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Radiofrequency thermal ablation (RFA) destroys tumoral tissue generating a local necrosis followed by marked inflammatory response with a dense T-cell infiltrate. In this study, we tested whether hepatocellular carcinoma thermal ablation can induce or enhance T-cell responses specific for hepatocellular carcinoma–associated antigens. Peripheral blood mononuclear cells derived from 20 patients with hepatocellular carcinoma were stimulated before and a month after RFA treatment with autologous hepatocellular carcinoma–derived protein lysates obtained before and immediately after RFA treatment. The effect of thermal ablation on memory T-cell responses to recall antigens [tetanus toxoid, protein purified derivative (PPD), Escherichia coli] was also assessed. T-cell reactivity was analyzed in an IFN-γ enzyme-linked immunospot assay and by intracellular IFN-γ staining. Treatment was followed by a significant increase of patients responsive either to tumor antigens derived from both the untreated hepatocellular carcinoma tissue (P < 0.05) and the necrotic tumor (P < 0.01) and by a higher frequency of circulating tumor-specific T cells. T-cell responses to recall antigens were also significantly augmented. Phenotypic analysis of circulating T and natural killer cells showed an increased expression of activation and cytotoxic surface markers. However, tumor-specific T-cell responses were not associated with protection from hepatocellular carcinoma relapse. Evidence of tumor immune escape was provided in one patient by the evidence that a new nodule of hepatocellular carcinoma recurrence was not recognized by T cells obtained at the time of RFA. In conclusion, RFA treatment generates the local conditions for activating the tumor-specific T-cell response. Although this effect is not sufficient for controlling hepatocellular carcinoma, it may represent the basis for the development of an adjuvant immunotherapy in patients undergoing RFA for primary and secondary liver tumors. (Cancer Res 2006; 66(2): 1139-46)

Transient T cell immunodeficiency is a common complication following hematopoietic stem cell transplantation. In breast cancer patients transplanted with autologous peripheral blood progenitor cells (PBPC) harvested after cytotoxic treatment with either cyclophosphamide or epirubicin plus paclitaxel, we evaluated T cells infused in grafts and in peripheral blood during the early reconstitution phase. We found that PBPC grafts harvested after treatment with epirubicin plus paclitaxel contained substantially larger numbers of T cells with less altered composition than after cyclophosphamide. Three months after high-dose cytotoxic chemotherapy, the numbers and the kinetics of circulating naive T cells, but not of memory and CD28- T cells, correlated positively with the number of naive T cells infused PBPC grafts. Finally, retrospective analysis of two cohorts of patients transplanted in different clinical settings with PBPC grafts harvested following cyclophosphamide or epirubicin plus paclitaxel showed apparently different susceptibilities to develop endogenous varicella zoster virus reactivation in the first year after high-dose cytotoxic chemotherapy. On the whole, these data indicate that number and composition of T cells in PBPC grafts vary according to the former cytotoxic therapy, and suggest that autologous transfer of T cells may accelerate the early T cell reconstitution phase and possibly ameliorate immune competence in patients rendered lymphopenic by high-dose chemotherapy.

Background In peripheral blood, myeloid markers identify a heterogeneous mixture of cells in transit from the bone marrow to peripheral tissues. Similarly, HLA-class II DR expression usually identifies mononuclear cells with the potential for developing antigen-presenting activity. We gathered putative antigen presenting cells bearing myeloid markers (My-APC) to study their composition by cell surface phenotype. Methods To gather and dissect My-APC phenotype while excluding lymphocytes and granulocytes, we developed a strategy based on staining red cell-lysed peripheral blood and gating cells bearing myeloid markers and physical parameters of large mononuclear cells. Results Phenotypic analysis within the My-APC gate showed three distinct populations. The largest fraction was constituted by CD14+ monocytes that extended into the other two populations, each expressing gradually lower levels of CD14 surface antigen along with increasing levels of CD16 and CD2, respectively. The CD16 and CD2 expression patterns extended from CD16+CD14+ or CD2+CD14+ double- positive intermediate cells toward each single positive subset, but they were reciprocally exclusive. Interestingly, CD2+CD14- cells within the My-APC gate were equivalent to myeloid dendritic cell precursors (pre-DC) defined previously by the absence of lineage markers and expression of HLA-DR and myeloid markers. Phenotypic analysis of each population revealed differences in the expression of costimulatory molecules and CD62L. Conclusions This novel analytical approach allowed us to distinguish circulating My-APC in three subsets and to identify relationships between monocytes and other related myeloid populations including DC. Cytometry 45:124–132, 2001. © 2001 Wiley-Liss, Inc.

