Pediatric Cancer Variant Pathogenicity Information Exchange (PeCanPIE): a cloud-based platform for curating and classifying germline variants.

Variant interpretation in the era of massively parallel sequencing is challenging. Although many resources and guidelines are available to assist with this task, few integrated end-to-end tools exist. Here, we present the Pediatric Cancer Variant Pathogenicity Information Exchange (PeCanPIE), a web- and cloud-based platform for annotation, identification, and classification of variations in known or putative disease genes. Starting from a set of variants in variant call format (VCF), variants are annotated, ranked by putative pathogenicity, and presented for formal classification using a decision-support interface based on published guidelines from the American College of Medical Genetics and Genomics (ACMG). The system can accept files containing millions of variants and handle single-nucleotide variants (SNVs), simple insertions/deletions (indels), multiple-nucleotide variants (MNVs), and complex substitutions. PeCanPIE has been applied to classify variant pathogenicity in cancer predisposition genes in two large-scale investigations involving >4000 pediatric cancer patients and serves as a repository for the expert-reviewed results. PeCanPIE was originally developed for pediatric cancer but can be easily extended for use for nonpediatric cancers and noncancer genetic diseases. Although PeCanPIE's web-based interface was designed to be accessible to non-bioinformaticians, its back-end pipelines may also be run independently on the cloud, facilitating direct integration and broader adoption. PeCanPIE is publicly available and free for research use.

Contrasting roles of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, 'epigenetic' drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors.

C11orf95-RELA fusions drive oncogenic NF-κB signalling in ependymoma.

Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells--the cell of origin of ependymoma--to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.

Germline Mutations in Predisposition Genes in Pediatric Cancer.

BACKGROUND: The prevalence and spectrum of predisposing mutations among children and adolescents with cancer are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis, direct patient care, and enable genetic counseling of patients and families. METHODS: In 1120 patients younger than 20 years of age, we sequenced the whole genomes (in 595 patients), whole exomes (in 456), or both (in 69). We analyzed the DNA sequences of 565 genes, including 60 that have been associated with autosomal dominant cancer-predisposition syndromes, for the presence of germline mutations. The pathogenicity of the mutations was determined by a panel of medical experts with the use of cancer-specific and locus-specific genetic databases, the medical literature, computational predictions, and second hits identified in the tumor genome. The same approach was used to analyze data from 966 persons who did not have known cancer in the 1000 Genomes Project, and a similar approach was used to analyze data from an autism study (from 515 persons with autism and 208 persons without autism). RESULTS: Mutations that were deemed to be pathogenic or probably pathogenic were identified in 95 patients with cancer (8.5%), as compared with 1.1% of the persons in the 1000 Genomes Project and 0.6% of the participants in the autism study. The most commonly mutated genes in the affected patients were TP53 (in 50 patients), APC (in 6), BRCA2 (in 6), NF1 (in 4), PMS2 (in 4), RB1 (in 3), and RUNX1 (in 3). A total of 18 additional patients had protein-truncating mutations in tumor-suppressor genes. Of the 58 patients with a predisposing mutation and available information on family history, 23 (40%) had a family history of cancer. CONCLUSIONS: Germline mutations in cancer-predisposing genes were identified in 8.5% of the children and adolescents with cancer. Family history did not predict the presence of an underlying predisposition syndrome in most patients. (Funded by the American Lebanese Syrian Associated Charities and the National Cancer Institute.).

Novel mutations target distinct subgroups of medulloblastoma.

Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood. One-hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma; several target distinct components of the epigenetic machinery in different disease subgroups, such as regulators of H3K27 and H3K4 trimethylation in subgroups 3 and 4 (for example, KDM6A and ZMYM3), and CTNNB1-associated chromatin re-modellers in WNT-subgroup tumours (for example, SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.

The genetic basis of early T-cell precursor acute lymphoblastic leukaemia.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

Targetable kinase-activating lesions in Ph-like acute lymphoblastic leukemia.

