IJCRIMPH Articles:

Peculiarities of Vascular Component of Communicative Systems in Rectal Adenomas 
International Journal of Collaborative Research on Internal Medicine & Public Health, 2009 Vol. 1 No. 1 (Pages 12-21)
Authors: A. V. Bychkov (1), A. E. Dorosevich (2)

(1) Smolensk Regional Institute of Pathology
(2) Smolensk Regional Institute of Pathology

Abstract  |  Full Text  |  PDF 

Paper review summary:
Paper submission date: February 10, 2009
Paper acceptance date: February 06, 2009
Paper publication date: March 2009

1. Introduction
1.1. The role of rectal adenomas in development of cancer
Colorectal cancer in recent years is considered as a dynamically and epidemiologically progressive pathology. In past 10 years the prevalence and mortality of this oncopathology has evidently increase globally. (Jemal,2008:7)
Advances in molecular biology have revealed the sequence of genetic alterations in development of various neoplasms. (Kern,2001:8) Molecular mechanism of colorectal cancer development is well-studied and adenomas are assumed to be the precursors. (Jass,2007:6) Incidence of rectal adenomas in general population is about 30%. (Hardy,2000: 4) The most important determinants of colorectal cancer risk include the size of adenoma, histological type and the degree of epithelial dysplasia. (O’Brien,1990:9)

It is well known that phenotypical changes in the development of cancer cells are manifested by molecular rearrangements in the epithelial cells. Recent studies have revealed that similar changes occur also in stromal cells. Occasionally, certain genetic alterations in the stroma may even precede genotypic changes in the epithelial cells of colorectal cancer. (Ishiguro,2006:5) Changes in the stromal component of a tumor are accompanied by the parenchymal rearrangements and sometimes they are the leading ones. On a genetic level, it is revealed that chromosomal and microsatellite instability is characteristic not only for epithelial cells but also for stromal cellular elements of CRA.

1.2. Conception of communicative systems
In the past two decades, in Smolensk regional institute of pathology, there has been an active study of various scientific projects that were focused on the evaluation of peculiarities of quantitative and qualitative combination of distribution of cell populations in pathological processes (mainly in the neoplastic) with the use morphometric methods. (Dorosevich,2007:1) These methods not only visually, but also independently of researcher (mathematical statistics) help to evaluate the peculiarities of mutual combinations of tissue elements. In the course of these studies, there had been an attempt to introduce a point of reference in the methodology of morphometric study. Also, an assumption was made about existence of fixed systems like histophysiological complexes, consisting of microcirculatory vessels with adjacent cell microenvironment, cell content which are localized close to the informative-trophic structures (objects of reference points), acquiring new properties (informational, configurational, etc.) by measures of approximation to the focus of pathological process. (Dorosevich,1998:3) On the basis of above mentioned and literature, the objects of reference points were considered to be microcirculatory vessels and vegetative nerve terminals. Microcirculatory vessels are the active parts of the cell redistributions in neoplastic growth and nerve elements are the integrative units between influencing factors and eventual reaction of the tissue on irritation. The cell population, which determines the peculiarities of regulation of trophic processes in the tissues in normal conditions and in pathological processes, is liable to a huge variability than vessels and nerves. Eventually, the adjacent cell microenvironment is a peculiar communicative system, which can be determined as follows. Communicative systems are open systems containing the whole structural and functional units: microcirculatory vessels, nerve terminals, cell microenvironment of these structures that are localized in the histophysiological correlation, providing a structural basis of homeostasis. (Dorosevich,2002:2) It is important to state the presentation of communicative systems as a dynamic totality of equivalent elements where each of them is taken in to account during the study of a pathological process.

Based on the definition of the communicative system, it can be assumed that the characteristic peculiarities of distribution of stromal cell population, which can be used as markers of pathological processes, should be the most expressed in the immediate proximity of the above mentioned tissue elements, with the help of which a structural homeostasis is maintained.

