Opposite Roles of Human Pancreatitis-Associated Protein and REG1A

Imaging, Diagnosis, Prognosis

Opposite Roles of Human Pancreatitis-Associated Protein and REG1A Expression in Hepatocellular Carcinoma: Association of Pancreatitis- Associated Protein Expression with Low-Stage Hepatocellular Carcinoma, B-Catenin Mutation, and Favorable Prognosis Ray-HwangYuan,1,4 Yung-Ming Jeng, 2,5 Hui-Ling Chen,3 Fon-Jou Hsieh,6 Ching-YaoYang,1 Po-Huang Lee,1,4 and Hey-Chi Hsu2,5

Abstract Purpose: Pancreatitis-associated protein (PAP) and regenerating protein 1 A (Reg1A) are up- regulated during the pancreas regeneration. This study is to investigate the clinicopathologic de- notation of their expression in hepatocellular carcinoma (HCC). Experimental Design: PAP and REG1A mRNA levels were measured in 265 surgically removed unifocal primary HCCs using reverse transcription-PCR. Results: PAP and REG1AmRNAs were detected in 97 (36.6%) and 55 (20.8%) HCCs, respectively, including 46 with coexpression but in none of the 219 nontumorous livers. HCCs with PAP expression correlated with low-stage tumors without evidence of vascular invasion (P =0.013) butthe REG1A expression did not.By a combination analysis, HCCs withPAP expression alone showed the lowest frequency of p53 mutation (P < 0.036), the highest rates of grade 1and low- stage tumors (P < 0.007 and P < 0.001, respectively), less frequent early tumor recurrence (P = 0.051), and hence a better 5-year survival (P = 0.044) than groups expressing PAP and REG1A, REG1A alone, and neither PAP or REG1A. Besides, PAP expressing HCCs had significantly frequent h-catenin mutation, regardless of REG1Aexpression, P < 0.00001. In the subset of HCCs that has no mutations of p53 and h-catenin but showed PAP expression, coexpression of REG1A and PAP was associated with more frequent vascular invasion than PAP expression alone (P < 0.005). Conclusions: These data suggest that PAP expression designate a subset of low-grade, low- stage HCC with frequent h-catenin mutation and hence more favorable prognosis, whereas further genetic or epigenetic alterations, such as p53 mutation and REG1Aexpression, lead to more advanced HCCs.

Hepatocellular carcinoma (HCC) is one of the most frequent oncogenes, inactivation of tumor suppressor genes, and over- malignancies in south China, Taiwan, southeastern Asia, and expression of growth factors, in the neoplastic transformation sub-Saharan Africa. HCCs are genetically heterogeneous neo- of liver cells (3–5). plasm and the genetic heterogeneity correlates with the variety Regenerating gene (Reg), which is expressed in regenerating of etiologic factors (1, 2). However, the molecular mechanisms pancreatic islet, was first identified in the screening of regenerat- remain largely unknown. Recent studies have unraveled many ing islet-derived cDNA library taken from 90% depancreatized aberrant gene expressions, including activation of cellular rat (6). Reg and Reg-related genes constitute a family within the superfamily of calcium-dependent lectin (C-type lectin; ref. 7). The C-type lectins are involved in several complex events, such as Authors’ Affiliations: Departments of 1Surgery,2Pathology, and 3Hepatitis human malignancy and other diseases (8, 9). The Reg gene 4 Research Center, National Taiwan University Hospital;, Departments of Surgery, family consists of a group of acute phase reactants, lectins, 5Graduate Institute of Pathology, and 6Obstetrics and Gynecology, College of Medicine, NationalTaiwan University, Taipei, Taiwan antiapoptotic factors, or growth factors for pancreatic islet cells, Received 5/10/04; revised 12/21/04; accepted 10/1/05. neural cells, and epithelial cells in the digestive system (10, 11). Grant support: National Health Research Institute, Department of Health of the Until now, 17 members of the Reg family have been identified Republic of China, Taiwan grantNHRI-GT-EX89B901L (H-C. Hsu) and National and classified into four classes (Reg I-IV; ref. 12). Science Council, Republic of China, Taiwan grantNSC-92-2320-B-002-141 (H-C. Hsu). Reg I protein has many other synonyms, such as pancreatic The costs of publication of this article were defrayed in part by the payment of page thread protein, pancreatic stone protein, pancreatic stone charges. This article must therefore be hereby marked advertisement in accordance protein, secretory, islet cells regeneration factor, etc. (13). with 18 U.S.C. Section 1734 solely to indicate this fact. There are two members of human REG I gene, REG1A and Requests for reprints: Po-Huang Lee, Department of Surgery, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan. Phone: REG1B. The REGIA gene encodes a 166-amino-acid protein 886-2-23123456; Fax: 886-2-23934358; E-mail: [email protected]. with a 22-amino-acid signal sequence. The REG1A protein is F 2005 American Association for Cancer Research. identical to the pancreatic thread protein, pancreatic stone

