Circulating mutant DNA to assess tumor dynamics

Abstract

The measurement of circulating nucleic acids has transformed the management of chronic viral infections such as HIV. The development of analogous markers for individuals with cancer could similarly enhance the management of their disease. DNA containing somatic mutations is highly tumor specific and thus, in theory, can provide optimum markers. However, the number of circulating mutant gene fragments is small compared to the number of normal circulating DNA fragments, making it difficult to detect and quantify them with the sensitivity required for meaningful clinical use. In this study, we applied a highly sensitive approach to quantify circulating tumor DNA (ctDNA) in 162 plasma samples from 18 subjects undergoing multimodality therapy for colorectal cancer. We found that ctDNA measurements could be used to reliably monitor tumor dynamics in subjects with cancer who were undergoing surgery or chemotherapy. We suggest that this personalized genetic approach could be generally applied to individuals with other types of cancer.

Figure 1. Measurement of ctDNA

The left side of the schematic depicts conventional Sanger sequencing of DNA derived from the subject’s tumor, representing the first step of the analysis. The approach for quantifying tumor-derived DNA in plasma samples is shown on the right. Real-time PCR is used to measure the total number of DNA fragments in the plasma, whereas BEAMing measures the ratio of mutant to wild-type fragments labeled with the fluorescent probes Cy5 and Cy3, respectively.

Figure 2. Representative flow cytometric data obtained from BEAMing

The four graphs illustrate the data obtained from subject 6 (APC G4189T) at different time points during treatment. The green and red dots represent beads bound to wild-type and mutant fragments, respectively. The blue dots represent beads bound to both wild-type and mutant fragments resulting from their inclusion in an emulsion microdroplet that contained both wild-type and mutant DNA templates. Numbers in each quadrant represent events for each population measured. (a) Before surgery, the fraction of mutant DNA fragments was 13.4%. (b) After surgery (day 3), the fraction of mutant DNA fragments dropped to 0.015%. (c) After surgery (day 48), the fraction of mutant DNA fragments increased to 0.11%, suggesting disease recurrence. (d) On day 244, the subject had progressive disease and the fraction mutant DNA fragments increased further to 0.66%.

Figure 3. Recurrence-free survival, as detected by ctDNA and CEA

(a) The difference in recurrence-free survival in subjects with detectable versus undetectable post-operative ctDNA levels (P = 0.006 by Mantel-Cox log-rank test). (b) The difference in recurrence-free survival in subjects with detectable versus undetectable post-operative CEA levels (P = 0.03 by Mantel-Cox log-rank test).

Figure 4. Comparison of ctDNA, CEA and imaging dynamics in individual study subjects

For each subject, the top, middle, and bottom graphs represent ctDNA level, tumor volume as assessed by imaging, and CEA level. The red lines represent the upper bound of the normal levels: one mutant DNA fragment per sample for ctDNA levels, 0.0 cm for tumor diameter, and 5.0 ng ml⁻¹ for CEA abundance. (a) Subject 8 had a sigmoid adenocarcinoma and solitary metastases in both hepatic lobes. The subject underwent a sigmoidectomy and left lateral hepatic sectorectomy (Surgery 1). A right-sided liver metastasis was left in place while the subject was treated with systemic chemotherapy (Chemotherapy 1). On day 120, a right hepatectomy was performed (Surgery 2). After surgery, the subject was treated for 4 months with systemic chemotherapy (Chemotherapy 2). (b) Subject 11 had a sigmoid adenocarcinoma and two liver metastases that were treated with systemic chemotherapy before surgery (Chemotherapy 1). The subject underwent a sigmoid colectomy, left hepatic lobectomy and RFA of a solitary right hepatic lesion (Surgery 1). Imaging studies at 2 months showed recurrence in the liver, and the subject underwent a right hepatectomy (Surgery 2). Given the high risk of recurrence, chemotherapy was reinitiated (Chemotherapy 2). At 8 months, imaging showed three recurrent liver lesions and a suspicious celiac lymph node. The subject underwent RFA of these lesions and resection of the celiac node (Surgery 3). After surgery, the subject received additional chemotherapy (Chemotherapy 3); however, later imaging revealed multiple pulmonary metastases.