The Evolutionary Landscape of Localized Prostate Cancers Drives Clinical Aggression.

The majority of newly diagnosed prostate cancers are slow growing, with a long natural life history. Yet a subset can metastasize with lethal consequences. We reconstructed the phylogenies of 293 localized prostate tumors linked to clinical outcome data. Multiple subclones were detected in 59% of patients, and specific subclonal architectures associate with adverse clinicopathological features. Early tumor development is characterized by point mutations and deletions followed by later subclonal amplifications and changes in trinucleotide mutational signatures. Specific genes are selectively mutated prior to or following subclonal diversification, including MTOR, NKX3-1, and RB1. Patients with low-risk monoclonal tumors rarely relapse after primary therapy (7%), while those with high-risk polyclonal tumors frequently do (61%). The presence of multiple subclones in an index biopsy may be necessary, but not sufficient, for relapse of localized prostate cancer, suggesting that evolution-aware biomarkers should be studied in prospective studies of low-risk tumors suitable for active surveillance.

A practical guide to cancer subclonal reconstruction from DNA sequencing.

Subclonal reconstruction from bulk tumor DNA sequencing has become a pillar of cancer evolution studies, providing insight into the clonality and relative ordering of mutations and mutational processes. We provide an outline of the complex computational approaches used for subclonal reconstruction from single and multiple tumor samples. We identify the underlying assumptions and uncertainties in each step and suggest best practices for analysis and quality assessment. This guide provides a pragmatic resource for the growing user community of subclonal reconstruction methods.

Parental legacy, demography, and admixture influenced the evolution of the two subgenomes of the tetraploid Capsella bursa-pastoris (Brassicaceae).

Allopolyploidy is generally perceived as a major source of evolutionary novelties and as an instantaneous way to create isolation barriers. However, we do not have a clear understanding of how two subgenomes evolve and interact once they have fused in an allopolyploid species nor how isolated they are from their relatives. Here, we address these questions by analyzing genomic and transcriptomic data of allotetraploid Capsella bursa-pastoris in three differentiated populations, Asia, Europe, and the Middle East. We phased the two subgenomes, one descended from the outcrossing and highly diverse Capsella grandiflora (CbpCg) and the other one from the selfing and genetically depauperate Capsella orientalis (CbpCo). For each subgenome, we assessed its relationship with the diploid relatives, temporal changes of effective population size (Ne), signatures of positive and negative selection, and gene expression patterns. In all three regions, Ne of the two subgenomes decreased gradually over time and the CbpCo subgenome accumulated more deleterious changes than CbpCg. There were signs of widespread admixture between C. bursa-pastoris and its diploid relatives. The two subgenomes were impacted differentially depending on geographic region suggesting either strong interploidy gene flow or multiple origins of C. bursa-pastoris. Selective sweeps were more common on the CbpCg subgenome in Europe and the Middle East, and on the CbpCo subgenome in Asia. In contrast, differences in expression were limited with the CbpCg subgenome slightly more expressed than CbpCo in Europe and the Middle-East. In summary, after more than 100,000 generations of co-existence, the two subgenomes of C. bursa-pastoris still retained a strong signature of parental legacy but their evolutionary trajectory strongly varied across geographic regions.

Hybrid origins and the earliest stages of diploidization in the highly successful recent polyploid Capsella bursa-pastoris.

Whole-genome duplication (WGD) events have occurred repeatedly during flowering plant evolution, and there is growing evidence for predictable patterns of gene retention and loss following polyploidization. Despite these important insights, the rate and processes governing the earliest stages of diploidization remain poorly understood, and the relative importance of genetic drift, positive selection, and relaxed purifying selection in the process of gene degeneration and loss is unclear. Here, we conduct whole-genome resequencing in Capsella bursa-pastoris, a recently formed tetraploid with one of the most widespread species distributions of any angiosperm. Whole-genome data provide strong support for recent hybrid origins of the tetraploid species within the past 100,000-300,000 y from two diploid progenitors in the Capsella genus. Major-effect inactivating mutations are frequent, but many were inherited from the parental species and show no evidence of being fixed by positive selection. Despite a lack of large-scale gene loss, we observe a decrease in the efficacy of natural selection genome-wide due to the combined effects of demography, selfing, and genome redundancy from WGD. Our results suggest that the earliest stages of diploidization are associated with quantitative genome-wide decreases in the strength and efficacy of selection rather than rapid gene loss, and that nonfunctionalization can receive a "head start" through a legacy of deleterious variants and differential expression originating in parental diploid populations.

