p53 Mutations in Lung Cancer following Radiation Therapy ... · lung cancer cases from the Netherlands and Ontario. These subjects were included in prior studies of Hodgkin’s disease

5ol. 5. 93-98. February /996 Cancer Epidemiology, Biomarkers & Prevention 93

p53 Mutations in Lung Cancer following Radiation Therapy forHodgkin’s Disease

Virna M. G. De Benedetti, Lois B. Travis,Judith A. Welsh, Flora E. van Leeuwen, Marilyn Stovall,E. Aileen Clarke, John D. Boice, Jr., andWilliam P. Bennett’

Laboratory of Human Carcinogenesis [V. M. G. D. B., J. A. W., W. P. B.l

and Radiation Epidemiology Branch [L. B. T.. J. D. B.J, National Cancer

Institute, Bethesda, Maryland 20892; The Netherlands Cancer Institute.

Department of Epidemiology. 1066 CX Amsterdam, The Netherlands

[F. E. v. Li: Department of Radiation Physics. The University of Texas,M. D. Anderson Cancer Center, Houston, Texas 77030 [M. S.]; and Ontario

Cancer Treatment and Research Foundation, Toronto, Ontario, Canada

M5G2L7 [E. A. C.[

Abstract

High risks of lung cancer occur after successful treatmentof Hodgkin’s disease. In addition to tobacco smoking,other risk factors include radiotherapy, chemotherapy,and immunosuppression, although the relativecontributions of each are unknown. We conducted p53mutational spectrum analysis in second lung cancersafter radiation therapy for Hodgkin’s disease in theNetherlands and in Ontario, Canada. Lung cancertissues from 1 1 patients were analyzed by p53immunohistochemistry and DNA sequence analysis. Allwere male cigarette smokers, all received radiationtherapy, and six also received chemotherapy. The lungcancers occurred 9.8 years (mean) after treatment.Radiation doses to lung lobes that developed the tumorsaveraged 5.7 Gy (range, 3.7-1 1.7 Gy). Sequence analysisshowed four missense and two silent p53 point mutationsin five patients. There were four G:C-*A:T transitions;three of four mutated deoxyguanines occurred on thecoding strand, and one was a CpG site. There were twotransversions: one G:C-*C:G and one A:T-sC:G.Despite moderate or heavy smoking histories in allpatients, the mutational spectrum appears to differ fromusual smoking-related lung cancers in which G:C-�T:Atransversions predominate. The absence of G:C-sT:Amutations and the prominence of G:C-�A:T transitions,which are characteristic of radiation and oxidativedamage, suggest that radiotherapy might have causedsome of the p53 mutations. These data illustrate thepotential of mutation analysis to determine causes ofhuman cancer. If confirmed in a larger series, these

Received 7/19/95; revised I 1/7/95; accepted I1/9/95.

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I To whom requests for reprints should be addressed, at Laboratory of Human

Carcinogenesis. National Cancer Institute. Building 37, Room 2C25. 37 Convent

DR MSC 4255. Bethesda. MD 20892-4255. Phone: (30l)-496-4668; Fax: (301)-

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results imply that some radiation-induced cancers can bedistinguished from those caused by other factors.

Introduction

Hodgkin’s disease patients have a 2-8-fold increased risk of

developing lung cancer ( 1-4) related in part to the mutageniceffects of cancer treatment (5, 6). Associations with therapeuticexternal beam radiation and lung cancer risk have been reportedfor patients with Hodgkin’s disease (7, 8) and breast cancer (9,10), further strengthening this association. The role of smokingin pulmonary neoplasia complicates the analysis of risk factorssuch as radiation. Epidemiological studies suggest that tobacco

and radiotherapy may interact in a more than additive mannerto produce lung cancer after Hodgkin’s disease (8) and breast

cancer (9, 10), although the evidence is not strong (1 1, 12).Mutations driving carcinogenesis in lung cancer afterHodgkin’s disease could be generated by radiation, tobacco,

oxidative damage, and/or chemotherapy in a setting of immu-

nosuppression (2, 3, 7). The relative contributions of these

factors have not been characterized.Mutations in the p.53 tumor suppressor gene occur in about

one-half of human lung cancers, and most are point mutations

in the highly conserved, DNA-binding domain (reviewed inRefs. 13, 14). In tobacco-related lung cancers, these mutations

are predominantly G:C-s’T:A transversions on the nontran-scribed, DNA-coding strand ( I 3, 1 5). Despite extensive smok-ing histories, lung cancers in uranium miners exposed to radon

gas have different mutation patterns than those seen in heavysmokers; these mutations may vary with the amount of radon

gas exposure (16, 17). These data suggest that radiation mightalter the p53 mutation spectrum, despite the influence of a

potent carcinogen, such as tobacco smoke. To determinewhether a distinctive mutation profile occurred among patientswho develop lung cancer after external beam radiotherapy for

