Atlas of Genetics and Cytogenetics in

Oral presentation Homozygous deletions are often seen in cancer as a means of inactivating tumour suppressor genes. However, such deletions can also result from genome instability and as such probably represent passenger events that do not contribute to carcinogenesis. These passenger deletions are often associated with known regions of genomic fragility (fragile sites). We have analysed approximately 800 cancer cell lines together with 466 normal DNAs using one of the latest high density SNP arrays (Affymetrix Genome-Wide Human SNP Array 6.0 containing over 1.8 million loci). This screen provides integrated analysis of copy number and genotyping information and is probably the largest analysis of its type performed to date. Over 10000 homozygous deletions were identified within the set of cancer cell lines. Of these >3400 deletions were putatively somatic. These somatic homozygous deletions collectively accounted for over 552Mb of genomic DNA (>18% of the genome). There were approximately 300 clusters of two or more overlapping homozygous deletions which accounted for ~2200 deletions. 14 clusters include known tumour suppressor genes and account for ~450 homozygous deletions, while ~300 deletions are within the defined footprint of the well mapped common fragile sites. Analysing the deletion patterns seen over known loci provides a means to classify previously unknown homozygous deletion clusters. There is evidence that a substantial proportion of these may be regions of cancer associated fragility, although a minority may be novel tumour suppressor genes. © Atlas of Genetics and Cytogenetics in Oncology and Haematology 11 European Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 3 Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Genetic changes in solid tumours Genomic profiling of breast cancers J Adelaide 1 *, P Finetti 1 *, I Bekhouche 1 , S Raynaud 1 , F Sircoulomb 1 , J Bonansea 1 , E Charafe-Jauffret 1,2 , J Jacquemier 1,2 , P Viens 3 , F Bertucci 1,3 , D Birnbaum 1 , M Chaffanet 1 1 Marseille Cancer Institute, Department of Molecular Oncology, UMR599 Inserm and Institut Paoli-Calmettes, Marseille, France. 2 Department of BioPathology, Institut Paoli-Calmettes, Marseille, France. 3 Department of Medical Oncology, Institut Paoli-Calmettes, Marseille, France. * J Adelaide and P. Finetti have equally contributed to this work. Published in Atlas Journal in October 2008 Abstract Oral presentationOral presentation Breast cancer (BC) is a complex, heterogeneous disease at the molecular level. Accumulation and combination of genetic and epigenetic alterations cause tumorigenesis, genetic instability, and acquisition of an increasingly invasive and resistant phenotype. High-throughput molecular analyses provide an unprecedented opportunity for resolving BC heterogeneity and identifying new classes biologically and clinically relevant. We recently reported the integrated genomic profiling of basal and luminal breast cancers (Adelaide et al, Cancer Res., 2007) by applying combined high resolution genomic analyses (244K aCGH [Agilent Technologies] + U133 Plus 2.0 human [Affymetrix]). The results support the existence of specific oncogenic pathways in basal and luminal BCs, involving several potential oncogenes and tumor suppressor genes (TSG). In basal tumors, 73 candidate oncogenes were identified in chromosome regions 1q21-23, 10p14, and 12p13, and 28 candidate TSG in regions 4q32-34 and 5q11-23. In luminal BCs, 33 potential oncogenes were identified in 1q21-23, 8p12-q21 11q13 and 16p12-13 and 61 candidate TSG in 16q12-13, 16q22-24 and 17p13. HORMAD1 (p=6.5 10) and ZNF703 (p=7 10) were the most significant basal and luminal potential oncogenes, respectively. Using the same strategy, we have now extended our study to various molecular subtypes and other types of BC associated with poor evolution and aggressiveness including inflammatory BC and young women BCs. The integrated genomic profiles were established to facilitate the search for specific molecular signatures and new cancer-associated genes. We hope that this analysis of the different aggressive forms of BCs will provide a better understanding of mammary carcinogenesis to help in the development of appropriate treatments. © Atlas of Genetics and Cytogenetics in Oncology and Haematology 11 European Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 