Leukemia/Lymphoma

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Components and General Properties of Blood

Seven kinds of formed elementsSeven kinds of formed elements Erythrocytes: red blood cells (RBCs)Erythrocytes: red blood cells (RBCs) Platelets (thrombocytes) (clotting cells)Platelets (thrombocytes) (clotting cells)

Cell fragments from special cell in bone marrowCell fragments from special cell in bone marrow Leukocytes: white blood cells (WBCs)-5 kindsLeukocytes: white blood cells (WBCs)-5 kinds

Components and General Properties of Blood

Five leukocyte types divided into two categoriesFive leukocyte types divided into two categories Granulocytes (with granules)Granulocytes (with granules)

• 3)Neutrophils3)Neutrophils• 4)Eosinophils4)Eosinophils• 5)Basophils5)Basophils

Agranulocytes (without granules)Agranulocytes (without granules)

• 6)Lymphocytes6)Lymphocytes• 7)Monocytes7)Monocytes

Components and General Properties of Blood

Neutrophil

Erythrocyte

Eosinophil

Monocyte

Neutrophil

Basophil

Neutrophil

Platelets

Monocyyte

Smalllymphocyte

Young (band)neutrophil

Smalllymphocyte

Largelymphocyte

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Leukocytes

Least abundant formed elementLeast abundant formed element– 5,000 to 10,000 WBCs/5,000 to 10,000 WBCs/LL

Protect against infectious microorganisms and other Protect against infectious microorganisms and other pathogenspathogens

Conspicuous nucleusConspicuous nucleus Spend only a few hours in the bloodstream before migrating Spend only a few hours in the bloodstream before migrating

to connective tissueto connective tissue Retain their organelles for protein synthesisRetain their organelles for protein synthesis GranulesGranules

All WBCs have lysosomes called nonspecific (azurophilic) granules: All WBCs have lysosomes called nonspecific (azurophilic) granules: inconspicuous so cytoplasm looks clearinconspicuous so cytoplasm looks clear

Granulocytes have specific granules that contain enzymes and other Granulocytes have specific granules that contain enzymes and other chemicals employed in defense against pathogenschemicals employed in defense against pathogens

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Types of Leukocytes

GranulocytesGranulocytes Neutrophils (60% to 70%): polymorphonuclear leukocytesNeutrophils (60% to 70%): polymorphonuclear leukocytes

Barely visible granules in cytoplasm; three- to five-lobed Barely visible granules in cytoplasm; three- to five-lobed nucleusnucleus

Eosinophils (2% to 4%) Eosinophils (2% to 4%) Large red-orange granules; Large red-orange granules;

Basophils (less than 1%) Basophils (less than 1%) Large, abundant, violet granulesLarge, abundant, violet granules

AgranulocytesAgranulocytes Lymphocytes (25% to 33%) Lymphocytes (25% to 33%)

Variable amounts of bluish cytoplasm (scanty to abundant); Variable amounts of bluish cytoplasm (scanty to abundant); ovoid/round, uniform dark violet nucleus ovoid/round, uniform dark violet nucleus

Monocytes (3% to 8%) Monocytes (3% to 8%) Largest WBC; generally ovoid, kidney-, or horseshoe-shaped Largest WBC; generally ovoid, kidney-, or horseshoe-shaped

nucleusnucleus

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Granulocytes

Neutrophils—increased numbers in bacterial infectionsNeutrophils—increased numbers in bacterial infections Phagocytosis of bacteriaPhagocytosis of bacteria Release antimicrobial chemicalsRelease antimicrobial chemicals

Eosinophils—increased numbers in parasitic infections, collagen Eosinophils—increased numbers in parasitic infections, collagen diseases, allergies, diseases of spleen and CNSdiseases, allergies, diseases of spleen and CNS Phagocytosis of antigen–antibody complexes, Phagocytosis of antigen–antibody complexes,

allergens, and inflammatory chemicalsallergens, and inflammatory chemicals Release enzymes to destroy large parasitesRelease enzymes to destroy large parasites

