Exploring the biology of ctDNA release in colorectal cancer.

BACKGROUND: Circulating tumor DNA (ctDNA) has emerged as a promising tool for early cancer detection and minimal residual disease monitoring. However, the biology underlying ctDNA release and its variation across cancer types and histologies remains poorly understood. This study investigated the biology behind ctDNA shedding in colorectal cancer. METHODS: The study included a local cohort of 747 stage I-III colorectal cancer patients. All patients had ctDNA measurement prior to treatment and extensive clinical data. Primary tumor RNA sequencing and whole exome sequencing was performed in 95 and 652 patients respectively. Additionally, the study evaluated 89 non-small cell lung cancer patients from the TRACERx cohort, comprising primary tumor RNA sequencing and ctDNA measurement. RESULTS: We found tumor size and proliferative capacity to be key factors associated with ctDNA shedding in colorectal cancer. Furthermore, we found that the secretory and CMS3 colorectal cancer subtypes exhibited lower ctDNA shedding, while microsatellite instability (MSI) tumors had higher levels of ctDNA. Mutational analysis did not reveal any genes or pathways associated with ctDNA shedding in colorectal cancer. A comparison of transcriptomic profiles across multiple cancer types demonstrated that colorectal cancer and lung squamous cell carcinoma tumors shared a high-proliferative ctDNA shedding phenotype, while lung adenocarcinoma tumors displayed a distinct low-proliferative subgroup. Additionally, proliferation levels correlated with ctDNA detection sensitivity across multiple cancer types. CONCLUSION: These findings suggest that tumor size and proliferative capacity are drivers of ctDNA release in colorectal cancer and provide insights into the biology of ctDNA shedding on a pan-cancer level.

Ultrasensitive plasma-based monitoring of tumor burden using machine-learning-guided signal enrichment.

In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole-genome sequencing (WGS). Here we now introduce MRD-EDGE, a machine-learning-guided WGS ctDNA single-nucleotide variant (SNV) and copy-number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGESNV uses deep learning and a ctDNA-specific feature space to increase SNV signal-to-noise enrichment in WGS by ~300× compared to previous WGS error suppression. MRD-EDGECNV also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1 Gb to 200 Mb, vastly expanding its applicability within solid tumors. We harness the improved performance to identify MRD following surgery in multiple cancer types, track changes in TF in response to neoadjuvant immunotherapy in lung cancer and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal-to-noise enrichment in MRD-EDGESNV enables plasma-only (non-tumor-informed) disease monitoring in advanced melanoma and lung cancer, yielding clinically informative TF monitoring for patients on immune-checkpoint inhibition.

Tumour-agnostic circulating tumour DNA analysis for improved recurrence surveillance after resection of colorectal liver metastases: A prospective cohort study.

PURPOSE: Nearly 50% of patients recur within two years after curatively intended resection of colorectal cancer liver metastasis (CRLM). The optimal surveillance strategy is unknown due to the lack of evidence. Here, we explored the potential for improving postoperative CRLM surveillance by performing serial circulating tumour DNA (ctDNA) assessments parallel to standard-of-care surveillance. EXPERIMENTAL DESIGN: 499 prospectively collected serial plasma samples from 96 patients undergoing CRLM resection were analysed using the tumour-agnostic methylation multiplex droplet-digital PCR test 'TriMeth'. RESULTS: Patients with ctDNA postoperatively or post adjuvant chemotherapy experienced a significant lower recurrence-free survival than patients without ctDNA (hazard ratio (HR) 4.5; P < 0.0001 and HR 8.4, P < 0.0001). ctDNA status was a stronger predictor of recurrence than standard clinical risk factors and carcinoembryonic antigen. Serial TriMeth analysis detected ctDNA before radiological recurrence in 55.6% of ctDNA-positive patients, with up to 10.6 months lead-time (median 3.1 months). During surveillance, 24% of patients had inconclusive CT scans, which was associated with a significant delay in recurrence diagnosis (median 3.5 months versus 1.0 month, P < 0.0001). Uniquely, ctDNA status at the time of inconclusive CT scans predicted recurrence with positive and negative predictive values of 100%, and 75% (P = 0.0003). Serial TriMeth analysis allowed ctDNA growth rate assessment and revealed that fast ctDNA growth was associated with poor overall survival (HR: 1.6, P = 0.0052). CONCLUSIONS: Serial postoperative ctDNA analysis has a strong prognostic value and is more sensitive for recurrence detection than standard-of-care CRLM surveillance tools. Altogether, TriMeth provides several opportunities for improving postoperative surveillance of CRLM patients.

Circulating tumor DNA for prognosis assessment and postoperative management after curative-intent resection of colorectal liver metastases.