4 Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Genetic changes in solid tumours Chromosome translocations in breast cancer K Howarth 1 , K Blood 1 , B Ng 2 , J Beavis 1 , Y Chua 1 , S Cooke 1 , JCM Pole 1 , S Chin 3 , K Ichimura 4 , VP Collins 4 , I Ellis 5 , C Caldas 3 , N Carter 2 , PAW Edwards 1 1 Hutchison-MRC Research Institute, Addenbrooke's Site, University of Cambridge, Hills Road, Cambridge, CB2 0XZ, UK 2 Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK 3 Cancer Research UK Cambridge Research Institute, Cambridge, UK 4 Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Addenbrooke’s, Hills Road, Cambridge, UK 5 Department of Histopathology, School of Molecular Medical Sciences, University of Nottingham, Nottingham, UK Published in Atlas Journal in October 2008 Abstract Oral presentationOral presentation Relatively little is known about chromosome translocations in the common epithelial cancers such as breast cancer, in spite of the central role played by translocations and consequent gene fusions in haematopoietic cancers. We present a comprehensive analysis by array painting of the chromosome translocations of breast cancer cell lines HCC1806, HCC1187 and ZR-75-30. In array painting, chromosomes are isolated by flow cytometry, amplified and hybridized to DNA microarrays. A total of 200 breakpoints were identified and all were mapped to 1Mb resolution on BAC arrays, then 40 selected breakpoints, including all balanced breakpoints, were further mapped on tiling-path BAC arrays or to around 2kb resolution using oligonucleotide arrays. Many more of the translocations were balanced than expected, either reciprocal (8 in total) or balanced for at least one participating chromosome (19 paired breakpoints). Many breakpoints were at genes that are plausible targets of oncogenic translocation, including CTCF and P300. Two gene fusions were also demonstrated, TAX1BP1-AHCY and RIF1PKD1L1. A preliminary screen of paraffin sections of breast tumours revealed breaks in several genes, including PKD1L1, so the rearrangements are not confined to cell lines. Our data establishes that array painting is a very effective way to map substantial numbers of translocation breakpoints and supports the emerging view that chromosome rearrangements that fuse, activate or otherwise alter genes at their breakpoints may play an important role in common epithelial cancers. © Atlas of Genetics and Cytogenetics in Oncology and Haematology 11 European Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 5 Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Genetic changes in solid tumours Patterns of genomic instability associated with cell cycle and DNA repair in EWING Sarcomas: Gene expression and a-CGH profiling BI Ferreira, J Alonso, J Carrillo, F Acquadro, C Largo, J Suela, MR Teixeira, N Cerveira, A Molares, G Gomez-Lopez, A Pestaña, A Sastre, P Garcia-Miguel, JC Cigudosa Molecular Cytogenetics Group, Centro Nacional de Investigaciones Oncologicas (CNIO), C/ Melchor Fernandez Almagro, 3, 28029 Madrid, Spain. Published in Atlas Journal in October 2008 Abstract Oral presentationOral presentation Ewing’s sarcoma (ES) is characterized by specific chromosome translocations, the most common being t(11;22)(q24;q12). Additionally, other type of genetic abnormalities may occur and be relevant for explaining the variable tumour biology and clinical outcome. We have carried out a high-resolution arrayCGH and expression profiling on 25 ES tumour samples to characterize the DNA copy number aberrations (CNA) occurring in these tumours and determine their association with gene-expression profiles and clinical outcome. CNA were observed in 84% of the cases. We observed a median number of three aberrations per case. Besides numerical chromosome changes, smaller aberrations were found and defined at chromosomes 5p, 7q and 9p. All CNA were compiled to define the smallest overlapping regions of imbalance (SORI). A total of 35 SORI were delimited. Bioinformatics analyses were conducted to identify subgroups according to the pattern of genomic instability. Unsupervised and supervised clustering analysis (using SORI as variables) segregated the tumours in two distinct groups: one genomically stable (3 CNA). The genomic unstable group showed a statistically significant shorter overall survival and was more refractory to