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Tab. S1: Genes covered in the applied sequencing Panel; Tab. S2: List of vaccine peptides

Melanoma often recurs after a latency period of several years, presenting a T cell–edited phenotype that reflects a role for CD8+ T cells in maintaining metastatic latency. Here, we report an investigation of a patient with multiple recurrent lesions, where poorly immunogenic melanoma phenotypes were found to evolve in the presence of autologous tumor antigen–specific CD8+ T cells. Melanoma cells from two of three late recurrent metastases, developing within a 6-year latency period, lacked HLA class I expression. CD8+ T cell–resistant, HLA class I–negative tumor cells became clinically apparent 1.5 and 6 years into stage IV disease. Genome profiling by SNP arrays revealed that HLA class I loss in both metastases originated from a shared chromosome 15q alteration and independently acquired focal B2M gene deletions. A third HLA class I haplotype-deficient lesion developed in year 3 of stage IV disease that acquired resistance toward dominant CD8+ T-cell clonotypes targeting stage III tumor cells. At an early stage, melanoma cells showed a dedifferentiated c-Junhigh/MITFlow phenotype, possibly associated with immunosuppression, which contrasted with a c-Junlow/MITFhigh phenotype of T cell–edited tumor cells derived from late metastases. In summary, our work shows how tumor recurrences after long-term latency evolve toward T-cell resistance by independent genetic events, as a means for immune escape and immunotherapeutic resistance. Cancer Res; 76(15); 4347–58. ©2016 AACR.

Additional information about experimental techniques and materials relevant to this study

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Supplementary Figures S1-S6. Supplementary Fig. S1, localization of T cells in the periphery of metastasis Ma-Mel-86f. Supplementary Fig. S2, re-expression of HLA class I on B2M-transfected melanoma cells. Supplementary Fig. S3, no enrichment of CD4+ T cells and NK cells in metastasis Ma-Mel-86f. Supplementary Fig. S4, similar PD-L1 expression on Ma-Mel-86a and Ma-Mel-86c cells. Supplementary Fig. S5, autologous CD8+ T cells kill Ma-Mel-86c cells. Supplementary Fig. S6, autologous CD8+ T cells kill Ma-Mel-86a cells.

Telomerase reverse transcriptase (TERT) promoter mutations occur frequently in cancer, have been associated with increasedTERTexpression and cell proliferation, and could potentially influence therapeutic regimens for melanoma. As the role ofTERTexpression in malignant melanoma and the non-canonical functions of TERT remain understudied, we aimed to extend the current knowledge on both types ofTERTalterations with respect to survival, further clinical and molecular parameters. Using multivariate models,TERTalterations were not consistently associated with survival in melanoma cohorts under immune checkpoint inhibition. The presence of CD4+ T cells increased withTERTexpression and correlated with the expression of exhaustion markers. While the frequency of promoter mutations did not change with Breslow thickness,TERTexpression was increased in metastases arising from thinner primaries. Enrichment analyses of single-cell RNA-seq showedTERTexpression is associated with genes involved in cell migration and dynamics of the extracellular matrix, supporting the role ofTERTduring invasion and metastasis. Co-regulated genes in several bulk tumors and single-cell RNA-seq cohorts also indicated non-canonical functions ofTERTrelated to mitochondrial DNA stability and nuclear DNA repair in line with increasedTERTexpression during chromothripsis (PCAWG cohort) and under hypoxic conditions (PCAWG and SKCM cohorts). Also in glioblastoma (Klughammer and PCAWG cohorts),TERTwas co-expressed with DNA repair genes. Our results thus indicate a relevance ofTERTexpression in melanoma metastasis, T cell dysfunction and DNA repair across cancer entities.SignificanceIn addition to the frequently occurringTERTpromoter mutations, we testTERTexpression with respect to clinical and molecular associates, extending the canonical role ofTERTin melanoma and other cancer entities.

Large genome-scale studies are deposited in various public sequence repositories. However, their access and analysis can be non-trivial to infrequent users. Here, we present a new database connecting whole transcriptomes with clinical data for straight-forward access and analysis of patient-specific samples. Users can perform association tests of survival and gene expression across different cohorts, identify cell-type expressions, or correlate the presence of immune cells. In summary, we present a new data hub for bench scientists to perform replication and discovery studies.

Purpose A wide therapeutic repertoire has become available to oncologists including radio- and chemotherapy, small molecules and monoclonal antibodies. However, drug efficacy can be limited by genetic heterogeneity. Here, we designed a webtool that facilitates the data analysis of the in vitro drug sensitivity data on 265 approved compounds from the GDSC database in association with a plethora of genetic changes documented for 1001 cell lines in the CCLE data. Methods The webtool computes odds ratios of drug resistance for a queried set of genetic alterations. It provides results on the efficacy of single compounds or groups of compounds assigned to cellular signaling pathways. Webtool availability: https://tools.hornlab.org/GDSC/. Results We first replicated established associations of genetic driver mutations in BRAF, RAS genes and EGFR with drug response. We then tested the ‘BRCAness’ hypothesis and did not find increased sensitivity to the assayed PARP inhibitors. Analyzing specific PIK3CA mutations related to cancer and mendelian overgrowth, we found support for the described sensitivity of H1047 mutants to GSK690693 targeting the AKT pathway. Testing a co-mutated gene pair, GATA3 activation abolished PTEN-related sensitivity to PI3K/mTOR inhibition. Finally, the pharmacogenomic modifier ABCB1 was associated with olaparib resistance. Conclusions This tool could identify potential drug candidates in the presence of custom sets of genetic changes and moreover, improve the understanding of signaling pathways. The underlying computer code can be adapted to larger drug response datasets to help structure and accommodate the increasingly large biomedical knowledge base.

