Hematopathological Concepts and Controversies in the Diagnosis and Classification of Myelodysplastic Syndromes

Although the myelodysplastic syndromes (MDS) were initially considered by many to be synonymous with “pre-leukemia,” this concept has given way to the realization that MDS is a heterogeneous group of diseases and that the majority of patients succumb to complications of bone marrow failure rather than to acute leukemia. The heterogeneity in the clinical and morphologic findings in MDS reflects the diversity and complexity of the underlying genetic defects, of which we are still largely ignorant. However, the feature common to all cases of MDS is ineffective hematopoiesis usually accompanied by morphologic dysplasia, often with increased numbers of blasts in the blood and/or bone marrow. In 1982, the French-American-British (FAB) cooperative group proposed guidelines for the diagnosis and classification of MDS based on the myeloid lineages morphologically affected by the dysplasia and the percentage of blood and bone marrow blasts.¹ Numerous published studies documented the clinical utility of the FAB scheme and thus also validated the contributions of a morphologically-based classification for predicting prognosis and evolution to acute leukemia. In 2001, the FAB guidelines were revised and updated in the World Health Organization (WHO) Classification of MDS.² The concept of the WHO system is that classification of hematopoietic neoplasms should utilize not only morphologic findings but also clinical, genetic, immunophenotypic, and biologic information to define disease entities.³ However because a single biologic or genetic marker that reliably identifies all cases of MDS has not yet been discovered, morphology remains the cornerstone of diagnosis in the WHO system. This discussion will focus on some of the problematic morphologic issues in the diagnosis and classification of MDS, as well as on some of the controversial aspects of the WHO classification.

Concordance of the diagnosis of MDS in cooperative clinical trials is generally reported to be ~ 80%, although concordance of subclassification among different observers is often considerably less.⁴ Although there are a number of issues that contribute to inconsistencies in diagnosis and classification, the quality of the specimen, inaccurate enumeration of blasts, and failure to consider other diseases in which non-clonal dysplastic hematopoiesis can occur often account for discordant results. Therefore, a brief review of basic guidelines for the diagnosis of MDS may be helpful.

Recently the National Comprehensive Cancer Network (NCCN) recommended that the minimal initial evaluation for patients clinically suspected to have MDS include a comprehensive history and physical examination, complete blood count with leukocyte differential, reticulocyte count, bone marrow aspiration and biopsy with iron stain and cytogenetic studies, erythropoietin levels, and iron studies.⁵ 

The blood and marrow aspirate smears should be examined for dysplasia, the percentage of blasts and monocytes, and for ringed sideroblasts. Cells counted as “blasts” include myeloblasts, monoblasts, and megakaryoblasts. Small, dysplastic megakaryocytes are not blasts, and erythroid precursors are also not counted as blasts, except in rare cases of “pure” erythroleukemia, in which primitive erythroblasts account for the majority of cells. In monocytic and myelomonocytic proliferations, promonocytes are included as “blast equivalents.”^2,3 Substitution of the percent of CD34⁺ cells determined by flow cytometry for a visual blast count is discouraged. Although hematopoietic cells that express CD34 are blasts, not all blasts express CD34. Furthermore, dilution of the marrow sample by peripheral blood as well as further processing of the sample may complicate the comparison between the visual estimate and the CD34 value obtained by flow cytometry.

A bone marrow biopsy specimen is not always necessary to establish a diagnosis of MDS, particularly if the patient is frail and elderly and the treatment options limited.⁶ Nevertheless, a bone marrow biopsy often offers valuable diagnostic and prognostic information. Dysplasia, particularly of megakaryocytes, evidence of disruption of the normal marrow architecture, such as abnormal localization of immature precursors (ALIP), and an estimate of the blast percentage are important diagnostic findings that can be evaluated in bone marrow biopsies that are well prepared. The biopsy also provides tissue for a number of immunohistochemical (IHC) or molecular procedures that can provide additional diagnostic and prognostic information. For example, not only the percentage of CD34⁺ cells but also their distribution in the bone marrow as determined by IHC is reported to have prognostic significance.⁷ An under-appreciated role of the biopsy is that it may provide evidence for another disease that can mimic MDS clinically, such as hairy cell leukemia, lymphoma, or metastatic tumor.

