[Normal organoids and their applications in cancer research]. / Les organoïdes normaux et leurs applications dans la recherche sur le cancer.

Three-dimensional (3D) culture of organoids from primary cells (wild type) or tumoroids from tumor cells, is used to study the physiological mechanisms in vivo, in order to model normal or tumor tissues more accurately than conventional two-dimensional (2D) culture. The features of this 3D culture, such as the three-dimensional structure, the self-renewal capacity and differentiation are preserved and appropriate to cancer study since their cellular characteristics are very similar to in vivo models. Here, we summarize the recent advances in the rapidly evolving field of organoids and their applications to cancer biology, clinical research and personalized medicine.

A novel 3D culture model recapitulates primary FL B-cell features and promotes their survival.

Non-Hodgkin B-cell lymphomas (B-NHL) mainly develop within lymph nodes as aggregates of tumor cells densely packed with their surrounding microenvironment, creating a tumor niche specific to each lymphoma subtypes. In vitro preclinical models mimicking biomechanical forces, cellular microenvironment, and 3D organization of B-cell lymphomas remain scarce, while all these parameters are key determinants of lymphomagenesis and drug resistance. Using a microfluidic method based on cell encapsulation inside permeable, elastic, and hollow alginate microspheres, we developed a new tunable 3D model incorporating lymphoma B cells, extracellular matrix (ECM), and/or tonsil stromal cells (TSC). Under 3D confinement, lymphoma B cells were able to form cohesive spheroids resulting from overexpression of ECM components. Moreover, lymphoma B cells and TSC dynamically formed self-organized 3D spheroids favoring tumor cell growth. 3D culture induced resistance to the classical chemotherapeutic agent doxorubicin, but not to the BCL2 inhibitor ABT-199, identifying this approach as a relevant in vitro model to assess the activity of therapeutic agents in B-NHL. RNA-sequence analysis highlighted the synergy of 3D, ECM, and TSC in upregulating similar pathways in malignant B cells in vitro than those overexpressed in primary lymphoma B cells in situ. Finally, our 3D model including ECM and TSC allowed long-term in vitro survival of primary follicular lymphoma B cells. In conclusion, we propose a new high-throughput 3D model mimicking lymphoma tumor niche and making it possible to study the dynamic relationship between lymphoma B cells and their microenvironment and to screen new anti-cancer drugs.

Model-Based Quantification of Impact of Genetic Polymorphisms and Co-Medications on Pharmacokinetics of Tamoxifen and Six Metabolites in Breast Cancer.

Variations in clinical response to tamoxifen (TAM) may be related to polymorphic cytochromes P450 (CYPs) involved in forming its active metabolite endoxifen (ENDO). We developed a population pharmacokinetic (PopPK) model for tamoxifen and six metabolites to determine clinically relevant factors of ENDO exposure. Concentration-time data for TAM and 6 metabolites come from a prospective, multicenter, 3-year follow-up study of adjuvant TAM (20 mg/day) in patients with breast cancer, with plasma samples drawn every 6 months, and genotypes for 63 genetic polymorphisms (PHACS study, NCT01127295). Concentration data for TAM and 6 metabolites from 928 patients (n = 27,433 concentrations) were analyzed simultaneously with a 7-compartment PopPK model. CYP2D6 phenotype (poor metabolizer (PM), intermediate metabolizer (IM), normal metabolizer (NM), and ultra-rapid metabolizer (UM)), CYP3A4*22, CYP2C19*2, and CYP2B6*6 genotypes, concomitant CYP2D6 inhibitors, age, and body weight had a significant impact on TAM metabolism. Formation of ENDO from N-desmethyltamoxifen was decreased by 84% (relative standard error (RSE) = 14%) in PM patients and by 47% (RSE = 9%) in IM patients and increased in UM patients by 27% (RSE = 12%) compared with NM patients. Dose-adjustment simulations support an increase from 20 mg/day to 40 and 80 mg/day in IM patients and PM patients, respectively, to reach ENDO levels similar to those in NM patients. However, when considering Antiestrogenic Activity Score (AAS), a dose increase to 60 mg/day in PM patients seems sufficient. This PopPK model can be used as a tool to predict ENDO levels or AAS according to the patient's CYP2D6 phenotype for TAM dose adaptation.

Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells.