Clin Cancer Res 2005;11(7) April 1, 20 05 2568 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Opposite Roles of PAP and REG1A in HCC protein, or protein X, and is highly represented in human transhepatic arterial embolization or chemotherapy before surgery. pancreatic secretion (14). The REG1B gene codes for a To study the tissue distribution, we also examined the mRNA transcript with 87% homology to the REG1A transcript, but expression of PAP and REG1A in multiple adult tissues and multiple the Reg1B protein has never been characterized and its fetal tissues obtained from autopsy. Histologic study and tumor staging. Tumor grade was divided into expression in the pancreas remains questionable (15). three groups, well differentiated (grade 1, 61 cases), moderately The secretory Reg I protein is synthesized in the regenerating h differentiated (grade 2, 102 cases), and poorly differentiated (grade 3 cells (16), and through the Reg I receptor (17), stimulates the and 4, 102 cases). HCC tends to spread in the liver via the portal vein h proliferation of pancreatic cells, leading to an increase in the invasion even in advanced stage. At the time of operation, no evidence of h cells mass in 90% depancreatized rats and nonobese diabetic regional lymph node or distant metastasis was noted, and minute HCC mice and hence amelioration of experimental diabetes (18, 19). (V2 cm) has excellent prognosis. HCC with complete fibrous Pancreatitis-associated protein (PAP), a member of the Reg encapsulation has a favorable four-survival, and vascular invasion, the III family, is also called HIP (hepatocarcinoma-intestine- most crucial step of intrahepatic tumor metastasis, is a crucial prognostic pancreas), PAP1 (islet neogenesis associated protein INGAP), factor for HCC (34). Therefore, a modified tumor staging with special PAP-H (pancreatic h cell growth factor, human pancreatitis- emphasis on the extent of vascular invasion, tumor size (V2 or >2 cm), associated protein), and REG-III. The human PAP cDNA and encapsulation was adopted (35, 36). This modified staging encodes a 175-amino-acid protein with 49% identity with the correlated with survival (P < 0.0001) in 781 cases of unifocal surgical HCCs analyzed (37). Stage I to II HCCs had no vascular invasion, human Reg protein (7). PAP protein is merely detectable in whereas stage III to IV HCCs had various extent of vascular invasion. normal pancreas but remarkably increased representing up to Stage I HCC (six cases) included completely encapsulated minute HCC 5% of secreted protein in acute pancreatitis and in some V2 cm with no liver invasion. Stage II HCC (115 cases) included minute chronic pancreatitis (20). HCC with liver invasion and/or microscopic satellite close to the main In human, the REG1A, REG1B, RS (REG-related sequence), tumor; or larger HCC without or with liver invasion and/or minute and PAP genes are clustered tandemly in a 95-kb region on satellite close to the main tumor. Stage IIIA HCC (43 cases) had invasion chromosome 2p12. This gene cluster may have arisen from the of thin-walled vessels in the tumor capsule but no portal vain invasion same ancestral gene by gene duplication (21). The REG I mRNA or satellite deep in the liver parenchyma. Stage IIIB HCC (33 cases) had was detected predominantly in the pancreas and at lower levels invasion of small portal vein in portal tract near the main tumor but no in gastric mucosa and kidneys but not in many other tissues invasion of major portal vein branch and satellite deep in the liver (22, 23). PAP mRNA is expressed in the normal small intestine, parenchyma. Stage IV HCC (68 cases) had invasion of major portal vein branches, satellites extending deeply into the surrounding liver, tumor pancreas, and pituitary gland but not in other tissues (7). REG I rupture, or invasion of the adjacent organs. mRNA was detected in gastric cancer (24, 25), colorectal cancer The intrahepatic tumor recurrence was based on imaging diagnosis (26), and cholangiocarcinoma (27). PAP mRNA can be detected with ultrasonography and/or computed tomography, supplemented in gastric cancer (28), colorectal cancer (26), pancreatic cancer with elevated serum A-fetoprotein. Among the 265 patients studied, (28, 29), and in about a quarter of primary human HCC (7, 30). 