A Novel Tool for the Risk Assessment and Personalized Chemo-/Immunotherapy Response Prediction of Adenocarcinoma and Squamous Cell Carcinoma Lung Cancer.

BACKGROUND: The prevalence and cancer-specific death rate of lung cancer (LC) have risen in recent decades. A universally applicable prognostic signature for both adenocarcinoma LC (LUAD) and squamous cell carcinoma LC (LUSC) is still lacking. METHODS: A total of 453 patients from The Cancer Genome Atlas (TCGA)-LUAD cohort and 452 patients from TCGA-LUSC cohort were enrolled, and a prognostic model was constructed using least absolute shrinkage and selection operator (LASSO) regression analysis based on the consensus prognostic genes in both cohorts. The newly defined pan-lung cancer risk count (PLCRC) of each patient was calculated via the summation formula. RESULTS: A total of 23 genes were selected for the calculation of the PLCRC. The PLCRC showed a moderate prognostic value in the entire (p < 0.001, HR: 2.75, AUC: 0.643), LUAD (p < 0.001, HR: 2.51, AUC: 0.636) and LUSC (p < 0.001, HR: 2.89, AUC: 0.656) cohorts. The PLCRC was an independent prognostic factor after adjusting the clinical features. The PLCRC was also effective in nine external validation cohorts and in patients with different clinical features. Activation of extracellular matrix pathways and infiltration of immunocytes promoted the tumorigenesis and development of both LUAD and LUSC. We generated a universally applicable prognostic signature, the PLCRC, which could dichotomize patients with significantly different clinical outcomes and guide the clinical treatment of LC patients. Chemotherapy is more suitable for patients with a low PLCRC, while anti-cytotoxic T-lymphocyte-associated protein 4 immunotherapy is more suitable for patients with a high PLCRC. CONCLUSION: We established and validated a newly defined prognostic signature, the PLCRC, for both LUAD and LUSC patients and provided clinical strategies for patients from different risk subgroups.

Polymorphisms of matrix metalloproteinases affect the susceptibility of esophageal cancer: Evidence from 20412 subjects, systematic review and updated meta-analysis.

BACKGROUND: The results of how matrix metalloproteinases (MMPs) polymorphisms affect esophageal cancer (EC) risk are not consistent, especially for MMP1,2,7 and 9. A meta-analysis focused on the impact of MMPs to digestive cancers, but not a precise analysis to EC, therefore, we designed the current study to make a clear understanding of the association between MMPs polymorphisms and EC. METHODS: Up to March 2020, we searched several databases to find case-control cohorts concerned about the risk of MMPs polymorphisms to EC risk. Odds ratios with 95% confidence intervals under five genetic models to generate the risk predicted value. The Q test and I2 statistics are used to estimate heterogeneity. Sensitivity analysis, Egger test, and Begg's funnel plot were employed to assess the results. In-silico analysis was performed to study the association between the polymorphism and mRNA expression. RESULTS: 19 case-control studies were enrolled, including 8371 EC patients and 12041 health controls. We observed the increased risk in BA vs. AA and BB + BA vs. AA models of MMP1-rs1799750 polymorphism. The protective effectiveness of EC was found in the MMP2 rs243865 polymorphism in B vs. A, BA vs. AA, and BB + BA vs. AA models. Meanwhile, the risk effect was also observed in the MMP7 rs11568818 polymorphism in most genetic models. In the furthermore bioinformatics analysis, we found that MMP1, MMP3, MMP7, MMP9, MMP12, MMP13 all increased in the tumor tissues, and the genetic alteration in the polymorphisms could impact the mRNA expression of the above MMPs. CONCLUSION: MMP1 rs1799705 and MMP7 rs1156818 polymorphisms will take part in the tumorigenesis of EC, while MMP2 rs243865 acts as a protective role to decrease the risk of EC.

MicroRNA-34a suppresses invasion and metastatic in esophageal squamous cell carcinoma by regulating CD44.

