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Background Genomic alterations of the anaplastic lymphoma kinase gene (ALK) occur recurrently in neuroblastoma, a pediatric malignancy of the sympathetic nervous system. However, information on their development over time has remained sparse. Methods ALK alterations were assessed in neuroblastomas at diagnosis and/or relapse from a total of 943 patients, covering all stages of disease. Longitudinal information on diagnostic and relapsed samples from individual patients was available in 101 and 102 cases for mutation and amplification status, respectively. Results At diagnosis, ALK point mutations occurred in 10.5% of all cases, with highest frequencies in stage 4 patients ALK alteration frequency increased by 70%, both in high-risk and non-high-risk cases. The increase was most likely due to de novo mutations, frequently leading to R1275Q substitutions, which are sensitive to pharmacological ALK inhibition. By contrast, the frequency of ALK amplifications did not change over the course of the disease. ALK amplifications, but not mutations, were associated with poor patient outcome. Conclusions The considerably increased frequency of ALK mutations at relapse and their high prevalence in young stage 4 patients suggest surveying the genomic ALK status regularly in these patient cohorts, and to evaluate ALK-targeted treatment also in intermediate-risk patients.

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Purpose:Pleomorphic dermal sarcoma (PDS) is a rare malignant cutaneous tumor with an unknown cell of origin. Locally defined tumors can be treated by curative excisions, whereas advanced stages of the disease are difficult to treat, using standard regimens.Experimental Design:We performed whole-exome sequencing on a cohort of 28 individuals and corresponding transcriptomic analysis on 21 patients, as well as quantitative IHC image analysis on 27 patients.Results:PDS exhibits a universally high mutational load (42.7 mutations/mega base) with an inflamed, immunogenic tumor microenvironment. Three cases of PDS showed response to immune checkpoint blockade. Local mutation rate variation together with mRNA expression data demonstrate that PDS form a distinct entity, with PDGFRB as a lineage marker. In addition, we found that PDS is of mesenchymal, fibroblastic differentiation.Conclusions:PDS is of fibroblastic differentiation and exhibits a strong susceptibility to immunotherapy, including a high mutational burden and an inflamed tumor microenvironment.

Quality measurements of exome sequencing

The mechanisms behind the evolution of complex genomic amplifications in cancer have remained largely unclear. Using whole-genome sequencing data of the pediatric tumor neuroblastoma, we here identified a type of amplification, termed 'seismic amplification', that is characterized by multiple rearrangements and discontinuous copy number levels. Overall, seismic amplifications occurred in 9.9% (274 of 2,756) of cases across 38 cancer types, and were associated with massively increased copy numbers and elevated oncogene expression. Reconstruction of the development of seismic amplification showed a stepwise evolution, starting with a chromothripsis event, followed by formation of circular extrachromosomal DNA that subsequently underwent repetitive rounds of circular recombination. The resulting amplicons persisted as extrachromosomal DNA circles or had reintegrated into the genome in overt tumors. Together, our data indicate that the sequential occurrence of chromothripsis and circular recombination drives oncogene amplification and overexpression in a substantial fraction of human malignancies.

The identification of the mutational processes operating in tumour cells has implications for cancer diagnosis and therapy. These processes leave mutational patterns on the cancer genomes, which are referred to as mutational signatures. Recently, 81 mutational signatures have been inferred using computational algorithms on sequencing data of 23,879 samples. However, these published signatures may not always offer a comprehensive view on the biological processes underlying tumour types that are not included or underrepresented in the reference studies. To circumvent this problem, we designed CaMuS (Cancer Mutational Signatures) to construct de novo signatures while simultaneously fitting publicly available mutational signatures. Furthermore, we propose to estimate signature similarity by comparing probability distributions using the Hellinger distance. We applied CaMuS to infer signatures of mutational processes in poorly studied cancer types. We used whole genome sequencing data of 56 neuroblastoma, thus providing evidence for the versatility of CaMuS. Using simulated data, we compared the performance of CaMuS to sigfit, a recently developed algorithm with comparable inference functionalities. CaMuS and sigfit reconstructed the simulated datasets with similar accuracy; however two main features may argue for CaMuS over sigfit: (i) superior computational performance and (ii) a reliable parameter selection method to avoid spurious signatures.

