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Immature tumor angiogenesis in high-grade and high-stage renal cell carcinoma
BASIC SCIENCE
IMMATURE TUMOR ANGIOGENESIS IN HIGH-GRADE AND HIGH-STAGE RENAL CELL CARCINOMA TOSHIAKI KINOUCHI, MASAYUKI MANO, IKUYO MATSUOKA, SAE KODAMA, TOMOMI AOKI, MINA OKAMOTO, HISAKO YAMAMURA, MICHIYUKI USAMI, AND KATSUHITO TAKAHASHI
ABSTRACT Objectives. To investigate the correlation between pathologic findings and maturation of the tumor neovasculature of renal cell carcinoma by immunohistochemical studies. Methods. Formalin-fixed and paraffin-embedded specimens from 25 randomly selected patients with renal cell carcinoma were stained with mouse monoclonal antibodies, anti-human CD31, anti-alpha smooth muscle actin (␣SMA), and anti-human calponin by the indirect immunoperoxidase method. The microvessels were counted in six areas with the higher number of microvessels in each patient at 200⫻ magnification (0.255 mm2 per area). Results. The number of CD31-positive microvessels in grade 3 tumors was significantly lower than those in grade 1 or 2 tumors (P ⫽ 0.003222 and P ⫽ 0.043217, respectively). The CD31-positive microvessel counts of those of higher stage, tumor size greater than 4.5 cm, or non-clear cell type were significantly lower than tumors of lower stage, size less than 4.6 cm, or clear cell type. In the grade 3 tumors, the expression ratio of the number of ␣SMA-positive microvessels to the number of CD31-positive microvessels was significantly decreased compared with grade 1 or 2 tumors (P ⫽ 0.000011 and P ⫽ 0.000000, respectively). The expression of calponin in the tumor neovasculature was not observed. The expression ratios of the number of ␣SMA-positive microvessels to the number of CD31-positive microvessels in higher stages, larger tumor sizes, or non-clear cell types were significantly decreased. Conclusions. The tumor neovasculature of high-grade and high-stage tumors was immature. These results imply that high-grade tumors of renal cell carcinomas may be susceptible to antiangiogenesis therapy inducing apoptosis of immature tumor vessels. UROLOGY 62: 765–770, 2003. © 2003 Elsevier Inc.
N
eovascularization is essential to the growth and metastasis of solid tumors. Solid tumors recruit blood vessels from the neighboring tissues by angiogenesis. To stimulate angiogenesis, tumors secrete growth factors that act on endothelial cells. Increased microvessel counts have been associated with early progression in a number of tumors, including breast, colon, and prostate.1–3 In general, the clear cell type of renal cell carcinoma is highly hypervascular, but the correlation between From the Departments of Urology, Pathology, and Medicine, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka, Japan Reprint requests: Toshiaki Kinouchi, M.D., Ph.D., Department of Urology, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan Submitted: December 9, 2002, accepted (with revisions): May 8, 2003 © 2003 ELSEVIER INC. ALL RIGHTS RESERVED
the microvessel count and pathologic factors in renal cell carcinoma is still controversial.4 – 8 The suppression and degradation of angiogenesis is the strategy for treating tumors. To accomplish this, it is necessary to discriminate tumor angiogenesis from normal blood vessels. The angiogenesis factor vascular endothelial growth factor (VEGF) is overexpressed in human prostate cancer and glioblastoma. Some fraction of angiogenesis in prostate cancer and glioblastoma involves immature vessels with the absence of associated pericytes or smooth muscle cells, both of which express alpha-smooth muscle actin (␣SMA), and these immature vessels are selectively obliterated by VEGF withdrawal.9 Because the tumor tissues of renal cell carcinoma express abundant VEGF, we investigated the correlation between the microvessel count and pathologic findings and the maturation of angiogenesis by im0090-4295/03/$30.00 doi:10.1016/S0090-4295(03)00512-0 765
TABLE I. Patient characteristics, histopathologic findings, and immunohistochemical staining with anti-CD31 and anti-␣SMA antibodies Pt. No./Sex/ Age (yr)
Cell Type
1/M/48 2/M/55 3/M/43 4/M/46 5/M/46 6/M/56 7/M/55 8/M/43 9/M/50 10/F/61 11/F/72 12/M/44 13/F/72 14/M/54 15/M/61 16/M/66 17/M/69 18/M/68 19/M/58 20/M/61 21/F/63 22/M/45 23/F/67 24/M/51 25/M/65
Unclassified Unclassified Papillary Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Unclassified Clear Unclassified
Grade
