Original Article | Published:

Lymphoma

Increased bone marrow microvascular density in haematological malignancies is associated with differential regulation of angiogenic factors

Leukemia volume 23, pages 162169 (2009) | Download Citation

Subjects

Abstract

Antiangiogenic drugs are currently tested in haematological malignancies. As these drugs target different angiogenic regulators, and as cancers are inherently heterogeneous, a detailed characterization of angiogenesis in individual cancers is needed. Hence, we measured bone marrow microvessel density (MVD), plasma concentrations of eight angiogenesis-related parameters and the expression in blood mononuclear cells of 40 angiogenesis-related mRNAs in 93 patients with haematological neoplasias (acute myeloid leukaemia; chronic lymphatic leukaemia; multiple myeloma (MM); or non-Hodgkin's lymphoma (NHL)) before start and after completion of cancer therapy. Compared with healthy individuals, the patients had significantly increased bone marrow MVD, especially patients with advanced stage disease. A novel finding was that patients with NHL also had increased bone marrow MVD. The plasma levels of vascular endothelial growth factor (VEGF), interleukin (IL)-6 and IL-8 were significantly increased. VEGF levels were highest in those who did not achieve complete remission after cancer therapy. The mRNA expression of IL-8 was upregulated 15-fold. Our data show that patients with haematological malignancies have increased bone marrow MVD; hence, supporting the notion that bone marrow angiogenesis plays a role in the pathogenesis and progression of these cancers. VEGF, IL-6 and IL-8 seem to contribute to the malignant phenotype.

Introduction

Emerging evidence suggests that bone marrow microvascular density (MVD) is increased in leukaemia and multiple myeloma (MM),1, 2, 3 and that this may have prognostic value.3, 4 However, whether the bone marrow MVD in non-Hodgkin's lymphoma (NHL) is increased compared with that of healthy individuals, has not been clarified.

Although current understanding of the complex regulation of angiogenesis emphasizes the importance of vascular endothelial growth factor (VEGF), several other pro-angiogenic factors may be involved—including fibroblast growth factor 2 (FGF2; or basic FGF), interleukin (IL)-6, IL-8, tumour necrosis factor-α (TNF-α), angiogenin and tissue factor (TF)5, 6—in addition to antiangiogenic factors, such as TF pathway inhibitor type 1 (TFPI-1).7

Despite advances in treatment, chronic lymphatic leukaemia (CLL), MM and indolent subtypes of NHL are still regarded to be incurable diseases, and only half of the patients with acute myeloid leukaemia (AML) and aggressive subtypes of NHL are alive after 5 years.8 Given the role of angiogenesis in haematological neoplasias, several angiogenesis-inhibitors have been tested in clinical trials, but many have not lived up to expectations.9 As these drugs have different targets, and as cancers are heterogeneous, a detailed characterization of angiogenesis in separate cancer diagnoses is needed.

The purpose of this prospective cohort study was to determine the bone marrow MVD in unselected patients with various haematological malignancies. In particular, we wanted to investigate whether MVD was increased, especially in NHL, and if MVD was associated with disease stage and remission status. To further characterize the angiogenic process in haematological malignancies, we measured plasma concentrations of eight angiogenesis-related markers, with regard to diagnosis, stage and remission, as well as longitudinally during treatment. Finally, as various blood cells secrete pro-angiogenic molecules,10 we also studied the mRNA expression of angiogenesis-related genes in peripheral blood mononuclear cells.

Materials and methods

Patients and controls

The patient material and study design have previously been described in detail.11 Briefly, we studied 93 patients (40 females, 53 males; median age 62 years, range 28–89 years) with AML (n=20), CLL (n=14), MM (n=11) and NHL (n=48), included in the ‘Angiogenesis and Haemostasis in Haematological Neoplasia’ study. Patients were newly diagnosed, apart from 10 patients with relapsed NHL. The malignancies were diagnosed according to internationally accepted criteria.12, 13, 14 From 7 December 2004 to 26 January 2006, patients were prospectively and consecutively recruited from Ullevål and Rikshospitalet University Hospitals, Oslo, Norway. Patients were excluded from the study in case of pregnancy, previous or current other form of cancer (except squamous cell carcinoma of the skin and stage 0 cervical cancer), the promyelocytic subtype of AML, incapability to consent, other bone marrow diseases, HIV, other uncontrolled infectious diseases, use of immunomodulatory drugs or having received chemotherapy within the previous 3 months.

