01. Overview of AML - Dr Ahmed Makboul PDF

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Dr Ahmed Makboul...

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Overview of Acute Myeloid Leukemia TERMINOLOGY .............................................................................................. 1 ETIOLOGY/PATHOGENESIS ........................................................................ 2 CLINICAL IMPLICATIONS ............................................................................. 5 MACROSCOPIC PATHOLOGY ..................................................................... 8 MICROSCOPIC PATHOLOGY ..................................................................... 10 ANCILLARY TESTS ..................................................................................... 15 REPORTING CRITERIA ............................................................................... 19 DIFFERENTIAL DIAGNOSIS .......................................................................20 DIAGNOSTIC CHALLENGES ...................................................................... 21 CLASSIFICATION OF AML .......................................................................... 23

OVERVIEW OF ACUTE MYELOID LEUKEMIA

OVERVIEW OF ACUTE MYELOID LEUKEMIA TERMINOLOGY: Synonyms: • Acute myelogenous leukemia.

Definition: • Clonal hematopoietic neoplasm. • Blasts/blast equivalents comprise ≥ 20% of peripheral blood (PB) &/or bone marrow (BM) cells. o Exceptions: § AML with recurring genetic abnormality [t(15;17), t(8;21), inv(16)/t(16;16)]. § Pure erythroid leukemia: ü 80% immature erythroid precursors with > 30% proerythroblasts.

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OVERVIEW OF ACUTE MYELOID LEUKEMIA ETIOLOGY/PATHOGENESIS: 1. Germline (Constitutional) Predisposition Disorders: • Down syndrome: o 10-20x increased risk for AML. o 500x increased risk for acute megakaryoblastic leukemia. o Separately classified in WHO classification. • Others: All are rare.

2. Environmental Exposures: • Radiation. • Chemotherapy. • Benzene.

3. Underlying Hematopoietic Neoplasm: • Variable risk of progression to AML. • Myelodysplasia (MDS), myeloproliferative neoplasms (MPNs), MDS/MPN disorders.

Pathogenesis of Leukemogenesis: • Accumulation of multiple genetic hits. • Class I and class II mutations. o Class I: Proproliferative signal. § e.g., FLT3, JAK2, KIT. o Class II: Impairment of cellular maturation. § e.g., PML-RARA, CEBPA, RUNX1-RUNX1T1.

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OVERVIEW OF ACUTE MYELOID LEUKEMIA • Complex series of genetic events: o AML genome shows fewer mutations compared with other cancers o Complex acquired gene mutations in AML: § FLT3 ITD: ü Seen in 20% of AML cases. ü Associated with poor prognosis. ü Not stable over time. § NPM1 mutations: ü Seen in 30% of AML cases. ü More common in karyotypically normal AML (50%). ü Associated with good prognosis if FLT3 wild type. ü Stable over time. § CEBPA mutations: ü Seen in 10% of AML cases. ü Biallelic mutations associated with good prognosis if FLT3 wild type. § RUNX1 mutations: ü Seen in 10% of AML cases. ü Associated with unfavorable prognosis. § IDH1 and IDH2 mutations: ü Seen in 5-10% of AML cases. ü Associated with favorable prognosis if NPM1 mutated and FLT3 wild type. § Others: ü TET2. ü ASXL1.

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OVERVIEW OF ACUTE MYELOID LEUKEMIA Myeloid Neoplasms with Germline Predisposition and Increased Risk of Myeloid Neoplasms: • Although small, subset of AML cases arise from associated underlying germline mutation. • It is important to note germline predisposition in such cases as part of diagnosis. • Detection of such mutations should prompt consideration of testing of related family members. • 3 major categories: o Myeloid neoplasm with germline predisposition without preexisting disorder or organ dysfunction. o Myeloid neoplasm with germline predisposition and preexisting platelet disorders. o Myeloid neoplasm with germline predisposition and other organ dysfunction: § Fanconi anemia. § Dyskeratosis congenita. • Gene mutations that may be familial (germline) in AML: o CEBPA: § Family history and biallelic mutation clue to familial inheritance. § 1% or less of all AML cases. o GATA2. o Others: § SRP72, DDX41, ETV6, TERT, TERC, etc.

