TO THE EDITOR
Treatment results of childhood acute lymphoblastic leukemia (ALL) have greatly improved over the last decades. Identification of patients at higher risk of treatment failure remains one major target of current clinical research in this field. In the last decade the Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP)1 and the Berlin–Frankfurt–Münster (BFM) study groups have emphasized the role of corticosteroid sensitivity of leukemic blasts at the time of diagnosis, a risk feature first described and subsequently assessed by the BFM group.2 Patients with ⩾1000 blast cells per μl of peripheral blood after 7 days of prednisone monotherapy and one injection of intrathecal methotrexate (IT-MTX) were considered to have a poor response to prednisone (PPR) and no more than a 35% chance of becoming long-term disease-free survivors on standard therapeutic protocols.3 Recently their outcome has improved when more intensive chemotherapy has been applied.4
Mechanisms underlying insufficient response to steroid in childhood ALL remain unclarified despite repeated attempts.5, 6 The in vivo effect of glucocorticoids is mediated by cell receptors. Studies performed to correlate steroid response in vivo and the numbers of glucocorticoid receptors detectable on the leukemic cells do not allow firm conclusions. Of the three isoforms of the steroids receptors, two, α and β, are more frequently expressed on the leukemic blasts of the B-lineage ALL, but over expression does not correlate with response to glucocorticoids in vivo nor glucocorticoids-induced apoptosis in vitro.7 Expression of the γ isoform appears possibly more related to in vivo response, although these data deserve confirmation.
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with the unique ability to over-ride the immunosuppressive effects of glucocorticoids including the inhibition of proliferation of lymphocytes. A single nucleotide polymorphism of the MIF promoter (−173 G/C) has been recently identified and has been shown to be functionally relevant with the MIF-173*C allele being associated with higher MIF expression both in vitro and in vivo. In patients with juvenile idiopathic arthritis, carriers of the MIF-173*C allele show poor response to systemic and intra-articular glucocorticoid treatment.8
On the basis of this rational, we decided to investigate whether in childhood ALL the MIF-173 polymorphism is associated with prednisone poor response in vivo, an adverse prognostic features which is usually observed in approximately 10% of the patients.
Steroid response was defined on the basis of leukemic blast cell count in peripheral blood, measured after 7 days of steroid monotherapy (prednisone, 60 mg/kg/day) plus one intrathecal dose of methotrexate (age-adjusted dose as follows: <1 year 6 mg, >1<2 years 8 mg, >2<3 years 10 mg, >3 years 12 mg). Patients with blast count <1000/mm3 on day 8 qualified as prednisone good responders (PGR), while those with ⩾1000 blasts/mm3 qualified as prednisone poor responders (PPR).3
Among patients enrolled in the multicenter AIEOP-ALL-2000 study and evaluable for steroid response, we selected 75 consecutive patients who were reported as prednisone-poor-responders (PPR), and 76 consecutive patients reported as prednisone-good-responders (PGR). Peripheral blood or remission bone marrow samples from the patients were obtained from the central AIEOP-ALL repository (G.B., Padova, Italy) upon approval of the study design by the AIEOP-ALL scientific committee. Informed consent for biologic material storage and use for research purposes had been previously obtained at the individual clinical centers by the attending physicians from the patients' legal guardians.
A population of 355 healthy white adult subjects was studied to define the frequency of carriers of the MIF-173*C allele.
Genomic DNA was obtained using the QuIAmp DNA Blood kit (Qiagen). A 365 bp fragment of the MIF promoter was amplified using specific primers and analyzed by DHLPC Wave analysis (Transgenomic, San Jose, CA, USA) utilizing conditions recommended by the WaveMaker 2.0 software and by enzymatic digestion with AluI (New England Biolabs). A total of 151 patients with childhood ALL was studied, and of them 34 (22.5%) were identified as carrier of the MIF-173*C allele. A similar percentage (22.0%) of carriers of this allele was found in healthy white controls.
The distribution of the MIF-173*C allele was also comparable among leukemic patients who were either PGR (18 carriers out of 77 PGRs; 21.6%) or PPR (16 carriers out of 74 PPRs; 23.4%). The MIF-173 C/C genotype (homozygous for the G-C transition) was found in 2 of 355 (0.6%) of controls and 0 of the 151 leukemic patients (Table 1).
