement as a distinct subtype of leukemia

Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are the two most common hematologic malignancies, challenging to treat and associated with high recurrence and mortality rates. This work aims to identify specific Raman biomarkers of ALL cells with the KMT2A gene rearrangement (KMT2A-r), representing a highly aggressive subtype of childhood leukemia with a poor prognosis. The proposed approach combines the sensitivity and specificity of Raman spectroscopy with machine learning and allows us to distinguish not only myelo- and lymphoblasts but also discriminate B-cell precursor (BCP) ALL with KMT2A-r from other blasts of BCP-ALL. We have found that KMT2A-r ALL cells fixed with 0.5% glutaraldehyde exhibit a unique spectroscopic profile that enables us to identify this subtype from other leukemias and normal cells. Therefore, a rapid and label-free method was developed to identify ALL blasts with KMT2A-r based on the ratio of the two Raman bands assigned to phenylalanine – 1040 and 1008 cm−1. This is the first time that a particular group of leukemic cells has been identified in a label-free way. The identified biomarker can be used as a screening method in diagnostic laboratories or non-reference medical centers.

The influence of cytotoxic drugs on the immunophenotype of blast cells in paediatric B precursor acute lymphoblastic leukaemia.

Flow cytometry plays is important in the diagnosis of acute lymphoblastic leukaemia (ALL) and when antigen-specific immunotherapy is indicated. We have investigated the effects of prednisolone, vincristine, daunorubicin, asparaginase and methotrexate on the antigen expression on blast cells that could influence the planning of antigen-specific therapy as well as risk-based treatment assignment. Patients aged ≤ 17 years with de novo B-cell ALL (B-ALL) were enrolled in the study. Blast cells were isolated and exposed in vitro to 5 individual cytotoxic drugs in logarithmically increasing concentrations. Then, the expression of CD10, CD19, CD20, CD27, CD34, CD45, CD58, CD66c and CD137 antigens was determined by quantitative flow cytometry. Cytotoxic drugs caused dose-dependent or dose-independent modulation of antigen expression. Daunorubicin caused a dose-dependent down-modulation of CD10, CD19, CD34, CD45 and CD58 and an up-modulation of CD137. Vincristine caused a dose-dependent down-modulation of CD19 and CD58 and an up-modulation of CD45. Daunorubicin also caused dose-independent down-modulation of CD27 and prednisolone down-modulation of CD10, CD19, CD27, CD34 and CD58. Down-modulation of CD20 was detected only in relation to the specific dose of daunorubicin. The results of the study have shown that cytotoxic drugs can alter the expression of antigens that are important for immunotherapy. Importantly, daunorubicin, prednisolone and vincristine caused down-modulation of CD19 and CD58, suggesting that these drugs are better avoided during bridging therapy prior to bispecific antibodies or CAR-T cell therapy. In addition, immunophenotypic changes on blast cells induced by different drugs could also influence risk-based treatment assignment.

Flow cytometric and cytomorphological definition of remission achievement in children with B-lineage acute lymphoblastic leukemia

The achievement of remission at the end of induction (EOI) chemotherapy in patients with acute lymphoblastic leukemia (ALL) is the key parameter of treatment effectiveness evaluation. The aim of the study – defining complete remission by multicolor flow cytometry (MFC) and bone marrow (BM) cytomorphology (CM) at the EOI chemotherapy in children with B-lineage ALL. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The study included patients of “ALL-MB 2008” and “ALL-MB 2015” trials for whom minimal residual disease (MRD) was evaluated by MFC at the EOI simultaneously with CM BM investigation. Less than 5% blasts in BM and MRD < 1% were established as the remission achievement criteria for CM and MFC respectively. The study group included 1498 children aged from 1 to 18 years (median age was 4 years and 11 months) with B-cell precursor ALL. The overall concordance of MFC and CM was found to be 96.1% (1440 of 1498 patients). In 36 (2.4%) children with MRD ≥ 1%, M1 BM status was observed. In contrast, in 22 (1.5%) patients with M2/M3 BM status by CM, MRD value was below 1%. Treatment outcome was analyzed in 522 patients of “ALL-MB 2008” trial. Children with M2/M3 BM, as well as with MRD ≥ 1% demonstrated dramatically inferior outcome, in comparison to those who achieved remission. The presence of at least one of the mentioned criteria (M2/M3 status by CM or MRD ≥ 1% by MFC) defined a group of 23 (4.4%) patients with very low event-free survival (34.9%, standard error 11.0%) and very high cumulative incidence of relapse (56.4%, standard error 12.0%). For the evaluation of remission achievement, MFC and CM should be applied simultaneously at the EOI. High leukemic burden found by any of these methods is the clear definition of induction failure. MRD detection at the EOI should be implemented in any modern treatment protocol as an obligatory stage of treatment response monitoring and final risk group stratification. Considering the crucial importance of the MRD detection results, this study must be performed only in the reference laboratories of the study groups.

Multimodal classification of molecular subtypes in pediatric acute lymphoblastic leukemia

Genomic analyses have redefined the molecular subgrouping of pediatric acute lymphoblastic leukemia (ALL). Molecular subgroups guide risk-stratification and targeted therapies, but outcomes of recently identified subtypes are often unclear, owing to limited cases with comprehensive profiling and cross-protocol studies. We developed a machine learning tool (ALLIUM) for the molecular subclassification of ALL in retrospective cohorts as well as for up-front diagnostics. ALLIUM uses DNA methylation and gene expression data from 1131 Nordic ALL patients to predict 17 ALL subtypes with high accuracy. ALLIUM was used to revise and verify the molecular subtype of 281 B-cell precursor ALL (BCP-ALL) cases with previously undefined molecular phenotype, resulting in a single revised subtype for 81.5% of these cases. Our study shows the power of combining DNA methylation and gene expression data for resolving ALL subtypes and provides a comprehensive population-based retrospective cohort study of molecular subtype frequencies in the Nordic countries.

