P53 Abnormalities and Genomic Instability in Primary Human Breast Carcinomas1

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[CANCER RESEARCH 55, 646-651, February 1, 1995] p53 Abnormalities and Genomic Instability in Primary Human Breast Carcinomas1

Jorunn E. EyfjÃ¶rd,2Steinunn Thorlacius, Margret Steinarsdottir, Rut Valgardsdottir, Helga M. Ã–gmundsdottir, and Kesara Anamthawat-Jonsson

Molecular and Cell Biology Research Laboratory, Icelandic Cancer Society, Skogarhlid 8, 105 Reykjavik [J. E. E., S. T., R. V., H. M. Ã–.], and Cytogenetics Laboratory, Department of Pathology, University of Iceland. Reykjavik [M. S., K. A-J.J, Iceland

ABSTRACT patients (12, 13). Studies of normal cells, non-neoplastic cells from patients with the Li-Fraumeni syndrome that are heterozygous (wild- Abnormalities in the p53 tumor suppressor gene have been shown to type/mutant) at the p53 gene locus, murine embryonic fibroblasts with affect cell cycle control and lead to genetic instability in cell lines of germ line p53 mutation, and homozygous p53 mutant derivatives murine and human origin. We have examined genetic instability in 183 primary human breast carcinomas with and without p53 abnormalities. indicated that a loss or alteration of both copies of the p53 gene was Mutation analysis was performed by constant dÃ©naturant gel electro- needed for amplification to occur (10, 3). This p53-dependent path phoresis and DNA sequencing, and abnormal protein expression was way is not the only way amplification is regulated, however, since examined by immunohistochemical staining methods. Genetic instability there are tumor cells with normal p53 that are able to undergo gene was studied by detection of gene amplification, allelic loss, karyotype amplification (10). analysis, and fluorescent in situ hybridization. We found a significant Nonrandom alterations of chromosomes are well known in leuke- association between /;5.? abnormalities and genetic instability detected by mias and lymphomas, and more recently in some solid tumors (14, these methods. 15). In some cases these changes have been found to involve partic ular oncogenes or tumor suppressor genes (16). Random changes or cytogenetic noise also occur in cancer cells, especially at later stages INTRODUCTION of the tumor progression. All these changes reflect the chromosomal Genomic instability is a common feature of cancer cells, but the instability of tumor cells. Relatively little is known about specific reasons for this are still largely unknown. Alterations of the p53 tumor chromosomal changes in breast carcinomas, but a number of chromo suppressor gene have been reported in a spectrum of human cancers, somes have been shown to be affected (17). Chromosomal changes suggesting that it plays a central role in the formation of tumors (1). can be detected by karyotype analysis based on G-banding, but more Proliferating cells have several mechanisms for repairing DNA recently FISH has become widely used. FISH provides additional damage, and it has been shown that at least two stages in the cell information, e.g., about translocations, amplifications, and alterations cycle, the G,-S and G2-M transitions, are regulated in response to in interphase cells (18). such damage. These transitions serve as checkpoints to allow neces Human cancer prone syndromes, like Fanconi's anaemia, Bloom's sary repair to take place (2). A number of studies have implicated the syndrome, and AT, that are characterized by chromosomal instability, p53 protein in the Gj-S arrest in response to DNA damage (3-5). The provide evidence that genetic instability can be heritable and that this content of p53 protein in the cell increases as a response to DNA is associated with a predisposition to cancer (19, 2). The AT gene(s) damage and recent studies have shown that p53 activates a MT21,000 has been implicated in the same signal transduction pathway as p53 protein (Cipl/WAFl/SDI) that has been shown to inhibit the activity that controls cell cycle arrest following DNA damage (4). Cells from of cyclin-dependent kinases and thus induce either arrest in G, or patients with the AT syndrome lack the increase in p53 protein levels apoptosis (6). p53 has also been shown to be involved in the control that is induced by ionizing radiation in normal cells. of DNA replication through interaction with PCNA3 (7). Cells with The recent findings concerning the role of the p21 protein suggest abnormal p53 do not activate p21 and do not show the normal GÃŒ a mechanism by which p53 could influence and perhaps coordinate arrest necessary for repair after exposure to DNA-damaging agents. cell cycle progression, DNA synthesis, and, possibly, DNA repair. Normal cells in culture generally have a stable karyotype and do not This may explain the apparent importance of p53 abnormalities in undergo amplification, whereas most transformed and immortal cells cancer. If p53 abnormalities cause genetic instability, this may give do (8, 9). Cells that lack wild-type p53 can undergo gene amplifica rise to genetic changes that occasionally result in neoplasia. tion and show conversion to aneuploidy (10). Germ line mutations in Do p53 abnormalities lead to increased genomic instability in the p53 gene are found in families with the Li-Fraumeni cancer primary tumor cells? We have examined genetic instability in primary syndrome (11). Normal somatic cells from individuals with the Li- human breast tumor samples with and without p53 abnormalities, Fraumeni syndrome are heterozygous and wild-type/normal at the p53 using four different approaches: (a) detection of gene amplification; locus, whereas tumors that arise have no wild-type p53 alÃele.Germ (b) detection of allelic loss; (c) karyotype analysis; and (d) FISH. line mutations have also been found in children with two different malignancies and in cancer-prone families that are not of the classical MATERIALS AND METHODS Li-Fraumeni type, but such mutations are very rare in breast cancer Materials. Tumor specimens were obtained from 183 patients with pri Received 8/29/94; accepted 11/29/94. mary, invasive breast cancer. Peripheral blood was collected from the same The costs of publication of this article were defrayed in part by the payment of page patients on the day of surgery. The tumor tissue was finely minced, half of it charges. This article must therefore be hereby marked advertisement in accordance with processed for tissue culture and direct harvest for chromosomal analysis, and 18 U.S.C. Section 1734 solely to indicate this fact. the other half was kept at -80Â°C for DNA analysis. The mean age of patients 1This work was supported by grants from The Icelandic Cancer Society Science Fund, The Nordic Cancer Union, The Icelandic Science Fund, the women's branch of the was 58.0 years (range, 29-94) compared to a mean age of 59.4 years for Icelandic Red Cross, The National Hospital Science Fund, and The Memorial Fund of Icelandic breast cancer patients over a period of 30 years (20). Information was Bergthora Magnusdottir and Jakob J. Bjarnason. collected on tumor type, tumor size, axillary lymph node involvement, estro 2 To whom requests for reprints should be addressed, at Molecular and Cell Biology gen and progesterone receptor status, and clinical outcome of the disease (data Laboratory, Icelandic Cancer Society, P.O. Box 5420, 125 Reykjavik, Iceland. 3 The abbreviations used are: PCNA, proliferating cell nuclear antigen; FISH, fluo not presented). rescent in situ hybridization; AT, ataxia telangiectasia; CDGE, constant dÃ©naturantgel DNA Analysis. DNA was extracted from breast tumor samples and blood electrophoresis. samples by conventional phenol/chloroform extraction. p53 mutation analysis 646

