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Revista Española de Enfermedades Digestivas

versión impresa ISSN 1130-0108

Rev. esp. enferm. dig. vol.97 no.1 Madrid ene. 2005

 

ORIGINAL PAPERS


DNA aneuploidy in colorectal adenomas. Role in the adenoma-carcinoma sequence

M. Alcántara Torres, R. Rodríguez Merlo1, A. Repiso Ortega, A. de Lucas Veguillas2, J. Valle Muñoz, R. Sánchez Simón1
and J. L. Martínez Potenciano

Departments of Gastroenterology and 1Pathology. Hospital Virgen de la Salud. Toledo. 2Service of Public Health. Instituto de
Ciencias de la Salud de Castilla La Mancha. Talavera de la Reina. Toledo, Spain

 

ABSTRACT

Introduction: aneuploidy has been observed in 6-27% of lesions known to be precursors of colorectal cancer, such as adenomas or ulcerative colitis. It has been suggested that aneuploidy may predispose to malignancy in these cases. However, its role in the adenoma-carcinoma sequence has not been definitely established. The objective of this study was to assess the incidence of aneuploidy in colon adenomas, as well as to study its possible role in the adenoma-carcinoma sequence.
Material and methods:
the study was performed on a series of 57 large bowel adenomas measuring 10 mm or more, collected from 54 consecutive patients. All specimens were obtained either by endoscopic or by surgical resection. There were 49 adenomas with low-grade dysplasia, two with high-grade dysplasia, two intramucous carcinomas, and four microinvasive carcinomas. A flow cytometric DNA analysis was performed in fresh specimens following Vindelov´s method.
Results:
aneuploid DNA was detected in five out of 49 low-grade dysplasia adenomas (10%), in all four high-grade dysplasia adenomas or intramucous carcinomas (100%), and in three out of four microinvasive carcinomas (75%). The association between aneuploidy and high-grade dysplasia adenomas, intramucous, or microinvasive carcinoma was statistically significant (p < 0.001). No association was found between aneuploidy and any of the following features: age, gender, clinical symptoms of patients, and size or location of adenomas.
Conclusions:
the incidence of aneuploidy in this series was 10% in low-grade dysplasia adenomas, and 87% in high-grade dysplasia adenomas or carcinomas, and this difference was statistically significant. These findings suggest that aneuploidy may play a role in the adenoma-carcinoma sequence.

Key words: Aneuploidy. Colon adenoma. Colon adenocarcinoma. Flow cytometry.


Alcántara Torres M, Rodríguez Merlo R, Repiso Ortega A, de Lucas Veguillas A, Valle Muñoz J, Sánchez Simón R, Martínez Potenciano JL. DNA aneuploidy in colorectal adenomas. Role in the adenoma-carcinoma sequence. Rev Esp Enferm Dig 2005; 97: 7-15.


Recibido: 12-02-04.
Aceptado: 11-05-04.

Correspondencia: Mariano Alcántara Torres. Servicio de Aparato Digestivo. Hospital Virgen de la Salud. Avda. de Barber, 30. 45004 Toledo. e-mail: malcantara@cht.insalud.es.


This article has been realized with the economic support of a grant of the Junta de Comunidades de Castilla La Mancha GC03012.

 

INTRODUCTION

Abnormal DNA amounts are most commonly caused by changes in the number of chromosomes and chromosomal deletions. Changes in the normal cellular DNA content give rise to aneuploidy, an abnormality that reflects chromosomal alterations that play an essential role in the development and progression of tumors (1).

Aneuploidy has been observed in 6-27% of large bowel lesions known to be precursors of colorectal carcinoma (CRC), such as adenomas or ulcerative colitis (2-5). In colorectal adenomas, the prevalence of aneuploidy increases in parallel to histopathological findings known to be associated with a higher risk of malignant transformation such as larger size, villous elements in the adenoma, or high-grade dysplasia (2-8). These observations suggest that DNA aneuploidy might play an important role in the adenoma-carcinoma sequence (2,4,9,10). However, some authors have found aneuploid DNA content in only 13% of colon adenomas harboring an invasive carcinoma, an observation that does not support an important role for aneuploidy in the adenoma-carcinoma sequence (11).

