Loss of heterozygosity (LOH) and copy number alteration (CNA) feature prominently in the somatic genomic landscape of tumors. As such, karyotypic aberrations in cancer genomes have been studied ...extensively to discover novel oncogenes and tumor-suppressor genes. Advances in sequencing technology have enabled the cost-effective detection of tumor genome and transcriptome mutation events at single-base-pair resolution; however, computational methods for predicting segmental regions of LOH in this context are not yet fully explored. Consequently, whole transcriptome, nucleotide-level resolution analysis of monoallelic expression patterns associated with LOH has not yet been undertaken in cancer. We developed a novel approach for inference of LOH from paired tumor/normal sequence data and applied it to a cohort of 23 triple-negative breast cancer (TNBC) genomes. Following extensive benchmarking experiments, we describe the nucleotide-resolution landscape of LOH in TNBC and assess the consequent effect of LOH on the transcriptomes of these tumors using RNA-seq-derived measurements of allele-specific expression. We show that the majority of monoallelic expression in the transcriptomes of triple-negative breast cancer can be explained by genomic regions of LOH and establish an upper bound for monoallelic expression that may be explained by other tumor-specific modifications such as epigenetics or mutations. Monoallelically expressed genes associated with LOH reveal that cell cycle, homologous recombination and actin-cytoskeletal functions are putatively disrupted by LOH in TNBC. Finally, we show how inference of LOH can be used to interpret allele frequencies of somatic mutations and postulate on temporal ordering of mutations in the evolutionary history of these tumors.
Aberrant activation or disruption of autophagy promotes tumorigenesis in various preclinical models of cancer, but whether the autophagy pathway is a target for recurrent molecular alteration in ...human cancer patient samples is unknown. To address this outstanding question, we surveyed 211 human autophagy-associated genes for tumor-related alterations to DNA sequence and RNA expression levels and examined their association with patient survival outcomes in multiple cancer types with sequence data from The Cancer Genome Atlas consortium. We found 3 (RB1CC1/FIP200, ULK4, WDR45/WIPI4) and one (ATG7) core autophagy genes to be under positive selection for somatic mutations in endometrial carcinoma and clear cell renal carcinoma, respectively, while 29 autophagy regulators and pathway interactors, including previously identified KEAP1, NFE2L2, and MTOR, were significantly mutated in 6 of the 11 cancer types examined. Gene expression analyses revealed that GABARAPL1 and MAP1LC3C/LC3C transcripts were less abundant in breast cancer and non-small cell lung cancers than in matched normal tissue controls; ATG4D transcripts were increased in lung squamous cell carcinoma, as were ATG16L2 transcripts in kidney cancer. Unsupervised clustering of autophagy-associated mRNA levels in tumors stratified patient overall survival in 3 of 9 cancer types (acute myeloid leukemia, clear cell renal carcinoma, and head and neck cancer). These analyses provide the first comprehensive resource of recurrently altered autophagy-associated genes in human tumors, and highlight cancer types and subtypes where perturbed autophagy may be relevant to patient overall survival.
Mexico is developing the basis for genomic medicine to improve healthcare of its population. The extensive study of genetic diversity and linkage disequilibrium structure of different populations has ...made it possible to develop tagging and imputation strategies to comprehensively analyze common genetic variation in association studies of complex diseases. We assessed the benefit of a Mexican haplotype map to improve identification of genes related to common diseases in the Mexican population. We evaluated genetic diversity, linkage disequilibrium patterns, and extent of haplotype sharing using genomewide data from Mexican Mestizos from regions with different histories of admixture and particular population dynamics. Ancestry was evaluated by including 1 Mexican Amerindian group and data from the HapMap. Our results provide evidence of genetic differences between Mexican subpopulations that should be considered in the design and analysis of association studies of complex diseases. In addition, these results support the notion that a haplotype map of the Mexican Mestizo population can reduce the number of tag SNPs required to characterize common genetic variation in this population. This is one of the first genomewide genotyping efforts of a recently admixed population in Latin America.
Malignant rhabdoid tumors (MRTs) are rare lethal tumors of childhood that most commonly occur in the kidney and brain. MRTs are driven by SMARCB1 loss, but the molecular consequences of SMARCB1 loss ...in extra-cranial tumors have not been comprehensively described and genomic resources for analyses of extra-cranial MRT are limited. To provide such data, we used whole-genome sequencing, whole-genome bisulfite sequencing, whole transcriptome (RNA-seq) and microRNA sequencing (miRNA-seq), and histone modification profiling to characterize extra-cranial MRTs. Our analyses revealed gene expression and methylation subgroups and focused on dysregulated pathways, including those involved in neural crest development.
