AN INTEGRATED ANALYSIS OF GENETIC AND EPIGENETIC ALTERATIONS IN GASTROINTESTINAL STROMAL TUMORS...

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AN INTEGRATED ANALYSIS OF GENETIC AND EPIGENETIC ALTERATIONS IN GASTROINTESTINAL STROMAL TUMORS REVEALS DISTINCT CLINICAL PHENOTYPES Sosipatros A. Boikos , Suzanne George, Katherine A. Janeway, Keith J. Killian, Su Young Kim, Michael P. LaQuaglia, Lauren Long, Paul S. Meltzer, Markku M. Miettinen, Karel Pacak, Alberto Pappo, Margarita Raygada, Joshua Schiffman, Constantine Stratakis, Jonathan Trent, Margaret Von Mehren, Christopher B. Weldon, Jennifer Wright, Lee J. Helman On behalf of the Consortium for Pediatric & wildtype GIST Research October 31, 2013 Connective Tissue Oncology Society

Transcript of AN INTEGRATED ANALYSIS OF GENETIC AND EPIGENETIC ALTERATIONS IN GASTROINTESTINAL STROMAL TUMORS...

Page 1: AN INTEGRATED ANALYSIS OF GENETIC AND EPIGENETIC ALTERATIONS IN GASTROINTESTINAL STROMAL TUMORS REVEALS DISTINCT CLINICAL PHENOTYPES Sosipatros A. Boikos,

AN INTEGRATED ANALYSIS OF GENETIC AND EPIGENETIC ALTERATIONS IN GASTROINTESTINAL STROMAL TUMORS

REVEALS DISTINCT CLINICAL PHENOTYPES

Sosipatros A. Boikos, Suzanne George, Katherine A. Janeway, Keith J. Killian, Su Young Kim, Michael P. LaQuaglia, Lauren Long, Paul S. Meltzer, Markku M. Miettinen, Karel Pacak, Alberto Pappo, Margarita Raygada, Joshua Schiffman, Constantine Stratakis, Jonathan Trent, Margaret Von Mehren, Christopher B. Weldon, Jennifer Wright, Lee J. Helman

On behalf of the Consortium for Pediatric & wildtype GIST Research

October 31, 2013Connective Tissue Oncology Society

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NO DISCLOSURES

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Overview

• Presentation of the most recent genetic and clinical findings of the NIH Pediatric and Wildtype GIST clinic.

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Overview

• Presentation of the most recent genetic and clinical findings of the NIH Pediatric and Wildtype GIST clinic.

• Correlation of the genetic and epigenetic findings with the clinical phenotype

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Overview

• Presentation of the most recent genetic and clinical findings of the NIH Pediatric and Wildtype GIST clinic.

• Correlation of the genetic and epigenetic findings with the clinical phenotype

• Discussion

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Wild-Type GIST

• 85% of GIST occurring in young population is lacking mutations in KIT or PDGFRA

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Wild-Type GIST

• 85% of GIST occurring in young population is lacking mutations in KIT or PDGFRA

• Stomach location, epithelioid histology

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Wild-Type GIST

• 85% of GIST occurring in young population is lacking mutations in KIT or PDGFRA

• Stomach location, epithelioid histology• May be syndromic (Carney Triad, Stratakis-

Carney Syndrome)

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Wild-Type GIST

• 85% of GIST occurring in young population is lacking mutations in KIT or PDGFRA

• Stomach location, epithelioid histology• May be syndromic (Carney Triad, Stratakis-

Carney Syndrome)• Tyrosine kinase inhibition is less effective

compared to KIT or PDGFRA mutant tumors

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Wild-Type GIST

• 85% of GIST occurring in young population is lacking mutations in KIT or PDGFRA

• Stomach location, epithelioid histology• May be syndromic (Carney Triad, Stratakis-

Carney Syndrome)• Tyrosine kinase inhibition is less effective

compared to KIT or PDGFRA mutant tumors• Mutations in Succinate Dehydrogenase (SDH)

genes have been found in some but not in all patients

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10 WT GIST clinics; 115 patients have been seen till An international clinic twice a year

115 patients have been seen in 10 clinics since 2008.

