From the literature it is clear that there are a number of questions which clinicians face on a daily basis to which there is as yet no satisfactory scientific explanation or rationale for a particular treatment decision. Three of these key questions are:
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In patients with rectal cancer is it possible to predict response to neoadjuvant chemoradiotherapy?
In patients with colorectal liver metastases is it possible to predict response to neoadjuvant chemotherapy?
Can biological information from the primary tumour be used to predict response to treatment in colorectal liver metastases?
The aim of this thesis is to begin to address these research questions.
1.12.1 Hypotheses
These research questions can be used to formulate a number of testable hypotheses investigated in this project:
1. Somatic non-synonymous mutations in the primary tumour correlate with those present in the liver metastasis and predict response to treatment.
2. The phenotype of the primary tumour is biologically similar to the liver metastasis, with markers present in the primary tumour which predict the response of the liver metastasis to neoadjuvant chemotherapy.
4. The phenotype of a rectal tumour changes with neoadjuvant chemoradiotherapy, and inter-patient variation in these changes predicts response to treatment.
5. Levels of expression of key proteins involved in the activation and metabolism of chemotherapeutics are comparable between primary and metastatic tumours and predict response to treatment.
6. Levels of expression of DNA base excision repair proteins in rectal tumours vary with treatment, and inter-patient variation in the expression of these proteins predicts response to neoadjuvant chemoradiotherapy.
1.12.2 Study plan
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1. Establish a research network across multiple sites to facilitate the collection of patient matched primary and metastatic tissue, alongside normal liver parenchyma and colonic mucosa, focussing on those receiving neoadjuvant chemotherapy.
2. Establish a research protocol to facilitate the collection of serial temporal biopsies from patients with rectal cancer, acquiring tissue prior to chemoradiotherapy, immediately following chemoradiotherapy and at the time of surgical resection.
3. Investigate alternative strategies to the standard mechanisms of tissue collection (i.e. liquid nitrogen, dedicated research technician and archiving facilities in a -80oC freezer)
in order to facilitate 1 and 2.
4. Subject tumour samples (alongside normal adjacent tissue) from patients undergoing synchronous resection to exome sequencing in order to establish the degree of
biological similarity between primary and metastatic tumours, specifically to identify whether the presence of somatic non-synonymous mutations in the primary tumour predicts the presence of the same mutations in the liver metastasis.
5. Perform isobaric tagging for relative quantification (iTRAQ) of patient matched primary and metastatic tumours as well as adjacent normal liver parenchyma and colonic mucosa on a discovery set of fresh tissue. Data will be analysed to assess the degree of biological similarity between primary and metastatic tumours, as well as identify potential biomarkers measurable in the primary tumour which will predict the response to chemotherapy in the liver metastases.
6. Validate any potential biomarkers by immunohistochemistry on a larger validation tissue set, with samples combined into a tissue micro-array. Any potential mechanisms for validated biomarkers will be explored.
7. Perform isobaric tagging for relative quantification (iTRAQ) of serial rectal cancer samples on a discovery set of fresh tissue. Data will be analysed to assess how the tumour phenotype changes with treatment, as well as identify potential biomarkers which may predict response of the primary tumour to neoadjuvant chemoradiotherapy.
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8. Validate any potential biomarkers by immunohistochemistry on a larger validation tissue set, with samples combined into a tissue micro-array. Any potential mechanisms for validated biomarkers will be explored.
9. Undertake a targeted analysis of the expression and function of key proteins involved in the activation and metabolism of oxaliplatin, 5-fluorouracil and irinotecan in patient matched primary and metastatic tumour samples. Data will be analysed to assess the correlation in expression and function between primary and metastatic tumours as well as how this information may predict response to treatment.
10. Undertake a targeted analysis of the expression of key proteins involved in DNA base excision repair in serial rectal cancer samples. Data will be analysed to assess how levels of expression vary with treatment and how this information may be used to predict response.
