APPROACH TO A CASE OF ACUTE LEUKEMIA

&CH LYMPHOPROLIFERATIVE DISORDER

Dr. Tejinder SinghDirector Professor

Department of PathologyMAMC, Delhi

Myeloid leukemia

Arises from these cells

Lymphoid leukemia

Arises from these cells

How is Leukemia Diagnosed?

Starts with history,physical examination

PS & BMA to assess morphology of leukemic cells, 5oo cells differential count on BM smears

Cytochemistry to distinguish AML from ALL

Blast immunophenotyping with lineage and maturation specific markers - Flow cytometry / BMB

Cytogenetics to identify genetic changes in leukemic cells

Molecular genetics to find the molecular abnormality

Stain is + if

Reactivity is seen in >3% of the blasts

NSE entailed >20% positivity for a separate positive designation

PAS -assessed as diffuse, block or granular

MPO stain

Unequivocal marker of myeloid differentiation

Myeloid lineage is confirmed when 3% blasts show positivity with MPO

SBB positivity goes hand in hand with MPO

1-3% of ALL show positivity with SBB but are negative for MPO Lipid droplets in lymphoblasts in these cases stain positive with SBB

Double esterase positivity

AML M4 - + for both CAE & NSE differentiating it from M5 + only for NSE

PAS

Block positivity in B lineage ALL

Acid phosphatase

+ in T lineage ALL

Oil red O

+ Vacuoles in L3 lymphoblasts

The French-American-British (FAB)

Classification of AML Mo: AML-Undifferentiated

M1: AML without maturation

M2: AML with granulocytic maturation

M3: Acute promyelocytic leukemia

M4: Acute myelomonocytic leukemia

M5: Acute monoblastic leukemia

M6: Acute Erythroid leukemia

M7: Acute Megakaryocytic leukemia

L1: Small, uniform lymphoblasts

L2: Large, pleomorphic lymphoblasts

L3: Burkitts type (vacuolated and deeply basophilic)

ALL: FAB Classification

L1 small cells

scant pale blue cytoplasm

Variation in size - mild

regular nuclear shape

homogenous chromatin Absent /small nucleoli

MPO ve

PAS +ve

ALL-L2 large heterogeneous

cells

moderate cytoplasm, often more basophilic

variable vacuoles

Irregular nuclear shape with clefting/ indentation

Less homogenous nuclear chromatin

Nucleoli are variabl

Precursor B/Tcell L/leuk-WHO

1-3% of ALL show positivity with SBB

e

ALL-L3 medium to large

homogenous cells

moderate cytoplasm intensely basophilic

prominent cytoplasmic vacuoles

round to oval nucleus,

finely stippled homogenous chromatin, at least one prominent , sharply defined nucleolus

Classified as Burkitt Leukemia variant

B-ALL subtypes

Precursor

B-ALL

Common

ALL

Pre-B-

ALL

Mature-B-

ALL

HLA-DR

cCD22

CD79a

CD19

Positive

TdT Positive Negative

CD10 Negative Positive Negative

cIgM Negative Positive Negative

sIg Negative Positive

T-ALL

Pro-T-

ALL

Pre-T-

ALL

Cortical-T-

ALL

Mature-T-

ALL

TdT Positive Negative

cCD3 Positive

CD7 Positive

CD2 Negative Positive

CD5 Negative Positive

CD4 Negative

CD8 Negative

Positive for

CD4 and

CD8

Positive for

CD4 or

CD8

CD1a Negative Positive Negative

sCD3 Negative Positive

T - ALL

15-20% of all ALL cases

Morphology of L1/L2

Acid phosphatase positivity

Worse prognosis

AML- definition (WHO)

When blasts 20% in PB/BM

< 20% if blasts contain Auer rods

20% acceptable in M6

< 20% acceptable in certain genetic abnormalities: t(8;21),inv(16), t(16;16), t(15;17)

For blasts All cells in the BM are counted and then theblasts% calculated except in M6

