Bastianina Canu

Pharmacodynamic and pharmacogenetic angiogenesis-related markers of first-line FOLFOXIRI plus bevacizumab schedule in metastatic colorectal cancer

Background:The identification of molecular and genetic markers to predict or monitor the efficacy of bevacizumab (BV) represents a key issue in the treatment of metastatic colorectal cancer (mCRC).Methods:Plasma levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble VEGF receptor 2 (sVEGFR-2) and thrombospondin-1 (TSP-1) were assessed by ELISA assay at different time points in a cohort of 25 patients enroled in a phase II trial of GONO-FOLFOXIRI plus BV as first-line treatment of mCRC. VEGF: −2578A/C, −1498C/T, −1154A/G, −634C/G and 936C/T; and VEGFR-2: −604A/G, +1192C/T and +1719A/T, polymorphisms were assessed in a total of 54 patients.Results:Treatment with GONO-FOLFOXIRI plus BV determined a prolonged and significant reduction in plasma free, biologically active VEGF concentration. Interestingly, VEGF concentrations remained lower than at baseline also at the time of PD. Conversely, PlGF levels increased during the treatment if compared with baseline, suggesting a possible role in tumour resistance; moreover, sVEGFR-2 increased at the time of PD, as well as TSP-1. No association of assessed polymorphisms with outcome was found.Conclusion:Our study suggested the possible mechanisms of resistance to combined therapy in those patients with a progressive disease to be tested in ongoing phase III randomised studies.

VEGF-A polymorphisms predict progression-free survival among advanced castration-resistant prostate cancer patients treated with metronomic cyclophosphamide.

Background:No data are available on the pharmacogenetics of metronomic chemotherapy in prostate cancer. The aim of this study was to evaluate the association between VEGF-A sequence variants and prostate-specific antigen (PSA) progression, progression-free survival (PFS) and overall survival (OS), in advanced castration-resistant prostate cancer patients treated with metronomic cyclophosphamide (CTX), celecoxib and dexamethasone.Methods:Forty-three patients were enrolled, and genomic DNA was extracted. VEGF-A gene SNPs (−2578A/C, −634C/G, +936C/T) were analysed using TaqMan PCR assays. Hardy–Weinberg equilibrium was tested for each SNP, and genetic effects were evaluated by Fisher’s exact test. PFS and OS were analysed with GraphPad Prism software, using the product limit method of Kaplan and Meier, and comparing survival curves using both the log-rank test and the Gehan–Wilcoxon test. We used Bonferroni correction to account for multiple testing, and a two-tailed P-value of <0.017 was considered statistically significant.Results:Overall, 20 patients (46%) experienced a reduction in PSA levels from baseline and, among them, 14 (32%) showed a confirmed PSA ≥50% decrease. In non-responders, the −2578CC genotype was more frequent (18.60% vs 2.33% in responders; P=0.0212) whereas the −634CC genotype frequency was 22.73% vs 0% in responders (P=0.0485). With regard to PFS, patients harbouring the −634CC genotype had a median PFS of 2.2 months whereas patients with the genotype −634CG/GG had a median PFS of 6.25 months (P=0.0042).Conclusion:The −634CC genotype is significantly associated with a shorter PFS in patients treated with a metronomic CTX schedule.

Metronomic 5-fluorouracil, oxaliplatin and irinotecan in colorectal cancer

Metronomic chemotherapy (the frequent, long term, low dose administration of chemotherapeutic drugs) is a promising therapy because it enhances the anti-endothelial activity of conventional chemotherapeutics, but with lower or no toxic effects compared to maximum tolerated dose administration. The aims of the present study were to compare, in vitro and in vivo, the antiangiogenic and antitumor activities of metronomic irinotecan (CPT-11), oxaliplatin (L-OHP) and 5-fluorouracil (5-FU) in colorectal cancer and to investigate the metronomic combination of these drugs. In vitro cell proliferation, combination studies and vascular endothelial growth factor (VEGF) secretion analyses were performed on endothelial (HMVEC-d) and colorectal cancer (HT-29) cells exposed for 144 h to metronomic concentrations of SN-38, the active metabolite of CPT-11, L-OHP and 5-FU. HT-29 human colorectal cancer xenograft model was used and tumour growth, microvessel density and VEGF quantification were performed in tumours after the administration of metronomic CPT-11, L-OHP, 5-FU and their simultaneous combination. Low concentrations of SN-38, but not 5-FU and L-OHP, preferentially inhibited endothelial cell proliferation. Simultaneous and continuous exposure of HT-29 and HMVEC-d cells to low concentrations SN-38+L-OHP+5-FU for 144 h showed a strong antagonism and an unfavorable dose-reduction index. Moreover, the ternary combination resulted in a significant increase of VEGF secretion in HT-29 cancer cells. In a xenograft model metronomic CPT-11, but not 5-FU and L-OHP, significantly inhibits HT-29 tumor growth and microvessel density in the absence of toxicity. On the contrary, metronomic 5-FU+L-OHP+CPT-11 therapy did not affect the microvascular count. The metronomic concept might not universally apply to every cytotoxic drug in colorectal cancer and metronomic combination regimens should be used with caution.

