5 RESULTADOS
5.1 Manuscrito 1: Submetido à revista Journal Pediatric Hematology/Oncology
parameters in medulloblastomas
Tânia Maria Vulcani-Freitas1,3; Njasla Saba-Silva, 1; Andréa Cappellano, 1; Sérgio Cavalheiro2; Sílvia Regina Caminada de Toledo1,3.
1Institute of Pediatric Oncology (GRAACC), Pediatric Department – Federal University of
São Paulo, São Paulo, Brazil; 2Neurology and Neurosurgery Department – Federal
University of São Paulo, São Paulo, Brazil; 3Morphology and Genetic Department –
Federal University Of São Paulo, São Paulo, Brazil Silvia Regina Caminada de Toledo
Rua Botucatu, 743, 8° andar
Vila Clementino, São Paulo-SP, Brazil CEP: 04023-062
silviatoledo@graacc.org.br Telephone: 551150808582 Fax: 551150808480
ABSTRACT
To investigate the expression of genes MYC, MYCN and TERT in tumor fragments of pediatric medulloblastoma and correlate gene expression profiles with clinical parameters. Analysis of gene expression was performed by quantitative PCR real time in 37 tumor samples and correlated with clinical and pathological data. All 37 samples overexpressed
MYCN gene (p= 0.001), 95% and 84% of the samples overexpressed TERT and MYC,
respectively (p<0.0001). Twenty nine (78%) of all samples had concomitant high expression of MYC, MYCN and TERT genes together. Seventeen (59%) were high-risk classification, 10 (34%) were metastatic (M+) stage, two (7%) were anaplastic or large- cell/anaplastic subtype, eight (28%) of patients relapsed, beyond thirteen (45%) suffered partial surgical resection. and fourteen (48%) died. We found correlation between MYC,
MYCN and TERT expression (p<0.0001). The identification of a subgroup with concomitant
overexpression of the three investigated genes suggests the possibility of using more than one aspect of molecular indicative of unfavorable prognosis that characterizes the group with poor outcome. However, in future this may be enhanced by targeted therapy for the product TERT as proposed in some neoplasms. The identification of molecular events in the medulloblastoma categorization aims to help at-risk groups moving towards individualized medicine.
INTRODUCTION
Medulloblastoma (MB) is the most common malignant solid tumors of central nervous system (CNS) in the childhood and comprises 15-20% of brain tumors of infancy
1,2. Usually, this neoplasm occurs in people under 18 years1 and has two peaks of
incidence between 3 and 4 years and 8 and 9 years old3. The 5-year survival rate after the
diagnosis for these children is around 50-60%4. Despite its sensibility to no-specific
therapeutic as chemotherapy and radiotherapy, the treatment is very aggressive and frequently results in neurological development and growth deficit, and endocrine dysfunction 2,3.
Currently, the most of main researches aim to understand the molecular mechanisms that underlie the pathogenesis of MB. Many molecular and histopathological biomarkers have been proposed in order to facilitate improved patient management and disease outcome, through the identification of prognostic factors that can guide new therapies, since the aggressiveness of treatments remain associated with poor survival in high-risk disease group (e.g, infant or metastatic disease) and it have a negative impact on long-term survivals5. These studies recognized characteristic cytogenetic or molecular abnormalities in medulloblastoma which can be used as targets in strategies for development of more individualized therapies based on disease-risk and for development of novel therapeutic approaches targeted against specific molecular defects, in order to increase survival and reduce the late effects.
MYC (Avian Myelocytomatosis Viral Oncogene Homolog) family genes, mainly MYC
and MYCN, are commonly deregulated in tumorigenesis6. Although MYC/MYCN amplification has been considered unusual event (<10-15%), its expression is found in up to 64% in medulloblastoma2, 6-8. MYC expression and amplification has been associated with dismal prognostic and with large cell/anaplastic subtype which is the most aggressive phenotype for patients carrying medulloblastoma7, 9, 10. Thence, MYC has become an important prognostic indicator of medulloblastoma malignancy. Another gene, frequently, expressed in human cancers is TERT (Telomerase Reverse Transcriptase). TERT is catalytic subunit of telomerase which expression is highly specific to cancer cells and it is associated with enzymatic activity of telomerase 11, 12. Telomerase activation is present in
expression plays a key role in cancer-specific telomerase activation, important enzyme to carcinogenesis and cellular immortalization12, 14. The increased activity of telomerase is associated with poor clinical outcomes15.
MYC and TERT are closely correlated. MYC has been considered very important
regulator of TERT transcription in the carcinogenesis12, inducing telomerase activity by transcriptional activation of TERT16. As telomere length is a limiting factor in the replicative
life span of a cell17, 18, an important function of MYC, during cell proliferation, is
maintenance of chromosomal integrity by stimulation of telomerase activity. Thus, increased MYC expression is correlated with increasing TERT RNA expression and telomerase activity19.