Purpose Genetic diagnostics of neurodevelopmental disorders with epilepsy (NDDE) are predominantly applied in children, thus limited information is available regarding adults or elderly. Methods We investigated 150 adult/elderly individuals with NDDE by conventional karyotyping, FMR1 testing, chromosomal microarray, panel sequencing, and for unresolved cases, also by exome sequencing (nsingle = 71, ntrios = 24). Results We identified (likely) pathogenic variants in 71 cases (47.3%) comprising fragile X syndrome (n = 1), disease-causing copy number (n = 23), and single-nucleotide variants (n = 49). Seven individuals displayed multiple independent genetic diagnoses. The diagnostic yield correlated with the severity of intellectual disability. Individuals with anecdotal evidence of exogenic early-life events (e.g., nuchal cord, complications at delivery) with alleged/unproven association to the disorder had a particularly high yield of 58.3%. Screening for disease-specific comorbidities was indicated in 45.1% and direct treatment consequences arose in 11.8% of diagnosed individuals. Conclusion Panel/exome sequencing displayed the highest yield and should be considered as first-tier diagnostics in NDDE. This high yield and the numerous indications for additional screening or treatment modifications arising from genetic diagnoses indicate a current medical undersupply of genetically undiagnosed adult/elderly individuals with NDDE. Moreover, knowledge of the course of elderly individuals will ultimately help in counseling newly diagnosed individuals with NDDE.

Endometrial mesonephric-like adenocarcinoma (ML-AC) represents a recently recognized subtype of endometrial adenocarcinoma (AC) associated with a subtle immunophenotype with a characteristic KRAS-mutation. Detailed clinico-pathologic analyses and prognostic data on ML-AC are limited. We report a series of four cases with histopathological, immunohistochemical, and molecular analyses. These cases as well as the data of previously published cases were reviewed for clinico-pathologic variables and clinical follow-up information. Forty cases of ML-AC were identified. ML-AC represents about 1% of all endometrial carcinomas. Similar to other types of endometrial AC, vaginal bleeding was the leading presenting symptom, and the mean age was 60.0 years (range 31–91). More than a half of the patients presented with locally advanced disease (≥ FIGO stage II) at time of diagnosis, developed a recurrence or died of the disease within a mean follow-up period of 24.7 months (range 3–144.5 months). The most common site of distant disease was pulmonary involvement. Microscopically, ML-ACs present with mixed morphology and show a co-expression of so-called mesonephric and Mullerian markers, suggesting a Mullerian origin of the tumors. Immunostaining for PD-L1 was negative in all tested cases, using different antibodies against PD-L1. Retained staining for mismatch repair proteins on immunohistochemistry and a POLE-mutation suggest a copy number low phenotype within the molecular classification of endometrial carcinomas. Almost all cases showed a KRAS-mutation at codon 12 (mostly G12V). Uterine ML-AC represents a distinct subtype of invasive endometrial AC, associated with KRAS-mutations and characteristic immunohistochemical findings. Clinically, ML-AC may show an aggressive behavior with a high rate of recurrent disease and a substantial risk for distant metastatic disease, especially to the lungs.

The Bering Land Bridge (BLB) last connected Eurasia and North America during the Late Pleistocene. Although the BLB would have enabled transfers of terrestrial biota in both directions, it also acted as an ecological filter whose permeability varied considerably over time. Here we explore the possible impacts of this ecological corridor on genetic diversity within, and connectivity among, populations of a once wide-ranging group, the caballine horses (Equus spp.). Using a panel of 187 mitochondrial and eight nuclear genomes recovered from present-day and extinct caballine horses sampled across the Holarctic, we found that Eurasian horse populations initially diverged from those in North America, their ancestral continent, around 1.0–0.8 million years ago. Subsequent to this split our mitochondrial DNA analysis identified two bidirectional long-range dispersals across the BLB ~875–625 and ~200–50 thousand years ago, during the Middle and Late Pleistocene. Whole genome analysis indicated low levels of gene flow between North American and Eurasian horse populations, which probably occurred as a result of these inferred dispersals. Nonetheless, mitochondrial and nuclear diversity of caballine horse populations retained strong phylogeographical structuring. Our results suggest that barriers to gene flow, currently unidentified but possibly related to habitat distribution across Beringia or ongoing evolutionary divergence, played an important role in shaping the early genetic history of caballine horses, including the ancestors of living horses within Equus ferus. © 2021 John Wiley & Sons Ltd 19-05-00477, AAAA-A19-119032590102-7; National Science Foundation, NSF: ARC-1417036; Gordon and Betty Moore Foundation, GBMF: 3804; University of California, Santa Cruz, UCSC; U.S. Bureau of Land Management, BLM; Horizon 2020 Framework Programme, H2020: 681605; Seventh Framework Programme, FP7: IEF-302617; Natural Sciences and Engineering Research Council of Canada, NSERC; Marie Curie; European Research Council, ERC; Svenska Forskningsrådet Formas: 2018-01640; Russian Foundation for Basic Research, РФФИ: 18-04-00327; Knut och Alice Wallenbergs Stiftelse; Vetenskapsrådet, VR; Science for Life Laboratory, SciLifeLab We are grateful to the placer gold mining community, the Tr’ondëk Hwëch’in First Nation, and the Vuntut Gwitchin First Nation for their collaboration and support with our research in Yukon. Tamara Pico, Sarah Crump and Paul Koch provided advice interpreting the palaeoclimate of Beringia. Data generation was supported with funds from NSF ARC-1417036, the Gordon & Betty Moore Foundation (no. 3804) and the American Wild Horse Campaign. A.O.V. was additionally supported by a UCSC Chancellor’s Dissertation Year Fellowship and the CANA Foundation. M.T. and D.G. were supported by the Russian Foundation for Basic Research (project 18-04-00327), and L.E. by a Marie-Curie Intra-European fellowship (FP7, IEF-302617). G.B. is supported by the Federal theme of Zoological Institute of the Russian Academy of Sciences no. AAAA-A19-119032590102-7. S.V. was funded by the Russian Foundation for Basic Research, Grant 19-05-00477. L.D. acknowledges support from FORMAS (project 2018-01640). D.F. was supported by the Natural Science and Engineering Research Council. P.G. and D.M. are supported by the Bureau of Land Management. We acknowledge support from Science for Life Laboratory, the Knut and Alice Wallenberg Foundation, the National Genomics Infrastructure funded by the Swedish Research Council, and Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. L.O. received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement 681605). We are grateful to the placer gold mining community, the Tr?ond?k Hw?ch?in First Nation, and the Vuntut Gwitchin First Nation for their collaboration and support with our research in Yukon. Tamara Pico, Sarah Crump and Paul Koch provided advice interpreting the palaeoclimate of Beringia. Data generation was supported with funds from NSF ARC-1417036, the Gordon & Betty Moore Foundation (no. 3804) and the American Wild Horse Campaign. A.O.V. was additionally supported by a UCSC Chancellor?s Dissertation Year Fellowship and the CANA Foundation. M.T. and D.G. were supported by the Russian Foundation for Basic Research (project 18-04-00327), and L.E. by a Marie-Curie Intra-European fellowship (FP7, IEF-302617). G.B. is supported by the Federal theme of Zoological Institute of the Russian Academy of Sciences no. AAAA-A19-119032590102-7. S.V. was funded by the Russian Foundation for Basic Research, Grant 19-05-00477. L.D. acknowledges support from FORMAS (project 2018-01640). D.F. was supported by the Natural Science and Engineering Research Council. P.G. and D.M. are supported by the Bureau of Land Management. We acknowledge support from Science for Life Laboratory, the Knut and Alice Wallenberg Foundation, the National Genomics Infrastructure funded by the Swedish Research Council, and Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. L.O. received funding from the European Research Council (ERC) under the European Union?s Horizon 2020 research and innovation programme (grant agreement 681605).

