Retinoblastoma-related cataract

Abstract

Purpose

The occurrence of cataract in an eye with retinoblastoma is rare. The aim of this study was to analyze the histopathology and the expression of transforming growth factor-β (TGF-β) in retinoblastoma with and without cataractous changes.

Methods

Twenty patients with unilateral retinoblastoma underwent enucleation. None of these patients had received preoperative chemo/radiotherapy. Formalin-fixed, paraffin-embedded tissue sections were examined histologically for the presence of Morgagnian globules or liquefaction of lens fibers; TGF-β was immunolocalized using anti-TGF-β antibody.

Results

Two globes each showed several Morgagnian globules and liquefaction of the lens fibers, representing cataractous changes. One patient had posterior subcapsular cataract, the other, anterior polar cataract. In both cases, prominent cytoplasmic immunoreactivity for TGF-β was detected in retinoblastoma cells. In contrast, three patients showed histologic evidence of minor cataractous changes. The globes with either minor or no cataractous changes revealed minimal to no expression for TGF-β.

Conclusions

These results suggest that TGF-β produced by retinoblastoma cells may induce cataract formation.

Clinical Relevance

The growth factors produced by retinoblastoma cells may lead to associated pathologies in the ocular structures such as cataracts. This study implies that when a child presents with a unilateral cataract, retinoblastoma should be excluded as primary diagnosis.

Retinoblastoma is a malignant ocular tumor that occurs in childhood. Radiation-induced cataract is one complication in the treatment of retinoblastoma.¹ In contrast, the simultaneous occurrence of cataract and retinoblastoma is rare,² with a frequency of less than 1 %.³

The cytokine transforming growth factor-β (TGF-β) plays an important role in epithelial-mesenchymal transition of lens epithelial cells and in the accumulation of extracellular matrix, which subsequently induces cataract formation.^4,5 Herein, we used immunohistochemical methods to examine the expression of TGF-β in enucleated eyes with retinoblastoma, with and without cataract.

Materials and Methods

Operative Specimens

The Institutional Review Board of the University of Southern California approved our use of human specimens. All procedures conformed to the Declaration of Helsinki for research involving human subjects. We collected 20 cases with retinoblastoma determined by enucleation using the medical records at Doheny Eye Institute from January 2006 through October 2007. All eyeballs had been fixed in 4% paraformaldehyde soon after enucleation. Formalin-fixed, paraffin-embedded tissue sections were processed for routine hematoxylin & eosin staining and periodic acid-Schiff (PAS) staining. Necrotic areas in retinoblastoma specimens were measured using a SPOT RT-SE™ digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI), and the data is presented as size and percentage of the tumor size.

Immunohistochemistry

Twenty cases were examined by immunohistochemistry to determine whether TGF-β expression is characteristic in retinoblastoma in eyes with concomitant posterior subcapsular or anterior polar cataract. The slides were dewaxed, rehydrated, and rinsed in phosphate-buffered saline, twice for 10 min. As a pretreatment, microwave-based antigen retrieval was performed in 10 mM citrate buffer (pH 6.0). These slides were incubated with 3% hydrogen peroxide for 10 min, then with normal goat serum for 30 min. Next, sections were incubated with anti-mouse TGF-β monoclonal antibody (dilution 1:50; 1D11, R&D Systems, Minneapolis, MN) at room temperature for 2 hr. Binding of the primary antibody was localized with the FITC-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min. Negative controls were incubated with FITC-conjugated mouse IgG without treatment of the primary antibody. Uveal melanoma tissues served as a positive control for TGF-β immunohistochemistry as previously reported.⁶ Sections were mounted in mounting medium including 4′6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Slides were examined using a Zeiss LSM510 (Zeiss, Thornwood, NY) confocal microscope. In the non-necrotic viable tumor tissues, 300 tumor cells were counted from 3 or 4 fields of the same slide for each specimen. The positive rate of immunopositive cells was shown as a percentage in viable tumor cells (%).

Statistical Analysis

Data is presented as mean ± standard deviation. Statistical evaluations were performed using the student's t-test. Accepted level of significance for all tests was P<0.05.

