ABSTRACT
The generation of retinal organoids from human pluripotent stem cells (hPSC) is now a well-established process that in part recapitulates retinal development. However, hPSC-derived photoreceptors that exhibit well-organized outer segment structures have yet to be observed. To facilitate improved inherited retinal disease modeling, we determined conditions that would support outer segment development in maturing hPSC-derived photoreceptors. We established that the use of antioxidants and BSA-bound fatty acids promotes the formation of membranous outer segment-like structures. Using new protocols for hPSC-derived retinal organoid culture, we demonstrated improved outer segment formation for both rod and cone photoreceptors, including organized stacked discs. Using these enhanced conditions to generate iPSC-derived retinal organoids from patients with X-linked retinitis pigmentosa, we established robust cellular phenotypes that could be ameliorated following adeno-associated viral vector-mediated gene augmentation. These findings should aid both disease modeling and the development of therapeutic approaches for the treatment of photoreceptor disorders.
Subject(s)
Organoids , Pluripotent Stem Cells , Antioxidants/pharmacology , Dietary Supplements , Humans , Lipids , Retina , Retinal Cone Photoreceptor CellsABSTRACT
A portable and automated IC system with a dual-capability for the analysis of both fresh and saline environmental waters has been developed. Detection of nitrate in complex matrices such as seawater was achieved by the employment of an automated two-dimensional (heart-cut) IC method utilised in tandem with on-column matrix elimination, using a sodium chloride eluent. The system also demonstrated the capability to switch to a second mode of analysis, whereby direct one-dimensional IC analysis was employed to rapidly detect nitrite and nitrate in freshwater, with direct UV LED based absorption detection in under 3 minutes. Calibration curves using a 195 µL sample loop were generated for both freshwater and artificial seawater samples. For marine analysis, an analytical range of 0.1 mg L-1 - 40 mg L-1 NO3- was possible, while an analytical range (0.1 mg L-1 - 15 mg L-1 NO2-, 0.2 - 30 mg L-1 NO3-) appropriate for freshwater analysis was also achieved. Chromatographic repeatability for both marine and freshwater analysis was verified over 40 sequential runs with RSD values of < 1% demonstrated for both peak area and retention times for each mode of analysis. The selectivity of both methods was demonstrated with interference tests with common anions present in environmental waters. Recovery analysis was carried out on marine samples from Tramore Bay, Co. Waterford, Ireland, and the systems analytical performance was compared with that of an accredited IC following environmental sample analysis.
Subject(s)
Chromatography , Environmental Monitoring , Fresh Water , Nitrates , Seawater , Environmental Monitoring/methods , Fresh Water/chemistry , Nitrates/analysis , Nitrites/analysis , Seawater/chemistry , Ultraviolet RaysABSTRACT
INTRODUCTION: The burden of hepatocellular carcinoma (HCC) occurring in patients with alcoholic liver disease (ALD) is increasing at an alarming rate. The aims of this study were to compare the patient and tumor characteristics of HCC occurring in ALD-alone relative to and in addition to other chronic liver diseases. METHODS: Patients diagnosed with HCC between 2000 and 2014 were identified at 5 US clinical centers. The patients were categorized as ALD-alone, ALD plus viral hepatitis, or a non-ALD etiology. Clinical and tumor characteristics among the 3 groups were compared, and survival probability was estimated by the Kaplan-Meier method. The frequency of noncirrhotic HCC was compared across the 3 groups. RESULTS: A total of 5,327 patients with HCC were analyzed. Six hundred seventy (12.6%) developed HCC due to underlying ALD. Ninety-one percent of ALD-related HCC arose in men, in contrast to non-ALD etiologies where men accounted for 70% of HCCs cases (P < 0.001). Patients with ALD-alone-related HCC were older at diagnosis and had tumors less likely to be detected as part of routine surveillance. The ALD-alone cohort was least likely to be within the Milan criteria and to undergo liver transplantation. Overall survival in the ALD-alone HCC cohort was lower than the other 2 groups (1.07 vs 1.31 vs 1.41 years, P < 0.001). HCC in the noncirrhotic ALD cohorts occurred in only 3.5% of the patients compared with 15.7% in patients with non-ALD etiologies (P < 0.001). DISCUSSION: HCC occurring in patients with ALD occurred mostly in older men and almost exclusively in a cirrhotic background. They present with advanced tumors, and their survival is lower than HCCs occurring in non-ALD.
