Fluorescence In Situ Hybridization Assay Research Articles

RETRACTED ARTICLE: Genomic amplification and high expression of EGFR are key targetable oncogenic events in malignant peripheral nerve sheath tumor

BackgroundThe dismal outcome of malignant peripheral nerve sheath tumor (MPNST) highlights the necessity of finding new therapeutic methods to benefit patients with this aggressive sarcoma. Our purpose was to investigate epidermal growth factor receptor (EGFR) as a potential therapeutic target in MPNSTs.Patients and methodsWe performed a microarray based-comparative genomic hybridization (aCGH) profiling of two cohorts of primary MPNST tissue samples including 25 patients treated at The University of Texas MD Anderson Cancer Center (MD Anderson) and 26 patients from Tianjin Medical University Cancer Institute & Hospital (TMUCIH). Fluorescence in situ hybridization (FISH) method was used to validate the gene amplification detected by aCGH analysis. Another independent cohort of 56 formalin fixed paraffin embedded (FFPE) MPNST samples was obtained to explore EGFR protein expression by immunohistochemical analysis. Cell biology detection and validation were performed on human MPNST cell lines ST88-14 and STS26T.ResultsaCGH and pathway analysis of the 51 MPNSTs identified significant gene amplification events in EGFR pathway, including frequent amplifications of EGFR gene itself, which was subsequently validated by FISH assay. High expression of EGFR protein was associated with poor disease-free and overall survival of human MPNST patients. In human MPNST cell lines ST88-14 and STS26T, inhibition of EGFR by siRNA or Gefitinib led to decreased cell proliferation, migration, and invasion accompanied by attenuation of PI3K/AKT and MAPK pathways.ConclusionThese results suggest that EGFR is a potential therapeutic target for MPNST.

Abstract A33: Aneuploidy due to inhibition of Aurora B induces p21 through activation of p38 by proteotoxic stress and ROS

Abstract Faithful transmission of genomic information to the two daughter cells requires highly coordinated cell cycle regulation. Aurora kinases have been implicated in mediating critical events during mitosis and cytokinesis. Aurora B is the enzymatic component of the chromosome passenger complex (CPC) which shows a characteristic localization during various phases of mitosis. Both the expression level and kinase activity of Aurora B is upregulated in several primary tumors including breast cancer and colon cancer and is associated with poor prognosis. Therefore, Aurora B is an attractive anti-cancer target and several chemical inhibitors of Aurora B are already in clinical trials. Inhibition of Aurora B results in cell cycle arrest. However, the signaling pathways responsible for the phenotypes observed after Aurora B inhibition have not yet been fully explored. We have recently shown that the p53 target gene p21 (CDKN1A) is activated both at protein and mRNA levels after pharmacological inhibition of Aurora B and that p21-activation is dependent on p53 and on the p38 MAP kinase. While the stabilization of p53 and its recruitment to the p21 promoter occur independently of p38, transcriptional elongation of p21 depends on active p38 after Aurora B is inhibited. We further show that partial inhibition of Aurora B is sufficient to induce p21. FISH (Fluorescence in-situ hybridization) assays indicate increased aneuploidy without inhibition of cytokinesis when Aurora B is partially inhibited. Increased aneuploidy is correlated with generation of reactive oxygen species (ROS) and proteotoxic stress, but not with DNA damage. In summary, our data suggest that aneuploidy due to partial inhibition of Aurora B results in proteotoxic stress and in the production of ROS, which contributes to the induction of p21 through activation of p38 MAPK. Significantly, Aurora B inhibitors and drugs that act on aneuploid cells (AICAR and 17AAG) synergize in the inhibition of cell proliferation and in inhibition of transformation. Thus, combining Aurora B inhibitors with drugs that act on aneuploid cells may result in a more effective cancer therapy than Aurora B inhibitors alone. Note: This abstract was not presented at the conference. Citation Format: Geeta Kumari, Tanja Ulrich, Stefan Gaubatz. Aneuploidy due to inhibition of Aurora B induces p21 through activation of p38 by proteotoxic stress and ROS. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A33.

