Editor’s Introduction

Upon its inception in 2006, Genocide Studies and Prevention: An International Jour- nal 1:1 published a special issue entitled ‘‘Genocide in Darfur.’’ At that time, the crisis in Darfur had already been the focus of international attention for some three years. That special issue provided an overview of the crisis, the crimes perpetrated against black African women and girls of Darfur by government of Sudan (GoS) troops and the Janjaweed, the legal implications of the US determination that the atrocities in Darfur constituted genocide, a comparative analysis of the atrocities perpetrated in Rwanda in 1994 and Darfur in the early 2000s, and a critical analysis of the US gov- ernment’s Atrocities Documentation Project whose data were used by the US govern- ment to make its determination of genocide

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

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

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

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

Editors\u27 Introduction

Indigenous peoples—those people who consider themselves, or are considered by others, to be Aboriginal, ‘‘First Nations,’’ native peoples, Fourth World peoples, or ‘‘original occupants’’ of specific places on the planet—have faced genocide, cultural destruction, and forced removal from their ancestral areas for thousands of years. Over the centuries, colonization—the expansion of populations into new areas and the exploitation of natural and human resources there—has led to significant declines in the populations of indigenous groups. As Patrick Brantlinger notes, ‘‘One of the main causes for these declines is not mysterious: violence, warfare, genocide.’’

The US Investigation into the Darfur Crisis and the US Government\u27s Determination of Genocide

This article examines the genesis and implementation of the Atrocities Documentation Project initiated by the US State Department as well as the US government’s determination that genocide had been perpetrated in Darfur, Sudan, between late 2003 and August 2004. In doing so, the author considers and analyzes the rationale for the investigation and the reasoning for the genocide determination, as given by various US officials. He also delineates and discusses the perceptions of various scholars vis-a`-vis the same issues, noting that many of the latter suspect there were ulterior motives behind the genesis and implementation of the investigation as well as the genocide determination. Finally, the author discusses the positive aspects of the investigation and the potential negative ramifications of the genocide determination

Editor\u27s Introduction

Fourteen years have passed since the 1994 Rwandan Genocide, during which an estimated 500,000 to 800,000 (or more) Tutsis and moderate Hutus died at the hands of extremists Hutus. Rwanda is still in the process of recovering from the genocide, which not only resulted in vicious and mass murder but virtually destroyed the country’s infrastructure. Like any nation reconstituting itself in the aftermath of genocide, Rwanda is experiencing growing pains. Survivors continue to suffer the ill effects of what they were subjected to, witnessed, and lost. Many of the women who were raped now have AIDS. Those who gave birth to what are commonly referred to as ‘‘rape babies’’ face additional psychological turmoil and, in many cases, are ostracized by neighbors, friends, and family members. Many of the babies have been maltreated, neglected, and even left to their own devices to eke out an existence on the streets. Orphans fill orphanages, where many of the youngest children are raised by the ‘‘older’’ (often teenage) orphans. Groups of widows have banded together to provide mutual support and get back on their feet while dealing with the absence of beloved husbands and children. Many individuals are so scarred by what they experienced and witnessed that they are not able to function and carry on normal lives. The medical and social-services communities are stretched so thin in attempting to provide assistance to those in need that people often fall through the cracks or simply do not receive the treatment they need in order to fully regain their health (whether physical or psychological). Some 100,000 alleged perpetrators still remain in Rwandan prisons. Three different court systems—the International Criminal Tribunal for Rwanda (Arusha, Tanzania), the national courts of Rwanda, and gacaca (the adaptation of precolonial mediation and reconciliation processes to try, today, those who are suspected of having carried out the killing and mass rapes) are currently in operation

Book Review: Samuel Totten, ed. Teaching about Genocide: Issues, Approaches, and Resources

There are only a few scholars writing about teaching genocide and the Holocaust, and Samuel Totten is truly one of the most prolific and most effective. His book Teaching about Genocide: Issues, Approaches, and Resources is the latest of a long list of publications for educators on the subject of genocide