E2F3 in Soft Tissue Sarcoma

Development

M. Scurr; A Feber; J Shipley; A Fletcher; N Dennis; I Judson; C Cooper

Active E2F3

RB E2F3

P

P

Cyclin DCDK

RB

Cyclin E, CDK2 G1/S G1/S TransitionTransition

Growth Factors

P

DNA synthesis (TS,TK,DHFR)

Cyclin A

E2F3 Overexpression and Cancer

Human Bladder Cancer (Feber et al)

E2F3 an oncogene activated by gene amplification and overexpression.E2F3 overexpression correlates with increasing grade and stage Prostate Cancer (Foster et al)

E2F3 overexpression an independent variable overall survival (P=.0014)cause specific survival (P=.0004)

Evaluation of Potential Role for E2F3 in STS

RB1 mutation/loss in STS

~1/3 of leiomyosarcomas

Up to 70% of primary tumours altered RB expression

Reports of cyclin D1/Cdk4 overexpression

Implicating RB-E2F Axis

STS Tissue Microarrays

165 patients with primary tumour samples and clinical data

Tumour TypeTumour Type Patient NPatient Noo

Leiomyosarcoma 118

MFH 97

Synovial Sarcoma 52

Fibrosarcoma 23

TOTAL 290

Fibrosarcoma

MFH

Leiomyosarcoma

Monophasic Synovial Sarcoma

Epithelial cell staining Spindle cell staining

E2F3 Nuclear Staining:Total STS TMA

38%

53.6%

5.6%2% 0.7%

0% 1-20% 21-40% 41-60% 61-80% 81-100%

Leiomyosarcoma Fibrosarcoma

MFH Synovial Sarcoma

44.7%52.8%

2.4%

neg upto 20% 21-40% 41-60% 61-80% 81-100%

32%

64.1%

3.9%

neg upto 20% 21-40% 41-60% 61-80% 81-100%

50%45.8%

4.2%

neg upto 20% 21-40% 41-60% 61-80% 81-100%

29.1%

40%

16.4%10.9%

3.4%

neg upto 20% 21-40% 41-60% 61-80% 81-100%

Synovial Sarcoma Prostate Cancer

29.1%

40%

16.4%10.9%

3.4%

33.3% 31.3%

9.5%13.6%

6.8% 5.4%

Clinical Correlations: E2F3 Staining and Prognostic Variables.

• Size

• Stage

• Grade

Clinical Correlations: E2F3 Staining and Prognostic Variables.

• Size No significant correlation

• Stage No significant correlation

• Grade

Clinical Correlations: E2F3 Staining and Prognostic Variables.

• Size No significant correlation

• Stage No significant correlation

• Grade P<0.001

Clinical Outcomes: Disease Free Survival

UNIVARIATE ANALYSIS size

stagegrade

MULTIVARIATE ANALYSIS stage

+ve E2F3 nuclear stainingHR 1.41 (95% CI .89-2.44) P=0.148

Clinical Outcomes: Disease Free Survival

UNIVARIATE ANALYSIS size

stagegrade

MULTIVARIATE ANALYSIS stage

+ve E2F3 nuclear stainingHR 1.45 (95% CI 0.93-2.25) P=0.1

Clinical Outcomes: Cause Specific Survival

UNIVARIATE and MULTIVARIATE ANALYSESsizestagegradesite

Clinical Outcomes: Cause Specific Survival

UNIVARIATE and MULTIVARIATE ANALYSESsizestagegradesite

+ve E2F3 nuclear staining HR 1.57 (95% CI 0.92-2.7) P=0.10

Assessment of biological function of E2F3 overexpression in STS

E2F3 in STS Cell LinesH

T1

08

0

HTB

-88

HTB

-11

4

SK

W

SS-S

Y-I

I

BA

X

FUJI

56

37

(+ve

contr

ol)

GAPDH

E2F3

E2F3 in STS Cell LinesH

T1

08

0

HTB

-88

HTB

-11

4

SK

W

SS-S

Y-I

I

BA

X

FUJI

56

37

(+ve

contr

ol)

GAPDH

E2F3

E2F3 siRNA Transfection: 24 hours

E2F3

GAPDH

Si-

1

Si-

2

Si-

3

Si-

all

Scr

-1

Scr

-2

Scr

-3

Scr

-all

contr

ol

Biological effect of “knocking down” E2F3 production

Si-3 and Scr-3 transfection of HT 1080

Analysis of effect on G1/S transition

BrdU Incorporation:Immunofluorescence Microscopyfluorescent activated cell sorting (FACS)

BrdU Incorporation Immunofluorescence Microscopy

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

0hrs 24hrs 48hrs 72hrs

Si-3

Scr-3

BrdU Incorporation: FACS

Si-3: 0 hrs Si-3: 24 hrs post

Scr-3: 0 hrs Scr-3: 24 hrs post

Conclusions

• 62% STS samples positive E2F3 nuclear staining• LMS/MFH and fibrosarcoma predominantly <20% intensity• Synovial sarcoma more intense staining similar pattern to prostate and

bladder cancer.

• +ve Nuclear staining correlates with higher grade• Non-significant trend to poorer disease free and cause specific survivals

• E2F3 knockdown studies demonstrate that inhibition of E2F3 expression inhibits cell cycle progression.

E2F3 OVEREXPRESSION HAS A ROLE IN THE MALIGNANT E2F3 OVEREXPRESSION HAS A ROLE IN THE MALIGNANT PHENOTYPE IN STSPHENOTYPE IN STS

AcknowledgementsInstitute of Cancer Research

Molecular CarcinogenesisColin CooperAndy FeberSandra EdwardsPenny FlohrToby RoeJeremy ClarkJanet ShipleyAnne FletcherNening Dennis

Cancer TherapeuticsJenny TitleyMike Walton

Royal Marsden Hospital

Sarcoma UnitIan JudsonOmar Al-Muderis

Histopathology DepartmentCyril Fisher

StatisticsRoger Ahern

University of LiverpoolDepartment of Pathology and Medical

GeneticsChris FosterAndy Dodson

Supported by: Royal Marsden NHS Trust Charitable Fund Grant

Jeannette Pomeraniec Sarcoma Research Fund