1 Department of Experimental Medicine, University of LAquila, LAquila; Divisions of 2 Oncologic Surgery and 3 Pathology and Cytology, S. Salvatore Hospital, LAquila; 4 Department of Oncology and Neurosciences, University G. D. Annunzio, Chieti, Italy
Received 4 July 2002; revised 14 November 2002; accepted 3 December 2002
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Abstract |
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Mutations in the p53 gene are the most common genetic alterations in human primary breast carcinoma and these mutations are often associated with worse prognosis and chemo/radioresistance.
Patients and methods:
The analysis of the p53 gene was performed by fluorescence-assisted mismatch analysis in 13 consecutive high-risk primary breast cancer (HR-BC) patients with 10 or more involved axillary nodes to evaluate its prognostic value.
Results:
Three p53 mutations (23%) and four allelic variants were detected. After a median follow-up of 52 months the HR-BC disease-free survival (DFS) was 51% and overall survival 79%. All patients harboring a p53 mutation (p53mut) relapsed within 10 months of the median DFS while 67% of those showing a wild-type p53 status (p53wt) survive disease-free at a median follow-up of 43 months. One p53mut patient is still alive while all the p53wt patients survive at 56 months median follow-up. Two out of the four p53wt relapsing breast cancer patients showed the Arg72Pro allelic variant; one of these died at 75 months.
Conclusions:
p53 mutations may help identify a subset of very high risk breast cancer patients (vHR-BC) with worse prognosis.
Key words: breast cancer, FAMA, p53, prognostic factors
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Introduction |
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High-risk, node-positive breast cancers (HR-BC) with 10 involved axillary lymph nodes show a 5-year disease-free survival (DFS) of 3040% and 5-year overall survival (OS) ranging between 50% and 71%, despite different adjuvant treatments [14]. The wide distribution of survival intervals (DFS and OS) may be due to the different extent of micrometastatic involvement at the time of diagnosis as well as to the biological aggressiveness and sensitivity of cancer cells [5, 6]. The identification of a different subset of very high risk (vHR-BC) patients using molecular determinants could help to better address therapeutic strategies (i.e. conventional, high-dose, experimental).
p53 gene inactivation represents a critical step in the development of human malignancies due to its pivotal role in multiple cellular pathways such as cell cycle control at the G1/S checkpoint, DNA repair, programmed cell death and neoangiogenesis [7]. p53 mutations characterize 2040% of breast cancers [8, 9]. Most reported studies, in which different molecular diagnostic approaches were used, have established that p53 molecular status is an independent marker of poor prognosis in breast cancer patients, while p53 status assessed by immunohistochemistry has shown conflicting results [1022].
Semiautomatic scanning approaches based on chemical cleavage of mismatch, such as fluorescence assisted mismatch analysis (FAMA), show optimal diagnostic accuracy in detecting mutations of large and complex genes such as BRCA1 [23, 24]. In the present study, complete analytical scanning of the p53 gene coding sequence (exons 211) and adjacent intronic regions was performed by FAMA in tumor DNA samples from 13 HR-BC patients in order to prospectively assess its prognostic value.
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Patients and methods |
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Quadrantectomy plus adjuvant radiotherapy or modified mastectomy combined with axillary dissection were the primary treatments. All patients received systemic adjuvant chemotherapy at the end of which patients with positive estrogen receptor (ER) status were also treated with hormone therapy (Tamoxifen). Recurrent cancers were treated with systemic chemotherapy. Clinical staging consisted of serum chemistry including tumor markers (CEA and CA 15-3), radiologic imaging using X-ray, ultrasounds and computed tomographic (CT) scans of the abdomen and chest. Bone scans were performed every year. All patients were seen every 36 months for 5 years according to standard follow-up procedures; after 5 years, they were seen on a yearly basis. Follow-up was carried out at the Division of Medical Oncology, S. Salvatore Hospital, LAquila.
Analysis of p53 status
Breast cancer tissue fragments and nearby normal breast tissue were immediately snap-frozen and kept at 80°C. Genomic DNA was obtained by proteinase K digestion and phenol/chloroform extraction [26]. Complete analytical scanning of the p53 coding frame (exons 211) was planned by FAMA, a semiautomatic scanning procedure based on chemical cleavage of mismatch [24], which has recently been demonstrated to guarantee optimal diagnostic accuracy in scanning large PCR amplicons, up to 1.4 kb in length [23], also at the somatic level [27]. Altogether, the p53 scanning strategy was based on fluorescent amplification of four DNA regions: two amplicons covering exons 59 (p53.1, 1264 bp, spanning exons 57 and adjacent introns, and p53.2, 831 bp, spanning exons 89 and flanking introns) were designed as already described [27]; one covering exons 24 (p53.3, 1078 bp), and one covering exons 1011 (p53.4, 1303 bp).
