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Circulating cell-free DNA (cfDNA) has spurred much interest as a biomarker in oncology. However, inter- and intra-individual cfDNA levels vary greatly. Consequently, in order to base clinical decisions on cfDNA measurements, normal reference intervals are essential to avoid that ordinary variation is confused with clinically relevant change. The lack of reference intervals may potentially explain the ambiguous results reported in the field. Our study aimed to establish reference intervals and to evaluate the association between cfDNA and demographic and clinical variables, including colorectal cancer (CRC). Plasma samples and clinical data from 2817 subjects were collected including 1930 noncancer individuals and 887 CRC patients. cfDNA was measured using droplet digital polymerase chain reaction (PCR). The large cohort combined with robust cfDNA quantification enabled establishment of reference intervals (<67 years: 775-4860 copies/mL; ≥67 years: 807-6561 copies/mL). A cfDNA level above the age-stratified 90% percentile was prognostic of reduced survival in both noncancer individuals and CRC patients, with HR values of 2.56 and 2.01, respectively. Moreover, cfDNA levels increased significantly with age, elevated BMI and chronic diseases. In CRC, the cfDNA level was increased for Stage IV, but not Stage I to Stage III cancer. In summary, the use of reference intervals revealed that high cfDNA levels were predictive of shorter survival in both noncancer individuals and CRC patients, and that CRC development did not affect the cfDNA level until metastatic dissemination. Furthermore, cfDNA levels were impacted by age and chronic diseases. Conclusively, our study presents reference intervals that will help pave the way for clinical utilization of cfDNA., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background and Objectives: 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)., Methods: Patients with CRC were recruited prospectively at six centers from 2006 to 2008. Preoperative plasma samples were available from 494 patients and from 328 of these patients at 6 months postoperatively. Determinations of intact soluble uPAR (suPAR) suPAR(I-III) and the cleaved forms suPAR(I-III) + (II-III) and uPAR(I) were performed. Clinical data were retrieved retrospectively., Results: In a multivariable model based on preoperative plasma samples suPAR(I-III) + (II-III) and uPAR(I) showed an independent statistically significant association to long term survival. When including the change in biomarker level between the pre- and postoperatively samples the hazard ratios were 3.06 (95% confidence interval [CI], 1.78-5.28; P < .0001) and 2.24 (95% CI, 1.59-3.16; P < .0001) for suPAR(I-III) + (II-III) and uPAR(I), respectively. A one-unit decrease in biomarker levels between the pre- and postoperative levels resulted in a 55% and 34% reduction in the risk estimate of death for suPAR(I-III) + (II-III) and uPAR(I), respectively., Conclusion: This study validates previously findings regarding the prognostic significance of suPAR in preoperative samples. The inclusion of postoperative samples added further prognostic information., (© 2019 Wiley Periodicals, Inc.)

Serological biomarkers may be an option for early detection of colorectal cancer (CRC). The present study assessed eight 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 (i) CRC and high-risk adenoma and (ii) CRC. Logistic regression was performed. Final reduced models were constructed selecting the four biomarkers with the highest likelihood scores. Subjects (N = 4,698) were consecutively included during 2010-2012. Colonoscopy detected 512 CRC patients, 319 colonic cancer and 193 rectal cancer. Extra colonic malignancies were detected in 177 patients, 689 had adenomas of which 399 were high-risk, 1,342 had nonneoplastic bowell disease and 1,978 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 the selection of CEA, hs-CRP, CyFra21-1 and Ferritin for the two endpoints; AUCs were 0.76 and 0.84, respectively. The postive predictive value at 90% sensitivity was 25% for endpoint 1 and the negative predictive value was 93%. For endpoint 2, the postive predictive value was 18% and the negative predictive value was 97%. Combinations of serological protein biomarkers provided a significant identification of subjects with high risk of the presence of colorectal neoplasia. The present set of biomarkers could become important adjunct in early detection of CRC., (© 2016 UICC.)

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., (© 2015 UICC.)