Characterizing Morphometric and Nanomechanical Malignant Cell Features in a Rare Paediatric γδ T-acute Lymphoblastic Leukaemia: Insights from a Single Case Study Using Atomic Force Microscopy

The most common cancer in paediatric age is acute lymphoblastic leukaemia (ALL) which accounts for nearly a quarter of all cases of paediatric cancer. ALL cases are classified as B-cell or T-cell precursor ALL, based on their immunophenotypical features. Patients with T-cell ALL (T-ALL) comprise 10–15% of all newly diagnosed cases and are more prevalent in boys and in older age compared to the overall incidence peak age of ALL. In this case report, we present the morphometric and nanomechanical features of T-lymphoblasts derived from an 11-month-old infant with a rare subtype of γδ T-ALL with aggressive biological behavior. To investigate these features, we employed atomic force microscopy (AFM) and compared the blast cell deviations found in their nanostructure and elasticity to those of normal lymphocytes from a healthy child. The malignant T-lymphoblasts exhibited reduced roughness and Young’s modulus values. This single case analysis demonstrates the potential of the AFM method to provide additional information regarding the characteristics of malignant cells and suggests its potential as a complementary approach for distinguishing neoplastic cells from normal cells. The application of AFM could potentially facilitate the introduction of in vitro tests to determine the efficacy of anti-leukemic treatments.

High-Hyperdiploid Acute Lymphoblastic Leukemia in Children with LZTR1 Germline Variants

Introduction Firstly described in 1967, hyperdiploidy (HD) is the most frequent genetic abnormality in B-cell- precursor acute lymphoblastic leukemia (BCP-ALL) in children, comprising about 25% of all cases. A not yet exactly defined proportion have predisposing pathogenic germline variants in DNA repair pathway genes, chromatin remodeling factors, transcription factors regulating B-cell development (particularly ETV6) or receptor tyrosine kinases pathway genes like RAS/RAF. Among the latter, mutations have mostly been described in PTPN11 and SOS1, but not yet in other components of this central regulatory hub of cellular communication. Germline loss-of-function (LOF) LZTR1 mutations are typically linked to hereditary nerve sheath tumors. However, it remains largely unknown, if other tumor entities are associated with LZTR1 LOF germline mutations, potentially broadening the spectrum of malignancies associated with RASopathies. Aim Our aim is to understand the frequency and the impact of LZTR1 germline variants in HD BCP-ALL of childhood. Material and Methods We analyzed WES data of 283 children with BCP-ALL for the presence of pathogenic variants in the LZTR1 cancer predisposition gene. For a part of the detected variants, we performed further functional analyses to investigate the effect of the alteration on protein function. We applied a Drosophila model that is particularly suited for functional evaluation of Ras pathway activity. Results We identified LZTR1 germline variants in 10/283 (3.5%) patients (Figure 1). Interestingly, in 6 out of 283 (2.1%) children the LZTR1 variants were classified as pathogenic/likely pathogenic (P/LP). The majority of patients (5/6) harboring a P/LP germline LZTR1 variant presented with a HD BCP-ALL. Only two of our patients showed concomitant phenotypic features indicative of an underlying syndromic condition. Patient P1 presented with a HD BCP-ALL at the age of 9 years and mild psychomotor delay. Sequencing revealed a likely pathogenic variant (p.Arg283Trp) that was not yet reported in association with RASopathy syndromes. The second child P8, also diagnosed with a HD BCP-ALL, carried a well-known autosomal dominant mutation described in Noonan syndrome (NS) variant (p.Gly248Arg). The child was characterized by facial dysmorphism, as well as mild developmental delay, although a diagnosis of NS was not established prior to development of the BCP-ALL. The other three patients had no clinical peculiarities other than HD BCP-ALL. We functionally characterized patient derived LZTR1 variants in dLztr1-depleted ISC. Like wild-type hLZTR1 wt, hLZTR1 p.Arg283Trp and hLZTR1 p.Lys761Arg restored control-like ISC lineage production, suggesting that hLZTR1 p.Arg283Trp and hLZTR1 p.Lys761Arg still regulate Ras ubiquitination. In contrast, hLZTR1 p.Tyr535Ter results in even 1.8-fold higher ISC lineage production than dLztr1-RNAi. Beyond ISC production as a readout, we employed a second experimental paradigm directly addressing Ras signalling activity with a translocating modified ERK sensor. In line with Ras activity control of LZTR1, hLZTR1 wt significantly reduced ERK activity, while the putatively LOF variant hLZTR1 p.Tyr535Ter variant significantly increased Ras pathway activation by 1.4-fold over controls. We also noticed that hLZTR1 p.Tyr535Ter induced extensive membrane blebbing and nuclear fragmentation of GFP positive cells likely indicating programmed cell death. Quantification of apoptotic cells revealed an 11.1-fold increase compared to controls, which was not observed for hLZTR1 p.Arg283Trp and hLZTR1 p.Lys761Arg. Most notably, forced expression of hLZTR1 p.Arg283Trp andhLZTR1 p.Tyr535Ter increased mitotic recombination by 4-fold and 3.4-fold, respectively, which was not observed for the hLZTR1 p. Lys761Arg variant. Conclusion In our cohort, approximately 2% of all children with BCP-ALL harboured a P/LP LZTR1 germline variation, not necessarily linked with clinical appearance of Noonan-syndrome-like features, but to the development of a high hyperdiploid karyotype. By applying a Drosophila model, we demonstrated that patient-derived LZTR1 germline variants affect RAS pathway activation, ERK accumulation, cell proliferation, DNA recombination and apoptosis. Figure 1: Depiction of the ten LZTR1 variants detected in an unselected pediatric cohort of 283 BCP-ALL patients

