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Molecular Biology of the Cell, 15, 3709- 3718

Does a sentinel or a subset of short telomeres determine replicative senescence?

Y. Zou, A. Sfeir, S.M. Gryaznov, J.W. Shay, W.E. Wright

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.

Genes Chromosomes Cancer, 37, 333- 345

A recurrent translocation breakpoint in breast and pancreatic cancer cell lines targets the Neuregulin/NRG I gene

J. Adélaide, H.-E. Huang, A. Murati, A.E. Alsop, B. Orsetti, M.-J. Mozziconacci, C. Popovici, C. Ginestier, A. Letessier, C. Basset, C. Courtay-Cahen, J. Jacquemier, C. Theillet, D. Birnbaum, P.A.W. Edwards, M. Chaffanet

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.

Cancer letters, 197, 29- 34

Disseminated tumour cells in the bone marrow - chances and consequences of microscopical detection methods

P.F. Ambros, G. Mehes, I.M. Ambros, R. Ladenstein

The detection of disseminated tumor cells (DTCs) in the hematopoetic system is important for various reasons. It is essential for tumor staging. According to the International Neuroblastoma Staging System (INSS) only the cytomorphological examination of bone marrow smears is accepted despite the fact that an infiltrate below 0.1%, can hardly be detected and even infiltrates of more than 10% are sometimes overlooked. Another important aspect is the monitoring of the disease response to cytotoxic drugs by quantifying DTCs. Moreover, bone marrow aspirates represent an ideal source to determine the genetic and biological make up of DTCs at diagnosis and during follow up. Key issues that can be tested on DTCs are: determination of the proliferation capacity, the apoptotic rate, the drug sensitivity etc. The prerequisite for such a bone-marrow diagnosis, however, is the unequivocal identification of disseminated tumor cells. Thus, in order to avoid false positive and false negative results, which are a risk in bone-marrow diagnostics, a system was developed to distinguish tumor cells from non-neoplastic cells and to facilitate the gain of insights into the biological make-up of tumor cells more easily.

Cancer Genet. Cytogenet., 142, 80- 82

Cytogenetic and molecular characterization of simultaneous chronic and acute myelotic leukemia.

L. Harder, S. Gesk, J.I. Martin-Subero, H. Merz, A. Hochhaus, E. Maa\ss, A. Feller, W. Grote, R. Siebert, S. Fetscher

We describe a patient initially diagnosed with a chronic myeloproliferative disorder in the accelerated phase. Cytogenetic analysis showed the presence of two independent clones. One clone contained a typical Philadelphia (Ph) chromosome due to t(9;22)(q34;q11), as the sole abnormality which was proven molecularly to result in the b2a2-BCR/ABL fusion. The other clone displayed a complex karyotype with several structural and numerical aberrations including trisomy 11 and 22 but lacking a t(9;22) or any other structural abnormalities involving chromosomes 9 and 22. Fluorescence in situ hybridization demonstrated that the t(9;22) was present only in cells with two copies of chromosomes 11 and 22. In contrast, cells with trisomies 11 and 22 lacked evidence for a BCR/ABL fusion. Based on the genetic findings, simultaneous chronic and acute myelocytic leukemias were diagnosed rather than a blastic phase of a chronic myelocytic leukemia.

Cell, 115, 523- 535

EMSY links the BRCA2 pathway to sporadic breast and ovarian cancer.

L. Hughes-Davies, D. Huntsman, M. Ruas, F. Fuks, J. Bye, S.-F. Chin, J. Milner, L.A. Brown, F. Hsu, B. Gilks, T. Nielsen, M. Schulzer, S. Chia, J. Ragaz, A. Cahn, L. Linger, H. Ozdag, E. Cattaneo, E.S. Jordanova, et al.