Each of the following fluorescent primers was selected in intronic DNA sequences (100 nucleotides away from exons) and contained a GG dinucleotide at the 5' end as a spacer between the fluorophore and the DNA sequence: p53.1 forward/5'-FAM-ggttgcaggaggtgcttaca-3'; p53.1 reverse/ 5'-HEX-ggtatggaagaaatcggtaaga-3'; p53.2 forward/5'-6-FAM-ggtcatcacatttccggcgg-3'; p53.2 reverse/5'-HEX-ggaagtaactccatcgtaagtc-3'; p53.3 forward/5'-6-FAM-gggaagtccctctctgattg-3'; p53.3 reverse/5'-HEX-gggtgtgatgggatggataa-3'; p53.4 forward/5'-6-FAM-gggcttttgatccgtcataa-3'; p53.4 reverse/5'-HEX-ggagcaagggttcaaagac-3'.
Polymerase chain reactions (35 cycles: 30 min at 90°C, 30 min at 57°C, 1.20 min at 72°C) were performed using 200 ng of genomic tumor DNA; 10 pmol of primers labeled at the 5' end with 6-FAM or HEX fluorophore; 200 µM dNTPs (Amersham Pharmacia, Buckinghamshire, UK); 1x PCR buffer [Tris 50 mM, pH 9.2, (NH4)2SO4 16 mM, MgCl2 2.25 mM] (Sigma, St Louis, MO, USA) and AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA, USA) 1.25 U/l, in a total volume of 25 µl.
The FAMA protocol for p53 mutation scanning was as previously described [27]. Briefly, after heteroduplex formation, 0.2 pmol of fluorescent PCR product were subjected to chemical cleavage reaction using either hydroxylamine or osmium tetroxide, which interact with cytosines and thymines, respectively, at the level of mismatched nucleotides along heteroduplex DNA molecules.
The reaction products were loaded onto a 4% polyacrylamide denaturing gel and electrophoresis was performed on an ABI PRISM 377 DNA Sequencer (Applied Biosystems). The electrophoretic fluorescent profiles were analyzed using the GeneScan 3.1 software. All the mismatches observed by FAMA analysis were confirmed by semiautomatic sequencing analysis using Big Dye Terminator Kit (Applied Biosystems) according to the manufacturers instructions.
Statistical analysis
Disease recurrence was defined as either local recurrence or metastatic disease. Time to recurrence, defined as the period from surgery to first recurrence, and survival time, defined as the interval from surgery to death, were calculated by the KaplanMeier method [28] and compared according to the p53 status (mutated versus wild-type).
Analysis of survival data was planned after the detection of metastatic disease in 50% of patients and performed each year thereafter.
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Results |
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Discussion |
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After a median follow-up of 52 months, the survival rates (51% DFS and 79% OS) relative to this small but specific cohort of HR-BC patients correspond to those reported in patients with 10 or more involved axillary nodes, treated with conventional and high-dose chemotherapy [3]. The role of high-dose chemotherapy in vHR-BC patients is currently under evaluation in randomized phase III trials and remains questionable.
The meta-analysis of published studies concerning the prognostic value of the molecular detection of p53 mutations shows a relative hazard estimate of death of 2.0 (CI 1.7% to 2.5%) in all breast cancer patients and 2.6 (CI 1.7% to 3.9%) in node-positive breast cancer patients [29]. The prognostic evaluation according to p53 molecular status (Table 2) in the cohort of HR-BC patients shows a statistically significant, worse prognosis in the p53mut patients in terms of DFS and OS at 52 months median follow-up, thus identifying a very high risk breast cancer subset: each of the three patients relapsed within 2 years; two out of three patients died within a median of 32 months. Conversely, in the p53wt subset 67% of patients are disease-free and all are alive at 52 months. High-risk breast cancers represent a wide range of different and unmeasurable micro-metastatic diseases [3]. Thus, in this subset of patients (vHR-BC) the detection of p53 mutations may exert its prognostic value as a molecular factor of biological aggressiveness as well as a clinical indicator of early metastatic disease [2931]. Furthermore, the present observation of long-surviving p53wt HR-BC patients as well as the comparable survival rates already reported in node-positive and node-negative p53wt breast cancer patients [22] requires the need to redefine the prognostic relevance of nodal involvement in the p53wt subset.
