Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 35. Acute leukemias

Woodlyne Roquiz, Pranav Gandhi, Ameet R. Kini

OUTLINE

Introduction

Classification Schemes for Acute Leukemias

Acute Lymphoblastic Leukemia

World Health Organization Classification

Morphology

Prognosis

Immunophenotyping

Genetics and Molecular Findings

Acute Myeloid Leukemia

Clinical Presentation

Subtypes of Acute Myeloid Leukemia and Related Precursor Neoplasms

Acute Leukemias of Ambiguous Lineage

Future Directions in the Classification of Acute Leukemias

Cytochemical Stains and Interpretations

Myeloperoxidase

Sudan Black B

Esterases

Objectives

After completion of this chapter, the reader will be able to:

1. Discuss the causes and development of acute leukemia.

2. Characterize the diagnostic criteria used for acute myeloid and acute lymphoblastic leukemias.

3. Compare and contrast acute lymphoblastic and myeloid leukemias by morphology, presenting signs and symptoms, laboratory findings, and prognosis.

4. Interpret the results of diagnostic tests for acute leukemias.

5. Discuss tumor lysis syndrome, including risk, cause, and laboratory findings.

6. Discuss the cell staining patterns for the following tests: myeloperoxidase, Sudan black B, and esterases.

CASE STUDY

After studying the material in this chapter, the reader should be able to respond to the following case study:

A 5-year-old child was seen by her family physician because of weakness and headaches. She had been in good health except for the usual communicable diseases of childhood. Physical examination revealed a pale, listless child with multiple bruises. The WBC count was 15 × 109/L, the hemoglobin was 8 g/dL, and the platelet count was 90 × 109/L. She had “abnormal cells” in her peripheral blood (Figure 35-1). Cytogenetic studies revealed hyperdiploidy.

1. What is the most likely diagnosis?

2. What characteristics of this disease indicate a positive prognosis?

3. What prognosis is associated with the hyperdiploidy?

Image 

FIGURE 35-1 Peripheral blood film for the patient in the case study (×1000). Source: (From Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

Introduction

The broad term leukemia is derived from the ancient Greek words leukos (λευκóç), meaning “white,” and haima (αἷμα), meaning “blood.”1 As defined today, acute leukemia refers to the rapid, clonal proliferation in the bone marrow of lymphoid or myeloid progenitor cells known as lymphoblasts and myeloblasts, respectively. When proliferation of blasts overwhelms the bone marrow, blasts are seen in the peripheral blood and the patient’s symptoms reflect suppression of normal hematopoiesis.

For most cases of acute leukemia, the causes directly related to the development of the malignancy are unknown. The exceptions that exist are certain toxins that can induce genetic changes leading to a malignant phenotype. Environmental exposures known to lead to hematopoietic malignancies include radiation and exposure to organic solvents, such as benzene. Rarely, leukemias can be seen in patients with known familial cancer predisposition syndromes. Alkylating agents and other forms of chemotherapy used to treat various forms of cancer can induce deoxyribonucleic acid (DNA) damage in hematopoietic cells, leading to therapy-related leukemias.

Regardless of the mechanism of initial genetic damage, the development of leukemia is currently believed to be a stepwise progression of mutations or “multiple hits” involving mutations in genes that give cells a proliferative advantage, as well as mutations that hinder differentiation.23 These mutations result in transformation of normal hematopoietic stem cells or precursors into leukemic stem cells (LSCs). The LSCs then initiate, proliferate, and sustain the leukemia.4

Classification schemes for acute leukemias

The French-American-British (FAB) classification of the acute leukemias was devised in the 1970s and was based on morphologic examination along with cytochemical stains to distinguish lymphoblasts from myeloblasts (). The use of cytochemical stains continues to be a useful adjunct for differentiation of hematopoietic diseases, especially acute leukemias. The details of the cytochemical stains are addressed at the end of this chapter. In addition to morphologic and cytochemical stains, techniques commonly used to diagnose hematopoietic malignancies include flow cytometry and genetic/molecular studies. Findings of these techniques are discussed throughout the chapter in relation to specific leukemias. Figure 35-2

Image 

FIGURE 35-2 A, Lymphoblasts (bone marrow, Wright stain, ×500). Cells have a diameter two to three times the normal lymphocyte diameter, scant blue cytoplasm, coarse chromatin, deeper staining than myeloblasts, and inconspicuous nucleoli. B,Myeloblasts (bone marrow, Wright stain, ×500). Cells have a diameter three to five times the lymphocyte diameter, moderate gray cytoplasm, uniform fine chromatin, two or more prominent nucleoli, and possibly Auer rods.

Hematologists and pathologists are now moving toward more precise classification of many of the leukocyte neoplasms based on recurring chromosomal and genetic lesions found in many patients. These lesions are related to disruptions of oncogenes, tumor suppressor genes, and other regulatory elements that control proliferation, maturation, apoptosis, and other vital cell functions. In 2001 the World Health Organization (WHO) published new classification schemes for nearly all of the tumors of hematopoietic and lymphoid tissues,5 and in some cases WHO melded the older morphologic schemes with the newer schemes. For instance, in the WHO classification scheme for acute myeloid leukemias (AMLs), there are some remnants of the old FAB classification, but new classifications were introduced for leukemias associated with consistently recurring chromosomal translocations. According to the WHO classification, a finding of at least 20% blasts in the bone marrow is required for diagnosis of the majority of acute leukemias, and testing must be performed to detect the presence or absence of genetic anomalies. In 2008 the WHO classification of hematologic malignancies was revised to reflect advances in the field.6 In-depth discussion of each of the subclassifications is beyond the scope of this book, so only the most common subtypes of acute lymphoblastic leukemia and acute myeloid leukemia are detailed here.

Acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is primarily a disease of childhood and adolescence, accounting for 25% of childhood cancers and up to 75% of childhood leukemia.7 The peak incidence of ALL in children is between 2 and 5 years of age.8 Although ALL is rare in adults, risk increases with age; most adult patients are older than 50 years of age. The subtype of ALL is an important prognostic indicator for survival.6Adults have a poorer outlook: 80% to 90% experience complete remission, but the cure rate is less than 40%.910

Patients with B cell ALL typically present with fatigue (caused by anemia), fever (caused by neutropenia and infection), and mucocutaneous bleeding (caused by thrombocytopenia). Lymphadenopathy, including enlargement, is often a symptom.11 Enlargement of the spleen (splenomegaly) and of the liver (hepatomegaly) may be seen. Bone pain often results from intramedullary growth of leukemic cells.11Eventual infiltration of malignant cells into the meninges, testes, or ovaries occurs frequently, and lymphoblasts can be found in the cerebrospinal fluid.12

In T cell ALL, there may be a large mass in the mediastinum leading to compromise of regional anatomic structures. Similar to B-ALL, T-ALL may present with anemia, thrombocytopenia, organomegaly, and bone pain, although the degree of leukopenia is often less severe.13

World health organization classification

B lymphoblastic leukemia/lymphoma (B-ALL) is subdivided into seven subtypes that are associated with recurrent cytogenetic abnormalities.14 These entities are linked with unique clinical, phenotypic, or prognostic features (Box 35-1). Cases of B cell ALL that do not exhibit the specific genetic abnormalities are classified as B lymphoblastic leukemia/lymphoma, not otherwise specified. Although 50% to 70% of patients with T lymphoblastic leukemia/lymphoma have abnormal gene rearrangements, none of the abnormalities is clearly associated with specific biologic features, and thus T-ALL is not further subdivided clinically.14

BOX 35-1

B Lymphoblastic Leukemia/Lymphoma with Recurrent Genetic Abnormalities (2008 World Health Organization Classification)

B lymphoblastic leukemia/lymphoma with t(9; 22)(q34; q11.2); BCR-ABL1

B lymphoblastic leukemia/lymphoma with t(v; 11q23); MLL rearranged

B lymphoblastic leukemia/lymphoma with t(12; 21)(p13; q22); TEL-AML1 (ETV6-RUNX1)

B lymphoblastic leukemia/lymphoma with hyperdiploidy

B lymphoblastic leukemia/lymphoma with hypodiploidy

B lymphoblastic leukemia/lymphoma with t(5; 14)(q31; q32); IL3-IGH

B lymphoblastic leukemia/lymphoma with t(1; 19)(q23; p13.3); E2A-PBX1(TCF3-PBX1)

From Swerdlow SH, Campo E, Harris NL, et al, editors: WHO classification of tumours of haematopoietic and lymphoid tissues, ed 4, Lyon, France, 2008, IARC Press.

Morphology

Lymphoblasts vary in size but fall into two morphologic types. The most common type seen is a small lymphoblast (1.0 to 2.5 times the size of a normal lymphocyte) with scant blue cytoplasm and indistinct nucleoli (Figure 35-2); the second type of lymphoblast is larger (two to three times the size of a lymphocyte) with prominent nucleoli and nuclear membrane irregularities (Figure 35-3).13 These cells may be confused with the blasts of acute myeloid leukemia (AML).

Image 

FIGURE 35-3 Acute lymphoblastic leukemia. Large lymphoblast with prominent nucleoli and membrane irregularities (peripheral blood, ×1000). Source: (From Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

Prognosis

Prognosis in ALL has improved dramatically over the past decades as a result of improvement in algorithms for treatment.13 The prognosis for ALL depends on age at the time of diagnosis, lymphoblast load (tumor burden), immunophenotype, and genetic abnormalities. Children rather than infants or teens do the best. Chromosomal translocations are the strongest predictor of adverse treatment outcomes for children and adults. Peripheral blood lymphoblast counts greater than 20 to 30 × 109/L, hepatosplenomegaly, and lymphadenopathy all are associated with worse outcome. The effects of other variables previously associated with a poorer prognosis, such as sex and ethnic group, have been eliminated when patients have been given equal access to treatment in trials carried out at a single institution.15

Immunophenotyping

Although morphology is the first tool used to distinguish ALL from AML, immunophenotyping and genetic analysis are the most reliable indicators of a cell’s origin. Because both B and T cells are derived from lymphoid progenitors, both usually express CD34, terminal deoxynucleotidyl transferase (TdT), and HLA-DR. Four types of ALL have been identified by immunologic methods: early B-ALL (pro-B, or pre-pre-B), intermediate (common) B-ALL, pre-B-ALL, and T-ALL (Table 35-1). B-ALL is characterized by specific B cell antigens that are expressed at different stages of B cell development. In general, B cells express CD19, CD20, CD22, CD24, C79a, CD10, cytoplasmic μ, and PAX-5 (B cell specific activator protein). The degree of differentiation of B-lineage lymphoblasts often correlates with genetics and plays an important role in treatment decisions.61316 In the earliest stage of differentiation (pre-pre B or pro-B), blasts express CD34, CD19, cytoplasmic CD22, and TdT. The incidence of pro-B ALL is about 5% in children and 11% in adults. In intermediate or common B-ALL, CD10 is expressed. The most mature B-ALL is called pre-B-ALL, in which CD34 is typically negative, but there is characteristic expression of cytoplasmic μ heavy chain. Pre-B-ALL accounts for 15% of childhood cases and 10% of adult B-ALL.17

TABLE 35-1

Immunophenotypic Characteristics of Acute Lymphoblastic Leukemia

ALL Subtype

Immunophenotype

Early (pro/pre-pre) B-ALL

CD34, CD19, cytoplasmic CD22, TdT

Intermediate (common) B-ALL

CD34, CD19, CD10, cytoplasmic CD22, TdT

Pre-B-ALL

CD34, CD19, cytoplasmic CD22, cytoplasmic μ, TdT (variable)

T-ALL

CD2, CD3, CD4, CD5, CD7, CD8, TdT

T-ALL is seen most often in teenaged males with a mediastinal mass, elevated peripheral blast counts, meningeal involvement, and infiltration of extra marrow sites.1819 The common T cell markers CD2, CD3, CD4, CD5, CD7, and CD8 are usually present. Most cases express TdT. A distinct subtype of T-ALL, ETP-ALL (early T cell precursor ALL), contains a characteristic immunophenotype (CD8-, CD5dim) and has a poor response to chemotherapy, low rates of remission, and overall poor survival.2021

Genetic and molecular findings

Cytogenetic abnormalities are seen in the majority of B and T cell ALL, which produce changes that affect normal B and T cell development and underlie the pathogenesis of these neoplasms. A majority of T-ALL have been shown to have gain-of-function mutations involving the NOTCH1 gene, which alters the Notch receptor signaling pathway responsible for normal T cell development.22

In T-ALL, however, the cytogenetic alterations show less specificity and less correlation with prognosis and treatment outcome than in B-ALL. B lymphoblastic leukemia/lymphoma with the t(9; 22)(q34; q11.2); BCR-ABL1 mutation (Philadelphia chromosome–positive ALL) has the worst prognosis among ALLs. It is more common in adults than in children. Imatinib, which has shown success in treating chronic myelogenous leukemia, has improved survival (Chapter 33).