Staging of patients were according to internationally accepted classifications.14, 15, 16 The following were considered as advanced stage disease: Binet stage C CLL, stage 3 MM and Ann Arbor stage IV NHL. Remission status was evaluated at completion of cancer therapy according to conventional criteria.14, 17, 18, 19 After cancer treatment, 44 of the 93 patients (47%) obtained complete remission (CR). For clinical features of consecutive patients with NHL, see Supplementary Table 1.

Archival bone marrow specimens from 16 patients were used as controls for the bone marrow MVD estimations. None of the patients had previous cancer, and neither were any diagnosed with cancer after the bone marrow biopsy (minimum follow-up: 1 year). The controls consisted of nine females and seven males; with median age of 54 years, range 32–90 years. As reference for the plasma levels and mRNA expressions, we used samples from 11 healthy individuals (ten males and one female; median age 23 years, range 21–29 years). All control individuals were subjected to the same exclusion criteria as the patients.

The protocol was approved by the Norwegian Regional Committee for Research Ethics in Health Region East. Written informed consent was obtained before inclusion.

Bone marrow microvessel staining

Bone marrow biopsy specimens were fixed, decalcified with EDTA and embedded in paraffin. Pretreatment bone marrow biopsies were evaluated in 72 of the patients (77%). The CD34 antigen specific for endothelial cells,20 was used as a marker of vascular endothelial cells. Immunohistochemical staining for CD34 was performed by a two-step polymer-based method run on a Dako Autostainer (Dako, Glostrup, Denmark). Specimens were deparaffinized in xylene and rehydrated in serially graded ethanol. Tissue sections were then pretreated in a microwave oven with a tris/EDTA solution, pH 9.1, for 24 min, followed by a 30 min cooling. Tissue sections were incubated with a 1:50 dilution of the primary monoclonal anti-CD34 (Monoclonal Mouse Anti-Human CD34 Class II Clone QBEnd 10, Dako) for 30 min at room temperature. The EnVision+ System-HRP detection kit (Dako) was used for antigen visualization. Sections were counterstained with haematoxylin. On each slide, paraffin sections of tonsils as positive control, and healthy bone marrow as normal control, were run.

Estimation of MVD

All counting was done in a blinded manner, by the simultaneous assessment of two investigators using a double-headed light microscope (Ernst Leitz, Wetzlar, Germany), as described.20 Briefly, slides were scanned at × 100 magnification to determine three ‘hot spots’ defined as areas with the maximum number of microvessels. At × 400 magnification, the field was set to cover the maximum number of microvessels within the hot spot, and microvessels were counted in one field (1.13 mm2) in each of the three hot spots. Both investigators agreed on what constituted a single microvessel before any vessel was included in the count. Microvessels were defined as endothelial cells, either single or clustered in nests or tubes, clearly separated from adjacent microvessels, with or without a lumen. Great care was taken to omit the counting of stained non-endothelial cells such as myeloid blasts. In Figure 1a stained bone marrow biopsy from a patient is shown; arrows point at examples of stained myeloid blasts that were not counted. We were unable to measure MVD in seven biopsies from patients with AML due to large numbers of stained myeloid blasts. Large vessels or vessels in the periosteum or bone were excluded. MVD is reported as the median of the three hot spots and expressed as the number of microvessels per mm2.

Figure 1
Figure 1

Bone marrow biopsy stained for CD34 (brown) for microvessel density (MVD) counting. Magnification at × 400. Patients with non-Hodgkin's lymphoma (NHL) with (a) diffuse large B cell subtype, stage II; (b) follicular lymphoma subtype, stage IV. Myeloid blasts that stained for CD34 (arrows point at examples) were not counted as microvessels.

Blood sampling and processing

Blood samples were collected prior to and after end of cancer therapy, and stored at −70 °C until assayed.

Peripheral blood mononuclear cells (PBMNC) were extracted from EDTA whole blood samples by density gradient centrifugation (Lymphoprep, Axis-Shield, Oslo, Norway) within 2 h of sampling. They were washed with 0.9% NaCl, lysed with lysis/binding buffer (MagNA Pure LC RNA Isolation kit, Roche Applied Sciences, Penzberg, Germany) and stored at −70 °C. Later, ribonucleic acid (RNA) was extracted with MagNA Pure LC Instrument (Roche Applied Sciences) using the MagNA Pure LC RNA Isolation kit (Roche Applied Sciences). The amount of RNA was quantified using the NanoDrop Spectrophotometre (NanoDrop Technologies, Wilmington, DE, USA).

