scholarly journals A straightforward molecular strategy to retrospectively investigate the spread of SARS-CoV-2 VOC202012/01 B.1.1.7 variant

2021 ◽  
Vol 15 (02) ◽  
pp. 242-246
Author(s):  
Gabriele Ibba ◽  
Rosangela Sau ◽  
Flavia Angioj ◽  
Marcello Abbondio ◽  
Salvatore Rubino ◽  
...  
Keyword(s):  
Gene Mutations ◽  
Cost Effective ◽  
Rt Pcr ◽  
Curve Pattern ◽  
Amplification Curve ◽  
Viral Genomes ◽  
S Gene ◽  
Thermo Fisher Scientific ◽  
S Genes ◽  
Fisher Scientific

The spread of new SARS-CoV-2 variants represents a serious threat worldwide, thus rapid and cost-effective methods are required for their identification. Since November 2020, the TaqPath COVID-19 assay (Thermo Fisher Scientific) has been used to identify viral strains of the new lineage B.1.1.7, since it fails to detect the S-gene with the ∆69/70 deletion. Here, we proposed S-gene mutations screening with the Allplex SARS-CoV-2 assay (Seegene), another widely used RT-PCR test that targets Sarbecovirus E, SARS-CoV-2 N, and RdRp/S genes. Accordingly, we evaluated the S gene amplification curve pattern compared to those of the other genes. Exploiting an Allplex assay-generated dataset, we screened 663 RT-PCR digital records, including all SARS-CoV-2 respiratory samples tested in our laboratory with the Allplex assay between January 1st and February 25th, 2021. This approach enabled us to detect 64 samples with peculiar non-sigmoidal amplification curves. Sequencing a selected group of 4 RNA viral genomes demonstrated that those curves were associated with B.1.1.7 variant strains. Our results strongly suggest that B.1.1.7 variant spread has begun in this area at least since January and imply the potential of these analytical methods to track and characterize the spread of B.1.1.7 strains in those areas where Allplex SARS-CoV-2 datasets have been previously recorded.

scholarly journals Mutational Analysis of MDS and AML Occurring after Treatment for Acute Promyelocytic Leukemia (APL). a Report of 9 Cases

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2861-2861
Author(s):  
Philippe Attias ◽  
Aline Renneville ◽  
Xavier Thomas ◽  
Cecile Bally ◽  
Sandrine Hayette ◽  
...  
Keyword(s):  
Research Funding ◽  
De Novo ◽  
Mutational Analysis ◽  
Gene Mutations ◽  
Advisory Committees ◽  
Npm1 Mutation ◽  
Thermo Fisher Scientific ◽  
Ion Proton ◽  
Next Generation Sequencing Ngs ◽  
Fisher Scientific

