Agena Bioscience Research Articles

Abstract 750: Detection of mutations in single circulating tumor cells using MALDI-TOF mass spectrometry

Abstract Background: As cancer therapeutics are increasingly selected based upon molecular genetic information, having reliable, accurate, rapid and inexpensive methods for mutational analysis are extremely desirable. Circulating tumor cells (CTCs) allow non-invasive “liquid biopsy” access to intact cells for molecular analysis. Here we demonstrate the successful detection of mutations in model CTCs individually isolated from blood (AccuCyte® - CyteFinder® system, RareCyte) using MALDI-TOF Mass Spectrometry (MassARRAY, Agena Bioscience). Methods: Breast (MDA-MD-231) and lung (NCI-H1975) cancer cells with a set of known mutations were spiked into blood and processed by AccuCyte onto microscope slides and stained on an automated immunostainer. Slides were imaged using the CyteFinder digital fluorescence scanning microscope and mCTCs were identified by positive nuclear, EpCAM, and cytokeratin staining, and negative CD45 staining. mCTCs and white blood cell (WBC) negative controls were picked from the slides and put into PCR tubes using the CytePicker® module. DNA from individual or small pools of cells (3-5) was amplified using the PicoPLEX® (Rubicon) whole genome amplification (WGA) kit; alternatively cells were lysed and directly entered into the ensuing iPLEX® Pro workflow. Specific regions surrounding 5 different mutations in each of the mCTC lines were amplified from the WGA product or the lysed cells and the products were detected and scored for the mutations using a single PCR reaction iPLEX® Pro panel that includes a combination of 10 common lung and breast cancer mutations using the MassARRAY® platform. Results: Five point mutations in four different genes (CDKN2A, EGFR, PIK3CA, and TP3) were measured in the NCI-H1975 lines and four point mutations in four genes (BRAF, KRAS, NF2, and TP53) and a deletion in one gene (CDKN2A) were measured in the MDA-MB-231 cells by iPLEX® Pro chemistry on the MassARRAY® system. All mutations were accurately detected in the WGA single and pooled cell samples and most were also detected in cells that did not undergo WGA before PCR with with iPLEX Pro panel. Allelic frequency observed was consistent with known zygosity of the mutation. Conclusions: MassARRAY successfully detected mutations in single model CTCs that were individually picked from a blood sample processed by the AccuCyte - CyteFinder system. Integrating CTC isolation with MassARRAY may be a practical way to identify and monitor known cancer mutations non-invasively. Citation Format: Jackie L. Stilwell, Ryan T. Birse, Arturo Ramirez, Melinda Duplessis, Brennan Enright, Darryl Irwin, Eric Kaldjian. Detection of mutations in single circulating tumor cells using MALDI-TOF mass spectrometry [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 750. doi:10.1158/1538-7445.AM2017-750

Mutation detection for biochemical genetics in the genomic era

Objective: Our laboratory offers specialist testing for lysosomal and peroxisomal disorders. Requests for molecular confirmation of an enzyme or analyte diagnosis are increasing. To review the efficiency of molecular testing in our laboratory using first-tier screening for a customised panel of 77 common mutations in 29 biochemical genetic genes, followed by Next Generation Sequencing. Methods: A MassArray Primer Extension custom panel was designed by Agena Bioscience with 3 multiplexed assays. False negative and positive results due to underlying SNPs at the primer binding sites required redesign of some assays. NGS was performed using the Illumina TruSightTM Inherited Disease (552 genes) and TruSightTM One (4813 genes) sequencing panels, depending of coverage of the relevant genes. Clinical exome testing is performed using the Roche medical exome. Libraries were sequenced on the Illumina MiSeq or NextSeq sequencing systems. Detection of variants of uncertain significance were regarded as a negative result. Results: 33 Massarray tests have been performed with 30 mutations detected out of 66 alleles. 215 ID tests have been performed with mutations detected in 139. 99 One tests have been performed with mutations detected in 30. 1 whole exome test has been performed without a diagnosis. The diagnosis rate was higher (97%) for patients with a known biochemical or clinical diagnosis but only 20% for patients with broad, less-defined phenotypes. No mutations were detected in 19 reproductive partners of carriers of an autosomal recessive disorder. Conclusion: Molecular testing has a high diagnostic yield in biochemical genetics patients with a known diagnosis. The Massarray panel increases efficiency by reducing the need for Next Generation Sequencing. Carrier testing of partners did not detect any mutations.

