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Frequent p16 inactivation by homozygous deletion or methylation is associated with a poor prognosis in Japanese patients with pleural mesothelioma
Lung Cancer (2008) 62, 120—125
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/lungcan
Frequent p16 inactivation by homozygous deletion or methylation is associated with a poor prognosis in Japanese patients with pleural mesothelioma Naruyuki Kobayashi a, Shinichi Toyooka a,∗, Hiroyuki Yanai b, Junichi Soh a, Nobukazu Fujimoto c, Hiromasa Yamamoto a, Shuji Ichihara a, Kentaro Kimura a, Kouichi Ichimura b, Yoshifumi Sano a, Takumi Kishimoto c, Hiroshi Date d a
Department of Cancer and Thoracic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan b Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan c Department of Internal Medicine, Okayama Rosai Hospital, Okayama, Japan d Department of Thoracic Surgery, Graduate School of Medicine, Kyoto University, Japan Received 14 December 2007; received in revised form 24 January 2008; accepted 14 February 2008
KEYWORDS Malignant pleural mesothelioma; P16 gene; Homozygous deletion; Methylation
∗
Summary This study examined the p16 expression status and the P16 gene deletion and methylation status in specimens from Japanese patients with malignant pleural mesothelioma (MPM). Immunohistochemical staining for p16 protein and fluorescence in situ hybridization for the P16 gene were performed using specimens from 30 Japanese patients with primary MPM. The methylation status of the P16 gene was examined in 13 patients whose frozen tumor specimens were available using a methylation-specific PCR assay. Among the 30 patients, the loss of p16 protein expression was observed in 24 patients (80.0%). Twenty-one patients had homozygous deletions, and 9 patients retained the P16 gene. None of the patients with P16 homozygous deletions exhibited p16-positive expression, and 3 patients who retained the P16 gene did not exhibit p16-positive expression. Aberrant P16 methylation was present in two patients with an intact P16 gene but without p16 expression. These results suggest that either a homozygous deletion or methylation is responsible for P16 inactivation. Regarding the prognosis, patients with p16-negative expression had a significantly shorter survival time than those with p16-positive expression (P = 0.040). Our study showed that P16 inactivation by homozygous deletions or methylation is a frequent event in Japanese patients with MPMs,
Corresponding author. Tel.: +81 86 235 7265; fax: +81 86 235 7269. E-mail address: [email protected] (S. Toyooka).
0169-5002/$ — see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2008.02.013
Frequent p16 inactivation in pleural mesothelioma
121
relating to poor prognosis. Homozygous deletion is the major cause of P16 inactivation, but methylation also lead to the inactivation of P16 when the P16 alleles are retained. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Malignant pleural mesothelioma (MPM) is an aggressive tumor that develops from the pleura or other mesothelial surfaces. Many MPMs have already progressed by the time they are discovered, resulting in a dismal prognosis [1]. The incidence of MPM has been increasing and has been recognized as a worldwide problem [2]. In particular, the increasing incidence of MPM in Japan has become a serious problem since 2005. Asbestos, especially amphiboles fibers, is a wellestablished carcinogen leading to MPM [3,4] but the molecular alterations involved in the pathogenesis of MPM are relatively unknown. Of interest, the disease has a long latency period (20—40 years, after the initial exposure to asbestos); but once diagnosed, the disease grows very aggressively. P16 IKN4a is a tumor suppressor gene, located on chromosome 9p21, that inhibits cyclin-dependent kinases (CDKs) and prevents CDK-mediated hyperphosphorylation and the inactivation of retinoblastoma protein (pRB) and leading to G1-phase cell cycle arrest [2]. The loss of p16 expression is observed in 48—100% of MPMs and is a well-known molecular alteration that has been reported in Western countries. Homozygous deletion of P16 is considered to be a major mechanism for the loss of p16 expression. The prevalence of P16 homozygous deletion ranges from 22% to 74% in MPM patients in the Western countries [5—7]. These results also suggest a discrepancy in the rate of P16 alterations among the studies. Other mechanisms leading to the loss of gene expression, such as epigenetic alterations like DNA promoter methylation, have also been well established [8]. DNA methylation in MPM samples has been reported in Western countries, and the aberrant methylation of several genes has been identified. A low frequency of DNA methylation of P16 has been reported in Western countries [9—11] but the status of P16 methylation in Japanese patients with MPM is unknown. In lung cancer, geographical differences in the frequency of EGFR, K-ras mutations, and the aberrant methylation of MGMT and GSTP1 have been reported [12]. The etiology of MPM in Western countries and Japan may also differ, since infection with simian virus 40 is thought to play an important role in the pathogenesis of MPMs in Western countries, but not in Japan [13—15]. Indeed, the aberrant methylation of insulin-like growth factor-binding protein-3 is significantly more frequent in MPMs from Japanese patients than in those from American patients [16]. Thus, we believed it was crucial to investigate the alteration of P16 in MPM specimens from Japanese patients. In this study, we evaluated p16 protein expression, P16 deletion, and the methylation status in MPM specimens from Japanese patients to investigate the impact of P16 on the carcinogenesis and clinicopathogical factors of MPMs.
