Acute leukemia; imtiaz

Introduction to Acute Leukemias

Dr Imtiaz Ahmed

Introduction to leukemia• Leukemia is a malignant disease characterized by

unregulated proliferation of immature cells in the bone marrow– It may involve any of the cell lines or a stem cell common to

several cell lines

– Leukemias are classified into 2 major groups:• Chronic: Onset is insidious, the disease is usually less

aggressive, and the cells involved are usually more mature cells

• Acute: Onset is usually rapid, the disease is very aggressive, and the cells involved are usually poorly differentiated with many blasts

• Both acute and chronic leukemias are further classified according to the prominent cell line involved in the expansion:

– If the prominent cell line is of the myeloid series it is a myeloid leukemia

– If the prominent cell line is of the lymphoid series it is a lymphocytic leukemia

– Four basic types of leukemia:» Acute myeloid leukemia – AML» Acute lymphoblastic leukemia – ALL» Chronic myeloid leukemia – CML» Chronic lymphocytic leukemia – CLL

Acute Leukemias

Introduction

• Malignant clonal disorder

• Hematopoietic stem cells

• Maturation arrest with excessive proliferation of immature blasts in bone marrow

• Rapidly progressive fatal course

Etiology

• Inherited Factors:Down syndromeBloom’s syndromeFanconi anemiaAtaxia talangiectasiaWiskott-Aldrich syndromeNF-1

Etiology

• Acquired factors:Ionising radiationChemical agents- benzene, alkylating agentsViruses- HTLV-1, EBVAcquired conditions- MPDs, PNH, MDS, Aplastic

anemiaAge- Children – ALL

Epidemiology

• Chronic myeloid leukemia: 40%• Acute lymphoblastic leukemia: 35%• Acute myeloid leukemia: 15%• Chronic lymphocytic leukemia: 10%

Pathogenesis

• Clonal disorder• Idiopathic• Mutations

Activation of a proto-oncogeneFormation of a chimeric transcription factorActivation of tyrosine kinase pathwayInactivation of tumor suppressor gene

• Chromosomal abnormalityHyperdiploidyHypodiploidy

Genetic abnormalities in Acute Leukemias

Classification of Acute Leukemias

1. Acute lymphoblastic leukemia

2. Acute myeloid leukemia

Acute leukemia1. FAB classification: morphology and cytochemistry

2. WHO Classification: cytogenetics

3. European Group for the immunological classification of leukemias (EGIL): immunophenotyping

FAB Classification: AML

M0: Acute myeloblastic leukemia with minimal differentiation

• Most common in adult patients• 5% of AML• Leukocytosis in 40% and > 50% with leukocytopenia• Diagnosis

Less than 3% of the blasts are positive for peroxidase or the Sudan black B reaction

Blasts are positive for the myeloid-associated markers CD13, CD14, CD15 or CD33, CD34 and negative for B or T lineage marker (CD3, CD10, CD19 and CD5)

Almost no mature myeloid cells are seen

• Morphology: The blasts are small to

medium-sized round cells with an eccentric nucleus

The nucleus has a flattened shape and sometimes lobulated or cleaved and contain fine chromatin with several distinct nucleoli

The cytoplasm is lightly basophilic without granules

Auer rods are not found

M1: Acute myeloblastic leukemia without maturation

• Highest incidence seen in adult and in infants less than a year old

• 10% AML cases• Predominant cell in the peripheral blood is usually a

poorly differentiated myeloblast with fine reticular chromatin and prominent nucleoli

• Auer rods are found in 50% of blasts• If no evidence of granules or Auer rods is present, the

blasts resemble L2 lymphoblast

• The myeloperoxidase or Sudan black B stains are positive in more than 3% of the blasts indicating granulocytic differentiation

• PAS and NSE are negative• CD13, 14, 15, 33 and CD34

myeloid antigens are frequently positive

• The most common cytogenetic abnormalities are: t (9; 22) (q34; q11)

M2: Acute myeloblastic leukemia with maturation

• Presenting symptoms for M2 AML are similar to those of the M1 type

• 30% to 45% of cases of AML• Blasts show azurophilic granules and

Auer rods• Evidence of maturation is present,

with >10% of the marrow cells being promyelocytes, myelocytes,and mature neutrophils and <20% being monocytes