B lymphoblastic leukemia/lymphoma with t(v; 11q23); MLL rearranged is more common in very young infants, and the translocation may even occur in utero.23 This leukemia has a very poor prognosis. About 25% of childhood ALL cases show a t(12; 21)(p13; q22); ETV6-RUNX1 translocation and appear to derive from a B cell progenitor rather than the hematopoietic stem cell.24 This translocation is rare in adults. In children, it carries an excellent prognosis, with a cure rate of over 90%. Hyperdiploidy in B lymphoblastic leukemia/lymphoma is common in childhood B-ALL, accounting for 25% of cases, but it is much less common in adults. This genotype is associated with a very favorable prognosis in children. Conversely, hypodiploidy (less than 46 chromosomes) conveys a poor prognosis in both children and adults.

Acute myeloid leukemia

AML is the most common type of leukemia in adults, and the incidence increases with age. AML is less common in children. The French-American-British (FAB) classification of AML was based on morphology and cytochemistry; the WHO classification relies heavily on cytogenetics and molecular characterization (Chapters 30 and 31).614

Clinical presentation

The clinical presentation of AML is nonspecific but reflects decreased production of normal bone marrow elements. Most patients with AML have a total WBC count between 5 and 30 × 109/L, although the WBC count may range from 1 to 200 × 109/L. Myeloblasts are present in the peripheral blood in 90% of patients. Anemia, thrombocytopenia, and neutropenia give rise to the clinical findings of pallor, fatigue, fever, bruising, and bleeding. In addition, disseminated intravascular coagulation and other bleeding abnormalities can be significant.25 Infiltration of malignant cells into the gums and other mucosal sites and skin also can be seen.

Splenomegaly is seen in half of AML patients, but lymph node enlargement is rare. Cerebrospinal fluid involvement in AML is rare and does not seem to be as ominous a sign as in ALL. Patients with AML tend to have few symptoms related to the central nervous system, even when it is infiltrated by blasts.

Common abnormalities in laboratory test results include hyperuricemia (caused by increased cellular turnover), hyperphosphatemia (due to cell lysis), and hypocalcemia (the latter two are also involved in progressive bone destruction). Hypokalemia is also common at presentation. During induction chemotherapy, especially when the WBC is quite elevated, tumor lysis syndrome may occur. Tumor lysis syndrome is a group of metabolic complications that can occur in patients with malignancy, most notably lymphomas and leukemias, with and without treatment of the malignancy. These complications are caused by the breakdown products of dying cancer cells, which in turn cause acute uric acid nephropathy and renal failure. Tumor lysis syndrome is characterized by hyperkalemia, hyperphosphatemia, hyperuricemia and hyperuricosuria, and hypocalcemia.26 The hyperkalemia alone can be life-threatening. Aggressive prophylactic measures to prevent or reduce the clinical manifestations of tumor lysis syndrome are critical.27

Subtypes of acute myeloid leukemia and related precursor neoplasms

Laboratory diagnosis of AML begins with a complete blood count, peripheral blood film examination, and bone marrow aspirate and biopsy specimen examination. The total WBC count may be normal, increased, or decreased; anemia is usually present, along with significant thrombocytopenia. The bone marrow is usually hypercellular, and greater than 20% of cells typically are marrow blasts, although if certain genetic abnormalities are present, the 20% blast threshold is not necessary for the diagnosis of AML.6 Each category is discussed, and a summary of the classification is presented in Box 35-2.

BOX 35-2

Acute Myeloid Leukemia and Related Precursor Neoplasms (2008 World Health Organization Classification)

Acute myeloid leukemia with recurrent genetic abnormalities

Acute myeloid leukemia with myelodysplasia-related changes

Therapy-related myeloid neoplasms

Acute myeloid leukemia, not otherwise specified

Myeloid sarcoma

Myeloid proliferations related to Down syndrome

Blastic plasmacytoid dendritic cell neoplasm

From Swerdlow SH, Campo E, Harris NL, et al, editors: WHO classification of tumours of haematopoietic and lymphoid tissues, ed 4, Lyon, France, 2008, IARC Press.

The 2008 WHO classification for myeloid malignancies has categorized AMLs with recurrent cytogenetic abnormalities into subgroups based on the primary cytogenetic aberrations ().Box 35-3614

BOX 35-3

Acute Myeloid Leukemia with Recurrent Genetic Abnormalities (2008 World Health Organization Classification)

AML with t(8; 21)(q22; q22); RUNX1-RUNX1T1

AML with inv(16)(p13.1q22) or t(16; 16)(p13.1; q22); CBFB-MYH11

APL with t(15; 17)(q22; q12); PML-RARA

AML with t(9; 11)(p22; q23); MLLT3-MLL

AML with t(6; 9)(p23; q34); DEK-NUP214

AML with inv(3)(q21q26.2) or t(3; 3)(q21; q26.2); RPN1-EVI1

AML with t(1; 22)(p13; q13) RBM15-MKL1

Provisional entity: AML with mutated NPM1

Provisional entity: AML with mutated CEBPA

From Swerdlow SH, Campo E, Harris NL, et al, editors: WHO classification of tumours of haematopoietic and lymphoid tissues, ed 4, Lyon, France, 2008, IARC Press.