GA101/obinutuzumab is a novel type II anti-CD20 monoclonal antibody (mAb), which is more effective than rituximab (RTX) in preclinical and clinical studies when used in combination with chemotherapy. Ca2+ signaling was shown to play a role in RTX-induced cell death. This report concerns the effect of GA101 on Ca2+ signaling and its involvement in the direct cell death induced by GA101. We reveal that GA101 triggered an intracellular Ca2+ increase by mobilizing intracellular Ca2+ stores and activating Orai1-dependent Ca2+ influx in non-Hodgkin lymphoma cell lines and primary B-Cell Chronic Lymphocytic Leukemia (B-CLL) cells. According to the cell type, Ca2+ was mobilized from two distinct intracellular compartments. In Raji, BL2, and B-CLL cells, GA101 induced a Ca2+ release from lysosomes, leading to the subsequent lysosomal membrane permeabilization and cell death. Inhibition of this calcium signaling reduced GA101-induced cell death in these cells. In SU-DHL-4 cells, GA101 mobilized Ca2+ from the endoplasmic reticulum (ER). Inhibition of ER replenishment, by blocking Orai1-dependent Ca2+ influx, led to an ER stress and unfolded protein response (UPR) which sensitized these cells to GA101-induced cell death. These results revealed the central role of Ca2+ signaling in GA101's action mechanism, which may contribute to designing new rational drug combinations improving its clinical efficacy.

Factors Affecting Tamoxifen Metabolism in Patients With Breast Cancer: Preliminary Results of the French PHACS Study.

In addition to the effect of cytochrome P450 (CYP) 2D6 genetic polymorphisms, the metabolism of tamoxifen may be impacted by other factors with possible consequences on therapeutic outcome (efficacy and toxicity). This analysis focused on the pharmacokinetic (PK)-pharmacogenetic evaluation of tamoxifen in 730 patients with adjuvant breast cancer included in a prospective multicenter study. Plasma concentrations of tamoxifen and six major metabolites, the genotype for 63 single-nucleotide polymorphisms, and comedications were obtained 6 months after treatment initiation. Plasma concentrations of endoxifen were significantly associated with CYP2D6 diplotype (P < 0.0001), CYP3A4*22 genotype (P = 0.0003), and concomitant intake of potent CYP2D6 inhibitors (P < 0.001). Comparison of endoxifen levels showed that the CYP2D6 phenotype classification could be improved by grouping intermediate metabolizer (IM)/IM and IM/poor metabolizer diplotype into IM phenotype for future use in tamoxifen therapy optimization. Finally, the multivariable regression analysis showed that formation of tamoxifen metabolites was independently impacted by CYP2D6 diplotype and CYP3A4*22, CYP2C19*2, and CYP2B6*6 genetic polymorphisms.

Selective dysfunction of p53 for mitochondrial biogenesis induces cellular proliferation in bronchial smooth muscle from asthmatic patients.

BACKGROUND: Increase of bronchial smooth muscle (BSM) mass is a crucial feature of asthma remodeling. The mechanisms of such an increased BSM mass are complex but involve enhanced mitochondrial biogenesis, leading to increased proliferation of BSM cells in asthmatic patients. The major tumor suppressor protein p53 is a key cell regulator involved in cell proliferation and has also been implicated in mitochondrial biogenesis. However, the role of p53 in BSM cell proliferation and mitochondrial biogenesis has not been investigated thus far. OBJECTIVE: We sought to evaluate the role of p53 in proliferation of BSM cells in asthmatic patients and mitochondrial biogenesis. METHODS: The expression of p53 was assessed both in vitro by using flow cytometry and Western blotting and ex vivo by using RT-PCR after laser microdissection. The role of p53 was assessed with small hairpin RNA lentivirus in both asthmatic patients and control subjects with BSM cell proliferation by using 5-bromo-2'-deoxyuridine and cell counting and in the expression of p21, BCL2-associated X protein, mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). RESULTS: Twenty-nine patients with moderate-to-severe asthma and 26 control subjects were enrolled in the study. p53 expression was increased in BSM from asthmatic patients both ex vivo and in vitro, with a decreased interaction with mouse double minute 2 homolog (Mdm2) and an increased phosphorylation of serine 20. p53 did not inhibit the transcription of both TFAM and PGC-1α in BSM cells from asthmatic patients. As a consequence, p53 is unable to slow the increased mitochondrial biogenesis and hence the subsequent increased proliferation of BSM cells in asthmatic patients. CONCLUSION: This study suggests that p53 might act as a new potential therapeutic target against BSM remodeling in asthmatic patients.

