Development of blood-based biomarker tests for early detection of colorectal neoplasia: Influence of blood collection timing and handling procedures.
Clinica chimica acta; international journal of clinical chemistry (2020 Aug)
p. 39-53 issn:1873-3492)
Blood-based, cancer-associated biomarkers are susceptible to a variety of well-known preanalytical factors. The influence of bowel preparation before a diagnostic colonoscopy on biomarker levels is, however, poorly investigated. The present study assessed the influence of bowel preparation on colorectal cancer-associated biomarkers. In addition, the effect of single versus double centrifugation of plasma biomarkers was assessed.
Forfattere: Lech Pedersen N, Mertz Petersen M, Ladd JJ, Lampe PD, Bresalier RS, Davis GJ, Demuth C, Jensen SØ, Andersen CL, Ferm L, Christensen IJ, Nielsen HJ
Enhanced Performance of DNA Methylation Markers by Simultaneous Measurement of Sense and Antisense DNA Strands after Cytosine Conversion.
Clinical chemistry (2020 May)
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.
Forfattere: Jensen SØ, Øgaard N, Nielsen HJ, Bramsen JB, Andersen CL
Biomarkers for Early Detection of Colorectal Cancer:The Early Detection Research Network, a Framework for Clinical Translation.
Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology (2020 Apr)
Early detection by screening significantly reduces mortality from colorectal cancer, but 40% of guideline-eligible patients are not screened as recommended in the US. Novel strategies to improve screening uptake overall, and efforts to deploy best-practices to underserved populations is a high priority for healthcare. This review focuses on existing biomarkers in practice and those in development with clinical relevance to early detection of colorectal neoplasia, with an emphasis on those developed by investigators of the National Cancer Institute’s Early Detection Research Network (EDRN). Aberrantly methylated DNA markers (blood and stool), stool-based markers (including FIT-DNA), and a variety of blood-based markers assays in development (protein markers, glycoproteins including mucins, cell-free DNA tests) are reviewed. Individual markers and biomarker panels, sample resources, and barriers to translating biomarkers to clinical practice are discussed.
Forfattere: Bresalier RS, Grady WM, Markowitz SD, Nielsen HJ, Batra SK, Lampe PD
Gel-Based Proteomics of Clinical Samples Identifies Potential Serological Biomarkers for Early Detection of Colorectal Cancer.
International journal of molecular sciences (2019 Dec)
Issue 23 issn:1422-0067)
The burden of colorectal cancer (CRC) is considerable-approximately 1.8 million people are diagnosed each year with CRC and of these about half will succumb to the disease. In the case of CRC, there is strong evidence that an early diagnosis leads to a better prognosis, with metastatic CRC having a 5-year survival that is only slightly greater than 10% compared with up to 90% for stage I CRC. Clearly, biomarkers for the early detection of CRC would have a major clinical impact. We implemented a coherent gel-based proteomics biomarker discovery platform for the identification of clinically useful biomarkers for the early detection of CRC. Potential protein biomarkers were identified by a 2D gel-based analysis of a cohort composed of 128 CRC and site-matched normal tissue biopsies. Potential biomarkers were prioritized and assays to quantitatively measure plasma expression of the candidate biomarkers were developed. Those biomarkers that fulfilled the preset criteria for technical validity were validated in a case-control set of plasma samples, including 70 patients with CRC, adenomas, or non-cancer diseases and healthy individuals in each group. We identified 63 consistently upregulated polypeptides (factor of four-fold or more) in our proteomics analysis. We selected 10 out of these 63 upregulated polypeptides, and established assays to measure the concentration of each one of the ten biomarkers in plasma samples. Biomarker levels were analyzed in plasma samples from healthy individuals, individuals with adenomas, CRC patients, and patients with non-cancer diseases and we identified one protein, tropomyosin 3 (Tpm3) that could discriminate CRC at a significant level ( = 0.0146). Our results suggest that at least one of the identified proteins, Tpm3, could be used as a biomarker in the early detection of CRC, and further studies should provide unequivocal evidence for the real-life clinical validity and usefulness of Tpm3.
Forfattere: Thorsen SF, Gromova I, Christensen IJ, Fredriksson S, Andersen CL, Nielsen HJ, Stenvang J, Moreira JMA
The concentration of the cleaved suPAR forms in pre- and postoperative plasma samples improves the prediction of survival in colorectal cancer: A nationwide multicenter validation and discovery study.
Journal of surgical oncology (2019 Dec)
Issue 8 p. 1404-1411 issn:1096-9098)
The aim was to evaluate the prognostic biomarker potential of the soluble urokinase-type plasminogen activator receptor (suPAR) in plasma samples collected pre- and postoperatively from patients resected for colorectal cancer (CRC).
Forfattere: Rolff HC, Christensen IJ, Svendsen LB, Wilhelmsen M, Lund IK, Thurison T, Høyer-Hansen G, Illemann M, Nielsen HJ,
Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer-a clinical biomarker discovery and validation study.
Clinical epigenetics (2019 Nov)
Issue 1 p. 158 issn:1868-7083)
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.
Forfattere: Jensen SØ, Øgaard N, Ørntoft MW, Rasmussen MH, Bramsen JB, Kristensen H, Mouritzen P, Madsen MR, Madsen AH, Sunesen KG, Iversen LH, Laurberg S, Christensen IJ, Nielsen HJ, Andersen CL
Evaluation of algorithm development approaches: Development of biomarker panels for early detection of colorectal lesions.
Clinica chimica acta; international journal of clinical chemistry (2019 Aug)
p. 108-115 issn:1873-3492)
Colorectal cancer (CRC) is the third most common cancer in the U.S. Early detection of CRC can substantially increase survival rates. Test compliance may be improved by offering a blood-based test option.
Forfattere: Gawel SH, Lucht M, Gomer H, Treado P, Christensen IJ, Nielsen HJ, Davis GJ,
Nature (2019 Jun)
Issue 7761 p. 385-389 issn:1476-4687)
Cell-free DNA in the blood provides a non-invasive diagnostic avenue for patients with cancer. However, characteristics of the origins and molecular features of cell-free DNA are poorly understood. Here we developed an approach to evaluate fragmentation patterns of cell-free DNA across the genome, and found that profiles of healthy individuals reflected nucleosomal patterns of white blood cells, whereas patients with cancer had altered fragmentation profiles. We used this method to analyse the fragmentation profiles of 236 patients with breast, colorectal, lung, ovarian, pancreatic, gastric or bile duct cancer and 245 healthy individuals. A machine learning model that incorporated genome-wide fragmentation features had sensitivities of detection ranging from 57% to more than 99% among the seven cancer types at 98% specificity, with an overall area under the curve value of 0.94. Fragmentation profiles could be used to identify the tissue of origin of the cancers to a limited number of sites in 75% of cases. Combining our approach with mutation-based cell-free DNA analyses detected 91% of patients with cancer. The results of these analyses highlight important properties of cell-free DNA and provide a proof-of-principle approach for the screening, early detection and monitoring of human cancer.
Forfattere: Cristiano S, Leal A, Phallen J, Fiksel J, Adleff V, Bruhm DC, Jensen SØ, Medina JE, Hruban C, White JR, Palsgrove DN, Niknafs N, Anagnostou V, Forde P, Naidoo J, Marrone K, Brahmer J, Woodward BD, Husain H, van Rooijen KL, Ørntoft MW, Madsen AH, van de Velde CJH, Verheij M, Cats A, Punt CJA, Vink GR, van Grieken NCT, Koopman M, Fijneman RJA, Johansen JS, Nielsen HJ, Meijer GA, Andersen CL, Scharpf RB, Velculescu VE
Increased serological, cancer-associated protein biomarker levels at diagnosis of large bowel adenoma: Risk of subsequent primary malignancy?
Acta Oncol. 2019
58(sup1):S42-S48. doi: 10.1080/0284186X.2018.1540885.
Epub 2018 Dec 7.
Blood-based, cancer-associated biomarkers may detect subjects at risk of having neoplastic diseases. The aim of the present study was to evaluate whether elevated serological protein biomarker levels may identify adenoma patients, who are at increased risk of being diagnosed with subsequent primary malignancy.
Forfattere: Piper TB, Jørgensen LN, Olsen J, Nielsen KT, Davis G, Johansen JS, Jarle Christensen I, Nielsen HJ
Clean Colorectum at Diagnostic Colonoscopy: Subsequent Detection of Extracolonic Malignancies by Plasma Protein Biomarkers?
Biomarkers in cancer (2018 )
p. 1179299X18776974 issn:1179-299X)
Most of the subjects undergoing diagnostic colonoscopy do not have neoplastic bowel lesions. Potentially, some of the symptoms may therefore be caused by extracolonic malignancy, and subjects with persisting symptoms may need subsequent examinations. Blood-based, cancer-associated biomarkers may aid in directing the examinations for other specific malignant diseases.
