ALBERT

Description: Analytical Chemistry DOI: 10.1021/acs.analchem.5b02378

Description: Oncolytic adenoviruses are a therapeutic alternative to treat cancer based on their ability to replicate selectively in tumor cells. However, their use is limited mainly by the neutralizing antibody (Nab) immune response that prevents repeated dosing. An alternative to facilitate the DNA access to the tumor even in the presence of anti-viral Nabs could be gold nanoparticles able to transfer DNA molecules. However, the ability of these nanoparticles to carry large DNA molecules, such as an oncolytic adenovirus genome, has not been studied. In this work, gold nanoparticles were functionalized with different amounts of polyethylenimine to transfer in a safe and efficient manner a large oncolytic virus genome. Their transfer efficacy and final effect of the oncolytic virus in cancer cells are studied. For each synthesized nanoparticle, (a) DNA loading capacity, (b) complex size, (c) DNA protection ability, (d) transfection efficacy and (e) cytotoxic effect were studied. We observed that small gold nanoparticles (70–80 nm in diameter) protected DNA against nucleases and were able to transfect the ICOVIR-15 oncolytic virus genome encoded in pLR1 plasmid. In the present work, efficient transgene RNA expression, luciferase activity and viral cytopathic effect on cancer cells are reported. These results suggest gold nanoparticles to be an efficient and safe vector for oncolytic adenovirus genome transfer.

Description: Background: Single nucleotide polymorphisms (SNPs) within the genes involving drug detoxification enzymes of anthracyclines could lead to interindividual differences in treatment outcome. Several studies suggested, in different kinds of cancer, that SNPs of genes coding anthracyclines metabolism may influence their effectiveness or toxicity, being well-known their association with cardiotoxicity. The impact of these polymorphisms in adult acute myeloid leukemia (AML) patients treated with the combination of cytarabine and anthracyclines for induction remains undetermined. Methods: SNPs of anthracycline metabolism genes previously associated with clinical significance in other malignances (CBR3:rs1056892, rs8133052, NQO1 rs1800566, NQO2 rs1143684, NOS3:rs1799983, rs2070744, MnSOD rs4880) were evaluated in 225 adult patients at initial diagnosis from AML using a Sequenom (iPLEX) mass spectrometry-based multiplex genotyping assay (Sequenom, San Diego, CA). All patients received induction chemotherapy consisting of idarubicin plus cytarabine (PETHEMA-LMA 99, 2007 and 2010 trials). Efficacy of first induction cycle was evaluated comparing complete remission (CR) vs. partial remission or resistance. Patients dying during induction were considered as no evaluable for efficacy. Based on WHO grading scale, toxicities were grouped as binary variables (grade 0-1 vs. grade 2-4). The grade of toxicity assigned to an organ group was the maximum grade of all the specific toxicities within that group. Hematologic toxicity was measured with the time to neutropenia and thrombocytopenia recovery since first day of chemotherapy. Genotypes were studied with co-dominant model. Association between variables was assessed using linear and logistic regression adjusting for age, gender, ECOG, leukocyte and platelet count at diagnosis (R® version 3.1.2). Results: The median age of patients was 51.1 years (16-78 years). There were no statistically significant differences in CR. Nevertheless, several associations were obtained between NQO1, NQO2, NOS3 and MnSOD polymorphisms and toxicities (significant toxicities were summarized in table 1 and 2). Table 1. Significant association between SNPs in gene metabolizers and different toxicities Toxicity Gene/SNP Genotypes Grade 0-1 n (%) Grade 2-4 n (%) OR (95%IC) P Cardiotoxicity NQO2 rs1143684 TT TC 119 (86.2) 74 (94.9) 19 (13.8) 4 (5.1) 0.26 (0.07-0.77) 0.025 Neurotoxicity NOS3 rs1799983 GG GT 71 (84.5) 101 (94.4) 13 (15.5) 6 (5.6) 0.28 (0.09-0.80) 0.022 Skin toxicity NOS3 rs1799983 GG GT TT 45 (53.6) 78 (72.9) 26 (76.5) 39 (46.4) 29 (27.1) 8 (23.5) 0.44 (0.24-0.82) 0.36 (0.14-0.88) 0.010 0.030 Skin toxicity NQO1 rs1800566 CC CT 78 (60.9) 64 (74.4) 50 (39.1) 29 (25.6) 0.53 (0.28-0.97) 0.042 Skin toxicity NQO2 rs1143684 TT CC 5.49 (1.19-38.9) 0.044 Gastrointestinal toxicity NQO2 rs1143684 TT CC 91 (65.9) 2 (25.0) 47 (34.1) 6 (75.0) 5.5 (1.19-38.99) 0.043 Mucositis NQO1 rs1800566 CC TT 119 (93.0) 8 (72.7) 9 (7.0) 3 (27.3) 6.1 (1.03-33.1) 0.035 Mucositis NQO2 rs1143684 TT CC 128 (92.8) 5 (62.5) 10 (7.2) 3 (37.5) 8.8 (1.53-45.60) 0.010 Nephrotoxicity MnSOD rs4880 TT CC 47 (81.0) 55 (94.8) 11 (19.0) 3 (5.2) 0.23 (0.05-0.86) 0.042 Nephrotoxicity NQO1 rs1800566 CC TT 114 (89.1) 8 (72.7) 14 (10.9) 3 (27.3) 