Characterisation and mechanisms of altered body composition and tissue wasting in cancer cachexia
Cancer cachexia has been defined as a multifactorial syndrome characterised by an ongoing loss of skeletal muscle that cannot be fully reversed by conventional nutritional support. Cachexia affects most patients with advanced cancer and is associated with reductions in treatment tolerance, response to therapy, quality of life, and survival. Thus, amelioration of cachexia would improve both quality of life and clinical outcome. However, the aetiology of cachexia is poorly understood, and there are no agreed diagnostic biomarkers or management strategy for patients with cancer cachexia. Recent advances in the field of cachexia research include the development of diagnostic criteria for cachexia, as well as computed tomography (CT) body composition analysis software, making the ability to detect clinically significant muscle wasting in obese patients in particular more accurate. Although muscle loss appears to be the most important and physiologically relevant event in cachexia, the importance of fat wasting is less understood. During cachexia, different adipose depots around the body demonstrate differential rates of wasting. Furthermore, recent studies from animal models have suggested that adipose tissue may be a key driver of muscle wasting through fat-muscle crosstalk. However, human studies in this area are lacking. The molecular mechanisms driving muscle loss in humans are also poorly understood, and the relationships between muscle and fat wasting, functional impairment and reduced survival are largely unknown. The prognostic significance of adipose wasting and investigations in tissue cross-talk therefore are now becoming more important whilst the quest for a cachexia related biomarker remains at the fore. The main aim of this thesis was to investigate specific mediators, mechanisms and biomarkers of cachexia in robustly phenotyped patients with upper gastrointestinal cancer (UGI) in whom cachexia is known to be prevalent. This thesis is comprised of several projects designed to investigate various areas of cachexia pathophysiology, diagnosis and staging. In order to recruit patients to clinical trials, drive cachexia research and identify those who would benefit from early intervention, it is important to understand how to screen and diagnose patients with cachexia. Many patients present to clinicians with unintentional weight loss (UWL). This can occur in patients with cachexia, sarcopenia and malnutrition. With increasing rates of obesity worldwide, as well as an ageing population, differentiating causes of UWL is difficult. Firstly therefore, in order to investigate the feasibility of screening for UWL a systematic review was undertaken in chapter 3 to determine which screening tools were able to assess cachexia, sarcopenia and malnutrition according to the consensus definitions for each. Each tool was judged against a reference method and psychometric evaluation carried out. No one tool was able to assess all three conditions simultaneously, and out of the 22 tools assessed, only 3 had been validated against the gold standard of CT cross-sectional imaging. Thus, the development of a novel tool that encompasses the consensus definition criteria and directs clinicians towards the underlying diagnosis would likely improve detection and outcomes. Secondly, building upon screening and methods for diagnosing low muscularity, chapter 4 uses CT body composition analysis to determine any age and sex-related variations in patients with UGI cancer. CT-based cut-offs for determining low skeletal muscle volume are sex and body mass index (BMI) specific and have been driven in order to predict mortality in these patients. As discussed above, the prevalence of obesity is increasing and the population is ageing therefore, many patients may be sarcopenic at diagnosis, making the assessment of clinically significant muscle wasting difficult. A retrospective, observational study was carried out on patients who had undergone potentially curative oncological and surgical treatment for oesophageal cancer. Analysis of both staging and post neoadjuvant chemotherapy (NAC) CT was performed in order to assess baseline characteristics and dynamic changes in body composition. Males had higher baseline muscle and visceral fat volume whereas females had higher subcutaneous fat volume. Patients of all ages and both sexes lost muscle volume though there was no difference in rates of wasting between groups. Older patients and females lost significantly more total fat during chemotherapy. This chapter therefore highlights the need for further investigation to define differences in adipose depots during cancer progression and their prognostic value. Chapter 5 showcases the main biological assessment of cancer associated muscle wasting in this thesis. As shown in chapter 4 all patients demonstrated some evidence of muscle wasting. A potential