BACKGROUND: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR-ABL1-positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults. METHODS: We performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL. We examined the functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors in mouse pre-B cells and xenografts of human Ph-like ALL. RESULTS: Ph-like ALL increased in frequency from 10% among children with standard-risk ALL to 27% among young adults with ALL and was associated with a poor outcome. Kinase-activating alterations were identified in 91% of patients with Ph-like ALL; rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB, PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions resulted in cytokine-independent proliferation and activation of phosphorylated STAT5. Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were sensitive to ruxolitinib, and the ETV6-NTRK3 fusion was sensitive to crizotinib. CONCLUSIONS: Ph-like ALL was found to be characterized by a range of genomic alterations that activate a limited number of signaling pathways, all of which may be amenable to inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL are needed to assess whether adding tyrosine kinase inhibitors to current therapy will improve the survival of patients with this type of leukemia. (Funded by the American Lebanese Syrian Associated Charities and others.).

Genetic alterations in uncommon low-grade neuroepithelial tumors: BRAF, FGFR1, and MYB mutations occur at high frequency and align with morphology.

Low-grade neuroepithelial tumors (LGNTs) are diverse CNS tumors presenting in children and young adults, often with a history of epilepsy. While the genetic profiles of common LGNTs, such as the pilocytic astrocytoma and 'adult-type' diffuse gliomas, are largely established, those of uncommon LGNTs remain to be defined. In this study, we have used massively parallel sequencing and various targeted molecular genetic approaches to study alterations in 91 LGNTs, mostly from children but including young adult patients. These tumors comprise dysembryoplastic neuroepithelial tumors (DNETs; n = 22), diffuse oligodendroglial tumors (d-OTs; n = 20), diffuse astrocytomas (DAs; n = 17), angiocentric gliomas (n = 15), and gangliogliomas (n = 17). Most LGNTs (84 %) analyzed by whole-genome sequencing (WGS) were characterized by a single driver genetic alteration. Alterations of FGFR1 occurred frequently in LGNTs composed of oligodendrocyte-like cells, being present in 82 % of DNETs and 40 % of d-OTs. In contrast, a MYB-QKI fusion characterized almost all angiocentric gliomas (87 %), and MYB fusion genes were the most common genetic alteration in DAs (41 %). A BRAF:p.V600E mutation was present in 35 % of gangliogliomas and 18 % of DAs. Pathogenic alterations in FGFR1/2/3, BRAF, or MYB/MYBL1 occurred in 78 % of the series. Adult-type d-OTs with an IDH1/2 mutation occurred in four adolescents, the youngest aged 15 years at biopsy. Despite a detailed analysis, novel genetic alterations were limited to two fusion genes, EWSR1-PATZ1 and SLMAP-NTRK2, both in gangliogliomas. Alterations in BRAF, FGFR1, or MYB account for most pathogenic alterations in LGNTs, including pilocytic astrocytomas, and alignment of these genetic alterations and cytologic features across LGNTs has diagnostic implications. Additionally, therapeutic options based upon targeting the effects of these alterations are already in clinical trials.

Tyrosine kinome sequencing of pediatric acute lymphoblastic leukemia: a report from the Children's Oncology Group TARGET Project.

One recently identified subtype of pediatric B-precursor acute lymphoblastic leukemia (ALL) has been termed BCR-ABL1-like or Ph-like because of similarity of the gene expression profile to BCR-ABL1 positive ALL suggesting the presence of lesions activating tyrosine kinases, frequent alteration of IKZF1, and poor outcome. Prior studies demonstrated that approximately half of these patients had genomic lesions leading to CRLF2 overexpression, with half of such cases harboring somatic mutations in the Janus kinases JAK1 and JAK2. To determine whether mutations in other tyrosine kinases might also occur in ALL, we sequenced the tyrosine kinome and downstream signaling genes in 45 high-risk pediatric ALL cases with either a Ph-like gene expression profile or other alterations suggestive of activated kinase signaling. Aside from JAK mutations and 1 FLT3 mutation, no somatic mutations were found in any other tyrosine kinases, suggesting that alternative mechanisms are responsible for activated kinase signaling in high-risk ALL.

A hybrid method of correcting CBCT for proton range estimation with deep learning and deformable image registration.