Four patients show the exon 4/Arg72Pro allelic variant and therefore we suggest considering the functional implications of specific p53 mutations, or allelic variants, in terms of prognosis and treatment-response prediction. The Arg72Pro polymorphism affects a proline-rich region of the p53 protein that is essential for triggering p53-dependent apoptosis [2]. Furthermore, in the presence of p53 conformational mutations, the allelic variant frequently reported at this level has been demonstrated to induce a gain of function of the p53 gene, through the inactivation of the p73 gene, that loses its role as a transcriptional regulator and apoptosis trigger [3]. In our cohort of HR-BC patients, two patients carrying a p53 pathologic mutation, associated to the allelic variant, relapsed and one died. Two patients showing the same allelic variant relapsed within the p53wt subset with a median DFS of 36 months (29 and 44 months); one of these died at 75 months. These results preliminarily suggest the possibility of identifying patients with an intermediate prognosis within the p53wt HR-BC subset.
Present data represent findings specifying the prognostic value according to p53 molecular status in HR-BC patients using a highly accurate molecular diagnostic approach. Further prospective studies will need to better define the prognostic and predictive role of the p53 molecular status in different subsets of breast cancer.
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Footnotes |
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References |
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2. Fisher B, Anderson S, Wickerham DL et al. Increased intensification and total dose of cyclophosphamide in a doxorubicincyclophosphamide regimen for the treatment of primary breast cancer: findings from National Surgical Adjuvant Breast and Bowel Project B-22. J Clin Oncol 1997; 15: 18581869.[Abstract]
3. Sledge GW, Miller K. Adjuvant high-dose chemotherapy in breast cancer: current status. ASCO educational book. American Society of Clnical Oncology 36th Annual Meeting, New Orleans, LA, 3023 May. 2000; 251256.
4. Hudis C, Fornier M, Riccio L et al. Five-year results of dose-intensive sequential adjuvant chemotherapy for women with high-risk node-positive breast cancer: a phase II study. J Clin Oncol 1999; 17: 1118.
5. Nemoto T, Vana J, Bedwani RN et al. Management and survival of female breast cancer: results of a national survey by the American College of Surgeons. Cancer 1980; 45: 29172924.[ISI][Medline]
6. Perrone F, Carlomagno C, Lauria R et al. Selecting high-risk early breast cancer patients: what to add to the number of metastatic nodes? Eur J Cancer 1996; 32: 4146.
7. Hartmann A, Blaszyk H, Kovach JS, Sommer SS. The molecular epidemiology of p53 gene mutations in human breast cancer. Trends Genet 1997; 13: 2733.[CrossRef][ISI][Medline]
8. Hernandez-Boussard T, Rodriguez-Tome P, Montesano R, Hainaut P. IARC p53 mutation database: a relational database to compile and analyze p53 mutations in human tumors and cell lines. International Agency for Research on Cancer. Hum Mutat 1999; 14: 18.[CrossRef][ISI][Medline]
9. Thorlacius S, Borresen AL, Eyfjord JE. Somatic p53 mutations in human breast carcinomas in an Icelandic population: a prognostic factor. Cancer Res 1993; 53: 16371641.[Abstract]
10. Thorlacius S, Thorgilsson B, Bjornsson J et al. TP53 mutations and abnormal p53 protein staining in breast carcinomas related to prognosis. Eur J Cancer 1995; 31: 18561861.[CrossRef]
11. Andersen TI, Holm R, Nesland JM et al. Prognostic significance of TP53 alterations in breast carcinoma. Br J Cancer 1993; 68: 540548.[ISI][Medline]
12. Elledge RM, Fuqua SA, Clark GM et al. The role and prognostic significance of p53 gene alterations in breast cancer. Memorial Symposium. Breast Cancer Res Treat 1993; 27: 95102.[ISI][Medline]
