<p>The HER2/neu gene (also known as ERBB2) is located on chromosome 17 (q11.2-q12) and encodes a glycoprotein known to be a member of the epidermal growth factor receptor family. Clinically, the determination of its amplification status is of utmost importance, as 10-35% of invasive human breast carcinomas come along with HER2/neu overexpression, and treatment has to be adjusted accordingly. Here a method to analyze HER2 FISH samples with digital microscopy, using the slide scanning -platform Metafer PV (MetaSystems, Altlussheim, Germany), is presented. Metafer PV is a system for the automation of HER2/neu FISH assay analysis of samples hybridized with the Abbott(™) PathVysion(®) probe kit.</p>

A 12-year-old male with pre-B-cell acute lymphoblastic leukemia with cryptic BCR/ABL rearrangement underwent sex-mismatched allogeneic bone marrow transplantation (allo-BMT). Contradictory results were provided by various chimerism analyses 3 months later. Y-chromosome-specific quantitative polymerase chain reaction and sex chromosome-specific interphase fluorescence in situ hybridization (i-FISH) showed complete donor chimerism. Analysis of autosomal short tandem repeats (A-STR), BCR/ABL i-FISH test, and X-STR haplotype indicated relapse. Metaphase-FISH and combined BCR/ABL and sex chromosome-specific i-FISH patterns revealed loss of the Y-chromosome and duplication of the X-chromosome in the host cells. Sex chromosome changes after allo-BMT can cause significant difficulties in chimerism analysis.

<p>Although aneuploidy has many possible causes, it often results from underlying chromosomal instability (CIN) leading to an unstable karyotype with cell-to-cell variation and multiple subclones. To test for the presence of CIN in high hyperdiploid acute lymphoblastic leukemia (HeH ALL) at diagnosis, we investigated 20 patients (10 HeH ALL and 10 non-HeH ALL), using automated four-color interphase fluorescence in situ hybridization (I-FISH) with centromeric probes for chromosomes 4, 6, 10, and 17. In HeH ALL, the proportion of abnormal cells ranged from 36.3% to 92.4%, and a variety of aneuploid populations were identified. Compared with conventional cytogenetics, I-FISH revealed numerous additional clones, some of them very small. To investigate the nature and origin of this clonal heterogeneity, we determined average numerical CIN values for all four chromosomes together and for each chromosome and patient group. The CIN values in HeH ALL were relatively high (range, 22.2-44.7%), compared with those in non-HeH ALL (3.2-6.4\), thus accounting for the presence of numerical CIN in HeH ALL at diagnosis. We conclude that numerical CIN may be at the origin of the high level of clonal heterogeneity revealed by I-FISH in HeH ALL at presentation, which would corroborate the potential role of CIN in tumor pathogenesis.</p>

In forensic sciences, short tandem repeat (STR) analysis has become the prime tool for DNA-based identification of the donor(s) of biological stains and/or traces. Many traces, however, contain cells and, hence, DNA, from more than a single individual, giving rise to mixed genotypes and the subsequent difficulties in interpreting the results. An even more challenging situation occurs when cells of a victim are much more abundant than the cells of the perpetrator. Therefore, the forensic community seeks to improve cell-separation methods in order to generate single-donor cell populations from a mixed trace in order to facilitate DNA typing and identification. Laser capture microdissection (LCM) offers a valuable tool for precise separation of specific cells. This review summarises all possible forensic applications of LCM, gives an overview of the staining and detection options, including automated detection and retrieval of cells of interest, and reviews the DNA extraction protocols compatible with LCM of cells from forensic samples.