The importance of rapid aneuploidy screening and prenatal diagnosis in the detection of numerical chromosomal abnormalities

ObjectivesEvaluation of Fluorescent in situ hybridization (FISH) as a tool for rapid aneuploidy screening (RAS) of high risk pregnancies, before its approval in the national antenatal screening and genetic diagnosis program in Egypt.MethodsThe cytogenetic data of prenatal specimens, and results of FISH of 100 patients performed between, January 2009 and December 2009, at the Medical Genetics Center (MGC) laboratory were retrieved and reviewed. AneuVysion Assay kit was used for detection of 13, 21, X, Y, 18 aneuploidies.ResultsMaternal age varied from 21 to 44 years (mean was 35.6 year). Ninety percent of pregnancies had normal chromosomes and 10% of the cases had numerical chromosomal abnormalities. Trisomy 21 was the most frequent chromosomal disorder across all indications (5%), followed by Turner syndrome (2%), trisomy 18 (2%), and trisomy 13 (1%). When comparing the FISH data with karyotype results for chromosomes 13, 18, 21, X, and Y in the 83 individual tested, no false positive or negative results were detected by the FISH assay. The result obtained by FISH and the banding cytogenetic were in complete accordance.ConclusionThis study supports the integration of amniotic fluid (AF) FISH as a RAS test, in to routine antenatal practice for identification of chromosome aneuploidies. There are trends towards delayed childbearing and most cases of Down Syndrome (DS) are currently detected post-nataly in the Egyptian population. Consequently, the live birth prevalence of DS has increased, which might lead to a serious negative public health effects.

Identification of a novel, recurrent MBTD1‐CXorf67 fusion in low‐grade endometrial stromal sarcoma

Endometrial stromal sarcomas (ESSs) are a genetically heterogeneous group of rare uterine neoplasms that are commonly driven by recurrent gene rearrangements. In conventional low-grade ESS, JAZF1-SUZ12, PHF1-JAZF1, EPC1-PHF1 and MEAF6-PHF1, and recently described ZC3H7-BCOR chimeric fusions have been reported in > 50% of cases. Conversely, oncogenic t(10;17)(q22;p13) translocation yields YWHAE-FAM22A/B chimeric proteins that are associated with histologically high-grade and clinically more aggressive ESS. Integrating whole-transcriptome paired-end RNA sequencing with fluorescence in situ hybridization (FISH) and banding cytogenetics, we identified MBTD1 (malignant brain tumor domain-containing 1) and CXorf67 (chromosome X open reading frame 67) as the genes involved in the novel reciprocal t(X;17)(p11.2;q21.33) translocation in two independent low-grade ESS of classical histology. The presence of the MBTD1-CXorf67 fusion transcript was validated in both cases using reverse-transcription polymerase chain reaction followed by Sanger sequencing. A specific FISH assay was developed to detect the novel t(X;17) translocation in formalin-fixed paraffin-embedded material, and resulted in identification of an additional low-grade ESS case positive for the MBTD1-CXorf67 fusion among 25 uterine stromal tumors [14 ESS and 11 undifferentiated endometrial sarcomas (UESs)] that were negative for JAZF1 and YWHAE rearrangements. Gene expression profiles of seven ESS (including three with YWHAE and two with JAZF1 rearrangements) and four UES without specific chromosomal aberrations indicated clustering of tumors with MBTD1-CXorf67 fusion together with low-grade JAZF1-associated ESS. The chimeric MBTD1-CXorf67 fusion identifies yet another cytogenetically distinct subgroup of low-grade ESS and offers the opportunity to shed light on the functions of two poorly characterized genes.

