AIM:To investigate the effect of ankyrin repeat domain 49(ANKRD49)on epithelial-mesenchy-mal transition(EMT)in NCI-H1299 cells,and to explore its mechanism.METHODS:The ANKRD49 was over-ex-pressed in NCI-H1299 cells.The morphological changes of ANKRD49-overpressing NCI-H1299 cells were observed under microscope.The mRNA and protein expression levels of EMT-related markers[E-cadherin,transforming growth factor-β1(TGF-β1),vimentin and α-smooth muscle actin(α-SMA)]and EMT-related transcription factors(Snail1,Slug,Twist and ZEB1)were detected by RT-qPCR Western blot.Immunofluorescence staining was performed to observe the localiza-tion and expression of E-cadherin and vimentin in ANKRD49-overexpressing cells or control cells.Immunohistochemical method was performed to examine the levels of E-cadherin,α-SMA,Snail,Slug and ZEB1 in lung tissues of nude mice in-oculated with ANKRD49-overexpressing H1299 cells or control cells.RESULTS:Compared with the control group,the ANKRD49 overexpressing cells showed mesenchymal cell morphology(fusiform and less tight connections).RT-qPCR and Western blot results showed that the mRNA and protein levels of mesenchymal markers vimentin and α-SMA in ANKRD49 overexpressing cells were significantly higher than those in cells of control group,while the mRNA and protein levels of epithelial marker E-cadherin were lower than those in cells of control group.Compared with control group,the im-munofluorescence intensity of E-cadherin of H1299 cells decreased in after ANKRD49 overexpression,while that of vimen-tin increased significantly.Snail,Slug and ZEB1 expression were significantly elevated in ANKRD49 overexpressing cells compared with control group.The levels of E-cadherin in lung tissues of nude mice inoculated with ANKRD49-overexpressing H1299 cells declined,while the levels of α-SMA,Snail,Slug and ZEB1 increased compared with those in control mice.CONCLUSION:ANKRD49 promoted EMT of NCI-H1299 cells by increasing the expression of Snail1,Slug and ZEB1 and consequent downreguation of E-cadherin and upregulation of vimentin and α-SMA.

BACKGROUND:Internal tension-reduction technique is to reconstruct the anterior cruciate ligament through high-strength suture system combined with tendon.It can effectively reduce graft relaxation and frets by sharing the internal load of the knee joint,and has achieved good biomechanical results and clinical efficacy.However,whether it can reduce cartilage degeneration after anterior cruciate ligament reconstruction through stress sharing reduction has not been studied. OBJECTIVE:To investigate the effect of internal tension-reduction technique on articular cartilage degeneration in southern Yunnan small-ear pigs undergoing anterior cruciate ligament reconstruction. METHODS:Ten adult female Yunnan small-ear pigs were selected,and the ipsilateral knee Achilles tendon was taken from the left knee joint for anterior cruciate ligament reconstruction(normal group,n=10),and the ipsilateral knee Achilles tendon from the right knee joint combined with internal tension-reduction and augmentation system for anterior cruciate ligament reconstruction(tension-reduction group,n=10).One year after surgery,the experimental pigs were sacrificed,and the left and right knee cartilage was taken for hematoxylin-eosin staining,Safranin O-fast green staining,Osteoarthritis Research Society International scoring,and immunohistochemistry staining of type Ⅱ collagen,interleukin-1β,and tumor necrosis factor-alpha in the cartilage. RESULTS AND CONCLUSION:Hematoxylin-eosin staining showed that in the tension-reduction group,there were mild pathologic changes of osteoarthritis,with a low number of empty bone lacunae and no obvious pathological changes such as fibrosis or cell layer breakage;in the normal group,more severe cartilage damage,with an increased number of empty bone lacunae,loss of chondrocytes near the bone and even the formation of fissures.Safranin O-fast green staining indicated that the tension-reduction group had normal cartilage tissue thickness,flat cartilage surface,a neat cell arrangement in a polar pattern,and no swelling or apoptosis,while in the normal group,the thickness of cartilage tissue was obviously thinner,the cell arrangement was disordered with no polarity,the number of cells was reduced,obvious cartilage fractures and cartilage vacuoles formed,and the absence of cells near the central bone was obvious.The Osteoarthritis Research Society International score was significantly lower in the tension-reduction group than in the normal group(P＜0.05).Immunohistochemical findings showed that the protein expression of type Ⅱ collagen in cartilage tissue of the tension-reducing group was higher than that of the normal group(P＜0.05),and the protein expression of interleukin 1β and tumor necrosis factor ɑ in cartilage tissue was lower than that of normal group(P＜0.05).To conclude,internal tension-reduction technique can delay the degeneration of articular cartilage in Yunnan small-eared pigs following anterior cruciate ligament reconstruction.

