Improved proteome coverage by using iTRAQ labelling and peptide OFFGEL fractionation

<p>Abstract</p> <p>Background</p> <p>The development of mass spectrometric techniques and fractionation methods now allows the investigation of very complex protein mixtures ranging from subcellular structures to tissues. Nevertheless, this work is particularly difficult due to the wide dynamic range of protein concentration in eukaryotic tissues. In this paper, we present a shotgun method whereby the peptides are fractionated using OFFGEL electrophoresis after iTRAQ labelling.</p> <p>Results</p> <p>We demonstrated that iTRAQ peptide labelling enhances MALDI ionisation and that the OFFGEL fractionation of the labelled peptides introduces a supplementary criterion (pI) useful for validation and identification of proteins. We showed that iTRAQ samples allowed lower-concentrated proteins identification in comparison with free-labelled samples.</p> <p>Conclusion</p> <p>The combined use of iTRAQ labelling and OFFGEL fractionation allows a considerable increase in proteome coverage of very complex samples prepared from total cell extracts and supports the low-concentrated protein identification.</p

A Proteomic Approach for Plasma Biomarker Discovery with iTRAQ Labelling and OFFGEL Fractionation

Human blood plasma contains a plethora of proteins, encompassing not only proteins that have plasma-based functionalities, but also possibly every other form of low concentrated human proteins. As it circulates through the tissues, the plasma picks up proteins that are released from their origin due to physiological events such as tissue remodeling and cell death. Specific disease processes or tumors are often characterized by plasma “signatures,” which may become obvious via changes in the plasma proteome profile, for example, through over expression of proteins. However, the wide dynamic range of proteins present in plasma makes their analysis very challenging, because high-abundance proteins tend to mask those of lower abundance. In the present study, we used a strategy combining iTRAQ as a reagent which improved the peptide ionization and peptide OFFGEL fractionation that has already been shown, in our previous research, to improve the proteome coverage of cellular extracts. Two prefractioning methods were compared: immunodepletion and a bead-based library of combinatorial hexapeptide technology. Our data suggested that both methods were complementary, with regard to the number of identified proteins. iTRAQ labelling, in association with OFFGEL fractionation, allowed more than 300 different proteins to be characterized from 400 μg of plasma proteins

Irinotecan induces steroid and xenobiotic receptor (SXR) signaling to detoxification pathway in colon cancer cells

<p>Abstract</p> <p>Background</p> <p>Resistance to chemotherapy remains one of the principle obstacles to the treatment of colon cancer. In order to identify the molecular mechanism of this resistance, we investigated the role of the steroid and xenobiotic receptor (SXR) in the induction of drug resistance. Indeed, this nuclear receptor plays an important role in response to xenobiotics through the upregulation of detoxification genes. Following drug treatments, SXR is activated and interacts with the retinoid X receptor (RXR) to induce expression of some genes involved in drug metabolism such as phase I enzyme (like CYP), phase II enzymes (like UGT) and transporters (e.g. MDR1).</p> <p>Results</p> <p>In this study, we have shown that endogenous SXR is activated in response to SN-38, the active metabolite of the anticancer drug irinotecan, in human colon cancer cell lines. We have found that endogenous SXR translocates into the nucleus and associates with RXR upon SN-38 treatment. Using ChIP, we have demonstrated that endogenous SXR, following its activation, binds to the native promoter of the CYP3A4 gene to induce its expression. RNA interference experiments confirmed SXR involvement in CYP3A4 overexpression and permitted us to identify CYP3A5 and MRP2 transporter as SXR target genes. As a consequence, cells overexpressing SXR were found to be less sensitive to irinotecan treatment.</p> <p>Conclusions</p> <p>Altogether, these results suggest that the SXR pathway is involved in colon cancer irinotecan resistance in colon cancer cell line via the upregulation of select detoxification genes.</p

Skin toxicity and quality of life in patients with metastatic colorectal cancer during first-line panitumumab plus FOLFIRI treatment in a single-arm phase II study

