signatures+mutaonnelles +et évolu-on+ clonaledes tumeurs++ · aca acc acg act cca ccc ccg cct gca...
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Signatures muta-onnelles et évolu-on clonale des tumeurs
Rencontres scien-fiques des Grands Causses
Eric Letouzé Equipe "Génomique Fonc-onnelle des Tumeurs
Solides", INSERM U1162
29 septembre 2015
PLAN
1. Signatures muta-onnelles
2. Evolu-on clonale des tumeurs 2.1) Introduc-on à l’évolu-on clonale des tumeurs 2.2) Approche 1 : plusieurs tumeurs d’un même pa-ent 2.3) Approche 2 : plusieurs prélèvements d’une même tumeur 2.4) Approche 3 : au sein d’un même échan-llon tumoral
1.1) Principe des analyses de signatures muta-onnelles 1.2) Applica-on aux cancers du foie
1. Signatures muta-onnelles
2. Evolu-on clonale des tumeurs 2.1) Introduc-on à l’évolu-on clonale des tumeurs 2.2) Approche 1 : plusieurs tumeurs d’un même pa-ent 2.3) Approche 2 : plusieurs prélèvements d’une même tumeur 2.4) Approche 3 : au sein d’un même échan-llon tumoral
1.1) Principe des analyses de signatures muta-onnelles 1.2) Applica-on aux cancers du foie
PLAN
Les spectres muta-onnels diffèrent en fonc-on du type tumoral (version 2002)
Pfeifer et al. 2002
A G C
T C G
A G C
T A G
A T C
T A G
DNA mispairing
DNA replica-on
Benzo[a]pyrene Benzo[a]pyrene adduct on guanine
G>T (or C>A) muta-on
TP53 muta-ons
! Different cancers have different muta-on rates:
Alexandrov et al., Nature 2013
Lawrence et al., Nature 2013
! Different cancers display different types of muta-ons:
Les spectres muta-onnels diffèrent en fonc-on du type tumoral (version 2015)
Le spectre muta-onnel d’une tumeur reflète l’accumula-on de plusieurs processus mutagènes
Helleday et al., Nat Rev Genet 2014
MÉTHODE : Extraire les signatures muta-onnelles à par-r de données de séquençage à haut débit
! The muta-onal landscape of a tumor results from the combina-on of several muta/onal processes opera-ve with different intensi/es.
Alexandrov et al., Cell Rep 2013
! The problem is to find the muta/onal processes (P) and the exposures (E) while only knowing the muta/onal pa:ern of each sample (M).
M
P
E
R
! This is similar to the cocktail party problem, and can be solved with the non-‐nega-ve matrix factoriza-on (NMF) algorithm.
M = P x E + R Alexandrov et al., Cell Rep 2013
! Applica-on of this method to >7,000 cancers iden-fies 21 signatures, associated with known or new oncogenic mechanisms.
Signature 10 : DNA polymerase E deficiency
Signature 4: Tobacco
Signature 16 : New mutagenic mechanism?
Alexandrov et al., Nature 2013
! Associa-on of signatures with tumor type and e-ology:
-‐> Most signatures remain to be explained!
1. Signatures muta-onnelles
2. Evolu-on clonale des tumeurs 2.1) Introduc-on à l’évolu-on clonale des tumeurs 2.2) Approche 1 : plusieurs tumeurs d’un même pa-ent 2.3) Approche 2 : plusieurs prélèvements d’une même tumeur 2.4) Approche 3 : au sein d’un même échan-llon tumoral
1.1) Principe des analyses de signatures muta-onnelles 1.2) Applica-on aux cancers du foie
PLAN
Le spectre muta-onnel des carcinomes hépatocellulaires
! Average muta-onal spectrum over 243 HCC analyzed by WES:
Spontaneous deamina-on of methylated CpGs
T>C muta-ons in an AT[AGT] context, characteris-c of liver
tumors
C>A muta-ons enriched in a subset of HCC
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
0.0
0.1
0.2C>A C>G C>T T>A T>C T>G
Signature 1A
• Ubiquitous in human cancers • Reflects the spontaneous deamina-on of methylated Cs at CpG sites • Associated with age
Signature 4
• Mostly found in lung and liver cancers • Not associated with smoking in our series.