6.66 (1.07-38.35) 0.033 Hepatotoxicity grades 3-4 NOS3 rs2070744 CC CT 19 (24.8) 100 (67.1) 20 (51.3) 49 (32.9) 0.44 (0.20-0.94) 0.035 Table 2. Significant association between SNPs in gene metabolizers and hematologic toxicities Hematologic toxicity Gene/SNP Genotypes Mean days Logarithm of the difference (95%IC) P Time to neutropenia recovery NOS3 rs2070744 CC TT 32.7 26.7 -0.17 (-0.35 to -0.01) 0.048 Time to thrombocytopenia recovery NOS3 rs1799983 GG GT TT 35.6 28.8 30.3 -0.17 (-0.17 to -0.06) -0.15 (-0.28 to -0.01) 0.002 0.034 Conclusions: This study reveals that, as in other cancers, there is a prognostic impact of anthracycline metabolism gene polymorphisms in adult AML patients. Further studies with larger population are needed to validate these associations, which could be useful biomarkers in clinical practice. Disclosures No relevant conflicts of interest to declare.

Description: Background: Several studies have suggested that genetic variability related with single nucleotide polymorphisms (SNPs) within the genes involving Ara C pathway could influence in treatment outcome of AML patients treated with this cytotoxic drug. However, the association of these polymorphisms with toxicity of combinations of Ara C and anthracyclines in AML patients remains undetermined. Methods: The SNPs of Ara C metabolism genes previously described in literature (DCK: rs2306744, rs11544786, rs4694362; CDA: rs2072671, rs3215400, rs532545, rs602950; NT5C2: rs11598702; RRM1: rs9937; NME1: rs2302254) were evaluated in 109 adult patients of a single center at initial diagnosis of AML, using a Sequenom (iPLEX) mass spectrometry–based multiplex genotyping assay (Sequenom, San Diego, CA). All patients were treated with intensive induction chemotherapy consisting of idarubicin plus Ara C (PETHEMA-AML 99, 2007 and 2010 trials). Genotypes were grouped as dichotomous variables (dominant and recessive model). Efficacy of first induction cycle was evaluated comparing complete remission (CR) vs. partial remission or resistance. Patients dying during induction were considered as no evaluable for efficacy. Based on WHO grading scale, toxicities were grouped as binary variables (grade 0-1 vs. grade 2-4). The grade of toxicity assigned to an organ group was the maximum grade of all the specific toxicities within that group. Overall toxicity was grouped with grade 3-4 assessment. Hematologic toxicity was measured with the time to neutropenia and thrombocytopenia recovery since first day of chemotherapy. Categorical and continuous variables were assessed using χ2 test with Yates correction if needed and Mann–Whitney U test, respectively. Multivariate analyses were performed using the Cox method. Results: The median age of patients was 53 years (17-78 years). Among the baseline characteristics analyzed (age, gender, leukocyte count, hemoglobin level, platelet count and percentage of peripheral or BM blasts) there was statistically significant difference in the genotype distributions of CDA polymorphisms regarding age (patients with variant alleles of rs2072671, rs532545, rs602950, and wild type of rs3215400 were older, P= 0.006, 0.025, 0.025 and 0.012, respectively) and gender (men had higher proportion of variant alleles than women for rs2072671, rs3215400 and rs602950, P= 0.04, 0.046 and 0.039). The variant homozygous of DCK SNP rs11544786, enzyme that catalyzes the limiting first phosphorylation in activation of Ara C, showed a trend towards a prolonged neutropenia duration (67.5 vs. 34.2 days, P=0.058). Concerning the CDA polymorphisms, the main inactivating enzyme in the Ara C, wild genotype of rs2072671 was associated with higher frequency of grade 2-4 liver toxicity (83.3% vs. 51.0%, P= 0.034) and time to thrombocytopenia recovery (36.1 vs. 20.6 days, P= 0.026) compared to variant alleles, which is consistent with the CDA activity reduction attributed to this SNP. Multivariable regression models including age and gender as covariates were performed in CDA SNPs analysis with similar results for the association between rs2072671 and liver toxicity (OR=0.209, 95%IC=0.043-1.002, P=0.026), but not for time to thrombocytopenia recovery. We did not found any association between any of the investigated cytarabine pathway gene polymorphisms and the CR rates. Conclusions: This study reveals associations between polymorphisms of metabolic genes of Ara C and the toxicity during induction therapy in adult AML patients. Further studies with larger population are needed to validate these associations, especially in SNPs with low variant allele frequency, such as in DCK. A better knowledge of the patient’s risk of toxicity related to genetic variability could improve the treatment outcomes in AML. Disclosures No relevant conflicts of interest to declare.

