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. Author manuscript; available in PMC: 2019 Mar 21.
Published in final edited form as: Curr Cancer Drug Targets. 2016;16(9):789–795. doi: 10.2174/1568009616666151222150359

Epigenetic Targeting of Platinum Resistant Testicular Cancer

Daniel Sonnenburg 1, Michael J Spinella 2, Costantine Albany 1,*
PMCID: PMC6428575  NIHMSID: NIHMS1007614  PMID: 26694252

Abstract

The involvement of epigenetic aberrations in the development and progression of tumors is now well established. However, little is known of the epigenetic alterations in testicular cancer and particularly in platinum refractory germ cell tumors. Germ cell derived testicular cancers, as compared to somatic tumors, appear to have a unique epigenetic profile that features more extensive DNA hypomethylation. Emerging data from clinical specimens suggest that epigenetic aberrations, especially DNA hypermethylation, can contribute to chemotherapy resistance and poor clinical outcomes in testicular germ cell tumors. Recent data indicate that testicular cancer cells, even those resistant to platinum, are highly sensitive to low doses of demethylating agents. Based on these promising preclinical studies, we suggest that DNA methylation inhibitors in combination with chemotherapeutic agents may offer a path to overcome acquired drug resistance in testicular cancer, laying the foundation and rationale for testing this class of epigenetic drugs in the clinical setting. In this mini-review we provide a brief overview of the promise of DNA methylation therapy to treat patients with refractory cancer of the testes.

Keywords: Testicular cancer, germ cell tumor, embryonal carcinoma, cisplatin resistance, epigenetics, DNA methyltransferase, decitabine, SGI-110

INTRODUCTION

Testicular cancer is the most common cancer in men aged 15 to 40 years in the United States, Canada, and Europe [1]. Ninety-five percent of testicular neoplasms are germ cell tumors (GCTs). GCTs consist of two histologically distinct subtypes: seminomas (40%) and non-seminomas (60%). Non-seminomatous GCTs are further divided into embryonal carcinoma (EC), teratoma, yolk sac tumor (YST), and choriocarcinoma [2]. The incidence of testicular GCTs has been increasing in the last 30 years. Since no strong behavioral or environmental risk factors have been identified, treatment and cure, rather than screening and prevention, is paramount to fighting this type of cancer [1].

The most important development in the treatment of advanced testicular cancer was the discovery of cisplatin [3]. Cisplatin was established as the essential component for the treatment of advanced disease and marked the beginning of the modern era of chemotherapy for GCTs [4]. This discovery increased the 5-year survival rate of patients with metastatic GCTs from 5% to over 80% [5, 6]. In 1997, the International Germ Cell Cancer Collaborative Group (IGCCCG) published an analysis from almost 6000 patients treated for metastatic GCTs. The IGCCCG risk stratification system classified patients with metastatic GCTs at the onset of diagnosis into three groups: good, intermediate and poor risk with cure rates of 90%, 75% and 50%, respectively [7]. The standard therapy for these patients is cisplatin-based combination chemotherapy. Patients who relapse after first line chemotherapy have approximately a 50% chance of achieving a cure through the use of salvage chemotherapy. This consists of either high-dose chemotherapy (HDCT) followed by a peripheral stem cell transplant or traditional chemotherapy [8]. Nearly all patients progressing after HDCT will ultimately die from progressive disease [6, 9]. Patients with platinum refractory disease are defined as progressing while on cisplatin based chemotherapy or within 6 weeks after completing chemotherapy or after HDCT. Multiple studies from Indiana University have shown that further chemotherapy after HDCT results in a response rate of less than 20%, with a median survival time of 8 months. All deaths occurred within 12 months of HDCT [10]. In addition, seven phase II clinical trials evaluated single-agent activity in 90 refractory GCT patients at Memorial Sloan Kettering Cancer Center. These trials showed a median progression free survival (PFS) of 4 weeks and a median overall survival (OS) of 4 months [9]. The failure of these trials highlights the need to explore other pathways and targets to combat the refractory form of this disease. Morbidity and mortality in this age group occur during the most productive years of a patient’s life. An average of more than 35 years of life is lost when a patient dies from this disease, well over a decade longer than any other adult malignancy.

EPIGENETIC ALTERATIONS IN TESTICULAR CANCER

Epigenetic features of neoplasms are being explored as possible novel therapeutic options. Epigenetic alterations are defined as changes in gene expression without alteration of the DNA sequence. This can include DNA methylation, histone modification, nucleosome repositioning, and posttranscriptional gene regulation by micro-RNA (miRNA) [11, 12]. In recent years, DNA methylation has increasingly become a focus of study in GCTs and will be the topic of this review.

DNA methylation can be defined as the addition of a methyl moiety to the cytosine-5 position within the context of a CpG dinucleotide, mediated by DNA methyltransferases (DNMTs) [11]. DNA methylation is an important regulator of gene transcription. Its role in carcinogenesis and resistance to therapy has been a topic of considerable recent interest. Hypermethylation represses transcription of CpG-rich promoter regions of tumor suppressor genes leading to gene silencing [13]. Hypomethylation is associated with gene activation whereas hypermethylation is associated with gene inactivation. Epigenetic regulation of gene expression is complex and also involves posttranslational modifications of histones which is in part mediated by recruitment of DNA binding proteins to methylated CpGs. The net effect is a regional condensation of chromatin structure. A unique feature of GCTs that may explain their exquisite sensitivity to platinum therapies compared to other solid somatic tumors is global CpG hypomethylation [14, 15].

