Cytogenetics

Leukemic blasts of the majority of patients with AML at diagnosis carry at least one clonal chromosome abnormality, i.e., an identical structural aberration or gain of the same, structurally intact chromosome (trisomy) found in at least two metaphase cells or the same chromosome missing (monosomy) from a minimum of three cells. Abnormal karyotypes are more frequent in children with de novo AML, being detected in 70-85% of patients compared with 55-60% of adults.8-13 Therapy-related (secondary) AML is usually characterized by a high proportion, 80-90%, of both adult and pediatric patients who carry chromosome abnormalities.12 Among AML patients with an abnormal karyotype, slightly more than one-half harbor only one chromosome aberration, whereas the remaining patients have two or more aberrations, including 10-15% of patients whose karyotype is complex, i.e., contains three or more aberrations.1213

From the cytogenetic standpoint, AML is heterogeneous, with more than 200 different structural and numerical aberrations identified as recurring in this disease.5 While many of the recurring aberrations are rare, being thus far detected in a few patients worldwide, others are more common (Table 2.2). Notably, cytogenetic findings at diagnosis constitute one of the most important independent prognostic factors for attainment of complete remission (CR), risk of relapse, and survival.8-14 Recent large collaborative studies8-10 have proposed cytogenetic risk systems categorizing AML patients into one of three risk groups (favorable, intermediate, or adverse) based upon cytogenetic findings at diagnosis (Table 2.2). Although some differences among the three prioritization schemata exist, pretreatment cytogenetic results are being used to stratify therapy.1516 Moreover, a recent report advocates the use of cytogenetic remission as one of the criteria of CR in AML.17 This is based on a recent study that demonstrated a significantly worse outcome for patients whose marrow on the first day of morphologically documented CR contained cytogenetically abnormal cells than those whose marrow showed an entirely normal karyotype.18

Major cytogenetic studies of AML agree that the prognosis of patients with inv(16)/t(16;16), t(8;21), and t(15;17) is relatively favorable (these subtypes of AML are discussed below), whereas the clinical outcome of patients with inv(3)(q21q26) or t(3;3) (q21;q26), -7, and a complex karyotype is adverse (Table 2.2). Complex karyotype has been defined as either greater than or equal to five unrelated cytoge-netic abnormalities or greater than or equal to three abnormalities. However, Byrd et al.10 have shown that although patients with three or four abnormalities [other than t(8;21), inv(16)/t(16;16), or t(9;11) (p22;q23)] had significantly better survival and a lower probability of relapse than those with greater than or equal to five abnormalities, their probabilities of achieving a CR, or obtaining prolonged CR and survival, were significantly worse than those of patients with a normal karyotype. These data seem to justify combining patients with three or four abnormalities with those with greater than or equal to five abnormalities into one complex karyotype category with greater than or equal to three abnormalities if three clinical prognostic groups are going to be used. Of note, patients with inv(16)/t(16;16), t(8;21), t(15;17), or t(9;11) and greater than or equal to three abnormalities are usually not included in this prognostically unfavorable complex karyotype category, because in these patients, the presence of a complex karyotype does not influence prognosis adversely.8,10

Patients with a normal karyotype of marrow cells at diagnosis constitute the largest cytogenetic subset of AML and are classified in the intermediate prognostic category by all major classification schemata.8-10 However, despite the absence of microscopically detectable chromosome aberrations, these patients can harbor submicroscopic genetic abnormalities discernible only by molecular genetic techniques, such as RT-PCR or direct sequencing. Among several such

The WHO classification of AMLsa Postulated

WHO category of AML cell origin

Morphology and cytochemistry

Cytogenetics

Immunophenotype

AML with t(8;21)(q22;q22), [AML1(RUNX1)/ETO(CBFA2T1)]

