Nonsynonymous SNPs (nsSNPs) in DNA repair genes may be important determinants of DNA damage and cancer risk. We applied a set of screening criteria to a large number of nsSNPs and selected a subset of SNPs that were likely candidates for phenotypic effects on DNA double-strand break repair (DSBR). In order to induce and follow DSBR, we exposed panels of cell lines to gamma irradiation and followed the formation and disappearance of gammaH2A.X foci over time. All panels of cell lines showed significant increases in number, intensity, and area of foci at both the 1-hour and 3-hour time points. Twenty four hours following exposure, the number of foci returned to preexposure levels in all cell lines, whereas the size and intensity of foci remained significantly elevated. We saw no significant difference in gammaH2A.X foci between controls and any of the panels of cell lines representing the different nsSNPs.

Karyotyping is currently the #gold##standard# test for the detection of human chromosome abnormalities. Over the past 40 years, changes in techniques have improved the band definition of chromosomes; however, very little has changed with respect to improvements through automation. In this study, we compare chromosome analysis by traditional microscopy with semi-automatic karyotyping using robotic equipment from MetaSystems (Altlussheim, Germany). Analysis using MetaSystems was significantly quicker than using the microscope with an average reduction in analysis time of 26.5 minutes; for the average analyst, this equates to a reduction of 27 percent. Analysis checking times using MetaSystems showed even greater improvement with an average reduction in checking time of 11.4 minutes; for the average checker, this equates to a reduction of 48 percent. The MetaSystems semi-automatic karyotyping equipment offers increased throughput of cases for karyotype analysis while maintaining accuracy.

BACKGROUND: Human 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. METHODS: The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark(R) 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 phosphatase (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. RESULTS: Sequential 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). CONCLUSION: Automated 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.

Food irradiation is the process of exposing food to ionising radiation in order to disinfect, sanitise, sterilise and preserve food or to provide insect disinfestation. Irradiated food should be adequately labelled according to international and national guidelines. In many countries, there are furthermore restrictions to the product-specific maximal dose that can be administered. Therefore, there is a need for methods that allow detection of irradiated food, as well as for methods that provide a reliable dose estimate. In recent years, the comet assay was proposed as a simple, rapid and inexpensive method to fulfil these goals, but further research is required to explore the full potential of this method. In this paper we describe the use of an automated image analysing system to measure DNA comets which allow the discrimination between irradiated and non-irradiated food as well as the set-up of standard dose-response curves, and hence a sufficiently accurate dose estimation.

<p>Among the various methods available for analyzing minimal residual disease, a new procedure for the cell-based approaches using consecutive phenotypic and genotypic analysis as revealed by immunofluorescent labeling and subsequent fluorescent in situ hybridization (FISH) has been developed. We are introducing a fluorescent microscopy-based technique by which not only cellular targets and immunological marker positivity, but also the FISH pattern was identified by automated scanning. For the latter one translocation-specific FISH pattern recognition was accomplished by using an automated scanning mode for the 3D determination of valid distances between FISH signals, to define the cutoff value for the shortest green-red spot distance differentiating positive cells from negative ones. The procedure was tested with CD10(+) acute lymphoblastic leukemia cell line harboring the t(12;21)(p13;q22) resulting in the ETV6/RUNX1 rearrangement (formerly TEL/AML1), as well as peripheral blood lymphocytes of healthy individuals. Using the combined, automated method, a sensitivity of 98.67% and a specificity of 99.97% were obtained. The mean false positivity + 2 standard deviations cutoff level (0.09%) allows detection of leukemic cells with high accuracy, even a bit below the tumor load dilution of 10(-3), a value reported to be critical in clinical decision making.</p>

Interleukin-6 (IL-6) is known to promote tumour growth and survival. We evaluated IL-6 gene amplification in tumours from 53 glioma patients using fluorescence in situ hybridisation. Amplification events were detected only in glioblastomas (15 out of 36 cases), the most malignant tumours, and were significantly associated with decreased patient survival.