Subject(s)
Carcinoma, Hepatocellular/epidemiology , Liver Diseases, Alcoholic/pathology , Liver Neoplasms/epidemiology , Liver/pathology , Aged , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/pathology , Disease Progression , Female , Humans , Liver Diseases, Alcoholic/mortality , Liver Neoplasms/diagnosis , Liver Neoplasms/pathology , Male , Middle Aged , Retrospective Studies , Survival Analysis , United States/epidemiologyABSTRACT
A cost-effective, automated and portable IC has been developed for in-situ analysis of nitrite and nitrate in natural waters. The system employed 3D printed pumps for eluent delivery and a deep-UV LED based optical detector. Isocratic separation and selective detection of nitrite and nitrate was achieved in under 3 min. The total weight of the analyser was ~11 kg, and included electronics along with a sample intake system for automated analysis. Linear calibration ranges were generated using different sample injection loops. Using a 150 µL loop, an analytical range (0.05-30 mg L-1 NO2-, 0.10-75 mg L-1 NO3-) suitable for freshwater analysis was generated, while using a 10 µL loop an analytical range (0.30-100 mg L-1 NO2-, 2.5-500 mg L-1 NO3-) suitable for effluent and domestic wastewater analysis was achieved. Chromatographic repeatability demonstrated by the system is graphically presented and RSD values of <4% were obtained in terms of peak area and retention time over 82 sequential runs. The system was deployed in-situ at multiple sites for varying deployment periods analysing septic tank water, effluent from a waste water treatment plant and stream water. The data generated by the in-situ system were comparable to grab sample data generated by accredited laboratory instrumentation.
ABSTRACT
A low cost, UV absorbance detector incorporating a 235â¯nm light emitting diode (LED) for portable ion chromatography has been designed and fabricated to achieve rapid, selective detection of nitrite and nitrate in natural waters. The optical cell was fabricated through micromilling and solvent vapour bonding of two layers of poly (methyl methacrylate) (PMMA). The cell was fitted within a 3D printed housing and the LED and photodiode were aligned using 3D printed holders. Isocratic separation and selective detection of nitrite and nitrate was achieved in under 2.5â¯min using the 235â¯nm LED based detector and custom electronics. The design of the new detector assembly allowed for effective and sustained operation of the deep UV LED source at a low current (<10â¯mA), maintaining consistent and low LED temperatures during operation, eliminating the need for a heat sink. The detector cell was produced at a fraction of the cost of commercial optical cells and demonstrated very low stray light (0.01%). For retention time and peak area repeatability, RSD values ranged from 0.75 to 1.10 % and 3.06-4.19 %, respectively. Broad dynamic linear ranges were obtained for nitrite and nitrate, with limits of detection at ppb levels. The analytical performance of the IC set up with optical cell was compared to that of an ISO-accredited IC through the analysis of five various water samples. Relative errors not exceeding 6.86% were obtained for all samples. The detector was also coupled to a low pressure, low cost syringe pump to assess the potential for use within a portable analytical system. RSD values for retention time and peak area using this simple configuration were <1.15% and <3.57% respectively, highlighting repeatability values comparable to those in which a commercial HPLC pump was used.