Adverse Prognostic Role of Copy Number Alterations and Mutations in Adults with Philadelphia Chromosome-Negative Acute Lymphoblastic Leukemia

Background: The impact of copy number alterations (CNAs) or mutations on outcomes has been broadly analyzed in pediatric acute lymphoblastic leukemia (ALL). However, the magnitude of the effect was often marginal, frequently restricted to specific genetic subtypes and rarely replicated across independent cohorts in adult ALL. Aim: We tried to analyze the prognostic relevance of genetic copy number alterations and gene mutations in patients with Ph-negative ALL who were treated with modified hyper-CVAD based chemotherapy followed by allogeneic hematopoietic cell transplantation (alllo-HCT). Methods: We investigated the role of CNAs or mutations to refine risk stratification in 128 adults with newly diagnosed Philadelphia chromosome (Ph)-negative B-cell precursor ALL treated with intensive chemotherapy followed by allo-HCT for post-remission therapy. We performed multiplex ligation-dependent probe amplification (MLPA) to detect deletions of 11 genes ( IKZF1, CDKN2A/B, EBF1, ETV6, PAX5, BTG1, JAK2, RB1, PAR1, ZFY) and high throughput sequencing (HTS) of 70 gene mutations. Results: MLPA analysis showed that IKZF1 and CDKN2A/B deletions were observed in 54 (42.2%) and 52 (40.6%) patients, respectively. The remaining gene deletions were: PAX5 in 29 (22.6%), ETV6 in 22 (17.2%), BTG1 in 15 (11.7%), EBF1 in 14 (10.9%), RB1 in 13 (10.1%), JAK2 in 8 (6.2%), and PAR1 in 4 (3.1%). By HTS, NRAS (n=21, 16.4%), KRAS (n=11, 8.6%), TP53 (n=14, 10.9%), and PTPN11 (n=12, 9.4%) mutations were frequently detected. Overall, multivariate analysis for disease-free survival (DFS) and cumulative incidence of relapse (Table 1) showed that TP53 mutations (HR 5.04, 95%CI 2.29-11.30, P<0.001) and CDKN2A/B deletions (HR 1.88, 95%CI 1.10-3.31, P=0.026) were significantly associated with a poorer DFS. Patients with TP53 mutations (HR 5.64, 95%CI 1.95-16.20, P<0.001) and IKZF1 deletions (HR 1.91, 95%CI 1.09-3.31, P=0.022) had a higher relapse risk. To better define the prognostic role of CNAs, further analyses were restricted to patients excluding 14 TP53 mutations (n=114). Here, we classified patients into 4 subgroups; (1) No deletions (n=45, 39.5%), (2) IKZF1 deletion-alone (n=11, 9.6%), (3) IKZF1plus (n=40, 35.1%), and (4) CDKN2A/B±other deletions without IKZF1 deletions (n=18, 15.8%). Patients with IKZF1plus or CDKN2A/B±other deletions had an inferior DFS ( P=0.023) and higher relapse incidence ( P=0.002) than patients having no deletions. The results were similarly reproduced in patients receiving allo-HCT in first complete remission. Conclusions: Our data showed that IKZF1 or CDKN2A/B deletions and TP53 mutations may confer a poor prognosis for adult Ph-negative B-cell precursor ALL in the setting of intensive chemotherapy and allo-HCT. The combination of genomic abnormalities and minimal residual disease response may further refine risk stratification and better select patients who could benefit from novel therapeutic approaches.

Inflammatory Signatures Define a New High-Risk T-Lineage ALL Subtype

T-lineage acute lymphoblastic leukemia (ALL) is an aggressive cancer comprising of diverse subtypes that are challenging to stratify using conventional immunophenotyping and have historically exhibited poor treatment outcomes in response to cytotoxic chemotherapy ( Nguyen, K et al. Leukemia 2008). Early T-cell Precursor ALL (ETP-ALL) represent a distinct subtype that displays a developmental block at the earliest stages of T-cell commitment, accompanied by the aberrant expression of myeloid and stem cell markers ( Coustan-Smith, E. et al. Lancet Oncol. 2009). The classification can be further complicated by T/Myeloid Mixed phenotype acute leukemia (T/My-MPAL), a rare and aggressive malignancy characterized by blasts with both T-cell lymphoid and myeloid markers ( George, B. S. et al. Biomedicines 2022). Accurate clinical diagnosis of T-lineage ALL is hindered by phenotypic variations among its subsets. Nevertheless, the precise diagnosis holds critical importance as drug sensitivity in preclinical models of T-lineage ALL is closely linked to the differentiation state, such as the sensitivity of ETP-ALL to the BCL-2 inhibitor venetoclax ( Chonghaile, T. N. et al. Nat Cancer 2021). This highlights the critical need to identify consistent phenotypic traits associated with unique therapeutic vulnerabilities to effectively tailor therapy to individual patients. To gain insights into subset-specific therapeutic vulnerabilities, we performed an integrative multiomic analysis of bone marrow (BM) samples (n=21) from newly diagnosed T-lineage ALL patients, including T-cell ALL (T-ALL), ETP-ALL, and T/My-MPAL. Leveraging cellular indexing of transcriptomes and epitopes in conjunction with T-cell receptor sequencing, we identified a distinct subset of patients spanning all three subtypes characterized by the high frequency of leukemic cells that most closely resemble healthy hematopoietic stem and progenitor cells (HSPC) by Symphony ( Kang, J. B. et al. Nat Commun 2021) query mapping, indicative of an earlier stage of differentiation arrest ( Figure 1A bottom). Pathway analysis revealed an inflammatory signature in patients with high frequencies of stem-like cells suggestive of an inflammatory microenvironment akin to high-risk AML ( Lasry, A. et al. Nat Cancer 2023), which we refer to as inflammatory T-lineage ALL. Next, we mined our multiomic data to generate a comprehensive geneset for inflammatory T-lineage ALL scoring in single cells and bulk samples ( Figure 1A top). Interestingly, Olink cytokine array analysis uncovered upregulation of production of cytokines IL-1β, IL-18, and/or OSM in inflammatory T-lineage ALL samples. Thus, this comprehensive multiomic analysis identified a subset of T-lineage ALL patients characterized by early differentiation arrest and inflammatory signatures. To explore the relationship between inflammatory T-lineage status and clinical response, we utilized the ssGSEA algorithm to score activity of our inflammatory signature in bulk RNA-sequencing data from the TARGET T-lineage ALL dataset (n=265, 74/265 inflammatory) ( Liu, Y. et al. Nat Genet 2017) and the Tran et al. study (n=27, 17/27 inflammatory) ( Tran, T. H. et al. Blood Adv 2022). We found a significant association between high inflammatory ssGSEA score and elevated MRD. To uncover the inflammatory T-lineage ALL mutational landscape, we performed whole exome sequencing on our study cohort, and compiled the reported mutations in the TARGET dataset. Interestingly, inflammatory T-lineage ALL patients, as predicted by inflammatory signature activity, had a significantly higher prevalence of mutations affecting cytokine signaling ( NRAS, JAK3), chromatin remodeling genes ( EZH2), and the WT1 oncogene. These analyses demonstrate a significant relationship between inflammatory status, genomic lesions, and MRD. Finally, we demonstrate that inflammatory, but not non-inflammatory, T-lineage ALL cells are significantly more sensitive to BCL-2 inhibitor venetoclax ex vivo (Figure 1B).Overall, our study has identified a new subtype of T-lineage ALL defined by an early arrest in T-cell development, inflammatory signaling, a unique mutational landscape, sensitivity to venetoclax, and an association with adverse clinical parameters.

