Research Article
10.2217/PGS.13.249 © 2014 Future Medicine Ltd
Pharmacogenomics (2014) 15(5), 619–627
ISSN 1462-2416
619
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Franca, Rebora, Athanasakis et al.
nephrotic syndrome revealed a higher frequency of the -308A allele and the AA genotype in the former group [8]; In human leukocyte antigen (HLA)-DR mismatched transplant patients, the TNF-a high producer genotypes were significantly associated with multiple and steroid-resistant rejection episodes [9]. By altering the transcriptional activity of the cytokine promoter, the -308G>A variant could therefore influence treatment response in ALL, particularly the steroid response. Earlier studies on primary ALL cells and cell lines showed that aberrant expression (high levels) of endogenous TNF-a may be associated with refractory disease [10–12]. However, case–control studies in childhood ALL patients could not find a correlation between the -308G>A variant and prednisone response or relapse [13–15], although these studies were limited in sample size and could therefore have underestimated the pharmacological importance of this polymorphism in ALL outcome. This study was aimed at investigating the impact of the TNF-a -308G>A variant on the risk of relapse in pediatric patients affected by ALL and cured according to the AIEOP-BFM (Associazione Italiana Ematologia Oncologia Pediatrica-Berlin Frankfurt Münster) ALL 2000 study protocol.
Methods Patients The present retrospective study is based on a cohort of 1999 Philadelphia-negative ALL patients (55.7% male, 1–18 years), newly diagnosed in Italy between September 2000 and July 2006, homogenous for ethnicity (Caucasians) and for risk-adapted treatment, being stratified and cured according to the AIEOP-BFM ALL 2000 study protocols (ClinicalTrials.gov identifier NCT00613457). The following criteria were used to identify patients as high-risk: Presence of t(4;11) or t(9;22); Prednisone poor response (≥1000 blasts/µl in the peripheral blood at day +8, after 7 days of daily prednisone and a single dose of intrathecal methotrexate at day +1); Inability to achieve complete remission in bone marrow aspirate at day +33; High burden (≥10 -3) of minimal residual disease (MRD) measured by PCR (PCR-MRD) at day +78. 620
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More intense regimens during consolidation (blocks) and re-induction phases are used for patients at high-risk compared with the others. The standard-risk group included patients who lacked high-risk criteria and were negative by PCR-MRD performed by using two sensitive markers (≥10 -4) at both day +33 and day +78. The intermediate-risk group included the remaining patients, and those not evaluated by PCR-MRD. Written informed consent was obtained from the parents or legal guardians before patient enrolment, while the protocol was approved by the ethics committee of each participating institution. Study design & statistical analysis 614 patients were selected to be genotyped according to a novel two-phase study design. The sampling strategy and the statistical analyses performed were described previously [16]. Briefly, the selected subcohort comprised all relapsed patients within the overall cohort and a subset of other patients in remission, randomly chosen within each of the three risk groups according to optimal strata specific sampling fractions. Representativeness of the whole population was recovered in the statistical analysis by appropriate weights (equal to the inverse of the sampling fractions). Patients were followed from diagnosis up to relapse, second malignancy, death or December 2008. Weighted estimate of the crude cumulative incidence of relapse was calculated from the date of diagnosis to the date of relapse accounting for competing risks (death and second malignancies). The comparisons of these curves were based on the Wald test in a Cox model with TNF-a genotype as covariate. To account for potential confounders the weighted Cox model for two-phase design was applied adjusting for immunophenotype (T-cell ALL [T-ALL] and B-cell precursor ALL [B-ALL]), risk group (standard-, medium-, high-risk group), age (1–5, 6–9, 10–17 years) and gender and including interaction terms between the SNP and these characteristics. Genotyping DNA of the AIEOP ALL patients was kindly provided by SSD Clinical and Experimental Hematology Bio Bank, Department of Paediatrics, University of Padua, Italy (AIEOP Bio Bank). Genotype analyses for the TNFa-308G>A variant were performed blinded to patient outcome on DNA extracted from blasts of bone marrow aspirates at first diagnosis or, when not available, at relapse by using allelic specific fluorescent probes with TaqMan® SNP future science group
Treatment prognostic genetic factors in childhood acute lymphoblastic leukemia
genotyping assays (Applied Biosystems, CA, USA). Subjects of this cohort had been previously genotyped for deletions of GST-M1 and GST-T1 genes [16].
Results The 614 patients of the AIEOP-BFM ALL 2000 cohort were selected to be genotyped according to the study design: among them, 363 patients were males (59.1%); 108 (17.6%) were T-ALL; 355 were preschooler (age less than 6 years, 57.8%) and 142 (23.1%) were older than 10 years; 345 fell in the medium-risk group (56.2%), whereas 78 (12.7%) and 191 (31.1%) were treated according to the standard- and the high-risk regimens, respectively. 5-year cumulative incidence of relapse is reported in Figure 1. The incidence of relapse was similar for wild-type patients and subjects carrying the minor allele genotypes (5-year estimates 16.4 ± 1.29% versus 23.0 ± 4.95%, respectively, p = 0.13, Figure 1A). Table 1 summarizes the TNF-a -308G>A variant distribution among the 614 AIEOP ALL genotyped patients, the weighted 5-year cumulative incidence of relapse, and the association between the genetic variations and risk of relapse analyzed by means of the weighted Cox model. As the SNP is very rare in homozygosis among Caucasians, analyses were performed considering a dominant model. In order to account for possible differential effects of the polymorphism according to patient distinct pharmacological profiles [17] and therapeutic approaches, we considered the following interaction terms in the Cox model (Table 2): Blast immunophenotype (T-ALL and B-ALL); Age class (with cut-off set at 6 and 10 years old for roughly dividing preschoolers, children and adolescents); Polychemotherapeutic protocols (standard-, medium- and high-risk); Early in vivo response to the steroid prephase (defined as prednisone good responders [PGR] and prednisone poor responders [PPR] at day +8 from diagnosis). A highly significant interaction was found between immunophenotype and -308G>A variant (p = 0.0007), with the minor allele genotypes being adverse genetic markers in B-ALL and protective genetic traits in T-ALL. Figure 1B & 1C report the crude cumulative incidence of relapse by -308G>A variant in B-ALL and T-ALL patients, respectively. These curves confirm the opposite effect of the SNP in the two future science group
Research Article
immunophenotypes. A significant interaction term was observed also among risk protocols (p = 0.0041) where the minor allele genotypes emerged as prognostic factors of relapse only in the standard risk group, and in the in vivo response to the steroid prephase (p = 0.035) with a trend of higher risk of the minor allele genotypes in the PGR group. Among the children considered, only one T-ALL patient fell in the standard risk (and generally in the AIEOPBFM LLA 2000 whole cohort, only 25 out of 244 T-ALL): therefore, the genetic adverse effect observed in the less intensive group is limited to the B-ALL immunophenotype. Instead, the minor allele genotypes were protective for T-ALL in the medium- and high- therapeutic arms. We also investigated the role of the polymorphism on prednisone response by means of a weighted logistic model adjusted by sex, age and immunophenotype (p = 0.97, odds ratio [OR]: 0.99, 95% CI: 0.53–1.85), revealing that it was not decisive to assess prednisone response itself.