Objectives of the Wildtype Clinic at NIH

•To bring together healthcare providers who have the most experience treating and studying GIST•To obtain clinical history, response to prior treatments, histopathologic results, radiographic assessments and genetic/molecular analyses•Continue long-term follow-up for these patients

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Summary of Mutation Analysis of 78 Patients with Wild-Type GIST Fully Analyzed for SDH, BRAF, and NF1 Mutations

78 patients

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NF1 (3)

BRAF (3) 78 patients

6 patients Have NF1 or BRAF mutations

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SDHA (22)

SDHB (10)

SDHC (11)

SDHD (1)

44 Patients Have Mutations in SDHA, B, C, D

78 patients

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None (28)

28 Patients Have No Detectable Mutation To Date

78 patients

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15U USDHA1 52 315 402 487 585 664

FAD binding CAP FAD Helical C

R31X H99Y R171C

R188W

A223fs R352X Splice site

T256I

T273I

S445L

A454E

S505fs

R512X

K598N

L511P

A454T

AA 1 28 40 133 176 206 280

SDHB 1 2 3 4 5 6 7 8U U

2Fe-2S 4Fe-4S

c.17_42dupR201fs

W200Cc189F

I127S

Splice siteS92TR46Q

R46X

1 2 3 4 5U USDHC

1L 2L 3L 4L 5L 3S 4S

R133X

p54TSplice Site

R15X G75DM1V

H127R

1 2 3 4U USDHD

1S 2S 3S 4S

D118fs

Mutations Distributed across all exons-90% Germline

78 patients

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Wildtype GIST can have negative SDHB staining regardless of the status of mutations in SDH genes

Janeway and Kim et al. 2011 PNAS 108:314

NF1 (3) BRAF (3) SDHA (22)SDHB (10) SDHC (11) SDHD (1)

None (28)

78 patients

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53 tumors had negative SDHB staining

9 had positive staining

NF1 (3) BRAF (3) SDHA (22)SDHB (10) SDHC (11) SDHD (1)

None (28)

1st CircleMutated Genes:

2nd CircleSDHB staining:

Negative (53)

Positive (9)

78 patients

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BRAF (3) SDHA (22)SDHB (10) SDHC (11) SDHD (1)

None (28)

1st CircleMutated Genes:

2nd CircleSDHB staining:

Negative (53)

Positive (9)

• Tumors with SDHA, B, C, D mutations have always negative SDHB staining.

NF1 (3)

78 patients

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BRAF (3) SDHA (22)SDHB (10) SDHC (11) SDHD (1)

None (28)

1st CircleMutated Genes:

2nd CircleSDHB staining:

Negative (53)

Positive (9)

• Tumors with SDHA, B, C, D mutations have always negative SDHB staining.

NF1 (3)

78 patients

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BRAF (3) SDHA (22)SDHB (10) SDHC (11) SDHD (1)

None (28)

1st CircleMutated Genes:

2nd CircleSDHB staining:

Negative (53)

Positive (9)

• Tumors with SDHA, B, C, D mutations have always negative SDHB staining.

• Tumors with negative SDHB staining and no mutations have probably alterations in SDH pathway.

NF1 (3)

78 patients

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BRAF (3) SDHA (22)SDHB (10) SDHC (11) SDHD (1)

None (28)

1st CircleMutated Genes:

2nd CircleSDHB staining:

Negative (53)

Positive (9)

• Tumors with SDHA, B, C, D mutations have always negative SDHB staining.

• Tumors with no mutations and negative SDHB staining have probably alterations in SDH pathway.

• Tumors with BRAF or NF1 mutations had SDHB positivity.

• Tumors with SDHB positivity and no mutations likely have alterations outside the SDH pathway.

NF1 (3)

78 patients

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Infinium 450 methylation dataKillian et al. Cancer Discovery 2013

Succinate Dehydrogenase Mutations lead to a hypermethylator phenotype in Gastrointestinal Stromal Tumor

78 patients

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NF1 (3)

BRAF (3)

SDHA (22)1st CircleMutated Genes:

Infinium 450 methylation dataKillian et al. Cancer Discovery 2013

Negative SDHB staining by IHC correlates with Hypermethylation in 66 tumors

78 patients

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How SDHB loss is causing hypermethylation?

