Subclonal NT5C2 mutations are associated with poor outcomes after relapse of pediatric acute lymphoblastic leukemia.

Activating mutations in cytosolic 5'-nucleotidase II (NT5C2) are considered to drive relapse formation in acute lymphoblastic leukemia (ALL) by conferring purine analog resistance. To examine the clinical effects of NT5C2 mutations in relapsed ALL, we analyzed NT5C2 in 455 relapsed B-cell precursor ALL patients treated within the ALL-REZ BFM 2002 relapse trial using sequencing and sensitive allele-specific real-time polymerase chain reaction. We detected 110 NT5C2 mutations in 75 (16.5%) of 455 B-cell precursor ALL relapses. Two-thirds of relapses harbored subclonal mutations and only one-third harbored clonal mutations. Event-free survival after relapse was inferior in patients with relapses with clonal and subclonal NT5C2 mutations compared with those without (19% and 25% vs 53%, P < .001). However, subclonal, but not clonal, NT5C2 mutations were associated with reduced event-free survival in multivariable analysis (hazard ratio, 1.89; 95% confidence interval, 1.28-2.69; P = .001) and with an increased rate of nonresponse to relapse treatment (subclonal 32%, clonal 12%, wild type 9%, P < .001). Nevertheless, 27 (82%) of 33 subclonal NT5C2 mutations became undetectable at the time of nonresponse or second relapse, and in 10 (71%) of 14 patients subclonal NT5C2 mutations were undetectable already after relapse induction treatment. These results show that subclonal NT5C2 mutations define relapses associated with high risk of treatment failure in patients and at the same time emphasize that their role in outcome is complex and goes beyond mutant NT5C2 acting as a targetable driver during relapse progression. Sensitive, prospective identification of NT5C2 mutations is warranted to improve the understanding and treatment of this aggressive ALL relapse subtype.

Aneuploidy in children with relapsed B-cell precursor acute lymphoblastic leukaemia: clinical importance of detecting a hypodiploid origin of relapse.

Aneuploidy is common in paediatric B-cell precursor acute lymphoblastic leukaemia (ALL). Specific subgroups, such as high hyperdiploidy (>50 chromosomes or DNA Index ≥1·16) and hypodiploidy (<45 chromosomes), predict outcome of patients after primary treatment. Whether aneuploidy has a prognostic value for relapsed disease is yet to be determined. Using DNA index and centromere screening by multiplex ligation-dependent probe amplification, we investigated aneuploidy in 413 children treated for first relapse of B-cell precursor ALL according to the ALL-REZ BFM 2002 protocol. Ten-year event-free survival of patients with high hyperdiploid relapses approached 70%, whereas it was only 40% in low hyperdiploid relapses. Three patients with apparent hyperdiploid relapse had TP53 mutations. In these cases, array-based allelotyping revealed a hypodiploid origin with absence of the hypodiploid founder clone (masked hypodiploidy). Collectively, patients with evident or masked hypodiploid relapses showed an extremely low event-free survival rate of 9%. Importantly, the current relapse risk stratification did not identify cases with masked hypodiploidy as high-risk patients, due to their favourable clinical presentation. In multivariate analysis, hypodiploidy proved to be an independent prognostic factor. This finding supports stratification of relapses with hypodiploid origin into high-risk arms in future trials or allocation of patients to alternative treatment approaches.

Pediatric T-cell lymphoblastic leukemia evolves into relapse by clonal selection, acquisition of mutations and promoter hypomethylation.

Relapsed precursor T-cell acute lymphoblastic leukemia is characterized by resistance against chemotherapy and is frequently fatal. We aimed at understanding the molecular mechanisms resulting in relapse of T-cell acute lymphoblastic leukemia and analyzed 13 patients at first diagnosis, remission and relapse by whole exome sequencing, targeted ultra-deep sequencing, multiplex ligation dependent probe amplification and DNA methylation array. Compared to primary T-cell acute lymphoblastic leukemia, in relapse the number of single nucleotide variants and small insertions and deletions approximately doubled from 11.5 to 26. Targeted ultra-deep sequencing sensitively detected subclones that were selected for in relapse. The mutational pattern defined two types of relapses. While both are characterized by selection of subclones and acquisition of novel mutations, 'type 1' relapse derives from the primary leukemia whereas 'type 2' relapse originates from a common pre-leukemic ancestor. Relapse-specific changes included activation of the nucleotidase NT5C2 resulting in resistance to chemotherapy and mutations of epigenetic modulators, exemplified by SUZ12, WHSC1 and SMARCA4. While mutations present in primary leukemia and in relapse were enriched for known drivers of leukemia, relapse-specific changes revealed an association with general cancer-promoting mechanisms. This study thus identifies mechanisms that drive progression of pediatric T-cell acute lymphoblastic leukemia to relapse and may explain the characteristic treatment resistance of this condition.

