Nicholas Willumsen

Serum biomarkers reflecting specific tumor tissue remodeling processes are valuable diagnostic tools for lung cancer

Extracellular matrix (ECM) proteins, such as collagen type I and elastin, and intermediate filament (IMF) proteins, such as vimentin are modified and dysregulated as part of the malignant changes leading to disruption of tissue homeostasis. Noninvasive biomarkers that reflect such changes may have a great potential for cancer. Levels of matrix metalloproteinase (MMP) generated fragments of type I collagen (C1M), of elastin (ELM), and of citrullinated vimentin (VICM) were measured in serum from patients with lung cancer (n = 40), gastrointestinal cancer (n = 25), prostate cancer (n = 14), malignant melanoma (n = 7), chronic obstructive pulmonary disease (COPD) (n = 13), and idiopathic pulmonary fibrosis (IPF) (n = 10), as well as in age‐matched controls (n = 33). The area under the receiver operating characteristics (AUROC) was calculated and a diagnostic decision tree generated from specific cutoff values. C1M and VICM were significantly elevated in lung cancer patients as compared with healthy controls (AUROC = 0.98, P < 0.0001) and other cancers (AUROC = 0.83 P < 0.0001). A trend was detected when comparing lung cancer with COPD+IPF. No difference could be seen for ELM. Interestingly, C1M and VICM were able to identify patients with lung cancer with a positive predictive value of 0.9 and an odds ratio of 40 (95% CI = 8.7–186, P < 0.0001). Biomarkers specifically reflecting degradation of collagen type I and citrullinated vimentin are applicable for lung cancer patients. Our data indicate that biomarkers reflecting ECM and IMF protein dysregulation are highly applicable in the lung cancer setting. We speculate that these markers may aid in diagnosing and characterizing patients with lung cancer.

Extracellular matrix specific protein fingerprints measured in serum can separate pancreatic cancer patients from healthy controls

BackgroundPancreatic cancer (PC) is an aggressive disease with an urgent need for biomarkers. Hallmarks of PC include increased collagen deposition (desmoplasia) and increased matrix metalloproteinase (MMP) activity. The aim of this study was to investigate whether protein fingerprints of specific MMP-generated collagen fragments differentiate PC patients from healthy controls when measured in serum.MethodsThe levels of biomarkers reflecting MMP-mediated degradation of type I (C1M), type III (C3M) and type IV (C4M, C4M12a1) collagen were assessed in serum samples from PC patients (n = 15) and healthy controls (n = 33) using well-characterized and validated competitive ELISAs.ResultsThe MMP-generated collagen fragments were significantly elevated in serum from PC patients as compared to controls. The diagnostic power of C1M, C3M, C4M and C4M12 were ≥83% (p < 0.001) and when combining all biomarkers 99% (p < 0.0001).ConclusionsA panel of serum biomarkers reflecting altered MMP-mediated collagen turnover is able to differentiate PC patients from healthy controls. These markers may increase the understanding of mode of action of the disease and, if validated in larger clinical studies, provide an improved and additional tool in the PC setting.

Levels of circulating MMP-7 degraded elastin are elevated in pulmonary disorders.

OBJECTIVES Elastin is a signature protein of the lungs. Matrix metalloproteinase-7 (MMP-7) is important in lung defence mechanisms and degrades elastin. However, MMP-7 activity in regard to elastin degradation has never been quantified serologically in patients with lung diseases. An assay for the quantification of MMP-7 generated elastin fragments (ELM7) was therefore developed to investigate MMP-7 derived elastin degradation in pulmonary disorders such as idiopathic pulmonary fibrosis (IPF) and lung cancer. DESIGN AND METHODS Monoclonal antibodies (mABs) were raised against eight carefully selected MMP-7 cleavage sites on elastin. After characterisation and validation of the mABs, one mAB targeting the ELM7 fragment was chosen. ELM7 fragment levels were assessed in serum samples from patients diagnosed with IPF (n=123, baseline samples, CTgov reg. NCT00786201), and lung cancer (n=40) and compared with age- and sex-matched controls. RESULTS The ELM7 assay was specific towards in vitro MMP-7 degraded elastin and the ELM7 neoepitope but not towards other MMP-7 derived elastin fragments. Serum ELM7 levels were significantly increased in IPF (113%, p<0.0001) and lung cancer (96%, p<0.0001) compared to matched controls. CONCLUSIONS MMP-7-generated elastin fragments can be quantified in serum and may reflect pathological lung tissue turnover in several important lung diseases.

