Van criekinge next_generation_epigenetic_profling_vvumc_uploaded

Next Generation Epigenetic Profiling

Wim Van Criekinge 14th november 2013, Boston (US) 20th november 2013, Amsterdam

Overview Epigene,cs

–  Introduc*on –  Methyla*on & Oncology –  Biomarkers

MDxHealth – NEXT-‐GENera*on Epigene*c Biomarkers – Methyla*on Based CDx

Actionable Epigenome

Outside Oncology ?

Historically, Cancer Was Considered to be Driven Mostly by Gene,c Changes

Example: Replica,on errors

GENETIC

Altered DNA/mRNA/proteins

Altered DNA sequence

X X

Oncogenesis

Tumor

§  Muta*ons in p53

§  Ac*va*ng muta*ons in RAS

§  Muta*ons or amplifica*ons of the

HER-‐2 gene

§  Chromosomal transloca*ons in

myeloid cells and the genera*on of

the BCR-‐ABL fusion protein

Epigene,c Changes are Important in Causing Cancer

Example: Replica,on errors

GENETIC EPIGENETIC

Example: Chroma,n modifica,on errors

Altered DNA/mRNA/proteins

Altered DNA sequence

Altered levels of mRNA/proteins

Altered chroma,n structure

X X

Oncogenesis

Tumor

Source: Schuebel et al 2007

0

20

40

60

80

100

120

Methylated Mutated

76-100 51-75 21-50 1-20

Dx

CDx

Example of Methyla,on vs Muta,on: Colon & Breast Cancer

MGMT Biology O6 Methyl-Guanine Methyl Transferase

Essential DNA Repair Enzyme Removes alkyl groups from damaged guanine bases Healthy individual:

-‐ MGMT is an essen*al DNA repair enzyme Loss of MGMT ac*vity makes individuals suscep*ble to DNA damage and prone to tumor development

Glioblastoma pa,ent on alkylator chemotherapy: -‐ Pa*ents with MGMT promoter methyla*on show have longer PFS and OS with the use of alkyla*ng agents as chemotherapy

MGMT Promoter Methylation Predicts Benefit form DNA-Alkylating Chemotherapy

Post-hoc subgroup analysis of Temozolomide Clinical trial with primary glioblastoma patients show benefit for patients with MGMT promoter methylation

0

5

10

15

20

25Median Overall Survival

21.7 months

12.7 months

radiotherapy

plus temozolomide

Methylated MGMT Gene

Non-Methylated MGMT Gene

radiotherapy

Adapted from Hegi et al. NEJM 2005 352(10):1036-‐8. Study with 207 pa*ents

Clinical Valida,on of MDxHealth assay RTOG 0525 Study

§  MGMT was measured prospec*vely in the RTOG 0525 study §  MGMT used to balance the arms of the study as there is an

advantage to overall survival and progression free survival being MGMT methyla*on

§  Physicans goal in this study was to compare current temozolomide schedule (Arm 1) vs a dose-‐dense administra*on of temozolomide(Arm2).

§  The dose dense schedule was hypothesized as being able to overcome the unmethylated MGMT tumors by overwhelming the tumor with alkyla*on.

§  The dose dense schedule did not work.

Outcomes by MGMT Status-‐ MDxHealth Assay

Ove

rall

Surv

ival

(%)

0

25

50

75

100

Months after Randomization0 12 24 36 48

Patients at RiskMethyUnmethy

244516

168295

104106

3728

66

Dead162433

Total244516

p (2-sided) =< 0.0001HR (95% CI) =1.74 (1.45, 2.08)

MethylatedUnmethylated

Prog

ress

ion-

free

Sur

viva

l (%

)

0

25

50

75

100

Months after Randomization0 12 24 36 48

Patients at RiskMethyUnmethy

244516

99108

5540

1914

34

Dead202486

Total244516

p (2-sided) =< 0.0001HR (95% CI) =1.63 (1.38, 1.92)

MethylatedUnmethylated

ASCO 2011, Mark R. Gilbert, MD 12 Confiden*al

However the study did show that the MDxHealth assay was able to show the benefit to OS and PFS with the MGMT methylated cohort.

