OBJECTIVE—Diabetes is associated with an increased risk of death in women. Oxidative stress due to chronic hyperglycemia leads to the generation of reactive oxygen species and loss of chromosomal integrity. To clarify whether diabetes is a premature aging syndrome, we determined telomere erosion dynamics and occurrence of structural chromosomal aberrations in women of the Ludwigshafen Risk and Cardiovascular Health (LURIC) Study. RESEARCH DESIGN AND METHODS—Telomere lengths and karyotypes were examined in peripheral blood mononuclear cells. Regarding these parameters, surviving and deceased type 2 diabetic women of the LURIC study were compared with nondiabetic LURIC women with or without coronary heart disease and with healthy female control subjects. RESULTS—Significantly enhanced telomere attrition was seen in all LURIC subjects compared with healthy control subjects. Although the average telomere-length loss is equivalent to well >10 years of healthy aging, telomere erosion was not associated with outcome within the LURIC cohort. However, strikingly high numbers of stable chromosomal aberrations were found in type 2 diabetic women but not in LURIC disease control subjects or in healthy individuals. Furthermore, within the younger age- groups, deceased type 2 diabetes patients had significantly more marker chromosomes than the surviving type 2 diabetic patients. CONCLUSIONS—All women at high risk for cardiovascular death have accelerated telomere erosion, not caused by type 2 diabetes per se but likely linked to other risk factors, including dyslipidemia. By contrast, the occurrence of marker chromosomes is associated with type 2 diabetes and is a novel risk factor for type 2 diabetes–related early death. Type 2 diabetes is characterized by increased morbidity and all-cause mortality (1,2). The combination of excess caloric intake and reduced physical activity leading to obesity, dyslipidemia, and hypertension increases the risk for diabetes and coronary heart disease (CHD). Recent data show that among diabetic men, the mortality rate has decreased significantly, whereas in diabetic women, no such trend was found (3). The all-cause mortality rate difference between diabetic and nondiabetic women is considerable. Therefore, the combination of diabetes with multiple risk factors identifies women at particularly high risk (2,4). The relative risk for morbidity and mortality in women with diabetes is increased compared with nondiabetic control subjects (2,5). Diabetes may therefore be regarded as a premature aging syndrome in which the overall metabolic shift leads to genotoxic stress that results in loss of chromosomal integrity (rev. in 6). Oxidative stress plays a crucial role in the pathogenesis of type 2 diabetes and in diabetes-associated complications. The generation of reactive oxygen species (ROS) is a common downstream mechanism whereby multiple by-products of glucose and (pro)inflammatory molecules exert adverse effects (7–11). DNA damage and telomere attrition can serve as markers of these processes and, consequently, mirror the pace of biological aging (rev. in 12–14). Hypothesizing along these lines, we studied telomere erosion dynamics and/or the occurrence of structural chromosomal aberrations in women with type 2 diabetes who were participants of the Ludwigshafen Risk and Cardiovascular Health (LURIC) prospective cohort study (15). Life expectancy within the LURIC female cohort falls short by ∼10 years compared with the general female population in Germany. Telomeric erosion was much further advanced in all LURIC women, irrespective of type 2 diabetes, compared with age-matched control subjects, the difference amounting to >10 life-years. We further found a strikingly enhanced number of structural chromosomal aberrations in the peripheral lymphocytes of women with type 2 diabetes that was diabetes specific and, within the younger age-groups, associated with mortality.

