Telomerase reverse transcriptase promoter€¦ · Barbara Heidenreich Characteristic sa m pl e nu m be r (n = 28 7) TE RT wil dt yp e TE RT m ut ati on O d d s ra ti o 95 % CI P-val

Telomerase reverse transcriptase promoter

(TERT) mutationsEduardo Nagore

Department of DermatologyInstituto Valenciano de Oncología

Valencia, Spain

Background

Oncogenic mutations

Delections

Amplifications

Diagnosis

Classification

Treatment

Molecular signaling pathways

Host immune elements

Host phenotype/genotype Environmental factors

Molecular model: therapy

PLoS ONE 2011; 6(3): e18257.

Primary molecular subtypes

Vidwans SJ, Flaherty KT, Fisher DE, Tenenbaum JM, Travers MD, et al. (2011) A Melanoma Molecular Disease Model. PLoS ONE 6(3): e18257. doi:10.1371/journal.pone.0018257

Secondary molecular subtypes

Vidwans SJ, Flaherty KT, Fisher DE, Tenenbaum JM, Travers MD, et al. (2011) A Melanoma Molecular Disease Model. PLoS ONE 6(3): e18257. doi:10.1371/journal.pone.0018257

Non-CSD

CSD

TERT coding region somatically mutated in 1%

Negative for CDKN2A/CDK4 mutations; no high risk MC1R variantsMedian age of onset 28 years in women and 41 years in men

Original discovery of TERT promoter mutations as germline mutations in a large melanoma family:

Multipoint linkage analysis: 2.2 MB linkage region on chromosome 5p (maximal LOD-scores of 2.35 at rs1379917 and 2.45 at rs 1968011

Target enriched high throuput sequencing

Variant no.

Location

Gene

Position hg19 (bp)

Reference

Variant

UCSC exon no.

Intron no.

bp in exon / intron

Min. distance to next exon (bp)

Nearby gene

1 Promoter

hTERT

1,295,161

T G 1 - 2 0 -

2 Intron

SLC9A3

501,288

C T - 1 22,938

9,102

-

3 Intron

NKD2

1,036,775

C G - 9 859

276

-

4 Intron

SLC12A7

1,073,496

C T - 17

251

251

-

5 Non-gene

- 1,140,303

C T - - 28,131

SLC12A7 (uc003jbu.3)

6 Non-gene

- 1,140,764

C T - - 28,592

SLC12A7 (uc003jbu.3)

7 Non-gene

- 1,557,614

C T - - 9,983

CR749689 (uc003jcn.3)

803

804

802 801

767

766

772

778

763 782

764

779

765

755

756 757 758

759 760 761

762

773

769 768

770

776 777

774775

780

753812

852

781

754

Index

Age onsetAge death

DNA sample

3031

****

*

L

LL

L L

L L

L

L

LL L L

L

L#

# 2050

42

3232

3940

36unk

3031

4242

28

4649

3049

1819

2020

2731

unk

772

TERT promoter mutation -57bp T>G*

Linkage analysisLHigh throughput sequencing

Not found in 34 Spanish families CDKN2A negatives

Science. 2013 Feb 22:339(6122):959-61

5´ GAGTTTCAGGCAGC 3´

3´ CTCAAAGTCCGTCG 5´

TERTTERT Promoter- 57bp A>C (T>G) mutation

Ets/TCF Binding

x2 increase of promoter activity

5´ GAGTTTCCGGCAGC 3´

TERTTERT Promoter

3´ CTCAAAGGCCGTCG 5´

TERT promoter mutation effect

-146 G>A

-124 G>A -57 T>G

-124,125 GG>AA

Tumor somatic mutations in TERT promoter

-UV-signature: G>A (C>T); GG>AA (CC>TT)-Creation of TCF binding motifs: CCGGAA/T


ATGTranscription start

E-Box E-Box EtsEtsEtsEts SP1SP1

Familial

-50 -60 ..|....|....|....|Ref AGCGCTGCCTGAAACTCGFamily ~~~~~~~~~G~~~~~~~~Ets ---------GGAA-----TCF -------CCGGAA-----

