Allele-specific Real-time Polymerase Chain Reaction Research Articles

Epidermal growth factor receptor mutations in non-small cell lung cancer undetected by high-sensitivity allele-specific real-time polymerase chain reaction-based assays.

Identifying epidermal growth factor receptor (EGFR) mutation status is critical for planning lung cancer treatment. Sanger sequencing detects both known and novel mutations but shows poor sensitivity. High-sensitivity allele-specific real-time polymerase chain reaction (ASRP)-based assays offer quick and reliable results, but may overlook uncommon mutations. We aimed to define the rate at which high-sensitivity ASRP-based assays missed uncommon EGFR mutations. Non-small cell lung cancer specimens that were diagnosed as EGFR wild-type (EGFR-WT) by high-sensitivity ASRP-based assays and had residual DNA samples were sent for Sanger sequencing. Patient characteristics and clinical features were evaluated by chart review, and outcomes of EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy were studied. Hundred DNA specimens diagnosed by high-sensitivity ASRP-based assays as EGFR-WT were rechecked by Sanger sequencing. Two samples which were re-biopsy specimens from patients with EGFR mutations were excluded from the analysis. Sanger sequencing was failed in 24 samples. Among the remaining 74 samples, 6 (8.1%) had EGFR mutations-one exhibited exon 19 deletion (delT751_I759insS), two exhibited substitution mutations (S768I+V769L and L861Q), and three exhibited exon 20 insertions (N771_P772insN, P772_H773insHP, and H773_V774insAH). Only the patient with the exon 19 deletion had received EGFR-TKI therapy. Although the best tumor response was only stable disease, this was maintained for >10 months. High-sensitivity ASRP-based assays can overlook uncommon mutations. This detection failure rate is worth noting, especially when treating patients from regions known to have a high prevalence of EGFR mutation. Patients carrying uncommon mutations may still benefit from EGFR-TKI therapy.

Subclonal NT5C2 mutations are associated with poor outcomes after relapse of pediatric acute lymphoblastic leukemia

AbstractActivating mutations in cytosolic 5′-nucleotidase II (NT5C2) are considered to drive relapse formation in acute lymphoblastic leukemia (ALL) by conferring purine analog resistance. To examine the clinical effects of NT5C2 mutations in relapsed ALL, we analyzed NT5C2 in 455 relapsed B-cell precursor ALL patients treated within the ALL-REZ BFM 2002 relapse trial using sequencing and sensitive allele-specific real-time polymerase chain reaction. We detected 110 NT5C2 mutations in 75 (16.5%) of 455 B-cell precursor ALL relapses. Two-thirds of relapses harbored subclonal mutations and only one-third harbored clonal mutations. Event-free survival after relapse was inferior in patients with relapses with clonal and subclonal NT5C2 mutations compared with those without (19% and 25% vs 53%, P < .001). However, subclonal, but not clonal, NT5C2 mutations were associated with reduced event-free survival in multivariable analysis (hazard ratio, 1.89; 95% confidence interval, 1.28-2.69; P = .001) and with an increased rate of nonresponse to relapse treatment (subclonal 32%, clonal 12%, wild type 9%, P < .001). Nevertheless, 27 (82%) of 33 subclonal NT5C2 mutations became undetectable at the time of nonresponse or second relapse, and in 10 (71%) of 14 patients subclonal NT5C2 mutations were undetectable already after relapse induction treatment. These results show that subclonal NT5C2 mutations define relapses associated with high risk of treatment failure in patients and at the same time emphasize that their role in outcome is complex and goes beyond mutant NT5C2 acting as a targetable driver during relapse progression. Sensitive, prospective identification of NT5C2 mutations is warranted to improve the understanding and treatment of this aggressive ALL relapse subtype.

HIgh accuracy of HLA-B*27 genotyping by allele-specific real-time polymerase chain reaction in a heterogeneous population

HIgh accuracy of HLA-B*27 genotyping by allele-specific real-time polymerase chain reaction in a heterogeneous population

Gender-specific differences in clinical profile of hypertrophic cardiomyopathy and their association with the polymorphic variant rs1739843 of the hspb7 gene

Objective. The aim of this study was to investigate gender-specific differences in the clinical profile of hypertrophic cardiomyopathy (HCM) and to determine the impact of polymorphic variant rs1739843 of the HSPB7 gene on clinical profile and outcomes in women and men with HCM. Design and methods . The study population consisted of 171 patients with HCM ≥ 18 years old. A novel disease pathway model was employed to assess clinical course of HCM. Single nucleotide polymorphism (SNP) rs1739843 of the HSPB7 gene was genotyped by allele-specific real-time polymerase chain reaction assay. Results. We found no significant gender-specific differences in clinical course of HCM in patients ≥ 18 years old during 10-year follow-up. High prevalence of T allele of rs1739843 of the HSPB7 gene was observed in women > 18 years old with HCM and chronic heart failure (CHF) with preserved ejection fraction (EF) ( ≥ 50 %) (C: Т , odds ratio (OR) = 0,213, 95 % confidence interval (CI) = 0,077-0,593, p 18 years old.