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.

Single-cell chromatin accessibility reveals malignant regulatory programs in primary human cancers.

To identify cancer-associated gene regulatory changes, we generated single-cell chromatin accessibility landscapes across eight tumor types as part of The Cancer Genome Atlas. Tumor chromatin accessibility is strongly influenced by copy number alterations that can be used to identify subclones, yet underlying cis-regulatory landscapes retain cancer type-specific features. Using organ-matched healthy tissues, we identified the "nearest healthy" cell types in diverse cancers, demonstrating that the chromatin signature of basal-like-subtype breast cancer is most similar to secretory-type luminal epithelial cells. Neural network models trained to learn regulatory programs in cancer revealed enrichment of model-prioritized somatic noncoding mutations near cancer-associated genes, suggesting that dispersed, nonrecurrent, noncoding mutations in cancer are functional. Overall, these data and interpretable gene regulatory models for cancer and healthy tissue provide a framework for understanding cancer-specific gene regulation.

The genomic and evolutionary landscapes of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations.

Central Nervous System Tuberculosis in a Murine Model: Neurotropic Strains or a New Pathway of Infection?

Tuberculosis (TB) of the central nervous system (CNS) is a lethal and incapacitating disease. Several studies have been performed to understand the mechanism of bacterial arrival to CNS, however, it remains unclear. Although the interaction of the host, the pathogen, and the environment trigger the course of the disease, in TB the characteristics of these factors seem to be more relevant in the genesis of the clinical features of each patient. We previously tested three mycobacterial clinical isolates with distinctive genotypes obtained from the cerebrospinal fluid of patients with meningeal TB and showed that these strains disseminated extensively to the brain after intratracheal inoculation and pulmonary infection in BALB/c mice. In this present study, BALB/c mice were infected through the intranasal route. One of these strains reaches the olfactory bulb at the early stage of the infection and infects the brain before the lungs, but the histological study of the nasal mucosa did not show any alteration. This observation suggests that some mycobacteria strains can arrive directly at the brain, apparently toward the olfactory nerve after infecting the nasal mucosa, and guides us to study in more detail during mycobacteria infection the nasal mucosa, the associated connective tissue, and nervous structures of the cribriform plate, which connect the nasal cavity with the olfactory bulb.

The relative role of plasticity and demographic history in Capsella bursa-pastoris: a common garden experiment in Asia and Europe.

The colonization success of a species depends on the interplay between its phenotypic plasticity, adaptive potential and demographic history. Assessing their relative contributions during the different phases of a species range expansion is challenging, and requires large-scale experiments. Here, we investigated the relative contributions of plasticity, performance and demographic history to the worldwide expansion of the shepherd's purse, Capsella bursa-pastoris. We installed two large common gardens of the shepherd's purse, a young, self-fertilizing, allopolyploid weed with a worldwide distribution. One common garden was located in Europe, the other in Asia. We used accessions from three distinct genetic clusters (Middle East, Europe and Asia) that reflect the demographic history of the species. Several life-history traits were measured. To explain the phenotypic variation between and within genetic clusters, we analysed the effects of (i) the genetic clusters, (ii) the phenotypic plasticity and its association to fitness and (iii) the distance in terms of bioclimatic variables between the sampling site of an accession and the common garden, i.e. the environmental distance. Our experiment showed that (i) the performance of C. bursa-pastoris is closely related to its high phenotypic plasticity; (ii) within a common garden, genetic cluster was a main determinant of phenotypic differences; and (iii) at the scale of the experiment, the effect of environmental distance to the common garden could not be distinguished from that of genetic clusters. Phenotypic plasticity and demographic history both play important role at different stages of range expansion. The success of the worldwide expansion of C. bursa-pastoris was undoubtedly influenced by its strong phenotypic plasticity.

