Probe Kit Research Articles

Metamaterial absorber sensor design by incorporating swastika shaped resonator to determination of the liquid chemicals depending on electrical characteristics

In this work, sensor abilities of the metamaterial absorber based on swastika shaped resonator are developed both theoretically and experimentally at X-band frequency range. The structure is consisted of a swastika shaped resonator on the top of dielectric layer and have an air gap to fill chemicals liquids between the copper plate and backside resonator. In this study, a full-wave EM solver CST Microwave Studio (Computer Simulation Technology) based on finite integrate technique has been used to simulate and investigate the absorption of the metamaterial structure with chemicals liquids depending on the electrical properties. A vector network analyzer 85070E probe kit has been used to measure the relative dielectric constants and loss tangent of some chemical liquids (ethanol content, methanol content, acetone, methanol, ethanol, Polyethylene Glycol 300, water) in the related frequency range. Absorption value of the sensor structure for selected chemicals placed in the air gaphas been investigated. It is obtained that there is a significant difference in absorption ratios between each chemicals and overall resonance frequency shifts have been observed which provides information to accurately estimate density rate of the sensed liquids. The absorption mechanisms of the metamaterial has been explained by using electric field, magnetic field and surface current distributions. Furthermore, the resonance absorption properties of the metamaterial based absorber sensor can be modified and adjusted easily by varying the dimensions of the resonator.Experimental and simulated results demonstrate that the resonance frequency of the swastika metamaterial based sensor is linearly related to the permittivity of selected chemicals which creates an appropriate approach for multipurpose sensor devise and electrochemical sensor.

PB1651 INFERIOR PROGNOSIS OF TLX3 GENE REARRANGEMENTS IN PEDIATRIC T‐ALL

Background:T‐cell acute lymphoblastic leukemia (T‐ALL) is a clinically and genetically heterogeneous disease that comprises 10‐15% of newly diagnosed pediatric ALL. Despite improved survival rates of children with T‐ALL, relapse occurs in almost 20% of patients and is associated with a dismal subsequent outcome. Therefore, it is of vital importance to identify children already early on during treatment who are at increased risk of such an event. Although several clinical and genetic factors were identified, prognostic implication of majority of them remains unclear. Therefore, the patients risk stratification is currently based only on minimal residual disease (MRD) monitoring.Aims:We performed retrospective and/or prospective comprehensive molecular cytogenomic analysis of bone marrow cells of children with T‐ALL with the aim to determine recurrent chromosomal aberrations and to identify those with significant impact on event free (EFS) and overall survival (OS).Methods:Samples of all patients were analyzed at the time of diagnosis with combination of conventional and molecular cytogenomic methods. For detection of the most frequent known chromosomal changes, i.e. rearrangements of TCR loci and TLX3 gene, deletion of CDKN2A and amplification of ABL1, interphase FISH with locus‐specific probes (Dako, Abbott Molecular) was used. MLPA analysis using SALSA MLPA ALL‐IKZF1 probemix (MRC‐Holland) was performed to detect deletions/amplifications of additional genes. Complex chromosomal rearrangements were proved with multicolor FISH and multicolor banding (24XCyte/XCyte Probe Kit; MetaSystems) or array CGH/SNP (SurePrint G3 Cancer CGH + SNP 4x180K, Agilent). For OS and EFS Kaplan‐Meier analysis with Mantel Cox test was done.Results:During the years 1996‐2017 we examined archived material of 68 children with T‐ALL (21 girls and 47 boys, median age 8.25 years), treated according to ALL Interfant and/or BFM based protocols. Median follow up was 9.5 years. In total, chromosomal aberrations were detected in 91% of patients. The most frequent were deletion of CDKN2A gene in 47/68 patients (27x homozygous, 20x heterozygous), rearrangements of TCR loci in 18/68 children (11x TRA/TRD, 7x TRB) and TLX3 gene rearrangement in 15/68 cases. Complex chromosomal aberrations were proved in 14/68 children. All chromosomal changes occurred in various combinations, however TLX3 aberrations never appeared along with TCR loci rearrangements. 48 patients are living in the first (45x)/second (3x) complete remission, 16 patients relapsed, 19 children died. Best outcome (EFS and OS) was associated with TRA/TRD locus translocations (p < 0.05). Conversely, patients with TLX3 rearrangement had significantly worse OS (p < 0.05).Summary/Conclusion:Considering the poor survival after relapse, it is of utmost importance to identify children at an increased risk of this event. MRD monitoring at different time points is a strong prognostic indicator and is used for risk stratification. In addition, rearrangements of TLX3 gene correlated with poor outcome in contrast to TRA/TRD translocations, which correlated with a more favorable course of the disease.Supported by grant RVO‐VFN64165, GACR P302/12/G157.

MicroRNA-214 contributes to regulation of necroptosis via targeting ATF4 in diabetes-associated periodontitis.

Diabetes and periodontal diseases have a mutual promoting relationship that induces severe tissue damage and cell death. The potential roles of microRNAs (miRNAs) and the type of cell death involved in diabetes-associated periodontitis are obscure. The gingival tissues of patients were obtained and MC3T3-E1 cells were costimulated with high glucose and lipopolysaccharide (LPS). Osseous morphometric analysis was evaluated with micro-CT, and histological characteristics were measured by hematoxylin/eosin and immunohistochemical staining. Cytokine secretion was confirmed by enzyme-linked immunosorbent assay, and reactive oxygen species (ROS) was measured using a DCFH-DA probe kit. Gene expression was measured by real-time quantitative reverse transcription PCR (qRT-PCR), and protein expression was assessed by Western blot and immunofluorescence analysis. The miR-214 level, receptor-interacting serine-threonine protein (RIP) 1, RIP3, and phospho-mixed lineage kinase domain-like (p-MLKL) protein expression were elevated in the inflamed gingival tissues of diabetes-associated periodontitis patients, with activating transcription factor 4 (ATF4) expression showing the opposite effect. The high glucose (22 mM) could not induce significant increase of RIP1, RIP3, and p-MLKL; however, the high glucose and LPS (500-1000 ng/mL) cotreatment resulted in increase in the number of RIP1, RIP3, and p-MLKL in MC3T3-E1 cells. NAC (ROS inhibitor) inhibited RIP1, RIP3, and increased ATF4; however, necrostatin-1 (Nec-1) (RIP1 inhibitor) specifically inhibited the protein expression of RIP1 and RIP3 and had no influence on ATF4. The use of antagomir-214 suppressed the expression of miR-214, RIP1, RIP3, and p-MLKL, but increased ATF4 protein level in glucose and LPS-induced cells. ATF4 knockdown by ATF4 small interfering RNA offset the effect of antagomir-214. RIP1- and RIP3-dependent necroptosis was confirmed in the inflamed gingival tissues of diabetes-associated periodontitis patients and high glucose- and LPS- cotreated cells. It was suggested that miR-214-targeted ATF4 participated in the regulation of necroptosis in vivo and in vitro.

Advancing mite phylogenomics: Designing ultraconserved elements for Acari phylogeny.

Mites (Acari) are one of the most diverse groups of life on Earth; yet, their evolutionary relationships are poorly understood. Also, the resolution of broader arachnid phylogeny has been hindered by an underrepresentation of mite diversity in phylogenomic analyses. To further our understanding of Acari evolution, we design targeted ultraconserved genomic elements (UCEs) probes, intended for resolving the complex relationships between mite lineages and closely related arachnids. We then test our Acari UCE baits in-silico by constructing a phylogeny using 13 existing Acari genomes, as well as 6 additional taxa from a variety of genomic sources. Our Acari-specific probe kit improves the recovery of loci within mites over an existing general arachnid UCE probe set. Our initial phylogeny recovers the major mite lineages, yet finds mites to be non-monophyletic overall, with Opiliones (harvestmen) and Ricinuleidae (hooded tickspiders) rendering Parasitiformes paraphyletic.

Tissue-mimicking materials for breast phantoms up to 50 GHz

Millimeter (mm)-wave imaging has been recently proposed as a new technique for breast cancer detection, based on the significant dielectric contrast between healthy and tumor tissues. Here we propose a procedure to fabricate, electromagnetically characterize and preserve realistic breast tissue-mimicking phantoms for testing mm-wave imaging prototypes. Low-cost, non-toxic and easy-to-produce mixtures made of sunflower oil, water and gelatin were prepared and their dielectric properties were for the first time measured in the (0.5–50) GHz frequency range using a coaxial probe kit. Different oil and gelatin percentages were tested. An alternative recipe based on a waste-oil hardener was also proposed. Finally, water and sunflower oil were investigated as preservation media. The mixtures electromagnetic properties were in good agreement with those of human breast ex vivo samples. By changing the ingredient concentrations or using different solidifying agents it was possible to mimic different tissue types. Besides, we show that sunflower oil represents an effective preservation medium for the developed materials. The first breast phantom mimicking a tumor mass into healthy tissues up to 50 GHz was also successfully fabricated. Results demonstrated the potential of the designed recipes to mimic breast tissues with different biological characteristics, preserving dielectric properties over time. Thus, this study represents a fundamental step towards the development of heterogeneous breast phantoms able to mimic the electromagnetic behavior of healthy and tumor tissues for mm-wave imaging applications.

ALK Expression in Angiomatoid Fibrous Histiocytoma: A Potential Diagnostic Pitfall.

We recently encountered a case of primary pulmonary angiomatoid fibrous histiocytoma (AFH), which was initially misdiagnosed as inflammatory myofibroblastic tumor (IMT) based in part on anaplastic lymphoma kinase (ALK) expression by immunohistochemistry (IHC). Prompted by this experience, we evaluated ALK expression in 11 AFH, 15 IMT, and 11 follicular dendritic cell sarcomas using 3 different antibody clones: D5F3, 5A4, and ALK1. ALK IHC positive cases were analyzed with fluorescence in situ hybridization (FISH) using dual color ALK break-apart probe kit. The majority of AFH cases studied were positive for ALK IHC with at least 1 antibody (9/11 D5F3, 6/9 5A4, 1/9 ALK1), most demonstrating moderate to strong cytoplasmic staining. AFH with positive ALK IHC showed no ALK gene rearrangement by FISH (0/8) with ALK copy number ranging from 1.6 to 2.1. Sixty-seven percent of IMT were ALK positive by IHC (10/15 D5F3, 8/15 5A4, 7/15 ALK1), and 9 of the 10 cases were positive for ALK gene rearrangement by FISH. All follicular dendritic cell sarcomas were negative for ALK by IHC (D5F3 and 5A4). Our results indicate that ALK expression in AFH is common, particularly with the highly sensitive D5F3 and 5A4 antibodies and enhanced detection systems, and may be a potential source of diagnostic confusion with IMT. The underlying mechanism of ALK expression in AFH is unclear, although it does not appear to be from ALK rearrangement or amplification.

Newborn screening for Prader-Willi syndrome is feasible: Early diagnosis for better outcomes.

Prader-Willi syndrome (PWS), is a complex genetic disease affecting 1/15,000 individuals, characterized by lack of expression of genes on the paternal chromosome 15q11-q13 region. Clinical features include central hypotonia, poor suck, learning and behavior problems, growth hormone deficiency with short stature, hyperphagia, and morbid obesity. Despite significant advances in genetic testing, the mean age for diagnosis in PWS continues to lag behind. Our goal was to perform a pilot feasibility study to confirm the diagnosis utilizing different genetic technologies in a cohort of 34 individuals with genetically confirmed PWS and 16 healthy controls from blood samples spotted and stored on newborn screening (NBS) filter paper cards. DNA was isolated from NBS cards, and PWS testing performed using DNA methylation-specific PCR (mPCR) and the methylation specific-multiplex ligation dependent probe amplification (MS-MLPA) chromosome 15 probe kit followed by DNA fragment analysis for methylation and copy number status. DNA extraction was successful in 30 of 34 PWS patients and 16 controls. PWS methylation testing was able to correctly identify all PWS patients and MS-MLPA was able to differentiate between 15q11-q13 deletion and non-deletion status and correctly identify deletion subtype (i.e., larger Type I or smaller Type II). mPCR can be used to diagnose PWS and MS-MLPA testing to determine both methylation status as well as the type of deletion or non-deletion status from DNA extracted from NBS filter paper. We propose that PWS testing in newborns is possible and could be included in the Recommended Uniform Screening Panel after establishing a validated cost-effective method.

P3.13-03 Real-World Anaplastic Lymphoma Kinase Testing Practices: Results from a Survey in the United States

Anaplastic lymphoma kinase (ALK) translocation is a clinically validated predictive biomarker in multiple cancer types including non-small cell lung cancer, and widespread adoption of diagnostic assays permits a review of test performance. This analysis presents ALK and epidermal growth factor receptor (EGFR) testing rates, expediency, and results from 4 large national laboratories. Deidentified data from January 2013 to December 2015 were compiled from 40 states and Washington DC and included in the final analysis. ALK status was evaluated with the Vysis ALK Break Apart FISH Probe Kit; EGFR status was evaluated by therascreen EGFR RGQ PCR Kit, cobas EGFR Mutation Test, or Sanger sequencing. Results from the most recent conclusive test were used. Collection to order (time from date of biopsy to date of biomarker test order), order to results (time from date of biomarker test order to date of available results), and overall turnaround times (collection to order + order to results) were evaluated. Results were analyzed by laboratories and US regions (Northeast, Midwest, South, and West). Data were summarized with descriptive statistics. s of 59,058 ALK and 50,992 EGFR tests were collected; 50,023 patients were tested for both ALK and EGFR, 9035 had results for ALK only, and 969 for EGFR only. Mean patient ages at ALK-positive (ALK+) and EGFR-positive (EGFR+) diagnoses were 62.5 and 69.0 years, respectively. Overall ALK+ and inconclusive rates were 2.8% and 6.5%, respectively, vs 10.4% and 6.2% for EGFR testing. ALK+ (2.3–5.1%) and inconclusive (1.1–17.4%) rates varied between test centers. In the western US, the ALK+ rate (3.3%) was comparably greater than in other regions (2.4–2.7%). The median turnaround time was 15 days, with a median of 10 days for collection to order and 4 days for order to results; some regional variation was seen in turnaround time (14–18 days). No correlation was seen between age and turnaround time. EGFR+ and ALK+ rates and age distributions were consistent with prior reports. An interesting geographic difference in ALK rates was seen that warrants further investigation. The average turnaround time was longer than current guidelines recommend, suggesting an opportunity to improve current testing practices.

P1.09-22 Detection of ALK Gene Rearrangement in FFPE Tissues of Non-Small Cell Lung Cancer Using in Situ RNA Hybridization

Approximately 4-7% of non-small cell lung cancer (NSCLC) harbor anaplastic lymphoma kinase (ALK) gene rearrangements which lead to overexpression of ALK fusion protein and constitutive activation of the kinase. The tumor with ALK gene rearrangements is sensitive to its inhibitor, so identification of this molecular feature from the patients accurately is important. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are conventional methods for testing ALK gene rearrangement and protein overexpression respectively; however it is challenge for distinguishing some rare variations by FISH, or determining the equivocal stain by IHC. A morphology related method under bright field about rearranged mRNA expression (RNAscope) of ALK gene fusion was developed, which is helpful to realize the biological behavior of ALK gene, together with FISH (break apart FISH probe kit, Abbott) and IHC (D5F3 clone, Ventana). We explored 7 cases of formalin-fixed paraffin-embedded (FFPE) samples from NSCLC patients by RNAscope® 2.5 Red assay Three of seven samples were found with ALK positive by RNAscope, which matched with FISH and IHC results. In these 3 ALK positive samples, exon 19-29 of ALK gene transcribed RNA signals were revealed by probe-Hs-ALK E1-E18 (score 1 to 2 in >50% cells), whereas exon 1-18 transcribed RNA signals were not shown by probe-Hs-ALK E19-E29, which confirmed by FISH results (with break apart signals). In 2 of 4 ALK RNAscope negative cases, ALK RNA signals were detected by both probe-Hs-ALK E1-E18 (score 2) and probe-Hs-ALK E19-E29 (score 2) in small regions (showed a small number of signals), which may indicate that ALK gene amplification and also confirmed by FISH results (with more than 2 fusion signals). And these 2 typical ALK RNAscope negative samples were considered close to be disomy by FISH. Our results demonstrate that RNAscope® 2.5 Red assay is a reliable and precise method to detect ALK mRNA expression caused by ALK gene fusion and also a one to discover the ALK gene amplification as well in FFPE samples of NSCLC, even though latter’s clinical significant is not clear. RNAscope® 2.5 Red assay is a both sensitive and specific method to provide information on the spatial distribution of ALK gene status and morphologic characteristic of tumors simultaneously under the bright field microscope.

P15 Prevalence of EML4- ALK Fusion Gene in Adenocarcinoma Lung Patients by Using Immuno Histo Chemistry

An oral ATP- competitive TKI of ALK and c- MET, Crizotinib has shown impressive clinical activity in advanced non–small cell lung cancers especially in the tumors harboring ALK rearrangements. With FISH as the mainstay for detection of ALK rearrangements, the ALK Break Apart FISH Probe Kit (Abbott Molecular, Des Plaines, IL) has become an FDA-approved companion diagnostic for targeted therapy with the ALK inhibitor crizotinib in lung cancers. The objective of this molecular epidemiological study is to estimate the prevalence of EML4-ALK fusion gene using IHC as a cost effective alternative to FISH for Indian patients with adenocarcinoma lung. Patients with NSCLC, adenocarcinoma histology, whose tumors had been tested for EML4-ALK fusion gene using IHC 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 (Ventana Medical Systems, Illkirch Graffenstaden, France). Detection was performed using a multimer-technology system with the UltraView Universal DAB detection kit. A total of 204 NSCLC adenocarcinoma patients were included in the study. There were 126 (61.7%) men and 78 (38.23%) women with a median age of 57 years. Of the 204 patients, 47 (23.03%) were non-smokers and 175 (85.78%) had stage-IV disease at the time of initial diagnosis. 48 (23.52%) blocks were positive for EGFR mutations whereas 156 (76.47%) were EGFR wild type. EML4-ALK fusion gene was present in 27 (13.23%) patients whereas 177 (86.76%) tumors were EML4-ALK negative. 25 out of the 27 patients with ALK positivity received Crizotinib therapy. The incidence of EML4-ALK gene fusions (13.23%) 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 cell signaling technology (Ventana) can accurately identify ALK rearranged lung ACA as compared to FISH. The inclusion of IHC for the detection of EML4-ALK gene fusions as a low cost alternative seems warranted.

Using Cultural Probes in the Sensitive Research Setting of Informal Caregiving. A Case Study

Abstract This case study report covers our experiences in using Cultural Probes during the first phase of our European and interdisciplinary research project TOPIC (The Online Platform for Informal Caregivers). In that stage of our research, we focused on two major issues: first, describing and analyzing the characteristics of the care and coordination work of elderly informal caregivers, and second, on first implications for design for the field of informal care. Although our general methodological approach was qualitative (ethnographic) with participant observation and interviewing, we included Cultural Probes as an additional method to ethnography for gaining insight information about the care practices by the means of self-observation of and reflection by the informal caregivers. The paper describes our adaptation of the Cultural Probes approach, the similarities and differences to Gaver et al. [1999. Interactions. 6(1): 21–29], the items of our TOPIC Cultural Probes Kit in detail, and re-constructs the design process of one of the items (the actimoClock). Based on the experiences of our participants with the probes kit and our analyses of that use, we also present lessons learned, pros and cons for including that method in the sensitive setting of informal caregiving.

Digital Probes Kit

Abstract (Cultural) probes have been used in various software design projects and HCI contexts over the last years. With the ubiquity of smartphones today, there are new ways to design probes material as digital probes. Smartphones allow to document everyday life in situ in mobile contexts and provide a permanent channel for communication between researchers and participants. Based on an analysis of strengths and weaknesses of traditional probes, we propose a concept for a Digital Probes Kit in the form of a digital diary and describe its potential.

Fabrication and Characterization of Flexible Polymer Iron Oxide Composite Substrate for the Imaging Antennas of Wearable Head Imaging Systems

Given the increased interest in wearable electromagnetic imaging systems, developing a low-cost, lightweight, flexible, and conformal customized substrate to accommodate the imaging antenna array is essential. The characterization and assessment of a custom-made composite substrate using a flexible polymer poly-di-methyl-siloxane (PDMS) and magnetite iron oxide ( ${\text{FeO.Fe}}_{2}{\text{O}}_{3}$ ) for wearable head imaging systems is presented. Micro-scale ${\text{FeO.Fe}}_{2}{\text{O}}_{3}$ particles are homogeneously combined with PDMS in different ratios to build the flexible engineered magneto-dielectric (MD) composite substrate. Besides the low cost, fabrication simplicity, and durability, the magnetite ${\text{FeO.Fe}}_{2}{\text{O}}_{3}$ particles can be used to control the relative permittivity and permeability over a wide range of values to suit the proposed application. The permittivity, permeability, and losses of the developed substrate are extracted using a custom-made two-port multilayer microstrip transmission line test fixture with the help of conformal mapping algorithms. The characterization is performed across the microwave frequency range 1.2–4 GHz, which is widely adopted for head imaging. The extracted permittivity is successfully verified by using a Keysight 85070E dielectric slim probe kit.

Relationship between HER2 gene status and selected potential biological features related to trastuzumab resistance and its influence on survival of breast cancer patients undergoing trastuzumab adjuvant treatment.

BackgroundThe aim of the study was to investigate if parameters associated with human epidermal growth factor receptor type 2 (HER2) status (HER2 gene copy number, HER2/CEP17 ratio or polysomy of chromosome 17) are related to various biological features potentially responsible for trastuzumab resistance (PTEN, IGF-1R, MUC4, EGFR, HER3, HER4, and mutation status of PIK3CA) as well as their influence on survival of HER2-positive breast cancer patients treated with adjuvant chemotherapy and trastuzumab.Patients and methodsThe investigated group consisted of 117 patients with invasive ductal breast cancer (T≥1, N≥0, M0) with overexpression of HER2, who underwent radical surgery between 2007 and 2014. Status of ER, PR, and HER2 expression was retrieved from patients’ files. HER2 gene copy number was investigated by fluorescence in situ hybridization using PathVysion HER-2 DNA Probe Kit II. Expression of PTEN, IGF-1R, MUC4, EGFR, HER3, and HER4 was assessed immunohistochemically on formalin-fixed paraffin-embedded tissue sections. PIK3C mutation status was determined by qPCR analysis.ResultsOverexpression of HER2 protein (IHC 3+) and ER negativity corresponded to higher HER22 copy number and HER2/CEP17 ratio (.<0.001). Tumors with polysomy were characterized by higher HER22 gene copy number but lower HER2/CEP17p ratio (p<0.026, p<0.001). Patients with tumors featuring HER3 immunonegativity or low HER2/CEP17 ratio (#4) were characterized by 100% metastasis-free survival (.=0.018, p=0.062).ConclusionPresence of both unfavorable factors, ie, HER3 expression and high HER2/CEP17 ratio, allowed to distinguish a group of patients with worse prognosis (.=0.001).

Killer cell immunoglobulin-like receptor genotypes in the Turkish population

One hundred eighty-seven healthy and unrelated volunteers from various regions of Turkey were selected for the study. Killer-cell immunoglobulin-like receptors (KIR) genotyping was performed by polymerase chain reaction using commercial sequence-specific oligonucleotide probe (SSOP) kits. Gene frequencies of the Turkish population were determined by direct counting of the positive and negative loci. The genotype data is publicly available in the Allele Frequencies Net Database under the population name “Turkey KIR pop 3” number “3399”.

Abstract P5-20-08: HER2 in-Situ hybridization positive breast cancers with HER2/CEP17 ratio ≥2.0 and average HER2 copy number &amp;lt;4.0 are frequently discordant with HER2 immunohistochemistry results: Implications for potential modification of testing algorithm

Abstract Background: The current ASCO/CAP guidelines for the determination of HER2 status indicate that dual-probe in-situ hybridization (ISH) samples demonstrating a HER2/CEP17 ratio ≥ 2.0 and average HER2 copy number &amp;lt;4.0 must be reported as positive. Patients with tumors showing these rare features were included in the first generation adjuvant trastuzumab clinical trials. In the absence of data showing lack of efficacy of HER2-targeted in this small subset of patients, these cases continue to be classified as positive without requiring additional work-up. Given that the HER2/CEP17 ratio is skewed in these cases by complex segmental deletions in chromosome 17, additional work-up may improve the final HER2 classification of these tumors. The objective of this study is to assess the potential impact of integrating HER2 IHC into the testing algorithm for these tumors by correlating the HER2 IHC results with ISH positive breast cancers demonstrating HER2/CEP17 ratio ≥2.0 and average HER2 copy number &amp;lt;4.0. Methods: All invasive mammary carcinomas demonstrating a HER2/CEP17 ratio ≥2.0 and average HER2 copy number &amp;lt;4.0 were identified from our database for years 2009-2017. All ISH testing was performed using the PathVysion HER2 DNA probe kit (Abbott Molecular). During the study interval, HER2 FISH was the institutional primary testing methodology to determine HER2 status. HER2 IHC (Ventana 4B5) was performed on the same sample for all identified cases and scored using 2013 ASCO/CAP scoring guidelines. Results: A total of 96 (1.4%) of 6,976 consecutive FISH cases met criteria for inclusion into the study. The majority of cases tested negative (67%) or equivocal (29%) by IHC. HER2 protein overexpression was observed in 4% of cases. The results of HER2 IHC testing are summarized below. HER2 IHC results for HER2 ISH positive tumors showing ratio≥2.0 and average HER2 copy number &amp;lt;4.0Cases (n)IHC Score 0IHC score 1+IHC score 2+IHC score 3+9612 (13%)52 (54%)28 (29%)4 (4%) Conclusions: HER2 ISH positive breast cancers demonstrating a HER2/CEP17 ratio ≥2.0 and average HER2 copy number &amp;lt;4.0 are rare (1.4% of cases in this study). HER2 IHC results for these tumors are negative in majority of cases. The observed HER2 protein overexpression rate was only 4% in this study. Integration of HER2 IHC into the testing algorithm for tumors demonstrating these uncommon ISH findings would result in reclassification of HER2 status to negative for most tumors. Citation Format: Livasy C, Johnson N, Domfeh A. HER2 in-Situ hybridization positive breast cancers with HER2/CEP17 ratio ≥2.0 and average HER2 copy number &amp;lt;4.0 are frequently discordant with HER2 immunohistochemistry results: Implications for potential modification of testing algorithm [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-20-08.

Utility of Alternate, Noncentromeric Chromosome 17 Reference Probe for Human Epidermal Growth Factor Receptor Fluorescence In Situ Hybridization Testing in Breast Cancer Cases.

Context PathVysion-a US Food and Drug Administration-approved dual-probe human epidermal growth factor receptor ( HER2) fluorescence in situ hybridization (FISH) assay-provides the HER2: CEP17 ratio, a centromeric enumeration probe ratio for determining HER2 status in breast cancers. However, pericentromeric amplifications might then skew the HER2: CEP17 ratio, underestimating the HER2 status, which calls into question the use of CEP17 as the reference probe. Objective To analyze the utility of a noncentromeric chromosome 17 reference locus ( D17S122) to assess HER2 gene status in cases showing "nonclassical" FISH patterns with the CEP17 probe. Design The HER2 status of breast cancers accessioned in the years 2015-2017, displaying "nonclassical" or "equivocal" results by the PathVysion (Abbott Molecular Inc, Des Plaines, Illinois) HER2 DNA Probe Kit were reflex tested using an alternate FISH probe (ZytoLight SPEC/D17S122, ZytoVision, Bremerhaven, Germany) and interpreted with American Society of Clinical Oncology/College of American Pathologists 2013 guidelines. Results Of 37 cases, 17 were FISH equivocal. With the alternate D17S122 probe, 13 (76.4%) were reclassified as amplified, 3 (17.6%) as nonamplified, and a single case retained an equivocal result. Of the 17 cases with a chromosome 17 polysomy pattern, disomy, polysomy, and monosomy patterns were seen with 14 cases, 2 cases, and 1 case, respectively. Within the 17 cases with polysomy pattern, 3 (17.6%) demonstrated an unusual colocalization pattern of HER2 and CEP17, which was not observed with the alternate probe. Conclusions The denominator-stable alternate probe is a useful adjunct in the diagnostic armamentarium to analyze HER2 status in cases with FISH equivocal and complex patterns.

Clinical outcome study of crizotinib in immunohistochemistry-proven echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion gene among Indian patients with adenocarcinoma lung.