Background Recurrence after PBSC transplantation in breast cancer (BC) patients may be related to the reinfusion of tumor cells contaminating the graft. We have developed a liquid culture (LC) method for the identification of viable epithelial tumor cells in PBSC collections. Methods Mononuclear fraction from PBSC harvests of BC patients undergoing high dose chemotherapy (HDCJ (adjuvant setting n =60, metastatic disease n =30) were seeded in petri dishes containing round cover slips. Cells were cultured for 3 weeks, then cover slips were stained with the pan-cytokeratin A45-B/B3 mAb and scored under a light microscope. Samples were considered positive when more than one adherent cell or a cluster of cells staining bright red was present. Results were compared with those obtained on cytospins prepared directly from the PBSC harvest. Specificity of the method was tested on lymphoma patients, collections: all were negative. The sensitivity, evaluated by serial dilutions of CG5 BC cell line, was 1 epithelial cell in 10 6 mononuclear cells. Results The percentage of positivity was superimposable in the two groups (adjuvant 25%, metastatic 24%). However, a significantly higher proportion of positive samples from metastatic vs adjuvant patients has shown the presence of tumor clusters (86% vs 33%, p =0,063), In 21% of all samples a discrepancy with the results obtained by immuno-cytochemical analysis (ICC) was found, mostly due to liquid-culture-positive/ICC-negative PBSCs. Discussion Our data suggest that LC assay may enhance the identification of viable disseminated epithelial tumor cells in PBSC grafts and might provide insights about their growth capacity.

Objective: The objective of this study was to assess the genetic effect of occupational exposure to antineoplastic agents. Method: The influence of occupational handling of cytotoxic drugs was investigated by monitoring the frequency of sister chromatid exchanges (SCE), the percentage of cells with high frequencies of SCE (high-frequency cells, HFC), and the frequency of somatic mutation at the T-cell receptor (TCR) locus in mononuclear cells of exposed hospital nurses. These parameters were also measured in healthy donors and in cancer patients at the time of the diagnosis and following the administration of high doses of cytotoxic drugs requiring stem cell support. Results: Our results show that (a) SCE and HFC values in occupationally exposed nurses do not differ from controls, (b) patients with newly diagnosed cancer or following chemotherapy show a number of SCE comparable to those of healthy donors, but a significantly different percentage of HFC, (c) cigarette smokers of all categories studied show higher frequencies of SCE and HFC as compared to nonsmokers, but the differences are not statistically significant, (d) the mutation frequency at the TCR locus in oncology nurses is higher, but not significantly different from the frequency in the control group, and (e) the increase of mutation frequency is statistically significant and seems to be dose dependent in patients treated with high-dose chemotherapy. Conclusions: Our data suggest that SCE frequency and HFC percentage are not reliable indicators of exposure to possible mutagenic/carcinogenic effects of antineoplastic drugs; on the contrary, our observations indicate that anticancer therapy induces somatic mutations at the TCR locus and suggest an association between exposure to cytotoxic agents and the increase in somatic mutations.

