Dose escalation in radical radio(chemo)therapy for cervical and upper thoracic esophageal cancer with 3DCRT/IMRT (ChC&UES): a multicenter retrospective study.

BACKGROUND: Cervical and upper thoracic esophageal cancer (ESCA) presents treatment challenges due to limited clinical evidence. This multi-center study (ChC&UES) explores radical radio(chemo)therapy efficacy and safety, especially focusing on radiation dose. METHOD: We retrospectively analyzed clinical data from 1,422 cases across 8 medical centers. According to the radiation dose for primary gross tumor, patients were divided into standard dose radiotherapy (SD, 50-55 Gy) or high dose (HD, > 55 Gy) radiotherapy. HD was further subdivided into conventional- high-dose group (HD-conventional, 55-63 Gy) and ultra-high-dose group (HD-ultra, ≥ 63 Gy). Primary outcome was Overall Survival (OS). RESULTS: The median OS was 33.0 months (95% CI: 29.401-36.521) in the whole cohort. Compared with SD, HD shown significant improved survival in cervical ESCA in Kaplan-Meier (P = 0.029) and cox multivariate regression analysis (P = 0.024) while shown comparable survival in upper thoracic ESCA (P = 0.735). No significant difference existed between HD-conventional and HD-ultra in cervical (P = 0.976) and upper thoracic (P = 0.610) ESCA. Incidences of radiation esophagitis and pneumonia from HD were comparable to SD (P = 0.097, 0.240), while myosuppression risk was higher(P = 0.039). The Bonferroni method revealed that, for both cervical and upper thoracic ESCA, HD-ultra enhance the objective response rate (ORR) compared to SD (P < 0.05). CONCLUSION: HD radiotherapy benefits cervical but not upper thoracic ESCA, while increasing bone marrow suppression risk. Further dose escalating (≥ 63 Gy) doesn't improve survival but enhances ORR.

Immune checkpoint inhibitors combined with or without radio(chemo)therapy for locally advanced or recurrent/metastatic esophageal squamous cell carcinoma.

OBJECTIVE: This study was designed to investigate the efficacy and prognostic factors for immune checkpoint inhibitors (ICIs) combined with or without radio(chemo)therapy and to evaluate their toxicity in patients with locally advanced or recurrent/metastatic esophageal squamous cell carcinoma (LA/RM ESCC). METHODS: In this study, 198 patients with locally advanced or recurrent/metastatic (LA/RM) ESCC who received ICIs combined with or without radiotherapy/chemotherapy in the Department of Radiotherapy of the Fourth Hospital of Hebei Medical University were retrospectively analyzed. Univariate and multivariate analyses were performed to determine the prognostic factors for overall survival (OS) and progression free survival (PFS). The factors affecting treatment response and the occurrences of treatment-related adverse events (trAEs) were analyzed. RESULTS: The median OS and PFS were 30.4 months (95% confidence interval [CI] 15.1-45.7 months) and 15.3 months (95% CI 12.8-17.8 months), respectively. Univariate and multivariate analysis showed that the number of ICI cycles, the intervention of radiotherapy and dysphagia were independent factors affecting OS (Hazard ratio [HR] = 0.39, 2.043 and 0.365, respectively; P = 0.018, 0.001 and 0.032, respectively). The intervention of radiotherapy was an independent factor for PFS (hazard ratio [HR] = 18.149, P = 0.013). The median OS and PFS for patients who had complete response and partial response (Objective response, ORR) were 50.8 months (95% CI 25.8-75.7 months) and 20.5 months (95% CI 14.1-27.0), respectively, which were significantly higher than those in the non-ORR group (OSnon-ORR:17.5 months, 95% CI 14.0-21.0; χ2 = 13.881, P < 0.001; PFSnon-ORR: 12.1 months, 95% CI 10.1-14.1, χ2 = 10.676, P = 0.001). The intervention of radiotherapy could improve treatment response (χ2 = 47.725, P = 0.000). In entire study population, 83 patients (41.9%) had ≥ grade 2 trAEs. CONCLUSIONS: ICIs combined with radiotherapy/chemotherapy are safe and effective in LA/RM ESCC patients. Intervention of radiotherapy, the number of immunotherapy cycles and occurrence of dysphagia affecting the overall survival of LR/RM ESCC patients. Intervention of radiotherapy was an independent prognosis factor for OS and PFS and associated with better treatment response.

YTHDF3 mediates HNF1α regulation of cervical cancer radio-resistance by promoting RAD51D translation in an m6A-dependent manner.

