Elevated Calprotectin and Abnormal Myeloid Cell Subsets Discriminate Severe from Mild COVID-19.

Blood myeloid cells are known to be dysregulated in coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2. It is unknown whether the innate myeloid response differs with disease severity and whether markers of innate immunity discriminate high-risk patients. Thus, we performed high-dimensional flow cytometry and single-cell RNA sequencing of COVID-19 patient peripheral blood cells and detected disappearance of non-classical CD14LowCD16High monocytes, accumulation of HLA-DRLow classical monocytes (Human Leukocyte Antigen - DR isotype), and release of massive amounts of calprotectin (S100A8/S100A9) in severe cases. Immature CD10LowCD101-CXCR4+/- neutrophils with an immunosuppressive profile accumulated in the blood and lungs, suggesting emergency myelopoiesis. Finally, we show that calprotectin plasma level and a routine flow cytometry assay detecting decreased frequencies of non-classical monocytes could discriminate patients who develop a severe form of COVID-19, suggesting a predictive value that deserves prospective evaluation.

SRSF2 plays an unexpected role as reader of m5C on mRNA, linking epitranscriptomics to cancer.

A common mRNA modification is 5-methylcytosine (m5C), whose role in gene-transcript processing and cancer remains unclear. Here, we identify serine/arginine-rich splicing factor 2 (SRSF2) as a reader of m5C and impaired SRSF2 m5C binding as a potential contributor to leukemogenesis. Structurally, we identify residues involved in m5C recognition and the impact of the prevalent leukemia-associated mutation SRSF2P95H. We show that SRSF2 binding and m5C colocalize within transcripts. Furthermore, knocking down the m5C writer NSUN2 decreases mRNA m5C, reduces SRSF2 binding, and alters RNA splicing. We also show that the SRSF2P95H mutation impairs the ability of the protein to read m5C-marked mRNA, notably reducing its binding to key leukemia-related transcripts in leukemic cells. In leukemia patients, low NSUN2 expression leads to mRNA m5C hypomethylation and, combined with SRSF2P95H, predicts poor outcomes. Altogether, we highlight an unrecognized mechanistic link between epitranscriptomics and a key oncogenesis driver.

TCTP regulates genotoxic stress and tumorigenicity via intercellular vesicular signaling.

Oncogenic intercellular signaling is regulated by extracellular vesicles (EVs), but the underlying mechanisms remain mostly unclear. Since TCTP (translationally controlled tumor protein) is an EV component, we investigated whether it has a role in genotoxic stress signaling and malignant transformation. By generating a Tctp-inducible knockout mouse model (Tctp-/f-), we report that Tctp is required for genotoxic stress-induced apoptosis signaling via small EVs (sEVs). Human breast cancer cells knocked-down for TCTP show impaired spontaneous EV secretion, thereby reducing sEV-dependent malignant growth. Since Trp53-/- mice are prone to tumor formation, we derived tumor cells from Trp53-/-;Tctp-/f- double mutant mice and describe a drastic decrease in tumori-genicity with concomitant decrease in sEV secretion and content. Remarkably, Trp53-/-;Tctp-/f- mice show highly prolonged survival. Treatment of Trp53-/- mice with sertraline, which inhibits TCTP function, increases their survival. Mechanistically, TCTP binds DDX3, recruiting RNAs, including miRNAs, to sEVs. Our findings establish TCTP as an essential protagonist in the regulation of sEV-signaling in the context of apoptosis and tumorigenicity.

DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability.

The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult-to-replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l-deficient cells are compromised in the repair of heterochromatin-associated double-stranded breaks, eliciting deletions in late-replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair-associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions.

Structural variation cooperates with permissive chromatin to control enhancer hijacking-mediated oncogenic transcription.

