Allele-specific Oligonucleotide PCR Research Articles

Association between Genetic Variants Linked to Premature Ovarian Insufficiency and Inflammatory Markers: A Cross-Sectional Study.

Premature menopause (PM) is the cessation of ovarian function before age 40. PM women are more likely to have cardiovascular diseases (CVDs), diabetes, and mental disorders. This is the first study that assessed the association of single nucleotide polymorphisms (SNPs) with anti-heat shock protein 27 (Hsp27), High-sensitivity C-reactive protein (hs- CRP), and PM and serum pro-oxidant-antioxidant balance (PAB), as putative risk factors for CVDs. We aimed to explore the association of oxidative stress markers with eight different SNPs shown to be related to premature menopause. In this cross-sectional research, we included 183 healthy women and 117 premature menopausal women. We determined baseline characteristics for all participants and measured serum hs-CRP, anti-HSP-27 antibody titer, and PAB levels using the established methods. Genotyping for eight SNPs was done using the tetra amplification refractory mutation system polymerase chain reaction (Tetra-ARMS PCR) and allele-specific oligonucleotide PCR (ASO-PCR) methods. We found a significant difference between mean serum PAB levels and the genetic variant of rs16991615 (P=0.03). ANCOVA showed a significant effect of the genotypes rs4806660 and rs10183486 on hs-CRP serum levels in the case and control groups, respectively (P=0.04 and P=0.007). ANCOVA also showed an association between rs244715 genotypes and anti-hsp27 serum levels in the case group (P=0.02). There was a significant effect of the genotypes of rs451417 on the serum hs-CRP level in the control group (P=0.03). There was a significant association of the genetic variants related to PM with oxidative stress and inflammatory markers (serum PAB, anti-hsp27 antibody, and hs-CRP). Accordingly, this seems to be an effective approach to predicting susceptible subjects for cardiovascular and mental disorders as well as various cancers.

Interleukin-10 promoter polymorphisms and haplotypes in patients with Guillain-Barré syndrome.

Interleukin-10 (IL-10) is a multifunctional cytokine that exerts both pro- and anti-inflammatory effects on the immune system as well as in the pathogenesis of Guillain-Barré syndrome (GBS). We investigated whether the three common polymorphisms -1082 G/A(rs1800896), -819 C/T(rs1800871), and -592 C/A(rs1800872) in the promoter region of IL-10 have any influence on the susceptibility, severity, and clinical outcome of GBS. IL-10 promoter polymorphisms were investigated in 152 patients with GBS and 152 healthy controls from Bangladesh using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP), and allele-specific oligonucleotide-PCR (ASO-PCR). Haplotype patterns and frequencies were analyzed using Heatmaply R-package, chi-square, and Fisher's exact test. The serum level of IL-10 was measured through enzyme-linked immunosorbent assays. p-values < 0.05 were considered statistically significant. IL-10 promoter polymorphisms -1082 G/A, -819 C/T, and -592 C/A were not associated with GBS susceptibility. The homozygous -819 TT genotype showed a tendency with susceptibility (p = 0.029; pc = 0.08) and was prevalent in axonal variants of GBS compared to demyelinating subtypes and controls (p = 0.042, OR = 8.67, 95% CI = 1.03-72.97; pc = 0.123 and p = 0.005, OR = 4.2, 95% CI = 1.55-11.40; pc = 0.015, respectively). Haplotype analysis revealed 19 patterns of genotypes and high IL-10 expression haplotype combinations (GCC/GTA, GCC/ATA, and GCC/GCA) may have influence on disease severity (p = 0.026; pc = 0.078). Serum expression of IL-10 was elevated in GBS patients ([GBS, 12.16 ± 45.71] vs. [HC, 0.65 ± 5.17] pg/mL; p = 0.0027) and varied with disease severity ([severe-GBS, 15.25 ± 51.72] vs. [mild-GBS, 3.59 ± 19.79] pg/mL, p = 0.046). The -819 TT genotypes influence axonal GBS, and high frequency of IL-10 expression haplotype combination with elevated serum IL-10 may play an important role in disease severity.