Poor technical quality of the specimen is a common obstacle in the accurate diagnosis of MDS. For example, hypogranular cytoplasm of neutrophils is a well-accepted feature of dysplasia, but visualization of neutrophil granules is critically dependent on an optimal stain. The diagnosis of MDS should never be based on “pale granulocytes” without other features to substantiate the diagnosis.⁶ Biopsies should be of adequate size for evaluation (at least 1–2 cm) and bone marrow aspirates should be cellular enough to assess at least 500 cells. ^3,6 

As mentioned above, the detection of CD34⁺ cells by flow cytometry or by immunohistochemical detection in biopsy sections may provide diagnostic and prognostic information. Studies using multiparameter flow cytometry can also provide evidence of abnormal maturation of the myeloid lineages. There is no single abnormality that is specific, but abnormal light scatter properties of dysplastic cells, abnormal antigen density, loss of antigens, and dyssynchronous expression of antigens that are normally co-expressed during myeloid maturation have all been reported in MDS and may even correlate with the grade of disease.^8,–10 However, the specificity of aberrant antigen expression for MDS as compared to diseases in which secondary dysplasia may be seen has not been extensively studied, and aberrant antigen expression has been documented in non-hematologic disorders, such as autoimmune disease, that can mimic MDS.¹¹ 

Morphologic dysplasia is not specific for MDS, but can be seen in a number of other conditions, including megaloblastic anemia, exposure to toxins such as arsenic and alcohol, and after cytotoxic and growth factor therapy, to name a few. Dyserythropoiesis is also common when there is brisk erythroid hyperplasia, i.e., “stress dyserythropoiesis,” due to hemolysis or regeneration of the bone marrow after transplantation or chemotherapy. Transient dysplasia is most problematic when only one lineage (usually the erythroid) is involved and there is no increase in blasts.

One of the most challenging problems is a persistent, unexplained cytopenia that is clinically suspicious for MDS, but that is associated with no or only mild morphologic abnormalities in the blood and bone marrow. Often the patient is an older adult with normocytic or macrocytic anemia for whom the diagnosis of refractory anemia (RA) is being considered. The bone marrow may show mild “megaloblastoid changes” in a small percentage of the cells, with no dysplasia in other lineages and no increase in blasts. In such cases, it may be easy to over-interpret a few dysplastic erythroid precursors. Inter-observer agreement among morphologists for recognition of dyserythropoiesis is notoriously poor,¹² and dyserythropoiesis is the most common form of secondary dysplasia. Furthermore, features such as multinuclearity, irregular nuclear membranes and “megaloblastoid” changes have been observed in up to 10% of erythroid precursors in bone marrow specimens of normal individuals.¹³ The guideline that 10% of the cells should be dysplastic to consider a lineage as dysplastic is therefore a reasonable rule of thumb.⁶ However, the morphologic diagnosis of RA in cases where there is no or minimal dyserythropoiesis may be nearly impossible if there are no accompanying cytogenetic abnormalities. Whether it is reasonable to assume that most cases of unexplained macrocytic anemia in the elderly usually represent an early form of MDS is an interesting but unanswered question. Mahmoud et al reported that of 124 patients 75 years of age or older with macrocytic anemia, a definitive cause was ascertained by non-invasive techniques in 60% of the cases.¹⁴ Of the remaining 49 patients, all underwent a bone marrow procedure, but in only 6 could the diagnosis of MDS be made. The authors suggested that the remaining 43 patients might have “MDS in evolution.” However, in another study, bone marrow samples from 9 elderly patients with “idiopathic macrocytic anemia” were analyzed for molecular and biologic parameters that have been linked to MDS.¹⁵ About one-half of the patients demonstrated hyper-methylation of the calcitonin gene area, whereas others had diminished growth of erythroid progenitors similar to that observed in typical MDS in in vitro assays. Yet, only 1 of these patients showed progression to unequivocal MDS with followup of up to 31 months. The finding of a clonal cytogenetic abnormality in a patient who has clinical and morphologic evidence of dysplasia is always reassuring when a diagnosis of MDS is made, although cytogenetic abnormalities are only found in 40–60% of cases of MDS, and are even less frequent in the lower-grade lesions.¹⁶ But in a patient suspected clinically to have MDS because of unexplained cytopenias, are cytogenetic abnormalities alone enough to make the diagnosis of MDS when the morphologic findings are absent? In one study of such cases, Steensma and associates reported that the cytopenia and chromosomal abnormalities usually persist and that morphologic evidence of MDS appears after a variable length of follow-up.¹⁷ It may well be that in the future more directed molecular testing may identify patients who have RA when the morphologic findings fall short of the criteria for that diagnosis. Currently, however, it is best to monitor such patients at regular intervals with non-invasive procedures whenever possible, and with genetic and immunophenotypic analysis as necessary, but to avoid the diagnosis of MDS until the morphologic criteria are satisfied.