The recurrence rate of colorectal liver metastases (CRLM) patients treated with curative intent is above 50%. Standard of care surveillance includes intensive computed tomographic (CT) imaging as well as carcinoembryonic antigen (CEA) measurements. Nonetheless, relapse detection often happens too late to resume curative treatment. This longitudinal cohort study enrolled 115 patients with plasma samples (N = 439) prospectively collected before surgery, postoperatively at day 30 and every third month for up to 3 years. Droplet digital PCR (ddPCR) was used to monitor serial plasma samples for somatic mutations. Assessment of ctDNA status either immediately after surgery, or serially during surveillance, stratified the patients into groups of high and low recurrence risk (hazard ratio [HR], 7.6; 95% CI, 3.0-19.7; P < .0001; and HR, 4.3; 95% CI, 2.3-8.1; P < .0001, respectively). The positive predictive value (PPV) of ctDNA was 100% in all postoperative analyses. In multivariable analyses, postoperative ctDNA status was the only consistently significant risk marker associated with relapse (P < .0001). Indeterminate CT findings were observed for 30.8% (21/68) of patients. All patients (9/21) that were ctDNA positive at the time of the indeterminate CT scan later relapsed, contrasting 42.6% (5/12) of those ctDNA negative (P = .0046). Recurrence diagnoses in patients with indeterminate CT findings were delayed (median 2.8 months, P < .0001). ctDNA status is strongly associated with detection of minimal residual disease and early detection of relapse. Furthermore, ctDNA status can potentially contribute to clinical decision-making in case of indeterminate CT findings, reducing time-to-intervention.

[Circulating tumour DNA in the treatment of cancer].

Circulating tumour DNA analysis has a potential to improve multiple aspects of cancer management. This includes: a) early cancer detection in asymptomatic individuals, b) identification of patients with residual disease after curative intended treatment, c) patient stratification in relation to treatment decisions like adjuvant therapy and intensity of radiological surveillance, d) monitoring treatment effect for optimised adaptive therapy, e) identification of actionable targets, and f) early recurrence detection. These points are summarised in this review.

Age-stratified reference intervals unlock the clinical potential of circulating cell-free DNA as a biomarker of poor outcome for healthy individuals and patients with colorectal cancer.

Circulating cell-free DNA (cfDNA) has spurred much interest as a biomarker in oncology. However, inter- and intra-individual cfDNA levels vary greatly. Consequently, in order to base clinical decisions on cfDNA measurements, normal reference intervals are essential to avoid that ordinary variation is confused with clinically relevant change. The lack of reference intervals may potentially explain the ambiguous results reported in the field. Our study aimed to establish reference intervals and to evaluate the association between cfDNA and demographic and clinical variables, including colorectal cancer (CRC). Plasma samples and clinical data from 2817 subjects were collected including 1930 noncancer individuals and 887 CRC patients. cfDNA was measured using droplet digital polymerase chain reaction (PCR). The large cohort combined with robust cfDNA quantification enabled establishment of reference intervals (<67 years: 775-4860 copies/mL; ≥67 years: 807-6561 copies/mL). A cfDNA level above the age-stratified 90% percentile was prognostic of reduced survival in both noncancer individuals and CRC patients, with HR values of 2.56 and 2.01, respectively. Moreover, cfDNA levels increased significantly with age, elevated BMI and chronic diseases. In CRC, the cfDNA level was increased for Stage IV, but not Stage I to Stage III cancer. In summary, the use of reference intervals revealed that high cfDNA levels were predictive of shorter survival in both noncancer individuals and CRC patients, and that CRC development did not affect the cfDNA level until metastatic dissemination. Furthermore, cfDNA levels were impacted by age and chronic diseases. Conclusively, our study presents reference intervals that will help pave the way for clinical utilization of cfDNA.

Enhanced Performance of DNA Methylation Markers by Simultaneous Measurement of Sense and Antisense DNA Strands after Cytosine Conversion.

BACKGROUND: Most existing DNA methylation-based methods for detection of circulating tumor DNA (ctDNA) are based on conversion of unmethylated cytosines to uracil. After conversion, the 2 DNA strands are no longer complementary; therefore, targeting only 1 DNA strand merely utilizes half of the available input DNA. We investigated whether the sensitivity of methylation-based ctDNA detection strategies could be increased by targeting both DNA strands after bisulfite conversion. METHODS: Dual-strand digital PCR assays were designed for the 3 colorectal cancer (CRC)-specific methylation markers KCNQ5, C9orf50, and CLIP4 and compared with previously reported single-strand assays. Performance was tested in tumor and leukocyte DNA, and the ability to detect ctDNA was investigated in plasma from 43 patients with CRC stages I to IV and 42 colonoscopy-confirmed healthy controls. RESULTS: Dual-strand assays quantified close to 100% of methylated control DNA input, whereas single-strand assays quantified approximately 50%. Furthermore, dual-strand assays showed a 2-fold increase in the number of methylated DNA copies detected when applied to DNA purified from tumor tissue and plasma from CRC patients. When the results of the 3 DNA methylation markers were combined into a ctDNA detection test and applied to plasma, the dual-strand assay format detected 86% of the cancers compared with 74% for the single-strand assay format. The specificity was 100% for both the dual- and single-strand test formats. CONCLUSION: Dual-strand assays enabled more sensitive detection of methylated ctDNA than single-strand assays.

Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer-a clinical biomarker discovery and validation study.

BACKGROUND: Early detection plays an essential role to reduce colorectal cancer (CRC) mortality. While current screening methods suffer from poor compliance, liquid biopsy-based strategies for cancer detection is rapidly gaining promise. Here, we describe the development of TriMeth, a minimal-invasive blood-based test for detection of early-stage colorectal cancer. The test is based on assessment of three tumour-specific DNA methylation markers in circulating cell-free DNA. RESULTS: A thorough multi-step biomarker discovery study based on DNA methylation profiles of more than 5000 tumours and blood cell populations identified CRC-specific DNA methylation markers. The DNA methylation patterns of biomarker candidates were validated by bisulfite sequencing and methylation-specific droplet digital PCR in CRC tumour tissue and peripheral blood leucocytes. The three best performing markers were first applied to plasma from 113 primarily early-stage CRC patients and 87 age- and gender-matched colonoscopy-verified controls. Based on this, the test scoring algorithm was locked, and then TriMeth was validated in an independent cohort comprising 143 CRC patients and 91 controls. Three DNA methylation markers, C9orf50, KCNQ5, and CLIP4, were identified, each capable of discriminating plasma from colorectal cancer patients and healthy individuals (areas under the curve 0.86, 0.91, and 0.88). When combined in the TriMeth test, an average sensitivity of 85% (218/256) was observed (stage I: 80% (33/41), stage II: 85% (121/143), stage III: 89% (49/55), and stage IV: 88% (15/17)) at 99% (176/178) specificity in two independent plasma cohorts. CONCLUSION: TriMeth enables detection of early-stage colorectal cancer with high sensitivity and specificity. The reported results underline the potential utility of DNA methylation-based detection of circulating tumour DNA in the clinical management of colorectal cancer.

Biomarker potential of ST6GALNAC3 and ZNF660 promoter hypermethylation in prostate cancer tissue and liquid biopsies.

Current diagnostic and prognostic tools for prostate cancer (PC) are suboptimal, leading to overdiagnosis and overtreatment. Aberrant promoter hypermethylation of specific genes has been suggested as novel candidate biomarkers for PC that may improve diagnosis and prognosis. We here analyzed ST6GALNAC3 and ZNF660 promoter methylation in prostate tissues, and ST6GALNAC3, ZNF660, CCDC181, and HAPLN3 promoter methylation in liquid biopsies. First, using four independent patient sample sets, including a total of 110 nonmalignant (NM) and 705 PC tissue samples, analyzed by methylation-specific qPCR or methylation array, we found that hypermethylation of ST6GALNAC3 and ZNF660 was highly cancer-specific with areas under the curve (AUC) of receiver operating characteristic (ROC) curve analysis of 0.917-0.995 and 0.846-0.903, respectively. Furthermore, ZNF660 hypermethylation was significantly associated with biochemical recurrence in two radical prostatectomy (RP) cohorts of 158 and 392 patients and remained significant also in the subsets of patients with Gleason score ≤7 (univariate Cox regression and log-rank tests, P < 0.05), suggesting that ZNF660 methylation analysis can potentially help to stratify low-/intermediate-grade PCs into indolent vs. more aggressive subtypes. Notably, ZNF660 hypermethylation was also significantly associated with poor overall and PC-specific survival in the RP cohort (n = 158) with long clinical follow-up available. Moreover, as proof of principle, we successfully detected highly PC-specific hypermethylated circulating tumor DNA (ctDNA) for ST6GALNAC3, ZNF660, HAPLN3, and CCDC181 in liquid biopsies (serum) from 27 patients with PC vs. 10 patients with BPH, using droplet digital methylation-specific PCR analysis. Finally, we generated a three-gene (ST6GALNAC3/CCDC181/HAPLN3) ctDNA hypermethylation model, which detected PC with 100% specificity and 67% sensitivity. In conclusion, we here for the first time demonstrate diagnostic biomarker potential of ST6GALNAC3 and ZNF660 methylation, as well as prognostic biomarker potential of ZNF660. Furthermore, we show that hypermethylation of four genes can be detected in ctDNA in liquid biopsies (serum) from patients with PC.