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Hum Reprod, 19, 685- 693

Fluorescence in situ hybridization analysis of two blastomeres from day 3 frozen-thawed embryos followed by analysis of the remaining embryo on day 5.

E.B. Baart, D. Van Opstal, F.J. Los, B.C.J.M. Fauser, E.Martini

BACKGROUND: Chromosomal mosaicism in human embryos may give rise to false positive or false negative results in preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). Therefore, we have investigated whether the results obtained from a 2-cell biopsy of frozen-thawed embryos and fluorescence in situ hybridization (FISH) analysis are representative for the chromosome constitution of the remaining embryo on day 5. METHODS: Cryopreserved day 3 embryos were thawed and from surviving embryos two blastomeres were biopsied. FISH analysis was performed for chromosomes 1, 7, 13, 15, 16, 18, 21, 22, X and Y. After biopsy, the embryos were cultured until day 5 and further analysed using the same probe panels. RESULTS: In all, 17 embryos were available with a diagnosis based on two blastomeres on day 3 and confirmatory studies on day 5. In 10 of these 17 cases the initial diagnosis could be confirmed. However, in only six cases cytogenetic results were concordant. Besides the 10 cases with a 'correct' diagnosis, there were six false positive results and one false negative, all involving mosaicism. CONCLUSIONS: Investigating the chromosomal constitution of two blastomere nuclei offers a good opportunity to study the incidence of chromosomal mosaicism in early embryo development. The confirmation rate of the results obtained on day 3 depends on the interpretation and is higher when considered from a clinical than from a cytogenetic point of view.

Nature Reviews - Genetics, 5, 11- 22

Systematic genome-wide screens of gene function.

A.E. Carpenter, D.M. Sabatini

By using genome information to create tools for perturbing gene function, it is now possible to undertake systematic genome-wide functional screens that examine the contribution of every gene to a biological process. The directed nature of these experiments contrasts with traditional methods, in which random mutations are induced and the resulting mutants are screened for various phenotypes. The first genome-wide functional screens in Caenorhabditis elegans and Drosophila melanogaster have recently been published, and screens in human cells will soon follow. These high-throughput techniques promise the rapid annotation of genomes with high-quality information about the biological function of each gene.

Cytogenet Genome Res, 104, 390- 393

mBAND: a high resolution multicolor banding technique for the detection of complex intrachromosomal aberrations

I. Chudoba, G. Hickmann, T. Friedrich, A. Jauch, P. Kozlowski, G. Senger

Precise breakpoint definition of chromosomal rearrangements using conventional banding techniques often fails, especially when more than two breakpoints are involved. The classic banding procedure results in a pattern of alternating light and dark bands. Hence, in banded chromosomes a specific chromosomal band is rather identified by the surrounding banding pattern than by its own specific morphology. In chromosomal rearrangements the original pattern is altered and therefore the unequivocal determination of breakpoints is not obvious. The multicolor banding technique (mBAND, see Chudoba et al., 1999) is able to identify breakpoints unambiguously, even in highly complex chromosomal aberrations. The mBAND technique is presented and illustrated in a case of intrachromosomal rearrangement with seven breakpoints all having occurred on one chromosome 16, emphasizing the unique analyzing power of mBAND as compared to conventional banding techniques.

International Journal of Oncology, 24, 127- 136

Breakpoint differentiation in chromosomal aberrations of hematological malignancies: identification of 33 previously unrecorded breakpoints

A. Heller, I.F. Loncarevic, M. Glaser, E. Gebhart, U. Trautmann, U. Claussen, T. Liehr

Routine cytogenetic analysis provides important information of diagnostic and prognostic relevance for hematological malignancies. In spite of this, poorly spread metaphase chromosomes and highly rearranged karyotypes with numerous marker chromosomes, are often difficult to interpret. In order to improve the definition of chromosomal breakpoints multicolor banding (MCB) was applied on 45 bone marrow samples from patients suffering from hematological malignancies like myelodysplastic syndrome (MDS), acute myelocytic leukemia (AML), chronic myelocytic leukemia (CML) or acute lymphoblastic leukemia (ALL). The breakpoints defined by GTG banding were confirmed by MCB in 8 cases, while in the remaining 37 cases the breakpoints had to be redefined. In 20/45 cases the breakpoints could only be characterized after application of MCB. In summary, 73 different breakpoints were characterized, thereof 33 were previously undescribed. Eleven cases showed known acquired aberrations and 21 cases had previously described aberration types such as del(5q-), del(7q-), del(13q-) or t(1;5) as sole rearrangement or in connection with other complex ones. In a total of 11 cases 19 breakpoints as described before were involved in hematological malignancies, while in 14 cases 33 breakpoints were identified which have not been described previously. Thus, MCB has proven to be a powerful and reliable method for screening of chromosomal aberrations, which considerably increased the accuracy of cytogenetic diagnosis.

