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BackgroundPatients with late, ≥18 months postdiagnosis, isolated central nervous relapse (iCNS-R) of B-acute lymphoblastic leukemia (ALL) have excellent outcomes with chemotherapy plus cranial radiotherapy, with 5-year overall survival (OS) approaching 80% in POG 9412. Subsequent relapse and radiation-related morbidity remain the causes of treatment failure and long-term sequelae. COG AALL02P2 aimed to maintain outcomes in patients with late iCNS-R using intensified chemotherapy and a decrease in cranial irradiation from 1800 to 1200 cGy.ProceduresCOG AALL02P2 enrolled 118 eligible patients with B-cell ALL (B-ALL) and late iCNS-R who received intensified systemic therapy, triple intrathecal chemotherapy, and 1200 cGy cranial irradiation delivered at 12 months, with maintenance chemotherapy continuing until 104 weeks postdiagnosis.ResultsThe 3-year event-free survival (EFS) and OS were 64.3% ± 4.5% and 79.6% ± 3.8%, with 46.1% (18/39) of second relapses including the CNS. Of the 112 patients who completed therapy, 78 received protocol-specified radiation. Study enrollment was closed after interim monitoring analysis showed inferior EFS compared to POG 9412. Patients with initial NCI standard-risk classification fared better than high-risk patients.ConclusionsCOG AALL02P2 showed inferior EFS but similar OS compared to POG 9412. Limitations included a small sample size, more intensive prior therapies, and a significant number of patients (34/118, 29%) who did not receive protocol-directed radiation due to early relapse prior to 1 year or did not otherwise follow the treatment plan. New approaches are needed to improve outcome for these patients and determine the optimal timing and dose of cranial radiation in the treatment of iCNS-R.

Reverse phase protein arrays (RPPA) can assess protein expression and activation states in large numbers of samples (n > 1000) and evidence suggests feasibility in the setting of multi-institution clinical trials. Despite evidence in solid tumors, little is known about protein stability in leukemia. Proteins collected from leukemia cells in blood and bone marrow biopsies must be sufficiently stable for analysis. Using 58 leukemia samples, we initially assessed protein/phospho-protein integrity for the following preanalytical variables: 1) shipping vs local processing, 2) temperature (4 °C vs ambient temperature), 3) collection tube type (heparin vs Cell Save (CS) preservation tubes), 4) treatment effect (pre- vs post-chemotherapy) and 5) transit time. Next, we assessed 1515 samples from the Children's Oncology Group Phase 3 AML clinical trial (AAML1031, NCT01371981) for the effects of transit time and tube type. Protein expression from shipped blood samples was stable if processed in ≤72 h. While protein expression in pre-chemotherapy samples was stable in both heparin and CS tubes, post-chemotherapy samples were stable in only CS tubes. RPPA protein extremes is a successful quality control measure to identify and exclude poor quality samples. These data demonstrate that a majority of shipped proteins can be accurately assessed using RPPA. SIGNIFICANCE: RPPA can assess protein abundance and activation states in large numbers of samples using small amounts of material, making this method ideal for use in multi-institution clinical trials. However, there is little known about the effect of preanalytical handling variables on protein stability and the integrity of protein concentrations after sample collection and shipping. In this study, we used RPPA to assess preanalytical variables that could potentially affect protein concentrations. We found that the preanalytical variables of shipping, transit time, and temperature had minimal effects on RPPA protein concentration distributions in peripheral blood and bone marrow, demonstrating that these preanalytical variables could be successfully managed in a multi-site clinical trial setting.

Outcomes after relapse of childhood B-acute lymphoblastic leukemia (B-ALL) are poor, and optimal therapy is unclear. Children's Oncology Group study AALL0433 evaluated a new platform for relapsed ALL. Between March 2007 and October 2013 AALL0433 enrolled 275 participants with late bone marrow or very early isolated central nervous system (iCNS) relapse of childhood B-ALL. Patients were randomized to receive standard versus intensive vincristine dosing; this randomization closed due to excess peripheral neuropathy in 2010. Patients with matched sibling donors received allogeneic hematopoietic cell transplantation (HCT) after the first three blocks of therapy. The prognostic value of minimal residual disease (MRD) was also evaluated in this study. The 3-year event free and overall survival (EFS/OS) for the 271 eligible patients were 63.6% +/- 3.0% and 72.3% +/- 2.8% respectively. MRD at the end of Induction-1 was highly predictive of outcome, with 3-year EFS/OS of 84.9 +/- 4.0% and 93.8 +/- 2.7% for patients with MRD