Detailed study of a pathological process is presumed by the morphological study of, first of all the effector element of integrative system (microcirculatory vessels and vegetative nerve terminals), and then its immediate cell microenvironment which includes epithelial cells and stromal structures. A conclusive step of such a study is the integration of all the mentioned components in dynamic system by the methods of biomedical statistics. This helps in revealing the changes that are characteristic for distribution of cell population and correlations between the contents of communicative system. Mathematical analysis of cellular microsurrounding of vascular and nerve component of communicative systems allows revealing on the light-microscopic level of those intercellular correlations, which usually are interpreted on a more deep level of investigation. And thus, micromorphometric indicators further prove to be prognostic factors. Also it is revealed that the changes in the stroma of a tumor may predict its behavior. Therefore a study of parenchymal and stromal interactions explains the mechanisms of tumor progression and further assumes the prognostic factors.
Our aim was to study the peculiarities of vascular component of communicative systems in different histological types of rectal adenomas.


2. Materials and Methods
A study of surgical and biopsy materials of tubular (TA), tubulovillous (TVA) and villous (VA) rectal adenomas — 30 cases of each histological type was performed. 20 biopsies of rectal mucosa without any evidently revealed atrophic, inflammatory or hyperplastic changes were studied as a control group. Clinical and endoscopic signs of these adenomas were taken into consideration.
The material was microscopically analyzed using Hematoxylin and Eosin and Picrofuchsin by van Gieson, taking in to account the histological type of tumor and the degree of epithelial dysplasia.
A review microscopy of parallel sections was done. Histotopographically distinct capillaries were revealed, the area around which did not overlap with the corresponding area of surrounding capillaries. Absolute number of cellular elements around the capillaries was counted in 10 different fields of view with a magnification of x 900 (oil immersion applicable).

Proliferative activity of epithelial cells (10 cases from each group) and structure of vascular bed (5 cases from each group) were estimated immunohistochemically. Expression of Ki-67 and CD31 was analysed in formalin fixed, paraffin-embedded sections with the method of indirect immunoperoxidase with the use of polyclonal rabbit antibodies to human antigen Ki-67 and CD31 (“Dako”, Denmark). Immunopositivity was revealed by detecting the dark-brown nuclei (Ki-67) and membranes (CD31) staining of antigen. In each case, Ki-67 immunopositive cells were counted among 1500 epithelial cells, and their ratio in multiples of 100, was calculated as an index of proliferative activity (IPA). Area of vascular bed was calculated by micromorphometry with the use of Glagolev’s ocular net. A relative area of vascular bed in 10 fields of view at x 400 was calculated by the method of squares.
Differences in distributions between variables were calculated using nonparametric methods (U-Test, Kruskal-Wallis test). Probability values < 0.05 were considered significant. Correlations were calculated using Spearman rank correlation coefficients ranging from -1 to +1. A positive correlation suggests that two variables vary in the same direction, while a negative correlation suggests that two variables vary in the opposite direction, p-values below 0.05 indicate a statistically significant correlation at the 95 percent level of confidence. Statistically independent variables have an expected correlation coefficient of zero. All data analysis was done with the statistical package Statgraphics Plus, version 5.0.


3. RESULTS
Study of case reports of patients revealed that adenomas were detected mainly in the age group 50–69 years (68.9% of cases). Age of patients varied from 19 to 83 years (mean 52.3±3.4 years). High specific proportion of females is related to their prevalence in later age groups, that apparently determines their long life span. In 67.8% of patients the process was revealed for the first time. Endoscopic investigation confirmed that the adenomas were localized mainly in the distal part of rectum (5–10 cm from anus), prevalent size was upto 1 cm (54.5%) and large tumors (more than 2 cm) were noted in 22.2% cases. The important morphological characteristics of adenomas are described briefly in Tables 1 and 2.
 

Localization of proliferating epithelial cells had the following distribution: normal mucosa in basal parts of crypts, TA in basal and middle parts of crypts, and in VA proliferation was active on the surface of villi and the upper third of the crypt (Fig. 1, A–D). Positive immunostaining (Ki-67) of epithelial cells was observed in villous structures of TVA, similarly as in VA and had a diffused pattern. The above mentioned not only explains the shift in the proliferative pool but also suggests a consequtive switch in mitotic rate of large number of cells along the length of crypt. In rare cases Ki-67 immunopositive cells were revealed in stroma, predominantly in the lymphatic follicles.

Figure 1: Proliferative Pool of Epithelial Cells

IPA in the control group comprised of 20.9±0.7, 26.6±0.3 in TA, 36.8±1.3 in TVA, 47.1±1.7 in VA. The reliable significant differences (p < 0.05) in proliferative activity of epithelial cells in the following adjacent pairs of tumors were revealed: TA–TVA, TVA–VA.

