TP53 mutation patterns in breast cancers: searching for clues of environmental carcinogenesis

seminars in CANCER BIOLOGY, Vol. 11, 2001: pp. 353–360 doi:10.1006/scbi.2001.0390, available online at http://www.idealibrary.com on

TP53 mutation patterns in breast cancers: searching for clues of environmental carcinogenesis Magali Olivier and Pierre Hainaut∗ rapid increase in age-specific incidence rates. In Japan, however, rates remain constant after the age of 45. These observations suggest that the risk of BC is in part determined by environmental and lifestyle factors. 3 Many chemical carcinogens can cause mammary tumours in rodents. However, in humans, the evidence for a role of exogenous carcinogens is controversial (see Reference 4 for review). Recent studies have investigated the association of BC risk with tobacco smoking, alcohol consumption, dietary fat intake, ionizing radiations and exposure to organochlorines. Consistent associations have been reported only with ionizing radiation (atomic bomb survivors and exposure to intense radiation therapy) and dietary fat intake. In the case of tobacco smoking, recent reports suggest that increased risk may correlate with the presence of specific polymorphisms in genes involved in carcinogen metabolism and detoxification. For example, an elevated risk has been reported among smoking post-menopausal women who are deficient in N-acetyltransferase 2 (NAT2). 5

Mutations in the tumour suppressor gene TP53 occur in about 30% of breast cancers. We have used the IARC TP53 mutation database to analyse the pattern of mutations in breast cancers (1392 mutations). The global pattern of mutations is similar to the one of most other cancers, but there is an excess of transversions on G bases in tumours from Western (USA and Europe) as compared to Eastern (Japan) countries. Moreover, the patterns of inherited TP53 mutations associated with breast cancer, differ from those of somatic mutations. These differences support the hypothesis that a fraction of breast cancer mutations occur as a consequence of environmental exposures. Key words: breast cancer / TP53 / mutations / carcinogens c 2001 Academic Press

Introduction: exogenous risk factors in breast cancers Breast cancer (BC) is the most common form of cancer in women in developed countries, with important geographic variations in incidence rates. The highest rates are observed in the US and in Western Europe (ASR: over 50/100 000/year), whereas incidences are three to four times lower in most parts of Japan and China. 1 These differences do not appear to reflect variations in genetic susceptibility, since Japanese women born and living in the US have rates approaching those of US Caucasians. 2 In all areas of the world, the first breast cancers arise in late adolescence, with a

TP53 mutation as ‘reporter’ of carcinogenic exposures The TP53 tumour suppressor gene (chromosome 17p13) is altered in a large spectrum of human cancers by loss of alleles, deletions, insertions or point mutations. TP53 mutations are also found in the germline, associated with Li–Fraumeni and Li–Fraumeni related syndromes. These syndromes are rare familial cancer syndromes with multiple primary neoplasms in children and young adults (including sarcoma, breast cancers, brain tumours, adrenal gland carcinoma and leukaemia). 6 The p53 protein is a transcription factor constitutively expressed in most cell types and activated in response to stress signals (including, in particular,

From the Group of Molecular Carcinogenesis, International Agency for Research on Cancer, World Health Organization, 150 Cours Albert Thomas, 69372 Lyon cedex08, France. *Corresponding author. E-mail: [email protected] c

2001Academic Press 1044–579X / 01 / 050353+ 08 / $35.00 / 0

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Table 1.

Mutations as carcinogen fingerprints

Mutation type

High prevalence in

Suspected agent or mechanism

G:C>T:A G : C > T : A (codon 249) A : T base pairs

Lung Liver (HCC) Oesophagus (SCC), Head and neck Liver (ASL) Bladder Many other cancers Skin (other than melanoma) Colon, brain, stomach, other cancers Head and neck Head and neck

PAH (Benzo(a)pyrene) Aflatoxins Acetaldehyde?