Radiotherapy, as an important primary treatment, has effectively improved the survival of patients with cervical cancer (CC). Some patients, however, do not benefit optimally from radiotherapy because of radio-resistance. Therefore, identifying radio-resistance biomarkers and unravelling the underlying mechanisms is of critical importance for these patients. In the present study, we found significant upregulation of hepatocyte nuclear factor 1-alpha (HNF1α) expression in radio-resistant cervical cancer tissues and cell lines. Depletion of HNF1α reduced and overexpression of HNF1α promoted the resistance of CC cells to irradiation in vitro and in vivo. HNF1α positively regulated DNA repair protein RAD51 homologue 4 (RAD51D) at the protein level but not at the mRNA level. Mechanistically, upregulation of HNF1α enhanced YTH domain-containing family protein 3 (YTHDF3) transcription, which in turn promoted RAD51D mRNA N6 -methyladenosine (m6A) modification. YTHDF3 mediates HNF1α regulation of cervical cancer radio-resistance by promoting RAD51D translation in an m6A-dependent manner. The HFN1α/YTHDF3/RAD51D regulatory axis was found to play a critical role in conferring radio-resistance of CC cells. In conclusion, dysregulation of the HFN1α/YTHDF3/RAD51D axis may promote the radio-resistance of CC cells. Blocking this pathway may provide therapeutic benefits against CC radio-resistance.

Construction of a predictive model of abdominal lymph node metastasis in thoracic esophageal squamous cell carcinoma and preliminary analysis of its effect on target for postoperative radiotherapy.

Purpose: To investigate the influencing factors of abdominal lymph node metastasis in thoracic esophageal squamous cell carcinoma (TESCC), and to construct its predictive model, in order to analyze the targets for postoperative radiotherapy. Methods and materials: From January 2008 to December 2014, the clinicopathological data of 479 patients who underwent radical resection for esophageal cancer in the Fourth Hospital of Hebei Medical University were collected and retrospectively analyzed. The influencing factors of postoperative abdominal lymph node metastasis were analyzed, and a predictive model was constructed based on their independent influencing factors. Receiver operating characteristic (ROC) curve was utilized to analyze the predictive value of this model; in the meantime, the postoperative locoregional recurrence (LRR) of this group was analyzed. Results: The postoperative pathology of all patients showed that the lymph node metastasis rate (LNR) was 39.7%, of which the abdominal lymph node metastasis rate was 22.0%. Logistic regression analysis revealed that the patient's lesion location, pN stage, vascular invasion, LND and mediastinal lymph node metastasis were independent risk factors for the positive rate of abdominal lymph nodes after surgery (P = 0.000, 0.000, 0.033, 0.000, 0.000). The probability of abdominal lymph node metastasis was Y = ex/(1 + ex), and X = -5.502 + 1.569 × lesion location + 4.269 × pN stage + 1.890 × vascular invasion + 1.950 × LND-4.248 × mediastinal lymph node metastasis. The area under the ROC curve (AUC) of this model in predicting abdominal lymph node metastasis was 0.962 (95% CI, 0.946-0.977). This mathematical model had a high predictive value for the occurrence of abdominal lymph node metastasis (P = 0.000), and the sensitivity and specificity of prediction were 94.6% and 88.3% respectively. The overall survival rate was significantly higher (X2 = 29.178, P = 0.000), while abdominal lymph node recurrence rate was lower in patients with negative abdominal lymph nodes than in those with negative lymph nodes (1.4%&7.7%, X2 = 12.254, P = 0.000). Conclusion: The lesion location, pN stage, vascular invasion, LND and mediastinal lymph node metastasis are independent influencing factors of abdominal lymph node metastasis in patients with TESCC. The mathematical model constructed by these indicators can accurately predict abdominal lymph node metastasis, which can help clinicians to choose the targets for postoperative radiotherapy.

Relationship between postoperative nodal skip metastasis of mid-thoracic esophageal squamous cell carcinoma and patient prognosis and its value in guiding postoperative adjuvant treatment.

Objective: To evaluate the predictive role of nodal skip metastasis (NSM) in the prognosis of lymph node-positive mid-thoracic esophageal squamous cell carcinoma, and to evaluate the significance of postoperative adjuvant treatment in patients with different sites of metastatic nodes. Methods: A retrospective analysis was performed on clinical data of 321 lymph node-positive mid-thoracic esophageal squamous cell carcinoma patients who underwent surgery in the Fourth Hospital of Hebei Medical University. Based on the site and condition of lymph node metastasis by postoperative pathology, the patients were divided into two groups: NSM group and non-NSM (NNSM) group. The propensity score matching (PSM) method was employed to match the two groups. The prognostic factors of patients before and after PSM as well as the effect of different adjuvant treatment modes on the prognosis of patients before and after PSM were analyzed. SPSS 29.0 statistical software was used for analysis. Results: PSM in a 1 : 1 matching ratio was performed, 103 patients were assigned to NSM group and NNSM group respectively. Significant differences were found in the 3- and 5-year OS and DFS between the two groups before PSM, the 3- and 5-year OS also showed a significant difference after PSM (P < 0.05). Multivariate analysis illustrated that gender, postoperative adjuvant treatment mode, N stage and lymph node metastasis were independent risk factors for OS and DFS after PSM (P < 0.05); for NSM patients, postoperative adjuvant chemotherapy and radiotherapy significantly prolonged OS and DFS before and after PSM (P < 0.05). But no significant difference was found in OS and DFS for NNSM patients after PSM (P > 0.05). Conclusion: Postoperative NSM is a good prognostic factor for patients with mid-thoracic esophageal squamous cell carcinoma, postoperative adjuvant chemoradiotherapy was recommended for those group, thereby gaining survival benefits.

