Allele-specific Real-time Quantitative PCR Research Articles

Genotyping Sri Lankan women with polycystic ovary syndrome (PCOS): towards a novel screening tool.

Polycystic ovary syndrome (PCOS) is the multigenic, endocrine disorder of young women. Inheritance of PCOS is likely to be oligogenic and genetic basis remains largely unknown. Screening the candidate genes of PCOS and their SNPs individually is time consuming. Hence, developing a tool that would help in screening multiple candidate genes simultaneously is essential to determine the exact genetic basis of PCOS. Objectives: This study aimed to develop a simple and cost-effective genetic screening tool to simultaneously genotype 16 single nucleotide polymorphisms (SNPs) of PCOS. Methods: The genetic screening tool was developed using allele specific real time quantitative PCR (AS-qPCR) in 96 well PCR plate. Eight SNPs identified in our previous study as well as 8 SNPs identified from other reported studies that had a strong association in the etiology of PCOS were used to develop the tool. Samples from our previous study were reanalyzed using the developed genetic screening tool. Genetic screening tool results were validated with Sanger sequencing. Results: Totally 10 AS-qPCR runs (160 reactions = 16SNPs*10runs) were performed using the developed tool and all except 3 genotype results agreed with Sanger sequencing. The tool showed 100% specificity and 96% sensitivity. Conclusion: The developed genetic screening tool has excellent potential in determining the genotype of multiple SNPs of PCOS simultaneously. This tool is highly suitable for developing countries as a cost effective and accurate early genetic screening test for PCOS. Thus, provides a reliable, fast and user-friendly genotyping method facilitating a wider implication in clinical practice.

Digital Droplet PCR: Next Generation KIT D816V Testing As Advanced Tool for Diagnosis and Prognostication in Mastocytosis

Background: The somatic mutation KIT D816V is a diagnostic criterion for systemic mastocytosis (SM) and can be found in the majority of patients. Therefore, sensitive detection of KIT D816V in blood and bone marrow (BM) is important for diagnosis of SM. Additionally, precise quantification of the KIT D816V allele burden is relevant clinically as it helps to predict multilineage involvement and prognosis in advanced SM and might be useful as a biomarker for treatment response to midostaurin or other interventional drugs. However, despite the high relevance of reliable KIT D816V allele burden measurement, no single assay for the detection and quantification of the KIT mutation has been accepted as a global standard. In addition, differences in calibration material and data normalization hamper the standardization of KIT D816V quantification and complicate the comparison of results obtained in different study groups. Digital droplet PCR (ddPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations.Methods: We performed a validation study of the PrimePCR ddPCR mutation assay for KIT wild-type and the KIT D816V point mutation using the QX-200 droplet-reader on 302 DNA samples from 156 patients with mastocytosis (median age 56 years; 85 females, 71 males) in comparison to melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) (Sotlar et al., 2003, AJP 162, 737-74) and allele-specific quantitative real-time PCR (qPCR) (Kristensen et al., 2011, JMD 13, 180-188). According to WHO criteria, 16 patients were diagnosed with cutaneous mastocytosis, 5 with mastocytosis in the skin, 105 with indolent SM, 7 with smoldering SM, 1 with BM mastocytosis, 8 with aggressive SM, 12 with SM with associated hematologic neoplasm, and 2 with mast cell leukemia. Data were analyzed using the Bland-Altman plot, ordinary least squares regression and Passing-Bablok regression for method comparison, Kaplan-Meier survival plots, and log-rank test.Results: ddPCR was found to be highly sensitive, specific and precise in detecting KIT D816V with a limit of detection of 0.005% variant allele fraction and a coefficient of variation <20%. ddPCR identified the mutation in 90% of all mastocytosis patients compared to 89% with qPCR and 70% with clamp-PCR indicating a superior diagnostic yield of ddPCR over clamp-PCR (p<0.001). In addition, ddPCR was also found to be suitable to detect and quantify KIT D816V in DNA isolated from formalin-fixed paraffin-embedded (FFPE) BM sections. In peripheral blood and BM aspirates, ddPCR for KIT D816V showed a high concordance to qPCR (r=0.99). No systematic deviation for the conversion from qPCR to ddPCR was found (slope of 1.00 of log-transformed data; 95% confidence interval 0.98 to 1.02). In a next step, we confirmed the clinical value of KIT D816V allele burden measurements by ddPCR. In these studies, patients with advanced SM showed a significantly higher KIT D816V allele burden (median 2.43%) compared to patients with indolent SM (median 0.14%; p<0.001). Moreover, ddPCR confirmed the prognostic significance of a high KIT D816V allele burden regarding survival of KIT D816V+ SM patients (n=115) using a cutoff level of 2% mutant alleles. In the group with <2% mutant allele burden the median survival was not reached, whereas in patients with a KIT D816V allele burden of ≥2% the median survival was 7.7 years (p<0.001).Conclusion: We performed a comprehensive validation of ddPCR for KIT D816V in peripheral blood and BM in order to establish a new reliable, quantitative qPCR technique for patients with mastocytosis. ddPCR is highly sensitive and specific for detection and quantification of KIT D816V, gives highly reproducible results even at a very low variant allele fraction level, and confirms the clinical value of KIT D816V allele burden measurement in blood and BM aspirates. In addition, ddPCR for quantification of KIT D816V can be performed in FFPE BM section material. Finally, ddPCR-based quantification of KIT D816V is not limited by the necessity to use generally accepted calibration material for assay standardization. In this regard, we propose to assess the ddPCR for KIT D816V in multicentric studies to increase comparability of allele burden data from different study groups. These studies will be a next crucial step towards the implementation of KIT D816V allele burden measurement as a global standard in clinical practice. [Display omitted] DisclosuresSperr:Teva: Honoraria; Novartis: Other: Register; Meda: Research Funding; Phadia: Research Funding; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Valent:Blueprint: Research Funding; BMS: Honoraria; Incyte: Honoraria; Ariad: Honoraria, Research Funding; Pfizer: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Deciphera: Honoraria, Research Funding; Teva: Honoraria. Hoermann:Gilead: Honoraria, Research Funding; Amgen: Honoraria; Ariad: Honoraria; Novartis: Honoraria.

