scholarly journals Liquid Biopsy Analysis of FGFR3, TERT Promoter and STAG2 Hotspot Mutations for Disease Surveillance in Bladder Cancer

2020 ◽  
pp. 1-7
Author(s):  
Victor Romanov ◽  
Dimitri Gnatenko ◽  
Edward Forsyth ◽  
Liang Xiaohui ◽  
Olga Povcher ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Sensitivity And Specificity ◽  
Liquid Biopsy ◽  
Disease Surveillance ◽  
Digital Pcr ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tert Promoter ◽  
Mutational Status ◽  
Muscle Invasive

Patients with non-muscle invasive bladder cancer (NMIBC) are followed by frequent cystoscopies. Innovative approaches partly replacing cystoscopy (uncomfortable, expensive, low sensitive procedure) are demanded. The current study aims to establish a fast, reliable, non-invasive, and inexpensive procedure for NMIBC patient surveillance. Liquid biopsy is a reliable source of biomarkers for cancer patient monitoring. Urine is the most suitable biological liquid to search for bladder cancer biomarkers. Cell-free DNA in urine represents tumor-related mutations for several cancers, including the bladder. We investigated mutations in FGFR3, TERT promoter, and STAG2 as markers for diagnostics and follow-up in NMIBC. Digital PCR was used to detect mutations in urine-derived cell-free DNA. The sensitivity and specificity of the markers in relation to clinical outcomes served as criteria of the assay efficiency. The sensitivity with a single marker (TERT) reached 87%, with a specificity of 77%. Combining two biomarkers (TERT+FGFR3) increased the specificity of the assay to 100% with a sensitivity of 72%. Different mutational status of STAG2 can indicate NMIBC presence or recurrence. Therefore, applying the suggested combination of biomarkers with simple detection procedures to larger patient cohorts will allow developing procedures for BC detection and surveillance with optimal sensitivity and specificity. Based on the results of this proof-in-concept study, we conclude that this simple, fast and inexpensive assay can add diagnostic and prognostic value to cystoscopy/cytology analysis of NMIBC patients.

scholarly journals Performance of the OncomineTM Lung cfDNA Assay for Liquid Biopsy by NGS of NSCLC Patients in Routine Laboratory Practice

2020 ◽  
Vol 10 (8) ◽  
pp. 2895 ◽  
Author(s):  
Giuseppa De Luca ◽  
Sonia Lastraioli ◽  
Romana Conte ◽  
Marco Mora ◽  
Carlo Genova ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Kinase Inhibitors ◽  
Limit Of Detection ◽  
Variant Calling ◽  
Plasma Samples ◽  
Cell Free Dna ◽  
Coverage Metrics ◽  
Free Dna ◽  
Mutational Status ◽  
Nsclc Patients

Targeted next-generation sequencing (NGS) based on molecular tagging technology allowed considerable improvement in the approaches of cell-free DNA (cfDNA) analysis. Previously, we demonstrated the feasibility of the OncomineTM Lung cell-free DNA Assay (OLcfA) NGS panel when applied on plasma samples of post-tyrosine kinase inhibitors (TKIs) non-small cell lung cancer (NSCLC) patients. Here, we explored in detail the coverage metrics and variant calling of the assay and highlighted strengths and challenges by analyzing 92 plasma samples collected from a routine cohort of 76 NSCLC patients. First, performance of OLcfA was assessed using Horizon HD780 reference standards and sensitivity and specificity of 92.5% and 100% reported, respectively. The OLcfA was consequently evaluated in our plasma cohort and NGS technically successful in all 92 sequenced libraries. We demonstrated that initial cfDNA amount correlated positively with library yields (p < 0.0001) and sequencing performance (p < 0.0001). In addition, 0.1% limit of detection could be achieved even when < 10 ng cfDNA was employed. In contrast, the cfDNA amount seems to not affect the EGFR mutational status (p = 0.16). This study demonstrated an optimal performance of the OLcfA on routine plasma samples from NSCLC patients and supports its application in the liquid biopsy practice for cfDNA investigation in precision medicine laboratories.

