Abnormalities of Tumor Suppressor Gene P16 in Pancreatic Carcinoma: Immunohistochemical and Genetic ﬁndings Compared with Clinicopathological Parameters

J Gastroenterol 2003; 38:663–671 DOI 10.1007/s00535-003-1119-6

Abnormalities of tumor suppressor gene p16 in pancreatic carcinoma: immunohistochemical and genetic ﬁndings compared with clinicopathological parameters

Koushiro Ohtsubo, Hiroyuki Watanabe, Yasushi Yamaguchi, Yu-Xin Hu, Yoshiharu Motoo, Takashi Okai, and Norio Sawabu

Department of Internal Medicine and Medical Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan

Background. Abnormalities of the tumor suppressor pression of p16 protein and any of the clinicopathologi- gene p16 have been reported in a variety of human cal parameters. However, there was a tendency for the tumors but are rare in pancreatic carcinoma except for tumor to be larger in patients with decreased expression cancer cell lines and xenografts. Their clinicopathologi- of p16 protein than in those with normal expression cal significance remains unknown. The purpose of this levels. In contrast, the tumor was significantly larger and study was to examine immunohistochemical and genetic the survival period significantly shorter for patients alterations of p16 in primary pancreatic carcinoma with pancreatic carcinoma with p16 mutation or tissues and to investigate the relation between abnor- hypermethylation than for those with pancreatic carci- malities of p16 and clinicopathological parameters to noma with an intact p16 gene (P Ͻ 0.05). Conclusions. elucidate their clinicopathological significance. Meth- These findings suggest that p16 alterations may partici- ods. We investigated p16 expression in 60 pancreatic pate in the aggressiveness of pancreatic carcinoma. carcinoma cases by immunohistochemistry using a monoclonal antibody clone G175-405. In addition, we Key words: p16, pancreatic carcinoma, immunohis- analyzed genetic alterations of the p16 gene using DNA tochemical staining, mutation, hypermethylation extracted from microdissected tissue of pancreatic carcinoma, by polymerase chain reaction, nonradioisotopic single-strand conformation polymorphism (non-RI-SSCP), DNA sequencing, and hypermethy- Introduction lation analyses using restriction enzymes. We compared the abnormalities of p16 alterations with clinicopatho- The carcinogenesis and progression of pancreatic logical parameters to elucidate their significance. Re- carcinoma (PC) have been elucidated and various sults. On immunohistochemical study, staining for p16 carcinoma-related genes reported in PC. For example, a protein was strongly positive in 22 (37%) of 60 pancre- K-ras mutation at codon 12 has been found in 75%– atic carcinoma cases, weakly positive in 24 (40%), and 100%, and a p53 mutation has been recognized in negative in 14 (23%). In contrast, p16 mutations were 40%–76% of PC tissues.1–6 To date, we have reported recognized in 9 (15%) of the 60 pancreatic carcinoma mutations of K-ras and p53 in pure pancreatic juice and cases. The incidence of p16 mutations was 2 (9%) in 22 duodenal juice for application to clinical practice.7–13 cases of pancreatic carcinoma with strongly positive The cell cycle is regulated by cyclin and cyclin- staining, 4 (17%) in 24 with weakly positive staining, dependent kinase (CDK).14,15 CDKs in turn are nega- and 3 (21%) in 14 with negative staining. Hypermethy- tively regulated by cyclin-dependent