Description: Single nucleotide polymorphisms (SNPs) in Pharmacogenetics can play an important role in the outcomes of the chemotherapy treatment in Neuroblastoma, helping doctors maximize efficacy and minimize toxicity. Employing AgenaBioscience MassArray, 96 SNPs were genotyped in 95 patients looking for associations of SNP with response to induction therapy (RIT) and grade 3–4 toxicities, in High Risk patients. Associations of SNPs with overall (OS) and event-free (EFS) survival in the whole cohort were also explored. Cox and logistic regression models with Elastic net penalty were employed. Association with grade 3–4 gastrointestinal and infectious toxicities was found for 8 different SNPs. Better RIT was correlated with rs726501 AG, rs3740066 GG, rs2010963 GG and rs1143684 TT (OR = 2.87, 1.79, 1.23, 1.14, respectively). EFS was affected by rs2032582, rs4880, rs3814058, rs45511401, rs1544410 and rs6539870. OS was influenced by rs 1801133, rs7186128 and rs1544410. Remarkably, rs1801133 in MTHFR (p = 0.02) and rs1544410 in VDR (p = 0.006) also added an important predictive value for OS to the MYCN status, with a more accurate substratification of the patients. Although validation studies in independent cohorts will be required, the data obtained supports the utility of Pharmacogenetics for predicting Neuroblastoma treatment outcomes.

Description: Background: Cytarabine (Ara C) is considered the most effective chemotherapeutic agent in acute myeloid leukemia (AML) treatment. Several studies suggest that single nucleotide polymorphisms (SNPs) within the genes involving metabolic pathway of Ara C could influence in treatment outcomes, although their clinical relevance remains undetermined. Methods: The SNPs of cytarabine pathway (DCK: rs2306744, rs11544786, rs4694362; CDA: rs2072671, rs3215400, rs532545, rs602950; NT5C2: rs11598702; RRM1: rs9937; NME1: rs2302254) were evaluated in 225 adult patients at initial diagnosis from AML using a mass spectrometry-based multiplex genotyping assay (Sequenom®). All patients received induction chemotherapy consisting of idarubicin plus cytarabine (PETHEMA 99, 2007 and 2010 trials). Efficacy of first induction cycle was evaluated comparing complete remission (CR) vs. partial remission (PR) or resistance (patients dying during induction excluded); and overall survival (OS). Induction death was defined as patients dying during induction against CR, excluding these patients with PR or resistance. Based on WHO grading scale, toxicities were grouped as binary variables (grades 0-1 vs. 2-4; or 0 vs. 1-4), assigning the maximum grade of all the specific toxicities within that group (evaluated in all patients). Genotypes were studied with co-dominant model. Association between variables was assessed using linear and logistic regression adjusting for age, gender, cytogenetic risk, ECOG, leukocyte and platelet count, hemoglobin, creatinine, bilirubin, albumin and LDH level at diagnosis (R® version 3.1.2). Kaplan-Meier method and Cox proportional were employed to OS estimates with the same covariates. Results: The median age of patients was 51.1 years (16-78 years). The variant allele of DCK SNP rs2306744, enzyme that catalyzes the limiting first phosphorylation in activation of Ara C, showed higher CR (OR:6.3; 95%CI 1.3-31.1; P=0.024), as well as higher mucositis (OR:3.3; 95%CI 1.1-10.0; P=0.038). CDA is the main inactivating enzyme of Ara C. The variant allele of rs602950 was related to higher CR (OR:3.0; 95%CI 1.02-8.8; P=0.045) and OS at 5 years (HR:0.4; 95%CI 0.2-0.9; P=0.012; Figure 1) and the variant homozygous of rs2072671 to higher OS at 5 years (HR:0.3; 95%CI 0.1-0.96; P=0.018; Figure 2), whereas the wild-type allele of rs532545 was associated to higher OS at 3 years (HR:1.5; 95%CI 1.01-2.4; P=0.039; Figure 3). In addition, variant alleles of rs532545 and rs602950 were related to skin toxicity (OR:2.2; 95%IC 1.1-4.3; P=0.033; OR:2.1; 95%IC 1.01-4.5; P=0.047, respectively). Variant allele of RRM1 (rs9937), enzyme directly associated with Ara C sensitivity, was associated to induction death (OR:0.2; 95%IC 