• Pseudopelger–Huet and hypogranular neutrophils

• Basophils, eosinophils, and mast cells may be increased

M3: Acute promyelocytic leukemia (APML)

• Younger adults• Median survival is about 18 months • Fusion of a truncated retinoic acid receptor alpha (RAR-alpha)

gene on chromosome 17 to a transcription unit called PML (for promyelocytic leukemia) on chromosome 15

• The blasts are large with abundant cytoplasm, completely occupied by closely packed large granules, staining bright pink, red or purple

• The nucleus is often bilobed or markedly indented with a prominent nucleolus

• Cells containing bundles of Auer rods "faggots" randomly distributed in the cytoplasm are characteristic, but are not present in all cases

• Cytochemistry: MPO and Sudan black B are strong

positive PAS is negative and NSE is weak

positive• IHC: positive for CD13, CD15, CD1

and CD33 myeloid antigens• Cytogenetic studies: 50% cases

show translocation t(15; 17)

M4: Acute myelomonocytic leukemia (AMML)

• 15% to 25%• Usually in the elderly • Sometimes in patients who have had preceding chronic

myelomonocytic leukemia• Both neutrophilic and monocytic cells and their precursors are

present, each constituting at least 20% of the marrow cells• Monocytosis (≥5×10⁹/l)• Serum and urine levels of muramidase (lysozyme) are usually

elevated because of the monocytic proliferation• Positive reactions for Sudan black B, MPO and both specific and

non-specific esterase• Positivity for CD13, CD33, CD11b and CD14• inv(16) (p13; q22) and del (16)(q22)

• Morphology: Monoblasts: large cells with

round nuclei, one or more prominent nucleoli and abundant basophilic cytoplasm, sometimes with fine azurophilic granules, vacuoles, and pseudopod formation

Promonocytes: less basophilic and more granulated cytoplasm, occasional vacuoles and azurophilic granules. The nuclei are irregular and indented

M5: Acute monoblastic leukemia (AMoL)

• M5a: (Acute monoblastic Leukemia) Granulocyte <20% and monocytes >80%; >80% monoblasts Children

• M5b: (Acute monocytic leukemia) Granulocyte <20% and monocytes >80%; <80%

monoblasts; all developmental stages of monocytes seen Adults

• Serum and urinary muramidase levels are often extremely high• Cytochemistry: NSE positive and PAS is negative• IHC: positivity with CD11b and CD14

M5a

M5b

M6: Acute erythroid leukemias• Erythroleukemia: 5% of AML cases

Both erythroid and myeloid cells At least 50% of the nucleated cells in the bone marrow are erythroid

and at least 20% of the non-erythroid cells are myeloblasts Erythroid cells are dysplastic, megaloblastoid nuclei, the cytoplasm

often possessing poorly delineated, coalescing vacuoles Myeloblasts are similar to those in AML with and without maturation

• Pure erythroid leukemia: very rare >80% of the marrow cells are erythroid Erythroblasts have deeply basophilic, often agranular, cytoplasm may

contain poorly delineated vacuoles. The nuclei have fine chromatin and one or more nucleoli

• Howell-Jolly bodies, ring sideroblast, megaloblastoid and dyserythropoietic changes

• Coarse positivity of PAS

• IHC: glycophorin A

M7: Acute megakaryoblastic leukemia (AMkL)

• Rare • 5% of AML• At least 50% of the blasts are from the megakaryocytic

lineage.• The megakaryoblasts are often pleomorphic and have a

basophilic, agranular cytoplasm that may demonstrate pseudopod and bleb formation, indicating budding platelets

• Dysplastic platelets may be visible in the blood• Circulating micromegakaryocytes and megakaryocyte

fragments

Antigen expression in AML by FAB classification categories

FAB classification of ALL

L1 L2 L3

Cell size Small Large, often heterogeneous

Large, homogeneous

Amount of cytoplasm Scant Moderately abundant

Moderately abundant

Nucleoli Inconspicuous Prominent Present, may be prominent

Cytoplasmic vacuoles Variable Variable Prominent

ALL-L1• Common in childhood, with 74%

of these cases occurring in children 15 years of age or younger

• Morphology: One population of cells, small cells with regular nuclear borders with occasional nuclear clefts. Cytoplasm is moderately basophilic.

