Endoscopic surveillance alone is feasible and safe in type I gastric neuroendocrine neoplasms less than 10 mm in diameter.

PURPOSE: Type I gastric neuroendocrine neoplasms (g-NENs) have a low risk of metastasis and a generally favourable prognosis. Patients with small type I g-NENs (≤10 mm) frequently require no treatment, whereas those with larger polyps usually undergo resection. We evaluated the safety and outcomes of endoscopic surveillance after no initial treatment in selected patients with type I g-NENs. METHODS: Retrospective analysis of type I g-NEN patients across two European Neuroendocrine Tumour Society Centers of Excellence 2003-2019. RESULTS: Following initial assessment, 87 of 115 patients with type I g-NEN (75 with polyps ≤10 mm) received no initial treatment and underwent endoscopic surveillance. 79/87 (91%) demonstrated no clinically meaningful change in tumour size or grade over a median 62 month follow up. Only two patients developed NEN progression that required a change in management and two other patients developed gastric adenocarcinoma/high grade dysplasia; all four initially had ≥11 mm g-NENs. CONCLUSIONS: Patients with ≤10 mm type I g-NENs were unlikely to develop clinically significant tumour progression and in most cases, resection was not needed. The endoscopic surveillance interval could therefore potentially be safely increased to every 2-3 years in such patients. However, lifelong surveillance is still advocated due to the additional risk of developing gastric adenocarcinoma.

New Developments in Gastric Neuroendocrine Neoplasms.

PURPOSE OF REVIEW: Gastric neuroendocrine neoplasms (g-NENs) are a rare type of stomach cancer. The three main subtypes have different pathogeneses, biological behaviours and clinical characteristics, so they require different management strategies. This article will provide an overview of g-NENs and highlight recent advances in the field. RECENT FINDINGS: Molecular profiling has revealed differences between indolent and aggressive g-NENs, as well as a new somatic mutation responsible for some familial type I g-NENs. Novel biomarkers have been developed which will hopefully improve diagnosis, treatment, risk stratification and follow-up. Patient treatment is also changing, as evidence supports the use of less aggressive options (e.g. endoscopic surveillance or resection) in some patients with more indolent tumours. g-NEN heterogeneity poses challenges in understanding and managing this rare disease. More basic science research is needed to investigate molecular pathogenesis, and future larger clinical studies will hopefully also further improve treatment and patient outcomes.

Plasma Metabolomics Identifies Lipid and Amino Acid Markers of Weight Loss in Patients with Upper Gastrointestinal Cancer.

Cachexia is a multifactorial wasting syndrome associated with high morbidity and mortality in patients with cancer. Diagnosis can be difficult and, in the clinical situation, usually relies upon reported weight loss. The 'omics' technologies allow us the opportunity to study the end points of many biological processes. Among these, blood-based metabolomics is a promising method to investigate the pathophysiology of human cancer cachexia and identify candidate biomarkers. In this study, we performed liquid chromatography mass spectrometry (LC/MS)-based metabolomics to investigate the metabolic profile of cancer-associated weight loss. Non-selected patients undergoing surgery with curative intent for upper gastrointestinal cancer were recruited. Fasting plasma samples were taken at induction of anaesthesia. LC/MS analysis showed that 6 metabolites were highly discriminative of weight loss. Specifically, a combination profile of LysoPC 18.2, L-Proline, Hexadecanoic acid, Octadecanoic acid, Phenylalanine and LysoPC 16:1 showed close correlation for eight weight-losing samples (≥5% weight loss) and nine weight-stable samples (<5%weight loss) between predicted and actual weight change (r = 0.976, p = 0.0014). Overall, 40 metabolites were associated with ≥5% weight loss. This study provides biological validation of the consensus definition of cancer cachexia (Fearon et al.) and provides feasible candidate markers for further investigation in early diagnosis and the assessment of therapeutic intervention.

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.

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.

