C transversion on p53 gene alterations in breast cancers from Taiwan

Cancer Letters 207 (2004) 59–67 www.elsevier.com/locate/canlet

High frequency of G/C transversion on p53 gene alterations in breast cancers from Taiwan Fang-Ming Chena,b, Ming-Feng Houb, Jaw-Yuan Wanga,b, Tsung-chi Chena, David C.P. Chenc, Sung-Yu Huangc, Yi-Shu Chungc, Shiu-Ru Lina,* a

MedicoGenomic Research Center, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan, ROC b Department of Surgery, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan, ROC c Asia Hepato Gene Co. Ltd, 10130 Sorrento Valley Road, Suite C, San Diego, CA 92121, USA Received 16 May 2003; received in revised form 1 December 2003; accepted 2 December 2003

Abstract p53 gene mutation is a very frequent event in many human cancers and is associated with a poor clinical outcome in breast cancer patients. Analysis of p53 gene mutations can also provide clues to the etiology of tumor formation. The present study was conducted to investigate the p53 mutations in patients with breast cancer from Taiwan. Tumor samples from 119 patients undergoing mastectomy for breast cancer were evaluated. The mutational status of the p53 gene (exons 5 – 8) was screened by polymerase chain reaction-single strand conformation polymorphism analysis followed by direct sequencing. Of all 119 cases of breast carcinoma, 26 mutations of the p53 gene were found in 22 cases (18.5%). Among these mutations, 78% (20/26) were point mutations with the majority of those being missense mutations (75%, 15 of 20 mutations) and the other 22% (6/26) were frameshift mutations. No significant correlation between p53 mutations and clinicopathological features was found, including HER2 status. Moreover, our results disclosed distinct mutation spectra in excess transversions to transitions (15/21, 71.4% vs. 6/21, 28.6%) with GC to CG dominant (6/15, 40%). Mutation hot spots we identified at codons 167, 185, 186, 210, 265 and 295 have rarely been documented in the literature. These findings showed that p53 gene mutation might contribute to the pathogenesis of breast carcinoma. Furthermore, the different mutation spectrum with high transversions in G:C to C:G may imply that the exogenous mutagens outweigh the endogenous processes in breast cancer in patients in Taiwan. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Breast cancer; p53; HER2

1. Introduction Breast cancer is the most frequent malignancy occurring in women worldwide, with 600,000 new cases arising each year. It accounts for nearly 20% of * Corresponding author. Tel.: þ 886-7-312-1101x7055; fax: þ 886-7-398-8864. E-mail address: [email protected] (S.-R. Lin).

all cancers among women [1]. Among Taiwanese women, breast cancer is the second most common form of malignancy and the fourth-leading cause of cancerrelated death. Death rates in females from breast cancer in 2001 were 11.36 per 100,000 people, an increase of 66% over 1989 [2]. The risk factors of getting breast cancer in Taiwan, a low incidence area, are similar to those in high to moderate-risk areas [3]. Family history

0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2003.12.005

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and the effects of reproductive hormones play the most significant role. However, when compared with the figures of breast cancer in Western countries [4], the peak of the age-specific incidence rates appeared 10 years younger [5] and the age-specific mortality rates peaked at ages 55 –59 and after 80 [2] in Taiwan. These divergences may suggest some distinct mechanisms regarding the tumorigenesis and carcinogenesis of breast cancer in Taiwan. The process of carcinogenesis involves the gain of oncogene activity and the loss of tumor suppressor gene function. Among the tumor suppressors, p53 gene abnormalities are the most frequent genetic events illustrated to date [6,7]. When in response to genotoxic [8] (irradiation, or chemical carcinogen) or non-genotoxic [9,10] (oncogene activation, and hypoxia) stress, p53, as a transcription factor, will be phosphorylated and exert a protective response either by regulating cell cycle at G1/S checkpoint for DNA repair [11] or by inducing apoptosis in genetically damaged cells [12]. The majority of p53 mutations occur as point mutations in the evolutionary conserved regions of the p53 gene, affecting the DNAbinding area of the protein [13]. The aberrations of p53 in breast cancer, either with p53 protein overexpression [14,15] or mutations of the p53 gene [16,17] in their tumors, were found to correlate strongly with tumor grade and may predict poor prognosis. Recent studies have also shown that patients with mutations within conserved residues of the DNA-binding surface appear to have increased resistance to doxorubicin-based therapy, but not to taxol [18 – 20]. Studies on p53 gene mutations may permit identification of a subset of breast cancer patients who respond poorly to certain adjuvant therapies and are at high risk of early recurrence and death. Furthermore, the observed patterns of mutation in the p53 gene can be used as an epidemiological tool to explore the contribution of exogenous mutagens vs. endogenous processes in particular cancers [21]. The published data has shown that the pattern of p53 mutations differs among 15 geographically and ethnically diverse populations [22]. The variations of p53 mutation patterns in breast cancer are consistent with a significant contribution by a diversity of exogenous mutagens. To clarify the mutation rate and pattern of p53 tumor suppressor gene in patients with breast cancer in

