Clinical Use of Cancer Biomarkers in Epithelial Ovarian Cancer: Updated Guidelines From the European Group on Tumor Markers.

OBJECTIVE: To present an update of the European Group on Tumor Markers guidelines for serum markers in epithelial ovarian cancer. METHODS: Systematic literature survey from 2008 to 2013. The articles were evaluated by level of evidence and strength of recommendation. RESULTS: Because of its low sensitivity (50-62% for early stage epithelial ovarian cancer) and limited specificity (94-98.5%), cancer antigen (CA) 125 (CA125) is not recommended as a screening test in asymptomatic women. The Risk of Malignancy Index, which includes CA125, transvaginal ultrasound, and menopausal status, is recommended for the differential diagnosis of a pelvic mass. Because human epididymis protein 4 has been reported to have superior specificity to CA125, especially in premenopausal women, it may be considered either alone or as part of the risk of ovarian malignancy algorithm, in the differential diagnosis of pelvic masses, especially in such women. CA125 should be used to monitor response to first-line chemotherapy using the previously published criteria of the Gynecological Cancer Intergroup, that is, at least a 50% reduction of a pretreatment sample of 70 kU/L or greater. The value of CA125 in posttherapy surveillance is less clear. Although a prospective randomized trial concluded that early administration of chemotherapy based on increasing CA125 levels had no effect on survival, European Group on Tumor Markers state that monitoring with CA125 in this situation should occur, especially if the patient is a candidate for secondary cytoreductive surgery. CONCLUSIONS: At present, CA125 remains the most important biomarker for epithelial ovarian cancer, excluding tumors of mucinous origin.

Validation of new cancer biomarkers: a position statement from the European group on tumor markers.

BACKGROUND: Biomarkers are playing increasingly important roles in the detection and management of patients with cancer. Despite an enormous number of publications on cancer biomarkers, few of these biomarkers are in widespread clinical use. CONTENT: In this review, we discuss the key steps in advancing a newly discovered cancer candidate biomarker from pilot studies to clinical application. Four main steps are necessary for a biomarker to reach the clinic: analytical validation of the biomarker assay, clinical validation of the biomarker test, demonstration of clinical value from performance of the biomarker test, and regulatory approval. In addition to these 4 steps, all biomarker studies should be reported in a detailed and transparent manner, using previously published checklists and guidelines. Finally, all biomarker studies relating to demonstration of clinical value should be registered before initiation of the study. SUMMARY: Application of the methodology outlined above should result in a more efficient and effective approach to the development of cancer biomarkers as well as the reporting of cancer biomarker studies. With rigorous application, all stakeholders, and especially patients, would be expected to benefit.

Design of tumor biomarker-monitoring trials: a proposal by the European Group on Tumor Markers.

A major application of tumor biomarkers is in serial monitoring of cancer patients, but there are no published guidelines on how to evaluate biomarkers for this purpose. The European Group on Tumor Markers has convened a multidisciplinary panel of scientists to develop guidance on the design of such monitoring trials. The panel proposes a 4-phase model for biomarker-monitoring trials analogous to that in use for the investigation of new drugs. In phase I, biomarker kinetics and correlation with tumor burden are assessed. Phase II evaluates the ability of the biomarker to identify, exclude, and/or predict a change in disease status. In phase III, the effectiveness of tumor biomarker-guided intervention is assessed by measuring patient outcome in randomized trials. Phase IV consists of an audit of the long-term effects after biomarker monitoring has been included into standard patient care. Systematic well-designed evaluations of biomarkers for monitoring may provide a stronger evidence base that might enable their earlier use in evaluating responses to cancer therapy.

Sampling and storage conditions influencing the measurement of parathyroid hormone in blood samples: a systematic review.