Evaluation of urovysion and cytology for bladder cancer detection

Urine cytology has been used for screening of bladder cancer but has been limited by its low sensitivity. UroVysion is a multiprobe fluorescence in situ hybridization (FISH) assay that detects common chromosome abnormalities in bladder cancers. For this study, the authors evaluated the effectiveness of multiprobe FISH and urine cytology in detecting urothelial cell carcinoma (UCC) in the same urine sample. In total, 1835 cases with the following criteria were selected: valid results from both the multiprobe FISH assay and urine cytology in the same urine sample, histologic and/or cystoscopic follow-up within 4 months of the original tests, or at least 3 years of clinical follow-up information. The results of FISH and cytology were correlated with clinical outcomes derived from a combination of histologic, cystoscopic, and clinical follow-up information. Of 1835 cases, 1045 cases were from patients undergoing surveillance of recurrent UCC, and 790 were for hematuria. The overall sensitivity, specificity, positive predictive value, and negative predictive value in detecting UCC were 61.9%, 89.7%, 53.9%, and 92.4%, respectively, for FISH and 29.1%, 96.9%, 64.4%, and 87.5%, respectively, for cytology. The performance of both FISH and cytology generally was better in the surveillance population and in samples with high-grade UCC. In 95 of 296 cases with atypical cytology that were proven to have UCC, 61 cases, mostly high-grade UCC, were positive using the multiprobe FISH assay. The UroVysion multiprobe FISH assay was more sensitive than urine cytology in detecting UCC, but it produced more false-positive results. The current data suggest that the use of FISH as a reflex test after an equivocal cytologic diagnosis may play an effective role in detecting UCC.

A Genome-Wide High-Resolution Array-CGH Analysis of Cutaneous Melanoma and Comparison of Array-CGH to FISH in Diagnostic Evaluation

Benign melanocytic nevi and cutaneous melanomas can be difficult to differentiate by means of routine microscopic analysis. Recent evidence has suggested that cytogenomic analysis may be a useful diagnostic method for evaluation of melanocytic proliferations. We investigated the array-based comparative genomic hybridization (aCGH) platform for DNA copy number analysis of formalin-fixed, paraffin-embedded (FFPE) tissues in melanocytic tumors and compared aCGH analysis with fluorescence in situ hybridization (FISH) assays in diagnosis of melanoma. aCGH findings and FISH results were interpreted independently in a blinded fashion. Positive findings were not noted in any benign nevi at aCGH analysis, whereas substantial unbalanced genomic aberrations were revealed in 92% of melanomas. Positive results were obtained in 72% of melanomas via the four-probe FISH assay (RREB1/MYB/CEP6/CCND1). A few additional FISH studies were performed to verify some aCGH findings of focal amplification of oncogenes and homozygous deletion of tumor suppressor genes. The overall concordance in aberrations detected using the two methods was 90%. Most discrepancies were due to a minor abnormal clone identified via FISH that was below analytical sensitivity of the FFPE aCGH test. Our study demonstrated that copy number analysis of FFPE tumor samples via aCGH isa robust and reliable method in diagnosis of melanoma and that aCGH and FISH tests should be used ascomplementary methods to improve the accuracy of genetic evaluation of melanocytic tumors.

Chromosomal gains measured in cytology samples from women with abnormal cervical cancer screening results

ObjectiveChromosomal gains at 3q26, 5p15 and 20q13 have been described in cervical precancer and cancer. We evaluated a novel fluorescence in situ hybridization (FISH) assay that detects gains at these three loci simultaneously as a possible biomarker for detecting cervical precancer. MethodsChromosomal copy numbers at 3q26, 5p15, 20q13 and the centromere of chromosome7 (cen7) in liquid-based cytology specimens from 168 women enrolled in the Biopsy Study were determined by FISH. The number of cells with ≥3 or ≥4 signals for a genomic locus was enumerated and diagnostic test performance measures were calculated using receiver operating characteristic (ROC) analyses. Sensitivity and specificity values were determined for the detection of CIN2+ and/or HSIL. ResultsThe median number of cells with ≥3 signals increased with the severity of cervical lesion for each genomic locus (p-trend<0.02 for each locus). ROC analysis for the number of cells with ≥3 signals resulted in area under the curve values of 0.70 (95% CI: 0.54–0.86), 0.67 (0.52–0.83), 0.67 (0.51–0.83) and 0.78 (0.64–0.92) for 3q26, 5p15, 20q13 and cen7, respectively, for the detection of CIN2+ and/or HSIL. Positivity for gains at multiple loci resulted in only slightly better test performance measures than those for the individual probes for four distinct combinations of probes. ConclusionsChromosomal gains at 3q26, 5p15, 20q13 and cen7 are associated with severity of cervical lesions. Further studies are required to quantify risk stratification of FISH assays for cervical cancer screening.