Abstract Background : 1 to 2% of APL treated with ATRA and chemotherapy (CT) develop MDS/ AML (other than APL) during follow-up, a problematic side effect for a now highly curable disease. Characteristics of those MDS/AML (usual preleukemic phase and deletion of chromosomes 5 and/or 7, often complex) are those of alkylator induced therapy-related (t) MDS/AML, although CT used for APL treatment generally consists of anthracycline +/- AraC, with or without maintenance CT with 6-mercaptopurine (6MP) and methotrexate (MTX). On the other hand, we recently found that, in AML with NPM1 mutation that evolved to NPM1-negative MDS , somatic mutations present at the MDS phase were already present at AML diagnosis, suggesting presence of an underlying MDS (with secondary acquisition of NPM1 mutation) (Morin et al, NEngl J Med, in press) . Because cases of APL secondary to MDS (or MPN) have also been reported, we wondered whether MDS/AML occurring during the evolution of APL wereactual t-MDS/AML, or were underlying myeloid disorders which had progressed to APL. Methods : Between 2006 and 2015, 956 patients with newly diagnosed APL were included in our APL 2006 trial and treated with ATRA plus idarubicin (Ida) and AraC induction chemotherapy, followed by 2 consolidation courses of Ida , with or without AraC, ATRA or ATO and 2-year maintenance with ATRA and CT with 6MP and MTX. 10 (1%) developed MDS or AML (without t(15 ;17) or PML-RARA rearrangement). Paired bone marrow samples collected at APL diagnosis and MDS/AML diagnosis from 9 of them were analyzed for gene mutations on genomic DNA in a selected panel of 30 genes (ASXL1, CALR, CBL, CSF3R, DNMT3A, ETV6, EZH2, FLT3-TKD, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, NRAS, MPL, NPM1, PHF6, PTPN11, RIT1, RUNX1, SETBP1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR2) by a next-generation sequencing (NGS) assay using the IonAmpliSeq Library Kit 2.0 (Thermo Fisher Scientific) and the Ion Proton system (Thermo Fisher Scientific). FLT3-internal tandem duplications were investigated by fragment analysis. Results : Median age of the 9 cases of MDS/AML was 52 years , and median interval from diagnosis of APL treatment to MDS/AML diagnosis was 2.8 years. No MDS morphological features were reported on APL diagnosis marrow aspirates. All the patients developed MDS/AML in first CR. Karyotype at APL diagnosis found no abnormalities in addition tot(15 ;17) in the 9 pts. Karyotype at MDS/AML diagnosis showed (table) : -7/del7q (n=6), del(5q-)/-5 (n=5), del (17p) (n=2), and was complex in 5 cases. Mutations identified at APL diagnosis (Table) were FLT3-ITD mutations (n=4), FLT3-TKD mutations (n=2), mutations in WT1 (n=2), NRAS, PHF6, DNMT3A (1 case each). At MDS/AML diagnosis, 7 patients had detectable mutations, while the remaining 2 pts had a complex karyotype typical of t-MDS/AML (table). The most frequently mutated gene at MDS/AML diagnosis was TP53 (3/9 cases), while other mutations involving ASXL1, CBL, DNMT3A, EZH2, GATA2, KRAS, PTPN11, RUNX1, TET2 ,and SMC1A were seen in one patient each, and 5 pts had several mutations. None of the mutations identified at APL diagnosis was found at MDS/AML diagnosis, and vice versa, strongly suggesting that APL and MDS/AML arose from distinct clones. Conclusion : No MDS type mutations were found at APL diagnosis in patients who developed subsequent MDS/AML (if one excepts DNMT3a mutation, however also seen in de novo APL). Cytogenetic and mutational profiles of those MDS/AML were suggestive ofalkylator type t- MDS/AML. Thus, MDS/AML occurring during the course of APL treated with ATRA and CT have characteristics of therapy-related cases. The fact that no MDS/AML has so far been reported in large trials of APL treated with ATRA and ATO without CT also supports this hypothesis, and provides further evidence to reduce or eliminate CT from APL treatment. Table Table. Disclosures Thomas: Pfizer: Consultancy. Park:Novartis: Research Funding. Ades:Celgene, Takeda, Novartis, Astex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fenaux:Celgene, Janssen,Novartis, Astex, Teva: Honoraria, Research Funding.

scholarly journals European Multicenter Evaluation of Commercial Enzyme Immunoassays for Detecting Norovirus Antigen in Fecal Samples

2007 ◽  
Vol 14 (10) ◽  
pp. 1349-1355 ◽  
Author(s):  
Jim J. Gray ◽  
Evelyne Kohli ◽  
Franco M. Ruggeri ◽  
Harry Vennema ◽  
Alicia Sánchez-Fauquier ◽  
...  
Keyword(s):  
Rt Pcr ◽  
Fecal Samples ◽  
Commercial Enzyme ◽  
Enzyme Immunoassays ◽  
Reverse Transcription Pcr ◽  
Thermo Fisher Scientific ◽  
Outbreak Investigations ◽  
Sporadic Cases ◽  
Screening Assays ◽  
Fisher Scientific

ABSTRACT A total of 2,254 fecal samples were tested in a European multicenter evaluation of commercially available norovirus antigen detection assays. Two commercial enzyme immunoassays, IDEIA Norovirus (Oxoid; Thermo Fisher Scientific, Ely, United Kingdom) and RIDASCREEN Norovirus (R-Biopharm, Darmstadt, Germany), were included in the evaluation, and their performance was compared with the results of reverse transcription-PCR (RT-PCR). Included in the evaluation were samples collected in sporadic cases of gastroenteritis, samples from outbreaks in which two or more samples were collected, well-characterized samples representing genotypes currently cocirculating within Europe, and samples collected from patients with gastroenteritis caused by a pathogen other than norovirus. The sensitivities and specificities of the IDEIA Norovirus and RIDASCREEN Norovirus assays were 58.93 and 43.81% and 93.91 and 96.37%, respectively, compared with RT-PCR. The sensitivities of both assays for outbreak investigations improved when six or more samples from an outbreak were examined. The IDEIA Norovirus assay exhibited reactivity to a broader range of norovirus genotypes than the RIDASCREEN Norovirus assay, which showed genotype-dependent sensitivities. The results indicate that, if used, these assays should serve as screening assays and the results should be confirmed by RT-PCR.