Myeloid sarcoma- equivalent to AML irrespective of blast%in PB/BM

BLAST cells

Myeloblasts:No paranuclear hofMPO, CD13, CD33,CD34, HLA-DR +ve

Monoblasts:CD34 ve, HLA-DR +veCo-expression of CD36/CD64

Megakaryoblasts:CD34 & HLA-DR veCD41/CD61 +ve

Promonocytes- are blast equivalents in M4 ,M5

Promyelocytes exclusively in Promyelocytic leukemia(M3)

Erythroblasts- exclusively in Proerythroid leukemia (M6b)

BLAST EQUIVALENTS

AML algorithmBlasts 20%

Blast equivalents

+nt

Bl + Bl equi 20%

T(8;21), inv(16),

t(16;16), t(15;17)

Erythroid 50% /

NEC blasts

MDS,MDS/MPN, MPN

AML

Y

Y

Y

Y

N

N

N

N

Y

Myeloid sarcoma- equivalent to AML irrespective of blast% in PB/BM

AML M0: with minimal evidence of

myeloid differentiation

Undifferentiated blasts, may resemble lymphoblasts

Large cells

Scanty grey blue cytoplasm, agranular cytoplasm

Round to slightly indented nuclear contours

Open nuclear chromatin

1-4 inconspicuous nucleoli

< 3% blasts: MPO, Sudan black positive

IHC: CD 13 and CD 33 help in establishing a myeloid lineage

AML-M0

AML-M1: AML without maturation

> 90% blasts in PS or Bone marrow

< 10% marrow NEC are maturing granulocytes

Large blasts with variable N/C ratio

1-4 nucleoli

Scant to moderate amount of pale, basophilic, agranular cytoplasm

Few cells contain fine azurophilic cytoplasmic granules

Very rare blast may demonstrate Auer rod

> 3% blasts are MPO/ Sudan black B positive

AML-M2: AML with maturation

MPO stain shows strong positivity in the blasts

Maturing myeloid precursors also positive

Auer rods highlighted by MPO staining

Hypergranular Promyelocytic leukemia Atypical/ leukemic promyelocytes:leukemia Blasts may be < 30%

Predominance of atypical leukemic promyelocytes

Known as PML-RARA positive

Moderate amount of cytoplasm

Densely packed, coarse red purple granules

Nuclei obscured by granules

Reniform/ folded/ bilobed nuclei,

Diffuse chromatin, 1-2 nucleoli

Prominence of Auer rods

Faggot cells: criss-crossing bundles of Auer rods

Presence of Phi bodies

Microgranular/ Hypogranular M3V

Shares the cytogeneticabnormality with hyper granular form, but morphologically different

Bilobed, buttock shaped or multinucleate/ multilobated nuclei

Sparse fine granules or agranular pale blue cytoplasm

Few cells may show Auer rodsor phi bodies

Intermediate cases: with features of both hyper and hypogranular variant may also be seen

AML-M4 Acute myelomonocytic

leukemia (AMML)

Variable morphologic features unlike the uniform morphology of M1, M2 and M3

Both myeloblast and monocytic components are present in varying proportions

Criteria for diagnosis:

Blasts > 30% of NEC

Monocytic component > 20% of NEC and Monocytes in Peripheral blood > 5000/mm3

Monocytic component must be confirmed by NSE, and Myeloid by MPO/ CAE

AML-M4 Eo.

Distinct entity with eosinophilic differentiation

Presence of inv. 16

Presence of eosinophilic Promyelocytes, myelocytes and metamyelocytes

Good prognosis

Abnormal eosinophils are a part of the leukemic clone as diff from M2Eo

AML-M5a: Acute Monoblastic leukemia

Propensity for extra-medullary infiltration into skin, gums etc

Monoblasts are the predominant cells, constituting

> 80% of the marrow monocytic component

Abundant pale basophilic cytoplasm

Cytoplasmic vacuolations may also be seen

Few fine azurophilic granules may be present

Round to oval, convoluted nuclei

Lacy, delicate chromatin

Prominent 2-4 nucleoli

Few promonocytes and monocytes may also be seen

Monoblasts are MPO/ Sudan black B negative

Promonocytes may show weak MPO positivity

Monoblasts are strongly positive for NSE fluoride sensitive

AML-M5A

Arrows =

monoblasts, rest

are

promonocytes

AML-M5b: Acute Monocytic leukemia

Promonocytes and monocytes are the predominant cells here

>20% cells are Blasts+promonos in Ac Monocytic leukemiaPromonocytes:

