Clinical value of next generation sequencing of plasma cell-free DNA in gastrointestinal stromal tumors

[Background] Gastrointestinal stromal tumor (GIST) initiation and evolution is commonly framed by KIT/PDGFRA oncogenic activation, and in later stages by the polyclonal expansion of resistant subpopulations harboring KIT secondary mutations after the onset of imatinib resistance. Thus, circulating tumor (ct)DNA determination is expected to be an informative non-invasive dynamic biomarker in GIST patients.[Methods] We performed amplicon-based next-generation sequencing (NGS) across 60 clinically relevant genes in 37 plasma samples from 18 GIST patients collected prospectively. ctDNA alterations were compared with NGS of matched tumor tissue samples (obtained either simultaneously or at the time of diagnosis) and cross-validated with droplet digital PCR (ddPCR).[Results] We were able to identify cfDNA mutations in five out of 18 patients had detectable in at least one timepoint. Overall, NGS sensitivity for detection of cell-free (cf)DNA mutations in plasma was 28.6%, showing high concordance with ddPCR confirmation. We found that GIST had relatively low ctDNA shedding, and mutations were at low allele frequencies. ctDNA was detected only in GIST patients with advanced disease after imatinib failure, predicting tumor dynamics in serial monitoring. KIT secondary mutations were the only mechanism of resistance found across 10 imatinib-resistant GIST patients progressing to sunitinib or regorafenib.[Conclusions] ctDNA evaluation with amplicon-based NGS detects KIT primary and secondary mutations in metastatic GIST patients, particularly after imatinib progression. GIST exhibits low ctDNA shedding, but ctDNA monitoring, when positive, reflects tumor dynamics.This research is supported by a Fero Fellowship Award (C.S.), Asociación Española Contra el Cáncer (J.P. Barcelona) (C.S.), and ISCIII PI16/01371 (C.S.). C.S. and A.V. acknowledge to the Cellex Foundation for providing facilities and equipment

Estudio preliminar sobre el estado de la salud periodontal de embarazadas que asisten al consultorio odontológico de la maternidad provincial de Córdoba

Nos propusimos evaluar la salud periodontal de las mujeres embarazadas que asisten al Servicio de Odontología de la Maternidad Provincial de Córdoba mediante la aplicación del Índicede Necesidad de Tratamiento Comunitario (INTC), y conocer la capacidad de respuesta inmunológica mediante el análisis de la concentración de Inmunoglobulina A (Ig A) e Ig G en saliva e identificar en las bolsas de mayor profundidad las bacterias periodontales patógenas más frecuentes. Se reclutaron 50 mujeres embarazadas que firmaron el consentimiento de participar en el proyecto (Registro REPIS 208/14), se les evaluaron los parámetros periodontales:Nivel de Inserción Clínica (NIC), Profundidad al Sondaje (PS); Hemorragia Superior (H sup), e inferior (H inf) e Índice de Placa superior e inferior(IP sup, IP inf).Se determinó el Índice de Necesidad de Tratamiento Comunitario (INTC). De un sitio de cada sectante se tomaron muestras para la identificación por biología molecular de A.actinomycetemcomitans P.gingivalis, T.forsythia, P.intermedia y T.denticola. Se tomaron muestras de saliva sin estimular para determinar la concentración de Inmunoglobulinas A (Ig A) y G (Ig G) por inmunoturbidimetría. Los pacientes fueron examinados por odontólogos previamente calibrados. Los datos se analizaron con la prueba de chi cuadrado o test T según corresponda, p< de 0,05 se consideró significativo.Observamos que el 74% correspondían a edades entre 18 a 25 años, el Índice de Masa Corporal fue un 38% de peso normal y 25.5% de sobrepeso, un 42% presentan entre 29 y 32 elementos en boca, 62% comentan que le sangran las encías a partir del embarazo. El INTC indicó que 47,5% presentan grado 3, lo que corresponde: Instrucción de higiene bucal y Raspaje y Alisado radicular como tratamiento. La frecuencia de Pg identificada en los sitios evaluados fue de 17,6%, de Pi 29%, Td 65,5 %,Tf 59,8% yAa 4%. La concentración de Ig A 62,2 mg/dL y de Ig 95,5 mg/dl.Podemos concluir que la población presenta en su mayoría, un código 3 de necesidad de tratamiento periodontal que indicaría una situación clínica periodontal reversible con la Terapia Básica.Los niveles elevados de Ig A y G en saliva estarían indicando una capacidad de respuesta positiva del huésped frente a la flora oral. Una alta frecuencia de Pi es característica del embarazo.Fil: Usín, María Matilde. Universidad Nacional de Córdoba. Facultad de Odontología. Cátedra de Periodoncia B; Argentina.Fil: Miazzo, Emilio Javier. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Cátedra de Bioquímica y Biología Molecular; Argentina.Fil: Artaza, M. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Cátedra de Bioquímica y Biología Molecular; Argentina.Fil: Tabares, Sandra. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Cátedra de Bioquímica y Biología Molecular; Argentina.Fil: Solari, Natalia. Universidad Nacional de Córdoba. Facultad de Odontología. Cátedra de Periodoncia B; Argentina.Fil: Salas, Leandro José. Universidad Nacional de Cuyo. Facultad de Odontología. Fundación Independencia; Argentina.Fil: Menso, Julieta. Universidad Nacional de Córdoba. Facultad de Odontología. Cátedra de Periodoncia B; Argentina.Fil: Ghirardi, Federico. Universidad Nacional de Córdoba. Facultad de Odontología. Cátedra de Periodoncia A; Argentina.Fil: Sembaj, Adela. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Cátedra de Bioquímica y Biología Molecular; Argentina.Otras Ciencias de la Salu

