József Tóvári

Identification and Clinical Significance of Circulating Endothelial Progenitor Cells in Human Non–Small Cell Lung Cancer

Until recently, it was generally accepted that vascularization of tumors arises exclusively from endothelial sprouting. Whether circulating bone marrow-derived endothelial progenitor cells (EPC) participate in the progression of non-small cell lung cancer (NSCLC) has not yet been evaluated. EPCs labeled with CD34, CD133, and vascular endothelial growth factor receptor-2 (VEGFR2) antibodies were counted by flow cytometry in the peripheral blood of 53 NSCLC patients. Furthermore, by means of a quantitative reverse transcription-PCR approach, we measured VEGFR2, CD133, CD34, and VE-cadherin mRNA in the peripheral blood samples of the same patient population. EPCs in tumor samples were identified by confocal microscopy using CD31, CD34, CD133, and VEGFR2 antibodies. Although immunofluorescent labeling of microvessels made clear that incorporation of EPCs is a rare phenomenon in NSCLC tissue (9 of 22 cases), circulating EPC levels before therapeutic intervention were increased in NSCLC patients (P < 0.002, versus healthy controls), and high pretreatment circulating EPC numbers correlated with poor overall survival (P < 0.001). Furthermore, in the subgroup of responders to treatment, the posttreatment EPC numbers in the peripheral blood were significantly lower compared with nonresponding patients. Interestingly, pretreatment mRNA levels of CD133, VE-cadherin, and CD34 were not significantly increased in NSCLC patients, whereas VEGFR2 expression was increased by 80-fold. Moreover, posttreatment VEGFR2 mRNA level in the peripheral blood was significantly higher in the subgroup of nonresponding patients when compared with posttreatment level of patients responding to antitumor therapy. Circulating levels of bone marrow-derived EPCs are significantly increased in NSCLC patients and correlate with clinical behavior.

Lymphangiogenesis Correlates with Lymph Node Metastasis, Prognosis, and Angiogenic Phenotype in Human Non–Small Cell Lung Cancer

Purpose: Recent experimental studies have revealed that lymphangiogenesis plays an important role in cancer progression, but its clinical significance in the case of non-small cell lung cancer (NSCLC) remains unclear. Our aim was to assess the lymphangiogenesis of human NSCLC, and to correlate this with angiogenic phenotype (angiogenic versus nonangiogenic growth pattern) and clinical behavior. Experimental Design: One hundred and three patients with NSCLC and complete follow-up information were included. Tumor samples were immunostained for vascular endothelial growth factor-C (VEGF-C), the lymphatic endothelial markers, LYVE-1 and D2-40/Podoplanin, and the panvascular marker, CD31. Lymphatic vessel density (LVD) and perimeters were evaluated within the tumor and peritumorally. Results: LVDs at the tumor periphery were significantly higher in lymph node metastatic tumors (P < 0.005) and high LVDs correlated with poor overall survival (P < 0.001). However, this tendency proved to be significant only in the angiogenic tumor group (P < 0.001). Although 68% of the patients with nonangiogenic tumors had lymph node metastasis (P = 0.0048 versus angiogenic tumors), in the patient group with nonangiogenic NSCLCs, there was no information from the LVDs in any investigated tumor area (P > 0.05). In contrast to angiogenic tumors, which had actively sprouting lymphatics in all of the investigated tumor areas, nonangiogenic tumors showed no Ki67 staining intratumorally. Conclusions: Our results reveal tumor lymphangiogenesis as a novel prognostic indicator for the risk of lymph node metastasis in NSCLC. Moreover, it also provides the first evidence that nonangiogenic NSCLCs mainly co-opt host tissue lymphatics during their growth, in contrast to most of the angiogenic tumors, which expand with concomitant lymphangiogenesis.

Circulating endothelial cells, bone marrow-derived endothelial progenitor cells and proangiogenic hematopoietic cells in cancer: From biology to therapy

Vascularization, a hallmark of tumorigenesis, is classically thought to occur exclusively through angiogenesis (i.e. endothelial sprouting). However, there is a growing body of evidence that endothelial progenitor cells (EPCs) and proangiogenic hematopoietic cells (HCs) are able to support the vascularization of tumors and may therefore play a synergistic role with angiogenesis. An additional cell type being studied in the field of tumor vascularization is the circulating endothelial cell (CEC), whose presence in elevated numbers reflects vascular injury. Levels of EPCs and CECs are reported to correlate with tumor stage and have been evaluated as biomarkers of the efficacy of anticancer/antiangiogenic treatments. Furthermore, because EPCs and subtypes of proangiogenic HCs are actively participating in capillary growth, these cells are attractive potential vehicles for delivering therapeutic molecules. The current paper provides an update on the biology of CECs, EPCs and proangiogenic HCs, and explores the utility of these cell populations for clinical oncology.