Objective. This study aimed to develop a novel method for generating synthetic CT (sCT) from cone-beam CT (CBCT) of the abdomen/pelvis with bowel gas pockets to facilitate estimation of proton ranges.Approach. CBCT, the same-day repeat CT, and the planning CT (pCT) of 81 pediatric patients were used for training (n= 60), validation (n= 6), and testing (n= 15) of the method. The proposed method hybridizes unsupervised deep learning (CycleGAN) and deformable image registration (DIR) of the pCT to CBCT. The CycleGAN and DIR are respectively applied to generate the geometry-weighted (high spatial-frequency) and intensity-weighted (low spatial-frequency) components of the sCT, thereby each process deals with only the component weighted toward its strength. The resultant sCT is further improved in bowel gas regions and other tissues by iteratively feeding back the sCT to adjust incorrect DIR and by increasing the contribution of the deformed pCT in regions of accurate DIR.Main results. The hybrid sCT was more accurate than deformed pCT and CycleGAN-only sCT as indicated by the smaller mean absolute error in CT numbers (28.7 ± 7.1 HU versus 38.8 ± 19.9 HU/53.2 ± 5.5 HU;P≤ 0.012) and higher Dice similarity of the internal gas regions (0.722 ± 0.088 versus 0.180 ± 0.098/0.659 ± 0.129;P≤ 0.002). Accordingly, the hybrid method resulted in more accurate proton range for the beams intersecting gas pockets (11 fields in 6 patients) than the individual methods (the 90th percentile error in 80% distal fall-off, 1.8 ± 0.6 mm versus 6.5 ± 7.8 mm/3.7 ± 1.5 mm;P≤ 0.013). The gamma passing rates also showed a significant dosimetric advantage by the hybrid method (99.7 ± 0.8% versus 98.4 ± 3.1%/98.3 ± 1.8%;P≤ 0.007).Significance. The hybrid method significantly improved the accuracy of sCT and showed promises in CBCT-based proton range verification and adaptive replanning of abdominal/pelvic proton therapy even when gas pockets are present in the beam path.

Setup Uncertainty of Pediatric Brain Tumor Patients Receiving Proton Therapy: A Prospective Study.

This study quantifies setup uncertainty in brain tumor patients who received image-guided proton therapy. Patients analyzed include 165 children, adolescents, and young adults (median age at radiotherapy: 9 years (range: 10 months to 24 years); 80 anesthetized and 85 awake) enrolled in a single-institution prospective study from 2020 to 2023. Cone-beam computed tomography (CBCT) was performed daily to calculate and correct manual setup errors, once per course after setup correction to measure residual errors, and weekly after treatments to assess intrafractional motion. Orthogonal radiographs were acquired consecutively with CBCT for paired comparisons of 40 patients. Translational and rotational errors were converted from 6 degrees of freedom to a scalar by a statistical approach that considers the distance from the target to the isocenter. The 95th percentile of setup uncertainty was reduced by daily CBCT from 10 mm (manual positioning) to 1-1.5 mm (after correction) and increased to 2 mm by the end of fractional treatment. A larger variation existed between the roll corrections reported by radiographs vs. CBCT than for pitch and yaw, while there was no statistically significant difference in translational variation. A quantile mixed regression model showed that the 95th percentile of intrafractional motion was 0.40 mm lower for anesthetized patients (p=0.0016). Considering additional uncertainty in radiation-imaging isocentricity, the commonly used total plan robustness of 3 mm against positional uncertainty would be appropriate for our study cohort.

Association of age at diagnosis and genetic mutations in patients with neuroblastoma.

