Correction: NG2 antigen is involved in leukemia invasiveness and central nervous system infiltration in MLL-rearranged infant B-ALL.

The original version of this Article contained an error in the spelling of the author Juan Carlos Rodriguez-Manzaneque, which was incorrectly given as J Carlos Rodríguez-Manzaneque. This has now been corrected in both the PDF and HTML versions of the Article.

NG2 antigen is involved in leukemia invasiveness and central nervous system infiltration in MLL-rearranged infant B-ALL.

Mixed-lineage leukemia (MLL)-rearranged (MLLr) infant B-cell acute lymphoblastic leukemia (iMLLr-B-ALL) has a dismal prognosis and is associated with a pro-B/mixed phenotype, therapy refractoriness and frequent central nervous system (CNS) disease/relapse. Neuron-glial antigen 2 (NG2) is specifically expressed in MLLr leukemias and is used in leukemia immunophenotyping because of its predictive value for MLLr acute leukemias. NG2 is involved in melanoma metastasis and brain development; however, its role in MLL-mediated leukemogenesis remains elusive. Here we evaluated whether NG2 distinguishes leukemia-initiating/propagating cells (L-ICs) and/or CNS-infiltrating cells (CNS-ICs) in iMLLr-B-ALL. Clinical data from the Interfant cohort of iMLLr-B-ALL demonstrated that high NG2 expression associates with lower event-free survival, higher number of circulating blasts and more frequent CNS disease/relapse. Serial xenotransplantation of primary MLL-AF4+ leukemias indicated that NG2 is a malleable marker that does not enrich for L-IC or CNS-IC in iMLLr-B-All. However, NG2 expression was highly upregulated in blasts infiltrating extramedullar hematopoietic sites and CNS, and specific blockage of NG2 resulted in almost complete loss of engraftment. Indeed, gene expression profiling of primary blasts and primografts revealed a migratory signature of NG2+ blasts. This study provides new insights on the biology of NG2 in iMLLr-B-ALL and suggests NG2 as a potential therapeutic target to reduce the risk of CNS disease/relapse and to provide safer CNS-directed therapies for iMLLr-B-ALL.

Identification of a genetically defined ultra-high-risk group in relapsed pediatric T-lymphoblastic leukemia.

In the search for genes that define critical steps of relapse in pediatric T-cell acute lymphoblastic leukemia (T-ALL) and can serve as prognostic markers, we performed targeted sequencing of 313 leukemia-related genes in 214 patients: 67 samples collected at the time of relapse and 147 at initial diagnosis. As relapse-specific genetic events, we identified activating mutations in NT5C2 (P=0.0001, Fisher's exact test), inactivation of TP53 (P=0.0007, Fisher's exact test) and duplication of chr17:q11.2-24.3 (P=0.0068, Fisher's exact test) in 32/67 of T-ALL relapse samples. Alterations of TP53 were frequently homozygous events, which significantly correlated with higher rates of copy number alterations in other genes compared with wild-type TP53 (P=0.0004, Mann-Whitney's test). We subsequently focused on mutations with prognostic impact and identified genes governing DNA integrity (TP53, n=8; USP7, n=4; MSH6, n=4), having key roles in the RAS signaling pathway (KRAS, NRAS, n=8), as well as IL7R (n=4) and CNOT3 (n=4) to be exclusively mutated in fatal relapses. These markers recognize 24/49 patients with a second event. In 17 of these patients with mostly refractory relapse and dire need for efficient treatment, we identified candidate targets for personalized therapy with p53 reactivating compounds, MEK inhibitors or JAK/STAT-inhibitors that may be incorporated in future treatment strategies.

A genome-wide association study identifies risk loci for childhood acute lymphoblastic leukemia at 10q26.13 and 12q23.1.