UGT1A1 genotype and irinotecan therapy: general review and implementation in routine practice.

Irinotecan is a major drug in the treatment of advanced colorectal cancer. Its active form is the SN38 metabolite, which is cleared by the biliary route after glucuronidation by uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1). UGT1A1 activity exhibits a wide intersubject variability, in part related to UGT1A1 gene polymorphisms. The present review on the impact of the deficient UGT1A1*28 variant on irinotecan efficacy and toxicity was produced by a French joint workgroup comprising the Group of Clinical Onco-pharmacology (GPCO-Unicancer) and the National Pharmacogenetics Network (RNPGx). It clearly emerges that for irinotecan doses at least equal to 180 mg/m(2) , patients homozygous for the UGT1A1*28 allele are at increased risk of developing hematological and/or digestive toxicities. Irinotecan dose reduction is thus recommended in homozygous *28/*28 patients. In addition, this personalized medicine strategy aims to secure high-dose irinotecan administration (≥240 mg/m(2) ) that have proven to be safe in homozygous *1/*1 patients only. The clinical relevance of this test is discussed in terms of treatment efficacy improvement, as increasing the irinotecan dose appears to be safe in patients not bearing a deficient allele. Best execution practices, cost-effectiveness, and result interpretation are discussed with the aim of facilitating the implementation of this analysis in clinical practice. The existence of networks of laboratories performing this test in routine hospital treatment, as in France, offers the prospect of widespread screening, thus guaranteeing equal access to safe treatment and optimized therapy for patients receiving irinotecan-based therapy in advanced colorectal cancer.

On the use of pharmacogenetics in cancer treatment and clinical trials.

There are an increasing number of studies devoted to the identification of associations between anticancer drug efficacy and toxicity and common polymorphisms present in the patients' genome. However, many articles presenting the results of such studies do not bring the simple and necessary background information allowing the evaluation of the relevance of the study, its significance and its potential importance for patients' treatment. This position paper first addresses clinical oncologists with the aim of giving them the basic knowledge on pharmacogenetics and on the potential use of gene polymorphisms as predictive biomarkers in routine and clinical research. A secondary objective is to give molecular biologists some recommendations on how to conceive protocols and how to publish their results when they develop pharmacogenetic studies appended to clinical trials or with autonomous goals.

[Interest of UGT1A1 genotyping within digestive cancers treatment by irinotecan]. / Intérêt du génotypage de l'UGT1A1 dans le cadre du traitement des cancers digestifs par irinotécan.

Irinotecan is a cytotoxic agent administered by IV infusion in the treatment of advanced colorectal cancer. Its anticancer activity results from its bioactivation into SN-38 metabolite, which is cleared through glucuronidation by the hepatic enzyme uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1). In the general population, there is wide inter-subject variability in UGT1A1 enzyme activity related to UGT1A1 gene polymorphisms. The French joint workgroup coming from the National Pharmacogenetic Network (RNPGx) and the Group of Clinical Oncologic Pharmacology (GPCO) herein presents an updated review dealing with efficacy and toxicity clinical studies related to UGT1A1 genetic variants. From a critical analysis of this review it clearly emerges that, for doses higher than 180 mg/m(2), hematologic and digestive irinotecan-induced toxicities could be prevented in daily clinical practice by generalizing the use of a simple pharmacogenetic test before starting treatment. The clinical relevance of this test is also discussed in terms of treatment efficacy improvement, with the possibility of increasing the irinotecan dose in patients not bearing the deleterious allele. This test involves using a blood sample to analyze the promoter region of the UGT1A1 gene (UGT1A1*28 allele). Best execution practices, laboratory costs, as well as results interpretation are described with the aim of facilitating the implementation of this analysis in clinical routine. The existence of a French laboratories network performing this test in clinical routine makes it possible to generalize UGT1A1 deficiency screening, so as to guarantee equal access to safe treatment and optimized irinorecan-based therapy for the many patients receiving irinotecan-based therapy in advanced colorectal cancer.

FOLFIRI® and bevacizumab in first-line treatment for colorectal cancer patients: safety, efficacy and genetic polymorphisms.