High accuracy of HLA-B*27 genotyping by allele-specific real-time polymerase chain reaction in a heterogeneous population compared to flow cytometry and single nucleotide polymorphism detection assays

HLA-B27 is a risk marker for ankylosing spondylitis and other associated seronegative spondyloarthropathies. We compared three methods of HLA-B*27 typing in a New South Wales (NSW) population: flow cytometry, rs4349859 single nucleotide polymorphism (SNP) detection assay, and allele-specific real-time polymerase chain reaction (RT-PCR) analysis of exons 2 and 3. Over a 5-month period, 543 samples underwent flow cytometric testing and RT-PCR high-resolution melt analysis of rs4349859 SNP and of exon 2 (5' fragment) and exon 3. In the third method, positive samples were further analysed with fluorescent resonance emission transfer (FRET) RT-PCR of exon 2 fragments, 2a and 2b. HLA-B*27 and other genotypes were confirmed by Sanger sequencing of a 600 base pair fragment of exons 2 and 3. In our cohort, the rs4349859 SNP method had 78.6% sensitivity and 98.7% specificity. Screening with exon 2 (5' fragment) and exon 3 RT-PCR provided 100% sensitivity. Further testing with exon 2a and 2b FRET RT-PCR produced 100% specificity. This cascade approach with allele-specific RT-PCR assays was able to differentiate all samples into HLA-B*27 subtypes. HLA-B*27 genotyping with allele-specific RT-PCR assays, to screen for and confirm HLA-B27 positive samples, was more sensitive and specific than flow cytometry and rs4349859 SNP assays. It is a potentially cost-effective method for differentiating HLA-B27 subtypes. Our cascade genetic testing approach is suitable for replacing the current flow cytometric HLA-B27 assay for the heterogeneous NSW population.

Polymorphic variant rs1739843 of heat shock protein beta-7 (HSPB7) gene and its relationship with on clinical profile and outcomes in patients with hypertrophic cardiomyopathy (results of a 10-year follow-up)

Aim. To determine the impact of polymorphic variant rs1739843 of the HSPB7 gene on clinical profile and outcomes in patients with hypertrophic cardiomyopathy (HCM).Material and methods. The study population consisted of 108 patients with HCM ≥45 years old. The control group included 192 healthy donors. The design of the study included an assessment of the clinical course, determining the outcome of HCM using a new methodological approach proposed by Rowin EJ, et al. (2017). Polymorphic variant rs1739843 of the HSPB7 gene was genotyped by allele-specific real-time polymerase chain reaction (PCR) assay.Results. It was found a significant increase in frequency of TT genotype of rs1739843 of the HSPB7 gene in patients with HCM — 20,4%, compared with control group — 4,2% (ТТ: ТС+СС, odds ratio (OR) =5,88, 95% confidence interval (CI) =2,52-13,75, p&lt;0,001). High prevalence of CC genotype of rs1739843 of the HSPB7 gene was observed in control group — 80,2% vs 31,5% in HCM group (CC: ТС+TT, OR=0,11, 95% CI=0,07-0,19, p&lt;0,001). The allele frequency (С:Т) also differs between HCM and control groups — 55,6:44,4% in HCM vs 88,02:11,98% in control group (OR=5,88, 95% CI=3,91-8,85, p&lt;0,001). It was also found a significant increase in frequency of TT genotype and T allele of rs1739843 of the HSPB7 gene in HCM patients with oligosymptomatic HCM course — 16,7%, compared with control group — 4,2% (ТТ: ТС+СС, OR=4,60, 95% CI=1,63-12,99, p&lt;0,001). HCM patients ≥45 years old showed a significant increase in T allele frequency in cases of presence of 2 (FC III-IV CHF (chronic heart failure)+AF (atrial fibrillation), 18,8% vs 6,6%) and 3 adverse pathways (FC III-IV CHF+AF+SCD (sudden cardiac death), 4,2% vs 1,6%).Conclusion. HCM progression along 2 and more adverse pathways in patients ≥45 years old has been characterized with adverse outcome. The T allele and TT genotype of rs1739843 of the HSPB7 gene were more frequent in patients with HCM ≥45 years old, compared with control group. It was also found a significant increase in frequency of TT genotype and T allele of rs1739843 of the HSPB7 gene in HCM patients with oligosymptomatic HCM course, compared with control group.Allele T of rs1739843 of the HSPB7 gene is associated with 2 and more adverse pathways of HCM progression.

