Characterizing genetic intra-tumor heterogeneity across 2,658 human cancer genomes.

Intra-tumor heterogeneity (ITH) is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the extent, origin, and drivers of ITH across cancer types are poorly understood. To address this, we extensively characterize ITH across whole-genome sequences of 2,658 cancer samples spanning 38 cancer types. Nearly all informative samples (95.1%) contain evidence of distinct subclonal expansions with frequent branching relationships between subclones. We observe positive selection of subclonal driver mutations across most cancer types and identify cancer type-specific subclonal patterns of driver gene mutations, fusions, structural variants, and copy number alterations as well as dynamic changes in mutational processes between subclonal expansions. Our results underline the importance of ITH and its drivers in tumor evolution and provide a pan-cancer resource of comprehensively annotated subclonal events from whole-genome sequencing data.

Universal Patterns of Selection in Cancer and Somatic Tissues.

Cancer develops as a result of somatic mutation and clonal selection, but quantitative measures of selection in cancer evolution are lacking. We adapted methods from molecular evolution and applied them to 7,664 tumors across 29 cancer types. Unlike species evolution, positive selection outweighs negative selection during cancer development. On average, <1 coding base substitution/tumor is lost through negative selection, with purifying selection almost absent outside homozygous loss of essential genes. This allows exome-wide enumeration of all driver coding mutations, including outside known cancer genes. On average, tumors carry ∼4 coding substitutions under positive selection, ranging from <1/tumor in thyroid and testicular cancers to >10/tumor in endometrial and colorectal cancers. Half of driver substitutions occur in yet-to-be-discovered cancer genes. With increasing mutation burden, numbers of driver mutations increase, but not linearly. We systematically catalog cancer genes and show that genes vary extensively in what proportion of mutations are drivers versus passengers.

A pan-cancer compendium of chromosomal instability.

Chromosomal instability (CIN) results in the accumulation of large-scale losses, gains and rearrangements of DNA1. The broad genomic complexity caused by CIN is a hallmark of cancer2; however, there is no systematic framework to measure different types of CIN and their effect on clinical phenotypes pan-cancer. Here we evaluate the extent, diversity and origin of CIN across 7,880 tumours representing 33 cancer types. We present a compendium of 17 copy number signatures that characterize specific types of CIN, with putative aetiologies supported by multiple independent data sources. The signatures predict drug response and identify new drug targets. Our framework refines the understanding of impaired homologous recombination, which is one of the most therapeutically targetable types of CIN. Our results illuminate a fundamental structure underlying genomic complexity in human cancers and provide a resource to guide future CIN research.

Signatures of copy number alterations in human cancer.

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 aneuploidy1,2. These copy number alterations contribute to cancer initiation, progression and therapeutic resistance3-5. 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.

Reconstructing extrachromosomal DNA structural heterogeneity from long-read sequencing data using Decoil.

Circular extrachromosomal DNA (ecDNA) is a form of oncogene amplification found across cancer types and associated with poor outcome in patients. ecDNA can be structurally complex and can contain rearranged DNA sequences derived from multiple chromosome locations. As the structure of ecDNA can impact oncogene regulation and may indicate mechanisms of its formation, disentangling it at high resolution from sequencing data is essential. Even though methods have been developed to identify and reconstruct ecDNA in cancer genome sequencing, it remains challenging to resolve complex ecDNA structures, in particular amplicons with shared genomic footprints. We here introduce Decoil, a computational method that combines a breakpoint-graph approach with LASSO regression to reconstruct complex ecDNA and deconvolve co-occurring ecDNA elements with overlapping genomic footprints from long-read nanopore sequencing. Decoil outperforms de novo assembly and alignment-based methods in simulated long-read sequencing data for both simple and complex ecDNAs. Applying Decoil on whole-genome sequencing data uncovered different ecDNA topologies and explored ecDNA structure heterogeneity in neuroblastoma tumors and cell lines, indicating that this method may improve ecDNA structural analyses in cancer.

The evolutionary history of 2,658 cancers.

Cancer develops through a process of somatic evolution1,2. Sequencing data from a single biopsy represent a snapshot of this process that can reveal the timing of specific genomic aberrations and the changing influence of mutational processes3. Here, by whole-genome sequencing analysis of 2,658 cancers as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)4, we reconstruct the life history and evolution of mutational processes and driver mutation sequences of 38 types of cancer. Early oncogenesis is characterized by mutations in a constrained set of driver genes, and specific copy number gains, such as trisomy 7 in glioblastoma and isochromosome 17q in medulloblastoma. The mutational spectrum changes significantly throughout tumour evolution in 40% of samples. A nearly fourfold diversification of driver genes and increased genomic instability are features of later stages. Copy number alterations often occur in mitotic crises, and lead to simultaneous gains of chromosomal segments. Timing analyses suggest that driver mutations often precede diagnosis by many years, if not decades. Together, these results determine the evolutionary trajectories of cancer, and highlight opportunities for early cancer detection.

