LEUKEMIAS

By

Dr Aamer Aleem

Consultant Hematologist

Prof. of Medicine

College of Medicine & KKUH

Riyadh, Saudi Arabia

Leukemias

Leukemias are a group of cancers of the blood/ bone marrow and are characterized by an abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes).

Leukemia is a broad term covering a spectrum of diseases. Any of various acute or chronic neoplastic diseases of the bone marrow in which unrestrained proliferation of white blood cells occurs and which is usually accompanied by anemia and thrombocytopenia

Classification of leukemiasTwo major types (4 subtypes) of leukemias

Acute leukemias

Acute lymphoblastic leukemia (ALL)

Acute myelogenous leukemia (AML)

(also "myeloid" or "nonlymphocytic")

Chronic leukemias

Chronic lymphocytic leukemia (CLL)

Chronic myeloid leukemia (CML)

(Within these main categories, there are typically several subcategories)

Myeloid maturation

myeloblast promyelocyte myelocyte metamyelocyte band neutrophil

MATURATIONMATURATION

Adapted and modified from U Va website

Hematopoieticstem cell

Neutrophils

Eosinophils

Basophils

Monocytes

Platelets

Red cells

Myeloidprogenitor

Lymphoidprogenitor

B-lymphocytesB-lymphocytes

T-lymphocytes

Plasmacells

naïve

ALLALL

AMLAML

Acute leukemia-definition

• Disorders with clonal expansion of precursor cells (myeloid or lymphoid) with reduced capacity to differentiate.

1. Maturation arrest at the blast stage

2. Bone marrow infiltration by blast cells causing suppression of normal precursors (bone marrow failure)

Myeloid vs Lymphoid

• Any disease that arises from the myeloid

elements (white cell, red cell, platelets) is a myeloid disease

….. AML, CML

• Any disease that arises from the lymphoid

elements is a lymphoid disease

….. ALL, CLL

Acute vs. chronic leukemia• Acute leukemias:• Young, immature, blast cells in the bone marrow (and often blood)• More fulminant presentation• More aggressive course

• Chronic leukemias:• Accumulation of mature, differentiated cells• Often subclinical or incidental presentation• In general, more indolent (slow) course• Frequently splenomegaly • Mature appearing cells in the B. marrow and blood

Acute vs. chronic leukemia• Leukemias are classified according to cell of origin:• Lymphoid cells ALL - lymphoblasts CLL – mature appearing lymphocytes• Myeloid cells AML – myeloblasts CML – mature appearing neutrophils

• On a CBC, if you see:• Predominance of blasts in blood consider an acute leukemia• Leukocytosis with mature lymphocytosis consider CLL• Leukocytosis with mature neutrophilia consider CML

Acute leukemiasAcute leukemias

Definition: Malignancies of immature hematopeotic cells.

(> 20% blast cells in the bone marrow)

Types: Acute Myeloid Leukaemia (AML)

Acute Lymphoblastic leukemia (ALL)

Groups: Childhood (< 15) > 80% ALL

Adult (> 15) > 80% AML

Elderly (> 60 years)

Epidemiology of AML

• Incidence – 2.7 per 100,000– 12.6 per 100,000 in those over 65 yrs– median age of presentation : 67 yrs

• More prevalent in:– Males– European descent– Hispanic/Latino background (promyelocytic

leukemia, AML-M3)

Etiology of acute leukemiasEtiology of acute leukemias• Mostly unidentifiable cause i.e., de nove (≥80%)• Drugs & chemicals • Alkylating agents (Chlorambucil, N mustard, Melphalan)

• Topoisomerase inhibitors (Etoposide)

• Benzene

• Ionizing radiation• Myelodysplastic syndrome• Myeloproliferative disorders• Genetic disorders

• Down’s syndrome• Bloom syndrome• Faconi anemia• Wiskott Aldrich syndrome

Clinical presentationClinical presentation

Symptoms• Usual 1-3 Month History : MDS – 1yr

• (Features of BM failure)

