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[CANCER RESEARCH 5K. 4260-426.1. October I. Advances in Brief Chromosomal Amplification Germ Cell Tumors1 Is Associated with Cisplatin Resistance of Human Male Pulivarthi H. Rao, Jane Houldsworth, Nallasivam Palanisamy, V. V. V. S. Murty, Victor E. Reuter, Robert J. Motzer, George J. Bosl, and R. S. K. Chaganti2 ¡Mhoralory of Cancer Genetics. Sloan-Kellering InstÃ-lalefar Cancer Research ¡P.H. R.. J. H.. N. P., R. S. K. CJ anil Departments of Pathology [V. E. R.I, Mediane ¡R.J. M.. C. J. H.¡.timi Httnuin Genetics ¡K.S. K. C.¡.Memorial Hospital. Memorial Sloan-Kellering Cancer Center. New York. New York 10021. und Department oj Pullioloi>\. College of Physicians antl Surgeons. Coliitnhia University, New York. New York 10032 ¡V.V. V. S. M.] through overexpression of cyclin D2, encoded by the CCND2 gene, mapped to this chromosomal arm (2). Although karyotypic analysis of GCTs occasionally identified the presence at sites other than 12p, of changes suggestive of gene amplification such as homogeneously staining regions and double minute chromosomes (3-5), their associ ation with the resistance phenotype has not been studied. To investi gate the possible association of chromosomal amplification with cis platin resistance, we subjected a panel of 34 GCT DNA samples derived of 17 tumors from relapse-free patients (sensitive) and 17 chemotherapy-resistant tumors belonging to multiple histologies, to CGH analysis. Our results identified chromosomal amplification (oth er than at 12pl 1.2-12) in the resistant tumors at the sites: lq31-32, 2p23-24, 7q21, 7q31. 9q22, 9q32-34, 15q23-24. and 20qll.2-12. These data demonstrate that chromosomal and. hence, gene amplifi cation may represent an important pathway in GCT resistance to cisplatin treatment. Abstract Chemotherapy resistance of tumors is an important biological and clinical problem. Studies Troni many tumor types have indicated that resistance may he hased on multiple genetic pathways. Human male (¡ermacell tumors (GCTs) are an especially good model system to study the genetic hasis of tumor sensitivity and resistance to chemotherapy. GCTs are exquisitely sensitive to treatment with DNA-damaging drugs such as cisplatin. rarely exhihit / /'5 <gene mutations, express normal p53 protein, and undergo p53-mcdiatcd apoptosis upon drug treatment. A small proportion of tumors (20-30% of metastatic lesions) escape the apoptotic response and result in treatment resistance. We have recently shown (J. Houldsworth, et al.. Oncogene. 16: 2345-2359. 1998) that in a suhset of such tumors, resistance is linked to TP53 gene mutations. In a further search for genetic mechanisms underlying resistance, we subjected a panel of 17 tumors from relapse-free patients (sensitive) and 17 chemo therapy-resistant tumors to comparative genomic hybridization analysis to identify possible amplified regions (implying amplified/overexpressed genes) associated with resistance. With the exception of 12pl 1.2-12, high Materials and Methods level amplification was not detected in any of the sensitive tumors. We have identified eight amplified regions (lq31-32, 2p23-24, 7q21, 7q31, 9q22. 9q32-34, 15q23-24. and 20ql 1.2-12) in five resistant tumors, which Patient Samples. Tumor biopsy specimens were obtained during diagnos tic evaluation from patients seen at the Memorial Sloan-Kettering Cancer suggests that chromosomal and. hence, gene amplification may comprise a pathway to drug resistance. Identification of amplified/overexpressed genes at these sites may elucidate new genetic pathways of chemotherapy resistance in GCTs and possibly also in other tumors. Center (New York). A total of 34 tumors from 32 patients comprised the study group. The sensitive cohort included 17 tumors (7 teratomas. 6 seminomas. 2 embryonal carcinomas. 