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Background: The negative consequences of the cytoplasmic localization of p16 in patients with high-grade astro- cytomas, on their prognosis, was investigated.
Ó Springer 2006

Journal of Neuro-Oncology (2006) 77: 273–277 DOI 10.1007/s11060-005-9037-5

Clinical–patient studies

Cytoplasmic, but not nuclear, p16 expression may signal poor prognosis in high-grade astrocytomas Muhamad Thohar Arifin1, Seiji Hama1, Yoshinori Kajiwara1, Kazuhiko Sugiyama1, Taiichi Saito1, Shinya Matsuura2, Fumiyuki Yamasaki1, Kazunori Arita1 and Kaoru Kurisu1 1 Department of Neurosurgery; 2Department of Radiation Biology, Research Institute for Radiation Biology and Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan

Key words: cytoplasmic p16, high-grade astrocytoma, immunohistochemical methods, prognosis Summary Background: The negative consequences of the cytoplasmic localization of p16 in patients with high-grade astrocytomas, on their prognosis, was investigated. Methods: p16 Expression was examined in 20 anaplastic astrocytoma and 42 glioblastoma patients by immunohistochemical analysis, and the relationship between both cytoplasmic and nuclear p16 expression and prognosis analyzed. Results: The cytoplasmic expression of p16 statistically correlated with poor prognosis. On the other hand, no correlation was observed between p16 nuclear expression and patient survival. Conclusion: The cytoplasmic immunoreactivity of p16 appears to be an unfavorable prognostic indicator in highgrade astrocytoma patients. The localization of p16 expression should be determined when evaluating the prognosis of these patients.

Introduction Despite advances in neurosurgery and clinical neurooncology, the prognosis of patients with malignant gliomas, the most frequent and malignant of brain tumors, remains poor. As surgical removal of the entire tumor is difficult, patients have been treated with radiation and chemotherapy using alkylating agents such as 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). However, the overall one-year survival rate of patients with highgrade astrocytomas (i.e., glioblastomas and anaplastic astrocytomas) is reported as only 40%, and is only slightly higher at 46% following combined radio- and chemotherapy [1]. Glioma cells display abnormalities in one or more genes that regulate their progression through the cell cycle. These include the well-characterized tumor suppressor gene p53, pRb, cyclin D, p21, and p16 [2–4]. As p16 inhibits the ability of CDK4 and CDK6 to regulate the phosphorylation status of pRb, p16 plays a role in progression to the S phase [5,6]. Homozygous deletion of the p16 locus has been reported in 4–52% of human malignant glioma specimens, and in 68–87% of glioma cell lines [5,7–10]. Therefore, restoring the normal function of p16 by gene therapy is an attractive goal in the treatment of human gliomas. We previously reported that radiation-induced non-apoptotic cell death was increased in glioma cells expressing p16. Our recent experiments showed that transfected glioma cells, containing DNA damaged by exposure to radiation, underwent late cell death over the course of 2 weeks

[11]. This suggests that their death was mediated by p16associated mechanisms [12]. We postulated that if the p16 gene is expressed, this would lead to enhanced radiosensitivity, an increase in radiation-induced cell death, and improvement in the prognosis of glioma patients. Therefore, we investigated the expression of p16 in cells from resected high-grade astrocytomas (stage III and IV). We found that there was an association between the expression of p16 and the prognosis of patients treated with surgery and radiation.

Materials and methods Clinical data and tumor specimen selection Prior written informed consent was obtained from all patients. We retrospectively studied the records of 62 patients (23 females, 39 males, aged 14–73 years, median average; 48.81 ± 17.51 years (mean ± SD)) with highgrade astrocytomas who underwent surgery and radiation at Hiroshima University Hospital, Japan, between March 1989 and June 2004. All tumor samples were derived from the initial operation. The patients’ followup data was obtained from hospital records (Table 1). To identify the prognostic factors of astrocytic tumors, we standardized the conditions that would influence the prognosis. We excluded brain stem- and cerebellar astrocytic tumors. Pilocytic astrocytomas were also excluded as their genetic changes during tumorigenesis do not correlate with those of other

274 Table 1. Clinical findings Age (year)

14–73

(48.81 ± 17.51)

Sex Follow up period (in days) Pathological diagnosis Anaplastic astrocytoma Gliobalstoma

F = 23 M = 39 (2:5) 70–3601

(815 ± 906.83)

20 42

astrocytic tumors. Other types of glioma such as oligodendroglial- and ependymal tumors, and gangliogliomas were excluded because they are pathologically distinct from astrocytic tumors.

