Leukemias

Etiology of Leukemias

General outlines

Acute leukemias Single cell mutation with “freezing” farther cell’s differentiation and maturation in early stages of development (e.g. stem cell)

Chronic leukemiasThe abnormal mutated (or transformed) cells will retain some capabilities to maturate (and differentiate) beyond the early cells (blasts) BUT they are all abnormal and useless malignant cells.

A systematic approach to diagnose different hematologic neoplasms:

1) CBC (Complete Blood Count), CBP (Complete Blood Picture)a) WBC total, differential count, left shift?, b) Peripheral blood film: evaluate the cellular constituents and search for abnormalities

(abnormal cells?).

2) Special staining (SBB, MPO, NSE, etc).

3) Bone marrow aspiration: • assess the cellularity• M:E ratio• percentage of blast cells• The maturation and differentiation of various cell lineage (Lymphoid, Myeloid).

4) Bone marrow biopsy (trephine biopsy):► in solid tumors (as lymphomas with invasion to Bone marrow) ► in cases that Bone marrow aspiration not possible (AML-M7 “due to fibrosis”, Hairy cell

Leukemias, etc)► in cases with a compact bone marrow “very high cellular proliferation and a dry tap”

5) Immunophenotyping: Use of cell surface, cytoplasmic, or nuclear markers to define cell’s character and origin

(phenotyping) BYFlow cytometry and Immunohistochemistry studies.

6) Genetics studies► Cytogenetics findings► Molecular genetics studies

Leukemia classification

►1976 French, American and British hematologists (FAB group) proposed a classification system (FAB classification): a) Morphologic assessmentb) Special staining techniques (if required)c) Limited use of Monoclonal antibodies (in selected cases)

► 2001WHO classification of Hematopoietic and Lymphoid malignancies.i) Adopting the FAB morphologic classification.ii) Limited use of special staining techniques.iii) Wider use of immunophenotyping (cell markers) studies (FC, IHC)iv) Cytogentics and Molecular genetics studies used heavily to define v) The character and prognosis of various disease entities.

► 2008An Updated WHO classification has been edited.

Differences between FAB and WHO

FAB-classification:1) Heavily used “Morphologic Findings”2) Special staining (SBB, MPO, NSE, etc), if required

WHO-classification:1) Morphologic findings 2) Special staining (decreased role)3) Immunophenotyping (in the form of FC and IHC) heavily used.4) Cytogentics and Molecular genetics studies frequently used.

Acute leukemias Chronic leukemias

Myeloid Lymphoid

L1L2L3

M0M1M2M3M4M5M6M7

Leukemias

The original FAB-Classification system of Acute Leukemias ,heavily based on morphologic findings

L1 L3L2

Myeloblast type IIMyeloblast type I

Types of blast cells in ALL and AML

Special staining methods in acute leukemias

FAB-classification of ALL

L1-lymphoblasts in the BM ALL-L1 L2-lymphobalsts in the BM ALL-L2

ALL-L3 L3-lymphoblasts in the BM

SBB (SHOULD BE –ve)MPO (SHOULD BE –ve)

FAB-classification of AML

M0 Undifferentiated blast cells (Myeloid origin by immunophenotyping), Special staining SBB-ve, MPO-ve

M1 Myeloblasts (≥ 20% in BM) SBB+ve, MPO+ve M2 Myeloblasts (≥20% in BM) SBB+ve, MPO+ve M3 Abnormal Promyelocytes SBB+ve, MPO+ve

M4 Myeloblasts (20-80%) + Monocytes (>20%), NSE +ve M5 (a): Monoblasts ≥80% NSE +ve (b): Monoblasts <80%, more promonocytes and monocytes in BM,NSE +ve

M6 Erythroleukemia, 50% of BM cells are of erythroid lineageM7 Blast cells are ≥20% of BM cells (they are identifiable as Megakaryoblasts).

2 important Practical points:

BM aspiration results in acute leukemia, although may be: i) dry tap or ii) difficult to aspirate (hypercellular marrow with excessive marrow blasts)

BUT is never ever an empty marrow.

Leukemoid reaction should be distinguished from Leukemias “of particular CML”

a) Neutrophil Alkaline Phosphatase Scoreb) C-reactive protein, ESR, and other inflammatory indicators.c) Blood film finding (left shift).d) Bone marrow aspiration, Bone marrow biopsy, immunophenotyping

and cytogentics studies may required accordingly.

The Current WHO classification has widely used the immunophenotyping to characterize leukemias and lymphomas

Frequently used Immunophenotyping & Genetics studies

The Panel of Antibodies recommended by the British Committee for Standards in Haematology (BCSH) for the Diagnosis and classification of acute leukemia are as follows:

B-Lymphoid markers:

CD19CD22 (cytoplasmic)CD79a (cytoplasmic)CD10Cytoplasmic µSurface membrane IgCD138

T-Lymphoid markers:

CD3 (cytoplasmic)CD2CD7

Myeloid markers:

CD13CD117anti-MPO (cytoplasmic)CD33CD41CD42CD61anti-Glycophorin A

Non-lineage restricted:

TdTCD45HLA-DR

Immunological subtypes of Acute Leukemia

►B-ALL (B-lineage markers are positive), ≈75% of ALL1) pro B-ALL 2) common ALL (CD19+/CD10+/TdT+/CD34+/-)…THE MOST COMMON3) B-ALL (the most mature) (CD34-ve/cytoplasmic and surface Ig positive)

►T-ALL (T-lineage markers are positive), ≈15% of ALL

►AML (Myeloid lineage markers (CD13/CD33, CD117, cyMPO) are positive

►Bi-lineage►Bi-Phenotypic

Cytogenetics/molecular genetics findings in ALL (as example)ONLY FOR YOUR REVIEW

ALL-L1

Small and homogenous blasts. These may closely resemble lymphocytes but are distinguished by their finer chromatin structure and the occasional presence of nucleoli

ALL-L2Lymphoblasts of varying size (small and large)

Large blast cells with marked cytoplasmic budding (blebing).The differential diagnosis will be: AML-M7 and ALL.

Farther cytochemical and immunophenotyping studies showed to be case of B-lineage ALL.

Mature B-ALL with prominent cytoplasmic vacuoles

Auer rods in AML (pathogmonomic for myeloid lineage origin),A case of AML-M3

Auer rods in malignant promyelocytes

Auer rods in an M1/M2 AML

Thank You