All assays were performed examiner-blind. Samples were run in batch by the end of the study with both samples for the same individual in each run.

Plasma assays

VEGF, IL-6, IL-8, FGF2 and TNF-α were assayed in EDTA plasma using the Luminex Multiplex assay with Bio-Plex Cytokine assay reagents (Bio-Rad Laboratories, Hercules, CA, USA) on a Bio-Plex Luminex xMAP (Bio-Rad Laboratories) as described by the manufacturer. Angiogenin was assayed in EDTA plasma with Human Angiogenin Flex Set reagents (BD Biosciences, San Diego, CA, USA) on a BD FACSArray Bioanalyzer (BD Biosciences). In citrated plasma TF antigen was assayed with the Imubind Tissue Factor ELISA kit (American Diagnostica, Stamford, CT, USA), TFPI-1 free and total antigen with Asserachrom Free TFPI and Asserachrom Total TFPI (Diagnostica Stago, Asnière, France) and TFPI-1 activity with an in-house method.21

In 89 (96%) patients all plasma results were available. Interassay coefficients of variation for individual assays were: VEGF 5.3%, IL-6 16%, IL-8 9.2%, FGF2 12%, TNF-α 12%, angiogenin 13%, TF 9.9%, total TFPI-1 antigen 2.3%, free TFPI-1 antigen 9.7% and TFPI-1 activity 3.5%.

Gene expression analyses

Equal amounts (750 ng) of RNA from PBMNC were reversely transcribed into cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). Equal amounts (150 ng) of cDNA from each sample were run with TaqMan Low Density Arrays (TLDA; Applied Biosystems) on an ABI PRISM 7900 HT Sequence Detection System RT-PCR (Applied Biosystems), according to the manufacturer's instructions.

After scrutinizing relevant reports, we chose to quantify 40 genes because of their possible influence on the angiogenic process. The relative quantification (RQ) of mRNA expressions were determined by employing the Comparative cycle threshold (Ct) method,22 calculated using RQ Manager 1.2 (Applied Biosystems) with a common threshold setting for each gene target. The Ribosomal Protein Large P0 (RPLP0) gene was selected as endogenous control as it showed the least scattering in Ct values when running cDNA from the 11 healthy controls and 21 randomly selected patients on a TLDA endogenous control plate (Applied Biosystems). All targets were normalized to the RPLP0 gene, to calculate the ΔCt. The normalized results were compared with the normalized results of a calibrator (RNA from the subject among the healthy controls with median value of RPLP0 expression) yielding the ΔΔCtCt (sample) −ΔCt (calibrator).

RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) with reagents from the RNA 6000 Nano LabChip kit (Agilent Technologies), calculating the RNA integrity number (RIN). Data were excluded if ΔRn was (fluorescence intensity minus background fluorescence) 1.35, if the amplification curve was non-optimal, or if both the RNA integrity number were <4.0 and the Ct>40. Consequently mRNA expression results were available in 81 (87%) patients. In 43 (46%) patients all 40 gene target expressions were available and in 12 (13%) patients 39 gene target expressions were available.

Statistical analyses

Variables that had normal distribution were reported as means and standard deviations. Medians and interquartile intervals were reported for the following variables whose distribution deviated markedly from the normal distribution: plasma levels of TNF-α, IL-6 and IL-8; and mRNA expression of angiogenic growth factor with G patch and FHA domains 1 (AGGF1), angiopoietin (ANGPT) 1 and 2, interferon-α (IFNA1), IL-6, platelet-derived growth factor (PDGF) A and B, placental growth factor (PGF) and TNF α-induced protein 2 (TNFAIP2). Comparisons between independent groups were performed with the t-test or the Mann–Whitney test, as appropriate. Differences between diagnostic groups were evaluated using one-way ANOVA or the Kruskal–Wallis test. Comparisons between values obtained before and after cancer therapy were evaluated with the paired samples t-test or Wilcoxon signed rank test. Correlation between continuous variables was studied with Pearson's correlation (r) or Spearman's rank correlation (rs). All tests were two-tailed and a 5% significance level was applied except for the correlation analyses in which a 1% significance level was applied. Statistical analyses were performed using the Statistical Package for the Social Sciences 14.02 (SPSS Inc., Chicago, IL, USA).

