DNA-methylation- and autoantibody- biomarker development strategies for minimal invasive diagnostics Andreas Weinhäusel, Matthias Wielscher, Johana Luna , Istvan Gyurjan, Walter Pulverer, Klemens Vierlinger, Christa Noehammer, Johannes Söllner * and Albert Kriegner Molecular Diagnostics, AIT- Austrian Institute of Technology GmbH, Vienna & * ..emergentec biodevelopment GmbH, Vienna, Austria, 4th Munich Biomarker Conference November 25th-26th, 2014
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DNA-methylation- and autoantibody- biomarker … and autoantibody- biomarker development strategies for minimal invasive diagnostics Andreas Weinhäusel, Matthias Wielscher, Johana
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DNA-methylation- and autoantibody- biomarker development strategies for
minimal invasive diagnostics
Andreas Weinhäusel, Matthias Wielscher, Johana Luna , Istvan Gyurjan, Walter Pulverer, Klemens Vierlinger, Christa Noehammer, Johannes Söllner* and Albert Kriegner
Molecular Diagnostics, AIT- Austrian Institute of Technology GmbH, Vienna & * ..emergentec biodevelopment GmbH, Vienna, Austria,
4th Munich Biomarker Conference November 25th-26th, 2014
AIM: improving cancer diagnostics
Breast, 4.570
Colon, 4461
Lung, 4,521
Prostate, 4.402
Other, 16,914
2%
33%
65%
13630 Colonoscopies of FOBT-positive Patients
carcinoma
polyps
healthy
Gsur A, Mach K. (2011) „Burgenländische Dickdarmkrebs –Initiative“
„the big 4“ breast, colon, lung, prostate cancer incidence EU25 1.2mio/y (2006) incidence AUSTRIA 18000/y (2008)
= 4x13% = 52%
low therapeutic treatment success early diagnosis improves survival „personalised medicine“
classical diagnostic methods are „inefficient“
Breast: Mammography (60€), MRT (400€), US (60€): ≈80% can be detected
PROTEIN (serum assays) protein -“serum”- cancer biomarkers
• 9 protein cancer biomarkers that have been approved by the FDA for clinical use • only PSA as a DIAGNOSTIC BM (AUC 0,66)
Autoantibody based lung cancer test EarlyCDT®-Lung test (Oncimmune): • seven antigens (p53, NY-ESO-1, CAGE, GBU4-5, SOX2, HuD, and MAGE A4) • sensitivity of 41% with a specificity of 91%;
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Aim: Improve cancer diagnostics
DNA methylation marker-developments • Big-4, thyroid ca, childhood ALL, Uveal Melanoma • Lung-cancer initial diagnostics as an example
Immuno-profiling on protein-& peptide-microarrays
• the big-4 cancer entities: breast-, colon-, lung-, and prostate cancer
Biomarkers for minimal invasive testing
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CpG360 uncovers Lung cancer marker panels
Bioinformatics
group samples (N=108)
AdenoCa 19 TU + 19 N
SqCCL 29 TU + 29 N
PB controls 8
Techn Contr. 4
select „top“ 24
for qPCR
Pyro-Sequencing
MSRE–qPCR independent samples
Weinhaeusel A, Pichler R, Nohammer C.
Lung Cancer Methylation Markers (2010).
EP2391728 (A1); WO2010086388 (A1)
100% correct
100% correct
70% correct
work out minimal invasive test for serum a/o sputum analyses
Isolation of cell-
free DNA
Volume reduction
and purification
Methylation sensitive
restriction digest
preAmplification
48x48 high-throughput
µ-fluidic qPCR (9 nl)
Dataanalysis
and
interptetation
MSREqPCR based marker validation
cut 10ng AciI(C^CGC= , Hin6I (G^CGC);
HpaII (C^CGG), HpyCH4IV (A^CGT)
48-plex - 22x
EvaGreen – ct & Tm
paralleled methylation analyses of 48
candidate markers x 48 samples
using cfDNA (400µl Heparin-plasma)
Lung Cancer plasma samples for cfDNA methylation analyses
conducted MSREqPCR (48.48) using 10ng of cfDNA setup: case vs control – at each array „cases – of a single specified
subtype“ vs „matched controls“ - 17 runs 48 analytically qualified markers 38 were suitable for cfDNA testing
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ENTITY patients gender age smoking female male <64 >64 current former never
cfDNA „Lung cancer“ case-control pilot study using first 96 out of 222 assays in 96.96 MSRE-HTqPCR
Spec Sens AUC
Cand 1 0.88 0.5 0.8
Cand 2 0.88 0.61 0.85
Cand 3 0.88 0.51 0.81
Cand 4 0.88 0.35 0.74
SHOX2* 0.9 0.62 0.78
* EpiProLung test marketed by Epigenomics
Combination of Candidates use logistic regression on 21 markers
AUC = 0.91-0.99 …
confirms suitability of tissue derived
candidate-methylation markers
Single Candidates
Summary - DNA Methylation biomarkers Lung-Cancer Methylation markers
