Identification of diagnostic upper gastrointestinal cancer tissue type-specific urinary biomarkers.

Several potential urinary biomarkers exhibiting an association with upper gastrointestinal tumour growth have been previously identified, of which S100A6, S100A9, rabenosyn-5 and programmed cell death 6-interacting protein (PDCD6IP) were further validated and found to be upregulated in malignant tumours. The cancer cohort from our previous study was subclassified to assess whether distinct molecular markers can be identified for each individual cancer type using a similar approach. Urine samples from patients with cancers of the stomach, oesophagus, oesophagogastric junction or pancreas were analysed by surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry using both CM10 and IMAC30 (Cu2+-complexed) chip types and LC-MS/MS-based mass spectrometry after chromatographic enrichment. This was followed by protein identification, pattern matching and validation by western blotting. We found 8 m/z peaks with statistical significance for the four cancer types investigated, of which m/z 2447 and 2577 were identified by pattern matching as fragments of cathepsin-B (CTSB) and cystatin-B (CSTB); both molecules are indicative of pancreatic cancer. Additionally, we observed a potential association of upregulated α-1-antichymotrypsin with pancreatic and gastric cancers, of PDCD6IP, vitelline membrane outer layer protein 1 homolog (VMO1) and triosephosphate isomerase (TPI1) with oesophagogastric junctional cancers, and of complement C4-A, prostatic acid phosphatase, azurocidin and histone-H1 with oesophageal cancer. Furthermore, the potential pancreatic cancer biomarkers CSTB and CTSB were validated independently by western blotting. Therefore, the present study identified two new potential urinary biomarkers that appear to be associated with pancreatic cancer. This may provide a simple, non-invasive screening test for use in the clinical setting.

Urinary diagnostic proteomic markers for dynapenia in cancer patients.

Dynapenia is defined as the age-related loss of muscle strength, and plays a significant role in the loss of physical function and increased risk of disability among older individuals. The need for an early diagnosis supports the search for a biomarker that reflects muscle 'weakening'. This has previously proven difficult due to patient heterogeneity at presentation and lack of understanding of the underlying molecular mechanisms. The aim of the present study was to identify potential urinary biomarkers of dynapenia in patients undergoing potentially curative surgery for upper gastrointestinal cancer. Maximum isometric knee extensor strength (strain gauge) and maximum leg extensor power (Nottingham power rig) measurements were taken. Cut-off values for dynapenia were based on the Allied Dunbar national fitness survey. Values below the 5th percentile for the population matched for age and sex on the Allied Dunbar national fitness survey were used to stratify the cohort into dynapenic or normal. Urine samples taken at induction of anaesthesia were analysed by SELDI-TOF mass spectrometry using CM10 and IMAC30 chip-types to establish statistically significant m/z peak fingerprint patterns, followed by in-gel LC-MS/MS to identify molecular constituents. Statistical analysis of decision-tree calculations using Biomarker Pattern software resulted in models with sensitivities of 86 and 96%, specificities of 81 and 89%, and overall correctness of 84 and 93%, when applied to the entire cohort for power and strength measurement-based stratifications using the IMAC30 chip-type and the CM10 chip-type, respectively. The molecular identities of 10 peaks of interest were further investigated. After subtraction of potentially unrelated proteins, they were identified as fragments of Annexin A1, collagen α-1 (XV), perlecan and myotrophin. These results demonstrate that urinary screening can be used to define cancer-associated muscle weakness, and the identification of potential biomarkers could be invaluable in establishing a rapid test to measure and assess dynapenia in the clinical setting.

Proteomic identification of potential markers of myosteatosis in human urine.