The spectrum of somatic genetic variation in colorectal adenomas caused by biallelic pathogenic germline variants in the MSH3 gene, was comprehensively analysed to characterise mutational signatures and identify potential driver genes and pathways of MSH3-related tumourigenesis. Three patients from two families with MSH3-associated polyposis were included. Whole exome sequencing of nine adenomas and matched normal tissue was performed. The amount of somatic variants in the MSH3-deficient adenomas and the pattern of single nucleotide variants (SNVs) was similar to sporadic adenomas, whereas the fraction of small insertions/deletions (indels) (21–42% of all small variants) was significantly higher. Interestingly, pathogenic somatic APC variants were found in all but one adenoma. The vast majority (12/13) of these were di-, tetra-, or penta-base pair (bp) deletions. The fraction of APC indels was significantly higher than that reported in patients with familial adenomatous polyposis (FAP) (p < 0.01) or in sporadic adenomas (p < 0.0001). In MSH3-deficient adenomas, the occurrence of APC indels in a repetitive sequence context was significantly higher than in FAP patients (p < 0.01). In addition, the MSH3-deficient adenomas harboured one to five (recurrent) somatic variants in 13 established or candidate driver genes for early colorectal carcinogenesis, including ACVR2A and ARID genes. Our data suggest that MSH3-related colorectal carcinogenesis seems to follow the classical APC-driven pathway. In line with the specific function of MSH3 in the mismatch repair (MMR) system, we identified a characteristic APC mutational pattern in MSH3-deficient adenomas, and confirmed further driver genes for colorectal tumourigenesis.

Purpose: Pleomorphic dermal sarcoma (PDS) is a rare malignant cutaneous tumor with an unknown cell of origin. Locally defined tumors can be treated by curative excisions, whereas advanced stages of the disease are difficult to treat, using standard regimens. Experimental Design: We performed whole-exome sequencing on a cohort of 28 individuals and corresponding transcriptomic analysis on 21 patients, as well as quantitative IHC image analysis on 27 patients. Results: PDS exhibits a universally high mutational load (42.7 mutations/mega base) with an inflamed, immunogenic tumor microenvironment. Three cases of PDS showed response to immune checkpoint blockade. Local mutation rate variation together with mRNA expression data demonstrate that PDS form a distinct entity, with PDGFRB as a lineage marker. In addition, we found that PDS is of mesenchymal, fibroblastic differentiation. Conclusions: PDS is of fibroblastic differentiation and exhibits a strong susceptibility to immunotherapy, including a high mutational burden and an inflamed tumor microenvironment.

The mechanisms behind the evolution of complex genomic amplifications in cancer have remained largely unclear. Using whole-genome sequencing data of the pediatric tumor neuroblastoma, we here identified a type of amplification, termed 'seismic amplification', that is characterized by multiple rearrangements and discontinuous copy number levels. Overall, seismic amplifications occurred in 9.9% (274 of 2,756) of cases across 38 cancer types, and were associated with massively increased copy numbers and elevated oncogene expression. Reconstruction of the development of seismic amplification showed a stepwise evolution, starting with a chromothripsis event, followed by formation of circular extrachromosomal DNA that subsequently underwent repetitive rounds of circular recombination. The resulting amplicons persisted as extrachromosomal DNA circles or had reintegrated into the genome in overt tumors. Together, our data indicate that the sequential occurrence of chromothripsis and circular recombination drives oncogene amplification and overexpression in a substantial fraction of human malignancies.

