How I Diagnose Low-Grade Myelodysplastic Syndromes (2024)

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Abstract

Case Presentations

Case 1

A 68-year-old man who recently moved to the area was referred to a local hematologist with isolated anemia (hemoglobin [Hb], 8.1 g/dL [reference range (RR), 12.0-18.0]; mean corpuscular volume [MCV], 95.1 fL [RR, 78.0-94.0]). He had had anemia for years, which was observed without treatment, but recently it worsened, and he had been receiving treatment with epoetin alfa. Because the anemia continued to be significant, a diagnosis of myelodysplastic syndrome (MDS) was being considered. A bone marrow biopsy and aspirate were performed, which showed a hypercellular, erythroid predominant marrow with dyserythropoiesis Image 1, including megaloblastoid changes, nuclear budding, and irregular nuclear contours but no ring sideroblasts on an iron stain. The myeloid and megakaryocyte lineages were morphologically unremarkable. There was no increase in blasts morphologically or by flow cytometry. Conventional cytogenetics showed a normal male karyotype in 20 metaphases. A next-generation sequencing (NGS) panel revealed a single pathogenic DNMT3A variant found at 4% variant allelic frequency.

Case 2

An 84-year-old man was referred to a hematologist for symptomatic isolated anemia (Hb, 7.3 g/dL [RR, 13.5-17.5]; MCV, 76.9 fL [RR, 80.0-100.0]). Iron studies were normal. The patient reported that he had a history of anemia but new onset of fatigue. A bone marrow biopsy and aspirate were performed, which showed a hypercellular marrow for age with erythroid predominance, left shift, and significant dyserythropoiesis manifesting as nuclear irregularities Image 2. No dysplasia was seen in granulocytes or megakaryocytes, but an iron stain on the bone marrow aspirate showed ring sideroblasts comprising 25% of erythroid elements. Subsequent to the bone marrow biopsy, results of hemoglobin electrophoresis came back showing elevated hemoglobin A₂ of 5.4% (RR, 2.0%-3.3%) and hemoglobin F of 2.7% (RR, 0%-0.9%), consistent with β-thalassemia. Conventional cytogenetics showed an abnormal 46, XY, del(20)(q11.2q13.3) karyotype in 18 of 20 metaphases. An NGS panel revealed a single pathogenic SF3B1 variant found at 8% variant allelic frequency.

Defining Myelodysplastic Syndromes

MDS are characterized by a combination of morphologic dysplasia of the various cell lines and ineffective hematopoiesis, leading to peripheral blood cytopenias. MDS have heterogeneous clinical behavior and many have a significant risk of transformation to acute myeloid leukemia (AML). The diagnosis of MDS first began to be defined by the 1982 French-American-British cooperative working group, in which five categories of MDS were proposed based entirely on morphologic criteria, including the bone marrow blast count.¹ Since then, the definitions have been modified and several subcategories have been added in the subsequent World Health Organization (WHO) classifications. In the current revised fourth edition, the WHO² describes the most up-to-date approach to the diagnosis of MDS, and here we discuss some of the diagnostic challenges and subtleties with a focus on low-grade MDS. Figure 1 presents a diagnostic algorithm for low-grade MDS based on the morphology and genetic testing results, assuming that the clinical situation has been taken into account and other causes of dysplasia considered. Specifically, low-grade MDS can be an especially difficult diagnosis to make, due to the differential diagnosis with the myriad nonneoplastic causes of cytopenia; interval rebiopsy of the patient may be required to establish a definitive diagnosis of MDS. It is particularly important to recognize that the presence of morphologic dysplasia does not necessarily equal a diagnosis of MDS. This article focuses on the diagnosis and differential diagnosis of MDS in adults and does not include specific details about pediatric MDS.