<p>Adaptive response (AR) is a term describing resistance to ionizing radiation-induced killing or formation of aberrant chromosomes that is mediated by pre-exposure to low ionizing radiation doses. The mechanism of AR remains elusive. Because cell killing and chromosome aberration formation derive from erroneous processing of DNA double-strand breaks (DSB), AR may reflect a modulation of DSB processing by nonhomologous end joining (NHEJ) or homologous recombination repair. Here, we use plasmid end-joining assays to quantify modulations induced by low ionizing radiation doses to NHEJ, the dominant pathway of DSB repair in higher eukaryotes, and investigate propagation of this response through medium transfer to nonirradiated bystander cells. Mouse embryo fibroblasts were conditioned with 10 to 1000 mGy and NHEJ quantified at different times thereafter by challenging with reporter plasmids containing a DSB. We show robust increases in NHEJ efficiency in mouse embryo fibroblasts exposed to ionizing radiation &gt;100 mGy, irrespective of reporter plasmid used. Human tumor cells also show AR of similar magnitude that is compromised by caffeine, an inhibitor of DNA damage signaling acting by inhibiting ATM, ATR, and DNA-PKcs. Growth medium from pre-irradiated cells induces a caffeine-sensitive AR in nonirradiated cells, similar in magnitude to that seen in irradiated cells. In bystander cells, γH2AX foci are specifically detected in late S-G(2) phase and are associated with Rad51 foci that signify the function of homologous recombination repair, possibly on DNA replication-mediated DSBs. The results point to enhanced NHEJ as a mechanism of AR and suggest that AR may be transmitted to bystander cells through factors generating replication-mediated DSBs. Cancer Res; 70(21); 8498-506. ©2010 AACR.</p>

PURPOSE: The backup pathway of nonhomologous end joining (B-NHEJ) enables cells to process DNA double-strand breaks (DSBs) when the DNA-PK-dependent pathway of NHEJ (D-NHEJ) is compromised. Our previous results show marked reduction in the activity of B-NHEJ when LIG4(-/-) mouse embryo fibroblasts (MEFs) cease to grow and enter a plateau phase. The dependence of B-NHEJ on growth state is substantially stronger than that of D-NHEJ and points to regulatory mechanisms or processing determinants that require elucidation. Because the different D-NHEJ mutants show phenotypes distinct in their details, it is necessary to characterize the dependence of their DSB repair capacity on growth state and to explore species-specific responses. METHODS AND MATERIALS: DSB repair was measured in cells of different genetic background from various species using pulsed-field gel electrophoresis, or the formation of ã-H2AX foci, at different stages of growth. RESULTS: Using pulsed-field gel electrophoresis, we report a marked reduction of B-NHEJ during the plateau phase of growth in KU and XRCC4, mouse or Chinese hamster, mutants. Notably, this reduction is only marginal in DNA-PKcs-deficient cells. However, reduced B-NHEJ is also observed in repair proficient, plateau-phase cells after treatment with DNA-PK inhibitors. The reduction of B-NHEJ activity in the plateau phase of growth does not derive from the reduced expression of participating proteins, is detectable by ã-H2AX foci analysis, and leads to enhanced cell killing. CONCLUSIONS: These results further document the marked dependence on growth state of an essential DSB repair pathway and show the general nature of the effect. Molecular characterization of the mechanism underlying this response will help to optimize the administration of DNA repair inhibitors as adjuvants in radiation therapy.

<p>Ionizing radiation (IR) induces a variety of DNA lesions among which DNA double-strand breaks (DSBs) are the biologically most significant. It is currently unclear if DSB repair is equally efficient after low and high doses. Here, we use gamma-H2AX, phospho-ATM (pATM), and 53BP1 foci analysis to monitor DSB repair. We show, consistent with a previous study, that the kinetics of gamma-H2AX and pATM foci loss in confluent primary human fibroblasts are substantially compromised after doses of 10 mGy and lower. Following 2.5 mGy, cells fail to show any foci loss. Strikingly, cells pretreated with 10 microM H(2)O(2) efficiently remove all gamma-H2AX foci induced by 10 mGy. At the concentration used, H(2)O(2) produces single-strand breaks and base damages via the generation of oxygen radicals but no DSBs. Moreover, 10 microM H(2)O(2) up-regulates a set of genes that is also up-regulated after high (200 mGy) but not after low (10 mGy) radiation doses. This suggests that low radical levels induce a response that is required for the repair of radiation-induced DSBs when the radiation damage is too low to cause the induction itself. To address the in vivo significance of this finding, we established gamma-H2AX and 53BP1 foci analysis in various mouse tissues. Although mice irradiated with 100 mGy or 1 Gy show efficient gamma-H2AX and 53BP1 foci removal during 24 h post-IR, barely any foci loss was observed after 10 mGy. Our data suggest that the cellular response to DSBs is substantially different for low vs. high radiation doses.</p>