•Extra-cranial malignant rhabdoid tumors (MRTs) exhibit molecular heterogeneity•Evidence is presented for epigenetic reprogramming of HOX genes•MRTs exhibit dysregulated expression of genes involved in neural crest development•Dysregulation of oncogenes and tumor suppressor genes is reported
Chun et al. perform integrated molecular analyses of extra-cranial malignant rhabdoid tumors (MRTs) and show that, although SMARCB1 loss drives nearly all MRTs, there are two subgroups of MRTs that are associated with patient age and differentially expressed genes.
Motivation: Next-generation sequencing (NGS) has enabled whole genome and transcriptome single nucleotide variant (SNV) discovery in cancer. NGS produces millions of short sequence reads that, once ...aligned to a reference genome sequence, can be interpreted for the presence of SNVs. Although tools exist for SNV discovery from NGS data, none are specifically suited to work with data from tumors, where altered ploidy and tumor cellularity impact the statistical expectations of SNV discovery. Results: We developed three implementations of a probabilistic Binomial mixture model, called SNVMix, designed to infer SNVs from NGS data from tumors to address this problem. The first models allelic counts as observations and infers SNVs and model parameters using an expectation maximization (EM) algorithm and is therefore capable of adjusting to deviation of allelic frequencies inherent in genomically unstable tumor genomes. The second models nucleotide and mapping qualities of the reads by probabilistically weighting the contribution of a read/nucleotide to the inference of a SNV based on the confidence we have in the base call and the read alignment. The third combines filtering out low-quality data in addition to probabilistic weighting of the qualities. We quantitatively evaluated these approaches on 16 ovarian cancer RNASeq datasets with matched genotyping arrays and a human breast cancer genome sequenced to >40× (haploid) coverage with ground truth data and show systematically that the SNVMix models outperform competing approaches. Availability: Software and data are available at http://compbio.bccrc.ca Contact: sshah@bccrc.ca Supplemantary information: Supplementary data are available at Bioinformatics online.
BackgroundBispecific T-cell engagers (TCEs) activate the immune system to Figureht cancer. TCEs that target peptides displayed on major histocompatibility complexes (pMHC) have shown promise for ...unlocking previously inaccessible intracellular tumor-specific antigens. Bispecific antibodies have the potential to overcome challenges associated with other pMHC-targeting modalities, such as soluble T cell receptors, by eliminating the need for extensive engineering of endogenous TCRs to produce molecules with the requisite affinity and specificity.1 Peptides derived from melanoma-associated antigen 4 (MAGE-A4) are presented by MHC class I (MHC-I) in many solid tumors, but rarely in healthy tissues. However, developing TCE therapies against MAGE-A4 pMHCs presents complex and unique challenges. The tumor-binding arm must bind specifically to small (~10 amino acid) MAGE-A4 peptides that are highly homologous to other proteins in the MAGE family, avoid substantial interactions with the MHC complex, bind tumor cells expressing very low levels of the target, and work in concert with the CD3-binding arm to activate T cells.To address these challenges, we have developed a technology platform to discover optimal TCEs that combines hundreds of diverse, fully human CD3-binding antibodies with antibody discovery, engineering, functional screening, and development capabilities. We discovered diverse and developable human antibodies that showed high specificity and affinity to a human MAGE-A4 peptide presented on MHC-I (HLA:02*01).We strategically selected and paired a panel of these MAGE-A4-pMHC-binding antibodies with diverse CD3-binders and used our high-throughput characterization platform to identify TCEs with optimal specificity and functional profiles.MethodsWe used our OrthoMab™ multispecifics platform and high-throughput expression to generate more than 400 MAGE-A4-pMHCxCD3 TCEs in bispecific and multispecific formats, and measured purity by mass spectrometry, aSEC, and CE-SDS. We assessed the functional activity of these TCEs in high-throughput T cell cytotoxicity and cytokine release assays. We investigated potential off-target cross-reactivity in MAGE-A4-pMHC-binding parental antibodies with a rationally designed screening library based on X-scan binding data. In-depth binding structural and kinetic assessments were also performed to map antibody-epitope interactions.ResultsWe identified a panel of MAGE-A4-pMHC-specific TCEs that induce T-cell dependent cellular cytotoxicity while maintaining low cytokine release in MAGE-A4-positive tumor cell lines. We mapped multiple binding parameters including target specificity, kinetics, and affinity to the functional properties of TCEs in different structural formats to identify candidates for further development.ConclusionsWe have identified a panel of functional and specific MAGE-A4-pMHCxCD3 TCEs as potential immunotherapies against solid tumors.ReferenceHolland CJ, Crean RM, Pentier JM, et al. Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA. J Clin Invest. 2020;130(5):2673–2688.
