Large-scale detection and characterization of interchromosomal rearrangements in normozoospermic bulls using massive genotype and phenotype data sets.

In this paper, we developed a highly sensitive approach to detect interchromosomal rearrangements in cattle by searching for abnormal linkage disequilibrium patterns between markers located on different chromosomes in large paternal half-sib families genotyped as part of routine genomic evaluations. We screened 5571 families of artificial insemination sires from 15 breeds and revealed 13 putative interchromosomal rearrangements, 12 of which were validated by cytogenetic analysis and long-read sequencing. These consisted of one Robertsonian fusion, 10 reciprocal translocations, and the first case of insertional translocation reported in cattle. Taking advantage of the wealth of data available in cattle, we performed a series of complementary analyses to define the exact nature of these rearrangements, investigate their origins, and search for factors that may have favored their occurrence. We also evaluated the risks to the livestock industry and showed significant negative effects on several traits in the sires and in their balanced or aneuploid progeny compared with wild-type controls. Thus, we present the most comprehensive and thorough screen for interchromosomal rearrangements compatible with normal spermatogenesis in livestock species. This approach is readily applicable to any population that benefits from large genotype data sets, and will have direct applications in animal breeding. Finally, it also offers interesting prospects for basic research by allowing the detection of smaller and rarer types of chromosomal rearrangements than GTG banding, which are interesting models for studying gene regulation and the organization of genome structure.

Durable disease control and refractory bullous pemphigoid after immune checkpoint inhibitor discontinuation in metastatic renal cell carcinoma: A case report.

Background: Immune checkpoint inhibitors deeply modified metastatic renal cell carcinoma's management, and confront us to adverse events that we were not used to with conventional anti-cancer therapies. We report the case of a patient who received nivolumab as second-line treatment of a metastatic clear cell renal cell carcinoma and who developed bullous pemphigoid four years after nivolumab introduction, with persistent exacerbations even after its discontinuation. Case presentation: A 66-year-old man was diagnosed with lung metastasis eight years after radical nephrectomy for a clear cell renal cell carcinoma. He firstly received an anti-angiogenic agent combination, and then received anti-programmed death 1 (PD1) nivolumab as second-line treatment. Nivolumab led to prolonged disease control, but after four years of exposure the patient developed skin lesions consistent with bullous pemphigoid. After seven years of nivolumab administration and perfect disease stability, nivolumab was discontinued and surveillance was proposed. Despite nivolumab discontinuation, the patient continued to develop bullous pemphigoid exacerbations. Metastatic renal cell carcinoma was still perfectly stable more than two years after immune checkpoint discontinuation with no further anti-cancer therapy. Discussion: We report the case of a refractory bullous pemphigoid which occurred four years after nivolumab introduction and lasted despite nivolumab discontinuation, in a patient whose metastatic renal cell carcinoma is still controlled after more than two years without any anticancer treatment. This highlights the potential association between immune-related adverse events and response to immune checkpoint inhibitors, and underlines the occurrence of late-onset and long-lasting immune-related adverse events even after discontinuation of treatment, which must encourage us to remain vigilant in the long term.

Genome-wide analysis of hybridization in wild boar populations reveals adaptive introgression from domestic pig.

The admixture of domestic pig into French wild boar populations has been monitored since the 1980s thanks to the existence of a cytogenetic difference between the two sub-species. The number of chromosomes is 2n = 36 in wild boar and 2n = 38 in pig, respectively. This difference makes it possible to assign the "hybrid" status to wild boar individuals controlled with 37 or 38 chromosomes. However, it does not make it possible to determine the timing of the hybridization(s), nor to guarantee the absence of domestic admixture in an animal with 2n = 36 chromosomes. In order to analyze hybridization in greater detail and to avoid the inherent limitations of the cytogenetic approach, 362 wild boars (WB) recently collected in different French geographical areas and in different environments (farms, free ranging in protected or unprotected areas, animals with 2n = 36, 37 or 38 chromosomes) were genotyped on a 70K SNP chip. Principal component analyses allowed the identification of 13 "outliers" (3.6%), for which the proportion of the genome of "domestic" origin was greater than 40% (Admixture analyses). These animals were probably recent hybrids, having Asian domestic pig ancestry for most of them. For the remaining 349 animals studied, the proportion of the genome of "wild" origin varied between 83% and 100% (median: 94%). This proportion varied significantly depending on how the wild boar populations were managed. Local ancestry analyses revealed adaptive introgression from domestic pig, suggesting a critical role of genetic admixture in improving the fitness and population growth of WB. Overall, our results show that the methods used to monitor the domestic genetic contributions to wild boar populations should evolve in order to limit the level of admixture between the two gene pools.

