Prevalence of NTRK Fusions in Canadian Solid Tumour Cancer Patients.

INTRODUCTION: Neurotrophic tyrosine receptor kinase (NTRK) gene fusions occur in ~ 0.3% of all solid tumours but are enriched in some rare tumour types. Tropomyosin receptor kinase (TRK) inhibitors larotrectinib and entrectinib are approved as tumour-agnostic therapies for solid tumours harbouring NTRK fusions. METHODS: This study investigated the prevalence of NTRK fusions in Canadian patients and also aimed to help guide NTRK testing paradigms through analysis of data reported from a national clinical diagnostic testing program between September 2019 and July 2021. RESULTS: Of 1,687 patients included in the final analysis, NTRK fusions were detected in 0.71% (n = 12) of patients representing salivary gland carcinoma (n = 3), soft tissue sarcoma (n = 3), CNS (n = 3), and one in each of melanoma, lung, and colorectal cancer. All three salivary gland carcinomas contained ETV6-NTRK3 fusions. Thirteen (0.77%) clinically actionable incidental findings were also detected. Two of the 13 samples containing incidental findings were NTRK fusion-positive (GFOD1-NTRK2, FGFR3-TACC3 in a glioblastoma and AFAP1-NTRK2, BRAF c.1799T>A in a glioma). The testing algorithm screened most patient samples via pan-TRK immunohistochemistry (IHC), whereas samples from the central nervous system (CNS), pathognomonic cancers, and confirmed/ putative NTRK fusion-positive samples identified under research protocols were reflexed straight to next-generation sequencing (NGS). CONCLUSION: These findings highlight the benefit and practicality of a diagnostic testing program to identify patients suitable for tumour-agnostic TRK inhibitor therapies, as well as other targeted therapies, due to clinically actionable incidental findings identified. Collectively, these findings may inform future guidance on selecting the appropriate testing approach per tumour type and on optimal NTRK testing algorithms.

Salt stress alters the spectrum of de novo mutation available to selection during experimental adaptation of Chlamydomonas reinhardtii.

The genetic basis of adaptation is driven by both selection and the spectrum of available mutations. Given that the rate of mutation is not uniformly distributed across the genome and varies depending on the environment, understanding the signatures of selection across the genome is aided by first establishing what the expectations of genetic change are from mutation. To determine the interaction between salt stress, selection, and mutation across the genome, we compared mutations observed in a selection experiment for salt tolerance in Chlamydomonas reinhardtii to those observed in mutation accumulation (MA) experiments with and without salt exposure. MA lines evolved under salt stress had a single-nucleotide mutation rate of 1.1 × 10 - 9 $1.1 \times 10^{-9}$ , similar to that of MA lines under standard conditions ( 9.6 × 10 - 10 $9.6 \times 10^{-10}$ ). However, we found that salt stress led to an increased rate of indel mutations, but that many of these mutations were removed under selection. Finally, lines adapted to salt also showed excess clustering of mutations in the genome and the co-expression network, suggesting a role for positive selection in retaining mutations in particular compartments of the genome during the evolution of salt tolerance. Our study shows that characterizing mutation rates and spectra expected under stress helps disentangle the effects of environment and selection during adaptation.

Inferring the distribution of fitness effects of spontaneous mutations in Chlamydomonas reinhardtii.

Spontaneous mutations are the source of new genetic variation and are thus central to the evolutionary process. In molecular evolution and quantitative genetics, the nature of genetic variation depends critically on the distribution of effects of mutations on fitness and other quantitative traits. Spontaneous mutation accumulation (MA) experiments have been the principal approach for investigating the overall rate of occurrence and cumulative effect of mutations but have not allowed the phenotypic effects of individual mutations to be studied directly. Here, we crossed MA lines of the green alga Chlamydomonas reinhardtii with its unmutated ancestral strain to create haploid recombinant lines, each carrying an average of 50% of the accumulated mutations in a large number of combinations. With the aid of the genome sequences of the MA lines, we inferred the genotypes of the mutations, assayed their growth rate as a measure of fitness, and inferred the distribution of fitness effects (DFE) using a Bayesian mixture model. We infer that the DFE is highly leptokurtic (L-shaped). Of mutations with absolute fitness effects exceeding 1%, about one-sixth increase fitness in the laboratory environment. The inferred distribution of effects for deleterious mutations is consistent with a strong role for nearly neutral evolution. Specifically, such a distribution predicts that nucleotide variation and genetic variation for quantitative traits will be insensitive to change in the effective population size.

Fungus Causing White-Nose Syndrome in Bats Accumulates Genetic Variability in North America with No Sign of Recombination.

Emerging fungal diseases of wildlife are on the rise worldwide, and the white-nose syndrome (WNS) epidemic in North American bats is a catastrophic example. The causal agent of WNS is a single clone of the fungus Pseudogymnoascus destructans. Early evolutionary change in this clonal population has major implications for disease ecology and conservation. Accumulation of variation in the fungus through mutation, and shuffling of variation through recombination, could affect the virulence and transmissibility of the fungus and the durability of what appears to be resistance arising in some bat populations. Our genome-wide analysis shows that the clonal population of P. destructans has expanded in size from a single genotype, has begun to accumulate variation through mutation, and presents no evidence as yet of genetic exchange among individuals. IMPORTANCE Since its discovery in 2006, the emerging infectious disease known as white-nose syndrome has killed millions of bats in North America, making it one of the most devastating wildlife epidemics in recorded history. We demonstrate that there has been as yet only spontaneous mutation across the North American population of P. destructans, and we find no indication of recombination. Thus, selective forces, which might otherwise impact pathogenic virulence, have so far had essentially no genetic variation on which to act. Our study confirmed the time of origin for the first and, thus far, only introduction of P. destructans to North America. This system provides an unprecedented opportunity to follow the evolution of a host-pathogen interaction unfolding in real time.

