Journal of Surgical Oncology 2003;82:111–120

p53 Alteration Is Not an Independent PrognosticIndicator, But Affects the Efficacy of AdjuvantChemotherapy in Human Pancreatic Cancer

MING DONG, MD, PhD,1 YOSHINORI NIO, MD, PhD,1* KUNIHIRO YAMASAWA, MD,1

TOMOKO TOGA, BEdu,1 LISHUANG YUE, MD,2 AND TAKAYUKI HARADA, MD, PhD2

1First Department of Surgery, Shimane Medical University, Izumo, Japan2Second Department of Pathology, Shimane Medical University, Izumo, Japan

Background and Objectives: Mutations in the p53 gene are found in more than 50%of human cancers and are observed in 60–80% of pancreatic cancers. The clinico-pathologic implications of p53 abnormalities and their effects on the efficacy of theadjuvant chemotherapy for pancreatic cancer remain controversial.Methods: We investigated the p53 status in core exon-4 to -9 (codon 33–331) bydirect DNA sequencing in a series of 72 pancreatic cancers and analyzed the effects ofp53 abnormalities on the patients’ survival and the efficacy of adjuvant chemotherapy.Results: p53 mutations were found in 62.5% (45/72) of cases, including 38 pointmutations and 7 frameshift mutations. The subtypes of p53 mutations included 68.9%(31/45) transitions and 15.6% (7/45) transversions. 39.5% (15/38) of point mutationswere CGT (Arg) to CAT (His) mutation at codon-273 of exon-8. 34.2% (13/38) ofpoint mutations were CGG (Arg) to TGG (Trp) mutation at codon-248 of exon-7.Of seven frameshift mutations, four were seen at exon-4, two at exon-5, and one atexon-6. Of overall cases, p53 abnormalities were not associated with a poorly dif-ferentiated grade and an advanced stage. The relationship of adjuvant chemotherapyto survival is approaching statistical significance. Univariate analysis showed that inthe p53 mutation group, the patients who received adjuvant chemotherapy had a bettersurvival ratio than that of patients who did not do. Multivariate analysis indicatedthat in the group with p53 mutations, the significant factors for survival were adju-vant chemotherapy, histologic grade, and clinical stage. However, in the group with awild-type p53 gene, only histologic grade was a significant factor. In addition, 34.7%(25/72) of the cases harbor p53 polymorphism mutation only at codon-72 of exon-4,which did not show any significant effect on the pathology, prognosis, and efficacy ofadjuvant chemotherapy of the pancreatic cancers.Conclusions: A p53 abnormality was not an independent factor for evaluating theprognosis of patients with pancreatic cancer, but was a beneficial indicator for select-ing a reasonable strategy of adjuvant chemotherapy against pancreatic cancer.J. Surg. Oncol. 2003;82:111–120. � 2003 Wiley-Liss, Inc.

KEY WORDS: p53; point mutation; polymorphism; pancreatic cancer;chemotherapy

The incidence of pancreatic cancer has risen markedlyduring the last half-century. Now, pancreatic cancer is thefourth or fifth leading cause of cancer death in the deve-loped Western countries [1] and Japan [2] and remainsthe most lethal of the common malignancies. The lack ofanatomic barriers to protect against local infiltration andthe biologic propensity to invade the lymphatics, nerves,

Ming Dong and Lishuang Yue are currently at the Department of Medicine,State University of New York, Stony Brook, NY.

*Correspondence to: Yoshinori Nio, MD, First Department of Surgery,Shimane Medical University 89-1, Enya-cho, Izumo, Shimane 693-8501,Japan. Fax: 81-853-20-2222.E-mail: fsurgery@shimane-med.ac.jp

Accepted 17 September 2002

DOI 10.1002/jso.10186

Published online in Wiley InterScience (www.interscience.wiley.com).

� 2003 Wiley-Liss, Inc.

and large vessels result in rapid progression of the tumor,and these are nearly insurmountable obstacles to achievea complete surgical eradication of this malignancy. Cur-rently, most patients are diagnosed in the advanced stages,which results in a poor prognosis. Pancreatic cancer re-mains a challenge to clinicians.

