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Hematologic Malignancies-Leukemia, Myelodysplastic Syndromes, and Allotransplant
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New Insight Into the Biology, Risk Stratification, and Targeted Treatment of Myelodysplastic Syndromes
Disclosures of potential conflicts of interest provided by the authors are available with the online article at asco.org/edbook.
Abstract
In myelodysplastic syndromes (MDS), somatic mutations occur in five major categories: RNA splicing, DNA methylation, activated cell signaling, myeloid transcription factors, and chromatin modifiers. Although many MDS cases harbor more than one somatic mutation, in general, there is mutual exclusivity of mutated genes within a class. In addition to the prognostic significance of individual somatic mutations, more somatic mutations in MDS have been associated with poor prognosis. Prognostic assessment remains a critical component of the personalization of care for patient with MDS because treatment is highly risk adapted. Multiple methods for risk stratification are available with the revised International Prognostic Scoring System (IPSS-R), currently considered the gold standard. Increasing access to myeloid gene panels and greater evidence for the diagnostic and predictive value of somatic mutations will soon make sequencing part of the standard evaluation of patients with MDS. In the absence of formal guidelines for their prognostic use, well-validated mutations can still refine estimates of risk made with the IPSS-R. Not only are somatic gene mutations advantageous in understanding the biology of MDS and prognosis, they also offer potential as biomarkers and targets for the treatment of patients with MDS. Examples include deletion 5q, spliceosome complex gene mutations, and TP53 mutations.
KEY POINTS
Over the past decade, high-throughput DNA sequencing methods have advanced the understanding of MDS biology.
Somatic MDS mutations occur in five major categories: RNA splicing, DNA methylation, activated cell signaling, myeloid transcription factors, and chromatin modfication.
Increasing access to myeloid gene panels and greater evidence for the diagnostic and predictive value of somatic mutations will soon make sequencing part of the standard evaluation of patients with MDS.
In the absence of formal guidelines for their prognostic use, well-validated mutations can still refine estimates of risk made with the IPSS-R.
Not only are somatic gene mutations advantageous in understanding the biology of MDS and prognosis, they also offer potential as biomarkers and targets for the treatment of patients with MDS.
MDS are bone marrow stem cell malignancies characterized by inefficient hematopoiesis, abnormal myeloid morphology, and cytopenias with risk of progression to secondary acute myeloid leukemia (AML). MDS is the most common hematopoietic myeloid cancer in adults with an average annual incidence of up to 75 per 100,000 persons 65 years or older.1,2 The diagnosis of MDS requires persistent cytopenias in the presence of dysplasia in one or more cell lineages and/or increased myeloblasts or clonal cytogenetic abnormalities and is classified according to the World Health Organization (WHO) criteria (Table 1).3 Over the last several years, many advances have been made in understanding the biology of MDS, most notably through the use of newer high-throughput DNA sequencing methods.
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The earliest known molecular alterations in MDS were cytogenetic abnormalities detected by metaphase cytogenetics.5 Approximately 45% of patients with MDS harbor a recurrent cytogenetic abnormality (Table 2).6,7 In contrast to AML, copy number alterations including chromosomal deletions and amplifications are more common than translocations. Certain cytogenetic findings in MDS are associated with changes in prognosis and are incorporated into the IPSS-R.8 Changes including complex karyotype (more than three cytogenetic abnormalities) and monosomal karyotype (one autosomal monosomy in the presence of a structural abnormality) have been associated with a poor prognosis.9,10 In addition to metaphase cytogenetics, fluorescence in situ hybridization may be used to detect recurrent cytogenetic alterations, providing increased sensitivity over conventional cytogenetics and permitting more accurate monitoring of disease burden in patients with MDS undergoing treatment.
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The advent of massively paralleled digital sequencing methods (often colloquially grouped as next-generation sequencing) has provided rapid growth in our understanding of the molecular biology of myeloid neoplasms.11-13 These methods allow for the sequencing of small gene panels, the exome (the coding portion of the genome), or the entire genome with high sensitivity and at minimal cost.14,15 Over the last 8 years, numerous studies have demonstrated the following: (1) approximately 90% of patients with MDS will harbor at least one mutation from a set of approximately 40 recurrently mutated MDS genes16-18 (Table 3; Fig. 1); (2) certain somatic mutations are associated with changes in prognosis19; (3) somatic mutations can be used to decipher the clonal architecture of MDS; and (4) a similar spectrum of somatic mutations can be seen in older patients without dysplasia (discussed in a later section), precluding the use of sequencing-based studies to replace morphologic evaluation.20,21 Sequencing-based “MDS gene panels” have now become commonplace in the clinical setting and can be used to better stratify patient risk and monitor clonal populations.22
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FIGURE 1. Summary of Recurrently Mutated Genes in MDS
Average reported mutation frequency for commonly mutated MDS genes from three NGS-based studies, including a total of 1,839 patients.16,18,23 Genes were included only when evaluated in at least two of the three studies. Abbreviations: MDS, myelodysplastic syndromes; NGS, next-generation exome sequencing.