A:T>T:A G:C>A:T CC > TT G : C > A : T at CpG

Small deletions Deletions

Vinyl chloride Alkylating agents? Aromatic amines? Radiations? UV Spontaneous deamination of methylated cytosines Polymerase slippage Irradiation?

Data compiled from References 8,9,12

genotoxic stress). Upon activation, p53 transactivates or transrepresses genes involved in cell-cycle control, apoptosis and DNA repair, thus exerting antiproliferative effects. Loss of p53 function is thought to suppress a mechanism of protection against accumulation of genetic alterations. 7 TP53 differs from other tumour suppressors such as RB1, APC or BRCA1, by the high prevalence of missense mutations. These mutations are scattered throughout the coding sequence of the gene, with a high density in exons 4–9, encoding the DNA binding domain. In this domain (residues 96 to 296), all codons are mutated at least once in human cancer. However, about 30% of mutations cluster at eight ‘hotspot’ codons (175, 176, 220, 245, 248, 249, 273 and 282). The diversity of the positions and chemical nature of missense mutations has made it possible to compare mutation patterns between types of cancers. In some instances, the mutation pattern is consistent with DNA damage inflicted by known carcinogens. Well-characterized examples are G to T transversions at codon 249 in hepatocellular carcinoma (in a context of exposure to aflatoxins and chronic HBV carriage), G to T transversions at several bases in lung cancers of smokers, and tandem CC to TT transitions in basal or squamous cell carcinoma of the skin after exposure to UV (reviewed in Reference 8). In general, however, it is difficult to ascribe a given mutation type to a specific carcinogen, as most mutations can arise through multiple mechanisms. Table 1 presents a simple key for interpretation of different types of mutations (see also References 8,9).

Transitions (purine to purine or pyrimidine to pyrimidine) at cytosines within pyrimidine repeats (CpG sites) can, in the first instance, be considered as resulting from an endogenous mutagenic process (spontaneous deamination of methylated cytosine). 10 Microdeletions, in particular in CG base repeats, are also thought to primarily result from an endogenous mechanism, polymerase slippage during replication. 11 In contrast, transversions (purine to pyrimidine or vice versa) at G bases (G : C to T : A or G : C to C : G) are often caused by exogenous carcinogens in experimental systems, and G : C to A : T transitions at non-CpG sites can be induced by many carcinogens, in particular N-nitroso compounds, oxidizing agents and alkylating agents (for review see References 12,13).

TP53 mutations in breast cancers: clinical and pathological significance TP53 mutation is the most common gene alteration in BC, with frequencies ranging from 12 to 60% (average: 29.7 ± 17.1%, calculated from 66 different studies). There is no significant variation according to geographic origin (Europe: 25.8%; Canada and USA: 34.5%; Japan 30.8%; References 14–16). Mutations are more frequent in advanced BC, suggesting that inactivation of TP53 is a ‘late’ event in mammary carcinogenesis. However, there is little information on the exact place of TP53 mutation in the sequence of genetic changes during the progression of BC. It cannot be ruled out that some TP53 mutations are 354