TFE3 Break-apart FISH Has a Higher Sensitivity for Xp11.2 Translocation–associated Renal Cell Carcinoma Compared With TFE3 or Cathepsin K Immunohistochemical Staining Alone

Renal cell carcinoma (RCC) associated with Xp11.2 translocation is uncommon, characterized by several different translocations involving the TFE3 gene. We assessed the utility of break-apart fluorescence in situ hybridization (FISH) in establishing the diagnosis for suspected or unclassified cases with negative or equivocal TFE3 immunostaining by analyzing 24 renal cancers with break-apart TFE3 FISH and comparing the molecular findings with the results of TFE3 and cathepsin K immunostaining in the same tumors. Ten tumors were originally diagnosed as Xp11.2 RCC on the basis of positive TFE3 immunostaining, and 14 were originally considered unclassified RCCs with negative or equivocal TFE3 staining, but with a range of features suspicious for Xp11.2 RCC. Seventeen cases showed TFE3 rearrangement associated with Xp11.2 translocation by FISH, including all 13 tumors with moderate or strong TFE3 (n=10) or cathepsin K (n=7) immunoreactivity. FISH-positive cases showed negative or equivocal immunoreactivity for TFE3 or cathepsin K in 7 and 10 tumors, respectively (both=3). None had positive immunohistochemistry but negative FISH. Morphologic features were typical for Xp11.2 RCC in 10/17 tumors. Unusual features included 1 melanotic Xp11.2 renal cancer, 1 tumor with mixed features of Xp11.2 RCC and clear cell RCC, and other tumors mimicking clear cell RCC, multilocular cystic RCC, or high-grade urothelial carcinoma. Morphology mimicking high-grade urothelial carcinoma has not been previously reported in these tumors. Psammoma bodies, hyalinized stroma, and intracellular pigment were preferentially identified in FISH-positive cases compared with FISH-negative cases. Our results support the clinical application of a TFE3 break-apart FISH assay for diagnosis and confirmation of Xp11.2 RCC and further expand the histopathologic spectrum of these neoplasms to include tumors with unusual features. A renal tumor with pathologic or clinical features highly suggestive of translocation-associated RCC but exhibiting negative or equivocal TFE3 immunostaining should be evaluated by TFE3 FISH assay to fully assess this possibility.

Canadian ALK (CALK): A pan-Canadian multicenter study to optimize and standardize ALK immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for ALK gene rearrangements.

8096 Background: ALK gene rearrangement (ALK+) has been found in 3-5% of advanced non-small cell lung cancer patients. FISH is considered the “gold standard” for identification of ALK+ tumors, but its cost-effectiveness and adoption as a screening assay has been debated. Recent reports suggested that ALK IHC may serve as an alternative screening or possibly a diagnostic method. In this context, CALK was initiated to assess the feasibility of implementing ALK IHC and/or FISH assays across Canadian hospitals. Methods: FISH-confirmed 22 ALK+ and 6 ALK- tumors were used as study samples. Unstained sections and scanned images of HE-stained slides from each tumor block were distributed to participating centres. IHC protocols with best signal to noise ratio using the 5A4 (Novocastra) or ALK-1 (Dako) antibodies were developed for various auto-stainers and implemented to suit the existing conditions of the participating centres. A common FISH protocol using the ALK break-apart probe (Abbott Molecular, Chicago, IL) was developed based on published reports. H-score was used to assess IHC. FISH signals were scored in 100 tumor cells/case by 2-3 pre-trained persons. A second round IHC study using newly distributed slides was completed by 8 centres. Results: Independent IHC scores from 12 centres and FISH scores from 11 centres were collected and analysed. The intraclass correlation coefficients (ICC) between centres for IHC and FISH were 0.84 and 0.68, respectively. Following the analysis of initial IHC results, a second round study resulted in improved ICC of 0.94. One of 23 tumors revealed IHC-/FISH+ discrepancy, with the FISH revealing unusual signal configurations that suggested an atypical rearrangement. However, the sensitivity and specificity of FISH results across centres using the 15 aberrant signals cut-off ranged from 86.7-100% and 100%, respectively. Conclusions: Standardization across multiple centres for ALK testing by IHC and FISH can be achieved. IHC detected all FISH+ ALK tumors, except for one discrepant case with atypical FISH finding of unknown clinical implication. The study was supported by a Pfizer Canada grant.

