acute leukemia lm754

Acute Leukemia

Ahmad A. Al-Qudah

Clinical Hematology II LM-754

Acute Leukemia- Introduction to Leukemia and Acute Leukemia- Revision of Special Stains and Cytochemistry related to Acute Leukemia Diagnosis .- Classification of Bone Marrow Cell Types

- AML : - Definition - Sign and Symptoms - Pathophysiology & Lab Findings - Classifications : - FAB - WHO

- ALL : - Definition - sign and symptoms - Pathophysiology & Lab Findings - Classifications : - FAB -WHO

- Is a group of malignant (neoplastic) disorders , characterized by the clonal expansion and accumulation of one or more blood cell line(s) , with eventual involvement of all hematopoietic organs and other organs.

Leukemia

• Prolonged life (immortal) resistant to apoptosis • Growth factor independent growth• Insensitivity to growth-inhibitory signals • Ability to invade and metastasize• Blockage of intracellular differentiation

Tow or more Mutations within the genome of HSC or multipotential progenitors/precursors

Activation of specific proto-oncogene De-activation of tumor suppressor genes

Leukemia

Clone of cells with characteristics of a malignant cell

LeukemiaBased on the clinical and path-physiologic characteristics:

• Acute: - Characterized by the clonal expansion and accumulation of immature leukocytes (blood cells) in hematopoietic organs .

• Chronic:- Clonal expansion and accumulation of mature, but abnormal leukocytes in hematopoietic organs .

Based on the affected blood cell lines: • Myelogenous: Myeloid cell line • Lymphocytic: Lymphoid cell line

Leukemia

Acute Chronic

Myeloid origin

Lymphoid origin

Acute Myeloid Leukemia (AML)

Acute Lymphoblastic Leukemia (ALL)

Chronic Myeloid Leukemia (CML)

Chronic Lymphocytic Leukemia (CLL)

Leukemia• Symptoms reflects B.M. failure to produce normally functioning blood cells :- Features of anemia: Weakness, shortness of breath and pallor .- Features of leukopenia: infections (septicemia, pneumonitis) - Features of thrombocytopenia: Bleeding and increased hemorrhagic

tendency .

• Symptoms reflects B.M. hyperplasia and excessive accumulation, infiltration and proliferation of malignant cells :- Bone and Joint pain - Hepatosplenomegaly - Lymphadenopathy - Gingival hypertrophy (hyperplasia) and oral lesions

Leukemia

Leukemia• CBC and blood film examination : - Normocytic normochromic anemia (mild to severe) - Platelets count: reduced to normal - WBC’s count: decreased mature leukocytes - The appearance of abnormally circulating blood cells

(malignant cells)

• BM aspirate and/or biopsy - B.M. hyperactivation and hyperplasia - Blasts : represents greater than 20 - 30% depends on the type

of Classification and type of Leukemia . - BM Cell Types : ANC , EC , NEC

Leukemia• Cytochemistry:

Blasts Identified Cellular Element Stained Cytochemical Reaction

Myeloblasts strong positive; monoblasts faint positiveLymphoblast Negative

Neutrophil primary granules Myeloperoxidase (MPO)

Myeloblasts strong positive; monoblasts faint positiveLymphoblast Negative

Phospholipids Sudan Black B (SBB)

Promyelocyte stage positive Cellular enzyme Specific esterase

Monoblasts strong positiveOthers Negative Cellular enzyme Nonspecific esterase

(NSE)

lymphoblast's and pronormoblasts Negative to Positive .Myeloblasts usually negative.Metamyelocyte & PMN Strong +ve

Glycogen and related substances Periodic acid-Schiff

Leukemia•Immunological markers (Surface, Cytoplasmic & Nuclear ) - TdT: Terminal Deoxynucleotidyl Transferase , differentiate Myeloblasts

from lymphoblasts (B & T precursors) - CD markers : e.g. AML: CD11/CD13/CD33/CD117 ALL (B-Cells): CD10/CD19/CD20/CD22 ALL (T-Cells): CD2/CD3/CD5/CD7

• Cytogenetics: specific Chromosomal abnormalities - Philadelphia chromosome (t(9;22)): common in CML - t(8;21): AML-M2 - t(1;19), t(8;22), t(11,14) ALL

Acute Myeloid Leukemia- Malignant neoplastic proliferation and accumulation of immature and nonfunctional myeloid line of blood cells in the bone marrow .