Somatic MDS mutations occur in five major categories, including RNA splicing, DNA methylation, activated cell signaling, myeloid transcription factors, and chromatin modifiers. Although many MDS cases harbor more than one somatic mutation, in general, there is mutual exclusivity of mutated genes within a class. In addition to the prognostic significance of individual somatic mutations, more somatic mutations in MDS have been associated with poor prognosis.
Mutations involving RNA splicing are present in up to 45% of MDS; however, they appear to be rare in de novo AML.13,24 These mutations affect 3′ splice recognition sites, although the exact mechanisms by which the mutations cause dysplasia and the RNA targets of aberrant gene splicing are unknown. Mutations in the splicing factor 3b, subunit 1 (SF3B1) gene are present in approximately 20% of MDS and are associated with ring sideroblast morphology, lower-grade disease, and better prognosis.25 Interestingly, mutations in SF3B1 have also been found in patients with chronic lymphocytic leukemia (CLL) and breast cancer.26-28 Patients with MDS with ring sideroblasts without mutated SF3B1 (approximately 20% of patients) are thought to have an inferior prognosis compared with patients with mutated SF3B1.29 Mouse models have demonstrated that mutated SF3B1 causes an MDS phenotype and that cells carrying the mutation are sensitive to spliceosome modulator drugs.30 SF3B1 mutations have also been associated with chronic lymphocytic leukemia.30 Mutations in the U2 small nuclear RNA auxiliary factor 1 gene (U2AF1 or U2AF35) are present in approximately 8% to 12% of MDS and are associated with a poor prognosis. U2AF1 mutations have been shown to alter splice recognition sites and specificity of precursor messenger RNA binding, eliciting changes in thousands of RNA transcripts; however, the exact mechanism by which U2AF1 mutations give rise to dysplasia have yet to be determined.31 Interestingly, in vitro and animal studies have demonstrated increased sensitivity to precursor messenger RNA splicing modulator drugs.32 Mutations in the Serine/arginine-rich splicing factor 2 (SRSF2) gene are present in approximately 10% of patients with MDS and are associated with a poor prognosis. SRSF2 mutations are enriched in chronic myelomonocytic leukemia (CMML) and are present in up to 40% to 50% of cases.33 Finally, mutations in zinc finger (CCCH type), RNA-binding motif, and serine/arginine rich 2 (ZRSR2) are present in 5% to 10% of patients with MDS and are associated with a poor prognosis. Mutations in ZRSR2 lead to impaired splicing of the U12-type introns, although the downstream RNA targets are unknown.34
Mutations involving DNA methylation including those in TET2, DNMT3A, and IDH1/2 are common in MDS and AML as well as in the recently defined clonal hematopoiesis of indeterminate potential (CHIP), which is discussed later in this article.35 In general, mutations of these genes are mutually exclusive within a patient. The most commonly mutated gene across MDS studies (present in over 20% of patients) is TET2, which encodes a protein involved in the conversion of 5-methyl-cytosine to 5-hydroxymethyl-cytosine and plays a key role in DNA demethylation.36 Although the TET family of proteins has three members (TET1, TET2, and TET3), only TET2 appears to be frequently mutated in MDS. TET2 mutations, unlike mutations in other DNA methylation–associated genes, do not appear to have a mutational hotspot and often involve insertions and deletions, making mutations difficult to detect outside of gene panel–based sequencing assays. TET2 mutations are generally associated with a normal karyotype and are of unclear prognostic significance.37 DNMT3A encodes a protein that catalyzes the transfer of methyl groups required for de novo methylation. Mutations in DNMT3A were originally described in AML and are among the most frequent mutations in AML as well as in CHIP; in MDS, DNMT3A is mutated in 12% to 18% of cases.38 The exact mechanism by which DNMT3A mutations contribute to MDS/AML pathogenesis is unknown, but it is thought to be an early event, and mutations have been shown to act in a dominant-negative manner.39,40 Most DNMT3A mutations involve the p.R882 codon. Mutations in DNMT3A are associated with a poor prognosis in MDS. Although DNMT3A mutations are an early event, they may persist during complete remission without adverse outcome.41 Mutations in IDH1 and IDH2 are generally more common in AML than in MDS, where they are present in fewer than 5% of cases. Isocitrate dehydrogenases catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate; mutations in the catalytic domains of IDH1/2 result in the accumulation of 2-hydroxyglutarate, resulting in DNA hypermethylation.42 IDH1 mutations occur almost exclusively in the p.R132 position, with IDH2 mutations generally occurring in the p.R140 and p.R172 positions. IDH1/2 mutations are also among the most common mutations in brain gliomas.43 IDH1/2 mutations are currently of uncertain prognostic significance in MDS.