Concepts Inc., Berkeley, CA). Differences of P < 0.05 were considered statistically significant. Survival time was recorded in years from the date of histological diagnosis at the time of surgery to the date of death or the last follow-up. Survival was plotted and the median survival time was estimated by the Kaplan–Meyer method. Associations between the relative risk of death and each prognostic variable (cytoplasmic and nuclear p16 expression) were evaluated according to the Cox proportional hazards regression model for censored data after dummy code-transformation of the predictor. Survival curves were computed with the Kaplan–Meyer product-limit method and compared using the log-rank test.

Tissue specimens and immunohistochemical staining Results The tumor specimens were fixed in 10% formalin and embedded in paraffin. Representative sections were stained with hematoxylin and eosin for standard histological diagnosis. Histological subtypes were defined according to WHO criteria by one of the authors (K.S.) who had no prior knowledge of the clinical and radiological data. p16 Expression was determined on formalin-fixed, paraffin-embedded tumor samples. We used a mouse anti-human monoclonal antibody (DAKO JAPAN, Kyoto, Japan) at a concentration of 1 lg/ml. Pathologic specimens were cut to 4 lm thickness and mounted on gelatin-coated slides. Sections were deparaffinized by immersion in xylene for 15 min, and then endogenous peroxidase was blocked by immersion in 3% hydrogen peroxidase in methanol for 30 min. Each specimen was rinsed 3 times for 5 min each in phosphatebuffered saline (PBS, pH 7.5) with gentle stirring. After overnight incubation (4 °C) with the primary antibody, we applied the streptavidin–biotin method using the histofine SAB (M) kit (Nichirei Co., Tokyo, Japan). After washing in PBS, the sections were incubated for 5 min with 0.1 mg/ml diaminobentizin tetra hydrochloride (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in 0.05 M Tris buffer (pH 7.6) containing 0.003% hydrogen peroxide. To facilitate visualization of the immunostained product in the cytoplasm, the slides were counterstained with Mayer’s hematoxylin. Experiments and control experiments were performed twice on different days using the same protocols and time exposures. Normal pituitary gland was the positive control [13]. In the negative controls the antibody was omitted. Evaluation of p16 expression The p16 expression of the gliomatous tumors was recorded as negative ()), and positive (+) by two authors (M.T. and S.H.) with no prior knowledge of the pathologic diagnosis or any clinical or radiological data. They also recorded whether p16 expression was cytoplasmic or nuclear.

Patient treatment and tumor pathology Tumor specimens from 62 patients were analyzed. According to the WHO classification, 20 of the tumors (32%) were anaplastic astrocytomas and 42 (68%) were glioblastomas. All patients had undergone craniotomy and all received adjuvant radiation therapy (40–60 Gy in 2-Gy fractions); 55 patients were also treated with ACNU (70 mg/m2 i.v. on days 1 and 28) and 26 patients received cis-diamminedichloplatinum (CDDP, 35 mg/ m2 i.v. on days 1, 14, and 28). Vincristine (0.7 mg/m2 i.v.) was administered to 9 patients showing regrowth or recurrence P16 immunohistochemistry The cytoplasm of 46 of the 62 tumors (74.2%) was immunopositive for p16, while nuclear immunostaining was observed in 25 of the 62 tumors (40.3%). Only the cytoplasm was immunopositive in 22 (35%) tumors, only the nucleus was immunopositive in 1 (2%) tumor, 24 (39%) tumors exhibited both nuclear and cytoplasmic immunopositivity, and 15 (24%) were negative for both cytoplasmic and nuclear staining (Tables 2 and 3, Figure 1). Effect of p16 expression on prognosis Next, we examined whether the localization of p16 expression had a bearing on the overall survival of patients with high-grade astrocytoma. As shown in Figure 2, a statistically significant correlation was observed between the cytoplasmic expression of p16 and the mortality rate (P = 0.0498). No such correlation with prognosis was observed for tumors with nuclear p16 expression (P = 0.363). These observations indicate that the cytoplasmic expression of p16 signals a poor prognosis in patients with high-grade astrocytoma.