<p>The BRCA2 gene is mutated in familial breast and ovarian cancer, and its product is implicated in DNA repair and transcriptional regulation. Here we identify a protein, EMSY, which binds BRCA2 within a region (exon 3) deleted in cancer. EMSY is capable of silencing the activation potential of BRCA2 exon 3, associates with chromatin regulators HP1beta and BS69, and localizes to sites of repair following DNA damage. EMSY maps to chromosome 11q13.5, a region known to be involved in breast and ovarian cancer. We show that the EMSY gene is amplified almost exclusively in sporadic breast cancer (13%) and higher-grade ovarian cancer (17%). In addition, EMSY amplification is associated with worse survival, particularly in node-negative breast cancer, suggesting that it may be of prognostic value. The remarkable clinical overlap between sporadic EMSY amplification and familial BRCA2 deletion implicates a BRCA2 pathway in sporadic breast and ovarian cancer.</p>

J. Appl. Genet., 44, 539- 546

Molecular cytogenetic techniques in detecting subtle chromosomal imbalances

B. Kaluzewski, M. Constantinou, E. Zajac

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.

Int J Mol Med, 11, 575- 577

Evidence for a new microdeletion syndrome in 15q21

T. Liehr, H. Starke, A. Heller, A. Weise, V. Beensen, G. Senger, G. Kittner, M. Prechtel, U. Claussen, J. Seidel

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.

Am J Pathol, 2003, 393- 399

Detection of disseminated tumor cells in neuroblastoma: 3 log improvement in sensitivity by automatic immunofluorescence plus FISH (AIPF) analysis compared with classical bone marrow cytology.

G. Méhes, A. Luegmayr, R. Kornmüller, I.M. Ambros, R. Ladenstein, H. Gadner, P.F. Ambros

<p>The sensitive detection of bone marrow involvement is crucial for tumor staging at diagnosis and for monitoring of the therapeutic response in the patient's follow-up. In neuroblastoma, only conventional cytomorphological techniques are presently accepted for the detection of bone marrow involvement, yet since the therapeutic consequences of the bone marrow findings may be far-reaching, the need for highly reliable detection methods has become evident. For this purpose, we developed an automatic immunofluorescence plus FISH (AIPF) device which allows the exact quantification of disseminated tumor cells and the genetic verification in critical cases. In this study, the power of the immunofluorescence technique is compared with conventional cytomorphology. 198 samples from 23 neuroblastoma patients (stages 4 and 4s) at diagnosis and during follow-up were investigated. At diagnosis, 45.6% of the samples (26 of 57) which were positive by AIPF investigation were negative by cytomorphology. During follow-up, 74.2% (49 of 66) of AIPF-positive samples showed no cytological signs of tumor cell involvement. False negative morphological results were found in up to 10% of tumor cell content. A tumor cell infiltrate below 0.1% was virtually not detectable by conventional cytomorphology. Using the sensitive immunofluorescence technique, the analysis of only two instead of four puncture sites did not lead to false negative results. Thus, the immunofluorescence technique offers an excellent tool for reliable detection and quantification of disseminated tumor cells at diagnosis and during the course of the disease.</p>

Int J Mol Med, 12, 139- 146

Detailed Hylobates lar karyotype defined by 25-color FISH and multicolor banding

K. Mrasek, A. Heller, N. Rubtsov, V. Trifonov, H. Starke, U. Claussen, T. Liehr

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.

European J Hum Genet, 11, 879- 883

Karyotyping of human synaptonemal complexes by cenM-FISH

M. Oliver-Bonet, T. Liehr, A. Nietzel, A. Heller, H. Starke, U. Claussen, M. Codina-Pascual, A. Pujol, C. Abad, J. Egozcue, J. Navarro, J. Benet

<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>

Neoplasia, 5, 23- 31

Cytogenetic profile of unknown primary tumors: clues for their pathogenesis and clinical management

D. Pantou, H. Tsarouha, A. Papandopoulou, L. Mahaira, I. Kyriazoglou, N. Apostolikas, S. Markidou, T. Trangas, N. Pandis, G. Bardi