The capital city of Prague is one of the most polluted areas of the Czech Republic. The impact of air pollution on the level of chromosomal aberrations was systematically studied: analyses were performed using fluorescence in situ hybridization (FISH) with whole-chromosome painting for chromosomes #1 and #4. In the present study, we analyzed the levels of stable (one-way and two-way translocations) and unstable (acentric fragments) chromosomal aberrations in 42 mothers living in Prague and in their newborns. The average age of the mothers was 29 years (range, 20-40 years). Blood samples were collected from October 2007 to February 2008. The average levels of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) and benzo[a]pyrene (B[a]P) in respirable particles (PM2.5), as determined by stationary monitoring, were 21.0+/-12.3ng/m(3) and 2.9+/-1.8ng/m(3), respectively. We did not observe any effect of either c-PAH or B[a]P exposure on the genomic frequency of translocations (per 100 cells, F(G)/100) in either group due to their similar exposure during the winter months. The mean values of F(G)/100 representing stable aberrations were 0.09+/-0.13 vs 0.80+/-0.79 (p

Cytosine arabinoside (a nucleoside analogue that inhibits the gap-filling step of excision repair), vinblastine (an aneugen that inhibits tubulin polymerisation), 5-fluorouracil (a nucleoside analogue with a steep response profile), and 2-aminoanthracene (a metabolism-dependent reference genotoxin) were tested in the in vitro micronucleus assay with L5178Y mouse lymphoma cells, without cytokinesis block. The four chemicals were independently evaluated in two Sanofi Aventis laboratories, one of which used an image analyser to score micronuclei, while the other scored micronucleated cells manually. Very similar results were obtained in the two laboratories, highlighting the robustness of the assay. The four test chemicals induced significant increases in the incidence of micronucleated cells at concentrations that produced no more than a 55+/-5\% reduction in survival growth, as measured with the three parameters recommended in the draft OECD Test Guideline on In Vitro Mammalian Cell Micronucleus Test (MNvit) for chemical testing, namely the relative increase in cell counts, relative population doubling, and the relative cell count. These results support the premise that the relative increase in cell counts and relative population doubling, that take into account both cell death and cytostasis, are appropriate measures of survival growth reduction in the in vitro micronucleus test conducted in the absence of cytokinesis block, as recommended in MNvit.

<p>Recently a high-throughput version of the comet assay was developed using a special 96-well multichamber plate (MCP) [1]. In this version, the electrophoresis is performed directly on the MCP, which makes transferring of cells to microscope slides unnecessary. In order to facilitate the scoring procedure we adapted an automated slide-scanning system (Metafer MetaCyte with CometScan) to enable unattended analysis of comets on the MCP. The results of the system were compared with the data obtained with two interactive comet-assay analysis systems. For induction of DNA damage in human fibroblasts methylmethane sulfonate (MMS) or H(2)O(2) was used. The three systems revealed similar, concentration-dependent results for all parameters tested: tail moment (tm), % DNA-in-tail and olive tail moment. Near the detection limit of 5-6% DNA-in-tail a significant difference with the untreated control was obtained by use of four parallel samples (p=0.01). With the newly developed automated analysis system, the evaluation of either 50 or 100 comets yielded similar standard errors for either treatment with MMS or H(2)O(2), thus showing that the method is suitable to reveal the crucial low-dose effects with high precision. The results also show that the time needed for automated evaluation of comets on the MCP was reduced by a factor of 10 when compared with the time required for interactive evaluation. In summary, the high-throughput version of the comet assay combined with the automated evaluating system increased the output by a factor up to 180 compared with the standard method.</p>

