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Histological expression of angiogenic factors: VEGF, PDGFRα, and HIF-1α in Hodgkin lymphoma
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Pathology – Research and Practice 205 (2009) 11–20 www.elsevier.de/prp
ORIGINAL ARTICLE
Histological expression of angiogenic factors: VEGF, PDGFRa, and HIF-1a in Hodgkin lymphoma Freda H. Passama,, Michael G. Alexandrakisc, Maria Kafousid, Marianthi Fotinoub, Katerina Darivianakid, George Tsirakisc, Paraskevi A. Roussoua, Efstathios N. Stathopoulosd, Nikolaos M. Siafakase a
3rd Department of Internal Medicine, Medical School of Athens, ‘‘Sotiria’’ Hospital, Athens, Greece Department of Pathology, ‘‘Sotiria’’ Hospital, Athens, Greece c Department of Hematology, University Hospital of Heraklion, Medical School of Crete, Greece d Department of Pathology, University Hospital of Heraklion, Medical School of Crete, Greece e Department of Thoracic Medicine, University Hospital of Heraklion, Medical School of Crete, Greece b
Received 9 March 2008; accepted 21 July 2008
Abstract Angiogenesis is a prerequisite for solid tumor growth, but there is relatively limited data regarding Hodgkin lymphoma. The purpose of this study was to examine the immunohistochemical expression of angiogenic and proliferation markers in Hodgkin biopsies in relation to clinical parameters. Immunostaining was performed on 65 Hodgkin biopsies with vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 alpha (HIF-1a), platelet-derived growth factor receptor alpha (PDGFRa), Ki-67, and p53. Microvessel density (MVD) was determined by CD31 staining. In all cases, neoplastic cells and reactive background cells were evaluated. The neoplastic population expressed VEGF in 48% of the cases, HIF-1a in 54% of the cases, and PDGFRa in 95% of the cases. Both Ki-67 and p53 were positive in neoplastic cells in over 60% of the cases. The MVD had a median of 2.6/0.0625 mm2 which was not different from normal lymph nodes. VEGF in the non-neoplastic compartment showed increased staining in Ann Arbor stage I–II versus III–IV. In conclusion, VEGF, HIF-1a, and predominantly PDGFRa are expressed in neoplastic cells in the majority of Hodgkin lymphomas. As microvessel formation is not increased in Hodgkin, additional functions of these angiogenic molecules should be investigated. r 2008 Elsevier GmbH. All rights reserved. Keywords: Hodgkin; Angiogenesis; VEGF; PDGFRa; HIF-1a
Introduction Corresponding author at: Department of Immunology, 2 South
Street, St. George Hospital, Kogarah, PC 2217 NSW, Australia. Tel.: +612 93502955; fax: +612 93503981. E-mail addresses: [email protected], [email protected] (F.H. Passam). 0344-0338/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2008.07.007
Angiogenesis is an adaptive mechanism of human tissues in response to hypoxia or increased metabolic demands. However, maladaptive angiogenesis is utilized in the pathogenesis of cancer as well as inflammatory and immune diseases [8]. Folkman [17,18] first showed
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that the establishment of new vessels is necessary for the expansion and dissemination of solid tumors, however, in hematological malignancies, there is not the same degree of evidence. Only recently, studies have been published which support a role for angiogenesis in the progression of leukemias and lymphomas [36,38,42, 45,54,55]. Hodgkin lymphoma is a common lymphoma especially affecting young ages [60]. The unique histology of this cancer is due to the presence of a small percentage of malignant cells in affected tissues amongst a reactive background of non-neoplastic inflammatory, accessory cells, and a varying amount of fibrosis [60]. The role of angiogenesis in this specific type of lymphoma is still unclear. Microvessel density [13,22,28,31,35] and immunohistochemical staining of various angiogenic molecules [2,11,13,15,19,27,29,35,37,41,50] in Hodgkin lymph nodes have been reported, often with conflicting results. The aim of this study was to examine novel angiogenic factors, such as HIF-1a and PDGFRa, in Hodgkin biopsies and their relation to the extent of angiogenesis and proliferation markers in affected lymph nodes as well as clinical parameters in the same patients.
Materials and methods Patients Lymph node biopsies from 65 patients with Hodgkin lymphoma were retrieved from the pathology archives of two tertiary hospitals. The study had the approval of the Ethical Committees of the institutes involved. The clinical characteristics of the patients are presented in Table 1. Histological classification was based on the 1999 World Health Organization classification of lymphomas into nodular lymphocyte predominant Hodgkin lymphoma and classical Hodgkin lymphoma (CHL) with its 4 subdivisions: nodular sclerosis (NS), mixed cellularity (MC), lymphocyte-rich (LR), and lymphocyte-depleted (LD) CHL. Staging was performed according to the Ann Arbor criteria [33]. Prognostic scoring was performed, where clinical data was available, according to the International Prognostic score (for advanced Hodgkin lymphoma) [24]. Other parameters taken into account included the presence of extranodular disease and the presence of B symptoms. Standard treatment consisted of 6–8 cycles of ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) with or without involved field irradiation in 55 patients and other modalities: COPP (cyclophosphamide, vincristine, procarbazine, prednisone)/ABVD for 8 and BEACOPP
Table 1.
Patients’ characteristics No.
%
Sex Male Female
40 25
62 38
Histology Nodular lymphocyte predominant Lymphocyte-rich classical HL Nodular sclerosis Mixed cellularity Lymphocyte-depleted
1 3 46 14 1
2 5 70 21 2
Ann Arbor stage I II III IV
2 45 4 14
3 69 6 22
IPS 0 1 2 3 4 5
6 24 13 7 3 5
10 41 23 12 5 9
Extranodal extension Present Absent
26 39
40 60
B symptoms Present Absent
41 24
63 37
Response to chemotherapy Complete Partial Resistant
43 13 3
73 22 5
Status Alive Deceased
43 16
73 27
Months of follow up Mean7sd
49725
(bleomycin, etoposide, adriamycin, cyclophosphamide, vincristine, procarbazine, prednisone) for 2 patients. After completion of therapy, patients were assessed for response.