Cancer has been suggested to result from interactions between genetic and environmental factors, and certain subgroups in the general population may be at increased risk because of their relatively higher susceptibility to environmental carcinogens. The current study, part of a large biomonitoring study conducted in Flanders from 2002 to 2006 (The Flanders Environment and Health Survey), aims to determine these susceptible subpopulations based on multiple genotypic differences between individuals. A random selection of 429 adolescents and 361 adults was genotyped for 36 polymorphisms in 23 genes selected because of their known role in carcinogen metabolism, DNA repair, and oxidative stress. In both age groups, relationships between endogenous exposure to organochloride substances (polychlorinated biphenyl, hexachlorobenzene, dichlorodiphenyl dichloroethane), metals (cadmium, lead), and urinary metabolites (1-hydroxypyrene, trans-trans muconic acid) versus genotoxic effects (Comet assay and micronuclei in lymphocytes, and urinary 8-hydroxydeoxyguanosine) were investigated. In addition, in the study among adults, the relationship of these exposures with several tumor markers (prostate-specific antigen, carcinoembryonic antigen, and p53) was tested. The impact of the genotype on established exposure-effect relationships was evaluated. Eight exposure-effect relationships were found, including three novel associations, with an impact of various genotypes, predominantly affecting biotransformation and oxidative stress response. This study shows that at least part of the interindividual differences in relationships between carcinogen exposure and genotoxic effect can be explained by genotypic differences, enabling the identification of more susceptible subgroups for environmental cancer risks. This may be of relevance for environmental health policy setting.

Trisomy 8 is one of the most frequent numerical chromosomal abnormalities observed in hematological malignancies, whereas tetrasomy 8 is a clonal aberration seen mainly in myeloid disorders such as acute myelod leukemia (AML) and myelodysplastic syndromes. In contrast to trisomy 8, tetrasomy 8 is a rare chromosomal aberration, in that only 17 reported AML cases with isolated tetrasomy 8 have been documented. Interestingly, the majority of reported cases were associated with monocytic-lineage leukemias. According to recent reports, tetrasomy 8 is regarded as a poor prognostic factor, and most patients having this abnormality relapsed and died within 1 yr. Here, we report a patient with acute monoblastic leukemia having tetrasomy 8 and a very aggressive disease course.

Renal oncocytosis is characterized by the presence of multiple tumors with oncocytic features, often associated with small clusters of tubule-like structures with oncocytic change. The morphologic features of the oncocytic nodules encompass a spectrum of appearances, with patterns typical of renal oncocytoma or classic chromophobe renal cell carcinoma, as well as 'hybrid' tumors with features resembling both oncocytoma and chromophobe renal cell carcinoma. We utilized interphase cytogenetic methods to study 11 tumors from the kidneys of a 45-year-old woman. The tumors included morphologically classical oncocytomas and 'hybrid' tumors with features reminiscent of chromophobe carcinoma. The kidneys also showed foci of oncocytic change in renal tubules. Fluorescence in situ hybridization was performed with centromeric probes for chromosomes 1, 2, 6, 10, and 17 in each of the 11 tumors to determine whether or not there were losses of the chromosomes that are most frequently lost in chromophobe renal cell carcinomas. Neoplastic nuclei from each tumor were evaluated for the number of hybridization signals and scored according to the percentage of nuclei with one, two, and three or more signals. The normal renal parenchyma surrounding the tumors was used as control tissue. All 11 tumors from this patient with renal oncocytosis showed no loss of any of the chromosomes 1, 2, 6, 10, or 17, a pattern identical to that found in normal control tissues. These observations weigh against the concept that hybrid tumors of oncocytosis are closely related to chromophobe renal cell carcinoma.

<p>Inappropriate expression of EVI1 (ecotropic virus integration-1), in particular splice form EVI1-1D, through chromosome 3q26 lesions or other mechanisms has been implicated in the development of high-risk acute myeloid leukemia (AML). To validate the clinical relevance of EVI1-1D, as well as of the other EVI1 splice forms and the related MDS1/EVI1 (ME) gene, real-time quantitative polymerase chain reaction was performed in 534 untreated adults with de novo AML. EVI1-1D was highly expressed in 6% of cases (n = 32), whereas 7.8% were EVI1(+) (n = 41) when all splice variants were taken into account. High EVI1 predicted a distinctly worse event-free survival (HR = 1.9; P = .002) and disease-free survival (HR = 2.1, P = .006) following multivariate analysis. Importantly, we distinguished a subset of EVI1(+) cases that lacked expression of ME (EVI1(+)ME(-); n = 17) from cases that were ME(+) (EVI1(+)ME(+); n = 24). The atypical EVI1(+)ME(-) expression pattern exhibited cytogenetically detectable chromosomal 3q26 breakpoints in 8 cases. Fluorescence in situ hybridization revealed 7 more EVI1(+)ME(-) cases that carried cryptic 3q26 breakpoints, which were not found in the EVI1(+)ME(+) group. EVI1(+)ME(-) expression predicts an extremely poor prognosis distinguishable from the general EVI1(+) AML patients (overall survival [OS]: P</p>

