Our study is the first that compares CNV profiles for primary tumors and metastases from patients with breast cancer samples globally and with high resolution. On the basis of large genomic clones with an average resolution of 50–100
kb per data point,16, 17, 18, 32
we applied a well established, thoroughly validated array covering ~99% of the human genome. The average resolution of our analysis is more than an order of magnitude higher than that used in the recent array-CGH analysis comparing ALN metastases and primary tumors.14
This is the most likely reason for discrepancies in results between the two studies. The experimental approach we took is also sensitive and robust, which is well illustrated by a strong correlation between findings from our work and previously published reports of breast cancers focusing on primary tumors.7, 8, 9, 10
Our methodology is also insensitive to DNA CNV that might be present in normal DNA of the studied patients.
On the basis of the analysis of global gene expression profiles, there is a debate in the field whether primary tumors from breast cancer patients and matched ALN metastases are different or not, as contradictory results have been published.23, 24, 25, 26, 33, 34
Many factors can be responsible for this incongruence: both related to the still poorly understood biology and a well-recognized heterogeneity of the disease, low numbers of studied patients as well as factors related to the differences in the methodology. Our results from the analysis of DNA copy number actually support both conclusions. The aberration class I cases (IDs 22 and 67) show essentially indistinguishable profiles and cases such as these should also display very similar profiles of gene expression in the primary tumor sample and corresponding metastasis. Although we have not investigated gene expression, the literature supports this assumption. Previous publications describing the parallel analysis of gene copy number changes and mRNA expression in primary tumors of breast cancer have reported a strong correlation between the findings derived from both types of analysis.7, 20, 21, 22, 31
On the other hand, the aberration class I cases are the minority in our study and all the remaining patients showed more or less pronounced differences in gene DNA copy number profiles. This would presumably result in dissimilarity in the levels of transcripts produced from regions affected by gains/amplifications or deletions. The aberration class I patients also raise questions regarding the timing and dynamics behind the process of ALN metastases and the number of cells derived from primary tumors that colonize a lymph node. The highly similar pattern of genetic aberrations in these matched samples may be a reflection of a large number of cells that colonized the lymph node and this metastasis has since then not increased considerably in cell number and therefore did not develop a different genomic profile.
We observed that the total number of aberrations was higher in primary tumors than in metastases and this may seem surprising at first glance. However, this finding likely reflects the heterogeneity of different clones of cells having different genetic profiles that are present within a primary tumor, which is related to the multistage process of cancer development. Our results and others from primary breast tumors suggest frequent intratumoral heterogeneity of genetic aberrations12, 15, 35, 36, 37, 38
implying a coexistence, within the same mass of primary tumor, of different cell subpopulations with different genetic profiles. Metastasis is likely established by a minority of cells from the primary tumor, which migrate to a single distant site. One can therefore envisage a bottleneck ‘purifying' effect for aberrations that a metastasis will contain at the time of its formation, which then may or may not develop into a profile differing from primary tumor by acquisition of additional genetic changes. It should also be emphasized that the above-mentioned previous studies of intratumoral heterogeneity of breast cancer require reanalysis using methodologies that fulfill the current standards of resolution for global genome analysis. The ‘flat' pattern of array-CGH profiles that was characteristic for two metastases (aberration class IV, IDs 35 and 127) also deserves a comment. This could simply be the result of heavy (>90%) non-cancerous cell contamination. This explanation is, however, less probable as the tissues were assessed for tumor cell content by a pathologist. The existence of such flat genomic profiles with no chromosomal alterations in primary tumors has been reported in the past.8, 10, 22
Furthermore, such findings are compatible with a newer model for parallel metastasis development. It has been shown that the early disseminated tumor cells, the potential metastatic progenitors, are genetically significantly less aberrant than the matched primary tumors.39, 40
The above issue requires further analysis in a considerably larger cohort of patients.
Although we studied a limited number of paired samples, we noticed numerous differences in amplifications/gains and deletions between matched samples, suggesting the differential activation of oncogenes and inactivation of tumor suppressor genes, respectively, in primary tumors versus
metastases. Aberrations that are detected in metastases and are not present in primary tumors, as well as changes that are more pronounced in metastases (eg higher level of amplification) compared with primary tumors, represent candidate biomarkers for disease progression and merit further study to delineate the specific gene (or genes) that may be involved. The above statement is based on a reasonable assumption that ALN metastases represent an expansion of a more aggressive clone of cells derived from a primary tumor. We observed many clear-cut differences that can be linked to progression of the disease. For instance, the DNA of case ID 23 displayed two high copy number gains on 8p and 11q that were more prominent in metastasis. The series of amplicons on 11q (64.47–78.3
Mb) that has been mentioned above involves many well-characterized cancer-related genes. Both 11q and 8p changes have been previously linked to poor survival of breast cancer patients.22, 41, 42
This and other examples of the known progression-related changes observed in our study suggest that our reasoning and approach toward finding biomarkers for breast cancer progression is correct. By analogy, the genetic aberrations described here that are acquired in metastases and that are not yet linked to poor patient survival, for example 1q (148–152
Mb), 6q (87–97
Mb), 10q (43–45
Mb) and 11p (56.06–56.51
Mb), might also be important and should be studied further. Moreover, we have recently completed the experimental phase of an ovarian cancer-related project. We performed a similar analysis comparing matched primary tumors and metastases to omentum, and the results also indicate frequent differences in DNA CNV profiles between such sample pairs (Poplawski et al
, in preparation). There also seems to be an overlap between progression-related genetic changes seen in a breast cancer study and those from ovarian cancer. In summary, the most valuable implication of this report is that our approach has the potential to enhance the characterization of specific genes that are linked to breast cancer progression, which forms the basis for the development of new anticancer drugs. The frequent genetic differences between primary tumors and metastases also question, at least to some extent, the role of primary tumors as a surrogate subject of study for the systemic disease, when the development of efficient new molecular therapy is considered.43, 44
Our study calls for an extension, using a considerably larger number of patients who have also been followed for a longer time. In an ideal scenario, it would be important to evaluate many samples from each patient; that is, several for the same primary tumor, several for different ALN metastases as well as samples derived from distant metastases.