Two-color Fluorescence In Situ Hybridization Research Articles

CD36 Neuronal Identity in the Olfactory Epithelium.

CD36 scavenger receptor is expressed in a subpopulation of olfactory sensory neurons (OSNs). These neurons express canonical olfactory signaling machinery; however, not all odorant receptors (ORs) are coexpressed with CD36. In situ hybridization (ISH) enables the detection of nucleic acids in tissues, cells, or isolated chromosomes. The development of nonradioactive and stable labeling probes almost 30years ago, allowed to routinely perform this technique employing different labeling strategies in one experiment. ISH is widely used in the field of neurobiology of the sense of smell, providing valuable neuroanatomical information regarding the molecular organization of the olfactory epithelium (OE). Here we show a method for studying CD36+-OSNs and provide a detailed protocol for chromogenic ISH, one- or two-color fluorescent ISH, which can be combined with immunofluorescence and are suitable for Cd36 mRNA probing simultaneous to other transcripts and/or proteins labeling.

Development and clinical significance of FISH technique for detecting PTPRZ1-MET fusion gene in glioma patients

Objective To establish a method for detecting the PTPRZ1-MET fusion gene in patients with glioma via fluorescence in situ hybridization (FISH), and to explore its feasibility and clinical application. Methods The expression of PTPRZ1-MET fusion gene in glioma patients was detected by two-color FISH method. The result comparison was performed between FISH and polymerase chain reaction/Sanger sequencing (PCR/Sanger) to analyze the correlation of these two methods. Results The results of PCR/Sanger and FISH methods showed that the percentage of PTPRZ1-MET fusion gene positive cells was greater than 20% in patients defined as PTPRZ1-MET positive with 30-cycle-PCR/Sanger; the percentage in patients defined as PTPRZ1-MET positive with 50-cycle-PCR/Sanger was about 10%; and the percentage was less than 5% in patients defined as PTPRZ1-MET negative in PCR/Sanger method. There was significant difference between any two groups (all P<0.05). Conclusions The detection of PTPRZ1-MET fusion gene in glioma patients with FISH method seems consistent with that of PCR/Sanger method, which indicates that the FISH method could serve as a supplementary tool in initial screening for the patient's diagnosis. Key words: Glioma; In situ hybridization, fluorescence; Polymerase chain reaction; PTPRZ1-MET fusion gene

Multicolor FISHs for simultaneous detection of genes and DNA segments on human chromosomes.

We have developed a convenient multicolor fluorescent in situ hybridization (FISH) (five-, four-, three-, and two-color FISHs) for detecting specific genes/DNA segments on the human chromosomes. As a foundation of multicolor FISH, we first isolated 80 bacterial artificial chromosome (BAC) probes that specifically detect the peri-centromeres (peri-CEN) and subtelomeres (subTEL) of 24 different human chromosomes (nos. 1~22, X, and Y) by screening our homemade BAC library (Keio BAC library) consisting of 200,000 clones. Five-color FISH was performed using human DNA segments specific for peri-CEN or subTEL, which were labeled with five different fluorescent dyes [7-diethylaminocoumarin (DEAC): blue, fluorescein isothiocyanate (FITC): green, rhodamine 6G (R6G): yellow, TexRed: red, and cyanine5 (Cy5): purple]. To observe FISH signals under a fluorescence microscope, five optic filters were carefully chosen to avoid overlapping fluorescence emission. Five-color FISH and four-color FISH enabled us to accurately examine the numerical anomaly of human chromosomes. Three-color FISH using two specific BAC clones, that distinguish 5' half of oncogene epidermal growth factor receptor (EGFR) from its 3' half, revealed the amplification and truncation of EGFR in EGFR-overproducing cancer cells. Moreover, two-color FISH readily detected a fusion gene in leukemia cells such as breakpoint cluster region (BCR)/Abelson murine leukemia viral oncogene homologue (ABL) on the Philadelphia (Ph') chromosome with interchromosomal translocation. Some other successful cases such as trisomy 21 of Down syndrome are presented. Potential applications of multicolor FISH will be discussed.

