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Changes in plasma vascular endothelial growth factor, angiopoietins, and their receptors following surgery for breast cancer
Cancer Letters 248 (2007) 131–136 www.elsevier.com/locate/canlet
Changes in plasma vascular endothelial growth factor, angiopoietins, and their receptors following surgery for breast cancer Graham J. Caine a, Paul S. Stonelake b, Gregory Y.H. Lip a, Andrew D. Blann a
a,*
Haemostasis, Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham B18 7QH, United Kingdom b Department of Surgery, City Hospital, Birmingham B18 7QH, United Kingdom Received 13 April 2006; received in revised form 20 June 2006; accepted 29 June 2006
Abstract Background: Vascular endothelial growth factor (VEGF), a major angiogenic growth factor, is involved in the pathogenesis of cancer. Plasma VEGF is raised in breast cancer and falls after successful surgery. Less is known about angiopoietins 1 and 2 (Ang-1, Ang-2). All three growth factors act on cells via receptors; Flt-1 for VEGF and Tie-2 for the angiopoietins. Cancer is also marked by abnormalities in platelet activation (marked by soluble P selectin) and inflammation (interleukin-6 [IL6]). We hypothesised altered plasma Ang-1, Ang-2, Flt-1 and Tie-2 in breast cancer that would normalize after 3 and 12 months treatment (i.e., surgery plus chemo/radiotherapy). Methods: Baseline venous blood was obtained from 40 women with breast cancer and 30 age-matched women with benign breast disease (BBD) also requiring surgery. Samples were taken again 3 months and 1 year later. Soluble P selectin, IL6, VEGF, Ang-1, Ang-2, Flt-1 and Tie-2 were measured in citrated plasma by ELISA. Results: Women with breast cancer had raised VEGF (7-fold), Ang-1 (50% higher) and Tie-2 (2-fold), but lower Flt-1 (to 26%), compared to the BBD women that broadly correlated with markers of platelet activation and inflammation. A level of Tie-2 or VEGF >95th percentile of the BBD group correctly identified 68% and 52% of the women with breast cancer. After 3 months of treatment, VEGF and Ang-1 normalized (as did IL6 and soluble P selectin) but Tie-2 was significantly lower only after 1 year. There were no significant changes in the women with BBD. Conclusions: Treatment for breast cancer (surgery followed by chemotherapy and/or radiotherapy) is effective in reducing plasma VEGF, Tie-2 and Ang-1. These may be linked pathogenically with coagulation and inflammation. Ó 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Breast cancer; VEGF; Angiopoietin; Tie-2; Flt-1; Soluble P selectin; Interleukin-6
1. Introduction
*
Corresponding author. Tel./fax: +44 121 507 5076. E-mail address: [email protected] (A.D. Blann).
Breast cancer is the leading cancer in women, accounting for 16% of female cancer deaths, translating to some 40,000 new cases and 12,000 deaths in the UK each year [1,2]. Characterised by
0304-3835/$ - see front matter Ó 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2006.06.011
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inappropriate and abnormal cell growth and potential to metastasize, a principal factor in this tumour enlargement is the requirement of an increased blood supply to feed the growing mass of cells. This process, orchestrated by growth factors such as vascular endothelial growth factor (VEGF) and the angiopoietins (all measurable in the blood), involves new blood vessel development (angiogenesis). However, the clinical relevance of increased levels of angiogenic growth factors in the blood is unclear [3–6]. Increased levels of the cell receptors (e.g., Flt-1, Tie-2) for these growth factors are present in the blood but, once more, implications for cell biology and clinical outcome need development [6]. Whilst the literature on VEGF is considerable, there are relatively little clinical data on angiopoietin-1 and angiopoietin-2. For example, raised plasma VEGF in breast cancer is commonly reported and is higher in metastatic disease [7–9], and fall 5 days and 3 weeks after surgery [10,11]. Furthermore, therapy aimed at VEGF is promising and provides a possible link with clinical outcome [12,13]. The majority of published work on the angiopoietins and its receptor (Tie-2) focuses on tissue culture and animal models, levels in tissues, or on RNA/DNA (e.g., [14–16]). More recently, changes in levels of cytokines such as interleukin-6 (IL-6) in breast cancer imply a degree of inflammation in these patients [17]. Furthermore, IL-6 and angiogenic growth factors can also be found within platelets although the significance of this is unknown [6,18]. Raised levels of the platelet marker soluble P selectin in cancer may be related to this finding [19,20]. We have previously reported altered levels of plasma VEGF, soluble P selectin, IL-6 and angiopoietins-1 and -2 and their receptors Flt-1 (VEGFR-1) and Tie-2 in cross-sectional studies of breast cancer [6,19–22]. In the present communication we tested the hypothesis that conventional treatment for breast cancer (surgery, followed by radiotherapy and/or chemotherapy) would, as it does for VEGF [10,11], normalize levels of plasma angiopoietins -1 and -2, their receptors, and that of VEGF 3 and 12 months after surgery. Thus, essentially, the broad objective was to determine whether or not the angiopoietins and receptors have anything more to offer than VEGF. We also measured the inflammatory marker IL-6 [17] and platelet marker soluble P selectin [19,20] to determine any relationship between tumour-debulking and subsequent therapy and markers.