Hodgkin’s disease, we studied all available histopathologicalmaterial for patients identified in two cohort studies. The mu-tational spectrum observed within our series suggests that ra-diation may cause genetic damage that initiates lung cancerafter treatment for Hodgkin’s disease.

Materials and Methods

All available tissue samples were obtained from second primary

lung cancer cases from the Netherlands and Ontario. Thesesubjects were included in prior studies of Hodgkin’s disease (4,

8, 18). For this study, the tissue samples came from malecigarette smokers, 8 of 3 1 cases from the Netherlands and 3 of

14 from Ontario. Only I I (24%) of 45 tumors in the combinedseries could be examined because many tumors were unresect-

able, and many diagnostic biopsy and cytology samples are toosmall to be fully tested using current methods. All received

radiation therapy, and six also received chemotherapy; thelung cancers occurred 9.8 years (mean) after treatment. In the

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94 p53 Mutations in Radiation-associated Lung Cancer

Table 1 Lung cancer following treatment for Hodgkin’s disease: patie nt summaries

HD’ Lung cancer

Total IntervalPt

.I. . .

Age /sex HD HD dtagnosts

(yr) stage (month/yr)

. .Initial therapy Subsequent therapy

radiation�

dose

(Gy)to lung

between

HD and

lung cancer

(yrs)

.Site of lung

cancer

.Histology

1” 40/Male I 5/73 RT: anterior and posterior upper

mantle

None 4.6 2.8 -‘ SCCA

2 37/Male Il I /66 RT: anterior and posterior chest

RT: left axilla

RT: anterior and posterior

epigastrium

RT: anterior and posterior pelvis

VLB. 6 months

CTX, VLB and PROC - 17 cycles

CTX. 44 months

5.6 10 Right upper lobe SCLC

3 33/Male III 7/74 RT: Anterior and posterior mantle

and abdomen

MOPP, 6 cycles

None 5.8 7.3 Left upper lobe SCCA

4 26/Male Ill 4/67 RT: mantle. inverted Y, inguinal

(left and right)

MOPP, 8 cycles 5. 1 12.2 Right upper lobe SCCA

5 49/Male II 1172 RT: mantle, inverted Y None 4.5 8.1 Right upper lobe SCCA

6 56/Male I 2/73 RT: mantle. para-aortic. spleen +

splenic hilumNone 5.6 9.5 Right upper lobe SCCA

7 38/Male N/A’ 6/62 RT: cervical (left): axillary (left) Cervical (right), mantle, para-aortic,

spleen and splenic hilum

MOPP, 7 cycles

6.0 17.8 Right bronchus SCLC

8 47fMale III 12/80 RT: Waldeyer ring, cervical (left +

right), jugular + mediastinal, para-aortic + spleen + splenic hilum

MOPP, 3 cycles

CTX. ADR, TENI - 3 cycles

None 3.7 9.3 Right upper lobe Adeno-

carcinoma

9 24/Male I I 1/65 RT: cervical (left) RT: cervical (left and right), axillary

(left), mediastinal4.3 15.3 Right upper lobe SCCA

10 50/Male I 9/80 RT: mantle, para-aortic. spleen +

splenic hilumNone I 1.7 I .3 Left upper lobe Adeno-

carcinoma

I I 46/Male II 10/74 RT: mantle, para-aortic. spleen +

splenic hilumRT: L4-L5, iliac (left) VLB +

PROC6.6 14.3 Left upper lobe SCCA

‘, HD, Hodgkin’s disease: CTX. cyclophosphamide: MOPP. mechlorethamine. oncovin, procarbazine, prednisone: RT, radiotherapy; SCLC, small cell lung cancer; SCCA,squamous cell carcinoma: TENI, teniposide: VLB, vinblastine.

I, Age at diagnosis of Hodgkin’s disease.,. Dose to lobe of lung in which second primary cancer later occurred. Radiotherapy was administered by Cobalt-60 (Patients 1-4). megavoltage (patients 5. 6, 8-1 1). or

orthovoltage and megavoltage (Patient 7).

d Patients 1-3 were contributed by the Ontario Cancer Treatment and Research Foundation, Toronto, Ontario; Patients 4-1 1 were contributed by the Netherlands Cancer

Institute, Amsterdam, The Netherlands.

