Bone Marrow Aspirate HematolymphoidNeoplasmSecond Basic Hematopathology CourseTMH, MumbaiJune 12, 2011

Dr Sumeet Gujral, MDAssociate ProfessorDepartment of PathologyTata Memorial Hospital, Mumbais_gujral@hotmail.com

Leukemia: Tumor cells in blood

Lymphoma - Tumor cells in other tissues

Leukemia/Lymphoma

Acute,Blastic,Precursor (lymphoid, myeloid)

Chronic,Mature(lymphoid, myeloid)

FAB ClassificationMorphology, cytochemistry and immunophenotyping

WHO classificationIt has classified myeloid and lymphoid malignanciesaccording to cell of origin, based on morphologic,immunologic, genetic and clinical characteristics

Evolution of leukemia classifications

A. Myeloid neoplasmsB. Precursor lymphoid neoplasmsC. Mature B cell neoplasmsD. Mature T- and NK- cell neoplasmsE. Hodgkin lymphomaF. Immunodeficiency associated LPDG. Histiocytic and dendritic cell neoplasms

2008 WHO Classification of Hematolymphoid Neoplasms

Few newer subtypes in WHO 2008Blastic plasmacytoid dendritic cell neoplasms

So many subtypes, however, there are a fewer treatment options

Role of a Pathologist

Establishing Diagnosis

Prognostication (sub-classification)

Predictive markers

Evaluation of Treatment Effectiveness (MRD)

Diagnosis

Clinical: History and Examination

Lab investigation

Radiology (X ray chest to PET-CT)

Lab Diagnosis

Initial Investigations: Complete blood counts Peripheral Blood Smear (200 cell counts), Bone Marrow Aspirate/ Imprint (500 cell counts) Common cytochemical stains: MPO, NSE, Iron and

TRAP

Immunophenotyping by flow cytometry, IHC

Cytogenetics (and FISH) & Molecular Diagnostics

Ancillary techniques

Immunohistochemistry, Flow Cytometry, Cytogenetics, Molecular Diagnostics

Diagnostic Prognostic Predictive

Pediatric LymphomasT-LLHD DLBCLBL ALCL

Adult LymphomasDLBCL HD SLLBL TCL - PTCL U, ALCL

Indian data

Pediatric LeukemiaB-ALL

Adult LeukemiaCMLAMLCLL

Presentation

Anemia, bicytopenia or pancytopenia Polycythemia Leucopenia or leucocytosis Thrombocytopenia or thrombocytosis Rarely normal counts

Start with a peripheral blood smear

Bone marrow aspirate and touch

Bone marrow biopsy

Leucocytosis

Leucocytosis

Pancytopenia

Morphology

What are blasts?

Acute Leukemia>20% blasts in peripheral blood or bone marrow

Morphology

Exceptions Small sizeGranular blastsAbnormal promyelocytes in AMLM3 Promonocytes in AMLM5

Blast - Look Alike:Hematogones, Erythroblasts, Regenerating cells,Lymphoma cells, etc

Other mimics:CLL vs ALL-L1MCL vs ALLRound cell tumor vs Burkitts lymphoma

Guess

?

Important stains:Giemsa / Wright

Myeloperoxidase

Non specific esterase

Iron

TRAP

Cytochemistry is extremely important

Cytochemistry is extremely important

Ancillary techniquesAncillary techniques

Flow Flow cytometrycytometryCytogeneticsCytogenetics, FISH, FISHMolecular diagnosticsMolecular diagnostics

Approach

Leukamoid reaction or a leukemia

Acute leukemia or chronic leukemia

ALL or AML

If ALL, is it T or B, or otherwise

If AML, is it APML or other

Cytogenetic workup for a final label

Morphology

Cytochemistry andFCM

Molecular genetics

For leukemia diagnosis, flow cytometry immunophenotyping is mandatory

Large panel Large panel of markers available (>200 markers)of markers available (>200 markers)

Lineage associated markersLineage associated markers

Lineage specific markersLineage specific markers-- CD3CD3-- CD22/CD79aCD22/CD79a-- AMPOAMPO

FCM

Acute leukemia panelsIndian Guidelines

National Guidelines, IJPM, 2008

B cell CD10, CD19

T cell CD7, CD5

Myeloid CD13, CD33, CD117

Other CD34, HLA-DR, CD45

Recommended minimal screening panel for immunophenotyping of AL (n = 10)

Preferred primary panel

It is further recommended to do all lineage specific cytoplasmic markers and Tdt with rest of the minimal panel.

B cell CD10, CD19, cCD22/cCD79a

T cell CD7, CD5, cCD3

Myeloid CD13, CD33, CD117, AMPO

Other CD34, HLA-DR, CD45, Tdt

cCD3, cCD22/cCD79a, anti-MPO (lineage specific markers),SIg (/), TdT, CD41, CD61, CD38, CD138, Kappa, Lambda CD56, CD43, CD4, CD123, CD64 CD2, CD4, CD8.

