Pharmacological effects of the simultaneous and sequential combinations of trifluridine/tipiracil (TAS-102) and 5-fluorouracil in fluoropyrimidine-sensitive colon cancer cells.

The aim of this study was to investigate possible synergistic effects in vitro of trifluridine/tipiracil (TAS-102) and 5-fluoruracil (5-FU) on fluoropyrimidine-sensitive colon cancer cell lines of different mutational status in order to build a rational basis for the future use of this combination therapy in adjuvant settings or as a first-line treatment for metastatic disease. Proliferation assays were performed on HT-29 (B-raf mutated), SW-620 (ras mutated), and Caco-2 (wild type) colon cancer cell lines exposed to 120-h treatments of 5-FU, TAS-102 and their different combination schedules (simultaneous, sequential and reverse) at equimolar and non-equimolar ratios. The synergistic, additive and antagonistic effects of 5-FU and TAS-102 were determined by the combination index (CI) and dose reduction index (DRI). Our preclinical in vitro results may suggest an apparently counterintuitive but strongly synergistic combination of 5-FU and TAS-102 in fluoropyrimidine-sensitive colon cancer cells allowing a marked theoretical reduction in the administered doses of both drugs. In particular, this association seems to be highly effective in wild-type colon cancer cells, both in sequential and simultaneous schedules. Together, these data may build a rational basis for the future use of TAS-102 combined with 5-FU in adjuvant settings, or as a first-line treatment for metastatic disease.

Pharmacological effects of vinorelbine in combination with lenvatinib in anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is a rare neoplasia with a poor prognosis. Proliferation and apoptosis assays were performed on ATC cell lines (8305C, 8505C) exposed to vinorelbine, lenvatinib, as well as to concomitant combinations. ABCB1, ABCG2 and CSF-1 mRNA expression was evaluated by real time PCR. The relative levels of pospho Akt were investigated as part of a human phospho-kinase array analysis, and CSF-1 and VEGFR-2 protein levels were measured by ELISA. The intracellular concentration of lenvatinib in ATC cells was measured by combined reversed-phase liquid chromatography-tandem mass spectrometry. An ATC subcutaneous xenograft tumor model in nude mice was treated with vinorelbine, lenvatinib, or vinorelbine plus lenvatinib. After treatment with vinorelbine, lenvatinib, a significant antiproliferative effect in ATC cell lines was observed. The concomitant treatment of vinorelbine and lenvatinib revealed synergism for all the fractions of affected cells. A decrease in ABCB1 expression was reported in both ATC cell lines treated with the lenvatinib plus vinorelbine combination, as was an increase in the intracellular concentration of lenvatinib. The combination caused a decrease in Akt, GSK3α/ß, PRAS40 and Src phosphorylation, and in both CSF-1 mRNA and protein levels. In the subcutaneous tumor model, the combination reduced the tumor volume during the treatment period. Our results establish the synergistic ATC antitumor activity of a vinorelbine and lenvatinib combination.

Pharmacokinetic analysis of metronomic capecitabine in refractory metastatic colorectal cancer patients.

The aim of the present study was to assess the pharmacokinetics (PK) of metronomic capecitabine and its metabolites in a population of refractory metastatic colorectal cancer (mCRC) patients. Thirty-four patients (M/F, 22/12) with a diagnosis of mCRC received capecitabine 800 mg p.o. twice a day and cyclophosphamide 50 mg/day p.o. Blood samples were collected at baseline, 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h and 5 h at day 1 after capecitabine administration. Plasma concentrations of capecitabine and its metabolites were measured by high performance liquid chromatography and the main PK parameters were calculated. Maximum plasma concentrations (Cmax) of capecitabine (11.51 ± 9.73 µg/ml) occurred at 0.5 h, whereas the Cmax of 5'-deoxy-5-fluorocytidine (5'-DFCR; 2.45 ± 2.93 µg/ml), 5'-deoxy-5-fluorouridine (5'-DFUR; 6.43 ± 8.2 µg/ml), and 5-fluorouracil (5-FU; 0.24 ± 0.16 µg/ml) were found at 1 h, 1.5 h and 1 h, respectively. Capecitabine, 5'-DFCR, 5'-DFUR and 5-FU AUCs at day 1 were 21.30 ± 10.78, 5.2 ± 4.6, 19.59 ± 3.83 and 0.66 ± 0.77 hxµg/ml, respectively. In conclusion, low doses of capecitabine were rapidly absorbed and extensively metabolized, achieving measurable plasma concentrations in a heavily pretreated population of patients.