Clinical Features

The reported incidence of MDS in the United States is approximately 4 per 100,000 and peaks in the eighth decade. Patients with MDS generally present with varying symptomatology due to cytopenias. At least one cytopenia is necessary for the diagnosis of MDS, although more than one is not uncommon. Anemia is the most common cytopenia, seen in up to 85% of cases, with or without concomitant thrombocytopenia and/or neutropenia.^3,4 Pancytopenia in MDS is less common, with only 15% of patients presenting with this finding Table 1.⁴ As anemia is the most common presentation, symptomatic patients generally experience fatigue, pallor, and/or weakness; bleeding can result from patients with thrombocytopenia.^2,6 Although the International Prognostic Scoring System^5,7 gives suggested prognostic cutoffs for cytopenias, a diagnosis of MDS may still be made with milder levels of cytopenia in patients with characteristic morphologic and/or cytogenetic findings.^2,8

Morphology and Immunohistochemistry

In the workup of a cytopenic patient for possible MDS, the evaluation generally starts with examination of a peripheral blood smear. The peripheral smear findings of low-grade MDS vary from isolated anemia (commonly macrocytic) with unremarkable RBC morphology to significant RBC anisopoikilocytosis and basophilic stippling. If abnormal leukocyte morphology exists, it generally manifests as hyposegmented neutrophils (pseudo-Pelger-Huët cells), with or without cytoplasmic hypogranularity. Thrombocytopenia may be seen, with either normal-appearing platelets or hypogranular forms (Table 1).

The core biopsy histology of low-grade MDS generally exhibits hypercellularity relative to the patient’s age.^9,10 While hypocellular marrows can be seen in a minority of MDS, this is more frequently seen in pediatric MDS and in MDS following cytotoxic therapy (therapy-related myeloid neoplasm). In normal marrow, blasts are not increased, and the few blasts present are mostly close to the bone trabeculae, while in MDS, there may be abnormal localization of immature precursors,¹¹ in which clusters of blasts are seen away from bone trabeculae. Erythroid and myeloid lineage dysplasia is evaluated mainly on the bone marrow aspirate, while megakaryocytes can exhibit more obvious dysplasia on either the aspirate or the core biopsy specimen. In cases with a hemodiluted or inadequate aspirate, a definitive diagnosis of low-grade MDS based on morphology is difficult and should be made with caution. In the assessment of dysplasia, at least 10% of a lineage should be dysplastic to diagnose MDS.² However, it must be recognized that there are a wide variety of causes of secondary morphologic dysplasia, including infections, medications, nutritional deficiencies, toxins, bone marrow lymphomas and plasma cell neoplasms, and autoimmune diseases, and this list continues to grow Table 2.¹² Possible secondary causes of cytopenia and dysplasia should be excluded prior to making a definitive diagnosis of MDS, through careful clinical examination of the clinical history and laboratory parameters.

Erythroid dysplasia has a wide spectrum of findings, including megaloblastoid change, nuclear budding, cytoplasmic bridging, multinucleation, and nuclear/cytoplasmic dysynchrony (megaloblastoid change, mimicking the erythroid findings seen in megaloblastic anemia). Furthermore, an iron stain (ideally performed on a well-spiculated aspirate smear) must be assessed for the presence of ring sideroblasts.¹⁰ Similar to the findings that can be seen in peripheral blood, the aspirate assessment for granulocytic dysplasia most frequently shows nuclear hypolobation and/or a subset of neutrophils with cytoplasmic hypogranularity. Dysplastic megakaryocytes are usually smaller than normal and have hypolobated or nonlobated nuclei, or separated nuclear lobes. Occasionally, if the megakaryocytes are particularly small (micromegkaryocytes), they may be difficult to visualize on the core biopsy specimen, and a CD61 stain can be helpful in their identification. Additional stains that should be considered are reticulin and CD34, as both increased fibrosis and clustering of CD34-positive blasts have been shown to portend a worse prognosis in MDS, including in patients without excess blasts.¹³ While there is significant interobserver variability in diagnosing MDS in general, the interobserver variability is highest among low-grade MDS compared with MDS with increased blasts.^14,15 Finally, it is very important to recognize that in some low-grade MDS cases, little to no overt morphologic dysplasia may be present. In MDS with ring sideroblasts (while the ring sideroblasts themselves constitute dysplastic erythroid cells), only minimal dyserythropoiesis may be visualized on the Wright-Giemsa–stained aspirate smear. In a subset of MDS-unclassifiable cases, significant morphologic dysplasia is absent, and the diagnosis is made by identifying an MDS-defining cytogenetic abnormality.

