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© 2017 Biogazelle. All rights reserved. 1
RNA capture sequencing enabled liquid biopsy screening
Jo Vandesompele, Biogazelle CSO
Revolutionizing next-generation sequencing
Antwerp, Belgium, March 21, 2017
© 2017 Biogazelle. All rights reserved. 2
Biogazelle
research & development stage biotech company deploying the power of RNA for next generation diagnostics and therapeutics in the field of oncology and other diseases
Dx (CRO) unit / Tx unit
focus on clinically relevant and challenging samples
http://bgzlle.com/2mIGo2L
presentation download
© 2017 Biogazelle. All rights reserved. 3
Unmet needs in oncology
• more effective and less toxic treatments for durable responses• combination therapies
• companion diagnostic tests > the right drug for the right patient
• better laboratory tests• early diagnosis
• monitoring of treatment effectivity
• early detection of relapse or recurrence
http://bgzlle.com/2mIGo2L
presentation download
© 2017 Biogazelle. All rights reserved. 4
Liquid biopsies are the holy grail of precision oncology• easy to obtain
• low risk for the patient
• serial profiling > longitudinal studies
• reflects entire tumor load
• full of biomarker potential• cell-free nucleic acids (DNA & RNA)
• circulating tumor cells
• extracellular vesicles
• tumor educated platelets
© 2017 Biogazelle. All rights reserved. 5
Active secretion and passive release of RNA into circulation• vesicles• exosomes, microvesicles, apoptotic bodies
• ribonucleoprotein complexes• AGO2, high-density lipoproteins
• extracellular RNA• intercellular communication
• biomarker potential
Wan et al., Nature Reviews Cancer, 2017
© 2017 Biogazelle. All rights reserved. 6
exRNA may offer sensitivity advantages
• Clinical Chemistry, 1972
• 10x higher concentration of RNA than DNA in plasma
© 2017 Biogazelle. All rights reserved. 7
RNA has great biomarker potential
• dynamic nature (time, location and condition specific)
• diverse• different types: messenger, micro, long non-coding, transfer, ribosomal, piwi,
sn(o)RNA, etc.
• varying abundance levels: 1 copy/cell > 100,000 copies/cell
• structural differences: splicing (incl. circRNA), isomiRs, fusion, SNV, indel, editing
• measurement technologies are state-of-the-art• RNA sequencing (discovery)
• quantitative and digital PCR (verification, validation, clinical-grade test)
• sensitive, high-throughput, large dynamic range
© 2017 Biogazelle. All rights reserved. 8
Challenges in mRNA sequencing (of clinical samples)• fragmented/degraded RNA
• abundant unwanted RNA (ribosomal, hemoglobin)
• low input (1 FFPE scroll, fine needle biopsy, 0.2 ml liquid biopsy)
• sense / antisense overlapping transcripts
• solution: probe based cDNA enrichment, RNA capture sequencing
© 2017 Biogazelle. All rights reserved. 9
RNA capture sequencing history
• Levin, 2009 467 genes
• Ueno, 2012 913 genes
• Mercer, 2012 2265 loci, 0.77 Mb
• Halvardson, 2013 exome
• Cabanski, 2014 exome on FFPE
• Biogazelle exome on plasma
• higher sensitivity
• higher transcriptome complexity
• fusion genes, variants
© 2017 Biogazelle. All rights reserved. 10
mRNA capture sequencing
fragmented RNA
random primed ds cDNA
adaptor ligation
PCR
2x capture
PCR
cleanup & quant
• 3 days of work; optimized conditions
• 21,415 genes - 214,122 exons – 425,437 probes
• 20M reads FFPE, 15M reads plasma
• +100 cases so far• colon, lung, ovarium, breast, esophageal
© 2017 Biogazelle. All rights reserved. 11
Colon & lung cancer samplessensitivity – detected genes
• matching tumor FFPE, plasma and serum (n=2)
• detected genes with FPKM>=1
• FFPE > 12,000 genes
• plasma > 8500 genes
• serum > 7500 genes
© 2017 Biogazelle. All rights reserved. 12
Colon & lung cancer samplesstrandedness, read distribution, coverage