Identification of the origin of marker chromosomes by two-color fluorescence in situ hybridization and polymerase chain reaction in azoospermic patients

Y chromosomal microdeletions at the azoospermia factor locus and chromosome abnormalities have been implicated as the major causes of idiopathic male infertility. A marker chromosome is a structurally abnormal chromosome in which no part can be identified by cytogenetics. In this study, to identify the origin of the marker chromosomes and to perform a genetic diagnosis of patients with azoospermia, two-color fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) techniques were carried out. The marker chromosomes for the two patients with azoospermia originated in the Y chromosome; it was ascertained that the karyotype of both patients was 46,X, ish del(Y)(q11)(DYZ3+, DXZ1-). The combination of two-color FISH and PCR techniques is an important method for the identification of the origin of marker chromosomes. Thus, genetic counseling and a clear genetic diagnosis of patients with azoospermia before intracytoplasmic sperm injection or other clinical managements are important.

Two-color fluorescent in situ hybridization using chromogenic substrates in zebrafish.

Two-color fluorescent in situ hybridization (FISH) is a widely used technique for comparing relative gene expression patterns. Current two-color FISH protocols are not ideal for detecting weakly expressed transcripts or monitoring signal strength and background levels during the course of the reaction. Here we describe an improved FISH protocol using the conventional highly sensitive chromogenic substrates nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Vector Red in zebrafish embryos. This protocol substantially improves on existing FISH techniques by combining the advantages of long reactivity of alkaline phosphatase, chromogenic monitoring of both developing reactions, and the ability to perform subsequent high-resolution fluorescent imaging. Although tested in zebrafish, a similar approach is expected to be applicable to ISH in any model organism.

ALK Status Testing in Non–Small-Cell Lung Carcinoma by FISH on ThinPrep Slides with Cytology Material

Oncogenic anaplastic lymphoma kinase (ALK) gene rearrangements in non-small-cell lung carcinomas (NSCLC) provide the basis for targeted therapy with crizotinib and other specific ALK inhibitors. Treatment eligibility is conventionally determined by the Food and Drug Administration-approved companion diagnostic fluorescence in situ hybridization (FISH) assay on paraffin-embedded tissue (PET). On limited samples such as fine needle aspiration-derived cytoblocks, FISH for ALK is often uninformative. FISH performed on liquid-based ThinPrep slides (ThinPrep-FISH) may represent a robust alternative. Two hundred thirty cytology samples from 217 patients with advanced NSCLC, including a consecutive series of 179 specimens, were used to generate matched ThinPrep slides and paraffin cytoblocks. The same ThinPrep slides used for cytologic diagnosis were assessed by standard ALK break-apart two-color probe FISH, after etching of tumor areas. Ultrasensitive ALK immunohistochemistry (IHC) on corresponding cytoblocks [D5F3 antibody, OptiView signal amplification] served as the reference data set. ThinPrep-FISH ALK signals were robust in 228 of 230 cases and not compromised by nuclear truncation inherent in paraffin-embedded tissue-FISH; only two samples displayed no signals. Nine of 178 informative cases (5%) in the consecutive series and 18 of 228 informative cases (7.8%) overall were ALK rearranged by ThinPrep-FISH. In 154 informative matched ThinPrep-FISH and cytoblock-IHC samples, 152 were concordant (10, 6.5% ALK status positive; 142, 92.2% ALK status negative), and two (1.3%) were ThinPrep-FISH positive but IHC negative (sensitivity 100%, specificity 98.6%, overall agreement 98.7%). Detection of ALK gene rearrangements in liquid cytology ThinPrep slides derived from patients with NSCLC can be confidently used for clinical ALK molecular testing.

