A Research On The Making Of Surgical Safety Margin In Limb Soft Tissue Sarcoma(STS) By Simultaneous Integrated Boost Intensity Modulated Radiation Therapy(SIB-IMRT)

Objective: The method of how preoperative SIB-IMRT makes surgical safety margin in STS will be given, and the short-term clinical effects and the possible untoward effects therein will be evaluated.To describe histopathologic, tumor cell proliferation and apoptosis, the microvascular density and its regulation in surgical margin after SIB-IMRT. To assess the effectiveness of SIB-IMRT making surgical margin in STS. Methods: 1) SIB-IMRT was applied to the 32 patients of locally advanced limb STS treated in our department from January 2012 to January 2015. Radiotherapy plan: GTVs(surgical margin target) 3.5 Gy/time, PTV(plan target) 3.0 Gy/time, 5 to 7 fields, 1 time/day, 13 times, 5 times per week(3weeks); the surgery would be processed in 3-6 weeks since the radiotherapy had been done. Preoperative two cycles of chemotherapy, postoperative adjuvant chemotherapy for 4 to 6 cycles.①Compare the limb circumference of pre-IMRT with the one of post-IMRT; ② Assess the curative effect by MRI according to RECIST standard; ③Analyze the margin area; ④Evaluate the relapse-free survival after 6 months and the tumor necrosis rate by sequential experiment design; ⑤Assess the radiation injury according to CTCAE4.0 and RTOG/EORTC standard. 2) The paraffin-embedded biopsy tissues before radiotherapy and the paraffin-embedded tissues in surgical margin after surgery from the 32 patients were extracted and matched.①Observe the histomorphological change after SIB-IMRT in the surgical margin through HE, Masson, PTAH; ②According to immunohistochemistry, detect Ki-67, Bcl-2 and Caspase-3 protein expression of STS cells in the surgical margin before and after SIB-IMRT; ③According to TUNEL, assess the STS cells apoptosis in the surgical margin before and after SIB-IMRT; ④According to RNA in situ molecular hybridization, detect Bcl-2 mRNA and Caspase-3 mRNA expression of STS cells in the surgical margin before and after SIB-IMRT; ⑤According to fluorescence in situ hybridization, detect the Bcl-2 gene amplification of STS cells in surgical margin before and after SIB-IMRT. 3) ①According to immunohistochemistry, detect CD34 and CD105 expression in the surgical margin before and after SIB-IMRT; compare the MVD of pre-SIB-IMRT with the MVD of post-SIB-IMRT in the surgical margin; ②According to immunohistochemistry, detect and compare the VEGF and HIF-1α protein expression of STS cells before and after SIB-IMRT in the surgical margin; ③Analyze the relevance of the VEGF and HIF-1α protein with MVD; compare the effects of clinical stages on the VEGF, HIF-1α protein and MVD of pre-SIB-IMRT with those of post-SIB-IMRT. Results: 1) The difference between pre-SIB-IMRT and post-SIB-IMRT of limb circumference was statistically significant(Z=-4.470, P<0.05); PR 14 patients, SD 18 cases; follow-up of 12 to 36 months there were 4 cases of pulmonary metastasis, 4 cases of death and 6 cases of relapses, the recurrence rate 18.7%. Relapse-free survival time was 5 to 32 months, the median relapse-free survival time was 15 months; the preoperative SIB-IMRT making the surgical margin of locally advanced limb STS showed the efficacy in more than 60% of cases; the necrosis rate of surgical margin was larger than tumor necrosis rate; the level 1 of acute radioactive skin injure was manifested in 26 cases(26/32), level 2 in 4 cases(4/32), and level 3 in 2 case(2/32). Late Skin toxicity: level 1 in 29 cases, level 2 in 3 cases; late subcutaneous tissue toxicity: level 1 in 31 cases, level 2 in 1case. late bone toxicity: level 0 in 27 cases, level 1 in 5 cases; late joint toxicity: level 0 in 30 cases, level 1 in 2 case. 2) According to HE, Masson and PTAH, it showed the STS cells of post-SIB-IMRT in surgical margin reduced, the necrosis increased, the fiber hyperplasia appeared, the pseudocapsule thickened and the striated muscle degenerated. The expression of Ki-67(proliferation index, PI) of STS cells in surgical margin was statistically significant difference(t=6.160, P<0.05) before and after SIB-IMRT; the expression of Bcl-2 and Caspase-3 of STS cells in surgical margin was statistically significant difference(t=4.828, P<0.05; t=-5.335, P<0.05) before and after SIB-IMRT. The apoptosis index(AI) of STS cells in surgical margin was statistically significant difference(t=-3.590, P < 0.05) before and after SIB-IMRT. The expression of Bcl-2mRNA and Caspase-3mRNA of STS cells in surgical margin was statistically significant difference(t=3.452, P<0.05; t=-3.611, P<0.05) before and after SIB-IMRT. The Bcl-2 gene amplification of STS cells in surgical margin was statistically significant difference(t=3.893, P<0.05) before and after SIB-IMRT. 3) The tumor microvascular density marked by CD34 in surgical margin was statistically significant difference(t=3.589, P<0.05) before and after SIB-IMRT; the tumor microvascular density marked by CD105 in surgical margin was statistically significant difference(t=2.539, P<0.05) before and after SIB-IMRT. The expression of VEGF of STS cells in surgical margin was statistically significant difference(t=5.301, P<0.05) before and after SIB-IMRT; the expression of HIF-1α of STS cells in surgical margin was statistically significant difference(t=3.198, P < 0.05) before and after SIB-IMRT. Conclusion: 1) The preoperative SIB-IMRT for making the surgical safety margin in the case of locally advanced limb soft tissue sarcoma increases the resection rate of R0, prolongs the relapse-free survival time and shortens the period of radiation therapy; the short-term effects are evident and the patient tolerance is good. 2) The preoperative SIB-IMRT can in surgical margin reduce tumor cell proliferation, increase apoptosis, produce fiber hyperplasia, and thicken pseudocapsule, which facilitate the excision. SIB-IMRT reduces Ki-67, Bcl-2 and Bcl-2mRNA expression of STS cells and increase Caspase-3 and Caspase-3mRNA expression of them so that the proliferation of STS cells is controlled. 3) The preoperative SIB-IMRT reduces HIF-1αand VEGF expression of STS cells in surgical margin, inhibits tumor angiogenesis and may reduce the risk of metastasis in the period of preoperative radiotherapy. The relevance of MVD with HIF-1α and VEGF expression can be set up. MVD, HIF-1αand VEGF may be the assessment indicators of the efficacy of the making surgical margin in locally advanced limb STS by SIB-IMRT and also may be the prognoses factors.