Allele-specific Oligonucleotide Research Articles

Analytical Validation of Patient-Specific PCR-Based MRD Assessment for Use As a Primary Endpoint in CLL Clinical Trials

Analytical Validation of Patient-Specific PCR-Based MRD Assessment for Use As a Primary Endpoint in CLL Clinical Trials

High-Sensitivity Genomic Minimal Residual Disease Detection Reveals Multiclonal Hematopoiesis and Is Associated with Survival in Adult AML

▪Introduction: Minimal residual disease (MRD) measurement is emerging as a prognostic marker of relapse-free survival (RFS) and overall survival (OS) in acute myelogenous leukemia (AML). In order to improve the technical feasibility of MRD detection and to enable broad, ultra-sensitive mutational profiling of residual AML, we optimized a droplet-based digital PCR (ddPCR) approach capable of detecting mutated alleles in most recurrently mutated genes in AML in as few as 5 in 100,000 cells. We subsequently analyzed bone marrow specimens of AML patients in early complete remission (CR) and correlated MRD levels with outcome.Methods: Fifty-two consecutively enrolled patients with AML at a single center who had paired diagnostic and CR bone marrow specimens were studied. First, genomic DNA from FACS-purified leukemic cells of diagnostic samples underwent Sanger resequencing of NPM1, IDH1, IDH2, NRAS, KRAS, FLT3, TP53, DNMT3A, TET2, ASXL1, BCORL1, CEBPA, and RUNX1. For cases with <2 mutations identified, an additional 36 recurrently mutated genes in AML were sequenced using droplet-based library preparation and massively parallel sequencing. Second, allele-specific fluorescent oligonucleotide probes using minor groove-binding or locked nucleic acid chemistries were developed for each mutation and optimized for allele discrimination using quantitative PCR. ddPCR (RainDance Technologies) was then performed on replicate DNA samples extracted from Ficoll-enriched bone marrow aspirates obtained in CR (median time from induction: 36d) to determine variant allele frequency (VAF) for all mutations.Results: In CR bone marrow samples, we detected residual gene mutations in 88% of cases (46 of 52) using a detection threshold of 0.005% VAF. Notably, DNMT3A and TET2 mutations persisted at >1% VAF in 85% (11 of 13) (range 1.6-34.6%) and 50% (5 of 10) (range 2.3-15.1%) of cases, respectively, including in patients with subsequent durable remissions (>1000 days). Conversely, 94% (15 of 16) and 100% (5 of 5) of NPM1 and NRAS mutations, respectively, were reduced to <1% VAF, and 44% (7 of 16) of NPM1 mutations and 60% (3 of 5) of NRAS mutations were undetectable.In cases for which ≥2 mutations were measured (N=28), two distinct patterns of residual allele burden were identified. In 32% (9 of 28) of cases, the VAFs of all genes measured were concordant (<1 log difference in VAF), but importantly, in 68% (19 of 28) of cases one or more VAFs were discordant (≥1 log difference), indicative of separate residual clones. In discordant cases, DNMT3A was the most common mutated gene of highest VAF (10 of 19, 53%), while NPM1 was the most frequent gene mutation of lowest VAF (10 of 19, 53%).Kaplan-Meier analysis of all patients in first CR (N=48) was performed using a cutoff of 0.01% VAF to define MRDhigh (N=33) and MRDlow/neg (N=15) groups. Due to frequent high-level persistence, DNMT3A mutations were excluded from the analysis. In cases where ≥2 mutations were measured, the highest VAF was used to assign that case. MRD burden was associated with RFS (MRDhigh, median RFS 9.9 mo; MRDlow/neg, median RFS not reached; p <0.01) and OS (MRDhigh, median OS 17.8 mo; MRDlow/neg, median OS not reached; p <0.01). In the exploratory subset of patients who proceeded to allogeneic stem cell transplantation in first CR (N=13), all patients who were MRDlow/neg (N=5) remained in a long-term remission, while 75% of MRDhigh (6 of 8) patients relapsed (median RFS 14.8 mo; p =0.01).Conclusions: High sensitivity measurement of residual clonal hematopoiesis with digital PCR is feasible for the majority of recurrently mutated genes in AML. Most DNMT3A and some TET2 and IDH1 mutations persist at high levels in remission even in patients with subsequent durable clinical remissions, further refining the hierarchical acquisition model of gene mutations in AML and implying they are present in pre-leukemic or non-blast differentiated cell populations. Due to high MRD levels, mutations in DNMT3A and possibly TET2 and IDH1 may not be suitable for use in MRD-based prognostication. The majority of cases with multiple measured gene mutations demonstrate discordance in VAF suggesting the simultaneous persistence of chemosensitive, chemoresistant and pre-leukemic populations. Ongoing work aims at identifying which mutation-defined subclones are responsible for relapse. Finally, in this cohort mutation-based MRD measured at initial CR is associated with RFS and OS. DisclosuresMalek:Abbvie: Equity Ownership; Gilead Sciences: Equity Ownership; Janssen Pharmaceuticals: Research Funding.

Phase Ib Trial of the mTOR Inhibitor Everolimus Given in Combination with Multiagent Chemotherapy in Relapsed Acute Lymphoblastic Leukemia