Circulating bone marrow-derived endothelial progenitor cells: Characterization, mobilization, and therapeutic considerations in malignant disease

Until recently, tumor vascularization was thought to occur exclusively through angiogenesis. However, recent studies using different animal models of cancer suggested the importance of bone marrow‐derived endothelial progenitor cells (EPCs) (i.e. postnatal vasculogenesis) in tumor vascularization and growth. EPCs are present in the peripheral blood, their levels are increased in response to certain signals/cytokines, and they home into the neovascular bed of malignant tissues. Furthermore, at the clinical level, evidence is emerging that changes in EPC levels might predict the efficacy of anticancer drug combinations that include antiangiogenic agents. On the basis of these observations, EPCs have attractive potential diagnostic and therapeutic applications for malignant diseases. In this paper, we review biological features of EPCs and speculate on the utility of these progenitor cells for medical oncology.

Clinical significance of genetic alterations and expression of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas

The significance of epidermal growth factor receptor (EGFR) signaling is well studied in a number of different tumors, but limited data is available with regard to head and neck squamous cell carcinoma (HNSCC). Since anti-EGFR therapies are currently under investigation in these malignancies as well, comprehensive information about the alteration of EGFR in HNSCC is necessary to design the most appropriate therapeutic protocols. We examined retrospectively the gene copy number of EGFR by FISH and the protein expression by immunohistochemistry using different epitope-specific antibodies in paraffin-embedded primary tumors of five different regions, from 71 HNSCC patients who had not been treated with anti-EGFR therapy. In seven cases corresponding lymph node metastases were also available for comparative analyses. We also determined the mutational status of tyrosine kinase (TK) domain (exon 19 and 21) and the extracellular deletion mutation (vIII) of EGFR, the KRAS mutation at codon 12 and the presence of HPV infection. Eight of the 71 cases (11.3%) showed EGFR gene amplification (most of them localized into the hypopharyngeal region) and the increased gene copy number (amplification+polysomy) was 43.7%. Despite pronounced intratumoral heterogeneity of EGFR protein expression being found, the high EGFR expression correlated with poor prognosis. On the other hand, the phosphorylation of EGFR was associated with prolonged survival. No mutations in the TK domain of EGFR were found in any of the HNSCC patients and only two cases were KRAS mutant at codon 12. We detected vIII deletion mutation of EGFR in 21% of the samples, but there was no statistically significant correlation between the presence of vIII mutant form and patient survival. EGFR vIII mutation was, however, associated with increased gene copy number. Fourteen of 71 cases (19.7%) were HPV-positive and the incidence of infection showed a decreasing tendency from the oral cavity towards the larynx. Interestingly, in contrast to previous findings, we could not observe improved survival in HPV-positive patients compared to non-infected patients, most probably due to the fact that the majority of these HNSCC patients were smokers and alcohol consumers. In conclusion, we found that increased EGFR protein levels and gene copy numbers (not gene amplification alone) have prognostic significance in the investigated HNSCC patient population. However, the relatively high incidence of the EGFR-vIII mutant form warrants careful therapeutic decision-making when choosing between different anti-EGFR treatment options.

Apelin Expression in Human Non-small Cell Lung Cancer: Role in Angiogenesis and Prognosis

Introduction: The recently discovered bioactive peptide, apelin, has been demonstrated to stimulate angiogenesis in various experimental systems. However, its clinical significance and role in tumor vascularization have not yet been investigated in a human malignancy. Therefore, our aim was to study whether apelin expression is associated with angiogenesis and/or tumor growth/behavior in human non-small cell lung cancer (NSCLC). Methods: A total of 94 patients with stage I–IIIA NSCLC and complete follow-up information were included. Apelin expression in human NSCLC samples and cell lines was measured by quantitative reverse-transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Effects of exogenous apelin and apelin transfection were studied on NSCLC cell lines in vitro. In vivo growth of tumors expressing apelin or control vectors were also assessed. Morphometric variables of human and mouse tumor capillaries were determined by anti-CD31 labeling. Results: Apelin was expressed in all of the six investigated NSCLC cell lines both at the mRNA and protein levels. Although apelin overexpression or apelin treatments did not increase NSCLC cell proliferation in vitro, increasing apelin levels by gene transfer to NSCLC cells significantly stimulated tumor growth and microvessel densities and perimeters in vivo. Apelin mRNA levels were significantly increased in human NSCLC samples compared with normal lung tissue, and high apelin protein levels were associated with elevated microvessel densities and poor overall survival. Conclusions: This study reveals apelin as a novel angiogenic factor in human NSCLC. Moreover, it also provides the first evidence for a direct association of apelin expression with clinical outcome in a human cancer.

