Acquired mutations in ASXL1 in acute myeloid leukemia: prevalence and prognostic value

Somatic mutations in the additional sex comb-like 1 (ASXL1) gene have been described in various types of myeloid malignancies, including acute myeloid leukemia. Analysis of novel markers, such as ASXL1 mutations, in independent clinical trials is indispensable before considering them for clinical decision-making. We analyzed 882 well-characterized acute myeloid leukemia cases to determine the prevalence and prognostic impact of ASXL1 exon12 mutations. Truncating ASXL1 mutations were present in 46 cases (5.3%). ASXL1 mutations were inversely associated with FLT3 internal tandem duplications and mutually exclusive with NPM1 mutations. ASXL1 mutations were an unfavorable prognostic factor as regards survival (median overall survival 15.9 months vs. 22.3 months; P=0.019), with a significantly lower complete response rate (61% vs. 79.6%; P=0.004). In multivariate analyses, ASXL1 mutations were independently associated with inferior poor overall survival (HR 1.52, P=0.032). In conclusion, ASXL1 mutations are common mutations in acute myeloid leukemia and indicate a poor therapy outcome.     

TET2 deletions are a recurrent but rare phenomenon in myeloid malignancies and are frequently accompanied by TET2 mutations on the remaining allele

TET2 mutations have been identified in 10-25% of patients with myeloid malignancies, but TET2 gene copy number alterations remain to be evaluated. We performed interphase and metaphase fluorescence in situ hybridization (FISH), using newly designed probes that span TET2, in 893 patients with acute myeloid leukaemia (AML) and chronic myeloid malignancies and validated the new assay by single nucleotide polymorphism arrays. Next-generation sequencing for molecular TET2 mutations was performed in 37/50 del(TET2) patients. We identified TET2 deletions in 50/893 patients (26 males, 24 females; 44-87 years) resulting in a 5.6% frequency [22/425 AML (5.2%), 15/217 chronic myelomonocytic leukaemia (CMML; 6.9%), 9/188 myelodysplastic syndromes (4.8%), 4/63 myeloproliferative neoplasms (6.3%)]. Cytogenetic alterations (mostly complex) were detected in 35/50 (70.0%) of del(TET2) cases. TET2 deletions were cytogenetically cryptic in 25/50 (50.0%). Sequencing detected TET2mut in 19/37 (51.4%) del(TET2) cases investigated, predominantly CMML (10/14; 71.4%). In de novo AML with intermediate cytogenetics, presence of any TET2 alteration was related to worse median overall survival (347 d vs. not reached; P = 0.052) and event-free survival (164 vs. 457 d; P = 0.024). JAK2(V617F) was found in 6/18 (33.3%) del(TET2) cases analysed. Thus, TET2 haploinsufficiency recurrently occurs in myeloid malignancies, frequently combined with TET2 mutations on the remaining allele. The prognostic impact of del(TET2) in myeloid malignancies should be further evaluated.     

Frequency, onset and clinical impact of somatic DNMT3A mutations in therapy-related and secondary acute myeloid leukemia

The recent identification of DNMT3A mutations in de novo acute myeloid leukemia prompted us to determine their frequency, patterns and clinical impact in a cohort of 98 patients with either therapy-related or secondary acute myeloid leukemia developing from an antecedent hematologic disorder. We identified 24 somatic mutations in 23 patients with a significantly higher frequency in secondary acute myeloid leukemia (35.1%) as compared to therapy-related acute myeloid leukemia (16.4%, P=0.0486). DNMT3A mutations were associated with a normal karyotype and IDH1/2 mutations, but did not affect survival. In contrast to de novo acute myeloid leukemia, most mutations did not affect arginine on position 882, but were predominantly found in the methyltransferase domain. All DNMT3A mutations identified in secondary acute myeloid leukemia were already present in the antecedent disorders indicating an early event. Reduction to homozygosity by uniparental disomy was observed in 2 patients with secondary acute myeloid leukemia during disease progression.     

