THE UMD TP53 MUTATION DATABASE UPDATES AND BENEFITS … · 2019. 12. 12. · Data-driven unbiased curation of the TP53 tumor suppressor gene mutation database and validation by ultradeep

THE UMD TP53 MUTATION DATABASE

UPDATES AND BENEFITS

Pr. Thierry Soussi

[email protected] [email protected]

TP53: 33 YEARS AND COUNTING

P. May

STRUCTURE FUNCTION RELATIONSHIP OF WILD AND MUTANT TP53

1983 Oren et al.

1984 Crawford et al.

1988 Soussi et al.

1987 Soussi et al.

1988 Soussi et al.

2000 Levine et al.

37°C

37°C

25°C

25°C

40°C

TP53: 33 YEARS AND COUNTING

P. May

STRUCTURE FUNCTION RELATIONSHIP OF WILD AND MUTANT TP53

1983 Oren et al.

1984 Crawford et al.

1988 Soussi et al.

1987 Soussi et al.

1988 Soussi et al.

2000 Levine et al.

2015 Soussi et al. A. Pompeii 60°C

Which TP53 residues are important for TP53 folding ? How these residues have evolved to ensure TP53 function from 0° to 60°C What is the status of these residues in human cancer ?

Wild type ? mutant ? associated with more deleterious clinical features ? http://p53.fr

37°C

37°C

25°C

25°C

40°C

TP53 MUTATIONS IN HUMAN CANCER

GENE MUTATION: FROM DISCOVERY TO APPLICATION

THE TP53 MUTATION DATABASE

TP53 MUTATION ANALYSIS IN THE NEXT DECADE


ONCE UPON A TIME

OR

A NEW MUTATED GENE IN HUMAN CANCER

IT iS STORY TIME!

A NEW MUTATED GENE IN HUMAN CANCER!

TP53!

Inci

denc

e

Discovery PHASE I Causes

Total number of variants (cumulated)

Discovery rate of novel variants

Variant detection

Inci

denc

e In

cide

nce

Discovery

I.F.

PHASE I Causes



Consequences

Variant detection

Publications Inconsistant

reports

Inci

denc

e

Discovery Consolidation PHASE II

Variant detection

Causes



Clinical value

No clinical value X

Inci

denc

e In

cide

nce

Discovery

I.F.

Publications

Consolidation PHASE II

Variant detection

Causes



Consequences

Inconsistant reports

Clinical value

No clinical value X

Inci

denc

e

Causes


Discovery Application Consolidation PHASE III

Variant detection


Inci

denc

e In

cide

nce

Causes


Discovery

Consequences I.F.


reports

Application Consolidation PHASE III

Variant detection






THE p53 MUTATION DATABASE

October 2015

3,900 PUBLICATIONS 60,000 MUTATIONS

"unbiased" compilation of "every" TP53 mutations published in the literature

+ TCGA and ICGC unpublished data

http://p53.fr

Bernard Leroy and Thierry Soussi

7 200 TP53 VARIANTS 1750 missense variants 5450 indel variants

TP53 MUTATION HETEROGENEITY AND DATABASE FREQUENCY

TP53 MUTANTS (1,750 VARIANTS)

R175H (1 600 x) R273H (1 100x)

800 variants 1x 400 variants 2x

hot spot variants

rare variants

WHAT IS THE BIOLOGICAL SIGNIFICANCE OF THESE RARE VARIANTS ????

2,314 p53 mutants representing all possible amino acid substitutions caused by a point mutation throughout the entire protein (5.9 substitutions per residue) have been constructed The transcriptional activity of all these mutants was analyzed in a yeast based functional assay For each p53 mutant, a quantitative value of the remaining activity is available

TP53 MUTATION HETEROGENEITY

0

25

50

75

100

125

150

175

1x 2x 3-5x 6-9x 10-49x 50-99x >99

AC

TIVI

TY (%

wt p

53)

FREQUENCY


ONLY TUMORS HAVE BEEN INCLUDED IN THE ANALYSIS

161_

+

81_1

60

41_8

0

21_4

09_

20 4-8

2_3 1

Tum

ors

cell l

ine

germ

line

-2

0

2

4

0

AC

TIVI

TY (%

)

100

REMAINING ACTIVITY OF MUTANT TP53 ACCORDING TO THEIR FREQUENCY IN THE DATABASE

FREQUENCY

NS

NS

NS

P<0.001


Are they randomly distributed in the

literature ?

