Immunohistochemical Analysis of Microvessel Density in Serous Ovarian Cancers

original papers Adv Clin Exp Med 2011, 20, 6, 737 743 ISSN 1230-025X Copyright by Wroclaw Medical University Radosław Blok 1, Julia Bar 2, Michał Jeleń 2, Łukasz Jagielski 1, Marian Gryboś 1 Immunohistochemical Analysis of Microvessel Density in Serous Ovarian Cancers Analiza unaczynienia nowotworów surowiczych jajnika na podstawie immunohistochemicznej oceny gęstości naczyniowej w surowiczym raku jajnika 1 Ist Department of Gynaecology and Obstetrics, Wroclaw Medical University, Poland 2 Division of Pathomorphology and Clinical Cytology, Wroclaw Medical University, Poland Abstract Background. Angiogenesis is an essential process involved in the normal growth and differentiation of cells. Excessive angiogenesis is also a crucial event in the progression of cancer because better oxygen and nutrition substances supply increases of tumor growth. Microvessel density (MVD) is believed to be a good prognostic indicator in ovarian neoplasms because by using this method the authors can estimate the velocity of neovascularization. Objectives. The aim of the study was to evaluate the expression of MVD in serous ovarian cancers in relation to the clinicopathological parameters of tumors. Material and Methods. The material consisted of 41 cases of serous ovarian cancer. For clinical examinations the authors chose only patients who underwent a primary surgical operation. The control group for CD34 levels were 15 patients with benign serous adenomas. The microscopic ( 200) evaluation of blood vessels in paraffin samples were done by showing the reaction of the endothelial CD34 antigen with DAKO serum (Monoclonal Mouse Anti Human CD34 class 1 N 1574 LSAB). Results. Immunohistochemical data revealed that the mean value of MVD was significantly higher in serous ovarian carcinomas than benign serous adenomas of the ovary (P = 0.0002). Whereas the differences between MVD expression and tumor grade were on a statistical borderline (p = 0.07) there were no statistically significant correlations between FIGO, levels of Ca125 after 3 and 6 courses of chemotherapy, and time of patient survival. Conclusions. The results revealed that the expression of the CD34 antigen characterizes biological features of serous ovarian carcinomas. The levels of CA125 antigen in serum blood positively correlate with microvessel density and the clinical stage of the tumor (Adv Clin Exp Med 2011, 20, 6, 737 743). Key words: serous ovarian carcinoma, microvessel density, immunohistochemistry. Streszczenie Wprowadzenie. Angiogeneza jest zasadniczym procesem zaangażowanym we wzrost oraz różnicowanie komórek i tkanek. Nadmierna angiogeneza jest również istotnym czynnikiem w procesie progresji nowotworu, która zależy od zaopatrzenia w tlen i substancje odżywcze. Ocena gęstości naczyniowej (MVD) może być uznana za prognostyk aktywnego wzrostu nowotworu, ponieważ za jej pomocą jest możliwa ocena nowotworzenia naczyń w tkankach nowotworowych. Cel pracy. Ocena ekspresji MVD w rakach surowiczych jajnika w odniesieniu do wskaźników klinicznych i histopatologicznych. Materiał i metody. Materiał badawczy obejmował 41 przypadków raka surowiczego jajnika. Do badań włączono jedynie pacjentki, u których pierwszym etapem leczenia był zabieg chirurgiczny i które nie miały obciążonego wywiadu onkologicznego. Grupę kontrolną stanowiło 15 przypadków gruczolaka surowiczego jajnika. Przeprowadzono mikroskopową analizę naczyń ( 200) w preparatach uzyskanych z bloczków parafinowych. Ekspresję antygenu CD34 uzyskano, posługując się surowicą monoklonalną (Monoclonal Mouse Anti Human CD34 class 1 N 1574 LSAB) firmy DAKO. Wyniki. Immunohistochemiczne dane ujawniły, że średnia wartość MVD była znacząco większa w surowiczych rakach jajnika niż w łagodnych gruczolakach surowiczych jajnika (P = 0,0002). Poziomy ekspresji MVD w zależności od stopnia zróżnicowania komórkowego (Grading) były na granicy istotności statystycznej (P = 0,07),

