I have read the journal's policy and the authors of this manuscript have the following competing interests: PJK has received royalties in the past two years from a license agreement with Correlagen, related to early-onset non-insulin-dependent diabetes mellitus. AG has received fees from Bristol Myers Squibb for leading webinar based teaching sessions organised by them for other doctors. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Current address: The Francis Crick Institute, London, United Kingdom
Current address: Millenium Health, San Diego, California, United States of America
Current address: Cancer Sciences Academic Unit, University of Southampton, Southampton, United Kingdom
Use of circulating tumour DNA (ctDNA) as a liquid biopsy has been proposed for potential identification and monitoring of solid tumours. We investigate a next-generation sequencing approach for mutation detection in ctDNA in two related studies using a targeted panel. The first study was retrospective, using blood samples taken from melanoma patients at diverse timepoints before or after treatment, aiming to evaluate correlation between mutations identified in biopsy and ctDNA, and to acquire a first impression of influencing factors. We found good concordance between ctDNA and tumour mutations of melanoma patients when blood samples were collected within one year of biopsy or before treatment. In contrast, when ctDNA was sequenced after targeted treatment in melanoma, mutations were no longer found in 9 out of 10 patients, suggesting the method might be useful for detecting treatment response. Building on these findings, we focused the second study on ctDNA obtained before biopsy in lung patients, i.e. when a tentative diagnosis of lung cancer had been made, but no treatment had started. The main objective of this prospective study was to evaluate use of ctDNA in diagnosis, investigating the concordance of biopsy and ctDNA-derived mutation detection. Here we also found positive correlation between diagnostic lung biopsy results and pre-biopsy ctDNA sequencing, providing support for using ctDNA as a cost-effective, non-invasive solution when the tumour is inaccessible or when biopsy poses significant risk to the patient.
Cell-free DNA (cfDNA) has been known to exist in blood since 1948, and is present in all people to some degree[
It is well-documented that solid tumours are not composed of a single oncogenic clone, but have extensive inter- and intra-tumoural genetic heterogeneity [
Previous NGS analysis of ctDNA has detected a wide range of mutant allele frequencies, from 52% [
Here we study two aspects of next-generation sequencing in ctDNA: first we identify factors influencing the concordance between mutations in melanoma tumours and circulating DNA. Second, we examine whether ctDNA sequencing can be used in lung cancer diagnosis, by sequencing plasma DNA taken prior to bronchoscopy.
This study, under the authority of the Oxford Radcliffe Biobank (ORB), was reviewed and approved by the South Central—Oxford C Research Ethics Committee (REC reference number 09/H0606/5+5), before the study began. Written informed consent was provided by participants (melanoma and suspected lung cancer patients) according to current ORB guidelines. Blood was drawn, stored at room temperature and processed within 6 hours. In the case of lung patients, blood samples were taken a few minutes prior to tumour biopsy.
In order to minimise lymphocyte lysis, blood samples were centrifuged at 2060 x g (3000 rpm in Beckman GS-6R centrifuge) for 10 minutes at room temperature without brake within 6 hours of collection [
Sequencing libraries were prepared with the Ion Ampliseq Cancer Hotspot Panel. The panel contains a collection of primers designed to interrogate hotspot regions in genes commonly mutated in cancer. Over the course of this study, the first version of the panel, which targeted 46 genes, was replaced by a new version targeting 50 genes (
The analysis was run with Torrent Variant Caller (TVC) v4.4.5, which was reported to be capable of detecting variants at frequencies of 0.5% [
Pearson’s product-moment correlation coefficients were computed using the R function ‘cor’. Permutation-adjusted (n = 10000) p-values were calculated using a two-tailed test in the R function ‘cor.test’.
Competitive Allele-Specific TaqMan® PCR (castPCR™) (ThermoFisher) is a quantitative PCR method that detects specific known mutations [
This study was a retrospective pilot investigation in melanoma patients to confirm that mutations discovered in the primary tumour were also detectable in ctDNA using the Ion Torrent™ platform, and to examine factors that may affect concordance of mutation detection (
Tumour and plasma DNA from melanoma patients were sequenced using Ampliseq Cancer Hotspot Panel (8–10 samples per 318 chip), and variant results were compared in relation to two factors, pre- or post-treatment sampling and time difference between sampling of tumour and plasma. (A) Pre- or post-treatment correlation. Blood samples taken before treatment are denoted by blue diamonds, dashed line, and samples taken post-treatment are shown by red squares, solid line; (B) Mutated genes in tumour and plasma DNA, marked in blue if plasma taken pre-treatment, red pattern if plasma was post-treatment; (C) Time difference correlation. Blood samples taken less than a year after biopsy are plotted as green triangles, dashed line, and samples taken more than a year after biopsy are shown as orange circles, solid line; (D) Mutated genes in tumour and plasma DNA, marked in green pattern if time difference between biopsy and blood sampling < 1 year, solid orange if time difference > 1 year (tumour biopsy date not available for patients 9 and 18, marked in blue stripe).
