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~ Journal of Cancer (1997) 7S($), 1119-1124 1997 Cancer Research C=zmplign p53 gene aberrations in non-small.cell lung carcinomas from a smoking population T Liloglou1, H Ross1, W Prime1, RJ Donnellyz, DA SpandidosZ, JR Gosney~ and JK Field1 ~Molecular Genetic~ and Oncology Group, Department of Clinical Dental Sciences, The University of Liverpool, liverpool L89 3BX, UK; =Cardiothoracic Centre, I~-oadgreen, liverpool, UK; =National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, Athens, Greece; 4Department of pathology, The University of liverpool, liverpool L6g 3BX, UK Summary We examined 46 non-small-cell lung carcinomas (NSCLCs) for the presence of p53 mutations in exons 4-9, positive p53 immunostaining and loss of heteroL'ygosity (LOH) in the TP53 locus, p5.3 mutations were detected in 13 tumour samples (28.3%), whereas overexpression of the p53 protein was found in 30 of 45 (67%) samples. Allelic loss was found in 9 of 38 (23.6%) informative cases. The statistical analysis revealed no significant correlation between p53 mutations and clinicopathological data, although mutations appear to occur more frequently in squamous cell carcinomas (7 of 18) than in adenocarcinomas (2 of 15). All but three individuals in this study group smoked, in contrast to previous reports, we found a higher prevalence of GC---,AT transitions than of GC--,.TA transversions, as expected in a smoking population. A trend was found between p53-po.sifive immunostaining and a history of heavy smoking (76-126 pack-years) and was inversely correlated with allelic deletion (LOH) at the TP53 locus. Eight of the 12 NSCLCs containing p53 mutations also had concomitant p53 overexpression, and it is of specific note that three of the four tumours containing p53 'mutations' with no overexpression of the p53 protein had either insertions or deletions in the p53 gene. No correlation was found between p53 mutations and fractional allele loss or ras mutations. p53 mutations in this Merseyside population in the UK do not appear to be as common as in other reports for NSCLC and exhibit predominance of GC-,-AT transitions preferentially at non-CpG sites, suggesting that other carcinogens in addition to those in tobacco smoke may be involved in NSCLC in the Merseyside area of the UK. Keyworde: p53 mutations; p53 expression; immunohistochemistry; loss of heterozygosity; lung tumours; non-small-cell lung carcinoma; GC-,AT transition; CpG dinucleofidas; smokers Lung cancer is one of the major causes of death in the Western world. Small-call lung carcinomas (SCLC) account for approxi- mately 25% of all lung tumours and non-small-cell carcinomas (N$CLCs) constitute the remaining 75% (Whitehouse. 1994). Lung cancer development is strongly related to environmental agents, and smoking appears to bc responsible for the majority of the cases. Lung cancer is considered to be the major cause of death among smokers in the United States (Shopland et al, 1991). Cigarette smoke contains a number of carcinogens, such as benzo- [a]pyrcne, which may act in the initiation and/or promotion of the disease (DeMarini. 1983). During the last decade, a number of the known oncogenes and turnout-suppressor genes have been shown to be altered in lung turnouts ('Field et al, 1994). The p53 gene encodes for a 53-kDa nuclear phosphoprotein which functions as a transcription factor and it is implicated in the regulation of the cell cycle and subsc- quendy in growth control, p53 acts as a tumour-suppressor'gene, arresting cells in the GJGI phase whenever DNA is damaged to give more time for the celts DNA repair mechanism to function, and, if unsuccessful, leads cells to apoptotic death (Levine et al, 1991; Lane, 1992; Yin et al, 1992). The p53 gene has been found to be mutated in a large range of human turnouts (Hollstein et al, 1991; Greenblatt ct al, 1994; Received 6 Februmy 1996 Revised 16 October 1996 Accepted 22 (~Lober 1996 Conespondence to: JK Re~d Sidransky and Hollst¢in. 1996). Many international research groups have contributed to the identification of p53 mutations and in a number of cases have correlated the presenc~ of p53 mutations with stage, histology, prognosis and exposure to certain environmenud agents. Mutagens can produce specific base substitutions at certain sites, and a mutation specu'um analysis may provide information about the origins of mutations that give rise to human turnouts. A reccm review of the published data on p53 mutations in human turnouts worldwide led to a number of hypotheses concerning the role of p53 in carcinogenesis (Gmcnblatt et al. 1994). Lung turnouts have been shown to contain various genetic aber- rations within the p53 gcne, including point mutations, insertions, deletions and loss of heterozygpsity at the TP53 locus (Chiba et al. 1990; Lehman ctal, 1991; Sameshima et al, 1992; Mitsudomi et al, 1992, 1993; Suzuki et al, 1992; Lohman et al, 1993; Takeshima et at, 1993; Westra ctal, 1993; Carbone et al, 1994; Zheng et at, 1994). With respect to point mutations, GC--*TA transversions have been shown to occur more frequendy in smokers than in non-smokers with lung cancer and may be the result of specific carcinogenic agents present in tobacco smoke, such as benzo- [a]pyrene (Cn'eenblatt ct al, 1994; Husgafuel-Pursianen et al, 1995; Ramet ¢t al, 1995). In the present study, we have examined p53 status in 46 patients with NSCLC for the presence of mutations in cxons 4-9, expres- sion of the p53 gone and all¢lic loss in the TP53 locus. All patients live within Merscyside, UK, where the incidence of the disease is Youngson and Williams, 1995 among the highest in Europe, especially in women (Williams, 1992). 1119 This article is for individual use only and may not be further reproduced or stored electronically without written permission from the copyright holder.

