Detection of methylation status of Epstein-Barr virus DNA C promoter in the diagnosis of nasopharyngeal carcinoma.

The detection of Epstein-Barr virus (EBV) DNA load in nasopharyngeal (NP) brushing samples for diagnosis of nasopharyngeal carcinoma (NPC) has attracted great attention. Further improvements that eliminate the need for clinical settings will greatly extend its application. A total of 250 participants were recruited to obtain NP brushing samples. Brush sampling both with and without the guide of endoscopy was conducted in 38 NPC patients. EBV DNA load, EBV RNA transcript and EBV DNA C promoter methylation status were, respectively, evaluated. Typical latency II transcripts were observed in brushing samples from NPC patients but not controls. Unlike in tissues, multiple lytic gene transcripts were observed not only in NPC patients but also in controls. Apart from EBV RNA transcript, samples from NPC patients also showed higher levels of EBV DNA load and C promoter methylation degree than their controls. Qualitative analysis further showed that EBV DNA C promoter was methylated in all NPC patients but in only 18.4% of the control group. Combined analysis of EBV DNA methylated degree and EBV DNA load increased the sensitivity to 100% in the detection of NPC. Using qualitative methylated type as the criteria, up to 89.5% of samples collected via blind brushing showed consistent results with samples collected via endoscopy-guided brushing from NPC patients. Detection of the methylation status of EBV DNA C promoter in NP brushing samples shows great potential in diagnosing NPC and may provide an appealing alternative for the non-invasive detection and screening of NPC without the need for clinical settings.

Tumor Cell Content and RNA Integrity of Surgical Tissues from Different Types of Tumors and Its Correlation with Ex Vivo and In Vivo Ischemia.

BACKGROUND: Tissues from tumor patients are important resources for promoting cancer research, and therefore many biobanks have been established to collect tumor tissues; however, the quality of tumor tissues after surgical resection has not been well documented. METHODS: A total of 896 cases of tissues from 12 types of tumors were chosen for this study. First, histopathological examination was conducted to evaluate the tumor cell content; second, microchip electrophoresis was used to determine the RNA integrity number (RIN) in 466 cases of tissues with a tumor cell content ≥ 75%; and, finally, a correlation test was used to analyze the effect of ischemia on RNA integrity in 384 cases of tissues with a recorded ischemia time. RESULTS: Tumor tissues from 12 different organs had different tumor cell contents and RNA integrity. The liver had the highest percentage (69.7%) of tissue samples with a tumor cell content ≥ 75%, and the highest percentage (96%) of samples with an RIN ≥ 7. RNA integrity was not correlated with limited ex vivo ischemia time (5-60 min) in any of the 12 types of tumors. In contrast, a significant correlation with in vivo ischemia time was observed in several types of tumors. CONCLUSIONS: Not every sample of excised tumor tissue has a sufficient amount of tumor cells and enough RNA integrity. In vivo ischemia has a more significant influence on RNA integrity, and tumor tissues have different tolerances to pre-analytical variables. Those conducting translational research should pay attention to pre-analytical variables when collecting and utilizing tumor tissues.

ELK1 Promotes Epithelial-Mesenchymal Transition and the Progression of Lung Adenocarcinoma by Upregulating B7-H3.

In previous studies, we found that B7 homolog 3 (B7-H3) was highly expressed in lung adenocarcinoma (LUAD) and promoted epithelial-to-mesenchymal transition (EMT) of LUAD cells. However, the underlying molecular mechanism is unclear. This study is aimed at evaluating the role of Ets-like protein 1 (ELK1) as a transcriptional regulator of B7-H3 for mediating the development and progression of LUAD in vitro and in vivo. We confirmed that ELK1 is highly expressed in LUAD and is associated with poor patient prognosis. ELK1 was found to promote proliferation, invasion, migration, and EMT of LUAD cells through in vivo and in vitro experiments. In terms of mechanism, ELK1 binds to the B7-H3 promoter region and induces the upregulation of B7-H3 in LUAD. Our data suggest that ELK1 plays an important role in the development of LUAD and could be used as a prognostic marker and therapeutic target for LUAD.

Association between circulating CD39+CD8+ T cells pre-chemoradiotherapy and prognosis in patients with nasopharyngeal carcinoma.

