Salvage Treatment with Lenalidomide and Dexamethasone In Patients with Relapsed Refractory Mantle Cell Lymphoma: Clinical Results and Modifications of Angiogenic Biomarkers

Abstract 966 Backgrounds Previous in vivo and in vitro studies have highlighted the activity of Lenalidomide (Len) in patients with relapsed or refractory mantle cell lymphoma (MCL), achieving a 53% overall response rate (ORR), which included 20% complete responses (CR) (Habermann et al. Br J Haematol. 2009) and the possible synergistic anti-proliferative effect of Dexamethasone (Dex). Purpose: On this basis we performed a prospective, multicenter, phase II study, to evaluate safety and efficacy of Len Dex combination for adult patients with MCL. Patients and Methods Patients had to have ≥1 prior treatment regimen, and were either not eligible for, or had relapsed after, more intensive treatments including stem cell transplantation (SCT). During the induction phase (month 1 to 3), patients received Len 25 mg/day on days 1 to 21 and Dex 40 mg/day on days 1, 8, 15, 22 of a 28-day cycle (Len Dex). Enoxaparin 4000 U/day was administered as anti-thrombotic prophylaxis. Patients who achieved a partial response (PR) or stable disease (SD) at the end of the induction phase continued to the consolidation phase, which consisted of treatment with Len Dex until disease progression or unacceptable toxicity, for a maximum of 12 months. Patients with a CR at the end of the induction phase, or those who achieved a CR during consolidation, received an additional 3 courses of Len Dex. The primary objective was to evaluate the ORR and CRR. Response data were correlated with the modification of angiogenic biomarkers by analyzing immunohistochemistry macrophage infiltration and the bone marrow (BM) microvessel counts, and by measuring plasma levels of VEGF, bFGF and HGF before and after therapy. Results Between July 2008 and July 2009, 33 patients were enrolled on this study. Patients' median age is 68 years (range 51–80); 30 have the classic histology while 3 patients have the blastoid variant; 10 patients previously received two lines of therapy, 10 patients had three lines and 13 patients had >3 prior lines (median 3; range 1–7). Twelve patients previously underwent an autologous SCT and 8 received prior therapy with Bortezomib. The number of patients who responded to induction phase was: OR= 22 (67%) including 5 CR (15%), SD= 1 (3%), no response (NR) or progression (PD)= 10 (30%); OR in patients previously treated with Bortezomib or an autologous SCT was 50% each. At present, 13 patients completed the therapeutic program, 15 discontinued therapy prematurely (14 NR or PD, 1 poor compliance) and 5 remain on therapy. The final response status at the end of the therapeutic program is: OR= 18 patients (55%) including 8 CR (24%), NR/PD= 15 (45%). After a median follow-up of 6 months (range 3–13 months) from the end of therapy, none of the CR patients had subsequent progression while 2 PR patients had progression 7 and 10 months after the end of therapy. The macrophage counts increased significantly in the BM after the first three months of therapy (P < 0.01; r2=0,8927). This was parallel to a significant increase in the microvessel counts (P < 0.05, r2= 0,1547). The within-group comparisons showed that both counts were always significantly correlated (P < 0.001). Regarding angiogenic plasma biomarkers, preliminary data of bFGF, VEGF and HGF concentrations showed a trend, albeit not significant, to decrease after the first three cycles of therapy. Most common Grade 3–4 adverse events were hematologic and included neutropenia (48%), thrombocytopenia (18%) and anemia (6%). Other events included 4 patients (12%) with Grade 3–4 neutropenic fever and 3 patients (9%) with Grade 3 bacterial pneumonia. Grade 3–4 hypotension and dyspnea developed in 1 and 3 patients respectively; no patient developed thrombo-embolic or neuropathic complications. Conclusions Results from this study confirm the high therapeutic activity of Len in association with Dex in patients with relapsed and refractory MCL with a favourable safety profile. The significant increase of the macrophage infiltration in the BM seems to be a possible immunomodulatory effect of Len. Perhaps, the increase in microvessel counts may be induced by the activated macrophages and be an example of “indirect angiogenesis”. On the other hand, angiogenic plasma biomarkers tend to be lower, suggesting only a limited effect of Len on the neovascularization. Disclosures: Off Label Use: Lenalidomide in Mantle Cell Lymphoma. Vitolo:Celgene: Membership on an entity's Board of Directors or advisory committees.

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.