Preliminary analysis of the benefits of different irradiation types on patients with postoperative locoregional recurrence of esophageal cell squamous carcinoma.

BACKGROUND: To explore the benefits of different types of irradiation on patients with postoperative locoregional recurrence (LRR) of thoracic esophageal squamous cell carcinoma (ESCC). METHODS: We analyzed the medical records of 344 patients with recurrent esophageal cancer (EC) at the Fourth Hospital of Hebei Medical University. All patients met an inclusion criteria that included having postoperative LRR (without distant metastasis), and having received either chemotherapy, radiotherapy, or chemoradiotherapy after LRR. Patients either received elective nodal irradiation (ENI) or involved field irradiation (IFI), with a stratified analysis performed on both groups. SPSS 19.0 software (IBM Corporation, Armonk, NY USA) was then used for statistical analysis. RESULTS: The median overall survival time of all patients after surgery was 33 months [95% confidence interval (CI): 28.3-37.7 months]; the median overall survival time of patients after recurrence after radiotherapy was 12.8 months (95% CI: 11.3-14.3 months). There were 276 cases (80.2%) of single local recurrence after surgery, and 68 cases (19.8%) of multiple local recurrence (≥2). The results of our multivariate analysis showed that the patient's gender, log odds of positive lymph nodes (LODDS), and the number of courses of chemotherapy were all independent factors affecting the patient's prognosis (P=0.003, P<0.001, and P<0.001). The results of stratified analysis showed that patients with esophageal lesion length <5.0 cm, stage N0, ≤9 surgically dissected lymph nodes, no positive regional lymph node metastasis (LNM), and LODDS ≤0.030 could benefit from ENI treatment (X2=4.208, P=0.032; X2=6.262, P=0.012; X2=10.359, P=0.001; X2=6.327, P=0.012; X2=6.026, P=0.014); and patients with ≥16 surgically dissected lymph nodes could benefit from IFI treatment (X2=4.429, P=0.035). CONCLUSIONS: Chemotherapy, radiotherapy, and chemoradiotherapy are all effective modes of treatment for patients with postoperative LRR of EC. Patients with shorter esophageal lesions determined by preoperative esophagography, earlier postoperative pathological N staging, lower LODDS scores, and fewer surgically dissected lymph nodes might benefit more from ENI treatment than from IFI. However, patients with a larger number of lymph nodes dissected during surgery might benefit more from IFI treatment. To further confirm this study's conclusions, multiple prospective studies should be undertaken in the future.

Effect of SIB-IMRT-based selective dose escalation of local tumor on the prognosis of patients with esophageal cancer.

BACKGROUND: To investigate the effect of SIB-IMRT-based selective dose escalation to local tumor on the prognosis of patients with esophageal cancer (EC). METHODS: A total of 302 EC patients were enrolled. The prognostic factors of the entire group were initially analyzed, and the composition ratios of the two groups and the different doses of each fraction for PTV were compared. The propensity-score matching (PSM) was carried out (1:1 ratio), and the prognostic factors for the two groups were analyzed according to the results of COX. RESULTS: The median overall survival (OS) for all patients was 30.0 months (23.495-36.505 months), and the median disease-free survival (DFS) was 21.3 months (7.698-24.902 months). In multivariate analysis, chemotherapy, cTNM stage and dose-per-fraction for the PTV were independent prognostic factors for OS (P = 0.013, 0.000, 0.028) and DFS (P = 0.033, 0.000, 0.047). Multivariate analysis of patients after PSM revealed that cTNM staging and dose-per-fraction were the independent prognostic factors for OS (P = 0.000, 0.015). Chemotherapy, cTNM staging and dose-per-fraction for the PTV were the independent prognostic factors for DFS (P = 0.025, 0.010, 0.015). There was no significant difference in grade ≥ 2 acute toxicities between the two groups. A subgroup analysis of patients with a single dose of 2 Gy and > 2 Gy in the SIB-IMRT group showed that OS and DFS of the latter were significantly better than those of the former. CONCLUSION: The selective dose escalation to local tumors based on SIB-IMRT technique can improve the survival of patients received radical radiotherapy without increasing toxicities.

Knockdown of FAM83D Enhances Radiosensitivity in Coordination with Irradiation by Inhibiting EMT via the Akt/GSK-3ß/Snail Signaling Pathway in Human Esophageal Cancer Cells.