Determining the Impact of Spatial Heterogeneity on Genomic Prognostic Biomarkers for Localized Prostate Cancer.

Localized prostate tumors show remarkably diverse clinical courses, with some being cured by local therapy alone, while others rapidly relapse and have a lethal course despite precision surgery or radiotherapy. Many genomic biomarkers have been developed to predict this clinical behavior, but these are confounded by the extreme spatial heterogeneity of prostate tumors: most are multifocal and harbor multiple subclonal populations. To quantify the influence of spatial heterogeneity on genomic prognostic biomarkers, we developed a case-control high-risk cohort (n = 42) using a prospective registry, risk matched by clinicopathologic prognostic indices. Half of the cohort had early biochemical recurrence (BCR; ie, ≤18 mo), while half remained without evidence of disease for at least 48 mo after radical prostatectomy. We then genomically profiled multiple tumor foci per patient, analyzing 119 total specimens. These data allowed us to validate three published genomic prognostic biomarkers and quantify their sensitivity to tumor spatial heterogeneity. Remarkably, all three biomarkers robustly predicted early BCR, and all three were robust to spatiogenomic variability. These data suggest that DNA-based genomic biomarkers can overcome intratumoral heterogeneity: single biopsies may be sufficient to estimate the risk of early BCR after radical treatment in patients with high-risk disease. PATIENT SUMMARY: We investigated whether heterogeneity between tumor regions within the prostate affects the accuracy of DNA-based biomarkers predicting early relapse after prostatectomy. We observed persistent accuracy in predicting disease relapse, suggesting that spatial heterogeneity may not hinder biomarker performance.

Transcriptional perturbation of protein arginine methyltransferase-5 exhibits MTAP-selective oncosuppression.

We hypothesized that small molecule transcriptional perturbation could be harnessed to target a cellular dependency involving protein arginine methyltransferase 5 (PRMT5) in the context of methylthioadenosine phosphorylase (MTAP) deletion, seen frequently in malignant pleural mesothelioma (MPM). Here we show, that MTAP deletion is negatively prognostic in MPM. In vitro, the off-patent antibiotic Quinacrine efficiently suppressed PRMT5 transcription, causing chromatin remodelling with reduced global histone H4 symmetrical demethylation. Quinacrine phenocopied PRMT5 RNA interference and small molecule PRMT5 inhibition, reducing clonogenicity in an MTAP-dependent manner. This activity required a functional PRMT5 methyltransferase as MTAP negative cells were rescued by exogenous wild type PRMT5, but not a PRMT5E444Q methyltransferase-dead mutant. We identified c-jun as an essential PRMT5 transcription factor and a probable target for Quinacrine. Our results therefore suggest that small molecule-based transcriptional perturbation of PRMT5 can leverage a mutation-selective vulnerability, that is therapeutically tractable, and has relevance to 9p21 deleted cancers including MPM.

Precision Radiotherapy: Reduction in Radiation for Oropharyngeal Cancer in the 30 ROC Trial.

BACKGROUND: Patients with human papillomavirus-related oropharyngeal cancers have excellent outcomes but experience clinically significant toxicities when treated with standard chemoradiotherapy (70 Gy). We hypothesized that functional imaging could identify patients who could be safely deescalated to 30 Gy of radiotherapy. METHODS: In 19 patients, pre- and intratreatment dynamic fluorine-18-labeled fluoromisonidazole positron emission tomography (PET) was used to assess tumor hypoxia. Patients without hypoxia at baseline or intratreatment received 30 Gy; patients with persistent hypoxia received 70 Gy. Neck dissection was performed at 4 months in deescalated patients to assess pathologic response. Magnetic resonance imaging (weekly), circulating plasma cell-free DNA, RNA-sequencing, and whole-genome sequencing (WGS) were performed to identify potential molecular determinants of response. Samples from an independent prospective study were obtained to reproduce molecular findings. All statistical tests were 2-sided. RESULTS: Fifteen of 19 patients had no hypoxia on baseline PET or resolution on intratreatment PET and were deescalated to 30 Gy. Of these 15 patients, 11 had a pathologic complete response. Two-year locoregional control and overall survival were 94.4% (95% confidence interval = 84.4% to 100%) and 94.7% (95% confidence interval = 85.2% to 100%), respectively. No acute grade 3 radiation-related toxicities were observed. Microenvironmental features on serial imaging correlated better with pathologic response than tumor burden metrics or circulating plasma cell-free DNA. A WGS-based DNA repair defect was associated with response (P = .02) and was reproduced in an independent cohort (P = .03). CONCLUSIONS: Deescalation of radiotherapy to 30 Gy on the basis of intratreatment hypoxia imaging was feasible, safe, and associated with minimal toxicity. A DNA repair defect identified by WGS was predictive of response. Intratherapy personalization of chemoradiotherapy may facilitate marked deescalation of radiotherapy.