CONTEXT: Neuroblastoma is diagnosed over a wide age range from birth through young adulthood, and older age at diagnosis is associated with a decline in survivability. OBJECTIVE: To identify genetic mutations that are associated with age at diagnosis in patients with metastatic neuroblastoma. DESIGN, SETTING, AND PATIENTS: Whole genome sequencing was performed on DNA from diagnostic tumors and their matched germlines from 40 patients with metastatic neuroblastoma obtained between 1987 and 2009. Age groups at diagnosis included infants (0-<18 months), children (18 months-<12 years), and adolescents and young adults (≥12 years). To confirm the findings from this discovery cohort, validation testing using tumors from an additional 64 patients obtained between 1985 and 2009 also was performed. Formalin-fixed, paraffin-embedded tumor tissue was used for immunohistochemistry and fluorescence in situ hybridization. Telomere lengths were analyzed using whole genome sequencing data, quantitative polymerase chain reaction, and fluorescent in situ hybridization. MAIN OUTCOME MEASURE: Somatic recurrent mutations in tumors from patients with neuroblastoma correlated with the age at diagnosis and telomere length. RESULTS: In the discovery cohort (n = 40), mutations in the ATRX gene were identified in 100% (95% CI, 50%-100%) of tumors from patients in the adolescent and young adult group (5 of 5), in 17% (95% CI, 7%-36%) of tumors from children (5 of 29), and 0% (95% CI, 0%-40%) of tumors from infants (0 of 6). In the validation cohort (n = 64), mutations in the ATRX gene were identified in 33% (95% CI, 17%-54%) of tumors from patients in the adolescent and young adult group (9 of 27), in 16% (95% CI, 6%-35%) of tumors from children (4 of 25), and in 0% (95% CI, 0%-24%) of tumors from infants (0 of 12). In both cohorts (N = 104), mutations in the ATRX gene were identified in 44% (95% CI, 28%-62%) of tumors from patients in the adolescent and young adult group (14 of 32), in 17% (95% CI, 9%-29%) of tumors from children (9 of 54), and in 0% (95% CI, 0%-17%) of tumors from infants (0 of 18). ATRX mutations were associated with an absence of the ATRX protein in the nucleus and with long telomeres. CONCLUSION: ATRX mutations were associated with age at diagnosis in children and young adults with stage 4 neuroblastoma. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00588068.

Pre- and Posttherapy Risk Factors for Vasculopathy in Pediatric Patients With Craniopharyngioma Treated With Surgery and Proton Radiation Therapy.

PURPOSE: Vasculopathy (VAS) is a significant complication associated with radiation therapy in patients treated for brain tumors. We studied the type, location, severity, timing, and resolution of VAS in children with craniopharyngioma treated with proton radiation therapy (PRT) and evaluated predictors of stenosis (STN) using a novel patient and imaging-based modeling approach. METHODS AND MATERIALS: Children with craniopharyngioma (n = 94) were treated with 54 Gy relative biological effectiveness PRT in a clinical trial, NCT01419067. We evaluated VAS type, location, severity, and resolution. VAS events were segmented and related to their location, operative corridor, PRT dose, and vascular territory to facilitate mixed effect logistic regression modeling of spatial predictors of STN events. RESULTS: Forty-five (47.9%) patients had 111 instances of confirmed VAS (pre-PRT n = 37, 33.3%). The median time to post-PRT VAS was 3.41 years (95% confidence interval, 1.86-6.11). STN events were observed post-PRT in 23.4% (n = 22) of patients. Post-PRT VAS was detected by cerebral angiogram in 9.6% (n = 9), severe in 4.3% (n = 4), and compensated on perfusion in 2.1% (n = 2). Revascularization was required for 5 (5.3%) patients. Postsurgical, pre-PRT VAS, and PRT dose to unperturbed vessels were predictive of STN. The effect of PRT on STN was negligible within the surgical corridor. CONCLUSIONS: VAS often precedes PRT and was the strongest predictor of post-PRT STN. The adverse effect of PRT on STN was only apparent in unperturbed vasculature beyond the operative corridor.

Revised clinical and molecular risk strata define the incidence and pattern of failure in medulloblastoma following risk-adapted radiotherapy and dose-intensive chemotherapy: results from a phase III multi-institutional study.

