Mitotic recombination is important for inactivating tumour suppressor genes by copy-neutral loss of heterozygosity (LOH). Although meiotic recombination maps are plentiful, little is known about mitotic recombination. The APC gene (chr5q21) is mutated in most colorectal tumours and its usual mode of LOH is mitotic recombination.

Papillary renal cell carcinoma (pRCC) is an important subtype of kidney cancer with a problematic pathological classification and highly variable clinical behaviour. Here we sequence the genomes or exomes of 31 pRCCs, and in four tumours, multi-region sequencing is undertaken. We identify BAP1, SETD2, ARID2 and Nrf2 pathway genes (KEAP1, NHE2L2 and CUL3) as probable drivers, together with at least eight other possible drivers. However, only ~10% of tumours harbour detectable pathogenic changes in any one driver gene, and where present, the mutations are often predicted to be present within cancer sub-clones. We specifically detect parallel evolution of multiple SETD2 mutations within different sub-regions of the same tumour. By contrast, large copy number gains of chromosomes 7, 12, 16 and 17 are usually early, monoclonal changes in pRCC evolution. The predominance of large copy number variants as the major drivers for pRCC highlights an unusual mode of tumorigenesis that may challenge precision medicine approaches.

Genomic analysis of multi-focal renal cell carcinomas from an individual with a germline VHL mutation offers a unique opportunity to study tumor evolution.

Understanding the cancer genome is seen as a key step in improving outcomes for cancer patients. Genomic assays are emerging as a possible avenue to personalised medicine in breast cancer. However, evolution of the cancer genome during the natural history of breast cancer is largely unknown, as is the profile of disease at death. We sought to study in detail these aspects of advanced breast cancers that have resulted in lethal disease.

Clear-cell renal cell carcinoma (ccRCC) exhibits a broad range of metastatic phenotypes that have not been systematically studied to date. Here, we analyzed 575 primary and 335 metastatic biopsies across 100 patients with metastatic ccRCC, including two cases sampledat post-mortem. Metastatic competence was afforded by chromosome complexity, and we identify 9p loss as a highly selected event driving metastasis and ccRCC-related mortality (p = 0.0014). Distinct patterns of metastatic dissemination were observed, including rapid progression to multiple tissue sites seeded by primary tumors of monoclonal structure. By contrast, we observed attenuated progression in cases characterized by high primary tumor heterogeneity, with metastatic competence acquired gradually and initial progression to solitary metastasis. Finally, we observed early divergence of primitive ancestral clones and protracted latency of up to two decades as a feature of pancreatic metastases.

Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumor-cell-intrinsic and microenvironmental features underpinning CPI sensitization. Here, we collated whole-exome and transcriptomic data for >1,000 CPI-treated patients across seven tumor types, utilizing standardized bioinformatics workflows and clinical outcome criteria to validate multivariable predictors of CPI sensitization. Clonal tumor mutation burden (TMB) was the strongest predictor of CPI response, followed by total TMB and CXCL9 expression. Subclonal TMB, somatic copy alteration burden, and histocompatibility leukocyte antigen (HLA) evolutionary divergence failed to attain pan-cancer significance. Dinucleotide variants were identified as a source of immunogenic epitopes associated with radical amino acid substitutions and enhanced peptide hydrophobicity/immunogenicity. Copy-number analysis revealed two additional determinants of CPI outcome supported by prior functional evidence: 9q34 (TRAF2) loss associated with response and CCND1 amplification associated with resistance. Finally, single-cell RNA sequencing (RNA-seq) of clonal neoantigen-reactive CD8 tumor-infiltrating lymphocytes (TILs), combined with bulk RNA-seq analysis of CPI-responding tumors, identified CCR5 and CXCL13 as T-cell-intrinsic markers of CPI sensitivity.

