NCSUniversity System

A Project of NCS Education System

Tanveer TaraSenior Lecturer

Bachelor of Science in MLTMasters of Science in Hematology

Acute leukemias

Leukemia:All leukemia's are stem cell/ and or precursor of HSC disorders characterized by malignant neoplastic proliferation and accumulation of immature and nonfunctional haemopoietic cells in the blood and bone marrow.

Conti…

• Leukemia's are cancer found in the blood cells.

• Acute leukemia are usually aggressive disease.

• They are classified by how quickly they progress and what type of cell they affect.

• Leukemia affects ability to produce normal blood cells.

• Bone marrow makes abnormally large number of immature white blood cells called blasts.

Hematopoieticstem cell

Neutrophils

Eosinophils

Basophils

Monocytes

Platelets

Red cells

Myeloidprogenitor

Lymphoidprogenitor

B-lymphocytesB-lymphocytes

T-lymphocytes

Plasmacells

naïve

ALLALL

AMLAML

Leukemia

Acute

Chronic

ALL

AML

CML

CLL

Comparison of acute and chronic leukemia

Features Features Acute Acute Chronic Chronic Age Age All ages All ages Adults Adults Clinical onset Clinical onset Sudden Sudden Insidious Insidious Course of Course of diseasedisease

Week or Week or monthsmonths

Months to Months to yearsyears

Predominant Predominant cell cell

Blast ,some Blast ,some mature formsmature forms

Mature formsMature forms

Anemia Anemia Mild – severeMild – severe Mild Mild Thrombocytopenia Thrombocytopenia Mild – severeMild – severe MildMildWBCWBC Variable Variable Increased Increased

Pathogenesis of acute leukaemia

• Acute leukaemias are usually aggressive diseases in which malignant transformation occurs in the haemopoietic stem cell or early progenitors.

• Genetic damage is believed to involve several key biochemical

a. an increased rate of proliferation;b.reduced apoptosis c. a block in cellular differentiation.

Acute Leukaemogenesis• Malignant transformation occurs as a result of the

accumulation of genetic mutations .

• Genes involved in the development of cancer are divided broadly into two groups:a. Oncogenesb. Tumour-suppressor genes

Clinical Manifestations

• Symptoms due to:– Marrow failure– Tissue infiltration– Leukostasis– Other (DIC)

Marrow failure• Neutropenia:

Infections, sepsis

• Anemia: Fatigue, pallor

• Thrombocytopenia:Bleeding

Infiltration of tissues/organs

• Enlargement of liver, spleen, lymph nodes

• Gum hypertrophy

• Bone pain

• Other organs: CNS, skin, testis, any organ

,

• Accumulation of blasts in microcirculation with impaired perfusion.

• Lungs: hypoxemia, pulmonary infiltrates• CNS: stroke

Constitutional symptoms• Fever and sweats………… common

• Weight loss ……………less common

Acute Myeloid Leukaemia

Acute myeloid leukaemia

• Acute myeloid leukaemia (AML) has an incidence of 2 – 3 per 100 000 per annum in children, rising to 15 per 100 000 in older

adults

Myeloid maturationmyeloblast promyelocyte myelocyte metamyelocyte band neutrophil

MATURATIONMATURATION

Adapted and modified from U Va website

Classification of Leukemia

WHO classification (Newer)• Incorporates and interrelates morphology, cytogenetics,

molecular genetics, and immunologic markers.• 20% blast in blood and bonemarrow.

FAB classification (Older)• based upon morphology as determined by the degree of

differentiation along different cell lines and the extent of cell maturation.

• 30% blast in blood and bone marrow.

FAB Classification of AML

M0: Minimally differentiatedM1: Myeloblastic leukemia without maturationM2: Myeloblastic leukemia with maturationM3: Hypergranular promyelocytic leukemiaM4: Myelomonocytic leukemiaM5: Monocytic leukemiaM6: Erythroleukemia (DiGuglielmo's disease)M7: Megakaryoblastic leukemia

WHO classification of AML1.Acute myeloid leukemia with recurrent

genetic abnormalities AML with t(8;21)(q22;q22), AML1

(CBF-a)/ETO Acute promyelocytic leukemias [AML with

t(15;17)(q22;q11) and variants, PML/RAR-a] AML with abnormal bone marrow

eosinophils inv(16)(p13q22) or t(16;16)(p13;q11), CBFb/MYH11X

AML with 11q23 (MLL) abnormalities

2 . AML with multilineage dysplasia

With previous myelodysplastic syndrome Without previous myelodysplastic syndrome

3. AML & myelodysplastic syndromes, therapy-related

Alkylating agent-related Topoisomerase type II inhibitor-related (some

may be lymphoid) Other types

4.AML not otherwise categorized AML minimally differentiated AML with maturation AML without maturation Acute myelomonocytic leukemia Acute monoblastic and monocytic

leukemia Acute erythroid leukemia Acute megakaryoblastic leukemia Acute basophilic leukemia Acute panmyelosis with myelofibrosis5. Acute Biphenotypic Leukemias