PURPOSE: To explore the effects of FAM83D on the proliferation, invasion and radiosensitivity of human esophageal cancer cells and to elucidate the mechanism involved in the regulation of the growth and metastasis of esophageal cancer cells. METHODS AND MATERIALS: This study included sixty-nine patients with esophageal cancer. The expression levels of FAM83D in the esophageal cancer tissues and paracarcinoma tissues of the sixty-nine patients were measured. We also examined FAM83D expression in five cell lines. We analyzed the effects of FAM83D on the proliferation, invasion and radiosensitivity of human esophageal cancer cells via MTS, Transwell, and colony formation assays. The effect of FAM83D knockdown on cell apoptosis was assayed by flow cytometry. In addition, we also examined changes in the expression of metastasis-related molecules at the protein and mRNA levels by qRT-PCR and Western blotting after silencing FAM83D expression, and we detected the expression of PI3K/Akt signaling-related proteins by Western blotting. RESULTS: The results demonstrated that the expression of FAM83D was obviously higher in esophageal cancer tissues and cell lines than that in human adjacent normal tissues and normal esophageal epithelial cell lines. FAM83D overexpression was positively associated with tumor size, tumor-node-metastasis (TNM) stage, T classification, N classification, distant metastasis and relapse and was negatively associated with patient survival rates. FAM83D shRNA transfection suppressed its expression. Compared to that in the control group, the proliferation of tumor cells in the FAM83D shRNA group was hindered after exposure to radiation in vitro and in vivo; in addition, FAM83D knockdown inhibited cell invasion, induced apoptosis and regulated apoptosis-related protein expression. Moreover, the radiosensitivity of esophageal cancer cells was increased after depletion of FAM83D. In addition, FAM83D silencing was associated with the reversion of EMT, as reflected by an increase in the epithelial marker E-cadherin and a decrease in the mesenchymal markers N-cadherin and vimentin. Further study showed that FAM83D depletion suppressed the signaling pathway involving p-Akt, p-GSK-3ß and Snail. CONCLUSION: The results reveal that FAM83D may be a potential therapeutic target for esophageal squamous cell carcinoma (ESCC) and that lower expression of FAM83D in coordination with irradiation promotes the radiosensitization of ESCC by inducing EMT through the Akt/GSK-3ß/Snail signaling pathway.

Dosimetric Predictors of Radiation Gastritis Due to Postoperative Intensity Modulated Irradiation Therapy in Patients with Esophageal Squamous Cell Carcinoma After Radical Esophagectomy.

Objective: To explore the association between the incidence of acute radiation gastritis attributed to postoperative intensity modulated irradiation therapy (IMRT) and the dose volume of intrathoracic stomach of patients with esophageal squamous cell carcinoma (ESCC) after radical esophagectomy. Methods: The authors retrospectively collected the data of 49 ESCC patients who participated in postoperative IMRT treatment after radical esophagectomy, and analyzed the incidence of acute radiation gastritis during the treatment. Results: Among all the 49 patients, acute grade ≥2 radiation gastritis was observed in 19 patients (39%). Receiver operating characteristic (ROC) curve analysis showed that the physical variables, such as stomach Dmax, Dmean, length of the whole stomach received 5-40 Gy (LSTT5-LSTT40), and V10-V50, were associated with acute radiation gastritis. Patients were grouped according to cutoff values in physical indicators obtained from the ROC curve. Other than V5, the incidence of acute grade ≥2 radiation gastritis was significantly higher in the group with indicators above cutoff values than that below cutoff values, and the between-group difference was statistically significant in terms of physical indicators. Multivariate analysis suggested that LSTT5 and V40 could be acted as indicators to predict the incidence of acute grade ≥2 radiation gastritis. Conclusions: In the postoperative IMRT treatment for ESCC patients, protection of intrathoracic stomach is strongly recommended. Dose-volume histogram is a preferable predictive indicator for the occurrence of acute radiation gastritis, especially for the stomach LSTT5 and V40. Nevertheless, a larger sample size is needed to provide insight into the relevant study.

Comparison of the effect of postoperative radiotherapy with surgery alone for esophagus squamous cell carcinoma patients: A meta-analysis.