Are they associated with other unusual features ?

What is the contribution of these "rare" mutations to tumorigenesis

passenger mutations ? PCR and sequencing artefacts ? Weak mutations ?


2,018 publications with 5 or more reported mutations

Frequency of each mutant in the db

Each publication was ranked according:

Remaining activity of each mutant

Number of tumors with multiple mutations

Number of tumors synonymous mutations

Tum 2x Tum 3x Tum >3x

Number of tumors with recurrent mutations

Edlund, K. et al. Data-driven unbiased curation of the TP53 tumor suppressor gene mutation database and validation by ultradeep sequencing of human tumors. Proc Natl Acad Sci U S A 109, 9551-9556 (2012).


Included >90% of the publications

Outliers 7 % of the publications (2 sigma) or 1% (5 sigma) Multiple tumours with more than 1 TP53 mutations Multiple tumours with rare variants Multiple tumours with a high frequency of synonymous variants

PARAFFIN EMBEDDED TISSUE


Only 2 papers describing TP53 mutations in lymphoma have been flagged Both papers used DNA from paraffin embeded tissue





THE TP53 PATHWAY: A COMPLEX NETWORK

1985-2005

2005-

TP53 activities are modulated by the ratio of the various isoforms

THE TP53 PATHWAY: A COMPLEX NETWORK

TP53 ISOFORMS: WHATS IMPORTANCE ?

6.6%

17.8%

p.P110L

TP53 exon 9 beta and gamma are poorly analyzed

They are not included in the majority of commercial exon selection kit

They are excluded from most analytical pipelines (labeled as intron)

They are excluded in custom arrays


Revised guidelines for screening of TP53 gene mutations Bernard Leroy , Mandy L. Ballinger , Gareth L. Bond , Antony Braithwaite , Nicole Concin , Lawrence A. Donehower , Wafik S. El-Deiry , Pierre Fenaux , Gianluca Gaidano , Anita Langerød , Eva Hellstrom-Lindberg , Richard Iggo , Jacqueline Lehmann-Che , Phuong L. Mai , David Malkin , Ute M. Moll , Jeffrey N. Myers , Kim E. Nichols , Sarka Pospisilova , Davide Rossi, Sharon A. Savage, Louise C. Strong , Patricia N. Tonin , Robert Zeillinger , Thorsten Zenz ,Joseph F. Fraumeni Jr. , Pierre Hainaut and Thierry Soussi


ASSESSING TP53 STATUS IN HUMAN CANCER

STRATEGY

MUTATION VALIDATION

METHODOLOGY

ANALYSIS OF TP53 MUTATIONS IN HUMAN CANCER

All coding exons with +/- 5 intronic extension Application phase

Intron 9

ANALYSIS OF TP53 MUTATIONS IN HUMAN CANCER

screening beta and gamma exons

Discovery phase

TP53 RE Int Promoter


Intron 4

Intron 4 promoter

Discovery phase

TP53 RE Int Promoter Intron 4

Intron 9


3'UTR

3’UTR ? Discovery phase


STRATEGY

MUTATION VALIDATION

METHODOLOGY


CONVENTIONAL SEQUENCING SANGER

HIGH-THROUGHPUT SEQUENCING

TUMOUR WITH MULTIPLE TP53 MUTATIONS

Multiple tumoral clones with different mutations

TUMOR HETEROGENEITY


STRATEGY

MUTATION VALIDATION

METHODOLOGY

Sequencing artefacts ? Passenger mutations ? Very rare neutral SNP ? Driver mutations ?

R72P R213R P47S

R283C R283H R156H P222L

?

N235S R290H

TP53 SNP CAN BE VERY UNFREQUENT

More than 150,000 TP53 gene have been sequenced

P36P

R72P R213R P47S

R283C R283H R156H P222L

?