In human esophageal squamous cell carcinoma (ESCC), miR-34a was downregulated and could inhibit in vitro cell proliferation and migration. However, the underlying mechanism was not clear yet. The expression levels of mRNA and protein were detected by quantitative real-time PCR or western blotting, respectively. MiR-34a was knocked down or overexpressed and transfected into human ESCC cell lines ECA109 and TE-13, respectively. Cell migration and wound healing assays were used to examine the effect on migration and invasion in vitro. Animal models were used to examine the role of miR-34a in metastasis in vivo. Luciferase assay was carried out to validate the potential target of miR-34a. CD44 was upregulated and miR-34a was downregulated in ESCC tissues and cell lines. The linear regression analysis showed that CD44 expression was negatively correlated with the level of miR-34a. Luciferase assay showed that miR-34a interacted with a putative binding site in the CD44 3'UTR. MiR-34a was found to negatively regulate the expression of CD44. In vitro experiment showed that miR-34a overexpression inhibited ESCC cell invasion and migration; whereas miR-34a knockdown showed reversed results. MiR-34a also inhibited esophagus tumor growth and metastasis in vivo; whereas miR-34a knockdown showed reversed results. Finally, we found that CD44 knockdown reversed the effects of miR-34a knockdown on ESCC cell invasion and migration in vitro. MiRNA-34a suppresses invasion and metastatic in ESCC by regulating CD44.

[Study on safety and feasibility of minimally invasive esophagectomy without the use of postoperative nasogastric tube decompression].

OBJECTIVE: To investigate the safety and feasibility of forgoing postoperative nasogastric tube decompression in minimally invasive esophagectomy for patients with esophagus carcinoma. METHODS: Clinical data of 90 eligible patients who underwent elective minimally invasive esophagectomy in our department from January 2012 to May 2013 by the same surgical team were retrospectively analyzed. Among them, 45 patients did not receive the use of postoperative nasogastric tube decompression and 45 patients received nasogastric tube decompression after operation. The observation parameters included the time to first flatus, the time to intake of fluid diet, the duration of postoperative hospitalization, pharyngalgia, vomiting, and postoperative complications, as well as the need for placing or replacing the nasogastric tube. RESULTS: The incidence of pharyngalgia was significantly higher in nasogastric tube group (100% vs 44.4%, P<0.001). The time to intake of fluid diet [median 2 d(2-4 d) vs. median 9 d(7-20 d), P<0.001] and the time to first flatus [median 3 d(3-8 d) vs. median 6 d(3-9 d), P<0.001] were all significantly shorter in non-nasogastric tube group as compared to nasogastric tube group. Compared with the nasogastric tube group, the non-nasogastric tube group had shorter postoperative hospital stay (P<0.001). There were no significant differences in the incidence of postoperative complications and vomiting between two groups. CONCLUSION: Minimally invasive esophagectomy without the use of postoperative nasogastric tube decompression is safe and feasible, which can improve recovery and shorten postoperative hospital stay.

Use of a fast-track surgery protocol on patients undergoing minimally invasive oesophagectomy: preliminary results.

OBJECTIVES: The aim of this study was to evaluate the safety and effectiveness of a fast-track surgery (FTS) protocol on patients undergoing minimally invasive oesophagectomy. METHODS: We retrospectively analysed the clinical data of 80 eligible patients who underwent elective minimally invasive oesophagectomy in our department from January 2012 to April 2013 by the same surgical team. Two groups of these patients were compared. The control group comprised patients treated with traditional methods. Clinical parameters were compared. The study group was formed by patients treated with the fast-track concept, such as (i) a semi-liquid meal was administered up to 6 h before surgery and the patients were made to drink 200 ml of 10% glucose solution 3 h before surgery; (ii) no nasogastric tube, no abdominal drainage tube and no draining sinus in the neck; (iii) the chest tube and catheter were removed as early as possible; (iv) prevention of hypothermia therapy; (v) an attempt at bedside rehabilitation on postoperative day (POD) 2; and (vi) early postoperative enteral nutrition, restrictive intravenous fluids intraoperatively and postoperatively, and oral feeding initiated 48 h after surgery. RESULTS: There were no significant differences between the two groups with regard to age, sex, pathologic tumor-node-metastasis stage, tumour location, pathology, American Society of Anesthesiologists score, preoperative albumin level, 30-day readmission or complications (P >0.05). Compared with the conventional group, the FTS group had earlier first flatus [(3 (3-4) vs 6 (6-7) days], less fluid transfusion [2.1 (2.06-2.2) vs 2.8 (2.7-2.9) l] and shorter postoperative hospital stay [7 (6-9) days vs 12 (10-16.5) days] (P <0.05). There was no difference between the two groups with regard to vomiting, but patients in the conventional group suffered from/experienced pharyngitis considerably more than the FTS group (P <0.001). CONCLUSIONS: FTS on patients with oesophageal cancer receiving minimally invasive oesophagectomy is safe, feasible and efficient, and can accelerate postoperative rehabilitation. Compared with the conventional protocol, its advantages were limited to short-term follow-up.

