Proc. Natl. Acad. Sci. USAVol. 90, pp. 4961-4965, June 1993Medical Sciences

p53 gene mutations and protein accumulation in humanovarian cancerJOLANTA KUPRYJA*CZYK*, ANN D. THOR*, ROBERTA BEAUCHAMPt, VICTOR MERRITTt,SUSAN M. EDGERTON*, DEBRA A. BELL*, AND DAVID W. YANDELLt,§*Department of Pathology, Harvard Medical School, and the James Homer Wright Laboratories of Massachusetts General Hospital, Boston, MA 02114;tDepartment of Cancer Biology, Harvard School of Public Health, Boston, MA 02115; and tMolecular Genetics Research Laboratory, MassachusettsEye and Ear Infirmary, Boston, MA 02114

Communicated by Ruth Sager, February 1, 1993

ABSTRACT Mutations of the p53 gene on chromosome17p are a common genetic change in the malignant progressionof many cancers. We have analyzed 38 malignant tumors ofovarian or peritoneal mullerian type for evidence of p53variations at either the DNA or protein levels. Genetic studieswere based on single-strand conformation polymorphism anal-ysis and DNA sequencing ofexons 2 through 11 of the p53 gene;mutations were detected in 79% of the tumors. These data showa statistically significant association between mutations at C-Gpairs and a history of estrogen therapy. Two of 20 patientswhose normal tissue could be studied carried germ-line muta-tions of p53. Immunohistochemical analysis of the p53 proteinwas carried out using monoclonal antibody PAb1801. Ninety-six percent of the missense mutations were associated withabnormal accumulation of p53 protein, but nonsense muta-tions, a splicing mutation, and most deletions did not result inp53 protein accumulation. A statistically significant associationbetween p53 protein accumulation in poorly differentiatedstage Im serous carcinomas and small primary tumor size atdiagnosis was found, perhaps suggesting that p53 proteinaccumulation accelerates the metastatic spread from a primarytumor. Overall, our findings indicate that alterations of p53play a major role in ovarian cancer, including predisposition tothe disease in some patients, and suggest a possible mechanismfor somatic mutations leading to this cancer.

The p53 tumor suppressor gene on chromosome 17p encodesa nuclear 393-aa phosphoprotein thought to play a role in cellcycle progression (1, 2). Mutations of p53 may result in abiologically altered protein, including increased stability andaccumulation detectable by immunohistochemical methods(3-6). Mutant p53 protein is characterized by loss of antipro-liferative activity and may have a dominant negative effectvia complex formation with the wild-type protein (7). Studiesindicate that p53 mutations occur most often in the evolu-tionarily conserved domains, particularly exons 5-9 (2, 8). Avariable mutation spectrum has been observed in humantumors, with indications that exposure to specific mutagensmay underlie the variations in some cases (9-12).The pathogenesis of ovarian cancer and the factors leading

to this disease are poorly understood. Although p53 genealterations are common in human tumors (2, 11, 13-15), theirrole in the etiology of ovarian cancer is unclear. Severalearlier studies have suggested that p53 may play a role inovarian tumor formation (16-18). We describe an exhaustiveanalysis of p53 gene alterations in 35 malignant ovariantumors and 3 peritoneal tumors of mullerian type. Thesestudies included single-strand conformation polymorphism(SSCP) analysis ofthe entire coding sequence of the p53 genefollowed by DNA sequencing of variants. We also performed

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immunohistochemical analysis on all samples for accumula-tion of the p53 protein and compared all of these data toepidemiologic, clinical, and histologic findings.

MATERIALS AND METHODSPatients and Clinical Samples. Tumor specimens were

derived from 38 patients treated for ovarian cancer at theMassachusetts General Hospital between 1985 and 1991; agesof these patients ranged from 38 to 89 years. The specifictumor types studied are shown in Table 1. Tumor 21 (amucinous adenocarcinoma) had a component of borderlinemalignancy (19). Two patients were diagnosed with synchro-nous endometrial cancers (patients 6 and 32).Most tissues studied were from the initial surgical resection