- Also known as acute myelogenous leukemia or acute nonlymphocytic leukemia (ANLL)

- Most common Acute Leukemia affecting adults.

- As an Acute Leukemia, AML progresses rapidly and is typically fatal within weeks or months if left untreated .

Hematopoieticstem cell

Neutrophils

Eosinophils

Basophils

Monocytes

Platelets

Red cells

Myeloidprogenitor

Lymphoidprogenitor

B-lymphocytes

T-lymphocytes

Plasmacells

naïve

ALL

AML

Acute Myeloid LeukemiaPathophysiology

- In AML, a single myeloblast genetic changes which "freeze" the cell in its immature state and prevent differentiation.

- In normal hematopoiesis, the myeloblast is an immature precursor of myeloid white blood cells; a normal myeloblast will gradually mature into a mature white blood cell .

- When such a "differentiation arrest" is combined with other mutations which disrupt genes controlling proliferation, the result is an uncontrolled growth of an immature clone of cells, leading to the clinical entity of AML .

Acute Myeloid Leukemia

- The clinical signs and symptoms of AML result from the growth of leukemic clone cells, which tends to displace or interfere with the development of normal blood cells in the bone marrow .

- This leads to neutropenia, anemia, and thrombocytopenia. The symptoms of AML are often due to the low numbers of these normal blood elements.

Pathophysiology

Acute Myeloid Leukemia*Specific Signs and Symptoms :- Most signs and symptoms of AML are general ( Same common signs of all Leukemia ) :- Features related to Pancytopenia . - Features related to infiltration and accumulation . - Some Specific Features : - Chloroma : a solid leukemic mass or tumor developed outside of the bone marrow .


A

B

C

Chloromas

NEJM 1998

Acute Myeloid LeukemiaLaboratory findings:

1- Peripheral Blood:

- The leukocyte count ranges from < 1X109 to >100X109 .

- Presence of blast on the blood smear.

- Neoplastic blast have few granules in the RNA rich cytoplasm. According to the FAB: 1- Type I: typical blast features without granules. 2- Type II: has < 20 granules. 3- Type III: has numerous granules.


- Erythrocyte decreased, Hb less than 10g/dl

- Slightly Macrocytic because of the inability to compete the neoplastic cell for folate and vitB12 or early release of Retic cells.

2- Bone marrow:

- Typically, hypocellular with increased fat content .

Laboratory findings:

- Special Stains and Percentage of Blasts aid in the Diagnosis .

Acute Myeloid LeukemiaClassification :

1- FAB Group Classification , Based on : - The Morphological criteria of PB and BM . - The Immunophenotyping of Leukemic cells . - The cytochemistry of Leukemic Cells . - For Diagnosis, Blast count in PB or BM is >30% .

2- WHO Classification : - Molecular/Genetic Features of Malignancies . - FAB Class. Incorporated into WHO Class. - For Diagnosis, Blast count in PB or BM is >20% .

FAB Classification of AMLAcute Myeloid Leukemia

- AML-M0 (Acute Myeloblastic Leukemia without differentiation ):

• AML with no evident morphological differentiation. ( no characteristic myeloid features )

• Bone marrow contains 30 to 90% blast forms with clear cytoplasm (granules free ).

• The blasts seen in AML and ALL are very similar; with a high nuclear cytoplasmic ratio, immature chromatin, nucleoli, and a variable amount of cytoplasm .