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1995 American Association for Cancer Research. p53 ABNORMALITIES AND OENOMIC INSTABILITY IN CARCINOMAS was carried out by PCR amplification and CDGE. Mutants were confirmed by majority of changes were single base substitutions leading to an amino direct DNA sequencing. PCR conditions, CDGE, and sequencing conditions acid change (Fig. 1). The sites most frequently affected, codons 248 were as described in Thorlacius et ai. (21) and BÃ¶rresen el al. (22). All the and 273, are within the proposed DNA-binding area (33). samples were screened for mutations in exons 5-8 of the p53 gene. Allelic loss Tumor samples were also studied with immunohistochemical meth was examined by standard Southern blotting and hybridization techniques with ods, and nuclear p53 staining was detected in 33.7% of cases. Nuclear 7 different chromosome 17 probes and 7 different PCR polymorphisms. The p53 staining was detected in all the cases that had a mutation leading probes used were: pl44D6 (D17S34); pYNZ22 (DI7S5); pYNH37.3 to an amino acid change but not in tumors with nonsense or frameshift (DÃŒ7S28);pTHH59 (DI7S4); pBHP53 (TP53); pCMM86 (DI7S74); and mutations. In addition there are p53 mutation-negative tumors that pRMU3 (D17S24). Three of the DNA markers map to 17pl3.3 (pl44D6, pYNH37.3 and pYNZ22), one maps to the p53 gene region (TP53), one maps show nuclear staining. When taken together, abnormalities, p53 mu close to the BRCA-I gene (pCMM86), and two of the probes map to 17q23-25 tations, and/or nuclear p53 staining were detected in 36% of the (pTHH59 and pRMU3). PCR polymorphisms used were: Accll polymorphism primary breast tumor samples. within the p53 gene (23); two different nucleotide repeats at the p53 locus Allelic Loss. Allelic loss was studied on chromosome 17 using (24);" three microsatellite markers, Mfdl88 (D17S579), THRA-1 (25), and four different probes and three different PCR polymorphisms for the MfdlS (DÃŒ7S250),close to the BRCA-l gene; and a marker close to the end of 17q, DI7S802. Amplification of the erbB2 oncogene was detected by Southern blotting with the probe ERB-B2 (26) and comparative PCR amplification (27). Am plification was measured by relative comparison of the signal strength between tumor sample and a normal control. A control probe for a different marker, NJ 4.1, was used to compare the amount of DNA loaded on the gel in the standard Southern blotting method. Immunohistochemistry. Five-|xm-thick sections from fixed, paraffin-em bedded tumors were stained with two different p53 antibodies, a monoclonal (DO-1) (28) detected by a streptavidin-biotin complex and immunoperoxidase labeling and a rabbit polyclonal antibody (CM-1) (29) detected with immunoperoxidase. Cell Culture and Chromosome Preparation. Tissue samples were minced in a Petri dish, digested with collagenase, and cultured in a special serum-free medium (30) at 37Â°Cfor 3â€”6days. The cells were arrested in mitosis by colchicine treatment and chromosomes were harvested by standard methods as described by Pandis et al. (31). In addition a small fraction of the minced tissue was used for direct harvesting of chromosomes. Karyotype Analysis. Chromosomes were G-banded with Wright's stain. Karyotype analysis was performed according to an International System for Human Cytogenetic Nomenclature (32). The number of cells analyzed ranged from 5 to 300. A sample was defined as having a clonal abnormality if it contained at least two cells with the same structural abnormality or the same additional chromosome, or at least three cells lacking the same Fig. 1. CDGE of PCR fragments in exon 8 (codons 265-301 ). The CDGE technique is chromosome. Single structural changes or up to four numerical alterations based on the melting behavior of DNA strands. It allows maximal separation between were defined as simple changes. All other aberrations were called complex. mutant and wild-type DNA fragments in a specific dÃ©naturantconcentration. The samples FISH. PCR-amplified whole chromosome probes for chromosomes 1, 3, are then sequenced to confirm the nature of Ihe mutation. Lane I. wild-type; Lane 2, Arg to Cys mutation in codon 273; Lane 3, Arg to Trp mutation in codon 282; Lane â€¢/,6-base 16, and 17 were used for in situ hybridization. These are the chromosomes deletion in codons 279-281 (Arg and Asp deleted). most frequently involved in changes in the breast carcinomas studied here.5 Labeled DNA probes were obtained from Cambio (Cambridge, England) and in situ hybridization was performed according to the company's protocol.