A chromosome study of two centromere differentiating Drosera species, D. arcturi and D. regia

Abstract Using sequential fluorescent staining method and fluorescence in situ hybridization (FISH) technique, karyomorphological and molecular cytogenetic investigations of two centromere differentiating Drosera species, D. arcturi and D. regia were carried out. Drosera arcturi had chromosome number of 2n = 58, while D. regia had chromosome number of 2n = 34. Many chromosome bands stained with CMA positive and DAPI positive (CMA+DAPI+) were the most common in both species. CMA positive and DAPI negative (CMA+DAPI–) sites were shown in two chromosomes of both species. Four sites stained with CMA+DAPI–appeared on both sides of the constrictions of two larger chromosomes in D. arcturi, while two CMA+DAPI–sites appeared at terminal positions of two chromosomes in D. regia. Two-color FISH of 5S and 45S rDNAs showed two regions with major 45S rDNA signals in the both species, and four sites with clear 5S rDNA signals in D. arcturi. Drosera arcturi did not show any primary constriction in all chromosomes, excep...

Chromosome mapping of 18S rDNA and 5S rDNA by dual-color fluorescence in situ hybridization in the half-smooth tongue sole (Cynoglossus semilaevis).

Half-smooth tongue sole (Cynoglossus semilaevis) is an important aquaculture flatfish in China. Cytogenetic analysis has revealed that its sex determination system is female heterogametic (ZZ/ZW). The W chromosome is morphologically larger and has been considered evolutionarily younger than any other chromosome in the set. However, the genetic origin and evolution process of this neo-chromosome remains unclear. In this study, 2 tandem arrays of rRNA genes were chosen to address this question. Both the major rDNA (18S rDNA) and the minor rDNA (5S rDNA) were located on the C. semilaevis chromosomes by fluorescence in situ hybridization (FISH). Six 18S rDNA signals were observed on the centromeric regions of 3 pairs of autosomes in both males and females. In females, there was an additional 18S rDNA signal mapping to the telomeric region of the W chromosome long arm. With respect to the 5S rDNA, 12 signals were mapped to the centromeric regions of six pairs of autosomes. Two-color FISH further confirmed that the two pairs of the 5S rDNA signals were correspondingly located at the same positions of the same autosomes as those of the 18S rDNA signals. These results allowed us to speculate about the evolution process of the W chromosome. Chromosome fusions and repetitive sequence accumulations might have occurred in C. semilaevis. The synteny and non-synteny of C. semilaevis 18S rDNA and 5S rDNA might imply the original and evolutionary characteristics of this species. These findings will facilitate studies on karyotype evolution of the order Pleuronectiformes.

Quantitative Analysis of Diagnostic Guidelines for HER2-Status Assessment

Human epidermal growth factor receptor 2 (HER2, alias ERBB2)-targeted therapy in breast and gastric cancers depends on the reliable assessment of HER2 protein expression and (in equivocal cases) the quantitative evaluation of HER2 gene amplification. Typically, HER2 and centromere 17 gene copy numbers are evaluated using in situ hybridization (ISH) to calculate ratios for which cutoff values dividing nonamplified and amplified cases have been proposed. Although several studies have investigated how laboratory procedures affect diagnostics, a rigorous quantitative assessment of the diagnostic guidelines for data analysis is still missing. Here, we analyze the dependence of the diagnosed HER2/chromosome 17 ratios on i) sample size (evaluated cells), ii) gene/chromosome signal distributions, and iii) the approach used for quotient calculation using Monte Carlo simulations. Our data show that the current recommendation may lead to statistical HER2/CHR17 ratio variations of up to 0.94 and may therefore lead to incorrect HER2 status diagnoses, given the ratio threshold of 2.0 defined by the Food and Drug Administration. Moreover, borderline cases may receive different amplification diagnoses, depending on the ratio calculation approach: Brightfield-silver ISH with aggregated signal counts may underestimate the HER2/CHR17 ratio compared with two-color fluorescence ISH. Our results provide a basis for quantitative rationales behind HER2 diagnostic guidelines that call for increased numbers of evaluated cells and emphasize the importance of well-designed data analysis methods in diagnostic pathology, especially for predictive clinical application.

