INTRODUCTION: Janus kinase 2 gene (JAK2) encodes for a cytoplasmic tyrosine kinase involved in normal hematopoietic growth factor signaling. Point mutations of the JAK2 gene on chromosome 9, specifically V617F, a point mutation at amino acid 617, are associated with myeloproliferative disorders (MPD). The V617F JAK2 mutation has been found in 90% of patients with polycythemia vera, 50–60% of patients with essential thrombocythemia or idiopathic myelofibrosis and 1–5% of patients with other MPD. To our knowledge, previous studies involving the V617F JAK2 mutation were not performed on a control population of normal individuals. Therefore, the prevalence of this mutation has not been established. In this study, we tested volunteer blood donors from a hospital-based blood donation center for the presence of the V617F JAK2 mutation. METHODS: Citrated whole blood was obtained from volunteer blood donors, age 17 and older, who presented to donate whole blood at a hospital-based blood donation center. The donors met all qualifications to donate blood as defined by FDA regulations. DNA was extracted using the QIAagen and QIAamp DNA extraction columns, quantified and diluted to 100ng/ul. DNA was simultaneously amplified and detected using allele specific minor groove binder probes and primers for the V617F JAK2 mutation. The resultant amplification was recorded by real-time, quantitative PCR using an ABI 7500 (Applied Biosystems, Foster City, CA). A 1% limit of detection, determined from sensitivity and specificity studies using a known cell line control, was set as the technically reproducible threshold sensitivity of the test. Samples were defined as negative for the V617F JAK2 mutation if only the wild type allele was detected. Samples that had a mutant allele detected above the 1% limit of detection were defined as positive for the V617F JAK2 mutation. Samples that had a mutant allele detected below the 1% limit of detection were defined as negative for the V617F JAK2 mutation. RESULTS: A total of 181 DNA samples from volunteer blood donors were tested for the V617F JAK2 mutation. The test group consisted of 104 males (mean age 44, range 17–77) and 77 females (mean age 42, range 18–71). Of the 181 donors tested, 171 had only wild type allele detected and were considered negative. Ten donors had high background of the mutant allele detected below the 1% limit of detection and were considered negative. DISCUSSION: To our knowledge, this is the first report documenting the prevalence of the V617F JAK2 mutation in a healthy blood donor population. In this study of 181 volunteer blood donors none had the V617F JAK2 mutation. Although 10 of the 181 donors were found to have mutant allele detected, they were below the 1% technically reproducible sensitivity threshold of the test and were considered negative. We recommend that mutations detected below the technical threshold of 1% of our assay be considered false positives. The results of this study suggest that the V617F JAK2 mutation is not present in a healthy blood donor population and is significant when detected by our method.
Introduction Next-generation sequencing has emerged as a clinical tool for the identification of actionable mutations to triage advanced colorectal cancer patients for targeted therapies. The literature is conflicted as to whether primaries or their metastases should be selected for sequencing. Some authors suggest that either site may be sequenced, whereas others recommend sequencing the primary, the metastasis, or even both tumors. Here, we address this issue head on with a meta-analysis and provide for the first time a set of sensible recommendations to make this determination. Methods From our own series, we include 43 tumors from 13 patients including 14 primaries, 10 regional lymph node metastases, 17 distant metastases, and two anastomotic recurrences sequenced using the 50 gene Ion AmpliSeq cancer NGS panel v2. Results Based on our new cohort and a meta-analysis, we found that ~ 77% of patient-matched primary-metastatic pairs have identical alterations in these 50 cancer-associated genes. Conclusions Low tumor cellularity, tumor heterogeneity, clonal evolution, treatment status, sample quality, and/or size of the sequencing panel accounted for a proportion of the differential detection of mutations at primary and metastatic sites. The therapeutic implications of the most frequently discordant alterations ( TP53 , APC , PIK3CA , and SMAD4 ) are discussed. Our meta-analysis indicates that a subset of patients who fail initial therapy may benefit from sequencing of additional sites to identify new actionable genomic abnormalities not present in the initial analysis. Evidence-based recommendations are proposed. Supplementary Information The online version contains supplementary material available at 10.1007/s40487-021-00151-7.
