Mutational spectra ofp53 in geographically localized esophageal squamous cell carcinoma groups in China

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BACKGROUNDEsophageal carcinoma is a particularly interesting tumor because of the dramatic difference in its incidence and geographic distribution among populations of similar ethnic origin. Epidemiologic data have suggested that many environmental
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  Mutational Spectra of  P53   in Geographically LocalizedEsophageal Squamous Cell Carcinoma Groupsin China Wei Cao,  M.D., Ph.D. 1,2 Xufeng Chen,  Ph.D. 3 Huifang Dai,  B.S. 3,4 Huizhen Wang,  R.N. 5 Binghui Shen,  Ph.D. 4 David Chu,  M.D. 4 Taylor McAfee,  Ph.D. 2 Zuo-Feng Zhang,  M.D., Ph.D. 2 1 Department of Pathology, Xinxiang Medical Col-lege, Xinxiang, Henan, People’s Republic of China. 2 Department of Epidemiology, University of Cali-fornia–Loc Angeles School of Public Health andJonsson Comprehensive Cancer Center, Los An-geles, California. 3 Zhejiang Cancer Research Institute, Hangzhou,Zhejiang, People’s Republic of China. 4 Department of Radiation Research and Divisionof Surgery, City of Hope National Medical Center,Duarte, California. 5 Department of Medicine, Xinxiang Central Hospi-tal, Xinxiang, Henan, People’s Republic of China.Supported in part by grant 954020900 from theHenan Natural Science Foundation and by a SPORELung Cancer Fellowship Award from the NationalInstitutes of Health (grant P50CA90388).The authors thank Fubao Chang from the Depart-ment of Surgery, Linzhou Esophageal Tumor Hos-pital and Youhua Jiang from the Division of Sur-gery, Zhejiang Cancer Hospital for their assistancein the collection of paired tumor samples and thefollow-up of patients. They also are grateful to Dr.Shu Zheng and Dr. Jianjian Li for their advice andsupport for this study. Address for reprints: Zuo-Feng Zhang, M.D., Ph.D.,Professor of Epidemiology, Department of Epide-miology, University of California–Los AngelesSchool of Public Health, 71-225 CHS, Box 951772,Los Angeles, CA 90095-1772; Fax: (310) 206-6039; E-mail: zfzhang@ucla.eduReceived January 23, 2004; revision received April21, 2004; accepted May 18, 2004. BACKGROUND.  Esophageal carcinoma is a particularly interesting tumor because of the dramatic difference in its incidence and geographic distribution among pop-ulations of similar ethnic srcin. Epidemiologic data have suggested that many environmental exposures may be associated with an increased risk of its formation. METHODS.  In this study, 92 samples of esophageal squamous cell carcinoma(ESCC) were collected from patients who resided in 2 geographic areas in China with different incidences of ESCC: Linxian and Zhejiang. Overexpression andmutations of the  p53   tumor-suppressor gene were examined by using immuno-histochemistry, single-strand conformation polymorphism analysis, and directsequencing. RESULTS.  The rates of point mutation and overexpression of p53 in the ESCCspecimens studied were 30.4% (29 of 92 specimens) and 51.1% (47 of 92 speci-mens), respectively. The overexpression of   p53   was associated with tumor metas-tasis and with 5-year case fatality. Significant differences were found in the rates of overexpression and mutations in patients with clinical T2 tumors between thespecimens from Linxian, which is a high-incidence geographic area, and thespecimens from Zhejiang, which is a low-incidence area. Furthermore, differentmutational spectra were found in the tumor samples from these two geographicareas: In tumor samples from Linxian, the most common substitution mutation was a transversion in exon 5, whereas the most common mutations in tumorsamples from Zhejiang were transitions in exon 7. CONCLUSIONS.  