Flow Cytometric Immunophenotyping

Immunophenotyping by flow cytometry serves as a useful adjunct for the diagnosis of MDS, particularly low-grade cases that may show subtle morphologic abnormalities. Although cytomorphology of the aspirate smear remains the definitive means of assessing blast percentage, flow cytometry provides valuable information about the overall immunophenotype of the blasts, such as aberrant expression of CD5, CD7, CD19, and/or CD56.^16,17 In addition, both myeloblasts and monocytic precursors have been shown to have aberrant expression of CD45, CD34, CD117, HLA-DR, CD13, and CD33, among others.¹⁷ The European LeukemiaNet Working group suggests a minimum panel of flow markers that can be used for the marrow assessment of MDS that evaluates myeloid progenitors and erythroid, neutrophil, and monocyte components for signs of aberrancy.¹⁷ It is recommended that in patients without significant morphologic dysplasia or cytogenetic findings diagnostic of MDS, finding three or more flow cytometric abnormalities warrants an interval repeat marrow analysis for further evaluation, as it raises strong suspicion of evolving MDS. In addition, it has been shown that multiparameter flow cytometry can be helpful in excluding MDS in patients with indeterminate morphology¹⁸ and in distinguishing MDS from benign mimics.^19-21 In patients who have an established diagnosis of MDS, Alhan et al²² showed that an MDS flow cytometric prognostic score can be applied that incorporates side scatter, CD117 expression on myeloid progenitors, and CD13 expression of monocytes. However, some overlap in the immunophenotypic profiles of MDS and non-MDS reactive conditions (particularly in maturing granulocytes) and a lack of consensus on which panels and criteria to use have limited the widespread application of flow cytometry to the diagnosis of MDS.²³

Conventional Cytogenetics

Conventional karyotyping is a cornerstone for both diagnosing MDS and assessing their prognosis. Specifically, certain specific karyotype abnormalities can establish clonality and allow a diagnosis of MDS in the absence of definitive morphologic dysplasia.² However, some of the most common cytogenetic abnormalities found in MDS, –Y, +8, and del(20q), also can be found in some nonneoplastic conditions such as aplastic anemia and are not considered diagnostic of MDS in the absence of sufficient morphologic dysplasia. Fluorescence in situ hybridization (FISH) studies assessing for the most common MDS-associated abnormalities can help confirm suspected karyotype abnormalities and allow for increased sensitivity in follow-up evaluations with known abnormalities. However, FISH studies are not necessary if 20 normal metaphases are obtained by conventional karyotyping.²⁴ Chromosomal abnormalities are found in approximately 50% of newly diagnosed MDS, with many cases exhibiting more than one abnormality; however, a normal karyotype is more common in some of the low-risk MDS subtypes, such as MDS with ring sideroblasts and MDS with single-lineage dysplasia. The only distinct WHO category based on a karyotype abnormality is MDS with isolated del(5q). This low-grade MDS subtype generally presents with anemia and thrombocytosis, an unusual combination in most other types of MDS. The bone marrow characteristically has abundant small, hypolobated megakaryocytes and no increase in blasts.² MDS with isolated del(5q) can be diagnosed with a single additional karyotypic abnormality, except for monosomy 7 or del(7q) (Figure 1).² Otherwise, the karyotype is mainly used to stratify patients with MDS into prognostic subgroups using the revised International Prognostic Scoring System^5,7 to estimate the median survival and risk of evolution to AML.^5,7,25

Molecular Genetic Findings

In addition to conventional cytogenetics, high-throughput sequencing technologies have led to an explosion of new information about acquired DNA variants in myeloid neoplasms. Specifically, rapidly increasing access to NGS (also called massively parallel sequencing) has contributed to the body of literature about molecular changes within myeloid neoplasms. While not currently used for diagnosis alone, DNA variants can help guide knowledge about pathogenesis, prognosis, and treatment in MDS. A relatively small number of genes (approximately 40) are mutated in up to 90% of patients with MDS; thus, whole-genome or whole-exome sequencing typically is not employed in clinical NGS panels. The variants affect genes involved in RNA splicing, transcription, DNA methylation, chromatin modification, signal transduction, and DNA repair. Identifying these somatic variants can be particularly useful to prove clonality in cases of cytogenetically normal suspected MDS.

