QIAxcel System Research Articles

Impact of Genemutations and Dynamics Methylation of CDKN2B and DLC-1 Promoters on Treatment Response with Azacitidine

Introduction: Azacitidine (AZA) is a DNA hypomethylating agent used to treat high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) in patients who are not eligible for intensive strategies. In view of the variability of drug response, last years it has been studied the genes mutations as possible response biomarkers. In addition, although there seems to be no association between promoter genes methylation and response, dynamics methylation before and after AZA treatment could be a predictable tool of clinical response. CDKN2B gene is a regulator of cell cycle and its aberrant hypermethylation is recurrent in myeloid diseases. DLC-1 gene plays a critical role in cell migration and proliferation and although its abnormal methylation is common in different kinds of cancers, its role in hematological malignancies is slightly studied.Aim: To assess the predictive value of gene mutations and dynamics methylation of CDKN2B and DLC-1 promoters on AZA response in a series of patients with myeloid neoplasms.Methods: We studied 61 patients with MDS and 16 patients with AML who received AZA treatment. According to the International Working Group (IWG) 2006 criteria for MDS, responders were defined as patients achieving complete remission (CR), bone marrow CR (mCR), partial remission (PR) or stable disease with hematologic improvement (SDHI). Ten genes with prior implication in the pathogenesis of myeloid diseases were analyzed: DNMT3A , IDH1 , IDH2 , ASXL1 , SETBP1 , TP53 , SF3B1 , SRSF2 , U2AF1 and ETNK1. For methylation assay, EZ DNA Methylation-Gold kit (Zymo Research) was employed for the bisulfite treatment of DNA. The methylation levels of CDKN2B and DLC-1 were studied with unmethylated and methylated primers, positive and negative controls and analysis of the PCR products in a QIAxcel System (Qiagen). In all patients CDKN2B and DLC-1 methylation were analyzed pre-AZA treatment and in 57 patients with post-AZA DNA sample the methylation assay was also possible at that point. P values <0.05 were considered as statistically significant .Results: Of the 77 patients analyzed, 30 patients (39%) showed AZA response (median cycles=6): CR=13, mCR=2, PR=8, and SDHI=7. Stable disease and treatment failure were observed in 19 (25%) and 28 patients (36%), respectively. Pre-treatment, genetic mutations were presented in 63 patients (82%) and methylationof CDKN2B and DLC-1 promoters were detected in 32 (42%) and 37 (48%) patients respectively (Figure 1). Patients with mutations in DNA methylators (DNMT3A , IDH1/2 ) showed a significant worse AZA response than the remaining patients (9% vs. 44%, P =0.026) and furthermore DNMT3A patients presented a lower overall survival (OS) compared with DNMT3A patients (8 vs . 19 months, P =0.042). All SETBP1 patients also did not show AZA response and moreover transformed to AML in a few months. On the other hand, TP53 mutations did not influence the drug response although they were associated with complex karyotypes (92% vs. 8%, P <0.001) and with a lower OS (10 vs. 21 months, P =0.043). Conversely, splicing genes mutations (SF3B1 , SRSF2 and U2AF1 ) were related to favourable karyotypes (78% vs. 54%, P =0.007), higher OS (28 vs. 13 months, P =0.038) and lower DLC-1 methylation (33% vs . 64%, P =0.010). Interestingly, patients with DLC-1 methylation showed higher levels (>5%) of bone marrow blasts (74% vs . 42%, P =0.005), higher frequency of complex karyotypes (29% vs. 9%, P =0.002) and lower OS (15 vs . 30 months, P =0.044). In addition,dynamics methylation of DLC-1 was significantly associated with the AZA response: 59% patients who reduced DLC-1 methylation levels post-treatment, responded favourably to the drug and 95% patients who maintained or increased them, did not respond (P =0.002; Figure 2). Finally, CDKN2B methylated patients had a significant co-occurrence with favourable karyotypes (80% vs. 45%, P =0.014), presented a higher OS (28 vs. 13 months, P =0.018) but its dynamics methylation was not associated with the AZA response (P =0.07).Conclusion: In our seriesof patients treated with AZA, DNMT3A and IDH1/2 mutations have a negative impact on drug response. DLC-1 dynamics methylation is associated with the AZA response and clinical outcome.The combination of point mutations and monitoring methylation could contribute to give light to the heterogeneity of AZA response in myeloid neoplasms. [Display omitted] DisclosuresSolano:Neovii: Honoraria.

Diagnostic test assessment. Validation study of an alternative system to detect microsatellite instability in colorectal carcinoma.

