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.

Previous studies have documented that Tumor necrosis factor alpha (TNFalpha) is a potent negative regulator of normal hematopoiesis. However, the mechanism by which TNFalpha acts at the cellular level is not totally understood. Although apoptotic cell killing appears to be the most common cellular effect of TNFalpha, other studies suggest that this cytokine may elicit other cellular responses such as prolonged growth inhibition. In this context, we have investigated whether TNFalpha may induce senescence in hematopoietic cells, which display intrinsic defect in the apoptotic machinery. The present study described that, in the leukemic KG1 cells, TNFalpha induced no apoptosis but a senescence state characterized by prolonged growth arrest, increased beta-galactosidase activity, p21WAF-1 induction, decreased telomerase activity, telomeric disturbances (shortening, losses, fusions), and additional chromosomal aberrations. Telomerase inhibition correlated with reduced levels of hTERT transcripts. GM-CSF prevented TNFalpha effects and allowed leukemic cells to recover growth capacity. Finally, our study shows for the first time that, at least in some hematopoietic cells, TNFalpha may induce senescence with important functional consequences, including sustained growth inhibition and genetic instability, and that this cellular response is efficiently regulated by hematopoietic growth factors.

The purpose of this study was to assess the feasibility of using rare event imaging system (REIS)-assisted analysis to detect occult tumor cells (OTCs) in peripheral blood (PB). The study also sought to determine whether REIS-assisted OTC detection presents a clinically viable alternative to manual microscopic detection to establish the true significance of OTC from solid epithelial tumors.We recently demonstrated proof of concept using a fluorescence-based automated microscope system, REIS, for OTC detection from the PB. For this study, the prototype of the system was adopted for high-throughput and high-content cellular analysis.The performance of the improved REIS was examined using normal blood (n = 10), normal blood added to cancer cells (n = 20), and blood samples obtained from cancer patients (n = 80). Data from the screening of 80 clinical slides from breast and lung cancer patients, by manual microscopy and by the REIS, revealed that as many as 14 of 35 positive slides (40\%) were missed by manual screening but positively identified by REIS. In addition, REIS-assisted scanning reliably and reproducibly quantified the total number of cells analyzed in the assay and categorized positive cells based on their marker expression profile.REIS-assisted analysis provides excellent sensitivity and reproducibility for OTC detection. This approach may enable an improved method for screening of PB samples and for obtaining novel information about disease staging and about risk evaluation in cancer patients.

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

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.

BACKGROUND AND OBJECTIVES: Nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL) showed recurrent rearrangement of the BCL6 which is gene detected in 48% of cases analyzed by interphase-fluorescent in situ hybridization (FISH). These findings point to a critical role for BCL6 in the development of this distinct Hodgkin's lymphoma. We present our results of metaphase-FISH analyses aimed at identifying and characterizing BCL6-related chromosomal translocations in NLPHL. DESIGN AND METHODS: Four NLPHL cases with available metaphase spreads obtained either at the time of diagnosis or during progression to diffuse large B-cell lymphoma (DLBCL) were collected. Extensive metaphase-FISH analysis was performed to identify the affected partner chromosomes and reciprocal breakpoints. RESULTS: Each of the analyzed NLPHL cases showed a different type of BCL6 rearrangement that included the t(3;22)(q27;q11) targeting immunoglobulin (IG) alpha chain locus, complex t(3;7;3;1) involving the 7p12/Ikaros gene region, t(3;9)(q27;p13) affecting an unknown gene in vicinity of PAX5, and t(3;4)(q27;q32) showing the alternative 3q27 breakpoint outside BCL6 and possibly, an internal deletion of BCL6. Retrospective interphase-FISH analysis of 2 cases with subsequent DLBCL showed the same type of BCL6 translocation as in NLPHL samples. INTERPRETATION AND CONCLUSIONS: The spectrum of BCL6 aberrations targeting IG as well as non-IG loci in NLPHL is similar to that found in DLBCL. These findings further support the hypothesis of a germinal center B-cell-derived origin of NLPHL and of a relationship between these two lymphoma entities. This latter issue is additionally illustrated in two NLPHL patients who subsequently developed DLBCL and showed the same type of BCL6 rearrangements in both tumors.

The proliferative life span of human cells is limited by telomere shortening, but the specific telomeres responsible for determining the onset of senescence have not been adequately determined. We here identify the shortest telomeres by the frequency of signal-free ends after in situ hybridization with telomeric probes and demonstrate that probes adjacent to the shortest ends colocalize with gammaH2AX-positive DNA damage foci in senescent cells. Normal BJ cells growth arrest at senescence before developing significant karyotypic abnormalities. We also identify all of the telomeres involved in end-associations in BJ fibroblasts whose cell-cycle arrest at the time of replicative senescence has been blocked and demonstrate that the 10% of the telomeres with the shortest ends are involved in >90% of all end-associations. The failure to find telomeric end-associations in near-senescent normal BJ metaphases, the presence of signal-free ends in 90% of near-senescent metaphases, and the colocalization of short telomeres with DNA damage foci in senescent interphase cells suggests that end-associations rather than damage signals from short telomeres per se may be the proximate cause of growth arrest. These results demonstrate that a specific group of chromosomes with the shortest telomeres rather than either all or only one or two sentinel telomeres is responsible for the induction of replicative senescence.

