Leukaemia

Leukaemia

BS967-7-SP: Session 6

Dr NJ Dodd

Leukaemia

 “White blood” – leucos/aemia

 1827 Alfred Velpeau

 63 year old florist, fever, weakness, urinary stones,

hepatosplenomegaly

 Blood “like gruel”

 1845 JH Bennett – patients with enlarged spleens and

changes in the colour of the blood – “Leucocythaemia”

 1856 Virchow observed the white cells with a

microscope – “Leukaemia”





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Classification of Leukaemia

 Acute

 Chronic



 Myeloid

 Lymphoid









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Acute Leukaemia

 Rapidly progressive

 Proliferation of primitive “blast” cells

 Acute myeloblastic (myeloid) leukaemia

 “Acute non-lymphoblastic leukaemia”

 Acute lymphoblastic leukaemia









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Clinical Features of Acute

Leukaemia

 Symptoms

 Anaemia: pallor, tiredness, breathlessness

 Neutropenia: mouth ulcers, infections

 Leucocytosis: general malaise, breathlessness,

confusion, pain, extra medullary tumours

 Thrombocytopenia: bruising, bleeding









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Clinical Features of Acute

Leukaemia 2

 Clinical Findings/signs

 Pallor

 Bruising, bleeding

 Mouth ulcers

 Lymphadenopathy: ALL>AML

 Hepato/splenomegaly: ALL>AML

 Masses: AML>ALL

 Central nervous system: ALL>AML

 Testicular involvement: ALL



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Cerebral bleed pallor and purpura hyphaema









Retinal bleeds gum infiltration skin infiltration

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AML or ALL?

 Morphology,

 Cytochemistry

 Flow cytometry









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Aetiology of AML

 Preceding bone marrow disorders

 Chemotherapy – especially alkylating agents,

epipodophyllotoxins and anthracyclines

 ?Occupational exposure – solvents/benzene

 Radiation

 Bad luck







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Genetic predisposition to AML/ALL

 Chromosomal instability

 Fanconi’s anaemia, ataxia telangiectasia,

neurofibromatosis, Bloom’s syndrome

 Germline mutations in AML-1/CBFA/RUNX1

gene

 Congenital immunedeficiency disorders

 Down’s syndrome (10-18 fold risk)





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Oncogenes in AML

 Proto-oncogenes

 Mutation produces oncogene

 i.e. FLT3 (fms-like tyrosine kinase)



 Translocation may lead to over expression of the

proto-oncogene product

 Translocation may produce adverse interaction with

other gene(s)

 i.e. t(15;17) RARA/APL fusion protein







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Epidemiology of AML

1.2% of cancer

deaths

Median age at

diagnosis 63 years

Male > female

North america,

europe, oceania >

asia, latin america









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WHO Classification of AML

AML with characteristic t (8;21) AML1/CBFA/ETO

Inversion 16 CBFB/MYH11

genetic abnormalities

t (15;17) RARA/PML

11q23 MLL abnormalities

AML with multi-lineage

dysplasia

AML and MDS – therapy

related

AML not otherwise

categorised

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FAB classification of AML

 M0: minimally differentiated

 M1: myeloblastic leukemia without maturation

 M2: myeloblastic leukemia with maturation

 M3: hypergranular promyelocytic leukemia

 M4: myelomonocytic leukemia

 M4Eo: variant, increase in marrow eosinophils

 M5: monocytic leukemia (M5a and M5b)

 M6: erythroleukemia (DiGuglielmo's disease)

 M7: megakaryoblastic leukemia



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Haematology’s Role in Diagnosis

 FBC and blood film morphology

 Coagulation screen

 Bone marrow aspirate and trephine biopsy









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AML morphology







m0 m1 m2 m3









m4 m4eo m5 m6 m7

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Diagnostic techniques in AML

 Flow cytometry

 Giemsa banded karyotype

 F.I.S.H.