Nonhematopoietic malignant cells may express receptors for hematopoietic growth factors and respond to these peptides. The aim of the present study was to investigate whether small cell lung cancer (SCLC) cells may be stimulated to proliferate by hematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), which are currently used in clinical trials in combination with cytotoxic chemotherapy. We studied two SCLC cell lines, H69 and N417. The effects of GM-CSF and IL-3 were evaluated by studying clonal growth, 3H-thymidine incorporation, BUDR/DNA bivariate flow cytometry, c-myc and N-myc oncogene expression, and myeloid surface markers. Our experiments show that both GM-CSF and IL-3 can increase 3H-thymidine incorporation and cloning efficiency and reduce DNA synthesis time of H69 and, to a lesser extent, N417 cells, supporting the hypothesis that hematopoietic growth factors can stimulate the growth of some malignant nonhematopoietic cells in vitro. Further in vitro and in vivo studies are needed to determine whether clinical trials applying these factors for bone marrow recovery after chemotherapy of solid tumors may be hazardous by potentially stimulating growth of remaining tumor tissue.

Tamoxifen (TAM)-induced changes in proliferation kinetics of the human breast cancer cell line MCF-7 were investigated using dual parameter flow cytometry (FCM) of bromodeoxyuridine (BrdU) immunolabelling and of the expression of cell-cycle related proteins (the proliferating cell nuclear antigen, PCNA, and the non proliferation-specific protein, Statin), versus the DNA content. Single-parameter FCM DNA histograms confirmed that after 96 hours of treatment with 10(-7) M TAM the fraction of S-phase cells decreased significantly, with a simultaneous accumulation of cells in the G(0)/G(1) range of DNA content. In dual-parameter FCM cytograms, the fraction of BrdU-positive cells after TAM exposure was significantly lower than in the controls, and no unlabeled S-phase cells were found. The TAM-induced block in G(0)/G(1) phase was paralleled by a decrease in the number of cells with a DNA content typical of the S-phase expressing PCNA, and by an increase in Statin-positive (G(0)) cells. Upon readdition of 10(-9) M 17 beta-estradiol (E2) to the TAM-treated cultures, BrdU-labelling as well as PCNA expression levels increased significantly, whereas the fraction of Statin-positive cells remained higher than in the controls. The results obtained confirm that the TAM-induced inhibition of cell growth is associated with major changes in the cell cycle parameters of MCF-7 cells, and provide experimental evidence that two main mechanisms are operating: the accumulation of cells in G(1), before the onset of S-phase, and the exit of some cells from the cycling compartment; however, some of those that are blocked at G(0); cannot be totally reversed by estrogens and may be permanent; These data should be taken into account in the attempt to combine the antiestrogen treatment with chemotherapy more effectively.

Recovery of total T cell numbers after in vivo T-cell depletion in humans is accompanied by complex perturbation within the CD8+ subset. We aimed to elucidate the reconstitution of CD8+ T cells by separate analysis of putative naive CD95− CD28+, memory CD95+ CD28+ and CD28− T cell compartments after acute maximal depletion by high-dose chemotherapy (HD-ChT) in women with high-risk breast cancer. We found that recovery of putative naive CD8+ CD95− CD28+ and CD4+ CD95− CD28+ T cells, was compatible with a thymus-dependent regenerative pathway since their recovery was slow and time-dependent, their values were tightly related to each other, and their reconstitution patterns were inversely related to age. By analysing non-naive T cells, a striking diversion between putative memory T cells and CD28− T cells was found. These latter increased early well beyond normal values, thus playing a pivotal role in total T-cell homeostasis, and contributed to reduce the CD4 : CD8 ratio. In contrast, putative memory T cells returned to values not significantly different from those seen in patients at diagnosis, indicating that this compartment may recover after HD-ChT. At 3–5 years after treatment, naive T cells persisted at low levels, with expansion of CD28− T cells, suggesting that such alterations may extend further. These findings indicate that CD28− T cells were responsible for ‘blind’ T-cell homeostasis, but support the notion that memory and naive T cells are regulated separately. Given their distinct dynamics, quantitative evaluation of T-cell pools in patients undergoing chemotherapy should take into account separate analysis of naive, memory and CD28− T cells.