Both sporadic and familial Alzheimer's disease (AD) patients exhibit increased chromosome aneuploidy, particularly trisomy 21, in neurons and other cells. Significantly, trisomy 21/Down syndrome patients develop early onset AD pathology. We investigated the mechanism underlying mosaic chromosome aneuploidy in AD and report that FAD mutations in the Alzheimer Amyloid Precursor Protein gene, APP, induce chromosome mis-segregation and aneuploidy in transgenic mice and in transfected cells. Furthermore, adding synthetic Abeta peptide, the pathogenic product of APP, to cultured cells causes rapid and robust chromosome mis-segregation leading to aneuploid, including trisomy 21, daughters, which is prevented by LiCl addition or Ca(2+) chelation and is replicated in tau KO cells, implicating GSK-3beta, calpain, and Tau-dependent microtubule transport in the aneugenic activity of Abeta. Furthermore, APP KO cells are resistant to the aneugenic activity of Abeta, as they have been shown previously to be resistant to Abeta-induced tau phosphorylation and cell toxicity. These results indicate that Abeta-induced microtubule dysfunction leads to aneuploid neurons and may thereby contribute to the pathogenesis of AD.

<p>DNA-, RNA-, and cell-based methods provide different biologic information for determining the presence of minimal residual disease (MRD). We monitored the responses of patients with pediatric acute lymphoblastic leukemia (pALL) using DNA markers, TEL/AML1 expression, and scanning fluorescence microscopy (SFM). Using SFM, 36% of patients exhibited 1.5-3.1 log and 2.9-4.2 log higher MRD levels compared with those based on DNA and RNA markers, respectively. CD10+ ancestor cells with germline antigen receptors, but silent TEL/AML1 expression, may reside in the lymphoid stem cell compartment of treated t(12;21)-positive patients and might act as a potential source of cells for late relapses.</p>

PURPOSE: In case of a large-scale radiation accident when hundreds of people may be exposed, it is important to distinguish the severely exposed individuals (> or =1 gray), who require early medical treatment, from those less exposed. The aim of our study was to develop a quick population triage method based on automated micronucleus (MN) scoring. MATERIALS AND METHODS: Using the MN software module developed by MetaSystems specifically for the Metafer4 platform, about 60 blood samples can be scored in one day. Standard dose response curves were determined for manual and automated MN scoring. RESULTS: The automated MN assay results were closely correlated with MN yields obtained with the manual procedure. A dose of 1 Gy can be estimated with an uncertainty of 0.2 Gy. Corrections for false positives and false negatives by visual inspection of the image gallery did not result in an improved accuracy or reproducibility. To test the automated MN assay in a multicenter setting, an inter-laboratory comparison was performed whereby irradiated blood samples were processed in Ghent University (Belgium) and BfS (Bundesamt fuer Strahlenschutz; Germany). Both laboratories obtained comparable results. CONCLUSIONS: These results confirm the efficacy of the automated MN assay for fast population triage in a multicenter setting, in the case of large radiation accidents.

Digital object identifier (DOI): 10.3109/09553000903264481

Dorsal root ganglia (DRG)-neurons are commonly characterized immunocytochemically. Cells are mostly grouped by the experimenter's eye as #marker-positive# and #marker-negative# according to their immunofluorescence intensity. Classification criteria remain largely undefined. Overcoming this shortfall, we established a quantitative automated microscopy (QuAM) for a defined and multiparametric analysis of adherent heterogeneous primary neurons on a single cell base.The growth factors NGF, GDNF and EGF activate the MAP-kinase Erk1/2 via receptor tyrosine kinase signalling. NGF and GDNF are established factors in regeneration and sensitization of nociceptive neurons. If also the tissue regenerating growth factor, EGF, influences nociceptors is so far unknown. We asked, if EGF can act on nociceptors, and if QuAM can elucidate differences between NGF, GDNF and EGF induced Erk1/2 activation kinetics. Finally, we evaluated, if the investigation of one signalling component allows prediction of the behavioral response to a reagent not tested on nociceptors such as EGF.We established a software-based neuron identification, described quantitatively DRG-neuron heterogeneity and correlated measured sample sizes and corresponding assay sensitivity. Analysing more than 70,000 individual neurons we defined neuronal subgroups based on differential Erk1/2 activation status in sensory neurons. Baseline activity levels varied strongly already in untreated neurons. NGF and GDNF subgroup responsiveness correlated with their subgroup specificity on IB4(+)- and IB4(-)-neurons, respectively. We confirmed expression of EGF-receptors in all sensory neurons. EGF treatment induced STAT3 translocation into the nucleus. Nevertheless, we could not detect any EGF induced Erk1/2 phosphorylation. Accordingly, intradermal injection of EGF resulted in a fundamentally different outcome than NGF/GDNF. EGF did not induce mechanical hyperalgesia, but blocked PGE2-induced sensitization.QuAM is a suitable if not necessary tool to analyze activation of endogenous signalling in heterogeneous cultures. NGF, GDNF and EGF stimulation of DRG-neurons shows differential Erk1/2 activation responses and a corresponding differential behavioral phenotype. Thus, in addition to expression-markers also signalling-activity can be taken for functional subgroup differentiation and as predictor of behavioral outcome. The anti-nociceptive function of EGF is an intriguing result in the context of tissue damage but also for understanding pain resulting from EGF-receptor block during cancer therapy.