<p>The acute myeloid leukemia (AML) subtype M4Eo occurs in 5% of all AML cases and is usually associated with either an inv(16)(p13.1q22) or a t(16;16)(p13.1;q22) chromosomal abnormality. At the molecular level, these abnormalities generate a CBFB-MYH11 fusion gene. Patients with this genetic alteration are usually assigned to a low-risk group and thus receive standard chemotherapy. AML-M4Eo is rarely found in infants. We describe clinical, conventional banding, and molecular cytogenetic data for a 12-month-old baby with AML-M4Eo and a chimeric CBFB-MYH11 fusion gene masked by a novel rearrangement between chromosomes 1 and 16. This rearrangement characterizes a new type of inv(16)(p13.1q22) masked by a chromosome translocation.</p>

Digital object identifier (DOI): 10.1016/j.cancergencyto.2007.12.014

<p>There is a steady search for procedure which could replace or at least reduce the frequency of the invasive cystoscopy in the surveillance of heterogeneous superficial transitional cell carcinoma (TCC) of the bladder. Recently, UroVysion FISH assay has been shown to provide with better sensitivity than the urine cytology except for the lowest stage pTa and grade I-II TCCs. Data indicate that this failure of the sensitive FISH might be due to mistargeting. Therefore, our aim was to elaborate a procedure enabling FISH analysis in phenotypically preselected urothelial cells, only. Cytokeratin 7 (CK-7) chromogenic immunolabeling was applied to various mixtures of negative and positive control cells as well as voided urine specimens. Cellular targets and CK-7 positive cells were identified by morphometric and pixel intensity indices using an automated microscope workstation. UroVysion FISH pattern was analyzed only in the subsequently relocalized CK-7 positive events. Automated phenotypical preselection of urothelial cells proved to have 97.3% sensitivity, 96.1% specificity, and 99.0% accuracy, whereas combined pheno- and genotyping revealed 93.3% sensitivity and 99.8% specificity, respectively. In clinical samples, the overall 20.4% FISH positivity gained by traditional target identification contrasted with the 55.6% positivity obtained by the combined method, by which the efficiency of identifying chromosomally aberrant cells proved to be two to threefold higher even in grade I lesions. FISH analysis of phenotypically preselected urothelial cells might represent a reliable asset in surveillance of low stage-low grade TCCs.</p>

Mazor, R., Korenstein-Ilan, A., Barbul, A., Eshet, Y., Shahadi, A., Jerby, E. and Korenstein, R. Increased Levels of Numerical Chromosome Aberrations after In Vitro Exposure of Human Peripheral Blood Lymphocytes to Radiofrequency Electromagnetic Fields for 72 Hours. Radiat. Res. 169, 28-37 (2008). We investigated the effects of 72 h in vitro exposure of 10 human lymphocyte samples to radiofrequency electromagnetic fields (800 MHz, continuous wave) on genomic instability. The lymphyocytes were exposed in a specially designed waveguide resonator at specific absorption rates (SARs) of 2.9 and 4.1 W/kg in a temperature range of 36-37 degrees C. The induced aneuploidy of chromosomes 1, 10, 11 and 17 was determined by interphase FISH using semi-automated image analysis. We observed increased levels of aneuploidy depending on the chromosome studied as well as on the level of exposure. In chromosomes 1 and 10, there was increased aneuploidy at the higher SAR, while for chromosomes 11 and 17, the increases were observed only for the lower SAR. Multisomy (chromosomal gains) appeared to be the primary contributor to the increased aneuploidy. The effect of temperature on the level of aneuploidy was examined over the range of 33.5-40 degrees C for 72 h with no statistically significant difference in the level of aneuploidy compared to 37 degrees C. These findings suggest the possible existence of an athermal effect of RF radiation that causes increased levels of aneuploidy. These results contribute to the assessment of potential health risks after continuous chronic exposure to RF radiation at SARs close to the current levels set by ICNIRP guidelines.

