1. ICCS e-Newsletter CSI Fall 2015 Hywyn R. O. Churchill, MD PhD Division of Hematopathology

2. eCSI: Clinical History A 10 year-old boy with no significant past medical history who presents with syncopy, headache and nausea. CBC showed marked leukocytosis (80K), anemia and thrombocytopenia. No hepatosplenomegaly. Flow cytometry was performed on the bone marrow.

3. eCSI: Original Morphology Large immature mononuclear cells with high N:C ratio, dispersed chromatin, prominent nucleoli, deeply basophilic cytoplasm.

4. eCSI Part 1: Flow Cytometric Analysis Tube 1 (CD34 FITC/CD14 PE/CD45 PerCP/CD38 APC) Tube 2 (CD7 FITC/CD13 PE/HLA-DR PerCP/CD34 APC) Tube 3 (CD15 FITC/CD33 PE/CD45 PerCP/CD34 APC) Tube 4 (CD36 FITC/CD64 PE/CD45 PerCP/CD34 APC) * four selected representative tubes from a 10 tube analysis

5. FSC SSC CD45 PerCPCD34 FITC CD117 PE CD38 APC CD14 PE CD34 APC CD33 PE CD13 PECD64 PE CD34 APC CD7 FITC CD36 FITC CD15 FITC HLA-DR PerCP eCSI Part 1: Flow Cytometric Analysis

6. eCSI Part 1: Final Diagnosis Immunophenotypic analysis reveals an 64% population of medium to large cells with the following immunophenotype CD2(-), surface and cytoplasmic CD3(-), CD4(partial +), CD5(partial dim +), CD7(+), CD11b(predominantly -), CD13(variably +), CD14(-), CD15(partial +), CD16(-), CD25(partial +), CD33(variably +), CD34(partial +), CD36(few +), CD38(variably +), CD45(moderate to dim +), CD56(-), CD64(-), CD117(variably +), CD123(+), HLA-DR(+), MPO(appears dim + to -), TdT(-). The findings reveal a population of immunophenotypically aberrant myeloid blasts consistent with acute myeloid leukemia. Cytogenetics: 46,XY.

7. eCSI: Follow-up Subsequent biopsies over the next four months show refractory disease in spite of intensive chemotherapy. At four months, the myeloblasts showed additional morphologic features not seen on the original material: – Part 2: bone marrow aspiration – Part 3: bone marrow biopsy

8. eCSI Part 2: Morphology Similar cytologic features to the original case, but now with rare cytoplasmic pseudopod formation and “zoned” granular and clear cytoplasm.

9. eCSI Part 2: Flow Cytometric Analysis Tube 1 (CD34 FITC/CD14 PE/CD45 PerCP/CD38 APC) Tube 2 (CD7 FITC/CD13 PE/HLA-DR PerCP/CD34 APC) Tube 3 (CD15 FITC/CD33 PE/CD45 PerCP/CD34 APC) Tube 4 (CD36 FITC/CD64 PE/CD45 PerCP/CD34 APC) Tube 5 (CD41 FITC/CD117 PE/CD45 PerCP/CD34 APC) Tube 6 (CD61 FITC/CD117 PE/CD45 PerCP/CD34 APC) * six selected representative tubes from a 9 tube analysis

10. FSC SSC CD45 PerCPCD34 FITC CD117 PE CD38 APC CD14 PE CD34 APC CD33 PE CD13 PECD64 PE CD34 APC CD7 FITC CD36 FITC CD15 FITC HLA-DR PerCP eCSI Part 2: Flow Cytometric Analysis

11. FSC SSC CD45 PerCPCD61 FITC CD41 PE CD34 APC CD117 PE eCSI Part 2: Flow Cytometric Analysis

12. eCSI Part 2: Final Diagnosis Immunophenotypic analysis reveals 40-50% medium-sized to large cells with the following immunophenotype: CD7(+), CD11b(-), CD13(variably +), CD14(-), CD15(partial +), CD16(-), CD33(+), CD34(partial +), CD36(partial +/approximately 25% of the total population is +), CD38(variably +), CD41(few +/approximately 17% of the total population is +), CD45(+), CD56(-), CD61(partial +/approximately 20% of the total population is +), CD71(variably +), CD117(+), HLA-DR(+), Gly-A(predominantly -). The findings reveal a population of immunophenotypically aberrant myeloid blasts with similar features to prior analyses consistent with a persistent acute myeloid leukemia. A proportion of the blasts show features suggestive of megakaryocytic differentiation. Cytogenetics: 46,XY.

13. eCSI Part 3: Morphology Medium to large immature mononuclear cells with high N:C ratio and prominent nucleoli and cytoplasmic pseudopod formation and “zoned” granular and clear cytoplasm with vacuoles.

