[Suspicion of constitutional abnormality at diagnosis of childhood leukemia: Update of the leukemia committee of the French Society of Childhood Cancers]. / Suspicion d'anomalie constitutionnelle au diagnostic de leucémie chez l'enfant : mise au point du comité leucémies de la Société française des cancers de l'enfant.

The spectrum of childhood leukemia predisposition syndromes has grown significantly over last decades. These predisposition syndromes mainly involve CEBPA, ETV6, GATA2, IKZF1, PAX5, RUNX1, SAMD9/SAMD9L, TP53, RAS-MAPK pathway, DNA mismatch repair system genes, genes associated with Fanconi anemia, and trisomy 21. The clinico-biological features leading to the suspicion of a leukemia predisposition are highly heterogeneous and require varied exploration strategies. The study of the initial characteristics of childhood leukemias includes high-throughput sequencing techniques, which have increased the frequency of situations where a leukemia predisposing syndrome is suspected. Identification of a leukemia predisposition syndrome can have a major impact on the choice of chemotherapy, the indication for hematopoietic stem cell transplantation, and screening for associated malformations and pathologies. The diagnosis of a predisposition syndrome can also lead to the exploration of family members and genetic counseling. Diagnosis and management should be based on dedicated and multidisciplinary care networks.

Single-cell analysis of megakaryopoiesis in peripheral CD34+ cells: insights into ETV6-related thrombocytopenia.

BACKGROUND: Germline mutations in the ETV6 transcription factor gene are responsible for familial thrombocytopenia and leukemia predisposition syndrome. Although previous studies have shown that ETV6 plays an important role in megakaryocyte (MK) maturation and platelet formation, the mechanisms by which ETV6 dysfunction promotes thrombocytopenia remain unclear. OBJECTIVES: To decipher the transcriptional mechanisms and gene regulatory network linking ETV6 germline mutations and thrombocytopenia. METHODS: Presuming that ETV6 mutations result in selective effects at a particular cell stage, we applied single-cell RNA sequencing to understand gene expression changes during megakaryopoiesis in peripheral CD34+ cells from healthy controls and patients with ETV6-related thrombocytopenia. RESULTS: Analysis of gene expression and regulon activity revealed distinct clusters partitioned into 7 major cell stages: hematopoietic stem/progenitor cells, common-myeloid progenitors (CMPs), MK-primed CMPs, granulocyte-monocyte progenitors, MK-erythroid progenitors (MEPs), progenitor MKs/mature MKs, and platelet-like particles. We observed a differentiation trajectory in which MEPs developed directly from hematopoietic stem/progenitor cells and bypassed the CMP stage. ETV6 deficiency led to the development of aberrant cells as early as the MEP stage, which intensified at the progenitor MK/mature MK stage, with a highly deregulated core "ribosome biogenesis" pathway. Indeed, increased translation levels have been documented in patient CD34+-derived MKs with overexpression of ribosomal protein S6 and phosphorylated ribosomal protein S6 in both CD34+-derived MKs and platelets. Treatment of patient MKs with the ribosomal biogenesis inhibitor CX-5461 resulted in an increase in platelet-like particles. CONCLUSION: These findings provide novel insight into both megakaryopoiesis and the link among ETV6, translation, and platelet production.

Cytological Diagnosis of Classic Myeloproliferative Neoplasms at the Age of Molecular Biology.

Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell-derived disorders characterized by uncontrolled proliferation of differentiated myeloid cells. Two main groups of MPN, BCR::ABL1-positive (Chronic Myeloid Leukemia) and BCR::ABL1-negative (Polycythemia Vera, Essential Thrombocytosis, Primary Myelofibrosis) are distinguished. For many years, cytomorphologic and histologic features were the only proof of MPN and attempted to distinguish the different entities of the subgroup BCR::ABL1-negative MPN. World Health Organization (WHO) classification of myeloid neoplasms evolves over the years and increasingly considers molecular abnormalities to prove the clonal hematopoiesis. In addition to morphological clues, the detection of JAK2, MPL and CALR mutations are considered driver events belonging to the major diagnostic criteria of BCR::ABL1-negative MPN. This highlights the preponderant place of molecular features in the MPN diagnosis. Moreover, the advent of next-generation sequencing (NGS) allowed the identification of additional somatic mutations involved in clonal hematopoiesis and playing a role in the prognosis of MPN. Nowadays, careful cytomorphology and molecular biology are inseparable and complementary to provide a specific diagnosis and to permit the best follow-up of these diseases.

SARS-CoV-2 spike protein induces a differential monocyte activation that may contribute to age bias in COVID-19 severity.