Seminomas have very little DNA methylation compared to normal cells and somatic tumors while non-seminomas appear to have an intermediate level of DNA methylation [16-20] (Fig. 1). Candidate approaches found certain tumor suppressors including RASSF1A, MGMT, HIC1, APOLD1, PCDH10 and RAG1 to be hypermethylated in GCTs and reactivation of gene expression occurred in GCT cell lines treated with demethylating agents [18, 21]. Recent detailed genome wide methylation studies in seminoma and nonseminoma cell lines and patient samples confirm the concept that GCTs may have originated from an erased embryonic germ cell, with decreased methylation of CpG islands, imprinted genes and repetitive elements [22, 23]. The hypo-methylation of seminomas correlates with hypersensitivity to chemotherapy. Within nonseminoma there is increased methylation associated with differentiation from pluripotent EC to teratoma that correlates with sensitivity to cisplatin [19, 21, 24, 25].

Fig. (1).

Fig. (1).

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Proposed distinctive patterns of DNA methylation among testicular germ cell tumors, normal cells and somatic solid cancers.

MiRNA IN GCTs

More recently discovered epigenetic changes in cancer involve miRNA. These small (19-22 nucleotides in length) non-coding RNAs regulate protein expression through posttranscriptional down-regulation and RNA silencing [26]. Interestingly, DNA methylation can regulate miRNA expression as shown in a study where a hypomethylating agent increased miRNA production [27]. Recently an important role for miRNAs in GCTs has been proposed. A variety of miRNAs have been found to be elevated in GCT patient serum including miR-302a-3p, 302b-3p, 302c-3p, 367-3p, 371a-3p, 372-3p and 373-3p [28]. In particular, miR-371a-3p was discovered to be both sensitive (84.7%) and specific (99%) when used as a serum tumor marker of GCTs while non-testicular malignancies had levels of miR- 371a-3p similar to control patients [28]. In GCT patients miR-371a-3p has been shown to increase with advancing disease and tumor bulk as well as decrease after tumor resection [28]. Importantly, miR-371a-3p outperformed current tumor markers human chorionic gonadotropin (HCG) and alpha-fetoprotein (AFP) in a study of 76 GCT patients [29]. This finding has high clinical significance as HCG and AFP are only elevated in 60% of GCT patients [30]. These studies show that epigenetic features of GCTs such as miRNAs could provide future biomarkers for this disease.

A MECHANISM FOR PLATINUM RESISTANCE IN TESTICULAR CANCER

Epigenetic silencing, such as hypermethylation, may play a role in resistance to platinum in refractory GCTs [18, 21, 31]. Cisplatin acts by forming platinum-DNA lesions in dividing cells, forcing them to undergo DNA repair or apoptosis. Hypomethylated DNA is generally thought to be more active, or “open”, allowing for easier integration of platinum into DNA leading to more cellular damage and apoptosis. When genes become hypermethylated this access is decreased, possibly leading to resistance. Hypermethylation is a reversible phenomenon and thus can be targeted [32].

Platinum refractory GCTs have been associated with hypermethylation in the promoter regions of tumor suppressor genes. Hypermethylation of RASSF1A and H1C1, was found to be associated with resistance in GCTs [21]. Additionally, cisplatin treatment of GCTs was found to induce de novo promoter hypermethylation in vivo [21]. Wermann et al. also showed a relationship between DNA methylation and chemotherapy resistance in GCTs by reporting that a cisplatin resistant seminoma was hypermethylated compared to sensitive seminoma [33]. These findings have implications as to how GCTs may become refractory to platinum based therapy.

Epigenetic silencing through methylation has been demonstrated to have similar effects to DNA mutations in tumorigenesis and chemotherapy resistance and has led to studies designed to reverse this potential mechanism in platinum refractory tumors [33]. De novo methylation of DNA involves DNMT3A and DNMT3B [35, 36]. Elevated DNMT3B levels are associated with tumor invasion in seminomatous GCT. Stage I and Stage II seminomas showed high expression of DNMT3B in 36 and 38% of tumors, respectively [37]. Stage III seminomas showed high expression of DNMT3B in 100% of tumors studied [37]. Additionally, if DNMT3B expression was not elevated, relapse occurred in only 7% of cases compared with over 24% of cases with elevated DNMT3B. Seminomas that had high expression levels of DNMT3B had a lower relapse-free survival rate [37].

PRECLINICAL EPIGENETIC DRUG STUDIES IN TESTICULAR CANCER

The nucleoside analogs 5-aza-deoxycytidine (5-aza-CdR, decitabine) and 5-aza-cytidine (5-aza-CR) are potent inhibitors of DNA methylation (Fig. 2). DNA hypomethylation is mediated through the covalent trapping of DNMTs [11, 38]. This class of therapeutic agents are known as DNA methyltransferase inhibitors (DNMTIs). Studies have shown that DNMTIs hit multiple targets including the tumor suppressor gene p53. Activation of this gene leads the cell down the DNA damage response pathway and to apoptosis in EC cells [39, 40]. We have shown that human pluripotent EC, the stem cells of nonseminoma GCTs, are highly sensitive to very low doses of decitabine, doses that are 100-1000 fold lower than those used to induce cytotoxicity of somatic cancer cells [41]. Low dose decitabine was effective in mediating global promoter demethylation, p53 target gene activation, cytotoxicity, and differentiation of EC cells [41, 42]. Low nanomolar concentrations of decitabine caused decreased cell proliferation and survival and the reexpression of genes silenced by DNA hypermethylation including MGMT, RASSF1A, and HOXA9.