2. Materials and methods We tested our hypothesis in blood samples from 40 women (mean [standard deviation] age 59 [10] yrs old, 7 pre-menopausal) about to undergo either mastectomy (n = 16) or wide local excision (n = 24) for biopsy-proven breast cancer, followed by standard best medical care (i.e., radiotherapy and/or chemotherapy with tamoxifen), as directed by clinical need alone. We have previously described cross-sectional studies on these women [6,19,22]. Blood samples were taken before surgery, and both 3 and 12 months later. Post-surgical treatment was a 3 month course of radiotherapy consisting of 12 cycles of 40 Gy to the affected breast (20 women) and/or 20 mg/day tamoxifen as secondary adjuvant therapy (23 women). None of the tamoxifen patients became intolerant. All patients had commenced tamoxifen by the 3 month follow-up appointment and were still taking tamoxifen at the 12 month follow-up appointment. Following surgery, excised tissue was examined by routine and established histological techniques. Lymphovascular invasion was deemed to be present in tissues from 5 women, lymph node involvement was present in 9, and 30 tumours were estrogen receptor positive. Sixteen of the tumours were Grade 1, 13 were Grade 2, and 11 were Grade 3. Tumour size was median 18 mm diameter (inter-quartile range 12.25–26.75 mm, minimum 6 mm, maximum 45 mm). The control group was 30 age-matched women (mean [standard deviation] age 57 [12] years, t test p = 0.518) undergoing surgery for relative benign reasons, i.e., Hadfield’s procedure in 2, mammillary fistula excision in 2, axillary hidradenitis in 1 and benign lump excision in the remaining 25. None of the excised tissue was neoplastic. Exclusion criteria for both groups were concurrent connective tissue or cardiovascular disease and use of antibiotics implying inflammation. The approval of the Research Ethics Committee of the Sandwell and West Birmingham NHS Trust was obtained, as was written informed consent from each participant. 2.1. Laboratory Venous blood was obtained into sodium citrated, centrifuged at 2500 rpm/1000 g for 20 min, and plasma stored at – 70 °C. Makers were measured by commercial ELISA (R&D Systems, Abingdon UK) [6,19,22]. 2.2. Data analysis Continuous data were subjected to the Ryan-Joiner test to assess distribution. Data are presented as mean and standard deviation (analysed by t test), or median and inter-quartile (IQR) range (by Mann–Whitney U test), as appropriate. Categorical data were compared using the v2 test. The Kruskal–Wallis test compared
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different groups where the data were not normally distributed and the one-way ANOVA for normally distributed data. Correlations were sought using Pearson’s method for normally distributed data, while Spearman’s method was used for non-normally distributed data. Serial samples were analysed by two-way (repeated measures) analysis of variance. P < 0.05 was taken to imply significance. All analyses were performed on Minitab release 13 (Minitab Inc, State College, PA, USA). 3. Results 3.1. Baseline case-control findings Table 1 shows baseline and follow-up differences in research indices. At baseline, the women with cancer had raised VEGF, IL-6, soluble P selectin, Ang-1 and Tie-2 with lower Flt-1 than the control women with benign breast disease (BBD). There was no difference in levels of Ang-2. There were no baseline differences in any of the seven research indices in the breast cancer group when analysed by type of surgery (wide local excision n = 24 versus mastectomy n = 16), by tertile of the
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size of the tumour, by the presence/absence of lymph node involvement (present in 9 women) or menopausal status (7 pre-menopausal). Only Tie-2 showed a relationship with positive estrogen receptor status (median 32 ng/mL [IQR 20-185] n = 9 versus 20 [13.5–26.3] ng/mL in women with negative estrogen receptor tumours, n = 30, p = 0.03). VEGF was higher in 5 women with lymphovascular involvement (350 [270–375,340] pg/mL) compared to the 35 free of involvement (150 [50–300] pg/mL, p = 0.022), as was IL-6 (18 [15.5–26.5] pg/mL versus 10 [5–12] pg/mL, p = 0.0074). VEGF was also different according to grade: 50 [12.5–30.5] pg/mL in 16 women with grade 1 tumours, 150 [65–270] pg/mL in 13 with grade 2 tumours, and 340 [220–350] pg/mL in 11 women with grade 3 tumours (p = 0.027). Taking the 95th percentile of the BBD group as a cut-off point, VEGF was raised in 26 (65%) of breast cancer cases (i.e., >100 pg/mL). Similarly, raised Tie-2 (>10 ng/mL) identified 34 (85%) of women with cancer, raised Ang-1 (>10.2 ng/mL) identified only 35% of women, whereas low Flt-1 (<0.5 ng/mL) correctly identified 23 (57.5%) women with breast cancer.