1120 T Liloglou et al Patient no. ( 18 52 37 I N T I I N T I I N T I Figure I Example of screening for point mutations in exon 5 of p53 by SSCP analysis. PCR products were denatured and run through an 8% native polyacrylamide gel, and the gels were visualized by silver staining. Bands with mobility shifts are indicated by arrows MATERIALS AND METHODS Tissue samples Turnouts were obtained from patients undergoing lung resection for bronchial tumours at the Cardiothoracic Centr¢, Liverpool, UK. All patients were Caucasians, and all but three were smokers. After resection the turnouts were snap frozen and stored at-70°C. Forty-six NSCLC specimens were analysed from 28 men and 18 women, of which 15 were adenocarcinomas, seven adenosquam- ous, tg squamous, three large-cell, one neuroendocfine, one carci- nosarcoma and one sarcomatoid carcinoma. Immunohistochemlstry The immunohistocbemical dcmoustration of p53 protein was performed using a standard ABC technique on formalin-paraffin- processed sections. The antigen was unmasked by micmwaving in citrate buffer pH 6.0 for 15 rain at full power in a 650-W microwave oven. The primary antibody, DO-1 monoclonal anti. body, was used at a concentration of 1:50 for'2 h at room tempera. ture. The secondary antibody (Vector elite standard kit) was used at a hi00 for 30 rain at room temperature. Diaminobenzidene used as the chromogen. The slides were scored as a percentage of positive cells per field (WP and JRG). DNA extraction and PCR Turnout tissues were microdissected before DNA extraction, which was undertaken with Nucleon II (Scotlab, Coatbridge, UK). The oligonucl~otides used for the polymerasc chain reaction (PCR) amplification of the p53 exons and the thermal profile of the amplification have been described previously (l_~hman et al, 1991). The macdon mixtta'e contained 16 mM ammonium sulphate, 67 mM Tris-HC1 pH 8.8, 0.1% Tween-20, dNTPs, 0.4 I~M of each primer, 2 raM magnesium chloride and 0.5 units of Biopro DNA polymcrase (Bioline, London, UK). SSCP analysis Single-strand conformation polymorphism (SSCP) analysis was undertaken as follows: 2-5 ~tl of the PCR product was mixed with I0 ~I of denaturing solution, which consisted of 50% formamide, 50 mM sodium hydroxide, I mM EDTA, 0.1% bromopbenoi blue and 0.1% xylcne cyanol FF. Samples were then beated at 95~C for 3 rain, chilled on ice and loaded onto an 3-10% native polyacryl- amide gel containing 5% glycerol. Gels were run at 10°C and, after elcctrophoresis, the bands were visualized by silver staining. Sequencing DNA samples that showed altered mobility in SSCP analysis were amplified using a :5' biotinylated upstream primer. The strands of the PCR product were then separated using streptavidin-conjugated Dynabeads M-280 (Dynal, Brombrough, UK). Sequencing reaction was performed using Sexluenase version 2.0 kit (Amersham Life Table I Sequence analysis results for mutations in the p53 gene in lung tumours Patient Age Sex, Hlstopethology p53 p53 mutation Sequence AA Allelic" no, (years) expression' change change imbalance Exon Codon at TP53 3 67 M Squamous No Data 5 136 cue > tea Gin > atop LOH 18 65 M Squamous ++ 5 131 sac > gac Asn> Asp No LOH 138 gcc>gtc Ale > Val 25 69 M Squamous +++ 7 248 egg > cag Atg > Gin No LOH 27 57 M Squamous +++ 7 247 ~an • sat Silent No LOH 41 65 M Squamous ++ 5 158 cgc • ccc Arg > Pro No LOH 43 65 F Squamous +++ " 8 273 Cgt > cat Arg • His No LOH 44 64 M Squamoua - 8 294 gag > fag Giu • atop No LOH 19 64 F Adenosquamous - 8 297 3-bp insertion His > Gin-Set NI 21 58 F Adenosquamous - 7 229--235 lg~bp deletion Truncation NI 34 73 M Adenocarcinoma ++ 7 245 ggc • tgc Gly • Cys MI 49 59 M Adenocaroinoma +++ 8 268 4-bp deletion Frameshift No LOH 28 72 F Large cell .H.+ 7 258 gas • aaa Glu > Lye No LOH 52 67 M Large cell - 5 intron 4 1-bp insertion Splicing? 