Robson Stage
pT Stage
Size (cm)
CD31-Positive Microvessels (n)
3 3 3 2 2 2 1 1 1 2 2 2 2 2 2 1 1 2 1 2 1 2 3 1 3
4 4 3 2 2 1 1 1 1 2 1 1 4 1 1 2 1 1 1 1 1 1 1 1 3
4 4 3 3 3 1 1 1 1 3 1 1 3 1 1 3 1 1 1 1 1 1 1 1 3
10 10 8 6 8 6 3 5 4.5 8 3.2 3.5 6.5 5 2.5 10 3 4 2.4 3 4.5 3 5.5 4 7
91.2 94.2 121.8 150 232.7 151.7 203.7 214.7 232.3 103.5 125.8 179.7 135.3 114 124.7 116.2 146.5 149.2 226.8 176.3 212.7 252.8 163.5 264.8 60.2
(68–121) (56–115) (101–146) (132–164) (199–307) (119–176) (157–230) (181–280) (178–280) (83–139) (107–152) (123–221) (119–149) (97–124) (106–152) (93–136) (112–176) (121–193) (198–264) (94–224) (155–265) (212–288) (104–216) (204–296) (48–70)
␣SMA-Positive Vessels/ CD31-Positive Vessels Ratio (%) 0 4.7 21.6 55.8 54.7 52.4 73.5 45.8 48.6 58.7 56.7 62.6 53.4 58.6 60.8 53 61.6 58 37.8 27.7 60.2 53.6 9.7 44.5 6.5
(0–0) (0–24) (4–64) (51–62) (50–64) (26–69) (62–96) (28–61) (30–79) (54–66) (52–64) (57–70) (44–61) (56–60) (56–64) (52–57) (53–69) (55–61) (20–49) (14–31) (31–80) (35–77) (0–28) (33–49) (5–13)
KEY: ␣SMA ⫽ alpha-smooth muscle actin; Pt. No. ⫽ patient number; M ⫽ male; F ⫽ female. Numbers in parentheses are the range.
munohistochemical staining against CD31, a specific marker of endothelial cells, and ␣SMA and calponin, differentiation markers of smooth muscle.10,11
the sections were autoclaved at 121°C for 5 minutes in 0.01 M citrate buffer for antigen retrieval of CD31 and calponin. The sections were incubated first with antibodies overnight at 4°C. Thereafter, assays were performed by the indirect immunoperoxidase method with a Vectastain ABC kit for mouse IgG (Vector Laboratory, Burlingame, Calif).
MATERIAL AND METHODS PATIENTS AND DEFINITION OF CHARACTERISTICS The surgical specimens from 25 randomly selected patients (20 men and 5 women) with renal cell carcinoma were examined. The patient characteristics, including age, sex, cell type, grade, Robson stage, pT stage, tumor size, CD31-positive microvessel count, and percentage of expression ratio of the number of ␣SM-positive microvessels to number of CD31positive microvessels, are shown in Table I. The 1997 TNM classification of malignant tumors was used.12 The stage and grade of renal cell carcinoma was based on the results of the World Health Organization-sponsored conference on renal cell carcinoma in 1997.13,14 The median tumor size was 4.5 cm, and patients were divided at this point.
IMMUNOHISTOCHEMICAL STAINING The formalin-fixed and paraffin-embedded sections of the renal cell carcinoma specimens were stained with mouse monoclonal antibodies, anti-human CD31 undiluted (IgG1, Dako, Carpinteria, Calif), anti-␣SMA diluted 1:2000 (IgG2a, Sigma, St. Louis, Mo), and anti-human calponin diluted 1:2000 (IgG1, Sigma). After deparaffinized sections were inactivated with endogenous peroxidase by hydrogen peroxide, 766
MICROVESSEL COUNTS Tumor sections without necrosis or cystic changes were selected from two to three paraffin blocks in each case to detect hot spots. The selected tumor section was examined under low magnification to detect areas with high microvessel density. A single microvessel was considered any endothelial cell or cell cluster with brown reaction product that was distinct and separable from adjacent microvessels, tumor cells, and other tissue elements. We did not require the presence of a vascular lumen for a structure to be defined as a microvessel. Microvessels were counted in six areas with the higher number of microvessels (0.255 mm2 per area) by one of us (T.A.) who did not know the patient characteristics. The microvessel count was expressed as the mean number of vessels in each area.
STATISTICAL ANALYSIS Statistical analysis of the relationship between the pathologic findings and the microvessel counts expressing CD31, ␣SMA, or calponin antigens was performed using Student’s t test. Statistical significance was considered as P ⬍0.05. UROLOGY 62 (4), 2003
FIGURE 1. Immunohistochemical staining of clear cell type in grade 1 (A, B, C) and unclassified cell type in grade 3 (D, E) tumors of renal cell carcinoma. (A) Anti-CD31 staining showed numerous fine microvessels. (B) Anti-␣SMA stained almost 60% of microvessels stained by anti-CD31. (C) Anti-calponin stained no microvessels. (D) Number of anti-CD31-positive microvessels in grade 3 tumors was significantly lower than that in grade 1 tumors. (E) Anti-␣SMA stained almost none of microvessels stained by anti-CD31. Original magnification ⫻100.