Introduction: Treatment of relapsed childhood acute lymphoblastic leukemia (ALL) remains a significant clinical challenge with a need for novel, more effective therapies. Pre-clinical studies have demonstrated that mTOR inhibitors have single agent activity in ALL and are synergistic with other chemotherapy agents typically used to treat ALL, including vincristine, corticosteroids, doxorubicin and L-asparaginase.Methods: Dana-Farber Cancer Institute Protocol 11-237 tested the combination of everolimus (given orally on days 1-32) with a standard reinduction regimen of prednisone (40 mg/m2/daily orally on days 4-32), weekly vincristine (1.5 mg/m2/dose, max 2 mg, on days 4,11,18 and 25), PEG-asparaginase (2,500 U/m2/dose IV on days 5 and 18) and doxorubicin (30 mg/m2/dose on days 4 and 5) with dexrazoxane pretreatment (300 mg/m2/dose on days 4 and 5). Intrathecal cytarabine was administered on day 1 and intrathecal methotrexate, cytarabine and hydrocortisone (IT-MAH) was administered on days 18 and 32; patients (pts) with cerebrospinal fluid blasts at study entry received additional IT-MAH on days 11 and 25. Pts with first bone marrow relapse ALL occurring greater than 18 months after continuous remission were eligible for participation. The primary aim was to identify a maximum tolerated dose of everolimus in combination with this backbone utilizing a classic "3 + 3" dose escalation design. Three dose levels (DL) of everolimus were tested (2-, 3-, and 5-mg/m2/day). 5 mg/m2/day (DL3) was the highest dose tested because it was the recommended phase 2 dose (RP2D) of single-agent everolimus in children with solid tumors. Secondary aims included assessment of second complete remission (CR2) rate and levels of end-reinduction minimal residual disease (MRD) assessed by allele-specific oligonucleotide PCR. On-target activity of everolimus was assessed 6 hours after the first dose of everolimus by examining phosphorylated S6 (pS6) levels in peripheral blood blasts by flow cytometry.Results: Thirteen pts with first relapse B-ALL were enrolled. (Table 1) Median age at enrollment was 10 yrs (range: 2-22 yrs). Median duration of first CR was 3.7 yrs (range 1.5-7.7 yrs). One pt treated at DL 3 stopped study drug early due to Grade 4 infection (meningitis due to Rothia mucilaginosa) and was inevaluable for dose limiting toxicity (DLT) assessment. Table 2 displays the most common Grade 2 or higher adverse events (AE's) attributable to everolimus. There were no Grade 5 events and no episodes of interstitial pneumonitis. No DLTs were observed at DL1 or DL2. One of 6 evaluable pts treated at DL3 experienced a DLT (Grade 4 hyperbilirubinemia). 12 of the 13 pts achieved CR (92%). Of the 12 pts who achieved CR, 9 (75%) had low end-reinduction MRD (²0.001). (Table 1) Of the 7 pts with evaluable samples (2 at DL1, 1 at DL2 and 4 at DL3), 6 (86%) had decrease in pS6 in peripheral blasts (>20% decrease in mean fluorescence intensity), consistent with on-target mTOR inhibition. Only 1 pt (treated at DL1) did not have evidence of pS6 inhibition; this pt achieved CR2 with low MRD.Discussion: Everolimus given in combination with a standard 4-drug reinduction was well tolerated at all dose levels tested in pediatric pts with first relapse ALL. CR2 rates were comparable to published re-induction regimens, with a promising proportion of pts with low end-reinduction MRD. An expansion cohort is currently enrolling to further assess toxicity of this combination, particularly infectious risks, in order to define the RP2D.Table 1Patients and Response to TherapyPt #Dose LevelAge (yrs)Duration of CR1 (yrs)DLT?CR?MRD0011107.7NoYesHigh0021203.5NoYesLow003183.1NoYesLow004221.5NoYesHigh0052225.1NoNoN/A006253.7NoYesLow0073125.7NoYesLow0083103.0N/AYesLow0093114.3NoYesLow0103214.8YesYesHigh011373.4NoYesLow0123135.0NoYesLow0133173.0NoYesLowTable 2Grade 2 or higher Adverse Events Attributable to Everolimus (Possible, Probable, Definite) at all dose levels; N=13 patientsToxicityGrade 2Grade 3Grade 4Grade 5Bacteremia0300Bacterial meningitis0010Invasive fungal disease0100Febrile Neutropenia0700Mucositis2200Anorexia3100AST increase1520ALT increase1520Bilirubin increase0210Hypophosphatemia2510 DisclosuresPauly:Seattle Genetics, Inc.: Research Funding. Stegmaier:Novartis Pharmaceuticals: Consultancy.

Prognostic Value of Sequencing-Based Minimal Residual Disease Detection in Patients with Multiple Myeloma Who Underwent Autologous Stem Cell Transplantation

Background: Autologous stem cell transplantation (ASCT) in conjunction with therapeutic drugs such as bortezomib, thalidomide, and lenalidomide can dramatically improve response rates and the prognosis of patients with multiple myeloma (MM). However, most patients with MM are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Here we utilized a next-generation sequencing (NGS) approach for MRD assessment, which offers at least 1 to 2 logs greater sensitivity (10-6) compared to allele-specific oligonucleotide PCR (ASO-PCR) and flow cytometry, respectively (Faham et al Blood 2012). Previous studies have shown that NGS-based MRD assessment 90 days post-ASCT has prognostic value (Martinez-Lopez et al Blood 2014). In this study, we compared the prognostic value of MRD assessment in autografts and bone marrow (BM) samples from MM patients in the ASCT setting.Methods: One hundred and twenty-three Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients received ASCT and were followed between June 15, 2004 and April 25, 2015. All patients had achieved a partial response (PR) or better after ASCT. Analyzed samples included: (1) BM slides from 96 MM patients at diagnosis, (2) fresh/frozen BM cells from 27 MM patients at diagnosis, (3) autografts and/or (4) post-ASCT BM cells obtained at the time of best response based on serum and urine tests. IGH-based ASO-PCR was performed as described previously (Methods Mol Biol 2009). NGS-based MRD assessment was performed using the immunosequencing platform (Adaptive Biotechnologies, South San Francisco, CA) (Martinez-Lopez et al Blood 2014).Results: We compared MRD results in 51 samples assessed by ASO-PCR and NGS. We observed a high correlation between NGS and ASO-PCR results at MRD levels of 10-5 or higher (r=0.86, P<0.0001). Twenty-seven samples were positive by NGS and negative by ASO-PCR, demonstrating the higher sensitivity of NGS (10-6 or higher) vs ASO-PCR (10-4-10-5). We evaluated the association of clinical outcome with post-ASCT BM MRD assessment. Patients who were MRD negative by NGS (defined as <10-6) in post-ASCT BM cases (N=21) showed a significantly better progression free survival (PFS) compared to MRD positive patients (N=31) (P =0.005) (Fig 1A). When restricting the analysis to the 39 patients in complete response (CR), patients who were MRD negative by NGS (N=20) showed a significantly better PFS than those that were MRD positive (N=19) (P =0.042). We also evaluated the association of clinical outcome with autograft MRD assessment. 53 patients received post-ASCT therapy using novel agents such as bortezomib/lenalidomide/thalidomide, and 45 patients did not. Among the 45 patients who did not receive post ASCT treatment, patients with NGS-based MRD negativity in the autograft (N=11) had a significantly better PFS (P=0.012) and tended to have a better OS (p=0.203) than those who were MRD positive (N=34), with prognosis clearly stratified by the quantitative level of MRD (Figs 1B, 1C). MRD assessment from the 53 patients who did receive post ASCT treatment tended to show a similar pattern. Finally, we studied whether clinical outcomes would have been altered by treatment in this cohort. Patients whose autografts were negative by NGS-based MRD assessment (N=19) had 100% PFS and OS at 5 years post ASCT irrespective of whether or not they received post ASCT treatment. Conversely, post ASCT treatment had a significant effect on the outcome of patients whose autografts were MRD positive by NGS. Specifically, the NGS-based MRD positive patients who received post ASCT treatment (N=45) had a significantly better PFS (P=0.003) and tended to have a better OS than those that were untreated (N=34) (Figs 1D, 1E).Conclusions: In this study, we show the prognostic value of NGS-based MRD assessment in autografts of patients with MM. The NGS platform has improved sensitivity compared with ASO-qPCR in detecting MRD in autografts. Importantly, this retrospective study suggests that therapeutic intervention based on NGS-based MRD positivity has a significant effect on patient outcome in the post-ASCT setting. [Display omitted] DisclosuresZheng:Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder.