Mutations in histone-modifier genes are common in MDS and appear to be enriched in CMML. The EZH2 gene encodes a H3K27 methyltransferase involved in histone H3 methylation leading to transcriptional repression.44 Mutations in EZH2 have been reported in 5% to 10% of patients with MDS and are associated with a poor prognosis independent of IPPS-R status.45,46 The ASXL1 gene is a member of the polycomb gene family and is involved in the epigenetic regulation of gene expression.47 ASXL1 is mutated in 15% to 25% of patients with MDS and is also commonly mutated in AML. Mutations in the ASXL1 gene are associated with a poor prognosis and have also been implicated in rare cases of familial MDS.48
Signal transduction genes as a group are less commonly mutated in MDS than in AML and myeloproliferative neoplasms. Acquisition and expansion clones containing signal transduction gene mutations have been associated with MDS progression to acute leukemia.24,49 The JAK2 gene is a nonreceptor tyrosine kinase that acts through the STAT signaling pathway and is also implicated in interferon receptor signaling.50 A single mutational hotspot, p. V617F, dominates the spectrum of JAK2 mutations. Although present in nearly all cases of polycythemia vera, JAK2 mutations are present in fewer than 5% of patients with MDS and are enriched in cases of refractory anemia with ring sideroblasts and thrombocytosis.51 The overall prognostic significance of JAK2 mutations is uncertain. Ras family members including NRAS, and less commonly KRAS, are mutated in 5% to 10% of patients with MDS and are more frequent in patients with CMML and juvenile myelomonocytic leukemia.52 Both NRAS and KRAS mutations occur at known hotspots, including p.G12, p.G13, and p.Q61. Mutations in NRAS confer a poor prognosis in MDS. Similar to the Ras-family members, CBL mutations are more common in patients with CMML and juvenile myelomonocytic leukemia and are present in less than 5% of patients with MDS.53,54 The CBL gene encodes an E3 ubiquitin-protein ligase involved in protein ubiquitination. Mutations in the CBL gene are of unknown prognostic significance.
Commonly mutated transcriptional regulation genes in MDS include TP53, RUNX1, GATA2, and MECOM. Mutations in TP53 have been reported in 5% to 18% of patients with MDS and are generally associated with higher-risk disease, including refractory anemia with excess blasts and therapy-related AML as well as complex cytogenetics and a subset of patients with del(5q).55,56 TP53 mutations are considered a poor prognostic factor in patients with MDS; however, recent studies have demonstrated that patients with MDS with TP53 mutations show a favorable response to decitabine.57 RUNX1 is a transcription factor that regulates myeloid development and is mutated in approximately 10% of patients with MDS. Mutations including copy losses in the RUNX1 gene are generally associated with a poor prognosis.58 The GATA2 gene encodes an important myeloid transcription factor and is mutated in approximately 1% to 2% of patients with MDS. Mutations in GATA2 are of unknown prognostic significance but may also be seen as germline mutations in cases of familial MDS or primary immunodeficiency.59 The MECOM gene (also known as EVI1) encodes a zinc finger protein that acts as a transcription factor. Somatic MECOM mutations are present in approximately 1% of patients with MDS and are generally considered to be a poor prognostic factor. Rearrangements including inv(3)(q21q26) and t(3;3)(q21;q26) also involve the MECOM gene and are seen rarely seen in patients with MDS and AML.3
Sequencing-based studies have demonstrated that MDS is composed of a founding clone. By analyzing the variant allele fractions (VAFs), fraction of reads containing a mutation generated by next-generation exome sequencing (NGS) methods, mutations can be assigned to clusters and can be used to determine the clonal composition of a given MDS. Most patients with MDS will harbor only 15 to 30 somatic mutations in the exome, a notably lower mutation rate than solid tumors, but similar to the number of mutations seen in AML.60 Reconstruction of MDS clonal architecture therefore requires nonbiased sequencing approaches such as exome or whole-genome sequencing of tumor and normal germline tissue to have a sufficient number of mutations for accurate clone assignment.61 The relationship between tumor clonality and disease progression is not well understood at present; the risk of MDS progression to AML has been associated with more subclones in at least one study.23
Serial sequencing of MDS bone marrow samples allows for more accurate assessment of tumor clonality by comparing the patterns of VAF changes over time. Serial bone marrow sequencing studies have also demonstrated that somatic mutations can be detected at low levels when MDS patients are in complete remission, and the presence of detectable somatic mutations 30 days after induction chemotherapy has been associated with adverse outcome in AML.62,63 However, the relationship between the depth of clonal clearance and recurrence has not been established. Recent studies have also shown a high concordance between blood and bone marrow samples in determining the clonal architecture of MDS, suggesting that more readily obtained peripheral blood samples could be an alternative to bone marrow–based assessment.64
MDS encompass a wide spectrum of clinical phenotypes that range from largely asymptomatic patients with mild cytopenias and long life expectancy to those with profound symptoms, severe cytopenias, and a very poor prognosis. Even patients with similar clinical presentations may see their disease evolve differently over time. This variability can make it challenging for physicians to determine the optimal timing and choice of treatment of their patients and how best to counsel them about their expected prognosis. Consensus treatment guidelines for MDS rely on an accurate estimation of risk to determine optimal treatment algorithms.65-67 Therapeutic choices for lower-risk patients share very little with the options recommended for patients of higher risk. Accurate determination of prognosis is therefore critical for individualizing risk-adapted therapy for patients.