(36 tumors); two samples involved only tumors that recurredafter chemotherapy (samples 29 and 33). All tumors werestaged according to the criteria of the International Federa-tion of Gynecologists and Obstetricians (20) and graded 1 to3 according to their nuclear features (21). Thirty-four tumorswere in stage III or VI, 2 were in stage II (tumors 37 and 38),and two were in stage I (tumors 21 and 33). All tumors werepoorly differentiated except for the two mucinous carcino-mas that were moderately differentiated. Medical records of36 patients were available for review, and 21 patients re-ported one or more close relatives with cancer. Thirteenpatients had one or more family members affected by ovariancancer, breast cancer, or multiple primary cancers. Fourpatients in this study had been treated previously for cancer(patient 9, squamous cell carcinoma of the vulva; patients 10and 24, basal cell carcinoma of the skin; patient 27, indepen-dent breast and colon carcinomas). Data on other risk factorswere also gathered, including: gravidity and parity (8 nullip-arous patients), peri- or postmenopausal estrogen therapy (6patients; Table 1), morbid obesity (4 patients), positivesmoking history (12 patients), and previous laparotomies (21patients). The median follow-up time was 19 months for thegroup as a whole and 21 months for survivors.To compare primary tumor sizes with other criteria, the

maximum tumor diameter involving the ovary was takenfrom the pathology reports. When bilateral ovarian tumorswere present, the diameter of the larger tumor was used. Forstatistical analysis, three size groups of tumors were arbi-trarily established that gave a roughly equal number ofsamples in each group: tumors c5 cm, >5 cm but <13 cm,and .13 cm.

Materials for DNA Analysis. For molecular genetic studies,tissues were snap-frozen in liquid nitrogen and stored at-80°C. These included 16 ovarian tumors, 17 omental met-

Abbreviations: SSCP, single-strand conformation polymorphism;LOH, loss of heterozygosity.§To whom reprint requests should be addressed at: Howe Labora-tory, Molecular Genetics Room 575, Massachusetts Eye and EarInfirmary, 243 Charles Street, Boston, MA 02114.

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Table 1. Histological tumor type and immunohistochemical and molecular genetics resultsMutation

Case Type Immunostaining Amino acid, no. Physical Functional LOH1 S + NMF NA2 S*- 332 C del Stop codon at aa3443 S* + 195 ATC to ACC Ile to Thr4 S NMF5 S - 192 CAG to TAG Gln to stop +6t E -, SC NMF7 S + 248 CGG to TGG§H Arg to Trp8 S + 220 TAT to TGT Tyr to Cys9 S - 342 CGA to TGA§ Arg to stop +lot S +, - intron 5, 5' splice CAG to CGG +11 S + 220 TAT to TGT Tyr to Cys -

12 S* + 214 CAT to CGT His to Arg -

13 S + 273 CGT to TGT§ Arg to Cys -

14 S NMF -

15 S* + 163 TAC to TGC Tyr to Cys -

16 S + 273 CGT to CAT§ Arg to His +17 S + 237 ATG to ATAI Met to Ile18t S/P -, SC NMF NA19 S/P + 165 CAG to CGG Gln to Arg +20 E + 273 CGT to CAT§ Arg to His +21 M + 277 TGTto TTT Cys to Phe +22 M NMF NA23 S* + 238 TGT to TAT Cys to Tyr24 S + 248 CGG to TGG§ Arg to Trp +25 S + 135 TGC to TAC Cys to Tyr +26 S + 175 CGC to CAC§H Arg to His +27t S* -, SC NMF NA28 TCC - 88 G del Stop codon at aa122 +29t S - 135 G del Stop codon at aa16930 S + 248 CGG to TGG§ Arg to Trp +31 S + 251 ATC to AGC Ile to Ser +32 S/P + 245 GGC to AGC§ Gly to Ser NA

279 G del Stop codon at aa344 NA33tt GCT +, SC NMF34 S + 176 TGC to CGC Cys to Arg35 TCC + 234 TAC to TGC Tyr to Cys36 S - 262, 5' splice GGT to AGT Gly to Ser +37 CS + 175 CGC to CAC§ Arg to His +38 U + 175 CGC to CAC§ Arg to His +