Basophils—increased numbers in chickenpox, sinusitis, Basophils—increased numbers in chickenpox, sinusitis, diabetesdiabetes Secrete histamine (vasodilator): speeds flow of blood to an injured Secrete histamine (vasodilator): speeds flow of blood to an injured

areaarea Secrete heparin (anticoagulant): promotes the mobility of other Secrete heparin (anticoagulant): promotes the mobility of other

WBCs in the areaWBCs in the area

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Agranulocytes

Lymphocytes—increased numbers in diverse Lymphocytes—increased numbers in diverse infections and immune responsesinfections and immune responses

Destroy cells (cancer, foreign, and virally infected cells)Destroy cells (cancer, foreign, and virally infected cells) ““Present” antigens to activate other immune cellsPresent” antigens to activate other immune cells Coordinate actions of other immune cellsCoordinate actions of other immune cells Secrete antibodies and provide immune memorySecrete antibodies and provide immune memory

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Agranulocytes

Monocytes—increased numbers in viral infections Monocytes—increased numbers in viral infections and inflammationand inflammation

Leave bloodstream and transform into macrophagesLeave bloodstream and transform into macrophages Phagocytize pathogens and debrisPhagocytize pathogens and debris ““Present” antigens to activate other immune cells—antigen-Present” antigens to activate other immune cells—antigen-

presenting cells (APCs)presenting cells (APCs)

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The Leukocyte Life Cycle

Leukopoiesis—production of white blood cellsLeukopoiesis—production of white blood cells Pluripotent stem cells (PPSCs)Pluripotent stem cells (PPSCs)

Myeloblasts—form neutrophils, Myeloblasts—form neutrophils, eosinophils, basophilseosinophils, basophils

Monoblasts—form monocytesMonoblasts—form monocytesLymphoblasts give rise to all forms of Lymphoblasts give rise to all forms of

lymphocyteslymphocytes

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Leukopoiesis

leaves

Lymphoblast

B prolymphocyteB lymphocyte

T lymphocyte

NK cell

T prolymphocyte

NK prolymphocyte

Monoblast Promonocyte Monocyte

Basophil

Eosinophil

Neutrophil

Pluripotentstem cell

Colony-formingunits (CFUs)

Precursorcells

Maturecells

Eosinophilicmyelocyte

Eosinophilicpromyelocyte

Eosinophilicmyeloblast

EosinophilicCFU

BasophilicCFU

NeutrophilicCFU

MonocyticCFU

LymphocyticCFU

Basophilicmyeloblast

Neutrophilicmyeloblast

Neutrophilicpromyelocyte

Basophilicpromyelocyte

Basophilicmyelocyte

Neutrophilicmyelocyte

What Is Leukemia?

Cancer of the white blood cellsCancer of the white blood cells Acute or ChronicAcute or Chronic Affects ability to produce normal blood Affects ability to produce normal blood

cellscells Bone marrow makes abnormally large Bone marrow makes abnormally large

number of immature white blood cells number of immature white blood cells called blastscalled blasts

History

Means “white blood” in GreekMeans “white blood” in Greek

Discovered by Dr. Alfred Velpeau in Discovered by Dr. Alfred Velpeau in France, 1827France, 1827

Named by pathologist Rudolf Virchow in Named by pathologist Rudolf Virchow in Germany, 1845Germany, 1845

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Leukocyte Disorders

Leukemia—cancer of hemopoietic tissue that usually Leukemia—cancer of hemopoietic tissue that usually produces an extraordinary high number of circulating produces an extraordinary high number of circulating leukocytes and their precursorsleukocytes and their precursors

Myeloid leukemia: uncontrolled granulocyte productionMyeloid leukemia: uncontrolled granulocyte production Lymphoid leukemia: uncontrolled lymphocyte or monocyte Lymphoid leukemia: uncontrolled lymphocyte or monocyte

productionproduction

Acute vs Chronic Leukemia

Acute leukemia: appears suddenly, progresses Acute leukemia: appears suddenly, progresses rapidly, death within months –blasts found in rapidly, death within months –blasts found in peripheral bloodperipheral blood

