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

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

Figure 2.
Hematoxylin-eosin staining and immunodetection for transforming growth factor β (TGF-β) in retinoblastoma and the detached retina adjacent to the tumor. A, Tumor cells diffusely proliferate without forming Flexner-Wintersteiner rosettes, representing undifferentiated retinoblastoma in case 1. Cytoplasmic immunoreactivity for TGF-β is observed in retinoblastoma cells (B) but not in the detached retina (D). C, Inner nuclear layer gets thin in the detached retina in conjunction with retinoblastoma tissue.

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

Table. 
Clinicopathologic Characteristics of Retinoblastoma Cases
Clinicopathologic Characteristics of Retinoblastoma Cases
1.
Brooks  HL  JrMeyer  DShields  JABalas  AGNelson  LBFontanesi  J Removal of radiation-induced cataracts in patients treated for retinoblastoma. Arch Ophthalmol 1990;108 (12) 1701- 1708
PubMedArticle
2.
Brown  GCShields  JAOglesby  RB Anterior polar cataracts associated with bilateral retinoblastoma. Am J Ophthalmol 1979;87 (3) 276
PubMed
3.
Balasubramanya  RPushker  NBajaj  MSGhose  SKashyap  SRani  A Atypical presentations of retinoblastoma. J Pediatr Ophthalmol Strabismus 2004;41 (1) 18- 24
PubMed
4.
Hales  AMChamberlain  CGDreher  BMcAvoy  JW Intravitreal injection of TGFβ induces cataract in rats. Invest Ophthalmol Vis Sci 1999;40 (13) 3231- 3236
PubMed
5.
Schulz  MWChamberlain  CGMcAvoy  JW Inhibition of transforming growth factor-beta-induced cataractous changes in lens explants by ocular media and alpha 2-macroglobulin. Invest Ophthalmol Vis Sci 1996;37 (8) 1509- 1519
PubMed
6.
Esser  PGrisanti  SBartz-Schmidt  K TGF-β in uveal melanoma. Microsc Res Tech 2001;52 (4) 396- 400
PubMedArticle
7.
Amaya  LTaylor  DRussell-Eggitt  INischal  KKLengyel  D The morphology and natural history of childhood cataracts. Surv Ophthalmol 2003;48 (2) 125- 144
PubMedArticle
8.
Pe'er  JNeufeld  MBaras  MGnessin  HItin  AKeshet  E Rubeosis iridis in retinoblastoma: histologic findings and the possible role of vascular endothelial growth factor in its induction. Ophthalmology 1997;104 (8) 1251- 1258
PubMedArticle
9.
Yu  XBSun  XHDahan  E  et al.  Increased levels of transforming growth factor-beta1 and -beta2 in the aqueous humor of patients with neovascular glaucoma. Ophthalmic Surg Lasers Imaging 2007;38 (1) 6- 14
PubMed
10.
Yamamoto  NItonaga  KMarunouchi  TMajima  K Concentration of transforming growth factor β2 in aqueous humor. Ophthalmic Res 2005;37 (1) 29- 33
PubMedArticle
11.
Saika  S TGFβ pathobiology in the eye. Lab Invest 2006;86 (2) 106- 115
PubMedArticle
Laboratory Sciences
November 10, 2008

Transforming Growth Factor β in Retinoblastoma-Related Cataract

Author Affiliations

Author Affiliations: Doheny Eye Institute (Drs Kase, Parikh, Youssef, Murphree, and Rao), Childrens Hospital Los Angeles (Drs Youssef and Murphree), and Department of Ophthalmology, Keck School of Medicine, University of Southern California (Drs Youssef, Murphree, and Rao), Los Angeles, California.

Arch Ophthalmol. 2008;126(11):1539-1542. doi:10.1001/archopht.126.11.1539
Abstract

Objective  To analyze the histopathology and 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 chemotherapy or 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 an anti-TGF-β antibody.

Results  Two globes 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, 3 patients showed histologic evidence of minor cataractous changes. The globes with either minor or no cataractous changes revealed minimal to no expression of 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, such as cataracts, in the ocular structures. This study implies that when a child presents with a unilateral cataract, retinoblastoma should be excluded as the primary diagnosis.