Interim Results of a Multicenter, Single-Arm Study to Assess Blinatumomab in Adult Patients (pts) with Minimal Residual Disease (MRD) of B-Precursor (BCP) Acute Lymphoblastic Leukemia (GMALL-MOLACT1-BLINA)

Interim Results of a Multicenter, Single-Arm Study to Assess Blinatumomab in Adult Patients (pts) with Minimal Residual Disease (MRD) of B-Precursor (BCP) Acute Lymphoblastic Leukemia (GMALL-MOLACT1-BLINA)

Rapid screening mutations of first-line-drug-resistant genes in Mycobacterium tuberculosis strains by allele-specific real-time quantitative PCR.

Tuberculosis (TB) is a worldwide health, economic, and social burden, especially in developing countries. Drug-resistant TB is the most serious type of this burden. Thus, it is necessary to screen drug-resistant mutations by using a simple and rapid detection method. A total of 32 pairs of allele-specific PCR (AS-PCR) primers were designed to screen mutation and/or wild-type alleles of 16 variations in four first-line drug-resistant genes (katG, rpoB, rpsL, and embB) of TB strains. A pair of primers was designed to amplify 16S rRNA gene and to verify successful amplification. Subsequently, we tested the specificity and sensitivity of these AS-PCR primers. The optimized condition of these AS-PCR primers was first confirmed. All mutations could be screened in general AS-PCR, but only 13 of 16 variations were intuitively investigated by using real-time quantitative PCR (qPCR) and AS-PCR primers. The results of specificity assay suggested that the AS-PCR primers with mutation and/or wildtype alleles could successfully amplify the corresponding allele under optimized PCR conditions. The sensitivity of nine pairs of primers was 500 copy numbers, and the other seven pairs of primers could successfully amplify correct fragments with a template comprising 103 or 104 copy numbers template. An optimized AS-qPCR was established to screen drug-resistant mutations in TB strains with high specificity and sensitivity.

Identification of selected genetic polymorphisms in polycystic ovary syndrome in Sri Lankan women using low cost genotyping techniques.

BackgroundPolycystic ovary syndrome (PCOS), the commonest endocrine disorder affecting young women, appears to be a multigenic trait with contributing genes being unclear. Hence, analysis of polymorphisms in multiple candidate genes is required. Currently available genotyping methods are expensive, time-consuming with limited analytical sensitivity.Aim(i) Develop and validate high resolution melting (HRM) assay and allele-specific real-time quantitative PCR (AS-qPCR) for genotyping selected SNPs associated with PCOS.(ii) Identify selected SNPs and their association with a Sri Lankan cohort of well-characterized PCOS.MethodsDNA was extracted from women with well-characterized PCOS from adolescence (n = 55) and ethnically matched controls (n = 110). FTO (Fat mass and obesity associated gene; rs9939609), FSHB (Follicle stimulating hormone beta subunit; rs6169), FSHR (Follicle stimulating hormone receptor; rs6165/rs6166), and INSR (Insulin receptor; rs1799817) genes were genotyped using HRM assay. GnRH1 (Gonadotropin releasing hormone; rs6185), LHB (Luteinizing hormone beta subunit; rs1800447/rs34349826) and LHCGR (Luteinizing hormone/choriogonadotropin receptor; rs2293275) genes were genotyped using AS-qPCR method. Genotyping results were validated using Sanger sequencing.ResultsA significant association was observed within FTO gene polymorphism (rs9939609) and PCOS. Genotype frequency of FTO gene (rs9939609)—cases versus controls were TT-36.4% vs.65.4% (p<0.05), AT-23.6% vs.20.9%, AA-40% vs.13.6% (p<0.05). Genotype frequencies of the SNPs GnRH1 (rs6185), FSHB (rs6169), FSHR (rs6165 & rs6166), LHB (rs1800447 & rs34349826), LHCGR (rs2293275) and INSR (rs1799817) were not significantly different between cases and controls (p>0.05). Only the mutant alleles were observed for LHB rs1800447 and rs34349826 SNPs in both groups. The HRM and AS-qPCR assay results had 100% concordance with sequencing results.ConclusionsFTO gene rs9939609 polymorphism is significantly more prevalent among Sri Lankan PCOS subjects while the other selected SNPs of HPG axis genes and INSR gene showed no association. HRM and AS-qPCR assays provide a reliable, fast and user-friendly genotyping method facilitating wider implication in clinical practice.

