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Review of evolving technologies in CUP Dr Natalie Cook

This review provides an overview of evolving liquid biopsy technologies in cancer of unknown primary (CUP), including circulating tumor cells (CTCs), circulating free DNA (cfDNA), and tumor-educated platelets (TEPs). It discusses their clinical applications in early detection, diagnosis/prognosis, minimal residual disease, therapy selection, response to therapy, resistance, and relapse. The diagnostic challenges of CUP and the potential role of liquid biopsies in determining tissue of origin and biomarker status are also explored.

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Review of evolving technologies in CUP Dr Natalie Cook

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  1. Review of evolving technologies in CUP Dr Natalie Cook Senior Clinical Lecturer in Experimental Cancer Medicine, University of Manchester Honorary Consultant in Medical Oncology Christie NHS Foundation Trust

  2. Talk overview • Liquid biopsy technologies to be discussed; • Circulating tumours cells (CTCs) • Circulating free DNA (cfDNA) • Tumour educated platelets (TEPs) • Current research in Manchester, UK

  3. Liquid biopsy Clinical Application Early detection Diagnosis/ prognosis Minimal residual disease Therapy selection Response to therapy Resistance Relapse Circulating microRNA Exosomes Microvesicles ctDNA CTC TEPs Blood Plasma Chromosomal aberrations Methylation patterns Amplifications/deletions Point mutations Translocations CTC derived organoids RNA expression Protein expression

  4. Diagnostic challenges of CUP • Delays in diagnosis & multiple (invasive) investigations to find primary site • Limited robust evidence that treating patients based on molecularly-predicted primary improves outcomes • Several research & commercial tissue of origin classifiers; not recommended for diagnosis of CUP • Without tissue of origin, no access to novel therapeutics or immunotherapy • 10-20% failure rate of molecularly profiling tumour tissue • How representative is a single small biopsy of 1 metastatic lesion? • Alternative = liquid biopsies

  5. Tissue of origin from liquid biopsy? RNA-Seq of tumor-educated platelets enables blood-based pan-cancer, multiclass and molecular pathway cancer diagnostics Best et al 2015, Cancer Cell, 28(5):666 Detection and localization of surgically resectable cancers with a multi-analyte blood test Cohen et al 2018, Science, 359(6378):926 Plasma DNA tissue mapping by genome-wide methylation sequencing for non-invasive prenatal, cancer, and transplantation assessments Sun et al 2015, PNAS, 112(40):E5503 Identification of methylation haplotype blocks aids in deconvolution of heterogeneous tissue samples & tumor tissue-of-origin mapping from plasma DNA Guoet al 2017, Nat Genetics, 49(4):635 Discovery of methylated circulating DNA biomarkers for comprehensive non-invasive monitoring of treatment response in metastatic colorectal cancer Baraultet al 2017, Gut, 67(11):1995

  6. Where could liquid biopsies fit? Diagnosis Management decisions Treatment response or resistance Determine Primary site Prognostic information Molecular subtype to stratify to treatment Early indicators or response/resistance Quicker diagnosis to direct investigations Understand the biology of CUP Treat as equivocal tumour type Targeted therapy Prognostic indicators of appropriateness of therapy Tissue of origin and biomarker status Immuno-therapy Chemo-therapy Palliative care

  7. Circulating Tumour Cells (CTCs)

  8. Circulating tumour cells are very rare • 1 ml of blood is about 1/5th of a teaspoon • 5,000,000,000 red blood cells/ml blood • 7,000,000 white blood cells/ml blood • <1-15 circulating tumour cells/ml blood <1 CTC per 500,000 WBC CTC

  9. What does 1 in 500,000 look like?

  10. What about CTCs in CUP? • Very limited published research (total of 21 patients evaluated across 2 studies) • Only CellSearch technology used (only identifies EpCam positive CTCs) • No cellular or molecular characterisation Summary of CTC counts in 7.5 mL of blood from patients with various types of carcinomas Allard et al 2004. Clin Cancer Res, 10(20):6897 Utility of measuring CTC counts to assess the efficacy of treatment for CUP Komineet al 2014. Anticancer Res, 34(6):3165

  11. The Manchester approach; the CUP BioBank HDSCA method ‘no cell left behind’ • Prospective recruitment of new CUP patients pre-treatment • Characterise CTCs & evaluate heterogeneity • Consent to: • Use of surplus biopsy • 2 additional blood samples for CTC banking and nucleic acid extraction • Clinical information HDSCA; High Definition Single Cell Assay Double spun plasma Cell pellet 12 x slides ~3 million cells per slide HDSCA slide preparation Parsortix method Total nucleic acids extracted Cells stored in glycerol

  12. Circulating Tumour DNA (ctDNA)

  13. Circulating tumour DNA • FoundationOne; tissue, >300 genes • FoundationOne Liquid, blood based, 70genes • Guardant 360; 73 genes, blood based • Oncologica; tissue, 505 genes • Local Academic Initiatives; eg TARGET Multiple potentials for molecular profiling from blood & tissue (none funded in the UK) • Targetable driver & actionable mutations • Copy number translocation • Tumour mutational burden in some cases Looks for

  14. Manchester approach Within the TARGET ctDNA trial DNA Next generation sequencing 360gene panel Molecular Tumour Board Stratified to treatment (Phase I) ctDNA & germline controls 650 somatic mutations All solid tumours Determine tissue of origin Tissue of origin directed therapy Copy number analysis Improved predictive power of mutational profile • Development and optimisation of cfDNA methylation assay • Combine mutational profile, methylation profile and copy number variation to evaluate prognostic and predictive biomarkers

  15. TARGET CUP patients so far 17 CUP TARGET patients recruited to date • Mutation data alone insufficient to determine tissue of origin • We can determine presence of tumour DNA • Majority of patients have at least 1 mutation (16/17) • 13/17 patients have VAF of >5%

  16. Tumour Educated Platelets (TEPs)

  17. Tumour Educated Platelets Best et al 2015. Cancer Cell 28(5):666-676

  18. Manchester CUP project overview Clinical and Experimental Pharmacology • Longitudinal cfDNA analysis of patients on treatment • Mutation & Copy Number analysis • Methylation patterns TARGET trial Nucleic acid biomarker team Phase 1 ECMT CUP project MCRC CUP Biobank • Advanced cultures • CUP biology & heterogeneity Preclinical Team Archival tissue Tissue Biomarker team • Immune markers • MSI/dMMR CTC team Cells & Proteins team • CTC enumeration • CTC biology TIIML

  19. Conclusions • Lots of new technologies in the field of liquid biomarkers • None routinely used; lack evidence of improved outcomes • Mostly research initiatives, but slowly being incorporated into trials • Future standard of care?

  20. Acknowledgements Supported by The Systemic Therapy Research Group @ The Christie NHS Foundation Trust STR • The patients and their families • Alicia Marie Conway • Claire Mitchell • Caroline Dive • Ged Brady • Experimental Cancer Medicine Team, Christie NHS Foundation Trust • Clinical and Experimental Pharmacology Group, Cancer Research UK Manchester Institute

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