Norbert Vey

Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia

The myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal haematological diseases characterized by ineffective haematopoiesis and predisposition to acute myeloid leukaemia (AML). The pathophysiology of MDSs remains unclear. A definition of the molecular biology of MDSs may lead to a better classification, new prognosis indicators and new treatments. We studied a series of 40 MDS/AML samples by high‐density array‐comparative genome hybridization (aCGH). The genome of MDSs displayed a few alterations that can point to candidate genes, which potentially regulate histone modifications and WNT pathways (e.g. ASXL1, ASXL2, UTX, CXXC4, CXXC5, TET2, TET3). To validate some of these candidates we studied the sequence of ASXL1. We found mutations in the ASXL1 gene in four out of 35 MDS patients (11%). To extend these results we searched for mutations of ASXL1 in a series of chronic myelomonocytic leukaemias, a disease classified as MDS/Myeloproliferative disorder, and found mutations in 17 out of 39 patients (43%). These results show that ASXL1 might play the role of a tumour suppressor in myeloid malignancies.

Outcome of Treatment in Adults With Acute Lymphoblastic Leukemia: Analysis of the LALA-94 Trial

PURPOSE We analyzed the benefits of a risk-adapted postremission strategy in adult lymphoblastic leukemia (ALL), and re-evaluated stem-cell transplantation (SCT) for high-risk ALL. PATIENTS AND METHODS A total of 922 adult patients entered onto the trial according to risk groups: standard-risk ALL (group 1), high-risk ALL (group 2), Philadelphia chromosome-positive ALL (group 3), and CNS-positive ALL (group 4). All received a standard four-drug/4-week induction course. Patients from group 1 who achieved a complete remission (CR) after one course of induction therapy were randomly assigned between intensive and less intensive postremission chemotherapy, whereas those who achieved CR after salvage therapy were then included in group 2. Patients in groups 2, 3, and 4 with an HLA-identical sibling were assigned to allogeneic SCT. In groups 3 and 4, autologous SCT was offered to all other patients, whereas in group 2 they were randomly assigned between chemotherapy and autologous SCT. RESULTS Overall, 771 patients achieved CR (84%). Median disease-free survival (DFS) was 17.5 months, with 3-year DFS at 37%. In group 1, the 3-year DFS rate was 41%, with no difference between arms of postremission randomization. In groups 2 and 4, the 3-year DFS rates were 38% and 44%, respectively. In group 2, autologous SCT and chemotherapy resulted in comparable median DFS. Patients with an HLA-matched sibling (groups 2 and 4) had improved DFS. Three-year DFS was 24% in group 3. CONCLUSION Allogeneic SCT improved DFS in high-risk ALL in the first CR. Autologous SCT did not confer a significant benefit over chemotherapy for high-risk ALL.

Impact of TET2 mutations on response rate to azacitidine in myelodysplastic syndromes and low blast count acute myeloid leukemias.

The impact of ten-eleven-translocation 2 (TET2) mutations on response to azacitidine (AZA) in MDS has not been reported. We sequenced the TET2 gene in 86 MDS and acute myeloid leukemia (AML) with 20–30% blasts treated by AZA, that is disease categories wherein this drug is approved by Food and Drug Administration (FDA). Thirteen patients (15%) carried TET2 mutations. Patients with mutated and wild-type (WT) TET2 had mostly comparable pretreatment characteristics, except for lower hemoglobin, better cytogenetic risk and longer MDS duration before AZA in TET2 mutated patients (P=0.03, P=0.047 and P=0.048, respectively). The response rate (including hematological improvement) was 82% in MUT versus 45% in WT patients (P=0.007). Mutated TET2 (P=0.04) and favorable cytogenetic risk (intermediate risk: P=0.04, poor risk: P=0.048 compared with good risk) independently predicted a higher response rate. Response duration and overall survival were, however, comparable in the MUT and WT groups. In higher risk MDS and AML with low blast count, TET2 status may be a genetic predictor of response to AZA, independently of karyotype.