• <3% of blasts are MPO and SBB positive • Blasts are positive for CD13 and CD33 markers

Acute Myeloid LeukemiaFAB Classification of AML

- AML-M1 (Acute Myeloblastic Leukemia, without maturation ):

• Poorly differentiated myeloblasts (more than 90% of NEB)• Blasts are Type I ( Agranular ) and Type II ( Granular ) • MPO & SBB are positive (>3 but < 50%) • CAE is with at least 10-20% positivity • Blasts Express CD13/CD33/CD117

• Auer rods (may present but rarely seen)


- AML-M2 (Acute Myeloblastic Leukemia, with granulocytic maturation ) :

• evidence of maturation up to the Promyelocyte stage (represents more than 10% of blasts) • > 50% of leukemic cells are MPO and SBB positive. • 10-20% positivity for CAE • Blasts Express CD13/CD33/CD117 , Also CD19/CD34• Specific Translocation ( t(8;21) ) . ( less specific t(6;9) )

• Auer rods are occasionally seen


- AML-M3 (Acute Promyelocytic leukemia )

• Promyelocytes with heavy granulation . ( >30% of NEC ) • Auer rods frequently seen (Faggot cells: cells with bundles of AR) • Cells are strongly positive with MPO and SBB • Negative with CAE • t(15;17) is a unique and common feature of AML-M3. • AML-M3 variant: showing the characteristic bilobed hypogranular Promyelocytes . • AML-M3 and DIC ( Thromboplastic Substances )


- AML-M4 (Acute Myelomonocytic leukemia ) :

• Cells are with granulocytic and monocytic differentiation

• 20-80% of cells are positive for MPO, SBB and NSE. • Differential diagnosis: serum lysozyme level > 3 times •16q deletion or inversion are common.

• AML-M4 with Eosinophilia - Eosinophils represents > 5% of nonerythroid cells - immature eosinophils positive for SE and PAS


- AML-M5 (Acute Monoblastic leukemia ):

• > 80% of NEC are monocytic lineage

• > 80% of cells are positive for NSE • Cells are negative for MPO and SBB (weakly positive in monoblast).• del (11q), t(9;11), t(11;19)

• Two subclasses: Poorly (M5a) & well (M5b) differentiated - M5a: Predominance of monoblasts - M5b: Predominance of promonocytes and monocytes


- AML-M6 (Acute Eythroblastic leukemia ) :

• Hypercellularity of B.M. with marked erythroid hyperplasia .• > 50% of ANC are of erythroid lineage .• Blasts commonly show megaloblastoid characterestic features including multi-nucleation, cytoplasm vaculation. • Most erythroid precursors are positive for PAS (Fine staining) • Negative to weakly positive for MPO, SBB and SE

• AML (M6) against MDS : - >50% or <50% Erythroblast of ANC:- With >30% Blast of ANC ---> AML (M6) - With <30% Blast of ANC ---> MDS


AML-M7 (Acute Megakaryoblastic leukemia ) :

• Uncommon form • Extensive proliferation of Megakaryoblasts and ~cyte. • >50% of blasts are megakaryoblasts

• Blasts identified by platelets peroxidase • Negative for MPO and SBB, while positive for PAS. • Blasts Express CD41/CD42• t(1;22) is common .

Acute Myeloid LeukemiaWHO Classification of AML

1- AML with recurrent Cytogenetic abnormalities - usually translocation, in most cases the chromosomal rearrangement create a fusion gene, encoding a novel fusion protein.

I. AML with t(8;21)(q22;q22);(ETO/AML1):- Present morphological as AML with maturation.

- Core binding factor (CBF) also called AML1 is a transcription factor critical for hematopoietic development.- It is believed that HSC is the origin of leukemia


- The t(8;21) translocation result in a chimeric protein containing the N- terminal portion of CBF (chromosome 21)and most of the ETO protein from chromosome 8 ( nuclear

protein involved in the regulation of transcription).

- The fusion protein blocks the normal function of CBF, and induce abnormal gene activation and gene repression, this will lead to increase proliferation with blocked differentiation.


II. AML with abnormal B.M eosinphils inv (16)(p13;q22) or t(16;16)(p13;q22)(CBFB/MYH11):

- Present morphology as AML with monocytic and granulocytic maturation and presence of abnormal eosinphils in B.M.

- Combination of acute myelomonocytic leukemia (AMML) with abnormal eosinphilis is morphologically AMML Eo.

