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J Neurooncol (2012) 106:71–79 DOI 10.1007/s11060-011-0647-9 LABORATORY INVESTIGATION - HUMAN/ANIMAL TISSUE Expression analysis of stem cell-related genes reveal OCT4 as a predictor of poor clinical outcome in medulloblastoma Carolina Oliveira Rodini • Daniela Emi Suzuki • Najsla Saba-Silva • Andréa Cappellano • Jorge Estefano Santana de Souza • Sérgio Cavalheiro Silvia Regina Caminada Toledo • Oswaldo Keith Okamoto • Received: 17 February 2011 / Accepted: 20 June 2011 / Published online: 2 July 2011 Ó Springer Science+Business Media, LLC. 2011 Abstract Aberrant expression of stem cell-related genes in tumors may confer more primitive and aggressive traits affecting clinical outcome. Here, we investigated expression and prognostic value of the neural stem cell marker CD133, as well as of the pluripotency genes LIN28 and OCT4 in 37 samples of pediatric medulloblastoma, the most common and challenging type of embryonal tumor. While most medulloblastoma samples expressed CD133 and LIN28, OCT4 expression was found to be more sporadic, with detectable levels occurring in 48% of tumors. Expression levels of OCT4, but not CD133 or LIN28, were significantly correlated with shorter survival (P B 0.0001). Median survival time of patients with tumors hyperexpressing OCT4 and tumors displaying low/undetectable OCT4 expression were 6 and 153 months, respectively. More importantly, when patients were clinically stratified according to their risk of tumor recurrence, positive OCT4 expression in primary tumor specimens could discriminate patients classified as average risk but which further deceased within 5 years of diagnosis (median survival time of 28 months), a poor clinical outcome typical of high risk patients. Our findings reveal a previously unknown prognostic value for OCT4 expression status in medulloblastoma, which might be used as a further indicator of poor survival and aid postoperative treatment selection, with a particular potential benefit for clinically average risk patients. Keywords Medulloblastoma CD133 LIN28 OCT4 Prognosis Stem cell markers Electronic supplementary material The online version of this article (doi:10.1007/s11060-011-0647-9) contains supplementary material, which is available to authorized users. C. O. Rodini D. E. Suzuki S. Cavalheiro Department of Neurology and Neurosurgery, Federal University of São Paulo, São Paulo, SP, Brazil C. O. Rodini D. E. Suzuki O. K. Okamoto (&) Centro de Estudos do Genoma Humano, Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277 Cidade Universitária, São Paulo, SP 05508-090, Brazil e-mail: [email protected] N. Saba-Silva A. Cappellano S. R. C. Toledo Institute of Pediatric Oncology (GRAACC), Pediatric Department, Federal University of São Paulo, São Paulo, SP, Brazil J. E. S. de Souza Ludwig Institute for Cancer Research, São Paulo Branch at Hospital Alemão Oswaldo Cruz, São Paulo, SP, Brazil Introduction Pediatric medulloblastoma is a malignant embryonal neuroepithelial tumor that accounts for approximately 16% of all pediatric brain tumors, typically occurring in children under the age 10. While treatable, about 15% of patients die from the disease within 2 years of diagnosis [1, 2]. Current treatment is based mainly on tumor resection, followed by craniospinal radiotherapy and treatment with conventional cytotoxic agents. However, these complementary therapies often elicit long-term adverse effects including secondary tumors, as well as hearing, cognitive, endocrinal, and vascular impairment [3]. Thus, to manage benefits and risks, the choice of postoperative treatment modality is based on clinical parameters including age at diagnosis, volume of the remaining tumor, and presence of metastasis. 