Prognostic factors for response and overall survival in 282 patients with higher-risk myelodysplastic syndromes treated with azacitidine

Prognostic factors for response and survival in higher-risk myelodysplastic syndrome patients treated with azacitidine (AZA) remain largely unknown. Two hundred eighty-two consecutive high or intermediate-2 risk myelodysplastic syndrome patients received AZA in a compassionate, patient-named program. Diagnosis was RA/RARS/RCMD in 4%, RAEB-1 in 20%, RAEB-2 in 54%, and RAEB-t (AML with 21%-30% marrow blasts) in 22%. Cytogenetic risk was good in 31%, intermediate in 17%, and poor in 47%. Patients received AZA for a median of 6 cycles (1-52). Previous low-dose cytosine arabinoside treatment (P = .009), bone marrow blasts > 15% (P = .004), and abnormal karyotype (P = .03) independently predicted lower response rates. Complex karyotype predicted shorter responses (P = .0003). Performance status ≥ 2, intermediate- and poor-risk cytogenetics, presence of circulating blasts, and red blood cell transfusion dependency ≥ 4 units/8 weeks (all P < 10(-4)) independently predicted poorer overall survival (OS). A prognostic score based on those factors discriminated 3 risk groups with median OS not reached, 15.0 and 6.1 months, respectively (P < 10(-4)). This prognostic score was validated in an independent set of patients receiving AZA in the AZA-001 trial (P = .003). Achievement of hematological improvement in patients who did not obtain complete or partial remission was associated with improved OS (P < 10(-4)). In conclusion, routine tests can identify subgroups of patients with distinct prognosis with AZA treatment.

Prognostic Score Including Gene Mutations in Chronic Myelomonocytic Leukemia

PURPOSE Several prognostic scoring systems have been proposed for chronic myelomonocytic leukemia (CMML), a disease in which some gene mutations-including ASXL1-have been associated with poor prognosis in univariable analyses. We developed and validated a prognostic score for overall survival (OS) based on mutational status and standard clinical variables. PATIENTS AND METHODS We genotyped ASXL1 and up to 18 other genes including epigenetic (TET2, EZH2, IDH1, IDH2, DNMT3A), splicing (SF3B1, SRSF2, ZRSF2, U2AF1), transcription (RUNX1, NPM1, TP53), and signaling (NRAS, KRAS, CBL, JAK2, FLT3) regulators in 312 patients with CMML. Genotypes and clinical variables were included in a multivariable Cox model of OS validated by bootstrapping. A scoring system was developed using regression coefficients from this model. RESULTS ASXL1 mutations (P < .0001) and, to a lesser extent, SRSF2 (P = .03), CBL (P = .003), and IDH2 (P = .03) mutations predicted inferior OS in univariable analysis. The retained independent prognostic factors included ASXL1 mutations, age older than 65 years, WBC count greater than 15 ×10(9)/L, platelet count less than 100 ×10(9)/L, and anemia (hemoglobin < 10 g/dL in female patients, < 11g/dL in male patients). The resulting five-parameter prognostic score delineated three groups of patients with median OS not reached, 38.5 months, and 14.4 months, respectively (P < .0001), and was validated in an independent cohort of 165 patients (P < .0001). CONCLUSION A new prognostic score including ASXL1 status, age, hemoglobin, WBC, and platelet counts defines three groups of CMML patients with distinct outcomes. Based on concordance analysis, this score appears more discriminative than those based solely on clinical parameters.