Probes for chromosomes 1 and 3 were biotin labeled, detected with Avidin- abnormalitiesCaseI23456789101112131415161718192(1212223ClonalTable 1 Clonal chromosomal changes related to p53 Texas Red, and amplified over anti-Avidin. Probes for chromosomes 16 and 17 typeex/s"cxFISH abnormalityExon typeSCX/SCXCXCXCX/Sscx/sCXCXCXcx/sCXp53abnormalityNegNegNegNegNegNegNegNegNegNegNegNegNegNcgNegNegNegNegNegNegNegNegNeg were directly labeled with fluorochrome FITC and the detection was amplified with anti-FITC. Each chromosome preparation was hybridized with probes for 8Exon 5Exon chromosomes 1 and 16 simultaneously, or with probes for chromosomes 3 and instCXCXcx/sCXsCXCXsCXcx/sssCXsp53 17. Chromosomes were counterstained with 4',6-diamidino-2-phenylindole- 5Exon 8Exon dihydrochloride hydrate (DAPI). 8Exon Statistical Analysis. When comparing two proportions, ic test with 5Exon Yates's correction was applied for testing the significance, or Fisher's exact 5Exon 5Pos. test, when appropriate. stainPos. stainPos. stainNegNegNegNegNegNegNegNegNegNegNegNegCase2425262728293031323334353637383940414243444546Clonal RESULTS