Tumor-Associated Macrophages: The Double-Edged Sword in Cancer Progression

Purpose Inflammation plays a critical role in cancer progression. In this study we investigate the pro-tumorigenic activities and gene expression profiles of lung cancer cells after interaction with macrophages. Materials and Methods We measured intratumoral microvessel counts and macrophage density in 41 lung cancer tumor specimens and correlated these with the patients' clinical outcome. The interaction between macrophages and cancer cell lines was assessed using a transwell coculture system. The invasive potential was evaluated by in vitro invasion assay. The matrix-degrading activity was assayed by gelatin zymography. The microarray was applied to a large-scale analysis of the genes involved in the interaction, as well as to monitor the gene expression profiles of lung cancer cells responding to anti-inflammatory drugs in cocultures. Results The macrophage density positively correlated with microvessel counts and negatively correlated with patient relapse-free survival (P < .05). After coculture with macrophages, lung cancer cell lines exhibited higher invasive potentials and matrix-degrading activities. We identified 50 genes by microarray that were upregulated more than two-fold in cancer cells after coculture. Northern blot analyses confirmed some gene expression such as interleukin-6, interleukin-8, and matrix metalloproteinase 9. The two-dimensional hierarchical clustering also demonstrated that the gene expression profiles of lung cancer cells responding to various anti-inflammatory drugs in cocultures are distinct. Conclusion The interaction of lung cancer cells and macrophages can promote the invasiveness and matrix-degrading activity of cancer cells. Our results also suggest that a great diversity of gene expression occurs in this interaction, which may assist us in understanding the process of cancer metastasis.

Microvessel quantification in benign and malignant ovarian tumors

Microvessel density (MVD) in 92 paraffin sections of ovarian samples of different histologic subtypes was correlated with microvessel counts from 58 corresponding frozen sections. Anti-human von Willebrand factor antibody was used as an endothelial marker. MVD was performed in neovascular hotspots using a Quantimet 500+ Image Analyzer. The highest vessel density (HVD) and average vessel density (AVD) of three fields at the × 200 and × 400 magnification were recorded. There was a strong correlation between the HVD and AVD at the × 200 and × 400 magnifications when comparing fixed with frozen sections (correlation coefficients at × 200 for the HVD was 0.37, P = 0.005 and AVD was 0.30, P = 0.02; correlation coefficients at × 400 for the HVD was 0.38, P = 0.003 and AVD was 0.37, P = 0.004). In the fixed tissue, the HVD and AVD at both these magnifications were significantly greater in the group containing functional cysts; this was also the case for the frozen sections. These findings are consistent with the development of a microcirculation necessary for the growth and maturation of such cysts, and this appears to be greater than that in tumors. The good correlation between MVD in fixed and frozen sections suggests that such observations represent a true reflection of ovarian angiogenesis in both physiologic and pathologic states.

Iron Deficiency Status in Polycythemia Vera Correlates Well with Bone Marrow Angiogenesis in Patients Treated with Phlebotomy.

Background: Polycythemia vera (PV) is a chronic myeloproliferative disorder derived from multipotent hematopoetic precursors. The pathogenesis of PV is still poorly understood; furthermore, it is not clear, why the bone marrow (BM) of patients with PV has an increased microvessel density. Aims: For these reasons, we aimed to investigate, if BM angiogenesis and pathogenesis in PV are associated, and if the increased angiogenesis can elucidate pathophysiologic mechanisms of PV. Methods: BM biopsies were taken from patients with PV before and during treatment. A total of 38 samples from 35 patients were analyzed. In three patients, we investigated biopsies in 12 months intervals. Also, eight patients with iron deficiency were studied. BM angiogenesis in thin sections was determined by immunostaining of endothelial cells (CD34, vWF) and counting the microvessels. The microvessel counts were correlated to hematologic features such as treatment modalities and iron metabolism parameters (zinc protoporphyrin (ZPP), ferritin). Iron deficiency was classified to be mild for ZPP ≤ 100 micromol/mol hem (normal value &lt; 40) and for ferritin 7 to 35 microgramm/l (normal value &gt; 35), and to be severe for ZPP &gt; 100 micromol/mol hem and for ferritin &lt; 7 microgramm/l. Results: When compared to healthy controls, the number of BM microvessels in patients with iron deficient anemia was indistinguishable from healthy individuals. As expected, the BM microvessel numbers of patients with PV at diagnosis (n= 6) or with only mild iron deficiency treated with various therapy schedules (n=20) showed no significant difference (p=n. s.). In contrast, in patients with severe iron deficiency, we detected a highly significant difference between two different treatment modalities: phlebotomy alone was associated with a normal microvessel density (n=7; mean 3.6/mm2, range 2.7 to 5.7/mm2), whereas other treatment modalites or a combination with phlebotomy were associated with a high microvessel density (n=10; mean 10.4/mm2; range 4.5 to 15.7/mm2; p=0.0004). The microvessel counts of the patients from who two sequential biopsies were investigated are in line with these results: the microvessel numbers in a patient who received an oral iron treatment increased from 9.3 to 15/mm2; the microvessels of another patient who was treated with hydroxy urea and phlebotomy showed almost unchanged microvessel counts, whereas, in a third patient, who was treated exclusively by phlebotomy, a clear decrease of BM microvessel density from 15.7 to normal value of 4.5/mm2 was observed. Conclusions: We conclude that i) iron deficiency per se does not induce increased microvessel density in the BM, ii) a severe iron deficiency induced by phlebotomy alone can reduce the BM microvessel density in patients with PV to normal. Provided that the increased BM angiogenesis is associated with pathogenesis in PV, which means that the multipotent hematologic precursors account for the increased angiogenesis, efficient iron depletion by phlebotomy represents the highly potent treatment for PV.