Mutations of ASXL1 gene in myeloproliferative neoplasms

measure epoxomicin uptake we utilized another P-gP substrate to determine if drug efflux is altered in KMS11R cells. Doxorubicin is an established substrate for P-gP and it can be easily measured in the cell because it fluoresces red. We compared the fluorescence of KMS11 and KMS11R cells treated with various concentrations of doxorubicin for 24 h. No differences in background fluorescence were observed and both cell lines could take up doxorubicin in a dose-dependent fashion (Figure 2b). However, the uptake observed was 3–5 fold lower in the resistant line. In addition, KMS11R cells are significantly resistant to doxorubicin-induced cell death (not shown). Finally, we determined the role of P-gP in the acquired resistance to epoxomicin by observing the effects of inhibiting PgP function with verapamil. Verapamil had no effect on the viability of either cell line nor did it alter the sensitivity of KMS11 cells to epoxomicin (Figure 3a) or doxorubicin (not shown). In contrast verapamil resensitized KMS11R to both drugs (Figure 3a and not shown). FACS analysis of cells treated with doxorubicin and verapamil revealed that the addition of verapamil to the KMS11R cells resulted in an increase in red fluorescence in doxorubicin-treated cells (Figure 3b). Given the promise of bortezomib it is not surprising that other proteasome inhibitors that are either irreversible and/or inhibit the other activities of the proteasome are currently being tested in the clinic. As proteasome inhibitors become more commonly used in newly diagnosed diseases it will be important to understand mechanisms of acquired resistance to this class of agents. In addition, we will need to know how acquired resistance to proteasome inhibitors alter the response to other therapeutics used to treat myeloma patients. Acquired expression of P-gP has been observed in myeloma patients and cell lines. P-gP is rarely seen in newly diagnosed patients, however, increased expression was observed in cells from approximately 75% of patients treated with vincristine, doxorubicin and dexamethasone. We observed that P-gP is expressed and the P-gP inhibitor verapimil sensitizes KMS11R to epoxomicin. These data suggest that the acquired resistance observed was the result of P-gP expression. This is the first report to demonstrate the acquisition of P-gP with a proteasome inhibitor. Although it is possible that the induction of P-gP could be the result of inhibition of protein turnover, this does not appear to be a likely mechanism. First epoxomicin does not acutely induce P-gP (Figure 2). More importantly we also observe increased expression of the MDR mRNA in these cells (not shown). Thus the mechanism is likely to be due to increased gene expression possibly because of gene amplification. This remains to be determined. Finally, our data suggest that carfilzomib could be ineffective in the treatment of P-gP-positive myeloma. Consistent with this possibility, a recent study demonstrated that the P-gP-positive cell line, RPMI8226-Dox40, is resistant to carfilzomib; however, they can be sensitized by co-treatment with verapamil. Unfortunately such resensitization of cells to P-gP substrates has not proven to be effective clinically. Several agents that inhibit drug efflux have been tested and either proven to be too toxic or had little effect on efficacy. In addition to confirming that epoxomicin-based compounds may be subject to this type of resistance mechanism we demonstrated that they can initiate resistance in this fashion. Therefore careful consideration of the use of these compounds both as single agents and in combination therapies will be needed to assure both efficacy of this therapy as well as subsequent treatment regimens.

Outcome of High-Risk Myelodysplastic Syndrome After Azacitidine Treatment Failure

PURPOSE Azacitidine (AZA) is the current standard of care for high-risk (ie, International Prognostic Scoring System high or intermediate 2) myelodysplastic syndrome (MDS), but most patients will experience primary or secondary treatment failure. The outcome of these patients has not yet been described. PATIENTS AND METHODS Overall, 435 patients with high-risk MDS and former refractory anemia with excess blasts in transformation (RAEB-T) were evaluated for outcome after AZA failure. The cohort of patients included four data sets (ie, AZA001, J9950, and J0443 trials and the French compassionate use program). RESULTS The median follow-up after AZA failure was 15 months. The median overall survival was 5.6 months, and the 2-year survival probability was 15%. Increasing age, male sex, high-risk cytogenetics, higher bone marrow blast count, and the absence of prior hematologic response to AZA were associated with significantly worse survival in multivariate analysis. Data on treatment administered after AZA failure were available for 270 patients. Allogeneic stem-cell transplantation and investigational agents were associated with a better outcome when compared with conventional clinical care. CONCLUSION Outcome after AZA failure is poor. Our results should serve as a basis for designing second-line clinical trials in this population.