Fig. (2).

Fig. (2).

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SGI-110 is a second generation hypomethylating agent. SGI-110 is a dinucleotide of decitabine and deoxyguanosine. Decitabine is rapidly eliminated by cytosine deaminase, limiting drug exposure in vivo. SGI-110 increases the in vivo exposure of decitabine by protecting it from deamination.

Decitabine hypersensitivity was associated with markedly high expression of pluripotency-associated DNMT3B compared with somatic tumor cells [41, 42]. Notably, hypersensitivity to decitabine also occurs in EC cells resistant to cisplatin. Further, low dose decitabine can restore cisplatin sensitivity to cisplatin resistant cells [41, 42]. This finding was recently confirmed by Wermann et al. who showed that decitabine restored sensitivity to cisplatin in a refractory seminoma cell line [33]. Knockdown of DNMT3B resulted in resistance to decitabine, suggesting that decitabine sensitivity in EC is mechanistically linked to, and dependent on, high levels of DNMT3B [41, 42]. That a variety of EC cell lines are sensitive to very low doses of decitabine with dependence on DNMT3B was also reported by Wongtrakoongate et al. [43]. Studies in ovarian cancer using a variety of DNMTIs also reported carboplatin resensitization after DNA hypomethylation [44-46].

CLINICAL EPIGENETIC STUDIES USING DEMETHYLATING AGENTS

Decitabine is approved for the treatment of myelodysplastic syndrome (MDS) and shows promise for the treatment of specific leukemias [47]. Recent experience with MDS suggests that decitabine should be given below the maximum tolerated dose and over many cycles since tumor responses usually do not occur until after the third cycle [47]. This may explain why DNA methylation inhibitors given at maximum tolerated doses incompatible with cell division were disappointing for solid tumors. In addition, recent preclinical and clinical studies have suggested that lower “biologically effective” doses of decitabine may be an effective clinical strategy to target tumor initiating cells in solid tumors [48, 49]. Two recent phase I/II studies showed that low dose DNMTI treatment could restore sensitivity to carboplatin in patients with heavily pretreated ovarian cancer and resistance to cisplatin was correlated with hypermethylation of ovarian tumor DNA [50, 51].

In a phase II trial of GCT patients who had progressed within four weeks of cisplatin based therapy and were then treated with 5-aza-cytidine, all patients had progressive disease. Fifteen of the seventeen died of progressive disease and one from sepsis [52]. However, high, cytotoxic doses (150 mg/m2) were used that are now known to be suboptimal for mediating demethylation. More recently Matei et al. conducted a phase I/II trial of a DNMTI followed by carboplatin, in ovarian cancer [50]. This study showed that sequential low dose decitabine followed by carboplatin was well tolerated. Additionally, this study demonstrated biologic response in the form of DNA-hypomethylation that correlated with reversed platinum resistance [53]. Similar data from other institutions corroborated these findings [54].

A currently ongoing phase II trial is based on a study of 17 patients with heavily pre-treated and platinum-resistant ovarian cancer. When this population is treated only with carboplatin, there was a small objective response of less than 10% [55]. However, pre-treating with a DNMTI before carboplatin administration led to a 35% objective response rate. Additionally, patients that were pre-treated with a DNMTI had a PFS of 10.2 months with over half (53%) of patients free of disease progression at 6 months. Interestingly, tumors biopsied after treatment found that patients who had PFS of more than 6 months had a greater number of demethylated genes involved in cancer pathways than patients who had PFS of less than 6 months. This suggests that demethylation with the DNMTI decitabine has an impact in mediating platinum resensitization [50]. Fu et al. showed similar findings in the same patient population with a DNMTI and carboplatin used in succession with a 13.8% overall response rate, as well as a disease control rate (partial response rate plus stable disease) of 45% [51]. From this data, a phase II study was designed to resensitize ovarian cancer to cisplatin using the novel DNMTI, SGI-110, in ovarian cancer (NCT01696032).

In addition to ovarian cancer, a combination phase I/II trial was conducted in patients with recurrent metastatic nonsmall cell lung cancer. The patients were heavily pre-treated with three lines of chemotherapy before receiving a DNMTI and a histone deacetylase inhibitor. A subset of patients then received further cytotoxic chemotherapy. Nearly 20% of the patients lived over 1 year with some living up to 4 years [48]. The expected survival of this patient population after receiving three lines of chemotherapy is 48% at 6 months [56]. The authors of this study suggest that the response may be attributable to prolonged stabilization, but they did document an unusually robust response to subsequent cytotoxic chemotherapy [48].

