In this study, 107 children with acute lymphoblastic leukemia (ALL) were analysed for the presence of hyperdiploidy by cytogenetics and interphase fluorescence in situ hybridisation (I-FISH). Structural aberrations in hyperdiploid cells were investigated by multiple colour FISH (mFISH). Clones with high hyperdiploidy (>50 chromosomes) (HeH) were found in 46 patients (43%). In nine of these (20%), the abnormal clone was present in <20% of the total cell population. There was no significant difference in EFS between those patients with HeH in 2.5-20% or >20% of cells. Structural rearrangements in the HeH clone were found in 10 patients (22%). In this study, HeH karyotypes containing structural aberrations were an indication of a poor prognosis in childhood ALL.

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.

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.

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.

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.

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.

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.

<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>

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.

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.

The 8p11-21 region is a frequent target of alterations in breast cancer and other carcinomas. We surveyed 34 breast tumor cell lines and 9 pancreatic cancer cell lines for alterations of this region by use of multicolor fluorescence in situ hybridization (M-FISH) and BAC-specific FISH. We describe a recurrent chromosome translocation breakpoint that targets the NRG1 gene on 8p12. NRG1 encodes growth factors of the neuregulin/heregulin-1 family that are ligands for tyrosine kinase receptors of the ERBB family. Breakpoints within the NRG1 gene were found in four of the breast tumor cell lines: ZR-75-1, in a dic(8;11); HCC1937, in a t(8;10)(p12;p12.1); SUM-52, in an hsr(8)(p12); UACC-812, in a t(3;8); and in two of the pancreatic cancer cell lines: PaTu I, in a der(8)t(4;8); and SUIT-2, in a del(8)(p). Mapping by two-color FISH showed that the breaks were scattered over 1.1 Mb within the NRG1 gene. It is already known that the MDA-MB-175 breast tumor cell line has a dic(8;11), with a breakpoint in NRG1 that fuses NRG1 to the DOC4 gene on 11q13. Thus, we have found a total of seven breakpoints, in two types of cancer cell lines, that target the NRG1 gene. This suggests that the NRG1 locus is a recurring target of translocations in carcinomas. PCR analysis of reverse-transcribed cell line RNAs revealed an extensive complexity of the NRG1 transcripts but failed to detect a consistent pattern of mRNA isoforms in the cell lines with NRG1 breakpoint.

Diagnostic possibilities of CGH and M-FISH techniques for detection of submicroscopic chromosomal imbalancies were compared on the basis of two cases of t(X;Y) and one case of marker chromosome. In cases with t(X;Y), the sequences specific for chromosome Y were detected by PCR and CGH, but the localisation of these sequences on the short arm of chromosome X was confirmed by the FISH technique, employing two Yp-specific probes for SRY and TSPY genes. Significant differences between above cases were revealed in the size of Yp chromosome fragments translocated on chromosome X. An extra material of chromosome marker could not be identified by classical banding and FISH techniques and it was only CGH and M-FISH techniques that enabled detecting the chromosomal origin of the marker. The applied CGH technique enabled finding subtle chromosomal imbalancies in the presented cases with a resolution of approximately 3 Mbp.

We report a 13-year-old female patient with multiple congenital abnormalities (microcephaly, facial dysmorphism, anteverted dysplastic ears and postaxial hexadactyly), mental retardation, and adipose-gigantism. Ultrasonography revealed no signs of a heart defect or renal abnormalities. She showed no speech development and suffered from a behavioural disorder. CNS abnormalities were excluded by cerebral MRI. Initial cytogenetic studies by Giemsa banding revealed an aberrant karyotype involving three chromosomes, t(2;4;11). By high resolution banding and multicolour fluoresence in-situ hybridisation (M-FISH, MCB), chromosome 1 was also found to be involved in the complex chromosomal aberrations, confirming the karyotype 46,XX,t(2;11;4).ish t(1;4;2;11)(q43;q21.1;p12-p13.1;p14.1). To the best of our knowledge no patient has been previously described with such a complex translocation involving 4 chromosomes. This case demonstrates that conventional chromosome banding techniques such as Giemsa banding are not always sufficient to characterise complex chromosomal abnormalities. Only by the additional utilisation of molecular cytogenetic techniques could the complexity of the present chromosomal rearrangements and the origin of the involved chromosomal material be detected. Further molecular genetic studies will be performed to clarify the chromosomal breakpoints potentially responsible for the observed clinical symptoms. CONCLUSION: This report demonstrates that multicolour-fluorescence in-situ hybridisation studies should be performed in patients with congenital abnormalities and suspected aberrant karyotypes in addition to conventional Giemsa banding.