• >99% strandedness
• coding regions enriched
• whole transcripts are covered
© 2017 Biogazelle. All rights reserved. 13
Colon & lung cancer samplessequencing depth
• at ~15 M saturation is reached and sequencing deeper will not lead to (many) more genes (representative plasma sample)
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© 2017 Biogazelle. All rights reserved. 14
Colon & lung cancer samplesreproducibility
• high reproducibility in plasma
• low abundant genes more variable in serum
© 2017 Biogazelle. All rights reserved. 15
Colon vs lung cancer plasmaspectral map
samples separate by cancer typeon the second principal component
0 5 10 15 200
510
1520
crc_p
nsclc_p
© 2017 Biogazelle. All rights reserved. 16
Colon vs lung cancer plasma
• PI3K-AKT signaling pathway
• cell cycle
• osteoclast differentiation
• oxidative phosphorylation
• focal adhesion
top 5 enriched significant KEGG pathways upon GSEA
© 2017 Biogazelle. All rights reserved. 17
Esophageal cancer plasma titrationexperimental design –platelet-free / platelet-poor plasma
cancer – male : 100%
healthy – female : 0%
50% cancer
10% cancer
2% cancer
2x replicates for each sample
FFPE from cancer tissue : 1x
© 2017 Biogazelle. All rights reserved. 18
Platelet-poor plasma (PPP) shows best reproducibility
PFP
PPP
0% cancer 2% cancer 10% cancer 50% cancer 100% cancer
© 2017 Biogazelle. All rights reserved. 19
Titration series to determine consistency of RNA abundance signal • if healthy > cancer then 0 > 2 > 10 > 50 > 100
• if cancer > healthy then 100 > 50 > 10 > 2 > 0
• bin the fold-changes • healthy vs. cancer
• determine fraction per bin that is titrating• showing correct order
100% cancer
healthy : 0%
50% cancer
10% cancer
2% cancer
© 2017 Biogazelle. All rights reserved. 20
Excellent titration response
platelet-free platelet-poor
titr
atin
g ge
ne fr
acti
on
titr
atin
g ge
ne fr
acti
on
© 2017 Biogazelle. All rights reserved. 21
Ovarian cancer longitudinal studyexperimental design – platelet-free plasma
FFPE tumor
patient 15
patient 17
diagnosisbefore treatment during treatment post-surgery relapse
15.1 15.2 15. 3 15.4 15.615.5
17.1 17.2 17. 3 17.4 17.617.5
© 2017 Biogazelle. All rights reserved. 22
RNA seq quality control
gene body coverage cumulative gene diversity
© 2017 Biogazelle. All rights reserved. 23
Plasma samples tend to cluster by time point
during treatment
before treatment
patient 15: relapse
cluster dendogram
© 2017 Biogazelle. All rights reserved. 24
Plasma samples tend to cluster by time point• samples after 1st chemotherapy cycle stand out
hemolytic sample
© 2017 Biogazelle. All rights reserved. 25
Changes upon 1st chemo treatment
patient 15
patient 17
diagnosis after 1 chemo
15.1 15.2
17.1 17.2
• low power, but reproducible changes
© 2017 Biogazelle. All rights reserved. 26
Changes upon 1st chemo treatment
• GSEA : lysome, lipid metabolism, ECM-receptor• lysosome
• glycerophospholipid metabolism
• alpha-linolenic acid metabolism
• linoleic acid metabolism
• ether lipid metabolism
• ECM-receptor interaction
• not clear yet what these changes effectively mean
• intriguing and first time that mRNA profiles in plasma provide insights
© 2017 Biogazelle. All rights reserved. 27
RNA seq variant calling pipeline
raw RNA seq reads(FASTQ)
(paired-end) RNA sequencing
map to referenceSTAR 2-pass
alignment (BAM)
basecalling, demultiplexing, QC, trimming, adaptor removal
remove duplicate reads & sort
analysis-ready reads
variant callingreads mpileup
VarScan
raw variants (VCF)
SNVs InDels
filtered variants
SNVs InDels
variant filtering(strand bias, base
quality, depth, allelic ratio, …)
variant annotationfunctional/genic annotation
dbSNP/COSMIC/CIViCRNAediting
HGVS/SIFT-PolyPhen
report annotated variants (VCF & CSV)
SNVs InDels