Reshaping MRD Detection Strategies: Next-Generation Sequencing and High DNA Input Identify Previously Missed Measurable Residual Disease in Peripheral Blood of B-Cell Precursor Acute Lmyphoblastic Leukemia

Background: In acute lymphoblastic leukemia (ALL), measurable residual disease (MRD) is the most important prognostic factor. MRD is traditionally quantified in bone marrow aspirates (BM) using real-time quantitative polymerase chain reaction (RQ-PCR) according to EuroMRD standards. Since the emergence of modern molecular methods, it has been a continuous effort to enable MRD assessment from peripheral blood (PB). The less invasive material collection would allow closer MRD monitoring and solve technical challenges of BM aspiration, such as skewed or non-representative MRD values due to hemodilution and unequal distribution of leukemic cells. While several studies demonstrated comparable MRD values of BM and PB in T-ALL, in B-cell precursor ALL (BCP-ALL) MRD determination from PB is still a challenge. Mean MRD levels in PB are considerably lower than in BM and frequently escape detection. Additionally, paired samples show a highly variable MRD level ratio in both pediatric and adult cases (Kotrova et al, Leukemia 2020). Objective: BCP-ALL cases with known MRD positivity in the BM likely also harbor MRD positive cells in the PB, even when missed by traditional methods. With more sensitive assays, either by increased DNA input or by application of amplicon-based next generation sequencing (NGS), MRD detection from PB may improve in these cases. Methods: We retrospectively selected PB - BM sample pairs of 74 BCP-ALL patients with BM MRD positivity (43 with quantifiable MRD positivity and 31 with positivity below quantitative range) and sufficient leftover DNA from a cohort of patients treated according to GMALL protocols, where conventional RQ-PCR of clonal immunoglobuline heavy chain (IGH) VJ/DJ gene rearrangements failed to detect PB MRD. MRD had been determined by standard RQ-PCR with 1.5 µg DNA in BM and PB, respectively, according to the EuroMRD guidelines (van der Velden et al, Leukemia 2007). PB samples were re-tested for MRD with high DNA input RQ-PCR using 6.6 µg DNA and amplicon-based NGS, each using 6.6 µg DNA, as well as standard input NGS. NGS analyses of corresponding PB and BM samples were performed with the EuroClonality amplicon-based NGS assay (Brüggemann et al, Leukemia 2019). Identification and quantification of leukemia-derived clonotypes was done using ARResT/Interrogate. Results: In total, 74 BCP-ALL patients with pre/c-B-ALL (n=60) and pro-B-ALL (n=14) were included. Patient age ranged from 19 - 71 years with a median of 44.5. In all included samples (56 in first-line therapy and 18 salvage treatment), BM was MRD positive and PB was MRD negative in standard RQ-PCR at a sensitivity of at least 10 -4. To assess the effect of high DNA input alone, RQ-PCR was repeated in PB with high DNA input for 59 patients and 63 IGH VJ/DJ markers. At least one positive signal was detected in 19/59 patients (32%) and 20/63 markers (32%) (FIGURE 1A). NGS was performed for 73 PB samples and 84 markers using standard and high DNA input. With standard DNA input, MRD could be detected for 19/73 patients (26%) and 21/84 markers (25%) (FIGURE 1A). In the high input approach, MRD was detected in 36/73 patients (49%) and 40/84 markers (48%) (FIGURE 1A). A quantifiable MRD value was identified in 17 patients and 19 markers. All markers with quantifiable NGS MRD positivity in PB showed higher MRD values in the corresponding BM sample and MRD values below 10 -4 (FIGURE 1B). No correlation of MRD in paired samples could be demonstrated (Pearson correlation coefficient: 0.09) (FIGURE 1B). Conclusion: In BCP-ALL, MRD detection from PB is challenging with conventional methods. Increased DNA input and amplicon-based NGS allowed detection of MRD in a subset of PB samples that were negative in standard RQ-PCR. With identical DNA input, NGS showed a higher sensitivity than RQ-PCR and best results were achieved when high DNA input and NGS were combined, identifying MRD in nearly half of the PB samples. These results demonstrate that PB can be a valuable tool for early MRD detection in BCP-ALL when adjusting traditional MRD assessment strategies. However, in half of the samples, MRD was still missed by high input NGS. Even with assays improved for sensitivity, PB therefore cannot fully replace BM for MRD assessment in BCP-ALL yet and should rather be considered to complement BM analyses or in cases where BM is not accessible.