Overview Epigene,cs

–  Introduc*on –  Methyla*on & Oncology –  Biomarkers

MDxHealth – NEXT-‐GENera*on Epigene*c Biomarkers

Can we rediscover MGMT ? What does the epigenome look like ?

MBD_Seq

DNA Sheared

Immobilized Methyl Binding Domain

Condensed Chroma*n

DNA Sheared

Immobilized Methyl binding domain

MgCl2

Next Gen Sequencing GA Illumina: 100 million reads

MBD_Seq

Confidential Information | ©2013 MDxHealth Inc. All rights reserved.

Quality evalua*on of Methyl Binding Domain based kits for enrichment DNA-‐methyla*on sequencing

De Meyer et al (2013) Plos One

MBD_Seq MGMT = dual core

BRCA1, a bidirec,onal promoter

18

19

Splice variants

Zooming into Exon 1

20

Zooming into Exon 1

21

Zooming into Exon 1

22

# samples

# markers

MBD_Seq

Genome-‐wide methyla,on …. by next genera,on sequencing

Discovery

1-‐2 million methyla*on

cores

GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

Where is the mC ?



25% 50% 25%



25% 50% 25%



Dense methylated needed for transcrip*onal silencing Are there alleles with all three posi4ons methylated ?

GCATCGTGACTTACGACTGATCGATGGATGCTAGCAT!

unmethylated alleles

less methyla*on methylated alleles

more methyla*on

Deep Sequencing

Deep Sequencing MGMT Heterogenic complexity

Data integra,on with DEEP Sequencing, Infinium, Reac,va,on, (direc,onal) Expression …

BRCA1

31

1 T47D 2 BT549 3 MCF7 4 MDAMB231 5 HS578T 6 NORMAL 7 NORMAL 8 NORMAL

9 BI T21 (BRCA1 MUT) 10 BI T22 (BRCA1 MUT) 11 BLC 12 BLC 13 BLC 14 BLC 15 BLC 16 BLC 17 BLC

13 BLC 14 BLC 15 BLC 16 BLC 17 BLC 18 BLC

19 IVM 20 DKO

# samples

# markers

MBD_Seq

BT_Seq

Genome-‐wide methyla,on …. by next genera,on sequencing

Discovery

Verifica*on

Valida*on


Gene,c tes,ng

107 106 105 104 103 102 101 1 108 109

Full genome bp

Whole-genome Bisulphite seq MSP Probes

(450-27K) Enrichment

Targeted Panels


Gene,c tes,ng

107 106 105 104 103 102 101 1 108 109

Full genome bp

G E N E T I C

Whole-genome sequencing

Enrichment seq (Exome)

PCR Enrichment Targeted Panels

Instrument and Assay providers

CLIA Lab service providers


Today

107 106 105 104 103 102 101 1 108 109

Full genome bp

Whole-genome Bisulphite seq

RUO sequencing

Clinical PCR

E P I G E N E T I C


Enrichment seq (MBD, RRBS)


MSP Probes (450-27K)


Enrichment Targeted Panels


New Methods…

107 106 105 104 103 102 101 1 108 109

Full genome bp


RUO sequencing

Clinical PCR

E P I G E N E T I C




MSP Probes (450-27K)




Deep Seq

Molecular Unifica,on

107 106 105 104 103 102 101 1 108 109

Full genome bp


E P I G E N E T I C




Probes (450-27K)



