A multi-platform reference for somatic structural variation detection.

Accurate detection of somatic structural variation (SV) in cancer genomes remains a challenging problem. This is in part due to the lack of high-quality, gold-standard datasets that enable the benchmarking of experimental approaches and bioinformatic analysis pipelines. Here, we performed somatic SV analysis of the paired melanoma and normal lymphoblastoid COLO829 cell lines using four different sequencing technologies. Based on the evidence from multiple technologies combined with extensive experimental validation, we compiled a comprehensive set of carefully curated and validated somatic SVs, comprising all SV types. We demonstrate the utility of this resource by determining the SV detection performance as a function of tumor purity and sequence depth, highlighting the importance of assessing these parameters in cancer genomics projects. The truth somatic SV dataset as well as the underlying raw multi-platform sequencing data are freely available and are an important resource for community somatic benchmarking efforts.

Fusion transcripts and their genomic breakpoints in polyadenylated and ribosomal RNA-minus RNA sequencing data.

BACKGROUND: Fusion genes are typically identified by RNA sequencing (RNA-seq) without elucidating the causal genomic breakpoints. However, non-poly(A)-enriched RNA-seq contains large proportions of intronic reads that also span genomic breakpoints. RESULTS: We have developed an algorithm, Dr. Disco, that searches for fusion transcripts by taking an entire reference genome into account as search space. This includes exons but also introns, intergenic regions, and sequences that do not meet splice junction motifs. Using 1,275 RNA-seq samples, we investigated to what extent genomic breakpoints can be extracted from RNA-seq data and their implications regarding poly(A)-enriched and ribosomal RNA-minus RNA-seq data. Comparison with whole-genome sequencing data revealed that most genomic breakpoints are not, or minimally, transcribed while, in contrast, the genomic breakpoints of all 32 TMPRSS2-ERG-positive tumours were present at RNA level. We also revealed tumours in which the ERG breakpoint was located before ERG, which co-existed with additional deletions and messenger RNA that incorporated intergenic cryptic exons. In breast cancer we identified rearrangement hot spots near CCND1 and in glioma near CDK4 and MDM2 and could directly associate this with increased expression. Furthermore, in all datasets we find fusions to intergenic regions, often spanning multiple cryptic exons that potentially encode neo-antigens. Thus, fusion transcripts other than classical gene-to-gene fusions are prominently present and can be identified using RNA-seq. CONCLUSION: By using the full potential of non-poly(A)-enriched RNA-seq data, sophisticated analysis can reliably identify expressed genomic breakpoints and their transcriptional effects.

Clinical Validation of Whole Genome Sequencing for Cancer Diagnostics.

Whole genome sequencing (WGS) using fresh-frozen tissue and matched blood samples from cancer patients may become the most complete genetic tumor test. With the increasing availability of small biopsies and the need to screen more number of biomarkers, the use of a single all-inclusive test is preferable over multiple consecutive assays. To meet high-quality diagnostics standards, we optimized and clinically validated WGS sample and data processing procedures, resulting in a technical success rate of 95.6% for fresh-frozen samples with sufficient (≥20%) tumor content. Independent validation of identified biomarkers against commonly used diagnostic assays showed a high sensitivity (recall; 98.5%) and precision (positive predictive value; 97.8%) for detection of somatic single-nucleotide variants and insertions and deletions (across 22 genes), and high concordance for detection of gene amplification (97.0%; EGFR and MET) as well as somatic complete loss (100%; CDKN2A/p16). Gene fusion analysis showed a concordance of 91.3% between DNA-based WGS and an orthogonal RNA-based gene fusion assay. Microsatellite (in)stability assessment showed a sensitivity of 100% with a precision of 94%, and virus detection (human papillomavirus), an accuracy of 100% compared with standard testing. In conclusion, whole genome sequencing has a >95% sensitivity and precision compared with routinely used DNA techniques in diagnostics, and all relevant mutation types can be detected reliably in a single assay.

Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material.

BACKGROUND: Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. METHOD: We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. RESULTS: Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data. CONCLUSION: Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.