Immune checkpoint therapy (ICT) provides substantial clinical benefits to cancer patients, but a large proportion of cancers do not respond to ICT. To date, the genomic underpinnings of primary resistance to ICT remain elusive. Here, we performed immunogenomic analysis of data from TCGA and clinical trials of anti-PD-1/PD-L1 therapy, with a particular focus on homozygous deletion of 9p21.3 (9p21 loss), one of the most frequent genomic defects occurring in ~13% of all cancers. We demonstrate that 9p21 loss confers "cold" tumor-immune phenotypes, characterized by reduced abundance of tumor-infiltrating leukocytes (TILs), particularly, T/B/NK cells, altered spatial TILs patterns, diminished immune cell trafficking/activation, decreased rate of PD-L1 positivity, along with activation of immunosuppressive signaling. Notably, patients with 9p21 loss exhibited significantly lower response rates to ICT and worse outcomes, which were corroborated in eight ICT trials of >1,000 patients. Further, 9p21 loss synergizes with PD-L1/TMB for patient stratification. A "response score" was derived by incorporating 9p21 loss, PD-L1 expression and TMB levels in pre-treatment tumors, which outperforms PD-L1, TMB, and their combination in identifying patients with high likelihood of achieving sustained response from otherwise non-responders. Moreover, we describe potential druggable targets in 9p21-loss tumors, which could be exploited to design rational therapeutic interventions.

The FDA has recently approved a high tumor mutational burden (TMB-high) biomarker, defined by ≥10 mutations/Mb, for the treatment of solid tumors with pembrolizumab, an immune checkpoint inhibitor (ICI) that targets PD1. However, recent studies have shown that this TMB-high biomarker is only able to stratify ICI responders in a subset of cancer types, and the mechanisms underlying this observation have remained unknown. The tumor immune microenvironment (TME) may modulate the stratification power of TMB (termed TMB power), determining if it will be predictive of ICI response in a given cancer type. To systematically study this hypothesis, we inferred the levels of 31 immune-related factors characteristic of the TME of different cancer types in The Cancer Genome Atlas. Integration of this information with TMB and response data of 2,277 patients treated with anti-PD1 identified key immune factors that determine TMB power across 14 different cancer types. We find that high levels of M1 macrophages and low resting dendritic cells in the TME characterized cancer types with high TMB power. A model based on these two immune factors strongly predicted TMB power in a given cancer type during cross-validation and testing (Spearman Rho = 0.76 and 1, respectively). Using this model, we predicted the TMB power in nine additional cancer types, including rare cancers, for which TMB and ICI response data are not yet publicly available. Our analysis indicates that TMB-high may be highly predictive of ICI response in cervical squamous cell carcinoma, suggesting that such a study should be prioritized.

In order to identify new genes with differential expression in early intestinal tumours, we performed mRNA (messenger ribonucleic acid) expression profiling of 16 human and 63 mouse adenomas. All individuals had germline APC mutations to ensure that tumorigenesis was driven by 'second hits' at APC. Using stringent filtering to identify changes consistent between humans and mice, we identified 60 genes up-regulated and 151 down-regulated in tumours. For 22 selected genes--including known Wnt targets--expression differences were confirmed by qRT-PCR (quantitative reverse transcription polymerase chain reaction). Most, but not all, differences were also present in colorectal carcinomas. In situ analysis showed a complex picture. Expression of up-regulated genes in adenomas was usually uniform/diffuse (e.g. ITGA6) or prominent in the tumour core (e.g. LGR5); in normal tissue, these genes were expressed at crypt bases or the transit amplifying zone. Down-regulated genes were often undetectable in adenomas, but in normal tissue were expressed in mesenchyme (e.g. GREM1/2) or differentiated cells towards crypt tops (e.g. SGK1). In silico analysis of TCF4-binding motifs showed that some of our genes were probably direct Wnt targets. Previous studies, mostly focused on human tumours, showed partial overlap with our 'expression signature', but 37 genes were unique to our study, including TACSTD2, SEMA3F, HOXA9 and IER3 (up-regulated), and TAGLN, GREM1, GREM2, MAB21L2 and RARRES2 (down-regulated). Combined analysis of our and published human data identified additional genes differentially expressed in adenomas, including decreased BMPs (bone morphogenetic proteins) and increased BUB1/BUB1B. Several of the newly identified, differentially expressed genes represent potential diagnostic or therapeutic targets for intestinal tumours.

