Bill H Diplas1,2, Heng Liu1,2, Rui Yang1,2, Landon J Hansen1,2, Alexis L Zachem1,2, Fangping Zhao3, Darell D Bigner1,4, Roger E McLendon1,2, Yuchen Jiao5, Yiping He1,2, Matthew S Waitkus1,2, Hai Yan1,2. 1. The Preston Robert Tisch Brain Tumor Center at Duke, Duke University Medical Center, Durham, North Carolina, USA. 2. Department of Pathology, Duke University Medical Center, Durham, North Carolina, USA. 3. Genetron Health Technologies, Inc., Research Triangle Park, North Carolina, USA. 4. Department of Neurosurgery, Duke University Medical Center, Durham, North Carolina, USA. 5. National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Abstract
BACKGROUND: Mutations in telomerase reverse transcriptase promoter (TERTp) and isocitrate dehydrogenase 1 and 2 (IDH) offer objective markers to assist in classifying diffuse gliomas into genetic subgroups. However, traditional mutation detection techniques lack sensitivity or have long turnaround times or high costs. We developed GliomaDx, an allele-specific, locked nucleic acid-based quantitative PCR assay to overcome these limitations and sensitively detect TERTp and IDH mutations. METHODS: We evaluated the performance of GliomaDx on cell line DNA and frozen tissue diffuse glioma samples with variable tumor percentage to mimic use in clinical settings and validated low percentage variants using sensitive techniques including droplet digital PCR (ddPCR) and next-generation sequencing. We also developed GliomaDx Nest, which incorporates a high-fidelity multiplex pre-amplification step prior to allele-specific PCR for low-input formalin-fixed paraffin embedded (FFPE) samples. RESULTS: GliomaDx detects the TERTp and IDH1 alterations at an analytical sensitivity of 0.1% mutant allele fraction, corresponding to 0.2% tumor cellularity. GliomaDx identified TERTp/IDH1 alterations in a cohort of frozen tissue samples with variable tumor percentage of all major diffuse glioma histologic types. GliomaDx Nest is able to detect these hotspot mutations with similar sensitivity from pre-amplified samples and was successfully tested on a cohort of clinical FFPE samples. Testing of a cohort of previously identified TERTpWT-IDHWT gliomas (by Sanger sequencing) revealed that 26.3% harbored low-percentage mutations. Analysis by ddPCR and whole exome sequencing of these tumors confirmed the low mutant fraction of these alterations and overall mutation-based tumor purity. CONCLUSIONS: Our results show that GliomaDx can rapidly detect TERTp/IDH mutations with high sensitivity, identifying cases that might be missed due to the lack of sensitivity of other techniques. This approach may facilitate more objective classification of diffuse glioma samples in clinical settings such as intraoperative diagnosis or in testing cases with low tumor purity.
BACKGROUND: Mutations in telomerase reverse transcriptase promoter (TERTp) and isocitrate dehydrogenase 1 and 2 (IDH) offer objective markers to assist in classifying diffuse gliomas into genetic subgroups. However, traditional mutation detection techniques lack sensitivity or have long turnaround times or high costs. We developed GliomaDx, an allele-specific, locked nucleic acid-based quantitative PCR assay to overcome these limitations and sensitively detect TERTp and IDH mutations. METHODS: We evaluated the performance of GliomaDx on cell line DNA and frozen tissue diffuse glioma samples with variable tumor percentage to mimic use in clinical settings and validated low percentage variants using sensitive techniques including droplet digital PCR (ddPCR) and next-generation sequencing. We also developed GliomaDx Nest, which incorporates a high-fidelity multiplex pre-amplification step prior to allele-specific PCR for low-input formalin-fixed paraffin embedded (FFPE) samples. RESULTS: GliomaDx detects the TERTp and IDH1 alterations at an analytical sensitivity of 0.1% mutant allele fraction, corresponding to 0.2% tumor cellularity. GliomaDx identified TERTp/IDH1 alterations in a cohort of frozen tissue samples with variable tumor percentage of all major diffuse glioma histologic types. GliomaDx Nest is able to detect these hotspot mutations with similar sensitivity from pre-amplified samples and was successfully tested on a cohort of clinical FFPE samples. Testing of a cohort of previously identified TERTpWT-IDHWT gliomas (by Sanger sequencing) revealed that 26.3% harbored low-percentage mutations. Analysis by ddPCR and whole exome sequencing of these tumors confirmed the low mutant fraction of these alterations and overall mutation-based tumor purity. CONCLUSIONS: Our results show that GliomaDx can rapidly detect TERTp/IDH mutations with high sensitivity, identifying cases that might be missed due to the lack of sensitivity of other techniques. This approach may facilitate more objective classification of diffuse glioma samples in clinical settings such as intraoperative diagnosis or in testing cases with low tumor purity.