Myosteatosis, the infiltration of fat in skeletal muscle, is associated with lower skeletal muscle density (SMD) as detected by computed tomography (CT). It increases with aging and obesity and is thought to play a role in the aetiology of insulin resistance and type II diabetes. The clinical significance of myosteatosis in cancer cachexia, however, remains to be determined. Along with demonstrable subcutaneous and visceral lipolysis, myosteatosis may also be a key component of the syndrome. We aimed to investigate the use of human urine as a non-invasive way to screen for molecular biomarkers of myosteatosis/reduced SMD using SELDI-TOF mass spectrometry. Pre-operative CT scans of patients undergoing surgery for upper gastrointestinal or hepatopancreaticobiliary cancer were analysed at the level of the third lumbar vertebrae. Myosteatosis was inferred as the presence of reduced SMD, which was defined as Hounsfield units for skeletal muscle <39.5 (two standard deviations below a normal healthy cohort). Urine was analysed by mass spectrometry using CM10 and IMAC30 SELDI-chips. Peaks observed in the CM10 and IMAC30 chip types, showed marked expressional differences between control and myosteatosis, were further investigated by mascot SELDI matrix matching. A total of 55 patients was recruited; 31 patients were found to be myosteatotic on CT scan. Application of the IMAC30-derived model to the entire cohort showed a sensitivity of 97%, specificity of 71% and an overall correctness of 85%. Application of the CM10 chipset-based model to the entire cohort, showed a 77% sensitivity, 67% specificity and 73% overall correctness. Analysis of the peaks of interest resulted in the identification of significant fragments of cathepsin C, argin, arylsulfatase A and glial fibrillary acidic protein. We identified several potential urinary molecular biomarkers associated with reduced SMD in cancer. Such markers are potentially useful in deriving a clinical screening test for myosteatosis.

Cancer-associated cachexia.

Cancer-associated cachexia is a disorder characterized by loss of body weight with specific losses of skeletal muscle and adipose tissue. Cachexia is driven by a variable combination of reduced food intake and metabolic changes, including elevated energy expenditure, excess catabolism and inflammation. Cachexia is highly associated with cancers of the pancreas, oesophagus, stomach, lung, liver and bowel; this group of malignancies is responsible for half of all cancer deaths worldwide. Cachexia involves diverse mediators derived from the cancer cells and cells within the tumour microenvironment, including inflammatory and immune cells. In addition, endocrine, metabolic and central nervous system perturbations combine with these mediators to elicit catabolic changes in skeletal and cardiac muscle and adipose tissue. At the tissue level, mechanisms include activation of inflammation, proteolysis, autophagy and lipolysis. Cachexia associates with a multitude of morbidities encompassing functional, metabolic and immune disorders as well as aggravated toxicity and complications of cancer therapy. Patients experience impaired quality of life, reduced physical, emotional and social well-being and increased use of healthcare resources. To date, no effective medical intervention completely reverses cachexia and there are no approved drug therapies. Adequate nutritional support remains a mainstay of cachexia therapy, whereas drugs that target overactivation of catabolic processes, cell injury and inflammation are currently under investigation.

The relationship between muscle protein content and CT-derived muscle radio-density in patients with upper GI cancer.

INTRODUCTION: Cancer cachexia is a multifactorial syndrome characterized by skeletal muscle loss. Cross-sectional analysis of CT scans is a recognized research method for assessing skeletal muscle volume. However, little is known about the relationship between CT-derived estimates of muscle radio-density (SMD) and muscle protein content. We assessed the relationship between CT-derived body composition variables and the protein content of muscle biopsies from cancer patients. METHODS: Rectus abdominis biopsies from cancer patients (n = 32) were analysed for protein content and correlated with phenotypic data gathered using CT body composition software. RESULTS: Skeletal muscle protein content varied widely between patients (median µg/mg wet weight = 89.3, range 70-141). There was a weak positive correlation between muscle protein content and SMD (r = 0.406, p = 0.021), and a weak positive correlation between protein content and percentage weight change (r = 0.416, p = 0.018). CONCLUSION: The protein content of skeletal muscle varies widely in cancer patients and cannot be accurately predicted by CT-derived muscle radio-density.

Alterations in the in vitro and in vivo regulation of muscle regeneration in healthy ageing and the influence of sarcopenia.