Tissue inhibitor of matrix metalloproteases 1 (TIMP-1) inhibits the proteolytic activity of matrix metalloproteases and hereby prevents cancer invasion. However, TIMP-1 also possesses other functions such as inhibition of apoptosis, induction of malignant transformation and stimulation of cell-growth. We have previously demonstrated that TIMP-1 is elevated in blood from colorectal cancer patients and that high TIMP-1 levels predict poor prognosis. To clarify the role of TIMP-1 in colorectal tumorigenesis, the expression pattern of TIMP-1 in benign and malignant colorectal tumors was studied. In all of 24 cases of colorectal adenocarcinoma TIMP-1 mRNA was detected by in situ hybridization. In all cases TIMP-1 expression was found in fibroblast-like cells located at the invasive front but was seen only sporadically in normal mucosa. No TIMP-1 mRNA was seen in any of the cases in benign or malignant epithelial cells, in vascular cells or smooth muscle cells. Comparison of sections processed for TIMP-1 in situ hybridization with sections immunohistochemically stained with antibodies against TIMP-1 showed good correlation between TIMP-1 mRNA and immunoreactivity. Combining TIMP-1 in situ hybridization with immunohistochemical staining for alpha-smooth muscle actin or CD68 showed TIMP-1 mRNA in myofibroblasts but not in macrophages. TIMP-1 mRNA was detected in 2 of 7 adenomatous polyps in the adenoma area: in both cases associated with focal stromal inflammation at the epithelial-stromal interface. In conclusion, TIMP-1 expression is a rare event in benign human colon tissue but is highly expressed by myofibroblasts in association with invading colon cancer cells.

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.

Flow cytometry was used to study the incidence of aneuploidy and to determine the significance of multiple sampling from colorectal tumors. DNA ploidy pattern has been proposed as a supplementary prognostic marker, but discrepancies in findings are major. DNA clonal heterogeneity, defined as two or more DNA aneuploid stemlines in the same tumor, is well established. However, most studies have been based on only one biopsy from each tumor. In our study multiple biopsies were taken from 163 patients (88 males and 75 females) electively operated for colorectal cancer. Tumor cells were harvested by fine needle aspiration from fresh frozen biopsies sampled at different sites of each tumor. DNA aneuploidy was detected in tumors from 145 patients (89%), and 18 patients (11%) had a solitary DNA diploid cell population. In a 79 month follow-up period 105 patients had died. Statistical analysis showed that distinction between diploidy and aneuploidy did not predict survival. However, grouping subpopulations into DNA diploid plus near diploid (DNA index (DI) 0. 97-1.15), DNA aneuploid with all aneuploid subpopulations in the interval 1.15-2.06, and DNA aneuploid with subpopulations with DI < 0.97 and/or DI > 2.06, showed a significant difference in survival in a Cox multivariate analysis including Dukes' stage P = 0.049 comparing the second group to the first and P = 0.01 comparing the third group to the first. In 21 (13%) patients only one subpopulation was found, 57 (35%) had two, 44 (27%) had three, and 41 (25%) had four or more different subpopulations. The association of DNA ploidy to survival is shown to be dependent on the number of biopsies analysed., (Copyright 1999 Wiley-Liss, Inc.)

We present a simple nonradioactive assay based on polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE), which allows comprehensive mutation scanning of the entire coding sequence and splice junctions of the p53 gene in one analytical step. The key features of the present method are (1) all fragments can be amplified using one common PCR protocol; (2) all fragments can be scanned for mutations on a single "broad-range" denaturing gradient gel; and (3) all fragments were tailored by the attachment of appropriate GC clamps and otherwise manipulated to consist of only two melting domains in order to maximize resolution of mutations by DGGE. The entire procedure starting with a sample of genomic DNA can be completed within 6 hr and has the potential to detect any sequence variation, irrespective of its location in the p53 gene. The mutation detection sensitivity was demonstrated by the analysis of 26 constructed control mutations distributed over the whole of the p53 gene. We have applied the method to genetic diagnosis in 43 cases of colorectal cancer. Overall, a point mutation, microdeletion, or microinsertion was found in 26 (61%) of the tumors. In addition to missense and frameshift mutations within exons 4-8, a 20-bp insertion in exon 11 was found in one sample, illustrating the importance of comprehensive gene scanning for reliable diagnosis.