Contrast-Enhancement of the Anterior Eye Segment in Patients with Retinoblastoma: Correlation between Clinical, MR Imaging, and Histopathologic Findings

Discussion

Three previous studies draw attention to abnormal AES enhancement, which is present in a substantial number of MR images of eyes harboring retinoblastoma.^6,8,18 Authors of these studies correlated AES enhancement with clinical and histopathologic findings. De Graaf et al⁶ reported the clinical presence of IA, hyperemia, or inflammation as possible explanations for AES enhancement. Galluzzi et al¹⁸ published a series of 16 eyes and found a correlation between histopathologic optic nerve and/or choroid invasion and AES enhancement on MR imaging; however, no correlation between clinical or histopathologic presence of IA and AES enhancement could be detected. On the other hand, Brisse et al⁸ could not confirm this correlation between AES enhancement and optic nerve invasion in 10 eyes. To our knowledge, the present investigation is the most comprehensive study so far in the search for an explanation for variable AES enhancement in retinoblastoma, which confirmed the role of IA in vivo. Additionally, our study indicates that abnormal AES enhancement in retinoblastoma is also related to tumor volume and optic nerve invasion.

In healthy human eyes, the uveal tract enhances after intravenous administration of gadolinium contrast material, with the choroid and ciliary body enhancing slightly more than the iris. In the literature,^21,22 there is evidence for an anterior diffusional pathway that causes diffusion of proteins (and gadolinium) from the choroid into the AC. This diffusion goes from the ciliary body vessels, via the ciliary body stroma, into the root of the iris and finally into the AC. A gradual signal-intensity increase in the AC, as a result of diffusion of gadolinium into the AC is physiologic (anterior diffusional pathway). However, this physiologic diffusion is much slower than the enhancement described earlier. First, this enhancement becomes detectable at the AC angle ∼12 minutes after injection and moves slightly toward the AC center (∼30 minutes), reaching a peak in ∼74 minutes.^21,22 However, in our patients, the MR images were obtained shortly after contrast material injection and showed an early signal-intensity increase in the AC of 69% of affected eyes. This early enhancement has been attributed to leakage of contrast material into the extravascular space through regions of BOB disruption.^18,23 In newly formed capillaries during IA, the BOB is poorly developed from the outset and becomes further compromised under hypoxic conditions, with VEGF as an important mediator.

Tumor hypoxia initiates compensatory mechanisms that prevent cell death, including up-regulation of proangiogenic factors, such as VEGF, which are highly expressed in areas of novel vasculature in eyes containing retinoblastoma.^12–14 We found a positive iris VEGF staining in most of the eyes, being more frequent at the anterior iris surface than in the iris stroma. However, no significant correlation between iris VEGF expression and AES enhancement could be detected. This could be explained by the fact that the study population did not include very early stages of retinoblastoma, because these patients are currently treated with eye-preserving strategies. A significant percentage of retinoblastoma eyes succumbing to enucleation demonstrate a high level of VEGF expression.^12–14 Different angiogenic mechanisms might be differentially important in various stages of tumor progression, and some data suggest that VEGF may be especially important in the initial stages.²⁴ Two human tyrosine kinases, Flt-1 and KDR, have been shown to function as specific receptors for VEGF-related angiogenesis.²⁵ In the present investigation, only Flt-1 was analyzed because the expression of Flt-1, but not that of KDR, is directly up-regulated by hypoxia.²⁶ Flt-1 expression is up-regulated by hypoxia via a HIF-1-dependent mechanism, and the expression is also potentiated by VEGF.²⁶ Hypoxia-induced up-regulation of Flt-1 could provide a mechanism by which local sensitivity to VEGF is enhanced, with increased angiogenesis and vascular permeability as a consequence. In the iris, a positive Flt-1 staining at the level of the iris stroma yielded a significant correlation with AES enhancement, which is a bit surprising because a receptor is expected to bind to cells. We assume that cellular protrusions are the basis for this staining, though staining of a soluble form of Flt-1 in the stroma cannot be excluded.

The degree of AES enhancement correlated significantly with vascular proliferation on the inner as well as on the outer surface of the anatomic anterior iris border.