Cellular Oncology, 26, 335- 345

DNA ploidy and chromosome (FISH) pattern analysis of peripheral nerve sheath tumors

A. Hruska, R. Bollmann, R.B. Kovács, M. Bollmann, M. Bodó, Z. Sápi

BACKGROUND AND METHODS: 44 peripheral nerve sheath tumors (PNST) (27 schwannomas, 9 neurofibromas and 8 malignant peripheral nerve sheath tumors (MPNST)) were analyzed to determine DNA ploidy pattern and to clarify the conflicting data in the literature concerning this topic (whether benign PNSTs are aneuploid or not). For further insight we analyzed 6 schwannomas, one atypical neurofibroma and five MPNSTs by fluorescence in situ hybridization (FISH) technique using centromeric chromosome probes (7, 17 and 18) and automatic image analysis station, Metafer 4. RESULTS: Benign schwannomas (including the problematic variants as ancient, cellular, neuroblastoma like and multiplex schwannomas) could be characterized by euploid-polyploidisation and by their 4c peak height value which was usually more than 10% of total cell number measured. These characters were not found among neurofibromas and MPNST-s. FISH analysis revealed and confirmed that the 'normal' euploid-polyploid cells are mainly eusomic-polysomic containing two, four, eight or sixteen signals for each chromosomes examined, but in a small proportion aneusomy was found among tumor cells of benign schwannomas (average: 2.58; range 1.33-3.44). In contrast, the atypical neurofibroma displayed marked aneusomy (18.44%) but it contained normal eusomic and polysomic cells too. Two diploid MPNSTs proved to be clearly aneusomic with trisomy of chromosome 17 and monosomy of chromosome 18. CONCLUSIONS: All these data suggest that ploidy pattern determination combined with FISH analysis may be a very useful supplementary tool for making a right diagnosis (to differentiate benign versus malignant schwannomas in problematic variants) and to understand better the malignant transformation in PNSTs.

Radiation Research, 161, 540- 548

Chromosome intrachanges and interchanges detected by multicolor banding in lymphocytes: searching for clastogen signatures in the human genome

C. Johannes, M. Horstmann, M. Durante, I. Chudoba, G. Obe

<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>

Blood, 104, 795- 801

Genomic DNA-chip hybridization in t(11;14)-positive mantle cell lymphomas shows a high frequency of aberrations and allows a refined characterization of consensus regions.

H. Kohlhammer, C. Schwaenen, S. Wessendorf, K. Holzmann, H.A. Kestler, D. Kienle, T.F.E. Barth, P. Möller, G. Ott, J. Kalla, B. Radlwimmer, A. Pscherer, S. Stilgenbauer, H. Döhner, P. Lichter, M. Bentz

Tumor samples of 53 patients with t(11;14)-positive mantle cell lymphomas (MCLs) were analyzed by matrix-based comparative genomic hybridization (matrix-CGH) using a dedicated DNA array. In 49 cases, genomic aberrations were identified. In comparison to chromosomal CGH, a 50% higher number of aberrations was found and the high specificity of matrix-CGH was demonstrated by fluorescence in situ hybridization (FISH) analyses. The 11q gains and 13q34 deletions, which have not been described as frequent genomic aberrations in MCL, were identified by matrix-CGH in 15 and 26 cases, respectively. For several genomic aberrations, novel consensus regions were defined: 8p21 (size of the consensus region, 2.4 megabase pairs [Mbp]; candidate genes: TNFRSF10B, TNFRSF10C, TNFRSF10D); 10p13 (2.7 Mbp; BMI1); 11q13 (1.4 Mbp; RELA); 11q13 (5.2 Mbp; CCND1); 13q14 (0.4 Mbp; RFP2, BCMSUN) and 13q34 (6.9 Mbp). In univariate analyses correlating genomic aberrations and clinical course, 8p- and 13q14- deletions were associated with an inferior overall survival. These data provide a basis for further studies focusing on the identification of pathogenetically or clinically relevant genes in MCL.