Multiple Ways to Detect IDH2 Mutations in Angioimmunoblastic T-Cell Lymphoma from Immunohistochemistry to Next-Generation Sequencing

Angioimmunoblastic T-cell lymphoma (AITL) is a peripheral T-cell lymphoma associated with chemoresistance and a poor prognosis. Various nonsynonymous mutations in the R172 residue of IDH2 are present in 20% to 30% of AITL patients. In addition to their diagnostic value, these mutations are potentially targetable, especially by isocitrate dehydrogenase (IDH) 2 inhibitor, and therefore their identification in a routine setting is clinically relevant. However, in AITL, the neoplastic cells may be scarce, making the identification of molecular anomalies difficult. We evaluated the diagnostic value of different methods to detect IDH2 mutations in formalin-fixed, paraffin-embedded tumor samples. Immunohistochemistry with an anti-IDH2 R172K antibody, Sanger sequencing, high-resolution melting PCR, allele-specific real-time quantitative PCR, and next-generation sequencing (NGS) were applied to biopsy specimens from 42 AITL patients. We demonstrate that the IDH2 R172K antibody is specific to this amino acid substitution and highly sensitive for the detection of the IDH2R172K variant, the most frequent substitution in this disease. In our study, NGS and allele-specific real-time quantitative PCR displayed a good sensitivity, detecting 96% and 92% of IDH2 mutations, respectively, in contrast to Sanger sequencing and high-resolution melting PCR, which showed a significantly lower detection rate (58% and 42%, respectively). These results suggest that a combination of immunohistochemistry and AS-PCR or NGS should be considered for the identification of IDH2 mutations in AITL in a routine setting.

Digital PCR: A Sensitive and Precise Method for KIT D816V Quantification in Mastocytosis.

The analytically sensitive detection of KIT D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the KIT D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. We performed a validation study of dPCR for KIT D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR (P < 0.001). Moreover, dPCR for KIT D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of KIT D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher KIT D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; P < 0.001). Moreover, dPCR confirmed the prognostic significance of a high KIT D816V VAF regarding survival (P < 0.001). dPCR for KIT D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of KIT D816V allele burden measurement. Thus, dPCR is suitable as a new method for KIT D816V testing in patients with mastocytosis.

Abstract 4727: Analysis of PIK3CA mutations and PI3K signaling proteins as prognostic biomarkers for advanced gastric cancer

Abstract Although surgery and chemotherapy has extended the survival of patients with advanced gastric cancer, some patients experience relapse. In cases of potentially curative gastrectomy, no visible tumor mass should be present at the time of post-operative chemotherapy. Therefore, relapse may be attributed to very small cancer cell populations that survive and develop drug resistance. Previously, we found that changes in the phosphatidylinositide 3-phosphate kinase (PI3K) pathway protein levels were responsible for the growth of drug-resistant cancer cells. In the present study, we postulated that the oncogenic mutations in PIK3CA and PI3K pathway proteins could be prognostic biomarkers for patients with advanced gastric cancer. A retrospective cohort of 160 patients with advanced gastric cancer, receiving potentially curative surgery with/without chemotherapy was investigated for PIK3CA mutations and PI3K pathway protein levels in the context of 5-year overall survival (OS) and relapse-free survival (RFS). Mutations were screened by direct sequencing followed by validation through allele-specific quantitative real-time PCR, digital PCR, and ultra-deep sequencing. Tissue microarrays were produced from tumor-rich areas of surgically removed specimens. Protein levels were assessed by incubation with specific primary antibodies against total and phosphorylated proteins including PI3K (p85), AKT, mTOR, and PTEN, followed by colorimetric detection. Samples were deemed to have positive staining when more than 5% of the cancer cells were stained. OS and RFS periods were estimated using the Kaplan-Meier method. A log-rank test and Cox proportional hazards model was used to compare the survival and hazards ratio, respectively. Subgroup analysis was also performed. Thirteen patients (13/111 11.7%) had PIK3CA mutations in codon 545, whereas one patient (1/94 1.1%) had a mutation in the PIK3CA codon 1047. Phosphorylated-AKT positive (p-AKT(+)) patients in the surgery-only group showed good prognosis in terms of OS and RFS. Both OS and RFS of PTEN(+) patients were higher than those of PTEN(-) patients in both surgery-only and chemotherapy groups although statistical significance was not confirmed. PTEN(+) patients showed similar survival curves for up to 2 years post-operation, and separated thereafter. No significant association was observed between PIK3CA mutations and PI3K pathway protein levels. Subgroup analysis demonstrated that both p-AKT(+) and PTEN(+) were better prognostic factors for survival. There was no interaction between the protein levels and any clinicopathological/mutational factors. This study revealed that: (i) PIK3CA hotspot mutations occurred with low frequency in gastric cancer; (ii) PIK3CA hotspot mutations were not directly associated with PI3K pathway activation; and (iii) p-AKT(+) may be a biomarker for better outcomes in gastric cancer patients undergoing gastrectomy. Citation Format: Satoshi S. Nishizuka, Chie Ito, Kohei Kume, Takeshi Iwaya, Keisuke Koeda, Akira Sasaki. Analysis of PIK3CA mutations and PI3K signaling proteins as prognostic biomarkers for advanced gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4727. doi:10.1158/1538-7445.AM2017-4727

