Anne Dawnay

Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry.

Glycation of proteins forms fructosamines and advanced glycation endproducts. Glycation adducts may be risk markers and risk factors of disease development. We measured the concentrations of the early glycation adduct fructosyl-lysine and 12 advanced glycation endproducts by liquid chromatography with tandem mass spectrometric detection. Underivatized analytes were detected free in physiological fluids and in enzymic hydrolysates of cellular and extracellular proteins. Hydroimidazolones were the most important glycation biomarkers quantitatively; monolysyl adducts (N(epsilon)-carboxymethyl-lysine and N(epsilon)-1-carboxyethyl-lysine) were found in moderate amounts, and bis(lysyl)imidazolium cross-links and pentosidine in lowest amounts. Quantitative screening showed high levels of advanced glycation endproducts in cellular protein and moderate levels in protein of blood plasma. Glycation adduct accumulation in tissues depended on the particular adduct and tissue type. Low levels of free advanced glycation endproducts were found in blood plasma and levels were 10-100-fold higher in urine. Advanced glycation endproduct residues were increased in blood plasma and at sites of vascular complications development in experimental diabetes; renal glomeruli, retina and peripheral nerve. In clinical uraemia, the concentrations of plasma protein advanced glycation endproduct residues increased 1-7-fold and free adduct concentrations increased up to 50-fold. Comprehensive screening of glycation adducts revealed the relative and quantitative importance of alpha-oxoaldehyde-derived advanced glycation endproducts in physiological modification of proteins-particularly hydroimidazolones, the efficient renal clearance of free adducts, and the marked increases of glycation adducts in diabetes and uraemia-particularly free advanced glycation endproducts in uraemia. Increased levels of these advanced glycation endproducts were associated with vascular complications in diabetes and uraemia.

Ovarian cancer screening and mortality in the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS): a randomised controlled trial

Summary Background Ovarian cancer has a poor prognosis, with just 40% of patients surviving 5 years. We designed this trial to establish the effect of early detection by screening on ovarian cancer mortality. Methods In this randomised controlled trial, we recruited postmenopausal women aged 50–74 years from 13 centres in National Health Service Trusts in England, Wales, and Northern Ireland. Exclusion criteria were previous bilateral oophorectomy or ovarian malignancy, increased risk of familial ovarian cancer, and active non-ovarian malignancy. The trial management system confirmed eligibility and randomly allocated participants in blocks of 32 using computer-generated random numbers to annual multimodal screening (MMS) with serum CA125 interpreted with use of the risk of ovarian cancer algorithm, annual transvaginal ultrasound screening (USS), or no screening, in a 1:1:2 ratio. The primary outcome was death due to ovarian cancer by Dec 31, 2014, comparing MMS and USS separately with no screening, ascertained by an outcomes committee masked to randomisation group. All analyses were by modified intention to screen, excluding the small number of women we discovered after randomisation to have a bilateral oophorectomy, have ovarian cancer, or had exited the registry before recruitment. Investigators and participants were aware of screening type. This trial is registered with ClinicalTrials.gov, number NCT00058032. Findings Between June 1, 2001, and Oct 21, 2005, we randomly allocated 202 638 women: 50 640 (25·0%) to MMS, 50 639 (25·0%) to USS, and 101 359 (50·0%) to no screening. 202 546 (>99·9%) women were eligible for analysis: 50 624 (>99·9%) women in the MMS group, 50 623 (>99·9%) in the USS group, and 101 299 (>99·9%) in the no screening group. Screening ended on Dec 31, 2011, and included 345 570 MMS and 327 775 USS annual screening episodes. At a median follow-up of 11·1 years (IQR 10·0–12·0), we diagnosed ovarian cancer in 1282 (0·6%) women: 338 (0·7%) in the MMS group, 314 (0·6%) in the USS group, and 630 (0·6%) in the no screening group. Of these women, 148 (0·29%) women in the MMS group, 154 (0·30%) in the USS group, and 347 (0·34%) in the no screening group had died of ovarian cancer. The primary analysis using a Cox proportional hazards model gave a mortality reduction over years 0–14 of 15% (95% CI −3 to 30; p=0·10) with MMS and 11% (−7 to 27; p=0·21) with USS. The Royston-Parmar flexible parametric model showed that in the MMS group, this mortality effect was made up of 8% (−20 to 31) in years 0–7 and 23% (1–46) in years 7–14, and in the USS group, of 2% (−27 to 26) in years 0–7 and 21% (−2 to 42) in years 7–14. A prespecified analysis of death from ovarian cancer of MMS versus no screening with exclusion of prevalent cases showed significantly different death rates (p=0·021), with an overall average mortality reduction of 20% (−2 to 40) and a reduction of 8% (−27 to 43) in years 0–7 and 28% (−3 to 49) in years 7–14 in favour of MMS. Interpretation Although the mortality reduction was not significant in the primary analysis, we noted a significant mortality reduction with MMS when prevalent cases were excluded. We noted encouraging evidence of a mortality reduction in years 7–14, but further follow-up is needed before firm conclusions can be reached on the efficacy and cost-effectiveness of ovarian cancer screening. Funding Medical Research Council, Cancer Research UK, Department of Health, The Eve Appeal.

