TARGETING NEW

ANGIOGENIC

FACTORS IN

RETINOBLASTOMAJESUS GARCIA

Background

Retinoblastoma(Rb)

Rapidly developing

cancer of the retina

Most common in children

Mutation in chromosome

13 in RB1

Invasive Retinoblastoma

represents the real

challenge in clinics

Background:

Angiogenesis

Angiogenesis is essential for tumor growth and metastasis

Pathological retinal angiogenesis occurs in several diseases characterized by retinal ischemia

Angiogenesis may complicate the clinical presentation by inducing glaucoma due to iris neovascularization(NVI) with secondary peripheral synechiae(PAS) formation

Inhibion of angiogenesis has been shown to kill retinoblastoma cells (harbour ophtalmic research)

Avastin (anti VEGF antibody)

Tyrosine kinase inhibitors

VEGF inhibition may also help as a chemosensityzer

Background: VEGF

VEGF is a hypoxia inducible

cytokine required for retinal

vascularization

Produced by Rb tumor

There are several pro-

angiogenic factors involved

in retinal angiogenesis

VEGF and its receptors are

expresed in non-

overlapping manner

(Prospero Ponce et al 2013)

High Risk Features

Lamina Cribosa

Optic Nerve

involvement

Choroidal

involvementLamina Cribosa

•Choroidal invasion

(‹3mm) + ANY optic

nerve involvement

High Risk Features

RPE

Choroid

Retina

Optic Nerve

involvement

Choroidal

involvement

Angiogenesis complicates RB

Neovascularization of the iris (NVI)

+/-

Peripheral synechia (PAS)

Neovascular Glaucoma

Relevant background

Neovascularization in retinoblastoma is associated with poorer prognosis and increased chances of invasion

Neovascularization as well as angiogenesis is driven by angiogenic factors.

VEGF is the most important and most studied angiogenic factor

Several anti-VEGF therapies have demonstrated good results in various types of cancer

No one has studied histopathological behavior of VEGF in Rb

VEGF is a promising therapy in Retinoblastoma

Hypothesis

HRF tumors will have increased secretion of VEGF

and its receptor VEGFR2

Objective

To analyze the expression of angiogenic factors in

the eye (retina, iris, and tumor ) in

retinoblastoma with high-risk features (HRF) and

non-high risk features (Non-HRF). To correlate the

expression of angiogenic factors in tumors with HRF and the expression of stem cell marker

Sox2. keep order same throughout. If possible,

use this order when discussing your results as well.

HRF= 6 cases

Non-HRF= 6 cases

Methods

AngiogenicFactors

•VEGF

•VEGFR2

•CD105 (endoglin)

Differentiation Factors

•Vimentin

•GFAP

Immuno-histochemistry

•Double Stain

Qualitative measure

•Grade 0 to 3

Quantitative measure

•Image J

•Total area

Methods

Differentiation Factors

Vimentin

Intermediate filament

associated with fibroblasts

and activated Muller cells

It can also be expressed

by RPE, cilliary body

epithelium, lens epithelium

GCL

INL

ONL

Differentiation Factors

Glial fibrillary acidic protein

(GFAP)

Normal retinaDamaged retina

•Intermediate

filament

•Associated to:

glial cells- Muller

cells

Up-regulated in

CNS and retinal

injury

ImmunohistochemistryCD10

5VEGFR-2

Iris. 40X magnification

Color deconvolution

Stain separation using Ruifrok and Johnston's method1

Measurements: Image J

[1] Ruifrok AC, Johnston DA. Quantification of histochemical staining by color deconvolution. Anal Quant Cytol Histol 23: 291-299, 2001

Vimentin VEGF

Analyzing particles

Measurements: Image J

8 bit

Binary

ResultsANGIOGENIC FACTORS AND HRF IN RB

A B C

D E F

100 μm100 μm100 μm

VEGF

Ret

ina

Tumor

Iris

0

2000

4000

6000

8000HRF

NON-HRF

p<0.05

Location

Pix

el

Are

a

Vimentin

Ret

ina

Tumor

Iris

0

5000

10000

15000

20000HRF

Non-HRF

p<0.05

Location

Pix

el

Are

a

VEGF+ Vimentin

Ret

ina

Tumor

Iris

0

2000

4000

6000

8000HRF

Non-HRFp<0.05

LocationP

ixel

Are

a

*

Figure 1.

A B C

D E F

100 μm

GFAP

HRF

Non-H

RF

0

1000

2000

3000

4000

5000

Tumor Type

Pix

el

Are

a

VEGF

HRF

Non-H

RF

0

1000

2000

3000

4000

5000

Tumor Type

Pix

el

Are

a

VEGF+GFAP

HRF

Non-H

RF

0

200

400

600

800

1000

Tumor TypeP

ixel

Are

a

**

Figure 2.

A B C

D E F

100 μm

VEGFR2

Ret

ina

Tumor

Iris

0

1000

2000

3000HRF

Non-HRFp<0.05

Location

Pix

el

Are

a

CD105

Ret

ina

Tumor

Iris

0

500

1000

1500HRF

Non-HRFp<0.05

Location

Pix

el

Are

a

VEGFR2 + CD105

Ret

ina

Tumor

Iris

0

50

100

150

200

250HRF

Non-HRF

Pix

el

Are

a

Figure 3.

*

*

*

*

*

TR

Figure 4.

A

F

C

ED

B

G

Summary

HRF tumor s express more VEGF and VEGFR2 than

non-HRF

Comparison with other stainings indicate that

VEGF secretion might be done by tumor cells

Neovascularization occures more in the iris

This expression is correlated with the expression of

stem cell marker SOX2

Conclusions

HRF tumors seem to be more “stem like”

They might regulate their invasiveness through a

VEGF feedback loop

Temporal expression of VEGF receptors should be

considered

Anti-VEGF therapy might be a promising therapy

for Rb and its side effects

Acknowledgements

Patricia Chevez-Barrios, MD

Rebecca Penland

Magdalena Arredondo

Claudia M. Prospero, MD