‘ -‘ Site unknown. Each lobe of lung had received 4-6 Gy.I N/A, not available.

Netherlands series (2), 31 lung cancers developed among 1939patients versus 8.3 expected, indicating a nearly 4-fold excessover the general population expectation.

Radiation dose to lobe of lung was estimated for eachpatient in whom a second cancer developed, using details ofradiotherapy abstracted from the treatment record. In regionsoutside the radiation beam, absorbed dose to the lung wascalculated, based on measurements in water phantoms ( 19). Forregions inside a treatment beam, doses were calculated using

conventional treatment planning techniques. All dose estimatestook account of lung blocking where appropriate. Data on

treatment fields, radiation energies, absorbed dose to lung,interval between Hodgkin’s disease and lung cancer, and other

pertinent clinical information are summarized in Table I.

p53 Immunohistochemistry. Immunochemical analysis wasperformed using conventional peroxidase methods as reportedpreviously (20). Tissue sections were incubated overnight at

4#{176}Cwith a saturating dilution of a polyclonal rabbit antiserum,CM-I, raised against full-length p53 protein (21). Localization

of the primary antibody was detected by an avidin-biotin,peroxidase system (Vectastain Elite kit; Vector Laboratories,

Burlingame, CA). A positive control section was included ineach experiment; nonneoplastic tissues within each sectionserved as internal negative controls. Results were analyzedsemiquantitatively and divided into four groups: Neg, no flu-clear staining present; 1 + , < 10% of tumor nuclei stained; 2+,10-70% of tumor cell nuclei stained; and 3+, >70% of tumornuclei stained.

Genomic DNA Extraction, p53 Amplification, and Se-quence Analysis. Paraffin-embedded tumor samples were de-waxed and microdissected from 50-j.tm sections. GenomicDNA was isolated by SDS/proteinase K treatment (final con-

centration, 1% SDS and 0.5 mg/ml of proteinase K) at 50#{176}Cfor12 h, followed by phenol-chloroform extraction, ethanol pre-

cipitation, and resuspension in 50 j.d of sterile water. Nonneo-

plastic tissues flanking the tumor were isolated separately forgermline analysis.

The most commonly mutated coding sequences (i.e. , ex-ons 5-8) were analyzed in all 1 1 tumors, and the occasionally

altered exons (i.e., exons 4, 9, and 10) were examined in tumorsamples that were positive by immunochemistry but negativefor mutation in exons 5-8. For each sample, there were two



Cancer Epidemiology, Biomarkers & Prevention 95

Table 2 p53 analysis in 1ung cancers after radiation ther apy for Hodgkin’s disease

Patient Smoking history (pack-yrs) Histology p53 IHC”p53 sequence analysis

Amino acid changeCodon Base change CpG site

I >�p�j5 SCCA 3+ w’r#{149}2 1-2 � SCLC 2 + I 45 CTG -� CGG No Lou -� Arg

3 Heavy. long-term” SCCA Neg WT

4 31 SCCA Neg WT

5,.

6

48

18

SCCA

SCCA

3 +

2+

278

293

l75�

CCT -= CGT

GGG -p GGA

CGC-�CAC

No

No

Yes

Pro -� Arg

Gly -* Gly

Arg-�His

7 27 SCLC Neg WT

8 35 Adenoca 3+ Wi”

9 25 SCCA 3 + 89� CCC -� TCC No Pro -� 5cr

I 0” 19 Adenoca 2+ Wi”

I I 35 SCCA Neg 285 GAG -� GAA No Glu - Glu

,‘ IHC. immunohistochemistry; SCCA. squamous cell carcinoma: SCLC. small cell lung carcinoma; adenoca, adenocarcinoma; ppd. packs per day: WT. wild type: Neg.

negative.F, Smoking histories for two patients were provided only as cigarettes/day: duration of smoking was not provided.

, In patients I. 8, 9, and 10, sequence analysis included exons 4, 9, and 10.

,1 Quantitative smoking history was not available for patient 3, who was described as a “heavy, long-term smoker.”‘. Heterozygous polymorphisms at codons 72 (CGC”5 -� CCC�m, exon 4) and 213 (CGA”5 -� CGG”5, exon 6) were detected in patients 10 and 5. respectively.

