Lenka Radová

Analysis of the prognostic impact of nestin expression in non-small cell lung cancer.

BACKGROUND Nestin is an intermediary filament protein, expressed in progenitor cells of neural and muscle origin and in activated endothelium. The expression of this protein in tumours can be associated with degree of differentiation, biological potential and/or neoangiogenesis. AIMS The aim of this study was to examine the immunohistochemical expression of nestin in primary non-small cell lung carcinomas (NSCLC) and to determine its prognostic significance. METHODS Immunohistochemical detection of nestin was carried out on tissue microarrays constructed from 114 formalin-fixed and paraffin-embedded NSCLC samples. These included 78 squamous cell carcinomas and 37 adenocarcinomas. Expression of nestin was also analysed in 35 primary tumour independent NSCLC brain metastasis. The H-score and degree of nestin positive microvascularisation were determined. Both parameters correlated with the clinicopathological characteristics including disease-free and overall survival. Results. We demonstrated that expression of nestin is not significantly higher in tumour cells of adenocarcinomas than in sqamous cell carcinomas despite the fact that adenocarcinomas were more frequently positive (P≤0.30). On the other hand, nestin positivity and nestin positive neovascularisation were significantly more often found in stage IIIa tumours than tumours in stages I and II (P≤0.04, P≤0.02). Nestin expression was also significantly higher in brain metastases of squamous cell carcinomas than brain metastases of adenocarcinomas (P≤0.003). The expression of nestin, in general, did not significantly correspond to disease-free or overall survival. CONCLUSION Nestin expression in NSCLCs is associated with poorer prognosis and with greater nestin-positive microvessel density.

Correlation between BRCA1 expression and clinicopathological factors including brain metastases in patients with non-small-cell lung cancer

BACKGROUND Previously identified as a breast and ovarian cancer susceptibility gene, BRCA1 has gained major scientific interest as a potential prognostic and/or predictive marker for various tumors, including non-small-cell lung cancer (NSCLC), the leading cause of cancer related mortality worldwide. BRCA1 plays a central role in DNA damage response (DDR. It undergoes phosphorylation by various DDR kinases at different serine residues, of which ser1524 is known to be specifically phosphorylated by ATM in response to genotoxic stress. METHODS We performed BRCA1 immunohistochemistry on several tissue microarrays (TMAs) of 113 early (I, II stage) and advanced (III, IV stage) NSCLCs, using MS110 antibody against the BRCA1 N-terminal and S1524 antibody against the phosphorylated form of BRCA1 protein at ser1524 (Abcam). Patients with III and IV stage disease were treated by adjuvant cisplatin-based chemotherapy. Staining results were correlated with overall survival (OS), disease free survival (DFS) and with the occurrence of brain metastases. RESULTS BRCA1 S1524 nuclear positivity was significantly correlated with longer OS and DFS in stage I and II patients (P<0.05), while OS and DFS were shorter in S1524 positive stage III and IV patients (P<0.05). No significant correlation was found with brain metastases. CONCLUSION The results show that BRCA1 phosphorylaton, at least in ser1524, differentiates the fate of early and advanced NSCLC as well as response to chemotherapy, but the underlying mechanisms are not completely understood. Detection of phosphorylated forms of BRCA1 might serve as a useful prognostic and predictive marker for patients with NSCLC.

Abstract B30: Colorectal cancer HCT116 cells resistant toward a novel Aurora kinase inhibitor CYC116: Molecular hallmarks of drug resistance and pharmacologic interventions restoring the drug sensitivity