RESULTS MICROVESSEL COUNTS STAINED IMMUNOHISTOCHEMICALLY BY ANTI-CD31 ANTIBODY IN RENAL CELL CARCINOMA The immunohistochemical staining of grade 1 and grade 3 renal cell carcinoma specimens by anti-CD31, anti-␣SMA, and anti-calponin are demonstrated in Figure 1. We examined whether the number of microvessels may depend on the pathologic findings. The results are summarized in Table II. The mean number of CD31-positive microvessels in grades 1, 2, and 3 was 202.2 (range 116 to 265), 158.0 (range 104 to 253), and 106.2 (range 60 to 164), respectively. The number of CD31positive microvessels in grade 3 tumors was significantly lower than those in grade 1 or 2 tumors (P ⫽ 0.003222 and P ⫽ 0.043217, respectively). The number of CD31-positive microvessels decreased depending on the tumor grade. The microvessel counts of Stage T3 and T4, Robson Stage III and IV, tumor size greater than 4.5 cm, and non-clear cell type were significantly lower than those of Stage T1 UROLOGY 62 (4), 2003
and T2, Robson Stage I and II, tumor size less than 4.6 cm, and clear cell type, respectively. NO EXPRESSION OF CALPONIN IN OF RENAL CELL CARCINOMA
TUMOR ANGIOGENESIS
The expression of calponin in the tumor neovasculature was not observed. Also, renal cell carcinoma cells did not stain immunohistochemically with the anti-human calponin antibody. DECREASED EXPRESSION RATIO OF NUMBER OF ␣SMAPOSITIVE MICROVESSELS TO NUMBER OF CD31POSITIVE MICROVESSELS IN HIGH-GRADE AND HIGHSTAGE RENAL CELL CARCINOMA We examined whether the expression ratio, defined by the number of ␣SMA-positive microvessels to the number of CD31-positive microvessels, may depend on the pathologic findings. The results are summarized in Table III. The expression ratio in the grade 3 tumors was significantly decreased compared with the expression ratio for grade 1 or 2 767
TABLE II. Number of microvessels in a 0.255-mm2 area of renal cell carcinoma tissues stained immunohistochemically by anti-CD31 antibody Factor Grade 1 2 3 T stage 1–2 3–4 Cell type Clear Nonclear Robson stage I–II III–IV Tumor size (cm) ⬍4.6 ⬎4.5
Mean CD31-Positive Microvessels (n)
P Value
202.2 (116–265) 158.0 (104–253) 106.2 (60–164)
G1/G3 0.003222 G1/G2 0.052771 G2/G3 0.043217
183.7 (114–265) 122.8 (60–233)
0.005616
175.7 (104–265) 106.2 (60–164)
0.009079
177.1 (104–265) 100.5 (60–135)
0.003423
191.3 (125–265) 134.5 (60–233)
0.007585
Numbers in parentheses are the range.
TABLE III. Ratio of ␣SMA-positive microvessels to CD31-positive microvessels in a 0.255-mm2 area of renal cell carcinoma tissues stained immunohistochemically by anti-␣SMA and anti-CD31 antibodies Factor Grade 1 2 3 T stage 1–2 3–4 Cell type Clear Nonclear Robson stage I–II III–IV Tumor size (cm) ⬍4.6 ⬎4.5
␣SMA Positive/CD31 Positive (%)
P Value
53.1 (37.8–73.5) 54.4 (27.7–62.6) 8.5 (0–21.6)
G1/G3 0.000011 G2/G3 0.000000 G1/G2 0.78065
50.8 (9.7–73.5) 34.3 (0–58.7)
0.054236
53.9 (27.7–73.5) 8.5 (0–21.6)
0.000000
51.7 (9.7–73.5) 12.2 (0–53.4)
0.000234
53.8 (27.7–73.5) 36.5 (0–58.7)
0.034358
KEY: ␣SMA ⫽ alpha-smooth muscle actin. Numbers in parentheses are the range.