Thoracoscopic purse string technique for minimally invasive Ivor Lewis esophagectomy.

OBJECTIVE: Thoracolaparoscopic esophagectomy with chest anastomosis (TLE-chest) is increasingly performed for middle and lower esophageal cancer; however, gastroesophageal anastomosis for this surgery remains both challenging and inefficient. To address this issue, we previously reported our MIE technique with Ivor-Lewis anastomosis. Here we present the video to introduce our TLE-chest operation procedures. METHODS: TLE-chest with a combined thoracoscopic and laparoscopic technique was performed by one group of surgeons. From October 2011 to September 2013, 80 esophageal cancer patients were treated with TLE-chest using this improved anastomotic technique. RESULTS: The surgery was successful for all patients, although the anastomosis in one patient required intraoperative manual repair. No patients required open conversion. In this video, dissociation of stomach, and dissection of lymph nodes, creation of gastric tube and staple line embedding, jejunostomy were carried out by laparoscopic surgery. Dissection of esophageal cancer and mediastinal lymph nodes were done through rib 3 or 4 by a 3-4 cm video-assisted right anterior minithoracotomy, then esophago-gastric anastomosis was performed in right thoracic cavity This video shows the R0 resection of T3N0M0 esophageal cancer. Totally, 36 lymph nodes were dissected, including 21 mediastinal lymph nodes and 15 abdominal lymph nodes. The patient recovered well and was discharged on day 8 after the surgery, with good short term outcomes. CONCLUSIONS: A safe, cost effective purse string stapled anastomotic technique has been presented for TLE-chest in our video. It is consistent with the oncology principles.

Minimally invasive esophagectomy-left neck anastomosis.

Minimally invasive esophagectomy (MIE) is supplementary to open surgery in the thoracic surgery. A 65-year-old male was identified with middle thoracic esophageal squamous cell carcinoma by gastroscopy. In preoperative examinations, neither obvious abnormality nor distant metastasis was noted, and he could tolerate the esophagectomy according to his heart and lung function tests. Chest computed tomography (CT) and endoscopic ultrasonography showed no visible swollen lymph node in the mediastinum. The cTNM classification was T2N0M0. Therefore, MIE was performed. The patient recovered well after the surgery.

[Effects of 5-Aza-2-deoxycytidine on DNA methylation of anti-oncogenes in non-small cell lung cancer cells].

OBJECTIVE: To observe the expression of SFRP1 gene methylation in non-small cell lung cancer (NSCLC), and study the effect of 5-Aza-2-deoxycytidine (5-Aza-CdR) on DNA methylation and expression of SFRP1, p16 and MGMT genes in the human lung cancer cell line SPC-A-1 cells. METHODS: SP immunohistochemistry and methylation-specific PCR were used to detect the SFRP1 methylation in 60 NSCLC cases, and 21 cases of benign lung diseases were used as control group. SPC-A-1 cells were cultured and treated with 5-Aza-CdR. The promoter methylation status of SFRP1, p16 and MGMT genes were detected by methylation-specific polymerase (MSP) chain reaction, and mRNAs were detected by real-time PCR. RESULTS: The positive rate of SFRP1 gene methylation in NSCLC was significantly higher than that in normal lung tissue (58.3% vs. 14.3%; χ(2) = 12.118, P = 0.001). SFRP1 gene methylation was closely correlated with lymph node metastasis and degree of differentiation in NSCLC (P < 0.05). SFRP1 protein expression was correlated with clinical stage, degree of differentiation and lymph node metastasis in NSCLC (P < 0.05). The positive expression of SFRP1 protein in 30 cases of NSCLC tissue containing SFRP1 gene methylation was significantly higher than that in non-methylated NSCLC (68.6% vs. 24.0%; χ(2) = 9.613, P = 0.002). SFRP1 gene methylation was closely correlated with SFRP1 gene protein expression in NSCLC (P < 0.05). Negative expression of SFRP1 protein was correlated with the differentiation, clinical stage, and lymph node metastasis in NSCLC (all P < 0.05). Without 5-Aza-CdR treatment, the expressions of methylation of SFRP1, p16 and MGMT genes and their mRNA were low. After 5-Aza-CdR treatment at different concentrations, their expressions were significantly elevated (all P < 0.05). CONCLUSIONS: SFRP1 gene methylation is closely associated with carcinogenesis and development of NSCLC. 5-Aza-CdR may reverse the methylation of SFRP1, p16 and MGMT genes, and facilitate the re-expression of the anti-oncogenes.