Despite the clinical relevance of the steroid response in childhood ALL has been known since many years,1, 2 investigators have not been able to identify its underlying genetic or functional basis. Even very recently, Tissing et al9 addressed this issue, showing that mRNA expression levels of hsp90, the hsp90/hsp70-based chaperone machinery, and cochaperones, do not correlate to glucocorticoid resistance in pediatric ALL Such studies are considered of interest for their potential therapeutic implications.
In this study, we explored to possibility that clinical resistance to steroid therapy documented in patients with arthritis and the MIF-173 G/C polymorphism may give us a hint to interpret the well-known finding that 10% of patients with childhood ALL show an insufficient response, that is, a clinical resistance, to steroid therapy.
To address this issue, we studied a large cohort of patients enrolled in the current AIEOP-ALL 2000 trial. Yet, the proportion of subjects with the MIF-173 G/C polymorphism detected among children with ALL was not different from that in the control population, and in particular, patients with ALL and PPR do not carry a higher proportion of such polymorphism.
Albeit negative our results have two implications. Since the −173 SNP has been shown to be functionally relevant in vitro and in vivo,8 our results suggest that MIF may not be relevant in the proliferation of leukemic blasts, nor in the protection from apoptosis, despite several observations that have involved MIF in protecting a variety of cell types from apoptosis. From a more practical point of view, the MIF-173 G/C polymorphism, as well as other previously investigated genetic variants, does not contribute to prednisone poor response in vivo in childhood acute lymphoblastic leukemia. Alternative biologic markers are worth to be investigated in such patients.
Conter V, Aricò M, Valsecchi MG, Basso G, Biondi A, Madon E et al. Long-term results of the Italian Association of Pediatric Hematology and Oncology (AIEOP) acute lymphoblastic leukemia studies, 1982–1995. Leukemia 2000; 14: 2196–2204.
Schrappe M, Reiter A, Zimmermann M, Harbott J, Ludwig WD, Henze G et al. Long-term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Berlin–Frankfurt–Munster. Leukemia 2000; 14: 2205–2222.
Riehm H, Reiter A, Schrappe M, Berthold F, Dopfer R, Gerein V et al. Corticosteroid-dependent reduction of leukocyte count in blood as a prognostic factor in acute lymphoblastic leukemia in childhood (therapy study ALL-BFM 83). Klin Pädiatr 1987; 199: 151–160.
Aricò M, Valsecchi MG, Conter V, Rizzari C, Pession A, Messina C et al. Improved outcome in high-risk childhood acute lymphoblastic leukemia defined by prednisone-poor response treated with double Berlin-Frankfurt-Muenster protocol. II. Blood 2002; 100: 420–426.
Lauten M, Cario G, Asgedom G, Welte K, Schrappe M . Protein expression of the glucocorticoid receptor in childhood acute lymphoblastic leukemia. Haematologica 2003; 88: 1253–1258.
Lauten M, Matthias T, Stanulla M, Beger C, Welte K, Schrappe M . Association of initial response to prednisone treatment in childhood acute lymphoblastic leukaemia and polymorphisms within the tumour necrosis factor and the interleukin-10 genes. Leukemia 2002; 16: 1437–1442.
Albuquerque LM, Garcia AB, Mengel JO, Falcao RP, de Castro M, Rego EM . The higher expression of alpha and beta isoforms of the human glucocorticoid receptor in leukemic B-progenitors compared to normal CD10+ BM cells does not correlate with methylprednisolone-induced apoptosis. Leukemia 2004; 18: 890–892.
De Benedetti F, Meazza C, Vivarelli M, Rossi F, Pistorio A, Lamb R, et al. British Paediatric Rheumatology Study Group. Functional and prognostic relevance of the −173 polymorphism of the macrophage migration inhibitory factor gene in systemic-onset juvenile idiopathic arthritis. Arthritis Rheumatism 2003; 48: 1398–1407.
Tissing WJ, Meijerink JP, den Boer ML, Brinkhof B, Pieters R . mRNA expression levels of (co)chaperone molecules of the glucocorticoid receptor are not involved in glucocorticoid resistance in pediatric ALL. Leukemia 2005; 19: 727–733.
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