- Abnormal immature ( basophilic ) granules in the eosinphils, promyelocyte, and myelocyte stages.

- The inv (16)(p13;q22) and t(16;16)(p13;q22) both result in the fusion of the CBFB gene (16q22) to the smooth muscle mysoin heavy chain gene SMMHC (MYH11)at (16p13).

- The CBFB/SMMHC fusion proetin binds to AML1/CBFa and represses it is function as transcription factor.


III . AML with t(15;17)(q22;q12)(PML/RARa) and variants:

- it is acute promyelocytic leukemia(APL), an AML in which abnormal promyelocyte predominate.

- Both hypergranular ( typical APL) and hypogranular or microgranular are seen.

- The presenting signs are DIC and bleeding .

- The typical t(15;17) gene rearrangement result in the fusion of (PML/RARa) gene and reciprocal ( RARa/PML) gene.

- (PML/RARa) mRNA has been identified in all APL patient while ( RARa/PML) mRNA in two third.

- The RARa protein is a nuclear hormone receptor that bind to specific DNA sequence and controls transcription.


- PML is growth suppressor nuclear protein normally found in complex macromolecular structure.

- The PML/RARa fusion protein leads to formation of co-repressor complex molecules that enhance the oncogenesis of APL .

- It is believed that HSC is the origin of leukemia


IV. AML with 11q23(MLL) abnormalities:

- These leukemia associated with monocytic features ( monoblasts and promonocyte).- Monoblast and promonocyte can have scattered azurophilic granules and vacuoles.

- The MLL gene (11q13) is involved in a number of leukemia associated translocation with different partner chromosome.

- The MLL protein is a DNA binding protein that interact with other nuclear protein and permits the association of transcription factor which regulate transcription.


2- AML with multilinage Dysplasia ( with or without prior MDS)

- It is an Acute leukemia ( >20% blast) with dysplasia in more than 50% of the cells in two or more myeloid cell lines.- It is occurs with or following MDS / MPD.- examples of dysplasia include: hypogranular PMNs, psuedo-pelger-Huet anomaly, megaloblastic erythrocyte, ringed sideroblast.- The cytogenetics abnormalities are variable and are similar to these in MDS.- HSC is the origin of leukemia- poor prognosis.


3- AML and Myelodysplastic syndrome, therapy releated:

- These disorder arise as a result of cytotoxic chemotherapy and / or radiation therapy.Two major subtypes:I. Alkylating agent/ radiation treatment: initially it start with

MDS and eventually evolving AML.II. Topoisomerase II inhibitor treatment.


4- Acute Myeloid leukemias not otherwise categorized:- Include all AML cases that not fulfill criteria for any of other described.- The subtypes of this AML are classified according to differentiated on morphology and cytochemical features.

I. AML Minimally DifferentiatedII. AML without MaturationIII. AML with MaturationIV. Acute Myelomonocytic leukemia (AMML)V. Acute monoblastic leukemia and Acute monocytic leukemiaVI. Acute Erythroid leukemia (AEL)VII. Acute Megakaryoblastic leukemia


VIII. Acute Basophilic leukemia

- The most characteristic feature by cytochemistry is metachromatic positivity with toluindine blue.

IX. Acute panmyelosis with mylofibrosis

- it is occur with or following chemo and radio therapy.

X. Myeloid Sarcoma

- a tumor of myeloblast or immature myeloid cells occures in the extramedullary site or in the bone.

Acute Lymphoid LeukemiaMalignant neoplastic proliferation and accumulation of immature and nonfunctional Lymphoid line of blood cells in the bone marrow .

The "lymphocytic" in acute lymphocytic leukemia refers to the white blood cells called lymphocytes, which ALL affects. Acute lymphocytic leukemia is also known as acute lymphoblastic leukemia.

