PCR-Fluo-ASXL1-FA: A fast, sensitive and inexpensive complementary method to detect ASXL1 mutations in haematological malignancies.

INTRODUCTION: The additional sex combs like 1 (ASXL1) gene is frequently mutated in a number of haematological neoplasms. The c.1934dupG, known to be the most common alteration in ASXL1, is associated with poor clinical outcome. A systematic determination of ASXL1 mutational status in myeloid malignancies is therefore necessary for prognostic stratification. METHODS: Because direct sequencing is not sensitive and next-generation sequencing (NGS) is time-consuming, expensive and sometimes does not allow the detection of the c.1934dupG, we have developed a fragment analysis assay, complementary to NGS, that allows the detection of c.1934dupG mutation in addition to other nearby insertions/deletions of ASXL1 located close to it. We called this assay the "PCR-Fluo-ASXL1-FA." RESULTS: First, we evaluated the efficiency of our approach compared to NGS and Sanger. We showed that "PCR-Fluo-ASXL1-FA" could detect all insertional mutations of ASXL1 located on its area, with a high sensitivity (1.5%). Then, we have illustrated the interest of this technique by three concrete cases. DISCUSSION: In summary, we have established a fragment analysis approach, which can detect most ASXL1 mutations, in particular the c.1934dupG, in a sensitive, fast and inexpensive manner. We therefore recommend the synchronous use of this method with NGS, to ensure complete detection of all clinically relevant ASXL1 mutations in patients suffering with myeloid neoplasms.

Biological Effects of BET Inhibition by OTX015 (MK-8628) and JQ1 in NPM1-Mutated (NPM1c) Acute Myeloid Leukemia (AML).

BET inhibitors (BETi) including OTX015 (MK-8628) and JQ1 demonstrated antileukemic activity including NPM1c AML cells. Nevertheless, the biological consequences of BETi in NPM1c AML were not fully investigated. Even if of better prognosis AML patients with NPM1c may relapse and treatment remains difficult. Differentiation-based therapy by all trans retinoic acid (ATRA) combined with arsenic trioxide (ATO) demonstrated activity in NPM1c AML. We found that BETi, similar to ATO + ATRA, induced differentiation and apoptosis which was TP53 independent in the NPM1c cell line OCI-AML3 and primary cells. Furthermore, BETi induced proteasome-dependent degradation of NPM1c. BETi degraded NPM1c in the cytosol while BRD4 is degraded in the nucleus which suggests that restoration of the NPM1/BRD4 equilibrium in the nucleus of NPM1c cells is essential for the efficacy of BETi. While ATO + ATRA had significant biological activity in NPM1c IMS-M2 cell line, those cells were resistant to BETi. Gene profiling revealed that IMS-M2 cells probably resist to BETi by upregulation of LSC pathways independently of the downregulation of a core BET-responsive transcriptional program. ATO + ATRA downregulated a NPM1c specific HOX gene signature while anti-leukemic effects of BETi appear HOX gene independent. Our preclinical results encourage clinical testing of BETi in NPM1c AML patients.

[Chronic lymphocytic leukemia with t(14 ;18) and trisomy 12: a case report]. / Leucémie lymphoïde chronique avec t(14;18) et trisomie 12 : un cas clinique.

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm defined by the presence of at least 5×109 G/L monoclonal B lymphocytes in the peripheral blood. It is the most common type of leukemia in adult patients from Western countries. CLL is characterized by a gradual accumulation of small, longliving, immunologically dysfunctional, morphologically mature-appearing B-lymphocytes in blood, bone marrow and lymphoid tissues. It has also been reported that CLL cells have a proliferation rate higher than previously recognized, particularly in the lymphoid tissues. The flow cytometry analysis of typical CLL identifies a monotypic B-cell population expressing a low level of surface immunoglobulins, light chain being either kappa or lambda-, CD5+, CD19+, CD23+, CD79b (dim), negative for FMC7 and CD10. Clinical presentation, course and outcome are highly variable. Interphase fluorescent in situ hybridization (I-FISH) identifies chromosomal abnormalities in about 80% of cases, most commonly involving 13q14 (55%), 11q22-23 (18%), or 17p13 deletions (7%) and trisomy 12 (16%). Therefore, five prognostic categories have been defined with a statistical model, showing the shortest median survival and treatment-free intervals in patients harboring 17p and 11q deletions, followed by trisomy 12 and a normal karyotype, whereas 13q deletion as the sole abnormality is associated with the best prognosis. We report here a rare case of CLL in a 54 year-old-man.

Postinduction Minimal Residual Disease Predicts Outcome and Benefit From Allogeneic Stem Cell Transplantation in Acute Myeloid Leukemia With NPM1 Mutation: A Study by the Acute Leukemia French Association Group.