Association of Glutathione S-Transferase P-1 (GSTP-1) rs1695 polymorphism with overall survival in glioblastoma patients treated with combined radio-chemotherapy.

Glioblastoma (GBM) is the most frequent malignant primary brain tumor in adults and, despite recent advances, the prognosis for this cancer remains dismal. The aims of this study were to test the influence of XRCC1 rs25487, XRCC3 rs861539, XRCC3 rs1799794, RAD51 rs1801320 and GSTP-1 rs1695 single nucleotide polymorphisms on progression free survival (PFS) and overall survival (OS) in GBM patients treated with radiotherapy (RT) and temozolomide (TMZ). Fifty GBM patients treated with upfront radio-chemotherapy (RT 60 Gy/30 sessions; TMZ 75 mg/m2 during RT and 200 mg/m2 days 1 â†’ 5 every 28 days) were enrolled. Survival curves were calculated using the Kaplan-Meier method, and the log-rank test was used to evaluate differences between curves. A trend to a statistically significant association with PFS in univariate and multivariate COX regression analysis was found with GSTP-1 rs1695 polymorphism (p = 0.087 and p = 0.097 on univariate and multivariate analyses, respectively). Conversely, the same GSTP-1 rs1695 SNP revealed a statistically significant association with OS (p = 0.007 and p = 0.042 on univariate and multivariate analysis, respectively). Our pharmacogenetic prospective study suggests that GSTP-1 rs1695 genotypes can be associated with different OS in GBM patients treated with RT and TMZ.

Impact of CTLA-4 blockade in conjunction with metronomic chemotherapy on preclinical breast cancer growth.

BACKGROUND: Although there are reports that metronomic cyclophosphamide (CTX) can be immune stimulating, the impact of its combination with anti-CTLA-4 immunotherapy for the treatment of cancer remains to be evaluated. METHODS: Murine EMT-6/P breast cancer, or its cisplatin or CTX-resistant variants, or CT-26 colon, were implanted into Balb/c mice. Established tumours were monitored for relative growth following treatment with anti-CTLA-4 antibody alone or in combination with; (a) metronomic CTX (ldCTX; 20 mg kg-1 day-1), b) bolus (150 mg kg-1) plus ldCTX, or (c) sequential treatment with gemcitabine (160 mg kg-1 every 3 days). RESULTS: EMT-6/P tumours responded to anti-CTLA-4 therapy, but this response was less effective when combined with bolus plus ldCTX. Anti-CTLA-4 could be effectively combined with either ldCTX (without a bolus), or with regimens of either sequential or concomitant gemcitabine, including in orthotopic EMT-6 tumours, and independently of the schedule of drug administration. Tumour responses were confirmed with CT-26 tumours but were less pronounced in drug-resistant EMT-6/CTX or EMT-6/DDP tumour models than in the parent tumour. A number of tumour bearing mice developed spontaneous metastases under continuous therapy. The majority of cured mice rejected tumour re-challenges. CONCLUSIONS: Metronomic CTX can be combined with anti-CTLA-4 therapy, but this therapy is impaired by concomitant bolus CTX. Sequential therapy of anti-CTLA-4 followed by gemcitabine is effective in chemotherapy-naive tumours, although tumour relapses can occur, in some cases accompanied by the development of spontaneous metastases.

Clinical, pharmacodynamic and pharmacokinetic results of a prospective phase II study on oral metronomic vinorelbine and dexamethasone in castration-resistant prostate cancer patients.