738 R. Blok et al. ale nie znaleziono korelacji między FIGO, stężeniem CA125 po 3 i 6 kursach chemioterapii i czasem przeżycia pacjentek. Wnioski. Własne wyniki pokazują, że ekspresja antygenu CD 34 charakteryzuje właściwości biologiczne surowiczych raków jajnika. Stężenie antygenu CA125 w surowicy krwi koreluje dodatnio z gęstością naczyniową (MVD) i stopniem zaawansowania guza (Adv Clin Exp Med 2011, 20, 6, 737 743). Słowa kluczowe: raki surowicze jajnika, gęstość naczyniowa, immunohistochemia. Ovarian neoplasms are a very important problem in medicine because they account for 23% of all female genital neoplasms and they are the cause of 47% of deaths among women suffering from gynecological cancers. There are many factors that increase the probability of incidence of ovarian tumors. Among them are genetic predispositions, ethnic and social factors, fertility, diet, viral infections, endometriosis and even the use of some cosmetics. Of course, a very important role is played by the biological features of the tumor. About 2/3 of cases are diagnosed in the advanced stages of the disease and only 25% of patients have 5 years survival time. Angiogenesis is an essential process involved in the normal growth and differentiation of cells. Excessive angiogenesis is a crucial event in the progression of cancer [1]. Microvessel density (MVD) is believed to be a good prognostic indicator in ovarian neoplasms because the velocity of neovascularization is an important factor in the process of tumor growth [2, 3]. Antigen CD34, which belongs to the cluster of differentiation antigens, was described for the first time in 1984 by Civin et al. in stem cells. This cell surface antigen, formerly known as hematopoietic progenitor cell antigen 1 (HPCA1), is also called gp105-120. It is recognized by monoclonal antibodies BI.3C5, ICH3, MY10, 188.27. The human CD34 gene, which maps to chromosome 1q32, spans 26 kb and has 8 exons. CD34 is a 67 kda transmembrane glycoprotein. The biological function of CD34 is still unknown. It is used as a surface marker for very early hematopoietic stem cells [4]. In the medical literature, antigen CD34 is also called microvessel density (MVD) [3]. It is used to mark endothelial tissues as well. Antigen CD34 is located on the surface and directed to the inside part of blood vessels [5]. The microscopic estimation expression of antigen CD34, based on a computer system of picture analysis, allows a quantitative representation of the vessels parameters in examined tissues [6, 7]. So far, the function of this glycoprotein isn t completely known. During estimation of angiogenesis, Schlingemann et al. researched the presence of CD34 in the processes of healing wounds and in areas where a new microvascular web was created. Additionally, the authors showed a relationship of this antigen with the spreading of tumor vessels because they found an increased expression of antigen CD34 at the top of new created blood vessels [8]. Other authors confirmed the connection between the increased expression of antigen CD34 in the endothelium and processes of neoangiogenesis. They suggested a relationship between antigen CD34 and the proliferative activity of cells. In addition, they found connections between neoangiogenesis and the process migrations of fibroblasts [9, 10]. In many articles about microvessel density in neoplasms, the tissue level of CD34 was accepted as an important predictive factor [10, 11]. The aim of the study was to evaluate the expression of MVD in serous ovarian carcinomas in relation to the clinicopathological parameters of tumors. Material and Methods The clinical material (41 cases) involved 34 women treated at the 1st Department and Clinic of Gynecology and Obstetrics, Wroclaw Medical University and 7 patients hospitalized at the Department of Oncology and Clinic of Oncological Gynecology, Wroclaw Medical University for serous carcinoma of the ovary. The age of the patients in the study group ranged from 31 to 74 years. The mean age of the patients was 50 years. The clinical staging and grading of the disease was as follows: FIGO I 7 cases, II 2 cases, III 28, IV 4 cases and G1 8, G2 13 and G3 20 cases. Only patients who underwent primary operation were qualified for the study. Cases with second-look operations, recurrence of the disease, metastases and previous chemotherapy were excluded. Moreover, patients who had received oncological treatment for other neoplastic diseases in the past were also excluded. The control group for CD34 microvessel density assessment was comprised of 15 patients with benign serous adenoma of the ovary. The investigations were carried out at the Immunocytological Laboratory of the Department of Pathological Anatomy, Wroclaw Medical University. All of the histochemical investigations were performed on 4 nm paraffin sections after placing them on gelatinous glass (2g KCr (SO 4 ) 2 12 H 2 O + 2.5 g of gelatin). Next they were cleansed of the admixture of gelatin by placing them in xylene and passing them through a number of alcohol

