Different Angiogenic Pathways in Human Cervical Cancers

GYNECOLOGIC ONCOLOGY ARTICLE NO.

68, 38 – 44 (1998)

GO974876

Different Angiogenic Pathways in Human Cervical Cancers Keizo Tokumo, M.D., Junichi Kodama, M.D., Noriko Seki, M.D., Yoshie Nakanishi, M.D., Yasunari Miyagi, M.D., Shigehito Kamimura, M.D., Mitsuo Yoshinouchi, M.D., Hiroyuki Okuda, M.D., and Takafumi Kudo, M.D. Department of Obstetrics and Gynecology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan Received February 3, 1997

acidic and basic fibroblast growth factor, transforming growth factor-a and transforming growth factor-b, vascular endothelial growth factor (VEGF), interleukin-8, tumor necrosis factor-a, and platelet-derived endothelial cell growth factor (PDECGF)/thymidine phosphorylase (dThdPase) [4 – 8]. Among these peptides, it is widely known that VEGF and PD-ECGF have angiogenic activity in vivo. VEGF is thought to be a selective growth factor for endothelial cells [9]. Four molecular isoforms of VEGF are generated by alternative splicing of mRNA, and are composed of 206, 189, 165, and 121 amino acid residues [10]. The 2 shorter isoforms, VEGF165 and VEGF121, are secreted proteins which may act as diffusible agents, whereas the longer isoforms remain cell associated [11, 12]. PD-ECGF was first isolated from platelets. The transfection of the PD-ECGF gene into ras-transformed NIH 3T3 cells resulted in a dramatic increase of angiogenecity in the nude mouse model [13]. Recently, it was reported that PD-ECGF is identical to dThdPase, an enzyme involved in nucleic acid metabolism [14, 15]. PD-ECGF stimulates chemotaxis of endothelial cells in vitro and exhibits angiogenic activity in vivo [16]. In accordance with this, it has been demonstrated that dThdPase has angiogenic activity, and that its enzymatic activity is essential for angiogenesis [17]. There have been few studies investigating angiogenic factors in cervical cancer. The purpose of this study was to examine the correlation of the expression of the angiogenic factors, VEGF, and PD-ECGF expression with clinicopathological features including microvasculature in cervical cancers. The association between microvessel density and clinicopathological features was also examined.

Objective. The objective of this study is to clarify the association between the expression of two types angiogenic factors, vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF)/thymidine phosphorylase (dThdPase) and clinicopathological features, including tumor angiogenesis, in cervical cancers. Methods. The expression of VEGF and PD-ECGF was evaluated by immunohistochemical staining of tumor specimens from 73 patients with stage Ib–IIb cervical cancer (51, squamous cell carcinoma; 19, adenocarcinoma; 3, adenosquamous carcinoma) who underwent radical hysterectomy. The microvessel density was assessed by immunostaining for factor VIII-related antigen in the most neovascularized area. Results. The microvessel density in adenocarcinomas was significantly higher than that in squamous cell carcinomas (P < 0.01). The intensity of VEGF expression in adenocarcinomas was significantly stronger than that in squamous cell carcinomas (P < 0.05). In contrast, the expression of PD-ECGF in squamous cell carcinomas was significantly higher than that in adenocarcinomas (P < 0.0001) and adenosquamous carcinomas (P < 0.01). There was an inverse relationship between VEGF expression and PDECGF expression among all patients studied (P < 0.001). The microvessel density was significantly correlated with the intensity of VEGF expression (P < 0.05). In contrast, there was no correlation between the microvessel density and the expression of PDECGF. Conclusions. The expression of VEGF appears to be involved in the promotion of angiogenesis in cervical cancers. Furthermore, we propose that angiogenic pathways may be different in different types of cervical cancers. © 1998 Academic Press

INTRODUCTION It is well known that angiogenesis, the development of new blood vessels, is essential in tissue development, reproduction, and wound healing [1]. In addition, solid tumors require angiogenesis for progression and metastasis. In fact, tumor growth beyond 1–2 mm is strictly dependent on angiogenesis [2]. Angiogenesis also contributes to the metastatic process, carrying cancer cells into the circulation [3]. Tumors are thought to secrete angiogenic factors which promote neovascularization around tumors [1]. Recently, several angiogenic factors have been identified. These include 0090-8258/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