BACKGROUND: Sarcopenia is defined as the age-related loss of skeletal muscle mass and function. While all humans lose muscle with age, 2-5% of elderly adults develop functional consequences (disabilities). The aim of this study was to investigate muscle myogenesis in healthy elderly adults, with or without sarcopenia, compared with middle-aged controls using both in vivo and in vitro approaches to explore potential biomarker or causative molecular pathways associated with sarcopenic versus non-sarcopenic skeletal muscle phenotypes during ageing. METHODS: Biomarkers of multiple molecular pathways associated with muscle regeneration were analysed using quantitative polymerase chain reaction in quadriceps muscle samples obtained from healthy elderly sarcopenic (HSE, n = 7) or non-sarcopenic (HENS, n = 21) and healthy middle-aged control (HMC, n = 22) groups. An in vitro system of myogenesis (using myoblasts from human donors aged 17-83 years) was used to mimic the environmental challenges of muscle regeneration over time. RESULTS: The muscle biopsies showed evidence of satellite cell activation in HENS (Pax3, P < 0.01, Pax7, P < 0.0001) compared with HMC. Early myogenesis markers Myogenic Differentiation 1 (MyoD1) and Myogenic factor 5 (Myf5) (P < 0.0001) and the late myogenesis marker myogenin (MyoG) (P < 0.01) were increased in HENS. In addition, there was a 30-fold upregulation of TNF-α in HENS compared with HMC (P < 0.0001). The in vitro system demonstrated age-related upregulation of pro-inflammatory cytokines (2-fold upregulation of interleukin (IL)-6, IL-8 mRNA, increased secretion of tumor necrosis factor-α (TNF-α) and IL-6, all P < 0.05) associated with impaired kinetics of myotube differentiation. The HSE biopsy samples showed satellite cell activation (Pax7, P < 0.05) compared with HMC. However, no significant upregulation of the early myogenesis (MyoD and Myf5) markers was evident; only the late myogenesis marker myogenin was upregulated (P < 0.05). Higher activation of the oxidative stress pathway was found in HENS compared with the HSE group. In contrast, there was 10-fold higher upregulation of HSPA1A a stress-induced chaperone acting upon misfolded proteins in HSE compared with the HENS group. CONCLUSIONS: Both pathological and adaptive processes are active in skeletal muscle during healthy ageing. Muscle regeneration pathways are activated during healthy ageing, but there is evidence of dysregulation in sarcopenia. In addition, increased cellular stress, with an impaired oxidative-stress and mis-folded protein response (HSPA1A), may be associated with the development of sarcopenia. The in vitro system of young and old myoblasts replicated some of the differences between young and old muscle.

Body composition measurement using computed tomography: Does the phase of the scan matter?

OBJECTIVES: The aim of this study was to determine, from the methodologic standpoint, the effect of the presence or absence of intravenous contrast on body composition variables obtained by analysis of computed tomography (CT) images. METHODS: Triphasic abdominal (noncontrast, arterial phase, and portovenous phase contrast) CT scans from 111 patients were analyzed by two independent assessors at the third lumbar vertebral level using SliceOmatic software (version 5.0, TomoVision, Montreal, Canada). Variables included skeletal muscle index (SMI), fat and fat-free mass (FM and FFM, respectively), and mean skeletal muscle Hounsfield units (SMHU). RESULTS: Mean SMHU was lowest in the noncontrast phase (29.4, standard deviation [SD] 8.9 HU), followed by arterial (32.4, SD 9.3 HU) then portovenous phases (34.9, SD 9.4 HU). The mean skeletal muscle attenuation was significantly different depending on the phase of the scan in which the images were obtained. Calculated FM was significantly lower in both arterial (28.6, SD 8.8 kg, P < 0.0001) and portovenous phase scans (28.5, SD 8.9 kg, P < 0.0001) when compared with noncontrast (29.2, SD 8.9 kg). The mean FFM was not significantly different as measured on noncontrast, arterial, or portovenous phase CT scans (48, SD 11.2; 48.1, SD 9.8; and 48.6, SD 10.2 kg, respectively). No difference was seen in SMI. Interobserver reliability was high. CONCLUSIONS: The definition of myosteatosis should include a standardized phase of CT for analysis and this should be incorporated within its definition. However, as the magnitudes of the differences were relatively small, the effect of the phase of the scan on predicting outcome needs to be determined.

Comprehensive proteome analysis of human skeletal muscle in cachexia and sarcopenia: a pilot study.