Genotyping data of French wild boar populations using porcine genome-wide genotyping array.

OBJECTIVE: The admixture of domestic pig into wild boar populations is controlled until now, by cytogenetic analysis. Even if a first-generation hybrid animal is discernable because of its 37-chromosome karyotype, the cytogenetic method is not applicable in the case of advanced intercrosses. The aim of this study is therefore to evaluate the use of SNP (Single Nucleotide Polymorphism) markers as an alternative technology to characterize recent or past hybridization between the two sub-species. The final goal would be to develop a molecular diagnostic tool. DATA DESCRIPTION: The Geneseek Genomic Profiler High-Density porcine beadchip (GGP70KHD, Illumina, USA), comprising 68,516 porcine SNPs, was used on a set of 362 wild boars with diverse chromosomal statuses collected from different areas and breeding environments in France. We generated approximately 62,192-64,046 genotypes per wild boar. The present dataset might be useful for the community (i) for developing molecular tools to evaluate the admixture of domestic pig into wild boar populations, and (ii) for genetic diversity studies including wild boar species or phylogeny analyses of Suidae populations. Raw data files and a processed matrix data file were deposited in the ArrayExpress at European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) data portal under accession number E-MTAB-10591.

Meiotic Silencing in Pigs: A Case Study in a Translocated Azoospermic Boar.

Carriers of balanced constitutional reciprocal translocations usually present a normal phenotype, but often show reproductive disorders. For the first time in pigs, we analyzed the meiotic process of an autosome-autosome translocation associated with azoospermia. Meiotic process analysis revealed the presence of unpaired autosomal segments with histone γH2AX accumulation sometimes associated with the XY body. Additionally, γH2AX signals were observed on apparently synapsed autosomes other than the SSC1 or SSC15, as previously observed in Ataxia with oculomotor apraxia type 2 patients or knock-out mice for the Senataxin gene. Gene expression showed a downregulation of genes selected on chromosomes 1 and 15, but no upregulation of SSCX genes. We hypothesized that the total meiotic arrest observed in this boar might be due to the silencing of crucial autosomal genes by the mechanism referred to as meiotic silencing of unsynapsed chromatin (MSUC).

ICOS is widely expressed in cutaneous T-cell lymphoma, and its targeting promotes potent killing of malignant cells.

The treatment of advanced-stage cutaneous T-cell lymphoma (CTCL) remains an unmet medical need. Mogamulizumab, anti-KIR3DL2, and brentuximab vedotin (BV), an anti-CD30 antibody-drug conjugate (ADC) coupled with monomethyl-auristatin-E (MMAE), provided encouraging results, but new targeted therapies are needed. Inducible T-cell costimulator (ICOS), a T-cell costimulatory receptor, is a promising therapeutic target, not only because it is expressed by malignant T cells in CTCL but also because of its connection with the suppressive activity of regulatory T (Treg) cells. Immunohistochemical analysis revealed that ICOS was widely expressed by malignant cells in skin biopsy specimens from 52 patients with mycosis fungoides and Sézary syndrome (SS), as well as in involved node biopsy specimens from patients with SS. Furthermore, flow cytometry demonstrated its strong expression by circulating tumor cells in all our patients with SS. Percentages of ICOS+ Treg cells were significantly higher in patients with SS than in healthy donors. We then investigated the preclinical efficacy of anti-ICOS ADCs generated by coupling murine anti-ICOS monoclonal antibodies with MMAE and pyrrolobenzodiazepine. In 3 CTCL cell lines (Myla, MJ, and HUT78), we observed a significant dose-dependent decrease in cell viability in the presence of anti-ICOS ADCs. In addition, anti-ICOS-MMAE ADCs had an in vitro and in vivo efficacy superior to BV in a mouse xenograft model (MyLa). Finally, we assessed the efficacy of anti-ICOS ADCs in ICOS+ patient-derived xenografts from patients with SS and angioimmunoblastic T-cell lymphoma. Collectively, our findings provide the preliminary basis for a therapeutic trial.

Stereotactic radiosurgery combined with anti-PD1 for the management of melanoma brain metastases: A retrospective study of safety and efficacy.