EGFR mutation detection in circulating cell-free DNA of lung adenocarcinoma patients: analysis of LUX-Lung 3 and 6

Background:In the Phase III LUX-Lung 3/6 (LL3/LL6) trials in epidermal growth factor receptor (EGFR) mutation-positive lung adenocarcinoma patients, we evaluated feasibility of EGFR mutation detection using circulating cell-free DNA (cfDNA) and prognostic and predictive utility of cfDNA positivity (cfDNA+).Methods:Paired tumour and blood samples were prospectively collected from randomised patients. Mutations were detected using cfDNA from serum (LL3) or plasma (LL6) by a validated allele-specific quantitative real-time PCR kit.Results:EGFR mutation detection rates in cfDNA were 28.6% (serum) and 60.5% (plasma). Mutation detection in blood was associated with advanced disease characteristics, including higher performance score, number of metastatic sites and bone/liver metastases, and poorer prognosis. In patients with common EGFR mutations, afatinib improved progression-free survival vs chemotherapy in cfDNA+ (LL3: HR, 0.35; P=0.0009; LL6: HR, 0.25; P<0.0001) and cfDNA− (LL3: HR, 0.46; P<0.0001; LL6: HR, 0.12; P<0.0001) cohorts. A trend towards overall survival benefit with afatinib was observed in cfDNA+ patients.Conclusions:Plasma cfDNA is a promising alternative to biopsy for EGFR testing. Detectable mutation in blood was associated with more advanced disease and poorer prognosis. Afatinib improved outcomes in EGFR mutation-positive patients regardless of blood mutation status.

Significance of KIT exon 17 mutation depends on mutant level rather than positivity in core-binding factor acute myeloid leukemia.

KIT exon 17 mutation is a poor prognostic factor in core-binding factor acute myeloid leukemia. However, the mutation detection method used for risk assessment is not assigned. It is necessary to verify the analytical and clinical performance before applying new methods. Herein, we firstly applied a highly sensitive allele-specific, real-time quantitative PCR (AS-qPCR) assay to analyze KIT mutations, which demonstrated excellent sensitivity and specificity. Much higher incidence of KIT mutations (62.2%, 69/111) and prevalence of multiple mutations (43.5%, 30/69) were observed using AS-qPCR, which meant the existence of multiple KIT mutant subclones. The relative KIT mutant level was variable (median, 0.3 per control allele 100 copies, 0.002–532.7) and was divided into two groups: high (⩾10, n=26) and low (<10) mutant level. Interestingly, rather than mutation positivity, mutant level was found to be associated with clinical outcome. High mutant level showed significantly inferior overall survival (P=0.005) and event-free survival (P=0.03), whereas low level did not influence the prognosis. The follow-up data showed that the mutant level were along with fusion transcripts in the majority (n=29), but moved separately in some cases, including the loss of mutations (n=5) and selective proliferation of minor clones (n=2) at relapse. This study highlighted that the KIT mutation should be analyzed using sensitive and quantitative techniques and set a cutoff level for identifying the risk group.

V617FJAK2-Positive Endothelial Cells Are Present in Bone Marrow Neovessels of Patients with Myelofibrosis and Could Derive from the Transdifferentiation of Mutated Hematopoietic Cells

Background. Extensive neoangiogenesis is a distinctive feature of both the spleen and the bone marrow (BM) of patients with myelofibrosis (MF). We and others have previously reported that mature endothelial cells obtained from microvessels of the spleen, either by laser microdissection or by cell sorting, harbor the V617FJAK2 mutation, whereas nothing is known about the existence of such mutated endothelial cells in the BM. The origin of the mutated endothelium has not been clarified up to now, although it has been proposed that it could derive from the transdifferentiation of a mutated CD34+ hematopoietic progenitor cells into an endothelial cell, an event similar to what already observed in solid tumors. Aim. To assess the presence of the V617FJAK2 mutation in mature endothelial cells from the BM of patients with MF and to evaluate the presence of transitional cells expressing both myeloid and endothelial cell markers in the BM of MF patients. Patients and Methods. Paraffin-embedded BM sections from 5 patients with MF, harboring a V617FJAK2 mutation in their granulocytes, were used both for isolating mature endothelial cells by laser microdissection and for immunostaining with antibodies directed against CD34, CD33, and CD144 (VE-cadherin) antigens. Sections from patients with lymphomas who underwent a BM biopsy for staging of the disease were used as controls (n=2). The images were obtained by confocal laser scanning microscopy (Olympus Fluoview FV10i, 60x objective) and processed by IMAGE J software. DNA was extracted from 50 microdissected endothelial cells by a commercial kit and the presence of V617FJAK2 mutation assessed by an allele-specific real-time quantitative PCR. Results. The V617FJAK2 mutation was detected in at least 1 sample of endothelial cells of 5/5 patients, indicating that a mutated endothelium, in addition to the spleen, is also detectable in the BM of patients with MF. The allelic burden ranged from 51.4% to 84.1% of mutated alleles, and was similar to the allelic burden of hematopoietic cells in 1 case, whereas it was lower in 4. Staining of BM sections from 4 patients with antibodies specific for hematopoiesis (CD33) and endothelium (VE-cadherin), as well as common to the 2 lineages (CD34), while confirming the presence of increased neoangiogenetic processes in samples from MF patients, allowed the detection of cells, usually located close to neovessels, co-expressing both hematopoietic (CD33, CD34) and endothelial (VE-cadherin, CD34) markers. These "triple positive" cells were found in all the slides of all patients whereas they were never observed in the BM sections of controls. Conclusions. V617FJAK2 positive endothelial cells can be detected in the BM of patients with MF, similarly to what observed in the spleen. These mutated endothelial cells could derive from the transdifferentiation of hematopoietic mutated cells as suggested by the detection of cells co-expressing both myeloid and endothelial markers. These observations support the hypothesis that the endothelial lineage is involved in the process of malignant transformation of MF, providing new perspectives in our understanding of the phenotype, evolution, and therapy of the disease. DisclosuresNo relevant conflicts of interest to declare.