Table 1 Longitudinal analysis of 40 women with breast cancer and 30 with benign breast disease, before and after surgery Baseline
3-Month
12-Month
P value
VEGF (pg/mL) Cancer Benign breast disease
200 (50–340)+++ 30 (24–50)
50 (10–150)* 27.5 (24–52)
50 (20–137)* 42.5 (24–60)
< 0.001 0.708
Flt-1 (ng/mL) Cancer Benign breast disease
0.4 (0.2–0.68)++ 1.5 (0.5–2.31)
0.5 (0.33–0.78) 0.75 (0.5–1.5)
0.5 (0.4–0.95) 0.75 (0.5–1.0)
0.645 0.290
Ang-1 (ng/mL) Cancer Benign breast disease
6.0 (4.0–23.5)+++ 4.0 (2.5–5.0)
5.0 (4.0–10.0)* 4.0 (2.0–8.0)
5.0 (4.0–7.7)* 4.0 (3.0–5.75)
<0.001 0.134
Ang-2 (ng/mL) Cancer Benign breast disease
1.62 (1.0–2.37) 1.3 (0.75–1.75)
1.5 (1.0–2.0) 1.5 (1.0–2.0)
1.3 (1.0–2.6) 1.5 (1.15–2.0)
0.750 0.450
Tie-2 (ng/mL) Cancer Benign breast disease
20 (15–31.0)+++ 7.5 (6.0–8.6)
15 (10.0–25.0) 8.0 (5.2–9.2)
10 (5.0–20.0)* 8.0 (5.9–9.1)
0.005 0.782
IL-6 (pg/mL) Cancer Benign breast disease
10 (5–15)+ 5.2 (5.0–7.5)
7.5 (5–10)* 6.25 (5.0–7.5)
7.5 (5–14.2)* 5.0 (5.0–7.6)
0.026 0.608
sPsel (pg/mL) Cancer Benign breast disease
65 (26)+++ 45 (9)
58 (13)* 38 (10)
57 (14)* 42 (16)
0.001 0.388
VEGF = vascular endothelial growth factor, Ang-1 = angiopoietin-1, Ang-2 = angiopoietin-2, IL-6 = interleukin-6, s-Psel = soluble P selectin. Data presented as median and inter-quartile range or as mean and standard deviation. Major p value by two-way analysis of variance. *p < 0.05 from baseline result. Baseline differences between cancer and benign breast disease patents +p = 0.003, ++p = 0.001, +++p < 0.001 (other time points not analysed).
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Table 2 Significant correlations between the research indices Pairs of indices
Tie-2 and Ang-2 sPsel and Ang-1 sPsel and VEGF VEGF and Flt-1 VEGF and Ang-1 Tie-2 and Ang-1 IL-6 and VEGF sPsel and IL-6 Il-6 and Tie-2
Breast cancer
Benign breast disease
r Value
p Value
0.637 0.621 0.541 0.505 0.503 0.477 0.456 0.451 0.450
<0.001 <0.001 <0.001 <0.001 <0.001 0.003 0.001 0.001 0.004
r Value 0.124 0.034 0.054 0.237 0.026 0.547 0.275 0.13 0.137
p Value 0.513 0.858 0.775 0.207 0.893 0.002 0.141 0.494 0.472
Data are Spearman’s correlation coefficient (r) and p value derived from n = 40 women with breast cancer and n = 30 women with benign breast disease.