Forfattere: Wilhelmsen M, Christensen IJ, Jørgensen LN, Madsen MR, Vilandt J, Hillig T, Klærke M, Nielsen KT, Laurberg S, Gawel S, Yang X, Davis G, Heijboer AM, Martens F, Nielsen HJ
European journal of surgical oncology : the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology (2018 10)
Issue 10 p. 1539-1541 issn:1532-2157)
Implementation of population screening for colorectal cancer by direct colonoscopy or follow-up colonoscopy after a positive fecal blood test has challenged the overall capacity of bowel examinations. Certain countries are facing serious colonoscopy capacity constraints, which have led to waiting lists and long-time latency of follow-up examinations. Various options for improvement are considered, including increased cut-off values of the fecal blood tests. Results from major clinical studies of blood-based, cancer-associated biomarkers have led to focus, however, on a triage concept for improved selection to colonoscopy. The triage test may include subject age, concentration of hemoglobin in a feces test and a combination of certain blood-based cancer associated biomarkers. Recent results have indicated that triage may reduce the requirements for colonoscopy by around 30%. Such results may be advantageous for the capacity, the heath budgets and in particular, the subjects, who do not need an unnecessary, unpleasant and risk-associated bowel examination.
Forfattere: Mertz-Petersen M, Piper TB, Kleif J, Ferm L, Christensen IJ, Nielsen HJ,
Oncotarget (2018 Feb)
Issue 12 p. 10247-10258 issn:1949-2553)
The aim was to evaluate serum levels of circulating cell-free nucleosomes (ccfn) containing a variety of epigenetic signals including 5-methylcytosine DNA, histone modifications H3K9Me3, H3K9Ac, H3S10PO4, H3K36Me3, H4K20Me3, H4PanAc and pH2AX, nucleosome variant H2AZ and nucleosome adducts with HMGB1 and EZH2 as well as ccfn per se, in addition to develop and evaluate predictor models based on the above mentioned ccfn and including serum levels of carcinoembryonic antigen (CEA), in early detection of colorectal cancer (CRC). Blood-samples were collected from 4,105 individuals undergoing colonoscopy. Serum levels of ccfn and CEA were determined using enzyme-linked immunosorbent assays platforms. Individual assessment of levels of ccfn showed area under the receiver operating characteristic curve (AUCROC) = 0.525-0.576 in discrimination of individuals with CRC from individuals with non-malignant findings. Predictor models including ccfn containing 5-methylcytosine DNA, CEA, age and gender improved results (AUCROC = 0.736, sensitivity = 0.37 at specificity = 0.90). Further improvement was achieved in discrimination of individuals with CRC from individuals with clean colorectum (AUCROC = 0.840, sensitivity = 0.57 at specificity = 0.90). The levels of ccfn among patients with CRC appeared to be stage-independent. In conclusion, the performance of the developed predictor models is potentially promising in early detection of CRC.
Forfattere: Rasmussen L, Christensen IJ, Herzog M, Micallef J, Nielsen HJ,
Science translational medicine (2017 Aug)
Issue 403 issn:1946-6242)
Early detection and intervention are likely to be the most effective means for reducing morbidity and mortality of human cancer. However, development of methods for noninvasive detection of early-stage tumors has remained a challenge. We have developed an approach called targeted error correction sequencing (TEC-Seq) that allows ultrasensitive direct evaluation of sequence changes in circulating cell-free DNA using massively parallel sequencing. We have used this approach to examine 58 cancer-related genes encompassing 81 kb. Analysis of plasma from 44 healthy individuals identified genomic changes related to clonal hematopoiesis in 16% of asymptomatic individuals but no alterations in driver genes related to solid cancers. Evaluation of 200 patients with colorectal, breast, lung, or ovarian cancer detected somatic mutations in the plasma of 71, 59, 59, and 68%, respectively, of patients with stage I or II disease. Analyses of mutations in the circulation revealed high concordance with alterations in the tumors of these patients. In patients with resectable colorectal cancers, higher amounts of preoperative circulating tumor DNA were associated with disease recurrence and decreased overall survival. These analyses provide a broadly applicable approach for noninvasive detection of early-stage tumors that may be useful for screening and management of patients with cancer.
Forfattere: Phallen J, Sausen M, Adleff V, Leal A, Hruban C, White J, Anagnostou V, Fiksel J, Cristiano S, Papp E, Speir S, Reinert T, Orntoft MW, Woodward BD, Murphy D, Parpart-Li S, Riley D, Nesselbush M, Sengamalay N, Georgiadis A, Li QK, Madsen MR, Mortensen FV, Huiskens J, Punt C, van Grieken N, Fijneman R, Meijer G, Husain H, Scharpf RB, Diaz LA, Jones S, Angiuoli S, Ørntoft T, Nielsen HJ, Andersen CL, Velculescu VE
Discovery and validation of a colorectal cancer classifier in a new blood test with improved performance for high-risk subjects.
Clinical proteomics (2017 )
p. 28 issn:1542-6416)
The aim was to improve upon an existing blood-based colorectal cancer (CRC) test directed to high-risk symptomatic patients, by developing a new CRC classifier to be used with a new test embodiment. The new test uses a robust assay format-electrochemiluminescence immunoassays-to quantify protein concentrations. The aim was achieved by building and validating a CRC classifier using concentration measures from a large sample set representing a true intent-to-test (ITT) symptomatic population.
Forfattere: Croner LJ, Dillon R, Kao A, Kairs SN, Benz R, Christensen IJ, Nielsen HJ, Blume JE, Wilcox B
Clinical cancer research : an official journal of the American Association for Cancer Research (2017 Jun)
We investigated if detection of circulating tumor DNA (ctDNA) after resection of colorectal cancer (CRC) 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 three year follow-up period.
Results: A total of 45 patients and 371 plasma samples were included. Longitudinal samples from 27 patients revealed ctDNA post-operatively in all relapsing patients (n=14), but not in any of the non-relapsing patients. ctDNA detected relapse with an average lead-time of 9.4 months compared to CT imaging. Of 21 patients treated for localized disease, six had ctDNA detected within 3 months post-surgery. All six later relapsed compared to four of the remaining patients (Hazard ratio (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 (Kappa=0.41, Spearman’s rho=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 post-operative management of CRC patients.
Forfattere: Schøler LV, Reinert T, Ørntoft MW, Kassentoft CG, Árnadóttir SS, Vang S, Nordentoft I, Knudsen M, Lamy P, Andreasen D, Mortensen FV, Knudsen AR, Stribolt K, Sivesgaard K, Mouritzen P, Nielsen HJ, Laurberg S, Ørntoft TF, Andersen CL
Scandinavian journal of gastroenterology (2017 Mar)
p. 1-3 issn:1502-7708)
FIT-based colorectal cancer screening has been implemented in many countries including Denmark, where 916 colorectal cancer and 4468 high- or medium-risk adenoma patients were identified within April-December 2014, among 16,806 subjects with a positive FIT test. Screening increases the overall requirements for colonoscopy, which may challenge the current capacity. Some countries have increased their initial FIT cut-off level in order to comply with lack of colonoscopy capacity. Many patients with neoplasia will not be detected, however, by using increased FIT cut-off levels. The number of patients with neoplastic lesions missed by increased cut-off levels appears to be much higher than expected. Therefore, tests that identify those patients missed by increased FIT cut-off levels must be developed. Preliminary results of determination of one of several biomarker entities currently under investigation show that nucleosome blood tests may be one option for identifying some of these patients. Implementation of a triage test consisting of FIT, blood-based biomarkers and plus/minus colonoscopy is suggested to identify subjects with FIT levels between the initial and the increased cut-off level that must be offered colonoscopy. In addition, triage may reduce the frequency of unnecessary colonoscopies by 25%.
Forfattere: Nielsen HJ, Christensen IJ, Andersen B, Rasmussen M, Friis-Hansen LJ, Bygott T, MiCallef J,
Metagenomic analysis of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer.
Gut (2017 01)
Issue 1 p. 70-78 issn:1468-3288)
To evaluate the potential for diagnosing colorectal cancer (CRC) from faecal metagenomes.
Forfattere: Yu J, Feng Q, Wong SH, Zhang D, Liang QY, Qin Y, Tang L, Zhao H, Stenvang J, Li Y, Wang X, Xu X, Chen N, Wu WK, Al-Aama J, Nielsen HJ, Kiilerich P, Jensen BA, Yau TO, Lan Z, Jia H, Li J, Xiao L, Lam TY, Ng SC, Cheng AS, Wong VW, Chan FK, Xu X, Yang H, Madsen L, Datz C, Tilg H, Wang J, Brünner N, Kristiansen K, Arumugam M, Sung JJ, Wang J
Detection of colorectal neoplasia: Combination of eight blood-based, cancer-associated protein biomarkers.