Parathyroid hormone (PTH) is relatively unstable: optimisation of pre-analytical conditions, including specimen type, sampling time and storage conditions, is essential. We have undertaken a systematic review of these pre-analytical conditions. An electronic search of the PubMed, Embase, Cochrane, Centre for Research and Dissemination and Bandolier databases was undertaken. Of 5511 papers identified, 96 underwent full text review, of which 83 were finally included. At room temperature PTH was stable in ethylenediaminetetraacetic acid (EDTA) preserved whole blood for at least 24 h and in EDTA plasma for at least 48 h after venepuncture. Losses were observed in clotted blood samples after 3 h and in serum after 2 h. At 4°C PTH was more stable in EDTA plasma (at least 72 h) than serum (at least 24 h). Central venous PTH concentrations were higher than peripheral venous concentrations. In the northern hemisphere, PTH concentrations were higher in winter than summer. PTH has a circadian rhythm characterised by a nocturnal acrophase and mid-morning nadir. Data related to frozen storage of PTH (-20°C and -80°C) were limited and contradictory. We recommend that blood samples for PTH measurement should be taken into tubes containing EDTA, ideally between 10:00 and 16:00, and plasma separated within 24 h of venepuncture. Plasma samples should be stored at 4°C and analysed within 72 h of venepuncture. Particular regard must be paid to the venepuncture site when interpreting PTH concentration. Further research is required to clarify the suitability of freezing samples prior to PTH measurement.

Roadmap for harmonization of clinical laboratory measurement procedures.

Results between different clinical laboratory measurement procedures (CLMP) should be equivalent, within clinically meaningful limits, to enable optimal use of clinical guidelines for disease diagnosis and patient management. When laboratory test results are neither standardized nor harmonized, a different numeric result may be obtained for the same clinical sample. Unfortunately, some guidelines are based on test results from a specific laboratory measurement procedure without consideration of the possibility or likelihood of differences between various procedures. When this happens, aggregation of data from different clinical research investigations and development of appropriate clinical practice guidelines will be flawed. A lack of recognition that results are neither standardized nor harmonized may lead to erroneous clinical, financial, regulatory, or technical decisions. Standardization of CLMPs has been accomplished for several measurands for which primary (pure substance) reference materials exist and/or reference measurement procedures (RMPs) have been developed. However, the harmonization of clinical laboratory procedures for measurands that do not have RMPs has been problematic owing to inadequate definition of the measurand, inadequate analytical specificity for the measurand, inadequate attention to the commutability of reference materials, and lack of a systematic approach for harmonization. To address these problems, an infrastructure must be developed to enable a systematic approach for identification and prioritization of measurands to be harmonized on the basis of clinical importance and technical feasibility, and for management of the technical implementation of a harmonization process for a specific measurand.

Variation in parathyroid hormone immunoassay results--a critical governance issue in the management of chronic kidney disease.

Renal physicians strive to maintain parathyroid hormone (PTH) concentrations for patients with chronic kidney disease (CKD) within guideline limits, but poor method comparability means there is currently serious risk of clinical misclassification. The potential for under- or over-treatment is significant, representing a major challenge to patient safety. In the short-term, raising awareness of clinical implications of method-related differences in PTH is essential. Agreeing and adopting assay-specific PTH action limits for CKD patients as an interim measure is highly desirable and has been achieved in Scotland. Establishing pre-analytical requirements for PTH is also a priority. In the longer term, re-standardization of PTH methods in terms of an appropriate International Standard is required. Provided commutability can be demonstrated, the recently established IS 95/646 for PTH (1-84) is a suitable candidate. Establishment of a well-characterized panel of samples of defined clinical provenance to enable manufacturers to determine appropriate reference intervals and clinical decision points is also recommended and will provide an invaluable clinical resource. Recent developments in mass spectrometry mean that a candidate reference measurement procedure for PTH is now achievable and will represent major progress. Concurrently, evidence-based recommendations on clinical requirements and performance goals for PTH are required. Improving the comparability of PTH results requires support from many stakeholders but is achievable.

Interaction of gonadal status with systemic inflammation and opioid use in determining nutritional status and prognosis in advanced pancreatic cancer.