Histol Histopathol, 19, 229- 237

Multicolour FISH probe sets and their applications

T. Liehr, H. Starke, A. Weise, H. Lehrer, U. Claussen

Multicolor fluorescence in situ hybridization (FISH) assays are nowadays indispensable for a precise description of complex chromosomal rearrangements. Routine application of such techniques on human chromosomes started in 1996 with the simultaneous use of all 24 human whole chromosome painting probes in multiplex-FISH (M-FISH) and spectral karyotyping (SKY). Since then different approaches for chromosomal differentiation based on multicolor-FISH (mFISH) assays have been described. Predominantly, they have been established to characterize marker chromosomes identified in conventional banding analysis. Their characterization is of high clinical impact and is the requisite condition for further molecular investigations aimed at the identification of disease-related genes. Here we present a review on the available mFISH methods including their advantages, limitations and possible applications.

Int J Cancer, 111, 358- 366

Intratumor chromosomal heterogeneity in advanced carcinomas of the uterine cervix.

H. Lyng, M. Beigi, D.H. Svendsrud, O.T. Brustugun, T. Stokke, G.B. Kristensen, K. Sundf\or, A. Skj\onsberg, P.M. De Angelis

Intratumor heterogeneity in chromosomal aberrations is believed to represent a major challenge in the treatment of cancer. The aim of our work was to assess the chromosomal heterogeneity of advanced cervical carcinomas and to distinguish aberrations that had occurred at a late stage of the disease from early events. A total of 55 biopsies, sampled from 2-4 different sites within 20 tumors, were analyzed by use of comparative genomic hybridization. Heterogeneous aberrations were identified as those present in at least 1 of the biopsies and which were not seen, nor seen as a tendency, in the others of the same tumor. The homogeneous aberrations were those seen in all biopsies of the tumor. The most frequent homogeneous aberrations were gain of 3q (65%), 20q (65%) and 5p (50%), indicating that these are early events in the development of the disease. Chromosomal heterogeneity was observed in 11 tumors. The most frequent heterogeneous aberrations were loss of 4p14-q25 (60% of 10 cases with this aberration), and gain of 2p22-pter (50% of 6 cases), 11qcen-q13 (33% of 9 cases) and 8q (27% of 11 cases), suggesting that these events promote progression at a later stage. Many of the heterogeneous regions contained genes known to influence the prognosis of cervical cancer, such as 7p (EGFR), 8q (c-MYC), 11qcen-q13 (CCND1) and 17q (ERBB2). Three evolution sequences for the subpopulations in the heterogeneous tumors were identified: a serial, a parallel and a mixed sequence. In 2 tumors with a serial sequence, it was indicated that the aberrations +8 and -X had occurred after the other heterogeneous aberrations and hence were the aberrations most recently formed. Our results suggest pronounced chromosomal instability in advanced cervical carcinomas. Moreover, aggressive and treatment-resistant subpopulations may emerge at a late stage and possibly contribute to a poor prognosis of the advanced stages.

Pathol Oncol Res, 10, 142- 148

Chromosomal aberrations accumulate in polyploid cells of high-grade squamous intraepithelial lesions (HSIL).

G. Méhes, N. Speich, M. Bollmann, R. Bollmann

<p>Persistant infection with human papillomavirus (HPV) of the uterine cervix is related with cytological atypia (SIL), the oncogenic potential of which is unclear in a given time point of monitoring. HPV-induced genetic instability result in polyploidization as well as in low frequency random chromosome aberrations in squamous cells. In the present work we analyzed whether highly polyploid/aneuploid cells reflect genomic changes at the chromosomal level. 13 samples with the cytological diagnosis of HSIL were analyzed for HPV type and nuclear DNA content measured by laser scanning cytometry (LSC). Hyperdiploid cells with &gt;5c and with &gt;9c DNA content were further analyzed for numerical aberrations of the chromosomes 3 and 17 by fluorescence in situ hybridization (FISH) following repositioning. Cells with &gt;5c DNA content were found more frequently than cells with &gt;9c DNA content (5-98 and 1-44 cells, respectively). The FISH analysis demonstrated frequent polysomies, however, the rate of aneusomy (other than 2, 4, 8 or 16 chromosome copies) was significantly higher in cells with &gt;9c DNA content than in cells with &gt;5c DNA content or the normal diploid cells. The imbalance of chromosome 3 and 17 copy number was also increased in cells with &gt;9c DNA content. Moreover, in three out of the 13 analyzed HSIL samples, recurrent abnormal chromosome 3/17 ratio was demonstrated in a significant part of the cells, indicating a common origin of these cells. Highly polyploid/aneuploid cells in HSIL accumulate cytogenetic aberrations detectable by FISH analysis. These cells may reflect early changes with tumorigenic potential in a very concentrated fashion.</p>