The novel detection of EGFR-T790M mutations in exhaled breath condensate.

9032 Background: The EGFR-T790M somatic mutation is the most common mechanism of resistance to Tyrosine Kinase Inhibitors (TKI) in NSCLC. However patients with advanced disease are not always amenable to repeat biopsy for further molecular analysis. Developing non-invasive methods to detect T790M in cell-free DNA (cfDNA), in the absence of tissue is being actively investigated. Furthermore these 'liquid biopsies' may also overcome the problem of tumour genomic heterogeneity. Unfortunately the sensitivity of plasma for T790M detection has been disappointing with a significant chance of a false negative result. Exhaled breath condensate (EBC) is an easily collected sample and is known to harbour cfDNA, including lung cancer mutations. We explored the potential of EBC as a novel method of T790M detection. Methods: We recruited 26 patients who were either 1) known T790M positive pre/during Osimertinib therapy or 2) other mEGFR positive patients on 1st/2nd generation TKI. We collected matched plasma and EBC samples in the majority of cases. EBC samples were collected using the RTube device. Plasma was collected using standard EDTA tubes and extracted within 90 minutes. Using UltraSEEK chemistry, a targeted PCR for ultra-sensitive somatic mutation profiling on the MassARRAY system (Agena Bioscience), we compared the performance of EBC to plasma for the detection of T790M. Results: See Table. Conclusions: In this pilot study we describe the first ever report of the successful and consistent detection of T790M in the EBC of patients with EGFR mutated NSCLC, using a commercially available targeted assay. Our results suggest EBC is responsive to recognised dynamic molecular changes that occur on TKI treatment. We believe this makes EBC analysis an attractive avenue for future research, to optimise the detection of T790M mutations in liquid biopsies. [Table: see text]

Genetic variants in SERPINA4 and SERPINA5, but not BCL2 and SIK3 are associated with acute kidney injury in critically ill patients with septic shock

BackgroundAcute kidney injury (AKI) is a multifactorial syndrome, but knowledge about its pathophysiology and possible genetic background is limited. Recently the first hypothesis-free genetic association studies have been published to explore individual susceptibility to AKI. We aimed to replicate the previously identified associations between five candidate single nucleotide polymorphisms (SNP) in apoptosis-related genes BCL2, SERPINA4, SERPINA5, and SIK3 and the development of AKI, using a prospective cohort of critically ill patients with sepsis/septic shock, in Finland.MethodsThis is a prospective, observational multicenter study. Of 2567 patients without chronic kidney disease and with genetic samples included in the Finnish Acute Kidney Injury (FINNAKI) study, 837 patients had sepsis and 627 patients had septic shock. AKI was defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria, considering stages 2 and 3 affected (severe AKI), stage 0 unaffected, and stage 1 indecisive. Genotyping was done using iPLEXTM Assay (Agena Bioscience). The genotyped SNPs were rs8094315 and rs12457893 in the intron of the BCL2 gene, rs2093266 in the SERPINA4 gene, rs1955656 in the SERPINA5 gene and rs625145 in the SIK3 gene. Association analyses were performed using logistic regression with PLINK software.ResultsWe found no significant associations between the SNPs and severe AKI in patients with sepsis/septic shock, even after adjustment for confounders. Among patients with septic shock (252 with severe AKI and 226 without AKI (149 with KDIGO stage 1 excluded)), the SNPs rs2093266 and rs1955656 were significantly (odds ratio 0.63, p = 0.04276) associated with stage 2–3 AKI after adjusting for clinical and demographic variables.ConclusionsThe SNPs rs2093266 in the SERPINA4 and rs1955656 in the SERPINA5 were associated with the development of severe AKI (KDIGO stage 2–3) in critically ill patients with septic shock. For the other SNPs, we did not confirm the previously reported associations.

