Myelodysplastic syndromes with 20q deletion: incidence, prognostic value and impact on response to azacitidine of ASXL1 chromosomal deletion and genetic mutations.

In myelodysplastic syndromes (MDS), the 20q deletion [del(20q)] may cause deletion of the ASXL1 gene. We studied 153 patients with MDS and del(20q) to assess the incidence, prognostic value and impact on response to azacitidine (AZA) of ASXL1 chromosomal alterations and genetic mutations. Additionally, in vitro assay of the response to AZA in HAP1 (HAP1WT ) and HAP1 ASXL1 knockout (HAP1KN ) cells was performed. ASXL1 chromosomal alterations were detected in 44 patients (28·5%): 34 patients (22%) with a gene deletion (ASXL1DEL ) and 10 patients (6·5%) with additional gene copies. ASXL1DEL was associated with a lower platelet count. The most frequently mutated genes were U2AF1 (16%), ASXL1 (14%), SF3B1 (11%), TP53 (7%) and SRSF2 (6%). ASXL1 alteration due to chromosomal deletion or genetic mutation (ASXL1DEL /ASXL1MUT ) was linked by multivariable analysis with shorter overall survival [hazard ratio, (HR) 1·84; 95% confidence interval, (CI): 1·11-3·04; P = 0·018] and a higher rate for acute myeloid leukaemia progression (HR 2·47; 95% CI: 1·07-5·70, P = 0·034). ASXL1DEL /ASXL1MUT patients were correlated by univariable analysis with a worse response to AZA. HAP1KN cells showed more resistance to AZA compared to HAP1WT cells. In conclusion, ASXL1 alteration exerts a negative impact on MDS with del(20q) and could become useful for prognostic risk stratification and treatment decisions.

Therapy-related acute myeloid leukemia developing 14 years after allogeneic hematopoietic stem cell transplantation, from a persistent R882H-DNMT3A mutated clone of patient origin.

BACKGROUND: Therapy-related acute myeloid leukemia (t-AML) develops in patients with prior exposure to cytotoxic therapies. Selection of a pre-existing TP53 mutated clone prone to acquire additional mutational events has been suggested as the main pathogenic mechanism of t-AML. Here, we report a unique case of t-AML which developed from a pre-existing DNMT3A mutated clone that persisted in the patient for more than 10 years despite treatment with intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (alloHSCT). CASE PRESENTATION: A 42-year-old male was diagnosed with AML harboring a normal karyotype and mutations in the NPM1 (c.863_864ins, p.W288 fs*12), DNMT3A (c.2645G > A, p.R882H), and IDH1 (c.395G > A, p.R132H) genes. He achieved complete remission with intensive chemotherapy and was subsequently submitted to alloHSCT. Eleven years later, he was given chemotherapy and radiotherapy to treat a lung carcinoma. Three years later, t-AML was diagnosed; the disease had arisen from a pre-existing DNMT3A mutated patient-origin clone that had subsequently acquired a TP53 mutation and a complex karyotype. Although a second transplantation was intended, the disease was refractory to induction chemotherapy, and the patient eventually died from disease complications. We retrospectively demonstrated the persistence and post-transplantation latency of the R882H-DNMT3A mutation using a real-time PCR allele-specific analysis at different time-points during the observation period. DISCUSSION AND CONCLUSION: The present case highlights the potential clinical implications of a R882H-DNMT3A mutated clone that persisted after conventional AML treatment, including alloHSCT. It also reinforces the notion of the importance of cell non-intrinsic factors, such as the hematopoietic-stress induced by chemotherapy and radiotherapy, as drivers of clonal expansion.

Coexistence of EGFR, KRAS, BRAF, and PIK3CA Mutations and ALK Rearrangement in a Comprehensive Cohort of 326 Consecutive Spanish Nonsquamous NSCLC Patients.

