BRCA2 and P53 Mutations in Primary Breast Cancer in Relation to Genetic Instability1

(CANCER RESEARCH 58. 859-862. March 1. 19981 Advances in Brief

BRCA2 and p53 Mutations in Primary Breast Cancer in Relation to Genetic Instability1

Solveig Gretarsdottir, Steinunn Thorlacius, Rut Valgardsdottir, Sigfridur Gudlaugsdottir, Stefan Sigurdsson, Margret Steinarsdottir, Jon Gunnlaugur Jonasson, Kesara Anamthawat-Jonsson, and Jorunn E. EyfjÃ¶rd2

Molecular and Cell Biology Research Laboratory. Icelandic Cancer Society Â¡S.Gr., S. T.. R. V.. S. Gu., S. S.. J. E. Â£./.â€¢Cylogenetics Laboratory Â¡M.S.] and Department of Pathology IM. S.. J. G. J.]. University Hospital of Iceland; and Biology Department. University of Iceland IK. A. J.j. Reykjavik. Iceland

Abstract population.' This enabled us to analyze over 400 consecutive breast cancer cases for somatic p53 mutations, as well as for the germ-line The products of the BRCA breast cancer susceptibility genes have been BRCA2 founder mutation. A subset of samples were studied by implicated in cell cycle control and DNA repair. It has been suggested that conventional cytogenetic methods. FISH,4 and flow cytometry. thus mutations in the p53 gene are a necessary step in tumorigenesis in BRCA tumors. We tested samples from 402 breast cancer patients for germ-line allowing analysis of genetic instability in relation to these two genes. BRCA2 and p53 mutations in tumors. p53 mutations are more frequent in BRCA2 mutation carriers than they are in controls. Tumors with muta Materialsand Methods tions in either gene had multiple chromosomal abnormalities, as shown by The study material consisted of 402 breast tumor samples from the Icelandic cytogenetic analysis. Cancer Society Tumor Specimen Bank. Samples were screened for mutations in exons 5-8 of the p53 gene with the Introduction constant dÃ©naturantgel electrophoresis method (19). p53 mutation status in Germ-line mutations in the BRCA I and BRCA2 genes have been relation to prognosis has been previously published for the majority of these shown to predispose to breast cancer (1-3), and the most common samples (20-22). BRCA2 exon 9 fragments were PCR-amplified and run on 6% denaturing polyacrylamide gels for mutation detection (17). Mutants were somatic changes found in breast tumors are mutations in the p53 gene identified by the presence of an extra alÃele. (4). Both the BRCA and p53 genes have been implicated in cell cycle Tumors were harvested directly for cytogenetic analyses or cultured on control (5-7). A number of studies have shown that p53 mutations are average for 6-7 days before harvesting (23). Analysis of karyotypes was done associated with genomic instability in tumors (8, 9), and recent studies according to an International System for Human Cytogenetic Nomenclature on the functions of the BRCA proteins suggest that they are also (24). Karyotypic clones with one to three numerical changes (i.e.. additional or involved in control of genomic integrity (10, 11). Both BRCA pro lacking chromosome) or a single structural change (i.e.. translocation or teins and p53 have been shown to interact with hRad51, a human deletion) were listed as having simple clonal changes. Clones with more than homologue of the yeast Rad51 protein that is involved in recombina three numerical changes and/or more than two structural changes were called tion and DNA double-strand repair (10-12). BRCA1 and BRCA2 complex. FISH was performed using PCR-amplified whole chromosome probes for deficiencies cause cell lethality in early mouse embryos (13, 14), and hypersensitivity to -y-irradiation is seen in BRCA2 â€”¿/-cells (11). chromosomes 1, 3, 16, and 17, as described by Anamthawat-Jonsson et al. (25), in which chromosomes 1 and 3 were labeled with red rhodamine and Recent studies show that BRCA1- and BRCA2-deficient embryos can chromosomes 16 and 17 were labeled with green fluorescein. be partially rescued by p53 or p21 null mutations (13, 14). This Flow cytometry was used to analyze the DNA content of the tumor cells. suggests that cells with nonfunctional BRCA can only survive if they DNA ploidy index and S-phase assessment were performed as described also have defective checkpoint control. Germ-line mutations in previously (26). Histograms were classified as diploid (DNA index = 1), if BRCA 1 or BRC A2 increase the risk of breast cancer, but even in these there was a single G,/G, peak, or aneuploid (DNA index ^ 1), if there were hereditary cases, breast cancer is a multistep process, and somatic at least two clearly distinct peaks, including multiploid, if more than two peaks mutations in other genes play a part in the tumor formation. Recent were seen. The median S-phase value of <7% was defined as low S-phase fraction, and Â£7% was defined as high S-phase fraction. publications suggest that p53 abnormalities are a necessary step in For statistical analysis, the jf test and Fisher's exact test were used as tumorigenesis in BRCA1 carriers (15, 16). We wanted to examine this appropriate. in an unselected group of breast cancer patients and test whether The germ-line mutation screening was approved by the Icelandic Cancer BRCA2 abnormalities were associated with genomic instability in Society Institutional Review Board. All DNA analyses were performed on tumors. samples without personal identification. Family and population studies indicate that there is only one mu tation in each BRCA gene in the Icelandic population, one rare Results BRCA1 mutation (D1692N) and a common BRCA2 founder mutation (999del5), found in 8% of all breast cancer cases in Iceland (17, 18). p53 and BRCA2 Mutation Analysis. Samples from unselected breast cancer patients (n = 402) were screened for BRCA2 and p53 There are no indications of other BRCA 1 or BRCA2 mutations in the mutations. The BRCA2 999del5 germ-line mutation was detected in samples from 34 patients (8.4%), and somatic mutations in the p53 Received 11/4/97; accepted 1/19/98. The cosls of publication of this article were defrayed in part by the payment of page gene were detected in 72 (17.9%) tumors. The p53 mutations were charges. This article must therefore be hereby marked advertisement in accordance with distributed as follows: 25 mutations in exon 5 (35%), 9 in exon 6 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by grants from The Icelandic Cancer Society Fund. Nordic (12%), 17 in exon 7 (24%), and 21 in exon 8 (29%). Cancer Union. Icelandic Research Fund for Higher Education. University of Iceland We compared the frequency and pattern of p53 mutations in tumors Science Fund, and Icelandic Science Council. 2 To whom requests for reprints should be addressed, at Molecular and Cell Biology Research Laboratory, Icelandic Cancer Society, P.O. Box 5420. 125 Reykjavik. Iceland. 1 Unpublished results. E-mail: [email protected]. 4 The abbreviation used is: FISH, fluorescence in situ hybridization. 859

