Pathology of Myelodysplastic Syndrome with Multilineage Dysplasia
Myelodysplastic syndrome with multilineage dysplasia (MDS-MLD) is one of the more common myelodysplastic syndromes (MDS). MDS are clonal disorders of myeloid stem cells. These syndromes are characterized by ineffective hematopoiesis manifested in morphologic dysplasia of hematopoietic precursors, one or more peripheral blood cytopenias, and a propensity to progress to acute myeloid leukemia (AML). [1, 2]
The 2016 update to the fourth edition of the World health Organization (WHO) Classification of Tumors of the Hematopoietic and Lymphoid Tissues renamed the entity previously diagnosed as refractory cytopenia with multilineage dysplasia (RCMD) as MDS-MLD, in part because the presence of a specific cytopenia (eg, anemia) does not necessarily correlate with the degree or type of dysplasia seen in the bone marrow and peripheral blood. 
The definition of myelodysplastic syndrome with multilineage dysplasia (MDS-MLD) is largely unchanged from the previous definition of refractory cytopenia with multilineage dysplasia (RCMD). MDS-MLD is defined by the World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues [3, 4] as a type of MDS with one or more cytopenias and dysplastic changes in two or more of the myeloid lineages (erythroid, granulocytic, or megakaryocytic). 
Cytopenias are defined as a hemoglobin level below 10 g/dL, an absolute neutrophil count of less than 1,800/μL (see the Absolute Neutrophil Count calculator), and a platelet count below 100,000/μL. A diagnosis of MDS-MLD requires that dysplastic features be present in more than 10% of cells from two or more lineages. There are less than 1% blasts in the peripheral blood and less than 5% blasts in the bone marrow. Auer rods are absent.
Cases previously diagnosed as RCMD with ring sideroblasts (same criteria as RCMD but with greater than 15% ring sideroblasts), as well as cases of MDS-MLD with more than 5% ring sideroblasts plus an accompanying mutation in SF3B1 will be classified as MDS with ring sideroblasts and multilineage dysplasia (MDS-RS-MLD) as well as grouped under the heading MDS with ring sideroblasts (MDS-RS) in the upcoming revision of the WHO classification.
Cases in which there are findings of MDS-MLD in conjunction with a history of cytotoxic chemotherapy and/or radiation therapy are better classified as therapy-related MDS (t-MDS).
Myelodysplastic syndrome (MDS) is an uncommon disorder; it has a higher incidence in elderly patients.  Approximately 10-15,000 new cases of MDS are diagnosed each year in the United States, with patients older than 60 years the largest age group affected.
The incidence of MDS has been reported to be between 22 and 45 cases per 100,000 people older than age 70 years. Cases previously classified as refractory cytopenia with multilineage dysplasia (RCMD) and RCMD with ring sideroblasts accounted for 26.8% and 12.7% of 1095 MDS cases, respectively, in a 2006 review,  and would roughly correlate with the reclassification as MDS-MLD and MDS-RS-MLD, respectively. The precise incidence of MDS-MLD is not known.
As noted earlier, patients with myelodysplastic syndrome with multilineage dysplasia (MDS-MLD) tend to be elderly. Most patients are asymptomatic at presentation, and MDS-MLD is often detected incidentally. However, some patients present with fatigue, easy bruising, or susceptibility to infection related to anemia, thrombocytopenia, or neutropenia, respectively. Laboratory findings include one or more cytopenias, as defined earlier.
Other potential etiologies for cytopenias, including nutritional deficiencies, the effects of medications (particularly chemotherapeutic agents) or toxins, infection, immunologic processes, or sequestration, must be excluded before a diagnosis of MDS is considered. If cytopenias are persistent and other causes are excluded, a bone marrow biopsy and aspiration are performed, usually with submission of material for cytogenetic and/or flow cytometric analysis.
