5-Fluorouracil(5-FU)is an anticancer drug extensively used for different cancers.Intracellular metabolic activation leads to several nucleoside and nucleotide metabolites essential to exert its cytotoxic activity on m...5-Fluorouracil(5-FU)is an anticancer drug extensively used for different cancers.Intracellular metabolic activation leads to several nucleoside and nucleotide metabolites essential to exert its cytotoxic activity on multiple cellular targets such as enzymes,DNA and RNA.In this paper,we describe the development of a method based on liquid chromatography coupled with high resolution mass spectrometry suitable for the simultaneous determination of the ten anabolic metabolites(nucleoside,nucleotide and sugar nucleotide)of 5-FU.The chromatographic separationwas optimized on a porous graphitic carbon column allowing the analysis of the metabolites of 5-FU as well as endogenous nucleotides.The detection was performed on an Orbitrap■tandem mass spectrometer.Linearity of the method was verified in intracellular content and in RNA extracts.The limit of detection was equal to 12 pg injected on column for nucleoside metabolites of 5-FU and 150 pg injected on column for mono-and tri-phosphate nucleotide metabolites.Matrix effect was evaluated in cellular contents,DNA and RNA extracts for nucleoside and nucleotides metabolites.The method was successfully applied to i)measure the proportion of each anabolic metabolite of 5-FU in cellular contents,ii)follow the consequence of inhibition of enzymes on the endogenous nucleotide pools,iii)study the incorporation of metabolites of 5-FU into RNA and DNA,and iv)to determine the incorporation rate of 5-FUrd into 18 S and 28 S sub-units of rRNA.展开更多
Osteosarcoma is a rare and highly malignant tumor that usually affects adolescents and young adults. Despite current management protocols, up to half of patients succumb to the disease. Moreover, there is no well-char...Osteosarcoma is a rare and highly malignant tumor that usually affects adolescents and young adults. Despite current management protocols, up to half of patients succumb to the disease. Moreover, there is no well-characterized molecular marker for diagnosis and prognosis. TP53 alterations have been associated with a poor prognosis in many cancers. The aim of this retrospective work was to find out whether TP53 functional status predicts response to neoadjuvant chemotherapy and thus may help treatment decision for osteosarcoma patients. Seventeen biopsies of osteosarcoma patients receiving primary metotrexate, cisplatin and adryamicin chemotherapy followed by surgery were analyzed. TP53 exons 5-9 mutations were screened. Among 17 biopsies, 4 (23.5%) displayed TP53 mutations: 3 deletions and one single-nucleotide substitution. The presence of TP53 gene mutation does not correlate with resistance to chemotherapy according to histological Rosen grade and nevertheless is associated with patient’s age in a significant manner (p 0.05). The presence of non-mutated TP53 is not entirely specific for a good prognosis. We found no evidence that TP53 mutations predict chemoresistance in osteosarcoma patients more over the overall survival curve, followed for more than 12 years, showing no difference between patients with tumors harboring wild type or mutated TP53 gene (p 0.5). Further analysis to identify other genes that can influence chemotherapy response and clinical outcome in osteosarcoma is needed to improve patient treatment.展开更多
基金supported by Institut National Du Cancer(grant number 2018-131).
文摘5-Fluorouracil(5-FU)is an anticancer drug extensively used for different cancers.Intracellular metabolic activation leads to several nucleoside and nucleotide metabolites essential to exert its cytotoxic activity on multiple cellular targets such as enzymes,DNA and RNA.In this paper,we describe the development of a method based on liquid chromatography coupled with high resolution mass spectrometry suitable for the simultaneous determination of the ten anabolic metabolites(nucleoside,nucleotide and sugar nucleotide)of 5-FU.The chromatographic separationwas optimized on a porous graphitic carbon column allowing the analysis of the metabolites of 5-FU as well as endogenous nucleotides.The detection was performed on an Orbitrap■tandem mass spectrometer.Linearity of the method was verified in intracellular content and in RNA extracts.The limit of detection was equal to 12 pg injected on column for nucleoside metabolites of 5-FU and 150 pg injected on column for mono-and tri-phosphate nucleotide metabolites.Matrix effect was evaluated in cellular contents,DNA and RNA extracts for nucleoside and nucleotides metabolites.The method was successfully applied to i)measure the proportion of each anabolic metabolite of 5-FU in cellular contents,ii)follow the consequence of inhibition of enzymes on the endogenous nucleotide pools,iii)study the incorporation of metabolites of 5-FU into RNA and DNA,and iv)to determine the incorporation rate of 5-FUrd into 18 S and 28 S sub-units of rRNA.
文摘Osteosarcoma is a rare and highly malignant tumor that usually affects adolescents and young adults. Despite current management protocols, up to half of patients succumb to the disease. Moreover, there is no well-characterized molecular marker for diagnosis and prognosis. TP53 alterations have been associated with a poor prognosis in many cancers. The aim of this retrospective work was to find out whether TP53 functional status predicts response to neoadjuvant chemotherapy and thus may help treatment decision for osteosarcoma patients. Seventeen biopsies of osteosarcoma patients receiving primary metotrexate, cisplatin and adryamicin chemotherapy followed by surgery were analyzed. TP53 exons 5-9 mutations were screened. Among 17 biopsies, 4 (23.5%) displayed TP53 mutations: 3 deletions and one single-nucleotide substitution. The presence of TP53 gene mutation does not correlate with resistance to chemotherapy according to histological Rosen grade and nevertheless is associated with patient’s age in a significant manner (p 0.05). The presence of non-mutated TP53 is not entirely specific for a good prognosis. We found no evidence that TP53 mutations predict chemoresistance in osteosarcoma patients more over the overall survival curve, followed for more than 12 years, showing no difference between patients with tumors harboring wild type or mutated TP53 gene (p 0.5). Further analysis to identify other genes that can influence chemotherapy response and clinical outcome in osteosarcoma is needed to improve patient treatment.