TP53 mutations in breast cancers

we eliminated mutations described in cell lines, metastases and recurrent tumours to extract 1392 mutations identified by sequencing in primary BC (described in 102 original publications, list available on request at [email protected] ). As a comparison group, we have used mutations from all primary cancers excluding breast (11 569 mutations). Figure 1(a) shows the pattern and codon distribution of TP53 mutations in breast tumours. BC shows a very similar profile to all other cancers [Figure 1(b)], with, however, less G : C to T : A transversions (10% versus 16% in all other cancers) and more A : T to G : C transitions (15% versus 11% in all other cancers). In cancers other than breast, the higher prevalence of G : C to T : A transversions is largely attributable to cancers in which carcinogens play an important role, such as lung cancers of smokers (prevalence of G : C to T : A transversions: 30%) and hepatocellular carcinoma in a context of dietary intoxication with aflatoxin B1 (45%) (see introduction). In cancers other than lung and liver, the prevalence of G : C to T : A transversions is 10%, similar to BC. In BC, 77% of G : C to T : A transversions are localized on the non-transcribed (coding) strand of the TP53 gene (that is, 77% of G : C to T : A mutations are G to T substitutions in the coding sequence). As the number of potentially mutable sites is approximately equal on both DNA strands, this asymmetry suggests that there is preferential repair of DNA damage on the transcribed strand. 12 The codon distribution in BC shows similar ‘hotspot’ mutations as in all other cancers, with, however, an overrepresentation of codon 163 (TAC to TGC). This codon is rarely mutated in most cancers (less than 1%), but accounts for over 2% of all BC mutations (26 mutations). Interestingly, codon 163 is a hotspot for TP53 mutation in BC of BRCA1/2 carriers, in which six of 63 mutations identified to date fall on that particular codon (data not shown). However, in this case the mutation preferentially occurs on the first base of the codon (TAC to AAC). Overall, this global analysis indicates that the TP53 mutation pattern in BC does not show any feature that significantly distinguishes it from most other human cancers.

present at early stages, in particular in aggressive cancers that rapidly progress towards advanced stages. BC is also the most frequent type of cancer in patients with inherited TP53 mutations, suggesting that mutation of one TP53 allele can predispose to early BC. Accumulation of p53 protein is commonly detected in over 50% of primary BC, 17 including cases that do not contain a TP53 mutation. 18,19 The functional status of this accumulated, wild-type p53 is not known. It has been proposed that wild-type p53 protein may be sequestered in the cytoplasm in inflammatory BC. 20 Overexpression of Mdm2 has been observed in a subset of BC, but whether this participates in p53 inactivation is not known. 21 Recently, Hall and collaborators 22 reported abnormal expression of ATM (a gene involved in the signalling of DNA damage to p53) in the majority of BC with wildtype TP53. Thus, several distinct mechanisms may contribute to the inactivation of the p53 pathway in the pathogenesis of BC. The p53 protein mediates apoptosis in response to many cytotoxic agents and the status of TP53 may affect responses to therapy. 23 While TP53 mutation is consistently described as a factor of poor prognosis in BC, 24 there is limited evidence that BC without TP53 mutation respond better to therapy than tumours with mutations. However, all mutations are not equivalent and TP53 mutants may differ by their biological and functional characteristics (stability, capacity to interact with other proteins, dominantnegative potential, residual wild-type activities). In 1996, Aas and coworkers 25 observed that tumours with mutations in specific loops of the DNA-binding domain (L2 and L3, involved in zinc-binding) had a poorer response to doxorubycin-based therapy than tumours with mutations located elsewhere in the protein structure. These findings were recently confirmed in larger cohort studies 26,27 but, to date, the data available are still too limited to identify which mutations may systematically have a worse prognosis for BC.

Global TP53 mutation pattern in sporadic breast cancer

TP53 mutation patterns in familial breast cancers

We have used the data compiled in the IARC TP53 mutation database (http://www.iarc.fr/p53) to analyse the pattern of TP53 mutations in sporadic and inherited BC. This database contains 14 051 somatic mutations and 196 germline mutations (version R4, April 2000). 28 From this dataset,

Constitutive alteration of the susceptibility genes BRCA1 or BRCA2 confers a substantial risk of developing breast, ovarian and some other cancers. 355

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(a) Breast Somatic Mutations (n=1392) 10 15%

5%

21%

% of single base substitutions

3% 2% 4% 12% A:T>C:G A:T>G:C A:T>T:A G:C>A:T 10% G:C>A:T at CpG G:C>C:G G:C>T:A 7% del ins other

8

4 163 179

2 0

21%

248 273

175

6

1

41

81

121

213

245 220 249

161 201 241 Codon number

282

281

321

361

321

361

(b) Other Somatic Mutations (n=11569) 3% 2% 4%

10 11% 5%

19%

% of single base substitutions

9% A:T>C:G A:T>G:C A:T>T:A 16% G:C>A:T G:C>A:T at CpG G:C>C:G G:C>T:A 8% del ins Other