Although the reason for the aggressiveness of thisdisease is unknown, oncologists have noted the impor-tant role of oncogenes and tumor suppressor genes in itscarcinogenesis. Previous studies have shown that morethan 50% of human cancers harbor a p53 tumor sup-pressor gene mutation [3,4]. The frequency and spectrumof p53 mutation, as well as the functions of p53, aredependent on the cell type, developmental stage, andtissue origin of the cancer [5–7]. Mutations in the p53gene have been observed in 60–80% of pancreaticcancers [8]. Some studies indicated that p53 mutationscould be associated with malignant progression and apoor prognosis in pancreatic cancer [9,10]. However,these studies did not investigate the effects of p53mutations on the survival of the patients, especially thechemosensitivity of the pancreatic cancer cells, possiblydue to the relatively small number of cases [3,11–13].

Because of a low resection rate and early recurrence,adjuvant chemotherapy (ACT) has been used extensivelyfor treating pancreatic cancer. However, clinical studiesdemonstrated that pancreatic cancers are extremely re-sistant to all anticancer agents and combination regimens[14]. Thus, an index of screening the chemosensitivity ofhuman cancers has become an important issue. The moststudies have focused on the effects of the multidrug-resistance 1 (MDR1) gene on the efficacy of ACT inhuman pancreatic cancers, but the conclusions still re-main controversial [15]. Since many anticancer agentswork by inducing apoptosis in tumor cells and p53 playsan important role in inducing programmed cell death inresponse to severe damage of cellular DNA [16–18], thep53 gene has been followed with interest for its possibleinfluence on the efficacy of ACT in human cancers.Experimentally, the status of the p53 gene either increas-ed or decreased the sensitivity of tumor cells to anticanceragents in various tumor cell lines [14,19,20]. Clinically,some investigators have reported that p53 mutations wereassociated with resistance to chemotherapy in colorectalcancer [21], breast cancer [22], and head and necksquamous cell carcinoma [23]. Another study, however,showed that p53 alterations increased the sensitivity oftumor cells to ACT in bladder cancer [24]. Expression ofp53 protein was also noted to affect the efficacy of ACTin invasive ductal carcinoma (IDC) of the pancreas andwas associated with a shorter or longer survival for pan-creatic cancer patients who received ACT [25–28].

The controversial findings prompted us to assess thesignificance of p53 alterations in the outcome of a series

of 72 patients with resectable IDCs of the pancreas, andin the efficacy of their postoperative ACT.

Previous studies indicated that polymorphism mutationin p53 gene was frequently found in some human cancers.The most studies did not show a significant relationshipbetween polymorphism mutation of the p53 and biologicbehavior of human cancer, such as laryngeal tumors [29],oral cancer [30], cutaneous squamous cell carcinoma[31], squamous cell carcinoma of the head and neck [32],and acute myeloid leukemia [33]. However, a few reportsindicated that p53 polymorphism might play a role inthe development of lung cancer [34], and esophaguscancer [35]. Effect of p53 polymorphism on pathology ofcervical cancer remains controversial [36–39]. Likewise,we also are interested in assessing effect of p53polymorphism on biologic behavior of pancreaticcarcinogenesis.

Concerning the methods used to screen and identifythe p53 mutations, the immunohistochemistry (IHC) iscommonly used to assess p53 function. However, theabsence of p53 expression is not synonymous with nor-mal p53 function, and when p53 is not stained by IHC,there might be p53 null status, deletion, frameshift ornonsense mutation [26]. Although single-strand confor-mation polymorphism (SSCP) analysis is widely used forscreening p53 status, the length of the PCR productscould limit the sensitivity of SSCP, especially if they arelonger than 300 base pairs [40]. A study indicated that in32 ovarian cancers strongly suspected of containing p53mutations, 25 (78%), 26 (81%), and 29 (91%) werepositive for p53 abnormalities by SSCP, IHC, and DNAsequencing, respectively [41]. Thus, we employed directDNA sequencing for assessing p53 status.

MATERIALS AND METHODS

Pancreatic Cancer Profile

We obtained informed consent of this study on thegenetic background of the patients from the patientsthemselves or their family according to the recom-mendation by the ethical committee of our departmentfrom 1999.

