Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma
Abstract Epidermal growth factor receptor (EGFR) amplification and EGFR variant III (EGFRvIII, deletion of exons 2–7) are of clinical interest for glioblastoma. The aim was to develop a digital PCR (dPCR)-based method using locked nucleic acid (LNA)-based hydrolysis probes, allowing the simultaneous...
| Main Authors: | , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2020-04-01
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| Series: | Acta Neuropathologica Communications |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s40478-020-00917-6 |
| _version_ | 1798044871235207168 |
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| author | Maxime Fontanilles Florent Marguet Philippe Ruminy Carole Basset Adrien Noel Ludivine Beaussire Mathieu Viennot Pierre-Julien Viailly Kevin Cassinari Pascal Chambon Doriane Richard Cristina Alexandru Isabelle Tennevet Olivier Langlois Frédéric Di Fiore Annie Laquerrière Florian Clatot Nasrin Sarafan-Vasseur |
| author_facet | Maxime Fontanilles Florent Marguet Philippe Ruminy Carole Basset Adrien Noel Ludivine Beaussire Mathieu Viennot Pierre-Julien Viailly Kevin Cassinari Pascal Chambon Doriane Richard Cristina Alexandru Isabelle Tennevet Olivier Langlois Frédéric Di Fiore Annie Laquerrière Florian Clatot Nasrin Sarafan-Vasseur |
| author_sort | Maxime Fontanilles |
| collection | DOAJ |
| description | Abstract Epidermal growth factor receptor (EGFR) amplification and EGFR variant III (EGFRvIII, deletion of exons 2–7) are of clinical interest for glioblastoma. The aim was to develop a digital PCR (dPCR)-based method using locked nucleic acid (LNA)-based hydrolysis probes, allowing the simultaneous detection of the EGFR amplification and EGFRvIII variant. Sixty-two patients were included. An exploratory cohort (n = 19) was used to develop the dPCR assay using three selected amplicons within the EGFR gene, targeting intron 1 (EGFR1), junction of exon 3 and intron 3 (EGFR2) and intron 22 (EGFR3). The copy number of EGFR was estimated by the relative quantification of EGFR1, EGFR2 and EGFR3 amplicon droplets compared to the droplets of a reference gene. EGFRvIII was identified by comparing the copy number of the EGFR2 amplicon to either the EGFR1 or EGFR3 amplicon. dPCR results were compared to fluorescence in situ hybridization (FISH) and next-generation sequencing for amplification; and to RT-PCR-based method for EGFRvIII. The dPCR assay was then tested in a validation cohort (n = 43). A total of 8/19 EGFR-amplified and 5/19 EGFRvIII-positive tumors were identified in the exploratory cohort. Compared to FISH, the EGFR3 dPCR assay detected all EGFR-amplified tumors (8/8, 100%) and had the highest concordance with the copy number estimation by NGS. The concordance between RT-PCR and dPCR was also 100% for detecting EGFRvIII using an absolute difference of 10.8 for the copy number between EGFR2 and EGFR3 probes. In the validation cohort, the sensitivity and specificity of dPCR using EGFR3 probes were 100% for the EGFR amplification detection compared to FISH (19/19). EGFRvIII was detected by dPCR in 8 EGFR-amplified patients and confirmed by RT-PCR. Compared to FISH, the EGFR2/EGFR3 dPCR assay was estimated with a one-half cost value. These results highlight that dPCR allowed the simultaneous detection of EGFR amplification and EGFRvIII for glioblastoma. |
| first_indexed | 2024-04-11T23:11:14Z |
| format | Article |
| id | doaj.art-b76c4e1ab7454e6a9684b1d901ce8e47 |
| institution | Directory Open Access Journal |
| issn | 2051-5960 |
| language | English |
| last_indexed | 2024-04-11T23:11:14Z |
| publishDate | 2020-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Acta Neuropathologica Communications |
| spelling | doaj.art-b76c4e1ab7454e6a9684b1d901ce8e472022-12-22T03:57:50ZengBMCActa Neuropathologica Communications2051-59602020-04-018111010.1186/s40478-020-00917-6Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastomaMaxime Fontanilles0Florent Marguet1Philippe Ruminy2Carole Basset3Adrien Noel4Ludivine Beaussire5Mathieu Viennot6Pierre-Julien Viailly7Kevin Cassinari8Pascal Chambon9Doriane Richard10Cristina Alexandru11Isabelle Tennevet12Olivier Langlois13Frédéric Di Fiore14Annie Laquerrière15Florian Clatot16Nasrin Sarafan-Vasseur17Inserm U1245, Normandie Univ, UNIROUEN, IRON group, Normandy Centre for Genomic and Personalized Medicine, Rouen University HospitalInserm U1245, Normandie Univ, UNIROUEN, Normandy Centre for Genomic and Personalized Medicine, Rouen University HospitalInserm U1245, Normandie Univ, UNIROUEN, Normandy Centre for Genomic and Personalized Medicine, Rouen University HospitalDepartment of Pathology, Rouen University HospitalInserm U1245, Normandie Univ, UNIROUEN, IRON