Purpose Somatic deletions that affect the lymphoid transcription factor–coding gene IKZF1 have previously been reported as independently associated with a poor prognosis in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL). We have now refined the prognostic strength of IKZF1 deletions by analyzing the effect of co-occurring deletions. Patients and Methods The analysis involved 991 patients with BCP ALL treated in the Associazione Italiana Ematologia ed Oncologia Pediatrica–Berlin-Frankfurt-Muenster (AIEOP-BFM) ALL 2000 trial with complete information for copy number alterations of IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, Xp22.33/Yp11.31 (PAR1 region; CRLF2, CSF2RA, and IL3RA), and ERG; replication of findings involved 417 patients from the same trial. Results IKZF1 deletions that co-occurred with deletions in CDKN2A, CDKN2B, PAX5, or PAR1 in the absence of ERG deletion conferred the worst outcome and, consequently, were grouped as IKZF1plus. The IKZF1plus group comprised 6% of patients with BCP ALL, with a 5-year event-free survival of 53 ± 6% compared with 79 ± 5% in patients with IKZF1 deletion who did not fulfill the IKZF1plus definition and 87 ± 1% in patients who lacked an IKZF1 deletion ( P ≤ .001). Respective 5-year cumulative relapse incidence rates were 44 ± 6%, 11 ± 4%, and 10 ± 1% ( P ≤ .001). Results were confirmed in the replication cohort, and multivariable analyses demonstrated independence of IKZF1plus. The IKZF1plus prognostic effect differed dramatically in analyses stratified by minimal residual disease (MRD) levels after induction treatment: 5-year event-free survival for MRD standard-risk IKZF1plus patients was 94 ± 5% versus 40 ± 10% in MRD intermediate- and 30 ± 14% in high-risk IKZF1plus patients ( P ≤ .001). Corresponding 5-year cumulative incidence of relapse rates were 6 ± 6%, 60 ± 10%, and 60 ± 17% ( P ≤ .001). Conclusion IKZF1plus describes a new MRD-dependent very-poor prognostic profile in BCP ALL. Because current AIEOP-BFM treatment is largely ineffective for MRD-positive IKZF1plus patients, new experimental treatment approaches will be evaluated in our upcoming trial AIEOP-BFM ALL 2017.

No abstract

Long-term treatment with 6-thioguanine (6-TG) for pediatric acute lymphoblastic leukemia (ALL) is associated with high rates of hepatic sinusoidal obstruction syndrome (SOS). Nevertheless, current treatment continues to use short-term applications of 6-TG with only sparse information on toxicity. 6-TG is metabolized by thiopurine methyltransferase (TPMT) which underlies clinically relevant genetic polymorphism. We analyzed the association between hepatic SOS reported as a serious adverse event (SAE) and short-term 6-TG application in 3983 pediatric ALL patients treated on trial AIEOP-BFM ALL 2000 (derivation cohort) and defined the role of TPMT genotype in this relationship. We identified 17 patients (0.43%) with hepatic SOS, 13 of which with short-term exposure to 6-TG (P < 0.0001). Eight of the 13 patients were heterozygous for low-activity TPMT variants, resulting in a 22.4-fold (95% confidence interval 7.1–70.7; P ≤ 0.0001) increased risk of hepatic SOS for heterozygotes in comparison to TPMT wild-type patients. Results were supported by independent replication analysis. All patients with hepatic SOS after short-term 6-TG recovered and did not demonstrate residual symptoms. Thus, hepatic SOS is associated with short-term exposure to 6-TG during treatment of pediatric ALL and SOS risk is increased for patients with low-activity TPMT genotypes.

The proteasome inhibitor bortezomib seems to sensitize gc-resistant childhood ALL cells for prednisolone-induced cell death.

No abstract

Background Characterization of clinical phenotypes in context with tumor and host genomic information can aid in the development of more effective and less toxic risk-adapted and targeted treatment strategies. To analyze the impact of therapy-related hyperbilirubinemia on treatment outcome and to identify contributing genetic risk factors of this well-recognized adverse effect we evaluated serum bilirubin levels in 1547 pediatric patients with acute lymphoblastic leukemia (ALL) and conducted a genome-wide association study (GWAS). Patients and methods Patients were treated in multicenter trial AIEOP-BFM ALL 2000 for pediatric ALL. Bilirubin toxicity was graded 0 to 4 according to the Common Toxicity Criteria (CTC) of the National Cancer Institute. In the GWAS discovery cohort, including 650 of the 1547 individuals, genotype frequencies of 745,895 single nucleotide variants were compared between 435 patients with hyperbilirubinemia (CTC grades 1-4) during induction/consolidation treatment and 215 patients without it (grade 0). Replication analyses included 224 patients from the same trial. Results Compared to patients with no (grade 0) or moderate hyperbilirubinemia (grades 1-2) during induction/consolidation, patients with grades 3-4 had a poorer 5-year event free survival (76.6 ± 3% versus 87.7 ± 1% for grades 1-2, P = 0.003; 85.2 ± 2% for grade 0, P < 0.001) and a higher cumulative incidence of relapse (15.6 ± 3% versus 9.0 ± 1% for grades 1-2, P = 0.08; 11.1 ± 1% for grade 0, P = 0.007). GWAS identified a strong association of the rs6744284 variant T allele in the UGT1A gene cluster with risk of hyperbilirubinemia (allelic odds ratio (OR) = 2.1, P = 7 × 10− 8). TT-homozygotes had a 6.5-fold increased risk of hyperbilirubinemia (grades 1-4; 95% confidence interval (CI) = 2.9-14.6, P = 7 × 10− 6) and a 16.4-fold higher risk of grade 3-4 hyperbilirubinemia (95% CI 6.1-43.8, P = 2 × 10− 8). Replication analyses confirmed these associations with joint analysis yielding genome-wide significance (allelic OR = 2.1, P = 6 × 10− 11; 95% CI 1.7-2.7). Moreover, rs6744284 genotypes were strongly linked to the Gilbert’s syndrome-associated UGT1A1*28/*37 allele (r2 = 0.70), providing functional support for study findings. Of clinical importance, the rs6744284 TT genotype counterbalanced the adverse prognostic impact of high hyperbilirubinemia on therapy outcome. Conclusions Chemotherapy-related hyperbilirubinemia is a prognostic factor for treatment outcome in pediatric ALL and genetic variation in UGT1A aids in predicting the clinical impact of hyperbilirubinemia. Trial registration http://www.clinicaltrials.gov; #NCT00430118.