Formalin-fixed and paraffin-embedded specimenswere obtained from 72 consecutive patients with primaryIDC of the pancreas, who underwent a pancreatectomyfor the purpose of treating the pancreatic cancer at theFirst Department of Surgery, Shimane Medical Univer-sity (Izumo, Japan) between May 1982 and March 2001.Their profiles are summarized in Table I. The patientsranged in age from 35 to 80 years, with an average ageof 65.4� 9.6 years. The surgical procedures included 38standard or pylorus-preserving pancreatoduodenectomies,21 distal pancreatectomies, and 13 total pancreatec-tomies. The median survival was 10.2 months. All data

112 Dong et al.

were analyzed according to the TNM stage classificationof the International Union Against Cancer (UICC) [42].Histopathologically, all specimens were verified to beIDC of the pancreas. None of the patients received anytype of treatment before the surgical procedures. Post-operative survival was defined as the time that elapsedfrom the surgery to a cancer-related death. Forty-onepatients received ACT after their surgery, but none ofthem received adjuvant radiotherapy.

Adjuvant Chemotherapy

In our department, we have no standard regimen forACT against pancreatic IDC, because there is no evidencesupporting the survival benefits of ACT for pancreaticIDC at present. Accordingly, the respective doctors with

the informed consent of the patients and/or their familydecided the use of ACT. Of the 72 patients, 26 receiv-ed surgery alone and 41 received ACT after surgery(excluding five cases of death due to other causes; seestatistical analysis below). The ACT mainly involvedoral UFT (a mixture of uracil and ftorafur in a pro-portion of 4:1) and/or oral cyclophosphamide (CPA).Oral UFT was administered in 41 and oral CPA in20 patients, respectively. A few patients were given intra-venous chemotherapy including mitomycin-C (MMC)in four, Adriamycin (ADR) or 40-epirubicin in seven,5-fluorouracil (5-FU) in eight, and/or cisplatin (CDDP) insix patients, respectively.

Genomic DNA Extraction and Amplification

In brief, six 5mm-thick tissue sections were cut from aparaffin-embedded block of each pancreatic cancer. Thecancer tissue was scraped out of the section and placedinto a 1.5 ml of Eppendorf tube. After deparaffinizationin xylene as well as rehydration in a graduated ethanolseries, the tissue was incubated in 300 ml of lysis buffer(10 mM Tris-HCl, pH 8.0; 10 mM EDTA, pH 8.0;150 mM NaCl; 0.5% sodium dodecyl sulfate (SDS); 30 mgproteinase K) with constant shaking at 488C for 36–48 hin a Shaking Bath BW100 (Yamato Science, Tokyo,Japan), until the supernatant became cloudy. During theincubation, 50 ml of TE (10 mM Tris-HCl, pH 8.0; 1 mMEDTA, pH 8.0) containing 50 mg of proteinase K wasadded each 12 h. The genomic DNA was extracted withphenol/chloroform and ethanol precipitation [43]. Anequal volume (450 ml) of phenol, chloroform and isoamylalcohol in a proportion of 25:24:1 was added to theEppendorf tube. The sample was mixed with a RotatorRT-50 (TAITEC, Tokyo, Japan) at 40 rpm at room tem-perature for 60 min, and was then centrifuged at 12,000gat room temperature for 5 min. The supernatant (around450 ml) was transferred to a new 1.5 ml of Eppendorftube. This step was repeated three to five times until noimpurity was seen between the aqueous and organicphases. The genomic DNA was precipitated with 3 Msodium acetate (pH 7.4) at �808C for at least 30 min.After being centrifuged at 12,000g at 48C for 10 min, thesupernatant was discarded and the precipitate was washedwith 80% ethanol twice. The sample was dried at 558Cfor 10 min in a DNA mini apparatus (Heto Lab Equip-ment, Gydevang, Denmark). After being treated withRNase A (working concentration: 20mg/ml), the extractedDNA was purified again using a similar method asdescribed above. For avoiding contamination betweenDNA samples, the microdissections for each sample wereprepared independently.

