Prognostic relevance of c-MYC gene amplification and polysomy for chromosome 8 in suboptimally-resected, advanced stage epithelial ovarian cancers: A Gynecologic Oncology Group study

Methods Women with suboptimally-resected, advanced stage EOC who participated in GOG-111, a multicenter randomized phase III trial of cyclophosphamide + cisplatin vs. paclitaxel + cisplatin, and who provided a tumor block through GOG-9404 were eligible. Fluorescence in situ hybridization (FISH) with probes for c-MYC and the centromere of chromosome 8 (CEP8) was used to examine c-MYC amplification (≥ 2 copies c-MYC /CEP8) and polysomy 8 (≥ 4 CEP8 copies). Results c-MYC amplification, defined as ≥ 2 copies c-MYC /CEP8, was observed in 29% (28/97) of EOCs and levels were ranged from 2.0–3.3 copies of c-MYC /CEP8. c-MYC amplification was not associated with patient age, race, GOG performance status, stage, cell type, grade, measurable disease status following surgery, tumor response or disease status following platinum-based combination chemotherapy. Women with vs. without c-MYC amplification did not have an increased risk of disease progression (hazard ratio [HR] = 1.03; 95% confidence interval [CI] = 0.65–1.64; p = 0.884) or death (HR = 1.08; 95% CI = 0.68–1.72; p = 0.745). c-MYC amplification was not an independent prognostic factor for progression-free survival (HR = 1.03, 95% CI = 0.57–1.85; p = 0.922) or overall survival (HR = 1.01, 95% CI = 0.56–1.80; p = 0.982). Similar insignificant results were obtained for c-MYC amplification categorized as ≥ 1.5 copies c-MYC /CEP8. Polysomy 8 was observed in 22 patients without c-MYC amplification and 3 with c-MYC amplification, and was associated with age and measurable disease status, but not other clinical covariates or outcomes. Conclusions c-MYC amplification and polysomy 8 have limited predictive or prognostic value in suboptimally-resected, advanced stage EOC treated with platinum-based combination chemotherapy. Keywords c-MYC FISH Ovarian cancer Polysomy 8 Gene amplification Introduction Ovarian cancer is the leading cause of cancer-related death among the gynecologic malignancies [1] . It is estimated that in the United States 21,850 new cases of ovarian cancer will be diagnosed, 68% of whom will present with advanced disease, and 15,520 women would die from their cancer in 2008 [1] . Currently, surgical staging followed by platinum-based chemotherapy is the standard of care for this disease [2,3] . Even though 70% of women with advanced stage disease respond to first-line platinum-based chemotherapy, five-year survival for women in this setting remains around 30% [1–3] . Researchers continue to study molecular and biochemical defects in epithelial ovarian cancer (EOC) with the hope of identifying biomarkers with prognostic and/or predictive value in this patient setting. c-MYC is a multifunctional proto-oncogene that exhibits diverse and at times, opposing functions [4–9] . c-MYC protein promotes tumorigenesis by inducing proliferation, inhibiting cells from exiting the cell cycle, stimulating blood vessel formation and cell migration, enhancing genomic instability, and helping tumor cells adapt and thrive in hypoxic environments [4,8,10–13] . In contrast, c-MYC inhibits tumorigenesis by sensitizing cells to apoptosis [14] . c-MYC is activated in about 20% of human cancers by several mechanisms including gene amplification, mutation or rearrangement, promoter insertion, transcriptional and post-translational [4–9] , and appears to be a feasible target for cancer therapeutics [15–16] . Amplification of c-MYC has been described in a variety of human cancers [17–32] including ovarian cancer [33–51] . Investigators have shown an association between c-MYC amplification and poor outcome in breast cancer [18–23] , prostate cancer [24–27] and chondrosarcoma [28] , but not in non-small cell lung cancer (NSCLC) [29–30] , colorectal cancer [31] or esophageal squamous cell carcinoma (SCC) [32] . In EOC, amplifications of c-MYC have been shown to be associated with stage [43] , cell type [37,51] and/or grade [41,43] , or conversely to not be associated with stage [46,51] , cell type [43,46] , grade [36,37,46,51] , progression-free survival (PFS) [45,46] , overall survival (OS) [45,46,48,49] and/or response to platinum-based chemotherapy [36] . The Gynecologic Oncology Group (GOG) undertook a study of c-MYC amplification in women with suboptimally-resected, advanced stage EOC who participated in a multicenter randomized phase III trial and provided a tumor block for research. Fluorescence in situ hybridization (FISH) with probes for c-MYC and the centromeric region of chromosome 8 (CEP8) was used to examine c-MYC amplification defined as ≥ 2 copies of c-MYC /CEP8 per cell [23,28,21] or ≥ 1.5 copies c-MYC /CEP8 [22,24] and polysomy for chromosome 8 defined as ≥ 4 copies of CEP8 per cell (as recommended by the manufacturer). Our results will be discussed in context with the other studies of c-MYC amplification in invasive EOC and our current understanding of the c-MYC proteins. Methods Patients Women who participated in GOG-111, a phase III treatment protocol, and provided a tumor block through GOG-9404 were eligible for this translational research study. Women on the GOG-111 protocol had to have previously-untreated, histologically-confirmed, surgically-staged EOC with evaluable or measurable stage III disease that was suboptimally-resected (> 1 cm residual disease) or stage IV disease, a GOG performance status of 0 to 2, and adequate bone marrow counts, renal function and hepatic function as previously described [52] . Women with a borderline tumor or optimally-resected stage III disease were specifically excluded from GOG-9404. Women were required to provide written informed consent, and participating institutions were required to obtain annual Institutional Review Board approval for GOG-111 and GOG-9404 consistent with federal, state and local requirements. Platinum-based combination chemotherapy Women on GOG-111 were randomly allocated to receive either 75 mg/m 2 cisplatin intravenously on day 1 and 750 mg/m 2 cyclophosphamide intravenously on day 1 every 3 weeks for a total of 6 cycles, or a 24-hour continuous intravenous infusion of 135 mg/m 2 paclitaxel on day 1 and 75 mg/m 2 cisplatin intravenously on day 2 every 3 weeks for a total of 6 cycles [52] . Treatment at the time of disease progression was left to the discretion of the treating physician and patient. Clinical end-points Women on GOG-111 were followed quarterly for 2 years, semi-annually for the next 3 years and then annually until death from the time they went off-treatment due to completion of protocol-specified therapy, toxicity, or disease progression [52] . Progression-free survival (PFS) was calculated as the time in months from enrollment on GOG-111 to disease progression or death (failure), or to the date of last contact for women who were still alive with no evidence of disease progression (censored). Overall survival (OS) was calculated as the time in months from enrollment on GOG-111 to death (regardless of cause) or to the date of last contact for women who were alive. Tumor response was evaluated after every 2 cycles of treatment in the women with measurable disease. Complete disappearance of all disease was required for a complete response. A partial response required a ≥ 50% reduction in tumor (product of perpendicular diameters). Progressive disease required a ≥ 50% increase in the dimensions of any lesion documented within 6 weeks of study entry or the appearance of new lesions within 8 weeks of study entry. Stable disease was defined as any condition not meeting the above categories. Women on GOG-111 who were clinically-free of disease after primary chemotherapy, or who had CA125 < 100 U/ml and were entered with non-measurable disease were required to undergo a reassessment laparotomy as specified in the protocol. Disease status was assessed following primary chemotherapy and classified as negative when the reassessment laparotomy showed no evidence of disease, or positive when disease progression was documented during treatment or when microscopic or macroscopic evidence of disease was observed during the reassessment laparotomy. Tumor specimens and fluorescence in situ hybridization Previously-untreated, primary tumor was excised during cytoreductive surgery, fixed in formalin and then embedded in paraffin. Unstained tissue sections, 5 μm in thickness, were prepared on charged glass slides. Dual-label FISH was used to quantify the number of copies of c-MYC and chromosome 8 in unstained slides as previously described [24] . Briefly, unstained sections were baked overnight at 60 °C, deparaffinized, dehydrated, treated for 10 min with 4% (w/v) pepsin at 45 °C, denatured for 10 min at 90 °C, hybridized for 12 to 16 h at 37 °C with a probe cocktail consisting of an alpha satellite probe to chromosome 8 (CEP8) directly labeled with Spectrum Green and a c-MYC region probe directly labeled with Spectrum Orange (Vysis Inc., Naperville, IL). The slides were post-washed for 5 min with 2 × SSC buffer (0.3 M NaCl and 30 mM sodium citrate tribasic dihyrate, pH 