| Abstract|| |
Acute Promyelocytic Leukemia (APL) is different from other forms of acute myeloid leukemia (AML), to the reason being the potential devastating coagulopathy and the sensitivity to all-trans retinoic acid (ATRA) and arsenic trioxide (As 2 O 3 ). We hereby present a case of APL, morphologically distinct from the hypergranular APL; however, the flow cytometry revealed a characteristic phenotype showing dim CD45, bright CD13, bright CD33 and dim CD117 positivity. These were negative for CD34, HLA-DR, B-lymphoid and T-lymphoid lineage markers. Conventional cytogenetics revealed a distinct karyotype of a male with translocation t(4;15)(q34.2:q26.3). However, interphase florescence-in-situ hybridization (FISH) revealed PML/RARA fusion signal on chromosome 15 in 90% cells. The cryptic translocations may be missed on conventional cytogenetics, however, need to be picked by other techniques as FISH.
Keywords: Acute promyelocytic leukemia, cytogenetics, flow cytometry, florescence-in-situ hybridization, cryptic
|How to cite this article:|
Goyal M, Dattatreya PS, Goud I, Murthy SS. Cryptic PML-RARα positive acute promyelocytic leukemia with unusual morphology and cytogenetics. Indian J Pathol Microbiol 2010;53:817-9
|How to cite this URL:|
Goyal M, Dattatreya PS, Goud I, Murthy SS. Cryptic PML-RARα positive acute promyelocytic leukemia with unusual morphology and cytogenetics. Indian J Pathol Microbiol [serial online] 2010 [cited 2020 Jul 15];53:817-9. Available from: http://www.ijpmonline.org/text.asp?2010/53/4/817/72097
| Introduction|| |
Acute Promyelocytic Leukemia (APL) is a subtype of acute myeloid leukemia (AML) with a defined clinical course and biology that is distinct from the other forms of AML. Acute promyelocytic leukemia is different from other forms of AML due to the potential devastating coagulopathy and the sensitivity to retinoid differentiating agents including all-trans retinoic acid (ATRA) and novel agents such as arsenic trioxide (As 2 O 3 ). ,,,,, Morphologically, APL is defined into four distinct forms - hypergranular APL (classical M 3 ), microgranular (M 3 v), hyperbasophilic and promyelocytic leukemia zinc finger/retinoic acid receptor-α (PLZF/RARα)(M 3 r) variants . Biologically, the cytogenetic changes define the syndrome, and APL is cytogenetically characterized by the reciprocal translocation t(15;17)(q22;q21), disrupting the PML and RARα genes, which are localized to chromosomes 15q and 17q, respectively.  The molecular consequence of this is crucial in leukemogenesis. 
| Case Report|| |
A 50-year-old male presented with complaints of gum bleeding and loss of appetite since one month. He had recently developed purpuric rash all over the body. On examination, there was marked pallor and had no lymphadenopathy or any organomegaly. Complete blood picture revealed hemoglobin of 3.0 g/dL, total leukocyte count of 10.9 × 10 9 /L and platelet count of 20 × 10 9 /L. His peripheral smear revealed marked prominence of abnormal promyelocytes. Bone marrow aspiration showed predominantly abnormal promyelocytes and few blasts. These promyelocytes were large, hypergranular and most were without Auer rods. Faggot cells were very occasional. These cells had round to oval nucleus with regular nuclear outline and evenly distributed chromatin and prominent nucleoli. There were few cells, which had coarse basophilic granules (Chediak-Higashi like inclusions) [Figure 1]. No maturing myeloid precursors were seen. Myeloperoxidase staining was intense and highlighted the Auer rods. Immunophenotyping analysis was performed on bone marrow sample by three-color multiparameter flow cytometry and the cells showed high forward scatter and side scatter. The cells were gated on CD45 and side scatter and the gated population was 90%. These revealed bright positivity for CD13, CD33 and dim positivity for CD117. These were negative for CD34, HLA-DR, CD19, CD22, CD2, CD3, CD7 and CD4.
|Figure 1: Photomicrograph of bone marrow aspirate showing hypergranular abnormal promyelocytes, with an occasional promyelocyte containing Chediak-Higashi like inclusions. (Leishman stain; ×1000)|
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Bone marrow sample was analyzed for metaphase karyotyping by GTG banding after 48 h of unstimulated culture. Twenty metaphases were counted and analyzed. The results revealed karyotype of a male with translocation t(4;15)(q34.2:q26.3) [Figure 2]. There was no t(15;17)(q22;q21) detected. Interphase florescence in-situ hybridization (FISH) was performed on the fixed bone marrow aspirate smear for PML/RARA translocation using directly labeled LSI PML (15q22) / LSI RARA (17q21.1) DNA probe (Vysis). PML/RARα fusion signal on chromosome 15 was detected in 90% cells [Figure 3].