The aim of the present study was to evaluate clinical activity, and the pharmacodynamic and pharmacokinetic profiles, of oral metronomic vinorelbine (VNR) plus dexamethasone (DEX) in metastatic castration-resistant prostate cancer (mCRPC) patients. Fourty-one patients (92 % chemotherapy-resistant) received 30 mg/day VNR p.o. thrice a week plus 1 mg/day DEX p.o. until disease progression. Plasma soluble B cell antigen 7 homolog 3 (sB7-H3), vascular endothelial growth factor (VEGF), and thrombospondin-1 (TSP-1), were measured by ELISA. Plasma VNR was detected using a LC-MS-MS system. The fraction of patients free of progression, defined by criteria of the Prostate Cancer Clinical Trials Working Group 2, at 3 months was 61 %. PSA decrease ≥50 % from baseline was observed in 35 % of patients. Median PFS and OS were 4 months (95 % CI, 2.8-6.9) and 17.5 months (95 % CI, 10.8-24.5), respectively. Toxicity was mild, and no grade 4 toxicities were found. The mean plasma VNR Cmax ranged from 1 to 2.7 ng/ml (Tmax 1.1 h) and no evidence of drug accumulation was found. A moderate relationship was found between plasma sB7-H3 and PSA values (r = 0.565; P = 0.0094) at the baseline. Increased PFS (11.3 vs. 2.8 months; P = 0.0298) was observed in patients with sB7-H3 levels <30.25 ng/mL. Plasma VEGF AUC0-24day increased in non-responders (P < 0.0001), whereas responders maintained higher plasma TSP-1 AUC0-24day (P = 0.0063). In conclusion, metronomic VNR plus DEX showed favourable activity, and a low toxicity profile, in mCRPC patients. Plasma sB7-H3, VEGF and TSP-1 levels are potential pharmacodynamic markers at the reached low plasma concentrations of vinorelbine metronomically administered.

Aflibercept administration in neovascular age-related macular degeneration refractory to previous anti-vascular endothelial growth factor drugs: a critical review and new possible approaches to move forward.

PURPOSE: The recent introduction of anti-VEGF drugs has widely changed the prognosis of exudative age-related macular degeneration (AMD), even if a variable percentage of patients showed an insufficient response. Aflibercept is a new anti-VEGF drug approved by FDA for the treatment of exudative AMD with a wider binding capacity than either bevacizumab or ranibizumab. Therefore, the purposes were as follows: (i) to report anatomical and functional outcomes of switching from bevacizumab/ranibizumab to aflibercept previously described in the scientific literature, (ii) to hypothesize the possible pathophysiological mechanisms of the resistance and tachyphylaxis to anti-VEGF drugs, and (iii) to suggest possible clinical actions to increase the chances of success for such difficult cases. METHODS: We reviewed the available scientific literature in Medline, Cochrane database, Current Contents, PubMed, and cross-referencing from identified articles, regarding the treatment of exudative AMD patients refractory to bevacizumab and/or ranibizumab and switched to aflibercept monotherapy. We included in this review all the cases in which the diagnosis of refractory or resistant exudative AMD was properly made, and the results of at least one aflibercept injection were described. FINDINGS: We reported the outcomes of 21 papers for a total of 1066 eyes affected by exudative AMD resistant to previous anti-VEGF drug injections and switched to aflibercept. Enrolled reports were divided into two groups: 5 prospective reports and 16 retrospective reports. All the reported papers conclude their analysis, stating that switching from bevacizumab/ranibizumab to aflibercept injections can improve outcomes successfully in refractory neovascular AMD patients. IMPLICATIONS: Analysis of the papers reported in this review demonstrates that switching from bevacizumab/ranibizumab to aflibercept injections can improve outcomes successfully in refractory neovascular AMD patients. The mechanism for these effects is not yet completely understood.

Docetaxel plus oral metronomic cyclophosphamide: a phase II study with pharmacodynamic and pharmacogenetic analyses in castration-resistant prostate cancer patients.