• Most common: 70%

ALL-L2• Age : 15 years and above• Morphology: Large cells with

an irregular nuclear shape, cleft in the nucleus are common, with one or more large nucleoli with nuclear membrane irregularities, hence confused with blasts of AML

• 27% cases of ALL

ALL-L3• Burkitt's lymphoma type• Morphology: Cells are large and

homogenous in size, nuclear shape is round or oval with one to three prominent nucleoli and sometimes upto 5 nucleoli are visible, cytoplasm is deeply basophilic with prominent vacuoles. high mitotic index is characteristic with presence of varying degrees of macrophage activity

• Mature B-lymphoid markers

WHO Classification: 2008

Acute myeloid leukemia

1. AML with recurrent genetic abnormalitiesa. AML with t(8;21)(q22;q22); RUNX1-RUNX1T1b. AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22);

CBFB-MYH11c. Acute promyelocytic leukemia with t(15;17)(q22;q12);

PML-RARAd. AML with t(9 ;11)(p22;q23); MLLT3-MLLe. AML with t(6;9)(p23;q34); DEK-NUP214f. AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2);g. Acute myeloid leukemia (megakaryoblastic) with t(1;22)

(p13;q13); RBMI5-MKL1 h. AML with mutated NPM1i. AML with mutated CEBPA

2. AML with dysplasia related changes3. AML with myelodysplastic syndromes, therapy

related4. AML not otherwise specified5. Myeloid sarcoma6. Myeloid proliferations related to Down syndrome

a. Transient abnormal myelopoiesisb. Myeloid leukemia associated with Down syndrome

7. Blastic plasmacytoid denderitic cell neoplasm

AML with recurrent genetic abnormalities

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

• 10% cases with AML M2• Younger patients• Myeloid sarcomas; BM studies may

be misleading• Large blasts with abundant basophilic

cytoplasm, often containing numerous azurophilic granules and perinuclear clearing or bots• Pseudo-Chediak-Higashi granules• Auer rods are frequently found• Abnormal nuclear segmentation

(pseudo-Pelger-Huet nuclei)• Immunophenotype: CD34, HLA-DR,

MPO positive• Good prognosis

AML with recurrent genetic abnormalities 2. AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22);

CBFB-MYH11

• 5-8% cases, found in younger patients• Monocytic and granulocytic

differentiation• Shows eosinophils in the BM, mainly

evident at the promyelocyte and myelocyte stages• Granules are often larger , purple-violet

and may obscure the cell cytoplasm• NSE is faintly positive• MPO is positive• IPT: CD34,CD 117, CD 13, (granulocytic-

CD33, CD15, MPO); (monocytic-CD14, CD4, CD11b, CD64, CD36, lysozyme)• Excellent prognosis in younger patients

AML with recurrent genetic abnormalities 3. Acute promyelocytic leukemia with t(15;17)(q22;q12);

PML-RARA

• Acute promyelocytic leukemia • 5-8% cases; adults in mid-life• Both hypergranular and hypogranular

variants exists• DIC• Abnormal large promyelocytes, bilobed,

densely packed granules• Auer rods present• MPO positive• IPT: CD33 strongly positive: HLA-DR, CD34-

weak expression or absent. Granulocyte diff markers(CD15. CD65) are negative

AML with recurrent genetic abnormalities 4. AML with t(9 ;11)(p22;q23); MLLT3-MLL

• Usually associated with monocytic features• More common in children (9-12%)• Monoblasts are large cells . with

abundant cytoplasm which can be moderately to intensely basophilic and may show pseudopod formation• Fine azurophilic granules• MPO negative• NSE positive• IPT: strong expression of CD33, CD65,

CD4, HLA-DR

AML with recurrent genetic abnormalities 5. AML with t(6;9)(p23;q34); DEK-NUP214

• AML with or without monocytic features that is often associated with basophilia and multilineage dysplasia• 0.7-1.8% cases; both children and adults

affected• Usually presents with anemia and

thrombocytopenia• AML with maturation and AMML (FAB)• Auer rods in 30% cases• MPO positive and NSE negative• IPT: MPO, CD13, CD33, CD38, HLA-DR