157 1-bp deletion Frameshift LOH 159 gcc • gtc Ale • 'M, male; F. female. =(-) negative staining, (++) or (+-H-) positive staining, q.OH, loss of heterozygosity; NI, non-informative; MI, microsstellite instability. All but one patient (19) with a mutation in the p53 gene had a history of smoking. British Journal of Cancer (1997) 75(8), 1119-1124 Cancer Research Campaign 1997 This article is for individual use ordy and may not be further roproduccd or stored electronically without written p~rmission from the copyright holder. rdnauthor~.-'d r~pcodtt~tion may rc~tdt in fitmacial and other ~¢nalitiee. (~} CHURCHILl. LdVINGSTON~ 0 CO O~ 0 Insertion point Rgure 2 Sequer GAG insertion wi" Sciences, Litti tropho .resed th were then fixc room temperatt Loss of hete The LOH anal: oligonucleotid~ (Huntsville, U 16 mM ammon 20,200 I~M tiN" and 0.5 units B were 94°C for cycles were t' trophorcsis on using silver sta Statistical a. In order to fin, and the clini employed, usi @ Cancer Re¢-

Patient no. 19 (exon 8) Turnout I C T A G '1 Normal ~'C T A Turnout C T A p53 aberrations in NSCLC 1121 Patient no, 18 (exon 5) Normal G I I C T A Inse~on pointl Figure 2 Sequence analysis of exert 8 of p$3 from patie~ no. 19 showing a GAG insertion which distorts the whole sequence upstream Sciences, Little Chaltbnt, UK). Reaction products were elec- trophoresed through a 6% denaturing polyacrylamide gel. Gels were then fixed, dried and exposed to Kodak XAR-50 films at room temperature. Loss of heterozygosity at the TP53 locus The LOH analysis of the TP53 (17p13.1) locus was undertaken with o]igonucleotide palmers purchased from Research Genetics (Hunmville, USA). The amplification reaction mixture contained 16 rn~ ammonium sulphate. 67 mM Tds-HCI pH 8.8, 0.I% Tween- 20, 200 ~u~ dNTPs, 0.4 laM of each palmer, 2 mM magnesium chloride and 0.5 units Biopm DNA polymerase. The amplification parameters were 94"C for 30 s, 590C for 30 s and 72°C for 30 s. Twenty-eight cycles were performed. PCR products were analysed by dec- trophoresis on 10% polyacrylamide gels. Bands were visualized using silver staining (Field et al, 1995, t996; Neville et al. 1996). Statistical analysis In order to find any correlations between the presence of mutations and the clinicopathological dam. the Fisher's exact test was employed, using the SAS software for PCs. Cancer-Research Campaign 1997 Figure 3 Sequence analysis of exon 5 of p53 in a DNA sample from patient no. 18, displaying a C~T transition, as indicated by an arrow RESULTS We examined 46 NSCLCs and their normal adjacent tissue samples for aberrations in the p53 gene. Initially, we screened all of the samples for p53 mutations using the SSCP technique (Figure 1). Positive turnouts were then subjected to direct sequencing to confirm the mutation and identify its exact nature. Sequence analysis revealed 16 mutations in 13 samples (28.3%), including three deletions (one l-bp deletion, one 4-bp deletion and one 19-bp deletion), two insertions (one 3-bp insertion and one l-bp inser- tion) and 11 base substitutions (examples of such mutations are shown in Figures 2 and 3). The base substitutions consisted of four C---*T transitions, two G--~T transversions, three G-,,A transitions, one A-~G transition and one G-'-,C transversion. Six samples were found to carry a polymorphism in exert 4, eodun 72 (CGC--,'CCC, Arg---*Pro). p53 mutations were found in exerts 5, 7 and 8 and. with the exception of one l-bp deletion, all were located in the coding regions Gable 1). No mutations were found in adjacent normal tissue samples. In order to eliminate the possibility of missing any of the p53 mutations because of SSCP false negatives, for each exoa we sequenced 10 DNA samples picked randomly from those that had demonstrated no abnormality in SSCP analysis. No sequence abnormalities were detected in these analyses. In all SSCP- positive findings the mutations were reconfirmed by sequencing PCR products amplified by a separate aliquot of the DNA sample. On examining the 11 base substitutions, one was silent, two nonsense and eight missense. The 1-bp