KRAS mutations are a strong predictive marker of resistance to anti-epidermal growth factor receptor (EGFR) antibodies in advanced colorectal cancer (CRC) but only a subset of wild-type (WT) KRAS patients are responders, suggesting the existence of additional markers of resistance to this treatment. The activation of EGFR downstream signaling pathways may be one of these ones. In a series of 42 patients with advanced CRC treated with cetuximab/panitumumab, for whom KRAS status was previously determined, we retrospectively analyzed the intratumor expression of EGFR downstream signaling phosphoproteins of the RAS/MAPK and PI3K/AKT pathways (pERK1/2, pMEK1, pAKT, pP70S6K and pGSK3b) using Bio-Plex V R phosphoprotein array. Association with tumor response, progression-free survival (PFS) and overall survival (OS) was assessed. The expression of all the phosphoproteins was higher in KRAS mutated tumors than in WT tumors. The expression of pP70S6K was lower in responders than in nonresponder patients. In univariate analysis, patients with high pMEK1 or pP70S6K expression had a shorter PFS than those with low expression. Patients with high pP70S6K expression also had a shorter OS. In multivariate analysis, PFS was shorter for patients with high pMEK1 or pP70S6K expression, independently of KRAS status, as OS for patients with high pP70S6K expression. Therefore, WT KRAS patients with high pP70S6K expression had a shorter survival than those with low expression. Our results suggest the importance of EGFR downstream signaling phosphoproteins expression in addition to KRAS status to define the subgroup of patients who will not benefit from anti-EGFR therapy.Recent progress has been made in the treatment of colorectal cancer (CRC) with the introduction of new therapies targeting the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor. Monoclonal antibodies represent one of the most important options for the inhibition of EGFR.

BACKGROUND:Human epidermal growth factor receptor (HER) downstream signaling kinases have important effects on tumor response to anti-HER monoclonal antibodies and tyrosine kinase inhibitors. We validated an assay that uses phosphoprotein arrays for measurement of HER downstream signaling functionality in breast carcinomas.

Polyethylenimine ( PEI ) derivatives are polycationic nonviral vectors for gene transfer. Previous results achieved in vitro in head and neck cancer cells demonstrated that glucosylated PEI yields higher gene transfer efficiency and longer transgene expression than unsubstituted PEI. Using glucosylated PEI, p53 gene transfer was successfully achieved with subsequent recovery of P53 protein expression and induction of spontaneous apoptosis. The present study reports in vivo data achieved in human head and neck squamous cell carcinoma xenografted mice. Using biotinylated PEI and histochemistry analysis, the vector was found to diffuse in the proliferating cells of the tumor tissue, sparing necrotic areas. No diffusion was observed inside keratinized area composed of nonproliferating, mature differentiated cells. Using green fluorescent protein ( GFP ) transfection and fluorescence microscopy, the transgene expression was mainly observed at the periphery of the tumor containing proliferating cells. GFP expression appeared lower inside the tumor depth. Quantitative transgene expression kinetics was then determined using luciferase as reporter gene. The maximal transgene expression was achieved 48 hours after intratumoral injection of glucosylated PEI / DNA complexes. The highest gene transfer efficacy was achieved 48 hours after two intratumoral injection. After transfection of wild -type p53, tumor growth inhibition was observed in tumor -bearing mice receiving intratumoral injection of glucosylated PEI / DNA complexes repeated twice weekly. Tumor growth inhibition was maintained under continuous treatment using the same schedule. In all experiments, no noticeable toxicity was observed. The present results demonstrate the feasibility and the tumor growth inhibition potency of nonviral gene transfer using glucosylated polyethylenimine.