Urinary Cell-Free DNA IQGAP3/BMP4 Ratio as a Prognostic Marker for Non–Muscle-Invasive Bladder Cancer

2019 ◽  
Vol 17 (3) ◽  
pp. e704-e711
Author(s):  
Yanjie Xu ◽  
Ye-Hwan Kim ◽  
Pildu Jeong ◽  
Xuan-Mei Piao ◽  
Young Joon Byun ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Prognostic Marker ◽  
Invasive Bladder Cancer ◽  
Muscle Invasive Bladder Cancer ◽  
Cell Free Dna ◽  
Free Dna ◽  
Muscle Invasive

scholarly journals Development of Sensitive Droplet Digital PCR Assays for Detecting Urinary TERT Promoter Mutations as Non-Invasive Biomarkers for Detection of Urothelial Cancer

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3541 ◽  
Author(s):  
Md Ismail Hosen ◽  
Nathalie Forey ◽  
Geoffroy Durand ◽  
Catherine Voegele ◽  
Selin Bilici ◽  
...  
Keyword(s):  
Urothelial Cancer ◽  
Digital Pcr ◽  
Kappa Coefficient ◽  
Droplet Digital Pcr ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna ◽  
Tert Promoter ◽  
Tert Promoter Mutations ◽  
Promoter Mutations

Somatic mutations in the telomerase reverse transcriptase (TERT) promoter regions are frequent events in urothelial cancer (UC) and their detection in urine (supernatant cell-free DNA or DNA from exfoliated cells) could serve as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumor-borne mutations in urine requires highly sensitive methods, capable of measuring low-level mutations. In this study, we developed sensitive droplet digital PCR (ddPCR) assays for detecting TERT promoter mutations (C228T, C228A, CC242-243TT, and C250T). We tested the C228T and C250T ddPCR assays on all samples with sufficient quantity of urinary DNA (urine supernatant cell-free DNA (US cfDNA) or urine pellet cellular DNA (UP cellDNA)) from the DIAGURO (n = 89/93 cases and n = 92/94 controls) and from the IPO-PORTO (n = 49/50 cases and n = 50/50 controls) series that were previously screened with the UroMuTERT assay and compared the performance of the two approaches. In the DIAGURO series, the sensitivity and specificity of the ddPCR assays for detecting UC using either US cfDNA or UP cellDNA were 86.8% and 92.4%. The sensitivity was slightly higher than that of the UroMuTERT assay in the IPO-PORTO series (67.4% vs. 65.3%, respectively), but not in the DIAGURO series (86.8% vs. 90.7%). The specificity was 100% in the IPO-PORTO controls for both the UroMuTERT and ddPCR assays, whereas in the DIAGURO series, the specificity dropped for ddPCR (92.4% versus 95.6%). Overall, an almost perfect agreement between the two methods was observed for both US cfDNA (n = 164; kappa coefficient of 0.91) and UP cellDNA (n = 280; kappa coefficient of 0.94). In a large independent series of serial urine samples from DIAGURO follow-up BC cases (n = 394), the agreement between ddPCR and UroMuTERT was (i) strong (kappa coefficient of 0.87), regardless of urine DNA types (kappa coefficient 0.89 for US cfDNA and 0.85 for UP cellDNA), (ii) the highest for samples with mutant allelic fractions (MAFs) > 2% (kappa coefficient of 0.99) and (iii) only minimal for the samples with the lowest MAFs (< 0.5%; kappa coefficient 0.32). Altogether, our results indicate that the two methods (ddPCR and UroMuTERT) for detecting urinary TERT promoter mutations are comparable and that the discrepancies relate to the detection of low-allelic fraction mutations. The simplicity of the ddPCR assays makes them suitable for implementation in clinical settings.

Comparison of circulating tumor cells and cell-free DNA in the molecular characterization of patients with head and neck cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e18521-e18521
Author(s):  
Santiago Cabezas-Camarero ◽  
Vanesa García-Barberán ◽  
Virginia De la Orden-García ◽  
Beatriz Mediero-Valeros ◽  
Isabel Díaz-Millán ◽  
...  
Keyword(s):  
Head And Neck Cancer ◽  
Parotid Gland ◽  
Head And Neck ◽  
Molecular Characterization ◽  
Neck Cancer ◽  
Liquid Biopsy ◽  
Digital Pcr ◽  
Cell Free Dna ◽  
Free Dna