Immunophenotype of AML subtypes

Antigen M0 M1 M2 M3 M4 M5 M6 M7 ALL HLA-DR ++ ++ + - ++ ++ + - + CD11b + + + - +++ +++ - - - CD13 +++ +++ +++ +++ +++ ++ ++ + +/- CD14 - + + - +++ +++ - - - CD15 - - +++ + + + - - - CD33 +++ +++ +++ +++ +++ +++ ++ + +/- CD41, CD61 - - - - - - - +++ -

Glycophorin A - - - - - - +++ - - TdT ++ + + - - - - - +++ CD117 +++ +++ ++ + ++ ++ + - CD2 + + - ++ ++ - - - +++ CD7 + + - - - - - ++ ++ CD19 + ++ - - - - - ++++ CD34 +++ ++ ++ - + - - + ++

Prognostic Factors in AML• Favorable

– younger age (<50)– WBC <30,000– t(8;21) – seen in >50% with AML M2– inv(16) – seen in AML M4 eos– t(15;17) – seen in >80% AML M3

• Unfavorable– older age (>60)– Poor performance status– WBC >100,000– Elevated LDH– prior MDS or hematogic malignancy– CD34 positive phenotype, MRD1 postive phenotype– del (5), del (7)– trisomy 8– t(6;9), t(9;22)– t(9;11) – seen in AML M5– FLT3 gene mutation (seen in 30% of patients)

Peripheral Blood AML• Most patients with AML present with anemia normocytic normochromic. • Thrombocytopenia

• Leukocytosis white blood cell count up to 200x109

• Large, sometimes hypogranular platelets can be seen, and functional defects can contribute to hemorrhagic manifestations.

• Most patients are neutropenic, and morphologic abnormalities (hypogranulation,

nuclear hyperlobulation, Pelger-Huët anomaly) are often noted in the remaining neutrophils.

• Blasts are predominant cells.

BM CHANGES IN AML

BM aspirates show Hypercellularity

Cells predominantly myeloblasts

Immature granulocytes, erythroblasts, modest increase in plasma cells, monocytes, megaloblastic erythroblasts,ring sideroblasts.

Erythropoeitic cells scanty, megaloblasts, ring sideroblasts found

Megakaryocytes reduced

Myelofibrosis can be seen

CYTOCHEMICAL FEATURES OF AML SUBTYPESUBTYPE MPO/MPO/

SBBSBBSESE NSENSE

AML-M0AML-M0 __ __ __AML-M1AML-M1 ++ ++ __AML-M2AML-M2 ++ ++ __AML-M3AML-M3 ++ ++ __AML-M4AML-M4 ++ ++ ++AML-M5aAML-M5a __ __ ++AML-M5bAML-M5b __ __ ++AML-M6AML-M6 ++ __ +/_+/_AML-M7AML-M7 __ _ _ Strongly + using Strongly + using

acetate as substrateacetate as substrate

Blast classificationType 1

Typical myeloblast with open chromatin and prominent nucleoli, immature deep blue cytoplasm without granules.

Type 2Similar to type one + presence of up to 20 discrete azurophilic granules.

Type 3Similar to type one + numerous azurophilic granules.

AML-MO

• Distinguished by absence of visible granules in cytoplasm of blast.

• Negative –ve reactions with cytochemical stains.

• Positive +ve for myeloid lineage markers.

CD13 CD33.

M0 AML

AML-M1• AML variant and is most common in adults and in infants

less then 1 year.• 50% cases show leucocytosis.• Lack of cellular maturation.• Predominant cell in peripheral blood is poorly

differentiated myeloblast.• Vacuoles may be present.• Platelet are generally decreased.• A few blast may have scanty azurophilic granules or Auer

rod is present.

PB film of a patient with M1 AML showing blasts,some of which are heavily vacuolated

AML-M2

• Presence of more differantiated cells in the bone marrow with maturation.• Condition is more common in adults.• Leucocytosis in 50% of cases.• Thrombocytopenia • Myeloblast are predominant cell type in peripheral blood.• Bone marrow is hypercellular.• Azurophilic granules in variable amount. • Auer rods a azurophilic granules are common.Phi bodies:

Phi bodies are variant of auer rods but are smaller and not necessarily in rod shaped.