Using comparative genomic hybridization (CGH), we present a genome-wide screening of a mixed mesenchymal-epithelial hepatoblastoma, its recurrence and 2 novel hepatoblastoma cell lines raised from the ascites, 18 (HepU1) and 23 (HepU2) months after diagnosis of a hepatoblastoma in a 35-month-old boy. Both cell lines were also characterized by GTG-banding, multicolor-fluorescence in situ hybridization (M-FISH) and multicolor banding (M-Band). On the basis of CGH, we compared the cytogenetics of histologically different tumor areas of the parental tumor and its recurrence with the hepatoblastoma cell lines. We found different CGH profiles in the parental tumor rev ish enh(1q31-q32,8p,12,17,20,X), dim(4q34-q35,18q23)[cp] and its recurrence rev ish enh(8q24,17,Xq26-q28), dim(7q11.2-q21,13q34)[cp]. Although both epithelial cell lines were obtained at different times and the clonal ancestor of HepU2 had been exposed to a higher cumulative dose of chemotherapy, HepU1 and HepU2 have an identical karyotype: 48-56,XY,+Y,dup(2)(q32-q34),t(3;4)(q21;q34),+8,+12,+13, +17,+t(18;19)(q21;q?),+20[cp] and identical CGH profiles: rev ish enh(2q24-q33,8,12,13q,17,20), dim(4q34-q35,18q22-q23). In common with previously described hepatoblastoma cell lines, HepU1 and HepU2 demonstrate a gain of chromosome 20. The in situ aberrations most closely resembling that of HepU1 and HepU2 were found in areas of fetal-embryonal differentiation of the primary tumor. Interestingly, both cell lines mimic this histology in their three-dimensional growth pattern in vitro. HepU1 and HepU2 are thus cytogenetically and phenotypically highly characteristic of fetal-embryonal hepatoblastoma.

<p>The purpose of this work was to adapt the recently described centromere-specific multicolour (cenM-) FISH technique to human meiotic cells, and evaluate the usefulness of this multiplex fluorescence method for karyotyping human synaptonemal complex (SC), previously analysed by immunocytogenetic approaches. The results obtained demonstrate that cenM-FISH is a reliable one-single-step method, which allows for the identification of all SC present in pachytene spreads. Moreover, when cenM-FISH is applied after immunocytogenetic analysis, the number and distribution of MLH1 foci per chromosome can be established and recombination analysis for each chromosome can be performed easily.</p>

A comprehensive and detailed comparative chromosome map of the white-handed gibbon (Hylobates lar = HLA) has been established by hybridizing the recently developed complete human multicolor banding (MCB) probe set on metaphase chromosomes of a male HLA lymphoblastoid cell line. Thus, it was possible to precisely determine the breakpoints and distribution plus orientation of specific DNA-regions in this cytogenetically highly rearranged species compared to Homo sapiens (HSA). In general, the obtained results are in concordance with previous molecular-cytogenetic studies. In this study all 71 breakpoints present in HLA compared to HSA could be determined exactly. This study is a valuable complement to our knowledge on the phylogeny of huminoid chromosomes.

We report on the fourth known case with an interstitial deletion in 15q21. In the present case the breakpoints have been determined by GTG-banding, microdissection and the recently developed multicolor banding (MCB) technique as 15q21.1-q21.3. Common features in all four cases are mental retardation, growth retardation, a beak-like nose with hypoplastic alae nasi and a thin upper lip. Additional frequent features are small hands and feet, hypotonia, low hair implantation, low set ears, clinodactyly and obesity. The possibility that a critical region for a new microdeletion-syndrome is situated in 15q21 is discussed.

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine tumor of the skin. Cytogenetic studies have indicated that deletions and unbalanced translocations involving chromosome 1 short arm material occur in 40% of the investigated cases. Recurrent chromosomal imbalances detected by comparative genomic hybridization (CGH) analysis were loss of 3p, 10q, 13q and 17p and gains of 1q, 3q, 5p and 8q. In order to study genomic aberrations occurring in MCC in further detail, we combined karyotyping, CGH and multiplex-fluorescence in situ hybridization (M-FISH), a strategy that proved to be successful in the analysis of other malignancies. Analysis of 6 MCC cell lines and 1 MCC tumor revealed mostly near-diploid karyotypes with an average of 5 chromosomal rearrangements. The observed karyotypic changes were heterogeneous, with 3-27 breakpoints per case, leading to imbalance of the involved chromosomal regions that was confirmed by CGH. Chromosomal rearrangements involving the short arm of chromosome 1, the long arm of chromosome 3 and gain of 5p material were the most frequently observed abnormalities in our study. In keeping with previous observations, this series of MCCs showed no evidence for high-level amplification. We provid a detailed description of chromosomal translocations occurring in MCC that could be useful to direct future intensive investigation of these chromosomal regions.