Rafael Bejar

Clinical effect of point mutations in myelodysplastic syndromes

BACKGROUND Myelodysplastic syndromes are clinically heterogeneous disorders characterized by clonal hematopoiesis, impaired differentiation, peripheral-blood cytopenias, and a risk of progression to acute myeloid leukemia. Somatic mutations may influence the clinical phenotype but are not included in current prognostic scoring systems. METHODS We used a combination of genomic approaches, including next-generation sequencing and mass spectrometry-based genotyping, to identify mutations in samples of bone marrow aspirate from 439 patients with myelodysplastic syndromes. We then examined whether the mutation status for each gene was associated with clinical variables, including specific cytopenias, the proportion of blasts, and overall survival. RESULTS We identified somatic mutations in 18 genes, including two, ETV6 and GNAS, that have not been reported to be mutated in patients with myelodysplastic syndromes. A total of 51% of all patients had at least one point mutation, including 52% of the patients with normal cytogenetics. Mutations in RUNX1, TP53, and NRAS were most strongly associated with severe thrombocytopenia (P<0.001 for all comparisons) and an increased proportion of bone marrow blasts (P<0.006 for all comparisons). In a multivariable Cox regression model, the presence of mutations in five genes retained independent prognostic significance: TP53 (hazard ratio for death from any cause, 2.48; 95% confidence interval [CI], 1.60 to 3.84), EZH2 (hazard ratio, 2.13; 95% CI, 1.36 to 3.33), ETV6 (hazard ratio, 2.04; 95% CI, 1.08 to 3.86), RUNX1 (hazard ratio, 1.47; 95% CI, 1.01 to 2.15), and ASXL1 (hazard ratio, 1.38; 95% CI, 1.00 to 1.89). CONCLUSIONS Somatic point mutations are common in myelodysplastic syndromes and are associated with specific clinical features. Mutations in TP53, EZH2, ETV6, RUNX1, and ASXL1 are predictors of poor overall survival in patients with myelodysplastic syndromes, independently of established risk factors. (Funded by the National Institutes of Health and others.).

Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes

Recent genetic analyses of large populations have revealed that somatic mutations in hematopoietic cells leading to clonal expansion are commonly acquired during human aging. Clonally restricted hematopoiesis is associated with an increased risk of subsequent diagnosis of myeloid or lymphoid neoplasia and increased all-cause mortality. Although myelodysplastic syndromes (MDS) are defined by cytopenias, dysplastic morphology of blood and marrow cells, and clonal hematopoiesis, most individuals who acquire clonal hematopoiesis during aging will never develop MDS. Therefore, acquisition of somatic mutations that drive clonal expansion in the absence of cytopenias and dysplastic hematopoiesis can be considered clonal hematopoiesis of indeterminate potential (CHIP), analogous to monoclonal gammopathy of undetermined significance and monoclonal B-cell lymphocytosis, which are precursor states for hematologic neoplasms but are usually benign and do not progress. Because mutations are frequently observed in healthy older persons, detection of an MDS-associated somatic mutation in a cytopenic patient without other evidence of MDS may cause diagnostic uncertainty. Here we discuss the nature and prevalence of CHIP, distinction of this state from MDS, and current areas of uncertainty regarding diagnostic criteria for myeloid malignancies.

Validation of a Prognostic Model and the Impact of Mutations in Patients With Lower-Risk Myelodysplastic Syndromes

PURPOSE A subset of patients with myelodysplastic syndromes (MDS) who are predicted to have lower-risk disease as defined by the International Prognostic Scoring System (IPSS) demonstrate more aggressive disease and shorter overall survival than expected. The identification of patients with greater-than-predicted prognostic risk could influence the selection of therapy and improve the care of patients with lower-risk MDS. PATIENTS AND METHODS We performed an independent validation of the MD Anderson Lower-Risk Prognostic Scoring System (LR-PSS) in a cohort of 288 patients with low- or intermediate-1 IPSS risk MDS and examined bone marrow samples from these patients for mutations in 22 genes, including SF3B1, SRSF2, U2AF1, and DNMT3A. RESULTS The LR-PSS successfully stratified patients with lower-risk MDS into three risk categories with significant differences in overall survival (20% in category 1 with median of 5.19 years [95% CI, 3.01 to 10.34 years], 56% in category 2 with median of 2.65 years [95% CI, 2.18 to 3.30 years], and 25% in category 3 with median of 1.11 years [95% CI, 0.82 to 1.51 years]), thus validating this prognostic model. Mutations were identified in 71% of all samples, and mutations associated with a poor prognosis were enriched in the highest-risk LR-PSS category. Mutations of EZH2, RUNX1, TP53, and ASXL1 were associated with shorter overall survival independent of the LR-PSS. Only EZH2 mutations retained prognostic significance in a multivariable model that included LR-PSS and other mutations (hazard ratio, 2.90; 95% CI, 1.85 to 4.52). CONCLUSION Combining the LR-PSS and EZH2 mutation status identifies 29% of patients with lower-risk MDS with a worse-than-expected prognosis. These patients may benefit from earlier initiation of disease-modifying therapy.

