Targeted Next-Generation Sequencing of Circulating-Tumor DNA for Tracking Minimal Residual Disease in Localized Colon Cancer

N. Tarazona; F. Gimeno-Valiente; V. Gambardella; S. Zuñiga; P. Rentero-Garrido; M. Huerta; S. Roselló; C. Martinez-Ciarpaglini; J. A. Carbonell-Asins; F. Carrasco; A. Ferrer-Martínez; G. Bruixola; T. Fleitas; J. Martín; R. Tébar-Martínez; D. Moro; J. Castillo; A. Espí; D. Roda; A. Cervantes

Disclosures

Ann Oncol. 2019;30(11):1804-1812. 

In This Article

Abstract and Introduction

Abstract

Background: A high percentage of patients diagnosed with localized colon cancer (CC) will relapse after curative treatment. Although pathological staging currently guides our treatment decisions, there are no biomarkers determining minimal residual disease (MRD) and patients are at risk of being undertreated or even overtreated with chemotherapy in this setting. Circulating-tumor DNA (ctDNA) can to be a useful tool to better detect risk of relapse.

Patients and methods: One hundred and fifty patients diagnosed with localized CC were prospectively enrolled in our study. Tumor tissue from those patients was sequenced by a custom-targeted next-generation sequencing (NGS) panel to characterize somatic mutations. A minimum variant allele frequency (VAF) of 5% was applied for variant filtering. Orthogonal droplet digital PCR (ddPCR) validation was carried out. We selected known variants with higher VAF to track ctDNA in the plasma samples by ddPCR.

Results: NGS found known pathological mutations in 132 (88%) primary tumors. ddPCR showed high concordance with NGS (r = 0.77) for VAF in primary tumors. Detection of ctDNA after surgery and in serial plasma samples during follow-up were associated with poorer disease-free survival (DFS) [hazard ratio (HR), 17.56; log-rank P = 0.0014 and HR, 11.33; log-rank P = 0.0001, respectively]. Tracking at least two variants in plasma increased the ability to identify MRD to 87.5%. ctDNA was the only significantly independent predictor of DFS in multivariable analysis. In patients treated with adjuvant chemotherapy, presence of ctDNA after therapy was associated with early relapse (HR 10.02; log-rank P < 0.0001). Detection of ctDNA at follow-up preceded radiological recurrence with a median lead time of 11.5 months.

Conclusions: Plasma postoperative ctDNA detected MRD and identified patients at high risk of relapse in localized CC. Mutation tracking with more than one variant in serial plasma samples improved our accuracy in predicting MRD.

Introduction

Colon cancer (CC) is a heterogeneous disease representing the second leading cause of cancer-related deaths.[1] Most patients are diagnosed at early stage, but up to 50% of them relapse despite optimal initial treatment.[2] Staging is based upon surgical pathology and drives treatment decisions; however, it provides rough estimates of minimal residual disease (MRD) after curative-intent surgical resection. Patients with stage III do benefit from adjuvant therapy, but that advantage is smaller in stage II patients. However, staging itself is not predictive of the potential effect of adjuvant therapy. Using stage as the only criteria may lead us to undertreat individuals with MRD or, on the contrary to overtreat patients already cured by surgery. A better definition of MRD is required for localized CC after potential curative resection. Moreover, early detection of relapses could improve survival.[3,4] Nevertheless, currently recommended follow-up programs, consisting of the use of imaging techniques plus plasma CEA monitoring are suboptimal, failing to detect MRD and mostly diagnosing only far advanced relapses.[5]

Plasma circulating-tumor DNA (ctDNA) status has proved to be an accurate prognostic biomarker in several tumor types and may represent an improved estimate for MRD in resected patients. Droplet digital PCR (ddPCR) along with BEAMing are the most sensitive techniques currently available for detecting known mutations present in plasma.[6] Moreover, tailored ddPCR assays to detect in plasma known somatic mutations from the primary tumor by next-generation sequencing (NGS) may identify MRD in patients at higher risk of relapse with great accuracy.[7–9] However, although several NGS panels are commercially available, these often contain multiple genes or hotspots that are not relevant for profiling a particular case, due to lack of interest or to their uncertain clinical significance.

In this study, we assessed the potential of a custom-targeted 29-gene NGS panel to detect mutations in primary tumors, in a prospectively collected cohort of CC patients resected with curative intent. In liquid biopsies, the two mutations with the highest allelic fractions in each patient were also studied and tracked in plasma by ddPCR to detect MRD along follow-up to correlate with clinical relapses.

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