Abnormalities in the pS3 Gene. 183 primary breast tumor sam ples were screened for mutations in exons 5-8 of the p53 gene by CDGE. Suspected mutants were confirmed by DNA sequencing. In our material p53 mutations were found in 17.6% of cases. The

4 M. Santibanez-Koref, personal communication. 5 Margret Steinarsdottir, Steinunn Snorradoltir, Ingibjorg Pelursdoltir. Kesara Anamathawat-Jonsson, Jorunn E. EyfjÃ¶rd, and Helga M. Ã–gmundsdottir. Different a CX, complex changes (>1 structural change and/or >4 numerical changes); S, simple karyolypic profiles detected in breast carcinomas by direct harvesting and short term culture, submitted for publication. changes (1 structural change, or s4 numerical changes): Neg, negative; Pos, positive. 647

Fig. 2. A, B, and C, two metaphases from tumor case 2 (left, normal; right, abnormal). A, 4',6-diamidino-2-phenylindole-dihydrochloride hydrate staining. B, Texas Red fluorescence of chromosome 3. C, FITC green fluorescence of chromosome 17. Small arrowheads, normal chromosomes detected by FISH. This tumor contained three related clones with complex chromosome rearrangements, and the abnormal cells were from near-diploid to pentaploid. FISH revealed several translocations involving chromosomes 3 and 17. One pair of translocated chromosomes (large arrowheads) contained segments from both chromosomes at opposite telomeric ends, indicating at least two translocation events prior to chromosomal duplication. More translocations and deletions can be seen. D and E, a metaphase from tumor case 4. D, 4',6-diamidino-2-phenylindole-dihydrochloride hydrate staining. E, red and