International journal of cancer (2016 Dec)
Serological biomarkers may be an option for early detection of colorectal cancer (CRC). The present study assessed 8 cancer-associated protein biomarkers in plasma from subjects undergoing first time ever colonoscopy due to symptoms attributable to colorectal neoplasia. Plasma AFP, CA19-9, CEA, hs-CRP, CyFra21-1, Ferritin, Galectin-3, and TIMP-1 were determined in EDTA-plasma using the Abbott ARCHITECT® automated immunoassay platform. Primary endpoints were detection of: 1: CRC and high risk adenoma and 2: CRC. Logistic regression was performed. Final reduced models were constructed selecting the 4 biomarkers with the highest likelihood scores. Subjects (N=4,698) were consecutively included during 2010-2012. Colonoscopy detected 512 CRC patients, 319 colonic cancer (CC) and 193 rectal cancer (RC). Extra colonic malignancies were detected in 177 patients, 689 had adenomas of which 399 were high-risk, 1342 had non-neoplastic bowell disease, and 1978 subjects had “clean” colorectum. Univariable analysis demonstrated that all biomarkers were statistically significant. Multivariate logistic regression demonstrated that the blood-based biomarkers in combination significantly predicted the endpoints. The reduced model resulted in selection of CEA, hs-CRP, CyFra21-1 and Ferritin for the two endpoints; AUC’s were 0.76 and, 0.84, respectively. The postive predictive value (PPV) at 90% sensitivity was 25% for endpoint 1 and the negative predictive value (NPV) was 93%. For endpoint 2 the PPV was 18% and the NPV was 97%. Combinations of serological protein biomarkers provided a significant identification of subjects with high-risk of presence of colorectal neoplasia. The present set of biomarkers could become important adjunct in early detection of CRC. This article is protected by copyright. All rights reserved.
Forfattere: Wilhelmsen M, Christensen IJ, Rasmussen L, Jørgensen LN, Madsen MR, Vilandt J, Hillig T, Klaerke M, Nielsen KT, Laurberg S, Brünner N, Gawel S, Yang X, Davis G, Heijboer AM, Martens F, Nielsen HJ
Increased serological cancer-associated biomarkers and risk of development of primary malignancy after large bowel endoscopy.
Scandinavian journal of gastroenterology (2016 Oct)
Issue 10 p. 1272 issn:1502-7708)
Forfattere: Nielsen HJ
Pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA or histone modification H3K9Me3.
Scandinavian journal of clinical and laboratory investigation (2016 Oct)
Issue 6 p. 448-53 issn:1502-7686)
To evaluate pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA (5mC) or histone modification H3K9Me3 (H3K9Me3).
Forfattere: Rasmussen L, Herzog M, Rømer E, Micallef J, Bulut O, Wilhelmsen M, Christensen IJ, Nielsen HJ
Protocol Outlines for Parts 1 and 2 of the Prospective Endoscopy III Study for the Early Detection of Colorectal Cancer: Validation of a Concept Based on Blood Biomarkers.
JMIR research protocols (2016 )
Issue 3 p. e182 issn:1929-0748)
Programs for population screening of colorectal cancer (CRC) have been implemented in several countries with fecal immunochemical testing (FIT) as the preferred platform. However, the major obstacle for a feces-based testing method is the limited compliance that reduces the clinical sensitivity for detection of participants with non-symptomatic CRC. Therefore, research approaches have been initiated to develop screening concepts based on biomarkers in blood. Preliminary results show that protein, genetic, epigenetic, and metabolomic components may be valuable in blood-based screening concepts, particularly when combinations of the various components appear to lead to significant improvements.
Forfattere: Rasmussen L, Wilhelmsen M, Christensen IJ, Andersen J, Jørgensen LN, Rasmussen M, Hendel JW, Madsen MR, Vilandt J, Hillig T, Klærke M, Münster AM, Andersen LM, Andersen B, Hornung N, Erlandsen EJ, Khalid A, Nielsen HJ
miRNA profiling of circulating EpCAM(+) extracellular vesicles: promising biomarkers of colorectal cancer.
Journal of extracellular vesicles (2016 )
Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. These contain biomolecules, including proteins and microRNAs (miRNAs). Both circulating EVs and miRNAs have received much attention as biomarker candidates for non-invasive diagnostics. Here we describe a sensitive analytical method for isolation and subsequent miRNA profiling of epithelial-derived EVs from blood samples of patients with colorectal cancer (CRC). The epithelial-derived EVs were isolated by immunoaffinity-capture using the epithelial cell adhesion molecule (EpCAM) as marker. This approach mitigates some of the specificity issues observed in earlier studies of circulating miRNAs, in particular the negative influence of miRNAs released by erythrocytes, platelets and non-epithelial cells. By applying this method to 2 small-scale patient cohorts, we showed that blood plasma isolated from CRC patients prior to surgery contained elevated levels of 13 EpCAM(+)-EV miRNAs compared with healthy individuals. Upon surgical tumour removal, the plasma levels of 8 of these were reduced (miR-16-5p, miR-23a-3p, miR-23b-3p, miR-27a-3p, miR-27b-3p, miR-30b-5p, miR-30c-5p and miR-222-3p). These findings indicate that the miRNAs are of tumour origin and may have potential as non-invasive biomarkers for detection of CRC. This work describes a non-invasive blood-based method for sensitive detection of cancer with potential for clinical use in relation to diagnosis and screening. We used the method to study CRC; however, it is not restricted to this disease. It may in principle be used to study any cancer that release epithelial-derived EVs into circulation.
Forfattere: Ostenfeld MS, Jensen SG, Jeppesen DK, Christensen LL, Thorsen SB, Stenvang J, Hvam ML, Thomsen A, Mouritzen P, Rasmussen MH, Nielsen HJ, Ørntoft TF, Andersen CL
Localization of active, dually phosphorylated extracellular signal-regulated kinase 1 and 2 in colorectal cancer with or without activating BRAF and KRAS mutations.
Human pathology (2016 Aug)
p. 37-46 issn:1532-8392)
Colorectal cancers (CRC) often show activating mutations of the KRAS or BRAF genes, which stimulate the extracellular signal-regulated kinase (ERK) pathway, thus increasing cell proliferation and inhibiting apoptosis. However, immunohistochemical results on ERK activation in such tumors differ greatly. Recently, using a highly optimized immunohistochemical method, we obtained evidence that high levels of ERK activation in rectal adenocarcinomas were associated with resistance to radiochemotherapy. In order to determine whether KRAS and/or BRAF mutations correlate to immunohistochemically detectable increases in phosphorylation of ERK (pERK), we stained biopsies from 36 CRC patients with activating mutations in the BRAF gene (BRAFV600E: BRAF(m)), the KRAS gene (KRAS(m)) or in neither (BRAF/KRAS(n)) with this optimized method. Staining was scored in blind-coded specimens by two observers. Staining of stromal cells was used as a positive control. BRAF(m) or KRAS(m) tumors did not show higher staining scores than BRAF/KRAS(n) tumors. Although BRAFV600E staining occurred in over 90% of cancer cells in all 9 BRAF(m) tumors, 3 only showed staining for pERK in less than 10% of cancer cell nuclei. The same applied to 4 of the 14 KRAS(m) tumors. A phophorylation-insensitive antibody demonstrated that lack of pERK staining did not reflect defect expression of ERK1/2 protein. Thus, increased staining for pERK does not correlate to BRAF or KRAS mutations even with a highly optimized procedure. Further studies are required to determine whether this reflects differences in expression of counterregulatory molecules, including ERK phosphatases.
Forfattere: Holck S, Bonde J, Pedersen H, Petersen AA, Chaube A, Nielsen HJ, Larsson LI
TIMP-1 is under regulation of the EGF signaling axis and promotes an aggressive phenotype in KRAS-mutated colorectal cancer cells: A potential novel approach to the treatment of metastatic colorectal cancer.
Oncotarget (2016 Aug)
It is now widely accepted that therapeutic antibodies targeting epidermal growth factor receptor (EGFR) can have efficacy in KRAS wild-type advanced colorectal cancer (CRC) patients. What remains to be ascertained is whether a subgroup of KRAS-mutated CRC patients might not also derive benefit from EGFR inhibitors. Metalloproteinase inhibitor 1 (TIMP-1) is a pleiotropic factor predictive of survival outcome of CRC patients. Levels of TIMP-1 were measured in pre-treatment plasma samples (n = 426) of metastatic CRC patients randomized to Nordic FLOX (5-fluorouracil and oxaliplatin) +/- cetuximab (NORDIC VII study). Multivariate analysis demonstrated a significant interaction between plasma TIMP-1 protein levels, KRAS status and treatment with patients bearing KRAS mutated tumors and high TIMP-1 plasma level (> 3rd quartile) showing a significantly longer overall survival if treated with cetuximab (HR, 0.48; 95% CI, 0.25 to 0.93). To gain mechanistic insights into this association we analyzed a set of five different CRC cell lines. We show here that EGFR signaling induces TIMP-1 expression in CRC cells, and that TIMP-1 promotes a more aggressive behavior, specifically in KRAS mutated cells. The two sets of data, clinical and in vitro, are complementary and support each other, lending strength to our contention that TIMP- 1 plasma levels can identify a subset of patients with KRAS-mutated metastatic CRC that will have benefit from EGFR-inhibition therapy.