Cervical cancer screening: hTERC gene amplification detection by FISH in comparison with conventional methods

Aim: To assess the clinical significance of hTERC amplification for cervical cancer screening detected by fluorescence in situ hybridization (FISH) and compare it with that of current screening methods within the same group. Methods: A total of one hundred and nine women were recruited in this study. All of them had liquid-based thin-prep cytologic test (TCT), human papillomavirus (HPV) DNA testing and hTERC gene amplification analysis using interphase two-color FISH. In addition, colposcopically directed biopsy and/or cone biopsy were conducted for definite histopathologic diagnosis for each case. The optimal threashold of hTERC gene amplification by fluorescence in situ hybridization (FISH) were assecced by receiver operating characteristic (ROC) curve. The results of hTERC gene amplification analysis were compared with the cytological analysis, HPV DNA testing and those of subsequent biopsies. Results: Among the 109 patients, 18 were benign lesion, 17 were LSIL, 66 were HSIL and 8 were invasive carcinoma of cervix (ICC). Of them, hTERC-positive cases were found in 0.0% (0/18) of normal specimens, 11.8% (2/17) of LSIL, 72.7% (48/66) of HSIL and 100.0% (8/8) of ICC, respectively. The positive rate of hTERC gene amplification was significantly higher in HSIL and ICC compared with normal and LSIL (all P < 0.01).The optimal cut-off point of percentages of cells with hTERC amplification was determined as 5.5%. Using this threshold the hTERC test reached a much higher specificity(94.3%, 33/35) and a relatively lower sensitivity(77.0%, 57/74) to distinguish benign lesion and LSIL from HSIL and ICC in comparison with HR-HPV test (51.4%; 91.9%) and TCT (74.3%; 81.1%). Area Under the Curve revealed that hTERC amplification test performed more accurately (area under the curve = 0.857) compared to HPV test (area under the curve = 0.717) and cytology(area under the curve = 0.777) to discriminate HSIL or higher from LSIL or lower. This study also found a significant positive correlation between positive hTERC gain and HR-HPV infection, abnormal cytological or histopathologic lesions (all P < 0.01) in patients with cervical diseases. Conclusion: hTERC amplification testing may be a promising adjunct to screen women for cervical precancer or cancer with high specificity and accuracy.

Combined use of molecular cytogenetic techniques to detect a small chromosomal translocation

Comprehensive use of molecular cytogenetic techniques for the detection of 1 case of small chromosome translocation. Following conventional chromosome preparation, G-banding karyotype analysis, spectral karyotyping (SKY), whole chromosome painting, two-color fluorescence in situ hybridization (FISH) and subtelomeric probe FISH were performed. G-banded karyotype was 46, XX, ?(22q11.3), SKY karyotype analysis was 46, XX, der (4)t(4;6) and found no abnormalities on chromosome 22, staining signal was not found with any abnormalities on chromosome 6. Two-color FISH indicated a chromosomal translocation segment of 22q13.3 to one end of the short arm of chromosome 4. Subtelomeric FISH probe showed the end of the long arm of chromosome 22 and the end of the short arm of chromosome 4 reciprocal translocation. High resolution G-banding and FISH result indicated 46, XX, t(4;22)(p15.3;q13.2). The testing of small chromosomal translocation should be combined with clinical information and integrated use of molecular cytogenetic techniques to improve the accuracy of diagnosis of chromosomal diseases.

Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems

BackgroundWhole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round.ResultsWe show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated.ConclusionSince POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.

Visualization of Fine-Scale Genomic Structure by Oligonucleotide-Based High-Resolution FISH

The discovery of complex structural variations that exist within individual genomes has prompted a need to visualize chromosomes at a higher resolution than previously possible. To address this concern, we established a robust, high-resolution fluorescence in situ hybridization (FISH) method that utilizes probes derived from high complexity libraries of long oligonucleotides (>150 mers) synthesized in massively parallel reactions. In silico selected oligonucleotides, targeted to only the most informative elements in 18 genomic regions of interest, eliminated the need for suppressive hybridization reagents. Because of the inherent flexibility in our probe design methods, we readily visualized regions as small as 6.7 kb with high specificity on human metaphase chromosomes, resulting in an overall success rate of 94%. Two-color FISH over a 479-kb duplication, initially reported as being identical in 2 individuals, revealed distinct 2-color patterns representing direct and inverted duplicons, demonstrating that visualization by high-resolution FISH provides further insight in the fine-scale complexity of genomic structures. The ability to design FISH probes for any sequenced genome along with the ease, reproducibility, and high level of accuracy of this technique suggests that it will be powerful for routine analysis of previously difficult genomic regions and structures.

