1,720,986 research outputs found
Diagnostic Validation of a Comprehensive Targeted Panel for Broad Mutational and Biomarker Analysis in Solid Tumors
No full textSIMPLE SUMMARY: The analysis of tumor-associated genetic variants and biomarkers is critical for therapy choice, as specific mutations allow for a personalized treatment. Because more and more mutation-treatment combinations become available, screening should be performed on many genes simultaneously. The use of large and comprehensive gene panel screenings in molecular diagnostics, however, requires an extensive and thorough validation to demonstrate the correctness of all clinically relevant data. Here, we describe such validation using a large number of samples and confirmed effective detection of several types of mutations for different validation parameters. Samples of tumor patients thus can be reliably tested with a comprehensive assay to maximize their personalized treatment regimen. ABSTRACT: The use of targeted Next Generation Sequencing (NGS) for the diagnostic screening of somatic variants in solid tumor samples has proven its high clinical value. Because of the large number of ongoing clinical trials for a multitude of variants in a growing number of genes, as well as the detection of proven and emerging pan-cancer biomarkers including microsatellite instability (MSI) and tumor mutation burden (TMB), the currently employed diagnostic gene panels will become vastly insufficient in the near future. Here, we describe the validation and implementation of the hybrid capture-based comprehensive TruSight Oncology (TSO500) assay that is able to detect single-nucleotide variants (SNVs) and subtle deletions and insertions (indels) in 523 tumor-associated genes, copy-number variants (CNVs) of 69 genes, fusions with 55 cancer driver genes, and MSI and TMB. Extensive validation of the TSO500 assay was performed on DNA or RNA from 170 clinical samples with neoplastic content down to 10%, using multiple tissue and specimen types. Starting with 80 ng DNA and 40 ng RNA extracted from formalin-fixed and paraffine-embedded (FFPE) samples revealed a precision and accuracy >99% for all variant types. The analytical sensitivity and specificity were at least 99% for SNVs, indels, CNVs, MSI, and gene rearrangements. For TMB, only values around the threshold could yield a deviating outcome. The limit-of-detection for SNVs and indels was well below the set threshold of 5% variant allele frequency (VAF). This validated comprehensive genomic profiling assay was then used to screen 624 diagnostic samples, and its success rate for adoption in a clinical diagnostic setting of broad solid tumor screening was assessed on this cohort
Novel gene fusion discovery in Spitz tumours and its relevance in diagnostics
No full textIn addition to morphologic analysis, molecular diagnostic work up of Spitz tumours is often of great value for their accurate diagnosis/classification. Nowadays, next-generation sequencing (NGS) is the predominant screening method in molecular diagnostics. Up to 80% of these melanocytic neoplasms comprise gene fusions as genetic anomalies for which the driver codes for a protein harbouring a kinase domain. However, because of the variety of fusion partners the use of PCR-based targeted enrichment NGS methods is not recommended. We describe a series of four Spitz tumour samples in which distinct gene fusions were detected by hybridisation-based capture NGS (TPM3::ALK, LIMA1::ROS1, LRRFIP2::ROS1 and MYO5A::RET). Two of these fusions are not previously described. All 4 fusions were confirmed by reverse transcription-PCR. These findings demonstrate the need for molecular analysis that can detect unknown fusions in Spitz neoplasms for optimal diagnosis.The authors would like to thank the medical laboratory technologists of the Laboratory for Molecular Diagnostics for their technical contributions. We also acknowledge Anne-Marie Delsupehe for assistance in scanning of the tissue slides
1037P Tumour mutational burden and HLA diversity by TruSight oncology 500 (TSO500) next generation sequencing panel and clinical outcome in non-small cell lung cancer
