High Circulating Vascular Endothelial Growth Factor Receptor-1 Correlates with Increased Microvessel Density and Inferior Survival in Newly-Diagnosed Patients with Multiple Myeloma Who Receive Frontline Therapy with Novel Agent-Based Regimens

Abstract 5082 Vascular Endothelial Growth Factor Receptor-1 (VEGF-R1) is a receptor tyrosine kinase specific for the angiogenic factors VEGF-A, VEGF-B and placenta growth factor (PlGF), which is also a member of the VEGF family. In contrast to VEGF, the role of PlGF and VEGFR-1 in neovascularization is less clear. Angiogenesis is increased in patients with multiple myeloma (MM) and correlated with inferior outcome in several studies. The role of PlGF and VEGFR-1 has not been evaluated in MM. Therefore, we measured the circulating levels of PlGF and VEGFR-1: i) in 64 patients with newly diagnosed myeloma: 16 with asymptomatic disease (7M/9F; median age 59 years, range 37–82 years) and 48 with symptomatic myeloma (30M/18F; median age 70 years, range 45–89 years; ii) in 8 patients with MGUS (4M/4F; median age 72 years, range 39–84 years); and iii) in 20 healthy, gender and age-matched controls. PlGF and sVEGFR-1 were measured in serum samples using an electrochemiluminescence immunoassays (ECLIA) on a cobas e411 immunology analyzer (Roche Diagnostics, Mannheim, Germany). We also explored possible correlations between PlGF and VEGFR-1 circulating levels with clinical and laboratory values of the patients including microvessel density (MVD) of the bone marrow trephine biopsies and survival. Immunohistochemical identification of microvascular endothelial cells was performed in the trephine biopsies of MGUS and MM patients using a human monoclonal antibody against CD34 (DAKO A/S, Glostrup, Denmark). In each biopsy sample, microvessels were counted in at least 3 independent hot spots per section and the MVD of a bone marrow specimen was calculated as the mean value of all independent readings and recorded as the number of microvessels per ×400 field. When the microvessel count was 1–2, angiogenesis was characterized as low grade, while intermediate grade angiogenesis was defined by the presence of a microvessel count of 3–6 and high grade angiogenesis by the presence of microvessel count ≥7. Compared to controls, patients with symptomatic MM had elevated circulating PlGF (median: 19.5 pg/ml, range: 6.7–91.3 pg/ml vs. 16.1 pg/ml, 10.9–25.0 pg/ml of control group; p<0.01) and elevated VEGFR-1 levels (88.6 pg/ml, 51.5–320 pg/ml vs. 73.3 pg/ml, 62.9–100.8 pg/ml; p<0.001). Patients with MGUS and asymptomatic MM had no differences compared to controls for PlGF and VEGFR-1. In myeloma patients there was a strong correlation between circulating PlGF and VEGFR-1 levels (r=0.62, p=0.009 for asymptomatic patients and r=0.36, p=0.01 for symptomatic MM). PlGF also correlated with IL-6 (r=0.68, p<0.01) and high sensitivity CRP (r=0.5031, p<0.01) in MM patients. Of 16 patients with asymptomatic myeloma, 11 (68%) had low grade angiogenesis in the trephine biopsies and 5 (31%) intermediate grade angiogenesis; no one had high grade angiogenesis. There was no difference for PlGF levels between patients with low and intermediate grade angiogenesis in asymptomatic myeloma. However, patients with asymptomatic myeloma and intermediate grade angiogenesis had elevated VEGFR-1 (98.3 pg/ml, 87.1–148.9 pg/ml) compared to patients with low grade angiogenesis (72.3 pg/ml, 63.7–95.8 pg/ml). Similar results were obtained for patients with symptomatic myeloma. Of those, 18 (37%) had low grade angiogenesis in the trephine biopsies, while 20 (41%) had intermediate grade and 10 (20%) high grade angiogenesis. The median values and ranges of VEGFR-1 for low, intermediate and high grade angiogenesis were: 75.1 pg/ml (51.5–109.1 pg/ml), 94.2 pg/ml (61.2–143.8 pg/ml) and 151.8 pg/ml (103.7–320.0 pg/ml), respectively (p-ANOVA<0.0001). All patients with symptomatic myeloma received frontline therapy with novel agent-based regimens: 25 with lenalidomide-based regimens, 16 with thalidomide-based regimens and 7 with bortezomib-based regimens. The median follow-up period was 18.8 months and 8/47 patients have died. The probability of survival was 86% at 1 year and 78% at 2 years. In the univariate analysis the VEGFR-1 as a continuous variable correlated with higher risk of death (HR: 1.011, 95% CI: 1.004–1.018, p=0.003). In conclusion our study suggests that myeloma patients have increased circulating PlGF and VEGFR-1. High levels of VEGFR-1 correlated with increased angiogenesis and inferior survival, supporting a significant role of VEGFR-1 in the biology of angiogenesis in MM. Disclosures: No relevant conflicts of interest to declare.