Myeloid stem cell with predominant neutrophil differentiation

AML with recurrent genetic abnormalities BM contains large blasts with abundant basophilic cytoplasm often with azurophilic granulation and/or single Auer rods, smaller blasts, promyelocytes, myelocytes, and mature neutrophils with variable dysplasia and homogeneous pink cytoplasm. There is an increase in eosinophil precursors, which do not have abnormalities seen in AML with inv(16)/t(16;16)

t(8;21), variant translocations, or insertions

CD34+, CD13+, CD33+, MPO+; frequently, CD19 present on a subset of the blasts; often CD56+; some cases are TdT+, with dim expression

AML with abnormal BM eosinophils and inv(16) (p13q22) or t(16;16) (p13;q22), (CBFB/MYH11)

Hematopoietic stem cell with potential to differentiate to granulocytic and monocytic lineages

In addition to myelomonocytic features (see below), marrow contains an increased number of abnormal eosinophils, with immature, large purple-violet eosinophil granules and faint positivity to naphthol ASD chloroacetate esterase reaction. Myeloblasts can have Auer rods. MPO activity is seen in 3% or more of blasts; monoblasts and promonocytes are usually NSE positive inv(16) or t(16;16)

CD13+, CD33+, MPO + ; often positive for some or all of the following: CD14, CD4, CD11b, CD11c, CD64, CD36, and lysozyme; may be CD2 +

AML with t(15;17)(q22; q12-21), (PML/RARA) and variants

Myeloid stem cell with potential to differentiate to granulocytic lineage

Hypergranular APL: Abnormal promyelocytes of variable size and irregular shape, often kidney-shaped or bilobed, with cytoplasm packed with pink, red, or purple large granules. Cells containing multiple Auer rods, which are usually larger than in other types of AML, are called Faggot cells. Myeloblasts with single Auer rods may be present. Strongly positive MPO reaction; NSE weakly positive in 25% of cases. Microgranular (hypogranular APL): predominantly bilobed promyelocytes with the apparent absence of or a few large granules. Rare Faggot cells and/or abnormal promyelocytes with visible granules. Higher than in hypergranular APL leukocyte count with numerous abnormal microgranular promyelocytes. Strongly positive MPO reaction t(15;17), variant translocations, or insertions

CD33+ homogeneously and brightly; CD13+ het-erogeneously; CD34 and HLA-DR generally absent, if expressed then only on a subset of cells; CD15- or dimly expressed; frequent coex-pression of CD2 and CD9

table continues continued

WHO category of AML

Postulated cell origin

Morphology and cytochemistry

Cytogenetics

Immunophenotype

AML with 11q23 (MLL) abnormalities

Hematopoietic stem cell with multilineage potential

Mostly monocytic and myelomonocytic morphology, although a minority of cases have morphological features of AML with or without maturation t(9;11)(p22; q23),t(6;11) (q27;q23), t(11;19)(q23; p13.1) and other translocations, inversions, and insertions involving band 11q23

No specific immunophen-toypic features; variable expression of CD13 and CD33; cases with monoblastic morphology are CD34— and CD14+, CD4+, CD11b+, CD11c+, CD64+, CD36+, and/or lysozyme+

May develop de novo or following an MDS/ myeloproliferative disease

Hematopoietic stem cell

AML with multilineage dysplasia Dysplasia present in at least 50% of the cells of two or more myeloid lineages

Aberrations similar to those occurring in MDS, mostly unbalanced: —5/del(5q), — 7/del(7q), +8, +11, -18, + 19, +21, del(11q), del(12p), del(20q). Less often der(1;7) (q10;p10), inv(3)/t(3;3), t(3;5)(q25;q34)

Blasts, which often constitute a subpopulation of cells, are CD34+, CD13+, and CD33 + . Frequent aberrant expression of CD56 and/or CD7; increased expression of MDR1 on the blasts

Alkylating agent/radiation-related type

Hematopoietic stem cell

AML and MDS, therapy related Panmyelosis, dysplastic changes, ringed siderob-lasts in up to 60% of cases

Often complex karyotypes with — 5/del(5q), — 7/del(7q), and del(17p)

Blasts, which often constitute a subpopulation of cells, are CD34+, CD13+, and CD33+. Aberrant expression of CD56 and/or CD7 is frequent, and expression of MDR1 on the blasts increased

