Muscle Wasting in Cancer (MWIC)
Muscle Wasting in Cancer (MWIC)
One way cancer affects people is through weight loss. During this weight loss (called
 cachexia), all types of body tissue are lost, but there is a greater rate of muscle loss than
 fat. Cancer patients with cachexia show decreased quality of life, decreased response to
 treatment (e.g. chemotherapy), increased complications from surgery, and shorter overall
 survival. The Investigators aim to identify molecular (and patient) factors within the
 tissues and bodily fluids of patients with cancer and cachexia in order to identify patients
 at risk of weight loss, and identify potential therapies. For this, the Investigators aim to
 take patient samples (muscle, fat, tumour, urine and blood) whilst patients are asleep
 (general anaesthesia) during their operation to remove the cancer. These samples, and similar
 samples taken in previous studies, will be analysed in the laboratory. Along with the sample
 taking, the Investigators aim to perform nutritional assessments of patients before and after
 surgery to get an accurate picture of their weight loss. This will include simple
 measurements (e.g. height/weight), and computer reanalysis of their initial diagnostic
 computed tomography (CT) scans (this study will not require any additional scans for
 patients). The Investigators also aim to assess how their muscles function, by asking them to
 perform walk tests and wear a physical activity meter, and assess their quality of life
 through questionnaires. The Investigators aim to perform nutritional and functional
 assessments pre-surgery and at 2-3 appointments post-surgery, up to a period of 12 months. At
 these timepoints, the Investigators also aim to take repeat blood and urine samples, and
 where possible, one additional thigh muscle biopsy. Repeated assessments allow comparison
 between "cancer" and "cured" states. The study will take 2 years for patient recruitment.

 For comparison, the Investigators also aim to examine similar tissue and fluid samples
 (except tumour) from non-cancer patients who are having surgery for benign conditions (e.g.
 hernia).
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.
Inclusion Criteria:

 -  Patients identified at multidisciplinary team meetings (MDT) as having
 oesophagogastric, hepatobiliary, and colorectal cancer suitable for resection

 -  Patients over 18 years of age

 -  Patients identified at surgical clinic as being planned for an abdominal operation for
 a non-cancer, non-inflammatory condition

 Exclusion Criteria:

 -  Patients with inflammatory conditions or other condition other than cancer that might
 cause muscle wasting

 -  Patients without the capacity to consent

 -  Members of vulnerable groups

 -  Patients not undergoing abdominal surgery

 -  Patients undergoing totally minimally invasive (laparoscopic) surgery, with no open or
 hand-assisted component affording easy access to rectus muscle biopsy