Pediatric acute promyelocytic leukemia: Insights from a tertiary care center

INTRODUCTION

Acute myeloid leukemia (AML) makes up about 1% of all cancers. Although AML can be diagnosed at any age, occurrence is rare before the age of 45 years, making it even rarer among the pediatric age group.^[1] The Lymphoma and Leukemia Society published updated data on blood cancers as received from the National Cancer Institute Surveillance, Epidemiology, and End Results Program from the years 2013 to 2017. The statistics depict incidence rates of AML in children (1-15 years) to be 0.4-1/100,000 population.^[2] In India, according to data published by Indian council of medical research (ICMR) in 2019, the incidence of AML is <1/100,000 population.^[3] Acute promyelocytic leukemia (APL) represents a unique subtype of AML, which is usually seen in the middle-aged group (>20 years). The annual incidence rate, as reported in the World Health Organization (WHO) edition 2017, is 0.08/100,000 population. In the pediatric population, it is even more rare.^[4] APL is characterized by the chromosomal translocation t(15;17), resulting in the chimeric fusion of the PML::RARA gene that hampers the differentiation of myeloid lineage and results in increased proliferation of immature blast cells and abnormal promyelocytes in the peripheral blood and bone marrow.

Due to its tendency to cause life-threatening complications like disseminated coagulopathy, it is essential to timely establish a correct diagnosis of APL, which requires characteristic cytomorphology and immunophenotype (IPT) for identification of such cases and mandatory chromosomal or molecular testing by fluorescence in situ hybridisation (FISH), reverse transcriptase polymerase chain reaction, or conventional cytogenetics for its confirmation.

While several studies have been published on pediatric APL globally, there is a considerable lack of literature in the Indian population.

A total of 28 AML cases were encountered in our institution over 1 year, from February 2022 to February 2023, of which seven were diagnosed with APL. Among these seven cases, six belonged to the pediatric age group. Four cases with adequate bone marrow aspirates (BMAs), flowcytometry (FC) data, and t(15;17)/PML::RARA verified by FISH were included in the study. Only newly diagnosed, untreated cases were included. Cases which were negative for t(15;17)/PML::RARA or cases where cytogenetics/molecular results were not available were excluded.

Giemsa-stained BMA aspirates and FC data were retrieved from the archive material, and cytomorphology and immunophenotyping of the cases were studied. The panel routinely employed for acute leukemia evaluation by FC included lineage-defining markers. The antibodies used were CD45, CD13, CD14, CD15, CD33, CD117, CD34, CD64, HLA-DR, CD10, surface and cytoplasmic CD3, CD4, CD8, CD7, CD19, CD20, CD79a, cytoplasmic MPO, and nuclear terminal deoxynucleotidyl transferase (TDT). An additional panel was performed in some cases. Here, we present four cases of pediatric APL discussing its clinical presentation, peripheral blood picture, cellular morphology and FC data.

CASE SERIES

Case 2

A 7-year-old female child presented with complaints of cough, fever, and generalized fatigue for the past 1 week. On examination, she was found to have an enlarged liver and spleen. There was no history of bleeding manifestations. A complete blood count report was obtained, which showed bicytopenia and marked leukocytosis with a TLC of >1 lac cells/cumm (Hb-6.5 g/dL and platelet count-16,000/cumm) [Table 1]. Smears examined from peripheral blood and BMA showed leukocytosis with >90% blast population, including abnormal promyelocytes [Figure 1b]. Occasional cells showed folded and convoluted nuclei, including the characteristic “buttock-shaped” nuclei and fine granules. MPO stain was strongly positive.

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Figure 1:

(a and b) Hypogranular/microgranular acute promyelocytic leukemia: Both figures depict the blast cell population, which is characterized by paucity of granules (solid white arrow), Giemsa stained ×100.

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Immunophenotyping done by FCM showed a cell population in the blast gate, which was positive for CD33, CD13, strong MPO, and CD117. They were negative for HLA-DR, CD34, CD14, and CD15, as well as for all the B- and T-cell markers [Table 2]. Thus, the final diagnosis was reported as APL, AML-M3(M3v), and microgranular variant.