78 patients

Yang M, Pollard PJ Cancer Cell 2013

NF1 (3)

BRAF (3)

SDHA (22)

SDHB (10)

SDHC (11)

SDHD (1)

None (28)

1st CircleMutated Genes:

2nd CircleSDHB staining:

Negative (53)

Positive (9)

Deviator (66)

Centrist (12)

3rd CircleMethylation

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Group A (n=12)

Positive SDHB IHCNormal Methylation pattern

Mutations in NF1, BRAF or other unknown genes

78 patients

Group A

NF1 (3)

BRAF (3)

SDHA (22)

SDHB (10)

SDHC (11)

SDHD (1)

None (28)

1st CircleMutated Genes:

2nd CircleSDHB staining:

Negative (53)

Positive (9)

Deviator (66)

Centrist (12)

3rd CircleMethylation

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Group B (n=22)

Negative SDHB staining by IHCHypermethylation (Deviator)

Not known mutations

78 patientsGroup B

NF1 (3)

BRAF (3)

SDHA (22)

SDHB (10)

SDHC (11)

SDHD (1)

None (28)

1st CircleMutated Genes:

2nd CircleSDHB staining:

Negative (53)

Positive (9)

Deviator (66)

Centrist (12)

3rd CircleMethylation

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Group C (n=44)

Negative SDHB staining by IHCHypermethylation (Deviator)

SDHA, B, C, D mutations

78 patients

Group C

NF1 (3)

BRAF (3)

SDHA (22)

SDHB (10)

SDHC (11)

SDHD (1)

None (28)

1st CircleMutated Genes:

2nd CircleSDHB staining:

Negative (53)

Positive (9)

Deviator (66)

Centrist (12)

3rd CircleMethylation

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Comparison of Group B and C

1. Group B=young age

Age

60

40

20

0B C

Years

2. All patients in Group B are females while in Group C 65% are females.

We have Carney Triad patients (chondroma, paraganglioma) or Carney-Stratakis Syndrome patients (paraganglioma) in both groups B and C.

At the moment we have no statistically significant differences in overall Survival, Recurrence free Survival.

78 patientsGroup B

Group C

NF1 (3)

BRAF (3)

SDHA (22)

SDHB (10)

SDHC (11)

SDHD (1)

None (28)

1st CircleMutated Genes:

2nd CircleSDHB staining:

Negative (53)

Positive (9)

Deviator (66)

Centrist (12)

3rd CircleMethylation

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Conclusions

Group A Group B Group C

Mutated genes NF1, BRAF or other genes

Unknown genes SDH genes

SDHB expression by ICH normal no no

SDH Function Normal Impaired Impaired

Methylation Pattern Centrist Deviator(hypermethylation)

Deviator(hypermethylation)

Gender Both Females Female predominance

Age Young adults, adults Pediatric, young adults

Young adults, adults

Location Gastric, small bowel Gastric Gastric

3 distinct groups of Wildtype GIST

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Group A Group B Group C

Mutated genes NF1, BRAF or other genes

Unknown genes SDH genes

SDHB expression by ICH normal no no

SDH Function Normal Impaired Impaired

Methylation Pattern Centrist Deviator(hypermethylation)

Deviator(hypermethylation)

Gender Both Females Female predominance

Age Young adults, adults Pediatric, young adults

Young adults, adults

Location Gastric, small bowel Gastric Gastric

ConclusionsGroup B tumors have loss of SDHB staining without SDH mutations.We assume these 20 tumors have alterations in SDH pathway.

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Discussion

Should we redefine Wildtype GIST as tumors having SDHB deficiency by IHC?

Should we use demethylating agents for SDHB deficient GIST?

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Acknowledgements

Our patients!

Dr. Lee Helman labLauren LongYeung ChohArnaldez FernandaErin GombosChristine HeskeAmy McCalla-MartinAnna NkrumahWan Xiaolin

Page 34: AN INTEGRATED ANALYSIS OF GENETIC AND EPIGENETIC ALTERATIONS IN GASTROINTESTINAL STROMAL TUMORS REVEALS DISTINCT CLINICAL PHENOTYPES Sosipatros A. Boikos,
Page 35: AN INTEGRATED ANALYSIS OF GENETIC AND EPIGENETIC ALTERATIONS IN GASTROINTESTINAL STROMAL TUMORS REVEALS DISTINCT CLINICAL PHENOTYPES Sosipatros A. Boikos,

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15U USDHA1 52 315 402 487 585 664

FAD binding CAP FAD Helical C

R31X H99Y R171C

R188W

A223fs R352X Splice site

T256I

T273I

S445L

A454E

S505fs

R512X

K598N

A454T

SDHA

SDHB

SDHC

SDHD

FAD Binding Domain

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15U USDHA1 52 315 402 487 585 664

