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Most children with acute lymphoblastic leukemia (ALL) can be cured, but the prognosis is dismal for the minority of patients who relapse after treatment. To explore the genetic basis of relapse, we performed genome-wide DNA copy number analyses on matched diagnosis and relapse samples from 61 pediatric patients with ALL. The diagnosis and relapse samples typically showed different patterns of genomic copy number abnormalities (CNAs), with the CNAs acquired at relapse preferentially affecting genes implicated in cell cycle regulation and B cell development. Most relapse samples lacked some of the CNAs present at diagnosis, which suggests that the cells responsible for relapse are ancestral to the primary leukemia cells. Backtracking studies revealed that cells corresponding to the relapse clone were often present as minor subpopulations at diagnosis. These data suggest that genomic abnormalities contributing to ALL relapse are selected for during treatment, and they point to new targets for therapeutic intervention.

Background Sequencing errors are key confounding factors for detecting low-frequency genetic variants that are important for cancer molecular diagnosis, treatment, and surveillance using deep next-generation sequencing (NGS). However, there is a lack of comprehensive understanding of errors introduced at various steps of a conventional NGS workflow, such as sample handling, library preparation, PCR enrichment, and sequencing. In this study, we use current NGS technology to systematically investigate these questions. Results By evaluating read-specific error distributions, we discover that the substitution error rate can be computationally suppressed to 10 −5 to 10 −4 , which is 10- to 100-fold lower than generally considered achievable (10 −3 ) in the current literature. We then quantify substitution errors attributable to sample handling, library preparation, enrichment PCR, and sequencing by using multiple deep sequencing datasets. We find that error rates differ by nucleotide substitution types, ranging from 10 −5 for A>C/T>G, C>A/G>T, and C>G/G>C changes to 10 −4 for A>G/T>C changes. Furthermore, C>T/G>A errors exhibit strong sequence context dependency, sample-specific effects dominate elevated C>A/G>T errors, and target-enrichment PCR led to ~ 6-fold increase of overall error rate. We also find that more than 70% of hotspot variants can be detected at 0.1 ~ 0.01% frequency with the current NGS technology by applying in silico error suppression. Conclusions We present the first comprehensive analysis of sequencing error sources in conventional NGS workflows. The error profiles revealed by our study highlight new directions for further improving NGS analysis accuracy both experimentally and computationally, ultimately enhancing the precision of deep sequencing.

A constitutive mutation in a cancer-susceptibility gene can have implications for clinical treatment and genetic counseling of family members. This study involving 1120 children and adolescents showed that 95 (8.5%) had such a mutation. The frequency of germline mutations in cancer-predisposition genes in children and adolescents with cancer and the implications of such mutations are largely unknown. Previous studies have relied mainly on candidate-gene approaches, which are, by design, limited. To better determine the contribution of germline predisposition mutations to childhood cancer, we used next-generation sequencing, including whole-genome and whole-exome sequencing, to analyze the genomes of 1120 children and adolescents with cancer. We describe the prevalence and spectrum of germline variants among 565 cancer-associated genes, with an emphasis on the analysis of 60 genes that have been associated with autosomal dominant cancer-predisposition syndromes. We . . .

Anna Andersson, Tanja Gruber, James Downing and colleagues report a genomic analysis of infant acute lymphoblastic leukemias with MLL rearrangements. They identify recurrent activating mutations in tyrosine kinase, phosphatidylinositol 3-kinase and RAS pathway genes but find that these mutations were often present in minor subclones and lost at the time of relapse. Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements ( MLL -R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL -R and 18 non– MLL -R cases) and 20 older children ( MLL -R cases) with leukemia. Our data show that infant MLL -R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL -R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10 −5 ) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL , was rarely mutated in infant MLL -R ALL.

To evaluate the potential of an integrated clinical test to detect diverse classes of somatic and germline mutations relevant to pediatric oncology, we performed three-platform whole-genome (WGS), whole exome (WES) and transcriptome (RNA-Seq) sequencing of tumors and normal tissue from 78 pediatric cancer patients in a CLIA-certified, CAP-accredited laboratory. Our analysis pipeline achieves high accuracy by cross-validating variants between sequencing types, thereby removing the need for confirmatory testing, and facilitates comprehensive reporting in a clinically-relevant timeframe. Three-platform sequencing has a positive predictive value of 97–99, 99, and 91% for somatic SNVs, indels and structural variations, respectively, based on independent experimental verification of 15,225 variants. We report 240 pathogenic variants across all cases, including 84 of 86 known from previous diagnostic testing (98% sensitivity). Combined WES and RNA-Seq, the current standard for precision oncology, achieved only 78% sensitivity. These results emphasize the critical need for incorporating WGS in pediatric oncology testing. Clinical oncology is rapidly adopting next-generation sequencing technology for nucleotide variant and indel detection. Here the authors present a three-platform approach (whole-genome, whole-exome, and whole-transcriptome) in pediatric patients for the detection of diverse types of germline and somatic variants.

