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LABORATORY INVESTIGATION

Prognostic value of the TP53 Arg72Pro single-nucleotidepolymorphism and susceptibility to medulloblastoma in a cohortof Brazilian patients

Raimundo M. Carvalho • Giovanny R. Pinto • France K. N. Yoshioka •

Patrıcia D. L. Lima • Carolina R. T. Souza • Adriana C. Guimaraes •

Letıcia M. Lamarao • Juan A. Rey • Rommel R. Burbano

Received: 6 December 2011 / Accepted: 28 July 2012! Springer Science+Business Media, LLC. 2012

Abstract Medulloblastoma is a highly cellular malignantembryonal neoplasm, being the most common malignant

pediatric brain tumor, accounting for 20–25 % of pediatric

central nervous system tumors. To investigate the effect ofthe TP53 Arg72Pro single-nucleotide polymorphism (SNP)

on clinicopathological and phenotypic parameters, we

performed a case-controlled study of 122 patients and 122healthy controls from Brazil. No significant associations

were found between the TP53 Arg72Pro genotypes and the

clinicopathological parameters studied. Compared withArg/Arg, which is the most common genotype in the study

population, both the Arg/Pro and Pro/Pro genotypes did not

influence the medulloblastoma development risk [oddsratio (OR) = 1.36 and P = 0.339 for the Arg/Pro geno-

type; OR = 1.50 and P = 0.389 for the Pro/Pro genotype].

With regard to prognosis, disease-free survival was not

significantly different among the TP53 Arg72Pro SNPgenotypes (P [ 0.05), but the less frequent genotype (Pro/

Pro) was associated with shorter overall survival of

medulloblastoma patients (P = 0.021). These data suggestthat, although there is no association between the TP53Arg72Pro SNP and medulloblastoma risk, the Pro/Pro

genotype is associated with shorter overall survival ofpatients submitted to adjuvant therapy. Nevertheless, due

to the interethnic composition of the Brazilian population,

future studies on larger populations from other parts of theworld are essential for a definitive conclusion on the

function of the TP53 Arg72Pro SNP.

Keywords Medulloblastomas ! TP53 Arg72Pro SNP !Risk ! Prognosis ! Adjuvant therapy

Introduction

Medulloblastoma is a highly cellular malignant embryonal

neoplasm, being the most common malignant pediatricbrain tumor, accounting for 20–25 % of pediatric central

nervous system tumors. Approximately 75 % of medullo-blastomas are diagnosed in patients younger than 15 years

of age, and the peak incidence occurs between 3 and

9 years of age [1]. In contrast, only 25 % of medullo-blastomas are diagnosed in patients between 15 and

44 years of age [2].

Over the last two decades, in addition to the classicalclinical prognostic factors (i.e., histological tumor grade,

[1.5 cm residual postoperative tumor, metastasis, and

\3 years of age at diagnosis), multiple molecular prog-nostic factors have been tested in an attempt to better refine

the clinical risk stratification and medulloblastoma treat-

ment options. To date, TrkC signaling alterations, Wnt

Raimundo M. Carvalho and Giovanny R. Pinto contributed equally tothis study.

R. M. CarvalhoDepartment of Neurosurgery, Ophir Loyola Hospital,Av. Governador Magalhaes Barata 992, Belem,PA 66063-240, Brazil

R. M. Carvalho ! P. D. L. Lima ! C. R. T. Souza !A. C. Guimaraes ! L. M. Lamarao ! R. R. BurbanoHuman Cytogenetics Laboratory, Federal University of Para,R. Augusto Correa 01, Belem, PA 66075-110, Brazil

G. R. Pinto (&) ! F. K. N. YoshiokaGenetics and Molecular Biology Laboratory, Federal Universityof Piauı, Av. Sao Sebastiao 2819, Parnaıba, PI 64202-020, Brazile-mail: pintogr@ufpi.edu.br; pintogr@gmail.com

J. A. ReyResearch Unit, Unidad de Investigacion, Hospital UniversiatrioLa Paz, Paseo Castellana 261, 28046 Madrid, Spain

123

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DOI 10.1007/s11060-012-0950-0

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signaling alterations, and a chromosome 6q deletion have

been identified as promising favorable markers. However,only four other molecular markers have been reproducibly

associated with poor outcome as follows: MYC amplifica-

tion, BIRC5 overexpression, ERBB2 overexpression, andTP53 mutation [3–8].