Pervasive chromosomal instability and karyotype order in tumour evolution.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

Refphase: Multi-sample phasing reveals haplotype-specific copy number heterogeneity.

Most computational methods that infer somatic copy number alterations (SCNAs) from bulk sequencing of DNA analyse tumour samples individually. However, the sequencing of multiple tumour samples from a patient's disease is an increasingly common practice. We introduce Refphase, an algorithm that leverages this multi-sampling approach to infer haplotype-specific copy numbers through multi-sample phasing. We demonstrate Refphase's ability to infer haplotype-specific SCNAs and characterise their intra-tumour heterogeneity, to uncover previously undetected allelic imbalance in low purity samples, and to identify parallel evolution in the context of whole genome doubling in a pan-cancer cohort of 336 samples from 99 tumours.

Mutually exclusive genetic interactions and gene essentiality shape the genomic landscape of primary melanoma.

Melanoma is a heterogenous malignancy with an unpredictable clinical course. Most patients who present in the clinic are diagnosed with primary melanoma, yet large-scale sequencing efforts have focused primarily on metastatic disease. In this study we sequence-profiled 524 American Joint Committee on Cancer Stage I-III primary tumours. Our analysis of these data reveals recurrent driver mutations, mutually exclusive genetic interactions, where two genes were never or rarely co-mutated, and an absence of co-occurring genetic events. Further, we intersected copy number calls from our primary melanoma data with whole-genome CRISPR screening data to identify the transcription factor interferon regulatory factor 4 (IRF4) as a melanoma-associated dependency. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Allele-specific multi-sample copy number segmentation in ASCAT.

MOTIVATION: Allele-specific copy number alterations are commonly used to trace the evolution of tumours. A key step of the analysis is to segment genomic data into regions of constant copy number. For precise phylogenetic inference, breakpoints shared between samples need to be aligned to each other. RESULTS: Here, we present asmultipcf, an algorithm for allele-specific segmentation of multiple samples that infers private and shared segment boundaries of phylogenetically related samples. The output of this algorithm can directly be used for allele-specific copy number calling using ASCAT. AVAILABILITY AND IMPLEMENTATION: asmultipcf is available as part of the ASCAT R package (version ≥2.5) from github.com/Crick-CancerGenomics/ascat/.

Appraising the relevance of DNA copy number loss and gain in prostate cancer using whole genome DNA sequence data.

A variety of models have been proposed to explain regions of recurrent somatic copy number alteration (SCNA) in human cancer. Our study employs Whole Genome DNA Sequence (WGS) data from tumor samples (n = 103) to comprehensively assess the role of the Knudson two hit genetic model in SCNA generation in prostate cancer. 64 recurrent regions of loss and gain were detected, of which 28 were novel, including regions of loss with more than 15% frequency at Chr4p15.2-p15.1 (15.53%), Chr6q27 (16.50%) and Chr18q12.3 (17.48%). Comprehensive mutation screens of genes, lincRNA encoding sequences, control regions and conserved domains within SCNAs demonstrated that a two-hit genetic model was supported in only a minor proportion of recurrent SCNA losses examined (15/40). We found that recurrent breakpoints and regions of inversion often occur within Knudson model SCNAs, leading to the identification of ZNF292 as a target gene for the deletion at 6q14.3-q15 and NKX3.1 as a two-hit target at 8p21.3-p21.2. The importance of alterations of lincRNA sequences was illustrated by the identification of a novel mutational hotspot at the KCCAT42, FENDRR, CAT1886 and STCAT2 loci at the 16q23.1-q24.3 loss. Our data confirm that the burden of SCNAs is predictive of biochemical recurrence, define nine individual regions that are associated with relapse, and highlight the possible importance of ion channel and G-protein coupled-receptor (GPCR) pathways in cancer development. We concluded that a two-hit genetic model accounts for about one third of SCNA indicating that mechanisms, such haploinsufficiency and epigenetic inactivation, account for the remaining SCNA losses.

Functional characterization of the mouse Serpina1 paralog DOM-7.

