[CANÅ’R RESEARCH 56. 1493-1497. April I. I996| Advances in Brief Genetic Divergence in the Clonal Evolution of Breast Cancer1 Hiroaki Fujii, Carla Marsh, Paul Cairns, David Sidransky, and Edward Gabrielson2 Department of Pathology ¡H.F.. C. M., E. G./. Oncology Center [D. S.. E. G./, and Head and Neck Cancer Research Laboratory ¡P.C., D. S.¡.Johns Hopkins University School of Medicine, Baltimore, Maryland 21224 Abstract The progression of ductal carcinoma in situ (DCIS) to infiltrating and metastatic cancer of the breast is thought to be a consequence of clonal expansions of neoplastic cells with progressively more genetic alterations. To study this progression, we first dissected multiple foci from each of 23 breast tumors with DCIS only and 20 cases with synchronous DCIS and infiltrating cancer. We then tested microsatellite markers by PCR for allelic losses in the individual foci for loci on chromosomes 6q, 9p. llq, 13q, 16q, I7i|. and 17p. The patterns of allelic losses identified in the in situ cancers were generally conserved in the synchronous infiltrating tumors, supporting the paradigm that the infiltrating tumors are clonally derived from the in situ lesions. However, in 8 (40%) of the 20 cases with synchronous in situ and invasive cancer, heterogeneous patterns of allelic loss at one or more chromosomal loci were observed in adjacent DCIS foci. Moreover, some of the allelic losses recognized in in situ portions of the tumors were not conserved in the clonal progression to the synchro nous invasive tumor. Such allelic loss heterogeneity was noted in only 1 of the 20 infiltrating tumors and only 3 of the 23 cases of DCIS without invasion that were studied in a similar manner. This heterogeneity indi cates genetic divergence during the clonal evolution of breast cancer, particularly at the time when in situ cancers progress to invasive cancers. Introduction Breast cancer is recognized by hisiological studies to progress through a series of in situ stages prior to the development of infiltrat ing and metastatic cancer. Although previous studies have identified chromosomal loci with nonrandom allelic losses in cases of invasive breast cancer [reviewed by Devilee et a/.(l)], the molecular changes responsible for progression to advanced disease are not yet well understood. Recently, tumors with synchronous /'/; situ and invasive cancer were analyzed (2, 3), and the allelic losses observed in the HI situ lesions were found to be conserved in the synchronous invasive cancers. These data supported the paradigm of clonal derivation of the invasive cancer from the in situ cancer. Most of the previous genetic studies on HI situ breast cancer have been performed on DNA from either single in situ lesions or aggre gates of multiple HI situ lesions. We reasoned that this approach may not be adequate to define the sequence of genetic events in the progression of the individual tumor, since distinct foci at various stages of progression may be present within one tumor mass. For this study, multiple tumor foci from 20 cases with DCIS' and synchronous invasive breast cancer and 23 cases with DCIS only were individually microdissected from sections of archival breast cancer tissues. Critical suppressor loci on chromosomes 6q, 9p. 1Iq, 13q, 16q, 17q, and 17p Received 12/19/95: accepted 2/15/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked culvenisemeni in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by Grant R21 CA/ES66204 from the National Cancer Institute. 2 To whom requests for reprints should be addressed, at Department of Pathology. Johns Hopkins Bayview Medical Center. 4940 Eastern Avenue, Baltimore, MD 21224. ' The abbreviations used are: DCIS. ductal carcinoma in situ: LOH, loss of heterozygosity. were then characterized for allelic loss using PCR-amplified micro- satellite markers for each of the individual neoplastic foci. Materials and Methods Paraffin-embedded breast cancer tissues with synchronous DCIS and inva sive ductal carcinoma, or DCIS only, were obtained from the surgical pathol ogy files of the Johns Hopkins Hospital and the Johns Hopkins Bayview Medical Center. All pathology was reviewed for consistency of diagnosis. For each tissue block, multiple sections 12 /j.m in thickness were deparaffini/ed. stained with H&E, visualized with an inverted microscope, and dissected using a 26-gauge needle. Tissue was digested overnight in butter containing 0.5% NP40 and 200 /xg/ml proteinase K at an approximate ratio of I /^l huftcr/20 cells. This lysate was used directly in PCR reactions. PCR amplifications of microsutellite markers were performed using primers for 6q21-25 (D6S292, D6S3II. D6S3IO. D6S473. and D6S255). 9p2l (D9S17I), llql3-23 (Int2. DIÃOES29. D1ÃOES35. and D11S528). I3ql2-14 (DI3S260, DI3S263. and DI3SI55), 16ql2-24 (DÃOE6S54ÃOE, D16S4I5. DJ6S265. and DI6S402). 17p (D17S513. CHRNBI. TP53. DI7S786, and D17S122). and 17ql2-24 (THRAI. DI7S579, and D17S588) obtained from Research Genetics (Huntsville. AL), and additional primers for microsalellite markers on 9p21 (D9SÃOE748. D9SI749, D9SÃOE75I,and D9S1752) were prepared as previously described (4). PCR reactions typically contained 1 /il lysate and other reagents as previously described (5). PCR products were then separated using denaturing gel electrophoresis, and allelic loss was determined by at least a 75% reduction in the relative intensity of one alÃ-elein the tumor compared to normal after autoradiography. Allelic loss was usually confirmed by obser vation of LOH at multiple informative markers mapped to the same chromo somal region. When only one informative marker was recognized for a chromosomal region in a particular tissue sample. LOH was confirmed by repeating the PCR amplification of that marker. Results We tested 7 chromosomal loci using 29 microsatellite markers in a total of 224 individual tumor foci from the 43 cases. Each tumor focus was tested for loss at a minimum of three chromosomal loci, and cases with heterogeneity were tested at all seven loci. Allelic loss of at least one of the chromosomal loci studied was recognized in each lesion, reaffirming the ability to isolate neoplastic foci free of significant germ line contamination. As expected, nonrandom allelic loss was recognized in HI situ as well as invasive cancers at each of the chromosomal loci examined (Table I ). Unexpectedly, we found heterogeneity with respect to allelic loss among different neoplastic foci of in situ cancer within 8 of the 20 tumors with synchronous HIsitu and invasive cancer. Two such cases are illustrated in Fig. 1. For case B25, loss of the same alÃ-elesat 13ql 2-14 (e.g., marker D13S263), llq. 6q. and 17q was seen in all of the in situ, invasive, and metastatic foci examined. In contrast, dif ferent alÃ-elesof 9p2l («•.#.. marker D9SI749) were found to be lost in distinct foci of in situ cancer of similar histológica! appearance. The three foci of HI situ cancer that lost the alÃ-elecorresponding to the lower marker of D9S1749 (IS4, IS5, IS6) also lost an alÃ-eleon chromosome 17p. whereas the foci that lost the upper allelic marker of D9SI749 retained 17p at the same loci examined. No heterogeneity was observed among the four foci of infiltrating cancer and one focus of metastatic cancer examined. These data are supportive of a model 1493 on June 2, 2015. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from