The data suggest that the mutation and overexpression of   p53   may play important roles in the development of ESCC. The changes in  p53   may reflectenvironmental exposure to the different combinations of mutagenic factors andgenetic instability demonstrated by the populations in Linxian and Zhejiang. Theoverexpression of p53 protein may have significance as a prognostic factor forpatients with esophageal carcinoma.  Cancer   2004;101:834–44. © 2004 American Cancer Society. KEYWORDS: esophageal squamous cell carcinoma, mutational spectra of  P53  ,immunohistochemistry, single-strand conformation polymorphism, sequence, p53protein, high-incidence area, low-incidence area. E pidemiologic studies successfully have identified several cancerrisk factors in the human population, including exposure to chem-ical and physical mutagens (e.g., cigarette smoke, alcohol, heterocy-clin amines, asbestos, and ultraviolet radiation), infection by certainviral and bacterial pathogens, occupation, hormones, and dietary nongenotoxic constituents (e.g., macronutrients and micronutri-ents). 1–4 However, the precise molecular mechanisms by which theseinfluences generate or promote the genetic events required for tu- 834 © 2004 American Cancer SocietyDOI 10.1002/cncr.20437Published online 23 June 2004 in Wiley InterScience (www.interscience.wiley.com).  msrcenesis have not been delineated. Molecular ep-idemiology of human malignancy has the challenging goal of determining the molecular events of interac-tion between environmental risk factors and geneticmaterials that subsequently lead to carcinogenesis.The focus on risk assessment requires a multidisci-plinary strategy to investigate interindividual variationin cancer risk and gene-environment interactions.Thus, esophageal carcinoma has become one of themost interesting tumors among the spectrum of hu-man malignancies in which to pursue molecular stud-ies, because there are dramatic geographic differencesin incidence and distribution among populations of similar ethnic srcin worldwide. 5,6 Dietary factors andcultural habits have been identified in association with risk differences for this highly fatal disease. 7–10 One area of intense interest in recent molecularepidemiologic studies in esophageal carcinoma hasbeen the  P53   tumor suppressor gene, 11,12  which en-codes a nuclear phosphoprotein with cancer-inhibit-ing properties. It is a multifunctional transcriptionfactor that is involved in the control of cell cycle pro-gression, DNA integrity, and cell survival in cells ex-posed to DNA-damaging agents.Loss of p53 activity predisposes cells to the acqui-sition of oncogenic mutations and may favor geneticinstability. Furthermore, it has been shown that the P53   gene is one of the genes that is mutated mostfrequently in human malignancies, i.e., point muta-tions in this gene have been found in more tumorsacross the spectrum of human malignancies than inany other gene. 13–15 It is noteworthy that approxi-mately 80% of   P53   mutations are missense mutationsthat lead to amino acid substitutions, subsequently,that alter the protein conformation and increase thestability of p53. 14  A high frequency of   P53   gene muta-tions has been found in esophageal carcinoma. 16–18 The  P53   point mutations in esophageal carcinomahave been correlated with patients’ endogenous andexogenous environmental exposures. 17,18 However,further studies of the frequency and timing of onset of mutations and mutational spectra of   P53   should pro-vide insight into the etiology and molecular pathogen-esis of esophageal carcinoma and may generate hy-potheses for future investigations. 19 There are several geographic areas with a highincidence of esophageal carcinoma, especially innorthern China. 20,21 In Linxian, a county in the Henanprovince with a population of 800,000, the age-ad- justed mortality rates for the incidence of esophagealcarcinoma are up to 169 per 100,000 population. 22 Thegrowing epidemiologic data have implicated severaletiologic possibilities, including nitrosamines, nutri-tional deficiencies, fermented and moldy foods, andinhalation of polycyclic aromatic hydrocarbons. All of the above-mentioned factors qualify as environmentalcauses of this highly fatal disease in the region. 