BACKGROUND: The mortality rate among patients with nasopharyngeal carcinoma (NPC) has improved significantly with the advent of chemoradiotherapy strategies. However, distant metastasis remains problematic. Tumor-specific reactivity in cancer patients has been detected exclusively in CD39+ T cells, particularly in CD39+CD103+ T cells. Circulating cancer-specific T cells are important for protecting against metastasis. This study aimed to evaluate the predictive value of circulating CD39+CD8+ T cells for metastasis in patients with NPC. METHODS: We performed a cross-sectional, longitudinal study of 55 patients with newly diagnosed NPC of stage III-IVa. All patients were initially treated with standard combined chemoradiotherapy. Blood samples were obtained from 24 patients before and at 1 month and 6 months after treatment. T cell expression of CD39 and CD103, together with the markers of T cell exhaustion programmed death-1 (PD-1)/T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) and markers of cell differentiation CD27/CC-chemokine receptor 7/CD45RA, was examined by flow cytometry. The Wilcoxon rank-sum test analysis was used to analyze the differences between two groups. Kaplan-Meier analysis was used for analysis of progression-free survival (PFS). RESULTS: The expression of circulating CD39+CD8+ and CD39+CD103+ CD8+ T cells was significantly higher in patients without distant metastasis (CD39+CD8+: 6.52% [1.24%, 12.58%] vs. 2.41% [0.58%, 5.31%], Z=-2.073, P=0.038 and CD39+CD103+CD8+: 0.72% [0.26%, 2.05%] vs. 0.26% [0.12%, 0.64%], Z=-2.313, P = 0.021). Most CD39+ T cells did not express PD-1 or Tim-3. Patients with high expression of CD39+CD103+CD8+ T cells had better PFS than patients with low expression (log rank value = 4.854, P = 0.028). CD39+CD8+ T cells were significantly elevated at 1-month post-treatment (10.02% [0.98%, 17.42%] vs. 5.91% [0.61%, 10.23%], Z = -2.943, P = 0.003). The percentage of advanced differentiated CD8+ T cells also increased at 1-month post-treatment compared with pre-treatment (33.10% [21.60%, 43.05%] vs. 21.00% [11.65%, 43.00%], Z = -2.155, P = 0.031). There was a significant correlation between elevated CD39+CD8+ T cells and increased effector memory T cells (intermediate stage: r = 0.469, P = 0.031; advanced stage: r = 0.508, P = 0.019). CONCLUSIONS: CD39+CD8+ circulating T cells have preserved effector function, contributing to an improved prognosis and a reduced risk of metastasis among NPC patients. These cells may thus be a useful predictive marker for a better prognosis in patients with NPC.

The Chinese Society of Clinical Oncology (CSCO) clinical guidelines for the diagnosis and treatment of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor originating in the nasopharynx and has a high incidence in Southeast Asia and North Africa. To develop these comprehensive guidelines for the diagnosis and management of NPC, the Chinese Society of Clinical Oncology (CSCO) arranged a multi-disciplinary team comprising of experts from all sub-specialties of NPC to write, discuss, and revise the guidelines. Based on the findings of evidence-based medicine in China and abroad, domestic experts have iteratively developed these guidelines to provide proper management of NPC. Overall, the guidelines describe the screening, clinical and pathological diagnosis, staging and risk assessment, therapies, and follow-up of NPC, which aim to improve the management of NPC.

Enriched HLA-E and CD94/NKG2A Interaction Limits Antitumor CD8+ Tumor-Infiltrating T Lymphocyte Responses.

Immunotherapy treatments with anti-PD-1 boost recovery in less than 30% of treated cancer patients, indicating the complexity of the tumor microenvironment. Expression of HLA-E is linked to poor clinical outcomes in mice and human patients. However, the contributions to immune evasion of HLA-E, a ligand for the inhibitory CD94/NKG2A receptor, when expressed on tumors, compared with adjacent tissue and peripheral blood mononuclear cells, remains unclear. In this study, we report that epithelial-derived cancer cells, tumor macrophages, and CD141+ conventional dendritic cells (cDC) contributed to HLA-E enrichment in carcinomas. Different cancer types showed a similar pattern of enrichment. Enrichment correlated to NKG2A upregulation on CD8+ tumor-infiltrating T lymphocytes (TIL) but not on CD4+ TILs. CD94/NKG2A is exclusively expressed on PD-1high TILs while lacking intratumoral CD103 expression. We also found that the presence of CD94/NKG2A on human tumor-specific T cells impairs IL2 receptor-dependent proliferation, which affects IFNγ-mediated responses and antitumor cytotoxicity. These functionalities recover following antibody-mediated blockade in vitro and ex vivo Our results suggest that enriched HLA-E:CD94/NKG2A inhibitory interaction can impair survival of PD-1high TILs in the tumor microenvironment.

HLA-A*30:01 and HLA-A*33:03 are the protective alleles while HLA-A*01:01 serves as the susceptible gene for cervical cancer patients in Xinjiang, China.