236 (89.1%) were eligible for the evaluation of early intrahepatic tumor A coordinate increase in pancreatic stone protein (Reg I) and PAP recurrence (V1 year). Twenty-nine patients who died within 1 year after (Reg III) was found in experimental acute pancreatitis (31, 32), resection and had no information or were negative for intrahepatic and in human colorectal cancer (26). In spite of these, the tumor recurrence were excluded from the evaluation of early recurrence. clinicopathologic role of REG1A and PAP expression and their Reverse transcription-PCR. Reverse transcription-PCR was used to interaction in HCC is not clear. In the present study, we show determine the expression of PAP and REG1A in 265 samples of HCCs and 219 corresponding nontumorous liver parenchyma, as was that PAP expression is associated with a subset of HCCs that is described elsewhere (38). S26 ribosomal protein mRNA, a housekeep- often low-grade, low-stage tumor and shows high frequency of ing gene, was used as the internal control (39). Two micrograms RNA h -catenin mutation, whereas a coexpression of REG1A leads to was transcribed to c-DNA in 20 AL reaction containing 0.5 Ag random more advanced disease and poor prognosis. hexamer. Two microliters synthesis buffer (10Â), 1 AL of 10 mmol/L deoxynucleotide triphosphate mix, 2 AL of 0.1 mol/LDTT, and 10 units Materials and Methods Moloney murine leukemia virus reverse transcriptase (EPICENTRE, Madison, WI) at 37jC for 60 minutes. Two microliters reverse Tissue samples. From January 1983 to December 1997, 1,033 transcription product, 1.25 units Pro Taq polymerase (Protech surgically resected primary and 188 recurrent HCCs were pathologically Technology Enterprise, Taipei, Taiwan), 1Â Pro Taq buffer, and 200 assessed at the National Taiwan University Hospital. The tissues were Amol/L (each) dATP, dCTP, dGTP, and dTTP were mixed with primer immediately cut into small pieces, snap frozen in liquid nitrogen, and pairs for target gene and internal control gene in a total volume of 30 stored in deep freezers. 80% of these cases had been followed for >8 AL. PCR was done in an automatic DNA thermal cycler 480 (Perkin- years or until death. Multifocal HCCs were excluded from this Elmer Co., Wellesley, MA), with initial heating at 94jC for 2 minutes correlation because of incomplete sampling of the tumor nodules for followed by 29 cycles of 94jC for 30 seconds, 60jC for 1 minute, 72jC genetic analysis and their variation in pathologic features. Of these, 265 for 1 minute, and final 72jC for 10 minutes for PAP. S26 primers were patients who already had mRNA samples taken from resected primary added at cycle 8. For REG1A, initial heating at 94jC for 2 minutes was HCC were selected for this study. The diagnosis of unifocal HCCs followed by 28 cycles of 94jC for 30 seconds, 58jC for 1 minute, 72jC consisting of multiple nodules was made by morphology and verified for 1 minute, and final 72jC for 10 minutes. S26 primers were added at by HBV integration patterns, A-fetoprotein mRNA expression, and cycle 7. The primers used are summarized in Table 1. PCR was stopped mutation pattern of h-catenin and p53 genes, as described previously at the exponential phase of the amplified genes, 29 cycles for PAP, 28 (33, 34). The 265 patients included 209 males and 56 females with a cycles for REG1A, and 22 cycles for S26. After PCR, 5 AL of the reaction mean age of 55.6 years (range, 14-88 years). Serum hepatitis B surface product were electrophoresed on a 2% agarose gel. antigen (HBsAg) was detected in 187 cases and anti-HCV antibody in Analysis of p53 and BB-catenin mutations. Mutations of the p53 79 cases, including 26 cases positive for both. All of the patients had tumor suppressor gene and h-catenin gene were detected by direct adequate liver function reserve at the time of surgery and all of the sequencing of exons 2 to 11 of p53 (40) and exon 3 of h-catenin (33). tumors were surgically resectable. None of these patients had received Cases with incomplete study were excluded from statistical analysis.

