Week 10: Leukemia

AMLAML ALLALL FAB classificationFAB classification WHO classificationWHO classification CytochemistriesCytochemistries MPOMPO SBBSBB PASPAS

OROORO CD markersCD markers FlowcytometryFlowcytometry CMLCML CLLCLL KaryotypeKaryotype Ph chromosomePh chromosome LAPLAP

Signs and Symptoms of AML

Insidious nonspecific onsetInsidious nonspecific onset Pallor due to anemiaPallor due to anemia Febrile due to ineffective WBCFebrile due to ineffective WBC Petechiae due to thrombocytopeniaPetechiae due to thrombocytopenia Mucus membrane and gum bleed in Mucus membrane and gum bleed in

M4 and M5M4 and M5 Bone painBone pain

Typical Labs of AML

LeukocytosisLeukocytosis BlastemiaBlastemia Leukemic hiatusLeukemic hiatus Auer rods in M2, M3, Auer rods in M2, M3,

M4M4 ThrombocytopeniaThrombocytopenia AnemiaAnemia >20% blasts in BM>20% blasts in BM

Other Findings

CD 13 and CD 33 in flowcytometryCD 13 and CD 33 in flowcytometry CytochemistriesCytochemistries

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

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 Meylomonocytic LeukemiaM4 -- Acute Meylomonocytic Leukemia M5 -- Acute Monocytic LeukemiaM5 -- Acute Monocytic Leukemia M6 -- Erythroleukemia (DiGuglielmo’s)M6 -- Erythroleukemia (DiGuglielmo’s) M7 -- Megakaryoblastic LeukemiaM7 -- Megakaryoblastic Leukemia

M1 and M2

Myeloperoxidase

(MPO)

p-Phenylene diamine + Catecol + H2O2

MPO > Brown black deposits

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

Double Esterase in M4

NSE with Fl inhibition

Histiocyte

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)

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)

•Antigens •B-Lineage •T-Lineage

•HLA Dr•Tdt•CD34

•+•+•+

•0 to +•+•0

•CD19•C 22•CD10•CD20•Cyt-•SIg

•+•cALL and older•cALL and older•Pre-B and older•Pre-B and older•B-ALL

•Pre-T•Pre-T•Pre-T•0•0•0

•CD7•CD3•CD5•CD2•CD1

•0•0•0•0•0

•+•+•+•T-ALL•T-ALL

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

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

CML

Bone marrow aspirateand biopsy

Pseudo-Gaucher’s cells in BM

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 segs and bandsCount 100 consecutive segs and 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 and 22 translocation almost specific to CML9 and 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

FAB (1982) Classification of Myeloproliferative Disease (MPD)

Chronic Myelocytic Leukemia (CML)Chronic Myelocytic Leukemia (CML) Polycythemia Vera (PV)Polycythemia Vera (PV) Essential Thrombocythemia (ET)Essential Thrombocythemia (ET) Agnogenic Myeloid Metaplasia with or without Agnogenic Myeloid Metaplasia with or without

Myelofibrosis (AMM)Myelofibrosis (AMM) Benign Leukemoid ReactionBenign Leukemoid Reaction

WHO Classification of MPS (1997)

CML becomesCML becomes CML, Ph + t(9;22) BCR/ABLCML, Ph + t(9;22) BCR/ABL Chronic Neutrophilic Leukemia (CNL)Chronic Neutrophilic Leukemia (CNL) Chronic Eosinophilic Leukemia and Hyper-Chronic Eosinophilic Leukemia and Hyper-

eosinophilic Syndrome (CEL/HES)eosinophilic Syndrome (CEL/HES) PV remains PVPV remains PV ET remains ETET remains ET AMM becomesAMM becomes

Chronic Idiopathic MyelofibrosisChronic Idiopathic Myelofibrosis

Myelofibrosis

Myelofibrosis

Chronic Lymphocytic Leukemia

Exclusive in elderlyExclusive in elderly Lyphocytosis unrelated to viral infectionLyphocytosis unrelated to viral infection Hyper-mature lymphocytes with highly Hyper-mature lymphocytes with highly

condensed nucleicondensed nuclei Smudge cells: preventable with a drop of Smudge cells: preventable with a drop of

bovine albuminbovine albumin

CLLPB and BM

Bone Marrow

WHO Lymphoid Neoplasms

B cell neoplasmsB cell neoplasms T/NK cell neoplasmsT/NK cell neoplasms Hodgkin lymphoma (disease)Hodgkin lymphoma (disease)

Mature B Cell Neoplasms

B cell CLL/SLLB cell CLL/SLL B prolymphocytic B prolymphocytic

leukemialeukemia Burkitt’s lymphoma / Burkitt’s lymphoma /

leukemialeukemia Splenic marginal zone Splenic marginal zone

B lymphomaB lymphoma Extranodal marginal B Extranodal marginal B

lymphomalymphoma

Hairy cell leukemiaHairy cell leukemia Lymphoplasmocytic Lymphoplasmocytic

leukemialeukemia Mantle cell lymphomaMantle cell lymphoma Plasma cell myeloma / Plasma cell myeloma /

plasmacytomaplasmacytoma Follicular lymphomaFollicular lymphoma Diffuse large B Diffuse large B

lymphomalymphoma

T/NK Cell Neoplasms

T prolymphocytic T prolymphocytic leukemialeukemia

T granular T granular lymphocytic leukemialymphocytic leukemia

Aggressive NK cell Aggressive NK cell leukemialeukemia

Adult T lymphoma / Adult T lymphoma / leukemialeukemia

Mycosis fungoides Mycosis fungoides (Sezary syndrome)(Sezary syndrome)

Anaplastic large cell Anaplastic large cell lymphomalymphoma

Hepatosplenic T Hepatosplenic T lymphomalymphoma

Peripheral T lymphomaPeripheral T lymphoma Immunoblastic T Immunoblastic T

lymphomalymphoma