Sara Nannizzi

Correlation of CDA, ERCC1, and XPD polymorphisms with response and survival in gemcitabine/cisplatin-treated advanced non-small cell lung cancer patients.

Purpose: Selecting patients according to key genetic characteristics may help to tailor chemotherapy and optimize the treatment in non–small cell lung cancer (NSCLC). Polymorphisms at the xeroderma pigmentosum group D (XPD), excision repair cross-complementing 1 (ERCC1), and cytidine deaminase (CDA) genes have been associated with alterations in enzymatic activity and may change sensitivity to the widely used cisplatin-gemcitabine regimen. Experimental Design: Analyses of CDA, XPD, and ERCC1 polymorphisms were done on blood samples of 65 chemotherapy-naïve, advanced NSCLC patients treated with cisplatin-gemcitabine. Furthermore, CDA enzymatic activity was evaluated by high-performance liquid chromatography analysis. Association between XPD Asp312Asn and Lys751Gln, ERCC1 C118T, and CDA Lys27Gln polymorphisms and response, clinical benefit, toxicity, time to progression (TTP), and overall survival (OS) was estimated using Pearsons χ2 tests, the Kaplan-Meier method, the log-rank test, and the Cox proportional hazards model. Results: The CDA Lys27Lys polymorphism significantly correlated with better clinical benefit (P = 0.04) and grade ≥3 neutropenia and thrombocytopenia, as well as with longer TTP and OS (P = 0.006 and P = 0.002, respectively), whereas no significant associations were found among ERCC1 and XPD polymorphisms and both response and clinical outcome. Finally, the enzymatic activity assay showed a significant lower mean in subjects harboring the CDA Lys27Lys polymorphism. Conclusions: Our data suggested the role of CDA Lys27Lys polymorphism as a possible predictive marker of activity, toxicity, TTP, and OS in advanced NSCLC patients treated with cisplatin and gemcitabine. These results may be explained by the lower enzymatic activity associated with the Lys27Lys CDA and offer a potential new tool for treatment optimization.

Molecular Mechanisms Underlying the Synergistic Interaction of Erlotinib, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, with the Multitargeted Antifolate Pemetrexed in Non-Small-Cell Lung Cancer Cells

Because the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and the multitargeted antifolate pemetrexed are registered in the treatment of second-line non-small-cell lung cancer (NSCLC), empirical combinations of these drugs are being tested. This study investigated molecular mechanisms underlying their combination in six NSCLC cell lines. Cells were characterized by heterogeneous expression of pemetrexed determinants, including thymidylate synthase (TS) and dihydrofolate reductase (DHFR), and mutations potentially affecting chemosensitivity. Pharmacological interaction was studied using the combination index (CI) method, whereas cell cycle, apoptosis induction, and EGFR, extracellular signal-regulated kinases 1 and 2, and Akt phosphorylation were studied by flow cytometry, fluorescence microscopy, and enzyme-linked immunosorbent assays. Reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, and activity assays were performed to assess whether erlotinib influenced TS. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays demonstrated that EGFR and k-Ras mutations were related to erlotinib sensitivity, whereas TS and DHFR expression were related to pemetrexed sensitivity. Synergistic cytotoxicity was found in all cells, most pronounced with pemetrexed + erlotinib (24 h) → erlotinib (48 h) sequence (CI, 0.09-0.40), which was associated with a significant induction of apoptosis. Pemetrexed increased EGFR phosphorylation and reduced Akt phosphorylation, which was additionally reduced by drug combination (-70.6% in H1650). Erlotinib significantly reduced TS expression and activity, possibly via E2F-1 reduction, as detected by RT-PCR and Western blot, and the combination decreased TS in situ activity in all cells. Erlotinib and pemetrexed showed a strong synergism in NSCLC cells, regardless of their genetic characteristics. Induction of apoptosis, modulation of EGFR and Akt phosphorylation, and changes in the expression of critical genes involved in pemetrexed activity contribute to this synergistic interaction and support the clinical investigation of these markers.

