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Detection of the ADGRG6 hotspot mutations in urine for bladder cancer early screening by ARMS‐qPCR

BACKGROUND: In bladder cancer, recurrent ADGRG6 enhancer hotspot mutations (chr. 6: 142,706,206 G>A, chr. 6:142,706,209 C>T) were reported at a high mutation rate of approximately 50%. Thus, ADGRG6 enhancer mutation status might be a candidate for diagnostic biomarker. METHODS: To improve test...

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Autores principales: Tan, Dan, Jiang, Wenqi, Hu, Rixin, Li, Zhuoran, Ou, Tong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10242345/
https://www.ncbi.nlm.nih.gov/pubmed/37081791
http://dx.doi.org/10.1002/cam4.5879
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author Tan, Dan
Jiang, Wenqi
Hu, Rixin
Li, Zhuoran
Ou, Tong
author_facet Tan, Dan
Jiang, Wenqi
Hu, Rixin
Li, Zhuoran
Ou, Tong
author_sort Tan, Dan
collection PubMed
description BACKGROUND: In bladder cancer, recurrent ADGRG6 enhancer hotspot mutations (chr. 6: 142,706,206 G>A, chr. 6:142,706,209 C>T) were reported at a high mutation rate of approximately 50%. Thus, ADGRG6 enhancer mutation status might be a candidate for diagnostic biomarker. METHODS: To improve test efficacy, an amplification refractory mutation system combined with quantitative real‐time PCR (ARMS‐qPCR) assay was developed to detect the ADGRG6 mutations in a patient as a clinical diagnostic test. To validate the performance of the ARMS‐qPCR assay, artificial plasmids, cell DNA reference standard were used as templates, respectively. To test the clinical diagnostic ability, we detected the cell free DNA (cfDNA) and sediment DNA (sDNA) of 30 bladder cancer patients' urine by ARMS‐qPCR comparing with Sanger sequencing, followed by the droplet digital PCR to confirm the results. We also tested the urine of 100 healthy individuals and 90 patients whose diagnoses urinary tract infections or urinary stones but not bladder cancer. RESULTS: Sensitivity of 100% and specificity of 96.7% were achieved when the mutation rate of the artificial plasmid was 1%, and sensitivity of 96.7% and specificity of 100% were achieved when the mutation frequency of the reference standard was 0.5%. Sanger sequencing and ARMS‐qPCR both detected 30 cases of bladder cancer with 93.3% agreement. For the remaining unmatched sites, ARMS‐qPCR results were consistent with droplet digital PCR. Among 100 healthy individuals, three of them carried hotspot mutations by way of ARMS‐qPCR. Of 90 patients with urinary tract infections or urinary stones, no mutations were found by ARMS‐qPCR. Based on clinical detection, the ARMS‐qPCR assay's sensitivity is 83.3%, specificity is 98.4%. CONCLUSION: We here present a novel urine test for ADGRG6 hotspot mutations with high accuracy and sensitivity, which may potentially serve as a rapid and non‐invasive tool for bladder cancer early screening and follow‐up relapse monitoring.
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spelling pubmed-102423452023-06-07 Detection of the ADGRG6 hotspot mutations in urine for bladder cancer early screening by ARMS‐qPCR Tan, Dan Jiang, Wenqi Hu, Rixin Li, Zhuoran Ou, Tong Cancer Med RESEARCH ARTICLES BACKGROUND: In bladder cancer, recurrent ADGRG6 enhancer hotspot mutations (chr. 6: 142,706,206 G>A, chr. 6:142,706,209 C>T) were reported at a high mutation rate of approximately 50%. Thus, ADGRG6 enhancer mutation status might be a candidate for diagnostic biomarker. METHODS: To improve test efficacy, an amplification refractory mutation system combined with quantitative real‐time PCR (ARMS‐qPCR) assay was developed to detect the ADGRG6 mutations in a patient as a clinical diagnostic test. To validate the performance of the ARMS‐qPCR assay, artificial plasmids, cell DNA reference standard were used as templates, respectively. To test the clinical diagnostic ability, we detected the cell free DNA (cfDNA) and sediment DNA (sDNA) of 30 bladder cancer patients' urine by ARMS‐qPCR comparing with Sanger sequencing, followed by the droplet digital PCR to confirm the results. We also tested the urine of 100 healthy individuals and 90 patients whose diagnoses urinary tract infections or urinary stones but not bladder cancer. RESULTS: Sensitivity of 100% and specificity of 96.7% were achieved when the mutation rate of the artificial plasmid was 1%, and sensitivity of 96.7% and specificity of 100% were achieved when the mutation frequency of the reference standard was 0.5%. Sanger sequencing and ARMS‐qPCR both detected 30 cases of bladder cancer with 93.3% agreement. For the remaining unmatched sites, ARMS‐qPCR results were consistent with droplet digital PCR. Among 100 healthy individuals, three of them carried hotspot mutations by way of ARMS‐qPCR. Of 90 patients with urinary tract infections or urinary stones, no mutations were found by ARMS‐qPCR. Based on clinical detection, the ARMS‐qPCR assay's sensitivity is 83.3%, specificity is 98.4%. CONCLUSION: We here present a novel urine test for ADGRG6 hotspot mutations with high accuracy and sensitivity, which may potentially serve as a rapid and non‐invasive tool for bladder cancer early screening and follow‐up relapse monitoring. John Wiley and Sons Inc. 2023-04-20 /pmc/articles/PMC10242345/ /pubmed/37081791 http://dx.doi.org/10.1002/cam4.5879 Text en © 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle RESEARCH ARTICLES
Tan, Dan
Jiang, Wenqi
Hu, Rixin
Li, Zhuoran
Ou, Tong
Detection of the ADGRG6 hotspot mutations in urine for bladder cancer early screening by ARMS‐qPCR
title Detection of the ADGRG6 hotspot mutations in urine for bladder cancer early screening by ARMS‐qPCR
title_full Detection of the ADGRG6 hotspot mutations in urine for bladder cancer early screening by ARMS‐qPCR
title_fullStr Detection of the ADGRG6 hotspot mutations in urine for bladder cancer early screening by ARMS‐qPCR
title_full_unstemmed Detection of the ADGRG6 hotspot mutations in urine for bladder cancer early screening by ARMS‐qPCR
title_short Detection of the ADGRG6 hotspot mutations in urine for bladder cancer early screening by ARMS‐qPCR
title_sort detection of the adgrg6 hotspot mutations in urine for bladder cancer early screening by arms‐qpcr
topic RESEARCH ARTICLES
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10242345/
https://www.ncbi.nlm.nih.gov/pubmed/37081791
http://dx.doi.org/10.1002/cam4.5879
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