Case 3

An 11-year-old male child presented with a history of high fever spikes and right foot gangrene. Examination did not reveal any hepatosplenomegaly or purpuric rash. However, the coagulation profile revealed mildly raised prothrombin time and international normalized ratio (PT/INR) and abnormal D-dimer levels. Complete hemogram was done, which showed bicytopenia with Hb of 8.6 g/dL and a reduced TLC of 1200/cumm. Platelet count was found to be adequate in this patient [Table 1]. Peripheral smear was examined and showed the presence of abnormal promyelocytes, comprising 56% of the total population of white blood cells (WBCs). The cytoplasm of these cells was granular and showed the presence of numerous Auer rods. BMA smears were cellular and showed >90% population of abnormal promyelocytes with folded and convoluted nuclei and granular cytoplasm, along with the presence of Auer rods [Figure 2a]. The cells were strongly positive for MPO by cytochemistry. Flow cytometry [Table 2] revealed an abnormal promyelocyte population with high side scatter (granulocyte gate) and showed bright, homogenous positivity for CD33, moderate positivity for CD13, CD117, and strong MPO. In addition, positive expression for markers, such as CD34, HLA-DR, and TDT, with dim intensity was noted in this case. However, other markers such as CD14, CD15, CD3, CD4, CD8, CD19, CD20, and CD79a were negative. Thus, based on the above findings, a diagnosis of APL (AML-M3 and hypergranular variant) was proposed.

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Figure 2:

(a) Hypergranular acute promyelocytic leukemia: Shows many abnormal promyelocytes whose cytoplasm contained densely packed large granules with the presence of many kidney-shaped or bilobed nuclei (orange circle), Giemsa stained ×100. (b-c) Bundles of Auer rods (faggot cells) in the cytoplasm of some of the cells (solid white arrow) were also noted, Giemsa stained ×100.

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DISCUSSION

APL has been classified as a rare and distinct subtype under the AML category by the WHO due to the presence of a characteristic translocation between chromosomes 15 and 17.^[4] Even though it accounts for 10-15% of all adult AML cases, in the younger age group, the incidence is extremely low.^[5] APL occurs equally in males and females among all age groups, but we observed a male preponderance.^[6]

The diagnosis and classification of leukemia rely on the simultaneous application of cytomorphology, cytochemistry, immunophenotyping by FC, and a mandatory cytogenetics or molecular technique for confirmation.^[7] Cytogenetic studies or molecular testing are required to depict the presence of PML::RARA fusion gene and confirm the diagnosis of APL. However, due to a lack of available facilities in resource-limited countries like India, FC proves to be an important tool in the timely diagnosis of acute leukemia, especially APL, since it is a medical emergency. In our institute, BMA smear examination and immunophenotyping using flow cytometry were performed to arrive at a conclusion and inform the clinician for prompt management of the patients. Since the advent of targeted therapy regimes involving the use of alltrans retinoic acid (ATRA) and arsenic trioxide (ATO), the prognosis of APL has drastically improved, making it one of the more curable forms of myeloid leukemia.^[8] However, APL is characterized by complications associated with coagulopathy, where both bleeding and thrombotic events can occur. They mainly arise due to the overproduction of promyelocytes and the release of procoagulant factors from their granules.^[9,10] Pediatric APL has high 5-year survival rates in developed nations, but coagulopathy still remains a significant challenge in developing countries like India, resulting in premature death.^[11,12]

A detailed coagulation profile is thus desirable to avoid such complications. All four of our patients did not present with any bleeding abnormalities. However, coagulation tests were found to be deranged in one of the patients who was diagnosed with the hypergranular variant of APL. Thus, an urgent and prompt diagnosis in such patients is extremely crucial to prevent mortality.

APL has favourable cytogenetics; however, additional risk groups have been defined to allow for better risk-adapted therapy. WBC count at diagnosis is considered the most effective predictor of outcome, with patients at high risk (HR) of relapse having TLC >10,000 cells/μL.^[13]

Studies have shown that pediatric patients are more likely to present with an elevated total WBC count at diagnosis.^[5] This was observed in three of our cases who presented with leukocytosis (Cases 1, 2, and 4). Unfortunately, one patient (case 2) with a total leukocyte count of >1 lac cells/cumm and microgranular APL succumbed to the disease within a month of diagnosis.

Our study comprised two hypergranular (classic) and two microgranular cases. BMA morphological findings were in accordance with the WHO. The hypergranular variant was distinguished by abnormal promyelocytes with cytoplasm containing densely packed large granules and kidney-shaped or bilobed nuclei. Bundles of Auer rods were also noted in the cytoplasm of some of the cells.

The microgranular or hypogranular variant was characterized by paucity of granules in the blast or abnormal promyelocytes. Occasional cells showed the presence of Auer rods.

The microgranular variant of APL, accounting for only 10-25% of all APL cases, has distinctive features and is known to show higher association with increased TLC and a short doubling time.^[14] It gets misdiagnosed as myelomonocytic or monocytic AML due to folded nuclei, lack of Auer rods, and faint granules.^[15] Both cases 1 and 2 were diagnosed as APL: AML-M3v variant, which is in alignment with the literature available. Patients under the HR category often require more intensive regimes to achieve better remission rates.^[16]

Relevant clinical features and laboratory tests, including peripheral smear and BMA findings, have been shown in Table 1.

