A predictive classifier of poor prognosis in transplanted patients with juvenile myelomonocytic leukemia: a study on behalf of the Société Francophone de Greffe de Moelle et de Thérapie Cellulaire.

Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric myeloproliferative neoplasm requiring hematopoietic stem cell transplantation (HSCT) in most cases. We retrospectively analyzed 119 JMML patients who underwent first allogeneic HSCT between 2002 and 2021. The majority (97%) carried a RAS-pathway mutation, and 62% exhibited karyotypic alterations or additional mutations in SETBP1, ASXL1, JAK3 and/or the RAS pathway. Relapse was the primary cause of death, with a 5-year cumulative incidence of 24.6% (95% CI: 17.1-32.9). Toxic deaths occurred in 12 patients, resulting in treatment-related mortality (TRM) of 9.0% (95% CI: 4.6-15.3). The 5-year overall (OS) and event-free survival were 73.6% (95% CI: 65.7-82.4) and 66.4% (95% CI: 58.2-75.8), respectively. Four independent adverse prognostic factors for OS were identified: age at diagnosis >2 years, time from diagnosis to HSCT ≥6 months, monocyte count at diagnosis >7.2x109/L, and the presence of additional genetic alterations. Based on these factors, we proposed a predictive classifier. Patients with 3 or more predictors (21% of the cohort) had a 5-year OS of 34.2%, whereas those with none (7%) had a 5-year OS of 100%. Our study demonstrates improved transplant outcomes compared to prior published data, which can be attributed to the synergistic impacts of a low TRM and a reduced, yet still substantial, relapse incidence. By integrating genetic information with clinical and hematologic features, we have devised a predictive classifier. This classifier effectively identifies a subgroup of patients who are at a heightened risk of unfavorable post-transplant outcomes who would benefit from novel therapeutic agents and post-transplant strategies.

Germline bi-allelic SH2B3/LNK alteration predisposes to a neonatal juvenile myelomonocytic leukemia-like disorder.

Juvenile myelomonocytic leukemia (JMML) is a rare, generally aggressive myeloproliferative neoplasm affecting young children. It is characterized by granulomonocytic expansion, with monocytosis infiltrating peripheral tissues. JMML is initiated by mutations upregulating RAS signaling. Approximately 10% of cases remain without an identified driver event. Exome sequencing of 2 unrelated cases of familial JMML of unknown genetics and analysis of the French JMML cohort identified 11 patients with variants in SH2B3, encoding LNK, a negative regulator of the JAK-STAT pathway. All variants were absent from healthy population databases, and mutation spectrum was consistent with a loss of function of the LNK protein. A stoploss variant was shown to affect both protein synthesis and stability. The other variants were either truncating or missense, the latter affecting the SH2 domain that interacts with activated JAK. Of the 11 patients, 8 from 5 families inherited pathogenic bi-allelic SH2B3 germline variants from their unaffected heterozygous parents. These children represent half of the cases with no identified causal mutation in the French cohort. They displayed typical clinical and hematological JMML features with neonatal onset and marked thrombocytopenia. They were characterized by absence of additional genetic alterations and a hypomethylated DNA profile with fetal characteristics. All patients showed partial or complete spontaneous clinical resolution. However, progression to thrombocythemia and immunity-related pathologies may be of concern later in life. Bi-allelic SH2B3 germline mutations thus define a new condition predisposing to a JMML-like disorder, suggesting that the JAK pathway deregulation is capable of initiating JMML, and opening new therapeutic options.

The prognostic value of IKZF1plus in B-cell progenitor acute lymphoblastic leukemia: Results from the EORTC 58951 trial.

BACKGROUND: IKZF1 gene deletion is an indicator of poor prognosis in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The AEIOP/BFM group proposed that the prognostic strength of IKZF1 deletion could be remarkably improved by taking into account additional genetic deletions and reported that among patients with an IKZF1 deletion those with deletions in CDKN2A/2B, PAX5, or PAR1 in the absence of ERG deletion, grouped as IKZF1plus , had the worst outcome. PROCEDURE: Between 1998 and 2008, 1636 patients under 18 years of age with previously untreated BCP-ALL were registered in the EORTC 58951 trial. Those with multiplex ligation-dependent probe amplification data were included in this analysis. Unadjusted and adjusted Cox model was used to investigate the additional prognostic value of IKZF1plus . RESULTS: Among 1200 patients included in the analysis, 1039 (87%) had no IKZF1 deletion (IKZF1WT ), 87 (7%) had an IKZF1 deletion but not IKZF1plus (IKZF1del ) and 74 (6%) had IKZF1plus . In the unadjusted analysis, both patients with IKZF1del (hazard ratio [HR] = 2.10, 95% confidence interval [CI]: 1.34-3.31) and IKZF1plus (HR = 3.07, 95% CI: 2.01-4.67) had a shorter event-free survival compared with IKZF1WT . However, although the IKZF1plus status was associated with patients' characteristics indicating poor prognosis, the difference between IKZF1plus and IKZF1del was not statistically significant (HR = 1.46, 95% CI: 0.83-2.57, p = .19). The results of the adjusted analysis were similar to the unadjusted analysis. CONCLUSIONS: In patients with BCP-ALL from the EORTC 58951 trial, the improvement of the prognostic importance of IKZF1 by considering IKZF1plus was not statistically significant.