Results

Increased MVD and pro-angiogenic markers before cancer therapy

Before start of cancer therapy, patients had significantly higher bone marrow MVD than controls (Table 1, Supplementary Table 2 for separate haematological malignancies). In addition, they had significantly increased plasma levels of VEGF, IL-6, IL-8, as well as elevated levels of both free TFPI-1 antigen and TFPI-1 activity (Table 1). On the other hand, we found no significant differences in plasma levels of FGF2, TNF-α, angiogenin and TF between patients and controls (Table 1, Supplementary Table 2 for separate haematological malignancies). The mRNA expression of IL-8 was upregulated 15-fold, whereas the following mRNAs were downregulated fourfold or more: notch homologue 4 (NOTCH4), VEGFC, ANGPT2, chemokine ligand 2 (CCL2), TFPI2 and TFPI1 (Figure 2, Supplementary Figure 1 for separate haematological malignancies). We have confined the presented data of mRNA expression to these most up/downregulated genes.

Table 1: Bone marrow MVD and plasma levels of angiogenic markers before cancer therapy
Figure 2
Figure 2

mRNA expression of angiogenesis-related genes in peripheral blood mononuclear cells (PBMNC) from patients with haematological malignancies (n=93) before cancer therapy. Variables having normal distribution are presented as mean (square) and 95% confidence interval (CI), whereas variables not having normal distribution are presented as median (circle) and 95% CI. All targets were normalized to the endogenous gene ribosomal protein large P0, and compared with mRNA from PBMNC from a healthy individual. The results were calculated using the Comparative Ct method, generating relative quantitation (RQ) data, which are presented on a log2 scale. Data are sorted according to RQ. AGGF, angiogenic factor with G patch and FHA domains; AKT, v-akt murine thymoma viral oncogene homologue; ANGPT, angiopoietin; ANGPTL4, ANGPT-like 4; ANG, angiogenin; CCL, chemokine C-Cmotif ligand; EPAS, endothelial PAS domain protein; EPH, ephrin receptor; FGF, fibroblast growth factor; FIGF, c-fos induced growth factor (VEGFD); HIF, hypoxia-inducible factor; IFN, interferon; IL, interleukin; JAG, jagged; NOTCH, notch homologue; PDGF, platelet-derived growth factor; PGF, placental growth factor; SERPIN, serpin peptidase inhibitor; SH2D2A, SH2 domain protein 2A; TF, tissue factor; TFPI, TF pathway inhibitor; TGF, transforming growth factor; TNF, tumour necrosis factor; TNFAIP, TNF α-induced protein; TNFSF15, TNF superfamily member 15; TP53, tumour protein p53; TYMP, thymidine phosphorylase (or endothelial cell growth factor); VEGF, vascular endothelial growth factor.

Owing to the apparent lack of reported data on bone marrow MVD measurements in NHL, we were specifically interested in bone marrow MVD in NHL patients. Before cancer therapy, these patients had higher MVD (mean 38 microvessels per mm2) than healthy controls (mean 20 microvessels per mm2, P<0.001).

Angiogenic markers before cancer therapy according to diagnosis

Haematological malignancies represent distinct diseases with specific characteristics. We found no significant statistical differences in bone marrow MVD between the diagnoses or in the plasma levels of VEGF (Figures 3a and b). There were significant differences between the diagnoses in plasma levels of FGF2, TNF-α, IL-6 and IL-8. Patients with CLL had the highest plasma levels of FGF2, whereas patients with NHL had the highest plasma levels of TNF-α, and patients with AML had the highest plasma levels of IL-6 and IL-8 (Figures 3c–f), and lowest plasma levels of total TFPI-1 (mean 72 ng/ml for AML, 87 ng/ml for CLL, 82 ng/ml for MM and 88 ng/ml for NHL; P=0.009). In contrast to the non-significant differences in bone marrow MVD between the diagnoses; 32 of the 40 mRNA expressions measured, were statistically significantly different among the diagnoses (data not shown).

