LEUKEMIAS

Dr SAPNA M

Leukemoid Reaction• A leukemoid reaction describes a high

WBC count with neutrophilia,usually in

response to infection.• The WBC count may be as high as 50,000

/microL and can easily mimic CML or

AML.

• Serum leukocyte alkaline phosphatase is normal or elevated in leukemoid reaction, but is depressed in chronic myelogenous leukemia.

• The bone marrow in a leukemoid reaction, if examined, may be hypercellular but is otherwise typically unremarkable.

Features Suggesting Leukemoid Reaction

• Toxic granulation.• High LAP score.• Presence of an obvious cause for the

neutrophilia.

• As noted above, a leukemoid reaction is typically a response to an underlying medical issue. Causes of leukemoid reactions include:• Hemorrhage• Drugs

– Use of Sulfa drugs– Use of Dapsone– Use of glucocorticoids– Use of G-CSF or related growth factors– All-trans retinoic acid (ATRA)

• Infections – Clostridium difficile– Tuberculosis– Pertussis– Infectious mononucleosis (lymphocyte predominant)– Visceral Larva Migrans (eosinophil predominant)

• Asplenia• Diabetic ketoacidosis• Organ necrosis

– Hepatic necrosis– Ischemic colitis

• As a feature of Trisomy 21 in infancy (incidence of ~10%)• As a paraneoplastic phenomenon (rare)

Leukemoid reaction

LAP score

What is leukemia?

Leukemia is cancer of the white blood cells. White blood cells help your body fight infection. Your blood cells form in your bone marrow. In leukemia, however, the bone marrow produces abnormal white blood cells. These cells crowd out the healthy blood cells, making it hard for blood to do its work.

Hematopoieticstem cell

Neutrophils

Eosinophils

Basophils

Monocytes

Platelets

Red cells

Myeloidprogenitor

Lymphoidprogenitor

B-lymphocytes

T-lymphocytes

Plasmacells

naïve

ALL

AML

Myeloid maturation

myeloblast

promyelocytemyelocytemetamyelocyte band neutrophil

MATURATION

Adapted and modified from U Va website

Definition • Leukemia: is a cancer of the blood or bone marrow

characterized by abnormal proliferation of blood cells,usually WBCs(Leukocytes).

• Acute leukemia: rapid increase of immature blood cells.

• Chronic leukemia: excessive build up of relatively mature,but still abnormal blood cells.

About the Disease

• Leukemia, lymphoma and myeloma are cancers that originate in the bone marrow (leukemia & myeloma) or in lymphatic tissues (lymphoma).

Different Types of Blood Cancers

• Leukemia• Non-Hodgkin Lymphoma• Hodgkin Lymphoma• Myeloma• Myelodysplastic Syndromes

What is Leukemia

• Greek word which means “white blood”• Leukemia is when cells spread rapidly and destroy living

tissue.• It grows/invades the bone marrow which is the factory of

blood and replaces normal blood elements with cancer cells.

• Cancer cells replace all bone marrow cells which causes infection and bleeding problems.

• Leukemia is basically white blood cells that don’t work well and cause trouble.

Signs and Symptoms• Most of the signs and symptoms are due to: 1-Anemia. 2-Leukopenia. 3-Thrombocytopenia.• Bicytopenia,Pancytopenia.• All symptoms associated with leukemia can be attributed to other diseases, consequently,leukemia is always diagnosed by laboratory investigations.

Causes• Leukemia,like other malignancies, results from somatic mutations in the DNA.• Certain mutations produce leukemia by activating oncogenes or deactivating tumor suppressor genes.• These mutations may occur spontaneously or as a result of exposure to radiation or carcinogenic substances,and likely to be influenced by genetic factors.

Causes-cont’d• Ionizing radiation• Viruses: Human T-lymphotropic virus (HTLV-1)• Chemicals: Benzene,chemotherapy.• Smoking: slight increase in leukemia

incidence.• Genetic predisposition toward developing

leukemia: Down syn.,Fanconi anemia

Classification• Multiple classification systems.• FAB classification:

French-American-British Classification.• FAB Classification relies on morphologic,

cytochemical,and immunophenotyping

criteria to define 8 major subtypes

(M0-M7)

FAB vs WHO Classification• French-American-British (FAB) Cx

– Cellular morphology and cytochemical stain– Acute leukemia as > 30% bone marrow blasts– Widely used

• World Health Organization Cx– Cellular morphology and cytochemical stain– Immunologic probes of cell markers, cytogenetics,

molecular abnormalities & clinical syndrome– Acute leukemia as > 20% bone marrow blasts– Standard for diagnosis

Acute myeloid leukemias (AML) Classification - FAB

1. M0: minimally differentiated

2. M1: myeloblastic leukemia without maturation

3. M2: myeloblastic leukemia with maturation

4. M3: hypergranular promyelocytic leukemia

5. M4: myelomonocytic leukemia

6. M4Eo: variant, increase in marrow eosinophils

7. M5: monocytic leukemia

8. M6: erythroleukemia (DiGuglielmo's disease)

9. M7: megakaryoblastic leukemia

AML classification - WHO

AML not otherwise categorized1. AML minimally differentiated2. AML without maturation3. AML with maturation4. Acute myelomonocytic leukemia5. Acute monocytic leukemia6. Acute erythroid leukemia7. Acute megakaryocytic leukemia8. Acute basophilic leukemia9. Acute panmyelosis with myelofibrosis