Pediatric randomized trial EORTC CLG 58951: Outcome for adolescent population with acute lymphoblastic leukemia.

Over the years, the prognosis of adolescents treated for acute lymphoblastic leukemia (ALL) has improved. However, this age group still represents a challenge with an overall survival (OS) of 60% compared to 85% in younger children. Herein, we report the outcome of adolescents treated in the European Organisation for Research and Treatment of Cancer (EORTC) 58951 clinical trial. EORTC 58951 clinical trial included patients with de novo ALL between 1998 and 2008. For this study, we analyzed data of all adolescents between 15 and under 18. Data from 97 adolescents were analyzed, 70 had B-lineage and 27 had T-lineage ALL. The 8-year event-free survival (EFS) and OS for the B-cell precursor ALL cases were 72.3% (59.4%-81.7%) and 80.8% (67.4%-89.1%), respectively. For the T-lineage, the 8-year EFS and OS were 57.4% (36.1%-74.0%) and 59.0% (36.1%-76.2%), respectively. "B-other" ALL, defined as BCP-ALL lacking any known recurrent genetic abnormalities were more frequent in our adolescent population (52.8%) than in younger children (27.1%). Outcome of adolescents in the EORTC 58951 study is supporting the findings that adolescents have better outcome in pediatric compared to adults' trials. Nevertheless, in pediatric studies, adolescents still have a worse prognosis than younger children. Despite the fact that specific unfavorable characteristics may be linked to the adolescent population, a careful study and characterization of adolescents "B-other" genetic abnormalities in ALL is critical to improve the outcome of this population.

Large Duplications Can Be Benign Copy Number Variants: A Case of a 3.6-Mb Xq21.33 Duplication.

Segmental aneusomies are usually associated with clinical consequences, but an increasing number of nonpathogenic cytogenetically visible as well as large cryptic chromosomal imbalances have been reported. Here, we report a 3.6-Mb Xq21.33 microduplication detected prenatally on a female fetus which was inherited from a phenotypically normal mother and grandfather. It is assumed that male patients harboring Xq or Xp duplication present with syndromic intellectual disability because of functional disomy of the corresponding genes. Female carriers are generally asymptomatic because of preferential inactivation of the abnormal X. In the present case, the 3.6-Mb-duplicated segment encompasses only 2 genes, DIAPH2 and RPL4A. Since the asymptomatic grandfather carries the duplication, we hypothesize that these genes are not dosage sensitive and/or involved in cognitive function. Our observation further illustrates that large copy number variants can be associated with a normal phenotype, especially where gene density is low. Reporting rare cases of large genomic imbalances without a phenotypic effect can be very helpful, especially for genetic counseling in the prenatal setting.

CD36 cell surface expression as a surrogate marker to identify ABL/JAK-class kinase fusions in pediatric BCP-ALL.

Genetic alterations are the cornerstone of risk stratification in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and their accurate identification is critical for optimal treatment. Most cases with ABL-class fusion are classified as high-risk yet display good responses to tyrosine kinase inhibitors (TKIs). Current clinical protocols recommend adding a TKI to chemotherapy as soon as possible, making it mandatory to rapidly identify these alterations. We investigated here whether the identification of immunophenotypic features associated with these molecular alterations could be a valuable screening tool. CD36 expression was shown to be a characteristic feature of ABL- or JAK-class kinase fusions. The main genetic subgroups clustering in the subset with Philadelphia (Ph)-like features were also found to display specific immunophenotypic characteristics. A predictive multiparameter scoring system was generated, segregating genetic subtypes with aberrant kinase activation (PAX5/CRLF2alt, BCR::ABL1, ABL/JAK-class). The most robust markers identified were the TSLPR with CD19/22/9/38/81/304 and CD49f. As TKI adjunction is currently limited to the ABL-class kinase fusions, immunophenotypes distinguishing ABL from JAK-class were also investigated. The flow cytometry method reported here, accessible to most hematology departments, is thus a new useful tool to quickly screen for Ph-like kinase fusion with a good sensitivity (95%) and specificity (96%).

Partitioning for Easy Multiplexing: A Versatile Droplet PCR Application for Clone Monitoring in Tumors.

