Reference DNA was extracted from peripheral blood cells of healthy people.                                                           

      cCGH: Tumor DNA and reference DNA were labeled by nick translation. The hybridization mixture contained of 400 ng tumor DNA and reference DNA, and 10 µg unlabeled human Cot-1 DNA (Gibco/BRL, Life Technologies, Gaithersburg, MD) dissolved in 10 mL hybridization buffer. The denatured hybridization mixture was hybridized to the slide with normal metaphase spreads.

      aCGH: Labeling the 6 µg of  tumor and reference DNA Cy3-dUTP and Cy5-dUTP.

      CGH was executed on individual and pooled DNA from the LMS samples, divided according to both grade and the presence of DNA copy number changes in 17p.

      At last, analyzed the hybridized slides using an Olympus fluorescence microscope and the ISIS digital image analysis system ( MetaSystems GmbH, Altlussheim, Germany ).Agilent G2565AA Feature Extraction Software.

      Array CGH was performed on the Agilent cDNA microarray consisted of  13,000 cDNA clones, and using the DNA pooled for cCGH. And the pooled method  could detect the biological meaning. Sample pooling reduced the effects of biological variation on gene expression arrays and comparable expression measurements from pools and individuals

      The array CGH smooth software determines breakpoints within chromosomes by performing maximum likelihood estimation.

       Obtains of DNA sequence copy number were most commonly observed in chromosomes 1 (43%), 5 (29%), 8 (29%), 17 (43%), and 20 (29%).                                                                                                                                                            

       Losses most frequently influenced chromosomes 2 (43%), 6 (50%), 10 (57%), 13 (71%), 16 (43%), and X (50%)

        From the Table 1, we could find out the clinical characteristics are shown above. The samples 1-7 are low-grade ( I & II ), 8-14 are high-grade( III & IV ). The IV

        Importantly, pooled aCGH displayed all the changes that were frequent in the nonpooled approach. Hence, the array results from the pooled approach can be interpreted as characteristic biologically significant changes in LMS, even when some less frequent changes may have remained undetectable in our pool. The pooled approach showed amplified genes only in the 17p amplicon pool but not in the pool without changes in 17p.

        All of 14 LMS samples showed changes with a mean value of 9.71 ± 1.61 aberrations per sample (range, 2–20). Gains of DNA copy number changes were less frequent than losses (gains/losses 5 1.0:1.3), with mean values of 3.86 ± 0.57 (range, 0–7) and 5.00 ± 1.17 (range, 0–13) changes per sample, respectively.

        Other fewer frequent gains, high-level amplifications, and losses are described in Fig. 1 and Fig. 2, for low- and high-grade LMS samples, respectively.

cCGH: 

        Despite of the tumor grade, the minimal common regions of gain in DNA pooled from all 14 LMS samples were narrowed down to 1cen~q21 (cases 8–10 in the high-grade LMS pool with aberrations in chromosome 17p, and cases 1–3 in the low-grade LMS pool with aberrations in chromosome 17p) as well as 19p (cases 1–3 in the low-grade LMS pool with changes in chromosome 17p).

aCGH:

        Despite of the tumor grade, the minimal common regions of gene amplifications in the DNA pools were narrowed down to 15q26~qter (50.0%; cases 1–3 in the low-grade LMS pool with aberrations in 17p, and cases 8–10 in the high-grade LMS pool with changes in 17p) as well as 17p13.1~q11 (50.0%; cases 1–3 in the low-grade LMS pool with aberrations in 17p, and cases 8–10 in the high-grade LMS pool with changes in 17p).                                                                                      

       The aCGH results Table 2 showed that the number of areas affected by gene copy number losses in the low-grade LMS pool (10 areas) was higher in comparison to the high-grade LMS pool (7 areas). And gains in high-grade tumors (9 areas) were threefold in comparison to low-grade tumors (3 areas). This advice an increasing trend in the number of gene DNA sequences during the progression of LMS.

       Array CGH results revealed 25 changed chromosomal regions (at least five consecutive genes gained or lost) involving a total of 2218 genes. Previous cCGH reports demonstrate that among the most significant DNA copy number changes in LMS are losses in 10q and 13q, as well as gains in 16p and gains and/or amplifications in 17p.

       The cCGH results did not reveal any novel amplicons or losses, but all previously mentioned changes were found in all samples, despite of the tumor grade. Therefore we suggest that these changes may harbor genes involved in the tumor genesis of LMS.

        E.g. 17p13.1~q11 means:

17: chromosome 17

q:  means chromosome  q arm (longer arm) below the centromere.

21: means two-one (part two-length one), not twenty-one.

Recombination rate

   Means the size of 1 recombination rate.