Ultra Deep

Sequencing RUO Clinical


Sample MGMTCt MGMTCopies ACTBCt ACTBCopies Ratio Ratio 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 10024052 41.31 0.48 27.57 7307.99 0.07 0.07 0.203461634 0.134093898 0.134207627 0.149994669 0.139766144 0.046749831 0.072289157 0.033080997 0.016988307 0.013341863 0.001741479 0.013412944 0.013420052 0.011507979 0.007207591 0.003937875 0.002423855 0.001229698 0.000767672 0.000305647 7.11E-0524053 38.5 2.79 28.19 4957.3 0.56 0.56 0.268719091 0.096532193 0.118750823 0.138454814 0.094861755 0.106824251 0.052981044 0.033079475 0.024697345 0.015500952 0.002005724 0.012465424 0.012267845 0.009771168 0.006256661 0.00340075 0.001766234 0.000808277 0.000622672 0.000185604 4.79E-0524293 40.67 1.07 29.69 1742.4 0.61 0.61 0.273467629 0.05097645 0.031421802 0.054104784 0.09506031 0.082864938 0.080510995 0.11379954 0.055289448 0.049053294 0.004092476 0.049935382 0.027303684 0.015913473 0.009477312 0.003420653 0.001820588 0.000835932 0.000400016 0.00019488 5.64E-0524279 38.82 4.63 27.98 5541.91 0.83 0.83 0.03197928 0.013280651 0.01213759 0.024922695 0.03955784 0.085590824 0.165737241 0.19406269 0.169959299 0.126747628 0.003951159 0.060727595 0.037760658 0.015507308 0.009547533 0.003838835 0.0022663 0.001387531 0.000568227 0.000356794 0.00011232424265 37.95 3.26 28.69 3351.3 0.97 0.97 0.056994056 0.019769411 0.015913984 0.029030715 0.054868511 0.106125435 0.169261488 0.201250483 0.132483115 0.088697805 0.003478165 0.042676531 0.045901654 0.015725354 0.007733856 0.006063785 0.002222161 0.001016765 0.000478478 0.000253041 5.52E-0524061 37.91 5.12 28.71 3782.94 1.35 1.35 0.081114929 0.042035796 0.069555085 0.034374609 0.086467414 0.109080712 0.141810915 0.143841919 0.066533081 0.084735888 0.005722749 0.035240372 0.055800885 0.025444725 0.008592291 0.005107458 0.002341372 0.001148906 0.000593511 0.000348483 0.00010890124031 35.41 18.98 27.96 5687.55 3.34 3.34 0.22421415 0.061830533 0.024938381 0.0316917 0.021541898 0.025374505 0.046559574 0.086782129 0.098934139 0.095616951 0.008557288 0.137114839 0.060462688 0.037704929 0.017781977 0.013506638 0.00453305 0.001493395 0.000806169 0.00044934 0.00010572724269 35.8 12.59 28.52 3733.97 3.37 3.37 0.273363925 0.058772205 0.046178491 0.014855782 0.011610407 0.013424678 0.008720501 0.007764893 0.009025187 0.012921483 0.002137423 0.017884182 0.028562064 0.022542194 0.033132363 0.069427004 0.136402666 0.142427152 0.072515419 0.017570263 0.00076171724027 36.23 11.44 29.37 2337.17 4.89 4.89 0.343141131 0.136608047 0.115115764 0.04157705 0.022085337 0.016795294 0.029763411 0.023216093 0.047041055 0.088665549 0.003455529 0.054481908 0.044589765 0.014644484 0.008816738 0.004562563 0.00266479 0.001447052 0.000822368 0.000442814 6.33E-0524289 35.94 21.6 28.75 3190.3 6.77 6.77 0.118282761 0.026462975 0.011443727 0.00820412 0.009077907 0.009174766 0.01706152 0.022743197 0.028058049 0.05531444 0.004791404 0.163839223 0.175639472 0.123544031 0.111226517 0.044787777 0.028004468 0.014716308 0.003837245 0.023301679 0.00048841424177 36.48 12.24 30.26 1402.59 8.72 8.72 0.584283402 0.079453886 0.030287836 0.105659946 0.016766952 0.010978571 0.011251276 0.010477146 0.011541574 0.011585559 0.002058482 0.014945987 0.015843274 0.014242232 0.019027763 0.01140962 0.03199444 0.014470953 0.002946972 0.000624582 0.00014954824274 34.37 74.97 28.13 5042.56 14.87 14.87 0.444145473 0.082331479 0.031013551 0.017166966 0.023866926 0.018942859 0.029124646 0.02636394 0.032584947 0.027090441 0.002862955 0.04430046 0.036895524 0.039849964 0.058437645 0.022069507 0.032084468 0.011710131 0.016166008 0.002819903 0.00017220824168 35.33 24.62 30.09 1563.27 15.75 15.75 0.232961742 0.094066234 0.077658094 0.0182452 0.011580373 0.009423552 0.011962832 0.013448782 0.018332978 0.03469723 0.004576975 0.092003461 0.163498313 0.062735902 0.035023261 0.02299773 0.030540334 0.015198064 0.049287121 0.001573727 0.00018809524022 33.31 68.96 28.62 3762.21 18.33 18.33 0.032670856 0.011702993 0.004962808 0.00493684 0.004977235 0.004654075 0.006145803 0.008583926 0.013093734 0.019877199 0.003416258 0.034393412 0.060494319 0.080189741 0.169465459 0.230265626 0.210561547 0.079791562 0.017923815 0.00158983 0.00030296224042 33.21 78.19 28.72 3533.77 22.13 22.13 0.153857528 0.038000887 0.017214709 0.007148625 0.006335377 0.008968482 0.006102208 0.006170452 0.008081302 0.012050865 0.00196772 0.017004288 0.032717615 0.028657059 0.043830116 0.03318964 0.091425062 0.134407807 0.211023783 0.124262105 0.01758436724201 28.82 1015.04 27.72 6244.94 162.54 162.54 0.029623825 0.010316194 0.004583096 0.003917899 0.004859322 0.003838977 0.005067901 0.00550197 0.006973296 0.01071644 0.001871572 0.016274783 0.019876995 0.02168092 0.022904206 0.023343913 0.043993213 0.086594021 0.250666607 0.335214301 0.0921805524297 33.46 104.52 31.32 608.72 171.71 171.71 0.09853738 0.023217538 0.007607208 0.007109487 0.004270217 0.003602819 0.004688755 0.005011142 0.006459057 0.009699897 0.001532753 0.014411276 0.018890762 0.020152031 0.021181408 0.021763967 0.034625522 0.130431094 0.304921779 0.197702567 0.06418334224167 31.32 283.22 30.87 952.56 297.32 297.32 0.100591975 0.035233834 0.011751911 0.009642406 0.010474529 0.006226323 0.006890561 0.007459908 0.00916795 0.012109577 0.001956219 0.017197206 0.020992854 0.024073168 0.029270287 0.087110125 0.100146716 0.267279323 0.184081636 0.054847116 0.003496376