Correlation between morphology and human telomerase gene amplification in bronchial brushing cells for the diagnosis of lung cancer

The aim of this study was to investigate the frequency of amplification of the human telomerase gene (TERC), as measured by fluorescence in situ hybridization (FISH), in routine liquid-based cytological preparations from bronchial brushing specimens, and to assess the associations between TERC amplification, cytological diagnosis, and cytological morphology, in order to obtain further insight into these associations. Bronchial brushings from 102 patients with lung carcinoma (52 squamous-cell carcinomas, 22 adenocarcinomas, 28 small cell lung carcinomas) and 40 patients with nonmalignant disease were used. Amplification of TERC was performed using a commercially available two-color FISH probe, and slides were prepared for the SurePath liquid-based Pap test (LPT) using the same samples. Amplification of TERC was significantly associated with histological diagnoses (P < 0.05). Patients with lung cancer, and especially those with nonsmall cell lung cancer, had significantly higher percentages of cells with amplification of TERC than did patients with nonmalignant disease (P < 0.05). Comparing the FISH and LPT results, there was no significant difference in diagnostic sensitivity between the two methods (P > 0.05). However the difference in diagnostic sensitivity of the two methods for squamous-cell carcinoma was significant (P < 0.01). FISH can be performed on bronchial brushing specimens to detect amplification of TERC. This test may be an adjunct to cytology screening, especially in squamous-cell carcinoma, and may provide an indication of the potential of individual lesions to progress.

Clinical significance of hTERC gene amplification detection by FISH in the screening of cervical lesions

This study evaluated the clinical significance of hTERC gene amplification detection by fluorescence in situ hybridization (FISH) in the screening of cervical lesions. Cervical specimens of 50 high risk patients were detected by thin liquid-based cytology. The patients whose cytological results were classified as ASCUS or above were subjected to the subsequent colposcopic biopsies. Slides prepared from these 50 cervical specimens were analyzed for hTERC gene amplification using interphase FISH with the two-color hTERC probe. The results of the cytological analysis and those of subsequent biopsies, when available, were compared with the FISH-detected hTERC abnormalities. It was found that the positive rates of hTERC gene amplification in NILM, ASCUS, LSIL, HSIL, and SCC groups were 0.00, 28.57%, 57.14%, 100%, and 100%, respectively. The positive rates of hTERC gene amplification in HSIL and SCC groups were significantly higher than those in NILM, ASCUS and LSIL groups (all P<0.05). The mean percentages of cells with hTERC gene amplification in NILM, ASCUS, LSIL, HSIL, and SCC groups were 0.00, 10.50%, 36.00%, 79.00%, and 96.50%, respectively. Patients with HSIL or SCC cytological diagnoses had significantly higher mean percentages of cells with hTERC gene amplification than did patients with NILM, ASCUS or LSIL cytological diagnoses (all P<0.05). It was concluded that two-color interphase FISH could detect hTERC gene amplification to accurately distinguish HSIL and ISIL of cervical cells. It may be an adjunct to cytology screening, especially high-risk patients.

Gain of the 3q26 region in cervicovaginal liquid-based pap preparations is associated with squamous intraepithelial lesions and squamous cell carcinoma