Tissues Immunohistochemical staining Three micrometer sections of formalin-fixed and paraffin wax-embedded tissues were used. Sections were first dewaxed and rehydrated in alcohol. An antibody to vascular endothelial growth factor (VEGF/sc-7269, mouse monoclonal, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was used at a dilution of 1:200
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and incubated for 1 h at room temperature (RT). An antibody to hypoxia inducible factor 1 alpha (HIF-1a/ sc-13515, mouse monoclonal, Santa Cruz) was used at a dilution of 1:20 and incubated overnight at 4 1C. An antibody to platelet-derived growth factor receptor alpha (PDGFRa, rabbit polyclonal, Cat no. RB-9027PO, Neomarkers, Lab Vision Corporation, Fremont CA, USA) was used at a dilution of 1:40. The tissue samples were pretreated with saponine at a concentration of 50 mg/100 ml distilled water and incubated for 30 min at RT. Antibody to p53 (Ab5/MS-186-PO, mouse monoclonal, Neomarkers) was used at a 1:100 dilution, and an antibody to Ki-67 (RM-9106-SO, clone SP6, rabbit monoclonal, Neomarkers) was used at a 1:200 dilution. Antigen retrieval in all cases was performed by microwave with citric buffer pH: 6.0. The Ultravision LP Detection System, AP Polymer with Fast Red chromogen (TL-015-AF) (Neomarkers) was used for detection of all antibodies. Omission of the primary antibody was used as a negative control in all cases, whereas, in accordance with the literature, a breast carcinoma biopsy, previously used in our laboratory and known positive for VEGF, HIF-1a, and PDGFRa, was used as a positive control [9,40,47,57]. Phenotypic markers CD3, CD20, CD30, CD15, and EMA were used as adjuvants for confirmation of the diagnosis and highlighting of Reed-Sternberg cells (RS), both diagnostic and their variants. Staining pattern In all cases, neoplastic cells, reactive background cells, and endothelial cells were evaluated. The staining for VEGF in the RS population was diffuse cytoplasmic or paranuclear (Golgi staining), and, in most cases, the intensity was strong. VEGF-positive reactive cells displayed cytoplasmic staining and consisted of macrophages, fibroblasts, and variable amounts of lymphocytes. HIF-1a staining was nuclear and of moderate intensity in RS cells.
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The staining of RS cells for PDGFRa was intense membranous and diffuse cytoplasmic. The reactive population had a variable amount of positive PDGFRa staining but, generally, of lesser intensity than the neoplastic RS population. Ki67 and p53 stained the nucleus of RS cells. Reactive lymphocytes displayed varying degrees of staining for Ki67 (usually o50% of the reactive population), and low numbers stained weakly for p53. The above patterns of staining were in accordance with the literature for Hodgkin and non-Hodgkin lymphomas [1,13,44,49,62]. Scoring For a case to be considered positive for the neoplastic or reactive population, more than 10% of the RS or reactive cell population had to react with the antibody (Figs. 1–5) as previously described [13]. The following scoring system was applied for the grouping of results in order to facilitate analysis: Score 1 (low positive) when 10–50% of cells displayed staining; score 2 (high positive) when 450% of cells stained. Immunostaining was evaluated blindly by two independent observers. The interobserver variability was less than 10%, and the mean value of their counts was considered the final count. In case of a discrepancy higher than 10%, the case was reevaluated, and the result was reported by consensus. Microvessel density An antibody to CD31/PECAM-1 (ab-1, clone JC/ 70A, mouse monoclonal, Cat no. MS-353-SO CD31, Neomarkers) was used for endothelial cell staining at a dilution of 1:400 after overnight incubation at 4 1C. Angiogenesis assessment in Hodgkin lymph nodes was based on the method of Weidner et al. [59] and an international consensus report [56]. The immunostained sections were scanned, using a light microscope equipped with an eyepiece graticule calibrated against a stage micrometer, at low magnification to locate three
Fig. 1. (a) VEGF ( 400), negative staining of RS (arrow) amongst positive reactive cells and background staining and (b) VEGF ( 400), positive Reed-Sternberg cells showing cytoplasmic reaction (arrow). Some positive reactive cells can also be distinguished.
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Fig. 2. (a) HIF-1a ( 400), negative staining of RS (arrow) and reactive cells. (b) HIF-1a ( 400), positive nuclear staining of RS (arrow) and of a few reactive cells.
Fig. 3. (a) PDGFRa ( 400), negative staining of RS (arrow) amongst a percentage of positive reactive cells. (b) PDGFRa ( 400), intense cytoplasmic staining of RS (arrow) and their variants, while reactive cells show less intense cytoplasmic staining.
Fig. 4. Ki-67 ( 630) nuclear staining of a RS cell (arrow).
Fig. 5. p53 ( 630) nuclear staining of RS cells (arrow) in the presence of a negative reactive cell population.
hot spots (areas showing the highest microvessel density). The microvessels in these areas were then counted at 400 magnification against the graticule projection (measuring 0.0625 mm2) on the field of view. The MVD
of each specimen was calculated as the mean value of all readings (Fig. 6). Five normal lymph nodes (non-tumor infiltrated lymph nodes resected during breast cancer surgery) were used as controls for MVD measurement.
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Fig. 6. (a) CD31 ( 100), vessels highlighted with CD31 in an example of Hodgkin biopsy with low microvessel density and (b) CD31 ( 100), example of Hodgkin biopsy with high microvessel density.
Serum measurements Nineteen patients had simultaneous pretreatment measurements of serum VEGF and VEGF receptor 2. Elisa measurements were performed using the Quantikine, R&D kits (Cat no. DVE00 for VEGF and DVR200 for VEGFR2, Minneapolis, MN, USA). Serum from ten sex- and age-matched healthy individuals were used as controls. All samples were run in duplicate.