MATERIALS AND METHODS Tissues and Patients Seventy-three patients with a diagnosis of stage Ib–IIb cervical cancer were included in this study. All patients underwent radical hysterectomy at the Department of Obstetrics and Gynecology at Okayama University Medical School between 1992 and 1995. Tumor specimens from the primary lesion were obtained at the time of surgery. Specimens were imme38

ANGIOGENIC FACTORS AND CERVICAL CANCER

39

FIG. 1. Microvessel immunostaining with anti-factor VIII-related mAb. Microvessels are represented by clusters, which stand out sharply from other tissues (magnification, 3100).

diately fixed in 10% neutral-buffered Formalin and embedded in paraffin. The histological cell types of tumors were assigned according to the WHO classification: 51 were classified as squamous cell carcinoma, 19 as adenocarcinoma, and 3 as adenosquamous carcinoma. Clinical staging was reviewed based on the International Federation of Gynecology and Obstetrics (FIGO) staging system: 22 were stage Ib, 3 were stage IIa, and 48 were stage IIb. Immunohistochemical Methods Expression of factor VIII-related antigen, VEGF, and PDECGF were assessed in paraffin-embedded sections with the ABC procedure as described elsewhere [18]. As primary antibody, anti-factor VIII-related monoclonal antibody (Dakopatts, Copenhagen, Denmark), anti-human VEGF monoclonal antibody (Pepro Tech, London, England), and anti-dThdPase (Rosche Institute, Kanagawa, Japan) monoclonal antibody were used in each staining. For positive controls, corpus luteum known to express VEGF was stained for VEGF and placenta was stained for

PD-ECGF [19, 20]. Negative controls were carried out using nonspecific IgG as the primary antibody. Staining Evaluation The number of microvessels was recorded by counting the positively stained endothelial cell or endothelial cell cluster as a single, countable microvessel in a 1003 microscopic field (0.618 mm2), selecting several of the most neovascularized areas. The top three counts were used as the microvessel density for each case. With regard to VEGF, the immunoreactivities were graded as negative (2), weak positive (6), and positive (1) according to the staining intensity. We assessed negative (2) when no detectable stain was found, positive (1) when the staining intensity was compatible with corpus luteum, and weak positive (6) when the staining intensity was weaker than that of corpus luteum. Heterogeneity in the intensity of tumor cell staining within specimens, although infrequently noted, was not included in the determination of the grading. With regard to PD-ECGF expression in cancer cells, we assessed positive (1) when unequivocal staining of cyto-

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TOKUMO ET AL.

TABLE 1 Microvessel Counts in Cervical Cancers

FIGO stage Ib II Histological cell type Squamous cell carcinoma Adenocarcinoma Adenosquamous carcinoma Cervical infiltration depth %1/2 .1/2 Pelvic lymph node metastasis Positive Negative

No. of cases

Microvessel count (mean 6 SD)

22 51

99.1 6 44.3 100.1 6 26.3

51 19 3

89.3 6 22.8 126.6 6 45.9 110.0 6 9.3

22 51

98.4 6 29.5 100.5 6 35.9

15 58

100.7 6 20.1 99.6 6 36.8

Note. Statistics (Mann–Whitney test): squamous cell carcinoma versus adenocarcinoma, P , 0.01.

plasm or nuclear compartment was seen in more than 10% cancer cells. Statistical Analyses Statistical analysis was performed using a Mann–Whitney test, a Fisher’s exact probability test, or a one-way analysis of variance (one-way ANOVA). Probability values less than 0.05 were considered statistically significant.

