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Volume: Barrett's Esophagus
Chapter: Dysplasia

What is the value of image-cytometry in detecting ploidy changes in Barrett's mucosa and in correlating aneuploidy with malignant changes?

K. Geboes (Leuven)

Barrett's esophagus is a well known premalignant condition [1]. The estimated incidence of adenocarcinoma varies from one in 52 to one in 441 cases per patient year. The risk of malignancy is probably lower in short-segment than in long-segment Barrett's esophagus [2-4]. Adenocarcinoma does not arise de novo develops through a multistep process with progressive worsening of a precursor lesion. The precursor lesion is known as "dysplasia" and can be recognized with morphological tools. Its development is associated with defects in genes in control of cell proliferation, cell death and cell shape. Secondary prevention of esophageal adenocarcinoma associated with Barrett's metaplasia aims at the detection and treatment of premalignant changes (dysplasia) or early malignancy (early or superficial cancer). At present dysplasia is the most widely used indicator for the identification of patients at risk. The identification of dysplasia depends upon the number and type of biopsy samples examined and upon the technique used for the analysis of the samples. The number of biopsies is influenced by sampling error. Sampling error can be decreased by obtaining more samples or using new endoscopic techniques. Biopsies are generally analyzed with light microscopy of routinely processed sections. Identification and grading of dysplasia is thus currently mainly based on subjective assessment of haematoxylin and eosin stained sections and hence prone to observer variation. Considerable intraobserver and interobserver variation in the diagnosis and grading of dysplasia exists, particularly at the indefinite/low-grade interface [5-7]. Better agreement (88%) is found when distinguishing high-grade dysplasia (HGD) and intramucosal carcinoma from low-grade and indefinite [5, 6]. Therefore more reliable and more objective indicators are sought.

Tools for objective measurements

The development of neoplasia and malignancy is associated with defects in genes in control of cell proliferation and death. Such defects are commonly associated with alterations in the DNA content of cells or aneuploidy.

The DNA content of cells can be quantified with flow-cytometry. This technique using isolated cells, is not influenced by features that may confuse a morphological diagnosis of dysplasia such as regenerative epithelial lesions and inflammation. It gives however no information on the morphology of the lesions. Using flow cytometry, aneuploidy has been detected in samples from patients which are histologically negative for dysplasia. Aneuploidy might therefore precede the development of dysplasia and even be more reliable for the identification of premalignant changes and patients at risk than dysplasia.

The prevalence of DNA aneuploidy increases with increasing histological grade of abnormality. Aneuploidy might therefore be also more reliable for grading the lesions. However, patients with HGD can have only diploid cell populations and the exact meaning of flow cytometric abnormalities in patients without dysplasia on histology is not known [8, 9].

In order to overcome or diminish the inter- and intraobserver variation which occurs with microscopic analysis and in order to combine morphology with more objective methods of analysis, other methods have been applied.Various studies on premalignant lesions of the gastrointestinal tract, such as gastric dysplasia and colorectal adenomas, have demonstrated the feasibility of such methods.

These methods include grossly:
1) (computerized) morphometry;
2) counting objects;
3) computerized morphometry and immunoquantitation (p53, Ki67);
4) digital image analysis;
5) (image) cytometry.

Morphometry is the quantitative description of features of any dimension. Following the discovery, in the thirthies, that the size of nuclei and nucleoli is increased in tumor cells and later that neoplasia is associated with loss of stratification, loss of polarity and other features, it became clear that dysplastic epithelium could be assessed quantitatively.

These principles, coupled with the application of modern technology such as cameras and computers can now be used for the analysis of microscopic sections in "computerized morphometry".

Counting objects of interest in tissue sections is another widely applied technique. Coupled with image analysis this can also be done in objective way. Most of the time, this is used for proliferation markers in tumors. Such markers can be identified with immunohistochemistry. One such marker is the Ki67, which detects a nuclear protein present in all cells in G1, G2 or S phase.