The primers for the amplification and sequencing ofthe genomic DNA are listed in Table II. All primers were

TABLE I. Patient Backgrounds and p53 Gene Mutation

Parameter No. of cases p53 mutation (%)

Overall 72 45/72 (62.5)

Age (yr)<65 28 21/28 (75.0)�65 44 24/44 (54.5)

GenderMale 34 23/34 (67.6)Female 38 22/38 (57.9)

SiteHead 48 30/48 (62.5)Body and/or tail 20 13/20 (65.0)Entire 4 2/4 (50.0)

GradeG1 35 22/35 (62.9)G2 32 19/32 (59.4)G3 5 4/5 (80.0)

Stage (pTNM)I 10 5/10 (50.0)II 4 2/4 (50.0)III 36 24/36 (66.7)VI 22 14/22 (63.6)

pTa

1 5 4/5 (80.0)2 25 16/25 (64.0)3 23 14/23 (60.9)4 19 11/19 (57.9)

Nodal involvement� 22 14/22 (63.6)þ 50 31/50 (62.0)

a1, limited to pancreas, <2.0 cm; 2, limited to pancreas, >2.0 cm; 3,extended to peripancreatic structures, including the duodenum, bileduct, mesentery, measocolon, omentum, and peritoneum; 4, extendedto adjacent structures, including the stomach, spleen, colon, portalvein, celiac artery, and the superior mesenteric and common hepaticarteries and veins.

p53 Mutations and Chemotherapy in Pancreatic Cancer 113

purchased from Wako Pure Chemical Industries, Osaka,Japan. The polymerase chain reaction (PCR) was car-ried out in a DNAThermal Cycler 480 (Perkin-Elmer) for35 cycles. A total of 0.5 mg of genomic DNA was sub-jected to PCR in a total volume of 50 ml of PCR reactionmixture. It contains 2.5 U of Taq polymerase, 5 ml of10�PCR buffer, 4 ml of dNTP mixture (2.5 mM each;TaKaRa, Kyoto, Japan), and 50 pmole of each primer.Each cycle consisted of denaturation at 948C for 1 min,annealing at 558C for 45 s, and extension at 728C for2 min. After the last cycle of amplification, the extensionwas continued for an additional 7 min at 728C. A secondPCR was performed in several samples, because of anunsatisfactory small yield on the first PCR. The condi-tions of the second PCR were the same with the first PCR,but only 20 cycles were performed.

DNA Sequencing

The PCR products were electrophoresed on a 2%agarose gel containing 0.5 mg/ml of ethidium bromide.The desired band was excised and purified with aGIAquick Gel Extraction Kit (QIAGEN GmbH, Hilden,Germany). A Microcon-100-Column (Millipore, Bedford,MA) was used to purify the PCR products without ectopicsites confirmed by electrophoreses.

For automated cycle sequencing, 20–40 ng of purifi-ed PCR product was subjected to sequencing PCR intotal volume of 20 ml of PCR reaction mixture contain-ing 3.2 pmole of p53 sequencing primer (sense), 8 ml ofBigDye Terminator Ready Reaction Mix (Applied Bio-systems). The sequencing reaction was carried out in aDNA Thermal Cycler 480 (Perkin Elmer) for 20 cyclesfor exon-5 and -6, or 25 cycles for exon-4, -7, -8, and -9.Each cycle consisted of denaturation at 968C for 30 s,

annealing at 508C for 15 s, and extension at 608C for4 min. Ethanol precipitation was used to purify the seq-uencing PCR products. After being denaturated at 958Cfor 3 min, the purified sample was electrophoresed on anABI Prim 310 Genetic Analyzer (PE Applied Bio-systems, Foster City, CA). Sequencing Analysis andSequence Navigator Software (Applied Biosystems)were used to analyze the sequence data.

Statistical Analysis

The data were analyzed with an w2 test (or Yates’correction test). The survival curves were calculated ac-cording to the Kaplan-Meier method and were comparedusing the Cox-Mantel test. A multivariate analysis of themaximum likelihood estimates, with the Cox propor-tional hazard risk model to obtain the conditional risk ofdeath due to IDC of the pancreas. Of the 72 patients, fivepatients (two died from bleeding, one from acutemyocardial infarction within one month after surgery,and the remaining two died from other diseases) wereincluded only in the frequency and the pattern of the p53mutations and were excluded from the survival ana-lysis. Statistically significant differences were accepted atP< 0.05.