Acute vs Chronic Leukemia

Acute Chronic

Age Children & young adults

Middle age and elderly

Onset Sudden insidious

Duration weeks to months years

WBC count Variable High

Acute vs Chronic Leukemia

Acute Chronic

Platelets Low Early: Normal/ HighLate: Low

Anemia High (>90%) None/ mild

Predomi-nant cells

Blast cells Mature cells

AML = myeloblastALL= lymphoblast

CML=granulocytesCLL=lymphocytes

Acute vs Chronic Leukemia

Acute Chronic

Marrow cellularity

>20% marrow blasts (WHO)> 30% marrow blasts (FAB)

>70% marrow cellularity (hypercellular); No dysplasia

Diagnosis PBS, BM exam, cytochemical stains, surface markers, EM,chromosome

PBS (peripheral blood smear)

Acute Myeloid Leukemia

Definition• Acute myeloid leukemia (AML): acute

myelogenous leukemia,acute non-

lymphocytic leukemia.• AML consists of a group of relatively well-

defined hematopoietic neoplasms

involving precursor cells commited

to the myeloid line(WBCs,RBCs,PLTs)

Chracteristics• AML is characterized by a clonal proli- feration of myeloid precursors with a reduced capacity to differentiate into mature cellular elements.• As a result,there is an accumulation of leukemic blasts or other immature forms in the BM,peripheral blood,and other tissues with a variable reduction in the production of normal RBCs,platelets, and mature granulocytes.

Specific:• M2 : Chloroma:-presents as a mass lesion

‘tumor of leukemic cells’ • M3 : DIC• M4/M5 : Infiltration of soft tissues,

gum infiltration, skin deposits ,Meningeal involvement-headache, vomiting, eye symptoms

A

B

C

Chloromas

NEJM 1998

Gum hypertrophy

Leukostasis

• accumulation of blasts in microcirculation with impaired perfusion

• lungs: hypoxemia, pulmonary infiltrates

• CNS: stroke• only seen with WBC >> 50 x 109/L

Pathological Features• CBC and differential.• Blood film (smear).• Bone marrow examination: BM aspirate

and trephine biopsy.

1-Morphology.

2-Immunephenotyping.

3-Cytogenetics and molecular biology.

Jemshidi trephine & Salah aspiration needle

Bone marrow in acute leukemia

• necessary for diagnosis• useful for determining type• useful for prognosis• Acute leukemias are defined by the

presence of > 20% blasts in bone marrow (% of nucleated marrow cells)

• Bone marrow aspirate & trephine: Hypercellular, –blast cells ( > 20%), –presence of Auer rods - AML type

• Cytochemistry : Special stains to differentiate AML from ALL ; Positivity with Sudan black & Myeloperoxidase (MPO) in AML

WBC Count in AML• WBC count in AML can be high,normal,or

low.• Median WBC count in AML is 15 000/uL.• 20% of patients have > 100 000/uL• 25-40% of patients have <5000/uL• 95% of patients have blast cells on blood

film.

Distinguishing AML from ALL

• light microscopy– AML: Auer rods, cytoplasmic granules– ALL: no Auer rods or granules.

• flow cytometry• special stains (cytochemistry)

Cytochemical Stains

• Since the early 20th century, cytochemical staining of cells has been a useful tool in differentiating hematopoietic diseases.

• Smears and imprints made from bone marrow, lymph nodes, spleen, or peripheral blood are preferred.– In enzymatic techniques, fresh smears are used to

ensure optimal enzyme activity

• Certain elements may be inhibited during the fixation of smears and imprints

Myeloperoxidase (MPX/MPO)• The proxidase enzyme reacts with H2O2 & release O2,

which oxidizes the indicator dye and produce orange-brown granules in the cells (3-amino-9-erythrocarbazol)

• Enzyme MPX is found in the 1o granules of granulocytes, neutrophils and precursors (from the promyelocyte stage on) & eosinophils

• Monocytes may be weakly pos

• Leukemic myeloblasts are usually pos and Auer rods stain very strongly

• Used for differentiating AML (+) from ALL (-)

• Normal bone marrow smear <5 days old used for control slides (promyelocyte - neutrophils)

MPO (right) & Sudan black (left) showing intense localised positivity in blasts

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 (NSE)• Nonspecific esterase liberates alpha-naphthyl from

the substrate alpha-naphthyl acetate. Alpha-naphthyl is couples with the dye molecule to form dark reddish-brown granules

• Monocytes, monblasts, macrophages, histiocytes, megakaryocytes and some carcinomas are NSE pos

• Abnormal erythroblasts are strongly pos

• Lymphocytes are neg or may show dot positivity

Non-Specific Esterase

Monocytic Line

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

NSE continued

• Used for differentiating myelomonocytic and monocytic leukemia (+) from granulocytic leukemia (-)

• Monocyte NSE are fluoride sensitive

• Peripheral smear with appreciable # of monocytes or a normal BM smear used for control slides

http://www.healthsystem.virginia.edu/internet/hematology/hessedd/malignanthematologicdisorders/leukemias/aml-m4.cfm

Double Esterase in M4

NSE with Fl inhibition

Histiocyte

Periodic Acid Schiff (PAS)• Periodic acid oxidizes glycogen, mucoproteins, and

other high-molecular weight carbohydrates to aldehydes.