Although thousands of solid tumors have been sequenced to date, a fundamental under-sampling bias is inherent in current methodologies. This is caused by a tissue sample input of fixed dimensions (e.g., 6 mm biopsy), which becomes grossly under-powered as tumor volume scales. Here, we demonstrate representative sequencing (Rep-Seq) as a new method to achieve unbiased tumor tissue sampling. Rep-Seq uses fixed residual tumor material, which is homogenized and subjected to next-generation sequencing. Analysis of intratumor tumor mutation burden (TMB) variability shows a high level of misclassification using current single-biopsy methods, with 20% of lung and 52% of bladder tumors having at least one biopsy with high TMB but low clonal TMB overall. Misclassification rates by contrast are reduced to 2% (lung) and 4% (bladder) when a more representative sampling methodology is used. Rep-Seq offers an improved sampling protocol for tumor profiling, with significant potential for improved clinical utility and more accurate deconvolution of clonal structure.

Regulatory T cells (Tregs) play a critical role in the maintenance of immune homeostasis but also protect tumors from immune-mediated growth control or rejection and pose a significant barrier to effective immunotherapy. Inhibition of MALT1 paracaspase activity can selectively reprogram immune-suppressive Tregs in the tumor microenvironment to adopt a proinflammatory fragile state, which offers an opportunity to impede tumor growth and enhance the efficacy of immune checkpoint therapy (ICT).

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy.

Genetic intra-tumour heterogeneity fuels clonal evolution, but our understanding of clinically relevant clonal dynamics remain limited. We investigated spatial and temporal features of clonal diversification in clear cell renal cell carcinoma through a combination of modelling and real tumour analysis. We observe that the mode of tumour growth, surface or volume, impacts the extent of subclonal diversification, enabling interpretation of clonal diversity in patient tumours. Specific patterns of proliferation and necrosis explain clonal expansion and emergence of parallel evolution and microdiversity in tumours. In silico time-course studies reveal the appearance of budding structures before detectable subclonal diversification. Intriguingly, we observe radiological evidence of budding structures in early-stage clear cell renal cell carcinoma, indicating that future clonal evolution may be predictable from imaging. Our findings offer a window into the temporal and spatial features of clinically relevant clonal evolution.

Testicular germ cell tumour (TGCT) is the most common cancer in young men. Here we sought to identify risk factors for TGCT by performing whole-exome sequencing on 328 TGCT cases from 153 families, 634 sporadic TGCT cases and 1,644 controls. We search for genes that are recurrently affected by rare variants (minor allele frequency <0.01) with potentially damaging effects and evidence of segregation in families. A total of 8.7% of TGCT families carry rare disruptive mutations in the cilia-microtubule genes (CMG) as compared with 0.5% of controls (P=2.1 × 10-8). The most significantly mutated CMG is DNAAF1 with biallelic inactivation and loss of DNAAF1 expression shown in tumours from carriers. DNAAF1 mutation as a cause of TGCT is supported by a dnaaf1hu255h(+/-) zebrafish model, which has a 94% risk of TGCT. Our data implicate cilia-microtubule inactivation as a cause of TGCT and provide evidence for CMGs as cancer susceptibility genes.

ADAPTeR is a prospective, phase II study of nivolumab (anti-PD-1) in 15 treatment-naive patients (115 multiregion tumor samples) with metastatic clear cell renal cell carcinoma (ccRCC) aiming to understand the mechanism underpinning therapeutic response. Genomic analyses show no correlation between tumor molecular features and response, whereas ccRCC-specific human endogenous retrovirus expression indirectly correlates with clinical response. T cell receptor (TCR) analysis reveals a significantly higher number of expanded TCR clones pre-treatment in responders suggesting pre-existing immunity. Maintenance of highly similar clusters of TCRs post-treatment predict response, suggesting ongoing antigen engagement and survival of families of T cells likely recognizing the same antigens. In responders, nivolumab-bound CD8+ T cells are expanded and express GZMK/B. Our data suggest nivolumab drives both maintenance and replacement of previously expanded T cell clones, but only maintenance correlates with response. We hypothesize that maintenance and boosting of a pre-existing response is a key element of anti-PD-1 mode of action.