0.03-0.9, P= 0.034). Variant allele of NT5C2 (rs11598702), responsible of nucleotide pools balance, showed higher hepatotoxicity (OR: 4.1; 95%IC 1.1-14.5; P=0.032). Conclusions: This study reveals the influence in Ara C efficacy of DCK and CDA polymorphisms in AML adult patients, previously suggested in other studies. In addition, novel associations between SNPs in metabolic Ara C genes and toxicities were detected. Further studies with larger population are needed to validate these associations. Figure 1 Figure 1. Figure 3 Figure 3. Figure 2 Figure 2. Disclosures Boluda: Instituto de Investigación Sanitaria La Fe: Employment.

Description: Background: The genetic variability related with single nucleotide polymorphisms (SNPs) within the genes involving drug transport or metabolism, DNA repair and oxidative stress, could lead to interindividual differences in treatment outcome. Several studies suggested, in different kinds of cancer, that SNPs of genes coding for drug detoxification enzymes of anthracyclines may influence their effectiveness or toxicity. However the association of these polymorphisms in adult acute myeloid leukemia (AML) patients treated with the combination of cytarabine and anthracyclines for induction remains undetermined. Methods: The SNPs of anthracycline metabolism genes previously associated with clinical significance in other malignances (CBR3:rs1056892, rs8133052, NQO1 rs1800566, NQO2 rs1143684, NOS3:rs1799983, rs2070744, MnSOD rs4880) were evaluated in 109 adult patients at initial diagnosis from AML using a Sequenom (iPLEX) mass spectrometry–based multiplex genotyping assay (Sequenom, San Diego, CA). All patients received induction chemotherapy consisting of idarubicin plus cytarabine (PETHEMA-LMA 99, 2007 and 2010 trials). Genotypes were grouped as dichotomous variables (dominant and recessive model). Efficacy of first induction cycle was evaluated comparing complete remission (CR) vs. partial remission or resistance. Patients dying during induction were considered as no evaluable for efficacy. Based on WHO grading scale, toxicities were grouped as binary variables (grade 0-1 vs. grade 2-4). The grade of toxicity assigned to an organ group was the maximum grade of all the specific toxicities within that group. Overall toxicity was grouped with grade 3-4 assessment. Hematologic toxicity was measured with the time to neutropenia and thrombocytopenia recovery since first day of chemotherapy. Categorical and continuous variables were assessed using χ2 test with Yates correction if needed and Mann–Whitney U test, respectively. Results: The median age of patients was 53 years (17-78 years). There was no statistically significant difference in baseline characteristics (age, gender, leukocyte count, hemoglobin level, platelet count and percentage of peripheral or BM blasts) between the patients with the genotype distributions. There were lower CR rates among patients harboring CBR3 SNPs homozygous variant (rs1056892, AA: 37.5% vs. GA/AA: 76.5%, P=0.050; rs8133052, GG: 52.6% vs. GA/AA: 78.4%, P=0.049), both genotypes previously associated with higher enzymatic activity of CBR3 and higher anthracycline clearance. The variant allele (TC/CC) of NQO2 polymorphism was also associated with lower CR rates compared to TT genotype (45.2% vs. 69.2%, P=0.034). Skin toxicity was higher in wild genotypes of NQO1 (36.5% vs. 15.2%, P=0.025) and NOS3 rs1799983 (45.7% vs. 18.9%, P=0.007). Gastrointestinal toxicity was related with variant allele of NQO1 (69.2% vs. 45.2%, P=0.034). Regarding hematologic toxicity, time to thrombocytopenia recovery was delayed with variant alleles of NQO1 (29.1 vs. 43.2 days, P=0.004) and MnSOD (31.5 vs. 42.3 days, P=0.043). Conclusions: This study shows that, as in other cancers, there is a prognostic impact of anthracycline metabolism gene polymorphisms in adult AML patients. We obtained new associations of these anthracyclines metabolism pathways SNPs in relation to effectiveness and toxicity, which could be useful biomarkers in clinical practice. Disclosures No relevant conflicts of interest to declare.

