Background
Protein biomarkers with associations with the activity and outcomes of diseases are being identified by modern proteomic technologies. They may be simple, accessible, cheap and safe tests ...that can inform diagnosis, prognosis, treatment selection, monitoring of disease activity and therapy and may substitute for complex, invasive and expensive tests. However, their potential is not yet being realised.
Design and methods
The study consisted of three workstreams to create a framework for research: workstream 1, methodology – to define current practice and explore methodology innovations for biomarkers for monitoring disease; workstream 2, clinical translation – to create a framework of research practice, high-quality samples and related clinical data to evaluate the validity and clinical utility of protein biomarkers; and workstream 3, the ELF to Uncover Cirrhosis as an Indication for Diagnosis and Action for Treatable Event (ELUCIDATE) randomised controlled trial (RCT) – an exemplar RCT of an established test, the ADVIA Centaur® Enhanced Liver Fibrosis (ELF) test (Siemens Healthcare Diagnostics Ltd, Camberley, UK) consisting of a panel of three markers – (1) serum hyaluronic acid, (2) amino-terminal propeptide of type III procollagen and (3) tissue inhibitor of metalloproteinase 1, for liver cirrhosis to determine its impact on diagnostic timing and the management of cirrhosis and the process of care and improving outcomes.
Results
The methodology workstream evaluated the quality of recommendations for using prostate-specific antigen to monitor patients, systematically reviewed RCTs of monitoring strategies and reviewed the monitoring biomarker literature and how monitoring can have an impact on outcomes. Simulation studies were conducted to evaluate monitoring and improve the merits of health care. The monitoring biomarker literature is modest and robust conclusions are infrequent. We recommend improvements in research practice. Patients strongly endorsed the need for robust and conclusive research in this area. The clinical translation workstream focused on analytical and clinical validity. Cohorts were established for renal cell carcinoma (RCC) and renal transplantation (RT), with samples and patient data from multiple centres, as a rapid-access resource to evaluate the validity of biomarkers. Candidate biomarkers for RCC and RT were identified from the literature and their quality was evaluated and selected biomarkers were prioritised. The duration of follow-up was a limitation but biomarkers were identified that may be taken forward for clinical utility. In the third workstream, the ELUCIDATE trial registered 1303 patients and randomised 878 patients out of a target of 1000. The trial started late and recruited slowly initially but ultimately recruited with good statistical power to answer the key questions. ELF monitoring altered the patient process of care and may show benefits from the early introduction of interventions with further follow-up. The ELUCIDATE trial was an ‘exemplar’ trial that has demonstrated the challenges of evaluating biomarker strategies in ‘end-to-end’ RCTs and will inform future study designs.
Conclusions
The limitations in the programme were principally that, during the collection and curation of the cohorts of patients with RCC and RT, the pace of discovery of new biomarkers in commercial and non-commercial research was slower than anticipated and so conclusive evaluations using the cohorts are few; however, access to the cohorts will be sustained for future new biomarkers. The ELUCIDATE trial was slow to start and recruit to, with a late surge of recruitment, and so final conclusions about the impact of the ELF test on long-term outcomes await further follow-up. The findings from the three workstreams were used to synthesise a strategy and framework for future biomarker evaluations incorporating innovations in study design, health economics and health informatics.
Trial registration
Current Controlled Trials ISRCTN74815110, UKCRN ID 9954 and UKCRN ID 11930.
Funding
This project was funded by the NIHR Programme Grants for Applied Research programme and will be published in full in
Programme Grants for Applied Research
; Vol. 6, No. 3. See the NIHR Journals Library website for further project information.
The use of cytochrome P4501A (CYP1A) and other measurements as biomarkers was investigated in liver of goby (
Z. ophiocephalus) and digestive gland of mussel (
M. galloprovincialis) from several ...sites in the Venice lagoon as part of the UNESCO-MURST Venice Lagoon Ecosystem Project. Most tissue contaminants (PAHs, PCBs, DDTs) and biochemical measurements varied seasonally. Elevated 7-ethoxyresorufin O-deethylase activity and CYP1A-protein levels in goby were correlated with high tissue contaminant levels at the industrial Porto Marghera site. On occasions, activities of the antioxidant enzymes catalase and putative DT-diaphorase (resorufin reductase activity) in male but not female goby were also higher at Porto Marghera than other sites, but no differences were seen in Superoxide dismutase (SOD) activity. A range of measurements (SOD, catalase, NADPH-cytochrome c reductase and glutathione
S-transferase activities, P450 and ‘418-peak’ contents) in mussel showed little difference between sites. However, indications were obtained of elevated levels of
CYP1A1-like mRNA, CYP1A-like protein and metabolism of benzoapyrene to free metabolites in mussels from the Venice lagoon compared to a site in the Adriatic Sea. The studies demonstrate the usefulness of CYP1A as a biomarker for organic pollution in fish and indicate some potential for its application in molluscs.