Detecting balanced translocations using tissue sections plays an important diagnostic role in cases of hematological malignancies. Manual scoring is often problematic due to truncation and overlapping of nuclei. Reports have described automated analysis using primarily tile sampling. The aim of this study was to investigate an automated fluorescent in situ hybridization analysis method using grid sampling on tissue sections, and compare the performance of dual-fusion (DF) and break-apart (BA) probes in this setting. Ten follicular, 10 mantle cell lymphoma, and 10 translocation-negative samples were used to set the threshold of false positivity using IGH/CCND1, IGH/BCL-2 DF, and IGH BA probes. The cut-off distances of red and green signals to define fusion signals were 0.5, 1.0, and 1.2 mum for the IGH/CCND1, IGH/BCL-2 DF, and IGH BA probes, respectively. The mean false positivity of grid units was 5.3, 11.4, and 28.1%, respectively. Ten to 14 additional samples analyzed blindly and were correctly classified using each probe. Discriminating positive and negative samples using automated analysis and grid sampling was possible with each probe, although different definitions of fusion signals were required due to the different physical distances between the DNA probes. Using the DF probes resulted in lower false positivity, which was less affected by signal numbers per grid units.

OBJECTIVE: To determine the precision and accuracy of an automated cell counting system when applied to counting aneuploidies in sperm samples. DESIGN: Prospective pilot study. SETTING: Andrology clinic and research laboratory in a university teaching hospital. PATIENT(S): Ten anonymous sperm donors of known fertility and two patients seeking infertility treatment. INTERVENTION(S): Semen samples were processed for detection of aneuploidies for chromosomes 13, 18, 21, X, and Y with use of fluorescent in situ hybridization. The detection of chromosome aneuploidy was performed both by manual counting and by the use of an automated cell counting system with manual review of aneuploid sperm. MAIN OUTCOME MEASURE(S): Semen samples were judged for the percent aneuploidy for chromosomes 13, 18, 21, X, and Y when counted manually or with the use of the automated cell counting system and review by a technician. RESULT(S): The sperm aneuploidy rates determined by the automated cell counting system and careful review were comparable with those obtained by manual counting by a trained technician. CONCLUSION(S): These preliminary data demonstrate that automated cell counting devices may be useful in increasing productivity in aneuploidy detection in sperm and may become an alternative to the labor-intensive manual counting by technicians.

Severe male infertility has been shown to be associated with improper meiotic recombination and elevated sperm chromosome aneuploidy. Elevated sperm aneuploidy increases the risk of embryo lethality or fetal anomalies. Although difficulties in interpreting aneuploidy data still exist, advances in fluorescent in situ hybridization (FISH) technology have facilitated the study of sperm from patients with severe spermatogenesis defects, which has demonstrated the prudence of evaluating sperm chromosome aneuploidy in men with severe male factor infertility, such as nonobstructive azoospermia or severe ultrastructure defects, especially in cases of previous repeated in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) failure. Testing is also advisable in men with chromosome translocations and unexplained recurrent pregnancy loss, and it may be beneficial in patients with unexplained, repeated IVF failure. Automated FISH imaging and analysis technology is now available and is beneficial in reducing technician time analyzing sperm aneuploidy. Emerging technologies, such comparative genomic hybridization, may be beneficial in further improving the quality of data derived from aneuploidy analysis and reducing the cost of the assay.

