Elsevier

Human Pathology

Volume 32, Issue 5, May 2001, Pages 537-544
Human Pathology

Original Contributions
Alterations of cell cycle regulators affecting the RB pathway in nonfamilial retinoblastoma*

https://doi.org/10.1053/hupa.2001.24325Get rights and content

Abstract

We undertook the present study to examine alterations affecting the RB pathway in the G1 checkpoint and to determine their potential clinical significance in children affected with nonfamilial retinoblastoma. Using immunohistochemistry, patterns of expression of pRB, p16/INK4A, and E2F1 were analyzed in tissue from a cohort of 86 well-characterized patients with nonfamilial retinoblastoma diagnosed at the “Instituto Nacional de Pediatria” in Mexico City. The relationship of these phenotypes to proliferative index was assessed by analysis of Ki67 antigen expression. pRB expression was found in 11 (13%) cases. Using a hypophosphorylated specific pRB antibody, we observed low levels of underphosphorylated pRB expression in only 1 of 9 evaluable positive cases. These data suggest that the detected pRB products were hyperphosphorylated and thus had decreased functional activity. Increased p16 nuclear expression was found in only 6 tumors. No tumors showed deletions or mobility shifts of the INK4A gene. Undetectable pRB levels were significantly associated with undetectable p16 expression (odds ratio, 10.8; 95% confidence interval, 1.4-81.3; P =.03). All tumors showed nuclear immunoreactivities for E2F1 and Ki67. Increased Ki67 proliferative index was associated with increased staining for E2F1 (r =.44; P =.008) and increasing clinical stage (P =.03). Among children with unilateral disease, the mean Ki67 proliferative index was significantly higher in children with advanced clinical disease (stages 3 and 4) (mean 81.25; SD 6.78) than in those with earlier stage disease (mean 69.50; SD 9.45) (P = 0.001). Among children with bilateral disease, however, the mean proliferative index was not significantly higher for children with advanced clinical stage. When examining all cases together, there was a significant trend toward increasing proliferative index with increasing clinical stage (P =.03). In unilateral tumors, we also found that presence of detectable pRB was associated with a lower percentage of cells expressing E2F1 (46.7% v 70.8%) (P = 0.05), whereas there was no association between presence of pRB and E2F1 among bilateral tumors. We have found that expression of some of the cell cycle markers examined varies according to laterality, suggesting underlying differences in the capacity for cell cycle regulation between these 2 forms of the disease. Differences in capacities for cell cycle regulation may account for some differences in clinical behavior. Thus, the inclusion of molecular markers may become useful adjuncts to clinicopathological staging and subsequent determination of therapy. HUM PATHOL 32:537-544. Copyright © 2001 by W.B. Saunders Company

Section snippets

Patient characteristics and tissues

A cohort of 86 patients with nonfamilial retinoblastoma was included in the study. Fresh and/or paraffin-embedded tissue was collected at the time of enucleation at the “Instituto Nacional de Pediatria” (INP) in Mexico City, Mexico. Children were excluded from the study if they were older than 5 years at diagnosis or had a positive family history of retinoblastoma. None of the children had received any local or systemic therapy before enucleation. Demographic data on the cohort are summarized

Results

Tissue from 86 children with sporadic retinoblastoma was examined by immunohistochemistry for markers of cell cycle progression related to the RB pathway. Detectable pRB in the nuclei of tumor cells was found in 11 of 86 (12.8%) cases (Fig 1).

. Photomicrographs illustrating the immunohistochemical staining patterns of (A, B) pRB (C,D) p16, (E,F) E2F-1, and (G,H) Ki67 expression. The photomicrographs A and B are of paraffin-embedded tissue sections; all others are of frozen tissue. (A) Complete

Discussion

We undertook the present study to examine patterns of expression of critical regulators of the RB pathway, including the phenotypes of pRB, p16, and E2F1 in tumors from a cohort of 86 well-characterized patients with nonfamilial retinoblastoma. We also analyzed the relationship between these phenotypes with Ki67 proliferative index and clinicopathologic parameters of poor outcome, including age at diagnosis, stage, and mortality. Our study shows significant relationships between lack of pRB and

Acknowledgements

The authors thank Dr Roberto Rivera Luna and the staff and patients of the Departments of Ophthalmology, Oncology, and Pathology at the Instituto Nacional de Pediatria, Mexico City, Mexico, for their support and assistance with this study.

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    *

    Supported by grants NCI-CA-47538 and NCI-CA-47179 (C.C.C.) and by the Bowen Brooks Foundation, New York Academy of Medicine (M.O.).

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