scholarly journals Feline coronavirus with and without spike gene mutations detected by real-time RT-PCRs in cats with feline infectious peritonitis

2019 ◽  
Vol 22 (8) ◽  
pp. 791-799 ◽  
Author(s):  
Laura Emmler ◽  
Sandra Felten ◽  
Kaspar Matiasek ◽  
Hans-Joerg Balzer ◽  
Nikola Pantchev ◽  
...  
Keyword(s):  
Real Time ◽  
Gene Mutations ◽  
Buffy Coat ◽  
Sample Type ◽  
Rt Pcr ◽  
Mesenteric Lymph Nodes ◽  
Feline Infectious Peritonitis ◽  
S Gene ◽  
Mesenteric Lymph ◽  
Pcr Targeting

Objectives Feline infectious peritonitis (FIP) emerges when feline coronaviruses (FCoVs) mutate within their host to a highly virulent biotype and the immune response is not able to control the infection. FCoV spike ( S) gene mutations are considered to contribute to the change in virulence by enabling FCoV infection of and replication in macrophages. This study investigated the presence of FCoV with and without S gene mutations in cats with FIP using two different real-time RT-PCRs on different samples obtained under clinical conditions. Methods Fine-needle aspirates (FNAs) and incisional biopsies (IBs) of popliteal and mesenteric lymph nodes, liver, spleen, omentum and kidneys (each n = 20), EDTA blood (n = 13), buffy coat smears (n = 13), serum (n = 11), effusion (n = 14), cerebrospinal fluid (n = 16), aqueous humour (n = 20) and peritoneal lavage (n = 6) were obtained from 20 cats with FIP diagnosed by immunohistochemistry. Samples were examined by RT-PCR targeting the FCoV 7b gene, detecting all FCoV, and S gene mutation RT-PCR targeting mutations in nucleotides 23531 and 23537. The prevalence of FCoV detected in each sample type was calculated. Results In 20/20 cats, FCoV with S gene mutations was present in at least one sample, but there was variation in which sample was positive. FCoV with mutations in the S gene was most frequently found in effusion (64%, 95% confidence interval [CI] 39–89), followed by spleen, omentum and kidney IBs (50%, 95% CI 28–72), mesenteric lymph node IBs and FNAs (45%, 95% CI 23–67), and FNAs of spleen and liver and liver IBs (40%, 95% CI 19–62). Conclusions and relevance In these 20 cats with FIP, FCoVs with S gene mutations were found in every cat in at least one tissue or fluid sample. This highlights the association between mutated S gene and systemic FCoV spread. Examining a combination of different samples increased the probability of finding FCoV with the mutated S gene.

scholarly journals P–274 Undetectable viral RNA in follicular fluid (FF), cumulus cells (CC) and endometrial tissue in SARS-CoV–2 positive patients

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
L Boudry ◽  
W Essahib ◽  
I Mateizel ◽  
H Va. d. Velde ◽  
D D Geyter ◽  
...  
Keyword(s):  
Protein Expression ◽  
Endometrial Tissue ◽  
Viral Rna ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
S Protein ◽  
Immature Oocytes ◽  
Corona Radiata ◽  
Thermo Fisher Scientific ◽  
Fisher Scientific