FAD binding CAP FAD Helical C

R31X H99Y R171C

R188W

A223Fs R352X Splice site

T256I

T273I

S445L

A454E

S505fs

R512X

K598N

A454T

SDHA

SDHB

SDHC

SDHD

K589N

L511P

H99AA454L

R188W

T256I

A454EA454T

R171C

T273I

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15U USDHA1 52 315 402 487 585 664

FAD binding CAP FAD Helical C

R31X H99Y R171C

R188W

A223fs R352X Splice site

T256I

T273I

S445L

A454E

S505fs

R512X

K598N

A454T

SDHA

SDHB

SDHC

SDHD

K589N

L511P

H99AA445L

R188W

T256I

A454EA454T

R171C

T273I

FAD domain mutations

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15U USDHA1 52 315 402 487 585 664

FAD binding CAP FAD Helical C

R31X H99Y R171C

R188W

A223fs R352X Splice site

T256I

T273I

S445L

A454E

S505fs

R512X

K598N

A454T

SDHA

SDHB

SDHC

SDHD

K589N

L511P

H99AA445L

R188W

T256I

A454EA454T

R171C

T273I

FAD domain mutations

Protein-truncating mutations

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15U USDHA1 52 315 402 487 585 664

FAD binding CAP FAD Helical C

R31X H99Y R171C

R188W

A223fs R352X Splice site

T256I

T273I

S445L

A454E

S505fs

R512X

K598N

A454T

SDHA

SDHB

SDHC

SDHD

K589N

L511P

H99AA445L

R188W

T256I

A454EA454T

R171C

T273I

FAD domain mutations

Protein-truncating mutations

Mutations disrupting the FAD domain

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15U USDHA1 52 315 402 487 585 664

FAD binding CAP FAD Helical C

R31X H99Y R171C

R188W

c.666delT R352X Splice site

T256I

T273I

S445L

A454E

S505fs

R512X

K598N

A454T

SDHA

SDHB

SDHC

SDHD

K589N

L511P

H99AA454L

R188W

T256I

A454EA454T

R171C

T273I

Correlation of Metastasis and site of mutation

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15U USDHA1 52 315 402 487 585 664

FAD binding CAP FAD Helical C

R31X H99Y R171C

R188W

c.666delT R352X Splice site

T256I

T273I

S445L

A454E

S505fs

R512X

K598N

A454T

SDHA

SDHB

SDHC

SDHD

K589N

L511P

H99AA454L

R188W

T256I

A454EA454T

R171C

T273I

Correlation of Metastasis and site of mutation

Similar analysis on SDHB, C and D subunits is ongoing

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AA

61 85 89 114 119 145 150 158

AA 1 28 40 133 176 206 280

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15U USDHA

1 2 3 4U U

SDHB

1 2 3 4 5U USDHC

1 2 3 4 5 6 7 8U U

SDHD

1 52 315 402 487 585 664

FAD binding CAP FAD Helical C

1S 2S 3S 4S

AA

33 44 63 91 95 104 110 139 144 160

1L 2L 3L 4L 5L 3S 4S

2Fe-2S 4Fe-4S

R31X H99Y

c.17_42dup

R171C

R188W

A223fs R352X Splice site

T256I

T273I

S445L

A454E

S505fs

R512X

K598N

L511P

A454T

R201fsW200C

c189F

I127S

Splice siteS92TR46Q

D118fs

R46X

R133X

p54TSplice Site

R15X G75DM1V

H127R

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Female Gender

Age

at d

iagn

osis

Group CSDHx mutations

Hypermethylator phenotype

Group BNo mutations

Hypermethylator phenotype

Wildtype GIST Phenotype

Paragangliomas

Chondromas

Carney Triad

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Female Gender

Age

at d

iagn

osis

Group CSDHx mutations

Hypermethylator phenotype

Group BNo mutations

Hypermethylator phenotype

Almost all patients in group B are younger and female in comparison with group C

Paragangliomas

Chondromas

Carney Triad

Age

60

40

20

0B C

Years

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Female Gender

Age

at d

iagn

osis

Group CSDHx mutations

Hypermethylator phenotype

Group BNo mutations

Hypermethylator phenotype

We have Carney Triad patients (paraganglioma) or Carney-Stratakis Syndrome (chondroma, paraganglioma) in both groups B and C

ParagangliomasChondromas

Carney Triad