Together, this pilot study with melanoma patients demonstrates that ctDNA could be a reliable surrogate for tumour biopsy when sampled in a similar time frame and before treatment. Although it was a retrospective study, the dearth of mutations detected post-treatment suggests that ctDNA sequencing could be an attainable and easy measure of whether patients are responding to therapy.
Based on results of the first study, our second study focused on collecting blood samples prospectively from suspected lung cancer patients just prior to diagnostic tumour biopsy (endobronchial or endoscopic ultrasound-guided biopsy). The advantage of this is that ctDNA could be sampled at the same time as the tumour biopsy and would be free from any potential contamination by tumour DNA released into the blood as a consequence of the procedure. Furthermore, because most patients were at relatively early stages in the cancer pathway and were treatment-naïve, the collected ctDNA would preserve the original mutational load and allelic frequencies. Among lung cancer patients, there were seven adenocarcinomas, three squamous carcinomas, one carcinoid tumour, and one small cell lung cancer. Sequencing of ctDNA from 12 lung cancer patients identified 22 non-synonymous variants (
ID | Histology | Somatic mutation | Variant | Tumour VAF % | ctDNA VAF% | JAX Clinical Knowledgebase (CKB) |
Implications for Treatment and CKB Reference Link |
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1178 | Adeno |
yes | 19 | 6.8 | CKB: Hotspot mutation in DNA-binding domain of TP53 (PMID: 22713868); ClinVar: probable pathogenic | Treatment approach: p53 activator, p53 gene therapy (gene-associated clinical trials available) |
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no | 37 | 44 | CKB: Lies in extracellular Sema ligand-binding domain, predicted loss of function (PMID: 19723643); ClinVar: benign/ likely benign | May confer resistance to MET targeted agents (PMID: 19723643) |
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1530 | Metastatic adeno (brain) | no | 49 | 50 | CKB: Gain of function; no increase in MET phosphorylation, but increased cellular protein phosphorylation and increased proliferation and migration of cultured cells (PMID: 14559814, 20670955, 22973954); ClinVar: conflicting: likely benign(2), uncertain sig(2) | Treatment approach: MET inhibitor (Gene-associated clinical trials available) |
|
yes | 26 | 6 | CKB: Gain of function; causes constitutive MET phosphorylation and activation of downstream signaling, and transforming in cell culture (PMID: 15064724, 24061647); not found in ClinVar | Treatment approach: MET inhibitor (Gene-associated clinical trials available) |
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yes | 41 | 8 | CKB: Hotspot mutation, inhibits GTPase activity of KRAS leading to increased activation of downstream signaling pathways promoting tumour formation (PMID: 16051643); ClinVar: pathogenic | Confers resistance to EGFR tyrosine kinase inhibitors; Treatment approach: Pan-MEK inhibitor, Pan-PI3K inhibitor, RAS inhibitor (gene-associated clinical trials ongoing) |
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yes | 34 | 7 | CKB: Hotspot residue in MH2 domain of SMAD4, with predicted loss of function (PMID: 21763698); ClinVar: pathogenic | Rare in lung cancer and for which there is little evidence for targeted therapies |
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1533 | Metastatic adeno | yes | 17 | 2 | CKB: Mutation impairs BRAF kinase activity but paradoxically activates MEK and ERK through CRAF transactivation (PMID: 20141835); ClinVar: pathogenic | Results in BRAF inactivation and insensitivity to BRAF inhibitors; Treatment approach: MEK1, MEK2 and pan-MEK inhibitors |
|
yes | 21 | 2 | Not found in CKB or ClinVar | none | |||
yes | 24 | 3 | Not found in CKB or ClinVar | none | |||
594 | Adeno | no | 50 | 52 | Not found in CKB or ClinVar | none | |
no | 39 | 48 | CKB: Conflicting reports: increase in MET phosphorylation (PMID: 25605252), or no effect (PMID: 20670955); ClinVar: non-pathogenic | none; |
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yes | 17 | 3 | CKB: Mutation in DNA-binding region of TP53 but uncharacterised so its effect is unknown; not found in ClinVar | none; |
