BackgroundUrine poses an attractive non-invasive means for obtaining liquid biopsies for oncological diagnostics. Especially molecular analysis on urinary DNA is a rapid growing field. However, optimal and practical storage conditions that result in preservation of urinary DNA, and in particular hypermethylated DNA (hmDNA), are yet to be determined.AimTo determine the most optimal and practical conditions for urine storage that result in adequate preservation of DNA for hmDNA analysis.MethodsDNA yield for use in methylation analysis was determined by quantitative methylation specific PCR (qMSP) targeting the ACTB and RASSF1A genes on bisulfite modified DNA. First, DNA yield (ACTB qMSP) was determined in a pilot study on urine samples of healthy volunteers using two preservatives (Ethylenediaminetetraacetic acid (EDTA) and Urine Conditioning Buffer, Zymo Research) at four different temperatures (room temperature (RT), 4°C, -20°C, -80°C) for four time periods (1, 2, 7, 28 days). Next, hmDNA levels (RASSF1A qMSP) in stored urine samples of patients suffering from bladder cancer (n = 10) or non-small cell lung cancer (NSCLC; n = 10) were measured at day 0 and 7 upon storage with and without the addition of 40mM EDTA and/or 20 μl/ml Penicillin Streptomycin (PenStrep) at RT and 4°C.ResultsIn the pilot study, DNA for methylation analysis was only maintained at RT upon addition of preserving agents. In urine stored at 4°C for a period of 7 days or more, the addition of either preserving agent yielded a slightly better preservation of DNA. When urine was stored at -20 °C or -80 °C for up to 28 days, DNA was retained irrespective of the addition of preserving agents. In bladder cancer and NSCLC samples stored at RT loss of DNA was significantly less if EDTA was added compared to no preserving agents (p<0.001). Addition of PenStrep did not affect DNA preservation (p>0.99). Upon storage at 4°C, no difference in DNA preservation was found after the addition of preserving agents (p = 0.18). The preservation of methylated DNA (RASSF1A) was strongly correlated to that of unmethylated DNA (ACTB) in most cases, except when PCR values became inaccurate.ConclusionsAddition of EDTA offers an inexpensive preserving agent for urine storage at RT up to seven days allowing for reliable hmDNA analysis. To avoid bacterial overgrowth PenStrep can be added without negatively affecting DNA preservation.

A significant proportion of colorectal cancer (CRC) patients develop peritoneal metastases (PM) in the course of their disease. PMs are associated with a poor quality of life, significant morbidity and dismal disease outcome. To improve care for this patient group, a better understanding of the molecular characteristics of CRC-PM is required. Here we present a comprehensive molecular characterization of a cohort of 52 patients. This reveals that CRC-PM represent a distinct CRC molecular subtype, CMS4, but can be further divided in three separate categories, each presenting with unique features. We uncover that the CMS4-associated structural protein Moesin plays a key role in peritoneal dissemination. Finally, we define specific evolutionary features of CRC-PM which indicate that polyclonal metastatic seeding underlies these lesions. Together our results suggest that CRC-PM should be perceived as a distinct disease entity.

Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy (CRS-HIPEC) may be curative for colorectal cancer patients with peritoneal metastases (PMs) but it has a high rate of morbidity. Accurate preoperative patient selection is therefore imperative, but is constrained by the limitations of current imaging techniques. In this pilot study, we explored the feasibility of circulating tumor (ct) DNA analysis to select patients for CRS-HIPEC. Thirty patients eligible for CRS-HIPEC provided blood samples preoperatively and during follow-up if the procedure was completed. Targeted Next-Generation Sequencing (NGS) of DNA from PMs was used to identify bespoke mutations that were subsequently tested in corresponding plasma cell-free (cf) DNA samples using droplet digital (dd) PCR. CtDNA was detected preoperatively in cfDNA samples from 33% of patients and was associated with a reduced disease-free survival (DFS) after CRS-HIPEC (median 6.0 months vs median not reached, p = 0.016). This association could indicate the presence of undiagnosed systemic metastases or an increased metastatic potential of the tumors. We demonstrate the feasibility of ctDNA to serve as a preoperative marker of recurrence in patients with PMs of colorectal cancer using a highly sensitive technique. A more appropriate treatment for patients with preoperative ctDNA detection may be systemic chemotherapy in addition to, or instead of, CRS-HIPEC.