J Cancer Res Clin Oncol DOI 10.1007/s00432-015-1952-z
ORIGINAL ARTICLE – CLINICAL ONCOLOGY
Immunohistochemical expression of endoglin offers a reliable estimation of bone marrow neoangiogenesis in multiple myeloma Michael G. Alexandrakis1,2 · Ioannis K. Neonakis2 · Constantina A. Pappa2 · Ioannis Konsolas3 · Maria Kokonozaki2 · Rodanthi Vyzoukaki2 · Stella Soundoulounaki1 · Athina Xekalou4 · Katerina Sfiridaki5 · George Tsirakis1
Received: 10 November 2014 / Accepted: 5 March 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose The aim of the present study was to evaluate CD105 tissue marker in the bone marrow (BM) of multiple myeloma (MM) patients. CD105 was evaluated using immunohistochemical method. An effort was made to correlate this marker with BM microvascular density (MVD) along with other known markers of angiogenesis in order to evaluate its clinical significance. Methods BM MVD was estimated by CD31. CD105 in BM was estimated by immunohistochemical method in 54 newly diagnosed patients with MM. Circulating levels of known angiogenic factors such as basic fibroblast growth factor (b-FGF) and soluble CD105 (sCD105) were measured by ELISA in the same group of patients. All these factors were also measured in 20 age- and sex-matched healthy controls. Results We found that CD105 MVD, along with the expected CD31 MVD, and serum levels of sCD105 and bFGF were increased, also in parallel with disease stage, and all were decreased after effective treatment. Moreover,
* Michael G. Alexandrakis [email protected] 1
Hematology Department, University Hospital of Heraklion, P.O. BOX 1352, 71110 Voutes, Heraklion, Greece
2
Hematology Laboratory, University Hospital of Heraklion, Heraklion, Greece
3
Hematology Department, General Hospital of Rhodes, Rhodes, Greece
4
Pathology Department, University Hospital of Heraklion, Heraklion, Greece
5
Hematology Laboratory, Venizeleion General Hospital of Heraklion, Heraklion, Greece
CD105 MVD correlated with all the aforementioned markers of angiogenesis. Conclusions Our results indicated that CD105 MVD is following the behavior of CD31 MVD in MM, suggesting being a valid marker of BM neoangiogenesis in MM. Its prognostic impact remains to be proven. Keywords Angiogenesis · Cytokines · Endoglin · CD105 · Immunohistochemistry · Microvascular density · Multiple myeloma
Introduction In multiple myeloma (MM), malignant plasma cells interact with bone marrow (BM) microenvironment’s components and modify it, favoring, in multiple manners, the expansion of the clone (Otjacques et al. 2011). In-depth study of MM biology has revealed many hallmarks participating on its growth. The genesis of new blood vessels, in order to provide the suitable conditions for the expanding malignant mass, is a dynamic and complex pathological phenomenon with strategic and major role in tumor growth, invasion, and dissemination. Its initial stimulus is tumor cells’ hypoxia, disturbing the homeostasis between pro-angiogenic and anti-angiogenic molecules, favoring the genesis of the new vascular plexus. As time goes by and tumoral microenvironment gets more autonomous, this process loses the control of normal homeostasis, and finally, the disease self-enhances (Alexandrakis and Tsirakis 2014). BM microvascular density (MVD), as a direct index of BM vascularity, is elevated in plasma cell dyscrasias, in parallel with the progression from monoclonal gammopathy of undetermined significance (MGUS) to inactive and subsequently active MM, as well in the relapse, whereas it is significantly
13
reduced after effective anti-MM treatment (Kumar et al. 2002, 2004b; Rajkumar et al. 2002; Sezer et al. 2001b; Tsirakis et al. 2012b; Vacca et al. 1994). Moreover, MVD is associated with plasma cell burden and the rate of their proliferation (Alexandrakis et al. 2004a, b, c; Kumar et al. 2004a; Moulopoulos et al. 2010; Otjacques et al. 2011; Sezer et al. 2001a; Tsirakis et al. 2012a; Pruneri et al. 2002) and has also been correlated with other prognostic markers of MM activity (Bhatti et al. 2006; Hillengass et al. 2007; Munshi and Wilson 2001). These observations suggest the important role of angiogenesis in MM progression. Tumoral MVD is estimated in tissue specimens using, immunohistochemically revealed, specific markers of blood vessels. CD34, CD31, and vWF are the more commonly used. In the last years, endoglin (ENG, CD105) has been proposed as a more reliable marker of