Table I Frequency- of p53 mutations in tumors from BKCA2 mutation carriers and (76.5%) and compared to the controls. There was, however, a clear noncarriersa difference observed in the complexity of the clonal changes. All of the p53wl p53mut clonal changes detected in the BRCA2 and p53 mutated tumors were BRCA2wt (n = 368) 306 62 complex as compared to about half of the clones in the mutant- BRCA2mul (n = 34) 0.09 24 10 negative tumors (Table 3). The chromosomal aberrations included "p53wt, wild-type p53; p53mut. mulated p53; BRCA2wt. wild-type BRCA2; both structural and numerical changes, they were not restricted to any BRCA2mul, mutated BRCA2. particular chromosome and polyploidy was highly prevalent. How ever, among the primary breast tumor samples, chromosomes 1, 3, 16, Table 2 p5J mutations found in tumors from BRCA2 carriers and 17 were most frequently abnormal and were, therefore, selected Sample123456789IOExon5555677888Codon182173174141ND">NDNDNDfc298-302NDMutation1-basefor FISH analysis with whole chromosome paint. The FISH analysis deletionGTG->ATGCATâ€”CTTTGCâ€”confirmed the structural instability detected by G-banding and, fur thermore, showed that chromosomal rearrangements, gains, and losses CGCLarge were common. Multiple rearrangements of the painted chromosomes were detected in samples with BRCA2 germ-line mutations, p53 deletionLarge deletion14-bp somatic mutations, and both BRCA2 and p53 mutations (Fig. l, B, D, and F). Tumors with both BRCA2 and p53 mutations were not more deletion complex than tumors with either mutation alone. " ND. not determined. S Phase. The proliferation rate of the tumors was examined. Both * Samples from paraffin-embedded material. Mutation was not verified by sequencing. BRCA2 and p53 mutations were associated with a very high prolif- The mutations in samples 5, 8, and 10 were clearly detectable with the constant dÃ©naturant gel electrophoresis method, which was used for the p53 mutation screening. This is a erative rate, with approximately 70% of tumors showing high mitotic highly sensitive method that has enabled us to detect mutations in tissue with as little as activity, indicated by a high S-phase fraction (S7.00). This associa 25% tumor cells. tion was significant in all groups (Table 3). from BRCA2 carriers and noncarriers. We found that 10 BRCA2 Discussion mutation carriers (29%) had p53 mutations in their tumors or nearly Here, we screened samples from 402 unselected breast cancer twice as many as in the BRCA2 wild-type group (17%; Table 1). This patients for BRCA2 germ-line mutation and p53 mutations in primary difference was, however, not significant. The p53 mutation distribu tion in the BRCA2-positive group was similar to that found in the tumor samples. The frequency of BRCA2 mutation carriers (8.4%) was in agreement with our previous studies on Icelandic breast cancer whole group (Table 2). patients (18). We detected mutations in exons 5-8 of the Â¡>53genein Flow Cytometry, Cytogenetic, and FISH Analyses. Flow cyto- 17.9% of the tumors. This was also in agreement with our previous metric analysis of DNA content was performed on 266 of the samples findings (20-22) and those of others (4, 27). (Table 3). Aneuploidy was detected in 65% of all samples, and the prevalence was similar to that in BRCA2 mutants and wild-type p53 mutations were found to be more common in tumors from BRCA2 samples. The same was true for the p53 mutated samples. Cytogenetic mutation carriers than they were in controls. However, the frequency was analysis was performed on fresh tumor samples from 60 patients not as high as those recently reported in breast tumors from BRCA1 (Table 3). There was no significant difference between samples from mutation carriers (15). In that study, p53 mutations were found in eight of tumors with BRCA2 mutation compared to wild type, based on the eight tumors with known BRCA1 mutations, and the majority of p53 occurrence of simple and/or complex clonal changes. The Cytogenetic mutations were located in exon 5. The distribution of p53 mutations and flow cytometry data combined showed that 73% of the samples found in our study was the same in BRCA2 mutation carriers as in the from BRCA2 carriers were aneuploid, compared to 63% of the whole group. None of the mutations reported in the BRCA1 carriers by BRCA2/p53 normal samples. The instability trend was further Crook et al. (15) were found. p53 protein overexpression in BRCA1 strengthened if BRCA2 and p53 mutated samples were pooled tumors from high-risk families also indicate a fairly high frequency of

