Radosław Blok
1, Julia Bar
2, Michał Jeleń
2, Łukasz Jagielski
1, Marian Gryboś
1Immunohistochemical 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 sub-stances 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 ovar-ian 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 correla-tions 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 den-sity 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 histo-patologicznych.
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 wywia-du 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 surowi-czych rakach jajnika niż w łagodnych gruczolakach surowisurowi-czych jajnika (P = 0,0002). Poziomy ekspresji MVD w zależności od stopnia zróżnicowania komórkowego (Grading) były na granicy istotności statystycznej (P = 0,07),
Adv Clin Exp Med 2011, 20, 6, 737–743 ISSN 1230-025X
ORIGINAL PAPERS
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 surowi-czych 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 prob-lem 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 infec-tions, endometriosis and even the use of some cos-metics. 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 pro-gression of cancer [1]. Microvessel density (MVD) is believed to be a good prognostic indicator in ovarian neoplasms because the velocity of neovas-cularization is an important factor in the process of tumor growth [2, 3]. Antigen CD34, which be-longs 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 recog-nized 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 glyco-protein. 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 sur-face and directed to the inside part of blood ves-sels [5]. The microscopic estimation expression of antigen CD34, based on a computer system of picture analysis, allows a quantitative represen-tation of the vessels parameters in examined tis-sues [6, 7]. So far, the function of this glycopro-tein 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 cre-ated 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 activ-ity of cells. In addition, they found connections between neoangiogenesis and the process migra-tions 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 ex-pression of MVD in serous ovarian carcinomas in relation to the clinicopathological parameters of tumors.
Material and Methods
The clinical material (41 cases) involved 34 wo-men treated at the 1st Departwo-ment and Clinic of Gy-necology and Obstetrics, Wroclaw Medical Univer-sity 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 un-derwent primary operation were qualified for the study. Cases with second-look operations, recur-rence of the disease, metastases and previous che-motherapy were excluded. Moreover, patients who had received oncological treatment for other neo-plastic diseases in the past were also excluded. The control group for CD34 microvessel density assess-ment was comprised of 15 patients with benign se-rous 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 (SO4)2 ×12 H2O
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 speci-mens were washed in TBS (0.05 M hydroxyme- thylaminomethane – TRIS pH 7.6 + 0.15 M NaCl + 1% bovine albumin). The activity of endog-enous 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 (Rab-bit Anti Human CD34 Antigen class 1 N 1574 LSAB), was used to demonstrate CD34 expres-sion in the malignant tissue. Every sample was incubated for 20 minutes at room temperature and next washed in TBS (2 × 5 minutes). The sec-ond 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’-di-aminebenzidine tetrahydrochloride) was used again as a substrate. Next, the CD34 preparations were stained with Mayer’s hematoxylin, dehy-drated and covered with a cover glass and
pre-served 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 pro-gram for picture analysis. Areas with the high-est 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 Micro-soft 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
Microscopic pictures of a specimen treated with the above-described method differences be-tween 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 ovar-ian 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
Number of cases (Liczba przypad-ków) N
Mean value MVD expression (Średnia wartość ekspresji MVD)
P value (Istotność statys- tyczna) Ovarian
carcino-mas – 41 15.26 + 5.56 0.0002
Normal tissues
– 15 9.17 + 4.04
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 Number of cases
(Liczba przypadków) Mean value MVD expression (Średnia wartość ekspresji MVD) P value (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
the time period between 1–12 months after sur-gery 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 nonspe-cific, which makes a proper diagnosis more dif-ficult 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 bet-ter 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 thera-py. 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].
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 signifi-cance 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 tu-mors, whereas in our study the analysis was per-formed 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 se-rous ovarian carcinomas. The levels of the CA125 antigen in serum blood positively correlates with microvessel density (MVD) and the clinical stage of the tumor.
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 signifi-cant, 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) Number of cases
(Liczba przypadków) Ca 125 mean MVD P value
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) Number of cases
(Liczba przypadków) Ca 125 mean MVD P value
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) Number of cases (Liczba przypadków) Mean value MVD expression (Średnia wartość ekspresji MVD) P value (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) Number of cases (Liczba przypadków) Mean value MVD expression (Średnia wartość ekspresji MVD) P value (Istotność statystyczna)
1–12 8 18.46 + 4.56 P = 0.097
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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: [email protected]
Conflict of interest: None declared