SGI-110 AS A NOVEL DEMETHYLATING AGENT

DNMTIs such as decitabine are subject to rapid degradation by hydrolytic cleavage and deamination by cytidine deaminase and are unstable after intravenous infusion limiting their potential as cancer therapeutics [57]. Decitabine has a promising dinucleotide analogue named SGI-110 (2′-deoxy-5-azacytidylyl-(3′-5′)-2′-deoxyguanosine sodium salt) (Astex pharmaceuticals Inc.) that is not subject to the same metabolism as other DNMTIs [58] (Fig. 2). Additionally, SGI-110 can be given as a subcutaneous injection allowing for a longer effective half-life and a more extended exposure window compared to intravenous infusion of decitabine [59]. This novel DNMTI was shown to achieve hypomethylation and was well tolerated in primates [60]. Additionally, in cancer xenograft models it has proved to be an effective DNA methylation inhibitor in vivo and reduced tumor growth if MDS cells [58]. In a phase I/II trial in acute myeloid leukemia and MDS patients, SGI-110 was shown to be better tolerated and demonstrated activity in patients who had progressed on decitabine [61].

Preclinical data from Indiana University showed that like decitabine, SGI-110 also re-sensitizes platinum resistant ovarian cancer cell lines [32]. Specifically, a greater than two-fold reduction in the inhibitory concentration of platinum was achieved with SGI-110 pre-treatment. Additionally, biopsies of subcutaneous ovarian cancer xenografts showed that SGI-110 treatment induced significant demethylation and subsequent transcriptional enhancement of tumor suppressor genes [32]. SGI-110 alone or in combination with carboplatin was well tolerated in non-tumor bearing mice.

Significant antitumor activity was observed with single agent SGI-110 as well as with SGI-110 given in combination with carboplatin treatment in both biweekly and daily regimens [32]. The results of this preclinical study supports the recently launched clinical trial NCT01696032 using SGI-110 in combination with carboplatin in patients with recurrent, platinum-refractory ovarian cancer [32, 62]. The study demonstrated that the combination of SGI-110 at 30 mg/m2 and carboplatin at AUC 4 is well tolerated and this regimen is currently being implemented in stage 2 of this trial [62]. Further SGI-110 was shown to inhibit GCT-derived EC equally well and with a similar mode of action as compared to decitabine in vitro and in xenografts (Fig. 3 and Albany and Spinella, unpublished).

Fig. (3).

Fig. (3).

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Low dose, SGI-110 dramatically effects growth of testicular germ cell tumor xenografts. Representative result of a testicular cancer xenograft treated with 2.0 mg/kg SGI-110 or vehicle for 5 days per week for 2 weeks (Albany and Spinella, unpublished).

FUTURE DIRECTIONS

DNA methyltransferase inhibitors have the ability to resensitize both platinum-refractory testicular germ cell tumors and platinum-refractory ovarian cancer to platinum based chemotherapy and studies indicate that GCT cells are exquisitely sensitive to low dose DNMTI therapy [41, 42, 49, 53]. The most recent clinical trial using a DNMTI to treat refractory GCT was unsuccessful in improving outcomes. However, in this trial 5-azacytidine was used at high doses as a non-specific cytotoxic agent and not at the low doses that are optimized to inhibit DNA methylation. The trial also did not test whether DNMTI therapy could chemosensitize refractory GCTs to platinum [52]. The aforementioned preclinical trials have shown DNMTIs to have success in resensitizing platinum refractory ovarian cancer cells. The progression of this basic science discovery to a clinical trial using SGI-110 as a DNMTI to sensitize platinum-refractory ovarian cancer is currently underway (NCT01696032). With the evidence that SGI-110 also acts to demethylate refractory GCT cell lines, restoring the chemotherapeutic effects of platinum, a clinical trial using this agent in these patients is warranted.

We are now conducting an open label proof of concept, single arm, phase I dose escalation study of SGI-110 in patients with refractory GCTs (NCT02429466) (Fig. 4). Based on the clinical trial in ovarian cancer noted above, patients will be treated with subcutaneous SGI-110 injections at 30 mg/m2 on a daily basis for days 1-5 followed by cisplatin 100 mg/m2 on day 8 every 21 days. We seek to bring forward the new concept of epigenetic targeting in refractory GCTs and set the stage for interventions targeting the refractory GCT epigenome, as well as guide and impact the design of future clinical investigations in refractory GCTs.

Fig. (4).

Fig. (4).

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Outline of the ongoing phase I protocol, NCT02429466, for the treatment of refractory testicular germ cell cancer patients.

CONCLUSION

The tactic of using DNMTIs for cancer treatment is no longer isolated to hematologic malignancies. Due to their unique germ cell origins, germ cell tumors are associated with distinct epigenetics that may be exploited to treat this malignancy. Micro RNAs, specifically miR-371a-3p, have been shown in preclinical and early clinical studies to be promising biomarkers that may increase the sensitivity and specificity to track disease. Preclinical data indicates that GCTs are hypersensitive to hypomethylating agents perhaps due to a high level of the germ cell marker DNMT3B and that refractory GCTs can be resensitized with these agents. Clinical trials in ovarian cancer have demonstrated safety of DNMTI and platinum combination therapy which is currently being further investigated in phase II trials. Clinical trials of DNMTIs in platinum refractory GCT are warranted. We are currently conducting a phase I trial using SGI-110 to re-sensitize platinum refractory GCT patients to combat this deadly disease.