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OVERVIEW OF ACUTE MYELOID LEUKEMIA CLINICAL IMPLICATIONS: Epidemiology: • Age Range: o All ages affected; median: 63 years. o Overall, proportion of acute leukemias that are AML increases with age. § 80% of adult acute leukemias are myeloid. o Some disease types are more prevalent in older adults. § e.g., AML with myelodysplasia-related changes. o Some AML types are more prevalent in younger patients. § AML with recurring genetic abnormality [e.g., t(15;17), t(8;21), inv(16)/t(16;16), t(9;11)] § AML with t(1;22) occurs in infants and children < 3 years • Incidence: o Age-adjusted incidence: 4.1 cases per 100,000 individuals per year (based on Surveillance, Epidemiology, and End Results Program data from 2009-2013). o 20,000 new cases in USA each year. o AML comprises 1% of all cancer diagnoses. o Age-adjusted number of deaths: 2.8 per 100,000 individuals per year.

Clinical Presentation: • Symptoms related to bone marrow failure: o Fatigue (anemia). o Bleeding (thrombocytopenia). o Infection (neutropenia). • Extramedullary involvement: o Skin lesions. o Gingival hyperplasia. o Myeloid sarcomas. • Abnormal CBC. 5

OVERVIEW OF ACUTE MYELOID LEUKEMIA Natural History: • Clinically aggressive disease. • Despite advances in classification and treatment, prognosis remains unfavorable for patients > 60 years.

Clinical Risk Factors: • Prognostic factors and risk: o Genetics (ELN risk stratification 2017): § Cytogenetics. § Molecular genetics. o Prior chemotherapy &/or radiation. o Underlying hematopoietic neoplasm, e.g., MDS. o Increasing age (> 60 years). o Elevated lactate dehydrogenase. o Performance status. Acute Myeloid Leukemia Genetic Risk Classification (ELN risk stratification): Risk Status

Cytogenetic

Molecular Abnormalities

Favorable:

o Core binding factor AML: AML with Normal karyotype with: t(8;21), AML with inv(16)/t(16;16). o NPM1 mutation and absence of FLT3-ITD o Acute promyelocytic leukemia with mutation. PML-RARA. o Biallelic CEBPA mutation.

Intermediate:

o AML with t(9;11). o Normal karyotype. o Other non-defined.

Unfavorable:

o Complex (> 3 clonal abnormalities). o AML with inv(3)/t(3;3) (GATA2MECOM). o t(11q23-KMT2A gene) other than t(9;11). o AML with t(6;9) (DEK-NUP214). o AML with t(9;22). o Monosomal karyotype. o Monosomy 5, del(5q). o Monosomy 7, del(7q).

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o Core binding factor AML with KIT mutation. o Mutated NPM1 with FLT3-ITD. Normal karyotype with: o o o o o

FLT3-ITD mutation. TP53 mutation. Mutated RUNX1. Mutated ASXL1. Wild type NPM1 with FLT3-ITD.

OVERVIEW OF ACUTE MYELOID LEUKEMIA Treatment: • Induction therapy for AML [excluding acute promyelocytic leukemia (APL)]: o For those in otherwise good health and < 60 years, intensive anthracycline and cytarabine regimen, "7 + 3," induction therapy remains standard of care o Goal: Morphologic remission § < 5% residual blasts • Induction therapy for APL: o Utilization of tretinoin [a.k.a. all-trans retinoic acid (ATRA)] ± arsenic trioxide o Early use of tretinoin decreases risk of APL-induced coagulopathy, development of disseminated intravascular coagulation (DIC), and mortality • Consolidation therapy: o Goal: Maintain remission prior to transplantation or as part of cure o Assessment of minimal residual disease may play role in predicting durable remission or impending relapse

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OVERVIEW OF ACUTE MYELOID LEUKEMIA MACROSCOPIC PATHOLOGY: General Features: • Myeloid sarcomas: o Soft. o White/yellow. o Fleshy. o Variable foci of necrosis.

Specimen Handling: • Required elements in work-up of AML: o Complete and accurate clinical history. o Prior therapy. o Prior hematologic neoplasm. o PB &/or BM microscopic examination. o Flow cytometry. o Chromosomal study.