Table 2 shows numerous significant (p < 0.01) Spearman rank correlations in both patient groups. Notably, the correlation between Tie-2 and Ang-1 was significant in both groups. All other correlations were significant in the cancer group alone. 3.2. Three and Twelve month follow-up findings The effect of surgery followed by chemo- and/or radiotherapy was to reduce VEGF, Ang-1, Tie-2, IL-6 and soluble P selectin in the women with breast cancer (Table 1). There were no changes following surgery in BBD group. 3.3. Response of VEGF to treatment in cancer patients A level of <100 pg/mL in our hands [6,19,22] defines a normal level and therefore a good response to combined treatment. Under this criterion, 26 of the 40 women (65%) had raised VEGF (P100 pg/mL) at baseline. Of these, 13 women (50%) had a reduction in their VEGF level to <100 pg/ml (i.e., were responders) at 3 months, whilst 16 (62%) were responders at 12 months. None of the demographic (age, menopausal status), tumour indices (size, grade, estrogen receptor status, lymph node involvement, lymphovascular invasion), type of surgery (wide local excision or mastectomy) or type of therapy (none, radiotherapy alone, tamoxifen alone, both treatments) predicted those women whose VEGF levels responded. Similarly, none of the other research indices predicted a VEGF response at 3 or 12 months, although baseline Ang-1 was significantly higher in the 10 women whose VEGF failed to normalized (32.5 [8.8–76.3] ng/mL) compared to the 16 women whose VEGF did normalize (6.0 [4–11.5] ng/mL, p = 0.017).
4. Discussion Of the angiogenic growth factors, VEGF currently has the highest profile. Levels are raised in
breast and other cancers and fall after treatment, and it is a promising target for therapy [6–14,23– 26], although not all have found raised levels in breast cancer [27]. Our present finding that VEGF levels are reduced 3 months and 12 months after surgery supports and extends similar data at 5 days and 3 weeks [10,11] although these two studies did not examine the late effects of subsequent chemoand radio-therapy. Angiopoietins-1 and -2, and the receptors for these growth factors may also be important in cancer [6,14–16,21,22]. Indeed, combined therapy aimed at VEGF receptor-2 may also be useful [28]. We report abnormal Ang-1 (raised), Flt-1 (lower) and Tie-2 (raised) in breast cancer compared to BBD. Furthermore, combined treatment normalized levels of Ang-1 3 months after surgery, and Tie-2 12 months after surgery. Changes in Flt-1 after surgery were minor and not-significant. Unsurprisingly, many indices correlated significantly (Table 2), although only that between Tie-2 and Ang-2 was significant in both groups, implying the relationship is physiological, not pathological. None of the growth factors or their receptors were related strongly to any clinical or tumour-related index. VEGF was higher in the 5 women with lymphovascular involvement (p = 0.022), but this may result from small number bias, and was weakly (p = 0.027) positively related to increasing grade. With our small numbers we are unable to confirm raised VEGF in the menopause [7]. Abnormalities in inflammatory cytokines (such as IL6), and markers of platelet activation (such as soluble P selectin) in breast and other cancers are established [17–20] and our data confirm this. The effect of treatment was to reduce levels of these
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markers in parallel to those of several of the growth factors and their receptors, prompting the possibility of a link. Indeed, numerous strong correlations (Table 2) imply inter-relationships. Cytokines and angiogenic growth factors are known to be present within platelets [6,18,29], VEGF may be involved in the coagulation process [30,31], and platelets may deliver VEGF to the site of surgical wounds after breast surgery [32]. Thus changes in the growth factors and receptors may not necessarily primarily reflect tumour de-bulking, but instead may be secondary to changes in platelet and/or inflammatory status. Whilst VEGF has a place in breast cancer research, our objective was to compare VEGF with the angiopoietins and receptors as interest in these are growing. For example, although soluble Flt-1 is said