Statistical analysis The SPSS software (v. 11.0 for Windows) was used for the statistical analysis. Differences in immunostaining scores for stage, prognostic score, histological group, and response to treatment were analyzed by nonparametric tests (chi-squared tests). For MVD and serum factors, the Mann–Whitney test was used to examine differences between patients and controls. Correlations between VEGF and other molecules were assessed using the Spearman’s rho correlation technique. Univariate analysis of survival was performed with the Kaplan–Meier method. p values less than 0.05 were considered as significant.
Results The percentage of Hodgkin cases with positive RS and reactive cells according to stage and histology subgroup is depicted in Table 2, whereas the immunohistochemical score of RS and reactive cells is shown in Table 3. Endothelial cell staining was negative for VEGF, HIF-1a, but positive for PDGFRa. VEGF was positive in the neoplastic population in nearly half of the cases, whereas in 30% of the cases, more than half of the RS cells stained (score 2). The
reactive population in the lymph node biopsy was positive in 60% of the cases. HIF-1a was positive in the neoplastic compartment in half of the cases examined but only in 1/3 of the cases in the reactive cells. PDGFRa was strongly positive in RS cells in the majority of cases (95% of cases were positive). Staining of RS cells for Ki-67 was found in 62% of the cases. In the majority of cases, o50% of reactive population stained for Ki67. p53 staining was positive in the RS compartment in 75% of the cases, but only 1/3 of the cases stained the reactive cells with p53. The MVD had a median of 2.6, which was not statistically different from control lymph nodes (median 3.0/0.0625 mm2). Angiogenesis in relation to:
Ann Arbor stage: VEGF in the non-neoplastic compartment differed significantly according to Ann Arbor stage with increased staining in stages I–II versus III and IV (p+0.03). Also, p53 in reactive cells showed more staining in stages I–II versus III and IV (p ¼ 0.03). Histology: Nodular sclerosis cases showed less Ki-67 staining (score 0–1) in reactive cells than in mixed cellularity cases (p ¼ 0.03). Also, nodular sclerosis showed more cases of positive staining in RS cells for PDGFRa than mixed cellularity (p ¼ 0.02). IPS score: An IPS score less than 2 was associated with a higher VEGF immunohistochemical score in reactive cells than IPS more than 2 (p ¼ 0.05). Cases with an IPS score X2 had a MVD4median more often than cases with an IPS o2 (p ¼ 0.05). Presence of necrosis: Areas of necrosis were found in 26% of biopsy samples. Increased MVD was associated with the presence of necrotic lesions in the material (p ¼ 0.05). Presence of B symptoms: B symptoms were more often in cases with strong staining for PDGFRa in reactive cells (score 2 versus 0–1) (p ¼ 0.05)
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2
VEGF RS (%) VEGF reactive HIF-1a RS HIF-1a reactive PDGFRa RS PDGFRa reactive Ki-67 RS Ki-67 reactive p53 RS p53 reactive
52 40 46 67 5 14 38 18 25 66
18 39 49 33 20 42 42 53 18 27
30 21 5 75 44 20 29 57 7
Score 0 ¼ negative staining (o10% cells), score 1 ¼ low positive (10–50% cells), and score 2 ¼ high positive (450% cells).
The median (range) of MVD in the Hodgkin biopsies and serum levels of the circulating angiogenic molecules are also depicted. a Single measurements.
0/2 31 66 27 1/2 66 33 45 1/2 76 33 90 1/2 71 100 50 2/2 95 100 100 1/2 52 33 45
2/2 60 33 45
33 50 40 27 0/1 60 80 70 61 0/1 75 75 90 78 1/1 62 100 80 61 0/1 95 100 80 100 1/1
Total LR MC NS LD Control I II III IV
906 (153–2200) 286a 1373 (308–2200) 906 (153–2200) – 292 (220–314) 286a 918 (307–2200) – 707 (153–2200)
7261 (779–16,000) 6720a 4862 (2442–7784) 7480 (779–16,000) – 6722 (5237–8479) 6720a 7300 (779–15,485) – 7289 (2442–16,000)
2.6 (1–5) 2.0 (1.6–2.6) 2.6 (1.6–3.3) 2.6 (1.0–4.3) 1.3a 3.0 (1.0–4.0) 2.0 2.3 (1.0–4.3) 2.3 (1.6–2.6) 3.0 (1.3–3.6)
48 50 70 37 1/1
54 50 60 59 0/1
HIF-1a VEGF
1/2 76 66 100
34 75 40 33 1/1 82 100 100 75 0/1
1
p53
86 75 90 75 1/1
Ki-67
0
Ki-67
PDGFRa
Immunohistochemistry score
PDGFRa VEGF Median (min–max) Median (min–max)
sVEGFR2 (pg/ml) sVEGF (pg/ml)
Table 3. Immunohistochemistry score of RS and reactive cells expressed as percentage of Hodgkin cases
HIF-1a
% cases with RS positive MVD/0.06 mm2
% cases with reactive cells positive
p53
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Table 2. Frequency of positive staining (scores 1 and 2) in the RS and reactive cell compartment of 65 Hodgkin biopsies according to histological classification and Ann Arbor stage
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Response to treatment: A higher VEGF and p53 immunohistochemical score in reactive cells (2 versus 0–1) correlated with increased incidence of complete response (po0.03). Survival analysis did not show any variable statistically associated with survival. Serum angiogenic factors: VEGF receptor 2 negatively correlated with VEGF staining in RS cells (r: 0.44, p ¼ 0.05).
Correlation of VEGF and HIF-1a of RS cells with other angiogenic factors: VEGF in RS cells correlates with VEGF in reactive cells (r: 0.43, p ¼ 0.001), VEGF in RS correlates with PDGFRa in RS cells (r: 0.28, p ¼ 0.02), HIF-1a in RS cells strongly correlates with p53 in RS cells (r: 0.43, p ¼ 0.001), MVD correlates with Ki-67 staining in reactive cells (r 0.28, p ¼ 0.03).