The TP53 gene, which is located at chromosome

17p13.1, is responsible for the transcription of a site-spe-cific DNA-binding protein and acts as a transcription factor

for cell growth regulatory genes. TP53 is mutated inapproximately 50 % of human tumors, pointing to the

complexity of antiproliferative pathways under the control

of the TP53 protein and demonstrating that TP53 has animportant role in tumorigenesis [9]. The current release of

the TP53 mutation database of the International Agency for

Research on Cancer (version R15; www-p53.iarc.fr)reports more than 27,500 cases of somatic TP53 missense

mutations corresponding to more than 2,300 distinct

mutant proteins [10].In a retrospective study, Tabori et al. [7] observed that

somatic TP53 mutation is associated with adverse out-

comes in patients with medulloblastoma. However, studiesof the role of TP53 mutation in the tumorigenesis of spo-

radic medulloblastoma and the value of TP53 mutation

status as a prognostic marker have produced contradictoryresults. Pfaff et al. [11] reported that TP53 mutations occur

at low frequency in medulloblastomas but are overrepre-

sented in the prognostically favorable subgroup featuringalterations in the Wnt pathway. In addition, because no

correlation between TP53 mutation status and patient out-

come was observed in more than 300 patients, theseauthors concluded that TP53 mutation is not a universal

prognostic marker for medulloblastoma. These results were

supported by Lindsey et al. [12] in an independent andrepresentative series of all major established clinical and

molecular subtypes of medulloblastomas. Nevertheless,

Gessi et al. [13] reported that TP53 expression in patientswith metastatic medulloblastoma is associated with rapid

disease progression and poor prognosis, with a statistically

significant inverse correlation between TP53 expressionand patient survival.

The wild-type TP53 protein prevents cells with genetic

damage from growing and dividing by two distinct path-ways: cell cycle arrest and apoptosis [14]. The proline-rich

region located between codons 64 and 92 of TP53 is a

critical site for apoptosis signaling. A frequent functionalsingle-nucleotide polymorphism (SNP) that leads to an

arginine–proline amino acid change (Arg72Pro) has been

reported at codon 72, which is found in exon 4. Dumontet al. [15] reported that the Arg72 allele when homozygous

has an apoptosis-inducing ability that is 15-fold higher than

the Pro72 allele. According to Leu et al. [16], the highapoptosis-inducing ability of the Arg72 allele is due, in

part, to its mitochondrial location, which makes it possible

for TP53 to directly interact with the proapoptotic BAKprotein. Functional studies of this SNP have been the basis

for testing its impact on the risk and progression of tumors,

and it has been found that the less apoptotic allele (Pro72)is associated with increased risk for development and poor

prognosis of several types of tumors, including central

nervous system tumors [17–20]. However, to our knowl-edge, there are no studies that show if an association exists

between the TP53 Arg72Pro SNP and medulloblastomarisk, medulloblastoma prognosis, or adjuvant therapy

response. Therefore, we investigated the role of the TP53Arg72Pro SNP as a potential risk factor and/or prognosticmarker of medulloblastoma in a Brazilian population by

performing a case–control analysis using a polymerase

chain reaction-restriction fragment length polymorphism(PCR–RFLP) approach.

