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Definitions

• Figs. 4A-4C show comparison of ctDNA, CEA and imaging dynamics in individual study subjects.
• the top, middle, and bottom graphs represent ctDNA level, tumor volume as assessed by imaging, and CEA level.
• the horizontal 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.
• Fig. 4C Patient 8 had a sigmoid adenocarcinoma and solitary metastases in both hepatic lobes.
• the subject underwent a sigmoidectomy and left lateral hepatic sectorectomy (Surgery 1).
• Fig. 15 shows total DNA fragments in plasma prior to and after surgery.
• the Wisker box plot shows the total number of DNA fragments in 2 ml plasma, estimated by real-time PCR at baseline (day 0), post-surgery (day 1), day of discharge (days 2-5), and at the 1 st follow-up (days 13-56).
• ctDNA levels reflect the total systemic tumor burden, in that ctDNA levels decreased upon complete surgery and generally increased as new lesions became apparent upon radiological examination.
• Radiographs are inaccurate, because lesions that are observed upon imaging are composed of live neoplastic cells, dead neoplastic cells and varying amounts of nonneoplastic cells (stromal fibroblasts, inflammatory cells, vasculature, and the like) 11 .
• the proportion of these cell types in any lesion is unknown.
• micrometastatic lesions that are smaller than a few millimeters, which in aggregate may make a large contribution to the total tumor burden, are not detectable by positron emission tomography, computed tomography or magnetic resonance imaging scans.
• Plasma samples were drawn in BD Vacutainer tubes with EDTA (Becton Dickinson, Franklin Lakes, NJ USA) from 16 of the 25 patients.
• Plasma was prepared by centrifugation of blood at 1380 g for 30 min. The supernatant was transferred to a fresh tube and re-centrifuged. After centrifugation, the plasma was transferred to a Millipore Ultrafree-MC 0.45 micron filter device (Millipore, Billerica, MA, USA) to remove remaining cellular debris. The filter device was subjected to centrifugation at 1380 g for 15 min. The cleared plasma was transferred to a new tube and stored at -20° C until processed.
• Millipore Ultrafree-MC 0.45 micron filter device Millipore Ultrafree-MC 0.45 micron filter device
• Each reaction consisted of 5* Phusion high fidelity buffer, 1.5 U of Hotstart Phusion polymerase (both NEB), 0.2 ⁇ M of each primer, 0.25 mM of each dNTP, and 0.5 mM MgCl 2 .
• Nested PCR reactions were performed for selected target regions; for the second amplification, 2 ⁇ l of the first PCR was added to a 20- ⁇ l PCR reaction of the same makeup as described above except that different primers were used.
• Primer sequences and cycling conditions are listed in Fig. 9.
• PCR products were pooled, diluted, and quantified using the PicoGreen dsDNA assay (Invitrogen). The BEAMing procedure has been described previously 10 and modifications used in the current study are described here.
• the first PCR was performed in a 10 ⁇ l reaction volume containing 50-100 genome equivalents (GEs) of template DNA (1 GE equals 3.3 pg of human genomic DNA), 0.5 U of Platinum Taq DNA Polymerase (Invitrogen), Ix PCR buffer (67 mM of Tris-HCl, pH 8.8, 67 mM of MgCl 2 , 16.6 mM of (NH 4 ) 2 SO 4 , and 10 mM of 2-mercaptoethanol), 2 mM ATP, 6% (v/v) DMSO, 1 mM of each dNTP, and 0.2 ⁇ M of each primer.
• GEs genome equivalents
• the second (nested) PCR reaction was temperature cycled using the following conditions: 2 min at 94°C; 15 cycles of 94 0 C for 15 s, 58°C for 30 s, 70°C for 15 s.
• the PCR products were purified using the AMpure system (Agencourt, Beverly, MA) and sequenced from both directions using BigDye Terminator v3.1 (Applied Biosystems).
• Patient 7 has a prior history of a resected T3N2M1 rectosigmoid adenocarcinoma.
• the patient underwent surgical excision of two recurrent liver lesions, and an additional 4 liver lesions were treated with radiofrequency ablation (Surgery).
• Post-operative imaging revealed no evidence of disease, however, imaging three months later revealed new liver disease and new lung metastases.
• the patient was started on irinotecan, cetuximab, and bevacizumab (Chemotherapy). Despite chemotherapy, on follow-up imaging the patient was noted to have persistent and progressing disease.

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