Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin’s lymphoma (Harris et al., 1994). Approximately one-third of all adult lymphomas are DLBCL (Swerdlow et al., 2008). DLBCL is associated with an aggressive natural history, with a median survival of less than 1 year in untreated patients (Rosenwald et al., 2002). In DLBCL, the International Prognostic Index is a clinical prognostic model that predicts outcome (Shipp 1993; Rosenwald et al., 2002). On the one hand, gene expression profiling (GEP) has uncovered distinct molecular signatures for DLBCL subtypes that have distinct clinical behaviors and prognoses (Lenz et al., 2008). There are two molecularly distinct forms of DLBCL, which have gene expression patterns indicative of different stages of B-cell differentiation. One type expressed genes characteristic of germinal center B cells (GCB, germinal center B-like DLBCL); the second type expressed genes normally induced during in-vitro activation of peripheral blood B cells (activated B-like DLBCL). Patients with GCB-like DLBCL had a significantly better overall survival than those with activated B-like DLBCL (Coiffier et al., 2002; Coiffier, 2005). The molecular classification of tumors on the basis of gene expression has not only contributed prognostic information but has also helped in the identification of new therapeutic targets (Feugier et al., 2005). Cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy has been the mainstay of therapy for several decades. On the other hand, the integration of antilymphoma monoclonal antibodies, notably rituximab (R), into combination therapies for DLBCL has markedly improved patient outcomes (Folkman, 1971; Coiffier et al., 2002; Feugier et al., 2005).
Angiogenesis is the propelling force for tumor growth and metastasis, and antiangiogenic therapy represents one of the most promising modalities for cancer treatment (Hanahan and Folkman, 1996; Brekken et al., 2002). Antibodies directed against panendothelial cells, such as anti-CD31 and anti-CD34 antibodies, have been used in the evaluation of angiogenesis (Hanahan and Folkman, 1996). Endoglin (CD105) has proven to be a useful marker of angiogenesis, whereas antibodies against panendothelial cells, such as anti-CD31 and anti-CD34 antibodies, have usually been used in the evaluation of angiogenesis. These panendothelial antibodies react not only with newly forming vessels but also with normal vessels trapped within tumor tissues (Guerrero-Esteo et al., 1999; Sanchez-Elsner et al., 2002). In contrast, endoglin (CD105) is predominantly expressed on cellular lineages within the vascular system and is overexpressed on proliferating endothelial cells that participate in tumor angiogenesis, with either a weak or negative expression in the vascular endothelium of normal tissues (Wang et al., 1994; Kumar et al., 1996; Guerrero-Esteo et al., 1999; Yancopoulos et al., 2000). Investigators have recently shown that CD105 is a more specific and sensitive microvessel marker compared with other commonly used panendothelial antibodies in malignant neoplasms of the brain, breast, colon, esophagus, urothelial bladder, and lung (Wang et al., 1994; Kumar et al., 1996; Li et al., 1998; Yancopoulos et al., 2000). CD105 (endoglin) is a proliferation-associated and hypoxia-inducible protein that is abundantly expressed in angiogenic endothelial cells. It is a receptor for transforming growth factors (TGFs) β1 and β3 and modulates TGF-β signaling by interacting with TGF-β receptors I and/or II (Fonsatti et al., 2001). Immunohistochemical studies revealed that CD105 is strongly expressed in blood vessels of tumor tissues. Intratumoral microvessel density (MVD), determined using antibodies to CD105, has been found to be an independent prognostic indicator, in which increased MVD correlates with shorter survival. CD105 is released into circulatory system, with elevated levels detected in patients with various types of cancer and positively correlated with tumor metastasis (Weidner et al., 1991; Tanigawa et al., 1997; Paydas et al., 2009).
From this perspective, we studied a series of 40 DLBCL patients to investigate the correlation between CD105 and CD34 expression and the prognosis of DLBCL.
Materials and methods
Patient samples and tissue collection
Surgical tissue samples from 40 patients with DLBCL were used in the present study. All tumor specimens were retrieved from the archives of the Department of Pathology, Suez Canal University Hospital, between the years 2000 and 2009. The clinical data of these 40 patients were also retrieved from these archives. They were previously classified as GC-like and non-GC-like (ABC-like) by immunostaining for BCL6, CD10, and MUM1.