Materials and methods

Study population

A total of 122 medulloblastomas that were surgically

resected from previously untreated patients under the careof the Neurosurgery Department of the Hospital Ophir

Loyola (Belem, Para, Brazil) between 1999 and 2010 were

analyzed. All patient samples were independently reviewedby two experienced pathologists according to the current

World Health Organization (WHO) criteria for diagnostic

accuracy, and the samples were graded histologically usingpublished criteria [21, 22]. The clinical outcome, including

length of survival, was obtained from patient records and

by contacting the general practitioner of each patient.Peripheral blood samples in ethylenediamine tetraacetic

acid (EDTA) tubes from 122 healthy individuals with no

family history of cancer in first-degree relatives were col-lected as controls in the clinical laboratory of the Univer-

sity Hospital Joao de Barros Barreto (HUJBB). The control

samples were matched for gender and mean age with thepatient samples. The mean age of both the patient and

control groups was 11.12 years (±5.52 years), and 55.7 %

of the patients and 54.1 % of the controls were 10 years oldor older. Informed consent was given by all of the recruited

subjects or from a parent or legal guardian. The Ethics

Committee of the HUJBB and the Federal University ofPara previously approved this study.

DNA extraction and genotyping

Genomic DNA from tumor samples was extracted from

formalin-fixed, paraffin-embedded tissue and fresh-frozenmaterial using the QIAamp Tissue Kit and Genomic DNA

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Buffer Set, while control DNA was obtained from

peripheral lymphocytes using the QIAamp Blood Kit(QIAGEN, Germany) according to the manufacturer’s

instructions. After isolation, the DNA concentration was

determined using a BioSpec-nano spectrophotometer(Shimadzu, Japan). After isolation, the DNA concentration

was determined using a BioSpec-nano spectrophotometer

(Shimadzu, Japan).Genotyping was performed as previously described [23].

For RFLP, the TP53 Arg72Pro SNP PCR products weredigested with BstUI (2 U at 60 "C for 4 h) (NEB, USA).

While BstUI generates two fragments of different sizes (52

and 100 bp) by recognizing a restriction site in the Arg72allele (CG.CG), Pro72 allele digestion yields just one

fragment (152 bp). The DNA fragments were electropho-

resed on a 10 % acrylamide:bisacrylamide gel (19:1) andthen stained with silver nitrate.

The genotypes of more than 10 % of the samples were

reassessed to confirm the results. Moreover, selected PCRproducts were purified and submitted to bidirectional

sequencing to further confirm the authenticity of the

genotype analysis. PCR products were purified with Exo-SAP-IT (USB, USA) followed by sequencing with the

DYEnamic ET Dye Terminator Kit (GE Healthcare, UK)

according to the manufacturers’ protocols. Sequencingreactions were performed on a MegaBACE 1000 (GE

Healthcare, UK).

TP53 immunohistochemical analysis

Of all the medulloblastoma cases, 96 were available forTP53 immunohistochemical (IHC) analysis as previously

described [6, 7]. Immunohistochemical TP53 staining was

determined and graded for strength (0 = none, 1 = weak,and 2 = strong) and distribution (\25, 25–50, and [50 %

of tumor cells) by two observers who were blinded to the

clinical outcome. Only strongly staining cells (score = 2)with distribution greater than 50 % were considered posi-

tive for that marker.

Statistical analysis

The v2 test was used to determine if the genotype distri-butions were in Hardy–Weinberg equilibrium. The

observed medulloblastoma genotype and allele frequencies

were compared with the controls using the v2 and Fisher’sexact tests, respectively. Multivariate analysis was per-

formed to analyze the association between the genotypes

and medulloblastoma risk after stratifying the subjectsaccording to the clinicopathological parameters. Survival

and adjuvant treatment (radiotherapy and/or chemother-

apy) data were available for all 122 patients. Kaplan–Meiercurves were constructed to assess the overall survival and

disease-free survival rates, and the differences among the

groups were analyzed by the log-rank test. The odds ratio(OR) and 95 % confidence interval (CI) were calculated

using a logistic regression model. Statistical significance

was set at P \ 0.05. Statistical analyses were performedwith S-Plus 2000 (Insightful, Inc.) and GraphPad Prism 5.0

(GraphPad Software, Inc.) software.