Unraveling the Molecular Pathophysiology of Myelodysplastic Syndromes

Somatically acquired genetic abnormalities lead to the salient features that define myelodysplastic syndromes (MDS): clonal hematopoiesis, aberrant differentiation, peripheral cytopenias, and risk of progression to acute myeloid leukemia. Although specific karyotypic abnormalities have been linked to MDS for decades, more recent findings have demonstrated the importance of mutations within individual genes, focal alterations that are not apparent by standard cytogenetics, and aberrant epigenetic regulation of gene expression. The spectrum of genetic abnormalities in MDS implicates a wide range of molecular mechanisms in the pathogenesis of these disorders, including activation of tyrosine kinase signaling, genomic instability, impaired differentiation, altered ribosome function, and changes in the bone marrow microenvironment. Specific alterations present in individual patients with MDS may explain much of the heterogeneity in clinical phenotype associated with this disease and can predict prognosis and response to therapy. Elucidation of the full complement of genetic causes of MDS promises profound insight into the biology of the disease, improved classification and prognostic scoring schemes, and the potential for novel targeted therapies with molecular predictors of response.

TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients

Only a minority of myelodysplastic syndrome (MDS) patients respond to hypomethylating agents (HMAs), but strong predictors of response are unknown. We sequenced 40 recurrently mutated myeloid malignancy genes in tumor DNA from 213 MDS patients collected before treatment with azacitidine (AZA) or decitabine (DEC). Mutations were examined for association with response and overall survival. The overall response rate of 47% was not different between agents. Clonal TET2 mutations predicted response (odds ratio [OR] 1.99, P = .036) when subclones unlikely to be detected by Sanger sequencing (allele fraction <10%) were treated as wild-type (WT). Response rates were highest in the subset of TET2 mutant patients without clonal ASXL1 mutations (OR 3.65, P = .009). Mutations of TP53 (hazard ratio [HR] 2.01, P = .002) and PTPN11 (HR 3.26, P = .006) were associated with shorter overall survival but not drug response. Murine-competitive bone marrow transplantation followed by treatment with AZA demonstrated that Tet2-null cells have an engraftment advantage over Tet2-WT cells. AZA significantly decreased this advantage for Tet2-null cells (P = .002) but not Tet2-WT cells (P = .212). Overall, Tet2 loss appears to sensitize cells to treatment with AZA in vivo, and TET2 mutations can identify patients more likely to respond to HMAs.

Somatic Mutations Predict Poor Outcome in Patients With Myelodysplastic Syndrome After Hematopoietic Stem-Cell Transplantation

PURPOSE Recurrently mutated genes in myelodysplastic syndrome (MDS) are pathogenic drivers and powerfully associated with clinical phenotype and prognosis. Whether these types of mutations predict outcome after allogeneic hematopoietic stem-cell transplantation (HSCT) in patients with MDS is not known. PATIENTS AND METHODS We used massively parallel sequencing to examine tumor samples collected from 87 patients with MDS before HSCT for coding mutations in 40 recurrently mutated MDS genes. RESULTS Mutations were identified in 92% of patients, most frequently in the ASXL1 (29%), TP53 (21%), DNMT3A (18%), and RUNX1 (16%) genes. In univariable analyses, only TP53 mutations were associated with shorter overall (OS; hazard ratio [HR], 3.74; P < .001) and progression-free survival (HR, 3.97; P < .001). After adjustment for clinical variables associated with these end points, mutations in TP53 (HR, 2.30; P = .027), TET2 (HR, 2.40; P = .033), and DNMT3A (HR, 2.08; P = .049) were associated with decreased OS. In multivariable analysis including clinical variables, complex karyotype status, and candidate genes, mutations in TP53 (HR, 4.22; P ≤ .001) and TET2 (HR, 1.68; P = .037) were each independently associated with shorter OS. Nearly one half of patients (46%) carried a mutation in TP53, DNMT3A, or TET2 and accounted for 64% of deaths. Three-year OS in patients without these mutations was 59% (95% CI, 43% to 72%), versus 19% (95% CI, 9% to 33%) in patients with these mutations. CONCLUSION Mutations in TP53, TET2, or DNMT3A identify patients with MDS with shorter OS after HSCT.