A Comparison of Minimal Residual Disease Detection Among ASO-PCR, Dd-PCR and Deep-Sequencing in Patients with Multiple Myeloma Who Underwent Autologous Stem Cell Transplantation

▪Background: Most patients with multiple myeloma (MM) are considered to be incurable, and relapse, due to minimal residual disease (MRD), is the main cause of death among these patients. Even though allele-specific oligonucleotide real-time quantitative PCR (ASO-qPCR) of immunoglobulin heavy chain gene rearrangement has been used to assess the MRD in MM due to its excellent sensitivity and specificity, ASO-qPCR has a major limitation in its relative quantification method because it needs a reference standard curve, which is usually made with dilutions of diagnostic myeloma DNA or from plasmids containing the target IgH rearrangement gene. Recently, droplet digital PCR (ddPCR) was developed to perform reliable absolute quantification of target genes. Based on the principle of single target gene detection, the sensitivity can be increased when the larger amount of DNA is analyzed. Here we assessed the prognostic value of MRD assessment in autografts from MM patients in the autologous stem cell transplantation (ASCT) setting using ASO-qPCR, ddPCR and next-generation sequencing (NGS) approaches.Methods: Twenty-three Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT without any post-ASCT therapy were retrospectively analyzed. Median age 57 (range 39-67); males 11, females 12; ISS 1 (n=7), 2 (n=12), 3 (n=3), not assessed (n=1). 11 patients were analyzed by G-banding and FISH (t(4;14), del17p, t(14;16)) and 4 patients showed high-risk chromosomal abnormalities (t(4;14) (n=2), t(14;16) (n=1), -13 by G-banding (n=1)). All patients had achieved a very good partial response (VGPR) or better after ASCT. Analyzed samples included: (1) BM slides from 20 MM patients at diagnosis, (2) fresh/frozen BM cells from 3 MM patients at diagnosis, and (3) obtained autografts. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). ddPCR was performed by the QX200 Droplet Digital PCR system (Bio-RAD Inc.) with a total 6000 ng of genomic DNA combined with the same ASO-primers and TaqMan-probes used in the ASO-qPCR. Droplets were generated by the QX200 droplet generator. End-point PCR (40 cycles) was performed on a C1000 Touch Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX200 droplet reader and analyzed by QuantaSoft 1.7.4 (Bio-Rad Inc). NGS-based MRD assessment was performed using the immunosequencing platform (Adaptive Biotechnologies, South San Francisco, CA) (Martinez-Lopez et al Blood 2014).Results: Nineteen patients could be analyzed by ASO-qPCR and ddPCR, while all 23 patients could be analyzed by NGS. We compared MRD results in autografts between ddPCR and NGS. We observed a high correlation between ddPCR and NGS results of MRD (r=0.82, P<0.0001). Although 19 samples were MRD negative by ASO-qPCR (MRDASO (-)), 12 samples were MRD positive by NGS (0.6-46 x 10-6, median 3.5 x 10-6) (MRDNGS (+)) and 7 samples were MRD positive by ddPCR (1.2-102 x 10-6, median 7.5 x 10-6) (MRDddPCR (+)), demonstrating the higher sensitivity of NGS (10-6 or higher) and ddPCR compared to ASO-qPCR (10-4 -10-5). Two high-risk chromosomal abnormality cases (t(4;14) (n=1), -13 by G-banding (n=1)) could achieve MRDNGS (-) in autograft. We evaluated the association of clinical outcome with MRD in autografts using ddPCR and NGS. To investigate the value of sensitive MRD detection by ddPCR and NGS, we compared PFS in 7 MRDddPCR (+) cases (Group 1) with 12 MRDddPCR (-) cases (Group 2) and 12 MRDNGS (+) cases (Group 3) with 11 MRDNGS (-) (Group 4). Group 2 and Group 4 showed significantly better PFS than Group 1 (P = 0.028) and Group 3 (P=0.033), respectively (Figure 1A and 1B). We also compared 5 MRDddPCR (-) & MRDNGS (+) cases (Group 5) with 11 MRDNGS (-) cases (Group 4). Group 4 tended to show better PFS than Group 5 (P=0.063, Figure 1C).Conclusions: In this study, we showed the prognostic value of ddPCR and NGS-based MRD assessment in autografts of patients with MM. Although the ddPCR had improved sensitivity in detecting MRD in autografts and demonstrated higher prognostic value compared with ASO-qPCR, the NGS platform showed the highest sensitivity and prognostic value among these methods. [Display omitted] DisclosuresZheng:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder.

Impact of glucocorticoid receptor gene (NR3C1) polymorphisms in Turkish patients with metabolic syndrome

The metabolic syndrome (MetS) is characterized by a cluster of metabolic factors, including insulin resistance and type-2 diabetes, abdominal obesity, dyslipidemia, hypertension and microalbuminuria. Impaired glucocorticoid receptor (GR) activity also plays an important role in the etiology of MetS. The objective of our study is to evaluate the effects of GR gene polymorphisms (BclI, N363S, TthIII1 and ER22/23EK) in Turkish patients with MetS. Seventy subjects with MetS and 185 healthy controls were enrolled in the study. PCR-RFLP analysis was used for genotyping. Results for each polymorphism have been verified by allele-specific oligonucleotide analysis. BclI GG genotype was significantly associated with an increased risk of MetS (p = 0.02). Also, only in women, the G allele carriers were significantly associated with higher C-peptide. T allele carriers of TthIII1 polymorphism were significantly associated with higher C-peptide, triglyceride, insulin and C-reactive protein (CRP, p value 0.048, 0.022, 0.005 and 0.022, respectively), and lower fasting blood glucose (FBG, p = 0.02). The combined carriers of BclI polymorphism G allele and TthIII1 polymorphism T allele were significantly associated with higher diastolic blood pressure in all patients, and lower FBG and postprandial blood glucose in only men. All the ER22/23EK polymorphisms coexisted with polymorphic variant of TthIII1 (p = 0.0058). The presence of homozygote polymorphic variant of BclI might be good predictive markers for the disease susceptibility. The BclI and the TthIII1 polymorphism are associated with sex-specific clinical parameters. Our findings also suggest that the combination of BclI and TthIII1 polymorphisms may play a protective role in blood glucose.