The MDS classification system created by the WHO defines MDS subtypes comprised of patients that share disease features, genetic findings, and responses to treatment, but does not serve as a prognostic tool.3 The criteria for WHO subtypes include the proportion of bone marrow blast cells, deletion of chromosome 5q, and, in the case of patients with few ring sideroblasts, the presence of a typical somatic mutation in the SF3B1 gene. Other molecular and cytogenetic abnormalities are not considered, limiting the prognostic value of WHO-defined MDS subtypes. Therefore, several prognostic scoring systems have been developed that incorporate disease-related risk factors, patient features, and genetic findings to more accurately risk stratify patients with MDS. Most of these risk assessment tools do not formally incorporate somatic mutations that have been shown to carry independent prognostic associations capable of refining the prediction of disease risk. This section will review the most widely used prognostic scoring systems for MDS and summarize how somatic mutation testing can be used to more accurately assign patients to appropriate risk groups.
The WHO Classification-Based Prognostic Scoring System (WPSS) takes advantage of the fact that MDS subtypes are, in part, defined by several prognostic features (Table 1).68
The WPSS assigns risk scores to subtypes based on their bone marrow blast proportions and adds consideration of cytogenetic abnormalities and the presence of severe anemia to determine a total risk score. These scores are then translated into one of five risk groups with significant differences in median overall survival and likelihood of progression to AML. Advantages of the WPSS include its ease-of-use and that it has been validated at times other than diagnosis, making it a dynamic scoring system. Its major disadvantage is the limited number of cytogenetic abnormalities explicitly considered, which may make it less precise for certain patients.69-71
IPSS was the clinical standard for MDS risk assessment until the revised version was published in 2013.72 It was derived by examining over 800 patients with MDS who never received disease-modifying therapies likely to impact overall survival. Patients with proliferative CMML and therapy-related MDS were excluded, but the IPSS included patients with 20% to 30% blasts now considered to have AML by WHO criteria. Like the WPSS, the IPSS is simple to use and considers the percentage of blasts in the bone marrow, a small number of cytogenetic abnormalities, and the presence of cytopenias as relevant risk factors. Patients are assigned to one of four risk groups. In practice (and in clinical guidelines), those with low or intermediate-1 IPSS risk are considered to have lower-risk MDS, while those with intermediate-2 or high IPSS risk are labeled higher risk. This is an important distinction, as clinical guidelines recommend very different treatment algorithms for each group.66,67,73,74
After its adoption, it became evident that the IPSS has some important limitations. It considers only the presence of cytopenias and not their severity, and it outweighs the impact of blast proportion compared with cytogenetic abnormalities.75,76 This leads to an underestimation or risk in many lower-risk patients with normal blast proportions.77
The MD Anderson Lower Risk Prognostic Scoring System (LRPSS) is designed specifically to address concerns with the IPSS.78 This model takes patients considered to have lower risk by the IPSS and restratifies them according to criteria that include age, bone marrow blast percentage, cytogenetics, and, unlike the IPSS, the severity of anemia and thrombocytopenia. Patients could be assigned to one of three risk categories. A quarter to a third of IPSS lower-risk patients fall into the highest-risk LRPSS category and have a median overall survival comparable to that of IPSS intermediate-2 patients, indicating that they should actually be considered to have higher-risk disease.78-80 However, there are few prospective studies of approved MDS therapies demonstrating clinical benefit in patients identified as having higher-than-perceived risk in this manner.81
The IPSS-R also addresses several limitations of the IPSS and offers other improvements that make it the clinical standard for risk assessment for patients with MDS today. Developed by studying over 7,000 untreated patients with MDS, the IPSS-R evaluates the same features as the IPSS, but does so in greater detail (Fig. 2).8 Patients with bone marrow blasts of at least 20% are excluded, and those with as few as 3% or 4% blasts are considered to be at increased risk. The range of cytogenetic risk scores is increased, and the number of explicitly considered karyotype abnormalities more than double (Table 2).82 And unlike the original IPSS, the severity of each cytopenia is taken into account. This results in a broader range of risk scores and assignment of patients into one of five groups (very low, low, intermediate, high, or very high). The division between lower and higher risk is made in the middle of the intermediate risk group with an IPSS-R score of no more than 3.5 defining patients as having lower risk.