• Additional elements to be considered: o Cytochemistry: § Useful with minimal differentiation of blasts by morphology or expression of limited myeloid markers by immunophenotyping. § Useful for rapid diagnosis of APL. o FISH: § When karyotyping is inadequate or when rapid diagnosis is required (e.g., APL). § Possibility of cytogenetically cryptic abnormality. ü e.g., CBFB-MYH11, KMT2A translocation, PML-RARA (rare).

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OVERVIEW OF ACUTE MYELOID LEUKEMIA o Molecular genetics: § Normal karyotype: ü FLT3, CEBPA, NPM1, others. § Abnormal karyotype: ü Varies by institution. ü Evolution based on new discoveries. ü KIT testing for core-binding factor AMLs.

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OVERVIEW OF ACUTE MYELOID LEUKEMIA MICROSCOPIC PATHOLOGY: Blasts and Blast Equivalents: General Features: • Meticulous attention must be paid to recognizing and counting blasts. • Which cells count as blasts/blast equivalents, and what are their cytologic features: o Myeloblasts: § Cytoplasmic azurophilic granules, Auer rods. o Monoblasts: § Cytoplasmic, very fine, azurophilic granules; abundant blue-gray cytoplasm. o Megakaryoblasts: § May see cytoplasmic blebbing/shedding but not specific. o Promonocytes: § Gray-blue cytoplasm, fine chromatin, variably conspicuous nucleoli, delicate nuclear groove/folds. o Promyelocytes (abnormal): § Single or bilobed nuclei, hypo- or hypergranular cytoplasm, may see cytoplasm packed with Auer rods. § Counted in APL only. o Erythroblasts: § Only enumerated when considering pure erythroid leukemia. § Deeply basophilic cytoplasm, circumferential cytoplasmic vacuolization. § 80% erythroid precursors of which > 30% are pronormoblasts.

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OVERVIEW OF ACUTE MYELOID LEUKEMIA Cell type

Key Morphologic Features

Myeloblast

- Nucleus: Dispersed chromatin, variable nucleoli, variable nuclear contours.

Cytochemistry MPO+

Immunophenotyping - CD34+, CD117 usually +. - CD13+, CD33+, MPO+.

- Cytoplasm: Scant to moderate, agranular to sparse granules, variable Auer rods.

- HLA-DR+. - vCD11c+. - wCD45+.

Promyelocyte

- Blast equivalent only in APL. - Nucleus: Variably dispersed chromatin, variable nucleoli, eccentric nucleus; some cases show folded nuclei (sliding plates).

Strong MPO+

- Nucleus: Round, dispersed chromatin, variable nucleoli.

- CD34 negative. - CD13+, CD33+, MPO+. - HLA-DR negative. - wCD45+.

- Cytoplasm: May exhibit paranuclear hof, generous numbers of granules, variable Auer rods.

Monoblast

uniform

o

NSE+

CD34 usually negative in hypergranular variant; often positive in hypogranular variant.

- CD34 negative. - CD13+, CD33 bright +

- Cytoplasm: Abundant, variable sparse fine granules.

- vCD4+, CD11c+ - CD36/CD64 coexpression - wCD45+.

Promonocyte

- Blast equivalent in all myeloid neoplasms.

NSE+

- Nucleus: Lightly folded, dispersed chromatin, nucleoli.

- CD34 negative. - CD13+, CD33 bright + - CD4+, CD14+, CD11c+

- Cytoplasm: Abundant, basophilic, variable, sparse, fine granules.

- CD36/CD64 coexpression - CD45+.

Erythroblast (pronormoblast)

- Blast equivalent only in acute pure erythroid leukemia.

PAS+

- CD117 often +.

- Nucleus: Round, moderately dispersed chromatin, variable nucleoli.

- Glycophorin A+, CD71+ - All myeloid antigens negative.

- Cytoplasm: Moderate, deeply basophilic, often vacuolated.

Megakaryoblast

- Overall size highly variable. - Nucleus: Highly variable chromatin; may be condensed. - Cytoplasm: Highly variable; may show blebbing.

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- CD34 negative

- CD45 negative. N.A.

