Somatic point mutations in RUNX1/CBFA2/AML1 are common in high-risk myelodysplastic syndrome, but not in myelofibrosis with myeloid metaplasia

OBJECTIVE: Acquired somatic point mutations in RUNX1/CBFA2/AML1 have recently been described in a subset of patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Given the importance of core-binding factor in megakaryocytic differentiation and platelet production, as well as the central role of megakaryocytes in the pathophysiology of myelofibrosis with myeloid metaplasia (MMM), we hypothesised that RUNX1 gene mutations might be common in MMM. In addition, it is unclear whether patients with MDS-associated acquired alpha thalassaemia (ATMDS), a special subgroup with a very high incidence of point mutations in the ATRX gene, have an especially high incidence of RUNX1 mutations. METHODS: We analysed samples from 78 patients for RUNX1 point mutations by denaturing high-performance liquid chromatography (DHPLC): 26 with MMM and 52 with MDS, including 18 with ATMDS. RESULTS: We found five RUNX1 mutations in MDS patients (9.6%), all of whom had RAEB-2 or a history of treated AML, but none in MMM patients. ATMDS patients did not have an increased risk of RUNX1 point mutations (2/18, 11.1%) when compared with MDS without thalassaemia (3/34, 8.8%; P = 0.58). CONCLUSION: RUNX1 point mutations are common in high-risk MDS, but not in MMM. DHPLC is a useful technique for high-throughput analysis of RUNX1 mutation status in myeloid disorders, and may be complementary to screening via other methods.     

Acquired somatic ATRX mutations in myelodysplastic syndrome associated with alpha thalassemia (ATMDS) convey a more severe hematologic phenotype than germline ATRX mutations

Acquired somatic mutations in ATRX, an X-linked gene encoding a chromatin-associated protein, were recently identified in 4 patients with the rare subtype of myelodysplastic syndrome (MDS) associated with thalassemia (ATMDS). Here we describe a series of novel point mutations in ATRX detected in archival DNA samples from marrow and/or blood of patients with ATMDS by use of denaturing high-performance liquid chromatography (DHPLC), a technique sensitive to low-level mosaicism. Two of the new mutations result in changes in amino acids altered in previously described pedigrees with germ line ATRX mutations (ATR-X syndrome), but the hematologic abnormalities were much more severe in the patients with ATMDS than in the corresponding constitutional cases. In one ATMDS case where DNA samples from several time points were available, the proportion of ATRX-mutant subclones correlated with changes in the amount of hemoglobin H. This study strengthens the link between acquired, somatic ATRX mutations and ATMDS, illustrates how molecular defects associated with MDS and other hematologic malignancies masked by somatic mosaicism may be detected by DHPLC, and shows that additional factors increase the severity of the hematologic phenotype of ATRX mutations in ATMDS.     

The diagnostic utility of targeted gene panel sequencing in discriminating etiologies of cytopenia

The diagnostic utility of somatic mutations in the context of cytopenias is unclear: clonal hematopoiesis can be found in healthy individuals, patients with aplastic anemia (AA), clonal cytopenia of undetermined significance (CCUS) and myelodysplastic syndrome (MDS). We examined a cohort of 207 well-characterized cytopenic patients with a 640-gene next generation sequencing (NGS) panel and compared its diagnostic utility with a “virtual” 41 gene panel. The TET2, SF3B1, ASXL1, and TP53 were the most commonly mutated genes (frequency > 10%). Mutations in the 640-gene panel show high sensitivity (98.3%) but low specificity (47.6%) for diagnosis of MDS. Notably, mutations of splicing factors and genes in the RAS pathway are relatively specific to MDS. Furthermore, high variant allele frequency (VAF) predicts MDS: when the VAF is set at 20%, the positive predictive value (PPV) for MDS is 95.9%, with a specificity of 95.3%. The presence of two or more somatic mutations with ≥10% VAF showed a PPV of 95.2%. While the “virtual” 41-gene panel showed a mild decrease in sensitivity (95.7% vs 98.3%), 100% specificity was observed when either VAF was set at ≥20% (100% vs 95.3%), or two or more somatic mutations had VAFs ≥ 10%. Our study shows targeted gene panel sequencing improves the diagnostic approach and accuracy for unexplained cytopenia, with its high sensitivity and high PPV for MDS when applying VAF cutoffs. Furthermore, a 41-gene panel was shown to have at least comparable performance characteristics to the large 640-gene panel.     