Structural variants (SVs) involving enhancer hijacking can rewire chromatin topologies to cause oncogene activation in human cancers, including hematologic malignancies; however, because of the lack of tools to assess their effects on gene regulation and chromatin organization, the molecular determinants for the functional output of enhancer hijacking remain poorly understood. Here, we developed a multimodal approach to integrate genome sequencing, chromosome conformation, chromatin state, and transcriptomic alteration for quantitative analysis of transcriptional effects and structural reorganization imposed by SVs in leukemic genomes. We identified known and new pathogenic SVs, including recurrent t(5;14) translocations that cause the hijacking of BCL11B enhancers for the allele-specific activation of TLX3 in a subtype of pediatric leukemia. Epigenetic perturbation of SV-hijacked BCL11B enhancers impairs TLX3 transcription, which are required for the growth of t(5;14) leukemia cells. By CRISPR engineering of patient-derived t(5;14) in isogenic leukemia cells, we uncovered a new mechanism whereby the transcriptional output of SV-induced BCL11B enhancer hijacking is dependent on the loss of DNA hypermethylation at the TLX3 promoter. Our results highlight the importance of the cooperation between genetic alteration and permissive chromatin as a critical determinant of SV-mediated oncogene activation, with implications for understanding aberrant gene transcription after epigenetic therapies in patients with leukemia. Hence, leveraging the interdependency of genetic alteration on chromatin variation may provide new opportunities to reprogram gene regulation as targeted interventions in human disease.

Genomic characterization of metastatic breast cancers.

Metastasis is the main cause of death for patients with breast cancer. Many studies have characterized the genomic landscape of breast cancer during its early stages. However, there is evidence that genomic alterations are acquired during the evolution of cancers from their early to late stages, and that the genomic landscape of early cancers is not representative of that of lethal cancers1-7. Here we investigated the landscape of somatic alterations in 617 metastatic breast cancers. Nine driver genes (TP53, ESR1, GATA3, KMT2C, NCOR1, AKT1, NF1, RIC8A and RB1) were more frequently mutated in metastatic breast cancers that expressed hormone receptors (oestrogen and/or progesterone receptors; HR+) but did not have high levels of HER2 (HER2-; n = 381), when compared to early breast cancers from The Cancer Genome Atlas. In addition, 18 amplicons were more frequently observed in HR+/HER2- metastatic breast cancers. These cancers showed an increase in mutational signatures S2, S3, S10, S13 and S17. Among the gene alterations that were enriched in HR+/HER2- metastatic breast cancers, mutations in TP53, RB1 and NF1, together with S10, S13 and S17, were associated with poor outcome. Metastatic triple-negative breast cancers showed an increase in the frequency of somatic biallelic loss-of-function mutations in genes related to homologous recombination DNA repair, compared to early triple-negative breast cancers (7% versus 2%). Finally, metastatic breast cancers showed an increase in mutational burden and clonal diversity compared to early breast cancers. Thus, the genomic landscape of metastatic breast cancer is enriched in clinically relevant genomic alterations and is more complex than that of early breast cancer. The identification of genomic alterations associated with poor outcome will allow earlier and better selection of patients who require the use of treatments that are still in clinical trials. The genetic complexity observed in advanced breast cancer suggests that such treatments should be introduced as early as possible in the disease course.

Author Correction: Genomic characterization of metastatic breast cancers.

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

Prognostic impact of genetic abnormalities in 536 first-line chronic lymphocytic leukaemia patients without 17p deletion treated with chemoimmunotherapy in two prospective trials: Focus on IGHV-mutated subgroups (a FILO study).

The potential prognostic influence of genetic aberrations on chronic lymphocytic leukaemia (CLL) can vary based on various factors, such as the immunoglobulin heavy variable (IGHV) status. We conducted an integrative analysis on genetic abnormalities identified through cytogenetics and targeted next-generation sequencing in 536 CLL patients receiving first-line chemo(immuno)therapies (CIT) as part of two prospective trials. We evaluated the prognostic implications of the main abnormalities, with specific attention to their relative impact according to IGHV status. In the entire cohort, unmutated (UM)-IGHV, complex karyotype, del(11q) and ATM mutations correlated significantly with shorter progression-free survival (PFS). Focusing on the subset of mutated IGHV (M-IGHV) patients, univariate analysis showed that complex karyotype, del(11q), SF3B1 and SAMHD1 mutations were associated with significant lower PFS. The prognostic influence varied based on the patient's IGHV status, as these abnormalities did not affect outcomes in the UM-IGHV subgroup. TP53 mutations had no significant impact on outcomes in the M-IGHV subgroup. Our findings highlight the diverse prognostic influence of genetic aberrations depending on the IGHV status in symptomatic CLL patients receiving first-line CIT. The prognosis of gene mutations and cytogenetic abnormalities needs to be investigated with a compartmentalized methodology, taking into account the IGVH status of patients receiving first-line BTK and/or BCL2 inhibitors.