INTRODUCTION: Molecular screening is crucial for the care of nonsquamous non-small-cell lung cancer (NSCLC) patients. The coexistence of mutations could have important consequences regarding treatment. We described the mutational patterns and coexistence among patients and their outcomes after targeted treatment. MATERIALS AND METHODS: Data from consecutive patients with newly diagnosed nonsquamous NSCLC were prospectively collected. Next-generation sequencing analysis of mutational hotspots in the EGFR, KRAS, PIK3CA, and BRAF genes and analysis of anaplastic lymphoma kinase (ALK) rearrangement were performed. RESULTS: A total of 326 patients with nonsquamous NSCLC were identified. Of the 326 patients, 240 (73.6%) had EGFR, 141 (43.3%) KRAS, 137 (42.0%) BRAF, 130 (39.9%) PIK3CA mutation and 148 (45.4%) ALK rearrangement determined. Of the 240 with EGFR determination, 24.1% harbored EGFR mutations. Of these, 16.3% were activating mutations (43.6%, exon 19 deletion; 46.1%, exon 21; and 10.3%, exon 18) and 7.9% were nonsensitizing EGFR mutations. Furthermore, 39.0% had KRAS mutations, 2.9% BRAF mutations, 10.0% PIK3CA mutations, and 8.8% ALK rearrangements. Of the 154 stage IV patients with ≥ 1 mutations, analysis showed 19 coexisting cases (12.3%). Of 8 patients receiving targeted treatment, 6 had no response. Both responders to targeted treatment had coexistent PIK3CA mutations. CONCLUSION: Driver mutations can coexist in nonsquamous NSCLC. In our cohort, 12.3% of cases with stage IV disease had multiple mutations. Targeted treatment might not be as effective in patients with coexisting mutations; however, coexistence with PIK3CA might not preclude a response.

Dietary polyunsaturated fatty acids may increase plasma LDL-cholesterol and plasma cholesterol concentrations in carriers of an ABCG1 gene single nucleotide polymorphism: study in two Spanish populations.

BACKGROUND: ABCG1 mediates cellular cholesterol transport, but there is very little known about the influence of ABCG1 polymorphisms on human plasma lipoprotein cholesterol concentrations or on the interactions of these polymorphisms with diet. OBJECTIVE: Our objective was to investigate whether interactions between PUFA intake and ABCG1 polymorphisms modulate associations with plasma total cholesterol (TC), LDL- and HDL-cholesterol in two Spanish populations. METHODS: We grounded our investigation on two general population-based studies: the Hortega study (population A) and the Pizarra study (population B). Participants included 1178 individuals (50.0% women, age range 21-85 years) and 763 individuals (66% women, age range 23-73 years) from populations A and B, respectively, without lipid lowering drugs. Subjects were genotyped for ABCG1 variants. Biochemical measurements were taken by standard procedures. Dietary intakes were estimated with a validated questionnaire. RESULTS: In population A, the A allele homozygotes of SNP rs4148102 had higher TC and LDLc concentrations in subjects on a high PUFA diet than did the carriers of the G allele (242.1 ± 38.9 vs. 198.0 ± 36.0mg/dL, p = 0.003, and 149.8 ± 37.9 vs. 111.4 ± 32.1mg/dL, p = 0.005, respectively), and significant gene-diet interactions were observed (p=0.020 and p = 0.013, respectively). In population B, similar differences in TC and LDLc concentrations were also found in association with this SNP under a high PUFA diet (253.2±24.9 vs. 197.7 ± 39.9 mg/dL, p = 0.009, and 171.8 ± 20.5 vs. 120.4 ± 34.2mg/dL, p = 0.004, respectively), but the gene-diet interactions observed were not significant (p = 0.379 and p = 0.422, respectively). In the pooled populations, differences in the TC and LDLc concentrations increased (246.8 ± 32.9 vs. 198.0 ± 37.5, p = 6 × 10(-5), and 159.0±32.6 vs. 114.3 ± 33.1, p = 3 × 10(-5), respectively), and significant gene-diet interactions were maintained (p = 0.006 and p = 0.003, respectively). CONCLUSION: In two Spanish populations, the ABCG1 polymorphism rs4148102 was associated with variations in plasma lipoprotein cholesterol concentrations in subjects with high PUFA intakes. Carriers of the AA genotype consuming high PUFA diet showed higher plasma LDLc concentrations.

Effect of physical fitness and endurance exercise on indirect biomarkers of growth hormone and insulin misuse: Immunoassay-based measurement in urine samples.

