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TMH proceedings 2010-2011,pdf
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Bone Marrow Aspirate HematolymphoidNeoplasmSecond Basic Hematopathology CourseTMH, MumbaiJune 12, 2011
Dr Sumeet Gujral, MDAssociate ProfessorDepartment of PathologyTata Memorial Hospital, [email protected]
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