ACKNOWLEDGEMENTS

This work was supported by a grant from the Alex’s Lemonade Stand Foundation (MJS and CA) and a Career Development Award from ASCO (CAx).

Biography

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C. Albany

Footnotes

CONFLICT OF INTEREST

The authors confirm that this article content has no conflicts of interest.

REFERENCES

  • [1].Huyghe E; Matsuda T; Thonneau P Increasing incidence of testicular cancer worldwide: a review. J. Urol, 2003, 170(1), 5–11. [DOI] [PubMed] [Google Scholar]
  • [2].Feldman DR; Bosl GJ; Sheinfeld J; Motzer RJ Medical treatment of advanced testicular cancer. JAMA, 2008, 299(6), 672–684. [DOI] [PubMed] [Google Scholar]
  • [3].Rosenberg B; Vancamp L; Krigas T Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode. Nature, 1965, 205, 698–699. [DOI] [PubMed] [Google Scholar]
  • [4].Einhorn LH; Donohue J Cis-diamminedichloroplatinum, vinblastine, and bleomycin combination chemotherapy in disseminated testicular cancer. Ann. Intern. Med, 1977, 87(3), 293–298. [DOI] [PubMed] [Google Scholar]
  • [5].Williams SD; Birch R; Einhorn LH; Irwin L; Greco FA; Loehrer PJ Treatment of disseminated germ-cell tumors with cisplatin, bleomycin, and either vinblastine or etoposide. N. Engl. J. Med, 1987, 316 (23), 1435–1440. [DOI] [PubMed] [Google Scholar]
  • [6].Einhorn LH Curing metastatic testicular cancer. Proc. Natl. Acad. Sci. USA, 2002, 99 (7), 4592–4595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].International Germ Cell Consensus Classification: a prognostic factor-based staging system for metastatic germ cell cancers. International Germ Cell Cancer Collaborative Group. J. Clin. Oncol, 1997, 15(2), 594–603. [DOI] [PubMed] [Google Scholar]
  • [8].El-Helw L; Coleman RE Salvage, dose intense and high-dose chemotherapy for the treatment of poor prognosis or recurrent germ cell tumours. Cancer Treat. Rev 2005, 3(3), 197–209. [DOI] [PubMed] [Google Scholar]
  • [9].Feldman DR; Patil S; Trinos MJ; Carousso M; Ginsberg MS; Sheinfeld J, Bajorin DF, Bosl GJ, Motzer RJ. Progression-free and overall survival in patients with relapsed/refractory germ cell tumors treated with single-agent chemotherapy: endpoints for clinical trial design. Cancer, 2012, 118(4), 981–986. [DOI] [PubMed] [Google Scholar]
  • [10].Porcu P; Bhatia S; Sharma M; Einhorn LH Results of treatment after relapse from high-dose chemotherapy in germ cell tumors. J. Clin. Oncol, 2000, 18(6), 1181–1186. [DOI] [PubMed] [Google Scholar]
  • [11].Jones PA; Baylin SB The epigenomics of cancer. Cell, 2007, 128(4), 683–692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Lopez J; Percharde M; Coley HM; Webb A; Crook T The context and potential of epigenetics in oncology. Br. J. Cancer, 2009, 100(4), 571–577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Albany C; Alva AS; Aparicio AM; Singal R; Yellapragada S; Sonpavde G; Hahn NM Epigenetics in prostate cancer. Prostate Cancer, 2011, 580318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Lind GE; Skotheim RI; Lothe RA The epigenome of testicular germ cell tumors. APMIS, 2007, 15(10), 1147–1160. [DOI] [PubMed] [Google Scholar]
  • [15].Hanna NH; Einhorn LH Testicular cancer--discoveries and updates. N. Engl. J. Med, 2014, 371(21), 2005–2016. [DOI] [PubMed] [Google Scholar]
  • [16].Kawakami T; Okamoto K; Kataoka A; Koizumi S; Iwaki H; Sugihara H; Reeve AE; Ogawa O; Okada Y Multipoint methylation analysis indicates a distinctive epigenetic phenotype among testicular germ cell tumors and testicular malignant lymphomas. Genes Chromosomes Cancer, 2003, 38(1), 97–101. [DOI] [PubMed] [Google Scholar]
  • [17].Ohm JE; McGarvey KM; Yu X; Cheng L; Schuebel KE; Cope L; Mohammad HP; Chen W; Daniel VC; Yu W; Berman DM; Jenuwein T; Pruitt K; Sharkis SJ; Watkins DN; Herman JG; Baylin SB A stem cell-like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing. Nat. Genet, 2007, 39(2), 237–242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Cheung HH; Lee TL; Davis AJ; Taft DH; Rennert OM; Chan WY Genome-wide DNA methylation profiling reveals novel epigenetically regulated genes and non-coding RNAs in human testicular cancer. Br. J. Cancer, 2010, 102(2), 419–427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Okamoto K Epigenetics: a way to understand the origin and biology of testicular germ cell tumors. Int. J. Urol, 2012, 19(6), 504–511. [DOI] [PubMed] [Google Scholar]
