Andrea Ladányi

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.

T-Cell Activation Marker Expression on Tumor-Infiltrating Lymphocytes As Prognostic Factor in Cutaneous Malignant Melanoma

The central role of T cells in antitumor immunity is well established. However, tumor progression, often seen in the presence of substantial lymphocytic infiltration, suggests that these T cells are not capable of mounting an effective immune response to control tumor growth. Evidence has accumulated that T lymphocytes infiltrating human neoplasms are functionally defective, incompletely activated, or anergic. Therefore, when characterizing the immune competent cells within lymphoid infiltrates of tumors, it is important to assess their activation state. We investigated the expression of two T-cell activation markers, interleukin 2 receptor α (CD25) and OX40 (CD134), by immunohistochemistry in primary cutaneous melanoma samples of 76 patients and analyzed it in relation to tumor stage and tumor progression (>5 years follow-up), as well as to patients’ survival. We found that the degree of infiltration by CD25+ and intratumoral OX40+ lymphocytes showed a tendency to decrease in thicker melanomas. The frequency of samples with high numbers of peritumoral CD25+ and OX40+ cells was significantly lower (P = 0.0009 and P = 0.0087, respectively) in melanomas developing distant visceral metastases, compared with nonmetastatic or lymph node metastatic tumors. For both activation markers studied, high peritumoral densities were associated with longer survival by univariate analysis (P = 0.0028 and P = 0.0255 for CD25 and OX40, respectively), whereas peritumoral OX40+ lymphocyte infiltration had an impact on survival also in multivariate analysis (P = 0.035). The results suggest that the presence of lymphocytes expressing the T-cell activation markers CD25 or OX40 shows correlation with tumor progression as well as with patients’ survival in cutaneous malignant melanoma.

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.

Effect of lentinan on macrophage cytotoxicity against metastatic tumor cells

We studied the effect of lentinan, a fungal polysaccharide immunomodulator, on mouse peritoneal macrophages. The i.p. treatment of mice with 10 mg/kg lentinan affected the number, plastic-adherence, and endogen peroxidase activity of peritoneal cells. The cytotoxicity of lentinan-stimulated peritoneal macrophages was determined against several murine and human metastatic tumor targets: Lewis lung carcinoma (LLT) and two human melanomas, and was found to be significantly higher than that of the macrophages from control animals. However, the highly metastatic variant of LLT (LLT-HH) was resistant to the cytolytic effect of resident and lentinan-activated macrophages as well, indicating that the stimulation for cytotoxicity depends not only on the functional activity of the effector but also on the sensitivity of the target.

Role for β3 integrins in human melanoma growth and survival

The role of αIIbβ3 integrin in regulating platelet function is well appreciated, whereas its role in tumor progression and metastasis is not. The purpose of our study was to determine a functional relevance to expression of αIIbβ3 integrin in cells derived from human solid tumors. A study of human melanoma biopsies (n = 24) showed that αIIbβ3 expression increased with tumor thickness, which is indicative of metastatic propensity. Expression of αIIbβ3 was 8% (±1.8), 33% (±10.4) and 62% (±5) in melanomas ranging in thickness from 0–1.5 mm, 1.5–4.0 mm and >4 mm, respectively; αvβ3 was equally high all categories. To determine biological function, we stably transfected αIIbβ3 into human melanoma cells that express αvβ3, but not αIIbβ3. Surface expression of αvβ3 remained unaltered between αIIbβ3 (+) and mock transfected counterparts. The αIIbβ3 (+) cells possessed increased ability to adhere, spread and migrate on fibrinogen. They had decreased ability to attach, spread and migrate on vitronectin. Immunocytochemistry showed that expression of αIIbβ3 displaced αvβ3 from focal contact points. When implanted subcutaneously into SCID mice, the αIIbβ3 (+) cells developed ∼4‐fold larger tumors when compared to their mock counterparts and the level of apoptosis was reduced within the tumors. Results suggest that co‐expression of the 2 β3 integrins, αvβ3 and αIIbβ3, in human melanoma cells enhanced cell survival and promoted growth in vivo.

In vitro and in vivo antitumor effect of 2-methoxyestradiol on human melanoma.

2‐methoxyestradiol (2ME2) is an endogenous metabolite of estradiol with estrogen‐receptor‐independent antitumor and antiangiogenic activity. We examined the effects of 2ME2 on the cellular proliferation of 8 human melanoma cell lines. We show that 2ME2 inhibited cell proliferation by inducing apoptosis and an arrest in the G2/M phase, and the mechanism of action involved microtubules, mitochondrial damage and caspase activation. In male SCID mice, 2ME2 was effective in reducing primary tumor weight and the number of liver metastases after intrasplenic injection of human melanoma cells. In the metastases, we found a significantly higher rate of apoptotic cells after 2ME2 treatment. These findings on the antitumor effect of 2ME2 in cell culture as well as in an animal model may have implications for designing alternative treatment options for patients with advanced malignant melanoma.

