Local production of the chemokines CCL5 and CXCL10 attracts CD8+ T lymphocytes into esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is a very common malignant tumor with poor prognosis in China. Chemokines secreted by tumors are pivotal for the accumulation of CD8⁺ T lymphocytes within malignant lesions in several types of cancers, but the exact mechanism underlying CD8⁺ T lymphocyte homing is still unknown in ESCC. In this study, we revealed that, compared with marginal tissues, the expression of both chemokine (C-C motif) ligand 5 (CCL5) and (C-X-C motif) ligand 10 (CXCL10) was upregulated in ESCC tissues. CCL5 expression was positively associated with the overall survival of patients. Meanwhile, RT-PCR data showed that the expression of CCL5 and CXCL10 was positively correlated with the local expressions of the CD8⁺ T lymphocyte markers (CD8 and Granzyme B) in tumor tissues. Correspondingly, CD8⁺ T lymphocytes were more frequently CCR5- and CXCR3-positive in tumor than in peripheral blood. Transwell analysis showed both CCL5 and CXCL10 were important for the chemotactic movement of CD8⁺ T lymphocytes. Our data indicate that CCL5 and CXCL10 serve as the key chemokines to recruit CD8⁺ T lymphocytes into ESCC tissue and may play a role in patient survival.     

Downregulation of HLA Class I molecules in the tumour is associated with a poor prognosis in patients with oesophageal squamous cell carcinoma

As antigenic peptides in the context of human leukocyte antigen (HLA) class I molecules are recognised by cytotoxic T lymphocytes (CTL), the downregulation of HLA class I molecules is one of the reasons why tumour cells can evade CTL-mediated anti-tumour immunity. In this study, we investigated HLA class I expression in oesophageal squamous cell carcinoma (ESCC) (n=70) and in their metastatic lesions (lymph nodes (n=40) and liver (n=3)), by immunohistochemistry with anti-HLA class I monoclonal antibody (EMR8-5). As a result, the downregulation of HLA class I expression in primary lesions of ESCC was observed in 43%, and that in metastatic lymph nodes was noted in 90%. Furthermore, patients with preserved HLA class I expression in primary tumours showed a better survival in comparison to those with downregulated HLA class I molecules (P<0.01). Furthermore, multivariate analysis using Cox's proportional hazards model revealed that the downregulated expression of HLA class I in primary lesions was an independent, unfavourable prognostic factor (P<0.01). In conclusion, the downregulation of HLA class I expression frequently occurred in primary tumour and, to a greater extent, in metastatic lesions of patients with ESCC and was associated with patient survival.     

Support vector machine-based nomogram predicts postoperative distant metastasis for patients with oesophageal squamous cell carcinoma

Background:

We aim to develop effective models for predicting postoperative distant metastasis for oesophageal squamous cell carcinoma (OSCC) for the purpose of guiding tailored therapy.

Methods:

We used data from two centres to establish training (n=319) and validation (n=164) cohorts. All patients underwent curative surgical treatment. The clinicopathological features and 23 immunomarkers detected by immunohistochemistry were involved for variable selection. We constructed eight support vector machine (SVM)-based nomograms (SVM1–SVM4 and SVM1''–SVM4''). The nomogram constructed with the training cohort was tested further with the validation cohort.

Results:

The outcome of the SVM1 model in predicting postoperative distant metastasis was as follows: sensitivity, 44.7% specificity, 90.9% positive predictive value, 81.0% negative predictive value, 65.6% and overall accuracy, 69.5%. The corresponding outcome of the SVM2 model was as follows: 44.7%, 92.1%, 82.9%, 65.9%, and 70.1%, respectively. The corresponding outcome of the SVM3 model was as follows: 55.3%, 93.2%, 87.5%, 70.7%, and 75.6%, respectively. The SVM4 model was the most effective nomogram in prediction, and the corresponding outcome was as follows: 56.6%, 97.7%, 95.6%, 72.3%, and 78.7%, respectively.Similar results were observed in SVM1'', SVM2'', SVM3'', and SVM4'', respectively.

Conclusion:

The SVM-based models integrating clinicopathological features and molecular markers as variables are helpful in selecting the patients of OSCC with high risk of postoperative distant metastasis.     

