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How I Treat in Brief: Using Molecular Genetic Tests to Evaluate Myelodysplastic Syndromes

December 30, 2021

David P. Steensma, MD, of Dana-Farber Cancer Institute and Harvard Medical School in Boston, discussed how to use results from molecular genetic tests in the diagnosis and evaluation of patients with myelodysplastic syndromes. Below, we summarize his approach.

This material was repurposed from "How I use molecular genetic tests to evaluate patients who have or may have myelodysplastic syndromes," published in the October 18, 2018, issue of Blood.

  • Next-generation sequencing (NGS) assays that test for subsets of MDS-associated gene mutations at the DNA level are widely available in clinical practice, but we are still learning how best to use these assays.
  • NGS results may aid in selecting treatment or transplant decision making for MDS patients in selected circumstances.
  • Caution should be used in interpreting NGS results, given the high degree of allelic heterogeneity in MDS-associated genes.
  • As NGS panels become less expensive, it seems likely that they will move earlier in the diagnostic testing algorithm for patients with cytopenias.


Myelodysplastic syndromes (MDS) are defined by the presence of persistent blood cytopenias associated with certain characteristic morphologic changes in blood and marrow cells. However, none of these morphologic findings is specific for MDS and the assessment of dysplasia is to some extent subjective. Therefore, to diagnose MDS, the 2016 World Health Organization (WHO) classification continues to require finding >10 percent dysplastic cells in more than one hematopoietic lineage, an abnormal karyotype, or an increase in myeloblasts (TABLE).

In the past 10 years, though, researchers have discovered more than 40 recurrent, MDS-associated gene mutations. This has raised hope that molecular genetic testing for these mutations might help clarify the diagnosis in ambiguous cases where patients present with cytopenias and nondiagnostic marrow morphologic findings.

Next-generation sequencing (NGS) assays that test for subsets of these MDS-associated gene mutations at the DNA level are now widely available in clinical practice and are frequently employed in diagnostic evaluation. As a hematology community, however, we are still learning how best to use these assays, both for the evaluation of patients with cytopenias who might have MDS and in patients with established MDS.

The following four cases illustrate some of the potential benefits and challenges related to the use of molecular genetic testing in clinical practice.

Case 1: Ruling out a Clonal Disorder

A 72-year-old woman was found to have macrocytic anemia after she saw a new primary-care physician and mentioned increased fatigue. Her white blood cell (WBC) count and absolute neutrophil count were within normal limits, and the blood smear showed no abnormal leukocytes. There was no obvious explanation for her cytopenias based on her medications or basic laboratory studies, including serum chemistries, vitamin B12 and red cell folate levels, and a serum protein electrophoresis.

Her primary-care physician requested that a marrow aspiration and biopsy be performed. Marrow morphology was nondiagnostic: 30 percent cellular (normocellular for age), with only rare dysplastic megakaryocytes representing <10 percent of cells in that lineage. Flow cytometry was reported as unremarkable, and the karyotype was normal female metaphase in 20 metaphases.

A 95-gene NGS panel showed no pathogenic single nucleotide variants or small insertions/deletions and read count analysis showed no copy number alterations. The patient was told that she had idiopathic cytopenia of undetermined significance (ICUS), and observation was recommended.

Commentary on Case 1: NGS panels can be useful for helping rule out a clonal disorder because they have a relatively high negative predictive value for MDS. A small proportion of patients with MDS will have negative results on an NGS panel; however, the finding of a negative result on a well-designed panel in an ambiguous case such as this one should prompt consideration of an alternative diagnosis. In this case, over time, it became clear that the patient had excessive alcohol consumption and experienced pathologic grief after the loss of her husband. After she was provided appropriate psychosocial support and discontinued use of alcohol, her blood counts returned to normal.

It can be argued that a patient like this one could be followed serially over time, sparing the expense of an NGS panel (and of a marrow biopsy). However, especially for patients with one or more cytopenias, there is often considerable worry about an evolving clonal disorder, which may co-exist with other potential causes of cytopenias, including alcohol use. When NGS panels are used, they must be interpreted in the context of the information obtained from morphology and conventional cytogenetic testing. As NGS panels become less expensive, it seems likely that they will move earlier in the diagnostic testing algorithm for patients with cytopenias.

Case 2: Identifying High-Risk CCUS

A 67-year-old man was referred to our center after he was found to have macrocytic anemia, which persisted for nine months and was slightly worse at the time of initial evaluation. His WBC count and differential were unremarkable, and his platelet count was at the lower end of the normal range (188×109/L). His previous blood counts had been normal except for mild normocytic anemia noted at the time of a bacterial pneumonia three years earlier, which eventually resolved. There were no medications or comorbid conditions that were felt likely be contributing to his cytopenias, and he did not drink alcohol.

The patient was a physician with extensive previous exposure to fluoroscopy that raised concern for an exposure-related MDS, and therefore, he underwent a bone marrow aspirate and biopsy. This showed a 40 percent cellular marrow with rare megaloblastoid or binucleate erythroid cells, representing <10 percent of nucleated erythroid precursors. Flow cytometry was unremarkable, and the karyotype was normal male metaphase in all 20 metaphases tested.

However, a molecular genetic panel showed a DNMT3A R882H mutation with 14.6 percent variant allele frequency (VAF). The patient was concerned that he was evolving to MDS.

Commentary on Case 2: When a mutation is present in an older patient with a cytopenia, especially a mutation commonly associated with clonal hematopoiesis of indeterminate potential (CHIP; including DNMT3A, TET3, or ASXL1 at a VAF<20%), it raises the possibility that the mutation and the cytopenia may be unrelated. Still, research has shown that patients with clonal cytopenias of indeterminate significance (CCUS; defined as the presence of unexplained persistent cytopenias and mutations in leukemia-associated driver genes yet do not meet WHO criteria for MDS or acute myeloid leukemia [AML]) have a substantial risk of progression to AML or MDS. The risk is greatest for those with a higher mutation burden (VAF >20%), more than one mutation, or a mutation in a splicing factor.