CS, carcinosarcoma; E, endometrioid; GCT, granulosa cell tumor; M, mucinous; S, serous; P, peritoneal; TCC,transitional cell carcinoma; U, undifferentiated; SC, single positive cells; del, deleted; NA, data not available; del, deletion;NMF, no mutation found.*Carcinoma was present on the ovarian surface with or without infiltration of the ovary.tTwo results of immunostaining reflect differences in the category of staining among tumor blocks from one patient.*Tumors from second-look surgeries.§G-C to A-T transition at a CpG pair.VPatient received peri- or postmenopausal estrogen therapy.

astatic tumors, and 5 tumors for which the specific site ofresection could not be established. Frozen sections werestained with hematoxylin/eosin to select tissue with thesmallest possible admixture of nonmalignant cells, and thenormal cell fraction was estimated for each sample. Theselected portion of each frozen specimen was trimmed andkept at -70°C until DNA extraction.Paraffin-embedded normal tissues from 20 patients were

available. To confirm that there was no contamination of thenormal specimens by tumor cells, tissue sections were ex-amined before and after taking material for DNA analysis.DNA from frozen and paraffin-embedded tissues was ex-tracted (22).SSCP Analysis. For analysis of genomic DNA by SSCP,

p53 exons 2-11 were amplified in 14 PCRs as described (23).Amplified fragments from exons 2, 4, and 10 were digestedwith an appropriate restriction endonuclease to increase thesensitivity of the SSCP analysis (23). PCR products were

analyzed twice on nondenaturing 6% polyacrylamide gels,one of which contained 10% (vol/vol) glycerol. Electropho-resis was performed at ambient temperature for 8-16 h.

Direct Genomic Sequencing. Direct genomic sequencing ofdouble-stranded PCR fragments was performed using proto-cols as described (24). In short, amplified DNA samples werepurified through Sepharose CL-6B (Pharmacia LKB) andsequencing reactions were carried out using the Sequenaseenzyme (United States Biochemical).Loss of heterozygosity (LOH) affecting the p53 gene

(Table 1) was estimated on the basis ofthe intensity of variantvs. normal bases in DNA sequencing data (including analysisof point mutations, neutral polymorphisms, or both) and thepathologist's quantitative estimate of normal cell content. Insamples 1, 18, 22, and 27, LOH could not be estimated intumors because of a lack of informative DNA markers; intumor 32, a high nonmalignant cell content made it impossibleto score LOH.

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Immunohistochemical Analysis. Two to five paraffin tissueblocks from each tumor were studied for p53 reactivity (124in total); in all but two tumors (tumors 29 and 33), at least oneblock from the primary tumor was studied. In 28 patients,both primary and metastatic tumors were studied. Immuno-staining was carried out using a mouse monoclonal IgGlantibody PAb1801 (Cambridge Research Biochemicals,Wilmington, DE), recognizing an epitope between aa 32 and79 of the p53 protein (25). The specific methodology andcontrol assays have been described in detail elsewhere (6).The immunohistochemical assays for p53 were scored

according to the following categories: N, negative; SC,nuclear staining in isolated single tumor cells; FP, focalpositivity, <5% positive tumor cells; LP, low positivity,5-40% positive tumor cells; HP, high positivity, 41-100%positive tumor cells.

Statistical Analysis. Associations of the immunohistochem-ical, molecular genetic, histopathological, or clinical param-eters were analyzed by Fisher's exact test (26). The associ-ation between p53 protein accumulation and overall survivaltime for patients with stage III serous carcinomas wasevaluated by the log-rank test.

RESULTSMolecular Genetic Analysis of p53. We found a total of 31

mutations in 30 of the 38 tumors we studied (79%); 27 werebase-pair substitutions and 4 were 1-bp deletions (Fig. 1 andTable 1). Most of these changes occurred within exons 5-8,including 1 mutation that was found in a splice site flankingexon 6. Three mutations were found outside conserveddomains of p53 (1 in exon 4 and 2 in exon 10).Of 31 mutations, 24 resulted in missense errors that led to

a change in the amino acid sequence. Two base-substitutionmutations resulted in a new stop codon; five additionalnonsense mutations were due to either single-base deletionsor a splice-site mutation. One mutation (sample 36), a G -3 Atransition at codon 262, apparently changes a glycine to aserine residue; however, this may affect splicing due tocreation of a new 5' consensus site within the exon. Amongbase substitutions, there was a prevalence of transitions (25of 27 or 93%) over transversions (2 of 27 or 7%). Mosttransitions were G-C to A-T changes (16, or 60%o of allsubstitution mutations), and 11 of these occurred at CpGdinucleotide pairs (Table 2). Ten mutations (32% of allmutations detected) occurred in codons 175, 245, 248, or 273,which are known mutational hot spots in the p53 gene (15).A mutated CpG dinucleotide was also found in exon 10, atcodon 342.