Objective:To investigate the recurrence-free survival (RFS) and influencing factors of intensity-modulated radiotherapy±chemotherapy (IMRT±C) for the upper thoracic esophageal cancer.Methods:The medical records of 168 patients with cervical and upper thoracic esophageal cancer who met the inclusion criteria from January 2011 to December 2015 were retrospectively analyzed. The RFS was calculated by the Kaplan-Meier method. Multivariate prognostic analysis was performed by Cox models. The recurrence factors were identified by the Logistics model. Results:The 1-, 3-, and 5-year RFS rates were 67.8%, 38.0%, and 20.4%, respectively, and the median RFS was 21.9 months. The locoregional recurrence rate was 47.6%(80/168). The recurrence sites were local esophagus ( n=63), regional lymph nodes ( n=7), and local esophagus+ regional lymph node recurrence ( n=10). Multivariate analysis showed that hoarseness, cTstaging, combined with chemotherapy, 95%PTV 1 exposure dose and GTV average exposure dose were the influencing factors of RFS ( P=0.029, <0.001, 0.031, 0.038, 0.020). Logistics model showed that cTstaging, cNstaging, short-term efficacy, irradiationmethod, GTV maximum transverse diameter and PTV average exposure dose were the influencing factors of recurrence ( P=0.046, 0.022, 0.001, <0.001, 0.012, 0.001). Conclusions:Patients with cervical and upper thoracic esophageal cancer treated with radical IMRT combined with/without chemotherapy have a higher locoregional recurrence rate, and the recurrence rate is mainly the esophagus. The independent factors that affect RFS are different from the risk factors of recurrence.

Objective:To explore the benefit groups of patients with locoregional recurrence of esophageal thoracic squamous cell carcinoma who received radiotherapy or chemoradiotherapy with different patterns of irradiation.Methods:Clinical data of 344 esophageal thoracic squamous cell carcinoma patients with postoperative recurrence who received intensity conformal radiotherapy or concurrent chemoradiotherapy in the Fourth Hospital of Hebei Medical University from 2009 to 2014 were retrospectively analyzed. The distribution of recurrence sites and prognostic factors were analyzed. A stratified analysis was carried out on the benefit groups of patients receiving the elective nodal irradiation (ENI) and involved field irradiation (IFI).Results:276 cases (80.2%) recurred at a single site and 68 cases (19.8%) recurred at more than two sites. The follow-up rate was 96.2%. The 1-, 3-and 5-year overall survival rates were 53.6%, 22.6% and 16.4%, respectively, with a median of 12.8 months (95% CI: 11.3-14.3 months). The 1-, 3-and 5-year local recurrence-free survival rates were 46.5%, 16.9% and 12.0%, respectively, with a median of 11.0 months (95% CI: 9.6-12.4 months). The 1-, 3-and 5-year progression-free survival rates were 39.8%, 11.3% and 6.7%, respectively, with a median of 7.9 months (95% CI: 5.8-10.0 months). Multivariate analysis showed that gender, the log odds of metastatic lymph nodes (LODDS) and the number of chemotherapy cycles were the independent prognostic factors ( P=0.003, <0.001, <0.001). Subgroup univariate analysis demonstrated that patients with an esophageal lesion length<5.0 cm, N 0 stage, the number of surgically-dissected lymph nodes of ≤9, the number of postoperative positive lymph node metastasis site of 0, and LODDS≤0.030 obtained benefits from ENI ( P=0.032, 0.012, 0.001, 0.012 and 0.014). Patients with the number of surgically-dissected lymph nodes of ≥16 achieved benefits from IFI ( P=0.035). Conclusions:Radiotherapy is an effective treatment mode for patients with local recurrence after esophageal cancer surgery. For patients with preoperative esophagography showing shorter esophageal lesions, earlier postoperative pathological N stage, lower LODDS score, and fewer surgically-dissected lymph nodes probably obtain more benefits from ENI than IFI. However, patients with more surgically-dissected lymph nodes may obtain more benefit from IFI compared with ENI.