Rectal mucosa and adenomas were exclusively abundant with capillaries (Figure 2, A). In adenomas, numerous microvascular structures were observed: blood and lymphatic capillaries and sinusoids. Capillary beds correspond to the three dimensional histoarchitecture of tumor, underlying crypts or covering connective tissue core of villi. Structure of capillaries in various types of adenomas retains the usual histology, however VA is characterized by increase in the number of vessels and accompanying haemorrhages. Venular branches were prominent in the core of villous structures with a formation of a connective tissue base (Figure 2, B). Arterioles were usually found in the stalk of large exophytic tumors (Figure 2, C–D).
 

Figure 2: Microcirculatory Bed in Rectal Adenomas

Area of vascular bed was characterized by following indices: 30.7±0.9 in control group, 41.1±1.1 in TA, 82.2±1.3 in TVA, 274.3±7.3 in VA. The reliable significant differences (p < 0.05) in the intensity of vascularization in the following adjacent pairs of tumors were revealed: TA–TVA, TVA–VA.
Micromorphometric study of cell population (Table 3) was carried out around the blood capillaries. This histotopographical zone explains the physiological role of the pericapillary zone that reveals the most dynamic change in cell population and realization of immune and antitumoral responses.

 

In the following sequence “normal mucosa → TA → TVA → VA” there is a gradual increase in the absolute number of all the studied cell populations. This situation reflects the activation of integrative systems of organism to retain the local control in the framework of progressive pathological process. Apparently, qualitative changes in the cellular content in pericapillary zones do not occur. At the same time an increase in the following positions (p < 0.05) in the adjacent pairs is statistically reliable: epithelial cells and fibroblasts (control — TA), epithelial cells, plasma cells and fibrocytes (TA — TVA), epithelial cells, lymphocytes, plasma cells and polymorphonuclear leukocytes (TVA — VA). As noted above, IPA at transformation from normal mucosa to TA does not change, i.e. possibly, the number of epithelial cells increase at this stage due to damages in apoptotic processes.
The peculiarities of distribution of cell populations in relation to surrounding structures were noted. Thus, plasma cells were arranged mainly in small groups formed by 3–5 cells. Eosinophils were localized in periglandular region with a tendency to spread through the basal membrane into the gland. Cells of mesenchymal origin, fulfilling the supportive function were localized adjacent to the spatial structures (crypts, vessels): fibrocytes — to the immediate proximity, fibroblasts — at some distance. The following typical feature of distribution was expressed by lymphocytes: 1) perivascular (10–15 µm), 2) subglandular, 3) intraepithelial.
Correlation analysis of cell interaction in capillary microenvironment helps to determine the following peculiarities of intrastromal intercellular relations (Figure 3). In control group there is a positive relation between haematogenous and histogenous cell populations, where fibroblasts, macrophages, lymphocytes and fibrocytes actively participate in its formation. This condition helps in responding operatively on the local changes in the homeostasis. In the initial stages of tumor growth (TA), an increase in the number of correlations and activation of cell regulator such as macrophage, which along with fibroblast encloses the number of positive cell relations, is noted. In TVA, as the number of correlations decrease, the macrophage undergoes a peculiar isolation from other cells. In VA, for the first time at the stages of tumor progression, the number of intercellular correlations reaches to minimum, but isolation between haematogenous and histogenous population does not occur. Interesting feature that combine all the benign epithelial colorectal tumors is the participation of population of polymorphonuclear leukocytes in the process of intercellular integration, which was not noted in the study of parenchymal-stromal interactions in the tumors with other localizations. (Dorosevich,2007:1)

4. Conclusion

A positive correlation between transitions from one morphological category to another exists with an evident increase in the absolute number of parenchymal and stromal cells. In the course of a tumor progression “normal mucosa → TA → TVA → VA” there is a gradual loss of correlations between cell elements of stroma that are isolated from each other. In a number of cases, according to the state of cellular infiltrate, degree of evident changes in the epithelial cells can be predicted. A full-value estimation of prognostic factors of rectal adenomas is possible only with a parallel study of parenchyma and stroma of the tumors.

References:

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This study was performed by the financial support of Russian Humanitarian Scientific Foundation in frame of scientific research project No. 07-06-58606a/C.