Subject(s)
Chromatography, High Pressure Liquid/instrumentation , Nitrates/analysis , Nitrites/analysis , Chromatography, High Pressure Liquid/methods , Ultraviolet RaysABSTRACT
In this chapter, we describe a novel ascorbate peroxidase (APEX)-based labeling method that in combination with mass spectrometry identifies proteins in the immediate vicinity of αSyn in living rat cortical neurons. To isolate these interactions, we transduced primary cortical neurons with a lentivirus encoding APEX2 tagged to the C-terminus of alpha-synuclein (αSyn) and under the control of a synapsin promoter. Neural protein lysates were then incubated with streptavidin magnetic beads, washed, eluted from the beads, and digested overnight. The desalted peptides were then labeled with iTRAQ (4-plex) reagents and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). Collected data were analyzed using Spectrum Mill software, ultimately shedding light on αSyn physiological function and abnormal behavior during pathology.
Subject(s)
In Situ Hybridization , Mass Spectrometry , Neurons/metabolism , Peroxidase/metabolism , alpha-Synuclein/chemistry , alpha-Synuclein/metabolism , Animals , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/metabolism , Data Analysis , Fluorescent Antibody Technique , Humans , Parkinson Disease/metabolism , Proteomics/methods , Rats , Staining and LabelingABSTRACT
BACKGROUND: The use of human pluripotent stem cell-derived retinal cells for cell therapy strategies and disease modelling relies on the ability to obtain healthy and organised retinal tissue in sufficient quantities. Generating such tissue is a lengthy process, often taking over 6 months of cell culture, and current approaches do not always generate large quantities of the major retinal cell types required. METHODS: We adapted our previously described differentiation protocol to investigate the use of stirred-tank bioreactors. We used immunohistochemistry, flow cytometry and electron microscopy to characterise retinal organoids grown in standard and bioreactor culture conditions. RESULTS: Our analysis revealed that the use of bioreactors results in improved laminar stratification as well as an increase in the yield of photoreceptor cells bearing cilia and nascent outer-segment-like structures. CONCLUSIONS: Bioreactors represent a promising platform for scaling up the manufacture of retinal cells for use in disease modelling, drug screening and cell transplantation studies.
Subject(s)
Bioreactors/standards , Organoids/metabolism , Photoreceptor Cells/metabolism , Pluripotent Stem Cells/metabolism , Retina/metabolism , HumansABSTRACT
Adeno-associated viral vectors are showing great promise as gene therapy vectors for a wide range of retinal disorders. To date, evaluation of therapeutic approaches has depended almost exclusively on the use of animal models. With recent advances in human stem cell technology, stem cell-derived retina now offers the possibility to assess efficacy in human organoids in vitro. Here we test six adeno-associated virus (AAV) serotypes [AAV2/2, AAV2/9, AAV2/8, AAV2/8T(Y733F), AAV2/5, and ShH10] to determine their efficiency in transducing mouse and human pluripotent stem cell-derived retinal pigment epithelium (RPE) and photoreceptor cells in vitro. All the serotypes tested were capable of transducing RPE and photoreceptor cells in vitro. AAV ShH10 and AAV2/5 are the most efficient vectors at transducing both mouse and human RPE, while AAV2/8 and ShH10 achieved similarly robust transduction of human embryonic stem cell-derived cone photoreceptors. Furthermore, we show that human embryonic stem cell-derived photoreceptors can be used to establish promoter specificity in human cells in vitro. The results of this study will aid capsid selection and vector design for preclinical evaluation of gene therapy approaches, such as gene editing, that require the use of human cells and tissues.
