informed consent form - translational
4. storage and collection of

Blood, urine and tissue samples should be stored at local trial centres in a -80°C freezer and transferred in batches as detailed in the NAXIVA laboratory manual to the University of Cambridge on dry ice unless part of the fresh tissue for immunophenotyping/ cell culture study. Samples which are part of the fresh tissue for immunophenotyping/ cell culture study MUST be stored at 4°C and couriered to University of Cambridge in insulated polystyrene containers with ice packs in situ as soon as possible after collection (certainly within 24 hours of collection but ideally same day). Further details for transfer of these samples can be found in the NAXIVA laboratory manual.
informed consent form - translational
4.1. translational sample schedule

| Within 28 days  prior to registration                                                                                                              | Week 1   |    | Week 3   |    | Week 5   |    | Week 7   | Nephrectomy   | 12 weeks post  surgery follow up   |
|----------------------------------------------------------------------------------------------------------------------------------------------------|----------|----|----------|----|----------|----|----------|---------------|------------------------------------|
| Visit/Assessment Percutaneous image guided biopsy of nonnecrotic region of primary renal tumour (5 cores, 2 to be sent to pathology for  formalin-fixation and paraffin embedding of  which 1 to be transferred to University  Cambridge; 1 core to be snap frozen in  liquid nitrogen and stored at -80°C; 2 to be  placed in tubes containing media and  couriered within 24 hours to University of  Cambridge)                                                                                                                                                    | X        |    |          |    |          |    |          |               |                                    |
| Nephrectomy tissue                                                                                                                                 | X        |    |          |    |          |    |          |               |                                    |
| 2 EDTA tube blood samples for whole  blood, double-spun to obtain plasma, buffy  coat and PBMCs at selected sites  (samples to be stored at -80°C) | X        | X  | X        | X  | X        | X  |          |               |                                    |
| Urine sample (1 container to be stored at - 80°C, 1 sample EDTA treated and spun                                                                   | X        | X  | X        | X  | X        | X  |          |               |                                    |
| sample at -80°C)                                                                                                                                   |          |    |          |    |          |    |          |               |                                    |  
4.2. Sample Tracking Sites will be required to record the collection of participants' samples by emailing a sample reporting form to the central lab at the University of Cambridge and SCTRU. SCTRU will provide courier account details for sites to organise transport of the samples.
informed consent form - translational
4.2.1. tracking of baseline samples

In order to minimise invasive procedures, patients will be consented for the trial prior to obtaining both the standard diagnostic biopsy sample and baseline translational study research biopsies. Patients will be asked to provide consent for the research samples to be sent to the University of Cambridge should histological examination of the diagnostic sample show that the patient is ineligible for NAXIVA. Collection of these samples should be notified to the SCTRU by emailing: NSS.NAXIVA@nhs.net The following information will be required:
Centre name Confirmation of written informed consent Trial ID
Patient's date of birth This notification will allow these samples to be tracked by the University of Cambridge and SCTRU.
informed consent form - translational
4.3 expedited reporting of fresh

All participating sites must notify SCTRU in advance of the date of planned biopsy or nephrectomy.  
Centres should notify SCTRU of the biopsy or nephrectomy, by completing the NAXIVA
expedited notification of tissue collection form (included within the NAXIVA laboratory manual) and emailing it to: NSS.NAXIVA@nhs.net SCTRU will then notify University of Cambridge (Mr Grant Stewart and Dr Sarah Welsh) to expect samples.  
4.4 **Tissue Sample processing at the University of Cambridge**
All samples will be catalogued in an electronic database at the University of Cambridge. The time from core biopsy collection to snap freezing and from ischaemia to snap freezing for nephrectomy specimens will be recorded on a database. All tissues will be stored in an alarmed and locked -80°C freezer (or liquid nitrogen tank for PBMCs and dissociated tumour samples) which are dedicated to the storage of clinical trial patient samples.  
4.4.1 Blood Specimens **(Whole blood, Plasma, Buffy coat, PBMC)**
Analysis of whole blood, plasma, buffy coat, and PBMCs for translational studies will be conducted at the University of Cambridge. Plasma will be analysed for biomarkers (including metabolites, circulating tumour (ct) DNA, and cytokines). DNA will be extracted from whole blood/ or buffy coat and PBMCs for sequencing of host DNA. PBMCs will be immunophenotyped using CyTOF.
informed consent form - translational
4.4.2. urine specimens

Analysis of urine samples will be conducted at University of Cambridge. Urine will be analysed for biomarkers (including metabolites, circulating tumour (ct) DNA, and cytokines).
informed consent form - translational
4.4.3 core biopsy and nephrectomy

Processing and analysis of core biopsy, nephrectomy and surgical specimens will be conducted at the University of Cambridge. Dissociation of fresh tumour, venous thrombus and normal kidney samples will be performed to separate malignant cells from associated nd 63 of 66 stromal and immune components, and immunophenotyping will be performed using CyTOF.  
Cell lines (tumour, stomal, lymphocyte) will be generated if sufficient tissue allows. RNA and DNA from each will be extracted from fresh-frozen samples according to Good Laboratory Practice standards. Immunohistochemistry and immunofluorescence staining will be performed on FFPE samples.
informed consent form - translational
5. analysis of biosamples