Figure 3
Figure 3

MVD and plasma levels of angiogenic markers before cancer therapy. Comparison among the four haematological malignancies (number given in parenthesis under each group); controls as reference. Variables having normal distribution (MVD, VEGF and FGF2) are presented as means and standard deviations, and variables not having normal distribution (TNF-α, IL-6 and IL-8) are presented as medians and interquartile intervals. P-values: (a) MVD, P=0.81; (b) VEGF, P=0.798; (c) FGF2, P=0.001; (d) TNF-α, P=0.040: (e) IL-6, P=0.041; (f) IL-8, P=0.012. Significant results marked with asterix (*). AML, acute myeloid leukaemia; CLL, chronic lymphatic leukaemia; MM, multiple myeloma; NHL, non-Hodgkin's lymphoma; MVD, microvessel density; VEGF, vascular endothelial growth factor; FGF2, fibroblast growth factor 2; TNF-α, tumour necrosis factor-α; IL, interleukin.

Angiogenic markers before cancer therapy according to disease stage

Patients with advanced stage disease had higher bone marrow MVD than patients with lower stage disease (mean 45 vs 35 microvessels per mm2, P=0.030). An example illustrating this is shown in Figure 1. Advanced stage disease patients had higher plasma levels of IL-6 (median 7.95 vs 3.44 pg/ml; P=0.004) and IL-8 (median 7.58 vs 6.46 pg/ml; P=0.038) before treatment, whereas there were no statistical differences according to stage for the other plasma levels measured (data not shown). Patients with advanced stage disease had lower mRNA expression of CCL2 than those with low stage disease (mean 9-fold downregulated vs 2-fold downregulated; P=0.035).

Angiogenic markers before cancer therapy according to remission status

We found no differences in pretreatment bone marrow MVD between patients who did not obtain CR compared with those who did (mean 39 vs 38 microvessels per mm2). Patients who did not obtain CR after cancer therapy had significantly higher pretreatment plasma levels of VEGF (mean 35.11 vs 27.07 pg/ml; P=0.038) and FGF2 (mean 18.76 vs 10.71 pg/ml; P=0.008) than patients who did obtain CR, whereas the other plasma levels were not significantly different (data not shown). For results in the separate haematological malignancies, see Supplementary Table 3. Additionally, non-CR patients had significantly lower pretreatment mRNA expression of IL-8 (mean 7-fold vs 30-fold upregulated; P=0.012) and NOTCH4 (mean 47-fold vs 10-fold downregulated; P=0.029).

Correlation of angiogenic markers before cancer therapy

In 79 (85%) patients, results were available from two or more of the parameters, namely bone marrow MVD, all plasma angiogenesis factors or all mRNA angiogenesis factors. Bone marrow MVD was not significantly correlated to any of the angiogenic markers measured in plasma, nor mRNA expressions (data not shown). There were no statistically significant correlations between plasma levels of angiogenic markers and their respective mRNA expressions in PBMNC (data not shown). There were, however, strong correlations between VEGFA mRNA expression and both hypoxia-inducible factor 1α (HIF1A) and endothelial PAS domain protein 1 (EPAS1, or HIF2A) mRNA expressions (r=0.7; P<0.001 for both).

Changes in angiogenic markers during cancer treatment

We next compared the levels of angiogenic markers before treatment with those measured after completion of treatment. There was an increase in plasma levels of total TFPI-1 antigen (mean 85.8 ng/ml before cancer therapy to mean 105.9 ng/ml after cancer therapy; P=0.014) and TFPI-1 activity (mean 124.6% before cancer therapy to mean 140.2% after cancer therapy; P<0.001). The other plasma levels did not change significantly during cancer treatment (data not shown). CCL2 was downregulated fourfold before cancer therapy, whereas after cancer therapy it was upregulated 1.2-fold (P<0.001). In addition, the mRNA expression of VEGFC increased during treatment from six- to threefold downregulation (P=0.003).

Angiogenic markers after cancer therapy

After cancer treatment, patients had higher plasma levels of IL-8 (median 7.12 pg/ml for patients vs 3.94 pg/ml for controls; P=0.001) and TFPI-1 activity (mean 140.2% for patients vs 110.6% for controls; P<0.001), whereas the remaining plasma levels were not significantly different from controls (data not shown). The relative mRNA expression of IL-8 was strongly upregulated (mean 20-fold) also after cancer therapy, and PGF with median 18-fold was upregulated. The most downregulated mRNA after cancer therapy was IL-6 (median 9-fold).