The arrival of bison in North America marks one of the most successful large-mammal dispersals from Asia within the last million years, yet the timing and nature of this event remain poorly determined. Here, we used a combined paleontological and paleogenomic approach to provide a robust timeline for the entry and subsequent evolution of bison within North America. We characterized two fossil-rich localities in Canada's Yukon and identified the oldest well-constrained bison fossil in North America, a 130,000-y-old steppe bison, Bison cf. priscus We extracted and sequenced mitochondrial genomes from both this bison and from the remains of a recently discovered, ∼120,000-y-old giant long-horned bison, Bison latifrons, from Snowmass, Colorado. We analyzed these and 44 other bison mitogenomes with ages that span the Late Pleistocene, and identified two waves of bison dispersal into North America from Asia, the earliest of which occurred ∼195-135 thousand y ago and preceded the morphological diversification of North American bison, and the second of which occurred during the Late Pleistocene, ∼45-21 thousand y ago. This chronological arc establishes that bison first entered North America during the sea level lowstand accompanying marine isotope stage 6, rejecting earlier records of bison in North America. After their invasion, bison rapidly colonized North America during the last interglaciation, spreading from Alaska through continental North America; they have been continuously resident since then.

Severe early onset epilepsies are often caused by de novo pathogenic variants. Few studies have reported the frequency of somatic mosaicism in parents of children with severe epileptic encephalopathies. Here we aim to investigate the frequency of mosaicism in the parents of children with epilepsy caused by alleged de novo variants. We tested parental genomic DNA derived from different tissues for 75 cases using targeted next-generation sequencing. Five parents (6.6%) showed mosaicism at minor allele frequencies of 0.8%-29% for the pathogenic variant detected in their offspring. Parental mosaicism was observed in the following genes: SCN1A, SCN2A, SCN8A, and STXBP1. One of the identified parents had epilepsy himself. Our results show that de novo events can occur already in parental tissue and in some cases can be detected in peripheral blood. Consequently, parents affected by low-grade mosaicism are faced with an increased recurrence risk for transmitting the pathogenic variant, compared to the overall recurrence risk for a second affected child estimated at approximately 1%. However, testing for parental somatic mosaicism will help identifying those parents who truly are at higher risk and will significantly improve genetic counseling in the respective families.

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification., Ohio State University Comprehensive Cancer Center Barretos Cancer Hospital. Grant Number: FINEP ‐ CT‐INFRA (02/2010) Breast Cancer Foundation of New Zealand Canadian Institutes of Health Research. Grant Number: PSR‐SIIRI‐701 Cancer Research UK. Grant Numbers: C8197/A16565, C5047/A8384, C1281/A12014, C12292/A11174, C1287/A10710, C1287/A10118, C1287/A16563, C5047/A10692, C5047/A15007 Department of Defence, USA. Grant Number: W81XWH‐10‐1‐0341 Helsinki University Hospital Research fund Scientific Foundation Asociación Española Contra el Cáncer Leiden University Medical Centre. Grant Number: Grant 30.925 Generalitat de Catalunya. Grant Numbers: PERIS_MedPerCan, URDCat, 2017SGR1282, 2017SGR496 Royal Society of New Zealand Cancer Council Victoria Netherlands Organization for Scientific Research (NWO). Grant Number: Grant 017.008.022 Breast Cancer Research Foundation Cancer Foundation of Western Australia EU H2020. Grant Number: 634935 Fundación Mutua Madrileña Seventh Framework Programme. Grant Numbers: 634935, 223175, 633784 Cancer Council South Australia Government of Galicia. Grant Number: Consolidation and structuring program: IN607B Cancer Council Tasmania Italian Association of Cancer Research. Grant Number: 15547 Queensland Cancer Fund AstraZeneca National Institute of Health (USA). Grant Numbers: 1U19 CA148065‐01, CA128978, CA192393, 1U19 CA148537, P50 CA1162091, CA116167, 1U19 CA148112 Newcastle University Dutch Cancer Society KWF. Grant Numbers: KWF/Pink Ribbon‐11704, UL2012‐5649 National Institute for Health Research. Grant Number: Manchester Biomedical Research centre (IS‐BRC‐1215 National Council of Technological and Scientific Development (CNPq) Instituto de Salud Carlos III. Grant Numbers: FIS PI15/00355, FIS PI13/01711, CIBERONC, FIS PI16/01218, PI16/00563 French National Institute of Cancer National Breast Cancer Foundation National Health and Medical Research Council. Grant Numbers: ID1061778, ID1104808 Carlos III National Health Centro de Investigación Biomédica en Red de Enferemdades Raras. Grant Number: ACCI 2016: ER17P1AC7112/2018 Cancer Council NSW Deutsche Krebshilfe. Grant Numbers: (#110837, #70111850 Fondazione Pisa. Grant Number: Grant “Clinical characterization of BRCA 1/2 Mis

