Sample Banking in Renal Cancer - A Consideration of Some Issues - Roz Banks Clinical and Biomedical Proteomics Group Cancer Research UK Clinical Centre St James’s University Hospital Leeds, UK www.proteomics.leeds.ac.uk Why Bank Samples? • Essential if furthering our knowledge of renal cancer and potentially developing new biomarkers (diagnostic, prognostic, predictive…….) or therapies for example • Investment for the future – expedites translation of findings. If high quality (samples, processing and clinical annotation), can be used in new studies as new technologies/results emerge • Need multiple banks for RCC to ensure representative and sufficient numbers of samples and multi-centre validation of findings BUT Any Centre Needs: a defined reason for doing it, realistic plans to use and/or release them and quality banking procedures – gift from the patient and non-trivial financial investment so various obligations CRUK LTHT NHS Trust Oncology Urology Pathology Clinical Chemistry Leeds University Patients Clinical & Biomedical Proteomics Group Research Tissue Bank Tissue Blood Protein and Gene Studies e.g. • CRUK Proteomics Programme • EU CAGEKID €10.5M (RCC whole genome sequencing etc) Clinical data Urine New pathways, biomarkers & drug targets Patient Benefit Individualisation of diagnosis & treatment Improved safety & quality of life Partner Benefit More efficient use of £££ Research ability/reputation IP A Sample Banking Pathway Clinical Trial Biomarker Study (internal & external) • Dedicated team/lab including QA Manager – GCLP development ongoing Ethics • Collaboration with clinical teams Research Nurses & Clinicians Patient Recruitment • Detailed study-specific SOPs Clinical Data • Bar-coding and secure databases Research Nurses & Technicians Sample Collection (Study-specific timing and type) QA Manager Research Technicians Sample Processing (Study-specific SOP) Sample Bank Sample Data Ethics/Regulatory Issues • Ensure compliance with relevant regulations/guidance e.g. Human Tissue Act, Data Protection Act, EU Directives, GCP and MHRA guidelines etc • International studies – be aware of differences in requirements between different countries • Need relevant ethics/IRB approval. Newer technologies e.g. whole genome sequencing potentially have major personal implications which need to have been explicitly covered. • Obtain broad patient consent to maximise potential future use of the samples – consider “research tissue bank” status with defined access policies Which Patient Groups? Depends on research questions – consider the bigger picture e.g. Leeds Proteomics Group – broad biomarker-based discovery/validation etc: RCC • Ongoing • >500 plasma/serum/urine at diagnosis • >350 matched normal and tumour frozen tissue • Patients in TKI trials (longitudinal) Nephrectomy • Population-based TMA under construction • Extensive clinical data and follow-up RCC/matched normal cortical tissue Sample Collection Others Annotation • Controls (healthy and benign urological disease) • Living donors (controls for nephrectomy effects) Frozen Tissue Bank • Nephrotoxicity study • Renal medicine • Clinical trials Proteomics Clinical history, histopathology, follow-up FFPE Tissue bank FFPE blocks Tissue microarrays How Should Samples Be Banked? Sample types - depend on the research needs and the practical issues Frozen • Tissue Derived primary cell lines Formalin-fixed paraffin-embedded (FFPE) • Fluids Blood Plasma Serum PBMC/Buffy Coat Urine Critical that samples are collected consistently according to Standard Operating Protocols (SOPs) together with high quality clinical data Samples should be logged and mirror-banked in secure alarmed freezers/liquid nitrogen banks Pre-Analytical Effects All may influence the outcome of the research studies • Biological (in vivo) Age, gender, diet, time of day, concomitant illness, ethnicity….. • Technical (in vitro) sample collection (op procedure/hypoxic time, venepuncture method effects) sample handling (delay, fixative/anticoagulant, pH, temp, time) storage (length & temp)………. Nucleic acids, proteins and their post-translational modifications vary tremendously in terms of stability Frozen tissue Key issues: time delays, method and rate of freezing, storage length and temperature, representative sampling Limited studies generally and none in RCC - need for more investigation and also stringent validation Tissue – time to freezing Skold et al (2007) – postmortem brains (no renal examples) Peptides t=0 mins t=10 mins Proteins 53/1500 proteins on 2D-DIGE changed by >1.5 fold Phosphorylation Heterogeneity of Tissue Samples - QC If extracting, check sections from the actual blocks used in analysis (ICGC) Uniform – mainly tumour cells Granular and clear cells, PBMCs Fibrosis – minimal tumour cells Mixed grade areas Formalin-fixed paraffin-embedded (FFPE) tissue May often be only option • DNA/RNA analyses Often used – limited to short amplicons. Can produce fixationinduced artefactual “mutations” • Protein analyses Care with IHC and antigen retrieval Cross-links prevent use of FFPE tissue in proteomic studies??? Be aware of dangers of analysis of phosphorylation status Many effects from fixative duration, fixative age, size of blocks etc Fluids • Type of tube Manufacturer, gel/no gel, anticoagulant type • Time Delay between venepuncture and processing Length of storage • Temperature Interval and centrifugation – “RT” or 4oC Storage - -20oC or -80oC Blood tube type (gel/non-gel) and temperature Neutrophil elastase complex – a marker of inflammation (similar results for VEGF measurements) Elastase complex (pg/ml) Serum 800 4 4 gel RT RT gel 700 600 500 400 300 200 100 0 1 2 3 4 5 6 Donor Serum also much higher than plasma Conclusions • Need for coordinated sample banking across multiple centres in RCC to facilitate translational research in RCC • Samples should be collected so that they are “fit for purpose” with QC processes and relevant clinical data • Samples and data should be stored securely • No single ideal method for sample processing - key is to be consistent (SOPs) and to record the various variables to avoid bias • More systematic studies of pre-analytical factors are needed Clinical Proteomics Group St James’s University Hospital - Leeds Pat Harnden – Pathology Medical Oncology team Urology team Renal Medicine team Peter Selby (co-PI) (www.proteomics.leeds.ac.uk) The Patients Funders CR-UK, DoH, NIHR, MRC, EU, Kidney Research UK , TSB