Discussion In many solid tumors and some hematological malignancies, microvessel density and VEGF expression define the angiogenic state and risk of progression [25,34,51,64], however, this does not seem to be the case in Hodgkin lymphoma. The results from microvessel density measurements in Hodgkin cannot be easily interpreted. In a study comparing biopsies, at diagnosis and relapse, from 11 patients with Hodgkin lymphoma, the authors described increased MVD with disease progression [35]. On the other hand, a large immunohistological study, including 286 Hodgkin biopsies, found maximal MVD at initial stages of disease [31]. However, both studies did not use controls. We found that MVD in Hodgkin specimens
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did not differ from control lymph nodes, leading to the conclusion of a limited role of microvessel density in the progression of Hodgkin lymphoma. This conclusion is also supported by a study by Citak et al. [11] on childhood lymphoma where the degree of microvessel formation was not significantly related to patient outcome. The key molecule implicated in microvessel formation is VEGF [16]. VEGF induces endothelial cell proliferation and migration through multiple pathways [37]. In Hodgkin lymphoma, some investigators describe positive immunostaining of RS cells for VEGF [2,11,13,63]; however, two other studies could not demonstrate VEGF staining [19,35]. This may be explained by the difference in primary antibody and antigen retrieval method used in the studies. We confirmed the histological expression of VEGF in RS cells although at a lower level (48% in our series versus 70% [13]). However, it was interesting to observe that increased immunostaining of VEGF in the reactive cell population was found in the earlier stages of disease and in patients with complete response to treatment. This is in contrast to the bad prognosis described for Hodgkin patients with increased serum VEGF [21], which decreases following effective treatment [4,43]. In our series, tissue VEGF did not correlate with serum VEGF but correlated negatively with circulating VEGF receptor 2. VEGF R2 mediates the growth and permeability signal of VEGF [37], and is coexpressed with VEGF on RS cells [2]. Thus, combining the aforementioned seemingly paradoxical results, one could speculate that increased tissue VEGF expression in the early stages of Hodgkin lymphoma may mean restriction of angiogenic activity to the lymph node with low levels of circulating VEGF and VEGF R2. Circulating VEGF and VEGF R2 may increase at a later stage when extranodal expansion becomes a feature of the disease. Furthermore, the lack of correlation of VEGF expression with MVD (also observed by other investigators [13]) leads to the assumption that VEGF may play additional roles in vessel formation in Hodgkin. VEGF attracts monocytes and inhibits dendritic cell function mediating the immune response [12,20], which may be relevant in a disease where immune dysregulation is a prominent feature [23]. The main stimulus for VEGF expression is hypoxia through the HIF-1a pathway [10,48]. HIF1 is an oxygen-dependent transcriptional activator that plays a crucial role in the angiogenesis of tumors [32]. In prostate cancer, HIF-1a staining correlates with tissue VEGF [6]. In non-Hodgkin lymphoma (NHL), HIF-1a is expressed weakly in 70% of the cases [49]. To our knowledge, there has been no description of HIF-1a staining in Hodgkin samples. Our results were similar to those previously described for NHL [49], with diffuse weak expression of HIF-1a in RS cells and no
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correlation of HIF-1a with the presence of necrotic areas. The absence of a correlation of HIF-1a with necrotic areas was unexpected given the positive correlation of increased MVD with necrosis in the same samples. Hodgkin lymphoma may utilize alternative hypoxic and angiogenic pathways compared to solid tumors and may be relatively independent of HIF-1a as far as angiogenesis is concerned. HIF-1a expression in RS cells may be serving an alternative role apart from response to the hypoxic stimulus; HIF also modulates apoptosis resistance [30], which is a proposed mechanism of Hodgkin cell survival [58]. VEGF expression is also induced by cytokines, such as PDGF, TNFa, IL-6, and oncogenes, such as p53. PDGF is a mitogen for connective tissue, especially fibroblasts during wound healing [3]. Additionally, the PDGF/PDGFRa system plays a role in the development of angiogenesis [5]. PDGFRa expression in Hodgkin samples has been reported in two studies [7,44]. We confirmed the strong PDGFRa staining in the majority of RS cells in a relatively large cohort of patients. This finding may have exciting therapeutic implications. Activating mutations in PDGFRa have been seen in gastrointestinal tumors, and inhibition of PDGFRa signaling by imatinib mesylate is exploited therapeutically for these tumors [26]. p53 plays a key role in the control of the cell cycle, cell differentiation, and programmed cell death. In NHL, VEGF expression correlates with p53 and Ki-67 expression [53]. p53 staining has been reported in RS of Hodgkin samples [15,39,52,61] at high levels in stage I cases [58] with good prognosis [62]. HIF1 and p53 both accumulate during hypoxia [46], which agrees with our finding of a positive correlation between HIF-1a and p53. Abele et al. [1] described an approximately 75% Ki-67 positivity in RS cells in 80 Hodgkin cases, which is in accordance with our results. We found a correlation of Ki-67 reactive cells with microvessel density, which could mean that new vessel formation in Hodgkin lymphoma may be serving proliferating cells of the reactive environment rather than the malignant cells themselves. In conclusion, we have described immunohistological expression of a panel of angiogenic factors in Hodgkin tissues which, in contrast to solid tumors, are not combined with increased microvessel density. From the findings of this study, it seems that the overexpression of angiogenic molecules in Hodgkin lymphoma may be serving roles other than vessel formation; i.e., it would be interesting to explore if VEGF is serving lymphangiogenesis in Hodgkin through its receptor VEGFR3 [16] or if the PDGF-PDGFRa system in Hodgkin is providing mitogenic, survival, or chemotactic signals to the neoplastic or reactive cells [3]. Although the results are not coupled with direct proof of angiogenic molecule production from the malignant Hodgkin cells, they
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provide strong evidence to pursue further study, especially regarding the role of PDGFRa in Hodgkin patients, given the potential therapeutic implications with PDGFa blocking agents.
[13]
Acknowledgments The authors would like to acknowledge the help of Dr. Spiros Miyakis in the statistical analysis of the results.
