Liquid biopsy biomarkers for early detection: general principles and specific examples Charlie Massie Early Detection and Urological Malignancies Programmes University of Cambridge, CRUK Cambridge Centre Lectures in Cancer Biology• 28 th February 2019
Early Detection paradigm
Effective ED: cervical screening (i) Mike Quinn et al. BMJ 1999;318:904
Effective ED: cervical screening (ii) Landy et al. Br J Cancer. 2016
Benefits by age group are more complicated… Mike Quinn et al. BMJ 1999;318:904
Early detection of in situ disease… Mike Quinn et al. BMJ 1999;318:904
How early do we want to detect cancer development? Yates, Martincorena et al. Campbell Science Nat.Rev.Gen 2015 2012 Sottoriva et al. Nat Genet. 2015
Liquid biomarkers and early detection • Challenges of biomarker development • Options for non-invasive/minimally invasive sampling and biomarkers • Examples and strategies for early detection
Liquid biomarkers and early detection • Challenges of biomarker development • Options for non-invasive/minimally invasive sampling and biomarkers • Opportunities and strategies for early detection
Cells, vesicles and cellular material are released from solid tissues • Cells and cellular material are released into circulation • Allows minimally invasive or non-invasive sampling • Blood samples can be used to detect: • proteins (troponin) • metabolites (creatinine) • RNA (HCV-RNA) • aneuploidy (trisomy 21) • fusions / SVs (BCR-ABL) • exosomes (fusion genes) • mutations (EGFR T790M ) • epigenetic marks (SEPT9) • … Diaz Jr and Bardelli, JCO 2014
Th The biomarker challenge: sp specifi ficity. From Ng, Chong et al., Gut 2009
Markers with overlapping distributions: miRNA expression to detect early metastasis in melanoma FP? FN? Ono et al. Oncotarget 2015
Continuous biomarker trade-off: determining sensitivity and specificity cut point(s) Kulasingam V, Diamandis EP. Nat Clin Pract Oncol. 2008 ;5(10):588-99.
Protein markers in blood serum/plasma • Prostate Specific Antigen (PSA) – not cancer specific False Late positives diagnosis Which patients need treatment? Source: Lin et al. Clin Ca Res. 2013 United States Preventive Services Task Force (USPSTF)
Liquid biomarkers and early detection • Challenges of biomarker development • Options for non-invasive/minimally invasive sampling and biomarkers • Opportunities and strategies for early detection
Types and sources of biomarkers Biomarker type/source • – Circulating Tumour Cells (CTC): single/clusters – Immune ’sponges’ and responses: Tumour educated platelets and T/B-cell receptor repertoire – Extracellular Vesicles: exosomes, microvesicles, apoptotic bodies, … – Cell-free markers: proteins, cfRNA (miRNA, lncRNA), cfDNA • Sample types – blood plasma/serum or urine – local sampling: stool, saliva, urine, nipple aspirates, seminal fluid, menstrual fluid, CSF, …
Types and sources of biomarkers Biomarker type/source • – Circulating Tumour Cells (CTC): single/clusters – Immune ’sponges’ and responses: Tumour educated platelets and T/B-cell receptor repertoire – Extracellular Vesicles: exosomes, microvesicles, apoptotic bodies, … – Cell-free markers: proteins, cfRNA (miRNA, lncRNA), cfDNA • Sample types – blood plasma/serum or urine – local sampling: stool, saliva, urine, nipple aspirates, seminal fluid, menstrual fluid, CSF, …
Circulating tumour cells CTCs • 0-1000 CTCs/ml • Often <10 • Limited dynamic range – newer technologies improve detection rates • More suitable for late stage disease • Opportunities for disease biology Metastatic breast cancer Dawson, Tsui et al., NEJM 2013
Immunological sources (i): immune components as vehicles for markers • Tumour educated platelets (TEP) • Provide a vehicle for capturing tumour signals • Not widely validated • Signal/noise for ED? Best et al. Ca Res 2018
Immunological sources (ii): amplified signal from tumour-response • TCR/BCR clone analysis (>10^6 cells) • Immune response to tumours • Evidence of signal in early cancers • Identifying tumour- specific T/B-cell clones is a major challenge Beausang et al. PNAS 2017\ Early-stage breast cancer
Extracellular Vesicles Skotland et al. Prog Lipid Res. 2017
Early detection using EVs/exosomes • High abundance (~10 9 EVs / ml) • Carry cell material (including RNA/DNA) • ExososomeDx Epi-test McKiernan et al. Eur Urol. 2018 – 3 gene urine test for prostate cancer – AUC -0.7 (better than PSA…)