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1995 American Association for Cancer Research. pS3 ABNORMALITIES AND GENOMIC INSTABILITY IN CARCINOMAS short arm and three different probes and four different PCR polymor of the erbB2 oncogene, allelic loss on chromosome 17, and clonal phisms for the long arm. On 17p alleile loss was detected in 70.8% of chromosomal abnormalities involving several chromosomes. the tumors with p53 mutation as compared with 45% of the mutation- Nuclear staining with p53 antibodies reflected the p53 mutations; negative tumors, whereas on 17q loss was found in 65% of the all tumors with mutations leading to amino acid changes showed samples with p53 mutation and 32% of the mutation-negative sam nuclear staining, but no staining was seen in tumors with nonsense or ples. In both cases there is a statistically significant difference frameshift mutations. In addition nuclear staining was detected in (P < 0.04; P < 0.007). When tumors rated abnormal on the basis of some tumors without proven p53 mutations. In these cases the protein protein staining were included there was also a difference, i.e., loss of might have been stabilized by complexing with other proteins or they heterozygosity for all sites was more frequent in the tumors with p53 may harbor mutations in p53 exons not included in our analysis. abnormalities, but this was not statistically significant. Cell line studies indicate that both copies of the p53 gene have Amplification of the erbB2 Oncogene. Amplification of the to be lost or inactivated for gene amplification to occur (3). In our erbB2 oncogene was detected with the probe ERB-B2 and PCR material we detected alÃeleloss on 17p in over 70% of the tumors comparative amplification in 25% of the tumors. Amplification was with a p53 mutation. This is in agreement with our earlier studies found in 41% of the samples with p53 abnormalities as compared to and those of some others (21, 34-36), whereas other investigators 15.9% of the samples without p53 abnormalities. This difference is have not found any correlation between alÃelelosson 17p and p53 statistically significant (P < 0.008). mutations in breast tumors (37). All but one of the tumors with a Cytogenetic Study. Fifty-six of the primarybreastcarcinomasam p53 mutation and erbB2 amplification that were informative (het ples screened for p53 abnormalities were put into culture to obtain erozygous) for one or more probes on 17p showed a loss of metaphase cells for cytogenetic analysis. In 46 cases successfully heterozygosity at one or more sites. We cannot conclude from our cultured, 30 showed clonal chromosomal abnormalities by karyotype data, however, whether the wild-type alÃelehas been lost in the analysis and FISH (Table 1). Although changes were found both in tumors with p53 mutations. The information on loss within the p53 samples with and without p53 abnormalities, those with the p53 gene is too limited and the more informative probes are too distant abnormalities (samples 1-11) exhibited a significantly higher propor from the gene for this to be possible. Deletions on 17p, large and tion of samples with chromosome aberrations than the samples small, are frequent in the tumors, however, and more so in the ones without p53 abnormalities (P < 0.04). Furthermore, the proportion with p53 mutations. with complex clonal changes was much higher in the p53 abnormal The association between p53 abnormalities and genetic instabil group: 82% as compared to 40% in the p53 mutation-negative group ity such as oncogene amplification may indicate that p53 plays a (P < 0.008). No specific clonal changes could be associated with all role early in mammary tumor formation. Abnormal p53 protein samples with p53 abnormalities. expression has been detected in mammary carcinoma in situ, Chromosome instability in breast tumor samples with p53 abnor implicating p53 in mammary tumor evolution from in situ to malities included numerical and structural changes: loss and gain of invasive disease. Interestingly, there was no association between whole or parts of chromosomes, as well as chromosome rearrange p53 abnormalities and amplification of the erbB2 oncogene in the ments. Chromosomes 1, 3, 16, and 17, which were most frequently in situ lesions, whereas such association was found in invasive involved in aberrations in our samples, were examined by the FISH carcinoma (38). technique, and some of the abnormalities are shown in Fig. 2. The In this study we present only DNA data on allelic loss on chromo FISH method also identified changes undetected by other means and some 17, but both the chromosome data and our preliminary data on allowed examination of interphase chromosomes. The tumor case 3 allelic imbalance on other chromosomes indicate that this is a more appeared normal when analyzed by G-banding, but FISH detected general phenomenon involving several different chromosomes. This is high frequency of deletions and translocations of chromosomes 16 in agreement with the view that p53 has a wide ranging monitoring and 17. DNA analysis showed allelic loss on 17p and amplification of function in the cell cycle. Specific chromosomes may be more fre the erbB2 oncogene on 17q in this tumor. Another tumor sample, case quently involved, however, in cytogenetically aberrant cells in the 10, showed extensive DNA amplification on chromosome 17 when tumors. Results from a more extensive study of chromosome insta examined by FISH on both metaphase and interphase chromosomes. bility in breast carcinomas5 showed that chromosomes 1, 3, 16, and 17 The pattern of the in situ hybridization signal suggested amplification had the highest frequency of alterations, and chromosomes 7 and 11 of dispersed repetitive DNA sequences in this tumor. Chromosome 17 were also relatively unstable. This is in fair agreement with the was also involved in translocation and duplication, while DNA anal findings of others (17, 39). Further studies will be required to verify ysis showed allelic loss on 17p and 17q and amplification of oncogene the importance of these chromosomes in breast cancer. The examina erbB2. tion of interphase nuclei will also be important for screening of chromosomal instability in tumors, especially for characterizing DISCUSSION heterogeneous cell populations. Our comparison of cytogenetic changes in breast tumors with Our results support earlier findings suggesting that p53 has a role in and without p53 abnormalities revealed significant differences. cell cycle control and that p53 abnormalities may lead to genetic Only 1 of the 11 tumor samples with p53 abnormalities that were instability. Moreover, the data indicate that this is true for primary successfully analyzed by cytogenetic methods did not show clonal cancer cells. Primary breast tumor cells with p53 abnormalities show chromosomal abnormalities. More important, complex changes significantly increased genetic instability measured as amplification were much more frequent in the samples with p53 abnormalities.