2.1. Characteristics of patient Thirty Japanese patients with MPM were evaluated in this study. Eighteen patients were diagnosed or treated at Okayama University Hospital between October 1994 and August 2007 and 12 patients were treated in Okayama Rosai Hospital between June 2004 and August 2007. All patients were male and the median age was 60.3 years (range, 38—74). History of asbestos exposure was collected by trained physicians and 4 patients were considered to be free of asbestos exposure. Histologic diagnosis of MPM was determined using biopsied or surgically resected specimens, consisting of 16 epithelial types, 8 biphasic types, 5 sarcomatous types, and 1 lymphohistiocytoid type. Among 30 patients, 20 patients received surgery, 9 patients received chemotherapy, and one patient received best supportive care. All MPM samples were paraffin-embedded tissue specimens and 13 of 30 specimens were also stored at −80 ◦ C at the time of surgery. Institutional review board permission and informed consent were obtained from all patients for analyses.
2.2. Immunohistochemical staining The expressions of p16 proteins were analyzed using immunohistochemical staining. Tumor samples were formalin-fixed and paraffin-embedded. In brief, for epitope retrieval, the specimens were exposed to 10 mM citrate buffer (pH 6.0) and heated in a microwave for 15 min. Tumor sections were incubated for 60 min with a monoclonal antibody specific to the full-length human p16 protein at a 1:20 dilution (clone 6H12; Novocastra Laboratories, Newcastle, UK). Antibody binding was detected using an Envision DAB kit (Dako, Glostrup, Denmark), and Mayers hematoxylin was used for counterstaining. Small cell lung cancer samples were used as external positive controls. Two investigators (N.K. and H.Y.) evaluated p16 staining under a light microscope at a 400× magnification. P16 immunoreactivity was nuclear and cytoplasmic and scored for intensity and extent using the following method: 0, no staining; 1+, weak staining; 2+, strong staining for intensity and focal (<5%) versus multifocal/diffuse for extent. Specimens were considered negative if 0 or 1+ focal and considered positive if 1+ multifocal/diffuse and all 2+.
2.3. Fluorescence in situ hybridizations (FISH) analysis Specimens were processed for the analysis of chromosomal aneuploidies as described in standard procedures. Briefly, paraffin-embedded tissue specimens were sliced 5-m-thick sections. They were deparaffinized, dehydrated, immersed in 0.2 N HCl, and boiled in a microwave in citrate buffer
122 (pH 6.0) for 30 min. Sections were then immersed in pepsin solution, and the tissues were fixed in 10% neutral-buffered formalin. In each samples, a dual-color FISH assay was performed using the LSI P16 (9p21) spectrumOrange/CEP9 spectrumGreen probe (Vysis, Downers Grove, IL) following manufacturer’s instructions. The samples were observed under a fluorescent microscopy (BZ-Analyzer) (Keyence, Osaka, Japan). In our case, at least 100 non-overlapping interphase nuclei per core were scored by 2 independent observers (N.K. and J.S.). Patients were classified according to the previous report; [17] samples containing ≥15 nuclei that lacked both signal for P16 and contained at least 1 signal for chromosome 9 centromere were considered positive for homozygous deletion.
2.4. Methylation-specific PCR (MSP) assay Among 30 cases, frozen tumor tissues were available for 13 cases to extract genomic DNA. Genomic DNAs were isolated from frozen tumor tissues by SDS/proteinase K digestion (Life Technologies, Inc., Rockville, MD), phenol—chloroform extraction, and ethanol precipitation. The methylation status of P16 genes was determined by MSP assay as previously described [18]. Briefly, 1 g of genomic DNA was modified by sodium bisulfite, which converts all unmethylated cytosines to uracils while methylated cytosines remain unchanged. PCR amplification was done with sodium bisulfite-treated DNA as template as previously described, using specific primers for the methylated and unmethylated forms of P16. Primers sequences are as follows; methylated form of P16: 5 -TTA TTA GAC GGG TGG GGC GGA TCG C-3 (sense), 5 -GAC CCC GAA CCG CGA CCG TAA-3 (antisense), and unmethy-
N. Kobayashi et al. lated form of P16: 5 -TTA TTA GAG GGT GGG GTG GAT TGT-5 (sense), 5 -GAC CCC GAA CCG CGA CCG TAA-3 (antisense) [18].