1 yolk sac tumor, and 1 mixed tumor) resected from patients that were relapse-free as a result of surgical resection alone, of chemotherapy alone, or of surgical resection combined with adjuvant chemo therapy (6). Of the 17 sensitive tumors, 9 were studied at diagnosis before chemotherapy and 8 were studied after chemotherapy. Although all of the sensitive patients presented with primary teslicular tumors. 6 of the specimens studied were testicular lesions and 11 were retroperitoneal masses. Tumors resected from patients who either did not respond to cisplutin-based chemo Introduction Human mule GCTs1 arise in spermatogenous cells, display an array of histopathological differentiation patterns that resemble stages of embryonal development, and exhibit an exquisite sensitivity to a variety of chemolherapeutic agents, notably cisplatin and related comp< ids ( 1). Presently, more than 90% of all newly diagnosed patiei -nd 70-80'7r of patients with metastatic disease are cured ( 1). therapy or responded and then relapsed comprised the resistant cohort (6). Of the 17 resistant cases (5 teratomas. 4 malignant transformations. 3 yolk sac tumors. 3 seminomas. and 2 choriocarcinomas) used in this study. 4 were studied pretreatment, and 13 were studied posttreatment. Five of the tumor specimens studied were primary mediastinal lesions, and four were primary testicular lesions. The remaining eight patients presented with primary testic ular lesions, but the specimens studied were derived from seven retroperitoneal masses and one inguinal mass. CGH. Tumor DNA was extracted from fro/en tissue and subjected to CGH analysis as described, with minor modifications (7). Briefly, tumor DNA and reference DNA (normal male ftbroblast cell line) were labeled by nick trans lation with fluorescein-12-dUTP and Texas-red-5-dUTP (NEN-Dupont, Bos The t ining are at risk for relapse and death because of resistant diseast The predominant cisplatin sensitivity, coupled with infre quent but potentially lethal resistance, make these tumors a useful model system to investigate chemotherapeutic agent sensitivity and resistance. Chemotherapy resistance is an important cause of cancer treatment failure and. hence, a major clinical problem in treating this disease (1). Virtually all GCTs exhibit increased copy number of 12p which has been suggested to play a role in the origin of these tumors possibly ton. MA), respectively. Approximately 2(X)ng each of tumor and reference DNAs were coprecipitated with 15 ftg of unlabeled human cot-1 DNA (Life Technologies. Inc., Gaithersburg. MD) and resuspended in the hybridi/ation mix before hybridization to human metaphase chromosome spreads prepared from phytohemagglutinin-stimulated lymphocytes from normal individuals. After hybridi/ation for 2-3 days, the slides were washed, and the chromo somes were counterslained with 4'.6-diamino-2-phenylindole to identify the Received 7/15/98: accepted 8/17/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by N1H Grants and grants from the Byrne Fund (to R. S. K. C.). 2 To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center. 1275 York Avenue. New York. NY 10021. 1The abbreviations used are: OCT. germ cell tumor: CGH, comparative hybridisation. genomic chromosomes. Digital Image Analysis. Blue, green, and red images were captured with a cooled-charge coupled device camera attached to a Nikon Microphot-SA 4260 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1998 American Association for Cancer Research. CHROMOSOMAL AMPLIFICATION IN RESISTANT OCTS of gains and losses between histológica! groups. Fig. 1 summarizes the recurring losses, gains, and amplifications noted in tumor speci mens from relapse-free patients (sensitive) and cisplatin-resistant tu microscope and processed using the Quantitative Image Processing System (Quips. Vysis, IL). Chromosomal imbalances were detected on the basis of the ratio profiles deviating from the balance value of greenrred ratio of 1.0. Ratio values of 1.20 and 0.80 were set as upper and lower thresholds to define gains and losses, respectively. These threshold levels were thoroughly tested as reported previously by us (7). Regions near the centromere of chromosomes 1, 9. 13-16. 