Abstract Study question Is there any indication for presence of viral RNA in FF, CC, immature oocytes or endometrial biopsy (EB) of SARS-CoV–2 patients undergoing ovarian stimulation? Summary answer Viral RNA is undetectable in FF, CC and EB with RT-PCR. However, S-protein expression on corona radiata cells suggests susceptibility to SARS-CoV–2 infection. What is known already The effects of a SARS-CoV–2 infection on the female reproductive system are still poorly understood. Theoretically, co-localisation of the angiotensin converting enzyme (ACE2) and transmembrane serine protease 2 (TMPRSS2) on human blastocysts implies susceptibility to viral infection, mediated by the coronavirus spike (S) protein. To date, SARS-CoV–2 RNA was undetectable in mature oocytes from COVID–19 patients, despite the expression of ACE2 and TMPRSS2. The presence of viral RNA in endometrial tissue, immature oocytes, CC or FF has not yet been investigated in samples from patients with positive nasopharyngeal SARS-CoV–2 test. Study design, size, duration This is a prospective, single-centre, observational study including ten patients with a positive nasopharyngeal swab for SARS-CoV–2, performed 48 hours before oocyte retrieval (OR), from September 2020 to January 2021. A patient was eligible if she preferred to continue treatment following adequate counselling of the unknown but presumably low risk for vertical transmission. Since a freeze-all strategy was applied, an EB was performed. Participants/materials, setting, methods During OR, all protective measures were taken. Pooled FF, CC and EB from each patient were tested for viral RNA presence with RealStar® SARS-CoV–2 RT-PCR-Kit1.0 (Altona-Diagnostics). Ct values <40 were considered positive. EB was collected for pathological evaluation and cultured to obtain endometrial stromal cells (EnSC). Immature oocytes and EnSC were tested for S-protein expression by immunohistochemistry with anti-S antibody (MA5–35958, Thermo-Fisher Scientific) followed by Alexa Fluor™ 488-donkey-anti-mouse (Thermo-Fisher Scientific) and visualized with confocal microscopy. Main results and the role of chance SARS-CoV–2 RNA was undetectable in the pooled FF, CC and EB from all patients included in the study. Histological analysis of the EB showed no pathological modifications, including inflammatory reaction, as compared to biopsies collected from swab negative patients. After staining with anti-S antibody, cultured EnSC and immature oocytes tested negative for the S-protein. However, the binding of anti-S antibody was demonstrated on the corona radiata cells remaining on the zona pellucida after oocyte denudation for intra-cytoplasmatic sperm injection, indicating presence of SARS-CoV–2. In that case, the explanation for the undetectable viral RNA in CC could be that the viral RNA concentration remained under the detection limit of the currently used RT-PCR test. Limitations, reasons for caution This study was conducted in a small population (ten patients included) with different viral load, with mild or without symptoms of COVID–19. Another important limitation is the absence of validation of the RT-PCR protocol for the investigation of other types of samples than nasopharyngeal swabs. Wider implications of the findings: The absence of SARS-CoV–2 RNA in all samples analysed represents a step further in reassuring a safe ART program for COVID–19 patients. However, the presence of S-protein on corona radiata cells warrants further investigation, since the theoretical possibility to infect human oocytes and/or embryos cannot be ruled out. Trial registration number NCT04425317

scholarly journals The Need of Non-traditional Techniques to Screen for the Virus

2020 ◽  
Vol 8 (T1) ◽  
pp. 1-2
Author(s):  
Davide Borroni ◽  
Kunal Gadhvi
Keyword(s):  
Early Stage ◽  
False Negative ◽  
Cost Effective ◽  
The United States ◽  
Viral Metagenomics ◽  
Present Moment ◽  
Rt Pcr ◽  
Viral Genomes ◽  
Reverse Transcription Pcr ◽  
Positive Rate

BACKGROUND: At the present moment, the etiological diagnosis of SARS-CoV-2 is based on the polymerase chain reaction (PCR). False negative cases are increasingly reported in several studies using reverse transcription-PCR (RT-PCR). For example, the positive rate of RT-PCR for throat swabs was reported to be about 60% in early stage of COVID-19. AIM: We aimed to present metagenomic next-generation sequencing (mNGS) as a potential tool to detect pathogens. METHODS: In the recent year, mNGS is shown the potential to detect pathogens without the need of hypothesis guided approach and is proven to be highly effective. RESULTS: A recent prospective study in the United States compared the diagnostic performance of routine diagnostic tests with mNGS and showed that mNGS detected a bacteria or virus in the CSF of 13 of 58 patients presenting with meningoencephalitis who were negative for or not assessed with routine diagnostic test including PCR. NGS also has the advantage to cover entire viral genomes. CONCLUSION: As viral metagenomics has significantly improved in recent years and become more cost effective, we think that a change in the approach toward a shot-gun metagenomic testing should be explored and could potentially aid the diagnosis of COVID-19 cases and the management of this pandemic.