Testicular germ cell tumor (TGCT), the most common cancer in men aged 18 to 45 years, has a strong heritable basis. Genome-wide association studies (GWAS) have proposed single nucleotide polymorphisms (SNPs) at a number of loci influencing TGCT risk. To further evaluate the association of recently proposed risk SNPs with TGCT at 2q14.2, 3q26.2, 7q36.3, 10q26.13 and 15q21.3, we analyzed genotype data on 3,206 cases and 7,422 controls. Our analysis provides independent replication of the associations for risk SNPs at 2q14.2 (rs2713206 at P = 3.03 × 10-2; P-meta = 3.92 × 10-8; nearest gene, TFCP2L1) and rs12912292 at 15q21.3 (P = 7.96 × 10-11; P-meta = 1.55 × 10-19; nearest gene PRTG). Case-only analyses did not reveal specific associations with TGCT histology. TFCP2L1 joins the growing list of genes located within TGCT risk loci with biologically plausible roles in developmental transcriptional regulation, further highlighting the importance of this phenomenon in TGCT oncogenesis.

The evolutionary features of clear-cell renal cell carcinoma (ccRCC) have not been systematically studied to date. We analyzed 1,206 primary tumor regions from 101 patients recruited into the multi-center prospective study, TRACERx Renal. We observe up to 30 driver events per tumor and show that subclonal diversification is associated with known prognostic parameters. By resolving the patterns of driver event ordering, co-occurrence, and mutual exclusivity at clone level, we show the deterministic nature of clonal evolution. ccRCC can be grouped into seven evolutionary subtypes, ranging from tumors characterized by early fixation of multiple mutational and copy number drivers and rapid metastases to highly branched tumors with >10 subclonal drivers and extensive parallel evolution associated with attenuated progression. We identify genetic diversity and chromosomal complexity as determinants of patient outcome. Our insights reconcile the variable clinical behavior of ccRCC and suggest evolutionary potential as a biomarker for both intervention and surveillance.

Tumor antigens can emerge through multiple mechanisms, including translation of noncoding genomic regions. This noncanonical category of tumor antigens has recently gained attention; however, our understanding of how they recur within and between cancer types is still in its infancy. Therefore, we developed a proteogenomic pipeline based on deep learning de novo mass spectrometry (MS) to enable the discovery of noncanonical MHC class I-associated peptides (ncMAP) from noncoding regions. Considering that the emergence of tumor antigens can also involve posttranslational modifications (PTM), we included an open search component in our pipeline. Leveraging the wealth of MS-based immunopeptidomics, we analyzed data from 26 MHC class I immunopeptidomic studies across 11 different cancer types. We validated the de novo identified ncMAPs, along with the most abundant PTMs, using spectral matching and controlled their FDR to 1%. The noncanonical presentation appeared to be 5 times enriched for the A03 HLA supertype, with a projected population coverage of 55%. The data reveal an atlas of 8,601 ncMAPs with varying levels of cancer selectivity and suggest 17 cancer-selective ncMAPs as attractive therapeutic targets according to a stringent cutoff. In summary, the combination of the open-source pipeline and the atlas of ncMAPs reported herein could facilitate the identification and screening of ncMAPs as targets for T-cell therapies or vaccine development.

Amyotrophic Lateral Sclerosis (ALS) causes motor neuron degeneration, with 97% of cases exhibiting TDP-43 proteinopathy. Elucidating pathomechanisms has been hampered by disease heterogeneity and difficulties accessing motor neurons. Human induced pluripotent stem cell-derived motor neurons (iPSMNs) offer a solution; however, studies have typically been limited to underpowered cohorts. Here, we present a comprehensive compendium of 429 iPSMNs from 15 datasets, and 271 post-mortem spinal cord samples. Using reproducible bioinformatic workflows, we identify robust upregulation of p53 signalling in ALS in both iPSMNs and post-mortem spinal cord. p53 activation is greatest with C9orf72 repeat expansions but is weakest with SOD1 and FUS mutations. TDP-43 depletion potentiates p53 activation in both post-mortem neuronal nuclei and cell culture, thereby functionally linking p53 activation with TDP-43 depletion. ALS iPSMNs and post-mortem tissue display enrichment of splicing alterations, somatic mutations, and gene fusions, possibly contributing to the DNA damage response.

Checkpoint inhibitor (CPI) immunotherapies have provided durable clinical responses across a range of solid tumor types for some patients with cancer. Nonetheless, response rates to CPI vary greatly between cancer types. Resolving intratumor transcriptomic changes induced by CPI may improve our understanding of the mechanisms of sensitivity and resistance.