SummaryThe American Society for Clinical Pathology (ASCP), College of American Pathologists (CAP), Association for Molecular Pathology (AMP), and the American Society of Clinical Oncology (ASCO) have been recently strongly recommended the evaluation of mismatch repair status (MMS) as molecular biomarkers in colorectal cancer for a better prognostic stratification of patients. This recommendation is emphasized by the recent evidence of Microsatellite Instability (MSI) as a predictive marker for chemotherapy and immunotherapy.In this scenario, the validation of molecular biomarker testing methods seems to be essential to design the most appropriate tailored therapy and the most suitable care strategy, respectively.In this study, we validated an alternative method based on capillary electrophoresis system label-free PCR (Qiaxcel system) to evaluate the MSI Bethesda Panel. We also parallel the results with a standard approach.Our data showed total concordance with the standard approach, with a highly time-efficient and easy procedure combined with high sensitivity for MSI detection.Alternative capillary electrophoresis based on label-free PCR such as the Qiaxel system is a very sensitive and specific method to detect MSI for the management of patients with colorectal cancer. This procedure is adequate and suitable in diagnostic routine for the evaluation of microsatellite repeats compared to standard procedures.

Accuracy of the QIAxcel Automated System for MIRU-VNTR Genotyping of Mycobacterium tuberculosis in Two Limited Resource Settings.

Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis complex (MTBC) isolates, based on 24 loci, is still widely used as the standard for routine molecular surveillance of tuberculosis (TB). QIAxcel system is proposed as an affordable tool that could replace conventional gel electrophoresis and provide high concordance with the reference methods regarding MIRU-VNTR typing of MTBC. We aimed to evaluate the QIAxcel accuracy for allele calling of MIRU-VNTR loci in two regional reference laboratories. A total of 173 DNA were used for the study. Results obtained with QIAxcel were compared to the reference results obtained with an ABI 3730 DNA analyzer. In Albania, the overall agreement with the reference method was 97.92%. A complete agreement result was obtained for 17 loci. In Tunisia, the overall agreement with the reference method was 98.95%. A complete agreement result was obtained for 17 loci. Overall agreement in both centers was 98.43%. In our opinion, use of QIAxcel technology has the potential to be reliable, given an optimized algorithm. Inaccuracies in sizing of long fragments should be solved, especially regarding locus 4052.

Impact of Clinical Features, Cytogenetics, Genetic Mutations and Methylation of CDKN2B and DLC-1 Promoters on Treatment Response to Azacitidine

