01 Approach to a Case Leukemia and Lymphoproliferative Disor
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Transcript of 01 Approach to a Case Leukemia and Lymphoproliferative Disor
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APPROACH TO A CASE OF ACUTE LEUKEMIA
&CH LYMPHOPROLIFERATIVE DISORDER
Dr. Tejinder SinghDirector Professor
Department of PathologyMAMC, Delhi
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Myeloid leukemia
Arises from these cells
Lymphoid leukemia
Arises from these cells
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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
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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
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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
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L1 small cells
scant pale blue cytoplasm
Variation in size - mild
regular nuclear shape
homogenous chromatin Absent /small nucleoli
MPO ve
PAS +ve
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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
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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
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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
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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
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T - ALL
15-20% of all ALL cases
Morphology of L1/L2
Acid phosphatase positivity
Worse prognosis
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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
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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
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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
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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
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IHC: CD 13 and CD 33 help in establishing a myeloid lineage
AML-M0
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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
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AML-M2: AML with maturation
MPO stain shows strong positivity in the blasts
Maturing myeloid precursors also positive
Auer rods highlighted by MPO staining
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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
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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
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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
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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
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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
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Monoblasts are MPO/ Sudan black B negative
Promonocytes may show weak MPO positivity
Monoblasts are strongly positive for NSE fluoride sensitive
AML-M5A
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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
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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
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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
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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
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AML-M7
CD 61 positivity in blasts
and platelets, confirms
their megakaryocytic
lineage
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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
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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
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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.
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Gating is important
Variable expression of CD1a, CD2, CD3, CD4, CD5, CD7, CD8
cCD 3 is lineage specific T-ALL
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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
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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
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MBL
Monoclonal B cell lymphocytosis
Presence of
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Flow cytometric IPT is particularly useful in subtyping B-cell lymphomas/leukemias
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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
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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
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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
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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
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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)
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T CELL LGL
CD 8
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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
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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
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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
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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