tumors. The expression ratio in tumors that were Robson Stage III and IV, greater than 4.5 cm, or non-clear cell type was significantly lower than the expression ratio in tumors that were Robson Stage I and II (P ⫽ 0.000234), less than 4.6 cm (P ⫽ 0.034358), or clear cell type (P ⫽ 0.000000), respectively. No statistically significant correlations of the expression ratios between lower and higher 768
T stages were found; however, the expression ratio in higher T stages tended to decrease. COMMENT We demonstrated that the number of CD31-positive microvessels in grade 3 tumors is significantly lower than the number in grade 1 and 2 tumors. UROLOGY 62 (4), 2003
The CD31-positive microvessel count in tumors of higher stage, greater than 4.5 cm, or non-clear cell types was significantly lower than the count in tumors of lower stage, less than 4.6 cm, or clear cell type. The expression ratio of the number of ␣SMApositive microvessels to the number of CD31-positive microvessels in grade 3 tumors was significantly decreased compared with in grade 1 or 2 tumors. We also showed that the expression of calponin, which is a more differentiated marker of smooth muscle cells than ␣SMA,15 was not found at all in tumor neovasculature expressing CD31 antigen. Furthermore, renal cell carcinoma cells did not stain immunohistochemically with the anti-calponin antibody, suggesting that the tumor neovasculature of high-grade and high-stage tumors were immature. Increased microvessel counts have been associated with early progression in a number of tumors, including breast, colon, and prostate.1–3 However, the correlations between the microvessel count and pathologic factors in renal cell carcinoma have been controversial. Nativ et al.6 quantified the number of microvessels using immunohistochemical staining of endothelial cells for factor VIII-related antigen and demonstrated an inverse correlation between microvessel count and tumor grade (P ⫽ 0.047), but no association was noted between the microvessel count and cell type or tumor size. Slaton et al.8 assessed microvessel density by antiCD34 immunohistochemical analysis and showed a tendency for correlation between microvessel density and histologic grade, tumor size, and DNA ploidy. MacLennan and Bostwick4 found no correlation between the microvessel count stained by the antibody against factor VIII-related antigen and pathologic stage and grade. The reason the results of the correlation between the microvessel count and pathologic findings are controversial remains unclear. One explanation is that renal cell carcinoma contains highly vascular angiogenesis, so the difference in tumor angiogenesis between pathologic findings is difficult to detect. In our study, it is evident that limitations exist because of intraobserver variability and the few number of cases examined. Endothelial cells migrate, proliferate, and assemble into tubes from pre-existing vessels. Periendothelial support cells are recruited to encase the endothelial tubes; such cells include pericytes for small capillaries and smooth muscle cells expressing calponin for large vessels. In our results, about one half of the tumor vessels in grade 1 and 2 renal cell carcinoma specimens contained periendothelial cells positive for ␣SMA, and less than 10% of those in grade 3 renal tumors contained these cells. The mechanism of the interactions among tumor cells, endothelial cells, and mesenchymal cells difUROLOGY 62 (4), 2003
ferentiating to pericytes and smooth muscle cells has been intensively studied.16 –19 The endothelial cells proliferated by VEGF secreted from tumor cells express Tie2, the receptor for angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2). Ang-2 competitively inhibits Ang-1-induced kinase activation of Tie2.18 Ang-1 produced by tumor cells stimulates endothelial cells, which then may produce platelet-derived growth factor, heparin-binding epidermal growth factor, and transforming growth factor-beta to recruit mesenchymal cells that differentiate to pericytes and smooth muscle cells.19 Small capillaries with few periendothelial support cells showed strong expression of Ang-2, and larger glioblastoma vessels with many periendothelial support cells showed little or no expression.16 The mechanism of constructing immature tumor vessels in renal cell carcinoma should be studied in future investigations, including the expressions of VEGF, Tie2, Ang-1, and Ang-2. In human prostate cancer, the constitutive production of VEGF by the glandular epithelium was suppressed as a consequence of androgen-ablation therapy, and VEGF loss led to selective apoptosis of endothelial cells in vessels devoid of periendothelial cells. These results suggest that tumors, including high-grade and high-stage renal cell carcinoma, may regress after inducing apoptosis of immature tumor vessels by VEGF ablation. CONCLUSIONS The number of microvessels in high-grade and high-stage renal cell carcinoma was significantly lower than the number in low-grade and low-stage tumors, and the tumor neovasculature of highgrade and high-stage tumors consisted of immature components. Tumors, including high-grade and high-stage renal cell carcinoma, may regress after inducing apoptosis of immature tumor vessels. REFERENCES 1. Weidner N, Folkman J, Pozza F, et al: Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst 84: 1875– 1887, 1992. 2. Weidner N, Carroll PR, Flax J, et al: Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol 143: 401–409, 1993. 3. Saclarides TJ, Speziale NJ, Drab E, et al: Tumor angiogenesis and rectal carcinoma. Dis Colon Rectum 37: 921–926, 1994. 4. MacLennan GT, and Bostwick DG: Microvessel density in renal cell carcinoma: lack of prognostic significance. Urology 46: 27–30, 1995. 5. Yoshino S, Kato M, and Okada K: Prognostic significance of microvessel count in low stage renal cell carcinoma. Int J Urol 2: 156 –160, 1995. 769
6. Nativ O, Sabo E, Reiss A, et al: Clinical significance of tumor angiogenesis in patients with localized renal cell carcinoma. Urology 51: 693–696, 1998. 7. Chang SG, Jeon SH, Lee SJ, et al: Clinical significance of urinary vascular endothelial growth factor and microvessel density in patients with renal cell carcinoma. Urology 58: 904 –908, 2001. 8. Slaton JW, Inoue K, Perrotte P, et al: Expression levels of genes that regulate metastasis and angiogenesis correlate with advanced pathological stage of renal cell carcinoma. Am J Pathol 158: 735–743, 2001. 9. Benjamin LE, Golijanin D, Itin A, et al: Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J Clin Invest 103: 159 –165, 1999. 10. Takahashi K, Hiwada K, and Kokubu T: Isolation and characterization of a 34000-Dalton calmodulin- and f-actinbinding protein from chicken gizzard smooth muscle. Biochem Biophys Res Commun 141: 20 –26, 1986. 11. Takahashi K, and Nadal-Ginard B: Molecular cloning and sequence analysis of smooth muscle calponin. J Biol Chem 266: 13284 –13288, 1991.
