DCIS = ductal carcinoma in situ; IDC = invasive ductal carcinoma; LOH = loss of heterozygosity; PCR = polymerase chain reaction. AbstractWe examined the stage specificity and heterogeneity of 18p11 alterations in a series of tumors representing 96 microdissected samples. Significant loss of heterozygosity (LOH) (63%) was found, with 56% occurring early in ductal carcinoma in situ. Although most cases indicated LOH was clonally inherited, heterogeneity for 18p LOH occurred in 27% of tumors. When compared with other LOH data, 18p LOH was found in conjunction with allelic deletion on 3p, 9p, 17p and 17q, while 13q, 16q, and 11p were less frequently associated. These analyses suggest chromosome 18p11 alteration is a common and early event in breast disease.

SUMMARY:Genetic heterogeneity in breast cancer has been observed both by cytogenetic and loss of heterozygosity (LOH) analyses; however, the frequency with which genetically heterogeneous clones arise is unknown. In this study, a panel of 115 breast carcinomas was analyzed to determine the extent of clonal divergence in tumor foci at progressive stages of tumor evolution. Intraductal, infiltrating, and metastatic tumor components were microdissected from each tumor and tested for LOH at 20 microsatellite markers on seven chromosomal arms. Of these cases, 24 (21%) demonstrated genetically divergent clones during tumor progression. Clonal divergence, inferred from discordant LOH patterns, was observed most commonly between intraductal and infiltrating tumor (18 cases), but was also demonstrated between infiltrating and metastatic tumor (11 cases). Discordant LOH was observed with markers on one chromosomal arm in 16 cases, on two in 7 cases, and on four in 1 case, and was observed most commonly with markers on 17p, 17q, and 16q. More detailed microdissection of four cases provided evidence for a specific chronology of genetic alterations occurring during the progression of each tumor. The results indicate that the different tumor components observed microscopically in breast cancer specimens often represent genetically divergent clones. (Lab Invest 2000, 80:291-301).E lucidation of the sequence of genetic events responsible for progression of breast cancer from in situ to infiltrating and metastatic carcinoma is an important goal of efforts to understand the biological basis of this common malignancy. Progression is believed to occur through the accumulation of genetic changes via a process of clonal evolution and clonal selection (Brenner and Aldaz, 1997;Nowell, 1976). Surgically resected breast carcinoma specimens provide a unique resource for analyzing the genetic changes that occur during progression, because specimens typically contain foci of tumor in various stages of progression, including in situ carcinoma, invasive tumor, and lymph node metastases. Morphologically normal epithelial constituents of the breast are usually represented in such specimens, and foci of benign proliferative epithelial lesions may also be present.Detailed studies of colon cancer have shown that adenomatous epithelium adjacent to carcinoma typically has some but not all of the genetic lesions present in the fully developed malignancy, consistent with direct progression from adenoma to carcinoma (Boland et al, 1995;Fearon and Vogelstein, 1990;Vogelstein et al, 1988). By analogy, it might be expected that a similar analysis of breast tumors in different stages of progression would likewise show the accumulation of genetic changes with progression. However, genetic analysis of breast cancer specimens has suggested that the individual foci of tumor identifiable by microscopic examination may not show the precursor-product relationship often observed in colon cancer. Cytogenetic studies in particular have revealed sufficient genetic...

Loss of heterozygosity (LOH), a genetic change frequently detected in cancer, can also occur in benign epithelial foci in the breast. To characterize LOH in benign breast tissue, 32 cases containing the various components of fibrocystic change in the absence of malignancy were studied. Microdissected foci of ductal hyperplasia, apocrine metaplasia, sclerosing adenosis, and morphologically normal terminal duct lobular units (TDLUs) were analyzed for LOH at 14 polymorphic loci representing seven chromosomal arms. LOH was detected in 22% of normal TDLUs (6/27), 17% of adenosis (4/23), 19% of hyperplasia (4/21), and 53% of apocrine metaplasia (10/19) specimens. Because of the high percentage of LOH in apocrine metaplasia in nonneoplastic specimens, the genetic relationship between apocrine metaplasia and cancer was studied in a panel of breast cancer cases. Of 14 examples of apocrine metaplasia adjacent to a carcinoma, seven were found to have LOH with at least one marker. In all seven cases, the tumor and apocrine metaplasia shared LOH at one or more markers. The results demonstrate that LOH occurs frequently in the components of fibrocystic change as well as in normal TDLUs and suggest that foci of apocrine metaplasia can share a genetically altered precursor cell with an associated carcinoma.