INTRODUCTION: FMS-like tyrosine kinase 3 gene (FLT3) encodes for a tyrosine kinase receptor located on hematopoietic progenitor cells in the bone marrow, lymph nodes and thymus. Activation of FLT3 results in increased proliferation of these hematopoietic cells. Mutations in the FLT3 gene, resulting in a constitutively activated FLT3 receptor, have been identified in 1/3 of patients with acute myelogenous leukemia and portend a worse prognosis. The most frequently occurring FLT3 mutations are internal tandem duplications (ITD) located in the juxtamembrane domain. To our knowledge, previous studies involving FLT3 mutations have not been performed on a control population of normal individuals. Therefore, the prevalence of this mutation has not been established. In this study, we tested volunteer blood donors from a hospital-based blood donation center for the presence of FLT3-ITD mutations. METHODS: Citrated whole blood was obtained from volunteer blood donors, age 17 and older, who presented to donate whole blood at a hospital-based blood donation center. The donors met all qualifications for donating blood as defined by FDA regulations. DNA was extracted using the QIAagen and QIAamp DNA extraction columns, quantified and diluted to 100ng/ul. DNA was amplified using two fluorescently labeled primers for the FLT3-ITD mutation (InVivo Scribe Technologies, San Diego, CA). Using an experimental assay with high sensitivity but high non-specific background, FLT3-ITD mutations were detected by means of size and color separation through capillary electrophoresis (Vidiera NsD, Beckman-Coulter, Fullerton, CA). Samples were defined as negative for a FLT3-ITD mutation if only a single 330 base pair (bp) peak was detected. Samples were defined as positive for a FLT3-ITD mutation if an additional second peak > 330 bp was detected. Samples that had a small second peak (<5000 relative fluorescent units (RFU)) on initial testing were repeated using a highly specific protocol with low non-specific background. Samples that on repeat testing did not demonstrate the second peak were defined as negative for a FLT3-ITD mutation. RESULTS: A total of 181 DNA samples from volunteer blood donors were tested for FLT3-ITD mutations. The test group consisted of 104 males (mean age 44, range 17–77) and 77 females (mean age 42, range 18–71). Of 181 donors, 177 were negative for FLT3-ITD on initial testing. Only 4 donors (2 females: 18 and 19 years old and 2 males: 51 and 59 years old) had very small (<5000 RFU) second peaks > 330 bp. However, these second peaks were not detected with repeat testing and were therefore considered negative. DISCUSSION: To our knowledge, this is the first study documenting the prevalence of FLT3-ITD mutations in a healthy population. In this study of 181 volunteer blood donors none had FLT3-ITD mutations. Although 4 of the 181 donors were initially found to have very small additional second peaks equivocal for FLT3-ITD mutations, all were negative with high specificity, low background repeat testing, indicating these were probably false positive results. Therefore, we recommend that very small additional second peaks should be confirmed with repeat testing. The results of this study indicate that FLT3-ITD mutations are not present in a healthy blood donor population and are therefore significant when detected in leukemic patients.
Purpose: Coumadin (Warfarin) is known for its high inter-individual dose requirements and narrow therapeutic index. Common SNPs in the CYP 2C9 and haplotypes in the VKORC-1 have been linked to lower warfarin dose requirements. Our initial study was designed to compare the current pharmacy protocol for warfarin dosing to a multi-variant pharmacogenomic (PGx) dosing algorithm including CYP2C9 and VKORC1 genotype, patient height, weight, age, current INR, target INR, primary indication, and interfering medications. Method: The strategy developed with our Pharmacy and therapeutics committee has been to genotype all in-patients put on sodium heparin for prospective warfarin sensitivity genotyping without altering current dosing protocols. Patients were genotyped for CYP 2C9 *1, *2 and *3 and for the VKORC-1 (-1639) SNP. The genotype and dose recommendations were reported from our clinical lab. Inclusion criteria were genotype, 4 or more doses of warfarin, concurrent INR and the initial dose selection per the current pharmacy protocol. The final patient therapeutic dose was compared to Pharmacy’s initial dose and to our PGx predicted initial dose using a scatter plot analysis with a trend line fitted to our results. Results: Of the initial 75 patients, 30 were not transitioned to warfarin while in the hospital and thus were excluded from the study analysis. 25 of the remaining 45 patients met all study inclusion criteria. The pharmacy dosing strategy when compared to the final therapeutic dose showed a correlation of 0.068 (1.4% R2 value). However, our pharmacogenomic dosing strategy showed a correlation of 0.44 (44.5% R2 value). Per the pharmacy protocol 72% of patients (18/25) were given initial doses 2 or more mg above or below the final therapeutic dose. The pharmacogenomic algorithm predicted the final therapeutic dose within 2 mg for 76% of patients tested. Overdosing by 2 mg or more occurred in 52% (13/25) with the current dosing method. Use of the Pharmacogenomic algorithm would have resulted in only 8% (2/25) of patients being overdosed by more than 2 mg. Conclusion: The combination of demographic and genetic information accounted for 44% of the inter-patient variation in warfarin dosing. This demonstrates a significant improvement over the current method which accounted for only 6.8% of inter-patient warfarin dosing variation. Under the current dosing strategy only 24% of patients receive initial doses within 2 mg of their final therapeutic dose by 2 mg or less compared to 76% using the PGx algorithm. With the current dosing, 52% of patients were overdosed by 2 mg or more compared to 8% using the PGx algorithm. The PGx algorithm would also have reduced the number of overdosed patients above 2 mg by 85%. Comparison of the genotype, daily dose and INR reveals that patients harboring 2 or more variant alleles have more fluctuations in INR and require more dose adjustments than patients with 0 or 1. By identifying these patients and initiating therapy with more accurate dosing we should reduce the fluctuations in INR and number of dose adjustments. Our data suggests adopting PGx algorithm warfarin dosing will lead to a reduction in dose adjustments, a reduction in time to therapeutic INR, and identify difficult to dose patients. Future open label prospective studies are needed to demonstrate and quantify the clinical effectiveness of this predictive algorithm.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.