<p>BACKGROUND: The use of human embryonic stem cells (hESCs) in research is increasing and hESCs hold the promise for many biological, clinical and toxicological studies. Human ESCs are expected to be chromosomally stable since karyotypic changes represent a pitfall for potential future applications. Recently, several studies have analysed the genomic stability of several hESC lines maintained after prolonged in vitro culture but controversial data has been reported. Here, we prompted to compare the chromosomal stability of three hESC lines maintained in the same laboratory using identical culture conditions and passaging methods. RESULTS: Molecular cytogenetic analyses performed in three different hESC lines maintained in parallel in identical culture conditions revealed significant differences among them in regard to their chromosomal integrity. In feeders, the HS181, SHEF-1 and SHEF-3 hESC lines were chromosomally stable up to 185 passages using either mechanical or enzymatic dissection methods. Despite the three hESC lines were maintained under identical conditions, each hESC line behaved differently upon being transferred to a feeder-free culture system. The two younger hESC lines, HS181 (71 passages) and SHEF-3 (51 passages) became chromosomally unstable shortly after being cultured in feeder-free conditions. The HS181 line gained a chromosome 12 by passage 17 and a marker by passage 21, characterized as a gain of chromosome 20 by SKY. Importantly, the mosaicism for trisomy 12 gradually increased up to 89% by passage 30, suggesting that this karyotypic abnormality provides a selective advantage. Similarly, the SHEF-3 line also acquired a trisomy of chromosome 14 as early as passage 10. However, this karyotypic aberration did not confer selective advantage to the genetically abnormal cells within the bulk culture and the level of mosaicism for the trisomy 14 remained overtime between 15%-36%. Strikingly, however, a much older hESC line, SHEF-1, which was maintained for 185 passages in feeders did not undergo any numerical or structural chromosomal change after 30 passages in feeder-free culture and over 215 passages in total. CONCLUSION: These results support the concept that feeder-free conditions may partially contribute to hESC chromosomal changes but also confirm the hypothesis that regardless of the culture conditions, culture duration or splitting methods, some hESC lines are inherently more prone than others to karyotypic instability.</p>

ABSTRACT: Malignant melanomas are characterized by increased karyotypic complexity, extended aneuploidy and heteroploidy. We report a melanoma metastasis to the peritoneal cavity with an exceptionally stable, abnormal pseudodiploid karyotype as verified by G-Banding, subtelomeric, centromeric and quantitative Fluorescence in Situ Hybridization (FISH). Interestingly this tumor had no detectable telomerase activity as indicated by the Telomere Repeat Amplification Protocol. Telomeric Flow-FISH and quantitative telomeric FISH on mitotic preparations showed that malignant cells had relatively short telomeres. Microsatellite instability was ruled out by the allelic pattern of two major mononucleotide repeats. Our data suggest that a combination of melanoma specific genomic imbalances were sufficient and enough for this fatal tumor progression, that was not accompanied by genomic instability, telomerase activity, or the engagement of the alternative recombinatorial telomere lengthening pathway.

The American Society of Clinical Oncologists and College of American Pathologists have recently released new guidelines for laboratory testing of HER2 status in breast cancer, which require high levels (95%) of concordance between immunohistochemistry positive (3+) and fluorescence in situ hybridization-amplified cases, and between immunohistochemistry negative (0/1+) and fluorescence in situ hybridization-nonamplified cases; these required levels of concordance are significantly higher than those found in most published studies. We tested the hypothesis that a modification of the HER2 immunohistochemistry scoring system could significantly improve immunohistochemistry and fluorescence in situ hybridization concordance. A total of 6604 breast cancer specimens were evaluated for HER2 status by both immunohistochemistry and fluorescence in situ hybridization using standard methodologies. Results were compared when the standard immunohistochemistry scoring system was replaced by a normalized scoring system in which the HER2 score was derived by subtracting the score on the non-neoplastic breast epithelium from that on the tumor cells. Among the 6604 tumors, using a non-normalized immunohistochemistry scoring system, 267/872 (30.6%) of the immunohistochemistry 3+ cases proved to be fluorescence in situ hybridization nonamplified, whereas using the normalized scoring system only 30/562 (5.3%) of immunohistochemistry 3+ cases proved to be 'false positive'. The concordance rate between immunohistochemistry 3+ and fluorescence in situ hybridization-amplified cases using the normalized scoring method was 94.7%, whereas the concordance using the non-normalized method was only 69.4%. Extremely high concordance between immunohistochemistry and fluorescence in situ hybridization assessment of HER2 status in breast cancer is achievable, but to attain this high level of concordance, modification of the FDA-approved immunohistochemistry scoring system is required.