Table 3 Association of BRCA2 and/or p53 mutated samples with flow cytometry and Cytogenetic data The mutated samples were compared to the wild type using the Fisher's exact test. Abbreviations are as in Table 1.

BRCA2mutPloidy P450.5 p53mul orp53mut6217(27.5)45 BRCA2wl/p53wt2040.17 (n =266)TotalDiploid

(%)Aneuploid 10(22)0.0635 77(38)127(62)1980.0009 (%)S-phase (78)420.04 (72.5)5717(30)40 (n =255)Total<7.00(%)27.00

13(31)0.00629 109(55)89 (%)Cytogenetics (69)110.7 (70)177(41)10(59)10010(100)6415(23)49(45)430.8 60)TotalNo (n =

<%)Clonalclonal changes 0.57(64)70.12 4 (36) (49)22(51)220.013 21 (%)Clonal changes 32)TotalSimplechanges (n =

(%)Complex 0.067(100)470.390 10(45)12 (%)Ploidy (55)2130.05 217)TotalNormaland cytogenetics (n =

(%)Abnormal 9(20)0.01738 79(37)134(63) (%)268(31)18(69)227(32)15(68)83(37)5(63)505(100)267(27)19(73)P(80)BRCA2mut (77)P

860

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Fig. 1. FISH with whole chromosome painting showing structural abnormalities in metaphases (ÃŸ.O,and F). The cells were counterslained with 4'.6-diamidino-2-phenylindo!e (A, C. and Â£7).&â€¢Â«/('bar.K) Â¿im.The chromosome abnormalities involved both structural and numerical changes, and they are of very complex type, based on G-handing and FISH analysis. A and R, near-triploid cell from a tumor from a BRCA2 mutant carrier after painting of chromosomes I and 16 (red and gMtii/btOtttetOCtt respectively). Chromosome I is involved in eight translocations and shows overall gain of material, but chromosome 16 is seen in one translocatton and shows loss of material. C and D, near-triploid cell from p53 mutated tumor after painting of chromosomes 1 and 16 (red and green fluorescence, respectively). Here, chromosome 1 lakes part in two trÃ¤nslocations, and chromosome 16 shows gain. E and F. near-diploid cell from a BRCA2 and p53 mutated tumor after painting of chromosomes 3 and 17 (red and green fluorescence, respectively). In this cell, four rearrangements can be seen, one of which is a translocation between the two painted chromosomes. 861