Picalm::MLLT10 can Identify a New Subgroup of Acute Leukemias of Ambiguous Lineage with Unique Extramedullary Disease and Treatment Response

Background: Mixed-phenotype acute leukemias (MPAL), which is included in acute leukemias of ambiguous lineage (ALAL), can be classified as four different subtypes based on recurring genetic alterations including BCR::ABL, KMT2A and ZNF384 gene rearrangement as well as BCL11B activation. The 2022 WHO classification has already mentioned PICALM::MLLT10 fusions are also enriched in MPAL but need more data. In 2022 ELN classification, PICALM::MLLT10 was identified as acute myeloid leukemia (AML) with other rare recurring translocations but not ALAL. In addition, there is no separate classification for PICALM::MLLT10 in the new ICC classification. Therefore, it is still confused whether PICALM::MLLT10 corresponds to AML or acute lymphoblastic leukemia (ALL) or ALAL. Methods: Fourteen PICALM::MLLT10 positive patients of 390 AL patients (14/390, 3.6%) were identified by RNA sequencing (RNA-seq) in our center from 2020-2022, including 8 female and 6 male, with a median age of 33 years (16-50 years). These patients were newly diagnosed AL according to bone marrow morphology and immunology including 5 ALAL, 5 T-ALL [ 4 early T-cell precursor ALL (ETP-ALL), 1 cortical T-ALL], 2 AML, 1 B-ALL with aberrant expression of myeloid antigen, and 1 B/T MPAL. Results: The mean white blood cell counts of these patients was 8.25×10 9/L (2.34-51×10 9/L) and platelet counts was 168.5×10 12/L (39-429×10 12/L) individually. It should be mentioned that extramedullary disease (EMD) was found in 7 cases (7/14), including mediastinum, tonsil, and skin. In terms of immunotyping, CD7 was identified in all patients (14/14) and CD33 in 71.4% (10/14) patients. The major concurrence mutations were PHF6 mutation (8/14), JAK3 mutation (5/14), and SUZ12 mutation (4/14). Characteristic cytogenetic abnormality t(10;11)(p12.3;q14.2) was only found in three cases (3/14) (Table 1). The PICALM breakpoints are mainly concentrated in exon 17 (n=6) and exon 19 (n=8). Exon 4 (n=8), exon 6 (n=2), exon 9 (n=2) and exon 10 (n=2) are the most commonbreakpointsof MLLT10. To our knowledge, this is the first report about the breakpoints and fusion gene forms of PICALM::MLLT10. For initial treatment, these patients individually received standard ALL induction chemotherapy (VDPCP, vincristine, idarubicin, pegaspargase, cyclophosphamide, prednisone) and AML induction chemotherapy (3+7 IA regimen including idarubicin, cytarabine). The initial complete remission (CR) rate was only 35.7% (5/14), and 9 patients showing no remission (NR). These NR patients subsequently received salvage chemotherapy. It is worth mentioning that 6 of them received combined chemotherapy regimen including low doses of cytarabine (LDAC), granulocyte colony-stimulating factor (G-CSF) and anthracyclines such as aclarubicin or idarubicin or homoharringtonine (CAG or IAG or HAG), and 2 of 6 patients also further received combination therapy with venetoclax (CAG or IAG +VEN). Finally, 5 of them dramatically achieved CR (5/6). In the other 3 patients, 1 patient who was failure to initial Hyper-CVAD A/B regimen also dramatically achieved CR after the more Hyper-CVAD A regimen combined with venetoclax, and 1 patient achieved CR while another still NR with both receiving CLAG (cladribine, cytarabine, G-CSF) regimen. Subsequently 11 patients (9 CR, 2 with refractory disease) received allogenic hematopoietic stem cell transplantation (allo-HSCT), and all the CR patients survived well after HSCT (follow up 3-24 months, median 15 months) without relapse, while 2 NR patients died soon after transplantation because of severe complications (Table 2). Conclusions: Our data suggested PICALM::MLLT10 positive AL should be more appropriately recognized as an independent ALAL entity and may benefit from LDAC, G-CSF and anthracyclines combination chemotherapy as well as venetoclax. Sequential HSCT after chemotherapy combined with venetoclax may further improve long-term survival in AL patients with CR even MRD positive.

The MEK Inhibitor Selumetinib in Combination with Dexamethasone Leads to Responses in Adult and Pediatric Patients with Relapsed RAS-Pathway Mutated Acute Lymphoblastic Leukemia: Results of a Phase 1/2 Study