Chronic leukemia: undetected for months, Chronic leukemia: undetected for months, survival time average of 3 yearssurvival time average of 3 years

Effects: normal cell percentages disrupted; Effects: normal cell percentages disrupted; impaired clotting; opportunistic infectionsimpaired clotting; opportunistic infections

Main Types

Acute Lymphocytic Leukemia (ALL)Acute Lymphocytic Leukemia (ALL)

Acute Myelogenous Leukemia (AML)Acute Myelogenous Leukemia (AML)

Chronic Lymphocytic Leukemia (CLL)Chronic Lymphocytic Leukemia (CLL)

Chronic Myelogenous Leukemia (CML)Chronic Myelogenous Leukemia (CML)

Demographics of Leukemia Patients (2001 Data)

ALL11%

CLL26%

AML31%

CML15%

others17%

Total Reported Cases = 31,500

CLL=Chronic Lymphocytic

ALL=Acute Lymphocytic

CML=Chronic Mylogenous

AML=Acute Mylogenous

Pictures Of Blood

Normal human blood

White Cell Red Cell

Platelet

Blood with leukemia

BlastsRed Cell

Platelet

White Cell

Development of Leukemia in the Bloodstream

Stage 1- Normal Stage 2- Symptoms Stage 3- Diagnosis

Stage 4- Worsening

Stage 5a- Anemia

Stage 5b- Infection

Legend

White Cell

Red Cell Platelet Blast Germ

Causes

High level radiation/toxin exposureHigh level radiation/toxin exposure

VirusesViruses

GenesGenes

ChemicalsChemicals

Mostly unknownMostly unknown

Signs and Symptoms of AML

Insidious nonspecific onsetInsidious nonspecific onset Pallor due to anemiaPallor due to anemia Febrile (fever) due to ineffective WBCFebrile (fever) due to ineffective WBC Petechiae (skin bruising) due to Petechiae (skin bruising) due to

thrombocytopeniathrombocytopenia Bone painBone pain

PetechiaePetechiae

Typical Labs of AML

LeukocytosisLeukocytosis BlastemiaBlastemia Leukemic hiatusLeukemic hiatus Auer rods – only found Auer rods – only found

in myelocytic blastsin myelocytic blasts ThrombocytopeniaThrombocytopenia AnemiaAnemia >20% blasts in Bone >20% blasts in Bone

MarrowMarrow

Auer Rods

Auer Rod

CD Markers

The The cluster of differentiationcluster of differentiation ( (cluster of cluster of designationdesignation) (often abbreviated as ) (often abbreviated as CDCD) is a ) is a protocol used for the identification and protocol used for the identification and investigation of cell surface molecules investigation of cell surface molecules providing targets for providing targets for immunophenotyping of cells. of cells.

The CD markers can be used to identify the The CD markers can be used to identify the type of cell.type of cell.

Other Findings

CD 13 and CD 33 in flowcytometryCD 13 and CD 33 in flowcytometry Cytochemistries-stains that can be used to Cytochemistries-stains that can be used to

differentiate leukemiasdifferentiate leukemias MyeloperoxidaseMyeloperoxidase Sudan black BSudan black B Choloroacetate esterase (specific)Choloroacetate esterase (specific) Nonspecific esteraseNonspecific esterase

Flow Cytometry

Large clustering of CD 33s shows presence of blasts

FAB (1976) Classification

M0 -- Undifferentiated AMLM0 -- Undifferentiated AML M1 -- AML without maturationM1 -- AML without maturation M2 -- AML with maturationM2 -- AML with maturation M3 -- Acute Promyelocytic LeukemiaM3 -- Acute Promyelocytic Leukemia M4 -- Acute Myelomonocytic LeukemiaM4 -- Acute Myelomonocytic Leukemia M5 -- Acute Monocytic LeukemiaM5 -- Acute Monocytic Leukemia M6 -- Erythroleukemia (DiGuglielmo’s)M6 -- Erythroleukemia (DiGuglielmo’s) M7 -- Megakaryoblastic LeukemiaM7 -- Megakaryoblastic Leukemia