248 273

8 6

175 245 249 179 213 220

4 2

282

0 23%

1

41

81

121

161 201 241 Codon number

281

Figure 1. Type and position of TP53 mutations in breast cancer (a) and other cancers (b). TP53 somatic mutations were extracted from the IARC TP53 Mutation Database (primary breast tumours: 1392 mutations; all other cancers: 11569 mutations). Mutation patterns are given as pie charts (left panels) showing the proportion of the different type of mutations. Right panels show the distribution of single base substitutions along the TP53 coding sequence (codon numbers of ‘hotspot’ mutations are indicated).

at A : T base pairs (mostly A : T to G : C and A : T to T : A). In contrast, they have a lower prevalence of G : C to A : T transitions at non-CpG sites. Although the number of TP53 mutations reported in BRCA1 or 2 carriers is still limited, these differences are significant (P < 0.001). They suggest that different mechanisms may be involved in the acquisition of TP53 mutations in BRCA1 or 2 carriers, compared to patients without known genetic susceptibility. This hypothesis is in agreement with the notion that BRCA1 and 2 may play defined roles in DNA repair. 30 Among the 196 TP53 germline mutations compiled in the database, 164 have been found in families with a history of multiple cancers. The pattern of these 164 mutations [Figure 2(b)] differs from the one of somatic mutations by a higher prevalence of G : C to A : T transitions at CpG sites (49% versus 21%, respectively) and a lower prevalence of G : C to T : A transversions (5% versus 10%) and G : C to A : T transitions at non-CpG sites (9% versus 21%). These differences are in agreement with the hypothesis that most germline mutations result from endogenous mutagenic processes (such as deamination of methylated cytosines, or polymerase

A total of 73 TP53 mutations in BC arising in BRCA1 (44) or BRCA2 (29) germline mutation carriers is reported in the R4 version of the IARC TP53 database. In BC arising in BRCA1 carriers, the prevalence of TP53 mutation appears to be slightly higher than in sporadic BC (53.8%, range 20–67%). In BRCA2 carriers, the reported prevalence of TP53 mutation is 28.6% on average (range 11–64). It should be noted that breast tumours arising in BRCA1 and BRCA2 carriers are genetically unstable and display a high degree of chromosomal aberrations. In a study by Eiriksdottir and colleagues, 29 loss of allele at chromosome 17p, encompassing the TP53 locus, has been observed in 85% (23/27) of BC from patients carrying the Icelandic BRCA2 founder mutation 999del5. Interestingly, most of these tumours had a wild-type TP53 gene, but overexpressed the p53 protein. This observation suggests that in BRCA2 carriers the p53 pathway may be deregulated by other mechanisms in addition to mutation. Figure 2(a) shows the pattern of TP53 somatic mutations in BRCA1/2-associated BC. Compared with sporadic BC, they show an excess of mutations 356

TP53 mutations in breast cancers

(a) Breast Somatic Mutations in BRCA1-2 carriers (n=73)

of endogenous origin, which did not occur in breast cells but are nonetheless capable of contributing to the pathogenesis of BC. Thus, somatic mutations that do not have an equivalent in the germline may result from mutagenic processes specifically occurring in breast cells. It is interesting to note that these mutations are mostly G : C to T : A transversions, a type of mutation which is often caused by environmental carcinogens.

1% 3% 3% 10% A:T>C:G A:T>G:C A:T>T:A G:C>A:T G:C>A:T at CpG G:C>C:G G:C>T:A de l ins Other

23% 12%

8%

10%

12%

18%

(b) Germline Mutations associated with LFS/LFL/FH (n=164) 2% 1% 2% 10% A:T>C:G A:T>G:C A:T>T:A G:C> A:T G:C>A:T at CpG G:C>C:G G:C>T:A del ins Other