123 72 Children under 3 years of age are usually not subjected to radiotherapy to avoid damage to the developing brain. Older patients with non-metastatic and totally or nearly totally resected tumors are considered as having ‘‘average risk’’ of tumor recurrence and are treated with low doses of radiation and chemotherapy. Patients with partial tumor resection and/or metastatic disease are classified as ‘‘high risk’’, regarding tumor recurrence, and thus subjected to a more aggressive postoperative treatment with radiation and chemotherapy at higher doses. Under this rational treatment stratification based on clinical prognostic criteria, the 5-year progression-free survival rate for medulloblastoma stands at around 40 and 70% for high and average risk patients, respectively [4]. Still, a significant number of patients do not respond well to standardized treatments. About 30% of average risk patients either present disease progression or succumb to the disease within 5 years. Although only a few alternative experimental treatments are available for high risk patients, an improved classification system could help to identify beforehand those clinically average risk patients with potential poor prognosis and select them to more aggressive treatments, as performed for their high risk counterparts. Under the cancer stem cell hypothesis, factors contributing to poor prognosis, such as resistance to conventional therapies, tumor recurrence, and metastasis, could be associated with a small subset of highly tumorigenic stemlike cells. In medulloblastoma, stem-like cells were first identified and isolated based on expression of CD133, a cell membrane antigen present in neural stem cells [5]. As an embryonal tumor, medulloblastoma cells could also display ectopic expression of typical pluripotency genes such as LIN28 and OCT4, which encode an RNA binding protein and a transcription factor containing a POU homeodomain, respectively [6]. In fact, OCT4 expression has been reported in germ cell tumors including seminoma, dysgerminoma, germinoma, and embryonal carcinoma [7], and its expression has been associated with a more primitive and malignant tumor phenotype [8]. In medulloblastoma, expression of stem cell-related genes might help distinguish tumors associated with distinct clinical outcomes, but, thus far, a possible correlation of such stem cell markers with prognosis has not been investigated in this important pediatric tumor. In this study, we have analyzed the expression of CD133, LIN28, and OCT4 genes in a series of 37 medulloblastoma samples and have found a significant correlation of OCT4 expression with overall survival. Interestingly, OCT4 expression could discriminate primary tumor specimens from patients clinically classified as average risk but which had a poor clinical outcome similar to that of high risk patients. 123 J Neurooncol (2012) 106:71–79 Materials and methods Tumor collection A total of 37 medulloblastoma specimens were obtained by surgical resection of patients admitted for diagnosis and treatment at the Institute of Pediatric Oncology (IOP/ GRAACC), Federal University of São Paulo, Brazil, from 1990 to 2009. The study was approved by the Institutional Ethics Committee (CEP/UNIFESP N8 CEP 1115/09) and followed the criteria established by the Helsinki convention. Informed consent was obtained from all patients/ guardians. All patients were classified according to the North American Children’s Oncology Group trial into two groups, based on age at initial diagnosis, resection extent, and Chang metastasis staging [9]. Average risk patients were those over 3 years of age at diagnosis, with nonmetastatic and totally or nearly totally resected tumors (B1.5 cm2 on post-operative magnetic resonance imaging). Patients not fulfilling these criteria were regarded as high risk. Gene expression analysis Fresh surgical samples of primary medulloblastoma and non-neoplastic brain tissues obtained from two patients with Arnold Chiari Type I malformation were immediately snap-frozen in liquid nitrogen upon surgical removal. Total cellular RNA was extracted using TRIzolÒ Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and the RNA integrity was evaluated based on the intensity of 28S and 18S rRNA bands in 1% agarose gel. A total of 1 lg of RNA was reverse transcribed with SuperScriptTM III Reverse Transcriptase Kit (Invitrogen) according to the manufacturer’s instructions. Expression levels of CD133 (NM_006017), OCT4 (NM_002701.4) and LIN28 (NM_024674.4) genes were evaluated by quantitative real-time PCR. Amplification of specific PCR products was detected with SYBR Green (Power