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The prognostic impact of microvessel density using angiogenic markers (CD105 and CD34) on patients with diffuse large B-cell lymphoma

F.M. Mansour, Sahar; Mohamed, Hala A.D.

doi: 10.1097/01.XEJ.0000422047.19617.e4
ORIGINAL ARTICLES
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Background Diffuse large B-cell lymphoma is a clinically and molecularly heterogeneous disease. Gene expression profiling studies have shown that the tumor microenvironment affects survival and that the angiogenesis-related signature is prognostically unfavorable. The contribution of microvessel density (MVD) to survival in diffuse large B-cell lymphomas treated with immunochemotherapy remains unknown. The purpose of this single-institution retrospective study is to assess the prognostic impact of MVD in patients with diffuse large B-cell lymphoma (DLBCL) using angiogenic markers CD105 and CD34.

Patients and methods A total of 40 patients with DLBCL diagnosed and managed at Suez Canal University Hospital between the years 2000 and 2009 were included. On the basis of the results of the previous gene expression profiling study, the current MVD analysis by immunohistochemically staining for the CD105 and CD34 antibodies was carried out. Microvessels were quantitated using anti-CD105 and anti-CD34 monoclonal antibodies and analyzed in conjunction with the clinicopathological characteristics of DLBCL. The international prognostic variables and the overall survival were evaluated.

Results Although the prognostic significance of MVD in DLBCL remains controversial, we considered the growth of DLBCL to be dependent on angiogenesis and MVD determined by the anti-CD105 monoclonal antibody to be a reliable prognostic marker in patients with DLBCL.

Conclusion 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 survival rate. Multivariate analysis showed that a higher CD105 MVD was a significant and independent factor predicting a poor prognosis in DLBCL, whereas CD34 MVD failed to show a significant prognostic value.

Department of Pathology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

Correspondence to Sahar F.M. Mansour, MD, Department of Pathology, Faculty of Medicine, Suez Canal University, 41511 Ismailia, Egypt Tel: +20 100 693 0393; fax: +0643328543; e-mail: sfmansour36@yahoo.com

Received September 9, 2012

Accepted September 24, 2012

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Introduction

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.

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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.

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Immunohistochemical staining

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).

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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.

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Statistical analysis

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.

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Results

Clinicopathological features

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.

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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.

Fig. 1

Fig. 1

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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.

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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).

Table 1

Table 1

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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).

Fig. 2

Fig. 2

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Discussion

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.

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Conclusion

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.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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