chemotherapy. Expression profile analysis revealed significant differences between both groups. Genes related with chromosome segregation, DNA repair pathways and cell-cycle control were upregulated in the genomically unstable group. This report elucidates, for the first time, data about genomic instability in ES, based on CNA and expression profiling, and shows that a genomically unstable group of Ewing’s tumours is correlated with a significant poor prognosis. © Atlas of Genetics and Cytogenetics in Oncology and Haematology 11 European Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 6 Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Genetic changes in solid tumours Are ER+PR+ and ER+PRbreast tumours genetically different? An array CGH study A Carracedo 1,2,3 , M Salido 1,2,3 , JM Corominas 4,5 , BI Ferreira 6 , I Tusquets 7 , C Corzo 8 , M Segura 9 , B Espinet 1,2,3 , JC Cigudosa 6 , J Albanell 5,7 , S Serrano 1,2,3 , F Solé 1,2,3 1 Servei de Patologia, Laboratori de Citogenetica i Biologia Molecular, Hospital del Mar, IMAS, IMIM, Universitat Autonoma de Barcelona, Spain. 2 Escola de Citologia Hematologica S. Woessner-IMAS, Barcelona, Spain. 3 Unitat de Recerca Translacional en Tumors Solidos-PRBB, Barcelona, Spain. 4 Unitat de Patologia Mamaria del Servei de Patologia, Hospital del Mar, Barcelona, Spain. 5 Biomarkers and molecular therapeutics in breast cancer, Research Cancer Program, IMIM-Hospital del Mar, Barcelona, Spain. 6 Grupo de Citogenética Molecular, CNIO, Madrid, Spain. 7 Servei d’Oncologia, Hospital del Mar, Barcelona, Spain. 8 Escola Bonanova-IMAS, Barcelona, Spain. 9 Unitat de Patologia Mamaria del Servei de Cirurgia, Hospital del Mar, Barcelona, Spain. Published in Atlas Journal in October 2008 Abstract Oral presentationOral presentation Estrogen receptor (ER) is an accepted predictor of response to endocrine therapy. More than 20% of ER+ breast cancers express progesterone receptor (PR). Clinical observations have indicated that ER+/PR-breast cancers could present a different pattern of hormone sensitivity than ER+/PR+ ones. Furthermore, ER+/PR-tumors are more likely to have and aggressive phenotype and it could be a link between their progesterone negativity and an hypereactive growth factor signaling. The aim of this study is to investigate the pattern of DNA copy number aberrations (CNAs) of these two subtypes of breast carcinoma, establish the smallest overlapping regions of imbalance (SORIs), and try to identify differences between them using a high-resolution array CGH (a-CGH). The agilent 44K oligonucleotide a-CGH has been applied to 25 ER+/PR+ and 25 ER+/PR-ductal infiltrating carcinomas (DICs) of the breast (histological grades I, II, and III, negative ERBB2 status). Data analysis and chromosome segmentation was performed with the InSilicoArray CCGH software. For each sample, FISH was used to validate and to define cut-off values for gains and losses. Preliminary results show: The total altered genome was 20,3% in ER+/PR+ and 31,7% in ER+/PR-. The % of gained genome was 9,4 in ER+/PR+ and 16,3 in ER+/PR-tumors. The % of lost genome was 10,9 in ER+/PR+ and 15,3 in ER+/PR-tumors. The most frequently gained chromosomes (chrs.) in ER+/PR-tumors were 1, 16, 8 and 11, and in ER+/PR-tumors were 1, 17, 11, 8 and 20. The most frequently lost chrs. in ER+/PR+ tumors were 16, 1, 6 and 11, and in ER+/PR-tumors were 17, 22, 16, 11, 1 and 8. Considering the most frequently altered chrs., we found some SORIs in common between the two subtypes of breast tumors: gains of 1q25.2-q31.3, 1q32.1, 8q24.3 and 11q13.3; also losses of 1p21.1, 8pter-p21.2, 11q14.2, 11q14.3-qter, and 16q13-qter. Despite their similar CNAs, we found that the ER+/PR-tumors showed some 11 th Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Carracedo A et al Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 7 frequently altered regions not present in the ER+/PR+ tumors, related to the chrs. 1, 8, 16, 17, 20 and 22. Chrs. 17 and 22 are the most differently altered in the ER+/PR-tumors compared to the ER+/PR+ ones, specifically gain of 17p13.3-p12 and loss of 22q11.23-qter. In our knowledge, this is one of the first study focused on the genetic comparison of ER+/PR+ and ER+/PR-breast tumors by aCGH technology. In a global analysis of the 41 DICs, CNAs were consistent with the results previously reported on CGH and aCGH studies. Taking