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.

<p>Aloysia triphylla a perennial, bushy plant originally from South America has long been used in traditional medicine. Its aqueous extract contains considerable amounts of polyphenolic compounds, namely flavonoids and phenolic acids. In view of the interest in natural phenolic compounds as antioxidant in preventive medicine, this study was undertaken to investigate the chemoprotective effects of cedron leaves infusion against the genetic damage induced by acrylamide (AA) by using the alkaline version of the comet assay technique. Mice were separated in nine groups (eight animals each): (I) untreated, (II) negative control, (III) treated with infusion of cedron leaves 5%, 20 days twice a day, (IV) treated with AA (5 mg/kg b.w.), (V) treated with AA (20 mg/kg b.w.), (VI) treated with AA (30 mg/kg b.w.), (VII) treated with AA (50 mg/kg b.w.), (VIII) pretreated with infusion and treated with AA (50 mg/kg b.w.) and (IX) positive control (cyclophosphamide, 20 mg/kg b.w.). Three hundred blast cells were digitally evaluated per animal from three different slides (100 each). Media of tail moment (TM) values were analyzed by ANOVA test. No statistical differences (p&gt;0.05) were found between untreated animals, negative control and infusion-treated mice. A single dose of AA-induced genetic damage as revealed by a statistically significant increase in TM values (p&lt;0.01). Pretreatment with infusion prior to AA injection significantly reduces the capacity of AA to induce genetic damage. In these conditions, tail moments values did not differ from data obtained in negative control (p&gt;0.05) and exhibit statistical differences from animals treated only with AA (p&lt;0.01). Cell viability was at least 90% in all cases as measured by the trypan blue exclusion method. The ferric reducing ability of plasma (FRAP) method reveals that the plasma of infusion-treated mice has a significantly higher antioxidant capacity than plasma from controls (p&lt;0.01). The results suggest that the infusion could exerts an in vivo chemo protective action, probably due to its scavenging potency towards free radicals.</p>

Chromosome aberration analysis in astronauts has been used to provide direct, biologically motivated estimates of equivalent doses and risk associated to cosmic radiation exposure during space flight. However, the past studies concentrated on measurements of dicentrics and translocations, while chromosome intrachanges (inversions) have never been measured in astronauts' samples. Recent data reported in the literature suggest that densely ionizing radiation can induce a large fraction of intrachanges, thus leading to the suspicion that interchanges grossly underestimate the cosmic radiation-induced cytogenetic damage in astronauts. We have analyzed peripheral blood lymphocytes from 11 astronauts involved in short- or long-term space flights in low-Earth orbit using high-resolution multicolor banding to assess the frequency of intrachromosomal exchanges in both pre- and post-flight samples. We did not detect any inversions in chromosome 5 from a total of 2800 cells in astronauts' blood. In addition, no complex type exchanges were found in a total of 3590 astronauts' lymphocytes analyzed by multifluor fluorescence in situ hybridisation. We conclude that, within the statistical power of this study, the analysis of interchanges for biological dosimetry in astronauts does not significantly underestimate the space radiation-induced cytogenetic damage, and complex-type exchanges or intrachanges have limited practical use for biodosimetry at very low doses.

SUMMARY: BACKGROUND: Secretory carcinoma (SC) of the breast is a rare and indolent tumor. Although originally described in children, it is now known to occur in adults of both sexes. Recently, the tumor was associated with the ETV6-NTRK3 gene translocation. CASE PRESENTATION: A 52-year-old male was diagnosed with secretory breast carcinoma and underwent a modified radical mastectomy. At 18 months the tumor recurred at the chest wall and the patient developed lung metastases. He was treated concurrently with radiation and chemotherapy without response. His tumor showed the ETV6-NTRK3 translocation as demonstrated by fluorescent in situ hybridization (FISH). CONCLUSION: SC is a rare slow-growing tumor best treated surgically. There are insufficient data to support the use of adjuvant radiation or chemotherapy. Its association with the ETV6-NTRK3 fusion gene gives some clues for the better understanding of this neoplasm and eventually, the development of specific therapies.

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.