The patients who did not obtain CR had significantly higher plasma levels than those who did obtain CR of IL-6 (median 6.56 vs 3.50 pg/ml; P=0.016) and IL-8 (median 9.02 vs 6.44 pg/ml; P<0.001). CCL2 was downregulated in non-CR patients (mean twofold) and upregulated in CR patients (mean twofold; P=0.021). In addition, non-CR patients had significantly lower expression of NOTCH4 (mean 19-fold vs 2-fold downregulated; P=0.002).

Discussion

In this study, we have demonstrated that patients with haematological malignancies had higher pretreatment bone marrow MVD than controls, and elevated pretreatment plasma levels of VEGF, IL-6 and IL-8. IL-8 was the most upregulated gene in PBMNC. There were no differences in bone marrow MVD between the haematological malignant diseases, but the bone marrow microvascularity seems to be differentially regulated in the various malignancies studied as we found significant differences between the diagnoses for the majority of angiogenesis-related markers studied.

The strengths of this study are that we investigated consecutive, unselected patients with various haematological malignancies longitudinally, and with several surrogate markers of angiogenesis. Evaluation of MVD is widely used in studies of angiogenesis. Its major limitation is that it usually involves a subjective selection of areas for microvessel enumeration and vessel evaluation.23 Although computerized MVD evaluation has been developed, several limitations are also associated with this technology.23 The grouping of different diagnoses might lead to the loss of important characteristics of separate diseases; and the separate sample sizes were limited as were the control groups. In addition, PBMNC contain different cell types, and the composition varies with the haematological malignancy according to whether malignant cells are present in peripheral blood. This limits the comparison between different diagnoses. There is increasing evidence for the role of the microenvironment in pathogenesis of haematological malignancies. For instance, in vitro data on AML suggest that osteoblasts release IL-8 thus enhancing angiogenesis.24 We were not able to investigate the role of the influence of the microenvironment on angiogenesis in this study. The lack of correlation between plasma levels and mRNA expression was somewhat expected, because of differences between mRNA and protein, and different sources (plasma vs PBMNC).

We found that patients had higher bone marrow MVD than controls, especially patients with advanced stage disease. Bone marrow MVD has, to our knowledge, not been reported for NHL, whereas increased bone marrow MVD previously has been demonstrated in patients with AML,1 CLL,3 and MM.25 Higher bone marrow MVD in patients with advanced stage disease has been demonstrated previously in patients with MM.2 We did not find any association between bone marrow MVD and CR, in accordance with some MM studies;26 but in contrast to other studies on MM,4 and CLL.3

Vascular endothelial growth factor is regarded as the most important pro-angiogenic factor in cancer.5 In this study, patients had higher pretreatment plasma levels of VEGF than controls, with highest levels among those who did not achieve CR. However, elevated pretreatment levels of VEGF does not directly imply that anti-VEGF drugs are effective, as pretreatment plasma levels of VEGF did not correlate with response to the addition of the anti-VEGF monoclonal antibody bevacizumab in metastatic colorectal cancer or advanced pancreatic cancer.27 We measured VEGF levels in plasma as 80–90% of the VEGF levels measured in serum originate from ex vivo activated platelets.28 Increased levels of circulating VEGF have been demonstrated previously in patients with the haematological malignancies in this study.29, 30, 31, 32 Higher pretreatment plasma levels of VEGF in patients who did not obtain CR has been demonstrated previously in AML29 and NHL.32, 33

Interleukin-6 is another activator of angiogenesis5 and an important growth factor for myeloma cells. IL-8 is a pro-angiogenic chemokine, which serves as a growth factor in several tumour models.5 Levels of IL-6 and IL-8 were elevated in plasma, with highest levels in patients with advanced stage disease. IL-6 and IL-8 plasma levels were higher among non-CR patients than CR patients after cancer therapy. We found IL-8 to be the most upregulated gene. IL-6 levels in plasma have previously been shown to be elevated in patients with NHL32 and CLL.34 Furthermore, elevated IL-6 plasma levels are apparently associated to more advanced CLL stage35 as well as to poor outcome in both NHL32 and CLL.34, 35 In contrast, plasma IL-6 reportedly did not correlate with treatment response in patients with MM,36 and no significant differences were found according to MM stage.37 Plasma IL-8 has been reported to be increased in MM,38 as well as increased in advanced stage CLL and associated to poorer survival.39

FGF2 is a heparin-binding growth factor with pro-angiogenic properties.5 In this study the plasma level of FGF2 was higher before cancer therapy in patients who did not obtain CR, which is in line with previous studies reporting an association between FGF2 levels and poor prognosis in NHL.33 TNF-α is a multifunctional pro-inflammatory cytokine that can induce production of pro-angiogenic factors,5 whereas angiogenin is known to promote endothelial cell invasiveness.5 However, in this study plasma levels of TNF-α and angiogenin were not systematically altered, neither according to disease stage nor remission status.