• Fatigue, malaise, dyspnea (anemia)

• Bleeding eg after dental procedure

Easy bruisability

Severe epistaxis

• Fever (infections)

• Bone Pain

Clinical PresentationClinical Presentation

SignsSigns Pallor Hemorrhage from the gums, epistaxis, skin,

fundus, GI tract, urinary tract Hepato-splenomegaly Enlarged lymph nodes Gum (hypertrophy) or skin infiltration (M5) Fever (sepsis, pneumonia, peri-rectal abscess)

Differential DiagnosisDifferential Diagnosis

1. Aplastic anemia

2. Myelodysplastic syndromes

3. Multiple myeloma

4. Lymphomas

5. Severe megaloblastic anemia

6. Leukemoid reaction

Laboratory TestsLaboratory Tests

1. CBC a. Anemiab.

Trombocytopeniac. WBC

High Normal Low

2. Coagulation Studies (M3-DIC)

3. Biochemical Studies (U/E, LFT)Cont..

Peripheral Blood Smear – blasts in almost all

cases

Bone Marrow Examination (>20% blasts)

Diagnosis of Acute LeukemiaDiagnosis of Acute Leukemia

4. Peripheral Blood smear – blasts in almost all

cases

5. Bone Marrow Examination (>20% blasts)

6. Flow cyometry

(Surface immunophenotype of blast cells)

4. Cytogenetics (chromosomal analysis)

5. CSF analysis (all ALL patients, some AML)

Diagnostic methods of importance

• Bone marrow aspirate & Romanowsky stain (morphology) Enumeration of blasts, maturing cells, recognition of dysplasia

• Cytochemistry Myeloperoxidase, Sudan Black B, esterases to determine involved lineages

• Immunophenotyping

Defines blast cell lineage commitment as myeloid, lymphoid or biphenotypic

• Cytogenetics & molecular studies (FISH, PCR)Detects clonal chromosomal abnormalities, including those of prognostic importance

FAB Classification of AML

• MO undifferentiated acute myeloblastic leukemia (5%)• M1 AML with minimal maturation (20%)• M2 AML with maturation (30%)

- t(8;21)• M3 Acute promyelocytic leukemia (5%)

- t(15;17)• M4 Acute myelomonocytic leukemia (20%)• M4 eos Acute myelomonocytic leukemia with eosinophilia (5%)

- inv (16)• M5 Acute monocytic leukemia (10%)

- t(9;11)• M6 Acute erythroid leukemia (3%)• M7 Acute megakaryoblastic leukemia (3%)

M3*

M4

M5M6M0, M1, M2

58%

7%

M0, M1, M2 M7 M6M5

M4

M3*

52%

9%

AdultsAdults ChildrenChildren

Frequency of Acute Myeloid Leukemia SubtypesFrequency of Acute Myeloid Leukemia Subtypes

WHO Classification of AML• AML with recurrent genetic abnormalities

- t(8;21), t(16), inv(16), chromosome 11 changes

- t(15;17) as usually seen with AML M3

• AML with multilineage dysplasia (more than one abnormal myeloid cell type is involved)

• AML related to previous chemotherapy or radiation

• AML not otherwise specified- undifferentiated AML (MO)

- AML with minimal maturation (M1)

- AML with maturation (M2)

- Acute myelomonocytic leukemia (M4)

- Acute monocytic leukemia (M5)

- Acute erythroid leukemia (M6)

- Acute megakaryoblastic leukemia (M7)

- Acute basophilic leukemia (M8)

- Acute panmyelosis with fibrosis

- Myeloid sarcoma (also known as granulocytic sarcoma or chloroma)

• Undifferentiated or biphenotypic acute leukemias (leukemias that have both lymphocytic and myeloid features. Sometimes called ALL with myeloid markers, AML with lymphoid markers, or mixed lymphoid lineage leukemias).