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
0.00
0.05
0.10C>A C>G C>T T>A T>C T>G
! Using the Wellcome Trust Sanger Ins-tute method, we iden-fied 8 dis/nct signatures in our HCC series:
Signature 16
• T>C muta-ons at AT[AGT] trinucleo-des • Liver-‐specific signature
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
0.00
0.05
0.10C>A C>G C>T T>A T>C T>G
G G T A T T
C C A T A A
5' 3'
3' 5'
transcribed strand
non-‐transcribed strand G G T A T T
C C A T A A
5' 3'
3' 5'
Transcrip-on coupled repair (TCR) G G T A T T
C C A T A A
5' 3'
3' 5'
T>C muta-on on the transcribed strand
Less efficient TCR
G G T A C T
C C A T A A
5' 3'
3' 5'
• Very strong transcrip-onal strand bias:
G G T A T T
C C A T A A
5' 3'
3' 5'
Bulky DNA adduct
Signature 23
• Found in a single tumor with > 6000 muta-ons • Non-‐cirrho-c liver but with black mineral deposits
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
0.0
0.1
0.2C>A C>G C>T T>A T>C T>G
Throrotrast?
Schulze, Imbeaud, Letouzé et al., Nat Genet 2015
Signature 24
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
CCA
CCC
CCG
CCT
GCA
GCC
GCG
GCT
TCA
TCC
TCG
TCT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
0.0
0.1
0.2C>A C>G C>T T>A T>C T>G
• 5 pa-ents, all from African countries • 3/5 pa-ents present the R249S muta-on of TP53
Aflatoxin B1
Deux nouvelles signatures iden-fiées dans les CHC
U-lité clinique des signatures muta-onnelles
! Example 1 : Iden-fy a mutagenic exposure
! Example 2 : Iden-fy a predisposing muta-on
Signature of a known DNA repair defect
Perc
enta
ge o
f mut
atio
ns
ACA
ACC
ACG
ACT
CC
AC
CC
CC
GC
CT
GC
AG
CC
GC
GG
CT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
CC
AC
CC
CC
GC
CT
GC
AG
CC
GC
GG
CT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
CC
AC
CC
CC
GC
CT
GC
AG
CC
GC
GG
CT
TCA
TCC
TCG
TCT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
ATA
ATC
ATG
ATT
CTA
CTC
CTG CTT GTA
GTC
GTG GTT TTA
TTC
TTG
TTT
0.000
0.105
0.210
C>A C>G C>T T>A T>C T>G
Adrenocor-cal carcinoma ACC39
Aflatoxine B1
1. Signatures muta-onnelles
2. Evolu-on clonale des tumeurs 2.1) Introduc-on à l’évolu-on clonale des tumeurs 2.2) Approche 1 : plusieurs tumeurs d’un même pa-ent 2.3) Approche 2 : plusieurs prélèvements d’une même tumeur 2.4) Approche 3 : au sein d’un même échan-llon tumoral
1.1) Principe des analyses de signatures muta-onnelles 1.2) Applica-on aux cancers du foie
PLAN
Le modèle d’expansion clonale
Nik-‐Zainal et al., Cell 2012
U-liser l'évolu-on clonale pour reconstruire l'ordre d'appari-on des altéra-ons géné-ques
! Comparing the profiles of different clones can help reconstruc-ng the order in which genomic altera-ons occured:
Normal'cell'Common'precursor'1'
Common'precursor'2'
Clone'1'
Clone'2'
Clone'3'
≠ tumors
≠ parts of a same tumor
≠ cell popula-ons within a same sample
Approche 1 : plusieurs tumeurs d’un même pa-ent
Primary tumor
Recurrence
Chromosome aberra-ons
Letouzé et al., Genome Biol 2010
! Common breakpoints in recurrent bladder cancers can be used to reconstruct the -ming of CNAs in cancer progression:
Analyse de couples adénome -‐> carcinome hépatocellulaire par séquençage exome
! Comparing the muta-ons in HCA and HCC from the same pa-ents reveals the importance of TERT muta-ons for the transforma-on of CTNNB1-‐mutated adenomas:
Adenomas Carcinomas
Adenoma Carcinoma
! Common sequence in HCA -‐> HCC progression:
Normal liver
CTNNB1 muta-on
TERT muta-on
Pila- et al., Cancer Cell 2014
Approche 2 : plusieurs prélèvements d’une même tumeur
! Mul--‐region sequencing reveals intra-‐tumor heterogeneity in 8/12 breast cancers:
-‐> Tumor heterogeneity should be considered when designing targeted therapy trials.
