This cohort study investigates the use of longitudinal data from ultradeep sequencing of cell-free DNA in Danish patients with colorectal cancer.

Purpose: Up to 30% of patients with breast cancer relapse after primary treatment. There are no sensitive and reliable tests to monitor these patients and detect distant metastases before overt recurrence. Here, we demonstrate the use of personalized circulating tumor DNA (ctDNA) profiling for detection of recurrence in breast cancer.Experimental Design: Forty-nine primary patients with breast cancer were recruited following surgery and adjuvant therapy. Plasma samples (n ¼ 208) were collected every 6 months for up to 4 years. Personalized assays targeting 16 variants selected from primary tumor whole-exome data were tested in serial plasma for the presence of ctDNA by ultradeep sequencing (average >100,000X).Results: Plasma ctDNA was detected ahead of clinical or radiologic relapse in 16 of the 18 relapsed patients (sensitivity of 89%); metastatic relapse was predicted with a lead time of up to 2 years (median, 8.9 months; range, 0.5-24.0 months). None of the 31 nonrelapsing patients were ctDNA-positive at any time point across 156 plasma samples (specificity of 100%). Of the two relapsed patients who were not detected in the study, the first had only a local recurrence, whereas the second patient had bone recurrence and had completed chemotherapy just 13 days prior to blood sampling.Conclusions: This study demonstrates that patientspecific ctDNA analysis can be a sensitive and specific approach for disease surveillance for patients with breast cancer. More importantly, earlier detection of up to 2 years provides a possible window for therapeutic intervention. Personalized profiling detects rising ctDNA ahead of clinical relapse. A-E, Plasma levels of ctDNA across serial plasma time points for five patients with breast cancer (one per panel). Mean VAFs are denoted by a dark blue circle, and solid lines represent the average VAF profile over time. The lead time is calculated as the time interval between clinical relapse (red triangle) and molecular relapse (blue triangle). CA 15-3 levels are graphed over time (teal circle), and the baseline levels (32 U/mL) are marked in light blue. F, Summary of percent VAF and number of targets detected at molecular and clinical relapse for all ctDNA-positive samples. Data are from 13 relapsed patients, excluding three patients with only one plasma time point. Coombes et al.

PURPOSE Novel sensitive methods for early detection of relapse and for monitoring therapeutic efficacy may have a huge impact on risk stratification, treatment, and ultimately outcome for patients with bladder cancer. We addressed the prognostic and predictive impact of ultra-deep sequencing of cell-free DNA in patients before and after cystectomy and during chemotherapy. PATIENTS AND METHODS We included 68 patients with localized advanced bladder cancer. Patient-specific somatic mutations, identified by whole-exome sequencing, were used to assess circulating tumor DNA (ctDNA) by ultra-deep sequencing (median, 105,000×) of plasma DNA. Plasma samples (n = 656) were procured at diagnosis, during chemotherapy, before cystectomy, and during surveillance. Expression profiling was performed for tumor subtype and immune signature analyses. RESULTS Presence of ctDNA was highly prognostic at diagnosis before chemotherapy (hazard ratio, 29.1; P = .001). After cystectomy, ctDNA analysis correctly identified all patients with metastatic relapse during disease monitoring (100% sensitivity, 98% specificity). A median lead time over radiographic imaging of 96 days was observed. In addition, for high-risk patients (ctDNA positive before or during treatment), the dynamics of ctDNA during chemotherapy was associated with disease recurrence ( P = .023), whereas pathologic downstaging was not. Analysis of tumor-centric biomarkers showed that mutational processes (signature 5) were associated with pathologic downstaging ( P = .024); however, no significant correlation for tumor subtypes, DNA damage response mutations, and other biomarkers was observed. Our results suggest that ctDNA analysis is better associated with treatment efficacy compared with other available methods. CONCLUSION ctDNA assessment for early risk stratification, therapy monitoring, and early relapse detection in bladder cancer is feasible and provides a basis for clinical studies that evaluate early therapeutic interventions.

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Background Numerous methods have been reported for the determination of artemether (ARM) and its metabolite dihydroartemisinin (DHA) in plasma. However, stability issues in patient plasma have not received enough attention. Results An LC–MS/MS method for simultaneous determination of ARM and DHA in human plasma (K3EDTA) turned out to be problematic: ARM and DHA were degraded partially or completely in some patient plasma samples as indicated by the stable isotope-labeled internal standards. We postulated iron II (Fe2+) in hemoglobin or its derived products from malaria patients causes degradation of the drugs, and found that hydrogen peroxide (H2O2) protected the drugs from degradation. Acidifying plasma increased recovery of ARM significantly. Using only 50 µl of plasma sample, the method has a LLOQ at 0.5 ng/ml for both ARM and DHA. Conclusion H2O2 is a stabilizing agent for artemisinin derivatives. The modified method is reliable and sensitive.