The induction of a cytochrome P450 with immunochemical similarities to CYP1A, and accompanying changes in microsomal NADPH-dependent benzoapyrene (BaP) metabolism, were examined in digestive gland of ...the common mussel (Mytilus galloprovincialis L.) with exposure to 20 ppb water-borne polychlorobiphenyl (PCB) mixture (Arochlor 1254) for 4 or 10 days, or 4 days after a single injection into the mantle cavity of the mixed-type inducer PCB congener 2,2′,3,4,4′,5′-hexachlorobiphenyl (CB-138; 2.5 μg g−1 wet weight). Whole animal tissue levels of PCB following water-column exposure or injection were similar to those for mussel species from polluted field sites, viz. 0.8 to 1.9 μg g−1 wet weight. Levels of microsomal CYP1A-immunopositive protein increased 59% (CB-138) and 72% (Arochlor 1254; 10 days exposure) as determined by Western blot analysis using polyclonal antibodies to hepatic CYP1A of perch (Perca fluviatilis). No changes were seen in levels of digestive gland CYP1A-like mRNA 4 days after injection of CB-138 as determined by Northern analysis using cDNA to hepatic CYP1A1 of rainbow trout (Oncorhynchus mykiss). The increases in levels of CYP1A-immunopositive protein were accompanied by a shift in microsomal NADPH-dependent BaP metabolism towards phenol and diol and away from dione production, the former increasing from 32 to 85% of total free metabolites. The marked decrease in dione production (which is the major BaP metabolite formed in control microsomes) resulted in no increase in total microsomal BaP metabolism with exposure to PCBs. The Type I ligand α-naphthoflavone markedly inhibited microsomal phenol but had no affect on dione production, whereas the Type II ligand clotrimazole markedly inhibited dione, but had much less effect on phenol production. The overall results are interpreted in terms of the existence of an inducible CYP1A-like enzyme catalysing predominantly 2-electron monooxygenation leading to epoxide (and hence phenol and diol) formation, and a constitutive non-inducible cytochrome P450 catalysing predominantly 1-electron oxidation leading to dione formation. Both Arochlor 1254 or CB-138 produced cellular damage in the digestive gland in the form of decreased epithelial digestive cell height and decreased lysosomal membrane stability.
The presence and expression of four gene families of the cytochrome P450 multi-gene family (CYP450) was investigated in the digestive gland of
M. edulis using Southern and Northern blot analysis. The ...cDNA probes employed were for CYP1A1 (pfP
1450-3′, 1.5kb from rainbow trout), CYP3A (phPCN12, human), CYP4A1 (2.1kb, rat) and CYP1A1 (1.9kb, human). Single hybridising mRNAs of approximately 2.1kb were seen for all probes, indicating presence and expression of genes from or similar to the four families. Hybridisation on Southern blots was demonstrated for CYP3A and CYP11A, confirming the presence of related CYP genes. The former gave a strong band at about 4.5kb and a weak one at 7.3kb, and the latter a single band at 13kb. The detection of CYP3A-like sequences is consistent with the reported presence of testosterone 6-β-hydroxylase activity in
M. edulis. Marked variation in levels of CYP1A-like mRNA were seen in mussels from several sites in the Venice area (UNESCO-MURST Venice Lagoon Project), indicating modulation of gene expression. Some correlation with contaminant exposure was seen, indicating potential for use of this measurement as a biomarker for organic pollution.
The presence and putative catalytic properties of a CYP1A-like enzyme in the digestive gland of
Mytilus edulis L. were investigated by molecular biological and seasonal studies. Reverse-transcriptase ...PCR using oligonucleotide primers to amplify a sequence around the conserved haem binding cysteine region of hepatic CYP1A1 of trout (
Oncorhynchus mykiss) produced several cDNA bands resolved by electrophoresis, including major bands of about 220 and 280 bp compared to the predicted size for
O. mykiss of 208 bp. Following Southern blotting and probing with a cDNA probe to
O. mykiss CYP1A1 (pfP
1450-3′ probe modified by removal of 3′-non-coding region by digestion by Cla1), a single band (280 bp) only was detected using moderate stringency conditions of sequence recognition (i.e. hybridization at 42 °C followed by washing at 55 °C in 1 × SSC containing 0.1% SDS), providing evidence for the presence of a CYP1A orthologous gene sequence. The seasonal variation in levels of putative CYP1A mRNA (Northern analysis using the modified cDNA probe for
O. mykiss CYP1A1) over 1 year showed some similarity to seasonal patterns of change in microsomal metabolism of
3H-benzoapyrene (BaP) to polar metabolites (dials, diones and phenols resolved by HPLC). Maxima for putative CYP1A mRNA and BaP metabolism levels were in late spring-early summer. However, differences were also apparent, possibly indicative of other P450s contributing to BaP metabolism. Overall the results indicate the existence of a CYP1A-like enzyme which is, at least, partly responsible for the mono-oxygenase activity of BaP metabolism.
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