Morphologically abnormal cells may be seen in the peripheral blood or bone marrow of patients with myelodysplastic syndrome with multilineage dysplasia (MDS-MLD). The morphologic findings characteristic of dysplasia vary depending on the lineage. A diagnosis of MDS-MLD requires that more than 10% of cells be present in two or more cell lineages. This is often best appreciated on bone marrow aspirate smears; peripheral blood and biopsy sections may offer additional information.
Morphologic findings of granulocytic dysplasia include abnormal nuclear segmentation, particularly with hypolobated nuclei, including pseudo–Pelger-Huet anomaly with bilobed nuclei in which the nuclear lobes are connected by a fine filament of chromatin. Hypersegmented nuclei may also be a dysplastic finding, although this feature is less common. Other forms of granulocytic dysplasia include abnormalities of the cytoplasmic granularity. In some cases, the dysplastic neutrophils are hypogranular and the cytoplasm takes on a pale blue tinge (water-clear cytoplasm) (see the image below).
In other cases, there is an increase in large, coarse, azurophilic granules (pseudo–Chediak-Higashi granules). Auer rods may also be a sign of dysplasia in the granulocyte lineage; however, the presence of Auer rods excludes a diagnosis of MDS-MLD. Rather, their presence indicates a more aggressive form of disease.
Erythroid dysplasia may present as nuclear or cytoplasmic abnormalities. Common forms of dyserythropoiesis include multinucleation with internuclear bridges, nuclear blebs or nuclear buds, and nuclear fragmentation (karyorrhexis). Megaloblastoid change, in which there is asynchrony between cytoplasmic hemoglobinization and nuclear maturation, may be present; the nuclear chromatin appears more immature than is appropriate for the degree of hemoglobinization. (See the images below.)
Dysplastic findings in the cytoplasm include vacuolization and periodic acid-Schiff positivity. The presence of ring sideroblasts is another abnormal cytoplasmic finding that may be demonstrated with a stain for iron. (See the following image.)
In the megakaryocyte lineage, dysplasia is recognized by the presence of small forms with nonlobated or hypolobated nuclei, as well as micromegakaryocytes. Dysplastic megakaryocytes may exhibit multiple discrete nuclei, a finding that has been described as “pawn ball megakaryocytes” because of the resemblance to the signs that were sometimes used to indicate pawnshops in the past. (See the images below.)
Morphologic findings of dysplasia may also be identified in histologic sections of the bone marrow biopsy or clot section. The cellularity of the bone marrow is usually normal or increased in MDS, although a form of hypoplastic MDS has been recognized. The presence of clusters of immature myeloid elements away from bone trabeculae or blood vessels (so-called abnormal localization of immature precursors) may also be noted, particularly in cases of MDS with excess blasts (MDS-EB).
It is important to note that the described morphologic changes are not sufficient for making a diagnosis of myelodysplasia. Rather, for a diagnosis of myelodysplasia, the findings must be seen in the appropriate clinical setting, and persistent cytopenia(s) must be present; in addition, other causes of morphologic “dysplasia” must be absent. 
The clinical differential diagnosis for myelodysplastic syndrome with multilineage dysplasia (MDS-MLD) includes virtually any entity that can lead to persistent cytopenias, including congenital and acquired conditions. Non-neoplastic conditions that can present with cytopenias and varying degrees of morphologic “dysplasia” include viral and other infections, nutritional deficiencies, autoimmune disorders, heavy metal toxicity, Fanconi anemia, congenital dyserythropoietic anemia, among others. Cytopenias with associated “dysplasia” can be the result of medications (especially chemotherapeutic agents). Aplastic anemia also presents with cytopenias and can raise concern for a hypoplastic MDS, but acquired aplastic anemia typically lacks significant morphologic dysplasia and has a normal karyotype.
Neoplastic marrow infiltrates including metastatic carcinoma, lymphoproliferative disorders, and acute myeloid leukemia can present with clinical cytopenias; however, the morphologic findings would not support a diagnosis of MDS-MLD. Chronic myelomonocytic leukemia (CMML) may also present with similar findings in the peripheral blood and bone marrow, however, the peripheral blood monocytosis seen with CMML (>1,000 monocytes per μL) excludes a diagnosis of MDS-MLD. ASXL1 mutation and somatic mutations may be potential indicators of progression of MDS with single or multilineage dysplasia to CMML. 