BACKGROUND: Cancer cachexia (cancer-induced muscle wasting) is found in a subgroup of cancer patients leaving the patients with a poor prognosis for survival due to a lower tolerance of the chemotherapeutic drug. The cause of the muscle wasting in these patients is not fully understood, and no predictive biomarker exists to identify these patients early on. Skeletal muscle loss is an inevitable consequence of advancing age. As cancer frequently occurs in old age, identifying and differentiating the molecular mechanisms mediating muscle wasting in cancer cachexia vs. age-related sarcopenia are a challenge. However, the ability to distinguish between them is critical for early intervention, and simple measures of body weight may not be sufficiently sensitive to detect cachexia early. METHODS: We used a range of omics approaches: (i) undepleted proteome was quantified using advanced high mass accuracy mass spectrometers in SWATH-MS acquisition mode; (ii) phospho epitopes were quantified using protein arrays; and (iii) morphology was assessed using fluorescent microscopy. RESULTS: We quantified the soluble proteome of muscle biopsies from cancer cachexia patients and compared them with cohorts of cancer patients and healthy individuals with and without age-related muscle loss (aka age-related sarcopenia). Comparing the proteomes of these cohorts, we quantified changes in muscle contractile myosins and energy metabolism allowing for a clear identification of cachexia patients. In an in vitro time lapse experiment, we mimicked cancer cachexia and identified signal transduction pathways governing cell fusion to play a pivotal role in preventing muscle regeneration. CONCLUSIONS: The work presented here lays the foundation for further understanding of muscle wasting diseases and holds the promise of overcoming ambiguous weight loss as a measure for defining cachexia to be replaced by a precise protein signature.

New genetic signatures associated with cancer cachexia as defined by low skeletal muscle index and weight loss.

BACKGROUND: Cachexia affects the majority with advanced cancer. Based on current demographic and clinical factors, it is not possible to predict who will develop cachexia or not. Such variation may, in part, be due to genotype. It has recently been proposed to extend the diagnostic criteria for cachexia to include a direct measure of low skeletal muscle index (LSMI) in addition to weight loss (WL). We aimed to explore our panel of candidate single nucleotide polymorphism (SNPs) for association with WL +/- computerized tomography-defined LSMI. We also explored whether the transcription in muscle of identified genes was altered according to such cachexia phenotype METHODS: A retrospective cohort study design was used. Analysis explored associations of candidate SNPs with WL (n = 1276) and WL + LSMI (n = 943). Human muscle transcriptome (n = 134) was analysed using an Agilent platform. RESULTS: Single nucleotide polymorphisms in the following genes showed association with WL alone: GCKR, LEPR, SELP, ACVR2B, TLR4, FOXO3, IGF1, CPN1, APOE, FOXO1, and GHRL. SNPs in LEPR, ACVR2B, TNF, and ACE were associated with concurrent WL + LSMI. There was concordance between muscle-specific expression for ACVR2B, FOXO1 and 3, LEPR, GCKR, and TLR4 genes and LSMI and/or WL (P < 0.05). CONCLUSIONS: The rs1799964 in the TNF gene and rs4291 in the ACE gene are new associations when the definition of cachexia is based on a combination of WL and LSMI. These findings focus attention on pro-inflammatory cytokines and the renin-angiotensin system as biomarkers/mediators of muscle wasting in cachexia.

Proteomic identification of potential cancer markers in human urine using subtractive analysis.

Urine is an ideal medium in which to focus diagnostic cancer research due to the non-invasive nature and ease of sampling. Many large-scale proteomic studies have shown that urine is unexpectedly complex. We hypothesised that novel diagnostic cancer biomarkers could be discovered using a comparative proteomic analysis of pre-existing data. We assembled a database of 100 published datasets of 5,620 urinary proteins, as well as 46 datasets of 8,620 non-redundant proteins derived from kidney and blood proteome analyses. The data were then used to either subtract or compare molecules from a novel urinary proteome profiling dataset that we generated. We identified 1,161 unique proteins in samples from either cancer-bearing or healthy subjects. Subtractive analysis yielded a subset of 44 proteins that were found uniquely in urine from cancer patients, 30 of which were linked previously to cancer. In conclusion, this approach is useful in discovering novel biomarkers in tissues where unrelated profiling data is available. Only a limited disease-specific novel dataset is required to define new targets or substantiate previous findings. We have shared this discovery platform in the form of our Large Scale Screening Resource database, accessible through the Proteomic Analysis DataBase portal (www.PADB.org).