Results

A total of 122 medulloblastoma patients and 122 control

subjects were included in this study. The mean age for boththe patient and control groups was 11.12 years (patients

ranged from 0.17 to 26 years old and controls ranged from

2 to 25 years old). Only four (3.28 %) of the patients wereyounger than 3 years at diagnosis and treated without

craniospinal radiation. Together, the patient and control

samples comprised 69 (56.6 %) males and 53 (43.4 %)females (M/F ratio = 1.3). Ninety-five (77.9 %) patients

had ‘‘classic’’ medulloblastoma, 16 (13.1 %) had ‘‘des-

moplastic’’ medulloblastoma, and 11 (9.0 %) had ‘‘largecell/anaplastic’’ medulloblastoma. Of the 122 patients, 10

(8.2 %) had undergone biopsy, 18 (14.8 %) had undergone

subtotal resection, 16 (13.1 %) had undergone near-totalresection, and 78 (63.9 %) had undergone gross total

resection. Adjuvant postsurgical treatment consisted of

radiotherapy and chemotherapy in 81 patients (66.4 %),radiotherapy alone in 26 patients (21.3 %), and chemo-

therapy alone in 15 patients (12.3 %). Information

regarding metastatic status was available for 117 patients,and 85 (72.6 %) were clinically classified as average risk

and 32 (27.4 %) were high risk, which was defined as

having [1.5 cm residual postoperative tumor, metastaticdisease at diagnosis, and/or patients younger than 3 years

old.

The treatment of patients in the average-risk grouptypically consisted of surgery followed by cranial and

neuroaxis radiation of 23–24 Gy and a booster radiation

dose delivered to the tumor bed, reaching a final radiationdose of 54–55.8 Gy by a maximum of 28 days after sur-

gery. Patients in this group received etoposide and vin-

cristine chemotherapy before radiation and six cycles ofcyclophosphamide, vincristine, and cisplatin after radio-

therapy. Patients in the high-risk group were treated with

three consecutive cycles of chemotherapy with cyclo-phosphamide, vincristine, cisplatin, and etoposide before

radiotherapy. Patients with leptomeningeal or cerebrospi-

nal fluid dissemination received intravenous methotrexate.The radiation treatment was initiated 28 days after the

recovery of the third cycle; the dose to the neuroaxis

increased to 36 Gy, and the final dose of the tumor volumewas 55.8 Gy. After radiotherapy, patients were also

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submitted to 6 months of treatment with oral etoposide

chemotherapy, with cycles of 21 days, followed byrecovery periods of 14–21 days. In cases of intolerance or

unacceptable adverse effects, the chemotherapy schemes

were changed individually according to the characteristicsof each patient. We did not observe significant differences

in the prognoses of patients who received different treat-

ment regimens, in either the average-risk or the high-riskgroup (P [ 0.05).

Information regarding recurrence status and TP53 IHCstaining was available for 111 and 96 patients, respectively.

Twenty-nine (26.1 %) patients experienced disease recur-

rence in the follow-up period, whereas TP53-positive IHCwas found in 17 (17.7 %) tumors (Fig. 1). Figure 2 shows

the restriction pattern of the three genotypes (Arg/Arg,

Arg/Pro, and Pro/Pro) upon digestion with BstUI. Table 1summarizes the clinicopathological parameters of the

patients, showing no significant difference between the

TP53 Arg72Pro genotype groups (P [ 0.05).The genotype and allele frequencies were in Hardy–

Weinberg equilibrium in all groups. The TP53 Arg72Pro

SNP allele and genotype frequencies did not significantlydiffer between the case and control groups. While the

Pro72 allele frequency in the control group was 32.0 %, it

was 37.7 % in the patient group (P = 0.459). In the con-trols, the Arg/Arg, Arg/Pro, and Pro/Pro genotype

frequencies were 49.2, 37.7, and 13.1 %, respectively. In

the patients, the Arg/Arg, Arg/Pro, and Pro/Pro genotype

frequencies were 41, 42.6, and 16.4 %, respectively(P = 0.423). Compared with Arg/Arg, which is the most

common genotype in the study population, both the Arg/

Pro and Pro/Pro genotypes did not influence medulloblas-toma development risk (OR = 1.36, 95 % CI = 0.79–