2.5. Statistical analyses The differences of significance among categorized groups were compared using Chi-square test or Fisher’s exact test when appropriate. Overall survival was defined as the time of diagnosis to the time of death from any cause or to the date the patient was last known to be alive. The univariate analysis of overall survival was carried out by the Kaplan—Meier method using the log—rank test. All data were analyzed using StatView® 5.0 Program for Windows (SAS Institute Inc., Cary, NC). All statistical tests were two-sided and probability values <0.05 were defined as being statistically significant.
3. Results 3.1. Immunohistochemical staining for p16 protein Of the 30 specimens that were examined, six (20%) were p16-positive and 24 (80%) were p16-negative. Representative examples of the immunohistochemical staining patterns are shown in Fig. 1. Regarding the histological subtypes, the loss of p16 expression was identified in 12 of 16 (75.0%) epithelial-type specimens, 6 of 8 (75.0%) biphasic-type specimens, all 5 of 5 sarcomatous-type specimens, and 1 of 1 lymphohistiocytoid-type specimens (Table 1).
Fig. 1 FISH and immunohistochemical staining. (A) FISH for Patient #1, tumor cells showing homozygous deletion of P16 genes (spectrum orange) with chromosome 9 aneuploidy (spectrum green). Normal lymphocytes retained P16 genes (upside). (B) Immunohistochemical staining for Patient #1, tumor cells showing no expression of p16 protein. (C) FISH for Patient #2, tumor cells retaining P16 genes (spectrum orange) with chromosome 9 aneuploidy (spectrum green). (D) Immunohistochemical staining for Patient #2, tumor cells showing expression of p16 protein.
Frequent p16 inactivation in pleural mesothelioma Table 1
123
The status of P16 in Japanese malignant pleural mesothelioma
Subtype
No.
P16 gene
p16 Protein
HD
Retained
Negative
Positive
Epithelial type Biphasic type Sarcomatous type Lymphohistiocytoid
16 8 5 1
11 4 5 1
5 4 0 0
12 6 5 1
4 2 0 0
Total
30
21
9
24
6
HD, homozygous deletion.
Fig. 2
Representative examples of MSP assay. P16 U; unmethylated form of P16, P16 M, methylated form of P16.
3.2. Deletion and DNA methylation of P16 gene Among the 30 specimens that were examined using a FISH assay, 21 (70%) exhibited homozygous deletions of the P16 gene and 9 (30%) retained the P16 gene. Representative examples of the FISH assay results are shown in Fig. 1. Regarding the histological subtypes, the homozygous deletion of P16 was identified in 11 of 16 (68.8%) epithelial-type specimens, 4 of 8 (50.0%) biphasic-type specimens, 5 of 5 sarcomatous-type specimens, and 1 of 1 lymphohistiocytoid-type specimens (Table 1). While none of the specimens with P16 homozygous deletions exhibited p16-positive expression, these specimens that were shown by FISH to retain the P16 gene did not exhibit p16-positive expression. Because we suspected that aberrant methylation might be responsible for the loss of p16 expression in these 3 specimens, the methylation status of the P16 gene was examined in 13 specimens that had been stored at −80 ◦ C. Two of the 3 patients that retained the P16 gene but did not express p16 were included in among the 13 available specimens. Aberrant methylation was detected in 2 of the 13 specimens using an MSP assay (Fig. 2). The p16 protein was not immunohistochemically detected in these 2 specimens. These results suggest that either homozygous deletions or aberrant methylation can lead to p16 inactivation. We examined the relationship between asbestos, p16 expression and P16 homozygous deletion. Among 4 patients with no history of asbestos exposure, 2 patients revealed loss of p16 expression with P16 homozygous deletion. No significant difference in molecular status between patients with asbestos exposure and those with no exposure.
We examined the relationship between the p16 status and patient survival. Despite the small sample size of this study, the patients with a p16-negative status had a shorter survival time (MST, 8.4 months) than those with a p16-positive status (MST, 12.7 months) (Fig. 3). No significant differences in sur-
3.3. Patient prognosis The follow-up data were documented for all 30 patients. A Kaplan—Meier plot for all the patients is shown in Fig. 3. The overall median survival time (MST) was 9.6 months.
Fig. 3 Kaplan—Meier plots of overall survival after diagnosis of MPM. (A) Overall survival for all 30 patients. (B) Overall survival according to the p16 protein status.
124 vival were seen according to the P16 homozygous deletion or methylation statuses. No differences in the p16 expression status or the P16 homozygous deletion status were seen between patients with curative resections and those who did not undergo surgery (P = 0.63). No differences in the prognosis, p16 expression status, or P16 homozygous deletion statuses were seen between cases treated at two institutions.
