Fibroblast Activation Protein (FAP)-Mediated Cleavage of Type III Collagen Reveals Serum Biomarker Potential in Non-Small Cell Lung Cancer and Spondyloarthritis.

Fibroblast activation protein (FAP) is a known promoter of tumor development and is associated with poor clinical outcome for various cancer types. Being specifically expressed in pathological conditions including multiple types of fibrosis and cancers, FAP is an optimal target for diagnostics and treatment. Treatment strategies utilizing the unique proteolytic activity of FAP are emerging, thus emphasizing the importance of biomarkers to directly assess FAP activity. FAP is a type II transmembrane serine protease that has been shown to cleave collagens and other ECM components. In this study, we developed an ELISA assay (C3F) targeting a circulating type III collagen fragment derived from FAP cleavage to reflect FAP activity. We demonstrated that C3F was specific to the neoepitope of the cleavage site and that the fragment was generated through FAP cleavage of type III collagen. We measured C3F in serum from a cohort of patients with non-small cell lung cancer (NSCLC) (n = 109) matched to healthy subjects (n = 42) and a cohort of patients with spondyloarthritis (SpA) (n = 17) matched to healthy subjects (n = 19). We found that C3F was significantly elevated in patients with NSCLC and in patients with SpA compared to healthy controls (p < 0.0001 and p = 0.0015, respectively). These findings suggest that C3F is a promising non-invasive biomarker reflecting FAP activity, which may aid in understanding tumor heterogeneity and potentially FAP-targeted therapies.

Levels of type XVII collagen (BP180) ectodomain are elevated in circulation from patients with multiple cancer types and is prognostic for patients with metastatic colorectal cancer.

BACKGROUND: Collagens are the major components of the extracellular matrix (ECM) and are known to contribute to tumor progression and metastasis. There are 28 different types of collagens each with unique functions in maintaining tissue structure and function. Type XVII collagen (BP180) is a type II transmembrane protein that provides stable adhesion between epithelial cells and the underlying basement membrane. Aberrant expression and ectodomain shedding of type XVII collagen have been associated with epithelial damage, tumor invasiveness, and metastasis in multiple tumor types and may consequently be used as a potential (non-invasive) biomarker in cancer and treatment target. METHOD: An ELISA targeting the type XVII collagen ectodomain (PRO-C17) was developed for use in serum. PRO-C17 was measured in a cohort of patients with 11 different cancer types (n = 214) and compared to healthy controls (n = 23) (cohort 1). Based on the findings from cohort 1, PRO-C17 and its association with survival was explored in patients with metastatic colorectal cancer (mCRC) treated with bevacizumab in combination with chemotherapy (n = 212) (cohort 2). RESULTS: PRO-C17 was robust and specific towards the ectodomain of type XVII collagen. In cohort 1, PRO-C17 levels were elevated (p < 0.05) in serum from patients with CRC, kidney, ovarian, bladder, breast, and head and neck cancer compared to healthy controls. PRO-C17 was especially good at discriminating between CRC patients and healthy controls with an AUROC of 0.904. In cohort 2, patients with mCRC and high levels (tertile 3) of PRO-C17 had shorter overall survival (OS) with a median OS of 390 days compared to 539 days for patients with low levels of PRO-C17. When evaluated by multivariate Cox regression analysis, high PRO-C17 was predictive for poor OS independent of risk factors and the tumor fibrosis biomarker PRO-C3. CONCLUSION: PRO-C17 measures the ectodomain of type XVII collagen in serum and is a promising non-invasive biomarker that can aid in understanding tumor heterogeneity as well as elaborate on the role of collagen XVII in tumor progression. Moreover, the findings in the study proposes PRO-C17 as novel biomarker of epithelial damage in specific cancer types including CRC.