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12. Sobin LH, and Wittekind CH (Eds): TNM Classification of Malignant Tumors, 5th ed. New York, John Wiley & Sons, 1997, pp 180 –182. 13. Storkel S, Eble JN, Adlakha K, et al: Classification of renal cell carcinoma. Workgroup No. 1. Cancer 80: 987–989, 1997. 14. Feffrey Medeiros L, Jones EC, Aizawa S, et al: Grading of renal cell carcinoma. Workgroup No. 2. Cancer 80: 990 – 991, 1997. 15. Owens G: Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 75: 487–517, 1995. 16. Stratmann A, Risau W, and Plate KH: Cell type-specific expression of angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis. Am J Pathol 153: 1459 – 1466, 1998. 17. Suri C, Jones PF, Patan S, et al: Requisite role of angiopoietin-1, a ligand for the Tie2 receptor, during embryonic angiogenesis. Cell 87: 1171–1180, 1996. 18. Hanahan D: Signaling vascular morphogenesis and maintenance. Science 277: 48 –50, 1997. 19. Folkman J, and D’Amore PA: Blood vessel formation: what is its molecular basis? Cell 87: 1153–1155, 1996.
UROLOGY 62 (4), 2003
IMMATURE TUMOR ANGIOGENESIS IN HIGH-GRADE AND HIGH-STAGE RENAL CELL CARCINOMA TOSHIAKI KINOUCHI, MASAYUKI MANO, IKUYO MATSUOKA, SAE KODAMA, TOMOMI AOKI, MINA OKAMOTO, HISAKO YAMAMURA, MICHIYUKI USAMI, AND KATSUHITO TAKAHASHI
ABSTRACT Objectives. To investigate the correlation between pathologic findings and maturation of the tumor neovasculature of renal cell carcinoma by immunohistochemical studies. Methods. Formalin-fixed and paraffin-embedded specimens from 25 randomly selected patients with renal cell carcinoma were stained with mouse monoclonal antibodies, anti-human CD31, anti-alpha smooth muscle actin (␣SMA), and anti-human calponin by the indirect immunoperoxidase method. The microvessels were counted in six areas with the higher number of microvessels in each patient at 200⫻ magnification (0.255 mm2 per area). Results. The number of CD31-positive microvessels in grade 3 tumors was significantly lower than those in grade 1 or 2 tumors (P ⫽ 0.003222 and P ⫽ 0.043217, respectively). The CD31-positive microvessel counts of those of higher stage, tumor size greater than 4.5 cm, or non-clear cell type were significantly lower than tumors of lower stage, size less than 4.6 cm, or clear cell type. In the grade 3 tumors, the expression ratio of the number of ␣SMA-positive microvessels to the number of CD31-positive microvessels was significantly decreased compared with grade 1 or 2 tumors (P ⫽ 0.000011 and P ⫽ 0.000000, respectively). The expression of calponin in the tumor neovasculature was not observed. The expression ratios of the number of ␣SMA-positive microvessels to the number of CD31-positive microvessels in higher stages, larger tumor sizes, or non-clear cell types were significantly decreased. Conclusions. The tumor neovasculature of high-grade and high-stage tumors was immature. These results imply that high-grade tumors of renal cell carcinomas may be susceptible to antiangiogenesis therapy inducing apoptosis of immature tumor vessels. UROLOGY 62: 765–770, 2003. © 2003 Elsevier Inc.
N
eovascularization is essential to the growth and metastasis of solid tumors. Solid tumors recruit blood vessels from the neighboring tissues by angiogenesis. To stimulate angiogenesis, tumors secrete growth factors that act on endothelial cells. Increased microvessel counts have been associated with early progression in a number of tumors, including breast, colon, and prostate.1–3 In general, the clear cell type of renal cell carcinoma is highly hypervascular, but the correlation between From the Departments of Urology, Pathology, and Medicine, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari-ku, Osaka, Japan Reprint requests: Toshiaki Kinouchi, M.D., Ph.D., Department of Urology, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan Submitted: December 9, 2002, accepted (with revisions): May 8, 2003 © 2003 ELSEVIER INC. ALL RIGHTS RESERVED
the microvessel count and pathologic factors in renal cell carcinoma is still controversial.4 – 8 The suppression and degradation of angiogenesis is the strategy for treating tumors. To accomplish this, it is necessary to discriminate tumor angiogenesis from normal blood vessels. The angiogenesis factor vascular endothelial growth factor (VEGF) is overexpressed in human prostate cancer and glioblastoma. Some fraction of angiogenesis in prostate cancer and glioblastoma involves immature vessels with the absence of associated pericytes or smooth muscle cells, both of which express alpha-smooth muscle actin (␣SMA), and these immature vessels are selectively obliterated by VEGF withdrawal.9 Because the tumor tissues of renal cell carcinoma express abundant VEGF, we investigated the correlation between the microvessel count and pathologic findings and the maturation of angiogenesis by im0090-4295/03/$30.00 doi:10.1016/S0090-4295(03)00512-0 765