4. Discussion The worldwide incidence of MPM is thought to be increasing, especially in Japan and non-Western counties where asbestos continued to be heavily used after its use had been discontinued in Western counties [1]. Thus, the urgent establishment of new preventive, diagnostic and treatment strategies for MPM is mandatory, drawing attention to the importance of understanding the molecular alterations in MPM. In fact, much less information regarding molecular biological alterations is available for MPM than for other solid neoplasms. In this study, we showed that the loss of p16 protein expression is common in MPM specimens from Japanese patients. Homozygous deletions were the major mechanism for p16 inactivation, but DNA methylation was also responsible for inactivation when the p16 allele was retained. This is the first study to show that the alteration of P16 is a major event in the pathogenesis of MPM in Japanese patients, as it is in Western countries. We did not examine the specific P16 mutations in this study. Among the 13 specimens in which p16 expression, P16 deletion and P16 methylation were completely examined, all patients with p16 negative expression showed either P16 homozygous deletion or aberrant P16 methylation, suggesting that these two mechanisms are critical for the loss of expression. Of note, P16 gene mutations are uncommon in MPMs [19,20]. In MSP assay for primary tumors, either the unmethylated band only or both methylated and unmethylated bands were present. Two major possibilities were responsible for this finding: (1) tumor tissues without microdissection consists of mixtures of tumor cells and non-malignant cells, therefore, the unmethylated band is derived from non-malignant cells; (2) there may be a heterogenesity of methylation status among two alleles in a cell or among each tumor cell. For this case, p16 expression can be preserved in tumor tissue. However, our result that methylated samples show that p16 expression did not support the latter possibility despite the small sample size. From a clinicopathological aspect, P16 homozygous deletions seemed to be more common in cases of sarcomatous-type (100%) than those of epithelial- or biphasic-type (62.5%), consistent with observations in Western countries [7,21]. In addition, although the number of patients in our study was not large, our results showed that lack of p16 expression was a significant negative prognostic factor among Japanese patients with MPM. Indeed, these findings have been reported in several studies conducted in Western countries [22—24]. Our results suggest the possibility of diagnostic and therapeutic applications focusing on P16 for Japanese MPMs. Illei and colleagues reported that the detection of homozygous deletions of the P16 gene in pleural effusions using a FISH
N. Kobayashi et al. assay was useful for distinguishing malignant mesothelial cells from reactive mesothelial cells and other malignant cells [25] indicating the usefulness of the P16 deletion as a diagnostic marker of MPM. Regarding therapeutic applications, cell cycle arrest was reportedly induced in MPM cells to which p16 expression constructs were introduced, suggesting the possibility of gene therapy utilizing P16 [26,27]. Regarding other molecular alterations in MPM, neurofibromatosis type 2 (NF2) has been shown to be an important target of gene mutation in MPMs in Western studies [28]. No information is available regarding NF2 mutations in Japanese patients with MPM other than that among 4 cell lines derived from Japanese patients, one carried a mutation [29]. It would be interesting to investigate the relationship between P16 and NF2 alterations from the viewpoint of the carcinogenesis of MPMs. Of note, mutations in other genes, including the P53, Ras, and RB genes (which have been found at high frequencies in other malignant tumors), are very rare in MPM [20,30,31]. In conclusion, our results showed that the loss of p16 expression is a common alteration that is related to a poor prognosis in Japanese patients with MPM. The homozygous deletion of P16 is a major mechanism for p16 protein depletion, although aberrant methylation is also involved in gene silencing.
Authors disclosures of potential conflicts of interest The authors indicated no financial relationship with companies whose products are mentioned in this article.
Acknowledgements This study was supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology, Japan and a Grant-in-Aid for Cancer Research from the Ministry of Health, Labor, and Welfare, Japan.