RESULTS

Representative results from the DNA sequencing areshown in Figure 1. The mutation profile of the p53 genein primary IDC of the pancreas is summarized in Table I.A p53 mutation was found in 62.5% (45/72) of pancreaticIDC cases including 38 point mutations and 7 frameshiftmutations. The patterns of the p53 mutation are sum-marized in Table III. With respect to the nature of the basechanges, the subtypes of p53 point mutations consisted of

TABLE II. Primers for Genomic DNA Amplification and Sequencing

Primer sequence Exon (codon included) Amplified fragment (bp)

Amplifying 50-TGAGGACCTGGTCCTCTGAC-30 4 (33–125) 41330-ACCTTGAAACCCTAAGGAGA-50

50-GTTTCTTTGCTGCCGTGTTC-30 5 (126–186) 32330-ACGGGTCCCAGGGGTCCGGA-50

50-TGGTTGCCCAGGGTCCCCAG-30 6 (187–224) 22330-ACCAACAGTCACCGGGAGG-50

50-ACCATCCTGGCTAACGGTGA-30 7 (225–261) 46630-AGACGAACGGCGACTGGGGA-50

50-TTGGGAGTAGATGGAGCCT-30 8, 9 (262–306, 307–331) 44530-TTTCAAAGGTCAGATTGTGA-50

Sequencing 50-TGCTCTTTTCACCCATCTAC-30 450-TTCAACTCTGTCTCCTTCCT-30 550-GCCTCTGATTCCTCACTGAT-30 650-CTTGCCACAGGTCTCCCCAA-30 750-TTCCTTACTGCCTCTTGCTT-30 From 8 to 9

114 Dong et al.

68.9% (31/45) transitions and 15.6% (7/45) trans-versions. For the transition, the base changes favoredpurine to purine (54.8%, 17/31) changes slightly morethan pyrimidine to pyrimidine (45.2%, 14/31). No spe-cial predilection, with respect to purine (pyrimidine) topyrimidine (purine) was seen for the transversions. p53abnormalities were not associated with a poorly dif-ferentiated grade (P¼ 0.674) and an advanced stage(P¼ 0.75) (Table I). When the different subtypes of point

mutations as well as frameshift mutations were comparedin a pair-wise combination, no statistical significance wasobserved between the subtype mutations and the patients’backgrounds. A correlation analysis also did not showany significant correlation between the p53 mutations andany clinicopathologic parameters, including age, gender,site of tumor, histologic grade, clinical stage, pT, pN, M,invasion of tumor to large vessel or adjacent tissues ororgans. In five cases excluded from the survival analysis,

TABLE III. Mutations of p53 Gene in Invasive Ductal Carcinoma of the Pancreas

Pattern No. of cases Exon Codon Nucleotide (amino acida) change

I. Point mutationTransition (n¼31, 68.9%) 1 5 184 GAT (Asp) to GGT (Gly)

13 7 248 CGG (Arg) to TGG (Trp)1 7 258 GAA (Glu) to AAA (Lys)

15 8 273 CGT (Arg) to CAT (His)1 9 317 CAG (Gln) to TAG (Termb)

Transversion (n¼7, 15.6%) 1 4 73 GTG (Val) to CTG; (Leu)1 4 113 TTC (Phe) to TTA (Leu)1 5 162 ATC (Ile) to TTC (Phe)1 8 281 GAC (Asp) to TAC (Tyr)1 9 324 GAT (Asp) to TAT (Tyr)1 9 327 TAT (Tyr) to TTT (Phe)1 9 331 CAG (Gln) to CAC (His)

II. Frameshift mutation 1 4 46 1-bp deletion(n¼7, 15.6%) 3 4 103–111 24-bp deletion

2 5 135–136 1-bp insertion1 6 191–194 12-bp deletion

aUnderline denotes nucleotide changed.bTerm denotes term codon.