Aberrant Methylation and Decreased Expression of NRIP1 in IGHV-Unmutated CLL

Aberrant DNA methylation patterning occurs in many cancers, including chronic lymphocytic leukemia (CLL). Methylation programming changes as B cells differentiate from immature progenitors to mature B cells. CLL clones originate from a continuum of B cell maturation states, which differ in their "methylome." Emerging evidence suggests that aberrant methylation programming occurs at numerous genes that are differentially expressed in IGHV-defined CLL subtypes. Nuclear receptor interacting protein 1 (NRIP1) reproducibly shows significant gene expression differences in IGHV unmutated (U-CLL) and mutated CLL (M-CLL), with lower transcript levels observed in U-CLL cells. Low expression of NRIP1 is associated with poorer overall survival and time-to-treatment. NRIP1 encodes a cofactor that interacts with various nuclear receptors and other proteins to regulate transcription of genes involved in cellular proliferation, survival, metabolism, inflammation, and other processes.Published data reveal several CpG dinucleotides near the NRIP1 promoter that undergo increasing methylation during the maturation and differentiation of normal peripheral blood B cells (NBC), concordant with a stepwise decrease in transcript levels of NRIP1 over the course of maturation. Interestingly, our analysis of publically available expression data revealed significantly lower NRIP1 expression in U-CLL cells (the less mature CLL subtype) than NBC, while M-CLL cells (the more mature CLL subtype) express levels comparable to NBC. Given these observations, our objective was to interrogate the promoter and regulatory regions at the NRIP1 locus for differences in CpG methylation in genomic DNA isolated from U-CLL, M-CLL, and NBC. We hypothesized that the NRIP1 locus is more highly methylated in U-CLL than in M-CLL and NBC, and that this increased methylation represses NRIP1 gene transcription, contributing to its aberrantly low expression in U-CLL compared to M-CLL and NBC.We bisulfite-converted genomic DNA from negatively-selected CD19+ treatment-naïve CLL samples (n=74 U-CLL, n=37 M-CLL) and NBC from healthy donors (n=5). We PCR-amplified regions in or near the CpG island of NRIP1 and used Agena Bioscience’s EpiTyper kit to prepare the samples for analysis on a MassARRAY spectrometer. EpiTyper software was used to detect mass differences that indicate either non-methylated or methylated CpG-containing fragments, and to calculate their relative frequency in each sample. For each CpG locus that was assayed, we fit a beta-regression model to detect differential methylation between M-CLL and U-CLL. We also computed Pearson correlation coefficients comparing methylation levels to gene expression levels.We assessed 47 CpGs mapping to the promoter and nearby regulatory elements of NRIP1 for methylation frequency and correlation with NRIP1 expression. From our comparison of methylation frequency in U-CLL and M-CLL, 3 adjacent CpG loci were significant at false discovery rate (FDR) =1% (CpG 58, p=0.00016; CpG 144, p=0.00062; and CpG 155, p=0.00287). The three most significant correlation coefficients arose from the same three CpG loci (CpG 58, R=0.677, p=2.2e-16; CpG 144, R=0.539, p=7.16e-10; CpG 155, R=0.651, p=5.77e-15). Unexpectedly, all 3 loci were methylated at higher levels in M-CLL and at lower levels in U-CLL (on average), and expression was positively correlated with methylation levels. These CpGs map near a documented binding site for the repressive transcription factor RUNX3, prompting us to hypothesize that differential methylation in U-CLL versus M-CLL may affect the binding affinity of RUNX3 and contribute to the robust NRIP1 expression differences in these CLL subtypes. Future work will test this hypothesis using gel-shift and luciferase reporter assays followed by chromatin immunoprecipitation experiments.Few studies have focused on aberrant methylation patterning in CLL and the consequences of deregulated gene expression. We are the first to look at NRIP1 for differences in DNA methylation across IGHV subtypes. Future studies aimed at understanding how methylation at this locus affects NRIP1 transcription, as well as how levels of NRIP1 affect cell survival pathways, will enhance our understanding of the role of this gene in CLL, and the contributions of deregulated methylation to disease pathophysiology. DisclosuresNo relevant conflicts of interest to declare.

The MassARRAY® System for Targeted SNP Genotyping.

Research to understand the genetic basis of disease, particularly complex disease, regularly involves single nucleotide polymorphism (SNP) genotyping. The use of genome-wide SNP genotyping arrays has become increasingly more commonplace for gene discovery. However, smaller-scale genotyping platforms capable of efficiently genotyping tens to hundreds of SNPs are still crucial for many aspects of this work, including replication of associations. The Agena Bioscience MassARRAY System is one such platform. Here, we provide a guide to using the MassARRAY System, from assay design, through mass spectrometry, to generation of genotype data.