Recommended additional panel(based on the results of the minimal panel)

Flow AlgorithmApproach to Acute Leukemia

Acute Leukemia

Auer Rods + Auer Rods

MPO and/or NSE + MPO and/or NSE

FCM

AML

National Guidelines, IJPM, 2008

FCM with first line panel

CD10, CD19, HLADR CD3 CD13, CD33, CD117 HLADR, CD34

Diagnosis

No diagnosis

IJPM, 2008

However, if lineage not established

FCM with second line panel

cCD22 cCD3, CD4, CD8 anti MPO, CD41, CD61

IJPM, 2008

All lymphoid cellsAll lymphoid cells CD45+ (LCA)CD45+ (LCA)

BB--cellscells CD19, CD10, CD19, CD10, cCD22cCD22

TT--cells cells CD3, CD5,CD3, CD5, cCD3cCD3

Myeloid cellsMyeloid cells CD13, CD33, CD117, CD13, CD33, CD117, anti MPOanti MPO

MegakaryocyticMegakaryocytic CD41, CD61CD41, CD61

Blasts Blasts CD34, CD34, TdtTdt, CD99, CD99

Other: HLAOther: HLA--DR, CD23, FMCDR, CD23, FMC--7, CD43, CD11c, CD25, CD103, CD38, 7, CD43, CD11c, CD25, CD103, CD38, CD138, CD20, CD79a, Kappa and Lambda light chains, TCR alpha beta, CD138, CD20, CD79a, Kappa and Lambda light chains, TCR alpha beta, TCR gamma delta, CD4, CD64, CD55, CD59TCR gamma delta, CD4, CD64, CD55, CD59

Common CD markers Common CD markers Leukemia labLeukemia lab

IJPM. 2008

Examples

Case 1

14 year old boy with fever and splenomegaly since 2 months

Peripheral blood smear is provided

Diagnosis

Diagnosis

Diagnosis

Diagnosis (on morphology)

CML - chronic phase

LAP score

Role of FCM

Confirmation: Molecular diagnosis for targeted therapy by conventional cytogenetics, FISH, PCR, RT-PCR

Case 256 year old female, case of Acute Leukemia

PBS: Blasts > 88%

Diagnosis ? Role of FCM, Cytogenetics

Diagnosis: AML-M2 with t(8;21)

Case 345 year old female, fever and weakness since 1 month

PBS is provided

Blasts 88%

Diagnosis

No Auer rods, MPO/NSE negative

What next ?

FCM: Myeloid markers (CD13, CD33, CD117, AMPO)

Diagnosis: AML (Non M3)

Can be labeled AMLM0 only once common cytogenetic markers have been done

Diagnosis

Case 4

der(17)

der(15):PML/RARA

der(17)

der(15):PML/RARA

Acute Promyelocytic Leukemia

Dr AmareDr Anuradha

Case 5

12 year old boy, fever, pallor, bleeding gums

Diagnosis

Case of Acute leukemiaMPO -, NSE ++

Role of FCM: CD13, CD33, CD117, CD11C, CD64, CD4, AMPO

Diagnosis: AML-M5

Can be labeled AMLM5 only once common cytogenetic markers have been done

Case 6

68 year old male, case of Acute leukemia (MPO, NSE negative)

FCM: Almost all markers were negative except CD13, CD33 Additional markers: CD41, CD61

Diagnosis: AML-M7Pediatric age, Downs syndrome

Case 7

5-year-old boy presented with fever - 1 month duration

On examination: Pallor, Hepatosplenomegaly

Peripheral Blood Smear Examination

DiagnosisMPO, NSE negative

CD19, CD10, HLADR positive blasts

CD34 negative

Tdt and light chain restriction not done

Diagnosis: B-cell ALL

1. B-cell ALLTdt, CD34

2. Burkitts LymphomaCD20++, Kappa, Lambda

Morphological differentials CD19, CD10, HLADR positive blasts

Diagnosis: B-cell ALL

Prognostication: Cytogenetic studies

Chemotherapy: MCP 841 protocol

Evaluation of Treatment: Day 18 BM

- Morphology

- MRD Lite by flow

Case 8

1-year-old boy, a case of acute leukemia

MPO negative Acute Leukemia

BlastsWeak CD45, CD20Express CD19, CD34, HLADR, Cyto CD79a, CD15CD10 negative

Diagnosis: It is B-cell ALL

Final impression is CD10 -, CD15 + Pro B-ALL

Adv: MLL gene rearrangement studiesMLL-AF4 translocations have poor prognosis (infants)