13. Bergh J, Norberg T, Sjogren S et al. Complete sequencing of the p53 gene provides prognostic information in breast cancer patients, particularly in relation to adjuvant systemic therapy and radiotherapy. Nat Med 1995; 1: 10291034.[ISI][Medline]
14. Seshadri R, Leong AS, McCaul K et al. Relationship between p53 gene abnormalities and other tumour characteristics in breast-cancer prognosis. Int J Cancer 1996; 69: 135141.[CrossRef][ISI][Medline]
15. de Witte HH, Foekens JA, Lennerstrand J et al. Prognostic significance of TP53 accumulation in human primary breast cancer: comparison between a rapid quantitative immunoassay and SSCP analysis. Int J Cancer 1996; 69: 125130.[CrossRef][ISI][Medline]
16. Falette N, Paperin MP, Treilleux I et al. Prognostic value of p53 gene mutations in a large series of node-negative breast cancer patients. Cancer Res 1998; 58: 14511455.[Abstract]
17. Soong R, Iacopetta BJ, Harvey JM et al. Detection of p53 gene mutation by rapid PCR-SSCP and its association with poor survival in breast cancer. Int J Cancer 1997; 74: 642647.[CrossRef][ISI][Medline]
18. Berns EM, van Staveren IL, Look MP et al. Mutations in residues of TP53 that directly contact DNA predict poor outcome in human primary breast cancer. Br J Cancer 1998; 77: 11301136.[ISI][Medline]
19. Gretarsdottir S, Tryggvadottir L, Jonasson JG et al. TP53 mutation analyses on breast carcinomas: a study of paraffin-embedded archival material. Br J Cancer 1996; 74: 555561.[ISI][Medline]
20. Sjogren S, Inganas M, Norberg T et al. The p53 gene in breast cancer: prognostic value of complementary DNA sequencing versus immunohistochemistry. J Natl Cancer Inst 1996; 88: 173182.
21. Kovach JS, Hartmann A, Blaszyk H et al. Mutation detection by highly sensitive methods indicates that p53 gene mutations in breast cancer can have important prognostic value. Proc Natl Acad Sci USA 1996; 93: 10931096.
22. Blaszyk H, Hartmann A, Cunningham JM et al. A prospective trial of midwest breast cancer patients: a p53 gene mutation is the most important predictor of adverse outcome. Int J Cancer 2000; 89: 3238.[CrossRef][ISI][Medline]
23. Ricevuto E, Sobol H, Stoppa-Lyonnet D et al. Diagnostic strategy for analytical scanning of BRCA1 gene by fluorescence-assisted mismatch analysis using large, bifluorescently labeled amplicons. Clin Cancer Res 2001; 7: 16381646.
24. Verpy E, Biasotto M, Meo T, Tosi M. Efficient detection of point mutations on color-coded strands of target DNA. Proc Natl Acad Sci USA 1994; 91: 18731877.[Abstract]
25. Yarbro JW, Page DL, Fielding LP et al. American Joint Committee on Cancer prognostic factors consensus conference. Cancer 1999; 86: 24362446.[CrossRef][ISI][Medline]
26. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory 1982.
27. Tessitore A, Di Rocco ZC, Cannita K et al. High sensitivity of detection of p53 somatic mutations using fluorescence assisted mismatch analysis (FAMA). Genes Chromosom Cancer 2002; 35: 8691.[CrossRef]
28. Kaplan EL, Meier P. Non parametric estimation from incomplete observation. J Am Stat Assoc 1958; 53: 457481.[ISI]
29. Pharoah PD, Day NE, Caldas C. Somatic mutations in the p53 gene and prognosis in breast cancer: a meta-analysis. Br J Cancer 1999; 80: 19681973.[CrossRef][ISI][Medline]
30. Elledge RM, Allred DC. Prognostic and predictive value of p53 and p21 in breast cancer. Breast Cancer Res Treat 1998; 52: 7998.[CrossRef][ISI][Medline]
31. Kirsch DG, Kastan MB. Tumor-suppressor p53: implications for tumor development and prognosis. J Clin Oncol 1998; 16: 31583168.[Abstract]
32. Venot C, Maratrat M, Dureuil C et al. The requirement for the p53 proline-rich functional domain for mediation of apoptosis is correlated with specific PIG3 gene transactivation and with transcriptional repression. EMBO J 1998; 17: 46684679.
33. Marin MC, Jost CA, Brooks LA et al. A common polymorphism acts as an intragenic modifier of mutant p53 behaviour. Nature Genet 2000; 25: 4754.[CrossRef][ISI][Medline]