The X-ray repair cross complementing 1 (XRCC1) protein is required for viability and efficient repair of DNA single-strand breaks (SSBs) in rodents. XRCC1-deficient mouse or hamster cells are hypersensitive to DNA damaging agents generating SSBs and display genetic instability after such DNA damage. The presence of certain polymorphisms in the human XRCC1 gene has been associated with altered cancer risk, but the role of XRCC1 in SSB repair (SSBR) in human cells is poorly defined. To elucidate this role, we used RNA interference to modulate XRCC1 protein levels in human cell lines. A reduction in XRCC1 protein levels resulted in decreased SSBR capacity as measured by the comet assay and intracellular NAD(P)H levels, hypersensitivity to the cell killing effects of the DNA damaging agents methyl methanesulfonate (MMS), hydrogen peroxide and ionizing radiation and enhanced formation of micronuclei following exposure to MMS. Lowered XRCC1 protein levels were also associated with a significant delay in S-phase progression after exposure to MMS. These data clearly demonstrate that XRCC1 is required for efficient SSBR and genomic stability in human cells.

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.

Long-lived, sensitive, and specific biomarkers of particular mutagenic agents are much sought after and potentially have broad applications in the fields of cancer biology, epidemiology, and prevention. Many clastogens induce a spectrum of chromosome aberrations, and some of them can be exploited as biomarkers of exposure. Densely ionizing radiation, for example, alpha particle radiation (from radon or plutonium) and neutron radiation, preferentially induces complex chromosome aberrations, which can be detected by the 24-color multifluor fluorescence in situ hybridization (mFISH) technique. We report the detection and quantification of stable complex chromosome aberrations in lymphocytes of healthy former nuclear-weapons workers, who were exposed many years ago to plutonium, gamma rays, or both, at the Mayak weapons complex in Russia. We analyzed peripheral-blood lymphocytes from these individuals for the presence of persistent complex chromosome aberrations. A significantly elevated frequency of complex chromosome translocations was detected in the highly exposed plutonium workers but not in the group exposed only to high doses of gamma radiation. No such differences were found for simple chromosomal aberrations. The results suggest that stable complex chromosomal translocations represent a long-lived, quantitative, low-background biomarker of densely ionizing radiation for human populations exposed many years ago.

<p>Genomic fingerprints of mutagenic agents would have wide applications in the field of cancer biology, epidemiology and prevention. The differential spectra of chromosomal aberrations induced by different clastogens suggest that ratios of specific aberrations can be exploited as biomarkers of carcinogen exposure. We have tested this hypothesis using the novel technique of multicolor banding in situ hybridization (mBAND) in human peripheral blood lymphocytes exposed in vitro to X rays, neutrons, heavy ions, or the restriction endonuclease AluI. In the heavy-ion-irradiated cells, we further analyzed aberrations in chromosome 5 using multicolor FISH (mFISH). Contrary to the expectations of biophysical models, our results do not support the use of the ratios of inter-/intrachromosomal exchanges or intra-/interarm intrachanges as fingerprints of exposure to densely ionizing radiation. However, our data point to measurable differences in the ratio of complex/simple interchanges after exposure to different clastogens. These data should be considered in current biophysical models of radiation action in living cells.</p>

The aim of the present study was to estimate the genotoxicity of desflurane, applied as a volatile anaesthetic. The potential genotoxicity was determined by the comet assay as the extent of DNA fragmentation in human peripheral blood lymphocytes in vitro. The comet assay detects DNA strand breaks induced directly by genotoxic agents as well as DNA fragmentation due to cell death. Another anaesthetic, halothane, already proved to be a genotoxic agent, was used as a positive control. Both analysed drugs were capable of increasing DNA migration in a dose-dependent manner under experimental conditions applied. The results of the study demonstrated that the genotoxicity of desflurane was comparable with that of halothane. However, considering the pharmacodynamics of both drugs, the genotoxic activity of desflurane may be connected with a less harmful effect on the exposed patients or medical staff.

OBJECTIVE: To study chromosome analysis by comparative genomic hybridization (CGH) compared with the conventional technique in early amniocentesis. MATERIAL AND METHOD: Cross-sectional descriptive study design was performed in 32 singleton pregnant women with gestational age between 12-15 weeks. Transabdominal amniocentesis was carried out under ultrasound guidance. The amniotic fluid samples were simultaneously investigated using CGH and the conventional cytogenetics study as a gold standard. RESULTS: Amniocentesis were done for advanced maternal age in all cases. The mean maternal age was 35.8 years (35-42 years). The mean gestational age was 13.7 weeks (12-15 weeks). The chromosome analysis by CGH technique of uncultured amniocyte showed 17 normal female chromosomes (53.1%) and 15 normal male chromosomes (46.9%). This finding was the same as the conventional cytogenetics method. The mean duration of the CGH method was 6 days and that of the conventional cytogenetics method was 13.7 days (10-23 days). CONCLUSION: The CGH technique is a reliable technique for a rapid prenatal diagnosis of chromosome study in early gestation.

