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. 2017 Jan 19:7:40737.
doi: 10.1038/srep40737.

Individualised multiplexed circulating tumour DNA assays for monitoring of tumour presence in patients after colorectal cancer surgery

Sarah B Ng  1 , Clarinda Chua  2 , Matthew Ng  2 , Anna Gan  3 , Polly Sy Poon  1 , Melissa Teo  4 , Cherylin Fu  5 , Wei Qiang Leow  6 , Kiat Hon Lim  6 , Alexander Chung  7 , Si-Lin Koo  2 , Su Pin Choo  2 , Danliang Ho  2 , Steve Rozen  8 , Patrick Tan  8   9 , Mark Wong  5 , William F Burkholder  1 , Iain Beehuat Tan  2   3   8
Affiliations

Individualised multiplexed circulating tumour DNA assays for monitoring of tumour presence in patients after colorectal cancer surgery

Sarah B Ng et al. Sci Rep. .

Abstract

Circulating tumour DNA (ctDNA) has the potential to be a specific biomarker for the monitoring of tumours in patients with colorectal cancer (CRC). Here, our aim was to develop a personalised surveillance strategy to monitor the clinical course of CRC after surgery. We developed patient-specific ctDNA assays based on multiplexed detection of somatic mutations identified from patient primary tumours, and applied them to detect ctDNA in 44 CRC patients, analysing a total of 260 plasma samples. We found that ctDNA detection correlated with clinical events - it is detectable in pre-operative but not post-operative plasma, and also in patients with recurrent CRC. We also detected ctDNA in 11 out of 15 cases at or before clinical or radiological recurrence of CRC, indicating the potential of our assay for early detection of metastasis. We further present data from a patient with multiple primary cancers to demonstrate the specificity of our assays to distinguish between CRC recurrence and a second primary cancer. Our approach can complement current methods for surveillance of CRC by adding an individualised biological component, allowing us not only to point to the presence of residual or recurrent disease, but also attribute it to the original cancer.

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Figures

Figure 1
Figure 1. Workflow for design and use of patient primary-tumour specific (PPS) assays to detect circulating DNA (ctDNA).
Figure 2
Figure 2. Error correction reduces non-reference base calls in negative (healthy) samples.
(a) Using paired-end consensus calling (blue, circles) and removal of noise as modelled in control samples (green, squares), the non-reference base call proportion is reduced from raw calls (orange, triangles). Points are plotted as the average of 3 samples over each target position (2619 bp after sites of common variation were removed), with zero-value points at the bottom. (b) Histogram representation of the data in (a).
Figure 3
Figure 3. Sensitivity and specificity of patient-specific amplicon-sequencing assays for detection of ctDNA in plasma.
Plasma from 6 patients (labelled A–E) and 3 healthy volunteers (H1,H4,H5) were assayed for ctDNA using 6 different patient-specific primer sets (labelled A–F). Primer set labels match that of the patient they were designed for. Plasma from volunteer H1 was taken from a later timepoint (H1B) and re-tested with the same primer set F. This sample H1B was not used for calculations of sensitivity and specificity.
Figure 4
Figure 4. Multiplex amplicon assays performed on serial dilutions of tumour DNA exhibit a linear correlation between observed and input mutation frequencies and can sensitively detect low-frequency variants.
(a) The expected vs observed variant frequency for each amplicon in the dilution series is shown (Pearson: r2 = 0.89) with the fitted linear model in orange. (b) Amplicons were binned by expected variant frequency and the proportion of positive amplicons (i.e. input mutation detected) is shown. The number of amplicons in the bin is indicated by size of the dot, and the grey ribbon indicates the expected proportion of positive amplicons in 3000 genomic equivalents based on the minimum and maximum of the expected frequencies in the bin under a binomial distribution. The cumulative proportion of positive amplicons at the expected variant frequency and higher (orange line) and lower (green line) are also plotted.
Figure 5
Figure 5. ctDNA fraction detected in metastatic patients across the course of chemotherapy.
Boxplots of the ctDNA fraction in plasma samples obtained from patients after recurrence and during treatment with chemotherapy is shown in the following subsets: treatment naïve (“pre-chemo”), during first-line chemotherapy (“chemo-1”), during first-line chemotherapy break (“break-1”), and during all further lines of chemotherapy or re-challenges (“chemo-2+”). These subsets reflect the change in patient treatment over time.
Figure 6
Figure 6. ctDNA fraction and CEA levels in patients with plasma from before recurrence was diagnosed.
Only patients with multiple timepoints are shown (9 of 15 events). Events which occurred in patients post-metastectomy are identified with an “a”. Filled circles refer to detection of ctDNA (>0) or elevated CEA (>5 ng/ml). Orange dashed line indicates day of clinical detection of recurrence.
Figure 7
Figure 7. Detection of ctDNA variants is highly specific to tumour origin.
(a) Variant frequency of 20 somatic tumour variants identified by targeted capture sequencing (upper 11 specific to the original CRC primary [P] and liver metastasis [M]; lower 9 specific to second cholangiocarcinoma [C]) as assayed by a patient-specific multiplex amplicon assay in tumour tissue DNA. (b) ctDNA variant fraction over multiple timepoints post-primary surgery. The 11 CRC primary/metastasis variants (upper panel, red) and 9 cholangiocarcinoma variants (lower, orange) are plotted separately. Each line refers to a separate variant. E1: detection of liver lesion. E2: surgery to remove second primary. E3: clinical detection of a lung metastasis by CT scan.
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