21, and 22 were not scored for CGH analysis because of the highly mors. Recurring gains and losses were noted in both groups. Several of these regions were common to both groups while others were unique to each group. Thus, +1, +2p, +7p, +8, +12p, +17q, +20q, - Ip, -4p, - 1Iq, - 13q, - 18q, and - 19q were noted in both groups. repeated sequences native to these sites. Chromosomal or subchromosomal gain or loss was considered recurrent if detected in two or more tumors by CGH. A region was considered amplified when the fluorescence intensity values exceeded 2.0 and the FITC showed a strong localized signal. For assignment of high level amplification to the chromosomal band, the peaks of the ratio profiles were compared to the corresponding 4'.6-diamino-2- Changes that were not shared between the two groups comprised +2q. +6p, + 13q, + 18q, - 6q, -lip, - 18p. and - 19p in sensitive tumors (Fig. IA) and +3, +6q, +14q, +20p. +21. -4q, and -9 in resistant tumors (Fig. Iß).High level regional amplification of 12pl 1.2-12 phenylindole us (7). was noted in two seminomas, one sensitive (287B) and one resistant (CG 950110-02). In both cases, this amplification was in addition to banding of individual chromosomes as previously described by an increase in the copy number of the entire 12p (Fig. 2). In contrast, high level amplification affecting chromosomal regions other than 12p were restricted to only five resistant tumors. These comprised: lq31-32 (249A), 2p23-24 (231B), 7q21 and 7q31 (203B), 9q22 (240A), 9q32-34 (243A), 15q23-24 (203B, 249A), and 20ql 1.2-12 Results CGH detected copy number changes in all 34 tumors studied. Overall, gains of chromosomal regions were more common than losses (142 versus 82). As expected, all tumors exhibited a gain of 12p. In the entire study group, recurring gains other than 12p, affected chromosomes 1, 2, 3, 6, 7p, 8. 13q, 14q, 17q, 18q, 20, and 21 whereas losses affected chromosomes Ip, 4, 6q, 9, 11, 13q, 18, and 19. Because of the small number of cases, no comparison was attempted Fig. 1. Ideograms comparing the recurring chro mosomal gains and losses detected by CGH in 17 sensitive GCTs (A) and 17 resistant GCTs (B). Thin vertical lines on either side of the chromosome ideogram indicate only recurrent gains (right) or losses (left} of a chromosome or a chromosomal region. Gains/losses were considered recurrent if observed in two or more tumors. Thick lines indi cate regions of high copy number (amplification) as defined in the text. 13 14 (243A) (Fig. 2). The histologies of these five tumors included three malignant transformations (203B, 23IB, 243A), one teratoma (240A), and one choriocarcinoma (249A). In the case of tumor 203B, it is interesting that the adjacent teratomatous lesion (203A) from which 15 16 17 18 19 20 21 22 B 45 13 14 15 67 20 21 22 4261 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1998 American Association for Cancer Research. CHROMOSOMAL the malignant transformation (rhabdomyosarcoma) hibit the same high level amplifications. AMPLIFICATION IN RESISTANT OCTS arose, did not ex 203B Discussion Understanding of the genetic mechanisms that underlie tumor cell sensitivity and resistance to chemotherapeutic agents is important from the biological as well as the clinical standpoint. Several path ways of drug resistance have been identified in tumor cells, e.g., 231B amplification and/or overexpression of membrane transporter genes that decrease the intracellular accumulation of drug, such as MDRl/ PGYÃŒ(8), MRP1 (9, 10), cMOAT (10), and LRP (9, 10); activation and/or overexpression of genes that act as agonists to apoptosis signals, such as BCL2 (11), and BCLXL (11); or inactivation of genes that regulate cell cycle, such as TP53 (12). GCTs are an excellent model system to study the resistance phenomenon. They exhibit an 240 exquisite sensitivity to cisplatin-based chemotherapy that is thought to result from the elevated expression of wild-type p53 observed in these tumors (13, 14). Their cellular response to DNA-damaging insult is apoptosis by a p53-dependent