kinase inhibitors lation of p16 was detected in the two pancreatic carci- (CDKIs).16,17 The p16 gene is the most important inhibi- noma cases with weakly positive staining, although tor of CDKIs. The p16 protein was identified in 1993 by homozygous deletions were not found in any case. Serrano et al.18 Soon afterward, MTS1 (multiple tumor There was no significant correlation between the ex- suppressor gene 1) was found to be a tumor suppressor gene, coding the p16 protein, in 9p21 by Kamb et al.19 and Nobori et al.20 Although a high incidence of p16 abnormalities has been reported in various human tu- Received: June 27, 2002 / Accepted: January 31, 2003 mors21–27 and in pancreatic cell lines and xenografts,1,28–30 Reprint requests to: N. Sawabu such abnormalities are relatively rare in primary PC 664 K. Ohtsubo et al.: p16 in pancreatic cancer tissues.31–34 In addition, there have been no reports of 5% of target cells were stained regardless of cytoplas- comparisons between the immunohistochemistry and mic staining. Surrounding stromal cells, such as lym- genetic alterations or between the abnormalities of phoid cells and acinar cells, were used as an internal p16 and clinicopathological parameters in primary PC control. Immunostaining as intense as the internal tissues. Therefore, the clinicopathological significance control was considered strongly positive. Less intense of p16 in PC remains unknown. immunostaining was regarded as weakly positive.38 A The purpose of this study was to examine immunohis- negative or weakly positive immunoreaction was con- tochemical and genetic alterations of p16 in primary PC sidered abnormal. tissues and to investigate the relation between abnormalities of p16 and clinicopathological parameters to DNA extraction elucidate their clinicopathological significance. Tumor sections were deparaffinized with xylene and ethanol and then microdissected to enrich for neoplastic Materials and methods tissues prior to DNA isolation. DNA was extracted with a phenol-chloroform mixture and precipitated with Pancreatic carcinoma tissues ethanol according to the method of Goelz et al.39 We studied 60 cases of PC resected at surgery (including two autopsy cases) and confirmed pathologically in Non-RI-SSCP analysis Kanazawa University Cancer Research Institute Hospi- For the nonradioisotopic single-strand conformation tal and affiliated hospitals from January 1987 to March polymorphism (non-RI-SSCP) analysis we used primers 2000. Informed consent was obtained from all patients of exons 1 and 2 of p16 according to Hussussian et al. (32 men, 28 women). Their ages ranged from 42 to 89 (exon 1)40 and Okajima et al. (exon 2).41 Exon 2 of p16 years (average 66.7 years). Tumor size was classified was divided into two fragments: 2A (5Ј fragment) and according to the criteria used by the Japan Pancreas 2B (3Ј fragment). The sequences of primers were as Society.35 A total of 7 PCs were classified as tumor size follows: exon 1 sense, 5Ј-GGGAGCAGCATGGAGC Յ Ͼ 1 (TS1) (greatest dimension 2cm), 31 as TS2 ( 2cm CG-3Ј; exon 1 antisense, 5Ј-AGTCGCCCGCCATCC Յ Ͼ Յ but 4cm greatest dimension), 13 as TS3 ( 4cm but CCT-3Ј; exon 2A sense, 5Ј-TCTCTCTGGCAGGTCA Ͼ 6cm greatest dimension), 6 as TS4 ( 6cm greatest di- TGAT-3Ј; exon 2A antisense, 5Ј-AGCACCACCAG mension), and 3 as unknown. Altogether, 40 tumors CGTGTCCAG-3Ј; exon 2B sense, 5Ј-TTCCTGGAC were from the head, 12 from the body, and 7 from the