63 Chapter 2
Evaluation of a tissue stabilisation gel (AllprotectTM) to facilitate clinical sampling for translational research in surgical trials
64 2.1 Introduction
2.1.1 Background
Successful translational research in surgery is dependent upon collaboration between scientists and clinicians. Central to this is the acquisition of tissue samples following surgical or endoscopic resection. In specialties such as thoracic surgery, neurosurgery and some gastrointestinal subspecialties such as hepatopancreaticobiliary, centralisation of services within large trusts or university teaching hospitals has facilitated routine collection of tissue through biobanks. Higher volume specialties performing oncological resections, such as colorectal surgery, have not been centralised. Indeed the majority of colorectal cancer resections are still performed in the non-teaching hospital setting, making the harvest and storage of fresh tissue problematic because of a lack of specialised facilities, such as a ready supply of liquid nitrogen and -80oC storage (Shabihkhani et al, 2014). Consequently, most
colorectal biobanks contain only formalin-fixed paraffin embedded (FFPE) samples which are of limited use for most bioanalytical applications.
2.1.2 The need for an alternative infrastructure
In order to facilitate the inclusion of high volume non-teaching hospitals in more translational research projects and clinical trials, or for more ad-hoc tissue sampling, an alternative method of tissue stabilisation is needed which does not require immediate processing and freezing. Indeed, a system that allowed transfer of samples by post or courier, without the need for specialist shipping conditions, would also increase the potential recruitment of patients for large scale trials and reduce costs associated with on- site processing. AllprotectTM is a new to the market tissue stabilisation gel which according
to the manufacturer overcomes the need for liquid nitrogen and -800C storage, allowing
tissue collection and stabilisation to be performed with limited infrastructure (Qiagen, Venlo, Netherlands). As AllprotectTM is a proprietary compound, details of its composition
are currently unavailable.
2.1.3 Relevance to this thesis
Clinical samples used for the analysis which has informed later chapters of this thesis will attest to the complex regional infrastructure which was created in order to facilitate the
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work. On Merseyside all resections of colorectal liver metastases are performed at Aintree University Hospital, a teaching hospital with a well developed research infrastructure. The hospitals referring patients to this institution however are less well equipped for research, and it is these institutions in which the vast majority of the primary tumours of the patients recruited to these studies were resected. As such, a regional network was established to facilitate the acquisition of patient matched clinical samples and corresponding clinical and demographic data. The work described in this chapter was devised in order to facilitate other translational work presented later in the thesis.
2.1.4 Aims and hypotheses
The aim of this study was to rigorously evaluate the use of AllprotectTM as an alternative to
liquid nitrogen for the short and long term storage of clinical samples from patients
undergoing surgical resection of colorectal primary and metastatic tumours. Tissue integrity under all storage conditions was assessed across a range of typical biomolecules including DNA, mRNA, microRNA and protein. In addition, retention of protein function following storage in AllprotectTM was determined by enzyme activity measurements. Specifically, we
hypothesise that AllprotectTM offers comparable tissue stabilisation to liquid nitrogen for
66 2.2 Methods
2.2.1 Sample collection
The study was performed to facilitate an existing translational research project, for which NHS Research Ethics Committee and Research and Development approval had already been obtained (12/NW/0011). Samples were obtained from patients undergoing primary
resection for colorectal cancer (n=5). Following delivery of the surgical specimen, the proximal staple line was incised and a linear cut was made down the anti-mesenteric border being cautious to stop either 1cm away from the tumour or upon approaching the mesorectum. Using forceps and a scalpel a peripheral section of tumour was excised. Samples were obtained from a second cohort of patients undergoing resection for colorectal liver metastases (n=5). Following delivery of the surgical specimen an incision was made through the resected surface to the liver metastasis, with care being taken not to breach the liver capsule, and peripheral samples of tumour were obtained as previously described. All tissue sampling was performed in the operating theatre under aseptic conditions.
2.2.2 Stability study
Each sample was cut into 10 discrete sections of approximately 4 mm3, 5 of which were
individually placed into sealed cryodorfs and processed immediately (within 5 minutes of specimen delivery) as follows: 1. Snap frozen in liquid nitrogen and transferred to tissue archiving freezer (-80oC) – control sample. 2. AllprotectTM at room temperature (190C). 3.
AllprotectTM in a fridge (80C). 4. AllprotectTM in a standard laboratory freezer (-200C) 5.