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unifocal primary HCCs. PAP and REG1A were overexpressed in 97 Ta b l e 1. Sense and antisense primers (36.6%) and 55 (20.8%) tumors, respectively. Among them, 46 tumors expressed both PAP and REG1A, with a high concordance Size of rate (77.4%, P < 0.00001), whereas only nine HCCs expressed Primer amplified REG1A alone. Both genes were not detectable in 219 nontumorous sequences DNA (bp) liver tissues (Fig. 1). PAP-F 5V-CCAAAATCCTGGACAGATGC-3V 200 Clinicopathologic correlation and relation to p53 and PAP-R 5V-CTACTCCACTCCCAACCTTC-3V b-catenin mutations in hepatocellular carcinoma. As shown REG1A-F 5V-CATGAATTCGGGCAACCTGG-3V 200 in Table 2, PAP overexpression in HCC showed a positive REG1A-R 5V-TTAACACTGCTTGGGGCTGG-3V correlation with low A-fetoprotein level (V200 ng/mL), and S26F 5V-CCGTGCCTCCAAGATGACAAAG-3V 300 low-stage (stages I-II) HCCs that had no vascular invasion, S26R 5V-GTTCGGTCCTTGCGGGCTTCAC-3V P = 0.039 and P = 0.013, respectively, whereas REG1A overexpression did not. The expression of PAP or REG1A did not correlate with age, gender, chronic hepatitis infection, Follow-up observation. During the follow-up period up to 175 tumor size, tumor grade, or early tumor recurrence. months, 213 patients (80.4%) had been followed for >10 years or until Mutations of p53 and h-catenin are the two major genetic death. At the end of follow-up, 64 patients remained alive, 18 of who alterations in HCCs. In this series, p53 mutation was found in had survived for >10 years. 99 of 221 cases (44.8%), whereas h-catenin mutation was Statistical analysis. The analyses are carried out using the Statistica detected in 37 of 248 tumors (14.9%). We found that PAP and for the Windows software (Statsoft, Inc., Chicago, IL). Two-tailed mm2 REG1A expression showed strong association with h-catenin and Fisher exact tests were used for univariate analysis. The cumulative mutation (P < 0.00001 and P = 0.00005, respectively) but not survival after tumor removal was calculated with log-rank test. Ps < 0.05 are considered statistically significant. with p53 mutation (Table 3). Opposite effects of pancreatitis-associated protein and REG1A expression in the tumor progression of hepatocellular carcinoma. Results Because of the frequent coexpression of PAP and REG1A, we did a combination analysis to further characterize the effects Expression of pancreatitis-associated protein and REG1A in of PAP and REG1A expression in the tumor progression of hepatocellular carcinoma and liver. We used reverse transcription- HCC. We divided these cases into four groups according to PCR for large-scale analysis of PAP and REG1A mRNA levels in 265 presence or absence of PAP and REG1A overexpression:

