Aberrant RB gene expression in routinely processed, archival tumor tissues determined by three different anti‐RB antibodies

Abstract

The retinoblastoma (RB) susceptibility gene encodes a nuclear phosphoprotein which is likely involved in cell cycle control and cell differentiation. The RB protein is mutated or absent in a variety of human malignancies. Its role as a molecular marker for clinical tumor behavior is under extensive investigation. However, studies on the status of the RB protein in primary or metastatic tumors and their precursor lesions have been slowed by the lack of availability of a sensitive, reliable assay which allows examination of RB expression in selected cell populations within archival tissues. Thus far, meaningful immunohistochemical analysis of RB protein in formalin-fixed, paraffin-embedded specimens has been achieved only with the polyclonal antibody RB-WL-I. We now describe a method which produced excellent staining results in formalin-fixed, routinely processed tissues, using commercially available monoclonal antibodies (MAbs) in conjunction with an antigen-retrieval step. The resulting stains were superior to those on frozen sections and comparable to those obtained with RB-WL-I. Twelve of 51 random invasive bladder cancers (24%) had abnormal expression of the RB gene, as determined by immunohistochemistry. Smaller cohorts of breast, prostate and lung carcinomas had incidences of aberrant RB gene expression ranging from 9% to 24%. Since the staining method was widely applicable to essentially all formalin-fixed, archival tissues, it may expedite studies on the biological and clinical significance of altered RB expression in human neoplasia.

Retinoblastoma Expression in Thyroid Neoplasms

“…Second, Holm et al used a different Rb antibody clone that may bind to different epitopes in the target Rb antigen that might be associated with nonspecific binding to other nuclear antigens. Differences between Rb clones have been documented previously by Geradts et al (33) and confirm our personal observations in our referral service. Third, interlaboratory differences in the handling of tissues and quality control procedures might be responsible for the differences in the results.…”

“…We did not observe nonspecific cytoplasmic binding as reported previously (32)(33)(34). The cytoplasmic element of staining was eliminated by the addition of a 10-min incubation with pronase in the antigen retrieval procedure after microwaving.…”

“…The intensity of staining for P-Rb within some thyroid lesions varied from cell to cell in our study, also similar to previous reports (29,33,43). Despite this variability, the distinction between positive and negative cases was always easy to assess at the 10% cutoff level that we used.…”

“…We introduce a modified technique that does not have the drawback of high cytoplasmic background as documented in the few studies evaluating Rb protein expression in paraffin sections (32)(33)(34). We stratified thyroid lesions into the major histologic subtypes to detect any heterogeneity in Rb-1 expression that might influence the diagnostic utility of this technique or further elucidate the pathogenesis of thyroid neoplasia.…”

RB family members as predictive and prognostic factors in human cancer

“…The clinical importance of Rb function demands a reproducible Rb immunohistochemical assay to distinguish Rb þ from RbÀ tumor cells and many attempts have been made in this direction (Bianchi et al, 1994;Geradts et al, 1994). The prognostic relevance of pRb alterations in breast cancer was disclosed by two independent studies.…”