Quality control and quantification in IG/TR next-generation sequencing marker identification: protocols and bioinformatic functionalities by EuroClonality-NGS

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ARResT/Interrogate: an interactive immunoprofiler for IG/TR NGS data.

Abstract Motivation The study of immunoglobulins and T cell receptors using next-generation sequencing has finally allowed exploring immune repertoires and responses in their immense variability and complexity. Unsurprisingly, their analysis and interpretation is a highly convoluted task. Results We thus implemented ARResT/Interrogate, a web-based, interactive application. It can organize and filter large amounts of immunogenetic data by numerous criteria, calculate several relevant statistics, and present results in the form of multiple interconnected visualizations. Availability and Implementation ARResT/Interrogate is implemented primarily in R, and is freely available at http://bat.infspire.org/arrest/interrogate/ Supplementary information Supplementary data are available at Bioinformatics online

Quality control and quantification in IG/TR next-generation sequencing marker identification: protocols and bioinformatic functionalities by EuroClonality-NGS

Assessment of clonality, marker identification and measurement of minimal residual disease (MRD) of immunoglobulin (IG) and T cell receptor (TR) gene rearrangements in lymphoid neoplasms using next-generation sequencing (NGS) is currently under intensive development for use in clinical diagnostics. So far, however, there is a lack of suitable quality control (QC) options with regard to standardisation and quality metrics to ensure robust clinical application of such approaches. The EuroClonality-NGS Working Group has therefore established two types of QCs to accompany the NGS-based IG/TR assays. First, a central polytarget QC (cPT-QC) is used to monitor the primer performance of each of the EuroClonality multiplex NGS assays; second, a standardised human cell line-based DNA control is spiked into each patient DNA sample to work as a central in-tube QC and calibrator for MRD quantification (cIT-QC). Having integrated those two reference standards in the ARResT/Interrogate bioinformatic platform, EuroClonality-NGS provides a complete protocol for standardised IG/TR gene rearrangement analysis by NGS with high reproducibility, accuracy and precision for valid marker identification and quantification in diagnostics of lymphoid malignancies.This work was supported by Ministry of Health of the Czech Republic, grant no. 16-34272A; computational resources were provided by the CESNET LM2015042 and the CERIT Scientific Cloud LM2015085, provided under the programme “Projects of Large Research, Development, and Innovations Infrastructures”. Analyses in Prague (JT, EF and MS) were supported by Ministry of Health, Czech Republic, grant no. 00064203, and by PRIMUS/17/MED/11. Analyses in the Monza (Centro Ricerca Tettamanti, SS, AG and GC) laboratory were supported by the Italian Association for Cancer Research (AIRC) and Comitato Maria Letizia Verga

IgCaller for reconstructing immunoglobulin gene rearrangements and oncogenic translocations from whole-genome sequencing in lymphoid neoplasms

Immunoglobulin (Ig) gene rearrangements and oncogenic translocations are routinely assessed during the characterization of B cell neoplasms and stratification of patients with distinct clinical and biological features, with the assessment done using Sanger sequencing, targeted next-generation sequencing, or fluorescence in situ hybridization (FISH). Currently, a complete Ig characterization cannot be extracted from whole-genome sequencing (WGS) data due to the inherent complexity of the Ig loci. Here, we introduce IgCaller, an algorithm designed to fully characterize Ig gene rearrangements and oncogenic translocations from short-read WGS data. Using a cohort of 404 patients comprising different subtypes of B cell neoplasms, we demonstrate that IgCaller identifies both heavy and light chain rearrangements to provide additional information on their functionality, somatic mutational status, class switch recombination, and oncogenic Ig translocations. Our data thus support IgCaller to be a reliable alternative to Sanger sequencing and FISH for studying the genetic properties of the Ig loci.We are indebted to the Genomics Core Facility of the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) for the technical support, to R. Siebert and D. Huebschmann for sharing the CSR regions, and to K. Stamatopoulos, E. Vlachonikola and F. Psomopoulos for their helpful comments on the manuscript. We thank R. Eils, P. Lichter, C. von Kalle, S. Fröhling, H. Glimm, M. Zapatka, S. Wolf, K. Beck, and J. Kirchhof for infrastructure and pipeline development within DKFZ-HIPO and NCT POP. This study was supported by the Instituto de Salud Carlos III and the European Regional Development Fund “Una manera de hacer Europa” (PMP15/00007 to E.C.), the “la Caixa” Foundation (CLLEvolution-LCF/PR/HR17/52150017, Health Research 2017 Program HR17-00221 to E.C.), the National Institute of Health “Molecular Diagnosis, Prognosis, and Therapeutic Targets in Mantle Cell Lymphoma” (P01CA229100 to E.C.), and CERCA Programme/Generalitat de Catalunya. F.N. is supported by a pre-doctoral fellowship of the Ministerio de Economía y Competitividad (BES-2016-076372). F.M. is supported by the Memorial Sloan Kettering Cancer Center NCI Core Grant (P30 CA 008748). E.C. is an Academia Researcher of the “Institució Catalana de Recerca i Estudis Avançats” (ICREA) of the Generalitat de Catalunya. This work was partially developed at the Centre Esther Koplowitz (CEK, Barcelona, Spain).Peer ReviewedPostprint (published version