Tissue samples were fixed in 10% buffered formalin, embedded in paraffin, and then were processed conventionally for histological and immunohistochemical study. The sections (5 μm) were stained using hematoxylin and eosin for the histological evaluations. The remaining unstained serial sections were used for immunohistochemical analysis. All specimens were histologically diagnosed according to the WHO criteria for lymphomas Swerdlow et al., 2008. Immunohistochemical studies were conducted on the paraffin sections using the peroxidase–avidin–biotin method (LASB kit; DakoCytomation, Carpinteria, California, USA) after heat-induced antigen retrieval. The primary antibodies were directed toward endoglin (CD105, dilution 1 : 50; Novocastra) and CD34 (dilution 1 : 30; Novocastra, USA).
Microvessel density counting
Tissue sections stained with CD105 and CD34 were used for evaluating MVD (designated MVD-CD105 and MVD-CD34, respectively) according to Weidner et al.’s (1991) and Tzankov et al.’s (2006) standards, with a minor modification. Five different fields were chosen from each of the slides, and the stained vessels were counted simultaneously by two researchers under a multiocular microscope. The average from the five areas was recorded as the MVD score.
All data were analyzed using the SPSS software program (SPSS16.0 for Windows 2010; SPSS Inc., Chicago, Illinois, USA). Student’s t-test and Pearson’s correlation coefficient were used for comparison of continuous variables among patients. Overall survival was computed using the Kaplan–Meier method, and comparisons among patients were made using the log-rank test.
The 40 patients with de-novo DLBCL evaluated in this study included 26 men and 14 women. The median age of the patients was 51 years (range 34–63 years). The median follow-up of the surviving patients was 12 months (range 2–29 months). Thirty patients received chemotherapy, five received chemoradiotherapy, and five did not receive any therapy.
Expressions of CD105 and CD34 and microvessel density scores in diffuse large B-cell lymphoma
Samples were immunostained with both anti-CD105 antibody and anti-CD34 antibody and showed three patterns of expression: sinusoid-like, branching, and small without apparent lumina (endothelial sprouts; Fig. 1). Comparisons of consecutive sections showed that few of the vessels that were highlighted by anti-CD34 were not highlighted by anti-CD105, whereas almost all the vessels highlighted by anti-CD105 were also highlighted by anti-CD34. MVD-CD105 and MVD-CD34 scores were 71.7±8.3 (SD) and 106.3±10.4 (SD), respectively.
Correlation between microvessel density and clinicopathological parameters
No significant correlations were found between either MVD-CD105 or MVD-CD34 and sex and age. In contrast, MVD-CD105 was significantly related to several pathological variables. The MVD-CD105 score showed significant correlations with the tumor, node, and metastasis stage, with higher MVD observed in tumors of a more advanced stage (stages I and II vs. stages III and IV, P=0.0599), and serum lactate dehydrogenase (LDH) levels, with lower MVD observed in patients with low serum LDH levels compared with high serum LDH levels (P=0.0235).
DLBCL of the GCB type showed mean values of MVD-CD105 that were significantly lower than those of ABC-type DLBCL (60.71±7.60 vs. 72.52±9.78, respectively; P=0.0197). In addition, DLBCL of the GCB type showed mean values of MVD-CD34 that were significantly lower than those of ABC-type DLBCL (88.04±5.78 vs. 109.29±8.77, respectively; P=0.0016). Furthermore, there was no significant correlation between MVD-CD34 and any of the other clinicopathological features. Thus, CD105 appears to be both specific and sensitive as a marker of angiogenesis.
Multivariate analysis of MVD-CD105 as a predictive factor
Multivariate analysis showed that a higher MVD-CD105 was a significant (P=0.024) and independent factor predicting a poor prognosis (Table 1). However, when the MVD-CD34 was used in the regression model, it failed to show a significant prognostic value (P=0.072).
Microvessel density and overall survival rates
The 2-year survival rate in DLBCL patients with a lower MVD-CD105 was 47.1%, which was significantly higher than the 13.5% rate in DLBCL patients with a higher MVD-CD105 (P=0.014). Although the 2-year survival rate (33.3%) in DLBCL patients with a lower MVD-CD34 (<94) was also higher than that in DLBCL patients with a higher MVD-CD34 (26.9%), the difference was not statistically significant (P=0.601; Fig. 2).