further evaluation, candidate selection, troubleshooting, …
fusion detectionFusionCatcher
© 2017 Biogazelle. All rights reserved. 28
Testing of the RNA seq variant pipeline
• HCC1143 DNA exome sequencing vs polyA+ RNA sequencing
• variant positions covered > 4 reads
• good concordance
19,447 2911 4270
• mono-allelic expression
• low coverage
• RNA editing
• low coverage
© 2017 Biogazelle. All rights reserved. 29
Testing of the RNA seq variant pipeline
• 16 matched tumor / normal FFPE from diagnostic cases with known mutation
• 15/16 correctly called• myxoid liposarcoma: FUS-DDIT3 fusion• rhabdomyosarcoma: FOXO1-PAX3 fusion• synovial sarcoma: SS18-SSX2 fusion• Ewing sarcoma: ESWR1-FLI1 fusion• colon cancer: KRAS SNV (n=2)• melanoma: BRAF SNV (n=2)• lung cancer: EGFR SNV + EGFR deletion of 15 bp • GIST: PDGFRA SNV + KIT deletion of 21 bp
© 2017 Biogazelle. All rights reserved. 30
RNA editing event
• variant present in colon cancer FFPE RNA and plasma RNA, not in FFPE DNA
© 2017 Biogazelle. All rights reserved. 31
Therapy response monitoring
FCGR2A mutation present in FFPE and plasma at diagnosisdisappears during treatment and pops up again at time point 4 -> sign of relapse?
15.1 15.2 15. 3 15.4 15.615.5
© 2017 Biogazelle. All rights reserved. 32
Platelet-free plasma has very high PCR duplicate levels• ok for gene expression; problematic for variant analyses
• ~1000 variants PFP; ~5000 variants PPP
0
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20 000 000
30 000 000
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50 000 000
T1 T2 T3 T4 T5 T6 FFPE# reads # uniquely mapped # non-duplicate, uniquely mapped
© 2017 Biogazelle. All rights reserved. 33
Cumulative unique read coverage
• while better coverage of variants in PPP, due to PCR duplicates, only median coverage of 15 unique reads per variant
• envisaged improvements
• UMI (Fu et al., 2014)
• larger plasma volume
• platelet-rich plasma
• precision vs recall
FFPE
platelet-poor plasma
platelet-freeplasma
© 2017 Biogazelle. All rights reserved. 34
More unique reads > more variants
0
100 000
200 000
300 000
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500 000
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700 000
0 200 400 600 800 1000 1200 1400 1600 1800
# R
eads
# Variants
Patient 15
Patient 17
Lineair (Patient 15)
Lineair (Patient 17)
platelet-free plasma
© 2017 Biogazelle. All rights reserved. 35
Cancer plasma mixed 1:1 (v/v%) with plasma from healthy individual• AB heterozygous variants (cancer) mixed with BB variants (healthy)
• semiquantitative allele ratio determination
100% cancer50/50
alle
le r
atio
© 2017 Biogazelle. All rights reserved. 36
Conclusions – high level
• RNA is a fascinating molecule with a lot of biomarker potential
• liquid biopsies are emerging as holy grail for precision medicine
• novel application of mRNA capture sequencing in body fluids
© 2017 Biogazelle. All rights reserved. 37
Conclusions - details
• plasma > serum
• platelet poor > platelet free plasma
• gene expression: ~10,000 genes reproducibly detected
• variants: ~5000 detected
• PCR duplicates require attention in variant calling• UMI, larger plasma volumes, platelets,…
• many biological questions remain• function circulating RNA, where does it come from, different cargo in different
plasma fractions (RNP, EV, platelet)
© 2017 Biogazelle. All rights reserved. 38
Acknowledgements (A-Z)• Carolina Fierro
• Manuel Luypaert
• Nele Nijs
• Pieter Mestdagh
• Sandra Steyaert
• Thomas Piofczyk
• Gary Schroth
• Scott Kuersten
• Anneleen Decock
• Annouck Philippron
• An Hendrix
• David Creytens
• Glen Vergauwen
• Isabelle Rottiers
• Jo Van Dorpe
• Joni Van der Meulen
• Kimberly Verniers
• Olivier De Wever
• Pieter Mestdagh
http://bgzlle.com/2mIGo2L
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