Implications and Prognostic Impact of Mass Spectrometry in Patients with Newly-Diagnosed Multiple Myeloma

Background: The minimally invasive technology mass spectrometry (MS) is emerging as a promising approach for detecting and monitoring monoclonal proteins in the peripheral blood of multiple myeloma (MM) patients. We aimed to define implications and prognostic value of MS in a large cohort of transplant-eligible patients with newly-diagnosed MM treated within the phase 3 multicenter GMMG-MM5 trial (EudraCT No. 2010-019173-16). Patients and Methods: For quantitative immunoprecipitation MS (QIP-MS), we included peripheral blood serum samples from 480 patients at baseline, from 444 of these patients after 3 cycles induction therapy, from 305 patients prior to lenalidomide maintenance treatment (max. 2 years) or observation (in case of complete response [CR] in arm B of the trial), and from 227 patients after 1 year (+/- 3 months) of lenalidomide maintenance / observation. QIP-MS was carried out using the automated EXENT® assays and system (in development; The Binding Site Group Ltd., UK). Minimal residual disease (MRD) in the bone marrow was detected using an allele-specific oligonucleotide (ASO) PCR with a sensitivity of at least 1x10-6. Kaplan-Meier method was used for survival analyses. Progression-free survival (PFS) time was measured from the respective landmark to progression or death from any cause, whichever occurred first. For analyses of sustained MS from start of maintenance / observation until 1 year (+/- 3 months), PFS was calculated from the second time point (after 1 year) and only patients who did not have a prior progression event were included. Results: MS negativity rates increased from 6% post induction to 31% and 44% prior to and after 1 year of maintenance / observation, respectively. MS negativity was associated with improved PFS at all investigated timepoints (post induction: hazard ratio [HR]=0.41; p=0.003), prior to maintenance / observation: HR=0.66, p=0.01 and after 1 year of maintenance / observation: HR=0.37, p<0.001. Next, we evaluated the prognostic value of MS in a multivariable model, which included age at diagnosis, gender, treatment arm, conventional response (CR vs. no CR), revised International Staging System (R-ISS) stages and gain1q21. MS negativity was an independent prognostic factor for PFS (prior to maintenance / observation: HR=0.60, p=0.01, Figure 1A; and after 1 year of maintenance / observation: HR=0.28, p<0.001, Figure 1B). The prognostic impact of MS could be improved by baseline disease features, such as high-risk cytogenetics. Combining the FISH high-risk markers of the R-ISS del(17p13), t(4;14), and t(14;16) as well as gain1q21 with MS before maintenance / observation, patient groups with excellent or dismal outcomes could be defined. The best outcome was seen for patients without high-risk cytogenetics and MS negativity (median PFS: 4.8 years), while patients with high-risk FISH and MS positivity had a median PFS of just 1.9 years from start of maintenance / observation. Next, we aimed to determine the prognostic value of sequential MS testing and chose the time points prior to maintenance / observation and after 1 year. The best outcome was seen for patients who converted from MS positivity to negativity (median PFS not reached). The worst outcome was seen for patients who converted from MS negativity to positivity with a median PFS of only 0.6 years. Sustained MS negativity was associated with improved PFS (median PFS: 3.5 years) as compared to sustained MS positivity (median PFS: 1.9 years, p=0.007). In a pilot study, we combined MS with ASO-PCR for detection of MRD in the bone marrow in 45 patients with suspected CR. Double (MS / MRD) positive patients had the worst outcome (median PFS: 2.2 years) whereas double-negative patients had a median PFS of 3.3 years and no overall survival events. Conclusions: Our study provides strong evidence that MS is superior to conventional response monitoring and should become a new standard to monitor monoclonal protein in MM. We recommend performing MS testing sequentially and combined with baseline disease features to improve prognostication. Disease relapse in MS / MRD double-negative patients in our study probably occurred due to limited duration of lenalidomide maintenance or observation, but also highlights that even a combined approach with a highly sensitive bone-marrow MRD tool was not sufficient to identify disease-free MM patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Digital Droplet PCR in Hematologic Malignancies: A New Useful Molecular Tool.

Digital droplet PCR (ddPCR) is a recent version of quantitative PCR (QT-PCR), useful for measuring gene expression, doing clonality assays and detecting hot spot mutations. In respect of QT-PCR, ddPCR is more sensitive, does not need any reference curve and can quantify one quarter of samples already defined as “positive but not quantifiable”. In the IgH and TCR clonality assessment, ddPCR recapitulates the allele-specific oligonucleotide PCR (ASO-PCR), being not adapt for detecting clonal evolution, that, on the contrary, does not represent a pitfall for the next generation sequencing (NGS) technique. Differently from NGS, ddPCR is not able to sequence the whole gene, but it is useful, cheaper, and less time-consuming when hot spot mutations are the targets, such as occurs with IDH1, IDH2, NPM1 in acute leukemias or T315I mutation in Philadelphia-positive leukemias or JAK2 in chronic myeloproliferative neoplasms. Further versions of ddPCR, that combine different primers/probes fluorescences and concentrations, allow measuring up to four targets in the same PCR reaction, sparing material, time, and money. ddPCR is also useful for quantitating BCR-ABL1 fusion gene, WT1 expression, donor chimerism, and minimal residual disease, so helping physicians to realize that “patient-tailored therapy” that is the aim of the modern hematology.