BACKGROUND: This meta-analysis was conducted to evaluate the effect of postoperative radiotherapy for patients having esophagus squamous cell carcinoma after radical surgery. METHODS: A comprehensive research was performed in Pubmed, Embase and Cochrane Library electronic databases from inception until December 10, 2017. We collected all published full articles about comparison of surgery plus postoperative radiotherapy with surgery alone. RESULTS: Four randomized-controlled trials (RCTs) with 1050 participants and 8 non-randomized-controlled trials with 3248 participants were included and evaluated separately. The risk ratio rate and its 95% confidence interval (CI) were calculated. Both RCTs and non-randomized-controlled trials (NRCTs) groups showed a significant increase in 3-year overall survival (OS) rate (RRRCT = 0.89, 95% CI: 0.80-0.99; RRNRCT = 0.82, 95% CI: 0.76-0.88) and decrease locoregional recurrence rate (RRRCT = 0.53, 95% CI: 0.43-0.66; RRNRCT = 0.47, 95% CI: 0.32-0.69) after postoperative radiotherapy compared with surgery alone. The 5-year OS rate in the group of NRCTs was markedly enhanced (RR = 0.87, 95% CI: 0.82-0.92), while that of the RCTs group was not enhanced in a significant way (RR = 0.84, 95% CI: 0.70-1.02). Subgroup analysis based on pathological lymph node status revealed that postoperative radiotherapy could improve OS regardless of pathological lymph node status (pathological lymph node positive patients: RR5-year os-RCT = 0.81, 95% CI: 0.70-0.93; RR5-year os-NRCT = 0.87, 95% CI: 0.80-0.94; Pathological lymph node negative patients: RR3-year os-RCT = 0.76, 95% CI: 0.59-0.96; RR3-year os-NRCT = 0.52, 95% CI: 0.30-0.89). No effect on distant recurrence rate was detected. Adverse effects induced by postoperative radiotherapy were comparatively modest and tolerable. CONCLUSION: Polled results yielded that postoperative radiotherapy was promising in improving OS and reducing the locoregional recurrence rate. More large-scale up-to-date RCTs are needed to further validate the use of postoperative radiotherapy in modern practice.

BMI-1 suppression increases the radiosensitivity of oesophageal carcinoma via the PI3K/Akt signaling pathway.

B-cell­specific Moloney murine leukaemia virus integration site-1 (BMI-1) contributes to the growth of tumour cells post-irradiation (IR). The aim of the present study was to characterize the effects of BMI-1 on cell viability, radiosensitivity and its mechanisms of action in oesophageal squamous cell cancer (ESCC). Western blotting and immunohistochemistry were employed to evaluate the protein expression of BMI-1 in ESCC cells and specimens, respectively. Additionally, the protein expression levels of BMI-1, H2AK119ub and γH2AX in ESCC cells were detected following different doses of IR and at different times after IR. The protein expression levels of MDC1 and 53BP1 were also measured. Flow cytometry and MTT assays were used to determine cell cycle progression, apoptosis and cell viability. The phosphatidylinositol 3-kinase inhibitor LY294002 and the agonist IGF-1 were employed to suppress or induce the phosphorylation of Akt to determine whether BMI-1 induces radioresistance in ESCC cells via activation of the PI3K/Akt pathway. The expression of BMI-1 was higher in ESCC tissues and cells compared with that in normal oesophageal tissues and cells. In addition, BMI-1 was positively related to tumour size and lymph node metastases and negatively to the overall survival of ESCC patients. IR induced the expression of BMI-1, H2AK119ub and γH2AX in a dose- and time-dependent manner. BMI-1 knockdown lowered the expression of γH2AX, MDC1 and 53BP1, suppressed cell viability and increased radiosensitivity. G2/M phase arrest was eliminated; this was followed by an increased proportion of cells entering the G0/G1 phase after IR and BMI-1 knockdown via the upregulation of P16 and downregulation of cyclin D2 and cyclin-dependent kinase-4. Moreover, BMI-1 knockdown increased cell apoptosis, downregulated MCL-1 and p-Akt and upregulated Bax. Additionally, the inhibitory effect of the downregulation of p-Akt by LY294002 on tumour cell viability was identical to that of BMI-1 knockdown, while the kinase agonist IGF-1 reversed the effects of BMI-1 knockdown on cell viability and radiosensitivity. Taken together, BMI-1 knockdown induces radiosensitivity in ESCC and significantly inhibits cell viability, which may contribute to an increased proportion of cells in the G0/G1 phase and cell apoptosis via suppression of the PI3K/Akt signalling pathway.

Analysis of the causes of failure after radical surgery in patients with PT3N0M0 thoracic esophageal squamous cell carcinoma and consideration of postoperative radiotherapy.

BACKGROUND: Five-year overall survival rate of TESCC after surgery is low (approximately 30% to 60%), so it is meaningful to discuss the significance of PORT. METHODS: We retrospectively collected the data of 227 patients with PT3N0M0 esophageal cancer (EC). The failure pattern after surgery was analyzed. Difference of adjuvant PORT in patients with PT3N0M0 TESCC and the appropriate population were explored based on the relevant studies. RESULTS: There were 58 cases with intrathoracic locoregional recurrence (LRR) after radical surgery and 27 cases with distant metastasis, including 10 cases of recurrence. The recurrence rate of mediastinal lymph nodes in the thoracic cavity was 50.0%. Univariate analysis revealed that compared with patients with middle and lower thoracic EC, the 3/5-year survival rate of patients with upper thoracic EC was significantly lower, accompanied with remarkably higher thoracic LRR. Compared with those with moderately- and well-differentiated TESCC, the 3/5-year survival rate of patients with poorly differentiated TESCC was significantly lower, whereas the distant metastasis rate was notably higher. Multivariate analysis revealed that different lesion locations and different pathologic differentiation were the independent prognostic factors. The lesion location and degree of differentiation were the independent influencing factors for thoracic LRR and distant metastasis, respectively. CONCLUSION: The intrathoracic LRR is the major failure pattern for patients with PT3N0M0 TESCC after conventional two-field lymphadenectomy. In addition, recurrence rate of PT3N0M0 TESCC was significantly higher in upper thoracic EC than in middle and lower thoracic EC. PORT is recommended to patients with PT3N0M0 upper TESCC.

