Comparison of the time to relapse detection using CEA and standard-of-care CT imaging for fourteen relapse patients.

List of Supplementary Tables

Detection of ctDNA in pre-operative plasma samples according to stage.

Comparison of serial ctDNA analysis performed using SSV and SPM ddPCR assays.

Abstract Purpose: We investigated whether detection of ctDNA after resection of colorectal cancer identifies the patients with the highest risk of relapse and, furthermore, whether longitudinal ctDNA analysis allows early detection of relapse and informs about response to intervention. Experimental Design: In this longitudinal cohort study, we used massively parallel sequencing to identify somatic mutations and used these as ctDNA markers to detect minimal residual disease and to monitor changes in tumor burden during a 3-year follow-up period. Results: A total of 45 patients and 371 plasma samples were included. Longitudinal samples from 27 patients revealed ctDNA postoperatively in all relapsing patients (n = 14), but not in any of the nonrelapsing patients. ctDNA detected relapse with an average lead time of 9.4 months compared with CT imaging. Of 21 patients treated for localized disease, six had ctDNA detected within 3 months after surgery. All six later relapsed compared with four of the remaining patients [HR, 37.7; 95% confidence interval (CI), 4.2–335.5; P < 0.001]. The ability of a 3-month ctDNA analysis to predict relapse was confirmed in 23 liver metastasis patients (HR 4.9; 95% CI, 1.5–15.7; P = 0.007). Changes in ctDNA levels induced by relapse intervention (n = 19) showed good agreement with changes in tumor volume (κ = 0.41; Spearman ρ = 0.4). Conclusions: Postoperative ctDNA detection provides evidence of residual disease and identifies patients at very high risk of relapse. Longitudinal surveillance enables early detection of relapse and informs about response to intervention. These observations have implications for the postoperative management of colorectal cancer patients. Clin Cancer Res; 23(18); 5437–45. ©2017 AACR.

Abstract MicroRNAs constitute a class of small cellular noncoding RNAs (typically 19-23 nt) which function as post-transcriptional regulators of gene expression. The involvement of microRNA in both normal development as well as disease is well established and microRNAs are considered important biomarkers in several diseases including cancer. Surprisingly cell free microRNAs are also found in several biofluids where they have been shown to be very stable, most likely because they are protected from degradation by protein complexes and other particles including exosomes. Exosomes are nanovesicles secreted into the extracellular environment by a wide range of cell types under normal and pathological conditions. As the profile of exosomal microRNAs may be a fingerprint of the releasing cell type and because they are released in easily accessible body fluids such as blood and urine, their microRNA content holds potential as biomarkers for early detection of malignancy. We have developed different technologies enabling the detection of prostate cancer microRNA biomarkers in exosomes derived from urine from non-prostate massaged men. The first of these technologies is a highly sensitive LNA™-based qPCR platform for microRNA detection, which enables profiling in biofluids where microRNA levels are extremely low. At this point we have already applied this platform on thousands of biofluid samples including serum/plasma and urine to establish normal reference ranges for circulating microRNAs as well as to identify biomarkers of disease. The second technology is a simple exosome precipitation system which only requires low speed centrifugation to harvest urine exosomes. Our developed technologies was applied to cell-free urine from three different cohorts including individuals with prostate cancer (PCa) and benign prostate hyperplasia, to identify several differentially regulated microRNAs in urine from PCa bearing individuals. PCa specific signatures were obtained by different combinations of these differentially regulated microRNAs. In conclusion, cell free urine samples holds potential as a liquid biopsy source for exosomal microRNA markers in prostate cancer. Data showing that small miRNA signatures (down to two miRNAs) hold strong diagnostic potential in PCa will be presented. Citation Format: Thorarinn Blondal, Anne I. Rasmussen, Anni R. Thomsen, Michael Borre, Jacob Fredsøe, Ditte Andreasen, Torben Falck Ørntoft, Karina D. Sørensen, Peter Mouritzen. A microRNA signature in urinary exosomes for diagnosis of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1943.

