Jeppe Kildsig

BACKGROUND: Increasingly, circulating tumor DNA (ctDNA) is proposed as a tool for minimal residual disease (MRD) assessment. Digital PCR (dPCR) offers low analysis costs and turnaround times of less than a day, making it ripe for clinical implementation. Here, we used tumor-informed dPCR for ctDNA detection in a large colorectal cancer (CRC) cohort to evaluate the potential for post-operative risk assessment and serial monitoring, and how the metastatic site may impact ctDNA detection. Additionally, we assessed how altering the ctDNA-calling algorithm could customize performance for different clinical settings. PATIENTS AND METHODS: Stage II-III CRC patients (N = 851) treated with a curative intent were recruited. Based on whole-exome sequencing on matched tumor and germline DNA, a mutational target was selected for dPCR analysis. Plasma samples (8 ml) were collected within 60 days after operation and-for a patient subset (n = 246)-every 3-4 months for up to 36 months. Single-target dPCR was used for ctDNA detection. RESULTS: Both post-operative and serial ctDNA detection were prognostic of recurrence [hazard ratio (HR) = 11.3, 95% confidence interval (CI) 7.8-16.4, P < 0.001; HR = 30.7, 95% CI 20.2-46.7, P < 0.001], with a cumulative ctDNA detection rate of 87% at the end of sample collection in recurrence patients. The ctDNA growth rate was prognostic of survival (HR = 2.6, 95% CI 1.5-4.4, P = 0.001). In recurrence patients, post-operative ctDNA detection was challenging for lung metastases (4/21 detected) and peritoneal metastases (2/10 detected). By modifying the cut-off for calling a sample ctDNA positive, we were able to adjust the sensitivity and specificity of our test for different clinical contexts. CONCLUSIONS: The presented results from 851 stage II-III CRC patients demonstrate that our personalized dPCR approach effectively detects MRD after operation and shows promise for serial ctDNA detection for recurrence surveillance. The ability to adjust sensitivity and specificity shows exciting potential to customize the ctDNA caller for specific clinical settings.

OBJECTIVE: Circulating tumor DNA (ctDNA) is a candidate biomarker of cancer with practice-changing potential in the detection of both early and residual disease. Disease stage and tumor size affect the probability of ctDNA detection, whereas little is known about the influence of other tumor characteristics on ctDNA detection. This study investigates the impact of tumor cell whole-genome doubling (WGD) on the detection of ctDNA in plasma collected preoperatively from newly diagnosed colorectal cancer (CRC) patients. METHODS: WGD was estimated from copy numbers derived from whole-exome sequencing (WES) data of matched tumor and normal DNA from 833 Danish CRC patients. To explore if tumor WGD status impacts ctDNA detection, we applied tumor-informed ctDNA analysis to preoperative plasma samples from all patients. RESULTS: Patients with WGD+ tumors had 53% increased odds of being ctDNA positive (OR = 1.53, 95%CI: 1.12-2.09). After stratification for UICC stage, the association persisted for Stage I (OR = 2.44, 95%CI: 1.22-5.03) and Stage II (OR = 1.76, 95%CI: 1.11-2.81) but not for Stage III (OR = 0.83, 95%CI: 0.44-1.53) patients. CONCLUSION: The presence of WGD significantly increases the probability of detecting ctDNA, particularly for early-stage disease. In patients with more advanced disease, the benefit of WGD on ctDNA detection is less pronounced, consistent with increased DNA shedding from these tumors, making ctDNA detection less dependent on the amount of ctDNA released per tumor cell.

BACKGROUND: Multiple initiatives aim to develop circulating tumour DNA (ctDNA) tests for early cancer detection in asymptomatic individuals. The few studies describing ctDNA-testing in both asymptomatic and symptomatic patients report lower ctDNA detection in the asymptomatic patients. Here, we explore if asymptomatic patients differ from symptomatic patients e.g. by including a 'low-ctDNA-shedding' and 'less-aggressive' subgroup. METHODS: ctDNA assessment was performed in two independent cohorts of consecutively recruited patients with asymptomatic colorectal cancer (CRC) (Cohort#1: n = 215, Cohort#2: n = 368) and symptomatic CRC (Cohort#1: n = 117, Cohort#2: n = 722). RESULTS: After adjusting for tumour stage and size, the odds of ctDNA detection was significantly lower in asymptomatic patients compared to symptomatic patients (Cohort#1: OR: 0.4, 95%CI: 0.2-0.8, Cohort#2: OR: 0.7, 95%CI: 0.5-0.9). Further, the recurrence risk was lower in asymptomatic patients (Cohort#1: sHR: 0.6, 95%CI: 0.3-1.2, Cohort#2: sHR: 0.6, 95%CI: 0.4-1.0). Notably, ctDNA-negative asymptomatic patients had the lowest recurrence risk compared to the symptomatic patients (Cohort#1: sHR: 0.2, 95%CI: 0.1-0.6, Cohort#2: sHR: 0.3, 95%CI: 0.2-0.6). CONCLUSIONS: Our study suggests that asymptomatic patients are enriched for a 'low-ctDNA-shedding-low-recurrence-risk' subgroup. Such insights are needed to guide ctDNA-based early-detection initiatives and should prompt discussions about de-escalation of therapy and follow-up for ctDNA-negative asymptomatic CRC patients.