Probe Kit Research Articles

Abstract 1737: ALK rearrangement analysis in circulating tumor cells of lung cancer patients

Abstract The evaluation of ALK rearrangement in non-small-cell lung cancer (NSCLC) is a significant tool when considering chemotherapy. It is not always possible to perform a tumor biopsy in patients. We suggest isolation and culturing of circulating tumor cells (CTCs) as an alternative tool to a tumor biopsy for the diagnosis of ALK rearrangement. From 22 patients with NSCLC harboring ALK rearrangement, blood samples were collected and divided into two parts: one for immunofluorescence staining of CTC marker and the other for culturing of CTCs. Both samples were processed by size-based filtration, and Cultured CTCs were analyzed for EML4-ALK translocation by fluorescence in situ hybridization (FISH) using Vysis ALK break apart FISH probe kit. CTC culturing was successful in 18 of 22 cases (81.8%). Among 18 cases of successful CTC cultures, 13 cases showed ALK rearrangement positivity (72.2%). Therefore, we suggest that the CTCs can be used as an alternative method to tissue biopsy for diagnosing ALK rearrangement. In addition, this method may have clinical applications including serial blood sampling for the development of personalized cancer therapy based on individual genomic information. Citation Format: Min Kyung Jeon, Young Hun Kim, Eunjoo Hwang, Hye Seon Lee, Ji-hyun Uh, Myoung Shin Kim, JooKyung Park, Byung Hee Jeon, Se-Hoon Lee. ALK rearrangement analysis in circulating tumor cells of lung cancer patients [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 1737. doi:10.1158/1538-7445.AM2017-1737

Effects of benzo(a)pyrene exposure on the ATPase activity and calcium concentration in the hippocampus of neonatal rats.

To investigate whether postnatal benzo(a)pyrene (B(a)P) exposure caused the impairments on the process of neurodevelopment and the alteration in the calcium medium in the neonatal rats. Eighty neonatal Sprague Dawley (SD) rats were randomly divided into 5 groups (untreated control group, vehicle group, 0.02 mg/kg, 0.2 mg/kg and 2 mg/kg B(a)P-exposed group). Rats were treated with B(a)P by the intragastric administration from postnatal day (PND) 4 to 25. Morris water maze (MWM) was employed to observe the spatial memory of rats. The activity of calcium adenosine triphosphatase (Ca2+-ATPase), sodium-potassium adenosine triphosphatase (Na+-K+-ATPase) and calcium-magnesium adenosine triphosphatase (Ca2+-Mg2+-ATPase) in the hippocampus were detected by commercial kits. Fura-2 pentakis(acetoxymethyl) (Fura-2/AM) probe and reactive oxygen species (ROS) reagent kit were used for measuring the concentration of Ca2+ and ROS in the hippocampus synapse, respectively. Rats exposed to B(a)P resulted in the deficits in the spatial memory manifested by the increased escape latency and decreased number of crossing platform and time spent in target quadrant in comparison with the control groups. Benzo(a)pyrene exposure caused the significant decrease in the ATPase activity in the hippocampus and caused Ca2+ overload in the synaptic, besides, the ROS concentration increased significantly which may further induce neurobehavioral impairment of the neonatal rats. Our findings suggest that postnatal B(a)P exposure may cause the neurobehavioral impairments in the neonatal rats, which were mediated by the decreased ATPase activity and elevated Ca2+ concentration. Int J Occup Med Environ Health 2017;30(2):203-211.

A comparison of interfraction setup error, patient comfort, and therapist acceptance for 2 different prostate radiation therapy immobilization devices