7.0) at 72 °C and counterstained with 4′,6′-diamidino-2-phenyindole (DAPI). Red staining for chromosome 8 and green staining for c-MYC were visualized using a Zeiss Axiophot fluorescence microscope (Carl Zeiss MicroImaging, Inc. Thornwood, NY, USA) with appropriate filters and an Applied Imaging Cytovision system (Pittsburgh, PA, USA), and quantified in at least 50 tumor cells per case by one or two technicians (CC and GV; see Acknowledgements) working under the direct supervision of one of the authors (JKB). Average copies of c-MYC and CEP8 per case were calculated for the 28% of equivocal cases scored by two reviewers. c-MYC amplification is defined as ≥ 2 copies of c-MYC /CEP8 per cell, and unless specifically stated otherwise, this is the definition used. Supportive analyses were done defining c-MYC amplification as ≥ 1.5 copies of c-MYC /CEP8 per cell based on studies in breast cancer [22] and prostate cancer [24] showing that low level amplification increased protein expression. Statistical methods Biomarker and clinical data were analyzed using SPSS versions 14.0 (SPSS Inc., Chicago, IL) and SAS ® version 9.1 software (SAS Institute, Inc. Cary, NC). All tests were two-sided and the level of significance was set at 0.05. Associations between categorical variables were evaluated using Fisher's Exact Test [53,54] . Estimates of survival probabilities were calculated using the Kaplan–Meier method [55] . Logrank tests [56] were used to test the equality of survival distributions between groups. Cox proportional hazards regression [57] was used to model associations of variables with PFS or OS. Results GOG-111 was a randomized phase III trial that enrolled 410 women between April 13, 1990 and March 2, 1992 [52] . Ninety-seven women on GOG-111 were enrolled on the companion protocol, GOG-9404 and provided primary tumor tissue for research. The patient characteristics for the 97 women in this cohort are summarized in Table 1 and are representative of that observed in the entire GOG-111 cohort [52] . At the time of the final analysis, five women were alive with no evidence of disease, four women were alive with disease progression and 88 women died due to disease progression. Median follow-up for the nine women who were still alive at the time of the final analysis was 127 (range 15 to 207) months including three women who were lost to follow up after 15, 24 or 87 months of enrollment. Twenty-eight (29%) of women had tumors that exhibited c-MYC amplification with levels ranging from the lower limit of 2.0 up to 3.3 copies of c-MYC /CEP8 per cell. Of the 69 (71%) of women with tumors without c-MYC amplification, the ratio of c-MYC /CEP8 ranged from 0.42 to 1.98 copies per cell. c-MYC amplification was not associated with patient age, race, GOG performance, stage, cell type, grade or measurable disease status following primary surgery ( Table 1 ). There was no difference in the PFS distributions ( Fig. 1 A , p = 0.885) or OS distributions ( Fig. 1 B, p = 0.745) for women with or without c-MYC amplification. Unadjusted Cox modeling demonstrated that women with c-MYC amplification did not have an increased risk of disease progression (hazard ratio [HR] = 1.03; 95% confidence interval [CI] = 0.65–1.64; p = 0.884) or death (HR = 1.08; 95% CI = 0.68–1.72; p = 0.745) compared with women without c-MYC amplification ( Table 2 ). After adjusting for patient age and stratifying by tumor stage, histologic cell type, tumor grade, measurable disease status, and treatment regimen, c-MYC amplification was not an independent prognostic factor for PFS (HR = 1.03, 95% CI = 0.57–1.85; p = 0.922) or OS (HR = 1.01, 95% CI = 0.56–1.80; p = 0.982) in women with suboptimally-resected, advanced stage EOC ( Table 2 ). c-MYC amplification was not associated with tumor response following platinum-based combination chemotherapy or disease status following platinum-based combination chemotherapy ( Table 3 ). c-MYC amplification using the ≥ 1.5 copies of c-MYC /CEP8 per cell yielded similar insignificant associations with clinical characteristics, PFS, survival, response, and disease status as reported using the ≥ 2.0 cut point ( Tables 1–3 ). Polysomy for chromosome 8, defined as a tumor with ≥ 4 copies of CEP8 per cell, was observed in 22 women without c-MYC amplification and 3 patients with c-MYC amplification. Polysomy 8 was not associated with any of the clinical covariates tested except for patient age at enrollment and measurable disease status ( Table 1 ). Although statistically significant, the relationship between polysomy 8 and age category was