- Email: [email protected]
Antibodies to endostatin in a multifocal glioblastoma patient
RESEARCH LETTERS
Antibodies to endostatin in a multifocal glioblastoma patient David Ratel, Valéry Nasser, Isabelle Dupré, Alim Louis Benabid, François Berger Endostatin, a C-terminal fragment of collagen XVIII is involved in the regulation of neovascularisation in solid tumours in mice. However, few data are available on the concentration of endostatin protein in patients with cancer. Paradoxical results obtained in this way prompted us to investigate an antibody to endostatin. We detected antibodies to endostatin in the serum and in the tumour brain tissue of a patient with a multifocal glioblastoma, and in the serum samples from two patients with aggressive tumours. These data suggest that endostatin overexpression by tumour tissue might induce a humoral immune response.
Endostatin has been initially reported as a collagen XVIII proteolytic fragment produced by haemangioendothelioma cells in mice.1 It is characterised by a strong antiangiogenic activity inhibiting endothelial-cell proliferation1,2 and migration.2 These properties provided clues for a new antitumour strategy for which the therapeutic effectiveness has been previously shown in numerous models of neoplasia.3 Faced with these preclinical results, therapeutic trials are underway. However, few data are available concerning the concentrations of circulating and tumour-tissue endostatin in patients with cancer. To address this issue, we investigated the concentration of circulating endostatin in several patients with cancer, using a competitive ELISA assay (Accucyte Human Endostatin, Cytimmune Sciences). In one patient, who had a multifocal glioblastoma, we observed a paradoxical zone effect consisting of an increasing endostatin concentration despite increasing sample dilutions. This finding suggested that an endostatin-binding product was present in the patient’s serum. We postulated that the glioblastoma consisted of an antibody raised against human endostatin. We assessed serum samples from nine patients with cancer (one multifocal glioblastoma [patient 1], one carcinoma of unknown primary site [patient 2], five grade IV glioma [patients 3, 4, 5, 8, and 9], one prostatic cancer [patient 6], one gastric cancer [patient 7], and eight healthy adults (four men, four women). We also assessed tumour brain tissue from patient 1 and non-tumour brain tissue from a patient presenting with pharmacoresistant cryptogenic epilepsy. We tested the immunoreactivity of serum samples and tissues against purified recombinant human endostatin (France Biochem, Meudon, France) and murine endostatin by western immunoblotting. 3 µg purified recombinant human (France Biochem) or murine endostatin (provided by T Bachelot, Lyon, France) was loaded into wells of a 10% sodium dodecyl sulfate polyacrylamide gel and electrophoresis was done. The gel was blotted onto polyvinylidenc fluoride membrane (PVDF, Millipore, Saint Quentin, France). The blot was blocked in Tris-buffered saline (TBS)-10% non-fat milk. PVDF strips were cut and reacted separately at 4°C overnight with each of the patients’ serum samples at 1/1000, tumour and non-tumour brain homogenates. The strips were washed two times, blocked in TBS-5% non-fat milk, and reacted with goat antibodies to human IgG conjugated to horseradish peroxidase (Sigma, Saint Quentin Fallavier, France). The blot was developed with Diaminobenzidine substrate (Vector Laboratories, Burlingame, CA, USA). The positive control was human endostatin exposed to antibodies to murine endostatin (R&D System, Abingdon, UK). The reactivity of the antibodies to murine endostatin with recombinant human
1656
Figure 1 : Reactivity of serum samples from patients with cancer, control serum samples, samples of tumour from patient 1, and non-tumour brain tissue with recombinant human endostatin Western immunoblot shows that serum samples of patients 1, 2, and 4, and patient 1’s tumour were strongly reactive to recombinant human endostatin, whereas non-tumour brain tissue and serum samples of other patients and controls were negative.