Human leukocyte antigen-E alleles and expression in patients with serous ovarian cancer.

Human leukocyte antigen-E (HLA-E) is one of the most extensively studied non-classical MHC class I molecules that is almost non-polymorphic. Only two alleles (HLA-E*0101 and HLA-E*0103) are found in worldwide populations, and suggested to be functional differences between these variants. The HLA-E molecule can contribute to the escape of cancer cells from host immune surveillance. However, it is still unknown whether HLA-E gene polymorphisms might play a role in cancer immune escape. To explore the association between HLA-E alleles and the susceptibility to serous ovarian cancer (SOC), 85 primary SOC patients and 100 healthy women were enrolled. Here, we indicated that high frequency of HLA-E*0103 allele existed in SOC patients by the allele-specific quantitative real-time PCR method. The levels of HLA-E protein expression in SOC patients with the HLA-E*0103 allele were higher than those with the HLA-E*0101 allele using immunohistochemistry analysis. The cell surface expression and functional differences between the two alleles were verified by K562 cells transfected with HLA-E*0101 or HLA-E*0103 allelic heavy chains. The HLA-E*0103 allele made the transfer of the HLA-E molecule to the cell surface easier, and HLA-E/peptides complex more stable. These differences ultimately influenced the function of natural killer cells, showing that the cells transfected with HLA-E*0103 allele inhibited natural killer cells to lysis. This study reveals a novel mechanism regarding the susceptibility to SOC, which is correlated with the HLA-E*0103 allele.

An Evaluation of Molecular Response in a Phase 2 Open-Label, Multicenter Confirmatory Study in Patients (pts) with Relapsed/Refractory B-Precursor Acute Lymphoblastic Leukemia (r/r ALL) Receiving Treatment with the BiTE® Antibody Construct Blinatumomab

An Evaluation of Molecular Response in a Phase 2 Open-Label, Multicenter Confirmatory Study in Patients (pts) with Relapsed/Refractory B-Precursor Acute Lymphoblastic Leukemia (r/r ALL) Receiving Treatment with the BiTE® Antibody Construct Blinatumomab

BLAST: A Confirmatory, Single-Arm, Phase 2 Study of Blinatumomab, a Bispecific T-Cell Engager (BiTE®) Antibody Construct, in Patients with Minimal Residual Disease B-Precursor Acute Lymphoblastic Leukemia (ALL)