e18521 Background: The role of liquid biopsy in diagnosis and therapy monitoring in patients with head and neck cancer has been much less studied compared to other cancers. Our aim was to evaluate the perfomance in the isolation and recovery for molecular characterization of circulating tumour cells (CTC) of a new immunoafinity-based method and to compare it with the molecular diagnostic yield of plasma cell-free DNA. Methods: Patients with recurrent/metastatic (RM) head and neck cancer (HNC) were enrolled prospectively. Forty mililiters (ml) of plasma were collected at one or several time-points. First blood draw was always collected before starting a new therapeutic intervention or at the time of radiologic progression. For CTC detection and isolation, either anti-EpCAM or both anti-EpCAM + anti-EGFR antibodies were used. Digital PCR and castPCR were used to study KRAS and PI3KCA mutations in non-squamous HNC. A 15-gene customized NGS panel was used to characterized both CTC and cfDNA in patients with squamous HNC. Results: Between February 2016 and October 2018, 14 patients with R/M HNC were included (n = 1 local-only disease, n = 10 local and distant disease, n = 3 distant-only disease). Squamous histology (S): n = 9. Non-squamous (NS): n = 5 (1 naso-ethmoidal intestinal-type adenocarcinoma, 1 parotid gland exadenoma pleomorfic carcinoma, 2 parotid-gland salivary duct carcinomas (SDC), 1 parotid-gland high-grade neuroendocrine carcinoma). Twenty-five CTC determinations were performed. In 5 patients serial CTC determinations were performed. Median CTC was 4 (min-max: 0-49). Median CTC among 11 CTC determinations in S-HNC was 4 (min-max: 0-49). Median CTC was 3 CTC (min-max: 0-26) among the 14 determinations performed in NS-HNC. Digital PCR unveiled mutations in CTC and in cfDNA in 2 of 4 patients tested with NS histology (KRAS, PIK3CA), with one of them being concordant for the specific mutation. NGS unveiled mutations in CTC in 7/9 patients and in cfDNA in 6/9 patients, with only one loci-concordant case between CTC and plasma. Conclusions: IsoFlux detected CTC in the majority of patients with R/M HNC, regardless of the histologic type, and allowed for molecular characterization of CTC using different techniques for mutational analysis. Both NGS and digital PCR allowed for the detection in cell-free DNA of commonly mutated genes in HNC. Liquid biopsy should be more actively studied in this disease in order to better define its role in diagnosis and therapeutic monitoring.

scholarly journals Urinary Cell-Free DNA in Bladder Cancer Detection

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 306
Author(s):  
Ryan Tsz-Hei Tse ◽  
Hongda Zhao ◽  
Christine Yim-Ping Wong ◽  
Carol Ka-Lo Cheng ◽  
Angel Wing-Yan Kong ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cancer Detection ◽  
Urinary Bladder Cancer ◽  
Molecular Classification ◽  
Cell Free Dna ◽  
Molecular Features ◽  
Promising Tool ◽  
Free Dna ◽  
Genetic Features ◽  
Muscle Invasive

Urinary bladder cancer is a common urological cancer. Although flexible cystoscopy is widely employed in bladder cancer detection, it is expensive, invasive, and uncomfortable to the patients. Recently, urinary cell-free DNA (ucfDNA) isolated from urine supernatant has been shown to have great potential in bladder cancer detection and surveillance. Molecular features, such as integrity and concentration of ucfDNA, have been shown to be useful for differentiating bladder cancer patients from healthy controls. Besides, bladder cancer also exhibits unique genetic features that can be identified from sequencing and expression of ucfDNA. Apart from bladder cancer detection, ucfDNA is also useful for molecular classification. For example, ucfDNA exhibits significant differences, both molecularly and genetically, in non-muscle-invasive and muscle-invasive bladder cancers. There is no doubt that ucfDNA is a very promising tool for future applications in the field of bladder cancer.

Superior molecular pathology of urothelial bladder cancer using urinary cell-free DNA.

2021 ◽  
Vol 39 (6_suppl) ◽  
pp. 491-491
Author(s):  
Ruiyun Zhang ◽  
Feng Xie ◽  
Yue Zhang ◽  
Yiqiu Wang ◽  
Zhixin Zhao ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Molecular Pathology ◽  
Predictive Value ◽  
Invasive Bladder Cancer ◽  
Urothelial Bladder Cancer ◽  
Muscle Invasive Bladder Cancer ◽  
Cell Free Dna ◽  
Free Dna ◽  
Urothelial Bladder ◽  
Muscle Invasive