Melanoma often recurs after a latency period of several years, presenting a T cell–edited phenotype that reflects a role for CD8+ T cells in maintaining metastatic latency. Here, we report an investigation of a patient with multiple recurrent lesions, where poorly immunogenic melanoma phenotypes were found to evolve in the presence of autologous tumor antigen–specific CD8+ T cells. Melanoma cells from two of three late recurrent metastases, developing within a 6-year latency period, lacked HLA class I expression. CD8+ T cell–resistant, HLA class I–negative tumor cells became clinically apparent 1.5 and 6 years into stage IV disease. Genome profiling by SNP arrays revealed that HLA class I loss in both metastases originated from a shared chromosome 15q alteration and independently acquired focal B2M gene deletions. A third HLA class I haplotype-deficient lesion developed in year 3 of stage IV disease that acquired resistance toward dominant CD8+ T-cell clonotypes targeting stage III tumor cells. At an early stage, melanoma cells showed a dedifferentiated c-Junhigh/MITFlow phenotype, possibly associated with immunosuppression, which contrasted with a c-Junlow/MITFhigh phenotype of T cell–edited tumor cells derived from late metastases. In summary, our work shows how tumor recurrences after long-term latency evolve toward T-cell resistance by independent genetic events, as a means for immune escape and immunotherapeutic resistance. Cancer Res; 76(15); 4347–58. ©2016 AACR.

The extinct ‘New World stilt-legged’, or NWSL, equids constitute a perplexing group of Pleistocene horses endemic to North America. Their slender distal limb bones resemble those of Asiatic asses, such as the Persian onager. Previous palaeogenetic studies, however, have suggested a closer relationship to caballine horses than to Asiatic asses. Here, we report complete mitochondrial and partial nuclear genomes from NWSL equids from across their geographic range. Although multiple NWSL equid species have been named, our palaeogenomic and morphometric analyses support the idea that there was only a single species of middle to late Pleistocene NWSL equid, and demonstrate that it falls outside of crown group Equus. We therefore propose a new genus, Haringtonhippus, for the sole species H. francisci. Our combined genomic and phenomic approach to resolving the systematics of extinct megafauna will allow for an improved understanding of the full extent of the terminal Pleistocene extinction event., eLife digest The horse family – which also includes zebras, donkeys and asses – is often featured on the pages of textbooks about evolution. All living horses belong to a group, or genus, called Equus. The fossil record shows how the ancestors of these animals evolved from dog-sized, three-toed browsers to larger, one-toed grazers. This process took around 55 million years, and many members of the horse family tree went extinct along the way. Nevertheless, the details of the horse family tree over the past 2.5 million years remain poorly understood. In North America, horses from this period – which is referred to as the Pleistocene – have been classed into two major groups: stout-legged horses and stilt-legged horses. Both groups became extinct near the end of the Pleistocene in North America, and it was not clear how they relate to one another. Based on their anatomy, many scientists suggested that stilt-legged horses were most closely related to modern-day asses living in Asia. Yet, other studies using ancient DNA placed the stilt-legged horses closer to the stout-legged horses. Heintzman et al. set out to resolve where the stilt-legged horses sit within the horse family tree by examining more ancient DNA than the previous studies. The analyses showed that the stilt-legged horses were much more distinct than previously thought. In fact, contrary to all previous findings, these animals actually belonged outside of the genus Equus. Heintzman et al. named the new genus for the stilt-legged horses Haringtonhippus, and showed that all stilt-legged horses belonged to a single species within this genus, Haringtonhippus francisci. Together these new findings provide a benchmark for reclassifying problematic fossil groups across the tree of life. A similar approach could be used to resolve the relationships in other problematic groups of Pleistocene animals, such as mammoths and bison. This would give scientists a more nuanced understanding of evolution and extinction during this period.

© 2014 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd. Polar bears are an arctic, marine adapted species that is closely related to brown bears. Genome analyses have shown that polar bears are distinct and genetically homogeneous in comparison to brown bears. However, these analyses have also revealed a remarkable episode of polar bear gene flow into the population of brown bears that colonized the Admiralty, Baranof and Chichagof islands (ABC islands) of Alaska. Here, we present an analysis of data from a large panel of polar bear and brown bear genomes that includes brown bears from the ABC islands, the Alaskan mainland and Europe. Our results provide clear evidence that gene flow between the two species had a geographically wide impact, with polar bear DNA found within the genomes of brown bears living both on the ABC islands and in the Alaskan mainland. Intriguingly, while brown bear genomes contain up to 8.8% polar bear ancestry, polar bear genomes appear to be devoid of brown bear ancestry, suggesting the presence of a barrier to gene flow in that direction.

This is a reply to the comments of Morey (2014) on our identification of Palaeolithic dogs from several European Palaeolithic sites. In his comments Morey (2014) presents some misrepresentations and misunderstandings that we remedy here. In contrast to what Morey (2014) propounds, our results suggest that the domestication of the wolf was a long process that started early in the Upper Palaeolithic and that since that time two sympatric canid morphotypes can be seen in Eurasian sites: Pleistocene wolves and Palaeolithic dogs. Contrary to Morey (2014), we are convinced that the study of this domestication process should be multidisciplinary.