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591 | Metastatic adeno | yes | 11 | 0.2 | CKB: Hotspot mutation in DNA-binding domain of TP53 (PMID: 22713868), decreased transactivation activity of TP53, and context-dependent transforming ability in cell culture (PMID: 20212049, PMID: 20538734); ClinVar: non-pathogenic | Treatment approach: p53 activator, p53 gene therapy (gene-associated clinical trials available) |
|
no | 47 | 54 | CKB: Mutation in protein kinase 1 domain of JAK3, confers gain of function and activation of JAK3/STAT3 pathway (PMID: 23689514); ClinVar: no information | Treatment approach: Pan-JAK inhibitor or JAK3 inhibitor |
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590 | Adeno | yes | 22 | not found | CKB: Mutation within ubiquitination recognition motif of CTNNB1 (PMID: 15064718), gain of function due to nuclear accumulation of CTNNB1 in liver cancer (PMID: 9671767); ClinVar: conflicting: pathogenic(1); uncertain sig(1) | Treatment approach: CTNNB1 inhibitor, PDPK1 inhibitor, Tankyrase inhibitor |
|
572 | Adeno | negative | |||||
463 | Small cell lung cancer | yes | not avail. | 4 | CKB: Hotspot mutation in DNA-binding domain of TP53 (PMID: 22713868), decreased activation of p21, and also confers a gain-of-function (PMID: 22214764); ClinVar: pathogenic | Treatment approach: p53 activator, p53 gene therapy (gene-associated clinical trials available) |
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no | 9 | CKB: Mutation in DNA-binding domain of TP53 (PMID: 22713868), decreased TP53 transactivation activity in cell culture (PMID: 16492679); ClinVar: pathogenic/likely pathogenic | |||||
593 | Squamous cell | yes | not avail. | 17 | Not found in CKB or ClinVar | none | |
yes | 21 | CKB: Results in premature truncation of PTEN protein, predicted loss of function (UniProt.org); not found in ClinVar | Treatment approach: Pan-AKT inhibitor, AKT1 inhibitor, AKT2 inhibitor, AKT3 inhibitor, Pan-PI3K inhibitor (gene-associated clinical trials available) |
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yes | 28 | CKB: Mutation in DNA-binding domain of TP53 (PMID: 22713868), decreased TP53 transactivation activity in cell culture (PMID: 16492679); ClinVar: pathogenic/likely pathogenic | |||||
no | 2 | CKB: Hotspot mutation in DNA-binding domain of TP53 (PMID: 22713868), decreased activation of |
Treatment approach: p53 activator, p53 gene therapy (gene-associated clinical trials available) |
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466 | Squamous cell | yes | not avail. | 2 | CKB: Hotspot mutation in DNA-binding domain of TP53 (PMID: 22713868), decreased activation of TP53 targets, inhibited AMPK signaling, and promoted tumour development in mouse models (PMID: 24857548); ClinVar: conflicting: likely benign(2); pathogenic(2) | Treatment approach: p53 activator, p53 gene therapy (gene-associated clinical trials available) |
|
538 | Squamous cell | yes | not avail. | 0.6 | CKB: Hotspot mutation in DNA-binding domain of TP53 (PMID: 17401432), loss of function, decreased TP53 transcriptional activity in cell culture (PMID: 16861262, 23630318); ClinVar: pathogenic | Treatment approach: p53 activator, p53 gene therapy (gene-associated clinical trials available) |
|
462 | Carcinoid tumour | negative | not avail. | negative |
Blood samples were drawn from patients prior to bronchoscopy. Plasma DNA and genomic DNA from each patient were sequenced using Ampliseq Cancer Hotspot Panel v2, using one plasma DNA:gDNA paired sample per 318 chip. Tumour DNA was sequenced when enough bronchoscopy material was available.
1Adeno, adenocarcinoma.
2CKB website content is for educational and research purposes only.
In the ctDNA of lung adenocarcinoma patients, we found mutations in
Variant allele frequency of mutations are shown in solid red bars for tumour and in hatched blue bars for ctDNA, for different types of lung cancer (Adenocarcinoma; Squamous, squamous cell carcinoma; Small cell, small cell lung cancer). Tumour DNA from squamous and small cell lung cancer patients was not available for sequencing. Mutations are denoted as somatic because they were not present in germline DNA from the same patients.