Preferential Binding to Elk-1 by SLE-Associated IL10 Risk Allele Upregulates IL10 Expression

Immunoregulatory cytokine interleukin-10 (IL-10) is elevated in sera from patients with systemic lupus erythematosus (SLE) correlating with disease activity. The established association of IL10 with SLE and other autoimmune diseases led us to fine map causal variant(s) and to explore underlying mechanisms. We assessed 19 tag SNPs, covering the IL10 gene cluster including IL19, IL20 and IL24, for association with SLE in 15,533 case and control subjects from four ancestries. The previously reported IL10 variant, rs3024505 located at 1 kb downstream of IL10, exhibited the strongest association signal and was confirmed for association with SLE in European American (EA) (P = 2.7×10-8, OR = 1.30), but not in non-EA ancestries. SNP imputation conducted in EA dataset identified three additional SLE-associated SNPs tagged by rs3024505 (rs3122605, rs3024493 and rs3024495 located at 9.2 kb upstream, intron 3 and 4 of IL10, respectively), and SLE-risk alleles of these SNPs were dose-dependently associated with elevated levels of IL10 mRNA in PBMCs and circulating IL-10 protein in SLE patients and controls. Using nuclear extracts of peripheral blood cells from SLE patients for electrophoretic mobility shift assays, we identified specific binding of transcription factor Elk-1 to oligodeoxynucleotides containing the risk (G) allele of rs3122605, suggesting rs3122605 as the most likely causal variant regulating IL10 expression. Elk-1 is known to be activated by phosphorylation and nuclear localization to induce transcription. Of interest, phosphorylated Elk-1 (p-Elk-1) detected only in nuclear extracts of SLE PBMCs appeared to increase with disease activity. Co-expression levels of p-Elk-1 and IL-10 were elevated in SLE T, B cells and monocytes, associated with increased disease activity in SLE B cells, and were best downregulated by ERK inhibitor. Taken together, our data suggest that preferential binding of activated Elk-1 to the IL10 rs3122605-G allele upregulates IL10 expression and confers increased risk for SLE in European Americans.Peer Reviewe

Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA

Objectives Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. Methods Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. Results The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10-4, OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10-7, OR 0.71; case-only pmeta=1.9×10-4, OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. Conclusions These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications

Genetic associations of leptin-related polymorphisms with systemic lupus erythematosus

Leptin is abnormally elevated in the plasma of patients with systemic lupus erythematosus (SLE), where it is thought to promote and/or sustain pro-inflammatory responses. Whether this association could reflect an increased genetic susceptibility to develop SLE is not known, and studies of genetic associations with leptin-related polymorphisms in SLE patients have been so far inconclusive. Here we genotyped DNA samples from 15,706 SLE patients and healthy matched controls from four different ancestral groups, to correlate polymorphisms of genes of the leptin pathway to risk for SLE. It was found that although several SNPs showed weak associations, those associations did not remain significant after correction for multiple testing. These data do not support associations between defined leptin-related polymorphisms and increased susceptibility to develop SLE