Angiogenesis has been shown to play a major role in the development, invasion, and metastasis of tumors. Tumor angiogenesis and its clinical significance have been investigated in a variety of solid tumors (Tzankov et al., 2006; Alshenawy, 2010). Recently, several studies have suggested that the growth of hematopoietic neoplasms is also dependent on angiogenesis (Tzankov et al., 2006; Gratzinger et al., 2007; Paydas et al., 2009). However, there are conflicting opinions on whether the degree of angiogenesis as measured by the MVD has a prognostic value in lymphomas (Wang et al., 1993; Tzankov et al., 2006; Gratzinger et al., 2007; Paydas et al., 2009; Alshenawy, 2010). MVD has been reported to be an independent prognostic indicator of outcome in a variety of human malignancies, with increased MVD being correlated with shorter overall and relapse-free survival rates (Takahashi et al., 2001; Tanaka et al., 2001; Akagi et al., 2002; Schimming and Marme, 2002; Mucci et al., 2009). However, a few investigators have failed to confirm these findings (Meert et al., 2002; Seon, 2002). Discrepancies among these studies may be because of the various methods of staining tissues using different panendothelial marker antibodies and different methods of counting microvessels. Panendothelial markers such as CD34, CD31, and von Willebrand factor stain endothelial cells well in large blood vessels but fail to localize some microvessels. The use of such markers would therefore underestimate the MVD in tumors (Seon et al., 1997; Tabata et al., 1999; Fonsatti et al., 2001). CD105 antibodies have shown a greater specificity for tumor vasculature in comparison with panendothelial markers (Tanaka et al., 2001; Taskinen et al., 2010).
In the present study, no significant correlations were found between either MVD-CD105 or MVD-CD34 and sex and age. In contrast, the MVD-CD105 score showed significant correlation with the tumor, node, and metastasis stage, with higher MVD observed in tumors of a more advanced stage (stages I and II vs. stages III and IV). A significant correlation with serum LDH levels was demonstrated, with lower MVD observed in patients having low serum LDH levels compared with high serum LDH levels. However, there was no significant correlation between MVD-CD34 and any of the other clinicopathological features.
On studying the correlation between the expression of MVD-CD105 and MVD-CD34 and GEP, we found that the mean values for MVD-CD105 in GCB-type DLBCL were significantly lower than those in ABC-type DLBCL. Moreover, DLBCL of the GCB type showed mean values for MVD-CD34 that were significantly lower than those for ABC-type DLBCL. ABC-type DLBCL arises from a post-GCB that is blocked during plasmacytic differentiation, whereas the GCB-type DLBCL subtype arises from a GCB. These two types of DLBCL also differ in their profile of genetic alterations and dysregulation of molecular pathways (Bea et al., 2005; Jørgensen et al., 2007). In particular, ABC-type DLBCL shows constitutive activation of NF-κB, which may be related to the presence of mutations in multiple genes regulating this pathway. The NF-κB transcription factor has been associated with multiple aspects of angiogenesis by regulating several genes involved in this process, such as VEGF, IL-8, and several metalloproteinases among others (Davis et al., 2001; Feuerhake et al., 2005; Compagno et al., 2009). Therefore, the higher MVD observed in ABC-type DLBCL is concordant with the NF-κB activation in these tumors (Feuerhake et al., 2005; Jørgensen et al., 2007). The prognostic value of the MVD in the current study agrees with the previously published GEP data on DLBCL (Davis et al., 2001; Bea et al., 2005; Feuerhake et al., 2005; Jørgensen et al., 2007; Compagno et al., 2009).
Kaplan–Meier analysis showed that MVD-CD105, but not MVD-CD34, was significantly correlated with the 2-year survival rate, and multivariate analysis showed that MVD-CD105, but not MVD-CD34, was a significant and independent prognostic factor, which is consistent with the results of retrospective studies on other tumors (Wang et al., 1995; Jørgensen et al., 2007; Pazgal et al., 2007; Tzankov et al., 2007).
Endoglin (CD105) is emerging as a prime vascular target of antiangiogenic cancer therapy (Jiang et al., 2000; Ding et al., 2001). Recent studies have shown the systemic administration of naked antihuman endoglin monoclonal antibodies to suppress established tumors, and its efficacy was markedly enhanced in combination with a chemotherapeutic drug with an antiangiogenic schedule of drug dosing (Jiang et al., 2000; Ding et al., 2001; Duff et al., 2003). Therefore, in the present study, the results of endoglin staining in DLBCL could eventually lead to the performance of therapeutic trials on antiangiogenic treatment for patients with DLBCL.
The use of the anti-CD105 monoclonal antibody proved to be an ideal means to quantify new vessels in DLBCL, and MVD-CD105 was a significant and independent prognostic factor for DLBCL. In addition, CD105 can be used as a potential target in DLBCL therapy.
Conflicts of interest
There are no conflicts of interest.