• Aldehydes react with colorless Schiff reagent, staining them a bright red-pink

• Megakaryocytes and platelets stain strongly pos

• Normoblasts will stain Pos

• Lymphoblasts in ALL show course and granular (block) positivity

PAS Continued

• Myeloblasts are Neg

• Aids in diagnosis of ALL, erythroleukemia, and megakaryoblastic leukemia

• Normal bone marrow smear used for control slides

http://www.pathologyoutlines.com/leukemia.html

Periodic Acid Schiff

Periodic acid + Glycogen oxidation > Aldehyde + Schiff reagent

(para-rosaniline, Na metabisulfite) > Red deposit

AML

AML

Auer rods in AML

ALL

P. Smear AML

MO: Minimally differentiated

• Undifferentiated Blasts (No maturation)

• Myeloid phenotype - CD13, CD33, CD34

• (-) SBB, MPO• Negative: Auer rods,

Esterase

M1 AML without maturation

> 30% myeloblasts Large cells, round nucleus Nucleoli (+) scanty cytoplasm >3% MPO, SBB (+) <20% NSE (+) CD 13, 33, 117

M1 AML without maturation

M2 AML with maturation

• Common type• >30% myeloblasts• >10% granulocyte• Kidney shape nucleus• Nucleoli (+)• (+) Auer rods • Eosinophilic granules• >50% MPO, SBB (+)• CD 13, 33

M2 AML with maturation

Auer Rods

M3 (hypergranular promyelocytic)

• Promyelocyte-predominant • Large, kidney shape • (+) Auer rods (faggot cells) • basophilic, bilobed nuclei• CD 13,33• High incidence of DIC

Acute myeloid leukemia with very abnormal cells (AML M3/ t15;17)

M4 Acute myelomonocytic

• >30% myeloblast (FAB)• >20% granulocyte• >20% promonocytes and

monocytes• CD 11, 13, 33,14• (+) Auer rods common• High serum lysozyme level

– M4Eo = w/ eosinophilia

M3

M5

M4

M5: acute monocytic leukemia

1. M5a – without maturation– Monoblasts , few promonocytes

2. M5b – with maturation – Blast, Promonocytes (BM), Monocytes

(Blood)

M5a

• Monoblast ameboid with round to oval nuclei,

• prominent nucleoli, • <20% promonocytes/mono• Vacuolated cytoplasm

AML M5a

M5b

• > 20% promonocytes, monocytes

• Promonocytes folded, convulated nucleus

• Azurophilic granules

AML M5b

M6 - erythroleukemia

Large, bizarre,round-to-oval cells

(+) nucleoli > 50% Erythroblasts > 30 % Myeloblasts CD 45,71 Glycophorin A CD 13, 15,33 myeloblast PAS (+)

M6 (erythroblast)

M6 (erythroblast)

M7 – acute megakaryoblastic

• >30% megakaryoblasts• platelet like granules on

PAS stain• NSE (but not BE) (+) • Myeloid blasts may show

SBB or MPO (+) • CD 41,42,61

M7 (Megakaryoblast)

Megakaryoblast

Acute Nonlymphocytic Leukemias

• Acute lymphoblastic leukemias

FAB Classification of ALL

L1: Small homogeneous blasts; mostly in children

L2: Large heterogeneous blasts; mostly in adults

L3: “Burkitt” large basophilic B-cell blasts with vacuoles

WHO Classification of Lymphoproliferative Syndromes

Precursor B Lymphoblastic Leukemia/Lymphoma (ALL/LBL) -- ALL in children (80-85% of childhood ALL); LBL in young adults and rare; FAB L1 or L2 blast morphology

Precursor T ALL/LBL -- 15% of childhood ALL and 25% of adult ALL

Burkitt Leukemia/Lymphoma (FAB L3)

• Confirmation:– Immunophenotyping–Molecular genetics–Cytogenetics: Chromosomal abnormalities

ALL L1

L3L2

Burkits / ALL L3

Prognostic factors

• High WBC – relapse in testis /cns• Infants <1 yr, children >10 yr poor outcome• L1- good prognosis• L2,3- bad prognosis

Prognosis• The response to treatment and overall survival of patients with AML are heterogenous.• Prognostic factors are related to patient and tumor characteristics: 1-Age 2-Performance status 3- Karyotype

Adverse Clinical Predictors

• Advanced age.• Poor performance status.• History of exposure to cytostatic agents or

radiotherapy.(Therapy-related AML).• History of MDS or other hematological

diseases

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