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![Measurements: Image J
Color deconvolution
Stain separation using Ruifrok and Johnston's method1
Vimentin
VEGF
[1] Ruifrok AC, Johnston DA. Quantification of histochemical staining by color deconvolution. Anal
Quant Cytol Histol 23: 291-299, 2001](https://image.slidesharecdn.com/vegfexpressioninhrf-131111154300-phpapp02/85/Vegf-expression-in-hrf-17-320.jpg)
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Vegf expression in hrf
This study analyzed the expression of angiogenic factors in retinoblastoma tumors with high-risk features (HRF) compared to those without. The results showed that HRF tumors expressed significantly higher levels of VEGF and VEGFR2 than non-HRF tumors. Staining indicated the VEGF was secreted by tumor cells. Neovascularization was also higher in the iris of HRF tumors. Expression of VEGF correlated with the stem cell marker SOX2, suggesting HRF tumors are more stem-like. The conclusions were that HRF tumors may regulate invasiveness through a VEGF feedback loop, and anti-VEGF therapy may be a promising treatment for retinoblastoma due to these findings.
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Vegf expression in hrf
- 1.
- 2. Background Retinoblastoma(Rb) Rapidly developing cancer ofthe retina Most common in children Mutation in chromosome 13 in RB1 Invasive Retinoblastoma represents the real challenge in clinics
- 3. Background: Angiogenesis Angiogenesis is essentialfor 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
- 4. Background: VEGF VEGF isa hypoxia inducible cytokine required for retinal vascularization Produced by Rb tumor There are several proangiogenic factors involved in retinal angiogenesis VEGF and its receptors are expresed in nonoverlapping manner (Prospero Ponce et al 2013)
- 5. High Risk Features LaminaCribosa Lamina Cribosa Optic Nerve involvement Choroidal involvement
- 6. High Risk Features Retina RPE Choroid Optic Nerve involvement Choroidal involvement •Choroidal invasion (‹3mm) + ANY optic nerve involvement
- 7. Angiogenesis complicates RB Neovascularizationof the iris (NVI) +/Peripheral synechia (PAS) Neovascular Glaucoma
- 8. Relevant background Neovascularization inretinoblastoma 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
- 9. Hypothesis HRF tumors willhave increased secretion of VEGF and its receptor VEGFR2
- 10. Objective To analyze theexpression 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.
- 11. HRF= 6 cases Non-HRF=6 cases
- 12. Methods Angiogenic Factors •VEGF •VEGFR2 •CD105 (endoglin) Differentiation Factors •Vimentin •GFAP Immunohistochemistry •Double Stain Qualitative measure •Grade 0to 3 Quantitative measure •Image J •Total area
- 13.
- 14. Differentiation Factors Vimentin GCL Intermediate filament associatedwith fibroblasts and activated Muller cells INL It can also be expressed by RPE, cilliary body epithelium, lens epithelium ONL
- 15. Differentiation Factors Glial fibrillaryacidic protein (GFAP) •Intermediate filament •Associated to: glial cells- Muller cells Up-regulated in CNS and retinal injury Damaged retina Normal retina
- 16.
- 17. Measurements: Image J Colordeconvolution Stain separation using Ruifrok and Johnston's method1 Vimentin VEGF [1] Ruifrok AC, Johnston DA. Quantification of histochemical staining by color deconvolution. Anal Quant Cytol Histol 23: 291-299, 2001
- 18. Measurements: Image J Analyzingparticles 8 bit Binary
- 19. Results ANGIOGENIC FACTORS ANDHRF IN RB
- 20. Figure 1. * 100 μm 100μm A B C VEGF 8000 VEGF+ Vimentin Vimentin p<0.05 8000 20000 HRF NON-HRF 6000 HRF Non-HRF 4000 p<0.05 10000 4000 E Location F Iri s Tu m a et in R Tu m et in R Tu m et in R Iri s Location Location or 0 Iri s 0 or 0 a 2000 or 5000 a 2000 D HRF Non-HRF p<0.05 6000 Pixel Area 15000 Pixel Area Pixel Area 100 μm
- 21. Figure 2. * * 100 μm A B GFAP C VEGF VEGF+GFAP 4000 800 2000 1000 0 F R F H on -H H on -H R R F R N N E N R F H 0 TumorType Tumor Type D 400 200 F 0 F 1000 600 R 2000 3000 on -H 3000 Pixel Area 1000 Pixel Area 5000 4000 Pixel Area 5000 Tumor Type F
- 22. Figure 3. * * * T * * 100 μm A B C VEGFR2 CD105 3000 VEGFR2+ CD105 1500 HRF Non-HRF p<0.05 1000 HRF Non-HRF 200 1000 Pixel Area 2000 250 HRF Non-HRF p<0.05 Pixel Area Pixel Area R 500 150 100 50 0 Location D E F Iri s Tu m or R et in a Iri s Tu m et in R Location or 0 a Iri s Tu m or R et in a 0
- 23.