to be an important negative regulator of VEGF-mediated angiogenesis [33], we found altered levels identified only slightly more than half the women with breast cancer. Whilst some report that Tie-2 immunoexpression by tumours is an independent prognostic indicator [34], we find that although increased plasma Tie-2 identified a higher proportion of women with breast cancer than did raised VEGF, levels failed to normalize after surgery as rapidly as did levels of VEGF. Finally, although overall Ang-1 levels were (like VEGF) also very significantly raised at baseline in the breast cancer women, an increased result was present in slightly over one third of women. References [1] G.A. Colditz, Epidemiology and prevention of breast cancer, Cancer Epidemiol. Biomarkers Prev. 14 (2005) 768–772. [2] F. Bray, P. McCarron, D.M. Parkin, The changing global patterns of female breast cancer incidence and mortality, Breast Cancer Res. 6 (2004) 229–239. [3] J. Folkman, What is the evidence that tumours are angiogenesis dependent?, J Natl. Cancer Inst. 82 (1990) 4–6. [4] D.C. Feldmeden, A.D. Blann, G.Y.H. Lip, Angiogenesis: basic pathophysiology and implications for disease, Eur. Heart J. 24 (2003) 586–603. [5] N. Ferrara, K. Alitalo, Clinical applications of angiogenic growth factors and their inhibitors, Nat. Med. 5 (1999) 1359–1364. [6] G.J. Caine, G.Y. Lip, A.D. Blann, Platelet-derived VEGF, Flt-1, angiopoietin-1 and P-selectin in breast and prostate cancer: further evidence for a role of platelets in tumour angiogenesis, Ann. Med. 36 (2004) 273–277. [7] R. Nishimura, K. Nagao, H. Miyayama, M. Matsuda, K. Baba, H. Yamashita, M. Fukuda, Higher plasma vascular endothelial growth factor levels correlate with menopause,
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Changes in plasma vascular endothelial growth factor, angiopoietins, and their receptors following surgery for breast cancer Graham J. Caine a, Paul S. Stonelake b, Gregory Y.H. Lip a, Andrew D. Blann a
a,*
Haemostasis, Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham B18 7QH, United Kingdom b Department of Surgery, City Hospital, Birmingham B18 7QH, United Kingdom Received 13 April 2006; received in revised form 20 June 2006; accepted 29 June 2006
Abstract Background: Vascular endothelial growth factor (VEGF), a major angiogenic growth factor, is involved in the pathogenesis of cancer. Plasma VEGF is raised in breast cancer and falls after successful surgery. Less is known about angiopoietins 1 and 2 (Ang-1, Ang-2). All three growth factors act on cells via receptors; Flt-1 for VEGF and Tie-2 for the angiopoietins. Cancer is also marked by abnormalities in platelet activation (marked by soluble P selectin) and inflammation (interleukin-6 [IL6]). We hypothesised altered plasma Ang-1, Ang-2, Flt-1 and Tie-2 in breast cancer that would normalize after 3 and 12 months treatment (i.e., surgery plus chemo/radiotherapy). Methods: Baseline venous blood was obtained from 40 women with breast cancer and 30 age-matched women with benign breast disease (BBD) also requiring surgery. Samples were taken again 3 months and 1 year later. Soluble P selectin, IL6, VEGF, Ang-1, Ang-2, Flt-1 and Tie-2 were measured in citrated plasma by ELISA. Results: Women with breast cancer had raised VEGF (7-fold), Ang-1 (50% higher) and Tie-2 (2-fold), but lower Flt-1 (to 26%), compared to the BBD women that broadly correlated with markers of platelet activation and inflammation. A level of Tie-2 or VEGF >95th percentile of the BBD group correctly identified 68% and 52% of the women with breast cancer. After 3 months of treatment, VEGF and Ang-1 normalized (as did IL6 and soluble P selectin) but Tie-2 was significantly lower only after 1 year. There were no significant changes in the women with BBD. Conclusions: Treatment for breast cancer (surgery followed by chemotherapy and/or radiotherapy) is effective in reducing plasma VEGF, Tie-2 and Ang-1. These may be linked pathogenically with coagulation and inflammation. Ó 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Breast cancer; VEGF; Angiopoietin; Tie-2; Flt-1; Soluble P selectin; Interleukin-6
1. Introduction
*
Corresponding author. Tel./fax: +44 121 507 5076. E-mail address: [email protected] (A.D. Blann).
