konstantin agelopoulos †1 daniel baumhoer 13 wolfgang e. berdel #1 regina besoke 9 thorsten buch 7...
TRANSCRIPT
![Page 1: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/1.jpg)
Konstantin Agelopoulos†1 Daniel Baumhoer13 Wolfgang E. Berdel#1 Regina Besoke9 Thorsten Buch7 Stefan Burdach2 Martin Dugas4 Kristina von Heyking2, Heribert Jürgens3 Hans-Ulrich Klein4 Gabriele Köhler11 Udo Kontny5 Eberhard Korsching6 Benjamin Moser1 Carsten Müller-Tidow#*1, 14 Kathrin Poos6 Günther HS Richter†*2 Claudia Rossig3, Eva Schmidt#1 Isabell Schulze1,14 Monika Stoll10 Eva Wardelmann12 Matthias Weckesser8, Anika Witten10
ANALYSIS OF GENOMIC ALTERATIONS IN EWING
SARCOMA REVEALS COOPERATING MUTATIONS
AND NOVEL THERAPY TARGETSUta Dirksen
![Page 2: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/2.jpg)
- Whole Genome Sequencing of 2 triplets (normal tissue, primary tumor, relapsed tumor)
NGS sequencing on HiSeq2000, 100 bp paired runs
- Exome discovery data set of 14 normal/tumor pairs
- Exome NGS of 52 EwS (incl.3 tumor/metastase samples)
Validation EwS
- Expression Analysis on 18 GeneST array (Affymetrix)
- Immunhistochemistry, PCR on 79 EwS
![Page 3: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/3.jpg)
Whole Exome Data
![Page 4: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/4.jpg)
Patient Tissue Mapped Seq (Gbp)
Genome called
Coverage
1 male Blood 286 .977 .974
36 years Relapse 1 281 .976 .974
DOD Relapse 2 278 .973 .962
2 female Blood 280 .975 .977
12years Primary 282 .973 .970
Relapse 1 279 .974 .972
DOD Relapse 2 291 .967 .954
![Page 5: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/5.jpg)
Gain of mutation in relapsed tumours
![Page 6: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/6.jpg)
Gain of mutation confirmed for several mutation types
![Page 7: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/7.jpg)
Validated mutations ( Sanger Sequencing)Gen Variation Patient Funtcion Other Inhibitor
FGFR1 N546K Pat. 1 TKR Glioblast.Lymph/Leuc
Ponatinib
PTEN G165E Pat. 1 R Tu suppr. Akt signaling
several -
ABCA8 T1335A Pat. 1 R MembranTransporter
-
SLC1A3 R47W Pat. 2 Glutamat transporter
- TBOA
ATP1A2 A599D Pat. 1 R ATPaseNa+/K+ Trsp.
-
AGRN G719V Pat. 1 Glycoprotein - -
CDH23 c.26delA Pat. 1 R Cadherin rel.
NCAM1 pQ3fs Pat 1 und 2 Ig-family -
SUZ12 Splice site loss Pat. 2 2ndR PRC2 Several
OVCH2 P127delV Pat. 1 - - -
![Page 8: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/8.jpg)
ABC A8 Allel- frequency: Clonal Evolution
Primary Tumor (T>C) Relapse (T>C) Blood (CNTR)
T1335A
![Page 9: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/9.jpg)
FGFR1 Mutation
Primary Tumor Relapse Blood (CNTR)
Mutation FGFR1 c.1638 G>C
![Page 10: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/10.jpg)
Intracellular signalling cascades downstream of FGFRs
I Ahmad , ToBiochimica et Biophysica Acta (BBA) – Molecular Cell Research, Volume 1823, Issue 4, 2012, 850 - 860
FGFR 1, 2, 3, 4
Amplification, Mutation, Translocation:
CarcinomaSarcomaM. MyelomLeukemiaLymphoma...
![Page 11: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/11.jpg)
Validation
• Validate that the mutation identified FGFR1 c.1638 G>C matters
• Validate that FGFR 1 matters
![Page 12: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/12.jpg)
FGFR1-N546K in NIH 3T3
Retroviral transduction using MSCV- constructs
MSCV-GFP-Mock
MSCV-GFP-EWS-Fli
MSCV-GFP-FGFR1-WT
MSCV-GFP-FGFR1-N546K
Transduction efficacy > 90% Proliferationassay
![Page 13: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/13.jpg)
Validation of FGFR1 mutation in NIH 3T3 cells
![Page 14: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/14.jpg)
Knockdown of FGFR1 impairs proliferation in the EwS celllines A673 and SK NMC
![Page 15: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/15.jpg)
Kaplan-Meier-Plot:
p < 0.03
p <
0.0
1
Tumor growth:
![Page 16: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/16.jpg)
Cells with FGFR1-mutation are sensitive to ponatinib
![Page 17: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/17.jpg)
Whole exome sequencing of 15 EwS samples Somatic mutations in tumor-normal pairs
14 patients m:f =1:17 relapsed7 primary1 additional relapse- sample
![Page 18: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/18.jpg)
FGFR copy number status and expression ( 62 EwS- samples primary and relapsed)
![Page 19: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/19.jpg)
Conclusions on FGFR• Trisomy 8 as well as amplifications of chromosome 8
are the most frequent cytogenetic finding in ES (Mackintosh et al., 2012; Lynn et al., 2013)
• A mutation in a female patient with ES, that was present at the time of diagnosis as well as in both relapses occurred in the tyrosine kinase domain (N546K) of FGFR-1 and was independently discovered as an activating mutation in glioblastoma.