green fluorescence of chromosomes 1 and 16, respectively. No normal chromosomes 1 and 16 were found. In this near-diploid clone, a reciprocal translocation 1(1;16) was detected (large arrowheads). The karyotype analysis showed the breakpoints to be Iq21 and 16ql2. F, a partial metaphase from tumor case 5 showing red fluoresence of chromosome 3 and green fluorescence of chromosome 17. Small arrowheads, normal chromosomes. This tumor showed complex chromosomal changes. Abnormal cells were hypopentaploid and most of the chromosomes were rearranged. When probed with chromosomes 3 and 17, two deleted chromosome 3 and three copies of normal chromosome 17, and 13 translocated chromosomes were seen. Similarly complex results were obtained using chromosome 1 and 16 probes. 649

This high frequency of complex clonal changes, along with the Jackman, J., Pietenpol, J. A., Burrel, M., Hill, D. E., Wang, Y., Wiman, K. G., Mercer, W. E., Kastan, M. B., Kohn, K. W., Elledge, S. J., Kinzler, K. W., and diversity of chromosomal aberrations, is likely to reflect a mal Vogelstein, B. WAF1/CIP1 is induced in p5^-mediated Gt arrest and apoptosis. function of the cell division process. Cancer Res., 54: 1169-1174, 1994. The mechanism by which p53 affects genetic stability is most 7. Waga, S., Hannon, G. J., Beach, D., and Stillman, B. The p21 inhibitor of cyclin- likely linked to its function as a control protein in the cell cycle. dependent kinases controls DNA replication by interaction with PCNA. Nature (Lond.), 369: 574-578, 1994. Normal cells arrest in G, before entering S phase in response to g Otto, E., McCord, S., and Tlsty, T. D. Increased incidence of CAD gene amplification DNA damage. This appears to be an important checkpoint to allow in tumorigenic rat lines as an indicator of genomic instability of neoplastic cells. J. necessary DNA repair to take place (2). It is not clear how p53 Biol. Chem., 264: 3390-3396, 1989. Tlsty, T. D. Normal diploid human and rodent cells lack a detectable frequency of senses DNA damage, but its content in the cell increases and it gene amplification. Proc. Nati. Acad. Sci. USA, 87: 3132-3136, 1990. induces a number of other genes including the p21 gene that has 10 Livingstone, L. R., White, A., Sprouse, J., Livanos E., Jacks, T., and Tlsty T. D. been shown to inhibit the cell cycle in Gt through direct interaction Altered cell cycle arrest and gene amplification potential accompany loss of wild-type with cyclin-dependent kinases. Cells lacking normal p53 do not p53. Cell, 70: 923-935, 1992. Malkin, D., Li, F. P., Strong, L. C., Fraumeni, J. F., Nelson, C. E., Kim, D. H., Kassel, show G[ arrest in response to DNA damage, and this may lead to J., Gryka, M. A., Bischoff, F. Z., Tainsky, M A., and Friend, S. H. Germ-line p53 accumulation of unrepaired lesions and increased mutation fre mutations in a familial syndrome of breast cancer, sarcomas and other neoplasms. quency. It is widely accepted that changes in a number of genes are Science (Washington DC), 250: 1233-1238, 1990. 