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.

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.

Muscle protein kinetics in cancer cachexia.

PURPOSE OF REVIEW: Skeletal muscle loss appears to be the most significant event in cancer cachexia and is associated with a poor outcome. The balance between mechanisms that control synthesis and degradation is fundamental when designing new therapies. This review aims to highlight the molecular mechanisms that are associated with protein kinetics. RECENT FINDINGS: The mechanisms that promote muscle synthesis have been explored in detail recently but moreover they have been the mechanisms behind degradation. Specific advances in cellular signalling molecules related to autophagy pathways including signal transducer and activators of transcription-3, activin type-2 receptor, TRAF6, and transcriptomic research have been given special attention in this review to highlight their roles in degradation and as potential targets for therapeutics. Ways to quantify muscle loss are badly needed for outcome measures; recent research using radiolabelled amino acids has also been discussed in this review. SUMMARY: Only by having an appreciation of the complex regulation of muscle protein synthesis and degradation, will potential new therapeutics be able to be developed. This review identifies known targets in molecular pathways of current interest, explores methods used to find novel genes which may be involved in muscle kinetics and also highlights ways in which muscle kinetics may be measured to assess the efficacy of such interventions.

Suppression of skeletal muscle turnover in cancer cachexia: evidence from the transcriptome in sequential human muscle biopsies.

PURPOSE: The mechanisms underlying muscle wasting in patients with cancer remain poorly understood, and consequently there remains an unmet clinical need for new biomarkers and treatment strategies. EXPERIMENTAL DESIGN: Microarrays were used to examine the transcriptome in single biopsies from healthy controls (n = 6) and in paired biopsies [pre-resection baseline (weight-loss 7%) and 8 month post-resection follow-up (disease-free/weight-stable for previous 2 months)] from quadriceps muscle of patients with upper gastrointestinal cancer (UGIC; n = 12). RESULTS: Before surgery, 1,868 genes were regulated compared with follow-up (false discovery rate, 6%). Ontology analysis showed that regulated genes belonged to both anabolic and catabolic biologic processes with overwhelming downregulation in baseline samples. No literature-derived genes from preclinical cancer cachexia models showed higher expression in baseline muscle. Comparison with healthy control muscle (n = 6) revealed that despite differences in the transcriptome at baseline (941 genes regulated), the muscle of patients at follow-up was similar to control muscle (2 genes regulated). Physical activity (step count per day) did not differ between the baseline and follow-up periods (P = 0.9), indicating that gene expression differences reflected the removal of the cancer rather than altered physical activity levels. Comparative gene expression analysis using exercise training signatures supported this interpretation. CONCLUSIONS: Metabolic and protein turnover-related pathways are suppressed in weight-losing patients with UGIC whereas removal of the cancer appears to facilitate a return to a healthy state, independent of changes in the level of physical activity.

Sexual dimorphism modulates the impact of cancer cachexia on lower limb muscle mass and function.

BACKGROUND & AIMS: There is a sparsity of data on the impact of cachexia on human muscle function. This study examined the relationship between cachexia, quality of life and the mass/function/mechanical quality of lower limb skeletal muscle in gastrointestinal cancer patients. METHODS: Quadriceps strength and lower limb power were measured in 54 patients with gastrointestinal cancer (n = 24 ≥ 10% weight-loss) and 18 healthy controls. Quadriceps cross-sectional area was measured in 33/54 patients and in all controls using MRI. Muscle mechanical quality was defined as quadriceps strength/unit quadriceps cross-sectional area. Quality of life was assessed using the EORTC QLQ-C30. Patients with weight-loss ≥ 10% were classified as cachectic. RESULTS: In male cachectic patients, quadriceps strength (p = 0.003), lower limb power (p = 0.026), quadriceps cross-sectional area (p = 0.019) and muscle quality (p = 0.008) were reduced compared with controls. In female cachectic patients, quadriceps strength (p = 0.001) and muscle quality (p = 0.001) were reduced compared with controls. Physical function (p = 0.013) and fatigue (p = 0.004) quality of life scores were reduced in male cachectic compared with non-cachectic patients, but not in females. CONCLUSIONS: Muscle quality is reduced in cancer patients. The degree of impairment of lower limb muscle mass, quality and function and the impact on quality of life varies with weight-loss and sex.