TET2 mutation is an independent favorable prognostic factor in myelodysplastic syndromes (MDSs)

Oncogenic pathways underlying in the development of myelodysplastic syndromes (MDS) remain poorly characterized, but mutations of the ten-eleven translocation 2 (TET2) gene are frequently observed. In the present work, we evaluated the prognostic impact of TET2 mutations in MDS. Frameshift, nonsense, missense mutations, or defects in gene structure were identified in 22 (22.9%) of 96 patients (95% confidence interval [CI], 14.5-31.3 patients). Mutated and unmutated patients did not significantly differ in initial clinical or hematologic parameters. The 5-year OS was 76.9% (95% CI, 49.2%-91.3%) in mutated versus 18.3% (95% CI, 4.2%-41.1%) in unmutated patients (P = .005). The 3-year leukemia-free survival was 89.3% (95% CI, 63.1%-97.0%) in mutated versus 63.7% (95% CI, 48.2%-75.4%) in unmutated patients (P = .035). In univariate analysis (Cox proportional hazard model), the absence of TET2 mutation was associated with a 4.1-fold (95% CI, 1.4-12.0-fold) increased risk of death (P = .009). In multivariate analysis adjusted for age, International Prognostic Scoring System, and transfusion requirement, the presence of TET2 mutation remained an independent factor of favorable prognosis (hazard ratio, 5.2; 95% CI, 1.6-16.3; P = .005). These results indicate that TET2 mutations observed in approximately 20% of patients, irrespective of the World Health Organization or French-American-British subtype, represent a molecular marker for good prognosis in MDS.

Mutations of IDH1 and IDH2 genes in early and accelerated phases of myelodysplastic syndromes and MDS/myeloproliferative neoplasms

Mutations of IDH1 and IDH2 genes in early and accelerated phases of myelodysplastic syndromes and MDS/myeloproliferative neoplasms

TET2 gene mutation is a frequent and adverse event in chronic myelomonocytic leukemia

Acquired somatic deletions and loss-of-function mutations in one or several codons of the TET2 (Ten-Eleven Translocation-2) gene were recently identified in hematopoietic cells from patients with myeloid neoplasms. This study shows that TET2 mutations are more frequent in chronic myelomonocytic leukemia than in other subgroups of myeloid neoplasms studied so far. Background Acquired somatic deletions and loss-of-function mutations in one or several codons of the TET2 (Ten-Eleven Translocation-2) gene were recently identified in hematopoietic cells from patients with myeloid malignancies, including myeloproliferative disorders and myelodys-plastic syndromes. The present study was designed to determine the prevalence of TET2 gene alterations in chronic myelomonocytic leukemias. Design and Methods Blood and bone marrow cells were collected from 88 patients with chronic phase chronic myelomonocytic leukemia and from 14 with acute transformation of a previously identified disease. Polymerase chain reaction analysis and direct sequencing were used to sequence exons 3 to 11 of the TET2 gene. Annotated single nucleotide polymorphisms were excluded. Survival curves were constructed by the Kaplan-Meier method. Results We detected TET2 mutations in 44 of 88 (50%) patients with chronic myelomonocytic leukemia, which suggests that TET2 gene mutations are especially frequent in this myeloid disease. A TET2 gene alteration was identified in 18 of the 43 patients studied at diagnosis and was associated with a trend to a lower overall survival rate; confining the analysis to the 29 patients with chronic myelomonocytic leukemia-1, according to the WHO classification, the difference in overall survival between patients with or without TET2 gene mutations became statistically significant. Conclusions TET2 gene alterations are more frequent in chronic myelomonocytic leukemia than in other subgroups of hematopoietic diseases studied so far and could negatively affect the patients’ outcome. The striking association between TET2 gene alterations and monocytosis, already observed in patients with systemic mastocytosis, could indicate a negative role of TET2 in the control of monocytic lineage determination.