deletion led to a frameshift, resulting in a stop eodon 12 amino acids after the deletion point; the 4-bp deletion led to a frameshift, again resulting in a stop codoa 36 amino acids after the deletion point; the 19-bp deletion led to a truncation of six amino acids and a frameshift, which generated a stop codon 11 amino acids after the deletion point; and the 3-bp insertion led to a substitution of valine for hisddine and the addidun of a serine without producing any frameshift changes. The 1-bp insertion in patient no. 52 occurs in the intronic region 24 bp before the first nucleotide of exon 5 and does not appear to affect splicing. Two of the turnouts (patient nos. 18 and 52) were found to carry more than one mutation, and all multiple mutations occurred in Bdtis~ Journal of Cancer (1997) 75(8), 1119-1124 uT~ ais ..a~ic!e is for individua/use only and may not be further reproduced or stored electr ' • umo~t ~pradu~n'oa may r~/¢ (a a .... :o, ......... omeally wzthout written permission from tbe copyright holder. ........ ~* ~t** uta~r I~na.t~. (el CHURCHILL LIVINGSTONE

1122 TLiloglou etal ) Table 2 Distribution of GC~AT transitions in the p53 genes in pdmary lung tumours, as found in the international datapasa0 compared with our results GC--AT CpC/GpG CpG Other Total GO--AT Present study 5 (71.4%) 2 (28.6%) - 7 Hollstein et al (1994) 33 (37%) 27 (30%) 29 (33%) 89 • Data from cell lines a~a not include~ in the above table. exon 5. In the case of patient no. 52, there was an additional 1-bp insertion in the intronic region, and the distance between the 1-bp deletion and the C'-*T substitution within exon 5 was only four nucleofides. Statistical analysis showed no correlation between the presence of p53 mutations and the clinicopathologicai data, i.e. age, stage, TNM stage, nodes at pathology, alcohol intake, cigarettes and clin- ical outcome (follow-up 18 months). Samples from 4 of the 18 female patients and 9 of 28 male patents cont~nedp53 mutations. However, p53 mutations occurred more frequently in squamous cell carcinomas (7 of 18) than in adenocarcinomas (2 of 15), but this was not statistically significant. Similarly, the presence of the minor allele (Pro) in codon 72 in exon 4, found in 6 out of 46 samples, did not correlate with any of the patticulax clinicopatho- logical parameters. The immunohistuchemical study of the p53 protein revealed that 15 of 45 (33%) samples had negative (-) staining, 5 of 45 (11%) samples had weak positive (+) staining and 25 of 45 (56%) samples had intense positive (++ or +++) staining for p53. We divided the patients into three classes with respect to the number of cigarettes they consumed in pack-years: light smokers (0-45 pack-years); moderate smokers (46-67.5 pack-years); and heavy smokers (76-126 pack-years). A trend was found between p53- positive staining and the patients' smoldng history; however, this was found to be not statistically significant (P = 0.13) (Table 3). In the 12 NSCLC specimens with p53 mutations for'which we also had p53 expression data (Table 1), eight had concomitant p33 mutation and p53-positive staining. However, it is noteworthy that three of the four turnouts containing p53 mutations that had no p53-positive staining (patient nos. 21, 44 and 52) had mutations that result in truncation of the expressed protein. The fourth sample (patient no. 19) has an inframe 3-bp insertion that did not result in mancation, and so the reason for non-lmmunoreactivity remains unclear. It is of interest that patient no. 19 was the only non-smoker with a p53 mutation. The TP53 LOH study indicated allelic loss in 9 of 38 (23.6%) of the informative cases. In the case of patient no. 34, microsatellite instability (MI) was observed at this locus. Statistical analysis showed no correlation between LOH and the clinicopathological parameters. However, an inverse correlation was found betw~n LOH and staining for p53, indicating that overexpression of the pS3 gene was