&lt;i&gt;STAT4&lt;/i&gt; rs7574865 G/T and &lt;i&gt;IRF5&lt;/i&gt; rs2004640 G/T polymorphisms as markers of predisposition to juvenile idiopathic arthritis. What can genetics give to understand its heterogeneity?

Juvenile idiopathic arthritis (JIA) is a multifactorial immune-mediated inflammatory disease in childhood, the most common type of rheumatic disease in children. It is characterized by the polygenic type of hereditary predisposition.Objective:to study the association of STAT4 rs7574865 G/T and IRF5 rs2004640 G/T polymorphisms with the predisposition to certain JIA subtypes in the Russian pediatric population.Patients and methods.The investigation enrolled 177 patients, including 66 patients diagnosed with JIA and 111 healthy unrelated volunteers (a control group). Of the 66 patients with JIA there were 30 (45%) with oligoarthritis: 20 (67%) with human leukocyte antigen B27(HLA-B27)-positive JIA (that was associated with enthesitis, HLA-B27 positive JIA (JIA-B27), 10 (33%) with anterior uveitis concurrent with antinuclear antibody-positive JIA (JIA-uveitis); 20 (30%) with polyarticular JIA (JIA-poly), seronegative for rheumatoid factor; and 16 (24%) with systemic JIA (JIA-sys). As a control for genotyping STAT4 rs7574865 G/T and IRF5 rs2004640 G/T polymorphisms, the investigators studied 103 and 111 DNA samples from healthy adult volunteers, respectively. STAT4 rs7574865 G/T and IRF5 rs2004640 G/T polymorphisms were investigated using allele-specific real-time polymerase chain reaction (RT-PCR).Results and discussion.In the oligoarticular JIA group, the frequency of the STAT4 T allele was significantly higher than that in the control group (38.3 and 20.4%, respectively; p=0.004). This allele was also significantly more common in the JIA-B27 (35.0 and 20.4%, respectively; p=0.044) and JIA-uveitis (45.0 and 20.4%, respectively; p=0.021) groups compared with the control one. No significant differences were found in the frequency of the mutant STAT4 T allele between the control group and the JIA-sys and JIA-poly groups. Regression analysis showed that the identification of the STAT4 T allele was associated with the high risk of a predisposition to oligoarticular JIA as a whole (odds ratio, OR 2.43; 95% confidence interval (CI) 1.23–4.70; p=0.007), as well as to the antinuclear antibody-positive oligoarticular JIA with uveitis (JIA-uveitis): the risk in T allele carriers was 3.2 times higher than that in the control (OR 3.19; 95% CI 1.09–9.06; p= ). A high risk for predisposition was also found in the JIA-B27 subgroup compared with the control (OR 2.10; 95% CI 0.38–4.60; p=0.070). There were no statistical differences in the frequency of genotypes and alleles of the IRF5 rs2004640 G/T polymorphism between the entire group of JIA as a whole and its individual clinical types, as well as the control group.Conclusion.This pilot study confirmed that the STAT4 rs7574865 G/T polymorphism was associated with the risk of oligoarticular JIA, mainly that of JIA-uveitis and JIA-B27.

Metaplastic thymoma: a distinctive thymic neoplasm characterized by YAP1-MAML2 gene fusions.

Metaplastic thymomas are rare biphasic thymic tumors that are characteristically well-circumscribed, confined to the thymus, and follow a benign to indolent clinical course. Their relationship to other thymic neoplasms remains unclear, and their molecular characteristics have not been defined. We report for the first time recurrent translocation events in metaplastic thymomas involving the Yes Associated Protein 1 (YAP1) and Mastermind Like Transcriptional Coactivator 2 (MAML2) genes. Eight metaplastic thymomas were retrieved from two institutions' archives over a 21-year period. Paraffin-embedded material from all cases underwent targeted DNA-based hybrid capture next-generation sequencing. Cases showing no somatic alterations subsequently underwent targeted RNA sequencing. Allele-specific real-time polymerase chain reaction was performed to detect GTF2I c.74146970T>A (p.L424H) mutations. All cases showed characteristic histologic features of metaplastic thymoma and demonstrated no local recurrence or distant metastatic disease at 1-22 years of follow-up. Six of eight cases were successfully sequenced, all showing YAP1-MAML2 fusions; in four cases the fusions were detected by DNA sequencing and in two cases by RNA sequencing. Two distinct products were identified: 5' YAP1 exon 1 fused to 3' MAML2 exons 2-5 or 5' YAP1 exons 1-5 fused to 3' MAML2 exons 2-5. All cases underwent allele-specific real-time polymerase chain reaction and demonstrated no GTF2I L424H mutations. Metaplastic thymoma is a distinct, clinically indolent thymic epithelial neoplasm characterized by YAP1-MAML2 fusion and lacking the GTF2I mutations found in Type A and AB thymomas.