BACKGROUND: Docetaxel plus prednisone is currently the standard first-line treatment in metastatic castration-resistant prostate cancer (mCRPC). The aim of this study was to assess the clinical activity and pharmacodynamic/pharmacogenetic profile of docetaxel plus prednisone in combination with metronomic cyclophosphamide in mCRPC patients. METHODS: Forty-one chemotherapy-naive patients received docetaxel (60 mg/m(2) intravenously every 3 weeks up to 12 cycles) and, from day 2, prednisone 10 mg/day, celecoxib 400 mg/day, and metronomic cyclophosphamide 50 mg/day, continuously. Plasma VEGF and bFGF were detected by ELISA. Real-time PCR-SNP analysis of VEGF gene was performed using an ABI PRISM 7900HT SDS and TaqMan SNP genotyping. RESULTS: Eighty-seven percent of patients were free of progression at 6 months. A decrease in prostate-specific antigen ≥50% was observed in 82% of 39 evaluable patients, with a median time to progression of 12.3 months. Grade 3 adverse events were neutropenia (5%), thrombocytopenia, diarrhea, and stomatitis (2.5%). Median PFS and OS were 14.9 months (95% CI, 9.2-15.3 months) and 33.3 months (95% CI, 23-35.6 months), respectively. Of 11 patients (28%) with evaluable disease, 5 (44%) achieved a complete response, 2 (11%) a partial response, and 2 (11%) stable disease, whereas 2 showed disease progression. The -1154A/G VEGF polymorphism, plasma VEGF, and bFGF after the first cycle of chemotherapy may represent useful pharmacodynamic markers to predict better outcomes. CONCLUSIONS: The combination of docetaxel and oral metronomic chemotherapy is effective and well tolerated in mCRPC patients and may deserve further evaluation.

Analysis of acquired resistance to metronomic oral topotecan chemotherapy plus pazopanib after prolonged preclinical potent responsiveness in advanced ovarian cancer.

An alternative or follow-up adjunct to conventional maximum tolerated dose (MTD) chemotherapy now in advanced phase III clinical trial assessment is metronomic chemotherapy--the close regular administration of low doses of drug with no prolonged breaks. A number of preclinical studies have shown metronomic chemotherapy can cause long term survival of mice with advanced cancer, including metastatic disease, in the absence of overt toxicity, especially when combined with targeted antiangiogenic drugs. However, similar to MTD chemotherapy acquired resistance eventually develops, the basis of which is unknown. Using a preclinical model of advanced human ovarian (SKOV-3-13) cancer in SCID mice, we show that acquired resistance can develop after terminating prolonged (over 3 months) successful therapy utilizing daily oral metronomic topotecan plus pazopanib, an oral antiangiogenic tyrosine kinase inhibitor (TKI). Two resistant sublines were isolated from a single mouse, one from a solid tumor (called KH092-7SD, referred to as 7SD) and another from ascites tumor cells (called KH092-7AS, referred to as 7AS). Using these sublines we show acquired resistance to the combination treatment is due to tumor cell alterations that confer relative refractoriness to topotecan. The resistant phenotype is heritable, associated with reduced cellular uptake of topotecan and could not be reversed by switching to MTD topotecan or to another topoisomerase-1 inhibitor, CPT-11, given either in a metronomic or MTD manner nor switching to another antiangiogenic drug, e.g. the anti-VEGFR-2 antibody, DC101, or another TKI, sunitinib. Thus, in this case cross resistance seems to exist between MTD and metronomic topotecan, the basis of which is unknown. However, gene expression profiling revealed several potential genes that are stably upregulated in the resistant lines, that previously have been implicated in resistance to various chemotherapy drugs, and which, therefore, may contribute to the drug resistant phenotype.

Clinical, pharmacokinetic and pharmacodynamic evaluations of metronomic UFT and cyclophosphamide plus celecoxib in patients with advanced refractory gastrointestinal cancers.

