The SPIDIA4P project will build on data of the STRATFix consortium covering central activities and topics within both projects such as solid tissue, fine needle aspirates (FNAs) and liquid biopsies. The close collaboration of experts throughout different areas will help to develop and assure clinical guidelines and standards for pre-analytical and analytical work with biological specimens.
In the STRATFix project, prototypes of integrated systems for sample collection, stabilisation and nucleic acid purification (CSP-systems) will be developed for implementation in routine pathology, based on work started within the SPIDIA project. Formaldehyde-free CSP-systems will be provided for solid tissue, FNAs and circulating cell free DNA (ccfDNA) in blood to enable genetic follow up characterisation with less invasive sample material. STRATFix is a joint effort of industry and academia and will involve significant cross-sector collaboration and knowledge transfer between them. All members of STRATFix can build upon excellent and unique expertise in their fields.
After many decades of cancer research, treatment is increasingly changing to become more tailored to individual patients' needs (often referred to as 'personalised' or 'stratified' medicine). Current preparation methods allow microscopic assessment of cells and tissue but often do not preserve nucleic acids well enough for comprehensive genetic tests that are necessary for personalised medicine. These pre-analytical workflows include unstandardised formalin-fixation of solid tissue and large biopsies and non-fixation or methanol-based treatment of small biopsies and FNAs. In the STRATFix project new pre-analytical workflows linked to new analytical technologies are developed, in order to help select the right treatment for individual patients. Therefore, STRATFix is developing new solutions to preserve cells and biomolecular analytes as DNA and RNA in aspirated cell-rich fluids (e.g. FNAs) and blood. The project coordinator QIAGEN is market leader in providing tools for sample collection, stabilisation and nucleic acid purification to cover the entire “sample-to-insight” workflow. By working in a UK-wide collaboration, STRATFix members have access to the widest possible range of blood, cell and tissue types. Because these developments will take place in multiple major, opinion-leading UK hospitals, this project has great opportunities that the project outcomes will be adopted by the whole UK pathology community.
STRATFix is divided into three major activities based on the different sample types. The business-led consortium assures the development of commercially relevant products, whilst the participation of a diverse geographical network of NHS partners guarantees the most effective uptake of the novel tools and products into routine clinical practice.
Implementation of PAXgene Tissue into routine clinical use
Commonly used tissue fixation methods based on formaldehyde chemically modify proteins and nucleic acids and are becoming the rate limiting step in the application of these techniques to clinical practice. It is estimated that suboptimal procedures during collection, handling, preparing or storing the specimens account for 60-70% of all problems occurring in laboratory diagnostics (Lippi G. et al. 2011). It is important to enable the reliable use of applications such as digital PCR or next generation sequencing for stratified medicine with nucleic acids from the same diagnostic sample that is used for morphological analysis. For this purpose, novel and formaldehyde free fixatives for improved pre-analytical pathology workflows are urgently needed.
Following a systematic evaluation, procedures routinely used in pathology for a broad range of cancers will be optimised for use with PAXgene Tissue treated samples. The PAXgene Tissue System was originally developed within the SPIDIA project. Basic studies performed by academic partners within SPIDIA demonstrated morphology, nucleic acid and protein (including phosphoprotein) preservation. However for implementation into clinical use, upfront a systematic evaluation and optimisation is necessary, since protocols for histochemical and IHC staining and FISH in clinical use currently were developed to work with formalin-fixed paraffin-embedded (FFPE) samples.
Development and validation of a CSP-system for fine needle aspiration (FNA) cytology samples
As a less invasive technique, FNA cytology sampling is expected to become more popular for diagnostic procedures in different applications like lung, pancreas, lymph node and thyroid. The standard procedure is to smear a part of the sample directly onto a slide followed by staining. Other techniques involve centrifugation, fixation of the cell pellets in formalin or methanol and preparation of paraffin embedded cell blocks. Both methods are of limited use for molecular analysis. Biomolecule purification from smears is inefficient and results in low yield and quality. Fixatives that contain methanol or formaldehyde compromise either cytology of the cells or integrity of biomolecules. To enable molecular analyses for stratified medicine from the same sample which is used for cytological analysis, there is a need for a fixative that simultaneously preserves cell morphology and biomolecules such as DNA and RNA. Starting with a reagent which was developed within SPIDIA, this activity will include testing and final formulation of the FNA fixative and development of the CSP-system components including protocols for cytopreparation methods. This system will be validated with cytological and immunocytological staining methods in comparison to other fixation alternatives e.g. based on methanol. DNA and RNA extraction will be established from liquid samples and paraffin cell blocks. The consortium will be the first to develop and validate a high-quality multi-purpose system for FNA samples.
Development and validation of a circulating cell free (ccfDNA) DNA CSP-system
Circulating cell-free DNA (ccfDNA) in blood is currently under investigation as a tool for screening, diagnosis, prognosis and surveillance of residual/progressive diseases. Blood samples for ccfDNA purification are usually collected in EDTA-tubes which inhibit coagulation but do not efficiently prevent the release of nucleic acids from cell lysis occurring around the time of blood draw. When using EDTA blood, plasma has to be prepared shortly after blood collection. At present alternatives in the market have impacts on the cell lysis and nucleic acid integrity. Therefore a system is needed for transportation into pathology labs, avoiding the need for immediate plasma generation at the site of collection, that preserves the ccfDNA profile by preventing blood cell lysis without damage and modification of the ccfDNA.
This activity will start with a blood cell preservation reagent, developed within SPIDIA, to prevent lysis or apoptosis and release of DNA into plasma. After a proof of principal study with blood from healthy donors, the whole CSP-system including a collection tube with a stabiliser and automated purification of ccfDNA from stabilised plasma will be developed by QIAGEN. In a study with clinical samples the system will be validated for time and cost effectiveness in routine analysis.