Recent advances in sequencing technologies supported the development of molecularly targeted therapy in cancer patients. Thus, genomic analyses are becoming a routine part in clinical practice and accurate detection of actionable mutations is essential to assist diagnosis and therapy choice. However, this is often challenging due to major problems associated with DNA from formalin-fixed paraffin-embedded tissue which is usually the primary source for genetic testing. Here we want to share our experience regarding major problems associated with FFPE DNA used for PCR-based sequencing as illustrated by the mutational analysis of ERBB4 in melanoma. We want to focus on two major problems including extensive DNA fragmentation and hydrolytic deamination as source of non-reproducible sequence artifacts. Further, we provide potential explanations and possible strategies to minimize these difficulties and improve the detection of targetable mutations. Genomic DNA from formalin-fixed paraffin-embedded tumor samples was isolated followed by PCR amplification, Sanger sequencing and statistical analysis. Analysis of Sanger sequencing data revealed a total of 46 ERBB4 mutations in 27 of 96 samples including the identification of 11 mutations at three previously unknown mutational hotspots. Unfortunately, we were not able to confirm any assumed hotspot mutation within repeated sequencing of relevant amplicons suggesting the detection of sequence artifacts most likely caused by DNA lesions associated with FFPE tissues. Since DNA from FFPE tissue is usually the primary source for mutational analyses, appropriate measures must be implemented in the workflow to assess DNA damage in formalin-fixed tissue to ensure accurate detection of actionable mutations and minimize the occurrence of sequence artifacts.

here is the tendency to assume that endangered species have been both genetically and demographically healthier in the past, so that any genetic erosion observed today was caused by their recent decline. The Iberian lynx (Lynx pardinus) suffered a dramatic and continuous decline during the 20th century, and now shows extremely low genome- and species-wide genetic diversity among other signs of genomic erosion. We analyze ancient (N = 10), historical (N = 245), and contemporary (N = 172) samples with microsatellite and mitogenome data to reconstruct the species' demography and investigate patterns of genetic variation across space and time. Iberian lynx populations transitioned from low but significantly higher genetic diversity than today and shallow geographical differentiation millennia ago, through a structured metapopulation with varying levels of diversity during the last centuries, to two extremely genetically depauperate and differentiated remnant populations by 2002. The historical subpopulations show varying extents of genetic drift in relation to their recent size and time in isolation, but these do not predict whether the populations persisted or went finally extinct. In conclusion, current genetic patterns were mainly shaped by genetic drift, supporting the current admixture of the two genetic pools and calling for a comprehensive genetic management of the ongoing conservation program. This study illustrates how a retrospective analysis of demographic and genetic patterns of endangered species can shed light onto their evolutionary history and this, in turn, can inform conservation actions., This research was funded by the Spanish Dirección General de Investigación Científica y Técnica, through projects CGL2006-10853/BOS, CGL2010-21540/BOS, and CGL2013-47755-P of the Spanish Ministerio de Ciencia e Innovación (MICINN) to J.A.G., and by the Fundación BBVA through a project on ancient lynx genetics to M.D. M.C. and E.M. received a JAE predoctoral grant from CSIC (Spanish National Research Council), M.L.P. was supported by PhD contracts from Programa Internacional de Becas “La Caixa-Severo Ochoa.” J.N. received financial support through projects HAR2014-55131 from the Ministerio de Ciencia e Innovación (MICINN) and SGR2014-108 from Generalitat de Catalunya.

We report here a new discovery of a cave bear left metatarsal 3 from Rochedane, an archaeological site near Montbeliard (French Jura) that yielded only Lateglacial and Holocene material, with no evidence of pre-LGM deposits, a context that made this bone a possible candidate for being a post-LGM cave bear in western Europe. To test this hypothesis, this bone was analyzed for mitochondrial DNA, which confirmed its attribution to cave bear of the Ursus spelaeus lineage, and a direct radiocarbon AMS dating on well preserved collagen (%C, %N and C/N well in the range of fresh collagen) yielded an age of 23,900 +110 -100 BP (28,730-28,500 cal BP, one sigma range). Its carbon and nitrogen isotopic values were similar to those of slightly older cave bears from the Swabian Jura, around 300 km to the East, suggesting that the ecological preferences of cave bears remained unchanged until the extirpation of this species in western Europe. Interestingly, the genetic type U. spelaeus was replaced by Ursus ingressus around 28,000 C-14 BP in the Swabian Jura. In contrast, the older type U. spelaeus apparently persisted in France ca. 3000 years longer. Traces left on the cave bear metapodium have been left by human activity on this bone, as it was the case for older cave bear bones from the Swabian Jura. This case study shows that cave bear remains found in post-LGM sites or layers may be candidates to be late survivors of this extinct species, but without direct radiocarbon AMS dated on well-preserved collagen (demonstrated by actual chemical composition results) and ancient DNA confirmation of the species attribution, such evidence can only be considered dubious. (C) 2013 Elsevier Ltd and INQUA. All rights reserved.

Cave bears are among the most well known extinct Pleistocene mammals. Their biogeography and taxonomy, along with the factors that led to their extinction, have been subject to long-standing controversy. Here, we reconstruct the phylogeography as well as the temporal and spatial population dynamics of cave bears across their range using mitochondrial DNA control region sequences from 77 published as well as 65 new cave bear samples, Our analyses reveal a dramatic loss of genetic diversity in cave bear populations after 30,000 years before present and provide evidence for a range decline from east to west towards the onset of the last glacial maximum. Our results also suggest that the three major haplogroups within cave bears, which may correspond to distinct species, were previously more widespread, with relict populations in remote and alpine areas still harbouring haplotypes that have disappeared from most of their previous range. Applying a phylogenetic dating approach, we estimated the age of the oldest of our samples, originating from the Yana River region in north-eastern Siberia, to be around 178,000 years, which confirms a previous estimate of a Middle Pleistocene age based on its stratigraphic position. Our results extend our knowledge about the evolutionary history of cave bears, but they also show that to unravel the complexities of cave bear evolution future ancient DNA studies on this Pleistocene species will need to go beyond short mitochondrial DNA fragments, including full mitochondrial genomes as well as nuclear DNA sequences.

After centuries of human hunting, the Eurasian beaver Castor fiber had disappeared from most of its original range by the end of the 19th century. The surviving relict populations are characterized by both low genetic diversity and strong phylogeographical structure. However, it remains unclear whether these attributes are the result of a human-induced, late Holocene bottleneck or already existed prior to this reduction in range. To investigate genetic diversity in Eurasian beaver populations during the Holocene, we obtained mitochondrial control region DNA sequences from 48 ancient beaver samples and added 152 modern sequences from GenBank. Phylogeographical analyses of the data indicate a differentiation of European beaver populations into three mitochondrial clades. The two main clades occur in western and eastern Europe, respectively, with an early Holocene contact zone in eastern Europe near a present-day contact zone. A divergent and previously unknown clade of beavers from the Danube Basin survived until at least 6000 years ago, but went extinct during the transition to modern times. Finally, we identify a recent decline in effective population size of Eurasian beavers, with a stronger bottleneck signal in the western than in the eastern clade. Our results suggest that the low genetic diversity and the strong phylogeographical structure in recent beavers are artefacts of human hunting-associated population reductions. While beaver populations have been growing rapidly since the late 19th century, genetic diversity within modern beaver populations remains considerably reduced compared to what was present prior to the period of human hunting and habitat reduction.