Convoluted nuclear configurationDelicate chromatin, prominent nucleoliAbundant, vacuolated cytoplasm, Few azurophilic granules

AML-M6: AML with erythroid differentiation

AML-M6a: Acute Erythroid leukemia

Criteria for diagnosis of AML-M6a:

Erythroblasts > 50% of BM nucleated cells

Myeloblasts > 20% of marrow NEC

Prominent erythroid component

Erythroblasts demonstrate bizzaremegaloblastic/ macronormoblastic Rxn

Marrow may show erythroblasts at all stages of development

AML-M6b: Pure erythroid leukemia

Early proerythroblasts: malignant component, comprise > 80% marrow cells

Large erythroblasts

Basophilic cytoplasm

Few cytoplasmic vacuoles

Round nuclei, fine chromatin

1-2 nucleoli

M7 - Ac. Megakaryocytic leukemiaBlasts > 30% of marrow

nucleated cells consisting of

myeloblasts and megakaryoblasts

>50% blasts should be of megakaryocytic lineage as per immunophenotyping CD41 / CD61

Micromgk not counted as blasts

Myelofibrosis is present

BM Aspirate is very scant

CD61 most specific marker

CD41 most sensitive marker

Infant ac megak leuk marrow fibrosis

ve

Blasts resemble lymphoblasts

AML-M7

CD 61 positivity in blasts

and platelets, confirms

their megakaryocytic

lineage

Indications for Flow cytometry in Acute Leukemia

In ALL

To know T-cell/B-cell lineage and the differentiation

Biphenotypic/ acute mixed lineage leukemia

Cytochemistry is inconclusive

Constellation of myeloid & lymphoid Ags

Flow cytometry diagnostic modality of choice

Diagnosis of AML M0

Diagnosis of ac undiff leukemia

Detection of minimal residual disease

Acute lymphoblastic leukemia vs B-cell lymphoma

Limited use in subtyping AML, e.g. APL lacks HLADR

and is CD117+

Candidate for immunotherapy (anti-CD33, Myelotarg)

80% of all leukemias can be diagnosed by morphology alone ,few cases require immunophenotyping for proper characterization

LEUKEMIA IMMUNOPHENOTYPING STRATEGY

Identify blasts/abnormal cells

Determine lineage (B, T-lymphoid or myeloid)

Determine immunological subtype (EGIL)

Search for leukemia aberrant phenotypes

Customise follow up panel for MRD

Multiparametric FCM is however preferred for IPT due to the following reasons:

Its ability to analyze a high number of cells in a short period of time

Provides simultaneous recording of information regarding expression of several antigens for an individual cell.

Allows the analysis of expression pattern of several antigens, both cytoplasmic and surface, which is crucial for lineage assignment in Mixed phenotypic acute leukemia.

Allows detection of aberrant phenotype and hence followup and detection of MRD.

Gating is important

Variable expression of CD1a, CD2, CD3, CD4, CD5, CD7, CD8

cCD 3 is lineage specific T-ALL

Lymphoproliferative Disorders

Tumours originating from lymphoid tissue mainly of lymph nodes and BM

Hodgkin`s (HD) is potentially curable with distinct histology, cl characteristics and biologic behavior. - -RS cells-clonal proliferation of lymphos , which express CD 15,CD 30