Fig. 1. PAPandREG1AmRNAexpressions in paired HCC (T) and nontumorous liver parenchyma (N). Reverse transcription-PCR measurementin theexponential phase showed PAP overexpression in four of six hepatocellular carcinoma specimens (A)andREG1A overexpression in one of six HCC specimens (B). Neither PAP nor REG1A was expressed in the nontumorous liver samples.

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Ta b l e 2 . PAP and REG1Aexpression in relation to clinicopathologic features of 265 unifocal HCCs

PAP present REG1Apresent Factor Total n (%) Odds ratio Pn(%) Odds ratio P Age V55 114 36 (31.6) 0.40-1.17 NS 20 (17.5) 0.36-1.36 NS >55 151 61 (40.4) 35 (23.2) Gender Male 209 76 (36.4) 0.50-1.83 NS 46 (22.0) 0.64-3.50 NS Female 56 21 (37.5) 9 (16.1) HbsAg Positive 187 67 (35.8) 0.50-1.60 NS 39 (20.9) 0.51-2.07 NS Negative 78 30 (38.5) 16 (20.5) Anti-HCV Positive 79 35 (44.3) 0.91-2.93 NS 19 (24.1) 0.72-2.92 NS Negative 168 55 (32.7) 30 (17.9) A-Fetoprotein (ng/mL) V200 139 59 (42.4) 1.00-2.97 0.039 31 (22.3) 0.64-2.31 NS >200 126 38 (30.2) 24 (19.0) Tu m o r s i z e (c m) V2 21 7 (33.3) 0.30-2.37 NS 1 (4.8) 0.01-1.28 NS >2 244 90 (36.9) 54 (22.1) Tu m o r g r a d e 1 61 28 (45.9) 0.89-3.09 NS 9 (14.8) 0.25-1.37 NS 2-4 204 69 (33.8) 46 (22.5) Tu m o r s ta g e I-II 121 54 (44.6) 1.11-3.24 0.013 23(19.0) 0.43-1.56 NS IIIA-IV 144 43 (29.9) 32 (22.2) Early recurrence Yes 109 36 (33.0) 0.49-1.54 NS 25 (22.9) 0.88-3.71 NS No 127 46 (36.2) 18 (14.2)

Abbreviation: NS, not significant.

PAP(+)/REG1A(+), PAP(+)/REG1A(À), PAP(À)/REG1A(+), difference in age, gender, A-fetoprotein level, and chro- and PAP(À)/REG1A(À). Among the four groups, HCCs nic hepatitis infection between these four groups (data not expressing PAP alone were associated with the highest shown). frequencies of low-grade (grade 1) and low-stage tumors In the two groups of HCCs with PAP expression, HCCs (P < 0.007 and P < 0.001, respectively) and hence the with coexpression of REG1A showed more frequent high-grade lowest early tumor recurrence (P = 0.051; Table 4). Consistent (84.8% versus 58.8%, P = 0.005), high-stage tumors (60.9% with results shown in Table 2, there was also no significant versus 29.4%, P = 0.002) and hence high-early tumor

Ta b l e 3 . PAPandREG1AmRNAexpressioninrelationtop53and-cateninh mutations in patients with resected unifocal primary HCC

PAP present REG1Apresent Factor Total n (%) Odds ratio Pn(%) Odds ratio P h-Catenin mutation Yes 37 28 (75.7) 3.29-19.10 <0.00001 17 (45.9) 1.91-9.56 0.00005 No 211 60 (28.4) 35 (16.6) p53 mutation Yes 99 29 (29.3) 0.34-1.13 NS 23 (23.2) 0.62-2.417 NS No 122 49 (40.2) 24 (19.7)

Abbreviation: NS, not significant.

