Marie Loosveld

Posttranscriptional deregulation of MYC via PTEN constitutes a major alternative pathway of MYC activation in T-cell acute lymphoblastic leukemia

Cumulative evidence indicates that MYC, one of the major downstream effectors of NOTCH1, is a critical component of T-cell acute lymphoblastic leukemia (T-ALL) oncogenesis and a potential candidate for targeted therapy. However, MYC is a complex oncogene, involving both fine protein dosage and cell-context dependency, and detailed understanding of MYC-mediated oncogenesis in T-ALL is still lacking. To better understand how MYC is interspersed in the complex T-ALL oncogenic networks, we performed a thorough molecular and biochemical analysis of MYC activation in a comprehensive collection of primary adult and pediatric patient samples. We find that MYC expression is highly variable, and that high MYC expression levels can be generated in a large number of cases in absence of NOTCH1/FBXW7 mutations, suggesting the occurrence of multiple activation pathways in addition to NOTCH1. Furthermore, we show that posttranscriptional deregulation of MYC constitutes a major alternative pathway of MYC activation in T-ALL, operating partly via the PI3K/AKT axis through down-regulation of PTEN, and that NOTCH1(m) might play a dual transcriptional and posttranscriptional role in this process. Altogether, our data lend further support to the significance of therapeutic targeting of MYC and/or the PTEN/AKT pathways, both in GSI-resistant and identified NOTCH1-independent/MYC-mediated T-ALL patients.

Identification of a new stromal cell type involved in the regulation of inflamed B cell follicles.

Identification of a new stromal cell type in mouse lymph nodes that can be activated by B cells to delineate the transient boundaries of B cell zones during inflammation

Effect of CYP2C19*2 and *17 Genetic Variants on Platelet Response to Clopidogrel and Prasugrel Maintenance Dose and Relation to Bleeding Complications

The present study was performed to compare the influence of cytochrome P459 2C19 (CYP2C19) *2 and *17 genetic variants on the platelet response to clopidogrel and prasugrel maintenance therapy and to assess the relation between platelet reactivity and bleeding complications. A total of 730 patients were included (517 patients treated with clopidogrel 150 mg/day and 213 discharged with prasugrel 10 mg). Platelet reactivity was assessed at 1 month with the platelet reactivity index vasodilator-stimulated phosphoprotein (PRI VASP). High on-treatment platelet reactivity was defined as PRI VASP >50% and low on-treatment platelet reactivity (LTPR) as PRI VASP <20%. The patients were classified according to their genotypes as poor metabolizers (*2/non *17), intermediate metabolizers (*2/*17 or non *2/non *17) and ultrametabolizers (non *2/*17). At 1 month, the prasugrel response was significantly better than the clopidogrel response in all groups of patients, with a lower incidence of high on-treatment platelet reactivity but a greater incidence of LTPR, regardless of the genetic variants. The genetic distribution had a significant effect on the mean PRI VASP values, the incidence of high on-treatment platelet reactivity, and LTPR with both clopidogrel and prasugrel (p <0.05 for all). LTPR identified a group of patients at a greater risk of bleeding (odds ratio 4.8, 95% confidence interval 2.7 to 8.3; p <0.0001). In conclusion, the present study showed that both clopidogrel and prasugrel have genetic modulation by CYP2C19 *2 and *17 alleles and that prasugrel provides greater platelet inhibition, regardless of the genotypes. In addition, LTPR was associated with a greater risk of bleeding.

Two Types of Procoagulant Platelets Are Formed Upon Physiological Activation and Are Controlled by Integrin α IIb β 3

Objective—Phosphatidylserine (PS) externalization by platelets upon activation is a key event in hemostasis and thrombosis. It is currently believed that strong stimulation of platelets forms 2 subpopulations, only 1 of which expresses PS. Methods and Results—Here, we demonstrate that physiological stimulation leads to the formation of not 1 but 2 types of PS-expressing activated platelets, with dramatically different properties. One subpopulation sustained increased calcium level after activation, whereas another returned to the basal low-calcium state. High-calcium PS-positive platelets had smaller size, high surface density of fibrin(ogen), no active integrin &agr;IIb&bgr;3, depolarized mitochondrial membranes, gradually lost cytoplasmic membrane integrity, and were poorly aggregated. In contrast, the low-calcium PS-positive platelets had normal size, retained mitochondrial membrane potential and cytoplasmic membrane integrity, and combined retention of fibrin(ogen) with active &agr;IIb&bgr;3 and high proaggregatory function. Formation of low-calcium PS-positive platelets was promoted by platelet concentration increase or shaking and was decreased by integrin &agr;IIb&bgr;3 antagonists, platelet dilution, or in platelets from kindlin-3–deficient and Glanzmann thrombasthenia patients. Conclusion—Identification of a novel PS-expressing platelet subpopulation with low calcium regulated by integrin &agr;IIb&bgr;3 can be important for understanding the mechanisms of PS exposure and thrombus formation.