Reaching Beyond Overall Response in Patients With Multiple Myeloma

The refinement of transplantation procedures and the development and application of novel agents has improved outcomes for patients with multiple myeloma, resulting in a shift of focus from disease stabilisation to attainment of complete response (CR). More recently, depth of response has been shown to exhibit prognostic significance in multiple myeloma; studies have reported significantly longer progression-free survival (PFS), overall survival, time to progression, and duration of response in patients who achieve CR compared with those who do not. This has been demonstrated in newly diagnosed disease (transplant-eligible and -ineligible patients), progressive or relapsed/refractory multiple myeloma, and following a variety of interventions. As increasingly effective therapies allow more patients to achieve deeper responses, new classifications have been endorsed: stringent CR, immunophenotypic CR, and molecular CR. However, even patients who respond to therapy may go on to relapse, possibly due to the presence of minimal residual disease (MRD) undetected by morphological assessment of bone marrow alone. The three main assays for detecting MRD in multiple myeloma are multiparameter flow cytometry (MFC), allele-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-PCR), and high-throughput sequencing. While these techniques differ in applicability, sensitivity, specificity, ease of use, and turnaround time, concordance rates between high-throughput sequencing and MFC of 83% and between high-throughput sequencing and ASO-PCR of 85% have been reported. The presence or level of MRD has been shown to predict outcomes in patients who have responded to therapy, including those who have achieved a CR. In the Myeloma IX trial, a significantly longer PFS was observed among intensively treated patients who were MRD-negative (by MFC) versus patients who were MRD-positive (median 28.6 vs 15.5 mo.; p<0.001); this was also true in patients who achieved CR (34.3 vs 14.1 mo.; p=0.007). In a prospective study in Spain, elderly patients who were ineligible for transplantation who attained an immunophenotypic CR (by MFC) also exhibited superior PFS versus patients who attained stringent or conventional CR. With increasing evidence of the prognostic implications of depth of response and improved detection techniques, eliminating MRD may become the new goal of myeloma therapy.

Polymorphisms in ERCC1 and ERCC2/XPD genes and carcinogen DNA adducts in human lung.

In this exploratory study, we aimed to investigate whether polymorphisms in excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2/xeroderma pigmentosum group D (ERCC2/XPD) in the nucleotide excision repair (NER) pathways associated with DNA adducts in human lung tissue. We also analyzed the association stratified by the major histologic subtypes of non-small cell lung cancer (NSCLC): adenocarcinoma (ADC) and squamous cell carcinoma (SQCC). The study population consisted of 107 early stage NSCLC patients from the Massachusetts General Hospital (MGH) in Boston who underwent curative surgical resection. Genotyping was completed for SNPs in ERCC1 [C8092A (rs3212986) and C118T (rs11615)] and ERCC2/XPD [Asp312Asn (rs1799793) and Lys751Gln (rs1052559)] using a PCR-RFLP method and the PCR with fluorescent allele-specific oligonucleotide probes (Taqman). DNA adduct levels were measured as relative adduct levels per 10(10) nucleotides by (32)P-postlabeling in non-tumor lung tissue. After adjusting for potential confounders, lung DNA adduct levels increased by 103.2% [95% confidence interval (CI), -11.5 to 366.6] for ERCC2/XPD rs1799793AA genotype compared with their corresponding wild type homozygous genotypes in overall NSCLC, but the difference did not reach statistical significance. When we stratified by the subtypes of NSCLC, we found that DNA adducts levels in lung increased by 204.9% (95% CI, 0.8 to 822.2, P=0.059) for ERCC2/XPD rs1799793AA genotype in subjects with SQCC and the trend was statistically significant (P for trend=0.0489). Polymorphisms in ERCC2/XPD Asp312Asn may be associated with increased DNA adduct levels in the lung, especially among subjects with SQCC. Further large scale studies are needed to confirm our findings.

Large cohort screening of G6PD deficiency and the mutational spectrum in the Dongguan District in Southern China.

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzymatic disorder of the erythrocytes that affects 400 million people worldwide. We developed a PCR-reverse dot blot (RDB) assay to screen twenty genotypes of seventeen Chinese G6PD mutations and investigate the spectrum of G6PD deficiency mutations in Dongguan District, Guangdong Province, in southern China.MethodThe PCR-RDB assay consists of multiplex PCR amplification of seven fragments in the G6PD target sequence of wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. A total of 16,464 individuals were analyzed by a combination of phenotypic screening and genotypic detection using the PCR-RDB assay and DNA sequence analysis.ResultsThe PCR-RDB assay had a detection rate of 98.1%, which was validated by direct sequencing in a blind study with 100% concordance. The G6PD deficiency incidence rate in Dongguan District is 4.08%. Thirty-two genotypes from 469 individuals were found. The two most common variants were c.1376G>T and c.1388G>A, followed by c.95A>G, c.871G>A, c.392G>T, and c.1024 C>T. In addition, two rare mutations (c.703C>A and c.406C>T) were detected by DNA sequencing analysis. In our study, 65 cases harbored the C1311T/IVS polymorphism and 67 cases were homozygote.ConclusionThe PCR-RDB assay we established is a reliable and effective method for screening G6PD mutations in the Chinese population. Data on the spectrum of mutations in the Dongguan District is beneficial to the clinical diagnosis and prevention of G6PD deficiency.

Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients

Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis.

96-POS

Objectives VEGF is key component in the differentiation, invasion, regulation of vascular remodeling and the survival of cytotrophoblasts. In preeclampsia expression of cytotrophoblasts is disregulated. Evidence supports the hypothesis that VEGF plays an important role in the pathogenesis of preeclampsia. In this study, we aimed to determine whether VEGF genetic polymorphisms are associated with risk of early onset of severe preeclampsia. Methods A cross-sectional study with observational analytic was conducted in 32 pregnant women with early onset severe preeclampsia and 32 normal pregnant women. After peripheral blood was obtained from all women and genomic DNA was isolated, we genotyped +405G>C polymorphisms in the 5′- untranslated region and +936C>T polymorphisms in 3′-untranslated region of the VEGF gene genotype using Allelic Specific Oligonucleotide–Polymerase Chain Reaction (ASO–PCR) methods. Results The distribution of G allele of VEGF +405 more dominant (60.9%) than C allele (39.1%) in early onset severe preeclampsia. Conversely in normal pregnant women, the frequency of C allele of +405 (53.1%) little bit higher than G allele (46.9%). The distribution of the T allele of +936 approximately was the same between early onset severe preeclampsia (35.9%) and normal pregnant women (31.3%). Overall, the genotype and allele frequency distribution of the two polymorphisms (+405 and +936) were not significantly different between women with and without the disease ( p > 0.05). Conclusions There was no evidence of an association between VEGF allele or genotype frequencies and early onset preeclampsia in our study. VEGF gene polymorphisms studied are unlikely to be major predisposing for early onset severe preeclampsia in the Indonesian population. Disclosures M. Susanna: None. E.G. Dachlan: None.