BM film of a patient with M2 AML showing unusually heavy granulation of neutrophils and precursors

AML-M3

• Typically seen in young adults.• Sudden and severe progression.• Cause acute DIC.• DLC shows predominance of promyelocytes.• Nucleus is very delicate sometime show foldings.• Most common clinical finding is bleeding.

Auer rods

• Auer rods are red staining, needle-like bodies seen in the cytoplasm of myeloblasts, and/or progranulocytes in certain leukemias. Auer rods are cytoplasmic inclusions which result from an abnormal fusion of the primary (azurophilic) granules.

•

Faggot cells:

Faggot cells:Cells with multiple auer rods sometime occuring in bundles.

AML-M4

• Both myelocytic and monocytic cells are present in peripheral blood and bone marrow.

• Infilteration of leukemic cells in extramedullary sites is more common.

• Serum and urine level of meuramidase are usually elevated because of monocytic proliferation.

• Anemia • Thrombocytopenia• Cytochemical stains will demonstrate two cells population in

bone marrow.

AML-M4

AML-M5• Usually seen in children and young adults.

• Degree of gum hypertrophy ,lymph node ,CNS and extra medullary infiltrates seen.

• Occasional episods of DIC.

• Moderately elivated serum and urine muramidase.

• More then 80% of non erythroid cells seen in BM are monocytic .

AML-M5

AML-M5a

• Poorly differentiated.

• Monoblast account for 80% or more of all monocytic cells.

• Remeining 20% are monocytes.

• The monoblast are larger.

• Azurophilic granules may be present.

AML-M5b

• Well differentiated.

• More then 80% of monocytic cells in nonerythroid marrow.

• The remaining cells are promonocytic or monocytic.

• The percentage of blast is less then 30%.

• Fine azeurophilic granules are present.

AML-M6• Predominant cells in the bone marrow is erythroblast.

• Predominant feature is anemia with striking poikilocytosis and anisocytosis.

• The diagnosis of erythroleucaemia can be done if more then 50% of bone marrow cells are erythroid and 30% of remaining are blast.

• True erythro leukemia occurs when BM is replaced by proliferating normoblast showing no maturation beyond basophilic normoblasts.

PB film in a patient with M6 AML showing an abnormal circulating erythroblast

AML-M7 Peripheral blood pancytopenia.

High peripheral blood blast count.

Micro megakaryocytes and undifferentiated blast.

Bone marrow reveals increased fibroblast.

Showing cytoplasmic budding.

AML-M7

Once again AML vs ALL

• Leukaemia is the most common childhood cancer and acute lymphoblastic leukaemia (ALL) is the most common subtype, accounting for 75 – 80% of all cases

FAB Classification of ALL

• L1—Mature-appearing lymphoblasts (T-cells or pre-B-cells) small blast with High N:C ration.

• L2—Immature and pleomorphic (variously shaped) lymphoblasts (T-cells or pre-B-cells) small and large blast present with moderate N:C ration.

• L3— Lymphoblasts (B-cells; Burkitt's cells) are large and uniform, deep basophilic cytoplasm, vaculation in cytoplasm and low N:C ratio.

FeaturesFeatures L1L1 L2L2 L3L3Cell sizeCell size Small, Small,

uniformuniformLarge, often Large, often

heterogeneousheterogeneousLarge, homogeneousLarge, homogeneous

NucleusNucleus Round, Round, regularregular

Oval to round, Oval to round, irregular irregular cleftingclefting

RoundRound

Amount of Amount of cytoplasmcytoplasm

ScantScant Moderately Moderately abundantabundant

Moderately abundantModerately abundant

Genetic Genetic materialmaterial

Dense, Dense, uniform uniform

variable variable finely stippled and finely stippled and uniform uniform

NucleoliNucleoli InconspicuousInconspicuous,,

smallsmall

Prominent, largeProminent, large1 - >11 - >1

Present, may be Present, may be prominentprominent

1- >1, vesicular1- >1, vesicularcytoplasmcytoplasmvacuolesvacuoles

OcassionalOcassional VariableVariable ProminentProminent

BasophiliaBasophilia SlightSlight VariableVariable PunctatePunctate

FrequencyFrequency 85%85% 15%15% 2%2%

FAB CLASSIFICATION ALL

• WHO has recognized just two groups of acute lymphoblastic leukemias,

• precursor B-cell and • precursor T-cell lymphoblastic

leukemia/lymphoma.

Precursor B lymphoblastic leukemia (B-ALL)/lymphoblastic lymphoma (B-LBL)

• B-ALL comprises approximately 85% of all childhood ALL, whereas B-LBL is a rare type of lymphoma and constitutes approximately 10% of lymphoblastic lymphoma cases.

