Abstract Objective To investigate the the FUS CHOP mRNA and of MDM2 and p53 protein expression in myxoid / round cell liposarcoma (MRCLs) organization. Methods formalin-fixed, 25 cases the paraffin embedded MRCLs tissue. As a control group of 18 cases of non MRCLs liposarcoma and other soft tissue sarcomas. Nested RT PCR method detecting FUS CHOP fusion gene mRNA and do DNA sequencing confirmed and β actin gene as a control. Immunohistochemical SP assay the 25 cases MRCLs of MDM2 and p53 protein expression. Results MRCLs group and negative control group β actin-positive rate of 72% (18/25) and 66.7% (12/18). 15 cases out of 25 cases MRCLs detection type Ⅱ the FUS CHOP fusion gene expression, to remove β actin-negative cases, the positive rate was 83.3% (14/18). 18 specimens of the control group were not detected in the fusion gene FUS CHOP. The MRCLs of MDM2, p53 protein expression was 56% (14/25) and 44% (11/25), round cell-based cases positive expression rate is the highest, the difference was statistically significant. FUS CHOP fusion gene with MDM2 and p53 protein expression was not correlated; MDM2 and p53 protein expression was positively correlated with statistically significant. Conclusion (1) FUS CHOP fusion gene MRCLs specific molecular genetic markers can be used for the diagnosis and differential diagnosis of the tumor. (2) MRCLs MDM2, p53 protein expression and its prognosis. (3) MRCLs FUS CHOP fusion gene with MDM2 and p53 protein expression was not correlated.
Key words myxoid / round cell liposarcoma; by TLS / FUS-CHOP; of MDM2 protein; p53 protein
Expression of FUS CHOP mRNA and MDM2, p53 Protein in Myxiod / round Cell Liposarcoma
WEI Wan li 1, WANG Yan1, RUAN Yong hua2, CHEN Yun1, YE En1
1.Department of Pathology, The 3nd Affiliated Hospital of Kunming Medical College, Kunming 650118, China; 2.Department of Pathology, Kunming Medical CollegeAbstract: Objective To explore the expression of FUS CHOP mRNA and MDM2, p53 protein in Myxiod / round cell Liposarcoma and their significance.Methods Twenty five formalin fixed, paraffin embedded MRCL samples and 18 control cases retrieved from the archival files were studied. Nested reverse transcription polymeras ase chain reaction (RT PCR) technique was employed to detect the FUS CHOP mRNA expression, followed by DNA sequencing to confirm the PCR product. β actin gene was used to assess the quality of the mRNA templates. All of the MRCL cases was available for immunohistochemical analysis of MDM2 and p53 status.Results β actin mRNA was detected in 18 MRCLs (18/25, 72%) and 12 negative control case (12/18, 66.7%). Type Ⅱ FUS CHOP mRNA was successfully detected in 15 out of 25 (60%) MRCLs. All of control cases were negative for the FUS CHOP fusion gene transcripts. The positive rate of MDM2 and p53 proteins were 56% (14/25) and 44% (11/25), respectively.both of them were significantly higher in round cell liposarcoma than them in myxiod and mixed liposarcoma, There was no correlation between FUS CHOP mRNA and the expression of p53, MDM2 protein, but there was a tendency of positive correlation between the expression of MDM2 and p53.Conclusion (1) FUS CHOP is considered as a specific molecular and genetic hallmark for MRCLs and is useful for the diagnosis of MRCLs. (2) The expression of MDM2 and p53 protein is associated with the progression of MRCLs. (3) There is a no correlation between MDM2, p53 and FUS CHOP in MRCLs.
Key words: Myxiod / round cell liposarcoma; TLS / FUS CHOP; p53 protein; MDM2 protein
Myxoid / round cell liposarcoma (myxiod / round cell liposarcoma, MRCLs) is one of the most common type of adult liposarcoma, accounting for about 50%, often occurs in the lower extremity deep soft tissue and retroperitoneal. Cellular and molecular genetics studies have shown that the presence of specific chromosomal translocation t (12; 16) (q13; p11), that is located in 12q13 CHOP gene and 16p11FUS of gene fusion to form more than 95% of the MRCLs the FUS CHOP fusion gene . Found on the human chromosome 12 at the same time there are fat-derived tumor development related genes, such as: MDM2 and CDK4 , one of the most constant amplification of MDM2, it can cause tumors in addition to their can act on the p53 gene which prompted MRCLs occur . Alava, etc. [4 5] found that in some specific chromosomal translocation occurs sarcoma, p53 was significantly negatively correlated with prognosis. The purpose of this experiment is to investigate the FUS CHOP mRNA specificity in MRCLs and relationship of MDM2 and p53 protein expression, and to explore the relationship between them and MRCLs development.