uncommon in s~mples with LOH at the TP53 locus (P ffi 0.005). In an extensive allelotype analysis of lung turnouts, the frac- tional allele loss (FAL) value was calculated for the 46 turnouts for which we had LOH data for 38 chromosome arms. The FAL value for this group of turnouts was found to be 0.09. (FAL was calcu- lated as the number of chromosome arms showing loss of heterozy- gosity divided by the number of informative chromosome arms; Field et el, 1996; Neville et ai, 1996.) We investigated whether Bdtish Jouma! of Cancer (1997) 75(8), 1119-1124 T~ble 3 p53 mutations and overexpression in relation to the smoking history of the patient Smoking history p53 mutations p53 overexpression (packl par day x yaarl smoking) 0-45 2 11 7 6 4~-67.5 5 10 11 4 72-126 4 4 7 1 The patients have been subdivided into light smokers (0-45 pack-years), moderate smokers (46-67,5 pack-years) and heavy smokers (72-126 pack-years), p53 mutation analysis of light smokers and heavy smokers, P > 0.05; p53-positive staining analysis of light smokers and heavy smokers, P = 0.13. there was a correlation betw~n the presence of p53 mutations and the FAL value, but no statistically significant correlation was found. Furthermore, there was no correlation between p53 aberra- tions and K-ras mutations (Neville et el, 1995a) or ailelic loss on chromosome 9 at 9p23, a site considered to contain a putative turnout-suppressor gene in these turnouts (Neville et el, 1995b). DISCUSSION p53 gene inactivation by mutation or allelic loss has been impli- cated in the d¢velopmeur of lung cancer (Greenblatt et ai, 1994). The identification of mutations that arise within the gene may lead to an understanding of the role of the p53 gene in the development of lung turnouts and may also help to elucidate the role of certain carcinogenic agents, e.g. those contained in tobacco or environ- menud pollutants, in this disease process. In this study, we found that 28% of the mmours contained mute- dons in the p53 gene. This incidence is significantly lower (P < 0.0002) than that reported in a major review of p53 mutations in 897 lung cancer padents who have smoked (Greenblatt et ai. 1994; Hollstein et al, 1994). On comparing our results with those of Suzuld et al 0992), who demonstratedp53 mutations in 47% of 30 NSCLCs investigated, no statistically significant difference was found (P = 0.082). The possibility that p53 mutations were missed by SSCP screening is very unlikely as SSCP-negative results were confirmed by sequencing 10 SSCP-negative samples per exon; however, even if we calculated that 10% of p53 muta- tions were missed, the total incidence would rise to only 31%, which is still lower than the average incidence of p53 mutations in lung cancer (56%), as r~ported by Greenblatt et al (1994). Our results indicate that p53 mutations in NSCLC may be caused by carcinogens other than those found in tobacco smoke. This may account for the population-specific mutation spectrum. Sixty-seven per cent of the NSCLC samples in this study demonstrated p53-positive staining. It is of note that all but one of the patients' turnouts with p53 mutations, excluding those with insertions or deletions, also demonstrated p53-posifive staining. This provides further evidence for the hypothesis that there is an increased p53 gene product in tumours containing mutations in the p53 gene. The p53-positive staining is considered to represent stable p53 protein overexpression that results from post-transla- tional modification and/or p53 protein complexing. However, there were also NSCLC turnouts that demonstrated p53-posidve staining but in which no mutations were found. These differences Cancer Research Campaign 1997 This article is for individual use only and may not b¢ further reproduced or stored electronically without written permission fi'om the copyright holder. U'nauff~on'z~t tvprodu'gttbn may result in ~nanclal cad other pcnalitics. (¢7 CHIJ~CHILL LIVINGSTON~ between molec