Une alpha thalassémie acquise chez un patient de 86 ans avec un syndrome myélodysplasique

BackgroundAlpha thalassemia-myelodysplastic syndrome (ATMDS) is one of the possible complications related to the genetic instability typical of clonal hemopoietic disorders such as myelodysplastic syndromes (MDS). Hemoglobin H acquisition, which is hemoglobin without alpha chains and with 4 beta chains is the hallmark of this disease.ObservationAn 86-year-old male with chronic, microcytic anemia was referred due to a fall in his hemoglobin level. The blood smear was remarkable for intense anisocytoses and poikilocytosis. Bone marrow analysis was followed by a diagnosis of MDS with a good prognostic score. Peripheral blood coloration with brilliant cresyl blue showed “golf ball-like” erythrocytes. Hemoglobin electrophoresis is notable for the presence of H hemoglobin. The new generation sequencing confirmed the diagnosis of ATMDS showing a non-sense mutation in the gene ATRX.ConclusionThe diagnosis of ATMDS should be considered in the presence of the association of MDS, microcytic anemia and marked blood smear abnormalities such as anisocytosis and poikilocytosis. A little less than 10% of all MDS are complicated by ATMDS.     

Deletion of the alpha-globin gene cluster as a cause of acquired alpha-thalassemia in myelodysplastic syndrome

Rarely, myelodysplastic syndrome (MDS) is complicated by an acquired form of alpha-thalassemia (alpha-thalassemia in myelodysplastic syndrome [ATMDS]) characterized by hypochromic, microcytic, anisopoikilocytic red blood cells with hemoglobin H (HbH) inclusions. Acquired mutations in ATRX, a chromatin remodeling gene, have recently been found in 12 patients with typical features of ATMDS, though they have not been detected in MDS patients with similar red blood cell findings but little HbH. The alpha-globin genes themselves have appeared normal in all ATMDS patients studied to date. Here we characterize the molecular defect in a unique MDS patient with rare HbH inclusions in which an abnormal clone lost a greater than 1.9-Mb segment of the telomeric region of the short arm of one allele of chromosome 16, including both alpha-globin genes. Red blood cell changes associated with this acquired somatic genotype (--/alpha alpha) are surprisingly severe, demonstrating that a minor globin chain imbalance may be unexpectedly deleterious during the abnormal erythropoiesis that occurs in the context of MDS.     

TP53 mutations predict decitabine‐induced complete responses in patients with myelodysplastic syndromes

To identify the molecular signatures that predict responses to decitabine (DAC ), we examined baseline gene mutations (28 target genes) in 109 myelodysplastic syndrome (MDS ) patients at diagnosis. We determined that TP 53 mutations predicted complete response (CR ), as 10 of 15 patients (66·7%) who possessed TP 53 mutations achieved a CR . Univariate and multivariate analyses showed that TP 53 mutations are the only molecular signatures predictive of a CR to DAC in MDS . Among the ten patients with TP 53 mutations who achieved a CR , nine presented with complex karyotypes due to abnormalities involving chromosome 5 and/or chromosome 7, and eight possessed monosomies. Although TP 53 mutations were associated with a higher frequency of CR s, they were not associated with improved survival. Poor outcomes were attributed to early relapses and transformation to acute myeloid leukaemia after CR . Post‐DAC therapy patient gene mutation profiles showed that most CR patients exhibited fewer gene mutations after achieving a CR . It seems that suppression of these gene mutations was facilitated by DAC , resulting in a CR . In summary, TP 53 mutations might predict decitabine‐induced complete responses in patients with MDS . DAC ‐induced responses may result from partial suppression of malignant clones containing mutated TP 53 genes.     

