Genomic–transcriptomic evolution in lung cancer and metastasis

Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy. 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 study. 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 worldwide. 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

The evolution of non-small cell lung cancer metastases in TRACERx

Metastatic disease is responsible for the majority of cancer-related deaths. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse

Antibodies against endogenous retroviruses promote lung cancer immunotherapy

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS). Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response

Book review: For the love of jungle trees

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Review of the relationship between Indigenous Australians, dingoes (Canis dingo) and domestic dogs (Canis familiaris)

Smith, BP ORCiD: 0000-0002-0873-3917Canids form a large part of Indigenous Australian life and myth- ology, an association first developed with the dingo, and later with the domestic dog. The relationship between canids and Indigenous Australians is intricate, but unique in that these peoples never domesticated the wild dingo. Neither were dingoes and dogs seen as a source of food nor in many cases considered practical hunting assistants, yet they were highly prized. Apart from featuring heavily in Indigenous Australian spirituality (The Dreaming), advantages of camp dingoes and dogs include them being protectors or guardians, "bed warmers," and companions. However, these benefits were weighed against the many associated social and economic costs incurred such as disruption to camp life and religious ceremony, burden on camp food supply and storage, and potential source of disease. This review explores the relationship between Indigenous Australians, dingoes and dogs, and attempts to explain why dingoes, and later dogs, were kept, yet not domesticated. By bringing together the many disparate observations made by early anthropologists, insight into traditional human-canid relationships may be gleaned

Recycling 115,369 mobile phones for gorilla conservation over a six-year period (2009-2014) at Zoos Victoria: A case study of 'points of influence' and mobile phone donations.

More than seven billion mobile phones are estimated to be in service globally, with more than a billion older phones likely to be retired. A major barrier to a sustainable circular economy for mobile phones is people's hoarding of their retired phones. Old mobile phones may be refurbished for re-use or ultimately dismantled for possible extraction of elements, including 'conflict' metals such as coltan (containing elements tantalum and niobium), mined in eastern Democratic Republic of Congo and threatening wild populations of eastern Grauer's gorillas (Gorilla beringei graueri). Zoos Victoria cares for western gorillas (Gorilla gorilla gorilla) who served as ambassadors for their Grauer's gorilla counterparts in this community-based social marketing initiative. Through tracking of barcodes on satchels of recycled mobile phones, efficiency of ten different points of influence could be calculated for the 'They're Calling on You' mobile phone recycling community campaign at Zoos Victoria in Australia. Over a six-year period (2009-2014), a total of 115,369 mobile phones were donated. The Courier Collect initiative resulted in 50,883 mobile phone donations (44% of total), followed by the Static Display at Melbourne Zoo, resulting in 29,778 mobile phone donations (26% of total). The number of phones collected for Keeper Talks (at Melbourne Zoo and Werribee Open Range Zoo) was 12,684 (11% of total), and in terms of fostering close connections between visitors and the conservation campaign, keeper talks were effective as one phone was donated for every four people attending a keeper talk at Werribee Open Range Zoo and one phone was donated for every 28 people who attended a keeper talk at Melbourne Zoo. We provide suggestions for future campaigns, so that accurate data capture can allow cost-benefit analyses to be conducted. Our results demonstrate that a conservation-based organisation, in partnership with corporate sponsors and community groups can effectively influenced people's mobile phone recycling behavior, paving the way for international collaborations to maximize scale and impact

Behavioral Monitoring of Big Cats Involved in ‘Behind-the-Scenes’ Zoo

While interactive tours have been argued to hold great conservation potential for zoo visitors, the influence on the participating animal’s behavior is often ignored. To investigate this, we observed the behavior of one Sumatran tiger (Panthera tigris sumatrae) and three African lions (Panthera leo leo) involved in a protected contact tour, as well as that of three cheetahs (Acinonyx jubatus) involved in a hands-on tour, at Zoos South Australia. Instantaneous scan sampling (30-s intervals) was used to record animal behavior before, during, and after behind-the-scenes tours, as well as for equivalent times on non-tour days, over a three-month period. Estimated proximity (close, < 2 m; moderate, 2-5 m; and distant, > 5 m) to humans was also recorded as an indirect measure of interaction. The animals in the protected contact tour displayed decreased inactivity and increased feeding and pacing during the tours, compared to before and after. We suggest that the increased pacing is more associated with the animals being fed during the tours, rather than the tours being a stressful experience. Those in the hands-on tour showed variation in proportions of multiple behavior categories and primarily these were shifts in species-typical behaviors. In contrast to those in the protected contact tour, they showed decreased pacing during the tour sessions. No aggressive or otherwise antagonistic behaviors directed at humans were observed by animals in either tour, with these animals typically spending more than half of their tour times in distant proximity to keepers and visitors. Combined, these findings indicate that large felid behavior may be altered by participation in interactive tours, but that these changes are not necessarily indicative of compromised wellbeing. Additional research is needed to determine the impact that these experiences are having on the welfare of the animals. This study reinforces the potential for behavioral monitoring to be used as a method for assessing the influence of visitors on zoo animals