Increased p53 immunopositivity in anaplastic medulloblastoma and supratentorial PNET is not caused by JC virus

The current World Health Organization classification for tumors of the nervous system includes medulloblastoma, medulloepithelioma, ependymoblastoma, supratentorial primitive neuroectodermal tumor (sPNET) and atypical teratoid/rhabdoid tumor (ATRT) in the category of embryonal brain neoplasms [1]. These tumors are united by their primitive cytological appearance and the ability to differentiate into multiple cell types. However, recent studies indicate that these lesions are genetically, and to some extent clinically, separable. ATRT are defined by the presence of rhabdoid cells, contain INI1 mutations, and cause particularly grim clinical outcomes [2]. Medulloblastomas commonly contain isochromosome 17q, but this chromosomal alteration is rarely detected in sPNET or ATRT [3]. Global gene expression profiles also suggest that medulloblastoma, sPNET and ATRT are distinct entities [4].

Little is known about the differences in p53 expression and function among the various embryonal brain tumor subtypes. Initial reports on the p53 tumor suppressor gene suggested it was mutated in 10% or less of medulloblastomas [5–8]. However, Frank and colleagues have recently shown that the p53 pathway is inactivated by mutation of p53, methylation of p14ARF, or deletion of INK4/ARF in 21% of medulloblastomas [9]. In their study, 5 of the 6 medulloblastomas with alterations abrogating p53 function had significant anaplasia. Large cell/anaplastic changes in medulloblastoma are prognostic of significantly worse clinical outcomes [10–12]. Interestingly, p53 protein accumulation, which is often associated with loss of functionality, has been found by some [13–15], but not others [16, 17], to predict shorter survival in medulloblastoma patients as well.

Several other lines of evidence also support a role for the p53 pathway in medulloblastomas. First, medulloblastomas sometimes arise in the context of Li Fraumeni syndrome, in which p53 germline mutations predispose patients to a wide range of neoplasms [18]. Second, inactivation of p53 accelerates the formation of medulloblastomas in transgenic mouse models [19]. Finally, inactivation of p53 and Rb simultaneously, either through genetic disruption or overexpression of viral T antigen, results in medulloblastomas in rodents [20–22].

It has been suggested that viral infection of human CNS tissues could promote formation of brain tumors by inhibiting p53 and Rb activity [23]. Some researchers have reported the presence of JC virus or other oncogenic polyomaviruses in human brain tumor specimens, including medulloblastomas [24, 25]. Large T antigen expressed by these viruses binds and inactivates p53 [26]. This process results in the accumulation and immunohistochemical detection of p53 protein. In human neural tissue this is best demonstrated in progressive multifocal leukoencephalopathy, in which JC virus infected oligodendroglial cells are strongly p53 immunopositive [27, 28]. It is therefore possible that the accumulation of p53 protein in some human medulloblastomas is caused by viral infection.

In order to confirm the association between p53 immunopositivity, clinical outcome, and embryonal tumor subtype, we stained a tissue array containing representative cores from 80 embryonal brain tumors for p53. We also investigated JC virus infection as a possible mechanism for accumulation of p53 protein by searching for viral sequences using a highly sensitive quantitative real time polymerase chain reaction (PCR) assay. We found an association between p53 immunoreactivity, clinical outcome, and tumor subtype, but did not detect JC virus in medulloblastoma or supratentorial PNET.

To investigate the prognostic potential and pathological role of p53 expression in embryonal brain tumors, we analyzed this protein in 75 medulloblastoma, ATRT and sPNET using immunohistochemistry. Overall, we found significant increases in p53 immunoreactivity in anaplastic medulloblastomas (45% positive) as compared to non-anaplastic ones (18% positive). The percentage of p53 immunopositive medulloblastomas in our study (27%) fell within the previously reported range of 3% to 53% [13, 16, 17, 33, 34]. This wide variation in published values is likely due to differences in antibodies used, their dilutions, and antigen retrieval protocols. Cuttoffs for a calling a tumor "positive" also varied among previous investigators, with some scoring only intensely positive lesions. Only 5% of our medulloblastomas fell into this strong staining category, and all of these were anaplastic. Mutation of the p53 gene often results in a stabilized protein of altered functionality that accumulates in the nucleus of tumor cells [35]. Our data are thus consistent with those recently reported by Frank and colleagues, who found that the TP53 pathway was frequently disrupted in large cell/anaplastic medulloblastomas [9].