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1998 American Association for Cancer Research. BRCA2 AND p53 MUTATIONS [N PRIMARY BREAST CARCINOMAS p53 abnormalities, 40 and 70%, respectively (16,27). p53 overexpression mutations in human tumors and cell lines. Nucleic Acids Res.. 22: 3551-3555, 1994. was not associated with BRCA2 mutation in our study (data not shown). Vaughn, J. P., Cirisano, F. D., Huper. G., Berchuck, A., Futreal, P. A., Marks, J. R., The samples analyzed in this study were from breast cancer patients and Iglehart, J. D. Cell cycle control of BRCA2. Cancer Res., 56: 4590-4594, 1996. unselected for family history. Ten of the BRCA2 mutation carriers were, Rajan, J. V., Marquis, S. T., Gardner, H. P.. Chodosh, L. A., Rajan, J. V., Wang, M., however, found to belong to known high-risk families (17). Of those, Marquis. S. T.. and Chodosh, L. A. Developmental expression of Brca2 colocalizes with Brea I and is associated with proliferation and differentiation in multiple tissues. only three had p53 mutations, and there was one additional case of p53 Dev. Biol.. 184: 385-401, 1997. Levine. A. J. p53, the cellular gatekeeper for growth and division. Cell, 88: 323-331, overexpression. Soboi et al. (16) suggest that specific mutations in the 1997. BRCAI gene are more often associated with p53 abnormalities as meas Livingstone, L. R., White, A.. Sprouse, J., Livanos, E., Jacks, T., and Tlsty, T. D. ured by abnormal staining. Because all of the BRCA2 mutation carriers Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell. 70: 923-935, 1992. in our study have the same 999del5 mutation, no conclusion can be drawn Eyfjord, J. E., Thorlacius, S., Steinarsdottir. M.. Valgardsdoltir. R.. Ã–gmundsdottir. about association between p53 abnormalities and BRCA2 mutations in H., and Anamthawat-Jonsson, K. p53 abnormalities and genomic instability in pri general. As mentioned previously, only one rare BRCAI mutation has mary human breast carcinomas. Cancer Res., 55: 645-651, 1995. 10. Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T., and been found in this population. A single case of this mutation was found Livingston. D. M. Association of BRCAI with Rad51 in mitotic and meiotic cells. in this cohort. Neither p53 mutation nor abnormal p53 staining was Cell, 88: 265-275, 1997. detected in a tumor sample from this patient. Sharan. S. K.. Morimalsu. M., Albrecht, U.. Lim. D. S.. Regel, E., Dinh. C.. Sands. A.. Eichele, G., Hasty. P., and Bradley, A. Embryonic lethality and radiation hypersensitivity We previously described a significant association between p53 abnor mediated by RadSI in mice lacking Brca2. Nature (Lond.). 386: 804-810, 1997. malities and genomic instability in primary breast tumors (9, 22). This 12. Buchhop. S.. Gibson, M. K., Wang, X. W., Wagner. P.. Sturzbecher. H. W., and Harris. C. C. Interaction of p53 with the human rad51 protein. Nucleic Acids Res., 25: study supports this and, furthermore, shows that BRCA2 mutated tumors 3868-3874, 1997. have complex chromosomal changes as well. Culturing primary breast Hakem. R., de la Pompa, J. L.. Elia, A., Potter, J., and Mak, T. W. Partial rescue of tumor cells is difficult, and in particular, it is hard to get cells with Brcal early embryonic lethality by p53 or p2l null mutation. Nat. Genet., 16: 298-302. 