Introduction : Azacitidine (AZA) is a DNA hypomethylating agent used in myeloid neoplasms, however approximately half of patients show treatment failure or relapse. Last years, several studies have showed that genetic mutations may influence on response and survival of the treated patients. Other biomarkers that have traditionally been associated with the response to AZA are the recovery of the platelet count and the presence of abnormalities in the chromosome 7. Finally, the methylation dynamics of genes promoters could be a useful tool to predict the clinical response. Aim: To assess the predictive value on response to AZA of clinical features, cytogenetics, genetic mutations and the methylation dynamics of CDKN2B and DLC-1 promoters in a series of patients with myeloid neoplasms. Methods: We studied 85 patients with myelodysplastic syndromes (MDS) and 28 patients with acute myeloid leukemia (AML) who received AZA. Treatment schedules included 5-azacytidine 75 mg/m2/day for 7 days (n=62) and 5-azacytidine 75 mg/m2/day for 5 days (n=51). According to the International Working Group (IWG) 2006 criteria for MDS, responders were defined as patients achieving complete remission (CR), bone marrow CR (mCR), partial remission (PR) or stable disease with hematologic improvement (SDHI). Sixteen genes with prior implication in the pathogenesis of myeloid diseases were analyzed: NPM1, FLT3, CEBPA, TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, RUNX1, SETBP1, TP53, SF3B1, SRSF2, U2AF1 and ETNK1. For methylation assay, EZ DNA Methylation-Gold kit (Zymo Research) was employed for the bisulfite treatment of DNA. Methylation of CDKN2B and DLC-1 were qualitatively studied pre- and post-AZA with unmethylated and methylated primers and analysis of the PCR products in a QIAxcel System (Qiagen). Overall survival (OS) was measured from start of AZA treatment until the last follow-up or death from any cause. P values &lt;0.05 were considered as statistically significant. Results: Of the 113 patients analyzed, 46 patients (41%) showed response to AZA (median cycles=6): CR=19, mCR=6, PR=12, and SDHI=9. Stable disease and treatment failure were observed in 20 (17%) and 47 patients (42%), respectively. Analysis of clinical features showed that platelet doubling after the first AZA cycle was significantly associated with a better response (58% vs. 31% responders, P=0.041). Forty-seven patients (42%) had chromosomal abnormalities. A positive co-occurrence between del(7q)/-7 and del(17p) was found (n=6, P&lt;0.001) and was significantly associated with a worse response to AZA (P=0.039). Pre-treatment, genetic mutations were presented in 98 patients (87%) and methylation of CDKN2B and DLC-1 promoters were detected in 50 (44%) and 37 (33%) patients respectively (Figure 1). Patients with SF3B1 mutations showed a better response to AZA (68% vs. 35% responders, P=0.008) and a higher OS (30 vs. 15 months, P=0.019) than the remaining patients. In contrast, patients with mutations in transcription factors (RUNX1, SETBP1, NPM1) showed a worse response to AZA (20% vs. 47% responders, P=0.014) and a lower OS (10 vs. 19 months, P=0.010). Splicing genes mutations (SF3B1, SRSF2 and U2AF1) were related to favourable karyotypes (90% vs. 59%, P&lt;0.001) and a lower DLC-1 methylation (31% vs. 57%, P=0.006). Interestingly, patients with DLC-1 methylation showed higher levels (&gt;5%) of bone marrow blasts (63% vs. 40%, P=0.016), higher frequency of complex karyotypes (40% vs. 15%, P=0.002), a positive co-occurrence with TP53 alterations (38% vs. 16%, P=0.009) and its methylation dynamics showed a strong trend towards predict the response to AZA: 57% of patients without DLC-1 methylation or who reduced it post-treatment responded favourably to the drug and 65% of patients who maintained or increased it did not respond (P=0.053). In addition, leukemia transformation was higher in MDS patients with DLC-1 methylation post-AZA than in the remaining MDS patients (58% vs. 21%, P=0.002). Conclusion: The combination of chromosomal alterations and genetic mutations together with the monitoring of platelet count and DLC-1 methylation could contribute to give light in myeloid neoplasms to the heterogeneity observed both in the response to AZA and in patient survival. Samples and data from patients included in this study were provided by the INCLIVA Biobank (PT13/0010/0004). Disclosures Hernandez Boluda: Incyte: Other: Travel expenses paid.

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease.

MIRU-VNTR Genotyping of Mycobacterium tuberculosis Strains Using QIAxcel Technology: A Multicentre Evaluation Study

BackgroundMolecular genotyping of M.tuberculosis is an important laboratory tool in the context of emerging drug resistant TB. The standard 24-loci MIRU-VNTR typing includes PCR amplification followed by the detection and sizing of PCR fragments using capillary electrophoresis on automated sequencers or using agarose gels. The QIAxcel Advanced system might offer a cost-effective medium-throughput alternative.MethodsPerformance characteristics of the QIAxcel Advanced platform for the standard 24 VNTR loci panel was evaluated at two centres on a total of 140 DNA specimens using automated capillary electrophoresis as a reference method. Additionally 4 hypervariable MIRU-VNTR loci were evaluated on 53 crude DNA extracts. The sizing accuracy, interlaboratory reproducibility and overall instrument’s performance were assessed during the study.ResultsAn overall concordance with the reference method was high reaching 98.5% and 97.6% for diluted genomic and crude DNA extracts respectively. 91.4% of all discrepancies were observed in fragments longer than 700bp. The concordance for hypervariable loci was lower except for locus 4120 (96.2%). The interlaboratory reproducibility agreement rates were 98.9% and 91.3% for standard and hypervariable loci, respectively. Overall performance of the QIAxcel platform for M.tuberculosis genotyping using a panel of standard loci is comparable to that of established methods for PCR fragments up to 700bp. Inaccuracies in sizing of longer fragments could be resolved through using in-house size markers or introduction of offset values. To conclude, the QiaXcel system could be considered an effective alternative to existing methods in smaller reference and regional laboratories offering good performance and shorter turnaround times.

Assessment of an automated capillary system for Plasmodium vivax microsatellite genotyping.