Radiosensitization of esophageal carcinoma cells by the silencing of BMI-1.

Radiotherapy (RT) has been widely used to treat cancer patients, particularly esophageal cancer patients. B-cell-specific Moloney murine leukemia virus integration site-1 (BMI-1) plays an important role in promoting the growth of cancer cells after exposure to irradiation. The present study aimed to characterize the effects of BMI-1 on the proliferation and invasion of cancer cells, as well as the mechanism involved in the regulation of the growth of esophageal cancer ECA109 and TE13 cells. The expression levels of the BMI-1 gene and protein in esophageal cancer ECA109 and TE13 cells were determined by quantitative PCR and western blotting after transfection. Co-immunoprecipitation (Co-IP) assay was employed to detect the interaction of BMI-1 with r-H2AX and H2AK119ub. We used flow cytometry to analyze the cell cycle distribution and apoptosis of transfected cells after irradiation or not, and examined cellular growth and invasion in vitro by MTS and Transwell assays. The results revealed that shRNA targeting the BMI-1 gene and protein downregulated BMI-1 expression after transfection for 24 h. The proliferation and invasion of tumor cells in the BMI-1­shRNA group were suppressed after RT. In addition, the interaction of BMI-1, H2AK119ub and r-H2AX was increased after exposure to IR, followed by an increased apoptosis rate and decreased percentage of cells arrested at the G2/M phase after irradiation and silencing of BMI-1 by shRNA. Knockdown of BMI-1 expression decreased the phosphorylation of H2AX, upregulated p16, and induced the radiosensitivity of esophageal cancer ECA109 and TE13 cells in vitro and significantly inhibited the growth and invasion of tumor cells. The mechanisms were found to be abrogation of cell cycle arrest at the G2/M stage and promotion of apoptosis.

Analysis of failure patterns in patients with resectable esophageal squamous cell carcinoma receiving chemoradiotherapy.

PURPOSE: This study investigates the failure pattern after chemoradiotherapy of patients with resectable esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: We retrospectively analyzed 92 patients with T1-2, N0-1, and M0 ESCC. These patients were inoperable because of poor performance, comorbidities, poor tumor region, or refusal of operation. RESULTS: Among the 92 patients, 29 cases displayed simple locoregional recurrence, 12 cases displayed simple distant metastasis, and 6 cases displayed distant metastasis with locoregional recurrence. Univariate analysis shows that the incidence of recurrence in the middle thoracic region was significantly higher than other regions (χ2 = 14.415, P = 0.001). For the 18 patients with distant metastasis, incidence of distant metastasis in the lower thoracic region was significantly higher than the other regions (= 39.359, P < 0.001). Among 35 cases with regional recurrence, 7 cases reached complete remission (14.6%) and 28 cases reached partial remission (PR; 63.6%) (χ2= 23.435, P < 0.001). Multivariate analysis shows that the patient age, tumor node metastasis (TNM) stage, and short-term efficacy were independent factors for locoregional recurrence. Patient age, TNM stage, X-ray length of the lesions, and short-term efficacy were the independent factors for distant metastases. CONCLUSION: The incidence of locoregional recurrence and distant metastasis in patients with upper thoracic esophageal cancer was lower than those who had middle thoracic and lower thoracic esophageal cancer. The incidence of locoregional recurrence and distant metastasis in patients who achieved complete response after treatment was low.

Radiosensitization of esophageal carcinoma cells by knockdown of RNF2 expression.