mechanism (15). In such a system, although resistance is rare, it provides opportunities to propose and test specific genetic pathways that may mediate resistance. We have 243A shown recently (15) that one such pathway in GCTs (predominantly teratomas) is inactivating missense mutations in the TP53 gene. In addition, in vitro studies of a resistant cell line with a TP53 missense mutation in one alÃ-eleshowed that although the non-mutated alÃ-ele apparently was intact, only the mutated form of the mRNA was transcribed (15). Although this predicted pathway has been validated in the present study, other mechanisms of resistance remain to be 249A identified. To this end, we decided to investigate the possible role of gene amplification in GCT resistance, a phenomenon that is observed in a number of tumor systems and is associated with tumor progres 15 sion and resistance (16). In the present study, we subjected a group of tumor specimens from relapse-free patients (sensitive) and from patients whose tumor ex CG 950110-02 hibited clinical resistance to cisplatin-based chemotherapy (resistant) to CGH analysis with the hypothesis that some of the resistant tumors may exhibit chromosomal amplification in evidence of possible gene amplification. Although CGH has been shown to be highly sensitive 12 in identifying amplified DNA sequences in tumors (7), this technique Fig. 2. Partial CGH karyotypes (left) and corresponding ratio profiles (righi) illustrating has thus far not been widely applied to identify amplified DNA high level amplification of chromosomal regions in six resistant GCTs. Hyhridired tumor regions in drug-resistant tumors. To our surprise, in the relatively DNA was visualized via F1TC (green), and control DNA was visualized via Texas Red (ml). The average greenired fluorescent signal ratio along the length of the chromosome is shown. small sample of GCTs studied here, we found several sites of chro The blue line in the ratio profile represents the mean of 8-10 chromosomes, and the yellowmosomal amplification other than 12p that were restricted to only the line represents the SD. The vertical red and green bars on the left and righi of the ideogram indicate threshold values of 0.80 and 1.20 for loss and gain, respectively. resistant tumors. These data, therefore, comprise the first evidence of chromosomal amplification in GCTs associated with resistance. Among the other sites amplified that were restricted to resistant tumors, several are of further interest. The ßCi-2-relatedgene, MFL1/ and overexpressed (64%) in breast cancer though its role in tumor BCL2AÃŒ,has been mapped to 15q23-24 (17), a region that was seen response is as yet unknown (20). Candidate genes that have been amplified in two cases in this study. Interestingly, the MFLI gene noted to be amplified in other tumors or that have been predicted to product has been demonstrated to suppress p53-induced apoptosis in play a role in cellular resistance have not been mapped to the remain a manner similar to that of the antagonists BCL2 and BCLXL (18). ing four sites found to be amplified in resistant GCTs. Further analysis This gene, therefore, becomes a good candidate for the gene amplified of all of the sites needs to be performed to test the possible amplifi in the two resistant GCTs that may contribute towards their observed cation of the candidate genes mentioned as well as to identify possible clinical resistance. The MYCN oncogene mapped to the 2p23-24 site, novel amplified genes. The regional amplification at 12pll.2-12 is also of interest. In noted here to be amplified in one resistant mixed GCT, has been shown to be frequently expressed in seminomas and embryonal car creased copy number of 12p, either as one or more copies of i( I2p) or cinomas, although with no evidence of amplification (19). Amplifi tandem duplications of 12p in situ or elsewhere in the genome, is a cation of MYCN is known to predict poor outcome in neuroblastoma nearly universal feature of GCTs (2). In a small proportion of tumors, additional amplification of 12pl 1.2-12 has been noted (21, 22). In one (16). The amplification/overexpression of the MDRI/PGYI gene, mapped to 7q21, has long been shown to be a mechanism whereby study, an yeast artificial chromosome clone has been identified that is tumor cells attain resistance to chemotherapeutic agents (8). A newly derived from this region (21 ). However, neither a candidate amplified gene nor the possible association of this amplification with progresidentified gene (AIBI) mapped to 20ql2 is frequently amplified (10%) 4262 § <.