BACKGROUND: Brain metastases can be effectively treated with stereotactic radiosurgery (SRS). Immune checkpoint inhibitors are now pivotal in metastatic melanoma care, but some concerns have emerged regarding the safety of their combination with radiation therapy. METHODS: We present a retrospective analysis of a cohort of patients treated by anti-PD1 and SRS as a sole modality of radiation therapy (no whole brain radiation therapy at any time) in a single institution. We included patients on anti-PD1 at the time of SRS or patients who started anti-PD1 within a maximum period of 3 months following SRS and were treated at least one year before the analysis. Clinical and serial imaging data were reviewed to determine the efficacy and the rate of adverse radiation effectss of the combination. RESULTS: A total of 50 patients were included. SRS targeted 1, 2 to 3 and >3 brain metastases in 17, 16 and 17 patients, respectively. Two patients died before the first evaluation. Nine patients presented with an increase in peritumoral oedema, three with intracranial haemorrhage and one patient with both oedema and haemorrhage. Median follow-up was 38.89 months (interquartile range 24.43; 45.28). Median overall survival from SRS was 16.62 months with 1-, 2- and 3-year rates of 60%, 40% and 35%, respectively. Median brain-Progression Free Survival was 13.2 months with 1, 2 and 3-year rates of 62.1%, 49.7% and 49.7%, respectively. CONCLUSIONS: This real-world cohort of patients treated with a homogeneous strategy combining upfront stereotactic radiosurgery and anti-PD1 shows remarkable survival rates and does not reveal unexpected toxicity.

Common origin of sequential cutaneous CD30+ lymphoproliferations with nodal involvement evidenced by genome-wide clonal evolution.

AIMS: This study sought to clarify the molecular pathways underlying the putative evolution from lymphomatoid papulosis (LyP) to cutaneous anaplastic large-cell lymphoma (c-ALCL) and lymph node invasion (LNI). METHODS AND RESULTS: We analysed nine sequential tumours from the same patient presenting with parallel evolution of LyP (n = 3) and c-ALCL (n = 1) with LNI (n = 1), combined with systemic diffuse large B-cell lymphoma (DLBCL) (n = 4). Clonality analysis showed a common clonal T-cell origin in the five CD30+ lesions, and a common clonal B-cell origin in the four DLBCL relapses. Array-comparative genomic hybridisation and targeted next-generation sequencing analysis demonstrated relative genomic stability of LyP lesions as compared with clonally related anaplastic large-cell lymphoma (ALCL) tumours, which showed 4q and 22q13 deletions involving the PRDM8 and TIMP3 tumour suppressor genes, respectively. The three analysed CD30+ lesions showed mostly private (specific to each sample) genetic alterations, suggesting early divergence from a common precursor. In contrast, DLBCL tumours showed progressive accumulation of private alterations, indicating late divergence. CONCLUSIONS: Sequential cutaneous and nodal CD30+ tumours were clonally related. This suggests that LyP, c-ALCL and LNI represent a continuous spectrum of clonal evolution emerging from a common precursor of cutaneous CD30+ lymphoproliferations. Therefore, nodal ALCL tumours in the context of LyP should be considered as a form of transformation rather than composite lymphoma.

Analysis of Meiotic Segregation Pattern and Interchromosomal Effects in a Bull Heterozygous for a 3/16 Robertsonian Translocation.

Robertsonian translocations are the most frequent chromosomal rearrangements detected in cattle. Here, we report on the detection of a new Robertsonian translocation between chromosomes BTA3 and BTA16. This rob(3;16) was dicentric, suggesting that its occurrence was recent. FISH analysis of decondensed sperm nuclei revealed a relatively low rate of unbalanced gametes produced by adjacent segregation (5.87%). In addition, and for the first time in bovines, a significant interchromosomal effect (ICE) was detected for 2 different autosomes: BTA17 (global disomy + nullisomy rate of 9%) and BTA20 (1.8%). These results suggest that ICE should be taken into consideration when assessing the putative effect of Robertsonian translocations on reproduction.

Intraindividual Variation of Meiotic Recombination Parameters in Pig Spermatocytes: A Preliminary Study.

Meiotic recombination parameters like crossover (CO) rate or synaptonemal complex (SC) length are known to vary strongly between individuals and between cells from the same individual. The origins of this variability remain elusive, and little is known about the variations that might occur between different samples and/or over time within the same individual. To document this question, pachytene cells from 3 boars of the Large White breed were analyzed twice, at a 1-year interval, using immunocytological techniques. CO rate, SC length, and MLH1 inter-foci distances varied significantly between the 3 individuals. CO rate and SC length differed significantly between the 2 sampling periods for 1 individual. However, no significant differences were observed between the 2 samples for CO distribution and inter-foci distances in the 3 boars studied.