Low Muscularity and Myosteatosis Is Related to the Host Systemic Inflammatory Response in Patients Undergoing Surgery for Colorectal Cancer.

OBJECTIVE: We examined the relationships between computed tomography (CT)-defined skeletal muscle parameters and the systemic inflammatory response (SIR) in patients with operable primary colorectal cancer (CRC). BACKGROUND: Muscle depletion is characterized by a reduced muscle mass (myopenia) and increased infiltration by inter- and intramuscular fat (myosteatosis). It is recognized as a poor prognostic indicator in patients with cancer, but the underlying factors remain unclear. METHODS: A total of 763 patients diagnosed with CRC undergoing elective surgical resection between 2006 and 2013 were included. Image analysis of CT scans was used to calculate Lumbar skeletal muscle index (LSMI), and mean muscle attenuation (MA). The SIR was quantified by the preoperative neutrophil to lymphocyte ratio (NLR) and albumin levels. Correlation and multivariate regression analysis was performed to identify independent relationships between patient SIR and muscle characteristics. RESULTS: Patients with NLR > 3 had significantly lower LSMI and lower MA than those with NLR < 3 [LSMI = 42.07 cmm vs 44.27 cmm (P = 0.002) and MA = 30.04 Hounsfield unit (HU) vs 28.36 HU (P = 0.016)]. Multivariate logistic regression analysis showed that high NLR [odds ratio (OR) = 1.78 (95% confidence interval [CI]: 1.29-2.45), P < 0.001] and low albumin [OR = 1.80 (95% CI: 1.17-2.74), P = 0.007] were independent predictors of reduced muscle mass. High NLR was significantly related with a low mean MA and hence myosteatosis [OR = 1.60 (95% CI: 1.03-2.49), P = 0.038]. CONCLUSIONS: These results highlight a direct association between myopenia, myosteatosis, and the host SIR in patients with operable CRC. A better understanding of factors that regulate muscle changes such as myopenia and myosteatosis may lead to the development of novel therapies that influence a more metabolically "healthy" skeletal muscle and potentially alter cancer outcomes.

The impact of sarcopenia and myosteatosis on outcomes of unresectable pancreatic cancer or distal cholangiocarcinoma.

BACKGROUND & AIMS: Patients with pancreatic cancer have a poor prognosis, are often cachectic, and frequently demonstrate features of systemic inflammation, which may contribute to the phenomenon of myosteatosis. Analysis of body composition from CT scans has been used to study sarcopenia and its association with prognosis in a number of types of cancer, particular in combination with obesity. It has also been suggested that myosteatosis, defined as attenuated mean skeletal muscle Hounsfield units (HU), is associated with reduced survival in cancer. This study aimed to assess the association between body composition (sarcopenia and myosteatosis) and outcome in patients with unresectable pancreatic cancer. METHODS: All patients diagnosed with unresectable pancreatic cancer at Nottingham University Hospitals NHS Trust between 2006 and 2013 were considered for the study. A total of 228 patients were included retrospectively. Body composition was assessed using cross-sectional CT analysis to calculate a skeletal muscle index (SMI) for sarcopenia and use mean skeletal muscle HU for myosteatosis. RESULTS: The prevalence of sarcopenia in the whole patient group at baseline was 60.5% (138/228). Overall, patients who were sarcopenic had no significant difference in overall survival versus those who were not (p = 0.779). However, patients who were overweight/obese and sarcopenic had a significantly lower survival (p = 0.013). Of the 58 patients who were overweight or obese and sarcopenic, 32 were also myosteatotic. The prevalence of myosteatosis overall at baseline was 55.3% (126/228) and this was associated with significant reduction in overall survival (p = 0.049). Univariate Cox regression revealed myosteatosis but not sarcopenia to be predictive of reduced survival, however this relationship was lost on multivariate testing. Myosteatosis was associated with significantly greater levels of systemic inflammation (white cell count, neutrophil-lymphocyte ratio and C-reactive protein), anaemia and worsening of baseline blood urea. This relationship was not seen with sarcopenia. CONCLUSIONS: This is the largest study on the association between body composition and survival in patients with unresectable pancreatic cancer and has shown that although sarcopenia alone did not have a bearing on survival, the presence of myosteatosis was associated significantly with the presence of systemic inflammation and reduced survival.