Discussion Due to the risk-adapted polychemotherapy, today’s cure rates for pediatric ALL reach more than 80% of success in the developed countries. Actually, the patients’ risk stratification is assessed mainly on the basis of MRD and other molecular methods [18,19], so that traditional adverse prognostic factors such as T-ALL immunophenotype and older age at diagnosis are outdated. The classification of T-ALL or B-ALL is now further defined by genomic alterations with potential prognostic or therapeutic relevance [20], and age is no longer associated with worse outcome. In the context of the AIEOP-BFM ALL 2000 protocol, within the whole cohort of 1999 patients enrolled, 306 children relapsed (15.3%, median follow-up: 4 years), among which 28 were in the standardrisk and 186 were in the medium-risk group, revealing therefore that the majority of them were treated according to the less intensive arms because of their favorable prognostic features. These absolute numbers emphasize the need of identifying additional criteria to refine the treatment stratification, particularly for those patients currently considered at lower risk. Individual genetic polymorphisms influencing drug disposition, metabolism and mechanisms of action might affect treatment efficiency and could therefore be potential predictors of response to take into account to personalize the chemotherapy [21]. However, it is unlikely www.futuremedicine.com
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A
Crude cumulative incidence of relapse
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Franca, Rebora, Athanasakis et al.
0.6
ALL Patients Wild type Non-wild-type
0.4
No. relapsed 5-year estimate (SE) 204 47
510 103
16.4% (1.29) 23% (4.95)
0.2 p = 0.13 0.0 1
0
4
3
2
5
B
Crude cumulative incidence of relapse
Time since diagnosis (years) 0.6 B-ALL
0.4
Wild type Non-wild-type
Patients
No. relapsed
421 81
154 42
5-year estimate (SE) 14.3% (1.12) 25.6% (3.82)
0.2 p = 0.0097 0.0
2
1
0
3
4
5
C
Crude cumulative incidence of relapse
Time since diagnosis (years) T-ALL 0.6
Patients Wild type Non-wild-type
86 22
No. relapsed 5-year estimate (SE) 49 5
35.6% (5.24) 11.4% (5.17)
0.4
0.2
p = 0.0105
0.0 0
1
2
3
4
5
Time since diagnosis (years)
Figure 1. Crude cumulative incidence of relapse according to TNF-a -308G>A variant genotypes in genotyped patients. (A) ALL, (B) B-ALL and (C) T-ALL genotyped patients. Hz+mut patients were analyzed together as minor allele genotypes. p-values refers to Wald test in univariate Cox model for twophase designs. ALL: Acute lymphoblastic leukemia; B-ALL: B-cell acute lymphoblastic leukemia; SE: Standard error; T-ALL: T-cell acute lymphoblastic leukemia.
that a single polymorphism could influence the outcome of such a complex disease as ALL and of such complex therapeutic approaches, 622
Pharmacogenomics (2014) 15(5)
particularly in the case of the TNF-a gene. This gene is located on chromosome 6p21.3, in the class III region of the human major histocompatibility complex (MHC) within the human leukocyte antigen (HLA). This part of the genome is essential to the immune system and is one of the most polymorphic in humans with a distribution of one SNP per 1.71 kb instead of the average one SNP per 1.9 kb found throughout the human genome [22,23]. Multiple SNPs identified in the promoter region and in the TNF-a gene itself are in linkage disequilibrium, and many of them are included in HLA haplotypes [24,25], but the TNF-a -308G>A variant is the one studied for the association with outcome in pediatric leukemia. The TNF-a -308G>A variant could be the nominal SNP of a clinical phenotype, rather than the truly and only causative genotype. Indeed, several studies investigated the possible concomitant role of other polymorphisms. For instance, the high-cytokine producer haplotype comprising the minor alleles of TNF-a -308G>A and lymphotoxin-a (LT-a) +252A>G (rs909253) is generally studied for the association with autoimmune and severe inflammatory disorders and in the pathogenesis and prognosis of hematologic malignancies. Stanulla et al. failed to observe any meaningful association between the high-producer TNF-a/LT-a haplotype in comparison with a low-producing one [14]. Also, Takeuchi et al. did not find an association between the haplotype and the immunophenotype, the response to chemotherapy, and the event-free survival in ALL [15]. The TNF-a -308G>A variant is also commonly studied with IL-10 -1082A>G (rs1800896), with conflicting results when analyzed for the association with ALL overall survival [13,26]. Genetic traits could be covariates of importance, together with the already known clinical and biological ones, for improving ALL prognosis. This study suggests that the presence of at least one -308A allele conferred a protective advantage and reduced risk of relapse in T-ALL, while it predicted a worse clinical outcome in B-ALL patients falling in the AIEOP-BFM ALL 2000 low risk arm. The exact reason for these discrepant results is unknown. Accumulating evidence suggests that the net effect of TNF-a action in vivo can be attributed to the biological context within which the cytokine is exerting its action, and is the result of a crosstalk between the various signaling pathways engaged, determined by timing and duration of TNF-a action, tissue type and TNF receptors composition [27]. future science group
Treatment prognostic genetic factors in childhood acute lymphoblastic leukemia
Research Article
Table 1. Genotype distribution of TNF-a -308G>A variant in AIEOP acute lymphoblastic leukemia patients genotyped subgroup and 5-year cumulative incidence of relapse weighted to the whole AIEOP-BFM ALL 2000 cohort.