Computerized morphometry and immunoquantitation can and have been combined and used for objective assessment of dysplasia in Barrett's esophagus; Using a combination of features associated with cellular differentiation and proliferation, such as a stratification index, p53 and Ki67, quantitative pathological analysis can reduce diagnostic variability in the grading of dysplasia [10, 11].

Digital image analysis for different morphometric parameters is also highly sensitive and specific in differentiating HGD from LGD in Barrett's esophagus. In a study of 60 biopsies from different patients it was shown that parameters such as nuclear area, optical density and length are significant discriminators of dysplasia grade [12].

Most of these techniques are reliable and objective and can be applied on routinely processed tissue material. The major disadvantages are the cost of the technical equipment for the analysis and time. Only few laboratories have indeed the necessary equipment and technical skills.


Cytometry refers to measuring the amount of a given substance in tissue, cells or nuclei. The observation of hyperchromatism in the nuclei of neoplastic cells, reflecting an increased DNA content, is the subjective counterpart of DNA-cytometry in all day pathology.

Cytometry depends on the possibility to detect the substance of interest by a specific dye and to measure the concentration of that dye. The Feulgen staining has proved to be specific for DNA, and the amount of stain is proportional to the amount of DNA present.

DNA cytometry measures the total amount of DNA per nucleus. For this purpose the complete nucleus needs to be present in the image (which is simple in cytological specimens but not in sections) and the nucleus needs to be discriminated from its environment (which is simple in cytological smears but difficult in tissue). Overlapping nuclei, a common feature of dysplastic epithelium precludes measurements.

Attempts have been made to overcome these problems (correction formulas to cope with incomplete nuclei...) and the use of confocal laser scanning microscopy allowing to measure the DNA content in three dimensional reconstructions of tissue and thick tissue sections with advanced image processing seems promising.

DNA image-cytometry has been shown to correlate well with the results obtained by flow cytometry for the assessment of aneuploidy in breast lesions. The advantages of image-cytometry over other methods are:
1) the ability to detect a single "rare event aneuploid cell",
2) the direct visualization of abnormal cells which can be related to morphological changes. Direct visualization is indeed not possible in DNA flow cytometry. In flow cytometry the results are however not influenced by overlapping cells.

However, as for other objective methods, DNA image cytometry is time consuming and overlapping nuclei can induce false measurements [13]. Therefore a cut off value has to be established, for instance three separate aneuploid cells

DNA image-cytometry has been applied to Barrett's mucosa [14]. Aneuploid cells were detected in specimens from six patients out of a series of 55 which were followed in a prospective endoscopic surveillance study. A total of 91 biopsies were obtained in this series. Four of the six patients subsequently developed dysplasia and adenocarcinoma. In this series, aneuploidy was always associated with some morphological abnormality varying from mild dysplasia to frank carcinoma. Aneuploidy was not found in material from one patient who had an esophagectomy for dysplasia nor in material from four patients diagnosed as having indefinite dysplasia.

In general, for Barrett's esophagus, DNA image-cytometry shows a good correlation between aneuploidy, dysplasia and the subsequent development of adenocarcinoma and the correlation is similar as the one seen with flow cytometry. Application of DNA imagecytometry is however not widely used because it is time consuming and the results can be influenced by overlapping nuclei, a phenomenon which is common in dysplasia in Barrett's esophagus [15].


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12. Buttar WS, Wang KK, Krishnadath KK, Lutzke LS, Anderson MA, Harmsen S, Darren RL, Zinsmeister AR, Burgart LJ, Sebo TJ. Digital image analysis is highly sensitive and specific in differentiating high from low grade dysplasia in Barrett's esophagus. Gastroenterology 2000;118:A684.

13. Wright TA. High-grade dysplasia in Barrett's oesophagus. Br J Surg 1997;84:760-766.

14. James PD, Atkinson M. Value of DNA image cytometry in the prediction of malignant change in Barrett's oesophagus. Gut 1989;30:899-905.

15. Geboes K, Van Eyken P. The diagnosis of dysplasia and malignancy in Barrett's oesophagus. Histopathology 2000;37:99-107.

Publication date: August 2003 OESO©2015