What to collect and is it fit for purpose?

Transcription

1 What to collect and is it fit for purpose? Gerry Thomas, Professor of Molecular Pathology, Imperial College, London Scientific Director, Wales Cancer Bank

2 The pragamatic approach... What do the studies need that your biobank will supply? Blood samples, tissue samples, other bodily fluids? Are there times when these samples would be taken for the diagnostic record? can your samples be taken at the same time? Are you asking for something to be made available that requires others to change their practice? understand the clinical pathway for your patients

3 Making the most of your resource Modern studies use only small amounts of material different studies use different types of material derived from cells By processing samples instead of giving out tissue blocks, tissue banks can support many different projects carried out by different researchers on material from the same patient

4 Systems biology throwing everything into the pot! Biologists now starting to realise that our inherited genes not only may make us more or less susceptible to disease, but also may work hand in hand with genetic changes acquired by diseased cells to make things worse. protein methylation mirna sdna gdna RNA

5 Need to collect biological samples from the same patient but in different formats e.g. Blood sample for DNA extraction (inherited susceptibility to disease or metabolic activity affecting drug toxicity/efficacy Tissue sample for somatic changes which predict prognosis or response to treatment Serum samples for marker detection

6 The possibilities are endless... Pre-operative Core biopsies (very useful for non-operable cancers e.g. Lung) Blood samples Whole blood germline DNA Serum/plasma Circulating tumour cells/rna/mirna/methylated DNA Other bodily fluids (urine etc)

7 Intra-operative Bile Pancreatic fluid Operative specimen Snap frozen tissue Tissue stored in stabiliser Fixed tissue

8 Patients with metastatic disease Blood (circulating tumour cells, DNA, RNA, mirna) Metastatic biopsies Cells from ascites/pleural effusions Post mortem biopsies particularly useful for non-clinically accessible sites e.g. brain

9 But... Not all hospitals work in the same way - clinical pathway may be different in different centres In academic centres there are often competing priorities Smaller hospitals may not have access to the basics (freezer, dry ice/liquid nitrogen...)

10 What are you going to do with your sample? Do your samples need to be handled differently from the way in which normal clinical samples for diagnosis are handled? Are there internationally agreed protocols for handling your samples (e.g. Fixation type and duration, processing to paraffin, separation of blood components) Is there any scientificevidence that differences in protocols affect your chosen marker?

11 The Importance of Quality Assurance The sample MUST be what we say it is It MUST be fit for purpose

12 Garbage in garbage out Diamonds in

13 How you collect your sample matters. TC C Villanueva et al., 2005 J Proteome Res 4:

14 Ensuring quality of samples Material must be collected, documented and stored according to written protocols (SOPs) Adherence to SOPs must be regularly checked If you can t control quality at input level, must control quality at output

15 The Importance of SOPs SOPs enable us to collect data on how a specimen is obtained and manipulated. If SOPs are too rigid or impractical, human nature means that either specimens will not get collected or people will not tell the truth. SOPs should be developed with team involved in collection of material to ensure they are practical.

16 Is it what you think it is? Pathology QA Frozen and FFPE tissue blocks received, barcoded Section taken to confirm presence of disease H and E s digitised available to researchers over the web eventually!

17 % tumour Is it tumour? How much of it is tumour? % cases Breast Colorectal n-520 Prostate n-103 Renal n-119 Head &Neck n-60 n

18 Is it fit for purpose? Microarray Affymetrix 3, exon array or Agilent cdna? Single gene expression RT-PCR or qrt-pcr? BAC acgh? Single proteins? Multiple proteins at the same time?

19 Frozen Tissue Format of choice for omics that require intact RNA and DNA DNA, RNA, mirna and protein can be potentially be extracted from the same piece of tissue, but individual yield will suffer Method of extraction will affect quality Need to consider stability of extracted material over time and ease of storage

20 Tissue type and RNA quality n/a < >7 % of cases Tumour N=107 breast Normal N=26 colorectal Tumour N=49 Normal N=24 prostate Cores (T/N) N=68 renal Tumour N=34 Normal N=24 RIN

21 Length of time in storage

22 Collection site B Freezer failure RIN Time of storage in years

23 Not the best RNA in the world, but can I still use it? Single gene expression RT- PCR or qrt-pcr?

24 Cold Ischaemic Time Expect RIN to be low for samples with high CIT Pattern shows there is no correlation between high CIT and low RIN

25 Effect of cold ischaemic time on protein biomarkers Time unknown 17 minutes t-vegfr (CST 2479) a SMA/ CD34 Courtesy of Prof C Womack, AstraZeneca

26 Fixation & RNA Quality RIN frozen Z7 Finefix NBF Fixative Median RIN for each group upper value of line is maximum value and lower minimum

27 mirna from FFPE tissue

28 QA for DNA from frozen tissue 10kb gel electrophoresis

29 DNA QA FFPE Multiplex PCR Failure rate 42/93 = 45% Van Beers et al., BJC, 2006

30 DNA QA FFPE Multiplex PCR 52 breast cancer cases (IDC) tested (all fixed in 10% NBF) Cases operated between 1996 and 2001 DNA extracted used Qiagen kit 8 showed only 1 or no bands QA failure rate: 15.38% EC GC

31 Take home messages Communication is paramount tissue banking is multi-disciplinary, and not everyone speaks the same language Decide what you want to collect and who you want to collect it for Design SOPs with the teams involved in collection and storage Build QA into every step so you can work out when something goes wrong where it is going wrong

32 Acknowledgements Prof Malcolm Mason Dr Alison Parry-Jones Mr D Naeh The Clinical and non-clinical Staff from the WCB sites particularly histopathology CRW and Welsh Assembly Government The patients of Wales who make it all possible Prof Robert Leonard Dr Kristian Unger Dr Bill Mathieson Dr Sileida Oliveros Dr A Galpine Dr S Mackay Dr S Pericleous Mrs J Bethel Miss Mahrokh Nohadani