Introduction
The molecular pathogenetic pathway of breast tumor progression is not yet clearly defined. Although LOH at chromosomes 1, 3p, 6q, 7q, 8p, 9p, 10q, 11, 13q, 16q, 17, 18q, 22q, and X have all been reported [
5,
6], their relationship to known genes and, more specifically, to different stages of breast disease is still under investigation. Studies in benign fibroadenomas and fibrocystic disease showed no LOH on 1p, 3p, 7q, 11p, 17p, 17q or 18q [
7]. However, 37% of ductal hyperplasias and 42% of atypical ductal hyperplasias exhibit LOH [
8], suggesting that these foci are benign lesions resulting from the alteration of tumor suppressor genes. Furthermore, the discovery that morphologically normal terminal duct-lobule units adjacent to breast cancers exhibit LOH in up to 60% of cases when analyzed with chromosome 3p markers indicates these genetic events can be cryptically present before histopathological abnormalities are identifiable [
9].
These aforementioned studies and other research support the idea that events affecting tumor suppressors can occur early and that preinvasive DCIS can be a direct precursor of invasive carcinoma, a paradigm supported pathologically by the frequency of DCIS lesions found in areas adjacent to invasive breast cancer [
10]. These same analyses also confirm the complexity of this disease. Examination of multiple lesions from individual tumors, for example, reveals the presence of intratumoral heterogeneity [
11,
12,
13]. Markers on chromosome 9p, in one study, demonstrate loss of opposite alleles in different ducts while examples of ducts retaining both alleles were also found [
14]. An extensive analysis of markers on chromosome 11p15 showed LOH early in breast disease (DCIS), although examples of late stage LOH without concomittant DCIS involvement were also found [
3]. This indicates that different components from an individual tumor can represent genetically divergent clones, even at the preinvasive stage of DCIS. The heterogeneous nature of breast cancer alterations could have important implications for the development of targeted therapies.
If LOH events define a localized region of increased risk for carcinoma, the presence of LOH may be clinically important. A complete understanding of all the genetic events involved in breast tumorigenesis and their heterogeneity in relation to stage specificity will therefore be critical to the development of successful treatments. This laboratory and other workers have reported the presence of a previously unidentified region of LOH in band 18p11.3 in non-small cell lung carcinomas, glioblastomas and a small series (14) of invasive breast carcinomas [
1,
2]. The current study uses informative loci in 18p11.3 to investigate, in microdissected material, the stage specificity and extent of molecular heterogeneity exhibited by chromosome 18p11 in breast cancer progression. A series of 30 tumors from which 96 multiple stages of disease have been microdissected, including normal, DCIS and IDC material, were studied. Associated metastases were also examined in some cases. These results were analyzed in conjunction with other LOH data available for markers on 3p, 11p, 13q, 16p, 17p, and 17q in an effort to place chromosome 18p alterations on the breast cancer progression pathway.
Discussion
Current knowledge about the progression of breast cancer reveals that, by analogy to colon and brain tumors, genetic aberrations can occur in a disease stage or grade specific manner. Clonal evolution is, however, a dynamic process from which clones that are not necessarily committed to a malignant phenotype can also emerge, resulting in genetic heterogeneity in the tissue. Such genetic heterogeneity has been described for multiple loci in breast cancer [
3,
11,
16,
19,
20], providing an understanding of the complex picture of breast cancer pathogenesis. Such an understanding is necessary if detection of LOH or other genetic abnormalities will ever allow for consistent and useful identification of preinvasive breast lesions at higher risk of progression.
The present study investigated stage specificity and level of genetic heterogeneity of LOH events in a new region (18p11.3) involved in breast cancer. Significant LOH for chromosome 18p11.3 (63%) was discovered using microdissected foci, suggesting the presence of a putative tumor suppressor gene(s) on 18p with an important role in the pathogenesis of breast cancer. The frequency reported in the present study (63%) is significantly higher than the original reported frequency of 21% [
1] and most probably reflects the superior separation of tumor cells from normal contaminating tissue using a microdissection protocol. A series of unrelated microdissected DCIS tumors for which no invasive component was present also show this high LOH frequency (58%) when measured at 18p11.3 (data not shown), suggesting the 63% frequency reported in the present study is not a consequence of analyzing samples containing multiple stages of disease. These measured allelic deletions represent alterations specific to 18p11.3 and not entire chromosome 18 events based on results using marker D18S452, although the presence of simultaneous but independent LOH on 18q was not examined.
It was discovered, by analyzing multiple foci occurring simultaneously within individual tumors, that chromosome 18p events occur at a significant frequency (56%) early in the tumorigenic process, making LOH at chromosome 18p one of the most common known events in DCIS tumors. For 73% of the cases in which multiple stages of disease arose simultaneously and for which information on at least one marker was available, 18p11.3 LOH in the DCIS component could also be found in the invasive component isolated for analysis (Fig.
2). This suggests that the majority of breast tumors progress in a clonal fashion. Four cases (27%) were, however, consistent with genetic heterogeneity at this chromosomal region. These latter observations argue that invasive foci of an individual tumor can represent genetically divergent clones rather than progressive stages of the disease. Such conclusions are further supported by flow cytometric and comparative genomic hybridization analyses on primary and metastatic disease components [
19,
20]. In the comparative genomic hybridization study [
20], 69% of metastatic lesions showed a high degree of clonal progression from the primary tumor, whereas 31% did not. These frequencies parallel the 70% clonal/30% heterogeneous progression frequencies reported for chromosome 18p events in the present study.
We are not yet at a stage in our understanding of breast disease such that genetic alterations can substitute for the histopathology of the lesion. A combinatorial histological and genetic approach with sufficient information on survival and response profiles may, however, make for more effective disease prevention and cure in the future. The power of genetic profiles in prognostic relevance has recently been shown by Emi
et al [
21]. Studying 15 loci in 264 women, Emi
et al found that LOH at markers 1p34, 13q12, 17p13.3 and 17q21.1, as well as two pairs of markers (1p34/17p13.3 and 13q12/17p13.3), had significant prognostic value and carried significant relative risk of death. It would be interesting, given the high frequency of LOH discovered for chromosomal region 18p11.3 in this report, to include this marker along with these regions to determine its usefulness as a predictive risk marker in breast cancer.
To that extent, it was interesting to view a partial genetic profile for other regions of LOH in these tumors when chromosome 18 LOH was present. Chromosome 18p LOH always occurred in conjunction with LOH on 3p, 9p, 17p and 17q independent of tumor stage, suggesting this group of loci were critical to the development of breast cancer. Allelic deletion on chromosomes 16q, 13q and 11p occurred less consistently (81, 55 and 46%, respectively), suggesting these regional events are more varied depending on the stage and/or type of tumor foci analyzed. Of further interest is the putative tumor suppressor gene, DAL-1, which has recently been mapped to chromosome band 18p11.3 [
22]. It will be important to learn more about the function of this gene and its potential mutational profile in breast cancer. Future studies will determine whether this or another tumor suppressor gene is the target of the high frequency of allelic deletion measured in this study for breast cancer.