P894The association of the polymorphic variant rs1739843 of the hspb7 gene with clinical profile and outcomes in patients with hypertrophic cardiomyopathy (10 years follow-up)

Abstract Purpose The aim of this study was to determine the impact of polymorphic variant rs1739843 of the hspb7 gene on clinical profile and outcomes in patients with hypertrophic cardiomyopathy (HCM). Methods The study population consisted of 108 patients with HCM ≥45 years old. The control group included 192 healthy donors. A novel disease pathway model, firstly designed in foreign outcome study (2017), was employed to assess clinical course of HCM. SNP rs1739843 of the hspb7 gene was genotyped by allele-specific real-time polymerase chain reaction (PCR) assay. Results It was found a significant increase in TT genotype frequency of rs1739843 of the hspb7 gene in patients with HCM (n=108) – 20.4%, compared with control group (n=192) – 4.2% (TT: TC+CC, odds ratio (OR) = 5.88, 95% confidence interval (CI) = 2.52–13.75, p&lt;0.001). High prevalence of CC genotype of rs1739843 of the hspb7 gene was observed in control group – 80.2% vs 31.5% in HCM (CC: TC+TT, OR = 0.11, 95% CI = 0.07–0.19, p&lt;0.001). The allele frequency (C: T) also differed between HCM and control groups – 55.6: 44.4% in HCM, vs 88.02: 11.98% in control group (OR = 5.88, 95% CI = 3.91–8.85, p&lt;0.001). It was also found a significant increase in TT genotype frequency of rs1739843 of the hspb7 gene in HCM patients with benign course free of adverse pathways (n=48) – 16.7%, compared with control group (n=192) – 4.2% (TT: TC+CC, OR= 4.60, 95% CI = 1.63–12.99, p&lt;0.001)). The allele frequency (C: T) in HCM patients with benign course free of adverse pathways was 56.3: 43.7% vs 88.02: 11.98% in control group (OR = 5.71, 95% CI= 3.44–9.49, p&lt;0.001). The mortality rate of HCM patients with 1, 2 or 3 adverse pathways was higher compared with HCM patients with benign course free of adverse pathways. HCM patients ≥45 years old showed a significant increase in T allele frequency in cases of presence of 2 (CHF (chronic heart failure) III–IV functional class (NYHA) + AF (atrial fibrillation)) and 3 adverse pathways (CHF III-IV functional class (NYHA) + AF + SCD (sudden cardiac death) of HCM progression. Conclusions The T allele and TT genotype of rs1739843 of the hspb7 gene were more frequent in patients with HCM ≥45 years old, compared with control group. It was also found a significant increase in frequency of TT genotype and T allele of rs1739843 of the hspb7 gene in HCM patients with benign course free of adverse pathways, compared with control group. HCM progression along 2 and more adverse pathways in patients ≥45 years old has been characterized with adverse outcome. Allele T of rs1739843 of the hspb7 gene was associated with 2 and more adverse pathways of HCM progression.

Association of NLRP3 and CARD8 Inflammasome Polymorphisms With Aseptic Loosening After Primary Total Hip Arthroplasty.