AIMS: To evaluate UFT and cyclophosphamide (CTX) based metronomic chemotherapy plus celecoxib (CXB) for the treatment of patients with heavily pre-treated advanced gastrointestinal malignancies. METHODS: Thirty-eight patients received 500 mg/mq(2) CTX i.v bolus on day 1 and, from day 2, 50 mg/day CTX p.o. plus 100 mg/twice a day UFT p.o. and 200 mg/twice a day CXB p.o. Tegafur, 5-FU, 5-FUH(2), GHB and uracil pharmacokinetics were assessed. Plasma vascular endothelial growth factor (VEGF), soluble VE-cadherin (sVE-C) and thrombospondin-1 (TSP-1) levels were detected by ELISA and real-time PCR of CD133 gene expression on peripheral blood mononuclear cell was also performed. RESULTS: Seventeen patients (45%) obtained stable disease (SD) with a median duration of 5.8 ms (range, 4.2-7.4). Median progression free survival (PFS) and overall survival (OS) were 2.7 ms (95% CI, 1.6-3.9 ms) and 7.1 ms (95% CI, 4.3-9.9 ms), respectively. No toxicities of grade >1 were observed. Pharmacokinetics of 27 patients (13/14, SD/progressive disease, PD) after the first treatment of UFT revealed that 5-FU AUC and C(max) values greater than 1.313 h × µg/ml and 0.501 µg/ml, respectively, were statistically correlated with stabilization of disease and prolonged PFS/OS. VEGF and sVE-C plasma levels were greater in the PD group when compared to SD group. CD133 expression increased only in the PD patients. CONCLUSION: Metronomic UFT and CTX with CXB in heavily pre-treated gastrointestinal patients were well tolerated and associated with interesting activity. Potential predictive pharmacokinetic parameters and pharmacodynamic biomarkers have been found.

The rs2071559 AA VEGFR-2 genotype frequency is significantly lower in neovascular age-related macular degeneration patients.

In this prospective, case-control genetic study, 120 consecutive neovascular age-related macular degeneration (AMD) cases and 78 controls were enrolled. Two SNPs (rs2071559 and rs1870377) of VEGF-A receptor-2 (VEGFR-2) gene were analyzed with the technique of Real-Time PCR to investigate a genetic link between AMD and VEGFR-2 gene polymorphisms in Italian patients. The frequency of the VEGFR-2 genotype rs2071559 AA was significantly lower (18.33%) in patients with AMD than in the control subjects (34.62%; P = 0.0095, chi-square test; P(corr) = 0.038; OR = 0.42, 95% CI 0.22 to 0.82). In conclusion, although with the limitations of a small sample size and the few SNPs studied, this study demonstrates a lower frequency of VEGFR-2 rs2071559 AA genotype in an AMD patient population, suggesting future studies on the role VEGFR-2 SNPs.

Active metronomic vinorelbine schedules decrease plasma interleukin-2 levels in mice with Lewis lung carcinoma.

The aim of our study was to investigate the effects of metronomic vinorelbine (mVNR) in a tumor model of Lewis Lung (LL) cancer in immunocompetent C57BL/6 mice, looking at the plasma levels of interleukin-2 (IL-2) and interleukin-8 (IL-8). mVNR caused a concentration-dependent antiproliferative effect in vitro on LL/2 cells. The in vivo experiment showed the significant antitumor effects of mVNR at the dose of 4 mg/Kg and 5 mg/Kg, 3 times/week, and the significant dose-dependent decrease of IL-2 concentrations in plasma samples. Conversely, such an effect was not observed for IL-8. A significant decrease in microvessel density was also found at both the active mVNR doses. In conclusion, our study confirmed the activity of mVNR in an immunocompetent model of lung carcinoma and suggest multiple mechanisms of action, including the modulation of IL-2 levels.

Pharmacodynamic biomarkers in metronomic chemotherapy: multiplex cytokine measurements in gastrointestinal cancer patients.