A risk of essential thrombocythemia in carriers of constitutional CHEK2 gene mutations

Germline mutations of the CHEK2 gene have been reported in some myeloid and lymphoid malignancies, but their impact on development of essential thrombocythemia has not been studied. In 16 out of 106 (15.1%) consecutive patients, newly diagnosed with essential thrombocythemia, we found one of four analyzed CHEK2 mutations: I157T, 1100delC, IVS2+1G>A or del5395. They were associated with the increased risk of disease (OR=3.8; P=0.002). The median age at ET diagnosis among CHEK2+/JAK2V617F+ patients was seven years lower than that among CHEK2-/JAK2V617F+ (52 vs. 59 years; P=0.04), whereas there was no difference in the medians of hematologic parameters between these groups. The results obtained suggest that CHEK2 mutations could potentially contribute to the susceptibility to essential thrombocythemia. The germline inactivation of CHEK2, as it seems, has no direct impact on the development of disease, but it could cause disruption of cell cycle checkpoints and initiate or support the cancerogenic process of essential thrombocythemia at a younger age.     

Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia

Genomic DNA from 97 cases of adult de novo acute myeloid leukaemia (AML) was screened using polymerase chain reaction (PCR) and conformation-sensitive gel electrophoresis (CSGE) for FLT3 exon 20 mutations. Initial sequencing of four cases, representing the spectrum of CSGE abnormalities, revealed changes affecting codon Asp835 in three cases and also an intron 20 A to G change. In order to identify all possible Asp835 alterations, as well as the frequency of the intronic change nucleotide 2541 + 57 A-->G, the patient PCR products were digested with EcoRV and NlaIII respectively. Seven cases (7.2%) possessed a mutation affecting Asp835; these were identified, following DNA sequencing, as Asp835Tyr (n = 5), Asp835His (n = 1) and Asp835del (n = 1). Alterations affecting Asp835 were not found in 80 normal control DNA samples. In contrast, the nucleotide 2541 + 57 A-->G change was shown to be a polymorphism, with an allelic frequency of 0.24 for the G and 0.76 for the A allele. This study reports, for the first time, point mutations in the human FLT3 gene that, because of their homology with other class III receptor tyrosine kinase mutations, probably result in constitutive activation of the receptor.     

The role of matched sibling donor allogeneic stem cell transplantation in pediatric high-risk acute myeloid leukemia: results from the AML-BFM 98 study

Background

The role of allogeneic stem cell transplantation in post-remission management of children with high-risk acute myeloid leukemia remains controversial. In the multi-center AML-BFM 98 study we prospectively evaluated the impact of allogeneic stem cell transplantation in children with high-risk acute myeloid leukemia in first complete remission.

Design and Methods

HLA-typed patients with high-risk acute myeloid leukemia, who achieved first complete remission (n=247), were included in this analysis. All patients received double induction and consolidation. Based on the availability of a matched-sibling donor, patients were allocated by genetic chance to allogeneic stem cell transplantation (n=61) or chemotherapy-only (i.e. intensification and maintenance therapy; n=186). The main analysis was done on an intention-to-treat basis according to this allocation.

Results

Intention-to-treat analysis did not show a significantly different 5-year disease-free survival (49±6% versus 45±4%, P_{log rank}=0.44) or overall survival (68±6% versus 57±4%, P_{log rank}=0.17) between the matched-sibling donor and no-matched-sibling donor groups, whereas late adverse effects occurred more frequently after allogeneic stem cell transplantation (72.5% versus 31.8%, P_Fischer<0.01). These results were confirmed by as-treated analysis corrected for the time until transplantation (5-year overall survival: 72±8% versus 60±4%, P_Mantel-Byar 0.21). Subgroup analysis demonstrated improved survival rates for patients with 11q23 aberrations allocated to allogeneic stem cell transplantation (5-year overall survival: 94±6% versus 52±7%, P_log-rank=0.01; n=18 versus 49) in contrast to patients without 11q23 aberrations (5-year overall survival: 58±8% versus 55±5%, P_log-rank=0.66).

