- Email: [email protected]
CD105 (Endoglin) expression in breast carcinoma effusions is a marker of poor survival
The Breast 19 (2010) 493e498
Contents lists available at ScienceDirect
The Breast journal homepage: www.elsevier.com/brst
Original article
CD105 (Endoglin) expression in breast carcinoma effusions is a marker of poor survival Ben Davidson a, b, *, Helene Tuft Stavnes a, Mette Førsund a, Aasmund Berner a, b, Anne Cathrine Staff b, c a
Division of Pathology, Norwegian Radium Hospital, Oslo University Hospital, N-0310 Oslo, Norway The Medical Faculty, University of Oslo, N-0316 Oslo, Norway c Department of Obstetrics and Gynaecology, Ulleval Hospital, Oslo University Hospital, N-0407 Oslo, Norway b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2010 Accepted 24 May 2010 Available online 17 June 2010
We analyzed the expression and clinical role of endoglin (CD105) in breast carcinoma effusions. Endoglin levels were measured in 36 effusion supernatants by ELISA and studied for association with the cancerassociated markers calprotectin, VEGF, and the VEGF receptor sFlt1. Endoglin expression was further studied in 46 effusions and 22 primary carcinomas using immunohistochemistry. The four secreted molecules were detected in all specimens and their levels significantly correlated (p < 0.001). In effusions, endoglin was localized to carcinoma cells and reactive mesothelium using immunohistochemistry. Tumor cell expression was higher in effusions compared to primary carcinomas (p ¼ 0.025), and in postchemotherapy compared to pre-chemotherapy effusions (p ¼ 0.017). Higher tumor endoglin expression was associated with poor overall (p ¼ 0.021) and disease-free (p ¼ 0.032) survival in univariate analysis, and was an independent predictor in Cox multivariate analysis (p ¼ 0.001 and p ¼ 0.038, respectively). Our data suggest that endoglin may be an important therapeutic target in metastatic breast cancer. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Breast carcinoma Effusions Angiogenic markers Survival
Introduction Breast cancer is the most common malignancy (23% of all cancers), with an estimated 1.15 million new cases worldwide in 2002, and is the leading cause of cancer mortality in women (14% of female cancer deaths).1 In the U.S., 61% of new breast cancer cases are diagnosed while localized, 31% are diagnosed in a regional stage, and 6% have already metastasized to distant sites at diagnosis (stage unknown in the remaining 2%).2 Breast cancer metastasizes most often to axillary lymph nodes, but may involve any organ. Metastasis to serosal surfaces, which occurs in approximately 50% of patients with metastatic disease, involves primarily the pleural cavity,3,4 and occasionally the pericardial and peritoneal cavities.5,6 Pleural effusions may occur at any point during the clinical course and may be the sole manifestation of metastatic disease.3,4,7 The prognosis of breast cancer is relatively good, with 5-year survival averaging 73% in developed countries and 57% in developing
* Corresponding author at: Division of Pathology, Norwegian Radium Hospital, Oslo University Hospital, Ullernchausseen 70, Montebello N-0310, Oslo, Norway. Tel.: þ47 22934871; fax: þ47 22508554. E-mail addresses: [email protected] (B. Davidson), helene.tuft.stavnes@ radiumhospitalet.no (H.T. Stavnes), [email protected] (M. Førsund), [email protected] (A. Berner), annetine.staff@uus. no (A.C. Staff). 0960-9776/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.breast.2010.05.013
countries.1 However, the presence of pleural effusion is associated with poor prognosis, with median survival of 6 and 13 months reported in 2 independent studies.3,4 Endoglin (CD105) is a transmembrane glycoprotein composed of two 95-kDa subunits that form a homodimeric 180-kDa protein. There are two splice isoforms of the protein, termed Short (S)endoglin and long (L)-endoglin based on differences in its cytoplasmic part. In addition, a soluble form of the protein (sEng) is probably formed by proteolytic shedding. Endoglin is primarily expressed on endothelial cells, but has been detected in various normal tissues of mesenchymal and hematopoietic origin, as well as in tumor cells in melanoma, ovarian carcinoma and prostate carcinoma.8,9 Endoglin is part of the transforming growth factorb pathway, and is able to bind both TGF-b and other ligands of this signaling pathway, such as activin-A and BMP family members. TGF-b binding to its receptor results in activin-like kinase (ALK) recruitment and signaling propagation to the nucleus via Smad proteins that act as transcription repressors or activators.8 Mutations in endoglin or its associated protein activin-like kinase-1 (ALK-1) are the molecular cause for hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu disease), a condition that is characterized by vascular malformations and severe bleeding episodes. sEng levels are elevated in the plasma of women suffering from pre-eclampsia compared to women with normal pregnancy, a finding that precedes disease development.10,11
494
B. Davidson et al. / The Breast 19 (2010) 493e498
Endoglin expression on endothelial cells and sEng serum or plasma concentration have been shown to be associated with the presence of metastasis, poor response to chemotherapy and poor survival in breast carcinoma.12e19 However, the presence and clinical role of sEng in breast carcinoma effusions has not been studied to date. We recently showed that sEng and calprotectin, member of the S100 family, are present at high concentrations in ovarian carcinoma effusion supernatants,20 suggesting a role in tumor biology at this anatomic site. In the present study, we measured sEng concentration in breast carcinoma effusion supernatants and studied the association between sEng concentration and the concentration of other secreted cancer-associated proteins in effusion supernatants. We further attempted to identify the cellular source of this molecule in breast carcinoma effusions, and studied the relationship between sEng levels and clinicopathologic parameters, including patient survival.
Table 1 Clinicopathologic data of 38 breast carcinoma patients. Parameter Age Histological type
Grade
Stage (TNM Classification)
Materials and methods Receptor status
Patients and material ELISA cohort The material used for ELISA analysis consisted of 36 effusions (31 pleural, 4 peritoneal, 1 pericardial) from 36 female patients aged 42e86 years (mean ¼ 62 years) with histologically verified breast cancer submitted for routine diagnostic purposes to the Division of Pathology at the Norwegian Radium Hospital during the period of 1999e2007. Slides from 31 cases were available for review. These consisted of 25 infiltrating ductal carcinomas, 4 lobular carcinomas and 2 infiltrating carcinomas of mixed ductal and lobular type. Histological grade was as follows: grade 2: 17 cases; grade 3: 11 cases; no surgical specimen available for definitive grading: 3 cases. Immunohistochemistry (IHC) cohort The material analyzed using IHC consisted of 46 effusions from 44 patients. Clinicopathologic data were available for 38 patients and are presented in Table 1. In addition, 18 patient-matched primary breast carcinomas were immunostained. All specimens underwent morphological evaluation by cytopathologists and were further characterized using immunohistochemistry, as previously detailed.21 Study approval was given by the Regional Committee for Medical Research Ethics in SouthEastern Norway.