Cancer research, 64, 6453- 6460

Genomic and expression profiling of chromosome 17 in breast cancer reveals complex patterns of alterations and novel candidate genes

B. Orsetti, M. Nugoli, N. Cervera, L. Lasorsa, P. Chuchana, L. Ursule, C. Nguyen, R. Redon, du Manoir, S., C. Rodriguez, C. Theillet

Chromosome 17 is severely rearranged in breast cancer. Whereas the short arm undergoes frequent losses, the long arm harbors complex combinations of gains and losses. In this work we present a comprehensive study of quantitative anomalies at chromosome 17 by genomic array-comparative genomic hybridization and of associated RNA expression changes by cDNA arrays. We built a genomic array covering the entire chromosome at an average density of 1 clone per 0.5 Mb, and patterns of gains and losses were characterized in 30 breast cancer cell lines and 22 primary tumors. Genomic profiles indicated severe rearrangements. Compiling data from all samples, we subdivided chromosome 17 into 13 consensus segments: 4 regions showing mainly losses, 6 regions showing mainly gains, and 3 regions showing either gains or losses. Within these segments, smallest regions of overlap were defined (17 for gains and 16 for losses). Expression profiles were analyzed by means of cDNA arrays comprising 358 known genes at 17q. Comparison of expression changes with quantitative anomalies revealed that about half of the genes were consistently affected by copy number changes. We identified 85 genes overexpressed when gained (39 of which mapped within the smallest regions of overlap), 67 genes underexpressed when lost (32 of which mapped to minimal intervals of losses), and, interestingly, 32 genes showing reduced expression when gained. Candidate genes identified in this study belong to very diverse functional groups, and a number of them are novel candidates.

Cytogenet. Genome Res., 104, 87- 94

Human fibroblasts expressing hTERT show remarkable karyotype stability even after exposure to ionizing radiation.

L.M. Pirzio, M.A. Freulet, Y. Bai, B. Fouladi, J.P. Murnane, L. Sabatier, C. Desmaze

Ectopic expression of telomerase results in an immortal phenotype in various types of normal cells, including primary human fibroblasts. In addition to its role in telomere lengthening, telomerase has now been found to have various functions, including the control of DNA repair, chromatin modification, and the control of expression of genes involved in cell cycle regulation. The investigations on the long-term effects of telomerase expression in normal human fibroblast highlighted that these cells show low frequencies of chromosomal aberrations. In this paper, we describe the karyotypic stability of human fibroblasts immortalized by expression of hTERT. The ectopic overexpression of telomerase is associated with unusual spontaneous as well as radiation-induced chromosome stability. In addition, we found that irradiation did not enhance plasmid integration in cells expressing hTERT, as has been reported for other cell types. Long-term studies illustrated that human fibroblasts immortalized by telomerase show an unusual stability for chromosomes and for plasmid integration sites, both with and without exposure to ionizing radiation. These results confirm a role for telomerase in genome stabilisation by a telomere-independent mechanism and point to the possibility for utilizing hTERT-immortalized normal human cells for the study of gene targeting.

Cytogenet Genome Res, 104, 383- 389

New developments in automated cytogenetic imaging: unattended scoring of dicentric chromosomes, micronuclei, single cell electrophoresis, and fluorescence signals.

C. Schunck, T. Johannes, D. Varga, T. Lörch, A. Plesch

The quantification of DNA damage, both in vivo and in vitro, can be very time consuming, since large amounts of samples need to be scored. Additional uncertainties may arise due to the lack of documentation or by scoring biases. Image analysis automation is a possible strategy to cope with these difficulties and to generate a new quality of reproducibility. In this communication we collected some recent results obtained with the automated scanning platform Metafer, covering applications that are being used in radiation research, biological dosimetry, DNA repair research and environmental mutagenesis studies. We can show that the automated scoring for dicentric chromosomes, for micronuclei, and for Comet assay cells produce reliable and reproducible results, which prove the usability of automated scanning in the above mentioned research fields.

Leukemia Research, 28, 1013- 1021

Dynamics of telomere erosion and its association with genome instability in myelodysplastic syndromes (MDS) and acute myelogenous leukemia arising from MDS: a marker of disease prognosis?