FIGURE 2. The Revised International Prognostic Scoring System
Patients with a risk score of 3.5 or below are considered to have lower risk. Patients with a score greater than 3.5 have higher-risk disease. An online calculator is available at www.ipss-r.com.
The IPSS-R has been extensively validated even in contexts for which it was not originally developed.22 This includes cohorts of patients treated with hypomethylating agents, lenalidomide, or allogeneic stem cell transplantation.9,83-88 It has also been examined at times other than diagnosis, where it continues to risk stratify patients well.89,90 The IPSS-R compares favorably to other risk stratification systems, making it the current gold standard for MDS risk assessment.70,71 However, it is important to explain to patients that survival estimates based on the IPSS-R were derived from a cohort that did not receive disease-modifying therapy. Survival estimates in treated patients may well be different, and, in practice, factors not considered by the IPSS-R should influence the final assessment of risk.
For example, patient age is not an element of the IPSS-R but figures heavily in the accurate estimation of prognosis. Age can be considered by adjusting the cutoffs for IPSS-R–defined risk groups in a model called the IPSS-RA. An online calculator (www.ipss-r.com) is available to help apply the IPSS-R and IPSS-RA.
Physicians should also consider other factors that can refine the prognosis predicted by the IPSS-R. For intermediate-risk patients near the border of lower- versus higher-risk disease, greater lactate dehydrogenase, ferritin, and bone marrow fibrosis have been shown to carry increased risk.8,91-93 Conversely, greater time since diagnosis may portend a better-than-predicted prognosis for initially higher-risk patients.89 Finally, comorbid conditions can impact longevity in the setting of MDS and should influence which therapeutic options are recommended for patients.91,94-96
Studies have repeatedly demonstrated the prognostic impact of somatic mutations in patients with MDS.17,97 These genetic events are present in nearly every patient with MDS and represent the pathophysiologic drivers of disease development and evolution (Fig. 1). This makes mutations potentially better disease biomarkers than clinical features alone. Mutations of several genes have prognostic significance independent of clinical scoring systems, including the IPSS-R.97-100 However, mutations have not been formally incorporated into these models to date as there is no consensus of how best to consider them. This is in part because mutations can co-occur in a wide variety of patterns and can be present in either the dominant clone or smaller subclones where they might have different impacts.17,101-103 Despite this complexity and lack of formal guidelines, somatic mutations can be used to refine the prognosis of patients with MDS today.
In general, a greater number of somatic mutations is associated with a shorter overall survival (Fig. 3A).18,100 Yet not all mutated genes carry equal prognostic significance; their independent prognostic value may depend upon the clinical context in which they are observed. For example, the splicing factor SF3B1 is the only recurrently mutated gene associated with a favorable prognosis.97,104,105 SF3B1 mutations are highly enriched in lower-risk patients, where they are associated with a longer overall survival even after adjustment for the IPSS-R (Fig. 3B).100 However, SF3B1 mutations lose their independent prognostic impact in those patients with rare mutated disease with higher blast proportions (Fig. 3C).