Expression of carbonic anhydrase 9, a potential intrinsic marker of hypoxia, is associated with poor prognosis in oesophageal squamous cell carcinoma

Carbonic anhydrase 9 (CA9) is a protein to be upregulated under exposure to hypoxic conditions. Hypoxic conditions are known to be associated with resistance to chemotherapy and radiotherapy, and with poor cancer prognosis. We examined CA9 expression in surgical specimens from oesophageal squamous cell carcinoma (ESCC) patients (n=127) using immunohistochemistry and real-time RT-PCR. We also examined CA9 expression and cell proliferation in ESCC cell lines (TE-2, TE-8 and TE-15) and an immortalised human oesophageal cell line (CHEK-1) using real-time RT-PCR, Western blotting, ELISA and MTT assay. Immunohistochemistry, high expression of CA9 was found in 63 of the 127 primary tumour specimens and was correlated with poor outcome (P=0.0003) and more aggressive/less favourable clinicopathological parameters (tumour size (P=0.0235), tumour depth (P<0.0001), regional lymph node metastasis (P=0.0031), distant lymph node metastasis (P=0.0077), stage (P<0.0001) and blood vessel invasion (P=0.006)). In vitro, CA9 expression in cultured cells and culture medium was also induced by hypoxia (P<0.01). CA9 is correlated with poor prognosis and malignant phenotype in patients with ESCC, and was upregulated by hypoxia. It is suggested that control of CA9 expression might improve the effectiveness of chemotherapy and radiotherapy in ESCC.     

PD-1、CTLA-4、BLTA在食管鳞状细胞癌患者外周血中的表达及临床意义

目的 检测食管鳞癌患者外周血中抑制性协同刺激因子受体PD-1、CTLA-4、BLTA表达情况,并分析其临床意义。方法 选取2016年6月—2017年4月河北医科大学第四医院胸外科90例食管鳞状细胞癌患者(其中50例患者行手术治疗)和40例健康对照者为研究对象,收集其外周血液标本,采用酶联免疫吸附方法检测血清中可溶性PD-1(sPD-1)、可溶性CTLA-4(sCTLA-4)及可溶性BLTA(sBLTA)的表达水平。结果 食管鳞癌组血清中sPD-1、sCTLA-4及sBLTA水平均明显高于对照组(P＜0.05);食管鳞癌组手术前后血清中sPD-1、sCTLA-4及sBLTA水平均无统计学差异(P＞0.05)。sPD-1和sBLTA的表达水平与临床病理特征无相关性,sCTLA-4的表达水平与TNM分期相关(P＜0.05),与T分期、N分期、肿瘤体积大小、肿瘤部位、组织分化程度、性别、年龄无相关性;血清中sPD-1、sCTLA-4及sBLTA两两间无相关性(P＞0.05)。结论 食管鳞癌患者血清中sCTLA-4较正常人表达升高,且sCTLA-4的表达水平与TNM分期相关,说明血清中sCTLA-4表达水平与病情发展变化有一定相关性。     

Prevalence of human papillomavirus among oesophageal squamous cell carcinoma cases: systematic review and meta-analysis

Background:

Oncogenic human papillomavirus (HPV) has been hypothesised as a risk factor for oesophageal squamous cell carcinoma (OSCC), but aetiological research has been limited by the varying methodology used for establishing HPV prevalence. The aims of this systematic review and meta-analysis were to estimate the prevalence of HPV DNA detected in OSCC tumours and the influence of study characteristics.

Methods:

Study-level estimates of overall and type-specific HPV prevalence were meta-analysed to obtain random-effects summary estimates.

Results:

This analysis included 124 studies with a total of 13 832 OSCC cases. The average HPV prevalence (95% confidence interval) among OSCC cases was 0.277 (0.234, 0.320) by polymerase chain reaction; 0.243 (0.159, 0.326) by in situ hybridisation; 0.304 (0.185, 0.423) by immunohistochemistry; 0.322 (0.154, 0.490) by L1 serology; and 0.176 (0.061, 0.292) by Southern/slot/dot blot. The highest HPV prevalence was found in Africa and Asia, notably among Chinese studies from provinces with high OSCC incidence rates.