Unknown primary tumors (UPTs) represent an entity of great clinical and biological interest, whose origin cannot be determined even after medical workup. To better understand their pathogenesis by outlining their genetic composition, 20 UPTs were investigated by G-banding, supplemented with Fluorescence In Situ Hybridization and Comparative Genomic Hybridization analyses. The data obtained were sufficient to reach a diagnosis in five cases-four lymphomas and one Ewing sarcoma-demonstrating that in a subset of UPTs, cytogenetics can be an adjunct for differential diagnosis. In the remaining 15 UPTs, an aggressive cytogenetic pattern was revealed. The most frequently rearranged chromosome regions were 1q21, 3p13, 6q15-23, 7q22, 11p12-5, and 11q14-24, pinpointing gene loci probably associated with the peculiar pathogenesis of UPTs. The preferential involvement of 4q31, 6q15, 10q25, and 13q22 in adenocarcinomas (whereas 11q22 is involved in the rest of the carcinomas)-in addition to the marked divergence in the mean average of chromosomal changes, 16 and 3, respectively-demonstrates genotypic differences between the two histologic subgroups. Furthermore, the significantly shorter survival in cases displaying massive chromosome changes compared with those having a few changes indicates that the cytogenetic pattern might be used as a tool to assess prognosis in UPTs, even without the detection of their primary site.

Radioprotection, 38, 323- 340

Comparaison de systèmes d'analyse d'images cytologiques en dosimétrie biologique (FRENCH)

L. Roy, M. Delbos, N. Paillole, V. Durand, P. Voisin

La technique de référence en dosimétrie biologique est basée sur le dénombrement des aberrations chromosomiques de type dicentrique induit par les rayonnements ionisants. Cet article présente divers systèmesd'analyse d'images utilisés en dosimétrie biologique pour aider la détection de ces aberrations. Les systèmes présentés sont le CYTOGEN de la société IMSTAR, le CYTOSCAN (APPLIED IMAGING) et le METAFER (METASYSTEM). Tous ne présentent pas les mêmes fonctionnalités et chacun peut être utilisé de façon plus ou moins automatique. Certaines fonctionnalités communes de ces systèmes sont comparées. L'aide apportée par les systèmes porte sur 3 points : (1) localisation automatique des métaphases sur les lames, dans ce cas on a un gain de temps d'un facteur 2 à 4 par rapport au comptage manuel ; (2) un outil d'aide au comptage qui apporte un confort de lecture et une meilleure fiabilité des résultats ; (3) la détection automatique des dicentriques est particulièrement utile en cas de tri de population. En effet, dans ce cas il faut estimer très rapidement la dose reçue par un nombre important de personnes. Par contre, l'estimation de dose n'a pas besoin d'être aussi précise que dans le cas de l'expertise individuelle. Des erreurs dans la détection des dicentriques est alors tolérée et une détection automatique des dicentriques est envisageable. Le gain de temps est très appréciable puisqu'il est possible de compter 300 cellules en une demie-heure (METAFER) contre 25 avec la seule aide du chercheur de métaphases. Cependant la qualité de la détection doit encore être améliorée puisque 50 % des dicentriques ne sont pas détectées. Le marquage des centromères par technique FISH devrait permettre d'améliorer la sensibilité de la technique. Les premiers résultats sont encourageant puisque 90 % des centromères sont correctements détectés mais d'autres expériences doivent êtres réalisées pour évaluer le gain de temps.

Int. J. Cancer, 105, 347- 352

Two novel in vitro human hepatoblastoma models, HepU1 and HepU2, are highly characteristic of fetal-embryonal differentiation in hepatoblastoma

S. Scheil, S. Hagen, S. Brüderlein, I. Leuschner, W. Behnisch, P. Möller

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.

Blood, 102, 2395- 2402

AML with 11q23/MLL abnormalities as defined by the WHO classification: incidence, partner chromosomes, FAB subtype, age distribution, and prognostic impact in an unselected series of 1897 cytogenetically analyzed AMl cases.

