1C3) compared to control/baseline array (vertebral disc) and 3-fold expression switch chondrosarcoma cell collection/baseline array

1C3) compared to control/baseline array (vertebral disc) and 3-fold expression switch chondrosarcoma cell collection/baseline array. interesting candidate genes whose differential expression likely plays a role in chordoma. hybridization Introduction Chordoma is usually a rare, low-malignant bone tumor. This unique bone tumor has both epithelial and mesenchymal characteristics (1). Chordomas arise along the spine with hot spots at the upper (skull base 20C30%) and lower (sacro-coccygeal 50C60%) end, and are therefore thought to originate from remnants of the notochord (2). Chordomas grow slowly. However, due to their location, (S)-Rasagiline mesylate it is difficult to obtain wide-margin resection. Frequently, these tumors recur after surgical treatment. Systemic treatments are largely ineffective and new therapeutic methods are therefore needed. To date, no targeted therapeutic strategies have been established for chordomas. Recently, however, a phase II study showed a modest antitumor activity of lapatinib in chordoma (3C6). Chordoma characteristically occurs in adolescence and is rarely found in children. Standard and molecular cytogenetic analyses revealed chromosomal gains of 7q and losses of 1p and 3p to be the most prominent alterations in chordoma (7). In addition, loss of heterozygosity (LOH) and genome-wide linkage studies have already been successfully used to thin down and define candidate regions for chordoma development on 1p36.13 and 7q33 (8,9). Some studies focused on gene expression analysis in chordoma. (S)-Rasagiline mesylate Brachyury (T) was one of these candidates (examined in ref. 10), which was knocked down in U-CH1, resulting in striking morphological changes in the tumor cells (11). However, many specific genes or altered transcripts have yet to be determined. This study comprises a genome-wide cytogenetic analysis of 33 chordomas using comparative genomic hybridization (CGH) and, in selected cases, additional transcript profiling by microarray analysis. We linked these with RT-PCR, immunohistochemistry and FACS analysis. We performed this comprehensive study to determine those genes most differentially expressed in chordoma and thus to establish which had the most promise for translation into clinically useful targets. Materials and methods Samples We examined 33 paraffin-embedded chordoma tumor samples (for 7 of which snap-frozen tissue samples were also available) obtained from 26 patients (8 male, 18 female; median age at diagnosis: 66 years), 6 fresh-frozen, standard chondrosarcomas (6 patients; 4 male, 2 female; median age at diagnosis: 54 years; 1 clivus, 3 femur, 2 pelvis; 3 grade 1, 3 grade 2) and pooled material of short-term cultures of 2 ISGF3G vertebral discs (both male; age 47 and 63 years) from your files of the Institute of Pathology, University or college Hospitals of Ulm, Germany, Department of Orthopedics, University or college of Dsseldorf, Germany, Department of Neuropathology, Ludwig-Maximilian University or college of Munich, Munich, Germany, and Department of Neurosurgery, University or college of Kiel, Kiel, Germany (Table I). Table I. Summary of selected clinical data, histopathologic characteristics. o Met3.35.5+++NDNDA/KY*3/F/70/PSacral303.814.4++++++KY*3R/F/71/RSacral65.314.2++++NDY3R/U-CH2SacralNDND++NDNDA/KY*4/F/69/PSacral22p MetsNDNDNDNDNDNDND*4R/F/70/RSacral1448.34.5ND+++NDND*5/F/46/PSacral7245.5ND+++NDND*5R/F/52/RSacral122.32.1ND+++NDND*6/F/74/RSacral45DOD8.34.5++++++NDY7/M/68/PSacral471.74.9ND++++NDND8/F/60/PSacral8DOD6.32.6ND+++NDND9/F/78/PSacral77DOD1.91.6NDNDNDNDND10/F/56/RSacral62DOD2.45NDNDNDNDND12/M/70/PSacral58R4.42.6ND+++++NDND13/F/66/RSacral58DOD7.93.3NDNDNDNDND14/F/66/RSacral2MDODND7ND++-NDND15//M/70/RSacral382.42.8ND+NDNDND16/F/63/PSacral23ND6.1ND+-NDND17/F/72/PSacral232.12.3ND+++NDND17R1/F/73/RSacral/vaginal4.431.5ND++++++NDND17R2/F73/MetAbdominal/perianal2.48.6ND++NDNDND18/M/65/R/MetAbdominal/sacral1218.85.5ND+++++NDND19/F/17/PSpinal36DOD5.32.7ND++-NDND20/M/78/PSpinal36R152.1NDNDNDNDND21/F/63/RClivus13NDND+++NDNDNDY22/M/52/RClivus525.710.7ND+++NDND23/F/57/PClivus26ND2ND++NDND24/F/67/PClivus122.72.8ND++NDND24R/F/68/RClivus31.312.3ND+++NDND*25/M/37/PClivus663.45.7NDNDNDNDND*26/F/58/PClivus10814.23.2NDNDNDNDND26R/F/67/RClivus3.39.3ND+++NDND (S)-Rasagiline mesylate Open in a separate windows M, male; F, female; P, main chordoma; R, recurrence and radiotherapy before surgery; Met, metastasis; oMet, bone metastasis; pMet, pulmonary metastasis; DOD, died of disease; CD24 IR, immunoreactivity (physique CD24); PI, proliferation index; ND, no data; A, Affymetrix U133A/B; K, Kappler (17); Y, yes; *CGH, FISH and Ki67-PI data published before Scheil (7). The (S)-Rasagiline mesylate chordoma cell lines U-CH1 and U-CH2 were established from sacral chordoma recurrences as explained previously (7,12). The chondrosarcoma cell collection U-CS2 was established from a chondrosarcoma of the distal femur in a 48-year-old female patient, operated in 2002. One and two years after primary diagnosis, the patient underwent surgery following pulmonary metastasis of the primary grade 2 chondrosarcoma. Immunohistochemistry and fluorescence-activated cell sorter analysis (FACS) Immunostaining was performed using a routine indirect peroxidase method. The following antibodies were applied: TP53 (Dako, Denmark), Ki-67 (Dako), and CD24 (clone 24C02, Dianova, Hamburg, Germany). These antibodies were used at a final concentration of 1C2 locus) and 22q11.2 (locus), and the indirect labeled probes assigned to loci 7cen, 1p36 (all probes by Q-Biogene, Illkirch Cedex, France), and the Her2/probe (Zytomed, Germany) were applied. Additionally, we used the following YAC clones obtained from the CEPH YAC library: 801_A_8 (3p14.2), 724_G_5 (karyotype enh(1p34.2Cp36.1,7,12p,15q,Xq), (on average 15 signals per cell) in case 18. All nuclei exhibited 10 signals per nucleus. Interestingly, this case was an abdominal metastasis of a sacral chordoma 9 years after (S)-Rasagiline mesylate main diagnosis. The tumor recurred twice during the following 7 months. We checked four further samples with a gain of chromosome 17 (nos. 9, 16, 17R1 and 17R2), but they did not reveal further amplifications of Her2/esophageal malignancy.