Conclusions:

Future research should focus on quantifying HPV in OSCC cases using strict quality control measures, as well as determining the association between HPV and OSCC incidence by conducting large, population-based case–control studies. Such studies will provide a richer understanding of the role of HPV in OSCC aetiology.     

The nm23-H1 gene as a predictor of sensitivity to chemotherapeutic agents in oesophageal squamous cell carcinoma.

Recently, nm23-H1, an anti-metastasis gene, has been reported to correlate with sensitivity to chemotherapeutic agents including cisplatin in human breast and ovarian carcinoma cells. The aim of this study was to evaluate a role for nm23-H1 in responsiveness to cisplatin-based chemotherapy in patients with oesophageal squamous cell carcinoma (OSCC). The expression of nm23-H1 protein was examined immunohistochemically in 32 eligible patients with OSCC who underwent adjuvant chemotherapy with cisplatin, etoposide, and 5-fluorouracil after tumour resection. Fifteen (46.9%) of 32 patients were positive for nm23-H1 staining and 17 (53.1%) were negative. Both disease-free survival and overall survival rates of nm23-H1-negative patients were significantly shorter than in nm23-H1-positive patients (P < 0.01 for both). There was no significant difference in clinicopathologic characteristics between nm23-H1-positive and nm23-H1-negative groups. Multivariate analysis also showed that nm23-H1 expression was the most significant factor for overall survival of OSCC patients included in this study (P = 0.0007). To further study the role of nm23-H1, a human OSCC cell line (YES-2) was transfected with a plasmid containing a fragment of the nm23-H1 cDNA in an antisense orientation. Reduced expression of nm23-H1 protein in the antisense-transfected (AS) clones was found by Western blot analysis as compared to wild-type YES-2 and YES-2/Neo (clone transfected with the neomycin resistance gene alone). MTT (3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide) assay showed that reduced expression of the nm23-H1 protein in AS clones was consistent with the degree of increased resistance to cisplatin but not etoposide or 5-fluorouracil. These data support the conclusion that reduced expression of nm23-H1 may be associated with resistance to cisplatin, suggesting the value of nm23-H1 expression as a prognostic marker for OSCC patients who are to undergo cisplatin-based chemotherapy.     

Reduced expression of Axin correlates with tumour progression of oesophageal squamous cell carcinoma

Axin is a negative regulator of the Wnt signalling pathway, and genetic alterations of AXIN1 have been suggested to be an important factor of carcinogenesis in some tumours. The objective of this study was to clarify the clinicopathologic and prognostic significance of Axin in oesophageal squamous cell carcinoma (SCC). Immunohistochemical staining for Axin was performed on surgical specimens obtained from 81 patients with oesophageal SCC. Western and Northern blottings were performed on proteins and RNA from oesophageal SCC cell lines. Then polymerase chain reaction-single-strand conformational analysis (PCR-SSCP) was performed on DNA from oesophageal SCC patients and cell lines. Axin expression was found to be correlated inversely with depth of invasion, lymph node metastasis, and lymphatic invasion. Although univariate analysis showed Axin to be a negative predictor, multivariate analysis showed that it was not an independent prognostic marker. In all but one of the seven cell lines examined, the levels of protein expression were equivalent to RNA expression. PCR-SSCP showed that five patients and three cell lines had polymorphisms in exon 4 or 5 of the AXIN1 gene, but none of the 81 patients with oesophageal SCC had mutations. Our findings suggest that reduced expression of Axin is correlated with tumour progression of oesophageal SCC. However, additional studies will be necessary to elucidate the mechanism responsible for loss of Axin expression in tumour cells.     

Dietary patterns and oesophageal squamous cell carcinoma: a systematic review and meta-analysis

Background/Objective:

Dietary patterns, which represent a complex integration of food and nutrients, have been used to explore the association between dietary factors and the risk of oesophageal cancer. However, the association remains unclear. This systematic review was performed to evaluate the relationship between dietary patterns and oesophageal squamous cell carcinoma (ESCC) by pooling available data from existing studies.

Methods:

Pertinent articles published up to the end of 2013 were systematically searched and retrieved. The most common dietary patterns with high loadings of foods/nutrients were selected. Adjusted odds ratios (ORs) were derived by comparing the highest with the lowest categories of dietary pattern scores and by using a random-effect model. Heterogeneity was tested using I² statistic.