On the other hand, aneuploidy can be detected in the normal colon mucosa, in hyperplastic polyps, in adenomas with low-grade dysplasia, and in ulcerative colitis without dysplasia (12,13). Thus, it has been suggested that aneuploidy is an early change in carcinogenesis (12).

The purpose of this study was to assess the prevalence of aneuploidy in colorectal adenomas and its possible role in the adenoma-carcinoma sequence.

MATERIAL AND METHODS

The study was done in a series of 57 large bowel adenomas measuring 10 mm or more, collected from 54 consecutive patients at "Virgen de la Salud" Hospital, Toledo (Spain). Adenoma samples were obtained either by endoscopical or surgical resection. Adenomas were distributed according to the Vienna classification (14) (Fig. 1). Specimens were stained with hematoxylin-eosin and other routine techniques, and then assessed by one single pathologist.


DNA cell content was determined by flow cytometry in fresh specimens or snap-frozen specimens preserved at -80 ºC, according to Vindelov's method (15). Briefly, fresh specimens were fragmented with a surgical blade, suspended in a citrate buffer, and filtered through a nylon mesh with 50 mm pores. The cell suspension was digested using trypsin and detergent for 10 minutes, and then digestion was interrupted with a trypsin inhibitor and RNAse. Cells were stained with propidium iodide and analysed in a Fac Sort (Becton-Dickinson) flow cytometer. Normal colonic mucosa was used as diploid control using the same procedure. An analysis was done with the CellFit software application on a minimum of 10,000 nuclei.

DNA content was considered aneuploid if there were two clearly separated peaks of G0/G1 cells (Fig. 2). The DNA index was calculated by obtaining the proportion between the mean DNA content of G0/G1 cells in the study cell group and in the normal dyploid cell group. In diploid histograms the DNA index was equal to 1. DNA content was considered tetraploid when there was a single G0/G1 peak and the DNA index was equal to 2. The proliferative fraction was determined based on the number of S-phase cells. Proliferative fractions equal to or higher than 10% were considered elevated and those lower than 10% were considered low (1). Histograms with a G0/G1 peak variation coefficient larger than six were considered invalid (16).


Clinical data such as age, gender, symptoms, presence of synchronous CRC, and tumor location were obtained from patient clinical records.

Student's T test was used for comparison of quantitative results, and χ2 and Fisher's exact tests were used for comparison of qualitative results. A two-tailed p value < 0.05 was considered statistically significant.

RESULTS

Forty-nine out of 57 adenomas included in this study were adenomas with low-grade dysplasia, two were adenomas with high-grade dysplasia, two adenomas harbored an intramucous carcinoma, and four adenomas had a microinvasive carcinoma. Regarding clinical features, patients studied included 32 men and 22 women with a mean age of 65.5 ± 8.9 years. Hematochezia prior to diagnosis was present in 51.8% of patients, although in most cases this symptom was not related to the polyps. Other clinico-pathological features are listed in table I.


Twelve out of 57 adenomas (21%) were aneuploid, and the remaining 45 were diploid. There were no cases with tetraploid DNA. When the correlation between aneuploidy and clinico-pathological features was assessed we observed that 50% of adenomas measuring 2.5 cm were aneuploid, whereas only 26% of adenomas smaller than 2.5 cm were aneuploid (p = 0.005). Aneuploid DNA was found in 87% of adenomas with high-grade dysplasia or harboring intramucous or microinvasive carcinomas, as compared to 10% in adenomas with low-grade dysplasia (p < 0.001). Aneuploid DNA was found in 71% of adenomas with an S-phase higher than 10 versus 5% in adenomas with an S-phase 10% (p < 0.001). There was a trend towards a statistically significant association between aneuploidy, male gender, and hematochezia. Other features such as age, synchronous CRC, or histology of adenomas were not associated with aneuploidy (Table II). The mean variation coefficient of G0/G1 peaks was 2.8 ± 0.99 (range: 1-5.9).


The presence of high-grade dysplasia or carcinoma inside adenomas was statistically associated with the magnitude of the proliferative fraction (Table III).