<p>The simultaneous detection of protein expression and gene copy number changes in patient samples, like paraffin-embedded tissue sections, is challenging since the procedures of immunohistochemistry (IHC) and Fluorescence in situ Hybridization (FISH) negatively influence each other which often results in suboptimal staining. Therefore, we developed a novel automated algorithm based on relocation which allows subsequent detection of protein content and gene copy number changes within the same cell. Paraffin-embedded tissue sections of colorectal cancers were stained for CD133 expression. IHC images were acquired and image coordinates recorded. Slides were subsequently hybridized with fluorescently labeled DNA probes. FISH images were taken at the previously recorded positions allowing for direct comparison of protein expression and gene copy number signals within the same cells/tissue areas. Relocation, acquisition of the IHC and FISH images, and enumeration of FISH signals in the immunophenotyped tumour areas were done in an automated fashion. Automated FISH analysis was performed on 13 different colon cancer samples that had been stained for CD133; each sample was scored for MYC, ZNF217 and Chromosome 6 in CD133 positive and negative glands. From the 13 cases four (31%) showed amplification for the MYC oncogene and seven of 13 (54%) cases were amplified for ZNF217. There was no significant difference between CD133 positive tumour and CD133 negative tumour cells. The technique and algorithm presented here enables an easy and reproducible combination of IHC and FISH based on a novel automated algorithm using relocation and automated spot counting.</p>

<p>Human epidermal growth factor receptor 2 (HER2)/neu, topoisomerase II alpha (TOP2A), and polysomy 17 may predict tumor responsiveness to doxorubicin (DOX) therapy.We identified neoadjuvant DOX/cyclophosphamide treated breast cancer patients in our registry from 1997 to 2008 with sufficient tissue for testing (n = 34). Fluorescence in situ hybridization (FISH) testing was done on deparaffinized tissue sections pretreated using vendor's standard protocol modification, and incubated with US Food and Drug Administration approved Abbott Diagnostics Vysis PathVysion™ probe set, including Spectrum-Green-conjugated probe to a-satellite DNA located at the centromere of chromosome 17 (17p11.1-q11.1) and a Spectrum-Orange-conjugated probe to the TOP2A gene. Morphometric analysis was performed using a MetaSystems image analysis system. Manual counting was performed on all samples in which autofluorescence and/or artifact prevented the counting of sufficient numbers of cells. A ratio &gt;2.0 was considered positive for TOP2A amplification. Polysomy 17 (PS17) presence was defined as signals of ≥2.5. Outcomes were pathological complete response (pCR), partial response (PR), and nonresponse (NR). Of 34 patients tested, one was TOP2A amplified (hormone receptor negative/HER2 negative, partial responder). The subset of TOP2A nonamplified, HER2 negative, and PS17 absent (n = 23) patients had treatment response: pCR = 2 (9%), PR = 14 (61%), and NR = 7 (30%). Including the two PS17 present and HER2-positive patients (n = 33), 76% of TOP2A nonamplified patients had pCR or PR. We observed substantial treatment response in patients lacking three postulated predictors that would be difficult to attribute to cyclophosphamide alone. Patients who are HER2 negative and lack TOP2A amplification and PS17 should not be excluded from receiving DOX-containing regimens.</p>