 PCR









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Flow cytometry









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AML phenotype

 CD 13, 33, 117 +ve

 CD 14 +ve Monocytic

 CD 34 +/-

 HLA-DR +/-

 Myelo-peroxidase

 Aberrant lymphoid antigen expression







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Giemsa banding









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Cytogenetics in AML

M0

M1

M2 t(8;21(q22;q22), t(6;9)

M3 t(15;17)(q22;q12)

M4 inv(16)(p13;q22), del(16q)

M4eo inv(16), t(16;16)

M5 del(11q), t(9;11), t(11;19)

M6

M7 t(1;22)

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FISH

Fluorescence in-situ Hybridisation









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Interphase FISH









Normal 15 & 17 t(15;17)



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Prognostic factors in AML

 Age and performance status

 Chromosomes:

 Good : t(8;21), t(15;17), inv (16)

 Intermediate: Normal, +8, +21, +22, del(7q),

abnormal 11q23

 Poor: -5, -7, del(5q), abnormal 3q, complex

 Genes:

 FLT-3 mutation





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Genes of interest in AML

 FLT-3 (CD 135)

 Nucleophosmin

 MLL

 RARA

 PML

 c-KIT (CD117)

 RAS



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Polymerase Chain Reaction

18000









16000









14000









12000









10000









dna copies

Series1



8000









6000









4000









2000









0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

replications









Heat cycling and taq polymerase

http://learn.genetics.utah.edu/content/labs/pcr/

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Sensitivity of analyses in AML

 Bone marrow visual differential – 5%

 Flow cytometry – 1%

 FISH - 0.5-1%

 PCR - 0.0001%









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Microarray technology









Specific DNA fragments fixed in wells, hybridise with labelled m-

RNA or c-DNA from normal and tumour cells

green:underexpressed yellow:normal red:overexpressed

http://learn.genetics.utah.edu/content/labs/microarray/

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DNA microarrays in AML









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Acute Lymphoblastic Leukaemia

 WHO classification:

 Precursor B cell ALL

 Precursor T cell ALL

 Burkitt’s mature B lymphoblastic

leukaemia/lymphoma

 Bi-phenotypic leukaemia

 FAB

 L1, L2, L3





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Clinical features of ALL

 Bone pain – children

 CNS involvement

 Lymphadenopathy









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Morphology of ALL









L2



L1







L3







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Cytogenetics in ALL

Translocation Genetics %

Cryptic t(12;21) TEL-AML1 25.4

t(1;19)(q23;p13) E2A-PBX 4.8

t(9;22)(q34;q11) BCR-ABL 1.6

t(4;11)(q21;q23) MLL-AF4 1.6

t(8;14)(q24;q32) IGH-MYC (Burkitt)

t(11;14)p13;q11) TCR-RBTN2 (T-ALL)





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Epidemiology of ALL





 Peak incidence in

children aged 4 years

 Rising incidence in old

age







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Prognostic factors in ALL

 Sex: female > male

 Ethnicity: caucasian > non-caucasian

 Age: 1 year old

 WBC 50, t(12;21)

 Intermediate

 Hyperdiploidy 47-50, Normal, del(6q),

rearrangments of 8q24

 Poor

 Hypodyploidy/near haploidy; near tetraploidy,

del(17p), t(9;22), t(11q23)



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Acute leukaemia references

 Acute myeloid leukaemia.

Estey E and Döhner H.

Lancet 2006; 368: 1894-1907.



 http://www.clevelandclinicmeded.com/medicalpubs/d

iseasemanagement/hematology-oncology/acute-

myelogenous-leukemia/



 Flow cytometric analysis of acute leukemias.

Diagnostic utility and critical analysis of data.

Kaleem Z, et al Arch Pathol Lab Med. 2003

Jan;127(1):42-8.



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 Questions?