AML with recurrent genetic abnormalities 6. AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2)

• May arise from a previous MDS• Associated with normal or elevated PB

platelet counts and has increased atypical BM megakaryocytes with mono- or bi-lobed nuclei• Multi-lineage dysplasia can be seen• Rare, 1-2 % cases, adult predilection• Subset of case have < 20% blasts at the

time of diagnosis• Morphology and cytochemistry of the

blasts matches with: M4 and M7 variant of FAB classification• IPT: CD13. CD33. HLA-DR. CD34 and

CD38, Megs marker CD41 and CD61.

AML with recurrent genetic abnormalities 7. Acute myeloid leukemia (megakaryoblastic) with t(1;22)

(p13;q13); RBMI5-MKL1• Rare < 1% cases,• Commonly occurs in infants without

Down syndrome; female preponderance• Marked organomegaly, especially

hepatosplenomegaly• M7 variant of FAB• The megakaryoblasts are usually of

medium to large size (12- 18 µ) with a round, slightly irregular or indented nucleus with fine reticular chromatin and one to three nucleoli. Cytoplasm is basophilic, agranular, blebs • IPT: CD41, CD61• Poor prognosis

AML with recurrent genetic abnormalities

8. AML with mutated NPM1

•Most common recurring genetic lesion in AML, 2-8 % of childhood AML, 45-64% of adult AML• No previous h/o MDS or MPN• Strong association with AMML or M4 (80-90%)• IPT: CD13, CD33, MPO; CD14, CD11b; NPM1

Prognosis of AML related to cytogenetics

WHO Classification: 2008

Acute Lymphoid leukemia

Acute lymphoblastic leukemiaWHO Classification: 2008

B lymphoblastic leukemia• B lymphoblastic leukemia(NOS)

• Disease of the children (<6 years of age)• Small to medium-sized blast cells

with scant cytoplasm with finely dispersed chromatin and inconspicuous nucleoli• Pseudopods may be present (Hand

mirror cells)• IPT: CD19, CD79a, CD22

B lymphoblastic leukemia• B ALL with recurrent genetic abnormalities

1. B lymphoblastic leukemia/lymphoma with t(9;22)(q34;q 112); BCR·ABL1 translocation

•More common in adults (25%)• Philadelphia chromosome• No unique morphological or

cytochemical characteristics• IPT: CD10, CD19, TdT, CD25;

myeloid markers like CD13, CD33 may be expressed


2. B Lymphoblastic leukemia/lymphoma with t(v;11q23); MLL rearranged

• Translocation between the MLL gene at band 11q23 and anyone of a large number of different fusion partners• Deletion at 11q23 do not qualify• In utero defect; disease of young infants• Very high TLC, (>100,000/cumm); frequent CNS involvement• No unique morphological identification features

• IPT: pro-B markers, CD19, CD15, chondroitin sulfate proteoglycan neuroglial antigen 2 (NG2)• Poor prognosis


3. B Lymphoblastic leukaemia/lymphoma with t(12;21) (p13;q22); TEL-AML1 (ETV6-RUNX1)

• Translocation between the TEL (ETV6) gene on chr12 with the AML 1(RUNX1) gene on chr21• Common in children; 25% cases of B-ALL ; not seen in infants• No unique morphological identification features

• IPT: positive for CD10, CD19, CD34 ; negative for CD9, CD20, CD66; myeloid marker(CD13) frequently expressed• Very favourable prognosis ; cure rates >90% of children


4. B Lymphoblastic leukaemia/lymphoma with hyperdiploidy

• Blasts contain >50 and usually <66 chromosomes without translocations• Common in children; 25% cases of B-ALL ; not seen in

infants• No unique morphological or cytological identification

features• IPT: pro B-cell markers positive; CD34+ and CD45-• Very favourable prognosis ; cure rates >90% of children


