Contributions
Abstract: P1263
Type: Poster Sessions
Abstract Category: Therapy - Tools for detecting therapeutic response
Cladribine (2-CdA) is converted in the active compound 2-chlorodeoxyadenosine 5'-monophosphate (2-CdAMP) through the phosphorylation activity of deoxycytidine kinase (DCK). This activity is balanced by 5'-nucleotidases (5'-NT) enzymes that prevent accumulation of 2-CdATP. Among the 5'NT genes, NT5C1A and NT5C2 have been observed as those who are able to metabolize 2-CdAMP. Low level of DCK mRNA and a higher level of 5'NT mRNA can be associated with 2CdA drug resistance. Other factors, such as viral infections, may affect the 2-CdA effectiveness interfering with its metabolism. In our previous work, we showed that Epstein-Barr nuclear antigen 2 (EBNA2) alleles variants associates with multiple sclerosis (MS).
Our aim is to assess a method fast and reliable to identify possible drug resistant MS patients, and to study whether EBNA2 variants may interfere with the 2-CdA metabolism. We evaluated the gene expression profile of DCK, NT5C1A and NT5C2 genes and the EBNA2 alleles in peripheral blood of relapsing-remitting MS patients (RRMS).
Peripheral blood mononuclear cells (PBMCs) were obtained from 40 RRMS patients free from therapy for at least 3 months. DNA and RNA were extracted from the same samples and cDNAs were obtained using commercial kits. Gene expression and EBV alleles identification were studied using digital droplet PCR approaches (QX200-Biorad). We observed comparable level of expression among DCK and NT5C2 genes and lower level of NT5C1A compared to the others. The DCK/NT5C1A ratio obtained are higher than 1 in all but 2 samples analyzed. The DCK/NT5C2 ratio show values range from 0,2 to 1,9. We performed a Spearman correlation analysis, obtaining a moderate level of correlation (r2=0,27; p=0,01) among the two ratios. To evaluate whether EBV variants can affect the 2-CdA metabolism, we stratified the expression data obtained in 13 sumples, according to the EBNA2 alleles: 1.2 (MS-associated; n=5) and 1.3 (HD-associated; n= 7), without observing statistically relevant differences.
Our results show that a protocol for 2-CdA metabolism evaluation in peripheral blood could be useful in patients management, given the degree of variability existing among the genes involved in this process. The lack of correlation with EBV alleles may be due to the reduced number of observations. Our preliminary data needs to be further explored in a larger population of patients under 2-CdA treatment and correlated with the clinical parameters.
Disclosure: Mechelli R receives research support from the Italian Multiple Sclerosis Foundation (FISM). Manfrè G reports no disclosures. Pellicciari G reports no disclosures. Reniè R reports no disclosures. Romano C reports no disclosures. Ristori G receives research support from the Italian Multiple Sclerosis Foundation (FISM). Visconti A reports no disclosures. Salvetti M, receives research support and has received fees as speaker from Sanofi Aventis, Biogen, Bayer Schering, Merck Serono. He also receives research support from the Italian Multiple Sclerosis Foundation (FISM).
Abstract: P1263
Type: Poster Sessions
Abstract Category: Therapy - Tools for detecting therapeutic response
Cladribine (2-CdA) is converted in the active compound 2-chlorodeoxyadenosine 5'-monophosphate (2-CdAMP) through the phosphorylation activity of deoxycytidine kinase (DCK). This activity is balanced by 5'-nucleotidases (5'-NT) enzymes that prevent accumulation of 2-CdATP. Among the 5'NT genes, NT5C1A and NT5C2 have been observed as those who are able to metabolize 2-CdAMP. Low level of DCK mRNA and a higher level of 5'NT mRNA can be associated with 2CdA drug resistance. Other factors, such as viral infections, may affect the 2-CdA effectiveness interfering with its metabolism. In our previous work, we showed that Epstein-Barr nuclear antigen 2 (EBNA2) alleles variants associates with multiple sclerosis (MS).
Our aim is to assess a method fast and reliable to identify possible drug resistant MS patients, and to study whether EBNA2 variants may interfere with the 2-CdA metabolism. We evaluated the gene expression profile of DCK, NT5C1A and NT5C2 genes and the EBNA2 alleles in peripheral blood of relapsing-remitting MS patients (RRMS).
Peripheral blood mononuclear cells (PBMCs) were obtained from 40 RRMS patients free from therapy for at least 3 months. DNA and RNA were extracted from the same samples and cDNAs were obtained using commercial kits. Gene expression and EBV alleles identification were studied using digital droplet PCR approaches (QX200-Biorad). We observed comparable level of expression among DCK and NT5C2 genes and lower level of NT5C1A compared to the others. The DCK/NT5C1A ratio obtained are higher than 1 in all but 2 samples analyzed. The DCK/NT5C2 ratio show values range from 0,2 to 1,9. We performed a Spearman correlation analysis, obtaining a moderate level of correlation (r2=0,27; p=0,01) among the two ratios. To evaluate whether EBV variants can affect the 2-CdA metabolism, we stratified the expression data obtained in 13 sumples, according to the EBNA2 alleles: 1.2 (MS-associated; n=5) and 1.3 (HD-associated; n= 7), without observing statistically relevant differences.
Our results show that a protocol for 2-CdA metabolism evaluation in peripheral blood could be useful in patients management, given the degree of variability existing among the genes involved in this process. The lack of correlation with EBV alleles may be due to the reduced number of observations. Our preliminary data needs to be further explored in a larger population of patients under 2-CdA treatment and correlated with the clinical parameters.
Disclosure: Mechelli R receives research support from the Italian Multiple Sclerosis Foundation (FISM). Manfrè G reports no disclosures. Pellicciari G reports no disclosures. Reniè R reports no disclosures. Romano C reports no disclosures. Ristori G receives research support from the Italian Multiple Sclerosis Foundation (FISM). Visconti A reports no disclosures. Salvetti M, receives research support and has received fees as speaker from Sanofi Aventis, Biogen, Bayer Schering, Merck Serono. He also receives research support from the Italian Multiple Sclerosis Foundation (FISM).