Ras pathway mutations are prevalent in relapsed childhood acute lymphoblastic leukemia and confer sensitivity to MEK inhibition.

For most children who relapse with acute lymphoblastic leukemia (ALL), the prognosis is poor, and there is a need for novel therapies to improve outcome. We screened samples from children with B-lineage ALL entered into the ALL-REZ BFM 2002 clinical trial (www.clinicaltrials.gov, #NCT00114348) for somatic mutations activating the Ras pathway (KRAS, NRAS, FLT3, and PTPN11) and showed mutation to be highly prevalent (76 from 206). Clinically, they were associated with high-risk features including early relapse, central nervous system (CNS) involvement, and specifically for NRAS/KRAS mutations, chemoresistance. KRAS mutations were associated with a reduced overall survival. Mutation screening of the matched diagnostic samples found many to be wild type (WT); however, by using more sensitive allelic-specific assays, low-level mutated subpopulations were found in many cases, suggesting that they survived up-front therapy and subsequently emerged at relapse. Preclinical evaluation of the mitogen-activated protein kinase kinase 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) showed significant differential sensitivity in Ras pathway-mutated ALL compared with WT cells both in vitro and in an orthotopic xenograft model engrafted with primary ALL; in the latter, reduced RAS-mutated CNS leukemia. Given these data, clinical evaluation of selumetinib may be warranted for Ras pathway-mutated relapsed ALL.

Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL.

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric patients and over 50% of adult patients with ALL do not achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most substantial challenge in the treatment of this disease. Using whole-exome sequencing, we identify mutations in the cytosolic 5'-nucleotidase II gene (NT5C2), which encodes a 5'-nucleotidase enzyme that is responsible for the inactivation of nucleoside-analog chemotherapy drugs, in 20/103 (19%) relapse T cell ALLs and 1/35 (3%) relapse B-precursor ALLs. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside-analog metabolism in disease progression and chemotherapy resistance in ALL.

Synergistic activity of bortezomib and HDACi in preclinical models of B-cell precursor acute lymphoblastic leukemia via modulation of p53, PI3K/AKT, and NF-κB.

PURPOSE: Relapse of disease and subsequent resistance to established therapies remains a major challenge in the treatment of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). New therapeutic options, such as proteasome and histone deacetylase inhibitors (HDACi) with a toxicity profile differing from that of conventional cytotoxic agents, are needed for these extensively pretreated patients. EXPERIMENTAL DESIGN: Antiproliferative and proapoptotic effects of combined HDACi/proteasome inhibitor treatments were analyzed using BCP-ALL monocultures, cocultures with primary mesenchymal stroma cells from patients with ALL, and xenograft mouse models. The underlying molecular mechanisms associated with combined treatment were determined by gene expression profiling and protein validation. RESULTS: We identified the proteasome inhibitor bortezomib as a promising combination partner for HDACi due to the substantial synergistic antileukemic activity in BCP-ALL cells after concomitant application. This effect was maintained or even increased in the presence of chemotherapeutic agents. The synergistic effect of combined HDACi/BTZ treatment was associated with the regulation of genes involved in cell cycle, JUN/MAPK, PI3K/AKT, p53, ubiquitin/proteasome, and NF-κB pathways. We observed an activation of NF-κB after bortezomib treatment and the induction of apoptosis-related NF-κB target genes such as TNFαRs after concomitant treatment, indicating a possible involvement of NF-κB as proapoptotic mediator. In this context, significantly lower NF-κB subunits gene expression was detected in leukemia cells from patients who developed a relapse during frontline chemotherapy, compared with those who relapsed after cessation of frontline therapy. CONCLUSION: These results provide a rationale for the integration of HDACi/BTZ combinations into current childhood BCP-ALL treatment protocols.

High VLA-4 expression is associated with adverse outcome and distinct gene expression changes in childhood B-cell precursor acute lymphoblastic leukemia at first relapse.

