New Frontiers in Pathology Hematopathology Breakout Session

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New Frontiers in PathologyHematopathology
Break-out Session

• Bryan Coffing, M.D.
• Hematopathology Fellow

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Disclaimer

• The cases reviewed here have been slightly
  modified for illustrative purposes
• Terms borrowed from the 2008 WHO blue book have
  been formatted to fit our lexicon
• (Leukaemia ? Leukemia, etc)

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Goals

• Examine two cases of acute leukemia with
  ambiguous lineage
• Discuss the specificity of lineage specific
  markers in acute leukemia
• Compare the European Group for the Immunological
  Characterization of Leukemia (EGIL) schema to the
  new WHO guidelines for lineage designation
• Summarize a few of the characteristics of the
  acute leukemias of ambiguous lineage

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Case Presentation 1

• 64 year old male with hypertension, diabetes,
  coronary artery disease
• Seen for routine follow-up in cardiology
• CBC WBC 4.4 Hgb 8.1 Plt 76

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Additional history

• Patient reports
• Fatigue for 2-3 months
• Persistent sinus infection for 4-6 weeks
• Easy bruising
• Referred to hematology for further workup
• Bone marrow biopsy, aspirate
• Flow cytometry
• Cytogenetic and molecular analysis

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74.2 blasts 3.6 granulocytic precursors beyond
promyelocytes 19.2 erythroid precursors 3
lymphocytes
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Ancillary Testing

• Molecular testing negative
• Cytogenetic analysis
• Complex abnormalities, multiple clones
• Hypotetraploid with del(5), del(21), add(13)
• Abnormality seen in AML as well as ALL

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Flow Cytometry
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Flow Cytometry
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Flow Summary

• Positive
• CD34 (mod)
• CD33 (dim, sub)
• CD117 (dim-mod)
• CD38 (dim)
• CD45 (dim)
• cCD22 (dim, sub)
• cCD79a (dim, sub)
• Tdt (dim, sub)
• 5-7 MPO () by IHC

• Negative
• CD19
• CD20
• CD10
• CD3 (surf cyto)
• Other T cell markers
• Monocytic markers

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Differential Diagnosis

• AML, without maturation (M1)
• B Lymphoblastic Leukemia/Lymphoma
• (with aberrant expression of MPO?)
• Acute leukemia of ambiguous lineage
• Biphenotypic/Bilineage (B-cell and myeloid)

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Acute Leukemias of Ambiguous Lineage

• WHO 2001
• 3 categories (Undiff, Biphenotypic, Bilineage)
• Pages 106-107
• WHO 2008
• Combined two categories (Biphenotypic and
  Bilineage)
• Pages 149-155
• Created 6 new categories

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Acute leukemias of ambiguous lineage
No lineage-specific antigens
Antigens of gt 1 lineage
Acute undifferentiated leukemia
Mixed phenotype acute leukemia (MPAL)
Separate blast populations of different
lineages OR One blast population expressing
antigens gt1 lineage
With additional genetic abnormalities
MPAL T/myeloid NOS
MPAL B/myeloid NOS
MPAL with t(922) BCR-ABL1
MPAL with t(v11q23) MLL rearr
Other
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Lineage Assignment

• European Group for the Immunological
  Characterization of Leukemias (EGIL)

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B Lymphoid Markers in AML

• CD7 and TdT expression in AML is well known
• CD79a expression in AML
• Cruse 2005 4/46 (8.7)
• Tiacci 2004 10/160 (1.6)
• El-Sissy 2006 1/34 (2.9)
• CD19 expression
• Tiacci 2004 10/160 (1.6)
• El-Sissy 2006 4/34 (11)
• CD22 (EGIL score 2)
• El-Sissy 2006 1/34 (2.9)

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Lymphoid markers in AML

• t(821)(AML1ETO)
• CD19
• cCD79a
• Weak TdT
• More than 30 show PAX5 expression

Pax5 in t(821) Gibson, 2006
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AML with cytogenetic abnormalities

• AML with inv(16) or t(1616)
• CD2 expression
• AML with t(1517)
• CD2 expression in microgranular variant
• AML with t(69)
• 50 TdT expression
• M1 and M2 (AML, NOS)
• 10-20 positive for CD2, CD4, CD19, or CD56

American Society of Hematopathology Image Bank
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Aberrant Markers in ALL