MDS-associated somatic mutations and clonal hematopoiesis are common in idiopathic cytopenias of undetermined significance

Establishing a diagnosis in patients suspected of having a myelodysplastic syndrome (MDS) can be challenging and could be informed by the identification of somatic mutations. We performed a prospective study to examine the frequency and types of mutations encountered in 144 patients with unexplained cytopenias. Based on bone marrow findings, 17% were diagnosed with MDS, 15% with idiopathic cytopenias of undetermined significance (ICUS) and some evidence of dysplasia, and 69% with ICUS and no dysplasia. Bone marrow DNA was sequenced for mutations in 22 frequently mutated myeloid malignancy genes. Somatic mutations were identified in 71% of MDS patients, 62% of patients with ICUS and some dysplasia, and 20% of ICUS patients and no dysplasia. In total, 35% of ICUS patients carried a somatic mutation or chromosomal abnormality indicative of clonal hematopoiesis. We validated these results in a cohort of 91 lower-risk MDS and 249 ICUS cases identified over a 6-month interval. Mutations were found in 79% of those with MDS, in 45% of those with ICUS with dysplasia, and in 17% of those with ICUS without dysplasia. The spectrum of mutated genes was similar with the exception of SF3B1 which was rarely mutated in patients without dysplasia. Variant allele fractions were comparable between clonal ICUS (CCUS) and MDS as were mean age and blood counts. We demonstrate that CCUS is a more frequent diagnosis than MDS in cytopenic patients. Clinical and mutational features are similar in these groups and may have diagnostic utility once outcomes in CCUS patients are better understood.

Recent developments in myelodysplastic syndromes.

Once thought to be rare disorders, the myelodysplastic syndromes (MDS) are now recognized as among the most common hematological neoplasms, probably affecting >30 000 patients per year in the United States. US regulatory approval of azacitidine, decitabine, and lenalidomide between 2004 and 2006 seemed to herald a new era in the development of disease-modifying therapies for MDS, but there have been no further drug approvals for MDS indications in the United States in the last 8 years. The available drugs are not curative, and few of the compounds that are currently in development are likely to be approved in the near future. As a result, MDS diagnoses continue to place a heavy burden on both patients and health care systems. Incomplete understanding of disease pathology, the inherent biological complexity of MDS, and the presence of comorbid conditions and poor performance status in the typical older patient with MDS have been major impediments to development of effective novel therapies. Here we discuss new insights from genomic discoveries that are illuminating MDS pathogenesis, increasing diagnostic accuracy, and refining prognostic assessment, and which will one day contribute to more effective treatments and improved patient outcomes.

Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS.

The casein kinase 1A1 gene (CSNK1A1) is a putative tumor suppressor gene located in the common deleted region for del(5q) myelodysplastic syndrome (MDS). We generated a murine model with conditional inactivation of Csnk1a1 and found that Csnk1a1 haploinsufficiency induces hematopoietic stem cell expansion and a competitive repopulation advantage, whereas homozygous deletion induces hematopoietic stem cell failure. Based on this finding, we found that heterozygous inactivation of Csnk1a1 sensitizes cells to a CSNK1 inhibitor relative to cells with two intact alleles. In addition, we identified recurrent somatic mutations in CSNK1A1 on the nondeleted allele of patients with del(5q) MDS. These studies demonstrate that CSNK1A1 plays a central role in the biology of del(5q) MDS and is a promising therapeutic target.

Toll-like receptor alterations in myelodysplastic syndrome

Recent studies have implicated the innate immunity system in the pathogenesis of myelodysplastic syndromes (MDS). Toll-like receptor (TLR) genes encode key innate immunity signal initiators. We recently identified multiple genes, known to be regulated by TLRs, to be overexpressed in MDS bone marrow (BM) CD34+ cells, and hypothesized that TLR signaling is abnormally activated in MDS. We analyzed a large cohort of MDS cases and identified TLR1, TLR2 and TLR6 to be significantly overexpressed in MDS BM CD34+ cells. Deep sequencing followed by Sanger resequencing of TLR1, TLR2, TLR4 and TLR6 genes uncovered a recurrent genetic variant, TLR2-F217S, in 11% of 149 patients. Functionally, TLR2-F217S results in enhanced activation of downstream signaling including NF-κB activity after TLR2 agonist treatment. In cultured primary BM CD34+ cells of normal donors, TLR2 agonists induced histone demethylase JMJD3 and interleukin-8 gene expression. Inhibition of TLR2 in BM CD34+ cells from patients with lower-risk MDS using short hairpin RNA resulted in increased erythroid colony formation. Finally, RNA expression levels of TLR2 and TLR6, as well as presence of TLR2-F217S, are associated with distinct prognosis and clinical characteristics. These findings indicate that TLR2-centered signaling is deregulated in MDS, and that its targeting may have potential therapeutic benefit in MDS.