Acute lymphocytic leukemia is the most common type of cancer in children

Acute Lymphoid Leukemia*The signs and symptoms of ALL are variable but follow from bone marrow replacement and/or organ infiltration :- Generalized weakness and fatigue- Anemia- Frequent or unexplained fever and infection- Weight loss and/or loss of appetite- Bone pain, joint pain (caused by the spread of "blast" cells to the surface of the bone or into the joint from the marrow cavity)- Breathlessness- Enlarged lymph nodes, liver and/or spleen- Pitting edema (swelling) in the lower limbs and/or abdomen- Petechiae, which are tiny red spots or lines in the skin due to low platelet levels

Acute Lymphoid Leukemia

- Damage to DNA that leads to uncontrolled cellular growth and spread throughout the body.

Pathophysiology

- This damage may be caused by environmental factors such as chemicals, drugs or radiation.

- Some evidence suggests that secondary leukemia can develop in individuals treated for other cancers with radiation and chemotherapy as a result of that treatment

- Damage can be caused through the formation of fusion genes, as well as the dysregulation.

Initial Laboratory finding characteristic of ALL

Peripheral Blood: 1- Leukocyte count usually increased but may be normal or decreased. 2 -Neutropenia

3 -Lymphoblast 4 -Thrombocytopenia

Bone Marrow: 1- Hypercellular 2> -20% lymphoblast ( WHO)

Other laboratory finding: 1- associated with increased cellular metabolism and turn over such: Hyperuricemia.

incrased serum LD. hypercalacemia due to increased BM resorption.

2 -Renal failure.

3 -increased CSF lymphblast.

Acute Lymphoid Leukemia- Terminal Deoxynucleotidyl transferase (TdT): - Lymphocyte can identified by immunophenotying and certain intracellular enzyme.- TdT, is the most important enzyme that is helpful in the identifying cellular subtypes.- TdT is a DNA polymerase found in cell nuclei , this enzyme not present in the normal mature lymphocyte but can be found in 65% of the thymic population of lymphocyte.It can be determined by direct enzyme assay and indirect immunofluorescence.

Acute Lymphoid LeukemiaClassification

- Based on FAB classification, ALL is categorized into three catergories ALL-L1, ALL-L2 and ALL-L3 .

- Immunohistochemistry and immunophenotyping are almost always necessary to distinguish ALL from AML.

- On other hand WHO identify of malignant cell as T, B and on the degree of maturation.

Acute Lymphoid Leukemia

- Blasts in ALL-L1 are with high N/C ratio.- Delicate diffuse chromatin pattern and small prominent nucleoli. - Scant cytoplasm . - Affects primarily children

FAB classificationALL-L1

Acute Lymphoid LeukemiaFAB classification

ALL-L2- The blasts are larger than those of L1, have more plentiful cytoplasm and are more pleomorphic. - Abundant cytoplasm, predominant nucleoli, nuclear clefting .

-Affects adults

Acute Lymphoid LeukemiaFAB classification

ALL-L3 : Burkitt's type- L3 blasts cells are fairly regular in shape with strong basophilic cytoplasm and prominent cytoplasmic vaculation .

Affects adults and children

FAB Classification of ALL

Morphology TdT CD10

ALL-L1 Small lymphocyte scant cytoplasm+ + ; Moderately clumped chromatin;

Inconspicuous nucleoli

ALL-L2 Small and medium size lymphblast+ + ; Mixed chromatin patterns;

Inconspicuous nucleoli

ALL-L3Burkitt- type Large lymphblast; large nucleus with-/+ - nucleoli; cytoplasm vacuolization;

Intense cytoplasm basophilia

Acute Lymphoid LeukemiaWHO classification

- WHO classification considers ALL and lymphblastic lymphoma to be single disease with different clinical presentation.

- Precursor T- and B-cell neoplasm with B.M and peripheral blood involvement are ALL, while precursor T- and B-cell neoplasm presenting solid tumors are lymphoma.


WHO classification defines two subgroups of ALL:

1- Precursor B- and T-cell neoplasm ( leukemia/ lymphoma) which include L1 and L2 . 2- Burkitt type ALL (L3).


1- Precursor B- cell leukemia:

- it is a neoplasm of lymphoblast committed to the B- cell linage, involve B.M, peripheral blood - some cases may present with primary involvement of lymph node and exranodal site ( lymphoma).- Rearrangement of immunoglobulin genes can be detected by molecular testing.- Additional markers of B linage commitment required for dignosis. CD10/CD19/CD20/CD22 , TdT = +ve


1- Precursor B- cell leukemia:

- Cytogenetic abnormalities associated with B-ALL include translocation, hypodiploidy, and hyperdiploidy.