BACKGROUND: We characterize the patterns of progression across medulloblastoma (MB) clinical risk and molecular subgroups from SJMB03, a Phase III clinical trial. METHODS: One hundred and fifty-five pediatric patients with newly diagnosed MB were treated on a prospective, multi-center phase III trial of adjuvant radiotherapy (RT) and dose-intense chemotherapy with autologous stem cell transplant. Craniospinal radiotherapy to 23.4 Gy (average risk, AR) or 36-39.6 Gy (high risk, HR) was followed by conformal RT with a 1 cm clinical target volume to a cumulative dose of 55.8 Gy. Subgroup was determined using 450K DNA methylation. Progression was classified anatomically (primary site failure (PSF) +/- distant failure (DF), or isolated DF), and dosimetrically. RESULTS: Thirty-two patients have progressed (median follow-up 11.0 years (range, 0.3-16.5 y) for patients without progression). Anatomic failure pattern differed by clinical risk (P = .0054) and methylation subgroup (P = .0034). The 5-year cumulative incidence (CI) of PSF was 5.1% and 5.6% in AR and HR patients, respectively (P = .92), and did not differ across subgroups (P = .15). 5-year CI of DF was 7.1% vs. 28.1% for AR vs. HR (P = .0003); and 0% for WNT, 15.3% for SHH, 32.9% for G3, and 9.7% for G4 (P = .0024). Of 9 patients with PSF, 8 were within the primary site RT field and 4 represented SHH tumors. CONCLUSIONS: The low incidence of PSF following conformal primary site RT is comparable to prior studies using larger primary site or posterior fossa boost volumes. Distinct anatomic failure patterns across MB subgroups suggest subgroup-specific treatment strategies should be considered.

Genomes for Kids: The Scope of Pathogenic Mutations in Pediatric Cancer Revealed by Comprehensive DNA and RNA Sequencing.

Genomic studies of pediatric cancer have primarily focused on specific tumor types or high-risk disease. Here, we used a three-platform sequencing approach, including whole-genome sequencing (WGS), whole-exome sequencing (WES), and RNA sequencing (RNA-seq), to examine tumor and germline genomes from 309 prospectively identified children with newly diagnosed (85%) or relapsed/refractory (15%) cancers, unselected for tumor type. Eighty-six percent of patients harbored diagnostic (53%), prognostic (57%), therapeutically relevant (25%), and/or cancer-predisposing (18%) variants. Inclusion of WGS enabled detection of activating gene fusions and enhancer hijacks (36% and 8% of tumors, respectively), small intragenic deletions (15% of tumors), and mutational signatures revealing of pathogenic variant effects. Evaluation of paired tumor-normal data revealed relevance to tumor development for 55% of pathogenic germline variants. This study demonstrates the power of a three-platform approach that incorporates WGS to interrogate and interpret the full range of genomic variants across newly diagnosed as well as relapsed/refractory pediatric cancers. SIGNIFICANCE: Pediatric cancers are driven by diverse genomic lesions, and sequencing has proven useful in evaluating high-risk and relapsed/refractory cases. We show that combined WGS, WES, and RNA-seq of tumor and paired normal tissues enables identification and characterization of genetic drivers across the full spectrum of pediatric cancers. This article is highlighted in the In This Issue feature, p. 2945.

Defining Optimal Target Volumes of Conformal Radiation Therapy for Diffuse Intrinsic Pontine Glioma.

PURPOSE: Optimal radiation therapy (RT) target margins for diffuse intrinsic pontine glioma (DIPG) are unknown. We sought to define disease progression patterns in a contemporary cohort treated with conformal RT using different clinical target volume (CTV) margins. METHODS AND MATERIALS: We reviewed 105 patients with newly diagnosed DIPG treated with conformal conventionally fractionated RT at our institution from 2006 to 2014. CTV margins were classified as standard (1 cm) for 60 patients and extended (2-3 cm) for 45 patients. Survival and cumulative incidence of progression in treatment groups were compared by log-rank and Gray's tests, respectively. Cox proportional hazard models identified predictors of survival. RESULTS: For 97 patients evaluated with magnetic resonance imaging at progression, the cumulative incidences of isolated local, isolated distant, and synchronous disease progression at 1 year were 62.6%, 12.3%, and 7.2%, respectively, and did not differ significantly according to the CTV margin. Central dosimetric progression (Vprogression95% ≥95%) was observed in 80 of 81 evaluable patients. Median progression-free survival and overall survival (OS) were 7.6 months (95% confidence interval, 6.9-8.2) and 11.3 months (95% confidence interval, 10.0-12.8), respectively, and did not differ significantly according to margin status. DIPG survival prediction risk group (standard vs high, P = .02; intermediate vs high, P = .009) and development of distant metastasis (P = .003) were independent predictors of OS. For the 41 patients (39%) with a pathologic diagnosis, H3.3 K27M mutation was associated with shorter OS (hazard ratio [HR], 0.41; P =.02), whereas H3.1 K27M and ACVR1 mutations were associated with longer OS (HR, 3.56; P =.004 and HR, 2.58; P =.04, respectively). CONCLUSIONS: All patients who experienced local failure showed progression within the high-dose volume, and there was no apparent survival or tumor-control benefit to extending the CTV margins beyond 1 cm. Given the increasing use of reirradiation, standardizing the CTV margin to 1 cm may improve retreatment tolerance.