Biosample analysis aims to correlate changes in biomarkers with toxicity and response to axitinib treatment. The techniques below may be used to explore this endpoint and are based on the current literature. Equivalent novel methods and technologies might be used at the time of analysis.  
5.1. Metabolomics It is well established that there is a strong metabolic basis to ccRCC. The presence of the Warburg effect has revealed several novel targets for treatment or tumour surveillance. Dr Frezza is an expert in metabolomics, working mainly on inherited forms of kidney cancer.  
Using LC-MS, metabolites will be assayed in urine, blood and tissue across the various time points of the NAXIVA study to assess the use of these molecules in patients with IVC VTT and with Axitinib therapy. This work will be discovery in nature assessing alterations in metabolic profile within protein from tumour, plasma and urine at sequential time points within the NAXIVA study.  
5.2. **Circulating Tumour DNA**
In view of intratumoural heterogeneity, the use of tumour tissue for the development of prognostic and predictive biomarkers in ccRCC is challenging. Circulating tumour DNA (ctDNA) has been established to represent the tumour biology and may be superior to tissue as heterogeneity will be negated. Variant analysis will be undertaken on multi-region sampled issue using exome and shallow whole genome sequencing. A custom capture panel for targeted higher-depth (and higher sensitivity) sequencing of plasma and urine samples will be developed for each patient to assess the feasibility of tracking circulating DNA molecules that harbour somatic mutations. The end-point of this analysis will be to determine the ability to identify variants in both tissue and plasma and the change in ctDNA profiles from baseline to treatment phase to posttreatment phase which may be informative regarding markers of tumour response and minimal residual disease. Further validation studies of any positive findings will be undertaken separately.  
5.3. **Proteomics**
Formalin fixed paraffin embedded biopsy and nephrectomy tissue will be utilised in an automated quantified immunohistochemical based analysis of putative markers of response to tyrosine kinase inhibitors. In addition to Ki67 measurement of effect of Axitinib on proliferation, other markers of response to tyrosine kinase inhibitors will be evaluated. In recent work, it has been established that modulation of CA9 protein levels occurs following sunitinib therapy in metastatic clear cell RCC (ccRCC); this effect will be validated using patient samples from NAXIVA. The outcome of this work will be to establish a link between expression of these proteins and reduction in height of the VTT with axitinib. If a correlation is established these proteins will be helpful as surrogate translational endpoints in future ccRCC neoadjuvant studies.  
5.4. **Immune studies**
It is known that RCC is a typical immunogenic tumour with immune mechanisms known to play an important role in its natural history. Recent studies have also shown that TKI treatment can influence the immune system and, importantly, changes in the immune response may affect response and survival. The immune studies aim to identify immuneand cytokine-based signatures in PBMCs, plasma and urine which predict response to nd 64 of 66 axitinib treatment in RCC patients using CyTOF and luminex-based cytokine assays. These studies will also provide important information about the effect of axitinib on the immune system in RCC, and vice versa, including providing comprehensive analysis of how peripheral immune cell populations and cytokines vary during treatment with axitinib, and whether they correlate with other markers of response and resistance in RCC patients. Importantly, it will also provide essential immunological information to inform biologically driven, rational, scheduling of future studies examining combinations of axitinib and immunotherapy.
informed consent form - translational
appendix 7- the principles of

1. Clinical trials should be conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki, and that are consistent with GCP and the applicable regulatory requirement(s).  
2. Before a trial is initiated, foreseeable risks and inconveniences should be weighed against the anticipated benefit for the individual trial subject and society. A trial should be initiated and continued only if the anticipated benefits justify the risks.  
3. The rights, safety, and well-being of the trial subjects are the most important considerations and should prevail over interests of science and society.  
4. The available nonclinical and clinical information on an investigational product should be adequate to support the proposed clinical trial.  
5. Clinical trials should be scientifically sound, and described in a clear, detailed protocol.  
6. A trial should be conducted in compliance with the protocol that has received prior institutional review board (IRB)/independent ethics committee (IEC) approval/ favourable opinion.  
7. The medical care given to, and medical decisions made on behalf of, subjects should always be the responsibility of a qualified physician or, when appropriate, of a qualified dentist.  
8. Each individual involved in conducting a trial should be qualified by education, training, and experience to perform his or her respective task(s).  
9. Freely given informed consent should be obtained from every subject prior to clinical trial participation.  
10. All clinical trial information should be recorded, handled, and stored in a way that allows its accurate reporting, interpretation and verification.  
11. The confidentiality of records that could identify subjects should be protected, respecting the privacy and confidentiality rules in accordance with the applicable regulatory requirement(s).  
12. Investigational products should be manufactured, handled, and stored in accordance with applicable good manufacturing practice (GMP). They should be used in accordance with the approved protocol.  
13. Systems with procedures that assure the quality of every aspect of the trial should be implemented.