Asxl1 loss cooperates with oncogenic Nras in mice to reprogram the immune microenvironment and drive leukemic transformation.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1-/- accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1-/- (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.

Cytokine-like protein 1-induced survival of monocytes suggests a combined strategy targeting MCL1 and MAPK in CMML.

Mouse models of chronic myeloid malignancies suggest that targeting mature cells of the malignant clone disrupts feedback loops that promote disease expansion. Here, we show that in chronic myelomonocytic leukemia (CMML), monocytes that accumulate in the peripheral blood show a decreased propensity to die by apoptosis. BH3 profiling demonstrates their addiction to myeloid cell leukemia-1 (MCL1), which can be targeted with the small molecule inhibitor S63845. RNA sequencing and DNA methylation pattern analysis both point to the implication of the mitogen-activated protein kinase (MAPK) pathway in the resistance of CMML monocytes to death and reveal an autocrine pathway in which the secreted cytokine-like protein 1 (CYTL1) promotes extracellular signal-regulated kinase (ERK) activation through C-C chemokine receptor type 2 (CCR2). Combined MAPK and MCL1 inhibition restores apoptosis of monocytes from patients with CMML and reduces the expansion of patient-derived xenografts in mice. These results show that the combined inhibition of MCL1 and MAPK is a promising approach to slow down CMML progression by inducing leukemic monocyte apoptosis.

ANKRD26 is a new regulator of type I cytokine receptor signaling in normal and pathological hematopoiesis.

Sustained ANKRD26 expression associated with germline ANKRD26 mutations causes thrombocytopenia 2 (THC2), an inherited platelet disorder associated with a predisposition to leukemia. Some patients also present with erythrocytosis and/or leukocytosis. Using multiple human-relevant in vitro models (cell lines, primary patients' cells and patient-derived induced pluripotent stem cells) we demonstrate for the first time that ANKRD26 is expressed during the early steps of erythroid, megakaryocyte and granulocyte differentiation, and is necessary for progenitor cell proliferation. As differentiation progresses, ANKRD26 expression is progressively silenced, to complete the cellular maturation of the three myeloid lineages. In primary cells, abnormal ANKRD26 expression in committed progenitors directly affects the proliferation/differentiation balance for the three cell types. We show that ANKRD26 interacts with and crucially modulates the activity of MPL, EPOR and G-CSFR, three homodimeric type I cytokine receptors that regulate blood cell production. Higher than normal levels of ANKRD26 prevent the receptor internalization that leads to increased signaling and cytokine hypersensitivity. These findings afford evidence how ANKRD26 overexpression or the absence of its silencing during differentiation is responsible for myeloid blood cell abnormalities in patients with THC2.

Caspase Inhibition Modulates Monocyte-Derived Macrophage Polarization in Damaged Tissues.

Circulating monocytes are recruited in damaged tissues to generate macrophages that modulate disease progression. Colony-stimulating factor-1 (CSF-1) promotes the generation of monocyte-derived macrophages, which involves caspase activation. Here, we demonstrate that activated caspase-3 and caspase-7 are located to the vicinity of the mitochondria in CSF1-treated human monocytes. Active caspase-7 cleaves p47PHOX at aspartate 34, which promotes the formation of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase complex NOX2 and the production of cytosolic superoxide anions. Monocyte response to CSF-1 is altered in patients with a chronic granulomatous disease, which are constitutively defective in NOX2. Both caspase-7 down-regulation and radical oxygen species scavenging decrease the migration of CSF-1-induced macrophages. Inhibition or deletion of caspases prevents the development of lung fibrosis in mice exposed to bleomycin. Altogether, a non-conventional pathway that involves caspases and activates NOX2 is involved in CSF1-driven monocyte differentiation and could be therapeutically targeted to modulate macrophage polarization in damaged tissues.

Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells.

The therapeutic efficacy of anthracyclines relies on antitumor immune responses elicited by dying cancer cells. How chemotherapy-induced cell death leads to efficient antigen presentation to T cells, however, remains a conundrum. We found that intratumoral CD11c(+)CD11b(+)Ly6C(hi) cells, which displayed some characteristics of inflammatory dendritic cells and included granulomonocytic precursors, were crucial for anthracycline-induced anticancer immune responses. ATP released by dying cancer cells recruited myeloid cells into tumors and stimulated the local differentiation of CD11c(+)CD11b(+)Ly6C(hi) cells. Such cells efficiently engulfed tumor antigens in situ and presented them to T lymphocytes, thus vaccinating mice, upon adoptive transfer, against a challenge with cancer cells. Manipulations preventing tumor infiltration by CD11c(+)CD11b(+)Ly6C(hi) cells, such as the local overexpression of ectonucleotidases, the blockade of purinergic receptors, or the neutralization of CD11b, abolished the immune system-dependent antitumor activity of anthracyclines. Our results identify a subset of tumor-infiltrating leukocytes as therapy-relevant antigen-presenting cells.

Genetic characterization of B-cell prolymphocytic leukemia: a prognostic model involving MYC and TP53.

B-cell prolymphocytic leukemia (B-PLL) is a rare hematological disorder whose underlying oncogenic mechanisms are poorly understood. Our cytogenetic and molecular assessments of 34 patients with B-PLL revealed several disease-specific features and potential therapeutic targets. The karyotype was complex (≥3 abnormalities) in 73% of the patients and highly complex (≥5 abnormalities) in 45%. The most frequent chromosomal aberrations were translocations involving MYC [t(MYC)] (62%), deletion (del)17p (38%), trisomy (tri)18 (30%), del13q (29%), tri3 (24%), tri12 (24%), and del8p (23%). Twenty-six (76%) of the 34 patients exhibited an MYC aberration, resulting from mutually exclusive translocations or gains. Whole-exome sequencing revealed frequent mutations in TP53, MYD88, BCOR, MYC, SF3B1, SETD2, CHD2, CXCR4, and BCLAF1. The majority of B-PLL used the IGHV3 or IGHV4 subgroups (89%) and displayed significantly mutated IGHV genes (79%). We identified 3 distinct cytogenetic risk groups: low risk (no MYC aberration), intermediate risk (MYC aberration but no del17p), and high risk (MYC aberration and del17p) (P = .0006). In vitro drug response profiling revealed that the combination of a B-cell receptor or BCL2 inhibitor with OTX015 (a bromodomain and extra-terminal motif inhibitor targeting MYC) was associated with significantly lower viability of B-PLL cells harboring a t(MYC). We concluded that cytogenetic analysis is a useful diagnostic and prognostic tool in B-PLL. Targeting MYC may be a useful treatment option in this disease.

Multilayer intraclonal heterogeneity in chronic myelomonocytic leukemia.

The functional diversity of cells that compose myeloid malignancies, i.e., the respective roles of genetic and epigenetic heterogeneity in this diversity, remains poorly understood. This question is addressed in chronic myelomonocytic leukemia, a myeloid neoplasm in which clinical diversity contrasts with limited genetic heterogeneity. To generate induced pluripotent stem cell clones, we reprogrammed CD34+ cells collected from a patient with a chronic myelomonocytic leukemia in which whole exome sequencing of peripheral blood monocyte DNA had identified 12 gene mutations, including a mutation in KDM6A and two heterozygous mutations in TET2 in the founding clone and a secondary KRAS(G12D) mutation. CD34+ cells from an age-matched healthy donor were also reprogrammed. We captured a part of the genetic heterogeneity observed in the patient, i.e. we analyzed five clones with two genetic backgrounds, without and with the KRAS(G12D) mutation. Hematopoietic differentiation of these clones recapitulated the main features of the patient's disease, including overproduction of granulomonocytes and dysmegakaryopoiesis. These analyses also disclosed significant discrepancies in the behavior of hematopoietic cells derived from induced pluripotent stem cell clones with similar genetic background, correlating with limited epigenetic changes. These analyses suggest that, beyond the coding mutations, several levels of intraclonal heterogeneity may participate in the yet unexplained clinical heterogeneity of the disease.