HT29 0.753566786 0.096042826 0.036705396 0.01508264 0.01079523 0.008066144 0.008954308 0.00800155 0.007460578 0.008704007 0.001711734 0.0105853 0.011715691 0.00939839 0.00650782 0.0031005 0.00185707 0.00088009 0.000524824 0.000201855 0.000137262

24025 Undetermined 0 28.34 4471.4 0 0 0.418789899 0.179009196 0.096823939 0.055595242 0.042328582 0.013686204 0.047079634 0.010675693 0.009400052 0.010692701 0.001916295 0.013516118 0.014462927 0.012359538 0.00860632 0.004921137 0.004546949 0.003974328 0.00454128 0.042322913 0.00475105224026 Undetermined 0 29.66 1944.13 0 0 0.516916773 0.202731325 0.059880115 0.046417299 0.037086771 0.021069827 0.012320727 0.011109419 0.011386289 0.012902154 0.002242649 0.015214021 0.0156847 0.013234398 0.009607398 0.004962899 0.003460878 0.002131901 0.00106595 0.000498366 7.61E-0524034 Undetermined 0 29.71 1879.44 0 0 0.383378528 0.21459944 0.178158129 0.094466966 0.033571401 0.01313284 0.011757902 0.00994102 0.008135049 0.008718852 0.001374938 0.010252017 0.010497542 0.008326013 0.006099923 0.002853542 0.00177869 0.000987555 0.000660188 0.000583803 0.000725662

