Michał Tenderenda1, 2, Dorota Kupnicka3, Jan Berner4, *Konrad Wroński5, 6
Immunohistochemical study on the prognostic value of CD44 antigen – marker of metastases – in gastric cancer and its correlation with selected molecular parameters
1Department of Surgical Oncology, Faculty of Medicine, University of Warmia and Mazury, Olsztyn, Poland
Head of Department: prof. Michał Tenderenda, MD, PhD
2Department of Oncology, Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology
Head of Department: prof. Michał Tenderenda, MD, PhD
3Department of Pathomorphology, Faculty of Medicine, Medical University, Łódź, Poland
Head of Department: prof. Radzisław Kordek, MD, PhD
4Department of Surgical Oncology, Faculty of Medicine, Medical University, Łódź, Poland
Head of Department: prof. Arkadiusz Jeziorski, MD, PhD
5Department of Oncology, Faculty of Medicine, University of Warmia and Mazury, Olsztyn, Poland
Head of Department: prof. Sergiusz Nawrocki, MD, PhD
6Department of Surgical Oncology, Hospital Ministry of Internal Affairs with Warmia and Mazury Oncology Centre, Olsztyn, Poland
Head of Department: Andrzej Lachowski, MD
Introduction. Classical prognostic factors basing on clinico-pathological features are being replaced, due to rapid development of molecular biology, by modern molecular factors of potential prognostic value, including antigen CD44 expression as a metastizing marker in various types of carcinoma. CD44 is a glycoprotein of 85 kDa molecular mass, making a receptor for hyaluronic acid and other components of extracellular matrix, found on the surface of lymphocytes, fibroblasts and epithelial cells.
Aim. The aim of the study was evaluation of the prognostic value of CD44 antigen expression level as a metastizing marker in resectable gastric cancer and analysis of some correlations between the expression of this antigen and selected histoclinical parameters, protein products of cell-cycle regulatory genes and proliferation, angiogenesis and apoptosis markers.
Material and methods. Immunohistochemical analysis was performed on specimens obtained from radical stomach resections in 80 patients treated at the Department of Surgical Oncology, Medical University of Lodz, in the period 1992-1997 for gastric cancer stage I-IIIB (TNM-UICC). For immunohistochemical examinations, the LSAB system was used, designed for assessment of antigen expression including monoclonal antibody anti-CD44 (DAKO). Similar immunohistochemical procedure was used for determination of other examined molecular parameters. To determine AI apoptosis index and show up apoptosis in tumour cells, the TUNEL method was used according to standard procedure. In statistical analysis, Fisher’s exact test as well as Pearson’s test and Spearman’s test were applied to evaluate correlations between the analyzed variables. Evaluation of their influence on post-operative overall survival and disease-free survival was done by the Cox regression model.
Results. In presented study there was a statistically significant difference in the length of overall and recurrence-free survival correlating to the protein CD44 expression level in cancer cells – the highest minimum 5 year survival probability occurred in the group with the lowest CD44 expression. Moreover, there was a positive correlation between CD44 expression and malignancy degree, and non-linear correlation with a histological type according to the Lauren classification. There was also observed a positive correlation between CD44 and Ki67, and a negative one between P21 and RB and CD44.
Conclusions. The above results demonstrate a big value of immunohistochemistry in determination of prognosis in gastric cancer using CD44 expression and existence of mutual correlations between CD44 and the selected cell-cycle regulators.
Over more than a two decades many research centers have been conducting studies on the factors that may potentially affect prognosis in gastric cancer, the prognosis which despite better diagnostic methods and improved therapies still remains unfavourable. Of great importance is the task of determining prognosis on individual basis since the rate of the so-called 5-year survivals greatly varies depending on many clinical and pathological features (1, 2). Identification of crucial prognostic factors directly entails the choice of best adjuvant therapy to increase the chances of survival. Classical prognostic factors basing on clinico-pathological features are being replaced, due to rapid development of molecular biology, by modern molecular factors of potential prognostic value, including antigen CD44 expression as a metastizing marker in various types of carcinoma (1-4). CD44 is a glycoprotein of 85 kDa molecular mass, making a receptor for hyaluronic acid and other components of extracellular matrix, including osteopontine, found on the surface of lymphocytes, fibroblasts and epithelial cells (5-7). It participates in inter-cellular processes and is considered an important molecule active in the formation of neoplastic metastases and in mediation of intra-lymphocyte and intra-macrophage information (1-4). The cells capable of metastizing exhibit on their surfaces a modified CD44 molecule where the part protruding over the membrane surface is bigger and strongly glycosylized (8). Usually, already at the point of making the clinical diagnosis of a tumour there is a population of cancer cells present in the bloodstream, capable of forming metastatic foci. Complexes of these cells circulating in the blood can, together with platelets, settle at various locations in the body, e.g. in the bone marrow or lymph nodes. The molecules such as CD44 play a significant role in this process, both at an early stage of dissemination and in the subsequent implantation at a new localization (9-14). CD44 protein has been found to occur in various isoforms which tend to possess similar ends but differ in the middle part that contains additional aminoacids totaling even up to 361, included in the already existing ones – positions 224-246. Thus enlarged CD44 molecules can change their adhesive properties and lose the ability of signal transmission, and these changes in the molecule structure have made the production of a new specific antibody possible, the antibody which has proved effective in preventing metastases in pancreatic cancer in rats (8). CD44v5 increased expression was observed in gastric cancer, especially in Goseki type I and III (14). The evaluation of expression of particular CD44 variants in various carcinomas yields different results depending on the technique used (CD44 expression evaluation of freezed or paraffin sections) and on the type of antibodies applied (1, 3, 4, 15). The use of modern molecular techniques, including PCR, is recommended not only for identification of increased CD44 expression in the metastatic tissues sections but also for CD44 presence in the systemic fluids which would enable early detection of carcionoma dissemination risk (1-4).
