Highly sensitive droplet digital PCR for detection of RET fusion in papillary thyroid cancer
Abstract Background Thyroid cancer is the most frequent malignancy of the endocrine system, of which papillary thyroid cancer (PTC) is the predominant form with a rapid increasing incidence worldwide. Rearranged during transfection (RET) fusions are common genetic drivers of PTC and the potent RET i...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-04-01
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| Series: | BMC Cancer |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12885-023-10852-z |
| _version_ | 1797841038433320960 |
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| author | Mengke Chen Junyu Xue Ye Sang Wenting Jiang Weiman He Shubin Hong Weiming Lv Haipeng Xiao Rengyun Liu |
| author_facet | Mengke Chen Junyu Xue Ye Sang Wenting Jiang Weiman He Shubin Hong Weiming Lv Haipeng Xiao Rengyun Liu |
| author_sort | Mengke Chen |
| collection | DOAJ |
| description | Abstract Background Thyroid cancer is the most frequent malignancy of the endocrine system, of which papillary thyroid cancer (PTC) is the predominant form with a rapid increasing incidence worldwide. Rearranged during transfection (RET) fusions are common genetic drivers of PTC and the potent RET inhibitor selpercatinib has been recently approved for treating advanced or metastatic RET fusion-positive thyroid cancer. In this study we aimed to develop a droplet digital PCR (ddPCR) system to accurately detect RET fusion in PTC samples. Methods The frequency and distribution of RET fusions in PTC were analyzed using genomic data of 402 PTC patients in The Cancer Genome Atlas (TCGA) database. To establish the ddPCR system for detecting CCDC6::RET fusion, a plasmid containing CCDC6::RET infusion fragment was constructed as standard template, the annealing temperature and concentrations of primers and probe were optimized. The analytical performance of ddPCR and quantitative reverse transcription PCR (qRT-PCR) were assessed in standard templates and tissue samples from 112 PTC patients. Sanger sequencing was performed in all the RET fusion-positive samples identified by ddPCR. Results RET fusions were observed in 25 (6.2%) of the 402 TCGA samples, and 15 (60%) of the RET fusion-positive patients had the CCDC6::RET fusion. Compared with qRT-PCR, the ddPCR method showed a lower limit of detection (128.0 and 430.7 copies/reaction for ddPCR and qRT-PCR, respectively). When applying the two methods to 112 tissue samples of PTC, eleven (9.8%) CCDC6::RET fusion-positive samples were detected by qRT-PCR, while ddPCR identified 4 additional positive samples (15/112, 13.4%). All the CCDC6::RET fusion-positive cases identified by ddPCR were confirmed by Sanger sequencing except for one case with 0.14 copies/uL of the fusion. Conclusion The accurate and sensitive ddPCR method reported here is powerful to detection CCDC6::RET fusion in PTC samples, application of this method would benefit more RET fusion-positive patients in the clinic. |
| first_indexed | 2024-04-09T16:24:27Z |
| format | Article |
| id | doaj.art-d679a23b0db241c29457eb68e599f432 |
| institution | Directory Open Access Journal |
| issn | 1471-2407 |
| language | English |
| last_indexed | 2024-04-09T16:24:27Z |
| publishDate | 2023-04-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Cancer |