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125 [20] Papp T, Schipper H, Pemsel H, Bastrop R, Muller KM, Wiethege T, et al. Mutational analysis of N-ras, p53, p16INK4a, p14ARF and CDK4 genes in primary human malignant mesotheliomas. Int J Oncol 2001;18:425—33. [21] Xio S, Li D, Vijg J, Sugarbaker DJ, Corson JM, Fletcher JA. Codeletion of p15 and p16 in primary malignant mesothelioma. Oncogene 1995;11:511—5. [22] Lopez-Rios F, Chuai S, Flores R, Shimizu S, Ohno T, Wakahara K, et al. Global gene expression profiling of pleural mesotheliomas: overexpression of aurora kinases and P16/CDKN2A deletion as prognostic factors and critical evaluation of microarray-based prognostic prediction. Cancer Res 2006;66:2970—9. [23] Borczuk AC, Taub RN, Hesdorffer M, Hibshoosh H, Chabot JA, Keohan ML, et al. P16 loss and mitotic activity predict poor survival in patients with peritoneal malignant mesothelioma. Clin Cancer Res 2005;11:3303—8. [24] Ladanyi M. Implications of P16/CDKN2A deletion in pleural mesotheliomas. Lung Cancer 2005;49(Suppl. 1):S95—8. [25] Robinson BW, Musk AW, Lake RA. Malignant mesothelioma. Lancet 2005;366:397—408. [26] Frizelle SP, Grim J, Zhou J, Gupta P, Curiel DT, Geradts J, et al. Re-expression of p16INK4a in mesothelioma cells results in cell cycle arrest, cell death, tumor suppression and tumor regression. Oncogene 1998;16:3087—95. [27] Piperno-Neumann S, Oudar O, Reynier P, Briane D, Cao A, Jaurand MC, et al. Transfer into a mesothelioma cell line of tumor suppressor gene p16 by cholesterol-based cationic lipids. Biochim Biophys Acta 2003;1611:131—9. [28] Sekido Y, Pass HI, Bader S, Mew DJ, Christman MF, Gazdar AF, et al. Neurofibromatosis type 2 (NF2) gene is somatically mutated in mesothelioma but not in lung cancer. Cancer Res 1995;55:1227—31. [29] Usami N, Fukui T, Kondo M, Taniguchi T, Yokoyama T, Mori S, et al. Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients. Cancer Sci 2006;97:387—94. [30] Kratzke RA, Otterson GA, Lincoln CE, Ewing S, Oie H, Geradts J, et al. Immunohistochemical analysis of the p16INK4 cyclindependent kinase inhibitor in malignant mesothelioma. J Natl Cancer Inst 1995;87:1870—5. [31] Papp T, Schipper H, Pemsel H, Unverricht M, Muller KM, Wiethege T, et al. Mutational analysis of the PTEN/MMAC1 tumour suppressor gene in primary human malignant mesotheliomas. Oncol Rep 2001;8:1375—9.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/lungcan
Frequent p16 inactivation by homozygous deletion or methylation is associated with a poor prognosis in Japanese patients with pleural mesothelioma Naruyuki Kobayashi a, Shinichi Toyooka a,∗, Hiroyuki Yanai b, Junichi Soh a, Nobukazu Fujimoto c, Hiromasa Yamamoto a, Shuji Ichihara a, Kentaro Kimura a, Kouichi Ichimura b, Yoshifumi Sano a, Takumi Kishimoto c, Hiroshi Date d a
Department of Cancer and Thoracic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan b Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan c Department of Internal Medicine, Okayama Rosai Hospital, Okayama, Japan d Department of Thoracic Surgery, Graduate School of Medicine, Kyoto University, Japan Received 14 December 2007; received in revised form 24 January 2008; accepted 14 February 2008
KEYWORDS Malignant pleural mesothelioma; P16 gene; Homozygous deletion; Methylation
∗
Summary This study examined the p16 expression status and the P16 gene deletion and methylation status in specimens from Japanese patients with malignant pleural mesothelioma (MPM). Immunohistochemical staining for p16 protein and fluorescence in situ hybridization for the P16 gene were performed using specimens from 30 Japanese patients with primary MPM. The methylation status of the P16 gene was examined in 13 patients whose frozen tumor specimens were available using a methylation-specific PCR assay. Among the 30 patients, the loss of p16 protein expression was observed in 24 patients (80.0%). Twenty-one patients had homozygous deletions, and 9 patients retained the P16 gene. None of the patients with P16 homozygous deletions exhibited p16-positive expression, and 3 patients who retained the P16 gene did not exhibit p16-positive expression. Aberrant P16 methylation was present in two patients with an intact P16 gene but without p16 expression. These results suggest that either a homozygous deletion or methylation is responsible for P16 inactivation. Regarding the prognosis, patients with p16-negative expression had a significantly shorter survival time than those with p16-positive expression (P = 0.040). Our study showed that P16 inactivation by homozygous deletions or methylation is a frequent event in Japanese patients with MPMs,
Corresponding author. Tel.: +81 86 235 7265; fax: +81 86 235 7269. E-mail address: [email protected] (S. Toyooka).