into account our preliminary results, ER+/PR-tumors present a higher chromosomal instability and have a different genetic profile compared to the ER+/PR+ ones. Acknowledgements: Grant PI05/0961 from Ministerio de Sanidad y consumo ISCIII © Atlas of Genetics and Cytogenetics in Oncology and Haematology 11 European Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 8 Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Genetic changes in solid tumours Genomic aberrations associated with poor survival in Malignant Peripheral Nerve Sheath Tumors HR Brekke, FR Ribeiro, M Eken, GE Lind, M Eknæs, KS Hall, B Bjerkehagen, E van den Berg, S Smeland, MR Teixeira, N Mandahl, RI Skotheim, F Mertens, RA Lothe Department of Cancer Prevention, Institute for Cancer Research, The Norwegian Radium Hospital, Rikshospitalet University Hospital, Montebello, Oslo, Norway. Published in Atlas Journal in October 2008 Abstract Oral presentationOral presentation Background : Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are rare neoplasias associated with a poor clinical outcome, for which few treatment options exist. Tumors may arise sporadically, but commonly develop in patients with the hereditary condition neurofibromatosis type1 (NF1). Due to the limited number of reported cases and the genomic complexity often observed, it is unclear which genetic aberrations are contributing to the initiation, progression and clinical aggressiveness, and whether MPNST etiology is similar in patients with and without NF1. Material and methods : As part of a long-standing international collaboration involving Norway, Sweden and the Netherlands, a series of fresh-frozen material from 48 MPNSTs and 10 neurofibromas from 51 patients with (n=31) and without (n=20) NF1 history was obtained. Chromosomal and array-based comparative genomic hybridization (aCGH) were performed to assess DNA copy number changes, and genome-wide expression data for a subset of samples (n=30) was later integrated to evaluate candidate target genes within regions recurrently affected by copy number aberrations. Results : Forty-four MPNSTs (92%) displayed copy number changes, whereas no aberrations were found in the nine neurofibromas. A small deletion at 9p was identified as the sole alteration in a plexiform neurofibroma. Most tumors presented complex profiles with a median of 16 aberrations per sample. Gains at 17q (69%), 8q (65%) and 7p (56%), and losses at 9p (46%), 11q (46%) and 17p (42%) are the most common. Several homozygous deletion could also be pinpointed, in particular 9p21 loss seen in five out of 12 samples by aCGH. Expression data confirmed significant differences in mRNA levels of several candidate genes, such as the lower expression of CDKN2A and CDKN2B at 9p21 or the very high levels of BIRC5 at 17q25. Interestingly, no significant differences were found in the genomic profiles of sporadic versus NF1-related MPNSTs. Several genomic changes showed prognostic significance independently of clinical variables or patient group. In particular, patients whose tumors displayed concurrent gains at chromosomal regions 7p and 17q, and losses at 9p, displayed a significantly worse prognosis (p=0.00008). Conclusions: Most MPNSTs display complex profiles that make it difficult to pinpoint primary genetic events, as virtually all chromosomes show recurrent gains and/or losses of genomic material. No differences could be found in the genetic profiles of sporadic tumors as compared to those from patients with NF1. However, the simultaneous occurrence of specific genetic aberrations (gains of 7p and 17q, and loss of 9p) was strongly associated with an aggressive clinical course independently of patient group or clinical variables. Putative targets in these regions are CDKN2A (9p) and BIRC5 (17q), confirmed to be differentially expressed in MPNSTs when compared to their benign counterparts. © Atlas of Genetics and Cytogenetics in Oncology and Haematology 11 European Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 9 Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Genetic changes in solid tumours Cytogenetic and molecular cytogenetic findings in lipoblastomas H Bartuma 1 , HA Domanski 2 , F Vult Von Steyern 3 , CM Kullendorff 4 , N Mandahl 1 , F Mertens 1 1 Department of Clinical Genetics, 2 Department of Cytology and Pathology, 3 Department of Orthopedics, 4 Department of Pediatric