Lymphangiogenesis in early and advanced gastric cancer: Is there any difference?

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1 bs_bs_banner doi: /jgh ORIGINAL ARTICLE Lymphangiogenesis in early and advanced gastric cancer: Is there any difference? Julia Rudno-Rudzińska,* Piotr Donizy, Ewelina Frejlich,* Krzysztof Kotulski,* Piotr Dzięgiel, Agnieszka Hałoń and Wojciech Kielan* *Second Department of General and Oncological Surgery, and Departments of Pathomorphology and Oncological Cytology and Histology and Embryology, Wrocław Medical University, Wrocław, Poland Key words advanced and early gastric cancer, D2-40, lymphangiogenesis, VEGF-C, VEGF-D. Correspondence Dr Julia Rudno-Rudzińska, Second Department of General and Oncological Surgery, Wrocław Medical University, Borowska 213, Wrocław, Poland. Conflict of interest: None. Abstract Background and Aim: Gastric cancer (GC) in Poland is on the third place of men s mortality and on the fifth place of women s mortality in malignant neoplasms, and the percentage of diagnosed early GC is less than 20%. In this study, the relationship among lymphatic vessel density, marked with D2-40, expression of vascular endothelial growth factor (VEGF)-C/D, VEGF receptor 3 VEGFR-3, and the stage of GC patient were investigated. Methods: This study examined the relationships between the peritumoral lymphatic vessels (PTL) density and intratumoral lymphatic vessels (ITL) density stained immunohistochemically with D2-40/podoplanin, the expression of VEGF-C/D and VEGFR-3, and the stage of 58 GC patients. Results: Lymphatic vessel density measured by D2-40 decreases outside the tumor (PTL) and increases within the tumor (ITL) as the staging grows from I to III, whereas in the case of patients belonging to stage IV group, lymphatic vessel density decreases outside the tumor as well as within the tumor in comparison with the stage III group (not statistically significant). We observed a difference between morphology of the vessels within the tumor (ITLs) and vessels that are located outside the tumor (PTLs). PTLs were enlarged and unsqueezed as opposed to ITLs, which were collapsed. Conclusions: (i) There is no significant correlation between the density of ITL nor PTL marked with D2-40 and the stage of GC. (ii) We did not observe relationship between expression of VEGF-C/D and VEGFR-3 and the stage of GC. (iii) Further studies are needed to fully determine the role of PTL and ITL. Introduction Despite progress in gastric cancer (GC) diagnostics, prophylaxis, as well as treatment methods, it is still one of the most dangerous diseases in the world. GC morbidity rate is decreasing since the 1970s. 1 However, this type of cancer remains the fourth most frequent cancer worldwide and second most common cause of cancer-related death. 2,3 Needless to say, there are demographic differences concerning GC prevalence. GC is the most frequent cancer in Japan and Korea and it is deppendent on the diet, Helicobacter Pylori infections. Usually it is located in the distal part of the somach and in Lauren clasiffication is enteric type. At the same time, in the case of European countries, there was an observed increase in proximal as well as esophagogastric junction (EG junction) morbidity rate. Obesity, gastroesophageal reflux disease, and genetic predispositions are supposed to be main causes concerning GC morbidity in Europe. 1 Diffuse-type cancer (based on Lauren classification) is the most common GC in Europe. It is worth pointing out that GC survival rate in Asian countries reaches 95%, whereas in Europe it is oscillating between 10% and 20%. There are several factors responsible for that difference in survival rate. The first one is the above-mentioned difference in cancer biology. Esophagogastric junction cancers with diffuse histopathological morphology have significantly worse survival rate. Prophylaxis programs implemented in Asian countries are the next key factor. Becouse of these programs the percentage of early gastric cancer diagnosis reaches 60 70% within those countries, whereas in Europe it is not greater than 5%. Taking into account the abovementioned differences, European GC and Japanese or Korean GC might be classified as almost different diseases. Therefore, one may ask whether the biological processes are the same in both cases. In Wrocław, a city located within Lower Silesia Region in southwestern Poland, the morbidity rate concerning is illustrated on the Figure 1. Lymphangiogenesis is the formation of lymphatic vessels within healthy tissues as well as in carcinogenesis. 4 Once those 107