0169-5002/$ — see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2008.02.013
Frequent p16 inactivation in pleural mesothelioma
121
relating to poor prognosis. Homozygous deletion is the major cause of P16 inactivation, but methylation also lead to the inactivation of P16 when the P16 alleles are retained. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Malignant pleural mesothelioma (MPM) is an aggressive tumor that develops from the pleura or other mesothelial surfaces. Many MPMs have already progressed by the time they are discovered, resulting in a dismal prognosis [1]. The incidence of MPM has been increasing and has been recognized as a worldwide problem [2]. In particular, the increasing incidence of MPM in Japan has become a serious problem since 2005. Asbestos, especially amphiboles fibers, is a wellestablished carcinogen leading to MPM [3,4] but the molecular alterations involved in the pathogenesis of MPM are relatively unknown. Of interest, the disease has a long latency period (20—40 years, after the initial exposure to asbestos); but once diagnosed, the disease grows very aggressively. P16 IKN4a is a tumor suppressor gene, located on chromosome 9p21, that inhibits cyclin-dependent kinases (CDKs) and prevents CDK-mediated hyperphosphorylation and the inactivation of retinoblastoma protein (pRB) and leading to G1-phase cell cycle arrest [2]. The loss of p16 expression is observed in 48—100% of MPMs and is a well-known molecular alteration that has been reported in Western countries. Homozygous deletion of P16 is considered to be a major mechanism for the loss of p16 expression. The prevalence of P16 homozygous deletion ranges from 22% to 74% in MPM patients in the Western countries [5—7]. These results also suggest a discrepancy in the rate of P16 alterations among the studies. Other mechanisms leading to the loss of gene expression, such as epigenetic alterations like DNA promoter methylation, have also been well established [8]. DNA methylation in MPM samples has been reported in Western countries, and the aberrant methylation of several genes has been identified. A low frequency of DNA methylation of P16 has been reported in Western countries [9—11] but the status of P16 methylation in Japanese patients with MPM is unknown. In lung cancer, geographical differences in the frequency of EGFR, K-ras mutations, and the aberrant methylation of MGMT and GSTP1 have been reported [12]. The etiology of MPM in Western countries and Japan may also differ, since infection with simian virus 40 is thought to play an important role in the pathogenesis of MPMs in Western countries, but not in Japan [13—15]. Indeed, the aberrant methylation of insulin-like growth factor-binding protein-3 is significantly more frequent in MPMs from Japanese patients than in those from American patients [16]. Thus, we believed it was crucial to investigate the alteration of P16 in MPM specimens from Japanese patients. In this study, we evaluated p16 protein expression, P16 deletion, and the methylation status in MPM specimens from Japanese patients to investigate the impact of P16 on the carcinogenesis and clinicopathogical factors of MPMs.
2.1. Characteristics of patient Thirty Japanese patients with MPM were evaluated in this study. Eighteen patients were diagnosed or treated at Okayama University Hospital between October 1994 and August 2007 and 12 patients were treated in Okayama Rosai Hospital between June 2004 and August 2007. All patients were male and the median age was 60.3 years (range, 38—74). History of asbestos exposure was collected by trained physicians and 4 patients were considered to be free of asbestos exposure. Histologic diagnosis of MPM was determined using biopsied or surgically resected specimens, consisting of 16 epithelial types, 8 biphasic types, 5 sarcomatous types, and 1 lymphohistiocytoid type. Among 30 patients, 20 patients received surgery, 9 patients received chemotherapy, and one patient received best supportive care. All MPM samples were paraffin-embedded tissue specimens and 13 of 30 specimens were also stored at −80 ◦ C at the time of surgery. Institutional review board permission and informed consent were obtained from all patients for analyses.
2.2. Immunohistochemical staining The expressions of p16 proteins were analyzed using immunohistochemical staining. Tumor samples were formalin-fixed and paraffin-embedded. In brief, for epitope retrieval, the specimens were exposed to 10 mM citrate buffer (pH 6.0) and heated in a microwave for 15 min. Tumor sections were incubated for 60 min with a monoclonal antibody specific to the full-length human p16 protein at a 1:20 dilution (clone 6H12; Novocastra Laboratories, Newcastle, UK). Antibody binding was detected using an Envision DAB kit (Dako, Glostrup, Denmark), and Mayers hematoxylin was used for counterstaining. Small cell lung cancer samples were used as external positive controls. Two investigators (N.K. and H.Y.) evaluated p16 staining under a light microscope at a 400× magnification. P16 immunoreactivity was nuclear and cytoplasmic and scored for intensity and extent using the following method: 0, no staining; 1+, weak staining; 2+, strong staining for intensity and focal (<5%) versus multifocal/diffuse for extent. Specimens were considered negative if 0 or 1+ focal and considered positive if 1+ multifocal/diffuse and all 2+.
2.3. Fluorescence in situ hybridizations (FISH) analysis Specimens were processed for the analysis of chromosomal aneuploidies as described in standard procedures. Briefly, paraffin-embedded tissue specimens were sliced 5-m-thick sections. They were deparaffinized, dehydrated, immersed in 0.2 N HCl, and boiled in a microwave in citrate buffer
122 (pH 6.0) for 30 min. Sections were then immersed in pepsin solution, and the tissues were fixed in 10% neutral-buffered formalin. In each samples, a dual-color FISH assay was performed using the LSI P16 (9p21) spectrumOrange/CEP9 spectrumGreen probe (Vysis, Downers Grove, IL) following manufacturer’s instructions. The samples were observed under a fluorescent microscopy (BZ-Analyzer) (Keyence, Osaka, Japan). In our case, at least 100 non-overlapping interphase nuclei per core were scored by 2 independent observers (N.K. and J.S.). Patients were classified according to the previous report; [17] samples containing ≥15 nuclei that lacked both signal for P16 and contained at least 1 signal for chromosome 9 centromere were considered positive for homozygous deletion.