Results

Definite Cataract Associated with Retinoblastoma in Two Cases

Table 1 summarizes the clinicopathologic profile revealed by this study. Two retinoblastoma cases showed histologic evidence of cataract. (Cases 1 and 2, Table 1). One 2-year-old girl (Case 1) and one 1-year-old girl (Case 2) diagnosed with retinoblastoma and neovascular glaucoma in the left and right eyes, respectively, underwent enucleation. No systemic anomalies or metabolic disorders were observed. None of the patients had cataract in the unaffected eye. In each of the affected eyes, the anterior chamber was shallow and the angle was closed. The iris revealed rubeosis iridis. Marked neovascularization of the iris was present with ectropion uvea. The vitreous cavity was partially obliterated by a grayish white mass arising from the retina. In Case 1, the lens showed significant cataractous changes, forming Morgagnian globules within the entire cortex with the exception of the anterior portion (Figure 1A, B). Posteriorly, wide liquefaction of the lens fibers was seen (Figure 1B, C) while the nucleus remained intact. The posterior lens capsule was thickened where lens epithelial cells migrated (Figure 1C). In Case 2, liquefaction was present beneath the anterior lens capsule (Figure 1D). No cataractous changes were observed in the posterior side of the lens. Examination of the tumors revealed undifferentiated retinoblastoma (Figure 2A) with a necrotic area and multiple foci of calcification in both cases. The retina adjacent to the tumor tissue was detached. Retinoblastoma cells invaded the vitreous cavity and were present in the vicinity of the posterior capsule (Figure 1C, arrow). Microscopic rupture of the lens capsule was not detected in either case.

Table 1.

Clinicopathological profiles in patients with retinoblastoma examined in this study.

No.	Age (year)	Gender	Eye	Lens	Morgagnian globules	Liquefaction	Tumor size (mm)	Diff.	NVG	Choroidal invasion	ON head invasion	TGF-β -positive cells (%)
1	2	F	L	PSC	++	++	12 × 9	Un	+	+	-	80
2	1	F	R	APC	+	+	12 × 10	Un	+	-	+	80
3	1	M	L	minor	+	-	16 × 11	Un	+	+	+	10>
4	2	M	L	minor	+	-	14 × 14	Un	-	+	+	10>
5	3	M	L	minor	-	+	14 × 13	Un	-	-	+	10>
6	1	F	R	-	-	-	16 × 12	Un	-	-	-	10>
7	3	M	R	-	-	-	7 × 5	Un	-	-	-	10>
8	1	F	L	-	-	-	6 × 2	Well	-	-	+	10>
9	0.5	F	L	-	-	-	12 × 9	Well	-	-	-	10>
10	2	F	L	-	-	-	17 × 13	Un	+	-	-	20
11	1	F	L	-	-	-	14 × 10	Well	+	+	-	10>
12	2	M	R	-	-	-	13 × 10	Well	-	-	-	10>

F, female; M, male; L, left; R, right; PSC, posterior subcapsular cataract; APC, anterior polar cataract; Diff, differentiation; Un, undifferentiated; NVG, neovascular glaucoma; ON, optic nerve; TGF, transforming growth factor.

Figure 1.

Hematoxylin & eosin staining (A, B, D-E), and periodic-acid Schiff (PAS) staining (C) in the lens of retinoblastoma cases. There are no apparent changes in the anterior capsule and cortex of the lens in Case 1 (A). The lens reveals significant cataractous changes forming Morgagnian globules within the posterior cortex (B, black arrows). Lens epithelial cells migrate beneath the posterior capsule (B). There is liquefaction of the lens fibers (B, blue arrow). PAS-positive posterior lens capsule is thickened with wide liquefied necrosis (c). Retinoblastoma cells in the vitreous cavity exist in the vicinity of the posterior capsule (C, arrow). There is liquefaction beneath the anterior lens capsule (Case 2) (D). Several Morgagnian globules exist near the posterior capsule (Case 3) (E). Focal liquefied necrosis is observed beneath the posterior capsule (Case 5) (F).

Figure 2.

Hematoxylin & eosin staining (A, C), and immunodetection for transforming growth factor beta (TGF-β) (B, D) in retinoblastoma (A, B) and the detached retina (C, D) adjacent to the tumor. Tumor cells diffusely proliferate without forming Flexner-Wintersteiner rosettes, representing undifferentiated retinoblastoma in Case 1 (A). Inner nuclear layer gets thin in the detached retina in conjunction with retinoblastoma tissue (C). Cytoplasmic immunoreactivity for TGF-β is observed in retinoblastoma cells (B), but not in the detached retina (D). The bar = 50μm.