1997. complex karyotypes to divide. The most abnormal cancer cells may, 14. Ludwig. T.. Chapman. D. L.. Papaioannou. V. E., and Efstratiadis. A. Targeted therefore, be lost in the process of cell culture and chromosome harvest mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of ing. The BRCA2 samples were, indeed, highly complex showing multi Brcal, Brca2. Brcal/Brca2, Brcal/p53. and Brca2/p53 nullizygous embryos. Genes Dev., //: 1226-1241, 1997. ple chromosomal rearrangements (e.g.. Fig. IÃŸ),which seems to fit the Crook. T.. Crossland. S.. Cromplon. M. R.. Osin. P.. and Gusterson, B. A. p53 mutations notion of BRCA2 involvement in double-strand DNA repair. in Brea I-associated familial breast cancer. Lancet, 350: 638-639. 1997. 16. Sobol, H.. Stoppa-Lyonnet, D., Bressac-De Paillerets, B., Peyrat, J. P., Guinebretiere, It is well known that p53 participates in cell cycle control by activating J-M.. Jacquemier. J.. Eisinger. F.. and Birnbaum. D. BRCAl-p53 relationship in p21 in response to DNA damage (29). It has been shown that the hereditary breast cancer. Int. J. Oncol., 10: 349-353, 1997. expression of the BRCA genes is cell cycle dependent (5, 30), and in a 17 Thorlacius. S.. Olafsdottir. G.. Tryggvadottir, L.. Neuhausen, S.. Jonasson, J. G., recent study, BRCAI was found to activate p21 in a p53-independent Tavtigian, S. V., Tulinius. H.. Ã–gmundsdottir. H. M., and Eyfjord, J. E. A single BRCA2 mutation in male and female breast cancer families from Iceland with varied manner (31). It is as yet unknown how BRCA2 participates in cell cycle cancer phenotypes. Nat. Genet.. 13: 117-119, 1996. control. We found that tumors from BRCA2 mutation carriers were 18 Thorlacius, S.. Sigurdsson, S.. Bjamadotlir. H.. Olafsdottir. G.. Jonasson. J. G.. Tryggvadottir. L., Tulinius. H., and Eyfjord, J. E. Study of a single BRCA2 mutation with highly proliferative, as judged by high S phase. This was also true for high carrier frequency in a small population. Am. J. Hum. Genet., 60: 1079-1084, 1997. tumors without additional p53 abnormalities, suggesting a possible role 19 Borresen, A. L., Hovig. E., Smilh-Sorensen, B., Malkin, D.. Lystad, S.. Andersen, for BRCA2 in cell cycle control. T. I.. Nesland, J. M.. Isselbacher. K. J., and Friend, S. H. Constant dÃ©naturantgel electrophoresis as a rapid screening technique for p53 mutations. Proc. Nail. Acad. In conclusion, our results support findings that implicate BRCA2 in Sci. USA. 88: 8405-8409, 1991. DNA repair and cell cycle control. Inactivation of p53 may be Thorlacius, S.. Thorgilsson. B.. BjÃ¶msson, J.. Tryggvadottir, L., BÃ¶rresen, A-L., Ã–gmundsdottir, H. M.. and EyfjÃ¶rd,J. E. TP53 mutaÃ»onsand abnormal p53 protein important in initiation of tumorigenesis in BRCA2 carriers. However, staining in breast carcinomas related to prognosis. Eur. J. Cancer, 31A: 1856-1861. 1995. our results show that two of three of the BRCA2 mutated tumors have 21 Gretarsdottir, S., Tryggvadottir, L.. Jonasson, J. G., Sigurdsson, H., Olafsdottir, K.. Agnarsson, normal p53 and, therefore, inactivation of other integrity control genes B. A.. Ã–gmundsdottir.H. M., and EyfjÃ¶rd,J.E. TP53 mutation analyses on breast carcinomas; a study of paraffin embedded archival material. 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Solveig Gretarsdottir, Steinunn Thorlacius, Rut Valgardsdottir, et al.

Cancer Res 1998;58:859-862.

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