Type VIII collagen is elevated in diseases associated with angiogenesis and vascular remodeling.

OBJECTIVES Type VIII collagen is involved in angiogenesis and remodeling of arteries. We hypothesized that type VIII collagen was upregulated in diseases associated with vascular remodeling, e.g. pulmonary fibrosis and cancer. In this paper we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) targeting type VIII collagen in serum and plasma. DESIGN AND METHODS A monoclonal antibody was raised against the C-terminal of type VIII collagen (C8-C) and a competitive ELISA was developed. The assay was evaluated in relation to epitope specificity, technical performance, and in relevant disease cohorts. The developed ELISA was applied for the assessment of type VIII collagen in serum from patients diagnosed with chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and various cancers. RESULTS The C8-C ELISA was technically stable and applicable for measurements in serum and plasma samples. Concentrations of C8-C was increased in serum from patients diagnosed with COPD (n=68) (p<0.0001), SCC lung- (n=10) (p<0.0001), breast- (n=13) (p<0.0001), colon- (n=7) (p=0.002), melanoma- (n=7) (p=0.001), non-small cell lung- (n=12) (p<0.0001), ovary- (n=10) (p=0.0001), pancreas- (n=5) (p=0.017), prostate- (n=14) (p=0.001) and small cell lung cancer (n=8) (p=0.0002) when compared to controls (n=43). Concentrations of C8-C were not significantly increased in serum from patients diagnosed with IPF. CONCLUSIONS The C8-C assay was technically robust and specific for type VIII collagen. Concentrations of C8-C were significantly elevated in serum from patients diagnosed with COPD and within 9 different cancer types, but not IPF. Further research is required to test C8-C as an efficacy marker for angiostatic treatments.

Excessive matrix metalloprotease-mediated degradation of interstitial tissue (type I collagen) independently predicts short-term survival in an observational study of postmenopausal women diagnosed with cancer

Extensive tissue remodeling mediated by matrix metalloproteases (MMPs) is an important part of cancer. The aim of this study was to investigate whether serum biomarkers reflecting MMP-mediated degradation of type I collagen (C1M), type IV collagen (C4M) and citrullinated vimentin (VICM) were predictive of cancer-specific mortality. Between 1999 and 2001, 5855 Danish postmenopausal women participated in The Prospective Epidemiologic Risk Factor (PERF I) study. Demographics and serum samples were collected at enrolment. Cancer diagnosis, and cause and time of death were obtained from Danish registries. C1M, C4M and VICM were measured by ELISA. Hazard ratios (HR) and Kaplan-Meier curves were applied to assess mortality at 3 and 12 years of follow-up for women diagnosed with cancer within 3 years from blood sampling. Within 3 years from blood sampling, 250 women had been diagnosed with cancer. C1M and VICM were associated with survival over time at 3 years of follow-up. Only C1M was predictive of mortality at 3 years follow-up: the adjusted HR was 2.65 [95% CI: 1.08-6.51]. In conclusion, C1M and VICM are associated with survival in postmenopausal women with cancer, and C1M is an independent risk factor for cancer-specific mortality. Thus, quantification of tissue remodeling is important in cancer.Extensive tissue remodeling mediated by matrix metalloproteases (MMPs) is an important part of cancer. The aim of this study was to investigate whether serum biomarkers reflecting MMP-mediated degradation of type I collagen (C1M), type IV collagen (C4M) and citrullinated vimentin (VICM) were predictive of cancer-specific mortality. Between 1999 and 2001, 5855 Danish postmenopausal women participated in The Prospective Epidemiologic Risk Factor (PERF I) study. Demographics and serum samples were collected at enrolment. Cancer diagnosis, and cause and time of death were obtained from Danish registries. C1M, C4M and VICM were measured by ELISA. Hazard ratios (HR) and Kaplan-Meier curves were applied to assess mortality at 3 and 12 years of follow-up for women diagnosed with cancer within 3 years from blood sampling. Within 3 years from blood sampling, 250 women had been diagnosed with cancer. C1M and VICM were associated with survival over time at 3 years of follow-up. Only C1M was predictive of mortality at 3 years follow-up: the adjusted HR was 2.65 [95% CI: 1.08-6.51]. In conclusion, C1M and VICM are associated with survival in postmenopausal women with cancer, and C1M is an independent risk factor for cancer-specific mortality. Thus, quantification of tissue remodeling is important in cancer.