not consistent: the proportion of women with polysomy 8 increased incrementally by age for women up to 69 years but none of the women who were ≥ 70 years old had polysomy 8 ( Table 1 ). The percentage of women with polysomy was statistically significantly higher in women with measurable disease (36%) than in those with non-measurable disease (14%). PFS distributions ( Fig. 1 C, p = 0.982) and OS distributions ( Fig. 1 D, p = 0.747) were very similar for women with vs. without polysomy 8. Unadjusted Cox modeling demonstrated that women with polysomy 8 did not have an increased risk of disease progression (HR = 0.99; 95% CI = 0.62–1.59; p = 0.982) or death (HR = 1.08; 95% CI = 0.67–1.74; p = 0.747) compared with women without polysomy 8 ( Table 2 ). After adjusting for patient age and stratifying by tumor stage, histologic cell type, tumor grade, measurable disease status, and treatment regimen, polysomy 8 was not an independent prognostic factor for PFS (HR = 1.07, 95% CI = 0.57–2.02; p = 0.823) or OS (HR = 1.04, 95% CI = 0.54–1.99; p = 0.916) in women with suboptimally-resected, advanced stage EOC ( Table 2 ). Finally, polysomy 8 was not associated with tumor response or disease status following platinum-based combination chemotherapy ( Table 3 ). Discussion Our study is unique because it is a multicenter study of c-MYC amplification, detected by FISH using probes for c-MYC and CEP8, in FFPE primary tumor specimens from women with suboptimally-resected, advanced stage EOC treated with platinum-based combination chemotherapy. The availability of detailed clinical, treatment and follow-up data was a major strength of this study. We not only examined the relationship between c-MYC amplification and a full spectrum of clinical covariates but also evaluated the association between c-MYC amplification and multiple measures of clinical outcome. Limited FISH data for c-MYC amplification is currently available in EOC. Our study examined FFPE primary tumor from women with advanced stage EOC ( N = 97), adjusted for copy number alterations in chromosome 8, used two different cut points for c-MYC amplification (≥ 2 and ≥ 1.5 copies of c-MYC /CEP8 copies) and demonstrated that c-MYC amplification was not associated with tumor stage, cell type, grade, PFS, OS, tumor response or disease status following platinum-based combination chemotherapy. Wang et al. studied mechanically- and enzymatically-dispersed, frozen, early and advanced stage, epithelial and non-epithelial, ovarian cancers, adjusted for copy number alterations in chromosome 8, used ≥ 1.5 c-MYC /CEP8 copies cut point for c-MYC amplification, and showed that c-MYC amplification was not associated with OS [48] . Dimova et al. studied FFPE tumor from women with invasive, early and advanced stage, epithelial and non-epithelial, ovarian cancers ( N = 280), did not adjust for copy number alterations in chromosome 8 or have access to any measures of clinical outcome, used > 2 copies c-MYC cut point for c-MYC gain/amplification, and reported that copy number increases in c-MYC were associated with histologic subtype but not with tumor grade or stage [51] . The disparity between our study and Dimova et al. with respect to the relationship of c-MYC amplification and tumor stage may be attributable at least in part to differences in tumor stages, histologic cell type and the adjustment for chromosome 8 copy number alterations. Our study demonstrated that c-MYC amplification was observed in 29% (28/97) of the women with suboptimally-resected advanced stage EOC. This result is similar to the 25 to 34% levels described in some ovarian cancer studies using Southern/dot/slot blotting [33,34,36,39,45] , polymerase chain reaction (PCR) [46] or comparative genome hybridization (CGH) [44,50] , but is higher than the 0 to 17% levels reported in some other studies that also used Southern/dot/slot blotting [35,40,42] , PCR [38] or CGH [47] , and is lower than the 38 to 55% levels provided in still other studies using Southern/dot/slot blotting [37,41,43] , PCR [49] , CGH [49] , or FISH [48,51] . Differences in the source, type and stage of the tumor specimens, method for detecting gene amplification and the definition for c-MYC amplification may offer some explanation for these disparities. In the 97 EOC studied herein, c-MYC amplification was not associated with tumor stage, cell type, grade, PFS, OS, tumor response or disease status after platinum-based combination chemotherapy. These findings are consistent with studies demonstrating that c-MYC amplification was not associated with tumor stage [46,51] , histologic cell type [43,46] , tumor grade [36,37,46,51] , progression-free survival (PFS) [45,46] , overall survival (OS) [45,46,48,49] , and/or response to platinum-based chemotherapy [36] but contradict studies showing that c-MYC amplification was associated with tumor stage [43] , histologic cell type [37,51] and/or tumor grade [41,43] . The disparities between studies with respect to the relationship of c-MYC amplification and tumor characteristics or outcome may be explained at least in part by differences in sample size, surgery, type of chemotherapy, tumor stage, clinical end-points, follow-up, type of tumor specimen, method for detecting c-MYC amplification, definition for c-MYC amplification and/or adjustment for copy number alterations in chromosome 8 in these studies. The lack of an association between c-MYC amplification and OS was also observed in other diseases including NSCLC [29,30] , colorectal cancer [31] and esophageal SCC [32] . In contrast, studies in breast cancer [18–23] , prostate cancer [24–27] and chondrosarcoma [28] demonstrated a strong association between c-MYC amplification and poor outcome. Wang et al. undertook a chromosome 8 centromere study of ovarian cancer using interphase FISH in mechanically- and enzymatically-dispersed, frozen, invasive ovarian cancers and demonstrated that ≥ 50% of both c-MYC amplified and non-amplified tumors exhibited polysomy 8 [48] . Despite the prevalence of alterations in the centromeric region of chromosome 8 in these 40 ovarian cancers, the presence of polysomy in chromosome 8 did not appear to be correlated with clinical presentation or disease progression [48] . Herein, we utilized a dual color FISH in 97 FFPE EOC and showed that 11% (3/28) of women with c-MYC amplified tumors and 32% (22/69) of women with non-amplified tumors exhibited polysomy 8. Despite the differences in the prevalence of polysomy 8 in women with amplified and non-amplified c-MYC , we confirmed that polysomy 8 was not associated with OS [48] and went on to demonstrate that polysomy 8 was not associated with PFS, tumor response and disease status following platinum-based combination chemotherapy. Unlike studies in prostate cancer [25,27] , hematologic malignancies [58] , NSCLC [29–30] or chondrosarcoma [28] , polysomy 8 does not appear to have prognostic value in ovarian cancer patients. c-MYC protein is a well recognized transcription factor with a basic helix–loop–helix leucine zipper motif responsible for sequence-specific DNA binding and protein–protein interactions [59–62] that binds to an estimated 25,000 sites in the human genome and regulates as many as 15% of human genes [63] . c-MYC not only up-regulates genes involved in cell cycle regulation, metabolism, ribosome biogenesis, protein synthesis, mitochondrial function and apoptosis and represses genes involved in cell growth arrest and adhesion [64–67] , but influences DNA replication, translation and chromatin structure [68–71] . In humans, the c-MYC gene encodes three isoforms: the transcription factors c-MYC-1 and c-MYC-2 as well as c-MYC-S, a dominant-negative inhibitor of c-MYC-1 and c-MYC-2 [72–77] . The level and function of the c-MYC isoforms can be influenced by binding other proteins [78,79] and by phosphorylation, ubiquitinylation and acetylation [80–83] . In conclusion, c-MYC amplification and polysomy 8 were not associated with PFS, OS, tumor response or disease status following platinum-based combination chemotherapy and have limited prognostic value in suboptimally-resected, advanced stage EOC. These results, however, do not rule out the potential that c-MYC has prognostic value when evaluated in a panel of biomarkers that exert epistatic interactions or in women with a borderline tumor or optimally-resected stage III EOC. In addition, the c-MYC isoforms which exhibit diverse and at times opposing functions, and the factors that affect their level and function have yet to be fully evaluated in EOC and may have prognostic value in this disease setting. Conflict of interest statement The authors declare that there is no conflict of interest with the exception of William Brady who received payment from Merck and Co, Inc. for working as an independent contractor in the past 2 years. Acknowledgments The authors thank Anne Reardon for her assistance in formatting and editing this manuscript, Suzanne Baskerville for coordinating the clinical data for this study, Dr. Mark Brady for his expert work as the statistician for GOG-111, and Christian Cao and Gary Veytsman for scoring the FISH staining for c-MYC and CEP8. Finally, we would like to thank Dr. Heather Lankes and the GOG Publications Subcommittee for their critical review of and thoughtful suggestions for the manuscript. References [1] A. Jemal R. Siegel E. Ward Y. Hao J. Xu T. Murray Cancer statistics, 2008. CA- A Cancer J. Clin. 58 2 2008 71 96 [2] T. Thigpen First-line therapy for ovarian carcinoma: what's next? Cancer Invest. 