Based on our preliminary experience, we have further evaluated the capacity of the paclitaxel/epirubicin combination (at the dose of 175 and 90 mg/m(2), respectively) plus G-CSF to mobilize hematopoietic progenitors into the circulation.The study was conducted in a homogeneous cohort of 25 stage IV breast cancer patients showing response to three cycles of the same chemotherapy regimen and who were included in a high-dose chemotherapy program.In most cases (68%) more than 5_10(6) CD34+ cells/kg b.w. (the threshold fixed in our study) were collected by a single leukapheresis, 28% and 4% of patients requiring 2 and 3 procedures, respectively. Based on the CD34+ cell count in the peripheral blood, most of the leukaphereses (53%) were performed on day 11 after chemotherapy. More than 50 CD34+ cells/mL along with a preleukapheresis WBC count between 10 and 20_10(9)/L predicted that only a single harvest would be required in 100% of cases. The evaluation of the clonogenic potential of collected cells showed that a large number of committed colony-forming cells (CFCs) and more primitive long-term culture-initiating cell (LTC-IC) hematopoietic progenitors were present in 20 harvests studied.These data demonstrate that the epirubicin/paclitaxel combination followed by G-CSF, besides being a very active regimen in MBC, is effective in releasing large amounts of progenitor cells into the circulation which can then be safely employed to support myeloablative regimens.

We describe a new procedure for large-scale CB processing in the collection bag, thus minimizing the risk of CB contamination. A solution of 6% hydroxyethyl starch (HES) was added directly to the CB containing bag. After RBC sedimentation at 4 degrees C, the WBC-rich supernatant was collected in a satellite bag and centrifuged. After supernatant removal, the cell pellet was resuspended and the percent recovery of total WBC, CD34+ progenitor cells, CFU-GM and cobblestone area-forming cells (CAFC) evaluated. Results obtained with three different types of CB collection bags (300, 600 and 1000 ml) were analyzed and compared with those of an open system in 50 ml tubes. CB processing procedures in 300 and 1000 ml bags were associated with better WBC, CFU, CD34+ cell and CAFC recovery (83-93%). This novel CB processing procedure appears to be easy, effective and particularly suitable for large-scale banking under GMP conditions.

The aim of this study was the survey of cathepsin D determination in a large group of patients enrolled at several centers, under the coordination of the Italian Committee for Quality Control in the Oncological Laboratory. Cathepsin D was measured with the same method-ology, under control of an intra and interlaboratory quality control program, in order to verify the comparability of cathepsin D results from different institutions and to analyze the frequency of cathepsin D positive cases in subgroups of patients stratified according to other prognostic parameters. This retrospective study included 2575 patients with primary breast cancer evaluat-ed in 10 institutions. Cytosol from tumor tissue was the substrate for biochemical cathepsin D, estrogen receptor and progesterone receptor determination, with an interlaboratory quality control survey provided by the E.O.R.T.C. Receptor Group and the Italian Committee for Quality Control in the Oncological Laboratory. The results of the present study can be summarized as follows: 1) Cathepsin D is independent of menopausal status; 2) In spite of standardization of tissue handling and assay methods, different results may be obtained by different institutions. It is therefore essential that each laboratory calculates its own positive/negative cutoff values prior to any routine clinical use of the parameter. This should be a serious consideration when a multicenter study is planned.

