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 involvement

Lamina Cribosa

• Choroidal invasion (‹3mm) + ANY optic nerve involvement

High Risk Features

RPE

Choroid

Retina

Optic Nerve involvement

Choroidal involveme

nt

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

Angiogenic Factors

• VEGF• VEGFR2• CD105 (endoglin)

Differentiation Factors

• Vimentin• GFAP

Immuno-histochemis

try• Double Stain

Qualitative measure

• Grade 0 to 3

Quantitative measure

• Image J• Total area

VEGF-Vimentin (Muller cells)

VEGFR-2-CD105 (Neovascularization)

VEGF-GFAP(Glial activation)

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 FactorsGlial fibrillary acidic protein

(GFAP)

Normal retinaDamaged retina

• Intermediate filament

• Associated to:glial cells- Muller

cells

Up-regulated in CNS and

retinal injury

ImmunohistochemistryCD1

05VEGFR-2

Iris. 40X magnification

Color deconvolutionStain 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

Retin

a

Tumor

Iris

0

2000

4000

6000

8000HRFNON-HRF

p<0.05

Location

Pix

el A

rea

Vimentin

Retin

a

Tumor

Iris

0

5000

10000

15000

20000HRFNon-HRF

p<0.05

Location

Pix

el A

rea

VEGF+ Vimentin

Retin

a

Tumor

Iris

0

2000

4000

6000

8000HRFNon-HRF

p<0.05

LocationP

ixel

Are

a

*

Figure 1.

A B C

D E F

100 μm

GFAP

HRF

Non-HRF

0

1000

2000

3000

4000

5000

Tumor Type

Pix

el A

rea

VEGF

HRF

Non-HRF

0

1000

2000

3000

4000

5000

Tumor Type

Pix

el A

rea

VEGF+GFAP

HRF

Non-HRF

0

200

400

600

800

1000

Tumor TypeP

ixel

Are

a

* *

Figure 2.

A B C

D E F

100 μm

VEGFR2

Retin

a

Tumor

Iris

0

1000

2000

3000HRFNon-HRF

p<0.05

Location

Pix

el A

rea

CD105

Retin

a

Tumor

Iris

0

500

1000

1500HRFNon-HRF

p<0.05

Location

Pix

el A

rea

VEGFR2 + CD105

Retin

a

Tumor

Iris

0

50

100

150

200

250HRFNon-HRF

Pix

el A

rea

Figure 3.

*

*

*

*

*T

R

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