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Loss of Heterozygosity in Benign Breast Epithelium in Relation to Breast Cancer Risk

David M. Euhus, Leslie Cler, Narayan Shivapurkar, Sara Milchgrub, George N. Peters, A. Marilyn Leitch, Shashank Heda, Adi F. Gazdar

Affiliations of authors: D. M. Euhus, L. Cler, G. N. Peters, A M. Leitch (Department of Surgery), N. Shivapurkar, S. Milchgrub, S. Heda, A. F. Gazdar (Department of Pathology), University of Texas Southwestern Medical Center at Dallas.

Correspondence to: David M. Euhus, M.D., Division of Surgical Oncology, University of Texas Southwestern Medical Center, E6.222, 5323 Harry Hines Blvd., Dallas, TX 75390-9155 (e-mail: david.euhus{at}UTSouthwestern.edu).

ABSTRACT

The multistage model of breast carcinogenesis suggests that errors in DNA replication and repair generate diversity in the breast epithelium (the mutator phenotype), resulting in selection and expansion of premalignant clones with an acquired survival advantage. We measured loss of heterozygosity (LOH) in breast epithelial cells obtained by random fine-needle aspiration (FNA) biopsy from 30 asymptomatic women whose risk of breast cancer had been defined by the Gail model. Polymorphic microsatellite markers were selected on the basis of their relevance to breast cancer. Breast epithelium of 11 (37%) of 30 women had normal cytology, and that of 19 (63%) had proliferative cytology (eight with atypia and 11 without atypia). LOH was detected in two women with normal cytology and in 14 women (seven with atypia and seven without atypia) with proliferative cytology (P = .007). The frequency of LOH was associated with the cytological diagnosis, as well. The mean proportion (range) of informative markers demonstrating LOH was 0.02 (0–0.20) for the 11 women with normal cytology, as compared with 0.15 (0–0.50) for the 19 women with proliferative cytology (P = .02). Mean lifetime risk for developing breast cancer, as calculated by the Gail model, was 16.7% for women with no LOH compared with 22.9% for women with any LOH (P = .05). These observations support a multistage model of breast carcinogenesis where the initiating events are those that result in genomic instability. Accurate individualized breast cancer risk assessment may be possible based on molecular analysis of breast epithelial cells obtained by random FNA.


Breast cancer is uniformly characterized by DNA alterations that include structural rearrangement of the chromosomes as well as changes in chromosome copy number and DNA content (1). These alterations are not terminal events in breast carcinogenesis, as they have been observed in ductal carcinoma in situ (DCIS) (2,3) and benign proliferative lesions (48), suggesting that the initiating events in breast carcinogenesis are those that generate general genomic instability.

Another marker of genomic instability is mutation in microsatellite DNA. Microsatellites are short repetitive nucleotide sequences that are dispersed throughout the genome, and because they show a higher spontaneous mutation rate than coding DNA, they can be used as a barometer of DNA stability (9). This study was designed to determine whether loss of heterozygosity (LOH) could be detected in benign breast epithelium obtained by random breast fine-needle aspiration (FNA), to measure the frequency of LOH, and to determine whether LOH is associated with validated markers of breast cancer risk such as cytological atypia and risk level calculated by the Gail model.

From June 4, 1998, through February 4, 1999, 60 women underwent breast cancer risk assessment, using interactive software we have developed (10). Fifty-four of these women also underwent breast epithelial cell sampling by random FNA in the upper outer quadrant of each breast as part of an institutional review board–approved protocol. Written informed consent was obtained from all participants. Papanicolaou-stained direct smears were evaluated by a cytologist and scored as normal, proliferative without atypia, or atypical. Normal cytology was defined as sheets of cohesive epithelial cells with abundant myoepithelial cells, good polarity at the margin, and nuclei all less than twice the diameter of adjacent red blood cells. Proliferative clusters without atypia showed nuclear overlapping, prominent nucleoli, and a relative paucity of myoepithelial cells with all nuclei less than twice the diameter of adjacent red blood cells. Clusters were designated as atypical if they showed dyscohesion, significant anisonucleosis, chromatin clumping, and some nuclear sizes more than twice the diameter of adjacent red blood cells. Breast epithelial cell clusters were diagnosed as atypical in eight (15%) of 54 women. All eight women with atypical cytology were selected for allelotyping, and an additional 22 women were selected on the basis of the abundance of epithelial cells retrieved by FNA. Cell yields range from 0 to 12 000 cells per sample. We chose samples with greater than 500 cells. There is no normal range for random sampling.

Selected breast epithelial cell clusters were microdissected using laser capture (PixCell; Arcturus Engineering, Mountain View, CA) and polymerase chain reaction (PCR)–based allelotyping performed as previously described (11). A panel of 20 highly polymorphic microsatellite markers (see Table 1Go) relevant to breast cancer were selected: D3S1597, D17S969, D3S1766, 8pNEFL, D4S404, D4S194, D4S1584, D3S1447, D17S787, D4S2366, D4S1652, D3S1612, D4S2397, D8S277, 9pIFNA, p53PENTA, p53CA, D17S855, D17S1322, and D17S1323. Markers were initially amplified in a multiplex PCR that included five or six primer pairs known to amplify well together and then in a uniplex PCR that included only one primer pair per reaction.