- CD34 negative - CD33 bright +, CD13 negative - CD41+/CD61+ - HLA-DR dim or negative

OVERVIEW OF ACUTE MYELOID LEUKEMIA • Enumeration of blast percentage: o In PB: § Percentage of circulating white blood cells. o In uncomplicated BM: § Percentage of all nucleated cells, excluding histiocytes, megakaryocytes, mast cells. o In BM complicated by 2nd hematologic neoplasm: § Percentage of all nucleated cells, excluding coexisting tumor cells (e.g., plasma cells in myeloma, lymphoid cells in chronic lymphocytic leukemia).

• Key diagnostic feature: Blast count o Requisite ≥ 20% blasts in PB &/or BM o Exceptions: Cases with t(8;21), inv(16) or PML-RARA do not require 20% blasts (Low blast count/Oligoblastic AML).

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OVERVIEW OF ACUTE MYELOID LEUKEMIA Peripheral Blood: • Abnormal CBC. • Cytopenias: o Anemia. o Thrombocytopenia. o Neutropenia. • Circulating blasts/blast equivalents. • Assess erythrocytes for evidence of DIC.

Bone Marrow Aspirate: • Requirements: o Well stained. o Adequate specimen: § Cellular. § Representative. § If fibrotic or dry tap, assess touch preparation. • Enumerate blasts. • Assess for dysplasia in all lineages: o Diagnostic criterion for AML with MDS-related changes § Cases must lack NPM1, biallelic CEBPA and RUNX1 mutations if diagnosis based solely on dysplasia. • Assess for increased/abnormal-appearing mast cells. o Mastocytosis may be concurrent.

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This case of Acute monoblastic leukemia has effaced the entire BM typical of the behavior of AML in general.

OVERVIEW OF ACUTE MYELOID LEUKEMIA Bone Marrow Core Biopsy: • Requirements: o Adequate specimen. o Thin section. • Identify blasts. o Utilize immunohistochemistry if needed. o Caveats: § Not all blasts are CD34(+), particularly APL, megakaryocytic, monocytic, and erythroid blasts. § Immunohistochemistry not as sensitive as flow cytometry. • Assess megakaryocytic dysplasia. • Evaluate for associated concurrent neoplasm: o Mastocytosis. o Other.

BM core biopsy illustrates features of APL, including abundant pink and heavily granulated cytoplasm. Although blasts may be < 20%, APL presents with complete BM effacement.

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OVERVIEW OF ACUTE MYELOID LEUKEMIA ANCILLARY TESTS: 1. Cytochemical stains • MPO: o If positive, confirms myeloid lineage. o If negative, does not exclude myeloid lineage. o 5% of acute monoblastic leukemias may show scattered MPO(+) granules.

• NSE: o If positive, confirms monocytic lineage. o If negative, does not exclude monocytic lineage.

In cases of suspected APL, particularly the microgranular variant, cytochemical MPO is useful in demonstrating the diffuse and abundant MPO granules.

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This case of suspected acute monoblastic leukemia shows NSE cytochemical positivity (BROWN COLOUR) confirming monoblasts. Any degree of positivity is sufficient for monocytic differentiation.

OVERVIEW OF ACUTE MYELOID LEUKEMIA 2. Flow cytometric immunophenotyping: • Should be performed in all new cases of AML as possible. o Establishes lineage o Establishes phenotype "fingerprint" for future monitoring • Blast markers: o CD34: Not all blasts are CD34(+). o CD117: Also stains pronormoblasts, mast cells. o TdT: Stains subset of AMLs. • Myeloid markers: o MPO, CD13, CD33. • Monocytic markers: o CD14, CD36/CD64 coexpression, CD163, CD4 (weak), CD33 (bright). • Megakaryocytic markers: o CD31, CD41, CD42b, CD61. • Erythroid markers: o Glycophorin A, hemoglobin A, CD71, e-cadherin.

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Immunophenotyping is considered standard of care in cute leukemia to establish lineage. Typical finding in AML, but by no means specific, include coexpression of CD34 and CD117 (ORANGE POPULATION).

Myeloid-associated antigens useful in supporting myeloid lineage in acute leukemia include CD13 and CD33 expression.