No abstract

991 Poster Board I-13 The response to initial glucocorticoid therapy in childhood acute lymphoblastic leukemia (ALL) reliably predicts the response to multi-agent chemotherapy. In a recent study, we identified the valosin-containing protein (VCP/p97), part of the ubiquitin proteasome degradation pathway (UPDP), as a differentially expressed protein in prednisone good (PGR) and poor responder (PPR) patients (Lauten et al. 2006). This may indicate that the UPDP is possibly altered and may be involved in multi-agent chemotherapy resistance in childhood ALL patients. The proteasome inhibitor bortezomib (VELCADE®, Millenium Pharmaceuticals, Cambridge, MA, USA) blocks proteasomal degradation and hence the UPDP. It may therefore be a useful tool to determine mechanistically, whether prednisone resistance is linked to ubiquitination of proteins like IkB, which can be chaperoned by VCP within the UPDP. Thus, the aim of the study was to investigate the association of differential VCP expression and glucocorticoid response in childhood ALL cell lines in order to find ways to overcome prednisone resistance by means of proteasomal inhibition. To investigate whether treatment of ALL with the proteasome inhibitor bortezomib acts synergistically with glucocorticoid treatment, human B-cell precursor leukemic cell lines MHH cALL 2 (PPR) and MHH cALL 3 (PGR) were treated with prednisone (6.2 μM) and various concentrations of bortezomib (1.5 nM-12 nM) for up to 96 hours. Cells were sampled every 24 h for subsequent analyses. Cell death rates were analyzed by propidium iodide (PI) and trypan blue stainings. Viability was quantified by the colorimetric WST-1 assay (11644807007, Roche). Moreover, apoptosis was determined by the Caspase-Glo 3/7® assay (G8091, Promega) and resulting data were normalized to the amount of viable cells, detected by WST-1 assay, carried out in parallel. To quantify the amount of expressed VCP and its known interaction partner IkB within the UPDP-associated NFkB pathway, real-time PCR and Western blot analysis were performed. For Western blot analyses, the mouse monoclonal anti-VCP antibody (65278, Progen) and the rabbit polyclonal anti-IkB-alpha (9242, Cell Signaling Technology) were used. Both cell lines showed a dose-dependent increase in apoptosis after bortezomib single treatment. Single therapy with 12 nM bortezomib induced in PGR a 42-fold and in PPR cells a 29-fold higher caspase activity compared to treatment with prednisone alone. But only the PGR cells showed an additive effect on apoptosis induction after combined treatment with prednisone and bortezomib. In the PPR cell line, the amount of PI-positive, lethal cells increased within 48 hours to 20-fold after combined treatment, using 7 nM bortezomib, compared to single prednisone and 3-fold compared to single bortezomib treatment. In contrast, apoptosis was reduced compared to bortezomib single treatment. Prednisone treatment induced an increased IkB and VCP expression predominantly in PPR, with a maximum at 48 and 96h, respectively. IkB expression increased to 2-fold within 24 hours in PGR cells, whereas it increased to 3.4-fold within 48 hours in PPR cells. VCP levels were only elevated in PPR cells to about 2-fold, whereas in PGR cells the amount remained largely unchanged. In agreement with the bortezomib-induced rise in caspase activity in PPR cells, the expression profiles of the target genes VCP and IkB were modified to levels comparable to those found in PGR cells after prednisone treatment. The altered VCP and IkB expression patterns indicate that the glucocorticoid-induced apoptosis mechanisms, mediated by the proteasomal NfkB pathway, are deregulated in prednisone resistant cells. The results of this study suggest that VCP expression modulates prednisone response in childhood ALL. In addition, proteasome inhibitors like bortezomib seem to be able to sensitize glucocorticoid-resistant childhood ALL cells for prednisone treatment. Therefore, drug targeting the proteasome may be a novel therapeutic option in resistant ALL. Figure 1: Increase of PI-positive cells in human B-cell precursor ALL cell lines, 48 hours after induction, (A) Prednisone poor responder (B) Prednisone good responder, n=3. Drug concentrations: 6.2 μM prednisone (PRED + ), 7 nM bortezomib (BORT ++ ). Figure 1:. Increase of PI-positive cells in human B-cell precursor ALL cell lines, 48 hours after induction, (A) Prednisone poor responder (B) Prednisone good responder, n=3. Drug concentrations: 6.2 μM prednisone (PRED + ), 7 nM bortezomib (BORT ++ ). Disclosures: No relevant conflicts of interest to declare.