All patients
TNF-a -308G>A
Patients (n)
Relapses (n)
5-year cum. inc. (%)
SE
Hazard ratio (95% CI)
p-value HWE p-value
wt
510
204
16.41
1.29
1.00
hz
100
46
–
–
– (–)
–
–
mut
3
1
22.96†
4.95
1.13 (0.75–1.72)
0.55
>0.05
NA
1
–
–
–
– (–)
–
–
Hazard ratio adjusted by risk group, age class, gender and immunophenotype. † hz+mut: analyses on the minor allele genotypes group. 5-year cum. inc. (%): Weighted 5-years cumulative incidence of relapse; NA: Not assessed; SE: Standard error; HWE: Hardy–Weinberg equilibrium.
The death-inducing capability of the cytokine is mediated by TNF membrane receptor type 1 (TNF-R1). Micheau et al. have demonstrated that the TNF-R1-mediated signal transduction includes a checkpoint for balancing cell functional fate between life and death [28]. Indeed, TNF-a activates the prosurvival transcription factor NF-kB through the ubiquitin-mediated degradation of its inhibitor, IkB, whereas it activates the caspase cascade in instances where this signal for NF-kB failed [29]. In light of such complex biological interplay, we hypothesized that TNF-a might behave as an amplifier of the prevailing stimuli acting on the cell, strengthening the antiapoptotic pathways when cell integrates mostly prosurvival signals, and supporting the death process if signals delivered concurrently on the cell are mainly apoptotic. It should be noted that in the AIEOP-BFM ALL 2000, T-ALL patients received a more aggressive consolidation phase in comparison with B-ALL, regardless of their risk class. This slightly intensive protocol might account for enhanced cytotoxic signals, likely further boosted in T-ALL carrying the minor allele genotypes because of putative higher TNF-a levels: these patients could be genetically predisposed to a better pharmacological response, and thus a better protection from relapse in such therapeutic condition. On the contrary, the studied polymorphism had an adverse effect in B-ALL standard-risk patients: in the AIEOPBFM ALL 2000, patients in the low-arm group were randomized and half of them received one third less dexamethasone, a known downregulator of TNF-a expression, and half pulses of anthracyclines during the reinduction phase in comparison to the medium-risk group. Interestingly, endogenous TNF-a was found to mediate resistance to doxorubicin-induced cytotoxicity [10]. This less intense protocol could be counterproductive for higher cytokine producers, and future science group
the minor allele genotypes could represent an adverse individual trait, leading to resistance and disease progression. These hypotheses are highly speculative and would have benefited of measuring TNF-a plasma levels at diagnosis and during therapy. However, being designed as a retrospective pharmacogenetic study, cytokine levels were not investigated. Subsets analysis makes sense in moving towards a personalized medicine, rendering the pediatric population more uniform, and results more informative for targeting patient subgroups. Noteworthy is that subgroups considered in the present study were still large in size because of the large number of the whole series genotyped. In a previous study with 135 children with ALL, treated according to BFM-protocols -86 and -90, subjects with the minor allele genotypes did also not show a statistically significant association with risk of relapse nor with prednisone response compared with wild-type subjects in overall analysis. However, in the subgroup analysis approach evaluating the TNF-a genotype exclusively in patients who had at least one known clinical risk factor for poor treatment outcome, the minor allele genotypes emerged as adverse genetic traits within 45 PPR [13]. Our subset of PPR patients included 131 patients and did not show a significant genetic effect of the TNF-a -308G>A variant. Replication of results is essential and needs to be performed before drawing any firm conclusion. However, when the genotyped population was divided into training (with 413 observations) and replication (with 197 observations) sets (by a random Bernoulli variable with parameter 2/3), the opposite effect of TNF-a -308G>A variant in B-ALL and T-ALL was observed in both subgroups, with the corresponding interaction term still significant (data not shown). Given that risk criteria adopted in the actual AIEOP protocols remain the basic guidelines www.futuremedicine.com
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Table 2. Impact of TNF-a -308G>A variant on relapse in acute lymphoblastic leukemia patients stratified by immunophenotype (Cox model adjusted by gender, risk and age class), by age group (Cox model adjusted by gender, risk class and immunophenotype), by risk group and in vivo prephase response (Cox model adjusted by gender, age and immunophenotype). TNF-a SNP rs1800629 (-308G>A) Patients (n)
Relapses (n)
5-year cum. inc. (%)
SE
wt
421
154
14.31
1.12
Non-wt
81
42
25.64
3.82
wt
86
49
35.58
5.24
22
5
11.36
5.17
HR†
95% CI
1.72
1.11–2.66
0.24
0.08–0.68
1.51
0.91–2.51
1.04
0.45–2.40
0.42
0.13–1.36
1.52
0.95–2.42
0.50
0.20–1.27
6.25
1.98–19.68
1.14
0.66–1.96
0.61
0.29–1.28
Immunophenotype (p‡ = 0.0007) B-ALL T-ALL
Non-wt
Age at diagnosis, years (p = 0.1363) ‡
1–5 6–9 10–17
wt
293
104
13.94
1.43
Non-wt
62
30
22.57
4.8
wt
90
41
18.75
3.37
Non-wt
26
13
33.12
9.80
wt
127
59
21.44
3.17
15
4
11.72
6.30
Non-wt
Prednisone response (p = 0.0350) ‡
PGR PPR
wt
401
159
14.71
1.11
Non-wt
80
40
23.18
4.27
wt
108
44
30.35
3.87
Non-wt
23
7
21.04
7.26
wt
63
13
3.66
1.06
Non-wt
14
9
20.55
8.55
wt
290
123
18.00
1.50
Non-wt
55
26
22.99
5.03
wt
157
68
32.82
3.20
Non-wt
34
12
25.70
6.69
Risk group (p = 0.0041) ‡
Standard Medium High
Patients with missing genotype or clinical data were omitted from the analysis. † Non-wt (hz+mut) versus wt. ‡ p-value for the interaction. 5-year cum. inc. (%): Weighted 5-year cumulative incidence of relapse; B-ALL: B-cell acute lymphoblastic leukemia; HR: Hazard ratio; PGR: Prednisone good responders; PPR: Prednisone poor responders; SE: standard error; T-ALL: T-cell acute lymphoblastic leukemia.
for stratification, if our results are confirmed in a validation cohort, the presence of at least one minor allele of the -308G>A variant could be discriminative to target B-ALL patients with all the best prognostic factors now used (i.e., MRD, PGR) into the medium group rather than into the standard one. In the current AIEOP-BFM ALL 2009 trial, the less intense therapeutic arm consists of half pulses of anthracyclines during the induction phase (rather than during the re-induction phase as in the AIEOP-BFM ALL 2000), and thus this SNP should still be investigated as an applicable 624
Pharmacogenomics (2014) 15(5)
prognostic tool. The possible concomitant role of other genes should also be considered. The final aim is to give all children the most appropriate intensive therapy, further improving the already outstanding success rate of pediatric ALL chemotherapy.