Metronomic chemotherapy has shown promising antitumor activity in a number of malignancies. We previously reported a phase II clinical trial of metronomic UFT (a 5-fluorouracil prodrug; 100 mg/twice per day p.o.) and cyclophosphamide (CTX; 500 mg/m2 i.v. bolus on day 1 and then 50 mg/day p.o.) plus celecoxib (200 mg/twice a day p.o.) in 38 patients with advanced refractory gastrointestinal tumors. The mechanisms of action of metronomic chemotherapy include inhibition of angiogenesis, direct cytotoxic effects on cancer cells, and, at least for drugs such as CTX, activation of the immune system. To further evaluate the latter, we carried out an immune system multiplex 14-cytokine profiling of plasma samples that were available (for day 0, day 28, and day 56) from 31 of the 38 patients in the above-noted clinical trial. Our results show that pre-treatment plasma-level cutoffs of interferon gamma (> 12.84 pg/ml), sCD40L (< 2168 pg/ml), interferon alpha 2 (> 55.11 pg/ml), and IL-17a (< 15.1 pg/ml) were predictive markers for those patients with better progression-free survival (p < .05 for each cytokine). After 28 days of metronomic therapy, the plasma levels of sCD40L, IL-17a, and IL-6 (< 130 pg/ml) could serve as predictors of improved progression-free survival, as could levels interferon gamma and sCD40L after 56 days of therapy. We observed minimal changes in cytokine profiles, from baseline, as a consequence of the metronomic therapy, with the exception of an elevation of IL-6 and IL-8 levels 28 days (and 56 days) after treatment started (p < 0.05). Our results indicate that a selective cytokine elevation involves IL-6 and IL-8, following metronomic chemotherapy administration. In addition, interferon gamma and sCD40L may be potential biomarkers for gastrointestinal cancer patients that are likely to benefit from metronomic chemotherapy. Our study contributes to our understanding of the mechanisms of action of metronomic chemotherapy, and the cytokine profiling we describe may guide future selection of gastrointestinal cancer patients for UFT/CTX/celecoxib combination metronomic chemotherapy.

Effects of Pazopanib Monotherapy vs. Pazopanib and Topotecan Combination on Anaplastic Thyroid Cancer Cells.

The purpose of this study was to examine pazopanib/topotecan combination activity vs. pazopanib monotherapy on anaplastic thyroid cancer (ATC) cells. Proliferation analyses were performed on ATC cell lines administered for 72 h with pazopanib and topotecan alone and to their simultaneous combination. Pazopanib and topotecan produced a strong synergism on ATC cells, calculated by the combination index, increasing the intracellular concentrations of topotecan lactone measured by high-performance liquid chromatography. Furthermore, a significantly decrease of the gene expression of ATP-binding cassette transporter G2 (ABCG-2), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1α (HIF-1α), and colony stimulating factor-1 (CSF-1) was presented in combination-treated ATC cells by real time PCR tests. In summary, the simultaneous association of pazopanib and topotecan established a highly synergistic ATC antiproliferative effect, suggesting a new possibility to translate this schedule into clinical trials.

Dual Kit/Aur Inhibitors as Chemosensitizing Agents for the Treatment of Melanoma: Design, Synthesis, Docking Studies and Functional Investigation.

Melanoma is the most serious form of skin cancer but its medication is still far from being safe and thoroughly effective. The search of novel therapeutic approaches represents therefore a health emergency to push through eagerly. In this study, we describe a novel class of dual c-Kit/Aur inhibitors, characterized by a 1,2,4-triazole core and developed by a structure-based optimization of a previously developed hit, and report the evidence of their significance as drug candidates for the treatment of melanoma. Compound 6a, merging the best inhibitory profile against the target kinases, showed anti-proliferative efficacy against the human melanoma cell lines A2058, expressing the BRAF V600D mutation, and WM266-4, expressing BRAF V600E. Significantly, it displayed also a highly synergistic profile when tested in combination with vemurafenib, thus proving its efficacy not only per se but even in a combination therapy, which is nowadays acknowledged as the cornerstone approach of the forthcoming tumour management.

Chemotherapeutic and antiangiogenic drugs beyond tumor progression in colon cancer: Evaluation of the effects of switched schedules and related pharmacodynamics.