PurposeThe Children's Oncology Group (COG) protocol AALL0434 evaluated the safety and efficacy of multi-agent chemotherapy with Capizzi-based methotrexate/pegaspargase (C-MTX) in patients with newly diagnosed pediatric T-cell lymphoblastic lymphoma (T-LL) and gained preliminary data using nelarabine in high-risk patients.Patients and methodsThe trial enrolled 299 patients, age 1-31 years. High-risk (HR) patients had ≥ 1% minimal detectable disease (MDD) in the bone marrow at diagnosis or received prior steroid treatment. Induction failure was defined as failure to achieve a partial response (PR) by the end of the 4-week induction. All patients received the augmented Berlin-Frankfurt-Muenster (ABFM) C-MTX regimen. HR patients were randomly assigned to receive or not receive 6 5-day courses of nelarabine incorporated into ABFM. Patients with induction failure were nonrandomly assigned to ABFM C-MTX plus nelarabine. No patients received prophylactic cranial radiation; however, patients with CNS3 disease (CSF WBC ≥ 5/μL with blasts or cranial nerve palsies, brain/eye involvement, or hypothalamic syndrome) were ineligible.ResultsAt end-induction, 98.8% of evaluable participants had at least a PR. The 4-year event-free survival (EFS) and overall survival (OS) were 84.7% ± 2.3% and 89.0% ± 2.0%. The 4-year disease-free survival (DFS) from end-induction was 85.9% ± 2.6%. There was no difference in DFS observed between the HR and standard-risk groups (P = .29) or by treatment regimen (P = .55). Disease stage, tumor response, and MDD at diagnosis did not demonstrate thresholds that resulted in differences in EFS. Nelarabine did not show an advantage for HR patients. CNS relapse occurred in only 4 patients.ConclusionCOG AALL0434 produced excellent outcomes in one of the largest trials ever conducted for patients with newly diagnosed T-LL. The COG ABFM regimen with C-MTX provided excellent EFS and OS without cranial radiation.

With modern chemotherapy, approximately 90% of patients with pediatric acute lymphoblastic leukemia are now cured. However, subsets of patients can be identified who remain at very high risk of relapse with expected 4-year disease-free survival rates

Germline SAMD9 and SAMD9L mutations cause a spectrum of multisystem disorders that carry a markedly increased risk of developing myeloid malignancies with somatic monosomy 7. Here, we describe 16 siblings, the majority of which were phenotypically normal, from 5 families diagnosed with myelodysplasia and leukemia syndrome with monosomy 7 (MLSM7; OMIM 252270) who primarily had onset of hematologic abnormalities during the first decade of life. Molecular analyses uncovered germline SAMD9L (n = 4) or SAMD9 (n = 1) mutations in these families. Affected individuals had a highly variable clinical course that ranged from mild and transient dyspoietic changes in the bone marrow to a rapid progression of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with monosomy 7. Expression of these gain-of-function SAMD9 and SAMD9L mutations reduces cell cycle progression, and deep sequencing demonstrated selective pressure favoring the outgrowth of clones that have either lost the mutant allele or acquired revertant mutations. The myeloid malignancies of affected siblings acquired cooperating mutations in genes that are also altered in sporadic cases of AML characterized by monosomy 7. These data have implications for understanding how SAMD9 and SAMD9L mutations contribute to myeloid transformation and for recognizing, counseling, and treating affected families.

There is increasing evidence from genome-wide association studies for a strong inherited genetic basis of susceptibility to acute lymphoblastic leukaemia (ALL) in children, yet the effects of protein-coding variants on ALL risk have not been systematically evaluated. Here we show a missense variant in CDKN2A associated with the development of ALL at genome-wide significance (rs3731249, P=9.4 × 10(-23), odds ratio=2.23). Functional studies indicate that this hypomorphic variant results in reduced tumour suppressor function of p16(INK4A), increases the susceptibility to leukaemic transformation of haematopoietic progenitor cells, and is preferentially retained in ALL tumour cells. Resequencing the CDKN2A-CDKN2B locus in 2,407 childhood ALL cases reveals 19 additional putative functional germline variants. These results provide direct functional evidence for the influence of inherited genetic variation on ALL risk, highlighting the important and complex roles of CDKN2A-CDKN2B tumour suppressors in leukaemogenesis.