Development of a methylation marker set for forensic age estimation using analysis of public methylation data and the Agena Bioscience EpiTYPER system

Individual age estimation has the potential to provide key information that could enhance and extend DNA intelligence tools. Following predictive tests for externally visible characteristics developed in recent years, prediction of age could guide police investigations and improve the assessment of age-related phenotype expression patterns such as hair colour changes and early onset of male pattern baldness. DNA methylation at CpG positions has emerged as the most promising DNA tests to ascertain the individual age of the donor of a biological contact trace. Although different methodologies are available to detect DNA methylation, EpiTYPER technology (Agena Bioscience, formerly Sequenom) provides useful characteristics that can be applied as a discovery tool in localized regions of the genome. In our study, a total of twenty-two candidate genomic regions, selected from the assessment of publically available data from the Illumina HumanMethylation 450 BeadChip, had a total of 177 CpG sites with informative methylation patterns that were subsequently investigated in detail. From the methylation analyses made, a novel age prediction model based on a multivariate quantile regression analysis was built using the seven highest age-correlated loci of ELOVL2, ASPA, PDE4C, FHL2, CCDC102B, C1orf132 and chr16:85395429. The detected methylation levels in these loci provide a median absolute age prediction error of ±3.07years and a percentage of prediction error relative to the age of 6.3%. We report the predictive performance of the developed model using cross validation of a carefully age-graded training set of 725 European individuals and a test set of 52 monozygotic twin pairs. The multivariate quantile regression age predictor, using the CpG sites selected in this study, has been placed in the open-access Snipper forensic classification website.

Natural killer cells and single nucleotide polymorphisms of specific ion channels and receptor genes in myalgic encephalomyelitis/chronic fatigue syndrome.

AimThe aim of this paper was to determine natural killer (NK) cytotoxic activity and if single nucleotide polymorphisms (SNPs) and genotypes in transient receptor potential (TRP) ion channels and acetylcholine receptors (AChRs) were present in isolated NK cells from previously identified myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS) patients.Subjects and methodsA total of 39 ME/CFS patients (51.69±2 years old) and 30 unfatigued controls (47.60±2.39 years old) were included in this study. Patients were defined according to the 1994 Centers for Disease Control and Prevention criteria. Flow cytometry protocols were used to examine NK cytotoxic activity. A total of 678 SNPs from isolated NK cells were examined for 21 mammalian TRP ion channel genes and for nine mammalian AChR genes via the Agena Bioscience iPlex Gold assay. SNP association and genotype was determined using analysis of variance and Plink software.ResultsME/CFS patients had a significant reduction in NK percentage lysis of target cells (17%±4.68%) compared with the unfatigued control group (31%±6.78%). Of the 678 SNPs examined, eleven SNPs for TRP ion channel genes (TRPC4, TRPC2, TRPM3, and TRPM8) were identified in the ME/CFS group. Five of these SNPs were associated with TRPM3, while the remainder were associated with TRPM8, TRPC2, and TRPC4 (P<0.05). Fourteen SNPs were associated with nicotinic and muscarinic AChR genes: six with CHRNA3, while the remainder were associated with CHRNA2, CHRNB4, CHRNA5, and CHRNE (P<0.05). There were sixteen genotypes identified from SNPs in TRP ion channels and AChRs for TRPM3 (n=5), TRPM8 (n=2), TRPC4 (n=3), TRPC2 (n=1), CHRNE (n=1), CHRNA2 (n=2), CHRNA3 (n=1), and CHRNB4 (n=1) (P<0.05).ConclusionWe identified a number of SNPs and genotypes for TRP ion channels and AChRs from isolated NK cells in patients with ME/CFS, suggesting these SNPs and genotypes may be involved in changes in NK cell function and the development of ME/CFS pathology. These anomalies suggest a role for dysregulation of Ca2+ in AChR and TRP ion channel signaling in the pathomechanism of ME/CFS.