Multiple introductions of a species are thought to enhance its invasion success by increasing genotypic diversity; this involves frequent crossing among different lineages. However, genetic diversity through crossing is less likely in autogamous species. To understand the impact of multiple introductions on the colonization success of autogamous species, we studied hairy bittercress, Cardamine hirsuta, which invaded Japan several decades ago. We detected temporal changes in its population structure using nine microsatellite markers amplified from leaf samples collected from 87 sites between 2009 and 2010, and herbarium specimens collected between 1988 and 2007. To examine whether the phenotypic variation corresponded with the genetic population structure, we also investigated the geographic variation in the lateral stamen number of this species across 49 sites. The present populations can be divided into three genetic groups, which are distributed in northern, eastern, and western Japan. This finding suggests that there are three invasive lineages (North, East, and West) in Japan. The geographic variation in lateral stamen number corresponded to the distributions of these lineages. The former distributions of the North and West lineages mostly corresponded to those found at present, but they were also historically found in eastern Japan. However, the East lineage has apparently expanded into eastern Japan, resulting in a change in dominant lineages over only a few decades. For the autogamous C. hirsuta, multiple introductions contributed toward colonization success over a wider range, which was associated with a local change in the dominant lineages.

Background Accurate and automated phenotyping of leaf images is necessary for high throughput studies of leaf form like genome-wide association analysis and other forms of quantitative trait locus mapping. Dissected leaves (also referred to as compound) that are subdivided into individual units are an attractive system to study diversification of form. However, there are only few software tools for their automated analysis. Thus, high-throughput image processing algorithms are needed that can partition these leaves in their phenotypically relevant units and calculate morphological features based on these units. Results We have developed MowJoe, an image processing algorithm that dissects a dissected leaf into leaflets, petiolule, rachis and petioles. It employs image skeletonization to convert leaves into graphs, and thereafter applies algorithms operating on graph structures. This partitioning of a leaf allows the derivation of morphological features such as leaf size, or eccentricity of leaflets. Furthermore, MowJoe automatically places landmarks onto the terminal leaflet that can be used for further leaf shape analysis. It generates specific output files that can directly be imported into downstream shape analysis tools. We applied the algorithm to two accessions of Cardamine hirsuta and show that our features are able to robustly discriminate between these accessions. Conclusion MowJoe is a tool for the semi-automated, quantitative high throughput shape analysis of dissected leaf images. It provides the statistical power for the detection of the genetic basis of quantitative morphological variations. Electronic supplementary material The online version of this article (10.1186/s13007-018-0290-y) contains supplementary material, which is available to authorized users.

Summary 1. Plants vary widely in the extent to which they defend themselves against herbivores. Because the resources available to plants are often site-specific, variation among sites dictates investment into defence, and may reveal a growth-defence trade-off. Moreover, plants that have evolved different life-history strategies in different environments may situate themselves on this trade-off curve differently. For instance, plants that flower later have a longer vegetative lifespan, and may accordingly defend themselves differently than those that flower earlier. 2. Here, we tested whether late-flowering plants, with a longer vegetative lifespan, invest more in defence than early-flowering plants, using recombinant genotypes of the annual herb Cardamine hirsuta that differ in flowering time as a result of differences in the activity of the major floral repressor Flowering Locus C (FLC). 3. We found that variation at FLC was mainly responsible for regulating flowering time and allocation to reproduction, but this partially depended on where the plants grew. We also found that variation at FLC mediated plant allocation to defence, with late- flowering plants producing higher levels of total glucosinolates and stress-related phytohormones. Nonetheless, plant growth and the qualitative values of plant defence and plant resistance against specialist herbivores were mainly independent from FLC. 4. Synthesis - Our results highlight pleiotropic effects associated with flowering-time genes that might influence plant defence and plant-herbivore interactions.

