Once-per-cycle pegfilgrastim in breast cancer patients treated with docetaxel/epidoxorubicin/cyclophosphamide.

The incidence of neutropenia following combination chemotherapy is significant in breast cancer and impairs patients' quality of life. Colony-stimulating factors significantly decrease the risk of febrile neutropenia (FN). Aim of the present study was to assess the efficacy and safety profile of once-per-cycle pegfilgrastim in reducing FN in breast cancer patients treated with docetaxel (75 mg/m(2)), epidoxorubicin (75 mg/m(2)), cyclophosphamide (500 mg/m(2)) administered every 3 weeks. Thirty-five breast cancer patients were enrolled. Chemotherapy was administered in adjuvant, neoadjuvant and metastatic setting respectively in 26, 4 and 5 patients. Toxicity was monitored with programmed clinical evaluation and blood sampling. All patients completed the therapeutic programme consisting of six cycles for overall 210 cycles. The FN appeared in 6 out of 35 patients (17%), requiring dose reduction in 3 patients. Hypertransaminasemia was registered in two patients. In one patient pegfilgrastim administration was stopped because of skin hypersensitivity reaction. In conclusion, pegfilgrastim was able to maintain doses and timing of docetaxel/epidoxorubicin/cyclophosphamide in almost all breast cancer patients treated in this series. The reduced need for daily administration of colony-stimulating factors, blood sampling, antibiotic therapy and hospitalization has a significant impact in terms of both quality of life and pharmaco-economic evaluations.

Vascular endothelial growth factor monitoring in advanced hepatocellular carcinoma patients treated with radiofrequency ablation plus octreotide: a single center experience.

Local therapies such as radiofrequency ablation (RFA) represent a valuable choice in limited hepatocellular carcinoma (HCC) and are increasingly used also in advanced tumors. Medical treatments generally gave frustrating results in advanced HCC especially if comorbidities exist. Several biologic non-chemotherapeutic drugs are currently tested in HCC and, among them, octreotide was evaluated in single series of HCC patients reporting conflicting results. We have treated a series of 35 patients affected by advanced HCC (26 M and 9 F; age range: 55-85 years, median: 73 years) with RFA followed by octreotide to primarily evaluate the safety of combined treatment and to give preliminary evaluation on its activity. We have also evaluated serum VEGF changes during the study. Child A and Child B represented 60% and about 34% of the cases, respectively. Only two patients with Child C compensated cirrhosis were included in this study. All patients have multiple liver HCC nodules and one had bone metastases. Two complete responses, 3 partial responses and 23 disease stabilization for at least three months were obtained (overall response rate, 14,2%; clinical benefit, 80%). Mean overall survival was 31.4 months. The combined treatment was well tolerated. Statistically significant correlation was found between serum VEGF and tumor progression. In conclusion, the combination of RFA and octreotide was active in advanced HCC, however, confirmation in a larger series is required.

Daily low-dose subcutaneous recombinant interleukin-2 by alternate weekly administration: antitumor activity and immunomodulatory effects.

A phase II clinical trial of subcutaneous recombinant Interleukin 2 (rIL-2) given by 5 days pulses followed by a 9 days rest has been performed in patients affected by renal cell carcinoma, malignant melanoma and colorectal cancer. A total of 25 patients entered the study, completed at least six courses of treatment, and were evaluable for toxicity and response to treatment. This schedule of subcutaneous rIL-2 was well tolerated and no World Health Organization grade 3 side effects were observed. A 33.3% response rate was recorded in patients affected by renal cell carcinoma, although no major responses were achieved in patients with malignant melanoma and colorectal cancer. A durable increase of natural killer activity retained by poeripheral blood mononuclear cells was demonstrated in these patients and was paralleled by increased serum levels of interferon gamma and tumor necrosis factor a without changes of circulating interleukin-1d. It is concluded that this schedule of pulse administration of subcutaneous rIL-2 has antitumor activity in renal cell carcinoma and produces durable biomodulatory effects.

Extracellular adenosine 5' triphosphate involvement in the death of LAK-engaged human tumor cells via P2X-receptor activation.