9,20,22,23 In Zhejiang, a province in eastern China, there hasbeen a low incidence of esophageal carcinoma. Ac-cording to the malignant tumor mortality survey of Chinese residents during 1974–1976, the adjustedmortality rate for esophageal carcinoma was 10.09 per100,000 population. 24  With regard to etiology, nostudy has been conducted on the risk factors foresophageal carcinoma in this low-incidence area.However, several studies 25–27 have been conducted onthe risk factors for large bowel carcinoma. These risk factors include low intake of crude fiber, high-lipiddiet, history of intestinal polyps, emotional trauma,and family history of cancer. These studies suggestthat there are different environmental and geneticfactors at work in Linxian and Zhejiang.In the current study, the expression of p53 proteinand mutations in the  P53   gene in samples of esopha-geal squamous cell carcinoma (ESCC) from patientsliving in Linxian and Zhejiang were studied. Experi-ments were performed using immunohistochemistry,single-strand conformation polymorphism (SSCP),and DNA sequencing. The objectives of this study  were to characterize p53 alterations in the carcinogen-esis and development of esophageal carcinoma and todetermine the geographic variations in  P53   muta-tional spectra in this disease. MATERIALS AND METHODS Tumor Samples Overall, 92 patients with ESCC who had undergoneesophagectomy at the Linxian Esophageal TumorHospital (47 patients) between 1979 and 1991 and atthe Zhejiang Cancer Hospital (45 patients) between1980 and 1990 were included in this study. There were64 men and 28 women and they ranged in age be-tween 37–71 years (mean age    standard deviation:51.3    9.2 years for males and 54.5    7.4 years forfemales). The tumors were staged according to theTNM staging system. 28 Data on the survival status(dead or alive) 5 years after surgery were collected. Alltumor samples were fixed in formalin and embeddedin paraffin. There were 194 tissue blocks, including 92primary ESCC lesions, 78 matched normal esophagealmucosa (5–10 cm adjacent to tumor site), and 24lymph nodes without metastases obtained from the 92patients. Each block was sectioned serially into 5-  m-thick sections, 1 of which was stained with hematox- ylin and eosin for histopathologic analysis. The othersections were used in the study of p53 protein expres-sion and gene mutation. P53   in Geographically Localized ESCC in China/Cao et al. 835  Immunohistochemistry  A conventional peroxidase method was used in theimmunohistochemical analysis, as described previ-ously, 29  with a modification of microwave oven heat-ing for technical enhancement. The primary antibody  was a 1:200 dilution of a monoclonal mouse antibody,DO-7, which was raised against an epitope betweenamino acids 1 and 45 in the C-terminal domain of human wild-type and mutant p53 (Pharmingen, SanDiego, CA). In each batch of immunostaining, we usedpositive esophageal carcinoma samples as controls.For negative controls, the primary antibody was re-placed with Tris buffered saline. Assessment was car-ried out according to the intensity of staining in thenuclei of neoplastic cells, and the results of immuno-staining in tissues were scored, with scores of 1  in-dicating weak staining, 2  indicating moderate stain-ing, and 3  indicating strong or dark-brown staining. A sample was defined to be positive when    10% of cells demonstrated staining intensity from at least 1  to 2  (weak to moderate). 