Taiwan, we analyzed p53 gene mutations in 119 cases. Moreover, we compared the mutation patterns in Asia, where generally possess low breast cancer risk.

2. Materials and methods 2.1. Specimen collections In the present investigation, samples obtained from 119 primary breast cancers removed surgically at the Department of Surgery of Kaohsiung Medical University Hospital were analyzed for somatic mutations in the p53 gene. None of the patients had undergone radiotherapy or chemotherapy before operation. There were 16 intraductal carcinomas, 78 infiltrating ductal carcinomas, 12 infiltrating lobular carcinomas, 5 medullary carcinomas, 3 tubular carcinomas, 2 mucinous carcinomas, 2 atypical medullary carcinomas and 1 invasive cribriform carcinoma. Immediately after surgery, the parts of the tumor samples were placed in liquid nitrogen, and then stored in 2 80 8C freezers until analyzed. The rest of unfrozen tissues were sent for routine histopathological diagnosis including HER2 expression study according to the usual criteria at the Kaohsiung Medical University Hospital, as described previously [23]. 2.2. DNA extraction Genomic DNA was isolated from frozen primary breast cancers using proteinase K (Stratagene, La Jolla, CA, USA) digestion and phenol/chloroform extraction procedure according to Sambrook’s method [24]. 2.3. PCR-SSCP analysis To search for subtle mutation of the p53 gene, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis was used as described previously [25]. The oligonucleotide primers for p53 coding regions were described below: Exon 5: SR5-1LT: 5 0 -CTTTGCTGCCGTCTTCCAGTTCG-30 and SR5-1RT, 50 -CTATCTGACCAGCGCTCATG-30 ; SR5-2LT: 50 -GCCATCTACAAGCAGTCA-30 and SR5-2RT, 50 -AGACCTAAGAGCAATGAGTG-30 ;

F.-M. Chen et al. / Cancer Letters 207 (2004) 59–67

Exon 6: SR6LT, 50 -AGGTCTGGCCCCCTCCTCAGC-30 and SR6RT, 50 -ACCTCAGGCGGC-TCATAGGGCA-30 ; Exon 7: SR7LT, 5 0 -TCTCCTAGGTTGGCTCTGAC-30 and SR7RT, 50 -CACAGCAGGCCAGTGTGCAG-30 ; Exon 8: SR8LT, 5 0 -ATGGGACAG-GTAGGACCTGA-30 and SR8RT, 50 -TGAATCTGAGGCATAACTGCACC-30 . The PCR conditions for each exon were as follows: 40 cycles of 1 min denaturation at 94 8C, 1 min annealing at 55 8C for exons 5, 6, 7 or 58 8C for exon 8, and 1 min extension at 72 8C, and 5 min final extension at 72 8C. The PCR products were heated at 94 8C for 4 min and loaded onto polyacrylamide gels of GeneGel Excel 12.5/24 kit (Pharmacia Biotech Inc., San Francisco, CA) using the Genephore electrophoresis unit (Pharmacia Biotech). Gels were run for 5 h at 200 V, and then they were stained with PlusOone DNA silver staining kit (Pharmacia Biotech). 2.4. Direct sequencing Sequencing analysis was performed to confirm the mobility shift bands detected on SSCP analysis. The PCR products were purified by the QIAEX II Gel Extraction Kit (QIAGEN Inc., Valencia, CA) and then subjected to sequencing using a double-strand cycle sequencing system (GIBCO BRL, Gaithersburg, MD). The purified products were then sequenced directly with T7 promoter/IRD800 (LI-COR, Lincoln, NE). An automated DNA electrophoresis system (Model 4200; LI-COR) with a laser diode emitting at 785 nm and fluorescence detection between 815 and 835 nm was used to detect and analyze the sequencing ladder. Data collection and image analysis utilized an IBM486 (Model 90) using the Base Image IR software supplied with the model 4200 DNA sequencer. 2.5. Statistical analysis All data were analyzed using the Statistical Package for the Social Sciences Ver 8.0 software (SPSS Inc., Chicago, IL). The two-sided Pearson x 2 test was used to compare the clinicopathological parameters between cancers with p53 mutations or