Most patients with MDS have normal or hypercellular bone marrow specimens, but a minority have hypocellular specimens (< 30% cellular in patients < 60 years old, < 20% cellularity in patients ≥ 60 years old). The low cellularity and poor aspirates usually obtained from such specimens render the identification of dyspoiesis and the enumeration of blasts difficult, and often it is virtually impossible to distinguish between aplastic anemia (AA) and MDS. In blood smears, macrocytic red cells are often found in both conditions, but neutrophils with pseudo Pelger-Huët nuclei and/or hypogranular cytoplasm favor MDS.^18,19 In the bone marrow, dysplastic granulopoiesis and megakaryocytopoiesis argue for MDS, but dyserythropoiesis has been reported in AA as well as MDS. A normal or increased percentage of CD34⁺ cells in the bone marrow is more likely in MDS, whereas CD34⁺ cells are markedly decreased in AA.^20,21 Additional studies, such as abnormal antigen expression of CD34⁺ cells measured by multiparameter flow cytometry, the number of HbF-containing erythroblasts, and the percentage of Ki-67⁺ cells, have also been reported to be helpful in separation of AA from MDS.^19,22,23 Although the identification of a clonal chromosomal abnormality at the time of presentation is generally considered as indicative of MDS, patients with clonal chromosomal abnormalities have been included in some series of AA.²⁴ On the other hand, AA and hypocellular MDS have a number of overlapping features that suggest a similar pathogenesis. In AA and in some cases of MDS there is evidence for T-cell–mediated myelosuppression that often responds to immunosuppressive therapy.²⁵ Clones of PNH-type cells can be found in AA and MDS, and their presence in MDS argues for a higher probability of response to immunosuppressive therapy and more favorable prognosis.²⁶ Furthermore, patients with AA successfully treated with immunosuppression may develop clonal hematopoiesis and MDS and/or AML years after the diagnosis of AA (~ 20% risk at 10 years). Whether this progression is part of the natural history of AA or is related to therapy is currently not clear, but it is unlikely that such a high level of risk is due merely to misdiagnosis of hypocellular MDS as AA.²⁴ 

The difficulty in distinguishing between MDS and AA is therefore understandable, and in some cases this distinction may be impossible. It is necessary to exclude other diseases with hypocellular bone marrow specimens that may masquerade clinically as AA or MDS, particularly hypocellular AML, hairy cell leukemia and large granular lymphocytosis.