explained by th gene outside ex overexpression some other fact, thus increase it, DO-I antibody wild-type p53 instability witht More than 1" residues, which et el, 1994). H~ found a higher mutations) thin be expected fr~ of GC--~TA showed a sign et ai (1995) rc smokers amon mutations wet only 18% ofth tO our own '~ whom G---'T detected in a el, 1994). GC- of benzo[a]p.x carbons, whic smoke. How~ samino)- 1-~ 3- lions and G( depending o~ Hoffmann. compounds. investigation whether or n, the smoking that, rather th many enviro~ syne~isticall: Strikingly. mutations CpG dinuclc Table 2). Cp( deamination ~ccurr~nce ol ktie is kno~ PO .:ells (Tashe~ C) transitions ~' rautagenesis O~ ' Nocorrek CO , and the stag. CO : considered O~ However, it /'Q ' could be hat : have a selec~ the late am[, status tumor smoker' in comparison non-smokin staining am © Cancer R

between molecular and immunohistochemical results may be explained by the possibility that there are mutations in the p53 gene outside exons 4-9, within the p53 promoter region, or that overexpression of p53 is not only due top53 mutations but also to some other factors, e.g. mdm2, which bind to the p53 protein and thus increase its half-life. Furthermore, it may be argued that the DO-1 antibody may also detect a stable eonformationally altered wild-type p53 protein which, in its own fight, may lead to genetic instability without an initial p53 mutation. More than half (6 of 11) of base substitutions occur at G residues, which is in agreement with previous reports (Greenblatt et al. 1994). However, in eonmast with most previous reports, we found a higher prevalence of GC'-,.AT transitions (37.5% of all mutations) than of GC--,'TA transversions (12.5%), which would be expected from a smoking population. Comparison of the ratio of GC--*TA to GC---~AT in this study and Greenblatt's review showed a significant difference (P =: 0.04). Husgafvel-Pursiainen et al (1995) reviewed the mutational profile in smokers and ex- smokers among lung cancer patients and found that 34% of p53 mutations were G-~T base substitutions. In this study, however, only 18% of the smokers had G--,.T transversions. Findings similar to our own were reported in NSCLC patients from Taiwan, in whom G--,'T mutations constituted only 6% of p53 mutations detected in a population sample comprising 61% smokers (Lee et al, 1994). GC-,,TA transversions have been attributed to the action of benzo[a]pyrene, a member of the polycyclie aromatic hydro- carbons, which represent the major carcinogens found in tobacco smoke. However, other carcinogens, such as 4-(methylnitro- samino)-l-(3-pyridyl)-l-butanone (NNK), cause GC--~AT transi- tions and GC--.TA transversions at CpG and non-CpG sites, depending on the experimental system used (Wynder and Hoffmann. 1994). Tobacco smoke is a complex mixture of compounds, and the mutational spectrum it causes needs further investigation (DeMadni et al, 1995). Hence, it remains unclear whether or not the prevalence of GC-,'AT transitions found in the smoking population in this study is a paradox. It is possible that, rather than one factor acting alone, a combination of one or many environmental components and genetic factors may act synergistically. Strikingly, in this study all four C--,'T and one out of three G-,.A mutations (five out of seven GC-,AT in total) occurred at non- CpG dinucleotides (in this case in CpC/GpG dinucleotides; Table 2). CpG sites can account for mutations due to spontaneous deamination of 5-methyleytosine residues, but the reason for the occurrence of GC--,.AT transitions at non-CpG sites is unclear. As litde is known about methylation at non-CpG sites in mammalian cells (Tasbeva and Roufa 1994), it is uncertain whether GC---~AT transitions at non-CpG sites represent induced or spontaneous mutagenesis (Yang et at, 1996). No correlation was found between the presence of mutations and the stage of tumour, indicating that p53 mutations may be considered