DISCUSSION

The prevalence of DNA aneuploidy in our series of colorectal adenomas was 21% when all cases were included, and 17% after excluding cases with invasive carcinoma inside the polyp. This prevalence is higher than in studies using formalin-fixed specimens (3,4), and similar to that in studies using fresh or frozen specimens (17). The reason is that the sensitivity to detect aneuploid peaks is higher when using fresh specimens (18). On the other hand, we have not included adenomas smaller than one centimeter, which have a lower prevalence of aneuploidy, a fact that can explain the observation of a larger number of aneuploid adenomas in our study (2,7,19).

An association exists between aneuploidy and the histopathological features that predispose to malignant change in adenomas, such as larger size, villous elements, and high-grade dysplasia (3,5). We also found an association between aneuploid DNA and size and degree of adenomatous dysplasia (7,17). Aneuploidy was also more common in adenomas with villous elements, although this association did not reach statistical significance, an observation similar to that in other studies (7).

Assessing the incidence of aneuploidy by gender, we observed a higher incidence in men versus women (29 vs. 9%). However, this difference did not reach statistical significance. The reason for a higher incidence of aneuploidy in men is not clear; one possible explanation is that in men colorectal adenomas are biologically more aggressive, and thus more prone to malignant transformation. This may explain the higher incidence of CRC in men versus women in epidemiological studies (21,22).

In subjects presenting with hematochezia, adenoma DNA aneuploidy was more often than in subjects without such symptom. In most cases, adenomas were not probably the cause of hematochezia; instead, the symptom was related to an associated condition such as CRC, which was present in 30% of patients in our series. One might think that adenomas associated with a synchronous CRC are biologically more aggressive and have a higher frequency of aneuploidy. However, we did not find such association between DNA aneuploidy and the presence of synchronous or metachronous CRC, an observation similar to that reported by other groups (3,11,22).

The prevalence of DNA aneuploidy in adenomas with high grade dysplasia, in situ carcinoma, or microinvasive carcinoma ranges from 12.5 to 37% in studies using formalin-fixed specimens (6,23), and is 72% for fresh or frozen specimens (17). In our study, the prevalence of aneuploidy in this group of adenomas was somewhat higher, reaching 87.5%. This prevalence of aneuploidy is even higher than in CRC, in which prevalence ranges from 50 to 68% using formalin-fixed specimens (24-26), and from 64 to 75% using fresh or frozen specimens (27-29). We do not know the reason for the high prevalence of aneuploidy in our study. It is possible that DNA ploidy heterogeneity, which in some studies is over 40% (30,31), was smaller in our study, thus making it more feasible that aneuploid peaks be detected. Taking into account the low prevalence of aneuploidy in low-grade dysplasia adenomas, and its high prevalence in high grade dysplasia adenomas, intramucosal, or microinvasive carcinoma, we think that aneuploidy plays a role in the adenoma-carcinoma sequence. Our results are in contradiction with the results of other authors, who found aneuploidy in only 17% of adenomas with microinvasive carcinoma (11). However, this study was done in formalin-fixed specimens, a method less sensitive to detect aneuploid peaks.

DNA aneuploidy has been observed in adenomas with low-grade dysplasia, in hyperplastic polyps, and in ulcerative colitis mucosa before the development of dysplasia (12,13,32), which suggests that aneuploidy is an early change in carcinogenesis (12). Although the prevalence of aneuploidy in our study was higher in adenomas with high-grade dysplasia or carcinoma, we found DNA aneuploidy in 11% of adenomas with low-grade dysplasia. These results support the hypothesis that aneuploidy is an early event in colorectal carcinogenesis.

The exact mechanism giving rise to aneuploidy is unclear. Cytogenetic and molecular genetic studies indicate that aneuploidy is the result of changes in the number of chromosomes, and of deletions in chromosomes (33,34). Chromosomal alterations more commonly found in association with aneuploidy are located in chromosomes 1, 7, 17, and 18 (34-37). In colorectal adenomas, a close relationship between 1p deletion and DNA aneuploidy has been observed (35).

In this study, we also observed a significant association between increased proliferative cell fraction and signs of malignant transformation in adenomas. However, this association did not add anything to the information provided by DNA ploidy.

In conclusion, although aneuploidy can be detected in low-grade dysplasia adenomas, which supports the hypothesis that aneuploidy is an early event in colorectal carcinogenesis, its prevalence remarkably increases when high-grade dysplasia or carcinoma appear within the adenoma. Therefore, we think that DNA aneuploidy plays an important role in the adenoma-carcinoma sequence.

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