BackgroundChromosomal aberrations have been documented in cervical carcinomas, especially chromosome 3q. The human telomerase RNA gene (hTERC) is located in the chromosome 3q26 region, and its product, telomerase, is involved in the maintenance of chromosome length and stability. Upregulation of telomerase is in general associated with tumorigenesis. In this study, cervicovaginal specimens were analyzed by fluorescence in situ hybridization (FISH) for gain of chromosome 3q26 containing hTERC, and FISH findings were compared with the cytologic and histologic diagnoses. MethodsSlides prepared from 66 liquid-based preparations from cervical specimens with cytologic diagnoses of negative for squamous intraepithelial lesion or malignancy (NILM, n=4), atypical squamous cells of undetermined significance (ASC-US, n=15), low-grade squamous intraepithelial lesion (LSIL, n=20), high-grade squamous intraepithelial lesion (HSIL, n=24), or cervical squamous cell carcinoma (SCCA, n=3) were analyzed for aberrations of 3q26 using a commercially available two-color FISH probe. The results of the cytologic analysis and those of concurrent or subsequent biopsies, when available, were compared with the FISH-detected 3q26 abnormalities. The Wilcoxon rank–sum test was used to assess associations between 3q26 gains and diagnoses. ResultsGain of 3q26 was significantly associated with the cytologic diagnosis (p<0.0001). Patients with HSIL or SCCA cytology diagnoses had significantly higher percentages of cells with 3q26 gain than did patients with NILM or ASC-US cytologic diagnoses. ConclusionsFISH can be performed on cervicovaginal liquid-based preparations to detect gain of 3q26. Gain of 3q26 is associated with HSIL and SCCA. This test may be an adjunct to cytology screening, especially high-risk patients.

Development of automated brightfield double In Situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence In Situ hybridization (FISH)

BackgroundHuman epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas.MethodsThe BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark® XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatise (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives.ResultsSequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 – 1.0000) and the ASCO/CAP scoring method with the FISH equivocal cases (95.7%, Simple Kappa = 0.8993%, 95% CI = 0.8068 – 0.9919) and without the FISH equivocal cases (100%, Simple Kappa = 1.0000%, 95% CI = 1.0000 – 1.0000).ConclusionAutomated BDISH applications for HER2 and CEN 17 targets were successfully developed and it might be able to replace manual two-color HER2 FISH methods. The application also has the potential to be used for other gene targets. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.

Del(6)(q22) and BCL6 Rearrangements in Primary Central Nervous System Lymphoma (PCNSL) Are Indicators of an Aggressive Clinical Course.

Purpose: Despite therapeutic advancements, biological markers that predict the natural history of primary central nervous system lymphoma (PCNSL) are lacking and age and performance status are the only consistently identified independent prognostic variables. BCL6 rearrangements and deletion of the tumor suppressor gene R-PTP-κ (PTPRK) at 6q22 are thought to be common genetic abnormalities in PCNSL but their prognostic significance is unknown. The aim of this study is to determine the prevalence and survival impact of del(6)(q22), BCL6, immunoglobulin heavy chain (IGH), and MYC gene rearrangements and prevalence of Epstein-Barr virus (EBV) infection in PCNSL affecting immunocompetent patients.Patients and methods: Seventy-six specimens from 76 HIV-negative, immunocompetent patients with PCNSL newly diagnosed and treated at Mayo Clinic between 1985 and 2006 were studied. Interphase fluorescence in-situ hybridization (FISH) was performed using two-color break apart probes (BAP) for BCL6 and MYC, a two-color dual-fusion probe for IGH-BCL6, and a two-color probe for del(6)(q22) on thin sections of paraffin-embedded tumor samples. Two-color IGH BAP FISH probes were also used to confirm IGH rearrangements in cases showing extra IGH signals without fusion using the IGH-BCL6 probe. In situ hybridization was performed using probes that recognize EBV-encoded RNA (EBER) on paraffin-embedded tumor samples. Survival data were analyzed for patients diagnosed after 1997 (n=53), corresponding to the change to high dose methotrexate as the standard of care. Survival was calculated from the date of tissue diagnosis to date of death or last contact. Survival curves were estimated using the Kaplan-Meier method. The log-rank test was used to compare survival across groups. Two-tailed p-values <0.05 were considered statistically significant. Clinical information for the 1985–2006 and 1997–2006 groups was comparable, including median age (67 years (y) vs 68 y), age range (26–87 y for both groups), percentage of deaths (67 vs 64) and median follow up for survivors (588 d vs 395 d).Results: Thirty-four (45%) cases showed del(6)(q22), 6 of which alsocontained a BCL6 rearrangement. Seventeen (22%) cases had a BCL6 rearrangement. Translocations involving IGH and an unknown partner gene (n=2) and MYC and an unknown partner gene (n=2) were also identified. All cases were EBER negative. Of the 53 patients diagnosed after 1997, 23 lacked del(6)(q22) or BCL6 rearrangement and had a median overall survival (MS) of 731 days (d). The 17 patients with an isolated del(6)(q22) had a MS of 90 d and the 13 patients with a BCL6 rearrangement had a MS of 442 d (p=0.0016).Conclusions: Del(6)(q22) and BCL6 rearrangements are common in PCNSL (45% and 22% of cases, respectively) and are associated with decreased survival, particularly del(6)(q22) seemingly independent of patient age and treatment time trends. IGH translocations were less frequent than in systemic diffuse large B-cell lymphoma (13% vs 51%), suggesting a distinct pathogenesis. MYC translocations and EBV infection are rare in PCNSL affecting immunocompetent patients.

