Search

Overexpression of the multi-drug transporter P-glycoprotein, encoded by the ABCB1 gene, is a clinically relevant problem in acute myeloid leukemia (AML). Polymorphisms in ABCB1 might contribute to cancer risk and therapeutic response. We therefore investigated the influence of polymorphisms G1199A, C1236T, G2677T/A and C3435T on cancer susceptibility, in vitro cytotoxicity and overall survival in 100 de novo AML patients with normal karyotype. Patients with 1236C/C or 2677G/G genotypes showed poorer survival than patients with other genotypes (P = 0.03 and P = 0.02, respectively). Both these genotypes were significant factors for survival in multivariate analysis, along with age, NPM1 and FLT3 mutation status. In vitro cytotoxicity studies demonstrated that leukemic cells from 1236T/T and 2677T/T patients were significantly more susceptible to mitoxantrone (P 0.02), and tended to be more susceptible to etoposide and daunorubicin (P = 0.07-0.09), but not to cytarabine. No significant difference in allele frequencies was found between patients and healthy volunteers (n = 400). Funding Agencies|Swedish Cancer Society||Swedish Research Council-Medicine||Cancer Society in Stockholm||Karolinska Institutet||County Council in Ostergotland

Drug resistance is a clinically relevant problem in the treatment of acute myeloid leukaemia (AML). We have previously reported a relationship between single nucleotide polymorphisms (SNPs) of ABCB1, encoding the multi-drug transporter P-glycoprotein, and overall survival (OS) in normal karyotype (NK)-AML. Here we extended this material, enabling subgroup analysis based on FLT3 and NPM1 status, to further elucidate the influence of ABCB1 SNPs. De novo NK-AML patients (n = 201) were analysed for 1199Ggreater thanA, 1236Cgreater thanT, 2677Ggreater thanT/A and 3435Cgreater thanT, and correlations to outcome were investigated. FLT3 wild-type 1236C/C patients have significantly shorter OS compared to patients carrying the variant allele; medians 20 vs. 49 months, respectively, P = 0.017. There was also an inferior outcome in FLT3 wild-type 2677G/G patients compared to patients carrying the variant allele, median OS 20 vs. 35 months, respectively, P = 0.039. This was confirmed in Cox regression analysis. Our results indicate that ABCB1 1236Cgreater thanT and 2677Ggreater thanT may be used as prognostic markers to distinguish relatively high risk patients in the intermediate risk FLT3 wild-type group, which may contribute to future individualizing of treatment strategies. Funding Agencies|Swedish Cancer Society; County Council of Ostergotland; AFA Insurance; Stockholm Cancer Society; Karolinska Institutet; Swedish Research Council

De novo acute myeloid leukemia with normal karyotype (NK-AML) comprises a large group of patients with no common cytogenetic alterations and with a large variation in treatment response. Single-nucleotide polymorphisms (SNPs) in genes related to the metabolism of the nucleoside analogue AraC, the backbone in AML treatment, might affect drug sensitivity and treatment outcome. Therefore, SNPs may serve as prognostic biomarkers aiding clinicians in individualized treatment decisions, with the aim of improving patient outcomes. We analyzed polymorphisms in genes encoding cytidine deaminase (CDA 79A>C rs2072671 and −451C>T rs532545), 5′-nucleotidase (cN-II 7A>G rs10883841), and deoxycytidine kinase (DCK 3′UTR 948T>C rs4643786) in 205 de novo NK-AML patients. In FLT3-internal tandem duplication (ITD)-positive patients, the CDA 79C/C and −451T/T genotypes were associated with shorter overall survival compared to other genotypes (5 vs. 24 months, P