A strong bias related to age is observed in COVID-19 patients with pediatric subjects developing a milder disease than adults. We hypothesized that a specific SARS-CoV-2 effect conjugated with preexisting differences in the immune systems may explain this. Using flow cytometry, we investigated basal immune differences in a cohort consisting of 16 non-infected young and 16 aged individuals and further leveraged an in vitro whole blood model of SARS-CoV-2 infection so that functional differences could be mined as well. In short, blood diluted in culture media was incubated 5 or 24 h with the trimeric spike protein or controls. Following unsupervised analysis, we first confirmed that the immune lymphoid and myeloid systems in adults are less efficient and prone to develop higher inflammation than those in children. We notably identified in adults a higher CD43 lymphocyte expression, known for its potentially inhibitory role. The spike protein induced different responses between adults and children, notably a higher increase of inflammatory markers together with lower monocyte and B cell activation in adults. Interestingly, CD169, a CD43 ligand overexpressed in COVID-19 patients, was confirmed to be strongly modulated by the spike protein. In conclusion, the spike protein exacerbated the preexisting lower immune responsiveness and higher inflammatory potential in adults. Altogether, some of the markers identified may explain the marked age bias and be predictive of severity.

Calcium Signaling Is Impaired in PTEN-Deficient T Cell Acute Lymphoblastic Leukemia.

PTEN (Phosphatase and TENsin homolog) is a well-known tumor suppressor involved in numerous types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). In human, loss-of-function mutations of PTEN are correlated to mature T-ALL expressing a T-cell receptor (TCR) at their cell surface. In accordance with human T-ALL, inactivation of Pten gene in mouse thymocytes induces TCRαß+ T-ALL development. Herein, we explored the functional interaction between TCRαß signaling and PTEN. First, we performed single-cell RNA sequencing (scRNAseq) of PTEN-deficient and PTEN-proficient thymocytes. Bioinformatic analysis of our scRNAseq data showed that pathological Ptendel thymocytes express, as expected, Myc transcript, whereas inference of pathway activity revealed that these Ptendel thymocytes display a lower calcium pathway activity score compared to their physiological counterparts. We confirmed this result using ex vivo calcium flux assay and showed that upon TCR activation tumor Ptendel blasts were unable to release calcium ions (Ca2+) from the endoplasmic reticulum to the cytosol. In order to understand such phenomena, we constructed a mathematical model centered on the mechanisms controlling the calcium flux, integrating TCR signal strength and PTEN interactions. This qualitative model displays a dynamical behavior coherent with the dynamics reported in the literature, it also predicts that PTEN affects positively IP3 (inositol 1,4,5-trisphosphate) receptors (ITPR). Hence, we analyzed Itpr expression and unraveled that ITPR proteins levels are reduced in PTEN-deficient tumor cells compared to physiological and leukemic PTEN-proficient cells. However, calcium flux and ITPR proteins expression are not defective in non-leukemic PTEN-deficient T cells indicating that beyond PTEN loss an additional alteration is required. Altogether, our study shows that ITPR/Calcium flux is a part of the oncogenic landscape shaped by PTEN loss and pinpoints a putative role of PTEN in the regulation of ITPR proteins in thymocytes, which remains to be characterized.

Isolation and enrichment of mouse splenic T cells for ex vivo and in vivo T cell receptor stimulation assays.

Specific antigen recognition by T cell receptor (TCR) activates TCR signaling pathway, leading to T cell proliferation and differentiation into effector and memory cells. Herein, we describe protocols for TCR stimulation assays, including procedures for the isolation and enrichment of mouse splenic T cells for ex vivo TCR stimulation with anti-CD3/CD28 antibodies, and the use of ovalbumin-OT-II mouse model for in vivo TCR stimulation. We applied this protocol to show that MYC protein is essential for T cell proliferation and differentiation. For complete details on the use and execution of this protocol, please refer to Nozais et al. (2021).

Novel partial loss-of-function variants in the tyrosyl-tRNA synthetase 1 (YARS1) gene involved in multisystem disease.

Cytoplasmic aminoacyl-tRNA synthetases (ARSs) are emerging as a cause of numerous rare inherited diseases. Recently, biallelic variants in tyrosyl-tRNA synthetase 1 (YARS1) have been described in ten patients of three families with multi-systemic disease (failure to thrive, developmental delay, liver dysfunction, and lung cysts). Here, we report an additional subject with overlapping clinical findings, heterozygous for two novel variants in tyrosyl-tRNA synthetase 1 (NM_003680.3(YARS1):c.176T>C; p.(Ile59Thr) and NM_003680.3(YARS1):c.237C>G; p.(Tyr79*) identified by whole exome sequencing. The p.Ile59Thr variant is located in the highly conserved aminoacylation domain of the protein. Compared to subjects previously described, this patient presents a much more severe condition. Our findings support implication of two novel YARS1 variants in these disorders. Furthermore, we provide evidence for a reduced protein abundance in cells of the patient, in favor of a partial loss-of-function mechanism.

Cell Analysis from Dried Blood Spots: New Opportunities in Immunology, Hematology, and Infectious Diseases.