PURPOSE: Hypogonadism has been linked with systemic inflammation and opioid use in males with advanced cancer. We aimed to investigate the interaction of gonadal status with systemic inflammation and opioids in determining nutritional status and prognosis in advanced pancreatic cancer. METHODS: One hundred and seventy-five patients (92 males, 83 postmenopausal females) with unresectable pancreatic cancer were studied. Serum sex steroids (total testosterone [TT], calculated free testosterone [cFT], oestradiol, sex hormone binding globulin), gonadotropins (follicle-stimulating hormone and luteinising hormone) and pro-inflammatory mediators (C-reactive protein [CRP], interleukin-6 [IL-6], soluble tumour necrosis factor receptor 75 [sTNFR75]) were measured, and nutritional assessment and opioid use recorded. RESULTS: Seventy-three percent of males were hypogonadal (by cFT definition). cFT correlated positively with BMI (r (2) =0.349; p< 0.001) and grip strength (r (2) = 0.229; p = 0.034) and inversely with weight loss (r (2) = -0.287; p = 0.007), CRP (r (2) = -0.426; p < 0.001) and IL-6 (r (2) = -0.303; p = 0.004). CRP (p= 0.007), opioid dosage (p = 0.009) and BMI (p = 0.005) were independent determinants of cFT on ANOVA. Hypogonadal males demonstrated worsened survival compared with eugonadal patients (TT: OR of death = 2.87; p < 0.001; cFT: OR = 2.26; p = 0.011). Furthermore, male opioid use was associated with decreased TT (p < 0.001) and cFT (p < 0.001) and worsened survival (OR = 1.96; p = 0.012). In contrast, 18% of postmenopausal females exhibited premenopausal ("hyperoestrogenic") oestradiol levels. Oestradiol correlated positively with sTNFR75 (r (2) = 0.299; p = 0.008). CRP (p < 0.001) was an independent determinant of oestradiol. Hyperoestrogenic females demonstrated worsened survival compared with eugonadal patients (OR = 2.43; p = 0.013). CONCLUSIONS: In males with pancreatic cancer, systemic inflammation and opioid use are associated with hypogonadism. Male hypogonadism and female hyperoestrogenism are associated with shortened survival in advanced pancreatic cancer.

National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for use of tumor markers in liver, bladder, cervical, and gastric cancers.

BACKGROUND: Updated National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for the use of tumor markers in the clinic have been developed. METHODS: Published reports relevant to use of tumor markers for 4 cancer sites--liver, bladder, cervical, and gastric--were critically reviewed. RESULTS: Alpha-fetoprotein (AFP) may be used in conjunction with abdominal ultrasound for early detection of hepatocellular carcinoma (HCC) in patients with chronic hepatitis or cirrhosis associated with hepatitis B or C virus infection. AFP concentrations >200 microg/L in cirrhotic patients with typical hypervascular lesions >2 cm in size are consistent with HCC. After a diagnosis of HCC, posttreatment monitoring with AFP is recommended as an adjunct to imaging, especially in the absence of measurable disease. Although several urine markers have been proposed for bladder cancer, none at present can replace routine cystoscopy and cytology in the management of patients with this malignancy. Some may, however, be used as complementary adjuncts to direct more effective use of clinical procedures. Although carcinoembryonic antigen and CA 19-9 have been proposed for use gastric cancer and squamous cell carcinoma antigen for use in cervical cancer, none of these markers can currently be recommended for routine clinical use. CONCLUSIONS: Implementation of these recommendations should encourage optimal use of tumor markers for patients with liver, bladder, cervical, or gastric cancers.

Differences in recognition of the 1st WHO international reference reagents for hCG-related isoforms by diagnostic immunoassays for human chorionic gonadotropin.

BACKGROUND: The 1st WHO International Reference Reagents (IRRs) for 6 human chorionic gonadotropin (hCG)-related molecular variants, highly purified and calibrated in substance concentrations by the IFCC Working Group for hCG, permit experimental elucidation of what commercially available hCG methods measure in molar terms and enable assessment of their fitness for clinical purposes. METHODS: Pools containing known amounts of the IRRs spiked into normal human serum were issued to participants through the UK National External Quality Assessment Service for hCG for a period of 7 years. Among 16 assays used, 4 recognized only hCG, whereas 6 recognized hCG and its free beta-subunit (hCGbeta), and 6 recognized hCG, hCGbeta, and the beta core fragment. RESULTS: Differences in calibration of current hCG assays are moderate. Mean recovery of the current International Standard (IS), hCG IS 75/589, was 107% (range 93% to 126%), whereas that of the IRR 99/688 for hCG was 139% (range 109%-164%). Between-method variation for the latter (CV 12.3%) was also greater than for IS 75/589 (CV 8.8%). Recognition of hCGbeta varied markedly (CV 37%). Most assays overestimated it, but 2 RIAs produced results that were slight underestimations. Recognition of the beta core fragment was even more variable (CV 57%) and was closest to equimolarity for the RIAs. CONCLUSIONS: Assays for hCG show considerable variation in their recognition of various forms of hCG, and this variability is the most important cause of method-related differences in hCG results in serum and an even more important cause of method-related differences in urine measurements. Equimolar recognition of the major hCG isoforms is essential if between-method comparability for hCG is to be improved.