ACGCTGGTGGT-3Ј; exon 2B antisense, 5Ј-GATC tail of the pancreas; 1 was of unknown origin. The ATCAGTCCTCACCTGA-3Ј. The size of the poly- pathology of PC tissues was diagnosed as papillary merase chain reaction (PCR) products was 204bp for adenocarcinoma in 4 cases, well-differentiated adeno- exon 1, 132bp for exon 2A, and 206 bp for exon 2B. The carcinoma in 17 cases, moderately differentiated PCR mixture was composed of template DNA (100ng), adenocarcinoma in 26 cases, poorly differentiated ad- 10 ϫ PCR buffer 2µl (Perkin-Elmer Cetus, Foster, enocarcinoma in 10 cases, adenosquamous carcinoma in CA, USA), dNTPs 4pmol (Takara, Kyoto, Japan), 2 cases, and unknown in 1 case. The staging of the PC AmpliTaq DNA polymerase 1U (Perkin-Elmer Cetus), was done according to the TNM classification as set and primer mixture 10pmol for exon 2A. For exons 1 down by the International Union Against Cancer36; it and 2B, we used 2µl 2 ϫ GC buffer I (Takara) and 1U was judged to be stage I for 12 patients, stage II for 10 TaKaRa LA Taq (Takara) instead of 10 ϫ PCR buffer patients, stage III for 25 patients, stage IV for 12 pa- and AmpliTaq DNA polymerase, respectively. Then tients, and unknown in 1 case. Lymph node and distant water was added to a total volume of 20µl. PCR was metastases were identified in 24 and 10 of 60 PC cases, performed using TaKaRa MP MT3000 (Takara) with respectively. denaturing at 95°C for 1min, annealing at 62°C (exons 1 All PC tissues were fixed in formalin and embedded and 2A) or 60°C (exon 2B) for 1min, and extension at µ in paraffin. Sections were cut 3–4 m thick for immuno- 72°C for 1min, for 40 cycles, and a final extension at µ histochemistry and 8–10 m thick for genetic analysis of 72°C for 6min. PCR products were electrophoresed us- p16. ing 2% NuSieve 3:1 agarose gel (FMC BioProducts, Rockland, ME, USA) and stained with SyBr Green I (Nippon Gene, Tokyo, Japan). After sample denatur- Immunohistochemical staining of p16 ation with 95% formamide for 5min at 95°C, electro- Immunohistochemical staining of p16 was performed phoresis was performed at 4°C and 150V for 6–24h according to the method of Hu et al.37 Immunoreactivity (exon 1, 24h; exon 2A, 6–12h; exon 2B, 18h) using 15% was regarded as positive when the nuclei of more than homogeneous polyacrylamide gel with a buffer circulat- K. Ohtsubo et al.: p16 in pancreatic cancer 665 ing thermocontrol system (ATTO, Tokyo, Japan), fol- Clinicopathological findings lowed by staining with SyBr Green II (Nippon Gene). We classified 60 PC cases by tumor size, localization, Additional bands that shifted differently from the wild- histological grade, clinical stage, nodal metastasis, and type bands were judged abnormal.42 For imaging analy- distant metastasis. We then compared the expression of sis, densitograph systems and software (ATTO) were p16 protein as well as p16 genetic alterations with the used. The abnormal bands were cut from the gel, and clinicopathological parameters. Furthermore, we ana- DNA was extracted from them. The DNA was used as lyzed survival period in 38 patients with an intact p16 the template for the second PCR. Second PCR products gene and 10 patents with a p16 mutation or hyper- obtained under the same conditions were purified with methylation. In this analysis, we eliminated the cases in SUPPEC-02 (Takara) and used as the template for se- which information about prognosis was not obtained. quence analysis.