The generation of authentic mouse-models for human α1-antitrypsin (A1AT)-deficiency is difficult due to the high complexity of the mouse Serpina1 gene locus. Depending on the exact mouse strain, three to five paralogs are expressed, with different proteinase inhibitory properties. Nowadays with CRISPR-technology, genome editing of complex genomic loci is feasible and could be employed for the generation of A1AT-deficiency mouse models. In preparation of a CRISPR/Cas9-based genome-engineering approach we identified cDNA clones with a functional CDS for the Serpina1-paralog DOM-7. Here, we show that DOM-7 functionally inhibits neutrophil elastase (ELANE) and chymotrypsin, and therefore needs to be considered when aiming at the generation of A1AT-deficient models.

CD25+ FoxP3+ Memory CD4 T Cells Are Frequent Targets of HIV Infection In Vivo.

UNLABELLED: Interleukin 2 (IL-2) signaling through the IL-2 receptor alpha chain (CD25) facilitates HIV replication in vitro and facilitates homeostatic proliferation of CD25(+) FoxP3(+) CD4(+) T cells. CD25(+) FoxP3(+) CD4(+) T cells may therefore constitute a suitable subset for HIV infection and plasma virion production. CD25(+) FoxP3(+) CD4(+) T cell frequencies, absolute numbers, and the expression of CCR5 and cell cycle marker Ki67 were studied in peripheral blood from HIV(+) and HIV(-) study volunteers. Different memory CD4(+) T cell subsets were then sorted for quantification of cell-associated HIV DNA and phylogenetic analyses of the highly variable EnvV1V3 region in comparison to plasma-derived virus sequences. In HIV(+) subjects, 51% (median) of CD25(+) FoxP3(+) CD4(+) T cells expressed the HIV coreceptor CCR5. Very high frequencies of Ki67(+) cells were detected in CD25(+) FoxP3(+) memory CD4(+) T cells (median, 27.6%) in comparison to CD25(-) FoxP3(-) memory CD4(+) T cells (median, 4.1%; P < 0.0001). HIV DNA content was 15-fold higher in CD25(+) FoxP3(+) memory CD4(+) T cells than in CD25(-) FoxP3(-) T cells (P = 0.003). EnvV1V3 sequences derived from CD25(+) FoxP3(+) memory CD4(+) T cells did not preferentially cluster with plasma-derived sequences. Quasi-identical cell-plasma sequence pairs were rare, and their proportion decreased with the estimated HIV infection duration. These data suggest that specific cellular characteristics of CD25(+) FoxP3(+) memory CD4(+) T cells might facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. The contribution of this cell population to plasma virion production remains unclear. IMPORTANCE: Despite recent advances in the understanding of AIDS virus pathogenesis, which cell subsets support HIV infection and replication in vivo is incompletely understood. In vitro, the IL-2 signaling pathway and IL-2-dependent cell cycle induction are essential for HIV infection of stimulated T cells. CD25(+) FoxP3(+) memory CD4 T cells, often referred to as regulatory CD4 T cells, depend on IL-2 signaling for homeostatic proliferation in vivo Our results show that CD25(+) FoxP3(+) memory CD4(+) T cells often express the HIV coreceptor CCR5, are significantly more proliferative, and contain more HIV DNA than CD25(-) FoxP3(-) memory CD4 T cell subsets. The specific cellular characteristics of CD25(+) FoxP3(+) memory CD4(+) T cells probably facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. However, the contribution of this cell subset to plasma viremia remains unclear.

Expitope: a web server for epitope expression.

MOTIVATION: Adoptive T cell therapies based on introduction of new T cell receptors (TCRs) into patient recipient T cells is a promising new treatment for various kinds of cancers. A major challenge, however, is the choice of target antigens. If an engineered TCR can cross-react with self-antigens in healthy tissue, the side-effects can be devastating. We present the first web server for assessing epitope sharing when designing new potential lead targets. We enable the users to find all known proteins containing their peptide of interest. The web server returns not only exact matches, but also approximate ones, allowing a number of mismatches of the users choice. For the identified candidate proteins the expression values in various healthy tissues, representing all vital human organs, are extracted from RNA Sequencing (RNA-Seq) data as well as from some cancer tissues as control. All results are returned to the user sorted by a score, which is calculated using well-established methods and tools for immunological predictions. It depends on the probability that the epitope is created by proteasomal cleavage and its affinities to the transporter associated with antigen processing and the major histocompatibility complex class I alleles. With this framework, we hope to provide a helpful tool to exclude potential cross-reactivity in the early stage of TCR selection for use in design of adoptive T cell immunotherapy. AVAILABILITY AND IMPLEMENTATION: The Expitope web server can be accessed via http://webclu.bio.wzw.tum.de/expitope.