I The mutation at codon I 75 was homozygous, indicating that the second allele was deleted. All others appeared heterozygous. although contamination with normal stroma

cannot be excluded as a source of the wild-type sequence.

sequential rounds of PCR using nested primers as described

previously (22). The first (35 cycles) and second (20 cycles)rounds consisted of denaturation at 94#{176}Cfor I mm, annealingat 60#{176}Cfor I mm, and polymerization at 78#{176}Cfor 30 s. Theprimers bound intron sequences and generated complete codingsequences, including splice sites. Negative and positive con-

trols were included in each set of reactions.After purification by electrophoresis on a 4% agarose gel

(3: 1 NuSieve), the PCR products were sequenced by a modi-fication of the dideoxy chain-termination method of Sanger et

a!. (23), as described previously (24). Each exon was sequencedin both directions; two separate, independent PCR productswere examined for each exon. Mutations were confirmed by

finding the same base change in two independent PCR prod-ucts. Cases with mutations were analyzed for germline muta-tions at the same site by using DNA from nonneoplastic tissues.

Results

Lung cancers from five Hodgkin’s disease survivors containedsix somatic, point mutations (four missense and two silentmutations); there were four G:C-sA:T transitions plus one

G:C-sC:G and one A:T-*C:G transversions (Table 2). Germ-line analysis of nontumor samples showed that all mutationswere somatic alterations. Missense and silent mutations oc-curred at codons 278 and 293, respectively, in the tumor from

patient 5. Clonal analysis indicated that both base substitutions

occurred on the same allele.Immunohistochemical analysis showed nuclear accumula-

tion of p53 protein in tumor tissues from 7 patients (Table 2).Tumors from patients 2, 5, and 6 were positive by immuno-

chemistry and contained missense mutations, but four tumorswhich were positive by immunochemistry contained wild-type

sequence in the commonly mutated regions within exons 5-8.

Because 15% of lung cancer mutations occur outside exons 5-8

(14), exons 4, 9, and 10 were examined in the four patients (I,

8, 9, and 10) who were positive for immunochemistry andnegative for mutation. A missense mutation in exon 4 (codon

89) was identified in patient 9. The mechanisms for proteinaccumulation in patients 1, 8, and 10 are probably nonmuta-tional; possibilities include binding and stabilization by DNA

tumor virus proteins or cellular proteins such as mdm-2 (25).

Discussion

We report the first p53 mutational spectrum analysis in lungcancers after radiotherapy for Hodgkin’s disease. The principalfinding is an unusual mutational profile dominated by

G:C-*A:T transitions at non-CpG sites, which are character-istic of radiation or oxidative damage (reviewed in Refs. 26,27). Despite moderate or heavy smoking histories in all 1 1subjects, these data suggest that radiation was the primarymutagen and that smoking, chemotherapy, and other influenceswere cofactors in the development of these secondary lungcancers. Although all lung cancers occurred an average of 9.8years after treatment, for patients I and 10, latencies wereunusually short, i.e. , 2.8 and 1 .3 years, respectively. However,neither of these patients had a p.53 mutation, so that excluding

these two cases would not change our results, except to increasethe mutation frequency within the residual cohort. Because ofthe short latency interval and the absence of G:C-�A:T tran-

sitions in patients I and 10, we do not believe that these two

lung cancers were radiogenic, and we point out that bothpatients were heavy smokers.

In tobacco-related lung tumors, the most common p53base changes are G:C-�T:A transversions, with a strand bias inwhich the mutated guanine typically occurs on the nontran-

scribed, DNA-coding strand (see Table 3; Ref. I 3). The fre-quency of G:C-*T:A transversions increases with increasingsmoking history, and conversely, the frequency of G:C-�A:T

transitions significantly decreases ( I 5). Although all I I patientsin this series were moderate or heavy smokers, not one

G:C’-T:A base change was detected, and 4 of 6 mutationswere G:C-�A:T transitions (patients 5, 6, 9, and I 1). Three offive mutated guanine residues occurred on the coding strand,

and only one of four G:C-�A:T transitions occurred at a CpGsite.2 This mutation profile appears to differ from smoking-related lung cancers in which G:C’-�A:T base changes at non-

2 CpG site is a shorthand notation for a cytosine linked to a guanine by a

phosphodiester bond. The significance in this context is that cytosine bases within

CpG sites are methylated frequently at the 5’ position. 5-Methyl-cytosine is

susceptible to spontaneous deamination, which produces a base change of cy-tosine to thymidine. This is a form of spontaneous or endogenous mutation that

does not require an exogenous mutagen.




ferase.

