This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Norberg, T. Articles by Bergh, J. Search for Related Content PubMed PubMed Citation Articles by Norberg, T. Articles by Bergh, J.

Increased p53 Mutation Frequency during Tumor Progression—Results from a Breast Cancer Cohort¹

Department of Oncology, Radiumhemmet, Karolinska Institute and Hospital, Stockholm, Sweden S-17176 [T. N., S. K., G. K., J. B.], and Department of Pathology [H. N.] and Regional Oncologic Center [L. H.], University of Uppsala, Akademiska sjukhuset, Uppsala, Sweden S-75185

	ABSTRACT

Top ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The mutational patterns of the p53 gene for exons 4–9 were analyzed in 30 recurring tumors compared with the p53 status of the corresponding 30 primary breast cancers. The prevalence of p53 mutations was higher, although not statistically significant (P = 0.07), in the evaluable recurring tumors compared with the corresponding primaries, 12 of 29 (41%) versus 7 of 30 (23%). Twenty-one of the patients had unchanged p53 mutation status in the recurring compared with the primary tumors, whereas 8 had an altered mutational status or pattern in the sequential tumor. These findings indicate that p53 mutations may be an important factor for tumor progression in human breast cancer.

	INTRODUCTION

Top ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The tumor suppressor p53 gene is located on chromosome 17p. The p53 gene product is a nuclear phosphoprotein, involved in cell-cycle regulation (1) . DNA damaging agents such as carcinogens, cytostatics, radiation and UV light will normally result in activation of the p53 gene (2) . Accumulating evidence suggest that the tumor suppressor p53 plays a central part in the intracellular resistance against malignant transformation and tumor progression (2, 3, 4, 5) . Mutations in the p53 gene are among the most common genetic abnormalities thus far described in human cancer. Studies on primary breast cancer have shown a worse outcome for patients with p53 protein overexpression (6 , 7) or mutations of the p53 gene (8, 9, 10) in their tumors. Previous studies have indicated that p53 mutations may be an early event in breast cancer development and have been found in 20–25% of invasive primary breast cancers (9 , 11) . The majority of these mutations are missense mutations (12) . Mutations in certain parts of the p53 gene, e.g., the evolutionarily conserved regions II and V and/or in the L2 and L3 regions, have been associated with a significantly worse prognosis (9 , 13 , 14) . However, little is known of the p53 status in recurring tumors and the longitudinal variation in the same individuals during tumor progression.

	PATIENTS AND METHODS

Top ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The patients in the present study were derived from a population-based cohort of 315 primary breast cancer patients who underwent breast cancer surgery during the years 1987–1989 in Uppsala County, Sweden (9) . Ninety-nine of the primary 315 patients suffered breast cancer relapse during the primary study period. Of those 99 patients, 30 had relapses that were diagnosed with biopsies, and from which tumor samples were available in the pathological archives. These 30 available tumor samples were used for p53 determinations (see Fig. 1 )

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Flowchart of the patient material used in this study

Sequence Determination.
The p53 gene in fresh frozen samples from the primary tumors was sequenced by cDNA based sequencing using the PCR and automated fluorescence electrophoresis, as previously described (9 , 15) . DNA was isolated from the microdissected recurring tumors by enzymatic digestion and then phenol extraction and precipitation. Exons 4–9 of the p53 gene were amplified using PCR with a full nested approach, where each exon individually was subjected to sequence analysis after the second round of PCR. With one of the primers in the second round of PCR modified with a biotin molecule, biotinylated PCR products were generated which were subsequently sequenced using the AutoLoad solid-phase sequencing kit from Amersham Pharmacia Biotech. The generated sequencing products were analyzed with an ALFexpress automated sequencer (Amersham Pharmacia Biotech), and the resulting base sequence from the samples were compared with the wild-type p53 sequence with the aid of the Mutation Analyser software (Amersham Pharmacia Biotech). Each nucleotide deviation that generated an amino acid change (substitution, deletion, insertion, or truncation) was considered as a mutation. Each scored mutation was confirmed by a complete rerun of PCR and sequence analysis starting from the corresponding DNA sample.