Yates et al., Nat Medicine 2015
Approche 3 : au sein d’un même échan-llon tumoral
! Low-‐amplitude copy-‐number changes and muta-ons with a low allelic frac-on reveal subclonal altera-ons:
Nik-‐Zainal et al., Cell 2012
Variant allele fraction
VAF = Number of mutated reads/Total number of reads
Analyse sous-‐clonale mul--‐échan-llons : Transforma-on adénome -‐> carcinome hépatocellulaire
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0.0 0.2 0.4 0.6 0.8
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CHC464T
CHC465T
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●CHC465T (HCC)
CHC464T (HCA)
-‐> There is clear evidence of mul-ple clones in the HCA. The HCC is derived from subclone 1, represen-ng around 18% of tumor cells in the HCA sample:
HCA-‐specific variants
HCC-‐specific variants
Subclone 1 -‐> clonal muta-ons
Histogram of tmp$Tumor_Varprop
tmp$Tumor_Varprop
Frequency
0.0 0.1 0.2 0.3 0.4 0.5 0.6
0200
400
600
800
Histogram of tmp$Tumor_Varprop
tmp$Tumor_Varprop
Frequency
0.0 0.1 0.2 0.3 0.4 0.5 0.6
0500
1000
1500
2000
2500
3000
Variant allele frac-on
Variant allele frac-on
Freq
uency
Freq
uency
clonal
subclone 1 (18%) subclone 2 (35%)
clonal
subclonal
Ordonner les duplica-ons chromosomiques à l'aide des muta-ons 6p
X
1q
14 13 8 16/17
CHC909T
5 10
0.0 0.2 0.4 0.6 0.8 1.0
−2−1
01
23
variant proportion
log
ratio
T/N
cov
erag
e
6p 6p
X
13/14
8/16/17
1q 1q
Normal copy-‐number
5 10
5 10
Variant allele frac-on
0.0 0.2 0.4 0.6 0.8 1.0
−10
12
3
variant proportion
log
ratio
T/N
cov
erag
e
Late
Early
Variant allele frac-on
0.0 0.2 0.4 0.6 0.8 1.0
−10
12
3
variant proportion
log
ratio
T/N
cov
erag
e
0.0 0.2 0.4 0.6 0.8 1.0
−10
12
3
variant proportion
log
ratio
T/N
cov
erag
e
Chr 10 (late) Chr 5 (early)
! The ra-o of duplicated/non-‐duplicated muta-ons depends on the earliness of each duplica-on
! ... and can be used to infer the molecular -me of each duplica-on:
0 100 Point muta-on -me
Muta-ons/chromosome copy (1309-‐16)/3 16
1309 16 Duplicated/non-‐duplicated muta-ons
Time = 16/(16+(1309-‐16)/3) = 4%
4%
Chr 5 duplica-on
0 20 40 60 80 100
+1+5 +6 +10
+17
+20 +Xloh
13loh
14loh
21
Point mutation time (%)
0 20 40 60 80 100
+1 +3+5+8 +11
+12
+15
+19 +X
Point mutation time (%)
0 20 40 60 80 100
+8 +X
Point mutation time (%)
0 20 40 60 80 100
+1+2 +8+15
Point mutation time (%)
! Timing chromosome duplica-ons in hepatocellular carcinomas:
+1 +10
+11
+12
+13
+15
+17
+19 +2 +20
+21 +3 +4 +5 +6 +7 +8 +X
0
20
40
60
80
100
Recurrent CNAs
Poin
t mut
atio
n tim
e
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! Molecular -me distribu-on for recurrent chromosome duplica-ons:
! Comprendre l'évolu-on clonale des tumeurs est capital pour l'u-lisa-on des marqueurs géné-ques en clinique
! Diverses approches permeyent de reconstruire l'évolu-on clonale et l'ordre d'appari-on des altéra-ons géné-ques chez un pa-ent
! Les données de séquençage génome complet nous offrent une vision dynamique du développement tumoral.
ÉVOLUTION CLONALE DES TUMEURS
! Les échan-llons tumoraux portent les traces des processus mutagènes auxquels ils ont été soumis
SIGNATURES MUTATIONNELLES
! Les signatures muta-onnelles sont un formidable ou-l pour comprendre les liens entre exposi-ons environnementales, prédisposi-ons géné-ques et cancer
! 31 signatures muta-onnelles ont été iden-fiées à ce jour, dont une bonne moi-é restent inexpliquées.
Conclusions