▪Introduction: In ALL, minimal residual disease (MRD) is defined as the detection of leukemic cells in bone marrow by PCR or flow cytometry in the presence of hematological complete remission (CR). Patients with persistent/recurrent MRD after induction therapy have a higher risk of relapse than those with no detectable MRD. Effective treatment of patients with MRD aims to avoid hematologic relapse, reduce MRD load, and provide a bridge to subsequent HSCT.Blinatumomab, an investigational BiTE® antibody construct, redirects CD3+ T cells to CD19+ target cells, resulting in serial lysis of CD19+ B cells. In a phase 2 study of blinatumomab in 21 patients with MRD+ ALL in first-line treatment, 80% of evaluable patients achieved a complete MRD response. BLAST, a confirmatory single-arm, phase 2 study evaluated efficacy, safety, and tolerability of blinatumomab in patients with MRD+ ALL in a larger population.Methods: Adults (≥18 years) with B-precursor ALL in hematologic CR (<5% blasts in bone marrow) after ≥3 intensive chemotherapy treatments and with MRD ≥10-3 were eligible. Patients with current CNS pathology or extramedullary disease, previous allogeneic HSCT, or Ph+ ALL eligible for treatment with tyrosine kinase inhibitors were excluded. Blinatumomab 15 µg/m²/day was given by continuous IV infusion for 4 weeks, followed by a 2-week treatment-free period (1 cycle). Responders could receive ≤4 cycles of treatment or undergo HSCT after ≥1 cycle. Patients with hematologic relapse discontinued treatment. Rate of complete MRD response (absence of MRD confirmed with a sensitivity of at least 10-4 after 1 cycle) was the primary endpoint. MRD was assessed by allele-specific quantitative real-time PCR for clonal rearrangements of immunoglobulin or T-cell receptor genes by a central laboratory. Overall survival (OS), relapse-free survival (RFS), duration of complete MRD response, and incidence and severity of adverse events (AEs) were secondary endpoints. Per protocol, OS and RFS will be analyzed after 18 months minimum follow-up.Results: 116 patients enrolled and received blinatumomab. Median (range) age was 45 (18–76) years; 15 (13%) patients were ≥65 years of age. 35% of the patients were treated in second or later remission (Table). As of February 2014, 106 patients had ended treatment: 74 had completed treatment (4 cycles or 1 cycle followed by HSCT) and 32 had discontinued treatment due to AEs, disease relapse, or investigators’ decision; 79 patients were still alive and being followed. Three patients were excluded from the efficacy analysis: one had no central lab assay and two had assays with a sensitivity of 5×10-4. Eighty-eight patients (78% [95% CI, 69%–85%]) had a complete MRD response after 1 cycle of treatment. The lower CI bound exceeded 44% (the null hypothesis response rate), confirming that the study met its primary objective. Two additional patients had a complete MRD response after >1 cycle of blinatumomab; across all cycles the complete MRD response rate was 80%. The rate of complete MRD response did not differ significantly across baseline age, sex, line of treatment, and MRD burden categories (Table). All patients experienced ≥1 AE. AEs occurring in ≥20% of patients included pyrexia (88%), headache (38%), tremor (29%), chills (25%), fatigue (24%), nausea (22%) and vomiting (22%). Serious AEs (SAEs) occurred in 60% of patients; 59% and 27% of patients had grade ≥3 and grade ≥4 AEs, respectively. SAEs occurring in ≥5% of patients were pyrexia (15%), tremor (7%), aphasia (5%), encephalopathy (5%) and overdose (5%). Two fatal AEs occurred on treatment: subdural hemorrhage and atypical pneumonia (the latter was deemed treatment-related).Conclusion: This is the largest prospective trial with an experimental compound in MRD+ ALL.Blinatumomab treatment resulted in complete MRD response across multiple patient demographics including patients in second-line treatment and those with high MRD burden. With a complete MRD response rate of 78%, the study met its primary objective. Among patients with a complete MRD response, 98% had a response within the first treatment cycle. In patients with MRD+ ALL following intensive therapy, rapid MRD response induced by blinatumomab has the potential to improve patient outcomes. [Display omitted] DisclosuresGoekbuget:Amgen Inc.: Consultancy, Honoraria, Research Funding. Off Label Use: This presentation will discuss the off-label use of blinatumomab, as this agent is not approved for use by the FDA, EMA or any other regulatory authorities.. Dombret:Amgen Inc.: Honoraria, Research Funding. Bonifacio:Amgen Inc.: Consultancy. Graux:Amgen Inc.: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Buss:Amgen Inc.: Reimbursement for participation in clinical studies, Reimbursement for participation in clinical studies Other; BMS: Travel support, Travel support Other; Novartis: Travel support Other; Pfizer: Reimbursements for participation in clinical studies, Reimbursements for participation in clinical studies Other. Bruggemann:Amgen Inc.: Consultancy, Research Funding. Stieglmaier:Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Wessels:Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Haddad:Amgen Ltd.: Employment. Zugmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Nagorsen:Amgen Inc.: Employment, Equity Ownership, Related to blinatumomab Patents & Royalties. Bargou:Amgen Inc.: Consultancy, Honoraria.

Occurrence of the JAK2 V617F mutation in patients with peripheral arterial disease.

The acquired JAK2 V617F mutation is common in patients with myeloproliferative neoplasms. We previously showed that JAK2 V617F is also found in coronary patients, most of them affected by coronary atherosclerosis. Peripheral arterial disease (PAD) is another important manifestation of atherosclerosis. However, prevalence of the JAK2 V617F mutation and its effect on clinical or hematologic characteristics is unknown in PAD patients. In the present study we determined the prevalence of JAK2 V617F in a cohort of 287 patients with sonographically proven PAD and compared mutation frequency with mutational status of 997 healthy people from the KORA F4 study. JAK2 V617F screening and quantification of allele burden in both cohorts was performed with same allele-specific quantitative real-time PCR method. From a total of 287 PAD patients, 9 individuals were tested positive for the JAK2 V617F mutation. One patient showed elevated hemoglobin values, indicating polycythemia vera. Observed JAK2 V617F frequency (3.1%) in PAD patients showed a 5-fold, highly significant increase compared with healthy people (P < 0.001). Furthermore, occurrence of the mutation in PAD patients was significantly decreased in patients using aspirin (P = 0.003). We conclude that the prevalence of JAK2 V617F mutation is significantly increased in PAD patients compared to the general population. Future studies are warranted to confirm our observations and to define the underlying mechanisms behind our findings.

The KIT D816V expressed allele burden for diagnosis and disease monitoring of systemic mastocytosis