Clinical cancer genomic profiling by three-platform sequencing of whole genome, whole exome and transcriptome.

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.

The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias.

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.

Outcomes of children with BCR-ABL1­like acute lymphoblastic leukemia treated with risk-directed therapy based on the levels of minimal residual disease.

PURPOSE: BCR-ABL1­like acute lymphoblastic leukemia (ALL) is a recently identified B-cell ALL (B-ALL)subtype with poor outcome that exhibits a gene expression profile similar to BCR-ABL1-positive ALL but lacks the BCR-ABL1 fusion protein. We examined the outcome of children with BCR-ABL1­like ALL treated with risk-directed therapy based on minimal residual disease (MRD) levels during remission induction. PATIENTS AND METHODS: Among 422 patients with B-ALL enrolled onto the Total Therapy XV study between 2000 and 2007, 344 had adequate samples for gene expression profiling. Next-generation sequencing and/or analysis of genes known to be altered in B-ALL were performed in patients with BCR-ABL1­likeALL who had available material. Outcome was compared between patients with and those without BCR-ABL1­like ALL. RESULTS: Forty (11.6%) of the 344 patients had BCR-ABL1­like ALL. They were significantly more likely to be male, have Down syndrome, and have higher MRD levels on day 19 and at the end of induction than did other patients with B-ALL. Among 25 patients comprehensively studied for genetic abnormalities, 11 harbored a genomic rearrangement of CRLF2, six had fusion transcripts responsive to ABL tyrosine kinase inhibitors or JAK inhibitors, and seven had mutations involving the Ras signaling pathway. There were no significant differences in event-free survival (90.0% +/- 4.7% [SE] v. 88.4% +/- .9% at 5 years; P = .41or in overall survival (92.5% +/- 4.2% v. 95.1% +/- 1.3% at 5 years; P = .41) between patients with and without BCR-ABL1­like ALL. CONCLUSION: Patients who have BCR-ABL1­like ALL with poor initial treatment response can be salvaged with MRD-based risk-directed therapy and may benefit from identification of kinase-activating lesions for targeted therapies.

RB1 gene inactivation by chromothripsis in human retinoblastoma.

Retinoblastoma is a rare childhood cancer of the developing retina. Most retinoblastomas initiate with biallelic inactivation of the RB1 gene through diverse mechanisms including point mutations, nucleotide insertions, deletions, loss of heterozygosity and promoter hypermethylation. Recently, a novel mechanism of retinoblastoma initiation was proposed. Gallie and colleagues discovered that a small proportion of retinoblastomas lack RB1 mutations and had MYCN amplification [1]. In this study, we identified recurrent chromosomal, regional and focal genomic lesions in 94 primary retinoblastomas with their matched normal DNA using SNP 6.0 chips. We also analyzed the RB1 gene mutations and compared the mechanism of RB1 inactivation to the recurrent copy number variations in the retinoblastoma genome. In addition to the previously described focal amplification of MYCN and deletions in RB1 and BCOR, we also identified recurrent focal amplification of OTX2, a transcription factor required for retinal photoreceptor development. We identified 10 retinoblastomas in our cohort that lacked RB1 point mutations or indels. We performed whole genome sequencing on those 10 tumors and their corresponding germline DNA. In one of the tumors, the RB1 gene was unaltered, the MYCN gene was amplified and RB1 protein was expressed in the nuclei of the tumor cells. In addition, several tumors had complex patterns of structural variations and we identified 3 tumors with chromothripsis at the RB1 locus. This is the first report of chromothripsis as a mechanism for RB1 gene inactivation in cancer.