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not. A probability of less than 0.05 was considered to be statistically significant. 3. Results In our study, there were 119 breast cancer patients enrolled for study. The clinicopathological characteristics are summarized in Table 1. All of the cancer Table 1 Patients’ characteristics and clinicopathological features and relationships to p53 Total patients (%)

Positive p53 No. (%)

P

Age ,40 40–49 50–59 60–69 .70

21(71.6) 54(45.4) 25(21.0) 11(9.2) 8(6.7)

4(23.8) 10(24.1) 5(24.0) 2(18.2) 1(12.5)

0.951

Location Right Left Bil

65(54.6) 52(43.7) 2(1.7)

13(26.2) 9(19.6) 0

0.659

Histological type Intraductal Infiltrating ductal Lobular Mucinous

16(14.8) 78(72.2) 12(11.1) 2(1.9)

2(18.8) 15(24.4) 3(25.0) 0

0.916

Histological grade Grade I Grade II Grade III

17(27.9) 28(46.0) 16(26.1)

4(23.5) 5(17.9) 2(12.5)

0.548

Tumor size (cm) T1 T2 T3

31(26.1) 80(67.2) 5(6.7)

5(19.4) 17(26.3) 0

0.538

No. of involved nodes 0 1–3 4–9 .10

37(31.1) 32(26.9) 22(18.5) 27(22.7)

5(16.2) 5(21.9) 6(31.8) 6(25.9)

0.663

ER status Positive Negative

68(63.0) 40(37.0)

10(17.6) 8(27.5)

0.261

6(39) 8(61)

0.59

HER2 status Positive Negative

34(35) 63(65)

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Fig. 1. PCR-SSCP analysis of p53 mutations in breast cancer. Representative samples are shown for exons 5, 6 and 8. An electrophoretic mobility shift of the bands between the tumor (T) and its paired normal tissue (N) implies a different conformation of the fragment, suggesting the presence of mutation in these exons. The PCR-SSCP analysis of exons 5, 6 and 8 showed an apparently mobility shift in cases 9, 5 and 39, 40 indicated by arrows.

tissues and their adjacent normal breast tissues were screened for the presence of p53 gene mutations in exons 5 – 8 using the PCR-SSCP analysis. DNA fragments containing a deletion, insertion, or singlebase change showed motility shift, which was distinct from those of normal strands in non-denaturing polyacrylamide gel electrophoresis (Fig. 1). The analysis of exon 5 showed different bands in cases

5, 7, 9, 18, 27, 33, 82, 87 and 90; exon 6 in cases 5, 28, 38, 55 and 108; exon 8 in cases 20, 34, 35, 39, 40, 49, 96 and 97; in addition to the normal bands. To identify the mutations in this single stranded DNA, PCR products displaying a mobility shift on SSCP analysis were directly sequenced. The results of direct sequencing were compared to those of controls (Fig. 2). The incidence of p53 gene mutation and its

Fig. 2. Sequencing analysis of the p53 tumor suppressor gene in breast cancer. Each mutation in the tumor cells (T) shown is matched to a normal adjacent breast tissue (N). The codon at which the mutation occurs is indicated. Each sequence is shown 50 (bottom) to 30 (top). Arrows point to bands corresponding to mutated basepairs. Sequencing gel showed a p53 point mutation at codon 185 in tumor of case 9 and resulted in a change in the encoding amino acid from serine to arginine; and a base deletion in tumor of case 5 resulting in protein truncating.

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Table 2 A summary of p53 gene mutations in the breast cancer Case no.

