Introduction
Early diagnosis of acute promyelocytic leukaemia (APL) is essential because of its associated life-threatening coagulopathy and unique response to ATRA therapy. However, laboratory detection of the reciprocal translocation t(15;17) either by conventional cytogenetics or by molecular analyses cannot give an immediate answer, and treatment is often initiated before the results are made available. The characteristic cell morphology supplemented by cytochemistry offer the most rapid means for diagnosis. Yet, variants e.g. microgranular variant M3 (M3v) exist and may cause difficulty in diagnosis. Here, we describe a case of M3v that poses particular diagnostic challenge because of the atypical morphology and immunophenotypic profile.
Case History
A 35-year-old previously healthy Chinese male presented with tongue haematoma, bilateral sub-conjunctival haemorrhage, peri-rectal bleeding and easy bruising. He also suffered from anaemic symptoms with fever and chills. Physical examination revealed multiple ecchymoses, right cervical lymphadenopathy, but no hepatosplenomegaly. Laboratory data showed haemoglobin 13.9 g/dL, while blood cell count (WBC) 92 x 109/L with circulating blasts, and platelet count 34 x 109/L. Coagulation studies suggested disseminated intravascular coagulopathy with PT 15.2 sec, APTT 27.5 sec, fibrinogen 1.05 g/L and D-Dimer 8-32 mg/L. The circulating blasts were large in size showing convoluted nuclear contour, open chromatin with distinct nucleolus and appreciable amount of basophilic cytoplasm. Bilobed forms were also present, and occasional ones showed hypergranulated cytoplasm (Figure 1).
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| Figure 1: Peripheral blood film showing circulating blasts, bilobed and occasionally hypergranulated cells. (Wright stain x 1000) | |
The bone marrow aspirate was markedly hypercellular and frankly leukaemic. Normal haematopoiesis was replaced by sheets of blasts with similar morphology as found in the peripheral blood. Again, there were occasional blasts containing coarse azurophilic granules, but no Auer rod was found. A prominent population of hand mirror blasts was also present (Figure 2).
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| Figure 2: Bone marrow smears, showing the presence of hand-mirror blasts. (Wright-Giemsa x 1000) | |
Cytochemically, the leukaemic cells were strongly positive for myeloperoxidase and Sudan black B (Figure 3). Immunophenotyping by flow cytometry revealed the blast cells expressing myeloid markers (cMPO, CD33, CD13) and CD34. They were negative for HLA-DR, CD117(c-kit) and CD15. They were also negative for B-cell (CD19, CD20, CD22, CD79a) and T-cell markers (CD2, CD3, CD7). The blasts also show weak expression of CD56. Trephine biopsy showed hypercellular marrow infiltrated by sheets of blasts and marked depression of normal haemopoiesis (Figure 4).
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| Figure 3: Cytochemistry result | |
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| Figure 4: Trephine biopsy showing sheets of blasts. (H&E x 1000) |
In view of the atypical blasts morphology and peculiar immunophenotype, the diagnosis of microgranular variant of acute promyelocytic leukaemia (M3v) was suspected. The patient was immediately started on all-trans retinoic acid (ATRA) and cytarabine. The WBC rose to 240 x 109/L despite cytarabine. He developed severe frontal headache one day after commencement of ATRA and an urgent plain CT scan confirmed massive right parieto-temporal intracerebral haemorrhage with mass effect. Decompressive crainiotomy and clot evacuation was performed but his condition further deteriorated and succumbed four days after initial presentation. Although conventional cytogenetics showed no growth, the definitive diagnosis of APL was later confirmed by the presence of PML-RAR demonstrated by interphase FISH (Figure 5) and RT-PCR (Figure 6) after the clinical deterioration.
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| Figure 5: Detection of PML-RARa gene
fusion by interphase FISH: Left: S-FISH (one wild type PML, one wild type RARa, and one yellow fusion signal); Right: D-FISH (one wild type PML, one wild type RARa, and two yellow fusion signals). |
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| Figure 6: Detection of PML-RARa fusion transcript by RT-PCR |
Discussion
This case report emphasizes the importance of a high index of suspicion for the diagnosis of APL, the microgranular variant in particular. A comparison of M3 and M3v is shown in Table 1.
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| Table 1: Comparison of Hypergranular and Microgranular/hypogranular Acute Promyelocytic Leukaemia (summarized from references 1 & 2) |
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Overall, M3 and M3v (under the FAB classification) constitute 5 - 8 % of cases of AML in different Western series1. Both APL subtypes share a common pathogenic pathway, namely presence of t(15;17)(q21;q22) translocation, and a similar clinical picture, namely consumptive coagulopathy at presentation (Table 1). They are responsive to differentiation therapy with all-trans-retinoic acid (ATRA). The resultant oncoprotein, being an aberrant nuclear receptor, represses retinoic acid targets causing differentiation block and contributes to the eventual development of leukaemia. Over the years, other morphological variations have been described. In 2000, a committee of morphologists3 proposed a new classification of APLs: (1) classical hypergranular M3, (2) microgranular M3v, (3) hyperbasophilic promyelocytic leukaemia, (4) acute eosinophilic leukaemia with PML/RAR positivity, and (5) promyelocytic leukaemia with basophil-like granules. The classification never gains popularity because of rarity of APL variants other than M3v. A hand-mirror variant of microgranular APL was described by Sun4 in 1991.
Although our patient presented with bleeding diathesis, the blasts have an atypical morphological appearance (frequent hand mirror forms, absent Auer rods/faggot cells in the marrow) and an unusual immunophenotype (CD34+). Absent or weak expression of stem cell markers (HLA-DR, CD34, CD117) and the more mature myeloid markers (CD15, CD11b) in the context of a myeloid phenotype (CD33, CD13) used to be the paradigm of the APL immunophenotype. However, in several subsequent studies, CD34 expression has been shown to be associated with hypogranular morphology and/or S-form (bcr-3 type) of PML/RARa transcript5-9. In addition, results from gene expression profiling suggest that, despite close clustering, the two morphological subtypes of APL (M3 and M3v) are clearly separable. The difference may lie in FLT3 which has been found to be mutated more frequently in M3v than M3, and more frequently in APL with the short (S-isoform, bcr3) rather than the long (L-isoform, bcr1) PML/RARa transcript form10. The prognostic significance of such phenotype/genotype is still controversial9,11.
Conclusion
In conclusion, the therapeutic option and prognostic implication in APL has made early diagnosis of paramount clinical significance. Although the cytogenetic signature should always be referred to in diagnosing APL, from the practical point of view, other parameters such as morphology, cytochemistry and immunophenotyping are still important tools for rapid recognition of APL.
References