We recently reported that in stroma-free cultures 11-33% of clonogenic cells derived from a bulk long-term culture [long-term culture-clonogenic cells (LTC-CC)] could be transduced by supernatant exposure or coculture of human CD34+ progenitors with MDR retroviral producer line A12M1. We reasoned that a stromal cell layer may generate niches in which LTC-CC could enter in the S-phase, thus becoming a more accessible target for gene delivery. In static culture studies in flasks, human engineered stromal cell line L87/4 or stromal murine M2-10B4 cells were used as feeder after irradiation, and CD34+ cells from either cord blood or peripheral blood of mobilized cancer patients were exposed to MDR supernatant for 7 consecutive days before 5-week culture for LTC-CC evaluation. In continuous flow perfusion culture studies, CD34+ cells were seeded over irradiated stromal murine M2-1OB4 cells and exposed to MDR supernatant for 7 days before LTC-CC evaluation. In mock-transduced controls,5% of LTC-CC were found to he viable after exposure to 10 ng/ml Taxol. In cells exposed to MDR supernatant in static stroma cultures, 68 +/- 4% of seeded LTC-CC were found to be drug resistant and express MDR mRNA as evaluated by reverse transcription-PCR analysis of single colonies. The addition of cytokines did not further enhance transfer efficiency. After MDR retroviral exposure in continuous flow cultures, 88 +/- 5% of LTC-CC were found to be drug resistant (P0.01 versus static stroma culture). P-glycoprotein expression in CD34+ cells was evaluated using flow cytometry and found to he higher after continuous flow versus static cultures. Finally, very high levels of P-glycoprotein expression after MDR supernatant exposure in the presence of stroma were confirmed by APAAP staining of cultured cells. We conclude that engineered stromal cell layers and continuous flow culture conditions can significantly enhance retroviral-mediated gene transfer into human hematopoietic progenitor cells.

In malignant cells multidrug resistance (MDR) is frequently associated with the expression of a 170 KDa P-glycoprotein (P-gp) in the plasma membrane. P-gp acts as an ATP-dependent efflux pump causing a decreased intracellular accumulation of structurally unrelated natural anticancer agents such as anthracyclines. Doxorubicin (DX) resistance is mostly related to the multidrug resistance gene product P-gp. In our experiments the revertant activity of medroxyprogesterone acetate (MPA) in comparison to that of the well known revertant agent verapamil (VRP) was investigated. In vitro tests were carried out on a DX-resistant variant (CG5/DX) obtained in our laboratory from the parental CG5 human breast cancer cell line by continuous exposure to the drug. The ability of MPA to modulate intracellular DX accumulation and to reverse MDR was evaluated. MPA appeared more active than VRP in reversing MDR, suggesting a possible role of this synthetic progestin as chemosensitizing agent in the clinical management of anthracycline-resistant breast cancer.

Hematopoietic growth factors (HGFs) are glycoproteins that control hemopoiesis. They have potential usefulness in a range of clinical conditions including the treatment of patients with myelosuppression induced by chemotherapy. Among HGFs, Stem Cell Factor (SCF) and Interleukin 6 (IL-6) are attracting interest for their capacity to stimulate early hematopoietic progenitors. Furthermore, their use in combination with late-acting growth factors with a more lineage-restricted potential (such as granulocyte colony-stimulating factor, G-CSF) might be expected to offer optimal marrow stimulation and usefulness in clinical oncology. Since non-hematopoietic malignant cells may express receptors for HGFs and respond to these peptides in vitro, we investigated clonal growth 3H-thymidine incorporation and cell cycle analysis by flow cytometry of 5 human solid tumor cell lines under the influence of SCF and IL-6 with or without G-CSF. Our experiments show that these cytokines have no effects on the proliferative capacity of the cell lines tested. Based on our and previously reported data, the use of these HGFs can be considered safe in cancer patients.

An MCF-7 human breast cancer line variant (MCF-7/MPA), resistant to medroxyprogesterone-acetate (MPA), was obtained by continuous exposure in vitro to the drug. MCF-7/MPA cells were grown in the presence of 12.5 x 10(-6) M MPA and were selected by increasing the concentration of the drug in the growth medium in a stepwise manner from 0.025 x 10(-6) M up to 12.5 x 10(-6) M. Comparative studies of cellular morphology, cytosolic steroid receptor content and P-Glycoprotein expression were performed on both MCF-7 parental line and MCF-7/MPA variant. MCF-7/MPA cells, when compared to the parental line, exhibit a different morphology in terms of membrane alterations, reduced content of cytosolic progesterone receptor, increased expression of P-glycoprotein along with reduction of Doxorubicin (Dx) activity on the growth of MCF-7/MPA resistant cells.