Unique insight into microenvironmental changes in colorectal cancer: Ex vivo assessment of matrix metalloprotease‑mediated molecular changes in human colorectal tumor tissue and corresponding non‑neoplastic adjacent tissue

Matrix metalloprotease (MMP)-mediated tissue remodeling is one of the malignant changes driving colorectal cancer. Measurement of altered MMP expression/activity is not sufficient to fully understand the effect of MMP-mediated tissue remodeling. Biomarkers are required that specifically reflect the dynamic processes of the MMP-mediated degradation of signature proteins from colorectal tissue. The aim of the present study was to profile and characterize the release of MMP-degraded type III collagen (C3M) and citrullinated and MMP-degraded vimentin (VICM) from tumor tissue and corresponding non-neoplastic adjacent tissue (NAT) in a human colorectal cancer ex vivo model. Colorectal tumor tissue and NAT biopsies from tissue removed during resection of colorectal cancer patients (n=13) were cut into pieces of 2 mm2 and cultured for 24 h in growth medium. C3M and VICM were evaluated by ELISA. As part of the characterization, C3M was determined subsequent to the tumor tissue being cleaved with recombinant MMP-2/-9 and trypsin. C3M was generated by MMP-2/-9, but not by trypsin. In addition, the level of C3M was significantly elevated in the conditioned medium from tumor tissues (3.7 ng/ml) compared with that observed in the conditioned medium from the NATs (2.2 ng/ml) and in the growth medium alone (1.9 ng/ml). The level of VICM was significantly elevated in the tumor tissues (0.51 ng/ml) and NATs (0.52 ng/ml) compared with that in the growth medium alone (0.03 ng/ml). No differences were detected between the tumor tissues and NATs. No correlation was observed between biomarker levels from the tumor tissue and corresponding NAT, and the biomarker levels did not correlate with tumor stage. In conclusion, the present study provided support of the concept that C3M and VICM are applicable as tools to investigate dynamic tissue changes of colorectal tumor tissue and corresponding NAT. By the assessment of these specific MMP-mediated molecular changes, the present study provides novel and relevant insight into the dynamic changes of colorectal tumor tissue and corresponding NAT.

Nidogen-1 Degraded by Cathepsin S can be Quantified in Serum and is Associated with Non–Small Cell Lung Cancer

Loss of basement membrane (BM) integrity is typically associated with cancer. Nidogen-1 is an essential component of the BM. Nidogen-1 is a substrate for cathepsin-S (CatS) which is released into the tumor microenvironment. Measuring nidogen-1 degraded by CatS may therefore have biomarker potential in cancer. The aim of this study was to investigate if CatS-degraded nidogen-1 was detectable in serum and a possible biomarker for cancer, a pathology associated with disruption of the BM. A competitive enzyme-linked immunosorbent assay (NIC) was developed with a monoclonal mouse antibody specific for a CatS cleavage site on human nidogen-1. Dilution and spiking recovery, inter- and intravariation, as well as accuracy were evaluated. Serum levels were evaluated in patients with breast cancer, small cell lung cancer (SCLC), and non-SCLC (NSCLC) and in healthy controls. The results indicated that the NIC assay was specific for nidogen-1 cleaved by CatS. Inter- and intraassay variations were 9% and 14%, respectively. NIC was elevated in NSCLC as compared to healthy controls (P < .001), breast cancer (P < .01), and SCLC (P < .5). The diagnostic power (area under the receiver operating characteristics) of NIC for NSCLC as compared to all other samples combined was 0.83 (95% confidence interval: 0.71-0.95), P < .0001. In conclusion, nidogen-1 degraded by CatS can be quantified in serum by the NIC assay. The current data strongly suggest that cathepsin-S degradation of nidogen-1 is strongly associated with NSCLC, which needs validation in larger clinical cohorts.

