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  Table of Contents    
CASE REPORT  
Year : 2012  |  Volume : 55  |  Issue : 4  |  Page : 574-577
CD34 positive-microgranular variant of acute promyelocytic leukemia in a child


1 Department of Molecular Biology, R & D, Triesta Reference Laboratory, HCG, Bangalore, Karnataka, India
2 Department of Paediatric Oncology, Health Care Global Enterpreises, Bangalore, Karnataka, India

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Date of Web Publication4-Mar-2013
 

   Abstract 

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) in which abnormal promyelocytes predominate. APL is rare in children (approximately 10% of childhood AML) and is characterized by a higher incidence of hyperleukocytosis, an increased incidence of microgranular morphology, the presence of balanced t(15;17)(q22;q11.2-12) translocation, and more frequent occurrence of the PML-RARα isoforms bcr 2 and bcr 3 compared to adults. The cytomorphology of microgranular variant blasts is obviously different from AML M3 blasts; these cells have a nongranular or hypogranular cytoplasm or contain fine dust-like cytoplasmic azurophil granules that may not be apparent by light microscopy. This case report emphasizes the importance of a high index of suspicion for the diagnosis of APL, the hypogranular variant in particular. They are responsive to differentiation therapy with all trans-retinoic acid and complete remission in seen in >80% cases.

Keywords: Acute promyelocytic leukemia, CD34, complete molecular remission, PML-RARα

How to cite this article:
Nargund AR, Patil GV, Raghuram C P, Venkataswamy E. CD34 positive-microgranular variant of acute promyelocytic leukemia in a child. Indian J Pathol Microbiol 2012;55:574-7

How to cite this URL:
Nargund AR, Patil GV, Raghuram C P, Venkataswamy E. CD34 positive-microgranular variant of acute promyelocytic leukemia in a child. Indian J Pathol Microbiol [serial online] 2012 [cited 2020 Sep 19];55:574-7. Available from: http://www.ijpmonline.org/text.asp?2012/55/4/574/107828



   Introduction Top


Acute promyelocytic leukemia (APL) is characterized by typical promyelocyte morphology, and is associated with coagulopathy and the presence of balanced t(15;17)(q22;q11.2-12) translocation and PML-RARa fusion transcripts. [1],[2] APL has a characteristic CD13+, CD33+, CD9+, HLA-DR–, CD14– immunophenotypic profile. The majority of patients with APL can achieve complete remission (CR) with all-trans retinoic acid (ATRA) with or without anthracycline, resulting in a favorable clinical outcome. [2]

Although the outcomes in APL patients are markedly improved by the above therapeutic approaches, some patients of this subtype still show a poor clinical course. Several clinical and biological parameters affect the CR rate and survival in APL patients. In general, younger age (under 30 years), lower white blood cell (WBC; <10 × 109/L), and the absence of purpura at diagnosis favor achieving CR. Importantly, rapid disappearance of PML-RARα fusion transcripts during post remission therapy is an independent predictor of a favorable prognosis. [2]

CD34 antigen is a 116-kDa glycophosphoprotein expressed on 1-2% of normal bone marrow (BM) cells, especially early hematopoietic precursor cells. The significance of CD34 expression in APL is unknown but likely identifies an immature form of APL. CD34 expression is clinically associated with leukocytosis, microgranular/hypogranular morphology, and expression of CD2 and bcr2 isoform. [3] We report a case of APL-microgranular variant that poses particular diagnostic challenge because of its atypical morphology.


   Case Report Top


A 2-year-old girl child presented with 1 week history of skin and gum bleeds. There was no history of pallor, fever, or sore throat. Her immunization history and developmental milestones were normal. On examination, the child had widespread purpura and ecchymoses. Abdomen revealed a huge hepatomegaly (15 cm below the right costal margin). She did not have any lymphadenopathy or splenomegaly.

Complete blood count showed a hemoglobin level of 6.9 g/dl, WBC count of 16,700 cells/μl, and platelet count 35 × 103/μl. The peripheral smear demonstrated an increased number of WBCs, 90% abnormal promyelocytes, and few blasts showing high nuclear-cytoplasmic ratio and conspicuous nucleoli. These promyelocytes had markedly lobulated and invaginated nuclei. The cytoplasm of the cells contained few clearly recognizable granules, but showed occasional  Auer rods More Details.

Coagulation parameters were slightly deranged with prothrombin time of 17.2 s [biological reference interval (BRI) 11-16 s], International Normalized Ratio (INR) 1.42, and partial thromboplastin time of 38.9 s (BRI 30.8-40.8 s). D-Dimer was elevated at 8.91 mcg/ml (BRI 0-0.5 mcg/ml). Chest X-ray and echocardiogram did not show any abnormality.