Surgery
Chemotherapyc
Radiotherapy
Antihormonal therapy
Site of first recurrence
Number of cases Mean; Range Ductal Lobular Mixed ductal and lobular NAa I II III NA I (T1N0M0) IIA (T1N1M0, T2N0M0) IIB (T2N1M0, T3N0M0) IIIA (T2N2M0, T3N1M0) IIIB (T4N2M0) IV (TXNXM1)b T3/T4, unknown N- and M-status Positive Negative NA No Yes (þadjuvant chemo) Yes (no adjuvant chemo) NA No Yes NA No Yes NA No Yes NA Lung/Pleurad Bonee Axilla/regional lymph nodes Disseminated/progressive Local Chest wall Distant skin
59; 33e83 31 2 2 3 20 10 5 3 5 5 9 6 2 8 3 23 14 1 4 12 20 2 17 19 2 17 17 4 17 20 1 15 7 5 5 3 2 1
a
NA ¼ Not available. Different T and N status, all with documented distant metastases at diagnosis. CMF (cyclophosphamide, methotrexate, 5-fluorouracil) or FEC (5-fluorouracil, epirubicin, methotrexate). d Including three patients with concomitant liver metastases, three patients with bone metastases, two patients with mediastinal metastases and two patients with cutaneous metastases. e Including one patient with concomitant recurrence in the chest wall and two with liver metastases. b
c
ELISA The levels of sEng, vascular endothelial growth factor (VEGF) and its soluble receptor Flt1 (sFlt1; VEGFR1) were measured in 36 fresh-frozen effusion supernatants with commercially available immunoassays according to the manufacturer’s instructions (R&D systems, Minneapolis, MN), as previously described.11,22 Calprotectin levels were measured in the same samples at the Blood Bank and Department of Immunology and Transfusion Medicine, Ulleval University Hospital using ELISA, as previously described.20 The technicians performing the tests were blinded to any clinicopathologic data related to the study cohort. The assays were done in duplicate and results represent the mean of the two analyses per woman.
sections were incubated for 30 min with a mouse monoclonal antiendoglin antibody (Novocastra/Leica, Newcastle Upon Tyne, UK), diluted 1:40. Visualization was achieved using the EnVisionÔ þ peroxidase system (Dako, Glostrup, Denmark). Negative controls consisted of sections that underwent similar staining procedures with a non-relevant antibody of the corresponding isotype (mouse IgG2a). Positive control consisted of normal colon. All specimens containing <100 tumor cells were excluded. Staining was considered positive when localized to the membrane or cytoplasm. Staining extent was scored on a scale of 0e4, as follows: 0 ¼ no staining, 1 ¼ 1e5%, 2 ¼ 6e25%, 3 ¼ 26e75%, 4 ¼ 76e100% of tumor cells. Slides were scored by two surgical pathologists with diagnostic experience in cytopathology (AB and BD).
Immunohistochemistry (IHC) Statistical analysis Sixty-eight specimens (46 effusions, 22 patient-matched primary carcinomas) were manually immunostained for endoglin. Following microwave pretreatment in Tris-EDTA buffer (pH ¼ 10),
Statistical analysis was performed applying the SPSS-PC package (Version 16.0, Chicago, IL). Probability of <0.05 was considered
B. Davidson et al. / The Breast 19 (2010) 493e498
significant. The association between angiogenic factor levels in effusion supernatants was analyzed using a two-sided T-test. Analysis of the association between the studied proteins and clinicopathologic parameters were performed using the ManneWhitney U Test. Clinicopathologic parameters consisted of patient age (60 vs. >60 years), disease stage (1e2 vs. 3e4), hormone receptor status in the primary tumor, surgical status (primary surgery vs. surgery following neoadjuvant chemotherapy or no surgery), previous chemotherapy (yes vs. no), previous radiotherapy (yes vs. no) and previous anti-hormone therapy (yes vs. no). Comparison of endoglin expression by IHC in matched primary carcinomas and effusions was performed using the Wilcoxon Signed Ranks Test. Comparison of this parameter in all effusions vs. primary carcinomas was done using the ManneWhitney U Test. Disease-free survival (DFS) and overall survival (OS) were calculated from the date of diagnosis to the date of recurrence/death or last follow-up. Univariate survival analyses were executed using the KaplaneMeier method and log-rank test. In this analysis, secreted angiogenic molecule expression level in effusions supernatants was categorized as high or low based on median expression values. IHC results were re-grouped as high (3e4) vs. low (0e2) score. The above-listed clinicopathologic parameters were additionally analyzed for their prognostic role. Multivariate survival analysis was performed using the Cox regression model. Parameters with p < 0.2 in univariate analysis were entered into the Cox analysis. Results Breast carcinoma effusion supernatants contain secreted cancerassociated molecules ELISA analysis of 36 effusion supernatants showed measurable sEng, calprotectin, VEGF and sFlt in all specimens, with levels as follows: sEng: range: 1.55e7.31 ng/ml, median ¼ 3.18 ng/ml; calprotectin: range: 301e9431 ng/ml, median ¼ 1155 ng/ml; VEGF: range: 61e2000 pg/ml, median ¼ 1473 pg/ml; sFlt: range: 85e2000 pg/ml, median ¼ 534 pg/ml. Significant direct association was seen between the levels of all factors (sEng-calprotectin: p < 0.001; sEng-VEGF: p ¼ 0.013; sEng-sFlt: p ¼ 0.002; calprotectinVEGF: p ¼ 0.002; calprotectin-sFlt: p ¼ 0.001; VEGF-sFlt: p < 0.001). With the exception of higher VEGF levels in effusions from women who received chemotherapy prior to tapping (p ¼ 0.036), no association was found between marker levels by ELISA and clinicopathologic parameters or survival (p > 0.05; data not shown). Carcinoma cells and reactive cells in breast carcinoma effusions commonly express endoglin The source of effusion endoglin was hypothesized to be at least in part the blood-borne soluble endoglin. However, we wished to investigate whether any of the cell populations in the effusions may express endoglin at the cell membrane, thereby suggesting local production and shedding as an additional contributing source. Immunostaining showed endoglin expression on carcinoma cells in 31/46 (67%) effusions, with the following staining score: 0: 15; 1: 11; 2: 5; 3: 7; 4: 8 specimens (Fig. 1A and B). Reactive mesothelial cells and macrophages expressed endoglin in 13/46 (28%) effusions (Fig. 1C and D). In order to evaluate whether tumor cells expressed endoglin in primary carcinomas, we stained 21 patient-matched tumors from patients with effusions. Endoglin was expressed in carcinoma cells in 10/21 (48%) tumors with the following staining score: 0: 11; 1: 8; 2: 1; 3: 0; 4: 1 specimens (Fig. 1EeG). Wilcoxon test showed a trend for higher expression in carcinoma cells in effusions compared to primary tumors (p ¼ 0.174), a difference that
495
became significant in comparative analysis of all effusions vs. primary carcinomas (p ¼ 0.025). No association was found between endoglin expression in effusions by IHC and clinicopathologic parameters (p > 0.05; data not shown). Positive and negative controls gave satisfactory results in all experiments, and endothelial cells were endoglin-positive in all primary carcinoma specimens (Fig. 1E), providing an internal control. Inter-observer agreement was good at 80%, with differences of one scoring level that were easily settled in consensus meetings. Cancer cell endoglin expression in effusions is an independent marker of poor survival in breast cancer The role of endoglin expression by IHC in predicting survival was analyzed for 38 patients with available clinical data. Follow-up period ranged from 2 to 393 months (mean ¼ 96 months, median ¼ 51 months). At the last follow-up, 2 patients were alive with disease and 36 were dead of disease. The parameters that were evaluated in univariate survival analysis were patient age (60 vs. >60 years), disease stage (1e2 vs. 3e4), hormone receptor status in the primary tumor, surgical status (primary surgery vs. surgery following neoadjuvant chemotherapy or no surgery), previous chemotherapy (yes vs. no), previous radiotherapy (yes vs. no), previous