Neoadjuvant Immunotherapy of Oral Squamous Cell Carcinoma Modulates Intratumoral CD4/CD8 Ratio and Tumor Microenvironment: A Multicenter Phase II Clinical Trial

PURPOSE To investigate the clinicopathologic effects of local neoadjuvant Leukocyte Interleukin Injection (LI) regimen in oral squamous cell carcinoma (OSCC) patients. Treatment regimen included LI 800 IU/d as interleukin-2 (IL-2), administered half peritumorally and half perilymphatically five times per week for 3 weeks; low-dose cyclophosphamide; indomethacin; zinc; and multivitamins. PATIENTS AND METHODS Thirty-nine patients diagnosed with T2-3N0-2M0 OSCC participated in the pathology portion of this phase II multicenter study (19 LI-treated patients and 20 historical controls). Clinical responses were determined by imaging. Paraffin-embedded tumor samples were obtained at surgery for all patients. Surgery for the LI-treated group was performed between days 14 and 54 after the end of treatment. Histologic evaluation, pathologic staging, necrosis, and American Joint Committee on Cancer grading were performed from hematoxylin and eosin sections. Immunohistochemistry and morphometry determined cellular infiltrate. RESULTS Two pathologically complete, two major (> 50%), and four minor responses (> 30% but < 50%) resulted from LI treatment (overall response rate, 42%). Histopathology showed that the intratumoral CD4+:CD8+ ratio was low (< 1) in patients not treated with LI (controls). An increase in tumor-infiltrating CD4+ and a decrease of CD8+ T cells was observed in LI-treated patients, leading to a significantly (P < .05) higher intratumoral CD4+:CD8+ ratio (> 2.5). This was paralleled by dendritic cell transition from tumor surface toward stromal interface (P < .05), with macrophage decrease and neutrophil accumulation, multifocal microscopic necrosis, and significant (P < .05) increase in tumor stroma of LI-treated patients compared with controls. CONCLUSION LI-treated OSCC patients were characterized by a markedly altered composition of tumor-infiltrating mononuclear cells, increased CD4+:CD8+ ratio, and increased tumor stroma to epithelial ratio, all of which were distinct from controls.

FOXP3+ Cell Density in Primary Tumor Has No Prognostic Impact in Patients with Cutaneous Malignant Melanoma

Regulatory T cells (Tregs) have been implicated as inhibitors of antitumor immune reactions. However, data on the relevance of their prevalence at tumor sites in influencing disease outcome are controversial. The aim of our study was to investigate the role in tumor progression and the prognostic impact of the density of lymphocytes expressing FOXP3, a transcription factor expressed predominantly by CD4+CD25+ Tregs, in primary cutaneous melanoma. We examined the infiltration of FOXP3+ cells by immunohistochemistry in tumor samples from 97 patients and evaluated in relation to patient and tumor parameters. The degree of infiltration by FOXP3+ cells did not show correlation with the thickness of melanomas. Moreover, no associations were found with metastasis formation during the 5-year follow-up period, patient survival, or any other clinicopathologic parameters studied. These results suggest that the presence of FOXP3+ lymphocytes in primary tumors is not of prognostic importance in human cutaneous melanoma.

Immune cell profile of sentinel lymph nodes in patients with malignant melanoma - FOXP3+ cell density in cases with positive sentinel node status is associated with unfavorable clinical outcome.

BackgroundBesides being a preferential site of early metastasis, the sentinel lymph node (SLN) is also a privileged site of T-cell priming, and may thus be an appropriate target for investigating cell types involved in antitumor immune reactions.MethodsIn this retrospective study we determined the prevalence of OX40+ activated T lymphocytes, FOXP3+ (forkhead box P3) regulatory T cells, DC-LAMP+ (dendritic cell-lysosomal associated membrane protein) mature dendritic cells (DCs) and CD123+ plasmacytoid DCs by immunohistochemistry in 100 SLNs from 60 melanoma patients. Density values of each cell type in SLNs were compared to those in non-sentinel nodes obtained from block dissections (n = 37), and analyzed with regard to associations with clinicopathological parameters and disease outcome.ResultsSentinel nodes showed elevated amount of all cell types studied in comparison to non-sentinel nodes. Metastatic SLNs had higher density of OX40+ lymphocytes compared to tumor-negative nodes, while no significant difference was observed in the case of the other cell types studied. In patients with positive sentinel node status, high amount of FOXP3+ cells in SLNs was associated with shorter progression-free (P = 0.0011) and overall survival (P = 0.0014), while no significant correlation was found in the case of sentinel-negative patients. The density of OX40+, CD123+ or DC-LAMP+ cells did not show significant association with the outcome of the disease.ConclusionsTaken together, our results are compatible with the hypothesis of functional competence of sentinel lymph nodes based on the prevalence of the studied immune cells. The density of FOXP3+ lymphocytes showed association with progression and survival in patients with positive SLN status, while the other immune markers studied did not prove of prognostic importance. These results, together with our previous findings on the prognostic value of activated T cells and mature DCs infiltrating primary melanomas, suggest that immune activation-associated markers in the primary tumor may have a higher impact than those in SLNs on the prognosis of the patients. On the other hand, FOXP3+ cell density in SLNs, but not in the primary tumor, was found predictive of disease outcome in melanoma patients.

Association of microvessel density with infiltrating cells in human cutaneous malignant melanoma

Vascularization and host response to malignant tumors may have common molecular regulators, therefore, we analyzed the relationship between microvessel density (MVD) and tumor infiltrating cells in cutaneous malignant melanoma. Density of lymphocyte subpopulations, macrophages, dendritic cells and CD34+ microvessels was determined by immunohistochemistry in primary tumor samples from fifty-two patients with melanoma thicker than 1 mm. Intratumoral MVD did not show significant association with infiltration for any of these cell types. In the case of peritumoral reactive cell densities analyzed in the whole patient population, a positive correlation of MVD was found with CD3+ T cell density. This association was stronger in melanomas >4.0 mm and in visceral metastatic tumors. In these subgroups similar phenomenon was observed for CD8+ cells. We found significant correlation of MVD with CD68+ macrophage density only in the highest thickness category, and weak associations with B-cell and dendritic cell infiltration in visceral metastatic cases. MVD did not vary significantly in tumors categorized according to thickness, localization, ulceration or histological type. However, both intratumoral MVD and macrophage infiltration were significantly higher in male patients compared to females. The correlation of immune cell density with tumor vascularization and gender differences in vascularity and macrophage infiltration of melanoma deserve further attention.