Forfattere: Tarpgaard LS, Ørum-Madsen MS, Christensen IJ, Nordgaard C, Noer J, Guren TK, Glimelius B, Sorbye H, Ikdahl T, Kure EH, Tveit KM, Nielsen HJ, Pfeiffer P, Brünner N, Moreira JM
Increased serological cancer-associated biomarker levels at large bowel endoscopy and risk of subsequent primary cancer (†).
Scandinavian journal of gastroenterology (2016 Jul)
Issue 7 p. 860-5 issn:1502-7708)
Frequently, subjects offered colonoscopy due to symptoms of colorectal neoplasia are diagnosed with diverticula. The symptoms may, however, also be related to extra-colonic neoplasia. The present retrospective study evaluated a possible association between increased levels of predefined biomarkers in subjects diagnosed with diverticula and risk of developing a primary malignant disease.
Forfattere: Hvolris MH, Piper TB, Hammer E, Jørgensen LN, Olsen J, Rahr HB, Nielsen KT, Laurberg S, Christensen IJ, Brünner N, Johansen JS, Davis GJ, Dowell BL, Nielsen HJ
Journal of cellular and molecular medicine (2015 Dec)
Issue 12 p. 2865-73 issn:1582-4934)
Olfactomedin 4 (OLFM4) is a secreted glycoprotein predominantly expressed in bone marrow and gastrointestinal tissues. Aberrant expression of OLFM4 has been shown in several cancers. However, the clinical significance hereof is currently controversial. OLFM4 has been proposed as a candidate biomarker of gastrointestinal cancers. To address this, we developed monoclonal antibodies against synthetic peptides representing various segments of OLFM4. We examined expression of OLFM4 in epithelial cells by immunohistochemistry and found that OLFM4 is highly expressed in proliferating benign epithelial cells and in some carcinoma cells. We developed an Enzyme Linked Immunosorbent Assay for OLFM4 and investigated whether plasma levels of OLFM4 reflect colorectal malignancies, but were unable to see any such association. Instead, we observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 ?g/ml, mean 0.028 ?g/ml while 10% of both normals and patients with cancers had OLFM4 between 4 and 60 ?g/ml, mean 15 ?g/ml. The levels were constant over time. The background for this high plasma level is not known, but must be taken into account if OLFM4 is used as biomarker for GI cancers.
Forfattere: Clemmensen SN, Glenthøj AJ, Heebøll S, Nielsen HJ, Koch C, Borregaard N
The prognostic and predictive value of soluble of type IV collagen in colorectal cancer: A retrospective multicenter study.
Clinical cancer research : an official journal of the American Association for Cancer Research (2015 Dec)
To investigate the prognostic and predictive biomarker value of type IV collagen in colorectal cancer (CRC).
Forfattere: Rolff HC, Christensen IJ, Vainer B, Svendsen LB, Loevendahl Eefsen R, Wilhelmsen M, Lund IK, Hoyer-Hansen G, Nielsen HJ, Illemann M
Intact and cleaved plasma soluble urokinase receptor in patients with metastatic colorectal cancer treated with oxaliplatin with or without cetuximab.
International journal of cancer. Journal international du cancer (2015 Feb)
Circulating forms of the urokinase plasminogen activator receptor (uPAR) are associated with prognosis in patients with colorectal cancer. Preclinical studies have shown that uPAR can influence the state of phosphorylation and signalling activity of the epidermal growth factor receptor (EGFR) in a ligand-independent manner. The purpose of the study was to evaluate whether plasma soluble intact and cleaved uPAR(I-III)+(II-III) levels could identify a subpopulation of patients with metastatic colorectal cancer (mCRC) where treatment with cetuximab would have a beneficial effect. Plasma samples were available from 453 patients treated in the NORDIC VII study. Patients were randomized between FLOX and FLOX?+?cetuximab. The levels of uPAR(I-III)+(II-III) were determined by time-resolved fluorescence immunoassay. We demonstrated that higher baseline plasma uPAR(I-III)+(II-III) levels were significantly associated with shorter progression-free survival (PFS) (HR?=?1.30, 1.14-1.48, p?=?0.0001) and overall survival (OS) (HR?=?1.75, 1.52-2.02, p?<?0.0001). Multivariate Cox analysis showed that plasma uPAR(I-III)+(II-III) was an independent biomarker of short OS (HR?=?1.45, 1.20-1.75, p?=?0.0001). There were no significant interactions between plasma uPAR(I-III)+(II-III) levels, KRAS mutational status and treatment either PFS (p?=?0.43) or OS (p?=?0.095). However, further explorative analyses indicated that patients with low levels of circulating suPAR and a KRAS wild-type tumor have improved effect from treatment with FLOX?+?cetuximab as compared to patients with KRAS wild-type and high levels of suPAR. These results thus support the preclinical findings and should be further tested in an independent clinical data set.
Forfattere: Tarpgaard LS, Christensen IJ, Høyer-Hansen G, Lund IK, Guren TK, Glimelius B, Sorbye H, Tveit KM, Nielsen HJ, Moreira JM, Pfeiffer P, Brünner N
Performance of the colorectal cancer screening marker Sept9 is influenced by age, diabetes and arthritis: a nested case-control study.
BMC cancer (2015 Oct)
p. 819 issn:1471-2407)
Annually, colorectal cancer (CRC) is diagnosed in >1.4 million subjects worldwide and incidence is increasing. Much effort has therefore been focused on screening, which has proven to reduce cancer-related mortality. The Sept9 DNA-methylation assay is among the most well studied blood-based screening markers. However, earlier reported performances may be misleading: the Sept9 test was recently examined in two screening based cohorts and yielded performances lower than expected. We hypothesize that comorbidities and/or demographic characteristics affect the results of the Sept9 test.
Forfattere: Ørntoft MB, Nielsen HJ, Ørntoft TF, Andersen CL,
Phospho-ERK1/2 levels in cancer cell nuclei predict responsiveness to radiochemotherapy of rectal adenocarcinoma.
Oncotarget (2015 Oct)
Issue 33 p. 34321-8 issn:1949-2553)
Locally advanced rectal adenocarcinoma is treated with radiochemotherapy (RCT) before surgery. The response to RCT is heterogeneous and consensus regarding reliable predictors is lacking. Since the ERK pathway is implicated in radioprotection, we examined pretreatment biopsies from 52 patients by immunohistochemistry for phosphorylated ERK (pERK). Immunostaining for pERK was considerably enhanced by use of alkaline demasking. Nuclear staining occurred in both cancer cells and stromal cells. Blind-coded sections were scored by 2 independent investigators. In patients showing no residual tumor after RCT (TRG1), staining for pERK in cancer, but not stromal, cell nuclei was significantly weaker than in patients showing a poor RCT response (TRG1 vs TRG4: p = 0.0001). Nuclear staining for pERK predicted poor responders, as illustrated by receiver operating characteristic curves with an area under curve of 0.86 (p = 0.0007) and also predicted downstaging (area under curve: 0.76; p = 0.01). A number of controls documented the specificity of the optimized staining method and results were confirmed with another pERK antibody. Thus, staining for pERK in cancer cell nuclei can predict the response to RCT and may help spare poor responders this treatment. These results also raise the question whether inhibitors of ERK activation may serve as response modifiers of RCT.
Forfattere: Holck S, Nielsen HJ, Pedersen N, Larsson LI
Biomarkers in cancer (2015 )
Soluble cancer-related protein biomarker levels may be increased in subjects without findings at large bowel endoscopy performed due to symptoms associated with colorectal cancer. The present study focused on a possible association between increased biomarker levels in such subjects and subsequent development of malignant diseases. In a major study of 4,990 subjects undergoing large bowel endoscopy, 691 were without pathology and comorbidity. Plasma levels of TIMP-1, CEA, CA19-9, and YKL-40 were determined in samples collected just before endoscopy and compared with subsequent development of a malignant disease within a period of 7-8 years. The upper 90% limits of the reference levels of every single protein were used to differentiate between normal and increased levels. The levels were separated into three groups: 0, none of the biomarkers increased; 1, one biomarker increased; 2, two or more biomarkers increased. A total of 43 subjects developed a primary malignant disease in the observation period. Univariatly, increase of all four biomarkers was significantly associated with subsequent development of a malignant disease. A multivariate analysis showed that increased biomarker levels were associated with subsequent development of a malignant disease (P = 0.002). The cumulative risk of developing malignant disease within the first 5 years after endoscopy was group 0, 3.3%; group 1, 5.8%; group 2, 7.8%. It is concluded that increased levels of plasma TIMP-1, CEA, CA19-9, and serum YKL-40 at large bowel endoscopy without findings may be associated with an increased risk of developing a subsequent malignant disease.
Forfattere: Kring TS, Piper TB, Jørgensen LN, Olsen J, Rahr HB, Nielsen KT, Laurberg S, Davis G, Dowell B, Johansen JS, Christensen IJ, Brünner N, Nielsen HJ
TIMP-1 and CEA as biomarkers in third-line treatment with irinotecan and cetuximab for metastatic colorectal cancer.
Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine (2015 Jan)
KRAS wild-type (wt) status determines indication for treatment with combination therapy, including epidermal growth factor receptor (EGFR) inhibitors, but still, the overall response rate in KRAS wt patients is less than 40 %. Consequently, the majority of patients will suffer from substantial side effects and no apparent benefit. Tissue inhibitor of metalloproteinases-1 is a glycoprotein, which regulates metalloproteinases and may consequently imply a central role in tumour progression. Furthermore, it is closely related to the EGFR regulation and has shown promising potential as a biomarker in colorectal cancer (CRC). The aim of the present study was to investigate the clinical value of TIMP-1 in patients with metastatic colorectal cancer (mCRC) treated with cetuximab and irinotecan. Patients with chemotherapy-resistant mCRC referred to third-line treatment with cetuximab (initial 400 mg/m(2) followed by weekly 250 mg/m(2))/irinotecan (350 mg/m(2) q3w) were prospectively included in the biomarker study, as previously published. Pre-treatment blood samples were collected, and plasma TIMP-1 was measured by a validated in-house ELISA assay. In addition, carcinoembryonic antigen (CEA) measurement was performed with a standardised method. A total of 107 patients were included in the biomarker study. The median baseline plasma TIMP-1 level was 271.1 ng/ml (range 65.9-1432 ng/ml) with no significant associations with baseline clinical characteristics. Median baseline plasma TIMP-1 levels were significantly higher in patients with early progression compared to patients who achieved disease control, 349 ng/ml (233-398 95 % confidence interval (CI)) and 215 ng/ml (155-289 95 % CI), respectively, p?=?0.03, suggesting some association with treatment efficacy. When dividing patients according to TIMP-1 tertiles, the median progression-free survival (PFS) in patients with a high level of TIMP-1 was 2.4 months (95 % CI 2.1-4.1) compared to 3.3 months (95 % CI 2.1-6.2) and 4.7 months (95 % 3.2-7.6) in patients with intermediate or low levels, respectively. Analysis of TIMP-1 as a continuous variable revealed a shorter PFS associated with increasing levels of TIMP-1 (hazard ratio (HR) 1.36). These results translated into a significantly lower overall survival (OS) in patients with a high baseline TIMP-1 level (4.5 months (95 % CI 3.4-5.4)), compared to those with intermediate or low TIMP-1 levels (7.8 months (95 % CI 4.4-13.7) and 12.0 months (95 % CI 10.1-14.3), respectively, p?<?0.0001). An 83 % higher hazard for death was revealed (HR?=?1.83) with each twofold increase in the TIMP-1 level. Pre-treatment levels of CEA were not associated with any of the baseline characteristics (except primary tumour localisation) or to differences in PFS or OS. The rank correlation between CEA and TIMP-1 was r?=?0.50, and a test for interaction between TIMP-1 and CEA (dichotomised at 5 ng/ml) in survival analysis was not significant (p?=?0.18). A multivariate analysis for PFS and OS resulted in a model with significant contributions from TIMP-1, KRAS, and the number of metastatic sites. We have confirmed the potential prognostic value of TIMP-1 measurement prior to palliative chemotherapy for mCRC. However, validation in randomised trials will be essential with the perspective of establishing a potential predictive role of plasma TIMP-1 in this setting.
Forfattere: Spindler KL, Christensen IJ, Nielsen HJ, Jakobsen A, Brünner N
Topoisomerase-1 and -2A gene copy numbers are elevated in mismatch repair-proficient colorectal cancers.
Molecular oncology (2015 Mar)
Topoisomerase 1 (TOP1) and 2A (TOP2A) are potential predictive biomarkers for irinotecan and anthracycline treatment, respectively, in colorectal cancer (CRC), and we have recently reported a high frequency of gene gain of the TOP1 and TOP2A genes in CRC. Furthermore, Mismatch Repair (MMR) subtypes of CRC have been associated with benefit from adjuvant chemotherapy of primary CRC. Given the involvement of the topoisomerase enzymes in DNA replication and repair, we raised the hypothesis that an association may exist between TOP gene copy numbers and MMR proficiency/deficiency in CRC.
Forfattere: Sønderstrup IM, Nygård SB, Poulsen TS, Linnemann D, Stenvang J, Nielsen HJ, Bartek J, Brünner N, Nørgaard P, Riis L
Serum YKL-40 in Risk Assessment for Colorectal Cancer: A Prospective Study of 4,496 Subjects at Risk of Colorectal Cancer.
Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology (2015 Mar)
issue 3 p. 621-6 (issn:1538-7755)
The aim of the present study was to test the hypothesis that high serum YKL-40 associates with colorectal cancer in subjects at risk of colorectal cancer. We measured serum YKL-40 in a prospective study of 4,496 Danish subjects [2,064 men, 2,432 women, median age 61 years (range, 18-97)] referred to endoscopy due to symptoms or other risk factors for colorectal cancer. Blood samples were collected just before large bowel endoscopy. Serum YKL-40 was determined by ELISA. Serum YKL-40 was higher (P < 0.0001, unadjusted for confounding covariates) in subjects diagnosed with colon cancer (median 126 ?g/L, 25%-75%: 80-206 ?g/L) and rectal cancer (104, 72-204 ?g/L) compared with subjects with adenoma (84, 53-154 ?g/L), other nonmalignant findings (79, 49-138 ?g/L), and no findings (62, 41-109 ?g/L). Serum YKL-40 independently predicted colorectal cancer [OR, 1.53; 95% confidence interval (CI), 1.40-1.67; AUC = 0.68, P < 0.0001]. Restricting the analysis to subjects with no comorbidity increased the OR for serum YKL-40 to predict colorectal cancer (OR, 1.82; 1.58-2.08; AUC = 0.73, P < 0.0001). Combining serum YKL-40 and CEA demonstrated that both were significant [(YKL-40, OR, 1.27; 95% CI, 1.16-1.40); (CEA, OR, 1.92; 1.75-2.10; AUC = 0.75, P < 0.0001; OR for a 2-fold difference in marker level)]. Multivariable analysis (YKL-40, CEA, age, gender, body mass index, and center) showed that serum YKL-40 was a predictor for colorectal cancer in individuals without comorbidity (OR, 1.25; 95% CI, 1.05-1.40; P = 0.012), whereas this was not the case for those with comorbidity (OR, 0.98; 95% CI, 0.84-1.14; P = 0.80). In conclusion, high serum YKL-40 in subjects suspected of colorectal cancer and without comorbidity associates with colorectal cancer. Determination of serum YKL-40 may be useful in combination with other biomarkers in risk assessment for colorectal cancer. Cancer Epidemiol Biomarkers Prev; 24(3); 621-6. ©2015 AACR.
Forfattere: Johansen JS, Christensen IJ, Jørgensen LN, Olsen J, Rahr HB, Nielsen KT, Laurberg S, Brünner N, Nielsen HJ
Frontiers in microbiology (2015 )
p. 20 issn:1664-302X)
The dysbiosis of the human intestinal microbiota is linked to sporadic colorectal carcinoma (CRC). The present study was designed to investigate the gut microbiota distribution features in CRC patients. We performed pyrosequencing based analysis of the 16S rRNA gene V3 region to investigate microbiota of the cancerous tissue and adjacent non-cancerous normal tissue in proximal and distal CRC samples. The results revealed that the microbial structures of the CRC patients and healthy individuals differed significantly. Firmicutes and Fusobacteria were over-represented whereas Proteobacteria was under-represented in CRC patients. In addition, Lactococcus and Fusobacterium exhibited a relatively higher abundance while Pseudomonas and Escherichia-Shigella was reduced in cancerous tissues compared to adjacent non-cancerous tissues. Meanwhile, the overall microbial structures of proximal and distal colon cancerous tissues were similar; but certain potential pro-oncogenic pathogens were different. These results suggested that the mucosa-associated microbiota is dynamically associated with CRC, which may provide evidences for microbiota-associated diagnostic, prognostic, preventive, and therapeutic strategies for CRC.
Forfattere: Gao Z, Guo B, Gao R, Zhu Q, Qi
Gut (2015 Feb)
To develop an affordable and robust pipeline for selection of patient-specific somatic structural variants (SSVs) being informative about radicality of the primary resection, response to adjuvant therapy, incipient recurrence and response to treatment performed in relation to diagnosis of recurrence.
Forfattere: Reinert T, Schøler LV, Thomsen R, Tobiasen H, Vang S, Nordentoft I, Lamy P, Kannerup AS, Mortensen FV, Stribolt K, Hamilton-Dutoit S, Nielsen HJ, Laurberg S, Pallisgaard N, Pedersen JS, Ørntoft TF, Andersen CL
IQGAP1 in rectal adenocarcinomas: localization and protein expression before and after radiochemotherapy.