Therefore, in order to investigate the association of MYC, MYCN and TERT genes expression profiles with prognosis in MB patients, we investigated the expression profiles of these genes, and correlated with clinical aspects.
MATERIALS AND METHODS Patients
We selected 37 MBs samples from patients attending the Pediatric Oncology Institute (IOP)/Grupo de Apoio ao Adolescente e a Criança com Câncer (GRAACC) – Federal University of São Paulo (UNIFESP) - Brazil. Informed consent was obtained from all patients/guardians according to the University’s IRB (CEP/UNIFESP Nº0155/06). All MB patients were classified according to the WHO (World Health Organization), as follows: M stage include M1 (tumor cells identified by cerebrospinal fluid (CSF) cytology only), M2 (intracranial metastatic tumor detectable by computed tomography (CT) or magnetic resonance imaging (MRI) and M3 (spinal metastatic tumor detectable by CT myelography or spine MRI)21.
RNA extraction and cDNA synthesis
Total RNA was isolated from frozen samples pulverized under nitrogen liquid and extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). To reduce the risk of genomic DNA contamination, DNase treatment was performed using deoxyribonuclease I
Amplification Grade (Invitrogen, Carlsbad, CA, USA). The concentration of RNA was determined by spectrophotometry and total RNA integrity was monitored by visualization of ribosomal RNAs (28S and 18S) on 1.0% agarose gel. One microgram of total RNA derived from 37 MB samples was reverse-transcribed using the SuperScript III Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Quantitative Real-Time RT-PCR
Oligonucleotides for MYC, MYCN, TERT and HPRT were designed using Primer Express Software from Applied Biosystems (Foster City, CA, USA), warranting that forward and reverse sequences were in different exons. We conducted a BLAST search to confirm the total gene specificity of the nucleotide sequences chosen for primers. Real-time PCR amplification and data analysis were performed using the ABI Prism 7500 Sequence Detector System Applied Biosystems (Foster City, CA, USA). Each cDNA sample was mixed with 12µl of Mastermix (SYBR® Green PCR Master Mix, Applied Biosystems). Cycling conditions consisted of two singles steps at 50°C and 95°C for 2min and 10min respectively, followed by 40 cycles of amplification at 95°C for 15s and at 60°C for 1min for annealing and elongation. Experiments were performed in triplicate for both target genes and the endogenous gene. For each sequence, a standard curve was constructed to determine the assay sensitivity. The data were averaged from the values obtained in each reaction. Relative expression was automatically calculated and analysed according to 2- Ct method, described previously22. HPRT (hypoxanthine-guanine phosphoribosyl transferase) was used as a housekeeping gene in order to standard its relative expression levels with relative expression levels of three genes studied. A no-template control was included in each amplification reaction as well as a reference RNA. The PCR efficiencies of the four genes were comparable ( 95%).
Statistical Analysis
Statistical analysis was performed using Graph Pad Prism 4 Program Software (San Diego, CA, USA). Clinical variable correlated with gene expression were: histological MB subtype, metastatic status at diagnosis (WHO classification), risk, surgical resection,
follow-up and relapse. Overall survival was defined as the time from diagnosis until date of either the more recent follow-up or death. Overall survival was generated by applying the Kaplan-Meier method. The t test was used to analyze the gene expression of each gene studied. The Kruskal-Wallis and Mann-Whitney tests were used to analysis the relationship between gene expressions and clinical characteristics. Categorical data were studied using Chi-Square and Fisher's exact test applying software VassarStats (Website for Statistical Computation). In all cases, p<0.05 was considered statistically significant.
RESULTS
All clinical variables are summarized in Table 1. The median age of patients was 8 years (range 5 months to 18 years old). Stages M1, M2 and M3 were included in just one group named M+ 32% (n=12). In the M+ stage was: 68% (n=25) stage M0, 6% (n=2) stage M1, 13% (n=5) stage M2 and 13% (n=5) M3. Eighteen of 37 patients died from disease or resulting complications from therapy and 19 patients were disease-free or in treatment. The most of our samples was classic MB (92%).
MYC was overexpressed in 30 (81%) samples, median 9.2 (p<0.0001) (cutoff 2.0).
By analyzing the clinical characteristics of these samples, we observed that 18/30 (60%) were high-risk, two (7%) were anaplastic or large-cell/anaplastic subtype, nine (30%) were relapsed, 15 (50%) died, 11 (37%) were metastatic (M+) stage, beyond that 13 (43%) suffered partial resection of tumor. No statistical association was found between clinical features and MYC overexpression. MYCN was highly expressed in all MB samples, median 31 (p=0.0018) (cutoff 2.0). MYCN overexpression was significantly correlated with died patient group (p=0.008).
