HER-2 expression and cell proliferation: prognostic markers in patients with node-negative breast cancer.

PURPOSE: We analyzed the clinical relevance of HER-2 expression, widely investigated in breast cancer but with contradictory results, in the largest case series of node-negative breast cancer patients investigated to date. PATIENTS AND METHODS: The pure prognostic value of HER-2 expression was investigated in 529 patients treated with locoregional therapy alone until early relapse. Proliferative activity was evaluated as [3H]thymidine labeling index and HER-2 expression by immunohistochemistry. All biologic determinations were conducted within the context of an intra- and interlaboratory National Quality Control Program. RESULTS: HER-2 expression was not related to relapse-free survival in the overall series but was a significant discriminant of prognosis in the subgroup of patients with rapidly proliferating tumors. Six-year rate of relapse was 40% for patients with highly (> or =30%) positive tumors and 26% for those with weakly HER-2-expressing tumors (P =.039). CONCLUSION: HER-2 expression in association with proliferative activity identifies a subgroup of node-negative breast cancer patients with the worst prognosis, who are candidates for specific intensive adjuvant therapy.

Development and characterization of a monoclonal antibody directed against human telomerase reverse transcriptase (hTERT).

Telomerase activity plays an important role in the two complementary processes of cellular immortalization and senescence. This enzyme is active in almost all tumors, but also in inflammatory and many normal proliferating cells. Therefore, the main limits of molecular determinations, such as telomeric repeat amplification protocol assay is that they are not able to discriminate between the enzymatic activity of tumor and normal cells. The most appropriate technique for this would be immunohistochemical determination using monoclonal antibodies. Very few monoclonal antibodies (Mabs) directed against the human telomerase reverse transcriptase (hTERT) are commercially available and in the present study, we developed a new Mab directed against this protein (TERT-3 36-10) to investigate the possibility of detecting immunoreactivity to this Mab by immunohistochemical and flow cytometric approaches. Immunohistochemical determination showed a lack of reactivity to the Mab in highly differentiated striated muscle tissue, a variable reactivity in dysplastic cervical epithelial tissue and similar and widespread immunoreactivity in cell lines and clinical tumors. Furthermore, we demonstrated the ability of this Mab to inhibit enzyme activity in cell extract from MCR bladder tumor cell line.

In Vitro schedule-dependent interactions between the multitargeted antifolate LY231514 and gemcitabine in human colon adenocarcinoma cell lines.

PURPOSE: Multitargeted antifolate (MTA) and gemcitabine (GEM) have shown preclinical and clinical activity in tumor histotypes such as colon, renal, small and non-small cell lung cancers, hepatomas and carcinoid tumors. In our study, we investigated the cytotoxic activity of MTA alone or in combination with GEM using different exposure schedules in three different colon cancer cell lines (LoVo, WiDr, and LRWZ). EXPERIMENTAL DESIGN: Cytotoxic activity was evaluated by sulforhodamine B assay, cell cycle perturbations and apoptosis were evaluated by flow cytometry, and thymidylate synthase expression was evaluated by immunohistochemical method. RESULTS: A 48-h exposure to MTA caused a minimal and no-dose-response effect on the three cell lines used. Flow cytometric analysis showed a cell accumulation in S phase that completely resolved in LoVo and LRWZ cell lines and persisted in WiDr cells after a 48-h washout. Moreover, a significant increase in thymidilate synthase expression was observed in all of the cell lines after MTA exposure. Among the different combinations tested, the highest synergistic interaction, assessed using Kern's method and expressed as the synergistic ratio index, was produced by pretreatment with GEM followed by MTA (ratio index: 1.3- 6.7). It is possible that the depletion of nucleotide pools induced by MTA and required for DNA synthesis prevented cells from repairing DNA damage caused by GEM. The type and degree of drug interactions were not paralleled by apoptosis, which was almost always negligible, or by the type and persistency of the cell cycle perturbations. CONCLUSIONS: Our results indicate that the sequential administration of GEM --> MTA provides the greatest benefit in the clinical treatment of colon cancer.

In vitro schedule-dependent interaction between docetaxel and gemcitabine in human gastric cancer cell lines.