Cancer letters (2015 Jan)
issue 2 Pt B p. 556-60 (issn:1872-7980)
Treatment of rectal adenocarcinoma includes total mesorectal excision, which is preceded by radiochemotherapy (RCT) in cases of advanced disease. The response to RCT varies from total tumor regression to no effect but this heterogeneous response is unexplained. However, both radiation and treatment with 5-fluorouracil may induce treatment resistance through upregulation of the mitogen-activated protein kinase (MAPK) cascade. IQGAP1 is a scaffold protein that appears to be essential to MAPK signaling in cancers. We have therefore studied IQGAP1 protein expression in rectal adenocarcinomas before and after RCT. We demonstrate that cancer cells show increased apical staining for IQGAP1 following RCT. Interestingly, this increase is significantly higher in patients showing poor RCT responses. Our results also suggest that low levels of apical IQGAP1-staining in biopsies may predict the RCT response. Together, these data suggest that both the level and localization of IQGAP1 may influence the treatment response.
Forfattere: Holck S, Nielsen HJ, Hammer E, Christensen IJ, Larsson LI
Cancer immunology, immunotherapy : CII (2015 Jan)
Issue 1 p. 41-50 issn:1432-0851)
Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Lack of symptoms results in late detection and increased mortality. Inflammation, including complement activation, plays an important role in tumorigenesis.
Forfattere: Storm L, Christensen IJ, Jensenius JC, Nielsen HJ, Thiel S,
Circulating intact and cleaved forms of the urokinase-type plasminogen activator receptor: biological variation, reference intervals and clinical useful cut-points.
Clinica chimica acta; international journal of clinical chemistry (2015 Jan)
p. 84-90 issn:1873-3492)
High levels of circulating forms of the urokinase-type plasminogen activator receptor (uPAR) are significantly associated to poor prognosis in cancer patients. Our aim was to determine biological variations and reference intervals of the uPAR forms in blood, and in addition, to test the clinical relevance of using these as cut-points in colorectal cancer (CRC) prognosis.
Forfattere: Thurison T, Christensen IJ, Lund IK, Nielsen HJ, Høyer-Hansen G
Scandinavian journal of gastroenterology (2014 Dec)
Issue 12 p. 1399-408 issn:1502-7708)
Despite intended curative resection, colorectal cancer will recur in ?45% of the patients. Results of meta-analyses conclude that frequent follow-up does not lead to early detection of recurrence, but improves overall survival. The present literature shows that several factors play important roles in development of recurrence. It is well established that emergency surgery is a major determinant of recurrence. Moreover, anastomotic leakages, postoperative bacterial infections, and blood transfusions increase the recurrence rates although the exact mechanisms still remain obscure. From pathology studies it has been shown that tumors behave differently depending on their location and recur more often when micrometastases are present in lymph nodes and around vessels and nerves. K-ras mutations, microsatellite instability, and mismatch repair genes have also been shown to be important in relation with recurrences, and tumors appear to have different mutations depending on their location. Patients with stage II or III disease are often treated with adjuvant chemotherapy despite the fact that the treatments are far from efficient among all patients, who are at risk of recurrence. Studies are now being presented identifying subgroups, in which the therapy is inefficient. Unfortunately, only few of these facts are implemented in the present follow-up programs. Therefore, further research is urgently needed to verify which of the well-known parameters as well as new parameters that must be added to the current follow-up programs to identify patients at risk of recurrence.
Forfattere: Wilhelmsen M, Kring T, Jorgensen LN, Madsen MR, Jess P, Bulut O, Nielsen KT, Andersen CL, Nielsen HJ
Scandinavian journal of gastroenterology (2014 Feb)
Issue 2 p. 191-201 issn:1502-7708)
Optimal management of colon cancer (CC) requires detailed assessment of extent of disease. This study prospectively investigates the diagnostic accuracy of 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography/computed tomography (PET/CT) for staging and detection of recurrence in primary CC.
Forfattere: Engelmann BE, Loft A, Kjær A, Nielsen HJ, Berthelsen AK, Binderup T, Brinch K, Brünner N, Gerds TA, Høyer-Hansen G, Kristensen MH, Kurt EY, Latocha JE, Lindblom G, Sloth C, Højgaard L
Urokinase-type plasminogen activator receptor (uPAR) on tumor-associated macrophages is a marker of poor prognosis in colorectal cancer.
Cancer medicine (2014 Aug)
Issue 4 p. 855-64 issn:2045-7634)
Patients were identified from a population-based prospective study of 4990 individuals with symptoms associated with colorectal cancer (CRC). A total of 244 CRC tissue samples were available for immunohistochemical staining of uPAR, semiquantitatively scored at the invasive front, and in the tumor core on cancer cells, macrophages, and myofibroblasts. In addition, the levels of the intact and cleaved uPAR-forms in blood from the same patients are evaluated in this study. In a univariate analysis, the number of uPAR-positive versus uPAR-negative macrophages (HR = 2.26, [95% CI: 1.39-3.66, P = 0.0009]) and cancer cells (HR=1.49, [95% CI: 1.01-2.20, P = 0.047]) located in the tumor core were significantly associated to overall survival. In a multivariate analysis, uPAR-positive versus uPAR-negative macrophages located in the tumor core showed the best separation of patients with positive score associated to poor prognosis (HR = 1.84 [95% CI: 1.12-3.04, P = 0.017]). In a multivariate analysis including clinical covariates and soluble uPAR(I), the latter was significantly associated to overall survival (HR = 2.68 [95% CI: 1.90-3.79, P < 0.0001]) and uPAR-positive macrophages in the tumor core remained significantly associated to overall survival (HR = 1.81 [95% CI: 1.08-3.01, P = 0.023]). Membrane-bound uPAR showed additive effects with the circulating uPAR(I) and stage, giving a hazard ratio of 12 between low and high scores. Thus, combining stage, uPAR(I) in blood and uPAR on macrophages in the tumor core increase the prognostic precision more than tenfold, as compared to stage alone.
Forfattere: Illemann M, Laerum OD, Hasselby JP, Thurison T, Høyer-Hansen G, Nielsen HJ, , Christensen IJ
BMC cancer (2014 )
p. 259 issn:1471-2407)
Cathepsin X is a cysteine protease involved in mechanisms of malignant progression. It is secreted from tumour cells as a proenzyme and may serve to predict the disease status and risk of death for cancer patients. In a previous, pilot, study on 77 colorectal patients we demonstrated the correlation of higher serum levels with shorter overall survival.
Forfattere: Vižin T, Christensen IJ, Wilhelmsen M, Nielsen HJ, Kos J
Ugeskrift for laeger (2014 Mar)
Issue 11B issn:1603-6824)
Screening programmes for colorectal cancer (CRC) are being implemented in various countries worldwide including Denmark. The majority of programmes rely on faecal occult blood testing with subsequent colonoscopy. This approach is challenged by limited compliance, which reduces the efficiency of the screening programme. Current research into improve-ments of screening of CRC includes biological markers identified in blood. Combining blood-based biological markers with clinical and demographical parameters have shown promising results, which may improve the present approach to screening.
Forfattere: Rasmussen L, Jørgensen LN, Madsen MR, Vilandt J, Klærke M, Andersen J, Nielsen KT, Khalid A, Laurberg S, Andersen CL, Christensen IJ, Brünner N, Nielsen HJ
Scientific reports (2014 )
p. 4313 issn:2045-2322)
Platinum chemotherapy remains part of standard therapies in the management of a variety of cancers. Severe side effects and a high degree of resistance to platinum drugs have led numerous researchers to search for predictive biomarkers, which could aid in identifying patients that are the most likely to respond to therapy. The ERCC1-ERCC4 endonuclease plays a critical role in the repair of platinum-DNA damage and has widely been studied in relation to sensitivity to platinum chemotherapy. The standard method to evaluate ERCC1 protein expression is through the use of immunohistochemistry with monoclonal antibody 8F1, an antibody that was recently found to bind an unrelated protein. The present study determines the specificity of a novel antibody, monoclonal antibody 4F9, and presents a method to evaluate ERCC1 expression in colorectal tumor specimens. Using relevant cell lines as controls, the specificity of antibody 4F9 was tested by immunoblotting, immunohistochemistry and immunofluorescence. Scoring guidelines to aid in the evaluation of ERCC1 tumor expression were developed and evaluated in archival formalin-fixed paraffin embedded colorectal cancer specimens. Antibody 4F9 was found to be specific by all methods applied and it was possible to evaluate the ERCC1 expression in the majority (85%) of colorectal cancer tumor specimens.
Forfattere: Smith DH, Fiehn AM, Fogh L, Christensen IJ, Hansen TP, Stenvang J, Nielsen HJ, Nielsen KV, Hasselby JP, Brünner N, Jensen SS
Positron emission tomography/computed tomography and biomarkers for early treatment response evaluation in metastatic colon cancer.
The oncologist (2014 Feb)
Issue 2 p. 164-72 issn:1549-490X)
Treatment options for metastatic colon cancer (mCC) are widening. We prospectively evaluated serial 2-deoxy-2-[18F]fluoro-d-glucose positron-emission tomography/computed tomography (PET/CT) and measurements of tissue inhibitor of metalloproteinases-1 (TIMP-1), carcinoembryonic antigen (CEA), and liberated domain I of urokinase plasminogen activator receptor (uPAR(I)) for early assessment of treatment response in mCC patients.