Objective: To evaluate the effect of different irradiation methods on the long-term prognosis of patients with clinical T_2-3N₀M₀ esophageal squamous cell carcinoma, aiming to select the optimal treatment for these patients. Methods: A total of 268 eligible patients admitted to our hospital from January 2007 to December 2012 were enrolled in this study. All patients were divided into the involved-field irradiation (IFI) and elective node irradiation (ENI) groups. The composition ratio, prognostic factors and adverse events were analyzed between two groups. Results: The median overall survival (OS) time was 35.5 months (95%CI : 30.12-40.88) and the median disease-free survival (DFS) time was 23.5 months (95%CI: 19.00-28.00). According to the multivariate analysis results, all patients were assigned into two groups at a ratio of 1 vs.1(n=86 in each group). Multivariate analysis after propensity score matching (PSM) demonstrated that irradiation method was the independent factor of OS (P=0.038), and T stage and radiotherapy were the independent factors affecting DFS (P=0.002, 0.032). The incidence of ≥grade Ⅱ adverse events did not significantly differ between two groups (P=0.819, 0.756). However, patients with combined chemotherapy experienced more adverse events. Conclusion ENI can prolong the OS and DFS of patients with clinical T_2-3N₀M₀ esophageal squamous cell carcinoma, and does not increase the incidence of severe adverse events.

Objective: To study the relationship between the dose-volume indexes and acute toxicity of intrathoracic stomach in esophageal cancer patients receiving radiotherapy after esophagectomy. Methods: A total of 104 patients treated with postoperative radiotherapy followed by radical esophagectomy were enrolled. The dose-volume indexes of intrathoracic stomach were collected from treatment planning system. The ROC curve and logistic regression were performed to analyze the relationship between acute toxicity of intrathoracic stomach and clinical parameters, dose-volume indexes. Results: A total of 29 patients (27.88 %) suffered from grade 2 or above acute toxicity. The ROC curve analysis showed that the dose-volume indexes including D_max, D_mean, L₅-L₄₅ and V₅-V₅₀were associated with occurrence of grade 2 or above acute toxicity. The univariate analysis showed that location, D_max, D_mean, L₅-L₄₅ and V₅-V₅₀ were significantly correlated with the incidence of grade 2 or above acute toxicity (P<0.05). The multivariate analysis showed that location, L₅ and V₃₅ were independent factors for incidence of grade 2 or above acute toxicity. The ROC curve analysis showed that cut-off values of L₅ and V₃₅ were 14.00 cm and 44.00%, respectively. And the rates of Grade 2 or above acute toxicity were 20.00% for L₅>14.00 cm and 38.64% for L₅≥14.00 cm (χ²=4.473, P<0.05), 14.08% for V₃₅<44.00% and 57.58% for V₃₅≥44.00% (χ²=7.263, P<0.05), respectively. The incidence of grade 2 or above acute toxicity was significantly higher in post-mediastinum stomach group than the other two groups (χ²=12.881, P<0.05). Conclusions Dose-volume index may be indicator to predict acute toxicity of intrathoracic stomach. It is recommended that post-mediastinum stomach should be chosen carefully if esophageal cancer patients require postoperative radiotherapy.