Breast cancer is the leading cancer in women, accounting for 16% of female cancer deaths, translating to some 40,000 new cases and 12,000 deaths in the UK each year [1,2]. Characterised by
0304-3835/$ - see front matter Ó 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2006.06.011
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inappropriate and abnormal cell growth and potential to metastasize, a principal factor in this tumour enlargement is the requirement of an increased blood supply to feed the growing mass of cells. This process, orchestrated by growth factors such as vascular endothelial growth factor (VEGF) and the angiopoietins (all measurable in the blood), involves new blood vessel development (angiogenesis). However, the clinical relevance of increased levels of angiogenic growth factors in the blood is unclear [3–6]. Increased levels of the cell receptors (e.g., Flt-1, Tie-2) for these growth factors are present in the blood but, once more, implications for cell biology and clinical outcome need development [6]. Whilst the literature on VEGF is considerable, there are relatively little clinical data on angiopoietin-1 and angiopoietin-2. For example, raised plasma VEGF in breast cancer is commonly reported and is higher in metastatic disease [7–9], and fall 5 days and 3 weeks after surgery [10,11]. Furthermore, therapy aimed at VEGF is promising and provides a possible link with clinical outcome [12,13]. The majority of published work on the angiopoietins and its receptor (Tie-2) focuses on tissue culture and animal models, levels in tissues, or on RNA/DNA (e.g., [14–16]). More recently, changes in levels of cytokines such as interleukin-6 (IL-6) in breast cancer imply a degree of inflammation in these patients [17]. Furthermore, IL-6 and angiogenic growth factors can also be found within platelets although the significance of this is unknown [6,18]. Raised levels of the platelet marker soluble P selectin in cancer may be related to this finding [19,20]. We have previously reported altered levels of plasma VEGF, soluble P selectin, IL-6 and angiopoietins-1 and -2 and their receptors Flt-1 (VEGFR-1) and Tie-2 in cross-sectional studies of breast cancer [6,19–22]. In the present communication we tested the hypothesis that conventional treatment for breast cancer (surgery, followed by radiotherapy and/or chemotherapy) would, as it does for VEGF [10,11], normalize levels of plasma angiopoietins -1 and -2, their receptors, and that of VEGF 3 and 12 months after surgery. Thus, essentially, the broad objective was to determine whether or not the angiopoietins and receptors have anything more to offer than VEGF. We also measured the inflammatory marker IL-6 [17] and platelet marker soluble P selectin [19,20] to determine any relationship between tumour-debulking and subsequent therapy and markers.
2. Materials and methods We tested our hypothesis in blood samples from 40 women (mean [standard deviation] age 59 [10] yrs old, 7 pre-menopausal) about to undergo either mastectomy (n = 16) or wide local excision (n = 24) for biopsy-proven breast cancer, followed by standard best medical care (i.e., radiotherapy and/or chemotherapy with tamoxifen), as directed by clinical need alone. We have previously described cross-sectional studies on these women [6,19,22]. Blood samples were taken before surgery, and both 3 and 12 months later. Post-surgical treatment was a 3 month course of radiotherapy consisting of 12 cycles of 40 Gy to the affected breast (20 women) and/or 20 mg/day tamoxifen as secondary adjuvant therapy (23 women). None of the tamoxifen patients became intolerant. All patients had commenced tamoxifen by the 3 month follow-up appointment and were still taking tamoxifen at the 12 month follow-up appointment. Following surgery, excised tissue was examined by routine and established histological techniques. Lymphovascular invasion was deemed to be present in tissues from 5 women, lymph node involvement was present in 9, and 30 tumours were estrogen receptor positive. Sixteen of the tumours were Grade 1, 13 were Grade 2, and 11 were Grade 3. Tumour size was median 18 mm diameter (inter-quartile range 12.25–26.75 mm, minimum 6 mm, maximum 45 mm). The control group was 30 age-matched women (mean [standard deviation] age 57 [12] years, t test p = 0.518) undergoing surgery for relative benign reasons, i.e., Hadfield’s procedure in 2, mammillary fistula excision in 2, axillary hidradenitis in 1 and benign lump excision in the remaining 25. None of the excised tissue was neoplastic. Exclusion criteria for both groups were concurrent connective tissue or cardiovascular disease and use of antibiotics implying inflammation. The approval of the Research Ethics Committee of the Sandwell and West Birmingham NHS Trust was obtained, as was written informed consent from each participant. 