HIF-1α effects on angiogenic potential in human small cell lung carcinoma.

BACKGROUND: Hypoxia-inducible factor-1 alpha (HIF-1α) maybe an important regulatory factor for angiogenesis of small cell lung cancer (SCLC). Our study aimed to investigate the effect of HIF-1α on angiogenic potential of SCLC including two points: One is the effect of HIF-1α on the angiogenesis of SCLC in vivo. The other is the regulation of angiogenic genes by HIF-1α in vitro and in vivo. METHODS: In vivo we used an alternative method to study the effect of HIF-1a on angiogenic potential of SCLC by buliding NCI-H446 cell transplantation tumor on the chick embryo chorioallantoic membrane (CAM) surface. In vitro we used microarray to screen out the angiogenic genes regulated by HIF-1a and tested their expression level in CAM transplantation tumor by RT-PCR and Western-blot analysis. RESULTS: In vivo angiogenic response surrounding the SCLC transplantation tumors in chick embryo chorioallantoic membrane (CAM) was promoted after exogenous HIF-1α transduction (p < 0.05). In vitro the changes of angiogenic genes expression induced by HIF-1α in NCI-H446 cells were analyzed by cDNA microarray experiments. HIF-1α upregulated the expression of angiogenic genes VEGF-A, TNFAIP6, PDGFC, FN1, MMP28, MMP14 to 6.76-, 6.69-, 2.26-, 2.31-, 4.39-, 2.97- fold respectively and glycolytic genes GLUT1, GLUT2 to2.98-, 3.74- fold respectively. In addition, the expression of these angiogenic factors were also upregulated by HIF-1α in the transplantion tumors in CAM as RT-PCR and Western-blot analysis indicated. CONCLUSIONS: These results indicated that HIF-1α may enhance the angiogenic potential of SCLC by regulating some angiogenic genes such as VEGF-A, MMP28 etc. Therefore, HIF-1α may be a potential target for the gene targeted therapy of SCLC.

Downregulation of a mitochondria associated protein SLP-2 inhibits tumor cell motility, proliferation and enhances cell sensitivity to chemotherapeutic reagents.

Results from tissue microarray in this study and our previous reports revealed that stomatin-like protein 2 (SLP-2) is notably associated with tumorigenesis and metastasis. Many members of stomatin family are involved in tumor as mitochondrial component, and recent study has revealed that SLP-2 may also function in mitochondria. To further investigate the function of SLP-2, we used siRNA target SLP-2. Data showed that knock-down of SLP-2 potently inhibited cell motility, proliferation and slightly altered cell cycle without any significant change of apoptosis. Moreover, by combined application with different chemotherapeutic reagents, we observed the enhancement of cell chemosensitivity by SLP-2 depletion. We also confirmed that, SLP-2 localizes in mitochondria, affects mitochondrial membrane potential (MMP) and ATP production. We conclude that, SLP-2 is a mitochondrial protein and therefore, functions in energy process by MMP maintenance, and subsequently affecting cell motility, proliferation and chemosensitivity.

XIAP is highly expressed in esophageal cancer and its downregulation by RNAi sensitizes esophageal carcinoma cell lines to chemotherapeutics.