The lymphocyte micronucleus test is the most standardized of all cellular biomarkers for genotoxic effects. Until recently, the analysis of micronuclei was done exclusively by visual microscopical inspection but recently great progress has been made in automation. In this article we compare micronucleus data obtained by 'classical' visual scoring with those obtained by automated scoring using an image analysis system with micronucleus software. The results show that the use of automation is perfectly reliable for this task, especially after conducting limited post-analysis corrections.

Novel recurrent gene fusions between the androgen-regulated gene TMPRSS2 and the ETS family members ERG, ETV1, or ETV4 have been recently identified as a common molecular event in prostate cancer development. We comprehensively analyzed the frequency and risk of disease progression for the TMPRSS2 and ETS family genes rearrangements in a cohort of 96 American men surgically treated for clinically localized prostate cancer. Using three break apart (TMPRSS2, ERG, ETV4) and one fusion (TMPRSS:ETV1) fluorescence in situ hybridization (FISH) assays, we identified rearrangements in TMPRSS2, ERG, ETV1, and ETV4 in 65, 55, 2, and 2% of cases, respectively. Overall, 54 and 2% of cases demonstrated TMPRSS2:ERG and TMPRSS2:ETV1 fusions, respectively. As intronic loss of genomic DNA between TMPRSS2 and ERG has been identified as a mechanism of TMPRSS2:ERG fusion, our assays allowed us to detect deletion of the 3' end of TMPRSS2 and the 5' end of ERG in 41 and 39% of cases rearranged for respective genes. Prostate cancers demonstrating TMPRSS2 gene rearrangement were associated with high pathologic stage (P=0.04). Our results confirm that recurrent chromosomal aberrations in TMPRSS2 and/or ETS family members are found in about 70% of prostate cancers. Importantly, we define a novel approach to study these gene fusions and identified cases where TMPRSS2 was rearranged without rearrangement of ERG, ETV1 or ETV4 and cases with ETS family gene rearrangement without TMPRSS2 rearrangement, suggesting that novel 5' and 3' partners may be involved in gene fusions in prostate cancer.

Urban air pollution resulting from traffic is a major problem in many cities in Asia, including Bangkok, Thailand. This pollution originates mainly from incomplete fossil fuel combustion, e.g. transportation, and the composition of which is very complex. Some of the compounds are carcinogenic in experimental animals and in man. Polycyclic aromatic hydrocarbons (PAHs) and benzene are among the major carcinogenic compounds found in urban air pollution from motor vehicle emissions. In major cities in Asia, the levels of PAHs and benzene are relatively high compared with those in Europe or in the United States and thus people are exposed to higher levels. Biomarkers of exposure and early biological effects have been used to study the potential health effects of exposure to PAHs and benzene in air pollution in school children attending schools in inner-city Bangkok compared to those attending schools in rural areas. Bangkok school children are exposed to total PAHs at levels 3.5-fold higher than those in the rural area. Urinary 1-hydroxypyrene, a metabolite of PAH, was also significantly higher, while PAH-DNA adducts in lymphocytes were five-fold higher in Bangkok school children than rural school children. Benzene exposure in Bangkok school children was approximately two-fold higher than in rural school children. This is in agreement with the levels of biomarkers of internal benzene dose, i.e. blood benzene and urinary t,t-muconic acid. The potential health risks from exposure to genotoxic substances were assessed through DNA-damage levels and DNA repair capacity. DNA strand breaks were significantly higher, whereas DNA repair capacity was significantly reduced in Bangkok children. Genetic polymorphisms have been detected in glutathione-S-transferases (GSTs) and cytochrome P450 (CYP450) enzymes involved in the metabolism of benzene and PAHs, but these polymorphisms had no significant effects on the biomarkers of PAH exposure. Our results indicate that children living in a mega city such as Bangkok may have an increased health risk of the development of certain diseases due to exposure to genotoxic substances in air pollution compared to children living in suburban/rural areas.