Radiotherapy has been widely used for the treatment of cancer patients, especially for esophageal cancer patients. Ring finger protein 2 (RNF2) plays an important role in promoting the growth of cancer cells after exposure to irradiation. The present study aims to characterize the proliferative effects of RNF2 on cancer cells, and its mechanisms on the growth of esophageal cancer cells. We demonstrate that expression of RNF2 was markedly upregulated in esophageal cancer cell lines and surgically resected cancer specimens. In addition, RNF2 expression level is positively correlated with the presence of tumor size, lymph node metastases and negatively correlated with patient survival rates, suggesting that it plays an important role in the progression of esophageal cancer. Furthermore, the expression of RNF2 at both mRNA and protein levels in esophageal cancer ECA109 and TE13 cells was detected by real-time PCR and western blot assay after shRNA targeting RNF2. Co-immunoprecipitation (Co-IP) assay and western blot analysis were employed to detect the interaction between RNF2 and r-H2AX, H2AK119ub, and the expression of proteins associated with cell cycle and apoptosis, respectively. We used flow cytometry assay to analyze cell cycle and apoptosis of transfected cells, and further examined cellular growth in vitro and in vivo. shRNA targeting RNF2 gene and protein downregulated RNF2 expression after transfection for 24 h. The proliferation of tumor cells in RNF2-shRNA group was suppressed after radiotherapy. In addition, the interaction of RNF2, H2AK119ub, r-H2AX was increased after exposure to IR, followed by increasing apoptosis rates and inducing the arrest at G0/G1 phase after irradiation and shRNA targeting RNF2. Expression of the short-hairpin RNA is also correlated with the upregulation of p16 and Bax, and the downregulation of cyclin D2, cyclin-dependent kinase (CDK)-4, H2AX and Bcl-2. RNF2 gene knockdown induces radiosensitivity of esophageal cancer cells in vitro and significantly inhibits the growth of tumor cells. The mechanisms include inducing the cell cycle arrest at G0/G1 phase and promoting apoptosis.

TKTL1 promotes cell proliferation and metastasis in esophageal squamous cell carcinoma.

Transketolase-like-1 (TKTL1), which is a rate-limiting enzyme in the non-oxidative part of the pentose-phosphate pathway, has been demonstrated to promote carcinogenesis through enhanced aerobic glycolysis. Dysregulation of TKTL1 expression also leads to poor prognosis in patients with urothelial and colorectal cancer. However, the expression pattern and underlying cellular functions in human esophageal squamous cell carcinoma (ESCC) remain largely unexplored. In this study, we measured TKTL1 expression in ESCC cell lines and paraffin-embedded ESCC tumor tissues. Our results revealed that TKTL1 expression was upregulated in all of the four ESCC cell lines and in 61.25% (98/160) of ESCC specimens detected, while only 27.5% (11/40) in normal epithelium. Silencing of TKTL1 expression decreased cell proliferation through inhibiting the expression of MKI67 and cyclins including Ccna2, Ccnb1, Ccnd1 and Ccne1. Meanwhile, down-regulation of TKTL1 also associated with increased apoptotic ratio and altered protein expression of Bcl-2 family in ESCC cells. Furthermore, knockdown of TKTL1 significantly reduced the invasive potential of ESCC cells through up-regulation of anti-metastasis genes (MTSS1, TIMP2 and CTSK) and down-regulation of pr-metastasis genes (MMP2, MMP9, MMP10 and MMP13). Taken together, our results indicate that TKTL1 is associated with a more aggressive behavior in ESCC cells and suppresses its expression or enzyme activity might represents a potential target for developing novel therapies in human ESCCs.

Pathological characteristics of esophageal cancer.

The pathological characteristics of esophageal squamous cell carcinoma, which include regularly occurring multiple carcinogenic lesions (MLs), severe dysplasia (SD) and direct intramural infiltration (DI), were investigated using large pathological sections. A total of 52 esophageal cancer patients underwent surgical resection and were diagnosed with esophageal squamous cell carcinoma. Large sections of the surgical resection specimens were then made for pathological examination. The actual length of the carcinoma was calculated during surgery from the length determined microscopically. ML, SD and DI were identified during pathological examination of the large sections by microscope and were statistically analyzed. The lesion lengths obtained by the various inspection methods differed from each other. ML, SD and DI were identified in 15, 28 and 41 patients, respectively. Furthermore, a higher incidence of DI was observed in patients with lymphatic infiltration or those with a later stage of disease. ML, SD and DI were identified as characteristics of esophageal squamous cell carcinoma, and ML and DI were found to correlate with lymphatic infiltration.

[Low dose volume histogram analysis of the lungs in prediction of acute radiation pneumonitis in patients with esophageal cancer treated with three-dimensional conformal radiotherapy].