New Alternatives for the Evaluation of Minimal Residual Disease (MRD) Detection By Next Generation Sequencing in Multiple Myeloma

New Alternatives for the Evaluation of Minimal Residual Disease (MRD) Detection By Next Generation Sequencing in Multiple Myeloma

Efficacy and Safety of Front-Line Venetoclax and Rituximab (VenR) for the Treatment of Young Patients with Chronic Lymphocytic Leukemia and an Unfavorable Biologic Profile. Preliminary Results of the Gimema Study ‘Veritas‘

Efficacy and Safety of Front-Line Venetoclax and Rituximab (VenR) for the Treatment of Young Patients with Chronic Lymphocytic Leukemia and an Unfavorable Biologic Profile. Preliminary Results of the Gimema Study ‘Veritas‘

A novel ultra-sensitive method for the detection of FGFR3 mutations in urine of bladder cancer patients \u2013 Design of the Urodiag\xae PCR kit for surveillance of patients with non-muscle-invasive bladder cancer (NMIBC)

BackgroundWe have recently developed a highly accurate urine-based test, named Urodiag®, associating FGFR3 mutation and DNA methylation assays for recurrence surveillance in patients with low-, intermediate-, and high-risk NMIBC. Previously, the detection of four FGFR3 mutations (G372C, R248C, S249C and Y375C) required amplification steps and PCR products were analyzed by capillary electrophoresis (Allele Specific-PCR, AS-PCR), which was expensive and time-consuming. Here, we present the development a novel ultra-sensitive multiplex PCR assay as called “Mutated Allele Specific Oligonucleotide-PCR (MASO-PCR)”, generating a cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in voided urine.MethodsComparative clinical performances of MASO-PCR and AS-PCR technologies were performed from 263 urine DNA samples (87 FGFR3 mutated and 176 FGFR3 wild-type). In the development of Urodiag® PCR Kit, we studied the stability and reproducibility of each all-in-one PCR master mix (single reaction mixture including all the necessary PCR components) for MASO-PCR and QM-MSPCR (Quantitative Multiplex Methylation-Specific PCR to co-amplify SEPTIN9, HS3ST2 and SLIT2 methylated genes) assays.ResultsComplete concordance (100%) was observed between the MASO-PCR and AS-PCR results. Each PCR master mix displayed excellent reproducibility and stability after 12 months of storage at − 20 °C, with intra-assay standard deviations lower than 0.3 Ct and coefficient of variations (CV) lower than 1%. The limit of detection (LoD) of MASO-PCR was 5% mutant detection in a 95% of wild-type background. The limit of quantification (LoQ) of QM-MSPCR was 10 pg of bisulfite-converted DNA.ConclusionsWe developed and clinically validated the MASO-PCR assay, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine. We also designed the Urodiag® PCR Kit, which includes the MASO-PCR and QM-MSPCR assays. Adapted to routine clinical laboratory (simplicity, accuracy), the kit will be a great help to urologists for recurrence surveillance in patients at low-, intermediate- and high-risk NMIBC. Reducing the number of unnecessary cystoscopies, it will have extremely beneficial effects for patients (painless) and for the healthcare systems (low cost).

PS1325 IDENTIFICATION AND TARGETING OF MOLECULAR SIGNATURES OF HYPOMETHYLATING AGENT RESISTANCE IN MYELODYSPLASTIC SYNDROME AND CHRONIC MYELOMONOCYTIC LEUKEMIA