The aim of the study was to evaluate the effects and the related pharmacological mechanisms of switched schedules of antiangiogenic and chemotherapeutic drugs beyond progression after a first-line treatment in a colorectal cancer preclinical model. In vivo studies were performed in nude mice subcutaneously transplanted with colon cancer cells. The treatments included drug combinations with a switch between chemotherapeutic (i.e., irinotecan and 5-fluorouracil) and/or antiangiogenic drugs (i.e., anti-VEGF antibodies and sunitinib) at the time of tumor progression. Proliferation assays were also achieved in vitro on different colon cancer cell lines exposed to SN-38 and sunitinib alone or in combination. ABCG2 gene expression was performed with real-time PCR and SN-38 intracellular concentrations were measured. The switch in the combined treatments, at the time of tumor progression, of the chemotherapeutic (from irinotecan to 5-fluoruracil), or the antiangiogenic drug (from anti-VEGF antibodies to sunitinib) or of both drugs induced a new response. Immunohistochemistry of stromal PDGF-C, PlGF, SD1-α, Tie-2, and VEGFR-2 showed statistical differences between tumors at the time of relapse and after the switched therapy. Moreover, the combination of SN-38 and sunitinib caused synergism on colon cancer cells, with significant inhibition of the ABCG2 gene expression and an increase of SN-38 intracellular concentrations. Our observations may be of clinical relevance, suggesting the switch of single chemotherapeutic or antiangiogenic drugs beyond progression of the disease to obtain a new tumor response due to a modulation of angiogenic factors and a direct effect on tumor cells with a possible variation of intracellular drug concentrations.

Metronomic vinorelbine is directly active on Non Small Cell Lung Cancer cells and sensitizes the EGFRL858R/T790M cells to reversible EGFR tyrosine kinase inhibitors.

Metronomic vinorelbine (mVNR) has been described primarily as an antiangiogenic therapy, and no direct effects of mVNR on Non Small Cell Lung Cancer (NSCLC) cells has yet been demonstrated. The aims of this study were i) to establish the direct activity of mVNR on NSCLC cells either EGFR wt or EGFRL858R/T790M, and ii) to quantify the synergism of the combination with reversible EGFR tyrosine kinase inhibitors (TKIs), investigating the underlying mechanism of action. Proliferation assays were performed on A-549 (wt EGFRhigh), H-292 (EGFR-wt), H-358 (EGFR-wt), H-1975 (EGFRL858R/T790M) NSCLC cell lines exposed to mVNR, its active metabolite deacetyl-VNR (D-VNR), gefitinib and erlotinib for 144 h treatments. The synergism between mVNR and EGFR TKIs was determined by the combination index (CI) in EGFR-wt and H-1975 NSCLC cells. Cyclin-D1 and ABCG2 genes expression and protein levels were measured by RT-PCR and ELISA assays, as well as the phosphorylation of ERK1/2 and Akt. Intracellular concentrations of EGFR TKIs and VNR were investigated with a mass spectrometry system. mVNR, and its active metabolite D-VNR, were extremely active on NSCLC cells, in particular on H-1975 (IC50 = 13.56 ±â€¯2.77 pM), resistant to TKIs. mVNR inhibited the phosphorylation of ERK1/2 and Akt and significantly decreased the expression of both cyclin-D1 and ABCG2 m-RNA and protein. The simultaneous combination of VNR and reversible EGFR TKIs showed a strong synergism on EGFR-wt NSCLC cells and on H-1975 cells (e.g. CI = 0.501 for 50% of affected cells), increasing the intracellular concentrations of EGFR TKIs (e.g. +50.5% vs. gefitinib alone). In conclusions, mVNR has direct effects on NSCLC cells and sensitizes resistant cells to EGFR TKIs, increasing their intracellular concentrations.

Optimization of biomarkers-based classification scores as progression-free survival predictors: an intuitive graphical representation.