Abstract P5-12-06: Comprehensive assessment of the effect of genetic polymorphisms in drug metabolizing enzymes and transporters on tamoxifen activation to endoxifen

Abstract Background: Tamoxifen is the most commonly prescribed hormonal drug for estrogen receptor positive breast cancer treatment. Tamoxifen itself has weak anti-estrogenic activity, but is bioactivated to the more potent inhibitor endoxifen. Recent data suggest inferior efficacy of tamoxifen treatment in patients who have low systemic endoxifen concentration. Genetic variability in drug metabolizing enzymes and transporters, particularly CYP2D6, are known to effect serum endoxifen concentration. The association of CYP2D6 genotype and endoxifen concentration is well established; however, there is a paucity of data regarding the effects of genetic variants in other drug metabolizing enzymes and transporters on endoxifen concentrations. The objective of our study was to comprehensively screen known, functionally consequential polymorphisms and copy number variations in genes of interest to detect additional pharmacogenetic predictors of endoxifen concentration during tamoxifen treatment. Methods: This analysis includes patients prospectively enrolled on the Lineberger Comprehensive Cancer Center 0801 trial. Patients had received tamoxifen for a minimum of 4 months prior to enrollment and were not concurrently taking strong or moderate CYP2D6 inhibitors. Samples were collected at enrollment for measurement of steady state endoxifen level and collection of germline DNA. Genotyping was performed for CYP2D6 using the Amplichip® CYP450 test (Roche Diagnostics) and for other candidate genes (CYP2C9, CYP3A4, CYP3A5, ABCB1, SLCO1B1, SULT1A1, SULT1A2, and UGT2B7) using the iPLEX® ADME PGx Pro Panel (Agena Bioscience). Activity phenotype for each gene was inferred from genotype data based on known activity of variant alleles or copy numbers. Metabolite concentrations were measured via LC/MS-MS assay at Indiana University and square root transformed prior to analysis to improve normality. Linear regression models were used to evaluate the association of each gene individually with endoxifen concentration, assuming an additive pharmacogenetic effect, after adjustment for CYP2D6 phenotype (EM/UM, IM or PM). Results: 304 Patients with steady-state endoxifen concentration and successful genotyping were included in the analysis. After transformation and adjustment, endoxifen concentration was significantly associated with carrying low-activity CYP2C9 variant alleles (*2, *3, *5, *6, *8, *11, *12) (p=0.016). Predicted endoxifen concentration based on CYP2C9 and CYP2D6 genotype can be found in. Predicted endoxifen concentration (ng/mL) based on CYP2C9 and CYP2D6 Phenotype CYP2D6 EM/UMCYP2D6 IMCYP2D6 PMCYP2C9 WT/WT9.716.553.41CYP2C9 WT/Var8.375.482.63CYP2C9 Var/Var7.134.481.96Abbreviations: WT= wild-type, Var=Variant allele for CYP2C9 Phenotype activity of other enzymes and transporters was not associated with endoxifen concentration (all p&amp;gt;0.05). Conclusions: Polymorphisms in CYP2C9 and CYP2D6, but not other enzymes or transporters, contribute to variation in endoxifen exposure. If endoxifen exposure is validated to predict tamoxifen efficacy, personalized tamoxifen dosing algorithms should include CYP2C9, in addition to CYP2D6 and clinical factors, to improve efficacy and minimize side effects. Citation Format: Hertz DL, Danko W, Deal A, Walko CM, Flockhart DA, McLeod HL, Ibrahim JG, Irvin Jr WJ. Comprehensive assessment of the effect of genetic polymorphisms in drug metabolizing enzymes and transporters on tamoxifen activation to endoxifen. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-12-06.

Digitally guided microdissection aids somatic mutation detection in difficult to dissect tumors

Molecular genetic testing on formalin fixed, paraffin embedded (FFPE) tumors frequently requires dissection of tumor from tissue sections mounted on glass slides. In a process referred to as "manual macrodissection," the pathologist reviews an H&E stained slide at the light microscope and marks areas for dissection, and then the laboratory performs manual dissection from adjacent sections without the aid of a microscope, using the marked reference H&E slide as a guide. Manual macrodissection may be inadequate for tissue sections with low tumor content. We compared manual macrodissection to a new method, digitally guided microdissection, on a series of 32 FFPE pancreatic cancer samples. KRAS hotspot mutation profiling was performed using the Sequenom MassARRAY system (Agena Bioscience). Digitally guided microdissection was performed on multiple smaller areas of high tumor content selected from within the larger areas marked for manual macrodissection. The KRAS mutant allele fraction and estimated neoplastic cellularity were significantly higher in samples obtained by digitally guided microdissection (p < 0.01), and 7 of the 32 samples (22%) showed a detectable mutation only with digitally guided microdissection. DNA quality and yield per cubic millimeter of dissected tissue were similar for both dissection methods. These results indicate a significant improvement in tumor content achievable with digitally guided microdissection.

High-throughput genotyping of Anopheles mosquitoes using intact legs by Agena Biosciences iPLEX.

Recent developments in genotyping technologies coupled with the growing desire to characterize genome variation in Anopheles populations open the opportunity to develop more effective genotyping strategies for high-throughput screening. A major bottleneck of this goal is nucleic acid extraction. Here, we examined the feasibility of using intact portions of a mosquito's leg as sources of template DNA for whole-genome amplification (WGA) by primer-extension preamplification. We used the Agena Biosciences MassARRAY(®) platform (formerly Sequenom) to genotype 78 SNPs for 265 WGA leg samples. We performed nucleic acid extraction on 36 mosquito carcasses and compared the genotype call concordance with their corresponding legs and observed full concordance. Using three legs instead of one improved genotyping success rates (96% vs. 89%, respectively), although this difference was not significant. We provide a proof of concept that WGA reactions can be performed directly on mosquito legs, thereby eliminating the need to extract nucleic acid. This approach is straightforward and sensitive and allows both species determination and genotyping of Anopheles mosquitoes to be performed in a high-throughput manner. Our protocol also leaves the mosquito body intact facilitating other experimental analysis to be undertaken on the same sample. Based on our findings, this method would also be suitable for use with other insect species.

Detection of KRAS, NRAS and BRAF by mass spectrometry - a sensitive, reliable, fast and cost-effective technique.

BackgroundAccording to current clinical guidelines mutational analysis for KRAS and NRAS is recommended prior to EGFR-directed therapy of colorectal cancer (CRC) in the metastatic setting. Therefore, reliable, fast, sensitive and cost-effective methods for routine tissue based molecular diagnostics are required that allow the assessment of the CRC mutational status in a high throughput fashion.MethodsWe have developed a custom designed assay for routine mass-spectrometric (MS) (MassARRAY®, Agena Bioscience) analysis to test the presence/absence of 18 KRAS, 14 NRAS and 4 BRAF mutations. We have applied this assay to 93 samples from patients with CRC and have compared the results with Sanger sequencing and a chip hybridization assay (KRAS LCD-array Kit, Chipron). In cases with discordant results, next-generation sequencing (NGS) was performed.ResultsMS detected a KRAS mutation in 46/93 (49 %), a NRAS mutation in 2/93 (2 %) and a BRAF mutation in 1/93 (1 %) of the cases. MS results were in agreement with results obtained by combination of the two other methods in 92 (99 %) of 93 cases.In 1/93 (1 %) of the cases a G12V mutation has been detected by Sanger sequencing and MS, but not by the chip assay. In this case, NGS has confirmed the G12V mutation in KRAS.ConclusionsMutational analysis by MS is a reliable method for routine diagnostic use, which can be easily extended for testing of additional mutations.

The suitability of small biopsy and cytology specimens for EGFR and other mutation testing in non-small cell lung cancer.

Patients with advanced non-small cell lung cancer (NSCLC) benefit from treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) when their tumor harbors an activating EGFR mutation. As the majority of NSCLC patients present with advanced disease, cytology and small biopsy specimens are frequently the only tissue available for mutation testing, but can pose challenges due to low tumor content. We aim to better define the suitability of these specimens for mutation testing. NSCLC cases referred to our institution for mutation testing over a 15-month period were retrospectively reviewed. Specimens were tested for mutations including EGFR, KRAS, and BRAF, using a multiplex PCR assay (OncoCarta Panel v1.0) and analyzed on the Agena Bioscience MassARRAY platform. A total of 146 specimens were tested, comprising 53 (36.3%) resection specimens (including 28 lung resection specimens), 55 (37.7%) small biopsy specimens and 38 (26%) cytology specimens. Of 142 cases with sufficient DNA for mutation testing, EGFR mutations were detected in 31 specimens (21.8%), KRAS mutations in 31 specimens (21.8%) and BRAF mutations in three specimens (2.1%). There was no significant difference in the EGFR mutation rate between lung resection (10 of 28 cases; 35.7%), small biopsy (9 of 53 cases; 17%), and cytology specimens (8 of 36 cases; 22.2%). Our results support the utility of small biopsy and cytology specimens for mutation testing. Careful evaluation of the adequacy of small specimens is required to minimize the risk of false negative or positive results.

Assessing the clinical value of targeted massively parallel sequencing in a longitudinal, prospective population-based study of cancer patients.

Introduction:Recent discoveries in cancer research have revealed a plethora of clinically actionable mutations that provide therapeutic, prognostic and predictive benefit to patients. The feasibility of screening mutations as part of the routine clinical care of patients remains relatively unexplored as the demonstration of massively parallel sequencing (MPS) of tumours in the general population is required to assess its value towards the health-care system.Methods:Cancer 2015 study is a large-scale, prospective, multisite cohort of newly diagnosed cancer patients from Victoria, Australia with 1094 patients recruited. MPS was performed using the Illumina TruSeq Amplicon Cancer Panel.Results:Overall, 854 patients were successfully sequenced for 48 common cancer genes. Accurate determination of clinically relevant mutations was possible including in less characterised cancer types; however, technical limitations including formalin-induced sequencing artefacts were uncovered. Applying strict filtering criteria, clinically relevant mutations were identified in 63% of patients, with 26% of patients displaying a mutation with therapeutic implications. A subset of patients was validated for canonical mutations using the Agena Bioscience MassARRAY system with 100% concordance. Whereas the prevalence of mutations was consistent with other institutionally based series for some tumour streams (breast carcinoma and colorectal adenocarcinoma), others were different (lung adenocarcinoma and head and neck squamous cell carcinoma), which has significant implications for health economic modelling of particular targeted agents. Actionable mutations in tumours not usually thought to harbour such genetic changes were also identified.Conclusions:Reliable delivery of a diagnostic assay able to screen for a range of actionable mutations in this cohort was achieved, opening unexpected avenues for investigation and treatment of cancer patients.

Blood group genotyping: the power and limitations of the Hemo ID Panel and MassARRAY platform

Abstract Matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS), is a sensitive analytical method capable of resolving DNA fragments varying in mass by a single nucleotide. MALDI-TOF MS is applicable to blood group genotyping, as the majority of blood group antigens are encoded by single nucleotide polymorphisms. Blood group genotyping by MALDI-TOF MS can be performed using a panel (Hemo ID Blood Group Genotyping Panel, Agena Bioscience Inc., San Diego, CA) that is a set of genotyping assays that predict the phenotype for 101 antigens from 16 blood group systems. These assays involve three fundamental stages: multiplex target-specific polymerase chain reaction amplification, allele-specific single base primer extension, and MALDI-TOFMS analysis using the MassARRAY system. MALDI-TOF MS–based genotyping has many advantages over alternative methods including high throughput, high multiplex capability, flexibility and adaptability, and the high level of accuracy based on the direct detection method. Currently available platforms for MALDI-TOF MS–based genotyping are not without limitations, including high upfront instrumentation costs and the number of non-automated steps. The Hemo ID Blood Group Genotyping Panel, developed and optimized in a collaboration between the vendor and the Blood Transfusion Service of the Swiss Red Cross in Zurich, Switzerland, is not yet widely utilized, although several laboratories are currently evaluating the MassARRAY system for blood group genotyping. Based on the accuracy and other advantages offered by MALDI-TOF MS analysis, in the future, this method is likely to become widely adopted for blood group genotyping, in particular, for population screening. Immunohematology 2015;31:75–80.

Examination of Single Nucleotide Polymorphisms (SNPs) in Transient Receptor Potential (TRP) Ion Channels in Chronic Fatigue Syndrome Patients

Background The transient receptor potential (TRP) superfamily in humans comprises 27 cation channels with permeability to monovalent and divalent cations. These channels are widely expressed within humans on cells and tissues and have significant sensory and regulatory roles on most physiological functions. Chronic fatigue syndrome (CFS) is an unexplained disorder with multiple physiological impairments. OBJECTIVES The purpose of this study was to determine the role of TRPs in CFS. Methods The study comprised 115 CFS patients (age = 48.68 ± 1.06 years) and 90 nonfatigued controls (age = 46.48 ± 1.22 years). CFS patients were defined according to the 1994 Center for Disease Prevention and Control criteria for CFS. A total of 240 single nucleotide polymorphisms (SNPs) for 21 mammalian TRP ion channel genes ( TRPA1, TRPC1, TRPC2, TRPC3, TRPC4, TRPC6, TRPC7, TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8, TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, and TRPV6) were examined via the Agena Biosciences iPLEX Gold assay. Statistical analysis was performed using the PLINK analysis software. Results Thirteen SNPs were significantly associated with CFS patients compared with the controls. Nine of these SNPs were associated with TRPM3 (rs12682832; P &lt; 0.003, rs11142508; P &lt; 0.004, rs1160742; P &lt; 0.08, rs4454352; P &lt; 0.013, rs1328153; P &lt; 0.013, rs3763619; P &lt; 0.014, rs7865858; P ≤ 0.021, rs1504401; P ≤ 0041, rs10115622; P ≤ 0.050), while the remainder were associated with TRPA1 (rs2383844; P ≤ 0.040, rs4738202; P ≤ 0.018) and TRPC4 (rs6650469; P ≤ 0.016, rs655207; P ≤ 0.018). Conclusion The data from this pilot study suggest an association between TRP ion channels, predominantly TRPM3 and CFS. This and other TRPs identified may contribute to the etiology and pathomechanism of CFS.