WHO Classification of AML

• AML not otherwise specified

- undifferentiated AML (MO)

- AML with minimal maturation (M1)

- AML with maturation (M2)

- Acute myelomonocytic leukemia (M4)

- Acute monocytic leukemia (M5)

- Acute erythroid leukemia (M6)

- Acute megakaryoblastic leukemia (M7)

- Acute basophilic leukemia (M8)

- Acute panmyelosis with fibrosis

- Myeloid sarcoma (also known as granulocytic sarcoma or

chloroma)

Blood Film-NormalBlood Film-Normal

Blood Film-NormalBlood Film-Normal

Normal BM cells

BM aspirate

AML

Bone marrow biopsy-Normal

Trephine biopsy in AML

AMLAML

AMLAMLAuer rods

AML-M3 (Acute promyelocytic leukemia)

ALL

Myeloperoxidase

(MPO)

p-Phenylene diamine + Catecol + H2O2

MPO > Brown black deposits

Chloracetate (Specific) Esterase

Myeloid Cell Line

Naphthol-ASD-chloracetate CAE > Free naphthol compounds

+ Stable diazonium salt (eg, Fast Corinth) > Red deposit

Non-Specific Esterase

Monocytic Line

Naphthyl acetate ANAE > Free naphthyl compounds +Stable diazonium salt (eg, Fast blue RR) > Brown deposits

BMT/Stem CellCord BloodThrombosisHemostasisLaboratoryMalignanciesPediatricsRed Cell DisordersTransfusion MedicineVeterinary

Plasma Cells, Acute myelomonocytic (M-4) leukemiaClump of Plasma Cells most of which are small with a deep basophilic blue cytoplasm. Two cells in the center are partially smudged and show a paler cytoplasm and less dense and redder staining nuclear chromatin. Acute myelomonocytic (M-4) leukemia. Marrow - 100X

                                                                                                                                                                                                                        

Image ID: 0147-094

            

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Immunophenotyping in acute leukemias

Immunophenotyping detects the presence or absence of white blood cell (WBC) antigens.

Most of the antigens that immunophenotyping detects are identified by a CD (clusters of differentiation or cluster designation) number, such as: CD1a, CD2, CD3, CD4, CD8, CD13, CD19, CD20, CD33, CD61, etc.

Immunophenotyping in acute leukemias

• Flow cytometry is performed by processing blood or marrow samples and adding specific antibodies tagged with fluorescent markers.

• These antibodies attach to corresponding antigens on the WBCs when the antigens are present, and are analyzed.

• Results are then graphed and compared to "normal" results and to patterns that are known to be associated with different leukemias

Flow Cytometry

• First labeled with fluorescent dyes.

• Forced through a nozzle in a single-cell stream passing through a laser beam

• The laser is focused to a known wavelength

• Excitation of a specific fluorochrome

• Photo-multiplier tubes detect the scattering of light and emission from the fluorescent dye

Flow cytometryForward and Side Scatter

Immunophenotyping in leukemias

Immunophenotyping in leukemias

Application of Cytogenetic, FISH and Microarray Analysis in the Diagnosis of Leukemia

ISCN: 46,XX,t(8;21)(q22;q22)[19]/46,XX[1]

Fluorescence in situ hybridization (FISH)

FISH with AML1/ETO –DF probe:

76% of cells show one fusion, two red and two green signals

Three way translocation of t(8;21)(q22;q22)

45,X,-Y,t(8;20;21)(q22;p13;q22),del(11)(q21q25)

FISH: Dual fusion probe for t (8;14): IgH (14), MYC (8), Cen8 Aqu

nl 8

der(8)t(3;8;14)

nl 8

der(14)t(3;8;14)

der(3)t(3;8;14)

der(18)t(14;18)

der(14)t(14;18)

Application of genomic array analysis in leukemias

(microarrays)

38 acute leukemias (27 ALL, 11 AML)

Microarray Profiling

Develop class predictor (50 genes)

Test on independent set (34 samples)

Strong predictions on 29/34; accuracy 100%

Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia

• N Engl J Med. 2004 Apr 15;350(16):1605-16.