Subject(s)
Dependovirus/physiology , Genetic Vectors/genetics , Photoreceptor Cells/cytology , Photoreceptor Cells/metabolism , Pluripotent Stem Cells/cytology , Retinal Pigment Epithelium/cytology , Viral Tropism , Animals , Cell Differentiation , Cells, Cultured , Dependovirus/classification , Fluorescent Antibody Technique , Gene Expression , Gene Transfer Techniques , Genes, Reporter , Humans , Mice , Organ Specificity/genetics , Promoter Regions, Genetic , Transduction, Genetic , TransgenesABSTRACT
PURPOSE OF REVIEW: A major cause of visual disorders is dysfunction and/or loss of the light-sensitive cells of the retina, the photoreceptors. To develop better treatments for patients, we need to understand how inherited retinal disease mutations result in the dysfunction of photoreceptors. New advances in the field of stem cell and gene editing research offer novel ways to model retinal dystrophies in vitro and present opportunities to translate basic biological insights into therapies. This brief review will discuss some of the issues that should be taken into account when carrying out disease modelling and gene editing of retinal cells. We will discuss (i) the use of human induced pluripotent stem cells (iPSCs) for disease modelling and cell therapy; (ii) the importance of using isogenic iPSC lines as controls; (iii) CRISPR/Cas9 gene editing of iPSCs; and (iv) in vivo gene editing using AAV vectors. RECENT FINDINGS: Ground-breaking advances in differentiation of iPSCs into retinal organoids and methods to derive mature light sensitive photoreceptors from iPSCs. Furthermore, single AAV systems for in vivo gene editing have been developed which makes retinal in vivo gene editing therapy a real prospect. SUMMARY: Genome editing is becoming a valuable tool for disease modelling and in vivo gene editing in the retina.
ABSTRACT
Ubiquitination is the first step of the ubiquitin-proteasome pathway that regulates cells for their homeostatic functions and is an enzymatic, protein post-translational modification process in which ubiquitin is transferred to a target protein substrate by a set of three ubiquitin enzymes (Weissman et al., 2011; Bhattacharyya et al., 2014; Ristic et al., 2014). Given the importance of this process, it is plausible that ubiquitination is under strict control by many factors and that the regulatory machineries are protein-specific. An assay for the detection of a specific protein ubiquitination will enable us to examine whether a factor has a function to regulate the ubiquitination of this protein. Here we describe a protocol that detects the ubiquitination status of the human REST4 protein in cultured cells, a neural alternative splicing isoform of REST (RE-1 silencing transcription factor), that antagonizes the repressive function of REST on neural differentiation and neuron formation. Using this protocol, we show that the telomere binding protein TRF2 stabilizes the expression of the human REST4 by inhibiting its ubiquitination. This indicates that TRF2 plays a positive role in neural differentiation (Ovando-Roche et al., 2014). This protocol is also useful for the detection of ubiquitination of other proteins of interest.
ABSTRACT
Telomere repeat binding factor 2 (TRF2) is a component of the shelterin complex that is known to bind and protect telomeric DNA, yet the detection of TRF2 in extra-telomeric regions of chromosomes suggests other roles for TRF2 besides telomere protection. Here, we demonstrate that TRF2 plays a critical role in antagonizing the repressive function of neuron-restrictive silencer factor, also known as repressor element-1 silencing transcription factor (REST), during the neural differentiation of human embryonic stem cells (hESCs) by enhancing the expression of a truncated REST splice isoform we term human REST4 (hREST4) due to its similarity to rodent REST4. We show that TRF2 is specifically upregulated during hESC neural differentiation concordantly with an increase in the expression of hREST4 and that both proteins are highly expressed in NPCs. Overexpression of TRF2 in hESCs increases hREST4 levels and induces their neural differentiation, whereas TRF2 knockdown in hESCs and NPCs reduces hREST4 expression, hindering their ability to differentiate to the neural lineage. Concurrently, we show that TRF2 directly interacts with the C-terminal of hREST4 through its TRF2 core binding motif [F/Y]xL, protecting hREST4 from ubiquitin-mediated proteasomal degradation and consequently furthering neural induction. Thus, the TRF2-mediated counterbalance between hREST4 and REST is vital for both the generation and maintenance of NPCs, suggesting an important role for TRF2 in both neurogenesis and function of the central nervous system.