2.34, and P = 0.339 for the Arg/Pro genotype; OR = 1.50,95 % CI = 0.70–3.20, and P = 0.389 for the Pro/Pro

genotype). The Arg/Pro and Pro/Pro genotypes were then

combined because of the low Pro/Pro frequency, and thecombination did not influence the risk of developing

medulloblastomas (OR = 1.39, 95 % CI = 0.84–2.31, and

P = 0.247) (Table 2).The median and mean follow-up period for all patients

were 59.30 and 62.32 months (range 7.23–148.93 months),

respectively. For the 103 patients who survived the follow-up period (censored patients), the mean follow-up time was

58.87 months, and the mean follow-up time was

81.02 months for the 19 patients who died during the fol-low-up period. While the disease-free survival was not

significantly different among the TP53 Arg72Pro SNP

genotypes (P [ 0.969), the less frequent Pro/Pro genotypewas associated with shorter overall survival of the medul-

loblastoma patients (P = 0.021). Likewise, the clinical

high-risk group and those with TP53 positivity by IHCwere independently associated with shorter overall survival

on multivariate analysis (P = 0.0001 and P = 0.0016,

respectively) (Fig. 3).

Discussion

The TP53 gene has a central role in maintaining genomic

stability, and it is a frequently mutated tumor suppressor inhuman cancers [24]. Because somatic TP53 mutations have

Fig. 1 Immunohistochemical staining of TP53. The panels showrepresentative samples deemed immunopositive (a) or immunoneg-ative (b) for the purposes of the current study. All images are shownat the original magnification (9400)

Fig. 2 Electrophoretic patterns of the three genotypes of TP53Arg72Pro separated on a 10 % acrylamide:bisacrylamide gel (19:1)and then stained with silver nitrate. Lane 1 50 bp molecular weightmarker. Lanes 2, 6, and 7 fragments of 100 and 52 bp (Arg/Arg).Lanes 3, 4, 5, and 8 fragments of 152, 100, and 52 bp (Arg/Pro). Lane9 fragment of 152 bp (Pro/Pro)

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been recently related to poor outcome in medulloblastoma

cases [7], it is reasonable to assume that a SNP in this gene

may also be associated with medulloblastoma risk and/or

prognosis. In particular, the TP53 Arg72Pro SNP has

attracted a considerable amount of attention from different

research groups because of a potential role in nervous

Table 1 TP53 Arg72Pro SNP genotypes in relation to clinicopathological parameters

Variable n (%) Genotype P value*

Arg/Arg (%) Arg/Pro (%) Pro/Pro (%)

Age group

\10 years 54 (44.3) 23 (18.9) 22 (18.0) 9 (7.4) 0.929

C10 years 68 (55.7) 27 (22.1) 30 (24.6) 11 (9.0)

Total 122 (100) 50 (41.0) 52 (42.6) 20 (16.4)

Gender

Female 53 (43.4) 19 (15.6) 24 (19.7) 10 (8.2) 0.575

Male 69 (56.6) 31 (25.4) 28 (23.0) 10 (8.2)

Total 122 (100) 50 (41.0) 52 (42.6) 20 (16.4)

Histological grade

Classic 95 (77.9) 40 (32.8) 38 (31.1) 17 (13.9) 0.121

Desmoplastic 16 (13.1) 3 (2.5) 11 (9.0) 2 (1.6)

Large cell/anaplastic 11 (9.0) 7 (5.7) 3 (2.5) 1 (0.8)

Total 122 (100) 50 (41.0) 52 (42.6) 20 (16.4)

IHC for TP53

Negative 79 (82.3) 32 (33.3) 41 (42.7) 6 (6.3) 0.242

Positive 17 (17.7) 10 (10.4) 5 (5.2) 2 (2.1)

Total 96 (100) 42 (43.8) 46 (47.9) 8 (8.3)

Clinical risk group**

Average risk 85 (72.6) 32 (27.4) 38 (32.5) 15 (12.8) 0.389

High risk 32 (27.4) 14 (12.0) 10 (8.5) 8 (6.8)

Total 117 (100) 47 (38.5) 51 (41.8) 24 (19.7)

Recurrence

No 82 (73.9) 38 (34.2) 33 (29.7) 11 (9.9) 0.627

Yes 29 (26.1) 13 (11.7) 10 (9.0) 6 (5.4)