Examination of Single Nucleotide Polymorphisms in Acetylcholine Receptors in Chronic Fatigue Syndrome Patients

Objective Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a disorder characterized by debilitating fatigue accompanied by pain and impairments in memory, cognition, and concentration. Acetylcholine (ACh) has a plethora of roles in neuronal and neuromuscular transmission. There are two types of ACh receptors, muscarinic and nicotinic, comprising 17 different subunits of the nicotinic ACh receptor (nAChR) and five different subtypes of the muscarinic receptor (mAChR) that have been identified in humans. The purpose of this study was to determine the role of ACh receptor (nAChRs and mAChRs) single nucleotide polymorphisms (SNPs) in CFS/ME patients. Methods One-hundred and fifteen CFS/ME patients (age = 48.68 ± 1.06 years) and 90 nonfatigued controls (age = 46.48 ± 1.22 years) participated in this study, where CFS/ME patients were defined according to the 1994 Center for Disease Prevention and Control (CDC) criteria. A total of 464 SNPs for nine mammalian ACh receptor genes ( M1, M2, M3, M4, M5, alpha 2, 5, 7, and 10) were examined via the Agena Biosciences iPLEX Gold assay. Statistical analysis was performed using the PLINK analysis software. Results Seventeen SNPs were significantly associated with CFS/ME patients compared with the controls. Nine of these SNPs were associated with mAChRM3 (rs4463655; P = 0.00281, rs589962; P = 0.00348, rs1072320; P = 0.00371, rs7543259; P = 0.00513, rs6661621; P = 0.00536 rs7520974; P = 0.0167, rs726169; P = 0.02349, rsrs6669810; P = 0.02361, rsrs6429157; P = 0.0375), while the remainder were associated with nAChR alpha 10 (rs2672211; P = 0.0107, rs2672214; P = 0.0108, rs2741868; P = 0.01185, rs2741870; P = 0.01281, rs2741862; P = 0.03043), alpha 5 (rs951266; P = 0.01153; rs7180002, P = 0.03682), and alpha 2 (rs2565048; P = 0.01403). Conclusion The data from this pilot study suggest an association between ACh receptors, predominantly M3 and CFS. ACh receptor SNPs may contribute to the pathomechanism of CFS/ME.

Blood group genotype analysis of Australian reagent red blood cell donors across three genotyping platforms: consistent detection of 7·0% phenotype genotype nonconcordance

Background and ObjectivesStandards mandate reagent red blood cells (RBCs) must be homozygous for certain antigens. Genotyping provides an enhanced approach to characterizing RBCs as mutations encoding weakened antigen expression may be detected. This study aimed to genotype reagent RBC donors and compare outcomes with serological phenotype. Genotyping was undertaken using three commercially available platforms.Materials and MethodsBlood samples were collected from donors (n = 300) who contribute to reagent RBC panels. Genotyping was performed on at least one of three commercial platforms; BLOODchip Reference v4.1 (Progenika); human erythrocyte antigen (HEA) and RHD BeadChip (BioArray Solutions Ltd); Hemo ID Blood Group Genotyping Panel (Agena Bioscience).ResultsGenotype and phenotype were concordant for 279 of 300 donors (21 of 300; 7·0% nonconcordance). Five donors carried RH mutations and fifteen donors carried FY mutations, including two donors who had an incorrect phenotype reported. One donor carried RH and FY mutations.ConclusionDiscrepancies between genotype and phenotype were detected in 7·0% of reagent RBC donors tested in clinically significant blood group systems. Genotyping of reagent RBC donors can improve transfusion safety by optimizing sensitivity of antibody screening and identification panels.