BACKGROUND: Over 50% of colorectal cancer (CRC) patients develop metastases. The aim of this study was to evaluate efficacy and tolerance of first-line FOLFIRI® + bevacizumab (B) treatment for metastatic CRC, and to assess genetic polymorphisms as potential markers. METHODS: Adult patients with histologically-proven, non-resectable metastatic CRC and ECOG ≤ 2 were included. 14-day cycles consisted of bevacizumab (5 mg/kg), irinotecan (180 mg/m2), bolus FU (400 mg/m2) and leucovorin (400 mg/m2), followed by 46-hour FU infusions (2400 mg/m2). Primary endpoint was response rate according to RECIST criteria. Secondary endpoints were overall (OS) and progression-free (PFS) survivals, response duration, and toxicity. Associations between clinical data, UGT1A1, thymidylate synthase, VEGFA polymorphisms and PFS, OS and toxicity were analyzed. RESULTS: Sixty-two patients were enrolled (median age 68y). 59/62 patients were eligible and evaluable for response at 6 months: 28 showed partial response (47.5%; 95% CI; 34.3-60.9), 20 stable disease (33.9%) and 11 progression (18.6%). Grade 3/4 toxicities were as follows: neutropenia 16.1%; diarrhea 11.3%; nausea-vomiting 1.6%. Median response duration was 9.5 months (range 2.7-20); median PFS 10.3 months (range 8.8-11.7); and median OS 25.7 months (range 20.2-29.7). 11/59 initially unresectable patients were resectable after treatment. VEGFA polymorphism (rs25648) was associated with better OS (HR: 3.61; 95% CI: 1.57-8.30). CONCLUSIONS: FOLFIRI® + bevacizumab is active with good response rate, long median OS, and a good safety profile. A VEGFA polymorphism might have a prognostic value in this malignancy. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00467142 (registration date: April 25, 2007).

No association between XRCC3 Thr241Met and XPD Lys751Gln polymorphisms and the risk of colorectal cancer in West Algerian population: a case-control study.

Colorectal cancer (CRC) is a complex and multifactorial disease, in which genetic and environmental factors both seem to play a part. Many epidemiological studies have explored the association between genetic polymorphisms of X-ray repair cross-complementing group 3 (XRCC3) (Thr241Met) and Xeroderma pigmentosum group D (XPD) lysine to glutamine at codon 751 (Lys751Gln) and risk of CRC in various populations; however, the results are controversial. We conducted this case-control study in a West Algerian population to assess the potential role of this genetic polymorphism on the risk of CRC in this population. Genomic DNA was extracted from blood samples collected from 129 sporadic CRC patients and 148 normal controls. The polymorphisms were determined by pyrosequencing technique. The distribution of XRCC3 Thr241Met and XPD Lys751Gln genotypes among controls did not differ significantly from those predicted by the Hardy-Weinberg distribution (p > 0.05). There were no significant differences in the genotypes distribution and allele frequencies between CRC patients and controls. A significant association was found between the combined heterozygous of XRCC3 and homozygous variant of XPD gene and CRC. This is the first study on DNA repair genetic polymorphisms in West Algerian population, and it suggests that the XRCC3 Thr241Met and XPD Lys751Gln polymorphisms may not be associated with the CRC risk in this population.

A functionalized heterobimetallic (99m)Tc/Re complex as a potential dual-modality imaging probe: synthesis, photophysical properties, cytotoxicity and cellular imaging investigations.

A novel bimodal fluorescent/radiolabelled probe based on a pyridyltriazole scaffold (known as pyta) is reported here. The final dual imaging agent combines carboxylate functionalization, for biomolecule conjugation, with two distinct metal chelating sites: a pyta-based tricarbonylrhenium moiety as a fluorescent probe and a (99m)Tc(CO3)(+) core through the tridentate chelating iminodiacetic acid (IDA) clamp as a SPECT reporter. The heterodinuclear (99m)Tc/Re complex , as well as its non-radioactive dirhenium analog , was prepared in six steps. The (99m)Tc/Re agent is water-soluble and stable against histidine challenge. Its structural characterization was achieved by HPLC comparison with the non-radioactive complex . Upon excitation in the MLCT band at 321 nm, the compound exhibits a bright green luminescence centered at 496 nm, with a quantum yield of 0.86% in Tris buffer, pH 7.4. Additionally, the influence of this compound on cell viability was tested on malignant cell lines (A549, HT29 and MCF-7 human lung, colon and breast carcinomas, respectively). Cell viability after 72 h incubation at 37 °C with 300 µmol of complex was >60% for all cell lines. Finally, cellular uptake studies of compound were performed by fluorescent microscopy, showing that the complex was clearly detected at the cellular level in A549 cells and to a lesser extent in HT29 cells. Taking into consideration the luminescent properties, the good radiochemical purity and the promising biological data (in vitro stability, non-toxicity, and cell tracking in two cell lines), the functionalized (99m)Tc/Re dinuclear compound can be considered a potential pre- and intraoperative diagnostic probe.