AML with recurrent genetic abnormalities

Acute myeloid leukemia with t(8; 21)(q22; q22); RUNX1/RUNX1T1. 

The t(8; 21)(q22; q22); RUNX1/RUNX1T1 mutation is found in about 5% of AML cases. Seen predominantly in children and young adults, AML with this translocation has myeloblasts with dysplastic granular cytoplasm, Auer rods, and some maturation (Figure 35-4), similar to the FAB M2 classification (see later in chapter). Various anomalies, such as pseudo–Pelger-Huët cells and hypogranulation, can be seen. Eosinophilia is possible. Prognosis is generally favorable but may be negatively impacted if unfavorable additional abnormalities, such as monosomy 7, occur.28 The diagnosis of this subtype is based on the genetic abnormality, regardless of blast count.6

Image 

FIGURE 35-4 Acute myeloid leukemia with t(8; 21). Myeloblasts with granular cytoplasm and some maturation (bone marrow, ×500). Source: (From Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

Acute myeloid leukemia with inv(16)(p13.1q22) or t(16; 16)(p13.1; q22); CBFB-MYH11. 

Accounting for approximately 5% to 8% of all AML cases, core-binding factor (CBF) AML occurs at all ages, but it is found predominantly in younger patients.6 The genetic aberration is sufficient for diagnosis regardless of blast count.629 Myeloblasts, monoblasts, and promyelocytes are seen in the peripheral blood and bone marrow. In the bone marrow there may be eosinophilia with dysplastic changes (Figure 35-5). The incidence of extramedullary disease is higher than in most types of AML, and the central nervous system is a common site for relapse.629 The remission rate is good, but only one half of patients are cured.29

Image 

FIGURE 35-5 Acute myeloid leukemia with inv(16). There is an increase in myeloid and monocytic lines. Eosinophilia may also be present (peripheral blood, ×1000). Source: (From Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

Acute myeloid leukemia with t(15; 17)(q22; q12); PML-RARA. 

Also known as acute promyelocytic leukemia (APL), AML with the t(15; 17)(q22; q12); PML-RARA mutation comprises 5% to 10% of AML cases. It occurs in all age groups but is seen most commonly in young adults. This disorder is characterized by a differentiation block at the promyelocytic stage. The abnormal promyelocytes are considered to be comparable to blasts for the purpose of diagnosis. Detection of the 15; 17 translocation is sufficient for diagnosis regardless of blast count.628 Characteristic of this presentation are the abnormal hypergranular promyelocytes, some with Auer rods (Figure 35-6). When promyelocytes release primary granule contents, their procoagulant activity initiates disseminated intravascular coagulation; however, thromboembolic events may occur at presentation and during treatment.30 In one variant of APL, the granules are so small that because of the limits of light microscopy, the cells give the appearance of having no granules. This microgranular variant, accounting for 30% to 40% of APL cases, may be confused with other presentations of AML, but the presence of occasional Auer rods, the “butterfly” or “coin-on-coin” nucleus, and the clinical presentation are clues. The treatment of APL is significantly different from all other types of acute myeloid leukemia, and it is therefore important to arrive at an accurate diagnosis. Treatment includes all- trans-retinoic acid (ATRA) and arsenic trioxide.31 ATRA is a vitamin A analogue and induces differentiation of the malignant promyelocytes. In adults who achieve a complete remission, the prognosis is better than for any other type of AML.28 There are a few variants inRARAtranslocations that confer a poor diagnosis because the cells do not respond to ATRA therapy.614

Image 

FIGURE 35-6 Acute myeloid leukemia with t(15; 17), or promyelocytic leukemia. A, Low-power view of the more common hypergranular variant (peripheral blood, ×500). B, Oil immersion view of the microgranular variant showing bilobed nuclear features (peripheral blood, ×1000). Source: (B from Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

Acute myeloid leukemia with t(9; 11)(p22; q23); MLLT3-MLL. 

AML with t(9; 11)(p22; q23); MLLT3-MLL represents a specific subgroup of the previous classification of AML with 11q23 abnormalities, and AMLs with other MLL abnormalities should not be placed in this group.14 AML with t(9; 11) is a rare leukemia (6% of AML cases) that presents with an increase in monoblasts and immature monocytes (Figure 35-7). The blasts are large with abundant cytoplasm and fine nuclear chromatin. The cells may have motility, with pseudopodia seen frequently. Granules and vacuoles can be observed in the blasts. Typically this disease occurs in children and may be associated with gingival and skin involvement and/or disseminated intravascular coagulation. The prognosis is intermediate to poor.28

Image 

FIGURE 35-7 Acute myeloid leukemia with t(9; 11) abnormalities. Both monoblasts and immature monocytes are increased (bone marrow, ×500).

Acute myeloid leukemia with t(6; 9)(p23; q34); DEK-NUP214, acute myeloid leukemia with inv(3)(q21q26.2) or t(3; 3)(q21; q26.2); RPN1-EVI1, and acute myeloid leukemia (megakaryoblastic) with t(1; 22)(p13; q13); RBM15-MKL1. 

These are rare leukemias included in the 2008 WHO classification. Detailed description of these entities is beyond the scope of this chapter.

Acute myeloid leukemia with myelodysplasia-related changes

AML with myelodysplasia affects primarily older adults and has a poor prognosis. This subcategory of AML with myelodysplasia-related changes incorporates leukemias with at least 20% blasts, multilineage dysplasia, a history of MDS or MDS/myeloproliferative neoplasm (MPN), or a specific MDS-associated cytogenetic abnormality and the absence of AML with recurrent genetic abnormalities.2832 Significant dysplastic morphology includes pancytopenia with neutrophil hypogranulation or hypergranulation, pseudo–Pelger-Huët cells, and unusually segmented nuclei. Erythrocyte precursors have vacuoles, karyorrhexis, megaloblastoid features, and ringed sideroblasts. There may be dysplastic micromegakaryocytes and dysplastic megakaryocytes. Genetic findings are similar to those found in MDS, with complex karyotypes and −7/del(7q) and −5/del(5q) being the most common.633

