M Jamal-Hanjani1, G A Wilson2, S Horswell3, R Mitter3, O Sakarya3, T Constantin4, R Salari4, E Kirkizlar4, S Sigurjonsson4, R Pelham4, S Kareht4, B Zimmermann4, C Swanton5. 1. Translational Cancer Therapeutics Laboratory, Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, London. 2. Translational Cancer Therapeutics Laboratory, Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, London Translational Cancer Therapeutics Laboratory, The Francis Crick Institute, London. 3. Department of Bioinformatics and Biostatistics, The Francis Crick Institute, London, UK. 4. Natera Inc., San Carlos, USA. 5. Translational Cancer Therapeutics Laboratory, Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, London Translational Cancer Therapeutics Laboratory, The Francis Crick Institute, London charles.swanton@crick.ac.uk.
Abstract
BACKGROUND: The aim of this pilot study was to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in cell-free DNA (cfDNA) from patients with early-stage non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Three stage I and one stage II primary NSCLC tumors were subjected to multiregion whole-exome sequencing (WES) and validated with AmpliSeq. A subset of ubiquitous and heterogeneous single-nucleotide variants (SNVs) were chosen. Multiplexed PCR using custom-designed primers, coupled with next-generation sequencing (mPCR-NGS), was used to detect these SNVs in both tumor DNA and cfDNA isolated from plasma obtained before surgical resection of the tumors. The limit of detection for each assay was determined using cfDNA from 48 presumed-normal healthy volunteers. RESULTS: Tumor DNA and plasma-derived cfDNA was successfully amplified and sequenced for 37/50 (74%) SNVs using the mPCR-NGS method. Twenty-five (68%) were ubiquitous and 12 (32%) were heterogeneous SNVs. Variant detection by mPCR-NGS and WES-AmpliSeq in tumor tissue was well correlated (R(2) = 0.8722, P < 0.0001). Sixteen (43%) out of 37 SNVs were detected in cfDNA. Twelve of these were ubiquitous SNVs with a variant allele frequency (VAF) range of 0.15-23.25%, and four of these were heterogeneous SNVs with a VAF range of 0.28-1.71%. There was a statistically significant linear relationship between the VAFs for tumor and cfDNA (R(2) = 0.5144; P = 0.0018). For all four patients, at least two variants were detected in plasma. The estimated number of copies of variant DNA present in each sample ranged from 5 to 524. The average number of variant copies required for detection (VCRD) was 3.16 (range: 0.2-7.6 copies). CONCLUSIONS: The mPCR-NGS method revealed intratumor heterogeneity in early-stage NSCLC tumors, and was able to detect both ubiquitous and heterogeneous SNVs in cfDNA. Further validation of mPCR-NGS in cfDNA is required to define its potential use in clinical practice.
BACKGROUND: The aim of this pilot study was to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in cell-free DNA (cfDNA) from patients with early-stage non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Three stage I and one stage II primary NSCLC tumors were subjected to multiregion whole-exome sequencing (WES) and validated with AmpliSeq. A subset of ubiquitous and heterogeneous single-nucleotide variants (SNVs) were chosen. Multiplexed PCR using custom-designed primers, coupled with next-generation sequencing (mPCR-NGS), was used to detect these SNVs in both tumor DNA and cfDNA isolated from plasma obtained before surgical resection of the tumors. The limit of detection for each assay was determined using cfDNA from 48 presumed-normal healthy volunteers. RESULTS:Tumor DNA and plasma-derived cfDNA was successfully amplified and sequenced for 37/50 (74%) SNVs using the mPCR-NGS method. Twenty-five (68%) were ubiquitous and 12 (32%) were heterogeneous SNVs. Variant detection by mPCR-NGS and WES-AmpliSeq in tumor tissue was well correlated (R(2) = 0.8722, P < 0.0001). Sixteen (43%) out of 37 SNVs were detected in cfDNA. Twelve of these were ubiquitous SNVs with a variant allele frequency (VAF) range of 0.15-23.25%, and four of these were heterogeneous SNVs with a VAF range of 0.28-1.71%. There was a statistically significant linear relationship between the VAFs for tumor and cfDNA (R(2) = 0.5144; P = 0.0018). For all four patients, at least two variants were detected in plasma. The estimated number of copies of variant DNA present in each sample ranged from 5 to 524. The average number of variant copies required for detection (VCRD) was 3.16 (range: 0.2-7.6 copies). CONCLUSIONS: The mPCR-NGS method revealed intratumor heterogeneity in early-stage NSCLC tumors, and was able to detect both ubiquitous and heterogeneous SNVs in cfDNA. Further validation of mPCR-NGS in cfDNA is required to define its potential use in clinical practice.
Authors: Jonathan C M Wan; Charles Massie; Javier Garcia-Corbacho; Florent Mouliere; James D Brenton; Carlos Caldas; Simon Pacey; Richard Baird; Nitzan Rosenfeld Journal: Nat Rev Cancer Date: 2017-02-24 Impact factor: 60.716
Authors: Nicholas McGranahan; Andrew J S Furness; Rachel Rosenthal; Sofie Ramskov; Rikke Lyngaa; Sunil Kumar Saini; Mariam Jamal-Hanjani; Gareth A Wilson; Nicolai J Birkbak; Crispin T Hiley; Thomas B K Watkins; Seema Shafi; Nirupa Murugaesu; Richard Mitter; Ayse U Akarca; Joseph Linares; Teresa Marafioti; Jake Y Henry; Eliezer M Van Allen; Diana Miao; Bastian Schilling; Dirk Schadendorf; Levi A Garraway; Vladimir Makarov; Naiyer A Rizvi; Alexandra Snyder; Matthew D Hellmann; Taha Merghoub; Jedd D Wolchok; Sachet A Shukla; Catherine J Wu; Karl S Peggs; Timothy A Chan; Sine R Hadrup; Sergio A Quezada; Charles Swanton Journal: Science Date: 2016-03-03 Impact factor: 47.728
Authors: Bernard Pereira; Christopher T Chen; Lipika Goyal; Charlotte Walmsley; Christopher J Pinto; Islam Baiev; Read Allen; Laura Henderson; Supriya Saha; Stephanie Reyes; Martin S Taylor; Donna M Fitzgerald; Maida Williams Broudo; Avinash Sahu; Xin Gao; Wendy Winckler; A Rose Brannon; Jeffrey A Engelman; Rebecca Leary; James R Stone; Catarina D Campbell; Dejan Juric Journal: Nat Commun Date: 2021-05-27 Impact factor: 14.919