MVD in Serous Ovarian Cancer 739 solutions with concentrations decreasing to pure water. Antigens of tissues fixed in formalin were determined by boiling the section specimens in a 700 W microwave oven in 0.01M citrate buffer at ph 6.0 for 30 minutes. After cooling, the specimens were washed in TBS (0.05 M hydroxymethylaminomethane TRIS ph 7.6 + 0.15 M NaCl + 1% bovine albumin). The activity of endogenous peroxidase was blocked by incubation in a 3% hydrogen peroxide solution for 5 minutes. A monoclonal Mouse antibody against the human CD34 antigen, manufactured by DAKO (Rabbit Anti Human CD34 Antigen class 1 N 1574 LSAB), was used to demonstrate CD34 expression in the malignant tissue. Every sample was incubated for 20 minutes at room temperature and next washed in TBS (2 5 minutes). The second antibody and marker complex was obtained from Universal LSAB 2 KITS HRP, Rabbit/Mouse (liquid DAB) K 0675 manufactured by DAKO. In order to demonstrate the reaction, DAB (3,3 -diaminebenzidine tetrahydrochloride) was used again as a substrate. Next, the CD34 preparations were stained with Mayer s hematoxylin, dehydrated and covered with a cover glass and preserved with Canada balsam. The internal positive control were tonsils. Negative control consisted of replacement of the primary antibody by 0.1 M Tris-buffer, ph 7.4. Obtained in such a way, the stained microscopic specimens of CD34 were characterized by distinct brown staining of the investigated vessels. The evaluation was carried out by means of MultiScan 98, a computer program for picture analysis. Areas with the highest density of active structures were searched for in the whole specimen using an Olympus BX 50 light microscope at 200-fold magnification of the image. Afterwards, the image was transferred to the computer screen. The mean value of MVD was assessed from six separate areas. Clinical data elicited from the case history as well as the findings obtained were ranked using a database from the computer program Microsoft Access. The statistical evaluation was carried out using a Student s-t test (for the comparison of mean values in a population divided into two parts), unifactoral variance analysis (for unequal populations divided into three or more subgroups) and, where necessary from the statistical viewpoint, non-parametric tests where used (χ²). A B Fig. 1. Immunohistochemical staining for CD34 in serous ovarian carcinomas. A. strong immunostaining for CD34 antigen. B. CD34 expression was limited to small number of vessels Ryc. 1. Immunohistochemiczne barwienie na CD34 w surowiczych rakach jajnika. A. silne barwienie na antygen CD34. B. ekspresja CD34 była ograniczona do niewielkiej liczby naczyń

740 R. Blok et al. Microscopic pictures of a specimen treated with the above-described method differences between MVD. Results Immunohistochemical data revealed that the mean value of MVD was significantly higher in serous ovarian carcinomas than benign serous adenomas of the ovary (p = 0.0002) (Table 1). In serous ovarian carcinomas, the immunostaining for CD34 was detected in a higher percentage of tumor tissue than in benign neoplasms. No correlation was observed between the mean value of MVD expression and FIGO stages (p > 0.05). Whereas the differences between MVD expression and tumor grade were on a statistical borderline (p = 0.07) (Table 2). The analysis of the impact of MVD on free survival time revealed that the levels of MVD don t correlate with 24 survival time of patients with ovarian cancer (Table 4). No correlation was found between the time of death and the value of MVD of patients with ovarian cancer (p > 0.09) Although, taking into account Table 1. Comparison of MDV in analyzed groups of patients, Student s-t test Tabela 1. Porównanie poziomu gęstości naczyniowej w grupie badanej i grupie kontrolnej, test t-studenta N Ovarian carcinomas 41 Normal tissues 15 Mean value MVD expression (Średnia wartość ekspresji MVD) (Istotność statystyczna) 15.26 + 5.56 0.0002 9.17 + 4.04 the time period between 1 12 months after surgery and between 13 24 months, the authors have found that the better prognosis was associated with a low value of MVD (Table 5). Discussion Concerning the complexity of mechanisms responsible for carcinogenesis, there are still no routine methods of early detection of ovarian cancer, and its biological behavior. Moreover, the symptoms of the early stage cancer are nonspecific, which makes a proper diagnosis more difficult and late. The most important prognostic factors are the clinical stage and histological grade and the type of the neoplasm. It is important to investigate new biomarkers which allow for a better prognosis of patients with ovarian neoplasms. Searching for new methods of