Clinical implications of the SETBP1 mutation in patients with primary myelodysplastic syndrome and its stability during disease progression

Mutations of the SET binding protein 1 (SETBP1) gene have been identified in patients with myeloid neoplasms, but the clinical relevance of this mutation and its association with other gene mutations in myelodysplastic syndrome (MDS) and the stability during disease progression remains unclear. Mutations in SETBP1 gene at exon 4 were analyzed by polymerase chain reaction and direct sequencing in 430 MDS patients. The results were correlated with clinical features, cytogenetics, gene mutations and treatment outcomes. SETBP1 mutations were identified in 14 (3.3%) of the 430 patients with primary MDS based on the FAB classification and 8 (2.4%) of the 333 patients based on the WHO classification. The SETBP1 mutation was closely associated with higher white blood cell counts, isochromosome of 17q, monosomy 7, and mutations of ASXL1, EZH2 and SRSF2. With a median follow‐up of 43.9 months, MDS patients, based on either the FAB or WHO classification, had a significantly poorer overall survival (OS) if they harbored SETBP1 mutation. Further, SETBP1 mutation was an independent poor prognostic factor for OS (HR = 1.842, CI 95%, 1.1018–3.332, P = 0.043) irrespective of age, sex, and the International Prognostic Scoring System. Sequential analysis showed that the original SETBP1 mutations in the eight SETBP1‐mutated patients studied were retained while two of the 101 SETBP1‐wild patients acquired novel SETBP1 mutations during follow‐ups. The SETBP1 mutation is associated with poor prognosis in MDS. The mutation can be acquired during the clinical course suggesting it may play a role in disease progression. Am. J. Hematol. 89:181–186, 2014. © 2013 Wiley Periodicals, Inc.     

The role of X-inactivation in the gender bias of patients with acquired α-thalassaemia and myelodysplastic syndrome (ATMDS)

Αlpha thalassaemia myelodysplastic syndrome (ATMDS) is an unusual complication of chronic myeloid malignancy that is associated with a striking red cell phenotype. It represents an acquired form of α-thalassaemia that most commonly arises in the context of myelodysplasia. It has recently been shown that this condition occurs in association with somatic mutations of a known X-encoded trans -acting regulator of α globin gene ( HBA ) expression, ATRX. There is an unexplained, strong male preponderance of individuals with the ATMDS phenotype with a >5:1 male–female ratio and furthermore, all the somatic ATRX mutations described to date have been in males. Here we report the identification, in a single centre, of two females with ATMDS and mutations in the ATRX gene, proving that ATMDS associated with such mutations may occur, albeit rarely, in females. It seemed possible that females might be less likely to develop ATMDS if the inactivated copy of the ATRX gene ( ATRX ) became progressively re-activated throughout life. This study ruled out this hypothesis by investigating the pattern of ATRX inactivation in a cross-sectional analysis of normal females at ages ranging from newborn to 90 years.     

HFE genotype and iron metabolism in Chinese patients with myelodysplastic syndromes and aplastic anemia

The incidence of HFE gene mutations in myelodysplastic syndrome (MDS) cases remains controversial. In this study, we examined the HFE C282Y and H63D mutations in 271 Chinese patients with MDS, 402 with aplastic anemia (AA) and 1,615 healthy controls by polymerase chain reaction–restriction fragment length polymorphism in combination with DNA sequencing. No C282Y mutations were observed in the entire cohort. The distribution of H63D heterozygous and homozygous genotypes was not significantly different between the AA cases and the controls (9.7% versus 10.2%, 0.25% versus 0.24%, respectively). While the H63D heterozygous genotype in MDS patients was significantly lower than that in the controls (4.1% versus 10.2%, p = 0.002), the H63D homozygous genotype was not detected in the MDS patients. The results suggest that HFE gene mutations are not common genetic factors in Chinese patients with MDS and AA. We also compared iron metabolic parameters, including serum ferritin, serum iron, and transferrin saturation values, between HFE mutant and HFE wild-type groups in the absence of transfusion iron overload, but no significant difference was found in either MDS or AA patients except that the level of serum iron in AA patients was significantly higher in mutant carriers than in those with wild-type HFE (p = 0.011). Similarly, there was no significant difference between HFE mutant and HFE wild-type MDS and AA patients in clinical indices such as alanine aminotransferase, aspartate aminotransferase, fasting blood sugar values, and electrocardiogram. The results suggest that H63D mutations may not have clinical significance in Chinese patients with MDS and AA.     