Several clinical studies have shown that recombinant human erythropoietin (rHuEpo) can ameliorate the anemia associated with hematologic malignancies and solid tumors. On the other hand, only a few studies have been performed to investigate whether rHuEpo can affect or modulate the growth of malignant cells.We studied the effects of rHuEpo (0.5 to 10 IU/mL) on clonogenic growth and cell kinetics in ten cell lines derived from both hematologic malignancies and solid tumors. Clonogenic assays were performed by plating 5 x 10(3) cells in agar, while the percentage of cells in S phase was assessed by DNA flow cytometry.rHuEpo did not affect either in vitro colony formation or S phase percentage in the human erythroid cell lines K-562 and HEL expressing erythropoietin receptors (40 receptors per cell). No effect of rHuEpo was observed in the remaining hematopoietic cell lines or in five solid tumor cell lines.These findings indicate that rHuEpo, even at very high concentrations, does not affect either clonogenic growth or DNA synthesis in the cell lines tested. Available evidence suggests that rHuEpo can be safely employed in all malignancies except acute myeloid leukemia.

We established and characterized two cell lines derived from glioblastoma multiforme. Both cell lines exhibited tumor cell morphology and growth kinetics and showed variable expression of glial fibrillary acidic protein (GFAP), S-100, fibronectin and vimentin. Cytofluorimetrical analysis of tumor samples showed a diploid DNA distribution, whereas permanent culture cells evolved to the hyperdiploid DNA content. Karyotype studies revealed cytogenetical abnormalities described in glial tumors including gain of chromosome 7, loss of chromosome 10 and presence of double minutes (DMs). Enhanced expression of Ha-ras and c-myc genes resulted from high p-21 and p-62 levels. The contemporary presence of TGF-alpha and EGF-Rc transcripts suggested an autocrine mechanism in the cell lines growth.

The significance of steroid receptors (SR) in human brain tumors is presently a field of intense investigation in order to clarify some aspects of the biological behavior of these neoplasms. We studied the relationship between the presence of steroid receptors and the production of metabolites of the arachidonic acid cascade which have been reported to have a role in the biological behavior of some human tumors. We found that some metabolites of arachidonic acid are produced in different amounts in brain tumors which either did or did not express some steroid receptors. In particular the PGE2 were higher in estrogen receptors (ER) positive meningiomas than in ER negative ones and 6-keto-PGF1 alpha, the stable metabolite of prostacyclin, is significantly higher in androgen receptors (AR) negative meningiomas than in AR positive ones. In neuroepithelial tumors the glucocorticoid receptors (GR) positive cases synthesized more TxB2 and less PGE2 than the GR negative ones. Our data seem to suggest that some correlations exist between the presence of some steroid receptors and arachidonic acid metabolite production.

Glucocorticoids, mainly dexamethasone (Dex) and methylprednisolone (Mp) are widely used as supportive therapy in the management of intracranial tumors. Their action on cells depends, in part, on the presence of a specific intracellular receptor (glucocorticoid receptor, GR) that binds the hormone leading to a sequence of biological events. In this study, we evaluated the presence of GR and its biological role in neuroepithelial tumors. Tumoral samples from 25 glioblastomas, 18 anaplastic astrocytomas and 14 astrocytomas were analyzed. Glucocorticoid specific binding proteins were detected in 38.6% of the cases using the dextran-coated charcoal method. A correlation was found between grade of anaplasia, sex, and GR positivity. The biological role of this receptor was investigated in 11 primary cell cultures derived from neuroepithelial tumors. For this purpose, different concentrations of Dex and Mp were added to the culture medium. When Dex doses ranging from 2 to 0.016 μg/ml were utilized, a significant stimulation of the cell growth was observed in receptor positive cultures. At the same doses, no modulation of the cell growth was observed in receptor negative cultures. Same responses were obtained when Mp was added to the culture medium.