Abstract Antibody based detection of circulating prostate specific antigen (PSA) remains an important test in the diagnosis of prostate cancer. Unfortunately the test identifies a high number of false positives which require further testing to confirm the presence of cancer including several biopsies taken from the prostate itself. Whereas the performance of biopsies on individuals is associated with discomfort and risk of serious side effects, the invasive test also represents an expense to health care systems. Given the high number of false positives, the PSA test is therefore the direct cause of unnecessary suffering of healthy individuals and costs to health systems. To investigate the possibilities for developing a non-invasive and more specific test, we have developed different technologies enabling the detection of prostate cancer microRNA biomarkers in exosomes derived from urine from non-prostate massaged men. The first of these technologies is a highly sensitive LNA™-based qPCR platform for microRNA detection, which enables profiling in biofluids where microRNA levels are extremely low. At this point we have already applied this platform on thousands of biofluid samples including serum/plasma and urine to establish normal reference ranges for circulating microRNAs as well as to identify biomarkers of disease. The second technology is a simple exosome precipitation system which only requires low speed centrifugation to harvest urine exosomes. We have combined our developed technologies and applied this on cell-free urine from two different cohorts of approximately 220 patients each, to identify a number of differentially regulated microRNAs in urine from prostate cancer bearing individuals. Prostate cancer specific signatures have been obtained by different combinations of these differentially regulated microRNAs. The smallest signature is composed of two microRNAs which allows constructing receiver operating characteristic curves with areas under the curve well above 0.8. Citation Format: Peter Mouritzen, Jacob Christian Fredsøe, Thorarinn Blondal, Anne Karin Rasmussen, Michael Borre, Christa Haldrup, Ditte Andreasen, Niels Tolstrup, Torben Falck Ørntoft, Karina Dalsgaard Sørensen. A two-microRNA signature in urinary exosomes for diagnosis of prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B40.

MicroRNAs constitute a class of small cellular RNAs (typically 19-23 nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the human cellular transcriptome is regulated by this small class of RNA (∼2000 miRNA). MicroRNAs have been shown to be actively exported from tissues into the circulation through a variety of mechanisms including complexing with RNA binding proteins or HDL and through active exosome transport. Exosomes are nanovesicles secreted into the extracellular environment by a wide range of cell types under normal and pathological conditions. As the profile of exosomal microRNAs may be a fingerprint of the releasing cell type and because they are released in easily accessible body fluids such as blood and urine, their microRNA content holds potential as biomarkers for early detection of malignancy. Cell free urine samples are an obvious liquid biopsy source for microRNA markers in prostate cancer and could serve as diagnostic tools as well as treatment response markers. Yet microRNA levels in urine samples are extremely sparse and cannot be detected reliable by conventional sample preparation methods. It has been reported that tumour derived exosomes carrying genetic information specific for prostate cancer can be measured in urine samples following prostate massage. However, a methodology eliminating the need for prostate massage of every patient to be tested in a clinical setting is highly desirable. The purpose of this study was to combine a simple exosomal enrichment method with our highly sensitive LNA™-based qPCR platform for detection of microRNAs and apply this to a cohort consisting of more than 300 cell free urine samples from prostate cancer cases and controls. In conclusion, cell free urine samples holds potential as a liquid biopsy source for exosomal microRNA markers in prostate cancer, showing miRNA signatures of strong diagnostic potential. Analysis is pending and data will be presented. Citation Format: Thorarinn Blondal, Anni R. Thomsen, Jorg Krummheuer, Michael Borre, Jacob Fredsoe, Christa Haldrup, Ditte Andreasen, Maria W. Teilum, Niels Tolstrup, Karina D. Sorensen, Torben F. Orntoft, Peter Mouritzen. Exosomal microRNA in cell-free urine samples as a source for liquid prostate cancer biopsy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3987. doi:10.1158/1538-7445.AM2015-3987

The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC). To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD), was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

Nat. Methods 11, 809–815 (2014); published online 29 June 2014; corrected after print 30 July 2014 In the version of this article initially published, the author Linda Wong was omitted from the author list. The error has been corrected in the HTML and PDF versions of the article.

Abstract microRNAs constitute a class of small cellular RNAs (typically 19-23 nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the human cellular transcriptome is regulated by this small class of RNA (∼2000 miRNA). The study of extracellular microRNAs and their potential as pathophysiological markers has greatly expanded in the last couple of years. microRNAs have been shown to be actively exported from tissues into the circulation through a variety of mechanisms including exosome and microvesicle transport, and complexing with RNA binding proteins or HDL. The high relative stability of microRNAs in common clinical source materials (FFPE blocks, plasma, serum, urine, saliva, etc.) and the ability of microRNA expression profiles to accurately classify discrete tissue types and specific disease states have positioned microRNAs as promising new biomarkers for diagnostic application in cancer. We have applied Exiqons highly sensitive LNA™-based qPCR platform for detection of microRNAs, which has enabled microRNA profiling in biofluids where levels are extremely low. The platform uses a single RT reaction to conduct full miRNome profiling and allows high-throughput profiling of microRNAs without the need for pre-amplification. Thousands of biofluid samples including serum/plasma and urine have been profiled to determine normal reference ranges for circulating microRNAs as well as to identify biomarkers of disease. Extensive data qualification and analysis methods have been developed and are central parameters to secure high quality data from biofluids. The methods can quickly and robustly be applied in biomarker discovery and validation projects. We will present examples from our collaborative cancer diagnostic projects. Also we have developed a new exosome enrichment method and will present a comparison of microRNA profiles obtained with this method to profiles obtained with different commercial available exosome isolation methods and standard profiles of whole plasma and serum. Citation Format: Thorarinn Blondal, Anni Thomsen, Jörg Krummheyer, Peter Mouritzen, Ditte Andreasen, Maria Theilum, Jan Stenvang, Claus L. Andersen, Hans J. Nielsen, Nils Brünner. MicroRNA in biofluid as robust biomarkers for cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5233. doi:10.1158/1538-7445.AM2014-5233