2.1. Laboratory Venous blood was obtained into sodium citrated, centrifuged at 2500 rpm/1000 g for 20 min, and plasma stored at – 70 °C. Makers were measured by commercial ELISA (R&D Systems, Abingdon UK) [6,19,22]. 2.2. Data analysis Continuous data were subjected to the Ryan-Joiner test to assess distribution. Data are presented as mean and standard deviation (analysed by t test), or median and inter-quartile (IQR) range (by Mann–Whitney U test), as appropriate. Categorical data were compared using the v2 test. The Kruskal–Wallis test compared
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different groups where the data were not normally distributed and the one-way ANOVA for normally distributed data. Correlations were sought using Pearson’s method for normally distributed data, while Spearman’s method was used for non-normally distributed data. Serial samples were analysed by two-way (repeated measures) analysis of variance. P < 0.05 was taken to imply significance. All analyses were performed on Minitab release 13 (Minitab Inc, State College, PA, USA). 3. Results 3.1. Baseline case-control findings Table 1 shows baseline and follow-up differences in research indices. At baseline, the women with cancer had raised VEGF, IL-6, soluble P selectin, Ang-1 and Tie-2 with lower Flt-1 than the control women with benign breast disease (BBD). There was no difference in levels of Ang-2. There were no baseline differences in any of the seven research indices in the breast cancer group when analysed by type of surgery (wide local excision n = 24 versus mastectomy n = 16), by tertile of the
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size of the tumour, by the presence/absence of lymph node involvement (present in 9 women) or menopausal status (7 pre-menopausal). Only Tie-2 showed a relationship with positive estrogen receptor status (median 32 ng/mL [IQR 20-185] n = 9 versus 20 [13.5–26.3] ng/mL in women with negative estrogen receptor tumours, n = 30, p = 0.03). VEGF was higher in 5 women with lymphovascular involvement (350 [270–375,340] pg/mL) compared to the 35 free of involvement (150 [50–300] pg/mL, p = 0.022), as was IL-6 (18 [15.5–26.5] pg/mL versus 10 [5–12] pg/mL, p = 0.0074). VEGF was also different according to grade: 50 [12.5–30.5] pg/mL in 16 women with grade 1 tumours, 150 [65–270] pg/mL in 13 with grade 2 tumours, and 340 [220–350] pg/mL in 11 women with grade 3 tumours (p = 0.027). Taking the 95th percentile of the BBD group as a cut-off point, VEGF was raised in 26 (65%) of breast cancer cases (i.e., >100 pg/mL). Similarly, raised Tie-2 (>10 ng/mL) identified 34 (85%) of women with cancer, raised Ang-1 (>10.2 ng/mL) identified only 35% of women, whereas low Flt-1 (<0.5 ng/mL) correctly identified 23 (57.5%) women with breast cancer.
Table 1 Longitudinal analysis of 40 women with breast cancer and 30 with benign breast disease, before and after surgery Baseline
3-Month
12-Month
P value
VEGF (pg/mL) Cancer Benign breast disease
200 (50–340)+++ 30 (24–50)
50 (10–150)* 27.5 (24–52)
50 (20–137)* 42.5 (24–60)
< 0.001 0.708
Flt-1 (ng/mL) Cancer Benign breast disease
0.4 (0.2–0.68)++ 1.5 (0.5–2.31)
0.5 (0.33–0.78) 0.75 (0.5–1.5)
0.5 (0.4–0.95) 0.75 (0.5–1.0)
0.645 0.290
Ang-1 (ng/mL) Cancer Benign breast disease
6.0 (4.0–23.5)+++ 4.0 (2.5–5.0)
5.0 (4.0–10.0)* 4.0 (2.0–8.0)
5.0 (4.0–7.7)* 4.0 (3.0–5.75)
<0.001 0.134
Ang-2 (ng/mL) Cancer Benign breast disease
1.62 (1.0–2.37) 1.3 (0.75–1.75)
1.5 (1.0–2.0) 1.5 (1.0–2.0)
1.3 (1.0–2.6) 1.5 (1.15–2.0)
0.750 0.450
Tie-2 (ng/mL) Cancer Benign breast disease
20 (15–31.0)+++ 7.5 (6.0–8.6)
15 (10.0–25.0) 8.0 (5.2–9.2)
10 (5.0–20.0)* 8.0 (5.9–9.1)
0.005 0.782
IL-6 (pg/mL) Cancer Benign breast disease
10 (5–15)+ 5.2 (5.0–7.5)
7.5 (5–10)* 6.25 (5.0–7.5)
7.5 (5–14.2)* 5.0 (5.0–7.6)
0.026 0.608
sPsel (pg/mL) Cancer Benign breast disease
65 (26)+++ 45 (9)
58 (13)* 38 (10)
57 (14)* 42 (16)
0.001 0.388
VEGF = vascular endothelial growth factor, Ang-1 = angiopoietin-1, Ang-2 = angiopoietin-2, IL-6 = interleukin-6, s-Psel = soluble P selectin. Data presented as median and inter-quartile range or as mean and standard deviation. Major p value by two-way analysis of variance. *p < 0.05 from baseline result. Baseline differences between cancer and benign breast disease patents +p = 0.003, ++p = 0.001, +++p < 0.001 (other time points not analysed).
