ICCS e-Newsletter CSI Fall 2010

David D. Grier, M.D.Department of Pathology.

Wake Forest University

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.

e-CSI - Peripheral Blood:

• CBC Normal 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)

e-CSI - Peripheral Blood:

• CBC Differential – Granulocytes: 11%– Bands: 1%– Lymphocytes: 6%– Monocytes: 1% – Basophils: 2% – Eosinophils: 0%– Blasts/Promyelocytes: 72%

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.

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

e-CSI - Flow Cytometric Analysis

CD45-PC7

SS

Lin

100

101

102

103

104

0

256

512

768

1024

Large population of atypical cells with increased side scatter. There are very few events in the “blast gate”.

e-CSI - Flow Cytometric Analysis

CD33-PE

CD

7-F

ITC

100

101

102

103

104

100

101

102

103

104

The atypical cell population has uniform expression of CD33

e-CSI - Flow Cytometric Analysis

CD13-PE

HL

AD

R-F

ITC

100

101

102

103

104

100

101

102

103

104

There is no expression of HLA-DR and variable expression of CD13.

e-CSI - Flow Cytometric Analysis

CD34_FITC

CD

2-P

E

100

101

102

103

104

100

101

102

103

104

There is expression of CD2 and CD34.

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.

Numerous atypical cells were seen. The nuclei were indented and overlapping.

Few cytoplasmic granules were seen. No Auer rods were identified.

The bone marrow core biopsy was 100% cellular with the

marrow space almost completely replaced by blasts.

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.

Two fused signals, one red and one green, indicating the

fused PML-RARA (yellow).

e-CSI Fall 2010– Diagnosis

• Acute promyelocytic leukemia (APL), microgranular variant (Acute myeloid leukemia with t(15;17)(q22;q12)).

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.

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.

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.

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.

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).

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

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)

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.