Future perspective In ALL, risk-adapted polychemotherapy has great success, and risk stratification criteria based mainly on minimal residual disease are highly reliable, but 10–15% of patients still do not respond adequately in terms of efficacy and future science group
Treatment prognostic genetic factors in childhood acute lymphoblastic leukemia
outcome. Pharmacogenomics is a relatively novel branch of investigation and represents an innovative frontier of medicine, standing at the basis of personalized treatment optimization. In the last decade, pharmacogenomics became more and more important because of the continuous advances in high-throughput DNA technology and bioinformatics methods. In the context of the AIEOP-BFM ALL protocols, the present work suggests that the low penetrance TNF-a -308G>A variant might be a useful genetic marker, although only in selected patients. Indeed, if our results are confirmed in a validation cohort, the minor allele genotypes could become discriminative to include B-ALL patients with the best prognostic factors into the medium-risk group rather than into the standard one. A future perspective will be to develop algorithms and informatics programs that will integrate demographic, clinical and pharmacogenetic data into user-friendly and cheap softwares for helping physicians in therapy personalization. Financial & competing interests disclosure All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on
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request from the corresponding author) and declare: no support from any organization for the submitted work, no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years and no other relationships or activities that could appear to have influenced the submitted work. Supporting foundations: Italian Ministry of Health, Fondazione Benefica Alberto e Kathleen Casali. R Franca and D Favretto are recipient fellowships from IRCCS Burlo Garofolo, Trieste. Grant from the European Commission (FP7-HEALTH-F2–2011 261474) to P Rebora and MG Valsecchi. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary AIEOP-BFM risk stratification of acute lymphoblastic leukemia patients needs to be improved In AIEOP-BFM acute lymphoblastic leukemia (ALL) protocols, patient risk stratification is based mainly on minimal residual disease. In the AIEOP-BFM ALL 2000 trial, 85% of pediatric patients were successfully cured with the risk-adapted polychemotherapeutic protocols employed. Among relapsed children, only 30% had adverse prognostic features and fell within the high-risk group. Patient genetic traits could be predictor markers of prognosis Polymorphisms in genes involved in drug disposition, metabolism and mechanisms of action might affect outcome. TNF-a is a cytokine involved in the regulation of immune cells. The TNF-a SNP rs1800629 in the promoter region has been associated with increased transcriptional activity and steroid resistance in pediatric inflammatory and autoimmune diseases; no correlation has been found in small-scale studies on ALL outcome. Innovative approach for a pharmacogenetic study on Italian AIEOP-BFM ALL 2000 cohort The novel two-phase design allows a considerable gain in precision of the estimate of genotype effect on relapse because it introduces sampling fractions optimized on the basis of a previous correlation between genotypes and outcome in a pilot study on the same AIEOP-BFM ALL 2000 whole cohort. With 614 patients genotyped, this study is probably one of the largest surveys performed on European ALL protocols. TNF-α SNP rs1800629 could influence outcome in specific pediatric ALL subgroups No significant association was found in the whole cohort. Highly significant interaction was found between immunophenotype and TNF-a SNP rs1800629 (p = 0.0007), with the non-wild-type genotype being an adverse genetic marker in non-T-ALL (hazard ratio [HR]: 1.72, 95% CI: 1.11–2.66) and a protective genetic trait in T-ALL (HR: 0.24, 95% CI: 0.08–0.68). A significant interaction term was also observed among risk protocols (p = 0.0041), where the non-wild-type genotype emerged as a prognostic factor of relapse for standard-risk patients (HR: 6.25, 95% CI: 1.98–19.68, only one T-ALL in the subgroup analyzed). Conclusion This study suggests that ALL therapeutic stratification could benefit a genotyping approach. In non-T-ALL standard-risk patients, whose risk has been assessed by AIEOP-BFM in-use criteria, TNF-a SNP rs1800629 could be a further tool to improve relapse prediction and target non-wild-type patients in the medium-risk group. Our results need confirmation in a validation cohort for understanding their clinical application.
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Affiliations
Raffaella Franca Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, UO Pediatric Hemato-Oncology, Via dell’Istria 65/1, 34137 Trieste, Italy
Paola Rebora Center of Biostatistics for Clinical Epidemiology, Department of Health
21 Stocco G, Franca R, Verzegnassi F, Londero
M, Rabusin M, Decorti G. Pharmacogenomic approaches for tailored anti-leukemic therapy in children. Curr. Med. Chem. 20(17), 2237–2253 (2013).
Detailed recent overview on genes and what might influence ALL treatment in terms of outcome and/or toxicity, and underlies the role of genetic variants, particularly SNPs, in predicting clinical response, with particular reference to the AIEOP-BFM ALL 2009 protocol.
future science group
Treatment prognostic genetic factors in childhood acute lymphoblastic leukemia
Sciences, University of Milano-Bicocca, Via Cadore 48, 20900 Monza (Milan), Italy
Emmanouil Athanasakis Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, Medical Genetic Service, Via dell’Istria 65/1, 34137 Trieste, Italy Diego Favretto Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, UO Pediatric Hemato-Oncology, Via dell’Istria 65/1, 34137 Trieste, Italy
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Research Article
Federico Verzegnassi Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, UO Pediatric Hemato-Oncology, Via dell’Istria 65/1, 34137 Trieste, Italy
Maria Grazia Valsecchi Center of Biostatistics for Clinical Epidemiology, Department of Health Sciences, University of Milano-Bicocca, Via Cadore 48, 20900 Monza (Milan), Italy
Giuseppe Basso Pediatric Clinic, Onco-Hematology, University of Padua, via Giustiniani 3, 35128 Padua, Italy
Giuliana Decorti Department of Life Sciences, University of Trieste, Via Fleming 22, 34127 Trieste, Italy
Alberto Tommasini Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, Laboratory of Immunopathology, Via dell’Istria 65/1, 34137 Trieste, Italy
Marco Rabusin Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, UO Pediatric Hemato-Oncology, Via dell’Istria 65/1, 34137 Trieste, Italy
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10.2217/PGS.13.249 © 2014 Future Medicine Ltd
Pharmacogenomics (2014) 15(5), 619–627
ISSN 1462-2416
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nephrotic syndrome revealed a higher frequency of the -308A allele and the AA genotype in the former group [8]; In human leukocyte antigen (HLA)-DR mismatched transplant patients, the TNF-a high producer genotypes were significantly associated with multiple and steroid-resistant rejection episodes [9]. By altering the transcriptional activity of the cytokine promoter, the -308G>A variant could therefore influence treatment response in ALL, particularly the steroid response. Earlier studies on primary ALL cells and cell lines showed that aberrant expression (high levels) of endogenous TNF-a may be associated with refractory disease [10–12]. However, case–control studies in childhood ALL patients could not find a correlation between the -308G>A variant and prednisone response or relapse [13–15], although these studies were limited in sample size and could therefore have underestimated the pharmacological importance of this polymorphism in ALL outcome. This study was aimed at investigating the impact of the TNF-a -308G>A variant on the risk of relapse in pediatric patients affected by ALL and cured according to the AIEOP-BFM (Associazione Italiana Ematologia Oncologia Pediatrica-Berlin Frankfurt Münster) ALL 2000 study protocol.