Total 111 (100) 51 (45.9) 43 (38.7) 17 (15.3)

IHC immunohistochemical

* P values obtained using v2 or Fisher’s exact tests

** Average risk, patients with total, or near total, tumor resection and nondisseminated disease; high-risk, [1.5 cm residual tumor on post-operative magnetic resonance imaging, metastatic disease at diagnosis, and/or patients younger than 3 years old

Table 2 Characteristics of medulloblastoma patients and healthy controls

Variable Cases, n = 122 (%) Controls, n = 122 (%) P value* OR (95 % CI) P value

Genotype

Arg/Arg 50 (41.0) 60 (49.2)

Arg/Pro 52 (42.6) 46 (37.7) 1.36 (0.79–2.34) 0.339

Pro/Pro 20 (16.4) 16 (13.1) 0.423 1.50 (0.70–3.20) 0.389

Arg/Pro ? Pro/Pro 72 (59.0) 62 (50.8) 1.39 (0.84–2.31) 0.247

Allelotype

Arg 152 (62.3) 166 (68.0)

Pro 92 (37.7) 78 (32.0) 0.459 0.78 (0.53–1.13) 0.217

OR odds ratio, CI confidence interval

* P values obtained using v2 or Fisher’s exact tests

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system tumors, particularly gliomas [17–20]. However, the

impact of the TP53 Arg72Pro SNP on risk and prognosishas never been evaluated in medulloblastomas. For this

reason, the major aim of this case–control study was to

investigate the relationship between this genomic variationand susceptibility to medulloblastoma and patient survival

in a Brazilian population.

The present study indicated that the genotype and allelefrequencies in the case and control groups were similar

(P = 0.423 and P = 0.459, respectively) and that they

showed no significant association with medulloblastomarisk (OR = 1.48, 95 % CI = 0.90–2.44, and P = 0.156).

These results suggested that the TP53 Arg72Pro SNP maynot significantly contribute to medulloblastoma suscepti-

bility in the Brazilian population. Compared with two other

studies by our group, which were also performed in theBrazilian population but with different tumors, these results

were consistent with those observed in a series of gliomas

(P = 0.488) [20] but inconsistent with those observed forextra-axial brain tumors (meningiomas and schwannomas)

(OR = 3.23, 95 % CI = 1.71–6.08, P = 0.003) [14].

These controversial results may be due to histological andcell function differences and to the dissimilar tumor his-

tological stratification among the studies. However, in

worldwide comparisons, the ethnic heterogeneity of theTP53 Arg72Pro SNP must also be considered, as Brazilians

form one of the most heterogeneous populations in the

world due to five centuries of interethnic crossing of peo-ples from three continents, including European colonizers

(mainly Portuguese), African slaves, and autochthonous

Amerindians [25]; For example, in Eastern Slovakia, Biroset al. [26] did not show any association with this SNP in

astrocytoma samples (P = 0.75). In India, however, Jha

et al. [19] reported that the Pro72 allele is associated withglioma risk (P = 0.037).

Parhar et al. [27] suggested a possible association

between the TP53 Arg72Pro SNP and susceptibility tobrain tumors, particularly high-grade astrocytomas (P =

0.002). However, in Portuguese patients with European

ethnic background, Lima-Ramos et al. [28] did not find anysignificant association between the TP53 Arg72Pro SNP

and histological glioma type. By correlating age and gen-

der in France, El Hallani et al. [18] found that the Pro/Progenotype is overrepresented in patients with glioblastomas

who are younger than 45 years old (P = 0.002), which

suggests that the TP53 Arg72Pro SNP is particularly crit-ical for glioblastoma oncogenesis in young patients.

However, these authors did not find a significant associa-

tion when comparing gender. In the present study, signif-icant association was not found between the TP53Arg72Pro SNP and medulloblastoma patient histological

grade, age or sex. With regard to the TP53 IHC analysis,our results are consistent with those reported by Wang

et al. [29] in ovarian carcinoma, as these authors did not

find a significant association between TP53 protein accu-mulation and the TP53 Arg72Pro SNP genotypes.