Strong Correlation Between Angiogenesis and Macrophage Counts in Waldenstrom’s Macroglobulinemia: Implications into the Biology of the Disease

Angiogenesis represents an essential step of disease progression in several hematological malignancies. A Mayo Clinic study reported that microvessel density (MVD) was increased (intermediate- or high- grade angiogenesis) in 30% of patients with Waldenstrom s Macroglobulinemia (WM), showed only weak correlation with marrow infiltration and had no impact on patients’ survival [Rajkumar et al, Semin Oncol2003;30:262-4]. Macrophage inflammatory protein-1 alpha (MIP-1alpha) is a potent chemoattractant for macrophages, which contributes to increased angiogenesis in malignant diseases, including multiple myeloma. Our group has reported that serum levels of MIP-1alpha are elevated in WM. To further elucidate the role of angiogenesis in WM, we investigated the association between MVD, MIP-1alpha expression and the macrophage numbers in trephine biopsies of 34 patients with newly-diagnosed WM (3 with asymptomatic disease) and 3 with IgM-Monoclonal Gammopathy of Undetermined Significance (MGUS). Bone marrow biopsies were studied using double immunohistochemical staining for CD34 (endothelial cells) and CD68 (macrophages/mast cells) using antibodies from Becton Dickinson, San Jose, CA, USA & Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, respectively. We have also used double immunohistochemical staining for CD20/MIP-1alpha and for CD138/MIP-1alpha using an anti-MIP-1alpha antibody from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) to evaluate the MIP-1alpha expression by WM cells. Thirteen patients (35%) showed intermediate-grade and 4 (10%) high-grade angiogenesis. All patients with IgM-MGUS and asymptomatic WM had a very low microvessel count (median: 1, range: 1–2), while the median microvessel count for symptomatic WM was 4 (range: 1–8, p&lt;0.01). There was a strong correlation between the grade of angiogenesis (as assessed by the microvessel counts) and the number of macrophages into the “hot-spots” (r=0.823, p&lt;0.0001). Furthermore, statistically significant correlations were observed between the percentage of lymphoplasmacytoid cell infiltration of the bone marrow with microvessel counts (r=0.554, p=0.002) and macrophage numbers into the “hot spots” (r=0.457, p=0.011). WM patients with intermediate or high grade angiogenesis had increased IgM levels (p=0.007), lower hemoglobin levels (p=0.024), and reduced platelet counts (p=0.043), while patients with high-grade angiogenesis had a tendency to higher incidence of lymphadenopathy compared with all others (3/4, 75% vs. 9/33, 27%; p=0.054). There was no correlation between angiogenesis and survival. We have also observed that WM cells of all patients produced MIP-1alpha. CD138 positive WM cells had higher expression of MIP-1alpha compared to CD20 positive WM cells (p=0.001). Patients with increased numbers of CD68 positive macrophages had increased expression of MIP-1alpha by their WM cells (r=0.732, p&lt;0.001). The results of our on going study suggest that WM cells produce MIP-1alpha to attract macrophages in their bone marrow microenvironment. These macrophages seem to play a significant role in the angiogenesis process and are possibly implicated into the biology of WM.

Dickkopf-3 As a New Potential Marker for Neoangiogenesis in Colorectal Cancer: Expression in Cancer Tissue and Adjacent Non-Cancerous Tissue

Gene expression of Dickkopf-3 (Dkk-3) has been shown to be upregulated in tumor endothelium of colorectal cancer (CRC). For the first time, we analyzed Dkk-3 protein expression in CRC and its potential as a marker for neoangiogenesis. We used tissue microarrays (TMAs) to investigate Dkk-3 in microvessels of 403 CRC samples, 318 appropriate adjacent non-cancerous samples and 127 normal colorectal samples. Of cancer samples with CD31-positive microvessels, 67.7% were positive for Dkk-3. Dkk-3 staining was demonstrated in endothelial cells of all microvessels in nearly all cases. Dkk-3-positive samples showed a higher mean microvessel count than did Dkk-3-negative samples (P=0.001). Dkk-3 expression increased with rising numbers of microvessels per sample (P<0.0001). In adjacent samples with CD31-positive microvessels, 56% were Dkk-3-positive in all microvessels. Similar to cancer samples, Dkk-3-positive adjacent samples had a higher mean microvessel count than did Dkk-3-negative samples (P<0.0001), and Dkk-3 expression also increased with rising numbers of microvessels (P<0.0001). All microvessels in normal mucosa samples were negative for Dkk-3.Dkk-3 can be considered a putative pro-angiogenic protein in neovascularization and may possibly be a marker for neoangiogenesis in CRC. Further investigations will elucidate whether Dkk-3 is a target structure for novel therapies.

Angiogenesis in squamous precancerous cervical lesions

Background/Aim. Vascularisation is one of basic tumor's characteristics. Neoangiogenesis starts at the stage of the dysplastic epithelial changes, thus before the progression into invasive lesion. This study was designed to determine the relation between stromal angiogenesis and the grade of cervical intraepithelial changes (CIN). Methods. The tissue sections of 50 cone biopsies were immunohistochemically stained for CD31 antigen, a marker for endothelial cells. All microvessels along the basement membrane subtending dysplastic epithelium were counted. The mean microvessel count was calculated from the three separate fields for each specimen. All the cases were devided into four groups: normal cervical epithelium (n = 5), CIN1 (n = 7), CIN2 (n = 13), and CIN3 (n = 25). Results. The mean microvessel count (MVC) under the dysplastic epithelium was 18.1. For the patients with CIN1 changes the mean MVC was 12.9, while this number was 18.72 and 19.24 for the patients with CIN2 and CIN3 grade of epithelial changes, respectively. In a subset of the high grade lesions vascular structures were also noted in the upper layer of the epithelium. The mean MVC in the cases with the presence of these structures was 22, while this number was 12.91 in the cases without intraepithelial vessels. Although we found an increase of the mean MVC with the increase of the CIN grade, statistically significant differences were found out between CIN1 and CIN3 lesions. The mean MVC of the patients with the presence of intraepithelial vessels was statistically higher than the mean MVC of the patients without these structures. Conclusion. On the basis of the obtained results, we can conclude that the mean MVC and CIN grade positively correlated, while the number of cases with intraepithelial vessels increased with the CIN grade.