BackgroundSeveral platforms have been used to generate the primary data for microsatellite analysis of malaria parasite genotypes. Each has relative advantages but share a limitation of being time- and cost-intensive. A commercially available automated capillary gel cartridge system was assessed in the microsatellite analysis of Plasmodium vivax diversity in the Peruvian Amazon.MethodsThe reproducibility and accuracy of a commercially-available automated capillary system, QIAxcel, was assessed using a sequenced PCR product of 227 base pairs. This product was measured 42 times, then 27 P. vivax samples from Peruvian Amazon subjects were analyzed with this instrument using five informative microsatellites. Results from the QIAxcel system were compared with a Sanger-type sequencing machine, the ABI PRISM® 3100 Genetic Analyzer.ResultsSignificant differences were seen between the sequenced amplicons and the results from the QIAxcel instrument. Different runs, plates and cartridges yielded significantly different results. Additionally, allele size decreased with each run by 0.045, or 1 bp, every three plates. QIAxcel and ABI PRISM systems differed in giving different values than those obtained by ABI PRISM, and too many (i.e. inaccurate) alleles per locus were also seen with the automated instrument.ConclusionsWhile P. vivax diversity could generally be estimated using an automated capillary gel cartridge system, the data demonstrate that this system is not sufficiently precise for reliably identifying parasite strains via microsatellite analysis. This conclusion reached after systematic analysis was due both to inadequate precision and poor reproducibility in measuring PCR product size.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0842-9) contains supplementary material, which is available to authorized users.

Molecular Strain Typing of Brucella abortus Isolates from Italy by Two VNTR Allele Sizing Technologies

Brucellosis, one of the most important re-emerging zoonoses in many countries, is caused by bacteria belonging to the genus Brucella. Furthermore these bacteria represent potential biological warfare agents and the identification of species and biovars of field strains may be crucial for tracing back source of infection, allowing to discriminate naturally occurring outbreaks instead of bioterrorist events. In the last years, multiple-locus variable-number tandem repeat analysis (MLVA) has been proposed as complement of the classical biotyping methods and it has been applied for genotyping large collections of Brucella spp. At present, the MLVA band profiles may be resolved by automated or manual procedures. The Lab on a chip technology represents a valid alternative to standard genotyping techniques (as agarose gel electrophoresis) and it has been previously used for Brucella genotyping. Recently, a new high-throughput genotyping analysis system based on capillary gel electrophoresis, the QIAxcel, has been described. The aim of the study was to evaluate the ability of two DNA sizing equipments, the QIAxcel System and the Lab chip GX, to correctly call alleles at the sixteen loci including one frequently used MLVA assay for Brucella genotyping. The results confirmed that these technologies represent a meaningful advancement in high-throughput Brucella genotyping. Considering the accuracy required to confidently resolve loci discrimination, QIAxcel shows a better ability to measure VNTR allele sizes compared to LabChip GX.

Validation of carbapenemase and extended-spectrum -lactamase multiplex endpoint PCR assays according to ISO 15189

To validate and accredit a set of three multiplex endpoint PCR assays, targeting the most important carbapenemase and minor extended-spectrum β-lactamase (ESBL) resistance genes, according to the international ISO 15189 particular requirements for the quality and competence of medical laboratories. Specific primers targeting ESBLs and carbapenemases were collected from the literature or designed internally. The multiplex PCRs were validated for sensitivity, specificity, intra- and inter-run reproducibility and accuracy by means of external quality control (EQC) using a collection of 137 characterized and referenced isolates. For each multiplex PCR assay, the presence of an extraction control ruled out false-negative results due to PCR inhibition or extraction faults. Amplicons were separated by capillary electrophoresis (QIAxcel system, Qiagen). The protocols and validation files were reviewed in the setting of an external audit conducted by the Belgian organization for accreditation (BELAC). Sensitivity, specificity and reproducibility for each targeted gene were 100%. All isolates from the three EQC panels were correctly identified by each PCR assay (accuracy 100%). The validation files were controlled by BELAC, and the PCR protocols were accepted as accredited according to ISO 15189. Three home-made multiplex PCRs targeting the major carbapenemases and four minor class A ESBL genes encountered in Gram-negative bacteria were accredited according to the ISO 15189 standards. This validation scheme could provide a useful model for laboratories aiming to accredit their own protocols.