Exon containing mutation

Codon mutated

Nucleotide change

Amino acid change

Types of mutation

5 5 7 9 18 18 20 20 27 28 33 34 35 38 38 39 40 49 55 77 82 87 90 96 97 108

5 6 5 5 5 5 8 8 5 6 5 8 8 6 6 8 8 8 6 6 5 5 5 8 8 6

185 215 169 185 180 186 274 276 168 208 165 264 293 194 212 265 265 267 213 210 186 167 167 295 295 210

Del.A(G)C Del.A(G)T ATG ! GTG AGC ! AGG GAG ! CAG GAT ! CAT GTT ! ATT GCC ! ACC CAC ! CAT GAC ! GAA CAG ! CACG CTA ! GGA GGG ! GGC CTT ! CGTT TTT ! ATT CTG ! ATG CTG ! ATG CGG ! CGT CGA ! TGA AAC ! ACC GAT ! CAT CAG ! CTG CAG ! CTG CCT ! ACCT CCT ! ACCT AAC ! AGC

Ser/– Ser/– Met/Val Ser/Arg Glu/Gln Asp/His Val/Ile Ala/Thr His/His Asp/Glu Gln/– Leu/Gly Gly/Gly Leu/– Phe/Ile Leu/Met Leu/Met Arg/Arg Arg/– Asn/Thr Asp/His Gln/Leu Gln/Leu Pro/– Pro/– Asn/Ser

Frameshift Frameshift Missense Missense Missense Missense Missense Missense Silent Missense Frameshift Missense Silent Frameshift Missense Missense Missense Silent Nonsense Missense Missense Missense Missense Frameshift Frameshift Missense

relation to histopathological features in breast cancer are shown in Table 1. There were 26 mutations detected in 22 tumor tissues. The mutation rate was 18.5% (22/119) in this investigation. No significant correlation between p53 mutations and age, tumor location, histological type and grade, tumor size, axillary lymph nodes, ER or HER2 status was found. Among the mutations, as shown in Table 2, the majority were point mutations (20/26, 78%) distributed through exons 5, 6, and 8. Only six mutations were frameshift (four insertions, two deletions). Most of the point mutations comprised of missense mutations (15/20, 75%). We identified several very rare mutations at codon 185, 210, 215, 265 and 293 in breast cancer when searching the IACR p53 mutation database [45]. In addition, recurring mutations were detected at codon 167 (CAG ! CTG), resulting in a change from glutamine to leucine substitution in

the protein; at codon 185 (AGC ! AGG and one base deletion, frameshift), resulting in a change from serine to arginine substitution and truncating in the protein; at codon 186 (GAT ! CAT), resulting in a change from aspartic acid to histidine substitution; at codon 210 (AAC ! AGC and AAC ! ACC), resulting in a change from asparagine to serine and threonine substitution; at codon 265 (CTG ! ATG), resulting in a change from leucine to methionine substitution and at codon 295 (CCT ! ACCT, frameshift), resulting in a change from glutamine to truncating in the protein. These mutations denoted hot spots of p53 mutations in this study. Amid the point mutations, four (19% of all point mutations) were GC to AT transitions, with one of the four occurring at the CpG sequence, and two of the point mutations were AT to GC transitions. Of the 15 transversions, six were GC to CG, two GC to TA, four AT to CG and three AT to TA (Table 3).

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Table 3 Summary of p53 point mutations Classes of mutations G:C/A:T at CpG no. G:C/A:T non-CpG no. A:T/G:C no. G:C/C:G no. G:C/T:A no. A:T/C:G no. A:T/T:A no. (%) (%) (%) (%) (%) (%) (%) (%) Transitions Transversions

1(4.8) –

3(14.3) –

2(9.5) –

4. Discussion With the application of PCR-SSCP, we identified 26 mutations in 22 tumors of 119 patients. The p53 mutation rate in our series was 18.5%. This figure is in concordance with the average mutation rate detected in breast cancer [26]. p53 is the most common gene alteration in breast cancer. In Asia, which generally possesses low incidence of breast cancer, the mutation rate of p53 is diverse among different areas [27 – 32] (Table 4). While the p53 mutation rate of breast cancer of Chinese either in Taiwan [27] or China [28] was lower, ranging from 18 to 22%, the higher mutation rates spanning from 36 to 71% were reported in Japan [30,33] and Korea [31]. Mutagenesis of p53 may play a significant but less predominant factor for breast cancer development in Taiwan. Our findings denoted no correlation between p53 gene mutation and clinicopathological features (Table 1). It is generally accepted that p53 gene mutation occurs