Approximately 15% of patients with MDS show significant reticulin fibrosis, and such cases often cause diagnostic problems. A marrow aspirate often cannot be obtained, so the extent of dysplasia and an accurate blast percentage is difficult to ascertain. In addition, some subtypes of AML, such as acute megakaryocytic leukemia and acute panmyelosis with fibrosis (APMF), as well some of the chronic myeloproliferative diseases (CMPDs), are frequently associated with marrow fibrosis, and their distinction from MDS is sometimes difficult. There is also considerable taxonomic confusion regarding some myeloid disorders with fibrosis, as they, as well as MDS-F, have been reported using a variety of similar and overlapping terms. For example, APMF is included in the WHO classification as a subtype of AML, but it likely encompasses diseases previously reported as malignant myelosclerosis, acute myelosclerosis, acute myelodysplastic syndrome, MDS-F, and even some cases of acute megakaryocytic leukemia (reviewed in ref. ²⁷). Whether MDS-F should be considered as a distinct clinical and biologic entity is not clear, largely because criteria for its recognition have not been well defined in the past. Most authors agree that the diagnosis of MDS-F should be made only when, on good-quality bone marrow specimens, there is diffuse, coarse reticulin fibrosis, associated with dysplasia in at least two cell lineages. The peripheral blood smear usually shows pancytopenia, and there is no significant organomegaly.^27,28 Findings that suggest AML, mast cell disease, chronic myelomonocytic leukemia, or CMPD exclude the diagnosis. One of the most important differential considerations is APMF. This entity is characterized in the marrow by significant myelofibrosis with panmyelosis, increased blasts (usually ~ 20–25%), and multilineage dysplasia, and in the peripheral blood by pancytopenia, rare blasts and normal erythrocyte morphology.^2,29 APMF is an aggressive disorder with median survival times even worse than those reported for MDS-F. It is also important to remember that a number of other diseases must be included in the differential diagnosis of MDS-F, including autoimmune diseases, metastatic tumors, malignant lymphoma and hairy cell leukemia.

Any new classification scheme is likely to be met with skepticism and criticism, particularly when it is suggested as a replacement for a proven system such as the FAB classification of MDS. In the first months after its publication, a number of criticisms surfaced regarding the utility of the WHO classification of MDS. Most of those criticisms centered on the new criteria for RA, RARS and RCMD and, particularly, on the elimination of the FAB group RAEBT. In addition, despite the claim that the WHO concept was to integrate morphologic, biologic, immunophenotypic and genetic characteristics into the classification, some critics pointed out that except for the category of the isolated del (5q) syndrome, there was little biologic or genetic information included. Over the past 3 years, however, a number of authors have reported that the WHO categories do correlate better with prognosis, response to therapy and progression to leukemia than did the FAB categories. The WHO classification scheme and criteria for its categories are shown in Table 1 , and the rationale for the classification is presented in detail in reference.³ 

Several studies have demonstrated that the concept of multilineage dysplasia in the lower-grade FAB groups of RA and RARS do make a difference with respect to survival and progression to acute leukemia. In a retrospective study of nearly 1600 cases, Germing and associates showed that patients classified as RA by the FAB criteria had a median survival time of 37 months.³⁰ When these patients were reclassified by WHO criteria as RA, with only dyserythropoiesis, or as RCMD, with multilineage dysplasia, the survival times were 69 months versus 33 months, respectively. A similar separation was observed when patients with FAB RARS and a median survival time of 50 months were reclassified as WHO RARS, median survival time of 69 months versus 32 months for those reclassified as WHO RCMD-RS. Other investigators have shown that the WHO classification reliably predicts therapeutic responses, that there is good concordance in the classification when the same specimen is viewed by different observers, and that when combined with cytogenetic studies, it is a useful guide to therapeutic decisions.^31 –33 

The controversy regarding RAEBT is somewhat less settled than the issues with the lower grade lesions. Some reports comparing survival times of patients diagnosed as RAEBT by the FAB criteria with survival of patients diagnosed as MDS-related AML and 30% or more blasts have shown no significant differences.^33,–34 However, some have cautioned that patients who have > 5% blasts in the blood but less than 20% in the marrow, and are thus considered as RAEB-2 in the WHO system, may have more aggressive disease than their WHO classification would indicate.³⁴ On the other hand, similar median survival times of MDS-associated AML and RAEBT do not necessarily prove synonymy between the two groups.

Within the last 5 years, there have been significant gains in the therapy of MDS and further understanding of its pathogenesis, and a workable classification system is necessary that will allow comparison of data from clinical trials and from scientific studies. Unfortunately, as the time for an update to the WHO classification draws near, there is still too little information for a more “genetically based” classification scheme free of morphologic subjectivity. The goal will be to keep the classification flexible and usable worldwide, so that, as new information and data accumulate, the WHO classification can adapt.