First-line metronomic chemotherapy in a metastatic model of spontaneous canine tumours: a pilot study

Metronomic chemotherapy—the low-dose, long term and frequently administered chemotherapy—has revealed in these years an important impact on the stabilization of cancer disease for its known antiangiogenic effects, prolonged clinical benefits and the improved quality of life of several cancer patients, without any high grade toxicity [1–3]. Both the low cost and the oral administration of the drugs are key characteristics of this schedule and may offer important social advantages [4]. Anecdotical case reports [5–7] and experiences in small subsets of patients enrolled in retrospective clinical studies [8–10] on metastatic cancers have been recently published about the use of metronomic therapy as a first-line treatment. These point out the possible importance of metronomic chemotherapy as an alternative approach to first-line therapy in frail patients requiring palliation or patients refusing the standard chemotherapy for its impact on the quality of life. However, no data of prospective clinical trials on first line metronomic chemotherapy are currently available in metastatic cancer human patients. The veterinary medical oncology has advanced dramatically over the past few decades, because of the successful application of a number of conventional chemotherapeutic drugs to the cancer conditions diagnosed in veterinary patients [11]. Veterinary oncology cases often present a unique opportunity to investigate novel drugs and treatment schedules providing many in vivo information to the larger medical community and giving new effective options for dogs themselves [12]. As well recently pointed out by Paoloni and Khanna [13], these studies may also have a great translational relevance, predicting new therapies and related surrogate markers in human beings because pet dogs with cancers might assist the transition between mouse models and human patients. Moreover, in the clinical practice, veterinarians and their clients are generally less willing to accept a high degree of side effects, which most often results in lower drug doses than the ones that are used in human oncology. The aim of the present pilot study was to test a first-line metronomic oral combination of cyclophosphamide (CTX) and celecoxib (CXB) in canine metastatic spontaneous tumours, characterizing possible biomarkers to translate in human clinical research.

Antiproliferative and proapoptotic activity of CLM3, a novel multiple tyrosine kinase inhibitor, alone and in combination with SN-38 on endothelial and cancer cells

AIMS To demonstrate the antiproliferative and pro-apoptotic activity of the novel pyrazolopyrimidine derivative multiple tyrosine kinase inhibitor CLM3, alone and in combination with SN-38 (the active metabolite of irinotecan), on endothelial and tumor cells and to show its mechanism of action. METHODS Proliferation and apoptotic assays were performed on microvascular endothelial (HMVEC-d) and lung (A549) and thyroid cancer (8305C, TT) cell lines exposed to CLM3 and to the simultaneous combination with SN38 for 72h. Cell-based phospho-VEGFR-2, phospho-EGFR and phospho-RET inhibition assays were performed and ERK1/2 and Akt phosphorylation were quantified by ELISA kits. Cyclin D1 gene expression was performed with real-time PCR and cyclin D1 intracellular concentrations were measured by ELISA. RESULTS A strong effect on antiproliferative and pro-apoptotic activity was found with the CLM3 on endothelial and cancer cells, synergistically enhanced by SN38. Phospho-VEGFR-2, phospho-EGFR and phospho-RET levels significantly decreased after CLM3 treatments in activated endothelial and cancer cells; ERK1/2 and Akt phosphorylation were significantly inhibited by lower concentrations of the pyrazolopyrimidine drug in endothelial cells if compared to cancer cells. Moreover, CLM3 treatment greatly inhibited the expression of the cyclin D1 gene in endothelial and cancer cells, decreasing the cyclin D1 protein intracellular concentration. CONCLUSIONS The pyrazolopyrimidine derivative CLM3 demonstrated a highly significant and promising antiproliferative and proapoptotic activity, alone and in combination with SN-38, for activated endothelial and cancer cells. These effects are mainly due to its inhibition of phosphorylation of VEGFR-2, EGFR and RET tyrosine kinases and their related signaling pathways.