Prognostic impact of RAS mutations in patients with myelodysplastic syndrome

RAS is an oncogene frequently mutated in human cancer. RAS mutations have been reported in 10–15% of cases of acute myeloid leukemia (AML) but they appear to be less frequent among patients with myelodysplastic syndrome (MDS). The impact of RAS mutations in patients with MDS is unclear. We conducted a retrospective study in 1,067 patients with newly diagnosed MDS for whom RAS mutational analysis was available. Overall, 4% of patients carried mutant RAS alleles. Notably, FLT3 mutations, which were found in 2% of patients, were mutually exclusive with RAS mutations. Patients with RAS mutations had a higher white blood cell count as well as bone marrow blasts compared with patients carrying wild‐type RAS. However, no differences were observed between both groups regarding the risk of AML transformation (9% vs. 7%) and overall survival (395 days vs. 500 days, P = 0.057). In summary, RAS mutations are infrequent in patients with MDS and do not appear to negatively impact their outcome. Am. J. Hematol. 88:365–369, 2013. © 2013 Wiley Periodicals, Inc.     

FLT3 mutations in myelodysplastic syndrome and chronic myelomonocytic leukemia

FMS‐like tyrosine kinase III (FLT3) mutations occur in one‐third of acute myeloid leukemia (AML) patients and predict poor outcome. The incidence and impact of FLT3 in myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) is unknown. We conducted a retrospective review to identify WHO MDS and CMML patients with FLT3 mutations at diagnosis. A total of 2,119 patients with MDS and 466 patients with CMML were evaluated at MD Anderson between 1997 and 2010. Of these, FLT3 mutation analysis was performed on 1,232 (58%) MDS and 302 (65%) CMML patients. FLT3 mutations were identified in 12 (0.95%) MDS patients: 9 (75%) had FLT3‐ITD mutation and 3 had FLT3‐tyrosine kinase domain (TKD) mutation. MDS patients with FLT3 mutations were younger (P = 0.02) and presented as RAEB (P = 0.03) more frequently. Median overall survival (OS) for FLT3‐mutated MDS patients was 19.0 months versus 16.4 months for FLT3‐nonmutated MDS patients (P = 0.08). FLT3 mutations were identified in 13 (4.3%) CMML patients: 8 had FLT3‐ITD mutation and 5 had FLT3‐TKD mutation. There were no significant differences in demographic and disease characteristics among CMML patients with and without FLT3 mutations. Median OS for FLT3‐mutated CMML patients was 10.8 months versus 21.3 months for FLT3‐nonmutated CMML patients (P = 0.12). FLT3 occurs in MDS and CMML at a lower frequency than AML and does not predict poor outcome. Am. J. Hematol. 2013. © 2012 Wiley Periodicals, Inc.     

Mutations of the TET2 and CBL genes: novel molecular markers in myeloid malignancies

Despite recent progress in molecular research in myeloid malignancies, in subsets of patients with myelodysplastic syndrome (MDS) so far no underlying mutation was identified. In the myeloproliferative neoplasms (MPNs), the JAK2V617F alone cannot explain the phenotypic heterogeneity. In acute myeloid leukemia (AML), clinical variability exists within distinct subgroups. Thus, the search for novel molecular markers continues. Recently, mutations of the tet oncogene family member 2 (TET2) and Casitas B-cell lymphoma (CBL) genes became the focus of interest. With diverse genetic methods, TET2 on chromosome 4q24 was identified as candidate tumor suppressor gene. Sequencing studies revealed heterogeneous mutations in 10–25% of patients with acute myeloid leukemia (AML), MDS, and MPNs, while the frequency might be higher in chronic myelomonocytic leukemia (CMML). The prognostic impact is being explored. The CBL gene is involved in the degradation of tyrosine kinases. In rare cases of human AML (<2%), CBL mutants were identified, with a higher frequency in core binding factor leukemias. Presence of these mutations was suggested to be involved in aberrant FLT3 expression. In the MPNs, a 2–8% frequency of CBL mutations was reported. These novel mutations deepened insights in the mechanisms of leukemogenesis, might contribute to the identification of new therapeutic targets, and improve diagnostics in the myeloid malignancies.     

Prognostic significance of SRSF2 mutations in myelodysplastic syndromes and chronic myelomonocytic leukemia: a meta-analysis

ABSTRACT

Objective: Serine/arginine-rich splicing factor 2 (SRSF2) mutations were detected frequently in myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) patients. However, its prognostic value has not yet been fully clarified.