Akagi K, Ikeda Y, Sumiyoshi Y, Kimura Y, Kinoshita J, Miyazaki M, Abe T. Estimation of angiogenesis with anti-CD105 immunostaining in the process of colorectal cancer development. Surgery. 2002;131:S109–S113
Alshenawy HA. Prognostic significance of vascular endothelial growth factor, basic fibroblastic growth factor, and microvessel density and their relation to cell proliferation in B-cell non-Hodgkin’s lymphoma. Ann Diagn Pathol. 2010;14:321–327
Bea S, Zettl A, Wright G, Salaverria I, Jehn P, Moreno V, et al. Diffuse large B-cell lymphoma subgroups have distinct genetic profiles that influence tumor biology and improve gene-expression-based survival prediction. Blood. 2005;106:3183–3190
Brekken RA, Li C, Kumar S. Strategies for vascular targeting in tumours. Int J Cancer. 2002;100:123–130
Coiffier B. Current strategies for the treatment of diffuse large B cell lymphoma. Curr Opin Hematol. 2005;12:259–265
Coiffier B, Lepage E, Briere J, Herbrecht R, Tilly H, Bouabdallah R, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med. 2002;346:235–242
Compagno M, Lim WK, Grunn A, Nandula SV, Brahmachary M, Shen Q, et al. Mutations of multiple genes cause deregulation of NF-kappaB in diffuse large B-cell lymphoma. Nature. 2009;459:717–721
Davis RE, Brown KD, Siebenlist U, Staudt LM. Constitutive nuclear factor kappaB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells. J Exp Med. 2001;194:1861–1874
Ding I, Sun JZ, Fenton B, Liu WM, Kimsely P, Okunieff P, Min W. Intratumoral administration of endostatin plasmid inhibits vascular growth and perfusion in MCa-4 murine mammary carcinomas. Cancer Res. 2001;61:526–531
Duff SE, Li C, Garland JM, Kumar S. CD105 is important for angiogenesis: evidence and potential applications. FASEB J. 2003;17:984–992
Feuerhake F, Kutok JL, Monti S, Chen W, LaCasce AS, Cattoretti G, et al. NFkappaB activity, function, and target-gene signatures in primary mediastinal large B-cell lymphoma and diffuse large B-cell lymphoma subtypes. Blood. 2005;106:1392–1399
Feugier P, Van Hoof A, Sebban C, Solal-Celigny P, Bouabdallah R, Fermé C, et al. Long-term results of the R-CHOP study in the treatment of elderly patients with diffuse large B-cell lymphoma: a study by the Groupe d’Etude des Lymphomes de l’Adulte. J Clin Oncol. 2005;23:4117–4126
Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston: tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285:1182–1186
Fonsatti E, Del Vecchio L, Altomonte M, Sigalotti L, Nicotra MR, Coral S, et al. Endoglin: an accessory component of the TGF-beta-binding receptor-complex with diagnostic, prognostic, and bioimmunotherapeutic potential in human malignancies. J Cell Physiol. 2001;188:1–7
Gratzinger D, Zhao S, Marinelli RJ, Kapp AV, Tibshirani RJ, Hammer AS, et al. Microvessel density and expression of vascular endothelial growth factor and its receptors in diffuse large B-cell lymphoma subtypes. Am J Pathol. 2007;170:1362–1369
Guerrero-Esteo M, Lastres P, Letamendia A, Perez-Alvarez MJ, Langa C, Lopez LA, et al. Endoglin overexpression modulates cellular morphology, migration, and adhesion of mouse fibroblasts. Eur J Cell Biol. 1999;78:614–623
Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86:353–364
Harris NL, Jaffe ES, Stein H, Banks PM, Chan JK, Cleary ML, et al. A revised European–American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood. 1994;84:1361–1392
Jiang YF, Yang ZH, Hu JQ. Recurrence or metastasis of HCC:predictors, early detection and experimental antiangiogenic therapy. World J Gastroenterol. 2000;6:61–65
Jørgensen JM, Sørensen FB, Bendix K, Nielsen JL, Olsen ML, Funder AM, d’Amore F. Angiogenesis in non-Hodgkin’s lymphoma: clinico-pathological correlations and prognostic significance in specific subtypes. Leuk Lymphoma. 2007;48:584–595
Kumar P, Wang JM, Bernabéu C. CD105 and angiogenesis. J Pathol. 1996;178:363–366
Lenz G, Wright G, Dave SS, Xiao W, Powell J, Zhao H, et al. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med. 2008;359:2313–2323
Li CG, Wilson PB, Bernabeu C, Raab U, Wang JM, Kumar S. Immunodetection and characterisation of soluble CD105-TGFbeta complexes. J Immunol Methods. 1998;218:85–93