PurposeOur purpose was to investigate interfraction setup error of the immobilization device required to implement transperineal ultrasound compared with the current, standard immobilization device. Patient comfort and radiation therapist (RT) satisfaction were also assessed. Methods and materialsCone beam computed tomography images were acquired before 4069 fractions from 111 patients (control group, n = 56; intervention group, n = 55) were analyzed. The intervention group was immobilized using the Clarity Immobilization System (CIS), comprising a knee rest with autoscan probe kit and transperineal ultrasound probe (n = 55), and control group using a leg immobilizer (LI) (n = 56). Interfraction setup errors were compared for both groups. Weekly questionnaires using a 10-point visual analog scale were administered to both patient groups to measure and compare patient comfort. RT acceptance for both devices was also compared using a survey. ResultsThere was no significant difference in the magnitude of interfraction cone beam computed tomography–derived setup shifts in the lateral and anteroposterior direction between the LI and CIS (P = .878 and .690, respectively). However, a significant difference (P = .003) was observed in the superoinferior direction between the 2 groups of patients. Patient-reported level of comfort and stability demonstrated no significant difference between groups (P = .994 and .132). RT user acceptance measures for the LI and CIS were ease of handling (100% vs 53.7%), storage (100% vs 61.1%), and cleaning of the devices (100% vs 64.8%), respectively. ConclusionsThe CIS demonstrated stability and reproducibility in prostate treatment setup comparable to LI. The CIS device had no impact on patient comfort; however, RTs indicated a preference for LI over the CIS mainly because of its weight and bulkiness.

Abstract P4-12-07: CCND1 amplification in early breast cancer patients: Correlation with subtypes and prognosis

Abstract Background: Mechanisms of progress and recurrence of early breast cancer (BC) have gained importance since several targeted therapeutic options in metastatic breast cancer have been introduced over the last years, especially in hormone receptor positive BC. This study investigates the amplification of cell cycle regulator cyclin D1 gene (CCND1) amplification as one possible progression mechanism. Patients & Methods: Patients from an unselected cohort of early BC patients were included into this study. A tissue microarray (TMA) was available for n=832 patients with early breast cancer. CCND1 amplification was assessed after FISH (Abbott Vysis LSI CCND1 SpectumOrange/CEP11 SpectrumGreen Probes Kit). A CCND1/CEP11-ratio ≥ 2.0 was considered as amplification. Staining was successful in 545 tumor cores. Amplification results were correlated with clinical patient and tumor characteristics and survival analyses were performed with regard to distant disease free survival and overall survival. Results: CCND1 amplification was found in 13.6% of patients. Triple negative, luminal A like, luminal B like and HER2 positive tumors were amplified in 7.5%, 7.8%, 18.6% and 15.7% respectively (p = 0.010). CCDN1 amplification was significantly associated with a higher grading and a higher body mass index. Furthermore an amplification was seen more frequently in lobular BC and ductal BC than other histological subtypes. Survival analysis showed a reduced DDFS for patients with CCDN1-amplification. 5 year DDFS rates were 90.6% for non-amplified tumors and 86.0% for amplified tumors (p, log-rank =0.066 ). 5 year OS rates were 93.0% for non-amplified tumors and 90.1% for amplified tumors (p, log-rank =0.119). Adjusted for age, tumor size, nodal status and molecular subtype, cox regression showed HR of 1.3 (95% CI: 0.76-2.5, p=0.46) for DDFS and a HR of 1.33 (95% CI: 0.7-2.53, p=0.38) for OS. Conclusion: With a 13.6% prevalence in all breast cancer patients, mainly present in luminal B like cancers, CCND1-amplification is a genetic aberration associated with an unfavorable prognosis. Within hormone receptor positive women CCDN1 amplification might play a role in treatment resistance mechanisms in early breast cancer patients. Citation Format: Hanf DC, Fasching PA, Villalobos IE, Gasparyan A, Wachter D, Santiago A, Guzman R, Weihbrecht S, Hanf V, Hartmann A, Beckmann MW, Press MF. CCND1 amplification in early breast cancer patients: Correlation with subtypes and prognosis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-07.

Abstract B35: A breast cancer susceptibility gene screening panel for variant discovery and gene exclusion in an African American cohort from Alabama

