Residency · Residency · Pathology

Acute Leukemias: Classification and Diagnosis

Overview

Acute leukemias are aggressive hematologic malignancies characterized by the clonal proliferation of immature hematopoietic cells (blasts) in the bone marrow and peripheral blood. The WHO 5th edition (2022) and the International Consensus Classification (ICC, 2022) have introduced parallel but not identical classification systems, both emphasizing the central role of genetics in defining disease entities. Accurate diagnosis requires the integration of morphology, immunophenotyping by flow cytometry, cytogenetics, and molecular studies.

Blast Enumeration and Thresholds

Definition of Blasts

Blasts are immature cells characterized by high nuclear-to-cytoplasmic ratios, fine chromatin, prominent nucleoli, and scant cytoplasm. They are counted on a 500-cell aspirate smear differential. The count includes myeloblasts, monoblasts, and promonocytes (in AML) or lymphoblasts (in ALL). Promonocytes are specifically counted as blast equivalents in acute monocytic and myelomonocytic leukemia because they retain the biological behavior of blasts.

Blast Threshold for AML

The traditional threshold for diagnosing AML has been 20% or greater blasts in the marrow or blood. However, the WHO 5th edition has lowered this to 10% or greater for AML with certain defining genetic abnormalities, including t(8;21), inv(16)/t(16;16), t(15;17), t(9;11), and other entities with defining fusions or mutations. The ICC maintains the 20% threshold for most cases but recognizes 10% or greater for specific entities with recurrent genetic abnormalities. Cases with 10-19% blasts and MDS-defining cytogenetics are classified differently between the two systems, creating a zone of divergence that clinicians and pathologists must navigate.

Blast Threshold for ALL

There is no formal minimum blast percentage for ALL. Diagnosis is based on the presence of lymphoblasts with an appropriate immunophenotype and clinical presentation. Typically more than 20-25% blasts are present, but lower percentages may suffice when supporting morphologic, immunophenotypic, and genetic evidence is present.

Acute Myeloid Leukemia (AML)

AML with Defining Genetic Abnormalities

AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1

This core-binding factor (CBF) AML shows morphologically distinctive blasts that are large with abundant cytoplasm, long thin Auer rods, a perinuclear hof, and salmon-colored granules. Maturation to the promyelocyte and myelocyte stage is typically present. Flow cytometry shows CD34+, CD117+, CD13+, and CD33+ blasts with aberrant CD19 and CD56 expression being common. This entity carries a favorable prognosis and can be diagnosed with 10% or greater blasts per the WHO 5th edition.

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

This is the other CBF AML, characterized morphologically by a monocytic component accompanied by abnormal eosinophils containing distinctive large basophilic granules. Flow cytometry demonstrates both myeloid and monocytic markers, and CD2 may be aberrantly expressed. It carries a favorable prognosis and is diagnostic at 10% or greater blasts.

APL: AML with t(15;17)(q24.1;q21.2); PML-RARA

Acute promyelocytic leukemia is a medical emergency due to disseminated intravascular coagulation at presentation, with early mortality from hemorrhage if treatment is delayed. The hypergranular variant shows heavily granulated promyelocytes with bilobed or reniform nuclei and bundles of Auer rods ("faggot cells"). The microgranular/hypogranular variant has bilobed nuclei with few visible granules and often presents with a high white blood cell count. Flow cytometry shows a characteristic pattern: CD13+, bright CD33, CD117+, but notably CD34-negative and HLA-DR-negative. Treatment with ATRA plus arsenic trioxide (a non-chemotherapy regimen for low/intermediate risk disease) has made this one of the most curable acute leukemias. Diagnosis requires 10% or greater blasts/abnormal promyelocytes, and critically, PML-RARA must be confirmed by FISH or RT-PCR. ATRA should be initiated immediately upon clinical and morphologic suspicion without waiting for karyotype results.

AML with t(9;11)(p21.3;q23.3); MLLT3-KMT2A

This KMT2A (MLL)-rearranged leukemia typically shows acute monocytic or myelomonocytic morphology. It carries an intermediate prognosis, though other KMT2A fusion partners confer variable outcomes. Flow cytometry shows CD33+, CD4+, variable CD14, CD64+, and HLA-DR+ blasts.

AML with NPM1 Mutation

NPM1 is the most commonly mutated gene in AML, present in approximately 30% of cases. Morphologically, it often shows monocytic differentiation with characteristic cup-like nuclear invaginations in blasts. Flow cytometry typically shows CD34-negative blasts with aberrant cytoplasmic NPM1 staining. When NPM1 mutation is isolated (without concurrent FLT3-ITD), prognosis is favorable. This entity can be diagnosed at 10% or greater blasts.

AML with Biallelic CEBPA Mutation

The WHO 5th edition requires at minimum an in-frame bZIP domain mutation. This entity carries a favorable prognosis and often presents with a normal karyotype.