Therapy-related myeloid neoplasms

Treatment with alkylating agents, radiation, or topoisomerase II inhibitors has been associated with the development of a secondary AML, MDS, or MDS/MPN.6283435 These therapy-related neoplasms account for 10% to 20% of AMLs, MDSs, and MDSs/MPNs. Generally these disorders occur following treatment for a prior malignancy, but they have also been associated with intensive treatment of patients with nonmalignant disorders requiring cytotoxic therapy.63436 Therapy-related myeloid neoplasms are similar in morphology to AML with myelodysplasia, monocytic/monoblastic leukemia, or AML with maturation, and the prognosis is generally poor, although therapy-related neoplasms with the t(15; 17) and inv(16) mutations behave more like the de novo counterparts.628

Acute myeloid leukemia, not otherwise specified

Because the leukemias in the “not otherwise specified” category do not fit easily into the WHO subtypes described earlier, they are grouped according to morphology, flow cytometric phenotyping (Chapter 32), and limited cytochemical reactions, as in the FAB classification. The FAB classification was based on the cell of origin, degree of maturity, cytochemical reactions, and limited cytogenetic features (Table 35-2).3738A blast percentage of at least 20% in the peripheral blood or bone marrow is required for diagnosis. This category accounts for about 25% of all AML, but as more genetic subgroups are recognized, the number in this group will diminish.14

TABLE 35-2

French-American-British Classification of the Acute Myeloid Leukemias

Subtype

Description

M0

Acute myeloid leukemia, minimally differentiated

M1

Acute myeloid leukemia without maturation

M2

Acute myeloid leukemia with maturation

M3

Acute promyelocytic leukemia

M4

Acute myelomonocytic leukemia

M4eo

Acute myelomonocytic leukemia with eosinophilia

M5a

Acute monocytic leukemia, poorly differentiated

M5b

Acute monocytic leukemia, well differentiated

M6

Acute erythroleukemia

M7

Acute megakaryocytic leukemia

Data from Bennett JM, Catovsky D, Daniel MT, et al: Proposals for the classification of the acute leukemias. French-American-British (FAB) co-operative group, Br J Haematol 33:451-458, 1976; and Bennett JM, Catovsky D, Daniel MT, et al: Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative Group, Ann Intern Med 103:620-625, 1985.

Acute myeloid leukemia with minimal differentiation. 

The blasts in AML with minimal differentiation are CD13+, CD33+, CD34+, and CD117+ (Figure 35-8).639 Auer rods typically are absent, and there is no clear evidence of cellular maturation. The cells yield negative results with the cytochemical stains myeloperoxidase and Sudan black B. These cases account for less than 5% of AML, and patients are generally either infants or older adults.

Image 

FIGURE 35-8 Acute myeloid leukemia, minimally differentiated (French-American-British classification M0). Blasts lack myeloid morphologic features and yield negative results with myeloperoxidase and Sudan black B staining. Auer rods are not seen. CD34 is frequently present (bone marrow, ×500). Source: (From Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

Acute myeloid leukemia without maturation. 

Closely aligned with the blasts in minimally differentiated AML, the blasts in AML without maturation are also CD13+, CD33+, and CD117+, and CD34 is present in about 70% of cases (Figure 35-9).6 Blasts may comprise 90% of nonerythroid cells in the bone marrow, and fewer than 10% of the leukocytes show maturation to the promyelocyte stage or beyond. Blasts have Auer rods and usually give positive results with myeloperoxidase or Sudan black B stains.628

Image 

FIGURE 35-9 Acute myeloid leukemia without maturation (French-American-British classification M1). Blasts constitute 90% of the nonerythroid cells; there is less than 10% maturation of the granulocytic series beyond the promyelocyte stage (bone marrow, ×500).

Acute myeloid leukemia with maturation. 

AML with maturation is a common variant that presents with greater than 20% blasts, at least 10% maturing cells of neutrophil lineage (), and fewer than 20% precursors with monocytic lineage. Auer rods and other aspects of dysplasia are present.Figure 35-106

Image 

FIGURE 35-10 Acute myeloid leukemia with maturation. Blasts constitute 20% or more of the nucleated cells of the bone marrow, and there is maturation beyond the promyelocyte stage in more than 10% of the nonerythroid cells (bone marrow, ×1000).

Acute myelomonocytic leukemia. 

Acute myelomonocytic leukemia is characterized by a significantly elevated WBC count and the presence of myeloid and monocytoid cells in the peripheral blood and bone marrow (). Monocytic cells (monoblasts and promonocytes) constitute at least 20% of all marrow cells. The monoblasts are large with abundant cytoplasm containing small granules and pseudopodia. The nucleus is large and immature and may contain multiple nucleoli. Promonocytes also are present and may have contorted nuclei. The cells are positive for the myeloid antigens CD13 and CD33 and the monocytic antigens CD14, CD4, CD11b, CD11c, CD64, and CD36. Nonspecific cytogenetic changes are found in most cases.Figure 35-116

Image 

FIGURE 35-11 Acute myelomonocytic leukemia. Both myeloid and monocytic cells are present. Monocytic cells comprise at least 20% of all marrow cells, with monoblasts and promonocytes present (peripheral blood, ×1000). Source: (From Carr JH, Rodak BF:Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

Acute monoblastic and monocytic leukemias. 

In these leukemias, which are divided into monoblastic and monocytic based on the degree of maturity of the monocytic cells present in the marrow and peripheral blood, more than 80% of the marrow cells are of monocytic origin. These cells are CD14+, CD4+, CD11b+, CD11c+, CD36+, CD64+, and CD68+. Blasts are large with abundant, often agranular cytoplasm and large prominent nucleoli (Figure 35-12A). When some evidence of maturation is present, the cells are called promonocytes. Promonocytes in monocytic leukemias with differentiation are considered to be blast equivalents (Figure 35-12B). Nonspecific esterase testing usually yields positive results. Acute monoblastic/monocytic leukemia comprises fewer than 5% of cases of AML and is most common in younger individuals. Extramedullary involvement, including cutaneous and gingival infiltration, and bleeding disorders are common. Nonspecific cytogenetic abnormalities are seen in most cases.640

Image 

FIGURE 35-12 A, Acute monoblastic leukemia. More than 80% of the bone marrow cells are of monocytic origin (bone marrow, ×500). B, Acute monocytic leukemia with promonocytes. Promonocytes are considered blast equivalents. Source: (B from Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

Acute erythroid leukemia. 