• is a malignancy where B lineage lymphoblasts predominate in the bone marrow (B lymphoblastic leukemia).

• Sometimes there is primary involvement of lymph nodes or extranodal sites (B lymphoblastic lymphoma).

• Greater than 20% of bone marrow cells

• However, the bone marrow aspirate typically consists of almost entirely lymphoblasts at diagnosis.

• When the leukemic process is limited to a mass lesion and less than 20% or fewer lymphoblasts are seen in the marrow, the designation lymphoma is used.

• The blood and bone marrow contains lymphoblasts• with L1 or L2 morphology

• B-ALL may also develop in adults, and the prognosis is generally much poorer in adults

Immunophenotype• The lymphoblasts in B-ALL/LBL are uniformly TdT positive and HLA-DR positive. • The flow cytometric immunophenotype in most cases is positive for CD 10, CD19,

CD20, CD24, CD22, and CD79a

Precursor T lymphoblastic leukemia/lymphoma

• is a malignancy of lymphoblasts with pre-T markers predominating in the bone marrow (T-ALL).

• When there is primary involvement of lymph nodes or extranodal sites, it is termed T lymphoblastic lymphoma.

• T-ALL represents approximately 15% to 20% of all childhood ALL, is more prevalent in adolescents than young children, and is seen more frequently in males than females.

• often have L2 morphology• less frequently, have L1 morphology

• The lymphoblasts in T-ALL are TdT, cytoplasmic CD3, CD7, CD1, CD2, , CD5 positive

Lymphoblast

Peripheral Blood ALL• WBCs

– Total cont elevated 50-60 x 109/L to 100 109/L– Rarely more than 100 x 109/L

– Lymphoblasts in large numbers• RBCs

– Normocytic normochromic anaemia. Thrombocytopenia.

BM CHANGES IN ALL

Marrow aspirates show: Blast cell predominace. They are lymphoblasts which are

earliest identifiable precursor of lymphoid cells Erythropeitic cells reduced Dyserythropoiesis Megakaryocytes reduced

ALL-L1• This is the most common form found in children

and it has the best prognosis.

ALL-L1

•This is the most frequent ALL found in adults.

ALL-L2

ALL-L2

ALL-L3

• This is the rarest form of ALL. • Burkit lymphoma type cells

ALL-L3

Aids in the diagnosis Classification of the leukemias

Identification of the chemical components of cells - is conducted to distinguish different types of leukemia.

Cytochemistry

Enzymatic Techniques Myeloperoxidases Esterases Phosphatases

Nonenzymatic Techniques Periodic Acid-Schiff Stain Sudan Black B Toluidine Blue

Leukemia,White Blood 75

Cytochemical differences in ALL & AML blasts

CYTOCHEMICAL STAINCYTOCHEMICAL STAIN ALL BLASTSALL BLASTS AML BLASTSAML BLASTS

MPOMPO NegativeNegative PositivePositive

SUDAN BLACK BSUDAN BLACK B NegativeNegative PositivePositive

PASPAS PositivePositive NegativeNegative

NON SPECIFIC NON SPECIFIC ESTERASEESTERASE

NegativeNegative PositivePositive

ACID PHOSPHATASEACID PHOSPHATASE PositivePositive NegativeNegative

Leukemia,White Blood 76

Biochemical tests• Serum uric acid raised due to breakdown of leukemic cells.

• Serum LDH may be raised

• Hypocalcaemia

• Hyperuricemia• Increase K+

Terminal Deoxynucleotidyl Transferase

Terminal deoxynucleotidyl transferase (TdT) is an intranuclear enzyme found in stem cells and immature lymphoid cells within the bone marrow, but not in mature B lymphocytes.

Significance of TdT

• It is present in 90% of acute lymphoblastic leukemias.

• Only 5%-10% of acute myeloblastic leukemias.

• It has also been demonstrated in 1/3 of cases of the blast crisis stage of chronic myelogenous leukemia and is a good prognostic indicator in these patients.

Imaging Studies

It is likely that the physician will want to perform imaging studies to determine whether the leukemia has invaded other organs within the body. Such studies will include:

• X-rays

• Computed tomography (CT or CAT)

• Magnetic resonance imaging (MRI)

• Radionuclide (radioactive atom)

• Ultrasound

CSF

• Look CSF for Blast cells

Karyotyping

• A karyotype is the characteristic chromosome complement of a eukaryote species.

• Its study is called karyotyping.

• The preparation and study of karyotypes is part of cytogenetics.

Thanks