tumoral angiogenesis, since it is highly expressed in vascular endothelial cells at sites of active neoangiogenesis. It must also be noted that endoglin is highly expressed in vascular smooth muscle cells, whereas its expression levels are low in resting cells. Furthermore, matrix metalloproteinase-14 (MMP14, MT-MMP) may partially shed the membrane-bound form of endoglin, resulting a soluble form (sCD105), being observed in the serum of patients with several malignancies and of pregnant women with preeclampsia (Nassiri et al. 2011). We have found that sCD105 is increased in the sera of MM patients, in parallel with the International Staging System (ISS) stage, and decreases after effective anti-MM treatment. Furthermore, we found that those levels correlate with BM MVD, estimated by CD31, with other markers of angiogenesis, such as serum levels of VEGF and angiogenin, as well as with the proliferative potential of malignant plasma cells, estimated by Ki-67 proliferation index (Pappa et al. 2013; Tsirakis et al. 2012b). In 2002, endoglin was used as an endothelial marker of MM BM, being correlated and also more sensitive than CD34 MVD, although not prognostic (Pruneri et al. 2002). These observations suggest that endoglin may be implicated in the angiogenic process during the progression of MM. The aim of the present study was to reveal microvascular components in the BM of MM patients, using endoglin as immunohistochemical marker. We tried to correlate this density with MVD estimated by CD31, and with other known markers of angiogenesis, such as basic fibroblast growth factor (b-FGF) and sCD105.
Materials and methods Patients Fifty-four patients with active MM were enrolled in the study, as well as 20 age- and sex-matched controls. Subjects
13
J Cancer Res Clin Oncol
with active inflammation, history of other malignancies, renal or liver impairment, or incapability to consent were excluded from the study. Twenty-four of the patients were male, with mean age of 66.3 ± 11.3 years. Thirty-three had IgG paraprotein, 16 IgA, and five light chain. According to ISS, 13 were in stage I, 19 in stage II, and 22 in stage III. The initial measurements were made before receiving any myeloma-related therapy. We also estimated 24 of them who responded to bortezomib-based treatments (10 in complete remission and 14 in very good partial remission, of them 10 had received PAD (bortezomib liposomial doxorubicin, dexamethasone), eight VCD (bortezomib, cyclophosphamide, dexamethasone), and six VMP (bortezomib, melphalan, methyl-prednisone) regimens). The study was performed according to the guidelines of the local ethics committee. Informed consent for the study was obtained from all subjects.
Methods Sera were collected from both patients and controls, stored at −70 °C and examined at the end of the study, in order to avoid inter-assay variability. Serum levels of b-FGF and sCD105 were measured with a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA), using monoclonal human antibodies against b-FGF and sCD105 from commercially available test kits (Quantikine®, R&D Systems Inc. Minneapolis MN, USA), according to manufacturer’s instructions. Immunohistochemistry was performed on formalinfixed paraffin-embedded BM sections, in order to reveal blood vessels. The estimation of BM MVD using CD31 as vascular marker has been described previously (Tsirakis et al. 2012b). For the case of CD105, the alkaline phosphatase–antialkaline phosphatase method was used (Ultarvision LP Detection System provided by Thermo Scientific). The monoclonal antibody CD105/endoglin/TGFbeta 1/3 receptor was used and obtained from Thermo Scientific, in dilution 1:100, with incubation for 1 h. Prior to incubation with the primary antibody, a step of microwave heating in a solution of sodium citrate with pH = 6 was performed. Positive control slides were used and included sections from normal placental tissue, known to be positive for CD105. In each specimen, three areas of neoplastic infiltration, containing the highest number of microvessels (capillaries and venules) and representing the most intense microvasculature (hot spots), were examined. Any cluster of red-stained endothelial cells with or without a (rudimentary or well formed) lumen was considered to be microvessels and was counted. As for the case of CD31 MVD, the mean microvessel count of the three hot spots was calculated and expressed as vessels/0.0625 mm2.