Methods: In this meta-analysis, Hazard Ratio (HR) and 95% confidence interval (CI) for overall-survival (OS) were chosen to evaluate the prognostic impact of SRSF2 mutations and to compare SRSF2 mutations to those with wild-type.

Results: A total of 2056 patients from 12 studies were obtained. The pooled HRs for OSsuggested that patients with MDS had a poorer prognosis (HR?=?1.780, 95% CI (1.410–2.249)), while analysis on SRSF2 mutations revealed no significant effect on the prognosis of CMML patients (HR?=?1.091, 95% CI (0.925–1.286)). The frequency of SRSF2 mutations was found to be 11.5% and 39.8% in patients with MDS and CMML, respectively.

Discussion: This meta-analysis suggests that SRSF2 has a poor prognosis in patients with MDS, but no prognosis impact on patients with CMML.

Conclusion: In conclusion, SRSF2 mutations were significantly related to the shorter OS in patients with MDS which may consider as an adverse prognostic risk factor. Whereas, analysis did not show any prognostic effect on OS of CMML patients with SRSF2 mutations.     

Phase I/II trial of the combination of midostaurin (PKC412) and 5‐azacytidine for patients with acute myeloid leukemia and myelodysplastic syndrome

We investigated the combination of midostaurin and azacitidine (AZA) in patients with acute myeloid leukemia (AML) and high risk myelodysplastic syndrome (MDS). Patients received AZA 75 mg m^?2 on days 1–7 and midostaurin 25 mg bid (in cohort 1 of phase I) or 50 mg bid (in cohort 2 of Phase I and in Phase II) orally on day 8–21 during the first cycle and continuously thereafter. Fourteen patients were enrolled in the phase I and 40 in the phase II. Overall response rate was 26%. The median remission duration (RD) was 20 weeks and was significantly longer in patients with FLT3 mutations not previously exposed to other FLT3 inhibitors (P = 0.05) and in patients not previously transplanted (P = 0.01). Thirty‐two (59%) patients have died, all of complications related to disease progression. G3‐4 nonhematological toxicity was reported in 38 (70%) patients, most frequently infections (56%), ejection fraction reduction (11%), and diarrhea or nausea/vomiting (9% each). The combination of midostaurin and AZA is an effective and safe regimen in patients with AML and high‐risk MDS. Patients with FLT3 mutations but not previously exposed to other FLT3 inhibitors and patients not previously transplanted derived the greatest benefit. Further studies with this combination are warranted. Am. J. Hematol. 90:276–281, 2015. © 2014 Wiley Periodicals, Inc.     

Mutations in idiopathic cytopenia of undetermined significance assist diagnostics and correlate to dysplastic changes

Cytopenia is common in the elderly population and etiology may be difficult to assess. Here, we investigated the occurrence of mutations in patients with idiopathic cytopenia of undetermined significance and the usefulness in improving diagnostics. We included 60 patients with persistent cytopenia > 6 months without definite diagnosis of hematological neoplasm after routine assessment. Bone marrow material underwent a blinded morphology review and DNA was sequenced with a targeted 20 gene panel representing the most commonly mutated genes in myelodysplastic syndrome. Thirty seven (62%) patients carried at least one mutation at inclusion, and of these 95% carried a mutation in TET2, ASXL1, SRSF2, or DNMT3A. The most commonly mutated gene was TET2 observed in 43% of all patients. During one to eight years follow‐up seven patients progressed to a myeloid neoplasm and six of these had a detectable mutation at study entry. Median time to progression was 53 months (range 10–78), and at time of progression each patient had at least two mutations detected. Mutations in TP53 and NRAS were not present in patients at inclusion, but identified as secondary hits triggering progression. The morphology review was concordant in 68% of all cases, and 93% of the cases reclassified into the group “highly suspicious for MDS” had a mutation. All patients who had a concordant review “highly suspicious for MDS” had at least two mutations detected. Overall, we show that morphology examination is challenging in this heterogeneous group and targeted sequencing helps identify patients at risk of progression. Am. J. Hematol. 91:1234–1238, 2016. © 2016 Wiley Periodicals, Inc.     

