Basic Research
Copyright ©2006 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. May 21, 2006; 12(19): 3020-3025
Published online May 21, 2006. doi: 10.3748/wjg.v12.i19.3020
Image cytometric DNA analysis of mucosal biopsies in patients with primary achalasia
I Gockel, P Kämmerer, J Brieger, UR Heinrich, WJ Mann, F Bittinger, VF Eckardt, T Junginger
I Gockel, P Kämmerer, T Junginger, Department of General and Abdominal Surgery, Johannes Gutenberg-University, Mainz
J Brieger, UR Heinrich, WJ Mann, Laboratory of Tumor Biology, Department of Otolaryngology, Johannes Gutenberg-University, Mainz
F Bittinger, Institute of Pathology, Johannes Gutenberg-University, Mainz
VF Eckardt, Department of Gastroenterology, German Diagnostic Clinic, Wiesbaden
Co-first-authors: I Gockel, P Kämmerer
Correspondence to: Ines Gockel, MD, Department of General and Abdominal Surgery, Johannes Gutenberg University, Langenbeckstr. 1, D-55101 Mainz, Germany.
Telephone: +49-6131-177291 Fax: +49-6131-176630
Received: November 16, 2005
Revised: December 13, 2005
Accepted: December 22, 2005
Published online: May 21, 2006

AIM: To determine DNA aneuploidy in mucosal biopsies of achalasia patients for subsequent rapid diagnosis.

METHODS: Biopsies from the middle third of the esophagus were obtained in 15 patients with achalasia. Immunohistochemical staining was carried out with monoclonal antibodies MIB-1 for Ki67 and PAb 1801 for p53, in addition to the conventional histologic examination for dysplasia. Nuclei of fresh biopsy material were enzymatically and mechanically isolated, and the DNA content was determined with image cytometry after Feulgen staining. DNA grading of malignancy was assessed according to Boecking to determine the variability of DNA values noted around the normal diploid peak. Further indices measured included the aneuploid rate, and the 5c-, 7c- and 9c-exceeding rate.

RESULTS: The histological examination did not demonstrate dysplasia; while MIB-1 (basal) showed a positive reaction in 8/15 achalasia specimens, p53 was negative in all specimens. Image cytometric DNA analysis detected aneuploidy in 4/15 (26.7%) specimens. Samples from 15 patients with squamous cell carcinoma as well as specimens obtained exclusively 2 cm proximal to the tumor served as reference tests. All carcinomas (15/15) as well as 9 of the peritumoral samples (9/15) were aneuploid. The comparison of biopsies from achalasia patients with peritumoral and carcinoma specimens revealed statistically significant differences regarding the aneuploid rate (diploid: P < 0.0001; tetraploid: P = 0.001), grading of malignancy according to Boecking (P < 0.0001) and the 5c- (P < 0.0001), 7c- (P < 0.0001), and 9c- (P = 0.0001) exceeding rate with progredient DNA alterations in the respective order.

CONCLUSION: The finding that DNA aneuploidy was identified by image cytometry in esophageal specimens of patients with achalasia, which may be due to specific chromosomal alterations presenting as precancerous lesions in 27% of patients, leads us to conclude that image cytometry represents a valuable screening tool.

Keywords: Achalasia, Precancerous epithelial alterations, Esophageal carcinoma, Image cytometric DNA analysis, Proliferation marker Ki67, Tumor suppressor gene p53