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Pathology – Research and Practice 205 (2009) 11–20 www.elsevier.de/prp
ORIGINAL ARTICLE
Histological expression of angiogenic factors: VEGF, PDGFRa, and HIF-1a in Hodgkin lymphoma Freda H. Passama,, Michael G. Alexandrakisc, Maria Kafousid, Marianthi Fotinoub, Katerina Darivianakid, George Tsirakisc, Paraskevi A. Roussoua, Efstathios N. Stathopoulosd, Nikolaos M. Siafakase a
3rd Department of Internal Medicine, Medical School of Athens, ‘‘Sotiria’’ Hospital, Athens, Greece Department of Pathology, ‘‘Sotiria’’ Hospital, Athens, Greece c Department of Hematology, University Hospital of Heraklion, Medical School of Crete, Greece d Department of Pathology, University Hospital of Heraklion, Medical School of Crete, Greece e Department of Thoracic Medicine, University Hospital of Heraklion, Medical School of Crete, Greece b
Received 9 March 2008; accepted 21 July 2008
Abstract Angiogenesis is a prerequisite for solid tumor growth, but there is relatively limited data regarding Hodgkin lymphoma. The purpose of this study was to examine the immunohistochemical expression of angiogenic and proliferation markers in Hodgkin biopsies in relation to clinical parameters. Immunostaining was performed on 65 Hodgkin biopsies with vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 alpha (HIF-1a), platelet-derived growth factor receptor alpha (PDGFRa), Ki-67, and p53. Microvessel density (MVD) was determined by CD31 staining. In all cases, neoplastic cells and reactive background cells were evaluated. The neoplastic population expressed VEGF in 48% of the cases, HIF-1a in 54% of the cases, and PDGFRa in 95% of the cases. Both Ki-67 and p53 were positive in neoplastic cells in over 60% of the cases. The MVD had a median of 2.6/0.0625 mm2 which was not different from normal lymph nodes. VEGF in the non-neoplastic compartment showed increased staining in Ann Arbor stage I–II versus III–IV. In conclusion, VEGF, HIF-1a, and predominantly PDGFRa are expressed in neoplastic cells in the majority of Hodgkin lymphomas. As microvessel formation is not increased in Hodgkin, additional functions of these angiogenic molecules should be investigated. r 2008 Elsevier GmbH. All rights reserved. Keywords: Hodgkin; Angiogenesis; VEGF; PDGFRa; HIF-1a
Introduction Corresponding author at: Department of Immunology, 2 South
Street, St. George Hospital, Kogarah, PC 2217 NSW, Australia. Tel.: +612 93502955; fax: +612 93503981. E-mail addresses: [email protected], [email protected] (F.H. Passam). 0344-0338/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2008.07.007
Angiogenesis is an adaptive mechanism of human tissues in response to hypoxia or increased metabolic demands. However, maladaptive angiogenesis is utilized in the pathogenesis of cancer as well as inflammatory and immune diseases [8]. Folkman [17,18] first showed
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that the establishment of new vessels is necessary for the expansion and dissemination of solid tumors, however, in hematological malignancies, there is not the same degree of evidence. Only recently, studies have been published which support a role for angiogenesis in the progression of leukemias and lymphomas [36,38,42, 45,54,55]. Hodgkin lymphoma is a common lymphoma especially affecting young ages [60]. The unique histology of this cancer is due to the presence of a small percentage of malignant cells in affected tissues amongst a reactive background of non-neoplastic inflammatory, accessory cells, and a varying amount of fibrosis [60]. The role of angiogenesis in this specific type of lymphoma is still unclear. Microvessel density [13,22,28,31,35] and immunohistochemical staining of various angiogenic molecules [2,11,13,15,19,27,29,35,37,41,50] in Hodgkin lymph nodes have been reported, often with conflicting results. The aim of this study was to examine novel angiogenic factors, such as HIF-1a and PDGFRa, in Hodgkin biopsies and their relation to the extent of angiogenesis and proliferation markers in affected lymph nodes as well as clinical parameters in the same patients.
Materials and methods Patients Lymph node biopsies from 65 patients with Hodgkin lymphoma were retrieved from the pathology archives of two tertiary hospitals. The study had the approval of the Ethical Committees of the institutes involved. The clinical characteristics of the patients are presented in Table 1. Histological classification was based on the 1999 World Health Organization classification of lymphomas into nodular lymphocyte predominant Hodgkin lymphoma and classical Hodgkin lymphoma (CHL) with its 4 subdivisions: nodular sclerosis (NS), mixed cellularity (MC), lymphocyte-rich (LR), and lymphocyte-depleted (LD) CHL. Staging was performed according to the Ann Arbor criteria [33]. Prognostic scoring was performed, where clinical data was available, according to the International Prognostic score (for advanced Hodgkin lymphoma) [24]. Other parameters taken into account included the presence of extranodular disease and the presence of B symptoms. Standard treatment consisted of 6–8 cycles of ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) with or without involved field irradiation in 55 patients and other modalities: COPP (cyclophosphamide, vincristine, procarbazine, prednisone)/ABVD for 8 and BEACOPP
Table 1.
Patients’ characteristics No.
%
Sex Male Female
40 25
62 38
Histology Nodular lymphocyte predominant Lymphocyte-rich classical HL Nodular sclerosis Mixed cellularity Lymphocyte-depleted
1 3 46 14 1
2 5 70 21 2
Ann Arbor stage I II III IV
2 45 4 14
3 69 6 22
IPS 0 1 2 3 4 5
6 24 13 7 3 5
10 41 23 12 5 9
Extranodal extension Present Absent
26 39
40 60
B symptoms Present Absent
41 24
63 37
Response to chemotherapy Complete Partial Resistant
43 13 3
73 22 5
Status Alive Deceased
43 16
73 27
Months of follow up Mean7sd
49725
(bleomycin, etoposide, adriamycin, cyclophosphamide, vincristine, procarbazine, prednisone) for 2 patients. After completion of therapy, patients were assessed for response.
