Douglas G. Ward

Identification of serum biomarkers for colon cancer by proteomic analysis

Colorectal cancer (CRC) is often diagnosed at a late stage with concomitant poor prognosis. Early detection greatly improves prognosis; however, the invasive, unpleasant and inconvenient nature of current diagnostic procedures limits their applicability. No serum-based test is currently of sufficient sensitivity or specificity for widespread use. In the best currently available blood test, carcinoembryonic antigen exhibits low sensitivity and specificity particularly in the setting of early disease. Hence, there is great need for new biomarkers for early detection of CRC. We have used surface-enhanced laser desorbtion/ionisation (SELDI) to investigate the serum proteome of 62 CRC patients and 31 noncancer subjects. We have identified proteins (complement C3a des-arg, α1-antitrypsin and transferrin) with diagnostic potential. Artificial neural networks trained using only the intensities of the SELDI peaks corresponding to identified proteins were able to classify the patients used in this study with 95% sensitivity and 91% specificity.

Results of the first international round robin for the quantification of urinary and plasma hepcidin assays: need for standardization

This study indicates that hepcidin levels reported by various methods vary considerably but analytical variance is generally low and similar for all methods. See related perspective article on page 1631. The recently discovered iron regulatory peptide hormone hepcidin holds promise as a novel biomarker in iron metabolism disorders. To date, various mass spectrometry and immunochemical methods have been developed for its quantification in plasma and urine. Differences in methodology and analytical performance hinder the comparability of data. As a first step towards method harmonization, several hepcidin assays were compared. Worldwide eight laboratories participated in a urinary and plasma round robin in which hepcidin was analyzed. For both urine and plasma: (i) the absolute hepcidin concentrations differed widely between methods, (ii) the between-sample variation and the analytical variation of the methods are similar. Importantly, the analytical variation as percentage of the total variance is low for all methods, indicating their suitability to distinguish hepcidin levels of different samples. Spearman correlations between methods were generally high. The round robin results inform the scientific and medical community on the status and agreement of the current hepcidin methods. Ongoing initiatives should facilitate standardization by exchanging calibrators and representative samples.

Fast targeted multidimensional NMR metabolomics of colorectal cancer

The study of small molecules in body fluids has become an important tool to monitor the state of biological organisms. Applications range from model studies using cell lines to applications where human body fluids are used to monitor disease states or drug responses. NMR spectroscopy has been an important tool for metabolomics although severe overlap of signals has limited the number of compounds, which can be unambiguously identified and quantified. Therefore, deconvolution of NMR spectra is one of the greatest challenges for NMR‐based metabolomics. This has commonly been achieved by using multidimensional spectra that have the disadvantage of requiring significantly longer acquisition times. Recently, a number of methods have been described to record NMR spectra much faster. Here, we explore the use of Hadamard‐encoded TOCSY spectra to simultaneously select multiple lines from crowded NMR spectra of blood serum samples to acquire pseudo‐two‐dimensional spectra in minutes which would otherwise require many hours. The potential of this approach is demonstrated for the detection of a signature for colorectal cancer from human blood samples. Copyright

SELDI-TOF-MS determination of hepcidin in clinical samples using stable isotope labelled hepcidin as an internal standard

BackgroundHepcidin is a 25-residue peptide hormone crucial to iron homeostasis. It is essential to measure the concentration of hepcidin in cells, tissues and body fluids to understand its mechanisms and roles in physiology and pathophysiology. With a mass of 2791 Da hepcidin is readily detectable by mass spectrometry and LC-ESI, MALDI and SELDI have been used to estimate systemic hepcidin concentrations by analysing serum or urine. However, peak heights in mass spectra may not always reflect concentrations in samples due to competition during binding steps and variations in ionisation efficiency. Thus the purpose of this study was to develop a robust assay for measuring hepcidin using a stable isotope labelled hepcidin spiking approach in conjunction with SELDI-TOF-MS.ResultsWe synthesised and re-folded hepcidin labelled with 13C/15N phenylalanine at position 9 to generate an internal standard for mass spectrometry experiments. This labelled hepcidin is 10 Daltons heavier than the endogenous peptides and does not overlap with the isotopic envelope of the endogenous hepcidin or other common peaks in human serum or urine mass spectra and can be distinguished in low resolution mass spectrometers. We report the validation of adding labelled hepcidin into serum followed by SELDI analysis to generate an improved assay for hepcidin.ConclusionWe demonstrate that without utilising a spiking approach the hepcidin peak height in SELDI spectra gives a good indication of hepcidin concentration. However, a stable isotope labelled hepcidin spiking approach provides a more robust assay, measures the absolute concentration of hepcidin and should facilitate inter-laboratory hepcidin comparisons.

