Clinical Observation And Experimental Study In Treatment Of Orbital Adenoid Cystic Carcinoma

Objective1.To summarize the treatment outcomes of orbital adenoid cystic carcinoma and to evaluate prognostic factors.2.To prepare and observe the character of Folate Receptor-mediated VCR-loaded Nanoparticles, which is abbreviated to FA-PLGA (VCR)-NPs. Study the inhibitive effect on ACC-2 cells in vitro and the xenograft tumor in BALB/c-nu mice.3.To observe the expression and distribution of CD44, CD133,and ABCG2 in orbital ACC and investigate their correlations with pathological type and prognosis.Method1.A retrospective case series study was performed on 75 patients with orbital adenoid cystic carcinoma.2.The modified W/O/W extraction-evaporation technique was chosen to prepare FA-PLGA (VCR)-NPs. These particles were characterized for size, modality, encapsulation efficiency, and release in vitro.The cytotoxicity of high concentration blank particles PLGA-NPs towards tumor cells was evaluated by MTT colorimetry. The tumor cells were divided into three groups:VCR,PLGA(VCR)-NPs and FA-PLGA(VCR)-NPs.Seven doses of VCR were tested: 0.05μg/ml,0.25μg/ml,0.5μg/ml,1μg/ml,5μg/ml, 10μg/ml,30μg/ml.After 1day, 2day,3day,4day,5day, the cells growth inhibition ratio were evaluated also by MTT colorimetry. PLGA-NPs and FA-PLGA-NPs were labeled by FITC. Conditions of those particles uptaked by ACC-2 cells with or without free FA were observed with fluorescence microscope. ACC xenograft tumor of nude mice orbit was built by injecting ACC cell suspension and divided into four groups: VCR, PLGA(VCR)-NPs, FA-PLGA(VCR)-NPs, and control group.The inhibition ratio of gross tumor volume was observed every two days. Residual concentrations of VCR in tumors were evaluated by HPLC.The feature of histopathology was observed by electron microscope and HE stain.3.Two stages method of immunohistochemical staining was employed in 33 cases paraffin embedded surgical specimens of human orbital ACC,5 cases recurrence samples,3 cases excisional lachrymal gland caused by neither inflammation nor tumor diseases, and 6 cases xenografts tumor in nude mice.Results1.The 2- and 5- year local recurrence rate of solid type orbital adenoid cystic carcinoma was significantly higher than that of the adeno-tubiform type. [2-year, 85%(17/20) vs 23.53%(8/34),χ2=19.14, p=0.000; 5-year,100%(19/19) vs 64.52% (20/31),Fisher's exact test, p=0.003] The regional extension and distant metastasis of solid type were more than those of adeno-tubiform type. The 5-year local recurrence rate treated by postoperative radiation was lower than that treated only by surgical excision [70%(14/20) vs 92.86%(13/14);Fisher's exact test, p=0.198].The 5-year local recurrence rate in patients initially treated by orbital evisceration during the first time was lower than that of cases which evisceration procedure was used after the recurrence. [25% (1/4)) vs 75% (6/8), Fisher's exact test, p=0.222].Tumors always extend into intracalvarium, nasal cavity and temporal fossa. They may spread to the lung, bone, liver and lymph node. The 5-year metastasis rate was 25.71% (9/35).Both of the lung and bone metastasis rates were 33.33% (3/9).The overall 5-year accumulative survival was 74.29% (26/35), mortality was 25.71% (9/35), and rate of survival without tumor recurrence was 37.14%(13/35).The 10-year disease free survival rate was 17.14% (6/35).Patients were most likely to die with intracranial extension. Surgical excision with postoperative radiation improved the 5-year survival rate to 80%(16/20).2.FA-PLGA (VCR)-NPs were smooth and spherical with a mean particle size 249.2nm. The drug loading rate was 4.53%.The release of VCR from PLGA nanoparticles persists for 14d. After blank particles PLGA-NPs and ACC-2 cells co-cultured for 5 days, cell viability had remained at more than 80 percent. The inhibitive effect of FA-PLGA (VCR)-NPs was more effective than VCR at 4th and 5th day. They were both in dose-dependent and time-dependent manner. Targeting particles could attach to tumor surface, via folate receptor. FA was competitive inhibitor of this recognization. The volume inhibition ratios of FA-PLGA(VCR)-NPs and PLGA(VCR)-NPs were significant higher than VCR. (p=0.016,p=0.029) The inhibition ratio of FA-PLGA(VCR)-NPs was higher than PLGA(VCR)-NPs. (p=0.376) There was signigicant different between residual concentrations of VCR at the 1st day,7th day and 14th day. (p=0.000) TEM pictures showed lots of electron-dense microspheres in tumor cells at the 14th day. Tumor necrosis was obviously, while surrounding tissues were normal.3.The positive rate of CD44 was 54.5% (18/33),with 76.9%(10/13)in solid type and 40% (8/20) in adeno-tubiform type. There wasn't statistically different between them (p=0.072).In solid type the positive expression part often located at the marginal part of the cancer nest. While in the adeno-tubiform type positive cells often located at the outer layer of the tubiform structure, which were the myoepithelial cells. It was also expressed in some normal tissues.The positive rate of CD133 was 57.6%(19/33),with 76.9%(10/13)in solid type and 45% (9/20) in adeno-tubiform type. There wasn't statistically different between them (p=0.087).Parts of the expression of CD133 antigen were both on the cytoplasm and the nucleus. The positive rate of ABCG2 was 21.2%(7/33),with 30.77% (4/13) in solid type and 15%(3/20) in adeno-tubiform type. There wasn't statistically different between them (p=0.393). Lots of positive cells surrounded vessels in tumor tissues.There were no significant difference between different prognosis groups of there surface phenotypes. The correlative analysis results of three surface phenotypes showed that CD44+ cells have positive correlation with CD133+ cells (Spearman, r,=0.416,p=0.016). Besides 1 case CD44+,1 case CD133+ and 4 cases ABCG2+ were detected in transplanted tumor of nude mice.Conclusions1.Orbital adenoid cystic carcinoma is one of the most malignant tumors in the orbit. They have a high local recurrence rate and low survival rate. Tumor histological types and treatment procedure can influence the prognosis. Combined therapy may decrease the recurrence and increase the survival rate.2.FA-PLGA(VCR)-NPs are stable and have high drug entrapment efficiency and high effect of growth inhibition in vitro and vivo.It is targeted and can be proposed as a potential controlled and targeted delivery system for the treatment of ACC.3.The expression of CD44, CD133 and ABCG2 in ACC should have some clinical significance in the progress of ACC.But it couldn't be regarded as one of the factors to evaluate the prognosis.