Our aim was to investigate the association of inflammasome polymorphisms NLRP3 rs35829419 (p. Q705K) and CARD8 rs2043211 (p. C10X) with aseptic loosening of total hip endoprostheses. We asked whether patients with the loosening of total hip arthroplasty earlier than 15 years after primary implantation had a higher proportion of the polymorphisms Q705K and C10X in comparison to subjects without loosening. A retrospective case-control study compared 36 patients with total hip endoprosthesis loosening earlier than 15 years after primary implantation and 51 control subjects with unloosened total hip endoprostheses, matched for gender, age, and follow-up period. Buccal mucosa samples were used for genomic DNA analysis and genotyped for NLRP3 rs35829419 and CARD8 rs2043211 using a fluorescence-based competitive allele-specific real-time polymerase chain reaction. The proportion of subjects with both wild-type NLRP3 and CARD8 (i.e., without Q705K or C10X) was considerably higher in the control group when compared with patients with early total hip arthroplasty loosening (49% vs. 28%; p = 0.05). After adjustment for gender, age, and follow-up, patients with combined wild type of both NLRP3 and CARD8 had significantly smaller odds for early implant loosening (odds ratio 0.33, p = 0.02). Investigated polymorphisms may influence several inflammatory pathways and contribute to the loosening of artificial implants with potential clinical significance for the appropriate selection of patients and endoprostheses when planning elective total hip arthroplasty. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:417-421, 2020.

JAK2V617F-positive clonal hematopoiesis of indeterminate potential in pregnant women

Aim: to assess the prevalence of V617F somatic mutation of the JAK2 gene in pregnant women.Materials and methods. This non-interventional study was performed in the framework of routine clinical practice and included 1532 samples of venous blood from pregnant women who applied for medical assistance at Krasnoyarsk Regional Clinical Center for Maternal and Child Welfare. We used blood samples left after all routine laboratory tests had been done. These leftovers were pooled in the way that ensured an equal ratio of nucleated cells. Each pool contained 7 separate blood samples. The unused samples that remained after the pooling were frozen and stored at –20°C until the end of entire testing procedure. The V617F JAK2 mutation was detected by the real-time allele-specific polymerase chain reaction test.Results. Among the examined pregnant women, 6 (0.4 %) were identified as carriers of V617F JAK2 mutation. Three women with this mutation suffered from infertility for 4, 5, and 10 years; two of them had repeated miscarriages in the first trimester of pregnancy. The 6 women – carriers of this mutations had no concomitant genetic polymorphisms typical of thrombophilia (factors FII, FV), and no abnormal coagulation characteristics. Analysis of their medical records showed that in the past, two of these women had gestational hypertension, one developed a clinical picture of preeclampsia, and another one (with the maximum presence of the mutant allele) had a history of acute lymphoblastic leukemia followed by stable remission.Conclusion. The routine laboratory detection of the V617F JAK2 mutation can facilitate timely identification of the increased risk of pregnancy pathology, as well as timely diagnosis of hematological cancer.

Assessment of a High Sensitivity Method for Identification of IDH1 R132x Mutations in Tumors and Plasma of Intrahepatic Cholangiocarcinoma Patients.

Hotspot codon 132 mutations (R132xIDH1m) are frequent in intrahepatic cholangiocarcinoma (ICC), are druggable by anti-IDH1m agents, and could represent a marker of disease progression. Developing an assay to identify R132xIDH1m would provide a useful tool to select patients benefitting from targeted treatments. We tested a quantitative real-time allele-specific polymerase chain reaction (qPCR)-based method to detect the main R132xIDH1m in an Italian ICC series (n = 61) of formalin-fixed paraffin-embedded (FFPE) samples, and on circulating-free DNA samples. The outcomes were compared with nested PCR/Sanger sequencing. Reconstitution experiments of plasmids harboring the different R132xIDH1m mixed with wild-type (WT) DNA demonstrated that qPCR is able to detect at least 2% of all mutated allele. High efficiency was also observed on patient-derived mutated DNA mixed with WT DNA (up to 10% and 0.3 ng of mutated template); qPCR detected 16.4% of mutated samples (one R132G, three R132C and six R132L) while nested PCR/Sanger sequencing only 8.2% (four R132L and one R132G). In a single patient with an R132C-mutated tumor, qPCR was also performed on plasma samples collected at four time-points, observing an increase correlating with disease progression. In conclusion, we developed a qPCR assay which could represent a fast, inexpensive and sensitive tool both for detection of R132xIDH1m in ICC samples and monitoring disease progression from liquid biopsy.

HLA-B*27 genotyping by real-time polymerase chain reaction is superior to rs4349859 single nucleotide polymorphism detection and flow cytometry in a heterogeneous population

Aim: HLA-B*27 is a risk marker for ankylosing spondylitis. We compared three methods of HLA-B*27 detection in a New South Wales (NSW) population – flow cytometry, rs4349859 single nucleotide polymorphism (SNP) assay, and allele-specific real-time polymerase chain reaction (RT-PCR) analysis of exons 2 and 3.