Indirect biomarkers of recombinant human growth hormone (rhGH), insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), insulin-like growth factor binding proteins (IGFBP-2 and IGFBP-3) and insulin (C-peptide) were measured together with urinary parameters of renal damage (beta(2)-microglobulin and proteinuria) by immunoassays, in house validated for the purpose, in 61 subjects (36 elite athletes, 18 recreational athletes and 7 sedentary individuals) with different levels of physical fitness and endurance exercise. Validation parameters were good for the evaluated assays, excluding a high inter-assay imprecision and inaccuracy of 24 and 26% obtained for GH assay. The range of concentrations found in urine samples under investigation was generally covered by the calibration curves of the studied immunoassays. However, for the samples below or above the calibration curve, opportune dilution or concentration were performed. Particularly, C-peptide samples had to be diluted 1:5 and beta(2)-microglobulin ones assayed using a triple sample volume, to fall within the calibration range. Urinary C-peptide was the only biomarker statistically higher in samples of elite athletes when compared to recreational athletes and sedentary individuals. Among elite athletes, tae-kwon-do athletes showed the highest IGF-II basal values while weightlifting athletes showed the lower IGF-I and IGFBP-3 basal values. The trend observed in weightlifters' basal samples was confirmed in their training samples: IGF-I, IGF-II, IGFBP-3 and beta(2)-microglobulin were lower in with respect to those from synchronised swimming. Over the training season, within athlete variability was observed for IGFBP-3 for weightlifting athletes. In the studied subjects, no direct associations were found between biomarkers of GH or insulin misuse and urinary parameters of renal damage, eventually due to high-workload endurance training. The variations observed in different biomarkers should be taken in consideration in the hypothesis of setting reference concentration ranges for doping detection.

Association of selected ABC gene family single nucleotide polymorphisms with postprandial lipoproteins: results from the population-based Hortega study.

The aim of the study was to determine the influence of twenty single nucleotide polymorphisms (SNPs) of the ABCA1, ABCG1, ABCG5 and ABCG8 genes on the plasmatic concentrations of total cholesterol (TC), HDL and LDL cholesterol (HDLc, LDLc) in the postprandial state with a representative Spanish Caucasian population (1473 individuals, 50.0% women, ages ranging 21-85 years). In men, subjects with the AA genotype of the ABCA1 rs2230806 (R219K) polymorphism were associated with increased plasma LDLc levels, while the ABCA1 haplotype, which included the rs2230806 A allele, was associated with higher TC and LDLc plasma concentrations. In women, significant relationships were found between rs1893590 polymorphisms (ABCG1 gene) and HDLc plasma concentrations (subjects with the AA genotype had lower HDLc levels). For the ABCG8 gene, the rs4148211 polymorphism was associated with higher plasma TC and LDLc concentrations in the total population. Moreover, an ABCG5-G8 haplotype, which included the rs6544718 T allele, was associated with higher HDLc plasma concentrations in women. In conclusion, different SNPs of the ABCA1, ABCG1 and ABCG5-ABCG8 genes were associated, some under gender-specific analysis, with variations in the plasma lipid levels under postprandial conditions in a representative Spanish population.

Evaluation of immunoassays for the measurement of insulin and C-peptide as indirect biomarkers of insulin misuse in sport: values in selected population of athletes.

Insulin and C-peptide have been proposed as possible biomarkers of human insulin hormone misuse in sport. An extended intra- and inter-laboratory validation of commercially available immunoassays was performed. Enzyme Amplified Sensitivity Immunoassay (EASIA) assays (Human Insulin-EASIA and C-peptide EASIA kits from BioSource) were evaluated for insulin and C-peptide in serum. The intra- and inter-laboratory precision and accuracy values were good for the evaluated assays with maximum imprecision and inaccuracy of 16% and 23%, respectively, obtained just for one day C-peptide assay evaluation. The range of concentrations found in serum samples under investigation was always covered by the calibration curves of the studied immunoassays. However, a 19.7% of the samples felt below the estimated insulin limit of quantification. High concordance between laboratory results was obtained for insulin assay (intraclass correlation coefficient -ICC=0.857), whereas that for C-peptide was lower (ICC=0.539). Evaluated immunoassays were used to measure serum concentrations of insulin and C-peptide in elite athletes of various sport disciplines at different moment of training season, in recreational athletes at baseline conditions and finally in sedentary individuals. Serum insulin was statistically lower both in recreational and elite athletes when compared to sedentary individuals. Among elite athletes, the specific sport affected serum insulin (e.g., weightlifting) and C-peptide (e.g., triathlon) concentrations. Over the training season, a within athletes variability was observed for taekwondo, swimming and weightlifting athletes. Variations due to those aspects should be taken in careful consideration in the hypothesis of setting reference concentration ranges for doping detection.

Pharmacokinetics of buprenorphine after intravenous administration of clinical doses to dogs.

