Laboratory tests of Myelodysplastic Syndromes



Laboratory Features

Blood Red Cells

Anemia is present in greater than 85 percent of patients.14,93,94 In approximately 4 percent of patients, the anemia results from erythroid aplasia.113 Mean cell volume often is increased. Red cell shape abnormalities include oval, elliptical, teardrop, spherical, and fragmented cells. Red cell findings occur in a spectrum. Some patients have only slight anisocytosis. Elliptical red cells sometimes dominate. Basophilic stippling of red cells occurs (Fig. 88–2). Nucleated red cells are seen in the blood film in approximately 10 percent of cases. Reticulocyte counts usually are low for the degree of anemia. Other abnormalities of red cells occur, such as an increased proportion of hemoglobin F114 and decreased red cell enzyme activities, especially acquired pyruvate kinase deficiency.115 Hemolysis has occurred in some patients with the latter deficiency. Enhanced sensitivity of membranes to complement116 and modification of red cell blood group antigens may be observed.117 Acquired hemoglobin H disease, a rare superimposition, results in red cell morphology similar to thalassemia (microcytosis, anisocytosis, basophilic stippling, poikilocytosis with target cells, fragmented cells, and tear-drop cells). Intracellular precipitates of -chain tetramers (identified by crystal violet stain) reflect an acquired decrease in the rate of -chain synthesis in erythroblasts.118–120 The decrease in -globin–chain synthesis is profound, involves each of the four -chain loci, and results from a transcription abnormality. No gross alterations in genes (e.g., insertions, deletions) are seen in these cases.118 Acquired hemoglobin H disease in this setting has been dubbed the -thalassemia–myelodysplastic syndrome and is the consequence of acquired mutations in ATRX, the gene associated with the X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome.120

Granulocytes and Monocytes


Neutropenia is present in approximately 50 percent of patients at the time of diagnosis.121 The proportion of monocytes often is increased, and monocytosis per se can be the dominant manifestation of the hematopoietic abnormality for months or years.122–124 Morphologic abnormalities of neutrophils can occur, sometimes resulting in the acquired Pelger-Huët anomaly (see Fig. 88–2E). In this condition, neutrophils have very condensed chromatin and unilobed or bilobed nuclei that often have a pince-nez shape. The neutrophils may be in the process of apoptosis.125 Ring-shaped nuclei also can occur in neutrophils (see Fig. 88–2F).126 Neutrophil alkaline phosphatase activity is decreased in some patients.14 Expression of normal surface antigens on neutrophils and monocytes is decreased, and abnormal surface antigen expression occurs in some cases.127 Defective primary granules of abnormal size and shape with decreased myeloperoxidase content can be present.128 Specific neutrophil granules can be decreased in number, producing hypogranular cells.129 Neutrophil granule membranes frequently are deficient in glycoprotein.130 Chemotactic, phagocytic, and bactericidal capability may be impaired.131–133 Formylleucyl-methionyl-phenylamine receptor signaling and actin polymerization can be abnormal.134,135 Muramidase (lysozyme) activity in blood and urine may be increased, reflecting granulocytic hyperplasia, heightened monocytopoiesis, and monocyte turnover.




Approximately 25 to 50 percent of patients have mild to moderate thrombocytopenia at the time of diagnosis.14,121 Mild thrombocytosis also can occur.14,121 Platelets may be abnormally large, have poor granulation, or have large, fused central granules (see Fig. 88–2H).136,137 Abnormal platelet function can contribute to a prolonged bleeding time, easy bruising, or exaggerated bleeding. Decreased platelet aggregation in response to collagen or epinephrine is a frequent functional abnormality.138