A Single-Arm Phase II Trial of Lenalidomide in Relapsing or Refractory Primary Cutaneous Large B-Cell Lymphoma, Leg Type.

Although the combination of rituximab and polychemotherapy has improved prognosis of primary cutaneous diffuse large B-cell lymphoma, leg type, the advanced age of patients limits therapeutic options in relapsing/refractory cases. A multicenter, single-arm, phase II trial was conducted to assess the benefits and safety of lenalidomide in refractory/relapsing primary cutaneous diffuse large B-cell lymphoma, leg type. The primary endpoint was the 6-month overall response rate. Secondary endpoints were 12-month overall response rate, overall and specific survival, duration of response, progression-free survival, safety, and identification of prognostic factors. Among the 19 patients included, the 6-month overall response rate was 26.3% (90% confidence interval [CI] = 11-47.6), including four complete responses and one partial response. At 12 months, there were still two complete responses and one partial response. Median progression-free survival was 4 months. Median overall and specific survivals were 19.4 and 23.8 months, respectively. Reduced doses tended to be associated with higher 6-month overall response rate and progression-free survival. Absence of the MYD88L265P mutation was associated with a higher overall response under treatment (80.0% vs. 33.3%; P = 0.05). The most common grade 3 adverse events were hematologic. Two grade 5 adverse events occurred (sepsis and pulmonary embolism). Lenalidomide at reduced doses may allow prolonged responses in a few patients and represents a therapeutic option in relapsing/refractory primary cutaneous diffuse large B-cell lymphoma, leg type.

Clinical, virological, and biological parameters associated with outcomes of Ebola virus infection in Macenta, Guinea.

BACKGROUND. The pathogenesis of Ebola virus (EBOV) disease (EVD) is poorly characterized. The establishment of well-equipped diagnostic laboratories close to Ebola treatment centers (ETCs) has made it possible to obtain relevant virological and biological data during the course of EVD and to assess their association with the clinical course and different outcomes of the disease. METHODS. We were responsible for diagnosing EBOV infection in patients admitted to two ETCs in forested areas of Guinea. The pattern of clinical signs was recorded, and an etiological diagnosis was established by RT-PCR for EBOV infection or a rapid test for malaria and typhoid fever. Biochemical analyses were also performed. RESULTS. We handled samples from 168 patients between November 29, 2014, and January 31, 2015; 97 patients were found to be infected with EBOV, with Plasmodium falciparum coinfection in 18%. Overall mortality for EVD cases was 58%, rising to 86% if P. falciparum was also present. Viral load was higher in fatal cases of EVD than in survivors, and fatal cases were associated with higher aspartate aminotransferase (AST) and alanine aminotransferase (ALT), C-reactive protein (CRP), and IL-6 levels. Furthermore, regardless of outcome, EVD was characterized by higher creatine kinase (CPK), amylase, and creatinine levels than in febrile patients without EVD, with higher blood urea nitrogen (BUN) levels in fatal cases of EVD only. CONCLUSION. These findings suggest that a high viral load at admission is a marker of poor EVD prognosis. In addition, high AST, ALT, CRP, and IL-6 levels are associated with a fatal outcome of EVD. Damage to the liver and other tissues, with massive rhabdomyolysis and, probably, acute pancreatitis, is associated with EVD and correlated with disease severity. Finally, biochemical analyses provide substantial added value at ETCs, making it possible to improve supportive rehydration and symptomatic care for patients. FUNDING. The French Ministry of Foreign Affairs, the Agence Française de Développement, and Institut Pasteur.

A Panel of Embryonic Stem Cell Lines Reveals the Variety and Dynamic of Pluripotent States in Rabbits.

Conventional rabbit embryonic stem cell (ESC) lines are derived from the inner cell mass (ICM) of pre-implantation embryos using methods and culture conditions that are established for primate ESCs. In this study, we explored the capacity of the rabbit ICM to give rise to ESC lines using conditions similar to those utilized to generate naive ESCs in mice. On single-cell dissociation and culture in fibroblast growth factor 2 (FGF2)-free, serum-supplemented medium, rabbit ICMs gave rise to ESC lines lacking the DNA-damage checkpoint in the G1 phase like mouse ESCs, and with a pluripotency gene expression profile closer to the rabbit ICM/epiblast profiles. These cell lines can be converted to FGF2-dependent ESCs after culture in conventional conditions. They can also colonize the rabbit pre-implantation embryo. These results indicate that rabbit epiblast cells can be coaxed toward different types of pluripotent stem cells and reveal the dynamics of pluripotent states in rabbit ESCs.

