J Interdiscipl Histopathol 2013; 1(3): 145-152
ISSN: 2146-8362
Original Research
Differences in the angiogenesis of benign and malignant
ovarian surface epithelial tumors demonstrated by
microvessel density and immunohistochemistry
Shelly Sehgal1, Prashant Goyal1, Reena Agarwal1, Sompal Singh1, Awanindra Kumar1,
Ruchika Gupta2, Vinita Kumar3, Dipti Agrawal4
1
Department of Pathology, Swami Dayanand Hospital, Dilshad Garden, New Delhi, India
2
Department of Pathology, Chacha Nehru Bal Chiktsalaya, New Delhi, India
3
Department of Gynecologic Oncology, Delhi State Cancer Institute, Dilshad Garden, New Delhi, India
4
Department of Obstetrics and Gynecology, Batra Hospital and Medical Research Centre, Tughlakabad
Institutional Area, New Delhi, India
Received: January 23, 2013
Accepted: February 04, 2013
Published Online: February 06, 2013
DOI: 10.5455/jihp.20130204114930
Corresponding Author:
Prashant Goyal,
Department of Pathology, Swami
Dayanand Hospital, Dilshad Garden,
New Delhi, India
[email protected]
Keywords: Ovarian, Angiogenesis,
MVD, CD34 intensity index, SMA
expression index
Abstract
Objectives: Angiogenesis plays a key role in tumor growth and metastasis. The analysis
of tumor vascularization by microvessel density (MVD) and its prognostic significance
has been evaluated in many tumors including ovary. However, very few studies have
tried to evaluate the characteristics of these vessels. This study aims to quantitatively
assess the characteristics of tumor vessels with the aid of immunohistochemistry and
thus, establish its role in difference in biological behavior of benign and malignant
surface epithelial ovarian tumors.
Methods: We examined 42 cases of ovarian surface epithelial tumors and divided them
in two groups (14 malignant and 28 benign tumors). Both study groups were compared
for smooth muscle and endothelial cells in tumor vessels using monoclonal antibodies
against smooth muscle actin (SMA) and CD34 (endothelial cell marker). MVD, SMA
expression index and CD34 intensity index was calculated in both groups.
Results: The malignant tumors showed higher MVD (34.5±12.9) as compared to benign
ovarian tumors (16.5±6.2) (p<0.001). Also, the blood vessels of malignant tumor
showed significantly poor SMA expression and intense CD34 expression compared with
vessels of benign tumors.
Conclusions: The present study shows higher MVD, greater endothelial cell expression
and poor muscle coat in tumor vessels of malignant ovarian surface epithelial tumors.
These results indicate higher tumor angiogenesis with thinner vessels, which facilitates
tumor spread.
© 2013 GESDAV
INTRODUCTION
Angiogenesis is a critical factor in tumor growth and
metastasis, because tumor proliferation is severely
limited by nutrient supply to proliferating tumor cells
[1]. Hence, the tumorigenesis of malignant neoplasm is
associated with extensive neovascularization. The
microvessel density (MVD) is an extensively studied
topic over past few years in variety of neoplasms
including ovarian surface epithelial tumors [2-7]. Many
studies have also established MVD as an important
145
prognostic factor in solid tumors. However, there is
paucity of literature regarding characteristics of tumor
blood vessels in surface epithelial tumors of the ovary.
We could find very few studies where nature of
proliferating
tumor
vessels
was
immunohistochemically assessed and compared
between benign and malignant ovarian surface
epithelial tumors [8-10]. Hence, there is lack of
knowledge regarding this aspect of tumor angiogenesis
till date.
Angiogenesis of benign and malignant ovarian tumors and MVD
The present study, thus, aims to evaluate not just the
density of microvessels in ovarian surface epithelial
tumors but also add to the limited existing literature
regarding maturity characteristics of proliferating
tumor
blood
vessels
with
the
aid
of
immunohistochemistry.
classified into four grades: high, moderate, low and
absent in comparison with positive control. The SMA
expression index was defined as ratio of tumor vessels
with high or moderate intensity of SMA to number of
all countable tumor vessels at 400X. These counts were
done in three hot spot areas and average SMA
expression index was calculated.
MATERIALS & METHODS
Similarly, the intensity of CD34 expression for
endothelial cells within the tumor vessels in each
ovarian tumor was also classified into 4 grades: high,
moderate, low and absent in comparison with each
positive control. CD34 intensity index was defined as
ratio of tumor vessels with high or moderate intensity
of CD34 to number of all countable tumor vessels at
400X. This ratio was also calculated in three hot spot
areas and average CD34 intensity index was calculated.
