Flow Cytometry Immunophenotypic Characteristics of Monocytic Population in Acute Monocytic Leukemia (AML-M5), Acute Myelomonocytic Leukemia (AML-M4), and Chronic Myelomonocytic Leukemia (CMML)

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Publisher Summary

Monocytic proliferations comprise a heterogeneous group of disorders ranging from reactive monocytosis to acute monocytic leukemia. Based on the cytomorphological and phenotypic features, differential diagnosis includes acute promyelocytic leukemia (especially microgranular variant), acute myeloid leukemia (AML) without maturation, minimally differentiated AML, chronic myelomonocytic leukemia (CMML), acute megakaryocytic leukemia, and acute myelomonocytic leukemia (AML-M4). Flow cytometric immunophenotyping is an accurate method for quantitative and qualitative evaluation of hematopoietic cells. It plays important role in diagnosis, classification, and monitoring of hematopoietic neoplasms, including acute leukemias. This chapter presents the phenotypic characteristic of monocytic populations from acute monocytic leukemia (AML-M5), CMML, and AML-M4. The chapter describes the different types of leukemia such as acute monocytic leukemia, chronic myelomonocytic leukemia, and acute myelomonocytic leukemia. Flow cytometry (FC) can identify and differentiate abnormal monocytes and complements cytomorphology and cytochemical staining in diagnosis of myeloid proliferations with monocytic differentiation.

Introduction

Monocytic proliferations comprise a heterogeneous group of disorders ranging from reactive monocytosis to acute monocytic leukemia. Based on the cytomorphological and phenotypic features, differential diagnosis includes acute promyelocytic leukemia (especially microgranular variant), acute myeloid leukemia (AML) without maturation, minimally differentiated AML, chronic myelomonocytic leukemia (CMML), acute megakaryocytic leukemia, and acute myelomonocytic leukemia (AML-M4). Extramedullary myeloid tumors with monocytic differentiation (monoblastic sarcoma) may be mistaken for large cell lymphoma, carcinoma, or sarcoma. Well-prepared fresh bone marrow aspirate with myeloperoxidase staining is helpful in differential diagnosis. Myeloblasts and abnormal promyelocytes are strongly MPO positive, whereas the monocytes are either weakly positive or negative. The monoblasts and promonocytes usually are positive with nonspecific esterase (NSE) staining, but a significant subset of acute monocytic leukemias is NSE negative. Therefore, the definite diagnosis often requires correlation of complete blood cell (CBC) count data, cytological features, and cytochemistry with additional techniques such as immunophenotyping by flow cytometry (FC), cytogenetics⧸fluorescence in situ hybridization (FISH), and molecular tests (e.g., polymerase chain reaction [PCR]). FC immunophenotyping is an accurate method for quantitative and qualitative evaluation of hematopoietic cells. It plays important role in diagnosis, classification, and monitoring of hematopoietic neoplasms, including acute leukemias (Baumgarth 2000, Borowitz 1997, Gorczyca 2002, Jennings 1997a, Jennings 1997b, Knapp 1994, Kotylo 2000, Kussick 2003, Manaloor 2000, Orfao 1999a, Orfao 1999b, Weir 2001, Weisberger 2000). This chapter presents the phenotypic characteristic of monocytic populations from acute monocytic leukemia (AML-M5), CMML and AML-M4.

Section snippets

Materials

Flow cytometric samples from IMPATH, Incorporated (New York division), containing abnormal monocytic populations were submitted for this study. FC data were reanalyzed and correlated with cytomorphology and⧸or bone marrow studies. All cases without firm morphological confirmation were excluded. The neoplasms were classified according to the World Health Organization (WHO) classification of hematopoietic neoplasms (Harris 2000a, Harris 2000b). There were 28 cases of AML-M5, 20 cases of CMML, and

Flow Cytometry Analysis

We used heparinized bone marrow aspirate, peripheral blood, and fresh tissue specimens for FC analysis and processed the specimens within 24 hours of collection. We obtained a leukocyte cell suspension from peripheral blood and bone marrow specimens after red blood cell (RBC) lysis with ammonium chloride lysing solution, followed by 5 minutes of centrifugation. The cell pellet was suspended with an appropriate amount of RPMI 1640 (GIBCO, New York). Fresh tissue samples were disaggregated with a

Acute Monocytic Leukemia

Acute monocytic (monoblastic) leukemia (AML-M5) is defined as myeloid leukemia in which 80% or more of the leukemic cells are of monocytic lineage (monoblasts, promonocytes, and monocytes). In this series (28 cases), patients ranged in age from 17 to 85 years. Figure 1 presents typical cytologic and phenotypic features of AML-M5. Leukemic cells have abundant cytoplasm that may show irregular borders with pseudopod and cytoplasmic vacuoles. NSE is positive in most cases, although it may be weak.

Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is mixed myelodysplastic⧸myeloproliferative disorder defined by persistent monocytosis (>1 by 109⧸L) in peripheral blood, fewer than 20% blasts, and dysplastic features in one or more myeloid lineages. Molecular⧸cytogenetic study results are negative for bcr-abl fusion gene (Philadelphia chromosome). The monocytes are usually mature with focal nuclear or cytoplasmic atypia. Based on the number of blasts, CMML is divided into two categories: CMML-1 (<5%

Acute Myelomonocytic Leukemia

Acute myelomonocytic leukemia (AML-M5) is an acute leukemia characterized by the proliferation of both neutrophil and monocyte precursors with 20% or more myeloblasts in the bone marrow. Both monocytic and granulocytic lineages must comprise at least 20% of marrow cells. FC analysis from AML-M4 cases shows distinct populations of blasts, monocytes, and residual (maturing) myeloid cells (Fig. 3). Monocytic cells in AML-M4 are always positive for CD4, CD11b, CD11c, CD13, CD14, CD33, CD45, CD64,

Differential Diagnosis

Figure 4 presents the expression of CD45 versus side scatter in different types of leukemia. Myeloblasts in AMLs (M0–M2) display moderate expression of CD45 and low side scatter (Fig. 4B). Blasts usually predominate and there are no or few other elements like granulocytes, monocytes, and lymphocytes (compare with normal marrow, Fig. 4A). Monoblasts in AML-M5 are characterized by bright CD45 and increased side scatter (blue dots, Fig. 4E). Note rare myeloblasts (green dots) and paucity of other

Conclusion

FC, particularly CD45 versus side scatter and expression of CD2, CD7, CD10, CD11b, CD11c, CD14, CD16, CD23, CD33, CD34, CD45, CD56, CD64, CD117, and HLA-DR, can identify and differentiate abnormal monocytes and complements cytomorphology and cytochemical staining in diagnosis of myeloid proliferations with monocytic differentiation.

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