- 24. Summary HRF tumor sexpress 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
- 25. Conclusions HRF tumors seemto 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
- 26. Acknowledgements Patricia Chevez-Barrios, MD RebeccaPenland Magdalena Arredondo Claudia M. Prospero, MD
Editor's Notes
- #5 Eyes with HRF show increased expression of VEGF and its receptor VEGFR-2 in both the tumor and the iris. CD105 expression(newly formed vessels) is elevated in tumors but is particularly higher in the iris of HRF eyes. Therefore in HRF eyes, treatments that target cells expressing angiogenic factors and/or the factors alone may be useful to prevent complications such as development of neovascular glaucoma.
- #21 VEGF expression in the retinawas lower in the non-HRF tumor compared to its expression in the retina of the non-HRF samples or to the HRF tumor. Its co-expression with the glial marker vimentin and the qualitative interpretation shows that a significant amount of VEGF is probably secreted by tumor cells. The VEGF Receptor 2 is highly expressed in the retina and the HRF tumor, unlike in the non-HRF tumor. The activated endothelial cell marker CD105 shows that the majority of angiogenesis takes place in the iris. Co-expression of VEGFR2 and CD105 seems to be predominant in the vessels of the tumor and the iris neovascular membranes. The neural stem cell marker SOX2 was more highly expressed in the perinecrotic areas of the tumor and the regions closest to the retina. It was also higher in HRFtumors compared to non-HRF tumors. Retina of what? THe HRF, non-HRF? The writing is very non-specific and thus hard to follow...
- #22 VEGF expression in the retinawas lower in the non-HRF tumor compared to its expression in the retina of the non-HRF samples or to the HRF tumor. Its co-expression with the glial marker vimentin and the qualitative interpretation shows that a significant amount of VEGF is probably secreted by tumor cells. The VEGF Receptor 2 is highly expressed in the retina and the HRF tumor, unlike in the non-HRF tumor. The activated endothelial cell marker CD105 shows that the majority of angiogenesis takes place in the iris. Co-expression of VEGFR2 and CD105 seems to be predominant in the vessels of the tumor and the iris neovascular membranes. The neural stem cell marker SOX2 was more highly expressed in the perinecrotic areas of the tumor and the regions closest to the retina. It was also higher in HRFtumors compared to non-HRF tumors. Retina of what? THe HRF, non-HRF? The writing is very non-specific and thus hard to follow...
- #24 VEGF expression in the retinawas lower in the non-HRF tumor compared to its expression in the retina of the non-HRF samples or to the HRF tumor. Its co-expression with the glial marker vimentin and the qualitative interpretation shows that a significant amount of VEGF is probably secreted by tumor cells. The VEGF Receptor 2 is highly expressed in the retina and the HRF tumor, unlike in the non-HRF tumor. The activated endothelial cell marker CD105 shows that the majority of angiogenesis takes place in the iris. Co-expression of VEGFR2 and CD105 seems to be predominant in the vessels of the tumor and the iris neovascular membranes. The neural stem cell marker SOX2 was more highly expressed in the perinecrotic areas of the tumor and the regions closest to the retina. It was also higher in HRFtumors compared to non-HRF tumors. Retina of what? THe HRF, non-HRF? The writing is very non-specific and thus hard to follow...tumors.
- #25 Conclusions and Relevance: Our results, for the first time, show that in HRF retinoblastoma eyes when compared with non-HRF eyes, there is overexpression of VEGFA and VEGFR2in the tumor cells, neovascularization of iris with increased amount of activated endothelial cells, and that overexpression of VEGFA is associated with an increase in SOX2-positive stem/progenitor cells in the tumor. The increasedexpression of angiogenic factors and concomitant increase in stem cells in the HRF tumors may partially explain the aggressiveness of these tumors. Further studies are needed to confirm this association. In non-HRF eyes the pattern of expression of VEGFA and VEGFR2 seems to be comparable to the recognized pattern of response of the non-neoplastic tissuesto injury . The complex interaction of pathways in these tumors, especially in HRF retinoblastoma, indicate that targeting vasculogenic tumor cells, genes, and signaling pathways may be necessary in anti-tumor vasculogenic and metastatic prevention drug development. ARE THERE CONTROLS?? I did not see them mentioned at any other point in the manuscript.