This study reports that extracellular ATP is a critical factor involved in LAK cell-mediated cytotoxicity. Human colon carcinoma LoVo cells were resistant to LAK cells as well as to ATP, while their multidrug resistant (MDR-1+) derivative, LoVo-Dx cells, were sensitive to both LAK and ATP. LoVo-Dx cells, became resistant to LAK cells and ATP after 48 h pretreatment with Phorbol 12-Myristate-13-Acetate (PMA), while 48 h pretreatment with verapamil in parallel sensitized LoVo cells to LAK cells and to ATP as well. The sensitivity to ATP and LAK cells was not related to the expression of extracellular ecto-ATPase activity on cell targets membranes. Conversely, apyrase, an enzyme with powerful ecto-ATPase activity, abolished the LAK- and ATP-mediated cytotoxicity. Furthermore, ADP-beta-S, an antagonist of ATP, abolished both LAK and ATP-mediated cell killing. Purine binding sites have been detected by radioreceptor assays with ADP-beta[35S] on the cell surface of ATP and LAK-sensitive LoVo-Dx cells. By contrast, no nucleotide receptor was found on the ATP and LAK-resistant cells. Such a putative cytotoxic purinoreceptor has been categorized as P2x purinergic receptor by a panel of synthetic nucleotides. These results demonstrate that extracellular ATP is needed for an efficient LAK cell-mediated killing of tumor cells. We propose that ATP acts as a natural amplifier of physical, or immune cytotoxic damages since it may be released in large amounts from target cells injured by several cytotoxic mediators secreted by LAK effectors.

Differential sensitivity to non-major histocompatibility complex-restricted recombinant interleukin 2-activated lymphocyte killing of human mammary epithelial MCF-10A cells overexpressing oncogenes or protein kinase A subunits.

The sensitivity of human tumor cells to activated lymphocytes is considered to play an essential role in the antitumor activity of recombinant interleukin-2 (rIL-2)-based immunotherapy. We have investigated the effects of several genes involved in the regulation of cell growth and transformation on the sensitivity of human mammary epithelial MCF-10A cells to non-MHC-restricted, rIL-2-activated lymphocytes. Therefore, the lysability of MCF-10A cells overexpressing activated oncogenes (Ha-ras, erbB-2, and a mutated p53), growth factors [transforming growth factor alpha (TGFalpha)], or cAMP-dependent protein kinase A subunits (RIalpha, RIIbeta, and Calpha) was evaluated comparatively at different effector:target ratios by a 51Cr release assay. Parental MCF-10A, MCF-10A p53-mutated, and MCF-10A RIIbeta cells showed an intermediate sensitivity. Lysability was increased significantly in MCF-10A Ha-ras, MCF-10A TGFalpha, and MCF-10A RIalpha cells, reduced in MCF-10A Calpha cells, and completely abrogated in MCF-10A erbB-2 cells. These differences could not be explained by simple changes in the cell surface expression of MHC class I and intercellular adhesion molecule-1 proteins or by secretion of TGFbeta. Treatment with TAb 250, a mouse anti-p185(erbB-2) monoclonal antibody, or down-regulation of p185(erbB-2) expression resulted in circumvention of MCF-10A erbB-2 cell resistance. We conclude that molecular changes at the single-gene level resulting in alterations of intracellular signaling and/or cell transformation modulate sensitivity of human mammary epithelial cells to non-MHC-restricted, rIL-2-induced cytotoxicity, regardless of MHC class I and/or intercellular adhesion molecule-1 expression or TGFbeta secretion. Furthermore, anti-p185(erbB-2) monoclonal antibodies may be useful as adjuncts to rIL-2 treatment in patients with erbB-2-overexpressing tumors.

alpha-Interferon potentiates epidermal growth factor receptor-mediated effects on human epidermoid carcinoma KB cells.

The molecular mechanisms underlying the growth inhibition of human tumor cells induced by recombinant interferon-alpha (IFN alpha) are mostly unknown. It has been proposed that this effect could be related to down-regulation and/or impaired function of peptide growth factor receptors (PGF-Rs) in tumor cells exposed to IFN alpha. However, we have previously described that IFN alpha-induced growth inhibition of human epidermoid carcinoma cells is paralleled by up-regulation of epidermal growth factor receptor (EGF-R). Here we report that an increase in EGF-R synthesis is detectable after 3 hr of exposure to cytostatic concentration of IFN alpha in epidermoid KB tumor cells. In these experimental conditions IFN alpha does not depress and even potentiates EGF-R function. IFN alpha-treated KB cells retain sensitivity to the cytotoxic activity of the anti-EGF-R 225 monoclonal antibody (MAb), which acts through receptor blockade, and are sensitized to the growth-promoting effect of EGF. EGF-induced tyrosine (tyr) phosphorylation both of total cellular protein extracts and of the immunoprecipitated EGF-R is increased in IFN alpha-treated cells. We conclude that a cross-talk between IFN alpha and EGF occurs in KB cells since IFN alpha, at cytostatic concentration, potentiates the effects mediated by the EGF-R.

Bryostatin 1 enhances lymphokine activated killer sensitivity and modulates the beta 1 integrin profile of cultured human tumor cells.