  • [20].Brait M; Maldonado L; Begum S; Loyo M; Wehle D; Tavora FF; Looijenga LH; Kowalski J; Zhang Z; Rosenbaum E; Halachmi S; Netto GJ; Hoque MO DNA methylation profiles delineate epigenetic heterogeneity in seminoma and non-seminoma. Br. J. Cancer, 2012, 106(2), 414–423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Koul S; McKiernan JM; Narayan G; Houldsworth J; Bacik J; Dobrzynski DL; Assaad AM; Mansukhani M; Reuter VE; Bosl GJ; Chaganti RS; Murty VV Role of promoter hypermethylation in cisplatin treatment response of male germ cell tumors. Mol, Cancer, 2004, 3, 16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Rijlaarsdam MA; Tax DM; Gillis AJ; Dorssers LC; Koestler DC; de Ridder J; Looijenga LH Genome wide DNA methylation profiles provide clues to the origin and pathogenesis of germ cell tumors. PLoS One, 2015, 10(4), e0122146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [23].van der Zwan YG; Rijlaarsdam MA; Rossello FJ; Notini AJ; de Boer S; Watkins DN' Gillis AJ; Dorssers LC; White SJ; Looijenga LH Seminoma and embryonal carcinoma footprints identified by analysis of integrated genome-wide epigenetic and expression profiles of germ cell cancer cell lines. PLoS One, 2014, 9(6), e98330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Furukawa S; Haruta M; Arai Y; Honda S; Ohshima J; Sugawara W; Kageyama Y; Higashi Y; Nishida K; Tsunematsu Y; Nakadate H; Ishii M; Kaneko Y Yolk sac tumor but not seminoma or teratoma is associated with abnormal epigenetic reprogramming pathway and shows frequent hypermethylation of various tumor suppressor genes. Cancer Sci, 2009, 100(4), 698–708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Krege S; Beyer J; Souchon R; Albers P; Albrecht W; Algaba F; and the European Germ Cell Cancer Consensus Group. European consensus conference on diagnosis and treatment of germ cell cancer: a report of the second meeting of the European Germ Cell Cancer Consensus Group (EGCCCG): part I. Eur. Urol, 2008, 55(3), 478–496. [DOI] [PubMed] [Google Scholar]
  • [26].Schickel R; Boyerinas B; Park SM; Peter ME MicroRNAs: key players in the immune system, differentiation, tumorigenesis and cell death. Oncogene, 2008, 27(45), 5959–5974. [DOI] [PubMed] [Google Scholar]
  • [27].Zhang L; Volinia S; Bonome T; Calin GA; Greshock J; Yang N; Liu CG; Giannakakis A; Alexiou P; Hasegawa K; Johnstone CN; Megraw MS; Adams S; Lassus H; Huang J; Kaur S; Liang S; Sethupathy P; Leminen A; Simossis VA; Sandaltzopoulos R; Naomoto Y; Katsaros D; Gimotty PA; DeMichele A; Huang Q; Bützow R; Rustgi AK; Weber BL; Birrer MJ; Hatzigeorgiou AG; Croce CM; Coukos G Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer. Proc. Natl. Acad. Sci. USA, 2008, 705(19), 7004–7009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Spiekermann M; Belge G; Winter N; Ikogho R; Balks T; Bullerdiek J; Dieckmann KP MicroRNA miR-371a-3p in serum of patients with germ cell tumours: evaluations for establishing a serum biomarker. Andrology, 2015, 5(1), 78–84. [DOI] [PubMed] [Google Scholar]
  • [29].Syring I; Bartels J; Holdenrieder S; Kristiansen G; Müller SC; Ellinger J Circulating serum miRNA (miR-367-3p, miR-371a-3p, miR-372-3p and miR-373-3p) as biomarkers in patients with testicular germ cell cancer. J. Urol, 2015, 705(1), 331–337. [DOI] [PubMed] [Google Scholar]
  • [30].Gillis AJ; Rijlaarsdam MA; Eini R; Dorssers LC; Biermann K; Murray MJ; Nicholson JC; Coleman N; Dieckmann KP; Belge G; Bullerdiek J; Xu T Bernard N Looijenga LH Targeted serum miRNA (TSmiR) test for diagnosis and follow-up of (testicular) germ cell cancer patients: a proof of principle. Mol. Oncol, 2013, 7(6), 1083–1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Giuliano CJ; Freemantle SJ; Spinella MJ Testicular Germ Cell Tumors: A Paradigm for the Successful Treatment of Solid Tumor Stem Cells. Curr. Cancer Ther. Rev, 2006, 2(3), 255–270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Fang F; Munck J; Tang J; Taverna P; Wang Y; Miller DF; Pilrose J; Choy G; Azab M; Pawelczak KS; VanderVere-Carozza P; Wagner M; Lyons J; Matei D; Turchi JJ; Nephew KP The novel, small-molecule DNA methylation inhibitor SGI-110 as an ovarian cancer chemosensitizer. Clin. Cancer Res, 2014, 20(24), 6504–6516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Wermann H; Stoop H; Gillis AJ; Honecker F; van Gurp RJ; Ammerpohl O; Richter J; Oosterhuis JW; Bokemeyer C; Looijenga LH Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance. J. Pathol, 2010, 227(4), 433–442. [DOI] [PubMed] [Google Scholar]