No full textBackground: The microbiota community is considered as an organ of the human body. Recent studies have found that gut microbiota may impact on the interaction between immune regulation and tumor treatment. The aim of this study is to characterize the gut microbiota in advanced NSCLC patients, and its relationship with response to immune checkpoint blockade (ICB). Methods: Stool samples from 84 advanced NSCLC patients were collected prior ICB as first or second line treatment. 16S rRNA gene sequencing and SILVA_release_132 database were used for taxonomic profiling. Diversity indices, including Chao1, Shannon and Inverse Simpson index (IDI), abundance of certain taxa, clinicopatho-logical characteristics, dietary habits and antibiotic usage were evaluated for association with clinical benefit (CB) [complete or partial response, stable disease vs. progressive disease (PD), according to RECIST1.1]. Continuous variables were stratified into high or low using median as cutoff. For survival analysis, Cox Regression and Kaplan Meier curves with log-rank test were performed. Results: From 84 NSCLC patients, 60 presented PD-L1 positive tumors and 39 were treated with ICB as first line. A total of 8872307 sequencing reads were obtained and clustered in 357 genera, being the most frequent Bacteroides (27.9%) and Alistipes (7.1%). Patients with CB exhibited higher IDI compared with those with PD (p¼0.004). Moreover, higher IDI was associated with prolonged progression-free survival (PFS) (p¼0.014). High abundance of Butyricimonas in the samples was correlated with increased RR (p¼0.01) and longer PFS (p¼0.011) compared to low abundance. On the other hand, high frequency of Dialister was associated with reduced RR (p¼0.002) and shorter PFS (p¼0.003). Finally, the expression of PD-L1 was associated with increased response rate (RR) (p¼0.020). Conclusions: This study shows that diversity of gut microbiota is related to the ICB treatment response. In addition, high abundance of Butyricimonas and low abundance of Dialister could be considered as potential predictors of clinical benefit and PFS. Further analyses are being undertaken to find a compositional signature with predictive value. Funded by CB16/12/00350 from CIBEROnc, Arnal Planelles Foundation , AMACMA, GIDO group and PI18/00226 from ISCIII. Background: This study investigated the role of programmed death-ligand 1 (PD-L1) expression in the sex-related differences in immune checkpoint inhibitor (ICI) efficacy. Methods: We first pooled individual patient-level data from prospective clinical trials to evaluate ICI efficacy between male and female stratified by PD-L1 expression. We further combined individual patient-level data with meta-analysis of randomized controlled trials (RCTs) to assess the efficacy of ICI versus chemotherapy in each of the sexes. Finally, we assessed sex associated with landscape of tumor microenvironment among PD-L1 expression-negative patients. Results: In total, 1,594 patients were included from five clinical trials, and nine RCTs with 4,718 patients were included in meta-analysis. Among patients with PD-L1 expression<1%, individual patient-level analysis showed that overall survival (OS) with ICI was significantly longer for female compared with male (HR 0.58, 95% CI 0.42-0.80; P<.001). The OS benefit of ICI over chemotherapy was significantly different in female (HR 0.57, 95% CI 0.38-0.85; P¼.006), but not in male. Among patients with PD-L1 expression1%, individual patient-level analysis combined with meta-analysis showed that the OS benefit of ICI over chemotherapy was significantly different in male (HR 0.76, 95% CI 0.67-0.85; P<.01), both in first-line patients (HR 0.79, 95% CI 0.65-0.97; P¼0.02) and subsequent-line patients (HR 0.73, 95% CI 0.62-0.85; P<.01); however, this benefit for female was only significant in subsequent-line patients (HR 0.77, 95% CI 0.62-0.96; P¼.02). Additionally, the central memory T cell was potentially correlated with the OS differences between the sexes at PD-L1 expression<1%. Conclusions: The association of sex with OS benefits of ICIs in cancer were greatly influenced by PD-L1 expression. At PD-L1 expression<1%, ICI should be recommended for female but not for male. At PD-L1 expression1%, ICI should be recommended for male regardless of treatment lines; whereas for female, ICI could be only recommended in subsequent-line setting. Sex and PD-L1 expression should be jointly considered in the clinical decision making for ICI in cancer. Legal entity responsible for the study: Herui Yao. Funding: Has not received any funding. Background: TMB predicts response to PD-1 immunotherapy (IO). High HLA class I diversity correlates with better responses in NSCLC. Standard to determine TMB and HLA diversity, is WES. We describe the correlation between TMB and HLA-diversity score by targeted NGS (TSO500) and outcome in immunotherapy treated NSCLC.Sponsor(s) of de conference :European Soc Med Oncol