491 Background: Both urinary and blood cell-free DNA (cfDNA) have been implicated in noninvasive detection and surveillance of urothelial bladder cancer (UBC). However, a direct comparison of their diagnostic performance in the real-world setting is lacking. Methods: 59 eligible cases with pathologically confirmed disease and accompanying tissue/urine pairs were prospectively enrolled and consented to Institutional Review Board-approved protocols. Baseline peripheral blood mononuclear cell (PBMC) and plasma specimens were collected during clinic visit. The 180-gene Predicine liquid biopsy assay was applied for ultra-deep targeted sequencing and somatic alteration identification in tumor tissue-based DNA (tDNA), urinary cfDNA (ucfDNA) and blood cfDNA (bcfDNA). Results: The 59 studied subjects constituted a natural UBC cohort of non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC), including 48 (81.4%) NMIBC and 42 (71.2%) male patients. Diverse quantitative metrics such as VAF (variant allele frequency) and TMB (tumor mutational burden) were invariably concordant between tDNA and ucfDNA, but not bcfDNA. The mutational landscape captured by tDNA or ucfDNA highly resembled each other and mirrored previously described genomic panorama of UBC, whereas a significant proportion of bcfDNA aberrations stemmed from clonal hematopoiesis. Using tDNA-informed variants as the ground truth, ucfDNA assays achieved a specificity of 99.3%, a sensitivity of 86.7%, a positive predictive value (PPV) of 67.2%, a negative predictive value (NPV) of 99.8%, and a diagnostic accuracy of 99.1%, which were generally lower in the case of bcfDNA analysis. Conclusions: Urine-based molecular pathology provides valid and complete genetic information about neoplastic lesions, and represents a faithful surrogate for genotyping and monitoring UBC.

scholarly journals Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma

Oncotarget ◽  
2017 ◽  
Vol 8 (45) ◽  
pp. 78890-78900 ◽  
Author(s):  
Ashleigh C. McEvoy ◽  
Leslie Calapre ◽  
Michelle R. Pereira ◽  
Tindaro Giardina ◽  
Cleo Robinson ◽  
...  
Keyword(s):  
Metastatic Melanoma ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tert Promoter ◽  
Tert Promoter Mutations ◽  
Pcr Method ◽  
Promoter Mutations

Evaluation of TERT promoter mutations in urinary cell-free DNA and sediment DNA for detection of bladder cancer

2019 ◽  
Vol 64 ◽  
pp. 60-63 ◽  
Author(s):  
Sebastian Stasik ◽  
Karsten Salomo ◽  
Ulrike Heberling ◽  
Michael Froehner ◽  
Ulrich Sommer ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tert Promoter ◽  
Tert Promoter Mutations ◽  
Promoter Mutations

scholarly journals PD47-09 LIQUID BIOPSY ANALYSIS OF TERT PROMOTER MUTATION IN URINARY CELL-FREE DNA IN PATIENTS WITH UROTHELIAL CARCINOMA

2020 ◽  
Vol 203 ◽  
pp. e928-e929
Author(s):  
Yujiro Hayashi* ◽  
Kazutoshi Fujita ◽  
Eisuke Tomiyama ◽  
Yoko Koh ◽  
Makoto Matsushita ◽  
...  
Keyword(s):  
Urothelial Carcinoma ◽  
Liquid Biopsy ◽  
Tert Promoter Mutation ◽  
Promoter Mutation ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tert Promoter

scholarly journals From Sampling to Sequencing: A Liquid Biopsy Pre-Analytic Workflow to Maximize Multi-Layer Genomic Information from a Single Tube

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3002
Author(s):  
Kendra K. Maass ◽  
Paulina S. Schad ◽  
Agnes M. E. Finster ◽  
Pitithat Puranachot ◽  
Fabian Rosing ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Pcr Amplification ◽  
Genomic Analysis ◽  
Digital Pcr ◽  
Great Promise ◽  
Sample Collection ◽  
Liquid Biopsies ◽  
Cell Free Dna ◽  
Dna And Rna ◽  
Free Dna

Liquid biopsies hold great promise for the management of cancer. Reliable liquid biopsy data depend on stable and reproducible pre-analytical protocols that comply with quality measures, irrespective of the sampling and processing site. We established a workflow for plasma preservation, followed by processing, cell-free nucleic acid isolation, quantification, and enrichment of potentially tumor-derived cell-free DNA and RNA. Employing the same input material for a direct comparison of different kits and protocols allowed us to formulate unbiased recommendations for sample collection, storage, and processing. The presented workflow integrates the stabilization in Norgen, PAX, or Streck tubes and subsequent parallel isolation of cell-free DNA and RNA with NucleoSnap and NucleoSpin. Qubit, Bioanalyzer, and TapeStation quantification and quality control steps were optimized for minimal sample use and high sensitivity and reproducibility. We show the efficiency of the proposed workflow by successful droplet digital PCR amplification of both cell-free DNA and RNA and by detection of tumor-specific alterations in low-coverage whole-genome sequencing and DNA methylation profiling of plasma-derived cell-free DNA. For the first time, we demonstrated successful parallel extraction of cell-free DNA and RNA from plasma samples. This workflow paves the road towards multi-layer genomic analysis from one single liquid biopsy sample.

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