Tissue factor is the main trigger of blood coagulation in vivo, and several reports have found that TF also exerts pro-angiogenic effects.6 In somewhat contrast to this we here found that TF was not significantly altered in the haematological malignancies under investigation. Moreover, we have recently reported that TF was apparently not responsible for the hypercoagulable state observed in this cohort of patients.11 The antiangiogenic molecule TFPI-1 inhibits endothelial cell proliferation.7 We found increased free TFPI-1 antigen and TFPI-1 activity before cancer therapy, and increased TFPI-1 activity after cancer therapy. Despite a shift in the balance between TF and its inhibitor TFPI-1 towards an antiangiogenic phenotype, we found increased angiogenesis in bone marrows.

Hypoxia is reportedly an important inducer of increased VEGF expression in cancer, and is mediated by the oxygen-sensitive transcription factors HIF1A and EPAS1.5 The role of hypoxia as inducer of VEGF and angiogenesis in haematological malignancies is less obvious than in solid tumours, as the malignant cells are dispersed in blood and bone marrow. Here we demonstrated a strong correlation between VEGFA mRNA and both HIF1A and EPAS1 expressions, thus corroborating findings in adult T-cell lymphoma.40

The chemokine CCL2 induces endothelial cell migration and increases the expression of pro-angiogenic factors such as VEGF and IL-8.41 However, the effect of CCL2 on neoplastic cells seems to depend on expression levels in the tumour environment: low expression promotes tumour growth and high expression promotes antitumour activities.41 We found a downregulated CCL2 expression, especially in patients with advanced stage disease, and after cancer therapy in those failing to obtain CR. Thus, low expression with subsequent promotion of tumour growth seems to have prevailed in these malignancies. Earlier studies in lymph node biopsies of NHL have shown that mRNA expression of CCL2 was as infrequent as in reactive lymphoid hyperplasia.42

NOTCH4 functions as a receptor that regulates cell-fate determination, and may regulate branching morphology in the developing vascular system.43 We found NOTCH4 to be the most downregulated gene, both before and after cancer therapy, especially in patients who did not obtain CR.

An apparent novel finding in this study was the increased bone marrow MVD in NHL. Also of note, we consistently found elevated bone marrow MVD among CLL patients. Of the circulating molecules investigated, VEGF, IL-6 and IL-8 seem to be of greatest importance to the haematological malignancies studied. The lack of correlation between bone marrow MVD and angiogenic markers in plasma or their mRNA expression can be explained by angiogenesis being the result of a complex network of cytokines, and that distinct subsets of haematological malignancies differ in their response to cytokines.44 Our results suggest that angiogenesis is differentially regulated by different angiogenesis-related factors in the various haematological malignancies, which has been reported earlier in acute lymphatic leukaemia and CLL.45 Further investigations into the complex regulation of angiogenesis in haematological malignancies are needed.

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Acknowledgements

The study was financially supported with grants from the Norwegian South-Eastern Health Authority Trust (fellowship for HFSN), Ullevål University Hospital Trust and the University of Oslo, Norway.

Author information

Affiliations

  1. Department of Haematology, Ullevål University Hospital Trust, Oslo, Norway

    • H F S Negaard
    • , P M Sandset
    • , B Steinsvik
    •  & P O Iversen
  2. Faculty of Medicine, University of Oslo, Oslo, Norway

    • H F S Negaard
    • , P M Sandset
    •  & P O Iversen
  3. Department of Medical Genetics, Ullevål University Hospital Trust, Oslo, Norway

    • N Iversen
  4. Department of Pathology, Ullevål University Hospital Trust, Oslo, Norway

    • I M Bowitz-Lothe
  5. Cancer Centre, Ullevål University Hospital Trust, Oslo, Norway

    • B Østenstad

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Corresponding author

Correspondence to H F S Negaard.

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DOI

https://doi.org/10.1038/leu.2008.255

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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