ctDNA as a biomarker of progression in oesophageal adenocarcinoma

Abstract

Background: The incidence of oesophageal adenocarcinoma (OAC) is rapidly increasing and despite improvements in treatment, the 5-year survival rate remains poor. Prognostic biomarkers that address the genomic heterogeneity in this highly complex disease will aid the development of precision therapeutics and improve patient survival. The aim of this study was to determine whether circulating tumour DNA (ctDNA) has prognostic significance as a biomarker in OAC patients.

Patients and methods: We profiled 209 blood and tumour samples from 57 OAC patients. Using a panel of 77 cancer genes, we sequenced ctDNA in plasma samples (n = 127) which were taken at multiple time points before and after therapy. In parallel, we sequenced matched tumour samples from 39 patients using the same gene panel. To assess whether the ctDNA profile reflected the tumour heterogeneity, we sequenced additional multi-region primary tumour samples in 17 patients. In addition, we analysed whole-genome and whole-exome sequencing data from primary tumours for a subset of 18 patients.

Results: Using a tumour-agnostic approach, we found that detectable ctDNA variants in post-treatment plasma samples were associated with worse disease-specific survival. To evaluate whether the ctDNA originated from the primary tumour, we carried out a tumour-informed analysis which confirmed post-treatment ctDNA variants were associated with worse survival. To determine whether ctDNA could be used as a clinical follow-up test, we assessed blood samples from multiple time points before and after treatment, in a subset of patients. Results showed that the variant allele frequency of ctDNA variants increased with disease recurrence.

Conclusion: This study demonstrates that ctDNA variants can be detected in patients with OAC and this has potential clinical utility as a prognostic biomarker for survival.

Keywords: biomarker; ctDNA; oesophageal adenocarcinoma; prognostic; sequencing; survival.

Figure 1

Study design. (A) Tumour-agnostic analysis on ctDNA variants from baseline (B1, orange squares) and post-treatment samples (B2, green squares). (B) Tumour-informed approach aligning the ctDNA variants to matched primary tumour samples (T1). For a subset of patients, an additional biopsy (T2) was taken from a different site within the one primary tumour. In the tumour-informed approach, a ctDNA variant was only considered positive if it was present in a matching primary tumour sample. (C) Analysis of ctDNA at multiple time points post treatment (B3 and B4). Blue squares represent pre-treatment tumour biopsies. Number of patients included in each analysis is indicated. ctDNA, circulating tumour DNA.

Figure 2

Patient overview. (A) Swimmer plot. The clinical timeline is shown for each patient. Triangles indicate the blood collection time points. Where possible, tumours were sampled at time of diagnosis. The colour of each bar represents the DSS. (B) Variants detected in ctDNA and tumour DNA using a pan-cancer gene panel. The top panel shows DSS in months. Stage and treatment type are indicated. The number of variants per patient is shown, range 0 to 9 variants. Mutated genes are plotted for each patient. Overall, 45% of patients harboured a TP53 variant. The analysis approach is represented using a colour code (tumour-informed or tumour-agnostic approach). CXRT, chemoradiotherapy; DSS, disease-specific survival; NSR, no sign of recurrence.

Figure 3

Overview of variants detected using a pan-cancer gene panel. VAF in tumour and blood samples analysed by WGS, WES and the AVENIO tumour and ctDNA platform. Samples run using WGS or WES are indicated. SOG066, SOG083 and SOG506 are not represented as they had no tumour variants detected using any platform. Biopsies (T1, T2 and T3) were taken from multiple sites within the primary sample. Blood samples were taken at baseline, B1 and after treatment—B2 and B3. Patient disease-specific survival is indicated in brackets. The asterisk indicates patients that are shedders.ctDNA, circulating tumour DNA; mo, months; NSR, no sign of recurrence; VAF, variant allele frequency; WES, whole-exome sequencing; WGS, whole-genome sequencing. ∗Significance of the P values.

Figure 4

Prognostic significance of ctDNA in post-treatment blood samples. (A). DSS analysis in the tumour-agnostic approach, showing patients with detectable ctDNA variants had worse survival (∗P = 0.0130). (B) DSS analysis in the tumour-informed approach assessing the post-treatment blood samples (∗∗∗P = 0.0007). (C) Analysis at the perioperative time point in patients with a measurable primary tumour using a tumour-informed approach (∗P = 0.0473). Patients described as shedders have ctDNA variants that are confirmed in the primary tumour. Non-shedders have primary tumour variants that are not detected in the blood samples. No variant detected indicates an absence of primary tumour variants. (D) Serial time point analysis of plasma-derived ctDNA variants. VAF of ctDNA detected across serial time points for 10 patients. The threshold for variant calling is 0.001, represented by the black horizontal line. Disease-specific survival is indicated for each patient in brackets. Recurrence time is indicated by the dotted line. Clinical time point is described where chemo is chemotherapy and CXRT is chemoradiotherapy. ctDNA, circulating tumour DNA; d, days; DSS, disease-specific survival; mo, months; NSR, no sign of recurrence; VAF, variant allele frequency.