22 suppl. 2 2004 21 28 [3] R.F. Ozols Systemic therapy for ovarian cancer: current status and new treatments Semin. Oncol. 22 suppl. 6 2006 S3 S11 [4] S.J. Garte The c-myc oncogene in tumor progression Crit. Rev. Oncogene. 4 4 1993 435 449 [5] P.J. Koskinen K. Alitalo Role of myc amplification and overexpression in cell growth, differentiation and death Sem. Cancer Biol. 4 1993 3 12 [6] L.M. Boxer C.V. Dang Translocations involving c-myc and c-myc function Oncogene 20 2001 5595 5610 [7] S. Pelengaris M. Khan G. Evan c-MYC: more than just a matter of life and death Nat. Rev. Cancer 2 10 2002 764 776 [8] S. Pelengaris M. Khan The many faces of c-MYC Arch. Biochem. Biophys. 416 2 2003 129 136 [9] S. Adhikary M. Eilers Transcriptional regulation and transformation by myc proteins Nat. Rev. Mol. Cell. Biol. 6 8 2005 635 645 [10] C. Arvantis D.W. Felsher Conditional transgenic models define how MYC initiates and maintains tumorigenesis Semin. Cancer Biol. 16 4 2006 313 317 [11] X.Y. Yin L. Grove N.A. Datta M.W. Long E.V. Prochownik C-myc overexpression and p53 loss cooperate to promote genomic instability Oncogene 18 1999 1177 1184 [12] C.V. Dang F. Li L.A. Lee Could MYC induction of mitochondrial biogenesis be linked to ROS production and genomic instability? Cell Cycle 4 11 2005 1465 1466 [13] J.D. Gordon C.B. Thompson M.C. Simon HIF and c-Myc: sibling rivals for control of cancer cell metabolism and proliferation Cancer Cell 12 2 2007 108 113 [14] N. Meyer S.S. Kim L.Z. Penn The Oscar-worthy role of Myc in apoptosis Semin. Cancer Biol. 16 4 2006 275 287 [15] R. Ponzielli S. Katz D. Barsyte-Lovejoy L.Z. Penn Cancer therapeutics: targeting the dark side of Myc Eur. J. Cancer 41 2005 2485 2501 [16] M. Vita M. Henricksson The Myc oncoprotein as a therapeutic target for human cancer Sem. Cancer Biol. 16 4 2006 318 330 [17] O. Brison Gene amplification and tumor progression Biochim. Biophys. Acta 1155 1 1993 25 41 [18] A. Borg B. Baldetorp M. Ferno H. Olsson H. Sigurdsson c-myc amplification is an independent prognostic factor in postmenopausal breast cancer Int. J. Cancer 51 5 1992 687 691 [19] E.M. Berns J.G. Klijn W.L. van Putten I.L. van Staveren H. Portengen J.A. Foekens c-myc amplification is a better prognostic factor than HER2/neu amplification in primary breast cancer Cancer Res. 52 5 1992 1107 1113 [20] S.L. Deming S.J. Nass R.B. Dickson B.J. Trock C-myc amplification in breast cancer: a meta-analysis of its occurrence and prognostic relevance Br. J. Cancer 83 12 2000 1688 1695 [21] C.M. Schlotter U. Vogt U. Bosse B. Mersche K. Wassmann C-myc, not HER-2/neu, can predict recurrence and mortality of patients with node-negative breast cancer Breast Cancer Res. 5 2 2003 R30 R36 [22] J. Blancato B.M. Singh A.M. Liu D.J. Liao R.B. Dickson Correlation of amplification and overexpression of the c-myc oncogene in high-grade breast cancer: FISH, in situ hybridization and immunohistochemical analyses Br. J. Cancer 90 2004 1612 1619 [23] S.M. Rodriquez-Pinilla R.L. Jones M.B. Lambros E. Arriola K. Savage M. James MYC amplification in breast cancer: a chromogenic in situ hybridization study J. Clin. Pathol. 60 9 2007 1017 1023 [24] R. Jenkins J. Qian M. Lieber D. Bostwick Detection of c-myc oncogene amplification and chromosomal anomalies in metastatic prostatic carcinoma by fluorescence in situ hybridization Cancer Res. 57 1997 524 531 [25] K. Sato J. Qian J.M. Slezak M.M. Lieber D.G. Bostwick E.J. Bergstralh Clinical significance of alterations of chromosome 8 in high-grade, advanced, nonmetastatic prostate carcinoma J. Natl. Cancer Inst. 91 18 1999 1574 1580 [26] H. Sato S. Minei T. Hachiva T. Yoshida Y. Takimoto Fluorescence in situ hybridization analysis of c-myc amplification in stage TNM prostate cancer in Japanese patients Int. J. Urol. 13 6 2006 761 766 [27] J. Dvorackova M. Uvirova A molecularly genetic determination of prognostic factors of the prostate cancer and their relationships to expression of protein p27kip1 Neoplasma 54 2 2007 149 154 [28] C. Morrison M. Radmacher N. Mohammed D. Suster H. Auer S. Jones Mayerson. MYC Amplification and polysomy 8 in chrondrosarcoma: array comparative genome hybridization, fluorescent in situ hybridization, and association with outcome J. Clin. Oncol. 