A constitutive BCL2 down-regulation aggravates the phenotype of PKD1-mutant-induced polycystic kidney disease.

The main identified function of BCL2 protein is to prevent cell death by apoptosis. Mouse knock-out for Bcl2 demonstrates growth retardation, severe polycystic kidney disease (PKD), grey hair and lymphopenia, and die prematurely after birth. Here, we report a 40-year-old male referred to for abdominal and thoracic aortic dissection with associated aortic root aneurysm, PKD, lymphocytopenia with a history of T cell lymphoblastic lymphoma, white hair since the age of 20, and learning difficulties. PKD, which was also detected in the father and sister, was related to an inherited PKD1 mutation. The combination of PKD with grey hair and lymphocytopenia was also reminiscent of Bcl2-/- mouse phenotype. BCL2 gene transcript and protein level were observed to be dramatically decreased in patient peripheral blood T-cells and in his aorta vascular wall cells, which was not detected in parents and sister T-cells, suggesting an autosomal recessive inheritance. Accordingly, spontaneous apoptosis of patient T-cells was increased and could be rescued through stimulation with an anti-CD3 antibody. Direct sequencing of BCL2 gene exons, promoter and 3'UTR region as well as BCL2 mRNA sequencing failed in identifying any pathogenic variant. Array-CGH was also normal and whole exome sequencing of the patient, parents and sister DNA did not detect any significant variant in genes encoding BCL2-interacting proteins. miRNA array identified an up-regulation of miR-181a, which is a known regulator of BCL2 expression. Altogether, miR-181a-mediated decrease in BCL2 gene expression could be a modifying factor that aggravates the phenotype of a PKD1 constitutive variant.

High prevalence of myeloid neoplasms in adults with non-Langerhans cell histiocytosis.

Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis that most commonly affects adults and is driven by a high frequency of mutations in BRAF, MAP2K1, and kinases promoting MAPK signaling. Because of the relative rarity of ECD, key clinical features of the disease may not be well defined. Across a multi-institutional cohort of 189 patients with ECD and ECD overlapping with Langerhans cell histiocytosis (so-called mixed histiocytosis [MH]), we identified an unexpected and heretofore undescribed frequent occurrence of myeloid neoplasms among patients with ECD and MH. Some 10.1% (19/189) of patients with ECD have an overlapping myeloid neoplasm, most commonly occurring as a myeloproliferative neoplasm (MPN), myelodysplastic syndrome (MDS), or mixed MDS/MPN overlap syndrome (including chronic myelomonocytic leukemia). Consistent with this, molecular analysis frequently detected hallmark driver mutations of myeloid neoplasms (such as JAK2V617F and CALR mutations) coexisting with those characteristic of histiocytosis (such as BRAFV600E and MAP2K1 mutations). Histiocytosis patients diagnosed with a concomitant myeloid malignancy were significantly older at diagnosis and more commonly presented with MH than those without a myeloid malignancy. In some cases, the presence of distinct kinase mutations in the histiocytosis and myeloid neoplasm resulted in discordant and adverse responses to kinase-directed targeted therapies. These data highlight the clinical importance of evaluating adults with histiocytosis for a concomitant myeloid neoplasm.

Downregulation of TREM-like transcript-1 and collagen receptor α2 subunit, two novel RUNX1-targets, contributes to platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia.