Evaluating potential biomarkers of cachexia and survival in skeletal muscle of upper gastrointestinal cancer patients.

BACKGROUND: In order to grow the potential therapeutic armamentarium in the cachexia domain of supportive oncology, there is a pressing need to develop suitable biomarkers and potential drug targets. This pilot study evaluated several potential candidate biomarkers in skeletal muscle biopsies from a cohort of upper gastrointestinal cancer (UGIC) patients. METHODS: One hundred seven patients (15 weight-stable healthy controls (HC) and 92 UGIC patients) were recruited. Mean (standard deviation) weight-loss of UGIC patients was 8.1 (9.3%). Cachexia was defined as weight-loss ≥5%. Rectus abdominis muscle was obtained at surgery and was analysed by western blotting or quantitative real-time-polymerase chain reaction. Candidate markers were selected according to previous literature and included Akt and phosphorylated Akt (pAkt, n = 52), forkhead box O transcription factors (n = 59), ubiquitin E3 ligases (n = 59, control of muscle anabolism/catabolism), BNIP3 and GABARAPL1 (n = 59, as markers of autophagy), myosin heavy-chain (MyHC, n = 54), dystrophin (n = 39), ß-dystroglycan (n = 52), and ß-sarcoglycan (n = 52, as markers of structural alteration in a muscle). Patients were followed up for an average of 1255 days (range 581-1955 days) or until death. Patients were grouped accordingly and analysed by (i) all cancer patients vs. HC; (ii) cachectic vs. non-cachectic cancer patients; and (iii) cancer patients surviving ≤1 vs. >1 year post operatively. RESULTS: Cancer compared with HC patients had reduced mean (standard deviation) total Akt protein [0.49 (0.31) vs. 0.89 (0.17), P = 0.001], increased ratio of phosphorylated to total Akt [1.33 (1.04) vs. 0.32 (0.21), P = 0.002] and increased expression of GABARAPL1 [1.60 (0.76) vs. 1.10 (0.57), P = 0.024]. ß-Dystroglycan levels were higher in cachectic compared with non-cachectic cancer patients [1.01 (0.16) vs. 0.87 (0.20), P = 0.007]. Survival was shortened in patients with low compared with high MyHC levels (median 316 vs. 1326 days, P = 0.023) and dystrophin levels (median 341 vs. 660 days, P = 0.008). CONCLUSIONS: The present study has identified intramuscular protein level of ß-dystroglycan as a potential biomarker of cancer cachexia. Changes in the structural elements of muscle (MyHC or dystrophin) appear to be survival biomarkers.

Efficacy of Intravenous Lidocaine for Postoperative Analgesia Following Laparoscopic Surgery: A Meta-Analysis.

BACKGROUND: Intravenous (IV) lidocaine has analgesic and anti-inflammatory properties. This study aims to evaluate the efficacy of IV lidocaine in controlling postoperative pain following laparoscopic surgery. METHODS: A meta-analysis of randomised controlled trials (RCTs) comparing IV lidocaine versus placebo/routine treatment for postoperative analgesia following laparoscopic surgery. The primary outcome was opiate requirement at 24 h. Secondary outcomes included cumulative opiate requirement, numerical pain scores (2, 12, 24, 48 h at rest and on movement), recovery indices (nausea and vomiting, length of stay, time until diet resumption, first flatus and bowel movement) and side effects (cardiac/neurological toxicity). Subgroup analyses were performed according to operation type and to compare IV lidocaine with intraperitoneal lidocaine. RESULTS: Fourteen RCTs with 742 patients were included. IV lidocaine was associated with a small but significant reduction in opiate requirement at 24 h compared with placebo/routine care. IV lidocaine was associated with reduced cumulative opiate requirement, reduced pain scores at rest at 2, 12 and 24 h, reduced nausea and vomiting and a shorter time until resumption of diet. The length of stay did not differ between groups. There was a low incidence of IV lidocaine-associated toxicity. In subgroup analyses, there was no difference between IV and intraperitoneal lidocaine in the measured outcomes. CONCLUSIONS: IV lidocaine has a multidimensional effect on the quality of recovery. IV lidocaine was associated with lower opiate requirements, reduced nausea and vomiting and a shorter time until resumption of diet. Whilst IV lidocaine appears safe, the optimal treatment regimen remains unknown. Statistical heterogeneity was high.