The identification of oncogene products that are highly expressed in malignant glial cells could permit its use both in the diagnosis and in the grading of malignant brain tumors. In this study monoclonal antibodies to the Ha-ras and c-myc gene products (i.e. p21 and p62 oncoprotein respectively) were used in conjunction with flow cytometry (FCM) to characterize and quantitate the oncoprotein expression in 2 human glioblastoma-derived primary cell lines, 5 fresh malignant gliomas and 2 nonneoplastic brain tisue biopsy sample. In these experiments, the levels of the p21 and p62 fluorescence (i.e. Ha-ras and c-myc oncogene expression) in the malignant cultured cells and in the brain tumors samples were more than three times that of the nonneoplastic cells. FCM was also used to relate the quantity of the Ha-ras and c-myc oncoproteins present in the cells to their cell cycle phase. In all neoplastic specimens (which showed diploid DNA content), there was an equal distribution of p21 in G0/ G1, S and G2-M phases of the cell cycle while, the p62 underwent a twofold increase as the cells progresed from G0/G1 to G2-M. The results support the hypotesis of Haras and c-myc activation in human glioblastomas, also, FCM permits a simultaneous examination of the cytokinetic characteristics of tumor cells and oncogene expression, providing a useful tool in the study of molecular biology of brain tumors.

✓ Tissue samples from 57 patients with neuroepithelial tumors (25 glioblastomas, 18 anaplastic astrocytomas, and 14 astrocytomas) were analyzed in order to evaluate the presence of estrogen, progesterone, glucocorticoid, and androgen receptors. Glucocorticoid- and androgen-specific binding proteins were present in 38.6% and 21.6% of the cases, respectively. Only a few tumors showed estrogen or progesterone receptors. A correlation was found between grade of anaplasia, patient's sex and age, and presence of glucocorticoid and androgen receptors. The biological role of these two receptors was investigated in 10 primary cell cultures derived from neuroepithelial tumors. For this purpose, dexamethasone and testosterone were added to culture medium at different concentrations (from 50 to 0.016 µg/ml). A significant stimulation of the cell growth was observed in four of five glucocorticoid receptor-positive cultures when dexamethasone in doses ranging from 2 to 0.016 µg/ml was added to the culture. No modulation of the growth was observed in glucocorticoid receptor-negative cultures at the same doses. Higher dexamethasone doses induced a significant decrease of the growth index independently from the glucocorticoid receptor status. All of the cultures tested for testosterone activity were negative for androgen receptors. This hormone induced an inhibition of the growth index at doses ranging from 50 to 0.4 µg/ml. The data suggest that neuroepithelial tumors contain specific glucocorticoid and androgen binding proteins. Glucocorticoid receptors modulate the growth of cultured neuroepithelial tumors in the presence of different concentrations of dexamethasone.

In an attempt to clarify the role of beta interferon in vitro cell systems, the metabolic expression of dihydrofolate reductase (DHFR), succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH), by histochemical methods was studied. DHFR was also quantified by flow cytometry. Glioblastoma cell line with or without beta interferon was used. Three days after the treatment the DHFR reaction was much less intense than in the control. Quantitative data confirmed these results. Immediately afterwards, most cells exhibited much more intense reaction. These facts underlined that, in this biological model in vitro, beta interferon could reduce the synthesis of these enzymes only for a short period.

An extremely rapid and complete hematopoietic reconstitution occurs in patients receiving high dose chemotherapy when circulating progenitor cells (CPC), collected by leukapheresis, rather than marrow-derived cells, are reinfused. The amount of progenitor cells collected, which correlates with the speed of bone marrow reconstitution, is usually evaluated by the number of CD34+ cells and/or the number of clonogenic cells (CFU-GM). Since not all investigators agree with the correlation between these two parameters, we have compared CFU-GM growth and CD34+ cells in 27 leukapheresis from patients with solid tumors undergoing CPC transplantation. In our study a clear correlation between the two assays was shown (see fig. below) and we conclude that there is no need to perform both of them. Since CD34+ assay is simpler, less time-consuming and can be completed in a few hours versus weeks, we now perform only immunophenotypic analysis for clinical decision making. Download : Download high-res image (48KB) Download : Download full-size image