• Bullinger L, Döhner K, Bair E, Fröhling S, Schlenk RF, Tibshirani R, Döhner H, Pollack JR.

• Source

• Department of Pathology, Stanford University, Stanford, Calif, USA.

• Abstract• RESULTS:

• Unsupervised analysis identified new molecular subtypes of AML, including two prognostically relevant subgroups in AML with a normal karyotype. Using the supervised learning algorithm, we constructed an optimal 133-gene clinical-outcome predictor, which accurately predicted overall survival among patients in the independent validation group (P=0.006), including the subgroup of patients with AML with a normal karyotype (P=0.046). In multivariate analysis, the gene-expression predictor was a strong independent prognostic factor (odds ratio, 8.8; 95 percent confidence interval, 2.6 to 29.3; P<0.001).

• CONCLUSIONS: The use of gene-expression profiling improves the molecular classification of adult AML.

• Hierarchical Cluster Analysis of Diagnostic AML Samples

• Outcome Prediction

• Age Above the age of 50 years the complete remission rate falls

progressively • Cytogenetics

Three risk groups defined– Good risk: patients with t(8;21), t(15;17) and inv/t(16)– Intermediate risk: Normal, +8, +21, +22, 7q-, 9q-,

abnormal 11q23, all other– Poor risk: patients with -7, -5, 5q-, abnormal 3q and

complex karyotypes

Acute Myeloid Leukaemia (AML)

Prognostic factors in AML

1. Wheatley K, Burnett AK, Goldstone AH et al. Br J Haem 1999; 107: 69-792. Grimwade D, Walker H, Oliver F et al. Blood 1998; 92: 2322-33

• Treatment response – Patients with >20% blasts in the marrow after first course of

treatment have short remissions (if achieved) and poor overall survival

• Secondary AML – Patients with AML following chemotherapy or myelodysplasia

respond poorly

• Trilineage myelodysplasia – Patients with trilineage myelodysplasia have a lower remission

rate

Acute Myeloid Leukaemia (AML)

Prognostic factors in AML

1. Wheatley K, Burnett AK, Goldstone AH et al. Br J Haem 1999; 107: 69-792. Grimwade D, Walker H, Oliver F et al. Blood 1998; 92: 2322-33

• Intensive chemotherapy – Patients < 55 years old: 80% remissions

– Patients > 55 years old: progressive reduction in remission rate

• Bone marrow (stem cell) transplantation – Autologous and allogeneic transplants reduce the relapse rate

• Importance of cytogenetics for prognosis in children and adults < 55 years old

• Good risk cytogenetic group – 91% remissions, 65% five year survival

Treatment and prognosis of AML

1. Wheatley K, Burnett AK, Goldstone AH et al. Br J Haem 1999;107: 69-792. Grimwade D, Walker H, Oliver F et al. Blood 1998; 92: 2322-33

• Intensive chemotherapy – Patients < 55 years old: 80% remissions

– Patients > 55 years old: progressive reduction in remission rate

• Bone marrow (stem cell) transplantation – Autologous and allogeneic transplants reduce the relapse rate

• Importance of cytogenetics for prognosis in children and adults < 55 years old

• Good risk cytogenetic group – 91% remissions, 65% five year survival

Acute Myeloid Leukaemia (AML)

Treatment and prognosis of AML

1. Wheatley K, Burnett AK, Goldstone AH et al. Br J Haem 1999;107: 69-792. Grimwade D, Walker H, Oliver F et al. Blood 1998; 92: 2322-33

Acute Lymphoblastic Leukemia (ALL)

Poor Prognostic Factors• Age < 2 yrs and > 10 yrs • Male sex• High WBC count ( > 50 х109/L)• Presence of CNS disease• Cytogenetics Good risk Poor risk