TABLE I. Patient characteristics, histopathologic findings, and immunohistochemical staining with anti-CD31 and anti-␣SMA antibodies Pt. No./Sex/ Age (yr)
Cell Type
1/M/48 2/M/55 3/M/43 4/M/46 5/M/46 6/M/56 7/M/55 8/M/43 9/M/50 10/F/61 11/F/72 12/M/44 13/F/72 14/M/54 15/M/61 16/M/66 17/M/69 18/M/68 19/M/58 20/M/61 21/F/63 22/M/45 23/F/67 24/M/51 25/M/65
Unclassified Unclassified Papillary Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Clear Unclassified Clear Unclassified
Grade
Robson Stage
pT Stage
Size (cm)
CD31-Positive Microvessels (n)
3 3 3 2 2 2 1 1 1 2 2 2 2 2 2 1 1 2 1 2 1 2 3 1 3
4 4 3 2 2 1 1 1 1 2 1 1 4 1 1 2 1 1 1 1 1 1 1 1 3
4 4 3 3 3 1 1 1 1 3 1 1 3 1 1 3 1 1 1 1 1 1 1 1 3
10 10 8 6 8 6 3 5 4.5 8 3.2 3.5 6.5 5 2.5 10 3 4 2.4 3 4.5 3 5.5 4 7
91.2 94.2 121.8 150 232.7 151.7 203.7 214.7 232.3 103.5 125.8 179.7 135.3 114 124.7 116.2 146.5 149.2 226.8 176.3 212.7 252.8 163.5 264.8 60.2
(68–121) (56–115) (101–146) (132–164) (199–307) (119–176) (157–230) (181–280) (178–280) (83–139) (107–152) (123–221) (119–149) (97–124) (106–152) (93–136) (112–176) (121–193) (198–264) (94–224) (155–265) (212–288) (104–216) (204–296) (48–70)
␣SMA-Positive Vessels/ CD31-Positive Vessels Ratio (%) 0 4.7 21.6 55.8 54.7 52.4 73.5 45.8 48.6 58.7 56.7 62.6 53.4 58.6 60.8 53 61.6 58 37.8 27.7 60.2 53.6 9.7 44.5 6.5
(0–0) (0–24) (4–64) (51–62) (50–64) (26–69) (62–96) (28–61) (30–79) (54–66) (52–64) (57–70) (44–61) (56–60) (56–64) (52–57) (53–69) (55–61) (20–49) (14–31) (31–80) (35–77) (0–28) (33–49) (5–13)
KEY: ␣SMA ⫽ alpha-smooth muscle actin; Pt. No. ⫽ patient number; M ⫽ male; F ⫽ female. Numbers in parentheses are the range.
munohistochemical staining against CD31, a specific marker of endothelial cells, and ␣SMA and calponin, differentiation markers of smooth muscle.10,11
the sections were autoclaved at 121°C for 5 minutes in 0.01 M citrate buffer for antigen retrieval of CD31 and calponin. The sections were incubated first with antibodies overnight at 4°C. Thereafter, assays were performed by the indirect immunoperoxidase method with a Vectastain ABC kit for mouse IgG (Vector Laboratory, Burlingame, Calif).
MATERIAL AND METHODS PATIENTS AND DEFINITION OF CHARACTERISTICS The surgical specimens from 25 randomly selected patients (20 men and 5 women) with renal cell carcinoma were examined. The patient characteristics, including age, sex, cell type, grade, Robson stage, pT stage, tumor size, CD31-positive microvessel count, and percentage of expression ratio of the number of ␣SM-positive microvessels to number of CD31positive microvessels, are shown in Table I. The 1997 TNM classification of malignant tumors was used.12 The stage and grade of renal cell carcinoma was based on the results of the World Health Organization-sponsored conference on renal cell carcinoma in 1997.13,14 The median tumor size was 4.5 cm, and patients were divided at this point.
IMMUNOHISTOCHEMICAL STAINING The formalin-fixed and paraffin-embedded sections of the renal cell carcinoma specimens were stained with mouse monoclonal antibodies, anti-human CD31 undiluted (IgG1, Dako, Carpinteria, Calif), anti-␣SMA diluted 1:2000 (IgG2a, Sigma, St. Louis, Mo), and anti-human calponin diluted 1:2000 (IgG1, Sigma). After deparaffinized sections were inactivated with endogenous peroxidase by hydrogen peroxide, 766
MICROVESSEL COUNTS Tumor sections without necrosis or cystic changes were selected from two to three paraffin blocks in each case to detect hot spots. The selected tumor section was examined under low magnification to detect areas with high microvessel density. A single microvessel was considered any endothelial cell or cell cluster with brown reaction product that was distinct and separable from adjacent microvessels, tumor cells, and other tissue elements. We did not require the presence of a vascular lumen for a structure to be defined as a microvessel. Microvessels were counted in six areas with the higher number of microvessels (0.255 mm2 per area) by one of us (T.A.) who did not know the patient characteristics. The microvessel count was expressed as the mean number of vessels in each area.