MYD88 (L265P) Mutation Confers Very Poor Response and Outcome after Second-Line Therapy in Patients with Diffuse Large B-Cell Lymphoma (DLBCL)

BACKGROUNDSomatic mutations in the myeloid differentiation primary response gene 88 (MYD88) have been described in B-cell lymphomas, including DLBCL. Our group has confirmed the remarkable site-specific occurrence of MYD88 mutations at some immune-privileged locations and, in addition, has described that DLBCL patients are at high risk of progression and death after first-line treatment, with independence of other well-known clinical prognostic factors (Leukemia 2014).AIMSTo further analyze the clinical responsiveness and outcome after second-line treatment in DLBCL patients carrying MYD88 L265P.METHODSA series of 175 patients with DLBCL diagnosed at our institution between 2000 and 2013 were included. Inclusion criteria were: full clinical data available, treatment with remission intention, enough material for DNA extraction and absence of HIV infection. The presence of MYD88 L265P mutation was assessed by allele-specific oligonucleotides (ASO)-PCR in DNA samples extracted from FFPE tissue.RESULTSIn 129 patients (74%) treatment consisted on rituximab plus CHOP or CHOP-like schedules, while the remaining cases received other treatments depending on their clinical requirements. With a median follow-up time of 57 months, progression free survival (PFS) at 5 years after first-line treatment was 57%. Twelve of these patients (9.3%) had MYD88 L265P mutation, and these patients had a significantly worse PFS that patients who did not (18% vs 59%, p=0.018). Overall survival (OS) at 5 years after first line treatment was 65% (17% vs 72%, with vs without MYD88 mutation, respectively; p=0.001).We further analyzed the outcome after second-line treatment in 32 cases: 6 patients had refractory disease (0 with MYD88 mutation) and 26 patients were in first relapse. Three out of 5 patients carrying MYD88 mutation and 25 out of 27 patients without MYD88 mutation were treated with chemotherapy. Only one out of 5 cases (20%) with MYD88 mutation responded to second-line therapy whereas response was observed in 16 out of 27 cases (59%) without MYD88 mutation(p=0.106). PFS at 4 years from starting second-line therapy was 0 % in cases with MYD88 DLBCL and 34% in those without MYD88 mutation (p=0.092). Moreover, OS at 4 years from starting second-line therapy was 0 % in cases with MYD88 DLBCL and 37% in those without MYD88 mutation (p=0.019)(Figure 1).CONCLUSIONOur study shows that MYD88 L265P in DLBCL patients is not only associated to worse respond after first -line therapy, but also to a very poor response to second-line therapy, and consequently to a dismal outcome. New treatments are urgently needed for these patients.Acknowledgments: This study was supported in part by 2014SGR567 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic Value of Sequencing-Based Minimal Residual Disease Detection in Multiple Myeloma

▪Background: Although molecular complete remission (mCR) in multiple myeloma (MM) can be assessed by allele-specific oligonucleotide (ASO)-PCR, this technique requires preparation of clonotype-specific primers for each individual, which is laborious and time-consuming. We utilized the LymphoSIGHTTM platform, which employs consensus primers and next-generation sequencing (NGS) to amplify and sequence all rearranged immunoglobulin gene segments present in a myeloma clone, to assess mCR. This technique has been shown to have 1-2 logs greater sensitivity compared to ASO-PCR and flow cytometry, respectively (Faham et al, Blood 2012). Usage of the sequencing method for minimal residual disease (MRD) detection in MM may provide increased sensitivity and specificity, while overcoming the challenges associated with ASO-PCR. We compared the LymphoSIGHTTM method with ASO-qPCR for MRD detection in autografts and bone marrow (BM) in the autologous peripheral blood stem cell (PBSC) transplantation (ASCT) setting.Methods: One hundred and nine Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients had achieved a partial response (PR) or better after ASCT. BM slides from 84 MM patients and fresh/frozen BM cells from 25 MM patients at diagnosis, as well as autografts/post-ASCT BM cells from each patient, were obtained for DNA extraction. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments, IGH-DJ, and IGK from genomic DNA. Amplified products were subjected to deep sequencing using NGS. Reads were analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency in BM samples.Results : Myeloma clonotypes could be identified in autografts/post-ASCT BM cells in 98 of 109 patients (90%) and by ASO-qPCR in 63 of 101 patients (62%). MRD by NGS was assessed in autografts of 89 patients. 70 of 89 patients (79%) were positive by NGS; 28 of 62 patients (45%) were positive by ASO-qPCR. Although we observed a high correlation between NGS and PCR MRD results at MRD levels of 10-5 or higher, the sensitivity of ASO-PCR was 10-4-10-5, whereas that of NGS was 10-6 or lower when a sufficient amount of DNA was available for analysis. Eight cases where MRD was not detected in the autograft by NGS (MRDNGS(-)) and 38 MRDNGS(+) cases received post-ASCT therapy using novel agents such as bortezomib/lenalidomide/thalidomide, while 11 MRDNGS(-) cases and 32 MRDNGS(+) cases were followed without post-ASCT therapy. The MRDNGS(-) cases without post-ASCT therapy showed significantly better progression-free survival (PFS) than the MRDNGS(+) cases without post-ASCT therapy (P = 0.012) (Figure 1A) although overall survival rates were comparable between these groups. To investigate the value of sensitive detection by NGS, we compared PFS in 11 MRDNGS(-) cases (Group 1) with the 12 MRDNGS(+) cases where MRD was not detected by ASO-qPCR (MRDASO(-)) (Group 2). The patients in both groups did not receive any post-ASCT therapy. Group 1 showed significantly better PFS than Group 2 (P = 0.027) (Figure 1B). Furthermore, 9 MRDNGS(-) in post-ASCT BM cases tended to show a better PFS than 18 MRDNGS(+) in post-ASCT BM cases (P = 0.075) (Figure 1C). In a multivariate analysis, post-ASCT therapy using novel agents (P <0.001) and MRDNGS(-) in autograft (P=0.025) were independently associated with superior PFS while ISS I/II vs III (P = 0.387) and MRDASO(-) in autograft (P=0.174) were not.Conclusions: In this study, we showed the prognostic value of MRD detection using the NGS-based LymphoSIGHT platform in autografts of patients with MM who received and in those who did not receive post-ASCT therapy with novel agents. The NGS platform has improved sensitivity compared with ASO-qPCR in detecting MRD in autografts. Patients with low level MRD detected by NGS but not by ASO-qPCR have worse prognosis compared to patients who are MRD negative by sequencing, which underscores the need for sensitive detection. [Display omitted] DisclosuresZheng:Sequenta, Inc.: Employment. Moorhead:Sequenta, Inc.: Employment. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

A Novel Method for the Detection of Minimal Residual Disease in B-Cell Malignancies: Ion Semiconductor Sequencing for the Evaluation of Igh Gene Rearrangements