2.4. Methylation-specific PCR (MSP) assay Among 30 cases, frozen tumor tissues were available for 13 cases to extract genomic DNA. Genomic DNAs were isolated from frozen tumor tissues by SDS/proteinase K digestion (Life Technologies, Inc., Rockville, MD), phenol—chloroform extraction, and ethanol precipitation. The methylation status of P16 genes was determined by MSP assay as previously described [18]. Briefly, 1 g of genomic DNA was modified by sodium bisulfite, which converts all unmethylated cytosines to uracils while methylated cytosines remain unchanged. PCR amplification was done with sodium bisulfite-treated DNA as template as previously described, using specific primers for the methylated and unmethylated forms of P16. Primers sequences are as follows; methylated form of P16: 5 -TTA TTA GAC GGG TGG GGC GGA TCG C-3 (sense), 5 -GAC CCC GAA CCG CGA CCG TAA-3 (antisense), and unmethy-
N. Kobayashi et al. lated form of P16: 5 -TTA TTA GAG GGT GGG GTG GAT TGT-5 (sense), 5 -GAC CCC GAA CCG CGA CCG TAA-3 (antisense) [18].
2.5. Statistical analyses The differences of significance among categorized groups were compared using Chi-square test or Fisher’s exact test when appropriate. Overall survival was defined as the time of diagnosis to the time of death from any cause or to the date the patient was last known to be alive. The univariate analysis of overall survival was carried out by the Kaplan—Meier method using the log—rank test. All data were analyzed using StatView® 5.0 Program for Windows (SAS Institute Inc., Cary, NC). All statistical tests were two-sided and probability values <0.05 were defined as being statistically significant.
3. Results 3.1. Immunohistochemical staining for p16 protein Of the 30 specimens that were examined, six (20%) were p16-positive and 24 (80%) were p16-negative. Representative examples of the immunohistochemical staining patterns are shown in Fig. 1. Regarding the histological subtypes, the loss of p16 expression was identified in 12 of 16 (75.0%) epithelial-type specimens, 6 of 8 (75.0%) biphasic-type specimens, all 5 of 5 sarcomatous-type specimens, and 1 of 1 lymphohistiocytoid-type specimens (Table 1).
Fig. 1 FISH and immunohistochemical staining. (A) FISH for Patient #1, tumor cells showing homozygous deletion of P16 genes (spectrum orange) with chromosome 9 aneuploidy (spectrum green). Normal lymphocytes retained P16 genes (upside). (B) Immunohistochemical staining for Patient #1, tumor cells showing no expression of p16 protein. (C) FISH for Patient #2, tumor cells retaining P16 genes (spectrum orange) with chromosome 9 aneuploidy (spectrum green). (D) Immunohistochemical staining for Patient #2, tumor cells showing expression of p16 protein.
Frequent p16 inactivation in pleural mesothelioma Table 1
123
The status of P16 in Japanese malignant pleural mesothelioma
Subtype
No.
P16 gene
p16 Protein
HD
Retained
Negative
Positive
Epithelial type Biphasic type Sarcomatous type Lymphohistiocytoid
16 8 5 1
11 4 5 1
5 4 0 0
12 6 5 1
4 2 0 0
Total
30
21
9
24
6
HD, homozygous deletion.
Fig. 2
Representative examples of MSP assay. P16 U; unmethylated form of P16, P16 M, methylated form of P16.
3.2. Deletion and DNA methylation of P16 gene Among the 30 specimens that were examined using a FISH assay, 21 (70%) exhibited homozygous deletions of the P16 gene and 9 (30%) retained the P16 gene. Representative examples of the FISH assay results are shown in Fig. 1. Regarding the histological subtypes, the homozygous deletion of P16 was identified in 11 of 16 (68.8%) epithelial-type specimens, 4 of 8 (50.0%) biphasic-type specimens, 5 of 5 sarcomatous-type specimens, and 1 of 1 lymphohistiocytoid-type specimens (Table 1). While none of the specimens with P16 homozygous deletions exhibited p16-positive expression, these specimens that were shown by FISH to retain the P16 gene did not exhibit p16-positive expression. Because we suspected that aberrant methylation might be responsible for the loss of p16 expression in these 3 specimens, the methylation status of the P16 gene was examined in 13 specimens that had been stored at −80 ◦ C. Two of the 3 patients that retained the P16 gene but did not express p16 were included in among the 13 available specimens. Aberrant methylation was detected in 2 of the 13 specimens using an MSP assay (Fig. 2). The p16 protein was not immunohistochemically detected in these 2 specimens. These results suggest that either homozygous deletions or aberrant methylation can lead to p16 inactivation. We examined the relationship between asbestos, p16 expression and P16 homozygous deletion. Among 4 patients with no history of asbestos exposure, 2 patients revealed loss of p16 expression with P16 homozygous deletion. No significant difference in molecular status between patients with asbestos exposure and those with no exposure.