PURPOSE: Uveal melanoma is a highly malignant disease with a mortality rate of 50% at 10 to 15 years. Previous studies have shown that chromosomal changes are associated with decreased survival of the patient. However, in these studies the small number of tumors analyzed did not allow robust statistical analysis. In the present study, the independent numerical changes in chromosomes 1, 3, 6, and 8 on disease-free survival (DFS) was assessed in a large series of patients with uveal melanoma. METHODS: One hundred twenty tumors from patients with uveal melanoma were analyzed for numerical changes in chromosomes 1, 3, 6, and 8, with cytogenetic analysis, fluorescent in situ hybridization, and/or comparative genomic hybridization. Data were correlated with disease outcome in univariate and multivariate analyses, by Kaplan-Meier and Cox regression analyses. RESULTS: At a mean follow-up time of 45 months, 42 patients had died or had metastatic disease. In the univariate analysis, loss of chromosome 3, gain of 8q, largest tumor diameter, or the presence of epithelioid cells was associated with a decreased DFS. In the multivariate analysis, the effect of monosomy 3 on survival was largely modified by changes in 1p36. Regarding all chromosomal changes, only the concurrent loss of the short arm of chromosome 1 and all of chromosome 3 was an independent prognostic parameter for disease-free survival (P < 0.001). CONCLUSIONS: In uveal melanoma, concurrent loss of the short arm of chromosome 1 and all of chromosome 3 is an independent predictor of decreased DFS.

<p>Recurrent chromosome breakpoints in tumour cells may point to cancer genes, but not many have been molecularly characterised. We have used multicolour-banding fluorescence in situ hybridisation (mbanding-FISH) on breast tumour cell lines to identify regions of chromosome break created by inversions, duplications, insertions and translocations on chromosomes 1, 5, 8, 12 and 17. We delineate a total of 136 regions of break, some of them occurring with high frequency. We further describe two examples of dual-colour FISH characterisation of breakpoints, which target the 1p36 and 5p11-12 regions. Both breaks involve genes whose function is unknown to date. The mbanding-FISH strategy constitutes an efficient first step in the search for potential cancer genes.</p>

Conventional mutation-selection theories have failed to explain (i) how cancer cells become spontaneously resistant against cytotoxic drugs at rates of up to 10(-3) per cell generation, orders higher than gene mutation, even in cancer cells; (ii) why resistance far exceeds a challenging drug-a state termed multidrug resistance; (iii) why resistance is associated with chromosomal alterations and proportional to their numbers; and (iv) why resistance is totally dependent on aneuploidy. We propose here that cancer-specific aneuploidy generates drug resistance via chromosomal alterations. According to this mechanism, aneuploidy varies the numbers and structures of chromosomes automatically, because it corrupts the many teams of proteins that segregate, synthesize, and repair chromosomes. Aneuploidy is thus a steady source of chromosomal variation from which, in classical Darwinian terms, resistance-specific aneusomies are selected in the presence of chemotherapeutic drugs. Some of the thousands of unselected genes that hitchhike with resistance-specific aneusomies can thus generate multidrug resistance. To test this hypothesis, we determined the rates of chromosomal alterations in clonal cultures of human breast and colon cancer lines by dividing the fraction of nonclonal karyotypes by the number of generations of the clone. These rates were about 10(-2) per cell generation, orders higher than mutation. Chromosome numbers and structures were determined in metaphases hybridized with color-coded chromosome-specific DNA probes. Further, we tested puromycin-resistant subclones of these lines for resistance-specific aneusomies. Resistant subclones differed from parental lines in four to seven specific aneusomies, of which different subclones shared some. The degree of resistance was roughly proportional to the number of these aneusomies. Thus, aneuploidy is the primary cause of the high rates and wide ranges of drug resistance in cancer cells.