Sequencing analysis Statistical analyses 2 For the cycle sequence reaction, the PCR mixture was The test was used to compare data between two made with Big Dye Terminator Cycle Sequence Reac- subgroups of patients classified based on the results of tion agent 8µl (Perkin-Elmer Cetus), template DNA immunohistochemical and genetic abnormalities of p16. 100 ng, and primer mixture 10pmol. Water was then The prognosis of patients was evaluated by the Kaplan- added to a total volume of 20µl. PCR was performed Meier method. Uninformative cases were eliminated using TaKaRa MP MT3000 with denaturing at 96°C for from this analysis. In addition, multivariate analyses 30s, annealing at 50°C for 15s, and extension at 60°C for were performed using the Cox hazard model. Probabil- Ͻ 4min, for 25 cycles. Reaction products were purified ity values of 0.05 (two-tailed) were regarded as statis- with a Centrisep spin column (Perkin-Elmer Cetus), tically significant. and sequence analysis was performed with the ABI PRISM 310 Genetic Analyzer (PE Biosystems Japan, Chiba, Japan). Results

The intensity of the immunohistochemical staining for Hypermethylation analysis p16 protein was uniform in 57 cases. In the remaining 3 The hypermethylation status of exon 1 of p16 was inves- PC cases, we selected 1000 cells from the visual field and tigated according to the method of Gonzalez-Zulueta et determined the immunoreactivity. According to the cri- al.43 All the DNA (1µg) samples were individually di- teria, the expression of p16 protein was positive in 22 gested for 12 h with 10 units of methylation-sensitive (37%), weakly positive in 24 (40%), and negative in 14 (Sac II, Hpa II; Roche, Mannheim, Germany) and (23%) of the 60 PC cases (Fig. 1). methylation-insensitive (Msp I; Roche) enzyme. We We obtained PCR bands in exon 2A in all 60 cases. used undigested DNA as a positive control. The se- However, under the same PCR conditions, we observed quences of the primers were as follows44: sense, 5Ј- PCR bands in exons 1 and 2B in only about half of the GGGAGCAGCATGGAGCCG-3Ј; antisense, 5Ј-CTG cases. Therefore, we used 2 ϫ GC buffer I and TaKaRa GATCGGCCTCCGACCGTA-3Ј. The PCR mixture LA Taq to raise the amplification efficacy of the PCR was composed of template DNA (100ng), 10 ϫ PCR products. Under these conditions, we obtained PCR buffer (1 µl), dNTPs (2pmol), AmpliTaq DNA poly- bands in all but two cases in exon 1. merase (0.5U), and primer mixture (5pmol). Water was With non-RI-SSCP analysis, mutant bands were ob- then added to a total volume of 10µl. PCR was per- served in nine cases (exon 1 in two cases and exon 2 in formed using the TaKaRa MP MT3000 with denaturing seven cases). In case 1 (Table 1), mutant bands were at 95°C for 1min, annealing at 62°C for 1min, and ex- recognized in both exon 2A and exon 2B. Sequencing tension at 72°C for 1 min, with a final extension at 72°C analysis was performed for the mutant bands of the nine for 6min. The optimal cycle number was determined as cases (total of 10 samples). The results were as follows: that giving a detectable band of minimal intensity from seven cases of missense mutation, two cases of silent the undigested template DNA and no detectable signal mutation, and one case of frame shift mutation that from Msp I-digested DNA. PCR products were produced a stop codon (TGA) (Table 1, Fig. 2). Case 1 electophoresed using 2% NuSieve 3: 1 agarose gel and involved a missense mutation in exon 2A and a silent stained with SyBr Green I. If PCR bands were obtained mutation in exon 2B. In case 9, the same silent mutation after treatment with methylation-sensitive but not me- was detected in normal pancreatic tissue as well as in PC thylation-insensitive enzymes, the case was determined tissue. Therefore, we determined case 9 to be polymor- to be positive for hypermethylation. phic. No hot spot of mutation was recognized except for 666 K. Ohtsubo et al.: p16 in pancreatic cancer

A,B C

Fig. 1. Immunohistochemical staining of p16 protein in pancreatic cancer (PC). A Strongly positive. B Weakly positive. C Negative. Cancer cells are indicated by arrows. A–C ϫ400

Table 1. The p16 mutations identiﬁed in pancreatic carcinomas Patient no. Exon Codon DNA sequencing alterations

Negative immunohistochemistry 12A80GAG Æ GGG (Glu Æ Gly) 2B 93 GGG Æ GGA (Gly Æ Gly) 22A75CAC Æ CCC (His Æ Pro) 32B139 GCC Æ ACC (Ala Æ Thr) Weakly positive immunohistochemistry 4137 GGT Æ G T (1 bp deletion) 52A75CAC Æ CCC (His Æ Pro) 62A78GCC Æ GTC (Ala Æ Val) 72B107 GTG Æ GAG (Val Æ Glu) Strongly positive immunohistochemistry 813CCT Æ TCT (Pro Æ Ser) 92B106 GGG Æ GGA (Gly Æ Gly) Glu, glutamic acid; Gly, glycine; His, histidine; Pro, proline; Ala, alanine; Thr, threonine; Val, valine; Ser, serine; 2A, 5Ј fragment of exon 2 in p16; 2B, 3Ј fragment of exon 2 in p16