We examined the relationship between the p16 status and patient survival. Despite the small sample size of this study, the patients with a p16-negative status had a shorter survival time (MST, 8.4 months) than those with a p16-positive status (MST, 12.7 months) (Fig. 3). No significant differences in sur-
3.3. Patient prognosis The follow-up data were documented for all 30 patients. A Kaplan—Meier plot for all the patients is shown in Fig. 3. The overall median survival time (MST) was 9.6 months.
Fig. 3 Kaplan—Meier plots of overall survival after diagnosis of MPM. (A) Overall survival for all 30 patients. (B) Overall survival according to the p16 protein status.
124 vival were seen according to the P16 homozygous deletion or methylation statuses. No differences in the p16 expression status or the P16 homozygous deletion status were seen between patients with curative resections and those who did not undergo surgery (P = 0.63). No differences in the prognosis, p16 expression status, or P16 homozygous deletion statuses were seen between cases treated at two institutions.
4. Discussion The worldwide incidence of MPM is thought to be increasing, especially in Japan and non-Western counties where asbestos continued to be heavily used after its use had been discontinued in Western counties [1]. Thus, the urgent establishment of new preventive, diagnostic and treatment strategies for MPM is mandatory, drawing attention to the importance of understanding the molecular alterations in MPM. In fact, much less information regarding molecular biological alterations is available for MPM than for other solid neoplasms. In this study, we showed that the loss of p16 protein expression is common in MPM specimens from Japanese patients. Homozygous deletions were the major mechanism for p16 inactivation, but DNA methylation was also responsible for inactivation when the p16 allele was retained. This is the first study to show that the alteration of P16 is a major event in the pathogenesis of MPM in Japanese patients, as it is in Western countries. We did not examine the specific P16 mutations in this study. Among the 13 specimens in which p16 expression, P16 deletion and P16 methylation were completely examined, all patients with p16 negative expression showed either P16 homozygous deletion or aberrant P16 methylation, suggesting that these two mechanisms are critical for the loss of expression. Of note, P16 gene mutations are uncommon in MPMs [19,20]. In MSP assay for primary tumors, either the unmethylated band only or both methylated and unmethylated bands were present. Two major possibilities were responsible for this finding: (1) tumor tissues without microdissection consists of mixtures of tumor cells and non-malignant cells, therefore, the unmethylated band is derived from non-malignant cells; (2) there may be a heterogenesity of methylation status among two alleles in a cell or among each tumor cell. For this case, p16 expression can be preserved in tumor tissue. However, our result that methylated samples show that p16 expression did not support the latter possibility despite the small sample size. From a clinicopathological aspect, P16 homozygous deletions seemed to be more common in cases of sarcomatous-type (100%) than those of epithelial- or biphasic-type (62.5%), consistent with observations in Western countries [7,21]. In addition, although the number of patients in our study was not large, our results showed that lack of p16 expression was a significant negative prognostic factor among Japanese patients with MPM. Indeed, these findings have been reported in several studies conducted in Western countries [22—24]. Our results suggest the possibility of diagnostic and therapeutic applications focusing on P16 for Japanese MPMs. Illei and colleagues reported that the detection of homozygous deletions of the P16 gene in pleural effusions using a FISH
N. Kobayashi et al. assay was useful for distinguishing malignant mesothelial cells from reactive mesothelial cells and other malignant cells [25] indicating the usefulness of the P16 deletion as a diagnostic marker of MPM. Regarding therapeutic applications, cell cycle arrest was reportedly induced in MPM cells to which p16 expression constructs were introduced, suggesting the possibility of gene therapy utilizing P16 [26,27]. Regarding other molecular alterations in MPM, neurofibromatosis type 2 (NF2) has been shown to be an important target of gene mutation in MPMs in Western studies [28]. No information is available regarding NF2 mutations in Japanese patients with MPM other than that among 4 cell lines derived from Japanese patients, one carried a mutation [29]. It would be interesting to investigate the relationship between P16 and NF2 alterations from the viewpoint of the carcinogenesis of MPMs. Of note, mutations in other genes, including the P53, Ras, and RB genes (which have been found at high frequencies in other malignant tumors), are very rare in MPM [20,30,31]. In conclusion, our results showed that the loss of p16 expression is a common alteration that is related to a poor prognosis in Japanese patients with MPM. The homozygous deletion of P16 is a major mechanism for p16 protein depletion, although aberrant methylation is also involved in gene silencing.
Authors disclosures of potential conflicts of interest The authors indicated no financial relationship with companies whose products are mentioned in this article.
Acknowledgements This study was supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology, Japan and a Grant-in-Aid for Cancer Research from the Ministry of Health, Labor, and Welfare, Japan.
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