Detection of the ADGRG6 hotspot mutations in urine for bladder cancer early screening by ARMS‐qPCR
Abstract 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 impro...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2023-05-01
|
| Series: | Cancer Medicine |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/cam4.5879 |
| _version_ | 1797810446804189184 |
|---|---|
| author | Dan Tan Wenqi Jiang Rixin Hu Zhuoran Li Tong Ou |
| author_facet | Dan Tan Wenqi Jiang Rixin Hu Zhuoran Li Tong Ou |
| author_sort | Dan Tan |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-03-13T07:07:55Z |
| format | Article |
| id | doaj.art-bacca5bd68704ed8a9b4f9af90633094 |
| institution | Directory Open Access Journal |
| issn | 2045-7634 |
| language | English |
| last_indexed | 2024-03-13T07:07:55Z |
| publishDate | 2023-05-01 |
| publisher | Wiley |
| record_format | Article |
| series | Cancer Medicine |
| spelling | doaj.art-bacca5bd68704ed8a9b4f9af906330942023-06-06T07:30:47ZengWileyCancer Medicine2045-76342023-05-011210115031151210.1002/cam4.5879Detection of the ADGRG6 hotspot mutations in urine for bladder cancer early screening by ARMS‐qPCRDan Tan0Wenqi Jiang1Rixin Hu2Zhuoran Li3Tong Ou4Medical Laboratory of Shenzhen Luohu Hospital Group Shenzhen 518000 Guangdong ChinaShenzhen Following Precision Medical Research Institute of Luohu Hospital Group Shenzhen 518000 Guangdong ChinaShenzhen Following Precision Medical Research Institute of Luohu Hospital Group Shenzhen 518000 Guangdong ChinaShenzhen Following Precision Medical Research Institute of Luohu Hospital Group Shenzhen 518000 Guangdong ChinaMedical Laboratory of Shenzhen Luohu Hospital Group Shenzhen 518000 Guangdong ChinaAbstract 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.https://doi.org/10.1002/cam4.5879ADGRG6ARMS‐qPCRbladder cancergenetic mutationliquid biopsy |
| spellingShingle | Dan Tan Wenqi Jiang Rixin Hu Zhuoran Li Tong Ou Detection of the ADGRG6 hotspot mutations in urine for bladder cancer early screening by ARMS‐qPCR Cancer Medicine ADGRG6 ARMS‐qPCR bladder cancer genetic mutation liquid biopsy |
| 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 | ADGRG6 ARMS‐qPCR bladder cancer genetic mutation liquid biopsy |
| url | https://doi.org/10.1002/cam4.5879 |
| work_keys_str_mv | AT dantan detectionoftheadgrg6hotspotmutationsinurineforbladdercancerearlyscreeningbyarmsqpcr AT wenqijiang detectionoftheadgrg6hotspotmutationsinurineforbladdercancerearlyscreeningbyarmsqpcr AT rixinhu detectionoftheadgrg6hotspotmutationsinurineforbladdercancerearlyscreeningbyarmsqpcr AT zhuoranli detectionoftheadgrg6hotspotmutationsinurineforbladdercancerearlyscreeningbyarmsqpcr AT tongou detectionoftheadgrg6hotspotmutationsinurineforbladdercancerearlyscreeningbyarmsqpcr |