Lars Bullinger, Jinghui Zhang, Jeffery Klco, James Downing and colleagues report a detailed genomic analysis of pediatric and adult core-binding factor acute myeloid leukemias (CBF-AMLs). They identify recurrent mutations in CCND2 , MGA , DHX15 and ZBTB7A and highlight dramatic differences in the landscape of cooperating mutations between different CBF-AML subtypes. Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric ( n = 87) and adult ( n = 78) samples, including cases with RUNX1 - RUNX1T1 ( n = 85) or CBFB - MYH11 ( n = 80) rearrangements, by whole-genome or whole-exome sequencing. In addition to known mutations in the Ras pathway, we identified recurrent stabilizing mutations in CCND2 , suggesting a previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1 - RUNX1T1 and CBFB - MYH11 AMLs demonstrated remarkably different spectra of cooperating mutations, as RUNX1 - RUNX1T1 cases harbored recurrent mutations in DHX15 and ZBTB7A , as well as an enrichment of mutations in epigenetic regulators, including ASXL2 and the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscapes of cooperating mutations for these related AML subtypes.

Studies of paediatric cancers have shown a high frequency of mutation across epigenetic regulators. Here we sequence 633 genes, encoding the majority of known epigenetic regulatory proteins, in over 1,000 paediatric tumours to define the landscape of somatic mutations in epigenetic regulators in paediatric cancer. Our results demonstrate a marked variation in the frequency of gene mutations across 21 different paediatric cancer subtypes, with the highest frequency of mutations detected in high-grade gliomas, T-lineage acute lymphoblastic leukaemia and medulloblastoma, and a paucity of mutations in low-grade glioma and retinoblastoma. The most frequently mutated genes are H3F3A , PHF6 , ATRX , KDM6A , SMARCA4 , ASXL2 , CREBBP , EZH2 , MLL2 , USP7 , ASXL1 , NSD2 , SETD2 , SMC1A and ZMYM3 . We identify novel loss-of-function mutations in the ubiquitin-specific processing protease 7 (USP7) in paediatric leukaemia, which result in decreased deubiquitination activity. Collectively, our results help to define the landscape of mutations in epigenetic regulatory genes in paediatric cancer and yield a valuable new database for investigating the role of epigenetic dysregulations in cancer. Proteins involved in epigenetic regulation are frequently mutated in several paediatric cancers. Here, Huether et al. characterize the somatic mutation frequency across 21 paediatric cancer subtypes by sequencing 633 epigenetic genes in over 1,000 tumours; generating a rich data set for investigating epigenetic dysregulation.

Ikaros protein in ALL The Philadelphia chromosome is a genetic abnormality associated with chronic myelogenous leukaemia and some types of acute lymphoblastic leukaemia. It produces an abnormal signalling protein causing bone marrow cells to multiply uncontrollably. Now Mullighan et al . identify another mutant protein that contributes to this destructive process. Over 80% of a group of acute lymphoblastic leukaemia patients with the Philadelphia chromosome also lacked the gene IKZF1 . The protein encoded by this gene is Ikaros, a lymphoid cell-specific transcription factor. The loss of Ikaros activity seems to cause a crucial malfunctioning that can contribute to the development of acute lymphoblastic leukaemia in the presence of the Philadelphia lesion. This paper reports the identification of high-frequency deletions in the Ikaros gene in acute lymphoblastic leukaemia cases that are characterized by BCR-ABL1 translocations. In contrast, BCR-ABL1 CML is not associated with Ikaros deletions in chronic phase patients, but are often acquired during progression to blast crisis. These deletions lead to expression of altered transcripts. In contrast to previous models suggesting that these transcripts result from aberrant alternative splicing, it is shown that the deletions found are due to aberrant RAG-mediated recombination. The Philadelphia chromosome, a chromosomal abnormality that encodes BCR–ABL1, is the defining lesion of chronic myelogenous leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL) 1 , 2 , 3 . To define oncogenic lesions that cooperate with BCR–ABL1 to induce ALL, we performed a genome-wide analysis of diagnostic leukaemia samples from 304 individuals with ALL, including 43 BCR–ABL1 B-progenitor ALLs and 23 CML cases. IKZF1 (encoding the transcription factor Ikaros) was deleted in 83.7% of BCR–ABL1 ALL, but not in chronic-phase CML. Deletion of IKZF1 was also identified as an acquired lesion at the time of transformation of CML to ALL (lymphoid blast crisis). The IKZF1 deletions resulted in haploinsufficiency, expression of a dominant-negative Ikaros isoform, or the complete loss of Ikaros expression. Sequencing of IKZF1 deletion breakpoints suggested that aberrant RAG-mediated recombination is responsible for the deletions. These findings suggest that genetic lesions resulting in the loss of Ikaros function are an important event in the development of BCR–ABL1 ALL.