Bryostatin 1 interferes with protein kinase C (PKC) signaling which is involved in the activation of human and murine cytotoxic T lymphocytes, and in the growth and differentiation of tumor cells. Bryostatin 1 has immunomodulating and antitumor properties as demonstrated by preclinical and clinical studies. Here we report that bryostatin 1 increases the susceptibility to lymphokine activated killers and modifies the pattern of beta 1 integrin expression of human tumor cells. On the basis of these results the use of bryostatin 1 in combination with immunostimulating cytokines such as interleukin-2 in the treatment of human cancer is suggested.

Phase I clinical study with 8-chloro-cAMP and evaluation of immunological effects in cancer patients

The site-selective cyclic AMP analogue 8-chloro-cAMP (8-Cl-cAMP) is able to inhibit the growth of a wide variety of cancer cell lines in vitro and in vivo. 8-Cl-cAMP has been extensively investigated as a new potential anticancer agent and, more recently, preclinical Phase I studies have been conducted in animal models to study its toxicity. We have conducted the first Phase I trial with 8-Cl-cAMP to define the maximum tolerated dose, toxicity, plasma drug levels, and immunological effects in patients with cancers refractory to standard treatments. We have administered 36 courses of 8-Cl-cAMP to 17 patients by continous i.v. infusion of the drug for 5 days/week for 2 weeks followed by a 1-week rest period. Six increasing dose levels, from 0.01 to 0.25 mg/kg/h, were explored. Drug plasma levels were determined and the expression of interleukin 2 receptor alpha amount of natural killer cells, and cytolytic activity against K562 cells were measured in peripheral blood lymphocytes. A grade 4 and a grade 3 increase in serum creatinine and a grade 2 increase in blood urea nitrogen observed in two patients were the dose-limiting toxicity. The maximum tolerated dose (0.2 mg/kg/h) determined a grade 1 increase in serum creatinine. An increase in calcium levels was observed in several patients. The 8-Cl-cAMP plasma concentrations obtained at the steady state were in the range previously shown to be effective for cancer cell growth inhibition in vitro. Interleukin 2 receptor alpha expression, natural killer cell number, and cytolytic activity from peripheral blood lymphocytes were markedly increased after 8-Cl-cAMP administration at all dose levels. In conclusion, at doses below the maximum tolerated dose, 8-Cl-cAMP was not toxic but reached plasma concentrations in the potential therapeutic range for growth inhibition. Moreover, 8-Cl-cAMP determined a marked biomodulatory effect and showed antitumor activity.

Alpha-interferon induces depletion of intracellular iron content and upregulation of functional transferrin receptors on human epidermoid cancer KB cells.

We have demonstrated that interferon-alpha (IFN alpha) upregulates the epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma cells. Here we report that IFN alpha induces growth inhibition and upregulation of transferrin receptor (TRF-R) on epidermoid cancer KB cells. IFN alpha does not alter TRF-R affinity for its ligand and induces a two-fold increase of TRF binding sites. IFN alpha does not modify receptor internalization and cycling. Intracellular iron levels are known to regulate TRF-R expression: we have, therefore, evaluated whether changes in the iron content could be determined by IFN alpha. Iron levels are transiently increased after addition of fresh growth medium in untreated controls but not in KB cells exposed for 48 h to IFN alpha. Iron depletion is however completely reversed 24 h later when maximal TRF-R upregulation occurs in IFN alpha-treated cells. We suggest that IFN alpha-induced iron depletion elicits a homeostatic cellular response through upregulation of TRF-R.

Transmembrane ion flux modifiers verapamil and ouabain modulate cytotoxic effects of extracellular ATP on human tumor-cells in-vitro.

Extracellular ATP can often induce tumor cell cytotoxicity; however, the molecular mechanisms of these effects are mostly unknown. We investigated whether modifications in transmembrane ion fluxes are involved in the determination of ATP-cytotoxicity. We have found that cultured human tumor cells derived from colon (LoVo) and lung (A549) carcinomas are resistant to ATP, while LoVo-Dx cells (a doxorubicin-resistant derivative of LoVo cells) and melanoma GLL-19 cells are highly sensitive to this nucleotide. 48 h exposure to 100 nM verapamil increases sensitivity of LoVo and A549 cells to ATP. This effect is completely reverted by the addition of the calcium ionophore A23187. Conversely, 4 h exposure to 100 nM ouabain, which blocks the Na+/K+ exchange pump, neutralyzes ATP cytotoxicity against LoVo-Dx and GLL-19 cells. Furthermore, ATP-mediated cytotoxic effects on these cells are completely antagonized by ADP-beta-S, a non hydrolyzable analogue of ATP. These findings suggest that transmembrane ion flux modifications play a critical role in ATP cytotoxicity and that ATP binding on surface receptors and probably nucleotide hydrolysis are needed for inducing cytotoxicity on human tumor cells.