Intramyocellular lipid droplets increase with progression of cachexia in cancer patients.

BACKGROUND: Intramyocellular lipids are an important source of fuel for mitochondrial fat oxidation and play an important role in intramuscular lipid homeostasis. We hypothesised that due to the phenotype associated with cancer cachexia, there would exist an association between increasing weight loss and the number/size of intramyocellular lipid droplets. METHODS: Nineteen cancer patients and 6 controls undergoing surgery were recruited. A rectus abdominis biopsy was performed and processed for transmission electron microscopy (TEM). The number of intramyocellular lipid droplets and lipid droplet diameter were calculated from the TEM images. CT scans, performed as part of patients' routine care, were analysed to determine amount of adipose (intermuscular, visceral and subcutaneous) and muscle tissue. RESULTS: Compared with controls, cancer patients had increased numbers of lipid droplets (mean (SD) 1.8 (1.9) vs. 6.4 (9.1) per ×2,650 field, respectively, p = 0.036). Mean (SD) lipid droplet diameter was also higher in cancer patients compared with controls (0.42 (0.13) vs. 0.24 (0.21) µm, p = 0.015). Mean lipid droplet count correlated positively with the severity of weight loss (R = 0.51, p = 0.025) and negatively with CT-derived measures of intermuscular fat (R = -0.53, p = 0.022) and visceral fat (R = -0.51, p = 0.029). CONCLUSIONS: This study suggests that the number and size of intramyocellular lipid droplets is increased in the presence of cancer and increases further with weight loss/loss of adipose mass in other body compartments.

Magnetic resonance imaging with k-means clustering objectively measures whole muscle volume compartments in sarcopenia/cancer cachexia.

BACKGROUND & AIMS: Sarcopenia and cachexia are characterized by infiltration of non-contractile tissue within muscle which influences area and volume measurements. We applied a statistical clustering (k-means) technique to magnetic resonance (MR) images of the quadriceps of young and elderly healthy women and women with cancer to objectively separate the contractile and non-contractile tissue compartments. METHODS: MR scans of the thigh were obtained for 34 women (n = 16 young, (median) age 26 y; n = 9 older, age 80 y; n = 9 upper gastrointestinal cancer patients, age 65 y). Segmented regions of consecutive axial images were used to calculate cross-sectional area and (gross) volume. The k-means unsupervised algorithm was subsequently applied to the MR binary mask image array data with resultant volumes compared between groups. RESULTS: Older women and women with cancer had 37% and 48% less quadriceps muscle respectively than young women (p < 0.001). Application of k-means subtracted a significant 9%, 14% and 20% non-contractile tissue from the quadriceps of young, older and patient groups respectively (p < 0.001). There was a significant effect of group (i.e., cancer vs healthy) when controlling for age as a covariate (p = 0.003). CONCLUSIONS: K-means objectively separates contractile and non-contractile tissue components. Women with upper GI cancer have significant fatty infiltration throughout whole muscle groups which is maintained when controlling for age.

Using transcriptomics to identify and validate novel biomarkers of human skeletal muscle cancer cachexia.