FIGURE 3. Somatic Mutations and Disease Risk: Data From the International Working Group for MDS Molecular Prognosis Committee Presented at the American Society of Hematology Annual Meetings in 2014 and 2015100,106
(A) Overall survival in 1,996 patients with MDS sequenced for mutations in 17 genes (ASXL1, CBL, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, SRSF2, TET2, TP53, U2AF1, and SF3B1). (B) Hazard ratio of death adjusted for IPSS-R risk group, for mutations in various genes for patients with less than 5% bone marrow blasts. The hazard ratio for each gene compares patients with a mutation in that gene to those without one in that gene. The size of the marker indicates the frequency with which the gene is mutated in this population. Genes plotted above the dotted red line show a significant association with prognosis that is independent of the IPSS-R, while those below do not reach statistical significance. Mutations in significant genes with a hazard ration of greater than 1 are adverse, while SF3B1 is the only prognostically favorable mutated gene.100 (C) Same as panel B but for patients with bone marrow blasts of 5% to 30%.100 (D) Overall survival in 286 complex karyotype patients with MDS stratified by TP53 mutation status.106 Abbreviations: IPSS-R, Revised International Prognostic Scoring System; MDS, myelodysplastic syndromes.
Similarly, there are several mutated genes considered prognostically adverse, primarily in patients with lower-blast proportion (Fig. 3B). These genes, which include ASXL1, U2AF1, and SRSF2, among others, lose their independent prognostic significance in patients with elevated blast proportions and higher-risk disease (Fig. 3C). Mutations of RUNX1, EZH2, and particularly TP53 remain prognostically adverse across risk groups. Mutations in independently prognostic genes are not rare. About one-third of patients with MDS will carry one or more mutations associated with greater-than-perceived risk by the IPSS-R.100 A similar fraction of lower-risk patients will carry a favorable SF3B1 mutation. This suggests that we may be both under- and overestimating risk in a substantial proportion of patients with MDS.
This applies even in patients with complex karyotypes who typically have IPSS-R high and very high–risk disease. Approximately 50% of these patients will harbor a mutation of TP53, which is independently associated with a dismal prognosis even after treatment or stem cell transplantation.84,106-109 However, complex karyotype patients without a TP53 mutation may have substantially longer overall survival than predicted by the IPSS-R (Fig. 3D), suggesting that the adverse prognostic weight given to the complex karyotype is driven largely by its frequent association with TP53 mutations.17,84,106
Prognostic assessment remains a critical component of the personalization of care for patients with MDS as treatment is highly risk adapted. Multiple methods for risk stratification are available, with the IPSS-R currently considered the gold standard. Increasing access to myeloid gene panels and greater evidence for the diagnostic and predictive value of somatic mutations will soon make sequencing part of the standard evaluation of patients with MDS. In the absence of formal guidelines for their prognostic use, well-validated mutations can still refine estimates of risk made with the IPSS-R. The impact of less-frequent mutations and how best to incorporate them in practice will await the publication of consensus guidelines based on studies of large cohorts of sequenced patients. These are in development and will improve how we assess and care for our patients with MDS.
The advent of NGS introduced an additional set of molecular genetic data that is now routinely applied in decision making for patients with MDS. Few mutations, such as SF3B1, are favorable and modify the unfavorable impact of other specific mutations such as DNMT3A on overall survival.17 Not only are somatic gene mutations advantageous in understanding the biology of MDS and prognosis, they also offer potential as biomarkers and targets for the treatment of patients with MDS.
The first genetic abnormality guiding management decisions in MDS were the interstitial deletion involving the long arm of chromosome 5 (del5q MDS). Lenalidomide selectively suppresses del(5q) clones by inducing ubiquitination of the haplodeficient casein kinase 1A1 (CK1α) encoded within the commonly deleted region by the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN, resulting in CK1α degradation and erythroid growth arrest.110 A multicenter, international U.S. registration trial evaluated 148 red blood cell transfusion–dependent patients with IPSS low/intermediate-1–risk MDS with chromosome 5q deletion.111 Treatment with lenalidomide yielded a 50% or greater reduction in transfusions in 76% of patients, while 67% achieved transfusion independence lasting a median duration of more than 2.7 years in patients with isolated del(5q).112 Overall, 45% of evaluable patients achieved a complete cytogenetic response and 28% a partial response, with resolution of cytologic dysplasia in 36% of patients. Interestingly, there was no significant difference in response rate between patients with isolated deletion 5q and those harboring one or more additional chromosomal abnormalities. Long-term follow-up of this study showed that patients achieving transfusion independence had a significantly longer overall survival and reduced risk of leukemia progression, suggesting that clonal suppression modifies the disease natural history.112
Immunosuppressive therapy (IST) in the form of antithymocyte globulin and cyclosporine can yield durable hematologic responses in a subset of lower-risk patients with MDS. The National Institutes of Health IST Response Model identifies those with the highest probability for response based upon variables that include HLA-DR15 phenotype, age, and duration of transfusion dependence.113 The impact of somatic gene mutations in predicting response was evaluated in a retrospective study of 66 lower-risk patients with MDS treated with antithymocyte globulin plus cyclosporine.114 The overall response rate was 42%, and among 40 patients evaluable by NGS, somatic gene mutations were not detected in 50% of patients. Those patients without mutations experienced a higher response to IST compared with those harboring gene mutations and had a longer duration of hematologic response. The presence of an SF3B1 gene mutation was associated with a significantly lower response rate (11%) compared with wild type (68%; p = .01). The rate of transformation to acute leukemia was higher in patients harboring any mutation other than SF3B1 versus patients without mutations, which was accompanied by reduced overall survival. These data demonstrate a role for consideration of somatic gene mutations in the selection of candidates for IST and further implies that immune-mediated MDS may display less genetic instability.