Topoisomerase II inhibitor-related type (some may be lymphoid)

Hematopoietic stem cell

A significant monocytic component, most cases have acute myelomonocytic or monoblastic leukemia. APL also reported

Balanced translocations involving 11q23 [t(9;11), t(6;11), t(11;19), etc.], t(8;21), t(3;21) (q26;q22), inv(16), t(8;16) (p11;p13), t(6;9)(p23;q34), and, in therapy-related APL, t(15;17)

AML, minimally differentiated

Hematopoietic stem cell at the earliest stage of myeloid differentiation/maturation

AML not otherwise categorized Medium-size blasts with round or slightly indented nuclei with dispersed chromatin and 1 or 2 nucleoli, and agranular cytoplasm with a varying degree of basophilia. Less often blasts are small and resemble lymphoblasts, with more condensed chromatin, inconspicuous nucleoli, and scanty cytoplasm. MPO, SBB, and naphthol ASD chloroacetate esterase reactions are negative

There is no consistent abnormality; recurrent aberrations include +4, +8, +13, -7, and complex karyotypes

CD13+, CD33+, and/or CD117+;cCD3-, cCD79a—, and cCD22-; often MPO-; most cases are CD34+, CD38+, and HLA-DR+; CD11b-, CD14-, CD15-, CD65-; TdT+ in > one-third cases; CD7, CD2, and CD19 may be expressed but with lower intensity than in lym-phoid leukemias

AML without maturation

Precursor hematopoietic cell at the earliest stage of myeloid differentiation

In a proportion of cases, myeloblasts with azurophilic granulation and/or Auer rods are present. Other cases have blasts similar to lymphoblasts, without azurophilic granulation. Variable (but always in >3% of blasts) MPO and SBB positivity

There is no consistent abnormality

Expression of at least two of the following: CD13, CD33, CD117, and/or MPO. Often CD34 + . CD11b- and CD14-

AML with maturation

Hematopoietic precursor cell at the earliest stage of myeloid development

Myeloblasts with azurophilic granulation and/or Auer rods are present, and myelocytes, promyelocytes, and mature neutrophils constitute >10% of BM cells. There is variable degree of dysplasia. Blasts and maturing neutrophils are lysozyme and MPO positive

Deletions and translocations involving 12p, t(6;9)(p23;q34), t(8;16)(p11;p13)

Expression of one or more of the following: CD13, CD33, and CD15. CD117, CD34, and HLA-DR also may be expressed

Acute myelomonocytic leukemia

Hematopoietic precursor cell with potential to differentiate into neu-trophil and monocytic lineages

The BM contains >20% neutrophils and their precursors and >20% monocytes, monoblasts, and promonocytes

The majority of cases are cyto-genetically abnormal, but no specific aberration has been identified

Variable expression of CD13 and CD33. CD4, CD14, CD11b, CD11c, CD36, CD64, and/or lysozyme may be expressed. Residual population of less differentiated myeloblasts expresses CD34 and panmyeloid markers

Acute monoblastic and monocytic leukemia

BM stem cell with some commitment to monocytic differentiation

80% or more of the leukemic cells are monoblasts, promonocytes, and monocytes; a neutrophil component is minor (<20%). In acute monoblastic leukemia, monoblasts constitute >80% of mono-cytic cells, whereas in acute monocytic leukemia, promonocytes predominate. Both monoblasts and promonocytes usually display NSE activity; monoblasts are MPO negative and promonocytes may show scattered MPO positivity

In cases with hemophagocyto-sis by leukemic cells, t(8;16)(p11;p13) is often detected

Variable expression of CD13, CD33, and CD 117. CD4, CD14, CD11b, CD11c, CD36, CD64, CD68, and/or lysozyme may be expressed. CD34 often negative table continues continued

WHO category of AML

Postulated cell origin

Morphology and cytochemistry

Cytogenetics

Immunophenotype

Erythroleukemia (erythroid/myeloid)

Acute erythroid leukemias (erythroid/myeloid and pure erythroleukemia)

