ICCS e-Newsletter CSI Fall 2010

1. ICCS e-Newsletter CSI Fall 2010 David D. Grier, M.D. Department of Pathology. Wake Forest University

2. e-CSI - Clinical History: • 61 year old man with no significant past medical history presented to his primary care physician complaining of fatigue, easy bruising, mild dyspnea on exertion, and headache for 3 weeks.

3. e-CSI - Peripheral Blood: • CBCNormal Range • WBC: 24.0 x 109/l (4.8 – 10.8) • RBC: 3.51 x 1012/l (4.7 – 6.1) • Hgb: 11.2 g/dl (14.0 – 18.0) • Hct: 31.5 % (42.0 – 52.0) • MCV: 89.8 fl (80.0 – 94.0) • MCH: 32.0 pg (27.0 – 31.0) • MCHC: 35.6 g/dl (33.0 – 37.0) • RDW: 15.4% (11.5 – 14.5) • Plts: 19.0 x 109/l (160 – 360)

4. e-CSI - Peripheral Blood: • CBC Differential • Granulocytes: 11% • Bands: 1% • Lymphocytes: 6% • Monocytes: 1% • Basophils: 2% • Eosinophils: 0% • Blasts/Promyelocytes: 72%

5. e-CSI – Clinical History • Bone marrow aspirate and biopsy were received for evaluation. • Flow cytometric immunophenotyping was performed on a portion of the bone marrow aspirate and the results from selected 3-color and 5-color tubes are provided for review.

6. e-CSI - Flow Cytometric Studies • Acquired with a FC500 and initially analyzed with CXP Analysis 2.0 and then with FSC Express version 3 • Tube 1: CD34~FITC/ CD2~PE/ CD117~PC5/ CD45~PC7 • Tube 2: CD7~FITC/ CD33~PE/ CD45~PC7 • Tube 3: HLA-DR~FITC/ CD13~PE/ CD45~PC7

7. e-CSI - Flow Cytometric Analysis Large population of atypical cells with increased side scatter. There are very few events in the “blast gate”.

8. e-CSI - Flow Cytometric Analysis The atypical cell population has uniform expression of CD33

9. e-CSI - Flow Cytometric Analysis There is no expression of HLA-DR and variable expression of CD13.

10. e-CSI - Flow Cytometric Analysis There is expression of CD2 and CD34.

11. e-CSI – Key immunophenotypic findings • The atypical cells have high side scatter and fall in the “myeloid gate”. • Few events are seen in the “blast gate”. • The cells express CD34, CD117, and CD2. • CD13 expression is heterogenous. • CD33 expression is homogenous. • There is no expression of HLA-DR.

12. Numerous atypical cells were seen. The nuclei were indented and overlapping. Few cytoplasmic granules were seen. No Auer rods were identified.

13. The bone marrow core biopsy was 100% cellular with the marrow space almost completely replaced by blasts.

14. Two fused signals, one red and one green, indicating the fused PML-RARA (yellow). Molecular cytogenetic analysis with DNA probes specific for the 15;17 translocation [PML-15q22 and RARA-17q21] was performed and revealed that a total of 82.5% of the interphase nuclei had a PML/RARA fusion event.

15. e-CSI Fall 2010– Diagnosis • Acute promyelocytic leukemia (APL), microgranular variant (Acute myeloid leukemia with t(15;17)(q22;q12)).

16. e-CSI – APL characteristic immunophenotype • Absent or low expression of HLA-DR, CD34, CD11a, CD11b, CD15, and CD64. • Homogenous expression of CD33 • Heterogenous expression of CD13 • Expression of CD117, but it may be weak. • CD56 is seen in approximately 20% of cases.

17. e-CSI – APL characteristic immunophenotype • Strong myeloperoxidase expression. • Microgranular variant can express CD34 and CD2. • By CD45/side scatter the leukemic cells are typical found in the “myeloid gate”. Occasionally there is a “hockey stick” configuration. Microgranular variant tends to have lower side scatter.

18. e-CSI – APL characteristic immunophenotype • Lack of HLA-DR and CD34 is not specific for APL and can be seen in other types of acute myeloid leukemia. • Immunophenotyping can suggest a diagnosis of APL, but it is NOT diagnostic.

19. e-CSI – APL clinical presentation • Fatigue, weakness, and dyspnea related to anemia. • Easy bruising or bleeding caused by thrombocytopenia or coagulopathy. • Most cases present with pancytopenia. • Microgranular variant can have very high WBC counts. • Risk of disseminated coagulopathy.

20. e-CSI – APL Diagnosis • Suspicion for APL is typically raised by morphologic assessment. • Abnormal promyelocyte morphology • Abundant cytoplasmic granules • Microgranular/hypogranular variant can have few to no visible granules. • Frequent Auer rods • Irregular nuclei: bilobed and kidney shaped. • More frequently seen in microgranular variant. • The diagnosis is confirmed by cytogenetics (FISH).

21. e-CSI – APL Differential diagnosis • Acute myeloid leukemia • HLA-DR negative leukemia is not limited to APL • Cytogenetics essential for diagnosis • APL is sometimes mistaken, especially the microgranular variant, for monocytic leukemia due to the nuclear features • Growth factor effect • Lower blast counts • Lacks atypical nuclei • No t(15;17) abnormality

22. e-CSI – APL Treatment • The correct diagnosis is essential since the treatment is very different from other types of AML. • Treatment consists of all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy for induction and ATRA plus arsenic trioxide for consolidation. • Some cytogenetic variants are ATRA resistant. • ZBTB16 (11q23), STAT5B (17q11.2)

23. e-CSI – references • Nagendra S, Meyerson H, Skallerud G, et al. Leukemias resembling acute promyelocytic leukemia, microgranular variant. American journal of clinical pathology 2002:117(4):651-657. • Orfao A, Chillon MC, Bortoluci AM, et al. The flow cytometric pattern of CD34, CD15 and CD13 expression in acute myeloblastic leukemia is highly characteristic of the presence of PML-RARalpha gene rearrangements. Haematologica 1999:84(5):405-412. • Redner RL. Variations on a theme: the alternate translocations in APL. Leukemia 2002:16(10):1927-1932.