Plasma Kallikrein-Activated TGF-ß Is Prognostic for Poor Overall Survival in Patients with Pancreatic Ductal Adenocarcinoma and Associates with Increased Fibrogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is a hard-to-treat cancer due to the collagen-rich (fibrotic) and immune-suppressed microenvironment. A major driver of this phenomenon is transforming growth factor beta (TGF-ß). TGF-ß is produced in an inactive complex with a latency-associated protein (LAP) that can be cleaved by plasma kallikrein (PLK), hereby releasing active TGF-ß. The aim of this study was to evaluate LAP cleaved by PLK as a non-invasive biomarker for PDAC and tumor fibrosis. An ELISA was developed for the quantification of PLK-cleaved LAP-TGF-ß in the serum of 34 patients with PDAC (stage 1−4) and 20 healthy individuals. Biomarker levels were correlated with overall survival (OS) and compared to serum type III collagen (PRO-C3) and type VI collagen (PRO-C6) pro-peptides. PLK-cleaved LAP-TGF-ß was higher in patients with PDAC compared to healthy individuals (p < 0.0001). High levels (>median) of PLK-cleaved LAP-TGF-ß were associated with poor OS in patients with PDAC independent of age and stage (HR 2.57, 95% CI: 1.22−5.44, p = 0.0135). High levels of PLK-cleaved LAP-TGF-ß were associated with high PRO-C3 and PRO-C6, indicating a relationship between the PLK-cleaved LAP-TGF-ß fragment, TGF-ß activity, and tumor fibrosis. If these preliminary results are validated, circulating PLK-cleaved LAP-TGF-ß may be a biomarker for future clinical trials.

Fibrotic activity quantified in serum by measurements of type III collagen pro-peptides can be used for prognosis across different solid tumor types.

Due to activation of fibroblast into cancer-associated fibroblasts, there is often an increased deposition of extracellular matrix and fibrillar collagens, e.g. type III collagen, in the tumor microenvironment (TME) that leads to tumor fibrosis (desmoplasia). Tumor fibrosis is closely associated with treatment response and poor prognosis for patients with solid tumors. To assure that the best possible treatment option is provided for patients, there is medical need for identifying patients with high (or low) fibrotic activity in the TME. Measuring unique collagen fragments such as the pro-peptides released into the bloodstream during fibrillar collagen deposition in the TME can provide a non-invasive measure of the fibrotic activity. Based on data from 8 previously published cohorts, this review provides insight into the prognostic value of quantifying tumor fibrosis by measuring the pro-peptide of type III collagen in serum of a total of 1692 patients with different solid tumor types and discusses the importance of tumor fibrosis for understanding prognosis and for potentially guiding future drug development efforts that aim at overcoming the poor outcome associated with a fibrotic TME.

Collagen Biomarkers Quantify Fibroblast Activity In Vitro and Predict Survival in Patients with Pancreatic Ductal Adenocarcinoma.

The use of novel tools to understand tumour-fibrosis in pancreatic ductal adenocarcinoma (PDAC) and novel anti-fibrotic treatments are highly needed. We established a pseudo-3D in vitro model including humane pancreatic fibroblasts (PFs) and pancreatic cancer-associated fibroblasts (CAFs) in combination with clinical collagen biomarkers, as a translational anti-fibrotic drug screening tool. Furthermore, we investigated the prognostic potential of serum collagen biomarkers in 810 patients with PDAC. PFs and CAFs were cultured in Ficoll-media. Cells were treated w/wo TGF-ß1 and the anti-fibrotic compound ALK5i. Biomarkers measuring the formation of type III (PRO-C3) and VI (PRO-C6) collagens were measured by ELISA in supernatant at days 3, 6, 9, and 12. PRO-C3 and PRO-C6, and their association with overall survival (OS), were evaluated in serum with PDAC (n = 810). PRO-C3 and PRO-C6 were upregulated in CAFs compared to PFs (p < 0.0001.). TGF-ß1 increased PRO-C3 in both PFs and CAFs (p < 0.0001). The anti-fibrotic compound ALK5i inhibited both PRO-C3 and PRO-C6 (p < 0.0001). High serum levels of PRO-C3 and PRO-C6 in patients with PDAC were associated with short OS (PRO-C3: HR = 1.48, 95%CI: 1.29-1.71, p < 0.0001 and PRO-C6: HR = 1.31, 95%CI: 1.14-1.50, p = 0.0002). PRO-C3 and PRO-C6 have the potential to be used both pre-clinically and clinically as a measure of tumor fibrosis and CAF activity.

Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein.