Correlation -0.378717418 0.749526573 0.817780411 0.715954841 0.721224123 0.725933015 0.871789771 0.95567526 0.930713611 0.913277334 0.73018162 0.80233553 0.819357467 0.852443607 0.831785309 0.853097602 0.922340853 0.663535204 0.755785968 0.866175664 0.975660054

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100ivm dko 78.19% 6.71% 2.65% 1.45% 1.16% 0.90% 0.97% 0.91% 0.93% 0.95% 0.17% 1.15% 1.22% 1.02% 0.69% 0.34% 0.24% 0.16% 0.09% 0.06% 0.03%dko 0.07 20.35% 13.41% 13.42% 15.00% 13.98% 4.67% 7.23% 3.31% 1.70% 1.33% 0.17% 1.34% 1.34% 1.15% 0.72% 0.39% 0.24% 0.12% 0.08% 0.03% 0.01%

24052 41.31 0.48 27.57 7307.99 0.07 0.56 26.87% 9.65% 11.88% 13.85% 9.49% 10.68% 5.30% 3.31% 2.47% 1.55% 0.20% 1.25% 1.23% 0.98% 0.63% 0.34% 0.18% 0.08% 0.06% 0.02% 0.00%0.61 27.35% 5.10% 3.14% 5.41% 9.51% 8.29% 8.05% 11.38% 5.53% 4.91% 0.41% 4.99% 2.73% 1.59% 0.95% 0.34% 0.18% 0.08% 0.04% 0.02% 0.01%0.83 3.20% 1.33% 1.21% 2.49% 3.96% 8.56% 16.57% 19.41% 17.00% 12.67% 0.40% 6.07% 3.78% 1.55% 0.95% 0.38% 0.23% 0.14% 0.06% 0.04% 0.01%0.97 5.70% 1.98% 1.59% 2.90% 5.49% 10.61% 16.93% 20.13% 13.25% 8.87% 0.35% 4.27% 4.59% 1.57% 0.77% 0.61% 0.22% 0.10% 0.05% 0.03% 0.01%1.35 8.11% 4.20% 6.96% 3.44% 8.65% 10.91% 14.18% 14.38% 6.65% 8.47% 0.57% 3.52% 5.58% 2.54% 0.86% 0.51% 0.23% 0.11% 0.06% 0.03% 0.01%3.34 22.42% 6.18% 2.49% 3.17% 2.15% 2.54% 4.66% 8.68% 9.89% 9.56% 0.86% 13.71% 6.05% 3.77% 1.78% 1.35% 0.45% 0.15% 0.08% 0.04% 0.01%3.37 27.34% 5.88% 4.62% 1.49% 1.16% 1.34% 0.87% 0.78% 0.90% 1.29% 0.21% 1.79% 2.86% 2.25% 3.31% 6.94% 13.64% 14.24% 7.25% 1.76% 0.08%4.89 34.31% 13.66% 11.51% 4.16% 2.21% 1.68% 2.98% 2.32% 4.70% 8.87% 0.35% 5.45% 4.46% 1.46% 0.88% 0.46% 0.27% 0.14% 0.08% 0.04% 0.01%6.77 11.83% 2.65% 1.14% 0.82% 0.91% 0.92% 1.71% 2.27% 2.81% 5.53% 0.48% 16.38% 17.56% 12.35% 11.12% 4.48% 2.80% 1.47% 0.38% 2.33% 0.05%8.72 58.43% 7.95% 3.03% 10.57% 1.68% 1.10% 1.13% 1.05% 1.15% 1.16% 0.21% 1.49% 1.58% 1.42% 1.90% 1.14% 3.20% 1.45% 0.29% 0.06% 0.01%