Sporadic

-120 -130 -140 -150 ...|....|....|....|....|....|....|Ref GCCCGGAGGGGGCTGGGCCGGGGACCCGGGAGGGSM ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A~~~~ 39%SM ~~~~~~~A~~~~~~~~~~~~~~~~~~~~~~~~~~ 32%SM ~~~~~~~~~~~~~~~~~~~~~AA~~~~~~~~~~~ 5%SM ~~~~~~~AA~~~~~~~~~~~~~~~~~~~~~~~~~ 4%SM ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A~~A~ 2%Ets ----GGAA-----------GGAA----GGAA---TCF --CCGGAA---------CCGGAA--CCGGAA---

* *

-100 -200

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Heidenreich

-124_125CC>TT

C>T transitions at dipyrimidinic sites and CC>TT tandem mutations indicate UV-signature

counting from ATG start site-124C>T = ,228G>A-138_139CC>TT = ,242_243GG>AA-146C>T = ,250G>A-57A>C = ,161T>G

Chr 5:1,295,xxx hg19 coordinate (Build 37)

A: TERT promoter: most frequent mutation.B: TERT promoter: concomitant mutation with BRAF and CDKN2A (blue: observed frequencies, red: expected frequencies).

Sequence of mutations: BRAF CDKN2A TERT Prom


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HeidenreichTERT promoter mutations in other neoplasias

Recent findings in other cancer types:

Heidenreich et al., Current Opinion in Genetics & Development 2014

Rachakonda et al., PNAS, 2013

Bladder Cancer: Better survival in patients with TERT promoter mutations if rs2853669 is present

Killela et al., Oncotarget, 2014

Glioma: TERT promoter mutations and IDH1 mutations allow grouping of histological subtypes and indicate survival

Pilati et al., Cancer Cell, 2014

Hepatocellular Adenoma/Carcinoma: TERT promoter mutations are required to promote full malignant transformation

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Melanocyte

BRAFNRAS

loss of tumor supressor (CDKN2A; PTEN)

increased CD1

E-cadherin loss

Reduced TRPM1

Absent TRPM1

Where do we place the TERT promoter mutations and what is their role in the clinical picture?

MetastasesBenign Nevus Dysplastic

Nevus

Radial-Growth Phase Vertical-Growth Phase

Metastatic Melanoma

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TERT promoter mutations in nevi

Sequencing of 92 nevi samples revealed that there are no TERT promoter mutations in nevi

Melanocyte MetastasesBenign Nevus

Dysplastic Nevus

Radial-Growth Phase

Vertical-Growth Phase

Metastatic Melanoma

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HeidenreichTERT promoter in primary cutaneous melanoma

287 primary melanoma samples were further investigated according to available information in ✓ sex, age at diagnosis; profession; sun-exposure behavior✓ phenotype (skin-, eye-, hair color; information on nevi; personal & family history; MC1R status)✓ tumor information (location; histological type; stage)✓ clinical-histopathological data (growth phase; Breslow thickness; presence of ulceration; regression; contiguous neval remnants; presence of solar elastosis)


Dysplastic Nevus

Radial-Growth Phase


Metastatic Melanoma

Valencia

Heidelberg227 FFPE60 fresh frozen tissues

PCR and sanger sequencing

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TERT promoter mutations

BRAF (n = 287)

OR 2.5; 95% CI 1.53 - 4.14; P = 0.0003

NRAS (n = 274)

OR 2.6; 95% CI 1.03 - 6.62; P = 0.04

CDKN2A (n = 60)

OR 3.65; 95% CI 0.96 - 13.90; P = 0.06


Dysplastic Nevus

Radial-Growth Phase


Metastatic Melanoma

TERT promoter in primary cutaneous melanoma

TERT promoter: 38%BRAF: 36%NRAS: 7%

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Characteristic

sample

number(n=287)

TERT wildtype

TERT mutation

Odds ratio

95% CI

P-value

Age at diagnosis (years)