30 DNA Extraction Paraffin embedded tissue sections were deparaf-finized, placed in cell lysis buffer (100 mM Tris-Cl, pH8.4; 0.5 mM ethylenediamine tetraacetic acid [EDTA];1% sodium dodecyl sulfate;, and 0.5 mg/mL protein-ase K), and incubated at 55 °C overnight. After phenoland chloroform extractions, DNA was precipitated inethanol and resuspended in sterile Tris-EDTA (TE)buffer (10 mM Tris-Cl, pH 8.0; 1 mM EDTA). Prior tothe polymerase chain reaction (PCR), an aliquot of DNA solution was examined by agarose gel electro-phoresis. PCR-SSCP Different regions of exons 5–8 of the  P53   gene wereamplified individually using PCR. PCR amplifications were performed in 25   L reaction mixture containing 200–500 ng of genomic DNA and 10 pmol of the fol-lowing sense and antisense primers, respectively, es-sentially as described previously  31 : exon 5, 5  -TTC CTCTTC CTG CAG TAC TCC-3  and 5  -GCC CCA GCT GCTCAC CAT CG-3  ; exon 6, 5  -CAC TGA TTG CTC TTA GGT CT-3  and 5  -AGT TGC AAA CCA GAC CTC AGG-3  ; exon 7, 5  -TCT CCT AGG TTG GCT CTG AC-3  and5  -CAA GTG GCT CCT GAG CTG CA-3  ; and exon 8,5  -CCT ATC CTG AGT AGT GGT AA-3   and 5  -GTCCTG CTT GCT TAC CTC G-3  . PCR parameters were asfollows: 1 cycle at 94 °C for 4 minutes followed by 36cycles at 94 °C for 1 minute, 57 °C for 1 minute, and 72°C for 1 minute. The reaction was terminated by a7-minute of extension at 72 °C. Amplified products were screened for sequencevariations using SSCP analysis, as described previous-ly. 32 Briefly, equal volumes of stop solution (96% for-mamide, 20 mM EDTA, and 0.05% bromophenol blue)and PCR products (6   L each) were mixed, heat de-natured at 98 °C for 5 minutes, and quickly chilled onice until loading. Denatured products were electro-phoresed on 8% native polyacrylamide gels supple-mented with 6% glycerol. Electrophoresis was per-formed at 50 watts in 0.5    Tris-borate-EDTA bufferfor 3 hours at 4 °C. After electrophoresis, gels weresilver stained and photographed. DNA Sequencing DNA samples with a mobility shift band present inSSCP analysis were reamplified using the correspond-ing primer set and the procedure described above.Thirty cycles of PCR reactions were employed. PCRproducts were purified with QIAquick PCR purifica-tion kit (QIAGEN, Valencia, CA), and eluted in 50   LTE buffer. Sequencing reactions were then performedin 10   L of the eluted DNA using one of the primersthat was used srcinally for PCR with a Taq sequencekit from Perkin Elmer. Automatic sequencing was per-formed on an ABI 377 Sequencer (Perkin Elmer, Nor- walk, NJ). Statistical Analysis To evaluate the correlations between p53 overexpres-sion and mutation and clinical variables, we com-pared p53 overexpression and mutation with age (  60 years vs.    60 years), gender (male vs. female),tumor stage at presentation (T1–T4), lymph node in-vasion (present vs. absent), and 5-year case fatality survival (dead vs. alive) using 2-tailed chi-square tests.Immunohistochemical comparisons of p53 overex-pression and mutation with clinical variables betweentwo geographic areas (Zhejiang and Linxian) and within each geographic area also were assessed by chi-square test. The FREQ procedure in SAS software(SAS Institute Inc., Cary, NC) was used. RESULTS Immunohistochemical Analysis Using the DO-7 monoclonal antibody, nuclear p53protein was detected in 47 of 92 ESCC samples (51.1%)(Fig. 1). The correlation of p53 expression with theclinical data and histopathologic characteristics issummarized in Table 1. The rate of positive p53 ex-pression was higher (28 of 47 samples; 68.3%) in pa-tients who had lymph node invasion compared withthe rate in patients who did not have lymph nodemetastasis (19 of 51 samples; 37.3%;  P     0.01). A significant difference in the rate of p53 expression also 836 CANCER August 15, 2004 / Volume 101 / Number 4   was observed between patients who survived for    5 years postoperatively (25 of 60 patients; 41.7%,) andpatients who survived for  5 years (20 of 27 patients;70.1%;  P     0.01). Conversely, the overexpression of p53 protein did not differ significantly among patients with respect to age, gender, or tumor classification. p53 Mutation in ESCC Strict criteria were used, and even the slightest shiftfrom normal mobility of the bands on the SSCP gel was tentatively identified as a