MicroRNAs (miRNAs) constitute a class of small cellular RNAs (typically 21-23 nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, although they are relatively few in number (less than 2000 human miRNAs).The high relative stability of miRNA in common clinical tissues and biofluids (e.g. plasma, serum, urine, saliva, etc.) and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNA quantification as a promising new tool for a wide range of diagnostic applications. Furthermore miRNAs have been shown to be rapidly released from tissues into the circulation with the development of pathology.To facilitate discovery and clinical development of miRNA-based biomarkers, we developed a genome-wide Locked Nucleic Acid (LNA (TM))-based miRNA qPCR platform with unparalleled sensitivity and robustness. The platform allows high-throughput profiling of miRNAs from important clinical sources without the need for pre-amplification.Using this system, we have profiled thousands of biofluid samples including blood derived plasma and serum. An extensive quality control (QC) system has been implemented in order to secure technical excellence and reveal any unwanted bias coming from pre-analytical or analytical variables. We present our approaches to sample and RNA QC as well as data QC and normalization. Specifically we have developed normal reference ranges for circulating miRNAs in serum and plasma as well as a hemolysis indicator based on microRNA expression. (C) 2012 Elsevier Inc. All rights reserved.

microRNAs represent the best described class of small RNAs (21-23nt) and have been shown to function as post-transcriptional regulators of gene expression. The high relative stability of microRNA in common clinical source materials and the ability of microRNA expression profiles to accurately classify discrete tissue types and specific disease states have positioned microRNA quantification as a promising new biomarker for a wide range of diagnostic applications We have developed a genome-wide LNA™-based microRNA qPCR platform with unparalleled sensitivity and robustness even in biofluids where microRNA levels are extremely low. Only a single cDNA synthesis reaction is required to conduct full miRNome profiling thereby facilitating high-throughput profiling in important clinical sources without the need for pre-amplification. Thousands of biofluid samples have been profiled including blood derived plasma/serum and urine to accurately determine normal reference ranges for circulating microRNAs. Procedures have been developed to control pre-analytical variables such as hemolysis in serum/plasma samples. In addition, a data QC system has been implemented to secure technical excellence and reveal any unwanted bias in the dataset. We are currently screening for and validating microRNAs as biomarkers for stage II colorectal cancer (CRC). microRNA profiling has been performed on plasma samples from a clinical trial conducted in 7 different hospitals. We show that hemolysis in this sample set correlates with hospital ID, and with the utilization of specific blood sample collection vials. Using a microRNA-based hemolysis signature, we eliminated hemolyzed samples and demonstrated that this step leads to a major improvement of CRC detection (ROC AUC increase from 0.67 to 0.80). We conclude that pre-analytical variables such as hemolysis can be a source of bias in samples of different origin, and that sample and data QC procedures can overcome this challenge and lead to improved miRNA biomarker performance.

microRNAs represent the best described class of small RNAs (21-23nt) and have been shown to function as post-transcriptional regulators of gene expression. The high relative stability of microRNA in common clinical samples such as serum/plasma, urine and other biofluids, and the ability of micro RNA expression profiles to accurately classify discrete tissue types and specific disease states have positioned microRNA quantification as a promising new biomarker for a wide range of diagnostic applications. We have developed a genome-wide LNATM-based microRNA qPCR platform with unparalleled sensitivity and robustness. A single cDNA synthesis reaction is sufficient for full microRNA analysis without the need for preamplification. The platform is thus highly suited to high-throughput microRNA profiling from challenging clinical source material. Thousands of biofluid samples, including blood derived serum/plasma and urine, have been profiled in-house allowing the development of focused microRNA assay sets for the discovery of micro RNA biomarkers for disease, toxicology and injury studies. Here we demonstrate the applicability of urine microRNA profiling for the discovery of potential biomakers for nephrotoxin induced kidney injury using the Toxicology Focus micro RNA PCR Panel. We also present our approach to the challenging aspect of data processing and normalization specifically for microRNA profiling in urine. Urine microRNA profiling to discover biomarkers for nephrotoxicity Jörg Krummheuer, Thorarinn Blondal, Ditte Andreasen, Maria Wrang Teilum, Kim Bundvig Barken, Niels Tolstrup, Nana Jacobsen, Ina K Dahlsveen and Peter Mouritzen Exiqon A/S, Skelstedet 16, 2950 Denmark