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Ta b l e 4 . Combination of PAP and REG1A expression status in relation to tumor progression and early recurrence in 265 patients with resected unifocal primary HCC

PAP/REG1A overexpression Factor (À)///(À), n = 159 (%) (+)///(À), n =51(%) (À)///(+), n = 9 (%) (+)//(+), n =46(%) P A-Fetoprotein (ng/mL) >200 84 (52.8) 18 (35.3) 4 (44.4) 20 (43.5) 0.158 Tu m o r s i z e (c m) >2 146 (91.8) 44 (79.5) 8 (88.9) 46 (100) 0.092 Tu m o r g r a d e 1 31 (19.5) 21 (41.2)* 2 (22.2) 7 (15.2)* <0.007 2-4 128(81.5) 30 (58.8) 7 (77.8) 39 (84.8) Tu m o r s ta g e I-II 62 (39.0) 36 (70.6)c 5 (55.6) 18 (39.1)c <0.001 IIIA-IV 97 (61.0) 15 (29.4) 4 (4 4.4) 28 (60.9) Early recurrence Yes 69 (47.6) 15 (31.3)b 4(44.4) 21(61.8)b 0.051 No 76 (52.4) 33 (68.7) 5 (55.6) 13(38.2)

Abbreviations: Presence (+) and absence (À). *P = 0.005. cP = 0.002. bP =0.006.

recurrence rate (61.8% versus 31.3%, P = 0.006) compared Discussion with HCCs showing PAP expression alone (Table 4). To elucidate the reasons for the more aggressive tumors and PAP and regenerating protein 1 A (Reg1A) are reported to be early tumor recurrence in PAP(+)/REG1A(+) HCCs than in expressed in various types of human cancer, such as gastric PAP(+)/REG1A(À) HCCs, we further analyzed the role of p53 cancers (24, 25), colorectal cancers (22, 26), pancreatic cancers and h-catenin mutations. As shown in Table 5, the former (29, 41), cholangiocarcinoma (27), and hepatocellular cancers group showed significantly higher frequency of p53 mutation (7, 30) but not in normal adult tissues, such as liver (7) and (46.2% versus 25.6%, P = 0.036), whereas the two groups did pancreas (29, 41). PAP can increase DNA synthesis and acts as a not differ significantly in h-catenin mutation. growth factor in vivo to enhance liver regeneration (42). PAP is Then, we further analyzed the role of p53 and h-catenin induced by tumor necrosis factor A and cells overexpressing mutations in PAP(+)/REG1A(+) and PAP(+)/REG1A(À) HCCs. PAP show significantly less apoptosis on exposure to tumor As shown in Table 6, HCCs coexpressed PAP and REG1A necrosis factor A (43). Therefore, PAP seems to have dual tended to have more frequent high-stage tumors but not of mitogenic and antiapoptotic functions. REG1A is also shown to statistical significance, and the majority of HCCs with h-catenin act as a factor to reduce epithelial apoptosis in inflammation mutation had low-stage tumors, regardless of the presence or absence of REG1A. Correlation of pancreatitis-associated protein and REG1A Ta b l e 5 . Mutations of p53 and h-catenin in relation to expression with tumor progression in hepatocellular carcinomas combination of PAP and REG1A expression status in without mutations of p53 and bb-catenin. Despite the frequent p53 and h-catenin mutations, approximately half of HCCs are patients with resected unifocal primary HCC negative for both mutations. We then analyzed potential role of PAP and REG1A expression in this subset of HCC. In the subset PAP/REG1A overexpression, n (%) of HCCs expressing PAP, the coexpression of REG1A had Factor (À)//(À)(À)//(+) (+)//(À)(+)//(+) P adverse effect in the groups of HCCs with PAP expression. As h-Catenin mutation shown in Table 6, HCCs with coexpression of REG1A and PAP Yes 9 (6.0) 0 (0) 11(24.4)* 17(39.5)* <0.0001 had >3-fold high-stage tumor than those with PAP expression No 142 (94.0) 9 (100) 34(75.6) 26(60.5) alone (P < 0.005). p53 mutation Prognostic significance of pancreatitis-associated protein and Yes 66(48.9) 4(50.0) 10(25.6)c 19 (4 6.2)c 0.071 REG1A expression. To elucidate the roles of PAP and REG1A No 69 (51.1) 4 (50.0) 29 (74.4) 20 (53.8) in HCC, we analyzed the survival rates of the four different groups of HCCs. According to the expression pattern of PAP Abbreviations: Presence (+) and absence (À). and REG1A, HCCs with PAP expression alone had the best *P = notsignificant. 5-year survival (P = 0.044) significantly better than HCC with cP =0.036. coexpression of PAP and REG1A (P < 0.0002; Fig. 2).