Genome-wide association studies (GWASs) have shown that common genetic variation contributes to the heritable risk of childhood acute lymphoblastic leukemia (ALL). To identify new susceptibility loci for the largest subtype of ALL, B-cell precursor ALL (BCP-ALL), we conducted a meta-analysis of two GWASs with imputation using 1000 Genomes and UK10K Project data as reference (totaling 1658 cases and 7224 controls). After genotyping an additional 2525 cases and 3575 controls, we identify new susceptibility loci for BCP-ALL mapping to 10q26.13 (rs35837782, LHPP, P=1.38 × 10-11) and 12q23.1 (rs4762284, ELK3, P=8.41 × 10-9). We also provide confirmatory evidence for the existence of independent risk loci at 9p21.3, but show that the association marked by rs77728904 can be accounted for by linkage disequilibrium with the rare high-impact CDKN2A p.Ala148Thr variant rs3731249. Our data provide further insights into genetic susceptibility to ALL and its biology.

Polymorphic variation in TPMT is the principal determinant of TPMT phenotype: A meta-analysis of three genome-wide association studies.

Thiopurine-related hematotoxicity in pediatric acute lymphoblastic leukemia (ALL) and inflammatory bowel diseases has been linked to genetically defined variability in thiopurine S-methyltransferase (TPMT) activity. While gene testing of TPMT is being clinically implemented, it is unclear if additional genetic variation influences TPMT activity with consequences for thiopurine-related toxicity. To examine this possibility, we performed a genome-wide association study (GWAS) of red blood cell TPMT activity in 844 Estonian individuals and 245 pediatric ALL cases. Additionally, we correlated genome-wide genotypes to human hepatic TPMT activity in 123 samples. Only genetic variants mapping to chromosome 6, including the TPMT gene region, were significantly associated with TPMT activity (P < 5.0 × 10-8 ) in each of the three GWAS and a joint meta-analysis of 1,212 cases (top hit P = 1.2 × 10-72 ). This finding is consistent with TPMT genotype being the primary determinant of TPMT activity, reinforcing the rationale for genetic testing of TPMT alleles in routine clinical practice to individualize mercaptopurine dosage.

Preexisting conditions in pediatric ALL patients: Spectrum, frequency and clinical impact.

INTRODUCTION: The etiology of acute lymphoblastic leukemia remains undisclosed in the majority of cases. A number of rare syndromic conditions are known to predispose to different forms of childhood cancer including ALL. The present study characterized the spectrum and clinical impact of preexisting diseases in a cohort of ALL patients from Germany, Austria and Switzerland with a focus on genetic diseases predisposing to cancer development. METHODS: Retrospective database and study chart review included all patients from Germany, Austria and Switzerland (n = 4939) enrolled into multicenter clinical trial AIEOP-BFM ALL 2000 between July 1999 and June 2009. Patients enrolled into study AIEOP-BFM ALL 2009 - which was initiated subsequent to AIEP-BFM ALL 2000 - who were reported with a cancer prone syndrome or chromosomal abnormality were additionally included in this study to increase conclusiveness of observations. RESULTS: A total of 233 patients with at least one reported condition could be identified. The following conditions were reported in more than one patient: Gilbert's disease (n = 13), neurofibromatosis type I (n = 8), ataxia telangiectasia (n = 8), thalassemia (n = 7), Nijmegen Breakage syndrome (n = 6), cystic fibrosis (n = 4), glucose-6-phosphate dehydrogenase deficiency (n = 4), Noonan syndrome (n = 2), Klinefelter syndrome (n = 2), alpha-1-antitrypsin deficiency (n = 2), primary ciliary dyskinesia (n = 2). Especially those syndromes with a known cancer predisposition (NF type I, Ataxia telangiectasia, Nijmegen Breakage syndrome etc.) were associated with certain general and ALL-related characteristics, high therapy-related toxicity and reduced survival. CONCLUSION: The spectrum of underlying diseases within ALL patients is dispersed. A small number of ALL patients are reported with cancer predisposition syndromes at initial diagnosis which are associated with high rates of therapy-related toxicity and a markedly reduced chance of survival. The true prevalence of these conditions within the ALL population remains unknown due to inapparent clinical presentation. A targeted clinical and/or genetic examination for certain diagnoses like NF type I, Ataxia telangiectasia or Nijmegen Breakage syndrome could identify patients who benefit from adjustment of antileukemic therapy or intensification of supportive care.

Prognostic value of rare IKZF1 deletion in childhood B-cell precursor acute lymphoblastic leukemia: an international collaborative study.