BACKGROUND: HER2 gene amplification and/or protein overexpression in breast cancer is associated with a poor prognosis and predicts response to anti-HER2 therapy. We examine the natural history of breast cancers in relationship to increased HER2 copy numbers in a large population-based study. PATIENTS AND METHODS: HER2 status was measured by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in approximately 1,400 breast cancer cases with greater than 15 years of follow-up. Protein expression was evaluated with two different commercially-available antibodies. RESULTS: We looked for subgroups of breast cancer with different clinical outcomes, based on HER2 FISH amplification ratio. The current HER2 ratio cut point for classifying HER2 positive and negative cases is 2.2. However, we found an increased risk of disease-specific death associated with FISH ratios of >1.5. An 'intermediate' group of cases with HER2 ratios between 1.5 and 2.2 was found to have a significantly better outcome than the conventional 'amplified' group (HER2 ratio >2.2) but a significantly worse outcome than groups with FISH ratios less than 1.5. CONCLUSION: Breast cancers with increased HER2 copy numbers (low level HER2 amplification), below the currently accepted positive threshold ratio of 2.2, showed a distinct, intermediate outcome when compared to HER2 unamplified tumors and tumors with HER2 ratios greater than 2.2. These findings suggest that a new cut point to determine HER2 positivity, at a ratio of 1.5 (well below the current recommended cut point of 2.2), should be evaluated.

<p>We aimed to directly align a chromosomal CGH (cCGH) pattern with the gene mapping data by taking advantage of the clustering of the GGCC motif at certain positions in the human genome. The alignment of chromosomal with sequence data was achieved by superimposition of (i) the fluorescence intensity of the sequence specific fluorochrome, Chromomycin A3 (CMA3), (ii) the cCGH fluorescence intensity profile of individual chromosomes and (iii) the GGCC density profile extracted from the Ensembl genome sequence database. The superimposition of these three pieces of information allowed us to precisely localize regions of amplification in the neuroblastoma cell line STA-NB-15. Two prominent cCGH peaks were noted, one at 2p24.3, the position 15.4 mega base (Mb), and the other at 2p23.2, 29.51 Mb. FISH and high resolution array CGH (aCGH) experiments disclosed an amplification of MYCN (16 Mb) and ALK (29.2-29.9 Mb), thus confirming the cCGH data. The combined visualization of sequence information and cCGH data drastically improves the resolution of the method to less than 2 Mb.</p>

The formin family of proteins mediates dynamic changes in actin assembly in eukaryotes, and therefore it is important to understand the function of these proteins in Entamoeba histolytica, where actin forms the major cytoskeletal network. In this study we have identified the formin homologs encoded in the E. histolytica genome based on sequence analysis. Using multiple tools, we have analyzed the primary sequences of the eight E. histolytica formins and discovered three subsets: (i) E. histolytica formin-1 to -3 (Ehformin-1 to -3), (ii) Ehformin-4, and (iii) Ehformin-5 to -8. Two of these subsets (Ehformin-1 to -3 and Ehformin-4) showed significant sequence differences from their closest homologs, while Ehformin-5 to -8 were unique among all known formins. Since Ehformin-1 to -3 showed important sequence differences from Diaphanous-related formins (DRFs), we have studied the functions of Ehformin-1 and -2 in E. histolytica transformants. Like other DRFs, Ehformin-1 and -2 associated with F-actin in response to serum factors, in pseudopodia, in pinocytic and phagocytic vesicles, and at cell division sites. Ehformin-1 and -2 also localized with the microtubular assembly in the nucleus, indicating their involvement in genome segregation. While increased expression of Ehformin-1 and -2 did not affect phagocytosis or motility, it clearly showed an increase in the number of binucleated cells, the number of nuclei in multinucleated cells, and the average DNA content of each nucleus, suggesting that these proteins regulate both mitosis and cytokinesis in E. histolytica.

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.