The activating KIT D816V mutation plays a central role in the pathogenesis, diagnosis, and targeted treatment of systemic mastocytosis (SM). For improved and reliable identification of KIT D816V, we have developed an allele-specific quantitative real-time PCR (RQ-PCR) with an enhanced sensitivity of 0.01-0.1 %, which was superior to denaturing high-performance liquid chromatography (0.5-1 %) or conventional sequencing (10-20 %). Overall, KIT D816 mutations were identified in 146/147 (99 %) of patients (D816V, n = 142; D816H, n = 2; D816Y, n = 2) with SM, including indolent SM (ISM, n = 63, 43 %), smoldering SM (n = 8, 5 %), SM with associated hematological non-mast cell lineage disease (SM-AHNMD, n = 16, 11 %), and aggressive SM/mast cell leukemia ± AHNMD (ASM/MCL, n = 60, 41 %). If positive in BM, the KIT D816V mutation was found in PB of all patients with advanced SM (SM-AHNMD, ASM, and MCL) and in 46 % (23/50) of patients with ISM. There was a strong correlation between the KIT D816V expressed allele burden (KIT D816V EAB) with results obtained from DNA by genomic allele-specific PCR and also with disease activity (e.g., serum tryptase level), disease subtype (e.g., indolent vs. advanced SM) and survival. In terms of monitoring of residual disease, qualitative and quantitative assessment of KIT D816V and KIT D816V EAB was successfully used for sequential analysis after chemotherapy or allogeneic stem cell transplantation. We therefore conclude that RQ-PCR assays for KIT D816V are useful complimentary tools for diagnosis, disease monitoring, and evaluation of prognosis in patients with SM.

CirculatingBRAFV600Ein the Diagnosis and Follow-Up of Differentiated Papillary Thyroid Carcinoma

Cell-free nucleic acids circulating in plasma are considered a promising noninvasive tool for cancer monitoring. BRAF(V600E) mutation in cell-free DNA (cfDNA) could represent an appropriate marker for papillary thyroid carcinoma (PTC). Our aim is to investigate the role of BRAF(V600E)-mutated allele in cfDNA as a marker for the diagnosis and follow-up of PTC. BRAF(V600E) allele was detected and quantified by an allele-specific real-time quantitative PCR assay in plasma from 103 patients affected by nodular goiter. As control populations, we enrolled 49 healthy subjects and 16 patients with non-nodular thyroid diseases. The percentage of circulating BRAF(V600E) was significantly different between patients and controls and throughout different cytological categories of ultrasound-assisted fine-needle aspiration. Patients with a histopathological diagnosis of PTC showed a higher percentage of circulating BRAF(V600E) (P = .035) compared to those with benign histology. In 19 patients, a second blood draw, taken 3-6 months after surgery, showed a lower percentage of BRAF(V600E) in cfDNA than the presurgical sample (P < .001). The diagnostic performance of circulating BRAF(V600E) was assessed by receiver operating characteristic curve analysis resulting in an area under the curve of 0.797. A cutoff value was chosen corresponding to maximum specificity (65%) and sensitivity (80%). On this basis, we evaluated the predictive value of BRAF(V600E) in Thy 3 patients with a resulting positive predictive value of 33% and a negative predictive value of 80%. The results of the present study provide encouraging data supporting the possibility to take advantage of circulating BRAF(V600E) in the management of PTC.

Extensive paternal mtDNA leakage in natural populations of Drosophila melanogaster

Strict maternal inheritance is considered a hallmark of animal mtDNA. Although recent reports suggest that paternal leakage occurs in a broad range of species, it is still considered an exceptionally rare event. To evaluate the impact of paternal leakage on the evolution of mtDNA, it is essential to reliably estimate the frequency of paternal leakage in natural populations. Using allele-specific real-time quantitative PCR (RT-qPCR), we show that heteroplasmy is common in natural populations with at least 14% of the individuals carrying multiple mitochondrial haplotypes. However, the average frequency of the minor mtDNA haplotype is low (0.8%), which suggests that this pervasive heteroplasmy has not been noticed before due to a lack of power in sequencing surveys. Based on the distribution of mtDNA haplotypes in the offspring of heteroplasmic mothers, we found no evidence for strong selection against one of the haplotypes. We estimated that the rate of paternal leakage is 6% and that at least 100 generations are required for complete sorting of mtDNA haplotypes. Despite the high proportion of heteroplasmic individuals in natural populations, we found no evidence for recombination between mtDNA molecules, suggesting that either recombination is rare or recombinant haplotypes are counter-selected. Our results indicate that evolutionary studies using mtDNA as a marker might be biased by paternal leakage in this species.

Mutations affecting BRAF, EGFR, PIK3CA, and KRAS are not associated with sporadic vestibular schwannomas

Sporadic vestibular schwannomas are benign tumors originating from the Schwann cells of the vestibular portion of the eigth cranial nerve. An important clinical hallmark of these tumors is their variable growth rate. Investigating vestibular schwannoma biology can help to clarify this variable growth rate and may offer targets for therapeutic treatment. A recent mutation analysis on sporadic non-head and neck schwannomas detected BRAF mutations in around 20 % of tumors. BRAF is part of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. MAPK/ERK activation is associated with an uncontrolled cell growth. Mutated BRAF can function as a target to inhibit this pathway. Mutations in BRAF and other members of the MAPK/ERK pathway have not been investigated in sporadic vestibular schwannomas before. The goal of this study was to investigate if these mutations are present in vestibular schwannomas and whether these mutations correlate with tumor growth. Tumor specimens of 48 patients surgically treated for a sporadic vestibular schwannoma were analyzed. An allele-specific quantitative real-time PCR assay was performed to detect the 13 most frequent mutations affecting BRAF, EGFR, PIK3CA, and KRAS. Radiologically measured tumor growth was included in the analysis to identify potential relationships between these mutations and tumor progression. No activating hotspot mutations in BRAF, EGFR, PIK3CA, or KRAS were detected. The 13 most frequent mutations affecting BRAF, EGFR, PIK3CA, and KRAS are not involved in sporadic vestibular schwannoma development. These results are in contrast to the recent detection of these BRAF mutations in non-head and neck schwannomas.