Myeloperoxidase

(MPO)

p-Phenylene diamine + Catecol + H2O2

MPO > Brown black deposits

Brown deposits considered to be a positive test-differentiates AML from other leukemias

M1 and M2

M3

M5

M4

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

FAB vs WHO Classifications of Hematologic Neoplasm FAB criteriaFAB criteria

MorphologyMorphology CytochemistryCytochemistry

WHO criteriaWHO criteria MorphologyMorphology ImmunophenotypingImmunophenotyping Genetic featuresGenetic features

KaryotypingKaryotyping Molecular testingMolecular testing

Clinical featuresClinical features

WHO Classification of AML

AML with recurrent cytogenic AML with recurrent cytogenic translocationstranslocations

AML with multi-lineage dysplasiaAML with multi-lineage dysplasia AML and myelodysplasia, therapy relatedAML and myelodysplasia, therapy related AML, not otherwise categorizedAML, not otherwise categorized

AML with Recurrent Cytogenetic Translocations (WHO 1995)

t(8;21) -- some maturation of neutrophilic line; t(8;21) -- some maturation of neutrophilic line; rare in older patients; AML1/ETO fusion protein; rare in older patients; AML1/ETO fusion protein; >90% FAB M2>90% FAB M2

t(15;17) -- APL (granular and microgranular t(15;17) -- APL (granular and microgranular variants); retinoic acid receptor (RAR) leukemias; variants); retinoic acid receptor (RAR) leukemias; middle aged adults; DICmiddle aged adults; DIC

inv(16) or t(16;16) -- monocytic and granulocytic; inv(16) or t(16;16) -- monocytic and granulocytic; abnormal eosinophilic componentabnormal eosinophilic component

11q23 -- monocytic; children; most common is 11q23 -- monocytic; children; most common is t(9;11)t(9;11)

Lymphocytic Leukemias

Can involve T or B lymphocytesCan involve T or B lymphocytes B lymphs mature in bone marrowB lymphs mature in bone marrow

Responsible for making antibodiesResponsible for making antibodies T lymphs go to thymus to matureT lymphs go to thymus to mature

Are cytotoxic cells of immune systemAre cytotoxic cells of immune system

FAB Classification of ALL

L1: Small homogeneous blasts; mostly in L1: Small homogeneous blasts; mostly in childrenchildren

L2: Large heterogeneous blasts; mostly in L2: Large heterogeneous blasts; mostly in adultsadults

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

L3L2

Periodic Acid Schiff

Periodic acid + Glycogen oxidation > Aldehyde + Schiff reagent

(para-rosaniline, Na metabisulfite) > Red deposit

ALL Cytochemistries

Oil Red OOil Red O: stains L3 vacuoles: stains L3 vacuoles Terminal deoxynucleotidyl transferaseTerminal deoxynucleotidyl transferase (Tdt): (Tdt):

DNA polymerase in early lymphoblastsDNA polymerase in early lymphoblasts Cell surface markersCell surface markers (CD’s) (CD’s) Cytoplasmic and surface immunoglobulinsCytoplasmic and surface immunoglobulins: :

B-cell lineB-cell line T-cell receptor T-cell receptor (TCR)(TCR)

WHO Classification of Lymphoproliferative Syndromes

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

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

Burkitt Leukemia/Lymphoma (FAB L3)Burkitt Leukemia/Lymphoma (FAB L3)

IndicatorsIndicators FavorableFavorable PoorPoor

WBCWBC < 50,000/< 50,000/LL 50,000/50,000/LL

AgeAge 1 - 101 - 10 < 1 or < 1 or 1010

GenderGender FemaleFemale MaleMale

BlastBlast B-cellB-cell T-cell and mixedT-cell and mixed

KaryotypeKaryotype HyperploidyHyperploidy

Trisomy 4, 10, 17Trisomy 4, 10, 17

t(12;21) (TEL/AML1)t(12;21) (TEL/AML1)