Abstract 2362: Detection of alk rearrangement in advanced non-small cell lung cancer using fluorescent in situ hybridization,immunohistochemistry,reverse transcription-polymerase chain reaction.

Abstract Background:ALK (anaplastic lymphoma kinase)gene fusions are detected in 3% to 13% of non-smallcell lung cancer(NSCLC). The ALK FISH assay is the current gold standard assay, but it can be technically challenging and costly.Therefore,other easy-to-use diagnostic modalities are needed to select the suitable patients for the target therapy. We investigated ALK fusions using immunohistochemistry (IHC), reverse transcription-polymerase chain reaction (RT-PCR), and fluorescent in situ hybridization (FISH) in order to compare the three methodologies for detection of ALK fusions. Methods:139 tumor samples with advanced NSCLC were collected from Cancer Institute/Hospital of Chinese Academy of Medical Sciences. All of these patients who previously received cell cytotoxic regimens therapy had progressive disease. ALK protein was detected by IHC using new clone antibody (D5F3, Cell Signal Techonology, USA). ALK gene rearrangement was evaluated by FISH using Vysis ALK Break Apart FISH Probe kit (Abbott, USA) and by RT-PCR detecting 9 EML4-ALK fusion variants with formalin-fixed paraffin-embedded tissue. Results:132 samples (95.0%)can be evaluated by FISH,137 samples(98.6%) can be evaluated by IHC and 122samples (87.8%)can be evaluated by RT-PCR in 139 cases.ALK positive samples were34.3% (47/137) by IHC, and ALK rearrangement samples were 32.6% (43/132) by FISH.EML4-ALK variants were detectable by RT-PCR in 32.4% (34 of 122) of specimens,and the variants 1 and variants 3 were the most common variant type.The concordance rate is 93.8% (kappa = 0864,p &amp;lt; 0.001) in the 130 paired samplesof FISH and IHC.75 of 76(98.7%)IHC 0 samples was FISH-,34 of 39 (87.2%) IHC 3+ samples were FISH +, 7 of 8 (87.5%) IHC 2+ samples were FISH +, whereas 1 of 7 (14.3%) IHC 1+ samples was FISH +. Considering FISH as the gold standard, the sensitivity and specificity of IHC were 95.3% and 93.1%, respectively, when 2+ and 3+ were regarded as IHC positive and 0 and 1+ as IHC negative.The concordance rate is 95.7% (kappa = 0.898,p &amp;lt; 0.001) in the 115 paired samples of FISH and RT-PCR . There are 4 negative cases by RT-PCR with ALK positive by FISH,and 1 positive case by RT-PCR with ALK negative by FISH. The sensitivity and specificity of RT-PCR were 89.2% and 98.7% respectively.But in the 120 paired samples of IHC and RT-PCRthe concordance rate is 90.8% (kappa = 0.788,p &amp;lt; 0.001),lower than IHC or RT-PCR with FISH. Conclusion:IHC can be the next frontier of molecular diagnostics to evaluate ALK fusions in NSCLC screening for ALK targeted therapy.RT-PCR showed relatively lower sensitivity and higher specificity for ALK rearrangement,it can be an alternative diagnostic assay for ALK detection. FISH was the most sensitive method for different ALK fusions. Citation Format: Yuan-kai Shi, Xiao-hong Han, Ning-ning Zhang, Yu-tao Liu, Xue-zhi Hao, Li Ma, Lin Wang, Dan li, Le Tang, Sheng Yang, Hua Lin. Detection of alk rearrangement in advanced non-small cell lung cancer using fluorescent in situ hybridization,immunohistochemistry,reverse transcription-polymerase chain reaction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2362. doi:10.1158/1538-7445.AM2013-2362

Comet‐FISH studies for evaluation of genetic damage of citalopram in somatic cells of the mouse