Methods Patients The present retrospective study is based on a cohort of 1999 Philadelphia-negative ALL patients (55.7% male, 1–18 years), newly diagnosed in Italy between September 2000 and July 2006, homogenous for ethnicity (Caucasians) and for risk-adapted treatment, being stratified and cured according to the AIEOP-BFM ALL 2000 study protocols (ClinicalTrials.gov identifier NCT00613457). The following criteria were used to identify patients as high-risk: Presence of t(4;11) or t(9;22); Prednisone poor response (≥1000 blasts/µl in the peripheral blood at day +8, after 7 days of daily prednisone and a single dose of intrathecal methotrexate at day +1); Inability to achieve complete remission in bone marrow aspirate at day +33; High burden (≥10 -3) of minimal residual disease (MRD) measured by PCR (PCR-MRD) at day +78. 620
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More intense regimens during consolidation (blocks) and re-induction phases are used for patients at high-risk compared with the others. The standard-risk group included patients who lacked high-risk criteria and were negative by PCR-MRD performed by using two sensitive markers (≥10 -4) at both day +33 and day +78. The intermediate-risk group included the remaining patients, and those not evaluated by PCR-MRD. Written informed consent was obtained from the parents or legal guardians before patient enrolment, while the protocol was approved by the ethics committee of each participating institution. Study design & statistical analysis 614 patients were selected to be genotyped according to a novel two-phase study design. The sampling strategy and the statistical analyses performed were described previously [16]. Briefly, the selected subcohort comprised all relapsed patients within the overall cohort and a subset of other patients in remission, randomly chosen within each of the three risk groups according to optimal strata specific sampling fractions. Representativeness of the whole population was recovered in the statistical analysis by appropriate weights (equal to the inverse of the sampling fractions). Patients were followed from diagnosis up to relapse, second malignancy, death or December 2008. Weighted estimate of the crude cumulative incidence of relapse was calculated from the date of diagnosis to the date of relapse accounting for competing risks (death and second malignancies). The comparisons of these curves were based on the Wald test in a Cox model with TNF-a genotype as covariate. To account for potential confounders the weighted Cox model for two-phase design was applied adjusting for immunophenotype (T-cell ALL [T-ALL] and B-cell precursor ALL [B-ALL]), risk group (standard-, medium-, high-risk group), age (1–5, 6–9, 10–17 years) and gender and including interaction terms between the SNP and these characteristics. Genotyping DNA of the AIEOP ALL patients was kindly provided by SSD Clinical and Experimental Hematology Bio Bank, Department of Paediatrics, University of Padua, Italy (AIEOP Bio Bank). Genotype analyses for the TNFa-308G>A variant were performed blinded to patient outcome on DNA extracted from blasts of bone marrow aspirates at first diagnosis or, when not available, at relapse by using allelic specific fluorescent probes with TaqMan® SNP future science group
Treatment prognostic genetic factors in childhood acute lymphoblastic leukemia
genotyping assays (Applied Biosystems, CA, USA). Subjects of this cohort had been previously genotyped for deletions of GST-M1 and GST-T1 genes [16].
Results The 614 patients of the AIEOP-BFM ALL 2000 cohort were selected to be genotyped according to the study design: among them, 363 patients were males (59.1%); 108 (17.6%) were T-ALL; 355 were preschooler (age less than 6 years, 57.8%) and 142 (23.1%) were older than 10 years; 345 fell in the medium-risk group (56.2%), whereas 78 (12.7%) and 191 (31.1%) were treated according to the standard- and the high-risk regimens, respectively. 5-year cumulative incidence of relapse is reported in Figure 1. The incidence of relapse was similar for wild-type patients and subjects carrying the minor allele genotypes (5-year estimates 16.4 ± 1.29% versus 23.0 ± 4.95%, respectively, p = 0.13, Figure 1A). Table 1 summarizes the TNF-a -308G>A variant distribution among the 614 AIEOP ALL genotyped patients, the weighted 5-year cumulative incidence of relapse, and the association between the genetic variations and risk of relapse analyzed by means of the weighted Cox model. As the SNP is very rare in homozygosis among Caucasians, analyses were performed considering a dominant model. In order to account for possible differential effects of the polymorphism according to patient distinct pharmacological profiles [17] and therapeutic approaches, we considered the following interaction terms in the Cox model (Table 2): Blast immunophenotype (T-ALL and B-ALL); Age class (with cut-off set at 6 and 10 years old for roughly dividing preschoolers, children and adolescents); Polychemotherapeutic protocols (standard-, medium- and high-risk); Early in vivo response to the steroid prephase (defined as prednisone good responders [PGR] and prednisone poor responders [PPR] at day +8 from diagnosis). A highly significant interaction was found between immunophenotype and -308G>A variant (p = 0.0007), with the minor allele genotypes being adverse genetic markers in B-ALL and protective genetic traits in T-ALL. Figure 1B & 1C report the crude cumulative incidence of relapse by -308G>A variant in B-ALL and T-ALL patients, respectively. These curves confirm the opposite effect of the SNP in the two future science group
Research Article
immunophenotypes. A significant interaction term was observed also among risk protocols (p = 0.0041) where the minor allele genotypes emerged as prognostic factors of relapse only in the standard risk group, and in the in vivo response to the steroid prephase (p = 0.035) with a trend of higher risk of the minor allele genotypes in the PGR group. Among the children considered, only one T-ALL patient fell in the standard risk (and generally in the AIEOPBFM LLA 2000 whole cohort, only 25 out of 244 T-ALL): therefore, the genetic adverse effect observed in the less intensive group is limited to the B-ALL immunophenotype. Instead, the minor allele genotypes were protective for T-ALL in the medium- and high- therapeutic arms. We also investigated the role of the polymorphism on prednisone response by means of a weighted logistic model adjusted by sex, age and immunophenotype (p = 0.97, odds ratio [OR]: 0.99, 95% CI: 0.53–1.85), revealing that it was not decisive to assess prednisone response itself.
