Human cancer arises through the accumulation of genetic alterations in multiple oncogenes and tumor suppressor genes. However, the exact timing of the majority of molecular genetic events during carcinogenesis and their correlation with defined histopathological stages are largely unknown. (1–7). Invasive ductal carcinoma (IDC) of the breast is the result of a multistep process, beginning with ductal hyperplasia and followed by atypical ductal hyperplasia, ductal carcinoma in situ (DCIS), invasive ductal carcinoma and metastatic disease (1–3). Previous studies in the literature (8–10) indicate that alterations in the p arm of chromosome 9 may be a common denominator in human cancer, and may have a role in the early stages of breast cancer, including ductal hyperplasia and DCIS (11–14). Of interest is the finding that loss of heterozygosity (LOH) in the p arm of chromosome 9 may be involved in the pathogenesis of breast cancer (15–19).

In the present study, laser capture microdissection (LCM) was used to analyze paraffin-embedded tissues of the normal breast, ductal hyperplasia, DCIS and IDC to obtain DNA from selected populations of cells for molecular genetic analysis (20–22). LCM was used in order to obtain cells with a high degree of purity in their phenotypes, without contamination of stromal, inflammatory or other cells that could interfere with final conclusions of molecular analysis. The isolated cells representing different stages of breast cancer progression were used for detecting LOH using five microsatellite markers: D9S199, D9S 157, D9S 171, D9S265 and D9S270. The present study was conducted in an attempt to investigate the intratumoral heterogeneity and to associate chromosomal alterations with morphologic findings and proliferation state of the tumor.

Invasive ductal carcinomas exhibited LOH for the five markers tested, and the marker at 9p22-23 (D9S157) was the most frequently identified, whereas the markers D9S171, D9S199, D9S265 and D9S270 (Fig. 1) were less frequently detected. LOH in the DCIS samples was found with 4/5 of the markers tested. D9S157 locus was also present in the majority of the samples, followed by 9p21 (D9S171), D9S199 and D9S265. There are several reports in the literature indicating that other tumor suppressor gene(s) may reside within different 9p loci, namely 9p22-23 (8,15,17,26–28). Notably, phenotypically normal breast tissues that were adjacent to IDC and DCIS also exhibited LOH at D9S157 (Fig. 2) and/or D9S171. The finding that LOH at these loci is also present in the normal tissue adjacent to either DCIS or IDC is an indication that microsatellite instability is an early event in the pathogenesis of breast cancer, and occurs even earlier than any morphological changes are able to be identified. The present study pursued further the validation of these observations by performing LCM of normal skin and lymphocytes from lymph nodes free of metastatic disease from 7 of the patients and was unable to detect LOH in these other normal tissues. (Fig. 3). This data supports previous observations reported in the literature (29). It is notable that the practical implications of these observations are of major importance in the evaluation of the resected margins of conservative breast surgery.

A novel finding was that LOH was heterogeneous in its distribution, as it was exhibited in certain foci, but not in all of the tumor foci studied (Fig. 4), suggesting that clones of cells with varied genetic composition co-exist in the same lesion.

The present data indicate that LOH at locus 9p22-23, (D9S157) and to a lesser degree at 9p21 (D9S171), occurs during the process of cancer initiation. More notably, clones of cells co-exist within a single tumor, indicating that they do not share a clonal origin and only those cells that have LOH at those loci may progress. The monoclonal origin of cancer has been suggested in the literature (30–32), and cytogenetic analyses have revealed that breast cancers are polyclonal (33–35). The use of LCM (36–38) has allowed the identification of more chromosomal aberrations than is possible using DNA isolated from tumor sections (39). By contrast to previous reports (7,40–42) that sustained clonal derivation from in situ cancer, the data presented in the current study support the findings of Fujii et al (8), who reported LOH heterogeneity in multiple foci of individual DCIS lesions.

In conclusion, the present study demonstrated that more than one clone of cells may exist in a simple lesion and that genetic divergence occurs during cancer initiation and progression.