STATISTICAL ANALYSIS Statistical analysis of the relationship between the pathologic findings and the microvessel counts expressing CD31, ␣SMA, or calponin antigens was performed using Student’s t test. Statistical significance was considered as P ⬍0.05. UROLOGY 62 (4), 2003
FIGURE 1. Immunohistochemical staining of clear cell type in grade 1 (A, B, C) and unclassified cell type in grade 3 (D, E) tumors of renal cell carcinoma. (A) Anti-CD31 staining showed numerous fine microvessels. (B) Anti-␣SMA stained almost 60% of microvessels stained by anti-CD31. (C) Anti-calponin stained no microvessels. (D) Number of anti-CD31-positive microvessels in grade 3 tumors was significantly lower than that in grade 1 tumors. (E) Anti-␣SMA stained almost none of microvessels stained by anti-CD31. Original magnification ⫻100.
RESULTS MICROVESSEL COUNTS STAINED IMMUNOHISTOCHEMICALLY BY ANTI-CD31 ANTIBODY IN RENAL CELL CARCINOMA The immunohistochemical staining of grade 1 and grade 3 renal cell carcinoma specimens by anti-CD31, anti-␣SMA, and anti-calponin are demonstrated in Figure 1. We examined whether the number of microvessels may depend on the pathologic findings. The results are summarized in Table II. The mean number of CD31-positive microvessels in grades 1, 2, and 3 was 202.2 (range 116 to 265), 158.0 (range 104 to 253), and 106.2 (range 60 to 164), respectively. The number of CD31positive microvessels in grade 3 tumors was significantly lower than those in grade 1 or 2 tumors (P ⫽ 0.003222 and P ⫽ 0.043217, respectively). The number of CD31-positive microvessels decreased depending on the tumor grade. The microvessel counts of Stage T3 and T4, Robson Stage III and IV, tumor size greater than 4.5 cm, and non-clear cell type were significantly lower than those of Stage T1 UROLOGY 62 (4), 2003
and T2, Robson Stage I and II, tumor size less than 4.6 cm, and clear cell type, respectively. NO EXPRESSION OF CALPONIN IN OF RENAL CELL CARCINOMA
TUMOR ANGIOGENESIS
The expression of calponin in the tumor neovasculature was not observed. Also, renal cell carcinoma cells did not stain immunohistochemically with the anti-human calponin antibody. DECREASED EXPRESSION RATIO OF NUMBER OF ␣SMAPOSITIVE MICROVESSELS TO NUMBER OF CD31POSITIVE MICROVESSELS IN HIGH-GRADE AND HIGHSTAGE RENAL CELL CARCINOMA We examined whether the expression ratio, defined by the number of ␣SMA-positive microvessels to the number of CD31-positive microvessels, may depend on the pathologic findings. The results are summarized in Table III. The expression ratio in the grade 3 tumors was significantly decreased compared with the expression ratio for grade 1 or 2 767
TABLE II. Number of microvessels in a 0.255-mm2 area of renal cell carcinoma tissues stained immunohistochemically by anti-CD31 antibody Factor Grade 1 2 3 T stage 1–2 3–4 Cell type Clear Nonclear Robson stage I–II III–IV Tumor size (cm) ⬍4.6 ⬎4.5
Mean CD31-Positive Microvessels (n)
P Value
202.2 (116–265) 158.0 (104–253) 106.2 (60–164)
G1/G3 0.003222 G1/G2 0.052771 G2/G3 0.043217
183.7 (114–265) 122.8 (60–233)
0.005616
175.7 (104–265) 106.2 (60–164)
0.009079
177.1 (104–265) 100.5 (60–135)
0.003423
191.3 (125–265) 134.5 (60–233)
0.007585
Numbers in parentheses are the range.
TABLE III. Ratio of ␣SMA-positive microvessels to CD31-positive microvessels in a 0.255-mm2 area of renal cell carcinoma tissues stained immunohistochemically by anti-␣SMA and anti-CD31 antibodies Factor Grade 1 2 3 T stage 1–2 3–4 Cell type Clear Nonclear Robson stage I–II III–IV Tumor size (cm) ⬍4.6 ⬎4.5
␣SMA Positive/CD31 Positive (%)
P Value
53.1 (37.8–73.5) 54.4 (27.7–62.6) 8.5 (0–21.6)
G1/G3 0.000011 G2/G3 0.000000 G1/G2 0.78065
50.8 (9.7–73.5) 34.3 (0–58.7)
0.054236
53.9 (27.7–73.5) 8.5 (0–21.6)
0.000000
51.7 (9.7–73.5) 12.2 (0–53.4)
0.000234
53.8 (27.7–73.5) 36.5 (0–58.7)
0.034358
KEY: ␣SMA ⫽ alpha-smooth muscle actin. Numbers in parentheses are the range.