anti-hormone therapy (yes vs. no) and endoglin expression by IHC. In univariate analysis, endoglin expression in a higher percentage (>25%) of tumor cells was associated with poor OS (p ¼ 0.021) and DFS (Fig. 2) (p ¼ 0.032). In addition, previous chemotherapy (48 vs. 163 months, p < 0.001) and radiotherapy (54 vs. 157 months, p ¼ 0.002) significantly correlated with poor OS, while advanced stage (p ¼ 0.095) and previous anti-hormone therapy (p ¼ 0.148) were associated with a trend for poor OS. Previous chemotherapy (25 vs. 101 months, p ¼ 0.002) and radiotherapy (28 vs. 100 months, p ¼ 0.002) were also the parameters that were significantly associated with poor DFS, whereas older age (p ¼ 0.199) and previous anti-hormone therapy (p ¼ 0.108) were associated with a trend for poor DFS. In Cox analysis of OS, higher endoglin expression (p ¼ 0.001; hazard ratio 11.2), previous chemotherapy (p ¼ 0.001; hazard ratio 11.7) and previous radiotherapy (p ¼ 0.004; hazard ratio 8.3) were independent markers of poor outcome. The same three parameters were independent predictors of poor DFS (endoglin: p ¼ 0.038, hazard ratio 4.3; chemotherapy: p ¼ 0.002, hazard ratio 9.3; radiotherapy: p ¼ 0.004, hazard ratio 8.3). Discussion Breast cancer patients diagnosed with malignant effusion constitute a patient group with extremely poor outcome in which curative treatment is essentially impossible to achieve. As such, they may benefit from targeted molecular therapy aimed at prolonging survival and hopefully providing improved quality of life. However, published studies focusing on the biology of breast carcinoma cells in effusions are rare. In recent years, we have documented the differential expression of cancer-associated molecules in breast carcinoma cells in effusions compared to primary tumors and reported on the prognostic role of several of these molecules.23e29 In the present study, we studied the expression and clinical role of endoglin, as well as the relationship between its secreted form and other secreted pro-angiogenic factors (VEGF and Flt1) and the pro-inflammatory S100 family member calprotectin. The latter molecule was included in the study in view of our previous observation that it is frequently expressed in ovarian carcinoma effusions,20 as well as the finding that
496
B. Davidson et al. / The Breast 19 (2010) 493e498
Fig. 1. Endoglin protein expression by immunohistochemistry in breast carcinoma. (AeD): Effusions: Endoglin-positive breast carcinoma cells (AeB) and reactive cells (macrophages and mesothelial cells, green arrows; CeD) in four effusions. Carcinoma cells in CeD (black arrows) are negative. (EeG): Primary carcinomas: Three examples of negative (E), focal (<5% of cells; F) and diffuse (>75%; G) endoglin expression in carcinoma cells (black arrows) in three primary tumors. Endothelial cells (red arrow in E) were positive in all specimens. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
chemokine receptors are expressed on both breast carcinoma cells and leukocytes in effusions,30 suggesting an interplay between cancer cells and the immune system. sEng, calprotectin, VEGF and sFlt were found in the effusion supernatant of all specimens. To date, data regarding the presence of sEng and calprotectin in effusions is limited to our recent study of ovarian carcinoma specimens.20 As in the latter study of ovarian cancer effusions, both secreted proteins were universally expressed in effusions from breast cancer patients, and their effusion concentration was unrelated to patient survival. The presence of secreted VEGF in all our specimens is in agreement with several previous reports in which this angiogenic molecule has been found in effusions, frequently with significantly higher levels in malignant compared to nonneoplastic effusions.31e35 The co-expression of VEGF and its receptors in effusions has previously been reported in benign mesothelium and malignant mesothelioma.35e37 Interestingly,
VEGF was shown to be significantly co-expressed with TGF-b in effusions,38 suggesting, together with our finding of such association between endoglin, VEGF and sFlt, co-activation of these pathways in an autocrine manner at this anatomic site in advanced cancer forms. The ubiquitous presence of endoglin in breast carcinoma effusions was assumed by us to originate at least in part from the patients’ blood. We had no access to the corresponding patient serum or plasma in this study. However, we additionally wished to assess whether host and tumor cells in the effusions express this protein. We observed endoglin expression in tumor cells in the majority of analyzed effusions, with significantly higher expression frequency and extent compared with primary tumors, suggesting that endoglin may promote metastasis in breast cancer. In addition, endoglin was expressed by mesothelial cells and macrophages, well in agreement with the ability of these cell classes to produce angiogenic factors. To the best of our knowledge, this is the first
B. Davidson et al. / The Breast 19 (2010) 493e498
497
reported in the present study must be regarded as preliminary in view of the small size of the analyzed series. However, it suggests that this protein may be a viable target for directed treatment against both tumor cells and the peri-tumoral vasculature. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements Financial acknowledgement: This work was supported by grants from the Norwegian Cancer Society and the Research Fund at the Norwegian Radium Hospital. We thank Ms. Lise Levy at Ulleval Hospital, Oslo University Hospital, for her competent help in organizing and performing the ELISA effusion analyses. References
Fig. 2. Endoglin expression in breast carcinoma effusions correlates with poor survival A: KaplaneMeier survival curve showing correlation between endoglin expression and disease-free survival (DFS) for 36 patients. Patients with effusions with higher expression (>25% of tumor cells) (n ¼ 11, dashed line) had mean DFS ¼ 25 months vs. 77 months for patients whose effusions showed low (25%) expression (n ¼ 25, solid line; p ¼ 0.032). Two patients had no data with respect to DFS. B: KaplaneMeier survival curve showing correlation between endoglin expression and overall survival (OS) for 38 patients. Patients with effusions with higher expression (>25% of tumor cells) (n ¼ 11, dashed line) had mean OS ¼ 48 months vs. 119 months for patients whose effusions showed low (25%) expression (n ¼ 27, solid line; p ¼ 0.021).
evidence of tumor cell endoglin expression in breast carcinoma. However, this finding concords with previous observations that endoglin is expressed in melanoma and ovarian carcinoma cells.39,40 We suggest that the soluble endoglin in effusions from breast carcinoma patients, in addition to its origin in the blood, also stems from shedding of membrane-bound endoglin molecules from tumor cells due to proteolytic cleavage of the receptor. We further report that endoglin expression in carcinoma cells is an independent prognostic marker of poor OS and DFS, in agreement with the finding in ovarian carcinoma.40 This suggests that endoglin may be a molecular therapeutic target in metastatic breast carcinoma, a modality that has already been investigated in the context of anti-angiogenic therapy.41 In conclusion, endoglin is frequently expressed in breast carcinoma cells in effusions and is most likely secreted into the effusion supernatant, together with other cancer-associated proteins. The association between tumor endoglin expression and poor survival