Introduction Relapsed acute lymphoblastic leukemia (ALL) is a major therapeutic challenge and novel treatment strategies are urgently sought. RAS-pathway activating mutations are found in 38% of relapsed pediatric B-cell precursor (BCP) ALL patients ( J Irving, Blood, 2014). Additional mutations that activate the RAS-signalling cascade (NF1, BRAF, IKZF2, IKZF3, JAK1) have also been found with varying frequencies in relapsed ALL. Selumetinib, a potent MEK1/2 inhibitor, works synergistically with dexamethasone in mouse models of RAS-pathway mutated ALL by enhanced upregulation of the proapoptotic protein, BIM ( EC Matheson, Haematologica, 2019). Methods We conducted an international, parallel cohort, dose-finding and expansion trial to evaluate the combination of selumetinib with dexamethasone in adult and pediatric patients with relapsed/refractory T- or BCP-ALL harbouring RAS-pathway activating mutations. Selumetinib was supplied free of charge by AstraZeneca. Details of in-/exclusion criteria, treatment and dosages are described by T. Menne, BMJ open, 2022. The trial was conducted according to the Declaration of Helsinki and after medical ethics committees of participating countries had approved the protocol. The trial was registered under NCT03705507 and ISRCTN92323261 and sponsored by the University of Birmingham. Results The trial opened to recruitment on 17-Apr-2018, the first patient was recruited on 18-May-2018. Twelve patients were included, 8 adults and 4 children. Six of the 8 adult patients had relapsed/refractory T-ALL, the remaining patients had BCP-ALL. Further patient characteristics are summarized in table 1. Pharmacokinetic measurements demonstrated lower maximum concentrations for pediatric patients on dose level -1, and no significant effect of CYP3A4 induction was observed. A total of 561 Adverse Events (AEs) were reported for 11/12 patients. For 1 patient who died on day 3 of treatment due to leukemia related events, no AEs were reported. 3/11 patients (27%) had AEs of a maximum grade 1-3, 5 patients (45%) had a maximum AE grade 4, and 3 patients (27%) suffered a grade 5 AE (2x sepsis, pneumonia). Further details of AEs are depicted in table 2. Sixteen serious adverse events (SAEs) were observed in 9 patients. Of those, 11 were due to infection, and all 9 patients had an SAE involving infection, most frequently neutropenic fever or sepsis. Three SAEs were fatal: one concerned a child with sepsis who had stopped trial treatment 14 days previously. Two patients were adults: one became septic 2 days after being admitted for myopathy/weakness and collapsed shortly after starting antibiotics. The 2 nd patient was admitted with fever, diagnosed with pneumonia and died 5 days later of cardiac arrest. All fatal events occurred before the last of 3 urgent safety measures were implemented in September 2019, changing the continuous dexamethasone dosing to pulsed doses and mandating antimicrobial prophylaxis. The 5 non-infection SAEs were anaemia, constipation, myopathy/weakness, fatigue and heart failure. Response data are available for 9/12 patients. Two patients died before reaching the 1 st bone marrow assessment after 28 days, and 1 patient stopped trial treatment after 11 days. Of the remaining 9 patients, 5 were non-responders (56%) and 4 (44%) had a complete remission (CR), or a CR with incomplete platelet recovery (3 adults, 1 child; response rate 44%). Of the 4 patients in CR, 2 adults had relapsed T-ALL and stayed on treatment for 3 to almost 5 months. Of the responding BCP-ALL patients, 1 patient died of pneumonia in cycle 2, and 1 pediatric patient progressed after cycle 2 then discontinued. Conclusion Recruitment to the trial was unexpectedly slow, due to competing new treatment options (commercial CAR-T cells, blinatumomab, inotuzumab), delays in opening international centres and recruitment stops due to COVID. Initially, the trial suffered from frequent SAEs concerning infections, which lead to a total of 3 urgent safety measures in 2018 and 2019, including lowering the dose of dexamethasone in the 2 nd half of cycle 1, mandating fluoroquinolone and co-trimoxazole prophylaxis and finally changing continuous dexamethasone to pulses. The response data with 44% of patients achieving a CR is promising and will need to be further evaluated in the context of targeted treatment approaches for RAS-altered leukemias, especially for relapsed T-ALL.

The Different Impacts of an MRD-Guided Protocol on Relapse of Childhood Acute Lymphoblastic Leukemia: The Report of Taiwan Pediatric Oncology Group (TPOG)-ALL-2013

Taiwan Pediatric Oncology Group (TPOG)-ALL-2013 protocol (TPOG-2013), modifying from St. Jude Total Therapy XV Study and Total Therapy XVI Study, was launched since January 2013. This is the first nationwide minimal residual disease (MRD)-directed protocol for treatment of childhood acute lymphoblastic leukemia (ALL) in Taiwan. According to TPOG-2013, two MRD measurements were scheduled on days 15-19 of induction (MRD1 time point, TP1) and days 35-42, end of induction (MRD2 time point, TP2) to make the definitive risk stratification to guide subsequent therapy. As of April 30, 2023, 814 newly diagnosed ALLs treated with TPOG-2013 and followed up ≥3 years were enrolled in the study. The outcomes were compared with those of TPOG-ALL-2002 protocol (TPOG-2002) (N=1350, between January 2002 and December 2012), which did not integrate the MRD monitoring. The 5-year event-free survival and overall survival were significantly improved from 76% (95% CI, 74-78) and 81% (80-84) of TPOG-2002 to 86% (83-88) and 89% (87-92) of TPOG-2013, respectively ( P< 0.0001). For further analysis of TPOG-2013, 585 (72%) patients with exact adherence (EA) to both TPs were assigned as MRD EA group; 225 patients who were non-adherence (NA) to either one of TPs as MRD NA group; and four patients were excluded for the comparative outcome analysis. For B-cell precursor (BCP) ALL, the 5-year cumulative incidence of relapse (CIR) significantly decreased from TPOG-2002 (N=1194) to TPOG-2013 MRD EA (N=527) ( P <0.0001). However, there was no significant improvement of CIR between TPOG-2002 and MRD NA (N=202) ( P= 0.21) (Figure 1). In MRD EA of BCP-ALL, compared with TPOG-2002, patients with ETV6::RUNX1 or BCR::ABL1 appeared to have significantly lower CIR. Patients without the common genetic subtypes , denoted as “negative” group, also had significant inferior CIR. And the trend of CIR decrease did not attain significant difference in high hyperdiploidy. In contrast, patients with T CF3::PBX1 or infant with KMT2A-rearrangement did not show CIR improvement (Table 1). In T-ALL, compared with TPOG-2002 (N=156), there was a significant decrease of CIR in TPOG-2013 MRD EA (N=58)( P= 0.01)(Table 1), but not in MRD NA (N=23)( P= 0.86). There was a longer duration to relapse in MRD EA of BCP-ALL. However, a delay of relapse was only demonstrated in patients with BCR::ABL1, which could be addressed by the introduction of MRD-monitoring and TKI inhibitor in TPOG-2013 (Table 1). Further, in term of early relapse before 1.5 year of remission, there was no significant difference in MRD EA of BCP-ALL, compared with TPOG-2002. In conclusion, this study demonstrated diffident impacts on relapse according to genetic subtypes and MRD adherence. In the negative group of BCP-ALL without the discovery of novel genetic alternations, the relapse could be much decreased with the MRD-directed treatment protocol.