National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for use of tumor markers in clinical practice: quality requirements.

BACKGROUND: This report presents updated National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines summarizing quality requirements for the use of tumor markers. METHODS: One subcommittee developed guidelines for analytical quality relevant to serum and tissue-based tumor markers in current clinical practice. Two other subcommittees formulated recommendations particularly relevant to the developing technologies of microarrays and mass spectrometry. RESULTS: Prerequisites for optimal use of tumor markers in routine practice include formulation of the correct clinical questions to ensure selection of the appropriate test, adherence to good clinical and laboratory practices (e.g., minimization of the risk of incorrect patient and/or specimen identification, tube type, or timing), use of internationally standardized and well-characterized methods, careful adherence to manufacturer instructions, and proactive and timely reactions to information derived from both internal QC and proficiency-testing specimens. Highly desirable procedures include those designed to minimize the risk of the reporting of erroneous results attributable to interferences such as heterophilic antibodies or hook effects, to facilitate the provision of informative clinical reports (e.g., cumulative and/or graphical reports, appropriately derived reference intervals, and interpretative comments), and when possible to integrate these reports with other patient information through electronic health records. Also mandatory is extensive validation encompassing all stages of analysis before introduction of new technologies such as microarrays and mass spectrometry. Provision of high-quality tumor marker services is facilitated by dialogue involving researchers, diagnostic companies, clinical and laboratory users, and regulatory agencies. CONCLUSIONS: Implementation of these recommendations, adapted to local practice, should encourage optimization of the clinical use of tumor markers.

National Academy of Clinical Biochemistry laboratory medicine practice guidelines for use of tumor markers in testicular, prostate, colorectal, breast, and ovarian cancers.

BACKGROUND: Updated National Academy of Clinical Biochemistry (NACB) Laboratory Medicine Practice Guidelines for the use of tumor markers in the clinic have been developed. METHODS: Published reports relevant to use of tumor markers for 5 cancer sites--testicular, prostate, colorectal, breast, and ovarian--were critically reviewed. RESULTS: For testicular cancer, alpha-fetoprotein, human chorionic gonadotropin, and lactate dehydrogenase are recommended for diagnosis/case finding, staging, prognosis determination, recurrence detection, and therapy monitoring. alpha-Fetoprotein is also recommended for differential diagnosis of nonseminomatous and seminomatous germ cell tumors. Prostate-specific antigen (PSA) is not recommended for prostate cancer screening, but may be used for detecting disease recurrence and monitoring therapy. Free PSA measurement data are useful for distinguishing malignant from benign prostatic disease when total PSA is <10 microg/L. In colorectal cancer, carcinoembryonic antigen is recommended (with some caveats) for prognosis determination, postoperative surveillance, and therapy monitoring in advanced disease. Fecal occult blood testing may be used for screening asymptomatic adults 50 years or older. For breast cancer, estrogen and progesterone receptors are mandatory for predicting response to hormone therapy, human epidermal growth factor receptor-2 measurement is mandatory for predicting response to trastuzumab, and urokinase plasminogen activator/plasminogen activator inhibitor 1 may be used for determining prognosis in lymph node-negative patients. CA15-3/BR27-29 or carcinoembryonic antigen may be used for therapy monitoring in advanced disease. CA125 is recommended (with transvaginal ultrasound) for early detection of ovarian cancer in women at high risk for this disease. CA125 is also recommended for differential diagnosis of suspicious pelvic masses in postmenopausal women, as well as for detection of recurrence, monitoring of therapy, and determination of prognosis in women with ovarian cancer. CONCLUSIONS: Implementation of these recommendations should encourage optimal use of tumor markers.

Improving the comparability of immunoassays for prostate-specific antigen (PSA): progress and problems.

BACKGROUND: Commutability of immunoassay test results is an important objective for laboratory medicine. METHODS: PSA is a clinically important analyte for which, as a consequence of a number of national and international initiatives over the last decade, considerable progress has been made towards improving method comparability. However, results from different assays are still not interchangeable, a situation that is only likely to improve once broad recommendations can be made about the most clinically relevant antibody combinations. CONCLUSIONS: Universal implementation of such recommendations would almost certainly improve between-method agreement substantially, provided careful attention were paid to assay design and use of appropriately pure secondary standards ensured.

