Charlie Massie Early Detection and Urological Malignancies - - PowerPoint PPT Presentation

Charlie Massie Early Detection and Urological Malignancies Programmes

University of Cambridge, CRUK Cambridge Centre

Lectures in Cancer Biology• 28th February 2019

Liquid biopsy biomarkers for early detection: general principles and specific examples

Early Detection paradigm

Effective ED: cervical screening (i)

Mike Quinn et al. BMJ 1999;318:904

Landy et al. Br J Cancer. 2016

Effective ED: cervical screening (ii)

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?

Martincorena et al. Science 2015 Yates, Campbell Nat.Rev.Gen 2012

Sottoriva et al. Nat Genet. 2015

• 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

Liquid biomarkers and 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 (EGFRT790M)
• epigenetic marks (SEPT9)
• …

Diaz Jr and Bardelli, JCO 2014

Th The biomarker challenge: sp specifi ficity.

From Ng, Chong et al., Gut 2009

Ono et al. Oncotarget 2015 FN? FP?

Markers with overlapping distributions:

miRNA expression to detect early metastasis in melanoma

Kulasingam V, Diamandis EP. Nat Clin Pract Oncol. 2008 ;5(10):588-99.

Continuous biomarker trade-off:

determining sensitivity and specificity cut point(s)

Protein markers in blood serum/plasma

• Prostate Specific Antigen

(PSA) – not cancer specific

Late diagnosis Which patients need treatment? False positives

Source: United States Preventive Services Task Force (USPSTF)

Lin et al. Clin Ca Res. 2013

• Challenges of biomarker development
• Options for non-invasive/minimally

invasive sampling and biomarkers

• Opportunities and strategies for early

detection

Liquid biomarkers and 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, …

• 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

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 (~109

EVs / ml)

• Carry cell material

(including RNA/DNA)

• ExososomeDx Epi-test

– 3 gene urine test for prostate cancer – AUC -0.7 (better than PSA…)

McKiernan et al. Eur Urol. 2018

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
• Tumour-specificity may be

limiting

– improved by local sampling (PCA3)

• May also be useful for

– Tumour of Unkown Primary (TUP) – localizing tumours from liquid biopsies

Hessels & Schalken Nat Rev Urol 2009 Tissue Urine

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…

• 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

• Expected signal range
• Input material: sampling limits (1/n)

Safe-SeqS Kinde et al. PNAS 2011 (see also: Duplex-seq, CAPP-seq, …)

Sequence specific genomic markers:

Counting individual molecules and broad molecular profiling

Boreal Genomics (106-fold mutation enrichment)

Methods for digital sequence analysis:

High-sensitivity quantitative and selective assays

Digital droplet PCR 1:200,000 sensitivity HTP-sequencing 1:25,000 sensitivity

ctDNA can detect minimal residual disease after surgery:

• pportunities for adjuvant/second-line treatments and POC for early detection

Early breast cancer Garcia-Murillas et al. STM 2015 Stage II colorectal cancer Tie et al. STM 2016

• 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

Wan et al. Nat Rev Cancer 2017

Cells, vesicles and cellular material are released into local fluids, blood circulation and excreted in urine/stool

Sampling routes for prostate cancer

Key considerations:

• 5000ml blood volume
• High background signals

Other non-invasive sample types: advantages of local sampling

• Plasma (>103/ml wt copies)
• Urine (Urological +)
• Saliva (Oral cancers)
• Local sampling

– Breadth, Stool, … – Cervical smear, menstrual fluid – Cerebrospinal fluid (CSF) – Oesophageal CytoSponge

Wang et al. Sci Trans Med 2015

…

Lower WT background, Enriched for tumour DNA

34

If ctDNA concentration in plasma is low, is cerebrospinal fluid (CSF) a better source of nucleic acids?

Emerging local sampling early detection tests: epigenetic markers for bladder cancer in urine

Bladder Cancer (UroMark Phase-III): Feber et al. Clin. Epigenetics 2017 Confirmed in multiple cohorts 150 CpG loci biomarker panel

• Challenges of biomarker development;

advantages of genomic biomarkers

• Non-invasive sampling and genomic

biomarkers

• Strategies for early detection

Liquid biomarkers and 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

Bettegowda, Diaz et al. Sci Transl Med 2014

ctDNA levels are low in patients with earlier stage cancers or low tumour burden (e.g. after treatment)

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?