Meiotic pairing and gene expression disturbance in germ cells from an infertile boar with a balanced reciprocal autosome-autosome translocation.

Individuals carrying balanced constitutional reciprocal translocations generally have a normal phenotype, but often present reproductive disorders. The aim of our research was to analyze the meiotic process in an oligoasthenoteratospermic boar carrying an asymmetric reciprocal translocation involving chromosomes 1 and 14. Different multivalent structures (quadrivalent and trivalent plus univalent) were identified during chromosome pairing analysis. Some of these multivalents were characterized by the presence of unpaired autosomal segments with histone γH2AX accumulation sometimes associated with the XY body. Gene expression in spermatocytes was studied by RNA-DNA-FISH and microarray-based testis transcriptome analysis. Our results revealed a decrease in gene expression for chromosomes 1 and 14 and an up-regulated expression of X-chromosome genes for the translocated boar compared with normal individuals. We hypothesized that the observed meiotic arrest and reproductive failure in this boar might be due to silencing of crucial autosomal genes (MSUC) and disturbance of meiotic sex chromosome inactivation (MSCI). Further analysis revealed abnormal meiotic recombination (frequency and distribution) and the production of a high rate of unbalanced spermatozoa.

Meiotic Recombination Analyses in Pigs Carrying Different Balanced Structural Chromosomal Rearrangements.

Correct pairing, synapsis and recombination between homologous chromosomes are essential for normal meiosis. All these events are strongly regulated, and our knowledge of the mechanisms involved in this regulation is increasing rapidly. Chromosomal rearrangements are known to disturb these processes. In the present paper, synapsis and recombination (number and distribution of MLH1 foci) were studied in three boars (Sus scrofa domestica) carrying different chromosomal rearrangements. One (T34he) was heterozygote for the t(3;4)(p1.3;q1.5) reciprocal translocation, one (T34ho) was homozygote for that translocation, while the third (T34Inv) was heterozygote for both the translocation and a pericentric inversion inv(4)(p1.4;q2.3). All three boars were normal for synapsis and sperm production. This particular situation allowed us to rigorously study the impact of rearrangements on recombination. Overall, the rearrangements induced only minor modifications of the number of MLH1 foci (per spermatocyte or per chromosome) and of the length of synaptonemal complexes for chromosomes 3 and 4. The distribution of MLH1 foci in T34he was comparable to that of the controls. Conversely, the distributions of MLH1 foci on chromosome 4 were strongly modified in boar T34Inv (lack of crossover in the heterosynaptic region of the quadrivalent, and crossover displaced to the chromosome extremities), and also in boar T34ho (two recombination peaks on the q-arms compared with one of higher magnitude in the controls). Analyses of boars T34he and T34Inv showed that the interference was propagated through the breakpoints. A different result was obtained for boar T34ho, in which the breakpoints (transition between SSC3 and SSC4 chromatin on the bivalents) seemed to alter the transmission of the interference signal. Our results suggest that the number of crossovers and crossover interference could be regulated by partially different mechanisms.

National survey of psoriasis flares after 2009 monovalent H1N1/seasonal vaccines.

BACKGROUND: Psoriasis is a common chronic immune-mediated disease resulting from interactions of a genetic background with environmental triggering factors such as stress and infection. So far, there is very limited information available about the impact of vaccine stimuli on psoriasis course. OBJECTIVE: To collect cases of psoriasis flares occurring after vaccination through a national survey. METHODS: We investigated cases of onset or flare of psoriasis occurring within 3 months following the 2009 monovalent H1N1/seasonal vaccination during the campaign of the 2009-2010 flu seasons in France. RESULTS: Ten patients, 6 men and 4 women with a median age of 44 years (range 9-88), were reported with a psoriasis of new onset (n = 7) or with a worsening of previously diagnosed psoriasis (n = 3) within a median time period of 8 days following vaccination. Nine of them presented with a mixed guttate/plaque clinical phenotype, and 1 showed 2 successive generalized pustular psoriasis (GPP) flares after 2 different vaccine injections. CONCLUSION: The short time interval between vaccination and onset of psoriasis flares, the lack of other triggers and the flaring sequence following 2 different vaccines in a GPP patient suggest a possible association between the 2009 monovalent H1N1/seasonal vaccination and psoriasis flaring in the collected cases. Nevertheless, the likely very low incidence of psoriasis following vaccination emphasizes the safe profile and the relevance of vaccination strategies in psoriasis patients, especially in candidates for immunosuppressive treatments.