A bone marrow aspirate (BMA) and a trephine biopsy were performed. The differential on the aspirate was: 70% promyelocytes which had morphology similar to those seen in the peripheral blood, 15% blasts, 5% myelocytes, and 10% lymphocytes. Normal hematopoiesis was suppressed. The promyelocytes were strongly positive for myeloperoxidase (MPO) stain [Figure 1]. A trephine biopsy showed marrow infiltrated by sheets of blasts and promyelocytes and a marked depression of normal hematopoiesis [Figure 2] and [Figure 3].
Figure 1: Peroxidase stain on peripheral blood in M3 variant AML

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Figure 2: Bone marrow trephine biopsy (high magnifaction)

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Figure 3: Bone marrow trephine biopsy (low magnifaction)

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Flow cytometry analysis on BMA was performed on Cyan ADP by using SSC/CD45 gating strategy. Majority of the gated cells strongly expressed CD45, CD33, CD13, CD117, and CD34, and were negative for CD14, CD10, CD19, CD7, CD3, HLA-DR, CD22, CD5, and CD4. There was coexpression of CD117 and CD34 [Figure 4].
Figure 4: Dot-plot histograms in APL

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In view of the morphology, HLA-DR negativity, and CD34 positivity, the possibility of APL-microgranular variant was suggested and confirmation by reverse transcriptase polymerase chain reaction (RT-PCR) was advised.

RT-PCR

BMA sample was obtained in ethylenediaminetetraacetic acid (EDTA) anticoagulant and total RNA was extracted by using TRI-Reagent (Sigma, MO, USA) as per the manufacturer's recommendations and converted to cDNA using the Fermentas kit for cDNA synthesis.

Multiplex RT-PCR to detect PML-RARα was performed using Seeplex® Leukemia PML-Rarα kit (Seegene, Seoul, South Korea). The kit is designed to detect the long, short, and variant forms of PML-RARa fusion transcript. Cycling conditions were as follows: 94°C for 15 min (1 cycle); 94°C for 30 s, 60°C for 1 min 30 s, 72°C for 1 min 30 s (37 cycles); and 72°C for 10 min (1 cycle). The PCR products were analyzed by 2% agarose gel electrophoresis at 70 V for 30 min.

RT-PCR analysis for PML-RARα rearrangement showed a unique bcr2 variant corresponding to 809 bp that reflects a break point which includes splicing of Exon 5 and an insertion in Intron 6 of the PML gene.


   Cytogenetics Top


Cytogenetic study was performed by initiating short-term (direct, 24 h and 48 h) unstimulated cultures from fresh BMA in Roswell Park Memorial Institute (RPMI) 1640 with l-glutamine (Gibco, NY, USA) supplemented with fetal bovine serum (20%) and penicillin-streptomycin (Gibco, NY, USA). The cultures were exposed to colcemid (0.01 mg/ml) (Gibco, NY, USA) for 25 min and harvested by hypotonic treatment in 0.075 M KCl (Merck, Darmstadt, Germany) and repeated fixations in methanol: Acetic acid (3:1). The slides were incubated at 60° C for 1 day and then G-banded with trypsin and giemsa (NICE, Kerala, India) stain. Twenty metaphases were analyzed which showed the karyotype 46, XX, t(15;17)(q24;q21) [Figure 5].
Figure 5: Karyotyping results showing the characteristic cytogenetic lesion 46, XX, t (15;17) (q24;q21) indicated by the arrow

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After the first course of treatment with ATRA, the gene expression levels were re-evaluated on BMA and found to be 4.7%, suggesting a major molecular response. The disease burden further dropped to 1.56% after the second course of treatment and complete molecular remission was observed after the third course. The BMA evaluation procedure was repeated after the 4 th month to confirm the complete molecular remission.

Response Evaluation

[Figure 6] shows the molecular response profile of the PML-RARα gene expression levels during the course of treatment. The initial estimate of the PML-RARα gene expression levels by quantitative RT-PCR on the BMA revealed 50% as the disease burden at the baseline.
Figure 6: Molecular response profile of the PML-RARa gene expression levels during the course of treatment. The initial estimate of the PML-RARa gene expression levels by quantitative RT-PCR on the BMA revealed 50% as the disease burden at the base line

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The patient was started on chemotherapy along with ATRA. In total, she received 4 cycles of chemotherapy with Cytarabine and anthracyclines (Mitoxantrone and Idarubicin), followed by maintenance chemotherapy with ATRA, Methotrexate, and 6 Mercaptopurine. She needed supportive treatment with intravenous fluids, blood and platelet transfusions; neutropenic fever was controlled with antibiotics and antifungals. Her BM at the end of first cycle was in morphological remission, and response was monitored with RT-PCR for PML-RARα as described above.


   Discussion Top


APL is a subtype of AML in which abnormal promyelocytes predominate. [1] APL is rare in children (approximately 10% of childhood AML) and is characterized by a higher incidence of hyperleukocytosis, an increased incidence of microgranular morphology, the presence of balanced t(15;17)(q22;q11.2-12) translocation, and by the more frequent occurrence of the PML-RARα isoforms bcr2 and bcr3, compared to adults. [4] APL in children is more common in girls and in obese children. [4],[5]

As a special entity, APL was first described in 1957 by a Swedish author, Hillestad, when he reported three patients characterized by "a very rapid fatal course of only a few weeks duration," with a WBC picture dominated by promyelocytes and a severe bleeding tendency. He concluded that the disease "seems to be the most malignant form of acute leukemia." More detailed features of APL were described by Bernard et al., [6] in 1959, and the severe hemorrhagic diathesis has been ascribed to disseminated intravascular coagulation (DIC) or hyperfibrinolysis. [1] In 1985, the introduction of ATRA opened a new page in the history of APL treatment. [7]

The cytomorphology of AML M3v blasts is obviously different from AML M3 blasts; these cells have a nongranular or hypogranular cytoplasm or contain fine dust-like cytoplasmic azurophil granules that may not be apparent by light microscopy. [1] This case report emphasizes the importance of a high index of suspicion for the diagnosis of APL, the hypogranular variant in particular. It is responsive to differentiation therapy with ATRA and CR in seen in >80% cases.