codon 75, in which a missense mutation [conversion tion between p16 expression and any clinicopathologi- from CAC (His) to CCC (Pro)] was observed in two cal parameter, although in the patients with decreased cases. expression of p16 protein the tumor tended to be larger For the hypermethylation analysis, we examined 35 than in those with normal expression levels (P ϭ 0.081). of the 60 cases (12 strongly positive, 16 weakly positive, In contrast, in the patients with a p16 mutation or 7 negative). In two cases with weakly positive immunos- hypermethylation, the tumor was significantly larger taining, we obtained a PCR band with or without diges- and the survival significantly shorter than for PC pa- tion by Sac II and Hpa II but no PCR band after tients with an intact p16 gene (tumor size, P ϭ 0.049; digestion by Map I. We considered these two cases posi- survival period, P ϭ 0.022), although there was no cor- tive for hypermethylation (Fig. 3). In neither case was a relation between a p16 mutation or hypermethylation mutation of p16 recognized by SSCP analysis. The other and tumor location, histological grade, clinical stage, or 33 cases were regarded as negative for hypermethyla- lymph node or distant metastasis (Table 2). With the tion because PCR bands were obtained only without a Kaplan-Meier analysis, we analyzed the survival period restriction enzyme. in 38 patients with an intact p16 gene and 10 patients Among 22 PC cases with normal immunohistochemi- with a p16 mutation or hypermethylation. Although cal staining of p16 protein, only 2 showed a genetic there was no significant correlation between p16 protein alteration. In contrast, among 38 cases with abnormal expression and prognosis, the survival period was p16 immunostaining, 9 showed genetic or epigenetic significantly shorter for PC patients with a p16 mutation alterations (7 cases of mutation, 2 cases of hyperme- or hypermethylation than for those with an intact p16 thylation). However, the difference between these two gene (P ϭ 0.028) (Fig. 4). groups was not significant (P ϭ 0.23). On the other hand, multivariate analysis revealed a We compared the relation between the p16 abnor- significant correlation between poor prognosis and ad- malities and various clinicopathological parameters. In vanced clinical staging (P ϭ 0.0048) or lymph node the univariate analysis, there was no significant correla- metastasis (P ϭ 0.019) but not tumor size, tumor loca- K. Ohtsubo et al.: p16 in pancreatic cancer 667

Fig. 2. A Nonradioisotopic single-strand conformation polymorphism (non-RI-SSCP) analyses of p16. B Sequence analyses of the mutant band shown in A. With non-RI-SSCP, mutant bands were found as extra bands (M arrowheads) compared with the wild type (W arrows). Sequence analyses showed a 1-bp deletion in exon 1 in case 4 and missense mutations in exon 2A in case 5 and exon 2B in case 3 (see Table 1)

Fig. 3. Hypermethylation analyses using restriction enzymes. Left In the control, hypermethylation analysis showed a polymerase chain reaction (PCR) band only in the undigested enzyme (arrow). Right In case 10, with weakly positive staining for p16, the hypermethylation analysis gave a positive result because the same PCR bands were obtained with or without the digestion by Sac II and Hpa II, whereas no PCR band was obtained after digestion by Msp I (arrow). U, undigested; S, Sac II; H, Hpa II; M, Msp I tion, histological grade, distant metastasis, or p16 p16 mutations in PC cell lines and xenografts by Caldas abnormalities. et al.28 described homozygous deletions and mutations at 44% and 38%, respectively. Other studies using PC cell lines and xenografts1,29,30 supported these results. However, genetic alterations are relatively rare in pri- Discussion mary PCs. Bartsch et al.31 and Huang et al.32 reported that only 12 (37%) of 32 cases and 8 (27%) of 30 cases Abnormalities of the p16 gene have been reported in a showed a homozygous deletion or somatic mutation in variety of human malignancies.21–27 The ﬁrst report of primary PC tissues, respectively. 668 K. Ohtsubo et al.: p16 in pancreatic cancer

Table 2. Correlation between clinicopathological parameters and alterations of the p16 gene in pancreatic carcinomas Parameters No. p16 alteration P