11%

5%

Geographic and age variations in TP53 mutation patterns of BC

5%

6%

9%

Geographic variations in BC incidence may reflect variations in undetermined environmental exposures (see introduction). To examine whether these variations are reflected in TP53 mutation patterns, we have compared the mutations in BC from Europe, Northern America (USA and Canada) and Japan. As the main difference in age-standardized rates between Western countries and Japan is observed after the age of 45, we have separated cancers occurring before and after that age. Figure 3 shows that there are geographic differences in the prevalence of G : C to A : T transitions (non-CpG sites), G : C to T : A transversions and deletions. G : C to A : T transitions and G : C to T : A transversions are more prevalent in Western countries than in Japan. Moreover, in Western countries, these two types of mutations are more prevalent before 45 years of age. In Europe, the rate of G : C to T : A transversions drops by at least five-fold after 45 years of age. In contrast, deletions are more frequent in BC from Japan, and their prevalence increases after 45 years of age both in Japan and in the US, but not in Europe. In experimental systems, there is evidence linking these three types of mutations with DNA-damage induced by environmental carcinogens (see Table 1 and introduction). These results suggest that carcinogen exposure may contribute to geographic differences in the mutation patterns of BC. The fact that Western tumours show a higher prevalence of mutations on G bases is interesting, since mutations of this type have been shown to occur in cancers associated with exposure to tobacco smoke (G : C to T : A transversions in lung cancer and G : C to A : T transitions in bladder cancers). However, the codon distribution of G : C to T : A transversions in BC from Western countries differ from the one observed in lung cancers from smokers. In the latter

49%

(c) Germline Mutations in families with more than 30% breast tumors (n=19) A:T>C:G A:T>G:C A:T>T:A G:C>A:T G:C>A:T at CpG G:C>C:G G:C>T:A de l ins Other

0% 5% 5% 0%

11%

21%

0% 47% 11%

Figure 2. TP53 mutation patterns in familial breast cancers. (a) TP53 somatic mutations in breast cancer of patients with a germline mutation in BRCA1 or 2 genes, (b) TP53 germline mutations linked to a family history of cancer (Li–Fraumeni syndrome, Li–Fraumenilike syndromes or other family history), (c) from the 164 mutations selected in (b), 19 mutations observed in families with a clustering of breast cancer were selected (defined as families with at least four tumours and more than 30% breast tumours).

errors during replication). BC represents about 30% of the neoplasms in TP53 germline mutation carriers. To analyse germline mutations specifically associated with BC, we selected a subset of 19 families with an excess of BC (more than four tumours reported, at least 30% of which were breast tumours). The pattern of mutations in these families is shown in Figure 2(c). When compared to all germline mutations, the ones found in families with an excess of BC showed an absence of transversions affecting G bases and an excess of mutations on A : T base pairs (32% compared with 18% in the 164 germline mutations). Germline mutations associated with BC can be considered as mutations 357

Percent of total mutations by country

M. Olivier and P. Hainaut

60 50 40 30 20 10 0

G:C>A:T

EU

20

NA

Japan

G:C>T:A

15 10 5 0 EU

30 25 20 15 10 5 0

NA

Japan

NA

Japan

Deletions

EU

Before 45

After 45

Figure 3. Comparison of TP53 mutations in breast cancer from Northern America, Europe and Japan. Three types of mutation are shown, G : C > A : T transitions at non-CpG sites, G : C > T : A transversions and deletions. Mutations are divided into two categories: mutations in tumours of individuals younger than 45 (before 45) and older than 45 (after 45). There is no significant variation in prevalence for other types of mutations. NA = North America (USA and Canada), EU = Europe.