Tissues Immunohistochemical staining Three micrometer sections of formalin-fixed and paraffin wax-embedded tissues were used. Sections were first dewaxed and rehydrated in alcohol. An antibody to vascular endothelial growth factor (VEGF/sc-7269, mouse monoclonal, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was used at a dilution of 1:200
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and incubated for 1 h at room temperature (RT). An antibody to hypoxia inducible factor 1 alpha (HIF-1a/ sc-13515, mouse monoclonal, Santa Cruz) was used at a dilution of 1:20 and incubated overnight at 4 1C. An antibody to platelet-derived growth factor receptor alpha (PDGFRa, rabbit polyclonal, Cat no. RB-9027PO, Neomarkers, Lab Vision Corporation, Fremont CA, USA) was used at a dilution of 1:40. The tissue samples were pretreated with saponine at a concentration of 50 mg/100 ml distilled water and incubated for 30 min at RT. Antibody to p53 (Ab5/MS-186-PO, mouse monoclonal, Neomarkers) was used at a 1:100 dilution, and an antibody to Ki-67 (RM-9106-SO, clone SP6, rabbit monoclonal, Neomarkers) was used at a 1:200 dilution. Antigen retrieval in all cases was performed by microwave with citric buffer pH: 6.0. The Ultravision LP Detection System, AP Polymer with Fast Red chromogen (TL-015-AF) (Neomarkers) was used for detection of all antibodies. Omission of the primary antibody was used as a negative control in all cases, whereas, in accordance with the literature, a breast carcinoma biopsy, previously used in our laboratory and known positive for VEGF, HIF-1a, and PDGFRa, was used as a positive control [9,40,47,57]. Phenotypic markers CD3, CD20, CD30, CD15, and EMA were used as adjuvants for confirmation of the diagnosis and highlighting of Reed-Sternberg cells (RS), both diagnostic and their variants. Staining pattern In all cases, neoplastic cells, reactive background cells, and endothelial cells were evaluated. The staining for VEGF in the RS population was diffuse cytoplasmic or paranuclear (Golgi staining), and, in most cases, the intensity was strong. VEGF-positive reactive cells displayed cytoplasmic staining and consisted of macrophages, fibroblasts, and variable amounts of lymphocytes. HIF-1a staining was nuclear and of moderate intensity in RS cells.
13
The staining of RS cells for PDGFRa was intense membranous and diffuse cytoplasmic. The reactive population had a variable amount of positive PDGFRa staining but, generally, of lesser intensity than the neoplastic RS population. Ki67 and p53 stained the nucleus of RS cells. Reactive lymphocytes displayed varying degrees of staining for Ki67 (usually o50% of the reactive population), and low numbers stained weakly for p53. The above patterns of staining were in accordance with the literature for Hodgkin and non-Hodgkin lymphomas [1,13,44,49,62]. Scoring For a case to be considered positive for the neoplastic or reactive population, more than 10% of the RS or reactive cell population had to react with the antibody (Figs. 1–5) as previously described [13]. The following scoring system was applied for the grouping of results in order to facilitate analysis: Score 1 (low positive) when 10–50% of cells displayed staining; score 2 (high positive) when 450% of cells stained. Immunostaining was evaluated blindly by two independent observers. The interobserver variability was less than 10%, and the mean value of their counts was considered the final count. In case of a discrepancy higher than 10%, the case was reevaluated, and the result was reported by consensus. Microvessel density An antibody to CD31/PECAM-1 (ab-1, clone JC/ 70A, mouse monoclonal, Cat no. MS-353-SO CD31, Neomarkers) was used for endothelial cell staining at a dilution of 1:400 after overnight incubation at 4 1C. Angiogenesis assessment in Hodgkin lymph nodes was based on the method of Weidner et al. [59] and an international consensus report [56]. The immunostained sections were scanned, using a light microscope equipped with an eyepiece graticule calibrated against a stage micrometer, at low magnification to locate three
Fig. 1. (a) VEGF ( 400), negative staining of RS (arrow) amongst positive reactive cells and background staining and (b) VEGF ( 400), positive Reed-Sternberg cells showing cytoplasmic reaction (arrow). Some positive reactive cells can also be distinguished.
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Fig. 2. (a) HIF-1a ( 400), negative staining of RS (arrow) and reactive cells. (b) HIF-1a ( 400), positive nuclear staining of RS (arrow) and of a few reactive cells.
Fig. 3. (a) PDGFRa ( 400), negative staining of RS (arrow) amongst a percentage of positive reactive cells. (b) PDGFRa ( 400), intense cytoplasmic staining of RS (arrow) and their variants, while reactive cells show less intense cytoplasmic staining.
Fig. 4. Ki-67 ( 630) nuclear staining of a RS cell (arrow).
Fig. 5. p53 ( 630) nuclear staining of RS cells (arrow) in the presence of a negative reactive cell population.
hot spots (areas showing the highest microvessel density). The microvessels in these areas were then counted at 400 magnification against the graticule projection (measuring 0.0625 mm2) on the field of view. The MVD
of each specimen was calculated as the mean value of all readings (Fig. 6). Five normal lymph nodes (non-tumor infiltrated lymph nodes resected during breast cancer surgery) were used as controls for MVD measurement.
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Fig. 6. (a) CD31 ( 100), vessels highlighted with CD31 in an example of Hodgkin biopsy with low microvessel density and (b) CD31 ( 100), example of Hodgkin biopsy with high microvessel density.
Serum measurements Nineteen patients had simultaneous pretreatment measurements of serum VEGF and VEGF receptor 2. Elisa measurements were performed using the Quantikine, R&D kits (Cat no. DVE00 for VEGF and DVR200 for VEGFR2, Minneapolis, MN, USA). Serum from ten sex- and age-matched healthy individuals were used as controls. All samples were run in duplicate.