candidate for sequencevariation in the amplified products (Fig. 2). Underthese conditions, band shifts were identified in 34 of 92 tumor samples (37.0%), and 29 mutations wereconfirmed in 28 of 92 samples (31.5%) by sequenceanalysis (Table 2). Amplified products from three tu-mor samples that yielded normal mobility patternsand from three corresponding adjacent normal tissuesamples were sequenced, and no mutations werefound in the sequence of the exon 5–8 regions.Twenty-seven mutations were single-base substi-tutions that resulted in amino acid substitutions orchain terminations. Among these mutations, 16 weretransversions (59.3%), and 11 were transitions (40.7%).The other two mutations were single-base insertionsthat led to a frameshift in amino acid sequence of thegene (Table 2). There were a number of mutationsclustered at sequences comprising codons 240–271, which lie within 1 of 2 conserved regions required forbinding to simian virus 40 (SV40) T antigen. 13 There were no correlations between the rate of   P53   genemutation and the variables of age, gender, or tumorclassification. It is worth noting that significantly higher mutation rates were found in patients who hadlymph node invasion and in patients who died within5 years after surgery (Table 1). Geographic Variation of p53 Overexpression andMutation The rate of p53 protein overexpression (29 of 47 tu-mors; 61.7%) was higher in the patients from Linxiancompared with the patients from Zhejiang (18 of 45tumors; 40.0%;  P   0.05). Particularly in patients withT2 tumors, the rate of p53 protein overexpression wassignificantly higher in the patients from Linxian (21 of 31 tumors; 67.7%) compared with the patients fromZhejiang (7 of 21 tumors; 33.3%;  P   0.05). There wasa correlation between the overexpression of p53 pro-tein and 5-year case fatality among the patients fromLinxian ( P   0.05). However, no such correlation wasfound in the patients from Zhejiang (Table 3). Signif-icant differences also were found in the mutation ratesin patients with T2 tumors between Linxian (15 of 31tumors; 48.4%) and Zhejiang (3 of 21 tumors; 14.3%;  P   0.05) (Table 4).Most of the  P53   gene mutations detected in thetumors from patients residing in Linxian were trans-version mutations (12 of 18 mutations; 66.7%). Among these mutations, 10 of 18 mutations (55.5%) occurredin exon 5. In contrast, transitions were the major mu-tation type found in Zhejiang (7 of 11 mutations;63.6%). The latter mostly occurred in exon 7 of the  P53  gene, and 3 were found at codon 260 (Fig. 3). It isnoteworthy that there were four mutations in tumorsfrom Linxian that occurred at a CpG dimer. No suchmutation was found in tumors from Zhejiang (Table2). Moreover,  P53   mutations in tumors from Linxian were dominated by GC  3   TA transversions (7 of 18mutations; 38.9%), whereas the most common  P53  mutations in tumors from Zhejiang were AT  3   GCtransitions (4 of 11 mutations; 36.4%) and GC 3   ATtransitions (3 of 11 mutations; 27.3%). P53 Overexpression and  P53   Mutation There was 69.57% agreement (64 of 92 samples) be-tween p53 overexpression and mutation, with 24 of 92samples (26.09%) that were positive for both and 40 of 92 samples (43.48%) that were negative for bothamong. Statistically, the overexpression of p53 wasassociated with mutations in the conserved regions ( P   0.05); however, no association was found with mu-tation types or specific locations. Geographically, theagreement between p53 overexpression and mutation was 75.5% (34 of 45 samples) and 63.8% (30 of 47 FIGURE 1.  