A candidate metastasis-associated DNA marker for ductal mammary carcinoma

The clinical outcome is generally positive for patients with node-negative breast carcinoma (i.e. those patients without detectable metastases in the lymph nodes) who have been treated with surgery or with surgery and radiation therapy. The disease spreads in about 13% of the patients, however, and their lives are at risk of death [1‐3]. Genetic tests capable of identifying patients at risk for metastatic spread and/or better treatment targeted to eradicate metastatic tumor deposits could have a dramatic impact on the overall survival of these patients.

The construction of a panel of molecular genetic markers for identifying the 13% lymph node-negative mammary carcinomas that are prone to develop metastases [3] would be valuable in indicating those cases in need of early aggressive therapy. There would also be considerable benefit to the 87% of women with mammary carcinomas who are not likely to metastasize by sparing them the physical, mental and financial costs of the treatment. In addition, further knowledge of the genetic mechanisms that play an important role in metastasis could ultimately lead to the development of improved therapeutic procedures.

The evidence for the role of gene alterations in promoting metastasis in general, and in mammary carcinomas in particular, is still accumulating. It is probable, however, that like tumorigenicity suppressor genes and oncogenes many more genes involved in metastasis remain to be discovered since the process is complex, involving a large number of pathways [4]. Most of the known genetic mechanisms involved in the progression of a tumor to the metastatic state involve the loss of function of genes that prevent cells from becoming invasive. These are similar to tumorigenicity suppressor genes, whose loss of wild-type growth regulatory function leads to unregulated or malignant growth. Similarly, the wild-type metastasis suppressor genes regulate the cell's mobility and its response to environmental messages, presumably keeping most cells localized and differentiated. Mutation or complete loss (deletion) of the wild-type counterpart of any such gene in a malignant cell may make that cell more motile, invasive or metastatic. In particular, mutations of genes regulating cell adhesion molecules have been reported to be metastasis-fostering alterations [5‐8]. In human breast cancers, several other genes have been found to be associated with metastasis, namely nm23 [9, 10], KAI1 [11, 12], mta1 [13], KiSS1 [14], PTEN/MMAC1 [15, 16] and BRMS1 [17].

Representational difference analysis (RDA), a DNA subtractive hybridization method [18], was used in the present study to test the working hypothesis that, to achieve the metastatic state, primary mammary carcinoma cells must acquire genetic changes in addition to those changes that led to transformation. With the RDA method, DNA from normal cells was compared with that of the metastatic cells of the same patient. The recovered differential sequences were then mapped on human chromosomes and used to screen DNA samples from normal, primary and metastatic cells of five additional ductal mammary carcinoma patients to determine whether these sequences were consistently associated with metastasis.

Reports show a statistically significant increase in loss of heterozygosity events involving unknown genes on chromosome 16q [26], on chromosome 10q [15] and on chromosome 14q [27], in metastatic breast cancers. Measuring loss of heterozygosity, O'Connell et al. [27] found that the majority of lymph node-negative primary breast tumors did not amplify a region linked to D14S62 and D14S51, while lymph node-positive breast tumors retained heterozygosity for these markers. These data could imply the existence of metastasis promoting gene(s) in that region or, alternatively, the observed molecular changes may be used as a marker of metastatic propensity [28].

In the present investigation we used RDA in an attempt to identify genetic alterations related to breast cancer metastasis by comparing normal cell DNA and metastatic cell DNA of a ductal breast carcinoma patient. Ten unique candidate MADS were identified and the sequence homologies found by searching the gene bank of these MADS are presented in Table 2. The search revealed 82–99% homology to known human gene sequences for nine of these MADS but MADS-IV was found to be novel, having no homology with the sequences in the gene banks. Further characterization of MADS-IX revealed interesting information on the genes and sequence tagged sites (STS) markers surrounding it. Within a range of 1.4 Mb of MADS-IX, we noticed two CDC2 genes (cell division cycle 2 protein, isoform 1 and isoform 2) and two splice variants of the ZWINT gene (without known exact function). In the range of 821 kb, there are nine STS markers (RH26942, SHGC-58267, SHGC-79901, AFM336XD1, SHGC-103705, SHGC-3856, SHGC-84937, STSG72171 and SHGC-81245).

Physical and cytological mapping of MADS-IX confirmed that it is located at 10q21.1. Screening of MADS-IX on DNA samples from five more sets of normal, primary and matched metastatic tumor samples (Fig. 4) revealed that MADS-IX is lost in the transition from normal cells to primary cells to metastasis cells in two of five cases (three of six cases if the case used in the RDA assay is included). PCR screening of PTEN on these five patient samples and screening of MADS-IX and PTEN on a breast carcinoma cell line that had loss of homozygosity of the PTEN gene revealed that MADS-IX is neither a part of the PTEN gene or localized in the homozygous loss region of chromosome 10q encompassing the PTEN gene. These findings together strongly suggest that MADS-IX is a candidate marker for a novel metastasis suppressor gene. Additional archival cases are being screened to determine whether MADS-IX is a reliable marker for metastasis in ductal breast cancers.