Fig. 1. Partial sequence of the p53 gene shows 24 base deletions between codon-102 and codon-112 in exon-4 (A), a base insertion betweencodon-135 and codon-136 in exon-5 (B), and a C to T transition at the first nucleotide of codon-248 in exon-7 with a CGG (Arg) to TGG (Trp)substitution (C).

p53 Mutations and Chemotherapy in Pancreatic Cancer 115

three were p53 mutant and two were p53 wild-type.The survival status of the patients, however, correlatedsignificantly with the clinical stage (P< 0.001), histolo-gic grade (P¼ 0.023), pT (P¼ 0.001), pN (P¼ 0.006),and vascular invasion (P¼ 0.027). The median survivalperiod grouped by p53 status, ACT and their interac-tions are summarized in Table IV. p53 mutation alonewas not associated with the prognosis of the patients(P¼ 0.377). However, the relationship of ACT aloneto survival was approaching statistical significance (P¼0.086) (Table IV, Fig. 2). Furthermore, their interactionshowed that in the p53 mutation group, the survival rateof the patients who received ACT was significantly higherthan that of the patients who did not receive ACT(P¼ 0.033) (Fig. 3). No significant differences in survivalwere found among the other subgroups.

A multivariate analysis with Cox proportional hazardrisk model was used to evaluate the significance of therisk factors related to the patients’ death due to IDC of thepancreas after surgery. Overall the results indicated thatsignificant risk factors responsible for the patients’survival were the clinical stage (P< 0.001) and histologicgrade (P¼ 0.002). Furthermore, we analyzed separatelythe potential risk factors related to the patients’ prognosisfor those groups with or without p53 mutation. In the p53mutation group, the significant risk factors were ACT(P¼ 0.012), clinical stage (P< 0.001), and histologicgrade (P¼ 0.006). However, in the group with a wild-type p53 gene, only the histologic grade was a significantrisk factor (P¼ 0.005) (Table V).

Polymorphisms, which altered the amino acid se-quence of p53, were found in 34.7% (25 of 72) of the

TABLE IV. Association of p53 Mutation and Adjuvant Chemotherapy With Prognosis in PancreaticCancer{

Subgroup No. of cases (%) Median survival (mo)

Wild-type p53 25 (37.3) 10.6Mutant type p53 42 (62.7) 10.2ACT (�) 26 (38.8) 9.7*ACT (þ) 41 (61.2) 11.3*Mutant type p53 ACT (þ) 28 (41.8) 13.9**Mutant type p53 ACT (�) 14 (20.9) 9.0**Wild-type p53 ACT (þ) 13 (19.4) 8.1Wild-type p53 ACT (�) 12 (17.9) 10.6

ACT, adjuvant chemotherapy.{Analysis excluded five patients who died of non-cancer-related diseases.*P¼ 0.086.**P¼ 0.033, by Cox-Mantel test.

Fig. 2. Separate analysis of the p53 status and adjuvant chemotherapy (ACT) and survival of patients with pancreatic cancer after surgery.

116 Dong et al.

specimens, all of which were located at codon-72 ofexon-4 for CGC (Arg) to CCC (Pro). The frequency forhomozygous arginine, homozygous proline, and hetero-zygous arginine/proline substitutions was 65.3% (47/72),8.3% (6/72), and 26.4% (19/72), respectively. In thepresent study, the polymorphism mutation at codon-72 ofthe p53 gene did not affect the survival of the patients in

comparison with the patients with a wild-type p53 gene(P¼ 0.628). Furthermore, in the group with polymor-phism mutations, the median survivals of the patientswith versus without ACT were 13.9 and 9 months, res-pectively (P¼ 0.136). Multivariate analysis showed norelationships between the patients’ survival and ACT inthe group with the polymorphism mutations (P¼ 0.227).

Fig. 3. Cooperative analysis of p53 status and adjuvant chemotherapy (ACT) and survival of patients with pancreatic cancer after surgery.

TABLE V. Multivariate Analysis on Clinicopathologic Variables and Patient Survival*

Variables Parameter estimate (SE) Conditional RR (95% confidence limit) P (chi-square)

Overalla (n¼ 67) Stage 0.6166 (0.1727) 1.8525 (1.3206–2.5987) 0.0004Grade 0.7878 (0.2548) 2.1985 (1.3341–3.6228) 0.0020ACT �0.3366 (0.3000) 0.7142 (0.3967–1.2856) 0.2617Age 0.0091 (0.0156) 1.0092 (0.9788–1.0405) 0.5583p53 status �0.1460 (0.3092) 0.8641 (0.4714–1.5841) 0.6367Gender 0.0111 (0.3048) 1.0111 (0.5564–1.8375) 0.9711