Description: Background: The intake of anthracyclines in blast cells could be affected by several transporters, including influx transporters (SLC22A16 and SLCO1B1) and efflux pumps of ABC family. Previous studies suggested that single nucleotide polymorphisms (SNPs) of genes coding for anthracyclines transporters may influence their effectiveness or toxicity, although their impact in acute myeloid leukemia (AML) induction therapy remains undetermined Methods: SNPs of anthracycline transporter genes (ABCB1: rs1128503, rs1045642, rs2032582 and haplotype; ABCC1: rs4148350; ABCC2: rs8187710; ABCG2: rs2231142, rs2231137; SLCO1B1: rs4149056; SLC22A16: rs12210538, rs714368) were evaluated in 225 adult patients at initial diagnosis from AML using a Sequenom (iPLEX) mass spectrometry-based multiplex genotyping assay (Sequenom, San Diego, CA). All patients received induction chemotherapy consisting of idarubicin plus cytarabine (PETHEMA-LMA 99, 2007 and 2010 trials). Efficacy of first induction cycle was evaluated comparing complete remission (CR) vs. partial remission or resistance. Patients dying during induction were considered as no evaluable for efficacy. Based on WHO grading scale, toxicities were grouped as binary variables (grade 0-1 vs. grade 2-4). The grade of toxicity assigned to an organ group was the maximum grade of all the specific toxicities within that group. Renal and hepatic toxicities were also evaluated with analytic markers (renal function with urea and creatinine; hepatic function with bilirubin, AST, ALT, FA and GGT). Hematologic toxicity was measured with the time to neutropenia and thrombocytopenia recovery since first day of chemotherapy. Genotypes were studied with co-dominant model. Association between variables was assessed using linear and logistic regression adjusting for age, gender, ECOG, leukocyte and platelet count at diagnosis (R® version 3.1.2). Results: The median age of patients was 51.1 years (16-78 years). There were no statistically significant differences in CR or hematologic toxicities. Nevertheless, several associations were obtained between transporter genes polymorphisms and toxicities (significant toxicities were summarized in table 1 and 2). Variant allele of ABCB1 SNPs was previously related with lower activity of ABCB1 pump in kidney cells and lower anthracycline clearance. Accordingly, we obtained nephrotoxicity associated with these SNPs. Association between cardiac toxicity and ABCG2 polymorphisms was found in agreement with previously published works. The ocular toxicity associated with SLCO1B1 was also correlated with low platelet count at diagnosis in multivariable analysis. Table 1. Significant association between SNPs in gene transporters and different toxicities Toxicity Gene/SNP Genotypes Grade 0-1 n (%) Grade 2-4 n (%) OR (95%IC) P Cardiotoxicity ABCG2 rs2231142 CC CA 171 (83.0) 9 (47.4) 35 (17.0) 10 (52.6) 5.64 (1.86-17.80) 0.002 Lung toxicity ABCG2 rs2231142 CC CA 174 (84.5) 12 (63.2) 32 (15.5) 7 (36.8) 3.37 (1.09-10.40) 0.031 Skin toxicity SLCO1B1 rs4149056 TT TC 112 (71.8) 21 (48.8) 44 (28.2) 22 (51.2) 2.43 (1.17-5.06) 0.015 Nephrotoxicity ABCB1 haplotype Other genotypes TT/TT/TT 175 (89.3) 21 (72.4) 21 (10.7) 8 (27.6) 3.74 (1.24-10.85) 0.002 Ocular toxicity ABCC1 rs4148350 GG GT/TT 205 (99.0) 16 (88.9) 2 (1.0) 2 (11.1) 13.7 (2.08-90.47)