Tumor size TS1, TS2 38 4 (11%) 0.049 TS3, TS4 19 6 (32%) Tumor location Head 40 6 (15%) 0.300 Body and tail 19 5 (26%) Pathological findings Pap, well 22 3 (14%) 0.540 Mod, poor 35 7 (20%) Clinical stage I/II 22 2 (9%) 0.150 III/IV 37 9 (24%) Survival Fig. 4. Survival curve for patients with PCs having a normal (n Ͼ 6 months 25 2 (8%) 0.022 ϭ 38; gray line) or abnormal (n ϭ 10; black line) p16 gene (by Յ 6 months 23 8 (35%) the Kaplan-Meier method). The survival period tended to be LN metastasis shorter in PCs with genetic or epigenetic abnormalities of p16 Absent 14 1 (7%) 0.067 than in PCs with an intact p16 gene (P ϭ 0.028) Present 24 8 (33%) Distant metastasis Absent 35 8 (23%) 0.850 Present 10 2 (20%) Gonzalez-Zulueta et al.43 clearly showed that there pap, papillary adenocarcinoma; well, well-differentiated adenocarcinoma; mod, moderately differentiated adenocarcinoma; poor, poorly was a highly significant correlation between the loss of differentiated adenocarcinoma; LN, lymph node p16 expression and hypermethylation of its promoter in bladder cancer but not a significant association between methylation of exon 2 and transcriptional repression of In this study, we identified somatic mutations in 9 of p16, indicating that methylation of exon 1, but not exon 60 cases (15%). The frequency of mutation was higher 2, of p16 is associated with transcriptional silencing. in exon 2 than exon 1 (seven cases in exon 2 compared Also in the PC tumors, hypermethylation of the p16 with two for exon 1). This result is consistent with pre- promoter was found in 11 of 27 PC cases without p16 vious reports.21,26,31,32,45 In addition, a polymorphism was expression and not in any PC cases (n ϭ 13) with observed in exon 2. However, a homozygous deletion p16 expression.45 We observed hypermethylation of the was not recognized in any of the 60 cases, possibly p16 promoter in only 2 of 23 PC patients with abnormal because of contamination by normal cells. immunostaining of p16, but not at all in PC patients with Much attention has recently been paid to the normal immunostaining. Thus, it is presumed that the hypermethylation of a tumor suppressor gene as the p16 gene is inactivated by hypermethylation of its epigenetic mechanism of transcriptional silencing.46 promoter also in a considerable number of PC tumors, This phenomenon induced genetic inactivation and although the incidence of p16 hypermethylation was carcinogenesis.47 Hypermethylation of p16 has been low in our study using restriction enzymes. reported in various human tumors.33,34,38,42,48–54 In PC tis- Previously we reported that 26 (42%) of 62 PC tissues sues, Sugimoto et al.33 found that none of 24 cases lost p16 expression.37 In that report, we classified the showed hypermethylation using restriction enzymes. In degree of immunohistochemical staining as positive or contrast, hypermethylation could be identified in 5 negative. In the present study, we classified staining as (14%) of 36 PC tumors by Ueki et al.34 and in 11 (28%) strongly positive, weakly positive, or negative. We clas- of 40 PC tumors by Gerdes et al.45 using methylation- sified strongly positive immunostaining as normal and specific PCR (MSP). In the present study, we analyzed weakly positive or negative staining as abnormal. The the status of hypermethylation with restriction en- results of immunohistochemical staining of the p16 pro- zymes.44 We detected only 2 (6%) of 35 PC tissues with tein were 22 (37%) strongly positive cases, 24 (40%) weakly positive immunostaining. MSP is more sensitive weakly positive cases, and 14 (23%) negative cases for detecting hypermethylation than the analysis with among the 60 PCs. We determined that 38 (63%) of 60 restriction enzymes. Hypermethylation of a single cell PC patients had abnormal expression of p16 protein. could be detected among 1000 normal cells by MSP.48 We detected 9 (24%) of 38 PC patients who harbored Given the MSP is available in this study, the incidence a mutation or hypermethylation with abnormal expres- of hypermethylation in PC cases may be found to be sion of p16 protein; there were only 2 (9%) of 22 PCs increased. with normal expression. Genetic alterations of p16 K. Ohtsubo et al.: p16 in pancreatic cancer 669 tended to be detected more in patients with immuno- patients with an intact p16 gene. The Kaplan-Meier histochemically p16-negative than p16-positive tumors. analysis showed that for PC patients with