Gains and losses of DNA are prevalent in cancer and emerge as a consequence of inter-related processes of replication stress, mitotic errors, spindle multipolarity and breakage–fusion–bridge cycles, among others, which may lead to chromosomal instability and aneuploidy^1,2. These copy number alterations contribute to cancer initiation, progression and therapeutic resistance^3–5. Here we present a conceptual framework to examine the patterns of copy number alterations in human cancer that is widely applicable to diverse data types, including whole-genome sequencing, whole-exome sequencing, reduced representation bisulfite sequencing, single-cell DNA sequencing and SNP6 microarray data. Deploying this framework to 9,873 cancers representing 33 human cancer types from The Cancer Genome Atlas⁶ revealed a set of 21 copy number signatures that explain the copy number patterns of 97% of samples. Seventeen copy number signatures were attributed to biological phenomena of whole-genome doubling, aneuploidy, loss of heterozygosity, homologous recombination deficiency, chromothripsis and haploidization. The aetiologies of four copy number signatures remain unexplained. Some cancer types harbour amplicon signatures associated with extrachromosomal DNA, disease-specific survival and proto-oncogene gains such as MDM2. In contrast to base-scale mutational signatures, no copy number signature was associated with many known exogenous cancer risk factors. Our results synthesize the global landscape of copy number alterations in human cancer by revealing a diversity of mutational processes that give rise to these alterations.

The nature and extent of immune cell infiltration into solid tumours are key determinants of therapeutic response. Here, using a DNA methylation-based approach to tumour cell fraction deconvolution, we report the integrated analysis of tumour composition and genomics across a wide spectrum of solid cancers. Initially studying head and neck squamous cell carcinoma, we identify two distinct tumour subgroups: ‘immune hot’ and ‘immune cold’, which display differing prognosis, mutation burden, cytokine signalling, cytolytic activity and oncogenic driver events. We demonstrate the existence of such tumour subgroups pan-cancer, link clonal-neoantigen burden to cytotoxic T-lymphocyte infiltration, and show that transcriptional signatures of hot tumours are selectively engaged in immunotherapy responders. We also find that treatment-naive hot tumours are markedly enriched for known immune-resistance genomic alterations, potentially explaining the heterogeneity of immunotherapy response and prognosis seen within this group. Finally, we define a catalogue of mediators of active antitumour immunity, deriving candidate biomarkers and potential targets for precision immunotherapy.

Background: Human papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) is an emerging disease, representing a distinct clinical and epidemiological entity. Understanding the genetic basis of this specific subtype of cancer could allow therapeutic targeting of affected pathways for a stratified medicine approach. Methods: Twenty HPV+ and 20 HPV- laser-capture microdissected oropharyngeal carcinomas were used for paired-end sequencing of hybrid-captured DNA, targeting 3,230 exons in 182 genes often mutated in cancer. Copy number alteration (CNA) profiling, Sequenom MassArray sequencing and immunohistochemistry were used to further validate findings. Results: HPV+ and HPV- oropharyngeal carcinomas cluster into two distinct subgroups. TP53 mutations are detected in 100% of HPV negative cases and abrogation of the G1/S checkpoint by CDKN2A/B deletion and/or CCND1 amplification occurs in the majority of HPV- tumors. Conclusion: These findings strongly support a causal role for HPV, acting via p53 and RB pathway inhibition, in the pathogenesis of a subset of oropharyngeal cancers and suggest that studies of CDK inhibitors in HPV- disease may be warranted. Mutation and copy number alteration of PI3 kinase (PI3K) pathway components appears particularly prevalent in HPV+ tumors and assessment of these alterations may aid in the interpretation of current clinical trials of PI3K, AKT, and mTOR inhibitors in HNSCC.

The integration of genomic and epigenomic data is an increasingly popular approach for studying the complex mechanisms driving cancer development. We have developed a method for evaluating both methylation and copy number from high-density DNA methylation arrays. Comparing copy number data from Infinium HumanMethylation450 BeadChips and SNP arrays, we demonstrate that Infinium arrays detect copy number alterations with the sensitivity of SNP platforms. These results show that high-density methylation arrays provide a robust and economic platform for detecting copy number and methylation changes in a single experiment. Our method is available in the ChAMP Bioconductor package: http://www.bioconductor.org/packages/2.13/bioc/html/ChAMP.html.

Background: Human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological condition compared with HPV-negative (HPV-) HNSCC. To test the possible involvement of epigenetic modulation by HPV in HNSCC, we conducted a genome-wide DNA-methylation analysis. Methods: Using laser-capture microdissection of 42 formalin-fixed paraffin wax-embedded (FFPE) HNSCCs, we generated DNA-methylation profiles of 18 HPV+ and 14 HPV- samples, using Infinium 450 k BeadArray technology. Methylation data were validated in two sets of independent HPV+/HPV- HNSCC samples (fresh-frozen samples and cell lines) using two independent methods (Infinium 450 k and whole-genome methylated DNA immunoprecipitation sequencing (MeDIP-seq)). For the functional analysis, an HPV- HNSCC cell line was transduced with lentiviral constructs containing the two HPV oncogenes (E6 and E7), and effects on methylation were assayed using the Infinium 450 k technology. Results and discussion: Unsupervised clustering over the methylation variable positions (MVPs) with greatest variation showed that samples segregated in accordance with HPV status, but also that HPV+ tumors are heterogeneous. MVPs were significantly enriched at transcriptional start sites, leading to the identification of a candidate CpG island methylator phenotype in a sub-group of the HPV+ tumors. Supervised analysis identified a strong preponderance (87%) of MVPs towards hypermethylation in HPV+ HNSCC. Meta-analysis of our HNSCC and publicly available methylation data in cervical and lung cancers confirmed the observed DNA-methylation signature to be HPV-specific and tissue-independent. Grouping of MVPs into functionally more significant differentially methylated regions identified 43 hypermethylated promoter DMRs, including for three cadherins of the Polycomb group target genes. Integration with independent expression data showed strong negative correlation, especially for the cadherin gene-family members. Combinatorial ectopic expression of the two HPV oncogenes (E6 and E7) in an HPV- HNSCC cell line partially phenocopied the hypermethylation signature seen in HPV+ HNSCC tumors, and established E6 as the main viral effector gene. Conclusions: Our data establish that archival FFPE tissue is very suitable for this type of methylome analysis, and suggest that HPV modulates the HNSCC epigenome through hypermethylation of Polycomb repressive complex 2 target genes such as cadherins, which are implicated in tumor progression and metastasis.