Abstract African American (AA) women are diagnosed with breast cancer (BC) at a higher rate under the age of 40 compared to Caucasian women and are more often diagnosed with triple-negative (TN) BC, a less treatable and more aggressive BC sub-type. Since an early age of onset is a key characteristic of hereditary BC, genetic risk factors are likely contributing towards these BC disparities. Genes that harbor genetic risk variants are referred to as BC susceptibility genes. Prior to searching for novel BC susceptibility genes, previously identified BC susceptibility genes should initially be excluded. Since the introduction of next-generation sequencing, gene-screening panels have been implemented; this innovative approach involves designing probes for target-capture of genes of interest, amplification, and sequencing on a massively parallel array. AA BC genetics is understudied; however, recently, Churpek et al. published the first article to screen AA hereditary/early onset/TN BC cases for a panel of known BC susceptibility genes. This study identified 68 pathogenic germline mutations in 65 of 289 (22%) AA cases; 18% had a BRCA1 or BRCA2 mutation. The authors acknowledged the paucity of their study and that more gene panel screening studies are needed. Currently, it appears that the majority and spectrum of BC susceptibility genes/risk variants that contribute towards the disproportionate number of aggressive and young onset AA BCs remain to be identified. Our group has custom-designed an Agilent Technologies (AT) HaloPlex probe kit, named BRAP (BReast And Prostate) that targets 87 known and candidate BC and prostate cancer (PC) susceptibility genes using the AT SureDesign program. PC genes were included on the panel since AA males are frequently diagnosed with PC in BC families, more susceptible to PC compared to Caucasians, and normally diagnosed at a younger age with larger tumors. Furthermore, men carrying BRCA1/2 mutations have a higher risk of PC, so further genetic overlap will likely be detected through BRAP screening. Ultimately, the probes were designed to generate 100 base pair (bp) reads on an Illumina platform. All coding exons as well as the 5' and 3' untranslated regions of the 87 genes were targeted, totaling 499.5 kbp. The designed probes covered 98.9% of the targeted region. Forty-six seemingly unrelated individuals (24 AA and 22 Caucasians) from Alabama who were diagnosed with BC under the age of 45 and/or had a BC family history were selected for the initial BRAP capture; the HaloPlex HS Target Enrichment System For Illumina Sequencing Protocol (Version C0) was followed. Each individually prepared and indexed library was quantified using a Bioanalyzer 2100. The targeted DNA from 44 of the 46 individuals were successfully captured and amplified. The successful samples were pooled for sequencing; the sample was loaded onto one flow cell lane and run on an Illumina HiSeqTM 2500 at the HudsonAlpha Genomic Services Laboratory. The sequencing data was downloaded and is being processed using an in-house, bioinformatics pipeline. Two of the screened individuals served as positive controls since upon recruitment one reported a mutation in BRCA1 and the other a mutation in BRCA2. The pipeline has been optimized using the control with BRCA1 p.M1775R, which has been successfully identified. Variant calling files are being processed for the remaining screened individuals. This data provides great insight towards the array of mutations in known BC and PC susceptibility genes that contributes towards hereditary AA BC. Furthermore, non-mutation individuals/families identified through BRAP screening will be whole-genome sequenced in order to identify novel, BC susceptibility genes that may contribute towards disease disparity. Citation Format: Madison R. Chandler, Kasey J. Shepp, Stephanie M. Spina, Nancy D. Merner. A breast cancer susceptibility gene screening panel for variant discovery and gene exclusion in an African American cohort from Alabama. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B35.

Microvolume Dielectric Spectroscopy and Molecular Dynamics of Amino Acids

Accurate knowledge of electromagnetic properties of biosystems is essential for the novel physical methods for therapy and diagnostics in medicine and biotechnology.To study the dynamics and electrical properties of biomolecules, we have designed, fabricated, and tested novel grounded coplanar waveguide-based chip for dielectric spectroscopy of liquid samples in 0.5 - 40 GHz band made on RO4350B substrate and we implemented a method for the permittivity extraction based purely on the extraction from measured scattering parameters.To our knowledge, the proposed bio sensing method represents an advantage over the current known sensing chips because the dielectric function of sample is obtained purely by using precision calibration techniques (NIST multi-line TRL) and computational optimization (CST Microwave Studio) without any a priori assumption about the dielectric model of measured sample. This chip is designed with well-defined active area to achieve the high sensitivity to dielectric change, enables perfect repeatability of sample position without the need of any microfluidics. Furthermore, due to its sensing size, the chip permits to work with only 250 microliter sample volume.In the pioneering experiments, we focused on dielectric (i.e. polarization) properties of solutions of amino acids, as building blocks of proteins. Alanine has been selected. The polar nature of amino acids determines their behavior in aqueous solutions and due to fact that at least one relaxation process is anticipated in microwave band, it allows us to study their dynamics and structure which employ using broadband dielectric spectroscopy. Data extracted from experiment are in an exact agreement in comparison with widely used commercial reflection method by coaxial probe (85070E Dielectric Probe Kit). Data show the trend of rising static permittivity at low frequencies with rising concentration of amino acids and also show the shift of relaxation times. We also performed molecular dynamics simulations to predict the complex permittivity of amino acid solution and obtained good agreement with experimental data. The presented procedure enables simple experimental verification of molecular dynamics of biomolecules by the dielectric spectroscopy using our microvolume chip.We acknowledge support by the Czech Science Foundation, project no. P102/15-17102S. Authors participate in COST Action BM1309 and bilateral exchange project between Czech and Slovak Academies of Sciences, no. SAV-15-22.

Analysis of Polarimetric Decomposition, Backscattering Coefficient, and Sample Properties for Identification and Layer Thickness Estimation of Silica Sand Distribution Using L-Band Synthetic Aperture Radar

Silica is one of the mineral needs of industry, especially in base materials for the primary or auxiliary raw materials. Silica sand on Rupat Island was identified on the northern coastline. Selected samples were measured to get the percentage of minerals/elements using X-ray fluorescence. Two adjacent scenes of ALOS PALSAR full-polarimetry data were used. The dielectric constant of samples was measured using a dielectric probe kit in the frequency range from 0.3 to 3.0 GHz and also used for calculating the backscattering coefficient. Freeman-Durdeen and Yamaguchi techniques were used to obtain the scattering decomposition of physical scattering from the incoherent object model base. Surface scattering is a very clear decomposition used to show the silica sand identification compared with other decompositions. From surface scattering, the backscattering coefficient value of silica sand resulted from −59 dB to −52 dB. Thickness estimation from satellite and field measurements show the minimum is 59 cm and the maximum is 74 cm, with the average differentiation between these measurements being 4.88 cm. The condition supported by the grain size of silica sand particles that have almost the same size and shape were conducted by using microscopic photograph testing. These values were caused by a slightly rough surface.

Superior Multiplexing Capacity of PlexPrimers Enables Sensitive and Specific Detection of SNPs and Clustered Mutations in qPCR

BackgroundWhilst qPCR provides an extremely powerful tool for genetic analysis, some applications such as multiplexing variant alleles (eg SNPs, point mutations or deletions), remain challenging using current primer/probe systems. The novel design features of PlexPrimers allow sensitive, multiplexed analysis of variant alleles even when these are tightly clustered.MethodPlexPrimers were combined with PlexZymes in qPCR assays for the detection of SNPs in human absorption, distribution, metabolism, and excretion (ADME) genes; clustered mutations in the 23S rRNA gene which confer antibiotic resistance to Mycoplasma genitalium; and deletions within the human epidermal growth factor receptor (EGFR) gene.ResultsThe combination of PlexPrimers and PlexZymes allowed robust multiplexing of targets which resulted in 100% concordance with results obtained using hydrolysis probe kits for 14 SNPs in the ADME genes. A 7-plex qPCR assay targeting M. genitalium, 5 clustered mutations associated with macrolide resistance and an internal control, allowed efficient amplification of all targets, with all 5 mutations detected in a single channel. Finally, the strategy was employed to analyse common EGFR mutants with high sensitivity, detecting deletions present at only 0.01%.ConclusionPlexPrime is a novel technology for the detection of genetic variants. Unlike previous strategies, the combination of PlexPrimers with PlexZymes enables both allele-specific detection and allele-specific amplification in qPCR. The study demonstrated highly sensitive and specific detection of mutations and SNPs, and superior multiplexing capacity. The ability to multiplex clustered genetic variants reduces the time to result providing more actionable information.

P1.02-030 Performance Evaluation of ALK/ROS1 Dual Break Apart FISH Probe Kit (RUO) in Non-Small-Cell Lung Cancer

ALK and ROS1 gene rearrangements are distinct molecular subsets of non-small-cell lung cancer (NSCLC), and they are strong predictive biomarkers of response to ALK/ROS1 inhibitors, such as crizotinib. Thus, it is clinically important to detect patients who will benefit from such treatment and develop an effective screening strategy. In this study, we aim to evaluate the diagnostic performance of ALK/ROS1 RUO FISH probes which can concurrently detect ALK and ROS1 rearrangements. The study populations were composed of three patient cohorts with histologically confirmed lung adenocarcinoma (ALK rearrangement, ROS1 rearrangement and both wild type). Patient specimens consisted of 12 ALK-positive, 9 ROS1-positive and 21 ALK/ROS1-wild type formalin-fixed paraffin-embedded samples obtained from surgical resection or excisional biopsy. ALK rearrangement status was determined by Vysis LSI Dual Color Break Apart Rearrangement Probe (Abbott Molecular, Abbott Park, IL, USA) and ROS1 rearrangement status was assessed by ZytoLight SPEC ROS1 dual color break apart probe (Zytovision. Bremerhaven, Germany). All specimens were re-evaluated by ALK/ROS1 Break Apart FISH RUO 4-color kit. FISH images were scanned via the BioView Duet and interpreted remotely via BioView SoloWeb. A total of 42 patient samples were evaluated. The concordance of results obtained from ALK/ROS1 Break Apart FISH RUO 4-color kit was evaluated relative to the ALK and ROS1 rearrangement status of the specimen, as previously determined. One ROS1-positive and 2 wild-type samples were excluded from analysis due to high background. Regarding 12 ALK-positive samples, 12 were ALK-positive by ALK/ROS1 RUO FISH, showing 100% (n=12/12) sensitivity to predict ALK rearrangement. Regarding 8 ROS1-positive samples, 6 cases were ROS1-positive by ALK/ROS1 RUO FISH, showing 75% (n=6/8) sensitivity to predict ROS1 rearrangement. Two cases showed weak ROS1 signals that could not be enumerated. Regarding 19 wild type cases, 18 cases were negative by ALK/ROS1 RUO FISH, showing 95% (n=18/19) specificity, while one case showed poor ROS1 signals which could not be properly enumerated. ALK/ROS1 RUO FISH can detect ALK and ROS1 rearrangements simultaneously in NSCLC. The fluorescence of ROS1 signal may be weakened by slide shipment and remote scoring.

Electromagnetic-Based Microfluidic Sensor Applications

In this study, Metamaterial Based Microfluidic Sensor (MBMS), which is designed by utilising a Split Ring Resonator (SRR) along with a patch has been employed to reveal the three different types of sensor applications by experimentally and numerically. These applications include purity demonstration of methanol, ethanol and acetone, different material sensing which is illustrated by poly ethylene glycol (PEG 300, PEG400) and temperature sensor application which is new in the current literature and proven by utilising pure ammonia solution. In the first step of the validation, these materials’ complex permittivity values were measured using an Agilent 85070E dielectric probe kit between 4 GHz and 6 GHz. Following this, a numerical simulation test was conducted by applying a FIT-based simulation programme. Finally, in order to verify the numerical simulation study, a proposed design has been manufactured prior to carrying out an experimental study on all of the applications. To support the simulation and experimental results, theoretical study carried out and obtained results were compared with similar studies. Both the numerical and experimental results reveal that the proposed Microfluidic sensor, which is based on a change in the complex permittivity, can be configured and applied to other sensing tools.

Assessment of topoisomerase II-alpha gene status by dual color chromogenic in situ hybridization in a set of Iraqi patients with invasive breast carcinoma.

The human epidermal growth factor receptor 2(HER2) proto-oncogene is overexpressed or amplified in approximately 15%-25% of invasive breast cancers. Approximately 35% of HER2-amplified breast cancers have coamplification of the topoisomerase II-alpha (TOP2A) gene encoding an enzyme that is a major target of anthracyclines. Hence, the determination of genetic alteration (amplification or deletion) of both genes is considered as an important predictive factor that determines the response of breast cancer patients to treatment. The aims of this study are to determinate TOP2A status gene amplification in a set of Iraqi patients with breast cancer that have had an equivocal (2+) and positive HER2/neu by immunohistochemistry (IHC) and to compare the results with estrogen receptor (ER) and progesterone receptor (PR) and HER2/neu status. A cross-sectional prospective study done on 53 patients with invasive breast carcinoma. Twenty-six out of total 53 cases were positive HER2/neu (3+), the remaining 27 equivocal HER2-IHC (2+) cases reanalyzed using dual-color chromogenic in situ hybridization (ZytoVision) probe kit for further identification of HER2/neu gene amplification. Using chromogenic in situ hybridization (CISH), TOP2A gene status determination was done for all cases. There is a direct significant correlation between TOP2A gene amplification and HER2/neu positivity, P < 0.05 in that 15 (39.4%) out of 38 positive HER2/neu cases were associated with topoisomerase gene amplification. Regarding relation of topoisomerase gene to hormone receptor status (ER and PR), there was a significant negative relationship between the gene and ER receptor status. The higher level of gene amplification was noticed in ER and PR negative cases in about 13 (43.3%) and 14 (48.2%) for ER and PR, respectively. TOP2A gene status has a significantly positive correlation with HER2/neu status while it has a significantly negative correlation with hormone receptor status.

Molecular epidemiological study of microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion gene using immunohistochemistry as a cost effective alternative to fluorescence in situ hybridization for Indian patients with adenocarcinoma lung

Abstract Background: With fluorescence in situ hybridization (FISH) as the main-stay for the detection of anaplastic lymphoma kinase (ALK) rearrangements, the ALK Break Apart FISH Probe Kit has become a Food and Drug Association-approved companion diagnostic for targeted therapy with the ALK inhibitor crizotinib in lung cancers. The objective of this molecular epidemiological study was to estimate the prevalence of microtubule-associated protein-like 4-ALK (EML4-ALK) fusion gene using immunohistochemistry (IHC) as a cost effective alternative to FISH for Indian patients with nonsmall-cell cancer (NSCC)-adenocarcinoma, favor adenocarcinoma lung and NSCC- Not otherwise specified (NOS). Materials and Methods: Patients with NSCC-adenocarcinoma, favor adenocarcinoma lung, and nonsmall cell lung cancer-NOS histology were considered for this study. Permission was obtained from the Ethics Committee before the start of the study. Clinical characteristics and treatment details were collected from the patient's medical records. IHC analysis was performed using a Ventana automated immunostainer (Benchmark XT). Detection was performed using OptiView DAB Detection and Amplification Kit. Results: A total of 200 NSCC-adenocarcinoma, favour adenocarcinoma and NSCC-NOS patients were included in the study. There were 122 (61%) men and 78 (39%) women with a median age of 57 years. Of the 200 patients, 43 (21.5%) were nonsmokers and 175 (87.5%) had Stage-IV disease at the time of initial diagnosis. 48 (24%) cases were positive for epidermal growth factor receptor mutations, whereas EML4-ALK fusion gene was present in 27 (13.5%) patients. 25 of the 27 patients with ALK positivity received crizotinib therapy. Conclusions: The incidence of EML4-ALK gene fusions (13.5%) in this Indian population is four-fold high than the previous reported incidences and supports the claim of several recent studies that a relatively new ALK clone, 5A4, and D5F3 from Leica/Novocastra and cell signaling technology/Ventana, respectively can accurately identify ALK rearranged lung adenocarcinoma. The inclusion of IHC for the detection of EML4-ALK gene fusions as a low cost alternative seems justified in low resource setting.

The status of Her2 amplification and Kras mutations in mucinous ovarian carcinoma

Jayson GC et al. remarked in Lancet that nearly 100% of mucinous ovarian cancer cases have Kras mutation as well as a high frequency of Her2 amplification. Using the Abbott PathVysion Her2 DNA Probe Kit and Kras mutant-enriched PCR Kits (FemtoPath®), 21 samples of primary ovarian mucinous cystadenocarcinomas from Taiwanese patients were examined to determine the status of Her2 amplification and Kras mutations. Our results showed the Her2 amplification rates were 33.33%, while the Kras mutation rates were 61.90%. We present here our results in order to enlighten the readership that the ~100% Kras mutant frequency and the high Her2 amplification rate reported by Jayson et al. may be too exaggerated to be applicable into all populations. Additionally, we report another 2 novel Kras mutations (A11V, V14I).

Multiplex Single Nucleotide Polymorphism Analyses.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strands, since the fluorescent signals increase as the reaction progresses. The availability of multiple probes that fluoresce at different wavelengths allows for multiplexing as this gives rise to amplicons with unique fluorescent signatures. Here, we describe a method using the SensiFAST and SensiFAST One-Step probe kits which allows simultaneous real-time quantitation of up to 5 amplicons.

Multiplex Analyses Using Real-Time Quantitative PCR.

Quantitative polymerase chain reaction (qPCR) is a routinely used method for the detection and quantitation of gene expression in real time. Multiplex qPCR requires the use of probe-based assays, in which each probe is labeled with a unique fluorescent dye, resulting in different observed colors for each assay. The signal from each dye is used to quantitate the amount of each target separately in the same tube or well. The availability to multiplex therefore allows the measurement of the expression levels of several targets or genes of interest quickly. Here, we describe a method using the SensiFAST and SensiFAST One-Step probe kits which allows simultaneous real-time quantitation of up to 5 amplicons.

Separating component parts of soil respiration under Robinia pseudoacacia plantation in the Taihang Mountains, China

Partitioning the respiratory components of soil surface CO2 efflux is important in understanding carbon turnover and in identifying the soil carbon sink/source function in response to land-use change. The sensitivities of soil respiration components on changing climate patterns are currently not fully understood. We used trench and isotopic methods to separate total soil respiration into autotrophic (RA) and heterotrophic components (RH). This study was undertaken on a Robinia pseudoacacia L. plantation in the southern Taihang Mountains, China. The fractionation of soil 13CO2 was analyzed by comparing the δ13C of soil CO2 extracted from buried steel tubes with results from Gas Vapor Probe Kits at a depth of 50 cm at the preliminary test (2.03‰). The results showed that the contribution of autotrophic respiration (fRA) increased with increasing soil depth. The contribution of heterotrophic respiration (fRH) declined with increasing soil depth. The contribution of autotrophic respiration was similar whether estimated by the trench method (fRA, 23.50%) or by the isotopic method in which a difference in value of 13C between soil and plant prevailed in the natural state (RC, 21.03%). The experimental error produced by the trench method was insignificant as compared with that produced by the isotopic method, providing a technical basis for further investigations.

Expanding anchored hybrid enrichment to resolve both deep and shallow relationships within the spider tree of life.

BackgroundDespite considerable effort, progress in spider molecular systematics has lagged behind many other comparable arthropod groups, thereby hindering family-level resolution, classification, and testing of important macroevolutionary hypotheses. Recently, alternative targeted sequence capture techniques have provided molecular systematics a powerful tool for resolving relationships across the Tree of Life. One of these approaches, Anchored Hybrid Enrichment (AHE), is designed to recover hundreds of unique orthologous loci from across the genome, for resolving both shallow and deep-scale evolutionary relationships within non-model systems. Herein we present a modification of the AHE approach that expands its use for application in spiders, with a particular emphasis on the infraorder Mygalomorphae.ResultsOur aim was to design a set of probes that effectively capture loci informative at a diversity of phylogenetic timescales. Following identification of putative arthropod-wide loci, we utilized homologous transcriptome sequences from 17 species across all spiders to identify exon boundaries. Conserved regions with variable flanking regions were then sought across the tick genome, three published araneomorph spider genomes, and raw genomic reads of two mygalomorph taxa. Following development of the 585 target loci in the Spider Probe Kit, we applied AHE across three taxonomic depths to evaluate performance: deep-level spider family relationships (33 taxa, 327 loci); family and generic relationships within the mygalomorph family Euctenizidae (25 taxa, 403 loci); and species relationships in the North American tarantula genus Aphonopelma (83 taxa, 581 loci). At the deepest level, all three major spider lineages (the Mesothelae, Mygalomorphae, and Araneomorphae) were supported with high bootstrap support. Strong support was also found throughout the Euctenizidae, including generic relationships within the family and species relationships within the genus Aptostichus. As in the Euctenizidae, virtually identical topologies were inferred with high support throughout Aphonopelma.ConclusionsThe Spider Probe Kit, the first implementation of AHE methodology in Class Arachnida, holds great promise for gathering the types and quantities of molecular data needed to accelerate an understanding of the spider Tree of Life by providing a mechanism whereby different researchers can confidently and effectively use the same loci for independent projects, yet allowing synthesis of data across independent research groups.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0769-y) contains supplementary material, which is available to authorized users.

Silica Sand Identification using ALOS PALSAR Full Polarimetry on The Northern Coastline of Rupat Island, Indonesia

Silica sand is one of the minerals which relatively abundant in Indonesia. One of the areas with abundant of silica sand distribution is the northern coastline of Rupat Island, Bengkalis district, Riau province, Indonesia. The distribution of silica sand in this island identified only on the northern coastline in this island. Some selected sample of silica sand was measured to get the percentage of silica sand mineral’s content using X-Ray Fluorescence (XRF). Two adjacent scenes of ALOS PALSAR full-polarimetry were used. The physical properties of silica sand sample such as dielectric constant were measured using dielectric probe kit in the frequency range from 0.3 to 3.0 GHz and used for calculating the backscattering coefficient and the difference characteristics of silica sand with another object. Freeman-Durdeen and Yamaguchi techniques were used to get the scattering decomposition of physical scattering from the incoherent object model based. Surface scattering is the clearest of Scattering decomposition to show silica sand identification compares with other decompositions. From surface scattering, the backscattering coefficient value of silica sand was calculated starting from -59 dB until -52 dB. These values were given by the surface roughness condition, where the roughness is slightly rough planar. The flat condition supported by the grain size of silica sand particles that have almost the same size and shape, that were conducted by using microscopic photograph testing.

