The treatment of SSs is still an unsolved issue. Although the specific chromosomal translocation characteristic to SSs and its outcome, the chimeric protein is well-known, appropriate targeted therapy could not be found. Its possible cause could be that the specific translocation is essential for the initiation of tumorgenesis, but in tumor progression (recurrence and metastasis) further genetic alterations also play a crucial role. Since these genetic alterations arise randomly (typical losses and gains of chromosomes or chromosomal segments are not known) [9, 10], measurement of the total DNA content gains high importance in estimating the prognosis of the disease and accordingly, in the selection of the aggressiveness of the therapy. A current report of a biphasic SS case with genomic instability revealed great cytogenetic heterogeneity within chromosome numbers and the recurrent presence of dicentric chromosomes . Nevertheless, whether and how the biological behavior of the tumor is affected by cytogenetic changes remains uncertain.
Some SSs recur or develop metastases after a long time, sometimes 5-10 years after the primary diagnosis while others are more aggressive. This can only be partly explained by "conventional" prognostic factors (patient's age, tumor size, extent of poorly differentiated areas and the resectability of the tumor). Karyotyping and CGH analyses provide excellent insight into the sum genetic alterations of the tumor, but they are considerably time consuming and expensive methods. Measurement of the sum DNA content by flow or image cytometry can be faster, easier and cheaper to perform. The aim of the study was to observe whether the frequently occurring diploid SSs could be divided into further subgroups which show correlation to simple or complex karyotype.
Although CGH is a screening method for DNA copy number changes of the entire genome, it only detects "average" genetic abnormalities of the tumor specimen. Furthermore, only aberrations involving losses or gains of DNA sequences can be detected, whereas balanced chromosome abnormalities, such as reciprocal translocations, inversions, and point mutations are not detectable . CGH cannot recognize changes if the fraction of normal cells is high or if cells are polyploid. Therefore, normal findings may be false negative. The samples in our population contained high fraction (at least 85%) of tumor cells.
We observed in our samples the most frequent chromosome alterations described in SS: gain of chromosome 2, 8, 12p, 12q and loss in 3p14 [9, 10]. The most likely candidate genes at 12q13-12q15 are MDM2, CDK2, ERBB3, SAS and CDK4; and RASSF1 at 3p21.3. A clear association was described between gain of SAS and poor prognosis .
Our results show a correlation between DNA-ploidy, fine-tuned DNA-ploidy and the HR-CGH results. Diploid complex cases show numerous genetic alterations whereas in diploid simple cases no detectable chromosome aberrations were found. The explanation of this phenomenon is not evident: the complex diploid tumors cannot be regarded as aneuploid, even if the complex karyotype shows evident aneuploidy (although at a more sensitive level). It may be explained as individual cells with 5c exceeding events (single cell aneuploidy) show that the case has complex karyotype usually developing some aneuploid cells. In contrary, in case of simple karyotype this phenomenon can not be detected. Little is known about the prognostic value of DNA ploidy (at flow and image cytometry level). El-Naggar et al. and Lopes et al. found that ploidy status (aneuploidy) correlated significantly with reduced patient survival in SS [19, 20]. However, the vast majority of SSs are diploid and these diploid tumors are aggressive in several cases. Furthermore, our previous results did not find ploidy (diploid, aneuploid group) as an independent prognostic factor . Conversely, promising results were published about the SS cases with complex karyotype that show an inverse correlation with prognosis.
We would like to find a feasible method to detect possible aberrations which may reflect the cytogenetic alterations within the diploid group. The detection of single cell aneuploidy in parallel to the diploid stemline was in correlation with the complex caryotype. Single cell aneuploidy is a well known phenomenon and its detection is applied e.g. in the diagnostics of cervical dysplasia-carcinoma. [21, 22]. Although the 3 groups (simple diploid - complex diploid - aneuploid)were significantly different, we cannot be completely sure in the unequivocally good prognoses of the simple diploid cases (12 out of 27 cases developed metastasis), still the tendency is unambiguous. By detecting single cell aneuploidy in the diploid group, we know that it means a complex karyotype at cytogenetic level and so there is a simple, inexpensive and fast tool available to provide important complementary data.
There are numerous prognostic factors in case of SSs to consider and some of them are still a matter of debate e.g. type of translocation (SSX1 or SSX2) and the histology type (monophasic or biphasic) [2, 4, 7, 9, 11, 20, 23]. The clearest prognostic factor is the size of the tumor (above or below 5 cm) but unfortunately in most of the cases (except the childhood SSs), at the time of diagnosis the tumor is larger than 5 cm as has happened in all of our examined cases.
In summary, measurement of DNA ploidy and separation of the diploid group based on single cell aneuploidy might provide us useful complementary data which reflects well the cytogenetic results and accordingly it may help the oncologists to select the appropriate therapy.