Marina N Nikiforova1, Abigail I Wald1, Melissa A Melan1, Somak Roy1, Shan Zhong1, Ronald L Hamilton1, Frank S Lieberman1, Jan Drappatz1, Nduka M Amankulor1, Ian F Pollack1, Yuri E Nikiforov1, Craig Horbinski1. 1. Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
Abstract
BACKGROUND: Identification of genetic changes in CNS tumors is important for the appropriate clinical management of patients. Our objective was to develop a next-generation sequencing (NGS) assay for simultaneously detecting the various types of genetic alterations characteristic for adult and pediatric CNS tumors that can be applied to small brain biopsies. METHODS: We report an amplification-based targeted NGS assay (GlioSeq) that analyzes 30 genes for single nucleotide variants (SNVs) and indels, 24 genes for copy number variations (CNVs), and 14 types of structural alterations in BRAF, EGFR, and FGFR3 genes in a single workflow. GlioSeq performance was evaluated in 54 adult and pediatric CNS tumors, and the results were compared with fluorescence in-situ hybridization, Sanger sequencing, and reverse transcription PCR. RESULTS: GlioSeq correctly identified 71/71 (100%) genetic alterations known to be present by conventional techniques, including 56 SNVs/indels, 9 CNVs, 3 EGFRvIII, and 3 KIAA1549-BRAF fusions. Only 20 ng of DNA and 10 ng of RNA were required for successful sequencing of 100% frozen and 96% formalin-fixed, paraffin-embedded tissue specimens. The assay sensitivity was 3%-5% of mutant alleles for SNVs and 1%-5% for gene fusions. The most commonly detected alterations were IDH1, TP53, TERT, ATRX. CDKN2A, and PTEN in high-grade gliomas, followed by BRAF fusions in low-grade gliomas and H3F3A mutations in pediatric gliomas. CONCLUSIONS: GlioSeq NGS assay offers accurate and sensitive detection of a wide range of genetic alterations in a single workflow. It allows rapid and cost-effective profiling of brain tumor specimens and thus provides valuable information for patient management.
BACKGROUND: Identification of genetic changes in CNS tumors is important for the appropriate clinical management of patients. Our objective was to develop a next-generation sequencing (NGS) assay for simultaneously detecting the various types of genetic alterations characteristic for adult and pediatric CNS tumors that can be applied to small brain biopsies. METHODS: We report an amplification-based targeted NGS assay (GlioSeq) that analyzes 30 genes for single nucleotide variants (SNVs) and indels, 24 genes for copy number variations (CNVs), and 14 types of structural alterations in BRAF, EGFR, and FGFR3 genes in a single workflow. GlioSeq performance was evaluated in 54 adult and pediatric CNS tumors, and the results were compared with fluorescence in-situ hybridization, Sanger sequencing, and reverse transcription PCR. RESULTS: GlioSeq correctly identified 71/71 (100%) genetic alterations known to be present by conventional techniques, including 56 SNVs/indels, 9 CNVs, 3 EGFRvIII, and 3 KIAA1549-BRAF fusions. Only 20 ng of DNA and 10 ng of RNA were required for successful sequencing of 100% frozen and 96% formalin-fixed, paraffin-embedded tissue specimens. The assay sensitivity was 3%-5% of mutant alleles for SNVs and 1%-5% for gene fusions. The most commonly detected alterations were IDH1, TP53, TERT, ATRX. CDKN2A, and PTEN in high-grade gliomas, followed by BRAF fusions in low-grade gliomas and H3F3A mutations in pediatric gliomas. CONCLUSIONS: GlioSeq NGS assay offers accurate and sensitive detection of a wide range of genetic alterations in a single workflow. It allows rapid and cost-effective profiling of brain tumor specimens and thus provides valuable information for patient management.