Multipotent stem cell with wide myeloid potential

Dysplastic erythroid precursors at all maturation stages may be present. There may be large multinucleated erythroid cells. The myeloblasts are similar to those in AML with and without maturation

Complex karyotypes with -5/del(5q) and -7/del(7q) are frequent

Erythroblasts lack myeloid-associated antigens, are MPO-, and react with antibodies to hemoglobin A and glycophorin A. Myeloblasts are usually CD13+, CD33+, CD117+, and MPO+. CD34 and class II HLA-DR expression are variable

Pure erythroleukemia

Primitive (BFU-E/CFU-E) stem cell with some degree of commitment to the erythroid lineage

Erythroblasts are medium to large in size and have basophilic cytoplasm. Infrequently, the blasts are smaller and resemble lymphoblasts. Erythroblasts are negative for MPO and SBB, but show reactivity with ANA esterase, acid phosphatase, and periodic acid Schiff

Complex karyotypes with -5/del(5q) and -7/del(7q) are frequent

In more differentiated forms, glycophorin A and hemoglobin A are expressed but MPO and other myeloid antigens are not. Glycophorin A is usually not expressed in more immature forms, which are positive for carbonic anhy-drase 1, CD36, and Gero antibody against the Gerbuch blood group

Acute megakaryoblastic leukemia

Hematopoietic precursor cell committed to the megakaryocyte lineage and possibly erythroid lineage and/or able to differentiate into these lineages

More than 50% of the blasts are of megakaryocyte lineage. Megakaryoblasts are usually medium to large in size but small blasts resembling lymphoblasts may also be present. Marrow fibrosis may occur in some patients

Translocation t(1;22)(p13;q13) is recurrent in children younger than 2 years. In some adults, inv(3) or t(3;3) is found, but they also occur in other AML types

Megakaryoblasts express CD36 and one or more of the following: CD41, CD61, and/or CD42 (less often). CD13 and CD33 may be positive, and CD34, CD45, and HLA-DR are often negative

Acute basophilic leukemia

Early myeloid cell committed to the basophil lineage

Medium-size blasts with moderately basophilic cytoplasm containing coarse basophilic granules. Characteristically, blasts are positive for cyto-chemical reaction with toluidine blue, and usually stain diffusely with acid phosphatase, but are negative for SBB, MPO, and NSE by light microscopy

No consistent abnormality

CD13+, CD33+, CD34+, class II HLA-DR+; usually, CD9+, some cases TdT+; negative for specific lymphoid markers

Acute panmyelosis with myelofibrosis

Myeloid hematopoietic stem cell. The fibroblastic proliferation is an epiphenomenon

Marked pancytopenia in the blood. BM aspirations often unsuccessful. BM biopsy hypercellular with variable hyperplasia of the granulocytes, megakaryocytes, and erythroid precursors. Variable degree of fibrosis, with increase in retic-ulin fibers

If analysis successful, the karyotype is usually complex, with -5/del(5q) and/or -7/ del(7q)

Phenotypic heterogeneity, with expression of one or more of the following: CD13, CD33, CD117, and MPO

Myeloid sarcoma

Primitive myeloid hematopoietic cell

Granulocytic sarcoma, the most common type, consists of myeloblasts, neutrophils, and neutrophil precursors, and is divided into three types based upon degree of maturation. The blastic type contains mainly myeloblasts, the immature type myeloblasts and promyelocytes, and the differentiated type promyelocytes and more mature neutrophils.

Less frequent monoblastic sarcoma is composed of monoblasts

In some cases of myeloid sarcoma, t(8;21) or inv(16)/ t(16;16); in monoblastic sarcoma 11q23 translocations

Most myeloid sarcomas express CD43. Granulocytic sarcoma myeloblasts are CD13+, CD33+, CD117+, and MPO+. The monoblasts in monoblastic sarcoma are CD14+, CD116+, CD11c+, and react with antibodies to lysozyme and CD68 by immunohistochemistry

Undifferentiated acute leukemia

Multipotent progenitor stem cell

Acute leukemia of ambiguous lineage The leukemic cells lack any differentiating features

Frequently abnormal cytoge-netically. Recurrent aberrations include del(5q) and +13, often as a sole abnormality

Often HLA-DR+, CD34+, CD38+, and may be TdT+ and CD7+.