SYBRÒ Green PCR Master Mix kit; Applied Biosystems, UK) according to the manufacturer’s protocol. All qPCR reactions had a total volume of 25 ll, containing 1 ll of cDNA sample, 10 pmol of each primer and 12.5 ll of SYBR Green. The thermal cycling was carried out by starting with 95°C for 10 min hold, followed by 50 amplification cycles of 95°C for 15 s and 68°C for 30 s. Reactions were performed in duplicate. Expression data analysis was performed in a real-time PCR system (MX3005PTM; Stratagene) and quantitation was based on standard curves. Each run was completed with a melting curve analysis to confirm amplicon specificity. J Neurooncol (2012) 106:71–79 The housekeeping gene encoding hypoxanthine guanine phosphoribosyltransferase (HPRT, NM_000194.2) was used as endogenous reference [10]. Normalized gene expression was calculated as target gene/HPRT. Relative gene expression was estimated as normalized gene expression in tumor/ non-neoplastic control. Primer sequences were designed at specific exon–exon junctions of the target gene to avoid DNA amplification and sequence specificity was confirmed by a BLAST search at the NCBI databank: OCT4 For: 50 -CCTGA AGCAGAAGAGGATCACC-30 , OCT4 Rev: 50 -AAAGCG GCAGATGGTCGTTTGG-30 ; LIN28 For: 50 -CCAGTGGA TGTCTTTGTGCACC-30 , LIN28 Rev: 50 -GTGACACGGATG GATTCCAGAC-30 ; HPRT For: 50 -CCTGGCGTCGTGAT TAGTGA-30 , HPRT Rev: 50 -GCCTCCCATCTCCTTCATC-30 ; CD133 For: 50 -CACTACCAAGGACAAGGCG TTC-30 , CD133 Rev: 50 -CAACGCCTCTTTGGTCTCC TTG-30 . Statistical analysis Correlation of clinical variables (metastasis, risk groups, surgical resection, relapse, and patient status) with gene expression data was determined by the Mann–Whitney test. Overall survival analysis from life-time data according to clinical risk group, presence or absence of gene expression, and relative gene expression levels (hyperexpression cutoff value of five-fold, relative to non-neoplastic control) was determined by the Kaplan–Meier product-limit estimation with log-rank (P \ 0.05). Overall survival was defined as time from diagnosis to the day of death or last follow-up. The Spearman’s rank correlation coefficient and the Fisher’s exact test to correlate numeric variables and categorical data, respectively, were performed with the software Statistical Package GraphPad Prism 5 (GraphPad Software, CA, USA). For correlation of gene expression in tumors, normalized expression (9107) were log transformed for graphical display. Significance was established at the P B 0.05 level in all analyses. Results Clinicopathological parameters Patients included in the study were mainly males (70%) over 3 years old (81%), and tumors were primarily of the classic histological subtype (89%). Based on age at initial diagnosis, resection extent, and Chang metastasis staging, 17 patients (46%) were clinically stratified as average risk and 20 patients (54%) as high risk (Table 1). As expected, significant differences (P = 0.0271) in overall survival were found according to clinical risk. The 5-year survival rates for average and high risk patients were 70 and 33%, 73 respectively. Among high risk patients, 60% had metastasis at diagnosis. Expression of stem cell markers and prognosis Expression of the neural stem cell marker CD133 could be detected in most high (94%) and average risk (90%) tumor samples. Similarly, the majority of high (85%) and average (88%) risk tumor samples had detectable expression of the pluripotency gene LIN28. In contrast, expression of another pluripotency-related gene, OCT4, was more scattered among tumors, being detected at low levels in 45 and 53% of high and average risk tumor samples, respectively. When analyzing the concurrent expression of these three genes in tumor samples, significant positive correlation were found only between LIN28 and OCT4 (P = 0.0014, Fig. 1a) and between LIN28 and CD133 (P = 0.0052, Fig. 1b). As shown in Fig. 2, detectable expression (presence or absence) of OCT4, but not CD133 or LIN28, was significantly correlated with overall survival. Patients with OCT4? tumors tend to have a shorter median survival time (24 months) than those whose tumors were negative for OCT4 expression (153 months). Such correlation