Antiproliferative and Proapoptotic Activity of Sunitinib on Endothelial and Anaplastic Thyroid Cancer Cells via Inhibition of Akt and ERK1/2 Phosphorylation and by Down-Regulation of Cyclin-D1.

CONTEXT Recent experimental evidence suggests a rationale for the use of multitarget tyrosine kinase inhibitors for the treatment of thyroid cancers. Sunitinib showed promising preliminary results against anaplastic thyroid cancer (ATC), and it has been used for some patients who are ineligible for clinical trials. OBJECTIVES The aims of this study were to investigate the in vitro and in vivo activity of sunitinib on ATC and on microvascular endothelial cells and the molecular mechanism for the observed sunitinib activity. METHODS Proliferation and apoptotic assays were performed on human dermal microvascular endothelial and on BRAF- or H-ras-mutated ATC cells (8305C and FB3, respectively) after in vitro exposure to sunitinib for 72 hours. Vascular endothelial growth factor receptor-2, epithelial growth factor receptor, ERK1/2, and Akt phosphorylation was quantified by ELISA and Western blot. Cyclin-D1 mRNA expression was evaluated by real-time PCR, and cyclin-D1 intracellular concentrations were measured by ELISA. 8305C tumor xenografts in nude mice were treated with sunitinib at 50 mg/kg/d (i.p.). RESULTS Antiproliferative and proapoptotic activity of sunitinib was observed in both endothelial and ATC cells. Phospho-vascular endothelial growth factor receptor-2 levels significantly decreased after sunitinib treatment in activated endothelial cells. Phospho-epidermal growth factor receptor, ERK1/2, and Akt phosphorylation was significantly inhibited by sunitinib treatment in endothelial and cancer cells, and cyclin-D1 mRNA and protein expression was inhibited. Sunitinib administration in vivo caused significant inhibition of tumor growth (P < .05). CONCLUSIONS Sunitinib is active in vitro and in vivo against activated endothelial and ATC cells via the inhibition of Akt and ERK1/2 phosphorylation and through the down-regulation of cyclin-D1.

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.

Abstract 4375: Sunitinib alone and in combination with SN-38 is active against anaplastic thyroid cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Anaplastic thyroid cancer (ATC) is the most aggressive thyroid gland malignancy. Recent experimental evidences suggest a rationale for the use of multitarget tyrosine kinase inhibitors (TKIs). Sunitinib showed evidences of activity in a phase I trial in different tumors, including thyroid cancers. TKIs are usually more effective in combination with standard chemotherapy. Among chemotherapeutic drugs, preclinical studies using irinotecan seem to be very promising in different types of thyroid carcinomas. The aim of this study is to determine the activity of sunitinib alone and in combination with irinotecan on endothelial and ATC cells. METHODS: Proliferation and apoptotic assays were performed on human dermal microvascular endothelial cells (HMVEC-d) and ATC (8305C, FB3) cell lines exposed to sunitinib (SU) and SN-38, the active metabolite of irinotecan, for 72h. The synergism was determined with the method by Chou and quantified by the combination index (CI), where CI 1 indicates synergism, additive effect, and antagonism, respectively. Cell-based phospho-VEGFR-2 assay was performed and ERK1/2 and Akt phosphorylation were quantified by ELISA kits. Cyclin D1 and CDK2 gene expression were performed with real-time PCR and cyclin D1 intracellular concentrations were measured by ELISA. 8305C xenografts in nude mice were treated with sunitinib (50 mg/kg/day) and tumour volumes were measured. RESULTS: A strong antiproliferative and pro-apoptotic activities were determined by SU on both endothelial and cancer cells, synergistically enhanced by SN38. Phospho-VEGFR-2 levels significantly decreased after SU treatments in activated endothelial cells; ERK1/2 and Akt phosphorylation was significantly inhibited by SU in endothelial and cancer cells, although at lower concentrations in the endothelial ones. Moreover, SU treatment greatly inhibited the expression of the cyclin D1 and CDK2 genes in both endothelial and cancer cells, decreasing the cyclin D1 protein intracellular concentration. In vivo administration of sunitinib was effective and determined a significant tumor regression. CONCLUSIONS: SU demonstrated a highly significant antiproliferative and proapoptotic activity, for activated endothelial and ATC cells. Moreover, the simultaneous combination of sunitinib and irinotecan determined a high synergism on endothelial and ATC cells, suggesting a possible translation of this schedule into the clinics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4375. doi:1538-7445.AM2012-4375