  • [34].Herman JG; Baylin SB Gene silencing in cancer in association with promoter hypermethylation. N. Engl. J. Med, 2003, 549(21), 2042–2054. [DOI] [PubMed] [Google Scholar]
  • [35].Okano M; Xie S; Li E Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat. Genet, 1998, 79(3), 219–220. [DOI] [PubMed] [Google Scholar]
  • [36].Okano M; Bell DW; Haber DA; Li E DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell, 1999, 99(3), 247–257. [DOI] [PubMed] [Google Scholar]
  • [37].Arai E; Nakagawa T; Wakai-Ushijima S; Fujimoto H; Kanai Y DNA methyltransferase 3B expression is associated with poor outcome of stage I testicular seminoma. Histopathology, 2012, 00(6B), E12–E18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].Friedman S The inhibition of DNA(cytosine-5)methylases by 5-azacytidine. The effect of azacytosine-containing DNA. Mol Pharmacol, 1981, 79(2), 314–320. [PubMed] [Google Scholar]
  • [39].Karpf AR; Moore BC; Ririe TO; Jones DA Activation of the p53 DNA damage response pathway after inhibition of DNA methyltransferase by 5-aza-2'-deoxycytidine. Mol. Pharmacol, 2001, 59(4), 751–757. [PubMed] [Google Scholar]
  • [40].Kerley-Hamilton JS, Pike AM, Li N, DiRenzo J, Spinella MJ. A p53-dominant transcriptional response to cisplatin in testicular germ cell tumor-derived human embryonal carcinoma. Oncogene, 2005, 24(40), 6090–6100. [DOI] [PubMed] [Google Scholar]
  • [41].Beyrouthy MJ; Garner KM; Hever MP; Freemantle SJ; Eastman A; Dmitrovsky E; Spinella MJ High DNA methyltransferase 3B expression mediates 5-aza-deoxycytidine hypersensitivity in testicular germ cell tumors. Cancer Res, 2009, 09(24), 9360–9366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [42].Biswal BK; Beyrouthy MJ; Hever-Jardine MP; Armstrong D; Tomlinson CR; Christensen BC; Marsit CJ; Spinella MJ Acute hypersensitivity of pluripotent testicular cancer-derived embryonal carcinoma to low-dose 5-aza deoxycytidine is associated with global DNA Damage-associated p53 activation, anti-pluripotency and DNA demethylation. PLoS One, 2012, 7(12), e53003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [43].Wongtrakoongate P; Li J; Andrews PW Aza-deoxycytidine induces apoptosis or differentiation via DNMT3B and targets embryonal carcinoma cells but not their differentiated derivatives. Br. J. Cancer, 2014, 770(8), 2131–2138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [44].Balch C Yan P Craft T Young S Skalnik DG Huang TH Nephew KP Antimitogenic and chemosensitizing effects of the methylation inhibitor zebularine in ovarian cancer. Mol. Cancer Ther, 2005, 4(10), 1505–1514. [DOI] [PubMed] [Google Scholar]
  • [45].Lenzi R; Frost P; Abbruzzese JL Modulation of cisplatin resistance by 2'-deoxy-5-azacytidine in human ovarian tumor cell lines. Anticancer Res, 1994, 74(1A), 247–251. [PubMed] [Google Scholar]
  • [46].Li Y; Hu W; Shen DY; Kavanagh JJ; Fu S Azacitidine enhances sensitivity of platinum-resistant ovarian cancer cells to carboplatin through induction of apoptosis. Am. J. Obstet. Gynecol, 2009, 200(2), 177 e1–9. [DOI] [PubMed] [Google Scholar]
  • [47].Santos FP; Kantarjian H; Garcia-Manero G; Issa JP; Ravandi F Decitabine in the treatment of myelodysplastic syndromes. Expert. Rev. Anticancer Ther, 2010, 70(1), 9–22. [DOI] [PubMed] [Google Scholar]
  • [48].Juergens RA; Wrangle J; Vendetti FP; Murphy SC; Zhao M; Coleman B; Sebree R; Rodgers K; Hooker CM; Franco N; Lee B; Tsai S; Delgado IE; Rudek MA; Belinsky SA; Herman JG; Baylin SB; Brock MV; Rudin CM Combination epigenetic therapy has efficacy in patients with refractory advanced non-small cell lung cancer. Cancer Discov, 2011, 7(7), 598–607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [49].Tsai HC; Li H; Van Neste L; Cai Y; Robert C; Rassool FV; Shin JJ; Harbom KM; Beaty R; Pappou E; Harris J; Yen RW; Ahuja N; Brock MV; Stearns V; Feller-Kopman D; Yarmus LB; Lin YC; Welm AL; Issa JP; Minn I; Matsui W; Jang YY; Sharkis SJ; Baylin SB; Zahnow CA Transient low doses of DNA-demethylating agents exert durable antitumor effects on hematological and epithelial tumor cells. Cancer Cell, 2012, 27(3), 430–446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [50].Matei D; Fang F; Shen C; Schilder J; Arnold A; Zeng Y; Berry WA; Huang T; Nephew KP Epigenetic resensitization to platinum in ovarian cancer. Cancer Res, 2012, 72(9), 2197–2205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [51].Fu S; Hu W; Iyer R; Kavanagh JJ; Coleman RL; Levenback CF; Sood AK; Wolf JK; Gershenson DM; Markman M; Hennessy BT; Kurzrock R; Bast RC Jr. Phase 1b-2a study to reverse platinum resistance through use of a hypomethylating agent, azacitidine, in patients with platinum-resistant or platinum-refractory epithelial ovarian cancer. Cancer, 2011, 777(8), 1661–1669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [52].Roth BJ; Elson P; Sledge GW Jr., Einhorn LH; Trump DL. 5-Azacytidine (NSC 102816) in refractory germ cell tumors. A phase II trial of the Eastern Cooperative Oncology Group. In-vest. New. Drugs, 1993, 77(2-3), 201–202. [DOI] [PubMed] [Google Scholar]