Retinoblastoma is a malignant ocular tumor that occurs in childhood. Radiation-induced cataract is 1 complication of 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 the 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.

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 specimens of eyes with retinoblastoma determined by enucleation using medical records at the Doheny Eye Institute from January 2006 through October 2007. All eyeballs had been fixed in paraformaldehyde, 4%, soon after enucleation. Formalin-fixed, paraffin-embedded tissue sections were processed for routine hematoxylin-eosin and periodic acid–Schiff staining. Necrotic areas in retinoblastoma specimens were measured using a SPOT RT-SE digital camera (Diagnostic Instruments Inc, Sterling Heights, Michigan).

IMMUNOHISTOCHEMISTRY

We examined 20 cases using 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 twice in phosphate-buffered saline for 10 minutes. As a pretreatment, microwave-based antigen retrieval was performed in 10mM citrate buffer (pH 6.0). These slides were incubated with hydrogen peroxide, 3%, for 10 minutes and then with normal goat serum for 30 minutes. Next, sections were incubated with anti-mouse TGF-β monoclonal antibody (dilution 1:50; 1D11, R&D Systems, Minneapolis, Minnesota) at room temperature for 2 hours. Binding of the primary antibody was localized with the fluorescein isothiocyanate–conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania) for 30 minutes. Negative controls were incubated with fluorescein isothiocyanate–conjugated mouse IgG without treatment of the primary antibody. Uveal melanoma tissues served as positive controls for TGF-β immunohistochemistry as previously reported.6 Sections were mounted in mounting medium, which included 46-diamidino-2-phenylindole (Vector Laboratories, Burlingame, California). Slides were examined using a Zeiss LSM510 confocal microscope (Zeiss, Thornwood, New York). In the non-necrotic viable tumor tissues, 300 tumor cells were counted from 3 or 4 fields of the same slide for each specimen.

STATISTICAL ANALYSIS

Statistical evaluations were performed using the t test. Significance for all tests was P < .05.

RESULTS
DEFINITE CATARACT ASSOCIATED WITH RETINOBLASTOMA IN 2 CASES

The Table summarizes the clinicopathologic profiles that were revealed in this study. Two retinoblastoma cases showed histologic evidence of cataract (cases 1 and 2). One 2-year-old girl (case 1) and one 1-year-old girl (case 2) with both diagnosed retinoblastoma and neovascular glaucoma in the left and right eyes, respectively, underwent enucleation. No systemic anomalies or metabolic disorders were observed. Neither of the patients had cataracts in the unaffected eyes. 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 and B). Posteriorly, wide liquefaction of the lens fibers was seen (Figure 1B and 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). Microscopic rupture of the lens capsule was not detected in either case.

MINOR CATARACTOUS CHANGES IN 3 CASES OF RETINOBLASTOMA

Three cases showed histopathologic evidence that could be classified as minor cataractous changes (cases 3-5). 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 with 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%) in 2 cases (cases 1 and 2), showing association of cataract with the retinoblastoma. Immunohistochemical analysis demonstrated cytoplasmic immunoreactivity for TGF-β in most of the tumor cells, including those in the vitreous cavity (Figure 2A and B). In contrast, no TGF-β was expressed in the detached retina adjacent to the tumor (Figure 2C and D). Compared with the findings seen in the definite cataract group, the rate of cases positive for TGF-β was low in those with retinoblastoma, representing minor cataractous changes, and in those with no cataractous changes (Table).

ASSOCIATION BETWEEN TUMOR SIZE, NECROSIS, AND CATARACTOUS CHANGE

In this study, statistical analysis was performed between retinoblastoma cases with (n = 5, cases 1-5) and without (n = 15, cases 6-20) cataractous changes. The mean tumor size, calculated by multiplying the anterior-posterior and horizontal dimensions, was 156.4 mm2 (standard deviation [SD], 39.6 mm2) in those with cataractous changes and 116.8 mm2 (SD, 65.5 mm2) in those without cataractous changes (P = .22). The mean necrotic area was 83.8 mm2 (SD, 40.9 mm2) (mean rate of immunopositive cells, 50.8% [SD, 15.5%]) in cases with cataractous changes and 55.1 mm2 (SD, 35.6 mm2) (mean rate of immunopositive cells, 44.2% [SD, 14.8%]) in those without cataractous changes (P = .06 in size, P = .45 in percentage).