was found with tumors displaying any level of OCT4 expression, as detected by real-time PCR, and its level of significance was similar to that of clinical risk and survival correlation. Furthermore, when considering relative OCT4 expression levels, a stronger correlation (P \ 0.0001; Fig. 3a) was found between hyperexpression of OCT4 (C5-fold, relative to non-neoplastic control) and lower survival rate. Median survival time of patients with tumors hyperexpressing OCT4 dropped to 6 months, while tumors displaying lower/undetectable OCT4 expression kept a median survival of 153 months. Likewise, OCT4 tumor expression levels in deceased patients were significantly higher (P = 0.0169) than in patients still alive by the end of the study (Fig. 3b). In an additional in silico analysis with distinct microarray and SAGE datasets publically available for medulloblastoma, OCT4 expression was also found to be increased in tumors associated with poor prognosis (supplementary material). No correlations were found between detectable expression of either of these three genes and metastasis. Since most medulloblastoma cases were of the classic subtype, it was not possible to evaluate associations between gene expression and histological subtypes. OCT4 expression is correlated with poor prognosis in clinically average risk patients The clinicopathological data of patients with OCT4? tumors is detailed in Table 2. Among them, nine (50%) 123 74 J Neurooncol (2012) 106:71–79 Table 1 Clinicopathological and gene expression features of medulloblastoma samples Clinicopathological features Number of cases CD133 expression LIN28 expression OCT4 expression Positive Positive Positive Undetectable Undetectable Undetectable Age at diagnosis (years) \3 7 (18.9%) 6 1 6 1 3 4 30 (81.1%) 28 2 26 4 15 15 Male 26 (70.3%) 24 2 23 3 13 13 Female 11 (29.7%) 10 1 9 2 5 6 33 (89.2%) 30 3 28 5 16 17 Desmoplastic 1 (2.7%) 1 – 1 – 0 1 Large-cell 2 (5.4%) 2 – 2 – 1 1 Anaplastic 1 (2.7%) 1 – 1 – 1 – 0 – – – – – – Yes 13 (32.4%) 11 2 11 2 4 9 No 24 (67.6%) 23 1 21 3 16 8 Partial 13 (35.1%) 11 2 10 3 6 7 Total 23 (62.2%) 22 1 21 2 12 11 1 (2.7%) 1 – 1 – – 1 [3 up to 18 Gender Histological subtype Classic Extensive nodularity Metastasis at diagnosis Tumor resection Not informative Relapse Yes 9 (24.3%) 7 2 7 2 5 4 No Patient status 28 (74.7%) 27 1 25 3 13 15 Deceased 19 (51.3%) 17 2 18 1 12 7 Alive 18 (48.7%) 17 1 15 3 6 12 High 20 (54.1%) 18 2 17 3 9 11 Average 17 (45.9%) 16 1 15 2 9 8 Risk group Fig. 1 Expression of stem cell-related genes in medulloblastoma. Significant positive correlations were found between expression of LIN28 and CD133 (a) as well as of OCT4 and LIN28 (b). The respective Spearman’s Rank correlation coefficients and significance 123 levels are shown in the plots. Each dot corresponds to the concurrent normalized gene expression level in each tumor sample, as determined by real-time PCR J Neurooncol (2012) 106:71–79 75 Fig. 2 Expression of stem cell-related genes and survival of patients with medulloblastoma. The Kaplan–Meier curves show overall survival rates with respect to detectable OCT4, LIN28 or CD133 expression. Survival curves of patients classified based on clinical prognostic criteria are also shown for comparison. Statistical differences were calculated by the log-rank test were clinically classified as average risk, three of which displaying OCT4 hyperexpressing tumors. When analyzing overall survival within this clinical subgroup, there was also a significant correlation between OCT4 expression and lower survival rate (P = 0.0112; Fig. 3c). All average risk patients deceased within 5 years of initial diagnosis had OCT4? tumors, an outcome typical of most clinically high risk patients. Median survival time of average risk patients with OCT4? tumors was 28 months. Among these average risk patients with shorter survival time, 60% had tumor relapse. From the stem cell markers evaluated in this study, only OCT4 was found to be significantly correlated with prognostic factors. Not all tumor specimens had detectable expression of CD133, LIN28 or OCT4. In fact, OCT4 was the most rarely expressed gene, being detected in less than a half the tumor collection examined