Patients with clonal hemopathies may have immunologic deficiencies, such as a decrease in natural killer cells in the blood but no decrease in large granular lymphocytes,139–142 a decrease in helper T lymphocytes,140 and a decrease in Epstein-Barr virus receptors on B lymphocytes.140–143 Antibody-dependent cellular cytotoxicity is normal.140 Thymidine incorporation after mitogenic stimulation144,145 and colony growth of T lymphocytes are decreased.140 Lymphocytes may have an increased sensitivity to irradiation.144 The defects in lymphoid cells could reflect the level of the somatic mutation in a primitive multipotential cell in different cases. Intrinsic, rather than secondary, alterations in lymphocytes are determined by whether no lymphocytes are generated from the clone, B cells are part of the clone, or B and T cells are part of the clone.63 Clonally derived, CD8+CD57+CD244+CD28–CD62L– T lymphocytes are present in marrow and to a lesser extent in blood in approximately 50 percent of patients, independent of type of myelodysplastic syndrome, age, and sex of the patient,64,65 as are NK and B cells (see “Pathogenesis” above).65


Plasma Abnormalities


Serum iron, transferrin, and ferritin levels may be elevated as a result of anemia and the shift of erythron iron to plasma and storage compartments. Lactic dehydrogenase and uric acid concentrations can be increased as a result of ineffective hematopoiesis and a high death fraction of maturing marrow precursors. Monoclonal gammopathy, polyclonal hypergammaglobulinemia, and hypogammaglobulinemia each occur with increased frequency.146,147 The frequency of autoantibodies was increased in one report133 but not in another.146 2-Microglobulin serum levels are increased in proportion to the prognostic category of the disease.148




Magnetic Resonance Imaging


Although rarely used clinically, magnetic resonance imaging scans of human femoral marrow correlate with disease severity. Approximately 85 percent of patients with refractory anemia have images consistent with fatty marrow, whereas approximately 85 percent of patients with oligoblastic leukemia have images showing marrow fat replaced by abnormal hematopoietic tissue.149




Marrow cellularity usually is normal or increased.14,150,151 Cellularity is decreased in approximately 15 percent of cases151 and may simulate hypoplastic or aplastic anemia.152 However, islands of dysmorphic cells, especially atypical megakaryocytes, usually are present (see Fig. 88–2L). An increased proportion of blast cells in this setting suggests hypoplastic myelogenous leukemia (see Chap. 89).




Erythroid hyperplasia is frequent. Very large or small erythroblasts, nuclear fragmentation, stippled erythroblasts, and poor hemoglobinization may be seen.14,150,151 Proerythroblasts may be present in excess, and the marrow may lack normal clusters or islets of erythroblasts. Erythroblasts may resemble megaloblasts that have nuclear-cytoplasmic maturation asynchrony, nuclear fragmentation, or cytoplasmic nuclear remnants. The asynchrony is manifest morphologically by nuclear immaturity with prominent euchromatin in cells with more advanced cytoplasmic maturation. This pattern is referred to as megaloblastoid erythropoiesis (see Fig. 88–2I). Erythroid aplasia seen in occasional cases results in a hypocellular marrow.113


Pathologic sideroblasts may be identified when the marrow is stained with Prussian blue stain (see Fig. 88–2J and 2K). The sideroblasts include erythroblasts with an increased number and size of siderosomes (cytoplasmic ferritin-containing vacuoles), referred to as intermediate sideroblasts, or erythroblasts with mitochondrial iron aggregates that take the form of a partial or complete circumnuclear ring of iron globules, referred to as ringed sideroblasts. Macrophage iron often is increased. Ringed sideroblasts are uncommon or present only in very low proportions in clonal myeloid disorders other than refractory anemia.




Granulocytic hyperplasia is frequent.14,121,150,151 Marrow monocytes may be increased in number. Abnormalities of granulocytes include hypogranulation, a monocytoid appearance of neutrophilic granulocytes, and the acquired Pelger-Huët nuclear abnormality of neutrophils.125,153 Progranulocytes and myelocytes may be increased. The proportion of blast cells is not increased in clonal hemopathies that are categorized as refractory anemia (i.e., <2%); a blast percentage of greater than 2 percent can be considered oligoblastic leukemia. Marrow biopsy may show abnormal localized immature precursors (ALIP),154,155 which are clusters of immature myeloid, CD34+ cells156 located centrally rather than subjacent to the endosteum. These clusters of atypical cells are present in almost all cases of oligoblastic leukemia where blast cells compose 3 percent or more of nucleated marrow cells (RAEB) and in approximately one-third of patients with refractory anemia, suggesting these patients have a disorder closely approaching oligoblastic leukemia. Patients with this abnormality are more prone to develop overt AML. Vascular endothelial growth factor and its receptor are expressed on cells forming ALIP clusters and has been proposed as providing an autocrine loop to promote leukemia progenitor cell formation.157 The number of plasma cells may be slightly increased. Marrow basophilia or eosinophilia occurs in approximately 1 in 7 patients and is associated with a higher probability of evolution to AML.158