Damage to DNA can lead to many different acute and chronic pathophysiological conditions, ranging from cancer to endothelial damage. The present study was designed to evaluate the DNA damage of an antidepressant drug, citalopram, at the recommended human doses in somatic cells of mice in vivo. Mice exposed to citalopram at varying oral doses of 12 or 24 mg kg(-1) for 7 days exhibited a significant increase in the level of DNA-strand breaking and micronuclei formation as detected by a bone marrow comet assay and micronucleus test, respectively. Furthermore, using fluorescence in situ hybridization (FISH) analysis with the centromeric mouse-satellite DNA-probe for erythrocyte micronuclei it could be shown that citalopram is aneugen as well as clastogen in somatic cells in vivo. Colchicine (COL) and mitomycin C (MMC) were used as positive controls and these compounds produced the expected responses. Both the clastogenic and the aneugenic potential of citalopram can give rise to the development of secondary tumours and abnormal reproductive outcomes. Overall, the results suggest that citalopram at the recommended human doses induces some genetic alterations, which can adversely affect the normal cellular functioning in mice. The mechanism(s) by which citalopram cause this adverse effect appear related, in part, to primary DNA strand breakage as detected by the comet assay as well as clastogenic and aneugenic events as detected by the FISH assay. Therefore, the clinical use of citalopram must be weighed against the risks of genetic damages in citalopram users.

The ALK translocation in advanced non‐small‐cell lung carcinomas: preapproval testing experience at a single cancer centre

To study the ALK translocation in patients with advanced non-small-cell lung carcinomas (NSCLCs) seen at a European cancer centre, and its association with EGFR mutations, KRAS mutations and MET amplification. The study included samples from 86 patients diagnosed with advanced NSCLC. ALK fluorescence in-situ hybridization (FISH) was performed using the ALK break-apart probe set (Vysis). ALK FISH-positive cases were defined as those with more than 15% break-apart signals or isolated red signals in 50 cells. EGFR and KRAS mutations were determined by direct sequencing. All ALK-positive cases were analysed retrospectively for MET amplification using a FISH assay, and for ALK mutations by sequencing. We found nine (10.5%) ALK-positive cases, all in adenocarcinomas and the majority in female patients (88.9%). Signet ring cells were observed in four (44.4%) of the nine patients. None of the ALK translocated cases showed MET amplifications or EGFR, KRAS and ALK mutations. The prevalence of ALK translocation in an unselected population of European patients with advanced NSCLCs was 10%. This alteration was mutually exclusive with EGFR and KRAS mutations, as well as with MET amplification. If multiplexing is considered at the preanalytical phase, lung biopsy specimens are sufficient for performing several predictive assays.

Bright-Field Microscopy for HER2 Gene Assessment

The human epidermal growth factor receptor gene ERBB2 (HER2) is located on chromosome 17 and amplified in approximately 20% of invasive breast carcinomas.1,2 Due to the availability of effective anti-HER2 therapies, the prognostic significance of HER2 gene amplification, and the predictive value of HER2 status with regard to response to chemotherapy, laboratory testing for protein overexpression and/or amplification is critical for clinical decision making in individual patients. Testing of HER2 on all newly diagnosed invasive breast carcinoma is standard of care. Although there is no gold standard for assessing HER2 status in breast cancer, immunohistochemistry (IHC) for protein expression and fluorescence in situ hybridization (FISH) for HER2 gene copy number are the preferred assays.3 Although no test is perfect, clinical utilization depends on a number of factors, including test accuracy but also consistency of results, as well as ease of performance and interpretation. Due to early standardization, consistency in reporting, and only a small number of equivocal results, the FISH assay became the preferred method for assessing HER2 gene status.4,5 FISH is also used as the reflex test in IHC equivocal (2+ score) cases. FISH on IHC equivocal cases can identify a small percentage of amplified cases but also aneuploidy and other difficult to assess abnormalities of chromosome 17. Despite its widespread use, the FISH assay does have some drawbacks. FISH is considered a morphology-based assay, but the fluorescent nature of the assay results in less than optimal histologic details (compared with bright-field microscopy), so the area of invasive tumor generally needs to be marked on a parallel H&E-stained section. The procedure …

Development of taxonomic rRNA-targeted probes of two harmful algae: Prorocentrum minimum and Kareniamikimotoi by fluorescence in situ hybridization