Authors: Jeremy Schwartzentruber; Andrey Korshunov; Xiao-Yang Liu; David T W Jones; Elke Pfaff; Karine Jacob; Dominik Sturm; Adam M Fontebasso; Dong-Anh Khuong Quang; Martje Tönjes; Volker Hovestadt; Steffen Albrecht; Marcel Kool; Andre Nantel; Carolin Konermann; Anders Lindroth; Natalie Jäger; Tobias Rausch; Marina Ryzhova; Jan O Korbel; Thomas Hielscher; Peter Hauser; Miklos Garami; Almos Klekner; Laszlo Bognar; Martin Ebinger; Martin U Schuhmann; Wolfram Scheurlen; Arnulf Pekrun; Michael C Frühwald; Wolfgang Roggendorf; Christoph Kramm; Matthias Dürken; Jeffrey Atkinson; Pierre Lepage; Alexandre Montpetit; Magdalena Zakrzewska; Krzystof Zakrzewski; Pawel P Liberski; Zhifeng Dong; Peter Siegel; Andreas E Kulozik; Marc Zapatka; Abhijit Guha; David Malkin; Jörg Felsberg; Guido Reifenberger; Andreas von Deimling; Koichi Ichimura; V Peter Collins; Hendrik Witt; Till Milde; Olaf Witt; Cindy Zhang; Pedro Castelo-Branco; Peter Lichter; Damien Faury; Uri Tabori; Christoph Plass; Jacek Majewski; Stefan M Pfister; Nada Jabado Journal: Nature Date: 2012-01-29 Impact factor: 49.962
Authors: Matthew Mistry; Nataliya Zhukova; Daniele Merico; Patricia Rakopoulos; Rahul Krishnatry; Mary Shago; James Stavropoulos; Noa Alon; Jason D Pole; Peter N Ray; Vilma Navickiene; Joshua Mangerel; Marc Remke; Pawel Buczkowicz; Vijay Ramaswamy; Ana Guerreiro Stucklin; Martin Li; Edwin J Young; Cindy Zhang; Pedro Castelo-Branco; Doua Bakry; Suzanne Laughlin; Adam Shlien; Jennifer Chan; Keith L Ligon; James T Rutka; Peter B Dirks; Michael D Taylor; Mark Greenberg; David Malkin; Annie Huang; Eric Bouffet; Cynthia E Hawkins; Uri Tabori Journal: J Clin Oncol Date: 2015-02-09 Impact factor: 44.544
Authors: Catherine Grasso; Timothy Butler; Katherine Rhodes; Michael Quist; Tanaya L Neff; Stephen Moore; Scott A Tomlins; Erica Reinig; Carol Beadling; Mark Andersen; Christopher L Corless Journal: J Mol Diagn Date: 2014-11-07 Impact factor: 5.568
Authors: Craig Horbinski; Marina N Nikiforova; Jill M Hagenkord; Ronald L Hamilton; Ian F Pollack Journal: Neuro Oncol Date: 2012-04-05 Impact factor: 12.300
Authors: Dominik Sturm; Hendrik Witt; Volker Hovestadt; Dong-Anh Khuong-Quang; David T W Jones; Carolin Konermann; Elke Pfaff; Martje Tönjes; Martin Sill; Sebastian Bender; Marcel Kool; Marc Zapatka; Natalia Becker; Manuela Zucknick; Thomas Hielscher; Xiao-Yang Liu; Adam M Fontebasso; Marina Ryzhova; Steffen Albrecht; Karine Jacob; Marietta Wolter; Martin Ebinger; Martin U Schuhmann; Timothy van Meter; Michael C Frühwald; Holger Hauch; Arnulf Pekrun; Bernhard Radlwimmer; Tim Niehues; Gregor von Komorowski; Matthias Dürken; Andreas E Kulozik; Jenny Madden; Andrew Donson; Nicholas K Foreman; Rachid Drissi; Maryam Fouladi; Wolfram Scheurlen; Andreas von Deimling; Camelia Monoranu; Wolfgang Roggendorf; Christel Herold-Mende; Andreas Unterberg; Christof M Kramm; Jörg Felsberg; Christian Hartmann; Benedikt Wiestler; Wolfgang Wick; Till Milde; Olaf Witt; Anders M Lindroth; Jeremy Schwartzentruber; Damien Faury; Adam Fleming; Magdalena Zakrzewska; Pawel P Liberski; Krzysztof Zakrzewski; Peter Hauser; Miklos Garami; Almos Klekner; Laszlo Bognar; Sorana Morrissy; Florence Cavalli; Michael D Taylor; Peter van Sluis; Jan Koster; Rogier Versteeg; Richard Volckmann; Tom Mikkelsen; Kenneth Aldape; Guido Reifenberger; V Peter Collins; Jacek Majewski; Andrey Korshunov; Peter Lichter; Christoph Plass; Nada Jabado; Stefan M Pfister Journal: Cancer Cell Date: 2012-10-16 Impact factor: 31.743
Authors: Simon A Forbes; David Beare; Prasad Gunasekaran; Kenric Leung; Nidhi Bindal; Harry Boutselakis; Minjie Ding; Sally Bamford; Charlotte Cole; Sari Ward; Chai Yin Kok; Mingming Jia; Tisham De; Jon W Teague; Michael R Stratton; Ultan McDermott; Peter J Campbell Journal: Nucleic Acids Res Date: 2014-10-29 Impact factor: 16.971
Authors: Aatur D Singhi; Christina A Arnold; Dora M Lam-Himlin; Marina N Nikiforova; Lysandra Voltaggio; Marcia I Canto; Kevin M McGrath; Elizabeth A Montgomery Journal: Mod Pathol Date: 2017-07-21 Impact factor: 7.842
Authors: Michael R Drumm; Karan S Dixit; Sean Grimm; Priya Kumthekar; Rimas V Lukas; Jeffrey J Raizer; Roger Stupp; Milan G Chheda; Kwok-Ling Kam; Matthew McCord; Sean Sachdev; Timothy Kruser; Alicia Steffens; Rodrigo Javier; Kathleen McCortney; Craig Horbinski Journal: Neuro Oncol Date: 2020-04-15 Impact factor: 12.300