2 Figure 1 Gastric cancer morbidity of the Lower Silesian patients in years , females;, males. Table 1 Feature Group characteristic All patients 58 Mean age 64 Sex Female 15 Male 43 Stage (5- th TNM) I 17 II 24 III 8 IV 8 n processes have been stopped, the size of the pathological tissue will not exceed few millimeters, and disease progress control will be enabled. 5 Research concerning lymphangiogenesis became possible a dozen or so years ago and were related to the discovery of lymphatic endothelial cells (LEC), specific lymphatic markers such as podoplanin, LYVE-1, and Prox-1. 4 These are not specific for only lymphatic vessels; however, they are used to distinguish blood vessels from lymphatic ones. Among the factors responsible for lymphatic vessels development are vascular endothelial growth factor C (VEGF-C), VEGF-D, platelet-derived growth factor BB, and hepatocyte growth factor. VEGF receptor 3 (VEGFR-3) is the receptor that is taking part in lymphangiogenesis. 4 Taking into account demographic differences and greater early cancer diagnosis ratio in Japan and Korea in comparison with European countries, the aim of this paper was to answer if there is a difference in lymphangiogenesis according to cancer stage. Methods Patients. The study was conducted using histopathological material from the Second Department and Clinic of General and Oncological Surgery, Wrocław Medical University, Poland. The analysis was performed retrospectively and covers patients that were operated between 1997 and There were 58 patients in the study 15 women and 43 men. The average patient age was 64. Patients were divided into groups according to fifth edition of the TNM classification there were 17 patients assigned to stage I, 24 to stage II, eight to stage III, and eight to stage IV. Every patient underwent total or subtotal gastrectomy with D2 lymphadenectomy. The average number of lymph node was 19. Analyzed group characteristic is given in Table 1. The study has been approved by Ethics Committee of Medical University of Wrocław, Poland. Tissue specimens. The tissue samples were fixed in 10% buffered formalin and embedded in paraffin. In each case, HE-stained slides were subjected to histopathological evaluation by two pathologists. Analyzed clinicopathological data was TNM stage. Immunohistochemistry. Immunohistochemical analyses were conducted at the laboratory of the Department of Pathomorphology in Lower Silesian Oncology Centre in Wrocław, Poland. Formalin-fixed, paraffin-embedded tissue was freshly cut (5 μm). The sections were mounted on Superfrost slides (Menzel Gläser, Braunschweig, Germany), dewaxed wtih xylene, and gradually hydrated. Activity of endogenous peroxidase was blocked by 5 min exposure to 3% H 2O 2. All the studied sections were boiled for 15 min at 250W in the Antigen Retrieval Solution (DakoCytomation, Dako Denmark A/S, Produktionsvej 42, DK-2600 Glostrup, Denmark). Then, immunohistochemical reactions were performed using: (i) the mouse monoclonal antibodies detecting D2-40 (dilution 1:100) (DakoCytomation); (ii) the mouse monoclonal antibodies detecting VEGF-C (dilution 1:150) (R&D Systems, 614 McKinley Place NE Minneapolis, MN 55413, USA); (iii) the mouse monoclonal antibodies detecting VEGF-D (dilution 1:150) (R&D Systems); and (iv) VEGFR-3 (dilution 1:150) (R&D Systems). Tested sections were incubated with antibodies for 1 h at room temperature. Subsequent incubations involved biotinylated antibodies (15 min, room temperature) and streptavidin-biotinylated peroxidase complex (15 min, room temperature) (LSAB+, HRP, DakoCytomation). NovaRed (Vector Laboratories, 3, Accent Park, Bakewell Road Orton Southgate, Peterborough, PE2 6XS, United Kingdom) was used as a chromogen (10 min, at room temperature). All the sections were counterstained with Meyer s hematoxylin. The control group for the antibodies studied was made up by healthy stomach specimens. Immunopathomorphological evaluation. The expression of the studied proteins was assessed under microscope OLYMPUS BX41 (New York Microscope Company Inc., Hicksville, NY, USA) using computer microscopic image analysis (software: AnalySIS DOCU, v. 3.2 for Windows 95/98/NT, developed by Soft Imaging GmbH, Olympus Soft Imaging Solutions GmbH, Münster, Germany, license No.: ). All the preparations were examined by two pathologists. They performed independently the immunopathomorphological evaluation based on analysis of large tissue slides (about 2 cm in diameter). D2-40 protein expression was evaluated using of a quantitative method a modified Weidner s method. 6 Vessel density was evaluated within neoplastic tissue (intratumoral lymphatic vessel [ITL] density) and within healthy tissue outside the tumor (peritumoral lymphatic vessel [PTL] density). A microscopic image from one preparation magnified 200 times was transferred 108