1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74e108. 2. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics, 2008. CA Cancer J Clin 2008;58:71e96. 3. Fentiman IS, Millis R, Sexton S, Hayword JL. Pleural effusion in breast cancer: a review of 105 cases. Cancer 1981;47:2087e92. 4. Raju RN, Kardinal CG. Pleural effusion in breast carcinoma: analysis of 122 cases. Cancer 1981;48:2524e7. 5. Wilkes JD, Fidias P, Vaickus L, Perez RP. Malignancy-related pericardial effusion. 127 cases from the Roswell Park Center Institute. Cancer (Phila) 1995;76: 1377e87. 6. Buck M, Ingle JN, Giuliani ER, Gordon JR, Therneau TM. Pericardial effusion in women with breast cancer. Cancer 1987;60:263e9. 7. Kamby C, Vejborg I, Kristensen B, Olsen LO, Mouridsen HT. Metastatic pattern in recurrent breast cancer. Special reference to intrathoracic recurrences. Cancer 1988;62:2226e33. 8. Dallas NA, Samuel S, Xia L, Fan F, Gray MJ, Lim SJ, et al. Endoglin (CD105): a marker of tumor vasculature and potential target for therapy. Clin Cancer Res 2008;14:1931e7. 9. ten Dijke P, Goumans MJ, Pardali E. Endoglin in angiogenesis and vascular diseases. Angiogenesis 2008;11:79e89. 10. Levine RJ, Lam C, Qian C, Yu KF, Maynard SE, Sachs BP, et al. Soluble endoglin and other circulating antiangiogenic factors in preeclampsia. N Engl J Med 2006;355:992e1005. 11. Staff AC, Braekke K, Johnsen GM, Karumanchi SA, Harsem NK. Circulating concentrations of soluble endoglin (CD105) in fetal and maternal serum and in amniotic fluid in preeclampsia. Am J Obstet Gynecol 2007;197:176.e1e6. 12. Kumar S, Ghellal A, Li C, Byrne G, Haboubi N, Wang JM, et al. Breast carcinoma: vascular density determined using CD105 antibody correlates with tumor prognosis. Cancer Res 1999;59:856e61. 13. Dales JP, Garcia S, Bonnier P, Duffaud F, Andrac-Meyer L, Ramuz O, et al. CD105 expression is a marker of high metastatic risk and poor outcome in breast carcinomas. Correlations between immunohistochemical analysis and long-term follow-up in a series of 929 patients. Am J Clin Pathol 2003;119: 374e80. 14. Beresford MJ, Harris AL, Ah-See M, Daley F, Padhani AR, Makris A. The relationship of the neo-angiogenic marker, endoglin, with response to neoadjuvant chemotherapy in breast cancer. Br J Cancer 2006;95:1683e8. 15. Dhakal HP, Naume B, Synnestvedt M, Borgen E, Kaaresen R, Schlichting E, et al. Vascularization in primary breast carcinomas: its prognostic significance and relationship with tumor cell dissemination. Clin Cancer Res 2008;14: 2341e50. 16. Li C, Guo B, Wilson PB, Stewart A, Byrne G, Bundred N, et al. Plasma levels of soluble CD105 correlate with metastasis in patients with breast cancer. Int J Cancer 2000;89:122e6. 17. Takahashi N, Kawanishi-Tabata R, Haba A, Tabata M, Haruta Y, Tsai H, et al. Association of serum endoglin with metastasis in patients with colorectal, breast, and other solid tumors, and suppressive effect of chemotherapy on the serum endoglin. Clin Cancer Res 2001;7:524e32. 18. Vo MN, Evans M, Leitzel K, Ali SM, Wilson M, Demers L, et al. Elevated plasma endoglin (CD105) predicts decreased response and survival in a metastatic breast cancer trial of hormone therapy. Breast Cancer Res Treat 2010;119: 767e71. 19. Zhou S, Wang GP, Liu C, Zhou M. Eukaryotic initiation factor 4E (eIF4E) and angiogenesis: prognostic markers for breast cancer. BMC Cancer 2006;6:231. 20. Ødegaard E, Davidson B, Engh V, Onsrud M, Staff AC. Assessment of endoglin and calprotectin as potential biomarkers in ovarian carcinoma and borderline tumors of the ovary. Am J Obstet Gynecol 2008;199:533.e1e8.
498
B. Davidson et al. / The Breast 19 (2010) 493e498
21. Davidson B, Nielsen S, Christensen J, Asschenfeldt P, Berner A, Risberg B, et al. The role of Desmin and N-cadherin in effusion cytology. A comparative study using established markers of mesothelial and epithelial cells. Am J Surg Pathol 2001;25:1405e12. 22. Staff AC, Braekke K, Harsem NK, Lyberg T, Holthe MR. Circulating concentrations of sFlt1 (soluble fms-like tyrosine kinase 1) in fetal and maternal serum during pre-eclampsia. Eur J Obstet Gynecol Reprod Biol 2005;122:33e9. 23. Davidson B, Reich R, Lazarovici P, Flørenes VA, Nielsen S, Nesland JM. Altered expression and activation of the nerve growth factor receptors TrkA and p75 provides the first evidence of tumor progression to effusion in breast carcinoma. Breast Cancer Res Treat 2004;83:119e28. 24. Davidson B, Konstantinovsky S, Nielsen S, Dong HP, Berner A, Vyberg M, et al. Altered expression of metastasis-associated and regulatory molecules in effusions from breast cancer patients- a novel model for tumor progression. Clin Cancer Res 2004;10:7335e46. 25. Elloul S, Bukholt Elstrand M, Nesland JM, Trope CG, Kvalheim G, Goldberg I, et al. Snail, Slug, and Smad-interacting protein 1 as novel parameters of disease aggressiveness in metastatic ovarian and breast carcinoma. Cancer 2005;103: 1631e43. 26. Konstantinovsky S, Nielsen S, Vyberg M, Kvalheim G, Nesland JM, Reich R, et al. Angiogenic molecule expression is downregulated in effusions from breast cancer patients. Breast Cancer Res Treat 2005;94:71e80. 27. Kleinberg L, Flørenes VA, Skrede M, Dong HP, Nielsen S, McMaster MT, et al. Expression of HLA-G in malignant mesothelioma and clinically aggressive breast carcinoma. Virchows Arch 2006;449:31e9. 28. Davidson B, Konstantinovsky S, Kleinberg L, Nguyen MTP, Bassarova A, Kvalheim G, et al. The mitogen-activated protein kinases (MAPK) p38 and JNK are markers of tumor progression in breast carcinoma. Gynecol Oncol 2006;102:453e61. 29. Kleinberg L, Flørenes VA, Nesland JM, Davidson B. Survivin, a member of the inhibitors of apoptosis (IAP) family, is down-regulated in breast carcinoma effusions. Am J Clin Pathol 2007;128:389e97. 30. Davidson B, Dong HP, Holth A, Berner A, Risberg B. The chemokine receptor CXCR4 is more frequently expressed in breast compared to other metastatic adenocarcinomas in effusions. Breast J 2008;14:476e82.
31. Hsu IL, Su WC, Yan JJ, Chang JM, Lai WW. Angiogenetic biomarkers in nonsmall cell lung cancer with malignant pleural effusion: correlations with patient survival and pleural effusion control. Lung Cancer 2009;65:371e6. 32. Duysinx BC, Corhay JL, Hubin L, Nguyen D, Henket M, Louis R. Diagnostic value of interleukine-6, transforming growth factor-beta 1 and vascular endothelial growth factor in malignant pleural effusions. Respir Med 2008;102:1708e14. 33. Atanackovic D, Cao Y, Kim JW, Brandl S, Thom I, Faltz C, et al. The local cytokine and chemokine milieu within malignant effusions. Tumour Biol 2008;29: 93e104. 34. Kraft A, Weindel K, Ochs A, Marth C, Zmija J, Schumacher P, et al. Vascular endothelial growth factor in the sera and effusions of patients with malignant and nonmalignant disease. Cancer 1999;85:178e87. 35. Thickett DR, Armstrong L, Millar AB. Vascular endothelial growth factor (VEGF) in inflammatory and malignant pleural effusions. Thorax 1999;54:707e10. 36. Strizzi L, Catalano A, Vianale G, Orecchia S, Casalini A, Tassi G, et al. Vascular endothelial growth factor is an autocrine growth factor in human malignant mesothelioma. J Pathol 2001;193:468e75. 37. Davidson B, Vintman L, Zcharia E, Bedrossian C, Berner A, Nielsen S, et al. Heparanase and basic fibroblast growth factor are co-expressed in malignant mesothelioma. Clin Exp Metastasis 2004;21:469e76. 38. Cheng D, Lee YC, Rogers JT, Perkett EA, Moyers JP, Rodriguez RM, et al. Vascular endothelial growth factor level correlates with transforming growth factorbeta isoform levels in pleural effusions. Chest 2000;118:1747e53. 39. Altomonte M, Montagner R, Fonsatti E, Colizzi F, Cattarossi I, Brasoveanu LI, et al. Expression and structural features of endoglin (CD105), a transforming growth factor beta1 and beta3 binding protein, in human melanoma. Br J Cancer 1996;74:1586e91. 40. Henriksen R, Gobl A, Wilander E, Oberg K, Miyazono K, Funa K. Expression and prognostic significance of TGF-beta isotypes, latent TGF-beta 1 binding protein, TGF-beta type I and type II receptors, and endoglin in normal ovary and ovarian neoplasms. Lab Invest 1995;73:213e20. 41. Matsuno F, Haruta Y, Kondo M, Tsai H, Barcos M, Seon BK. Induction of lasting complete regression of preformed distinct solid tumors by targeting the tumor vasculature using two new anti-endoglin monoclonal antibodies. Clin Cancer Res 1999;5:371e82.