Harmful algal blooms recently have been under the spotlight throughout the world, because of their negative impact on the marine environment, aquaculture, fisheries as well as public health. The development of methods for rapid and precise identification and quantification of causative species is essential for the warning andmonitoring of blooms, among which the techniques based on taxonomic probes are themost favored. In this study, two harmful algae, i.e., Prorocentrum minimum and Karenia mikimotoi were taken into consideration. The partial large subunit rDNA (D1-D2) of both species were firstly PCR-amplified, cloned and sequenced. The obtained sequences were then introduced to carry out alignment analysis for gene specific regions. Three respective candidate probes for each species were designed and used to screen the optimal probe by performing fluorescence in situ hybridization (FISH) tests. The results showed that the probes Pmin0443 and Kmik0602 displayed the best hybridization for P. minimum and K. mikimotoi, respectively. Both the specific (taxonomic) (Pmin0443 and Kmik0602) and the control probes (UniC0512 and UniR0499) were used for cross-reactivity tests with other microalgae in our laboratory. The probes Pmin0443 and Kmik0602 are specific and could be served as taxonomic probes introduced into the techniques targeting rRNA, such as FISH, sandwich hybridization, and DNA-microarray assay of P. minimum and K. mikimotoi in the future. Finally, FISH analyses with both probes were performed on the simulated field samples. The probes could hybridize exclusively with the target cells well, and no significant difference (p > 0.05) was observed in the cell densities of the samples determined by FISH and light microscopy (LM). All suggest that the probes are specific and could be introduced into FISH for the monitoring of both harmful algae.

EGFR status in oral squamous cell carcinoma: comparing immunohistochemistry, FISH and CISH detection in a case series study

ObjectivesTo compare the immunohistochemistry (IHC) expression of epidermal growth factor receptor (EGFR) in oral squamous cell carcinomas (OSCC) with the gene amplification evaluated by fluorescence in situ hybridization (FISH) and...

Genomic Rearrangements of PTEN in Prostate Cancer

The phosphatase and tensin homolog gene (PTEN) on chromosome 10q23.3 is a negative regulator of the PIK3/Akt survival pathway and is the most frequently deleted tumor suppressor gene in prostate cancer. Monoallelic loss of PTEN is present in up to 60% of localized prostate cancers and complete loss of PTEN in prostate cancer is linked to metastasis and androgen-independent progression. Studies on the genomic status of PTEN in prostate cancer initially used a two-color fluorescence in situ hybridization (FISH) assay for PTEN copy number detection in formalin fixed paraffin embedded tissue preparations. More recently, a four-color FISH assay containing two additional control probes flanking the PTEN locus with a lower false-positive rate was reported. Combined with the detection of other critical genomic biomarkers for prostate cancer such as ERG, androgen receptor, and MYC, the evaluation of PTEN genomic status has proven to be invaluable for patient stratification and management. Although less frequent than allelic deletions, point mutations in the gene and epigenetic silencing are also known to contribute to loss of PTEN function, and ultimately to prostate cancer initiation. Overall, it is clear that PTEN is a powerful biomarker for prostate cancer. Used as a companion diagnostic for emerging therapeutic drugs, FISH analysis of PTEN is promisingly moving human prostate cancer closer to more effective cancer management and therapies.

Chromosome-Specific DNA Repeats: Rapid Identification in Silico and Validation Using Fluorescence in Situ Hybridization

Chromosome enumeration in interphase and metaphase cells using fluorescence in situ hybridization (FISH) is an established procedure for the rapid and accurate cytogenetic analysis of cell nuclei and polar bodies, the unambiguous gender determination, as well as the definition of tumor-specific signatures. Present bottlenecks in the procedure are a limited number of commercial, non-isotopically labeled probes that can be combined in multiplex FISH assays and the relatively high price and effort to develop additional probes. We describe a streamlined approach for rapid probe definition, synthesis and validation, which is based on the analysis of publicly available DNA sequence information, also known as “database mining”. Examples of probe preparation for the human gonosomes and chromosome 16 as a selected autosome outline the probe selection strategy, define a timeline for expedited probe production and compare this novel selection strategy to more conventional probe cloning protocols.