Negative for markers specific for a given lineage, such as cCD79a, cCD22, CD3, and MPO. Generally, do not express more than one lineage-associated marker.

Bilineal acute leukemia

Multipotent progenitor stem cell

May present as monoblastic or poorly differentiated myeloid leukemia, or as ALL. In some cases, blasts are morphologically undifferentiated; in others, populations of small blasts resembling lymphoblasts may coexist with larger blasts

Typically abnormal cytogeneti-cally. Cases with B lymphoid component often have t(9;22) (q34;q11.2), t(4;11), or other 11q23 aberrations; these aberrations are not found in cases with T lymphoid component

Coexistence of two populations of blasts, each of which expresses antigens of a distinct lineage, which is myeloid and lymphoid or B and T

Biphenotypic acute leukemia

Multipotent progenitor stem cell

May present as monoblastic or poorly differentiated myeloid leukemia, or as ALL. In some cases, blasts are morphologically undifferentiated; in others, populations of small blasts resembling lymphoblasts may coexist with larger blasts

Usually abnormal cytogeneti-cally. Cases with B lymphoid component often have t(9;22), t(4;11), or other 11q23 aberrations; these aberrations are not found in cases with T lymphoid component

Blasts coexpress myeloid and T- or B-lineage-specific markers or, concurrently, T- and B-specific antigens a Data from Jaffe et al.1 and Mitelman et al.36

Table 2.2 Prognostic significance of the more common chromosome aberrations in AML

Chromosome aberration3

Cytogenetic risk category inv(16)(p13q22)/t(16;16) Favorable (p13;q22); t(8;21)(q22;q22); t(15;17)(q22;q12-21)

none (normal karyotype);Intermediate

—Y;del(7q)b; del(9q)b; del(11q)c; del(20q)d; isolated +8e; +11, +13, +21, t(9;11)(p22;q23)c complex karyotype with >3 Unfavorable abnormalities; inv(3) (q21q26)/t(3;3)(q21;q26);

— 7;del(5q)f; -5; t(6;9) (p23;q34)g; t(6;11)(q27;q23)h; t(11;19)(q23;p13.1)h a Chromosome aberrations whose prognostic impact is agreed on by major studies8-10 are indicated by bold type. b Classified in the adverse-risk category by SWOG/ECOG.9 c Would be included in "abn 11q" group and classified in the adverse-risk category by SWOG/ECOG.9 d Would be included in "abn 20q" group and classified in the adverse-risk category by SWOG/ECOG.9

eClassified in the adverse-risk category with regard to overall survival by CALGB.10

f Classified in the intermediate-risk category with regard to probability of achievement of CR and survival by CALGB if not part of a complex karyotype.

g Classified in the intermediate-risk category by virtue of being "other structural" abnormality by MRC8 and by CALGB (but intermediate only with regard to probability of achievement of CR).10 h Would be included in "abnormal 11q23" group and classified in the intermediate-risk category by MRC8 and by CALGB (but only with regard to probability of achievement of CR).10

mutations associated with AML, internal tandem duplication (ITD) of the FLT3 gene (FLT3 ITD), partial tandem duplication (PTD) of the mixed lineage leukemia (MLL) gene (MLL PTD), and point mutations of the CCAAT/enhancer-binding protein a (CEBPA) gene have been recently found to be of prognostic significance in patients with AML and a normal karyotype (Table 2.3). Likewise, adverse prognosis has been associated with overexpression of the brain and acute leukemia, cytoplasmic (BAALC) gene.19-21

Later in the chapter we will discuss in greater detail the four AML categories delineated primarily because of cytogenetic and molecular genetic findings, followed by data on major molecular genetic rearrangements relevant to AML pathogenesis, some of which are associated with clinical outcome.

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