Conclusions

Our analyses defined a genetic subgroup of children with high-risk acute myeloid leukemia who benefited from allogeneic stem cell transplantation in the prospective multi-center AML-BFM 98 study. For the remainder of the pediatric high-risk acute myeloid leukemia patients the prognosis was not improved by allogeneic stem cell transplantation, which was, however, associated with a higher rate of late sequelae.     

Graft-versus-host disease and graft-versus-leukaemia effects in secondary acute myeloid leukaemia: a retrospective,multicentre registry analysis from the Acute Leukaemia Working Party of the EBMT

We assessed the susceptibility of secondary acute myeloid leukaemia (sAML) to graft-versus-leukaemia effects. Data from 2414 sAML patients in first (n = 2194) or second (n = 220) complete remission were included. They were given grafts from human leucocyte antigen (HLA)-matched sibling (MSD, n = 1085), 10/10 unrelated donor (MUD, n = 1066) or 9/10 mismatched unrelated donor (MMUD, n = 263). The 100-day incidence of grade II-IV acute graft-versus-host disease (GVHD) was 25% while 2-year incidence of chronic GVHD was 38%. Relapse rates declined steadily by duration of follow-up and were significantly lower in patients with chronic GVHD (P < 0·001). Limited (hazard ratio [HR] = 0·66, P < 0·001) and extensive (HR = 0·52, P < 0·001) chronic GVHD were associated with a lower incidence of relapse. Each grade III-IV acute (HR = 7·04, P < 0·001) as well as limited (HR = 1·42, P = 0·03) and extensive (HR = 3·97, P < 0·001) chronic GVHD were associated with higher non-relapse mortality (NRM). This translated to better overall survival (OS; HR = 0·61, P < 0·001) in patients with limited chronic GVHD. In contrast, grade III-IV acute and extensive chronic GVHD were associated with worse OS (HR = 3·16, P < 0·001 and HR = 1·21, P = 0·03, respectively). Further, in comparison to HLA-identical sibling recipients, MUD recipients had a lower risk of relapse (HR = 0·82, P = 0·03) but higher NRM (HR = 1·38, P = 0·004). In conclusion, these data demonstrate that sAML is susceptible to graft-versus-leukaemia effects.     

Activating internal tandem duplication mutations of the fms-like tyrosine kinase-3 (FLT3-ITD) at complete response and relapse in patients with acute myeloid leukemia

FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations are among the most frequent molecular aberrations in patients with acute myeloid leukemia. We retrospectively analyzed 324 patients with acute myeloid leukemia treated with front-line induction chemotherapy between October 2004 and March 2010. Fifty-six patients had FLT3-ITD mutation at diagnosis. Fifty-one (91%) patients with FLT3-ITD achieved complete remission. Thirteen patients had FLT3 analysis at complete remission. None had FLT3-ITD. Twenty-five (49%) patients with FLT3-ITD relapsed. Of these, 13 (52%) had FLT3-ITD at relapse (3 negative and 9 not done). Among the 201 patients without FLT3-ITD at diagnosis who achieved complete remission, 77 (38%) relapsed among whom 8 (10%) patients acquired FLT3-ITD clone. We conclude that FLT3-ITD mutations are unstable at follow up and may occur for the first time at relapse. Therefore, FLT3-ITD is not a reliable marker for minimal residual disease in acute myeloid leukemia.     