Mutant type p53 (n¼ 42) Stage 1.1291 (0.2817) 3.0928 (1.7807–5.3719) 0.0001Grade 0.8703 (0.3167) 2.3877 (1.2835–4.4416) 0.0060ACT �0.9736 (0.3864) 0.3777 (0.1771–0.8056) 0.0118Gender �0.2756 (0.3947) 0.7591 (0.3502–1.6454) 0.4850Age �0.0013 (0.0181) 0.9987 (0.9639–1.0348) 0.9435

Wild-type p53 (n¼ 25) Grade 1.5240 (0.5452) 4.5904 (1.5768–13.3636) 0.0052Gender 0.7999 (0.6514) 2.2252 (0.6207–7.9777) 0.2195ACT 0.3746 (0.5006) 1.4544 (0.5452–3.8797) 0.4543Stage 0.1621 (0.2474) 1.1760 (0.7242–1.9097) 0.5121Age 0.0168 (0.0282) 1.0169 (0.9621–1.0748) 0.5530

ACT, adjuvant chemotheraphy.SE, standerd error; RR, risk ratio.*Dependent variable, survival month; and censoring variable, death due to pancreatic cancer.aAnalysis excluded five patients died of non-cancer-related diseases.

p53 Mutations and Chemotherapy in Pancreatic Cancer 117

DISCUSSION

We examined p53 mutations from exon-4 to -9 andtheir clinicopathologic implications in 72 patients withIDC of the pancreas by the direct DNA sequencing. Assummarized in Table III, 86.7% (39/45) of the mutations(33 point mutations and 6 frameshift mutations) werelocated in the core domain (residues 102–292), whichwas consistent with previous reports [9,44]. In addition,some of the sequencing data contained heterogeneouscomponents, probably due to the fact that the genomicDNA contained normal stroma, and the PCR productswere not cloned before the sequencing. Another causemay be that not all of the tumor cells harbored p53mutation.

p53 abnormalities appears to be an independentbiologic marker for evaluating the patients’ pathogenesisand prognosis in some human cancers, such as breastcancer [45,46], lung cancer [47], and bladder cancer[48], although other authors have reported contradic-tory conclusions [49]. These arguments can be attributedto differences in the type of cancer, original tissue,site, number of samples, and subtype of p53 mutation[50–52]. Previous studies indicated that p53 abnormal-ities either occur in harboring germline mutations ofsubjects [53,54] or in only a few cells of pancreaticadvanced cancer [9,13], suggesting that p53 alterationsmight represent either an early or a late event in thedevelopment and progression of pancreatic cancer. In thepresent study, the frequency of p53 mutation was higherin both the poorly-differentiated grade (80% for G3) andthe advanced stage (65.5%, a subset of stage III and stageVI) than in the well-differentiated grade (61.2%, a subsetof G1 and G2) and the early stage (50%, a subset of stageI and stage II), respectively, although no statistical dif-ferences were observed between them. These resultssuggest that p53 mutations may occur over the entireprocess of pancreatic carcinogenesis. We also comparedthe differences in survival between the subgroups by p53mutation, ACT, and subtype mutation (transition, trans-versions, and frameshift). Both univariate and multi-variate analyses, however, did not indicate any significantrelationship between p53 abnormalities and the clinico-pathologic parameters of IDC of the pancreas (data notshown). These findings suggested that the p53 mutationmay be involved in pancreatic carcinogenesis, but maynot be an independent indicator for evaluating thebiologic behavior of this disease. Previous findings alsoindicated that pancreatic carcinogenesis might be relatedto multiple cancer-related genes. Mutations in more thantwo of K-ras, p53, p16, and DPC4 were found in up to76% of pancreatic cancers, indicating that their interac-tions may be beneficial to evaluating the biologic charac-teristics of pancreatic cancer [55–57]. This may be one of

the explanations for the controversial effect of p53 statuson pancreatic cancer.