12. Malkin, D., Jolly, K. W., Barbier, N., Look, A. T., Friend, S. H., Gebhardt, M. C, necessary for cancer formation and progression, and this fits the Andersen, T. L, BÃ¶rresen, A-L., Li, F. P., Garber, J., and Strong, L. C. Germline presumed role of p53 in predisposition to cancer, spontaneous mutations of the p53 tumor-suppressor gene in children and young adults with second tumorigenesis and disease progression. malignant neoplasms. N. Engl. J. Med., 326: 1309-1315, 1992. To the best of our knowledge, the association between p53 abnor- 13 BÃ¶rresen,A-L., Andersen, T. L, Garber, J., Malkin, D., Pireaux, N. B., Thorlacius, S., EyfjÃ¶rd,J. E., Ottestad, L., Smith-SÃ¶rensen, B., Hovig, E., Malkin, D., and Friend, malities and genomic instability found in this study has not been S. H. Screening for germ line TP53 mutations in breast cancer patients. Cancer Res., demonstrated previously in primary tumor cells. p53 thus appears to 52: 3234-3236, 1992. be a major factor in the control of genetic stability but is clearly not 14 Mitelman, F. Catalog of Chromosome Aberrations in Cancer, Ed. 4. New York: Wiley-Liss, 1991. the only one, since chromosomal alterations were also detected in 15 Sandberg, A. A. The Chromosomes in Human Cancer and Leukemia, Ed. 2. New tumors without p53 abnormalities. In an earlier study we examined York: Elsevier Science Publishing Co., Inc. 1990. p53 mutations in breast tumors in relation to clinical data and found 16 Daley, G. Q., Van Etten, R. A., and Baltimore, D. Induction of chronic myelogenous leukemia in mice by the P210 (bcrlabl) gene of the Philadelphia chromosome. a highly significant association between p53 mutations and poor Science (Washington DC), 247: 824-830, 1990. short-term survival (21). The same is also true when patients are 17 Hainsworth, P. J., and Garson, O. M. Breast cancer cytogenetics and beyond. Aust. included that have strong abnormal p53 nuclear staining in the tumors NZ J. Surg., 60: 327-336, 1990. but no detectable p53 mutations.6 A number of studies have found a 18. Gray, J. W., and Pinkel, D. Molecular cytogenetics in human cancer diagnosis. Cancer (Phila.), 69: 1536-1542, 1992. similar association between p53 abnormalities, both mutation and 19 Barnes, D. E., Lindahl, T., and Sedgwick, B. DNA repair. Curr. Opin. Cell Biol., 5: abnormal staining, and poor survival in breast cancer (40, 36, 41, 42). 424-433, 1993. 20. Tulinius, H., Bjarnason, O., Sigvaldason, H., Bjarnadottir, G., and Olafsdottir, G. In this study we conclude that p53 abnormalities lead to increased Tumours in Iceland. Malignant tumours of the female breast. APMIS, 96: 229-238, genomic instability and thus an accumulation of different genetic 1988. alterations which could explain the observed association with more 21 Thorlacius, S., BÃ¶rresen,A-L., and EyfjÃ¶rd,J. E. Somatic p53 mutations in human breast carcinomas in an Icelandic population: a prognostic factor. Cancer Res., 53: aggressive disease. 1637-1641, 1993. 22. BÃ¶rresen, A.-L., Hovig, E., Smith-SÃ¶rensen, B., Malkin, D., Lystad, S., Andersen, T. L, Nesland, J. M., Isselbacher, K. J., and Friend, S. H. Constant dÃ©naturantgel electrophoresis (CDGE) as a rapid screening technique for p53 mutations. Proc. Nati. ACKNOWLEDGMENTS Acad. Sci. USA, 88: 8405-8409, 1991. 23. Ã„ra,S., Lee, P. S. Y., Hansen, M. F., and Saya, H. Codon 72 polymorphism of the We thank the Departments of Pathology and Oncology of the University TP53 gene. 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Jorunn E. Eyfjörd, Steinunn Thorlacius, Margret Steinarsdottir, et al.

Cancer Res 1995;55:646-651.

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