The effect of sex on the repeatability of evolution in different environments.

The adaptive function of sex has been extensively studied, while less consideration has been given to the potential downstream consequences of sex on evolution. Here, we investigate one such potential consequence, the effect of sex on the repeatability of evolution. By affecting the repeatability of evolution, sex could have important implications for biodiversity, and for our ability to make predictions about the outcome of environmental change. We allowed asexual and sexual populations of Chlamydomonas reinhardtii to evolve in novel environments and monitored both their change in fitness and variance in fitness after evolution. Sex affected the repeatability of evolution by changing the importance of the effect of selection, chance, and ancestral constraints on the outcome of the evolutionary process. In particular, the effects of sex were highly dependent on the initial genetic composition of the population and on the environment. Given the lack of a consistent effect of sex on repeatability across the environments used here, further studies to dissect in more detail the underlying reasons for these differences as well as studies in additional environments are required if we are to have a general understanding of the effects of sex on the repeatability of evolution.

Experimental adaptation to marine conditions by a freshwater alga.

The marine-freshwater boundary has been suggested as one of the most difficult to cross for organisms. Salt is a major ecological factor and provides an unequalled range of ecological opportunity because marine habitats are much more extensive than freshwater habitats, and because salt strongly affects the structure of microbial communities. We exposed experimental populations of the freshwater alga Chlamydomonas reinhardtii to steadily increasing concentrations of salt. About 98% of the lines went extinct. The ones that survived now thrive in growth medium with 36 g⋅L(-1) NaCl, and in seawater. Our results indicate that adaptation to marine conditions proceeded first through genetic assimilation of an inducible response to relatively low salt concentrations that was present in the ancestors, and subsequently by the evolution of an enhanced inducible response to high salt concentrations. These changes appear to have evolved through reversible and irreversible modifications, respectively. The evolution of marine from freshwater lineages is an example that clearly indicates the possibility of studying certain aspects of major ecological transitions in the laboratory.

Repeatability of adaptation in experimental populations of different sizes.

The degree to which evolutionary trajectories and outcomes are repeatable across independent populations depends on the relative contribution of selection, chance and history. Population size has been shown theoretically and empirically to affect the amount of variation that arises among independent populations adapting to the same environment. Here, we measure the contribution of selection, chance and history in different-sized experimental populations of the unicellular alga Chlamydomonas reinhardtii adapting to a high salt environment to determine which component of evolution is affected by population size. We find that adaptation to salt is repeatable at the fitness level in medium (Ne = 5 × 10(4)) and large (Ne = 4 × 10(5)) populations because of the large contribution of selection. Adaptation is not repeatable in small (Ne = 5 × 10(3)) populations because of large constraints from history. The threshold between stochastic and deterministic evolution in this case is therefore between effective population sizes of 10(3) and 10(4). Our results indicate that diversity across populations is more likely to be maintained if they are small. Experimental outcomes in large populations are likely to be robust and can inform our predictions about outcomes in similar situations.

Evolutionary rescue of sexual and asexual populations in a deteriorating environment.

The environmental change experienced by many contemporary populations of organisms poses a serious risk to their survival. From the theory of evolutionary rescue, we predict that the combination of sex and genetic diversity should increase the probability of survival by increasing variation and thereby the probability of generating a type that can tolerate the stressful environment. We tested this prediction by comparing experimental populations of Chlamydomonas reinhardtii that differ in sexuality and in the initial amount of genetic diversity. The lines were serially propagated in an environment where the level of stress caused by salt increased over time from fresh water to the limits of marine conditions. In the long term, the combination of high diversity and obligate sexuality was most effective in supporting evolutionary rescue. Most of the adaptation to high-salt environments in the obligate sexual-high diversity lines had occurred by midway through the experiment, indicating that positive genetic correlations of adaptation to lethal stress with adaptation to sublethal stress greatly increased the probability of evolutionary rescue. The evolutionary rescue events observed in this study provide evidence that major shifts in ways of life can arise within short time frames through the action of natural selection in sexual populations.

Overshooting dynamics in a model adaptive radiation.

The history of life is punctuated by repeated periods of unusually rapid evolutionary diversification called adaptive radiation. The dynamics of diversity during a radiation reflect an overshooting pattern with an initial phase of exponential-like increase followed by a slower decline. Much attention has been paid to the factors that drive the increase phase, but far less is known about the causes of the decline phase. Decreases in diversity are rarely associated with climatic changes or catastrophic events, suggesting that they may be an intrinsic consequence of diversification. We experimentally identify the factors responsible for losses in diversity during the later stages of the model adaptive radiation of the bacterium Pseudomonas fluorescens. Proximately, diversity declines because of the loss of biofilm-forming niche specialist morphotypes. We show that this loss occurs despite the presence of strong divergent selection late in the radiation and is associated with continued adaptation of resident niche specialists to both the biotic and abiotic environments. These results suggest that losses of diversity in the latter stages of an adaptive radiation may be a general consequence of diversification through competition and lends support to the idea that the conditions favouring the emergence of diversity are different from those that ensure its long-term maintenance.