Megakaryocytes are present in normal or increased numbers.14,150,151 Micromegakaryocytes (dwarf megakaryocytes) may occur.150,159,160 Megakaryocytes with unilobed or bilobed nuclei may be increased, and hypersegmented and hyposegmented megakaryocytes may be present (see Fig. 88–2L). Clusters of megakaryocytes may be seen. Megakaryocytes may be distributed laterally from their usual parasinusoidal location.161


Fibrosis and Angiogenesis


An increase in reticulin and collagen fibers of varying degree is common (approximately 15% of cases), especially in oligoblastic myelogenous leukemia.156 When fibrosis is prominent, the disorder can resemble primary myelofibrosis, although, in contrast to the latter, splenomegaly usually is not marked. Because primary myelofibrosis is an oligoblastic leukemia with striking dysmorphogenesis of cells, some confusion in classification with other fibrotic clonal myeloid disorders may occur.162 Marrow fibrosis is correlated with higher blast counts and poor-risk cytogenetics.156 Some physicians have proposed a category of myelofibrotic myelodysplasia, but all clonal myeloid diseases, including AML, chronic myelogenous leukemia, and chronic myelomonocytic leukemia may have within their spectrum of expression occasional cases with intense myelofibrosis. Like numerous other epiphenomena that occur in the expression of hematopoietic stem cell diseases, extending the general classifications is not warranted.


Increased angiogenesis is a feature of MDS. Microvessel density increases with more advanced stages of the disease.163 Mast cell frequency and mast cell tryptase activity are highly correlated with microvessel density.164 Circulating endothelial cells are also increased in concentration in patients with MDS and their concentration is correlated with marrow neoangiogenesis (microvessel density).165


Cell Culture


Clonal growth of marrow progenitors in soft agar or other viscous culture systems usually is abnormal in patients with clonal hemopathies.166 Most reports indicate growth of multipotential (colony-forming unit–granulocyte-erythrocyte-monocyte-megakaryocyte) and erythroid progenitors (burst forming unit–erythroid, colony forming unit–erythroid) in the blood or marrow is markedly decreased in subjects with clonal myeloid disorders.166–169 Biochemical abnormalities of erythroid precursors also have been found. Colony-forming units for granulocytes and monocytes (CFU-GM) are decreased.166,167 Very small colonies or clusters with impaired maturation often dominate the cultures. Abnormally small and infrequent CFU-GM may be found when blood neutrophil and monocyte counts are nearly normal. Occasionally, overabundant growth is present. Usually, cell culture results become more abnormal as the blood cell abnormalities in the patient worsen.


In overt AML, CFU-GM colony growth usually is absent. Some studies indicate very abnormal growth of progenitors in culture (decreased colonies or predominance of small clusters) is a poor prognostic sign and may be a harbinger of overt leukemia.170,171 Growth occurring in clonal myeloid hemopathies (and AML) usually remains dependent on growth factors such as erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF).172 Colony growth in children with the monosomy 7 syndrome may occur without added growth factors, supporting the view of autocrine and paracrine stimulation of progenitor cells.173–176 Colony-forming unit–blast cell progenitors may be increased in patients with oligoblastic leukemia.169 The long-term marrow initiating cell is decreased in some patients,175,176 and the ability of marrow stromal layers to support in vitro hematopoiesis can be impaired.177


Circulating monocyte colony-stimulating factor (M-CSF) is increased in some patients with MDS, AML, and other hematologic malignancies.178 IL-1 and GM-CSF levels have been undetectable in most patients. IL-6, G-CSF, and erythropoietin concentrations have been variable. TNF has been inversely related to hematocrit.179 Stem cell factor, a multilineage hematopoietin, has been decreased in some patients.180 The FLT-3 ligand, another multilineage growth factor, is increased in patients with indolent clonal hemopathies but not oligoblastic leukemia.181 The inverse relationship between platelet count and thrombopoietin levels is maintained in clonal anemia but not oligoblastic leukemia.182