Clinical genome-wide next-generation sequencing (NGS) has brought new challenges to genetic laboratories. The identification of numerous patient-specific variants that may require to be screened for on multiple other samples poses an issue when striving for time and cost-effectiveness. Here, we propose d-multiSeq, a straightforward method utilizing the advantages of droplet PCR for multiplexing combined with amplicon-based NGS. By comparing d-multiSeq with a standard multiplex amplicon-based NGS, it was shown that partitioning prevents the amplification competition seen when multiplexing and leads to a homogeneous representation of each target in the total read count for up to a 40-target multiplex without the need for prior adjustment. Variant allele frequency was reliably evaluated with a sensitivity of 97.6% for variant allele frequency up to 1%. The applicability of d-multiSeq was also tested on cell-free DNA with the successful amplification of an eight-target multiplex panel. Preliminary application of the technique to assess the clonal evolution in a childhood leukemia harboring high interpatient variability in its somatic variants is shown. d-multiSeq represents a turnkey solution for analyzing large sets of patient-specific variants on low DNA amounts and cell-free DNA.

Distinct subsets of multi-lymphoid progenitors support ontogeny-related changes in human lymphopoiesis.

Changes in lymphocyte production patterns occurring across human ontogeny remain poorly defined. In this study, we demonstrate that human lymphopoiesis is supported by three waves of embryonic, fetal, and postnatal multi-lymphoid progenitors (MLPs) differing in CD7 and CD10 expression and their output of CD127-/+ early lymphoid progenitors (ELPs). In addition, our results reveal that, like the fetal-to-adult switch in erythropoiesis, transition to postnatal life coincides with a shift from multilineage to B lineage-biased lymphopoiesis and an increase in production of CD127+ ELPs, which persists until puberty. A further developmental transition is observed in elderly individuals whereby B cell differentiation bypasses the CD127+ compartment and branches directly from CD10+ MLPs. Functional analyses indicate that these changes are determined at the level of hematopoietic stem cells. These findings provide insights for understanding identity and function of human MLPs and the establishment and maintenance of adaptative immunity.

Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment.

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood, initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however, the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome, RNA-seq), Colony forming assay and xenograft studies, we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones, both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells, JMML-PCs are not always restricted to this compartment, highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML, and the identity of JMML-PCs, and provides robust models to monitor the disease and test novel therapeutic approaches.

Correction: Despite mutation acquisition in hematopoietic stem cells, JMML-propagating cells are not always restricted to this compartment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

B-ALL With t(5;14)(q31;q32); IGH-IL3 Rearrangement and Eosinophilia: A Comprehensive Analysis of a Peculiar IGH-Rearranged B-ALL.

Background: B-cell acute lymphoblastic leukemia associated with t(5;14)(q31;q32); IGH-IL3 is an exceptional cause of eosinophilia. The IGH enhancer on 14q32 is juxtaposed to the IL3 gene on 5q31, leading to interleukin-3 overproduction and release of mature eosinophils in the blood. Clinical, biological and outcome data are extremely scarce in the literature. Except for eosinophilia, no relevant common feature has been highlighted in these patients. However, it has been classified as a distinct entity in the World Health Organization classification. Cases Presentation: Eight patients with t(5;14)(q31;q32) treated by French or Austrian protocols were retrospectively enrolled. Array comparative genomic hybridization, multiplex ligation-dependent probe amplification or genomic PCR search for IKZF1 deletion were performed in 7. Sixteen patients found through an exhaustive search in the literature were also analyzed. For those 24 patients, median age at diagnosis is 14.3 years with a male predominance (male to female ratio = 5). Eosinophilia-related symptoms are common (neurologic in 26%, thromboembolic in 26% or pulmonary in 50%). Median white blood cells count is high (72 × 109/L) and linked to eosinophilia (median: 32 × 109/L). Peripheral blasts are present at a low level or absent (median: 0 × 109/L; range: 0-37 × 109/L). Bone marrow morphology is marked by a low blast infiltration (median: 42%). We found an IKZF1 deletion in 5 out of 7 analyzable patients Outcome data are available for 14 patients (median follow-up: 28 months): 8 died and 6 are alive in complete remission. Some of these features are concordant with those seen in patients with other IGH-rearranged B-cell acute lymphoblastic leukemias: young age at onset, male sex, low blast count, high incidence of IKZF1 deletion and intermediate prognosis. Conclusion: Based on shared epidemiological and biological features, B-cell acute lymphoblastic leukemia with t(5;14)(q31;q32) is a peculiar subset of IGH-rearranged B-cell acute lymphoblastic leukemia with an intermediate prognosis and particular clinical features related to eosinophilia.

Neonatal Marfan Syndrome: Report of a Case with an Inherited Splicing Mutation outside the Neonatal Domain.

We report a child and her mother affected by Marfan syndrome. The child presented with a phenotype of neonatal Marfan syndrome, revealed by acute and refractory heart failure, finally leading to death within the first 4 months of life. Her mother had a common clinical presentation. Genetic analysis revealed an inherited FBN1 mutation. This intronic mutation (c.6163+3_6163+6del), undescribed to date, leads to exon 49 skipping, corresponding to in-frame deletion of 42 amino acids (p.Ile2014_Asp2055del). FBN1 next-generation sequencing did not show any argument for mosaicism. Association in the same family of severe neonatal and classical Marfan syndrome illustrates the intrafamilial phenotype variability.