Results:

From nine available case–control studies, in which smoking and other confounding factors were considered, three most common dietary patterns were selected: western pattern, healthy pattern, and drinker/alcohol pattern. Healthy pattern was significantly associated with a decreased risk of ESCC (OR=0.36, 95% confidence interval (CI): 0.23, 0.49); drinker/alcohol pattern was related to a significantly increased risk (OR=2.34, 95% CI: 1.22, 3.45), while no significant association with western pattern was observed (OR=1.29, 95% CI: 0.83, 1.75).

Conclusions:

Based on available studies, though limited in number, this meta-analysis suggests that some dietary patterns may be associated with the risk of ESCC.     

食管鳞癌患者血清TK1与SCCA表达及其预后价值

目的食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)是我国食管癌的主要病理类型,患者预后较差,5年生存率低,影响ESCC患者对治疗的应答以及预后的相关因素仍不清楚。本研究旨在探讨血清细胞质胸苷激酶1(thymidine kinase 1,TK1)与鳞状细胞癌抗原(squamous cell carcinoma antigen,SCCA)在ESCC同步放化疗前的表达及其预后预测价值。方法选取2013-1-30-2016-10-19淮北矿工总医院收治的96例ESCC患者和100名门诊健康体检者为研究对象,分为ESCC组和对照组。采用酶联免疫吸附实验法(enzyme-linked immunosorbent assay,ELISA)检测入组对象的血清TK1、SCCA的表达,比较2组血清TK1和SCCA表达差异,分析血清TK1和SCCA的表达与ESCC患者临床病理特征的相关性。采用ROC曲线评价血清TK1、SCCA对诊断ESCC价值,分析其与同步放化疗后24个月预后的关系。结果 ESCC组血清TK1表达高于对照组,差异有统计学意义,t=24.665,P<0.05。血清TK1表达量与年龄(t=1.783)、性别(t=0.467)、肿瘤位置(t=1.263)无统计学意义的关联,P>0.05。血清TK1水平病变长度≥5cm高于病变长度<5cm患者,t=3.481,P<0.05;Ⅲ期+Ⅳ期高于Ⅰ+Ⅱ患者,F=6.509,P<0.05;存在淋巴结转移高于无淋巴结转移患者,t=3.613,P<0.05。ESCC组血清SCCA表达高于对照组,差异有统计学意义,t=34.852,P<0.05。血清SCCA表达量与年龄(t=1.473)、性别(t=1.338)、肿瘤位置(t=1.280)无统计学意义的关联,P>0.05。血清SCCA水平病变长度≥5cm高于病变长度<5cm患者,t=3.229,P<0.05;Ⅲ期+Ⅳ期高于Ⅰ+Ⅱ期患者,F=8.513,P<0.05;存在淋巴结转移高于无淋巴结转移患者,t=3.029,P<0.05。血清TK1诊断的临界值为1.97,其对应的灵敏度为75.0%,特异度为85.4%,ROC曲线下面积为0.808,95%CI为0.750~0.896;中位无进展生存期(progression-free survival,PFS)TK1≥1.97pmol/L为9.3个月,<1.97pmol/L为13.6个月,P=0.004;SCCA诊断的临界值为3.54,其对应的灵敏度为83.3%,特异度为66.7%,ROC曲线下面积为0.798,95%CI为0.728~0.869。SCCA≥3.54ng/mL PFS为9.1个月,SCCA<3.54ng/mL为13.6个月,P=0.001。血清TK1、SCCA与患者PFS差异有统计学意义,P<0.05。Cox多因素回归分析显示,临床分期(HR=5.836,95%CI为1.585~21.486)、血清TK1(HR=1.024,95%CI为1.000~1.049)及SCCA(HR=1.879,95%CI为1.183~2.985)水平为影响ESCC患者PFS的独立危险因素,差异有统计学意义,P<0.05,临床分期(HR=1.060,95%CI为1.021~1.100)为影响ESCC患者总生存时间(overall survival,OS)的独立危险因素,差异有统计学意义,P<0.05。结论血清TK1与SCCA在ESCC中的表达受分期、肿瘤病变长度和淋巴结转移影响,二者联合检测对于ESCC具有一定诊断价值,血清TK1与SCCA高表达可以作为评估ESCC患者预后的潜在指标。     