C. Schoch, S. Schnittger, M. Klaus, W. Kern, W. Hiddemann, T. Haferlach

Acute myeloid leukemia (AML) cases with 11q23 abnormalities involving the MLL gene comprise one category of recurring genetic abnormalities in the WHO classification. In an unselected series of 1897 AML cases, 54 patients with an 11q23/MLL rearrangement were identified, resulting in an incidence of 2.8%. The incidence of AML with MLL rearrangement was significantly higher in therapy-related AML (t-AML) than in de novo AML (9.4% vs 2.6%, P <.0001). The frequency of MLL rearrangements was significantly higher in patients younger than 60 years (5.3% vs 0.8%, P <.0001). While the incidence of MLL rearrangements in AML M4, M5a, and M5b was 4.7%, 33.3%, and 15.9%, respectively, it was found in only 0.9% of all other French-American-British (FAB) subtypes (P <.0001). Compared with AML with intermediate karyotype, AML with 11q23/MLL rearrangement had a worse outcome, which was rather comparable with AML with unfavorable karyotype. Compared with t-AML, the median overall survival (OS) of de novo AML with MLL rearrangement was significantly better (2.5 vs 10 months, P =.0143). No significant differences in median OS were observed between cases with t(9;11) compared with all other MLL rearrangements (10.0 vs 8.9 months, P =.36). In conclusion, the category AML with 11q23/MLL abnormalities accounts for 2.8% of unselected AML, is closely associated with monocytic differentiation, and has a dismal prognosis.

Eur J Pediatr, 162, 582- 588

A multiple translocation event in a patient with hexadactyly, facial dysmorphism, mental retardation and behaviour disorder characterised comprehensively by molecular cytogenetics. Case report and review of the literature.

J. Seidel, A. Heller, G. Senger, H. Starke, I. Chudoba, C. Kelbova, H. Tönnies, H. Neitzel, C. Haase, V. Beensen, F. Zintl, U. Claussen, T. Liehr

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.

Int. J. Oral and Maxillofacial Surgery, 32, 198- 200

Search for deleteion 22q11.2 in interphase nuclei of buccal mucosa of patients ascertained by isolated cleft palate: a new diagnostic approach

N. Shouman, B. Pabst, M. Arslan-Kirchner, A. Eckardt, R. Schönweiler, M. Ptok, Y. Mehraein, J. Schmidtke, K. Miller

A new approach for the detection of chromosome deletion 22q11.2 in interphase nuclei from buccal mucosa cells obtained by a non-invasive procedure is described. FISH analysis has been performed on samples from a group of 101 patients that presented consecutively for speech therapy and/or surgical correction of cleft palate. A normal result has been obtained in 98 patients; a deletion 22q11.2 was present in three patients (2.8%) with cleft palate.

American Journal of Medical Genetics, 116, 26- 30

First Patient with trisomy 21 accompanied by an aditional der(4)(:p11->q11:) plus partial uniparental disomy 4p15-16

H. Starke, B. Mitulla, A. Nietzel, A. Heller, V. Beensen, G. Grosswendt, U. Claussen, von Eggeling, F., T. Liehr

We report on a rare additional numerical chromosomal aberration in a child with Down syndrome due to free trisomy 21. The karyotype showed 48,XY,+21,+mar after GTG banding, with the marker present in 80% of cells. The supernumerary marker chromosome (SMC) was as small as approximately one-third of 18p, and with the recently developed centromere-specific multi-color fluorescence in situ hybridization (cenM-FISH) technique, it was shown that the SMC was a derivative chromosome 4. The SMC was not specifically stained by arm-specific probes for chromosome 4; thus, it has been described as der(4)(:p11 --> q11:). Microsatellite analysis resulted in a partial maternal uniparental isodisomy (UPD) for chromosome 4p15-16 and a maternal origin for two chromosomes 21. Until now only two similar cases have been described in the literature, but without clarifying the origin of the SMC and without looking for an additional UPD. This is the only reported case of a UPD 4p in a liveborn child.