The Cancer Research UK Stratified Medicine Programme: Phases One and Two Dr Emily Shaw

The Cancer Research UK Stratified Medicine Programme: Phases One and Two Dr Emily Shaw

Introduction The CRUK Stratified Medicine Programme: SMP1 rationale, design and implementation The role of the cellular pathologist Learnings from SMP1 Planning and implementation of SMP2

Challenges in implementing stratified medicine Requirement for and scope of tissue-based predictive molecular analysis likely to increase Variable access across UK Need to ensure accuracy and reproducibility of results Requirement to work with formalindegraded nucleic acids Lack of consensus on approach to testing Clinical interpretation of the results

SMP1: 2011-2013 B ham 9,000 cancer patients Clinical data (National Cancer Registration Service) Compiled data Anonymised data extract Clinical Hubs Researchers Partners NHS Leeds Man Edin Glas Camb Card RMH Samples Genetic results Research infrastructure Service delivery Cardiff B ham ICR Genetic Technology Hubs

SMP1 gene list and technology Tumour Colorectal carcinoma Breast carcinoma Prostate carcinoma Lung carcinoma Ovarian carcinoma Malignant melanoma Genes of interest KRAS BRAF NRAS PIK3CA TP53 mutation PIK3CA TP53 BRAF PTEN mutation + PTEN LOH by microsatellite analysis PTEN BRAF mutation TMPRSS2-ERG fusion by FISH + PTEN LOH by microsatellite analysis EGFR KRAS BRAF DDR2* mutation ALK rearrangement by FISH TP53 PTEN PIK3CA BRAF mutation + PTEN LOH by microsatellite analysis BRAF KIT NRAS PIK3CA mutation * Squamous cell carcinoma subtype only

SMP1 progress 2011-2013 12000 Target for samples sent Patients consented Samples sent for testing Results returned 10754 patients consented and 9010 samples sent for testing 10000 8000 Target: 9000 samples 6000 4000 2000 0

The approach is acceptable to patients

SMP1 findings Critical role of pathology department in managing tissue samples Impact of differing sample volume/ flow in technology hubs Value of quality assurance scheme Highly variable sample quality and test failure rates Complexities of NHS IT systems Automated data extraction from electronic patient records is not yet reliable

The crucial role of cellular pathology Histological subtyping of tumour based on morphology +/- immunohistochemistry Confirm that tumour tissue being analysed and provide estimate of percentage tumour cell vs. non-tumour cell nuclei Success in mutation analysis likely to be dependent on specimen handling processes such as fixation and processing Interpretation of returned tumour genotype and issue integrated histopathology report

There is no such thing as a pathology SOP * Fixation Tissue processing Tissue economy in small biopsies Microtomy for molecular work Blade cleaning Block storage Different approaches to EBUS sample handling * Standard operating procedure

This is a truly multiprofessional and multidisciplinary endeavour Surgery Operational manager Oncology Physician Cellular pathology Research nurse Molecular genetics Biomedical scientist Cytogenetics Clinical scientist Biobanking Clinical data manager Informatics Bioinformatician Clinical trials assistant Administrative & clerical staff

The importance of information standards: clinical data Benefits of using existing NHS information standard (e.g. COSD, SNOMED, TNM) Define attributes (NHS data dictionary) Assist with mapping Facilitates aggregated data analysis

The importance of information standards: molecular data Efforts to agree list of polymorphisms for tumour suppressor gene screens at outset Standardised language for reporting genetic aberrations Nucleotide +/- amino acid change One letter or three letter abbreviation, stop codon PTEN loss of heterozygosity Agree approach to repeat analysis

Stratified Medicine Programme Phase Two (SMP2) Pre-screening for molecular abnormalities linked to stratification for clinical trials Sole focus on lung cancer Procurement and implementation of a single panel test delivered using next generation sequencing technology Support stratified medicine research by facilitating recruitment to a linked UK clinical trial Plan to maintain the existing network and infrastructure from phase one and expand nationally to incorporate further centres Longitudinal sampling element