This is a response to the comments of Crockford and Kuzmin (2012) on our identification of Palaeolithic dogs from different European Palaeolithic sites. In their comments Crockford and Kuzmin (2012) present some errors, misunderstandings and misrepresentations that we remedy here. In our opinion, the early wolf domestication must be regarded as an intimate relationship between humans and canids including the breeding of the latter by prehistoric people, resulting in the European Palaeolithic dogs.

// Mathias Stiller 1, 2, 3 , Antje Sucker 2 , Klaus Griewank 2 , Daniela Aust 4 , Gustavo Bruno Baretton 4 , Dirk Schadendorf 2 , Susanne Horn 1, 2 1 Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, D-04103 Leipzig, Germany 2 Department of Dermatology, University Hospital, West German Cancer Center, University Duisburg-Essen, and German Consortium for Translational Cancer Research (DKTK), D-45147 Essen, Germany 3 Department for Translational Skin Cancer Research, University Duisburg-Essen, and German Consortium for Translational Cancer Research (DKTK), D-45141 Essen, Germany 4 Departments of Surgery and Pathology, Technical University Dresden, D-01307 Dresden, Germany Correspondence to: Susanne Horn, email: Susanne.Horn@uk-essen.de Mathias Stiller, email: m.stiller@dkfz-heidelberg.de Keywords: high-throughput sequencing, library preparation, formalin-fixed paraffin embedded (FFPE) tissue, cancer, whole exome sequencing Received: April 27, 2016 Accepted: June 30, 2016 Published: July 24, 2016 ABSTRACT DNA derived from formalin-fixed and paraffin-embedded (FFPE) tissue has been a challenge to large-scale genomic sequencing, due to its low quality and quantities. Improved techniques enabling the genome-wide analysis of FFPE material would be of great value, both from a research and clinical perspective. Comparing a single-strand DNA library preparation method originally developed for ancient DNA to conventional protocols using double-stranded DNA derived from FFPE material we obtain on average 900-fold more library molecules and improved sequence complexity from as little as 5 ng input DNA. FFPE DNA is highly fragmented, usually below 100bp, and up to 60% of reads start after or end prior to adenine residues, suggesting that crosslinks predominate at adenine residues. Similar to ancient DNA, C > T substitutions are slightly increased with maximum rates up to 3% at the ends of molecules. In whole exome sequencing of single-strand libraries from lung, breast, colorectal, prostate and skin cancers we identify known cancer mutations. In summary, we show that single-strand library preparation enables genomic sequencing, even from low amounts of degraded FFPE DNA. This method provides a clear advantage both in research and clinical settings, where FFPE material (e.g. from biopsies) often is the only source of DNA available. Improving the genetic characterization that can be performed on conventional archived FFPE tissue, the single-strand library preparation allows scarce samples to be used in personalized medicine and enables larger sample sizes in future sequencing studies.

The Ice Free Corridor has been invoked as a route for Pleistocene human and animal dispersals between eastern Beringia and more southerly areas of North America. Despite the significance of the corridor, there are limited data for when and how this corridor was used. Hypothetical uses of the corridor include: the first expansion of humans from Beringia into the Americas, northward postglacial expansions of fluted point technologies into Beringia, and continued use of the corridor as a contact route between the north and south. Here, we use radiocarbon dates and ancient mitochondrial DNA from late Pleistocene bison fossils to determine the chronology for when the corridor was open and viable for biotic dispersals. The corridor was closed after ∼23,000 until 13,400 calendar years ago (cal y BP), after which we find the first evidence, to our knowledge, that bison used this route to disperse from the south, and by 13,000 y from the north. Our chronology supports a habitable and traversable corridor by at least 13,000 cal y BP, just before the first appearance of Clovis technology in interior North America, and indicates that the corridor would not have been available for significantly earlier southward human dispersal. Following the opening of the corridor, multiple dispersals of human groups between Beringia and interior North America may have continued throughout the latest Pleistocene and early Holocene. Our results highlight the utility of phylogeographic analyses to test hypotheses about paleoecological history and the viability of dispersal routes over time.

Melanocytic tumors originating in the central nervous system (MT-CNS) are rare tumors that generally have a favorable prognosis, however malignant tumors do occur. Pathogenetically MT-CNS are not well characterized. Similar to uveal melanoma and blue nevi, they frequently harbor activating GNAQ or GNA11 mutations. Rare NRAS mutations have also been reported. Other mutations have not yet been described. We analyzed 19 MT-CNS, 7 uveal melanomas and 19 cutaneous melanomas using a targeted next generation sequencing approach analyzing 29 genes known to be frequently mutated in other melanocytic tumors (in particular uveal and cutaneous melanomas). In concordance with previous studies, cutaneous melanoma samples showed frequent NRAS or BRAF mutations, as well as mutations in other genes (e.g. NF1, RAC1, PIK3CA, ARID1A). Metastasized uveal melanomas exhibited mutations in GNAQ, GNA11 and BAP1. In contrast, MT-CNS almost exclusively demonstrated mutations in GNAQ (71 %) or GNA11 (12 %). Interestingly both GNA11 mutations identified were detected in MT-CNS diagnosed as intermediate grade melanocytomas which also recurred. One of these recurrent cases also harbored an inactivating BAP1 mutation and was found to have lost one copy of chromosome 3. Our findings show that while MT-CNS do have GNAQ or GNA11 mutations, they rarely harbor other recurrent mutations found in uveal or cutaneous melanomas. Considering chromosome 3 and BAP1 loss are robust markers of poor prognosis in uveal melanoma, it will prove interesting to determine whether these genomic alterations are also of prognostic significance in MT-CNS.

