Development of a Flow Cytometric Method to Determine DNA Ploidy of Oesophageal Cancer Cells Obtained by Forceps Biopsy Samples During Oesophago-Gastro-Duodenoscopy

Abstract

Background: The DNA content of oesophageal tumour cells is a prognostic factor in untreated patients. To investigate whether DNA ploidy is useful to select patients for neoadjuvant therapy it is of interest to develop a method allowing reliable flow cytometric analysis of the DNA content of tumour cells obtained by forceps biopsy during endoscopy before start of therapy. Methods: Freshly frozen forceps biopsy samples from 30 patients with oesophageal cancer were disaggregated. DNA was stained with propidium iodide and ploidy was determined by flow cytometry. To enhance sensitivity epithelial cells were simultaneously labelled with anti-cytokeratin antibodies. Results were compared with image analysis. To evaluate the sampling error, parallel measurements were done in 10 patients by image analysis on forceps biopsies obtained during endoscopy before surgery and on the resected tumour. Results: The sensitivity to detect aneuploidy was lower for standard flow cytometry than for image analysis (13 versus 33%). The overall sensitivities were identical using a double labelling technique with additional cytokeratin-staining of the epithelial cells, but divergent results were obtained in 2 cases, where detection of aneuploidy was either possible with image analysis or with double labelling flow cytometry only. DNA content of samples gained by forceps biopsies and surgically resected tumours was concordant in 8 of 10 cases. In 2 patients, aneuploidy was detected only in the surgically resected tumour but not in the pre-operatively obtained forceps biopsies. Conclusions: A flow cytometric method for routine determination of the DNA ploidy of cells obtained by forceps biopsies from patients with oesophageal cancer was developed and evaluated against image analysis. The technique allows the prediction of DNA content before tumour resection, and might be used for optimising therapy and the patient’s quality of live.

Comparison of DNA histograms by standard flow cytometry and image cytometry on sections in Barrett's adenocarcinoma

“…In contrast to FC, high fidelity ICS identified all BAC tissues as aneuploid and the histograms showed lack of diploid peaks as also reported previously [ 10 , 17 ]. The main reason for these findings may be related to the fact that in ICS selected tumor cells, primarily, are included for DNA content analysis.…”

“…The main reason for the false negative aneuploidy result in BAC by standard FC appears to be the inclusion of disproportionately large number of non-cancerous diploid cells in the samples. This view is supported by the studies of Rickes and colleagues [ 10 ] who reported that many cases of esophageal squamous cell carcinoma or adenocarcinoma that were diploid on standard FC were aneuploid by the double-labeled FC in which esophageal cancer cells were labeled with cytokeratin-19 for squamous cell carcinoma and cytokeratin-18 for adenocarcinoma [ 10 ]. Similarly, all cases of BAC were found to be aneuploid by image cytometry that performs DNA content analysis on identified abnormal cells [ 17 , 20 ].…”

“…Moreover, unselected dispersed cells from tissues represent mixed populations of abnormal cells (cells of interest) and non-epithelial cells. The proportion of abnormal epithelial and non-epithelial cells varies with the source (e.g., superficial mucosal biopsy vs. deep surgical section) and the cellular composition of the tissue [ 10 ]. Therefore, studies on unselected dispersed cells are highly dependent on tissue selection.…”

“…However, in standard FC, proportion of cancer and non-cancer cells in the sample remains unknown. A comparison of standard FC and double labeled FC, using samples enriched with cancer cells, showed that many BAC that were diploid by standard FC were found to be aneuploid by the double-labeled FC [ 10 ]. This observation suggests that the high rate false negative aneuploidy by standard FC may be due to poor selection of cancer cells in the sample.…”

High-fidelity DNA histograms in neoplastic progression in Barrett's esophagus

“…DNA histograms obtained with standard flow cytometry (FC) suffer from inclusion of unknown quantities of the non-epithelial cells in the test sample. In recent years, attempts at better definition of the sample to be tested by FC have included labeling of the cells of interest 14 or selecting laser dissected areas of interest. In ICS, the selected test sample includes visually identified the epithelial cells in an area of interest.…”