Background: Integument-related toxicities are common during epidermal growth factor receptor (EGFR)-targeted therapy. Panitumumab is a fully human monoclonal antibody targeting the EGFR that significantly improves progression-free survival when added to chemotherapy in patients with metastatic colorectal cancer who have wild-type (WT) KRAS tumours. Primary efficacy and tolerability results from a phase II single-arm study of first-line panitumumab plus FOLFIRI in patients with metastatic colorectal cancer have been reported. Here we report additional descriptive tolerability and quality of life data from this trial. Methods: Integument-related toxicities and quality of life were analysed; toxicities were graded using modified National Cancer Institute Common Toxicity Criteria. Kaplan-Meier estimates of time to and duration of first integument-related toxicity were prepared. Quality of life was measured using EuroQoL EQ-5D and EORTC QLQ-C30. Best overall response was analysed by skin toxicity grade and baseline quality of life. Change in quality of life was analysed by skin toxicity severity. Results: 154 patients were enrolled (WT KRAS n = 86; mutant KRAS n = 59); most (98%) experienced integument-related toxicities (most commonly rash [42%], dry skin [40%] and acne [36%]). Median time to first integument-related toxicity was 8 days; median duration was 334 days. Overall, proportionally more patients with grade 2+ skin toxicity responded (56%) compared with those with grade 0/1 (29%). Mean overall EQ-5D health state index scores (0.81 vs. 0.78), health rating scores (72.5 vs. 71.0) and QLQ-C30 global health status scores (65.8 vs. 66.7) were comparable at baseline vs. safety follow-up (8 weeks after completion), respectively and appeared unaffected by skin toxicity severity. Conclusions: First-line panitumumab plus FOLFIRI has acceptable tolerability and appears to have little impact on quality of life, despite the high incidence of integument-related toxicity

Phase II study of preoperative radiation plus concurrent daily tegafur-uracil (UFT) with leucovorin for locally advanced rectal cancer

<p>Abstract</p> <p>Background</p> <p>Considerable variation in intravenous 5-fluorouracil (5-FU) metabolism can occur due to the wide range of dihydropyrimidine dehydrogenase (DPD) enzyme activity, which can affect both tolerability and efficacy. The oral fluoropyrimidine tegafur-uracil (UFT) is an effective, well-tolerated and convenient alternative to intravenous 5-FU. We undertook this study in patients with locally advanced rectal cancer to evaluate the efficacy and tolerability of UFT with leucovorin (LV) and preoperative radiotherapy and to evaluate the utility and limitations of multicenter staging using pre- and post-chemoradiotherapy ultrasound. We also performed a validated pretherapy assessment of DPD activity and assessed its potential influence on the tolerability of UFT treatment.</p> <p>Methods</p> <p>This phase II study assessed preoperative UFT with LV and radiotherapy in 85 patients with locally advanced T3 rectal cancer. Patients with potentially resectable tumors received UFT (300 mg/m/²/day), LV (75 mg/day), and pelvic radiotherapy (1.8 Gy/day, 45 Gy total) 5 days/week for 5 weeks then surgery 4-6 weeks later. The primary endpoints included tumor downstaging and the pathologic complete response (pCR) rate.</p> <p>Results</p> <p>Most adverse events were mild to moderate in nature. Preoperative grade 3/4 adverse events included diarrhea (n = 18, 21%) and nausea/vomiting (n = 5, 6%). Two patients heterozygous for dihydropyrimidine dehydrogenase gene (<it>DPYD</it>) experienced early grade 4 neutropenia (variant IVS14+1G > A) and diarrhea (variant 2846A > T). Pretreatment ultrasound TNM staging was compared with postchemoradiotherapy pathology TN staging and a significant shift towards earlier TNM stages was observed (p < 0.001). The overall downstaging rate was 42% for primary tumors and 44% for lymph nodes. The pCR rate was 8%. The sensitivity and specificity of ultrasound for staging was poor. Anal sphincter function was preserved in 55 patients (65%). Overall and recurrence-free survival at 3 years was 86.1% and 66.7%, respectively. Adjuvant chemotherapy was administered to 36 node-positive patients (mean duration 118 days).</p> <p>Conclusion</p> <p>Preoperative chemoradiotherapy using UFT with LV plus radiotherapy was well tolerated and effective and represents a convenient alternative to 5-FU-based chemoradiotherapy for the treatment of resectable rectal cancer. Pretreatment detection of DPD deficiency should be performed to avoid severe adverse events.</p

Place des outils pharmacogénétique, biochimique et pharmacocinétique dans l'optimisation thérapeutique de l'irinotécan