20 Background: MicroRNAs function as post-transcriptional regulators of gene expression. Their high relative stability in common clinical source materials (FFPE blocks, plasma, serum, urine, saliva, etc.) and the ability of microRNA expression profiles to accurately classify discrete tissue types and specific disease states have positioned microRNAs as promising new biomarkers for diagnostic application. Furthermore microRNAs have been shown to be rapidly released from tissues into the circulation with the development of pathology. Methods: Thousands of biofluid samples were profiled including blood derived plasma/serum and urine using a genome-wide LNA-based microRNA qPCR platform, which has unparalleled sensitivity and robustness even in biofluids with extremely low microRNA levels. Only a single RT reaction is required to conduct full miRNome profiling thereby facilitating high-throughput profiling without the need for pre-amplification. Results: Normal reference ranges for circulating microRNAs were determined in several biofluids, allowing development of qPCR arrays containing only relevant microRNA subsets present in various biofluids together with tissue specific microRNA markers. Procedures were developed to control pre-analytical variables, for quality checking and qualifying biofluid samples in particular serum and plasma but also urine and other biofluids. An extensive QC system was implemented in order to secure technical excellence and reveal any unwanted bias in the dataset. We currently screen and validate microRNAs biomarkers for cancer with the aim of developing minimal invasive tests to be applied in early detection population screens. Conclusions: The qPCR panels support development of robust biomarkers in disease, toxicology, and injury studies. We will demonstrate how panels may be quickly and robustly applied in biomarker discovery/validation projects using the specific case early detection of colorectal cancer in blood. Close attention is required on pre-analytical parameters. Hemolysis and cellular contamination affect miRNA profiles in biofluids and control is required.

A variety of physiological processes are associated with changes in microRNA (miRNA) expression. Analysis of miRNA has been applied to study normal physiology as well as diseased states including cancer. One major challenge in miRNA research is to accurately and practically determine the expression level of miRNAs in various experimental systems. Many genome-wide miRNA expression profiling studies have relied on microarrays technology, and frequently differentially expressed miRNAs have subsequently been confirmed with real-time quantitative PCR studies. Here, we describe how different primer strategies for first-strand cDNA synthesis and PCR amplification can affect measurements of miRNA expression levels. Overcoming the small nature of miRNAs is a difficult task as the short sequence available does not allow for designing primers using standard PCR primer design guidelines. Finally, we demonstrate how to determine differentially expressed miRNAs using a locked nucleic acid-based real-time PCR approach.

microRNAs are small regulatory RNAs that are currently emerging as new biomarkers for cancer and other diseases. In order for biomarkers to be useful in clinical settings, they should be accurately and reliably detected in clinical samples such as formalin fixed paraffin embedded (FFPE) sections and blood serum or plasma. These types of samples represent a challenge in terms of microRNA quantification. A newly developed method for microRNA qPCR using Locked Nucleic Acid (LNA™)-enhanced primers enables accurate and reproducible quantification of microRNAs in scarce clinical samples. Here we show that LNA™-based microRNA qPCR enables biomarker screening using very low amounts of total RNA from FFPE samples and the results are compared to microarray analysis data. We also present evidence that the addition of a small carrier RNA prior to total RNA extraction, improves microRNA quantification in blood plasma and laser capture microdissected (LCM) sections of FFPE samples.

Mouse channel activating proteases 1, 2, and 3 (mCAP1, mCAP2, and mCAP3) were described recently as regulators of the epithelial sodium channel (ENaC). The mCAP are membrane-bound serine proteases that are synthesized as inactive proenzymes. To mature into active proteases, they undergo intramolecular cleavage by auto- and/or heterocatalytic processing. Specific antibodies against each mCAP were developed to distinguish between proenzyme and active protease by Western blot analysis. Various point mutations were introduced in the catalytic or protein-protein interacting domains of mCAP and wild-type and mutant enzymes were expressed in the Xenopus oocyte expression system to test for ability to activate ENaC. In mCAP3, an intact catalytic triad was necessary for activation of ENaC but not for intramolecular cleavage of the protease. This suggests a heterocatalytic mechanism. Mutating the catalytic triad of mCAP2 not only abolished ENaC activation completely but also impeded cleavage of the protease. Processing of mCAP2 therefore seems to be autocatalytic. Furthermore, mutations in conserved residues of mCAP2 located in two protein-protein interacting domains significantly modulated ENaC activation. Surprisingly, mCAP1 catalytically inactive mutants were still able to fully activate ENaC, and no evidence of mCAP1 intramolecular cleavage was seen. The presence of an intact glycosylphosphatidylinositol anchor, however, was required. It is concluded that auto- and heterocatalytic requirements are specific for each CAP and that endogenous partners are a necessity for activation of ENaC by mCAP in the Xenopus oocyte expression system.