Germline RUNX1 mutations lead to thrombocytopenia and platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia (AML). Multiple aspects of platelet function are impaired in these patients, associated with altered expression of genes regulated by RUNX1 We aimed to identify RUNX1-targets involved in platelet function by combining transcriptome analysis of patient and shRUNX1-transduced megakaryocytes (MK). Down-regulated genes included TREM-like transcript (TLT)-1 (TREML1) and the integrin subunit alpha (α)-2 (ITGA2) of collagen receptor α2-beta (ß)-1, which are involved in platelet aggregation and adhesion, respectively. RUNX1 binding to regions enriched for H3K27Ac marks was demonstrated for both genes using chromatin immunoprecipitation. Cloning of these regions upstream of the respective promoters in lentivirus allowing mCherry reporter expression showed that RUNX1 positively regulates TREML1 and ITGA2, and this regulation was abrogated after deletion of RUNX1 sites. TLT-1 content was reduced in patient MK and platelets. A blocking anti-TLT-1 antibody was able to block aggregation of normal but not patient platelets, whereas recombinant soluble TLT-1 potentiated fibrinogen binding to patient platelets, pointing to a role for TLT-1 deficiency in the platelet function defect. Low levels of α2 integrin subunit were demonstrated in patient platelets and MK, coupled with reduced platelet and MK adhesion to collagen, both under static and flow conditions. In conclusion, we show that gene expression profiling of RUNX1 knock-down or mutated MK provides a suitable approach to identify novel RUNX1 targets, among which downregulation of TREML1 and ITGA2 clearly contribute to the platelet phenotype of familial platelet disorder with predisposition to AML.

Presence of atypical thrombopoietin receptor (MPL) mutations in triple-negative essential thrombocythemia patients.

Mutations in signaling molecules of the cytokine receptor axis play a central role in myeloproliferative neoplasm (MPN) pathogenesis. Polycythemia vera is mainly related to JAK2 mutations, whereas a wider mutational spectrum is detected in essential thrombocythemia (ET) with mutations in JAK2, the thrombopoietin (TPO) receptor (MPL), and the calreticulin (CALR) genes. Here, we studied the mutational profile of 17 ET patients negative for JAK2V617F, MPLW515K/L, and CALR mutations, using whole-exome sequencing and next-generation sequencing (NGS) targeted on JAK2 and MPL. We found several signaling mutations including JAK2V617F at very low allele frequency, 1 homozygous SH2B3 mutation, 1 MPLS505N, 1 MPLW515R, and 2 MPLS204P mutations. In the remaining patients, 4 presented a clonal and 7 a polyclonal hematopoiesis, suggesting that certain triple-negative ETs are not MPNs. NGS on 26 additional triple-negative ETs detected only 1 MPLY591N mutation. Functional studies on MPLS204P and MPLY591N revealed that they are weak gain-of-function mutants increasing MPL signaling and conferring either TPO hypersensitivity or independence to expressing cells, but with a low efficiency. Further studies should be performed to precisely determine the frequency of MPLS204 and MPLY591 mutants in a bigger cohort of MPN.

Acquired TET2 mutation in one patient with familial platelet disorder with predisposition to AML led to the development of pre-leukaemic clone resulting in T2-ALL and AML-M0.

Familial platelet disorder with predisposition to acute myeloid leukaemia (FPD/AML) is characterized by germline RUNX1 mutations, thrombocytopaenia, platelet dysfunction and a risk of developing acute myeloid and in rare cases lymphoid T leukaemia. Here, we focus on a case of a man with a familial history of RUNX1R174Q mutation who developed at the age of 42 years a T2-ALL and, 2 years after remission, an AML-M0. Both AML-M0 and T2-ALL blast populations demonstrated a loss of 1p36.32-23 and 17q11.2 regions as well as other small deletions, clonal rearrangements of both TCRγ and TCRδ and a presence of 18 variants at a frequency of more than 40%. Additional variants were identified only in T2-ALL or in AML-M0 evoking the existence of a common original clone, which gave rise to subclonal populations. Next generation sequencing (NGS) performed on peripheral blood-derived CD34+ cells 5 years prior to T2-ALL development revealed only the missense TET2P1962T mutation at a frequency of 1%, which increases to more than 40% in fully transformed leukaemic T2-ALL and AML-M0 clones. This result suggests that TET2P1962T mutation in association with germline RUNX1R174Q mutation leads to amplification of a haematopoietic clone susceptible to acquire other transforming alterations.