Objective: To evaluate the effect of locoregional risk factors of esophageal cancer on the recurrence of gross tumor volume (GTV) in patients with N₀ esophageal squamous cell carcinoma after radical intensity-modulated radiotherapy (IMRT) and to evaluate its effect on the 10-year survival of patients. Methods: Clinical data of 374 patients with clinical N₀ esophageal squamous cell carcinoma who underwent radical IMRT in the Fourth Hospital of Hebei Medical University from 2005 to 2010 were retrospectively analyzed. Involved-field irradiation was performed in 284 patients and selective lymph node irradiation was given in 90 patients. Concurrent radiochemotherapy was conducted in 69l patients and sequential radiochemotherapy was performed in 38 patients. The survival analysis was carried out by Kaplan-Meier method. The prognosis analysis was performed by multivariate Cox’s regression model. Results: A total of 143 patients (38.2%) had recurrence in GTV. The maximum transverse diameter (GTV-D), GTV volume (GTV-V) and GTV volume/length (GTV-V/L) of GTV patients were significantly longer than those without recurrence in GTV (P=0.008, 0.043, 0.001). ROC curve analysis showed that the optimal diagnostic thresholds for GTV-D, GTV-L, GTV-V and GTV-V/L for GTV recurrence were 3.5 cm, 5.5 cm, 24.0 cm³ and 4.6 cm², respectively (P=0.000, 0.003, 0.000 and 0.000), and the ratio of recurrence within GTV in the patient group was significantly greater than that in the smaller group (P=0.000, 0.002, 0.001 and 0.000). GTV-L and GTV-V/L were the independent risk factors of recurrence in GTV (P=0.021 and 0.009). The 3-, 5-and 10-year survival rates of all patients in the whole group were 42.9%, 23.2% and 7.9%, respectively. Multivariate analysis demonstrated that age, T stage, concurrent radiochemotherapy, GTV-D and GTV-V/L were the independent risk factors of survival (P=0.027, 0.000, 0.018, 0.009 and 0.034). The main cause of death in patients with a survival time of more than 5 years was still associated with cancer. Conclusions The locoregional risk factors of esophageal cancer exert significant effect on the recurrence of GTV in patients with N₀ esophageal squamous cell carcinoma undergoing radical radiochemotherapy, which can be utilized as the predicting markers. Both GTV-D and GTV-V/L are significantly correlated the 10-year survival of patients.

Objective:To study the effects of immunity index and blood inflammatory markers pre- and post-radiotherapy on prognosis of esophageal cancer patients with clinical stage Ⅲ.Methods:A total of 84 esophageal cancer patients with clinical stage Ⅲ (T 4N 1M 0) in Fourth Hospital of Hebei Medical University were analyzed, from May 2010 to April 2012. Intensity-modulated radiotherapy was delivered with a dose of 56-66 Gy/1.8-2.0 Gy per fraction. Flow cytometry was used to analyze the distribution of T-lymphocyte subsets (CD3, CD4, CD8, CD4/CD8) and natural killer cells (CD56) in the peripheral blood pre- and post- radiotherapy. Neutrophil-lymphocyte ratio (NLR) pre- and post- radiotherapy were also tested. The correlation of immunity index and blood inflammatory markers with prognosis was analyzed by univariate and multivariate analysis. Results:For all patients, the 1-, 3- and 5-year overall survival (OS) rates were 78.57%, 34.52% and 19.59%, respectively. The median OS time was 22.60 months. The 1-, 3- and 5-year progression free survival (PFS) rates were 69.05%, 27.38% and 12.09%, respectively. The median PFS time was 21.20 months. The objective response rate was 61.90%, with 11 patients of complete remission (CR) and 41 patients of partial remission (PR). Univariate analysis revealed that NLR before radiotherapy, T-lymphocyte subsets (CD3, CD4 and CD4/CD8) after radiotherapy were significantly associated with OS and PFS (OS: χ 2=7.851, 4.443, 8.381, 5.972, P<0.05, PFS: χ 2= 7.475, 6.290, 9.659, 8.738, P<0.05). Multivariate COX regression analysis showed that NLR before radiotherapy, T-lymphocyte subsets (CD4, CD4/CD8) after radiotherapy were independent prognostic factors for OS (χ 2=10.464, 4.292, 5.507, P<0.05). The NLR before radiotherapy and CD4/CD8 after radiotherapy were independent prognostic factors for PFS (χ 2=10.835, 8.545, P<0.05). Conclusions:Radiotherapy may influence the immune function. NLR before radiotherapy and CD4/CD8 after radiotherapy are of great value in predicting the prognosis of esophageal cancer patients.