AML with FLT3-ITD

The internal tandem duplication of FLT3 is present in approximately 25% of AML cases and is associated with leukocytosis and normal karyotype. It confers adverse prognosis, though this is mitigated when concurrent NPM1 mutation is present. FLT3-ITD is targetable with midostaurin (added to induction chemotherapy) and gilteritinib (in relapsed/refractory disease).

AML with Mutated TP53

TP53-mutated AML often has a complex karyotype and may be therapy-related. It carries adverse prognosis with poor response to standard chemotherapy and can be diagnosed at 10% or greater blasts per the WHO 5th edition.

AML Defined by Differentiation

When no defining genetic abnormality is identified, AML is classified by its pattern of differentiation: AML with minimal differentiation, AML without maturation, AML with maturation, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroid leukemia (requiring 80% or greater erythroid precursors with 30% or greater proerythroblasts), and acute megakaryoblastic leukemia. These categories are used only after genetic workup is negative for defining abnormalities.

Therapy-Related Myeloid Neoplasms

Therapy-related myeloid neoplasms arise after prior chemotherapy or radiation. Alkylating agent and radiation-related cases have a 5-7 year latency and often present as MDS with del(5q), del(7q), or complex karyotype. Topoisomerase II inhibitor-related cases have a shorter 1-3 year latency, present with balanced translocations involving KMT2A or RUNX1, and often manifest as overt AML rather than MDS. Both carry generally adverse prognosis.

Acute Lymphoblastic Leukemia/Lymphoma (ALL)

B-Lymphoblastic Leukemia/Lymphoma (B-ALL)

Immunophenotype

B-ALL blasts express CD19, CD10 (in most cases), CD22, TdT, and variable CD34. Cytoplasmic CD79a is lineage-defining for B-cell origin. Surface immunoglobulin is usually absent, reflecting arrest at the pre-B stage of development.

Genetic Subtypes

B-ALL with t(9;22)(BCR-ABL1, Philadelphia-positive) is the most common subtype in adult ALL. It responds to tyrosine kinase inhibitors (imatinib, dasatinib) and historically carried adverse prognosis, now substantially improved with TKI-containing regimens. B-ALL with KMT2A rearrangement is common in infants, carries poor prognosis, and shows a CD10-negative pro-B immunophenotype. B-ALL with hyperdiploidy (greater than 50 chromosomes) is the most common childhood ALL and carries favorable prognosis. B-ALL with hypodiploidy (fewer than 44 chromosomes) carries adverse prognosis, particularly the near-haploid and low-hypodiploid subtypes. B-ALL with t(12;21) (ETV6-RUNX1) is the most common translocation in childhood ALL and has favorable prognosis. B-ALL with t(1;19) (TCF3-PBX1) has intermediate prognosis. B-ALL with iAMP21 (intrachromosomal amplification of chromosome 21) is adverse in children. Ph-like ALL shows a gene expression profile resembling Philadelphia-positive ALL but without BCR-ABL1; it harbors targetable kinase fusions in ABL-class, JAK-STAT, and CRLF2 pathways and carries high-risk prognosis.

MRD Assessment

Minimal residual disease assessment by flow cytometry (at 0.01% sensitivity) or PCR-based detection of immunoglobulin/T-cell receptor gene rearrangements is the single most powerful prognostic tool in ALL. MRD positivity at end-of-induction or consolidation is the strongest prognostic factor and guides decisions about therapy intensification and allogeneic transplant.

T-Lymphoblastic Leukemia/Lymphoma (T-ALL)

Immunophenotype

T-ALL blasts express cytoplasmic CD3 (lineage-defining), CD7, TdT, and variable CD1a (reflecting cortical thymocyte stage). CD4/CD8 double-positive expression characterizes cortical-type T-ALL, while some cases show single-positive CD4 or CD8. CD34 is variable.

Genetic Subtypes

Early T-cell precursor (ETP) ALL is characterized by CD1a-negativity, CD8-negativity, and weak CD5, with expression of myeloid and stem cell markers (CD34, CD117, CD13/33). It carries adverse prognosis. NOTCH1/FBXW7 mutations are present in more than 50% of T-ALL and confer favorable prognosis. TAL1, LMO1/2, TLX1/3, and HOXA rearrangements define distinct molecular subgroups with varying clinical behavior.

Mixed Phenotype Acute Leukemia (MPAL)

Definition

MPAL is diagnosed when leukemic blasts express markers of more than one lineage meeting WHO criteria for lineage assignment. Myeloid lineage is established by MPO expression (by flow cytometry or cytochemistry) or by monocytic differentiation demonstrated by at least 2 monocytic markers (NSE, CD14, CD11c, CD64, lysozyme). B-lineage is established by strong CD19 with at least one of CD79a, cytoplasmic CD22, or CD10, or by weak CD19 with at least 2 of those markers. T-lineage is most definitively established by cytoplasmic CD3, with surface CD3 as an alternative. MPAL subtypes include B/myeloid and T/myeloid; B/T is extremely rare. Before diagnosing MPAL, known entities such as CML in blast crisis and ALL with aberrant myeloid marker expression must be excluded.