HypoploidyHypoploidy

Trisomy 5Trisomy 5

t(1;19 (E2A/PBX1)t(1;19 (E2A/PBX1)

Mixed lineage leukemiaMixed lineage leukemia

T(9;22) (Ph)T(9;22) (Ph)

BM blast count BM blast count during inductionduring induction

Mkd reduction at day 7Mkd reduction at day 7 Mild reduction at day 7Mild reduction at day 7

Prognosis

Burkitt’s Lymphoma

CML

Typical Labs in CML

Leukocytosis with blastemiaLeukocytosis with blastemia ThrombocytosisThrombocytosis BasophiliaBasophilia Micro-megakaryocytesMicro-megakaryocytes Low LAP score (intermediate if infected)Low LAP score (intermediate if infected) About 10% blasts in BMAbout 10% blasts in BM Philadelphia chromosomePhiladelphia chromosome

Bone marrow aspirateand biopsy

Conventionally, a leukocytosis exceeding Conventionally, a leukocytosis exceeding 50,000 WBC/mm50,000 WBC/mm33 with a significant with a significant increase in early neutrophil precursors is increase in early neutrophil precursors is referred to as a leukemoid reaction.referred to as a leukemoid reaction.

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

Leukemoid reactions are generally benign Leukemoid reactions are generally benign and are not dangerous in and of themselves, and are not dangerous in and of themselves, although they are often a response to a although they are often a response to a significant disease statesignificant disease state

Historically, various clues including the Historically, various clues including the leukocyte alkaline phosphatase score and leukocyte alkaline phosphatase score and the presence of basophilia were used to the presence of basophilia were used to distinguish CML from a leukemoid reaction. distinguish CML from a leukemoid reaction. However, at present the test of choice in However, at present the test of choice in adults to distinguish CML is an assay for the adults to distinguish CML is an assay for the presence of the Philadelphia chromosome, presence of the Philadelphia chromosome, either via cytogenetics and FISH, or via either via cytogenetics and FISH, or via PCR for the BCR/ABL fusion gene. PCR for the BCR/ABL fusion gene.

Leukocyte Alkaline Phosphatase (LAP)

Naphthol AS-MX phosphate LAP at pH8.6 >Naphthol AS-MX + Diazonium salt

(eg, Fast blue RR) > Insoluble pigment

LAP Score

Count 100 consecutive segmented neutrophils Count 100 consecutive segmented neutrophils and bandsand bands

Score:Score:

0 = no granules0 = no granules

1+ = occasional diffuse granules1+ = occasional diffuse granules

2+ = moderate number of granules2+ = moderate number of granules

3+ = many strongly positive granules3+ = many strongly positive granules

4+ = confluent strongly positive granules4+ = confluent strongly positive granules

0 1+

2+ 3+ 4+

LAP Score

Example:

0 x 35 cells = 01+ x 30 cells = 302+ x 20 cells = 403+ x 10 cells = 304+ x 5 cells = 20

120 LAP Score

Philadelphia Chromosome

9 ;22 translocation almost specific to CML9 ;22 translocation almost specific to CML Karyotype to visualize Ph chromosomeKaryotype to visualize Ph chromosome Produces BCR/c-abl fusion oncogeneProduces BCR/c-abl fusion oncogene Gene product p190 is a hyperactive tyrosine Gene product p190 is a hyperactive tyrosine

kinasekinase Ph chromosome seen in ALL produces p210 and Ph chromosome seen in ALL produces p210 and

chronic neutrophilic leukemia produces p230chronic neutrophilic leukemia produces p230

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

FISH showing the BCR (green), ABL (orange), and BCR-ABL fusion signals (arrow): A=positive (contains a residual ABL signal), B=normal

Chronic Lymphocytic Leukemia

Exclusive in elderlyExclusive in elderly Lymphocytosis unrelated to viral infectionLymphocytosis unrelated to viral infection Hyper-mature lymphocytes with highly Hyper-mature lymphocytes with highly

condensed nucleicondensed nuclei Smudge cellsSmudge cells

CLLPB and BM

Smudge cell

Bone Marrow