¿> Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1998 American Association for Cancer Research. CHROMOSOMAL AMPLIFICATION sion, resistance, or other clinical features of GCTs has been addressed thus far. Our finding of 12pl 1.2-12 amplification in a sensitive as well as a resistant tumor suggests that this amplification may not be associated with resistance. Although male GCTs exhibit a high sensitivity to cisplatin-based chemotherapy, studies directed at the understanding of the molecular mechanisms of invariable resistance of the remaining tumors are under way. Mutations of TP53 gene have now been identified in a subset of such clinically resistant tumors (15). Among the group of tumors studied, TP53 gene status was available for 15 of the 17 resistant cases; four had mutations (203A, 203B, 228A, 349A). We now have identified a possible second mechanism for resistance: gene amplification. Five tumors in the panel of 17 exhibited high level amplification; however, the gene(s) involved and its role in resistance remains to be confirmed/identified. Between the two studies, only one of these tumors (203B) showed both chromosomal amplification (7q21, 7q31, 15q22-24) and TP53 mutation (15). Tumors 203A and 203B represented discrete but contiguous teratoma and transformed rhabdomyosarcoma in the same patient (23). Although both lesions had the TP53 gene mutation (15), the 7q21, 7q31, and 15q22-24 amplifications were noted only in the rhabdomyosarcoma, suggesting that TP53 gene mutation may be proximal to amplification in the resistance pathway. Further studies aimed at examining other cellular features associated with resistance to DNA-damaging agents, such as ratios of BCL2 family members and cellular differentiation, may reveal additional mechanisms whereby resistance can be achieved. Such studies will also aid in the understanding of clinical resistance in other tumor systems. Acknowledgments We thank Geralyn Higgins for assistance in clinical data management. 20. 1. Bosl, G. J., and Motzer. R. J. Testicular germ-cell cancer. N. Engl. J. Mea., 337: 242-253. 1997. 2. Chaganti. R. S. K.. and Houldsworth. J. The cylogenelic theory of the pathogenesis of human adult male germ cell tumors. APMIS. 106: 80-84. 1998. 3. Samaniego, F.. Rodrigue?.. E.. Houldsworth, J.. Murty. V. V. V. S.. Ladanyi. M.. Lele, K. P., Chen. Q.. Dmitrovsky, E.. Geller, N. L.. Reuter. V.. Jhanwar, S. C., Bosl, G. J., and Chaganti. R. S. K. Cytogenetic and molecular analysis of human male germ cell tumors: chromosome 12 abnormalities and gene amplification. Genes Chromosomes Cancer. /: 289-300. 1990. 4. Albrecht, S.. Armstrong, D. L.. Mahoney. D. H.. Cheek, W. R., and Cooley, L. D. Cytogenetic demonstration of gene amplification in a primary intracranial germ cell tumor. Genes Chromosomes Cancer, 6: 61-63. 1993. 5. Chaganti, R. S. K.. Murty. V. V. V. S., Houldsworth. J.. Rao. P. H., Rodriguez. E.. and Bosl. G. J. Molecular genetics in germ cell tumours. Adv. Biosci., 91: 107-111, 1994. GCTS 6. Motzer. R. J.. Mazumdar. M., Bajorin. D. F.. Bosl. G. J.. Lyn. P.. and Vlamis, V. High-dose carboplatin. etoposide. and cyclophosphamide with aulologous bone mar row transplantation in first-line therapy for patients with poor-risk germ cell tumors. J. Clin. Oncol.. 15: 2546-2552. 1997. 7. Rao, P. H.. Houldsworth. J.. Dyomina. K.. Parsa. N. Z.. Cigudosa. J. C, Louie. D. C, Popplewell, L.. Offit. K.. Jhanwar. S. C.. and Chaganti. R. 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