OBJECTIVE: To investigate the predictive value of low dose volume of the lung on acute radiation pneumonitis (RP) in patients with esophageal cancer treated with three-dimensional conformal radiotherapy (3D-CRT) only, and to analyze the relation of comprehensive parameters of the dose-volume V5, V20 and mean lung dose (MLD) with acute RP. METHODS: Two hundred and twenty-two patients with esophageal cancer treated by 3D-CRT have been followed up. The V5-V30 and MLD were calculated from the dose-volume histogram system. The clinical factors and treatment parameters were collected and analyzed. The acute RP was evaluated according to the RTOG toxicity criteria. RESULTS: The acute RP of grade 1, 2, 3 and 4 were observed in 68 (30.6%), 40 (18.0%), 8 (3.6%) and 1 (0.5%) cases, respectively. The univariate analysis of measurement data:The primary tumor length, radiation fields, MLD and lung V5-V30 had a significant relationship with the acute RP. The magnitude of the number of radiation fields, the volume of GTV, MLD and Lung V5-V30 had a significant difference in whether the ≥ grade 1 and ≥ grade 2 acute RP developed or not. Binary logistic regression analysis showed that MLD, Lung V5, V20 and V25 were independent risk factors of ≥ grade 1 acute RP, and the radiation fields, MLD and Lung V5 were independent risk factors of ≥ grade 2 acute RP. The ≥ grade 1 and ≥ grade 2 acute RP were significantly decreased when MLD less than 14 Gy, V5 and V20 were less than 60% and 28%,respectively. When the V20 ≤ 28%, the acute RP was significantly decreased in V5 ≤ 60% group. When the MLD was ≤ 14 Gy, the ≥ 1 grade acute RP was significantly decreased in the V5 ≤ 60% group. When the MLD was >14 Gy, the ≥ grade 2 acute RP was significantly decreased in the V5 ≤ 60% group. CONCLUSIONS: The low dose volume of the lung is effective in predicting radiation pneumonitis in patients with esophageal cancer treated with 3D-CRT only. The comprehensive parameters combined with V5, V20 and MLD may increase the effect in predicting radiation pneumonitis.

Radiosensitization of esophageal cancer cells ECA109 by knockdown of H2AX.

BACKGROUND: Genetic studies on mice have demonstrated that the key regulator of DNA damage in mammalian cells is the histone H2A variant, H2AX. We hypothesize that knockdown of H2AX will cause DNA damage pathway defects and may be able to increase the sensitivity to radiotherapy. METHODS: The formation of foci and the interaction of several important proteins in esophageal cancer ECA109, triggered by irradiation, were detected by immunofluorescence staining and Co-immunoprecipitation (Co-IP) assay before and after H2AX silencing. Clone formation assay was employed to detect cell radiosensitivity and cloning formation ability also before and after H2AX silencing. Cell cycle distribution and apoptosis were detected by flow cytometry. We constructed a nude mice esophageal cancer model and detected the above contents in vivo. RESULTS: H2AX and several proteins could form foci in nuclear triggered by irradiation and establish a relationship in vitro. The foci reduced after H2AX silencing. H2AX silencing could lead to radiosensitization via a colony-forming test. The apoptosis rate increased and the cell cycle was blocked in G2-M stage after H2AX silencing in vivo. The tumor volume was decreased in the H2AX silenced group after irradiation, while the tumor only slowed down the growth rate in the control groups. CONCLUSIONS: Knockdown of H2AX induced radiosensitization of esophageal cancer ECA109 cells both in vitro and in vivo. The mechanisms include defective cell cycle checkpoints and abolishment of foci formation for several important mediator and effector proteins in the DNA damage response to irradiation (IR).

[Relationship of gross tumor volume with lymph node metastasis and prognosis of esophageal carcinoma].

OBJECTIVE: To explore whether there is a relationship between gross tumor volume (GTV) and pathologic lymph node metastasis or prognosis of esophageal carcinoma, and to provide a new prognosis reference for esophageal carcinoma (EC). METHODS: Six hundred and seven patients received radical resection of thoracic esophageal carcinoma from May 2002 to June 2006 in our hospital, and their pre-operative CT images were transmitted to the three-dimensional conformal radiotherapy planning system by the network in digital format. Esophageal GTV targets were outlined and their GTV volumes were calculated. To analyze whether there is a relationship between GTV volume and pathologic lymph node metastasis or prognosis. RESULTS: In the 607 cases of esophageal carcinoma, the GTV volume was (22.5 ± 16.8) cm(3) in 374 stage N0 EC patients, significantly different from that of (30.4 ± 20.1) cm(3) in 233 stage N1 EC cases (P < 0.001). There is a significant difference between the GTV volumes of the groups with and without lymph node metastasis (P < 0.05). There was a significant difference of the GTV volumes of EC patients with one lymph node metastasis and those with ≥ 4 lymph node metastasis (P < 0.05). There was a positive correlation between GTV volume and the number of lymph node metastasis (r = 0.230, P < 0.001). The 1-, 3-, 5-year survival rates since the surgery date were 83.8%, 53.5%, and 36.4%, respectively. There was a significant difference between the survival rates of stage N0 (48.5%) and stage N1 patients (18.2%, P < 0.001), and there was a significant difference between the survival rats of patients with 0, 1 and ≥ 2 lymph node metastasis (P < 0.01). Cox regression model analysis showed that GTV volume, number of lymph node metastasis, pathological type, and lesion site were independent prognostic factors (all P < 0.05). CONCLUSION: The GTV volume of esophageal carcinoma is positively correlated with the number of pathologic lymph node metastasis, and it is an independent prognostic factor for this cancer.