scholarly journals Variant Prediction by Analyzing RdRp/S Gene Double or Low Amplification Pattern in Allplex SARS-CoV-2 Assay

Diagnostics ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1854
Author(s):  
Min-Kyung So ◽  
Sholhui Park ◽  
Kyunghoon Lee ◽  
Soo-Kyung Kim ◽  
Hae-Sun Chung ◽  
...  
Keyword(s):  
Gene Amplification ◽  
Diagnostic Testing ◽  
Pcr Amplification ◽  
Cost Effective ◽  
Rdrp Gene ◽  
Genomic Sequencing ◽  
Amplification Curve ◽  
S Gene ◽  
Cost Constraints ◽  
Global Threat

The spread of delta variants (B.1.671.2) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a severe global threat. Multiplex real-time PCR is a common method for confirming SARS-CoV-2 infection, however, additional tests, such as whole genomic sequencing, are required to reveal the presence or type of viral mutation. Moreover, applying whole genomic sequencing to all SARS-CoV-2 positive samples is challenging due to time and cost constraints. Here, we report that the double or low amplification curve observed during RNA-dependent RNA polymerase (RdRp) gene/S gene amplification in the Allplex SARS-CoV-2 Assay is related to delta/alpha variants. We analyzed the RdRp/S gene amplification curve using 94 samples confirmed as SARS-CoV-2 infection by the Allplex SARS-CoV-2 Assay from January to August, 2021. These positive samples identified variant types using the Novaplex SARS-CoV-2 Variants I and IV Assays. Overall, 17 samples showing a double curve and 11 samples showing a low amplification pattern were associated with alpha-/delta-type strains with variants in the P681 region. The double or low curve shown in the RdRp gene amplification curve had 100% sensitivity and 100% specificity for diagnosing delta/alpha variants. During the SARS-CoV-2 virus diagnostic RT-PCR test using the Allplex SARS-CoV-2 Assay, we could consider the presence of delta/alpha variants in the samples with double or low amplification curve of the RdRp/S gene channel. This PCR amplification curve abnormality enables rapid and cost-effective variant type prediction during SARS-CoV-2 diagnostic testing in clinical laboratories.

Phylogeny of 2019-nCoVs and SARS-like CoVs of Human, Bat and Pangolin Origin

2020 ◽  
Keyword(s):  
N Gene ◽  
Whole Genome ◽  
Human Origin ◽  
Gene Sequences ◽  
M Gene ◽  
S Gene ◽  
Shared Identity ◽  
Novel Coronavirus ◽  
S Genes

A novel coronavirus first broke out in Wuhan, China in December, 2019 has been declared a pandemic by WHO on March, 2020. This work aimed to search for probable ancestor of the virus, phylogeny of 2019-nCoVs and similar SL-CoVs based on the whole genome, M, N, ORF1ab, orf3a, and S gene sequences (n=84) obtained from GenBank using BLASTn software in the NCBI was done. Nucleotides of ORF3a and S-genes among 2019-nCoVs are identical, whereas its similar on the whole genome (99.9-100%), M-gene (99.7-100%), N-gene (99.9-100%) and ORF1ab-gene (99.7-100%). nCoVs are similar to bat CoV/RaTG13 on the whole genome (96.2%), M-gene (95.0%), N-gene (97%), ORF1ab-gene (95.3%), ORF3a-gene (99.1%) and S-gene (90.7%). Likewise, nCoVs exhibited homology to bat-CoVZXC21 on M-gene (93.2%), N-gene (91.5%), ORF1ab-gene (93.1%) and ORF3a-gene (94.4%). The emergent viruses shared identity to bat-CoVZC45 on N-gene (91.3%), ORF1ab-gene (92.8%) and ORF3a-gene (94.0%). In addition, pangolin-CoV/MP789 exhibited common sequences on M-gene (91.0%), N-gene (96.3%) and ORF3a-gene (93.3%) to nCoV. Furthermore, pangolin-CoV/MP789 is analogous to bat CoV/RaTG13 (91.3%) and bat-SL-CoVZXC21 (92.2%) on M-gene and to bat CoV/RaTG13 (94.8%) on N-gene. Nevertheless, nCoVs are distinct from the previously identified SL-CoVs of human origin. The present analysis indicates that nCoVs may have transmitted from bats, pangolin and/or unidentified hosts.