Background:Minimal residual disease (MRD) detection is of high clinical relevance in patients with B-cell malignancies and is generally a surrogate parameter to evaluate treatment response and long-term prognosis. IgH gene rearrangements can be used as molecular marker in approximately 80% of lymphoma and myeloma patients since they represent lineage-specific markers and the complementarity determining region 3 (CDR3) is unique to each clone. To date, allele specific oligonucleotide polymerase chain reaction (ASO-PCR) and real-time quantitative polymerase chain reaction (RQ-PCR) are considered the most sensitive and widely applicable methods for MRD detection. A major disadvantage of ASO-PCR and RQ-PCR assays, is the use of specific primers and probes for every individual patient. Clone-specific primers and probes are not only expensive but also time-consuming to design and to test, which limits their wide applicability in the clinical setting. The recent major improvements in next generation sequencing (NGS) technologies, provide the opportunity to identify and quantify clonotypes with consensus primers combining the benefits of high sensitivity and universal applicability. The present work was designed to overcome ASO-PCR and RQ-PCR limitations by developing a feasible method for rearranged IgH genes amplification, NGS and analysis using Ion Torrent Personal Genome Machine (IT-PGM).Methods:To define a multiplex PCR protocol, DNA from 7 CLL patients, previously shown to bare a family specific clonal VDJ rearrangement, was amplified with a pool of the seven different family-specific IgH-V primers, and a consensus JH primer (Voena et al., Leukemia 1997). After Sanger sequencing, results were compared to the ones obtained with singleplex PCR protocol. Once validated, the multiplex PCR protocol was used to amplify DNA from patients and serially diluted (up to 10-8 ) DNA from Namalwa cell line (bearing a known IgH rearrangement) and subsequently sequenced on the IT-PGM using the 316 Ion-chip. NGS data were analyzed by using the IMGT-High V-Quest web server tool and the statistical software R. RQ-PCR was used to quantify the specific VDJ rearrangement in the serially diluted Namalwa DNA solutions and in DNA from patients as previously described (Farina et al, Haematologica 2009). RQ-PCR data were analyzed through a relative quantification procedure.Results:The multiplex PCR reactions we have tested, demonstrated the same specificity as the standard singleplex PCR protocol and therefore was used to construct the DNA library required for IT-PGM-based sequencing. The IT-PGM sequencing output is represented by at least 400000 reads per sample with a minimum average coverage of the VDJ repertoire of 500x. The IMGT-High V-quest tool allows a user-friendly web based analysis and a deep molecular characterization of the IgH recombinatorial repertoire. Namalwa clonal CRD3 sequences were detected up to a dilution of 10-5 without the need for specific CDR3 primers. Comparability of NGS and ASO RQ-PCR results was assessed. The use of CDR3 specific primers, along with the specific IgH-V family fluorescent probe, enabled the identification of clonal VDJ rearrangements with a sensitivity up to 10-5 (2/3 replicates) and 10-6 (just 1/3 replicates) in Namalwa Cell Line. Similar results were obtained when we characterized the IgH recombination repertoire of two CLL patients over time.Conclusions:IgH sequencing with the IT-PGM platform showed at least the same level of sensitivity as ASO RQ-PCR, without the need for patient-specific reagents. It also allows specific and detailed molecular characterization of the clonal rearrangements and could be easily incorporated into clinical laboratories for routine testing of MRD in B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Minimal Residual Disease Detection Using High-Throughput Sequencing Predicts Clinical Outcome in Patients with Pediatric B-Lineage Acute Lymphoblastic Leukemia

Background: Minimal residual disease (MRD) assessment at the end of induction is critical for risk stratification of B-lineage acute lymphoblastic leukemia (B-ALL) patients. There are multiple techniques for molecular MRD assessment, including next-generation sequencing (NGS) and allele-specific oligonucleotide PCR (ASO-PCR) amplification of immunoglobulin (Ig) or T-cell receptor rearrangements. Leukemia-specific rearrangements can be identified in >97% of B-ALL patients using the NGS-based LymphoSIGHT™ method, while ASO-PCR can typically identify leukemia-specific rearrangements in only 80-90% of B-ALL patients. In this report, we assessed the ability of the NGS method to predict event-free survival (EFS) in a cohort of B-ALL patients who were inevaluable by ASO-PCR.Methods: All patients were treated between 2005-2011 on a clinical trial conducted by the Dana-Farber Cancer Institute (DFCI) ALL Consortium (05-001) on which high levels of end-induction MRD (≥10-3) led to intensified treatment. The 37 pediatric B-ALL patients included in this study were inevaluable by ASO-PCR, and therefore this cohort represents a subset of patients in whom a decision to intensify therapy per protocol could not be made. Archived DNA samples from diagnosis and Day 32 were provided for MRD assessment using the NGS method. Using universal primer sets, we amplified the immunoglobulin (IGH-VDJ, IGH-DJ and IGK) and T cell receptor (TRB, TRD, TRG) loci in diagnostic bone marrow samples. Leukemia-specific clonotypes were identified in the diagnostic sample of each patient based on high-frequency within the B-cell and/or T-cell repertoire. The presence of leukemia-specific clonotypes was then assessed in the end-induction (Day 32) sample. NGS MRD assessment was performed blinded to all clinical and ASO-PCR information on these patients. The association of NGS MRD results and clinical outcome (EFS) was examined, and significance assessed using the log-rank test.Results: Of 37 patients who were inevaluable for end-induction MRD by ASO-PCR, leukemia-specific clonotypes were successfully identified in 35 (95%) by the NGS method. The use of a threshold of 10-3, which is the ASO-PCR threshold used for risk-stratification on DFCI protocols, demonstrated significantly improved EFS between the MRD negative and MRD positive groups (p<0.0001) (Figure 1A). A similar analysis with a threshold of 10-4 also demonstrated significantly improved EFS between the MRD negative and MRD positive groups (p<0.0001) (Figure 1B).Conclusions: This study demonstrates the prognostic significance of NGS-based MRD assessment in pediatric patients with B-ALL. This NGS-based assay led to successful assessment of MRD in 95% of patients who were inevaluable by ASO-PCR. The NGS method represents an alternative approach for clinical MRD monitoring that could fundamentally improve the ability to identify high-risk B-ALL patients and potentially improve their outcome. [Display omitted] [Display omitted] DisclosuresZheng:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Clinical Meanings of Quantitative PCR of Patient-Specific Immunoglobulin VDJ Regions Using Various Materials of Myeloma Patients

Introduction; The PCR with patient-specific, allele-specific oligonucleotide primers for individual IgH VDJ regions (ASO-PCR) is considered the most sensitive method to detect minimal residual disease (MRD) levels in patients with multiple myeloma (MM). In this study, we quantified the ASO-PCR products by using peripheral blood mononuclear cells (PBMNCs) as well as bone marrow mononuclear cells (BMMNCs). We also quantified the ASO-PCR products in mRNAs from CD20+38- B-cells in BM to examine whether there are clonogenic cells in relatively earlier B-cell fraction as well as cell-free DNAs from the sera to examine whether there are DNA fragments from MM cells in PB.Materials and methods; We have analyzed 30 MM cases. After an informed consent, patient-oriented PCR primers were designed from sequence information of immunoglobulin heavy chain (IgH) variable regions of myeloma cells of each patient.Results; The median age in this cohort was 64.5 years (36-83); the ratio of men to women was 16:14; and the numbers of IgG-, IgA-, and Bence Jones protein (BJP)- type patients were 18, 7, and 5, respectively. The patient-specific ASO primers could be designed in 25 cases, but not in 3 BJP-type cases or 2 IgG-type cases. We could quantify the ASO-PCR products in 20 of 30 BMMNCs samples at diagnosis. The ASO-PCR levels (IgH/β−actin levels)in BMMNCs correlated with those in PBMNCs, but not to the percent of plasma cells in BM or the values for M-protein. However, the ASO-PCR levels were decreased after the treatment and reflected tumor burden well individually. The ASO-PCR levels in PBMNCs showed a statistically significant correlation with those in BMMNCs at diagnosis (Spearman’s ρ= 0.98, P<0.001), 6 months (Spearman’s ρ= 0.83, P = 0.020) and 12 months (Spearman’s ρ= 1.90, P = 0.001). Therefore, ASO-PCR using PBMNCs as well as BMMNCs is suitable for MRD evaluation. We could detect the patient-specific IgH DNA sequences in cell-free DNA extracted from the sera and quantify the ASO-PCR products. The sequences of the ASO-PCR product were identical to the originally designed sequence, suggesting that detection of the ASO-PCR products in cell-free DNA could reflect the persistence of myeloma cells somewhere in body. The ASO-PCR products for CD20+CD38- B-cells in BM were relatively low but were clearly detected in 17 cases at diagnosis. The ASO-PCR levels in CD20+CD38- B-cells in BM showed good correlation with both values of ASO-PCR in BMMNCs (Spearman’s ρ= 1.35, P <0.001) and in PBMNCs (Spearman’s ρ= 1.09, P <0.001). Thus, the evaluation of relatively earlier B-cell stages of myeloma cells seems to be of interst, including for the possible existence of MM clones in CD20+CD38- B-cell population in BM.Discussion; Our findings that we could quantify the ASO-PCR products in PBMNCs, BM CD20+CD38- B-cells, cell-free DNA as well as BMMNCs strongly suggest wide sources of clinical materials to analyze. There were statistically significant correlations in values of the ASO-PCR products between BMMNCs and PBMNCs, therefore suggesting the possibility that clonogenic plasma cells or myeloma precursor cells might circulate in peripheral blood. We expect the possibility that PBMNCs will be a good source to monitor MRD. We could also detect and quantify the ASO-PCR products in cell-free DNA from the sera. To the best of our knowledge, this is the first report that IgH DNA fragments from MM cells circulate in the sera. Furthermore, we could detect and quantify the ASO-PCR products in CD20+CD38- B-cells in BM at diagnosis. These results indicated that clonogenic MM cells could exist not only in the CD20-CD38high plasma cell fraction but also in the CD20+CD38- B-cell fraction, which might include myeloma stem or initiating cells. Thus, we could consider the treatment strategies to include anti-CD20 antibodies against the clonogenic CD20+CD38- B-cell population. In conclusion, our ASO-PCR using various clinical materials is supposed to be useful for detecting MRD in the patients with MM as well as for clarifying the pathogenesis of MM. DisclosuresSata:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Shibayama:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Habuchi:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Fukushima:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Fujita:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Ezoe:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Tadokoro:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Maeda:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Mizuki:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Oritani:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Kanakura:Janssen: Research Funding; Takeda: Research Funding; Celgen: Research Funding.

Complete Clinical, Histopathologic and Molecular Remission of Primary Myelofibrosis with Long-Term Treatment with the JAK1/2 Inhibitor Ruxolitinib

The JAK1/2 inhibitor ruxolitinib (RUX) is effective in decreasing symptomatic splenomegaly, and constitutional symptoms in patients with myelofibrosis (MF). Long-term data suggest that treatment with RUX is associated with a survival benefit, and may delay/reverse bone marrow fibrosis. Further, a ≥ 20% reduction in JAK2 V617F allele burden with RUX has been described (Vannucchi et al. Blood. 2012). Yet, many uncertainties surrounding the full clinical potential of RUX persist. The conceivable disease-modifying impact including the achievement of a JAK2 V617F molecular remission under sustained JAK1/JAK2 inhibition in a patient with primary myelofibrosis (PMF) is presented.Patient and Methods: the diagnosis of JAK2 V617F-positive PMF according to the 2008 WHO criteria was made in a 50-years old caucasian male in February 2009. On the first consultation in November, 2009, he complained of fatigue and night sweats. Spleen was 12 cm below costal margin by palpation. Laboratory results were: Hb 153 g/L, WBC 18.9 x109/L, platelets 268 x109/L, peripheral blasts 2%, a leucoerythroblastic blood picture, LDH 10.6 µkat/L [2.8x upper limit of normal]. With an IPSS of 2 points (night sweats and peripheral blasts > 1%) the IPSS risk category for survival was intermediate-II. After signing informed consent, the patient was included in the phase 3, multicenter COMFORT-II trial and randomized to treatment with RUX at a dose of 20mg bid based on platelets count. By week (w) 4, the dose was increased to 25mg bid per protocol (< 40% reduction is palpable spleen length). For efficacy and safety evaluations, serial clinical, spleen volume [by magnetic resonance imaging (MRI)], blood picture, blood chemistry, bone marrow trephine biopsy, and JAK2 V617F allele burden from both peripheral blood samples as well as bone marrow assessments were conducted. The histology of the biopsies was evaluated according to the WHO criteria by experienced pathologists (Table). The JAK2 V617F allele burden was measured from blood samples using allele-specific oligonucleotide quantitative real-time polymerase chain reaction (qPCR) with a dynamic detection range from 0.1 to100% mutated allele (Lange et al. Haematologica 2013) and from bone marrow samples via qPCR followed by pyrosequencing for determining the allelic burden with a sensitivity of 5% of mutated DNA. If necessary, blocking of the non-mutated allele for sensitivity enhancement from 5% up to 0,001% of mutated DNA was done (Siebolts et al. J Clin Pathol 2010).Results: After a follow-up of 240 weeks, treatment with 25mg bid is ongoing. No dose modification/interruption because of adverse events was required. Clinical response was rapid with a 52% reduction in baseline spleen volume from 3.77 L to 1.8 L and improvement in constitutional symptoms at w 12. This was followed by a further decline in spleen volume. By w 216, spleen volume was 0.5 L which corresponded to 85.5% reduction from baseline (Figure). WBC and LDH normalized by w 24 and w 84 respectively. Histologic improvement in marrow cellularity, megakaryocytic, and granulocytic lineages was first evident at w 126 with further reversal of MF-related abnormalities including marrow fibrosis by w 216 (Table). JAK2 V617F allele burden of < 10% in a peripheral blood sample and < 1% in the bone marrow were first documented at w 108 and w 132 respectively. As of w 216, an allele burden <1% was sustained in both blood and marrow samples (Figure). Conclusions: The rapid symptomatic improvement with ruxolitinib could be followed by a considerable MF-modifying response with the potential to alter the course of disease and prognosis. Unlike targeted therapy for chronic myeloid leukemia, ruxolitinib-delivered molecular and histologic remissions could be gradual, and progressive. Whether such deep long-term responses are dose-dependent is not yet known. Thus, the full potential of a sustained JAK1/JAK2 inhibition needs to be elucidated by carefully analyzing the possible correlation between dose, short-term spleen responses and long-term molecular and histologic responses; both in retro- and prospective studies.Changes in marrow histology and spleen volume under ruxolitinib Table 1Marrow Cellularity %MegakaryocyteGranulocytic ProliferationG:EMarrow Fibrosis (EUMNET)Spleen volume (L)ProliferationAtypiaBaseline100%+++++++++8:1PMF13.8Week 4880%+++++++5:1PMF21.2Week 12630%+++-1:3PMF10.6Week 21640%-+-3:1PMF00.5 [Display omitted] DisclosuresAl-Ali:Novartis: Honoraria, Research Funding. Lange:Novartis: Consultancy, Honoraria, Research Funding. Prashanth:Novartis: Employment. Niederwieser:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Gentium: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau.