1 Materials and Methods
Soft tissue tumor classification Diagnostics 1.1 data collection Yunnan Provincial Tumor Hospital Pathology from 1999 to 2007 archive MRCLs cases of 25 cases, reference Fletch CDM written , is divided into nine cases mainly of mucus, mucus round cells mixed nine cases, seven cases of round cell component-based, specimens were confirmed by 10% neutral formalin-fixed, paraffin-embedded. Other subtypes of liposarcoma control group of 18 patients, respectively, four cases (including one cases of well-differentiated, go to the differentiation of type 1 cases and pleomorphic type 2 cases), 3 cases of synovial sarcoma, low-grade malignant fibrous sarcoma five cases, malignant two cases of fibrous histiocytoma, mucous lipoma fat neuroblastoma cases of schwannoma with mucus change cases and mucus chondrosarcoma patients. All cases were confirmed by the review.
1.2 FUS CHOP fusion gene detection RNA extraction: extraction method with reference to the relevant literature . Collecting 5μm thick paraffin-embedded tissue sections 21 to 25, in order to prevent contamination, and 70% ethanol was used to scrub the slicing knife and toolholder xylene dewaxing to anhydrous ethanol and after adding a lysis solution (20 mmol / L Tris-HCl, dried, pH8.0, 20mmol / L edta, 2% SDS) 200μl. 20mg/ml proteinase K to a final concentration of 100μg/ml overnight at 55 ° C warm bath. Organization completely dissolved after 95 ° C for 10min inactivated protease K. The following operations RNA extraction kit instructions by extracting RNA produced in strict accordance with the the Takara biological engineering company.
Nested PCR: Takara bio-engineering company to produce the two-step RT PCR kit. Strictly follow the instructions. RT reaction conditions: 42 ℃ 30min, 99 ℃ 5min. Refer to the literature , the experimental primers used by Shanghai Biological Engineering Co., Ltd. synthesizer used in the PCR primers, see Table 1. Primer amplification conditions: 94 ° C denaturation 2min cycles of denaturation at 94 ° C for 30 s, 61 ° C annealing 30s, 72 ° C extension 1min, after 40 cycles, 72 ℃ for 7min. Learn from the first round PCR products the 1μl plus 9μl distilled water as the second round of PCR template, the second round of the annealing temperature to 65 ° C, the same as the first round. With distilled water as the first round of the blank control, with a first wheel blank control product as a second round of the blank control. Positive control as sections containing the FUS CHOP fusion gene cases were confirmed by DNA sequencing. Within quality control choice of β actin detect RNA the reaction system and amplification conditions as above, but only for the first round of the PCR reaction. Taking a second round of PCR product line 2% agarose gel electrophoresis, and observe the results of electrophoresis with a gel imaging system. The PCR products were cloned and sequenced confirmed (by Dalian Takara Biological Engineering Co., Ltd. to assist in the completion of). Table 1 PCR primers
1.3 immunohistochemistry immunohistochemistry SP method, reagents were purchased from Fuzhou step new reagents. Instead of primary antibody with PBS as a negative control, known positive piece as a positive control, steps in accordance with the instructions.
10%，肿瘤细胞总数判定为阳性。">The immunohistochemical results judged with reference to the literature , the positive nuclei of tumor cells> 10%, the total number of tumor cells judged as positive.
1.4 statistical methods of data analysis SPSS11.0 statistical software.
2.1 clinicopathological features of 25 cases MRCLs, 15 males and 10 females (male: female = 3:2), the minimum age is 5 years old, the maximum age of 80 years old, with an average age of 50.2 years, the peak age of onset in 45 to 50 years old ( 58%). Occurred mainly in parts of the lower extremities, a total of 10 cases (40%), followed by pelvic and peritoneal. The 25 cases MRCLs microscopy showed different stages of differentiation the fat mother cells, significantly intertwined the capillary network and myxoid matrix. The round cell based MRCLs can see more mitotic activity, myxoid stroma and vascular network less, as shown in Figure 1, 2.
2.2 FUS the 25 cases MRCLs of the results of the detection CHOP β actin-positive, 18 cases, the positive rate of 72%. Of β actin positive in 12 patients of the control group, 6 patients did not express β actin, including three cases of low-grade fibrosarcoma, patients with liposarcoma, 1 case of synovial sarcoma, schwannomas associated with mucoid degeneration. β actin fragment size of 343bp, as shown in Figure 3.
The 18 cases β actin positive MRCLs, 15 cases detected the type Ⅱ FUS CHOP mRNA, the positive rate was 83.3%, the fragment size of 103bp, shown in Figure 4, failed to detect the type Ⅰ FUS CHOP fusion gene expression . 0.05）。">The positive rate with the χ2 test exact test analysis showed no significant difference (χ2 = 2.107, P = 0.460> 0.05). Control group, tumors were not detected FUS CHOP fusion gene are shown in Table 2. To Table 2 MRCLs and control group FUS CHOP fusion gene mRNA expression
2.3 immunohistochemistry results are shown in Figure 6, 7, Table 3. Mainly to mucus type case group of MDM2 and p53 protein positive cases were lower than the hybrid and predominant round cell group, the difference was statistically significant (χ 2 = 8.090, P = 0.015 <0.05; χ 2 = 8.666, P = 0.01 <0.05).