Tumor tissue methylation analyses enables almost perfect classification Plasma testing plasma samples up to 15y old are useful (age had no effect)
SUBTYPING lung caner entity based on cfDNA feasible AUC≈0.8-0.92 (using classifiers from targeted CpG360 screening) AUCs up to 0.99 based on genomic screening
option for improvements using more sample ≥ 10ng DNA; we used cfDNA from 400µl plasma Illumina‘s 450k array is currently most cost effective tool for BM development
sample size >20 per group needed for discovery
MethPipe: efficient combination of methylation analyses tools for BM development 450k discovery, BSP based confirmation (pyroseq / IonTorrent), MSRE-qPCR enables confirmation and classifier-refinement
LC480 (EvaGreen) & 48.48 / 96.96 high-throughput 0.1-1% LOD & paralleled analyses of 48 (96) markers works with bisulfite based discovery efficient tool (costs & time) for qualification of methylation markers
and bringing these forward to serum-cfDNA testing
•Autoantibodies in serum against tumor-associated antigens (TAAs) have been found in asymptomatic stage of cancer
early / non invasive / simple == best suited for diagnosis
DISCOVERY Macromembranes (38k fetal brain library) based serological identification of antigenic clones, SEREX, phage display (M13 random peptide phage library & NGS)
Pathway analyses - Diseased vs(Benign and Healthy)
- based on 473 distinct genes
Motif enrichment search:
E-value 3.4e-015
E-value 1.7e-005
E-value 2.6e-003
Zn-finger proteins
diverse proteins (e.g. MUC5), redundant sequences
Zn-finger proteins
enriched in Malignant samples:
healthy
Discovery Set
Discovery & Training
& Validation-SET
Bioinformatics
Y Y Fluoreszently anti-human IgG
detection antibody
IgG – Patient vs. Control
immobilised Antigen
custom 179.000
peptide array
200+ plex Luminex-
bead array
16k Protein-chip Classifiers,
Serex
COSMIC
Phage-Display Peptides patient
PR
OT
EIN
P
eptides confirm
ation & validation 2x 100-plex Luminex-ASSAYS – using
biotinylated peptides on avidin coated
magnetic beads … almost ready
….The technologies are ready … cooperations are welcome….
analyse 92 markers from 1µl of serum
Oncology
Cardio-Vascular / CVD
Inflammation
DNA Methylation work Matthias Wielscher, Rudolf Pichler, Elisabeth Reithuber, Markus Sonntagbauer, Walter Pulverer, Manuela Hofner, Christa Noehammer Immunoprofiling work: Istvan Gyurjan, Johana Luna, Stefanie Brezina, Regina Soldo, Tina Malovits, Regina Linhard, Roman Kreuzhuber, Lisa Milchrahm, Olga Peinando, Sandra Rosskopf, Roni Kulovics, Gabriel Beikircher, Silvia Schönthaler, Michael Stierschneider Assay design pipelines and bioinformatics Business development; IPR and technology marketing Albert Kriegner, Ilhami Visne, Rainer Kallmeyer, Uwe von Ahsen, Stefan Pabinger, Fabian Schröder, and Klemens Vierlinger Head of AIT-Molecular Diagnostics: Martin Weber Cooperations: Funding & Support:
Acknowledgement
T. Liloglou, J. Field; Roy Castle Lung Cancer Foundation, Liverpool, UK
M. Hajduch, Molecular and Translational Medicine, University Hospital Olomouc, Czech Republic
C. Singer, A. Gsur. & P. Hofer, R. Ziesche Medical Univ. Vienna F. Längle, J. Hofbauer, LKH Wr. Neustadt G. Leeb & K. Mach, LKH Oberpullendorf C. Jungbauer, Austrian Red Cross J. Söllner, Emergentec
Antigenic / Immunomics -profile based Biomarkers
microarrays improve discovery
30µl serum / plasma are sufficient…
IgG preparation from serum/plasma wide range of linearity
microarrays & bioinformatics - well established & suited for discovery
bead arrays established for validation studies
from proteins to peptides
direct peptide based screening - using phage display & NGS
tiling design and high density peptide arrays are efficient for defining chemically synthesizable peptides
specifying antigenic epitopes
peptide arrays improve classification success
peptide array data are biologically meaningful
….The technologies are ready.
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DNA methylation results (MSREqPCR) – Plasma & Sputum
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• Deep Sputum DNA: n=96 (48 TU vs 48 contr.; Liverpool - J. Field / T Liloglou)
• (Heparin)-Plasma cfDNA: n=200 (100 LuCa vs. 100 matched controls)
AUC values depicted:
high AUC in all Lung cancer entities
performance of plasma-DNA better than „deep sputum“-DNA