It is important to remember that more than one process affect the peripheral blood counts at a given time. For example, although infection can be associated with dysplastic findings, patients with MDS are predisposed to infection due to the low number of granulocytes. In addition, patients may have an MDS in the setting of a lymphoproliferative disorder.
To firmly establish a diagnosis of MDS, other potential etiologies for cytopenias and the morphologic changes must be excluded.
Additional laboratory findings that help confirm that cytopenias and morphologic dysplasia represent an MDS include increased numbers of blasts, flow cytometric demonstration of aberrant maturation patterns or aberrant antigen expression (such as lymphoid markers on myeloid cells), or clonal cytogenetic abnormalities.
Myelodysplastic syndrome (MDS) has largely been diagnosed on the basis of morphologic and clinical features; in some cases, the diagnosis is supported by abnormal cytogenetic findings. The use of immunohistochemical analysis and flow cytometry in the diagnosis of MDS is an area of active investigation. Some tests have proved useful in establishing this diagnosis.
Immunohistochemical analysis of bone marrow biopsy and clot sections has centered on the identification of immature myeloid precursors, or blasts, especially with the use of antibodies to the progenitor cell antigen CD34. It is important to note that CD34 is not expressed in all populations of myeloid blasts, and its expression is also not limited to myeloid blasts: CD34 is expressed in vascular endothelium, as well as in some immature B-cell precursors (hematogones).
Other markers (eg, monocytic markers) have been shown to have limited utility in establishing the diagnosis of MDS. However, immunohistochemical markers may help to delineate the distribution and proportions of erythroid (glycophorin, hemoglobin), megakaryocytic (CD61), monocytic (CD68, CD14), and myeloid (myeloperoxidase) elements in the bone marrow. 
Immunophenotyping by flow cytometry is used with increasing frequency in the evaluation of bone marrows for MDS. The phenotypic characteristics of myeloid blasts may be readily assessed by flow cytometry. Aberrant patterns of antigen expression can help to separate regenerative blast populations from abnormal blast populations. For example, expression of CD7 (a T-cell–associated antigen) and CD56 (a natural killer [NK]-cell–associated antigen) by myeloid blasts is considered aberrant, because it is not seen in normal blast populations. Other findings that may be of value in the diagnosis of myelodysplasia include abnormal patterns of antigen expression in maturing myeloid and monocytic cells, decreased cytoplasmic granularity in granulocytes, and decreased numbers of immature B-cell precursors. 
The precise molecular mechanisms of myelodysplastic syndrome (MDS) are not fully understood. However, in many cases of MDS, there is an increase in apoptosis among bone marrow precursors, which accounts for the ineffective hematopoiesis.
Cytogenetic abnormalities are relatively common in MDS with multilineage dysplasia (MDS-MLD); abnormal karyotypes are identified in up to 50% of cases.  The more frequently encountered abnormalities include deletions of the long arm of chromosome 5 or 7, monosomy of chromosome 5 or 7, deletions of chromosome 20, additional copies of chromosome 8, loss of chromosome Y, and some structural rearrangements or a complex karyotype with multiple abnormalities.
Cytogenetic findings have been shown to have prognostic significance. Analysis of large patient populations in a multicenter study allowed detailed subdivision of cytogenetic prognostic groups categorized as “very good” (del 11q, -Y); “good” (normal cytogenetics, del(5q), del(12p), del(20q)); “intermediate” (del(7q), +8, i(17q), +19, any “other” abnormalities); “poor” (inv(3)/t(3q)/del(3q), any two abnormalities including -7/del(7q), or any 3 abnormalities); or “very poor” (complex karyotype with >3 abnormalities). 