14. eCSI Part 3: Flow Cytometric Analysis Tube 1 (CD34 FITC/CD14 PE/CD45 PerCP/CD38 APC) Tube 2 (CD7 FITC/CD13 PE/HLA-DR PerCP/CD34 APC) Tube 3 (CD15 FITC/CD33 PE/CD45 PerCP/CD34 APC) Tube 4 (CD36 FITC/CD64 PE/CD45 PerCP/CD34 APC) Tube 5 (CD41 FITC/CD117 PE/CD45 PerCP/CD34 APC) Tube 6 (CD61 FITC/CD117 PE/CD45 PerCP/CD34 APC) * six selected representative tubes from a 10 tube analysis

15. FSC SSC CD45 PerCPCD34 FITC CD117 PE CD38 APC CD14 PE CD34 APC CD33 PE CD13 PECD64 PE CD34 APC CD7 FITC CD36 FITC CD15 FITC HLA-DR PerCP eCSI Part 3: Flow Cytometric Analysis

16. FSC SSC CD45 PerCP CD61 FITC CD41 PE CD34 APC CD117 PE eCSI Part 3: Flow Cytometric Analysis

17. eCSI Part 3: Final Diagnosis The cellular events include a 2.2% population (accounting for 70% of the events when debris is removed) that shows the following immunophenotype: CD7(+), CD11b(-), CD13(+), CD14(-), CD15(predominantly -), CD16(-), CD33(+), CD34(predominantly +), CD36(+), CD38(slightly variably +), CD41(+), CD45(+), CD56(-), CD61(+), CD64(-), CD71(+), CD117(+), HLA-DR(+), and GlyA(-). The findings reveal a population of immunophenotypically aberrant myeloid blasts showing evidence of megakaryocytic differentiation. Notably, the immunophenotype of the blasts in this case show uniform expression of markers of megakaryocytic differentiation compared to the concurrently submitted aspirate specimen and prior analyses.

18. eCSI: AML with Megakaryocytic Differentiation This case represents an example of antigenic evolution of AML toward megakaryocytic differentiation. Our case shows similarities and differences to the conventional immunophenotype for AML with megakaryocytic differentiation: – Similarities: aberrant expression of CD7, variable expression of CD13 and CD71, and expression of CD33, CD36, CD41, and CD61, with lack of expression of CD14 and CD64. – Differences: expression of CD34, CD38, CD45, HLA-DR (often negative in conventional cases), and lack of CD56 expression. – CD42 was not evaluated, but has been reported to be expressed in more mature megakaryoblasts.

19. eCSI: Immunophenotype and Morphology in AML with Megakaryocytic Differentiation The morphologic changes in the myeloblast population suggested the addition of megakaryocytic markers CD41 and CD61 to the antibody panel. What about the increasing level of CD36 expression?

20. eCSI: CD36 in Flow Cytometry CD36 is expressed in a variety of cell lineages. – Erythroid precursors, monocytes, megakaryocytes and platelets. CD36 expression increases as megakaryocytes mature. However, CD36 “expression” may be mistaken as platelets adhering to other cell types giving false-positive signals. Are CD36-positive platelets adhering to CD36-negative blasts in our case, or is it true CD36 expression? – Look at the granulocytes!

21. eCSI: CD36 in Flow Cytometry The significance of CD36, CD41, and CD61 staining on blast populations can be difficult to determine since platelet satellitism/adherence can result in false-positive staining. To help account for this phenomenon, staining for these markers on granulocytes can be assessed. In the following two slides, an example of pseudo-CD36 staining on granulocytes and myeloblasts from a normal bone marrow is shown (top row). Compare this to the staining pattern of CD36 for parts 1 and 2 of our case (bottom row). Notice that while the blasts in our case stain positive for CD36, the granulocytes do not. This finding suggests that the blasts truly express CD36. The same can be performed for CD41 and CD61.

22. FSC SSC CD36 FITC CD45 PerCP eCSI Part 1: CD36 Non-Specific Binding CD45 PerCP SSC CD64 PE FSC SSC CD36 FITC CD45 PerCP SSC CD64 PE Our case: part 1 Reference case: non-specific CD36 signal

23. FSC SSC CD36 FITC CD64 PE eCSI Part 2: CD36 Non-Specific Binding CD45 PerCP SSC CD45 PerCP FSC SSC CD36 FITC CD64 PE CD45 PerCP SSC CD45 PerCP Reference case: non-specific CD36 signal Our case: part 2

24. eCSI: CD36, CD41, and CD61 In our flow cytometry facility, CD36 is a part of our basic immunophenotyping panel for acute myeloid leukemia. As such, it can be used as a screening marker for potential cases of megakaryocytic differentiation in myeloid neoplasms. Importantly, some myeloid neoplasms with megakaryocytic differentiation may lack expression of CD36, as it is believed to be expressed at a later stage of megakaryocyte development. In contrast, the megakaryocytic markers CD41 and CD61 are not a part of our basic immunophenotyping panel for acute myeloid leukemia and are added only as needed. Therefore, correlation with the morphology of the myeloblasts remains important and may indicate the necessity of adding megakaryocytic markers (such as CD41 and/or CD61) in a reflex tube.

25. eCSI: Conclusions This case represents an example of antigenic evolution of acute myeloid leukemia toward megakaryocytic differentiation. The original diagnostic material showed very little CD36 staining on myeloblasts. Notably, specific CD36 staining of blasts in our case suggested megakaryocytic differentiation, which was confirmed with the addition of CD41 and CD61. If CD36 is used as a screening antigen for megakaryocytic differentiation, identification of platelet satellitism/adhesion representing false-positive staining is important.

26. eCSI: References Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (Eds.): WHO Classification of Tumours of the Haematopoietic and Lymphoid Tissues. IARC: Lyon 2008. Cherian S, Wood B: Flow Cytometry in the Evaluation of Hematopoietic Neoplasms. CAP Press: Northfield 2012. Wood BL. Myeloid Malignancies: Myelodysplastic Syndromes, Myeloproliferative Disorders, and Acute Myeloid Leukemia. 2007. Clin Lab Med 27: 551–575. Ortolani C. Flow Cytometry of Hematological Malignancies. Wiley-Blackwell, 2011. Bunting ST. Case Study Interpretation-New Orleans: Case 1. 2013. Cytometry Part B 84B:339-341.