Deletions in IKZF1 are found in ~15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of IKZF1 deletions affecting exons 4-7 and exons 1-8, but evidence for the remaining 33% of cases harboring other variants of IKZF1 deletions is lacking. In an international multicenter study we analyzed the prognostic value of these rare variants in a case-control design. Each IKZF1-deleted case was matched to three IKZF1 wild-type controls based on cytogenetic subtype, treatment protocol, risk stratification arm, white blood cell count and age. Hazard ratios for the prognostic impact of rare IKZF1 deletions on event-free survival were calculated by matched pair Cox regression. Matched pair analysis for all 134 cases with rare IKZF1 deletions together revealed a poor prognosis (P<0.001) that was evident in each risk stratification arm. Rare variant types with the most unfavorable event-free survival were DEL 2-7 (P=0.03), DEL 2-8 (P=0.002) and DEL-Other (P<0.001). The prognosis of each type of rare variant was equal or worse compared with the well-known major DEL 4-7 and DEL 1-8 IKZF1 deletion variants. We therefore conclude that all variants of rare IKZF1 deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.

Effects of p38α/ß inhibition on acute lymphoblastic leukemia proliferation and survival in vivo.

P38α/ß has been described as a tumor-suppressor controlling cell cycle checkpoints and senescence in epithelial malignancies. However, p38α/ß also regulates other cellular processes. Here, we describe a role of p38α/ß as a regulator of acute lymphoblastic leukemia (ALL) proliferation and survival in experimental ALL models. We also report first evidence that p38α/ß phosphorylation is associated with the occurrence of relapses in TEL-AML1-positive leukemia. First, in vitro experiments show that p38α/ß signaling is induced in a cyclical manner upon initiation of proliferation and remains activated during log-phase of cell growth. Next, we provide evidence that growth-permissive signals in the bone marrow activate p38α/ß in a novel avian ALL model, in which therapeutic targeting can be tested. We further demonstrate that p38α/ß inhibition by small molecules can suppress leukemic expansion and prolong survival of mice bearing ALL cell lines and primary cells. Knockdown of p38α strongly delays leukemogenesis in mice xenografted with cell lines. Finally, we show that in xenografted TEL-AML1 patients, ex vivo p38α/ß phosphorylation is associated with an inferior long-term relapse-free survival. We propose p38α/ß as a mediator of proliferation and survival in ALL and show first preclinical evidence for p38α/ß inhibition as an adjunct approach to conventional therapies.

Bortezomib Treatment can Overcome Glucocorticoid Resistance in Childhood B-cell Precursor Acute Lymphoblastic Leukemia Cell Lines.

BACKGROUND: The response to initial glucocorticoid (gc) treatment is a reliable stratification factor in childhood acute lymphoblastic leukemia (ALL) and may predict the response to multi-agent chemotherapy. In a former study we detected that the valosin-containing protein (VCP, cdc48), a member of the ubiquitin proteasome degradation system (UPS), is altered in gc-resistant leukemic cells suggesting that the associated pathways might be involved in chemotherapy resistance in childhood ALL. METHODS: Human B-cell precursor leukemia cell lines, gc-resistant MHH-cALL-2 and gc-sensitive MHH-cALL-3, were treated with prednisolone and various concentrations of bortezomib. Viability and apoptosis rates were determined. RESULTS: Both cell lines showed a dose-dependent increase in caspase activity after bortezomib single treatment. The gc-sensitive cells showed an additive effect after combined treatment with prednisolone and bortezomib. In contrast, both cell lines showed a reduced viability and enhanced propidium iodide positivity after combined treatment as determined by flow cytometry. Western blot analyses of poly-(ADP-ribose) polymerase 1 (PARP-1) suggested that combined treatment promote necrotic cleavage of PARP-1 in gc-resistant cells. Furthermore, after prednisolone treatment the UPS associated proteins VCP and NFκB-inhibitor IκBα were differentially modulated in gc-resistant cells. CONCLUSIONS: The proteasome inhibitor bortezomib seems to sensitize gc-resistant childhood ALL cells for prednisolone-induced cell death.