Surgery, Lund University Hospital, SE-221 85 Lund, Sweden. Published in Atlas Journal in October 2008 Abstract Poster presentationPoster presentation Lipoblastoma is a benign lipomatous tumor, which arises from embryonic adipose tissue and occurs primarily in children younger than three years of age. Microscopically, it consists of small irregular lobules of adipocytes at different maturation stages separated by connective tissue septa and primitive mesenchymal areas with a loose myxoid matrix. Here, we present a review of available cytogenetic data and the karyotypes of ten new cases of lipoblastoma, of which seven could be further studied by fluorescence in situ hybridization (FISH) concerning the involvement of the PLAG1 gene. All seven tumors with clonal aberrations harbored breakpoints in 8q11-13, in agreement with literature data; including previously published cases, 33 of 40 (82%) lipoblastomas had rearrangement of the 8q11-13 region. These rearrangements target the PLAG1 gene, which becomes up-regulated through promoter swapping. FISH revealed that five of seven cases in our series had a rearrangement of the PLAG1 gene. Occasionally, there can be difficulties in distinguishing a lipoblastoma from a conventional lipoma or a myxoid liposarcoma. As rearrangements of 8q11-13 have only been reported in 3% of conventional lipomas and never in myxoid liposarcomas, cytogenetic analysis or FISH for the PLAG1 gene can provide useful differential diagnostic information. © Atlas of Genetics and Cytogenetics in Oncology and Haematology 11 European Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 10 Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Genetic changes in solid tumours Characterization of NCI-H69 and NCI-H69AR Small Cell Lung Cancer (SCLC) cell lines by Spectral Karyotype (SKY) M Salido 1,3 , E Arriola 2 , A Carracedo 1 , A Rovira 4 , B Espinet 1,3 , F Rojo 4 , M Arumi 1,4 , I Calzadas 4 , S Serrano 1 , J Albanell 2,4 , F Solé 1,3 1 Pathology Department, Cytogenetic and Molecular Laboratory, IMAS, GRETNHE, IMIM-Hospital del Mar, Barcelona, Spain. 2 Servei de Oncologia del Hospital del Mar, Barcelona Spain. 3 Escola de Citologia Hematologica S. Woessner-IMAS, Barcelona, Spain. 4 Molecular Therapeutics and Biomarkers in Breast Cancer Program, IMIM-Hospital del Mar, Barcelona, Spain. Published in Atlas Journal in October 2008 Abstract Poster presentationPoster presentation Small cell lung cancer (SCLC) represents about 15% of all lung cancers, is invariably associated with cigarette smoking and distant metastases are often present at diagnosis. SCLC shows an excellent sensitivity to the chemotherapy at the beginning of the treatment, but develops resistance after few months. Research cellular models in vitro to study SCLC are based on assays using sensitive and resistant to chemotherapy cell lines. NCIH69 (sensitive) and NCI-H69AR (resistant) cell lines, purchased from ATCC (American Type Culture Collection) are used. NCI-H69 cell line was originally obtained from an untreated male patient diagnosed as SCLC. The NCI-H69AR was derived from the first one and shows resistance to topoisomerase II alpha inhibitors (doxorubicin). The G-banding karyotype of H69 parental cell line was previously described (Whang-Peng et al, 1982), showing several chromosome aberrations that include del(11)(q23), der(19)t(9;19), del(17)(p12) and dmins containing N-MYC (2p24.1) amplification. No hsr-bearing chromosomes were identified in parental H69 cell line. In addition, unbalanced t(5;16) was described (Slovak et al, 1991) by G-banding analysis. The analysis of NCI-H69AR revealed substantial karyotypic changes, including der(16) chromosome, hsr regions and dmins presented 16p13.1 amplification, which locates multi-drug resistance gene (MRP gene). Multi-color FISH technique (Spectral Karyotype-SKY) in combination with G-banding was used in order to screen cytogenetic aberrations of H69 and H69AR cell lines to find marker chromosomes responsible of sensitivity to topoIIa inhibitors. The current SKY protocol has been used with minor modifications to improve the spectral image. We have analysed a minimum of ten metaphases per karyotype and they have been described according to the International System for Human Cytogenetic Nomenclature (ISCN, 2005). The SKY of H69 cell line revealed translocations not previously defined by conventional