Rapid and Reliable One-Step ABO Genotyping Using Direct Real-Time Allele-Specific PCR and Melting Curve Analysis Without DNA Preparation

ABO genotyping is a molecular diagnostic technique important for transfusion and transplantation in medicine, and human identification in forensic science. Because ABO genotyping are labor intensive and time consuming, the genotyping cannot be firstly used to resolve the serological ABO discrepancy in blood bank. For rapid one-step ABO genotyping, we developed direct, real-time, allele-specific polymerase chain reaction (PCR), and melting curve analysis (DRAM assay) without DNA preparation. In DRAM assay, we used a special PCR buffer for direct PCR, a rapid RBC lysis buffer, white blood cells as template without DNA preparation, allele-specific primers for discriminating three ABO alleles (261G/del, 796C/A, and 803G/C), and melting curve analysis as a detection method. There was 100% concordance among the results of ABO genotyping by the DRAM assay, serologic typing, PCR-RFLP and PCR-direct sequencing of 96 venous blood samples. We were able to reduce the number of manual steps to three and the hands-on time to 12min, compared to seven steps and approximately 40min for conventional ABO genotyping using allele-specific PCR with purified DNA and agarose gel electrophoresis. We have established and validated the DRAM assay for rapid and reliable one-step ABO genotyping in a closed system. The DRAM assay with an appropriate number of allele-specific primers could help in resolving ABO discrepancies and should be valuable in clinical laboratory and blood bank.

Phase II Trial of Bortezomib-Based Induction, Autologous Stem Cell Transplantation, Bortezomib-Based Consolidation, and Bortezomib Maintenance in Newly Diagnosed Multiple Myeloma in Japan

Background: Three drug combinations are the standard treatment for newly diagnosed multiple myeloma (NDMM). However, induction, consolidation, and maintenance therapy have not been standardized in Japan. Therefore, in this single arm Phase II study, we evaluated bortezomib-based induction, autologous stem cell transplantation (ASCT), bortezomib-based consolidation, and bortezomib maintenance in transplant eligible NDMM patients and assessed clinical outcomes as well as the minimal residual disease (MRD) status.Methods: Patients received four cycles of CyBorD induction therapy with bortezomib 1.3 mg/m2 and cyclophosphamide 300 mg/m2 on day 1, 8, 15, and 22 and dexamethasone 40 mg on day 1-3, 8-10, 15-17, and 22-24 for the first two cycles and day 1, 8, 15, and 22 for the last two cycles of four 28-day cycles. Peripheral blood stem cells were collected after cyclophosphamide 2 g/m2 for 2 days, which was followed by melphalan 200 mg/m2 and ASCT. Three months after ASCT, patients received consolidation treatment with three cycles of CyBorD identical to the last two cycles of the induction therapy followed by maintenance therapy with bortezomib 1.3 mg/m2 on day 1 and 15 of a 28-day cycle, for 24 months. The primary end-point was the complete response (CR) rate after consolidation therapy and assessed as an interim analysis. Responses were assessed according to the International Myeloma Working Group (IMWG) criteria. MRD was assessed using an allele-specific oligonucleotide real-time quantitative polymerase chain reaction (qPCR). The toxicity was evaluated according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE).Results: From August 2013 to May 2016, 42 (25 male and 17 female) patients with a median age of 58 (range 42-65) years with NDMM were found eligible and enrolled in 15 centers in Japan. The International Staging System (ISS) values were 1 in 17 (40%), 2 in 20 (48%), and 3 in 5 patients (12%). Adverse cytogenetics of 17p deletion, t(4;14), and t(14;16) were observed in 20%, 18%, and 3% of the evaluable patients, respectively. Following four induction cycles of CyBorD, the overall response rate (ORR) was obtained in 71% of patients, including a CR/sCR of 10% and very good partial response (VGPR) of 26%. One of the evaluable 11 patients showed MRD negativity after induction therapy. Four patients discontinued the protocol during the induction therapy because of grade 4 interstitial pneumonia (n = 2), prolonged grade 3 drug eruption (n = 1), and grade 1 delirium (n = 1). Four patients discontinued the protocol due to doctor judgement (inadequate efficacy, n = 2; repetitive infection, n = 1; grade 3 neutropenia, n = 1). A total of 26 of the 42 patients completed ASCT following the protocol and 18 achieved VGPR/CR, including CR in 10 patients. Three of the evaluable 10 patients showed MRD negativity after ASCT. The 2-year overall and progression-free survival rates were 93% (95% confidence interval [CI], 76%-98%) and 81% (95% CI, 51%-94%), respectively (Figure 1).Conclusions: CyBorD with ASCT for NDMM resulted in relatively high CR rates in the investigated Japanese population, although a relatively high incidence of discontinuation of therapy was observed. Dose and schedule modification of induction therapy may be necessary in Japanese populations. Clinical trial information: UMIN000010542. [Display omitted] DisclosuresImada:Celgene: Honoraria; Bristol-Meyers Squibb: Honoraria; Takeda: Honoraria; Novartis: Honoraria; Nippon Shinyaku: Honoraria; MSD: Honoraria; Chugai: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Kyowa Hakko Kirin: Honoraria; Shire Japan: Honoraria; Ono: Honoraria; Mundipharma: Honoraria. Takaori-Kondo:Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Janssen Pharmaceuticals: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria.