here. Information regarding expression of these stem cellrelated genes in medulloblastoma is rather limited. Highly tumorigenic CD133? stem-like cells have originally been isolated from medulloblastoma [5]. In the DAOY medulloblastoma cell line, CD133? cells were reported to be more resistant to radiation than CD133-negative cells [11]. Furthermore, in oligodendroglial tumors and glioblastomas, CD133 expression is an indicator of shorter survival [12, 13]. Based on what is known for these high-grade gliomas, association between CD133 expression and poor prognosis was also expected in medulloblastoma. In our study, however, expression of this neural stem cell marker was not found to be correlated with either metastasis or survival. In normal tissue, OCT4 and LIN28 expression are more restricted to embryonic stem cells (ESC) and early embryonic structures, decreasing dramatically with the ESC differentiation [14, 15]. However, several recent studies have Discussion The clinicopathological characteristics of the patient cohort included in the study were typical. Clinical stratification of patients yielded 54% as high risk and 46% as average risk. In agreement with the literature, the 5-year survival rates for high and average risk patients were 33 and 70%, respectively. Patient stratification solely based on clinical parameters was not enough to prospectively identify average risk patients with poor prognosis, once more reinforcing the need for additional indicators of prognosis. 123 76 Fig. 3 Correlation of OCT4 expression levels and poor survival. a Kaplan–Meier curve showing overall survival rates according to the level of OCT4 expression (high or low/undetectable). b Median normalized OCT4 expression levels with respect to patient status. Only OCT4? tumors were included in this analysis. c Overall survival rates according to OCT4 expression status (positive or undetectable) in the subgroup of clinically average risk patients. All patients with OCT4? tumors were deceased within 5 years of initial diagnosis. In sharp contrast, all patients whose tumors had no detectable expression of OCT4 were still alive during the same time frame. Significant correlations with overall survival and with patient status were determined by the log-rank and Mann–Whitney tests, respectively 123 J Neurooncol (2012) 106:71–79 reported the involvement of both OCT4 and LIN28, as well as other embryonic associated genes, with tumor formation [16–19]. Although both genes are required for pluripotency and, despite the fact that all OCT4? medulloblastoma specimens had concomitant expression of LIN28, only the former gene was significantly correlated with poor overall survival. Of note, OCT4 expression was not restricted to a particular clinical staging, being detected at a similar frequency in tumor specimens from either average or high risk patients. In both clinical groups, patients whose tumors had any detectable expression of OCT4 had a significant lower survival time than patients with OCT4-negative tumors. Moreover, the higher the relative OCT4 expression level, the lower the survival time. Of particular interest was the OCT4 expression pattern in clinically average risk patients. Considering the 5-year survival rate calculated for this clinical group in our cohort, all deceased patients (corresponding to 30% of total average risk patients) had OCT4? tumors, while most of the living patients had OCT4-negative tumors. The practical meaning of this finding is that, in terms of 5-year survival rating, average risk patients with OCT4? tumors had a clinical outcome comparable to that of typical high risk patients, displaying a median survival time of 28 months. The positive correlation found between OCT4 expression and poor survival in medulloblastoma is in agreement with recent studies in other cancers, where expression of OCT4 and other pluripotency genes is correlated with poorly differentiated and more aggressive tumors. Although not previously addressed in medulloblastomas, expression of OCT4 was reported to be associated with higher grade gliomas, namely glioblastoma, when compared with oligodendroglioma and astrocytomas of grades II and III [20]. Similarly, expression