cytogenetics [del(5)(q13), add(12)(p?), del(15)(q22qter), add(18)(q?), der(19)t(12;13;19)(?;?;q12), der(20)t(1;20)(q21;p13), der(20)t(3;20)(?;q11) and der(22)t(12;22)(p11;q11)]. The analysis of H69AR cell line showed several chromosome translocations (25~30) that were difficult to define by G-banding, and the SKY technique confirmed structural abnormalities not observed in H69 karyotype [der(1)t(1;12)(p12;q11), der(3)t(3;4)(p21;?), der(4)t(3;4)(?;p15), der(5)t(5;15)( ?;?), der(7)t(1;7)(q25;q22), der(7)t(7;14)(p22;?), der(9)t(9;21)(q34;?), der(10)t(4;10)(q21;p11)x2, der(13)t(13;18)(q?;q?),der(15)t(X;15)(q11;p11),der(16)t(3;16;18;5;18),der(19)t(9;19)(q13;q12) and 11 th Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Salido M et al Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 11 der(21)t(12;21)]. Of interest, a der(16) with a complex translocation t(3;16;18;5;18) showed a hsr region with 18q amplification, and a hsr in der(13) with 18q amplification was observed. Whole chromosome painting probes (WCP) were used to confirm the complex translocation t(12;13;19) in the H69 and the structural abnormalities detected by SKY from the H69ARThe application of SKY technique provides a useful complementary technique to routine conventional cytogenetics for the accurate characterisation of SCLC cell lines. This technique allows defining new chromosomal aberrations not well defined previously, in order to set rules for future studies. The study of chromosomal aberrations in other resistant cell lines like H69AR could help to find marker regions of chemoresistance. Acknowledgements: Grant PIOP50961 from Ministerio de Sanidad y consumo ISCIII © Atlas of Genetics and Cytogenetics in Oncology and Haematology 11 European Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 12 Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Genetic changes in solid tumours Identification of novel oncogene candidates present in the highly amplified region 22q11-12 in laryngeal cancer cell lines preliminary results M Kostrzewska-Poczekaj 1 , M Giefing 1 , M Jarmuz 1 , D Brauze 1 , JI Martin-Subero 3 , R Siebert 3 , R Grenman 4 , K Szyfter 1,2 1 Institute of Human Genetic, Polish Academy of Sciences, Poznan, Poland. 2 Department of Otolaryngology, Poznan University of Medical Sciences, Poland. 3 Institute of Human Genetics, University Hospital Schleswig-Holstein Campus Kiel, Germany. 4 Department of Otorhinolaryngology -Head and Neck Surgery and Department of Medical Biochemistry, Turku University Central Hospital and Turku University, Finland. Published in Atlas Journal in October 2008 Abstract Poster presentationPoster presentation The diverse group of head and neck cancers includes laryngeal squamous cell carcinomas (LSCC). In the European Union the average, age adjusted incidence for this cancer is 9.1 / 100 000 for men and 0.6 / 100 000 for women. LSCC is a major health issue in European countries because the average 5-year survival equals only 62%. Amplification of oncogenes is a common event in human cancers, thus the present study was focused on an identification of the copy number amplifications in LSCC cancer cell lines to delineate novel oncogenes. We tested three cell lines (UT-SCC 11,22,34) derived from squamous cell carcinoma of the larynx at the University of Turku with array-CGH technique (Agilent -Human Genome CGH 44A), that spans the human genome with an average spatial resolution of approximately 75 kb. The obtained copy number profiles showed several copy number changes including in UT-SCC 11 cell line the highly amplified 22q11-12 region harbouring the putative oncogene: CRKL. CRKL (v-CRK avian sarcoma virus CT10-homolog-like) has been shown to activate the RAS and JUN kinase signalling pathways and transform fibroblasts in a RAS-dependent fashion. To determine possible amplification of CRKL gene in nine further cell lines (UT-SCC 8,18,19A,23,29,35,38,42,50) and three LSCC cell lines already tested by array-CGH, we applied real-time quantitative PCR both with SYBR Green I and FRET hydrolysis probes. The beta-2-microglobulin (B2M) gene was used as a reference; neither amplification nor deletions of B2M DNA were observed. The real-time analysis showed a CRKL gene amplification in 3 of 12 analyzed cell lines. We identified a gain of 5 copies CRKL gene over the ploidy level in UT-SCC 11 cell line as compared to the reference B2M gene