Increased expression of cellular retinol-binding protein 1 in laryngeal squamous cell carcinoma

Abstract

Purpose: To investigate the genomic alterations in larynx carcinomas (LaCa) tissues and its prognostics values in predicting survival.

Methods: To analyse the aberrations in the genome of LaCa patients, we used array comparative genomic hybridization in 19 human laryngeal tumour samples. DNA samples were also subjected to detect human papillomavirus (HPV) sequences by polymerase chain reaction (PCR). Copy number gain was confirmed by real-time PCR. The cellular retinol-binding protein 1 (CRBP-1) gene expression was also confirmed by immunohistochemistry assay on LaCa tissues. To identify prognostic feature, CRBP-1 gene gain was correlated to patient survival.

Results: The most common gains were detected for CRBP-1 and EGFR genes, while DNA lost in RAF-1 gene. Immunohistochemistry assay was revealed strong expression of CRBP1 protein in those cases with CRBP-1 gene gain. The CRBP-1 gene gain and its expression correlated significantly with survival (P = 0.003). Cox regression analysis indicated that CRBP-1 expression level was a factor of survival (P = 0.008). HPV sequences were detected in 42% of the samples, and did not show any relationship with specific gene alterations.

Conclusion: Our data shows that CRBP-1 gene gain can be determined by immunohistochemistry on routinely processed tissue specimens, and could support as a potential novel marker for long-term survival in laryngeal squamous cell carcinoma.

Fig. 1

Ideogram of gene alterations in laryngeal squamous cell carcinoma. The data were analysed and visualised by ISCN2 matrix. The lanes represent each sample, the row are each clone printed on the array starting from up to down from chromosome 1, 2, etc. The yellow strip represents the HPV-positive cases. L1, L2, etc, represent each larynx cancer sample. L1 and L2 were the benign lesions, the remaining were the invasive lesions. The keys are grouping the CRBP-1 gene gain samples

Fig. 2

DNA copy number quantitation of CRBP-1 gene in laryngeal tumours by using quantitative real-time PCR. CRBP-1 showed a significant increase in gene copy number above normal in L4, L9, L10 laryngeal cancer samples. For L5 sample no copy number gain was detected. Values above the cut-off line, being assigned as increased gene copy number compared with normal laryngeal epithelium. L5 tumour harboured normal CRBP-1 gene, in contrast L4, L9 and L10 presented CRBP-1 gene gain by array CGH

Fig. 3

CRBP1 protein immunodetection in the human larynx tissues. CRBP-1 expression was observed in the basal cell layer of the normal larynx tissue (a), as well as, the immunodetection in the transformed cells of larynx carcinoma tissue harbouring the CRBP-1 gene gain (L5 and L10). The brownish reaction was detected in the cellular cytoplasm (b, c). Some larynx carcinoma samples negative (L5 and L15) for CRBP-1 gene gain did not show any reaction (d, e). All tissue sections were haematoxylin counterstained. a, b, d, original amplification ×200; c, e, original amplification ×400

Fig. 4

Survival probabilities of larynx cancer patients with CRBP-1 or EGFR gene alterations. a CRBP-1 gene gain is significantly associated with survival probability (P = 0.003 at Kaplan–Meier analysis). b EGFR gene did not show any correlation with survival probability (P = 0.627)