of OCT4 and NANOG, another pluripotency-related gene, were detected in high grade lung adenocarcinoma, and their ectopic expression in tumor cells induced stem cell-like properties and enhanced tumorigenicity [21]. Interestingly, an opposed association between OCT4 levels and prognosis was found in hypopharyngeal squamous cell carcinoma, where it was correlated with lower tumor recurrence and longer patient survival [22]. Thus, both expression and predictive value of OCT4 may vary according to the type of cancer and the expressing cell. In human ESC, down-regulation of LIN28 expression reduced OCT4 protein levels suggesting that LIN28 can regulate OCT4 expression at a post-transcriptional level [23]. In our study, although all OCT4? medulloblastoma samples also expressed LIN28, several tumor specimens had detectable expression of LIN28 but not of OCT4, suggesting alternative mechanisms for OCT4 expression regulation in this type of cancer. In murine neural stem cells, a decreased expression of OCT4 along the embryonal 180 18 194 156 3 4 5 6 77 216 156 48 72 122 200 89 28 29 30 31 32 33 34 20 35 25 26 27 35 84 20 24 108 19 24 198 18 23 84 17 62 93 16 118 76 15 22 62 14 21 64 138 10 11 13 177 78 9 12 99 156 8 0.76 108 2 7 3 Age at diagnosis (months) 1 Patient Large-cell Classic Classic Classic Desmoplastic Classic Classic Classic Classic Classic Classic Classic Classic Classic Classic Classic Classic Classic Classic Classic Classic Classic Classic Classic Classic Large-cell Anaplastic Classic Classic Classic Classic Classic Classic Classic Histological subtype 68 75 97 103 123 0.5 41 63 4 15 6 19 41 41 153 31 59 27 45 61 40 94 10 60 18 24 11 5 28 6 3 10 2 7 Survival (months) M0 M0 M0 M0 M0 NI NI M0 NI M0 M3 M3 M1 M2 M3 M1 M0 M0 M0 M0 M1 M0 M0 M0 M0 M0 M0 M0 M0 M0 M2 M0 M1 M1 Metastasis T T T T T T NI P P T P P P P P T T P P T T T P P T T T T T T P T T P Tumor resection Table 2 Clinicopathological parameters of patients stratified by OCT4 expression C?V?CP C?V?CP C?V?CP IFO?ETO IFO?ETO POD NI C?V?CP?ETO POD C?V?CP?ETO POD POD C?V?CP?ETO C?V?CP?ETO EP NI C?V?CP NI EP C?V?CP C?V?CP C?V?CP EP EP C?V?CP C?V?CP C?V?CP POD IFO?ETO POD POD C?V?CP POD POD Treatment No No No No No No Yes No No No No Yes No No Yes Yes No Yes No No Yes No No No No Yes No No Yes No No Yes No No Tumor recurrence A A A A A D D A D A D D A A D D A D A A D A D A A D D D D D D D D D Status AR AR AR AR AR HR HR HR HR HR HR HR HR HR HR HR AR HR HR AR HR AR HR HR AR AR AR HR AR AR HR AR HR HR Clinical risk ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 7.78 39.90 76.36 93.10 155.41 311.79 504.17 1,064.09 1,018.46 1,120.18 1,354.15 3,861.32 5,263.13 7,372.39 9,662.98 21,756.45 22,715.48 317,464.43 OCT4 expression (910-7) – – – – – – – – – – – – – – – – 0.01 0.06 0.12 0.14 0.24 0.49 0.80 1.68 1.61 1.77 2.14 6.11a 8.33a 11.67a 15.30a 34.45a 35.97a 502.69a Relative OCT4 expression (OCT4/normal) J Neurooncol (2012) 106:71–79 77 123 123 P partial, T total, D deceased, A alive, HR high risk, AR average risk, V vincristine, C cisplatin, CP cyclophosphamide, ETO etoposide, IFO ifosfamide, EP experimental protocol, POD postoperative death, ND not detectable, NI not informed a OCT4 hyperexpressing tumors – – ND AR No 91 37 Classic 41 M0 T C?V?CP A ND AR ND AR No A No T C?V?CP M0 M0 52 50 98 36 Classic 112 35 Classic T C?V?CP A OCT4 expression (910-7) Clinical risk Status Tumor recurrence Treatment Tumor resection Metastasis Survival (months) Histological subtype Age at diagnosis (months) Patient Table 2 continued – J Neurooncol (2012) 106:71–79 Relative OCT4 expression (OCT4/normal) 78 development has been associated with a gradual methylation in its promoter region [24]. Further studies on the methylation status of OCT4 promoter should help understand the expression pattern found for medulloblastoma patients with poor prognosis. In conclusion, our findings reveal a predictive prognostic value for OCT4 expression in medulloblastoma. Expression of OCT4, but not CD133 or LIN28 stem cellrelated genes, was significantly correlated with shorter survival in either clinically high or average risk patients. More importantly, positive OCT4 expression in primary tumor specimens could