Programmed cell death 6 interacting protein (PDCD6IP) and Rabenosyn-5 (ZFYVE20) are potential urinary biomarkers for upper gastrointestinal cancer.

PURPOSE: Cancer of the upper digestive tract (uGI) is a major contributor to cancer-related death worldwide. Due to a rise in occurrence, together with poor survival rates and a lack of diagnostic or prognostic clinical assays, there is a clear need to establish molecular biomarkers. EXPERIMENTAL DESIGN: Initial assessment was performed on urine samples from 60 control and 60 uGI cancer patients using MS to establish a peak pattern or fingerprint model, which was validated by a further set of 59 samples. RESULTS: We detected 86 cluster peaks by MS above frequency and detection thresholds. Statistical testing and model building resulted in a peak profiling model of five relevant peaks with 88% overall sensitivity and 91% specificity, and overall correctness of 90%. High-resolution MS of 40 samples in the 2-10 kDa range resulted in 646 identified proteins, and pattern matching identified four of the five model peaks within significant parameters, namely programmed cell death 6 interacting protein (PDCD6IP/Alix/AIP1), Rabenosyn-5 (ZFYVE20), protein S100A8, and protein S100A9, of which the first two were validated by Western blotting. CONCLUSIONS AND CLINICAL RELEVANCE: We demonstrate that MS analysis of human urine can identify lead biomarker candidates in uGI cancers, which makes this technique potentially useful in defining and consolidating biomarker patterns for uGI cancer screening.

Habitual Myofibrillar Protein Synthesis Is Normal in Patients with Upper GI Cancer Cachexia.

PURPOSE: Skeletal muscle wasting and weight loss are characteristic features of cancer cachexia and contribute to impaired function, increased morbidity, and poor tolerance of chemotherapy. This study used a novel technique to measure habitual myofibrillar protein synthesis in patients with cancer compared with healthy controls. EXPERIMENTAL DESIGN: An oral heavy water (87.5 g deuterium oxide) tracer was administered as a single dose. Serum samples were taken over the subsequent week followed by a quadriceps muscle biopsy. Deuterium enrichment was measured in body water, serum alanine, and alanine in the myofibrillar component of muscle using gas chromatography-pyrolysis-isotope ratio mass spectrometry and the protein synthesis rate calculated from the rate of tracer incorporation. Net change in muscle mass over the preceding 3 months was calculated from serial CT scans and allowed estimation of protein breakdown. RESULTS: Seven healthy volunteers, 6 weight-stable, and 7 weight-losing (≥5% weight loss) patients undergoing surgery for upper gastrointestinal cancer were recruited. Serial CT scans were available in 10 patients, who lost skeletal muscle mass preoperatively at a rate of 5.6%/100 days. Myofibrillar protein fractional synthetic rate was 0.058%, 0.061%, and 0.073%/hour in controls, weight-stable, and weight-losing patients, respectively. Weight-losing patients had higher synthetic rates than controls (P = 0.03). CONCLUSION: Contrary to previous studies, there was no evidence of suppression of myofibrillar protein synthesis in patients with cancer cachexia. Our finding implies a small increase in muscle breakdown may account for muscle wasting.

The energy balance in cancer cachexia revisited.

PURPOSE OF REVIEW: To review new putative mechanisms involved in the pathophysiology of a disturbed energy balance in cancer cachexia, which can lead to novel targets for clinical cachexia management. In the context of rapid developments in tumour treatment with potential systemic consequences, this article reviews recent data on energy requirements. Furthermore, we focus on new insights in brown adipose tissue (BAT) activity and reward processing in the brain in relation to the cachexia process. RECENT FINDINGS: Nearly no new data have been published on energy requirements of cancer patients in the light of comprehensive new therapies in oncology. New developments, such as the introduction of staging with 18F-fluorodeoxyglucose PET-computed tomography scanning, led to the observation that BAT activation may contribute to impaired energy balance in cancer cachexia. Animal and human data to date provide an indication that BAT activation indeed occurs, but its quantitative impact on the degree of cachexia is controversial. The peripheral and central nervous system is known to influence satiation, with a possible role for impaired food reward processing in the brain. To date, there are limited confirmatory data, but this is an interesting new area to explore for better understanding and treating cancer-induced anorexia. SUMMARY: The multimodal approach to counteract cancer cachexia should expand its targets to BAT and food reward processing in the brain.