Contents lists available at ScienceDirect
The Breast journal homepage: www.elsevier.com/brst
Original article
CD105 (Endoglin) expression in breast carcinoma effusions is a marker of poor survival Ben Davidson a, b, *, Helene Tuft Stavnes a, Mette Førsund a, Aasmund Berner a, b, Anne Cathrine Staff b, c a
Division of Pathology, Norwegian Radium Hospital, Oslo University Hospital, N-0310 Oslo, Norway The Medical Faculty, University of Oslo, N-0316 Oslo, Norway c Department of Obstetrics and Gynaecology, Ulleval Hospital, Oslo University Hospital, N-0407 Oslo, Norway b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2010 Accepted 24 May 2010 Available online 17 June 2010
We analyzed the expression and clinical role of endoglin (CD105) in breast carcinoma effusions. Endoglin levels were measured in 36 effusion supernatants by ELISA and studied for association with the cancerassociated markers calprotectin, VEGF, and the VEGF receptor sFlt1. Endoglin expression was further studied in 46 effusions and 22 primary carcinomas using immunohistochemistry. The four secreted molecules were detected in all specimens and their levels significantly correlated (p < 0.001). In effusions, endoglin was localized to carcinoma cells and reactive mesothelium using immunohistochemistry. Tumor cell expression was higher in effusions compared to primary carcinomas (p ¼ 0.025), and in postchemotherapy compared to pre-chemotherapy effusions (p ¼ 0.017). Higher tumor endoglin expression was associated with poor overall (p ¼ 0.021) and disease-free (p ¼ 0.032) survival in univariate analysis, and was an independent predictor in Cox multivariate analysis (p ¼ 0.001 and p ¼ 0.038, respectively). Our data suggest that endoglin may be an important therapeutic target in metastatic breast cancer. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Breast carcinoma Effusions Angiogenic markers Survival
Introduction Breast cancer is the most common malignancy (23% of all cancers), with an estimated 1.15 million new cases worldwide in 2002, and is the leading cause of cancer mortality in women (14% of female cancer deaths).1 In the U.S., 61% of new breast cancer cases are diagnosed while localized, 31% are diagnosed in a regional stage, and 6% have already metastasized to distant sites at diagnosis (stage unknown in the remaining 2%).2 Breast cancer metastasizes most often to axillary lymph nodes, but may involve any organ. Metastasis to serosal surfaces, which occurs in approximately 50% of patients with metastatic disease, involves primarily the pleural cavity,3,4 and occasionally the pericardial and peritoneal cavities.5,6 Pleural effusions may occur at any point during the clinical course and may be the sole manifestation of metastatic disease.3,4,7 The prognosis of breast cancer is relatively good, with 5-year survival averaging 73% in developed countries and 57% in developing
* Corresponding author at: Division of Pathology, Norwegian Radium Hospital, Oslo University Hospital, Ullernchausseen 70, Montebello N-0310, Oslo, Norway. Tel.: þ47 22934871; fax: þ47 22508554. E-mail addresses: [email protected] (B. Davidson), helene.tuft.stavnes@ radiumhospitalet.no (H.T. Stavnes), [email protected] (M. Førsund), [email protected] (A. Berner), annetine.staff@uus. no (A.C. Staff). 0960-9776/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.breast.2010.05.013
countries.1 However, the presence of pleural effusion is associated with poor prognosis, with median survival of 6 and 13 months reported in 2 independent studies.3,4 Endoglin (CD105) is a transmembrane glycoprotein composed of two 95-kDa subunits that form a homodimeric 180-kDa protein. There are two splice isoforms of the protein, termed Short (S)endoglin and long (L)-endoglin based on differences in its cytoplasmic part. In addition, a soluble form of the protein (sEng) is probably formed by proteolytic shedding. Endoglin is primarily expressed on endothelial cells, but has been detected in various normal tissues of mesenchymal and hematopoietic origin, as well as in tumor cells in melanoma, ovarian carcinoma and prostate carcinoma.8,9 Endoglin is part of the transforming growth factorb pathway, and is able to bind both TGF-b and other ligands of this signaling pathway, such as activin-A and BMP family members. TGF-b binding to its receptor results in activin-like kinase (ALK) recruitment and signaling propagation to the nucleus via Smad proteins that act as transcription repressors or activators.8 Mutations in endoglin or its associated protein activin-like kinase-1 (ALK-1) are the molecular cause for hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu disease), a condition that is characterized by vascular malformations and severe bleeding episodes. sEng levels are elevated in the plasma of women suffering from pre-eclampsia compared to women with normal pregnancy, a finding that precedes disease development.10,11
494
B. Davidson et al. / The Breast 19 (2010) 493e498
Endoglin expression on endothelial cells and sEng serum or plasma concentration have been shown to be associated with the presence of metastasis, poor response to chemotherapy and poor survival in breast carcinoma.12e19 However, the presence and clinical role of sEng in breast carcinoma effusions has not been studied to date. We recently showed that sEng and calprotectin, member of the S100 family, are present at high concentrations in ovarian carcinoma effusion supernatants,20 suggesting a role in tumor biology at this anatomic site. In the present study, we measured sEng concentration in breast carcinoma effusion supernatants and studied the association between sEng concentration and the concentration of other secreted cancer-associated proteins in effusion supernatants. We further attempted to identify the cellular source of this molecule in breast carcinoma effusions, and studied the relationship between sEng levels and clinicopathologic parameters, including patient survival.
Table 1 Clinicopathologic data of 38 breast carcinoma patients. Parameter Age Histological type
Grade
Stage (TNM Classification)
Materials and methods Receptor status
Patients and material ELISA cohort The material used for ELISA analysis consisted of 36 effusions (31 pleural, 4 peritoneal, 1 pericardial) from 36 female patients aged 42e86 years (mean ¼ 62 years) with histologically verified breast cancer submitted for routine diagnostic purposes to the Division of Pathology at the Norwegian Radium Hospital during the period of 1999e2007. Slides from 31 cases were available for review. These consisted of 25 infiltrating ductal carcinomas, 4 lobular carcinomas and 2 infiltrating carcinomas of mixed ductal and lobular type. Histological grade was as follows: grade 2: 17 cases; grade 3: 11 cases; no surgical specimen available for definitive grading: 3 cases. Immunohistochemistry (IHC) cohort The material analyzed using IHC consisted of 46 effusions from 44 patients. Clinicopathologic data were available for 38 patients and are presented in Table 1. In addition, 18 patient-matched primary breast carcinomas were immunostained. All specimens underwent morphological evaluation by cytopathologists and were further characterized using immunohistochemistry, as previously detailed.21 Study approval was given by the Regional Committee for Medical Research Ethics in SouthEastern Norway.