Chromosomal aberrations are a hallmark of acute lymphoblastic leukaemia (ALL) but alone fail to induce leukaemia. To identify cooperating oncogenic lesions, we performed a genome-wide analysis of leukaemic cells from 242 paediatric ALL patients using high-resolution, single-nucleotide polymorphism arrays and genomic DNA sequencing. Our analyses revealed deletion, amplification, point mutation and structural rearrangement in genes encoding principal regulators of B lymphocyte development and differentiation in 40% of B-progenitor ALL cases. The PAX5 gene was the most frequent target of somatic mutation, being altered in 31.7% of cases. The identified PAX5 mutations resulted in reduced levels of PAX5 protein or the generation of hypomorphic alleles. Deletions were also detected in TCF3 (also known as E2A ), EBF1 , LEF1 , IKZF1 ( IKAROS ) and IKZF3 ( AIOLOS ). These findings suggest that direct disruption of pathways controlling B-cell development and differentiation contributes to B-progenitor ALL pathogenesis. Moreover, these data demonstrate the power of high-resolution, genome-wide approaches to identify new molecular lesions in cancer. Cancer genomics High-resolution genome-wide profiling of DNA copy number abnormalities using SNP microarrays has been used to identify genetic lesions associated with acute lymphoblastic leukaemia, the most common cancer of childhood. The analysis of leukaemic blast cells from 242 patients revealed deletion, amplification, point mutation and structural rearrangement in genes encoding key regulators of B lymphocyte development in 40% of B-progenitor acute lymphoblastic leukaemia cases. PAX5 was the most frequent target. The data highlight small-molecule inducers of differentiation that can by-pass the block caused by these genetic lesions as a focus for research in new therapeutics, and more generally demonstrate the potential for similar genome-wide approaches as a means of identifying new molecular lesions in cancer. The cover image shows fluorescence in situ hybridization of leukaemia cells showing fusion of PAX5 at chromosome 9p13 (red), to ZNF521 at 18q11.2 (green). A genome-wide, high-resolution single nucleotide polymorphism array analysis of a large number of acute lymphoblastic leukaemia samples identifies a number of genomic changes. These include alterations in the genes encoding Pax5 and other regulators of B-cell development and differentiation.

About 20% of children with acute lymphoblastic leukemia (ALL) that arises from a B-cell progenitor have a relapse after induction chemotherapy. This study investigated genetic abnormalities in high-risk and low-risk ALL. Copy-number variations and mutations of IKZF1 were reproducibly found in high-risk ALL. The evidence suggests that the loss of function of IKZF1 has a role in resistance to chemotherapy in ALL. This study investigated genetic abnormalities in children with high-risk and low-risk acute lymphoblastic leukemia (ALL). The evidence suggests that the loss of function of IKZF1 has a role in resistance to chemotherapy in ALL. Cure rates among children with acute lymphoblastic leukemia (ALL) now exceed 80%, 1 but current therapies have substantial toxic effects, and up to 20% of patients with ALL have a relapse after initial therapy. 2 Risk stratification in B-cell–progenitor ALL is based on a number of recurring chromosomal abnormalities, including hyperdiploidy, hypodiploidy, translocations t(12;21)( ETV6-RUNX1 ), t(9;22)( BCR-ABL1 ), and t(1;19)( TCF3-PBX1 ), and rearrangement of the mixed-lineage leukemia ( MLL ) gene. Treatment failure is common in BCR-ABL1– rearranged and MLL- rearranged ALL, but relapse occurs in all subtypes. Moreover, the biologic basis of resistance to therapy in ALL is poorly . . .