Le cancer colorectal pose un véritable problème de santé publique avec 33 000 nouveaux cas par an. Le traitement d'environ la moitié de ces patients relève d'une chimiothérapie. Si le 5-fluorouracile (5-FU) reste une molécule majeure, de nouveaux médicaments comme l'irinotécan (CPT-11)sont maintenant utilisés. Le CPT-11 est un promédicament dont le métabolite actif est le SN-38. Les toxicités majeures sont la diarrhée et la myélosuppression, souvent de survenue et d'intensité peu prédictibles, dues à une forte variabilité inter- et intra-individuelle du métabolisme du CPT-11. Nous avons donc étudié cette variabilité par différentes approches : pharmacogénétique, biochimique et pharmacocinétique sur l'ensemble des voies enzymatiques impliquées dans le métabolisme du CPT-11. Nous avons mis au point différentes méthodes, utilisables en pratique courante, permettant d'appréhender, chez chaque individu, l'existence d'un risque de toxicité grave à l'irinotécan. Ce travail constitue une étape incontournable dans l'individualisation des traitements par l'irinotécan.Colorectal cancer is a main public health problem with 33 000 new cases every year. Approximately one half of the patients would receive a chimiotherapeutic treatment. If 5-fluorouracil (5-FU) is a major molecule for this treatment, news drugs have been developed such as irinotecan (CPT-11). CPT-11 is a prodrug and SN-38 is its active metabolite. Toxicities are represented by profuse diarrhoea and myelosuppression, of which arising and intensivity are poorly predictable, due to inter- and intra-individual CPT-11 metabolism variability. We have then studied this variability by different approaches : pharmacogenetic, biochemical and pharmacokinetic on the whole enzymatic pathways implicated in CPT-11 metabolism. Globally, this work would allow to prevent sever toxic event arising and to optimise individually CPT-11 administration.ANGERS-BU Médecine-Pharmacie (490072105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Intensification thérapeutique dans les cancers colorectaux par des études pharmacogénétiques et pharmacogénomiques

Le cancer colorectal représente un défi thérapeutique compte tenu de sa fréquence (38 000 nouveaux cas annuels dans notre pays), et de sa gravité (moins de 10 % de survie à 5 ans en situation métastatique). Sa prise en charge adéquate est avant tout multidisciplinaire associant les nouvelles techniques de chirurgie et de radiologie à la chimiothérapie. Celle-ci est représentée par trois cytotoxiques majeurs : le 5-Fluorouracile, pierre angulaire du traitement, l'oxaliplatine (L-OHP) et enfin l'irinotecan (CPT-11), auxquels se sont ajoutées ces dernières années les biothérapies ciblant le facteur angiogénique VEGF (bevacizumab) ou le récepteur épithélial de surface EGFR (cetuximab, panitumumab). Notre travail de Thèse d'Université a cherché à explorer, chez des patients atteints d'un cancer colorectal métastatique, le retentissement sur les taux de réponses, les survies et les toxicités, de génotypes enzymatiques impliqués dans les métabolismes des trois cytotoxiques principalement utilisés dans cette indication. Nos études translationnelles montrent l'optimisation des traitements à base de 5-FU par l'approche pharmacocinétique associée au dépistage préthérapeutique d'une population à haut risque de toxicité (DPYD mutés et/ou rapport UH2/U abaissé). Par ailleurs apparaissent de mauvais pronostic sur la survie globale et en cas de monochimiothérapie par 5-FU, les patients 3R/3R pour TYMS et homozygote sauvage pour MTHFR 1298 A>C ou 677 C>T, facteurs de risques génotypiques disparaissant avec l'adjonction de CPT-11. Concernant ce dernier, un schéma de traitement adapté au statut UGT 1A1, et combiné au cetuximab, aboutit à des taux de maladie contrôlée et de survie sans progression inégalés en deuxième ligne de traitement. Enfin, les patients homozygotes T/T pour ERCC1 118 C>T et C/C XPD 751 A>C apparaissent à risque de neurotoxicité chroniques à l'oxaliplatine. Considérant le génome tumoral, outre Kras et Braf, PI3KCA muté apparaît pour la première fois comme un facteur possible de résistance aux traitements anti-EGFR. En conclusion, la prise en compte des facteurs pharmacogénétiques et pharmacogénomiques devrait permettre à l'avenir une meilleure rationalisation des traitements de chimiothérapies en optimisant leurs efficacités tout en limitant leurs toxicités.Colorectal cancer is one of the commonest tumours in France with 38,000 cases annually diagnosed. Treatment options for metastatic colorectal cancer (mCRC) patients have rapidly increased in the past years, but 50-70% of mCRC patients are still unlikely to undergo radical resection of metastases and are candidates for palliative chemotherapy only. Since 1996 the chemotherapeutic armamentarium has grown beyond 5-fluorouracil -corner stone of treatment-, to include irinotecan (CPT-11) and oxaliplatin (L-OHP) as well as three targeted monoclonal antibodies (Moabs): bevacizumab (an anti-vascular endothelial growth factor Moab) and cetuximab/panitumumab, both anti-epidermal growth factor receptor inhibitors. The purpose of our Thesis was to analyse the possible correlation between germinal polymorphism implicated in metabolisms of these drugs on efficacy and toxicity of 5-FU based chemotherapy regimens in mCRC patients. Our results confirm the interest in optimizing treatment for the pre-therapeutic detection of genetic factors such as DPYD following by 5-FU pharmacokinetic monitoring. We isolated a population (TYMS 3R/3R genotype associated with MTHFR C/C for 677 C>T or A/A for 1298 A>C) with a higher risk of response and survival failure under exclusive 5-FU treatment. This risk disappeared with added CPT-11. Tailored FOLFIRI regimen (5-FU plus irinotecan) with cetuximab and adapted doses according to pharmacokinetic monitoring for 5-FU and UGT 1A1 genotype status for irinotecan, showed excellent response and survival rates never previously observed in second line of treatment to our knowledge. For oxaliplatin, we identified a subgroup of patients with a higher risk of late neurotoxicity: T/T for 118 C>T ERCC1 or C/C for XPD 751 A>C. Finally regarding tumoral polymorphisms, in addition to Kras and Braf status, mutated PI3KCA appeared as a potential predictive biomarker of anti-EGFR resistance. In conclusion pharmacogenetic, pharmacokinetic and pharmacogenomic approaches should be considered in order to optimize chemotherapy treatments in mCRC.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Influence des paramètres pharmacogénétiques sur la tolérance et l'efficacité du 5-fluorouracile dans le traitement du cancer colo-rectal