Prospective Study Using the Risk of Ovarian Cancer Algorithm to Screen for Ovarian Cancer

PURPOSE To evaluate prevalence screening in the first prospective trial of a new ovarian cancer screening (OCS) strategy (risk of ovarian cancer or ROC algorithm) on the basis of age and CA125 profile. PATIENTS AND METHODS Postmenopausal women, > or = 50 years were randomly assigned to a control group or screen group. Screening involved serum CA125, interpreted using the ROC algorithm. Participants with normal results returned to annual screening; those with intermediate results had repeat CA125 testing; and those with elevated values underwent transvaginal ultrasound (TVS). Women with abnormal or persistently equivocal TVS were referred for a gynecologic opinion. RESULTS Thirteen thousand five hundred eighty-two women were recruited. Of 6,682 women randomly assigned to screening, 6,532 women underwent the first screen. After the initial CA125, 5,213 women were classified as normal risk, 91 women elevated, and 1,228 women intermediate. On repeat CA125 testing of the latter, a further 53 women were classified as elevated risk. All 144 women with elevated risk had TVS. Sixteen women underwent surgery. Eleven women had benign pathology; one woman had ovarian recurrence of breast cancer; one woman had borderline; and three women had primary invasive epithelial ovarian cancer (EOC). The specificity and positive predictive value (PPV) for primary invasive EOC were 99.8% (95% CI, 99.7 to 99.9) and 19% (95% CI, 4.1 to 45.6), respectively. CONCLUSION An OCS strategy using the ROC algorithm is feasible and can achieve high specificity and PPV in postmenopausal women. It is being used in the United Kingdom Collaborative Trial of Ovarian Cancer Screening and in the United States in both the Cancer Genetics Network and the Gynecology Oncology Group trials of high-risk women.

The definition of acute kidney injury and its use in practice.

Acute kidney injury (AKI) is a common syndrome that is independently associated with increased mortality. A standardized definition is important to facilitate clinical care and research. The definition of AKI has evolved rapidly since 2004, with the introduction of the Risk, Injury, Failure, Loss, and End-stage renal disease (RIFLE), AKI Network (AKIN), and Kidney Disease Improving Global Outcomes (KDIGO) classifications. RIFLE was modified for pediatric use (pRIFLE). They were developed using both evidence and consensus. Small rises in serum creatinine are independently associated with increased mortality, and hence are incorporated into the current definition of AKI. The recent definition from the international KDIGO guideline merged RIFLE and AKIN. Systematic review has found that these definitions do not differ significantly in their performance. Health-care staff caring for children or adults should use standard criteria for AKI, such as the pRIFLE or KDIGO definitions, respectively. These efforts to standardize AKI definition are a substantial advance, although areas of uncertainty remain. The new definitions have enabled the use of electronic alerts to warn clinicians of possible AKI. Novel biomarkers may further refine the definition of AKI, but their use will need to produce tangible improvements in outcomes and cost effectiveness. Further developments in AKI definitions should be informed by research into their practical application across health-care providers. This review will discuss the definition of AKI and its use in practice for clinicians and laboratory scientists.

Profound Mishandling of Protein Glycation Degradation Products in Uremia and Dialysis

The aim of this study was to define the severe deficits of protein glycation adduct clearance in chronic renal failure and elimination in peritoneal dialysis (PD) and hemodialysis (HD) therapy using a liquid chromatography-triple quadrupole mass spectrometric detection method. Physiologic proteolysis of proteins damaged by glycation, oxidation, and nitration forms protein glycation, oxidation, and nitration free adducts that are released into plasma for urinary excretion. Inefficient elimination of these free adducts in uremia may lead to their accumulation. Patients with mild uremic chronic renal failure had plasma glycation free adduct concentrations increased up to five-fold associated with a decline in renal clearance. In patients with ESRD, plasma glycation free adducts were increased up to 18-fold on PD and up to 40-fold on HD. Glycation free adduct concentrations in peritoneal dialysate increased over 2- to 12-h dwell time, exceeding the plasma levels markedly. Plasma glycation free adducts equilibrated rapidly with dialysate of HD patients, with both plasma and dialysate concentrations decreasing during a 4-h dialysis session. It is concluded that there are severe deficits of protein glycation free adduct clearance in chronic renal failure and in ESRD on PD and HD therapy.

Risk Algorithm Using Serial Biomarker Measurements Doubles the Number of Screen-Detected Cancers Compared With a Single-Threshold Rule in the United Kingdom Collaborative Trial of Ovarian Cancer Screening

Purpose Cancer screening strategies have commonly adopted single-biomarker thresholds to identify abnormality. We investigated the impact of serial biomarker change interpreted through a risk algorithm on cancer detection rates. Patients and Methods In the United Kingdom Collaborative Trial of Ovarian Cancer Screening, 46,237 women, age 50 years or older underwent incidence screening by using the multimodal strategy (MMS) in which annual serum cancer antigen 125 (CA-125) was interpreted with the risk of ovarian cancer algorithm (ROCA). Women were triaged by the ROCA: normal risk, returned to annual screening; intermediate risk, repeat CA-125; and elevated risk, repeat CA-125 and transvaginal ultrasound. Women with persistently increased risk were clinically evaluated. All participants were followed through national cancer and/or death registries. Performance characteristics of a single-threshold rule and the ROCA were compared by using receiver operating characteristic curves. Results After 296,911 women-years of annual incidence screening, 640 women underwent surgery. Of those, 133 had primary invasive epithelial ovarian or tubal cancers (iEOCs). In all, 22 interval iEOCs occurred within 1 year of screening, of which one was detected by ROCA but was managed conservatively after clinical assessment. The sensitivity and specificity of MMS for detection of iEOCs were 85.8% (95% CI, 79.3% to 90.9%) and 99.8% (95% CI, 99.8% to 99.8%), respectively, with 4.8 surgeries per iEOC. ROCA alone detected 87.1% (135 of 155) of the iEOCs. Using fixed CA-125 cutoffs at the last annual screen of more than 35, more than 30, and more than 22 U/mL would have identified 41.3% (64 of 155), 48.4% (75 of 155), and 66.5% (103 of 155), respectively. The area under the curve for ROCA (0.915) was significantly (P = .0027) higher than that for a single-threshold rule (0.869). Conclusion Screening by using ROCA doubled the number of screen-detected iEOCs compared with a fixed cutoff. In the context of cancer screening, reliance on predefined single-threshold rules may result in biomarkers of value being discarded.

Pre- and post-analytical factors that may influence use of serum prostate specific antigen and its isoforms in a screening programme for prostate cancer.

In 1995 the Standing Group on Health Technology Assessment commissioned two systematic reviews on screening for prostate cancer. The authors of these reports concluded that, at the time of the writing, the evidence available did not support the introduction of a screening programme. The main reasons given for this conclusion were that: (1) the serum prostate speci®c antigen (PSA) test in observational studies had resulted in a number of false positives and false negatives; (2) there were concerns about the potential costs (®nancial, social and psychological) of a screening programme; (3) there were concerns about the clinical and cost-effectiveness of treatments for localized disease; and (4) there was a need to look for prognostic markers that would identify those tumours likely to progress more rapidly and hence require more active treatment. Furthermore, there were overall concerns about the quality of the studies undertaken to date. At the time of the reviews referred to earlier, serum PSA was the biochemical marker of choice for prostate malignancy. Older markers such as prostatic acid phosphatase had been dropped and other potential markers had not been suf®ciently well developed to warrant further consideration. Since that time it has been recognized that there are several isoforms of PSA in the circulation. Furthermore, there are new methods of assay calibration and a recognition that several pre-analytical factors may affect the circulating level of PSA. The present review was commissioned by a steering group of the National Screening Committee to assess the preand post-analytical issues surrounding the measurement of PSA; the ®ndings should be taken into account in any future proposals for the implementation of a screening programme. An electronic search was undertaken using Medline and Embase, supported by handsearching of the most relevant journals and review articles.

Plasma tissue inhibitor of metalloproteinase-1 levels are elevated in essential hypertension and related to left ventricular hypertrophy

BACKGROUND Essential hypertensive patients have an increased heart and arterial collagen concentration. Increased collagen synthesis can be assessed using procollagen III N peptide (PIIINP) and reduced collagen degradation measured using tissue inhibitor of metalloproteinase-1 (TIMP-1). METHODS Plasma TIMP-1 and PIIINP levels were measured in 31 patients with essential hypertension and in 17 normotensive control subjects. The hypertensive patients were either treatment naive (n = 18) or had been without treatment for 1 month (n = 13). Both groups of patients were screened to exclude other fibrotic diseases. RESULTS In the hypertensive patients, TIMP-1 levels were significantly (P < .0002) elevated (median 380 ng/ mL, range 160 to 1,560 ng/mL) compared with those of the normotensive control subjects (median 178 ng/mL, range 99 to 330 ng/mL). In hypertensive subjects who had never received antihypertensive therapy there were significant correlations between TIMP-1 and left ventricular posterior wall thickness in diastole (LVPWd) (r = 0.58) (P < .02) and left ventricular mass index (r = 0.58) (P < .02). There was no difference in PIIINP levels (mean +/- 2 SD) between the hypertensive (0.56 U/mL +/- 0.3) and normotensive groups (0.52 U/mL +/- 0.2). CONCLUSIONS The increased tissue collagen III levels found in the heart and vessels of hypertensive patients is due to a reduction in collagen degradation because of high TIMP-1 levels, rather than an increase in synthesis of collagen type III. The tissue source of this TIMP-1 is unclear.

Microarray Glycoprofiling of CA125 Improves Differential Diagnosis of Ovarian Cancer

The CA125 biomarker assay plays an important role in the diagnosis and management of primary invasive epithelial ovarian/tubal cancer (iEOC). However, a fundamental problem with CA125 is that it is not cancer-specific and may be elevated in benign gynecological conditions such as benign ovarian neoplasms and endometriosis. Aberrant O-glycosylation is an inherent and specific property of cancer cells and could potentially aid in differentiating cancer from these benign conditions, thereby improving specificity of the assay. We report on the development of a novel microarray-based platform for profiling specific aberrant glycoforms, such as Neu5Acα2,6GalNAc (STn) and GalNAc (Tn), present on CA125 (MUC16) and CA15-3 (MUC1). In a blinded cohort study of patients with an elevated CA125 levels (30-500 kU/L) and a pelvic mass from the UK Ovarian Cancer Population Study (UKOPS), we measured STn-CA125, ST-CA125 and STn-CA15-3. The combined glycoform profile was able to distinguish benign ovarian neoplasms from invasive epithelial ovarian/tubule cancer (iEOCs) with a specificity of 61.1% at 90% sensitivity. The findings suggest that microarray glycoprofiling could improve differential diagnosis and significantly reduce the number of patients elected for further testing. The approach warrants further investigation in other cancers.

A genetic polymorphism of a human urinary mucin

We report here a novel genetically determined polymorphism of a human urinary mucin which is demonstrable by the separation technique of SDS polyacrylamide gel electrophoresis, followed by detection with radio‐iodinated lectins. The mucins are demonstrable using various lectins but the polymorphism is most easily recognized using peanut agglutinin and we therefore propose to designate this new genetic locus PUM (peanut‐reactive urinary mucin). Four common alleles have been identified and an autosomal codominant mode of inheritance has been found in the families studied so far.