Correlation of Vascular Endothelial Growth Factor Expression to Overall Survival in Feline Invasive Mammary Carcinomas

Samples from feline invasive mammary carcinomas (FMCs) were used to determine the prognostic significance of the immunohistochemical expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD). Forty-eight queens bearing FMCs were included in a 2-year follow-up study. Mammary tumors were classified according to the World Health Organization system and graded on the basis of histologic criteria. Tumor sections were immunostained using anti-VEGF and anti-von Willebrand factor (vWf) antibodies. VEGF expression was quantified on the basis of the percentage of positive cells. MVD of vWf-positive microvessels was determined by both mean microvessel counts and highest microvessel counts. Normal mammary gland tissues showed an inconspicuous VEGF staining. In FMCs the proportion of VEGF-positive cells was significantly higher in papillary and solid carcinomas than in tubular and papillary cystic tumors. An increased number of cells expressing VEGF was also observed in poorly differentiated FMCS. Sixteen (33.3%) of the queens bearing invasive carcinomas were still alive at the end of the 2-year follow-up period, and 32 (66.7%) had died. The VEGF expression was significantly correlated with the clinical outcome, but no correlation was observed with the invasion of lymphatic vessels. A correlation between the higher percentage of VEGF-positive cells and the unfavorable prognosis was demonstrated by the estimation of curves for overall survival ( P = 0.03). Univariate analysis showed that MVD did not correlate with the overall survival. The results of our study demonstrated that VEGF expression, although not associated with increased angiogenesis, is a prognostic indicator in feline mammary tumors. In contrast, there is no support for a role of neovascularization as an indicator of survivability.

αvβ3 and αvβ5 Integrin Expression in Meningiomas

OBJECTIVE Integrins are emerging as alternative receptors capable of mediating several biological functions, such as cell-matrix and cell-cell adhesion, cell migration, signal transduction, and angiogenesis. Two αv integrins, i.e., αvβ3 and αvβ5, play critical roles in mediating these activities, particularly in tumors. No data are available on the expression of these integrins in meningiomas. METHODS Using Western blot and immunohistochemical analyses with LM609 and PG32, two monoclonal antibodies capable of recognizing the functional integrin heterodimer, we evaluated the expression of αvβ3 and αvβ5 integrins in a series of 34 meningiomas of different histological subtypes and grades. We studied their expression in tumor cells and vasculature, as well as the expression of their related angiogenic factors (fibroblast growth factor 2 and vascular endothelial growth factor) and the αvβ3 ligand vitronectin. RESULTS αvβ3 and αvβ5 integrins were expressed by neoplastic vasculature and cells. αvβ3 and αvβ5 expression was associated and correlated with that of their respective growth factors (fibroblast growth factor 2 and vascular endothelial growth factor) and microvessel counts and densities. αvβ3 was more strongly expressed than αvβ5 in two cases of histologically benign meningiomas with aggressive clinical behavior. αvβ3 expression was associated with that of its related ligand vitronectin and was also evident in small vessels of brain tissue closely surrounding meningiomas. CONCLUSION Our data demonstrate the expression of αvβ3 and αvβ5 integrins in meningioma cells and vasculature. Our findings suggest a role for both of these integrins, and particularly αvβ3, in meningioma angiogenesis.