The UCP2 -866G>A promoter region polymorphism is associated with nonalcoholic steatohepatitis.

Uncoupling protein 2 - UCP2 - regulates mitochondrial lipid fluxes and reactive oxygen species production by the respiratory chain. The -866 G>A UCP2 promoter region polymorphism has been linked to insulin resistance and lipid metabolism. The aim of this study was to assess whether the -866 G>A UCP2 polymorphism predisposes to nonalcoholic steatohepatitis in patients at risk, and the relationship with lipid metabolism and hepatic UCP2 expression. We considered 688 Italian patients who underwent liver biopsy for suspected NASH, and 232 healthy controls. The UCP2 -866 G>A polymorphism was determined by allele specific oligonucleotide probes, hepatic UCP2 mRNA levels by quantitative real-time PCR. UCP2 A/A genotype was associated with a reduced risk of nonalcoholic steatohepatitis (Odds Ratio 0.49, 95% C.I. 0.26-0.90; P = 0.02; adjusted for age, sex, BMI, impaired fasting glucose or diabetes, PNPLA3 I148M alleles and recruitment centre). The A/A genotype was associated with reduced risk of steatosis grade G2-G3 and nonalcoholic steatohepatitis in patients without (P = 0.003 and P = 0.01 respectively), but not in those with (P = NS) impaired fasting glucose/diabetes. The UCP2 A/A genotype was associated with higher hepatic UCP2 mRNA levels (adjusted P = 0.008). Concerning the metabolic traits, the UCP2 A/A genotype was associated with higher total serum cholesterol levels (adjusted P = 0.03), but not with serum HDL, triglycerides or impaired fasting glucose/diabetes. UCP2 -866 A/A genotype is associated with increased hepatic UCP2 expression and reduced risk of nonalcoholic steatohepatitis, particularly in subjects with normal fasting glucose.

Fast and low-cost decentralized surveillance of transmission of tuberculosis based on strain-specific PCRs tailored from whole genome sequencing data: a pilot study

Molecular epidemiology has transformed our knowledge of how tuberculosis (TB) is transmitted. Whole genome sequencing (WGS) has reached unprecedented levels of accuracy. However, it has increased technical requirements and costs, and analysis of data delays results. Our objective was to find a way to reconcile speed and ease of implementation with the high resolution of WGS. The targeted regional allele-specific oligonucleotide PCR (TRAP) assay presented here is based on allele-specific PCR targeting strain-specific single nucleotide polymorphisms, identified from WGS, and makes it possible to track actively transmitted Mycobacterium tuberculosis strains. A TRAP assay was optimized to track the most actively transmitted strains in a population in Almería, Southeast Spain, with high rates of TB. TRAP was transferred to the local laboratory where transmission was occurring. It performed well from cultured isolates and directly from sputa, enabling new secondary cases of infection from the actively transmitted strains to be detected. TRAP constitutes a fast, simple and low-cost tool that could modify surveillance of TB transmission. This pilot study could help to define a new model to survey TB transmission based on a decentralized multinodal network of local laboratories applying fast and low-cost TRAPs, which are developed by central reference centres, tailored to the specific demands of transmission at each local node.

Molecular and genetic studies of the predisposition to the development of psoriasis among the population of the Russian Federation: a study of polymorphisms of TNFAIP3, TNIP1, TYK2 and REL genes

Goal. To study the role of polymorphic variants of genes encoding proteins of the signaling pathway of the nuclear transcription factor kappa-B, NF-kB (TNFAIP3 (rs610604), TNIP1 (rs17728338), TYK2 (rs12720356) and REL (rs702873)) in the predisposition to psoriasis among the Caucasian population of the Russian Federation. Materials and methods. The authors studied markers of the predisposition to the development of psoriasis such as TNFAIP3 (rs610604), TNIP1 (rs17728338), TYK2 (rs12720356) and REL (rs702873) using Real-Time PCR methods and allele-specific oligonucleotide probes and PFLP (Restriction Fragment Length Polymorphism) assay in whole blood samples taken from psoriatic patients (n = 301). To compare the frequency of genotypes, the authors used data obtained as a result of the genotyping of DNAs taken from healthy volunteers (n = 109). Results. The authors analyzed particular features of the distribution of polymorphic variants of TNFAIP3 (rs610604), TNIP1 (rs17728338), TYK2 (rs1272035) и REL (rs702873) genes in a sample of 301 psoriatic patients and 109 healthy volunteers from the Russian Federation. They revealed reliable differences in the frequency of registration of genotypes of these genes. The authors also determined genotypes considered as risk factors with regard to the development of psoriasis (TNFAIP3-A/C, TNIP1-A/G, TNIP1-A/A and TYK2-T/T) as well as protector genotypes (TNFAIP3-A/Ä, REL-A/A and TYK2-T/G)