< 45 70 48 22

1.00 Ref.

46 - 65 119 80 39

1.06

0.56 - 2.00

0.85

> 66 98 50 48

2.10

1.10 - 3.98

0.02

Presence of solar lentigines at melanoma site

no 159

109 50

1.00 Ref.

yes 118 64 54

1.84

1.12 - 3.01

0.02

Pattern of sun exposure at melanoma site

rarely exposed 52 42 10

1.00 Ref.

usually exposed

195

118 77

2.74

1.30 - 5.78

0.008

infrequently exposed 35 17 18

4.44

1.70 - 11.5

0.002

Histological type

Superficial spreading M

158

103 55

1.00 Ref.

Nodular M 76 34 42

2.31

1.3 -

4.00.03

Acral lentiginous M 24 23 1Lentigo maligna M 13 10 3

Growth phase

radial 39 33 6

1.00 Ref.

vertical 186

108 78

3.97

1.60 - 9.94

0.003

Breslow Thickness [mm]

< 1102 77 25

1.00 Ref.

1 - 2 58 35 232.0

1.0 -

4.00.05

2.1 - 4 51 26 252.9

1.5 -

6.00.003

> 4 60 29 313.3

1.7 -

6.5

0.0006

Presence of ulceration

no 194

133 61

1.00 Ref.

yes 86 43 43

2.18

1.30 - 3.67

0.003

Stage

in situ 12 11 1 1.00

Ref. localized

191

124 67

locoregional disease 74 38 36

1.90

1.17 - 3.38

0.012 distant

metastasis 4 1 3


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Multivariate analysis of TERT promoter mutations and various patient and tumour characteristics

OR 95% CI P-value

Age, years

< 65 1.00 Ref.> 65 1.97 1.03 - 3.79 0.04

Presence of solar lentigines at melanoma site

No 1.00 Ref.Yes 1.58 0.81 - 3.06 0.2

Sun exposure

Rarely 1.00 Ref.Infrequently

exposed3.01 1.07 - 8.46

0.05Usually

exposed4.94 1.34 - 18.2

Histological typea

SSM 1.00 Ref.LMM 0.38 0.08 - 1.86

0.04NM 0.86 0.38 - 1.96

ALM0.04 0.004 -

0.42Breslow thickness, mm

< 2 1.00 Ref.> 2 2.83 1.24 - 6.43 0.01

Presence of ulceration

No 1.00 Ref.Yes 2.34 1.04 - 5.26 0.04

StageIn situ +

localized 1.00 Ref.Locoregional

disease + distant metastasis

0.90 0.42 - 1.96 0.8

BRAF

Wildtype 1.00 Ref.Mutation 2.35 1.27 - 4.33 0.006

Age and sun-exposure

Association with subtype depends on growth phase, sunexposure and Breslow thickness at diagnosis

Association with stage influenced by Breslow thickness

Association of TERT promoter mutations with parameters linked to poor outcome of disease


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HeidenreichTERT mRNA expression in relation to TERT promoter mutations

relative expression compared to housekeeping gene (GUSB; sybr green)

TERT mRNA expression is significantly higher in tumors that carry TERT promoter mutations.

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Dysplastic Nevus

Radial-Growth Phase


Metastatic Melanoma

locoregional cutaneous metastasis

soft tissue metastasis

primary melanoma tumor

TERT promoter mutations in melanoma metastases

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✓ ...appear as somatic mutations with high frequency in primaries and tumor metastases

✓ ...are not present in nevi and associated with increased Breslow thickness → late events?

✓ ...are associated with the presence of BRAF/NRAS mutations and alterations at the 9p21 (CDKN2A) locus

✓ ...are associated with clinical factors known to indicate poor outcome (Breslow thickness; Ulceration)

✓ ...show UV-signature and are influenced by sunexposure (sunexposure; lentigines; age)

✓ ...cause upregulation of TERT mRNA

✓ do they have potential as markers in disease management?✓ are they associated with survival?

Conclusions: TERT promoter mutations…

Non-CSD CSD

Acknowledgements:

Barbara HeidenreichNadem SoufirZaida García-CasadoCarlos GuillénVirtudes SorianoJürgen BeckerKari HemminkiKrishna RachakondaRajiv Kumar

Thank you

28/06/2014

Documents

Telomerase reverse transcriptase promoter€¦ · Barbara Heidenreich Characteristic sa m pl e nu m be r (n = 28 7) TE RT wil dt yp e TE RT m ut ati on O d d s ra ti o 95 % CI P-val