There is incomplete understanding of genetic heterogeneity and clonal evolution during cancer progression. Here we use deep whole-exome sequencing to describe the clonal architecture and evolution of 20 pediatric B-acute lymphoblastic leukaemias from diagnosis to relapse. We show that clonal diversity is comparable at diagnosis and relapse and clonal survival from diagnosis to relapse is not associated with mutation burden. Six pathways were frequently mutated, with NT5C2, CREBBP, WHSC1, TP53, USH2A, NRAS and IKZF1 mutations enriched at relapse. Half of the leukaemias had multiple subclonal mutations in a pathway or gene at diagnosis, but mostly with only one, usually minor clone, surviving therapy to acquire additional mutations and become the relapse founder clone. Relapse-specific mutations in NT5C2 were found in nine cases, with mutations in four cases being in descendants of the relapse founder clone. These results provide important insights into the genetic basis of treatment failure in ALL and have implications for the early detection of mutations driving relapse.

ImportanceWith cure rates of childhood acute lymphoblastic leukemia (ALL) exceeding 85%, there is a need to mitigate treatment toxicities that can compromise quality of life, including peripheral neuropathy from vincristine treatment.ObjectiveTo identify genetic germline variants associated with the occurrence or severity of vincristine-induced peripheral neuropathy in children with ALL.Design, setting, and participantsGenome-wide association study of patients in 1 of 2 prospective clinical trials for childhood ALL that included treatment with 36 to 39 doses of vincristine. Genome-wide single-nucleotide polymorphism (SNP) analysis and vincristine-induced peripheral neuropathy were assessed in 321 patients from whom DNA was available: 222 patients (median age, 6.0 years; range, 0.1-18.8 years) enrolled in 1994-1998 in the St Jude Children's Research Hospital protocol Total XIIIB with toxic effects follow-up through January 2001, and 99 patients (median age, 11.4 years; range, 3.0-23.8 years) enrolled in 2007-2010 in the Children's Oncology Group (COG) protocol AALL0433 with toxic effects follow-up through May 2011. Human leukemia cells and induced pluripotent stem cell neurons were used to assess the effects of lower CEP72 expression on vincristine sensitivity.ExposureTreatment with vincristine at a dose of 1.5 or 2.0 mg/m2.Main outcomes and measuresVincristine-induced peripheral neuropathy was assessed at clinic visits using National Cancer Institute criteria and prospectively graded as mild (grade 1), moderate (grade 2), serious/disabling (grade 3), or life threatening (grade 4).ResultsGrade 2 to 4 vincristine-induced neuropathy during continuation therapy occurred in 28.8% of patients (64/222) in the St Jude cohort and in 22.2% (22/99) in the COG cohort. A SNP in the promoter region of the CEP72 gene, which encodes a centrosomal protein involved in microtubule formation, had a significant association with vincristine neuropathy (meta-analysis P = 6.3×10(-9)). This SNP had a minor allele frequency of 37% (235/642), with 50 of 321 patients (16%; 95% CI, 11.6%-19.5%) homozygous for the risk allele (TT at rs924607). Among patients with the high-risk CEP72 genotype (TT at rs924607), 28 of 50 (56%; 95% CI, 41.2%-70.0%) developed at least 1 episode of grade 2 to 4 neuropathy, a higher rate than in patients with the CEP72 CC or CT genotypes (58/271 patients [21.4%; 95% CI, 16.9%-26.7%]; P = 2.4×10(-6)). The severity of neuropathy was greater in patients homozygous for the TT genotype compared with patients with the CC or CT genotype (2.4-fold by Poisson regression [P

While childhood acute lymphoblastic leukaemia (ALL) is now highly curable, the dismal prognosis for children who relapse warrants novel therapeutic approaches. Previously, using an integrated genomic analysis of matched diagnosis-relapse paired samples, we identified overactivation of the Wnt pathway as a possible mechanism of recurrence. To validate these findings and document whether Wnt inhibition may sensitize cells to chemotherapy, we analysed the expression of activated β-catenin (and its downstream target BIRC5) using multiparameter phosphoflow cytometry and tested the efficacy of a recently developed Wnt inhibitor, iCRT14, in ALL cell lines and patient samples. We observed increased activation of β-catenin at relapse in 6/10 patients. Furthermore, treatment of leukaemic cell lines with iCRT14 led to significant downregulation of Wnt target genes and combination with traditional chemotherapeutic drugs resulted in a synergistic decrease in viability as well as a significant increase in apoptotic cell death. Finally, pre-treatment of purified blasts from patients with relapsed leukaemia with the Wnt inhibitor followed by exposure to prednisolone, restored chemosensitivity in these cells. Our results demonstrate that overactivation of the Wnt pathway may contribute to chemoresistance in relapsed childhood ALL and that Wnt-inhibition may be a promising therapeutic approach.