Aim: To construct classification scores based on a combination of cancer patient plasma biomarker levels, for predicting progression-free survival. Methods: The approach is based on the optimization of the biomarker cut-off values, which maximize the statistical differences between the groups with values lower or larger than the cut-offs, respectively. An intuitive visualization of the quality of the classification score is also proposed. Results: Even if there are only weak correlations between individual biomarker levels and progression-free survival, scores based on suitably chosen combination of three biomarkers have classification power comparable with the Response Evaluation Criteria in Solid Tumors criteria classification of response to treatments in solid tumors. Conclusion: Our approach has the potential to improve the selection of the patients who will benefit from a given anticancer treatment.

Melanocortin Receptor-4 and Glioblastoma Cells: Effects of the Selective Antagonist ML00253764 Alone and in Combination with Temozolomide In Vitro and In Vivo.

Currently, no description of melanocortin receptor-4 (MC4R) expression or activity is available in human cancer cells, including glioblastoma (GBM). The aim of this study is to evaluate the presence of MC4Rs in GBM cells and the selective inhibition of their activity through the MC4R antagonist ML00253764 alone and in association with temozolomide in vitro and in vivo. MC4R genotyping and gene expression were performed on human GBM cells (U-87 and U-118) with real-time PCR. MC4R western blotting, immunohistochemistry, and immunofluorescence were obtained in both cell lines and in human tissues. Proliferation, cell cycle, and apoptotic assays were performed with ML00253764, whereas the synergism of the simultaneous combination with temozolomide was evaluated by the combination index method. ERK1/2 and Akt phosphorylation were quantified by ELISA. In vivo experiments were performed in U-87 xenografted nude mice. Both GBM cell lines and tumor tissues expressed MC4R receptors. The selective antagonist ML00253764 determined an antiproliferative and proapoptotic activity through the inhibition of the phosphorylation of ERK1/2 and Akt. Moreover, the simultaneous combination of temozolomide and ML00253764 determined a highly synergistic effect on GBM cells. The same combination in vivo showed a strong and significant decrease of GBM tumor volumes if compared to the single drug treatments, with an excellent tolerability profile. In conclusion, MC4R is present in GBM cells and its selective inhibition determined antiproliferative and proapoptotic effects, through the inhibition of ERK1/2 and Akt phosphorylation, and the synergistic enhancement of temozolomide effects in vitro and in vivo.

Melanocortin Receptor-4 Gene Polymorphisms in Glioblastoma Patients Treated with Concomitant Radio-Chemotherapy.

Melanocortins are peptides with well-recognized antiinflammatory and neuroprotective activity. No data are currently available on melanocortin receptor-4 (MC4R) gene polymorphisms and tumors, including glioblastomas (GBMs), or their relationship with radiotherapy or chemotherapy. The aim of this study was to evaluate the possible predictive/prognostic role of the MC4R SNPs on GBM patients. Fifty-five patients with a proven diagnosis of GBM, treated with radiotherapy and temozolomide, were consecutively enrolled. MC4R gene SNPs (rs17782313, rs489693, rs8087522, rs17700633) were analyzed by a validated TaqMan® SNP genotyping assays. Univariate and multivariate analyses were performed. A P < 0.0125 (Bonferroni's correction) was considered significant ( Clinicaltrial.gov identifier NCT02458508). The median progression-free survival (PFS) and median overall survival (OS) of these patients were 9.54 (95% CI 5.4-14.3) months and 24.9 (95% CI 17.8-34.6) months, respectively. The MC4R rs489693 AA genotype was significantly associated with a shorter PFS and OS. Indeed, with regard to PFS, patients harboring the rs489693 AA genotype had a median PFS of 2.99 months whereas patients with AC/CC genotypes had a median PFS of 10.82 months (P = 0.009). Interestingly, the rs489693 AA patients also had a lower median OS as compared with the median OS of the AC/CC genotypes (10.75 vs. 29.5 months, respectively, P = 0.0001). This study suggests that the MC4R rs489693 AA genotype is significantly associated with a shorter PFS and OS in patients treated with radiotherapy and temozolomide. These findings represent a relevant effort to identify novel clinical markers for RT-CT therapy in GBM to be validated in future pharmacogenetic clinical trials.