Circular extrachromosomal DNA promotes tumor heterogeneity in high-risk medulloblastoma.

Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. A subset of tumors harbored multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative 'enhancer rewiring' events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA.

Targeted in silico characterization of fusion transcripts in tumor and normal tissues via FusionInspector.

Here, we present FusionInspector for in silico characterization and interpretation of candidate fusion transcripts from RNA sequencing (RNA-seq) and exploration of their sequence and expression characteristics. We applied FusionInspector to thousands of tumor and normal transcriptomes and identified statistical and experimental features enriched among biologically impactful fusions. Through clustering and machine learning, we identified large collections of fusions potentially relevant to tumor and normal biological processes. We show that biologically relevant fusions are enriched for relatively high expression of the fusion transcript, imbalanced fusion allelic ratios, and canonical splicing patterns, and are deficient in sequence microhomologies between partner genes. We demonstrate that FusionInspector accurately validates fusion transcripts in silico and helps characterize numerous understudied fusions in tumor and normal tissue samples. FusionInspector is freely available as open source for screening, characterization, and visualization of candidate fusions via RNA-seq, and facilitates transparent explanation and interpretation of machine-learning predictions and their experimental sources.

3D genome mapping identifies subgroup-specific chromosome conformations and tumor-dependency genes in ependymoma.

Ependymoma is a tumor of the brain or spinal cord. The two most common and aggressive molecular groups of ependymoma are the supratentorial ZFTA-fusion associated and the posterior fossa ependymoma group A. In both groups, tumors occur mainly in young children and frequently recur after treatment. Although molecular mechanisms underlying these diseases have recently been uncovered, they remain difficult to target and innovative therapeutic approaches are urgently needed. Here, we use genome-wide chromosome conformation capture (Hi-C), complemented with CTCF and H3K27ac ChIP-seq, as well as gene expression and DNA methylation analysis in primary and relapsed ependymoma tumors, to identify chromosomal conformations and regulatory mechanisms associated with aberrant gene expression. In particular, we observe the formation of new topologically associating domains ('neo-TADs') caused by structural variants, group-specific 3D chromatin loops, and the replacement of CTCF insulators by DNA hyper-methylation. Through inhibition experiments, we validate that genes implicated by these 3D genome conformations are essential for the survival of patient-derived ependymoma models in a group-specific manner. Thus, this study extends our ability to reveal tumor-dependency genes by 3D genome conformations even in tumors that lack targetable genetic alterations.

Variant Review with the Integrative Genomics Viewer.

Manual review of aligned reads for confirmation and interpretation of variant calls is an important step in many variant calling pipelines for next-generation sequencing (NGS) data. Visual inspection can greatly increase the confidence in calls, reduce the risk of false positives, and help characterize complex events. The Integrative Genomics Viewer (IGV) was one of the first tools to provide NGS data visualization, and it currently provides a rich set of tools for inspection, validation, and interpretation of NGS datasets, as well as other types of genomic data. Here, we present a short overview of IGV's variant review features for both single-nucleotide variants and structural variants, with examples from both cancer and germline datasets. IGV is freely available at https://www.igv.org Cancer Res; 77(21); e31-34. ©2017 AACR.

Cohesin Loss Eliminates All Loop Domains.

The human genome folds to create thousands of intervals, called "contact domains," that exhibit enhanced contact frequency within themselves. "Loop domains" form because of tethering between two loci-almost always bound by CTCF and cohesin-lying on the same chromosome. "Compartment domains" form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.

Discovery and saturation analysis of cancer genes across 21 tumour types.

Although a few cancer genes are mutated in a high proportion of tumours of a given type (>20%), most are mutated at intermediate frequencies (2-20%). To explore the feasibility of creating a comprehensive catalogue of cancer genes, we analysed somatic point mutations in exome sequences from 4,742 human cancers and their matched normal-tissue samples across 21 cancer types. We found that large-scale genomic analysis can identify nearly all known cancer genes in these tumour types. Our analysis also identified 33 genes that were not previously known to be significantly mutated in cancer, including genes related to proliferation, apoptosis, genome stability, chromatin regulation, immune evasion, RNA processing and protein homeostasis. Down-sampling analysis indicates that larger sample sizes will reveal many more genes mutated at clinically important frequencies. We estimate that near-saturation may be achieved with 600-5,000 samples per tumour type, depending on background mutation frequency. The results may help to guide the next stage of cancer genomics.

Mutations causing medullary cystic kidney disease type 1 lie in a large VNTR in MUC1 missed by massively parallel sequencing.