The mean number of iris vessels located directly beneath the anterior surface was higher in the abnormal AES enhancement groups than in the group with normal AES enhancement. Furthermore, increased AES enhancement showed a similar relationship with the histopathologic stage of IA on the outer surface. This relationship showed a borderline significance for the entire study group but reached statistical significance with immunohistochemical analysis, probably because CD31 antibody staining is supposed to be more sensitive for the detection of vessels than H&E staining,²⁷ providing a more accurate discrimination of IA subgroups.

Clinically, retinoblastoma-associated IA, for which enucleation is usually performed, especially in patients with unilateral disease, is considered a bad prognostic factor for globe salvage and vision. However, routine ophthalmoscopy has a low sensitivity for detecting early or subtle stages of IA, a finding confirmed in our study (2 eyes with IA detected clinically, compared with 14 eyes detected histopathologically).^18,28 The degree of AES enhancement might be a valuable diagnostic parameter in the choice between enucleation and conservative treatment strategies. Normal AES enhancement seems to be an indication for considering globe salvage rather than enucleation. Abnormal AES enhancement, especially in eyes showing a strong enhancement, represents a sign of IA and could be considered an additional argument in favor of enucleation.

Tumors with increased angiogenesis have a greater potential for tumor progression, invasion of surrounding structures, and metastasis.^9,10 In our study, patients with an advanced stage of disease, indicated by a large tumor volume or the presence of optic nerve invasion, showed a higher degree of AES enhancement, compared with those with early-stage disease. All eyes containing large tumors showed some degree of abnormal AES enhancement, whereas this finding was uncommon in very small and small tumors.

Optic nerve invasion beyond the lamina cribrosa is one of the most important risk factors for extraocular relapse; and as observed before,¹⁸ an abnormal AES enhancement should prompt a radiologist to be suspicious of optic nerve invasion. In the absence of abnormal AES enhancement, there was no tumor invasion of the optic nerve to a postlaminar degree.

Hypoxic conditions in retinoblastoma, secondary to the overgrowth of its vasculature, cause tumor necrosis and enhance angiogenic activity in the posterior segment as well as the AES, with the induction of IA and BOB disruption as a consequence. Pe'er et al¹² found a strong association between higher stages of IA in eyes containing tumors with greater amounts of necrosis. Our study material showed no differences between the amounts of necrosis in the various AES enhancement groups. Theoretically, retinal hypoxia secondary to retinal detachment may also cause BOB disruption and IA. Although retinal detachment was a frequent finding in our study population (74%), with a substantial amount of total detachments, we failed to demonstrate a significant association with AES enhancement.

A shallow AC was clinically demonstrated in 7 patients, whereas evaluation of MR imaging depicted it in 12 patients. In univariate statistical analysis, AES enhancement correlated statistically significantly only with a shallow AC as detected on MR imaging. Possibly, MR imaging is more sensitive in detecting minor anterior displacement of the lens-iris diaphragm, because this parameter can be judged on transverse cross-sectional images.

Inflammation and angiogenesis are linked processes, regulated by inflammatory cytokines and VEGF.^25,29 An increased uveal enhancement, including the AES, was reported as a sign of choroidal inflammation.⁶ Surprisingly, we found a significant inverse correlation between AES enhancement and choroidal inflammation. Angiogenesis and inflammation are multistep processes influenced by an imbalance between positive and negative regulators produced by both tumor cells and normal cells. Regulatory proteins other than VEGF, which show mutually opposing functions of stimulating angiogenesis and inhibiting inflammation, might play an additional role.^30,31 Furthermore, the AES is not always involved in posterior uveitis; therefore, differences in the extent and severity of uveal contrast enhancement depend on the severity of BOB breakdown in the AES.³²

Limitations to this study are primarily related to the retrospective design. Currently, the use of orbital surface coils is recommended for retinoblastoma imaging to increase spatial resolution and signal–to-noise ratio.^5,7 Future research on retinoblastoma-associated BOB impairment and quantification of leakage across the endothelium should be performed with dynamic contrast-enhanced MR imaging, which is widely used for tumor imaging.³³ Contrast-enhanced MR imaging seems to be a useful tool for noninvasive detection of retinoblastoma-related angiogenesis and could define a subgroup of patients who would potentially profit from vascular targeting drugs in the future.^11,34

In conclusion, the degree of abnormal AES enhancement on MR imaging in retinoblastoma reflects angiogenesis in the iris. AES enhancement is also a hallmark of advanced retinoblastoma because its degree correlates with tumor volume and optic nerve invasion.