|Figure 2: Karyotype of a G-banded metaphase cell obtained from a bone marrow sample showing translocation between the long arms of one chromosome no. 4 and other chromosome no. 15, thus with a karyotype 46, XY t(4;15)(q34.2:q26.3)|
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|Figure 3: Interphase FISH performed demonstrates the PML/RARα translocation (yellow / green-red fusion signal) in most cells using directly labeled LSI PML (15q22) / LSI RARα (17q21.1) DNA probe (Vysis): The green signal marks the RARα locus and red signal marks PML locus|
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The patient was planned for Daunorubicin and Cytosine arabinoside treatment along with ATRA. The patient expired before he could receive the treatment.
| Discussion|| |
Acute Promyelocytic Leukemia (APL) is a subtype of acute myeloid leukemia (AML), which is distinct from the other forms of AML. The key clinical features that set APL apart are the potential devastating coagulopathy and the sensitivity to retinoid differentiating agents including all-trans retinoic acid (ATRA) and to novel agents such as arsenic trioxide (As 2 O 3 ). ,,,,, Morphologically, hypergranular APL (classical M 3 ) are characterized by heavily granulated cells with folded, twisted nuclei.  Other morphological variants are the microgranular (M 3 v), hyperbasophilic and promyelocytic leukemia zinc finger/retinoic acid receptor-α (PLZF/RARα) (M 3 r).  Biologically, the cytogenetic changes define the syndrome, and APL is cytogenetically characterized by the reciprocal translocation t(15;17)(q22;q21), disrupting the PML and RARα genes, which are localized to chromosomes 15q and 17q, respectively, which is responsible for leukemogenesis. 
The present case had the classical clinical features, bicytopenia and normal leukocyte count. Pancytopenia is the most common finding; however, an increased leukocyte count may be seen in 10 to 30%, mostly in the microgranular variant.  The morphological classification of the blasts and abnormal promyelocytes by Sainty et al, was based on the appearance of nuclear outline (regular or irregular), degree of cytoplasmic granularity (hyper- or hypogranular) and number of Auer rods (0, 1 to 2, 3 or more).  Based on this three-parameter classification system, 12 categories of abnormal promyelocytes were identified. Three additional categories were also included with special features and these were - cells with basophilic granules, with Chediak-Higashi like inclusions and Pelger-like maturing cells.  The present case showed marked prominence of promyelocytes with regular nuclear outline, hypergranular cytoplasm and no Auer rods. There were minor populations with presence of Auer rods and cells with Chediak-Higashi like inclusions. Such abnormal cells were found predominantly in t(11;15)(q23;q21) or PLZF/RARα group cases, cases lacking evidence of a RARα rearrangement and very occasionally in cases lacking classical t(15;17)(q22;q21) but with PML/RARα rearrangement. This morphological pattern is used to define the PLZF/RARα group (M 3 r). 
A distinctive immunophenotypic pattern in APL as compared to other AMLs is well-known: positivity for the myeloid antigens - CD33, CD13 and CD117, negativity for HLA-DR and low frequency of CD34 expression.  A similar pattern was noted in our case.
Based on the cytogenetic and molecular studies, cases of APL are categorized into those with classical t(15;17), cases lacking classical t(15;17)(q22;q21) but with PML-RARα rearrangement and cases lacking PML/RARα rearrangements.  The last group is further categorized into cases with PLZF/RARα rearrangements, those with t(5;17) and the ones that lack evidence of RARα rearrangements.  In addition to t(15;17), other additional chromosomal abnormalities can be found in 30 to 40% of patients with APL. , The most common of these are trisomy 8 and isochromosome 17. , The translocation t(4;15)(q34.2:q26.3) is rare or probably unknown to the best of our knowledge.
Classical APL are characterized by the reciprocal translocation t(15;17)(q22;q21), disrupting the PML and RARα genes, which are localized to chromosomes 15q and 17q, respectively. Masked translocations, in which pieces of chromosome 15 and 17 are transposed but escape detection by conventional techniques, are reported in 9% patients with cases of morphologic APL. , These are associated with the formation of the PML/RARα fusion gene, created by insertion events or more complex rearrangements.  Such mechanisms occur in approximately 4 and 2% of cases of APL respectively and typically lead to the formation of PML/RARα at its usual location on 15q and, less commonly at the site of reciprocal fusion gene on 17q or alternative chromosomal location.  Expression of the fusion gene product ultimately results in the clinical syndrome identified with APL despite the lack of gross chromosomal changes.
Insertion (15;17) or (17;15) are characterized by the presence of fusion or co-localization signal reflecting the formation of PML/RARα on 15q most often or rarely on 17q.  The chromosomes 15q22 and 17q21 are normal by conventional karyotyping. Complex chromosomal changes are those in which the fusion gene is formed as a result of complex rearrangements involving at least three chromosomes. These are of three types: 1) complex variant t(15;17) due to a three-way balanced translocation involving 15q22,17q21, and another chromosome; 2) simple variant t(15;17), apparently involving either 15q22 or 17q21 with another chromosome and finally 3) very complex cases involving multiple abnormalities.  The present case using the above criteria can possibly be categorized in the insertion group; however, dual color/dual fusion probes for PML/RARA by FISH and advanced techniques as spectral karyotyping are required for confirmation.