Most anticancer agents kill cancer cells through theapoptosis activated by the wild-type p53 gene, whichcontrols the G1-S checkpoint and promotes the correctrepair of DNA damage [16,17]. However, basic and cli-nical studies indicated that p53 abnormalities could eitherdecrease or increase the sensitivity of tumor cells toanticancer agents [21,24,58,59]. On the one hand, loss ofp53 function decreases the ability of the cells to arrest inthe G1 phase of the cell cycle during apoptosis and pro-vides a basis for the tumor selective cytotoxicity of drug,resulting in resistance of the cancer cells to anticanceragents [60]. These findings were also verified clinically incolorectal cancer [21] and breast cancer [61].

In contrast, p53 mutations may also make cancer cellsmore sensitive to anticancer agents. A study and addi-tional reviews noted that a disruption of the cell cyclecheckpoint could have dramatic effects on the response toanticancer agents in mammalian cells [62]. p53 inactiva-tion may render the cancer cells more sensitive to anti-cancer agents through the cytotoxic effects of DNA cross-linking agents, which could be aggravated by a mutatedp53 [58,63,64]. Mueller et al. proposed that the effect ofthe mutated p53 on chemosensitivity might depend on therespective chemotherapeutic regimen and/or anticancerdrugs administered. For example, mutant p53 increasedthe cytotoxic effect of cis-platinum, Taxol, and Pento-xifylline, but decreased the cytotoxic effect of fluorour-acil, Adriamycin, and etoposide to tumor cells [64]. In thepresent study, although a separate analysis of the p53status and ACT by an univariate analysis did not signi-ficantly predict the patients’ survival, their interactionsshowed that the p53 status might affect the efficacy ofACT against IDC of the pancreas. In p53 mutant group,the patients who received ACT had a better survival ascompared with those patients who did not receive ACT(P¼ 0.033) (Table IV, Fig. 3). In the p53 wild-type group,however, ACT did not improve the patients’ survival(P¼ 0.789). Furthermore, after multivariate analysis forthe overall patients, the histologic grade and clinical stagewere the only significant parameters for the patients’prognosis. However, when the patients were divided intotwo groups according to their p53 status, ACT was asignificant factor for improving the survival of the pati-ents with a p53 mutation (P¼ 0.012) (Table V), but notfor those patients with a wild-type p53 gene (P¼ 0.454)(data not shown). In the present study, the majority ofpatients received UFT, which is a fluoropyrimidine andexerts its anticancer effect by inhibiting DNA synthesis.CPA, which is an alkylating agent, and CDDP, whichis a DNA cross-linking agent, were also used. Whenpancreatic cancer cells harbor a p53 mutation, theirphysiologic functions might be affected by damage-

118 Dong et al.

induced checkpoints, resulting in enhanced chemosensi-tivity. Taken together, effect of p53 abnormalities on thesensitivity of tumor cells to anticancer agents depends onthe cancer cell type, the environment of the tumor, anti-cancer agents and dose, and the treatment regimen.

In the present study, polymorphism mutation wasfound only at p53 codon-72 of exon-4 with a CGC (Arg)to CCC (Pro) substitution. This polymorphism mutationhad been noted having significant effect on some humancarcinogenesis as previously described. Our data, how-ever, did not show a significant relationship between thispolymorphism and the clinicopathologic parameters, aswell as the efficacy of ACT using both univariate andmultivariate analysis, suggesting that the polymorphismat p53 codon-72 might have no significant implications inthe biologic behavior of pancreatic cancer. Combined ourdata with previous reviews, we inferred that the biologicimplications of polymorphisms at p53 codon-72 might bedifferent in various cancers.

The present study indicated that p53 abnormality didnot represent an independent factor for evaluating theprognosis of patients with IDC of the pancreas, but wasa useful indicator for selecting a reasonable strategy ofACT against pancreatic cancer. Polymorphism at p53codon-72 of exon-4 might have no significant implica-tions in the biologic behavior of pancreatic cancer.

However, our study included mutations in the hot spotarea of exons of the p53 gene in a relatively small numberof patients. Moreover, the delivery of ACT was notrandomized. These disadvantages may cause a bias in thepatient selection and a difference in the analysis ofpatients’ survival. Further studies in all exons of the p53gene and an increased number of patients using a rigorousresearch design are necessary to clarify the overall effectsof p53 mutations on the carcinogenesis and progressionof pancreatic IDC in the future.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Ms. MiyukiIshihara, Ms. Yasuko Sonoyama, and Ms. Yuka Maniwafor their assistance in the experiment.

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