group, Normandy Centre for Genomic and Personalized Medicine, Rouen University HospitalInserm U1245, Normandie Univ, UNIROUEN, IRON group, Normandy Centre for Genomic and Personalized Medicine, Rouen University HospitalInserm U1245, Normandie Univ, UNIROUEN, Normandy Centre for Genomic and Personalized Medicine, Rouen University HospitalDepartment of Pathology, Rouen University HospitalDepartment of Genetics, Inserm U1245, Normandie Univ, UNIROUEN, Normandy Centre for Genomic and Personalized Medicine, Rouen University HospitalDepartment of Genetics, Inserm U1245, Normandie Univ, UNIROUEN, Normandy Centre for Genomic and Personalized Medicine, Rouen University HospitalDepartment of Statistics and Clinical Research Unit, Cancer Centre Henri BecquerelDepartment of Medical Oncology, Cancer Centre Henri BecquerelDepartment of Medical Oncology, Cancer Centre Henri BecquerelDepartment of Neurosurgery, Rouen University HospitalInserm U1245, Normandie Univ, UNIROUEN, IRON group, Normandy Centre for Genomic and Personalized Medicine, Rouen University HospitalInserm U1245, Normandie Univ, UNIROUEN, Normandy Centre for Genomic and Personalized Medicine, Rouen University HospitalInserm U1245, Normandie Univ, UNIROUEN, IRON group, Normandy Centre for Genomic and Personalized Medicine, Rouen University HospitalInserm U1245, Normandie Univ, UNIROUEN, IRON group, Normandy Centre for Genomic and Personalized Medicine, Rouen University HospitalAbstract Epidermal growth factor receptor (EGFR) amplification and EGFR variant III (EGFRvIII, deletion of exons 2–7) are of clinical interest for glioblastoma. The aim was to develop a digital PCR (dPCR)-based method using locked nucleic acid (LNA)-based hydrolysis probes, allowing the simultaneous detection of the EGFR amplification and EGFRvIII variant. Sixty-two patients were included. An exploratory cohort (n = 19) was used to develop the dPCR assay using three selected amplicons within the EGFR gene, targeting intron 1 (EGFR1), junction of exon 3 and intron 3 (EGFR2) and intron 22 (EGFR3). The copy number of EGFR was estimated by the relative quantification of EGFR1, EGFR2 and EGFR3 amplicon droplets compared to the droplets of a reference gene. EGFRvIII was identified by comparing the copy number of the EGFR2 amplicon to either the EGFR1 or EGFR3 amplicon. dPCR results were compared to fluorescence in situ hybridization (FISH) and next-generation sequencing for amplification; and to RT-PCR-based method for EGFRvIII. The dPCR assay was then tested in a validation cohort (n = 43). A total of 8/19 EGFR-amplified and 5/19 EGFRvIII-positive tumors were identified in the exploratory cohort. Compared to FISH, the EGFR3 dPCR assay detected all EGFR-amplified tumors (8/8, 100%) and had the highest concordance with the copy number estimation by NGS. The concordance between RT-PCR and dPCR was also 100% for detecting EGFRvIII using an absolute difference of 10.8 for the copy number between EGFR2 and EGFR3 probes. In the validation cohort, the sensitivity and specificity of dPCR using EGFR3 probes were 100% for the EGFR amplification detection compared to FISH (19/19). EGFRvIII was detected by dPCR in 8 EGFR-amplified patients and confirmed by RT-PCR. Compared to FISH, the EGFR2/EGFR3 dPCR assay was estimated with a one-half cost value. These results highlight that dPCR allowed the simultaneous detection of EGFR amplification and EGFRvIII for glioblastoma.http://link.springer.com/article/10.1186/s40478-020-00917-6GlioblastomaDigital PCREGFR amplificationEGFRvIII variantCost-effectiveness |
| spellingShingle | Maxime Fontanilles Florent Marguet Philippe Ruminy Carole Basset Adrien Noel Ludivine Beaussire Mathieu Viennot Pierre-Julien Viailly Kevin Cassinari Pascal Chambon Doriane Richard Cristina Alexandru Isabelle Tennevet Olivier Langlois Frédéric Di Fiore Annie Laquerrière Florian Clatot Nasrin Sarafan-Vasseur Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma Acta Neuropathologica Communications Glioblastoma Digital PCR EGFR amplification EGFRvIII variant Cost-effectiveness |
| title | Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma |
| title_full | Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma |
| title_fullStr | Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma |
| title_full_unstemmed | Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma |
| title_short | Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma |
| title_sort | simultaneous detection of egfr amplification and egfrviii variant using digital pcr based method in glioblastoma |
| topic | Glioblastoma Digital PCR EGFR amplification EGFRvIII variant Cost-effectiveness |
| url | http://link.springer.com/article/10.1186/s40478-020-00917-6 |
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