A key problem in biology is whether the same processes underlie morphological variation between and within species. Here, by using plant leaves as an example, we show that the causes of diversity at these two evolutionary scales can be divergent. Some species like the model plant Arabidopsis thaliana have simple leaves, whereas others like the A. thaliana relative Cardamine hirsuta bear complex leaves comprising leaflets. Previous work has shown that these interspecific differences result mostly from variation in local tissue growth and patterning. Now, by cloning and characterizing a quantitative trait locus (QTL) for C. hirsuta leaf shape, we find that a different process, age-dependent progression of leaf form, underlies variation in this trait within species. This QTL effect is caused by cis-regulatory variation in the floral repressor ChFLC, such that genotypes with low-expressing ChFLC alleles show both early flowering and accelerated age-dependent changes in leaf form, including faster leaflet production. We provide evidence that this mechanism coordinates leaf development with reproductive timing and may help to optimize resource allocation to the next generation.

A major goal in biology is to identify the genetic basis for phenotypic diversity. This goal underpins research in areas as diverse as evolutionary biology, plant breeding and human genetics. A limitation for this research is no longer the availability of sequence information but the development of functional genetic tools to understand the link between changes in sequence and phenotype. Here we describe Cardamine hirsuta, a close relative of the reference plant Arabidopsis thaliana, as an experimental system in which genetic and transgenic approaches can be deployed effectively for comparative studies. We present high-resolution genetic and cytogenetic maps for C. hirsuta and show that the genome structure of C. hirsuta closely resembles the eight chromosomes of the ancestral crucifer karyotype and provides a good reference point for comparative genome studies across the Brassicaceae. We compared morphological and physiological traits between C. hirsuta and A. thaliana and analysed natural variation in stamen number in which lateral stamen loss is a species characteristic of C. hirsuta. We constructed a set of recombinant inbred lines and detected eight quantitative trait loci that can explain stamen number variation in this population. We found clear phylogeographic structure to the genetic variation in C. hirsuta, thus providing a context within which to address questions about evolutionary changes that link genotype with phenotype and the environment., This work was supported by Biotechnology and Biological Sciences Research Council grants BB/D010977/1 and BB/F012934/1 to M.T., BB/H01313X/1 to A.H., Deutsche Forschungsgemeinschaft ‘Adaptomics’ grant TS 229/1-1 to M.T. and A.H., Max Planck Society core grant to M.T., Human Frontier Science Program grant to A.H., Natural Environment Research Council NE/E00489X/1 to D.F., Czech Science Foundation grant P501/10/1014 to M.L., Spanish Ministerio de Economía y Competitividad – FEDER grant BIO2011-23489 to J.M. and a Genetics Society Heredity Fieldwork grant to E.C. A.H. was supported by the Max Planck Society W2 Minerva programme and a Royal Society University Research Fellowship, and M.T. by a Royal Society Wolfson Merit award, Cluster of Excellence on Plant Sciences, and the Gatsby Charitable Foundation.

Leaves show considerable variation in shape, and may be described as simple, when the leaf is entire, or dissected, when the leaf is divided into individual leaflets. Here, we report that the SIMPLE LEAF3 (SIL3) gene is a novel determinant of leaf shape in Cardamine hirsuta - a dissected-leaved relative of the simple-leaved model species Arabidopsis thaliana. We show that SIL3 is required for leaf growth and leaflet formation but leaf initiation is less sensitive to perturbation of SIL3 activity. SIL3 is further required for KNOX (knotted1-like homeobox) gene expression and localized auxin activity maxima, both of which are known to promote leaflet formation. We cloned SIL3 and showed that it encodes RLI2 (RNase L inhibitor 2), an ATP binding cassette-type ATPase with important roles in ribosome recycling and translation termination that are conserved in eukaryotes and archaea. RLI mutants have not been described in plants to date, and this paper highlights the potential of genetic studies in C. hirsuta to uncover novel gene functions. Our data indicate that leaflet development is sensitive to perturbation of RLI2-dependent aspects of cellular growth, and link ribosome function with dissected-leaf development. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