Background: De novo AML with normal karyotype (NK-AML) comprise a large group of patients without common cytogenetic alterations but with a large variation in treatment response. New tools such as the analysis of nucleophosmine 1 (NPM1), FMS-like tyrosine kinase 3 (FLT3) and CCAAT/enhancer binding protein (CEBPA) mutations has emerged as clinical markers of prognosis, but there is still a need for additional tools to explain the variation in outcome. Cytarabine (Ara-C), a cornerstone in AML chemotherapy, is activated intracellularly by deoxycytidine kinase (dCK), and deactivated by cytidine deaminase (CDA) and poor treatment outcome or development of chemotherapy resistance can be due to altered activity of these enzymes. Single nucleotide polymorphisms (SNPs) of CDA genes have previously been reported to result in lower CDA allelic expression and to affect the outcome of treatment. In addition, pharmacological inhibition of CDA has been shown to reduce the degree of DNA methylation. DNA methylation may be an important regulatory mechanism for the expression of genes related to drug effect, and we therefore aimed to investigate the relationship between CDA polymorphisms and outcome in NK-AML, DNA methylation, and in vitro drug cytotoxicity. Method: 207 Swedish NK-AML patients were genotyped for the CDA SNPs 79A>C (rs2072671) and -451C>T (rs532545) and the results were correlated to treatment response and overall survival (OS) in the material as a whole as well as stratified based on FLT3 and NPM1 status. Cells from a subset of the patients were also investigated for genotype association with in vitro drug cytotoxicity (n=56) and global DNA methylation (n=82). Results: CDA 79C/C or -451T/T genotype was associated to a shorter OS compared to other genotypes (0.44 vs. 1.86 years, p=0.004; and 0.45 vs. 1.79 years, p=0.051) for FLT3-ITD+ patients, and this was even more pronounced in the FLT3-ITD+/NPM1+ cases. There was significantly less DNA methylation in cells heterozygous for both polymorphisms (A/C+C/T) compared to homozygous A/A+C/C cells (p=0.018). There was also a lower degree of methylation in homozygous C/C+T/T cells, but due to the small number of samples in this group significance was not reached. An altered in vitro sensitivity towards topoisomerase inhibitory drugs, but not towards nucleoside analogues was seen for both CDA SNPs with homozygous C/C or T/T cells being more sensitive. Conclusions: We found a significant correlation between CDA genotype and OS in NK-AML, the degree of DNA methylation in AML cells, and in vitro drug sensitivity. The change in methylation related to CDA genotype may be associated with differences in drug activation and subsequent sensitivity, making these polymorphisms potential markers of use for future individual chemotherapy decisions such as dose adjustments. Citation Format: Ingrid Jakobsen Falk, Anna Fyrberg, Monica Hermanson, Martin Höglund, Hareth Nahi, Lars Palmqvist, Christer Paul, Esbjörn Paul, Richard Rosenquist, Dick Stockelberg, Yuan Wei, Henrik Green, Kourosh Lotfi. Correlation between cytidine deaminase single nucleotide polymorphisms and in vitro drug sensitivity, DNA methylation and outcome in normal karyotype acute myelogenous leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1170. doi:10.1158/1538-7445.AM2013-1170

The nucleoside analog cytosine arabinoside (Cytosar®, AraC) is one of the corner stones in the treatment of acute myeloid leukemia (AML). Activated AraC is metabolized to inactive compounds by several intracellular enzymes. The levels of some of these enzymes have been correlated to effect of AraC and clinical outcome in patients with AML. This study was designed to evaluate if single nucleotide polymorphisms (SNPs) in genes coding for enzymes involved in the inactivation of AraC affects the therapeutic response in AML-patients treated with AraC in combination with anthracyclines. Three SNPs were investigated in 96 de novo AML patients with normal karyotype and correlated to response and overall survival. Two SNPs were located in the cytidine deaminase gene (CDA, A79C, rs2072671 and C-451T, rs532545), deaminating and inactivating AraC to its corresponding uridine derivative, AraU. The third SNP (A7G, rs10883841) was in the NT5C2 gene coding for the cytosolic 5′-nucleotidase II (cN-II), responsible for dephosphorylation and hence inactivation of AraC monophosphate. All SNPs were in Hardy-Weinberg equilibrium and the allele frequencies were 34.4, 34.4 and 18.8% for CDA A79C and C-451T, and NT5C2 A7G, respectively. Patients that were wild type (A/A) for NT5C2 A7G were found to have a borderline significant higher response rate (51 complete responders of 60 patients with A/A vs 20 complete responders of 30 with A/G or G/G, p=0.06) and longer survival (median survival 1.66 years compared to 0.87 years, log-rank p=0.08) compared to the rest of the patients. The two CDA SNPs (A79C, C-451T) were found to be in linkage disequilibrium. Although statistically not significant, patients that were found to be homozygous for the alternate genetic variant of the CDA SNP had a shorter survival compared to patients carrying the wild type or heterozygous genetic variants. In conclusion, our findings suggest that certain NT5C2 (cN-II) genotypes might affect the survival after chemotherapy and may provide useful information for treatment strategies, classification and individualized chemotherapy, and eventually used as prognostic marker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2757.