 Tea/coffee break









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Chronic Leukaemias

 Myeloid

 Myelo-proliferative disorders

 Chronic

 Myeloid, neutrophilic, eosinophilic, basophilic

 Myelodysplastic

 Chronic myelo-monocytic leukaemia

 Lymphoid

 Chronic lymphocytic leukaemia

 Prolymphocytic leukaemia

 Hairy cell leukaemia





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CML and Myeloproliferative diseases

 The myeloproliferative diseases

 clonal stem cell disorders

 leukocytosis,





 Polycythemia vera (PV), essential

thrombocytosis (ET), myelofibrosis and chronic

myeloid leukemia (CML)





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 CML results from a somatic mutation in a

pluripotential lymphohematopoietic cell

 CML is a MPD characterized by increased

granulocytic cell line, associated with erythroid

and platelet hyperplasia

 The disease usually envolves into an

accelerated phase that often terminates in

acute phase



chronic phase 3-5 years

accelerated phase

blastic phase 3-6 months

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Aetiology





 Exposure to high- dose ionizing radiation

 Chemical agents have not been established as a cause









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Epidemiology





 CML accounts for approximately 15 percent of all

cases of leukemia and approximately 3 percent of

childhood leukemias

 The median age of onset is 53 years









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Pathogenesis

Hematopoietic abnormality

 Expansion of granulocytic progenitors and a decreased

sensitivity of the progenitors to regulation – increased white cell

count

 Megakaryocytopoiesis is often expanded

 Erythropoiesis is usually deficient

 Function of the neutrophils and platelets is nearly normal









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Pathogenesis

Genetic abnormality

 CML is the result of an acquired genetic abnormality

 A translocation between chromosome 9 and 22 [t(9;22)] –

the Philadelphia chromosome

 The oncogene BCR-ABL encodes an enzyme – tyrosine

phosphokinase (usually p210)









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Translocation t(9;22)(q34;q11)









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Philadelphia Chromosome

• More than 95% of patients with CML has Philadelphia (Ph)

chromosome

 A subset of patients with CML lack a detectable Ph

chromosome but have the fusion product for the bcr/abl

translocation detectable by reverse transcriptase- polymerase

chain reaction (RT-PCR)









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The bcr/abl fusion protein

 Uncontrolled kinase activity

● Deregulated cellular proliferation

● Decreased adherence of leukemia cells to the bone marrow

stroma

● Leukemic cells are protected from normal programmed cell

death (apoptosis)









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Clinical features

 30 percent of patient are asymptomatic at the time of

diagnosis

 Symptoms are gradual in onset:

fatigue, malaise, anorexia, abdominal discomfort, weight

loss, excessive sweating

● Less frequent symptoms:





Night sweats, heat intolerance, gouty arthitis, symptoms of

leukostasis (tinnitus, stupor), splenic infartion (left upper-

quadrant and left shoulder pain), urticaria (result of

histamine release)

● Physical signs:





Pallor, splenomegaly, sternal pain



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Laboratory features



 Anaemia

 NRBCs

 WBC raised

 Granulocytes at all

stages of maturation

 Basophilia

 Platelets normal or

increased









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Laboratory features (2)



 The marrow is hypercellular (granulocytic hyperplasia)

 Reticulin fibrosis

 Hyperuricemia and hyperuricosuria

 Serum vitamin B12-binding proteine and serum vitamin B12

levels are increased

 Pseudohyperkalemia, and spurious hypoxemia and hypoglycemia

 Cytogenetic test- presence of the Ph chromosome

 Molecular test – presence of the BCR-ABL fusion gene









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Differential diagnosis

 Polycythemia vera

 Myelofibrosis

 Essential

thrombocytemia

 Reactive leukocytosis

 Chronic eosinophilic

leukaemia

 Chronic neutrophilic

leukaemia

 Chronic Myelo-

monocytic leukaemia



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Chronic Lymphocytic

Leukemia

Definition

 Clonal B cell malignancy.

 Progressive accumulation of long lived mature

lymphocytes.

 Increase in anti-apoptotic protein bcl-2.

 Intermediate stage between pre-B and mature B-

cell.







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Epidemiology

 Most common leukemia of Western world.

 Less frequent in Asia and Latin America.

 Male to female ratio is 2:1.

 Median age at diagnosis is 65-70 years.

 In US population incidence is similar in different

races.





