TEACHING CASE 14

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Figure 1: Wright-Giemsa-stained slide showing leukemic blasts with clefted nuclei.

Figure 2: Wright-Giemsa-stained slide showing rare cytoplasmic granules and bi-lobed nuclei.

Figure 3: Myeloperoxidase (MPO) cytochemical staining showing uniform positivity in the leukemic cells.

Figure 4: Flow cytometric histograms showing a leukemic blast population (highlighted in red) with low to negative CD34, low-intermediate CD117, CD13, and CD64, bright CD33, low CD38 and CD45, very low CD15, CD71, and CD123, near-uniform loss of HLA-DR, and aberrant low CD2.

Figure 5: Fluorescence in situ hybridization study for the t(15;17). The presence of dual fusion signals (highlighted by red-green signals with yellow overlap) within each leukemic blast indicates a reciprocal translocation involving the PML and RARA genes on chromosomes 15 and 17, respectively.

Figure 6: Illustration of dual color, dual fusion FISH for identification of PML/RARA (i.e., t(15:17) translocation.

Clinical Summary:
A 47-year-old male who presents with anemia, thrombocytopenia, leukocytosis, and petechiae.

Summary of Morphologic, Flow Cytometric, and In Situ Hybridization Findings:
The bone marrow aspirate shows extensive replacement by an apparent blast population with high N:C ratios and frequent clefted nuclei, with rare cytoplasmic granules (Figure 1). Auer rods are not readily identifiable and faggot cells are not seen. Rare enlarged cells showing bi-lobed nuclei and cytoplasmic granules are noted (Figure 2). Cytochemical staining shows uniform myeloperoxidase positivity in the blast-like cells, with focal needle-like positivity (Figure 3).

Immunophenotyping by flow cytometry shows that the leukemic population expresses variably low to negative CD34, low-intermediate CD117, CD9, CD13, and CD64, bright CD33, low CD38, CD45, aberrant CD2, very low CD15, CD71, and CD123, and near-uniform loss of HLA-DR (Figure 4). Fluorescence in situ hybridization studies show dual fusion signals characteristic of a reciprocal translocation involving the PML gene on chromosome 15 and the RARA-alpha gene on chromosome 17 (i.e., the t(15:17)) (Figure 5). Figure 6 schematically illustrates the use of dual color, dual fusion probes to identify PML/RARA translocations.

Final Diagnosis:
Acute promyelocytic leukemia, microgranular variant, positive for t(15;17) by in situ hybridization.

Discussion:
Acute promyelocytic leukemia [AML with t(15;17)(q22;q12), or APL] is an acute myeloid leukemia in which the leukemic cells are arrested at the promyelocytic stage. APL comprises 5-8% of AMLs, and is most common in middle-aged adults. About 70% of cases present with a hypergranular morphology, often with numerous Auer rods consistent with "faggot cells." About 30% of cases show a hypogranular morphology (microgranular variant), as seen in this case. The myeloperoxidase reaction is typically strongly positive in the leukemic cells, even in the microgranular variant, which can be very helpful in distinguishing the variant from other forms of acute leukemia, particularly leukemias with monocytic differentiation. Characteristic immunophenotypic findings in APL include very low to negative CD34, absent HLA-DR, homogeneous and bright CD33, heterogeneous CD117 and CD13, and low to negative CD15 (much lower than on normal promyelocytes).

Clinically, both forms of APL are associated with disseminated intravascular coagulation. Therapeutically, it is important to distinguish APL from other forms of AML, as most APL patients respond to all-trans-retinoic acid (ATRA), which leads to a differentiation of the leukemic cells.

APLs invariably contain a translocation involving the retinoic acid receptor alpha (RARA) on chromosome 17. In >90% of APLs, the RARA gene is fused to the PML gene on chromosome 15, but in a small minority of cases, RARA is fused to a different gene, which typically lessens the responsiveness to ATRA.

References:

1. ES Jaffe et al., WHO Classification of Tumours: Pathology & Genetics: Tumours of the Haematopoietic and Lymphoid Tissues, IARC Press (Lyon, France) 2001.
2. Kaleem Z, Crawford E, Pathan MH, Jasper L, Covinsky MA, Johnson LR, White G. Flow cytometric analysis of acute leukemias. Diagnostic utility and critical analysis of data. Arch Pathol Lab Med. 2003 Jan;127(1):42-8.
3. Falini B, Flenghi L, Fedeli L, Broe MK, Bonino C, Stein H, Durkop H, Bigerna B, Barbabietola G, Venturi S, et al. In vivo targeting of Hodgkin and Reed-Sternberg cells of Hodgkin's disease with monoclonal antibody Ber-H2 (CD30): immunohistological evidence. Br J Haematol. 1992 Sep;82(1):38-45.
4. Lin P, Hao S, Medeiros LJ, Estey EH, Pierce SA, Wang X, Glassman AB, Bueso-Ramos C, Huh YO. Expression of CD2 in acute promyelocytic leukemia correlates with short form of PML-RARalpha transcripts and poorer prognosis. Am J Clin Pathol. 2004 Mar;121(3):402-7.
5. Amare PS, Baisane C, Saikia T, Nair R, Gawade H, Advani S. Fluorescence in situ hybridization: a highly efficient technique of molecular diagnosis and predication for disease course in patients with myeloid leukemias. Cancer Genet Cytogenet. 2001 Dec;131(2):125-34.