Recombinant Human Erythropoietin α Targets Intratumoral Blood Vessels, Improving Chemotherapy in Human Xenograft Models

Recombinant human erythropoietin (rHuEPO) is widely used for correction of hemoglobin level in cancer patients. However, apart from hematopoiesis, rHuEPO reportedly has an effect on endothelial cells. We describe here how rHuEPOalpha can modulate tumor vasculature in human squamous cell (A431) and colorectal carcinoma (HT25) xenograft models. In vivo rHuEPO treatment of xenografts at human-equivalent dose significantly increased the proliferation index of the tumor-associated endothelial cells and the size of CD31-positive intratumoral blood vessels, whereas the pericyte coverage became fragmented. Moreover, rHuEPO administration resulted in decreased expression of vascular endothelial growth factor both by cancer cells and tumor stroma, measured by quantitative PCR. Due to the morphologic alterations in tumoral microvessels, DNA-binding agents (Hoechst and Doxorubicin) labeled significantly larger areas in the tumor mass. Furthermore, rHuEPO treatment led to a significantly improved efficacy of 5-fluorouracil (5-FU) chemotherapy in the case of both tumor xenografts. Meanwhile, rHuEPO had no effect on the in vitro proliferation of erythropoietin receptor-positive tumor cells, and did not interfere with the effects of 5-FU either. These data reveal a new effect of rHuEPO administration: remodeling tumoral microvessels, suppressing vascular endothelial growth factor expression, thereby augmenting antitumor effects of a cancer drug, 5-FU, even in erythropoietin receptor-positive human cancer xenografts.

Platelet-Mimicry of Cancer Cells: Epiphenomenon with Clinical Significance

Stem cell mimicry of cancer cells has been known for a long time and is considered to be responsible for ectopic gene expressions. The stem cell characteristics of tumor cells are shown to be involved in epithelial-mesenchymal transition and in the phenomenon of vascular mimicry. Certain cancer types acquire a geno-phenotype closely resembling the platelets and express several megakaryocytic genes (adhesion receptors αIIbβ3, thrombin receptor and PECAM/CD31 and/or platelet-type 12-LOX) able to activate the coagulation cascade or the platelets themselves. Here we define these potentials as platelet mimicry of cancer cells typical of pancreatic, breast, prostate, colorectal and urogenital cancers and melanoma. Data all support that platelet mimicry of certain cancer types is an important factor in their hematogenous dissemination and provides an attractive therapeutic target. Besides the long-available preclinical data, clinical trials have only recently provided evidence that targeting platelet mimicry of cancers is an efficient way to prevent tumor progression. The systematic discovery of the markers of platelet mimicry in various cancer types and their molecular targeting may provide new supportive therapeutic modalities for the management of the progressing disease.

High VEGFR-3-positive Circulating Lymphatic/Vascular Endothelial Progenitor Cell Level Is Associated with Poor Prognosis in Human Small Cell Lung Cancer

Purpose: The newly identified bone marrow–derived cell population, called lymphatic/vascular endothelial progenitor cells (LVEPC), has been shown to contribute to lymph capillary growth in experimental tumor systems. The clinical significance of these cells has not yet been investigated in a human malignancy. Our aim was to study whether peripheral blood circulating LVEPCs participate in the progression of human small cell lung cancer (SCLC). Experimental Design: A total of 88 patients with limited-stage SCLC and 32 tumor-free control subjects were included. Peripheral blood circulating LVEPC labeled with CD34 and vascular endothelial growth factor receptor-3 (VEGFR3) antibodies and the serum levels of the key lymphangiogenic molecule VEGF-C were measured by flow cytometry and ELISA, respectively. Results: CD34-positive/VEGFR3-positive LVEPC levels were significantly increased in patients (versus controls; P < 0.01), and there was also a significant relationship between LVEPC counts and lymph node metastasis (P < 0.01). High pretreatment circulating LVEPC numbers correlated with poor overall survival (P < 0.01). Although we observed significantly elevated VEGF-C concentrations in patients (versus controls; P < 0.01), there was no significant correlation between VEGF-C and LVEPC levels. Moreover, no significant differences in peripheral blood VEGF-C levels were seen between patients subgrouped by clinicopathologic variables including tumor and lymph node stages and survival. Conclusions: Peripheral blood levels of bone marrow–derived LVEPCs are significantly increased in patients with SCLC and correlate with lymphatic involvement and prognosis. This is the first study that shows evidence of increased numbers of circulating LVEPC in patients with a malignant tumor.