Major efficacy of trabectedin in 2 metastatic osteosarcoma patients with wild-type Asp1104 ERCC5 tumor status.

BACKGROUND: Treatment of osteosarcoma of the extremities consists of surgical resection preceded and followed by chemotherapy, including high-dose methotrexate or adriamycin-based protocols. When distant relapse occurs, therapeutic options are scarce. Trabectedin, a DNA-binding agent, is indicated for the treatment of patients with advanced soft tissue sarcomas after failure of anthracyclines and ifosfamide. In this indication, the 6-month progression-free survival is about 35-40%. Recent reports showed that some specific single nucleotide polymorphisms (SNPs) from DNA repair genes could be associated with sensitivity to trabectedin in soft tissue sarcomas. CASE REPORTS: We report our experience of 2 metastatic, heavily pre-treated osteosarcoma patients who were treated with trabectedin. Pyrosequencing analyses of tumors from both patients for several SNPs of the ERCC1, ERCC5 and BRAC1 genes were performed. Both patients showed major response to trabectedin, which was interestingly related with homozygoty of the common guanine allele of ERCC5 (G/G genotype; Asp/Asp) after pyrosenquencing analysis of tumors from both patients. This polymorphism was previously shown to be associated with better outcome in soft tissue sarcoma patients treated with trabectedin. CONCLUSION: Homozygoty for the wild-type Asp1104 SNP of the ERCC5 gene was found in 2 cases of relapsed osteosarcoma, who responded to trabectedin.

Prognostic value of PLAGL1-specific CpG site methylation in soft-tissue sarcomas.

Soft tissue sarcomas (STS) are rare, complex tumors with a poor prognosis. The identification of new prognostic biomarkers is needed to improve patient management. Our aim was to determine the methylation status of the 118 CpG sites in the PLAGL1 tumor-suppressor gene P1 CpG island promoter and study the potential prognostic impact of PLAGL1 promoter methylation CpG sites in STS. Training cohorts constituted of 28 undifferentiated sarcomas (US) and 35 leiomyosarcomas (LMS) were studied. PLAGL1 mRNA expression was investigated by microarray analysis and validated by RT-qPCR. Pyrosequencing was used to analyze quantitative methylation of the PLAGL1 promoter. Associations between global promoter or specific CpG site methylation and mRNA expression were evaluated using Pearson's product moment correlation coefficient. Cox univariate and multivariate proportional hazard models were used to assess the predictive power of CpG site methylation status. Sixteen CpG sites associated with PLAGL1 mRNA expression were identified in US and 6 in LMS. Statistical analyses revealed an association between CpG107 methylation status and both overall and metastasis-free survival in US, which was confirmed in a validation cohort of 37 US. The exhaustive study of P1 PLAGL1 promoter methylation identified a specific CpG site methylation correlated with mRNA expression, which was predictive for both metastasis-free and overall survival and may constitute the first US-specific biomarker. Such a biomarker may be relevant for identifying patients likely to derive greater benefit from treatment.

Evaluation of antiproliferative and antioxidant activities of the organic extract and its polar fractions from the Mediterranean gorgonian Eunicella singularis.

The present study was conducted to evaluate the antiproliferative and antioxidant activities of organic extract and its polar fractions from Eunicella singularis (Esper 1794). Organic extract and two fractions of E. singularis (F2 and F3) were screened for the presence of phenolic compounds, terpenoids and glycosides. The antiproliferative activity of E. singularis organic extract and its polar fractions was evaluated on human cancer cell lines (A549, lung cell carcinoma; HCT15, colon cell carcinoma and MCF7, breast adenocarcinoma), using the MTT colorimetric method and clonogenic assay, as well as the antioxidant activity, using the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the FRAP assays. The fractions F2 and F3 showed significant total phenolic content (40 and 35.72mg gallic-acid equivalent/g dried sample), respectively, and important antiproliferative properties against the cancer cell lines. The IC50 values, ranged from 36 to 274µg/ml for A549; 93 to 426µg/ml for HCT15; and 52 to 225µg/ml for MCF7 and in the clonogenic inhibition assay from 18 to 134µg/ml for A549; 43 to 357µg/ml for HCT15; and 17 to 160µg/ml for MCF7. Using the DPPH method, the fraction F2 exhibited the strongest radical scavenging activity, with IC50 0.08mg/ml, which approaches the activity of the powerful antioxidant standard, ascorbic acid (IC50=0.064mg/ml). The reducing power of the samples was in the following order: F2>organic extract>F3. These results suggest that E. singularis fractions might be used as a potential source of natural antioxidant and antitumor agents. The purification and determination of the chemical structures of compounds in these active fractions are under investigation. The results could provide a compound(s) with a promising role in future medicines.