Recent genomic profiling of childhood acute lymphoblastic leukemia (ALL) identified a high-risk subtype with an expression signature resembling that of Philadelphia chromosome-positive ALL and poor prognosis (Ph-like ALL). However, the role of inherited genetic variation in Ph-like ALL pathogenesis remains unknown. In a genome-wide association study (GWAS) of 511 ALL cases and 6,661 non-ALL controls, we identified a susceptibility locus for Ph-like ALL (GATA3, rs3824662; P = 2.17 × 10(-14), odds ratio (OR) = 3.85 for Ph-like ALL versus non-ALL; P = 1.05 × 10(-8), OR = 3.25 for Ph-like ALL versus non-Ph-like ALL), with independent validation. The rs3824662 risk allele was associated with somatic lesions underlying Ph-like ALL (CRLF2 rearrangement, JAK gene mutation and IKZF1 deletion) and with variation in GATA3 expression. Finally, genotype at the GATA3 SNP was also associated with early treatment response and risk of ALL relapse. Our results provide insights into interactions between inherited and somatic variants and their role in ALL pathogenesis and prognosis.

The B cell-specific transcription factor BACH2 is required for affinity maturation of B cells. Here we show that Bach2-mediated activation of p53 is required for stringent elimination of pre-B cells that failed to productively rearrange immunoglobulin VH-DJH gene segments. After productive VH-DJH gene rearrangement, pre-B cell receptor signaling ends BACH2-mediated negative selection through B cell lymphoma 6 (BCL6)-mediated repression of p53. In patients with pre-B acute lymphoblastic leukemia, the BACH2-mediated checkpoint control is compromised by deletions, rare somatic mutations and loss of its upstream activator, PAX5. Low levels of BACH2 expression in these patients represent a strong independent predictor of poor clinical outcome. In this study, we demonstrate that Bach2(+/+) pre-B cells resist leukemic transformation by Myc through Bach2-dependent upregulation of p53 and do not initiate fatal leukemia in transplant-recipient mice. Chromatin immunoprecipitation sequencing and gene expression analyses carried out by us revealed that BACH2 competes with BCL6 for promoter binding and reverses BCL6-mediated repression of p53 and other cell cycle checkpoint-control genes. These findings identify BACH2 as a crucial mediator of negative selection at the pre-B cell receptor checkpoint and a safeguard against leukemogenesis.

BackgroundAcute lymphoblastic leukemia (ALL) is the most common cancer in children and the incidence of ALL varies by ethnicity. Although accumulating evidence indicates inherited predisposition to ALL, the genetic basis of ALL susceptibility in diverse ancestry has not been comprehensively examined.MethodsWe performed a multiethnic genome-wide association study in 1605 children with ALL and 6661 control subjects after adjusting for population structure, with validation in three replication series of 845 case subjects and 4316 control subjects. Association was tested by two-sided logistic regression.ResultsA novel ALL susceptibility locus at 10p12.31-12.2 (BMI1-PIP4K2A, rs7088318, P = 1.1 × 10(-11)) was identified in the genome-wide association study, with independent replication in European Americans, African Americans, and Hispanic Americans (P = .001, .009, and .04, respectively). Association was also validated at four known ALL susceptibility loci: ARID5B, IKZF1, CEBPE, and CDKN2A/2B. Associations at ARID5B, IKZF1, and BMI1-PIP4K2A variants were consistent across ethnicity, with multiple independent signals at IKZF1 and BMI1-PIP4K2A loci. The frequency of ARID5B and BMI1-PIP4K2A variants differed by ethnicity, in parallel with ethnic differences in ALL incidence. Suggestive evidence for modifying effects of age on genetic predisposition to ALL was also observed. ARID5B, IKZF1, CEBPE, and BMI1-PIP4K2A variants cumulatively conferred strong predisposition to ALL, with children carrying six to eight copies of risk alleles at a ninefold (95% confidence interval = 6.9 to 11.8) higher ALL risk relative to those carrying zero to one risk allele at these four single nucleotide polymorphisms.ConclusionsThese findings indicate strong associations between inherited genetic variation and ALL susceptibility in children and shed new light on ALL molecular etiology in diverse ancestry.

Genomic profiling has identified a subtype of high-risk B-progenitor acute lymphoblastic leukemia (B-ALL) with alteration of IKZF1, a gene expression profile similar to BCR-ABL1-positive ALL and poor outcome (Ph-like ALL). The genetic alterations that activate kinase signaling in Ph-like ALL are poorly understood. We performed transcriptome and whole genome sequencing on 15 cases of Ph-like ALL and identified rearrangements involving ABL1, JAK2, PDGFRB, CRLF2, and EPOR, activating mutations of IL7R and FLT3, and deletion of SH2B3, which encodes the JAK2-negative regulator LNK. Importantly, several of these alterations induce transformation that is attenuated with tyrosine kinase inhibitors, suggesting the treatment outcome of these patients may be improved with targeted therapy.