N235S R290H

TP53 SNP CAN BE VERY UNFREQUENT

More than 150,000 TP53 gene have been sequenced

P36P

R72P R213R P36P


STRATEGY

MUTATION VALIDATION

METHODOLOGY

Sequencing artefacts ? Passenger mutations ? Very rare neutral SNP ? Driver mutations ?

Inci

denc

e In

cide

nce

Causes


Discovery

Consequences I.F.


reports

Application Consolidation PHASE III

Variant detection


TP53 MUTATION HETEROGENEITY AND DATABASE FREQUENCY

TP53 MUTANTS (1,750 VARIANTS)

R175H (1 600 x) R273H (1 100x)

800 variants 1x 400 variants 2x

hot spot variants

rare variants

cut off: TP53 frequeny Remaining activity Frequency in outliers studies


http://p53.fr

TP53 Variants 7,200 Variants

TP53 Mutation 60,000 mutations

Each publication are flagged with a statistical index

cDNA variant

c.524G>A

Polyphen: Damaging Mutassessor: Damaging Condel: Damaging Provean: Damaging Splice assessor: Splice Neutral Variant

PREDICTION

wt

p.R175H

RESIDUAL ACTIVITY

Comment Isoforms

This mutation targets the 12 TP53 isoforms

Comment Frequency

This mutation is very frequent

Comment Activity

No activity

Comment Prediction

Damaging

Comment Outliers

No specific association

Comment Splice

No defect predicted

Frequency

2307

138

Hemopathies

38

CLL

14

MDS

for ERIC

Protein variant

p.R175H

ALL ISOFORMS

p.R175H p.R175H p.R136H p.R136H p.R136H p.R43H p.R43H p.R43H p.R16H p.R16H p.R16H

cDNA variant

c.375G>T

Protein variant

p.T125T

p.T125T p.T125T p.T86T p.T86T p.T86T p.= p.= p.= p.= p.= p.=

wt

p.T125T

Polyphen: No data Mutassessor: No data Condel: No data Provean: No data Splice assessor: Splice Neutral Variant

PREDICTION RESIDUAL ACTIVITY

ALL ISOFORMS

Frequency

74

4

Hemopathies

4

CLL

0

MDS

for ERIC

Comment Frequency

This mutation is not frequent

Comment Activity

No data

Comment Isoforms

This mutation targets 6 TP53 isoforms

Comment Prediction

No data

Comment Outliers


Comment Splice

This exonic mutation, close to a splice site, has been predicted to impair splicing; high confidence index

No data

cDNA variant

c.704A>G

Protein variant

p.N235S

p.N235S p.N235S p.N196S p.N196S p.N196S p.N103S p.N103S p.N103S p.N76S p.N76S p.N76S

wt

p.N235S

Polyphen: Tolerated Mutassessor: Low Condel: Benign Provean: Damaging Splice assessor: Splice Neutral Variant

PREDICTION RESIDUAL ACTIVITY

ALL ISOFORMS

Frequency

39

8

Hemopathies

5

CLL

0

MDS

for ERIC

Comment Frequency

This mutation is no frequent

Comment Activity

Fully active

Comment Isoforms

This mutation targets the 12 TP53 isoforms

Comment Prediction

Probably Benign

Comment Outliers


Comment Splice

No defect predicted

TP53 MUTATIONS ARE HETEROGENOUS

EXON 4 -9

R175H deficient for apoptosis and cell cycle arrest R175P deficient for apoptosis only

17p deletion Notch deletion association with another TP53 mutation early versus late

TP53 MUTATIONS AND ERIC

TP53 MUTATIONS IN CLL

ERIC program

Other parameters can be discussed

Ola Larsson

Thierry Soussi

Adam Ameur

Ignas Bunikis

Ulf Gyllensten

Bernard Leroy Jean L. Fournier

Karolina Edlund

Magnus Sundström

Patrick Micke

Johan Botling

ACKNOWLEDGEMENTS

Documents

THE UMD TP53 MUTATION DATABASE UPDATES AND BENEFITS … · 2019. 12. 12. · Data-driven unbiased curation of the TP53 tumor suppressor gene mutation database and validation by ultradeep