Milagros Frómeta

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.

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.

Immunorecognition of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Some Normal and Malignant Tissues from Genitourinary System

N-glycolyl neuraminic acid has been considered as a tumour-associated antigen forming both glycolipid and glycoprotein, expressed in some human malignant cells. In this work, we evaluate the 14F7 Mab (an IgG1 murine highly specific to N-glycolyl GM3 ganglioside) reactivity in a variety of genitourinary-system-derived tumors as well as in their normal counterparts. Immunohistochemical assays with 14F7 followed by anti-mouse biotinylated antibody and ABC/HRP system using formalin-fixed and paraffin-embedded tissues were carried out. In normal tissues, 14F7 was reactive only in renal tubules of kidney (2/6) and in the stromal component and blood vessels of ovary (3/5). Tumors of kidney (12/38), urinary bladder (8/9), breast (41/42), ovary (21/34), testis (4/5), prostate (17/20), and uterus (5/14) as well as prostatic nodular hyperplasia (5/8) were stained with 14F7. N-glycolyl GM3 recognized by 14F7 could be considered as one attractive target for both active and passive immunotherapy of genitourinary malignancies expressing this molecule.

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 μg/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.

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.

Flow Cytometric Measurement of Aneuploid DNA Content Correlates with High S-Phase Fraction and Poor Prognosis in Patients with Non-Small-Cell Lung Cancer

Cellular DNA content (ploidy) and proliferation activity (e.g., S-phase fraction) measured by flow cytometry have been usually related to the biologic aggressiveness of various neoplasms. In this study, these parameters were analyzed in paraffin-embedded tumor specimens from 43 cases of resected non-small-cell lung cancer (NSCLC). Additionally, the correlation of them with both prognosis and a variety of clinic-pathological features were investigated. The stage and the appearance of both local recurrence and metastasis were related to overall survival of patients. Twenty-two tumors (51.2%) had a diploid DNA distribution, while 21 were aneuploid (48.8%). The mean of aneuploidy was 1.6% ± 0.3%. A correlation was found between ploidy and survival as well as with the appearance of local recurrence and/or metastasis. The mean values of S-phase fraction of diploid and aneuploid tumors were 16.7 ± 11.3% and 32.9 ± 12.1%, respectively, which were significantly different (). Similar results were obtained analyzing the proliferation index (sum of cells in S and G2/M phases of cell cycle) (). However, no correlation between these parameters and both overall survival of patients and clinicopathological features was observed. Our results could suggest the potential use of ploidy analysis as a useful complement of TNM stage in NSCLC.

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.

Tumor Expression of the Carcinoembryonic Antigen Correlates with High Mitotic Activity and Cell Pleomorphism Index in Lung Carcinoma

At present, some research efforts are focusing on the evaluation of a variety of tumor associated antigens (TAAs) for a better understanding of tumor biology and genetics of lung tumors. For this reason, we evaluated the tissue expression of carcinoembryonic antigen (CEA) and ior C2 (a cell surface O-linked glycoprotein carbohydrate chain TAA) in lung carcinomas, as well as its correlation with a variety of clinicopathological features. The tissue expression of CEA was evidenced in 22/43 (51.16%) lung carcinomas and it was correlated with mitotic activity, cell pleomorphism indexes, and age of patients. The expression of ior C2 was observed in 15/43 (34.88%) tumors but no correlation with the clinicopathological features mentioned above was obtained. No correlation between both CEA and ior C2 antigens expression and the overall survival (OS) of non-small-cell lung cancer patients was also observed. However, CEA-negative patients displayed higher OS rates as compared with positive ones (69.74 versus 58.26 months). Our results seem to be in agreement with the role of CEA expression in tumor cell proliferation, inhibition of cell polarizations and tissue architecture distortion. The significance of ior C2 antigen in these malignancies and it potential use in diagnosis, prognosis, and/or immunotherapy must be reevaluated.

Tissue Expression of Low and High Molecular Weight Cytokeratins in Lung Carcinoma Sections: Its Correlation with Some Clinic-Pathological Features

The tissue expression of low (LMW) and high (HMW) molecular weight cytokeratins and Ber-EP4 antigen in both small (SCLC) and non-small (NSCLC) cell lung carcinomas, as well as its correlation with a variety of clinic-pathological features, was evaluated. In general, 43/52 (82.7%) of NSCLC sections showed the expression of at least one type of cytokeratin while only 7/16 (43.7%) of SCLC were stained with both LMW cytokeratin and pan-cytokeratins antibodies. Remarkably, 18/52 (34.6%) of NSCLC were positive to both types of cytokeratins. However, none of SCLC showed this pattern of expression. In NSCLC patients, the increasing levels of HMW cytokeratins expression, as shown by 34βE12 antibody, correlated with the occurrence of disease recurrence (; Fisher’s exact test). Consequently, the expression of HMW cytokeratins was found to be associated with a poor 4-year overall survival of NSCLC patients (; Log rank test), not taking into account the histopathological classification of tumors. Similar results were obtained when 8-year overall survival was assessed (; Log rank test). Our results could suggest the assessment of HMW cytokeratins in a larger series of NSCLC samples in order to confirm the potential prognostic value of them.