cancers, G : C to T : A ‘hotspots’ occur at codons 157, 158, 245, 248, 249 and 273. All these codons, except codon 249, are demonstrated sites of adduct formation by metabolites of benzo(a)pyrene, one of the main tobacco carcinogens. 31 In BC from Europe, only codons 245 and 249 are frequent sites for G : C to T : A transversions (over 5%; other mutations being scattered over 44 different codons). Thus, there is no evidence that the pattern of G : C to T : A transversions in breast cancers resembles the one of lung cancers of smokers. In 1992, Sommer and coworkers 32 have reported that the TP53 mutation pattern in BC of women residing in the Midwestern region of the US was different from the one of patients from other US regions and Europe, with excess frameshift deletions, splice site mutations and nonsense mutations. The data available at that time were limited and these

observations are not substantiated in the current dataset. However, we observed that in tumours from the US, the proportion of small deletions (one to four nucleotides) was higher (80%) than in tumours from Europe and Japan (60% in both regions). The reason why small deletions appear to be more frequent in the US is not known and there is insufficient information available to determine whether they correlate with the place of residence within the US.

Conclusions and perspectives Several studies have proposed that environmental factors play a role in the aetiology of BC (see introduction). In this paper, we have analysed the pattern of TP53 mutations in BC in order to identify possible fingerprints of such environmental 358

TP53 mutations in breast cancers

At present, there is no molecular basis to interpret the base specificity of transversions detected in BC. Nevertheless, the fact that these transversions are more frequent in Europe and among young women is highly suggestive of the involvement of specific, exogenous factors of risk. Genetic susceptibility is likely to play an important role in shaping the TP53 mutation pattern of BC. In future studies, it will be important to further stratify BC patients according to detailed information on individual exposures as well as polymorphisms in genes encoding carcinogen-metabolizing enzymes. In addition, it will be important to further analyse mutations in patients with constitutive alterations in BC predisposing genes. Indeed, the data currently available indicate that mutation patterns in BRCA1/2 carriers differ from those of sporadic BC, suggesting a role of these genes as ‘biological filters’ in the control of DNA damage and repair. We therefore believe that TP53 mutations in BC have not yielded all their secrets and that more global approaches are needed to understand the relationships between risk factors, genetic susceptibility and mutation patterns.

exposures. When analysed as a single group, the pattern of somatic TP53 mutations in BC does not show significant differences with the one of a comparison group made of all other cancers. However, differences appear after stratification by age (tumours occurring before or after the age of 45) and by geographic origin (tumours from Western countries as compared with those from Japan). These differences lie in the prevalence of G : C to A : T transitions and of G : C to T : A transversions (higher in Western than in Japanese BC, particularly in tumours occurring before 45) and in the prevalence of deletions (higher in Japanese BC, particularly in tumours occurring after 45). Another interesting observation is derived from comparison between patterns of somatic and germline TP53 mutations associated with familial BC. The germline pattern lacks G : C to T : A transversions, suggesting that the presence of such mutations in the somatic pattern is the consequence of mutagenic events occurring specifically within breast cells. Thus, a limited subset of BC mutations may be induced in response to exposure to environmental carcinogens. Most of the other mutations probably result from endogenous processes such as spontaneous deamination of methylated cytosine, or polymerase errors during DNA replication or repair. The lack of detailed information on individual exposures (in particular smoking status) precludes the association of mutation patterns with suspected risk factors. Radiation damage can promote the formation of deletions (among other DNA changes). It is therefore interesting to note that the prevalence of deletions is high in Japanese women of over 45 years of age. However, there is no direct evidence that these patients are atom bomb survivors or have received particularly high levels of radiation. A high prevalence of deletions is also detected in older US women, but most of them are small deletions that may result from polymerase slippage during replication rather than exposure to carcinogens. G : C to T : A transversions are often induced by exposure to carcinogens forming bulky DNA adducts such as mycotoxins, PAHs and aromatic amines. In lung cancers, G : C to T : A transversions preferentially occur on the coding strand of DNA and at bases identified as sites of adduction for metabolites of benzo(a)pyrene in cultured bronchial cells, supporting a direct, causative role of this tobacco carcinogen. 33 Although G : C to T : A transversions in BC also occur preferentially on the coding strand, they have a different codon distribution.

Acknowledgements MO is the recipient of a Special Training Award of the International Agency for Research on Cancer. The IARC database is supported in part by EC contract No QLG1-1999-00273

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