X-linked inhibitor of apoptosis protein (XIAP) is the most potent member of the IAP gene family in terms of its ability to inhibit caspases and suppress apoptosis. In this study, we investigated the expression of XIAP in esophageal cancer tissues and cell lines, and found the elevated expression of XIAP in esophageal cancer tissues compared with normal tissues. Then we used small interfering RNA (siRNA) to block XIAP expression while evaluating the effect of XIAP siRNA on cell apoptosis, and the combined effects with Paclitaxel, Cisplatin, Fluorouracil and Etoposide in XIAP high expression ESCC cell line KYSE150 and EC9706. The results showed that XIAP siRNA efficiently decreased XIAP expression and induced cell apoptosis. Treatment with XIAP siRNA in combination with Paclitaxel, Cisplatin, Fluorouracil and Etoposide enhanced chemosensitivity. These results suggest that XIAP might be helpful for diagnosis of ESCC and XIAP siRNA combined with Paclitaxel, Cisplatin, Fluorouracil and Etoposide may be a feasible strategy to enhance the effects of chemotherapy in patients with ESCC.

Functional studies of a novel oncogene TGM3 in human esophageal squamous cell carcinoma.

AIM: To investigate the role of transglutaminase 3 (TGM3) gene in human esophageal squamous cell carcinoma (ESCC), and analyze its relationship with clinicopathological parameters. METHODS: Gene expression of TGM3 in fresh esophageal cancer tissues and their corresponding normal mucosas was detected immunohistochemically (IHC) by means of tissue microarray (TMA). Its correlation with clinical characteristics was evaluated and analyzed by univariate analysis. All statistical analyses were performed by SPSS version 10.0. RESULTS: Expression rate of TGM3 was reduced to 81.8% in ESCC. Expression of TGM3 was significantly inversely correlated with histological grade of esophageal carcinoma (in grade I, II and III tumors, the reduced expression was 4/7, 57/71, and 20/21, respectively, P < 0.05), whereas it had no obvious correlations with lymph node metastasis and depth of invasion. CONCLUSION: Reduced expression of TGM3 may play an important role in esophageal carcinogenesis.

Stomatin-like protein 2 is overexpressed in cancer and involved in regulating cell growth and cell adhesion in human esophageal squamous cell carcinoma.

PURPOSE: Stomatin-like protein 2 (SLP-2) is a novel and unusual stomatin homologue of unknown functions. It has been implicated in interaction with erythrocyte cytoskeleton and presumably other integral membrane proteins, but not directly with the membrane bilayer. We show here the involvement of SLP-2 in human esophageal squamous cell carcinoma (ESCC), lung cancer, laryngeal cancer, and endometrial adenocarcinoma and the effects of SLP-2 on ESCC cells. EXPERIMENTAL DESIGN: Previous work of cDNA microarray in our laboratory revealed that SLP-2 was significantly up-regulated in ESCC. The expression of SLP-2 was further evaluated in human ESCC, lung cancer, laryngeal cancer, and endometrial adenocarcinoma by semiquantitative reverse transcription-PCR, Western blot, and immunohistochemistry. Mutation detection of SLP-2 exons was done by PCR and automated sequencing. Antisense SLP-2 eukaryotic expression plasmids were constructed and transfected into human ESCC cell line KYSE450. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, clonogenecity assay, flow cytometry assay, nude mice tumorigenetic assay, and cell attachment assay were done to investigate the roles of SLP-2 gene. RESULTS: All tumor types we tested showed overexpression of SLP-2 compared with their normal counterparts (P < or = 0.05). Moreover, immunohistochemistry analysis of mild dysplasia, severe dysplasia, and ESCC showed that overexpression of SLP-2 occurred in premalignant lesions. Mutation analysis indicated that no mutation was found in SLP-2 exons. KYSE450 cells transfected with antisense SLP-2 showed decreased cell growth, proliferation, tumorigenecity, and cell adhesion. CONCLUSIONS: SLP-2 was first identified as a novel cancer-related gene overexpressed in human ESCC, lung cancer, laryngeal cancer, and endometrial adenocarcinoma. Decreased cell growth, cell adhesion, and tumorigenesis in the antisense transfectants revealed that SLP-2 may be important in tumorigenesis.