Molecular discrimination of Panax ginseng cultivar K-1 using pathogenesis-related protein 5 gene

BackgroundThe mixed-cultivation of different Panax ginseng cultivars can cause adverse effects on stability of yield and quality. K-1 is a superior cultivar with good root shape and stronger disease resistance. DNA markers mined from functional genes are clearly desirable for K-1, as they may associate with major traits and can be used for marker-assisted selection to maintain the high quality of Korean ginseng. MethodsFive genes encoding pathogenesis-related (PR) proteins of P. ginseng were amplified and compared for polymorphism mining. Primary, secondary, and tertiary structures of PR5 protein were analyzed by ExPASy-ProtParam, PSSpred, and I-TASSER methods, respectively. A coding single nucleotide polymorphism (SNP)–based specific primer was designed for K-1 by introducing a destabilizing mismatch within the 3′ end. Allele-specific polymerase chain reaction (PCR) and real-time allele-specific PCR assays were conducted for molecular discrimination of K-1 from other cultivars and landraces. ResultsA coding SNP leading to the modification of amino acid residue from aspartic acid to asparagine was exploited in PR5 gene of K-1 cultivar. Bioinformatics analysis showed that the modification of amino acid residue changed the secondary and tertiary structures of the PR5 protein. Primer KSR was designed for specific discrimination of K-1 from other ginseng cultivars and landraces. The developed real-time allele-specific PCR assay enabled easier automation and accurate genotyping of K-1 from a large number of ginseng samples. ConclusionThe SNP marker and the developed real-time allele-specific PCR assay will be useful not only for marker-assisted selection of K-1 cultivar but also for quality control in breeding and seed programs of P. ginseng.

A model for predicting effect of treatment on progression-free survival using MRD as a surrogate end point in CLL

AbstractOur objective was to evaluate minimal residual disease (MRD) at the end of induction treatment with chemoimmunotherapy as a surrogate end point for progression-free survival (PFS) in chronic lymphocytic leukemia (CLL) based on 3 randomized, phase 3 clinical trials (ClinicalTrials.gov identifiers NCT00281918, NCT00769522, and NCT02053610). MRD was measured in peripheral blood (PB) from treatment-naïve patients in the CLL8, CLL10, and CLL11 clinical trials, and quantified by 4-color flow cytometry or allele-specific oligonucleotide real-time quantitative polymerase chain reaction. A meta-regression model was developed to predict treatment effect on PFS using treatment effect on PB-MRD. PB-MRD levels were measured in 393, 337, and 474 patients from CLL8, CLL10, and CLL11, respectively. The model demonstrated a statistically significant relationship between treatment effect on PB-MRD and treatment effect on PFS. As the difference between treatment arms in PB-MRD response rates increased, a reduction in the risk of progression or death was observed; for each unit increase in the (log) ratio of MRD− rates between arms, the log of the PFS hazard ratio decreased by −0.188 (95% confidence interval, −0.321 to −0.055; P = .008). External model validation on the REACH trial and sensitivity analyses confirm the robustness and applicability of the surrogacy model. Our surrogacy model supports use of PB-MRD as a primary end point in randomized clinical trials of chemoimmunotherapy in CLL. Additional CLL trial data are required to establish a more precise quantitative relationship between MRD and PFS, and to support general applicability of MRD surrogacy for PFS across diverse patient characteristics, treatment regimens, and different treatment mechanisms of action.

Methods and clinical values for minimal residual disease detection in patients with multiple myeloma

The complete response (CR) rates in patients with multiple myeloma (MM) have been rapidly increasing after the approval of novel agents such as proteasome inhibitors, immunomodulatory drugs, histone deacetylase inhibitors, and monoclonal antibodies. Recently, patients administered with a combination of novel agents with autologous stem-cell transplantation achieved a 70% CR rate. The assessment of minimal residual disease (MRD) is necessary for effective stratification of the CR cases and the generation of accurate prognoses. This review summarizes recent topics pertaining to MRD detection methods (multiparameter flow cytometry, allele-specific oligonucleotide real-time quantitative polymerase chain reaction, droplet digital PCR, and next-generation sequencing) and the prognostic value of MRD assessment in patients with MM.