Discussion Due to the risk-adapted polychemotherapy, today’s cure rates for pediatric ALL reach more than 80% of success in the developed countries. Actually, the patients’ risk stratification is assessed mainly on the basis of MRD and other molecular methods [18,19], so that traditional adverse prognostic factors such as T-ALL immunophenotype and older age at diagnosis are outdated. The classification of T-ALL or B-ALL is now further defined by genomic alterations with potential prognostic or therapeutic relevance [20], and age is no longer associated with worse outcome. In the context of the AIEOP-BFM ALL 2000 protocol, within the whole cohort of 1999 patients enrolled, 306 children relapsed (15.3%, median follow-up: 4 years), among which 28 were in the standardrisk and 186 were in the medium-risk group, revealing therefore that the majority of them were treated according to the less intensive arms because of their favorable prognostic features. These absolute numbers emphasize the need of identifying additional criteria to refine the treatment stratification, particularly for those patients currently considered at lower risk. Individual genetic polymorphisms influencing drug disposition, metabolism and mechanisms of action might affect treatment efficiency and could therefore be potential predictors of response to take into account to personalize the chemotherapy [21]. However, it is unlikely www.futuremedicine.com
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A
Crude cumulative incidence of relapse
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0.6
ALL Patients Wild type Non-wild-type
0.4
No. relapsed 5-year estimate (SE) 204 47
510 103
16.4% (1.29) 23% (4.95)
0.2 p = 0.13 0.0 1
0
4
3
2
5
B
Crude cumulative incidence of relapse
Time since diagnosis (years) 0.6 B-ALL
0.4
Wild type Non-wild-type
Patients
No. relapsed
421 81
154 42
5-year estimate (SE) 14.3% (1.12) 25.6% (3.82)
0.2 p = 0.0097 0.0
2
1
0
3
4
5
C
Crude cumulative incidence of relapse
Time since diagnosis (years) T-ALL 0.6
Patients Wild type Non-wild-type
86 22
No. relapsed 5-year estimate (SE) 49 5
35.6% (5.24) 11.4% (5.17)
0.4
0.2
p = 0.0105
0.0 0
1
2
3
4
5
Time since diagnosis (years)
Figure 1. Crude cumulative incidence of relapse according to TNF-a -308G>A variant genotypes in genotyped patients. (A) ALL, (B) B-ALL and (C) T-ALL genotyped patients. Hz+mut patients were analyzed together as minor allele genotypes. p-values refers to Wald test in univariate Cox model for twophase designs. ALL: Acute lymphoblastic leukemia; B-ALL: B-cell acute lymphoblastic leukemia; SE: Standard error; T-ALL: T-cell acute lymphoblastic leukemia.
that a single polymorphism could influence the outcome of such a complex disease as ALL and of such complex therapeutic approaches, 622
Pharmacogenomics (2014) 15(5)
particularly in the case of the TNF-a gene. This gene is located on chromosome 6p21.3, in the class III region of the human major histocompatibility complex (MHC) within the human leukocyte antigen (HLA). This part of the genome is essential to the immune system and is one of the most polymorphic in humans with a distribution of one SNP per 1.71 kb instead of the average one SNP per 1.9 kb found throughout the human genome [22,23]. Multiple SNPs identified in the promoter region and in the TNF-a gene itself are in linkage disequilibrium, and many of them are included in HLA haplotypes [24,25], but the TNF-a -308G>A variant is the one studied for the association with outcome in pediatric leukemia. The TNF-a -308G>A variant could be the nominal SNP of a clinical phenotype, rather than the truly and only causative genotype. Indeed, several studies investigated the possible concomitant role of other polymorphisms. For instance, the high-cytokine producer haplotype comprising the minor alleles of TNF-a -308G>A and lymphotoxin-a (LT-a) +252A>G (rs909253) is generally studied for the association with autoimmune and severe inflammatory disorders and in the pathogenesis and prognosis of hematologic malignancies. Stanulla et al. failed to observe any meaningful association between the high-producer TNF-a/LT-a haplotype in comparison with a low-producing one [14]. Also, Takeuchi et al. did not find an association between the haplotype and the immunophenotype, the response to chemotherapy, and the event-free survival in ALL [15]. The TNF-a -308G>A variant is also commonly studied with IL-10 -1082A>G (rs1800896), with conflicting results when analyzed for the association with ALL overall survival [13,26]. Genetic traits could be covariates of importance, together with the already known clinical and biological ones, for improving ALL prognosis. This study suggests that the presence of at least one -308A allele conferred a protective advantage and reduced risk of relapse in T-ALL, while it predicted a worse clinical outcome in B-ALL patients falling in the AIEOP-BFM ALL 2000 low risk arm. The exact reason for these discrepant results is unknown. Accumulating evidence suggests that the net effect of TNF-a action in vivo can be attributed to the biological context within which the cytokine is exerting its action, and is the result of a crosstalk between the various signaling pathways engaged, determined by timing and duration of TNF-a action, tissue type and TNF receptors composition [27]. future science group
Treatment prognostic genetic factors in childhood acute lymphoblastic leukemia
Research Article
Table 1. Genotype distribution of TNF-a -308G>A variant in AIEOP acute lymphoblastic leukemia patients genotyped subgroup and 5-year cumulative incidence of relapse weighted to the whole AIEOP-BFM ALL 2000 cohort.