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Table 2 Significant correlations between the research indices Pairs of indices
Tie-2 and Ang-2 sPsel and Ang-1 sPsel and VEGF VEGF and Flt-1 VEGF and Ang-1 Tie-2 and Ang-1 IL-6 and VEGF sPsel and IL-6 Il-6 and Tie-2
Breast cancer
Benign breast disease
r Value
p Value
0.637 0.621 0.541 0.505 0.503 0.477 0.456 0.451 0.450
<0.001 <0.001 <0.001 <0.001 <0.001 0.003 0.001 0.001 0.004
r Value 0.124 0.034 0.054 0.237 0.026 0.547 0.275 0.13 0.137
p Value 0.513 0.858 0.775 0.207 0.893 0.002 0.141 0.494 0.472
Data are Spearman’s correlation coefficient (r) and p value derived from n = 40 women with breast cancer and n = 30 women with benign breast disease.
Table 2 shows numerous significant (p < 0.01) Spearman rank correlations in both patient groups. Notably, the correlation between Tie-2 and Ang-1 was significant in both groups. All other correlations were significant in the cancer group alone. 3.2. Three and Twelve month follow-up findings The effect of surgery followed by chemo- and/or radiotherapy was to reduce VEGF, Ang-1, Tie-2, IL-6 and soluble P selectin in the women with breast cancer (Table 1). There were no changes following surgery in BBD group. 3.3. Response of VEGF to treatment in cancer patients A level of <100 pg/mL in our hands [6,19,22] defines a normal level and therefore a good response to combined treatment. Under this criterion, 26 of the 40 women (65%) had raised VEGF (P100 pg/mL) at baseline. Of these, 13 women (50%) had a reduction in their VEGF level to <100 pg/ml (i.e., were responders) at 3 months, whilst 16 (62%) were responders at 12 months. None of the demographic (age, menopausal status), tumour indices (size, grade, estrogen receptor status, lymph node involvement, lymphovascular invasion), type of surgery (wide local excision or mastectomy) or type of therapy (none, radiotherapy alone, tamoxifen alone, both treatments) predicted those women whose VEGF levels responded. Similarly, none of the other research indices predicted a VEGF response at 3 or 12 months, although baseline Ang-1 was significantly higher in the 10 women whose VEGF failed to normalized (32.5 [8.8–76.3] ng/mL) compared to the 16 women whose VEGF did normalize (6.0 [4–11.5] ng/mL, p = 0.017).
4. Discussion Of the angiogenic growth factors, VEGF currently has the highest profile. Levels are raised in
breast and other cancers and fall after treatment, and it is a promising target for therapy [6–14,23– 26], although not all have found raised levels in breast cancer [27]. Our present finding that VEGF levels are reduced 3 months and 12 months after surgery supports and extends similar data at 5 days and 3 weeks [10,11] although these two studies did not examine the late effects of subsequent chemoand radio-therapy. Angiopoietins-1 and -2, and the receptors for these growth factors may also be important in cancer [6,14–16,21,22]. Indeed, combined therapy aimed at VEGF receptor-2 may also be useful [28]. We report abnormal Ang-1 (raised), Flt-1 (lower) and Tie-2 (raised) in breast cancer compared to BBD. Furthermore, combined treatment normalized levels of Ang-1 3 months after surgery, and Tie-2 12 months after surgery. Changes in Flt-1 after surgery were minor and not-significant. Unsurprisingly, many indices correlated significantly (Table 2), although only that between Tie-2 and Ang-2 was significant in both groups, implying the relationship is physiological, not pathological. None of the growth factors or their receptors were related strongly to any clinical or tumour-related index. VEGF was higher in the 5 women with lymphovascular involvement (p = 0.022), but this may result from small number bias, and was weakly (p = 0.027) positively related to increasing grade. With our small numbers we are unable to confirm raised VEGF in the menopause [7]. Abnormalities in inflammatory cytokines (such as IL6), and markers of platelet activation (such as soluble P selectin) in breast and other cancers are established [17–20] and our data confirm this. The effect of treatment was to reduce levels of these
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