Absence of correlation between serum CRP levels and mitochondrial D-loop DNA mutations in gastro-oesophageal adenocarcinoma.

INTRODUCTION: Both inflammation and mitochondrial DNA (mtDNA) mutation are thought to play a role in the many human cancers. The aim of this study was to evaluate the relationship between inflammation and accumulation of mitochondrial DNA (mtDNA) mutations in the D-loop region in carcinogenesis of gastro-oesophageal adenocarcinomas. MATERIALS AND METHODS: Blood samples of 20 patients with gastro-oesophageal adenocarcinoma were taken for measurement of serum C-reactive protein (CRP) concentration. Direct sequencing of mtDNA in the D-loop region was done in the 20 adenocarcinoma samples and their corresponding surrounding non-cancerous tissue. Sequences were compared with existing mtDNA databases to identify mutations. RESULTS: mtDNA mutations in the D-loop region occur commonly with almost identical frequency in both non-cancerous tissue (3.0 ± 1.6) and adenocarcinoma (3.1 ± 1.9) (P = 0.916, paired t-test). CRP levels are not predictive of the number of D-loop mutations in both adenocarcinoma (ß: -0.131; 95% CI: -2.354-1.364; P = 0.583) and non-cancerous tissue samples (ß: 0.130; 95% CI: -1.125-1.933; P = 0.586). Five new mutations were identified that were not recorded previously in mtDNA databases. CONCLUSION: D-loop mtDNA mutations are common in both gastro-oesophageal adenocarcinoma and surrounding non-cancerous tissue. However, the accumulation of such mutations appears to occur independent of systemic inflammation. The frequency of D-loop mutations is likely not useful as a marker for carcinogenesis in gastro-oesophageal adenocarcinoma.

Clinical classification of cancer cachexia: phenotypic correlates in human skeletal muscle.

BACKGROUND: Cachexia affects the majority of patients with advanced cancer and is associated with a reduction in treatment tolerance, response to therapy, and duration of survival. One impediment towards the effective treatment of cachexia is a validated classification system. METHODS: 41 patients with resectable upper gastrointestinal (GI) or pancreatic cancer underwent characterisation for cachexia based on weight-loss (WL) and/or low muscularity (LM). Four diagnostic criteria were used >5%WL, >10%WL, LM, and LM+>2%WL. All patients underwent biopsy of the rectus muscle. Analysis included immunohistochemistry for fibre size and type, protein and nucleic acid concentration, Western blots for markers of autophagy, SMAD signalling, and inflammation. FINDINGS: Compared with non-cachectic cancer patients, patients with LM or LM+>2%WL, mean muscle fibre diameter was reduced by about 25% (p = 0.02 and p = 0.001 respectively). No significant difference in fibre diameter was observed if patients had WL alone. Regardless of classification, there was no difference in fibre number or proportion of fibre type across all myosin heavy chain isoforms. Mean muscle protein content was reduced and the ratio of RNA/DNA decreased in patients with either >5%WL or LM+>2%WL. Compared with non-cachectic patients, SMAD3 protein levels were increased in patients with >5%WL (p = 0.022) and with >10%WL, beclin (p = 0.05) and ATG5 (p = 0.01) protein levels were increased. There were no differences in phospho-NFkB or phospho-STAT3 levels across any of the groups. CONCLUSION: Muscle fibre size, biochemical composition and pathway phenotype can vary according to whether the diagnostic criteria for cachexia are based on weight loss alone, a measure of low muscularity alone or a combination of the two. For intervention trials where the primary end-point is a change in muscle mass or function, use of combined diagnostic criteria may allow identification of a more homogeneous patient cohort, reduce the sample size required and enhance the time scale within which trials can be conducted.