and a gain of one CRKL copy in the UT-SCC 35 and UT-SCC 18 cell lines. The next step on the way is to check an expression of mRNA for CRKL gene. In any case, the obtained results are strongly indicative for an oncogenic character of CRKL in LSCC. © Atlas of Genetics and Cytogenetics in Oncology and Haematology 11 European Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 13 Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Genetic changes in solid tumours Array-CGH identifies tumor suppressor gene loci in laryngeal cancer cell lines M Giefing 1,2 , JI Martin-Subero 2 , K Kiwerska 1 , J Malgorzata 1 , R Grenman 3 , R Siebert 2 , K Szyfter 1,4 1 Institute of Human Genetics, Polish Academy of Sciences, 60-479 Poznan, Poland. 2 Institute of Human Genetics, University Hospital Schleswig-Holstein Campus Kiel, Christian-Albrechts University 24105 Kiel, Germany. 3 Department of Otorhinolaryngology Head and Neck Surgery and Department of Medical Biochemistry, Turku University Central Hospital and Turku University, P.O. Box 52, FIN-20521 Turku, Finland. 4 Department of Otolaryngology, University of Medical Sciences, 60-355 Poznan, Poland. Published in Atlas Journal in October 2008 Abstract Poster presentationPoster presentation Historically, the majority of classical tumor suppressor genes (TSG) like CDKN2A or RB1 were identified by the delineation of bi-allelic losses called homozygous deletions. To identify small, hitherto undetected homozygous deletions in laryngeal squamous cell carcinoma cell lines (LSCC) and to unravel novel putative tumor suppressor genes three LSCC cell lines were screened with the high resolution array Comparative Genomic Hybridization (array-CGH) technique (Agilent -Human Genome CGH 44B). Altogether, 31 candidate regions for homozygous deletions were identified by array-CGH. Out of these, 12 regions overlapped with known polymorphic sites delineated in the Database of Genomic Variants and Human Structural Variation Database and thus were excluded from the analysis. The remaining 19 candidate regions were further tested by multiplex PCR and 5 regions were verified as homozygous deletions. Among others, these homozygous deletions affected the apoptosis inducing STK17A gene in one cell line and the tumor suppressor CDKN2A in two cell lines. To assess the frequency of the identified deletions in a larger panel of samples we investigated the affected sites in 9 additional LSCC cell lines. In 5 of the 9 cell lines the CDKN2A gene was found homozygously lost. Thus, CDKN2A was homozygously deleted in a total of 7 of 12 analyzed cell lines. No other recurrent homozygous deletions were found. Summing up, in this study, we show homozygous deletions to be a frequent mechanism of CDKN2A inactivation in laryngeal cancer cell lines. Moreover, we present several other genes, including the putative tumor suppressor STK17A, which may be inactivated by homozygous deletions and thus, potentially implicated in laryngeal squamous cell carcinoma development. © Atlas of Genetics and Cytogenetics in Oncology and Haematology 11 European Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 14 Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Genetic changes in solid tumours Narrowing the breakpoint in deletion del(8)(q12.1q22.1) detected in cell lines derived from larynx cancer M Jarmuz 1,2 , A Abramowska 1 , M Giefing 1 , R Grenman 3 , K Szyfter 1,4 1 Institute of Human Genetics, Polish Academy of Sciences, 60-479 Poznan, Poland. 2 Department of Hematology, University of Medical Sciences, 60-355 Poznan, Poland. 3 Department of Otorhinolaryngology Head and Neck Surgery and Department of Medical Biochemistry, Turku University Central Hospital and Turku University, P.O. Box 52, FIN-20521 Turku, Finland. 