Finding causal relationships between genotypic and phenotypic variation is a key focus of evolutionary biology, human genetics and plant breeding. To identify genome-wide patterns underlying trait diversity, we assembled a high-quality reference genome of Cardamine hirsuta, a close relative of the model plant Arabidopsis thaliana. We combined comparative genome and transcriptome analyses with the experimental tools available in C. hirsuta to investigate gene function and phenotypic diversification. Our findings highlight the prevalent role of transcription factors and tandem gene duplications in morphological evolution. We identified a specific role for the transcriptional regulators PLETHORA5/7 in shaping leaf diversity and link tandem gene duplication with differential gene expression in the explosive seed pod of C. hirsuta. Our work highlights the value of comparative approaches in genetically tractable species to understand the genetic basis for evolutionary change.

SummaryMorphological diversity is often caused by altered gene expression of key developmental regulators. However, the precise developmental trajectories through which morphologies evolved remain poorly understood. It is also unclear to what degree genetic changes contributing to morphological divergence were fixed by natural selection. Here we investigate these problems in the context of evolutionary developmental transitions that produced the simple unlobed leaf of the model species Arabidopsis thaliana. We demonstrate that A. thaliana leaf shape likely derived from a more complex lobed ancestral state that persists in extant Arabidopsis species. We also show that evolution of the unlobed leaf form in A. thaliana involved loss of expression of the knotted1-like homeobox gene SHOOTMERISTEMLESS (STM) in leaves and that cis-regulatory divergence contributed to this process. Further, we provide evidence for a selective sweep at the A. thaliana STM locus, indicating that loss of STM expression in A. thaliana leaves may have been fixed by positive selection. In summary, our data provide key information as to when and how the characteristic leaf form of A. thaliana evolved.

Spatial regulation of C-function genes controlling reproductive organ identity in the centre of the flower can be achieved by adjusting the level of their expression within the genuine central expression domain in Antirrhinum and Petunia. Loss of this control in mutants is revealed by enhanced C-gene expression in the centre and by lateral expansion of the C-domain. In order to test whether the level of central C-gene expression and hence the principle of 'regulation by tuning' also applies to spatial regulation of the C-function gene AGAMOUS (AG) in Arabidopsis, we generated transgenic plants with enhanced central AG expression by using stem cell-specific CLAVATA3 (CLV3) regulatory sequences to drive transcription of the AG cDNA. The youngest terminal flowers on inflorescences of CLV3::AG plants displayed homeotic features in their outer whorls indicating ectopic AG expression. Dependence of the homeotic feature on the age of the plant is attributed to the known overall weakening of repressive mechanisms controlling AG. Monitoring AG with an AG-I::GUS reporter construct suggests ectopic AG expression in CLV3::AG flowers when AG in the inflorescence is still repressed, although in terminating inflorescence meristems, AG expression expands to all tissues. Supported by genetic tests, we conclude that upon enhanced central AG expression, the C-domain laterally expands necessitating tuning of the expression level of C-function genes in the wild type. The tuning mechanism in C-gene regulation in Arabidopsis is discussed as a late security switch that ensures wild-type C-domain control when other repressive mechanism starts to fade and fail.

Item does not contain fulltext It is commonly thought that deep phylogenetic conservation of plant microRNAs (miRNAs) and their targets indicates conserved regulatory functions. We show that the blind (bl) mutant of Petunia hybrida and the fistulata (fis) mutant of Antirrhinum majus, which have similar homeotic phenotypes, are recessive alleles of two homologous miRNA-encoding genes. The BL and FIS genes control the spatial restriction of homeotic class C genes to the inner floral whorls, but their ubiquitous early floral expression patterns are in contradiction with a potential role in patterning C gene expression. We provide genetic evidence for the unexpected function of the MIRFIS and MIRBL genes in the center of the flower and propose a dynamic mechanism underlying their regulatory role. Notably, Arabidopsis thaliana, a more distantly related species, also contains this miRNA module but does not seem to use it to confine early C gene expression to the center of the flower.