Perspective and priorities for improvement of parathyroid hormone (PTH) measurement - A view from the IFCC Working Group for PTH.

Parathyroid hormone (PTH) measurement in serum or plasma is a necessary tool for the exploration of calcium/phosphate disorders, and is widely used as a surrogate marker to assess skeletal and mineral disorders associated with chronic kidney disease (CKD), referred to as CKD-bone mineral disorders (CKD-MBD). CKD currently affects >10% of the adult population in the United States and represents a major health issue worldwide. Disturbances in mineral metabolism and fractures in CKD patients are associated with increased morbidity and mortality. Appropriate identification and management of CKD-MBD is therefore critical to improving clinical outcome. Recent increases in understanding of the complex pathophysiology of CKD, which involves calcium, phosphate and magnesium balance, and is also influenced by vitamin D status and fibroblast growth factor (FGF)-23 production, should facilitate such improvement. Development of evidence-based recommendations about how best to use PTH is limited by considerable method-related variation in results, of up to 5-fold, as well as by lack of clarity about which PTH metabolites these methods recognise. This makes it difficult to compare PTH results from different studies and to develop common reference intervals and/or decision levels for treatment. The implications of these method-related differences for current clinical practice are reviewed here. Work being undertaken by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) to improve the comparability of PTH measurements worldwide is also described.

Common decision limits --The need for harmonised immunoassays.

The main aim of clinical guidelines is to encourage the best clinical outcome for patients and the best use of resources, no matter where patients are investigated or managed. Where guidelines incorporate decision limits based on levels of analytes in serum, plasma or urine these may determine whether or not to treat or may be used to tailor further treatment. Consideration should be given to the effect of method-related differences in results when implementing common decision limits. Available evidence suggests that for some analytes the implications for the patient may be serious, e.g. in terms of missed biopsies or unnecessary prostatic biopsies when prostate specific antigen is measured. Major causes of between-method differences are reviewed and means of addressing them considered.

The harmonisation of growth hormone measurements: taking the next steps.

For over 20 years differences in results of growth hormone (GH) measurement have been recognised as being significant enough to lead to misdiagnosis and inappropriate management of patients with GH-related disorders. Whilst issues of method standardisation, variable antibody specificity, use of different reporting units with different conversion factors, and interference from GH binding protein have been acknowledged as contributing to the discrepancies, inconsistent approaches to method harmonisation have hampered opportunities to enhance the evidence base for GH measurements. Amongst the first steps to be taken, international collaboratives recommended the universal adoption of the International Standard 98/547 and the reporting of results in mass units. Whilst inter-method variability may have improved over the last 10 years, clinically significant differences remain. A more recently recognised issue contributing to the discrepancies may be the differences in the matrix materials used by kit manufacturers to assign values to their calibrants. The establishment of an international harmonisation oversight group is recommended: its key roles to include identification of a commutable matrix reference material, assessing the clinical significance of assay interferents, the evaluation of liquid chromatography-mass spectrometry as a reference measurement procedure and the provision of acceptance criteria for the clinical application of GH methods.

External quality assessment of hormone determinations.

Hormone determinations are of central importance to the practice of Clinical Endocrinology, and ensuring their correct use and performance is a multidisciplinary responsibility involving clinicians, laboratory staff, manufacturers of diagnostic systems and healthcare regulatory agencies. All these professional groups have, therefore, an interest in external quality assessment (EQA) as an audit tool that can identify areas where use of tests in routine practice requires improvement to reduce risks to patients. This chapter reviews the principles of EQA, and outlines its strengths and limitations, illustrated with example data from the UK National External Quality Assessment Service (UK NEQAS). The immunological nature of many hormone assays, often further complicated by heterogeneity of analyte structure and lack of suitable calibrators, presents special problems for the designers of EQA schemes in ensuring that specimens are appropriate and that target values are accurate. Laboratory users of EQA should have sufficient knowledge of the characteristics of the EQA schemes in which they participate to make informed interpretation of their data. The trend since the 1980s for in-house assays designed in individual laboratories to be superseded by automated assays provided by a small number of diagnostics manufacturers places a special responsibility on manufacturers to ensure reliable assay design and calibration. In collaboration with other parties EQA can help identify priorities for improved assay design and calibration. Although traditionally the focus of EQA has been on assessing the analytical phase it can also make some assessment of other important aspects of performance, e.g. the consistency of reference ranges and how results are interpreted. Overall, EQA has a valuable role both in laboratory accreditation and as an educational resource, thereby helping to ensure and improve the quality of laboratory services that support patient care.