AllprotectTM in a tissue archiving freezer (-800C). Those samples processed in AllprotectTM
were submerged in a 10:1 ratio by volume as per manufacturer’s instructions; samples snap frozen in liquid nitrogen were done so without additives. The remaining 5 sections per sample were left exposed to the environment on a theatre trolley until the end of the procedure (approximately 1 hour for both primary and metastatic resections) in an effort to replicate ‘real world’ tissue sampling in the absence of a designated research technician. Once the skin had been closed, the remaining samples were processed in an identical manner to that already described. Samples remained under stabilisation conditions for 7 days prior to proceeding with biomolecule extraction. A schematic outline of the study design can be seen in Figure 2.1.
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Figure 2.1 – A schematic outline of the stability study. The available tissue was divided into 10 fragments, 5 of which were stabilised immediately in one of the storage conditions shown and the remaining 5 in a similar manner but at the end of the procedure. The study was designed to ask two specific questions: can sampling wait until
the end of the procedure, and what is the optimal storage condition?
2.2.3 Archiving study
Further cohorts of patients undergoing resection for primary colorectal cancer (n=5) or liver metastases (n=5) were sampled in the same manner and again tissue was divided into 10 sections. One sample was placed into liquid nitrogen and then transferred to a tissue archiving freezer (-80oC) and the remaining 9 samples were submerged in AllprotectTM and
refrigerated (at 8oC). A single sample was removed following storage for 1, 2, 3, 4, 8, 12, 16,
20 and 24 weeks, and processed for biomolecule extraction. A schematic outline of the study design can be seen in Figure 2.2.
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Figure 2.2 – A schematic outline of the archiving study. The available tissue was divided into 10 fragments, one of which was immediately snap frozen in liquid nitrogen and transferred to a -80oC freezer (control sample) and one was archived in AllprotectTM for
each of the time points shown in the figure.
2.2.4 Biomolecule extraction
DNA and RNA extraction was performed using the Qiagen miRNeasy Mini Kit and EZ1 DNA Tissue Kit respectively as per manufacturer’s instructions (Qiagen, Venlo, Netherlands). Protein extraction was performed by a combination of both mechanical and ultrasonic homogenisation in phosphate-buffered saline. Following centrifugation the protein concentration of the supernatant was established using a Bradford assay (Bradford, 1976). In addition, a section of tissue was taken from each of the samples, fixed in formalin and embedded in paraffin. A slide was stained with haemotoxylin and eosin and reviewed by a pathologist who was blind to the details of the study. Extracted biomolecules were used to perform each of the following assays in triplicate on all samples.
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2.2.5 DNA assays
DNA concentration was measured using the NanoDropTM ND-1000 spectrophotometer
(Thermo Scientific, Waltham, USA) and quality assessed using the Human DNAOK! kit (Microzone, Haywards Heath, UK). KRAS mutational analysis was performed using an in- house pyrosequencing assay capable of detecting all somatic mutations in codons 12, 13 and 61 of the KRAS gene. The lower limit of detection for the assay is 5% of mutated alleles.
2.2.6 RNA Assays
Extracted RNA was initially subjected to quantification and quality control using the NanodropTM spectrophotometer and reverse transcription was performed with the RT2
First Strand Kit (Qiagen, Venlo, Netherlands). Further quality control assays were performed using a Bioanalyzer (Agilent, Santa Clara, USA) and the commercially available RT2TM RNA
QC PCR Array (SABiosciences, Venlo, Netherlands). In order to assess the functional integrity of the extracted RNA under different storage conditions, RT-PCR was performed on two genes (HMGB1 and CES1) and two microRNAs (mir-122 and let7d) relevant to an on- going translational project conducted within the Medical Research Council Centre for Drug Safety Science. Specific primers were designed for HMGB1, CES1, miR122 and let7d (Eurofins Scientific, Luxembourg, Luxembourg) and qPCR performed using the ABI-7900 (Applied Bioscience, Foster City, USA).
2.2.7 Protein expression by western blotting
Western blots for HMGB1 and CES1 as representative proteins were performed on both primary colorectal cancer tissue and liver metastases. 10% SDS-PAGE gels were transferred to a nitrocellulose membrane and blocked in 10% milk. Following overnight incubation with the primary antibody (Abcam: ab18256 {HMGB1} or ab45957 {CES1}) membranes were incubated with a LI-COR IRDye 680 LTsecondary antibody (Biosciences, Lincoln USA) before semi-quanitative analysis using the Oydssey scanner (LI-COR Biosciences, Lincoln USA).