23 26 2005 936 976 [29] H. Kubokura K. Koizumi M. Yamamoto S. Tanaka Chromosome 8 copy numbers and the c-myc gene amplification in non-small cell lung cancer. Analysis by interphase cytogenetics Nippon Ika Daigaku Zasshi 66 2 1999 107 112 [30] H. Kubokura T. Tenjin H. Akiyama K. Koizumi H. Nishirmura M. Yamamoto Relations of the c-myc gene and chromosome 8 in non-small cell lung cancer: analysis by fluorescence in situ hybridization Ann. Thorac. Cardiovasc. Surg. 7 4 2001 197 203 [31] K. Al-Kuraya H. Novotny P. Bavi A.K. Siraj S. Uddin A. Ezzat HER2, TOP2A, CCND1, EGFR and C-MYC oncogene amplification in colorectal cancer J. Clin. Pathol. 60 7 2007 768 772 [32] M. Bitzer M. Stahl J. Arjumand M. Rees B. Klump H. Heep C-myc gene amplification in different stages of oesophageal squamous cell carcinoma: prognostic value in relation to treatment modality Anticancer Res. 23 2B 2003 1489 1493 [33] O.M. Serova Amplification of c-myc proto-oncogene in primary tumors, metastases and blood leukocytes of patients with ovarian cancer Eksp. Onkol. 9 1 1987 25 27 [34] D.J. Zhou N. Gonzalez-Cadavid H. Ahuja H. Battifora G.E. Moore M.J. Cline A unique pattern of proto-oncogene abnormalities in ovarian adenocarcinomas Cancer 62 1988 1573 1576 [35] D.M. Smith D.E. Groff R.K. Pokul J.L. Bear G. Delgado Determination of cellular oncogene rearrangement or amplification in ovarian adenocarcinomas Am. J. Obstet. Gynecol. 161 1989 911 915 [36] V.V. Baker M.P. Borst D. Dixon K.D. Hatch M.H. Singleton D. Miller c-myc amplification in ovarian cancer Gynecol. Oncol. 38 1990 340 342 [37] H. Sasano C.T. Garrett D.S. Wilkson S. Silverberg J. Camerford J. Hyde Protooncogene amplification and tumor ploidy in human ovarian neoplasms Hum. Pathol. 21 1990 382 391 [38] G. Schreiber L. Dubeau C-myc proto-oncogene amplification detected by polymerase chain reaction in archival ovarian carcinomas Am. J. Pathol. 137 1990 653 658 [39] E.M.J.J. Berns J.G.M. Klijn S.C. Henzen_Logmans C.J. Rodenburg M.E.L. va der Burg J.A. Foekens Receptors for hormones and growth factors and (onco)-gene amplification in human ovarian cancer Int. J. Cancer 52 1992 218 224 [40] B. Csokay J. Papp I. Besznyak P. Bosze Z. Sarosi J. Toth Oncogene patterns in breast and ovarian carcinomas Eur. J. Surg. Oncol. 19 suppl. 1 1993 593 599 [41] X.Y. Xin The amplification of c-myc, N-ras, c-erb B oncogenes in ovarian malignancies Zhonghua Fu Chan Ke Za Zhi 28 7 1993 405 407 [42] A. Bellacosa D. de Feo A.K. Godwin D.W. Bell J.Q. Cheng D.A. Altomare Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas Int. J. Cancer 64 1995 280 285 [43] B. Meilu Q. Fan S. Huang J. Ma J. Lang Amplifications of proto-oncogenes in ovarian carcinoma Chin. Med. J. (Engl) 108 11 1995 844 848 [44] H. Iwabuchi M. Sakamoto H. Sakunaga Y.Y. Ma M.L. Carcangiu D. Pinkel Genetic analysis of benign, low-grade and high-grade ovarian tumors Cancer Res. 55 1995 6172 6180 [45] D. Katsaros C. Theillet P. Zola G. Louason B. Sanfilippo E. Isaia Concurrent abnormal expression of erb B-2, myc and ras genes is associated with poor outcome of ovarian cancer patients Anticancer Res. 15 1995 1501 1510 [46] J. Diebold B. Suchy G.B. Baretton S. Blasenbreu W. Meier M. Schmidt ploidy and MYC DNA amplification in ovarian carcinomas. Correlations with p53 and bcl-2 expression, proliferative activity and prognosis Virchows Arch. 429 1996 221 227 [47] G. Sonoda J. Palazzo S. du Manoir A.K. Godwin M. Feder Yakushiji, Testa JR. Comparative genomic hybridization detects frequent overrepresentation of chromosomal material from 3q26, 8q24, and 20q13 in human ovarian carcinomas Genes Chromosomes Cancer 20 1997 320 328 [48] Z.R. Wang W.H. Liu S.T. Smith R.S. Parrish S.R. Young c-myc and chromosome 8 centromere studies of ovarian cancer by interphase FISH Exp. Mol. Pathol. 66 1999 140 148 [49] S. Suzuki D.H. Moore D.G. Ginzinger T.E. Godfrey J. Barclay B. Powell An approach to analysis of large scale correlations between genome changes and clinical end points in ovarian cancer Cancer Res. 60 2000 5382 5385 [50] V. Shridhar J. Lee A. Pandita Genetic analysis of early- versus late-stage ovarian tumors Cancer Res. 61 2001 5895 5904 [51] I. Dimova S. Raitcheva R. Dimitrov N. Doganov D. Toncheva Correlations between c-myc gene copy-number and clinicopathological parameters of ovarian tumors Eur. J. Cancer 42 2006 674 679 [52] W. McGuire W. Hoskins M. Brady Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer N. Engl. J. Med. 334 1996 1 6 [53] R.A. Fisher On the interpretation of χ 2 from contingency tables, and the calculation of p J. R. Stat. Soc. 85 1 1992 87 94 [54] C.R. Mehta N.R. Patel A network algorithm for performing Fisher's exact test in r × c contingency tables J. Am. Stat. Assoc. 