Changes in the serum proteome associated with the development of hepatocellular carcinoma in hepatitis C-related cirrhosis

Early diagnosis of hepatocellular carcinoma (HCC) is the key to the delivery of effective therapies. The conventional serological diagnostic test, estimation of serum alpha-fetoprotein (AFP) lacks both sensitivity and specificity as a screening tool and improved tests are needed to complement ultrasound scanning, the major modality for surveillance of groups at high risk of HCC. We have analysed the serum proteome of 182 patients with hepatitis C-induced liver cirrhosis (77 with HCC) by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI). The patients were split into a training set (84 non-HCC, 60 HCC) and a ‘blind’ test set (21 non-HCC, 17 HCC). Neural networks developed on the training set were able to classify the blind test set with 94% sensitivity (95% CI 73–99%) and 86% specificity (95% CI 65–95%). Two of the SELDI peaks (23/23.5 kDa) were elevated by an average of 50% in the serum of HCC patients (P<0.001) and were identified as κ and λ immunoglobulin light chains. This approach may permit identification of several individual proteins, which, in combination, may offer a novel way to diagnose HCC.

Proteomic profiling of urine for the detection of colon cancer

BackgroundColorectal cancer is the second most common cause of cancer related death in the developed world. To date, no blood or stool biomarkers with both high sensitivity and specificity for potentially curable early stage disease have been validated for clinical use. SELDI and MALDI profiling are being used increasingly to search for biomarkers in both blood and urine. Both techniques provide information predominantly on the low molecular weight proteome (<15 kDa). There have been several reports that colorectal cancer is associated with changes in the serum proteome that are detectable by SELDI and we hypothesised that proteomic changes would also be detectable in urine.ResultsWe collected urine from 67 patients with colorectal cancer and 72 non-cancer control subjects, diluted to a constant protein concentration and generated MALDI and SELDI spectra. The intensities of 19 peaks differed significantly between cancer and non-cancer patients by both t-tests and after adjusting for confounders using multiple linear regressions. Logistic regression classifiers based on peak intensities identified colorectal cancer with up to 78% sensitivity at 87% specificity. We identified and independently quantified 3 of the discriminatory peaks using synthetic stable isotope peptides (an 1885 Da fragment of fibrinogen and hepcidin-20) or ELISA (β2-microglobulin).ConclusionChanges in the urine proteome may aid in the early detection of colorectal cancer.

The effect of carbohydrate ingestion on plasma interleukin-6, hepcidin and iron concentrations following prolonged exercise.

The aim of our study was twofold, firstly to examine the relationship between plasma concentrations of IL-6, hepcidin and iron following prolonged exercise and secondly, to assess the effect of carbohydrate ingestion on circulating hepcidin concentration post-exercise. The study was a randomised double-blind cross-over design, with participants consuming either a carbohydrate (CHO) or an isovolumetric placebo drink throughout the trial. Nine healthy, trained males completed a treadmill run at 60% vVO(2max) for 120 min followed by a 5 km time trial. Plasma concentrations of both IL-6 and hepcidin significantly increased post-exercise following both trials (p<.05) and returned to baseline by 24 h post (p>.05). A positive correlation between hepcidin and IL-6 was demonstrated immediately following exercise during PLA while there was a trend for a moderate correlation during CHO (PLA trial rho=0.81, p<0.001; CHO trial rho=0.36, p=0.07). Plasma iron was unaffected immediately post-exercise but significantly reduced by 24 h post-exercise compared to baseline. CHO ingestion significantly reduced post-exercise IL-6 (p<.05) but this had no effect on plasma hepcidin or iron concentration. Our data demonstrate CHO supplementation does not alter the rapid hepcidin response associated with exercise and does not prevent a subsequent fall in plasma iron concentration. This finding adds further support to the theory that an exercise-induced, up-regulation of hepcidin activity is a mechanism causing iron deficiency in endurance athletes.