discriminate patients clinically classified as average risk but which deceased within 5 years of diagnosis, a clinical outcome typical of high risk patients. Thus, OCT4 expression status might be used as a further indicator of poor prognosis to aid postoperative treatment selection, with a particular potential benefit for clinically average risk patients. Acknowledgments This work was supported by grants from INCTCélulas Tronco em Doenças Genéticas Humanas, FAPESP, CNPq, and CAPES. C.O.R. and D.E.S. were recipients of fellowships from CAPES and CNPq. References 1. Guessous F, Li Y, Abounader R (2008) Signaling pathways in medulloblastoma. J Cell Physiol 217:577–583 2. Kouprina N, Pavlicek A, Collins NK et al (2005) The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein. Hum Mol Genet 14:2155–2165 3. Mueller S, Chang S (2009) Pediatric brain tumors: current treatment strategies and future therapeutic approaches. Neurotherapeutics 6:570–586 4. Carlotti CG Jr, Smith C, Rutka JT (2008) The molecular genetics of medulloblastoma: an assessment of new therapeutic targets. Neurosurg Rev 31:359–369 5. Singh SK, Clarke ID, Terasaki M et al (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63:5821–5828 6. Okamoto OK (2009) Cancer stem cell genomics: the quest for early markers of malignant progression. Expert Rev Mol Diagn 9:545–554 7. Cheng L, Sung MT, Cossu-Rocca P et al (2007) OCT4: biological functions and clinical applications as a marker of germ cell neoplasia. J Pathol 211:1–9 8. Gidekel S, Pizov G, Bergman Y, Pikarsky E (2003) Oct-3/4 is a dosedependent oncogenic fate determinant. Cancer Cell 4: 361–370 9. Chang CH, Housepian EM, Herbert C (1969) An operative staging system and a megavoltage radiotherapeutic technic for cerebellar medulloblastoma. Radiology 93:1351–1359 10. Valente V, Teixeira SA, Neder L et al (2009) Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR. BMC Mol Biol 3:10–17 11. Blazek ER, Foutch JL, Maki G (2007) Daoy medulloblastoma cells that express CD133 are radioresistant relative to CD133cells, and the CD133? sector is enlarged by hypoxia. Int J Radiat Oncol Biol Phys 67:1–5 J Neurooncol (2012) 106:71–79 12. Beier D, Wischhusen J, Dietmaier W et al (2008) CD133 expression and cancer stem cells predict prognosis in high-grade oligodendroglial tumors. Brain Pathol 18:370–377 13. Zhang M, Song T, Yang L et al (2008) Nestin and CD133: valuable stem cell-specific markers for determining clinical outcome of glioma patients. J Exp Clin Cancer Res 24:27–85 14. Richard M, Tan SP, Tan JH et al (2004) The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells 22:51–64 15. Darr H, Benvenisty N (2009) Genetic analysis of the role of the reprogramming gene LIN-28 in human embryonic stem cells. Stem Cells 27:352–362 16. Marie SK, Okamoto OK, Uno M et al (2008) Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas. Int J Cancer 122:807–815 17. Viswanathan SR, Powers JT, Einhorn W et al (2009) Lin28 promotes transformation and is associated with advanced human malignancies. Nat Genet 41:843–848 18. Lu L, Katsaros D, Shaverdashvili K et al (2009) Pluripotent factor lin-28 and its homologue lin-28b in epithelial ovarian cancer and their associations with disease outcomes and expression of let-7a and IGF-II. Eur J Cancer 45:2212–2218 79 19. Dangi-Garimella S, Yun J, Eves EM et al (2009) Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7. EMBO J 28:347–358 20. Ben-Porath I, Thomson MW, Carey VJ et al (2008) An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet 40:499–507 21. Chiou SH, Wang ML, Chou YT et al (2010) Co-expression of Oct4 and Nanog enhances malignancy in lung adenocarcinoma by inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiation. Cancer Res 70:10433–10444 22. Ge N, Lin HX, Xiao XS et al (2010) Prognostic significance of Oct4 and Sox2 expression in hypopharyngeal squamous cell carcinoma. J Transl Med 8:94 23. Qiu C, Ma Y, Wang J et al (2010) Lin28-mediated post-transcriptional regulation of Oct4 expression in human embryonic stem cells. Nucleic Acids Res 38:1240–1248 24. Lee SH, Jeyapalan JN, Appleby V et al (2010) Dynamic methylation and expression of Oct4 in early neural stem cells. J Anat 217:203–213 123