Fluorescence in situ hybridization (FISH) may be used to assess interphase cells for common cytogenetic abnormalities, particularly in cases in which there are insufficient metaphase cells for routine cytogenetic analysis. However, its utility in the setting of a morphologically adequate karyotyping study is limited.  FISH may also be of value in the follow-up of known abnormalities.
Analysis with next-generation sequencing has been able to detect recurrent molecular abnormalities in genes involved in epigenetic regulation, RNA splicing, DNA repair, transcriptional regulation, signal transduction, and the cohesin complex. DNA repair gene expressions are associated with bone marrow cellularity in MDS.  Next-generation sequencing also identifies somatic mutations that are prognostic of malignancy.  However, because some mutations may be detected in otherwise healthy older patients without myelodysplasia (“clonal hematopoiesis of indeterminate potential”), the utility of molecular testing in the diagnosis of MDS remains under investigation, and results of molecular testing must be interpreted in the context of clinical and morphologic findings. 
It is noteworthy that the number of molecular abnormalities (especially driver mutations) in a patient with a diagnosis of MDS does correlate with outcome. One example of the use of molecular testing in MDS classification involves mutations in SF3B1 (an RNA splicing factor). These SF3B1 mutations have been correlated with the presence of ring sideroblasts (and a better prognosis), and they would support classifying a case with morphologic features of MDS-MLD as MDS-MLD-RS even if the number of ring sideroblasts is lower than 15%. 
Different variables have been assessed for their usefulness in determining the prognosis of patients with various types of myelodysplastic syndrome (MDS), such as MDS with single lineage dysplasia (MDS-SLD), MDS with ringed sideroblasts (MDS-RS), and MDS with multilineage dysplasia (MDS-MLD). [18, 19]
The International Prognostic Scoring System (IPSS), first described in 1997,  allows cases to be stratified on the basis of the percentage of blasts in the bone marrow, the karyotype, and the number of cytopenias (see the Myelodysplastic Syndrome International Prognostic Scoring System calculator). On the basis of the combination of these findings, cases are divided into four risk groups: low, intermediate-1, intermediate-2, and high. These groups were found to have significant differences in overall survival and risk of progression to acute myeloid leukemia.
Other prognostic scoring systems have been proposed and applied to patients with myelodysplasia, including the revised international prognostic score (IPSS-R), originally proposed in 2012.  The IPSS-R has been validated by several different groups and represents an improvement on earlier prognostic systems. 
Results from a study by Chu et al indicated that in patients with MDS, especially those with MDS-MLD, the flow cytometric scoring system (FCSS) can also aid in determining prognosis. 
In a separate study, Kharfan-Dabaja et al found that TP53 and IDH2 somatic mutations in those with MDS are prognostic for inferior 3-year overall survival following allogeneic hematopoietic cell transplantation. 
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John P Hunt, md Associate Pathologist, Assistant Professor, Department of Anatomic and Clinical Pathology, Tufts University School of Medicine
John P Hunt, md is a member of the following medical societies: American Society for Clinical Pathology, College of American Pathologists, New England Society of Pathologists, United States and Canadian Academy of Pathology
Disclosure: Received income in an amount equal to or greater than $250 from: Ventana Medical Systems.
Sung-hee Oh, MD Resident Physician, Department of Pathology, Baystate Medical Center
Sung-hee Oh, MD is a member of the following medical societies: American Society for Clinical Pathology, American Society of Hematology, Association for Molecular Pathology, College of American Pathologists, European Hematology Association
Disclosure: Nothing to disclose.
Christine G Roth, MD Director of Hematopathology, Medical Director of Flow Cytometry, Department of Pathology, Baylor-St Luke’s Medical Center; Senior Faculty, Department of Pathology and Immunology, Baylor College of Medicine
Christine G Roth, MD is a member of the following medical societies: American Medical Association, American Society for Clinical Pathology, American Society of Hematology, College of American Pathologists, International Clinical Cytometry Society, Society for Hematopathology, United States and Canadian Academy of Pathology
Disclosure: Nothing to disclose.
Pathology of Myelodysplastic Syndrome with Multilineage Dysplasia
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