Sampling rare events in low volume disease: hard limits are above assay sensitivity

~5000ml >10M amplifiable copies of cfDNA ~9ml ~4ml >8K copies (>0.01%) ~1ml ~2K copies (>0.05%) 1:200,000 sensitivity (>0.0005-0.04% ) 1:25,000 sensitivity (>0.0015-0.2% )

Options to increase detection in early /

low-burden disease

• High copy targets

– Multiple events, mtDNA, …

• Enrichment methods

– Fragment size, preferred ends, …

• Selective assays

– SV junctions, viral genomes…

When ctDNA levels are low sample size limits sensitivity and accuracy

NEG NEG NEG POS (e.g. 1/100) WT DNA BRAF V600E

Detecting multiple rare mutations negates hard limits on sensitivity

POS POS POS POS (e.g. (1*10)/100) WT DNA BRAF V600E Mut.2 Mut.3 Mut.4 Mut.5 Mut.6 Mut.7 Mut.8 Mut.9 Mut.10

Established epigenetic ctDNA blood tests for cancer

MeDIP antibody IP 5-methyl cytosine

Increasing sensitivity using more markers: Higher copy targets (mtDNA)

Ju et al. Elife 2014

mtDNA: 100s – 1000’s more copies than gDNA

Jiang et al. PNAS 2015

Epigenetic changes are often early and recurrent

Massie et al. ERC 2015

• DNA methylation in PrCa

– Shared 5mC changes between tumour foci – Over 100 recurrent, early events

Epigenetic changes are often early and recurrent

• DNA methylation targets

– 100-1000s of early and recurrent differentially methylated loci – Filter candidates for ctDNA analysis (e.g.Epigenome Roadmap)

17%

chr1:

1 kb 2,461,000 2,461,500 2,462,000

DNA Methylation by Reduced Representation Bisulfite Seq from ENCODE/HudsonAlpha

HES5

Options to increase detection in early /

low-burden disease

• High copy targets

– Multiple events, mtDNA, …

• Enrichment methods

– Epitopes, Fragment size, …

• Selective assays

– SV junctions, viral genomes…

Extracellular vesicle affinity enrichment

• Surface protein antigens
• Physical properties
• Advantages

– enriching signal/noise – simplifying downstream analysis

• Disadvantages

– loss of material/signal – Sensitivity/specificity of epitopes – cost and complication of upstream sample prep.

Ko et al. Sci Reports 2016

Enrichment of cfDNA by size selection

2018

Jaing et al 2018

Emerging options to increase detection in early / low-burden disease

• High copy targets

– Multiple events, mtDNA, …

• Enrichment methods

– Fragment size, preferred ends, …

• Selective assays

– SV junctions, viral genomes…

Chan et al. N Engl J Med 2017

Opportunities and challenges for liquid markers and ED

• Technical challenges

– signal/noise

• Other major challenges

– Clinical utility

• Trials, diagnostic pathway…

– Cost/complexity – Consequences of a positive and a negative result…

Summary

• Early(ier) detection to improve outcomes

– Stage-shift to reduce cancer mortality rates

• Significant challenges

– Technical, clinical and social…

• Reasons for optimism

– Previous and recent success stories – New technologies and concepts

• We need engagement from the next generation
• f scientists to take this forward

17%

International Summer School 2019

“Discovery and Development of Diagnostics for Early Detection of Cancer”

15th-18th July 2019, Robinson College, Cambridge

Acknowledgements

Urological Malignancies Early Detection Group

Gahee Park Anne Babbage Sabrina Rossi Robert Hanson Jelena Belic Radek Lach Sara Pita

Early Detection Programme

Rebecca Fitzgerald, Sarah Bohndiek, Daniel Munoz-Espin, Jamie Blundell, Wendy Alderton, Catherine Aitkins, Lieneke Makaske

ICGC Group

Andy Lynch, Dan Brewer, David Wedge, Chris Foster, Colin Cooper, Ros Eeles, David Neal

CRUK CI

Nitzan Rosenfeld, Chris Smith, James Morris, Wing Kit Leung

MRC-CU

Shamith Samarajiwa, Christian Frezza, Sakari Vanharanta

Hutch Staff

Jennifer Furman, Lab Management, IT, H&S, Reception, Porters

Urological Malignancies

Vincent Gnanapragasam, Grant Stewart Brendan Berry, Satoshi Hori, Anne George, Lorraine Starling

Pathology

Anne Warren, Tissue Bank

Oncology

Simon Pacey Vicki Sparkes, Bruce Daniels, Barbara Wall, Meg Horobin, Hayley Woffendin, Richard Hill, Jian Li, Lin Cheung

Radiology

Tristan Barrett, Iosif Mendichovszky, Luigi Aloj

ICGC/Practical:

Ian Mills, Zsofia Kote-Jarai

Stanford/Canary

Tanya Stoyanova, James Brooks, Olivier Gevaert, John Leppert

Astra Zeneca

Barry Davies, Eli Leo, Antonio Garcia-Trinidad, A. Galbiati

EpiEnrich Collaborators

Francesco Crea, Paul Millner, Tingting Zhu, Nicholas Leslie, Pedro Estrela, Vincent Gnanapragasam and VolitionRx https://www.earlydetectioncambridge.org.uk/ https://twitter.com/Massie_Lab https://www.facebook.com/cfDNA/