Cancer statastics 2000; CA J Clin 2000;

50:7-33

Etiology & Risk factors

 High familial risk with two-fold to seven-fold

higher risk.

 No documented association with environmental

factors.

 No established viral etiology.









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Diagnostic Criteria

 Defined by WHO

 Persistent lymphocytosis.

 Absolute count more than 5 x 109/l

 Mature appearing B-cells with 50%.

 5-10% have small monoclonal peak.

 Positive Coombs’ test in 30% .

 Autoimmune hemolytic anemia &

thrombocytopenia in 15 x 109/l)

Stage 1 Lymphocytosis and lymphadenopathy

Stage 2 Lymphocytosis and splenomegaly with

or without lymphadenopathy

Stage 3 Lymphocytosis and anemia (Hgb 13 years.

 Intermediate-risk: stage I & II with MS about 8

years.

 High-risk: stages III & IV with MS about

3 years









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The Binet Staging System

Stage A No anemia, no thrombocytopenia,

=3 involved nodal areas

Stage C Anemia (Hgb < 10 g/dL) and/or

thrombocytopenia (Plt <

100 x 109/l)





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Other Prognostic Features

 Bone marrow pattern of lymphoid infiltration.

 Lymphocyte doubling time.

 Serum beta-2-microglobulin.

 Mutational status of immunoglobulin heavy

chain gene (mutated is better)

 CD 38 and ZAP70 expression





Blood 94: 1848-1854, 1999

Hairy Cell Leukemia









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Introduction

 Hairy cell leukemia (HCL) is a B-cell malignancy comprising

2% of all leukemias



 It is highly sensitive to but not curable by treatment with

cladribine and pentostatin,with complete remission (CR) rates

of 80% to 95%



Lack of plateau in the disease-free survival curve suggests the

eventual need for additional treatment in many patients









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Introduction



 Clonal mature B-cell lymphoproliferative

disorder

 Low hairy cell proliferative rate

 A positive TRAP stain in conjunction

with a characteristic bone marrow biopsy is

essentially diagnostic of hairy cell leukemia

 Clonal cytogenetic abnormalities in 66%

of patients

 involvement of chromosomes 1, 2, 5, 6,

11, 14, 19, and 20

 Chromosome 5 abnormality (in 40% of

patients) with trisomy 5 and pericentric

inversions and interstitial deletions of band

5q13





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Clinical and Laboratory Features



Splenomegaly

Pancytopenia

Monocytopenia

CD20/CD22, CD25, CD11C, CD103

+ve









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Hairy Cell Leukemia Variant

• 10% of hairy cell leukemia (HCL) cases



• Morphology of variant cells intermediate

between that of classic HCL and prolymphocytic

leukemias



• Patients typically present with leukocytosis

rather than leukopenia, and

often lack the neutropenia, anemia, and/or

thrombocytopenia



• B-cell antigens FMC7, CD11c, CD20, CD22 and

surface immunoglobulin are strongly positive in

both classic HCL and HCLv,



•HCLv differs from classic HCL by lack of CD25, HC-

2 and CD123, and by expression of CD27

Blood Arons et al. 0 (2009)

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HCLv Cont….

• CD103 is usually positive in both but can be negative in HCLv 2



• HCLv lacking both CD25 and CD103 may be difficult to

differentiate from splenic marginal zone lymphoma/splenic

lymphoma with villous lymphocytes (SMZL or SLVL), without

relying also on morphologic differences



•Response to purine analogs pentostatin and cladribine in

patients with HCLv is limited to partial responses in approximately

50% of patients









Blood Arons et al. 0 (2009)

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Other Chronic Lymphoid

Leukaemias

 Adult T cell

leukaemia/lymphoma

 Prolymphocytic

leukaemia

 Large granular

lymphocytic leukaemia



 Leukaemic lymphomas



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Chronic Leukaemia Resources

 http://www.clevelandclinicmeded.com/medical

pubs/diseasemanagement/hematology-

oncology/chronic-leukemias/

 http://www.cancerindex.org/geneweb/gsitema

p.htm

 http://atlasgeneticsoncology.org/







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