to play a role in the early development of lung cancer. However, it may also be a.r~ued that cells containing pS3 mutations could be harbouring a mutation in another gene that may merely have a selective growth advantage in vivo, and this would result in the late amplification of a p53-mutant subset of cells in a mixed- status turnout cell population. The presence of only one 'non- smoker" in our samples did not allow us to undertake any comparison of p53-positive staining and a history of smoking vs non-smoking; however, a trend was found between p53 immuno- staining and a history of heavy smoking (76--126 pack-years) Cancer Research Campaign 1997 p53 aberrations in NSCLC 1123 compared with light smoking (0-45 pack-years), but this was found to be not statistically significant (P = 0.13). p53 mutations did not correlate with age. alcohol consumption or prognosis (median 18 months" follow-up). However, a trend was found between the prevalence of p53 mutations and squamous carcinomas, which is in agreement with a previous report by Mitsudomi et al (1993). The absence of a correlation between p53 mutations and various elinieopathological parameters indicates that a mutation in the p53 gene may not by itself lead to aggressive cell growth. Recently, an inverse correlation between clinical outcome and p53-positiv¢ immunostaining was found; however, no correlation was found between p53 mutations and survival (Carbone et at, 1994). It is also probable that these tumours carry additional alterations in genes that function as oneoganes or tumour-soppressor genes; indeed, the allelotype study for this set of turnouts showed a median FAL value of 0.09. indicating allelic imbalance throughout the genome 0'qeville et al, 1996). In partic- ular, LOH at the TP53 locus, as shown in this study, was found in 25% of these tumours. It is of note that only two out of ten samples with LOH at TP53 were found to contain a mutation in p53. implying that another candidate turnout-suppressor gene may also exist at this locus. Additionally, four mutations in cedon 12 of the K-ras gene have also been demonstrated in these tumours (Neville et al, 1995a). Only one of the tumours was found 1o contain muta- tions in both the K-ms and the p53 genes. In conclusion, p53 mutations in the population of the Merseyside region of the UK do not appear to be as common as in other reports of NSCLC. GC--,'AT transitions in p53, preferentially occurring at non-CpG sites, suggest that different environmental carcinogens may be involved in this geographic area. ACKNOWLEDGEMENTS This work was supported by the Roy Castle Foundation, Liverpool, UK. The authors would like to thank Professor David Lane for generously providing the DO-I antibody. REFERENCES Catbon¢ DP. Mitsudomi T. Chib=. I. Piantadosi S, R.usch V. Nowak JA. Mclntir¢ D, Slamon D. Gazdar A and Minna J ([994) p53 immunostaining posltivity is associated with reduced survival and i:; imperfectly con'elated with gene mu~,~dons in resected non-small ¢¢11 lung cancer, A prclimlnary report ot'LCSG 871. Chest 166:377S~38 IS Chiba [. Takahashi T and Nan MM (1990) Mutations in the p53 gene primary, reacted non-small cell lung cancer. Oncngene $: DeMa~'ini DM (1983) Ganotoxicity of tobacco-smoke and tobacco-smoke condensate. Murat Rex 114:59-89 DcM~rini DM. Sheltnn ML and Levinn LG (1995) Mutation specm;m of cigamtt~ smoke conjugate in Salmonella: comparison to mutations in smoking- msoeiated minnows. Carcinogenesis 16:~';35-2542 Field IK. Stocker C J. Neville EN and Taylor L~ (1994-) Molecular mechanisms in lung cancer. In International Con&re, for Lung Cancer. Antypa.s G pp. 29-24. Moeduzzi Editor International Proc¢¢~Jings Division: Bologna Field JK. Kim'is H. Risk JM. Tsiriyotis C. Adamson R. Zoumpourlis Taylor K. Whittaker L Howard P. Beime JC. Gosney JR. Woolger L Vaughan ED. Sp~didos DA and Jones AS (1995) Allelotypo of squamous cai'clnom~ of the headt and neck: fractional allele loss correlates