Recombinant Human Erythropoietin alpha Improves the Efficacy of Radiotherapy of a Human Tumor Xenograft, Affecting Tumor Cells and Microvessels

Background and Purpose:Tumor-induced anemia often occurs in cancer patients, and is corrected by recombinant human erythropoietins (rHuEPOs). Recent studies indicated that, besides erythroid progenitor cells, tumor and endothelial cells express erythropoietin receptor (EPOR) as well; therefore, rHuEPO may affect their functions. Here, the effect of rHuEPOα on irradiation in EPOR-positive human squamous cell carcinoma xenograft was tested.Material and Methods:A431 tumor-bearing SCID mice were treated from the tumor implantation with rHuEPOα at human-equivalent dose. Xenografts were irradiated (5 Gy) on day 14, and the final tumor mass was measured on day 22. The systemic effects of rHuEPOα on the hemoglobin level, on tumor-associated blood vessels and on hypoxia-inducible factor-(HIF-)1α expression of the tumor xenografts were monitored. The proliferation, apoptosis and clonogenic capacity of A431 cancer cells treated with rHuEPOα and irradiation were also tested in vitro.Results:In vitro, rHuEPOα treatment alone did not modify the proliferation of EPOR-positive A431 tumor cells but enhanced the effect of irradiation on proliferation, apoptosis and clonogenic capacity. In vivo, rHuEPOα administration compensated the tumor-induced anemia in SCID mice and decreased tumoral HIF-1α expression but had no effect on tumor growth. At the same time rHuEPOα treatment significantly increased the efficacy of radiotherapy in vivo (tumor weight of 23.9 ± 4.7 mg and 34.9 ± 4.6 mg, respectively), mediated by increased tumoral blood vessel destruction.Conclusion:rHuEPOα treatment may modulate the efficacy of cancer radiotherapy not only by reducing systemic hypoxia and tumoral HIF-1α expression, but also by destroying tumoral vessels.Hintergrund und Ziel:Patienten mit Krebserkrankungen leiden oft an tumorinduzierter Anämie, die mit rekombinantem menschlichen Erythropoetin (rHuEPO) korrigiert wird. Viele Studien berichteten, dass sowohl die erythroiden Vorläuferzellen als auch Tumor- und Endothelzellen den Erythropoetinrezeptor (EPOR) exprimieren, der die Funktionen dieser Zellen beeinflussen könnte. In der vorliegenden Arbeit wurde die Wirkung von rHuEPOα auf die Bestrahlung des xenotransplantierten EPOR-positiven menschlichen Plattenepithelkarzinoms untersucht.Material und Methodik:Mit A431-Zellen infiltrierte SCID-Mäuse wurden nach der Tumoreinimpfung mit rHuEPOα in menschlicher Äquivalentdosis behandelt. Die xenotransplantierten Tumoren wurden am 14. Tag bestrahlt (5 Gy), und am 22. Tag wurde die endgültige Tumormasse gemessen. Zur Kontrolle der systemischen Wirkungen von rHuEPOα wurden die Hämoglobinwerte, die tumorassoziierten Gefäße und die HIF-1α-Expression (hypoxieinduzierbarer Faktor) der Tumoren bestimmt. Außerdem wurden die Proliferation, Apoptose und Klonbildungsfähigkeit der mit rHuEPOα und Bestrahlung behandelten A431-Tumorzellen in vitro untersucht.Ergebnisse:In vitro hatte die rHuEPOα-Behandlung keine Wirkung auf die Proliferation EPOR-positiver A431-Tumorzellen, aber die Wirkung der Bestrahlung auf Proliferation, Apoptose und Klonbildungsfähigkeit wurde erhöht. In vivo kompensierte die rHuEPOα-Gabe die tumorinduzierte Anämie der SCID-Mäuse und verminderte die HIF-1α-Expression des Tumors, hatte aber keine Wirkung auf das Tumorwachstum. Gleichzeitig steigerte die rHuEPOα-Behandlung die Wirkung der Strahlentherapie in vivo (Tumorgewicht 23,9 ± 4,7 mg bzw. 34,9 ± 4,6 mg) durch verstärkte Zerstörung der Tumorgefäße.Schlussfolgerung:Eine Behandlung mit rHuEPOα reguliert die Wirksamkeit der Strahlentherapie nicht nur durch verminderte systemische Hypoxie und HIF-1α-Expression, sondern auch durch Zerstörung der Tumorgefäße.