mechanism of this was therefore investigated further by looking at the role of the neuromuscular junction (NMJ). The NMJ provides the link between myelinated motor nerves and skeletal muscle. Very little is known about the structure of the NMJ in human health or in disease. Experimental denervation is a recognised model for studying muscle wasting in vivo, and as a result experimental evidence for the role of the NMJ in cachexia is dependent upon animal models. Recent data, however have shown that rodent and human NMJs are markedly different. NMJ morph, an imageJ-based package was used for morphometric analysis of the NMJ in UGI cancer patients with or without cachexia and non-cancer controls. No significant differences were found between groups in any of the major pre- or post-synaptic variables measured suggesting that the NMJ remains structurally intact in cancer cachexia, and thus, the denervation of skeletal muscle is not a major driver of the disease. Whilst it is recognised that muscle mass plays a significant role in the syndrome of cancer cachexia, as shown in chapter 4 through body composition analysis the importance of fat wasting and the effect of metabolic mediators on fat volume requires attention. In murine tumour models, loss of fat volume may predate the loss of muscle volume. Fatty acids, leptines, cytokines and other adipokines may cause lipotoxic effects in skeletal muscle. Adipokines have been reported to induce insulin resistance, impair muscle development, alter muscle lipid amino acid metabolism and modify signalling thus affecting skeletal muscle volume. Clinical studies have shown that adipokines from murine models are also measurable in patients with cancer cachexia. In chapter 6 through the use of transcriptomics, subcutaneous (SAT) and visceral adipose tissue (VAT) depots were analysed from UGI cancer patients with and without cachexia and healthy controls to elucidate the biochemistry of fat wasting in cancer cachexia. Over 2000 genes differed between cachexia VAT and SAT. The gene that showed the largest difference in expression between cancer VAT and control was Intelectin-1 (ITLN1), a novel adipocytokine. Genes involving inflammation were upregulated in cancer whereas genes involved in energy metabolism and fat browning were down regulated. VAT, therefore, may be a target for therapeutic manipulation in cancer. Further investigation is required in to the role of Intelectin-1 as a biomarker in cachexia. Finally, in previous searches for biomarkers of cancer, likely responsiveness to treatment and the presence of cachexia, plasma has been used as a readily available biofluid for investigation. However, no robust cachexia biomarker has been found. Although as work continues it seems that individual biomarker targets should be replaced by an array of markers. Chapter 7 used liquid chromatography mass spectrometry (LC/MS)-based metabolomics to investigate the metabolic profile of weight loss from plasma samples taken at the time of anaesthesia from patients under-going UGI resectional surgery. This showed two distinct profiles based on percentage weight loss in accordance with the consensus definition. There were 40 metabolites associated with cachexia with six of those being highly discriminative of weight loss. Specifically, a combination profile of LysoPC 18.2, 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). In particular ,many of the metabolites discovered were involved in lipid metabolism, lending credence again to the importance of understanding adipose wasting in cachexia. In summary, the role of adipose wasting as investigated through imaging and biochemical results has been shown to be important in the aetiology of cancer cachexia. It has been demonstrated that there is currently no adequate way to screen for conditions which present with UWL and that the adaptation of a tool in order to do this would drive further research and the content of complex interventions. Previously published CT-derived cut points are BMI and sex specific, however, it has been shown that there is a growing need to develop these in order to define patients by age also. In doing so this would define stricter criteria for clinical trials and lead to improved end points. Potential novel biomarkers of lipid wasting have been discovered in this thesis. ITLN1 which has corresponded with weight loss previously in other groups warrants further investigation as it may be a target for future therapeutic manipulation. Those biomarkers discovered in the metabolomics study show that it is possible to separate patients based on weight loss alone. Although this was a pilot study after further investigation and development, biomarkers of lipid wasting may be useful as inclusion criteria or outcome measures in clinical trials. The discoveries of the lack of fat browning and the stability of the NMJ in cachectic patients also importantly highlights the need for patient rather than animal based research.