Platelet reactivity in diabetic patients undergoing coronary stenting for acute coronary syndrome treated with clopidogrel loading dose followed by prasugrel maintenance therapy

BACKGROUND Diabetes has been identified as a risk factor for impaired clopidogrel response, and these patients might have greater benefit with new P2Y12 blockers such as prasugrel. The present study was designed to assess response to thienopyridine in diabetic patients undergoing PCI for ACS. METHODS AND RESULTS 107 diabetic patients undergoing PCI for ACS were included and treated by clopidogrel 600 mg loading dose and switched to prasugrel 10mg daily after PCI. Platelet reactivity was assessed by PRI VASP. High-on-treatment platelet reactivity (HTPR) was defined by PRI VASP>50% and Low-on-treatment platelet reactivity (LTPR) as PRI VASP below the 75th percentile (PRI VASP<20%). After clopidogrel, mean PRI VASP was 47 ± 21% and 54 patients (50%) were non responders. At one month, mean PRI VASP on prasugrel 10mg daily was 31 ± 13%, 9 patients (8%) had HTPR and 23 patients (22%) had LTPR. In multivariate analysis, factors associated with platelet reactivity were waist circumference for HTPR on clopidogrel and body weight for HTPR and LTPR on prasugrel. 10 patients (9%) suffered from BARC bleeding complications. Patients with bleeding complications had significantly lower PRI VASP values: 22 ± 9 vs. 32 ± 13, p=0.02 and ROC curves identified a cut-off value of VASP=28% to predict bleeding complications. CONCLUSION The present study confirmed that many diabetic patients treated with clopidogrel for ACS have inadequate platelet inhibition. Switch to prasugrel is effective with acceptable safety in this specific population. We observed a significant relationship between on-treatment platelet reactivity and bleeding complications.

MYC Fails to Efficiently Shape Malignant Transformation in T-cell Acute Lymphoblastic Leukemia

MYC is a potent oncogene involved in ∼70% of human cancers, inducing tumorigenesis with high penetrance and short latency in experimental transgenic models. Accordingly, MYC is recognized as a major driver of T‐cell acute lymphoblastic leukemia (T‐ALL) in human and zebrafish/mouse models, and uncovering the context by which MYC‐mediated malignant transformation initiates and develops remains a considerable challenge. Because MYC is a very complex oncogene, highly dependent on the microenvironment and cell‐intrinsic context, we generated transgenic mice (tgMycspo) in which ectopic Myc activation occurs sporadically (<10−6 thymocytes) within otherwise normal thymic environment, thereby mimicking the unicellular context in which oncogenic alterations initiate human tumors. We show that while Myc+ clones in tgMycspo mice develop and initially proliferate in thymus and the periphery, no tumor or clonal expansion progress in aging mice (n = 130), suggesting an unexpectedly low ability of Myc to initiate efficient tumorigenesis. Furthermore, to determine the relevance of this observation in human pathogenesis we analyzed a human T‐ALL case at diagnosis and relapse using the molecular stigmata of V(D)J recombination as markers of malignant progression; we similarly demonstrate that despite the occurrence of TAL1 and MYC translocations in early thymocyte ontogeny, subsequent oncogenic alterations were required to drive oncogenesis. Altogether, our data suggest that although central to T‐ALL, MYC overexpression per se is inefficient in triggering the cascade of events leading to malignant transformation.

Major impact of an early bone marrow checkpoint (day 21) for minimal residual disease in flow cytometry in childhood acute lymphoblastic leukemia.

The early persistence of minimal residual disease (MRD) is considered a poor prognostic factor indicative of chemoresistance in acute lymphoblastic leukemia. In French children, chemosensitivity is assessed at day 21 post‐induction by cytomorphology. Here, it was investigated whether a more precise evaluation could be obtained at this time point with multiparameter flow cytometry (MFC).

Lineage switch from B acute lymphoblastic leukemia to acute monocytic leukemia with persistent t(4;11)(q21;q23) and cytogenetic evolution under CD19-targeted therapy