Table 3 p53 Mutational spectra in lung canc ers from Hodgkin’s disease patie nts. uranium miners, tub acco smoker s, and in vit ro model systems

Human lung cancers

- .

Genetic targetsequence

Primary. .

radiation

class

it

G:C A:TCpG (%)

Deletion/.insertion

(%)A:T(%) T:A(%) C:G(%) T:A(%) G:C(%) C:G(%)

Post-Hodgkin’s p53 y 6 67�’ 0 17 0 0 17 17 0

Uranium miners’ p53 a 8 2.5 37.5 12.5 12.5 0 0 0 25

Uraniuni miners” /)53 a 30 17 6Y 0 0 3 0 40 17

Sn#{236}okers� j�53 None 502 24 40 9 5 7 3 9 1 1

/�t iitro models

Lymphoblasts” HPRT’ y I 14 5 2 3 4 2 1 NR 83

Lymphobla.sts’ supF y 919 53 19 13 2 1 1 NR II

,‘ Percentage of mutations that are G:C A:T transitions at CpG dinucleotides.

F, Three of four guanines occurred on the coding strand; one of four was a CpG site., p = 0.10. FET (Fisher’s Exact Test): comparison of G:C - A:T frequency among post-Hodgkin’s lung cancers and smokers with lung cancer.

,i Uranium miners from New Mexico. reported by Vahakangas et al. ( 16)

‘. Uranium miners from the Colorado plateau. reported by Taylor et al. ( I 7)

I 16 of 19 G:C -* T:A transversions occurred at codon 249, AGG”#{176}’ .� ATG�’�’.

5. Composite data from Greenblatt et al. (14)

I, Composite data from Refs. 41-44; radiation doses were 2 Gy. All studies used human B-lymphoblastoid cell lines.

, HPRT, hypoxanthine-guanine phosphoribosyl transferase gene: NR, not reported.

J Conipesite data from Refs. 28. 29. 45-47. Alt studies used human lymphoid cells and shuttle plasmid. pZ/89. containing E. coIl supF gene as mutational target. Radiation

doses were 5-l6() Gy.

CpG sites account for only 15% of p53 mutations (P = 0.10,Fisher’s Exact Text; Table 3; reviewed in Ref. 14).

Related data come from analyses of lung cancers fromuranium miners exposed to radon, an a emitter. In these tumors,the p53 mutational spectrum is also remarkably different fromthat observed in tobacco-related lung tumors, despite heavysmoking histories among these subjects (16, 17). Among ura-

nium miners from New Mexico, 3 of 6 base substitutions wereG:C-�T:A, but no guanine occurred on the nontranscribed,DNA-coding strand (16). The most striking observation is a

mutational hotspot at the second position of codon 249, AGG�-5 ATG”#{176} (17) which occurred among Colorado plateau mm-ers with very high level exposure to radon (i.e., > 1000 Work-ing Level Month). Although this site is a minor mutationalhotspot in smoking-related lung cancers (14), 16 of 25 (64%)base changes reported in lung tumors from Colorado plateauminers were G:C-sT:A transversions at this codon ( I 7). Mu-tation hotspots are characteristic of radiation mutagenesis (28,29): typical mechanisms include sequence context and genestructure/function features. It is possible that the differences in

mutation spectrum between the uranium miners and Hodgkin’sdisease patients can be explained by factors including variationin amount of absorbed radiation, different types of radiation (yversus a), temporal pattern, and modality of exposure (severalhigh-dose fractions over 6 weeks versus low-dose, chronic

exposure over many years). genetic background, and environ-mental factors.

Although the mutational spectra of ionizing radiation varydepending on target locus. chromosomal context to locus, dose,

dose rate, and DNA repair mechanism, large and small dele-tions are the most common lesions (reviewed in Refs. 30, 3 1).However, several in vitro model systems have reported basesubstitutions and many show a predominance of G:C-sA:Ttransitions. We reviewed data generated by two model systems,the HPRT3 locus and a shuttle vector (Table 3); both systemsuse human cells to avoid interspecies differences in DNArepair. Mutations in the HPRT locus are selected by their

S The abbreviation used is: HPRT. hypoxanthine-guanine phosphoribosyl trans-

interruption of the coding sequence (further described in Ref.