This was a retrospective study comprising 42 cases of
ovarian surface epithelial tumors diagnosed over a
period of 3 years [2010-2012]. The histological
sections were reviewed and two study groups were
made comprising of 28 benign cases and 14 malignant
cases. In both the study groups, the average MVD was
calculated and characteristics of smooth muscles and
endothelium in tumor vessels were assessed
immunohistochemically. The monoclonal antibodies
used were anti-α-Smooth Muscle Actin (SMA) (clone
1A4, Biogenex, USA) for smooth muscle cells and
anti-CD-34 (clone QBEnd10, Biogenex, USA) for
endothelial cells in the tumor vessels.
Immunohistochemistry was performed using standard
protocols with microwave heat-induced antigen
retrieval in citrate buffer [pH 6.0] and streptavidinbiotin complex technique with 3,3'-diaminobenzidine
tetrahydrochloride (DAB) as the chromogen. Negative
control was set comprising of omission of each primary
antibody. Positive control was section from normal
ovary. In each case, the value of MVD, SMA
expression index and CD34 intensity index was semiquantitatively expressed (as described below) by two
pathologists (ShS and PG) and mean value was
calculated and compared between benign and
malignant group.
Measuring MVD
The CD34 stained sections were examined at 100X
magnification to delineate “hot spots” i.e. areas of
maximal MVD. In three such hot spots in each case, all
microvessels (any brown staining endothelial cell
clearly separated from adjacent microvessels, tumor
cells, or other connective tissue elements was
considered to be single countable microvessel) were
counted at 400X magnification by two pathologists
independently. The average MVD of three fields at
400X magnification (per 0.375 mm2) was calculated.
Large vessels with thick muscular wall were excluded
from the count.
Measuring SMA expression and CD 34 intensity
index
Density of smooth muscle cells, which was exhibited
by SMA staining in tumor vessels of each tumor, was
Statistical test (student t-test) was applied to assess the
significance of difference between benign and
malignant groups. A p value of <0.05 was taken as
statistically significant.
RESULTS
Forty two cases of ovarian surface epithelial neoplasms
were reviewed by two pathologists (ShS and PG). Out
of these, 28 were benign tumors (Fig. 1a, 1b) and 14
were malignant (Fig. 2a-2d). The histopathological
types of these tumors and their numbers are shown in
Table 1.
MVD
The average micro vessel count of malignant group was
34.5±12.9 per 0.375 mm2 and that of benign group was
16.5±6.2 per 0.375 mm2 (Table 2). Average MVD of
malignant tumors was statistically significantly higher
than that of benign tumors (p<0.001) (Fig. 3a, 3b).
SMA expression and CD34 intensity index
The average SMA expression index in malignant and
benign ovarian tumors was 7.7±1.2 and 50.1±5.5
percent respectively (p <0.001) (Table 3). The intensity
of SMA expression in the vessels of malignant ovarian
tumors was significantly lower than that in benign
tumors. Moreover, the tumor vessels in benign ovarian
tumors displayed a constant high density of smooth
muscle cells that was identified by the α-SMA
immunostaining, whereas tumor vessels in malignant
ovarian tumors demonstrated an attenuated or
discontinuous density of these cells (Fig. 4a-4c).
Conversely, the average CD34 intensity index in
malignant ovarian tumors (57.54±8.15) was
significantly higher than that in benign tumors
(9.6±3.3) (p <0.001) (Table 4) (Fig. 5a, 5b).
J Interdiscipl Histopathol 2013; 1(3): 145-152
146
Sehgal et al.
Figure 1. A. Ovarian serous adenofibroma with prominent stromal component. B. Mucinous cystadenoma of the ovary (H&E, x100
for A and B).
Figure 2. A. Serous cystadenocarcinoma of the ovary showing complex papillary architecture. B. Clear cell carcinoma of the ovary
showing papilla lined by clear & vacuolated cells. C. Carcinosarcoma of the ovary with both epithelial and mesenchymal
components. D. Transitional cell carcinoma of the ovary showing broad papillae (H&E, x 100 for A and C; x200 for B and D).
147
J Interdiscipl Histopathol 2013; 1(3): 145-152
Angiogenesis of benign and malignant ovarian tumors and MVD
Figure 3. A. Benign ovarian surface epithelial tumor showing lower density of microvessels as indicated by arrows. B. Malignant
ovarian surface epithelial tumor showing higher MVD (CD34 with DAB, x200 for A and B).