endostatin was tested and confirmed by western blot analysis. We also used control proteins: myoglobin, lactoferrin, and carbonic anhydrase (Sigma, France). Sections of tumour from patient 1 and non-tumour brain tissue were prepared for immunohistochemistry. Sections were fixed in 4% paraformaldehyde and stained with polyclonal goat antibodies to murine endostatin (R&D System) revealed with biotinylated antibodies to goat immunoglobulin (Dako, Trappes) and horseradish peroxidase-extravidin complex or tetramethylrhodamineisothiacyanate-extravidin (Sigma) complex. To demonstrate the specificity of the antibody to endostatin, it was reacted with proteins from tumour and non-tumour brain homogenates. Only human endostatin was recognised ozn immunoblot (data not shown). Other serial sections were used for immunostaining with fluorescein isothiocyanate-conjugated goat antibodies to human IgG (Sigma), monoclonal rabbit antibodies to human v3 protein (Chemicon International, Temecula, CA, USA) revealed with biotinylated antibodies to rabbit immunoglobulin (Boehringer Mannheim, Meylan, France), and isothiocyanate-extravidin complex (Sigma) and rabbit antibodies to human factor VIII protein (Dako) revealed with biotinylated antibodies to rabbit immunoglobulin (Boehringer Mannheim, France) and TRITC-extravidin complex (Sigma). Sections without primary antibody were used as controls. The serum samples of three patients were strongly reactive to recombinant human endostatin at 1/1000 dilution, whereas the serum samples of the six other patients and the eight healthy adults were negative (figure 1). Moreover, patient 1’s serum was positive at 1/10 000 dilution (data not shown). An immunoreactivity against human endostatin was detected using tumour brain homogenate (figure 1). No immunoreactivity against control proteins was detected. Immunohistochemistry showed an intense endostatin immunoreactivity in tumour vessels (figure 2) expressing factor VIII. Double staining with antibodies to endostatin and to human immunoglobulin showed the colocalisation of immunoglobulin deposits and endostatin (figure 2), and factor VIII protein (figure 2). Conversely, we didn’t observe immunoglobulin deposits and only low endostatin immunoreactivity in the brain control (figure 2). Our results show the presence of human endogenous endostatin antibodies in several patients with cancer. This autoimmune response is seen in the context of an aggressive multifocal glioblastoma and the documented analysis of brain tissue and serum samples indicates the existence of both circulating and tumoural endostatin antibodies. Among two other positive patients, one harboured a different kind of
THE LANCET • Vol 356 • November 11, 2000
For personal use only. Not to be reproduced without permission of The Lancet.
RESEARCH LETTERS
Association between acute hypobaric hypoxia and activation of coagulation in human beings Bjørn Bendz, Morten Rostrup, Knut Sevre, Trine 0 Andersen, Per Morten Sandset The risk of venous thrombosis is thought to be increased by flying. In a study of 20 healthy male volunteers who were suddenly exposed to a hypobaric environment similar to that encountered within aeroplane cabins, markers of activated coagulation transiently increased by two-fold to eight-fold. We suggest that hypobaric hypoxia, with sedentariness and dehydration, may cause this increased risk of venous thrombosis.
tumour (CUP syndrome). Interestingly, all positive patients had an aggressive tumour. The local deposits of immunoglobulins, as well as the antibody activity of these immunoglobulins taken from the tumour itself, reinforce the hypothesis of a local immunological stimulus, mediated in neoangiogenic area. Early reports suggested that the structure of endostatin might exhibit immunogenic properties.4 Immunoreactivity has already been reported against overexpressed mutated and non-mutated proteins in tumours, such as p535 or L-Myc. Thus, production of endostatin in the area of angiogenesis could be the trigger for local engendering of endostatin antibodies. Further analysis will be necessary to determine the prevalence of such antibodies in patients with cancer. Description of endostatin antibodies raises questions about their functional role in tumour progression and neoangiogenesis. Such antibodies could reduce effectiveness of endostatin therapy and provide an explanation as to how tumours can escape the effects of antiangiogenic molecules. David Ratel was supported by grants from the Ligue Nationale Contre le Cancer (Comité de l’Isère). 1
2
3
4
5
O’Reilly MS, Boehm T, Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997; 88: 277–85. Dhanabal M, Ramchandran R, Volk R, et al. Endostatin: yeast production, mutants, and antitumor effect in renal cell carcinoma. Cancer Res 1999; 59: 189–97. Blezinger P, Wang J, Gondo M, et al. Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene. Nat Biotechnol 1999; 17: 343–48. Hohenester E, Sasaki T, Olsen BR, Timpl R. Crystal structure of the angiogenesis inhibitor endostatin at 1.5 A resolution. EMBO J 1998; 17: 1656–64. Lubin R, Zalcman G, Bouchet L, et al. Serum p53 antibodies as early markers of lung cancer. Nat Med 1995; 1: 701–02.