GeneBreak: detection of recurrent DNA copy number aberration-associated chromosomal breakpoints within genes.

Development of cancer is driven by somatic alterations, including numerical and structural chromosomal aberrations. Currently, several computational methods are available and are widely applied to detect numerical copy number aberrations (CNAs) of chromosomal segments in tumor genomes. However, there is lack of computational methods that systematically detect structural chromosomal aberrations by virtue of the genomic location of CNA-associated chromosomal breaks and identify genes that appear non-randomly affected by chromosomal breakpoints across (large) series of tumor samples. 'GeneBreak' is developed to systematically identify genes recurrently affected by the genomic location of chromosomal CNA-associated breaks by a genome-wide approach, which can be applied to DNA copy number data obtained by array-Comparative Genomic Hybridization (CGH) or by (low-pass) whole genome sequencing (WGS). First, 'GeneBreak' collects the genomic locations of chromosomal CNA-associated breaks that were previously pinpointed by the segmentation algorithm that was applied to obtain CNA profiles. Next, a tailored annotation approach for breakpoint-to-gene mapping is implemented. Finally, dedicated cohort-based statistics is incorporated with correction for covariates that influence the probability to be a breakpoint gene. In addition, multiple testing correction is integrated to reveal recurrent breakpoint events. This easy-to-use algorithm, 'GeneBreak', is implemented in R ( www.cran.r-project.org) and is available from Bioconductor ( www.bioconductor.org/packages/release/bioc/html/GeneBreak.html).

Effective Therapeutic Intervention and Comprehensive Genetic Analysis of mTOR Signaling in PEComa: A Case Report.

BACKGROUND/AIM: Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal neoplasms. The exact genetic alterations underlying the pathophysiology of PEComas are largely unknown, although it has been shown that activation of the Mammalian target of rapamycin (mTOR) signaling pathway plays a pivotal role. Herein we describe the successful treatment of a patient with metastatic PEComa with the mTOR inhibitor everolimus and a comprehensive analysis to identify mechanisms for response. MATERIALS AND METHODS: Immunohistochemistry, array comparative genomic hybridization (aCGH) and genetic analyses were performed. RESULTS: Immunohistochemistry confirmed constitutive activation of mTOR. aCGH revealed a hyperdiploid karyotype affecting large regions of the genome. Next-generation sequencing did not reveal any tumor-specific mutations in mTOR-related genes. CONCLUSION: Our results show the complexity of determining causal genetic alterations that can predict responsiveness to mTOR inhibition, even for a tumor with a complete remission to this specific treatment.

Targeted next-generation sequencing: a novel diagnostic tool for primary immunodeficiencies.

BACKGROUND: Primary immunodeficiency (PID) disorders are a heterogeneous group of inherited disorders caused by a variety of monogenetic immune defects. Thus far, mutations in more than 170 different genes causing PIDs have been described. A clear genotype-phenotype correlation is often not available, which makes a genetic diagnosis in patients with PIDs complex and laborious. OBJECTIVE: We sought to develop a robust, time-effective, and cost-effective diagnostic method to facilitate a genetic diagnosis in any of 170 known PID-related genes by using next-generation sequencing (NGS). METHODS: We used both targeted array-based and in-solution enrichment combined with a SOLiD sequencing platform and a bioinformatic pipeline developed in house to analyze genetic changes in the DNA of 41 patients with PIDs with known mutations and 26 patients with undiagnosed PIDs. RESULTS: This novel NGS-based method accurately detected point mutations (sensitivity and specificity >99% in covered regions) and exonic deletions (100% sensitivity and specificity). For the 170 genes of interest, the DNA coverage was greater than 20× in 90% to 95%. Nine PID-related genes proved not eligible for evaluation by using this NGS-based method because of inadequate coverage. The NGS method allowed us to make a genetic diagnosis in 4 of 26 patients who lacked a genetic diagnosis despite routine functional and genetic testing. Three of these patients proved to have an atypical presentation of previously described PIDs. CONCLUSION: This novel NGS tool facilitates accurate simultaneous detection of mutations in 161 of 170 known PID-related genes. In addition, these analyses will generate more insight into genotype-phenotype correlations for the different PID disorders.

