Paclitaxel and CYC3, an aurora kinase A inhibitor, synergise in pancreatic cancer cells but not bone marrow precursor cells.

BACKGROUND: Amplification of aurora kinase A (AK-A) overrides the mitotic spindle assembly checkpoint, inducing resistance to taxanes. RNA interference targeting AK-A in human pancreatic cancer cell lines enhanced taxane chemosensitivity. In this study, a novel AK-A inhibitor, CYC3, was investigated in pancreatic cancer cell lines, in combination with paclitaxel. METHODS: Western blot, flow cytometry and immunostaining were used to investigate the specificity of CYC3. Sulforhodamine B staining, time-lapse microscopy and colony-formation assays were employed to evaluate the cytotoxic effect of CYC3 and paclitaxel. Human colony-forming unit of granulocyte and macrophage (CFU-GM) cells were used to compare the effect in tumour and normal tissue. RESULTS: CYC3 was shown to be a specific AK-A inhibitor. Three nanomolar paclitaxel (growth inhibition 50% (GI(50)) 3 nM in PANC-1, 5.1 nM in MIA PaCa-2) in combination with 1 μM CYC3 (GI(50) 1.1 μM in MIA PaCa2 and 2 μM in PANC-1) was synergistic in inhibiting pancreatic cell growth and causing mitotic arrest, achieving similar effects to 10-fold higher concentrations of paclitaxel (30 nM). In CFU-GM cells, the effect of the combination was simply additive, displaying significantly less myelotoxicity compared with high concentrations of paclitaxel (30 nM; 60-70% vs 100% inhibition). CONCLUSION: The combination of lower doses of paclitaxel and CYC3 merits further investigation with the potential for an improved therapeutic index in vivo

Gemcitabine diphosphate choline is a major metabolite linked to the Kennedy pathway in pancreatic cancer models in vivo.

BACKGROUND: The modest benefits of gemcitabine (dFdC) therapy in patients with pancreatic ductal adenocarcinoma (PDAC) are well documented, with drug delivery and metabolic lability cited as important contributing factors. We have used a mouse model of PDAC: KRAS(G12D); p53(R172H); pdx-Cre (KPC) that recapitulates the human disease to study dFdC intra-tumoural metabolism. METHODS: LC-MS/MS and NMR were used to measure drug and physiological analytes. Cytotoxicity was assessed by the Sulphorhodamine B assay. RESULTS: In KPC tumour tissue, we identified a new, Kennedy pathway-linked dFdC metabolite (gemcitabine diphosphate choline (GdPC)) present at equimolar amounts to its precursor, the accepted active metabolite gemcitabine triphosphate (dFdCTP). Utilising additional subcutaneous PDAC tumour models, we demonstrated an inverse correlation between GdPC/dFdCTP ratios and cytidine triphosphate (CTP). In tumour homogenates in vitro, CTP inhibited GdPC formation from dFdCTP, indicating competition between CTP and dFdCTP for CTP:phosphocholine cytidylyltransferase (CCT). As the structure of GdPC precludes entry into cells, potential cytotoxicity was assessed by stimulating CCT activity using linoleate in KPC cells in vitro, leading to increased GdPC concentration and synergistic growth inhibition after dFdC addition. CONCLUSIONS: GdPC is an important element of the intra-tumoural dFdC metabolic pathway in vivo

Modelling prognostic factors in advanced pancreatic cancer

Pancreatic cancer is the fifth most common cause of cancer death. Identification of defined patient groups based on a prognostic index may improve the prediction of survival and selection of therapy. Many prognostic factors have been identified often based on retrospective, underpowered studies with unclear analyses. Data from 653 patients were analysed. Continuous variables are often simplified assuming a linear relationship with log hazard or introducing a step function (dichotomising). Misspecification may lead to inappropriate conclusions but has not been previously investigated in pancreatic cancer studies. Models based on standard assumptions were compared with a novel approach using nonlinear fractional polynomial (FP) transformations. The model based on FP-transformed covariates was most appropriate and confirmed five previously reported prognostic factors: albumin, CA19-9, alkaline phosphatase, LDH and metastases, and identified three additional factors not previously reported: WBC, AST and BUN. The effects of CA19-9, alkaline phosphatase, AST and BUN may go unrecognised due to simplistic assumptions made in statistical modelling. We advocate a multivariable approach that uses information contained within continuous variables appropriately. The functional form of the relationship between continuous covariates and survival should always be assessed. Our model should aid individual patient risk stratification and the design and analysis of future trials in pancreatic cancer

A preclinical evaluation of pemetrexed and irinotecan combination as second-line chemotherapy in pancreatic cancer