Near-early T-cell precursor acute lymphoblastic leukemia following a diagnosis of essential thrombocythemia.

Near-early T-cell precursor acute lymphoblastic leukemia following a diagnosis of essential thrombocythemia.

Flow Sorting, Whole Genome Amplification and Next-Generation Sequencing as Combined Tools to Study Heterogeneous Acute Lymphoblastic Leukemia.

Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell receptor (TR) gene rearrangement analysis in acute lymphoblastic leukemia (ALL) and lymphoma (LBL). These methods likely constitute faster and more sensitive approaches to analyze heterogenous cases of ALL/LBL, yet it is not known whether gene rearrangements constituting low percentages of the total sequence reads represent minor subpopulations of malignant cells or background IG/TR gene rearrangements in normal B-and T-cells. In a comparison of eight cases of B-cell precursor ALL (BCP-ALL) using both the EuroClonality NGS method and the IdentiClone multiplex-PCR/gene-scanning method, the NGS method identified between 29% and 139% more markers than the gene-scanning method, depending on whether the NGS data analysis used a threshold of 5% or 1%, respectively. As an alternative to using low thresholds, we show that IG/TR gene rearrangements in subpopulations of cancer cells can be discriminated from background IG/TR gene rearrangements in normal B-and T-cells through a combination of flow cytometry cell sorting and multiple displacement amplification (MDA)-based whole genome amplification (WGA) prior to the NGS. Using this approach to investigate the clonal evolution in a BCP-ALL patient with double relapse, clonal TR rearrangements were found in sorted leukemic cells at the time of second relapse that could be identified at the time of diagnosis, below 1% of the total sequence reads. These data emphasize that caution should be exerted when interpreting rare sequences in NGS experiments and show the advantage of employing the flow sorting of malignant cell populations in NGS clonality assessments.

Abstract 24 Development of an In Vitro B-Cell Differentiation Model for the Characterization of the Origin and Causation of ETV6::RUNX1+ Preleukemic Cells

Abstract Introduction Pregnant women are exposed to various carcinogenic substances that can subsequently affect their progeny. Recurrent preleukemic chromosomal translocations characteristic for childhood acute lymphoblastic leukemia (ALL) frequently emerge before birth in utero. The most common translocation t (12;21) occurs in 25% of B-cell precursor ALL and results in the formation of the chimeric oncogenic transcription factor ETV6::RUNX1. Objectives We aim to analyze factors promoting the generation of preleukemic ETV6::RUNX1 translocations, investigate the biological origin of these preleukemic cells, and develop an in vitro model of early B-cell differentiation to analyze the mechanisms underlying the persistence of ETV6::RUNX1+ preleukemic cells and leukemia evolution in children. Methods Genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) (Schafer et. al., 2018) was applied to identify ETV6::RUNX1+ cord blood units (CBU). Screening results were validated by Sanger sequencing and quantified by real-time PCR (ipsogen ETV6-RUNX1 Kit, Qiagen). ETV6::RUNX1+ CBUs were thawed, sorted for CD34+ and CD19+ cells, and analyzed by qPCR. CD34+ were differentiated towards B-cell lineage on MSC feeder lines according to a modified protocol (Ichii et. al., 2008). Results We screened a cohort of 1405 cord bloods collected in 2004 and discovered that 103 (7.3%) carried the ETV6::RUNX1 fusion gene. The preleukemic translocation was detected in CD34+ stem and early B progenitor cells. For the establishment of an in vitro B-cell differentiation model, bone marrow (BM-MSCs) and CB (USSCs, CB-MSCs) stromal feeder cells were compared and the effect of different media (SCGM, DMEM/5 % FCS, MSCBM/10 % FCS) in the presence of stem cell factor (SCF), Flt-3, IL3, +/- IL7 was assessed. BM-MSCs demonstrated the highest ability to support B-lymphopoieses towards CD19+ cell differentiation in MSCBM media (~30%), primarily due to the release of IL-7 by the feeder layer. Currently, factors that may promote generation of preleukemic translocations in utero e.g. nitrosamines contained in food products, are being tested in this model. Discussion We identified the HSC/early B progenitor state as the origin of ETV6::RUNX1 translocations in CB. The establishment of an in vitro B-cell differentiation system will allow to test the impact of environmental carcinogens on the generation of ETV6::RUNX1+ pre-leukemic cells and their leukemic evolution.

Functional Characterization of Transforming Growth Factor-β Signaling in Dasatinib Resistance and Pre-BCR+ Acute Lymphoblastic Leukemia.