Statistical Methods.
Distribution of p53 mutations in primary and recurring tumors were compared in a 2 x 2 table and tested with McNemar’s test with the null hypothesis that primaries and relapses had the same p53 status. Change of p53 status by mode of treatment was tested with Fisher’s exact test. All calculations were executed in the SAS software, version 3.16, from the SAS Institute, Inc.

	RESULTS

Top ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
To avoid possible new primary tumors, we excluded two patients with recurrences in the contralateral breast (patients 20 and 21; Table 1 ), showing P = 0.07 by McNemar’s test on 27 evaluable tumor pairs (Table 2A) . In the 27 evaluable recurrences (including double recurrences as below), a total of 12 different mutations were found. One of the recurrences had two different p53 mutations, whereas none of the primary tumors had more than one mutation. Of the 12 mutations in the recurring tumors, 4 were the same as in the corresponding primary tumors, and 8 were new mutations (Table 1 ; Fig. 2 ).

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Schematic representation of the p53 gene in the paired primary (left column) and recurring (right column) tumors containing a mutation. The unpaired recurring samples (samples 29, 15, 22, 2, and 14) were not mutated in the primary analysis. Sample 6 and 28 had two recurring tumors each. Solid line, a point mutation that generates a stop codon or affects a splice site; hatched line, all other point mutations. , deletions; number inside the , the number of bases deleted; S inside the , a deleted splice site. Numbers above each line or , the codon affected by the mutation in question. within the figures, the extent of the genetic analysis that has been performed. Black line with roman numerals, the conserved regions of the p53 gene.

When we included the two patients with recurrences in the contralateral breast, 7 of 30 primary and 12 of 29 evaluable recurring tumors had a positive mutational status (Table 1) , and McNemar’s test on 29 evaluable patients gave a P of 0.07 (Table 2B) . For patients with two recurrences available for analysis, mutation in any of the two recurrences were counted as a positive mutational status.

The occurrence of a changed p53 mutation status in the recurring tumor compared with the primary tumor was not related to systemic treatment that was given (hormonal or chemotherapy), P = 1.0 by Fisher’s exact test. Primary tumors exposed to adjuvant radiotherapy resulted in an increased frequency of altered p53 status in the corresponding recurring tumors (5 of 12), compared with the nonirradiated tumors (2 of 15). However, this increase was not statistically significant; P = 0.139 by Fisher’s exact test (data not shown).

	DISCUSSION

Top ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The major finding in our study is that the prevalence of p53 mutations was doubled in the relapses compared with the primary tumors, indicating that p53 may not only play an important role in tumor development, but also during tumor progression and metastasis. The possibility of treatment-induced clonal selection, as described previously (16) cannot be clearly evaluated in the present study because of the lack of power.

Another potentially important finding is that molecular analysis of p53 may aid in discriminating between metastatic disease and new primary tumors, which sometimes is impossible using conventional histopathology. However, differences in mutational status or patterns in sequential tumors may occur after the time of metastasis, and p53 status preferably should be combined with information from other markers.

Studies have been published addressing the "genetic relationship" between primary and recurring disease. In a small longitudinal study on four women with relapse of ductal carcinoma in situ, the authors studied the loss-of-heterozygosity profile on paired samples, primary tumor versus the recurrence (17) . In accordance with our findings, there was additional loss of heterozygosity during tumor progression (17) . In another study on 10 patients with small cell lung cancer, using comparative genomic hybridization, an increase of genetic alterations was found during tumor progression (18) .

The major advantage of using paraffin-embedded, formalin-fixed tumor biopsies is the great number of archived clinical samples available for future studies. For molecular analysis, however, these 10–12-year-old samples were challenging. The quality of the DNA is very poor in formalin-fixed tissues, and one of the samples had to be excluded from the study. We could not generate results for all fragments because of insufficient material (or inhibiting substances) for the PCR amplification in four samples. Our strategy to rerun each sample scored with a mutation, starting from the DNA sample as described in "Patients and Methods," proved to be of great importance. Nine mutation candidates could not be confirmed when the samples were reanalyzed multiple times. A reasonable explanation for this could be PCR enzyme fidelity in combination with very few intact DNA targets.