The HER2 gene, amplified in 10 to 35% of invasive human breast carcinomas, has prognostic and therapeutic implications. Fluorescent in situ hybridization is one method currently used for assessing HER2 status, but fluorescent in situ hybridization involves the time-consuming step of manual signal enumeration. To address this issue, Vysis has developed an automated signal enumeration system, Vysis AutoVysion. A multicenter, blinded study was conducted on 39 formalin-fixed, paraffin-embedded invasive breast carcinoma specimens, including 20 HER2 nonamplified and 19 HER2 amplified (weakly to highly amplified), provided in duplicate to each study site for analysis. Calculation of the HER2/CEP17 ratio and the hands-on time of both manual and automated enumeration approaches were compared. Overall agreement of HER2 classification results (positive and negative) was 92.5% (196 of 212). The Vysis AutoVysion System requires manual enumeration for cases with scanner results within the ratio range of 1.5 to 3.0. When the data in this range are excluded, the agreement between manual and scanner results is 98.8% (169 of 171). The average Vysis AutoVysion System hands-on time per slide was 4.59 versus 7.47 minutes for manual signal enumeration (savings of 2.88 minutes/slide). These data suggest that the Vysis AutoVysion System can correctly classify specimens and may increase the overall efficiency of HER2 testing.

Food irradiation is the process of exposing food to ionising radiation in order to disinfect, sanitise, sterilise and preserve food or to provide insect disinfestation. Irradiated food should be adequately labelled according to international and national guidelines. In many countries, there are furthermore restrictions to the product-specific maximal dose that can be administered. Therefore, there is a need for methods that allow detection of irradiated food, as well as for methods that provide a reliable dose estimate. In recent years, the comet assay was proposed as a simple, rapid and inexpensive method to fulfil these goals, but further research is required to explore the full potential of this method. In this paper we describe the use of an automated image analysing system to measure DNA comets which allow the discrimination between irradiated and non-irradiated food as well as the set-up of standard dose–response curves, and hence a sufficiently accurate dose estimation.

The present study comprised a biomonitoring study in 95 workers occupationally exposed to styrene and 98 unexposed controls, employing an integrated approach involving biomarkers of exposure, effect, and susceptibility. Airborne styrene was evaluated at workplace, and urinary styrene metabolites, mandelic acid (MA), phenylglyoxylic acid (PGA), vinylphenols (VPTs) and phenylhydroxyethylmercapturic acids (PHEMAs), were measured as biomarkers of internal dose. Cytogenetic alterations were evaluated by analysing the frequency of chromosomal aberrations (CAs) and micronucleated binucleated cells (MNBN) in peripheral blood lymphocytes. The micronucleus assay was coupled with centromeric fluorescence in situ hybridization to distinguish micronuclei (MN) arising from chromosomal breakage (C- MN) from those harboring whole chromosomes (C+ MN). The possible influence of genetic polymorphisms of xenobiotic-metabolizing enzymes involved in styrene biotransformation (EPHX1, GSTT1, GSTM1, GSTP1) and NAT2 on the cytogenetic endpoints was investigated. The exposed workers showed a significantly higher frequency of MNBN (13.8+/-0.5% versus 9.2+/-0.4%; P<0.001) compared to control subjects. The effect appeared to concern both C- and C+ MN. A positive correlation was seen between the frequency of C+ MN and urinary level of MA+PGA (P<0.05) and VPTs (P<0.001). Chromosome-type CAs positively correlated with airborne styrene level and VPTs (P<0.05), whereas chromatid-type CAs correlated with PHEMAs (P<0.05). Workers bearing GSTM1 null genotype showed lowered levels of PHEMAs (P<0.001). The GSTT1 null genotype was associated with increased MNBN frequencies in the exposed workers (P<0.05) and the fast activity EPHX genotype with a moderate decrease in both MNBN and CAs in the controls. Our results suggest that occupational exposure to styrene has genotoxic effects that are potentiated by the GSTT1 gene deletion. These observations may have relevance considering the risk of lymphatic and haematopoietic malignancies tentatively associated with styrene exposure.