Cell-free markers • Proteins, metabolites, metal ions, miRNA, cfRNA, cfDNA… • Marker stability/half-life in blood • Signal / noise ratio – high background from blood cells and other tissues – low(er) signal and absolute counts in early cancers
RNA markers and signatures • Tissue specific mRNAs – Tumour specific is more challenging (as for proteins) • Gene expression signatures – Multi-gene panels may increase sensitivity and specificity (Epi Test…) – Surrogates for phenotypes (prognostic) • Fusion genes…
Non-coding RNAs: miRNA and lncRNA • Tissue -specific markers Tissue Urine • Tumour-specificity may be limiting – improved by local sampling (PCA3) • May also be useful for – Tumour of Unkown Primary Hessels & Schalken Nat Rev Urol 2009 (TUP) – localizing tumours from liquid biopsies
Cell-free DNA markers (ctDNA) • High analytical specificity – digital genomic assays • Mutations –EGFR KRAS • CNA – only higher burden disease? • Epigenetics (nucleosomes and methylation) – CUP, tumour loclisation – abundant markers…
Sequence specific genomic markers: Counting individual molecules and broad molecular profiling • Allele specific probes or HTP-sequence reads • Digital signal: targets are either WT or mutant • Breadth and depth required – genomic coverage • Homogeneity of target population • Specificity of individual markers – required sensitivity Safe-SeqS • Expected signal range Kinde et al. PNAS 2011 • Input material: sampling limits (1/n) (see also: Duplex-seq, CAPP-seq, …)
Methods for digital sequence analysis: High-sensitivity quantitative and selective assays Digital droplet PCR HTP-sequencing 1:200,000 sensitivity 1:25,000 sensitivity Boreal Genomics (10 6 -fold mutation enrichment)
ctDNA can detect minimal residual disease after surgery: opportunities for adjuvant/second-line treatments and POC for early detection Stage II colorectal cancer Early breast cancer Tie et al. STM 2016 Garcia-Murillas et al. STM 2015
Types and sources of biomarkers Biomarker type/source • – Circulating Tumour Cells (CTC): single/clusters – Immune ’sponges’ and responses: Tumour educated platelets and T/B-cell receptor repertoire – Extracellular Vesicles: exosomes, microvesicles, apoptotic bodies, … – Cell-free markers: proteins, cfRNA (miRNA, lncRNA), cfDNA • Sample types – blood plasma/serum or urine – local sampling: stool, saliva, urine, nipple aspirates, seminal fluid, menstrual fluid, CSF, …
Cells, vesicles and cellular material are released into local fluids, blood circulation and excreted in urine/stool Sampling routes for prostate cancer Wan et al. Nat Rev Cancer 2017 Key considerations: • 5000ml blood volume • High background signals
Other non-invasive sample types: advantages of local sampling • Plasma (>10 3 /ml wt copies) • Urine (Urological +) • Saliva (Oral cancers) • Local sampling – Breadth, Stool, … – Cervical smear, menstrual fluid Wang et al. Sci Trans Med 2015 – Cerebrospinal fluid (CSF) – Oesophageal CytoSponge Lower WT background, … Enriched for tumour DNA
If ctDNA concentration in plasma is low, is cerebrospinal fluid (CSF) a better source of nucleic acids? 34
Emerging local sampling early detection tests: epigenetic markers for bladder cancer in urine 150 CpG loci biomarker panel Confirmed in multiple cohorts Bladder Cancer (UroMark Phase-III): Feber et al. Clin. Epigenetics 2017
Liquid biomarkers and early detection • Challenges of biomarker development; advantages of genomic biomarkers • Non-invasive sampling and genomic biomarkers • Strategies for early detection
Multi-modal tests for Early Detection • Combining advantages of different biomarkers – while minimizing disadvantages… • Comprehensive studies to select core markers • Summary score / test result • Cost and ease of use à implementation
FDA approved multiplex stool test Protein marker (haemoglobin) • • 7 point mutations (KRAS) 2 methylation markers • (NDRG4, BMP1)
Protein markers • ctDNA •
ctDNA levels are low in patients with earlier stage cancers or low tumour burden (e.g. after treatment) Bettegowda, Diaz et al. Sci Transl Med 2014
Multi-modal markers: are we caught between a rock and a hard place? • Abundant low specificity markers – Proteins, metabolites, RNA-species • Limiting specific markers – ctDNA: mutations, methylation • How to bridge the gap?
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