Statistical analysis The SPSS software (v. 11.0 for Windows) was used for the statistical analysis. Differences in immunostaining scores for stage, prognostic score, histological group, and response to treatment were analyzed by nonparametric tests (chi-squared tests). For MVD and serum factors, the Mann–Whitney test was used to examine differences between patients and controls. Correlations between VEGF and other molecules were assessed using the Spearman’s rho correlation technique. Univariate analysis of survival was performed with the Kaplan–Meier method. p values less than 0.05 were considered as significant.
Results The percentage of Hodgkin cases with positive RS and reactive cells according to stage and histology subgroup is depicted in Table 2, whereas the immunohistochemical score of RS and reactive cells is shown in Table 3. Endothelial cell staining was negative for VEGF, HIF-1a, but positive for PDGFRa. VEGF was positive in the neoplastic population in nearly half of the cases, whereas in 30% of the cases, more than half of the RS cells stained (score 2). The
reactive population in the lymph node biopsy was positive in 60% of the cases. HIF-1a was positive in the neoplastic compartment in half of the cases examined but only in 1/3 of the cases in the reactive cells. PDGFRa was strongly positive in RS cells in the majority of cases (95% of cases were positive). Staining of RS cells for Ki-67 was found in 62% of the cases. In the majority of cases, o50% of reactive population stained for Ki67. p53 staining was positive in the RS compartment in 75% of the cases, but only 1/3 of the cases stained the reactive cells with p53. The MVD had a median of 2.6, which was not statistically different from control lymph nodes (median 3.0/0.0625 mm2). Angiogenesis in relation to:
Ann Arbor stage: VEGF in the non-neoplastic compartment differed significantly according to Ann Arbor stage with increased staining in stages I–II versus III and IV (p+0.03). Also, p53 in reactive cells showed more staining in stages I–II versus III and IV (p ¼ 0.03). Histology: Nodular sclerosis cases showed less Ki-67 staining (score 0–1) in reactive cells than in mixed cellularity cases (p ¼ 0.03). Also, nodular sclerosis showed more cases of positive staining in RS cells for PDGFRa than mixed cellularity (p ¼ 0.02). IPS score: An IPS score less than 2 was associated with a higher VEGF immunohistochemical score in reactive cells than IPS more than 2 (p ¼ 0.05). Cases with an IPS score X2 had a MVD4median more often than cases with an IPS o2 (p ¼ 0.05). Presence of necrosis: Areas of necrosis were found in 26% of biopsy samples. Increased MVD was associated with the presence of necrotic lesions in the material (p ¼ 0.05). Presence of B symptoms: B symptoms were more often in cases with strong staining for PDGFRa in reactive cells (score 2 versus 0–1) (p ¼ 0.05)
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2
VEGF RS (%) VEGF reactive HIF-1a RS HIF-1a reactive PDGFRa RS PDGFRa reactive Ki-67 RS Ki-67 reactive p53 RS p53 reactive
52 40 46 67 5 14 38 18 25 66
18 39 49 33 20 42 42 53 18 27
30 21 5 75 44 20 29 57 7
Score 0 ¼ negative staining (o10% cells), score 1 ¼ low positive (10–50% cells), and score 2 ¼ high positive (450% cells).
The median (range) of MVD in the Hodgkin biopsies and serum levels of the circulating angiogenic molecules are also depicted. a Single measurements.
0/2 31 66 27 1/2 66 33 45 1/2 76 33 90 1/2 71 100 50 2/2 95 100 100 1/2 52 33 45
2/2 60 33 45
33 50 40 27 0/1 60 80 70 61 0/1 75 75 90 78 1/1 62 100 80 61 0/1 95 100 80 100 1/1
Total LR MC NS LD Control I II III IV
906 (153–2200) 286a 1373 (308–2200) 906 (153–2200) – 292 (220–314) 286a 918 (307–2200) – 707 (153–2200)
7261 (779–16,000) 6720a 4862 (2442–7784) 7480 (779–16,000) – 6722 (5237–8479) 6720a 7300 (779–15,485) – 7289 (2442–16,000)
2.6 (1–5) 2.0 (1.6–2.6) 2.6 (1.6–3.3) 2.6 (1.0–4.3) 1.3a 3.0 (1.0–4.0) 2.0 2.3 (1.0–4.3) 2.3 (1.6–2.6) 3.0 (1.3–3.6)
48 50 70 37 1/1
54 50 60 59 0/1
HIF-1a VEGF
1/2 76 66 100
34 75 40 33 1/1 82 100 100 75 0/1
1
p53
86 75 90 75 1/1
Ki-67
0
Ki-67
PDGFRa
Immunohistochemistry score
PDGFRa VEGF Median (min–max) Median (min–max)
sVEGFR2 (pg/ml) sVEGF (pg/ml)
Table 3. Immunohistochemistry score of RS and reactive cells expressed as percentage of Hodgkin cases
HIF-1a
% cases with RS positive MVD/0.06 mm2
% cases with reactive cells positive
p53
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Table 2. Frequency of positive staining (scores 1 and 2) in the RS and reactive cell compartment of 65 Hodgkin biopsies according to histological classification and Ann Arbor stage
16
Response to treatment: A higher VEGF and p53 immunohistochemical score in reactive cells (2 versus 0–1) correlated with increased incidence of complete response (po0.03). Survival analysis did not show any variable statistically associated with survival. Serum angiogenic factors: VEGF receptor 2 negatively correlated with VEGF staining in RS cells (r: 0.44, p ¼ 0.05).
Correlation of VEGF and HIF-1a of RS cells with other angiogenic factors: VEGF in RS cells correlates with VEGF in reactive cells (r: 0.43, p ¼ 0.001), VEGF in RS correlates with PDGFRa in RS cells (r: 0.28, p ¼ 0.02), HIF-1a in RS cells strongly correlates with p53 in RS cells (r: 0.43, p ¼ 0.001), MVD correlates with Ki-67 staining in reactive cells (r 0.28, p ¼ 0.03).