Background: Waldenström macroglobulinemia (WM) is a subset of lymphoplasmacytic lymphoma (LPL) with IgM paraprotein. Genetic hallmark of WM is MYD88 L265P mutation which is important for its diagnosis and pathophysiology of this disease. Other genetic changes including CXCR4 mutation, which is associated with WHIM syndrome, are reported to be common in WM. Whereas comprehensive information on genetic changes of LPL with other paraprotein like IgG is scarce. Formalin-fixed paraffin-embedded (FFPE) samples are available for genomic studies, but it contains abundant non-tumor cells. Aims: In this report, we aim to evaluate, first, genetic difference between WM and LPL with IgG paraprotein (IgG type) using targeted next-generation sequencing (NGS) from FFPE DNA and second, the usefulness of allele-specific oligonucleotide (ASO) PCR and peptide nucleic acid (PNA) PCR clamp method compared with next-generation sequencing. Methods: Twenty patients with pathologically confirmed LPL (10 of WMs and 10 of IgG type LPLs) were subjected to this study after informed consent. There were include 8 DNA extracted from bone marrow (BM) FFPE samples and 12 DNA extracted from BM cells. MYD88 L265P was detected by ASO-PCR. CXCR4 mutations were detected by PNA PCR clamp method with 18-mer PNA, which enables to detect the most frequently mutated amino acid: S338. Using the amplification products from that, detected mutations were confirmed by Sanger sequencing. We also tried detecting CXCR4 mutations by only Sanger sequencing. The mutations in 12 genes were detected by targeted sequencing on coding sequence with NGS. Mann-Whitney U test and Chi-squared test were used for statistical analysis. This study was approved by the research ethics committee of Gunma University Hospital. Results: Using NGS, we found the somatic mutations in WMs and the IgG type LPLs: MYD88 (4 vs. 5), CXCR4 (3 vs. 2), NOTCH2 (0 vs. 1), ARID1A (9 vs. 8), RAG2 (5 vs. 5), KMT2D (10 vs. 10), TP53 (0 vs. 0), MYBBP1A (0 vs. 1), CD79B (0 vs. 0), PRDM1 (0 vs. 1), CD274 (0 vs. 0), PDCD1LG2 (2 vs. 0); there were no statistical differences, suggesting genetic similarity between these two subsets of LPL, IgM type and IgG type. MYD88 L265P was found in 9/10 (90%) of WM cases and 6/10 (60%) of the IgG type by ASO-PCR (p = 0.40); However, it was found only in 4/10 (40%) of WM cases and 5/10 (50%) of the IgG type by NGS (p = 0.05). In addition, A260 V was found in 1 of IgG type LPL case. All MYD88 L265P mutations detected by NGS were also detected by ASO-PCR. CXCR4 mutations were found in 1/9 (11%) of WM cases (T318 frameshift) and 2/9 (22%) of IgG type (T318 frameshift and S338∗) by PNA PCR clamp method (p = 0.22). Whereas, these were found in 3/10 (30%) of WM cases and 2/10 (20%) of IgG type by NGS (p = 0.67). NGS enabled to detect the three CXCR4 mutations detected by PNA PCR clamp method and the mutations in outside of target area of PNA (E345 frameshift and L305F). Summary/Conclusion: Our NGS analysis suggest genetic similarity between two subsets of LPL, WM and IgG type. ASO-PCR is superior sensitive to NGS in detection of MYD88 L265P mutation, and PNA PCR clamp method enabled to detect the CXCR4 mutations in FFPE samples even if they were the frameshift mutations. Meanwhile, NGS analysis uncovered mutations other than ASO-PCR and PNA PCR clamp method targets.

Whole genome sequencing-based analysis of tuberculosis (TB) in migrants: rapid tools for cross-border surveillance and to distinguish between recent transmission in the host country and new importations.

BackgroundThe analysis of transmission of tuberculosis (TB) is challenging in areas with a large migrant population. Standard genotyping may fail to differentiate transmission within the host country from new importations, which is key from an epidemiological perspective.AimTo propose a new strategy to simplify and optimise cross-border surveillance of tuberculosis and to distinguish between recent transmission in the host country and new importationsMethodsWe selected 10 clusters, defined by 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), from a population in Spain rich in migrants from eastern Europe, north Africa and west Africa and reanalysed 66 isolates by whole-genome sequencing (WGS). A multiplex-allele-specific PCR was designed to target strain-specific marker single nucleotide polymorphisms (SNPs), identified from WGS data, to optimise the surveillance of the most complex cluster.ResultsIn five of 10 clusters not all isolates showed the short genetic distances expected for recent transmission and revealed a higher number of SNPs, thus suggesting independent importations of prevalent strains in the country of origin. In the most complex cluster, rich in Moroccan cases, a multiplex allele-specific oligonucleotide-PCR (ASO-PCR) targeting the marker SNPs for the transmission subcluster enabled us to prospectively identify new secondary cases. The ASO-PCR-based strategy was transferred and applied in Morocco, demonstrating that the strain was prevalent in the country.ConclusionWe provide a new model for optimising the analysis of cross-border surveillance of TB transmission in the scenario of global migration.

‘Gatekeeper’ mutation in patients with chronic myeloid leukemia resistant to imatinib therapy: effect on survival

Background T315I is the most difficult type of point mutation that appears during treatment of chronic myeloid leukemia (CML). It represents kind of a ‘gatekeeper’ that controls access of small molecule inhibitors and confers resistance to the most known tyrosine kinase inhibitors. Aim The aim of the present study was to assess the characteristics of Egyptian patients with CML harboring T315I mutation and to evaluate its effect on the patients’ survival. Patients and methods A total of 45 patients with CML in chronic phase resistant to imatinib mesylate (IM) treatment were enrolled in this study. Allele-specific oligonucleotide-PCR was used to detect T315I BCR-ABL gene mutation in all patients at the time of IM resistance. Results T315I mutation was present in 11 (24.4%) patients. The level of resistance was heterogeneous. Responses were observed in two resistant T315+ patients with IM dose escalation and three patients who were shifted to second-generation tyrosine kinase inhibitor. There was no significant difference in patient characteristics and overall survival between T315I+ and T315I− resistant patients. Overall survival is significantly associated with the duration of response and the phase of the disease at the time of resistance. Conclusion Evaluation of T315I mutation is recommended in all patients with CML, even in the early phase of the disease, which may be subsequently associated with resistance or progression of their disease in the future and requires the need for an alternative therapeutic option(s).

Development of Novel Human Anti-HLA-Monoclonal Antibodies for Clinical Applications Using Peripheral Blood B Cells Derived from Anti-HLA Antibody-Positive Donors

Introduction: The development of monoclonal antibodies (mAbs) such as anti-CD20 mAb (rituximab) and anti-CD38 mAb (daratumumab) has revolutionized the treatment of lymphoid malignancies. Potential efficacy of HLA allele-specific mAbs in treating malignant lymphoma has been shown by several studies. However, treatment of B-cell malignancies with humanized mAbs against HLA-DR alleles is associated with infusion-related toxicities (Lin et al., Leuk Lymphoma, 2009). In addition, previous attempts to develop mAbs specific to some HLA alleles (e.g., HLA-B61) by immunizing mice with recombinant HLA proteins have failed, likely because of a wide variety of polymorphisms of HLA molecules. To overcome these problems, we recently created a novel method to develop mAbs using microarray technology and human peripheral blood (PB) B lymphocytes derived from donors who are positive for anti-HLA antibodies. The process took approximately 1 month to produce human anti-HLA mAbs.Methods: Approximately 20 ml of PB was collected from anti-HLA antibody-positive donors, and mononuclear cells (MNCs) isolated using the Ficoll-Hypaque method were cultured in RPMI-1640 + 10% FCS containing 5 μg/ml R-848, 1000 IU/ml hIL2, 2.5 μg/ml CpG2006, 2.5 μg/ml anti-CD40 antibody, 100 ng/ml hIL21, 2 ng/ml hIL17, 10 ng/ml hIL4, and 100 ng/ml hBAFF for 6 days. CD138+ cells were then isolated using anti-CD138-antibody-conjugated microbeads. A microwell array chip was manufactured using micromachining techniques, as previously described (Jin et al., Nature Medicine, 2009). Microwells (diameter, 10 μm; depth, 15 μm) were formed on a silicon surface. The chip was coated with a PBS-containing purified HLA antigen (10 μg/ml) and incubated overnight at 4°C. After removing the antigen solution, 50 μl of the CD138+ cell suspension was added to the chip, and the mixture was incubated for 4 h at 37°C. After gentle washing with PBS, 2 μg/ml of anti-human IgG Fc-Cy3 solution was added to the microwells, and the plate was left at room temperature for 15 min. Finally, the cells were stained with 1 μM Oregon Green for 2 min. The microwells were screened for the antigen-specific antibodies released from single cells under a fluorescence microscope. Antigen-specific antibody-secreting cells (ASCs) from individual wells were isolated using a micromanipulator fitted with capillaries under the fluorescence microscope and were then expelled to microtubes for reverse transcription. The antibody cDNA fragments for VH and VL fragments were amplified using the single-cell 5′-RACE method and inserted into expression vectors containing the complete constant region of cDNA for heavy or light chains. Thereafter, CHO cells were transfected with both the heavy and light chain expression vectors to obtain a supernatant containing complete antibody molecules. The antigen specificity of the recombinant antibodies was examined using ELISA and flow cytometry (FCM). The frequency of ASCs in the PB-MNCs of donors was quantified using allele-specific oligonucleotide PCR. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of the mAbs were assessed using conventional methods.Results and Discussion: Two novel human mAbs specific to HLA-A24 and HLA-B61 were successfully generated. The antigen specificity of the mAbs was confirmed using ELISA and FCM. The mAbs bound to normal and malignant blood cells that expressed corresponding HLA alleles and showed CDC/ADCC activities against five B lymphoblastoid cell lines but not against lymphocytes/monocytes/granulocytes derived from five healthy donors, probably because of low expression levels of the target HLA alleles. The frequency of ASCs in PB-MNCs of donors was less than 0.001%.Conclusions: The present method enabled the generation of mAbs specific to HLA alleles, which can be used for detecting minimal residual diseases of hematological malignancies as well as for treating B-cell lymphoma, within 1 month. DisclosuresTakamatsu:Celgene: Honoraria; Janssen Pharmaceuticals: Honoraria. Kawai:Wakunaga Pharmaceutical Co., Ltd.: Employment. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

LGALS3 and AXIN1 gene variants playing role in the Wnt/ β-catenin signaling pathway are associated with mucinous component and tumor size in colorectal cancer

The Wnt pathway alterations have been identified in colorectal and many other cancer types. It has been reported that galectin-3 (which is encoded by the LGALS3 gene) alters the signaling mechanism in the Wnt/β-catenin pathway by binding to β-catenin in colon and other cancers. AXIN1 is mainly responsible for the assembly of the β-catenin destruction complex in the Wnt pathway. This study investigated the relationship of rs4644 and rs4652 variants of the LGALS3 gene and rs214250 variants of the AXIN1 gene to histopathological and clinical properties. Our study included a total of 236 patients, of whom 119 had colorectal cancer (42 women, 77 men) and 117 were healthy controls. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and allele-specific oligonucleotide (ASO) PCR methods were used. In addition, the serum galectin-3 level was studied with the enzyme-linked immunosorbent assay (ELISA) method. For the rs4644 variant of the LGALS3 gene, the CC genotype a mucinous component was significantly more common than those without a mucinous component (p=0.026). C allele frequency of the rs214250 variant of the AXIN1 gene was significantly correlated to tumor size in the advanced tumor stage (p=0.022). The CCAACT haplotype was more common in colorectal cancer patients (p=0.022). Serum galectin-3 level was higher in the patient group compared to the control group (5.9± 0.69 ng/ml vs. 0.79±0.01 ng/ml; p<0.001). In conclusion, variants of LGALS3 and AXIN1 genes affect tumor sizes and the mucinous component via Wnt/β-catenin pathway in the pathogenesis of colorectal cancer.

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.

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.

Ikaros Gene Deletion Significantly Predicts Relapse in Pediatric B-ALL Patients with Low End-Induction Minimal Residual Disease