shortening the time, a diagnosis in neoplasm diseases is very important because it gives hope for quick and effective therapy. One of the main goals is also finding additional factors that can improve therapy effectiveness and extend the period of remission and survival time [1, 12 14]. The higher vascular density in serous ovarian carcinomas than in benign serous adenomas revealed in this study is in agreement with Amis et al. [15], who observed the difference between benign tumors and malignant epithelial. Additionally, Wang et al. [16] found differences between benign, borderline and malignant. Whereas, Shen et al. [17] did not observe any differences between ovarian tumor presence at different stages of malignancy. Similarly to earlier data [18, 19], the authors also did not observe differences between CD34 expression and FIGO and tumor grade. According to Sonmezer et al. [20], the current study did not reveal an association between the 2-year follow-up of patients with serous ovarian cancer and MVD. Table 2. Comparison of MVD expression with FIGO stage and grade of serous ovarian cancers, test χ² Tabela 2. Porównanie gęstości naczyniowej w zależności od stopnia zaawansowania klinicznego i stopnia zróżnicowania komórkowego, test χ² FIGO Mean value MVD expression (Średnia wartość ekspresji MVD) (Istotność statystyczna) I II 9 14.25 + 5.89 p > 0.05 III IV 32 15.56 + 3.97 Grade (Stopień) 1 8 13.56 + 2.01 0.07 2 13 13.26 + 4.43 3 20 17.25 + 6.38

MVD in Serous Ovarian Cancer 741 Table 3. Association between the value of MVD and the levels of Ca125 after 3 and 6 courses of treatment of patients with ovarian cancer. Observed differences between Ca125 and MVD levels were not statistical significant, Student s-t test Tabela 3. Korelacja między stężeniem Ca125 po 3 i po 6 kursach chemioterapii i gęstością naczyniową MVD, test t-studenta Time of observation after 3 courses of chemotherapy (Czas obserwacji po 3 cyklach chemioterapii) Ca 125 mean MVD 23 < 35 ml/u 13.88 + 5.72 0.11 15 > 35 ml/u 16.84 + 5.04 38 mean score 15.36 + 5.38 Time of observation after 6 courses of chemotherapy (Czas obserwacji po 6 cyklach chemioterapii) Ca 125 mean MVD 27 < 35 ml/u 14.06 + 5.45 p = 0.34 8 > 35 ml/u 16.09 + 4.66 35 mean score 15.07 + 5.055 Table 4. Relationship between 24-month survival and microvessel density, Student s-t test Tabela 4. Ocena zależności między 24-miesięcznym przeżyciem a gęstością naczyniową, test t-studenta Survival 24-months (24-miesięczne przeżycie) Mean value MVD expression (Średnia wartość ekspresji MVD) (Istotność statystyczna) > 24 months 23 14.51 + 5.72 P = 0.13 < 24 months 18 16.23 + 5.09 Table 5. Time of death dependent on microvessel density, Student s-t test Tabela 5. Analiza czasu zgonu w zależności od gęstości naczyniowej, test t-studenta Time of death months (Czas zgonu miesiące) Mean value MVD expression (Średnia wartość ekspresji MVD) (Istotność statystyczna) 1 12 8 18.46 + 4.56 P = 0.097 13 24 10 14.44 + 4.99 Moreover, the patients with a high mean value of MVD showed a higher risk to death than those with a low value of MVD. The borderline significance observed between the analyzed group might be a result of the number of cases. Ogawa et al. [21] suggested that high levels of MVD in tumor tissue might be an independent prognostic factor in the early stages of ovarian tumor development. The lack of a correlation observed between CA125 levels and mean values of MVD in patients with serous ovarian cancer in clinical response observed in the present study might be partially compared with data presented by van Dalen et al. [22], who performed the analysis on advanced stages of tumors, whereas in our study the analysis was performed on patients with low and advanced stages of tumors. In this group, a high level of CA125 was accompanied by a high mean value of MVD. This result suggests that vascularization is important to shedding of tumor markers to circulation. Present results revealed that the expression of the CD34 antigen characterizes the biological features of serous ovarian carcinomas. The levels of the CA125 antigen in serum blood positively correlates with microvessel density (MVD) and the clinical stage of the tumor.

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MVD in Serous Ovarian Cancer 743 Address for correspondence: Radosław Blok Ist Department of Gynaecology and Obstetrics Wroclaw Medical University Chałubińskiego 3 50-368 Wrocław Poland Tel. +48 600 32 68 29, +48 71 784 23 47 E-mail: radblok@o2.pl Conflict of interest: None declared Received: 7.06.2011 Revised: 2.08.2011 Accepted: 5.10.2011