Killer-cell immunoglobulin-like receptors genotyping of 127 individuals from Terceira Island, Azores, Portugal

One hundred and twenty-seven unrelated Azorean individuals were randomly selected to study the gene frequencies of Killer-cell immunoglobulin-like receptors (KIR) in the Azorean (Terceira) population. KIR genotyping was performed by polymerase chain reaction using commercial sequence-specific oligonucleotide probe kits. All loci were in HWE, showing no locus-level deviations. The genotype data is available in the Allele Frequencies Net Database under the population name “Azores Terceira Island KIR”.

Abstract 439: Detection of aurora kinase A (AURKA) focal amplification in plasma samples of patients with recurrent ovarian cancer

Abstract Purpose: Aurora kinases are associated with acquired resistance to therapy in human cancers. We hypothesized that AURKA amplification is a late event in ovarian cancer (OC) progression and that frequency of amplification might be underestimated if assessed in tumor samples collected at the time of initial diagnosis. To test this hypothesis, we determined AURKA amplification levels in patients with recurrent OC and compared those to AURKA amplification levels in the same patients at the time of diagnosis. Methods: Samples were collected from 33 patients with taxane- and platinum-resistant OC enrolled in a first-in-human (FIH) study that evaluated monotherapy with AMG 900, an orally administered pan-aurora kinase inhibitor (NCT00858377). We performed a targeted analysis of the AURKA locus and surrounding regions using next-generation paired-end sequencing (Illumina HiSeq2500 and 100 bp paired-end reads). By using extremely high sequence coverage (average coverage of &amp;gt; 2,000-fold), we were able to detect and quantify circulating tumor DNA (ctDNA) in plasma and to identify chromosomal alterations characteristic of tumor DNA. Sequencing was performed at Personal Genome Diagnostics (PGDx, Baltimore, MD). In addition, we performed AURKA (20q13) / 20q11 FISH in archival FFPE sections from the same patients. The AURKA (20q13) / 20q11 dual color FISH probe kit was purchased from Kreatech (now Leica Biosystems, Buffalo Grove, IL). Amplification states were defined as follows: amplified: AURKA (20q13) / 20q11 signal &amp;gt; 2.0; borderline: AURKA (20q13) / 20q11 signal &amp;lt; 2.0 and &amp;gt; 1.5; non-amplified: AURKA (20q13) / 20q11 signal &amp;lt; 1.5. FISH analyses were performed at Mosaic Laboratories (Lake Forest, CA). Survival analysis was performed using the Kaplan-Meier method. Results: Plasma samples were available from all patients. Analysis of rearrangements surrounding the AURKA locus in plasma samples identified 11 of the 33 patients as AURKA amplified. Archival FFPE sections were available from 27 of the 33 patients, and 21 of these 27 patients were evaluable by FISH for AURKA amplification. FISH only identified 1 patient as AURKA amplified and 1 additional patient as borderline amplified. Patients with AURKA amplification in plasma had received a median (min, max) of 6 (2,18) prior lines of chemotherapy, while patients without AURKA amplification had received a median (min, max) of 3 (1, 14) lines of prior chemotherapy. AURKA amplification as determined in plasma ctDNA at the time of AMG 900 study entry was an indicator of shorter PFS (median 9.4 vs 19.9 weeks; P = 0.04) and OS (median 9.4 vs 27.6 weeks; P = 0.013). Conclusions: Our results suggest that AURKA amplification is a late event in ovarian cancer progression,and that monitoring AURKA focal amplification in plasma ctDNA allows the non-invasive assessment of acquired resistance to cancer therapy. Citation Format: Gloria Juan, Katherine Paweletz, Abraham Anderson, Erick Gamelin, Gregory Friberg, Robert Loberg, Florian Vogl. Detection of aurora kinase A (AURKA) focal amplification in plasma samples of patients with recurrent ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 439.