2.2.8 Protein expression by immunohistochemistry
In order to assess the effect of AllprotectTM on tissue morphology, and the feasibility of
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was established by immunohistochemistry. 4µm sections were de-waxed in xylene and rehydrated with ethanol solutions of decreasing concentrations. After blocking with 3% hydrogen peroxide in 100% methanol, antigens were retrieved by microwaving for 20 minutes in 10mM citrate buffer and further blocked with 10% goat serum in 0.1% tris- buffered saline with tween. Slides were incubated with the primary antibody (Abcam, ab18256) for 2 hours, and a 1:200 horseradish peroxidase conjugated secondary antibody (Dako UK Ltd, E0432) for 30 minutes. Following incubation with the Vectastain Elite® ABC reporter system (Vectorlabs, Burlingame, USA), slides were developed with
diaminobenzidine tetrahydrochloride (DAB) and counterstained with haematoxylin. Slides were reviewed by light microscopy and HMGB1 expression was established using a previously reported scoring system (Liu et al, 2012). Two reviewers independently scored five fields at 400x magnification with a total cell count not less than 1000. The proportion of positive cells allowed classification into one of four categories: 1 (<25%), 2 (25-50%), 3 (51- 75%) or 4 (>75%). The intensity of nuclear staining was also classified: 1 (no
staining/background of negative controls), 2 (weak staining detectable above background), 3 (moderate staining) or 4 (intense staining). The index was then calculated by multiplying the intensity and percentage scores to give a total score out of 16.
2.2.9 Protein function by mass spectrometry
The activity of carboxylesterase was determined with irinotecan as a substrate in order to assess protein function. Tissue homogenate (250µg protein) was incubated with 100nM irinotecan (Sigma Aldrich, Poole, UK) for 30 minutes prior to termination of the reaction using a methanol/acetonitrile solution. The supernatant was subjected to a functional assay optimised to quantify the active metabolite, SN-38, by liquid-chromatography mass-
spectrometry (Jones et al, 2013).
2.2.10 Statistical analysis
Statistical analysis was based on the comparison of different methods of tissue
stabilisation/archiving to the current gold standard, i.e. tissue snap frozen in liquid nitrogen and transferred to a tissue archiving freezer (-80oC). One-way ANOVA with post-hoc
Dunnett’s test was used to compare each sample in turn to the reference sample. Analysis was performed using Stats Direct 2.7.9 (Stats Direct Ltd, Altrincham, UK).
71 2.3 Results
2.3.1 Assessment of tissue morphology
All samples were confidently identified as adenocarcinoma on review of the H&E slide, irrespective of the method of tissue stabilisation used or length of time archived in
AllprotectTM (Figure 2.3). A degree of clear cell change/vacuolation was seen in all samples
which was slightly more apparent in those samples exposed to AllprotectTM, although this
did not vary significantly with the length of exposure. Tumour sampling did not compromise a complete pathological assessment of the remaining surgical specimen in any of the cases.
Figure 2.3 – Representative micrographs of H&E stained sections of colorectal liver metastases showing a) tissue snap frozen in liquid nitrogen and b) tissue stabilised in AllprotectTM. Both are clearly identifiable as adenocarcinoma, although contain a degree of clear cell change/vacuolation (X). Histological appearances did not change significantly
with the length of the time the sample was exposed to AllprotectTM. Micrographs were taken at 200x magnification.
2.3.1 Short term (0 - 1 week) maintenance of biomolecule expression and function
DNA
Automated extraction of DNA from the snap frozen and AllprotectTM stabilised samples
yielded comparable quantities (2.1-3.3µg/mg tissue) of high quality DNA with 260:280 ratios of 1.8 - 1.9 and 260:230 ratios of 2.0 - 2.4. There was no correlation between the
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spectrophotometric quality ratios and the method of stabilisation. The samples
immediately stabilised were also comparable to those stabilised after a one hour delay. All samples passed the internal quality control check utilised by the EZ1 Advanced (Qiagen, Venlo, Netherlands). Extracted DNA was successfully utilised for downstream qPCR analysis of KRAS status at codons 12, 13 and 61 with 100% concordance between all samples originating from the same biological specimen for all patients and both tissue types (Table 2.1).
73 Sample Stabilisation Method Processing Concentration
(ng/µl)
260/280 260/230 Quality Check
KRAS Codon 12 KRAS