5. B Lymphoblastic leukaemia/lymphoma with hypodiploidy (Hypodiploid ALL)

• Blasts contain <46 chromosomes• Rare disease; <5% of ALL cases• Seen in both children and adult: haploid ALL (23-29) seen in

children• C/F similar to other ALL• No unique morphological or cytological identification

features

• IPT: B-precursor phenotype• Poor prognosis


6. B Lymphoblastic leukaemia/lymphoma with t(5;14)(q31;q32); IL3-IGH

• Translocation between the IL3 gene and the IGH gene. resulting in a variable eosinophilia• Rare disease; 1% of ALL cases• Patients may present with an asymptomatic eosinophilia,

and blasts may not be present on PBS• Eosinophils are part of reactive population and are not

neoplastic

• IPT: CD19, CD10+

B lymphoblastic leukemia• B-ALL with recurrent genetic abnormalities

7. B-lymphoblastic leukaemia/lymphoma with t(1;19) (q23;p I3.3); E2A·PBX1 (TCF3-PBX1)

• Translocation between the E2A(TCF3) gene on chromosome 19 and the PBX1 gene on chromosome 1• Common in children• No unique morphological or cytological identification

features

• IPT: CD9, CD19, CD10+ ; cytoplasmic µ(cµ) heavy chain positive pre B-cell phenotype• Good prognosis

T lymphoblastic leukemia• Neoplasm of Iymphoblasts committed to T-cell lineage• 15% of childhood ALL; adolescent > children; males > females• 25% cases of adult ALL• Sites of involvement: Bone marrow, thymus, lymph node, skin,

tonsils, spleen, testis etc• C/F: high leukocyte count, mediastinal mass, hepatosplenomegaly• Morphology: indistinguishable from blasts of B-ALL, medium sized

cells with high N:C , dispersed nuclear chromatin with 1-2 prominent nucleoli.

• In BM bx, mitosis is often numerous• IPT: TdT+,CD99, CD34; CD1a, CD2, CD3, CD4, CD5, CD7 and CD8; • Prognosis is worse than B-ALL

Prognosis of ALL related to cytogenetics

Usual cytology of AML and ALL

AML ALL

Cytochemical profiles in acute leukemia

European Group for the Immunological Characterization of Leukemias (EGIL)

Biphenotypic Acute Leukemia• Single population of blasts coexpressing markers of two

different lineages• Rare • EGIL Scoring system• Biphenotypic acute leukemia is defined when scores are >2

for the myeloid lineage and >1 for the lymphoid lineage• The prognosis of biphenotypic acute leukemia patients is poor

when compared with de novo acute myeloid leukemia or acute lymphoblastic leukemia

• Higher incidence of CD34 antigen expression, complexabnormal karyotype, extramedullary infiltration, relapse, and resistance to therapy after relapse

Guidelines for using the revised WHO classification of myeloid neoplasms

1. Specimen requirements

• PB and BM specimens collected prior to any definitive therapy• PB and cellular BM aspirate smears and/or touch preparations

stained with Wright-Giemsa or similar stain• BM biopsy, at least 1.5 cm in length and at right angles to the

cortical bone• BM specimens for complete cytogenetic analysis or flow

cytometry


2. Assessment of blasts

• Blast percentage in PB and BM is determined by visual inspection

• Myeloblasts, monoblasts, promonocytes, megakaryoblasts (but not dysplastic megakaryocytes) are counted as blasts when summing blast percentage for diagnosis of AML; count abnormal promyelocytes as blast equivalents in APML

• Proerythroblasts are not counted as blasts except in rare instances of pure acute erythroleukemia

• Flow cytometric assessment of CD34+ cells is not recommended as a substitute for visual inspection


3. Assessment of blast lineage

• Multiparameter flow cytometry (at least 3 colors) is recommended; panel should be sufficient to determine lineage as well as aberrant antigen profile of neoplastic population

• Cytochemistry, such as myeloperoxidase or nonspecific esterase, may be helpful, particularly in AML, NOS, but it is not essential in all cases

• IHC on biopsy


4. Assessment of genetic features

• Complete cytogenetic analysis from BM at initial diagnosis when possible

• Additional studies, such as FISH, RT-PCR, mutational status• Mutational studies for mutated NPM1, CEBPA, and FLT3

5. Correlation/reporting of data• All data should be assimilated into one report that states the

WHO diagnosis

Thank You

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Acute leukemia; imtiaz