Brazilian samples

The “Brazilian cohort” of samples was prospectively collected in the city of Cuiaba, capital of the state of Mato-Grosso, Brazil. The author of this thesis participated directly in the process of sample collection along with a colleague (Dr Lenuce Ydy, Surgical Oncologist). Clinicians from different hospitals and cancer centres were asked to identify patients with a recent diagnosis of CRC for whom a surgical resection of the tumour was scheduled. Then, the patient was interviewed, the nature of the research was explained and they were invited to participate. If they agreed, “informed consent” was obtained and a “clinico-epidemiological data form” was

94 completed. On the day of the surgery, before anaesthetic induction, blood was collected in tubes with and without EDTA (BD Vacutainer, Becton and Dickinson). These blood samples were processed within 5 hours according to the procedures detailed in section 2.9. Serum and plasma were extracted and stored at -80°C. Immediately after the surgical resection, the specimen was opened through the intestinal wall opposite to the tumour. Fragments from the tumour and from the apparently normal adjacent mucosa (at least 10cm from the tumour) were collected in 10% formalin in phosphate buffered saline (PBS) pH 7.4, incubated for 24 hours, and then processed into paraffin blocks for future histological work. In order to obtain tissues for RNA expression analysis, we also collected tumour and adjacent samples in cryovials, and these were immediately frozen in liquid nitrogen. When this research project started, the investigator had already collected 50 pairs of samples (tumour and adjacent). However, although a specialised company was hired to transfer the material from Brazil to the UK, all samples defrosted during the shipment due to delays in the customs authority and insufficient dry ice in the package. After experiencing these problems, we decided to use a preservative solution (Allprotect®, Qiagen) instead of nitrogen snap- freezing to guarantee the preservation of tissues. Therefore, all the samples used in the mRNA expression analysis were placed immediately after collection in Allprotect®, incubated overnight at 4°C and stored at -80°C until they were used. Samples from cancer patients were collected between January 2013 and August 2015. After excluding samples which did not contain representative tissues and those which experienced technical problems during storage or transportation, the number of cancer samples suitable for analysis was:

i. Formalin-fixed, paraffin-embedded samples: 32 cases (tumour and adjacent mucosa)

ii. Allprotect®-treated tissue samples: 25 cases (tumour and adjacent mucosa).

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b. Polyp-bearing individuals and normal controls

In order to obtain samples from individuals with colorectal polyps, we established a partnership with a colonoscopy clinic in Cuiaba (Dr Wladimyr Dias Moreno, Gastrocenter). Subjects referred to the clinic to undergo colonoscopy for a variety of symptoms (intestinal bleeding, constipation, diarrhoea, etc) or for CRC screening (asymptomatic) were identified, the research was explained and they were invited to participate. If they agreed, “informed consent” was obtained. During the colonoscopy, if one or more polyps were identified and no other alteration in the intestine was found, blood samples were collected and processed as explained in section 2.9. The polyps were then removed, immersed in formalin and sent for pathological analysis. This is the standard protocol after the identification of polyps and was not a research-related procedure. After the pathological assessment had been completed and the final report was released, the paraffin blocks were retrieved from the pathology laboratory for future histological and immunohistochemical evaluations. Alternatively, if no polyp, tumour or any other lesion was apparent, the individual was recruited as a normal control. Blood samples were collected and processed. Fragments from the normal mucosa were taken and placed into two flasks, one containing 10% buffered formalin (later processed into paraffin blocks) and the other with Allprotect® as explained above. Unfortunately, we were not able to collect fresh polyp samples either in liquid N2 or in Allprotect® because the diagnostic routine demands that all polyps removed be sent for pathological analysis. The number of samples collected from these individuals was:

i. Polyp-bearing individuals: 18 adenoma samples. ii. Normal controls: 10 intestinal mucosa samples.

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c. Samples from cancer patients with more than 4 years follow-up for prognostic assessment - the prognostic cohort

To test whether or not a correlation existed between the expression of candidate biomarkers and clinical outcomes in CRC patients, we decided to perform an analysis of the expression of the proteins in a larger retrospective cohort. To do so, we established a partnership with a Brazilian pathologist (Dr Ivana Menezes) and a pathology laboratory in Cuiaba (Laboratorio Sao Nicolau). This is the reference laboratory for immunohistochemistry in that region. It has extensive expertise in this procedure and highly trained technical personnel.

The first phase of the collection was a survey in the records of two pathology laboratories (Laboratorio Sao Nicolau and the Julio Muller University Hospital Pathology Lab) aiming to identify cases of CRC who had undergone surgery more than four years ago. Next, we tracked the health service where the patient was initially evaluated to find whether the patient was followed up in that service or in any other service in which clinical information was traceable. Alternatively, if no clinical information was available, we checked the Brazilian electronic death database – Mortality Information System (“Sistema de Informação de Mortalidade” – SIM), a database with all deaths and its causes in the country. Lastly, we retrieved the paraffin blocks from these patients from the pathology lab archives. In total, we collected blocks from 96 patients. Twenty-one cases were excluded due to lack of tumour tissue in the block or because death occurred within 30 days from the operation, thus suggesting postoperative complication as the cause. Consequently, 75 cases were included in the final prognostic analysis (detailed in Chapter 6).

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