PD-ECGF Expression and Clinicopathological Features Figure 3 illustrates a representative PD-ECGF immunostaining in the cancer cells. We found some cases which showed PD-ECGF-positive cells scattered in the stroma. Overall, 49 of the 73 cervical cancers expressed PD-ECGF in cancer cells (67.1%). The expression of PD-ECGF in squamous cell carcinomas was significantly higher than that in adenocarcinomas and adenosquamous carcinomas (Table 3). We could find no correlation between the PD-ECGF expression and other clinical features. Taking these results together, we found an inverse correlation between VEGF and PD-ECGF expression among all patients studied (Table 4). Relationship between VEGF or PD-ECGF Expression and Microvessel Density The microvessel density was significantly correlated with the intensity of VEGF expression (Table 5). In contrast, there was no correlation between the microvessel density and the expression of PD-ECGF (Table 5). DISCUSSION It has been reported that microvessel density has some relation to metastases and predicts patient prognosis in diverse types of malignancies arising from breast, lung, prostate, and head and neck [21–24]. These reports imply that angiogenesis is stimulated during tumor progression. There are conflicting studies on whether microvessel density predicts prognosis in cervical cancer. Several studies demonstrated that high mi-

RESULTS Microvessel Density and Clinicopathological Features Representative factor VIII staining is shown in Fig. 1. In most cases, the microvessel density was higher at the invasive edge of the tumor than within the tumor. Microvessel counts varied from 54 to 264 counts/3 fields (1003 microscopic field). The average microvessel counts in 73 cervical cancers was 99.8 6 33.9. There was a close correlation between the microvessel density and histological cell types. The microvessel density in adenocarcinomas was significantly higher than that in squamous cell carcinomas (Table 1). No correlation was found between the microvessel density and other clinical features. VEGF Expression and Clinicopathological Features Figure 2 illustrates a specimen of adenocarcinoma with positive staining for VEGF. Overall, 53 of the 73 cervical cancers expressed VEGF (72.6%). The intensity of VEGF expression in adenocarcinomas was significantly stronger than that in squamous cell carcinomas (Table 2). We did not find any correlation between the VEGF immunoreactivity and other clinical features.

TABLE 2 VEGF Expression in Cervical Cancers VEGF expression

FIGO stage Ib II Histological cell type Squamous cell carcinoma Adenocarcinoma Adenosquamous carcinoma Cervical infiltration depth %1/2 .1/2 Pelvic lymph node metastasis Positive Negative

(2)

(6)

(1)

7 13

8 20

7 18

17 3 0

20 7 1

14 9 2

12 8

20 8

19 6

4 16

8 20

3 22

Note. Statistics (Mann–Whitney test): squamous cell carcinoma versus adenocarcinoma, P , 0.05.

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ANGIOGENIC FACTORS AND CERVICAL CANCER

FIG. 2. Immunohistochemical staining of VEGF in adenocarcinoma. Cytoplasmic staining of VEGF (1) is clearly seen in tumor cells (original magnification, 3100).

crovessel density is a useful indicator for the prognosis in cervical cancer [25–28]. Rutgers et al. reported no correlation between vascularity and prognosis in squamous cell carcinoma of the cervix [29]. Furthermore, Kainz et al. found that patients with cervical cancer showing low microvessel density had a significant poorer recurrence-free interval [30]. In our study, the observation period was too short and only seven cases was recurred. Therefore, survival data are not yet available. Interestingly, this study found that the microvessel density of adenocarcinomas is higher than that in squamous cell carcinomas. Recently, it was shown that microvessel density varies with histological cell types in epithelial ovarian cancers [31]. Takahashi et al. reported that vessel count is higher in intestinaltype than in diffuse-type gastric cancers [32]. It is postulated from these findings that angiogenesis would be stimulated in different manners depending on histological cell types. It is widely known that prognosis is worse for adenocarcinoma than for squamous cell carcinoma. Therefore, prognosis analysis should be made in homogeneous histological cell type to determine whether the microvessel density is an important prognostic factor in cervical cancers.

TABLE 3 PD-ECGF Expression in Cervical Cancers PD-ECGF expression

FIGO stage Ib II Histological cell type Squamous cell carcinoma Adenocarcinoma Adenosquamous carcinoma Cervical infiltration depth %1/2 .1/2 Pelvic lymph node metastasis Positive Negative

(2)

(1)

6 18

16 33

8 13 3

43 6 0

17 7

34 15

3 21

12 37

Note. Statistics (Fisher’s exact probability test): squamous cell carcinoma versus adenocarcinoma, P , 0.0001; squamous cell carcinoma versus adenosquamous carcinoma, P , 0.01.

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TOKUMO ET AL.

FIG. 3. Immunohistochemical staining for dThdPase in squamous cell carcinoma. There is a cytoplasmic and nuclear staining of cancer cells (original magnification, 3100).