Immunophenotyping by Flow Cytometry

Lineage Assignment Panel

Flow cytometry assigns lineage using key markers: MPO, CD13, CD33, and CD117 for myeloid; CD19, CD22, CD79a, and CD10 for B-lymphoid; cytoplasmic CD3 and CD7 for T-lymphoid; and TdT as a marker of immaturity present in both lymphoid and sometimes myeloid blasts.

Prognostic and Therapeutic Markers

Several markers identified by flow cytometry or molecular testing now serve as therapeutic targets. CD20 is targeted by rituximab in Philadelphia-positive B-ALL. CD33 is targeted by gemtuzumab ozogamicin in AML. CD123 is targeted by tagraxofusp in blastic plasmacytoid dendritic cell neoplasm and is being explored in AML. FLT3 is targeted by midostaurin and gilteritinib in FLT3-mutated AML. IDH1 and IDH2 are targeted by ivosidenib and enasidenib, respectively, in IDH-mutated AML.

<image>A medical illustration showing the morphologic features of key acute leukemia subtypes on bone marrow aspirate smears (Wright-Giemsa stain). Panel A: AML with t(8;21) showing large blasts with abundant cytoplasm, prominent Auer rods (arrow), and salmon-colored granules with a perinuclear hof. Panel B: APL (hypergranular variant) showing promyelocytes packed with dense azurophilic granules, bilobed nuclei, and a cell containing bundles of Auer rods (faggot cell, arrow). Panel C: B-lymphoblastic leukemia showing uniform medium-sized blasts with high N:C ratio, fine dispersed chromatin, inconspicuous nucleoli, and scant agranular basophilic cytoplasm. Panel D: Acute monocytic leukemia showing monoblasts and promonocytes with folded/lobulated nuclei, fine lacy chromatin, and abundant gray-blue cytoplasm with fine azurophilic granules.</image>

<image>A diagnostic algorithm flowchart for the workup of acute leukemia. Starting from peripheral blood or bone marrow with increased blasts, branching into morphology assessment (Auer rods suggesting myeloid, lymphoblast morphology suggesting lymphoid), then flow cytometry for lineage assignment (myeloid vs. B-lymphoid vs. T-lymphoid vs. MPAL), followed by cytogenetics and molecular testing to define specific genetic subtypes. The algorithm highlights the emergency pathway for suspected APL (immediate FISH for PML-RARA and ATRA initiation) and shows how blast percentage thresholds differ between WHO 5th edition and ICC for AML with defining genetic abnormalities.</image>

Clinical Pearls

APL is a medical emergency because DIC-related hemorrhage is the leading cause of early death. ATRA must be initiated immediately upon clinical and morphologic suspicion, before molecular confirmation arrives. The microgranular variant of APL can be morphologically deceptive with few visible granules and bilobed nuclei resembling monocytes; a markedly elevated WBC and the characteristic flow pattern (CD34-negative, HLA-DR-negative, bright CD33) should trigger urgent FISH for PML-RARA. The CD34-negative, HLA-DR-negative blast immunophenotype is classic for APL, and this entity must be excluded whenever this pattern is encountered. FLT3-ITD testing should be performed rapidly in AML because it affects both prognosis and the decision to add midostaurin to induction chemotherapy. NPM1 mutation with absent FLT3-ITD confers favorable risk, while NPM1 wild-type with FLT3-ITD confers adverse risk -- the combination of these two markers matters more than either alone. Ph-like ALL is increasingly recognized as a high-risk subtype with potentially targetable kinase fusions, and gene expression profiling or fusion panel testing should be considered in all B-ALL. MRD assessment is the single most powerful prognostic tool in ALL, with flow cytometric MRD at end-of-induction guiding risk stratification and transplant decisions.

References

• Khoury JD, et al. The 5th edition of the WHO Classification of Haematolymphoid Tumours: Myeloid and Histiocytic/Dendritic Neoplasms. Leukemia. 2022;36(7):1703-1719.
• Arber DA, et al. International Consensus Classification of Myeloid Neoplasms and Acute Leukemias. Blood. 2022;140(11):1200-1228.
• Dohner H, et al. Diagnosis and management of AML in adults: 2022 ELN recommendations. Blood. 2022;140(12):1345-1377.
• Alaggio R, et al. The 5th edition of the WHO Classification of Haematolymphoid Tumours: Lymphoid Neoplasms. Leukemia. 2022;36(7):1720-1748.
• Bene MC, et al. Proposals for the immunological classification of acute leukemias (EGIL). Leukemia. 1995;9(10):1783-1786.

Read this lecture as Markdown