Meert AP, Paesmans M, Martin B, Delmotte P, Berghmans T, Verdebout JM, et al. The role of microvessel density on the survival of patients with lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer. 2002;87:694–701
Mucci LA, Powolny A, Giovannucci E, Liao Z, Kenfield SA, Shen R, et al. Prospective study of prostate tumor angiogenesis and cancer-specific mortality in the health professionals follow-up study. J Clin Oncol. 2009;27:5627–5633
Paydas S, Seydaoglu G, Ergin M, Erdogan S, Yavuz S. The prognostic significance of VEGF-C and VEGF-A in non-Hodgkin lymphomas. Leuk Lymphoma. 2009;50:366–373
Pazgal I, Boycov O, Shpilberg O, Okon E, Bairey O. Expression of VEGF-C, VEGF-D and their receptor VEGFR-3 in diffuse large B-cell lymphomas. Leuk Lymphoma. 2007;48:2213–2220
Rosenwald A, Wright G, Chan WC, Connors JM, Campo E, Fisher RI, et al. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med. 2002;346:1937–1947
Sanchez-Elsner T, Botella LM, Velasco B, Langa C, Bernabeu C. Endoglin expression is regulated by transcriptional cooperation between the hypoxia and transforming growth factor-beta pathways. J Biol Chem. 2002;277:43799–43808
Schimming R, Marme D. Endoglin (CD105) expression in squamous cell carcinoma of the oral cavity. Head Neck. 2002;24:151–1516
Seon BK. Expression of endoglin (CD105) in tumor blood vessels. Int J Cancer. 2002;99:310–311
Seon BK, Matsuno F, Haruta Y, Kondo M, Barcos M. Long-lasting complete inhibition of human solid tumors in SCID mice by targeting endothelial cells of tumor vasculature with antihuman endoglin immunotoxin. Clin Cancer Res. 1997;3:1031–1044
Shipp MA. The International Non-Hodgkin’s Lymphoma Prognostic Factors Project. A predictive model for aggressive non-Hodgkin’s lymphoma. N Engl J Med. 1993;329:987–994
Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. WHO classification of tumors of hematopoietic and lymphoid tissues. 20084th ed. Lyon IARC
Tabata M, Kondo M, Haruta Y, Seon BK. Antiangiogenic radioimmunotherapy of human solid tumors in SCID mice using (125)I-labeled anti-endoglin monoclonal antibodies. Int J Cancer. 1999;82:737–742
Takahashi N, Kawanishi-Tabata R, Haba A, Tabata M, Haruta Y, Tsai H, Seon BK. 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;7:524–532
Tanaka F, Otake Y, Yanagihara K, Kawano Y, Miyahara R, Li M, et al. Evaluation of angiogenesis in non-small cell lung cancer: comparison between anti-CD34 antibody and anti-CD105 antibody. Clin Cancer Res. 2001;7:3410–3415
Tanigawa N, Amaya H, Matsumara M, Shimomatsuya T. Association of tumour vasculature with tumour progression and overall survival of patients with non-early gastric carcinomas. Br J Cancer. 1997;75:566–571
Taskinen M, Jantunen E, Kosma VM, Bono P, Karjalainen-Lindsberg ML, Leppa S. Prognostic impact of CD31-positive microvessel density in follicular lymphoma patients treated with immunochemotherapy. Eur J Cancer. 2010;46:2506–2512
Tzankov A, Heiss S, Ebner S, Sterlacci W, Schaefer G, Augustin F, et al. Angiogenesis in nodal B cell lymphomas: a high throughput study. J Clin Pathol. 2006;60:476–482
Tzankov A, Heiss S, Ebner S, Sterlacci W, Schaefer G, Augustin F, et al. Angiogenesis in nodal B cell lymphomas: a high throughput study. J Clin Pathol. 2007;60:476–482
Wang JM, Kumar S, Pye D, van Agthoven AJ, Krupinski J, Hunter RD. A monoclonal antibody detects heterogeneity in vascular endothelium of tumours and normal tissues. Int J Cancer. 1993;54:363–370
Wang JM, Kumar S, Pye D, Haboubi N, al-Nakib L. Breast carcinoma: comparative study of tumor vasculature using two endothelial cell markers. J Nat Cancer Inst. 1994;86:386–388
Wang JM, Kumar S, van Agthoven A, Kumar P, Pye D, Hunter RD. Irradiation induces up-regulation of E9 protein (CD105) in human vascular endothelial cells. Int J Cancer. 1995;62:791–796
Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis – correlation in invasive breast carcinoma. N Engl J Med. 1991;324:1–8
Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular-specific growth factors and blood vessel formation. Nature. 2000;407:242–248