78 1983 427 434 [55] E.L. Kaplan P. Meier Nonparametric estimation from incomplete observations J. Am. Stat. Assoc. 53 1958 457 481 [56] N. Mantel Evaluation of survival data and two new rank order statistics arising in its consideration Cancer Chemother. Rep. 50 1966 163 170 [57] D.R. Cox Regression model and life tables J. R. Stat. Soc., B. 34 1972 187 220 [58] V. Beyer D. Muhlematter V. Parlier C. Cabrol S. Bougeon-Mamin M. Solenthaler Polysomy 8 defines a clinico-cytogenetic entity representing a subset of myeloid hematologic malignancies associated with a poor prognosis: report on a cohort of 12 patients and review of 105 published cases Cancer Genet. Cytogenet. 160 2 2005 97 119 [59] B. Amati S. Dalton M.W. Brooks T.D. Littlewood G.I. Evan H. Land Transcriptional activation by the human c-Myc oncoprotein in yeast requires interaction with Max Nature 359 1992 423 426 [60] B. Amati T.D. Littlewood G.I. Evan H. Land The c-Myc protein induces cell cycle progression and apoptosis through dimerization with Max EMBO J. 12 1993 5083 5087 [61] L.M. Facchini S. Chen W.W. Marhin J.N. Lear L.Z. Penn The Myc negative autoregulation mechanism requires Myc–Max association and involves the c-myc P2  minimal promoter Mol. Cell. Biol. 17 1997 100 114 [62] S.K. Oster D.Y.L. Mao J. Kennedy L.Z. Penn Functional analysis of the N-terminal domain of the Myc oncoprotein Oncogene 22 2003 1998 2010 [63] P.J. Hurlin J. Huang The MAX-interacting transcription factor network Semin. Cancer Biol. 16 4 2006 265 274 [64] V.H. Cowling M.D. Cole Mechanism of transcriptional activation by the Myc oncoproteins Semin. Cancer Biol. 16 4 2006 242 252 [65] C.V. Dang K.A. O'Donnell K.I. Zeller T. Nguyen F. Osthus RC Li The c-Myc target gene network Semin. Cancer Biol. 16 4 2006 253 264 [66] D. Kleine-Kohlbrecher S. Adhikary M. Eilers Mechanisms of transcriptional repression by Myc Curr. Top. Microbiol. Immunol. 302 2006 51 60 [67] L.A. Lee C.V. Dang Myc target transcriptomes Curr. Top. Microbiol. Immunol. 302 2006 145 167 [68] D. Dominguez-Sola C.Y. Ying C. Grandori L. Ruggiero B. Chen M. Li Non-transcriptional control of DNA replication by c-Myc Nature 448 7152 2007 445 451 [69] R. Lebofsky J.C. Waler New Myc-anisms for DNA replication and tumorigenesis? Cancer Cell. 12 2 2007 102 103 [70] E.V. Schmidt The role of c-myc in regulation of translation initiation Oncogene 23 2004 3217 3221 [71] P.S. Knoepfler Myc goes global: new tricks for an old oncogene Cancer Res. 67 11 2007 5061 5063 [72] S.R. Hann M.W. King D.L. Bentley C.W. Anderson R.N.A. Eisenman non-AUG translational initiation in c-myc exon 1 generates an N-terminally distinct protein whose synthesis is disrupted in Burkitt's lymphomas Cell 52 1988 185 195 [73] S.R. Hann Methionine deprivation regulates the translation of functionally-distinct c-Myc proteins Adv. Exp. Med. Biol. 375 1995 107 116 [74] G.D. Spotts S.V. Patel Q. Xiao S.R. Hann Identification of downstream-initiated c-Myc proteins which are dominant-negative inhibitors of transactivation by full-length c-Myc proteins Mol. Cell. Biol. 17 1997 1459 1468 [75] Q. Xiao G. Claassen J. Shi S. Adachi J. Sedivy S.R. Hann Transactivation-defective c-MycS retains the ability to regulate proliferation and apoptosis Genes Dev. 12 24 1998 3803 3808 [76] S.K. Hirst C. Grandori Differential activity of conditional MYC and its variant MYC-S in human mortal fibroblasts Oncogene 19 45 2000 5189 5197 [77] S.K. Oster D.Y.L. Mao J. Kennedy L.Z. Penn Functional analysis of the N-terminal domain of the Myc oncoprotein Oncogene 22 2003 1998 2010 [78] P.J. Hurlin S. Dezfouli Functions of Myc:Max in the control of cell proliferation and tumorigenesis Int. Rev. Cytol. 238 2004 183 226 [79] J. Hurlin P.J. Huang The MAX-interacting transcription factor network Semin. Cancer Biol. 16 4 2006 265 274 [80] S.Y. Kim A. Herbst K.A. Tworkowski S.E. Salghetti W.P. Tansey Skp2 regulates Myc protein stability and activity Mol. Cell. 11 5 2003 1177 1188 [81] J. Vervoorts J. Luscher-Firzlaff B. Luscher The ins and outs of MYC regulation by posttranslational mechanisms J. Biol. Chem. 281 46 2006 34725 34729 [82] S.R. Hann Role of post-translational modifications in regulating c-Myc proteolysis, transcriptional activity and biological function Semin. Cancer Biol. 16 4 2006 288 302 [83] M.S. Dai Y. Jin J.R. Gallegos H. Lu Balance of Yin and Yang: ubiquitylation-mediated regulation of p53 and c-Myc Neoplasia 8 8 2006 630 644