Certain recurrent cytogenetic abnormalities are diagnostic of a specific neoplasm and may portend prognosis. As conventional cytogenetics may not reveal a neoplastic clone, and unfixed material for fluorescence in situ hybridization may be unavailable, performing fluorescence in situ hybridization on fixed tissues is diagnostically and prognostically valuable. Manual interpretation of fluorescence in situ hybridization signals may be difficult on paraffin-embedded tissue sections due to truncated nuclei. Therefore, we investigated the use of an automated image acquisition and analysis system (MetaSystems) for interpretation of fluorescence in situ hybridization signals in tissue sections from dual fusion translocation probes. Three probe sets were analyzed on archival specimens with a confirmed diagnosis of mantle cell lymphoma, follicular lymphoma or Burkitt lymphoma. 100% of mantle cell lymphomas (7/7) were positive for t(11;14), 91% of follicular lymphomas (10/11) for t(14;18) and 100% of Burkitt lymphomas (9/9) for t(8;14). Successful hybridization was achieved using various tissue fixatives and fluorescence in situ hybridization interpretation was blinded with respect to the underlying diagnosis. Based on these results, automated analysis of fluorescence in situ hybridization on fixed tissues is accurate and valuable in the evaluation of B-cell lymphoma, and may provide pertinent diagnostic and prognostic information.

<p>Switching from a replicative to a translesion polymerase is an important step to further continue on replication at the site of DNA lesion. Recently, RAD18 (a ubiquitin ligase) was shown to monoubiquitinate proliferating cell nuclear antigen (PCNA) in cooperation with RAD6 (a ubiquitin-conjugating enzyme) at the replication-stalled sites, causing the polymerase switch. Analyzing RAD18-knockout (RAD18-/-) cells generated from human HCT116 cells, in addition to the polymerase switch, we found a new function of RAD18 for S phase-specific DNA single-strand break repair (SSBR). Unlike the case with polymerase switching, PCNA monoubiquitination was not necessary for the SSBR. When compared with wild-type HCT116 cells, RAD18-/- cells, defective in the repair of X-ray-induced chromosomal aberrations, were significantly hypersensitive to X-ray-irradiation and also to the topoisomerase I inhibitor camptothecin (CPT) capable of inducing single-strand breaks but were not so sensitive to the topoisomerase II inhibitor etoposide capable of inducing double-strand breaks. However, such hypersensitivity to CPT observed with RAD18-/- cells was limited to only the S phase due to the absence of the RAD18 S phase-specific function. Furthermore, the defective SSBR observed in S phase of RAD18-/- cells was also demonstrated by alkaline comet assay.</p>

Determination of HER2 status by fluorescence in situ hybridization (FISH) in breast carcinoma correlates well with response to targeted therapy and prognosis. However, manual time consuming methods and quantification aspects of the procedure may be challenging for some laboratories. We examined the feasibility of automating these components of the FISH assay using a tissue microarray (TMA-118 clinically annotated cases) and a series of 41 whole sections. An in situ hybridization automated staining workstation was used to automate a programmed overnight start, on line baking, deparaffinization, cell conditioning, protease digestion, and prehybridization buffer washing. Dual label probe/target codenaturation/hybridization and stringency washing were done off line. The HER2 and CEP17 spot counts were quantified, and the HER2/CEP17 ratio calculated, via an imaging workstation. Results were benchmarked against manual counts for whole sections, and bright field in situ hybridization [silver in situ hybridization (SISH)] for the TMA. Automated FISH results using whole sections correlated well with manual results: HER2/CEP17 ratio correlation coefficient r = 0.9154, r = 0.8380, P < 0.0001. Correlation between automated and manual TMA FISH results was also excellent, and disease-free survival was significantly shorter (P < 0.001) for the HER2 amplified cases. Automation of the laborious manual prehybridization and image quantification components of FISH using directly labeled probes is feasible. Operational gains and enhanced consistency are inherent in this automated approach to HER2 clinical FISH testing.