Subject(s)
Cell Differentiation/physiology , Neural Stem Cells/cytology , Neurogenesis/physiology , Repressor Proteins/metabolism , Telomeric Repeat Binding Protein 2/metabolism , Cell Line , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Flow Cytometry , Humans , Immunoblotting , Immunohistochemistry , In Situ Hybridization, Fluorescence , Neural Stem Cells/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transduction, Genetic , Up-RegulationABSTRACT
The importance of HER2 status in breast cancer management has focused attention on the ability of clinical assays to correctly assign HER2 amplification status. There is no consensus as to the best method for assessing HER2 status. Disadvantages of fluorescence in situ hybridization (FISH) testing include longer time required for staining and scoring slides, requirements for specialized training and fluorescence microscopy, and loss of the signal due to quenching of the fluorescent dye. Silver-enhanced in situ hybridization (SISH) is a rapid fully automated assay providing permanently stained slides that are interpreted by conventional bright field microscopy which enables pathologists to evaluate slides within the context of tissue morphology. This study evaluates the concordance between SISH and FISH assays in determining the status of HER2 gene amplification in a cohort of 298 primary invasive breast carcinomas. Furthermore, we assessed in detail the variables contributing to interobserver interpretive reproducibility of HER2 SISH among 10 pathologists. HER2 was quantified using the ratio of HER2 to CHR17 signals using the conventional historical interpretation scale and also by the American Society of Clinical Oncology/College of American Pathologists reporting scheme. For SISH status determined by consensus among 10 pathologists, overall concordance between SISH and FISH was identified in 288 of 298 cases (96.6%) using the conventional Food and Drug Administration approved criteria. Overall agreement was observed in 282 of 285 cases (98.9%) using the American Society of Clinical Oncology/College of American Pathologists result reporting scheme (with equivocal cases removed). In conclusion, SISH represents a novel approach for the determination of HER2 status in breast cancer. The overall concordance between SISH and FISH is excellent, and the interpretation of SISH results by pathologists is most reproducible using the HER2/CHR17 ratio.
Subject(s)
Breast Neoplasms/genetics , Genes, erbB-2 , In Situ Hybridization/methods , Nucleic Acid Amplification Techniques/methods , Silver Compounds , Female , Humans , In Situ Hybridization, Fluorescence , Observer Variation , Reproducibility of ResultsABSTRACT
A vast colour palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolour labelling and whole-body imaging techniques. In the present paper, we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.
Subject(s)
Fluorescent Dyes , Luminescent Proteins , Amino Acid Sequence , Animals , Cell Line , Diagnostic Imaging/methods , Embryo, Nonmammalian , Fluorescence , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Molecular Sequence Data , Sequence Alignment , Xenopus laevis , Red Fluorescent ProteinSubject(s)
Antibodies, Monoclonal/immunology , Breast Neoplasms/diagnosis , Immunohistochemistry/methods , In Situ Hybridization, Fluorescence/methods , Receptor, ErbB-2/immunology , Animals , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Female , Guideline Adherence/standards , Humans , Immunohistochemistry/standards , In Situ Hybridization, Fluorescence/standards , Rabbits , Receptor, ErbB-2/genetics , Reproducibility of ResultsABSTRACT
Metallographic methods, in which a target is visualized using a probe or antibody that deposits metal selectively at its binding site, offers many advantages for bright-field in situ hybridization (ISH) detection as well as for other labeling and detection methods. Autometallographically enhanced gold labeling procedures have demonstrated higher sensitivity than conventional enzyme chromogens. Enzyme metallography, a novel procedure in which an enzymatic probe is used to deposit metal directly from solution, has been used to develop bright-field ISH methods for HER2 gene determination in breast cancer and other biopsy specimens. It provides the highest level of sensitivity and resolution, both for visualizing endogenous gene copies in nonamplified tissues and for resolving multiple gene copies to allow copy enumeration in amplified tissues without the need for oil immersion or fluorescence optics. An automated enzyme metallography procedure, silver ISH, has been developed for use in slide-staining instruments. Metallographic staining also provides excellent results for immunohistochemistry and may be combined with other staining procedures for the simultaneous detection of more than one gene or combinations of genes and proteins.