Immunohistochemical analysis of p53 expression in esophagealsquamous cell carcinoma cells. Positive immunostaining for p53 protein waslocalized in nuclei of tumor cells (arrow). The tumor nuclei were stained,whereas the adjacent stroma tissue was unstained. The slided, paraffinembedded tissues were stained with peroxidase-labeled streptavidin-biotinimmunostaining methods using the monoclonal antibody of DO-7 (srcinalmagnification  400). P53   in Geographically Localized ESCC in China/Cao et al. 837  samples) in the Zhejiang and Linxian areas, respec-tively. DISCUSSION Tumsrcenesis occurs when a cell loses its regulatedgrowth cycle and clonally expands beyond control.This requires a series of critical molecular events thatcause the cell to divide and escape from normal pro-liferative control. 33 ESCC may follow this model. Thismalignancy is of particular interest, because epidemi-ologic data suggest that multiple environmental expo-sures are associated with increased incidence. 20 More-over, its clustered geographic distribution suggeststhat specific social, dietary, or heritable characteristicsconfined to a particular region are involved in itsdevelopment. 34,35 Intense research efforts are aimed at identifying alterations in genes involved in the regulation of cellgrowth and differentiation. The product of the  P53  gene plays an important role in the negative regula-tion of cell growth. The wild-type p53 protein binds tospecific DNA sequences as a transcriptional factor thatregulates the expression of particular genes in the cell.Consequently, it blocks cell progression through thelate G1 phase of the cell cycle. 13 Some mutant proteinsfail to block this progression, whereas others can gaina novel function and actually promote cellular prolif-eration. 36 There are significant geographic differences in the P53   mutation rates in esophageal carcinoma. Severalinvestigators have reported  P53   mutations in esopha-geal carcinoma around the world. Audrezet et al. re-ported  P53   mutations in 84% of ESCCs from France. 16 Tamura et al. reported P53  mutations in 38% of esoph-ageal carcinomas from Japan. 37 Gates et al. andGamieldien et al. reported that 67% and 17% of esoph-ageal tumors were associated with  P53   mutations incoastal South Carolina 38 and South Africa, 39 respec-tively. Previously, Bennett et al. reported  P53   muta-tions in 50% of tumors from China. 29 In the currentstudy, p53 protein expression and mutations were ex-amined in patients from two separate geographic re-gions with very different ESCC incidences in China.Overexpression and mutations of   P53   were noted in 47of 92 tumor samples (51.1%) and 28 of 92 tumor sam-ples (30.4%) from patients with ESCC in Linxian andZhejiang, respectively. No obvious differences werefound in the mutation rates between the patients fromthese two different geographic areas. This may havebeen due in part to the fact that mutation detection was focused on 1 of 4 conserved regions in the  P53  gene: exons 5–8. 36 However, the mutation rate in  P53  TABLE 1Correlation of p53 Over Expression/Mutation with Clinical Data and Histopathologic Characteristics  Variable No.p53 Protein over expression  P53   Mutation (%)    Positive %  P   value     Positive %  P   value Summary 92 47 45 51.1 — 29 a 64 30.4 — Age  60 yrs 53 31 22 58.5   0.05 15 38 28.3   0.05  60 yrs 39 16 23 41.0 2.74 b 13 26 33.3 0.267 b GenderMale 64 36 28 56.3   0.05 16 48 25.0   0.05Female 28 11 17 39.3 2.24 b 12 16 42.9 2.93 b Tumor classification c T1 14 6 8 42.9   0.05 1 13 7.1   0.05T2 52 28 24 53.9 18 34 34.6T3 25 13 12 52.0 8 17 32.0T4 1 1 0 1 0Lymph node invasionPresent 41 28 13 68.3   0.01 d 19 22 46.3   0.01 d  Absent 51 19 32 37.25 8.76 b 9 42 17.6 8.84 b 5-yr case fatality  Alive 60 25 35 41.7   0.01 d 14 46 23.3   0.05 d Dead 27 20 7 70.1 7.831 b 13 14 48.1 5.01 b  : Positive:  : negative. a Mutations in two codons of   P53   were found in one patient. b Statistically significant (chi-square test). c Classification of primary esophageal carcinoma: T1, invades lamina propria or submucosa; T2, invades muscularis propria; T3, invades adventitia; T4, invades adjacent structures. d  A significant association was found in the statistical analysis. 838 CANCER August 15, 2004 / Volume 101 / Number 4
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