Guanidinoacetate methyltransferase deficiency: First steps to newborn screening for a treatable neurometabolic disease

BackgroundGAMT deficiency is an autosomal recessive disorder of creatine biosynthesis resulting in severe neurological complications in untreated patients. Currently available treatment is only successful to stop disease progression, but is not sufficient to reverse neurological complications occurring prior to diagnosis. Normal neurodevelopmental outcome in a patient, treated in the newborn period, highlights the importance of early diagnosis. MethodsTargeted mutation analysis (c.59G>C and c.327G>A) in the GAMT gene by the QIAxcel system and GAA measurement by a novel two-tier method were performed in 3000 anonymized newborn blood dot spot cards. ResultsNone of the targeted mutations were detected in any newborn. Two novel heterozygous variants (c.283_285dupGTC; p.Val95dup and c.278_283delinsCTCGATGCAC; p.Asp93AlafsX35) were identified by coincidence. Carrier frequency for these insertion/deletion types of GAMT mutations was 1/1475 in this small cohort of newborns. GAA levels were at or above the 99th percentile (3.12μmol/l) in 4 newborns. Second-tier testing showed normal results for 4 newborns revealing 0.1% false positive rate. No GAMT mutations were identified in 4 of the newborns with elevated GAA levels in the first tier testing. ConclusionThis is the first two-tier study to investigate carrier frequency of GAMT deficiency in the small cohort of newborn population to establish evidence base for the first steps toward newborn screening for this treatable neurometabolic disorder.

Incidence of Helicobacter pylori infection and their clarithromycin-resistant strains in otitis media with effusion regarding phenotypic and genotypic studies

Helicobacter pylori (H. pylori) are pathogenic bacteria that infect a half of the human population, colonize gastric mucosa and can be found in gastric juice. Reflux of gastric juice has been suggested to be associated with glue ear in children. It has been suggested that tonsil and adenoid tissues are potential reservoirs of H. pylori infection. These observations raise the question as to whether H. pylori infection might have a role in otitis media with effusion (OME) in children. The objectives of this research were to evaluate the incidence and possible role of H. pylori in the pathogenesis of OME in children and to evaluate the clarithromycin-resistant strains. Molecular assessment was done to evaluate the culture results vs. molecular study. A total of 60 children, who were prone to ventilation tube insertion, adenoidectomy and/or tonsillectomy were included in the study. The control group consisted of 40 children who underwent adenoidectomy and/or tonsillectomy without the history of OME. Samples of the middle ear fluid and mucosa, adenoid tissue, tonsillar tissue and gastric lavage were cultured and underwent polymerase chain reaction (PCR) analysis then were assembled by using QIAxcel System as capillary electrophoresis for H. pylori detection. There was significant difference between the results of cultures and PCR (P<0.05). Middle ear fluid culture was positive for H. pylori in 40% of the patients vs. 56.7% PCR results while middle ear mucosa culture was positive in 20% vs. 26.7% PCR results. Gastric lavage culture was positive in 46.6% of the patients and PCR was positive in 63.3% of the patients. Adenoid culture and PCR were positive in 56.3% for each, while tonsil culture was positive in 70% and PCR was positive in 90%. H. pylori presence in the gastric lavage, the tonsillar and adenoid tissues by culture and PCR was significantly more frequent in the study group compared to the control group. The minimum inhibitory concentration (MIC) values of clarithromycin-resistant isolates ranged from 1.5 to 8μg/ml. This study showed the presence of H. pylori in around 50% of the patients with OME. PCR revealed its sensitivity than culture techniques. The incidence of clarithromycin resistance was found to be high among the isolates (39.6%).

A new electrophoresis technique to separate microsatellite alleles.

Analysis of large numbers of SSR (simple sequence repeats: microsatellites) reactions can be tedious, time-consuming and expensive. The objective of this study was to report a new electrophoresis method to analyze and visualize SSR data quickly and accurately and compare it to the ability of four other electrophoresis methods. Individual PCR reactions consisting of DNA from several Cornus florida L. (flowering dogwood) cultivars and two SSR primer pairs were assembled for analysis using the following three methods: agarose gel, polyacrylamide gel and QIAxcel System. Two separate PCR reactions consisting of the same components plus a fluorescent-labeled primer were set up for analyses using the CEQ TM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer. These five electrophoretic methods were assessed for advantages and disadvantages. Polyacrylamide gels had highest resolution of alleles, whereas agarose gels had the lowest. However, with both separation media, it was difficult to score the size of alleles. Capillary electrophoresis with the CEQ TM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer easily separated products and determined allelic size, but was more expensive than electrophoresis using either agarose or polyacrlamide gels. The QIAxcel System had lower resolution than CEQ TM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer. However, QIAxcel System was rapid and cost effective compared to the two widely used capillary sequencers, and also provided a computer generated gel image. For researchers in small to intermediate-sized laboratories, the QIAxcel System using a twelve channel, sieving-gel cartridge is an affordable device for SSR assays used for mapping and population diversity analysis.