Fig. 3 Overall survival curves of patients with medulloblastoma treatedwith adjuvant therapy according to a TP53 Arg72Pro genotypes(P = 0.021), b clinical risk group; average risk, patients with total, ornear total, tumor resection and nondisseminated disease; high risk,[1.5 cmresidual tumor on postoperative magnetic resonance imaging, metastaticdisease at diagnosis, and/or patients younger than 3 years old (P = 0.0001),and c TP53 immunohistochemical (IHC) analysis (P = 0.0016)

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With respect to prognosis, this is the first study to our

knowledge to examine the relationship between the TP53Arg72Pro SNP and the survival of medulloblastoma

patients subjected to adjuvant therapy. The less frequent

Pro/Pro genotype was associated with shorter overall sur-vival of medulloblastoma patients (P = 0.021), which was

not in agreement with a previous study by Lima-Ramos

et al. [28], who showed that glioma patients who harbor thePro72 allele (Arg/Pro ? Pro/Pro) have significantly better

response to adjuvant therapy (P = 0.027). To achievebetter data comparison, we performed a survival analysis

with Arg/Arg versus Arg/Pro ? Pro/Pro patients. How-

ever, the results from this survival analysis did not agreewith those reported by Lima-Ramos et al. [28] (P =

0.123). These authors argued that glioma cells harboring a

Pro72 allele have a survival disadvantage under hypoxicconditions compared with cells harboring an Arg72 allele.

Previously, we reported that TP53 Arg72Pro genotypes are

not associated with survival in glioma patients and patientswith extra-axial nervous system tumors from Brazil [17,

23]. These last results confirmed those of El Hallani et al.

[18], who studied a series of gliomas.Tabori et al. [7] reported that medulloblastoma patients

who have somatic TP53 mutations outside of codon 72 do

not survive long term (P \ 0.0001), which suggests thatTP53 mutations in medulloblastoma have a role in resis-

tance to conventional therapies, thus requiring alternative

treatments. It has been proposed that the link betweenTP53 abnormalities and poor prognosis occurs because an

intact TP53 pathway may be needed so that radiotherapy

and chemotherapy can achieve their full cytotoxic effects[30]. These observations corroborate the finding that TP53

IHC staining was significantly associated with shorter

overall survival in our cohort (P = 0.0016) and are con-sistent with the observations reported by Gessi et al. [13].

In contrast, Pfaff et al. [11] reported that TP53 mutation

status was not associated with unfavorable prognosis inmedulloblastomas (P = 0.63) and was not linked to 17p

allelic loss, but it was overrepresented in the prognostically

favorable well-defined medulloblastoma subgroup featur-ing alterations in the Wnt pathway as the result of CTNNB1mutations (7 of 35 TP53-mutated tumors versus 14 of 271

TP53 wild-type tumors; P = 0.005) and in tumors carryinga high level of MYCN amplification (7 of 21 TP53-mutated

tumors versus 14 of 282 TP53 wild-type tumors; P =

0.01). These results are consistent with those publishedby Lindsey et al. [12], who reported that the TP53 muta-

tions in medulloblastomas are not universally associated

with poor prognosis and can occur in the context ofWnt-subtype disease, which has favorable prognosis.

Recently, Northcott et al. [31] studied a large cohort of

medulloblastomas to determine how many subgroups of thedisease exist, how they differ, and the extent of overlap

between the subgroups. These authors suggest that the

histologic entity of medulloblastoma comprises four dis-tinct molecular variants that are demographically, clini-

cally, transcriptionally, and genetically distinct and can be

distinguished through IHC for DKK1 (Wnt pathway),SFRP1 (SHH pathway), NPR3 (group C), and KCNA1

(group D) in formalin-fixed, paraffin-embedded tumor

material. Because many of the samples assayed in ourstudy contained only a small amount of tumor tissue, we

were unable to identify molecular subgroups of medullo-blastomas; we did not evaluate the presence of somatic

mutations in TP53 or any other gene, and we did not per-

form IHC for markers other than TP53. This can be con-sidered a limitation of our study; therefore, further studies

in independent cohorts stratified into subgroups are needed

to better evaluate the impact of the TP53 Arg72Pro SNP inmedulloblastoma.

