CONIPHER: a computational framework for scalable phylogenetic reconstruction with error correction.

Intratumor heterogeneity provides the fuel for the evolution and selection of subclonal tumor cell populations. However, accurate inference of tumor subclonal architecture and reconstruction of tumor evolutionary histories from bulk DNA sequencing data remains challenging. Frequently, sequencing and alignment artifacts are not fully filtered out from cancer somatic mutations, and errors in the identification of copy number alterations or complex evolutionary events (e.g., mutation losses) affect the estimated cellular prevalence of mutations. Together, such errors propagate into the analysis of mutation clustering and phylogenetic reconstruction. In this Protocol, we present a new computational framework, CONIPHER (COrrecting Noise In PHylogenetic Evaluation and Reconstruction), that accurately infers subclonal structure and phylogenetic relationships from multisample tumor sequencing, accounting for both copy number alterations and mutation errors. CONIPHER has been used to reconstruct subclonal architecture and tumor phylogeny from multisample tumors with high-depth whole-exome sequencing from the TRACERx421 dataset, as well as matched primary-metastatic cases. CONIPHER outperforms similar methods on simulated datasets, and in particular scales to a large number of tumor samples and clones, while completing in under 1.5 h on average. CONIPHER enables automated phylogenetic analysis that can be effectively applied to large sequencing datasets generated with different technologies. CONIPHER can be run with a basic knowledge of bioinformatics and R and bash scripting languages.

Spatial and temporal intra-tumoral heterogeneity in advanced HGSOC: Implications for surgical and clinical outcomes.

Limited evidence exists on the impact of spatial and temporal heterogeneity of high-grade serous ovarian cancer (HGSOC) on tumor evolution, clinical outcomes, and surgical operability. We perform systematic multi-site tumor mapping at presentation and matched relapse from 49 high-tumor-burden patients, operated up front. From SNP array-derived copy-number data, we categorize dendrograms representing tumor clonal evolution as sympodial or dichotomous, noting most chemo-resistant patients favor simpler sympodial evolution. Three distinct tumor evolutionary patterns from primary to relapse are identified, demonstrating recurrent disease may emerge from pre-existing or newly detected clones. Crucially, we identify spatial heterogeneity for clinically actionable homologous recombination deficiency scores and for poor prognosis biomarkers CCNE1 and MYC. Copy-number signature, phenotypic, proteomic, and proliferative-index heterogeneity further highlight HGSOC complexity. This study explores HGSOC evolution and dissemination across space and time, its impact on optimal surgical cytoreductive effort and clinical outcomes, and its consequences for clinical decision-making.

Genomic-transcriptomic evolution in lung cancer and metastasis.

Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy1. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study2,3. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic-transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary-metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis.

The evolution of lung cancer and impact of subclonal selection in TRACERx.

Lung cancer is the leading cause of cancer-associated mortality worldwide1. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource.

Therapy sculpts the complex interplay between cancer and the immune system during tumour evolution.

Cancer development is an evolutionary process. A key selection pressure is exerted by therapy, one of the few players in cancer evolution that can be controlled. As such, an understanding of how treatment acts to sculpt the tumour and its microenvironment and how this influences a tumour's subsequent evolutionary trajectory is critical. In this review, we examine cancer evolution and intra-tumour heterogeneity in the context of therapy. We focus on how radiotherapy, chemotherapy and immunotherapy shape both tumour development and the environment in which tumours evolve and how resistance can develop or be selected for during treatment.

MYO10-filopodia support basement membranes at pre-invasive tumor boundaries.

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer. During invasion, the encapsulating DCIS basement membrane (BM) is compromised, and tumor cells invade the surrounding stroma. The mechanisms that regulate functional epithelial BMs in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing protein associated with metastasis and poor clinical outcome in invasive breast cancer (IBC). We identify elevated MYO10 expression in human DCIS and IBC, and this suggests links with disease progression. MYO10 promotes filopodia formation and cell invasion in vitro and cancer-cell dissemination from progressively invasive human DCIS xenografts. However, MYO10-depleted xenografts are more invasive. These lesions exhibit compromised BMs, poorly defined borders, and increased cancer-cell dispersal and EMT-marker-positive cells. In addition, cancer spheroids are dependent on MYO10-filopodia to generate a near-continuous extracellular matrix boundary. Thus, MYO10 is protective in early-stage breast cancer, correlating with tumor-limiting BMs, and pro-invasive at later stages, facilitating cancer-cell dissemination.

Potential Use of Shared Frameshift Mutations in 'Off-the-Shelf' Neoantigen Vaccines.

Neoantigens derived from frameshift mutations in microsatellite unstable tumours are more commonly shared between different patients' cancers compared with neoantigens arising from missense mutations. A recent study by Roudko et al. evaluates the immunogenicity of shared frameshift neoantigens, which could potentially be used in 'off-the-shelf' neoantigen vaccines.

Preperitoneal Fat Grafting Inhibits the Formation of Intra-abdominal Adhesions in Mice.

BACKGROUND: Adhesion formation contributes to postoperative complications in abdominal and gynaecological surgery. Thus far, the prevention and treatment strategies have focused on mechanical barriers in solid and liquid form, but these methods are not in routine use. As autologous fat grafting has become popular in treatment of hypertrophic scars because of its immunomodulatory effects, we postulated that fat grafting could also prevent peritoneal adhesion through similar mechanisms. METHODS: This was a control versus intervention study to evaluate the effect of fat grafting in the prevention on peritoneal adhesion formation. An experimental mouse model for moderate and extensive peritoneal adhesions was used (n = 4-6 mice/group). Adhesions were induced mechanically, and a free epididymal fat graft from wild type or CAG-DsRed mice was injected preperitoneally immediately after adhesion induction. PET/CT imaging and scaling of the adhesions were performed, and samples were taken for further analysis at 7 and 30 days postoperation. Macrophage phenotyping was further performed from peritoneal lavage samples, and the expression of inflammatory cytokines and mesothelial layer recovery were analysed from peritoneal tissue samples. RESULTS: Fat grafting significantly inhibited the formation of adhesions. PET/CT results did not show prolonged inflammation in any of the groups. While the expression of anti-inflammatory and anti-fibrotic IL-10 was significantly increased in the peritoneum of the fat graft-treated group at 7 days, tissue-resident and repairing M2 macrophages could no longer be detected in the fat graft at this time point. The percentage of the continuous, healed peritoneum as shown by Keratin 8 staining was greater in the fat graft-treated group after 7 days. CONCLUSIONS: Fat grafting can inhibit the formation of peritoneal adhesions in mice. Our results suggest that fat grafting promotes the peritoneal healing process in a paracrine manner thereby enabling rapid regeneration of the peritoneal mesothelial cell layer.