• Park et al, 1992
• 137 ALL cases in adults
• Phenotyped by immunofluoresence
• B-ALL more often
• CD13 (1) and CD33 (2)
• CD2 (1) and CD5 (1)
• T-ALL
• CD13 (8.3)
• CD10 (16.7)

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Myeloid markers in ALL

• Kalina et al, 2005
• 381 B-ALL pediatric patients
• Flow cytometric immunophenotyping
• CD33 (23)
• CD15 (20)
• CD13 (16)

• Vitale et al, 2007
• 374 adult ALL patients
• Flow cytometric immunophenotyping of CD13 and/or
  CD33
• T-ALL (24)
• B-ALL (38)

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Myeloid Markers in ALL

• MPO in pediatric ALL
• Austin 1998 (COG) 32/57 (56) pediatric ALL
  expressed MPO at protein or mRNA level
• All cases negative for t(922)
• MPO in adult ALL
• Arber 2001 19/67 (23) positive
• 8/19 (42) with bcr/abl
• Polyclonal MPO antibody
• Immunocytochemistry with monoclonal MPO negative

pMPO in ALL, Arber 2001
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Most specific lineage markers

• Distinctly difficult to find lymphoblastic
  leukemias with aberrant MPO
• Burkitt like ALL
• Myeloid leukemia with aberrant CD3 also
  distinctly uncommon
• B cell markers seem to be most promiscuous

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Myeloid lineage

• Monocytic
• differentiation

MPO
OR

• Flow cytometry
• Immunohistochemistry
• Cytochemistry

At least 2 NSE CD11c CD14 CD64 lysozyme
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T lineage
Cytoplasmic CD3

• Surface
• CD3

OR

• Flow cytometry
• Immunohistochemistry

Rare in MPAL
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B lineage
Strong CD19

• Weak
• CD19

OR
At least TWO (strong) CD79a Cytoplasmic
CD22 CD10

• At least ONE (strong)
• CD79a
• Cytoplasmic CD22
• CD10

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MPAL - Exclusions

• recurrent AML-associated translocations
• - t(821) freq expresses B cell markers
• - t(1517)
• - inv (16)
• FGFR1 mutation associated leukemias
• CML blast crisis
• MDS-related AML
• therapy-related AML

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Case Presentation 2

• 47 year old male with cellulitis
• Transferred to University of Michigan for
  pancytopenia and blasts on blood smear
• CBC WBC 2.9 Hb 8.6 Plt 71

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38.6 blasts 5.2 progranulocytes 16.4 other
granulocytic precursors 5 erythroid
precursors 6.4 lymphocytes 0.8
eosinophils 23.8 basophils 3.4 monocytes 0.4
plasma cells
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Ancillary Studies

• Molecular
• Negative for bcr/abl
• Cytogenetics
• 11q deletion, del(18)
• FISH for MLL (11q23) rearrangements negative

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Flow Cytometry
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Flow Cytometry
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Flow Summary

• Positive
• Cytoplasmic CD3
• CD5
• CD7
• Tdt
• CD11c
• CD13
• CD33
• CD34
• CD38
• CD45
• CD117
• Cytoplasmic MPO

• Negative
• CD2
• CD3 surface
• CD4
• CD8
• CD10
• CD14
• CD19
• CD20
• CD33
• CD56

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Differential Diagnosis

• AML with maturation (M2)
• T Lymphoblastic Leukemia/Lymphoma
• (with aberrant expression of myeloid markers)
• Acute leukemia of ambiguous lineage
• Biphenotypic/Bilineage (T-cell and myeloid)

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References

• Arber, Daniel et al. Myeloperoxidase
  Immunoreactivity in Adult Acute Lymphoblastic
  Leukemia. Am J Clin Pathol 200111625-33.
• Austin G et al. Prevalence of myeloperoxidase
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  leukemia. Am J Clin Pathol. 1998110575-581.
• Cruse, Julius et al. Aberrant expression of CD7,
  CD56, and CD79a antigens in acute myeloid
  leukemias. Experimental and Molecular Pathology
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• El-Sissy et al. Aberrant Lymphoid Antigen
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• Gibson, et al. Expression of the B
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• Park et al. Acute Leukemias with Unusual
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• Tiacci et al. PAX5 Expression in Acute Leukemias
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• Valbuena, Jose et al. Expression of B
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• Vitale, Antonella et al. Absence of prognostic
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