Previously we have demonstrated a very high consistency of p53 mutation detection in our primary patient material, whereas 22 of 23 p53 exon mutations were identified in a comparison between sequencing of cDNA and genomic DNA derived from microdissected frozen material (19) . In the present study, we microdissected four different parts of the tumor from two different biopsies, confirming a stable p53 pattern within the examined biopsies.

In conclusion, we have demonstrated that p53 mutations increase in frequency during metastasis, suggesting that the p53 gene may potentially be involved in tumor dissemination and progression.

	ACKNOWLEDGMENTS

 
We acknowledge Marit Holmqvist for skillful statistical assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by grants from the Swedish Cancer Society. The time of Drs. S. Klaar and T. Norberg for this study was supported by Eurona Medical AB, Uppsala, Sweden.

² Former affiliation: Department of Oncology, University of Uppsala, Akademiska sjukhuset, S-75185 Uppsala, Sweden.

³ To whom requests for reprints should be addressed, at Department of Oncology, Radiumhemmet, Karolinska Institute and Hospital, S-17176 Stockholm, Sweden.

Received 4/ 5/00. Accepted 9/18/01.

	REFERENCES

Top ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Levine A. J. p53, the cellular gatekeeper for growth and division. Cell, 88: 323-331, 1997.[Medline]
2. Harris C. Structure and function of the p53 tumor suppressor gene: clues for rational cancer therapeutic strategies. J. Natl. Cancer Inst., 88: 1442-1455, 1996.[Abstract/Free Full Text]
3. Sidransky D., Hollstein M. Clinical implications of the p53 gene. Annu Rev Med., 47: 285-301, 1996.[Medline]
4. Sidransky D., Mikkelsen T., Schwechheimer K., Rosenblum M. L., Cavanee W., Vogelstein B. Clonal expansion of p53 mutant cells is associated with brain tumour progression. Nature (Lond.), 355: 846-847, 1992.[Medline]
5. Lane D. P. Cancer. p53, guardian of the genome. Nature (Lond.), 358: 15-16, 1992.[Medline]
6. Borg Å., Lennerstrand J., Stenmark-Askmalm M., Ferno M., Brisfors A., Ohrvik A., Stal O., Killander D., Lane D., Brundell J. Prognostic significance of p53 overexpression in primary breast cancer; a novel luminometric immunoassay applicable on steroid receptor cytosols. Br. J. Cancer, 71: 1013-1017, 1995.[Medline]
7. Andersen T. I., Holm R., Nesland J. M., Heimdal K. R., Ottestad L., Børresen A. L. Prognostic significance of TP53 alterations in breast carcinoma. Br. J. Cancer, 68: 540-549, 1993.[Medline]
8. Aas T., Børresen A. L., Geisler S., Smith-Sorensen B., Johnsen H., Varhaug J. E., Akslen L. A., Lonning P. E. Specific P53 mutations are associated with de novo resistance to doxorubicin in breast cancer patients. Nat. Med., 7: 811-814, 1996.
9. Bergh J., Norberg T., Sjögren S., Lindgren A., Holmberg L. Complete sequencing of the p53 gene provides prognostic information in breast cancer patients, particularly in relation to adjuvant systemic therapy and radiotherapy. Nat. Med., 10: 1029-1034, 1995.
10. Silvestrini R., Veneroni S., Benini E., Daidone M. G., Luisi A., Leutner M., Maucione A., Kenda R., Zucali R., Veronesi U. Expression of p53, glutathione S-transferase-, and Bcl-2 proteins and benefit from adjuvant radiotherapy in breast cancer. J. Natl. Cancer Inst., 89: 639-645, 1997.[Abstract/Free Full Text]
11. Davidoff A. M., Kerns B. J., Pence J. C., Marks J. R., Iglehart JD. p53 alterations in all stages of breast cancer. J. Surg. Oncol., 48: 260-267, 1991.[Medline]
12. Beroud C., Soussi T. p53 gene mutation: software and database. Nucleic Acids Res., 26: 200-204, 1998.[Abstract/Free Full Text]
13. Børresen A. L., Andersen T. I., Eyfjord J. E., Cornelis R. S., Thorlacius S., Borg A., Johansson U., Theillet C., Scherneck S., Hartman S., Cornelisse C. J., Hovig E., Devilee P. TP53 mutations and breast cancer prognosis: particularly poor survival rates for cases with mutations in the zinc-binding domains. Genes Chromosomes Cancer, 14: 71-75, 1995.[Medline]
14. Cho Y., Gorina S., Jeffrey P. D., Pavletich N. P. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science (Wash. DC), 265: 346-355, 1994.[Abstract/Free Full Text]
15. Sjögren S., Inganäs M., Norberg T., Lindgren A., Nordgren H., Holmberg L., Bergh J. The p53 gene in breast cancer: prognostic value of complementary DNA sequencing versus immunohistochemistry. J. Natl. Cancer Inst., 88: 173-182, 1996.
16. Moll U. M., Ostermeyer A. G., Ahomadegbe J. C., Mathieu M. C., Riou G. p53 mediated tumor cell response to chemotherapeutic DNA damage: a preliminary study in matched pairs of breast cancer biopsies. Hum. Pathol., 26: 1293-1301, 1995.[Medline]
17. Lininger R. A., Fujii H., Man Y. G., Gabrielson E., Tavassoli F. A. Comparison of loss heterozygosity in primary and recurrent ductal carcinoma in situ of the breast. Mod. Pathol., 11: 1151-1159, 1998.[Medline]
18. Schwendel A., Langreck H., Reichel M., Schrock E., Ried T., Dietel M., Petersen I. Primary small-cell lung carcinomas and their metastases are characterized by a recurrent pattern of genetic alterations. Int. J. Cancer, 74: 86-93, 1997.[Medline]
19. Williams C., Norberg T., Ahmadian A., Ponten F., Bergh J., Inganäs M., Lundeberg J., Uhlen M. Assessment of sequence-based p53 gene analysis in human breast cancer: messenger RNA in comparison with genomic DNA targets. Clin. Chem., 44: 455-462, 1998.[Abstract/Free Full Text]