with survival. Br J C.ancer 72: l i B0-1 lgg Field JK, Neville EM. Stewart MP. Swift A, Liloglou T. Risk JM. Ross H, Gosney JR and Donnelly R$ (1996) Fractional allele loss data indicate distinct genetic populations in t.hn development of non-sraall cell lung cancer. Br J Cancer'/4: 1968-1974 Greenblatt MS. Bennett WP, Hollstein M and Hart'is CC (199~) Mutations in the p53 tumor suppressor gane: Clues to cancer etiology and molecular pathogenesi~. Cancer Res $4:4855..4878 British Journal of Cancer (1997) 75(8), 1119--1124 This article is for individual ~ only and may not be further reproduced or Stored electronically without written permission from the copyright holder. L1.v~,~-.~r2r.~d /'~'p,~'~gdu~L;on may ~.~uJt .;n f'Jnanc'aJ and O~er pe~JJ;';.e~. (~) C.F//_.rR~,/..HLL LIVI"~GSTONI~ "

benzoapyrene-DNA adducts in carcinoma cell lines. Careinogenesh: 16: 2H7-2124 SameshJma Y. Matsuno Y. Hirohashi S, Shimo~am Y, Mizoguchi H, Sugimura T. Terada M and Yokota J (I 992) Alterations of the #3 gene am common and eriti.eal events for the maintenance of malignant pheuofyp¢~ in small.¢ell lung ¢aramoma. Oncogene "/: 451 -a,57 Shopland Dl~ Eyre HI and P~ehacek TF ( 199 I) Smoking-atLfibutable cancer mortality in 1991: is lung cancer the leading cause of death among smokets in the United gta~s?dNatl Cancerln~¢~: 1142-114~ Sidr'~asky D and Hollstcin M (1996) Clinical impllcadons of the p53 gone. Ann R~ Med 47:285-301 Suzuki I~ Takahrahi To Kuroishi T. Suyama M. Ariyosbl Y, Tak~hashi T and Ueda R (1992) p53 mutations in non-small cell lung cancer in Japan: association between mutations and smoking. Cancer Res 52:734-736 T~c.eshim~ ¥. Sea, area T. Bennett WPo Akiyama Mo Tokuoka S. lnai K. Mabuchi K. Land CE and Harris CC (1993) p53 mutations in lung cancers fmra nonsmoking atzmi¢ bomb sm'vivors. Lancet 342:1520-1521 Toshevn F_,S and Roufa DJ ~ 1994) Densely methylated DNA islands in mammalian chromosomal replication origins. Mol Cell Biol 14:5636-5644 Wnstra F,S. Offeeaaus GJA~ Goodman gnu Slebos RJC. Polak Mo Bacs IO. Rodenhuis $ and Hruban RH (1993) Overexpression of the p53-tumour- supressor gene-pr~tuct in primal3, lung adenocarciuomas ia associated with cigaretm smoking, Ant d Sur~ Patho117: 213-220 Whitehouso JMA (1994.) Management of Lung Cancer Cunent Clinical Preatices 1994, 'Col. 1. pp. 1-58. Department of Hea/th: London Williams EMI. Youngson J. Ashby D and Donelly R.I f 1992l Lung cancer bulletin - A framework for action. Metseyside and Cheshire Cancer Registry: lAvetgool Wynder EL and Hoffmann D (1994) Smoking and lung cancer, scientific challenges and opportunities. CancerRes ~4:5284--5295 Yang AS. Gouzalgo MI~ Zingg J-M. Mill~" RP. Backley JD and Jones PA {1996) The rate of CpG mutations in Alu repetitive elements within the p53 mmour suppressor gane in the primate germline, d Mol Bio1255:240-250 Yin YX, Tainsky MA. Bischuff FZ and Strong LC { 1992) Wild-type p53 restores cell-cycle control and inhibits gene amplification in cells with mutant p53 • lleles. Cell 70:937-948 Youngson .I and Williams EMI 11995) Incidence of lung cancer in Merseyside and Cheshire (1089-1993). Met,seydde and Cheshire Cancer Regist~: Liveq~nol ~eng L ghn QR Li ZH. T~o JL. Weiss LM and Shiheta D ( 19941 Patterns of p53 mutations in ~quamo~s-ce]l carcinoma of the lung - acquisition at a relatively early age. Am J Pathol 145:1444-1449 British Joumal of Cancer (1997) 75(8), 1119-1124 ¢) Cancer Research Campaign 1997 ~dtisn journal of Ca 1997 Cancer Res¢ K.ras ; unfav( non-sr y Fukuyams ~0epartment of ~ Informatics, Fac: Summary W~ a mutation of (p53÷). There (2.5%) with r~ P<0.05). The 52 advanced- 94 cases with the pathologk mutations we Keywords: ~ Lung cancer the various early recurrc metastases l measuring k to distinguis prognosis b~ Recent at identify vat' involved in small-cell h and p53 tu genetic alto: A ras gc and 80% ol (Rodenhui~ Mitsudomi ---~ frequ ;but ; 'Mits~ Is mt 990: lutatl the, id tu '<11 ~,2~ .<12" Medicine, K