Forfattere: Engelmann BE, Loft A, Kjær A, Nielsen HJ, Gerds TA, Benzon EV, Brünner N, Christensen IJ, Hansson SH, Holländer NH, Kristensen MH, Löfgren J, Markova E, Sloth C, Højgaard L
Assessment of the topoisomerase I gene copy number as a predictive biomarker of objective response to irinotecan in metastatic colorectal cancer.
Scandinavian journal of gastroenterology (2014 Jan)
issue 1 p. 84-91 (issn:1502-7708)
DNA topoisomerase I is a putative biomarker of irinotecan efficacy with clinical associations previously demonstrated at the protein level. The purpose of the present study was to perform the first clinical investigation of the association between the DNA topoisomerase I gene (TOP1) copy number and objective response following irinotecan treatment in patients with metastatic colorectal cancer.
Forfattere: Nygård SB, Christensen IJ, Nielsen SL, Nielsen HJ, Brünner N, Spindler KL
Underpinning the repurposing of anthracyclines towards colorectal cancer: assessment of topoisomerase II alpha gene copy number alterations in colorectal cancer.
Scandinavian journal of gastroenterology (2013 Dec)
Issue 12 p. 1436-43 issn:1502-7708)
OBJECTIVE. We propose a repurposing strategy where anthracyclines are reintroduced to a subgroup of patients with metastatic colorectal cancer with the highest likelihood of response. In breast cancer, DNA topoisomerase II alpha gene (TOP2A) alterations predict incremental benefit of anthracyclines, but this association has not been investigated in colorectal cancer. Frequency analysis of TOP2A gene alterations in colorectal cancer and the association with prognosis are evaluated and the challenges of using a TOP2A/CEN-17 FISH probe combination are addressed. MATERIAL AND METHODS. Formalin-fixed, paraffin-embedded material from 154 stage III colorectal cancer patients included in the RANX05 clinical trial was retrospectively assessed for TOP2A gene alterations using FISH. The TOP2A/CEN-17 ratio as well as the TOP2A gene copy number alone was used to define gene alterations and associations between gene status and outcomes were analyzed. RESULTS. TOP2A gene gain was a frequent finding with 9.8 % having a total of ?4 TOP2A copies per cell. According to the TOP2A/CEN-17 ratio, 10.5 % had TOP2A gene gain. Polysomy or gain of the centromere region of chromosome-17 was not as frequent as reported in breast cancer. No prognostic characteristic of TOP2A was identified. CONCLUSION. TOP2A gene gain is present in numbers relevant to identify a subgroup of patients who may benefit from anthracycline therapy. Based on the present findings, we will initiate a prospective clinical trial designed to evaluate this hypothesis in patients with metastatic colorectal cancer who have failed 5-fluorouracil and oxaliplatin chemotherapy.
Forfattere: Nygård SB, Christensen IJ, Smith DH, Nielsen SL, Jensen NF, Nielsen HJ, Vainer B, Brünner N
An explorative analysis of ERCC1-19q13 copy number aberrations in a chemonaive stage III colorectal cancer cohort.
BMC cancer (2013 )
p. 489 issn:1471-2407)
Platinum-based chemotherapy has long been used in the treatment of a variety of cancers and functions by inducing DNA damage. ERCC1 and ERCC4 are involved in the removal of this damage and have previously been implicated in resistance to platinum compounds. The aim of the current investigation is to determine the presence, frequency and prognostic impact of ERCC1 or ERCC4 gene copy number alterations in colorectal cancer (CRC).
Forfattere: Smith DH, Christensen IJ, Jensen NF, Markussen B, Müller S, Nielsen HJ, Brünner N, Nielsen KV
Detection of serological biomarkers by proximity extension assay for detection of colorectal neoplasias in symptomatic individuals.
Journal of translational medicine (2013 )
p. 253 issn:1479-5876)
Although the potential of biomarkers to aid in early detection of colorectal cancer (CRC) is recognized and numerous biomarker candidates have been reported in the literature, to date only few molecular markers have been approved for daily clinical use.
Forfattere: Thorsen SB, Lundberg M, Villablanca A, Christensen SL, Belling KC, Nielsen BS, Knowles M, Gee N, Nielsen HJ, Brünner N, Christensen IJ, Fredriksson S, Stenvang J, Assarsson E
PloS one (2013 )
Issue 9 p. e73317 issn:1932-6203)
MASP-1 is the first protein in the activation of the lectin pathway and MASP-1 is, like its isoforms MASP-3 and MAp44, encoded by the MASP1 gene. Our aim was to explore associations between polymorphisms in MASP1 and corresponding concentrations of MASP-1, MASP-3, and MAp44 in plasma as well as the genetic contribution to the equilibrium between the three proteins.
Forfattere: Ammitzbøll CG, Steffensen R, Jørgen Nielsen H, Thiel S, Stengaard-Pedersen K, Bøgsted M, Jensenius JC
The Journal of biological chemistry (2013 Aug)
Issue 32 p. 23407-20 issn:1083-351X)
Collectins are pattern recognition molecules of the innate immune system showing binding to carbohydrate structures on microorganisms in a calcium-dependent manner. Recently, three novel collectins, collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1 and CL-11), and collectin placenta 1 (CL-P1), were discovered. The roles of these three collectins remain largely unknown. Here, we present a time-resolved immunofluorometric assay for quantification of CL-L1. The concentration of CL-L1 in donor plasma (n = 210) was distributed log-normally with a median value of 3.0 ?g/ml (range 1.5-5.5 ?g/ml). We observed on average 30% higher concentrations of CL-L1 in plasma as compared with serum. Size analysis by gel-permeation chromatography showed CL-L1 in serum to elute as large 700-800-kDa complexes and smaller 200-300-kDa complexes. CL-L1 showed specific binding to mannose-TSK beads in a Ca(2+)-dependent manner. This binding could be inhibited by mannose and glucose, but not galactose, indicating that CL-L1 binds via its carbohydrate-recognition domain and has ligand specificity similar to that of mannan-binding lectin. Western blot analysis of CL-L1 showed the presence of several oligomeric forms in serum. Ontogeny studies showed CL-L1 to be present at birth at near adult levels. CL-L1 levels exhibit low variation in healthy adults over a 1-year period. During acute-phase responses, the CL-L1 levels display only minor variations. In serum, CL-L1 was found in complexes with mannan-binding lectin-associated serine proteases, suggesting a role in the lectin pathway of complement activation. The presented data establish a basis for future studies on the biological role of CL-L1.
Forfattere: Axelgaard E, Jensen L, Dyrlund TF, Nielsen HJ, Enghild JJ, Thiel S, Jensenius JC
Energy intake, nutritional status and weight reduction in patients one year after laparoscopic sleeve gastrectomy.
SpringerPlus (2013 )
p. 352 issn:2193-1801)
Laparoscopic sleeve gastrectomy (LSG) is increasingly popular due to its efficiency in reducing excess weight, however little is known about the nutritional status in patients after surgery.
Forfattere: Gjessing HR, Nielsen HJ, Mellgren G, Gudbrandsen OA
Tissue inhibitor of metalloproteinases-1 as a biological marker in colorectal cancer: influence of smoking on plasma levels?
The International journal of biological markers ( )
Issue 2 p. 226-30 issn:1724-6008)
At present plasma tissue inhibitor of metalloproteinases-1 (TIMP-1) is undergoing validation as a biological marker in colorectal cancer (CRC). The clinical implementation of plasma TIMP-1 in prognosis, prediction, screening and monitoring CRC requires robust information as to the influence of preanalytical factors, including inter- and intrapersonal biological variations. The aim of the present study was to evaluate the possible effects of smoking on the level of TIMP-1 in plasma from healthy subjects.
Forfattere: Rasmussen L, Ladelund S, Brünner N, Jørgensen LN, Nielsen HJ, Sørensen LT
Mechanisms of topoisomerase I (TOP1) gene copy number increase in a stage III colorectal cancer patient cohort.
PloS one (2013 )
Issue 4 p. e60613 issn:1932-6203)
Topoisomerase I (Top1) is the target of Top1 inhibitor chemotherapy. The TOP1 gene, located at 20q12-q13.1, is frequently detected at elevated copy numbers in colorectal cancer (CRC). The present study explores the mechanism, frequency and prognostic impact of TOP1 gene aberrations in stage III CRC and how these can be detected by fluorescent in situ hybridization (FISH).
Forfattere: Smith DH, Christensen IJ, Jensen NF, Markussen B, Rømer MU, Nygård SB, Müller S, Nielsen HJ, Brünner N, Nielsen KV
Early detection of recurrence after curative resection for colorectal cancer - obstacles when using soluble biomarkers?
Scandinavian journal of gastroenterology (2013 Mar)
Issue 3 p. 326-33 issn:1502-7708)
Results from monitoring studies using biomarkers in blood samples aiming at early detection of recurrent colorectal cancer (CRC) are presently evaluated. However, some serological biomarker levels are influenced by the surgical trauma, which may complicate translation of the levels in relation to recurrence. The primary purpose of the present study was to evaluate the frequency of postoperative surgical interventions during a follow-up period of patients who have undergone surgery for primary CRC.