The aim of the study was evaluation of the prognostic value of CD44 antigen expression level as a metastizing marker in resective gastric cancer and analysis of some correlations between the expression of this antigen and selected histoclinical parameters, protein products of cell-cycle regulatory genes and proliferation, angiogenesis and apoptosis markers.
Material and methods
Following total or subtotal resection with regional lymphadenectomy, the resected specimens were fixed in 10% buffered formalin, then after dehydration in alcohols and acetone, put into carboxylin and xylenes and embedded in paraffin blocks. The paraffin sections were then used to make routine preparations stained with haematoxylin and eosin and determined, in cooperation with the Department of Tumor Pathology, for the histologic type of tumour according to the Lauren classification, the stage of histologic malignancy according to the 3-grade scale (G1-G3), and evaluated for local lymph nodes involvement. For the immunohistochemical studies, the LSAB – labelled streptavidin-biotin (DAKO) system designed for the assessment of antigene expression was used. The antibodies used in the analysis were at dilution from 1:20 to 1:100 (depending on their type) against: CD44 (DAKO), cyclin D1 (Santa Cruz, USA), cyclin E, P21, P27, RB, CD34 (Novocastra, UK), Ki67, P53 (DAKO, Denmark). After washing with a buffer of pH 7.6 and after application of chromogen and cell nuclei staining with haematoxylin, the immunohistochemical reaction was evaluated under the microscope. All preparations were assessed by two independent researchers in 10 fields (magnification x 400) in the areas displaying the highest degree of reaction („hot spots”). This degree – corresponding to the expression of the examined antigens, i.e. CD44, cyclin D1, cyclin E, P53, P21, P27, RB, was classified using the semiquantitative method as: negative (-) if staining of the assessed cellular structures was detected in less than 10% of cells in microscopic field, as weakly positive (+) with 10-50% of immunostained cells and as strongly positive (++) with over 50% of immunostained cells. For statistical analysis, I defined the above mentioned parameters were defined, with the subgroups classified according to the degree of reaction as dichotomous variables, whereas the three other molecular parameters – proliferation index, apoptosis index and tumour vascularization degree, as continuous variables. Nuclear reaction was observed where antibodies were used against cyclin D1, cyclin E, P53, P21, P27, Ki67, RB; membranous reaction was observed in CD44 positive cases. An increase in angiogenesis in tumour cells was measured with the use of anti-CD34 monoclonal antibody, determining the mean number of blood vessels in 10 consecutive fields x 400. Proliferation index Ki67 was designated as the mean percentage of Ki67 positive cells in 10 consecutive fields (x 400). In order to show up apoptosis in tumour cells and to determine apoptosis AI index, the TUNEL (TdT mediated dUTP Nick End Labeling) method was used according to the recommended procedure (Apoptag-Boehringer-Mannheim). Deparaffinized and dehydrated sections of tumour tissue were treated with trypsin for 20 minutes at room temperature and washed with 3% H2O2 solution for 10 minutes to inhibit endogenous peroxidase. After rinsing in phosphate buffer, they were incubated for 10 minutes at room temperature. Control of immunohistochemical reaction was being prepared simultaneously, with the TdT enzyme replaced by distilled water. The cells revealing apoptosis were characterized by lower cytoplasm content, nuclear condensation and presence of the so-called apoptotic bodies. The apoptotic index AI was defined as the number of positive reacting cells against the total number of assessed cells in 10 consecutive fields (x 400).
Assessment of correlations between prognostic factors: since the examined variables (risk factors) were evaluated on the nominal, ordinal and interval scale, various statistical tools were used to determine the correlations. If both the variables were on the ordinal or nominal scale, Fisher’s exact test was applied to determine their correlation and Mantel-Haenschel test to assess their colinearity (Fisher and van Belle, 1993).
Assessment of impact of risk factors on patients’ survival: the impact of examined risk factors on overall post-operation survival and disease-free survival was assessed with the Cox proportional hazards model to assess the impact of particular risk factors on survival.
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