Unité 318 INSERM, Laboratoire de Neurosciences PrécliniquesUJFG, Centre Hospitalier Universitaire Michallon, Grenoble, France (D Ratel PhS, V Nasser MD, I Dupré PhS, Prof A L Benabid MD, F Berger MD) Correspondence to: Dr David Ratel, Unité 318 INSERM, Pavillon B, Centre hospitalier Universitaire Michallon BP 217, 38 043 Grenoble cedex, France. (e-mail: [email protected])
THE LANCET • Vol 356 • November 11, 2000
Concentration of prothrombin fragments 1 and 2 (nmol/L)
A: immunostaining of endostatin in brain tumour of patient 1. Recombinant human endostatin blocked staining (scale bar 90 µm). B: endostatin vascular deposition in brain tumour of patient 1 compared with vessels of non-tumour tissue, which present a lower staining for endostatin protein (C). D and E: endostatin and IgG deposits into entire vascular wall respectively. F: IgG deposits seemed to co-localise with endostatin protein (yellow in superimposed images). G: immunostaining of factor VIII on lumen side of vascular wall. H: IgG vascular deposits. H: Factor VIII and IgG signals are superimposed. Scale bars for C to I=30 µm.
Aeroplane flights are thought to increase the risk of venous thrombosis, but the mechanism behind this effect is unknown. Suggested risk factors are a hypobaric and hypoxic environment, sedentariness, and dehydration.1 In an experimental study, we investigated whether acute exposure to reduced air pressure, similar to that encountered in aeroplane cabins, activates coagulation. The study was done in a hypobaric chamber, built as a comfortable modern apartment, in Sjumilskogen, Trysil, Norway. The facilities were built at 440 m above sea level (ambient air pressure about 96·3 kPa), but the air pressure within could be lowered to simulate that encountered at 5500 m above sea level (about 50·7 kPa). The air temperature was kept at between 20ºC and 22ºC. The scientific staff lived outside the chamber, but were sluiced in and out during the experiments. 20 healthy male volunteers were recruited. After acclimatising to the ambient air
Concentration of thrombin-antithrombin complex (µg/L)
Figure 2 : Endostatin immunostaining (a, b, and c) and Immunofluorescence analysis of serial slices of tumour brain tissue (patient 1)
76 kPa 4
3
2
1
0 125 100 75 50 25 0 0
1
2
4
8
16
Time (h) Figure 1: Concentrations of prothrombin fragments 1 and 2 and thrombin-antithrombin complex in 20 healthy volunteers exposed to 76 kPa air pressure Points=mean, bars=SE. Filled circles represent eight volunteers who returned to ambient air pressure (96·3 kPa).
1657
For personal use only. Not to be reproduced without permission of The Lancet.
Antibodies to endostatin in a multifocal glioblastoma patient David Ratel, Valéry Nasser, Isabelle Dupré, Alim Louis Benabid, François Berger Endostatin, a C-terminal fragment of collagen XVIII is involved in the regulation of neovascularisation in solid tumours in mice. However, few data are available on the concentration of endostatin protein in patients with cancer. Paradoxical results obtained in this way prompted us to investigate an antibody to endostatin. We detected antibodies to endostatin in the serum and in the tumour brain tissue of a patient with a multifocal glioblastoma, and in the serum samples from two patients with aggressive tumours. These data suggest that endostatin overexpression by tumour tissue might induce a humoral immune response.
Endostatin has been initially reported as a collagen XVIII proteolytic fragment produced by haemangioendothelioma cells in mice.1 It is characterised by a strong antiangiogenic activity inhibiting endothelial-cell proliferation1,2 and migration.2 These properties provided clues for a new antitumour strategy for which the therapeutic effectiveness has been previously shown in numerous models of neoplasia.3 Faced with these preclinical results, therapeutic trials are underway. However, few data are available concerning the concentrations of circulating and tumour-tissue endostatin in patients with cancer. To address this issue, we investigated the concentration of circulating endostatin in several patients with cancer, using a competitive ELISA assay (Accucyte Human Endostatin, Cytimmune Sciences). In one patient, who had a multifocal glioblastoma, we observed a paradoxical zone effect consisting of an increasing endostatin concentration despite increasing sample dilutions. This finding suggested that an endostatin-binding product was present in the patient’s serum. We postulated that the glioblastoma consisted of an antibody raised against human endostatin. We assessed serum samples from nine patients with cancer (one multifocal glioblastoma [patient 1], one carcinoma of unknown primary site [patient 2], five grade IV glioma [patients 3, 4, 5, 8, and 9], one prostatic cancer [patient 6], one gastric cancer [patient 7], and eight healthy adults (four men, four women). We also assessed tumour brain tissue from patient 1 and non-tumour brain tissue from a patient presenting with pharmacoresistant cryptogenic epilepsy. We tested the immunoreactivity of serum samples and tissues against purified recombinant human endostatin (France Biochem, Meudon, France) and murine endostatin by western immunoblotting. 