Funding
Has not received any funding
HLA-I diversity and tumor mutational burden by comprehensive next-generation sequencing as predictive biomarkers for the treatment of non-small cell lung cancer with PD-(L)1 inhibitors
Get PDFObjectives: Immune checkpoint inhibitors (ICIs) improved outcomes in non-small cell lung cancer (NSCLC) patients. We report the predictive utility of human leukocyte antigen class I (HLA-I) diversity and tumor mutational burden (TMB) by comprehensive next-generation sequencing. Methods: 126 patients were included. TMB high was defined as >= 10 nonsynonymous mutations/Mb. Patients exhibit high HLA-I diversity if at least one locus was in the upper 15th percentile for DNA alignment scores. Results: No difference in response rate (RR; 44.4% versus 30.9%; p = 0.1741) or 6-month survival rate (SR; 75.6% versus 77.8%; p = 0.7765) was noted between HLA-I high diversity and low diversity patients. HLA-I high diversity patients did significantly more often exhibit durable clinical benefit (DCB), defined as response or stable disease lasting minimally 6 months (64.4% [29/45] versus 43.2% [35/81]; p = 0.0223).TMB high patients exhibited higher RR (49.1% versus 25.4%; p = 0.0084) and SR 6 months after start ICI (85.5% versus 70.4%; p = 0.0468) than TMB low patients. The proportion of patients with DCB, did not differ significantly between TMB high and low subgroups (60.0% [33/55] versus 42.3% [30/71]; p = 0.0755).Patients with combined dual high TMB and HLA-I diversity had higher RR (63.2% versus 22.2%; p = 0.0033), but SR at 6 months did not differ significantly (84.2% versus 64,4%; p = 0.1536). A significantly higher rate of patients experienced DCB in dual high compared to the dual low group (73.7% [14/19] versus 35.6% [16/45]; p = 0.0052). Triple positive patients (high TMB and HLA-I diversity and PD-L1 positive) had higher RR (63.6% versus 0.0%; p = 0.0047) and SR at 6 months (100% versus 66.7%; p = 0.0378) compared to triple-negative patients. Conclusion: HLA-I diversity was able to predict durable clinical benefit in ICI treated NSCLC patients, but failed to confirm as a predictor of response or survival. TMB confirmed as a predictive biomarker.</p
Reference genes for qPCR assays in toxic metal and salinity stress in two flatworm model organisms
No full textThe flatworm species Schmidtea mediterranea
and Macrostomum lignano have become new and innovative model organisms in stem cell, regeneration and tissue
homeostasis research. Because of their unique stem cell
system, (lab) technical advantages and their phylogenetic
position within the Metazoa, they are also ideal candidate
model organisms for toxicity assays. As stress and biomarker screenings are often performed at the transcriptional level, the aim of this study was to establish a set of
reference genes for qPCR experiments for these two model
organisms in different stress situations. We examined the
transcriptional stability of nine potential reference genes
(actb, tubb, ck2, cox4, cys, rpl13, gapdh, gm2ap, plscr1) to
assess those that are most stable during altered stress
conditions (exposure to carcinogenic metals and salinity
stress). The gene expression stability was evaluated by
means of geNorm and NormFinder algorithms. Sets of best
reference genes in these analyses varied between different
stress situations, although gm2ap and actb were stably
transcribed during all tested combinations. In order to
demonstrate the impact of bad normalisation, the stressspecific gene hsp90 was normalised to different sets of
reference genes. In contrast to the normalisation according
to GeNorm and NormFinder, normalisation of hsp90 in
Macrostomum lignano during cadmium stress did not show
a significant difference when normalised to only gapdh. On
the other hand an increase of variability was noticed when
normalised to all nine tested reference genes together.