OVERVIEW OF ACUTE MYELOID LEUKEMIA 3. Immunohistochemistry: • Useful if flow cytometry inadequate or not performed. • In general, fewer antibodies are available compared with flow cytometry. o Some are unique to IHC, however o CD68: Myeloid and monocytic. o Lysozyme: Monocytic. o CD31: Megakaryocytic lineage.

CD34 IHC may identify increased blasts in a core biopsy; however, not all blasts in AML may be CD34+

Pure erythroid leukemia may be tricky to characterize as they often have a limited and non-specific antigenic expression.

4. Conventional Cytogenetic Studies: • Should be performed in all new cases of AML as possible. o Diagnostic: e.g., AML with recurring genetic abnormality. o Prognostic: Favorable, intermediate, and unfavorable risk groups.

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Karyotyping is required in the present day work-up of AML. A normal karyotype is seen in 40 – 50% of all de novo cases of AML.

OVERVIEW OF ACUTE MYELOID LEUKEMIA 5. Fluorescence in Situ Hybridization (FISH): • Perform as needed. o Depending on morphologic suspicion: § Monocytic differentiation: investigate for CBFB-MYH11 and KMT2A translocations. o Confirmation of cytogenetic findings.

FISH is useful technique to confirm the presence of a suspected genetic abnormality in AML. In this example, FISH for the PML and RARA genes in APL shows the classic fusion partner confirming the diagnosis of APL with PML-RARA.

6. Molecular genetics: • Perform as per your institution, protocol requirements, anticipated minimal residual disease testing. • FLT3 should be assessed in all AMLs. • Karyotypically

normal

AMLs

should

be

may

be

evaluated

for FLT3,

CEBPA,

and NPM1 mutations. • Additional

molecular

mutations

identified

and

(e.g., IDH1, IDH2, TET2, ASXL1). • KIT should be assessed in core binding factor-mutated cases.

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require

testing

OVERVIEW OF ACUTE MYELOID LEUKEMIA REPORTING CRITERIA: Minimum Requirements: • Use WHO classification as possible. • Utilize synoptic reporting as possible. • Follow established clinical practice guidelines. • Ancillary studies: Report results &/or if pending. • Consider issuing integrated report to incorporate all diagnostic and prognostic data.

Communication of Results: • New diagnosis, unsuspected relapse, suspected APL, associated DIC. • Prompt, verbal notification of clinician(s).

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OVERVIEW OF ACUTE MYELOID LEUKEMIA DIFFERENTIAL DIAGNOSIS: 1. Granulocyte Colony-Stimulating Factor: • Blast count may exceed 20% in hypocellular specimen. • Transient phenomenon, non-clonal, no Auer rods.

On occasion, treatment with G-CSF may yield PB or BM features of increased blasts mimicking AML. However, the increased blasts is a transient phenomenon and is non-clonal.

2. Blast Phase of Preexisting Myeloid Neoplasm: • Clinical history required.

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OVERVIEW OF ACUTE MYELOID LEUKEMIA DIAGNOSTIC CHALLENGES: 1. Use of G-CSF as Component of Acute Myeloid Leukemia Chemotherapy (Rarely Done): • Determination of residual disease blast count unreliable.

2. Low Blast Count Acute Myeloid Leukemia (< 20%): • Diagnosis established by detecting recurring genetic abnormality: [t(15;17), t(16;16)/inv16, t(8;21)].

3. Hypocellular Acute Myeloid Leukemia: • Document ≥ 20% blasts by immunohistochemistry.

4. Marked Increase in Erythroid Precursors: • High-grade MDS vs. erythroleukemia. o Pure erythroid leukemia: § 80% erythroid precursors. § 30% of erythroid precursors are pronormoblasts. o MDS with abundant erythroid precursors: § Myeloid blast count determined as percent of all nucleated elements. § MDS often shows admixture of all stages of erythroid differentiation. • Exclude non-neoplastic disorders: o Nutritional deficiency (vitamin B12, folate, copper). o Erythropoietin therapy.

5. Fibrosis: • Often inaspirable. • More accurate blast count may require IHC: CD34, CD117. • Underlying ...