Identification of macrophage migration inhibitory factor and human neutrophil peptides 1–3 as potential biomarkers for gastric cancer

Background:Proteomic methods have the potential to meet the urgent need for better cancer biomarkers. We have used a range of proteomic analyses of serum and tissue from gastric cancer patients and relevant controls to discover biomarkers for gastric cancer.Methods:Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI) and antibody arrays were used to compare protein expression in 21 pairs of gastric cancer tissue and adjacent normal mucosa and serum from 51 gastric cancer patients and 29 patients with benign gastric diseases. Expression differences were confirmed by enzyme-linked immunosorbent assay.Results:Tissue analysis shows human neutrophil peptides 1–3 (HNPs 1–3) elevated 10-fold (P=0.001) in gastric cancer relative to adjacent normal mucosa. Macrophage migration inhibitory factor (MIF) was increased five-fold (P=1.84 × 10−7) in the serum of gastric cancer patients relative to individuals with benign gastric disease. The large increase in MIF concentration in serum gives an area under the receiver operating characteristic curve of 0.85.Conclusions:Proteomic analyses of serum and tissue indicate that HNPs 1–3 and MIF have potential as biomarkers for gastric cancer. In particular MIF may be useful, either alone or in combination with other markers, for diagnosing and monitoring gastric cancer.

Functional Analysis of a Unique Troponin C Mutation, GLY159ASP, that Causes Familial Dilated Cardiomyopathy, Studied in Explanted Heart Muscle

Background—Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca2+ sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca2+-regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation. Methods and Results—Mass spectroscopy showed that the mutant cTnC was expressed approximately equimolar with wild-type cTnC. Contraction was compared in skinned ventricular myocytes from the cTnC G159D patient and nonfailing donor heart. Maximal Ca2+-activated force was similar in cTnC G159D and donor myocytes, but the Ca2+ sensitivity of cTnC G159D myocytes was higher (EC50 G159D/donor=0.60). Thin filaments reconstituted with skeletal muscle actin and human cardiac tropomyosin and troponin were studied by in vitro motility assay. Thin filaments containing the mutation had a higher Ca2+ sensitivity (EC50 G159D/donor=0.55±0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca2+ sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca2+ sensitivity was increased (EC50 P/unP=4.7±1.9) but not with cTnC G159D troponin (EC50 P/unP=1.2±0.1). Conclusions—We propose that uncoupling of the relationship between phosphorylation and Ca2+ sensitivity could be the cause of the dilated cardiomyopathy phenotype. The differences between these data and previous in vitro results show that native phosphorylation of troponin I and troponin T and other posttranslational modifications of sarcomeric proteins strongly influence the functional effects of a mutation.

Combined proteome and transcriptome analyses for the discovery of urinary biomarkers for urothelial carcinoma

Background:Proteomic discovery of cancer biomarkers in body fluids is challenging because of their low abundance in a complex background. Altered gene expression in tumours may not reflect protein levels in body fluids. We have tested combining gene expression profiling of tumours with proteomic analysis of cancer cell line secretomes as a strategy to discover urinary biomarkers for bladder cancer.Methods:We used shotgun proteomics to identify proteins secreted by three bladder cancer cell lines. Secreted proteins with high mRNA levels in bladder tumours relative to normal urothelium were assayed by ELISA in urine samples from 642 patients.Results:Midkine and HAI-1 were significantly increased in bladder cancer patients, with the highest levels in invasive disease (area under the receiver operating characteristic curve 0.89 vs non-cancer). The urinary concentration of both proteins was too high to be explained by bladder cancer associated haematuria and most likely arises by direct tumour secretion.Conclusions:This ‘dual-omic’ strategy identified tumour secreted proteins whose urine concentrations are increased significantly by bladder cancer. Combined secretome-transcriptome analysis may be more useful than direct proteomic analysis of body fluids for biomarker discovery in both bladder cancer and other tumour types.