3 Table 2 IRS (immunoreactive score) modified by authors Table 3 Basic statistic data Percentage of positive cells Points Intensity of reaction Points Number positive cells 0 No reaction 0 < 25% positive cells 1 Weak color reaction % positive cells 2 Moderate intensity % positive cells 3 Intense reaction 3 > 75% positive cells 4 IRS score according to Remmele et al. modified by authors. to a computer program, which automatically counted all positively stained vessels from three random fields of view with the highest density. Each individual vessel or cluster of endothelial cells was regarded as a hot point and counted as one microvessel. Afterward, the average number of vessels from the three fields of view was calculated. The expression of VEGF-C, VEGF-D, and VEGFR-3 proteins was calculated using a semiquantitative method. Two immunohistochemical reaction parameters were considered when evaluating the expression of the above proteins: the percentage of cells with a positive cytoplasmic reaction (the percentage of reactive tissue) and the intensity of a reaction. The ultimate immunohistochemical reaction results are expressed in the semiquantitative IRS (immunoreactive score) scale according to Remmele. 7 The scale assigns a score for a percentage of cells demonstrating reaction (0 4 points) and for the intensity of a reaction (0 3 points). The final result is the product of the scores for the parameters analyzed (0 12 points) and is referred to as an IRS factor (Table 2). Group parameter I II III IV II-IV D2-40 (ITL) Mean Std Median D2-40 (PTL) Mean Std Median VEGF-C Mean Std Median VEGF-D Mean Std Median VEGF-R3 Mean Std Median ITL, intratumoral lymphatic vessel; PTL, peritumoral lymphatic vessel; VEGF-C, vascular endothelial growth factor C; VEGF-D, vascular endothelial growth factor D; VEGFR-3, vascular endothelial growth factor receptor 3. a b Statistical analysis. Patients were divided into groups in accordance with fifth TNM edition I (group 1), II (2), III (3), and IV (4). In this study, Wilcoxon rank-sum test was used to assess whether there is a significant difference between the PTL and ITL density measured by D2-40 immunoreactivity, and parameters of VEGF-C, VEGF-D, and VEGFR-3 expression in tissue samples. The differences between 1 and 4 group and 1 and group consisting in patients from groups 2, 3, and 4 together was examined. Basic statistical data are given in the Table 3. c d Results Density and distribution of lymphatic vessels stained with D2-40. Lymphatic vessels stained with D2-40 marker were observed in ITL and PTL compartments. The mean ITL (Fig. 2a) and PTL (Fig. 2b) density was counted in every stage separately and is given in the Table 3. Expression of VEGF-C/D and VEGFR-3 in GC specimens. The mean IRS of VEGF-C (Fig. 2c), VEGF-D (Fig. 2d), and VEGFR-3 are given in the Table 3. Relationship among lymphatic vessel density, expression of VEGF-C/D, VEGFR-3, and GC staging. Performed analyses have revealed that lymphatic vessel density measured by D2-40 decreases the outside tumor and increases within the tumor as the staging grows from I to III, whereas in the Figure 2 Lymphatic vessels stained with D2-40 (a) intratumoral lymphatic vessel (ITL) compartment 100 and (b) peritumoral lymphatic vessel (PTL) zone 100; (c) vascular endothelial growth factor (VEGF-C), 100; and (d) VEGF-D, 100. case of patients belonging to stage IV group, lymphatic vessel density decreases outside the tumor as well as within the tumor in comparison with the stage III group (not statistically significant). The average lymphatic vessel density within the border of the tumor is greater than average lymphatic vessel density within the tumor and decreases as the staging decreases (Fig. 3). Differences in density are distinct but not statistically significant. Additionally, we observed a difference between morphology of the vessels within the tumor (ITLs) and vessels that are located outside the tumor (PTLs). PTLs were enlarged, uncompressed, and unsqueezed as opposed to ITLs. 109