Abstract P1-07-04: Comparison of HER2 expression by immunohistochemistry (IHC) using automated imaging system and fluorescence in situ hybridization (FISH). A retrospective analysis of 2853 cases

Abstract Background: Accurate assessment of HER2 status is critical for selecting patients who will benefit from trastuzumab therapy. There is still no consensus regarding the optimal method to assess HER2 status. Computerized image analysis has been shown to provide a more accurate and objective way for quantification of HER2 expression by IHC than manual evaluation. It has been suggested that the use of image analysis may help to resolve some of the discrepancies between IHC and FISH assay. We compared the results of HER2 expression by IHC using automated image analysis with fluorescent in situ hybridization (FISH) assay. Design: Testing for HER2 expression by IHC and FISH was performed on 2853 specimens at UT Southwestern Medical Center between the years 2002 to 2011. Quantification of IHC HER2 expression was done by image analysis and scored as positive (&amp;gt;2.0), borderline (1.5 to 2.0) and negative. (&amp;lt;1.5). The PathVysion kit was used for FISH assay to evaluate HER2 amplification. Ratios &amp;gt;2.0, 1.8 to 2.0 and &amp;lt;1.8 were defined positive, borderline and negative amplification respectively. Results: IHC compared to FISH Conclusion: Despite improvements in IHC testing, the FISH assay may be a better method for determining HER2 status. Factors such as tissue fixation, scoring methods and choice of antibodies may contribute to the lower specificity of IHC. In the amplified group, the gene amplification ratio correlated with protein expression, being highest in the IHC positive cases and lowest in those that were negative. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-04.

Detection of dengue group viruses by fluorescence in situ hybridization

BackgroundDengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays.MethodsOligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae). The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy.ResultsThe three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes.ConclusionBased on the FISH technique, we propose a way to design and use oligonucleotide probes to detect arboviruses. Results showed that this method was successfully implemented to specifically detect DENV in a mosquito cell line, as well as in mosquito salivary glands for the DENV-2 serotype. In addition, we emphasize that FISH could be an alternative method to detect arboviruses in host tissues, also offering to circumvent the discontinuity of antibodies used in immunofluorescent assays.

Molecular Confirmation of t(6;11)(p21;q12) Renal Cell Carcinoma in Archival Paraffin-embedded Material Using a Break-apart TFEB FISH Assay Expands its Clinicopathologic Spectrum

A subset of renal cell carcinomas (RCCs) is characterized by t(6;11)(p21;q12), which results in fusion of the untranslated Alpha (MALAT1) gene to the TFEB gene. Only 21 genetically confirmed cases of t(6;11) RCCs have been reported. This neoplasm typically demonstrates a distinctive biphasic morphology, comprising larger epithelioid cells and smaller cells clustered around basement membrane material; however, the full spectrum of its morphologic appearances is not known. The t(6;11) RCCs differ from most conventional RCCs in that they consistently express melanocytic immunohistochemical (IHC) markers such as HMB45 and Melan A and the cysteine protease cathepsin K but are often negative for epithelial markers such as cytokeratins. TFEB IHC has been proven to be useful to confirm the diagnosis of t(6;11) RCCs in archival material, because native TFEB is upregulated through promoter substitution by the gene fusion. However, IHC is highly fixation dependent and has been proven to be particularly difficult for TFEB. A validated fluorescence in situ hybridization (FISH) assay for molecular confirmation of the t(6;11) RCC in archival formalin-fixed, paraffin-embedded material has not been previously reported. We report herein the development of a break-apart TFEB FISH assay for the diagnosis of t(6;11)(p21;q12) RCCs. We validated the assay on 4 genetically confirmed cases and 76 relevant expected negative control cases and used the assay to report 8 new cases that expand the clinicopathologic spectrum of t(6;11) RCCs. An additional previously reported TFEB IHC-positive case was confirmed by TFEB FISH in 46-year-old archival material. In conclusion, TFEB FISH is a robust, clinically validated assay that can confirm the diagnosis of t(6;11) RCC in archival material and should allow a more comprehensive clinicopathologic delineation of this recently recognized neoplastic entity.