3 µg purified recombinant human (France Biochem) or murine endostatin (provided by T Bachelot, Lyon, France) was loaded into wells of a 10% sodium dodecyl sulfate polyacrylamide gel and electrophoresis was done. The gel was blotted onto polyvinylidenc fluoride membrane (PVDF, Millipore, Saint Quentin, France). The blot was blocked in Tris-buffered saline (TBS)-10% non-fat milk. PVDF strips were cut and reacted separately at 4°C overnight with each of the patients’ serum samples at 1/1000, tumour and non-tumour brain homogenates. The strips were washed two times, blocked in TBS-5% non-fat milk, and reacted with goat antibodies to human IgG conjugated to horseradish peroxidase (Sigma, Saint Quentin Fallavier, France). The blot was developed with Diaminobenzidine substrate (Vector Laboratories, Burlingame, CA, USA). The positive control was human endostatin exposed to antibodies to murine endostatin (R&D System, Abingdon, UK). The reactivity of the antibodies to murine endostatin with recombinant human
1656
Figure 1 : Reactivity of serum samples from patients with cancer, control serum samples, samples of tumour from patient 1, and non-tumour brain tissue with recombinant human endostatin Western immunoblot shows that serum samples of patients 1, 2, and 4, and patient 1’s tumour were strongly reactive to recombinant human endostatin, whereas non-tumour brain tissue and serum samples of other patients and controls were negative.
endostatin was tested and confirmed by western blot analysis. We also used control proteins: myoglobin, lactoferrin, and carbonic anhydrase (Sigma, France). Sections of tumour from patient 1 and non-tumour brain tissue were prepared for immunohistochemistry. Sections were fixed in 4% paraformaldehyde and stained with polyclonal goat antibodies to murine endostatin (R&D System) revealed with biotinylated antibodies to goat immunoglobulin (Dako, Trappes) and horseradish peroxidase-extravidin complex or tetramethylrhodamineisothiacyanate-extravidin (Sigma) complex. To demonstrate the specificity of the antibody to endostatin, it was reacted with proteins from tumour and non-tumour brain homogenates. Only human endostatin was recognised ozn immunoblot (data not shown). Other serial sections were used for immunostaining with fluorescein isothiocyanate-conjugated goat antibodies to human IgG (Sigma), monoclonal rabbit antibodies to human v3 protein (Chemicon International, Temecula, CA, USA) revealed with biotinylated antibodies to rabbit immunoglobulin (Boehringer Mannheim, Meylan, France), and isothiocyanate-extravidin complex (Sigma) and rabbit antibodies to human factor VIII protein (Dako) revealed with biotinylated antibodies to rabbit immunoglobulin (Boehringer Mannheim, France) and TRITC-extravidin complex (Sigma). Sections without primary antibody were used as controls. The serum samples of three patients were strongly reactive to recombinant human endostatin at 1/1000 dilution, whereas the serum samples of the six other patients and the eight healthy adults were negative (figure 1). Moreover, patient 1’s serum was positive at 1/10 000 dilution (data not shown). An immunoreactivity against human endostatin was detected using tumour brain homogenate (figure 1). No immunoreactivity against control proteins was detected. Immunohistochemistry showed an intense endostatin immunoreactivity in tumour vessels (figure 2) expressing factor VIII. Double staining with antibodies to endostatin and to human immunoglobulin showed the colocalisation of immunoglobulin deposits and endostatin (figure 2), and factor VIII protein (figure 2). Conversely, we didn’t observe immunoglobulin deposits and only low endostatin immunoreactivity in the brain control (figure 2). Our results show the presence of human endogenous endostatin antibodies in several patients with cancer. This autoimmune response is seen in the context of an aggressive multifocal glioblastoma and the documented analysis of brain tissue and serum samples indicates the existence of both circulating and tumoural endostatin antibodies. Among two other positive patients, one harboured a different kind of
THE LANCET • Vol 356 • November 11, 2000
For personal use only. Not to be reproduced without permission of The Lancet.