Hyperdiploid (>50 ch) Hypodiploid,

t(9:22), t(4:11)• Bone Marrow: Blasts present on day 14• Day 28:No complete response

Prognostic factors in ALL

ALLALL

Bone Marrow-ALLBone Marrow-ALL

Treatment of acute leukemiasTreatment of acute leukemias

1. Specific therapy (chemotherapy)

2. Supportive treatment

Stages of Therapya. Induction

b. Consolidation

c. Maintenance

(Treatment of acute leukemias)(Treatment of acute leukemias)

InductionInductionObtained by using high doses of chemotherapy1. Severe bone marrow hypoplasia

2. Allowing regrowth of normal residual stem cells to regrow faster than leukemic cells.

Remission Normal neutrophil count Normal platelet count Normal hemoglobin level

Remission defined as < 5% blast in the bone marrow

(Treatment of acute leukemias)(Treatment of acute leukemias)

ConsolidationConsolidation

• Repeated cycles of different or same drugs to those used during induction

• Higher doses of chemotherapy

• Advantage: Delays relapse and improved survival

(Treatment of acute leukemias)(Treatment of acute leukemias)

MaintenanceMaintenance

• Smaller doses for longer period

• Produce low neutrophil counts & platelet counts

• Objective is to eradicate progressively any remaining leukemic cells.

(Treatment of acute leukemias)(Treatment of acute leukemias)

Supportive CareSupportive Care

1. Vascular access (Central line)

2. Prevention of vomiting

3. Blood products (Anemia, ↓Plat)

4. Prevention & treatment of infections

(antibiotics)

5 Management of metabolic complications

ALL vs AMLALL vs AML

ALL

Induction

Consolidation

Maintenance

CNS prophylaxis all patients

AML

Induction

Consolidation

No maintenance

CNS – Selected group only

Definition: Neoplastic proliferations of mature haemopoeitic cells.

Types: Chronic lymphocytic leukemia (CLL)

Chronic myeloid leukemia (CML)

CHRONIC LEUKEMIASCHRONIC LEUKEMIAS

Neoplastic proliferations of mature lymphocytes. Distinguished from ALL by

a. Morphology of cells.b. Degree of maturation of cells.c. Immunologically immature blasts in

ALL.d. CLL affects mainly elderly.

CHRONIC LYMPHOCYTIC CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL)LEUKAEMIA (CLL)

SYMPTOMS OF CLLSYMPTOMS OF CLL

May be entirely absent in 40%

Weakness, easy fatigue, vague sense

of being ill

Night sweats

Feeling of lumps

Infections esp pneumonia

PHYSICAL EXAMINAITON-CLLPHYSICAL EXAMINAITON-CLL

Pallor

Lymphoadenopathy

a. Cervical, supraclavicular nodes more commonly involved than axillary

or inguino-femoral

b. Non-tender, not painful, discrete, firm, easily movable on palpation

Splenomegely, mild to moderate

Hepatomegaly

Stage

(0-1) - lymphocytosis LNS.

(II) - above + hepatosplenomagely.

(III-IV) - Anaemia. Hb< 10 g/l Thrombocytopenia.

Platelet count : <100x109/L.

CLINICAL STAGING-CLLCLINICAL STAGING-CLL

LABORATORY TESTS-CLLLABORATORY TESTS-CLL

CBC Lymphocyte count > 5 x 109/L (5 -500 x 109/L ). Platelets may be decreased Hb may be low Blood film PB immunophenotyping Bone marrow biopsy (needed before starting

treatment) Imaging

Observation Chemotherapy. Oral chlorambucil

Fludarabine, Cyclo Immunotherapy Anti-CD 20 (Rituximab), Anti-CD 52 (Alemtuzumab) FC-R is the current standardIndications for starting chemotherapy

a. Progressive Symptoms

b. Progressive Anemia or Thrombocytopenia

c. Bulky LN, large spleen

d. Recurrent Infections

TREATMENT OF CLLTREATMENT OF CLL

CML is a clonal stem cell disorder characterised by increased proliferation of myeloid elements at all stages of differentiation.

