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PurposeRapid advances in the understanding of cancer biology have transformed drug development thus leading to the approval of targeted therapies and to the development of molecular tests to select patients that will respond to treatments. KRAS status has emerged as a negative predictor of clinical benefit from anti-EGFR antibodies in colorectal cancer, and anti-EGFR antibodies use was limited to KRAS wild type tumors. In order to ensure wide access to tumor molecular profiling, the French National Cancer Institute (INCa) has set up a national network of 28 regional molecular genetics centers. Concurrently, a nationwide external quality assessment for KRAS testing (MOKAECM) was granted to analyze reproducibility and costs.Methods96 cell-line DNAs and 24 DNA samples from paraffin embedded tumor tissues were sent to 40 French laboratories. A total of 5448 KRAS results were collected and analyzed and a micro-costing study was performed on sites for 5 common methods by an independent team of health economists.ResultsThis work provided a baseline picture of the accuracy and reliability of KRAS analysis in routine testing conditions at a nationwide level. Inter-laboratory Kappa values were >0.8 for KRAS results despite differences detection methods and the use of in-house technologies. Specificity was excellent with only one false positive in 1128 FFPE data, and sensitivity was higher for targeted techniques as compared to Sanger sequencing based methods that were dependent upon local expertise. Estimated reagent costs per patient ranged from €5.5 to €19.0.ConclusionThe INCa has set-up a network of public laboratories dedicated to molecular oncology tests. Our results showed almost perfect agreements in KRAS testing at a nationwide level despite different testing methods ensuring a cost-effective equal access to personalized colorectal cancer treatment.

The regulation of cell metabolism by the surrounding environment is deeply altered by the posttranslational nonenzymatic modifications of extracellular proteins that occur throughout lifespan in vivo and modify their structural and functional properties. Among them are protein adducts formed by components generated from oxidative processes, such as malondialdehyde (MDA). We have investigated here the effects of MDA-binding to proteins on cultured fibroblast functions. Type I collagen and/or serum proteins were incubated with 0-100 mM MDA for 3 h before use in fibroblast cultures. In tridimensional lattice cultures, MDA-treated collagen inhibited the contracting activity of fibroblasts. A similar inhibition of lattice contraction was reproduced by the addition of MDA-treated serum to the culture medium. In monolayer cultures, the addition of MDA-modified serum proteins completely inhibited fibroblast multiplication without effect on initial adhesion steps. MDA-modified proteins decreased the proliferative capacities of cells, strongly altered cell cycle progression by blocking passage to G2/M phases, and induced apoptotic features in fibroblasts. Our results show, for the first time, that MDA-modified proteins are potentially as deleterious as free MDA, and could be involved in aging as well as in degenerative complications of diseases with increased oxidative stress such as diabetes mellitus or atherosclerosis.

Expression of three major resistance genes MDR1, MRP1 and LRP was investigated in small cell lung cancer, non-small cell lung cancer and metastasis. Single biopsies of bronchoscopy from 73 patients were performed to investigate expression of these three resistance genes by reverse transcriptase-polymerase chain reaction. Relations between gene expression and patient age, smoking status, histology, and chemotherapy were evaluated. A more frequent expression of MDR1 (77 versus 66%), MRP1 (91 versus 72%) and LRP (77 versus 63%) genes was detected in the malignant biopsies than in the non-malignant, respectively. In the metastasis biopsies, expression of these genes was markedly increased. No significant difference was observed between specimens before and after chemotherapy. Biopsies from progressing cancer showed higher MDR1, MRP1 and LRP gene expression. In conclusion, these data reveal a major role of MRP1 in intrinsic resistance and the high gene expression of MDR1 and MRP1 in relapsed diseases.

A method for detection of cells with reduced drug retention was evaluated in solid tumours. After a 1 h incubation with daunorubicin (DNR), the right angle scatter (RAS), forward angle scatter (FAS), and specific fluorescence (Fluo) were measured in sensitive and resistant cells; only Fluo was related qualitatively, but not quantitatively, to resistance. Various incubation conditions were examined. When the pH of the incubation medium increased, the DNR retention increased in sensitive and resistant cells. In contrast, when the cell concentration increased, the DNR retention decreased. Using sensitive and resistant cell lines, a proportion of resistant cells lower than 10% can be detected in a mixture. To analyse cells from solid tumours, the cells were dissociated by repeated fine needle aspirations. Tumours from 22 patients have been processed with this technique; 8 samples were classified as S (sensitive); 2 as R (resistant); and 12 as I (intermediate). Further experiments were run to study and improve the method. Another method of detection of dead cells was tested. The intra‐assay variability of the technique was found to be less than 10%. When the study was performed with different fragments of the same tumour, the variation, corresponding to the tumour heterogeneity, rose to 21 to 36%. The inter‐assay reproducibility was too high, so a variant of this technique has been adapted, using verapamil or cyclosporin A, which is able to block DNR efflux; this new method allows tumour cells to be used as their own controls. © 1992 Wiley‐Liss, Inc.