Cyclin D1, TP53 and nitrotyrosine expression in gastroesophageal reflux-induced esophagitis, Barrett's esophagus and esophageal adenocarcinoma

L Geldenhuys MD^a, Z Zheng MD^a, KA Gallant BSc^a, NM Vaninetti BSc^a, SC Evans BSc^a, PJ Veugelers PhD^b, DL Guernsey PhD^a, AG Casson FRCSC^c

^aDepartment of Pathology, ^bCommunity Health and Epidemiology, ^cSurgery; Dalhousie University, Halifax, Nova Scotia, Canada

AIM: To investigate the molecular pathogenesis of esophageal adenocarcinoma (EADC), we evaluated tissue distribution of cyclin D1, TP53, and nitrotyrosine (NTS), a stable reaction product of nitric oxide and a marker for cellular protein damage, in a well defined series of patients with gastroesophageal reflux-induced esophagitis, Barrett’s esophagus (BE) and EADC. METHODS: Esophageal tissues were obtained, with informed consent, from patients enrolled in a case-control study between 02/2001 and 02/2003, who underwent esophagogastroscopy and biopsy. Tissues comprised reflux-induced esophagitis (n=81), BE, characterized by intestinal metaplasia (n=130), and primary EADC, defined according to strict clinicopathologic criteria (n=57), each with matched histologically normal epithelia (internal control). Indirect immunoperoxidase assays were used to evaluate protein distribution in formalin-fixed, paraffin-embedded tissue sections using monoclonal antibodies for cyclin D1 (DCS-6, 1:200 dilution, Dako), TP53 (DO-7, 1:50, Dako), and NTS (HM-11, 1:50, Zymed). Stringent criteria were used to evaluate immunoreactivity, based on a composite score of intensity of immunoreactivity and percentage of immunopositive cells in tissue sections, as determined by two independent investigators. Statistical differences between groups were tested using Pearson’s chi-square test or Fisher’s exact test where appropriate. RESULTS: Expression of cyclin D1, TP53 and NTS was localized to the basal and parabasal layers in reflux-induced esophagitis, but was heterogenous throughout BE and EADC tissues. The presence of cell nuclear cyclin D1 immunopositivity varied between each tissue type, with overexpression in 31 (38%) esophagitis, 21 (16%) BE and 14 (25%) EADC. These differences were statistically significant (p=0.001). TP53 was overexpressed in 7 (9%) esophagitis, 16 (12%) BE and 31 (54%) EADC (p<0.001). Cytoplasmic NTS staining was seen in 18 (22%) esophagitis, 28 (22%) BE and 23 (40%) EADC (p=0.018). The combined expression of TP53 and NTS within the same tissue also varied significantly between reflux-induced esophagitis, BE and EADC (p<0.001). CONCLUSIONS: This study supports the hypothesis that chronic stress, possibly as a result of gastroesophageal reflux-induced esophagitis, may result in local overproduction of nitric oxide resulting in increased nitration of tyrosine residues in the distal esophageal mucosa, thus enhancing the rate of molecular alterations in oncogenes and tumor suppressor genes. This study further implicates alterations in cyclin D1 and TP53 as early molecular aberrations in the molecular pathogenesis of EADC.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 897 (Precursor lesions).