Figure 4. A. Normal ovary showing SMA expression acting as positive control. B. Benign ovarian surface epithelial tumor showing
complete and intense SMA expression. C. Malignant ovarian surface epithelial tumor showing incomplete and weak SMA
expression as indicated by arrow (SMA with DAB, x200 for A; x400 for B and C).
J Interdiscipl Histopathol 2013; 1(3): 145-152
148
Sehgal et al.
Figure 5. A. Benign ovarian surface epithelial tumor showing lower CD34 expression. B. Malignant ovarian surface epithelial tumor
showing higher CD34 expression (CD34 with DAB, x 200 for A; x400 For B).
Table 1. The histopathological types of ovarian tumors
BENIGN
(n)
23
5
28
Serous tumor
Mucinous tumor
Clear cell carcinoma
Carcinosarcoma
Transitional cell carcinoma
TOTAL
MALIGNANT
(n)
9
2
1
1
1
14
Table 2. Average microvessel density in benign and malignant ovarian tumors
Benign tumors
Serous adenoma
Serous adenofibroma
Mucinous adenoma
TOTAL
Malignant tumors
Serous carcinoma
Mucinous carcinoma
Clear cell carcinoma
Transitional cell carcinoma
Carcinosarcoma
TOTAL
149
Number of cases
(n)
MVD
(Mean±SD)
19
4
5
28
17.2 ± 5.1
7.9 ±1.6
20.4 ±6.5
16.5 ± 6.2
9
2
1
1
1
14
34.2 ± 15.1
44.3 ± 3.3
26
32
27
34.5 ± 12.9
J Interdiscipl Histopathol 2013; 1(3): 145-152
Angiogenesis of benign and malignant ovarian tumors and MVD
Table 3. Average SMA Expression in benign and malignant ovarian tumors
Number of cases
(n)
Average SMA expression index (%)
(Mean±SD)
Serous adenoma
19
49.9 ± 5.3
Serous adenofibroma
4
45.2 ± 4.7
Mucinous adenoma
5
54.8 ± 2.8
TOTAL
28
50.1 ± 5.5
9
9.3 ± 2.1
Mucinous carcinoma
2
9.1 ± 0.2
Clear cell carcinoma
1
6.5
Transitional cell carcinoma
1
7.5
Carcinosarcoma
1
9.8
TOTAL
14
7.7 ± 1.9
Benign tumors
Malignant tumors
Serous carcinoma
Table 4. Average CD34 expression in benign and malignant ovarian tumors
Number of cases
(n)
Average CD34 intensity index (%)
(Mean ± SD)
Serous adenoma
19
9.5 ± 2.5
Serous adenofibroma
4
6.6 ± 1.4
Mucinous adenoma
5
11.9 ± 5.2
TOTAL
28
9.6 ± 3.3
Benign tumors
Malignant tumors
Serous carcinoma
9
58 ± 6.3
Mucinous carcinoma
2
45.5 ± 5.7
Clear cell carcinoma
1
72
Transitional cell carcinoma
1
50
Carcinosarcoma
1
56
TOTAL
14
57.5± 8.2
DISCUSSION
The growth of solid tumors beyond 2-3 mm in greatest
dimension depends on development of new vessels
(tumor angiogenesis). It has been suggested that tumor
angiogenesis can facilitate the expansion of primary
neoplasm and increases its proliferative rate. However,
the nature of proliferating blood vessels and its role in
regulating the biological behavior of a neoplasm
(benign/malignant) has not been explicitly discussed in
literature. The degree of angiogenesis of a tumor, as
assessed by MVD has emerged as a powerful candidate
for prognosis and as a predictive tool. Earlier studies
done in breast cancer [2] and cutaneous melanoma [11]
have established MVD as an important prognostic
indicator in these tumors. Few studies have shown a
link between MVD and prognosis in several solid
tumors also such as those of lung, prostate, ovary, head
and neck, cervix, esophagus, colon and non-small cell
lung carcinoma [2-7, 11-13]. A high MVD was
associated with both low relapse free and overall
survival in these studies.