4 Department of Otolaryngology, University of Medical Sciences, 60-355 Poznan, Poland. Published in Atlas Journal in October 2008 Abstract Poster presentationPoster presentation The head and neck squamous cell carcinomas (HNSCC) are characterized by a huge variety of numerical and structural chromosome aberrations. At this stage it is a major difficulty to categorize chromosome aberrations and to select those attributed to HNSCC progression. Ten cell lines derived from laryngeal cancer were analyzed by GTG banding and comparative genomic hybridization (CGH). The number of chromosomes ranged from near-diploid to near-tetraploid but most frequently a near-triploid chromosome content was found. In the studied cell lines the following alterations were established as the most frequent: amplification of 11q13, deletions of 9p21-212, 18q, 3p12-24, gains of 3q, loss of 13q, 14q, loss or rearrangement of chromosome Y. Less frequent were the gains of 1q, 5p, 7p, 8q, 20q and losses of 8p, 7q22-qter, 22, 21q11-q21. The interstitial deletion of long arm of chromosome 8 was established by GTG in three cell lines. The aim of this study was focused on determine if any gene is damaged by this interstitial deletion. In UT-SCC-11 cell line the latter deletion was confirmed by array CGH. A further narrowing of the breakpoint by FISH using clones (BACs and fosmids) points to the region where the PDGP gene (plasma glutamate carboxypeptidase) is located. This gene can be affected by the identified 8q chromosome interstitial deletion. An involvement of this gene in prostate cancer progression was already suggested. The results of our study seem to suggest its involvement into laryngeal squamous cell carcinoma as well. We also hypothesize that because of different anatomical locations and histological types of prostate cancer and HNSCC a function of PDGP gene in carcinogenesis is not restricted to a particular cancer type or location. © Atlas of Genetics and Cytogenetics in Oncology and Haematology 11 European Workshop on Cytogenetics and Molecular Genetics of Solid Tumours Bilbao, Spain, September 6-9, 2008 Atlas Genet Cytogenet Oncol Haematol 2008, 12: suppl 1 15 Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS Genetic changes in solid tumours A papillary thyroid tumor of the follicular variant harboring RET/PTC and PAX8/PPARg gene fusion in different clones P Caria 1 , T Dettori 1 , DV Frau 1 , G Tallini 2 , R Vanni 1 1 Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Cagliari, Cagliari, Italia. 2 Dipartimento Clinico di Scienze Radiologiche e Istocitopatologiche, Università di Bologna, Bologna, Italia. Published in Atlas Journal in October 2008 Abstract Poster presentationPoster presentation Thyroid cancer is the most common endocrine malignancy and accounts for the majority of endocrine cancer related deaths each year. Recent molecular studies have described several abnormalities associated with the pathogenesis and the progression of thyroid lesions, leading to a consistent progress in the understanding of the biology of this tumor. In particular, several chromosomal and molecular mutations have been well-identified in the two commonest types of differentiated thyroid carcinoma, papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) (1). Activating, mutually exclusive, mutations of the BRAF, RET, or RAS genes are found in up to 70% of PTC (with almost all RAS mutations in the PTC follicular variant, PTC-FV), whereas point mutations of the RAS genes and PAX8-PPARg rearrangement are the most frequent genetic alterations in FTC. Due to their almost exclusive association, these molecular changes are making their way into use for clinical diagnostic testing, by RT-PCR and direct sequencing. Understanding the efficacy level of these tools may be important. An aggressive thyroid tumor in a 50 years old man was diagnosed as an invasive PTC-FV, i.e. a PTC showing both areas with papillary thyroid carcinoma type nuclear changes (PTC-NC), and areas with follicular architectural features. RT-PCR in microdissected areas with PTC-NC showed RET/PTC3 activation (2). Cultured cells from the excised tumor showed normal male karyotype. Nuclei from touch preparations subjected to fluorescence in situ hybridization with DNA probes on purpose, revealed two separate clones: one with RET gene disruption and the other with PAX8-PPARg gene fusion. Our results demonstrate that genetic changes usually associated with different types of thyroid carcinoma, may coexist inside the same tumor lesion, and are confined to different cell clones. Since RET/PTC and PAX8-PPARg are usually associated with diverse types of differentiated thyroid lesions, the unexpected finding of the two changes in the same nodule may be explained by the histological mixed morphology of the lesion, possibly driven by the dissimilar genetic background developed in different cells. Acknowledgements: We thank M Rocchi for the DNA probes. Supported by Fondazione Banco Sardegna and FIRB-MIUR project No RBIP0695BB.