In the future, it is possible that patients with certain mutation patterns may be able to be diagnosed as "MDS without dysplasia," given their shortened life expectancy and a natural disease history that is similar to that observed with MDS diagnosed using current WHO criteria.

Case 3: Evaluating a Patient With an Established MDS Diagnosis

A 64-year-old woman who had not had laboratory tests in more than five years presented with pancytopenia and underwent marrow aspiration and biopsy, which was consistent with MDS with excess blasts type I. Multilineage dysplasia was present, including 10 percent ring sideroblasts. The karyotype showed monosomy 7 in 6/20 metaphases, del20q and loss of the X chromosome in 8/20 metaphases, and 6 normal female metaphases. Mutation testing showed SF3B1 K700E (VAF=26.8%) and TP53 V197M (VAF=16.0%).

Commentary on Case 3: Molecular testing in patients with an established diagnosis of MDS can provide information that may influence risk stratification and, in some cases, treatment selection. For example, the presence of a mutation in TP53ETV6ASXL1EZH2, or RUNX1 effectively raises an MDS patient's International Prognostic Scoring System risk stratification by one risk group and has been associated with poor overall survival. With the exception of rare targetable mutations, such as the IDH1/IDH2 mutations for which U.S. Food and Drug Administration–approved drugs are available (approved for patients with relapsed or refractory, IDH1– or IDH2-mutated AML), though, NGS results have relatively little influence on drug selection outside the context of a clinical trial.

Case 4: Revealing Alternate Diagnoses

A 72-year-old man had moderate splenomegaly, liver enlargement, and a few retroperitoneal lymph nodes that were also enlarged in the 2- to 3-cm range. His blood counts included a hemoglobin of 11.1 g/dL, mean corpuscular volume 93 fL, platelets of 140×109/L, and WBC of 6.65×109/L with 31 percent monocytes, 36 percent neutrophils, and 24 percent lymphocytes.

Bone marrow biopsy was hypercellular for age and consistent with chronic myelomonocytic leukemia type 1 (CMML-1). Karyotype was normal, and an MDS-focused fluorescent in situ hybridization (FISH) was also unrevealing. Single-gene testing for JAK2 was wild type. Biopsy of the retroperitoneal lymph nodes demonstrated fibrosis, which was interpreted as reactive, and biopsy of the enlarged liver showed focal irregular fibrosis and areas of portal effacement.

The patient was then referred to our center to assess whether his hepatic fibrosis might be paraneoplastic due to the CMML. Molecular typing of blood demonstrated ASXL1 G642fs* in 70.6 percent of 119 sequences reads, KIT D816V in 41.8 percent of 593 reads, TET2 Q866* in 45.4 percent of 1,442 reads, and TET2 Y1255* in 46.7 percent of 302 reads.

After noting the KIT mutation, a pathologist requested outside liver biopsy, and lymph node blocks and immunoperoxidase studies revealed small aggregates of spindled cells within the fibrosis that were positive for mast cell tryptase and C-Kit and also aberrantly co-expressed CD25. The patient’s serum tryptase level was found to be >500 ng/mL and he had no mast cell mediator symptoms. He was then treated on a compassionate-use protocol with midostaurin and experienced clinical improvement. After he became resistant to midostaurin, he was treated with avapritinib (BLU-285), also by compassionate use, and regained response.

Commentary on Case 4: Occasionally, NGS panels may uncover an unexpected finding that reveals an additional diagnosis or an alternative diagnosis. In this patient, the discovery of a KIT D816 mutation commonly associated with systemic mastocytosis (SM) led to additional studies, which confirmed a diagnosis of SM with associated hematologic disease and allowed selection of a targeted therapy that improved the patient's symptoms.

The likelihood of finding an additional diagnosis depends on the composition of the sequencing panel. For example, for logistic and workflow reasons, our institution uses a single NGS panel with genes associated with lymphoid, plasma cell, and myeloid disorders.

Conversely, patients who are being evaluated for a plasma cell neoplasm or lymphoproliferative disorder may be found to have a CHIP-associated mutation, which may complicate the evaluation, because these mutations are also frequently associated with myeloid neoplasms. Thus, there may be uncertainty about whether there is concomitant MDS or another myeloid neoplasm being masked by the lymphoid or plasma cell disorder, and this uncertainty may influence eligibility for a myeloma or lymphoma treatment protocol.

Caveats of Interpreting NGS Results

When NGS results are interpreted or the consideration of obtaining molecular typing in a patient with MDS or possible MDS is being assessed, caution is indicated. In most publications to date, mutations have been treated as a binary variable – wild-type or mutant – but many MDS-associated genes have a high degree of allelic heterogeneity, and the specific allele may influence phenotype or clinical behavior.

Also, mutations are often considered in isolation, but combinations of cooperating mutations may influence outcomes differently than single mutations, and mutations need to be interpreted in the context of other clinical and pathologic data.

Importantly, some observed variants detected on NGS panels are germline rather than somatic, especially those with a VAF near 50 percent. The somatic-vs.-germline distinction is important for many reasons, including allogeneic transplant donor selection, monitoring of patients for nonhematologic complications, and family counseling.

There is great variability associated with the use of molecular testing to evaluate MDS – in the available panels' sensitivity and number of genes assayed, as well as in clinicians' comfort and skill in interpreting results. Ongoing education is important, and molecular pathologists signing out reports can help clinicians with clear, up-to-date interpretations of variants.


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