Only one variant is currently used to help classify one of the low-grade MDS subtypes—SF3B1. In the workup of MDS, if anemia is accompanied by erythroid dysplasia and 5% or more ring sideroblasts are identified, in conjunction with a pathogenic SF3B1 variant, a diagnosis of MDS with ring sideroblasts can be made (Figure 1)²; in the absence of an SF3B1 mutation, at least 15% ring sideroblasts would be required. SF3B1-mutated cases of low-grade MDS have a particularly favorable prognosis.^26,27

It is also important to note that many of the most common somatic DNA variants in MDS also can be found in healthy patients without overt evidence of MDS. In clonal hematopoiesis of indeterminate potential (CHIP), DNA variants (at a variant allele fraction [VAF] of at least 2%) can be identified in the peripheral blood or bone marrow of older individuals without cytopenias or other evidence of MDS. The rate of progression to MDS in patients with CHIP is approximately 1% to 2% per year. The most common genes seen in CHIP are DNMT3A, ASXL1, and TET2.²⁸ Additional genes less frequently involved are SF3B1, JAK2, TP53, BCORL1, and GNAS.²⁸ Clonal cytopenia of undetermined significance (CCUS) is similar to CHIP in the presence of somatic DNA variants, but these patients also manifest at least one unexplained peripheral blood cytopenia. In both CHIP and CCUS, examination of the bone marrow must lack sufficient morphologic evidence for a diagnosis of MDS and there must be no MDS-defining cytogenetic abnormality.²⁸ The risk of CCUS progression to MDS appears to be highest in patients with high mutated VAF (at least 10%) and with a DNMT3A, ASXL1, or TET2 variant in combination with at least one additional variant or spliceosome mutations such as SF3B1, U2AF1, SRSF2, or ZRSR2.²⁹

Interpretive Challenges

The most common challenges as well as errors encountered in the diagnosis of low-grade MDS center on (1) the certainty of whether sufficient morphologic dysplasia is actually present and (2) whether any dysplasia is being caused by MDS rather than a secondary cause (Table 2). The inherent subjectivity of dysplasia interpretation, even among very experienced hematopathologists, compounds the difficulties in the interpretation of bone marrows in patients with persistent cytopenias. Finally, our expanding knowledge of the molecular genetic findings in myeloid neoplasms can be difficult to integrate with the morphologic diagnosis. In the following section, some of the most common questions the authors have received about the diagnosis of low-grade MDS are discussed.

Questions for the Experts

• 1. How did the WHO writers come up with the 10% cutoff for dysplasia in each of the cell lineages? Have you ever considered increasing the cutoff?

The cutoff of 10% dysplasia in any of the marrow lineages (erythroid, granulocyte, and megakaryocyte) is admittedly arbitrary, as more than 10% dysplasia can be seen in association with nonmyeloid neoplasms involving the bone marrow, in many nonneoplastic conditions, and even in healthy volunteers. Furthermore, interobserver variability in assessing morphologic dysplasia is also a significant confounding variable, even among experts. When morphologic dysplasia is borderline (near 10%) or only in one cell lineage, caution should be exercised in making a definitive diagnosis of MDS.

A 30% to 40% dysplasia cutoff has been proposed for megakaryocytes, which has been shown in some studies to improve the specificity of dysmegakaryopoiesis in diagnosing MDS; however, it is impossible to derive a perfect cutoff, since sensitivity will be lost with higher cutoffs.^10,30 The current 10% threshold for a diagnosis of dysplasia appears to be an acceptable minimal level and has withstood the historic “test of time.” Nevertheless, it must be used with the recognition that many non-MDS causes of cytopenia may be associated with higher levels of dysplasia in one cell line (usually erythroid) and even occasionally in two cell lines.

• 2. Sometimes, my CD34 immunostain appears to stain megakaryocytes—what does that mean? Can it happen in normal bone marrow?

While CD34 is generally considered a marker of hematopoietic stem cells as well as endothelial cells and stromal cells^31,32 and can be used to help enumerate blasts in MDS cases with dry tap or a hemodilute aspirate, it has also been reported to stain megakaryocytes in myeloid neoplasms such as MDS (most common) Image 3, myeloproliferative neoplasms (MPN), and MDS/MPN, as well as in normal and reactive marrows.^33-36 Insuasti-Beltran et al³⁷ demonstrated that the presence of 30% or more strongly stained CD34-positive megakaryocytes can reliably differentiate between neoplastic myeloid neoplasms and benign conditions such as immune thrombocytopenia and autoimmune disease. Furthermore, high numbers of CD34-positive megakaryocytes in MDS correlate with a significantly lower platelet count than cases with fewer CD34-positive megakarytocytes³⁷ and have been shown to be an independent poor prognostic marker in MDS, being associated with more severe cytopenias, high-risk cytogenetics, increased blasts, and shorter overall survival.³⁵ Thus, a high number CD34-positive megakaryocytes (particularly if the latter exhibit clear dysplastic features) can help support a diagnosis of MDS.