Diffuse gliomas are the most common primary malignant brain tumor. Although extracranial metastases are rarely observed, recent studies have shown the presence of circulating tumor cells (CTCs) in the blood of glioma patients, confirming that a subset of tumor cells are capable of entering the circulation. The isolation and characterization of CTCs could provide a non-invasive method for repeated analysis of the mutational and phenotypic state of the tumor during the course of disease. However, the efficient detection of glioma CTCs has proven to be challenging due to the lack of consistently expressed tumor markers and high inter- and intra-tumor heterogeneity. Thus, for this field to progress, an omnipresent but specific marker of glioma CTCs is required. In this article, we demonstrate how the recombinant malaria VAR2CSA protein (rVAR2) can be used for the capture and detection of glioma cell lines that are spiked into blood through binding to a cancer-specific oncofetal chondroitin sulfate (ofCS). When using rVAR2 pull-down from glioma cells, we identified a panel of proteoglycans, known to be essential for glioma progression. Finally, the clinical feasibility of this work is supported by the rVAR2-based isolation and detection of CTCs from glioma patient blood samples, which highlights ofCS as a potential clinical target for CTC isolation.

Linkage disequilibrium between the CYP2C19*17 allele and wildtype CYP2C8 and CYP2C9 alleles: identification of CYP2C haplotypes in healthy Nordic populations.

PURPOSE: To determine the distribution of clinically important CYP2C genotypes and allele frequencies in healthy Nordic populations with special focus on linkage disequilibrium. METHODS: A total of 896 healthy subjects from three Nordic populations (Danish, Faroese, and Norwegian) were genotyped for five frequent and clinically important CYP2C allelic variants: the defective CYP2C8*3, CYP2C9*2, CYP2C9*3, and CYP2C19*2 alleles, and the CYP2C19*17 allele that causes rapid drug metabolism. Linkage disequilibrium was evaluated and CYP2C haplotypes were inferred in the entire population. RESULTS: Ten CYP2C haplotypes were inferred, the most frequent of which (49%) was the CYP2C wildtype haplotype carrying CYP2C8*1, CYP2C9*1, and CYP2C19*1. The second most frequent haplotype (19%) is composed of CYP2C19*17, CYP2C8*1, and CYP2C9*1. This predicted haplotype accounts for 99.7% of the CYP2C19*17 alleles found in the 896 subjects. CONCLUSION: CYP2C19*17 is a frequent genetic variant in Nordic populations that exists in strong linkage disequilibrium with wildtype CYP2C8*1 and CYP2C9*1 alleles, which effectively makes it a determinant for a haplotype exhibiting an efficient CYP2C substrate metabolism.

The effects of human CYP2C8 genotype and fluvoxamine on the pharmacokinetics of rosiglitazone in healthy subjects.

AIMS: To determine the effect of CYP2C8 genotype and of fluvoxamine on the pharmacokinetics of rosiglitazone. METHODS: Twenty-three healthy subjects with the following genotypes were included in a two-phase, open-label, cross-over trial: CYP2C8*3/ *3 (n = 3), CYP2C8*1/ *3 (n = 10) and CYP2C8*1/ *1 (n = 10). In Phase A, the subjects were given 4 mg rosiglitazone as a single oral dose. In Phase B, the subjects were treated with multiple oral doses of 50 mg fluvoxamine maleate for 3 days prior to the single oral administration of 4 mg rosiglitazone. Plasma concentrations of rosiglitazone and relative amounts of N-desmethylrosiglitazone were measured in both phases for 24 h after drug administration. RESULTS: The pharmacokinetics of rosiglitazone and N-desmethylrosiglitazone were not significantly different between the CYP2C8 genotypic groups. Fluvoxamine caused a statistically significant (P = 0.0066) increase in the AUC(0-infinity) of rosiglitazone, with a geometric mean ratio of 1.21 [95% confidence interval (CI) 1.06-1.39]. The elimination half-life (t(1/2)) was also significantly higher (P = 0.0203) with a geometric mean ratio of 1.38 [95% CI 1.06-1.79]. The coadministration of fluvoxamine had no influence on the pharmacokinetics of N-desmethylrosiglitazone. CONCLUSION: The importance of the CYP2C8*3 mutation in the in vivo metabolism of rosiglitazone could not be confirmed. Fluvoxamine increased the AUC(0-infinity) and t(1/2) of rosiglitazone moderately and hence may be a weak inhibitor of CYP2C8.