a mutation or Gerdes et al.45 reported that p16 alterations (muta- hypermethylation of p16 there was a tendency for a tion or hypermethylation) were recognized in 10 of 20 poor prognosis (P ϭ 0.028). Our results are considered (50%) short-term survivors (median survival 6 months) to be in line with the reports in the literature.45,56,57 and 6 of 20 (30%) long-term survivors (median survival The K-ras mutation is thought to occur during the 36 months), indicating that the presence of p16 alter- early stage of pancreatic carcinogenesis because it is ations in PC patients is associated with a worse progno- observed in mucous cell hyperplasia, which is supposed sis. Furthermore, they identified somatic mutations of to be a precancerous lesion.58 On the other hand, p53 p16 in 6 (15%) cases and hypermethylation in 11 (28%) mutation is thought to occur during the late stages.59,60 of 40 PC tumors, respectively. The frequency of muta- Wilentz et al.61 proposed a progression model for PC, tions found by them is identical with that of the present where K-ras is mutated first, followed by p16, and finally study (15%, 9/60 cases). However, the frequency of p16 p53 and DPC4. However, in our study p16 abnor- hypermethylation in the former is higher than in the malities were associated with a large tumor and short latter (28% vs 6%). This difference is thought to be survival period and were thought to occur late in pan- due to the lower sensitivity of the restriction enzyme creatic carcinogenesis. In this regard, our results suggest method we used for the analysis of p16 hyper- that p16 may participate in the late events of pancreatic methylation. We did not observe any mutations or carcinogenesis. hypermethylation in the remaining 29 cases with abnor- In summary, we have reported the analysis of K-ras mal p16 expression, which might be due to undetected and p53 mutations in pancreatic juice for application to mutations or hypermethylation on the basis of limita- daily practice.7–13 We also investigated mutations of tions of PCR-SSCP for mutation analysis and the re- p16 in pancreatic juice from several patients with PC. striction enzyme method for hypermethylation analysis. Among them, one patient showed a nonsense mutation More likely, the loss of p16 expression in these PC (GAG (Gly)ÆTAG (stop codon)) in exon 2B (data not tumors could be caused by homozygous deletion, which shown). From now on, we will evaluate the abnormali- could not be found chiefly due to insufficient elimina- ties of p16 in the pancreatic juice from many PC cases. tion of normal cells. Gerdes et al.45 also reported that The p16 abnormalities in pancreatic juice may become a mutation or hypermethylation was not recognized in marker for a poor prognosis in PC patients. any PC patients with positive immunostaining. How- ever, one case with normal expression of p16 protein Acknowledgments. We thank Dr. Masahiro Tsutsumi (De- showed a missense mutation in the present study. partment of Oncological Pathology, Cancer Center, Nara Medical University) for constant cooperation; Drs. Fujitsugu It is known from the literature that the genetic or Matsubara (Department of Pathology, Tatsunokuchi Hoju epigenetic alterations of oncogenes are not always re- Memorial Hospital) and Yasuyuki Asada (Department of flected in the abnormal expression of oncogene protein Surgery, Fukui Saiseikai Hospital) for donating tumor in cancer tissues.55 We speculated that the reasons samples; and Drs. Toshinari Minamoto (Division of Diag- for this are as follows: First, the genetic or epigenetic nostic Molecular Oncology, Cancer Research Institute, Kanazawa University) and Atsuhiro Kawashima (Depart- variation may exist only in a part of the PC tumor; ment of First Pathology, Kanazawa University) for pathologi- consequently, the expression of p16 protein does cal diagnoses. not decrease. Second, the conformational changes due to p16 mutation may not have influenced the epitope of the p16 antigen for the p16 antibody. References In neuroblastoma56 and lung adenocarcinoma,57 the expression of p16 is related to advanced clinical staging 1. Rozenblum E, Schutte M, Goggins M, Hahn SA, Panzer S, and a poor prognosis. Recently, Gerdes et al.45 reported Zahurak M, et al. Tumor-suppressive pathways in pancreatic that the presence of p16 alterations in resected tumors carcinoma. Cancer Res 1997;57:1731–4. 2. 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