Published online before print August 17, 2007
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* Hematology and
Imaging Divisions, Department of Internal Medicine, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil; and
Federal University of Santa Maria, Santa Maria, RS, Brazil
2 Correspondence: Hematology Division, Department of Internal Medicine, Medical School of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, CEP 14048-900, Ribeirão Preto, SP, Brazil. E-mail: emrego{at}hcrp.fmrp.usp.br
ABSTRACT
The use of all trans-retinoic acid (ATRA) is the basis of treatment of acute promyelocytic leukemia (APL) and represents the paradigm of differentiation therapy. In general, ATRA is well-tolerated but may be associated with a potentially lethal side-effect, referred to as retinoic acid or differentiation syndrome (DS). The cellular and molecular mechanisms of DS are poorly understood and involve changes in the adhesive qualities and cytokine secretion of leukemic cells during ATRA-induced differentiation. As leukocyte extravasation is a key event in DS pathogenesis, we analyzed the association between the polymorphisms at Exon 4 (G241R) and Exon 6 (E469K) of ICAM-1 and Exon 3 (L125V) of PECAM-1 genes with DS development in APL patients treated with ATRA and anthracyclines. DS was diagnosed in 23/127 (18.1%) APL patients at an average of 11.5 days after the start of ATRA. All patients presented respiratory distress associated with increased ground-glass opacity in chest radiographies. Other accompanying symptoms were: fever not attributable to infection (65.2%), generalized edema (37.5%), weight gain (37.5%), and impairment of renal function (8.6%). We detected an association between development of DS and the AA genotype at Codon 469 of ICAM-1 (odds ratio of 3.5; 95% confidence interval: 1.2–10.2). Conversely, no significant association was detected between G241R or L125V polymorphisms at Exon 4 of ICAM-1 and Exon 3 of PECAM-1, respectively. Our results suggest that susceptibility to DS in APL patients may be influenced by genetic variation in adhesion molecule loci.
Key Words: acute myelogenous leukemia • all trans-retinoic acid • adhesion molecules
Acute promyelocytic leukemia (APL) is invariably associated with gene rearrangements involving the retinoic acid receptor 
(RAR) locus on Chromosome 17. In the majority of cases, RAR is translocated and fused to the promyelocytic leukemia (PML) gene on Chromosome 15 as a consequence of the t(15;17)(q22;q21). Morphologically, two variants of APL are recognized: the granular and the hypogranular, corresponding to the M3 and M3v subtypes of the FAB classification, respectively. APL treatment is based on the use of all trans-retinoic acid (ATRA), which induces terminal granulocytic differentiation of blasts and clinical remission. ATRA is generally well-tolerated but may be associated with a potentially lethal side-effect, referred to as RA or differentiation syndrome (DS) [1
, 2
]. Initially described by Frankel et al. [3
], this syndrome is characterized by fever, pleural and pericardial effusion, respiratory distress, weight gain, and pulmonary infiltrates noted on chest radiography. Six percent to 27% of APL patients develop DS, and mortality rates range from 1% to 7% [4
5
6
]. Previously reported, predictive factors for development of DS include high leukocyte counts at diagnosis, hypogranular variant, PML/RAR bcr3 isoform, and a pattern of CD13 and CD33 expression [1
, 2
, 6
7
8
]; however, the existence of these associations is controversial.
The cellular and molecular mechanisms of DS are poorly understood, and the proposed mechanisms involve changes in the adhesive qualities and cytokine secretion during ATRA-induced differentiation [9 ]. The ICAM-1 (CD54) and PECAM-1 (CD31) are members of the Ig superfamily. ICAM-1 is widely expressed at basal levels on the surface of endothelial cells and leukocytes [10 ] and in the APL cell line NB4 [11 ] and can be up-regulated by proinflammatory cytokines and by ATRA. PECAM-1 is constitutively expressed by most circulating leukocytes, platelets, and endothelial cells. Both adhesion molecules are involved in endothelium integrity and extravasation of cells from the blood compartment into the vessel and underlying tissue [12 ]. As leukocyte extravasation is involved in DS pathogenesis, we decided to analyze the association between the polymorphisms at Exon 4 (G241R) and Exon 6 (E469K) of ICAM-1 and Exon 3 (L125V) of PECAM-1 genes and DS development in APL patients treated with ATRA and anthracyclines.
