BMC Cancer 2006, 6:2 http://www.biomedcentral.com/1471-2407/6/2 Background Treatment of human malignant gliomas often includes surgical resection followed by chemotherapy and radia- tion; however, it is common for such tumors to recur despite adjuvant therapy [1]. The recurrent tumor is often resistant to further therapy with the same agent, suggest- ing that cells which survive treatment and repopulate the tumor mass have an intrinsic genetic advantage. We have previously demonstrated that cells selected for resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in vitro or in vivo (recurrent tumor) were near diploid, with over- representation of part or all of chromosomes 7 and 22 [2]. Whereas over-representation of chromosome 7 is com- mon in gliomas, chromosome 22 is not typically over-rep- resented, and in fact it is often under-represented in untreated gliomas [3-7]. However, when a sufficient number of karyotypes are done it is possible to identify cells with over-representation of chromosome 22 in untreated tumors. These cells represent a very minor pro- portion of the cells in the primary, untreated tumor, but become a major subpopulation after treatment. We were able to demonstrate that this is likely due to selection of these cells through the identification of karyotypic mark- ers in cells from the primary and recurrent tumors [2]. Thus, selection for cells with over-representation of chro- mosome 22 sequences by BCNU treatment suggests the presence on this chromosome of a gene or genes that con- fer a selective advantage to these cells. We originally analyzed the expression of platelet-derived growth factor because the genes encoding the A and B chains of this growth factor are mapped to chromosome regions 7p22 and 22q13.1, respectively. Whereas we dem- onstrated increased expression of these genes in some BCNU-resistant cells, it was likely that this over-expres- sion provided a growth advantage and was not directly involved in resistance [8]. We also analyzed the expres- sion of glutathione-S-transferase theta 1, a gene mapped Table 1: Patient information to 22q11.23. Over-expression of this gene at the RNA level was not found in the majority of our BCNU resistant cells [9]. The specific over-representation of chromosome 22 sequences provides strong evidence that a gene(s) on this chromosome is important for survival after therapy and/ or therapy resistance. The availability of samples from tumors that recurred following therapy with BCNU and radiation provided us with a unique opportunity to exam- ine cells that survived therapy in vivo. To assist in the iden- tification of the gene(s) involved in the growth of BCNU resistant cells, we established sets of 4 cell lines from each of three patients (Table 1); cells from the primary tumor, cells from the primary tumor selected for resistance to 10 µ g/ml of BCNU in vitro, cells from recurrent tumor (in vivo selection), and cells from recurrent tumor selected for resistance to 10 µ g/ml of BCNU. These cells were analyzed for the retention of chromosome 22 by karyotypic analy- ses and the specific regions that were retained were identi- fied using fluorescent in situ hybridization with bacterial artificial chromosome (BAC) probes. Methods Cell culture and selection for drug resistant cell populations The availability of samples from tumors that recurred after therapy provided us with a unique opportunity to exam- ine cells that survived therapy in vivo. Patients from whom we have cell lines derived from both primary and recur- rent tumor samples were used for this study (Table 1). The time between surgeries and overall survival for patients this age was not unusual. All three patients received gross total resections of their primary tumor and all were then treated with BCNU and radiation. All three showed increased enhancement within 3 months of the primary surgery, demonstrating rapid progression of the tumors. Tumor codea Ageb Sex Diagnosisc Treatmentd DI/DIR LX/LXR ME/MER 38 48 49 F M F GBM Gliosarc./GBMe GBM Irradiation and BCNU Irradiation and BCNU Irradiation and BCNU Days between primary and secondary surgery 111 203 138 Survival (days) 426 466 285 a The tumor code is a random 2 letter code. Recurrent tumor from the same patient receives the same code with the addition of R. For each patient the primary and recurrent tumors were diagnosed by the same neuropathologist. b Age in years at diagnosis of primary tumor. c GBM = glioblastoma multiforme; gliosarc. = gliosarcoma. d BCNU = 1,3-bis(2-chloroethyl)-1-nitrosourea. e Different diagnoses of primary and recurrent tumors. Gliosarcoma is considered a variant of glioblastoma multiforme and the gliomatous and sarcomatous areas have similar genetic profiles and are thought to have a monoclonal origin [22,23]. Tumor LX was primarily glial, with a small sarcomatous element. Page 2 of 10 (page number not for citation purposes)