Prognostic Significance of N-Glycolyl GM3 Ganglioside Expression in Non-Small Cell Lung Carcinoma Patients: New Evidences.

The prognostic role of N-glycolyl GM3 ganglioside (NeuGcGM3) expression in non-small cell lung carcinoma (NSCLC) still remains controversial. In this study, the NeuGcGM3 expression was reevaluated using an increased number of NSCLC cases and the 14F7 Mab (a highly specific IgG1 raised against NeuGcGM3). An immunohistochemical score integrating the percentage of 14F7-positive cells and the intensity of reaction was applied to reassess the relationship between NeuGcGM3 expression, some clinicopathological features, and the overall survival (OS) of NSCLC patients. The double and the triple expression of NeuGcGM3 with the epidermal growth factor receptor (EGFR) and/or its ligand, the epidermal growth factor (EGF), were also evaluated. NeuGcGM3 expression correlates with both S-Phase fraction (p = 0.006) and proliferation index (p = 0.000). Additionally, NeuGcGM3 expression was associated with a poor OS of patients in both univariate (p = 0.020) and multivariate (p = 0.010) analysis. Moreover, the double and/or the triple positivity of tumors to NeuGcGM3, EGFR, and/or EGF permitted us to identify phenotypes of NSCLC with a more aggressive biological behavior. Our results are in agreement with the negative prognostic significance of NeuGcGM3 expression in NSCLC patients. However, standardization of techniques to determine the expression of NeuGcGM3 in NSCLC as well as the implementation of a universal scoring system is recommended.

Tissue Reactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Tumors of Neuroectodermal, Mesodermal, and Epithelial Origin.

The expression of N-glycolylneuraminic acid forming the structure of gangliosides and/or other glycoconjugates (Hanganutziu-Deicher antigen) in human has been considered as a tumor-associated antigen. Specifically, some reports of 14F7 Mab (a highly specific Mab raised against N-glycolyl GM3 ganglioside) reactivity in human tumors have been recently published. Nevertheless, tumors of epithelial origin have been mostly evaluated. The goal of the present paper was to evaluate the immunohistochemical recognition of 14F7 Mab in different human tumors of neuroectodermal, mesodermal, and epithelial origins using an immunoperoxidase staining method. Samples of fetal, normal, and reactive astrocytosis of the brain were also included in the study. In general, nontumoral tissues, as well as, low-grade brain tumors showed no or a limited immunoreaction with 14F7 Mab. Nevertheless, high-grade astrocytomas (III-IV) and neuroblastomas, as well as, sarcomas and thyroid carcinomas were mostly reactive with 14F7. No reaction was evidenced in medulloblastomas and ependymoblastomas. Our data suggest that the expression of N-glycolyl GM3 ganglioside could be related to the aggressive behavior of malignant cells, without depending on the tumor origin. Our data could also support the possible use of N-glycolyl GM3 as a target for both active and passive immunotherapies of malignancies expressing this molecule.

Immunohistochemical Characterization of Three Monoclonal Antibodies Raised against the Epidermal Growth Factor and Its Receptor in Non-Small-Cell Lung Cancer: Their Potential Use in the Selection of Patients for Immunotherapy.

Adequate methods to identify which lung cancer patients are most likely to benefit from the targeted drugs against both epidermal growth factor receptor/epidermal growth factor (EGFR/EGF) are needed. For this reason, we evaluated both the tissue reactivity of ior egf/r3 monoclonal antibody (Mab) in human lung carcinomas and its biological activity in NCI-H125 cells. Additionally, we assessed the tissue expression of EGF using two Mabs, CB-EGF1 and CB-EGF2. The overexpression of EGFR was detected in 33.33% and 62.71% of small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), respectively. The ability of ior egf/r3 Mab to bind the extracellular domain of EGFR inhibiting cell proliferation and inducing apoptosis in NCI-H125 cells was also demonstrated. The EGF expression was observed in about 17% and 70% of SCLC and NSCLC, respectively. However, differences in the reactivity of CB-EGF1 and CB-EGF2 were evidenced. A dual expression of EGFR and EGF was observed in 16.67% and 57.63% of SCLC and NSCLC patients, respectively. But, a correlation between them was only obtained in NSCLC. Our results permit to recommend the development of diagnostic kits using ior egf/r3 and/or CB-EGF1 Mabs in order to achieve a better selection of patients to EGFR/EGF-targeting treatment.

Immunoreactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Primary Lymphoid Tumors and Lymph Node Metastasis.