FcrR3A-158 Polymorphism and Stromal Tumor-Infiltrating Lymphocytes and Survival among Patients with Metastatic HER2-Positive Breast Cancer Receiving Trastuzumab-Based Treatment.

PurposeThe prognosis of human epidermal growth factor receptor 2 (HER2)-positive breast cancer has markedly improved since the introduction of trastuzumab. We aimed to evaluate the association between stromal tumor-infiltrating lymphocyte (sTIL) or FcrR polymorphisms and survival among patients with metastatic HER2-positive breast cancer who were treated with trastuzumab.MethodsA total of 56 women with recurrent or metastatic HER2-positive breast cancer who received the trastuzumab-taxane combination as first-line treatment were included in this retrospective analysis. The single-step multiplex allele-specific real-time polymerase chain reaction technique was employed for FcrR3A genotyping. sTILs were identified via immunohistochemical analysis of surgical (n=34, 60.7%) or biopsy specimens of metastatic lesions (n=22, 39.3%).ResultsWe classified patients based on the sTIL level (≤10% [n=44] or >10% [n=12]); high sTIL counts were more commonly observed in patients with hormone receptor-negative tumors than in those with hormone receptor-positive tumors (34.8% vs. 12.1%, p=0.02). There was a significant association between high sTIL levels and longer progression-free survival in comparison to low sTIL levels (median, 28.4 months vs. 16.8 months; p=0.03). With regard to the FcrR3A-158 genotype, patients were classified into the Phenylalanine/Phenylalanine group (23 patients, 41.1%), Phenylalanine/Valine group (23 patients, 41,1%), or Valine/Valine group (10 patients, 17.9%); these classifications were not associated with clinical outcomes.ConclusionHigh sTIL expression may be associated with better efficacy of trastuzumab-containing therapy in patients with metastatic HER2-positive breast cancer. However, this finding warrants further evaluation in the larger population.

Genetic and Epigenetic Mechanisms of Endometral Reciptivity Disorder in Patients Born with Low Weight

The aim is to study genetic and intrauterine epigenetic mechanisms of endometrial receptivity disorders in patients with reproductive failures. 96 girls of adolescents with disturbed menstrual function as anomalous uterine bleeding (AUB PP) and 210 women of reproductive age, suffering from uterine infertility or miscarriage, due to thin endometrium were examined. All the patients were born on the deadline. Within the main cohorts, the patients were stratified according to their birth weight. Clinical anamnestic data and molecular genetic studies of polymorphic variants of sex steroids receptor genes, endothelial function regulation genes, angiogenesis and thrombophilia were analyzed using the real-time allele-specific polymerase chain reaction with melting curves of the amplification products using a set of reagents and protocols of the company Test Gen LTD (Russia). The risk of reproductive failures due to impaired receptivity of the endometrium is associated with the carrier of the polymorphic allele A of the VEGF2578C gene> A: for patients with infertility (OR = 2.73 (1.36-5.45), p = 0.01) and miscarriage OR = 4.61 (2.19-9.71), p = 0.01) and bearing the polymorphic allele 675 4G of the PAI-1 gene 675 5G> 4G: for patients with infertility (OR = 8.45 (3, 96-18,21), p = 0,001) and miscarriage (OR = 9.98 (4.61-21.74), p = 0.001). The carrier of polymorphism pVull-СС of the gene ESR1 397T> C is associated with an increased risk of disruption of the development of the menstrual function, manifested by abnormal uterine bleeding of the pubertal period (OR = 4.58 (0.97-21.68) p = 0.04), and the risk of developing in the reproductive age of miscarriage, caused by a thin endometrium (OR = 6.79 (1.94-23.75), p = 0.01). In women born with low weight, a significantly higher frequency of genotypes containing polymorphic allele 786C of the gene for endothelial NO-synthase NOS3 786 T> C was determined: for women with infertility OR = 6.173 (1.83-20.83); p = 0.001; for women with miscarriage OR = 4.902 (1.69-14.08); p = 0.002; for girls OR = 2.56 (1.01-6.50); p = 0.04. The genetic network containing the described variable alleles coordinates the pathological nature of the regulation of the endometrial function, which can lead to the formation of a thin non-receptive endometrium in response to traumatic injury.