PURPOSE: The purpose of this study was to assess the activity of clinically used drugs and to define the most effective treatment scheme in human gastric cancer cell lines. EXPERIMENTAL DESIGN: Cytotoxic activity was evaluated by sulforhodamine B assay, potential clinical activity was estimated by relative antitumor activity, and the type of drug interaction was assessed using the method of Chou and Talalay. Cell cycle perturbations and apoptosis were evaluated by flow cytometry, mitotic index by microscopic analysis, bax, bcl-2, and p53 by immunohistochemistry, and cyclin B expression by Western blot. RESULTS: Gemcitabine (GEM) and docetaxel (DOC) were the most potent of the seven drugs tested, with maximum relative antitumor activity values in all of the cell lines. Simultaneous treatment with GEM and DOC, and the sequence GEM-->DOC caused an antagonistic interaction, as shown by the combination index >1, at all levels of killed cell fraction. Conversely, the sequential treatment DOC-->GEM produced a synergistic interaction (combination index < 1). On the basis of cell cycle perturbations, it can be hypothesized that the antimetabolite (GEM) attacks cells recovering rapidly from an M block induced by DOC as they progress to the S phase, producing a powerful cytocidal effect, as shown by the increase from 15 to 75% of apoptotic cells. CONCLUSIONS: Our findings suggest that the interaction of DOC and GEM is highly schedule dependent and has been used recently to plan a Phase I-II clinical protocol.

Basic fibroblast growth factor and vascular endothelial growth factor serum levels in breast cancer patients and healthy women: useful as diagnostic tools?

INTRODUCTION: The aim of the present study was to analyze the relationship between the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in breast cancer cells and the corresponding serum levels in individual patients. The study also evaluated the potential of serum levels of the two growth factors as diagnostic markers in a case-control study. METHODS: VEGF expression and bFGF expression were determined in 62 and 63 tumor samples, respectively. Serum VEGF and bFGF levels were determined in 54 and 65 healthy women and in 69 and 73 breast cancer patients, respectively, using a quantitative sandwich enzyme immunoassay technique. RESULTS: A direct correlation was observed between VEGF expression and bFGF expression in individual tumors (P = 0.001) and between serum levels (P = 0.038) in individual patients, but not between tumor cell expression and the corresponding serum level for either growth factor. Median values of serum levels in healthy women and breast cancer patients were not different for VEGF (P = 0.055), but were significantly different for bFGF (P < 0.001). The receiver operating characteristic curve identified a serum bFGF concentration of 1.0 pg/ml, with 84.9% sensitivity and 63.1% specificity, as the best cut-off value to discriminate between healthy women and breast cancer patients. An age-based subgroup analysis showed that serum values of patients older than 70 years of age mainly contributed to the high accuracy. CONCLUSIONS: Our data repropose bFGF as a noninvasive diagnostic tool for breast cancer.

Vascular endothelial growth factor and prognosis in patients with node-negative breast cancer.

Vascular endothelial growth factor (VEGF) has emerged as one of the most important angiogenic growth factors from experimental in vitro and in vivo studies. In the present study, we investigated the relationship between VEGF expression and microvessel density (MVD) and defined their prognostic relevance on a series of 242 patients with node-negative breast cancer, using immunohistochemical methods. In parallel, estrogen and progesterone receptors were quantitatively assessed using the dextran-charcoal technique and cell proliferation was evaluated as S-phase cell fraction according to (3)H-thymidine-labeling index (TLI). The percentage of VEGF-expressing cells varied from 0-95% in the different tumors and was unrelated to menopausal status, tumor size or steroid receptor status. Conversely, a significant inverse relation was observed with patient age or tumor cell proliferation, albeit with very poor correlation coefficients. A significant relation was observed between VEGF expression and MVD (r(s) = 0.55, p < 0.001). Clinical outcome analyzed as a function of high and low VEGF expression showed slight differences in terms of both disease-free survival (DFS) and overall survival (OS) that never reached statistical significance. Moreover, the trend was paradoxically in favor of patients with highly VEGF-expressing tumors. Finally, DFS and OS curves, when analyzed as a function of VEGF expression or MVD, were superimposable. In conclusion, our study did not highlight a prognostic relevance of VEGF expression in patients with node-negative breast cancer, as already observed for MVD.

Adjuvant immunotherapy with tumor infiltrating lymphocytes and interleukin-2 in patients with resected stage III and IV melanoma.