Subject(s)
Gold Colloid/chemistry , In Situ Hybridization/methods , Nucleic Acids/chemistry , Silver Compounds/chemistry , Silver Staining/methods , Adenocarcinoma/chemistry , Adenocarcinoma/diagnosis , Adenocarcinoma/genetics , Breast Neoplasms/chemistry , Breast Neoplasms/diagnosis , Breast Neoplasms/genetics , Enzymes/chemistry , Female , Gold Colloid/immunology , Humans , Receptor, ErbB-2/analysis , Receptor, ErbB-2/genetics , Silver Compounds/immunologyABSTRACT
The 2 methodologies in current clinical use to assess HER2 status in breast cancer are: fluorescence in situ hybridization (FISH) (gene amplification) and immunohistochemistry (protein over-expression). A consistent finding has been that 3% to 15% of breast cancers over-express HER2 protein without evidence for gene amplification. Accurate determination of the HER2 status has implications for selecting patients most likely to respond to trastuzumab. We report here our preliminary experience with a new anti-HER2 rabbit monoclonal antibody, 4B5. The evaluation of HER2 status in 2 different cohorts of breast cancer cases (Single Institution (SI) and Multinational (MN)) with a total of 322 breast cancer cases was performed on an automated staining system (Ventana Medical Systems, Inc, Tucson, AZ) and scored by 3 pathologists (0-3+), for comparison with CB11 staining results (PATHWAY) and FISH (PathVysion). Interlaboratory reproducibility of automated staining results and interpretation was determined on a subset of the SI cohort at 3 separate laboratories. Rabbit monoclonal 4B5 demonstrated sharper membrane staining with less cytoplasmic and stromal background staining than CB11. In the SI cohort, the staining results for 4B5 were highly comparable with those obtained for CB11 with an overall concordance of 93.3%. In the multinational cohort, the overall concordance with CB11 was 84.7%. This lower level of concordance was associated with a much higher overall agreement of 4B5 with FISH (89.5%), compared with agreement of CB11 with FISH (81.2%). The difference in the performance of CB11 in the MN cohort versus the SI cohort may be due to differences in tissue fixation and processing in a centralized, high volume laboratory in an academic medical center versus multiple sites in the international community with potentially nonstandardized techniques. The staining results with 4B5 indicate that it has a more robust performance than CB11 because the correlation of 4B5 with FISH was nearly equivalent (88.2% MN; 89.3% SI) in both cohorts. Interlaboratory reproducibility was also excellent (kappa 1.0). RMoAb 4B5 provides excellent sensitivity, specificity, and interlaboratory reproducibility for the detection of HER2 status in breast cancer.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Breast Neoplasms/diagnosis , Receptor, ErbB-2/analysis , Animals , Coloring Agents , Female , Humans , Immunohistochemistry/methods , Immunohistochemistry/standards , In Situ Hybridization, Fluorescence/standards , Methods , Rabbits , Receptor, ErbB-2/immunology , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Molecular morphologic tools exist for simultaneously visualizing immunophenotype and genotype of tumors, but are frequently hampered by a delicate balance between removing sufficient amount of the protein blocking full access of the probe to hybridize to target nucleic acids while still preserving sufficient target antigen for immunophenotyping. The result is often suboptimal, with either insufficiently visualized gene deletions and amplifications due to masking protein, or overdigestion of the protein target. Our purpose was to design and validate a gated genotyping assay that enables optimal and concomitant detection of both gene and protein. Using the proliferating endothelial cell compartment within gliomas organized in a tissue microarray (TMA), we tested the hypothesis that tyramide signal amplification (TSA) with deposition of a fluorochrome could be used during immunophenotyping, permitting sufficient protein digestion while insuring probe accessibility to nucleic acid target. The method was successfully validated using a TMA containing 38 glioma cases previously genotyped for EGFR amplification. CD31 positive endothelial cells were segregated via TSA-based Alexa-Fluor 647 immunofluorescence for analysis of EGFR amplification of the gliomas organized in the TMA. Enhanced immunoFISH (TSA) successfully segregates immunophenotypically-defined cell populations for gated genotyping.