Remodeling of the Tumor Microenvironment Predicts Increased Risk of Cancer in Postmenopausal Women: The Prospective Epidemiologic Risk Factor (PERF I) Study

Background: An altered tumor microenvironment is one of the earliest signs of cancer and an important driver of the disease. We have seen previously that biomarkers reflecting tumor microenvironment modifications, such as matrix metalloproteinase (MMP)-degraded type 1 collagen (C1M), MMP-degraded type IV collagen (C4M), and citrullinated and MMP-degraded vimentin (VICM), were higher in the serum of cancer patients than in healthy controls. However, it is not known if these biomarkers could predict an increased risk of cancer. The aim of this study was to investigate whether C1M, C4M, and VICM were elevated prior to diagnosis of solid cancers in a large prospective study. Methods: Between 1999 and 2001, 5,855 postmenopausal Danish women ages 48 to 89 years enrolled in the Prospective Epidemiologic Risk Factor study. Baseline demographics and serum were collected at the time of registration. Follow up cancer diagnoses were obtained from the Danish Cancer Registry in 2014. Serum C1M, C4M, and VICM levels were measured by competitive ELISAs. Results: A total of 881 women were diagnosed with solid cancers after baseline. C1M, C4M, and VICM levels were significantly elevated in women diagnosed less than 1 year after baseline. C1M and VICM, but not C4M, were independent predictors of increased risk of cancer. Conclusion: C1M, C4M, and VICM are elevated prior to cancer diagnosis. C1M and VICM are both independent predictors of increased cancer risk. Impact: C1M and VICM are predictors for increased risk of cancer. Cancer Epidemiol Biomarkers Prev; 25(9); 1348–55. ©2016 AACR.

Age-related collagen turnover of the interstitial matrix and basement membrane: Implications of age- and sex-dependent remodeling of the extracellular matrix

The extracellular matrix (ECM) plays a vital role in maintaining normal tissue function. Collagens are major components of the ECM and there is a tight equilibrium between degradation and formation of these proteins ensuring tissue health and homeostasis. As a consequence of tissue turnover, small collagen fragments are released into the circulation, which act as important biomarkers in the study of certain tissue-related remodeling factors in health and disease. The aim of this study was to establish an age-related collagen turnover profile of the main collagens of the interstitial matrix (type I and III collagen) and basement membrane (type IV collagen) in healthy men and women. By using well-characterized competitive ELISA-assays, we assessed specific fragments of degraded (C1M, C3M, C4M) and formed (PINP, Pro-C3, P4NP7S) type I, III and IV collagen in serum from 617 healthy men and women ranging in ages from 22 to 86. Subjects were divided into 5-year age groups according to their sex and age. Groups were compared using Kruskal-Wallis adjusted for Dunn’s multiple comparisons test and Mann-Whitney t-test. Age-specific changes in collagen turnover was most profound for type I collagen. PINP levels decreased in men with advancing age, whereas in women, the level decreased in early adulthood followed by an increase around the age of menopause (age 40–60). Sex-specific changes in type I, III and IV collagen turnover was present at the age around menopause (age 40–60) with women having an increased turnover. In summary, collagen turnover is affected by age and sex with the interstitial matrix and the basement membrane being differently regulated. The observed changes needs to be accounted for when measuring ECM related biomarkers in clinical studies.

Bringing cancer serological diagnosis to a new level: focusing on HER2, protein ectodomain shedding and neoepitope technology

Cancer is a heterogeneous disease and consequently an exact diagnosis is as important as the actual therapy. Therefore, identification of novel diagnostic biomarker targets is urgently needed. Physiological and pathological changes are reflected by post-translational modifications of proteins. Each post-translational modification (e.g., proteolytic cleavage) is the result of a specific local process and may produce disease-specific neoepitopes. Neoepitopes have been successfully used as biomarkers in many diseases, and may also serve as promising tools in the development of future diagnostic assays within oncology. By specifically targeting neoepitopes, more information regarding disease-type and -state may be obtained and future research into neoepitopes will provide important and novel means for the diagnosis, prognosis and treatment efficacy in cancer. In this paper, we focus on protein ectodomain shedding and the generation of neoepitopes as future noninvasive (serological) cancer biomarkers. We use the protein ectodomain shedding of the human epidermal growth factor receptor 2, which is associated with breast cancer, as an example. We assess the current status of measuring human epidermal growth factor receptor 2 and discuss how this potentially could be improved. Furthermore, we expand the discussion to include examples of other cancer associated proteins.