Z. Sieglová, S. Zilovcová, J. Cermák, H. Ríhová, D. Brezinová, R. Dvoráková, M. Marková, J. Maaloufová, J. Sajdová, J. Brezinová, Z. Zemanová, K. Michalová

Telomere length was evaluated by terminal repeat fragment method (TRF) in 50 patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) arising from MDS and in 21 patients with untreated primary AML to ascertain, whether telomere erosion was associated with progression of MDS towards overt leukemia. Heterogeneity of TRF among MDS FAB subgroups (P=0.004) originated from its shortening in increased number of patients during progression of the disease. Chromosomal aberrations were present in 32% MDS patients with more eroded telomeres (P=0.022), nevertheless a difference between mean TRF in the subgroups with normal and abnormal karyotype diminished during progression of MDS. A negative correlation between individual TRF and IPSS value (P=0.039) showed that telomere dynamics might serve as a useful prognostic factor for assessment of an individual MDS patient’s risk and for decision of an optimal treatment strategy.

Am J Clin Pathol, 122, 875- 882

Frequent gains of the short arm of chromosome 9 in Multiple Myeloma with normal G-banded karyotype detected by comparative genomic hybridization.

J. Tchinda, S. Volpert, M. Kropff, W.E. Berdel, J. Kienast, F. Meinhardt, J. Horst

A number of genetic abnormalities have been detected in multiple myeloma (MM) using cytogenetic techniques. The prominent abnormalities are deletions of 13q and translocations affecting the IgH locus on 14q32. The recurrence of chromosomal abnormalities in MM suggests a specific role for them concerning its pathogenesis. We performed comparative genomic hybridization (CGH) on samples from 53 patients with MM and 4 with monoclonal gammopathies of undetermined significance. In 31 cases (54%), normal ratio profiles were found, whereas 26 cases (46%) had aberrant profiles. The most common aberrations were gains of 9p (n = 14), 11 (n = 9), and 21q (n = 5) and loss of 22 (n = 7). In earlier reports on cytogenetics of lymphomas, gains of 9p are described as characteristic of primary mediastinal B-cell lymphoma, but the consensus region is smaller than in the present study (9p23pter vs 9p13pter). Therefore, we suggest a stronger genetic affinity between MM and primary mediastinal B-cell lymphoma than MM and other B-cell lymphomas. To support this suggestion, more molecular cytogenetic techniques and expression analyses have to be performed.

Digital object identifier (DOI): 10.1309/5KWK-P6UK-GNXX-HMYH

Mutagenesis, 19, 391- 397

An automated scoring procedure for the micronucleus test by image analysis

D. Varga, T. Johannes, S. Jainta, S. Schuster, U. Schwarz-Boeger, M. Kiechle, B.P. Garcia, W. Vogel

The micronucleus assay (MNT) in human lymphocytes is frequently used to assess chromosomal damage as a consequence of environmental mutagen exposure, to assess the effect of mutagens or to search for reduced DNA repair capacity after a mutagenic challenge. We have established an automated scoring procedure for the cytokinesis blocked MNT based on computerized image analysis (Metasystems Metafer 4 version 2.12). To evaluate the results we used the reproducibility of counts, established a dose-response curve for gamma-irradiation and used the ability of the system to differentiate between breast cancer patients and controls as a biological reference, a difference which we had observed before by visual counting. Blood cultures were irradiated with gamma-rays (2 Gy) at the beginning and treated with cytochalasin B during the last 24 h. The slides were stained with Giemsa for visual counting and with DAPI for automated analysis. Our test sample consisted of 73 persons (27 with breast cancer and 26 female and 20 male controls). A comparison between visual counting (controls, mean MN frequency 313) and automated counting (mean MN frequency 106) in slides from the same culture revealed a large drop for the automated counts. However, the automated counts were as reproducible as the visual counts [coefficient of variation (CV) on the sample approximately 20%; CV on repeated counts of the same slides approximately 5%] and both counts were highly correlated. Furthermore, the discrimination between cases and controls improved for automated counting of slides from the same cultures [visual odds rato (OR) < or = 4.0, P = 0.009; automated OR > 16, P < 0.0001], with a strong dependence on the set of parameters used. This improvement was confirmed in a validation sample of an additional 21 controls and 20 cases (OR = 11, P = 0.0018) performed as a prospective or diagnostic test.