ANGERS-BU Médecine-Pharmacie (490072105) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

INFLUENCE DU SEXE ET DE L'AGE SUR LA CLAIRANCE DU 5 FLUOROURACILE (DES ONCOLOGIE MEDICALE)

ANGERS-BU Médecine-Pharmacie (490072105) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Etude pharmacogénétique et pharmacogénomique du métabolisme des fluoropyrimides (cartographie métabolique et optimisation des traitements des cancers colorectaux)

Les fluoropyrimidines sont les anticancéraux les plus utilisés en chimiothérapie, le 5-fluorouracile (5-FU) étant le médicament de référence dans le traitement des cancers colorectaux. Dans l'organisme, le 5-FU est soit transformé en métabolites actifs par la voie anabolique, responsable de son activité cytotoxique, soit éliminé par la voie catabolique. Des toxicités sévères, pouvant engager le pronostic vital du patient, ont été rencontrées lors de traitements conventionnels et ont été attribuées à un déficit en dihydropyrimidine déshydrogénase (DPD), enzyme initiale du catabolisme du 5-FU. Afin de dépister avant traitement les patients présentant un risque de toxicité, plusieurs approches ont été étudiées : la détection de mutations sur le gène de la DPD, la mesure de l'expression des ARNm de la DPD leucocytaire et le dosage plasmatique de l'uracile endogène, substrat de la DPD, et de son métabolite, le 5,6-dihydrouracile. L'étude de la corrélation entre ces différents paramètres et la tolérance au traitement a permis de mettre au point une stratégie de dépistage des patients déficitaires en DPD permettant d'adapter les doses de 5-FU dès la première cure de chimiothérapie. Par ailleurs, un dosage plasmatique du ftorafur (prodogue du 5-FU administrée sous forme d'UFT®) et de deux de ses métabolites a été développé dans le but d'individualiser les traitements par UFT® en fonction des capacités métaboliques du patient.Fluoropyrimidines are the most commonly anticancer agents used in chemotherapy, 5-fluorouracile (5-FU) being the reference in colorectal cancer treatment. 5-FU is either transformed in active metabolites by anabolism, or eliminated by catabolic pathway. Severe side effects, including life-threatening toxicity, have been encountered during conventional treatments and have beeb attributed to a genetic deficiency of dihydropyrimidine dehydrogenase (DPD) activty, the initial enzyme of 5-FU catabolism pathway. Several approaches have been developed in an attempt to detect patients at high risk of 5-FU toxicity prior to treament : detection of mutations of DPD gene, measurement of DPD mRNA expression in leukocytes and determination of plasma levels of endogenous uracil, a DPD substrate, and of its metabolite, 5,6-dihydrouracile. Study of the correlation between these parameters and to adapt 5-FU doses at the first course of treatment. Moreover, a sensitive LC/MSMS method has been set up for plasma measurement of ftorafur (oral 5-FU prodrug administered as UFT®) and two metabolites in an attempt to individualize UFT® treatments to the patient's metabolic capacities.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF