Probe Kit Research Articles

Anchored enrichment dataset for true flies (order Diptera) reveals insights into the phylogeny of flower flies (family Syrphidae).

BackgroundAnchored hybrid enrichment is a form of next-generation sequencing that uses oligonucleotide probes to target conserved regions of the genome flanked by less conserved regions in order to acquire data useful for phylogenetic inference from a broad range of taxa. Once a probe kit is developed, anchored hybrid enrichment is superior to traditional PCR-based Sanger sequencing in terms of both the amount of genomic data that can be recovered and effective cost. Due to their incredibly diverse nature, importance as pollinators, and historical instability with regard to subfamilial and tribal classification, Syrphidae (flower flies or hoverflies) are an ideal candidate for anchored hybrid enrichment-based phylogenetics, especially since recent molecular phylogenies of the syrphids using only a few markers have resulted in highly unresolved topologies. Over 6200 syrphids are currently known and uncovering their phylogeny will help us to understand how these species have diversified, providing insight into an array of ecological processes, from the development of adult mimicry, the origin of adult migration, to pollination patterns and the evolution of larval resource utilization.ResultsWe present the first use of anchored hybrid enrichment in insect phylogenetics on a dataset containing 30 flower fly species from across all four subfamilies and 11 tribes out of 15. To produce a phylogenetic hypothesis, 559 loci were sampled to produce a final dataset containing 217,702 sites. We recovered a well resolved topology with bootstrap support values that were almost universally >95 %. The subfamily Eristalinae is recovered as paraphyletic, with the strongest support for this hypothesis to date. The ant predators in the Microdontinae are sister to all other syrphids. Syrphinae and Pipizinae are monophyletic and sister to each other. Larval predation on soft-bodied hemipterans evolved only once in this family.ConclusionsAnchored hybrid enrichment was successful in producing a robustly supported phylogenetic hypothesis for the syrphids. Subfamilial reconstruction is concordant with recent phylogenetic hypotheses, but with much higher support values. With the newly designed probe kit this analysis could be rapidly expanded with further sampling, opening the door to more comprehensive analyses targeting problem areas in syrphid phylogenetics and ecology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0714-0) contains supplementary material, which is available to authorized users.

Assessment of HER2 amplification status in breast cancer using a new automated HER2 IQFISH pharmDx™ (Dako Omnis) assay

In breast cancer the human epidermal growth factor receptor 2 (HER2) is an important target for a number of different HER2 inhibitors. Different slide-based assays are available for assessment of treatment eligibility, which include fluorescence in situ hybridization (FISH) or other in situ hybridization (ISH) methods for assessment of the HER2 gene status. Here we report a summary of the validation data on HER2 IQFISH pharmDx™ (Dako Omnis), a newly developed assay for the automated staining platform Dako Omnis. The assay uses a non-toxic buffer that significantly reduces the hybridization time, which results in a total turnaround time of 3½ to 4h from deparaffinization to counting of the gene and centromere signals. The data reported in the current summary covers method comparison, assessment of staining quality, observer-to-observer reproducibility as well as reproducibility within and between laboratories. Based on data from the different studies it was concluded that HER2 IQFISH pharmDx (Dako Omnis) is a reliable and robust assay with a high precision that is at least comparable to the manual HER2 IQFISH pharmDx™ assay and the PathVysion®HER-2 DNA Probe Kit.

IRate: Initial Video Bitrate Selection System for HTTP Streaming

Many HTTP streaming video systems have been developed and widely deployed in recent years. Previous efforts were mainly spent on improving the caching of videos or proposing mid-stream measurement methods to update the best bitrate. However, since the video length is often short, the mid-stream measurement may not even converge to the best bitrate due to insufficient bandwidth estimates. On the other hand, because of diversified Web infrastructure, estimating the actual network quality at the pre-stream stage is increasingly challenging for video service providers. In this paper, we propose IRate, which enables video service providers to proactively profile clients’ streaming performance by carrying out pre-stream measurement in the Content Delivery Network (CDN). With the measurement results, the video stream can start at the best video quality at the onset of streaming. This is especially beneficial to short video clips, which are very popular in the Internet today. IRate is composed of a probe kit and a quality oracle. The probe kit utilizes the pre-stream time window (e.g., user’s think time and pre-roll advertisement) for measuring network quality by running a lightweight measurement script on the Web page to induce probe packets from the IRate middlebox on the server side. With the measurement results, the quality oracle estimates the clients’ streaming performance by determining the highest initial bitrate with a pre-trained decision tree. Our testbed results show that IRate is able to achieve 80% accuracy in determining the bitrate within 10s. By having a better estimate of the best initial bitrate, the buffering time and rebuffering events are significantly reduced in HTTP streaming. Furthermore, the stability and the efficiency in dynamic adaptive streaming over HTTP streaming are also improved by about 40% and 36%, respectively. Our user quality of experience (QoE) experiment further validates that IRate can improve the QoE by more than 6% and the perceived quality of initial quality by 24% in the actual Internet environment.

Venetoclax Approved for CLL

Abstract Venetoclax, a BCL2 inhibitor, has been approved for patients with chronic lymphocytic leukemia (CLL) who lack part of chromosome 17. The FDA also approved a companion diagnostic, the Vysis CLL FISH probe kit, to detect the 17p deletion in patients' peripheral blood.

Differential responses of HER2-positive breast cancer cells to anti-HER2 treatment.

e12095 Background: HER2 is a membrane tyrosine kinase receptor which promotes cells growth by activating downstream kinase pathways: RAS/MEK/ERK and PI3K/AKT. Given the fact that HER2 gene is amplified in certain population of breast cancer patients, therapeutic strategy has evolved to target HER2 function. However, it has been long controversial as to which patients are truly benefited from such treatment. In this study, we evaluate impact of HER2 overexpression on response to anti-HER2 treatment using a panel of breast cancer cell lines, and compared FISH vs. RT-PCR for their accuracy in predicting drug responses. Methods: Human breast cancer lines were obtained from ATCC or from cell repository bank at MD Anderson Cancer Center. RhuMAB HER2 was provided by Genentech. Her2 gene amplification was evaluated by FISH method using the PathVysion HER2/NeuDNA probe kit form Abbott. HER2 mRNA expression was analyzed by RT-PCR method. Growth inhibitory and cytotoxic effects of anti-HER2 antibody were determined by MTS assay. Apoptotic cell death was visualized with DAPI staining and quantified by Annexin V-based flow cytometric detection. Results: A total of 12 cell lines are chosen for this study, 4 of them are HER2-negative and 8 are HER2-positive as determined by FISH testing. Anti-HER2 antibody treatment inhibits growth of all cell lines tested, among them, 3 HER2-positive cell lines are identified to undergo apoptotic cell death. Re-culture in fresh medium restores cell growth potential in those non-apoptotic cells, but not in the apoptotic cell lines. FISH score has poor correlation with apoptotic cell death; in contrast, cell lines those expressing more than 20,000 copies of HER2 mRNA, as measured in 1 ng of total RNA, all show apoptosis. More interestingly, apoptotic death appears more severe as HER2 mRNA number increases. Conclusions: Accurate measurement of HER2 expression is essential for improving the outcome of anti-HER2 treatment. Our study suggests that RT-PCR technique seems to better fulfill the aim, and warrants future clinical trials of using RT-PCT to predict a positive response to anti-HER2 therapies.

The FlexISH assay brings flexibility to cytogenetic HER2 testing.

Fluorescence in-situ hybridization (FISH) is the method of choice for quantitative human epidermal growth factor receptor 2 (HER2) (also known as ERBB2) gene testing in invasive breast cancer. HER2 testing has great clinical impact, and is often claimed to expeditiously complete the entire diagnostic procedure for an individual patient. Against this background, the aim of this study was to evaluate the usefulness and performance of a novel dual-colour HER2/cen17 FISH assay designed to facilitate flexible (overnight) and rapid (<2 h of hybridization) FISH. We quantitatively and qualitatively compared counting results and the performance of the FlexISH SPEC ERBB2/CEN 17 dual-colour hybridization kit with well-established HER2FISH assays, by using 90 malignant (polysomic and non-polysomic) and 19 benign paraffin-embedded breast tissue specimens. We used long (overnight) and short (2 h) hybridization periods, and found an excellent correlation between the FISH results obtained with FlexISH, ZytoLight and PathVysion hybridization probes. The results obtained with both the short-run and long-run application of FlexISH are in excellent accordance with the results obtained with other commercially available FISH kits. This appears to be true in all relevant respects: signal counts, signal-to-noise ratio, brightness, and distinctness of HER2 and cen17 signals. As FlexISH probes can be equivalently used as a short-run or long-run application, the FlexISH probe kit provides the highest flexibility in terms of time and laboratory management. If required, a reliable HER2 finding can be delivered within 4.5 h, but the standard workflow (including overnight hybridization) does not negatively affect the performance, specimen quality or diagnostic result.

Nanosized hybrid electromagnetic wave absorbing coatings

Abstract In this study, electromagnetic wave absorbing coating materials were prepared by using nano ferrite and nanosized polyaniline as filler and 2K bisphenol-A epoxy resin as binder. Polyaniline powder (PANI) was synthesized by chemical polymerization method. Coating material was prepared by mixing several ratios of ferrite/polyaniline as nanofiller with an epoxy binder matrix and their microwave absorption capability were compared. In addition, conductivity of the coating was measured by using a dielectric probe kit system with the Cole–Cole method. The microwave absorption measurements were performed by using Transmission/Reflection principle via the waveguide method (S 11 ) in the frequency range of 8–12 GHz. Dielectric permittivity and magnetic permeability measurements were also executed in the same frequency range. The results were compared in terms of electromagnetic wave absorption performance and characteristics. Finally, structural and morphological properties were also investigated. It was found that nanosized ferrite/PANI filled coatings have good electromagnetic absorbing properties. However, the nanosized PANI-filled sample with good conductivity showed superior electromagnetic absorbing performance.

Association of HLA-DP variants with the responsiveness to Hepatitis B virus vaccination in Korean Infants

Recently, HLA-DP single nucleotide polymorphisms (SNPs) have been reported to be related to responsiveness to hepatitis B virus (HBV) vaccination. The aim of this study was to investigate associations between HLA-DP SNPs and responsiveness to HBV vaccine in Korean infants. A total of 290 healthy Korean infants who were registered to Seoul Metropolitan Public Cord Blood Bank during the period of February 2007 to December 2011 were enrolled. Anti-HBs antibody level was analyzed after three doses of HBV vaccination. Genotyping of HLA-DPA1 SNPs (rs3077 and rs3830066) and HLA-DPB1 SNPs (rs7770370, rs7770501, rs3128961, and rs9277535) were performed by PCR-sequencing. HLA-A, -B, and –DRB1 genotyping was also performed by PCR-sequence-specific oligonucleotide probe kits. HLA-DPB1 SNPs (rs7770370, rs7770501, rs3128961, and rs9277535) were associated with HBV vaccine response. Allele frequencies of rs7770370 A, rs7770501 C, rs3128961 G, and rs9277535 A were significantly higher in responders than in non-responders (all p<0.01). Anti-HBs antibody levels were different according to genotypes of DPB1 rs7770370, rs7770501, rs3128961, and rs9277535 (all p<0.01). In multivariate analysis, HLA-DPB1 rs7770370 AA genotype was significantly associated with HBV vaccine response (relative risk, RR=2.5, p=0.033) and high-titer vaccine response (RR=2.7, p<0.001). In conclusion, HLA-DPB1 SNPs were significantly associated with responses to HBV vaccination in Korean infants.

Calibrated complex impedance of CHO cells and E. coli bacteria at GHz frequencies using scanning microwave microscopy

The application of scanning microwave microscopy (SMM) to extract calibrated electrical properties of cells and bacteria in air is presented. From the S11 images, after calibration, complex impedance and admittance images of Chinese hamster ovary cells and E. coli bacteria deposited on a silicon substrate have been obtained. The broadband capabilities of SMM have been used to characterize the bio-samples between 2 GHz and 20 GHz. The resulting calibrated cell and bacteria admittance at 19 GHz were Ycell = 185 μS + j285 μS and Ybacteria = 3 μS + j20 μS, respectively. A combined circuitry-3D finite element method EMPro model has been developed and used to investigate the frequency response of the complex impedance and admittance of the SMM setup. Based on a proposed parallel resistance–capacitance model, the equivalent conductance and parallel capacitance of the cells and bacteria were obtained from the SMM images. The influence of humidity and frequency on the cell conductance was experimentally studied. To compare the cell conductance with bulk water properties, we measured the imaginary part of the bulk water loss with a dielectric probe kit in the same frequency range resulting in a high level of agreement.

First-trimester spontaneous pregnancy loss - molecular analysis using multiplex ligation-dependent probe amplification.

Spontaneous miscarriages are the most frequent complications of pregnancy and, in at least half of cases, are caused by chromosomal abnormalities, mainly aneuploidies. We present the preliminary results of the implementation of multiplex ligation-dependent probe amplification (MLPA) in the detection of chromosomal aberrations in the tissue derived from first-trimester miscarriage and evaluate the limitations and requirements of the method. We studied 181 MLPA analyses with subtelomeric and subcentromeric probe kits for all chromosomes (SALSA P070 and SALSA P181) performed on the first-trimester spontaneous miscarriage products in our Department of Genetics between September 2012 and December 2014. Conclusive MLPA results were obtained in 97.2% of samples. Chromosomal aberrations were detected in 40.3% of samples: 61.8% samples of good quality and 12.6% samples of poor quality (p < 0.001). The normal female karyotype was detected in 14.7% of good quality samples and 84.8% of poor quality samples (p < 0.001). MLPA is a useful tool for the detection of chromosomal aberrations in first-trimester miscarriage products. However, the tissue has to be well prepared before testing and the results 46,XX should be interpreted with caution.

Interobserver reproducibility for HER2/neu immunohistochemistry: A comparison of reproducibility for the HercepTest™ and the 4B5 antibody clone

BackgroundIHC results for HER2/neu vary with replicate testing using the same antibody clone and when alternate clones are utilized. A number of factors appear to be responsible for this variability, including fixation times, equipment utilized and training and experience of staff. A number of studies have documented interobserver variability for a single antibody clone but few have evaluated reproducibility between antibody clones and which clones demonstrate the highest degree of interobserver reproducibility. DesignWe studied a series of 93 cases stained by both the HercepTest™ and the 4B5 clone for interobserver reproducibility. Formalin-fixed, paraffin-embedded sections were stained by the immunohistochemical technique using the manufactures directions for both the HercepTest™ and the 4B5 clone. FISH testing was performed on formalin-fixed paraffin embedded sections according to the PathVysion HER-2 DNA probe kit instructions. ResultsAbsolute agreement rate for Hercep was 85%. Absolute agreement for 4B5 was 69%. This difference was statistically significant (p<0.0001). The chance-corrected agreement (weighted kappa) for the HercepTest™ was 79% and 71% for 4B5 (p<0.0001). Absolute agreement between antibody clones was 58% with the chance corrected agreement being 51%. Absolute agreement of 4B5 with FISH was significantly greater than that of the HercepTest™ (54% vs 35%). ConclusionAgreement between evaluators was greater with the HercepTest. However, agreement with FISH results was superior for the 4B5 clone. Interobserver agreement was less than the 95% agreement threshold recommended by the ASCO/CAP guidelines for development of a new testing method for HER2 evaluation.

Low Representation of ETV6-RUNX1 Fusion Oncogene in Pediatric Acute Lymphoblastic Leukemia Patients from Saudi Arabia and Strong Association with Clinical Response to Day-14 of Remission Induction Therapy

Abstract:Introduction: t(12; 21) leading to ETV6-RUNX1 fusion oncogene is associated with better prognosis and excellent treatment outcome in pediatric ALL (pALL) patients have good prognosis as compared to other genetic abnormalities1. Accordingly, frequency of ETV6-RUNX1 has a huge implication in treatment strategies of pALL in a given population. Different ethnic groups from various geographical regions have different frequencies of ETV6-RUNX1 ranging from 10% (Southeast Asia) to 33% (Australia) 2,3. Therefore, aim of this study was to determine ETV6-RUNX1 status of Saudi Arabian pALL patients and its association with clinical parameters and early remission.Patients & Methods: Pediatric Acute Lymphoblastic Leukemia (pALL) patients at King Abdulaziz Medical City represent pure Saudi Arabian population. Clinical parameters and ETV6-RUNX1 status (using FISH technique) of pALL patients attending Pediatric Oncology Clinic, King Abdulaziz Medical City Riyadh from 2006 to 2011 were studied using Vysis ETV6/RUNX1 DF FISH Probe Kit (Abbot Laboratories, Illinois, USA)4. CCG1991 protocol was used for standard risk patients while CCG1961 protocol was used for high risk patients. Number of blasts at day 14 and day 29 of the treatment were also calculated as a part of routine clinical follow-up.Comparison between ETV6-RUNX1 positive and negative groups done using chi-square test or Fisher's exact test. All Statistical analysis was performed using SAS version 9.2 (SAS Institute, Inc., Cary, NC).Results: Out of 54 patients, 33 were male and 21 were females (ration1.57:1). B- and T-cell lineage was found in 47 (87%) and 7 (13%) patients respectively. Only 5 (9.3%) patients with ETV6-RUNX1 positive while 49(80.7%) were ETV6-RUNX1 negative. All ETV6-RUNX1 patients (100%) were of B-cell lineage and 80% (4/5) were in 3-7 year age group. None of ETV6-RUNX11 patients had ≥5% blasts (no remission) at Day 14 as compared to 9% patients from ETV6-RUNX1 negative group (Table 1).Discussion: Frequency of ETV6-RUNX1 positive patients (less than 10%) in our pALL patients is much lower than reported in most of the European countries, North America, Australia and Japan while it is in accordance with ETV6-RUNX1 frequencies from Egypt (11.6%), Pakistan (10%), and India (5-7%) 2,3,5,6. This diversity in frequencies of ETV6-RUNX1 among pALL can be attributed to level of industrialization and/or westernized lifestyle. Moreover, ethnic differences in frequencies of this and other prognostically important genetic abnormalities can have a significant bearing on global pediatric ALL management strategies7 which necessitates further large scale studies in this regard.

Characterization of NixZn1−xFe2O4 and Permittivity of Solid Material of NiO, ZnO, Fe2O3, and NixZn1−xFe2O4 at Microwave Frequency Using Open Ended Coaxial Probe

This paper describes a detailed study on the application of an open ended coaxial probe technique to determine the permittivity of NixZn1-xFe2O4 in the frequency range between 1 GHz and 10 GHz. The x compositions of the spinel ferrite were 0.1, 0.3, 0.5, 0.7, and 0.9. The NixZn1-xFe2O4 samples were prepared by 10-hour sintering at 900°C with 4°C/min increment from room temperature. Particles showed phase purity and crystallinity in powder X-ray diffraction (XRD) analysis. Surface morphology measurement of scanning electron microscopy (SEM) was conducted on the plane surfaces of the molded samples which gave information about grain morphology, boundaries, and porosity. The tabulated grain size for all samples was in the range of 62 nm–175 nm. The complex permittivity of Ni-Zn ferrite samples was determined using the Agilent Dielectric Probe Kit 85070B. The probe assumed the samples were nonmagnetic homogeneous materials. The permittivity values also provide insights into the effect of the fractional composition of x on the bulk permittivity values NixZn1-xFe2O4. Vector Network Analyzer 8720B (VNA) was connected via coaxial cable to the Agilent Dielectric Probe Kit 85070B.

Abstract 4244: Comparison of different ALK tests in non-small cell lung cancer (NSCLC) patients treated with crizotinib and their clinical outcome

Abstract Background: The screening algorithm for detection of ALK-rearranged NSCLC is still under investigation. The Break-Apart ALK FISH is the standard diagnostic test for the treatment with crizotinib. However, immunohistochemistry (IHC) with different antibodies shows excellent results when compared to ALK FISH. The efficacy of crizotinib is studied in relation to ALK FISH. We compared IHC outcome and the number of ALK breaks estimated with FISH with clinical outcome. Methods: Patients treated with crizotinib who had biopsies with sufficient tumor tissue were selected. Tumour response was assessed by CT using RECIST. Fluorescence in situ hybridization (FISH) was performed with Vysis LSI ALK Break Apart FISH Probe KIT (Abbott Molecular) and immunohistochemistry with Ventana ALK IHC Kit using D5F3 antibody. Results: Twenty one patients with NSCLC were selected, median (range) age 54 yrs (21-75); 19 had adenocarcinoma, 2 had large-cell carcinoma. We confirmed the presence of EML4-ALK fusions in all samples either by FISH or IHC. Median percentage of ALK breaks was 47% (6-76). Tumour response occurred in 12 pts having a median of 51 (6-76) breaks and 9 non-responders having a median of 27 (15-64) breaks. Of the 12 responders, 8 pts had positive IHC (67%), 3 were negative and 1 was not done; the FISH negative, IHC positive patient was one of the responders. Of the 9 non-responders 2 had positive IHC (22%), 3 negative (33%), and 1 was equivocal and 3 not done. Median PFS for positive IHC is 7.9 mo (95% CI., 5.5 - 10.3) and for negative IHC 1.6 mo (1.3 - 1.8), n = 17, p&amp;lt;0.0001. OS survival data are 6.5 mo (95% CI., 0 - 17.8) and 18.3 mo (95% CI., 11.1 - 25.6) respectively, n = 17 p = 0.052. PFS above 30% number of breaks was 7.0 mo (95% CI., 1.8 - 12.2) versus PFS below is 1.8 mo (95% CI., 0.7 - 2.9), n = 21, p = 0.05. OS survival data are 4.1 mo (95% CI., 0 - 9.3) and 16.1 mo (95% CI., 9.0 - 23.2) respectively, n = 21 p = 0.003. Therefore PFS and OS is more prolonged in those tumors who have ALK breaks above 30%. IHC difference are only significant for PFS. Conclusions: Both ALK FISH and IHC identify NSCLC patients who benefit from crizotinib treatment. Patients with ALK breaks above 30% have a better PFS and OS. IHC only differentiates for PFS. Citation Format: Anthonie J. van der Wekken, Thijo JN Hiltermann, Wim Timens, Ed Schuuring, Harry JM Groen. Comparison of different ALK tests in non-small cell lung cancer (NSCLC) patients treated with crizotinib and their clinical outcome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2015-4244

AMPLIFICATION AND OVEREXPRESSION OF HER2/NEU IN GASTRIC CANCER ASSESSED BY FLUORESCENCE IN SITU HYBRIDIZATION AND IMMUNOHISTOCHEMISTRY

Background: HER2/neu is a predictive biomarker for treatment of gastric cancer using trastuzumab in combination with chemotherapy. This study aimed to: (1) assess the amplification and the overexpression of HER2/neu using fluorescence in situ hybridization (FISH) and immunohistochemistry in gastric cancer; (2) survey the association between HER2/neu and clinicopathologic characteristics of gastric cancer. Patients and methods: one hundred and sixty gastric cancer patients were assessed HER2/neu overexpression by IHC using Ventana anti-HER-2/neu (4B5) kit and were assessed HER2/neu gene amplification by FISH using PathVysionTM HER-2 DNA Probe kit with biopsy specimens. Results: HER2/neu protein expression rates of IHC 0, 1+, 2+ and 3+ were 70%, 10.6%, 10.6% and 8.8%, respectively. HER2/neu gene amplification was identified in gastric cancer from 21 out of 160 (13.1%) patients. The concordance between IHC and FISH was 90.0%. The HER2/neu-positive rate assessed by both techniques was 13.1%. There was a significant difference in HER2/neu-positivity between cardia gastric cancer and non-cardia gastric cancer (36.4% vs 11.4%, p = 0.040); between intestinal type and diffuse type (20.7% vs 5.9%, p = 0.010); between well, moderately differentiated and poorly differentiated gastric cancer (18.6% and 23.8% vs 5.8%, p = 0.047). Conclusion: We applied successfully FISH and IHC technique with biopsy samples in gastric cancer detecting HER2/neu positivity in order to select patients that benefit from trastuzumab in combination with chemotherapy. HER2/neu status associated with tumor location, Lauren classification and differentiated grading. Keywords: gastric cancer, fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), HER-2/neu

Clear cell myoepithelial carcinoma of salivary glands showing EWSR1 rearrangement: molecular analysis of 94 salivary gland carcinomas with prominent clear cell component.

This study examines the presence of the EWSR1 rearrangement in a variety of clear cell salivary gland carcinomas with myoepithelial differentiation. A total of 94 salivary gland carcinomas with a prominent clear cell component included 51 cases of clear cell myoepithelial carcinomas de novo (CCMC), 21 cases of CCMCs ex pleomorphic adenoma (CCMCexPA), 11 cases of epithelial-myoepithelial carcinoma (EMC), 6 cases of EMC with solid clear cell overgrowth, and 5 cases of hyalinizing clear cell carcinoma of minor salivary glands. In addition, 10 cases of myoepithelial carcinomas devoid of clear cell change and 12 cases of benign myoepithelioma were included as well. All the tumors in this spectrum were reviewed, reclassified, and tested by fluorescence in situ hybridization (FISH) for the EWSR1 rearrangement using the Probe Vysis EWSR1 Break Apart FISH Probe Kit. The EWSR1 rearrangement was detected in 20 of 51 (39%) cases of CCMC, in 5 of 21 (24%) cases of CCMCexPA, in 1 of 11 (9%) cases of EMC, and in 4 of 5 (80%) cases of hyalinizing clear cell carcinoma. The 25 EWSR1-rearranged CCMCs and CCMCexPAs shared similar histomorphology. They were arranged in nodules composed of compact nests of large polyhedral cells with abundant clear cytoplasm. Necrosis, areas of squamous metaplasia, and hyalinization were frequent features. Immunohistochemically, the tumors expressed p63 (96%), cytokeratin CK14 (96%), and S100 protein (88%). MIB1 index varied from 10% to 100%, with most cases in the 20% to 40% range. Clinical follow-up information was available in 21 cases (84%) and ranged from 3 months to 15 years (mean 5.2 y); 4 patients were lost to follow-up. Ten patients are alive with no evidence of recurrent or metastatic disease in the follow-up period from 3 months to 15 years (mean 5 y), 3 patients are alive with recurrent and metastatic disease, and 8 died of disseminated cancer 9 months to 16 years after diagnosis (mean 6 y). Lymph node metastasis appeared in 5 patients within 5 months to 4 years after diagnosis (mean 22 mo), distant metastases were noted in 7 patients with invasion of orbit (2 cases), and in 1 case each metastasis to the neck soft tissues, liver, lungs, mediastinum, and thoracic vertebra was noted. We describe for the first time EWSR1 gene rearrangement in a subset of myoepithelial carcinomas arising in minor and major salivary glands. The EWSR1-rearranged CCMC represents a distinctive aggressive variant composed predominantly of clear cells with frequent necrosis. Most EWSR1-rearranged CCMCs of salivary glands are characterized by poor clinical outcomes.

Evaluation of HER2 gene amplification status in invasive breast cancer patients by Fluorescence in Situ Hybridization analysis and its correlation with clinical features

Precise assessment of HER2 gene status as an important biomarker plays a significant role in identifying the eligible patients for Trastuzumab therapy and determining their clinical outcomes. In this study, the researchers assigned HER2 amplification status in invasive breast cancer specimens by Fluorescence in Situ Hybridization (FISH) and determined its association with other clinical features. Formalin-fixed paraffin embedded tumor tissue specimens of 46 patients with invasive breast cancer were collected from November 2011 till May 2012. HER2status was evaluated by FISH. The Zytolight SPEC HER2/CEN17 dual color probe kit was applied for assessment of HER2status. HER2 gene amplification was defined as HER2/CEP17 ratio>2.2.The association between HER2status and clinical features like tumor grade, tumor type, tumor size, axillary lymph node involvement and age of patient was done using Chi squared test at the 0.05 level of significance (p value). Amplification of HER2 gene was detected in twelve cases (26%). On statistical analysis HER2status showed correlation with tumor grade (p=0.02).There was no correlation between HER2status and tumor type, tumor size, lymph node status and age of patients. The results of this study are consonant with the findings of other studies about the presence of HER2 gene amplification in invasive breast cancer. Statistical analysis showed patients with HER2 amplified gene have tumors with higher grade. In these patients the probability of increased proliferation and metastasis is high therefore evaluation of HER2 gene amplification status in breast cancer patients specially in high grade tumor with an accurate method such as FISH is essential

Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): A feasibility study

In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet.

Molecular cytogenetic analyses of hTERC (3q26) and MYC (8q24) genes amplifications in correlation with oncogenic human papillomavirus infection in Czech patients with cervical intraepithelial neoplasia and cervical carcinomas.

It is known that cervical cancer develop from precancerous intraepithelial neoplasia (CIN) which is characterized by series of genetic abnormalities. The progression of CIN to cervical carcinoma has been associated especially with the genomic integration of oncogenic human papilloma virus (HPV) and gain of the human telomerase RNA gene hTERC (3q26) and MYC (8q24). In this study, cytology specimens of cervical intraepithelial neoplasia and cervical carcinoma from 74 Czech women were analyzed using the triple-color Cervical FISH Probe Kit designed for identification of HPV infected cells and copy number aberration of the hTERC and MYC genes. HPV-positivity exhibited 70% of patients with premalignant lesions (CIN I- CIN III, carcinoma in situ), chromosomal changes were found in 53.3% of cases - MYC amplification had 33.3% of women with CINI- CIN III and 50% with carcinoma in situ. Amplification of hTERC was detected in 16.7% of patient with CIN I, in 50% with CIN II, in 58.3% with CIN III and in 66.7% with carcinoma in situ. Based on HPV-positivity and the occurrence of chromosomal aberrations, patients were divided into high-, intermediate- and low-risk group. Among women with cervical carcinomas, HPV infection was detected in 90.1% of specimens and chromosomal aberrations were found in 87.5% of samples. Amplification of MYC gene was detected in 25% and hTERC gene in 62.5% of patients. According to the histopathological grade of tumors, MYC gene amplification occurred more frequently in specimens of spinocellular carcinoma than adenocarcinoma (p=0.029). We found no association between the frequency of cytogenetic lesions and the incidence of lymphangiogenesis or lymph node metastases in cervical carcinoma patients. Simultaneous hTERC and MYC genes amplification was significantly more frequent in samples of cervical carcinomas than in premalignant lesions (p=0.008).In acohort of 26 patients with cervical carcinoma we used oligo-based GGH+SNP microarray technique for the high resolution mapping of copy number changes of hTERC and MYC genes. We found that recurrent gain of genetic material in chromosome 3q26 area carrying hTERC gene of size 43.6 Mb between 3q25.1-3qter and duplication of 3q were the most common genomic identifications of amplified gene. In MYC locus array-CGH profiling identified duplication of 8q and trisomy 8 as frequent genomic changes.Our work confirmed that in cervical carcinoma gains of hTERC and MYC genes are specific genomic changes associated with developing of malignant phenotype. We also showed that in premalignant stages HPV-FISH assay can be used as an effective diagnostic procedure to identify patients carrying highly risking HPV infection and chromosomal aberrations associated with this malignancy. cervical cancer, cervical dysplasia, HPV infection, hTERC amplification, MYC amplification, FISH, array-CGH.

Clinical and epidemiological study of EGFR mutations and EML4-ALK fusion genes among Indian patients with adenocarcinoma of the lung

BackgroundMutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a common feature observed in lung adenocarcinoma. A fusion gene between echinoderm microtubule-associated protein-like 4 (EML4) and the intracellular domain of anaplastic lymphoma kinase (ALK), named EML4-ALK, has been identified in a subset of non-small-cell lung cancer (NSCLC) tumors. The objective of this study was to determine the prevalence of EGFR mutations and EML4-ALK fusions in Indian patients with NSCLC (adenocarcinoma) as well as evaluate their clinical characteristics.Patients and methodsPatients with NSCLC, adenocarcinoma histology, whose tumors had been tested for EGFR mutational status, were considered for this study. ALK gene rearrangement was detected by fluorescence in situ hybridization using the Vysis ALK Break Apart Rearrangement Probe Kit. ALK mutation was tested in samples that were negative for EGFR mutation.ResultsA total of 500 NSCLC adenocarcinoma patients were enrolled across six centers. There were 337 (67.4%) men and 163 (32.6%) women with a median age of 58 years. One hundred and sixty-four (32.8%) blocks were positive for EGFR mutations, whereas 336 (67.2%) were EGFR wild-type. Of the 336 EGFR-negative blocks, EML4-ALK fusion gene was present in 15 (4.5%) patients, whereas 321 (95.5%) tumors were EML4-ALK negative. The overall incidence of EML4-ALK fusion gene was 3% (15/500).ConclusionThe incidence of EGFR mutations (33%) in this Indian population is close to the reported incidence in Asian patients. EML4-ALK gene fusions are present in lung adenocarcinomas from Indian patients, and the 3% incidence of EML4-ALK gene fusion in EGFR mutation-negative cases is similar to what has been observed in other Western and Asian populations. The mutual exclusivity of EML4-ALK and EGFR mutations suggests implementation of biomarker testing for tumors harboring ALK rearrangements in order to identify patients that can benefit from newer targeted therapies.