Subject(s)
Immunohistochemistry/methods , Immunophenotyping/methods , In Situ Hybridization, Fluorescence/methods , Neoplasms/genetics , Neoplasms/pathology , Tyramine , ErbB Receptors/metabolism , Genotype , Humans , Neoplasms/immunology , Neoplasms/metabolism , Phenotype , Platelet Endothelial Cell Adhesion Molecule-1/metabolismABSTRACT
Implementation of interphase fluorescence in situ hybridization (FISH) assays in the clinical laboratory requires validation against established methods. Validation tools in common use include exchange of consecutive sections with another institution that has already established the FISH assay, comparison with conventional banded metaphase cytogenetics, confirmation of specificity using probed normal metaphases, consecutive paraffin sections of a validation set tested by a reference laboratory, and specificity assessment against well characterized cell lines. We have investigated the feasibility of using tissue microarrays (TMA) constructed from murine xenografts as a preliminary specificity-screening tool for validation of interphase FISH assays. Cell lines currently in use for FISH controls are used to generate xenografts in SCID mice which are fixed in formalin and paraffin embedded. A TMA is constructed using duplicate donor cores from the xenograft blocks. Xenografts used represent a wide range of translocations used routinely for formalin fixed paraffin embedded sections evaluated by FISH. Probe cocktails (Abbott-Vysis), for several non-random translocations associated with hematologic neoplasms and soft tissue sarcomas have been used in this manner. On-line deparaffinization, cell conditioning, and prehybridization steps are automated using a staining workstation (Ventana Discovery XT); hybridization and stringency washes are performed manually offline. FISH-probed TMAs are tracked using a Metasystems image scanner and analyzed using classifiers specifically developed for each molecular abnormality. FISH results for each xenograft in the TMA correspond exactly to the genotype previously established for the parent cell line from which the xenograft was prepared. Moderate complexity tissue microarrays constructed from murine xenografts are excellent validation tools for initial assessment of interphase FISH probe specificity.
Subject(s)
DNA Probes/genetics , Formaldehyde , In Situ Hybridization, Fluorescence/methods , Paraffin Embedding/methods , Tissue Array Analysis/methods , Xenograft Model Antitumor Assays/methods , Animals , Cell Line, Tumor , Humans , Mice , Neoplasms/geneticsABSTRACT
CONTEXT: Correct assessment of human epidermal growth factor receptor 2 (HER2) status is essential in managing patients with invasive breast carcinoma, but few data are available on the accuracy of laboratories performing HER2 testing by immunohistochemistry (IHC). OBJECTIVE: To review the results of the 2004 and 2005 College of American Pathologists HER2 Immunohistochemistry Tissue Microarray Survey. DESIGN: The HER2 survey is designed for laboratories performing immunohistochemical staining and interpretation for HER2. The survey uses tissue microarrays, each consisting of ten 3-mm tissue cores obtained from different invasive breast carcinomas. All cases are also analyzed by fluorescence in situ hybridization. Participants receive 8 tissue microarrays (80 cases) with instructions to perform immunostaining for HER2 using the laboratory's standard procedures. The laboratory interprets the stained slides and returns results to the College of American Pathologists for analysis. In 2004 and 2005, a core was considered "graded" when at least 90% of laboratories agreed on the result--negative (0, 1+) versus positive (2+, 3+). This interlaboratory comparison survey included 102 laboratories in 2004 and 141 laboratories in 2005. RESULTS: Of the 160 cases in both surveys, 111 (69%) achieved 90% consensus (graded). All 43 graded cores scored as IHC-positive were fluorescence in situ hybridization-positive, whereas all but 3 of the 68 IHC-negative graded cores were fluorescence in situ hybridization-negative. Ninety-seven (95%) of 102 laboratories in 2004 and 129 (91%) of 141 laboratories in 2005 correctly scored at least 90% of the graded cores. CONCLUSION: Performance among laboratories performing HER2 IHC in this tissue microarray-based survey was excellent. Cores found to be IHC-positive or IHC-negative by participant consensus can be used as validated benchmarks for interlaboratory comparison, allowing laboratories to assess their performance and determine if improvements are needed.