Thirteen of the 30 tumors with mutations were heterozy-gous at p53 (Table 1). Heteroallelic point mutations wereidentified in one tumor (tumor 32). In the 16 remainingtumors, LOH was observed; these samples included roughlyequal proportions of primary and metastatic tumors.

B

A

Variant-_

Table 2. p53 gene base substitutions in ovarian cancerType Total number % of total

Total substitution mutations 27 100Transition

Total 25 93G-C -- AT 16 60CpG - TpG 11 41A-T G-C 9 33

TransversionTotal 2 7GC T.A 1 3.5T*A G*C 1 3.5

Denominator for all percentages is total substitution mutations.

A neutral genetic polymorphism (Arg -- Arg) at codon 213(27) was found in 3 of 38 tumors (8%). In 8 tumors (21%), nogenetic alterations were detected in the studied sequences.

p53 Mutations and Clinicopathological Data. Data regardingestrogen treatment were available on 36 of the 38 patients.Five of 11 patients whose tumors carried mutations at CpGdinucleotides and 1 of the remaining 25 patients had takenestrogens perimenopausally or postmenopausally (Table 1).The association between mutations at CpG dinucleotides anda history of estrogen therapy was statistically significant withP < 0.01. There was no association between the presence ofp53 mutations and a family history of cancer (n = 21/36),nulliparity (n = 8/36), or previous laparotomies (n = 21/36)nor were differences apparent in the p53 mutation spectrumbetween patients who had smoked (n = 12/36) and those whonever smoked. We did not find any association betweenmolecular genetic abnormalities and primary tumor size,grade, histologic type, or clinical stage.

Immunohistochemistry and Clinicopathological Data. A tu-mor was positive for p53 accumulation (Fig. 2A) if, atminimum, focal nuclear staining was present. By this defi-nition, 26 of the 38 tumors were positive (68%), 9 werenegative, and 3 were indeterminate. In 33 of 38 tumors,staining was the same among sections prepared from differentblocks from the same case. A discrepancy was seen in 1tumor (tumor 10), which was scored positive. Three tumorswith some negative and some single-cell positive sectionswere called indeterminate (Fig. 2B). One other tumor (tumor33) had sections with both single positive cells and focalpositivity and was scored positive. Within each specimen,including the positive control cell line MDA-MB-231, weobserved heterogeneity of staining among nuclei (Fig. 2A).Metastatic tumors (n = 28) did not differfrom their respectiveprimary tumors in p53 immunopositivity.

In the only stage I carcinoma studied (tumor 21), whichdeveloped in a borderline tumor (19), p53 protein was accu-mulated in both the borderline and malignant components.p53 positivity was observed in all types ofcarcinoma studied,in both carcinomatous and sarcomatous components of thecarcinosarcoma, and in the granulosa cell tumor.

Normal Variant

C T A G C T A G

1 2 3 4 5 6

TACACA

T-.- A

C/AACA

FIG. 1. (A) SSCP analysis of exon 8 ofthe p53 gene in DNA derived from ovariantumor specimens (lanes 1-6). Bands comi-grating with normal PCR fragments aremarked by an arrowhead; a variant allele inlane 3 is marked by an arrow. (B) DNAsequence analysis of the variant SSCP bandseen in A.

