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.

The mutational landscape of normal human endometrial epithelium.

All normal somatic cells are thought to acquire mutations, but understanding of the rates, patterns, causes and consequences of somatic mutations in normal cells is limited. The uterine endometrium adopts multiple physiological states over a lifetime and is lined by a gland-forming epithelium1,2. Here, using whole-genome sequencing, we show that normal human endometrial glands are clonal cell populations with total mutation burdens that increase at about 29 base substitutions per year and that are many-fold lower than those of endometrial cancers. Normal endometrial glands frequently carry 'driver' mutations in cancer genes, the burden of which increases with age and decreases with parity. Cell clones with drivers often originate during the first decades of life and subsequently progressively colonize the epithelial lining of the endometrium. Our results show that mutational landscapes differ markedly between normal tissues-perhaps shaped by differences in their structure and physiology-and indicate that the procession of neoplastic change that leads to endometrial cancer is initiated early in life.

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.

AmplificationTimeR: an R package for timing sequential amplification events.

MOTIVATION: Few methods exist for timing individual amplification events in regions of focal amplification. Current methods are also limited in the copy number states that they are able to time. Here we introduce AmplificationTimeR, a method for timing higher level copy number gains and inferring the most parsimonious order of events for regions that have undergone both single gains and whole genome duplication. Our method is an extension of established approaches for timing genomic gains. RESULTS: We can time more copy number states, and in states covered by other methods our results are comparable to previously published methods. AVAILABILITY AND IMPLEMENTATION: AmplificationTimer is freely available as an R package hosted at https://github.com/Wedge-lab/AmplificationTimeR.

Single-cell and spatial transcriptomics analysis of non-small cell lung cancer.

Lung cancer is the second most frequently diagnosed cancer and the leading cause of cancer-related mortality worldwide. Tumour ecosystems feature diverse immune cell types. Myeloid cells, in particular, are prevalent and have a well-established role in promoting the disease. In our study, we profile approximately 900,000 cells from 25 treatment-naive patients with adenocarcinoma and squamous-cell carcinoma by single-cell and spatial transcriptomics. We note an inverse relationship between anti-inflammatory macrophages and NK cells/T cells, and with reduced NK cell cytotoxicity within the tumour. While we observe a similar cell type composition in both adenocarcinoma and squamous-cell carcinoma, we detect significant differences in the co-expression of various immune checkpoint inhibitors. Moreover, we reveal evidence of a transcriptional "reprogramming" of macrophages in tumours, shifting them towards cholesterol export and adopting a foetal-like transcriptional signature which promotes iron efflux. Our multi-omic resource offers a high-resolution molecular map of tumour-associated macrophages, enhancing our understanding of their role within the tumour microenvironment.

Self-sustaining long-term 3D epithelioid cultures reveal drivers of clonal expansion in esophageal epithelium.

Aging epithelia are colonized by somatic mutations, which are subjected to selection influenced by intrinsic and extrinsic factors. The lack of suitable culture systems has slowed the study of this and other long-term biological processes. Here, we describe epithelioids, a facile, cost-effective method of culturing multiple mouse and human epithelia. Esophageal epithelioids self-maintain without passaging for at least 1 year, maintaining a three-dimensional structure with proliferative basal cells that differentiate into suprabasal cells, which eventually shed and retain genomic stability. Live imaging over 5 months showed that epithelioids replicate in vivo cell dynamics. Epithelioids support genetic manipulation and enable the study of mutant cell competition and selection in three-dimensional epithelia, and show how anti-cancer treatments modulate competition between transformed and wild-type cells. Finally, a targeted CRISPR-Cas9 screen shows that epithelioids recapitulate mutant gene selection in aging human esophagus and identifies additional drivers of clonal expansion, resolving the genetic networks underpinning competitive fitness.

Genomic evolution shapes prostate cancer disease type.

The development of cancer is an evolutionary process involving the sequential acquisition of genetic alterations that disrupt normal biological processes, enabling tumor cells to rapidly proliferate and eventually invade and metastasize to other tissues. We investigated the genomic evolution of prostate cancer through the application of three separate classification methods, each designed to investigate a different aspect of tumor evolution. Integrating the results revealed the existence of two distinct types of prostate cancer that arise from divergent evolutionary trajectories, designated as the Canonical and Alternative evolutionary disease types. We therefore propose the evotype model for prostate cancer evolution wherein Alternative-evotype tumors diverge from those of the Canonical-evotype through the stochastic accumulation of genetic alterations associated with disruptions to androgen receptor DNA binding. Our model unifies many previous molecular observations, providing a powerful new framework to investigate prostate cancer disease progression.

Notch1 mutations drive clonal expansion in normal esophageal epithelium but impair tumor growth.

NOTCH1 mutant clones occupy the majority of normal human esophagus by middle age but are comparatively rare in esophageal cancers, suggesting NOTCH1 mutations drive clonal expansion but impede carcinogenesis. Here we test this hypothesis. Sequencing NOTCH1 mutant clones in aging human esophagus reveals frequent biallelic mutations that block NOTCH1 signaling. In mouse esophagus, heterozygous Notch1 mutation confers a competitive advantage over wild-type cells, an effect enhanced by loss of the second allele. Widespread Notch1 loss alters transcription but has minimal effects on the epithelial structure and cell dynamics. In a carcinogenesis model, Notch1 mutations were less prevalent in tumors than normal epithelium. Deletion of Notch1 reduced tumor growth, an effect recapitulated by anti-NOTCH1 antibody treatment. Notch1 null tumors showed reduced proliferation. We conclude that Notch1 mutations in normal epithelium are beneficial as wild-type Notch1 favors tumor expansion. NOTCH1 blockade may have therapeutic potential in preventing esophageal squamous cancer.