Short curcumin treatment modulates oxidative stress, arginase activity, aberrant crypt foci, and TGF-ß1 and HES-1 transcripts in 1,2-dimethylhydrazine-colon carcinogenesis in mice.

This study investigated the effect of short curcumin treatment, a natural antioxidant on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) in mice. The incidence of aberrant crypt foci (ACF) was 100%, with 54 ± 6 per colon, 10 weeks after the first DMH injection and reached 67 ± 12 per colon after 12 weeks. A high level of undifferentiated goblet cells and a weak apoptotic activity were shown in dysplastic ACF. The morphological alterations of colonic mucosa were associated to severe oxidative stress ratio with 43% increase in malondialdehyde vs. 36% decrease in GSH. DMH also increased inducible nitric synthase (iNOS) mRNA transcripts (250%), nitrites level (240%) and arginase activity (296%), leading to nitrosative stress and cell proliferation. Curcumin treatment, starting at week 10 post-DMH injection for 14 days, reduced the number of ACF (40%), iNOS expression (25%) and arginase activity (73%), and improved redox status by approximately 46%, compared to DMH-treated mice. Moreover, curcumin induced apoptosis of dysplastic ACF cells without restoring goblet cells differentiation. Interestingly, curcumin induced a parallel increase in TGF-ß1 and HES-1 transcripts (42% and 26%, respectively). In conclusion, the protective effect of curcumin was driven by the reduction of arginase activity and nitrosative stress. The up regulation of TGF-ß1 and HES-1 expression by curcumin suggests for the first time, a potential interplay between these signalling pathways in the chemoprotective mechanism of curcumin.

Absence of transcriptomic signature of response to chemotherapy in metastatic colorectal carcinoma patients.

AIM: Tumor gene-expression profiling may define signatures capable of discriminating between responders and nonresponders to chemotherapy. PATIENTS & METHODS: Fifty seven metastatic colorectal cancer patients were prospectively included and 40 tumors were analyzed. Patients were treated in first line with 5-fluorouracil associated with irinotecan or oxaliplatin. Response was evaluated using WHO criteria every 2 months after chemotherapy. Gene-expression profiling was performed using Applied Biosystems microarrays (Human Genome Survey Microarray v2.0; Paris, France). Data were analyzed using Bioconductor packages. Differential-expression analysis was performed by fitting a linear model. Moderated t-statistics were computed and p-values were adjusted for false-discovery rate. Pearson correlations tests were evaluated between gene expression and progression-free and overall survival. RESULTS: Nonsupervised analysis did not show any clustering of expression levels according to treatment response. Supervised analysis compared expression levels between responders and nonresponders, within each treatment group and independently from treatment. No genes were identified as differentially expressed at a p-value of 10(-3) and false-discovery rate of 30%. No correlation between expression levels and survival data was found. CONCLUSION: These negative results show that the determinants of response to chemotherapy should be sought not only in the tumor characteristics, but also among the processes leading to drug availability to the tumor.

Deciphering the role of the ERCC2 gene polymorphism on anticancer drug sensitivity.