14.87 44.41% 8.23% 3.10% 1.72% 2.39% 1.89% 2.91% 2.64% 3.26% 2.71% 0.29% 4.43% 3.69% 3.98% 5.84% 2.21% 3.21% 1.17% 1.62% 0.28% 0.02%15.75 23.30% 9.41% 7.77% 1.82% 1.16% 0.94% 1.20% 1.34% 1.83% 3.47% 0.46% 9.20% 16.35% 6.27% 3.50% 2.30% 3.05% 1.52% 4.93% 0.16% 0.02%18.33 3.27% 1.17% 0.50% 0.49% 0.50% 0.47% 0.61% 0.86% 1.31% 1.99% 0.34% 3.44% 6.05% 8.02% 16.95% 23.03% 21.06% 7.98% 1.79% 0.16% 0.03%22.13 15.39% 3.80% 1.72% 0.71% 0.63% 0.90% 0.61% 0.62% 0.81% 1.21% 0.20% 1.70% 3.27% 2.87% 4.38% 3.32% 9.14% 13.44% 21.10% 12.43% 1.76%

162.54 2.96% 1.03% 0.46% 0.39% 0.49% 0.38% 0.51% 0.55% 0.70% 1.07% 0.19% 1.63% 1.99% 2.17% 2.29% 2.33% 4.40% 8.66% 25.07% 33.52% 9.22%171.71 9.85% 2.32% 0.76% 0.71% 0.43% 0.36% 0.47% 0.50% 0.65% 0.97% 0.15% 1.44% 1.89% 2.02% 2.12% 2.18% 3.46% 13.04% 30.49% 19.77% 6.42%297.32 10.06% 3.52% 1.18% 0.96% 1.05% 0.62% 0.69% 0.75% 0.92% 1.21% 0.20% 1.72% 2.10% 2.41% 2.93% 8.71% 10.01% 26.73% 18.41% 5.48% 0.35%

ivm 0.30% 0.27% 0.29% 0.36% 0.34% 0.47% 0.71% 1.01% 1.16% 1.45% 0.18% 1.56% 1.84% 1.92% 1.87% 1.73% 2.33% 3.56% 7.80% 22.58% 48.27%

0%

20.00%

40.00%

60.00%

80.00%

Amount

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 9095

100

Percentage Methylation

IVM

DKO0.07

0.560.61

0.83

1.350.97

3.343.374.89

6.778.7214.8715.7518.3322.13162.54171.71

297.32

MSP M

GMT Cop

ies / A

CTB C

opies


Deep Seq


107 106 105 104 103 102 101 1 108 109

Full genome bp


E P I G E N E T I C




Probes (450-27K)



Ultra Deep

Sequencing RUO Clinical

Epi-‐health: comprehensive epigene,c profiling

§ Key genes

– For all know epigene*c genes – For all important gene classes (CT genes, immune-‐response)


440 cancer-‐related genes genes are known to be epigene*cally altered in human solid cancers based on recent scien*fic and clinical literature.

44

Heyn, H. H. & Esteller, M. M. DNA methylation profiling in the clinic: applications and challenges. Nat. Rev. Genet. 13, 679–692 (2012).

Epigenetic Alterations Associated with Cancer Therapy Response

PCFT – folate transport (MTX)

WRN

Kinases, CT genes ….

46


§ Key genes


§ Key regions –  Iden*fied by mining epigenomes

Data integra,on Correla,on tracks

methylation methylation

expression expression

Corr =-1 Corr = 1

Correla,on track in GBM @ MGMT

+1

-1


§ Key genes


§ Key regions –  Iden*fied by mining epigenomes

§ Approach – Acceptable amounts of clinical material – TAT/cost

§ Analysis – Ac*onable interpreta*on – Get gene*c data as a bonus

Sequencing

Deep Seq


107 106 105 104 103 102 101 1 108 109

Full genome bp


RUO Clinical

E P I G E N E T I C




Probes (450-27K)



Ultra Deep

Education

Van criekinge next_generation_epigenetic_profling_vvumc_uploaded