  • [53].Fang F; Balch C; Schilder J; Breen T; Zhang S; Shen C; Li L; Kulesavage C; Snyder AJ; Nephew KP; Matei DE A phase 1 and pharmacodynamic study of decitabine in combination with carboplatin in patients with recurrent, platinum-resistant, epithelial ovarian cancer. Cancer, 2010, 770(17), 4043–4053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [54].Appleton K; Mackay HJ; Judson I; Plumb JA; McCormick C; Strathdee G; Lee C; Barrett S; Reade S; Jadayel D; Tang A; Bellenger K; Mackay L; Setanoians A; Schätzlein A; Twelves C; Kaye SB; Brown R Phase I and pharmacodynamic trial of the DNA methyltransferase inhibitor decitabine and carboplatin in solid tumors. J. Clin. Oncol, 2007, 25(29), 4603–4609. [DOI] [PubMed] [Google Scholar]
  • [55].See HT; Freedman RS; Kudelka AP; Burke TW; Gershenson DM; Tangjitgamol S; Kavanagh JJ Retrospective review: re-treatment of patients with ovarian cancer with carboplatin after platinum resistance. Int. J. Gynecol Cancer, 2005, 75(2), 209–216. [DOI] [PubMed] [Google Scholar]
  • [56].Girard N; Jacoulet P; Gainet M; Elleuch R; Pernet D; Depierre A; Dalphin JC; Westeel V Third-line chemotherapy in advanced non-small cell lung cancer: identifying the candidates for routine practice. J. Thorac. Oncol, 2009, 4(12), 1544–1549. [DOI] [PubMed] [Google Scholar]
  • [57].Jabbour E; Issa JP; Garcia-Manero G; Kantarjian H Evolution of decitabine development: accomplishments, ongoing investigations, and future strategies. Cancer, 2008, 112(11), 2341–2351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [58].Yoo CB; Jeong S; Egger G; Liang G; Phiasivongsa P; Tang C; Redkar S; Jones PA. Delivery of 5-aza-2'-deoxycytidine to cells using oligodeoxynucleotides. Cancer Res, 2007, 57(13), 6400–6408. [DOI] [PubMed] [Google Scholar]
  • [59].Kantarjian HM, R.G., Rizzieri DA, Stock W, O'Connell CL, Griffithes EA, Yee K, Tibes R., Garcia-Manero G, Ravandi F, Walsh K, Feldman E, Ritchie E, Rao A, Decastro C, Schimmer AD, Mesa RA, Syed I, Choy G, Organesian A, Taverna P, Azab M, Chung W, Issa JP. Results from the dose escalation phase of a randomized phase 1-2 first-in-human (FIH) study of SGI-110, a novel low volume stable subcutaneous (SQ) second generation hypomethylating agent (HMA) in patients with replapsed/refractory MDS and AML. In: Proceedings of the 54th Annual American Socitety of Hematology Meeting, Atlanta, Grorgia, December 8-11, 2012. [Google Scholar]
  • [60].Lavelle D; Saunthararajah Y; Vaitkus K; Singh M; Banzon V; Phiasivongsva P; Redkar S; Kanekal S; Bearss D; Shi C; Inloes R; DeSimone J S110, a novel decitabine dinucleotide, increases fetal hemoglobin levels in baboons (P. anubis). J. Transl. Med, 2010, 8, 92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [61].Issa JP; Roboz G; Rizzieri D; Faderl S; O'Connell CL; Stock W; Tibes R; Griffiths E; Yee K; Chung W; Choy G; Organesian A; Teverna P; Azab M; Kantarjian H Interim results from a randomized Phase 1-2 first-in-human study of PK/PD guided escalating doses of SGI-110, a novel subcutaneous second generation hypomethylating agent in relapsed/refractory MDS and AML. In: Proceedings of the 103rd Annual American Association of Cancer Research Meeting, March 31- April 4, Chicago, IL, Cancer Res, 2012, 72(Suppl 8), LB–214. [Google Scholar]
  • [62].Fleming G; Ghamande S; Lin Y; Secord AA; Nemunaitis J; Markham MJ; Nephew K; Fang F; Gupta S; Naim S; Choy G; Jueliger S; Taverna P; Hao Y; Keer H; Azab M; Matei D Clinical epigenetic resensitization of platinum-resistant, recurrent ovarian cancer patients with SGI-110, a novel second-generation, subcutaneously administered hypomethylating agent (HMA). In: Proceedings of the 105rd Annual American Association of Cancer Research Meeting, April 5-8, San Diego, CA, Cancer Res, 2014, 74(Suppl 19), LB-2320. [Google Scholar]

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