The lack of association between classical poor outcome

variables (i.e., histological tumor grade and patient age)and survival in our cohort requires explanation. In our

study, it is noteworthy that nine (82 %) patients with large

cell/anaplastic medulloblastoma, which was notably asso-ciated with poor outcome [32], were alive after the follow-

up period. This observation might be related to the small

sample size and the short follow-up period for patients withthis histological subtype. The mean follow-up period for

large cell/anaplastic medulloblastoma patients was only

32.32 months (range 7.23–67.75 months), and two patientswere clinically followed for less than 1 year. In addition, a

recent study by von Hoff et al. [33] demonstrated that the

prognosis of patients with large cell/anaplastic medullo-blastoma may not be unfavorable in all cases, and MYCamplification and predominant large cell histology were

confirmed to be negative prognostic parameters. Indeed, inour study, the presence of a significant or predominant

large cell component was found in only 4 (36.4 %) out of

11 samples. With regard to patient age, patients youngerthan 3 years experienced poor clinical outcome in com-

parison with older patients, particularly because they are

frequently treated without craniospinal radiation. Thislimitation has been set because of the high susceptibility of

the immature brain to this therapeutic modality in inducing

neurocognitive defects, which have been shown to increaseover time after treatment [34]. Only four (3.28 %) of the

patients were younger than 3 years in our cohort, and this

small proportion of patients may be the reason for thenoninterference of age observed for survival. Two of these

patients are still alive after the follow-up period.

According to previously established criteria [35],patients younger than 3 years with [1.5 cm residual post-

operative tumor and/or metastatic disease were grouped

into a high-risk category. Our results demonstrate that theclinical risk group is strongly associated with shorter

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overall survival in patients with medulloblastoma (P =

0.0001). These results are consistent with those previouslyreported [32, 33, 36–38].

Because this study was retrospectively designed, we

cannot rule out the risk of introducing bias as DNA fromtumor tissue samples from patients with medulloblastoma

was used for analysis whereas peripheral blood was used as

the control DNA source. The different tissues used as DNAsource in cases and controls may raise concerns about

introducing bias toward a specific allele originating fromsomatic mutations in medulloblastoma samples versus

germline polymorphisms in peripheral blood in controls.

However, studies have shown that TP53 is rarely mutatedin medulloblastoma [7, 30], and that for most of the

genotypes, use of tumor tissue samples is a valid alterna-

tive to peripheral blood and vice versa [39–41]; Forexample, Kweekel et al. [39] compared 11 genotypes,

including TP53 Arg72Pro SNP, in DNA isolated from

blood and formalin-fixed, paraffin-embedded colorectalcancer tissue and found that none of the individual geno-

types demonstrated discordance between the tumor and

blood samples that was significantly different from 0.0 %with the exception of the GSTP1 Ile105Val SNP (95 %

CI = 0.1–5.9 %). Therefore, in this study, the use of ret-

rospective medulloblastoma samples, which are routinelyarchived in many clinical trials, may not have significantly

affected our ability to identify germline variants in TP53Arg72Pro.

In conclusion, this is the first analysis of the TP53Arg72Pro SNP in medulloblastoma patients. Although we

were unable to find an association between this geneticvariation and clinicopathological parameters and medul-

loblastoma risk, the Pro/Pro genotype seems to be associ-

ated with shorter overall survival of patients submitted toadjuvant therapy. Nevertheless, due to the interethnic

composition of the Brazilian population, future studies of

larger populations from other parts of the world areessential for a definitive conclusion of the function of the

TP53 Arg72Pro SNP in medulloblastomas.

Acknowledgments This study was supported by the NationalCounsel of Technological and Scientific Development (ConselhoNacional de Desenvolvimento Cientıfico e Tecnologico, CNPq; grantnos. 302774/2009-2 and 484282/2010-7) and by the Coordination ofImprovement of Higher Education (Coordenacao de Aperfeicoamentode Pessoal de Nıvel Superior; CAPES). G.R.P. and R.R.B. havereceived fellowships granted by CNPq.

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