Objective:To investigate the prognosis and failure mode of patients with esophageal squamous cell carcinoma receiving selective lymph node irradiation (ENI).Methods:A total of 179 eligible patients with esophageal squamous cell carcinoma were retrospectively analyzed. The prognostic value of tumor-related factors, the influencing factors of short-term curative effect and prognosis of patients, and the single and multi factor indexes of affecting the overall survival rate (OS), progression free survival rate (PFS) and recurrence of patients were analyzed.SPSS 19.0 software was used for statistical analysis.Results:The 1, 3- and 5-year OS of the whole group were 77.1%, 40.1% and 26.0%, respectively, and 1-, 3- and 5-year PFS were 62.6%, 30.6%, and 20.3%, respectively. Multivariate analysis showed that hoarseness, cN stage, cTNM stage, GTV-transverse diameter (GTV-D) and GTV-volume/length (GTV-V/L) were independent factors affecting OS ( P<0.05). The sonar, cTNM staging, and short-term efficacy were independent factors affecting PFS ( P<0.05). Recurrence occurred in 75 patients (41.9%) in the whole group, and 61 patients (34.1%) had distant metastases. Among them, 9 patients (10.6%) had both recurrence and distant metastasis. Of the 75 patients with recurrence, 64(85.3%) had simple esophageal recurrence, 4(5.3%) had lymph node recurrence, and 7 (9.3%) had both. Recurrence occurred in 18 of the 63 patients who achieved CR after treatment. Only 2 patients had lymph node recurrence. Logistic multivariate analysis showed that the surrounding tissue/organ invasion, GTV-D and short-term were independent factors affecting the recurrence rate ( P<0.05). Conclusions:ENI is feasible in patients with esophageal squamous cell carcinoma, and the main mode of failure is esophageal recurrence. Pre-treatment sonar, larger GTV-D and GTV-V/L, more advanced clinical stage and poorer short-term efficacy are indicators of poor prognosis, while the peripheral tissue involvement, GTV-D and short-term efficacy are the independent factors that influence failure.

Objective:To evaluate the recurrence patterns of esophageal squamous cell carcinoma (ESCC) after radical surgery and analyze the predictors of post-recurrence survival (PRS).Methods:A total of 382 ESCC patients who developed recurrence after radical surgery were retrospectively reviewed from January 2007 to December 2011. All of them received salvage treatment after recurrence. The Univariate analysis was determined using the Kaplan-Meier method and log-rank test. Multivariate prognostic analysis was determined using the Cox proportional hazard model.Results:The 1-, 3-, 5-year overall survival (OS) rates were 80.9%, 35.6% and 20.4%, respectively. The median survival time and recurrence time were 29.2 months and 15.0 months. The 1-, 3-, 5-year PRS rates were 37.7%, 14.6% and 11.9%, respectively. The median PRS time was 10.2 months. Locoregional recurrence was noted at 462 sites among 382 patients including 20.5% (95/462) of sites in supraclavicular LN (lymph node), 10.0% (46/462) in anastomosis, 56.1% (259/462) in mediastinal regional LN and 13.4% (62/462) in upper abdominal LN. There were 156, 43, 82 and 101 patients treated with salvage radiotherapy, chemotherapy, chemoradiotherapy and palliative care after recurrence, respectively. The univariate analysis showed that T stage, N stage, TNM stage, recurrence site, number of recurrent regions and treatment regimen after recurrence were significantly correlated with PRS ( χ2=5.336, 4.292, 8.067, 7.482, 4.093, 76.426, P<0.05). Patients receiving salvage radiotherapy with the median dose of 60 Gy, were divided into <60 Gy group and ≥ 60 Gy group. The ≥60 Gy group had a better PRS than <60 Gy group ( χ2=8.320, P<0.05). Multivariate analysis showed that number of recurrent regions and treatment regimen were independent prognostic factors of PRS. Conclusions:Mediastinal and supraclavicular regions were the most common sites of recurrence in ESCC patients after radical surgery. Salvage radiotherapy and chemoradiotherapy could significantly improve PRS in recurrent ESCC patients.