- The most common is t(12;21)(p13;q22)- Produce TEL-AML1 fusion gene.- Hyperdiploid B-ALL have a mutation in the receptor tyrosine Kinase FLT-3 , resulting in constitutive activation of the receptor.- t(1;19) PBX1-E2A , t(4;11) AF4-MLL- Precursor B- cell has good prognosis.


2- Precursor T- cell leukemia:

- It is a neoplasm of lymphoblasts committed to T- cell linage, involving B.M and peripheral blood. - High WBC count, lymphoblast and cytochemistry similar to B-cell, but acid phosphatase shoe intense positivity in T-ALL.

- T- cell linage can be detected by rearrangement of the T cell receptor genes by molecular studies.- There are four TCR genes capable of rearranging, coding for the a ,B, Y and & chain of the TCR.


- Cytogenetic studies show translocation involving alpha and Delta TCR loci (14q11.2), the beta locus (7q35) or gamma locus (7p14-15).

- The translocation involve a variety of partner genes including transcription factors.

- CD2/CD3/CD5/CD7/CD10 = +ve

2- Precursor T- cell leukemia:


3- ALL- Burkitt type :

- Cell Large in size.- Nuclear chromatin : fine and homogeneous- Nuclear shape : Regular, oval to round.- Nucleoli prominent, one or more.- Cytoplasm amount : Moderately Abundant.- Vaculation Often prominent.- This tumor has a high proliferation rate and many mitotic figures may be seen in the B.M smear.


- Burkitt's cell show clonal rearrangement of the immunoglobulin heavy and light chain genes

3- ALL- Burkitt type :

All cases have translocation of the MYC gene (8q14) to the:1. heavy chain region on chromosome 14{t(8;14)} or2. light chain loci on chromosome 2p12{t(2;8)} or3. chromosome 22q11{t(8;22)}.

AML ALL

Common in adult Common in children Age

Anemia, neutropenia, thrompocytopenia,

myeloblast, Promyelocyte .Anemia, neutropenia, thrompocytopenia,

Lymphoblast, Prolymphcytes

Hematologic prsentation

Medium to large myeloblast with distinct nucleoli, fine nuclear chromatin and abundant basophilic

cytoplasm, Auer rod can be present

Small to medium lymphoblast, fine chromatin with scanty to abundant cytoplasm, indistinct

nucleoli

Prominent cell morphology

PAS negative, Peroxidase and SBB are positive,TdT may

positive or negative

PAS and TdT are Positive, Peroxidase and SBB are

negative Cytochemistry

CD2CD3CD5CD7

CD10 CD19CD20CD22

CD13CD33

TdT

- - - + - AML

- + + - + B cell

+ -/+ - - + T cell

Acute LeukemiaPrognosis :

1- AML :

- Prognosis Varies from Poor to Good According to : 1- type of Cytogenetic abnormalities 2- MDS 3- Prognostic markers ( Serum LD ) 4- Expectation of cure

Risk Category Abnormality

Good t(8;21), t(15;17), inv(16)

Intermediate Normal, +8, +21, +22, del(7q), del(9q), Abnormal 11q23, all other structural or numerical changes

Poor -5, -7, del(5q), Abnormal 3q, Complex cytogenetics

Acute LeukemiaPrognosis :

1- ALL :- Prognosis Varies from Poor to Good According to : 1- Type of Cytogenetic abnormalities 2- Diploidy , Hyper or Hypo 3- Metastasis

Cytogenetic change Risk categoryt(4;11)(q21;q23) Poor prognosist(8;14)(q24.1;q32) Poor prognosis

Complex karyotype (more than four abnormalities) Poor prognosis

Low hypodiploidy or near triploidy Poor prognosis

High hyperdiploidy (specifically, trisomy 4, 10, 17) Good prognosis

del(9p) Good prognosis

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acute leukemia lm754

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