Pharmacogenetics of anticancer drug sensitivity in pancreatic cancer

Chemotherapy has produced unsatisfactory results in pancreas cancer and novel approaches, including treatment tailoring by pharmacogenetic analysis and new molecular-targeted drugs, are required. The scarcity of effective therapies may reflect the lack of knowledge about the influence of tumor-related molecular abnormalities on responsiveness to drugs. Advances in the understanding of pancreas cancer biology have been made over the past decade, including the discovery of critical mutations in oncogenes (i.e., K-Ras) as well as the loss of tumor suppressor genes, such as TP53 and p16INK4. Other studies showed the dysregulation of the expression of proteins involved in the control of cell cycle, proliferation, apoptosis, and invasiveness, such as Bcl-2, Akt, mdm2, and epidermal growth factor receptor. These characteristics might contribute to the aggressive behavior of pancreatic cancer and influence response to treatment. Indeed, the inactivation of p53 may explain the relative resistance to 5-fluorouracil, whereas Bcl-2 overexpression is associated with reduced sensitivity to gemcitabine. However, the future challenge of pancreas cancer chemotherapy relies on the identification of molecular markers that help in the selection of drugs best suited to the individual patient. Recent pharmacogenetic studies focused on genes encoding proteins directly involved in drug activity, showing the role of thymidylate synthase and human equilibrative nucleoside transporter-1 as prognostic factor in 5-fluorouracil- and gemcitabine-treated patients, respectively. Finally, inhibitors of signal transduction and angiogenesis are under extensive investigation, and several prospective trials have been devoted to this area. Pharmacogenetics is likely to play a central role in the personalization of treatment, to stratify patients based on their likelihood of response to both standard agents (i.e., gemcitabine/nucleoside transporters) and targeted treatments (i.e., epidermal growth factor receptor gene mutations and/or amplification and tyrosine kinase inhibitors), Thus, molecular analysis should be implemented in the optimal management of the patient affected by pancreatic adenocarcinoma. [Mol Cancer Ther 2006;5(6):1387–95] [Mol Cancer Ther 2006;5(6):1387-95]

Correlation between cytidine deaminase genotype and gemcitabine deamination in blood samples.

Cytidine deaminase (CDA) is the major enzyme of gemcitabine inactivation. The aim of this study was to determine whether the CDA Lys27Gln polymorphism influenced gemcitabine deamination in blood samples from 90 lung cancer patients. The polymorphism was studied with Taqman probes-based assay; CDA activity was evaluated by HPLC in cytoplasmic extracts from red blood cells. Mean enzymatic activity was significantly lower in patients carrying the CDA Lys27Lys than in patients with the Lys27Gln or Gln27Gln protein (P < 0.05). CDA genotyping may be useful in screening patients before gemcitabine treatment, in order to identify subjects with lower CDA activity and potentially better clinical outcomes after gemcitabine-based chemotherapy.

In vitro synergistic cytotoxicity of gemcitabine and pemetrexed and pharmacogenetic evaluation of response to gemcitabine in bladder cancer patients

The present study was performed to investigate the capability of gemcitabine and pemetrexed to synergistically interact with respect to cytotoxicity and apoptosis in T24 and J82 bladder cancer cells, and to establish a correlation between drug activity and gene expression of selected genes in tumour samples. The interaction between gemcitabine and pemetrexed was synergistic; indeed, pemetrexed favoured gemcitabine cytotoxicity by increasing cellular population in S-phase, reducing Akt phosphorylation as well as by inducing the expression of a major gemcitabine uptake system, the human equilibrative nucleoside transporter-1 (hENT1), and the key activating enzyme deoxycytidine kinase (dCK) in both cell lines. Bladder tumour specimens showed an heterogeneous gene expression pattern and patients with higher levels of dCK and hENT1 had better response. Moreover, human nucleoside concentrative transporter-1 was detectable only in 3/12 patients, two of whom presented a complete response to gemcitabine. These data provide evidence that the chemotherapeutic activity of the combination of gemcitabine and pemetrexed is synergistic against bladder cancer cells in vitro and that the assessment of the expression of genes involved in gemcitabine uptake and activation might be a possible determinant of bladder cancer response and may represent a new tool for treatment optimization.