Overexpression of Ets-like protein 1 in human esophageal squamous cell carcinoma.

AIM: To study the expression pattern of Ets-like protein 1 (Elk-1) in human esophageal squamous cell carcinoma (ESCC) and to analyze its relationship with clinicopathologic parameters. METHODS: The expression of Elk-1 in fresh esophageal cancer tissues and their corresponding normal mucosae was detected immunohistochemically (IHC) by means of tissue microarray (TMA). Its correlation with clinical characteristics was evaluated and analyzed by univariate analysis. All statistical analyses were performed by SPSS version 13.0. RESULTS: Expression level of transcription factor Elk-1 increased in 78.5% (84/107) ESCC tissues compared with their matched normal esophageal epithelium. However, the expression of Elk-1 did not show any obvious correlation with degree of differentiation of esophageal carcinoma (in well-differentiated, moderately-differentiated and poorly-differentiated tumors, the increased expression was 7/8, 60/74, and 19/25, respectively, P > 0.05). Moreover, no obvious correlation was found with lymph node metastasis and depth of invasion. CONCLUSION: Increased expression of transcription factor Elk-1 may play an important role in esophageal carcinogenesis.

5-Aza-2'-deoxycytidine induces retinoic acid receptor-beta(2) demethylation and growth inhibition in esophageal squamous carcinoma cells.

Esophageal squamous cell carcinoma (ESCC) is one of the most common tumors in human. Previous studies showed that multiple genetic and epigenetic alterations involved in carcinogenesis of esophagus, whereas the molecular mechanisms are poorly understood. So far, more and more ESCC-related genes have been found and retinoic acid receptor beta(2) (RARbeta(2)) is such a gene which was recognized as a putative tumor suppressor gene since reduced RARbeta(2) mRNA expression has been observed in several solid tumors, including ESCC. A growing evidence indicated that RARbeta(2) was required for the growth inhibitory effect of retinoic acid (RA). However, the molecular mechanism of its inactivation remained obscure in ESCC. The RARbeta2 methylation status was assessed by methylation-specific PCR (MSP) in 12 ESCC cell lines and compared with their mRNA and protein expression level. Bisulfite sequencing of RARbeta(2) promoter region was performed to confirm the MSP results. After 5-aza-2'-deoxycytidine (5-aza-dc) treatment the expression of RARbeta(2) was reversed in two RARbeta(2)-downregulated cell lines. Therefore, hypermethylation of the promoter regions of RARbeta2 gene is a major mechanism of transcriptional inactivation and might be involved in tumor development of esophagus in some ESCC cell lines suggesting that multiple mechanisms contribute to the loss of RARbeta(2) expression in ESCC cell lines. Furthermore, the methylation status of RARbeta(2) promoter region and its expression was analyzed in 51 ESCC tissue samples with their adjacent normal epithelia and two normal esophageal epithelia. The results showed that there was a statistically significant correlation between methylation status of RARbeta(2) and tumor grade; Moreover, a relationship between methylation status and decreased RARbeta(2) expression was found only in G(2) stage tumors. After 5-aza-dc treatment, RARbeta(2) restoration was accompanied by growth inhibition and this might be one of the mechanisms but not the only mechanism for the tumor cell growth inhibition by 5-aza-dc. This study may have clinical applications for ESCC therapy and prevention.

Allelic imbalance of chromosome 1q in esophageal squamous cell carcinomas from China: a novel region of allelic loss and significant association with differentiation.

Our previous work has shown that a number of genes locating on 1q21 were down-regulated in esophageal squamous cell carcinomas (ESCC) and they may involve in carcinogenesis. To determine whether chromosome 1q LOH occurs in ESCC, we analyzed LOH in 61 ESCCs using 18 microsatellite markers on chromosome 1q. Forty-six of 61 (75.4%) tumors presented LOH at one or more loci. A significant association was found between chromosome 1q LOH and histopathological grade. LOH on D1S3466 had a negative correlation with family history, whereas LOH on D1S2777 positively correlated with smoking. These results suggest this region harbor putative tumor suppressor gene(s) contributing to tumorigenesis and differentiation in ESCC.