BACKGROUND: Resistance to therapy and subsequent relapse remain major challenges in the clinical management of relapsed childhood acute lymphoblastic leukemia. As the bone marrow environment plays an important role in survival and chemotherapy resistance of leukemia cells by activating different signaling pathways, such as the VLA-4 and PI3K/Akt pathways, we studied the prognostic and biological impact of VLA-4 expression in leukemia cells from children with relapsed B-cell precursor acute lymphoblastic leukemia and its influence on the sensitivity of the leukemia cells to drugs. DESIGN AND METHODS: VLA-4 expression was quantified by real-time polymerase chain reaction in leukemia cells from 56 patients with relapsed acute lymphoblastic leukemia enrolled in the ALL-REZ BFM 2002 trial of the Berlin-Frankfurt-Münster study group. Gene expression changes related to VLA-4 expression were investigated by microarray-based mRNA profiling. The effect of VLA-4 signaling on proliferation and drug resistance was studied in co-cultures of leukemia and stromal cells. RESULTS: High expression of VLA-4 at first relapse was associated with adverse prognostic factors, poor molecular response to therapy and significantly worse probabilities of event-free and overall survival. VLA-4 expression was an independent prognostic parameter. Comparing gene expression profiles of leukemia cells with high versus low VLA-4 expression, we identified 27 differentially expressed genes primarily involved in the PI3K/Akt, ephrin and Rho GTPase pathways. Blocking of VLA-4 signaling in combination with cytarabine treatment abolished the growth supportive effect of stromal cells. CONCLUSIONS: Our results show that high VLA-4 expression is a marker of poor prognosis and a potential therapeutic target in children with relapsed acute lymphoblastic leukemia and confirm that cellular interactions and biological effects related to VLA-4 play a decisive role in the survival of leukemia cells and response to therapy. (ClinicalTrials.gov identifier: NCT00114348).

Mutations and deletions of the TP53 gene predict nonresponse to treatment and poor outcome in first relapse of childhood acute lymphoblastic leukemia.

PURPOSE: In the clinical management of children with relapsed acute lymphoblastic leukemia (ALL), treatment resistance remains a major challenge. Alterations of the TP53 gene are frequently associated with resistance to chemotherapy, but their significance in relapsed childhood ALL has remained controversial because of small studies. PATIENTS AND METHODS: Therefore, we systematically studied 265 first-relapse patients enrolled in the German Acute Lymphoblastic Leukemia Relapse Berlin-Frankfurt-Mü nster 2002 (ALL-REZ BFM 2002) trial for sequence and copy number alterations of the TP53 gene by using direct sequencing and multiplex ligation-dependent probe amplification. RESULTS: We observed copy number and sequence alterations of TP53 in 12.4% (27 of 218) of patients with B-cell precursor ALL and 6.4% (three of 47) of patients with T-cell ALL relapse. Backtracking to initial ALL in 23 matched samples revealed that 54% of all TP53 alterations were gained at relapse. Within B-cell precursor ALL, TP53 alterations were consistently associated with nonresponse to chemotherapy (P < .001) and poor event-free survival (P < .001) and overall survival rates (P = .002). TP53 alterations also had a significant impact on survival within intermediate-risk (S2) and high-risk (S3/S4) relapse patients (P = .007 and P = .019, respectively). This prognostic significance of TP53 alterations was confirmed in multivariate analysis. Besides their clinical impact, TP53 alterations were associated with a higher fraction of leukemic cells in S/G(2)-M phase of the cell cycle at relapse diagnosis. CONCLUSION: Alterations of the TP53 gene are of particular importance in the relapse stage of childhood ALL, in which they independently predict high risk of treatment failure in a significant number of patients. Therefore, they will aid in future risk assessment of children with ALL relapse.

Dissociation of obesity and insulin resistance in transgenic mice with skeletal muscle expression of uncoupling protein 1.

We evaluated the effect of skeletal muscle mitochondrial uncoupling on energy and glucose metabolism under different diets. For 3 mo, transgenic HSA-mUCP1 mice with ectopic expression of uncoupling protein 1 in skeletal muscle and wild-type littermates were fed semisynthetic diets with varying macronutrient ratios (energy % carbohydrate-protein-fat): HCLF (41:42:17), HCHF (41:16:43); LCHF (11:45:44). Body composition, energy metabolism, and insulin resistance were assessed by NMR, indirect calorimetry, and insulin tolerance test, respectively. Gene expression in different organs was determined by real-time PCR. In wild type, both high-fat diets led to an increase in body weight and fat. HSA-mUCP1 mice considerably increased body fat on HCHF but stayed lean on the other diets. Irrespective of differences in body fat content, HSA-mUCP1 mice showed higher insulin sensitivity and decreased plasma insulin and liver triglycerides. Respiratory quotient and gene expression indicated overall increased carbohydrate oxidation of HSA-mUCP1 but a preferential channeling of fatty acids into muscle rather than liver with high-fat diets. Evidence for increased lipogenesis in white fat of HSA-mUCP1 mice suggests increased energy dissipating substrate cycling. Retinol binding protein 4 expression in white fat was increased in HSA-mUCP1 mice despite increased insulin sensitivity, excluding a causal role in the development of insulin resistance. We conclude that skeletal muscle mitochondrial uncoupling does not protect from the development of obesity in all circumstances. Rather it can lead to a "healthy" obese phenotype by preserving insulin sensitivity and a high metabolic flexibility, thus protecting from the development of obesity associated disturbances of glucose homeostasis.