Quantifying the influence of mutation detection on tumour subclonal reconstruction.

Whole-genome sequencing can be used to estimate subclonal populations in tumours and this intra-tumoural heterogeneity is linked to clinical outcomes. Many algorithms have been developed for subclonal reconstruction, but their variabilities and consistencies are largely unknown. We evaluate sixteen pipelines for reconstructing the evolutionary histories of 293 localized prostate cancers from single samples, and eighteen pipelines for the reconstruction of 10 tumours with multi-region sampling. We show that predictions of subclonal architecture and timing of somatic mutations vary extensively across pipelines. Pipelines show consistent types of biases, with those incorporating SomaticSniper and Battenberg preferentially predicting homogenous cancer cell populations and those using MuTect tending to predict multiple populations of cancer cells. Subclonal reconstructions using multi-region sampling confirm that single-sample reconstructions systematically underestimate intra-tumoural heterogeneity, predicting on average fewer than half of the cancer cell populations identified by multi-region sequencing. Overall, these biases suggest caution in interpreting specific architectures and subclonal variants.

A community effort to create standards for evaluating tumor subclonal reconstruction.

Tumor DNA sequencing data can be interpreted by computational methods that analyze genomic heterogeneity to infer evolutionary dynamics. A growing number of studies have used these approaches to link cancer evolution with clinical progression and response to therapy. Although the inference of tumor phylogenies is rapidly becoming standard practice in cancer genome analyses, standards for evaluating them are lacking. To address this need, we systematically assess methods for reconstructing tumor subclonality. First, we elucidate the main algorithmic problems in subclonal reconstruction and develop quantitative metrics for evaluating them. Then we simulate realistic tumor genomes that harbor all known clonal and subclonal mutation types and processes. Finally, we benchmark 580 tumor reconstructions, varying tumor read depth, tumor type and somatic variant detection. Our analysis provides a baseline for the establishment of gold-standard methods to analyze tumor heterogeneity.

Genomic Classifier for Guiding Treatment of Intermediate-Risk Prostate Cancers to Dose-Escalated Image Guided Radiation Therapy Without Hormone Therapy.