<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>

PURPOSE: To investigate if deviations from DNA-proportional distribution of radiation-induced chromosomal aberrations are individually variable. MATERIALS AND METHODS: Peripheral blood lymphocytes were collected from seven healthy donors and exposed to different doses of gamma rays. Chromosomes 2, 8 and 14 were painted in different colors and aberrations scored with the help of an image-analysis system. RESULTS: Chromosome 2 was generally less sensitive than expected on the basis of DNA-proportional distribution and the extent of inter-donor variability was minimal. A higher than expected frequency of aberrations was found in chromosome 14 of five donors, while a higher than expected frequency of aberrations was found in chromosome 8 of two donors. CONCLUSIONS: Inter-donor variability may explain some of the controversies regarding the inter-chromosomal distribution of radiation-induced aberrations.

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.

<p>Standard cytomorphological examination of bone marrow (BM) aspirates does not appear to be sensitive enough to detect single neuroblastoma cells. The SIOPEN Neuroblastoma Bone Marrow Committee developed a sensitive and reproducible anti-GD2 immunocytochemical assay and introduced morphological and immunocytological criteria for the interpretation of results. Fixed cytospins were incubated with a commercially available anti-GD2 monoclonal antibody and an APAAP kit. Cells fulfilling all morphological and immunocytological criteria were called criteria-positive cells (CPCs). Not convincingly interpretable cells fulfilled some, but not all, criteria, and negative cells displayed only exclusion criteria. The genetic profile of doubtful cells was checked by fluorescence in situ hybridization. Ideally, 3 x 10(6) cells were analyzed to reach a 95% probability of detecting one tumor cell in 1 x 10(6) mononuclear cells. Four quality control rounds were organized to validate the method. A total of 111 quality control samples were analyzed. Two main improvements were achieved: in discordant cases, the range between the lowest and highest reported result was reduced by half, and discordant results were only found in samples with less than 10 CPCs per 1 x 10(6). This article describes the first internationally standardized protocol to detect and quantify rare neuroblastoma cells by immunocytochemistry. This method is an indispensable tool for multicenter studies evaluating the clinical significance of minimal residual disease in neuroblastoma.</p>

<p>Jurkat cells in culture were exposed to oxidative stress in the form of continuously generated hydrogen peroxide, obtained by the addition of glucose oxidase to the medium. This treatment induced a rapid, dose-dependent increase in the ICIP (intracellular calcein-chelatable iron pool). Early destabilization of lysosomal membranes and subsequent nuclear DNA strand breaks were also observed, as evaluated by the Acridine Orange relocation test and the comet assay respectively. Somewhat later, these effects were followed by a lowered mitochondrial membrane potential, with release of cytochrome c and apoptosis-inducing factor. These events were all prevented if cells were pretreated with the potent iron chelator DFO (desferrioxamine) for a period of time (2-3 h) long enough to allow the drug to reach the lysosomal compartment following fluid-phase endocytosis. The hydrophilic calcein, a cleavage product of calcein acetoxymethyl ester following the action of cytosolic esterases, obviously does not penetrate intact lysosomal membranes, thus explaining why ICIP increased dramatically following lysosomal rupture. The rapid decrease in ICIP after addition of DFO to the medium suggests draining of cytosolic iron to the medium, rather than penetration of DFO through the plasma membrane. Most importantly, these observations directly connect oxidative stress and resultant DNA damage with lysosomal rupture and the release of redox-active iron into the cytosol and, apparently, the nucleus.</p>

Synovial sarcomas (SSs) account for 5% of soft tissue tumors and carry a balanced translocation t(X;18)(p11.2;q11.2), detectable in over 90% of cases. This translocation brings together portions of two genes: SYT and SSX. Detecting interruption of the SYT gene on chromosome 18 would be useful as a diagnostic tool. We describe a scoring method to detect disruption of SYT with breakapart probe fluorescence in situ hybridization (FISH) and the application of this method for identification of SS within a sarcoma tissue microarray. After optimization, SYT disruption was identified in 22 of 23 (96%) of known SS tumor samples but was not in 23 of 23 (100%) of non-SS sarcoma samples. Ten of 11 (91%) blinded test SS tumor samples were also correctly identified. For comparison, commercially available FISH and chromogenic in situ hybridization (CISH) probes were tested. The commercial FISH probes identified SYT disruption in 81% of the SS tumor samples but in none of the non-SS samples. The CISH probes produced signals too weak to interpret. The use of breakapart FISH probes is a relatively quick procedure for detection of synovial sarcoma translocations and can be applied to archival specimens in tissue microarrays.