An altered number or form of chromosomes occur in approximately 50 percent of patients with clonal hemopathies, depending on the severity of the syndrome.183–185 The chromosome abnormalities are nonrandom and often involve chromosomes that are abnormal in patients with AML, although certain chromosomal rearrangements seen in AML, such as t(15;17), t(8;21), and inv16, only are seen rarely (see Chap. 11).186–188


Chromosomal abnormalities involving virtually every chromosome have been noted in marrow cells, and approximately 60 percent of chromosomal abnormalities are very uncommon, occurring in less than 2 percent of patients in large series.168–170 The most common abnormalities are 5q–, -7/7q–, +8, –18/18q–,and 20q–. Losses of part or all of chromosomes 5 and 7 and complex chromosome aberrations are particularly common in the oligoblastic myelogenous leukemias associated with prior treatment with cytotoxic drugs, radiation, or a high exposure to benzene.32,42,185,189 In this circumstance, the deletion in 5q is at band 5q31.1, in distinction to the classic 5q– syndrome (see “5q– Syndrome” below) in which the deletions occur at band q32–33.3. The Philadelphia (Ph) chromosome t(9;22) and a variety of other chromosome abnormalities not characteristically seen in MDS have been described on rare occasion.190


Categories of cytogenetic abnormalities correlated with median survival have been determined. The more favorable risk category (survival longer than 3 years) includes a normal karyotype and isolated deletions of 5q32–33.3, 20q, or loss of the Y chromosome. Loss of the Y chromosome is an age-dependent variable, usually evident in a proportion of cell metaphases in older men. Only an absence of the Y chromosome in all metaphases is indicative of a clonal myeloid disorder.191 The poor-risk category (median survival less than 1 year) includes –5q31.1, –7 or 7q–, and complex chromosomal abnormalities (three or more abnormalities). The intermediate-risk group (median survival about 2 years) includes deletions of 17p, 11q translocations, trisomy 9 or 19, and 3q abnormalities, among other alterations. In treatment-induced MDS, complex cytogenetic abnormalities are very common, whereas in de novo MDS such abnormalities occur in approximately 15 percent of cases.42,190


The proportion of cases with chromosome abnormalities differs depending on the severity of clinical manifestations. Chromosome abnormalities are more frequent in patients with oligoblastic myelogenous leukemia (RAEB) than in patients with clonal (refractory) anemia. In general, prevalence of chromosome abnormalities and the likelihood of progression to overt AML are a function of the number of cell lines involved, the severity of the cytopenias, and the proportion of blast cells present.


Allelotype analysis of chromosomes of patients with MDS using microsatellite markers mapped to most of the arms of autosomes have found loss of heterozygosity on chromosomes 5q, 7q, 17p, and 20q, in keeping with the most prevalent chromosome abnormalities in MDS patients. Loss of heterozygosity on three other segments, 1p, 1q, and 18q, also were identified. These segments are presumed to contain tumor suppressor genes that may play a role in the initiation of this neoplasm.192


Gene expression studies using primitive multipotential cells (CD34+) from patients with MDS and confirmed by real-time polymerase chain reaction studies have identified 11 selected genes by hierarchical clustering that differ from the CD34+ cells of normal persons.193 In addition, distinctions in gene expression were identified that distinguished high-risk (more blast cells) from low-risk MDS patients. In low-risk patients three genes (retinoic acid-induced gene; radiation-inducible, immediate early response gene; stress-induced phosphoprotein 1 gene) were downregulated. Apparently, MDS patients accumulate gene defects that interfere with hematopoietic regulation.


MDS Stem Cells


MDS stem and progenitor cells do not engraft well in nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice.194 CD34+ cells circulate in MDS and may increase in number before AML transformation.195 In CD34+ CD38– cells in 5q– MDS, few distinct differences were found between MDS and normal stem cells.196 BMI1 was upregulated, and CEBPA was downregulated.