Although genetic lesions responsible for some mendelian disorders can be rapidly discovered through massively parallel sequencing of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing and de novo assembly did we find that each of six families with MCKD1 harbors an equivalent but apparently independently arising mutation in sequence markedly under-represented in massively parallel sequencing data: the insertion of a single cytosine in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (∼1.5-5 kb), GC-rich (>80%) coding variable-number tandem repeat (VNTR) sequence in the MUC1 gene encoding mucin 1. These results provide a cautionary tale about the challenges in identifying the genes responsible for mendelian, let alone more complex, disorders through massively parallel sequencing.

Reconstruction of the posterolateral corner of the knee.

PURPOSE: To describe a technique for reconstruction of the posterolateral corner (PLC) of the knee and report the results of a prospective series of patients. TYPE OF STUDY: Case series. METHODS: Twenty-two patients with PLC injuries underwent reconstruction; 15 patients had multiligamentous knee injuries and 7 had isolated PLC injuries. We used the modified 2-tailed technique that reconstructs the popliteofibular ligament and fibulocollateral ligament. The technique includes use of an allograft tendon placed through transtibial and transfibular bone tunnels and around a screw on the lateral femoral condyle. All patients have been followed-up prospectively with clinical examinations, Lysholm knee scores, KT-2000 ligament arthrometer examinations, and evaluation of work and recreational functional status. RESULTS: There were 15 male and 7 female patients (mean age, 32 years; range, 17 to 55 years). Follow-up was a minimum of 24 months (mean, 29.5 months; range, 24 to 38 months). Mean range of motion is extension of 0.2 degrees (range, 0 degrees to 5 degrees) and flexion of 133.4 degrees (80 degrees to 144 degrees). The range of motion for the multiligamentous knees was 0.3 degrees to 129 degrees compared with 0 degrees to 143 degrees for knees with isolated corner injuries. Mean Lysholm knee scores were 90 for the entire group with a score of 92 for the multiligamentous knees and 88 for the isolated corners. Stability was clinically graded on a scale of 0 to 3 for both varus stress and external rotation, with a score of 2 or 3 indicating a failed PLC reconstruction. The mean score for varus stress was 0.2 for the whole group, with 0.3 in the multiligamentous knee and 0.1 for the isolated injuries. Similarly, the mean score for external rotation was 0.4, with a 0.5 for multiligamentous knee and 0.3 for isolated PLC injuries. There were 2 failures in the multiligamentous knee injury group (13%), compared with no failures in the isolated PLC group. The failure rate for the whole study was 9%. CONCLUSIONS: Reconstruction of the PLC using an allograft reconstruction of the popliteus, popliteofibular, and fibulocollateral ligaments yielded a stable reconstruction with excellent functional results. Predictably, range of motion and incidence of failure were both better for patients with isolated PLC injuries than for those with multiligamentous knees. Both groups, however, showed excellent overall functional results. LEVEL OF EVIDENCE: Level IV, case series.

Vascular injuries in knee dislocations: the role of physical examination in determining the need for arteriography.

BACKGROUND: Popliteal artery injury is frequently associated with knee dislocation following blunt trauma, an injury that is being seen with increasing frequency. The primary purpose of the present study was to evaluate the use of physical examination to determine the need for arteriography in a large series of patients with knee dislocation. The secondary purpose was to evaluate the correlation between physical examination findings and clinically important vascular injury in the subgroup of patients who underwent arteriography. METHODS: One hundred and thirty consecutive patients (138 knees) who had sustained an acute multiligamentous knee injury were evaluated at our level-1 trauma center between August 1996 and May 2002 and were included in a prospective outcome study. Four patients (four knees) were lost to follow-up, leaving 126 patients (134 knees) available for inclusion in the study. The results of the physical examination of the vascular status of the extremities were used to determine the need for arteriography. The mean duration of follow-up was nineteen months (range, eight to forty-eight months). Physical examination findings, magnetic resonance imaging findings, and surgical findings were combined to determine the extent of ligamentous damage. RESULTS: Nine patients had flow-limiting popliteal artery damage, for an overall prevalence of 7%. Ten patients had abnormal findings on physical examination, with one patient having a false-positive result and nine having a true-positive result. The knee dislocations in the nine patients with popliteal artery damage were classified, according to the Wascher modification of the Schenck system, as KD-III (one knee), KD-IV (seven knees), and KD-V (one knee). CONCLUSIONS: Selective arteriography based on serial physical examinations is a safe and prudent policy following knee dislocation. There is a strong correlation between the results of physical examination and the need for arteriography. Increased vigilance may be justified in the case of a patient with a KD-IV dislocation, for whom serial examinations should continue for at least forty-eight hours.