According to the WHO classification, there are two subtypes of acute erythroid leukemia, based on the presence of a significant component of myeloblasts. The first is acute erythroleukemia (erythroid/myeloid), in which 50% or more of nucleated bone marrow cells are normoblasts and greater than 20% are myeloblasts. In the FAB classification, this subtype was known as M6.

The second type is pure erythroid leukemia. In this type, 50% or more nucleated cells are pronormoblasts and 30% or more are basophilic normoblasts. Together, these two erythroid components comprise more than 80% of the bone marrow. The myeloblast component is not significant. Complex rearrangements and hypodiploid chromosome number are common. Chromosomes 5 and 7 are frequently affected.6

The red blood cell (RBC) precursors have significant dysplastic features, such as multinucleation, megaloblastoid asynchrony, and vacuolization. The nucleated RBCs in the peripheral blood may account for more than 50% of the total number of nucleated cells. Ringed sideroblasts, Howell-Jolly bodies, and other inclusions may be present (). Abnormal megakaryocytes may be seen. Both types of erythroid leukemia have an aggressive and rapid clinical course.Figure 35-136

Image 

FIGURE 35-13 Acute erythroid leukemia. Erythroid precursors showing dysplastic features, including multinucleation and megaloblastic asynchrony (bone marrow, ×500). Source: (From Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

Acute megakaryoblastic leukemia. 

Patients with acute megakaryoblastic leukemia usually have cytopenias, although some may have thrombocytosis. Dysplastic features are often present in all cell lines. Diagnosis requires the presence of at least 20% blasts, of which at least 50% must be of megakaryocyte origin. This category excludes AML with MDS-related changes and Down syndrome–related cases, as well as those with recurrent genetic abnormalities, as discussed previously.

Megakaryoblast diameters vary from that of a small lymphocyte to three times their size. Chromatin is delicate with prominent nucleoli. Immature megakaryocytes may have light blue cytoplasmic blebs (, Figure 35-14 A). Megakaryoblasts are identified by immunostaining, employing antibodies specific for cytoplasmic von Willebrand factor or platelet membrane antigens CD41 (glycoprotein IIb), CD42b (glycoprotein Ib) (Figure 35-14B), or CD61 (glycoprotein IIIa).6

Image 

FIGURE 35-14 Acute megakaryocytic leukemia. A, Note heterogeneity of blasts, one small with scant cytoplasm, two with cytoplasmic blebbing, and one quite large (peripheral blood ×1000). B, Positive reaction for CD42b (bone marrow, ×1000). Source: (A from Carr JH, Rodak BF: Clinical hematology atlas, ed 4, St. Louis, 2013, Saunders.)

Myeloid sarcoma

Myeloid sarcoma refers to extramedullary proliferation of blasts of one or more myeloid lineages that disrupts tissue architecture. Tissue architecture must be effaced for the neoplasm to qualify for this diagnosis.628

Myeloid proliferations related to down syndrome

Unique patterns of malignancy occur in persons with trisomy 21 resulting in Down syndrome. Somatic mutations of the GATA1 gene have also been detected and are linked to both leukemogenesis and high cure rates.41 Approximately 10% of newborns with Down syndrome present with transient abnormal myelopoiesis, which is morphologically indistinguishable from AML. Spontaneous remission generally occurs within a few months. Among individuals with Down syndrome, there is a fiftyfold increased incidence of AML during the first 5 years of life compared with individuals without Down syndrome. The leukemia is of megakaryocytic lineage, and young children respond well to chemotherapy, although older children do not fare as well.641

Blastic plasmacytoid dendritic cell neoplasm

Blastic plasmacytoid cell neoplasm is a rare clinically aggressive tumor derived from precursors of plasmacytoid dendritic cells. It presents with skin lesions and may ultimately progress to involve peripheral blood and bone marrow.614

Acute leukemias of ambiguous lineage

Acute leukemias of ambiguous lineage (ALALs) include leukemia in which there is no clear evidence of differentiation along a single cell line and are commonly referred to as acute undifferentiated leukemias(AULs). Other cases of ALAL that demonstrate a multiplicity of antigens where it is not possible to determine a specific lineage are called mixed phenotype acute leukemias (MPALs). The 2008 WHO classification significantly revised the criteria for this designation and is shown in Box 35-4.6

BOX 35-4

Classification of Acute Leukemia of Ambiguous Lineage (ALAL) (2008 World Health Organization Classification)

Acute undifferentiated leukemia (AUL)—synonyms: ALAL without differentiation, primitive acute leukemia, stem cell leukemia

Mixed phenotype acute leukemia (MPAL)—synonyms: biphenotypic acute leukemia, bilineal leukemia, mixed lineage acute leukemia, dual lineage acute leukemia, hybrid acute leukemia:

MPAL with t(9; 22)(q34; q11.2); BCR-ABL1

MPAL with t(v; 11q23); MLL rearranged

MPAL B/myeloid, not otherwise specified

MPAL T/myeloid, not otherwise specified

From Swerdlow SH, Campo E, Harris NL, et al, editors: WHO classification of tumours of haematopoietic and lymphoid tissues, ed 4, Lyon, France, 2008, IARC Press.