The association of the expression of angiogenic factors and the clinicopathological features including microvessel density has scarcely been examined in cervical cancers. It is, of course, important to determine which angiogenic factor predominantly mediates angiogenesis because it could offer novel opportunities for therapeutic intervention of this disease. In this study, we explored whether the expression of VEGF and PD-ECGF correlate with vascularization and clinicopathological features in cervical cancers. TABLE 4 Association between VEGF Expression and PD-ECGF Expression in Cervical Cancers VEGF expression PD-ECGF expression

(2)

(6)

(1)

(2) (1)

2 18

8 20

14 11

Note. Statistics (Mann–Whitney test): P , 0.001.

We demonstrated that VEGF expression was stronger in adenocarcinomas, while PD-ECGF expression was higher in squamous cell carcinomas. Recently, we reported that VEGF expression is stronger in mucinous cystadenocarcinomas than in endometrioid carcinomas and clear cell carcinomas of the ovary [33]. O’Brien et al. demonstrated that VEGF and PDECGF expression is up-regulated in bladder cancer but is differentially expressed. VEGF expression is higher in superficial tumors compared to invasive tumors and the reverse is true for PD-ECGF [34]. Saeki et al. showed that the incidence of PD-ECGF expression is higher in polypoid growth colon carcinomas than in nonpolypoid ones [35]. Takahashi et al. reported that VEGF is higher in intestinal-type than diffusetype gastric cancers [32]. These data strongly suggest that angiogenic pathways vary with histological cell types. In addition, we noticed an inverse correlation between VEGF expression and PD-ECGF expression in this series of cervical cancers. A similar inverse correlation was observed, even if the histological cell types were defined to squamous cell carcinomas or adenocarcinomas (data not shown). We also found that only two cases expressed neither VEGF nor PD-ECGF. These

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TABLE 5 Association between VEGF or PD-ECGF Expression and Microvessel Counts in Cervical Cancers

VEGF (2) (6) (1) PD-ECGF (2) (1)

No. of cases

Microvessel count (mean 6 SD)

20 29 24

88.6 6 32.0 97.7 6 20.0 111.7 6 44.7

24 49

111.9 6 44.6 93.9 6 25.8

Note. Statistics (one-way ANOVA): VEGF, P , 0.05.

facts show that VEGF and/or PD-ECGF expression might play a crucial role in angiogenesis of cervical cancer. Several kinds of growth factors and cytokines were reported to induce the expression of PD-ECGF [36]. Similarly, hypoxic conditions, mutant p53, and mutant ras were known to induce VEGF expression [37–39]. The regulation of VEGF and PD-ECGF expression is still under investigation. We have demonstrated that the microvessel density was significantly correlated with the intensity of VEGF expression, but there was no statistical correlation between the microvessel density and PD-ECGF expression. This was compatible with the finding that strong VEGF mRNA expression is associated with increased microvessel density in squamous intraepithelial lesions and invasive squamous cell carcinomas of the cervix [40]. These findings indicate that VEGF exhibits a potent angiogenic effect in cervical cancers. The role of angiogenic factors in vivo is very complex and other angiogenic factors and inhibitors in vascularization should be investigated further to elucidate the ambiguity of angiogenesis in cervical cancers. It was reported that anti-VEGF monoclonal antibody potently inhibited both primary and metastatic tumor growth with no marked side effects in nude mice [41]. In addition, 59-deoxy-5-fluorouridine (59-DFUR) is a prodrug of 5-fluorouracil (5-FU) and is converted to 5-FU by PD-ECGF [42]. 59-DFUR may be effective with less toxicity in patients with PD-ECGF-positive cervical cancer. Thus, VEGF and PD-ECGF can be an important target of antitumor agents in cervical cancer. In conclusion, the expression of VEGF is involved in the promotion of angiogenesis in cervical cancers. Furthermore, it was supposed that angiogenic pathways may be different in different types of cervical cancers. ACKNOWLEDGMENTS This work was partially supported by Grants-in Aid for Scientific Research (09771277) from the Ministry of Education, Science and Culture, Japan. We thank Ms. Kasumi Oomori for her help with histochemistry and section cutting and Dr. Adriana Dusso for assistance in the preparation of the manuscript.

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