Loss of heterozygosity for Rb locus and pRb immunostaining in laryngeal cancer: a clinicopathologic, molecular and immunohistochemical study.

Abstract

Several risk factors for the development of laryngeal cancer have been identified, such as smoking and alcohol consumption, but the molecular mechanisms related to the carcinogenesis in the larynx remain under investigation. In this context, deregulations of the cell-cycle-controling mechanisms, Rb-pathway in particular, have been suggested to be involved in the pathogenesis of laryngeal carcinoma. Our purpose was to investigate 13q14 LOH and the expression of Rb protein and their possible prognostic value in laryngeal cancer. The group of 67 patients with laryngeal cancer, surgically treated with minimum 5 years observation, was multi-variously analysed. LOH for Rb was investigated by PCR-based techniques using two microsatellite markers, D13S263 and D13S126, flanking the Rb locus. Amplification products from each polymorphism were fractionated by denaturing gel electrophoresis and detected by audioradiography. Immunohistochemical staining of paraffin specimens of laryngeal cancers was supervised by the use of monoclonal mouse antibodies IgG1 (Anti-Human Retinoblastoma Gene Product of Dako) in dilution of 1:50. Inactivation of Rb protein was assumed to represent the expression in < or =10% tumour cells. The results of each examined individual factor were compared with clinicopathologic features and the results were statistically transformed (Chi-square test with Yates' correction, Mann-Whitney test). The Kaplan and Meier model was used for overall and disease free survival curves. Only p value of less than 0,05 was considered significant. 13q14 LOH was detected in 7/67 (10,4%) of informative tumours. No correlations were found between Rb genetic alteration (LOH) and gender, age, TNM staging, histological differentiation, nodal and local recurrences (p>0.05). There was a strong association between the loss of Rb and supraglottic localisation of tumour in the larynx (p<0.01). By univariate analysis 13q14 LOH proved to be significantly related to the overall survival whereas it was not related to the quicker relapse (p=0.01, p>0.05 respectively). The genetic data were correlated with the expression of the Rb protein (p=0.001). All tumours with Rb-LOH were immunohistochemically Rb-negative. Inactivation of Rb protein was observed in 9/67 cases (13.49%) and was significantly correlated with the polymorphism of cancer cells, but not with the histological grading. We also found the correlation between reduction of Rb protein and the size of primary tumour (T) (p=0.03) and local recurrence (p=0.035). There was no significant dependence between the level of Rb protein and other histopathological and clinical features (p>0.05). To conclude, analysis of 13q14 LOH enables the assessment of biology of laryngeal cancer and it can be a prognostic factor in overall survival. Immunohistochemical analysis of Rb protein expression in neoplastic cells made it easier to evaluate the mechanisms of cancerogenesis in laryngeal cancer and is closely related to genetic alteration in Rb locus.