All patients
TNF-a -308G>A
Patients (n)
Relapses (n)
5-year cum. inc. (%)
SE
Hazard ratio (95% CI)
p-value HWE p-value
wt
510
204
16.41
1.29
1.00
hz
100
46
–
–
– (–)
–
–
mut
3
1
22.96†
4.95
1.13 (0.75–1.72)
0.55
>0.05
NA
1
–
–
–
– (–)
–
–
Hazard ratio adjusted by risk group, age class, gender and immunophenotype. † hz+mut: analyses on the minor allele genotypes group. 5-year cum. inc. (%): Weighted 5-years cumulative incidence of relapse; NA: Not assessed; SE: Standard error; HWE: Hardy–Weinberg equilibrium.
The death-inducing capability of the cytokine is mediated by TNF membrane receptor type 1 (TNF-R1). Micheau et al. have demonstrated that the TNF-R1-mediated signal transduction includes a checkpoint for balancing cell functional fate between life and death [28]. Indeed, TNF-a activates the prosurvival transcription factor NF-kB through the ubiquitin-mediated degradation of its inhibitor, IkB, whereas it activates the caspase cascade in instances where this signal for NF-kB failed [29]. In light of such complex biological interplay, we hypothesized that TNF-a might behave as an amplifier of the prevailing stimuli acting on the cell, strengthening the antiapoptotic pathways when cell integrates mostly prosurvival signals, and supporting the death process if signals delivered concurrently on the cell are mainly apoptotic. It should be noted that in the AIEOP-BFM ALL 2000, T-ALL patients received a more aggressive consolidation phase in comparison with B-ALL, regardless of their risk class. This slightly intensive protocol might account for enhanced cytotoxic signals, likely further boosted in T-ALL carrying the minor allele genotypes because of putative higher TNF-a levels: these patients could be genetically predisposed to a better pharmacological response, and thus a better protection from relapse in such therapeutic condition. On the contrary, the studied polymorphism had an adverse effect in B-ALL standard-risk patients: in the AIEOPBFM ALL 2000, patients in the low-arm group were randomized and half of them received one third less dexamethasone, a known downregulator of TNF-a expression, and half pulses of anthracyclines during the reinduction phase in comparison to the medium-risk group. Interestingly, endogenous TNF-a was found to mediate resistance to doxorubicin-induced cytotoxicity [10]. This less intense protocol could be counterproductive for higher cytokine producers, and future science group
the minor allele genotypes could represent an adverse individual trait, leading to resistance and disease progression. These hypotheses are highly speculative and would have benefited of measuring TNF-a plasma levels at diagnosis and during therapy. However, being designed as a retrospective pharmacogenetic study, cytokine levels were not investigated. Subsets analysis makes sense in moving towards a personalized medicine, rendering the pediatric population more uniform, and results more informative for targeting patient subgroups. Noteworthy is that subgroups considered in the present study were still large in size because of the large number of the whole series genotyped. In a previous study with 135 children with ALL, treated according to BFM-protocols -86 and -90, subjects with the minor allele genotypes did also not show a statistically significant association with risk of relapse nor with prednisone response compared with wild-type subjects in overall analysis. However, in the subgroup analysis approach evaluating the TNF-a genotype exclusively in patients who had at least one known clinical risk factor for poor treatment outcome, the minor allele genotypes emerged as adverse genetic traits within 45 PPR [13]. Our subset of PPR patients included 131 patients and did not show a significant genetic effect of the TNF-a -308G>A variant. Replication of results is essential and needs to be performed before drawing any firm conclusion. However, when the genotyped population was divided into training (with 413 observations) and replication (with 197 observations) sets (by a random Bernoulli variable with parameter 2/3), the opposite effect of TNF-a -308G>A variant in B-ALL and T-ALL was observed in both subgroups, with the corresponding interaction term still significant (data not shown). Given that risk criteria adopted in the actual AIEOP protocols remain the basic guidelines www.futuremedicine.com
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Table 2. Impact of TNF-a -308G>A variant on relapse in acute lymphoblastic leukemia patients stratified by immunophenotype (Cox model adjusted by gender, risk and age class), by age group (Cox model adjusted by gender, risk class and immunophenotype), by risk group and in vivo prephase response (Cox model adjusted by gender, age and immunophenotype). TNF-a SNP rs1800629 (-308G>A) Patients (n)
Relapses (n)
5-year cum. inc. (%)
SE
wt
421
154
14.31
1.12
Non-wt
81
42
25.64
3.82
wt
86
49
35.58
5.24
22
5
11.36
5.17
HR†
95% CI
1.72
1.11–2.66
0.24
0.08–0.68
1.51
0.91–2.51
1.04
0.45–2.40
0.42
0.13–1.36
1.52
0.95–2.42
0.50
0.20–1.27
6.25
1.98–19.68
1.14
0.66–1.96
0.61
0.29–1.28
Immunophenotype (p‡ = 0.0007) B-ALL T-ALL
Non-wt
Age at diagnosis, years (p = 0.1363) ‡
1–5 6–9 10–17
wt
293
104
13.94
1.43
Non-wt
62
30
22.57
4.8
wt
90
41
18.75
3.37
Non-wt
26
13
33.12
9.80
wt
127
59
21.44
3.17
15
4
11.72
6.30
Non-wt
Prednisone response (p = 0.0350) ‡
PGR PPR
wt
401
159
14.71
1.11
Non-wt
80
40
23.18
4.27
wt
108
44
30.35
3.87
Non-wt
23
7
21.04
7.26
wt
63
13
3.66
1.06
Non-wt
14
9
20.55
8.55
wt
290
123
18.00
1.50
Non-wt
55
26
22.99
5.03
wt
157
68
32.82
3.20
Non-wt
34
12
25.70
6.69
Risk group (p = 0.0041) ‡
Standard Medium High
Patients with missing genotype or clinical data were omitted from the analysis. † Non-wt (hz+mut) versus wt. ‡ p-value for the interaction. 5-year cum. inc. (%): Weighted 5-year cumulative incidence of relapse; B-ALL: B-cell acute lymphoblastic leukemia; HR: Hazard ratio; PGR: Prednisone good responders; PPR: Prednisone poor responders; SE: standard error; T-ALL: T-cell acute lymphoblastic leukemia.
for stratification, if our results are confirmed in a validation cohort, the presence of at least one minor allele of the -308G>A variant could be discriminative to target B-ALL patients with all the best prognostic factors now used (i.e., MRD, PGR) into the medium group rather than into the standard one. In the current AIEOP-BFM ALL 2009 trial, the less intense therapeutic arm consists of half pulses of anthracyclines during the induction phase (rather than during the re-induction phase as in the AIEOP-BFM ALL 2000), and thus this SNP should still be investigated as an applicable 624
Pharmacogenomics (2014) 15(5)
prognostic tool. The possible concomitant role of other genes should also be considered. The final aim is to give all children the most appropriate intensive therapy, further improving the already outstanding success rate of pediatric ALL chemotherapy.