Surgery
Chemotherapyc
Radiotherapy
Antihormonal therapy
Site of first recurrence
Number of cases Mean; Range Ductal Lobular Mixed ductal and lobular NAa I II III NA I (T1N0M0) IIA (T1N1M0, T2N0M0) IIB (T2N1M0, T3N0M0) IIIA (T2N2M0, T3N1M0) IIIB (T4N2M0) IV (TXNXM1)b T3/T4, unknown N- and M-status Positive Negative NA No Yes (þadjuvant chemo) Yes (no adjuvant chemo) NA No Yes NA No Yes NA No Yes NA Lung/Pleurad Bonee Axilla/regional lymph nodes Disseminated/progressive Local Chest wall Distant skin
59; 33e83 31 2 2 3 20 10 5 3 5 5 9 6 2 8 3 23 14 1 4 12 20 2 17 19 2 17 17 4 17 20 1 15 7 5 5 3 2 1
a
NA ¼ Not available. Different T and N status, all with documented distant metastases at diagnosis. CMF (cyclophosphamide, methotrexate, 5-fluorouracil) or FEC (5-fluorouracil, epirubicin, methotrexate). d Including three patients with concomitant liver metastases, three patients with bone metastases, two patients with mediastinal metastases and two patients with cutaneous metastases. e Including one patient with concomitant recurrence in the chest wall and two with liver metastases. b
c
ELISA The levels of sEng, vascular endothelial growth factor (VEGF) and its soluble receptor Flt1 (sFlt1; VEGFR1) were measured in 36 fresh-frozen effusion supernatants with commercially available immunoassays according to the manufacturer’s instructions (R&D systems, Minneapolis, MN), as previously described.11,22 Calprotectin levels were measured in the same samples at the Blood Bank and Department of Immunology and Transfusion Medicine, Ulleval University Hospital using ELISA, as previously described.20 The technicians performing the tests were blinded to any clinicopathologic data related to the study cohort. The assays were done in duplicate and results represent the mean of the two analyses per woman.
sections were incubated for 30 min with a mouse monoclonal antiendoglin antibody (Novocastra/Leica, Newcastle Upon Tyne, UK), diluted 1:40. Visualization was achieved using the EnVisionÔ þ peroxidase system (Dako, Glostrup, Denmark). Negative controls consisted of sections that underwent similar staining procedures with a non-relevant antibody of the corresponding isotype (mouse IgG2a). Positive control consisted of normal colon. All specimens containing <100 tumor cells were excluded. Staining was considered positive when localized to the membrane or cytoplasm. Staining extent was scored on a scale of 0e4, as follows: 0 ¼ no staining, 1 ¼ 1e5%, 2 ¼ 6e25%, 3 ¼ 26e75%, 4 ¼ 76e100% of tumor cells. Slides were scored by two surgical pathologists with diagnostic experience in cytopathology (AB and BD).
Immunohistochemistry (IHC) Statistical analysis Sixty-eight specimens (46 effusions, 22 patient-matched primary carcinomas) were manually immunostained for endoglin. Following microwave pretreatment in Tris-EDTA buffer (pH ¼ 10),
Statistical analysis was performed applying the SPSS-PC package (Version 16.0, Chicago, IL). Probability of <0.05 was considered
B. Davidson et al. / The Breast 19 (2010) 493e498
significant. The association between angiogenic factor levels in effusion supernatants was analyzed using a two-sided T-test. Analysis of the association between the studied proteins and clinicopathologic parameters were performed using the ManneWhitney U Test. Clinicopathologic parameters consisted of patient age (60 vs. >60 years), disease stage (1e2 vs. 3e4), hormone receptor status in the primary tumor, surgical status (primary surgery vs. surgery following neoadjuvant chemotherapy or no surgery), previous chemotherapy (yes vs. no), previous radiotherapy (yes vs. no) and previous anti-hormone therapy (yes vs. no). Comparison of endoglin expression by IHC in matched primary carcinomas and effusions was performed using the Wilcoxon Signed Ranks Test. Comparison of this parameter in all effusions vs. primary carcinomas was done using the ManneWhitney U Test. Disease-free survival (DFS) and overall survival (OS) were calculated from the date of diagnosis to the date of recurrence/death or last follow-up. Univariate survival analyses were executed using the KaplaneMeier method and log-rank test. In this analysis, secreted angiogenic molecule expression level in effusions supernatants was categorized as high or low based on median expression values. IHC results were re-grouped as high (3e4) vs. low (0e2) score. The above-listed clinicopathologic parameters were additionally analyzed for their prognostic role. Multivariate survival analysis was performed using the Cox regression model. Parameters with p < 0.2 in univariate analysis were entered into the Cox analysis. Results Breast carcinoma effusion supernatants contain secreted cancerassociated molecules ELISA analysis of 36 effusion supernatants showed measurable sEng, calprotectin, VEGF and sFlt in all specimens, with levels as follows: sEng: range: 1.55e7.31 ng/ml, median ¼ 3.18 ng/ml; calprotectin: range: 301e9431 ng/ml, median ¼ 1155 ng/ml; VEGF: range: 61e2000 pg/ml, median ¼ 1473 pg/ml; sFlt: range: 85e2000 pg/ml, median ¼ 534 pg/ml. Significant direct association was seen between the levels of all factors (sEng-calprotectin: p < 0.001; sEng-VEGF: p ¼ 0.013; sEng-sFlt: p ¼ 0.002; calprotectinVEGF: p ¼ 0.002; calprotectin-sFlt: p ¼ 0.001; VEGF-sFlt: p < 0.001). With the exception of higher VEGF levels in effusions from women who received chemotherapy prior to tapping (p ¼ 0.036), no association was found between marker levels by ELISA and clinicopathologic parameters or survival (p > 0.05; data not shown). Carcinoma cells and reactive cells in breast carcinoma effusions commonly express endoglin The source of effusion endoglin was hypothesized to be at least in part the blood-borne soluble endoglin. However, we wished to investigate whether any of the cell populations in the effusions may express endoglin at the cell membrane, thereby suggesting local production and shedding as an additional contributing source. Immunostaining showed endoglin expression on carcinoma cells in 31/46 (67%) effusions, with the following staining score: 0: 15; 1: 11; 2: 5; 3: 7; 4: 8 specimens (Fig. 1A and B). Reactive mesothelial cells and macrophages expressed endoglin in 13/46 (28%) effusions (Fig. 1C and D). In order to evaluate whether tumor cells expressed endoglin in primary carcinomas, we stained 21 patient-matched tumors from patients with effusions. Endoglin was expressed in carcinoma cells in 10/21 (48%) tumors with the following staining score: 0: 11; 1: 8; 2: 1; 3: 0; 4: 1 specimens (Fig. 1EeG). Wilcoxon test showed a trend for higher expression in carcinoma cells in effusions compared to primary tumors (p ¼ 0.174), a difference that
495
became significant in comparative analysis of all effusions vs. primary carcinomas (p ¼ 0.025). No association was found between endoglin expression in effusions by IHC and clinicopathologic parameters (p > 0.05; data not shown). Positive and negative controls gave satisfactory results in all experiments, and endothelial cells were endoglin-positive in all primary carcinoma specimens (Fig. 1E), providing an internal control. Inter-observer agreement was good at 80%, with differences of one scoring level that were easily settled in consensus meetings. Cancer cell endoglin expression in effusions is an independent marker of poor survival in breast cancer The role of endoglin expression by IHC in predicting survival was analyzed for 38 patients with available clinical data. Follow-up period ranged from 2 to 393 months (mean ¼ 96 months, median ¼ 51 months). At the last follow-up, 2 patients were alive with disease and 36 were dead of disease. The parameters that were evaluated in univariate survival analysis were patient age (60 vs. >60 years), disease stage (1e2 vs. 3e4), hormone receptor status in the primary tumor, surgical status (primary surgery vs. surgery following neoadjuvant chemotherapy or no surgery), previous chemotherapy (yes vs. no), previous radiotherapy (yes vs. no), previous anti-hormone therapy (yes vs. no) and endoglin expression by IHC. In univariate analysis, endoglin expression in a higher percentage (>25%) of tumor cells was associated with poor OS (p ¼ 0.021) and DFS (Fig. 2) (p ¼ 0.032). In addition, previous chemotherapy (48 vs. 163 months, p < 0.001) and radiotherapy (54 vs. 157 months, p ¼ 0.002) significantly correlated with poor OS, while advanced stage (p ¼ 0.095) and previous anti-hormone therapy (p ¼ 0.148) were associated with a trend for poor OS. Previous chemotherapy (25 vs. 101 months, p ¼ 0.002) and radiotherapy (28 vs. 100 months, p ¼ 0.002) were also the parameters that were significantly associated with poor DFS, whereas older age (p ¼ 0.199) and previous anti-hormone therapy (p ¼ 0.108) were associated with a trend for poor DFS. In Cox analysis of OS, higher endoglin expression (p ¼ 0.001; hazard ratio 11.2), previous chemotherapy (p ¼ 0.001; hazard ratio 11.7) and previous radiotherapy (p ¼ 0.004; hazard ratio 8.3) were independent markers of poor outcome. The same three parameters were independent predictors of poor DFS (endoglin: p ¼ 0.038, hazard ratio 4.3; chemotherapy: p ¼ 0.002, hazard ratio 9.3; radiotherapy: p ¼ 0.004, hazard ratio 8.3). Discussion Breast cancer patients diagnosed with malignant effusion constitute a patient group with extremely poor outcome in which curative treatment is essentially impossible to achieve. As such, they may benefit from targeted molecular therapy aimed at prolonging survival and hopefully providing improved quality of life. However, published studies focusing on the biology of breast carcinoma cells in effusions are rare. In recent years, we have documented the differential expression of cancer-associated molecules in breast carcinoma cells in effusions compared to primary tumors and reported on the prognostic role of several of these molecules.23e29 In the present study, we studied the expression and clinical role of endoglin, as well as the relationship between its secreted form and other secreted pro-angiogenic factors (VEGF and Flt1) and the pro-inflammatory S100 family member calprotectin. The latter molecule was included in the study in view of our previous observation that it is frequently expressed in ovarian carcinoma effusions,20 as well as the finding that