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Table 1. Performance of Polymorphic Microsatellite DNA Markers Used for Allelotyping Breast Epithelial Cell Clusters Obtained by Fine-Needle Aspiration Biopsy from 30 Women*
 
Limitations in the amount of available material precluded amplification of every marker for every cluster. The number of clusters evaluated per woman ranged from one to 11, with a mean of four. The number of markers evaluated per cluster ranged from two to 11, with a mean of six. A marker was designated as informative if both the maternal and paternal alleles could be distinguished in the lymphocyte DNA. The frequency of LOH was expressed as a fractional allelic-loss index (FALI), which is the proportion of all informative markers for all clusters from a given individual that demonstrated loss of one of these alleles as compared with DNA from that individual's lymphocytes.

Means were compared using two-tailed Student's t tests, and proportions were compared using two-sided Fisher's exact test.

The women ranged in age from 30 to 60 years, with a mean age of 46.8 years. On the basis of calculations as per the Gail model (12), two participants were at low risk for breast cancer (h10% lifetime risk), seven were of average risk (10%–15%), 13 were at slightly increased risk (16%–25%), and eight were at high risk (g25% lifetime risk). Eighteen participants were premenopausal, eight were postmenopausal, and four were perimenopausal. Twenty-five (83%) of the women had a family history of breast cancer, but only two had a BRCA gene mutation probability greater than 10%, as calculated by the BRCAPRO model (13).

Allelotyping was performed on a total of 116 epithelial cell clusters from the 30 women (Table 1Go). Cytologically abnormal clusters containing at least 60 cells were selected. Loss of heterozygosity was detected in at least one cluster for at least one marker in 16 (53%) of the 30 women. In 12 instances, LOH was identified for the same marker in different clusters from the same woman. In each case, the same allele (upper or lower) was lost, suggesting that these clusters arose from the same progenitor.

LOH was identified in 14 of the 19 women with proliferative cytology (seven of 11 without atypia and seven of eight with atypia) but in only two women with normal cytology (P = .007). The amount of LOH identified was related to the cytologic diagnosis as well. Mean (range) FALI was 0.02 (0–0.2) for women with normal cytology as compared with 0.15 (0–0.5) for the women showing proliferating changes in their FNAs (P = .02). For comparison, a series of 12 FNAs taken from breast cancer patients tested using the same panel had a mean FALI of 0.50 (0.1–1.0) (Fig. 1Go). Mean lifetime risk for developing breast cancer, as calculated by the Gail model, was 16.7% for women with no LOH as compared with 22.9% for women with any LOH (P = .05).



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Fig. 1. Fractional allelic-loss index (FALI) by cytological diagnosis of breast epithelial cell clusters obtained by fine-needle aspiration biopsy. FALI is the proportion of all informative markers for all epithelial cell clusters from a given woman who demonstrated loss of heterozygosity. The biopsies were taken in random manner in the sense that they were not directed by a clinical abnormality of any kind. Closed circles are the FALIs for given individuals, and horizontal lines are the mean FALIs for all individuals with a given histology. Mean FALI was 0.02 for women with normal clusters and 0.15 for women whose clusters showed proliferative changes (P = .02). A series of 12 breast cancer fine-needle aspirates allelotyped using the same markers is shown for comparison (mean FALI = 0.5).

 
Results from the cytologic assessment were discussed with the participants. Three participants have been lost to follow-up. Follow-up for the remaining 27 participants ranged from 11 to 33 months, with a median of 23 months. Three women have started a 5-year course of tamoxifen to reduce their breast cancer risk, and three were placed on raloxifene by their gynecologists. There have been no breast cancers diagnosed during the follow-up period.

LOH results from the deletion of chromosomal material during DNA replication or repair. Although LOH is commonly used to identify putative tumor suppressor genes, we have used it to measure persistence and expansion of abnormal clones in benign breast epithelium from women whose risk of breast cancer has been defined by mathematical models. The women in our study are not representative of women in the general population, as 19 (63%) of the 30 were at increased risk for breast cancer, based on the Gail model. This series is also enriched with women whose random breast FNAs were characterized cytologically as atypical. Nevertheless, identification of LOH in benign breast epithelium obtained by random FNA is strongly associated with cytologic features of typical and atypical proliferation and nominally associated with calculated risk level. Prior studies have confirmed that the breast cancer risk for women with cytologically atypical cells identified by random FNA (14) or nipple duct aspiration (15) is similar to that associated with atypical ductal hyperplasia identified on tissue biopsies performed for breast lumps or imaging abnormalities (16).