Discussion In many solid tumors and some hematological malignancies, microvessel density and VEGF expression define the angiogenic state and risk of progression [25,34,51,64], however, this does not seem to be the case in Hodgkin lymphoma. The results from microvessel density measurements in Hodgkin cannot be easily interpreted. In a study comparing biopsies, at diagnosis and relapse, from 11 patients with Hodgkin lymphoma, the authors described increased MVD with disease progression [35]. On the other hand, a large immunohistological study, including 286 Hodgkin biopsies, found maximal MVD at initial stages of disease [31]. However, both studies did not use controls. We found that MVD in Hodgkin specimens
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did not differ from control lymph nodes, leading to the conclusion of a limited role of microvessel density in the progression of Hodgkin lymphoma. This conclusion is also supported by a study by Citak et al. [11] on childhood lymphoma where the degree of microvessel formation was not significantly related to patient outcome. The key molecule implicated in microvessel formation is VEGF [16]. VEGF induces endothelial cell proliferation and migration through multiple pathways [37]. In Hodgkin lymphoma, some investigators describe positive immunostaining of RS cells for VEGF [2,11,13,63]; however, two other studies could not demonstrate VEGF staining [19,35]. This may be explained by the difference in primary antibody and antigen retrieval method used in the studies. We confirmed the histological expression of VEGF in RS cells although at a lower level (48% in our series versus 70% [13]). However, it was interesting to observe that increased immunostaining of VEGF in the reactive cell population was found in the earlier stages of disease and in patients with complete response to treatment. This is in contrast to the bad prognosis described for Hodgkin patients with increased serum VEGF [21], which decreases following effective treatment [4,43]. In our series, tissue VEGF did not correlate with serum VEGF but correlated negatively with circulating VEGF receptor 2. VEGF R2 mediates the growth and permeability signal of VEGF [37], and is coexpressed with VEGF on RS cells [2]. Thus, combining the aforementioned seemingly paradoxical results, one could speculate that increased tissue VEGF expression in the early stages of Hodgkin lymphoma may mean restriction of angiogenic activity to the lymph node with low levels of circulating VEGF and VEGF R2. Circulating VEGF and VEGF R2 may increase at a later stage when extranodal expansion becomes a feature of the disease. Furthermore, the lack of correlation of VEGF expression with MVD (also observed by other investigators [13]) leads to the assumption that VEGF may play additional roles in vessel formation in Hodgkin. VEGF attracts monocytes and inhibits dendritic cell function mediating the immune response [12,20], which may be relevant in a disease where immune dysregulation is a prominent feature [23]. The main stimulus for VEGF expression is hypoxia through the HIF-1a pathway [10,48]. HIF1 is an oxygen-dependent transcriptional activator that plays a crucial role in the angiogenesis of tumors [32]. In prostate cancer, HIF-1a staining correlates with tissue VEGF [6]. In non-Hodgkin lymphoma (NHL), HIF-1a is expressed weakly in 70% of the cases [49]. To our knowledge, there has been no description of HIF-1a staining in Hodgkin samples. Our results were similar to those previously described for NHL [49], with diffuse weak expression of HIF-1a in RS cells and no
17
correlation of HIF-1a with the presence of necrotic areas. The absence of a correlation of HIF-1a with necrotic areas was unexpected given the positive correlation of increased MVD with necrosis in the same samples. Hodgkin lymphoma may utilize alternative hypoxic and angiogenic pathways compared to solid tumors and may be relatively independent of HIF-1a as far as angiogenesis is concerned. HIF-1a expression in RS cells may be serving an alternative role apart from response to the hypoxic stimulus; HIF also modulates apoptosis resistance [30], which is a proposed mechanism of Hodgkin cell survival [58]. VEGF expression is also induced by cytokines, such as PDGF, TNFa, IL-6, and oncogenes, such as p53. PDGF is a mitogen for connective tissue, especially fibroblasts during wound healing [3]. Additionally, the PDGF/PDGFRa system plays a role in the development of angiogenesis [5]. PDGFRa expression in Hodgkin samples has been reported in two studies [7,44]. We confirmed the strong PDGFRa staining in the majority of RS cells in a relatively large cohort of patients. This finding may have exciting therapeutic implications. Activating mutations in PDGFRa have been seen in gastrointestinal tumors, and inhibition of PDGFRa signaling by imatinib mesylate is exploited therapeutically for these tumors [26]. p53 plays a key role in the control of the cell cycle, cell differentiation, and programmed cell death. In NHL, VEGF expression correlates with p53 and Ki-67 expression [53]. p53 staining has been reported in RS of Hodgkin samples [15,39,52,61] at high levels in stage I cases [58] with good prognosis [62]. HIF1 and p53 both accumulate during hypoxia [46], which agrees with our finding of a positive correlation between HIF-1a and p53. Abele et al. [1] described an approximately 75% Ki-67 positivity in RS cells in 80 Hodgkin cases, which is in accordance with our results. We found a correlation of Ki-67 reactive cells with microvessel density, which could mean that new vessel formation in Hodgkin lymphoma may be serving proliferating cells of the reactive environment rather than the malignant cells themselves. In conclusion, we have described immunohistological expression of a panel of angiogenic factors in Hodgkin tissues which, in contrast to solid tumors, are not combined with increased microvessel density. From the findings of this study, it seems that the overexpression of angiogenic molecules in Hodgkin lymphoma may be serving roles other than vessel formation; i.e., it would be interesting to explore if VEGF is serving lymphangiogenesis in Hodgkin through its receptor VEGFR3 [16] or if the PDGF-PDGFRa system in Hodgkin is providing mitogenic, survival, or chemotactic signals to the neoplastic or reactive cells [3]. Although the results are not coupled with direct proof of angiogenic molecule production from the malignant Hodgkin cells, they
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provide strong evidence to pursue further study, especially regarding the role of PDGFRa in Hodgkin patients, given the potential therapeutic implications with PDGFa blocking agents.
[13]
Acknowledgments The authors would like to acknowledge the help of Dr. Spiros Miyakis in the statistical analysis of the results.
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