SMP2: 2013 - now Genetic and clinical data Patients with lung cancer Data repository Clinical Hubs (ECMC network) Samples Anonymised data Cardiff B ham RMH Genetic Technology Hubs Genetic results Researchers Partners NHS Eligible patients National Lung Matrix Trial (NLMT)

SMP2 Molecular Pre-Screening Primarily patients with advanced disease Samples derived from biopsy/ cytology cell block rather than resections Aim to screen 2,000 samples per year across the national network Use of an accredited multiplexed technology solution that can be developed during the course of the programme

Pathology and sample standards for SMP2 (1) Histological subtypes: Adenocarcinoma, squamous cell carcinoma and other non-small cell primary lung carcinoma Not metastases from non-lung primaries, carcinoid/neuroendocrine tumours or mesothelioma Nature of samples: Slide-mounted sections Predominantly biopsies from primary or site of metastasis Cytology specimens accepted as cell block sections including EBUS-FNA and malignant effusions Sample size requirement: 50ng DNA required (~5-10x 5um sections)

Pathology and sample standards for SMP2 (2) Laboratory handling: Formalin; fixation time 6-24 hours; encourage tissue economy Decalcification: EDTA recommended Tumour content: > 20% tumour nuclei content in tissue from which DNA to be extracted Turnaround times: Material should be dispatched from lab as soon as possible

Challenges of SMP2 Sample size and DNA quantity Formalin-degraded nucleic acids Move to NGS technology Change in gene list and scope of tests More than just mutations Sequencing data interpretation pipeline Delivering acceptable turnaround times for clinical trial enrolment

Challenges of working with FFPE tissue Risk of exhausting small tissue samples for standard of care tests Requirement for macrodissection Ensuring exclusion of paraffin wax during extraction Length of digestion: yield vs. utility for sequencing DNA fragmentation and formalin-induced sequencing artefacts

Pathologists aren t accustomed to counting cells Tumour content assessment informs sample adequacy decisions, need for macrodissection and even confidence of NGS variant calling (wild type status for NLMT)

Tumour content assessment Percentage tumour cell nuclei vs. other nuclei e.g. stromal or inflammatory cell In whole section or area marked for macrodissection only? How accurate eyeball estimate to nearest 10% or digital image analysis?

Tumour estimates vs. digital TCD

Overall SMP2 Study Attrition Rates for 2015

Study Attrition Rates for 2016

Ongoing work 1 Increase input quantity of DNA: Obtain more tissue, where appropriate and safe to do so and/or research protocol biopsy as part of matrix trial Promote tissue economy in pathology laboratory Efficient use of tissue in genetics laboratory, including link to established NHS testing processes Explore possible alternatives to formalin (STRATFix) Multivariate analysis of pre-analytical sample handling data Attempt to work towards establishing optimized tissue handling protocols for current and future analysis requirements (CM-Path, GEL/ 100KGP, RCPath )

Ongoing work 2 Develop NGS protocols for low input quantities of fragmented FFPE-derived nucleic acids (Illumina) Ascertain best QC step as predictor of sample performance (Illumina, GEL) Define and address implications of stratified medicine implementation for workforce, education and training (with CM-Path, RCPath, MRC, ACGS, ABPI POI, NHSE, HEE, GEL, UK NEQAS and others )

Awareness of the broader operational context Competing pressures of NHS and other research activities Consultation, discussion and listening to inform decisionmaking Share data and successful practice to stimulate discussion, innovation and maintain engagement Establish and advance the evidence base Minimise and justify changes to the programme processes e.g. reporting or analysis

The many roles of cellular pathology Situated at the interface of clinical and laboratory medicine Experts in tissue handling Experts in morphological, immunohistochemical and increasingly molecular characterisation of disease Integrators of multiple inputs of clinical and laboratory data Familiarity with use of classification systems, reporting terminology and data handling Central role in patient management for different cancer types via clinical report and MDT membership

Acknowledgements The patients who have consented to participate in the programme to date Lead investigators and teams at the clinical and technology hubs CRUK Stratified Medicine Programme team, past and present Funding partners AstraZeneca and Pfizer Illumina for collaboration to develop and validate NGS platform Wider network of advisory groups CM-Path initiative