The purpose of this study was to evaluate plasma concentrations and pharmacokinetic parameters of buprenorphine in dogs following intravenous (IV) administration of clinical doses of the opioid. An IV bolus of 0.02mg/kg buprenorphine was administered to six healthy Beagles and blood samples were collected through a jugular catheter before and at 1, 5, 10, 15, 20, 30 and 45 min, and 1, 2, 4, 6, 8 and 12h after administration. Plasma buprenorphine concentrations, measured using a commercial radioimmunoassay (RIA), decreased following a three-exponential curve. The two distribution and the elimination half-lives were 2.9+/-1.8min, 16.5+/-3.7min, and 266.6+/-82.0min, respectively; the clearance was 329.6+/-62.2mL/min, and the steady state volume of distribution was 83.7+/-26.5L. The results demonstrated the feasibility of the RIA assay to analyse buprenorphine in dog plasma samples. Following IV administration buprenorphine showed a three-compartment kinetic profile, as has been described previously in humans, rabbits and cats. The relationship between plasma concentrations and dynamic effects in dogs remains to be established.

Immunoassays for the measurement of IGF-II, IGFBP-2 and -3, and ICTP as indirect biomarkers of recombinant human growth hormone misuse in sport. Values in selected population of athletes.

Insulin-like growth factor-II (IGF-II), insulin-like growth factor binding proteins (IGFBPs) -2 and -3 and C-terminal telopeptide of type I collagen (ICTP) have been proposed, among others, as indirect biomarkers of the recombinant human growth hormone misuse in sport. An extended intra- and inter-laboratory validation of commercially available immunoassays for biomarkers detection was performed. ELISA assays for total IGF-II, IGFBP-2 and IGFBP-3 (IGF-II/ELISA1: DSLabs, IGFBP-2/ELISA2: Biosource, and IGFBP-3/ELISA3: BioSource) and an EIA assay for ICTP (ICTP/EIA: Orion Diagnostica) were evaluated. The inter- and intra-laboratory precision values were acceptable for all evaluated assays (maximum imprecision of 30% and 66% were found only for the lowest quality control samples of IGF-II and IGFBP-3). Correct accuracy was obtained for all inter-laboratory immunoassays and for IGFBP-2 intra-laboratory immunoassay. The range of concentrations found in serum samples under investigation was always covered by the calibration curves of the studied immunoassays. However, 11% and 15% of the samples felt below the estimated LOQ for IGF-II and ICTP, respectively, in the zone where lower precision was obtained. Although the majority of evaluated assays showed an overall reliability not always suitable for antidoping control analysis, relatively high concordances between laboratory results were obtained for all assays. Evaluated immunoassays were used to measure serum concentrations of IGF-II, IGFBP-2 and -3 and ICTP in elite athletes of various sport disciplines at different moments of the training season; in recreational athletes at baseline conditions and finally in sedentary individuals. Serum IGF-II was statistically higher both in recreational and elite athletes compared to sedentary individuals. Elite athletes showed lower IGFBP-2 and higher IGFBP-3 concentration with respect to recreational athletes and sedentary people. Among elite athletes, serum IGFBP-3 (synchronized swimming), and ICTP (rhythmic gymnastics) concentrations were sport-dependent. Over the training season, within athlete variability was observed for IGFBP-2 in case of taekwondo and IGFBP-2 and -3 in case of weightlifting. Variations due to those aspects should be taken in careful consideration in the hypothesis of setting reference concentration ranges for doping detection.

Intermittent hypoxia exposure in a hypobaric chamber and erythropoietin abuse interpretation.

The aim of this study was to assess the effect of intermittent hypoxia exposure on direct and indirect methods used to evaluate recombinant human erythropoietin (rhEPO) misuse. Sixteen male triathletes were randomly assigned to either the intermittent hypoxia exposure group (experimental group) or the control normoxic group (control group). The members of the experimental group were exposed to simulated altitude (from 4000 to 5500 m) in a hypobaric chamber for 3 h per day, 5 days a week, for 4 weeks. Blood and urine samples were collected before and after the first and the final exposures, and again 2 weeks after the final exposure. While serum EPO significantly increased after the first [from a mean 8.3 IU x l(-1) (s = 3.2) to 16.6 IU x l(-1) (s = 4.7)] and final exposures [from 4.6 IU x l(-1) (s = 1.4) to 24.8 IU x l(-1) (s = 9.3)], haemoglobin, percentage of reticulocytes, and soluble transferrin receptor were not elevated. Second-generation ON/OFF models (indirect rhEPO misuse detection) were insensitive to intermittent hypoxia exposure. The distribution of the urinary EPO isoelectric profiles (direct rhEPO misuse detection) was altered after intermittent hypoxia exposure with a slight shift towards more basic isoforms. However, those shifts never resulted in misinterpretation of results. The intermittent hypoxia exposure protocol studied did not produce any false-positive result for indirect or direct detection of rhEPO misuse in spite of the changes in EPO serum concentrations and urinary EPO isoelectric profiles, respectively.