Prognostic role of Helicobacter pylori infection in acute coronary syndrome: a prospective cohort study

In a prospective cohort study, we evaluated the effect of Helicobacter pylori seropositivity on the risk of future adverse cardiovascular outcomes among patients with acute coronary syndrome (ACS). In 433 patients, IgA and IgG antibodies to H pylori, along with classic risk factors, including hypertension, diabetes, hyperlipidaemia, smoking and family history of coronary artery disease (CAD) were determined. Shortand long-term follow-up information on adverse outcomes, defined as recurrence of unstable angina, myocardial infarction, coronary angioplasty, coronary artery bypass graft surgery, and sudden cardiac death was obtained. None of the classic CAD risk factors correlated with incidence of either short- or long-term outcomes. Seropositivity for H pylori was significantly associated with risk of short-term adverse outcomes, and independently predicted their incidence in multivariate regression (R = 3.05, p < 0.001). Results failed to show such an association between H pylori seropositivity and long-term adverse outcomes. H pylori infection may affect short-term prognosis in patients with ACS. Randomised trials are needed to evaluate the role of H pylori eradication in these patients.     

FLT3 internal tandem duplication mutations in adult acute myeloid leukaemia define a high-risk group

Genomic DNA from 106 cases of adult de novo acute myeloid leukaemia (AML) was screened by polymerase chain reaction (PCR) and gel electrophoresis for FLT3 internal tandem duplication (ITD) mutations within the juxtamembrane (JM) domain. FLT3 mutations were detected in 14 cases (13.2%) and occurred in FAB types M1 (4 out of 14 cases), M3 (1 out of 10 cases), M4 (5 out of 37 cases) and M5 (4 out of 11 cases). Sequence analysis of four cases with abnormal PCR electrophoretic patterns revealed in frame duplications in the region of exon 11 of between 27 and 111 base pairs. Three are predicted to result in the tandem duplication of adjacent amino acid residues and one to result in a tandem duplication plus insertion of a novel amino acid motif. Statistical analysis showed the FLT3 mutations to be a strong prognostic factor, with patients lacking the mutation surviving significantly longer from diagnosis (mean 29.1 months) than those with an ITD (mean 12.8 months; P = 0.0002). Thirteen of the 14 patients with FLT3 mutations died within 18 months of diagnosis. FLT3 mutations were of prognostic significance in good risk disease (P = 0.04), as well as in patients with standard risk disease (P = 0.0096). This study demonstrates that the FLT3 ITD mutation occurs in a significant percentage of adult AML cases and is an important adverse prognostic factor that appears independent of conventional karyotypic findings.     

Use of CBL exon 8 and 9 mutations in diagnosis of myeloproliferative neoplasms and myelodysplastic/myeloproliferative disorders: an analysis of 636 cases

We analyzed 636 patients with diverse myeloproliferative neoplasms or myelodysplastic/myeloproliferative neoplasms for mutations of the Casitas B-cell lymphoma gene (CBL^mut) in exons 8 and 9 and performed correlations to other genetic alterations. CBL^mut were detected in 63 of 636 (9.9%) of these selected patients. CBL^mut were more frequent in myelodysplastic/myeloproliferative neoplasms than myeloproliferative neoplasms (51 of 328, 15.5% vs. 12 of 291, 4.1%; P<0.001). Frequency was 48 of 278 (17.3%) in chronic myelomonocytic leukemia and 3 of 33 (9.1%) in unclassifiable myelodysplastic/myeloproliferative neoplasms. CBL^mut was not detected in polycythemia vera, primary myelofibrosis, essential thrombocythemia, or refractory anemia with ring sideroblasts and marked thrombocytosis. CBL^mut were underrepresented in JAK2^V617F mutated as compared to JAK2V617^wt cases (P<0.001), and mutually exclusive of JAK2exon12^mut and MPLW515^mut. CBL^mut were associated with monosomy 7 (P=0.008) and TET2^mut (P=0.003). In chronic myelomonocytic leukemia, CBL^mut had no significant impact on survival outcomes. Therefore, CBL^mut are frequent in chronic myelomonocytic leukemia, absent in classical myeloproliferative neoplasms, and are only exceptionally found in coincidence with JAK-STAT pathway activating mutations.