496
B. Davidson et al. / The Breast 19 (2010) 493e498
Fig. 1. Endoglin protein expression by immunohistochemistry in breast carcinoma. (AeD): Effusions: Endoglin-positive breast carcinoma cells (AeB) and reactive cells (macrophages and mesothelial cells, green arrows; CeD) in four effusions. Carcinoma cells in CeD (black arrows) are negative. (EeG): Primary carcinomas: Three examples of negative (E), focal (<5% of cells; F) and diffuse (>75%; G) endoglin expression in carcinoma cells (black arrows) in three primary tumors. Endothelial cells (red arrow in E) were positive in all specimens. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
chemokine receptors are expressed on both breast carcinoma cells and leukocytes in effusions,30 suggesting an interplay between cancer cells and the immune system. sEng, calprotectin, VEGF and sFlt were found in the effusion supernatant of all specimens. To date, data regarding the presence of sEng and calprotectin in effusions is limited to our recent study of ovarian carcinoma specimens.20 As in the latter study of ovarian cancer effusions, both secreted proteins were universally expressed in effusions from breast cancer patients, and their effusion concentration was unrelated to patient survival. The presence of secreted VEGF in all our specimens is in agreement with several previous reports in which this angiogenic molecule has been found in effusions, frequently with significantly higher levels in malignant compared to nonneoplastic effusions.31e35 The co-expression of VEGF and its receptors in effusions has previously been reported in benign mesothelium and malignant mesothelioma.35e37 Interestingly,
VEGF was shown to be significantly co-expressed with TGF-b in effusions,38 suggesting, together with our finding of such association between endoglin, VEGF and sFlt, co-activation of these pathways in an autocrine manner at this anatomic site in advanced cancer forms. The ubiquitous presence of endoglin in breast carcinoma effusions was assumed by us to originate at least in part from the patients’ blood. We had no access to the corresponding patient serum or plasma in this study. However, we additionally wished to assess whether host and tumor cells in the effusions express this protein. We observed endoglin expression in tumor cells in the majority of analyzed effusions, with significantly higher expression frequency and extent compared with primary tumors, suggesting that endoglin may promote metastasis in breast cancer. In addition, endoglin was expressed by mesothelial cells and macrophages, well in agreement with the ability of these cell classes to produce angiogenic factors. To the best of our knowledge, this is the first
B. Davidson et al. / The Breast 19 (2010) 493e498
497
reported in the present study must be regarded as preliminary in view of the small size of the analyzed series. However, it suggests that this protein may be a viable target for directed treatment against both tumor cells and the peri-tumoral vasculature. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements Financial acknowledgement: This work was supported by grants from the Norwegian Cancer Society and the Research Fund at the Norwegian Radium Hospital. We thank Ms. Lise Levy at Ulleval Hospital, Oslo University Hospital, for her competent help in organizing and performing the ELISA effusion analyses. References
Fig. 2. Endoglin expression in breast carcinoma effusions correlates with poor survival A: KaplaneMeier survival curve showing correlation between endoglin expression and disease-free survival (DFS) for 36 patients. Patients with effusions with higher expression (>25% of tumor cells) (n ¼ 11, dashed line) had mean DFS ¼ 25 months vs. 77 months for patients whose effusions showed low (25%) expression (n ¼ 25, solid line; p ¼ 0.032). Two patients had no data with respect to DFS. B: KaplaneMeier survival curve showing correlation between endoglin expression and overall survival (OS) for 38 patients. Patients with effusions with higher expression (>25% of tumor cells) (n ¼ 11, dashed line) had mean OS ¼ 48 months vs. 119 months for patients whose effusions showed low (25%) expression (n ¼ 27, solid line; p ¼ 0.021).
evidence of tumor cell endoglin expression in breast carcinoma. However, this finding concords with previous observations that endoglin is expressed in melanoma and ovarian carcinoma cells.39,40 We suggest that the soluble endoglin in effusions from breast carcinoma patients, in addition to its origin in the blood, also stems from shedding of membrane-bound endoglin molecules from tumor cells due to proteolytic cleavage of the receptor. We further report that endoglin expression in carcinoma cells is an independent prognostic marker of poor OS and DFS, in agreement with the finding in ovarian carcinoma.40 This suggests that endoglin may be a molecular therapeutic target in metastatic breast carcinoma, a modality that has already been investigated in the context of anti-angiogenic therapy.41 In conclusion, endoglin is frequently expressed in breast carcinoma cells in effusions and is most likely secreted into the effusion supernatant, together with other cancer-associated proteins. The association between tumor endoglin expression and poor survival
1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74e108. 2. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics, 2008. CA Cancer J Clin 2008;58:71e96. 3. Fentiman IS, Millis R, Sexton S, Hayword JL. Pleural effusion in breast cancer: a review of 105 cases. Cancer 1981;47:2087e92. 4. Raju RN, Kardinal CG. Pleural effusion in breast carcinoma: analysis of 122 cases. Cancer 1981;48:2524e7. 5. Wilkes JD, Fidias P, Vaickus L, Perez RP. Malignancy-related pericardial effusion. 127 cases from the Roswell Park Center Institute. Cancer (Phila) 1995;76: 1377e87. 6. Buck M, Ingle JN, Giuliani ER, Gordon JR, Therneau TM. Pericardial effusion in women with breast cancer. Cancer 1987;60:263e9. 7. Kamby C, Vejborg I, Kristensen B, Olsen LO, Mouridsen HT. Metastatic pattern in recurrent breast cancer. Special reference to intrathoracic recurrences. Cancer 1988;62:2226e33. 8. Dallas NA, Samuel S, Xia L, Fan F, Gray MJ, Lim SJ, et al. Endoglin (CD105): a marker of tumor vasculature and potential target for therapy. Clin Cancer Res 2008;14:1931e7. 9. ten Dijke P, Goumans MJ, Pardali E. Endoglin in angiogenesis and vascular diseases. Angiogenesis 2008;11:79e89. 10. Levine RJ, Lam C, Qian C, Yu KF, Maynard SE, Sachs BP, et al. Soluble endoglin and other circulating antiangiogenic factors in preeclampsia. N Engl J Med 2006;355:992e1005. 11. Staff AC, Braekke K, Johnsen GM, Karumanchi SA, Harsem NK. Circulating concentrations of soluble endoglin (CD105) in fetal and maternal serum and in amniotic fluid in preeclampsia. Am J Obstet Gynecol 2007;197:176.e1e6. 12. Kumar S, Ghellal A, Li C, Byrne G, Haboubi N, Wang JM, et al. Breast carcinoma: vascular density determined using CD105 antibody correlates with tumor prognosis. Cancer Res 1999;59:856e61. 13. Dales JP, Garcia S, Bonnier P, Duffaud F, Andrac-Meyer L, Ramuz O, et al. CD105 expression is a marker of high metastatic risk and poor outcome in breast carcinomas. Correlations between immunohistochemical analysis and long-term follow-up in a series of 929 patients. Am J Clin Pathol 2003;119: 374e80. 14. Beresford MJ, Harris AL, Ah-See M, Daley F, Padhani AR, Makris A. The relationship of the neo-angiogenic marker, endoglin, with response to neoadjuvant chemotherapy in breast cancer. Br J Cancer 2006;95:1683e8. 15. Dhakal HP, Naume B, Synnestvedt M, Borgen E, Kaaresen R, Schlichting E, et al. Vascularization in primary breast carcinomas: its prognostic significance and relationship with tumor cell dissemination. Clin Cancer Res 2008;14: 2341e50. 16. Li C, Guo B, Wilson PB, Stewart A, Byrne G, Bundred N, et al. Plasma levels of soluble CD105 correlate with metastasis in patients with breast cancer. Int J Cancer 2000;89:122e6. 17. Takahashi N, Kawanishi-Tabata R, Haba A, Tabata M, Haruta Y, Tsai H, et al. Association of serum endoglin with metastasis in patients with colorectal, breast, and other solid tumors, and suppressive effect of chemotherapy on the serum endoglin. Clin Cancer Res 2001;7:524e32. 18. Vo MN, Evans M, Leitzel K, Ali SM, Wilson M, Demers L, et al. Elevated plasma endoglin (CD105) predicts decreased response and survival in a metastatic breast cancer trial of hormone therapy. Breast Cancer Res Treat 2010;119: 767e71. 19. Zhou S, Wang GP, Liu C, Zhou M. Eukaryotic initiation factor 4E (eIF4E) and angiogenesis: prognostic markers for breast cancer. BMC Cancer 2006;6:231. 20. Ødegaard E, Davidson B, Engh V, Onsrud M, Staff AC. Assessment of endoglin and calprotectin as potential biomarkers in ovarian carcinoma and borderline tumors of the ovary. Am J Obstet Gynecol 2008;199:533.e1e8.