We attempted to validate the variants identified in melanoma and lung ctDNA using castPCR as an orthogonal method (
Blood was collected from patients with known melanomas for a retrospective pilot study, and we examined factors that may influence whether the confirmed mutations in the tumour were also detectable in plasma DNA. We only observe correlation between tumour and ctDNA variant allele frequency when the blood for ctDNA isolation was collected before treatment had started, or if collected less than one year after the tumour biopsy. With respect to treatment effects, although we did not do a time course study with successive samples taken from the same patient, we did observe a nearly complete absence of mutations in samples that were taken after treatment was started, which is most likely to be explained by the patients responding to targeted treatment. It is interesting to note that no further mutations were seen in all but one of these patient samples. Future longitudinal studies of ctDNA are warranted.
In the study with lung cancer patients, we specified in the standard operating procedure that blood draw for plasma DNA extraction occurred shortly (typically less than an hour) before tumour biopsy. This criterion was important because for several forms of cancer (e.g., breast, prostate, and lung), biopsies have been reported to increase the incidence of tumour cell seeding [
Collectively, these findings support using mutation analysis in ctDNA to provide a tumour profile helpful for diagnostic, predictive and prognostic analysis that does not require invasive procedures. Furthermore, the use of ctDNA also has potential for significant health economic, safety, and logistic benefits, whether as a means of obtaining repeat “liquid biopsies” from patients who are on treatment to allow monitoring of their mutanome, providing an early indication of whether or not patients are responding to targeted treatment, or after relapse of disease (where the standard of care is to undertake re-biopsy of the tumour to determine the presence of new mutations that may be actionable). Targeted therapies are costly and early identification of non-response (prior to symptomatic relapse) could save significant drug costs, as well as prevent unnecessary side effects. Panel ctDNA sequencing on the Ion Torrent PGM™ costs between £300–740 per sample, whereas transbronchial needle aspiration (TBNA) and endobronchial ultrasound (EBUS) cost £1365 in 2011, excluding tumour sequencing costs (NICE lung cancer costing report CG121, 2011). Lung cancers are often inaccessible to a bronchoscopically-guided tumour sampling, necessitating CT-guided biopsy that has inherent risks such as pneumothorax. Even in experienced hands, the amount of tumour material obtained by endobronchial or EBUS sampling may be insufficient or unsuitable for analysis. Further, these procedures are costly in terms of human resource, equipment and consumables. Our data suggest that sequencing plasma DNA would be a safer, cost effective yet just as informative a method to employ. Our findings also support the undertaking of further prospective studies of sequential ctDNA and tumour sequencing for patients throughout their treatment pathway. Identification of early disease progression or relapse, assessment of response and use within a screening programme are all potential applications of this relatively simple procedure.
In summary, we reveal two factors that influence concordance of mutation detection between primary tumour and ctDNA sequencing in melanoma: treatment status at time of blood sampling and time lag between sampling of the primary tumour and blood for ctDNA extraction. The findings provide evidence to support the use of plasma DNA sequencing to assess effectiveness of treatment, monitor cancer patients in remission, and provide an early indication of emerging mutations that could be amenable to targeted therapy. Additionally we find good but not perfect (80%) concordance of mutations in lung diagnostic biopsy and contemporaneous ctDNA, suggesting that liquid biopsy analysis could be a valuable first-line approach to confirm the diagnosis of lung cancer, especially when the tumour is inaccessible or when biopsy poses significant risk to the patient.
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We chose castPCR as an orthogonal platform to attempt validation of the Ion Ampliseq sequencing results. Nine castPCR assays were available to assess mutations in melanoma and lung cancer patients who had plasma DNA available for validation (less than the recommended 15–20ng DNA/well for the castPCR assay; assays were run in singlicate). X-axis, patient number and mutation tested; y-axis, percentage mutation. (A) Melanoma patient samples, (B) Lung patient samples. KIT M541L is a UCSC common polymorphism and not included in
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Blood samples were taken from melanoma patients who were known to have cancer (some patients were sampled pre-treatment, some were post-treatment). Tumour and plasma DNA were sequenced using Ampliseq Cancer Hotspot Panel (8–10 samples per 318 chip), and variant results were compared. For patients 9–14, ctDNA was also sequenced at higher coverage (using 1 ctDNA:gDNA paired sample per 318 chip).
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Blood samples were drawn from patients prior to bronchoscopy. These patients were subsequently found to be negative for cancer. Plasma DNA and genomic DNA from each were sequenced using Ampliseq Cancer Hotspot Panel v2.
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We are very grateful to the patients for their participation, and thank the research nurses Julianne Hollidge and Emma-Jayne Muir for their expertise and dedication in collecting and transporting samples. Sequences reported in this paper have been submitted to Sequence Read Archive (SRA) under accession number SRP066676. The views expressed in this paper are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.