TERT overexpression was detected in 35 (95%) samples, median 322, (p=0.0110)
(cutoff 2.0). By analyzing the clinical characteristics of these samples, 20/35 (57%) were high-risk, three (9%) were anaplastic or large-cell/anaplastic subtype, eight (23%) were relapsed, 17 (49%) were death, 11 (31%) were all metastatic (M+) stage, beyond that 16 (46%) suffered partial resection of tumor.
Twenty nine (78%) of all samples showed concomitant high expression of MYC,
MYCN and TERT genes together. Between them, 17 (59%) were high-risk classification,
subtype, eight (28%) of patients relapsed, beyond 13 (45%) suffered partial surgical resection and 14 (48%) died. Statistical analyses found correlation between MYC, MYCN and TERT expression (p<0.0001) (figure 1). However, we not found any relationship between the expression of all three genes studied and the clinical aspects of patients. The results of MYC, MYCN and TERT expression levels from each patient, together with their clinical characteristics are summarized in figure 2. No significant association in MYC,
MYCN and TERT expression and Kaplan- Meier survival curves.
DISCUSSION
Medulloblastoma is the most common solid tumor in children1. In spite of the sophisticated techniques of neurosurgery, radiotherapy and chemotherapy, this tumor has a high mortality rate3,4. Furthermore, the most of the survivors suffer a lost of normal- appearing white matter, an associated decline in intellectual function, growth deficit, and endocrine dysfunction2, 23-27. Usually, the method for stratification of patients between standard and high risk groups is based on age, metastatic stage at diagnosis, and, some studies extent of surgical resection 28-30. Moreover, in order to define different prognostics, histological subtypes are evaluated also24.
Recently, many studies evaluate the significance of molecular prognostic makers in MB children survival. Together, the molecular information and conventional diagnostic would identify patients with aggressive tumors for whom therapies more intensified would be appropriate, those who would survive with less toxic therapies of reduced intensity24. Since MB is heterogeneous disease at histopathological and molecular level, aggressiveness and response to treatment of tumors vary widely from individual to individual5, 31.
We analyze three of genes related with the molecular pathogenesis of MB: MYC,
MYCN and TERT. MYC acts within the MYC/MAD/MAX signaling pathway modulating cell
cycle progression, differentiation, and apoptosis 32, 33. Oncogenic activation of MYC occurs through a variety of events including gene amplification, translocation and DNA rearrangement, rise of transcript regulation, and enhanced translation32, 34-36. MYC
expression is found in up to 64% of cases with MB independent of gene amplification and has been associated with a poor prognosis37-39. MYC and MYCN amplification is an
unusual event (<10-15%) in this tumor2, 6-8 and this rate increases up to 17% in high-risk patients9.
In our study, we observed MYC overexpression in 81% of all samples while others studies reported 64%6. Similarly, MYCN was overexpressed in all our samples and rates higher than 68% found by Eberhart et al (2004)9. However, we did not find any association between MYC expression and clinical parameters. Additionally, we found statistical correlation between MYCN overexpression and died patient group (p=0.008). Moreover, we found a correlation tendency between relapsed patients and MYCN increased expression (p=0.0632).
In the meantime, many types of tumor present abnormal telomerase activity in about 90% of the cases12. This maintains tumor growth and increase potential proliferative cell12,
40, which also allow that telomerase be associated with dismal clinical outcomes15.
Therefore, we verify their expression and correlated with clinical aspects, individually and associated with each other. Our studied showed TERT overexpression in 95% of samples. It was much higher than reported by Fan et al (2003) that showed 42%15. Despite of increased expression, we just found correlation between TERT expression and relapse (p<0.05). There was statistical significance between the expression of the three genes studied (p<0.0001), however any association was found between concomitant expression of the genes and the clinical features evaluated. Nevertheless, due to the important roles played by these three genes in the tumorigenesis process, the presence of overexpression profiles could be collaborating for a poor clinical outcome.
The correlation between TERT mRNA expression and telomerase activity is not stronger enough42. This can be partly due to post-translation modification but also results of the formation of splice variants. There are, at least, four of these variants that can likely to alter protein activity43, 44. Although MYC has direct action in the TERT promoter region, the TERT control is undoubtedly very complex and likely a number of other transcription factors influence its expression12, 45. Hence, we could not find any association between
MYC and TERT expression. The MYC overexpression is not always associated with high
MYC protein activity, that can be a post-translation alterations, as protein inactivation or disrupted protein, or even protein degradation by a downstream regulator41.
Our results suggest that supplementing conventional diagnostics with molecular information, such as investigation of MYC, MYCN and TERT, should help to identify patients with aggressive tumors, since risk assessment until now continues to be solely based on clinical parameters. The molecular investigation of these biomarkers can provide appropriate treatment for those who need of maximal therapy and others who might survive with less toxic adjuvant therapy of reduced intensity. Moreover, they can be useful molecular markers of prognosis in the clinical evaluation of these patients.
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