2.4 FUS CHOP fusion gene and MDM2 and p53 protein expression and correlation analysis of the MRCLs
MRCLs in FUS CHOP fusion gene MDM2, p53 protein expression Spearman correlation analysis: FUS CHOP mRNA and MDM2 and p53 protein expression had no correlation (χ2 = 0.5000, P = 0.667). MDM2, p53 protein expression was a significant positive correlation (rs = 0.995, P = 0.0000).
3.1 FUS CHOP
This experiment Hisaoka  reported successfully detected from 15 cases MRCLs organizations FUS CHOP mRNA expression (all type Ⅱ fusion), the results are consistent with Hisaoka  reported confirmed FUS CHOP fusion gene showed specific expression in MRCLs, FUS CHOP fusion gene formation can be used as MRCLs specific molecular markers for MRCLs diagnosis
MDM2, p53 protein expression in 25 cases MRCLs MDM2, p53 protein expression was 56% (14/25) and 44% (11/25), respectively, in nine cases of mucus-based cases were 33.3% (3/9) and 11.1% (1/9), 9 cases of mixed cases are 44.4% (4/9), 7 cases round cell-based cases were 100% (7/7) and 85.7% (6/7); in Table 3 MRCLs of MDM2, p53 protein expression
The study found that more than 95% of myxoid and round cell liposarcoma presence of the t (12; 16) (q13; p11) chromosomal translocation results CHOP gene on chromosome 12 (also known as GADD153 mainly related to the 2nd exon FUS gene) and chromosome 16 (2,14 16,27) (also known as TLS genes mainly involved 5,7,8 exon) fusion FUS CHOP fusion gene. Positive rate was 60% (15/25), The negative cases removal of β actin-positive rate of 83.3% (15/18). 18 control cases were not detected the FUS CHOP fusion gene expression, and differential diagnosis. MRCLs round cell is the manifestations of poor differentiation, the circular cell number the more indicative of the higher its invasiveness. This group of patients, 11 cases of 16 cases of mixed round cell the MRCLs predominant expression the type Ⅱ FUS CHOP mRNA, and nine cases of mucus-based cases only three cases were type Ⅱ FUS CHOP mRNA results with previous consistent round cell liposarcoma fusion gene for type II, support FUS CHOP mRNA Ⅱ type may be associated with poor prognosis . Fusion gene isoforms and clinicopathological parameters MRCLs no significant correlation studies reported . About the FUS CHOP fusion gene isoforms with MRCLs biology behavior needs further study.
3.2 MDM2 and p53
The display the liposarcoma characteristic ring and a huge marker chromosome 12q13 15 amplified from this region is the most constant amplification of MDM2 gene . The MDM2 protein main function is to directly combine to form the p53-MDM2 complex, thereby inhibiting the function of wild-type p53-mediated transcriptional activation and p53 protein acidic activation domain. In addition, acidic activation domain of the MDM2 gene trans-activation This transcriptional regulation . As About MDM2, p53 expression in MRCLs reported in the literature varies. Smith and Goldblum, etc.  MRCLs MDM2 and p53 protein detected by immunohistochemical methods almost not expressed. By contrast, Dei Tos et al  in all the MRCLs of experimental samples detected the expression of p53, MDM2-positive rate of 56%. The experimentally observed MRCLs MDM2 and p53 positive rate were 56% and 44%, p53 and MDM2 expression was highly correlated (χ2 = 0.995, P = 0.0000), and Taubert et al , Wang Yalan et al  reported similar, suggesting MRCLs there MDM2 and p53 abnormal expression, MDM2 may be one of the factors of the abnormal expression of p53. The results also show that the round cell-based difference the the differentiation MRCLs in MDM2 and p53 protein expression was significantly higher than the well differentiated mucus the predominant and mixed MRCLs to, the difference was statistically significant (χ 2 = 8.090, P = 0.015 <0.05; χ2 = 8.666, P = 0.01 <0.05), to prompt MDM2 and p53 overexpression may be related to poor prognosis with MRCLs.
3.3 FUS CHOP MDM2, p53 relationship
Although the MDM2 gene and the the CHOP gene are located in the 12q13 15 chromosomal regions, but the results of this experiment are not prompted FUS CHOP fusion gene with MDM2 and p53 protein expression exists correlation (χ2 = 0.5000, P = 0.667). Chromosome region 12q13 15 gene mutation would not affect the formation of FUS-CHOP fusion gene for further study.