Muscle Wasting in Cancer (MWIC)

Patients: Patients with upper and lower gastrointestinal cancer for planned resectional surgery will be recruited. Patients will be identified by the clinical team at the relevant multidisciplinary team meeting and then approached for study information and consent in the surgical clinic. Age-matched, healthy controls undergoing elective surgery for benign conditions (e.g. hernias) will also be recruited.

Patient Phenotyping: CT scans performed as a routine part of diagnosis and staging will be used for analysis of body composition. All patients recruited will have their CT scans analysed where present. Control patients (e.g. hernias) may not have CT scans, but where available, will be analysed. A transverse CT image from the third lumbar vertebrae will be assessed and tissue volumes estimated by a single trained observer. Cross-sectional area for muscle and adipose tissue will be normalized for stature (cm2/m2). Estimates of whole body stores will be generated from raw data (cm2) using regression equations. Cutoffs for low muscularity are based on CT-based sarcopenia plus obesity studies of cancer patients. In a previous study, the median time to muscle biopsy after CT scan was 18 days. Phenotyping will also involve measuring basic physical attributes (e. g. weight, height etc) and self-reported physical abilities (through questionnaires).

Muscle function/Physical activity assessment: An objective assessment of physical ability will be undertaken in the clinic for all patients. This will involve a "timed up and go" test and a "6 minute walk" test. These tests have been well-validated in the literature and have good inter-rater reliability. Whilst they do add a slight burden of inconvenience to the patient clinic visit this is minimal. Additionally, a physical activity meter will be applied to the patients' thigh under a waterproof dressing. The Investigators have validated these meters in cancer patients, and they are small (35x53x7mm) and light (20g). When used in previous studies as above the patients have found them unobtrusive and they have been well-tolerated. Where possible, the Investigators will ask the patients to repeat the "timed up and go" and "6 minute walk test" in postoperative clinic up to 12 months after surgery.

Patient self-reported assessment: At routine clinic, all patients will be asked to complete validated questionnaires regarding their physical health, and their perceptions of this. These will be used to assess severity of co-morbidity, and also to assess frailty. Example questionnaires include the Scottish Co-Morbidity Severity Score, the Charlson Co-morbidity Index, and the Edmonton Frail Scale. Other questionnaires relating to self-reported appetite and function will be considered but the overall aim will be to ask select questions to reduce the burden of questioning and prevent questionnaire fatigue. The Investigators will ask participants to repeat these questionnaires in routine clinic up to 12 months post surgery.

Blood and urine sampling: At routine National Health Service (NHS) clinic, all patients will be asked to provide a blood and a urine sample. Where this is not possible, the research team will ask for permission to take these samples under general anaesthetic at planned surgery via urinary and vascular catheters inserted as part of routine care. In the postoperative phase the Investigators will ask participants to provide blood and urine samples, where possible, in routine clinic up to 12 months following surgery.

Muscle Biopsy: A biopsy of rectus abdominis muscle will be taken at surgery by the operating surgeon for all patients. This will be taken from the incision performed for the planned surgery and requires no further incisions. Where possible, an additional needle biopsy of quadriceps (thigh muscle) will also be sampled for comparison. Samples will be cleaned of fats, blood/fibrous tissue, snap frozen in liquid nitrogen and stored at —80°C, For immunohistochemistry (IHC), muscle specimens will be stitched onto a cork, treated with optimum cutting temperature (OCT) and then lowered into cooled isopentane solvent (-190°C) prior to storage at -80°C.

Fat biopsy: A biopsy of subcutaneous fat, and where possible, visceral fat will be collected at the time of surgery for all patients by the operating surgeon. This procedure does not require any additional incisions. These samples will be snap frozen in liquid nitrogen and stored at -80°C.

Tumour biopsy: For the cancer patients, once the resection specimen has been removed at surgery, this will be taken fresh to the pathology lab where the duty pathologist will retrieve sections of tumour. This will be done without compromising clinical staging and in accordance with current procedures. The tumour samples will be divided into aliquots and snap frozen in liquid nitrogen at -80°C. This will not apply to the control group.

Repeat sampling: At follow-up NHS clinic, all patients will be asked to provide repeat blood and urine samples which will be divided into aliquots and snap frozen in liquid nitrogen at -80°C. Where the patient is amenable, they will also be asked to provide a single repeat needle biopsy of the quadriceps muscle. This will be performed in clinic under local anaesthetic using standard aseptic technique. There is some discomfort associated with the injection of local anaesthetic but this is transitory. There is also some discomfort associated with the muscle biopsy but this is short-lived and in the past has been well-tolerated. Quadriceps biopsy will be requested at routine follow-up (at around 6-8 months after surgery) only. Where possible, repeat blood and urine samples will be requested at subsequent routine follow-up appointments for the duration of the study. The Investigators would anticipate this to take place for up to 12 months postoperatively.

Immunohistochemistry (IHC) and Western Blots: These will be performed as per previous publications. For IHC, frozen muscle sections will be co-stained for laminin and myosin heavy chain or lla to distinguish fibre type. Paraffin sections may also be used and stained. The distribution of fibre types, cross-sectional area of individual fibres, and the distribution of nuclei (central versus peripheral location to assess level of regeneration) will be assessed by a proprietary image analysis platform. Potential markers for measurement will include pathways members of autophagy (beclin, lamp, anti-thymocyte globulin (ATG)); apoptosis (poly adenosine di-phosphate ribose polymerase (PARP), caspase-3); oxidative stress (nitrosylated 4-Hydroxynonenal (HNE) and ubiquitinated [K48, K63] proteins); cell stress (Nuclear factor (erythroid derived 2)-like 2 Kelch-like erythroid cell-derived protein with Cap 'N' Collar homology-associated protein 1 (NRF2KEAP1), Heat Shock Protein (HSP), endoplasmic reticulum (ER)-stress); inflammation (IkB kinase (IKK), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB)-9p65, p.38, extracellular signal-related kinase (erk)1/2, c-Jun N-terminal kinase (jnk)); satellite cell maturation (Paired Box (Pax)3, Pax7, Myogenic factor (MYF)5, M-cadherin); myogenesis (myogenin, desmin); sma/mothers against decapentaplegic(SMAD) (SMAD3, phosphoSMAD3); and P-selectin.

C - reactive protein (CRP) Analysis: Systemic inflammation (SI) will be assessed using plasma CRP concentration, which will be measured with an automated immunoturbidimetric assay by the Department of Clinical Chemistry, Royal Infirmary of Edinburgh (RIE).

These analyses will be carried out both in the University of Edinburgh and at the premises of international collaborators. Any tissue transferred out of the care of the University will be fully anonymised using unique study identifiers.

This current study is a continuation of a previous study funded and supported by Cancer Research United Kingdom (UK). As part of that study tissue samples were collected, some of which remain in the University. The Investigators intend to use these remaining samples to add power to the new samples the Investigators collect by increasing the cohort size and thus the likelihood of any potential biomarker identified being significant rather than erroneous. In addition, including these samples in the analysis will add to their value, over and above that already gained by inclusion in the previous project.