OBJECTIVE: To determine whether it is possible to stratify patients with superficial bladder cancer into low- and high-risk groups for tumour recurrence/progression based on the chromosomal pattern detected by fluorescence in situ hybridization (FISH) in one urine cytology specimen used for follow-up testing. PATIENTS AND METHODS: Voided urine samples from 47 consecutive patients with urinary tract neoplasms (13 with no history of urothelial malignancy and 34 under follow-up after complete transurethral resection of superficial urothelial carcinoma of the bladder) were evaluated by liquid-based cytology (ThinPrep(R), CYTYC Corp., Boxborough, MA, USA) and UroVysion FISH (Vysis-Abbott, Downers Grove, IL). RESULTS: Of the 34 patients under surveillance, the UroVysion test was negative in four, 17 had loss of 9p21 sequences either alone or combined with low-frequency trisomy/ies or tetrasomy/ies of chromosomes 3, 7 and 17 in single cells (low-risk FISH), and 13 also had complex aneusomies of the remaining chromosomes (high-risk FISH). One of the four FISH-negative neoplasms, four of the 17 low-risk FISH cases and five of the 11 informative high-risk FISH-positive patients developed recurrence. Progression occurred only in patients with high-risk FISH results, showing high-frequency complex chromosomal polysomies (four of 11). CONCLUSION: The results from this pilot study indicate that the UroVysion FISH test may help to individually assess the clinical behaviour of superficial bladder cancer, based on the chromosomal pattern of exfoliated tumour cells in follow-up urinary cytology. It might be of use to identify those patients likely to progress at earlier and curable stages of disease, and lengthen the surveillance period in those with persistent or recurrent low-risk disease.

OBJECTIVE: A variety of methods have been used to select and identify fetal cells from maternal blood. In this study, a commonly used 3-step selection method is compared with selection directly from whole blood. Identification of fetal origin by XY FISH of male cells was also evaluated. METHODS: Maternal blood was drawn either before invasive chorion villus sampling (pre-CVS) or after (post-CVS) from women carrying a male fetus. Fetal cells were isolated either by density gradient centrifugation succeeded by CD45/CD14 depletion and CD71-positive selection from CD45/CD14-negative cells, or by CD71-positive selection directly from whole blood. The true origin of fetal cells recovered by the two methods was established by two rounds of XY chromosome FISH in reverse colors, in some instances combined with anti-zeta (zeta) or anti-zeta/anti-gamma (gamma) antibody staining. RESULTS: In blood samples taken post-CVS and enriched by CD71 selection directly from whole blood, fetal cells were identified with a frequency that was almost four orders of magnitude higher than in post-CVS samples enriched by the 3-step method. In blood samples taken pre-CVS and enriched by the 3-step procedure, no fetal cells were identified by reverse color FISH in 371 ml of blood. In similar samples enriched by CD71 selection on whole blood, two fetal cells were identified in 27 ml of blood. Rehybridization with X and Y chromosome probes with reverse colors was necessary to exclude false Y chromosome signals. Not all fetal cells identified by the presence of a true Y chromosome signal stained with anti-zeta antibody. CONCLUSIONS: Selection of fetal NRBCs from maternal blood by CD71-positive selection directly from whole blood is superior to density gradient centrifugation succeeded by CD45/CD14 depletion and CD71 selection of CD45/CD14-negative cells. Combining two markers for fetal origin is recommended for unambiguously identifying a cell as fetal.

<p>To benefit from the fluorescence-based automatic microscope (FLAME), we have adapted a PNA FISH technique to automatically determine telomere length in interphase nuclei. The method relies on the simultaneous acquisition of pan-telomeric signals and reference probe signals. We compared the quantitative figures to those for existing methods, i.e. Southern blot analysis and quantitative FISH (Q-FISH). Quantitative-FISH on interphase nuclei (IQ-FISH) allows the exact quantification of telomere length in interphase nuclei. Thus, this enables us to obtain not only exact information on the telomere length, but also morphological and topological details. The automatic measurement of large cell numbers allows the measurement of statistically relevant cell populations.</p>

In myelodysplastic syndromes (MDS), the karyotype is one of the most significant prognostic markers with profound impact on differential diagnosis and therapeutic decisions. In a retrospective study, we examined karyotypes of bone marrow specimens of an oligocentric cohort comprising 529 patients with MDS to address the question how many metaphases need to be analyzed to detect even small cell clones with an appropriate expenditure. We found a statistically significant difference of the frequency of normal karyotypes in the patient group with 19 or less analyzed metaphases compared to the group with 20 or more metaphases analyzed (56% versus 47%, p=0.041). Furthermore, we demonstrate that the analysis of 25 or more metaphases can further improve the sensitivity of karyotype analysis and leads to the identification of additional clinically relevant abnormal clones or subclones in a substantial proportion of patients. In summary, our data suggest the examination of at least 20 metaphases in MDS.