Blood cell analysis is a major pillar of biomedical research and healthcare. These analyses are performed in central laboratories. Rapid shipment from collection site to the central laboratories is currently needed because cells and biomarkers degrade rapidly. The dried blood spot from a fingerstick allows the preservation of cellular molecules for months but entire cells are never recovered. Here leucocyte elution is optimized from dried blood spots. Flow cytometry and mRNA expression profiling are used to analyze the recovered cells. 50-70% of the leucocytes that are dried on a polyester solid support via elution after shaking the support with buffer are recovered. While red blood cells lyse upon drying, it is found that the majority of leucocytes are preserved. Leucocytes have an altered structure that is improved by adding fixative in the elution buffer. Leucocytes are permeabilized, allowing an easy staining of all cellular compartments. Common immunophenotyping and mRNAs are preserved. The ability of a new biomarker (CD169) to discriminate between patients with and without Severe Acute Respiratory Syndrome induced by Coronavirus 2 (SARS-CoV-2) infections is also preserved. Leucocytes from blood can be dried, shipped, and/or stored for at least 1 month, then recovered for a wide variety of analyses, potentially facilitating biomedical applications worldwide.

Cytogenetics of Pediatric Acute Myeloid Leukemia: A Review of the Current Knowledge.

Pediatric acute myeloid leukemia is a rare and heterogeneous disease in relation to morphology, immunophenotyping, germline and somatic cytogenetic and genetic abnormalities. Over recent decades, outcomes have greatly improved, although survival rates remain around 70% and the relapse rate is high, at around 30%. Cytogenetics is an important factor for diagnosis and indication of prognosis. The main cytogenetic abnormalities are referenced in the current WHO classification of acute myeloid leukemia, where there is an indication for risk-adapted therapy. The aim of this article is to provide an updated review of cytogenetics in pediatric AML, describing well-known WHO entities, as well as new subgroups and germline mutations with therapeutic implications. We describe the main chromosomal abnormalities, their frequency according to age and AML subtypes, and their prognostic relevance within current therapeutic protocols. We focus on de novo AML and on cytogenetic diagnosis, including the practical difficulties encountered, based on the most recent hematological and cytogenetic recommendations.

MYC deficiency impairs the development of effector/memory T lymphocytes.

In the thymus, T cell progenitors differentiate in order to generate naive T lymphocytes which migrate in the periphery where they will fulfill their function in the adaptive immune response. During thymopoiesis, genomic alterations in thymocytes can promote leukemia development. Among recurrent alteration is PTEN inactivation, which is associated to MYC overexpression. Herein, we used conditional Pten and Myc knockout mouse models and single-cell RNA-sequencing approach, to investigate the impact of MYC loss on physio-pathological development of PTEN-proficient or PTEN-deficient T lymphocytes. First, our results confirm that MYC is mandatory for PTEN loss-mediated leukemogenesis, while it is not required for terminal steps of thymopoiesis. In contrast, we uncovered that Myc ablation in CD4+CD8+ thymocytes disrupts T lymphocytes homeostasis in the spleen, notably by drastically reducing the number of MYC-deficient effector/memory T cells. Collectively, our data show that besides naive T cells proliferation, MYC is essential for effector/memory differentiation.

GATA1 pathogenic variants disrupt MYH10 silencing during megakaryopoiesis.

BACKGROUND: GATA1 is an essential transcription factor for both polyploidization and megakaryocyte (MK) differentiation. The polyploidization defect observed in GATA1 variant carriers is not well understood. OBJECTIVE: To extensively phenotype two pedigrees displaying different variants in the GATA1 gene and determine if GATA1 controls MYH10 expression levels, a key modulator of MK polyploidization. METHOD: A total of 146 unrelated propositi with constitutional thrombocytopenia were screened on a multigene panel. We described the genotype-phenotype correlation in GATA1 variant carriers and investigated the effect of these novel variants on MYH10 transcription using luciferase constructs. RESULTS: The clinical profile associated with the p.L268M variant localized in the C terminal zinc finger was unusual in that the patient displayed bleeding and severe platelet aggregation defects without early-onset thrombocytopenia. p.N206I localized in the N terminal zinc finger was associated, on the other hand, with severe thrombocytopenia (15G/L) in early life. High MYH10 levels were evidenced in platelets of GATA1 variant carriers. Analysis of MKs anti-GATA1 chromatin immunoprecipitation-sequencing data revealed two GATA1 binding sites, located in the 3' untranslated region and in intron 8 of the MYH10 gene. Luciferase reporter assays showed their respective role in the regulation of MYH10 gene expression. Both GATA1 variants significantly alter intron 8 driven MYH10 transcription. CONCLUSION: The discovery of an association between MYH10 and GATA1 is a novel one. Overall, this study suggests that impaired MYH10 silencing via an intronic regulatory element is the most likely cause of GATA1-related polyploidization defect.

Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy.

Chimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19neg) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure.