Allele-specific Oligonucleotide Polymerase Chain Reaction Research Articles

Carrying SNP rs17506395 (T > G) in TP63 gene and CCR5Δ32 mutation associated with the occurrence of breast cancer in Burkina Faso.

Genetic alterations in the TP63 (GenBank: NC_000003.12, ID: 8626) and CCR5 (receptor 5 chemokine co-receptor) (GenBank: NC_000003.12, ID: 1234) genes may increase the risk of developing breast cancer. The aim of this study was to investigate the probable involvement of polymorphisms rs17506395 in the TP63 (tumour protein 63) gene and the CCR5Δ32 mutation in the occurrence of breast cancer in Burkina Faso. This case-control study included 72 patients and 72 controls. Genotyping of SNP rs17506395 (TP63) was performed by polymerase chain reaction-restriction fragment length polymorphism, and genotyping of the CCR5Δ32 mutation was performed by allele-specific oligonucleotide polymerase chain reaction. For SNP rs17506395 (TP63), the genotypic frequencies of wild-type homozygotes (TT) and heterozygotes (TG) were, respectively, 27.72 and 72.22% in cases and 36.11 and 63.89% in controls. No mutated homozygotes (GG) were observed. For the CCR5Δ32 mutation, the genotypic frequencies of wild-type homozygotes (WT/WT) and heterozygotes (WT/Δ32) were 87.5 and 13.5%, respectively, in the cases and 89.29 and 10.71%, respectively, in the controls. No mutated homozygotes (Δ32/Δ32) were observed. None of the polymorphisms rs17506395 of the TP63 gene (OR = 1.47, 95% CI = 0.69-3.17, P = 0.284) and the CCR5Δ32 mutation (OR = 1.32, 95% CI = 0.46-3.77; P = 0.79) were associated with the occurrence of breast cancer in this study.

Современные подходы к оценке минимальной остаточной болезни при множественной миеломе (плазмоклеточной миеломе)

The treatment of multiple myeloma is inextricably linked to the need for assessment and monitoring of the minimal residual disease (MRD). Assessment of the MRD allows evaluating the efficacy of therapy and obtaining significant prognostic information; it is an indicator of the degree of eradication of the tumor clone. The methods for detecting residual tumor cells evolve constantly, which translates into updates of the criteria reflecting the scale of response to therapy. There is no single MRD detection technique; common recommendations suggest seeking for pathological cells both intramedullary and extramedullary. This review describes current MDR determination methods, including imaging, next generation multiparametric flow cytometry, and methods based on DNA analysis — allele-specific oligonucleotide polymerase chain reaction and next generation sequencing. We compare their advantages, limitations, disadvantages, clinical significance, and show the necessary sensitivity thresholds of the described methods and the conditions that make this or that approach ideal in the context of detection of MRD.

Heterogeneity of Mycobacterium tuberculosis Strains Circulating in Panama's Western Region.

Tuberculosis remains a challenge in both rural and urban areas. Although a majority of countries display a higher burden in urban areas compared with rural areas, Panama continues to report the highest mortality rate in Central America. Urban areas, such as Panama City, report a high tuberculosis burden, whereas Panama's western region, including the provinces of Chiriquí, Bocas del Toro (both semiurban) and Ngäbe-Bugle (rural), show a lower burden. We aimed to identify highly transmitted Mycobacterium tuberculosis strains within rural and semiurban settings of Panama's western region during a 3-year period (2017, 2019, 2021). We randomly selected 87 M. tuberculosis isolates from a biobank from Panama's western region and analyzed them using allele-specific oligonucleotide polymerase chain reaction and 24-mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). Our results show only 11.7% (10/85) of M. tuberculosis strains identified as prevalent A-Beijing, B-Haarlem, or C-LAM Strains. We found a low prevalence of A, B, and C M. tuberculosis strains in both rural and semirural settings compared with isolates collected from the Eastern Colon Province. MIRU-VNTR genotyping revealed a high degree of diversity with no clusters with single loci variation of ≥ 2 loci. These results support the notion that tuberculosis prevalence in the rural and semiurban western region of Panama are not due to previously described highly transmitted strains but is influenced instead by other health determinants, including poor health system access and a lack of systematic transmission chain monitoring. For remote rural and semiurban settings, we recommend allocating resources to reinforce efforts to prevent tuberculosis spread.

Chronic Lymphocytic Leukemia (CLL) Clonal Growth Rate Is Slower Following Venetoclax-Rituximab (VenR): Results from a Minimal Residual Disease (MRD) Model from the Randomized Phase 3 Murano Trial

Chronic Lymphocytic Leukemia (CLL) Clonal Growth Rate Is Slower Following Venetoclax-Rituximab (VenR): Results from a Minimal Residual Disease (MRD) Model from the Randomized Phase 3 Murano Trial

Deciphering the association of intronic single nucleotide polymorphisms of crystallin gene family with congenital cataract.

Purpose:Introns play an important role in gene regulation and expression. Single nucleotide polymorphisms (SNPs) in introns have the potential to cause disease and alter the genotype–phenotype association. Hence, this study aimed to decipher the association of SNPs in the introns of the crystallin gene in congenital cataracts.Methods:SNPs in the introns of crystallin gene family – CRYAA (rs3788059), CRYAB (rs2070894), CRYBA4 (rs2071861), and CRYBB2 (rs5752083, rs5996863) – were genotyped in 248 participants consisting of 141 congenital cataracts and 107 healthy controls by allele-specific oligonucleotide polymerase chain reaction method. Around 10% of samples for each SNPs were sequenced to confirm the genotypes. The allele, genotype, and haplotype frequency were evaluated by the SHEsis online tool.Results:Using dominant model, the “A” allele of rs3788059 was found to have an increased risk toward congenital cataract development whereas the “G” allele was found to be protective (AA + AG vs. GG; odds ratio [OR] 95% confidence interval [CI] = 3.73 [1.71, 8.15], P = 0.0009). The “A” allele of both rs2070894 (AA + AG vs. GG; OR [95% CI] = 0.49 [0.29, 0.84], P = 0.012) and rs5752083 (AA + AC vs. CC; OR [95% CI] = 0.25 [0.08, 0.76], P = 0.016) were suggested to have a protective role by the dominant model. The A-C-T haplotype (rs2071861, rs5752083, and rs5996863) was found to be a significant risk factor for the development of congenital cataract.Conclusion:Intronic SNPs in crystallin genes may play a role in the predisposition toward congenital cataract. However, the present findings need to be replicated in a large cohort with more number of samples.

Five-Year Analysis of Murano Study Demonstrates Enduring Undetectable Minimal Residual Disease (uMRD) in a Subset of Relapsed/Refractory Chronic Lymphocytic Leukemia (R/R CLL) Patients (Pts) Following Fixed-Duration Venetoclax-Rituximab (VenR) Therapy (Tx)

Five-Year Analysis of Murano Study Demonstrates Enduring Undetectable Minimal Residual Disease (uMRD) in a Subset of Relapsed/Refractory Chronic Lymphocytic Leukemia (R/R CLL) Patients (Pts) Following Fixed-Duration Venetoclax-Rituximab (VenR) Therapy (Tx)

Association of promoter methylation of RASSF1A and KRAS mutations in non-small cell lung carcinoma in Kashmiri population (India)

BackgroundNon-small cell lung carcinoma (NSCLC) incidence and progression is increasing because of genetic and epigenetic changes. The mutations in the Kirsten rat sarcoma (KRAS) are the most frequently oncogene aberrations in lung carcinoma patients. A candidate tumor suppressor gene (TSG) Ras Association Domain Family 1 Isoform A (RASSF1A), is silenced by promoter hypermethylation in several human malignancies including non-small cell lung carcinoma (NSCLC). We hypothesized that RASSF1A methylation and KRAS mutations may play an important role in NSCLC. MethodsNon-small cell lung carcinoma patients (n = 100) and equal number of healthy controls were assessed for activating KRAS (exon 2) mutations using allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) and promoter hypermethylation of RASSF1A using methylation specific PCR. ResultsThe frequency of mutations in Kirsten rat sarcoma (KRAS) were found in 31% of NSCLC patients in the Kashmiri population and occur most commonly, but not exclusively, in adenocarcinoma histology and life-long smokers. The NSCLC patients in advanced stage reported the higher frequency of mutation in KRAS (exon 2). A significant higher frequency of this mutation was reported in patients with NSCLC (29.16%) who are positive for metastasis (P < 0.03). The frequencies of promoter hypermethylation at Ras Association Domain Family 1 Isoform A (RASSF1A) were 41% in cases and 3% in control samples. The frequency of KRAS mutation and RASSF1A promoter methylation were significantly different between adenocarcinomas (ADC) and squamous cell carcinomas (SCC) patients with NSCLC (P < 0.03). In addition, we reported that NSCLC patients having RASSF1A promoter methylation was significantly associated with smoking (P = 0.01). It was identified that NSCLC patients with RASSF1A promoter region hypermethylation had poorer survival and faster disease progression compared with those without hypermethylation of RASSF1A promoter region (P = 0.0001). The Median survivals among with cases containing promoter region hypermethylation of RASSF1A were 17.20 and 42.13 months for patients without promoter region hypermethylation of RASSF1A and the patients with KRAS mutation with or without hypermethylation of the promoter region of RASSF1A a tumor suppressor gene had poorer survival compared with those patients with wild type KRAS gene, with or without hypermethylation of RASSF1A promoter region. These differences were statistically significant based on Log-rank (Mantel-cox) test (P = 0.0001). The median survivals among patients with mutation in KRAS protooncogene were 16 months and 42 months for NSCLC patients with wild type KRAS gene. ConclusionsThe aberrant RASSF1A gene promoter methylation with the subsequent mutation in KRAS gene (exon 2) plays a significant role in the pathogenesis and disease progression of non-small cell lung carcinoma (NSCLC).

Primary Analysis of Anti-CD19 Tafasitamab (MOR208) Treatment in Combination with Idelalisib or Venetoclax in R/R CLL Patients Who Failed Prior BTK Inhibitor Therapy (COSMOS Trial)

Primary Analysis of Anti-CD19 Tafasitamab (MOR208) Treatment in Combination with Idelalisib or Venetoclax in R/R CLL Patients Who Failed Prior BTK Inhibitor Therapy (COSMOS Trial)

PB2200 PREVALENCE OF JAK2V617F, CALR AND MPL MUTATIONS IN PATIENTS OF MYELOPROLIFERATIVE NEOPLASMS AND SPLANCHNIC VEIN THROMBOSIS IN INDIA

Background:Recurrent somatic mutations in the JAK2, MPL, and CALR genes have been described in patients diagnosed with Philadelphia‐negative Myeloproliferative neoplasms (MPNs), including Polycythemia vera (PV), Essential thrombocythemia (ET) and Primary myelofibrosis (PMF). MPN is also the most common etiology in splanchnic vein thrombosis (SVT). MPNs account for approximately 40% of Budd Chiari syndrome (BCS) and 31% of portal vein thrombosis (PVT).The present study was aimed at studying the prevalence of JAK‐2V617F, MPLW515L/K and CALR exon 9 mutations in BCR‐ABL1 negative myeloproliferative neoplasms (MPNs) and splanchnic vein thrombosis (SVT) patients. There is paucity of such data in the Indian patients.Aims:The study had two aims: Identification of incidence of JAK2V617F, CALR exon 9 mutations and MPLW515L/K mutations in BCR‐ABL1 negative MPNs in India Identification of incidence of JAK2V617F, CALR exon 9 mutations and MPLW515L/K mutations in Indian patients of SVT MethodsA total number of 60 patients of SVT and 59 MPN patients (10 PV, 19 ET, and 30 PMF) were included in the study. The mutation analysis for JAK2V617F, CALR exon 9 and MPLW515L/K mutations were performed using the Allele specific oligonucleotide‐polymerase chain reaction (ASO‐PCR) was used to detect JAK‐2, CALR and MPL mutations.Results:Among 59 patients of MPNs and 60 SVT; JAK‐2 mutation was found in 61.0% (36/59) and 23.3% (14/60) respectively. On sub‐classification, JAK‐2 mutation was detected in 80.0% (8/10) of PV; 42.1% (8/19) of ET and 66.7% (20/30) of PMF patients.All the patients of MPNs and SVT were subjected to CALR mutation analysis. The overall rate of CALR mutations in PMF and ET was 10% (3/30) and 21.0% (4/19) respectively. Limiting the analysis to JAK2V617F negative patients of PMF and ET, it was seen that 30% (3/10) PMF and ∼36.36% (4/11) ET cases were CALR mutated. Of these 7 CALR mutations; L367fs∗46 (type‐1) and K385fs∗47 (type‐2) mutations were found in 5 and 2 patients respectively. MPL mutation was identified in 5.26% (1/19) ET cases. Triple negative ET and PMF were ∼31.5% (∼6/19) and ∼23.3% (7/30) respectively. None of the SVT patients were observed to be CALR mutated and none showed the MPLW515L/K mutations.Summary/Conclusion:Indian patients exhibit a lower incidence of CALR mutations in Primary myelofibrosis than that seen in the western literature. However, the incidence of CALR mutations is the same as that reported earlier in Indian patients of Essential thrombocythemia. Triple negative cases constitute 31.5% of all cases of Essential thrombocythemia and 23.3% of Primary myelofibrosis Indian patients show a Testing for CALR mutations and MPLW515L/K mutations in splanchnic vein thrombosis is not justified.

Effect of fixed-duration venetoclax plus obinutuzumab (VenG) on progression-free survival (PFS), and rates and duration of minimal residual disease negativity (MRD–) in previously untreated patients (pts) with chronic lymphocytic leukemia (CLL) and comorbidities.

7502 Background: The multinational, open-label, phase 3 CLL14 trial compared fixed-duration targeted VenG treatment with chlorambucil-obinutuzumab (ClbG) in previously untreated pts with CLL and comorbidities. Here we present endpoint analyses with particular emphasis on MRD− and PFS. Methods: Pts with a CIRS score &gt;6 and/or an estimated creatinine clearance &lt;70 mL/min were randomized 1:1 to receive equal duration treatment with 12 cycles (C) of standard Clb or Ven 400 mg daily in combination with G for first 6 C. Primary endpoint was PFS. MRD− in peripheral blood (PB) or bone marrow (BM) 3 months (mo) after treatment completion was a key secondary endpoint. MRD was analyzed serially from C4 every 3 mo by an allele-specific oligonucleotide polymerase chain reaction assay (ASO-PCR; cut-off, 10-4) and by next generation sequencing (NGS; cut-offs, 10-4, 10-5,10-6). Results: 432 pts were enrolled; 216 in each treatment group (intent-to-treat population). After 29 mo median follow-up, superior PFS was observed with VenG vs ClbG (HR 0.35; 95% CI 0.23–0.53; P&lt;0.0001). MRD− by ASO-PCR was significantly higher with VenG vs ClbG in both PB (76% vs 35% [P&lt;0.0001]) and BM (57% vs 17% [P&lt;0.0001]) 3 mo after treatment completion. Overall, 75% of VenG MRD-negative pts in PB were also MRD-negative in BM vs 49% in the ClbG group. Landmark analysis for this timepoint by PB MRD status showed that MRD− was associated with longer PFS. Higher MRD− rates were achieved early and were more sustainable with VenG: 81% (VenG) vs 27% (ClbG) of pts were MRD-negative 12 mo after treatment completion; HR for MRD conversion 0.19; 95% CI 0.12–0.30 (median time off-treatment: 19 mo). MRD− rates by NGS confirmed these results; 78% (VenG) vs 34% (ClbG) of pts had MRD− at &lt;10-4, 31% vs 4% at &lt;10-6 and 35% vs 15% at ≥10-6–&lt;10-5, respectively. Conclusions: Fixed-duration VenG induced deep (&lt;10-6 in 1/3 of pts), high, and long lasting MRD− rates (with a low rate of conversion to MRD+ status 1 year after treatment) in previously untreated pts with CLL and comorbidities, translating into improved PFS. Clinical trial information: NCT02242942.

Involvement of Interferon-γ + 874A/T Polymorphism in the Pathogenesis of and Therapeutic Response to Immune Thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by symptoms of thrombocytopenia and bleeding due to production of autoantibodies against platelets. Recently, the occurrence of polymorphisms has been identified as one of the main causes of disease onset. To conduct this study, we recruited 140 patients and control individuals with no history of platelet loss. After collection of specimens, the prevalence of interferon-γ polymorphism was evaluated using the allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) technique and confirmed by sequencing techniques. The results showed that the frequency of the AA genotype was higher in the control group, compared with patients with ITP; however, in the acute and chronic groups, the frequency of the AT genotype was higher than that of the AA genotype. We also discovered that there was no significant correlation between platelet counts before and after treatment, nor in its related parameters with interferon (IFN)-γ polymorphism. rs2430561 does not seem to have any role in ITP pathogenesis and treatment response.

Benefits of the early detection of M351T mutation, by allele-specific oligonucleotide polymerase chain reaction, in imatinib-resistant chronic myelogenous leukemia (CML) - A retrospective analysis

&lt;p class="s4"&gt;&lt;strong&gt;Introduction:&lt;/strong&gt; BCR-ABL kinase domain mutations represent the most important disease-related factor in chronic myelogenous leukemia (CML) resistance. Highly resistant clones may pre-exist and emerge rapidly. Patients with CML can acquire more than one BCR-ABL1 mutation, which may result in increased oncogenicity. &lt;strong&gt;Materials and Methods:&lt;/strong&gt; Retrospective analysis of 50 patients of imatinib resistance was done in GCRI, from January 2014 to May 2014. Response to imatinib was defined according to the European LeukemiaNet 2009 criteria. Allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) was performed on genomic DNA, extracted from peripheral blood mononuclear cells. Results: Average age was 40.75 years, 33 were males and 17 females. 47 (94%) were in chronic phase, 2 (4%) in accelerated phase, and 1 (2%) in blastic crisis. 29/50 were having low EUTOS score, whereas SOKAL score was low in 20, intermediate in 21 while only 9 had high SOKAL at presentation. Median duration of imatinib was 48 months. 43/50 had one or more than 1 mutation, T315I mutation in 5 (10%) patients, and M351T in 32% (16/50). &lt;strong&gt;Conclusion:&lt;/strong&gt; The presence of M351T mutation in mutant clone leads to the development of T315I mutations development, and the detection of M351T mutation in the initial months of the therapy has a prognostic significance. ASO-PCR is more sensitive method of the detection of such mutations as compared to direct sequencing. We report low cytogenetic response (25%) and durability of response to 600 mg of imatinib, even in M351T mutation, after 400 mg of imatinib for median period of 4 years.&lt;/p&gt;

Ibrutinib plus Venetoclax for the Treatment of Mantle-Cell Lymphoma

BackgroundBoth the BTK inhibitor ibrutinib and the BCL2 inhibitor venetoclax are active as monotherapy in the treatment of mantle-cell lymphoma. Complete response rates of 21% have been observed for each agent when administered as long-term continuous therapy. Preclinical models predict synergy in combination.MethodsWe conducted a single-group, phase 2 study of daily oral ibrutinib and venetoclax in patients, as compared with historical controls. Patients commenced ibrutinib monotherapy at a dose of 560 mg per day. After 4 weeks, venetoclax was added in stepwise, weekly increasing doses to 400 mg per day. Both drugs were continued until progression or an unacceptable level of adverse events. The primary end point was the rate of complete response at week 16. Minimal residual disease (MRD) was assessed by flow cytometry in bone marrow and by allele-specific oligonucleotide–polymerase chain reaction (ASO-PCR) in blood.ResultsThe study included 24 patients with relapsed or refractory mantle-cell lymphoma (23 patients) or previously untreated mantle-cell lymphoma (1 patient). Patients were 47 to 81 years of age, and the number of previous treatments ranged from none to six. Half the patients had aberrations of TP53, and 75% had a high-risk prognostic score. The complete response rate according to computed tomography at week 16 was 42%, which was higher than the historical result of 9% at this time point with ibrutinib monotherapy (P<0.001). The rate of complete response as assessed by positron-emission tomography was 62% at week 16 and 71% overall. MRD clearance was confirmed by flow cytometry in 67% of the patients and by ASO-PCR in 38%. In a time-to-event analysis, 78% of the patients with a response were estimated to have an ongoing response at 15 months. The tumor lysis syndrome occurred in 2 patients. Common side effects were generally low grade and included diarrhea (in 83% of the patients), fatigue (in 75%), and nausea or vomiting (in 71%).ConclusionsIn this study involving historical controls, dual targeting of BTK and BCL2 with ibrutinib and venetoclax was consistent with improved outcomes in patients with mantle-cell lymphoma who had been predicted to have poor outcomes with current therapy. (Funded by Janssen and others; AIM ClinicalTrials.gov number, NCT02471391.)

Molecular detection of minimal residual disease in multiple myeloma.

Despite the significantly higher complete remission rates and improved survival achieved in the last decade, multiple myeloma (MM) patients continue to relapse due to persistence of minimal residual disease (MRD). Generally, MRD refers to persistence of low levels of disease in the order of one tumour cell in ≥105 normal cells. Currently, molecular and immunophenotypic techniques are employed for MRD detection. This review focuses on MRD detection by molecular techniques, including allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), next-generation sequencing (NGS) and digital PCR (dPCR), in addition to a brief description of, and comparison with, multiparameter flow cytometry. The basic principles, technical advantages and limitations, and the clinical impact of all three molecular techniques are reviewed and compared. They all have a sensitivity of at least 10-5 , among which ASO real-time quantitative PCR is the most well-standardized, and NGS carries the highest sensitivity and applicability, while dPCR is still under investigation. Furthermore, molecular MRD negativity is a favourable prognostic factor for survival of patients with MM. However, several challenges inherent to molecular detection of MRD still remain to be overcome, particularly false negativity and failure to detect extramedullary disease. Finally, detection of MRD from peripheral blood remains challenging.

Molecular screening and the clinical impacts of BCR-ABL KD mutations in patients with imatinib-resistant chronic myeloid leukemia.

The present study aimed to detect the frequency of kinase domain (KD) mutations in order to evaluate their clinical significance and functional importance in 45 patients with chronic myeloid leukemia (CML) who were resistant to imatinib therapy. Sanger sequencing was used (45 patients), along with allele-specific oligonucleotide polymerase chain reaction (ASO-PCR; 3 patients), for the screening of mutations. BCR/ABL KD was amplified by nested PCR and sequencing was performed. Secondly, ASO-PCR was performed to confirm the results of the sequence analysis for E255K mutations. Mutations were detected in 11/45 patients (24.44%) via Sanger sequencing. D241G (4.4%), C369C (4.4%), K285N (2.2%), A380T (2.2%) and A366V (2.2%) mutations were detected. E255K (8.8%) was detected by ASO-PCR and Sanger sequencing. Mutations are a primary reason for suboptimal responses, loss of response and resistance to imatinib. In particular, the E255K mutation, which is characterized by resistance to imatinib and nilotinib, was detected in four patients. Analyzing the mutations and monitoring patients with CML may improve their prognosis and survival rate. ASO-PCR assays will be beneficial for the routine monitoring of mutations.

Prognostic value of sequencing-based minimal residual disease detection in patients with multiple myeloma who underwent autologous stem-cell transplantation.

BackgroundMost patients with multiple myeloma (MM) are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper response are required.Patients and methodsWe retrospectively analyzed 125 patients with MM who underwent high-dose melphalan plus autologous stem-cell transplantation (ASCT) to detect MRD in autograft/bone marrow (BM) cells using a next-generation sequencing (NGS)-based method and allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR).ResultsNGS-based method was applicable to 90% and this method had at least one to two logs greater sensitivity compared to ASO-PCR. MRD negative by NGS [MRDNGS(−)] (defined as <10−6) in post-ASCT BM cases (n = 26) showed a significantly better progression-free survival (PFS) (96% at 4 years, P < 0.001) and overall survival (OS) (100% at 4 years, P =0.04) than MRDNGS(+) in post-ASCT BM cases (n = 25). When restricting the analysis to the 39 complete response cases, patients who were MRDNGS(−) (n = 24) showed a significantly better PFS than those that were MRDNGS(+) (n = 15) (P =0.02). Moreover, MRDNGS(−) in post-ASCT BM cases (n = 12) showed significantly a better PFS than MRDNGS(+) cases (n = 7) where MRD was not detected by ASO-PCR (P = 0.001). Patients whose autografts were negative by NGS-based MRD assessment (<10−7) (n = 19) had 92% PFS and 100% OS at 4 years post-ASCT. Conversely, the NGS-based MRD positive patients who received post-ASCT treatment using novel agents (n = 49) had a significantly better PFS (P = 0.001) and tended to have a better OS (P= 0.214) than those that were untreated (n = 33).ConclusionsLow level MRD detected by NGS-based platform but not ASO-PCR has significant prognostic value when assessing either the autograft product or BM cells post-ASCT.

Cytogenetic and molecular genetic studies of number of chronic mylogenous leukemia in Erbil province

Chronic myeloid leukemia (CML) is amyeloprolirative disorder characterized by the Philadelphia chromosome originates from the translocation of chromosome 9 and 22 t (9;22) (q34;q11), creating a BCR-ABL fusion gene which results in the constitutive activation BCR-ABL tyrosine kinase. Patients of BCR-ABL fusion gene positive in (CML) are treated by an effective therapy called Imatinib (Gleevec). Point mutations alter the conformation of the ATP binding site that disturbs the binding of therapy to its target which leading to imatinib resistance. Fifty CML patients were collected from Nanakali Hospital in Erbil city which were diagnosed by physicians. The age of the patients from 9 to 80 years, 62% were male and 39% were female, median age of the patients were 38 years. The present study deals with two different aspects; conventional cytogenetic (G-banding) analysis to diagnose the Philadelphia chromosome as a (CML) marker and using Allele Specific Oligonucleotide Polymerase Chain Reaction (ASO-PCR) assay for screening the mutant allele at the three codons 351, 311 and 315 of the BCR-ABL ATP binding domain in Imatinib resistant CML patients. The phenotype of fifteen CML patients were successfully analyzed, thirteen patients were in complete cytogenetic response state (Philadelphia 0%) only two of those patients showed the Philadelphia chromosome. Three point mutations T1052C, T932C and C944T were identified by allele specific oligonucleotide polymerase chain reaction (ASO-PCR). One patient showed T1052C mutation, T932C was detected in three patients and two patients showed the C944T ATP binding domain mutation. In conclusion, conventional cytogenetic and molecular genetic tests are complementary techniques to show the exact types of abnormalities, which allow better evaluation of the genomic aberration involved in CML patients, specifically for kinase domain mutation that plays an important role in different diagnosis, prognosis, therapy treatment and drug resistant management of chronic myeloid leukemia.

Interleukin-8 251- A/T polymorphism related to peptic ulcer disease in H. pylori infected patient

Objectives This study includes the investigation of antifungal activity of the local propolis against dermatophytes and yeast. Methods A total of 92 tissue samples were taken from patients infected with H. pylori and 102 from uninfected individuals has been included. Genetic method used for the detection of H. pylori infection by polymerase chain reaction (PCR) for glM gene identification. IL-8- 251 A/T polymorphism was detected by allele-specific oligonucleotide polymerase chain reaction (ASO- PCR). Results Among the studied samples, the results improve that genetic polymorphism of IL-8-251 A/T genotype was found in a higher frequency with confidence interval 95% in duodenal ulcer H. pylori infected patient than control. Conclusion IL-8-251 A allele notice that it has been associated with severe inflammation lead to increase severity of disease.

Triple Negative Myeloproliferative Neoplasms (MPNs) Patients Show Low MPN10 Score and Lower Grades of Bone Marrow Fibrosis

Introduction: The vast majority ofmyeloproliferative neoplasms (MPNs) patients are characterized by a molecular genetic background and by variable symptoms reflecting disease burden that may correlate with prognosis.Aim: To study the impact of triple negative status of driver gene mutations: Janus kinase 2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia virus oncogene (MPL) on disease burden and its correlation with symptom severity (MPN10 score) and degree of bone marrow (BM) fibrosis in MPNs patients.Patients and Methods: MPN Symptom Assessment Form Total Symptom Score (MPN-SAF TSS) was assessed as median of 10 items: fatigue, concentration, early satiety, inactivity, night sweats, itching, bone pains, abdominal discomfort, weight loss and fever. JAK2V617F and MPL W515exon 10 mutations were performed by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) while CALR exon 9 insertion/deletion was detected by high-resolution melting (HRM) curve analysis.Results: 100 MPNs patients (54 males and 46 females): 18 polycythemia vera (PV), 41 essential thrombocythemia (ET), 24 primary myelofibrosis (PMF), 10 Post-ET/PV-myelofibrosis (post-ET/PV-MF) and 7 myelodysplastic/myeloproliferative neoplasms (MDS/MPN) were included. Median age at diagnosis was 55 years (17-75) and was lower in ET than PV and PMF patients; 44 (19-75) years vs. 56 (34-70) years and 56 (20-75) years, respectively (p=0.06). JAK2 mutation was positive in 15 (85%) PV patients, 14 (34%) ET patients, 15 (62%) PMF patients, 8(80%) post-ET/PV-MF patients and zero (0%) MDS/MPN patients (p<0.001). CALR mutation was positive in zero (0%) PV patients, 10 (24%) ET patients, 4 (17%) PMF patients, zero (0%) Post-ET/PV-MF patients and zero (0%) MDS/MPN patients (p<0.05). MPL mutation was positive in zero (0%) PV patients, 2 (5%) ET patients, 1(4%) PMF patients, zero (0%) Post ET/PV-MF patients and zero (0%) MDS/MPN patients. Twenty four/93 (26%) patients were triple negative; 15 ET (16%), 3 PV (3%), 6 PMF (6%). Median MPN10 score was 21 (4-45) in ET versus 37.5 (25-56) in PV, 54 (15-80) in PMF and 59 (45-75) in Post-ET/PV-MF (p<0.001). Median MPN10 score was 25 (10-50) in triple negative patients vs. 40 (4-80) in MPNs patients showing at least one driver mutation positivity (p<0.001). BM fibrosis was present in 6 (15%) patients with triple negative vs. 33 (85%) patients showing at least one molecular marker positivity (p=0.007). Out of 52 patients having splenomegaly; seven (13.5%) patients were triple negative vs. 45 (87%) patients with at least one gene mutation (p<0.001). Out of the 24 triple negative patients, 19 (80%), 4 (16%), 1 (3%) and 0(0%) had BM fibrosis grades 0, 1, 2 and 3 vs. 36 (52%), 7 (10%), 12 (17%), 14 (20%) out of 69 patients with at least one gene mutation, respectively (p=0.002). After a median follow-up period of 16 months (3-151), overall survival (OS) was 95%. OS in PV and ET patients was 100 % versus 83 % in PMF patients (p=0.08). OS in triple negative group was 100% versus 94% in the gene mutations group (p=0.387).Conclusion: Driver gene mutations show an impact on disease symptoms and burden. Triple negative MPNs patients in our cohort have significantly low MPN10 score and less BM fibrosis which may indicate a more indolent disease course. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification of the Dominant Genotypes of Phytophthora infestans in Canada Using Real-Time PCR with ASO-PCR Assays.

Phytophthora infestans, a pathogenic oomycete that is the causal agent of potato and tomato late blight, has devastating effects worldwide. The genetic composition of P. infestans populations in Canada has changed considerably over the last few years, with the appearance of several new genotypes showing different mating types and sensitivity to the fungicide metalaxyl. Genetic markers allowing for a rapid assessment of genotypes from small amounts of biological material would be beneficial for the early detection and control of this pathogen throughout Canada. Mining of the P. infestans genome revealed several regions containing single-nucleotide polymorphisms (SNP) within both nuclear genes and flanking sequences of microsatellite loci. Allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) assays were developed from 14 of the 50 SNP found by sequencing. Nine optimized ASO-PCR assays were validated using a blind test comprising P. infestans and other Phytophthora spp. The assays revealed diagnostic profiles unique to each of the five dominant genotypes present in Canada. The markers developed in this study can be used with environmental samples such as infected leaves, and will contribute to the genomic toolbox available to assess the genetic diversity of P. infestans at the intraspecific level. For late blight management, early warning about P. infestans genotypes present in potato and tomato fields will help growers select the most appropriate fungicides and application strategies.