KRAS and CREBBP mutations: a relapse-linked malicious liaison in childhood high hyperdiploid acute lymphoblastic leukemia.

High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relatively low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole-exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in receptor tyrosine kinase (RTK)/Ras pathway and chromatin-modifying genes. The relapse clones consisted of reappearing as well as new mutations, and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18-30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone.

Genomic profiling of thousands of candidate polymorphisms predicts risk of relapse in 778 Danish and German childhood acute lymphoblastic leukemia patients.

Childhood acute lymphoblastic leukemia survival approaches 90%. New strategies are needed to identify the 10-15% who evade cure. We applied targeted, sequencing-based genotyping of 25 000 to 34 000 preselected potentially clinically relevant single-nucleotide polymorphisms (SNPs) to identify host genome profiles associated with relapse risk in 352 patients from the Nordic ALL92/2000 protocols and 426 patients from the German Berlin-Frankfurt-Munster (BFM) ALL2000 protocol. Patients were enrolled between 1992 and 2008 (median follow-up: 7.6 years). Eleven cross-validated SNPs were significantly associated with risk of relapse across protocols. SNP and biologic pathway level analyses associated relapse risk with leukemia aggressiveness, glucocorticosteroid pharmacology/response and drug transport/metabolism pathways. Classification and regression tree analysis identified three distinct risk groups defined by end of induction residual leukemia, white blood cell count and variants in myeloperoxidase (MPO), estrogen receptor 1 (ESR1), lamin B1 (LMNB1) and matrix metalloproteinase-7 (MMP7) genes, ATP-binding cassette transporters and glucocorticosteroid transcription regulation pathways. Relapse rates ranged from 4% (95% confidence interval (CI): 1.6-6.3%) for the best group (72% of patients) to 76% (95% CI: 41-90%) for the worst group (5% of patients, P<0.001). Validation of these findings and similar approaches to identify SNPs associated with toxicities may allow future individualized relapse and toxicity risk-based treatments adaptation.

Minimal residual disease-based treatment is adequate for relapse-prone childhood acute lymphoblastic leukemia with an intrachromosomal amplification of chromosome 21: the experience of the ALL-BFM 2000 trial.

BACKGROUND: Recently, the UK CCLG and COG reported that an intrachromosomal amplification of chromosome 21 (iAMP21) in acute lymphoblastic leukemia (ALL) loses its adverse prognostic impact with intensified therapy. PATIENT AND METHODS: We evaluated the prognosis of iAMP21 among patients from the ALL-BFM (Berlin-Frankfurt-Münster) 2000 trial with 46 of 2 637 (2%) patients iAMP21+. RESULTS: 8-year event-free-survival (EFS, 64 ± 8% vs. 81 ± 1%, p=0.0026) and cumulative incidence of relapse (CIR, 29 ± 8% vs. 14 ± 1%, p=0.008) of the iAMP21 cases were significantly worse compared with non-iAMP21 patients. Within the MRD low-risk group, iAMP21 cases (n=14) had an inferior 8-year EFS (76 ± 12% vs. 92 ± 1%, p=0.0081), but no increased CIR (10 ± 10% vs. 6 ± 1%, p=0.624). Within the MRD intermediate-risk group, iAMP21 cases (n=27) had a worse 8-year EFS (56 ± 11% vs. 78 ± 2%, p=0.0077) and CIR (44 ± 11% vs. 20 ± 2%, p=0.003) with 6/10 relapses occurring after 2 years. CONCLUSIONS: Conclusively, we believe that there is no necessity for enrolling all iAMP21 patients into the high-risk arm of ongoing ALL-BFM trials because MRD low-risk patients have a moderate relapse risk under current therapy. Whether the increased relapse risk in MRD intermediate-risk patients can be avoided by late treatment intensification remains to be answered by the AIEOP-BFM ALL 2009 trial randomly using protracted pegylated L-asparaginase during delayed intensification and early maintenance.

Prediction of outcome by early response in childhood acute lymphoblastic leukemia.