Imetelstat Rapidly Induces and Maintains Substantial Hematologic and Molecular Responses in Patients with Essential Thrombocythemia (ET) Who Are Refractory or Intolerant to Prior Therapy: Preliminary Phase II Results

AbstractAbstract 179 Background:Myeloproliferative neoplasms (MPNs), such as essential thrombocythemia (ET), are driven by neoplastic progenitor cells. The JAK2 V617F mutation can be detected in approximately 50% of patients (pts) with ET, and the JAK2 V617F allele burden can be used to measure the treatment-induced molecular response (MR) over time. Telomerase is upregulated in neoplastic progenitor cells and sustains indefinite replication. Imetelstat is a first in class, potent, specific inhibitor of telomerase which selectively distributes to bone marrow and inhibits thrombopoiesis. In vitro studies demonstrate that imetelstat selectively inhibits spontaneous megakaryocytic colony-forming unit (CFU-Meg) growth from the blood of pts with ET but not from healthy individuals. Phase I studies have demonstrated that imetelstat inhibits telomerase activity in pts at doses of 7.5 mg/kg and above.Therefore, unlike conventional cytoreductive therapy and JAK2 kinase inhibitors, imetelstat may be uniquely able to selectively inhibit proliferation of neoplastic clonogenic cells in pts with ET and modify the biology and progression of the disease. Methods:A phase II study enrolled pts with ET who had failed or were intolerant to at least one prior therapy, or who refused standard therapy. Pts were treated with imetelstat 7.5 mg/kg or 9.4 mg/kg IV weekly. After attainment of best platelet response in the induction phase, maintenance dosing with imetelstat was commenced with dosing based upon platelet count. Primary endpoint was best overall hematologic response (HR) with complete response (CR) defined as platelet count <400 × 103/μl maintained for at least 4 consecutive weeks in the absence of new thromboembolic events. A key secondary endpoint was rate of MR in patients with JAK2 V617F molecular mutations. JAK2 V617F allele burden was measured by allele-specific quantitative real-time PCR with a limit of detection of 0.1%. CFU-Meg growth pre- and post-treatment and tolerability were also assessed. Results:As of July 9, 2012, 13 pts were treated. Median age was 60 yrs (range 21–83) with a median of 2 prior treatments (range 1–3). Median years since initial diagnosis were 5.8 (range 0.3 to 24.9) and initial platelet count was 809 × 103/μl (range 601 to 1359 × 103/μl).Best overall HR was 100%, with 11 of 13 pts achieving a confirmed CR after a median of 6.1 weeks (range 5.1 to 14.1 wks). Twelve of 13 pts remain on maintenance therapy (median time on study 26.1 weeks) and despite transient elevations of platelets above best response, pts continue to be responsive to imetelstat. Four pts have reached 1 year of therapy and continue to be treated with ongoing HR. Dosing frequency on maintenance therapy was generally reduced with time.A substantial decrease in JAK2 V617F allele burden was demonstrated in all 5 JAK2 V617F-positive pts (mean allele burden reduction of 82%; range of 59–94%, see table below). Four pts who were eligible for MR assessment by LeukemiaNetcriteria (initial JAKV617F allele burden >10%) reached molecular partial responses (PR): one pt after 12 weeks, which has been maintained through 1 year, and 3 other pts at 24, 36 and 48 weeks of therapy. One additional pt with JAK2 V617F levels of 4.8% prior to therapy has also had a 75% reduction after 12 weeks of treatment.A reduction in the spontaneous growth of CFU-Meg was also observed in the 2 pts tested, with 93% and 96% reduction from baseline, respectively.Long-term administration of imetelstat was generally well tolerated. Common adverse events reported on therapy were mild to moderate gastrointestinal toxicities, reductions in neutrophil counts, and fatigue.JAK2 V617F Allele BurdenPt #Baseline3 mo6 mo9 mo12 mo126.4%7.7%6.1%1.8%3.4%253.3%52.2%43.7%44.5%21.8%310.8%11.2%14.3%2.4%431.1%14.8%2.0%54.8%1.2% Conclusions:Imetelstat rapidly induces and maintains hematologic responses in pts with ET who have failed or are intolerant to conventional therapies. Importantly, substantial MR is observed in all JAK2 V617F-positive pts and inhibition of the neoplastic clonogenic growth ex-vivo is demonstrated. The reduction in JAK2 V617F allele burden and cytokine-independent growth of CFU-Meg suggests that imetelstat has a relatively selective inhibitory effect on the growth of the neoplastic clone(s) which drive ET and has the potential to modify the underlying biology of MPNs. Additional data will be presented from this ongoing study. Disclosures:Baerlocher:Geron Corporation: Research Funding. Oppliger Leibundgut:Geron Corporation: Research Funding. Ayran:Geron Corporation: Employment. Blaney:Geron Corporation: Employment. Burington:Geron Corporation: Employment. Morfeld:Geron Corporation: Employment. Odenike:Sanofi Aventis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Reddy:Geron Corporation: Employment. Roeth:Geron Corporation: Research Funding. Stuart:OncoMed Pharmaceuticals: Consultancy; Geron Corporation: Consultancy, Employment.