Statistical analysis

Discussion

Statistical analyses were performed by Student’s t-test using Stat ViewTM for Windows, version 5.0 (Abacus

The p16 gene, the product of which is thought to exert negative control on the progression of eukaryotic cells

275 Table 2. Immunopositivity of p16 expression in high grade astrocytoma

p16 Immunopositivity

Cytoplasm

Nucleus

46 (74.2%)

25 (40.3%)

Table 3. Distribution of location of p16 expression in high-grade astrocytoma

Categorical values are number of patients (percentage).

through G1 arrest in the cell cycle, has been shown to manifest homozygous deletions or inactivation via methylation, and to be associated with the tumorigenesis and progression of glioma [5,7,14]. The homozygous deletion of the p16 gene is a significant unfavorable prognosticator for the survival of glioblastoma patients [15]. We postulated that the prognosis of patients with p16-expressing gliomas treated with radiation was better than that of patients whose tumors did not express p16.

We tested this hypothesis by analyzing 62 resected highgrade astrocytomas (stages III and IV) by immunohistochemical staining for p16. We found that cytoplasmic,

Figure 1. Immunohistochemical staining for p16 expression of the gliomatous tumors was recorded as negative ()), and positive (+). Also recorded was whether p16 expression was cytoplasmic or nuclear. We used normal pituitary gland as the positive control. (a) Negative, (b) solely cytoplasmic positive, (c) solely nuclear positive, (d) nuclear and cytoplasmic positive, (e) normal pituitary gland (positive control).

276

Figure 2. Kaplan–Meyer curves for the overall survival rates of high-grade astrocytoma patients with cytoplasmic (a) or nuclear (b) expression of p16. The median survival of patients with cytoplasmic p16 expression was lower than that of patients without cytoplasmic p16 expression (P = 0.0498 log-rank test). Nuclear p16 expression had no statistically significant effect on overall survival (P = 0.363).

but not nuclear, expression of p16 was statistically correlated with poor prognosis. While nuclear p16 protein is known as a cell-cycle inhibitor, the cytoplasmic localization of p16 was considered non-specific in human gliomas. However, Evangelou et al. [16] who demonstrated that cytoplasmic p16 staining is specific in non-small cell lung carcinoma cell lines, suggested that it represents a mechanism of p16 inactivation. Furthermore, the cytoplasmic expression of p16 has also been demonstrated in breast cancer [17,18], and in those studies, solely cytoplasmicand nuclear-plus-cytoplasmic reactivity were unfavorable prognostic indicators. On the other hand, studies examining the predictive value of p16 immunoreactivity in high-grade astrocytomas have yielded inconsistent results [19]; according to the univariate analysis of Labuhn et al. [20] p16 immunoreactivity correlated with poor prognosis. However, unlike the present study, their studies did not distinguish between cytoplasmic and nuclear p16 expression. Our results are the first to demonstrate specifically that exclusively cytoplasmic immunoreactivity is an unfavorable prognostic indicator. p16, Expressed not only in the nucleus but also the cytoplasm of cancer cells [21,22], plays an importance role in the regulation of vascular endothelial growth factor (VEGF) expression and cell senescence [23]. While the mechanism(s) leading to the cytoplasmic

accumulation of p16 remains unclear, p16 mutations may affect cytoplasmic-nuclear shuttling in a manner similar to that reported for BRCA1. In fact, the mutation of nuclear localization signals (NLS) and the HN2terminal (nuclear export signal, NES) shifts BRCA1 to the cytoplasm [24]. With few reports on p16 mutations in gliomas, the possibility of alternative mechanisms cannot be excluded. p16 Lacks NLS and NES, and thus the mutation may involve p16’s nuclear chaperone groups. Efforts underway in our laboratory to test this hypothesis may reveal the existence of deregulated chaperone protein(s) that may interfere with vital components of the cellular checkpoint machinery. Alternatively, cytoplasmic p16 may represent a protective response to the over-expression of CDK4. According to this scenario, a portion of p16 binds to CDK4 in the cytoplasm and the large size of the resulting complex inhibits passage into the nucleus, while the unbound protein can enter the nucleus to exert its oncosuppressor activity [25].

Conclusion In patients with high-grade astrocytoma, cytoplasmic, but not nuclear, p16 immunoreactivity appears to be an unfavorable prognostic indicator. The sequestration of p16 in the cytoplasm may represent an alternative

277 mechanism of p16 inactivation. Based on our observations, we suggest that the cytoplasmic or nuclear localization of p16 expression should be determined when considering the prognosis of patients with high-grade astrocytoma. Acknowledgement This study was supported in part by grants-in-aid from the Ministry of Education, Science and Culture, Japan.

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