This study presents a cohesive review of the existing radiometric data as well as morphological and genetic analysis of bear remains from ten high-alpine caves, mostly from the Middle Wurmian Interstadial complex, roughly corresponding to the marine isotope stage (MIS) 3 and dating back between 65,000–30,000 years before present. Today these caves are located in an area without any vegetation, which could not provide the herbivorous bears with sufficient food resources. It therefore can be concluded that the Middle Wurmian in the Alps had to be warmer than it is today. Furthermore, congruent and conflicting data from soil formation in loess sequences as well as sinter data in caves are discussed in more detail to evaluate this hypothesis.

Palaeogenetic investigations in three geographically close caves (Hohle Fels, Geisenklosterle, and Sirgenstein) in the Ach Valley near Blaubeuren (Swabian Jura) document the sudden replacement of Ursus spelaeus by Ursus ingressus around 28,000 14 C BP. New radiocarbon dates suggest an earlier immigration of Ursus ingressus and at least a partial coexistence with Ursus spelaeus some 4500 years before the ultimate replacement. These two genetic types of cave bears used the same caves for hibernation and had the same herbivorous diet, as shown by the stable isotope results. In contrast, sympatric brown bears (Ursus arctos) exhibited a clearly different ecology, as shown by the carnivorous pattern of their isotopic signatures, and probably did not use the caves as dens before the Last Glacial Maximum (LGM). Once established, the younger cave bear (Ursus ingressus) remained the only cave bear for only another circa 2000 years after the last appearance of the classical cave bear (Ursus spelaeus) in the Ach Valley and elsewhere. The final appearance of cave bear (sensu lato) is now dated to 25,560 � 130 BP, disproving a refuge area of this species in the Swabian Jura. After the extinction of cave bears (sensu lato), brown bears took over their cave dens and their nutritional niche as they shift to a diet dominated by plant food. 2011 Elsevier Ltd and INQUA.

In the Austrian caves of Gamssulzen and Ramesch, two genetically distinct cave bears, Ursus ingressus and Ursus spelaeus eremus, apparently lived side by side for 15,000 years, together with brown bears Ursus arctos. The possible ecological partitioning of these three types of bears was investigated using multi-isotopic tracking of organic (δ13Ccoll, δ15Ncoll) and inorganic (δ13Ccarb, δ18Ocarb, δ18OPO4) fractions of bone. The cave bears from Ramesch, Ursus spelaeus eremus, were ecologically distinct from the cave bears from Gamssulzen, Ursus ingressus, both being ecologically distinct from brown bears from Ramesch, Ursus arctos. Both cave bear types were purely herbivorous but likely consumed different plant types and/or plants from different habitats, while brown bears included some animal proteins in their diet. Bone apatite δ18O values strongly suggest that both types of cave bears used isotopically distinct water sources, indicating that they may not have occupied the same landscape, either separated in space or in time due to climatic shifts. Therefore, the influence of environmental conditions strongly constrained the genetic structure of these bears.

The Pleistocene to Holocene transition was accompanied by a worldwide extinction event affecting numerous mammalian species. Several species such as the woolly mammoth and the giant deer survived this extinction wave, only to go extinct a few thousand years later during the Holocene. Another example for such a Holocene extinction is the Don-hare, Lepus tanaiticus, which inhabited the Russian plains during the late glacial. After being slowly replaced by the extant mountain hare (Lepus timidus), it eventually went extinct during the middle Holocene. Here, we report the phylogenetic relationship of L. tanaiticus and L. timidus based on a 339-basepair (bp) fragment of the mitochondrial D-loop. Phylogenetic tree- and network reconstructions do not support L. tanaiticus and L. timidus being different species. Rather, we suggest that the two taxa represent different morphotypes of a single species and the extinction of ‘L. tanaiticus’ represents the disappearance of a local morphotype rather than the extinction of a species.

The causes of the late Pleistocene megafaunal extinctions are still enigmatic. Although the fossil record can provide approximations for when a species went extinct, the timing of its disappearance alone cannot resolve the causes and mode of the decline preceding its extinction. However, ancient DNA analyses can reveal population size changes over time and narrow down potential causes of extinction. Here, we present an ancient DNA study comparing late Pleistocene population dynamics of two closely related species, cave and brown bears. We found that the decline of cave bears started approximately 25,000 years before their extinction, whereas brown bear population size remained constant. We conclude that neither the effects of climate change nor human hunting alone can be responsible for the decline of the cave bear and suggest that a complex of factors including human competition for cave sites lead to the cave bear's extinction.

Using multivariate techniques, several skulls of fossil large canids from sites in Belgium, Ukraine and Russia were examined to look for possible evidence of the presence of Palaeolithic dogs. Reference groups constituted of prehistoric dogs, and recent wolves and dogs. The fossil large canid from Goyet (Belgium), dated at c. 31,700 BP is clearly different from the recent wolves, resembling most closely the prehistoric dogs. Thus it is identified as a Palaeolithic dog, suggesting that dog domestication had already started during the Aurignacian. The Epigravettian Mezin 5490 (Ukraine) and Mezhirich (Ukraine) skulls are also identified as being Palaeolithic dogs. Selected Belgian specimens were analyzed for mtDNA and stable isotopes. All fossil samples yielded unique DNA sequences, indicating that the ancient Belgian large canids carried a substantial amount of genetic diversity. Furthermore, there is little evidence for phylogeographic structure in the Pleistocene large canids, as they do not form a homogenous genetic group. Although considerable variation occurs in the fossil canid isotope signatures between sites, the Belgian fossil large canids preyed in general on horse and large bovids.

Analyses of plant DNA in feces provides a promising, yet largely unexplored, means of documenting the diets of elusive primates. Here we demonstrate the promise and pitfalls of this approach using DNA extracted from fecal samples of wild western gorillas (Gorilla gorilla) and black and white colobus monkeys (Colobus guereza). From these DNA extracts we amplified, cloned, and sequenced small segments of chloroplast DNA (part of the rbcL gene) and plant nuclear DNA (ITS-2). The obtained sequences were compared to sequences generated from known plant samples and to those in GenBank to identify plant taxa in the feces. With further optimization, this method could provide a basic evaluation of minimum primate dietary diversity even when knowledge of local flora is limited. This approach may find application in studies characterizing the diets of poorly-known, unhabituated primate species or assaying consumer-resource relationships in an ecosystem.