Bone marrow or peripheral blood (PB) specimens from 127 patients with APL were collected at diagnosis after informed consent was obtained. The genetic confirmation of the PML/RAR rearrangement was done by RT-PCR using the BIOMED-1 protocol [13
]. PB samples from 249 healthy volunteers were used as controls. Patients were classified based on their white blood cell (WBC) and platelet counts at diagnoses as high (WBC>10,000/µl), low (WBC
10,000/µl, and platelets>40,000/µl), or intermediate risk (WBC10,000/µl, and platelets40,000/µl) of relapse, according to the PETEHMA criteria [14
]. PCR conditions and the restriction enzyme digestion used for ICAM-1 and PECAM-1 polymorphism detection were described elsewhere [15
, 16
].
Treatment was based on the APL93 protocol of the European APL Group [5
], using ATRA associated with chemotherapy during induction. Daunorubicin was initiated within 7 days of ATRA treatment. DS was defined clinically by the presence of respiratory distress, and suggestive pulmonary infiltrates were associated with fever, pleural/pericardial effusion, or weight gain, initiated after at least one dose of ATRA in patients with normal chest radiography and absence of the listed symptoms (except for fever) at diagnosis. For each polymorphism, allele and genotype frequencies were calculated, and the Hardy-Weinberg equilibrium was evaluated using the Fisher’s exact test. The association between DS and ICAM-1/PECAM-1 allelic isoforms was determined by 
2 analysis.
DS was diagnosed in 23/127 (18.1%) APL patients. This frequency is higher than that described by de Botton et al. [5 ] in the APL93 study for the treatment arm using ATRA and anthracycline simultaneously (9.2%) but similar to that described for patients using anthracyclines after complete remission was obtained with ATRA monotherapy (18%). Discrepancies in the incidence of DS reported in literature may reflect the lack of objective diagnostic criteria. In the present study, only patients with respiratory distress accompanied by pulmonary infiltrates were considered to present DS. The syndrome developed in patients at an average of 11.5 days (between the 4th and 20th days) after the start of ATRA, and other accompanying symptoms were: fever not attributable to infection (65.2%), generalized edema (37.5%), weight gain (37.5%), and impairment of renal function (8.6%). Table 1 presents clinical and laboratorial features at diagnosis of APL patients, with and without DS. No significant differences were detected between the two groups. It is worthwhile to point out that WBC counts and the frequency of cases presenting with more than 10,000 leukocytes/µl were not significantly higher in the group of patients with DS. Our results corroborate those by de Botton et al. [2 ] and Vahdat et al. [6 ], suggesting that high WBC counts are not a predictive factor for DS development. Chest radiographs of all patients showed increased ground-glass opacity and an increased vascular pedicle width. Pleural effusion was detected in 14 patients. No nodule formation nor septal lines were observed. These findings are consistent with those of Jung et al. [17 ].
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Table 1. Demographics and Complete Blood Counts of APL Patients with or without DS at Diagnosis
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Table 2. Genotype and Allele Distribution of the Polymorphisms Analyzed in APL Patients and Healthy Controls
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of 0.05. The samples sizes needed for the observed risk estimated for ICAM-1 G241R and E469K polymorphisms at the same test conditions would be 400 and 70 subjects in each group, respectively. It is unclear how the E469K polymorphism may affect ICAM-1 multiple functions. Nevertheless, Amodu et al. [18
] demonstrated that the presence of the G allele at position 469 was associated with increased risk (3.6 times) of severe malaria. In addition, ICAM-1 gene polymorphisms have been associated with several inflammatory disorders such as multiple sclerosis [21
], Type 1 diabetes [22
], ulcerative colitis, and Crohn’s disease [23
]. As Position 469 is located in the extracellular portion of ICAM-1, it is conceivable that it may affect interactions with LFA-1 and the adhesion of leukocytes [24
]. To our knowledge, the present study is the first to demonstrate an association between ICAM-1 E469K polymorphism and the development of DS, and our results support the concept that genetic-determined variations in adhesion molecules may be an important factor in determining susceptibility to several inflammatory diseases. |
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Table 3. Analysis of Association of ICAM-1 and PECAM-1 Polymorphisms with the Development of DS in APL Patients
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ACKNOWLEDGEMENTS
B. A. A. S-L. is a recipient of Fundação de Apoio a Pesquisa do Estado de São Paulo (FAPESP) grant No. 06/58136-1. This work was supported by a FAPESP grant (No. 98/14247-6). The authors are grateful to Prof. Lewis Greene for critically reviewing the manuscript.
FOOTNOTES
1 These authors contributed equally to this work. 
Received February 6, 2007; revised July 12, 2007; accepted July 13, 2007.
REFERENCES
isoform, and CD13 expression in patients treated with all-trans retinoic acid Blood 84,3843-3849 fusion mRNA type with pretreatment hematologic characteristics but not treatment outcome in acute promyelocytic leukemia: an intergroup molecular study Blood 90,1656-1663
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