| spelling | doaj.art-d679a23b0db241c29457eb68e599f4322023-04-23T11:20:18ZengBMCBMC Cancer1471-24072023-04-012311910.1186/s12885-023-10852-zHighly sensitive droplet digital PCR for detection of RET fusion in papillary thyroid cancerMengke Chen0Junyu Xue1Ye Sang2Wenting Jiang3Weiman He4Shubin Hong5Weiming Lv6Haipeng Xiao7Rengyun Liu8Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-Sen UniversityDepartment of Endocrinology, The First Affiliated Hospital, Sun Yat-Sen UniversityInstitute of Precision Medicine, The First Affiliated Hospital, Sun Yat-Sen UniversityInstitute of Precision Medicine, The First Affiliated Hospital, Sun Yat-Sen UniversityDepartment of Endocrinology, The First Affiliated Hospital, Sun Yat-Sen UniversityDepartment of Endocrinology, The First Affiliated Hospital, Sun Yat-Sen UniversityDepartment of Breast and Thyroid Surgery, The First Affiliated Hospital, Sun Yat-Sen UniversityDepartment of Endocrinology, The First Affiliated Hospital, Sun Yat-Sen UniversityInstitute of Precision Medicine, The First Affiliated Hospital, Sun Yat-Sen UniversityAbstract Background Thyroid cancer is the most frequent malignancy of the endocrine system, of which papillary thyroid cancer (PTC) is the predominant form with a rapid increasing incidence worldwide. Rearranged during transfection (RET) fusions are common genetic drivers of PTC and the potent RET inhibitor selpercatinib has been recently approved for treating advanced or metastatic RET fusion-positive thyroid cancer. In this study we aimed to develop a droplet digital PCR (ddPCR) system to accurately detect RET fusion in PTC samples. Methods The frequency and distribution of RET fusions in PTC were analyzed using genomic data of 402 PTC patients in The Cancer Genome Atlas (TCGA) database. To establish the ddPCR system for detecting CCDC6::RET fusion, a plasmid containing CCDC6::RET infusion fragment was constructed as standard template, the annealing temperature and concentrations of primers and probe were optimized. The analytical performance of ddPCR and quantitative reverse transcription PCR (qRT-PCR) were assessed in standard templates and tissue samples from 112 PTC patients. Sanger sequencing was performed in all the RET fusion-positive samples identified by ddPCR. Results RET fusions were observed in 25 (6.2%) of the 402 TCGA samples, and 15 (60%) of the RET fusion-positive patients had the CCDC6::RET fusion. Compared with qRT-PCR, the ddPCR method showed a lower limit of detection (128.0 and 430.7 copies/reaction for ddPCR and qRT-PCR, respectively). When applying the two methods to 112 tissue samples of PTC, eleven (9.8%) CCDC6::RET fusion-positive samples were detected by qRT-PCR, while ddPCR identified 4 additional positive samples (15/112, 13.4%). All the CCDC6::RET fusion-positive cases identified by ddPCR were confirmed by Sanger sequencing except for one case with 0.14 copies/uL of the fusion. Conclusion The accurate and sensitive ddPCR method reported here is powerful to detection CCDC6::RET fusion in PTC samples, application of this method would benefit more RET fusion-positive patients in the clinic.https://doi.org/10.1186/s12885-023-10852-zRET fusionddPCRMolecular diagnosisThyroid cancer |
| spellingShingle | Mengke Chen Junyu Xue Ye Sang Wenting Jiang Weiman He Shubin Hong Weiming Lv Haipeng Xiao Rengyun Liu Highly sensitive droplet digital PCR for detection of RET fusion in papillary thyroid cancer BMC Cancer RET fusion ddPCR Molecular diagnosis Thyroid cancer |
| title | Highly sensitive droplet digital PCR for detection of RET fusion in papillary thyroid cancer |
| title_full | Highly sensitive droplet digital PCR for detection of RET fusion in papillary thyroid cancer |
| title_fullStr | Highly sensitive droplet digital PCR for detection of RET fusion in papillary thyroid cancer |
| title_full_unstemmed | Highly sensitive droplet digital PCR for detection of RET fusion in papillary thyroid cancer |
| title_short | Highly sensitive droplet digital PCR for detection of RET fusion in papillary thyroid cancer |
| title_sort | highly sensitive droplet digital pcr for detection of ret fusion in papillary thyroid cancer |
| topic | RET fusion ddPCR Molecular diagnosis Thyroid cancer |
| url | https://doi.org/10.1186/s12885-023-10852-z |
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