Authors: Cameron W Brennan; Roel G W Verhaak; Aaron McKenna; Benito Campos; Houtan Noushmehr; Sofie R Salama; Siyuan Zheng; Debyani Chakravarty; J Zachary Sanborn; Samuel H Berman; Rameen Beroukhim; Brady Bernard; Chang-Jiun Wu; Giannicola Genovese; Ilya Shmulevich; Jill Barnholtz-Sloan; Lihua Zou; Rahulsimham Vegesna; Sachet A Shukla; Giovanni Ciriello; W K Yung; Wei Zhang; Carrie Sougnez; Tom Mikkelsen; Kenneth Aldape; Darell D Bigner; Erwin G Van Meir; Michael Prados; Andrew Sloan; Keith L Black; Jennifer Eschbacher; Gaetano Finocchiaro; William Friedman; David W Andrews; Abhijit Guha; Mary Iacocca; Brian P O'Neill; Greg Foltz; Jerome Myers; Daniel J Weisenberger; Robert Penny; Raju Kucherlapati; Charles M Perou; D Neil Hayes; Richard Gibbs; Marco Marra; Gordon B Mills; Eric Lander; Paul Spellman; Richard Wilson; Chris Sander; John Weinstein; Matthew Meyerson; Stacey Gabriel; Peter W Laird; David Haussler; Gad Getz; Lynda Chin Journal: Cell Date: 2013-10-10 Impact factor: 41.582
Authors: Roel G W Verhaak; Katherine A Hoadley; Elizabeth Purdom; Victoria Wang; Yuan Qi; Matthew D Wilkerson; C Ryan Miller; Li Ding; Todd Golub; Jill P Mesirov; Gabriele Alexe; Michael Lawrence; Michael O'Kelly; Pablo Tamayo; Barbara A Weir; Stacey Gabriel; Wendy Winckler; Supriya Gupta; Lakshmi Jakkula; Heidi S Feiler; J Graeme Hodgson; C David James; Jann N Sarkaria; Cameron Brennan; Ari Kahn; Paul T Spellman; Richard K Wilson; Terence P Speed; Joe W Gray; Matthew Meyerson; Gad Getz; Charles M Perou; D Neil Hayes Journal: Cancer Cell Date: 2010-01-19 Impact factor: 31.743
Authors: D Williams Parsons; Siân Jones; Xiaosong Zhang; Jimmy Cheng-Ho Lin; Rebecca J Leary; Philipp Angenendt; Parminder Mankoo; Hannah Carter; I-Mei Siu; Gary L Gallia; Alessandro Olivi; Roger McLendon; B Ahmed Rasheed; Stephen Keir; Tatiana Nikolskaya; Yuri Nikolsky; Dana A Busam; Hanna Tekleab; Luis A Diaz; James Hartigan; Doug R Smith; Robert L Strausberg; Suely Kazue Nagahashi Marie; Sueli Mieko Oba Shinjo; Hai Yan; Gregory J Riggins; Darell D Bigner; Rachel Karchin; Nick Papadopoulos; Giovanni Parmigiani; Bert Vogelstein; Victor E Velculescu; Kenneth W Kinzler Journal: Science Date: 2008-09-04 Impact factor: 47.728
Authors: Jeanette E Eckel-Passow; Daniel H Lachance; Annette M Molinaro; Kyle M Walsh; Paul A Decker; Hugues Sicotte; Melike Pekmezci; Terri Rice; Matt L Kosel; Ivan V Smirnov; Gobinda Sarkar; Alissa A Caron; Thomas M Kollmeyer; Corinne E Praska; Anisha R Chada; Chandralekha Halder; Helen M Hansen; Lucie S McCoy; Paige M Bracci; Roxanne Marshall; Shichun Zheng; Gerald F Reis; Alexander R Pico; Brian P O'Neill; Jan C Buckner; Caterina Giannini; Jason T Huse; Arie Perry; Tarik Tihan; Mitchell S Berger; Susan M Chang; Michael D Prados; Joseph Wiemels; John K Wiencke; Margaret R Wrensch; Robert B Jenkins Journal: N Engl J Med Date: 2015-06-10 Impact factor: 176.079
Authors: Bill H Diplas; Xujun He; Jacqueline A Brosnan-Cashman; Heng Liu; Lee H Chen; Zhaohui Wang; Casey J Moure; Patrick J Killela; Daniel B Loriaux; Eric S Lipp; Paula K Greer; Rui Yang; Anthony J Rizzo; Fausto J Rodriguez; Allan H Friedman; Henry S Friedman; Sizhen Wang; Yiping He; Roger E McLendon; Darell D Bigner; Yuchen Jiao; Matthew S Waitkus; Alan K Meeker; Hai Yan Journal: Nat Commun Date: 2018-05-25 Impact factor: 14.919
Authors: Ganesh M Shankar; Ameya R Kirtane; Julie J Miller; Hormoz Mazdiyasni; Jaimie Rogner; Tammy Tai; Erik A Williams; Fumi Higuchi; Tareq A Juratli; Kensuke Tateishi; Mara V A Koerner; Shilpa S Tummala; Alexandria L Fink; Tristan Penson; Stephen P Schmidt; Gregory R Wojtkiewicz; Aymen Baig; Joshua M Francis; Mikael L Rinne; Julie M Batten; Tracy T Batchelor; Priscilla K Brastianos; William T Curry; Fred G Barker; Justin T Jordan; A John Iafrate; Andrew S Chi; Jochen K Lennerz; Matthew Meyerson; Robert Langer; Hiroaki Wakimoto; Giovanni Traverso; Daniel P Cahill Journal: Proc Natl Acad Sci U S A Date: 2018-08-06 Impact factor: 12.779
Authors: Casey J Moure; Bill H Diplas; Lee H Chen; Rui Yang; Christopher J Pirozzi; Zhaohui Wang; Ivan Spasojevic; Matthew S Waitkus; Yiping He; Hai Yan Journal: Mol Cancer Res Date: 2019-07-10 Impact factor: 5.852
Authors: Marta Dratwa; Barbara Wysoczańska; Piotr Łacina; Tomasz Kubik; Katarzyna Bogunia-Kubik Journal: Front Immunol Date: 2020-11-19 Impact factor: 7.561