Incidence increases with age, M > F.

CHRONIC MYELOID LEUKEMIACHRONIC MYELOID LEUKEMIA

CML is characterised by 3 distinct phases

a) Chronic Phase:

Proliferation of myeloid cells, which show a full range of maturation.

b) Accelerated Phase decrease in myeloid differentiation occurs.

c) Blast crisis (acute leukemia)

Symptoms

Asymptomatic (50% of patients) Fatigue Weight loss Abdominal fullness and anorexia Abdominal pain, esp splenic area Increased sweating Easy bruising or bleeding

CLINICAL PRESENTAITON OF CMLCLINICAL PRESENTAITON OF CML

Splenomegaly (95%)

(50% of patients have a palpable spleen ≥ 10 cm BCM,

Usually firm and non-tender)

Hepatomegaly (50%)

SIGNS OF CMLSIGNS OF CML

Chronic phase

Peripheral blood – neutrophil leukocytes 20,000 - >500, 000/ L

basphilia LAP

scoreblasts < 5% Nucleated RBCsThrombocytosis

Anaemia

DIAGNOSIS OF CMLDIAGNOSIS OF CML

CYTOGENETICS OF CMLCYTOGENETICS OF CML

Philadelphia (Ph) chromosome is an acquired cytogenetic abnormality in all leukaemia cells in CML

Reciprocal translocation of

chromosomal material between

chromosome 22 and chromosome 9.

t(9;22)

Karyotype 46,XX,t(9;22)(q34;q11.2) -- Ph chromosome

CML-Treatment Response Criteria

• Hematological response Normalisation of blood count• Cytogenetic response Major cytogenetic response 1-35% Ph +ve cells in metaphase Minor cytogenetic response 36-65% Ph +ve cells in metaphase• Molecular response Absence of BCR/ABL gene

CML-Principles of Treatment

• Control & prolong chronic phase (non-curative)

- Tyrosine kinase inhibitors-Imatinib (Glivec)

- Alpha-Interferon

- Oral chemotherapy (Hydroxyurea, ARA-C)• Eradicate malignant Clone (curative)

- Allogeneic BM/stem cell transplantation

- Alpha Interferon?

- Imatinib? 2nd line TKIs

• Tyrosine kinase inhibitor (TKI) Imatinib (Glivec) is the first line treatment

• In resistent cases 2nd line TKIs (Nilotinib,

Dasatinib, Bosutinib) very useful (approved as 1st line)

• Allogenic bone marrow trasnsplantation can be curative in pts resisrant to TKIs but has significant complications & mortality

Accelerated and blast phaseImatinib, 2nd line TKIs

Treat like AML or ALL followed by BMT

TREATMENT OF CMLTREATMENT OF CML

1. LA P Score

2. Philadelphia Chromosome

3. Basophilia

4. Splenomegaly

CML VS LEUKEMOID CML VS LEUKEMOID REACTIONREACTION

Bone marrow or PBSC Bone marrow or PBSC transplantation in leukemiastransplantation in leukemias

Types of transplant1. Autologous transplant

2. Allogeneic Transplant

Purpose of transplant

Autologous -To deliver a high dose of chemo to kill any residual cancer

(lymphoma, multiple myeloma)

Allogeneic

-To eradicate residual leukemia cells

-Graft vs leukemia effect

Bone marrow or PBSC Bone marrow or PBSC transplantation in leukemiastransplantation in leukemias

Technique of transplantationMHC + HLA matchingChemotherapyTotal body irradiationGVHD prophylaxis

Complications of transplantation• Prolonged BM suppression (graft failure)

• Serious infections

• Mucositis

• Graft versus host disease (GVHD)

Complications of BMT

Lung toxicity13%

Other Organ toxicity

4%

Hemorrhage5%

Other5%

Relapse12%GVHD

29%

Infection26%

VOD6%