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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. Sieglov{\'a}, S. Zilovcov{\'a}, J. Cerm{\'a}k, H. R{\'i}hov{\'a}, D. Brezinov{\'a}, R. Dvor{\'a}kov{\'a}, M. Markov{\'a}, J. Maaloufov{\'a}, J. Sajdov{\'a}, J. Brezinov{\'a}, Z. Zemanov{\'a}, K. Michalov{\'a}

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.

Genes Chromosomes Cancer, 37, 333- 345

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

J. Ad{\'e}laide, 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.

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, 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.

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.

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. Br{\"u}derlein, I. Leuschner, W. Behnisch, P. M{\"o}ller

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.

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. T{\"o}nnies, 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.

Cancer Genet. Cytogenet., 138, 153- 156

Cryptic t(X;18), ins(6;18), and SYT-SSX2 gene fusion in a case of intraneural monophasic synovial sarcoma

V.S. Lestou, J.X. O'Connell, M. Robichaud, C. Salski, J. Mathers, J. Maguire, I. Chudoba, P.H.B. Sorensen, W. Lam, D.E. Horsman

A 54-year-old male presented with a spontaneous peroneal nerve palsy and a diagnosis of monophasic synovial sarcoma (SS) was rendered by histologic examination. Cytogenetic analysis revealed a complex abnormal karyotype without evidence of the typical t(X;18)(p11;q11) associated with SS. Subsequent reverse transcriptase polymerase chain reaction analysis showed the presence of an SYT/SSX2 fusion transcript, confirming the presence of a cyptic t(X;18). In light of -X, -18 and marker chromosomes evident in the G-band karyotype, it was suspected that a cryptic chromosomal rearrangement involving the marker chromosomes would harbor an X;18 fusion. Multi-colored karytotyping (M-FISH) revealed a previously unrecognized t(X;18) and t(5;19) in the marker chromosomes as well as unrecognized ins(6;18) and t(16;20). The addition of M-FISH analysis in this case led to the identification of complex inter-chromosomal rearrangements, thus providing an accurate karyotype.

Genes Chromosomes Cancer, 35, 204- 218

Reciprocal translocations in breast tumor cell lines: cloning of a t(3;20) that targets the FHIT gene

C. Popovici, C. Basset, F. Bertucci, B. Orsetti, J. Ad{\'e}laide, M.-J. Mozziconacci, N. Conte, A. Murati, C. Ginestier, E. Charafe-Jauffret, S.P. Ethier, M. Lafage-Pochitalof, C. Theillet, D. Birnbaum, M. Chaffanet

All molecular alterations that lead to breast cancer are not precisely known. We are evaluating the frequency and consequences of reciprocal translocations in breast cancer. We surveyed 15 mammary cell lines by multicolor fluorescence in situ hybridization (M-FISH). We identified nine apparently reciprocal translocations. Using mBanding FISH and FISH with selected YAC clones, we identified the breakpoints for four of them, and cloned the t(3;20)(p14;p11) found in the BrCa-MZ-02 cell line. We found that the breakpoint targets the potential tumor-suppressor gene FHIT (fragile histidine triad) in the FRA3B region; it is accompanied by homozygous deletion of exon 5 of the gene and absence of functional FHIT and fusion transcripts, which leads to the loss of FHIT protein expression. Additional experiments using comparative genomic hybridization provided further information on the genomic context in which the t(3;20)(p14;p11) reciprocal translocation was found.

Genes, Chromosomes {\&} Cancer, 30, 274- 282

Molecular cytogenetic and clinical findings in ETV6/ABL1-positive leukemia

H. Van Limbergen, H.B. Beverloo, van Drunen, E., A. Janssens, K. H{\"a}hlen, B. Poppe, N. Van Roy, P. Marynen, de Paepe, A., R. Slater, F. Speleman

Rearrangements of 12p, resulting from deletions or translocations, are common findings in hematologic malignancies. In many cases, these rearrangements target the ETV6 gene (previously called TEL) located at 12p13. Various partner genes have been implicated in the formation of fusion genes with ETV6. These include PDGFRB, JAK2, NTRK3, ABL2, and ABL1, each of which encodes for proteins with tyrosine kinase activity. To date, ETV6/ABL1 transcripts have been detected in only four patients with a leukemic disorder. Here, we describe one adult with chronic myeloid leukemia and a child with T-cell acute lymphocytic leukemia with ETV6/ABL1. Molecular cytogenetic analysis confirmed that formation of an ETV6/ABL1 fusion in these patients required at least three chromosomal breaks and showed that each of these translocations is the result of a complex chromosomal rearrangement. Molecular analysis showed the presence of two fusion transcripts in both patients as the result of alternative splicing, questioning the suggested role of these transcripts in the lineage specificity. Clinical findings of these patients were compared to those of previously reported cases, and the possible clinical and biological similarities between ETV6/ABL1 and other fusion genes leading to increased tyrosine kinase activity are discussed.