498
B. Davidson et al. / The Breast 19 (2010) 493e498
21. Davidson B, Nielsen S, Christensen J, Asschenfeldt P, Berner A, Risberg B, et al. The role of Desmin and N-cadherin in effusion cytology. A comparative study using established markers of mesothelial and epithelial cells. Am J Surg Pathol 2001;25:1405e12. 22. Staff AC, Braekke K, Harsem NK, Lyberg T, Holthe MR. Circulating concentrations of sFlt1 (soluble fms-like tyrosine kinase 1) in fetal and maternal serum during pre-eclampsia. Eur J Obstet Gynecol Reprod Biol 2005;122:33e9. 23. Davidson B, Reich R, Lazarovici P, Flørenes VA, Nielsen S, Nesland JM. Altered expression and activation of the nerve growth factor receptors TrkA and p75 provides the first evidence of tumor progression to effusion in breast carcinoma. Breast Cancer Res Treat 2004;83:119e28. 24. Davidson B, Konstantinovsky S, Nielsen S, Dong HP, Berner A, Vyberg M, et al. Altered expression of metastasis-associated and regulatory molecules in effusions from breast cancer patients- a novel model for tumor progression. Clin Cancer Res 2004;10:7335e46. 25. Elloul S, Bukholt Elstrand M, Nesland JM, Trope CG, Kvalheim G, Goldberg I, et al. Snail, Slug, and Smad-interacting protein 1 as novel parameters of disease aggressiveness in metastatic ovarian and breast carcinoma. Cancer 2005;103: 1631e43. 26. Konstantinovsky S, Nielsen S, Vyberg M, Kvalheim G, Nesland JM, Reich R, et al. Angiogenic molecule expression is downregulated in effusions from breast cancer patients. Breast Cancer Res Treat 2005;94:71e80. 27. Kleinberg L, Flørenes VA, Skrede M, Dong HP, Nielsen S, McMaster MT, et al. Expression of HLA-G in malignant mesothelioma and clinically aggressive breast carcinoma. Virchows Arch 2006;449:31e9. 28. Davidson B, Konstantinovsky S, Kleinberg L, Nguyen MTP, Bassarova A, Kvalheim G, et al. The mitogen-activated protein kinases (MAPK) p38 and JNK are markers of tumor progression in breast carcinoma. Gynecol Oncol 2006;102:453e61. 29. Kleinberg L, Flørenes VA, Nesland JM, Davidson B. Survivin, a member of the inhibitors of apoptosis (IAP) family, is down-regulated in breast carcinoma effusions. Am J Clin Pathol 2007;128:389e97. 30. Davidson B, Dong HP, Holth A, Berner A, Risberg B. The chemokine receptor CXCR4 is more frequently expressed in breast compared to other metastatic adenocarcinomas in effusions. Breast J 2008;14:476e82.
31. Hsu IL, Su WC, Yan JJ, Chang JM, Lai WW. Angiogenetic biomarkers in nonsmall cell lung cancer with malignant pleural effusion: correlations with patient survival and pleural effusion control. Lung Cancer 2009;65:371e6. 32. Duysinx BC, Corhay JL, Hubin L, Nguyen D, Henket M, Louis R. Diagnostic value of interleukine-6, transforming growth factor-beta 1 and vascular endothelial growth factor in malignant pleural effusions. Respir Med 2008;102:1708e14. 33. Atanackovic D, Cao Y, Kim JW, Brandl S, Thom I, Faltz C, et al. The local cytokine and chemokine milieu within malignant effusions. Tumour Biol 2008;29: 93e104. 34. Kraft A, Weindel K, Ochs A, Marth C, Zmija J, Schumacher P, et al. Vascular endothelial growth factor in the sera and effusions of patients with malignant and nonmalignant disease. Cancer 1999;85:178e87. 35. Thickett DR, Armstrong L, Millar AB. Vascular endothelial growth factor (VEGF) in inflammatory and malignant pleural effusions. Thorax 1999;54:707e10. 36. Strizzi L, Catalano A, Vianale G, Orecchia S, Casalini A, Tassi G, et al. Vascular endothelial growth factor is an autocrine growth factor in human malignant mesothelioma. J Pathol 2001;193:468e75. 37. Davidson B, Vintman L, Zcharia E, Bedrossian C, Berner A, Nielsen S, et al. Heparanase and basic fibroblast growth factor are co-expressed in malignant mesothelioma. Clin Exp Metastasis 2004;21:469e76. 38. Cheng D, Lee YC, Rogers JT, Perkett EA, Moyers JP, Rodriguez RM, et al. Vascular endothelial growth factor level correlates with transforming growth factorbeta isoform levels in pleural effusions. Chest 2000;118:1747e53. 39. Altomonte M, Montagner R, Fonsatti E, Colizzi F, Cattarossi I, Brasoveanu LI, et al. Expression and structural features of endoglin (CD105), a transforming growth factor beta1 and beta3 binding protein, in human melanoma. Br J Cancer 1996;74:1586e91. 40. Henriksen R, Gobl A, Wilander E, Oberg K, Miyazono K, Funa K. Expression and prognostic significance of TGF-beta isotypes, latent TGF-beta 1 binding protein, TGF-beta type I and type II receptors, and endoglin in normal ovary and ovarian neoplasms. Lab Invest 1995;73:213e20. 41. Matsuno F, Haruta Y, Kondo M, Tsai H, Barcos M, Seon BK. Induction of lasting complete regression of preformed distinct solid tumors by targeting the tumor vasculature using two new anti-endoglin monoclonal antibodies. Clin Cancer Res 1999;5:371e82.