Few studies have also tried to assess and compare
MVD of benign and malignant ovarian surface
epithelial neoplasms [9, 14, 15]. These studies
concluded that average MVD was significantly higher
in malignant ovarian surface epithelial neoplasms and
higher MVD was associated with transformation and
acquisition of invasive phenotype of advanced
epithelial ovarian cancers. However, there are also few
contradictory studies [8, 16, 17] in literature, which
have found no significant difference in MVD values of
benign and malignant ovarian surface epithelial tumors
J Interdiscipl Histopathol 2013; 1(3): 145-152
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Sehgal et al.
and believe that rather than spreading via vasculature,
these tumors generally spread via peritoneal
dissemination and tumor angiogenesis is unlikely to
play any role in such kind of spread. In our study,
average MVD was significantly higher in malignant
neoplasms as compared to benign tumors similar to
Wang et al [9] and contradictory to Thomassin et al
[16] findings. We forward that despite same
methodology adopted in various studies to measure
average MVD, there are conflicting results in literature.
This could be accounted to tumor heterogeneity with
respect to MVD between different areas of same
section or between corresponding areas in different
sections and between different blocks from same
tumor. We could not ascertain long term prognosis of
patients in our study because of lack of follow- up data.
Hence, larger and prospective studies are needed to
evaluate utility and prognostic value of MVD in
ovarian surface epithelial malignancies.
In our study, we also tried to assess periendothelial
support covering (pericyte and smooth muscle cells) of
blood vessels which apart from MVD, is also important
to distinguish between vessels of benign and malignant
ovarian surface epithelial tumors. This periendothelial
support covering is now coming into focus as important
regulator of angiogenesis and blood vessel function
[18]. We performed α-SMA immunostain to highlight
smooth muscle cells in our study. We found that
vessels of malignant neoplasms have reduced SMA
expression as compared to vessels of benign tumors.
Similarly, in our study we also tried to examine the
endothelial cells within tumor blood vessels and
concluded that intensity of CD34 expression in vessels
of malignant tumor was significantly higher as
compared to benign tumors. Thus, malignant ovarian
tumors statistically showed higher production of
immature blood vessels with paucity of smooth muscle
support and endothelial cell proliferation as compared
to benign tumors. These results suggest that tumor
vessels in malignant tumors are thin-walled, which
allows easier vascular invasion and dissemination.
There is a paucity of existing literature on maturity
characteristics of tumor blood vessels in surface
epithelial ovarian neoplasms. There are very few
studies where immunohistochemistry has been used to
ascertain smooth muscle covering and endothelial cell
proliferation in neo- vessels of benign and malignant
ovarian surface epithelial tumors [8-10]. Emoto et al.
[8] correlated preoperative intratumoral vascularization
assessed by color Doppler ultrasound with the paucity
of smooth muscle support in malignant ovarian tumors.
The vessels of malignant tumors significantly
demonstrated poor SMA expression and intense CD34
expression compared with the vessels of benign tumors.
Our results were in concordance with this study. Thus,
this aspect of angiogenesis is important in malignant
151
neoplasms which are characterized by not just higher
micro-vessel density of neo-vessels but also
proliferation of more immature, leaky and fenestrated
capillaries which allows better permeability of tumor
cells at invasive front.
To conclude, malignant surface epithelial ovarian
tumors are associated with higher vascular proliferation
as compared to benign lesions. Also, the angiogenesis
in malignant neoplasms is more primitive type where
there is paucity of periendothelial support cells and
thus, higher permeability for tumor cells. This can
attribute to different biological behavior of malignant
tumors in form of local invasion, omental and distal
metastasis. In view of these results, new therapy should
target not only MVD of malignant tumors but also
attempt to alter the ratio of immature/ mature vessels
for successful outcomes [19, 20]. Further larger studies
on this topic are warranted.
CONFLICTS INTEREST
The authors declare that they have no conflict interest.
SOURCE OF SUPPORT
None
REFERENCES
1. Bamberger ES, Perrett CW. Angiogenesis in epithelial
ovarian cancer. Mod Pathol 2002; 55:348-359.
2. Toi M, Kashitami J, Tominaga T. Tumour angiogenesis is
an independent prognostic indicator of breast carcinoma.
Int J Cancer 1993; 55:371-374.
3. Weidner N, Carroll PR, Flax J, et al. Tumor angiogenesis
correlates with metastasis in invasive prostate carcinoma.
Am J Pathol 1993; 143:401-409.
4. Macchiarini P, Fontanini G, Hardin MJ, Blumfeld W,
Folkman J. Relation of neovascularization to metastasis
of non-small-cell lung cancer. Lancet 1992; 340:145-146.