Future perspective In ALL, risk-adapted polychemotherapy has great success, and risk stratification criteria based mainly on minimal residual disease are highly reliable, but 10–15% of patients still do not respond adequately in terms of efficacy and future science group
Treatment prognostic genetic factors in childhood acute lymphoblastic leukemia
outcome. Pharmacogenomics is a relatively novel branch of investigation and represents an innovative frontier of medicine, standing at the basis of personalized treatment optimization. In the last decade, pharmacogenomics became more and more important because of the continuous advances in high-throughput DNA technology and bioinformatics methods. In the context of the AIEOP-BFM ALL protocols, the present work suggests that the low penetrance TNF-a -308G>A variant might be a useful genetic marker, although only in selected patients. Indeed, if our results are confirmed in a validation cohort, the minor allele genotypes could become discriminative to include B-ALL patients with the best prognostic factors into the medium-risk group rather than into the standard one. A future perspective will be to develop algorithms and informatics programs that will integrate demographic, clinical and pharmacogenetic data into user-friendly and cheap softwares for helping physicians in therapy personalization. Financial & competing interests disclosure All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on
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request from the corresponding author) and declare: no support from any organization for the submitted work, no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years and no other relationships or activities that could appear to have influenced the submitted work. Supporting foundations: Italian Ministry of Health, Fondazione Benefica Alberto e Kathleen Casali. R Franca and D Favretto are recipient fellowships from IRCCS Burlo Garofolo, Trieste. Grant from the European Commission (FP7-HEALTH-F2–2011 261474) to P Rebora and MG Valsecchi. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary AIEOP-BFM risk stratification of acute lymphoblastic leukemia patients needs to be improved In AIEOP-BFM acute lymphoblastic leukemia (ALL) protocols, patient risk stratification is based mainly on minimal residual disease. In the AIEOP-BFM ALL 2000 trial, 85% of pediatric patients were successfully cured with the risk-adapted polychemotherapeutic protocols employed. Among relapsed children, only 30% had adverse prognostic features and fell within the high-risk group. Patient genetic traits could be predictor markers of prognosis Polymorphisms in genes involved in drug disposition, metabolism and mechanisms of action might affect outcome. TNF-a is a cytokine involved in the regulation of immune cells. The TNF-a SNP rs1800629 in the promoter region has been associated with increased transcriptional activity and steroid resistance in pediatric inflammatory and autoimmune diseases; no correlation has been found in small-scale studies on ALL outcome. Innovative approach for a pharmacogenetic study on Italian AIEOP-BFM ALL 2000 cohort The novel two-phase design allows a considerable gain in precision of the estimate of genotype effect on relapse because it introduces sampling fractions optimized on the basis of a previous correlation between genotypes and outcome in a pilot study on the same AIEOP-BFM ALL 2000 whole cohort. With 614 patients genotyped, this study is probably one of the largest surveys performed on European ALL protocols. TNF-α SNP rs1800629 could influence outcome in specific pediatric ALL subgroups No significant association was found in the whole cohort. Highly significant interaction was found between immunophenotype and TNF-a SNP rs1800629 (p = 0.0007), with the non-wild-type genotype being an adverse genetic marker in non-T-ALL (hazard ratio [HR]: 1.72, 95% CI: 1.11–2.66) and a protective genetic trait in T-ALL (HR: 0.24, 95% CI: 0.08–0.68). A significant interaction term was also observed among risk protocols (p = 0.0041), where the non-wild-type genotype emerged as a prognostic factor of relapse for standard-risk patients (HR: 6.25, 95% CI: 1.98–19.68, only one T-ALL in the subgroup analyzed). Conclusion This study suggests that ALL therapeutic stratification could benefit a genotyping approach. In non-T-ALL standard-risk patients, whose risk has been assessed by AIEOP-BFM in-use criteria, TNF-a SNP rs1800629 could be a further tool to improve relapse prediction and target non-wild-type patients in the medium-risk group. Our results need confirmation in a validation cohort for understanding their clinical application.
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Affiliations
Raffaella Franca Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, UO Pediatric Hemato-Oncology, Via dell’Istria 65/1, 34137 Trieste, Italy
Paola Rebora Center of Biostatistics for Clinical Epidemiology, Department of Health
21 Stocco G, Franca R, Verzegnassi F, Londero
M, Rabusin M, Decorti G. Pharmacogenomic approaches for tailored anti-leukemic therapy in children. Curr. Med. Chem. 20(17), 2237–2253 (2013).
Detailed recent overview on genes and what might influence ALL treatment in terms of outcome and/or toxicity, and underlies the role of genetic variants, particularly SNPs, in predicting clinical response, with particular reference to the AIEOP-BFM ALL 2009 protocol.
future science group
Treatment prognostic genetic factors in childhood acute lymphoblastic leukemia
Sciences, University of Milano-Bicocca, Via Cadore 48, 20900 Monza (Milan), Italy
Emmanouil Athanasakis Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, Medical Genetic Service, Via dell’Istria 65/1, 34137 Trieste, Italy Diego Favretto Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, UO Pediatric Hemato-Oncology, Via dell’Istria 65/1, 34137 Trieste, Italy
future science group
Research Article
Federico Verzegnassi Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, UO Pediatric Hemato-Oncology, Via dell’Istria 65/1, 34137 Trieste, Italy
Maria Grazia Valsecchi Center of Biostatistics for Clinical Epidemiology, Department of Health Sciences, University of Milano-Bicocca, Via Cadore 48, 20900 Monza (Milan), Italy
Giuseppe Basso Pediatric Clinic, Onco-Hematology, University of Padua, via Giustiniani 3, 35128 Padua, Italy
Giuliana Decorti Department of Life Sciences, University of Trieste, Via Fleming 22, 34127 Trieste, Italy
Alberto Tommasini Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, Laboratory of Immunopathology, Via dell’Istria 65/1, 34137 Trieste, Italy
Marco Rabusin Institute for Maternal & Child Health (I.R.C.C.S) Burlo Garofolo, UO Pediatric Hemato-Oncology, Via dell’Istria 65/1, 34137 Trieste, Italy
www.futuremedicine.com
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