Testing appropriate reference genes is therefore strongly
advisable in every new experimental condition.This work was supported by PhD grants for Michelle Plusquin and Olivier DeGheselle from Hasselt University BOF (Bijzonder OnderzoeksFonds: BOF05N02 and BOF08G01) and Hasselt University tUL-impulsfinanciering, Andromeda Van Roten
was supported by Hasselt University tUL-impulsfinanciering (IMPF2PR). The authors gratefully acknowledge Dr. P. Ladurner (University of Innsbruck), Dr. M. Willems and S. Mouton (Ghent University) for providing us with cultures of the animals, and for their advice concerning maintenance of the cultures. They wish to thank Natascha Stefanie and Ria Vanderspikken for their skilful technical assistance. Dr. Nikki Watson (Australia) is greatly acknowledged for her critical reading of, and the linguistic comments on the manuscript
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Het effect van cadmium op het moleculaire niveau bij M.lignano
No full textCadmium is een toxisch metaal dat bij zowel mens als dier ernstige schade kan veroorzaken
aan ondermeer de longen, de nier, de lever en het botweefsel. Deze schade is het gevolg van
de effecten op cellulair niveau, waar het metaal oxidatieve stress induceert. Oxidatieve stress
is een onevenwicht tussen de pro- en anti-oxidanten in de cel, waardoor een overschot aan
pro-oxidanten ontstaat. Dit leidt tot de oxidatie van proteïnen, lipiden en DNA. Een cel
beschikt over verschillende beschermingsmechanismen, waaronder het anti-oxidatief systeem
met enzymen, zoals catalase en superoxidedismutase, en metabolieten, zoals glutathion en
vitamine E. Verder kunnen er ook heat-shock proteïnen geïnduceerd worden om de cel te
beschermen, en herstellen indien nodig.
In deze scriptie werd onderzoek verricht naar de effecten van blootstelling aan Cd op het
cellulaire niveau bij het modelorganisme Macrostomum lignano. Momenteel wordt M.
lignano reeds gebruikt als modelorganisme in ontogenetische studies, in onderzoek naar
regeneratie en stamceldynamiek en in evolutionaire studies. Dankzij een EST-databank, die
pas recent publiek beschikbaar werd gesteld, kan het onderzoek naar achterliggende,
moleculaire effecten gestart worden. Het onderzoek dat in deze scriptie wordt voorgesteld
vormt een eerste verkennende studie van moleculaire effecten veroorzaakt door Cd, een type
onderzoek dat nog bij geen enkele platwormsoort werd uitgevoerd.
In eerste instantie was het noodzakelijk de lysis van het weefsel en de RNA extractie te
optimaliseren. Hieruit kon besloten worden dat het mogelijk is om RNA te isoleren uit kleine
hoeveelheden materiaal door de stalen te malen en vervolgens gebruik te maken van de
‘RNeasy mini kit’ (Qiagen, Venlo). Als tweede werd de LC50-waarde van Cd bij M. lignano
bepaald over 1 dag en 5 dagen. Vervolgens werd er gezocht naar genen die stabiel blijven
tijdens de behandeling met Cd met geNormTM en NormFinder, en dus het meest aangewezen
zijn om gebruikt te worden als referentiegenen in dit experiment. Hieruit blijkt dat -tubuline,
caseïne kinase 2 en cytochroom c oxidase subeenheid IV de meest geschikte referentiegenen
zijn. Bij analyse van de genexpressie van catalase, calmoduline, heat-shock proteïne 60 en 90
werden er geen significante verschillen gevonden na Cd blootstelling. Tenslotte werden er
reactieve zuurstofvormen in vivo gedetecteerd bij M. lignano. H2O2 komt voornamelijk aan de
meest frontale lichaamswand en in de vesicula seminalis voor, O2
-° is meer verspreid over het
hele lichaam, maar ontbreekt in de gonaden
- …