<p>A better understanding of the underlying mechanisms of DNA repair after exposure to ionizing radiation represents a research priority aimed at improving the outcome of clinical radiotherapy. Because of the close association with DNA double strand break (DSB) repair, phosphorylation of the histone H2AX protein (gamma-H2AX), quantified by immunodetection, has recently been used as a method to study DSB induction and repair at low and clinically relevant radiation doses. However, the lack of consistency in literature points to the need to further validate the role of H2AX phosphorylation in DSB repair and the use of this technique to determine intrinsic radiosensitivity. In the present study we used human mammary epithelial MCF10A cells, characterized by a radiosensitive phenotype due to reduced levels of the Ku70 and Ku80 repair proteins, and investigated whether this repair-deficient cell line displays differences in the phosphorylation pattern of H2AX protein compared to repair-proficient MCF10A cells. This was established by measuring formation and disappearance of gamma-H2AX foci after irradiating synchronized cell populations with (60)Co alpha-rays. Our results show statistically significant differences in the number of gamma-H2AX foci between the repair-deficient and -proficient cell line, with a higher amount of gamma-H2AX foci present at early times post-irradiation in the Ku-deficient cell line. However, the disappearance of those differences at later post-irradiation times questions the use of this assay to determine intrinsic radiosensitivity, especially in a clinical setting.</p>

The international radiation biodosimetry community has recently been engaged in activities focused on establishing cooperative networks for biodosimetric triage for radiation emergency scenarios involving mass casualties. To this end, there have been several recent publications in the literature regarding the potential for shared scoring in such an accident or incident. We present details from a medical irradiation case where two independently validated laboratories found very different yields of dicentric chromosome aberrations. The potential reasons for this disparity are discussed, and the actual reason is identified as being the partial-body nature of the radiation exposure combined with differing criteria for metaphase selection. In the context of the recent networking activity, this report is intended to highlight the fact that shared scoring may produce inconsistencies and that further validation of the scoring protocols and experimental techniques may be required before the networks are prepared to deal satisfactorily with a radiological or nuclear emergency. Also, the findings presented here clearly demonstrate the limitations of the dicentric assay for estimating radiation doses after partial-body exposures and bring into question the usefulness of rapid #triage##mode# scoring in such exposure scenarios.

Gene fusions play a critical role in cancer progression. The mechanisms underlying their genesis and cell type specificity are not well understood. About 50\% of human prostate cancers display a gene fusion involving the 5' untranslated region of TMPRSS2, an androgen-regulated gene, and the protein-coding sequences of ERG, which encodes an erythroblast transformation-specific (ETS) transcription factor. By studying human prostate cancer cells with fluorescence in situ hybridization, we show that androgen signaling induces proximity of the TMPRSS2 and ERG genomic loci, both located on chromosome 21q22.2. Subsequent exposure of the cells to gamma irradiation, which causes DNA double-strand breaks, facilitates the formation of the TMPRSS2-ERG gene fusion. These results may help explain why TMPRSS2-ERG fusions are restricted to the prostate, which is dependent on androgen signaling.

<p>Gene fusions play a critical role in cancer progression. The mechanisms underlying their genesis and cell type specificity are not well understood. About 50% of human prostate cancers display a gene fusion involving the 5' untranslated region of TMPRSS2, an androgen-regulated gene, and the protein-coding sequences of ERG, which encodes an ETS transcription factor. Studying human prostate cancer cells by fluorescence in situ hybridization, we show that androgen signaling induces proximity of the TMPRSS2 and ERG genomic loci, both located on chromosome 21q22.2. Subsequent exposure of the cells to gamma-irradiation, which causes DNA double strand breaks, facilitates the formation of the TMPRSS2-ERG gene fusion. These results may help explain why TMPRSS2-ERG fusions are restricted to the prostate, which is dependent on androgen signaling.</p>