Although azacitidine treatment improves overall survival compared with conventional care, up to 50% of patients will not respond to treatment with hypomethylating agents.115-117 Decitabine and azacitidine inhibit DNA methyltransferases to decrease cytosine methylation, raising the question whether somatic gene mutations involving epigenetic regulators might serve as biomarkers for response to these agents. This notion was first supported by a retrospective study that found 85% of patients with TET2 mutations responded to azacitidine, a response rate nearly twofold greater than that in the overall cohort of 86 patients.118 In a larger retrospective study, 213 patients with MDS receiving treatment with hypomethylating agents were investigated by NGS.119 In this larger cohort, 94% of patients carried at least one mutation, with ASXL1 (46%) the most frequent, followed by TET2 (27%), RUNX1 (20%), TP53 (18%), and DNMT3A (16%). Though there was a trend favoring a higher response rate in patients with TET2 mutations, it was not significant until the analysis was limited to those patients with allele frequencies greater than 10%. At the higher variant allele frequency, TET2 mutations were associated with a significantly higher response rate (60%) compared with wild type (43%; p = .036). Furthermore, the presence of mutated TET2 and wild-type ASXL1 had the highest response rate, while those patients with mutated ASXL1 and wild-type TET2 had a trend toward a lower response rate (p = .051). Neither of these gene mutations impacted overall survival with azanucleoside treatment; however, TP53 and PTPN11 were each associated with a significantly inferior overall survival (p = .007 and .006, respectively). Of particular importance, a complex karyotype with a TP53 mutation was associated with poor overall survival, while complex karyotypes with wild-type TP53 had the same survival as noncomplex karyotypes. Interestingly, mutations involving RUNX1, ASXL1, EXH2, and ETV6 did not significantly influence prognosis, suggesting that treatment with hypomethylating agents may modify the unfavorable impact of these mutations. More importantly, although this study identified gene mutation profiles that may impact azanucleoside response, it did not identify specific mutations linked to primary resistance, thereby limiting their usefulness for treatment selection.
Interestingly, another retrospective study of NGS gene mutations from 134 patients with higher-risk MDS treated with azacitidine revealed a significant association between karyotype and mutation profile with overall survival.120 High-risk IPSS cytogenetics were negatively associated with survival (p < .001), while mutations involving histone modifiers, including ASXL1, EZH2, and MLL, were positively associated with prolonged survival (p = .001). Specifically, patients with mutations in histone modifiers without high-risk cytogenetics had a response rate of 79% and median survival of 29 months compared with a response rate of 49% and 10-month median survival in the same patients with high-risk cytogenetics (p < .001). TP53 mutations were again a significant unfavorable covariate for overall survival (p = .001).
Spliceosome complex gene mutations, including SRSF2, U2AF1, and SF3B1, are the most commonly identified mutations in MDS and occur almost exclusively in a heterozygous state. Murine models indicate that these mutations promote the expansion of hematopoietic stem and progenitor cells and alter messenger RNA splicing and stability.121 Mouse models show that SRSF2-mutated cells survive only in the presence of a wild-type allele, while pharmacologic inhibition of the wild-type protein with the spliceosome inhibitor E7107 fosters selective clonal suppression by virtue of synthetic lethality. Treatment of human AML xenografts with mutant SRSF2 in nonobese diabetic (NOD) scid gamma mice with E7107 demonstrated significant reduction in leukemia burden compared with wild-type xenografts. A phase I/II study is currently underway to evaluate the splicing inhibitor H3B-8800 in patients with splicing gene–mutant MDS and AML (NCT02841540).