Future directons in the classification of acute leukemias

A number of recent studies have shown the importance of gene mutations in the pathogenesis of acute leukemias.4243 These mutations also have prognostic importance and are likely to be incorporated into future classifications. Important mutated genes include KIT, FLT3, ASXL1, TP53, CEBPA, and NPM1. AML with mutated NPM1 and AML with mutated CEBPA are provisional entities in the 2008 WHO classification.6

Cytochemical stains and interpretations

Techniques such as flow cytometry, cytogenetic analysis, and molecular testing are now commonly used in the diagnosis of acute leukemias. However, older techniques such as cytochemical stains still retain their importance. An advantage of cytochemical stains is that they are relatively cheap and can be performed by laboratories throughout the world, including in areas where resources and access to advanced techniques are limited. The cytochemical stains are summarized in . Table 35-3

TABLE 35-3

Acute Leukemia Cytochemical Reaction Chart

Condition

MPO

SBB

NASDA

ANBE

ANAE

ALL

−/+ (focal)

−/+ (focal)

AML

+

+

+

AMML

+

+

+

+ (diffuse)

+ (diffuse)

AMoL

−/+

+ (diffuse)

+ (diffuse)

Megakaryocytic leukemia

+ (localized)

+, Positive reaction; −, negative reaction; −/+, negative or positive reaction; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; AMML, acute myelomonocytic leukemia; AMoL, acute monocytic leukemia; ANAE, α-naphthyl acetate esterase; ANBE, α-naphthyl butyrate esterase; MPO, myeloperoxidase; NASDA, naphthol AS-D chloroacetate esterase; SBB, Sudan black B.

Myeloperoxidase

Myeloperoxidase (MPO) ( and Figures 35-1535-16) is an enzyme found in the primary granules of granulocytic cells (neutrophils, eosinophils, and, to a certain extent, monocytes). Lymphocytes do not exhibit MPO activity. This stain is useful for differentiating the blasts of acute myeloid leukemia (AML) from those of acute lymphoblastic leukemia (ALL).

Image 

FIGURE 35-15 Positive reaction to myeloperoxidase stain in early myeloid cells. Note Auer rod at arrow (bone marrow, ×1000).

Image 

FIGURE 35-16 Strong positive reaction to myeloperoxidase stain in leukemic promyelocytes from a patient with acute promyelocytic leukemia (bone marrow, ×1000).

Interpretation

MPO is present in the primary granules of most granulocytic cells, beginning at the promyelocyte stage and continuing throughout maturation. Leukemic myeloblasts are usually positive for MPO. In many cases of the AMLs (without maturation, with maturation, and promyelocytic leukemia), it has been found that more than 80% of the blasts show MPO activity. Auer rods found in leukemic blasts and promyelocytes test strongly MPO positive.

In contrast, lymphoblasts in ALL and lymphoid cells are MPO negative. It is important that the reaction only in the blast cells be used as the determining factor for the differentiation of acute leukemias. This is true for MPO and for the other cytochemical stains used in determining cell lineage that are mentioned in this chapter. The fact that maturing granulocytes are MPO positive is normal and has little or no diagnostic significance.

Sudan black B

SBB staining () is another useful technique for the differentiation of AML from ALL. SBB stains cellular lipids. The staining pattern is quite similar to that of MPO; SBB staining is possibly a little more sensitive for the early myeloid cells. Figure 35-17

Image 

FIGURE 35-17 Sudan black B reaction. The positivity increases with the maturity of the granulocytic cell (bone marrow, ×1000).

Interpretation

Granulocytes (neutrophils) show a positive reaction to SBB from the myeloblast through the maturation series. The staining becomes more intense as the cell matures as a result of the increase in the numbers of primary and secondary granules. Monocytic cells can demonstrate negative to weakly positive staining due to various changes that occur during differentiation. Lymphoid cells generally do not stain. In ALL, fewer than 3% of the blast cells show a positive reaction.44-46

Esterases

Esterase reactions are used to differentiate myeloblasts and neutrophilic granulocytes from cells of monocytic origin. Nine isoenzymes of esterases are present in leukocytes. Two substrate esters commonly used are α-naphthyl acetate and α-naphthyl butyrate (both nonspecific). Naphthol AS-D chloroacetate (specific) also may be used. “Specific” refers to the fact that only granulocytic cells show staining, whereas nonspecific stains may produce positive results in other cells as well.

Interpretation

Esterase stains can be used to distinguish acute leukemias that are granulocytic from leukemias that are primarily of monocytic origin. When naphthol AS-D chloroacetate is used as a substrate, the reaction is positive in the granulocytic cells and negative to weak in the monocytic cells (). Chloroacetate esterase is present in the primary granules of neutrophils. Leukemic Figure 35-18 myeloblasts generally show a positive reaction. Auer rods show positivity as well.

Image 

FIGURE 35-18 Positive reaction to AS-D chloroacetate esterase stain in two granulocytic cells (bone marrow, ×1000).

α-Naphthyl acetate, in contrast to naphthol AS-D chloroacetate, reveals strong esterase activity in monocytes that can be inhibited with the addition of sodium fluoride.4446 Granulocytes and lymphoid cells generally show a negative result on nonspecific esterase staining (Figure 35-19).

Image 

FIGURE 35-19 A, Positive reaction to α-naphthyl acetate esterase stain in monocytes (bone marrow, ×1000). B, Same specimen with addition of sodium fluoride. The esterase reaction in the monocytes is inhibited (bone marrow, ×1000).

A diffuse positive α-naphthyl butyrate esterase reaction is seen in monocytes. α-Naphthyl butyrate is less sensitive than α-naphthyl acetate, but it is more specific. Granulocytes and lymphoid cells generally show a negative reaction (), although a small positive dot may be seen in lymphocytes. In myelomonocytic leukemia, positive AS-D chloroacetate activity and positive α-naphthyl butyrate or α-naphthyl acetate activity should be seen because myeloid and monocytic cells are present. In myelomonocytic leukemia, at least 20% of the cells must show monocytic differentiation that is nonspecific esterase positive and is inhibited by sodium fluoride. In the pure monocytic leukemias, 80% or more of the blasts are nonspecific esterase positive and specific esterase negative. Figure 35-20

Image 

FIGURE 35-20 α-Naphthyl butyrate esterase positivity in cells of monocytic origin from a patient with acute monoblastic/monocytic leukemia. Note the negativity of myeloid and erythroid precursors (bone marrow, ×1000).

Summary

• The development of leukemia is currently believed to be a stepwise progression of mutations, or “multiple-hits,” involving mutations that give leukemic stem cells a proliferative advantage and also hinder differentiation.

• For most acute leukemias, causes directly related to the development of the malignancy are unknown, but a few exceptions exist. Some known causes include environmental toxins, certain viruses, previous chemotherapy, and familial predisposition.