Minor Cataractous Changes in Three Cases with Retinoblastoma

Three cases showed histopathologic evidence that could be classified as minor cataractous changes (Cases 3-5, Table 1). Several Morgagnian globules existed near the posterior capsule (Figure 1E). The number of Morgagnian globules was low, and the extent of the lens change was small in this group compared to that in the definite group. Liquefaction was observed beneath the posterior capsule (Figure 1F), but the area of liquefied change was limited in this group.

Immunodetection for TGF-β in Retinoblastoma Tissues

The number of TGF-β-immunopositive tumor cells was markedly high (80%, Table 1) in two cases (Cases 1 and 2) showing association of cataract with the retinoblastoma. Immunohistochemical analysis demonstrated cytoplasmic immunoreactivity for TGF-β in a majority of the tumor cells, including those in the vitreous (Figure 2A, B). In contrast, no TGF-β was expressed in the detached retina adjacent to the tumor (Figure 2C, D). In comparison to the findings seen in the definite cataract group, the TGF-β-positive rate was low in retinoblastoma cases representing minor cataractous changes and in those cases with no cataractous changes (Table 1).

Association Between Tumor Size, Necrosis and Cataractous Change

In this study, statistical analysis was performed between retinoblastoma cases with cataractous (N=5, Case 1-5) and without cataractous changes (N=15, Case 6-20). The tumor size, measured by multiplication of anterior-posterior dimension and horizontal dimension, was 156.4 ± 39.6 mm² in those cases with cataractous changes and 116.8 ± 65.5 mm² in those without cataractous changes (P=0.22). Necrotic area was 83.8 ± 40.9 mm² (50.8 ± 15.5%) in cases with cataractous changes and 55.1 ± 35.6 mm² (44.2 ± 14.8 %) in those without cataractous changes (P=0.06 in size, P=0.45 in percentage).

Discussion

The two conditions, retinoblastoma and congenital cataract, are believed to be unrelated²; however, anterior polar cataract has been reported to occur in association with retinoblastoma. ⁷ Because it is very unlikely that congenital cataracts of any type are related to retinoblastoma, ² the presence of the cataracts found in the series is unusual. In the present study, two patients with retinoblastoma showed the presence of definite cataracts (Cases 1 and 2) in eyes containing retinoblastoma. Case 1 had a posterior subcapsular cataract intermingled with Morgagnian globules in the left eye, implying that the histological findings of the cataract are typical.

It is likely that retinoblastoma cells upregulate vascular endothelial growth factor, which causes neovascularization of the iris.⁸ This indicates that retinoblastoma cells can produce bioactive growth factors. Hales et al.⁴ demonstrated that intravitreal TGF-β injection induced posterior and anterior subcapsular cataractous changes in vivo. In retinoblastoma cases with cataract (Cases 1 and 2), immunoreactivity for TGF-β was detected in tumor cells, including cells floating in the vitreous, whereas the detached retina in conjunction with the tumor tissue showed no TGF- β expression. These results indicate that TGF-β was upregulated in retinoblastoma tissue in these cases. Moreover, the immunohistochemical results in retinoblastoma cases examined here revealed that the number of TGF-β-positive tumor cells was low in almost all cases without cataractous changes. Taken together, these findings indicate that TGF-β produced by retinoblastoma cells may induce cataract formation, suggesting that the two conditions might be related.

This study also showed that seven of the 20 eyes had neovascular glaucoma and that three of the seven had increased expression of TGF-β with positive neoplastic cells ranging from 20% to 80% (Table 1). It was demonstrated that TGF-β concentrations were high in the aqueous humor of patients with open angle glaucoma and neovascular glaucoma secondary to other pathologies.^9,10 Indeed, TGF-β is a multifunctional growth factor involved in various ocular changes.¹¹ These results suggest that TGF-β produced by tumor cells may play a role not only in the pathogenesis of cataract development, but also in neovascular glaucoma associated with retinoblastoma.

As shown in table 1, the three patients with retinoblastoma and only minimal cataractous changes (Case 3-5) demonstrated no upregulation of TGF-β. The pathogenesis of minor cataractous changes in eyes containing retinoblastoma is unclear from the current study. Size of the tumor and extent of necrosis did not correlate with the lens changes when comparing eyes with and without cataractous changes, since there was no statistically significant difference in size of the tumor (P= 0.22) and extent of necrosis (P=0.06 in size, P=0.45 in percentage). These results suggest that the extent of tumor growth and necrosis in retinoblastoma may not be associated with pathogenesis of cataractous changes. However, expression of TGF-β may play a role in the development of the lens changes with typical histologic features of well-developed cataract.