3 1); this explains the predominance of deletions and insertions,which account for 83% of lesions (see Table 3). By using y-raydoses of 2 Gy, the HPRT base substitutions include all mutation

classes with a slight excess of G:C-sA:T transitions. Thesecond system uses the pZ189 shuttle vector with the Esche-

richia co/i supF gene as a mutational target. The architecture of

this plasmid is biased toward base substitutions (89%) and

against insertions and deletions (1 1%; described in Ref. 29);

this resembles the p53 mutation profile that selects for basesubstitutions (reviewed in Ref. 14). ‘y-radiation (5-160 Gy) ofthe shuttle vector, followed by passage through human lym-phoblastoid cells for DNA repair, produced mostly G:C-�A:T

transitions (53%; Table 3), which are commonly attributed tothe mutagenic activity of free radicals produced by the radi-olysis of water (reviewed in Refs. 26, 27).

The minimum latency period for solid cancers caused by

radiation alone is typically 10 years or more (32). However, therisk of lung cancer begins to increase in the 5-9-year periodafter diagnosis of Hodgkin’s disease (4, 33), and this acceler-

ated onset suggests an additional factor(s) expediting the pro-cess. Factors that might shorten the latency period includetobacco, immunosuppression (34), chemotherapy, and hostvariables. Our data are consistent with primary mutagenesis byradiation and free radicals, and statistical analysis of the patient

series showed that lung cancer risk among smokers stronglycorrelated with increasing radiation dose (P trend 0.01 ; Ref. 8).

However, it is also possible that radiation and tobacco mayinteract to cause the unusual p.53 mutational spectrum observedin our series. Weak but supporting evidence for an interaction

between these two mutagens comes from epidemiological stud-ies of breast cancer patients (1 1, 35) and Hodgkin’s diseasepatients (8) treated with radiation. Among underground miners,the interaction between radon and tobacco use is less thanmultiplicative but more than additive (36).

Combination chemotherapy includes many alkylating

agents, including procarbazine, which is frequently used to

treat Hodgkin’s disease. Procarbazine commonly produces

G:C-sA:T transitions (37), which occurred in four tumors inour series. Among these four patients, three received radiation

therapy alone, and one had radiation therapy plus vinblastine



Cancer Epidemiology, Biomarkers & Prevention 97

and procarbazine. Therefore, one of four G:C-sA:T transitionsmight be related to procarbazine and/or radiotherapy.

Silent base changes at codons 293 and 285 of the p.53 gene

occurred in lung tumors from patients 5 and 1 1 , respectively.Silent mutations4 are uncommon in p53 because of selection for

missense mutations, but there is some evidence that thesemutations can influence gene expression. For example, uncom-mon aminoacyl-tRNA species can become rate limiting forprotein synthesis under some circumstances, e.g. , when two ormore rare codons occur consecutively and the gene is expressedat a high level (38, 39). It was reported that the insertion of 2-5consecutive low-usage arginine codons reduced the synthesis offull-length protein in bacteria; this effect was not produced byhigh-usage arginine codons (40). These data support a model inwhich consecutive rare codons reduce protein synthesis through

frameshift or premature termination caused by an upstreamtranslating ribosome “bumping” a stalled, downstream ribo-

some (40).

We provide the first molecular analysis of lung cancerfollowing radiotherapy for Hodgkin’s disease. Despite activesmoking by all patients and a small sample series, the mutationpattern appears to differ from the usual tobacco-related spec-trum and may implicate radiation as the primary mutagen.These findings illustrate the potential value of mutational anal-yses in determining the etiology of cancer. If confirmed in alarger series, these data would imply that some radiation-in-

duced cancers can be distinguished from those caused by other

factors.

Acknowledgments

We thank Dr. Curtis C. Harris (Bethesda, MD) for critical review of the manu-

script: Dr. Dolph Hatfield (Bethesda. MD) for helpful discussion on the biological

function of rare codons; Alexandra van den Belt and Rebecca Albert for technical

support; Virginia Hunter (Ontario, Canada) for administrative assistance; and

Rama Modali (Gaithersburg. MD) for expert technical assistance.

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1996;5:93-98. Cancer Epidemiol Biomarkers Prev V M De Benedetti, L B Travis, J A Welsh, et al. Hodgkin's disease.p53 mutations in lung cancer following radiation therapy for

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p53 Mutations in Lung Cancer following Radiation Therapy ... · lung cancer cases from the Netherlands and Ontario. These subjects were included in prior studies of Hodgkin’s disease