ERCC2 [Xeroderma pigmentosum (XP) group D] belongs to the nucleotide excision repair pathway. It is also part of the TFIIH transcription complex and is required for the association of the cyclin-dependent kinase (CDK)-activating kinase (CAK) subcomplex with TFIIH. Using the NCI-60 panel of human tumor cell lines, we had shown that the ERCC2 gene variant Gln(751) was significantly associated to increased taxanes sensitivity and decreased ERCC2 gene expression. Since TFIIH is involved in both DNA repair and cell cycle progression, we hypothesized that quantitative or qualitative ERCC2 alterations might cause CAK liberation, allowing its activation of the G(2)/M transition. Enhancing mitosis entry would lead to hypersensitivity to spindle poisons, explaining the effect of ERCC2 polymorphisms on taxane sensitivity. Starting from ERCC2-deficient XP6BE, we generated several isogenic clones differing only by the Lys751Gln variation. Wild-type and variant ERCC2-expressing clones recovered ultraviolet radiation and cisplatin resistance but presented similar sensitivity to paclitaxel, demonstrating that the amino acid change was not involved in paclitaxel differential sensitivity in the NCI-60 panel. Using small interfering RNA approach, we knocked down ERCC2 expression and observed a block in the G(2)/M phase, with a consistent increase in paclitaxel sensitivity and no change in cisplatin sensitivity. We observed in addition an increase in CDK1 activity, as evaluated by histone H1 phosphorylation. We evaluated messenger RNA (mRNA) half-life in the isogenic lines and observed a more rapid degradation in cells bearing the variant construct. We concluded that the increased paclitaxel sensitivity of ERCC2 variant cell lines is a consequence of lower gene expression, likely due to decreased stability of the variant ERCC2 mRNA.

Role of DNA repair gene polymorphisms in the efficiency of platinum-based adjuvant chemotherapy for non-small cell lung cancer.

BACKGROUND: Cisplatin-based adjuvant treatment of non-small cell lung cancer (NSCLC) has become standard, thanks to the studies that have shown a significant survival advantage. The identification of patients who could benefit from this adjuvant treatment would allow ineffective and toxic administrations to be avoided. Immunohistochemical expression of the excision repair cross-complementation group (ERCC)-1 protein has been associated with response to platinum-based chemotherapy in patients with NSCLC, and some polymorphisms of the genes involved in DNA repair have been shown to be associated with survival in advanced NSCLC. OBJECTIVE: The aim of our study was to evaluate the progression-free survival and tolerability of adjuvant treatment with platinum-based chemotherapy in patients with NSCLC, according to common DNA repair gene polymorphisms and ERCC1 expression. METHODS: We investigated the association of three DNA repair gene polymorphisms - Asn118Asn in ERCC1 (rs11615), Lys751Gln in ERCC2 (rs13181), and Asp1104His in ERCC5 (rs17655) - with the progression-free survival of 85 patients treated with platinum-based chemotherapy after surgery for NSCLC. RESULTS: We did not find significant associations between any of these polymorphisms and progression-free survival, nor did we observe any difference in progression-free survival according to ERCC1 expression. CONCLUSION: The previously reported impact of DNA repair gene polymorphisms on platinum-based chemotherapy treatment of advanced NSCLC was not observed in our study in the adjuvant setting.

Cytochrome P450 1B1 gene polymorphisms as predictors of anticancer drug activity: studies with in vitro models.

Cytochrome P450 1B1 (CYP1B1) is found in tumor tissue and is suspected to play a role in oncogenesis and drug resistance. CYP1B1 gene polymorphisms have been associated with the risk of developing lung and other cancers. They may be associated with tumor response to anticancer drugs. We have determined 4 frequent nonsynonymous gene polymorphisms of CYP1B1 in the human tumor cell lines panels of the National Cancer Institute (NCI) and the Japanese Foundation for Cancer Research (JFCR): rs10012 (R48G), rs1056827 (A119S), rs1056836 (L432V), and rs1800440 (N453S). Numerous anticancer drugs have been tested against these panels that offer the opportunity to detect associations between gene polymorphisms and drug sensitivity. CYP1B1 single nucleotide polymorphisms were in marked linkage disequilibrium. The L432V allelic variants were significantly associated with reduced sensitivity to DNA-interacting anticancer agents, alkylators, camptothecins, topoisomerase II inhibitors, and some antimetabolites. For instance, in the NCI panel, cell lines homozygous for the V432 allele were globally 2-fold resistant to alkylating agents (P = 5 × 10(-10)) and 4.5-fold to camptothecins (P = 6.6 × 10(-9)) than cell lines homozygous for the L432 allele. Similar features were exhibited by the JFCR panel. Cell lines homozygous for the V432 allele were globally less sensitive to DNA-interfering drugs than cell lines having at least 1 common allele. There was no significant association between mRNA expression of CYP1B1 and CYP1B1 genotype, and no significant association between CYP1B1 mRNA expression and drug cytotoxicity. These observations open the way to clinical studies exploring the role of CYP1B1 gene polymorphisms for predicting tumor sensitivity to chemotherapy.