5. Tanigawa N, Amaya H, Matsumura M, Shimomatsuya T,
Horiuchi T, Muraoka R, Iki M. Extent of tumor
vascularization
correlates
with
prognosis
and
hematogenous metastasis in gastric carcinomas. Cancer
Res 1966; 56:2671-2676.
6. Takebayashi Y, Aklyama S, Yamada K, Akiba S, Aikou
T. Angiogenesis as an unfavorable prognostic factor in
human colorectal carcinoma. Cancer 1966; 78:226-231.
7. Tanigawa N, Amaya H, Matsumura M, Lu C, Kitaoka A,
Matsuyama K, Muraoka R. Tumor angiogenesis and
mode of metastasis in patients with colorectal cancer.
Cancer Res 1997; 57:1043-1046.
8. Emoto M, Iwasaki H, Mimura K, Kawarabayashi T,
Kikuchi M. Differences in the angiogenesis of benign and
J Interdiscipl Histopathol 2013; 1(3): 145-152
Angiogenesis of benign and malignant ovarian tumors and MVD
malignant ovarian tumors, demonstrated by analyses of
color doppler ultrasound, immunohistochemistry, and
microvessel density. Cancer 1997; 80:899-907.
9. Wang J, Lv F, Fei X, Cui Q, Wang L, Gao X, Yuan Z,
Lin Q, Lv Y, Liu A. Study on characteristics of contrast
enhanced ultrasound and its utility in assessing
microvessel density in ovarian tumors or tumor like
conditions. Int J Biol Sci 2011; 7:600-606.
10. Kobayashi H, Tsuruchi N, Sugihara K, Kaku T, Saito T,
Kamura T, Tsukamoto N., Nakano H., Taniguchi S.
Expression of alpha- smooth muscle actin in benign or
malignant ovarian tumors. Gynecol Oncol 1993; 48: 308313.
11. Srivastava A, Laidler P, Davies RP, Horgan K, Hughes
LE. The prognostic significance of tumour vascularity in
intermediate thickness (0.76–4.0 mm thick) skin
melanoma: a quantitative study. Am J Pathol 1988;
133:419-423.
12. Albo D, Granick MS, Jhala N, Atkinson B, Solomon MP.
The relationship of angiogenesis to biological activity in
human squamous cell carcinomas of the head and neck.
Ann Plast Surg 1994; 32:588-594.
13. Ohta Y, Endo Y, Tanaka M, Shimizu J, Oda M, Hayashi
Y, Watanabe Y, Sasaki T. Significance of vascular
endothelial growth factor messenger RNA expression in
primary lung cancer. Clin Cancer Res 1996; 2:1411-1416.
14. Rossochacka-Rostalska B, Gisterek IJ, Suder E,
Szelachowska JK, Matkowski RA, Lacko A, Kornafel JA.
Prognostic significance of microvessel density in ovarian
cancer. Wiad Lek 2007; 60: 129-137.
15. Orre M, Miri L, Mamers P, Rogers PA. Increased
microvessel density in mucinous compared with
malignant serous and benign tumors of the ovary. Br J
Cancer 1998; 77:2204-2209.
16. Thomassin Naggara I, Bazot M, Darai E, Callard P,
Thomassin J, Cuenod CA. Epithelial ovarian tumors:
Value of Dynamic Contrast enhanced MR imaging and
correlation with tumor angiogenesis. Radiology 2008;
248:148-159.
17. Alvarez AA, Krigman HR, Whittaker RS, Dodge RK,
Rodriguez GC. The prognostic significance of
angiogenesis in epithelial ovarian cancer. Clin Cancer
Res 1999; 5:587-591.
18. Nagy JA, Brown LF, Senger DR, Lanir N, Van de Water
L, Dvorak AM, Dvorak HF. Pathogenesis of tumour
stroma generation: a critical role for leaky blood vessels
and fibrin deposition. Biochim Biophys Acta 1988;
948:305-326.
19. Parangi S, O’Reilly MS, Christofori G, Holmgren L,
Grosfeld J, Folkman J, Hanahan D. Antiangiogenic
therapy of transgenic mice impairs de novo tumour
growth. Proc Natl Acad Sci USA 1996; 93:2002-2007.
20. Witte L, Hicklin DJ, Zhu Z, Pytowski B, Kotanides H,
Rockwell P, Böhlen P. Monoclonal antibodies targeting
the VEGF receptor-2 (Flk1/KDR) as an anti-angiogenic
therapeutic strategy. Cancer Metastasis Rev 1998;
17:155-161.
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