– 6(28.6)

– 2(9.5)

– 4(19.0)

– 3(11.5)

mainly in advanced stage and might relate to high histological grade [32,34], negative productive hormone receptors status [35], positive HER2 overexpression [36], tumor progression [37] and poor survival [38,39]. Further survival analysis will be deployed to determine if the p53 gene mutation leads to poor prognosis of breast cancer in our series. Our direct sequencing data depicted a unique mutation pattern of p53 in breast cancer when compared with earlier data by Lou et al. [27], also from Taiwan, or results of other Asian areas (Table 4). Our results showed an excessive proportion of transversions (15/27, 55.5%) over transitions (6/27, 22.2%) in p53 mutations. Of the transversions, the G:C to C:G was predominant (6/27, 22.2%), and of the transitions, only 4% was G:C to A:T at CpG site. The pattern was much different from the results of previous studies. Lou et al. investigated the tumors of women from Northern Taiwan and revealed excess of G:C to A:T transition at CpG site (30.8%).

Table 4 Comparison of mutations in p53 gene in breast cancers in Asia Origin of patients No. of Del. or Classes of mutations (%) mutations (%) ins. (%) Transitions

Transversions

G:C/A:T at CpG G:C/A:T non-CpG A:T/G:C G:C/C:G G:C/T:A A:T/C:G A:T/T:A Taiwan South North [27] China Hong Kong [28] Japan Tokushima [29] Sapporo [30] Tokyo [32] Korea [31]

27(18.5) 26(22.8)

19 11.5

4 30.8

5(21.7)

0 19 0 21

26(23) 17(71) 29(25) 39(34.6)

50.1

12 19.2

8 15.4

0

40

40

0

35 41 17

11 17 21

8 18 24

10

–

8.5

23 7.7

8 7.7

15 3.9

11 3.9

20

0

0

4 6 0

4 0 3

8 12 7

11 6 7

–

18.3

–

–

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A similar pattern was also observed in studies of breast cancers in Northern Japan [29,30]. By using the IARC TP53 mutation database to analyze the global pattern of mutations in breast cancers, Olivier [26] found that there is an excess of transversions on G bases in tumors from Western (USA and Europe) as compared to Eastern (Japan) countries. Transitions at CpG dinucleotides are hot spots of p53 gene mutations [40], which reflect methylation and deamination of cytosine [41] or endogenous process. By contrast, the classical epidemiological studies such as G:C to A:T transversions in lung cancer of smokers [42] and hepatocellular carcinoma associated with aflatoxin B1 [43] and experimental studies [44] implicate transversions (purine to pyrimidine or vice versa) at G bases are often caused by exogenous carcinogens. The excess of transversions over transitions in our series may suggest more environmental or exogenous process being involved in breast cancer formation in Taiwan. Besides, the analysis of the p53 mutation patterns in breast cancer of 15 populations showed distinct patterns of mutation [22]. The results were interpreted as different mutagens are associated with breast cancer in different populations. Our study and the investigation from Lou et al. may further suggest that different mutagens are involved in breast cancer formation of the same population in same area when in different districts. Blaszyk et al. [30] has observed similar results. They investigated the molecular epidemiology of breast cancers in northern and southern Japan and disclosed multiple differences in acquired p53 mutations between the two Japanese cohorts. All the mutation codons we identified were rarely reported in IACR TP53 mutation database [45]. Except for codon 213 which had 28 events in 1407 mutations of the IACR database with a frequency of 2%, the rest of mutation codons we recognized were reported in less than 1% of IACR database. Mutation at codon 185, one of the recurring mutation codons in our series, has the proportion of one in 1905 mutations of the breast cancer category in IACR database. However, we identified two distinct mutation types at codon 185 (Fig. 2). AGC ! AGG missense mutation resulted in a change from serine to arginine substitution and AGC ! AG one base deletion led to truncating protein. Currently, the correlation between

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the mutations, protein structure and functional features are unclear. In conclusion, our result indicated that p53 gene mutation plays a significant role in the genesis of breast cancer in Taiwan. Furthermore, the different mutation spectrum with high transversions in G:C to C:G may imply the exogenous mutagens outweighing the endogenous processes in breast cancer from Taiwan. Continuous investigation will be necessary to clarify the impact of p53 gene mutation on the prognosis of breast cancer in Taiwan.

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