The reactivity of the 14F7 Mab, a highly specific IgG1 against N-glycolyl GM3 ganglioside (NeuGcGM3) in normal tissues, lymphomas, lymph node metastasis, and other metastatic sites was assessed by immunohistochemistry. In addition, the effect of chemical fixation on the 14F7 Mab staining using monolayers of P3X63Ag.653 cells was also evaluated. Moreover, the ability of 14F7 to bind NeuGcGM3 ganglioside inducing complement-independent cytotoxicity by a flow cytometry-based assay was measured. The 14F7 Mab was reactive in unfixed, 4% paraformaldehyde, 4% formaldehyde, and acetone fixed cells. Postfixation with acetone did not alter the localization of NeuGcGM3, while the staining with 14F7 Mab was significantly eliminated in both cells fixed and postfixed with methanol but only partially reduced with ethanol. The staining with 14F7 Mab was evidenced in the 89.2%, 89.4%, and 88.9% of lymphomas, lymph node metastasis, and other metastatic sites, respectively, but not in normal tissues. The treatment with 14F7 Mab affected both morphology and membrane integrity of P3X63Ag.653 cells. This cytotoxic activity was dose-dependent and ranged from 24.0 to 84.7% (10-1000 µ g/mL) as compared to the negative control. Our data could support the possible use of NeuGcGM3 as target for both active and passive immunotherapy against malignancies expressing this molecule.

Immunoreactivity of the 14F7 Mab (Raised against N-Glycolyl GM3 Ganglioside) as a Positive Prognostic Factor in Non-Small-Cell Lung Cancer.

Lung carcinoma is the leading cause of cancer-related mortality worldwide. Therefore, numerous studies are focusing on the assessment of other biological and molecular prognostic factors in these tumors. We evaluated the relationship between 14F7 Mab reactivity, pathological features, DNA-content and S-phase fraction (SPF), and their impact in the survival of NSCLC patients. Hematoxylin and eosin staining and immunohistochemistry optical microscopy assays as well as DNA content and SPF measuring using flow cytometry were performed. The 14F7 reactivity was widely observed in NSCLC sections, no depending of the clinicopathological characteristics. We also obtained differences in the intensity of reaction with 14F7 as well as in the SPF between diploid and aneuploid carcinomas. Patients with diploid tumors showing higher SPF and 14F7 reaction joint to a low mitotic index displayed higher survival rates. Our results are in agreement with the assumption of the possible positive prognostic value of 14F7 staining in NSCLC.

Phenotypic heterogeneity in the NCI-H125 cell line affects biological activity using the epidermal growth factor receptor as target.

We evaluated the influence of some morphological changes of the NCI-H125 cell line in surface expression of the epidermal growth factor receptor (EGFR) and their impact on some biological activity assays using this molecule as target. Hematoxylin and eosin (H/E) staining, light microscopy immunocytochemistry, flow cytometric antibody-receptor binding test, cell viability determination and cell cycle analysis were performed. Phenotypic changes and inconsistency in EGFR expression were detected in NCI-H125 cell cultures. A significant reduction in the growth rate, mainly characterized by cell cycle arrest in the G0-G1 phase, was also evidenced. Differential distribution of cell viability in NCI-H125 subpopulations and its relationship with the EGFR surface expression were determined. Nuclear alterations observed in NCI- -H125 were not apoptosis related. A lack of control of cell cultures affects the reliability and reproducibility of biomedical and biotechnological research using EGFR as target. Therefore, rigorous control of the above mentioned parameters in these experiments is recommended.

Immunoreactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Epithelial Malignant Tumors from Digestive System.

The limited expression of N-Glycolyl GM3 (NeuGcGM3) ganglioside in human normal tissues, as well as its presence in melanoma and breast carcinoma using 14F7 Mab (anti-NeuGcGM3), has been previously reported. In this work we evaluated for the first time the 14F7 Mab immunorecognition in some digestive system tumors. Immunohistochemical assays were made with 14F7, followed by anti-mouse biotinylated antibody and ABC/HRP system in normal and pathological human tissues were made. No immunoreaction was evidenced in normal tissues. The reactivity of 14F7 was detected in all adenocarcinomas of the stomach (12/12), colon (12/12), and pancreas (11/11). A finely granular immunorecognition in esophageal tumors (5/15), epidermoid carcinoma of the rectum (5/7), and basaloid carcinoma (4/5) of the latter as well as in hepatocellular carcinoma (13/14) was also observed. Our results are in agreement with the assumption that NeuGcGM3 ganglioside may be considered as target for passive and active immunotherapy in digestive system malignancies expressing this molecule.

Immunohistochemical Reactivity of the 14F7 Monoclonal Antibody Raised against N-Glycolyl GM3 Ganglioside in Some Benign and Malignant Skin Neoplasms.