The individual and combined effects of obesity- and ageing-induced systemic inflammation on human skeletal muscle properties.

BACKGROUND/OBJECTIVES: The purpose of this study was to determine whether circulating pro-inflammatory cytokines, elevated with increased fat mass and ageing, were associated with muscle properties in young and older people with variable adiposity. SUBJECTS/METHODS: Seventy-five young (18-49 yrs) and 67 older (50-80 yrs) healthy, untrained men and women (BMI: 17-49 kg/m(2)) performed isometric and isokinetic plantar flexor maximum voluntary contractions (MVCs). Volume (Vm), fascicle pennation angle (FPA), and physiological cross-sectional area (PCSA) of the gastrocnemius medialis (GM) muscle were measured using ultrasonography. Voluntary muscle activation (VA) was assessed using electrical stimulation. GM specific force was calculated as GM fascicle force/PCSA. Percentage body fat (BF%), body fat mass (BFM), and lean mass (BLM) were assessed using dual-energy X-ray absorptiometry. Serum concentration of 12 cytokines was measured using multiplex luminometry. RESULTS: Despite greater Vm, FPA, and PCSA (P0.05), while IL-8 correlated with VA in older but not young adults (r⩾0.378, P⩽0.027). TNF-alpha correlated with MVC, lean mass, GM FPA and maximum force in older adults (r⩾0.458; P⩽0.048). CONCLUSIONS: The age- and adiposity-dependent relationships found here provide evidence that circulating pro-inflammatory cytokines may play different roles in muscle remodelling according to the age and adiposity of the individual.International Journal of Obesity accepted article preview online, 29 August 2016. doi:10.1038/ijo.2016.151

PI3K Signaling in Normal B Cells and Chronic Lymphocytic Leukemia (CLL).

B cells provide immunity to extracellular pathogens by secreting a diverse repertoire of antibodies with high affinity and specificity for exposed antigens. The B cell receptor (BCR) is a transmembrane antibody, which facilitates the clonal selection of B cells producing secreted antibodies of the same specificity. The diverse antibody repertoire is generated by V(D)J recombination of heavy and light chain genes, whereas affinity maturation is mediated by activation-induced cytidine deaminase (AID)-mediated mutagenesis. These processes, which are essential for the generation of adaptive humoral immunity, also render B cells susceptible to chromosomal rearrangements and point mutations that in some cases lead to cancer. In this chapter, we will review the central role of PI3K s in mediating signals from the B cell receptor that not only facilitate the development of functional B cell repertoire, but also support the growth and survival of neoplastic B cells, focusing on chronic lymphocytic leukemia (CLL) B cells. Perhaps because of the central role played by PI3K in BCR signaling, B cell leukemia and lymphomas are the first diseases for which a PI3K inhibitor has been approved for clinical use