Analytical error and interference in immunoassay: minimizing risk.

Although generally robust, immunoassays remain vulnerable to occasional analytical errors that may have serious implications for patient care. Sporadic errors that occur as a result of properties of the specimen are particularly difficult to detect. They may be due to the presence of cross-reacting substances, antianalyte antibodies or antireagent antibodies, all of which may lead to erroneously high or low results. Low results may be observed for tumour markers due to high-dose hooking in the presence of very high analyte concentrations. Erroneous results can occur unexpectedly with any specimen and there is no practical means of identifying specimens likely to cause problems in immunoassays. The possibility of interference should always be considered when results do not appear to be in accord with the clinical picture. Errors can occur in even the best-managed laboratories and their early investigation is always desirable. If there is any doubt whatsoever about a result, clinical staff should be encouraged to contact the laboratory. Investigations for possible interference that can be undertaken in most laboratories include testing for linearity on dilution, recovery experiments, treatment with heterophilic blocking tubes and confirmation using a different method. It may be desirable to consult specialist laboratories if more complex studies are necessary. Informing clinical and laboratory staff of the ever-present possibility of unexpected interference, ensuring brief clinical details are available to laboratory staff, and above all facilitating excellent communication between laboratory and clinical staff are key to minimizing the risk of clinical mismanagement due to unsuspected interference.

The National Institute for Health and Clinical Excellence (NICE) guidelines for early detection of ovarian cancer: the pivotal role of the clinical laboratory.

The National Institute for Health and Clinical Excellence (NICE) has recently reviewed optimal means of early recognition and initial management of ovarian cancer, the leading cause of gynaecological death in the UK. The NICE guidelines state that general practitioners should measure serum CA125 in women presenting with persistent and continuous symptoms suggestive of ovarian cancer (e.g. abdominal distension, early satiety and loss of appetite or pelvic pain). If CA125 is ≥35 kU/L, the general practitioner should arrange an ultrasound scan of the abdomen and pelvis to enable calculation of the risk of malignancy score (RMI). Women with an RMI score of ≥250 should then be referred to a specialist multidisciplinary team. Successful implementation of these guidelines requires close liaison between primary care and laboratory medicine to ensure that CA125 is requested as a diagnostic aid only for women meeting the criteria stated in the guidelines. Preanalytical criteria must also be met and it is essential that both requestors and patients be aware of other possible causes of increases in CA125, as well as the fact that it is not raised in all patients with ovarian cancer. Guidance is being prepared to enable appropriate interpretation and implementation of the NICE guidelines. Continuous audit of the outcomes will also be essential to determine the effectiveness of this approach to early detection of ovarian cancer.

Mass spectrometric detection of candidate protein biomarkers of cancer cachexia in human urine.

Increased membrane permeability and myofibrillar protein breakdown are established features of cancer cachexia. Proteins released from cachectic muscle may be excreted in urine to act as biomarkers of the cachectic process. One-dimensional SDS polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption/ionisation or liquid chromatography tandem mass spectrometry was used to compare the protein content of urine from cachectic (>10% weight loss) (n=8) and weight-stable (n=8) gastro-oesophageal cancer patients and healthy controls (n=8). Plasma creatine kinase concentration was used as a marker of gross muscle breakdown. The number of protein species identified in cachectic samples (median 42; range 28-61; total 199) was greater than that identified in weight-stable cancer (median 15; range 9-28; total 79) and control samples (median 12.5; range 5-18; total 49) (P<0.001). Many of the proteins identified have not been reported previously in the urine of cancer patients. Proteins identified specifically in cachectic samples included muscle (myosin species), cytoskeletal (alpha-spectrin; nischarin) and microtubule-associated proteins (microtubule-actin crosslinking factor; microtubule-associated protein-1B; bullous pemphigoid antigen 1), whereas immunoglobulin kappa-light chain and zinc alpha-2 glycoprotein appeared to represent markers of cancer. The presence of myosin in urine (without an increase in plasma creatine kinase) is consistent with a specific loss of myosin as part of the cachectic process. Urinary proteomics using mass spectrometry can identify muscle-specific and non-muscle-specific candidate biomarkers of cancer cachexia.