KRAS mutation detection represents a crucial issue in metastatic colorectal cancer (mCRC). The optimization of KRAS mutation detection delay enabling rational prescription of first-line treatment in mCRC including anti-EGFR-targeted therapy requires robust and rapid molecular biology techniques. Routine analysis of mutations in codons 12 and 13 on 674 paraffin-embedded tissue specimens of mCRC has been performed for KRAS mutations detection using three molecular biology techniques, that is, high-resolution melting (HRM), polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and allelic discrimination PCR (TaqMan PCR). Discordant cases were assessed with COBAS 4800 KRAS CE-IVD assay. Among the 674 tumor specimens, 1.5% (10/674) had excessive DNA degradation and could not be analyzed. KRAS mutations were detected in 38.0% (256/674) of the analysable specimens (82.4% in codon 12 and 17.6% in codon 13). Among 613 specimens in whom all three techniques were used, 12 (2.0%) cases of discordance between the three techniques were observed. 83.3% (10/12) of the discordances were due to PCR-RFLP as confirmed by COBAS 4800 retrospective analysis. The three techniques were statistically comparable (κ > 0.9; P < 0.001). From these results, optimization of the routine procedure consisted of proceeding to systematic KRAS detection using HRM and TaqMan and PCR-RFLP in case of discordance and allowed significant decrease in delays. The results showed an excellent correlation between the three techniques. Using HRM and TaqMan warrants high-quality and rapid-routine KRAS mutation detection in paraffin-embedded tumor specimens. The new procedure allowed a significant decrease in delays for reporting results, enabling rational prescription of first-line-targeted therapy in mCRC.

Cetuximab (Erbitux) is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody whose activity is related to the inhibition of EGFR downstream signaling pathways. P53 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) have been reported to control the functionality of PI3K/AKT signaling. In this study we evaluated whether reintroducing P53 using non-viral gene transfer enhances PTEN-mediated inhibition of PI3K/AKT signaling by cetuximab in PC3 prostate adenocarcinoma cell line bearing p53 and pten mutations. Signaling phosphoproteins expression was analyzed using Bio-Plex phosphoprotein array and western blot. Apoptosis induction was evaluated from BAX expression, caspase-3 activation and DNA fragmentation analyses. The results presented show that p53 and pten gene transfer additionally mediated cell growth inhibition and apoptosis induction by restoral of signaling functionality, which enabled the control of PI3K/AKT and MAPKinase signaling pathways by cetuximab in PC3 cells. These results highlight the interest of the analysis of signaling phosphoproteins expression as molecular predictive markers for response to cetuximab and show that p53 and pten mutations could be key determinants of cell response to cetuximab through the functional impact of these mutations on cell signaling.

Abstract. MAP kinase signaling proteins have major implications in the molecular oncogenesis of breast cancers and have been extensively investigated as putative targets for therapy. This study reports the investigation of the expression of P38 MAPK and its phosphorylated form (p-P38 MAPK) in clinical specimens of invasive breast carcinomas and their correlation with estrogen receptor (ER) and HER2 expression, as well as MAPK and PI3 kinase-AKT pathway signaling phosphorylated proteins. Expression levels of P38 MAPK and p-P38 MAPK as well as p-AKT, p-GSK3β, p-S6 kinase, p-MEK1 and p-ERK1/2 were quantitatively assessed using multiplex bead immunoassay in frozen specimens from 45 invasive ductal breast cancers. Twenty-nine specimens were ER + , 15 were HER2 + and 10 were triple-negative breast cancers (TNBCs). P38 MAPK was found to be expressed in all tumor specimens and was significantly (P= 0.002) overexpressed in ER + tumors. P38 MAPK expression was lower in TNBCs than in all of the other tumors.

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The activity of S9788, recently synthetized as a modulator of multidrug resistance (MDR), was compared with verapamil and cyclosporine A in normal sensitive and MDR K562 cell lines, then in samples from 33 patients with hematological malignancies, using flow cytometry with simultaneous detection of P-glycoprotein and determination of intracellular daunorubicin fluorescence. This technique was compared and correlated with a tritiated daunorubicin accumulation method. In K562 cell lines, S9788 exhibited a significantly higher reversing activity than verapamil and cyclosporine A, and allowed a complete restoration of the accumulation of daunorubicin when used at 5 mumol/L. In the clinical samples, the three compounds were evaluated at equimolar concentration (5 mumol/L) using concomitant exposure to daunorubicin and to the reversing agent. In P-glycoprotein-negative samples, no significant effect on intracellular daunorubicin fluorescence of any of the reversing agents was noted. In the 15 P- glycoprotein-positive samples, a significant increase in daunorubicin fluorescence, by at least one reversing agent, was seen in 10 cases, among which S9788 reversing activity was higher than that of the two other agents in seven cases. Complete reversal was only achieved in one case with S9788.