Additional chromosomal abnormalities do not have a negative effect on the overall prognosis. , Secondary cytogenetic changes do not confer a poor prognosis in APL patients treated with anthracycline/cytarabine (Ara-C)-based chemotherapy. , A highly significant relationship exists between the PML-RAR alpha 5 isoform (intron 3 PML genomic breakpoint) and secondary cytogenetic changes in APL.  However, in the present case it is difficult to ascertain the role of the t(4;15)(q34.2:q26.3) in the morphological variation, which mimicked PLZF/RARα group.
The present case is an ideal example, where a limited diagnostic approach would lead to missing of cryptic translocations as PML/RARα, thus, emphasizing the importance of complete work-up. However, there is a need for further evaluation to assess whether this particular additional abnormality has any prognostic and therapeutic implication.
| Acknowledgement|| |
We would like to thank Dr Sanjana Dutt, PhD, Scientific manager and Professor and S.A. Hussain, Consultant Cytogenecist, Oncquest, New Delhi for their technical support for performing FISH and cytogenetics for the case.
| References|| |
|1.||Grimwade D, Biondi A, Mozziconzcci MJ,Hagemeijer A, Berger R, Neat M, et al. Characterization of acute promyelocytic leukemia cases lacking the classic the classic t (15;17): Results of the European Working Party. Blood 2000;96:1297-308. |
|2.||Talman MS, Kwaan HC. Reassessing the hemostatic disorder associated with acute promyelocytic leukemia. Blood 1992;79:543-53. |
|3.||Barbui T, Finazzi G, Falanga A. The impact of all-trans retinoic acid on the coagulopathy of acute promyelocytic leukemia. Blood 1998;91:3093-102. |
|4.||Degos L, Dombret H, Chomienne C, Daniel MT, Miclea JM, Chastang C et al. All trans retinoic acid as a differentiating agent in the treatment of acute promyelocytic leukemia. Blood 1995;85:2643-53. |
|5.||Shen ZX, Chen GQ, Ni JH, Li XS, Xiong Sm, Qui QY, et al. Use of arsenic trioxide (As 2 O 3 ) in the treatment of acute promyelocytic leukemia (APL): II, Clinical efficacy and pharmacokinetics in relapsed patients. Blood 1997;89:3354-60. |
|6.||Soignet SL, Maslak P, Wang ZG, Jhanwar S, Calleja E, Dardashti LJ, et al. Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N Engl J Med 1998;339:1341-8. |
|7.||Sainty D, Liso V, Cantu-Rajnoldi, Head D, Mozziconacci MJ, Arnoulet C, et al. A new morphological classification system for acute promyelocytic leukemia distinguishes cases with underlying PLZF-RARA rearrangements. Blood 2000;96:1287-96. |
|8.||Melnick A, Licht JD. Deconstructing a disease: RARα, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood 1999;93:3167-215. |
|9.||Golomb HM, Rowley JD, Vardiman JW, Testa JR, Butler A. Microgranular acute promyelocytic leukemia: A distinct clinical, ultrastructural, and cytogenetic entity. Blood 1980;55:253-9. |
|10.||Paietta E, Andersen J, Gallagher R, Bennett J, Yunis J, Cassileth P, et al. The immunophenotype of acute promyelocytic leukemia (APL): An ECOG. Leukemia 1994;8:1108-12. |
|11.||Johannson B, Mertens F, Mitelman F. Secondary chromosomal abnormalities in acute leukemias. Leukemia 1994;8:953-62. |
|12.||Slack JL, Arthur DC, Lawrence D, Mrozek K, Mayer RJ, Davey FR, et al. Secondary cytogenetic changes in acute promyelocytic leukemia: Prognostic importance in patients treated with chemotherapy alone and association with intron 3 breakpoint of the PML gene: A Cancer and Leukemia Group B study. J Clin Oncol 1997;15:1786-95. |
|13.||Grimwade DF, Gorman P, Duprez E, Howe K, Langabeer S, Oliver F, et al. Characterization of cryptic rearrangements and variant translocations in acute promyelocytic leukemia. Blood 1997;90:4876-85. |
|14.||Schoch C, Haase D, Haferlach T, Freund M, Link H, Lengfelder E, et al. Incidence and implication of additional chromosome aberrations in acute promyelocytic leukemias with t (15;17)(q22;q21): A report on 50 patients. Br J Hematol 1996;94:493-500. |
Department of Laboratory Medicine, Indo-American Cancer Institute and Research Centre, Road No. 14, Banjara Hills, Hyderabad
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]