Recently the presence of steroid hormone receptors (SHR) have been reported in a series of nervous system human tumors, mainly in meningiomas. The possibility of characterizing brain tumors deserves attention because of the poor knowledge of endocrine-related behaviour of such tumors. The reliability of biochemical characterization of various receptor types should allow for better understanding of biological profile of primary and metastatic brain tumor, together with new avenues for combined modality treatment. In the future the highest priority might be assigned to the definition of the cut-off value of positivity for single receptors and to the receptor-endocrine modulation complex relationship for individual patients. A clear definition of the role of SHR in human brain tumors might improve our therapeutic potentials.

Tissue samples derived from two neuroepithelial tumours and five meningiomas were obtained at surgery from seven patients and cultured in order to study the effect of dexamethasone (DEX) and testosterone acetate (TA) on cell proliferation. Glucocorticoid and androgen receptors (GR, AR) were determined both on tissue samples (7 cases) and on five out of the seven cell cultures obtained by tumours. GR and AR were present respectively in 5 and in 4 out of the tumour specimens assayed and in 4/5 and 2/3 of the tested cell cultures. DEX activity on cell growth was tested on six cell cultures. Four of them showed a significant growth inhibition at the highest drug concentration. On the contrary, a significant growth stimulation was observed in four out of the five cultures, where GR were present, using low hormone concentrations. Treatment with pharmacological doses of TA caused a significant cytotoxicity in all the tested cultures. Low TA concentrations inhibited cell growth in one out of the two cell cultures which contained AR, but were ineffective in cultures lacking AR. Our preliminary results suggest a possible role in growth regulation by DEX and TA in intracranial tumours, on the basis of the presence of specific hormone receptors.

Steroid hormone receptors in intracranial tumors have been studied by many authors in recent years. Most of the research has been conducted on the oncotype meningioma while few data are available on gliomas.

The importance of evaluating receptors for estrogen and progestin in human breast cancer has been pointed out by many authors. In the absence of a reference standard, receptor assays must be controlled by intra and interlaboratory quality control programs. Much interlaboratory variability exists due to non-uniform analytical protocols, non-uniform ligands, intrinsic errors and also errors in computation methods. The goals of our Italian Qualtiy Control Program on Multicenter Trials are to standardize the anaytical procedures and computation methods. Twenty Italian laboratories participated in the Quality Control Program. Each specimen was assayed for steroid receptor content according to the standardized dextran-coated-charcoal method. Data were subjected to computerized analyses by 5 different methods of calculation (Scatchard plot, direct plot, Lineweaver-Burk method, Brunauer-Emmet-Teller analysis, single-point approach). The results were than evaluated to identify intra- and inter-assay variation coefficients and to define other statistical parameters. The authors suggest different calculation methods depending on the specific experimental and/or physiopathological conditions.

Tamoxifen is known to inhibit the growth of some human mammary carcinoma cells; this effect is accompanied by a decrease in the proportion of cells synthesizing DNA. In this work, flow cytometry of DNA and of bromodeoxyuridine labeling and the evaluation of the cell cycle-related antigens Ki-67, PCNA, and statin were used to investigate the changes in the proliferation kinetics of MCF-7 cells before and after treatment with 10(-7) M TAM. The treatment with TAM induced a significant decrease in the fraction of S-phase cells and an increase in those with a DNA content typical of G0/1 phase. The TAM-induced block in G0/1 is paralleled by a decrease in the frequency of cells expressing Ki-67 and PCNA, and by an increase in statin-positive (G0) cells. These results confirmed that the TAM-induced inhibition of cell growth is associated with major changes in the cell cycle parameters of MCF-7 cells, and provide the first experimental evidence that two main mechanisms are operating: the accumulation of cells in G1, before the onset of S-phase, and the exit of some cells from the cycling compartment.