Purpose This study assessed the prognostic impact of postinduction NPM1-mutated ( NPM1m) minimal residual disease (MRD) in young adult patients (age, 18 to 60 years) with acute myeloid leukemia, and addressed the question of whether NPM1m MRD may be used as a predictive factor of allogeneic stem cell transplantation (ASCT) benefit. Patients and Methods Among 229 patients with NPM1m who were treated in the Acute Leukemia French Association 0702 (ALFA-0702) trial, MRD evaluation was available in 152 patients in first remission. Patients with nonfavorable AML according to the European LeukemiaNet (ELN) classification were eligible for ASCT in first remission. Results After induction therapy, patients who did not achieve a 4-log reduction in NPM1m peripheral blood-MRD (PB-MRD) had a higher cumulative incidence of relapse (subhazard ratio [SHR], 5.83; P < .001) and a shorter overall survival (OS; hazard ratio [HR], 10.99; P < .001). In multivariable analysis, an abnormal karyotype, the presence of FLT3-internal tandem duplication (ITD), and a < 4-log reduction in PB-MRD were significantly associated with a higher relapse incidence and shorter OS. In the subset of patients with FLT3-ITD, only age, white blood cell count, and < 4-log reduction in PB-MRD, but not FLT3-ITD allelic ratio, remained of significant prognostic value. In these patients with nonfavorable AML according to European LeukemiaNet, disease-free survival and OS were significantly improved by ASCT in those with a < 4-log reduction in PB-MRD. This benefit was not observed in those with a > 4-log reduction in PB-MRD, with a significant interaction between ASCT effect and PB-MRD response ( P = .024 and .027 for disease-free survival and OS, respectively). Conclusion Our study supports the strong prognostic significance of early NPM1m PB-MRD, independent of the cytogenetic and molecular context. Moreover, NPM1m PB-MRD may be used as a predictive factor for ASCT indication.

Dasatinib and low-intensity chemotherapy in elderly patients with Philadelphia chromosome-positive ALL.

Prognosis of Philadelphia-positive (Ph(+)) acute lymphoblastic leukemia (ALL) in the elderly has improved during the imatinib era. We investigated dasatinib, another potent tyrosine kinase inhibitor, in combination with low-intensity chemotherapy. Patients older than age 55 years were included in the European Working Group on Adult ALL (EWALL) study number 01 for Ph(+) ALL (EWALL-PH-01 international study) and were treated with dasatinib 140 mg/day (100 mg/day over 70 years) with intrathecal chemotherapy, vincristine, and dexamethasone during induction. Patients in complete remission continued consolidation with dasatinib, sequentially with cytarabine, asparaginase, and methotrexate for 6 months. Maintenance therapy was dasatinib and vincristine/dexamethasone reinductions for 18 months followed by dasatinib until relapse or death. Seventy-one patients with a median age of 69 years were enrolled; 77% had a high comorbidity score. Complete remission rate was 96% and 65% of patients achieved a 3-log reduction in BCR-ABL1 transcript levels during consolidation. Only 7 patients underwent allogeneic hematopoietic stem cell transplantation. At 5 years, overall survival was 36% and up to 45% taking into account deaths unrelated to disease or treatment as competitors. Thirty-six patients relapsed, 24 were tested for mutation by Sanger sequencing, and 75% were T315I-positive. BCR-ABL1(T315I) was tested by allele-specific oligonucleotide reverse transcription-quantitative polymerase chain reaction in 43 patients and detection was associated with short-term relapses. Ten patients (23%) were positive before any therapy and 8 relapsed, all with this mutation. In conclusion, dasatinib combined with low-intensity chemotherapy was well-tolerated and gave long-term survival in 36% of elderly patients with Ph(+) ALL. Monitoring of BCR-ABL1(T315I) from diagnosis identified patients with at high risk of early relapse and may help to personalize therapy.

BET inhibitor OTX015 targets BRD2 and BRD4 and decreases c-MYC in acute leukemia cells.

The bromodomain (BRD) and extraterminal (BET) proteins including BRD2, BRD3 and BRD4 have been identified as key targets for leukemia maintenance. A novel oral inhibitor of BRD2/3/4, the thienotriazolodiazepine compound OTX015, suitable for human use, is available. Here we report its biological effects in AML and ALL cell lines and leukemic samples. Exposure to OTX015 lead to cell growth inhibition, cell cycle arrest and apoptosis at submicromolar concentrations in acute leukemia cell lines and patient-derived leukemic cells, as described with the canonical JQ1 BET inhibitor. Treatment with JQ1 and OTX15 induces similar gene expression profiles in sensitive cell lines, including a c-MYC decrease and an HEXIM1 increase. OTX015 exposure also induced a strong decrease of BRD2, BRD4 and c-MYC and increase of HEXIM1 proteins, while BRD3 expression was unchanged. c-MYC, BRD2, BRD3, BRD4 and HEXIM1 mRNA levels did not correlate however with viability following exposure to OTX015. Sequential combinations of OTX015 with other epigenetic modifying drugs, panobinostat and azacitidine have a synergic effect on growth of the KASUMI cell line. Our results indicate that OTX015 and JQ1 have similar biological effects in leukemic cells, supporting OTX015 evaluation in a Phase Ib trial in relapsed/refractory leukemia patients.

Methylenetetrahydrofolate reductase polymorphism in the etiology of Down syndrome.

A methylenetetrahydrofolate reductase polymorphism (677 C/T mutation) was recently implicated in the etiology of Down syndrome. We studied a cohort of 85 women carrying fetuses with Down syndrome and found no difference in the frequencies of the three groups of subjects (C/C, C/T, T/T) between Down syndrome mothers and controls.