5-Fluorouracil (5-FU) is the chemotherapeutic drug of choice for the treatment of metastatic colorectal cancer, but resistance to 5-FU remains a major obstacle to successful therapy. We generated 5-FU-resistant derivatives of the HCT116 human colon cancer cell line by serial passage of these cells in the presence of increasing 5-FU concentrations in an attempt to elucidate the biological mechanisms involved in resistance to 5-FU. Two resultant resistant derivatives, HCT116 ResB and ResD, were characterized for resistance phenotypes, genotypes, and gene expression using cells maintained long-term in 5-FU-free media. Compared to parental HCT116 cells that respond to 5-FU challenge by inducing high levels of apoptosis, ResB and ResD derivatives had significantly reduced apoptotic fractions when transiently challenged with 5-FU. ResB and ResD cells were respectively 27- and 121-fold more resistant to 5-FU, had increased doubling times, and significantly increased plating efficiencies compared to the parental cells. Both resistant derivatives retained the wild-type TP53 genotype, TP53 copy number and CGH profile characteristic of the parental line. Alterations in gene expression in the resistant derivatives compared to the parental line were assessed using oligonucleotide microarrays. Overall, the 5-FU-resistant derivatives were characterized by reduced apoptosis and a more aggressive growth phenotype, consistent with the observed up-regulation of apoptosis-inhibitory genes (e.g., IRAK1, MALT1, BIRC5), positive growth-regulatory genes (e.g., CCND3, CCNE2, CCNF, CYR61), and metastasis genes (e.g., LMNB1, F3, TMSNB), and down-regulation of apoptosis-promoting genes (e.g., BNIP3, BNIP3L, FOXO3A) and negative growth-regulatory genes (e.g., AREG, CCNG2, CDKN1A, CDKN1C, GADD45A). 5-FU metabolism-associated genes (e.g., TYMS, DTYMK, UP) and DNA repair genes (e.g., FEN1, FANCG, RAD23B) were also up-regulated in one or both resistant derivatives, suggesting that the resistant derivatives might be able to overcome both 5-FU inhibition of thymidylate synthase and the DNA damage caused by 5-FU, respectively. Development of 5-FU resistance thus appears to encompass deregulation of apoptosis-, proliferation-, DNA repair-, and metastasis-associated regulatory pathways.

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.

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.

A clonal origin of hematopoiesis was studied by investigation of X-chromosome inactivation patterns (XCIP) in isolated granulocyte, CD14(+) and CD3(+) subpopulations obtained from bone marrow and peripheral blood of 36 female patients with primary myelodysplastic syndrome (MDS). Clonality was assessed by PCR amplification of polymorphic short tandem repeats of the human androgen receptor (HUMARA) gene and by investigation of silent polymorphism of iduronate sulphatase (IDS) or p55 genes. On the basis of results in a control group of 20 healthy age related females, a ratio of at least 9:1 between the two alleles was considered a significant marker of monoclonal hematopoiesis. Ten of the 11 patients with advanced forms of MDS (RAEB, RAEB-T, CMML) had clonal granulocytes and CD14(+) cells in peripheral blood. In patients with early disease, only 2 out of 11 patients (18%) with RA or RARS, according to WHO classification, had clonal granulocytes and CD14(+) cells in peripheral blood and bone marrow and 2 other patients with 5q-syndrome exhibited extremely oligoclonal granulocyte subpopulation in bone marrow. In contrast, we found clonal granulocytes in 12 out of 14 patients (86%) with refractory cytopenia with multilineage dysplasia (RCMD) and 8 of them simultanously exhibited clonal CD14(+) cells. Estimated 3 years survival of patients with early disease and clonal cell subpopulations was 61% as compared with 88% in patients without clonal hematopoiesis. Karyotype abnormalities were detected in 11 of the 25 females with early disease. Clonal patterns were present in 7 out of 8 patients with abberations diagnosed by routine cytogenetics, nevertheless, FISH revealed 5q deletion in 3 patients without signs of clonality in XCIP assay. No correlation was found between the presence of clonal subpopulations and the degree of telomere shortening in early MDS. Despite some limitations, the measurement of XCIP remains a sensitive tool for diagnosis of the first transforming mutation in the clonal development of MDS especially when combined with FISH and when an age related group is used to establish an appropriate allele ratio to exclude constitutional or acquired skewing. The occurrence of clonal cell subpopulations in most of the RCMD patients in contrast to RA may reflect a proposed multistep pathogenesis of MDS with dysplastic changes limited to erythropoiesis in early step and with subsequent development of multilineage dysplasia. The results also support the usefulness of separation of RCMD from 'pure' RA; however, a more complex insight combining different molecular techniques performed in a large number of patients is needed for refined classification of MDS on the basis of new molecular prognostic factors and for indication of more effective targeted therapy.

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.

No abstract available.

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.