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frequent early tumor recurrence, and the best 5-year survival Ta b l e 6 . Significance of REG1A coexpression in rela- compared with the other three groups (P = 0.051 and P = 0.044, tion to tumor stage in HCCs presence or absence of respectively). These data suggest that PAP expression is p53 and h-catenin mutations associated with a subset of less aggressive HCC with favorable prognosis, whereas REG1A is associated with more advanced PAP/REG1A expression disease and may contribute to tumor progression of HCC. This (+)///(À), % (+)///(+), % P suggestion is further supported by the finding that HCCs with coexpression of PAP and REG1A exhibited more frequent high- h -Catenin mutation (+)* grade and high-stage tumors and more frequent early tumor Tu m o r s ta g e recurrence than HCCs expression PAP alone (P = 0.005, I-II 10 (90.9) 13 (76.5) NS P = 0.002, and P = 0.006, respectively). This result is consistent IIIA-IV 1 (9.1) 4 (23.5) c with the observation that Reg I alone or coexpression of Reg I p53 mutation (+) with PAP have a significantly worse survival in early-stage Tu m o r s ta g e colorectal cancer after curative surgery (46). I-II 5 (50.0) 6 (31.6) NS The p53 and h-catenin are two most frequently mutated genes IIIA-IV 5(50.0) 13(68.4) in HCC (33, 40). HCCs have been classified into two major h -Catenin (À) and p53 mutation (À) groups: one is characterized by chromosome stability and h- Tu m o r s ta g e catenin mutation; the other is characterized by chromosomal I-II 16 (80.0) 4 (26.7) <0.005 instability and p53 mutation (47). Inactivation of p53 results in IIIA-IV 4 (20.0) 11(73.3) centrosome hyperamplification, leading to aberrant mitosis and chromosomal instability (48). We have shown that p53 NOTE: Presence (+) and absence (À). mutation is associated with more aggressive HCC (49, 50), Abbreviations: NS, not significant. whereas h-catenin mutation is associated with low-grade, low- *Includes cases with p53 mutation. cIncludes cases with h-catenin mutation. stage HCC, and better 5-year survival, and may possess tumor metastasis suppression activity (33). In this study, we showed that PAP and REG1A expression were closely associated with more frequent h-catenin mutation in HCCs (P < 0.00001 and (44). These observations indicate that PAP and REG1A play P = 0.00005, respectively) but not with p53 mutation. Indeed, important roles in the tumorigenesis of various types of human majority of HCCs expressing PAP alone, which had frequent cancer, including HCC. Their clinicopathologic significance in h-catenin mutation, had low tumor stage (70.6%) and better HCCs remains largely unknown because of the small number prognosis (P = 0.044). However, HCCs coexpressing PAP and of cases analyzed. In this study of 265 surgically resected REG1A, which also had high frequent h-catenin mutation, had unifocal primary HCCs, PAP was expressed in 97 HCCs more frequent p53 mutation, high-stage tumors, and poor (36.6%) and REG1A in 55 (20.8%) but not in any of the 219 prognosis, suggesting that the REG1A expression abolishes the nontumorous livers. Furthermore, the interrelation between beneficial effects of PAP. Further analysis showed that the PAP and REG1A expressions and their significance in the tumor majority of HCCs with h-catenin mutation had low-stage progression are not clear. tumors, regardless of the presence or absence of REG1A (13 of In contrast to the view that PAP overexpression is an indicator 17 versus 10 of 11, P > 0.05). This finding suggests that mutant of tumor aggressiveness in pancreatic cancer (29, 41), we showed that PAP expression was associated with more frequent low-stage (stages I and II) HCCs that had no vascular invasion (P = 0.013). REG protein expression has been shown to correlate with frequent vascular invasion, lymph node metas- tases, and worse prognosis in gastric cancers (24, 25). Other investigators have shown that REG 1 protein is more frequently expressed in well-differentiated cholangiocarcinoma (27), and Reg gene is up-regulated during the differentiation of colorectal cancer cell lines (45). In this study, we failed to show a positive correlation of REG1A expression with tumor progression. The reasons for these discrepancies need to be further explained. We showed that PAP expression closely correlated with REG1A expression (P < 0.00001), with a concordance of 77.4%, and 46 of 55 HCCs (83.6%) coexpressed REG1A and PAP. Coexpression of PAP and REG 1 has been shown in colorectal cancer (46). These data suggest a close interrelation between PAP and REG1A expressions. However, the significance is not fully understood. We then did a combination analysis of the four expression patterns of both genes in HCC. Fig. 2. Cumulative survival curves for 265 patients with primary unifocal HCC in We showed that HCCs with PAP expression alone had the relation to the expression status of PAP and REG1A.P was from comparison of all four groups (log-rank test, P = 0.044). HCC that expressed PAP alone had the best highest frequencies of well differentiated (grade 1) and low- survival rate, significantly better than HCC with coexpression of PAP and REG1A stage tumors (P < 0.007 and P < 0.001, respectively), the least (P <0.0002).