tumors. The expression ratio in tumors that were Robson Stage III and IV, greater than 4.5 cm, or non-clear cell type was significantly lower than the expression ratio in tumors that were Robson Stage I and II (P ⫽ 0.000234), less than 4.6 cm (P ⫽ 0.034358), or clear cell type (P ⫽ 0.000000), respectively. No statistically significant correlations of the expression ratios between lower and higher 768
T stages were found; however, the expression ratio in higher T stages tended to decrease. COMMENT We demonstrated that the number of CD31-positive microvessels in grade 3 tumors is significantly lower than the number in grade 1 and 2 tumors. UROLOGY 62 (4), 2003
The CD31-positive microvessel count in tumors of higher stage, greater than 4.5 cm, or non-clear cell types was significantly lower than the count in tumors of lower stage, less than 4.6 cm, or clear cell type. The expression ratio of the number of ␣SMApositive microvessels to the number of CD31-positive microvessels in grade 3 tumors was significantly decreased compared with in grade 1 or 2 tumors. We also showed that the expression of calponin, which is a more differentiated marker of smooth muscle cells than ␣SMA,15 was not found at all in tumor neovasculature expressing CD31 antigen. Furthermore, renal cell carcinoma cells did not stain immunohistochemically with the anti-calponin antibody, suggesting that the tumor neovasculature of high-grade and high-stage tumors were immature. Increased microvessel counts have been associated with early progression in a number of tumors, including breast, colon, and prostate.1–3 However, the correlations between the microvessel count and pathologic factors in renal cell carcinoma have been controversial. Nativ et al.6 quantified the number of microvessels using immunohistochemical staining of endothelial cells for factor VIII-related antigen and demonstrated an inverse correlation between microvessel count and tumor grade (P ⫽ 0.047), but no association was noted between the microvessel count and cell type or tumor size. Slaton et al.8 assessed microvessel density by antiCD34 immunohistochemical analysis and showed a tendency for correlation between microvessel density and histologic grade, tumor size, and DNA ploidy. MacLennan and Bostwick4 found no correlation between the microvessel count stained by the antibody against factor VIII-related antigen and pathologic stage and grade. The reason the results of the correlation between the microvessel count and pathologic findings are controversial remains unclear. One explanation is that renal cell carcinoma contains highly vascular angiogenesis, so the difference in tumor angiogenesis between pathologic findings is difficult to detect. In our study, it is evident that limitations exist because of intraobserver variability and the few number of cases examined. Endothelial cells migrate, proliferate, and assemble into tubes from pre-existing vessels. Periendothelial support cells are recruited to encase the endothelial tubes; such cells include pericytes for small capillaries and smooth muscle cells expressing calponin for large vessels. In our results, about one half of the tumor vessels in grade 1 and 2 renal cell carcinoma specimens contained periendothelial cells positive for ␣SMA, and less than 10% of those in grade 3 renal tumors contained these cells. The mechanism of the interactions among tumor cells, endothelial cells, and mesenchymal cells difUROLOGY 62 (4), 2003
ferentiating to pericytes and smooth muscle cells has been intensively studied.16 –19 The endothelial cells proliferated by VEGF secreted from tumor cells express Tie2, the receptor for angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2). Ang-2 competitively inhibits Ang-1-induced kinase activation of Tie2.18 Ang-1 produced by tumor cells stimulates endothelial cells, which then may produce platelet-derived growth factor, heparin-binding epidermal growth factor, and transforming growth factor-beta to recruit mesenchymal cells that differentiate to pericytes and smooth muscle cells.19 Small capillaries with few periendothelial support cells showed strong expression of Ang-2, and larger glioblastoma vessels with many periendothelial support cells showed little or no expression.16 The mechanism of constructing immature tumor vessels in renal cell carcinoma should be studied in future investigations, including the expressions of VEGF, Tie2, Ang-1, and Ang-2. In human prostate cancer, the constitutive production of VEGF by the glandular epithelium was suppressed as a consequence of androgen-ablation therapy, and VEGF loss led to selective apoptosis of endothelial cells in vessels devoid of periendothelial cells. These results suggest that tumors, including high-grade and high-stage renal cell carcinoma, may regress after inducing apoptosis of immature tumor vessels by VEGF ablation. CONCLUSIONS The number of microvessels in high-grade and high-stage renal cell carcinoma was significantly lower than the number in low-grade and low-stage tumors, and the tumor neovasculature of highgrade and high-stage tumors consisted of immature components. Tumors, including high-grade and high-stage renal cell carcinoma, may regress after inducing apoptosis of immature tumor vessels. REFERENCES 1. Weidner N, Folkman J, Pozza F, et al: Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst 84: 1875– 1887, 1992. 2. Weidner N, Carroll PR, Flax J, et al: Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol 143: 401–409, 1993. 3. Saclarides TJ, Speziale NJ, Drab E, et al: Tumor angiogenesis and rectal carcinoma. Dis Colon Rectum 37: 921–926, 1994. 4. MacLennan GT, and Bostwick DG: Microvessel density in renal cell carcinoma: lack of prognostic significance. Urology 46: 27–30, 1995. 5. Yoshino S, Kato M, and Okada K: Prognostic significance of microvessel count in low stage renal cell carcinoma. Int J Urol 2: 156 –160, 1995. 769
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