• There are several classification schemes for leukocyte neoplasia, including the FAB system, based primarily on morphology and cytochemical staining, and the WHO system, which retains some elements of the FAB scheme but emphasizes molecular and cytogenetic changes.

• Only half of patients with ALL have leukocytosis, and many do not have circulating lymphoblasts, but neutropenia, thrombocytopenia, and anemia are usually present.

• In children ALL is a disease in which the “good prognosis” subtypes are associated with a 95% rate of complete remission, but adults with ALL have a poorer outlook.

• Infiltration of malignant cells into the meninges can occur, with lymphoblasts found in the cerebrospinal fluid, testes, and ovaries.

• Prognosis in ALL depends primarily on age at the time of diagnosis, lymphoblast load (tumor burden), and immunophenotype. Chromosomal translocations seem to be the strongest predictor of adverse treatment outcomes for children and adults.

• The t(12; 21) marker is found in a significant number of patients with childhood ALL.

• There are two main subtypes of ALL according to the WHO classification system: B lymphoblastic leukemia/lymphoma and T lymphoblastic leukemia/lymphoma.

• Tumor lysis syndrome is an increasingly common complication of treatment, especially in patients with a high tumor burden.

• Although morphology is the first tool in distinguishing ALL from AML, immunophenotyping is often the only reliable indicator of a cell’s origin.

• The incidence of AML in adults increases with age.

• The clinical presentation of a patient with AML is nonspecific and reflects the decreased production of normal bone marrow elements, an elevated WBC count, and the presence of myeloblasts. Anemia, thrombocytopenia, and neutropenia give rise to the clinical findings of pallor, fatigue, bruising and bleeding, and fever with infections.

• The classification of AML is complicated by the presence or absence of multiple cell lines defined as “myeloid” in origin, specific cells within these cell lines, and specific karyotype abnormalities.

• Leukemias with ambiguous lineage include leukemias in which there is no clear evidence of differentiation along a single cell line.

• Cytochemical techniques are often used in conjunction with morphologic analysis, immunohistochemical methods, flow cytometry, cytogenetic analysis, and molecular biologic techniques in establishing a diagnosis.

• Cytochemical reactions may be enzymatic or nonenzymatic. Fresh smears must be used to detect enzymatic activity, whereas nonenzymatic procedures may be performed on specimens that have been stored at room temperature.

• MPO stains primary granules and is useful in differentiating granulocytic from lymphoid cells.

• SBB stains lipids and results parallel those with the MPO stain.

• Esterases help differentiate granulocytes and their precursors from cells of monocytic origin. Butyrate esterase testing gives positive results in monocytes but not in granulocyte precursors, whereas naphthol AS-D chloroacetate esterase stains granulocyte precursors.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. According to the WHO classification, except in leukemias with specific genetic anomalies, the minimal percentage of blasts necessary for a diagnosis of acute leukemia is:

a. 10%

b. 20%

c. 30%

d. 50%

2. A 20-year-old patient has an elevated WBC count with 70% blasts, 4% neutrophils, 5% lymphocytes, and 21% monocytes in the peripheral blood. Eosinophils with dysplastic changes are seen in the bone marrow. AML with which of the following karyotypes would be most likely to be seen?

a. AML with t(8; 21)(q22; q22)

b. AML with t(16; 16)(p13; q22)

c. AML with t(15; 17)(q22; q12)

d. AML with t(9; 11)(p22; q23)

3. Which of the following would be considered a sign of potentially favorable prognosis in children with ALL?

a. Hyperdiploidy

b. Presence of CD 19 and 20

c. Absence of trisomy 8

d. Presence of BCR/ABL gene

4. Signs and symptoms of cerebral infiltration with blasts are more commonly seen in:

a. AML with recurrent cytogenetic abnormalities

b. Therapy-related myeloid neoplasms

c. AML with myelodysplasia-related changes

d. ALL

5. An oncology patient exhibiting signs of renal failure with seizures after initial chemotherapy may potentially develop:

a. Hyperleukocytosis

b. Tumor lysis syndrome

c. Acute leukemia secondary to chemotherapy

d. Myelodysplasia

6. Disseminated intravascular coagulation is more often seen in association with leukemia characterized by which of the following mutations?

a. t(12; 21)(p13; q22)

b. t(9; 22)(q34; q11.2)

c. inv(16)(p13; q22)

d. t(15; 17)(q22; q12)

7. Which of the following leukemias affects primarily children, is characterized by an increase in monoblasts and monocytes, and often is associated with gingival and skin involvement?

a. Pre-B lymphoblastic leukemia

b. Pure erythroid leukemia

c. AML with t(9; 11)(p22; q23)

d. AML with t(15; 17)(q22; q12)

8. A 20-year-old patient presents with fatigue, pallor, easy bruising, and swollen gums. Bone marrow examination reveals 82% cells with delicate chromatin and prominent nucleoli that are CD14+, CD4+, CD11b+, and CD36+. Which of the following acute leukemias is likely?

a. Minimally differentiated leukemia

b. Leukemia of ambiguous lineage

c. Acute monoblastic/monocytic leukemia

d. Acute megakaryoblastic leukemia

9. Pure erythroid leukemia is a disorder involving:

a. Pronormoblasts only

b. Pronormoblasts and basophilic normoblasts

c. All forms of developing RBC precursors

d. Equal numbers of pronormoblasts and myeloblasts

10. A patient with normal chromosomes has a WBC count of 3.0 × 109/L and dysplasia in all cell lines. There are 60% blasts of varying sizes. The blasts stain positive for CD61. The most likely type of leukemia is:

a. Acute lymphoblastic

b. Acute megakaryoblastic

c. Acute monoblastic

d. AML with t(15; 17)

11. SBB stains which of the following component of cells?

a. Glycogen

b. Lipids

c. Structural proteins

d. Enzymes

12. The cytochemical stain α-naphthyl butyrate is a nonspecific esterase stain that shows diffuse positivity in cells of which lineage?

a. Erythroid

b. Monocytic

c. Granulocytic

d. Lymphoid

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