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For evaluation of the association between primary tumorsize at time of surgery and p53 accumulation, only serous

carcinomas at stage III were used (19 tumors exclusive ofsurface and peritoneal carcinomas). The association betweensmall primary tumor size and p53 immunopositivity was

statistically significant (P < 0.01). There was no associationbetween p53 accumulation and tumor grade, clinical stage, or

overall survival of patients with stage III or IV serous

carcinomas with follow up information (n = 24 patients).Molecular Genetics and Immunohistochemistry. The overall

fraction of genetic alterations that were detected by immu-nohistochemistry was 80% (24 of 30 tumors with p53 muta-tions). However, 96% (24 of 25) of the missense mutationswere detected, and the one apparent missense mutation thatdid not result in accumulation of p53 protein was a pointmutation of codon 262 near the 5' splice site (tumor 36) thatcreates a new splice consensus sequence. The only tumorwith both immunohistochemically positive and negative tu-mor blocks (tumor 10) had a splicing mutation in the 5' spliceconsensus sequence for exon 5.Two nonsense mutations and three of four deletions that

resulted in a stop codon (tumors 2, 5, 9, 28, and 29) did notcause the accumulation of p53 protein. In tumor 2, themutation was a single-base deletion in codon 332; in tumor 32,the mutation was a single-base deletion in codon 279. Bothdeletions resulted in frameshift errors and a new stop codondownstream at codon 344. Only tumor 32 showed accumu-lation of the protein, most likely because this tumor carriedheteroallelic p53 mutations, with the second being a missensemutation at codon 245 (Table 1).There was no correlation between the specific mutation or

LOH at p53 and the category of staining in immunohis-tochemically positive tumors. In one serous ovarian carci-noma (tumor 1), staining was scored as high positive, but no

genetic alterations were detected by SSCP or DNA sequenc-ing. Genetic alterations were not detected in tumor 33 (agranulosa cell tumor), having single cells positive in someblocks and focal positive staining in others, nor in any tumorwith single immunohistochemically positive cells.

p53 Analysis of Benign Tissues. Paraffin-embedded normaltissues were available from 20 of the 30 patients with p53genetic abnormalities in malignant tissue. In two patients(tumors 10 and 32), a mutation identified in the tumor was

found in histologically reviewed normal tissues, consistentwith a germ-line mutation (unpublished data). All benigntissues tested gave negative immunohistochemical staining,including both patients with germ-line p53 nutations.

DISCUSSIONStudies of ovarian cancer have suggested that p53 mutationmay represent an important step in disease progression, butour frequency of p53 mutation (79%) is substantially higherthan the 30-50% reported for ovarian cancer (16-18). One

FIG. 2. p53 protein accu-mulation in serous ovarian car-cinomas (paraffin sections,PAb18O1 monoclonal anti-body, streptavidin-biotin-per-oxidase complex, and methylgreen counterstaining). (A)Highly positive reaction withheterogeneous cellular stain-ing. (X40.) (B) Single positivecell. (x160.)

possible explanation for this discrepancy may be that wehave used a combination of techniques to detect p53 alter-ations including both immunohistochemistry and moleculargenetic analysis of the entire coding sequence (exons 2-11) ofthe p53 gene. This explanation is supported by our detectionof genetic alterations outside of the highly conserved regionsof p53 that most other genetic studies of p53 have ignored.This includes two nonsense mutations and three of fourdeletions that resulted in premature stop codons and wouldnot have been detected by immunohistochemical screeningwith PAb1801 alone.A comparison of our data on molecular genetic vs. immu-

nohistochemical analysis of p53 using antibody PAb1801provides clarification as to the effectiveness of the latterapproach for screening tumors. The majority of mutations weidentified by immunohistochemistry were missense errors inthe conserved domains of p53, and a strong concordancebetween abnormal p53 accumulation and the presence of amissense mutation was observed. The only missense muta-tion that did not cause accumulation of p53 protein is morelikely to result in a gross error in splicing than a single-aminoacid substitution and, hence, may be better classified as anonsense mutation. In another tumor, a single-base substi-tution in a splice consensus sequence was associated withinconsistent immunostaining among specimens from thesame tumor. Splice site mutations may affect transcription inways that cannot always be predicted on the basis of DNAanalysis, leading to the absence of detectable p53 protein inat least some tumors (28). Mutations that are present in onlya small subclone of cells (which may represent the earlieststages of malignant progression in a tumor) may appear asfocal and/or single-cell positivity by immunohistochemicalstaining but would not be detected as changes at the geneticlevel using the methodology we have applied. Tumor 33 maybe such an example, in which single positive cells and focalp53 protein accumulation were observed, but no moleculargenetic abnormalities were detected. Our combined immu-nohistochemical and molecular genetic analyses showed thatp53 mutations were present in both primary and metastatictumor in all patients where both were available for compar-ison. We also observed p53 immunopositivity in a borderlinecomponent of one tumor, and in both carcinomatous andsarcomatous components of a carcinosarcoma. Thus, thesedata suggest that p53 mutations may often be early events inthe progression of ovarian tumors.These data have clinical relevance. Since increased levels