BACKGROUND: In the ALL-BFM studies for treatment of acute lymphoblastic leukemia, reduction of leukemic blasts in peripheral blood after a one-week prednisone pre-phase - the so-called prednisone response - has been used for risk stratification since the 1980s and has been one of the most relevant factors for identification of high-risk patients. In the trial ALL-BFM 95, early cytomorphological marrow response on day 15 of induction therapy was prospectively evaluated and its prognostic value was analyzed in comparison to the prednisone response and other established prognostic factors. RESULTS: Compared to prednisone response, day 15 marrow response was superior in outcome prediction - yet with differential effect depending on blast lineage. Outcome was poor in T cell leukemia patients with prednisone poor-response independent of day 15 marrow response, whereas among patients with prednisone good-response different risk groups could be identified by day 15 marrow response. In contrast, prednisone response lost prognostic significance in precursor B cell leukemia when stratified by day 15 marrow response. CONCLUSIONS: Selective addition of day 15 marrow response to conventional stratification criteria applied on ALL-BFM 95 may significantly improve risk-adapted treatment delivery. Even though cutting-edge trial risk stratification is meanwhile dominated by minimal residual disease evaluation, an improved conventional risk assessment, as presented here, could be of great importance to countries lacking the technical and/or financial resources associated with the application of minimal residual disease analysis.

Key treatment questions in childhood acute lymphoblastic leukemia: results in 5 consecutive trials performed by the ALL-BFM study group from 1981 to 2000.

Between 1981 and 2000, 6 609 children (<18 years of age) were treated in 5 consecutive trials of the Berlin-Frankfurt-Münster (BFM) study group for childhood acute lymphoblastic leukemia (ALL). Patients were treated in up to 82 centers in Germany, Austria, and Switzerland. Probability of 10-year event-free survival (survival) improved from 65% (77%) in study ALL-BFM 81-78% (85%) in ALL-BFM 95. In parallel to relapse reduction, major efforts focused on reducing acute and late toxicity through advanced risk adaptation of treatment. The major findings derived from these ALL-BFM trials were as follows: 1) preventive cranial radiotherapy could be safely reduced to 12 Gy in T-ALL and high-risk ALL patients and eliminated in non-high-risk non-T-ALL patients, if it was replaced by high-dose and intrathecal methotrexate; 2) omission of delayed reintensification severely impaired outcome of low-risk patients; 3) 6 months less maintenance therapy caused an increase in systemic relapses; 4) slow response to an initial 7-day prednisone window was identified as adverse prognostic factor; 5) condensed induction therapy resulted in a significant improvement of outcome; 6) the daunorubicin dose in induction could be safely reduced in low-risk patients; 7) intensification of consolidation/reintensification treatment led to considerable improvement of outcome in high-risk patients.

Poor prognosis for P2RY8-CRLF2 fusion but not for CRLF2 over-expression in children with intermediate risk B-cell precursor acute lymphoblastic leukemia.

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has achieved an 80% cure rate as a result of a risk-adapted therapy largely based on minimal residual disease (MRD) monitoring. However, relapse is still the most frequent adverse event, occurring mainly in the patients with intermediate MRD levels (intermediate risk, IR), emphasizing the need for new prognostic markers. We analyzed the prognostic impact of cytokine receptor-like factor 2 (CRLF2) over-expression and P2RY8-CRLF2 fusion in 464 BCP-ALL patients (not affected by Down syndrome and BCR-ABL negative) enrolled in the AIEOP-BFM ALL2000 study in Italy. In 22/464 (4.7%) samples, RQ-PCR showed CRLF2 over-expression (≥20 times higher than the overall median). P2RY8-CRLF2 fusion was detected in 22/365 (6%) cases, with 10/22 cases also showing CRLF2 over-expression. P2RY8-CRLF2 fusion was the most relevant prognostic factor independent of CRLF2 over-expression with a threefold increase in risk of relapse. Significantly, the cumulative incidence of relapse of the P2RY8-CRLF2 + patients in the IR group was high (61.1% ± 12.9 vs 17.6% ± 2.6, P<0.0001), similar to high-risk patients in AIEOP-BFM ALL2000 study. These results were confirmed in a cohort of patients treated in Germany. In conclusion, P2RY8-CRLF2 identifies a subset of BCP-ALL patients currently stratified as IR that could be considered for treatment intensification.