The transforming growth factor-β (TGF-β) superfamily are potent regulators of erythropoiesis with a pathobiologic role in the ineffective erythropoiesis of MDS. TGF-β ligands trigger receptor-mediated phosphorylation and activation of the inhibitory Smad2/3 transcription factors that lead to suppression of terminal erythroid differentiation. Therapeutic agents that act as ligand traps by competitively binding several TGF-β superfamily ligands can diminish the effects of this inhibitory pathway.122 Sotatercept is a recombinant human fusion protein that contains the extracellular domain of the human activin receptor IIA (ACRIIA) that recognizes and neutralizes multiple TGF-β ligands such as activin-A and growth-and-development factor (GDF)-11. Phase I trials demonstrated sustained increases in hemoglobin in healthy volunteers, whereas treatment of mice with the murine analog RAP-011 demonstrated inhibition of ACRIIA/SMAD signaling with rapid and significant rise in hemoglobin as a result of derepression of late-stage erythroid precursor maturation.123,124 In a murine thalassemia intermedia model, RAP-536, a murine fusion protein including a site-specific mutated extracellular binding domain of the ActRIIB receptor, promoted erythroid differentiation while reducing hemolysis.122 Luspatercept is the recombinant human counterpart that contains the modified extracellular domain of the activin receptor IIB, which interacts with several cognate TGF-β ligands, including GDF11, GDF8, activin-B, and bone morphogenic protein (BMP)-6 and BMP10. The luspatercept PACE-MDS trial was a phase I/II multicenter, open-label, dose-finding study of 58 patients with IPSS low/intermediate-1 MDS (27 in dose escalation and 31 in expansion phase). In the dose-escalation phase, transfusion independence was achieved in 35% of patients receiving higher doses of treatment (0.75 to 1.75 mg/kg subcutaneously every 21 days). Notably, there was a higher erythroid response rate in patients with ring sideroblasts (55% vs. 29% in ring sideroblasts–negative) and 60% of those with a SF3B1 gene mutation.125 This has led to a phase III, randomized, double-blind study comparing luspatercept to placebo in transfusion-dependent, low/intermediate-risk patients with MDS with ring sideroblasts, referred to as the MEDALIST trial (NCT02631070).
Although TP53 mutations are uncommon in patients with MDS, they nevertheless represent one of the most unfavorable mutations impacting outcome.126 Although more often associated with complex, monosomal karyotypes and del(5q) chromosome abnormalities, the clone size as measured by VAF is critical to guiding prognostic implications.101 NGS performed on specimens of 219 patients with MDS and secondary AML showed that patients with a TP53 mutation VAF greater than 40% had a median overall survival of 124 days, while it was not reached in those with a TP53 VAF less than 20%, which was indistinguishable from that for wild-type cases. Two recent studies suggest that TP53-mutant MDS/AML may be more susceptible to clonal suppression by decitabine. In a retrospective study evaluating 109 patients with MDS treated with decitabine, TP53 mutations were identified in 13.8% of patients.127 TP53 was the only somatic gene mutation predictive for complete response (CR), with 10 of 15 patients with TP53 mutations (66.7%) achieving CR versus 20 (21%) of 94 with wild type (p = .001). Of those with monosomies, 80% achieved CR. Median overall survival remained disappointing at 14 months. Similar results were reported in a study of 116 patients with MDS/AML treated with a 10-day course of decitabine every 28 days.57 Patients with a TP53 mutations had a significantly higher overall response rate compared with wild type (21 [100%] of 21 patients vs. 32 [41%] of 78 patients; p < .001) and higher rate of complete remission/incomplete marrow recovery (CR/CRi; 13 [62%] of 21 patients vs. 26 [33%] of 78 patients; p = .04). Gene sequencing at sequential time points revealed selective suppression of the TP53 mutant clone; however, there was no discernible relationship between response and changes in cytosine methylation.
Whether TP53 mutant clones display exclusive sensitivity to decitabine compared with azacitidine is not clear. A retrospective analysis of a 54-patient cohort suggests differential sensitivity of mutant TP53 cases compared with wild type; however, this merits further investigation in larger numbers of patients.128
Allogeneic hematopoietic stem cell transplantation remains the only curative treatment strategy for patients with MDS. Recent investigations show that somatic gene mutations also influence the probability of relapse post-transplantation.84 In a study of 401 patients with MDS or secondary AML who underwent allogeneic hematopoietic stem cell transplantation, the number of somatic mutations and specific gene mutations significantly affected outcome.109 Mutations involving RUNX1, ASXL1, or TP53 were independent covariates for relapse after transplantation. Patients with TP53 mutations had a particularly poor outcome and should be considered for novel investigational studies to mitigate the relapse risk.
With the increase in understanding of genetic mutations specific to MDS, molecular data is being utilized in clinical practice for risk stratification and in some cases, to guide treatment recomendations. Specifically, data support the use of lenalidomide in deletion 5q, wth other mutations requiring further confirmation for thier impact on treatment selection. With ongoing investigation, this set of information can evolve and offer more personalized treatment options for patients with MDS.
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