4 I II III IV Figure 3 Density of lymhatic vessels inside (ITL) and outside (PTL) the tumor in different stages of gastric cancer., D2-40 inside the tumor;, D2-40 outside the tumor. Table 4 Wilcoxon rank-sum test results for analyzed data set I versus IV I versus II-IV Z P Z P D2-40 (ITL) D2-40 (PTL) VEGF-C VEGF-D VEGF-R ITL, intratumoral lymphatic vessel; P, statistical validity; PTL, peritumoral lymphatic vessel; VEGF-C, vascular endothelial growth factor C; VEGF-D, vascular endothelial growth factor D; VEGFR-3, vascular endothelial growth factor receptor 3; Z, standard score. case of breast, prostate, and cervical cancer As opposed to advanced GC, in early GC more taking notice of ITL is observed. ITL is concerned as predictor for lymph nodes metastases presence in GC. 15 Therefore, it is probable that in the early GC lymphatic vessels ITLs are responsible for cancer spreading. On the other hand, as the tumor mass and diameter growths, ITLs become non-functional, and their role is taken by PTLs. Aside from lymphatic vessel density, expression of growth factors VEGF-C, VEGF-D, and VEGFR-3 were also analyzed. There are studies that have proved that VEGF-C is a predictor and is correlated to cancer stage (presence of lymph node metastases and unfavorable prognosis) in GC, 16,17 as well as in, for instance, esophagogastric junction cancer. 18 Moreover, some studies have proved that VEGF-C level within serum is correlated with metastases in lymph nodes 19 and may be used in order to monitor treatment. Needless to say, its level decreases after gastrectomy and GC treatment. 20 However, papers concerning VEGF-C are not firm. In some studies VEGF-C is not concern as a predictor. Such a role is given to the VEGF-D, which also denotes patients survival. 21 Similar discrepancies concern receptor VEGFR-3. In some cases, it is assumed that VEGFR-3 is a prognostic factor. 22 In the early cancer it has no prognostic significance, whereas in advanced cancer it has. 23 In the case of our research, no significant difference in VEGF-C, VEGF-D, and VEGFR-3 expression according to particular GC stage were discovered. Therefore, no demographic differences impact on lymphangiogeness could be stated. However it may be caused to small number of analysed cases. Further studies are needed to fully determine the clinicopathological impact of PTL and ITL density and expression of VEGF-C/D and VEGFR-3 on GC patients prognosis and determine their roles in gastric carcinogenesis. Conducted tests have not shown significant difference (at the significance level set to 95%) in analyzed parameters medians between patients from described groups in case the density of lymphatic vessels outside the tumor and expression of VEGF-C, VEGF-D, and VEGFR-3. The results are given in the Table 4. Discussion In order to measure lymphatic vessels, D2-40 was used, which is their well-known marker. Under the right conditions, it prevents cells adhesion, is involved in the shape of podocyte foot process and maintenance of gromelular permeability, and is involved in lymph vessels, but it does not influence the formation of blood vessels. 8 Performed analyses have shown that lymphatic vessel density increases within the tumor as the staging growths. An opposite result has been reported in other papers concerning GC. 9 ITLs were collapsed and squeezed, which is probably correlated with the mass effect. Sudden ITL density decrease within stage IV group might be caused by a small number of patients within this group. PTLs were expanded and their quantity decreases as the cancer staging growths. Their morphology suggests that those lymphatic vessels may probably play a more significant role in the metastases process within lymph nodes as well as in cancer spreading in the case of advanced cancer. Such a hypothesis was confirmed by other analyses, which have pointed out greater PTL endothelial tissue activity. 10,11 Similar results were obtained in the Acknowledgments The study was supported by a grant from the Medical University of Wrocław, Poland. There is no disclousure of any commercial interest that the authors may have in the subject of study or the source of any financial or material support. The authors would like to thank you Jędrzej Kabarowski for statistical analysis. References 1 Crew KD, Negut AI. Epidemiology of gastic cancer. World J. Gastroenterol. 2006; 12: Parkin DM, Bray FI, Devesa SS. Cancer burden in the year The global picture. Eur. J. Cancer 2001; 37 (Suppl 8): S Parkin DM. International variation. Oncogene 2004; 23: Folkman J. The role of angiogenesis. 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