The Basal Cell Nevus Syndrome, an autosomal dominant condition, manifests in a multisystemic way, predominantly affecting the jaws. The presence of multiple Keratocysts at an early age is a key ...indicator, forming part of the major diagnostic criteria. Mutation of the Patched tumor suppressor gene is responsible for the predisposition to develop Basal Cell Carcinomas and difficult-to-manage Keratocysts, among other phenotypic features, at early ages. The Keratocysts interfere with the development of dental pieces, proper facial growth, as documented in a sample of 3 children, who exhibited the presence of Keratocysts before the age of 5 as their initial manifestation. The accurate diagnosis and decompressive-surgical treatment allowed for satisfactory aesthetic and functional outcomes in them during a follow-up period exceeding 15 years.
Next generation sequencing has now enabled a cost-effective enumeration of the full mutational complement of a tumor genome-in particular single nucleotide variants (SNVs). Most current computational ...and statistical models for analyzing next generation sequencing data, however, do not account for cancer-specific biological properties, including somatic segmental copy number alterations (CNAs)-which require special treatment of the data. Here we present CoNAn-SNV (Copy Number Annotated SNV): a novel algorithm for the inference of single nucleotide variants (SNVs) that overlap copy number alterations. The method is based on modelling the notion that genomic regions of segmental duplication and amplification induce an extended genotype space where a subset of genotypes will exhibit heavily skewed allelic distributions in SNVs (and therefore render them undetectable by methods that assume diploidy). We introduce the concept of modelling allelic counts from sequencing data using a panel of Binomial mixture models where the number of mixtures for a given locus in the genome is informed by a discrete copy number state given as input. We applied CoNAn-SNV to a previously published whole genome shotgun data set obtained from a lobular breast cancer and show that it is able to discover 21 experimentally revalidated somatic non-synonymous mutations in a lobular breast cancer genome that were not detected using copy number insensitive SNV detection algorithms. Importantly, ROC analysis shows that the increased sensitivity of CoNAn-SNV does not result in disproportionate loss of specificity. This was also supported by analysis of a recently published lymphoma genome with a relatively quiescent karyotype, where CoNAn-SNV showed similar results to other callers except in regions of copy number gain where increased sensitivity was conferred. Our results indicate that in genomically unstable tumors, copy number annotation for SNV detection will be critical to fully characterize the mutational landscape of cancer genomes.
Primary triple-negative breast cancers (TNBCs), a tumour type defined by lack of oestrogen receptor, progesterone receptor and ERBB2 gene amplification, represent approximately 16% of all breast ...cancers. Here we show in 104 TNBC cases that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some having only a handful of coding somatic aberrations in a few pathways, whereas others contain hundreds of coding somatic mutations. High-throughput RNA sequencing (RNA-seq) revealed that only approximately 36% of mutations are expressed. Using deep re-sequencing measurements of allelic abundance for 2,414 somatic mutations, we determine for the first time-to our knowledge-in an epithelial tumour subtype, the relative abundance of clonal frequencies among cases representative of the population. We show that TNBCs vary widely in their clonal frequencies at the time of diagnosis, with the basal subtype of TNBC showing more variation than non-basal TNBC. Although p53 (also known as TP53), PIK3CA and PTEN somatic mutations seem to be clonally dominant compared to other genes, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal, cell shape and motility proteins occurred at lower clonal frequencies, suggesting that they occurred later during tumour progression. Taken together, our results show that understanding the biology and therapeutic responses of patients with TNBC will require the determination of individual tumour clonal genotypes.
Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B cells. Targeted resequencing studies have revealed mutations in various genes ...encoding proteins in the NF-κB pathway that contribute to the activated B-cell (ABC) DLBCL subtype, but thus far few GCB-specific mutations have been identified. Here we report recurrent somatic mutations affecting the polycomb-group oncogene EZH2, which encodes a histone methyltransferase responsible for trimethylating Lys27 of histone H3 (H3K27). After the recent discovery of mutations in KDM6A (UTX), which encodes the histone H3K27me3 demethylase UTX, in several cancer types, EZH2 is the second histone methyltransferase gene found to be mutated in cancer. These mutations, which result in the replacement of a single tyrosine in the SET domain of the EZH2 protein (Tyr641), occur in 21.7% of GCB DLBCLs and 7.2% of FLs and are absent from ABC DLBCLs. Our data are consistent with the notion that EZH2 proteins with mutant Tyr641 have reduced enzymatic activity in vitro.
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