COMMENT

These 2 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 this series is unusual. In the present study, 2 patients showed the presence of definite cataracts (cases 1 and 2) in their eyes that contained retinoblastoma. Case 1 had a posterior subcapsular cataract intermingled with morgagnian globules in her 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 al4 demonstrated that an 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 cavity, 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 the retinoblastoma cases we examined 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 2 conditions are related.

Seven of the 20 eyes had neovascular glaucoma, of which 3 had increased expression of TGF-β with positive neoplastic cells ranging from 20% to 80% (Table). It was demonstrated that TGF-β concentrations were high in the aqueous humor of patients with open-angle 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 the Table, the 3 patients with retinoblastoma and only minimal cataractous changes (cases 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, as there was no statistically significant difference in size of the tumor (P = .22) and extent of necrosis (P = .06 in size, P = .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.

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Article Information

Correspondence: Narsing A. Rao, MD, Doheny Eye Institute, 1450 San Pablo St, Room DRVC 211, Los Angeles, CA 90033 (nrao@usc.edu).

Submitted for Publication: December 17, 2007; final revision received March 24, 2008; accepted April 25, 2008.

Financial Disclosure: None reported.

Funding/Support: This study was supported by grant EY03040 from the National Institutes of Health.

References
1.
Brooks  HL  JrMeyer  DShields  JABalas  AGNelson  LBFontanesi  J Removal of radiation-induced cataracts in patients treated for retinoblastoma. Arch Ophthalmol 1990;108 (12) 1701- 1708
PubMedArticle
2.
Brown  GCShields  JAOglesby  RB Anterior polar cataracts associated with bilateral retinoblastoma. Am J Ophthalmol 1979;87 (3) 276
PubMed
3.
Balasubramanya  RPushker  NBajaj  MSGhose  SKashyap  SRani  A Atypical presentations of retinoblastoma. J Pediatr Ophthalmol Strabismus 2004;41 (1) 18- 24
PubMed
4.
Hales  AMChamberlain  CGDreher  BMcAvoy  JW Intravitreal injection of TGFβ induces cataract in rats. Invest Ophthalmol Vis Sci 1999;40 (13) 3231- 3236
PubMed
5.
Schulz  MWChamberlain  CGMcAvoy  JW Inhibition of transforming growth factor-beta-induced cataractous changes in lens explants by ocular media and alpha 2-macroglobulin. Invest Ophthalmol Vis Sci 1996;37 (8) 1509- 1519
PubMed
6.
Esser  PGrisanti  SBartz-Schmidt  K TGF-β in uveal melanoma. Microsc Res Tech 2001;52 (4) 396- 400
PubMedArticle
7.
Amaya  LTaylor  DRussell-Eggitt  INischal  KKLengyel  D The morphology and natural history of childhood cataracts. Surv Ophthalmol 2003;48 (2) 125- 144
PubMedArticle
8.
Pe'er  JNeufeld  MBaras  MGnessin  HItin  AKeshet  E Rubeosis iridis in retinoblastoma: histologic findings and the possible role of vascular endothelial growth factor in its induction. Ophthalmology 1997;104 (8) 1251- 1258
PubMedArticle
9.
Yu  XBSun  XHDahan  E  et al.  Increased levels of transforming growth factor-beta1 and -beta2 in the aqueous humor of patients with neovascular glaucoma. Ophthalmic Surg Lasers Imaging 2007;38 (1) 6- 14
PubMed
10.
Yamamoto  NItonaga  KMarunouchi  TMajima  K Concentration of transforming growth factor β2 in aqueous humor. Ophthalmic Res 2005;37 (1) 29- 33
PubMedArticle
11.
Saika  S TGFβ pathobiology in the eye. Lab Invest 2006;86 (2) 106- 115
PubMedArticle
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