• 3. Currently, there is no information in the WHO on how to incorporate NGS variants into the diagnosis of MDS. Are there instances where pathogenic variants can be diagnostic of MDS?

At the current time, mutations alone cannot be used to diagnose MDS in the absence of dysplasia or a defining cytogenetic abnormality. In healthy individuals with normal blood counts and with no history of hematologic malignancy, a somatic DNA variant is classified as CHIP. Patients with persistent and unexplained cytopenias but no significant morphologic evidence of dysplasia are considered with the group of CCUS.²⁹ It is noted that some comutational patterns and high mutation VAFs (discussed above) identify patients virtually certain to develop MDS, even in the absence of morphologic dysplasia on the initial bone marrow examination.²⁹ We use DNA somatic mutations to support a borderline pathogenic finding; for example, the presence of multiple mutations at high VAF could bolster a definitive diagnosis of MDS with single- or multilineage dysplasia when the number of dysplastic cells is near 10% and if other possible nonneoplastic causes of cytopenic have been excluded clinically. In addition, the presence of the SF3B1 mutation accompanied by ring sideroblasts strongly supports a neoplastic rather than nonneoplastic etiology of anemia. Conversely, the absence of mutations, even in the presence of more than 10% myelodysplasia, tends to argue against MDS and should lead to a careful search for other causes of cytopenia and dysplasia (such as a medication-induced cytopenia secondary dysplasia). That being said, 10% to 15% of patients with MDS are reported to lack detectable DNA variants by clinical NGS testing; thus, a lack of detectable mutations does not exclude a diagnosis of MDS if definitive morphologic features, such as unexplained excess of blasts and clear-cut dysplasia, are present. At the current time, a diagnosis of MDS should not be based solely on mutations, no matter how compelling the mutation profile may be. When we encounter such cases, we recommend close clinical follow-up and repeat bone marrow biopsy at a later date, by which time sufficient morphologic dysplasia may be present to establish a definitive diagnosis.

Case Diagnosis

1. Upon discussion with the hematologist, you learn that the patient has a history of hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu disease) and has frequent epistaxis as a cause of his anemia. The findings of erythroid predominance and dysplasia in the marrow are therefore interpreted as secondary changes due to the frequent and significant bleeding episodes. The single DNMT3A variant is likely an incidental CHIP mutation in the absence of evidence of primary myelodysplasia or other clonal abnormalities.

2. The presence of a known inherited cause for anemia (β-thalassemia) and microcytosis tended to discourage a diagnosis of MDS in this patient. Patients with β-thalassemia often show erythroid hyperplasia and may exhibit dyserythropoiesis and even ring sideroblasts on bone marrow examination.³⁸ Subsequent review of the patient’s chart documented that he had been anemic for at least 5 years. However, longitudinal examination of CBC parameters showed that the patient had recently developed worsening of his anemia coincident with a rising MCV Figure 2; thus, although the patient’s anemia was microcytic, it was actually macrocytic relative to his baseline MCV. Finally, although the del(20q) cytogenetic abnormality can be seen in non-MDS conditions, the SF3B1 mutation favors a neoplastic etiology for the ring sideroblasts. The comprehensive morphologic, clinical, and genetic data, in the clinical context of worsening unexplained anemia in a patient with lifelong β-thalassemia minor, lead to a final pathologic diagnosis of MDS with ring sideroblasts and single-lineage dysplasia.

Conclusions

The diagnosis of low-grade MDS is one of the most challenging areas of hematopathology, as there is high interobserver variability in the morphologic assessment of dysplasia and a myriad of non-MDS causes of cytopenia and dysplasia in elderly populations most commonly affected. In addition, the integrative aspect of the MDS evaluation, which incorporates peripheral blood findings, bone marrow assessment, flow cytometry, cytogenetics, and molecular genetic studies, can be difficult to consolidate. In the era of increasing knowledge of the molecular genetic underpinnings of these clonal diseases, our assessment of MDS is becoming increasingly complex. When this complex array of information gives “mixed messages,” the pathologist should step back and look at the entire case carefully, always considering potential nonneoplastic MDS mimics. There is no shame in rendering a descriptive diagnosis and recommending close clinical follow-up and a repeat bone marrow sample if a definitive conclusion cannot be made on the available data.

References

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