CYC116 is a novel pan-Aurora kinase and VEGFR2 inhibitor, which has been discovered and developed by Cyclacel Ltd. In preclinical studies it displayed broad anticancer activity and it has been recently evaluated in a Phase I clinical study. Emergence of drug resistance towards CYC116 as well as other Aurora kinase inhibitors in the clinic is highly likely. Our work was focused on identification and characterization of potential cancer cell resistance mechanisms towards CYC116, alongside with ZM447439, an experimental Aurora kinase inhibitor. We generated several HCT116 resistant clones on both p53+/+ and −/− backgrounds by exposing cells to cytotoxic concentrations of CYC116 and ZM447439. HCT116:CYC116, HCT116p53−/−:CYC116, HCT116:ZM447439, HCT116p53−/−:ZM447439 clones were 9 to 82 folds, 36 to 64 folds, 18 to >83 folds and 33 to 39 folds more resistant to selecting agents, respectively. Resistant clones also displayed cross-resistance to other clinical Aurora kinase inhibitors and multidrug resistance to some anticancer agents tested. CYC116 clones, but not ZM447439 acquired polyploidy during the selection. As expected, all the resistance clones did not show up-regulation of common drug transporters including PgP and MRP1. Also we did not observe significant changes in Aurora kinases expression. ZM447439, but not CYC116 induced three novel mutations in Aurora B, namely I216L, L152S, and N76V. Structural modeling studies revealed that L152S may significantly affect the ZM447439 binding. Pangenomic microarray expression studies revealed that 885, 1085, 224, and 212 number of gene sets were differentially expressed (ANOVA p<0.001) in p53+/+:CYC116, p53−/−:CYC116, p53+/+:ZM447439, and p53−/−:ZM447439 groups compared to paternal cell lines, respectively. 23 most relevant genes were selected from all the groups for qRT-PCR validation studies on human primary tumors in vitro sensitive/resistant to CYC116. Interestingly, majority of cell line findings were confirmed also on primary human cells, suggesting validity of these genes as biomarkers of drug susceptibility or resistance. An apoptotic gene Bcl-xL was found to be significantly up-regulated in CYC116 resistant clones, particularly in p53+/+ cells. Knock-down of Bcl-xL using RNAi technology partially reversed the resistance to CYC116. Moreover, Bcl-xL overexpressing p53+/+ CYC116 clones were highly sensitive to a synthetic Bcl-xL inhibitor, ABT-263, compared to the parent cells. Our data cumulatively provide a genetic basis of resistance to Aurora kinase inhibitors, which could be used to predict and monitor clinical response and also to select patients who might benefit from Aurora kinase inhibition. Moreover, our study suggest a role of bcl-2 protein family inhibitors for reversal of drug resistance against Aurora kinase inhibitors and their possible significance for therapy of tumors primarily or secondary resistant to these drugs.

Combination of prednisolone and low dosed dexamethasone exhibits greater in vitro antileukemic activity than equiactive dose of prednisolone and overcomes prednisolone drug resistance in acute childhood lymphoblastic leukemia

INTRODUCTION Glucocorticoids, particularly prednisone/ prednisolone and dexamethasone, play a prominent role in the treatment of pediatric patients with acute lymphoblastic leukemia due to their ability to induce apoptosis in susceptible cells. Current therapeutic protocols use prednisone for both the prophase and the induction phase of the therapy because the greater antileukemic activity of dexamethasone is compromised by its high frequency of serious adverse reactions. AIM To compare, for the first time, the in vitro antileukemic activity of prednisolone alone to that of a combination of prednisolone and dexamethasone using dexamethasone at a very low and presumably safe dosage (1/50 w/w). METHODS Lymphoblasts were isolated from bone marrow and/or blood samples from children with newly diagnosed acute lymphoblastic leukemia. The cytotoxic activity of prednisolone, dexamethasone and the prednisolone/dexamethasone combination against isolated leukemia cells was analyzed using the MTT cytotoxicity assay. RESULTS We observed differences in the in vitro antileukemic activity of prednisolone and dexamethasone in 21% of the tested patients. 3% of the children were prednisolone sensitive but dexamethasone resistant, while 18% were prednisolone resistant and dexamethasone sensitive. 32% were sensitive to both glucocorticoids and 18% were resistant to both. Cells from patients with good in vivo responses to prednisone monotherapy were more responsive to prednisolone in vitro than were cells from patients with poor prednisone responses (P<0.07). Importantly, we demonstrated that the use of even a minimal dose (1/50 w/w) of dexamethasone with prednisolone dramatically increases the in vitro anti-leukemic activity of prednisolone (P<0.0006). CONCLUSION The high inter-individual variability of acute lymphoblastic leukemia responses to glucocorticoids suggest that either patients should be selected for prednisone or dexamethasone treatment on the basis of predictive biomarkers or that prednisone should be used directly in combination with a very low and safe dose of dexamethasone to potentiate its antileukemic activity. The latter option is likely to be cheaper and more efficient, and therefore warrants further clinical investigation to assess its efficacy and safety in treating childhood acute lymphoblastic leukemia.