• FGFR-1 is located on chromosome 8p• FGFR1 is highly expressed in ES as indicated by CNV,
RNA expression and immunohistochemistryOngoing:- treatment of FGFR1 overexpressing celllines
with Ponatinib
![Page 20: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/20.jpg)
- Whole Genome Sequencing of 2 triplets (normal tissue, primary tumor, relapsed tumor)
NGS sequencing on HiSeq2000, 100 bp paired runs - Exome discovery data set of 14 normal/tumor pairs
- Exome NGS of 52 EwS (incl.3 tumor/metastase samples)
Validation EwS- Expression Analysis on 18 GeneST array (Affymetrix)
in comparison to a normal body map (NBA)
- Immunhistochemistry, PCR on 79 EwS
![Page 21: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/21.jpg)
Disease
p = 0.0494
Whole exome sequencing analysis of ES
p < 0.1724
A B
C D
![Page 22: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/22.jpg)
ES006R ES014 ES014R
ES018 ES022
![Page 23: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/23.jpg)
name gene description recurrenceFreq (%) p-value*
expression (FC)** comment
ANKRD30A ankyrin repeat domain 30A 5 9.6 4.82E-02 1.02 ubiquitious
CCDC19coiled-coil domain
containing 19 6 11.5 4.39E-04 0.92 ubiquitious
KIAA0319 - 4 7.7 1.20E-02 0.57 increased in fetal brain
KIAA1522 - 4 7.7 1.60E-02 0.69increased in brain, fetal
brainLAMB4 laminin, beta 4 5 9.6 3.57E-02 1.03 ubiquitious
SLFN11 schlafen family member 11 4 7.7 1.60E-02 10.91 increase in ES
STAG2 stromal antigen 2 6 11.5 4.11E-05 2.10increase in ES, also in
cerebellum
TP53 tumor protein p53 3 5.7 4.46E-04 1.79 increase in ES
UNC80 unc-80 homolog 4 7.7 4.32E-02 0.36 increased in brainZNF98 zinc finger protein 98 4 7.7 1.08E-02 1.34 ubiquitious
Recurrent mutations across 52 Ewing sarcoma
* based on MutSigCV algorithm (presumed variations in patient-specific mutation frequency and gene-specific background mutations; http://www.broadinstitute.org/cancer/cga/mutsig)** FC: fold change of expression of 18 EwS samples compared to a normal body map (GSE45544)
![Page 24: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/24.jpg)
Further conclusions
•NGS confirmed low mutation frequency•Mutation frequency increased with relapse•EwS NGS sequencing identified STAG2 driver mutations in males in addition to the typical EWS/ETS translocation •CNV analysis confirmed previous findings
![Page 25: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/25.jpg)
RWTH Aachen (Freiburg)Udo Kontny
USB BaselDaniel Baumhoer
Fulda (Münster)Gabriele Köhler
WWU MünsterKonstantin AgelopoulosEva SchmidtBenjamin MoserHans-Ulrich KleinMartin DugasKathrin PoosEberhard KorschingMatthias WeckesserEva WardelmannClaudia RossigHeribert JürgensWolfgang E. Berdel
WWU Münster & MLU Halle Carsten Müller-Tidow
TU MünchenGünther RichterKristina von HeykingStefan BurdachThorsten Buch
![Page 26: Konstantin Agelopoulos †1 Daniel Baumhoer 13 Wolfgang E. Berdel #1 Regina Besoke 9 Thorsten Buch 7 Stefan Burdach 2 Martin Dugas 4 Kristina von Heyking](https://reader035.vdocuments.pub/reader035/viewer/2022062804/56649d345503460f94a0b916/html5/thumbnails/26.jpg)
Sarcoma Conference 2015
26th- 28th February 2015Mövenpick Hotel Münster
Kardinal-von-Galen-Ring 6548149 Münster
Telefon 0251 89020