Aims:The anaplastic lymphoma kinase (ALK) Break Apart FISH Probe Kit and Ventana anti-ALK (D5F3) CDx immunohistochemistry (IHC) assay are the Food and Drug Administration-approved companion diagnostic for targeted therapy with the ALK inhibitor crizotinib in lung cancers. The aim of this study was to assess the efficacy and safety of twice daily crizotinib tablet (250 mg) in IHC-proven echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene among Indian patients with adenocarcinoma lung in the routine clinical practice.Subjects and Methods:Patients with nonsmall cell lung cancer (NSCLC), adenocarcinoma histology, whose tumors were found to be positive for EML4-ALK fusion gene using IHC, were considered for this study. 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 25 NSCLC adenocarcinoma patients were included in the study. There were 14 (56%) women and 10 (44%) men with a median age of 53 years. All patients had Stage IV disease at the time of initiation of crizotinib therapy. One patient achieved complete response and 20 achieved response rate (PR) for an overall PR of 84%. The median progression-free survival (PFS) was 11.8 months and median overall survival (OS) was 20.6 months. Two (8%) patients experienced severe hepatotoxicity requiring permanent discontinuation of crizotinib therapy.Conclusions:A very high PR, PFS, and OS achieved in our study population indicates that IHC can accurately identify EML4 ALK fusion gene mutations in lung adenocarcinoma patients who are responsive to ALK inhibitors such as crizotinib. IHC should be considered as a cost-effective alternative to FISH, especially in low-resource countries.

Resveratrol attenuates high glucose-induced endothelial cell apoptosis via mediation of store-operated calcium entry.

The aim of this study was to evaluate the influence of resveratrol on HG-induced calcium entry in islet microvascular (MS-1) endothelial cells. MS-1 cells were pretreated with resveratrol or 2-APB (an inhibitor of store-operated calcium entry) and then incubated with high glucose. Cell viability was determined using the cell counting kit-8 method. Reactive oxygen species, endothelial apoptosis, and NO production were detected by DHE probe, TUNEL detection, and nitrate reductase assay kit. Protein levels of SOCE were detected by western blotting. Pretreatment with resveratrol significantly attenuated HG-induced endothelial apoptosis and improved cell viability. However, pretreatment with resveratrol and 2-APB abolished this effect, suggesting that the attenuation of HG-induced apoptosis by resveratrol may be associated with SOCE. Subsequent analyses indicated that HG induced the SOCE-related proteins, including TRPC1, Orai1, and Stim1. These results suggest that resveratrol pretreatment is associated with relieved HG-induced endothelial apoptosis at least partly via inhibition of SOCE-related proteins.

Validation of ALK/ROS1 Dual Break Apart FISH Probe probe in non-small-cell lung cancer

BackgroundALK 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 develop an effective screening strategy to detect patients who will benefit from such treatment. In this study, we aimed to validate analytical performance of Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) in NSCLC. MethodsStudy population composed of three patient cohorts with histologically confirmed lung adenocarcinoma (patients with ALK rearrangement, patients with ROS1 rearrangement and patients with wild-type ALK and ROS1). Specimens consisted of 12 ALK-positive, 8 ROS1-positive and 21 ALK/ROS1-wild type formalin-fixed paraffin-embedded samples obtained from surgical resection or excisional biopsy. ALK rearrangement was previously assessed by Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Abbot Park, IL, USA) and ROS1 rearrangement was previously assessed by ZytoLight® SPEC ROS1 Break Apart Probe (ZytoVision, GmbH). All specimens were re-evaluated by Vysis ALK/ROS1 Dual Break Apart Probe Kit. FISH images were scanned on BioView AllegroPlus system and interpreted via BioView SoloWeb remotely. ResultsFor a total of 41 patient samples, the concordance of the results by Vysis ALK/ROS1 Dual Break Apart Probe Kit was evaluated and compared to the known ALK and ROS1 rearrangement status of the specimen. Of the 12 ALK-positive cases, hybridization with Vysis ALK/ROS1 Dual Break Apart Probe Kit was successful in 10 cases (success rate 10/12, 83%) and of these 10 cases, all showed ALK rearrangement (100% concordance with the results of Vysis ALK Break Apart FISH Probe Kit). Two of the ALK+ cases were excluded due to weak ROS1 signals that could not be enumerated. Of the 8 ROS1-positive cases, 6 cases were successfully evaluated using Vysis ALK/ROS1 Dual Break Apart Probe Kit. The success rate was 75% (6/8), and of these 6 cases, all showed ROS1 rearrangement, giving a 100% concordance with ZytoLight® SPEC ROS1 Break Apart Probe. Two of the cases were excluded due to weak ROS1 gold signal or high background. In the cohort of 21 wild-type cases, the success rate using Vysis ALK/ROS1 Dual Break Apart FISH Probe Kit was 85% (18/21) and the concordance with ALK and ROS1 probe kit was 100% (18/18). ConclusionVysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) can detect ALK and ROS1 rearrangement simultaneously in NSCLC.