X-exome sequencing identifies a HDAC8 variant in a large pedigree with X-linked intellectual disability, truncal obesity, gynaecomastia, hypogonadism and unusual face.

BACKGROUND: We present a large Dutch family with seven males affected by a novel syndrome of X-linked intellectual disability, hypogonadism, gynaecomastia, truncal obesity, short stature and recognisable craniofacial manifestations resembling but not identical to Wilson-Turner syndrome. Seven female relatives show a much milder expression of the phenotype. METHODS AND RESULTS: We performed X chromosome exome (X-exome) sequencing in five individuals from this family and identified a novel intronic variant in the histone deacetylase 8 gene (HDAC8), c.164+5G>A, which disturbs the normal splicing of exon 2 resulting in exon skipping, and introduces a premature stop at the beginning of the histone deacetylase catalytic domain. The identified variant completely segregates in this family and was absent in 96 Dutch controls and available databases. Affected female carriers showed a notably skewed X-inactivation pattern in lymphocytes in which the mutated X-chromosome was completely inactivated. CONCLUSIONS: HDAC8 is a member of the protein family of histone deacetylases that play a major role in epigenetic gene silencing during development. HDAC8 specifically controls the patterning of the skull with the mouse HDAC8 knock-out showing craniofacial deformities of the skull. The present family provides the first evidence for involvement of HDAC8 in a syndromic form of intellectual disability.

Dominant missense mutations in ABCC9 cause Cantú syndrome.

Cantú syndrome is characterized by congenital hypertrichosis, distinctive facial features, osteochondrodysplasia and cardiac defects. By using family-based exome sequencing, we identified a de novo mutation in ABCC9. Subsequently, we discovered novel dominant missense mutations in ABCC9 in 14 of the 16 individuals with Cantú syndrome examined. The ABCC9 protein is part of an ATP-dependent potassium (K(ATP)) channel that couples the metabolic state of a cell with its electrical activity. All mutations altered amino acids in or close to the transmembrane domains of ABCC9. Using electrophysiological measurements, we show that mutations in ABCC9 reduce the ATP-mediated potassium channel inhibition, resulting in channel opening. Moreover, similarities between the phenotype of individuals with Cantú syndrome and side effects from the K(ATP) channel agonist minoxidil indicate that the mutations in ABCC9 result in channel opening. Given the availability of ABCC9 antagonists, our findings may have direct implications for the treatment of individuals with Cantú syndrome.

Chromothripsis is a common mechanism driving genomic rearrangements in primary and metastatic colorectal cancer.

BACKGROUND: Structural rearrangements form a major class of somatic variation in cancer genomes. Local chromosome shattering, termed chromothripsis, is a mechanism proposed to be the cause of clustered chromosomal rearrangements and was recently described to occur in a small percentage of tumors. The significance of these clusters for tumor development or metastatic spread is largely unclear. RESULTS: We used genome-wide long mate-pair sequencing and SNP array profiling to reveal that chromothripsis is a widespread phenomenon in primary colorectal cancer and metastases. We find large and small chromothripsis events in nearly every colorectal tumor sample and show that several breakpoints of chromothripsis clusters and isolated rearrangements affect cancer genes, including NOTCH2, EXO1 and MLL3. We complemented the structural variation studies by sequencing the coding regions of a cancer exome in all colorectal tumor samples and found somatic mutations in 24 genes, including APC, KRAS, SMAD4 and PIK3CA. A pairwise comparison of somatic variations in primary and metastatic samples indicated that many chromothripsis clusters, isolated rearrangements and point mutations are exclusively present in either the primary tumor or the metastasis and may affect cancer genes in a lesion-specific manner. CONCLUSIONS: We conclude that chromothripsis is a prevalent mechanism driving structural rearrangements in colorectal cancer and show that a complex interplay between point mutations, simple copy number changes and chromothripsis events drive colorectal tumor development and metastasis.