Using microsatellite DNA analysis, we have identified a subgroup of women at increased risk for breast cancer who express a mutator phenotype. It is likely that the initiating events in breast carcinogenesis are those that give rise to genomic instability. Allelotyping performed using multiplex PCR, as we have reported here, is labor intensive and, therefore, not suitable for the high-throughput clinical laboratory. Recent developments in oligonucleotide array technology may, however, provide a means for rapid, genome-wide allelotyping in the future (17,18). Molecular analysis of benign breast epithelial cells may provide an approach for accurate, individualized breast cancer risk assessment and may provide clues to the earliest steps in breast carcinogenesis.

NOTES

Supported by an American Society of Clinical Oncology Career Development Award.

REFERENCES

1 Dutrillaux B, Gerbault-Seureau M, Remvikos Y, Zafrani B, Prieur M. Breast cancer genetic evolution: I. Data from cytogenetics and DNA content. Breast Cancer Res Treat 1991;19:245–55.[Medline]

2 Nielson KV, Anderson JA, Blichert-Toft M. Chromosome changes of in situ carcinomas in the female breast. Eur J Surg Oncol 1987;13:225–9.[Medline]

3 Nielson KV, Blichert-Toft M, Anderson J. Chromosome analysis of in situ breast cancer. Acta Oncol 1989;28:919–22.[Medline]

4 Lundin C, Mertens F. Cytogenetics of benign breast disease. Breast Cancer Res Treat 1998;51:1–15.[Medline]

5 Bonsing BA, Corver WE, Fleuren GJ, Cleton-Jansen AM, Devilee P, Cornellisse CJ. Allelotype analysis of flow-sorted breast cancer cells demonstrates genetically related diploid and aneuploid subpopulation in primary tumors and lymph node metastases. Genes Chromosomes Cancer 2000;28:173–83.[Medline]

6 Visscher DW, Wallis TL, Crissman JD. Evaluation of chromosome aneuploidy in tissue sections of preinvasive breast carcinomas using interphase cytogenetics. Cancer (Cancer Cytopathol) 1996;77:315–20.[Medline]

7 Lundin CP, Mertens F, Rizou H, Idvall I, Georgiou G, Ingvar C, et al. Cytogenetic changes in benign proliferative as well as non-proliferative lesions of the breast. Cancer Genet Cytogenet 1998;107:118–20.[Medline]

8 Crissman JD, Visscher DW, Kubus J. Image cytophotometric DNA analysis of atypical hyperplasias and intraductal carcinomas of the breast. Arch Pathol Lab Med 1990;114:1249–53.[Medline]

9 Loeb LA. Microsatellite instability: marker of a mutator phenotype in cancer. Cancer Res 1994;54:5059–63.[Medline]

10 Euhus DM. Understanding mathematical models for breast cancer risk assessment and counseling. Breast J 2001;7:224–32.[Medline]

11 Euhus DM, Maitra A, Wistuba II, Ashfaq R, Alberts A, Gibbons D, et al. Use of archival fine needle aspirates for the allelotyping of tumors. Cancer (Cancer Cytopathol) 1999;87:372–9.[Medline]

12 Gail MH, Brinton LA, Byar DP, Corle DK, Green SB, Schairer C, et al. Projecting individualized probabilities of developing breast cancer for white females who are being examined annually. J Natl Cancer Inst 1989;81:1879–86.[Abstract]

13 Berry DA, Parmigiani G, Sanchez J, Schildkraut J. Probability of carrying a mutation of breast-ovarian cancer gene BRCA1 based on family history. J Natl Cancer Inst 1997;89:227–38.[Abstract/Free Full Text]

14 Fabian C, Kimler BF, Zalles CM, Klemp JR, Kamel S, Zeiger S, et al. Short-term breast cancer prediction by random periareolar fine-needle aspiration cytology and the Gail risk model. J Natl Cancer Inst 2000;92:1217–27.[Abstract/Free Full Text]

15 Wrensch MR, Petrakis NL, King EB, Miike R, Mason L, Chew KL et al. Breast cancer incidence in women with abnormal cytology in nipple aspirates of breast fluid. Am J Epidemiol 1992;135:130–41.[Abstract]

16 Dupont WD, Parl FF, Hartman WH, Brinton LA, Winfield AC, Worrell JA, et al. Breast Cancer risk associated with proliferative breast disease and atypical hyperplasia. Cancer 1993;71:1258–65.[Medline]

17 Lindblad-Toh K, Tanenbaum DM, Daly MJ, Winchester E, Lui WO, Villapakkam A, et al. Loss-of-heterozygosity analysis of small-cell lung carcinomas using single nucleotide polymorphism arrays. Nat Biotechnol 2000;18:1001–5.[Medline]

18 Mei R, Galipeau PC, Prass C, Berno A, Ghandour G, Patil N, et al. Genome-wide detection of allelic imbalance using human SNPs and high-density DNA arrays. Genome Res 2000;10:1126–37.[Abstract/Free Full Text]

Manuscript received September 18, 2001; revised February 28, 2002; accepted March 29, 2002.


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