J Cancer Res Clin Oncol Table 1 Values of microvascular density (MVD) when used CD31 and CD105 as vascular markers, and serum levels of soluble endoglin (sCD105) and basic fibroblast growth factor (b-FGF) in patients with active multiple myeloma (MM), before initiation of any treatment, and in controls
CD31 MVD CD105 MVD sCD105 (pg/ml) b-FGF (pg/ml)
MM patients
Controls
8.4 ± 4.3 4.9 ± 2.7 10.7 ± 5.9
2.1 ± 1.0 2.2 ± 0.5 6.5 ± 2.9
9.4 ± 6.8
1.7 ± 1.0
p
ORIGINAL ARTICLE – CLINICAL ONCOLOGY
Immunohistochemical expression of endoglin offers a reliable estimation of bone marrow neoangiogenesis in multiple myeloma Michael G. Alexandrakis1,2 · Ioannis K. Neonakis2 · Constantina A. Pappa2 · Ioannis Konsolas3 · Maria Kokonozaki2 · Rodanthi Vyzoukaki2 · Stella Soundoulounaki1 · Athina Xekalou4 · Katerina Sfiridaki5 · George Tsirakis1
Received: 10 November 2014 / Accepted: 5 March 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose The aim of the present study was to evaluate CD105 tissue marker in the bone marrow (BM) of multiple myeloma (MM) patients. CD105 was evaluated using immunohistochemical method. An effort was made to correlate this marker with BM microvascular density (MVD) along with other known markers of angiogenesis in order to evaluate its clinical significance. Methods BM MVD was estimated by CD31. CD105 in BM was estimated by immunohistochemical method in 54 newly diagnosed patients with MM. Circulating levels of known angiogenic factors such as basic fibroblast growth factor (b-FGF) and soluble CD105 (sCD105) were measured by ELISA in the same group of patients. All these factors were also measured in 20 age- and sex-matched healthy controls. Results We found that CD105 MVD, along with the expected CD31 MVD, and serum levels of sCD105 and bFGF were increased, also in parallel with disease stage, and all were decreased after effective treatment. Moreover,
* Michael G. Alexandrakis [email protected] 1
Hematology Department, University Hospital of Heraklion, P.O. BOX 1352, 71110 Voutes, Heraklion, Greece
2
Hematology Laboratory, University Hospital of Heraklion, Heraklion, Greece
3
Hematology Department, General Hospital of Rhodes, Rhodes, Greece
4
Pathology Department, University Hospital of Heraklion, Heraklion, Greece
5
Hematology Laboratory, Venizeleion General Hospital of Heraklion, Heraklion, Greece
CD105 MVD correlated with all the aforementioned markers of angiogenesis. Conclusions Our results indicated that CD105 MVD is following the behavior of CD31 MVD in MM, suggesting being a valid marker of BM neoangiogenesis in MM. Its prognostic impact remains to be proven. Keywords Angiogenesis · Cytokines · Endoglin · CD105 · Immunohistochemistry · Microvascular density · Multiple myeloma
Introduction In multiple myeloma (MM), malignant plasma cells interact with bone marrow (BM) microenvironment’s components and modify it, favoring, in multiple manners, the expansion of the clone (Otjacques et al. 2011). In-depth study of MM biology has revealed many hallmarks participating on its growth. The genesis of new blood vessels, in order to provide the suitable conditions for the expanding malignant mass, is a dynamic and complex pathological phenomenon with strategic and major role in tumor growth, invasion, and dissemination. Its initial stimulus is tumor cells’ hypoxia, disturbing the homeostasis between pro-angiogenic and anti-angiogenic molecules, favoring the genesis of the new vascular plexus. As time goes by and tumoral microenvironment gets more autonomous, this process loses the control of normal homeostasis, and finally, the disease self-enhances (Alexandrakis and Tsirakis 2014). BM microvascular density (MVD), as a direct index of BM vascularity, is elevated in plasma cell dyscrasias, in parallel with the progression from monoclonal gammopathy of undetermined significance (MGUS) to inactive and subsequently active MM, as well in the relapse, whereas it is significantly
13
reduced after effective anti-MM treatment (Kumar et al. 2002, 2004b; Rajkumar et al. 2002; Sezer et al. 2001b; Tsirakis et al. 2012b; Vacca et al. 1994). Moreover, MVD is associated with plasma cell burden and the rate of their proliferation (Alexandrakis et al. 2004a, b, c; Kumar et al. 2004a; Moulopoulos et al. 2010; Otjacques et al. 2011; Sezer et al. 2001a; Tsirakis et al. 2012a; Pruneri et al. 2002) and has also been correlated with other prognostic markers of MM activity (Bhatti et al. 2006; Hillengass et al. 2007; Munshi and Wilson 2001). These observations suggest the important role of angiogenesis in MM progression. Tumoral MVD is estimated in tissue specimens using, immunohistochemically revealed, specific markers of blood vessels. CD34, CD31, and vWF are the more commonly used. In the last years, endoglin (ENG, CD105) has been proposed as a more reliable marker of tumoral angiogenesis, since it