Regulatory T Cells Phenotype in Different Clinical Forms of Chagas' Disease

CD25High CD4+ regulatory T cells (Treg cells) have been described as key players in immune regulation, preventing infection-induced immune pathology and limiting collateral tissue damage caused by vigorous anti-parasite immune response. In this review, we summarize data obtained by the investigation of Treg cells in different clinical forms of Chagas' disease. Ex vivo immunophenotyping of whole blood, as well as after stimulation with Trypanosoma cruzi antigens, demonstrated that individuals in the indeterminate (IND) clinical form of the disease have a higher frequency of Treg cells, suggesting that an expansion of those cells could be beneficial, possibly by limiting strong cytotoxic activity and tissue damage. Additional analysis demonstrated an activated status of Treg cells based on low expression of CD62L and high expression of CD40L, CD69, and CD54 by cells from all chagasic patients after T. cruzi antigenic stimulation. Moreover, there was an increase in the frequency of the population of Foxp3+ CD25HighCD4+ cells that was also IL-10+ in the IND group, whereas in the cardiac (CARD) group, there was an increase in the percentage of Foxp3+ CD25High CD4+ cells that expressed CTLA-4. These data suggest that IL-10 produced by Treg cells is effective in controlling disease development in IND patients. However, in CARD patients, the same regulatory mechanism, mediated by IL-10 and CTLA-4 expression is unlikely to be sufficient to control the progression of the disease. These data suggest that Treg cells may play an important role in controlling the immune response in Chagas' disease and the balance between regulatory and effector T cells may be important for the progression and development of the disease. Additional detailed analysis of the mechanisms on how these cells are activated and exert their function will certainly give insights for the rational design of procedure to achieve the appropriate balance between protection and pathology during parasite infections

Standardized next-generation sequencing of immunoglobulin and T-cell receptor gene recombinations for MRD marker identification in acute lymphoblastic leukaemia; a EuroClonality-NGS validation study

Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) wa

1,1,2-Trichloroetan

1,1,2-Trichloroetan (1,1,2- TCE) jest bezbarwną, niepalną cieczą o słodkim zapachu, zbliżonym do zapachu chloroformu. Jest stosowany jako rozpuszczalnik: tłuszczów, wosków, naturalnych żywic i alkaloidów oraz wielu innych materiałów organicznych, a także jako produkt pośredni (półprodukt) w produkcji: chlorku winylidenu, rurek teflonowych i klejów. Powyżej 95% tego związku produkowanego w USA zużywa się do produkcji chlorku winylidenu. Według danych Głównej Inspekcji Sanitarnej w Polsce na 1,1,2- -trichloroetan nie było w 2010 r. narażonych pracowników powyżej wartości NDS (45 mg/m3). Mediana dawki śmiertelnej 1,1,2-trichloroetanu dla szczurów po podaniu do żołądka (DL50) wynosi – 837 mg/kg, po narażeniu inhalacyjnym (CL50) – 9000 mg/m3. U królików wartość DL50 wynosi 5,38 g/kg po podaniu przez skórę. Substancja działa drażniąco na: skórę, oczy, górne drogi oddechowe oraz żołądek. W badaniach na zwierzętach wykazano niewielkie właściwości immunomodulujące 1,1,2-trichloroetanu. Związek ten jest dobrze wchłaniany do organizmu wszystkimi drogami narażenia. 1,1,2-Trichloroetan jest metabolizowany głównie w wątrobie i wydalany z organizmu z: powietrzem wydychanym, moczem oraz kałem. 1,1,2-Trichloroetan nie wykazywał działania mutagennego dla szczepów Salmonella Typhimurium: TA1535, TA1537, TA1538, TA98, TA100 zarówno bez aktywacji metabolicznej, jak i z aktywacją metaboliczną. Działanie mutagenne 1,1,2-trichloroetanu wykazano u Saccharomyces cerevisiae. Związek ten powodował zwiększenie liczby mikrojąder w ludzkich limfocytach (w warunkach in vitro). Nie znaleziono danych wskazujących na działanie rakotwórcze 1,1,2-trichloroetanu na ludzi. Istnieją ograniczone dowody na potwierdzenie jego rakotwórczego działania na zwierzęta. 1,1,2-Trichloroetan podawany myszom B6C3F1 w dawkach 195 lub 390 mg/kg przez 78 tygodni wywołał raki wątroby. Chromochłonne nowotwory nadnerczy wykazano tylko po podaniu 1,1,2-trichloroetanu w dawce 390 mg/kg. U szczurów 1,1,2-trichloroetan nie powodował istotnego zwiększenia liczby nowotworów. W dostępnym piśmiennictwie i specjalistycznych bazach danych nie znaleziono informacji dotyczących działania embriotoksycznego, teratogennego i wpływu 1,1,2-trichloroetanu na rozrodczość ludzi. Nie wykazano również działania teratogennego 1,1,2-trichloroetanu na myszy. W IARC zaklasyfikowano 1,1,2-trichloroetan do grupy 3. związków kancerogennych (substancja nie może być sklasyfikowana pod względem działania rakotwórczego na ludzi). Wartość NOAEL dla 1,1,2-trichloroetanu ustalono na podstawie wyników 90-dniowych badań na samcach i samicach myszy, którym podawano związek w wodzie do picia. Skutkiem krytycznym było zwiększenie aktywności fosfatazy zasadowej w surowicy krwi u samic i samców myszy, którym podawano największą dawkę 1,1,2-trichloroetanu, tj. odpowiednio dla samic 384 oraz samców 305 mg/kg m.c./dzień. Zmian aktywności fosfatazy zasadowej w surowicy krwi samic nie obserwowano po podaniu dawki 44 mg/kg m.c./dzień związku, a samcom dawki 46 mg/kg m.c./dzień. Dawkę 44 mg/kg m.c./dzień wyznaczoną u samic przyjęto za wartość NOAEL i po zastosowaniu odpowiednich współczynników niepewności zaproponowano przyjęcie stężenia 40 mg/m3 za wartość NDS 1,1,2-trichloroetanu. Substancję należy oznaczyć literami: Carc. Cat. 3 – substancja o możliwym działaniu rakotwórczym na człowieka. Z uwagi na fakt, iż 1,1,2-trichloroetan jest wchłaniany przez skórę, należy wprowadzić dodatkowe oznaczenie literami „Sk”. Nie ma podstaw merytorycznych do ustalenia wartości najwyższego dopuszczalnego stężenia chwilowego (NDSCh) oraz dopuszczalnego stężenia w materiale biologicznym (DSB) 1,1,2-trichloroetanu.1,1,2-Trichloroethane (1,1,2 - TCE) is a colorless, inflammable liquid with a sweet odor, similar to the smell of chloroform. It is used as a solvent for fats, waxes, natural resins, alkaloids, and many other organic materials. It is also used as an intermediate in the production of vinylidene chloride, Teflon tubing and adhesives. Over 95% of the compound produced in the USA is used in the production of vinylidene chloride. According to the Chief Sanitary Inspectorate in Poland, 1,1,2-trichloroethane workers have not been exposed to values over the TWA (45 mg/m3). The median lethal dose of 1,1,2-trichloroethane for rats after administration to the stomach (LD50) is 837 mg/kg, after inhalation exposure (LC50) 9000 mg/m3. LD50 value for rabbits is 5.38 g / kg dose through the skin. The substance is irritating to the skin, eyes, upper respiratory tract and stomach. Animal studies showed slight immunomodulatory properties of 1,1,2-trichloroethane. This chemical compound is well absorbed into the body from all routes of exposure. 1,1,2-Trichloroethane is metabolized primarily in the liver and excreted from the body with the exhaled air, urine and faeces. 1,1,2-Trichloroethane was not mutagenic to Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100, either with or without metabolic activation. Mutagenicity of 1,1,2-trichloroethane was found in Saccharomyces cerevisiae. This compound was shown to increase the number of micronuclei in human lymphocytes (in vitro). There is no evidence of carcinogenic activity of 1,1,2- -trichloroethane in humans. There is limited evidence of its carcinogenicity in animals. 1,1,2-Trichloroethane administered to B6C3F1 mice at doses of 195 or 390 mg/kg for 78 weeks caused liver cancer. Chromaffin adrenal tumors were observed only after administration of 1,1,2-trichloroethane at a dose of 390 mg/kg. In rats, 1,1,2-trichloroethane does not cause a significant increase in the number of cancers. Information about embryotoxic, teratogenic and reproductive toxicity of 1,1,2-trichloroethane for humans has not been found in the available literature and specialist databases. No teratogenic effects were found for 1,1,2-trichloroethane in mice, either. The IARC classified 1,1,2-trichloroethane to group 3 of carcinogenic compounds (a substance cannot be classified in terms of its carcinogenicity to humans). NOAEL for 1,1,2-trichloroethane was established on the basis of a 90-day study in male and female mice given the compound in drinking water. The critical effect was the increase in alkaline phosphatase in serum in male and female mice given the highest dose of 1,1,2-trichloroethane, respectively, for females and males, 384 and 305 mg/kg bw/day. No changes in alkaline phosphatase in the serum of females were observed after a dose of 44 mg/kg/day of the compound, or in the serum of females after a dose of 46 mg/kg bw/day. The dose of 44 mg/kg bw/day in females was taken as the NOAEL and after applying appropriate uncertainty factors it is proposed to adopt it for the value of 40 mg/m3 TWA 1,1,2-trichloroethane. The substance should be labeled Carc. Cat. 3 - a possible carcinogen to humans. As 1,1,2-trichloroethane is absorbed through the skin, the "Sk" notation should be used. There is no factual basis for determining the maximum instantaneous concentration (TWA) or the admissible concentration in biological material (DSB)