Hematologic response to four weeks of intermittent hypobaric hypoxia in highly trained athletes.

We investigated changes induced by four weeks of intermittent hypobaric hypoxia (IHH) at a simulated altitude of 4000-5500 m in highly trained athletes. Serum erythropoietin increased significantly (p<0.001) after the sessions of IHH, but reticulocyte and red cell parameters did not. Our IHH protocol stimulated endogenous erythropoietin secretion without producing the subsequent erythropoietic response.

Evaluation of immunoassays for the measurement of insulin-like growth factor-I and procollagen type III peptide, indirect biomarkers of recombinant human growth hormone misuse in sport.

Insulin-like growth factor-I (IGF-I) and procollagen type III peptide (P-III-P) have been proposed as indirect biomarkers for the detection of the misuse of recombinant human growth hormone in sport. An extended intra- and inter-laboratory validation of commercially available immunoassays was carried out. For total IGF-I, two radioimmunoassay (RIA) kits (IGF-I/RIA1, Nichols Institute Diagnostics and IGF-I/RIA2, Mediagnost) and one enzyme-linked immunosorbent assay (ELISA) (R&D) were evaluated. For P-III-P, two RIA kits (P-III-P/RIA3, Cis-bioInternational and P-III-P/RIA4, Orion Diagnostica) were studied. The intra-laboratory precision and accuracy values for all IGF-I assays were better than 15%. The IGF-I/ELISA showed the lowest limit of quantification (LOQ) and its calibration curve covered the range of concentrations found in human serum samples. Higher agreement between laboratory results was obtained for IGF-I/ELISA and IGF-I/RIA1. Low inter-technique correlation was obtained for the three assays; the only comparable results were obtained between IGF-I/ELISA and IGF-I/RIA1. For P-III-P, intra-laboratory precision and accuracy values better than 15% were obtained for both assays in almost all cases. The calibration curve for P-III-P/RIA4 covered the range of concentrations of serum samples, while 30% of the values for P-III-P/RIA3 were below the calibration sample with the lowest concentration. Inter-laboratory correlation was also higher for P-III-P/RIA4. In summary, ELISA and RIA4 were the most suitable assays for measurement of IGF-I and P-III-P, respectively, in serum samples. However, the validation studies carried out show the need for harmonization of immunoassay parameters to improve the reproducibility and comparability of results between different laboratories and in different studies.

Evaluation of immunoassays for the measurement of erythropoietin (EPO) as an indirect biomarker of recombinant human EPO misuse in sport.

The measurement of serum erythropoietin (EPO) has been proposed as one of the indirect biomarkers for the detection of recombinant human EPO misuse in sport. An extended inter-laboratory validation of two commercial immunoassays for EPO measurement is described. A chemiluminescent immunoassay kit (CHEM) and an enzyme-linked immunosorbent assay kit (ELISA) were evaluated. The CHEM assay showed intra-laboratory precision better than 6% and correct accuracy values for all quality control samples tested. Precisions and accuracies better than 7 and 13%, respectively, were obtained for the ELISA assay for most of the quality control samples. The limit of quantification estimated for CHEM assay was lower than for the ELISA assay. Inter-laboratory concordance was good for both the assays, with lower dispersion shown by the CHEM assay. Results obtained with the ELISA assay were always lower than those of the CHEM assay. However, a good inter-technique correlation was obtained ([ELISA]=0.76 [CHEM]+0.06, r2=0.92). Quality control samples had a good stability after one and two freeze/thaw cycles and in simulated transportation conditions. In conclusion, CHEM and ELISA assays showed similar characteristics regarding intra-laboratory validation. Better inter-laboratory results were obtained with the CHEM assay and, hence, it is considered the recommended assay for anti-doping control analysis.