Detection of enteroviruses in marine waters by direct RT-PCR and cell culture

1995 ◽  
Vol 31 (5-6) ◽  
pp. 323-328 ◽  
Author(s):  
K. A. Reynolds ◽  
C. P. Gerba ◽  
I. L. Pepper
Keyword(s):  
Cell Culture ◽  
Polymerase Chain Reaction Amplification ◽  
Cost Effective ◽  
The United States ◽  
Water Runoff ◽  
Rt Pcr ◽  
Marine Waters ◽  
Storm Water Runoff ◽  
Routine Monitoring ◽  
Reaction Amplification

Sewage outfalls and storm water runoff introduces pathogenic human enteric viruses into marine coastal waters, which may pose a potential public health risk. Although members of the enterovirus group have been suggested as possible indicators of sewage pollution in marine waters, the lack of rapid, sensitive and cost effective methods have prevented routine monitoring in the United States. This study compared traditional cell culture and direct RT-PCR (reverse transcriptase-polymerase chain reaction) amplification for detection of an enterovirus. Poliovirus could be recovered from 100 L of artificial seawater with an average efficiency of 77%, using adsorption and elution from electronegative filters. Viruses were eluted from the filters with 1.5% beef extract for viruses (BEV) adjusted to pH 9.5 and reconcentrated by organic flocculation to a volume of 30 mL. Substances which interfered with detection by RT-PCR were removed by treatment of the concentrates with sephadex and chelex resins. Direct RT-PCR could detect 2.5 and 0.025 PFU (plaque forming units) for single (25 cycles) and double PCR (2 × 25 cycles) in 10 μL of pure culture poliovirus samples, respectively. These methods are currently being applied to assess the occurrence of enteroviruses at marine bathing beaches influenced by sewage discharges.

scholarly journals FGFR3 overexpression is a useful detection tool for FGFR3 fusions and sequence variations in glioma

2020 ◽  
Author(s):  
Jens Schittenhelm ◽  
Lukas Ziegler ◽  
Jan Sperveslage ◽  
Michel Mittelbronn ◽  
David Capper ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Growth Factor Receptor ◽  
Gene Mutations ◽  
Diagnostic Tools ◽  
Wild Type ◽  
Rt Pcr ◽  
Fgfr3 Gene ◽  
Detection Tool ◽  
Sequence Variations ◽  
Gene Panel Sequencing

Abstract Background Fibroblast growth factor receptor (FGFR) inhibitors are currently used in clinical development. A subset of glioblastomas carries gene fusion of FGFR3 and transforming acidic coiled-coil protein 3. The prevalence of other FGFR3 alterations in glioma is currently unclear. Methods We performed RT-PCR in 101 glioblastoma samples to detect FGFR3-TACC3 fusions (“RT-PCR cohort”) and correlated results with FGFR3 immunohistochemistry (IHC). Further, we applied FGFR3 IHC in 552 tissue microarray glioma samples (“TMA cohort”) and validated these results in two external cohorts with 319 patients. Gene panel sequencing was carried out in 88 samples (“NGS cohort”) to identify other possible FGFR3 alterations. Molecular modeling was performed on newly detected mutations. Results In the “RT-PCR cohort,” we identified FGFR3-TACC3 fusions in 2/101 glioblastomas. Positive IHC staining was observed in 73/1024 tumor samples of which 10 were strongly positive. In the “NGS cohort,” we identified FGFR3 fusions in 9/88 cases, FGFR3 amplification in 2/88 cases, and FGFR3 gene mutations in 7/88 cases in targeted sequencing. All FGFR3 fusions and amplifications and a novel FGFR3 K649R missense mutation were associated with FGFR3 overexpression (sensitivity and specificity of 93% and 95%, respectively, at cutoff IHC score > 7). Modeling of these data indicated that Tyr647, a residue phosphorylated as a part of FGFR3 activation, is affected by the K649R mutation. Conclusions FGFR3 IHC is a useful screening tool for the detection of FGFR3 alterations and could be included in the workflow for isocitrate dehydrogenase (IDH) wild-type glioma diagnostics. Samples with positive FGFR3 staining could then be selected for NGS-based diagnostic tools.

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