The multi-kinase inhibitor dasatinib has been implicated to be effective in pre-B-cell receptor (pre-BCR)-positive acute lymphoblastic leukemia (ALL) expressing the E2A-PBX1 fusion oncoprotein. The TGFβ signaling pathway is involved in a wide variety of cellular processes, including embryonic development and cell homeostasis, and it can have dual roles in cancer: suppressing tumor growth at early stages and mediating tumor progression at later stages. In this study, we identified the upregulation of the TGFβ signaling pathway in our previously generated human dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells using global transcriptomic analysis. We confirm the upregulation of the TGFβ pathway member SMAD3 at the transcriptional and translational levels in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Hence, dasatinib blocks, at least partially, TGFβ-induced SMAD3 phosphorylation in several B-cell precursor (BCP) ALL cell lines as well as in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Activation of the TGFβ signaling pathway by TGF-β1 leads to growth inhibition by cell cycle arrest at the G0/G1 stage, increase in apoptosis and transcriptional changes of SMAD-targeted genes, e.g. c-MYC downregulation, in pre-BCR+/E2A-PBX1+ ALL cells. These results provide a better understanding about the role that the TGFβ signaling pathway plays in leukemogenesis of BCP-ALL as well as in secondary drug resistance to dasatinib.

Analysis of non-alcoholic fatty liver disease during induction therapy for B-cell precursor acute lymphoblastic leukemia in children and adolescents

A prospective evaluation of non-alcoholic fatty liver disease (NAFLD) during induction therapy for acute lymphoblastic leukemia (ALL) has not been performed. Herein, we prospectively investigated the frequency, risk factors, and outcomes of NAFLD during induction therapy in children and adolescents with B-cell precursor ALL (BCP-ALL). This study enrolled 74 newly diagnosed BCP-ALL cases aged 1 year and older who were admitted to our department between January 2011 and December 2020. Median age was 6.6 years (1.3-17.5 years). Plain computed tomography (CT) of the upper abdomen was performed before induction therapy, and on days 15 and 29 after initiation of induction therapy. Patients with a liver/spleen CT ratio <0.9 were defined as having NAFLD. The frequency of NAFLD was 73%. Patients with NAFLD had a higher rate of hypertriglyceridemia. There was no significant difference in 5-year overall survival and event-free survival (EFS) between patients with and without NAFLD. However, after restricting the target age to 10 years and older, 5-year EFS was significantly higher in patients with NAFLD than in those without (88.5 vs. 42.9%, respectively, P=0.037). Similarly, 5-year cumulative incidence of relapse (CIR) was significantly lower in patients with NAFLD than in those without it (5-year CIR, 6.3 vs. 57.1%, respectively, P=0.013). Patients with NAFLD exhibit better outcomes including 5-year EFS and CIR. Further studies are necessary.

PersonALL: a genetic scoring guide for personalized risk assessment in pediatric B-cell precursor acute lymphoblastic leukemia

BackgroundRecurrent genetic lesions provide basis for risk assessment in pediatric acute lymphoblastic leukemia (ALL). However, current prognostic classifiers rely on a limited number of predefined sets of alterations.MethodsDisease-relevant copy number aberrations (CNAs) were screened genome-wide in 260 children with B-cell precursor ALL. Results were integrated with cytogenetic data to improve risk assessment.ResultsCNAs were detected in 93.8% (n = 244) of the patients. First, cytogenetic profiles were combined with IKZF1 status (IKZF1normal, IKZF1del and IKZF1plus) and three prognostic subgroups were distinguished with significantly different 5-year event-free survival (EFS) rates, IKAROS-low (n = 215): 86.3%, IKAROS-medium (n = 27): 57.4% and IKAROS-high (n = 18): 37.5%. Second, contribution of genetic aberrations to the clinical outcome was assessed and an aberration-specific score was assigned to each prognostically relevant alteration. By aggregating the scores of aberrations emerging in individual patients, personalized cumulative values were calculated and used for defining four prognostic subgroups with distinct clinical outcomes. Two favorable subgroups included 60% of patients (n = 157) with a 5-year EFS of 96.3% (excellent risk, n = 105) and 87.2% (good risk, n = 52), respectively; while 40% of patients (n = 103) showed high (n = 74) or ultra-poor (n = 29) risk profile (5-year EFS: 67.4% and 39.0%, respectively).ConclusionsPersonALL, our conceptually novel prognostic classifier considers all combinations of co-segregating genetic alterations, providing a highly personalized patient stratification.

Steroid-induced ocular hypertensive response in pediatric patients with acute lymphoblastic leukemia.

To report the characteristics of the steroid-induced ocular hypertensive response in pediatric patients with acute lymphoblastic leukemia (ALL) treated with prednisolone (PSL) during induction therapy and with dexamethasone (DEX) during reinduction therapy. Retrospective. This study included pediatric patients diagnosed with B-cell precursor ALL and treated with systemic corticosteroids sometime during the period from 2016 to 2018 at Shizuoka Children's Hospital. Data were extracted from the hematology/oncology records related to the type, dose, and duration of systemic corticosteroids as well as to the ophthalmologic examination findings, intraocular pressure (IOP) data, symptoms of high IOP, and antiglaucoma medications obtained during corticosteroid administration. The maximal IOPs of the PSL and DEX groups were compared. Twenty-eight patients (18 boys and 10 girls; mean age 5.5years) were treated with systemic corticosteroids. Twelve of the 22 courses of PSL and 33 of the 44 courses of DEX were found to be associated with high IOP. The maximal IOP was higher with the use of DEX than with the use of PSL, including in those who received prophylactic therapy (PSL 25.2mmHg, DEX 33.6mmHg; P = 0.02). Antiglaucoma medication was given to 21 patients; 6 patients had symptoms of ocular hypertension. The maximal IOPs were 52.8mmHg and 70.8mmHg in the PSL and DEX groups, respectively. Both groups of patients reported severe headache. Increased IOP was frequently observed during systemic corticosteroid therapy in pediatric patients with ALL. Although most patients were asymptomatic, they occasionally presented with severe systemic symptoms. Regular ophthalmologic examinations should be included in the treatment guidelines for ALL.