Non -Hodgkins (NHL) - a clonal expansion of B/T cells

-B cells origin -85%

-T cell origin 15%

-Follicular pattern less aggressive than diffuse

CLL , PLL ,HCL , HCL-V , SLVL

T-lymphoproliferative disorders

Guidelines for IPT of hematolymphoid neoplasms by FCM

Gujral et al IJPM 51: 161,2008

For CLPD/Peripheral lymphomas

A panel of 9 abs

CD 3

CD 5

CD 19

CD 20

CD 23

FMC 7

CD 10 Kappa

Lambda

HCL Additonal CD 25,CD 103

Plama cell dyscrasias CD 38 ,CD 138

T/NK cell -CD16 ,CD56

MBL

Monoclonal B cell lymphocytosis

Presence of

Flow cytometric IPT is particularly useful in subtyping B-cell lymphomas/leukemias

Guidelines

Gating strategies

CD 45 SS -gating

CD 19 gating - for B cell CLPD

CD 3 gating - for T cell CLPD

Cd 138 gating for P cell neoplasms

OTHER LYMPHOPROLIFERATIVE DISORDERS DIAGNOSTIC MORPHOLOGY

Prolymphocytic leukemia

Hairy cell leukemia

Leukemic phase of NHL

SLVL

Marginal zone lymphomas

Mantle cell lymphoma

Follicular small cleaved lymphoma

Large cell lymphoma

A dis of old ageSplenomegalyPancytopeniaHairy cells +-

Why pancytopeniaB.M failure

Splenomegaly sequestration of blood cells

Hairy cells express TNF alpha which

suppresses normal hematopoeisis

HCL Classic

>50yrs, Indian pts younger

Assoc with A.I. dis

CD25+ve part of receptor for key immunoregulatory hormone and are responsive to immune system hormones

Post therapy Bald lymphocytes

Hairy cell leukemia

S L V L

M/C small B-cell lymphoma

of spleen; usually > 50 yr age

Arise from post follicular

memory B-cells

Villous projections in 10 -

30% lymphocytes

PAS +ve granules in 10- 80%

cells

Indolent course: 70% cases

>10 yr survival

May progress to Large cell

lymphoma

T-cell granular lymphocytic leukemia

Chronic, indolent LPD

Clonal population of CTLs blood and bm.

Cytopenias,may be associated with A.I.D.

CD 8+CD3+CD4-, TIA +, granzyme B+

BMB - linear arrays of intravascular lymphocytes

Interstitial infiltration in b.m.

(Morice et al, Blood,Jan.02)

T CELL LGL

CD 8

CLPD why is FCM neededMorphologically similar entities

CLL, atyp CLL , MCL and SLL in leukemia phase

PLL and HCL-V

HCL,HCL-V,SLVL & Lymphoma spillover

To establish the clonality of cells (Kappa or lambda)

To differentiate B from rare T -CLPDs

To diagnose and asses prognostic parameterse.g.CD38 expression and ZAP70 expression

CONCLUSIONSApproach to a case of leukemia/LPD

Take into account history and clinical examination

Study the morphology of blood & bone marrow

Carry out cytochemistry of leukemic cells

BMB for IPT in cytopenias/inadeq BMA

Flow cytometery on P,blood /BM aspirate -identify the abn cells gate them

Apply the pr panel of MCAs based on morphology

Apply the secondary panel of MCAs if necessary

Report should take all the above points into consideration

CONCLUSIONS

FCM offers multiparameter evaluation and simultaneousrecording of expression of several antigens for an individual cell.

Differentiation of leukemic cells in ALL lineage B/T cell

Useful for diagnosis of AML M0 ,biphenotypic leukemia ,MRD and aberrant phenotype

To diagnose and assess prognosis in CLPDs

FCM is rapid, with high specificity and sensitivity

FCM

Basics of FCM Dr Shipra Panel of MCAs-which ones ? Dr Prashant How to process the sample Dr Garima Which cells to gate Dr Neha ALL-T/B-Differentiation Dr Gayathri AML/ Biphenotypic/Mo/M4-5/M7 Dr Prashant Red cells - CD 55/CD 59 Dr Renu Saksena Platelets-Gp IIb/IIIa,Ib/IX CLPD - morphology mimics Dr Pati Plasma cell disorders Dr Sanjeev Dr Pati MRD Dr Chatterjee How much have you learnt! Interactive session