of mutated p53 protein may be necessary for inactivation ofwild-type p53 protein (29-31), the biological consequences ofdifferent mutations may vary. Even among mutations leadingto accumulation of p53 protein, there is variable malignantpotential (2, 7, 28). When the presence or absence of amutation was used as a variable, no correlation with theclinical parameters studied, including survival, was apparent.When immunopositivity was considered, an association wasseen with small primary tumor size at diagnosis among ahomogeneous group of ovarian serous carcinomas. Oneinterpretation of this finding is that tumors that do notaccumulate p53 protein may reach a larger size before theyare first symptomatic and diagnosed, whereas tumors carry-ing missense p53 mutations may metastasize earlier and are,hence, symptomatic earlier due to metastasis.The spectrum ofDNA damage we observed is of particular

interest. The majority of the p53 mutations we found (52%)were G-C to A-T transitions, and a majority of these occurredat CpG dinucleotides; transversions accounted for only 7% ofthe mutations. This physical mutation spectrum is verysimilar to that reported by others for colon cancer but differsfrom data obtained on breast or other cancers (15); it is alsodramatically different from our study (23) of p53 in 127sarcomas, which was based on identical molecular genetic

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analysis and, hence, is appropriate for direct comparison.These findings strongly support the supposition that physicaland functional aspects of p53 mutations may be highlyvariable from one tumor type to another, due to either afunctional selection in some tissues or, alternatively, due todivergent mutagen exposure in different organ systems.Eleven mutations detected in our tumors occurred at CpG

dinucleotides, and we found a statistically significant asso-ciation between mutated CpGs and a history of estrogentreatment among these patients. CpG dinucleotide pairs area hot spot for inherited mutations in humans (32), due tospontaneous deamination of 5-methylcytosine at methylatedCpG pairs (33). It has also been reported that the apparentmutational hot spots at p53 codons 175, 248, and 273 may bedue to this mechanism (34). The correlation we have ob-served between estrogen treatment and mutations at CpGsthus may not be coincidental, as it has been shown thatestrogens influence the level of methylation at 5-methylcy-tosine in human DNA (35) and that the carcinogenic effectsof methylating mutagens may be mediated by estrogenicaction in some cases (36). However, our study is based on arelatively small sample size and our findings do not neces-sarily indicate a cause-and-effect relationship.The analysis of normal tissues from 20 of these patients

revealed germ-line p53 mutations in two patients (10%).Germ-line mutations of p53 were first found in families withLi-Fraumeni syndrome (37, 38). Although ovarian cancer isnot a common component tumor of the Li-Fraumeni syn-drome (39), significant evidence suggests that germ-line p53gene mutations may be associated with a more variableclinical presentation than suggested by the definition ofLi-Fraumeni syndrome (40-42). Unfortunately, limited datawere available on both germ-line carriers in this study,though neither carrier reported a remarkable family or per-sonal history of cancer.Thus, our data demonstrate that p53 gene mutations are

common in ovarian cancer. Alterations resulting in an accu-mulation of protein represented the majority of mutations,and comparison of molecular genetic and immunohistochem-ical assays using antibody PAb1801 showed excellent con-cordance between the presence of missense mutations andpositive staining. Further studies are needed to determine thebiological role ofp53 gene alterations in the development andprogression of this disease, and the potential prognostic valueof p53 analysis for ovarian cancer patients.

We thank Dr. R. E. Scully for advice and critical reading of themanuscript, Dr. J. Bovari for assisting in tissue collection, Drs. D.Schoenfeld and T. Flotte for assistance with statistical analysis, andC. Connery for help in preparing the manuscript. This research wassupported in part by National Institutes of Health Grant CA-44768,and a grant from the Center for Radiation Therapy. J.K. (whileaffiliated with the Medical Academy, Warsaw) was the recipient ofa Fulbright-Hayes Fellowship.

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