Natalya Oskina1, Igor Oscorbin2, Evgeniy Khrapov2, Ulyana Boyarskikh2,3, Dmitriy Subbotin3, Irina Demidova4, Evgeny Imyanitov5,6,7,8, Maxim Filipenko2,3. 1. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk, 630090, Russia. nattasha.o@gmail.com. 2. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8 Lavrentiev Avenue, Novosibirsk, 630090, Russia. 3. Novosibirsk State University, Novosibirsk, Russia. 4. City Oncological Hospital, No. 62, Moscow, Russia. 5. N.N. Petrov Institute of Oncology, St. Petersburg, Russia. 6. St. Petersburg Pediatric Medical University, St. Petersburg, Russia. 7. I.I. Mechnikov North-Western Medical University, St. Petersburg, Russia. 8. St. Petersburg State University, St. Petersburg, Russia.
Abstract
BACKGROUND: Analysis of EGFR mutations is becoming a routine clinical practice but the optimal EGFR mutation testing method is still to be determined. METHODS: We determined the nucleotide sequence of deletions located in exon 19 of the EGFR gene in lung tumor samples of patients residing in different regions of Russia (153 tumor DNA specimens), using Sanger sequencing. We developed a droplet digital polymerase chain reaction assay capable of detecting all common EGFR deletions in exon 19. We also compared the therascreen amplification refractory mutation system assay with a droplet digital polymerase chain reaction assay for the detection of all the deletions in our study. RESULTS: The droplet digital polymerase chain reaction assay demonstrated 100% sensitivity against polymerase chain reaction fragment length analysis and detected all possible types of deletions revealed in our study (22 types). At the same time, the therascreen EGFR RGQ PCR Kit was not able to detect deletions c.2252-2276>A and c.2253-2276 and showed low performance for another long deletion. CONCLUSION: Thus, we can conclude that the extraordinary length of deletions and their atypical locations (shift at the 3'-region compared to known deletions) could be problematic for the therascreen EGFR RGQ PCR Kit and should be taken into account during targeted mutation test development. However, droplet digital polymerase chain reaction is a promising and reliable assay that can be used as a diagnostic tool to genotype formalin-fixed paraffin-embedded cancer samples for EGFR or another clinically relevant somatic mutation.
BACKGROUND: Analysis of EGFR mutations is becoming a routine clinical practice but the optimal EGFR mutation testing method is still to be determined. METHODS: We determined the nucleotide sequence of deletions located in exon 19 of the EGFR gene in lung tumor samples of patients residing in different regions of Russia (153 tumor DNA specimens), using Sanger sequencing. We developed a droplet digital polymerase chain reaction assay capable of detecting all common EGFR deletions in exon 19. We also compared the therascreen amplification refractory mutation system assay with a droplet digital polymerase chain reaction assay for the detection of all the deletions in our study. RESULTS: The droplet digital polymerase chain reaction assay demonstrated 100% sensitivity against polymerase chain reaction fragment length analysis and detected all possible types of deletions revealed in our study (22 types). At the same time, the therascreen EGFR RGQ PCR Kit was not able to detect deletions c.2252-2276>A and c.2253-2276 and showed low performance for another long deletion. CONCLUSION: Thus, we can conclude that the extraordinary length of deletions and their atypical locations (shift at the 3'-region compared to known deletions) could be problematic for the therascreen EGFR RGQ PCR Kit and should be taken into account during targeted mutation test development. However, droplet digital polymerase chain reaction is a promising and reliable assay that can be used as a diagnostic tool to genotype formalin-fixed paraffin-embedded cancer samples for EGFR or another clinically relevant somatic mutation.
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Authors: C R Newton; A Graham; L E Heptinstall; S J Powell; C Summers; N Kalsheker; J C Smith; A F Markham Journal: Nucleic Acids Res Date: 1989-04-11 Impact factor: 16.971
Authors: Silvia Querings; Janine Altmüller; Sascha Ansén; Thomas Zander; Danila Seidel; Franziska Gabler; Martin Peifer; Eva Markert; Kathryn Stemshorn; Bernd Timmermann; Beate Saal; Stefan Klose; Karen Ernestus; Matthias Scheffler; Walburga Engel-Riedel; Erich Stoelben; Elisabeth Brambilla; Jürgen Wolf; Peter Nürnberg; Roman K Thomas Journal: PLoS One Date: 2011-05-05 Impact factor: 3.240