Interestingly, supratentorial PNET and ATRT were also more commonly p53 immunopositive than non-anaplastic medulloblastoma in our study. While extracerebellar PNET were included in several earlier studies, p53 immunoreactivity was not reported separately for these lesions [14, 15]. Ho and colleagues documented p53 mutations in 6 of 14 sPNET but did not examine protein expression [36]. In another relatively large series, only 1 of 12 sPNET contained a mutation in the p53 gene [37]. Finally, Postovsky and colleagues described an unusual p53 mutation in a case report of a sPNET [38]. With regard to rhabdoid lesions, Berrak and colleagues documented faint p53 immunoreactivity in 6 of the 7 ATRT of the CNS they examined [39]. Malignant rhabdoid tumors arising outside the CNS are also commonly p53 immunopositive, and mutations predicted to inactivate p53 function have been documented in some [40]. Thus while the number of sPNET and ATRT we examined was relatively low, our data, combined with earlier reports, suggests that p53 function may be commonly altered in embryonal tumors arising outside the cerebellum.

Clinical outcomes were significantly worse for embryonal brain tumor patients in our study whose lesions were p53 immunopositive. However, cases most commonly positive for p53 (anaplastic medulloblastomas, sPNET, and ATRT) are all more clinically aggressive than non-anaplastic medulloblastomas, making it difficult to infer causality resulting from p53 accumulation. p53 expression did not predict outcome within the group of medulloblastoma patients, although all 3 strongly p53 immunopositive tumors were severely anaplastic and associated with quite short survival. Interestingly, in a recently published report, Ray and colleagues found that p53 immunoreactivity was the only biological marker predictive of poor outcome on both univariate and multivariate analyses in a group of 112 medulloblastoma patients [41].

We also examined the potential role of viral infection as a mechanism for p53 protein stabilization in medulloblastoma. None of the four p53-positive cases from our tissue array with frozen material available contained viral sequences. We also tested nine additional samples of histological subtypes that were more commonly associated with p53 immunopositive staining (anaplastic medulloblastomas and sPNETs) where p53 staining was not performed. None of these tumors were JCV positive. While sample availability precluded complete testing of all confirmed p53 positive tumors for JCV, the available data do not support a causal role for JCV infection in p53 accumulation and development of embryonal brain tumors.

The impact of viruses on medulloblastoma pathogenesis is controversial. The most commonly implicated agents are the polyomavirus family members JC virus, BK virus and SV40 virus. Of these, JC virus, which infects approximately 80% of the pediatric population and can cause CNS disease in immunosuppressed patients, has been most studied. It was shown decades ago that inoculation of JC virus into rodents resulted in formation of medulloblastoma-like cerebellar tumors [20, 22, 42, 43]. Transgenic mice containing JC virus early region sequences also develop medulloblastomas [44]. Del Valle and colleagues isolated JC virus large T antigen sequences from 11 of 23 human medulloblastomas they examined, and suggested inactivation of p53 and Rb by viral T antigen could be important in the pathogenesis of human embryonal brain tumors [24]. A second gene (Agno) from JC virus was later reported to be present in 11 of 16 medulloblastoma samples by the same group [25].

We failed to detect JC virus sequences in the 33 cases examined, using a sensitive and specific technique which should identify as few as 10 viral genomes per PCR reaction. This suggests the p53 immunopositivity we observe is not caused by JC virus infection in the majority of cases. Our data are also not consistent with the hypothesis that ongoing JC virus infection is common in medulloblastoma. Other recent studies have also reported a lack of JC virus DNA in medulloblastomas. Hayashi and colleagues failed to identify JC viral sequences in 13 medulloblastomas [45]. Kim and colleagues similarly failed to identify JC virus in 15 medulloblastomas, 5 sPNET and 2 medulloblastoma cell lines [46]. Rollison and colleagues examined 225 brain tumors, including 20 medulloblastomas, for JC, BK and SV40 viruses at two different laboratories [47]. No tumor tested positive in both laboratories. Finally, Weggen and colleagues failed to detect JC virus sequences in any of 116 medulloblastomas analyzed, although 2 of these cases were SV40 positive [48]. These reports do not rule out the possibility of a "hit and run" process in which virus participates initially in the formation of a lesion and is then lost. They do strongly suggest that ongoing JC virus infection is not common in human medulloblastomas, as was initially suggested. Interestingly, it has recently been shown that the putative SV40 infection of human mesotheliomas can be accounted for by contamination of samples with small amounts of common laboratory plasmids containing regions of the T-antigen gene [49], calling into question the true association of SV40 with human cancers, including brain tumors.

In summary, we find significantly increased p53 protein levels in anaplastic medulloblastomas, sPNET, and ATRT as compared to classic and nodular medulloblastoma. JC virus was not detected in the 33 tumors examined, suggesting that T-antigen binding does not appear to be an ongoing factor in the pathobiology or p53 protein accumulation of embryonal brain tumors.