Adoptive immunotherapy with tumor infiltrating lymphocytes (TIL) and interleukin (IL)-2 is reasonably effective in the treatment of patients with advanced melanoma. However, theoretically it should be of greater benefit as adjuvant therapy, especially in high-risk stages (resected stages III and IV). In a preliminary study, 25 patients (aged 23-72 years) with stage III-IV melanoma who underwent resection of metachronous metastases were reinfused with TIL cultivated and expanded in vitro with IL-2 from surgically removed metastases. IL-2 (starting dose 12 x 10 IU/m ) was co-administered as a continuous infusion according to West's scheme. A total of 8/22 (36.3%) evaluable patients were disease-free (DF) at a median follow-up of 5 years. DF survival (DFS) and overall survival (OS) rates were 44% and 37%, respectively, at 2 years, and 52% and 45% at 3 years. The CNS was the only site of disease recurrence in 57% of patients who relapsed. DF patients received a higher median dose of IL-2 than those who progressed (total dose 110 x 10 versus 86 x 10 IU/m, respectively). The progressive reduction in IL-2 dosage allowed all patients to complete treatment without permanent grade 4 toxicity. Analysis of tumor immunosuppression factors in lymphocytes inside the tumor (TCR zeta and epsilon chains, p56, FAS, and FAS-ligand) confirmed that the immunologic potential of TIL, depressed at the time of metastasectomy, was significantly restored after in vitro culture with IL-2. Adoptive immunotherapy with TIL and IL-2 could improve DFS and OS, although further work is required to determine its role in the treatment of patients with high-risk melanoma.

Immunosuppression in renal cancer: differential expression of signal transduction molecules in tumor-infiltrating, near-tumor tissue, and peripheral blood lymphocytes.

Alterations in the expression of signal activation molecules, such as the T-cell receptor (TCR) zeta and epsilon chains and p56lck tyrosine kinase, are described in tumor-infiltrating lymphocytes (TIL). The aim of this study was to ascertain if such molecules were present in near-tumor-tissue lymphocytes (NTTL) and peripheral blood lymphocytes (PBL), as well as TIL, of renal cell carcinoma patients, to verify whether this tumor induces immunosuppression only locally or affects distant lymphocytes as well. Tissue from the tumor and from healthy nearby sites, as well as blood samples, were obtained from 27 consecutive patients who had undergone radical nephrectomy for renal cell carcinoma. Phenotype analysis and immunohistochemical staining of the TCR zeta and epsilon chains and p56lck were performed with standard techniques on TIL, NTTL, and PBL, and values were compared for each patient. Low expression of the TCR zeta chain and an almost complete absence of TCR epsilon chain and p56lck expression was observed in TIL (median values: 10% for zeta chain and 0% for epsilon and p56lck). In NTTL, these signal transduction molecules were expressed by a higher percentage of cells (60%, 50%, and 60%, respectively; p=0.000 vs. TIL), whereas PBL showed an almost normal expression of zeta and epsilon chains (80% and 90%, respectively; p=0.000 vs. TIL). Conversely, p56lck was detected in a greater proportion of NTTL than PBL (50% vs. 10%; p=0.001). The absence or the very low expression of signaling activation molecules in TIL compared with NTTL and PBL in renal cancer patients suggest that tumor-induced immunosuppression generally occurs or starts locally.

p27/kip1 expression in normal epithelium, benign and neoplastic breast lesions.

The development of cancer in the breast and in other sites is a complex process requiring a number of different genetic and epigenetic alterations. The accumulation of the genetic changes is thought to underlie the progression from precancerous lesions to carcinomas. The expression of p27/kip1 protein, a cyclin-dependent kinase inhibitor, was investigated by immunohistochemistry in normal epithelial specimens, benign alterations, and malignant lesions of the breast. The number of p27/kip1-positive cells ranged from none to more than 98% in the overall series. Wide ranges of p27/kip1-positive cells were consistently observed within each histological category, but the median value progressively decreased in typical hyperplasia and fibroadenoma, with an even more marked reduction in malignant lesions, compared with normal epithelium. Moreover, the percentage of cells expressing p27/kip1 in tumours was about three times lower in invasive than in in situ lesions and was inversely related to tumour size, but not to lymph node involvement. In conclusion, the degree to which p27 expression is altered in typical hyperplastic lesions and fibroadenomas indicates that the deregulation of p27 may occur very early on during breast cell transformation, but the usefulness of its determination to categorize subgroups of lesions at different risk of evolution remains somewhat doubtful.