Abstract 2648 Poster Board II-624 Background: Multidrug resistance and expression of the ATP-dependent drug transporting protein ABCB1 is a clinically relevant problem in the treatment of acute myeloid leukaemia. Several single nucleotide polymorphisms (SNPs) in the ABCB1 have been associated with altered P-glycoprotein expression and phenotype. These SNPs might influence the clinical outcome in AML and predict individual differences in response to therapy with ABCB1 substrates. Aims: To investigate the impact of the ABCB1 SNPs in exon 11, 12, 21 and 26 on treatment response, survival and in vitro drug sensitivity in AML patients. Methods: PCR and Pyrosequencing were used to determine the genotype of the SNPs G1199T/A, C1236T, A1308G, G2677T/A and C3435T in 100 de novo AML patients with normal karyotype treated at Linköping University Hospital or Karolinska University Hospital. Almost all patients were treated with one anthracycline and Ara-C during the induction regime. The affect of the genetic variants in ABCB1 on survival were analysed by Kaplan-Meier Log-rank tests and multivariant analysis by Cox regression. Patients receiving transplantation were censored at that point in the analysis. A Nordic reference material of 400 healthy volunteers of equal age and sex distribution was also included. NPM1 and FLT3-ITD mutations were determined by PCR. Leukemic cells were isolated and drug sensitivity was measured after 4 days culturing by a bioluminescence ATP-assay. Results: The survival of the AML patients was significantly correlated to the ABCB1 genotypes C1236T (P=0.02) and G2677T (P=0.02), with a borderline significance for G1199A (p=0.06). For the C1236T SNP the mean survival was 0.7, 1.3 and 1.8 years for the wild type, heterozygous and homozygous variants, respectively. The mean survival for patients with G/G, G/T and T/T genotype of SNP G2677T/A was 0.7, 1.2 and 1.7 years, respectively. Only the wild type of A1308T was found in the material and C3435T did not correlate to survival. Multivariate analysis showed that 1236T/T and 2677T/T were independent factors for survival (hazard ratio 0.24 and 0.22). Comparison of allele frequencies between AML patients and healthy volunteers showed no significant difference. In vitro testing showed that leukemic cells from patients carrying 1236T/T or 2677T/T were significantly more susceptible for mitoxantrone and borderline susceptible to daunorubicine and etoposide, substrates for Pgp but not to Ara-C. Conclusions: Our findings suggest that ABCB1 SNPs do not affect the development of the disease but the survival after chemotherapy possibly by impact on drug sensitivity. The correlation between ABCB1 genotype and the overall survival of AML patients might provide useful information for treatment strategies and individualized chemotherapy. Disclosures: Paul: Aprea AB: Consultancy, Research Funding.

Background: The p53 protein is strongly regulated by the E3 ligase HDM-2, which specifically binds to p53 and causes proteosomal degradation. The p14ARF protein activates p53 by binding to and inhibiting HDM-2. Design and methods: To further study the prognostic impact of p14ARF in AML, leukemic cells from 57 adult patients with normal karyotype de novo AML were analysed for p14ARF mRNA expression level using real-time PCR. We also measured the cytotoxicity against conventional cytostatics and PRIMA-1, a novel small molecule shown to activate mutated p53 (Nature Medicine2002; 8: 282–288) using an ATP-assay. Intracellular p53 protein was measured by FACS after incubation with PRIMA-1 in combination with the HDM-2 blocking agent RITA (Nature Medicine2004; 10: 1321–1328) Results: Patients whose cells expressed more p14ARF mRNA than the 75th percentile (0.26) showed significantly better survival compared with those with lower levels, 61% vs. 30% 3-year survival (p=0.033). The difference remained significant also when NPM1/FLT3 status was considered. The mean effects of all tested conventional antileukemic drugs were greater in leukemic cell samples expressing p14ARF mRNA ≥0.26, but the differences did not reach statistical significance. In contrast, PRIMA-1 had a significantly greater effect on leukemic cell samples with low levels of p14ARF mRNA and PRIMA-1 and RITA significantly increased intracellular p53 levels. Conclusions: Low level of p14ARF mRNA in leukemic cells from patients with normal karyotype AML is a strong marker for poor prognosis and decreased sensitivity to conventional cytostatics. Treatment with drugs targeting p53 can be a future possibility to improve the outcome for these patients.

The p53 protein exerts a significant role in growth control of cells and impaired function of p53 by mutation or otherwise is regarded to be important for development of many human cancers. The p53 protein is strongly regulated by the E3-ligase HDM-2 that specifically binds to p53 and causes proteosomal degradation. P14ARF encoded from the INK 4a/ARF gene locus of chromosome 9p, activates the p53 pathway by binding to and inhibiting HDM-2. The objective was to study if the level of mRNA for p14ARF in leukemic cells from patients with AML was related to in vitro drug resistance and clinical outcome. Leukemic cells (>90% pure) isolated from 46 adult patients with normal karyotype de novo AML were incubated with antileukemic drugs (daunorubicin, mitoxantrone, etoposide and Ara-C) cultured for 4 days. The effect was determined by bioluminiscence measuring of intracellular ATP compared to an untreated control. We also tested the activity against PRIMA (p53-dependent reactivation and induction of massive apoptosis), a novel, small molecule shown to activate down regulated p53. mRNA for p14ARF was determined by real time PCR. Results: Patients whose leukemic cells expressed high level of p14ARF mRNA in the leukemic cells (≥0.2 compared to the housekeeping gene) had a significantly better survival compared to those with low level (