细胞角蛋白20在宫颈鳞癌中的表达及意义

目的:检测宫颈鳞癌癌灶组织中细胞角蛋白20（CK20）的表达并探讨其临床意义。方法:采用免疫组化SP方法,检测43例宫颈鳞癌癌灶组织和30例正常宫颈组织中CK20的表达,并分析其表达与临床病理因素之间的关系。结果:CK20在宫颈鳞癌癌灶组织的阳性表达率(46.5%)明显高于对照组（P<0.01）。CK20的高表达与临床分期、淋巴结转移有相关性,与病理分化程度无相关性（P>0.05）。结论:检测CK20对宫颈鳞癌的临床分期及判断淋巴结转移等有一定参考价值。     

Human papillomavirus in high- and low-risk areas of oesophageal squamous cell carcinoma in China

To examine the potential roles of human papillomavirus (HPV) in oesophageal squamous cell carcinoma (ESCC) development, we examined the presence of HPV DNA in paraffin-embedded ESCC tissues collected from two areas with different ESCC incidence rates in China, that is, Gansu (n=26) and Shandong (n=33), using PCR with SPF10 primers, or PCR with GP5+/GP6+ primers combined with Southern blot hybridisation. HPV genotype was determined by the INNO-LiPA HPV genotyping kit. HPV DNA was detected in 17 cases (65%) in Gansu, where ESCC incidence is much higher than in Shandong, where HPV was positive in two samples (6%). HPV genotypes 16 and 18 were detected in 79 and 16% of HPV-positive samples, respectively. Real-time PCR analysis suggested the presence of integrated form of HPV DNA in all the HPV-16-positive samples, but its viral load was estimated to be only <1-2 copies cell(-1). We could not detect HPV 16/18 E6 protein expression by immunostaining in any of the HPV-16-positive samples. Neither p16(INK4a) nor p53 expression was related to HPV presence in ESCCs. Further studies seem warranted to examine the possible aetiological roles of HPV in ESCC.     

The contribution of smoking and smokeless tobacco to oesophageal squamous cell carcinoma risk in the African oesophageal cancer corridor: Results from the ESCCAPE multicentre case-control studies

Tobacco use is a well-established risk factor for oesophageal squamous cell carcinoma (ESCC) but the extent of its contribution to the disease burden in the African oesophageal cancer corridor has not been comprehensively elucidated, including by type of tobacco use. We investigated the contribution of tobacco use (smoking and smokeless) to ESCC in Tanzania, Malawi and Kenya. Hospital-based ESCC case-control studies were conducted in the three countries. Incident cases and controls were interviewed using a comprehensive questionnaire which included questions on tobacco smoking and smokeless tobacco use. Logistic regression models were used to estimate odds ratios (OR) and their 95% confidence intervals (CI) of ESCC associated with tobacco, adjusted for age, sex, alcohol use, religion, education and area of residence. One thousand two hundred seventy-nine cases and 1345 controls were recruited between August 5, 2013, and May 24, 2020. Ever-tobacco use was associated with increased ESCC risk in all countries: Tanzania (OR 3.09, 95%CI 1.83-5.23), and in Malawi (OR 2.45, 95%CI 1.80-3.33) and lesser in Kenya (OR 1.37, 95%CI 0.94-2.00). Exclusive smokeless tobacco use was positively associated with ESCC risk, in Tanzania, Malawi and Kenya combined (OR 1.92, 95%CI 1.26-2.92). ESCC risk increased with tobacco smoking intensity and duration of smoking. Tobacco use is an important risk factor of ESCC in Tanzania, Malawi and Kenya. Our study provides evidence that smoking and smokeless tobacco cessation are imperative in reducing ESCC risk.     

Serum microRNA expression profile: miR-1246 as a novel diagnostic and prognostic biomarker for oesophageal squamous cell carcinoma

Background:

Recent studies have demonstrated that microRNAs (miRNAs) are stably detectable in blood and can serve as useful biomarkers for cancer.

Methods:

We performed an miRNA array using serum samples obtained from oesophageal squamous cell carcinoma (ESCC) patients or healthy controls. MiR-1246 was the most markedly elevated in ESCC patients. Therefore, miR-1246 was selected as a candidate for further analysis. The serum miR-1246 level in 46 healthy controls and 101 ESCC patients was evaluated and compared among various clinicopathological characteristics. MiR-1246 expressions in tissue, exosomal, and cellular samples were also examined.

Results:

Serum miR-1246 alone yielded an receiver-operating characteristic curve area of 0.754, with 71.3% sensitivity and 73.9% specificity for distinguishing ESCC patients from healthy controls. Serum miR-1246 was significantly correlated with the TNM stage and showed to be the strongest independent risk factor for poor survival (HR, 4.032; P=0.017). Unlike the tendency shown in previous reports, miR-1246 was not upregulated in ESCC tissue samples. Furthermore, exosomal miR-1246 did not reflect the abundance in the cell of origin.

Conclusion:

These data support our contention that serum miR-1246 has strong potential as a novel diagnostic and prognostic biomarker in ESCC, and its releasing mechanism is selective and independent of tissue miRNA abundance.     

Aberrant activation of the mTOR pathway and anti-tumour effect of everolimus on oesophageal squamous cell carcinoma

Background:

The mammalian target of rapamycin (mTOR) protein is important for cellular growth and homeostasis. The presence and prognostic significance of inappropriate mTOR activation have been reported for several cancers. Mammalian target of rapamycin inhibitors, such as everolimus (RAD001), are in development and show promise as anti-cancer drugs; however, the therapeutic effect of everolimus on oesophageal squamous cell carcinoma (OSCC) remains unknown.

Methods:

Phosphorylation of mTOR (p-mTOR) was evaluated in 167 resected OSCC tumours and 5 OSCC cell lines. The effects of everolimus on the OSCC cell lines TE4 and TE11 in vitro and alone or in combination with cisplatin on tumour growth in vivo were evaluated.

Results:

Mammalian target of rapamycin phosphorylation was detected in 116 tumours (69.5%) and all the 5 OSCC cell lines. Everolimus suppressed p-mTOR downstream pathways, inhibited proliferation and invasion, and induced apoptosis in both TE4 and TE11 cells. In a mouse xenograft model established with TE4 and TE11 cells, everolimus alone or in combination with cisplatin inhibited tumour growth.

Conclusion:

The mTOR pathway was aberrantly activated in most OSCC tumours. Everolimus had a therapeutic effect both as a single agent and in combination with cisplatin. Everolimus could be a useful anti-cancer drug for patients with OSCC.     

食管鳞状细胞癌组织中CCL26的表达及其临床意义

背景与目的：趋化因子在癌症的发生、发展过程中起着重要的作用,但趋化因子CCL26在食管鳞状细胞癌组织中的表达功能报道甚少。本研究旨在探讨趋化因子CCL26在人食管鳞状细胞癌组织及正常食管黏膜组织中的表达水平,分析其与食管鳞状细胞癌相关临床病理指标之间的关系。方法：利用组织微阵列技术制作组织芯片,采用免疫组织化学ABC法分析197例食管鳞状细胞癌组织和相应癌旁正常食管黏膜组织中趋化因子CCL26的表达水平,并对食管鳞状细胞癌中趋化因子CCL26的表达与患者年龄、性别、临床分期、淋巴结转移及5年生存率等临床病理指标的相关性进行分析。结果：①趋化因子CCL26在食管鳞状细胞癌组织及癌旁正常组织中均有表达,在食管鳞状细胞癌组织中趋化因子CCL26的阳性表达率明显高于癌旁正常组,差异有统计学意义(61.8% vs 20.6%,P<0.05);②趋化因子CCL26的表达与淋巴结转移有关,而与患者的年龄、性别、组织学类型、肿瘤细胞分化程度和大体分型等无关(P>0.05);③趋化因子CCL26阳性表达的食管鳞状细胞癌患者5年生存率明显低于趋化因子CCL26阴性表达的食管鳞状细胞癌患者。结论：趋化因子CCL26高表达可能与食管鳞状细胞癌的发生、发展相关,导致5年生存率下降,检测趋化因子CCL26可能为食管鳞状细胞癌预后判断提供依据。