BACKGROUND: Cancer cachexia is a multi-organ tissue wasting syndrome that contributes to morbidity and mortality in many cancer patients. Skeletal muscle loss represents an established key feature yet there is no molecular understanding of the disease process. In fact, the postulated molecular regulators of cancer cachexia originate largely from pre-clinical models and it is unclear how these translate to the clinical environment. METHODS: Rectus abdominis muscle biopsies were obtained from 65 upper gastrointestinal (UGI) cancer patients during open surgery and RNA profiling was performed on a subset of this cohort (n = 21) using the Affymetrix U133+2 platform. Quantitative analysis revealed a gene signature, which underwent technical validation and independent confirmation in a separate clinical cohort. RESULTS: Quantitative significance analysis of microarrays produced an 83-gene signature that was able to identify patients with greater than 5% weight loss, while this molecular profile was unrelated to markers of systemic inflammation. Selected genes correlating with weight loss were validated using quantitative real-time PCR and independently studied as general cachexia biomarkers in diaphragm and vastus lateralis from a second cohort (n = 13; UGI cancer patients). CaMKIIbeta correlated positively with weight loss in all muscle groups and CaMKII protein levels were elevated in rectus abdominis. TIE1 was also positively associated with weight loss in both rectus abdominis and vastus lateralis muscle groups while other biomarkers demonstrated tissue-specific expression patterns. Candidates selected from the pre-clinical literature, including FOXO protein and ubiquitin E3 ligases, were not related to weight loss in this human clinical study. Furthermore, promoter analysis identified that the 83 weight loss-associated genes had fewer FOXO binding sites than expected by chance. CONCLUSION: We were able to discover and validate new molecular biomarkers of human cancer cachexia. The exercise activated genes CaMKIIbeta and TIE1 related positively to weight-loss across muscle groups, indicating that this cachexia signature is not simply due to patient inactivity. Indeed, excessive CaMKIIbeta activation is a potential mechanism for reduced muscle protein synthesis. Our genomics analysis also supports the view that the available preclinical models do not accurately reflect the molecular characteristics of human muscle from cancer cachexia patients.

Frequency of the mitochondrial DNA 4977bp deletion in oesophageal mucosa during the progression of Barrett's oesophagus.

PURPOSE: The mechanisms of the progression of Barrett's oesophagus (BO) to oesophageal adenocarcinoma (OA) are poorly understood. The frequency of the 4977bp deletion in mitochondrial DNA (mtDNA) was investigated in specimens ranging from normal oesophageal tissue to OA in order to investigate whether this deletion represents a useful biomarker of disease progression. METHODS: The presence of the 4977bp deletion was screened by PCR amplification from 70 specimens in total. RESULTS: The frequency of specimens with the 4977bp deletion increased in relation to the degree of dysplasia (8.3% in normal squamous epithelium; 15.4% in BO; 40% in low grade dysplasia (LGD); 69.2% in high-grade dysplasia and 90% in para-tumoural tissue). However, the frequency of the deletion reduced sharply in OA specimens (16.7%; p<0.001). CONCLUSION: The mtDNA 4977bp deletion may be useful as a biomarker to detect the severity of dysplasia but not the presence of OA.

Cachexia, survival and the acute phase response.

PURPOSE OF REVIEW: To review current knowledge of the relationship between cytokines, the acute phase response (APR) and the development of cachexia. RECENT FINDINGS: Cytokines important in the initiation of the APR are also important in the genesis of cachexia. The presence of an APR or high circulating levels of pro-inflammatory cytokines are known to be related to adverse outcome in cancer patients. Studies of host cytokine genotype have demonstrated that specific host cytokine polymorphisms are associated with both the development of cachexia and reduced patient survival. The desire to be able to predict accurately survival in cancer patients has led to the description of various APR-based prognostic scoring systems. SUMMARY: Cachexia is an important clinical problem affecting up to two thirds of cancer patients. It results from anorexia and metabolic change and leads to loss of both adipose tissue and lean body mass, particularly in the skeletal muscle compartment. An APR is seen in half of cancer patients at presentation, and is most often determined clinically by raised plasma C-reactive protein concentrations. Adverse outcome and shortened survival have been linked to the presence of an APR. This article explores the cause and consequences of the APR in cancer cachexia.