Case 9

45 year old male, case of acute leukemia

Auer Rods seen

Cytochemistry: MPO positive

Diagnosis: AML-M2

MPO positive AML

Role of flow cytometry

Blasts:Weak CD45 Express CD34, CD117, CD19, CD56, CD15, HLADRNegative for CD33

Diagnosis: AML M2 with CD19 & CD56

Prognostic markers: t(8;21) studies goodCD56 expression - bad

Approach to CLPDs

Mostly B cell type

CD19+ B-cell CLPD

CD3+ T-cell CLPD

CD138+, CD38+, CD19-, CD3- Plasmacytic tumors

CD3-, CD16+, CD56 + NK-LGL Leukemias

CD2, CD3, CD4, CD5, CD16+, CD56+ T/NK cell LGL Leukemias

CD19 positive B cell lymphomas

- CLL (Chronic lymphocytic leukemia)- Mantle cell lymphoma- Follicular lymphoma- Hairy cell leukemia - Splenic marginal zone lymphoma- Others

All subtypes mimic CLL on morphology, except HCL

B cell lymphomas expressing CD5 are CLL and MCL

T cell lymphomas are rare

CD3, CD5,

CD19, CD23, CD10,

CD20,

FMC7,

Kappa, Lambda

Recommended minimal screening panelfor CLPD / Mature lymphomas, (n= 9)

Additional markers for CLPDs may include CD45, CD43, CD2, CD4, CD7, CD8, TCR/b, TCR /, CD11c, IgM, IgD, CD25, CD103, cyclin D1, CD38, CD138, CD16, CD56, CD57ZAP70

In cases of primary effusion lymphomas, we recommend CD38 and CD138 in addition to the CLPD panel.

Recommended additional panel (based on the results of the minimal panel)

IJPM, 2008

FCM and Lymphomas

Diagnosis and sub-typing of NHL Clonality: LCR, v-beta repertoire Assessment of therapeutic targets (CD20) Staging Prognostication (CD38 and/or ZAP-70 in CLL) Minimal residual disease: CLL and HCL

CD - 23391 INDULKAR (BM).003

FSC - Height

SSC

- H

eigh

t

0 256 512 768 10240

256

512

768

1024

Small cell Lymphoma

Large cell Lymphoma

CD5+ CD5-

CD23+CD43+FMC7 -

CD23 FMC7+, CD43+

Cyclin D1+

CD10+ CD10-

SLL MCL FL, ALL, BL SMZLHCL

CD11c,CD25+, CD103+

CD25-,CD43-

CD19+ B-cell lymphomas

Newer IHC classification of DLBCLs GCB, non GCB, etc

CD10 Mum1

BCL6

GCBCD10+ >30 cells

GCBCD10-, MUM1-BCL6+, LMO2+

Non GCB

Non GCBMum1+FOXP1+

Case 1

CLL

B-CLPD 6 color panel

CD45, CD20, CD19, CD5, CD23, CD22wk, CD20, Kappa

LC, CD200

Impression: B-cell lymphoma, SLL

CLL

PLL

Subtyping of Small B cell lymphomas

Case 2

45 year old male,

Fever and left cervical LN since 1 month

Lymph node FNAC done

FNAC

CD19+, CD5+, CD23-Diagnosis

CyclinD1 Not available for flow

Case 3

45 year old male, cervical lymphadenopathy

Bone marrow aspirate

High grade lymphoma with plasmacytoid morphology

Tumor cells were negative for

CD3, CD4, CD8 CD20, CD19 CD13, CD33 Tdt, CD34

Additional stains done

Gating of plasma cells

SSC vs FSC CD138 and CD38

Hazards of small panels

Gated plasma cells showing CD56+ and CD19-

IPT pattern of Classical Myeloma,Plasmablastic Lymphoma

T-cell lymphomas - Rare

Need an elaborate T/NK cell panel

Diagnosis of T-cell lymphomas

DPT / DNT (CD4/CD8) CD7, CD5, CD3, CD2 (loss of T cell markers) Tdt CD30 CD16, CD56 CD4, CD25 HTLV-1 V-beta repetoire

Case 4:42 year old male, suspected case of lymphoma BM shows 28% lymphoid cells

Loss of CD5 and CD7

Peripheral T-cell lymphoma - NOS (BM)

Diagnosis: Peripheral T-cell lymphoma NOS

(Loss of CD7 and CD5)

BM Bx in blastic neoplasms

Acute Leukemia Do we need bone marrow biopsy ??

AML Non M3 AML M3

B-cell ALL

Burkitts lymphoma

ALL

ALL

CD34

Tdt Mic2

Final comprehensive report

Morphology Cytochemistry Flow cytometry / IHC Cytogenetics Molecular Diagnostics

Important

Auer rods Stains: Giemsa, MPO, NSE, Iron, TRAP APML and Burkitt Lymphoma are medical

emergencies Preserve the smears Trephine for IHC etc

Hematopathology Lab

Hematolymphoid Group - TMHR NairH MenonM SengarN KhattaryA Joshi

S BanavaliB AroraA Nahar

S LaskarM Muckaden

T ShetS EpariPG SubramanianS Gujral

PA Amare and teamAshokBadrinathSitaramAnuradha

Scientists, Technologists, Residents and Fellows

100bpladder

1 2 3 4 5 6

RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) detects diagnostic translocation markers like BCR/ABL in CML -BCR/ABL: b3a2 437bp