is highly expressed in vascular endothelial cells at sites of active neoangiogenesis. It must also be noted that endoglin is highly expressed in vascular smooth muscle cells, whereas its expression levels are low in resting cells. Furthermore, matrix metalloproteinase-14 (MMP14, MT-MMP) may partially shed the membrane-bound form of endoglin, resulting a soluble form (sCD105), being observed in the serum of patients with several malignancies and of pregnant women with preeclampsia (Nassiri et al. 2011). We have found that sCD105 is increased in the sera of MM patients, in parallel with the International Staging System (ISS) stage, and decreases after effective anti-MM treatment. Furthermore, we found that those levels correlate with BM MVD, estimated by CD31, with other markers of angiogenesis, such as serum levels of VEGF and angiogenin, as well as with the proliferative potential of malignant plasma cells, estimated by Ki-67 proliferation index (Pappa et al. 2013; Tsirakis et al. 2012b). In 2002, endoglin was used as an endothelial marker of MM BM, being correlated and also more sensitive than CD34 MVD, although not prognostic (Pruneri et al. 2002). These observations suggest that endoglin may be implicated in the angiogenic process during the progression of MM. The aim of the present study was to reveal microvascular components in the BM of MM patients, using endoglin as immunohistochemical marker. We tried to correlate this density with MVD estimated by CD31, and with other known markers of angiogenesis, such as basic fibroblast growth factor (b-FGF) and sCD105.
Materials and methods Patients Fifty-four patients with active MM were enrolled in the study, as well as 20 age- and sex-matched controls. Subjects
13
J Cancer Res Clin Oncol
with active inflammation, history of other malignancies, renal or liver impairment, or incapability to consent were excluded from the study. Twenty-four of the patients were male, with mean age of 66.3 ± 11.3 years. Thirty-three had IgG paraprotein, 16 IgA, and five light chain. According to ISS, 13 were in stage I, 19 in stage II, and 22 in stage III. The initial measurements were made before receiving any myeloma-related therapy. We also estimated 24 of them who responded to bortezomib-based treatments (10 in complete remission and 14 in very good partial remission, of them 10 had received PAD (bortezomib liposomial doxorubicin, dexamethasone), eight VCD (bortezomib, cyclophosphamide, dexamethasone), and six VMP (bortezomib, melphalan, methyl-prednisone) regimens). The study was performed according to the guidelines of the local ethics committee. Informed consent for the study was obtained from all subjects.
Methods Sera were collected from both patients and controls, stored at −70 °C and examined at the end of the study, in order to avoid inter-assay variability. Serum levels of b-FGF and sCD105 were measured with a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA), using monoclonal human antibodies against b-FGF and sCD105 from commercially available test kits (Quantikine®, R&D Systems Inc. Minneapolis MN, USA), according to manufacturer’s instructions. Immunohistochemistry was performed on formalinfixed paraffin-embedded BM sections, in order to reveal blood vessels. The estimation of BM MVD using CD31 as vascular marker has been described previously (Tsirakis et al. 2012b). For the case of CD105, the alkaline phosphatase–antialkaline phosphatase method was used (Ultarvision LP Detection System provided by Thermo Scientific). The monoclonal antibody CD105/endoglin/TGFbeta 1/3 receptor was used and obtained from Thermo Scientific, in dilution 1:100, with incubation for 1 h. Prior to incubation with the primary antibody, a step of microwave heating in a solution of sodium citrate with pH = 6 was performed. Positive control slides were used and included sections from normal placental tissue, known to be positive for CD105. In each specimen, three areas of neoplastic infiltration, containing the highest number of microvessels (capillaries and venules) and representing the most intense microvasculature (hot spots), were examined. Any cluster of red-stained endothelial cells with or without a (rudimentary or well formed) lumen was considered to be microvessels and was counted. As for the case of CD31 MVD, the mean microvessel count of the three hot spots was calculated and expressed as vessels/0.0625 mm2.
J Cancer Res Clin Oncol Table 1 Values of microvascular density (MVD) when used CD31 and CD105 as vascular markers, and serum levels of soluble endoglin (sCD105) and basic fibroblast growth factor (b-FGF) in patients with active multiple myeloma (MM), before initiation of any treatment, and in controls
CD31 MVD CD105 MVD sCD105 (pg/ml) b-FGF (pg/ml)
MM patients
Controls
8.4 ± 4.3 4.9 ± 2.7 10.7 ± 5.9
2.1 ± 1.0 2.2 ± 0.5 6.5 ± 2.9
9.4 ± 6.8
1.7 ± 1.0
p