This article has been cited by other articles:

				F. Tort, J. Bartkova, M. Sehested, T. Orntoft, J. Lukas, and J. Bartek Retinoblastoma Pathway Defects Show Differential Ability to Activate the Constitutive DNA Damage Response in Human Tumorigenesis Cancer Res., November 1, 2006; 66(21): 10258 - 10263. [Abstract] [Full Text] [PDF]

				K. Collett, G. E. Eide, J. Arnes, I. M. Stefansson, J. Eide, A. Braaten, T. Aas, A. P. Otte, and L. A. Akslen Expression of Enhancer of Zeste Homologue 2 Is Significantly Associated with Increased Tumor Cell Proliferation and Is a Marker of Aggressive Breast Cancer Clin. Cancer Res., February 15, 2006; 12(4): 1168 - 1174. [Abstract] [Full Text] [PDF]

				A. Goldhirsch, J. H. Glick, R. D. Gelber, A. S. Coates, B. Thurlimann, H.-J. Senn, and and Panel Members Meeting Highlights: International Expert Consensus on the Primary Therapy of Early Breast Cancer 2005 Ann. Onc., October 1, 2005; 16(10): 1569 - 1583. [Abstract] [Full Text] [PDF]

				W. E. Criss Molecular Mechanisms of Toxic Chemicals Indoor and Built Environment, December 1, 2003; 12(6): 395 - 399. [Abstract] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Norberg, T. Articles by Bergh, J. Search for Related Content PubMed PubMed Citation Articles by Norberg, T. Articles by Bergh, J.