Refractory anemia with ring sideroblasts and RARS with thrombocytosis

Disease Overview : Ring sideroblasts (RS) are erythroid precursors with abnormal perinuclear mitochondrial iron accumulation. Two myeloid neoplasms defined by the presence of RS, include refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS‐T). Diagnosis : RARS is a lower risk myelodysplastic syndrome (MDS) with dysplasia limited to the erythroid lineage, <5% bone marrow (BM) blasts and ≥15% BM RS. RARS‐T is a provisional entity in the MDS/MPN (myeloproliferative neoplasm) overlap syndromes, with diagnostic features of RARS, along with a platelet count ≥450 × 10(9)/L and large atypical megakaryocytes similar to those observed in BCR‐ABL1 negative MPN. Mutations and Karyotype : Mutations in the SF3B1 gene are seen in ≥80% of patients with RARS and RARS‐T, and strongly correlate with the presence of BM RS; RARS‐T patients have additional mutations such as, JAK2V617F (～60%), MPL (<5%), and CALR (<5%). Cytogenetic abnormalities are uncommon in both RARS and RARS‐T. Risk stratification : Most patients with RARS are stratified into lower risk groups by the International Prognostic Scoring System (IPSS) for MDS and the revised IPSS. Disease outcome in RARS‐T is better than that of RARS, but worse than that of essential thrombocytosis. Both RARS and RARS‐T have a low risk of leukemic transformation. Treatment : Anemia and iron overload are complications in both diseases and are managed similar to lower risk MDS. Aspirin therapy is reasonable in RARS‐T, especially in the presence of JAK2V617F, but the value of platelet‐lowering drugs is uncertain. Case reports of RARS‐T therapy with lenalidomide warrant additional studies. Am. J. Hematol. 90:550–559, 2015. © 2015 Wiley Periodicals, Inc.     

CSNK1A1 mutations and gene expression analysis in myelodysplastic syndromes with del(5q)

Mutations of CSNK1A1, a gene mapping to the commonly deleted region of the 5q‐ syndrome, have been recently described in patients with del(5q) myelodysplastic syndromes (MDS). Haploinsufficiency of Csnk1a1 in mice has been shown to result in β‐catenin activation and expansion of haematopoietic stem cells (HSC). We have screened a large cohort of 104 del(5q) MDS patients and have identified mutations of CSNK1A1 in five cases (approximately 5%). We have shown up‐regulation of β‐catenin target genes in the HSC of patients with del(5q) MDS. Our data further support a central role of CSNK1A1 in the pathogenesis of MDS with del(5q).     

Postallogeneic monitoring with molecular markers detected by pretransplant next‐generation or Sanger sequencing predicts clinical relapse in patients with myelodysplastic/myeloproliferative neoplasms

Relapse is the major cause of treatment failure after allogeneic stem‐cell transplantation (AHSCT) for patients with myelodysplastic syndrome/myeloproliferative syndrome neoplasms (MDS/MPN). We evaluated the impact of molecular mutations on outcome and the value of molecular monitoring post‐transplantation. We screened 45 patients with chronic myelomonocytic leukemia (n = 39 patients, including seven with transformed‐acute myeloid leukemia), MDS/MPN unclassifiable (n = 5), and atypical BCR‐ABL1‐negative CML (n = 1) for mutations in ASXL1, CBL, NRAS, and TET2 genes by molecular genetics including a sensitive next‐generation sequencing (NGS) technique. In 36 patients, sufficient DNA was available for molecular analyses. In particular, TET2 and CBL mutations were screened applying amplicon deep sequencing. In 89% of cases, at least one mutation could be detected: ASXL1: n = 18 (50%); CBL: n = 7 (19%); TET2: n = 15 (42%); and NRAS: n = 11 (32%). Survival after AHSCT at 5 yr was 46% (95% CI 28–64%) and was not influenced by any mutation. After a median of 6 months after AHSCT in 33% of the patients, one of the molecular markers was still detectable, resulting in a higher incidence of relapse than in patients with undetectable mutations (50% vs. 15%, P = 0.04). In conclusion, pretransplant molecular mutation analysis can help to detect biomarkers in patients with MPN/MDS, which may be subsequently used as minimal residual disease markers after AHSCT.