Gemcitabine (GEM)-based chemotherapy is regarded as the standard treatment of pancreatic adenocarcinoma, but yields a very limited disease control. Very few studies have investigated salvage chemotherapy after failure of GEM or GEM-containing chemotherapy and preclinical studies attempting to widen the therapeutic armamentarium, not including GEM, are warranted. MIA PaCa2, CFPAC-1 and Capan-1 pancreatic cancer cell lines were treated with GEM, fluouracil (5-FU), docetaxel (DCT), oxaliplatin (OXP), irinotecan (CPT-11), pemetrexed (PMX) and raltitrexed (RTX) as single agent. Pemetrexed, inducing apoptosis with IC50s under the Cmax in the three lines tested, appeared the most effective drug as single agent. Based on these results, schedule- and concentration-dependent drug interactions (assessed using the combination index) of PMX/GEM, PMX/DCT and PMX–CPT-11 were evaluated. The combinatory study clearly indicated the PMX and CPT-11 combination as the most active against pancreatic cancer. To confirm the efficacy of PMX–CPT-11 combination, we extended the study to a panel of 10 pancreatic cancer cell lines using clinically relevant concentrations (PMX 10 μM; CPT-11 1 μm). In eight of 10 lines, the PMX–CPT-11 treatment significantly reduced cell recovery and increased both the subG1 and caspase 3/7 fraction. After a 5-day wash out period, an increased fraction of subG1 and caspase3/7 persisted in PMX–CPT-11-pretreated cell lines and a significant reduction in the clonogenicity capacity was evident. Finally, in vivo, the PMX/CPT-11 combination showed the ability to inhibit xenograft tumours growth as second-line therapy after GEM treatment. The PMX and CPT-11 combination displays a strong schedule-independent synergistic cytotoxic activity against pancreatic cancer, providing experimental basis for its clinical testing as salvage chemotherapy in pancreatic cancer patients

Matrix metalloproteinases 2 and 9 (gelatinases A and B) expression in malignant mesothelioma and benign pleura

Matrix metalloproteinases (MMPs), in particular the gelatinases (MMP-2 and -9), play a significant role in tumour invasion and angiogenesis. The expression and activities of MMPs have not been characterised in malignant mesothelioma (MM) tumour samples. In a prospective study, gelatinase activity was evaluated in homogenised supernatants of snap frozen MM (n = 35), inflamed pleura (IP, n = 12) and uninflammed pleura (UP, n = 14) tissue specimens by semiquantitative gelatin zymography. Matrix metalloproteinases were correlated with clinicopathological factors and with survival using Kaplan-Meier and Cox proportional hazard models. In MM, pro- and active MMP-2 levels were significantly greater than for MMP-9 (P = 0.006, P<0.001). Active MMP-2 was significantly greater in MM than in UP (P=0.04). MMP-2 activity was equivalent between IP and MM, but both pro- and active MMP-9 activities were greater in IP (P=0.02, P=0.009). While there were trends towards poor survival with increasing total and pro-MMP-2 activity (P=0.08) in univariate analysis, they were both independent poor prognostic factors in multivariate analysis in conjunction with weight loss (pro-MMP-2 P = 0.03, total MMP-2 P = 0.04). Total and pro-MMP-2 also contributed to the Cancer and Leukemia Group B prognostic groups. MMP-9 activities were not prognostic. Matrix metalloproteinases, and in particular MMP-2, the most abundant gelatinase, may play an important role in MM tumour growth and metastasis. Agents that reduce MMP synthesis and/or activity may have a role to play in the management of MM. © 2003 Cancer Research UK

A novel biweekly pancreatic cancer treatment schedule with gemcitabine, 5-fluorouracil and folinic acid

Pancreatic adenocarcinoma is a common disease considered to be poorly responsive to antiblastic treatment. Recent clinical and preclinical results suggest that a combined treatment of gemcitabine (GEM), 5-flurouracil (5-FU) and folinic acid (FA) offers a clinical benefit in patients with advanced pancreas adenocarcinoma. The aim of this phase II clinical trial was to evaluate the antitumour activity and toxicity of a novel biweekly schedule of this combination in patients with pancreatic adenocarcinoma. A total of 42 patients received a 30 min infusion of FA (100 mgm2) and 5-FU (400 mgm2) (FUFA) on days 1–3, and GEM 1000 mgm2 on day 1 every 15 days. We observed 13 objective responses (two complete, 11 partial) and 23 stable diseases. The median time to progression was 9.75 months (95% Confidence Interval (CI), 6.88–12.62) and the median overall survival was 13.10 months (95% CI 9.64–16.56). There were seven cases of each grade III gastroenteric and haematological toxicity. The GEM plus FUFA combination appears to be well tolerated and very active in patients with pancreatic carcinoma