The classic IPT of APL (hypergranular) has been described in detail by the WHO and characteristically shows high side scatter [Figure 3a] and CD13, CD33, CD117, and bright MPO positivity, while CD34, HLA-DR, and CD2 markers are negative. CD64 is frequently positive.^[17] Expression of lymphoid and monocytic markers is rare.

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Figure 3:

CD45 versus side scatter density plot. (a) Case 3 APL, hypergranular variant showing characteristic high side scatter (tear drop) of abnormal blast and promyelocytes population. (b) Case 2 APL, M3v (microgranular variant) show characteristic low side scatter of the blast and promyelocytes.

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In our study, all four cases showed positive expression for CD13, CD33, CD117, and cytoplasmic MPO.

Four flow cytometry patterns of APL have been described in the literature, the most common being the classical pattern observed in the hypergranular variant. On the contrary, the microgranular variant differs from the classical pattern due to frequent overexpression of CD34 and CD2.^[17]

Hypogranular variant shows low side scatter and fall in blast gate [Figure 3b], similar to the findings observed in our study. Among both the cases of APL: AML-M3v (cases 1 and 2), case 1 showed expression of CD34, while case 2 did not, which is in accordance with the existing data.

Microgranular APL generally exhibits a similar IPT as hypergranular APL, with the notable exception of CD34 and CD2 positivity.^[18] Expression of these two markers has been associated with poor prognosis.^[17-19]

The complete immunophenotyping profile of case 2, displaying a population in blast gate with positive expression for CD33, CD13, strong MPO, and CD117, is shown in Figure 4. The cells were negative for HLA DR, CD34, CD14, and CD15 as well as for all the B- and T-cell markers [Figure 4].

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Figure 4:

Flow cytometry data images of case 2 (Acute promyelocytic leukemia M3v), Dot plots-gated population (green color) depicts blast/abnormal promyelocytes population with positive expression for (a) CD13, (b) CD33 vs CD117, (c) cytoplasmic MPO and negative expression of (d) CD34 vs HLA-DR, (e) CD4 vs CD3 and (f) CD10 vds CD19.

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Aberrant dim expression for other markers such as CD34, HLA-DR, and TDT was noted in case 3. The case was positive for HLA-DR and also positive for CD34 and CD117, revealing it to be an early myeloid progenitor. Expression of HLA DR, CD34, and TDT has been associated with poor prognosis in a few studies.^[20,21] Simultaneous presence of HLA-DR and TDT is extremely uncommon, yet documented in the literature. A unique case of coexistence of promyelocytic and lymphoid markers has been described in a study by Paietta et al., wherein exposure of the cultured leukemic cells to retinoic acid led to maturation of both myeloid and lymphoid lineages.^[22] A study conducted by Sun et al. on 143 cases of APL revealed elevated expression of CD34 and HLA-DR in relapsed cases.^[23] On the contrary, Kankhaw et al. conducted a detailed review of 170 APL cases and identified negative expression of HLA DR and MPO positivity as the most significant indicators for APL diagnosis, with a sensitivity of 87.5%, specificity of 95.49%, and an overall accuracy of 94.87%.^[24]

Thus, larger studies are warranted to resolve the inconsistencies in existing literature and to elucidate the immunophenotypic variations in APL and their prognostic relevance.

IPT by flow cytometry of all four cases has been shown in Table 2.

To reduce early mortality in pediatric APL patients, the treatment guidelines recommend initiating tretinoin therapy promptly based on clinical presentation and smear morphology. Transfusion of FFP and cytoreductive therapy is administered to manage coagulopathy and hyperleukocytosis.^[25]

One of our patients (case 2) did not survive due to treatment refusal stemming from financial constraints.

CONCLUSIONS

The true incidence of APL among the pediatric population remains largely unknown. Extensive data are available from the developed nations about the epidemiology, treatment strategies, and the outstanding outcomes of APL cases, including the younger age group. However, there is a major paucity of literature from the developing countries. Our case series aims to help in bridging this gap by providing additional data on APL cases in the pediatric population.

The introduction of ATRA and ATO has drastically changed the outcome of APL, from a highly fatal leukemia to a curable disease, with high long-term survival rates exceeding 90% in some studies. Despite the outstanding cure rates, APL represents a unique disease with peculiarities in terms of clinical presentation and risk factors, including coagulopathy, thus making it a medical emergency. Confirmation of diagnosis relies on cytogenetics or molecular tests; however, flow cytometry is a crucial tool that proves beneficial in the prompt diagnosis of APL, especially in underdeveloped countries like India.

The study emphasizes the importance of cytomorphology, cytochemistry, and flow cytometry as a swift and supplementary tool in preliminary identification of APL and expediting treatment initiation. Several flow cytometry patterns have been described in APL and include rare expression of markers such as CD34, HLA-DR, and CD2. CD34 positivity in particular has been associated with the less common, microgranular variant of APL. Understanding the various IPT variations of APL is important for the correct diagnosis of this rare entity.