The expression of CD34 was initially considered uncommon. Recent studies have shown that CD34 positivity occurs in about 20-30% of newly diagnosed cases. The significance of CD34 expression in APL is unknown but likely identifies an immature form of APL. CD34 expression is clinically associated with leukocytosis, microgranular/hypogranular morphology, and expression of CD2 and bcr2 isoform. [8]

CD34 antigen is a 116-kDa glycophosphoprotein expressed on 1-2% of normal BM cells, especially early hematopoietic precursor cells. The gene for this antigen is located on the long arm of chromosome 1 and its function is not yet clear. [8],[9]

In this particular case, we found aberrant expressions of myeloid markers for a promyelocytic acute leukemia, i.e., CD34, that suggest an immature phenotype, frequently associated with microgranular variant. Thus, clinicians and pathologists must pay attention to the morphologic diagnosis without misdiagnosing as the M2 subtype. [2]

We consider that a diagnosis of FAB M3v may be based on morphologic findings, which are extremely characteristic. Moreover, the demonstration of t(15;17) with RAR and PML rearrangements represents an important diagnostic step.

Complete hematological response was achieved after induction course and confirmed by BMA. Complete molecular response was also achieved and she experienced a good outcome, despite the initial poor prognosis factors. The absence of DIC, a frequent complication in APL at the onset, was a particular feature of the case.

The clinical and hematologic characteristics, particularly the morphologic and cytochemical, and molecular studies enable a rapid diagnosis to be made. Often, the precipitous clinical course and the prognosis seem worse than in typical forms of APL, and as in an adult setting may be attributed to the marked hyperleukocytosis and severe DIC. New therapeutic approaches may open new perspectives in this rare and dramatic form of leukemia.

To conclude, the therapeutic option and prognostic implication in APL-variant has made early diagnosis of paramount clinical significance. The expression of CD34 on APL cells at diagnosis may identify patients at a higher risk for relapse. Different treatment regimens may need to be considered for these immature/high WBC variants.


   Acknowledgment Top


We would like to acknowledge Shilpa Prabhudesai, Vidya H Veldore, Chandra R. Juvva, Prasanna Kumari, Intezar Mehdi for their thoughtful discussion and help with the figure.

 
   References Top

1.Jain D, Singh T, Arora P. Down syndrome with microgranular variant of acute promyelocytic leukemia in a child: A case report. J Med Case Rep 2007;1:147.  Back to cited text no. 1
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2.Lee JJ, Cho D, Chung IJ, Cho SH, Park KS, Park MR, et al. CD34 Expression is associated with poor clinical outcome in patients with acute promyelocytic leukemia. Am J Hematol 2003;73:149-53.  Back to cited text no. 2
[PUBMED]    
3.Foley R, Soamboonsrup P, Carter RF, Benger A, Meyer R, Walker I. CD34-Positive acute promyelocytic leukemia is associated with leukocytosis, microgranular/hypogranular morphology, expression of CD2 and bcr3 Isoform. Am J Hematol 2001;67:34-41.  Back to cited text no. 3
    
4.Mantadakis E, Samonis G, Kalmanti M. A comprehensive review of acute promyelocytic leukemia in children. Acta Haematol 2008;119:73-82.  Back to cited text no. 4
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5.Maule MM, Dama E, Mosso ML, Magnani C, Pastore G, Merletti F. High incidence of acute promyelocytic leukemia in children in northwest Italy, 1980-2003: A report from the Childhood Cancer Registry of Piedmont. Leukemia 2008;22:439-41.  Back to cited text no. 5
    
6.Yoo ES. Recent advances in the diagnosis and management of childhood acute promyelocytic leukemia. Korean J Pediatr 2011;54:95-105.  Back to cited text no. 6
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7.Wang ZY, Chen Z. Acute promyelocytic leukemia: From highly fatal to highly curable. Blood 2008;111:2505-15.  Back to cited text no. 7
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8.Sutherland DR, Keating A. The CD34antigen: Structure, biology, and potential clinical applications. J Hematother 1992;1:115-29.  Back to cited text no. 8
[PUBMED]    
9.Krause DS, Fackler MJ, Civin CI, May WS. CD34: Structure, biology, and clinical utility. Blood 1996;87:1-13.  Back to cited text no. 9
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Correspondence Address:
Ashwini R Nargund
E-204, Purva Fairmont Apartments, Parangipalya, Bangalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.107828

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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