www.aacrjournals.orgClin Cancer Res 2005;11(7) April 1,2573 2005 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Imaging, Diagnosis, Prognosis h-catenin exerts stronger negative effect on tumor progression expression might provide a valuable indicator for the identifi- and can not be abolished by REG1A expression. We then cation of patients at increased risk of tumor recurrence after exclude the interferences of p53 and h-catenin mutations and curative surgery for HCC and other cancers (46). The molecular analyzed the roles of PAP and REG1A expression in HCCs mechanisms for the opposite roles of PAP and REG1A in tumor without mutation of these two genes. In this subset of HCCs, progression and metastasis need further studies for clarification. the coexpression of PAP and REG1A was associated with more In conclusion, these observations provide in vivo evidence frequent high-stage tumors than PAP expression alone that PAP and REG1A have divergent effects in the tumor (P < 0.005). Although small in number of cases, these data progression of HCC. PAP expression plays a role in the further strengthen our observations that PAP and h-catenin development of subset of HCCs with less aggressive behavior mutations are associated with low-stage HCCs with favorable and frequent association with h-catenin mutation, whereas outcome, whereas REG1A possesses opposite effects that coexpression of REG1A had a deleterious effect leading to more abolish the beneficial effects of PAP in this subset of HCC, advanced disease and poor prognosis. resulting in more advanced disease. These data highlight the importance of combined analysis to better understand the Acknowledgments significance of PAP and REG1A expressions in various disease processes, particularly the tumor progression. Thus, the REG1A We thank Po-Lin Lai and Dr. Chiao-Ying Lin for their technical support.

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www.aacrjournals.orgClin Cancer Res 2005;11(7) April 1,2575 2005 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Opposite Roles of Human Pancreatitis-Associated Protein and REG1A Expression in Hepatocellular Carcinoma: Association of Pancreatitis-Associated Protein Expression with Low-Stage Hepatocellular Carcinoma, β-Catenin Mutation, and Favorable Prognosis

Ray-Hwang Yuan, Yung-Ming Jeng, Hui-Ling Chen, et al.

Clin Cancer Res 2005;11:2568-2575.

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