The evaluation of 14F7 Mab (anti-N-glycolyl GM3 ganglioside) immunorecognition in normal skin, cutaneous malignant melanoma (CMM), and in lymph node metastases (LNM) has been previously reported. In this work we extended the study to benign (BMN) and dysplastic (DMN) melanocytic nevi, basal (BCC), and squamous cell carcinoma (SCC). Immunohistochemical assays with 14F7 followed by a biotinylated link universal and streptavidin-AP in normal and pathological tissues were made. No reaction of 14F7 in normal skin (0/10) as well as a low reactivity in BMN (2/11) and DMN (1/7) was detected. A limited staining in BCC (2/13) and in SCC (4/8) was also evidenced, while 14F7 Mab were mostly reactive in CMM (28/28) and in LNM (6/7). These results suggest that 14F7 reactivity could be closely related with the more aggressive biological behavior of CMM and also support the use of NeuGcGM3 as target for both passive and active melanoma immunotherapy.

Pharmacodynamic trial of nimotuzumab in unresectable squamous cell carcinoma of the head and neck: a SENDO Foundation study.

PURPOSE: To assess the pharmacodynamic effects of nimotuzumab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody with intermediate affinity for the receptor, in skin and tumor tissues from head and neck cancer patients. EXPERIMENTAL DESIGN: Pharmacodynamic study in patients with advanced squamous cell carcinoma of the head and neck, unsuitable for chemoradiotherapy, enrolled in a single-center trial. Patients received 8 weekly infusions of nimotuzumab. The first nimotuzumab infusion was administered 1 week before starting radiation, whereas the remaining doses were administered concomitantly with irradiation. Paired biopsies were taken from skin and primary tumors, before (pretherapy) and 1 week (on single-agent therapy) after first infusion. Immunohistochemistry was conducted to assay the effects of nimotuzumab on total and phosphorylated EGFR, phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), p-AKT, and proliferation (Ki-67). RESULTS: Nimotuzumab was well tolerated and there was no evidence of skin rash. Objective response was achieved in 9 of 10 patients. The pharmacodynamic assays showed inhibition of p-EGFR in both skin and tumor (P = 0.042 in skin and P = 0.034 in tumor). No significant changes in p-ERK1/2, p-AKT, or Ki-67 were detected in skin. In addition, lymphocytic infiltrates, folliculitis, or perifolliculitis were not observed. In tumor samples, there was an upregulation of p-AKT (P = 0.043), a reduction in proliferation index (P = 0.012), and a nonsignificant trend toward a decrease of p-ERK1/2 (P = 0.091). CONCLUSIONS: The pharmacodynamic data confirmed the ability of nimotuzumab to decrease EGFR phosphorylation. Downstream effects were observed in tumor cells but not in skin, a finding that may help to explain the lack of skin rash in patients treated with nimotuzumab.

Immunohistochemical recognition of the epidermal growth factor receptor by the h-R3 antibody in the skin of experimental animals.

The h-R3 is a humanized monoclonal antibody (Mab) that binds to the external domain of the human epidermal growth factor receptor (EGF-R). It has being used for the treatment of head and neck tumors. Since it is known that different animal species have different EGF-R amino acid sequences, it raises the handicap that, a priori, the animal species relevant for the pharmacodynamic, pharmacokinetic, and toxicologic studies of this Mab are unknown. To elucidate relevant laboratory animal species, the authors investigated the immunohistochemical recognition by h-R3 Mab of EGF-R in small skin biopsy samples obtained from NMRI nu/nu, C57Bl/6, and OF-1 mice; Sprague-Dawley rats; Hartley guinea pigs; New Zealand rabbits; and Cercopithecus aethiops and Macaca fascicularis monkeys. Additionally, three human skin biopsies were obtained from trauma victims. The immunolocalization of EGF-R in different tissue sections was performed using the avidin-biotin peroxidase complex (ABC) technique. The skin sections from different tissues were tested with the biotinylated h-R3 Mab or with a biotinylated irrelevant Mab, used as negative control. The staining intensity was qualified as:-, no staining; +, weak staining; ++, moderate staining; +++, strong staining. Macaca fascicularis monkey, New Zealand rabbit, and OF-1 mouse skins had a strong staining intensity; Cercopithecus aethiops monkey and Sprague-Dawley rat skins showed a moderate to strong staining intensity; C57Bl/6 mouse skins showed a moderate staining intensity. No staining was observed in the skins from Hartley guinea pigs and MNRI nu/nu mice. The fact that h-R3 Mab recognizes other animal EGF receptors in addition to the human EGF-R makes it possible to perform relevant pharmacodynamic, pharmacokinetic, and toxicologic studies in some laboratory animals.