Background:Deletions in the Ikaros gene (IKZF1) have been associated with a poor outcome in pediatric B-ALL, but the interaction of IKZF1 deletions with other established prognostic factors, such as minimal residual disease (MRD), is still under investigation. Here we report the IKZF1 deletion status of 405 patients (pts) with acute lymphoblastic leukemia (ALL) and its association with other presenting features, end-induction MRD, and outcome.Methods:Between 2005-2011, 794 eligible pts (aged 1-18 yrs) with newly diagnosed pediatric ALL (B-ALL: 697; T-ALL: 97) were enrolled on DFCI ALL Consortium Protocol 05-001. End-induction MRD was assessed prospectively on all pts by allele-specific oligonucleotide-PCR assay. DNA was extracted from archived diagnostic blood or bone marrow samples from 399 B-ALL and 6 T-ALL pts with sufficient available material. Using a commercially available kit (SALSA MLPA P202 IKZF1, MRC-Holland), IKZF1 deletion status was assessed by multiplex ligation-dependent probe amplification (MLPA). Event free survival (EFS) and relapse free survival (RFS) were estimated using the method of Kaplan and Meier.Results:IKZF1 deletion was detected in 69 (17%) of the 405 evaluated pts. IKZF1 deletion was more common in pts over 10 years old (p<0.0001), males (p=0.05), Hispanic/Latinos (p=0.002) and those with presenting white blood cell count (WBC) at diagnosis ≥ 50K/ul (p<0.0001). IKZF1 deletions were detected in 7 of 13 (54%) pts with the Philadelphia chromosome (Ph+). Significantly more IKZF1 pts experienced induction failure (7% vs 1%, p=0.009) and relapse (25% vs 7%, p<0.0001) than pts without IKZF1 deletions. There was no difference in rates of treatment-related mortality. With a median follow-up of 5.3 years, the 5-year event free survival (EFS) for patients without IKZF1 deletion was 87% and 61% for those with IKZF1 deletion (p<0.0001; Figure 1). There was no significant difference in EFS based on type of IKZF1 deletion: 1) deletions of exons 4-7 or 4-8 (42% of cases, 5-yr EFS 51%) or 2) whole gene (49% of cases, 5-yr EFS 69%) (p=0.23).315 Ph-negative B-ALL pts achieving complete remission (CR) had assessable end-induction MRD. Significantly more IKZF1 non-deleted pts had low MRD compared with IKZF1-deleted pts (95% vs 82%, p=0.006). For the 293 MRD-low pts, relapse occurred in 7% of IKZF1 non-deleted and 24% of IKZF1-deleted pts (p=0.004); for 22 MRD-high pts (all of whom received intensified therapy), there was no significant difference in relapse between IKZF1 non-deleted and IKZF1-deleted pts (21% vs 37%, p=0.62). The 5-yr relapse-free survival (RFS) was significantly worse for MRD-low pts with IKZF1 deletions versus those without these deletions (Figure 1). In multivariable analysis, IKZF1 status, but not MRD, was significantly associated with RFS for Ph-negative B-ALL pts. (Table 1)Conclusions:IKZF1 deletion is associated with higher rates of induction failure and relapse, and is a significant predictor of inferior outcome in pediatric B-ALL. IKZF1 deletion was associated with significantly higher rates of relapse in Ph-negative B-ALL patients with low end-induction MRD, and thus may be useful factor to consider when stratifying treatment intensity in this subset of pts. We conclude that IKZF1 deletion should be incorporated into risk stratification for pediatric B-ALL.Table 1Prognostic Factors for RFS for B-ALL, Ph-negative pts with evaluable end-induction MRDUnivariateMultivariableHR [95% CI]p-valueHR [95% CI]p-valueAge, ≥10 vs <101.40 [0.68-2.91]0.361.19 [0.56-2.51]0.65IKZF1, del vs. not3.23 [1.61-6.45]0.00092.53 [1.20-5.30]0.01MRD, high vs. low2.51 [1.04-6.03]0.041.63 [0.64-4.11]0.30WBC, ≥50 vs. <502.50 [1.27-4.94]0.0081.99 [0.98-4.03]0.06[HR=Hazard ratio] [Display omitted] [Display omitted] 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.

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.

Real-time PCR assay to distinguish Phytophthora ramorum lineages using the cellulose binding elicitor lectin (CBEL) locus

Phytophthora ramorum is a pathogenic oomycete that causes sudden oak death in the Western USA and sudden larch death in the UK. Until recently, three genetically divergent clonal lineages of P. ramorum were known (EU1, NA1 and NA2), named according to the continent on which they were first detected. In 2009, a fourth lineage named EU2 was discovered in the UK. Sequencing and microsatellite genotyping revealed that the EU2 lineage is genetically distinct from all other lineages. Allele-specific oligonucleotide-PCR (ASO-PCR) assays using real-time PCR were developed in this study, allowing for the identification of the EU2 lineage. Also, a combination of ASO-PCR assays targeting the cellulose binding elicitor lectin (CBEL) locus was validated to rapidly identify all four lineages. Sequencing of the CBEL locus revealed eight single nucleotide polymorphisms (SNPs) that distinguished EU2 from the other three lineages. Two ASO-PCR assays were developed from these SNPs, providing the ability to rapidly identify EU2 individuals relative to EU1, NA1 and NA2 individuals. These new assays were combined with two existing assays targeting the same locus to allow rapid and simple identification of all four lineages. Blind tests performed on a panel of representative samples revealed diagnostic profiles unique to each lineage. These markers can be used with diseased field samples, making them well suited for routine procedures in diagnostic laboratories to identify P. ramorum.

Sensitivity, Reproducibility and Clinical Utility Of Next-Generation Sequencing (NGS) for BCR-ABL1 Kinase Domain Mutation Screening: Results From The CML Work Package Of The Iron-II (Interlaboratory RObustness Of Next-Generation Sequencing) International Study

Background and AimsIn chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL1 mutation status is an essential component of the therapeutic decision algorithm. Capillary Sanger sequencing (SS) is currently the gold standard for mutation screening of the BCR-ABL1 kinase domain (KD), despite key technical limitations including limited sensitivity and no discrimination between compound and polyclonal mutations. Benchtop next-generation sequencers have recently been introduced as potential diagnostic platforms and there is growing interest in their clinical application. In the framework of the IRON-II (Interlaboratory RObustness of Next-generation sequencing) international consortium, 10 laboratories from 7 countries (Italy, Germany, United Kingdom, Spain, Austria, Turkey, Czech Republic) have engaged in the set-up, standardization and validation of a laboratory-developed screening assay for BCR-ABL1KD mutations based on the Roche 454 amplicon deep-sequencing technology. MethodsFusion primers were designed to generate four partially overlapping amplicons by nested reverse transcription (RT)-polymerase chain reaction (PCR), the first amplification step needed to select for the translocated ABL1 allele. Fusion primers were barcoded with multiplex identifiers (MIDs) consisting of 10-base pair tags allowing multiplexing of twelve clinical samples (forty-eight amplicons) in a single NGS run. The assay was designed in a ready-to-use 96-well plate format containing lyophilized oligonucleotide primers. ResultsDifferent primer designs and primer-MID combinations were evaluated for their performances. Sequencing runs generated an average of 97,432 reads (range, 59,459-151,335). For the primer design selected for further evaluation, the coverage per amplicon ranged between 1,449 and 5,997 sequencing reads. To explore the sensitivity and accuracy of the assay, serial dilutions of BaF3 cell lines harboring four different known mutations (Y253F, E255K, T315I, M351T) into an unmutated BaF3 cell line (50%:50%; 25%:75%; 10%:90%; 5%:95%; 2%:98%; 1%:99%) were sequenced in parallel in two distinct laboratories (Bologna and Jena). In both centers, results showed a high linearity of mutation calling and accuracy of mutation detection and quantitation over the entire range of dilutions, down to 1% mutation abundance. Intra-run reproducibility and inter-run reproducibility were confirmed by a series of experiments in which a set of samples was resequenced in the same and in independent runs, respectively, with and without repetition of the RT and PCR steps. Importantly, we demonstrated that reproducibility could be maintained over a wide dynamic range of amplicon coverage (from 100 to 5,000 independent sequencing reads). A total of 554 clinical samples (2,216 amplicons) were analyzed by the 10 laboratories - including 517 clinical samples analyzed in parallel by NGS and SS and 30 clinical samples analyzed in parallel by NGS, SS and conventional pyrosequencing. Three hundred and ninety-four of 398 (99%) variants detected by SS were also detected by NGS. In addition, comparison between NGS, SS and conventional pyrosequencing results showed very good concordance with respect to the estimation of variant abundance. NGS allowed to detect additional, low level mutations (>1% but<10-15%, i.e. undetectable by SS) in 294/554 (53%) samples. In a subset of twenty randomly selected samples, low level mutations were confirmed by independent methods (restriction fragment length polymorphism or allele-specific oligonucleotide-PCR). Compound mutations as against polyclonality could be resolved in all the clinical samples harboring multiple mutations mapping 450 bp apart or closer. Longitudinal retrospective analysis of CML and Ph+ ALL clinical samples showed that NGS could have identified TKI-resistant mutations earlier than SS, thus allowing more timely therapeutic intervention. ConclusionsOur results indicate the technical feasibility, accuracy and robustness of NGS for BCR-ABL1 KD mutation screening and represent an important step forward towards its routine application in a clinical setting. An international ring trial to test inter-laboratory reproducibility of BCR-ABL1 mutation detection by NGS is now about to start. Disclosures:Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Machova Polakova:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Lion:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Hochhaus:ROCHE: Research Funding. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Evaluation Of The JAK2-V617F Gene Mutation In Egyptian Patients With Essential Thrombocythemia and Polycythemia Vera

An activating mutation of Janus kinase 2 (JAK2-V617F) was previously described in chronic myeloproliferative disorders (MPD). In previously published studies, the frequency of the JAK2-V617F mutation was determined to be 80–90 % for patients with polycythemia vera (PV) and 40–70 % for essential thrombocythemia (ET). In this study, we analyzed the relationship between the JAK2-V617F mutation and clinical-hematological parameters in Egyptian patients with MPD and compared these findings with published studies from other geographic regions and previous studies in EGYPT. A total of 56 patients were studied; of which, 32 were diagnosed with PV and 24 with ET. The mutation status of JAK2 was determined using allele-specific oligonucleotide (ASO) PCR assay. We found that 53% of the PV group and 79% of the ET group were positive for the JAK2-V617F mutation. When all patients were analyzed; patient age, levels of WBCs, levels of hemoglobin, levels of platelets and splenomegaly were significantly different in patients with the JAK2-V617F mutation (p < 0.05). The JAK2-V617F mutation is frequently detected in the Egyptian patients with MPD, and especially in patients with ET. Hence, it would be useful to include JAK2 mutation screening in the initial evaluation of patients suspected to have MPD especially ET. Disclosures:No relevant conflicts of interest to declare.