Dear Editor, We here report a case of B-acute lymphoblastic leukemia (ALL) with KMT2A rearrangement that underwent a lineage switch to acute monocytic leukemia following CD19-targeted therapy. A 15-year-old male patient presented with leukocytosis (white blood cells, 210 × 10/L, anemia (hemoglobin, 78 g/L), thrombocytopenia (platelets, 46 × 10 /L) and 97% peripheral blasts. Bone marrow (BM) aspirate (Fig. 1a) and flow cytometry diagnosed B1-ALL with positivity for cCD79a, CD19, CD22low and CD34 and negativity for CD10 and anymyeloid markers (Fig. 1d, purple). Cytogenetic analysis showed the translocation t(4;11)(q21;q23) leading to KMT2AAFF1 (also referred as MLL-AF4) rearrangement as the sole abnormality (Fig. 1e). The patient was first enrolled in the FRALLE 2000 protocol. Three months later, because of refractory disease, a CD19-targeted therapy using blinatumomab was initiated at 5 μg/m/day for the first 7 days and then had been escalated to 15 μg/m2/day starting from day 8 to day 29 according to the RIALTO protocol. At day 28, BM aspirate revealed monocytic blasts (Fig. 1b) positive for CD33 (low), CD65 and CD15 and negative for B lineage markers (cCD79a, CD19, CD22, CD34) (Fig. 1d, yellow). Review of flow cytometry analysis prior to blinatumomab treatment demonstrated no monocytic subclones (with a sensitivity of 0.1%). Moreover, blasts showed a cytogenetic evolution to a near-triploid karyotype associated with the persistent t(4;11)(q21;q23) (Fig. 1f). Deep sequencing of the IGH gene in BM aspirate revealed persistence of the malignant clone (JH4) identified before blinatumomab treatment associated with persistence of the KMT2A-AFF1 transcript (Fig. 1c). These findings suggest that the lymphoid blasts underwent a lineage switch to acute monocytic leukemia or that lymphoid and monocytic blasts were clonally related. Despite the use of acute myeloid leukemia protocol, the patient died of disease progression. Although rare, lineage switch may occur following therapy or at relapse. Lineage switch is also a characteristic feature of KMT2A-rearranged acute leukemia cases [1]. Gene expression profiles of KMT2A-rearranged leukaemias resemble those of early hematopoietic progenitors, supporting the idea that the essential transformation event targets an early hematopoietic progenitor that is still able to differentiate whatever the lineage [2]. However, other mechanisms explaining the biology of lineage switch are the subject of ongoing debates: cell reprogramming, de-differentiation or clonal selection [3]. In our case, the persistence of the translocation t(4;11)(q21;q23) associated with cytogenetic evolution suggests that the monocytic leukemia results from an evolution of the original clone. Moreover, the persistence of the IGH rearrangement in the myeloid blast cells might exclude the * Marie Loosveld marie.loosveld@ap-hm.fr

Early morphological diagnosis of Farber disease.

A 6-week-old infant girl was hospitalized for prolonged fever. This North African child was born at term from consanguineous parents without relevant family history. Pregnancy, birth and the neonatal period were uneventful until 6 weeks of age. On admission, her body weight was 3 5 kg [>1 standard deviation (SD) below mean for age] and height was 53 cm (>1 SD below mean for age). A hoarse cry was noted. Moderate hepatosplenomegaly and peripheral blood features of haemophagocytic syndrome were observed: hyperferritinaemia (1656 lg/l), hypertriglyceridaemia (6 84 mmol/l), elevated aspartate transaminase (97 iu/l) and normocytic aregenerative anaemia (haemoglobin concentration 77 g/l) without other cytopenias. Cerebrospinal fluid analysis showed an elevated protein concentration without any organisms. Cytomegalovirus polymerase chain reaction (PCR) was positive (5040 copy/ml) and a primary infection was suspected. A bone marrow aspirate (May-Gr€ unwald-Giemsa stained) showed a highly cellular bone marrow with haemophagocytic histiocytes and large cells with round to oval nuclei and foamy cytoplasm, arranged in clusters and resembling granulomas (images). Fever, hepatosplenomegaly and cytopenia are relatively nonspecific. When associated with inappropriate activation of macrophages, diagnoses to be considered include: visceral leishmaniasis, primary or secondary haemophagocytic lymphohistiocytosis (HLH) and storage disorders. No intrahistiocytic parasites were observed in the bone marrow, excluding the diagnosis of leishmaniasis. Normal natural killer cell degranulation and perforin expression excluded primary HLH. The diagnosis of a storage disease was strongly suspected. At 5 months of age, persistent fever was associated with noisy respiration and retardation in motor development. Physical examination showed hypotonia, hyporeflexia, nodular deformities of fingers, periarticular swelling in elbows and knees and marked flexion contractures of major joints. The diagnosis of type I Farber disease (classical form) was confirmed by determination of acid ceramidase activity and by sequencing of the human acid ceramidase gene (ASAH1). The patient died few weeks later. Farber disease, also called Farber lipogranulomatosis, describes one of a rare group of inherited metabolic disorders called lipid storage diseases with a high clinical variability between patients. In this patient the discovery of very suggestive storage histiocytes in the bone marrow aspirate in an appropriate clinical context led to the identification of an ASAH1 mutation and definitive diagnosis.

Interphase FISH for BCR-ABL1 rearrangement on neutrophils: A decisive tool to discriminate a lymphoid blast crisis of chronic myeloid leukemia from a de novo BCR-ABL1 positive acute lymphoblastic leukemia

Discrimination between lymphoid blast crisis of chronic myeloid leukemia (CML) and de novo BCR‐ABL1 positive acute lymphoblastic leukemia (ALL) represents a diagnostic challenge because this distinction has a major incidence on the management of patients. Here, we report an uncommon pediatric case of ALL with cryptic ins(22;9)(q11;q34q34) and p190‐type BCR‐ABL1 transcript. We performed interphase fluorescence in situ hybridization (FISH) for BCR‐ABL1 rearrangement on blood neutrophils, which was positive consistent with the diagnosis of lymphoid blast crisis of CML. This case illustrates the major interest of interphase FISH for BCR‐ABL1 rearrangement on blood neutrophils as a decisive method to discriminate a lymphoid blast crisis of CML from a de novo BCR‐ABL1 positive ALL.