Intercellular extrachromosomal DNA copy-number heterogeneity drives neuroblastoma cell state diversity.

Neuroblastoma exhibits significant inter- and intra-tumor genetic heterogeneity and varying clinical outcomes. Extrachromosomal DNAs (ecDNAs) may drive this heterogeneity by independently segregating during cell division, leading to rapid oncogene amplification. While ecDNA-mediated oncogene amplification is linked to poor prognosis in various cancers, the effects of ecDNA copy-number heterogeneity on intermediate phenotypes are poorly understood. Here, we leverage DNA and RNA sequencing from the same single cells in cell lines and neuroblastoma patients to investigate these effects. By analyzing ecDNA amplicon structures, we reveal extensive intercellular ecDNA copy-number heterogeneity. We also provide direct evidence of how this heterogeneity influences the expression of cargo genes, including MYCN and its downstream targets, and the overall transcriptional state of neuroblastoma cells. Our findings highlight the role of ecDNA copy number in promoting rapid adaptability of cellular states within tumors, underscoring the need for ecDNA-specific treatment strategies to address tumor formation and adaptation.

Crowd-sourced benchmarking of single-sample tumor subclonal reconstruction.

Subclonal reconstruction algorithms use bulk DNA sequencing data to quantify parameters of tumor evolution, allowing an assessment of how cancers initiate, progress and respond to selective pressures. We launched the ICGC-TCGA (International Cancer Genome Consortium-The Cancer Genome Atlas) DREAM Somatic Mutation Calling Tumor Heterogeneity and Evolution Challenge to benchmark existing subclonal reconstruction algorithms. This 7-year community effort used cloud computing to benchmark 31 subclonal reconstruction algorithms on 51 simulated tumors. Algorithms were scored on seven independent tasks, leading to 12,061 total runs. Algorithm choice influenced performance substantially more than tumor features but purity-adjusted read depth, copy-number state and read mappability were associated with the performance of most algorithms on most tasks. No single algorithm was a top performer for all seven tasks and existing ensemble strategies were unable to outperform the best individual methods, highlighting a key research need. All containerized methods, evaluation code and datasets are available to support further assessment of the determinants of subclonal reconstruction accuracy and development of improved methods to understand tumor evolution.

Elimusertib has Antitumor Activity in Preclinical Patient-Derived Pediatric Solid Tumor Models.

The small-molecule inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), elimusertib, is currently being tested clinically in various cancer entities in adults and children. Its preclinical antitumor activity in pediatric malignancies, however, is largely unknown. We here assessed the preclinical activity of elimusertib in 38 cell lines and 32 patient-derived xenograft (PDX) models derived from common pediatric solid tumor entities. Detailed in vitro and in vivo molecular characterization of the treated models enabled the evaluation of response biomarkers. Pronounced objective response rates were observed for elimusertib monotherapy in PDX, when treated with a regimen currently used in clinical trials. Strikingly, elimusertib showed stronger antitumor effects than some standard-of-care chemotherapies, particularly in alveolar rhabdomysarcoma PDX. Thus, elimusertib has strong preclinical antitumor activity in pediatric solid tumor models, which may translate to clinically meaningful responses in patients.

Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer.

DNA amplifications in cancer do not only harbor oncogenes. We sought to determine whether passenger coamplifications could create collateral therapeutic vulnerabilities. Through an analysis of >3,000 cancer genomes followed by the interrogation of CRISPR-Cas9 loss-of-function screens across >700 cancer cell lines, we determined that passenger coamplifications are accompanied by distinct dependency profiles. In a proof-of-principle study, we demonstrate that the coamplification of the bona fide passenger gene DEAD-Box Helicase 1 (DDX1) creates an increased dependency on the mTOR pathway. Interaction proteomics identified tricarboxylic acid (TCA) cycle components as previously unrecognized DDX1 interaction partners. Live-cell metabolomics highlighted that this interaction could impair TCA activity, which in turn resulted in enhanced mTORC1 activity. Consequently, genetic and pharmacologic disruption of mTORC1 resulted in pronounced cell death in vitro and in vivo. Thus, structurally linked coamplification of a passenger gene and an oncogene can result in collateral vulnerabilities. SIGNIFICANCE: We demonstrate that coamplification of passenger genes, which were largely neglected in cancer biology in the past, can create distinct cancer dependencies. Because passenger coamplifications are frequent in cancer, this principle has the potential to expand target discovery in oncology. This article is featured in Selected Articles from This Issue, p. 384.