Non-incidental coamplification of Myc and ERBB2, and Myc and EGFR, in gastric adenocarcinomas

This study was conducted to assess the frequencies of protein overexpression and gene amplification of Myc and to identify the mechanisms of Myc gene amplification, especially with regards to its possible coamplification with ERBB2 or EGFR in gastric adenocarcinomas. By immunohistochemical analysis of a total of 300 formalin-fixed and paraffin-embedded gastric adenocarcinomas, the nuclear overexpression of MYC was found in 47 tumors (16%). A fluorescence in situ hybridization (FISH) analysis revealed that nine (19%) of the 47 tumors with protein overexpression had cancer cells with high levels of Myc amplification, whereas only seven (6%) of the 122 tumors without protein overexpression showed high-level Myc gene amplification. Such Myc amplification was significantly correlated with positive nuclear protein overexpression. The coamplification of ERBB2 or EGFR with Myc that was found in six and four cases, respectively, is believed to be non-incidental because those frequencies were significantly higher than the individual frequencies observed for the total examined cases (ERBB2: 7%; EGFR: 4%). The high levels of gene amplification of these three genes, as visualized by FISH, could be broadly classified into two typical types, namely, ‘multiple scattered signals' and ‘large clustered signals'. Using two-color FISH, the coexistence of coamplified Myc and ERBB2, or Myc and EGFR, within single nuclei in various combinations of amplification types and copy numbers, could be ascertained in all nine cases, including one in which the synchronous ‘multiple scattered type' coamplification of Myc and ERBB2 was observed. In three tumors, coamplification of ERBB2 and EGFR was found; however, ERBB2- and EGFR-amplified cell populations were separate and mutually exclusive. We propose that the non-incidental coamplification of Myc and either ERBB2 or EGFR occurred through translocation and subsequent rearrangement.

Cell-type specific proximity of centromeric domains of one homologue each of chromosomes 2 and 11 in nuclei of cerebellar Purkinje neurons

In Purkinje neurons of the mouse cerebellum, the centromeres of several chromosomes are placed in close proximity to form a distinct pattern of clusters and exhibit reproducible spatial redistributions during development. In granule neurons, an adjacent cell type in the cerebellum, the pattern, size, and number of centromeric aggregations are different from those of Purkinje neurons. The present work was undertaken to test the hypothesis that the same chromosomes form part of one aggregate in a cell-type-specific manner. Fluorescence in situ hybridization (FISH) with chromosome-specific paracentromeric probes was used to identify centromeric regions of individual chromosomes in cerebellar Purkinje and granule neurons of the adult mouse. When pairs of centromeric probes were used in two-color FISH, one homologue each of chromosomes 2 and 11 were routinely found close to each other in Purkinje neurons but not in granule neurons. This finding of specific proximity was limited to the pair 2 and 11, out of the ten chromosome pairs that were randomly selected and studied. Our results indicate that, in adult Purkinje neurons, a cell-type-specific spatial proximity is present between centromeric domains of one homologue each of chromosomes 2 and 11.