Forfattere: Nielsen HJ, Jess P, Aldulaymi BH, Jørgensen LN, Laurberg S, Nielsen KT, Madsen MR, Brünner N, Christensen IJ
Metabolomics : Official journal of the Metabolomic Society (2013 Feb)
Issue 1 p. 3-8 issn:1573-3882)
We have recently shown that fluorescence spectroscopy of plasma samples has promising abilities regarding early detection of colorectal cancer. In the present paper, these results were further developed by combining fluorescence with the biomarkers, CEA and TIMP-1 and traditional metabolomic measurements in the form of (1)H NMR spectroscopy. The results indicate that using an extensive profile established by combining such measurements together with the biomarkers is better than using single markers.
Forfattere: Bro R, Nielsen HJ, Savorani F, Kjeldahl K, Christensen IJ, Brünner N, Lawaetz AJ
Topoisomerase 1(TOP1) gene copy number in stage III colorectal cancer patients and its relation to prognosis.
Molecular oncology (2013 Feb)
Issue 1 p. 101-11 issn:1878-0261)
A Topoisomerase 1 (Top1) poison is frequently included in the treatment regimens for metastatic colorectal cancer (mCRC). However, no predictive biomarkers for Top1 poisons are available. We here report a study on the TOP1 gene copy number in CRC patients and its association with patient prognosis and tumor cell proliferation.
Forfattere: Rømer MU, Nygård SB, Christensen IJ, Nielsen SL, Nielsen KV, Müller S, Smith DH, Vainer B, Nielsen HJ, Brünner N
Non-synonymous polymorphisms in the FCN1 gene determine ligand-binding ability and serum levels of M-ficolin.
PloS one (2012 )
Issue 11 p. e50585 issn:1932-6203)
The innate immune system encompasses various recognition molecules able to sense both exogenous and endogenous danger signals arising from pathogens or damaged host cells. One such pattern-recognition molecule is M-ficolin, which is capable of activating the complement system through the lectin pathway. The lectin pathway is multifaceted with activities spanning from complement activation to coagulation, autoimmunity, ischemia-reperfusion injury and embryogenesis. Our aim was to explore associations between SNPs in FCN1, encoding M-ficolin and corresponding protein concentrations, and the impact of non-synonymous SNPs on protein function.
Forfattere: Ammitzbøll CG, Kjær TR, Steffensen R, Stengaard-Pedersen K, Nielsen HJ, Thiel S, Bøgsted M, Jensenius JC
Scandinavian journal of gastroenterology (2012 Oct)
Issue 10 p. 1234-41 issn:1502-7708)
A prospective cross-sectional study was designed to test if total levels of TIMP-1 in saliva and plasma correlated with the diagnosis of colorectal cancer (CRC) in a population with symptoms consistent with this disease.
Forfattere: Holten-Andersen L, Christensen IJ, Jensen SB, Reibel J, Laurberg S, Nauntofte B, Brünner N, Nielsen HJ
Radiology and oncology (2012 Sep)
Issue 3 p. 207-12 issn:1581-3207)
Up-regulation of lysosomal cysteine protease cathepsin X (Cat X) is associated with disorders of the immune system and neurodegenerative diseases, while its role in the development and progression of cancer is less understood. Enhanced secretion of pro-Cat X was observed in malignant processes, and therefore, the level of total serum Cat X rather than the active enzyme may better reflect the tumour status.
Forfattere: Vizin T, Christensen IJ, Nielsen HJ, Kos J
Scandinavian journal of gastroenterology (2011 Nov)
Issue 11 p. 1283-94 issn:1502-7708)
Emerging results indicate that screening improves survival of patients with colorectal cancer. Therefore, screening programs are already implemented or are being considered for implementation in Asia, Europe and North America. At present, a great variety of screening methods are available including colono- and sigmoidoscopy, CT- and MR-colonography, capsule endoscopy, DNA and occult blood in feces, and so on. The pros and cons of the various tests, including economic issues, are debated. Although a plethora of evaluated and validated tests even with high specificities and reasonable sensitivities are available, an international consensus on screening procedures is still not established. The rather limited compliance in present screening procedures is a significant drawback. Furthermore, some of the procedures are costly and, therefore, selection methods for these procedures are needed. Current research into improvements of screening for colorectal cancer includes blood-based biological markers, such as proteins, DNA and RNA in combination with various demographically and clinically parameters into a “risk assessment evaluation” (RAE) test. It is assumed that such a test may lead to higher acceptance among the screening populations, and thereby improve the compliances. Furthermore, the involvement of the media, including social media, may add even more individuals to the screening programs. Implementation of validated RAE and progressively improved screening methods may reform the cost/benefit of screening procedures for colorectal cancer. Therefore, results of present research, validating RAE tests, are awaited with interest.
Forfattere: Nielsen HJ, Jakobsen KV, Christensen IJ, Brünner N,
Plasma TIMP-1 and CEA in detection of primary colorectal cancer: a prospective, population based study of 4509 high-risk individuals.
Scandinavian journal of gastroenterology (2011 Jan)
Issue 1 p. 60-9 issn:1502-7708)
The combination of plasma tissue inhibitor of metalloproteinases-1 (TIMP-1) and carcinoembryonic antigen (CEA) may be valuable biomarkers for early detection of colorectal cancer (CRC). A prospective, population based study was performed to validate this hypothesis.
Forfattere: Nielsen HJ, Brünner N, Jorgensen LN, Olsen J, Rahr HB, Thygesen K, Hoyer U, Laurberg S, Stieber P, Blankenstein MA, Davis G, Dowell BL, Christensen IJ
Plasma TIMP-1 and CEA as Markers for Detection of Primary Colorectal Cancer: A Prospective Validation Study Including Symptomatic and Non-symptomatic Individuals.
Anticancer research (2015 Sep)
Issue 9 p. 4935-41 issn:1791-7530)
The combination of plasma tissue inhibitor of metalloproteinases-1 (1) and CEA has been shown to have utility in early detection of colorectal cancer (2). A prospective study was performed to validate previous findings.
Forfattere: Christensen IJ, Brünner N, Dowell B, Davis G, Nielsen HJ, Newstead G, King D
Intact and cleaved forms of the urokinase receptor enhance discrimination of cancer from non-malignant conditions in patients presenting with symptoms related to colorectal cancer.
British journal of cancer (2009 Sep)
Issue 6 p. 992-7 issn:1532-1827)
Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality in developed countries. It is known that early detection results in improved survival, and consequently there is a need for improved diagnostic tools in CRC. The plasma level of soluble urokinase plasminogen activator receptor (suPAR) was proposed as a marker in CRC patients. This study was undertaken to evaluate the individual molecular forms of suPAR as discriminators in a group of patients undergoing endoscopical examination following symptoms related to colorectal cancer.
Forfattere: Lomholt AF, Høyer-Hansen G, Nielsen HJ, Christensen IJ
Molecular & cellular proteomics : MCP (2008 Oct)
Issue 10 p. 2061-6 issn:1535-9484)
With the increasing demand of providing personalized medicine the need for biobanking of biological material from individual patients has increased. Such samples are essential for molecular research aimed at characterizing diseases at several levels ranging from epidemiology and diagnostic and prognostic classification to prediction of response to therapy. Clinically validated biomarkers may provide information to be used for diagnosis, screening, evaluation of risk/predisposition, assessment of prognosis, monitoring (recurrence of disease), and prediction of response to treatment and as a surrogate response marker. Many types of biological fluids or tissues can be collected and stored in biorepositories. Samples of blood can be further processed into plasma and serum, and tissue pieces can be either frozen or fixed in formalin and then embedded into paraffin. The present review focuses on biological fluids, especially serum and plasma, intended for study of protein biomarkers. In biomarker studies the process from the decision to take a sample from an individual to the moment the sample is safely placed in the biobank consists of several phases including collection of samples, transport of the samples, and handling and storage of samples. Critical points in each step important for high quality biomarker studies are described in this review. Failure to develop and adhere to robust standardized protocols may have significant consequences as the quality of the material stored in the biobank as well as conclusions and clinical recommendations based on analysis of such material may be severely affected.
Forfattere: Schrohl AS, Würtz S, Kohn E, Banks RE, Nielsen HJ, Sweep FC, Brünner N
Journal of surgical oncology (2004 Dec)
Issue 4 p. 269-75 issn:0022-4790)
The role of the haemostatic system in colorectal cancer (CRC) is reviewed. Correlations between the activation of the haemostatic system and overall survival have been suggested. Experimental studies indicate that the haemostatic system plays a key role in growth, invasion and dissemination of tumour cells, and in tumour related angiogenesis. Additional activation by the surgical trauma and postoperative infections are discussed. Finally, anti-cancer modalities directed against regulation of the haemostatic system in CRC are considered.
Forfattere: Lykke J, Nielsen HJ