// Simone L. Scholz 1 , Susanne Horn 2 , Rajmohan Murali 6, 7 , Inga Moller 2 , Antje Sucker 2 , Wiebke Sondermann 2 , Mathias Stiller 2, 5 , Bastian Schilling 2 , Elisabeth Livingstone 2 , Lisa Zimmer 2 , Henning Reis 3 , Claudia H. Metz 1 , Michael Zeschnigk 4 , Annette Paschen 2 , Klaus-Peter Steuhl 1 , Dirk Schadendorf 2 , Henrike Westekemper 1 , Klaus G. Griewank 2 1 Department of Ophthalmology, University Hospital Essen, University Duisburg-Essen, West German Cancer Center and the German Cancer Consortium (DKTK), Essen Germany 2 Department of Dermatology, University Hospital Essen, University Duisburg-Essen, West German Cancer Center and the German Cancer Consortium (DKTK), Essen Germany 3 Institute of Pathology, University Hospital Essen, University Duisburg-Essen, West German Cancer Center and the German Cancer Consortium (DKTK), Essen Germany 4 Department of Human Genetics, University Hospital Essen, University Duisburg-Essen, West German Cancer Center and the German Cancer Consortium (DKTK), Essen Germany 5 University Duisburg-Essen and the German Cancer Consortium (DKTK), Essen Germany 6 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York NY, USA 7 Marie-Josee and Henry R. Kravis Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, New York NY, USA Correspondence to: Klaus Griewank, e-mail: klaus.griewank@uk-essen.de Keywords: melanoma, SDHD, promoter mutations Abbreviations: NGS = next generation sequencing; ETS = E26 transformation-specific; SDHD = Succinate dehydrogenase complex subunit D Received: May 09, 2015 Accepted: July 15, 2015 Published: July 27, 2015 ABSTRACT Objectives: Recently, recurrent mutations in regulatory DNA regions, such as promoter mutations in the TERT gene were identified in melanoma. Subsequently, Weinhold et al. reported SDHD promoter mutations occurring in 10% of melanomas and being associated with a lower overall survival rate. Our study analyzes the mutation rate and clinico-pathologic associations of SDHD promoter mutations in a large cohort of different melanoma subtypes. Methods: 451 melanoma samples (incl. 223 non-acral cutaneous, 38 acral, 33 mucosal, 43 occult, 43 conjunctival and 51 uveal melanoma) were analyzed for the presence of SDHD promoter mutations by Sanger-sequencing. Statistical analysis was performed to screen for potential correlations of SDHD promoter mutation status with various clinico-pathologic criteria. Results: The SDHD promoter was successfully sequenced in 451 tumor samples. ETS binding site changing SDHD promoter mutations were identified in 16 (4%) samples, of which 5 mutations had not been described previously. Additionally, 5 point mutations not located in ETS binding elements were identified. Mutations in UV-exposed tumors were frequently C>T. One germline C>A SDHD promoter mutation was identified. No statistically significant associations between SDHD promoter mutation status and various clinico-pathologic variables or overall patient survival were observed. Conclusions: Melanomas harbor recurrent SDHD promoter mutations, which occur primarily as C>T alterations in UV-exposed melanomas. In contrast to the initial report and promoter mutations in the TERT gene, our analysis suggests that SDHD promoter mutations are a relatively rare event in melanoma (4% of tumors) of unclear clinical and prognostic relevance.

Viruses preserved in ancient materials provide snapshots of past viral diversity and a means to trace viral evolution through time. Here, we use a metagenomics approach to identify filterable and nuclease-resistant nucleic acids preserved in 700-y-old caribou feces frozen in a permanent ice patch. We were able to recover and characterize two viruses in replicated experiments performed in two different laboratories: a small circular DNA viral genome (ancient caribou feces associated virus, or aCFV) and a partial RNA viral genome (Ancient Northwest Territories cripavirus, or aNCV). Phylogenetic analysis identifies aCFV as distantly related to the plant-infecting geminiviruses and the fungi-infecting Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 and aNCV as within the insect-infecting Cripavirus genus. We hypothesize that these viruses originate from plant material ingested by caribou or from flying insects and that their preservation can be attributed to protection within viral capsids maintained at cold temperatures. To investigate the tropism of aCFV, we used the geminiviral reverse genetic system and introduced a multimeric clone into the laboratory model plant Nicotiana benthamiana. Evidence for infectivity came from the detection of viral DNA in newly emerged leaves and the precise excision of the viral genome from the multimeric clones in inoculated leaves. Our findings indicate that viral genomes may in some circumstances be protected from degradation for centuries.

The genus Equus is richly represented in the fossil record, yet our understanding of taxonomic relationships within this genus remains limited. To estimate the phylogenetic relationships among modern horses, zebras, asses and donkeys, we generated the first data set including complete mitochondrial sequences from all seven extant lineages within the genus Equus. Bayesian and Maximum Likelihood phylogenetic inference confirms that zebras are monophyletic within the genus, and the Plains and Grevy’s zebras form a well-supported monophyletic group. Using ancient DNA techniques, we further characterize the complete mitochondrial genomes of three extinct equid lineages (the New World stilt-legged horses, NWSLH; the subgenus Sussemionus; and the Quagga, Equus quagga quagga). Comparisons with extant taxa confirm the NWSLH as being part of the caballines, and the Quagga and Plains zebras as being conspecific. However, the evolutionary relationships among the non-caballine lineages, including the now-extinct subgenus Sussemionus, remain unresolved, most likely due to extremely rapid radiation within this group. The closest living outgroups (rhinos and tapirs) were found to be too phylogenetically distant to calibrate reliable molecular clocks. Additional mitochondrial genome sequence data, including radiocarbon dated ancient equids, will be required before revisiting the exact timing of the lineage radiation leading up to modern equids, which for now were found to have possibly shared a common ancestor as far as up to 4 Million years ago (Mya).