Epigenetic mechanisms of irinotecan sensitivity in colorectal cancer cell lines

Irinotecan is a topoisomerase-I (Top-I) inhibitor used for the treatment of colorectal cancer. DNA demethylating agents, including 5-azacytidine (5-aza), display synergistic antitumor activity with several chemotherapy drugs. 5-Aza may enhance irinotecan cytotoxicity by at least one of the following mechanisms: (a) Top-I promoter demethylation, (b) activation of genes involved in Top-I transcriptional regulation (p16 or Sp1), and (c) modulation of the cell cycle and apoptosis after DNA damage. The growth-inhibitory effects of SN38, the active metabolite of irinotecan, 5-aza, and their combinations, were studied in four colorectal cancer cell lines. The effects of treatments on cell cycle were analyzed by flow cytometry, and apoptosis was measured by fluorescence microscopy. Top-I, Sp1, and p53 expression modulated by 5-aza were measured by real-time PCR. Methylation of Top-I, p16, 14-3-3σ, and hMLH1 promoters before and after 5-aza treatment were measured by MethyLight PCR and DNA bisulfite sequencing. Low-dose 5-aza significantly enhanced the apoptotic effect of irinotecan in all colorectal cancer cells, whereas a synergistic cytotoxic effect was observed only in p53-mutated cells (HT29, SW620, and WiDr). This synergistic effect was significantly correlated with Top-I up-regulation by 5-aza, and coupled to p16 demethylation and Sp1 up-regulation. p16 demethylation was also associated with enhanced cell cycle arrest after irinotecan treatment. In contrast, 5-aza down-regulated Top-I expression in the p53 wild-type LS174T cells in a p53-dependent manner, thereby reducing SN38 cytotoxicity. In conclusion, 5-aza modulates Top-I expression by several mechanisms involving Sp1, p16, and p53. If confirmed in other models, these results suggest that p16 and p53 status affects the 5-aza–irinotecan interaction. [Mol Cancer Ther 2009;8(7):1964–73]

Laser microdissection and primary cell cultures improve pharmacogenetic analysis in pancreatic adenocarcinoma.

A key focus of research on pancreatic ductal adenocarcinoma (PDAC) is identifying new techniques to tailor gemcitabine and 5-fluorouracil treatments. Availability of tumor tissue is critical for the accurate assessment of gene expression, and laser microdissection (LMD) and primary cell cultures may be useful tools to separate tumor cells from the stromal reaction. The aim of this study was (1) to address the genetic profile relevant to drug activity and (2) to evaluate differences between microdissected and non-microdissected tumors, normal tissues, and primary cell cultures. Quantitative PCR of seven key genes was performed on mRNA from 113 microdissected and 28 non-microdissected tumors, a pool of normal tissues and four established primary cell lines. Protein expression was evaluated by western blot and immunocytochemistry and cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. LMD allowed the analysis of 110 samples and revealed significant differences in mRNA levels between microdissected tumors and normal tissues, as well as between non-microdissected and microdissected tumors from the same patients. In contrast, primary cell lines showed similar expression profiles with respect to their respective microdissected tumors. In particular, expression levels of human equilibrative nucleoside transporter-1 and thymydilate synthase were significantly related to gemcitabine and 5-fluorouracil cytotoxicity. We conclude that LMD is a reliable technique for mRNA extraction, and allows detection of significant differences in the expression of specific target genes when compared to non-microdissected specimens and normal tissues. Moreover, expression levels in microdissected tumors are similar to those observed in primary tumor cell cultures, both at mRNA and protein level, and are related to drug chemosensitivity. The use of these ex vivo techniques for molecular analysis of tumors therefore appears to be of some value in implementing the clinical management of PDAC.

Synergistic cytotoxicity and molecular interaction on drug targets of sorafenib and gemcitabine in human pancreas cancer cells

Background: Current treatments have a modest impact on survival of pancreatic cancer patients and this study investigates the interaction between sorafenib and gemcitabine and the molecular pharmacodynamics of this combination. Methods: The pancreatic cancer cells were treated with sorafenib and gemcitabine, alone or in combination. The effects of treatments were evaluated on cell proliferation, cell cycle, apoptosis, phosphorylation of Akt, c-Kit, ERK and VEGFR2, and expression of genes related to drug activity. Results: Gemcitabine and sorafenib synergistically interacted on the inhibition of cell proliferation, and assessment of apoptosis demonstrated that drug associations increased the apoptotic index. Sorafenib reduced c-Kit, ERK and VEGFR2 activation and on the other hand, gemcitabine inhibited Akt phosphorylation. Moreover, quantitative PCR showed that sorafenib modulated the expression of genes related to gemcitabine activity, while gemcitabine induced the expression of RKIP. Conclusion: These data demonstrate that gemcitabine and sorafenib combination displays a synergistic effect in pancreatic cancer cells.

Single nucleotide polymorphisms and clinical outcome in biliary tract carcinoma treated with epirubicin, cisplatin and capecitabine

14558 Background: Biliary tract carcinoma is an uncommon malignancy. The activity of systemic chemotherapy is very low and there is no current therapy considered a gold standard. The poor prognosis...