RESEARCH LETTERS
Association between acute hypobaric hypoxia and activation of coagulation in human beings Bjørn Bendz, Morten Rostrup, Knut Sevre, Trine 0 Andersen, Per Morten Sandset The risk of venous thrombosis is thought to be increased by flying. In a study of 20 healthy male volunteers who were suddenly exposed to a hypobaric environment similar to that encountered within aeroplane cabins, markers of activated coagulation transiently increased by two-fold to eight-fold. We suggest that hypobaric hypoxia, with sedentariness and dehydration, may cause this increased risk of venous thrombosis.
tumour (CUP syndrome). Interestingly, all positive patients had an aggressive tumour. The local deposits of immunoglobulins, as well as the antibody activity of these immunoglobulins taken from the tumour itself, reinforce the hypothesis of a local immunological stimulus, mediated in neoangiogenic area. Early reports suggested that the structure of endostatin might exhibit immunogenic properties.4 Immunoreactivity has already been reported against overexpressed mutated and non-mutated proteins in tumours, such as p535 or L-Myc. Thus, production of endostatin in the area of angiogenesis could be the trigger for local engendering of endostatin antibodies. Further analysis will be necessary to determine the prevalence of such antibodies in patients with cancer. Description of endostatin antibodies raises questions about their functional role in tumour progression and neoangiogenesis. Such antibodies could reduce effectiveness of endostatin therapy and provide an explanation as to how tumours can escape the effects of antiangiogenic molecules. David Ratel was supported by grants from the Ligue Nationale Contre le Cancer (Comité de l’Isère). 1
2
3
4
5
O’Reilly MS, Boehm T, Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997; 88: 277–85. Dhanabal M, Ramchandran R, Volk R, et al. Endostatin: yeast production, mutants, and antitumor effect in renal cell carcinoma. Cancer Res 1999; 59: 189–97. Blezinger P, Wang J, Gondo M, et al. Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene. Nat Biotechnol 1999; 17: 343–48. Hohenester E, Sasaki T, Olsen BR, Timpl R. Crystal structure of the angiogenesis inhibitor endostatin at 1.5 A resolution. EMBO J 1998; 17: 1656–64. Lubin R, Zalcman G, Bouchet L, et al. Serum p53 antibodies as early markers of lung cancer. Nat Med 1995; 1: 701–02.
Unité 318 INSERM, Laboratoire de Neurosciences PrécliniquesUJFG, Centre Hospitalier Universitaire Michallon, Grenoble, France (D Ratel PhS, V Nasser MD, I Dupré PhS, Prof A L Benabid MD, F Berger MD) Correspondence to: Dr David Ratel, Unité 318 INSERM, Pavillon B, Centre hospitalier Universitaire Michallon BP 217, 38 043 Grenoble cedex, France. (e-mail: [email protected])
THE LANCET • Vol 356 • November 11, 2000
Concentration of prothrombin fragments 1 and 2 (nmol/L)
A: immunostaining of endostatin in brain tumour of patient 1. Recombinant human endostatin blocked staining (scale bar 90 µm). B: endostatin vascular deposition in brain tumour of patient 1 compared with vessels of non-tumour tissue, which present a lower staining for endostatin protein (C). D and E: endostatin and IgG deposits into entire vascular wall respectively. F: IgG deposits seemed to co-localise with endostatin protein (yellow in superimposed images). G: immunostaining of factor VIII on lumen side of vascular wall. H: IgG vascular deposits. H: Factor VIII and IgG signals are superimposed. Scale bars for C to I=30 µm.
Aeroplane flights are thought to increase the risk of venous thrombosis, but the mechanism behind this effect is unknown. Suggested risk factors are a hypobaric and hypoxic environment, sedentariness, and dehydration.1 In an experimental study, we investigated whether acute exposure to reduced air pressure, similar to that encountered in aeroplane cabins, activates coagulation. The study was done in a hypobaric chamber, built as a comfortable modern apartment, in Sjumilskogen, Trysil, Norway. The facilities were built at 440 m above sea level (ambient air pressure about 96·3 kPa), but the air pressure within could be lowered to simulate that encountered at 5500 m above sea level (about 50·7 kPa). The air temperature was kept at between 20ºC and 22ºC. The scientific staff lived outside the chamber, but were sluiced in and out during the experiments. 20 healthy male volunteers were recruited. After acclimatising to the ambient air
Concentration of thrombin-antithrombin complex (µg/L)
Figure 2 : Endostatin immunostaining (a, b, and c) and Immunofluorescence analysis of serial slices of tumour brain tissue (patient 1)
76 kPa 4
3
2
1
0 125 100 75 50 25 0 0
1
2
4
8
16
Time (h) Figure 1: Concentrations of prothrombin fragments 1 and 2 and thrombin-antithrombin complex in 20 healthy volunteers exposed to 76 kPa air pressure Points=mean, bars=SE. Filled circles represent eight volunteers who returned to ambient air pressure (96·3 kPa).
1657
For personal use only. Not to be reproduced without permission of The Lancet.