PURPOSE: The National Comprehensive Cancer Network (NCCN) has recently endorsed the stratification of intermediate-risk prostate cancer (IR-PCa) into favorable and unfavorable subgroups and recommend the addition of androgen deprivation therapy (ADT) to radiation therapy (RT) for unfavorable IR-PCa. Recently, more accurate prognostication was demonstrated by integrating a 22-feature genomic classifier (GC) to the NCCN stratification system. Here, we test the utility of the GC to better identify patients with IR-PCa who are sufficiently treated by RT alone. METHODS AND MATERIALS: We identified a novel cohort comprising 121 patients with IR-PCa treated with dose-escalated image guided RT (78 Gy in 39 fractions) without ADT. GC scores were derived from tumors sampled in diagnostic biopsies. Multivariable analyses, including both NCCN subclassification and GC scores, were performed for biochemical failure (prostate-specific antigen nadir + 2 ng/mL) and metastasis occurrence. RESULTS: By NCCN subclassification, 33 (27.3%) and 87 (71.9%) of men were classified as having favorable and unfavorable IR-PCa, respectively (1 case unclassifiable). GC scores were high in 3 favorable IR-PCa and low in 60 unfavorable IR-PCa. Higher GC scores, but not NCCN risk subgroups, were associated with biochemical relapse (hazard ratio, 1.36; 95% confidence interval [CI], 1.09-1.71] per 10% increase; P = .007) and metastasis (hazard ratio, 2.05; 95% CI, 1.24-4.24; P = .004). GC predicted biochemical failure at 5 years (area under the curve, 0.78; 95% CI, 0.59-0.91), and the combinatorial NCCN + GC model significantly outperformed the NCCN alone model for predicting early-onset metastasis (area under the curve for 5-year metastasis of 0.89 vs 0.86 [GC alone] vs 0.54 [NCCN alone]). CONCLUSIONS: We demonstrated the accuracy of the GC for predicting disease recurrence in IR-PCa treated with dose-escalated image guided RT alone. Our findings highlight the need to evaluate this GC in a prospective clinical trial investigating the role of ADT-RT in clinicogenomic-defined IR-PCa subgroups.

The Immune Microenvironment, Genome-wide Copy Number Aberrations, and Survival in Mesothelioma.

INTRODUCTION: Results of recent clinical studies of immune checkpoint inhibitors in malignant pleural mesothelioma (MPM) have dampened initial enthusiasm. However, the immune environment and targets of these treatments such as programmed cell death protein 1 and its ligand programmed death ligand 1 (PD-L1) have not been well characterized in MPM. Using a large cohort of patients, we investigated PD-L1 expression, immune infiltrates, and genome-wide copy number status and correlated them to clinicopathological features. METHODS: Tissue microarrays were constructed and stained with PD-L1(clone E1L3N [Cell Signaling Technology, Danvers, MA]), cluster of differentiation 4, cluster of differentiation 8, and forkhead box P3 antibodies. PD-L1 positivity was defined as at least 5% membranous staining regardless of intensity, and high PD-L1 positivity was defined as at least 50%. Genomic DNA from a representative subset of 113 patients was used for genome-wide copy number analysis. The percent genome alteration was computed as a proxy for genomic instability, and statistical analyses were used to relate copy number aberrations to other variables. RESULTS: Among 329 patients evaluated, PD-L1 positivity was detected in 130 of 311 (41.7%), but high PD-L1 positivity was seen in only 30 of 311 (9.6%). PD-L1 positivity correlated with nonepithelioid histological subtype and increased infiltration with cluster of differentiation 4-positive, cluster of differentiation 8-positive, and forkhead box P3-positive lymphocytes. High PD-L1-positive expression correlated with worse prognosis (hazard ratio = 2.37, 95% confidence interval: 1.57-3.56, p < 0.001) in univariate analysis but not in multivariate analysis. Higher percent genome alteration was associated with epithelioid histological subtype and poorer survival (hazard ratio = 1.59, 95% confidence interval: 1.01-2.5, p = 0.04) but not PD-L1 expression. CONCLUSIONS: PD-L1 expression was associated with nonepithelioid MPM, poor clinical outcome, and increased immunological infiltrates. Increased genomic instability did not correlate with PD-L1 expression but was associated with poorer survival.

Development of a chitinase and v-cathepsin negative bacmid for improved integrity of secreted recombinant proteins.

The application of the baculovirus-insect cell expression system for the production of integral membrane and secreted proteins is often more troublesome than for cytoplasmic proteins. One protein expressed at low levels in insect cells is the Theileria parva sporozoite surface protein p67. Theileria parva is a protozoan parasite, which causes the tick-transmitted disease East Coast fever in cattle. Baculovirus vectors were engineered to produce a secreted form of p67 by replacing the signal peptide of p67 with the honeybee mellitin signal sequence and deleting a putative membrane anchor from the C-terminus. Furthermore, the chitinase and v-cathepsin genes were deleted from the baculovirus expression vector in a bacmid setup, allowing broad scale application of this novel vector. Deletion of the chitinase and v-cathepsin gene had a positive effect on the integrity of both the intracellular and secreted recombinant protein.