OBJECTIVE: This study aims to investigate the distribution of HLA-A genes and identify alleles related to cervical cancer. MATERIALS AND METHODS: A total of 252 cervical cancer patients (56 Han ethnic and 196 Uyghur ethnic) and 213 controls (103 Han ethnic and 110 Uyghur ethnic) were recruited in this study. HLA-A alleles were examined by polymerase chain reaction with sequence-specific primers. The frequencies of different HLA-A alleles were compared between the two ethnic groups as well as patients and controls. The correlation of HLA-A frequencies with various clinical characteristics and short-term treatment efficacy was analyzed. RESULTS: (1) Significantly higher frequencies of HLA-A*03:01 and HLA-A*03:02 and lower frequencies of HLA-A*11:01, HLA-A*24:02, and HLA-A*30:01 were observed in the Uyghur control groups than in Han control groups (P ≤ 0.05). (2) The frequency of HLA-A*01:01 in patients was significantly higher than controls. In contrast, the frequencies of HLA-A*30:01 and HLA-A*33:03 were lower in patients (P ≤ 0.05). (3) The frequency of HLA-A*30:01 in Han patients was lower than Han control group (P ≤ 0.05). However, there was no statistically significant in the frequency of HLA-A between Uyghur patients and controls (P > 0.05). (4) There was no significant association between HLA-A alleles and HPV16 or squamous cell carcinoma antigen levels (P > 0.05). (5) The frequency of HLA-A*30:01 allele in complete response + partial response group was higher than stable disease + progressive disease group (P ≤ 0.05). CONCLUSIONS: People from two ethnic groups displayed different HLA-A gene distribution. HLA-A*30:01 and HLA-A*33:03 alleles are the protective factors to cervical cancer patients from Xinjiang while HLA-A*01:01 serves as the susceptible gene.

B7-H3 promotes metastasis, proliferation, and epithelial-mesenchymal transition in lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma is the most common pathological type of lung cancer. However, the mechanisms underlying its development are still poorly understood. B7-H3 was discovered as a new member of the B7 costimulatory family. METHODS: We detected the expression status of B7-H3 protein in lung adenocarcinoma tissues, and evaluated the relationship of B7-H3 expression and patients' prognosis. Then, we silenced its expression in A549 cells by transient siRNA transfection to ascertain the function of B7-H3 in lung adenocarcinoma cells. Western blotting was used to detect the expression of epithelial-mesenchymal transition (EMT) related proteins. RESULTS: We found that B7-H3 overexpressed in lung adenocarcinoma. It is correlated with lymph node metastasis, distant metastasis, and disease stage. The Cox regression analysis showed that B7-H3 might serve as an independent prognostic marker of lung adenocarcinoma. We also found that B7-H3 promoted proliferation, invasion and migration of A549 cells in vitro. B7-H3 also could promote EMT progression by regulating EMT-related molecules. CONCLUSION: B7-H3 is a potential target for the treatment of lung adenocarcinoma.

Human Leukocyte Antigen-A Allele Distribution in Nasopharyngeal Carcinoma Patients Showing Anti-Melanoma-Associated Antigen A or Synovial Sarcoma X-2 T Cell Response in Blood.

BACKGROUND: Development of innovative immunotherapy is imperative to improve the poor survival of the nasopharyngeal carcinoma (NPC) patients. In this study, we evaluated the T cell response to melanoma-associated antigen (MAGE)-A1, MAGE-A3, or synovial sarcoma X-2 (SSX-2) in the peripheral blood of treatment-naive NPC patients. The relationship of responses among the three proteins and the human leukocyte antigen (HLA)-A types were analyzed to provide evidence of designing novel therapy. METHODS: Sixty-one NPC patients admitted into the Tumor Hospital affiliated to the Xinjiang Medical University between March 2015 and July 2016 were enrolled. Mononuclear cells were isolated from the peripheral blood before any treatment. HLA-A alleles were typed with Sanger sequence-based typing technique. The T cell response to the MAGE-A1, MAGE-A3, or SSX-2 was evaluated with the Enzyme-Linked ImmunoSpot assay. Mann-Whitney U-test was used to compare the T cell responses from different groups. Spearman's rank correlation was used to analyze the relationship of T cell responses. RESULTS: HLA-A*02:01, A*02:07, and A*24:02 were the three most frequent alleles (18.9%, 12.3%, and 11.5%, respectively) among the 22 detected alleles. 31.1%, 19.7%, and 16.4% of the patients displayed MAGE-A1, MAGE-A3, or SSX-2-specific T cell response, respectively. The magnitudes of response to the three proteins were 32.5, 38.0, and 28.7 SFC/106 peripheral blood mononuclear cells, respectively. The T cell response against the three proteins correlated with each other to different extent. The percentage of A*02:01 and A*24:02 carriers were significantly higher in patients responding to any of the three proteins compared to the nonresponders. CONCLUSION: MAGE-A1, MAGE-A3, or SSX-2-specific T cell responses were detectable in a subgroup of NPC patients, the frequency and magnitude of which were correlated.

Associations of Human Leukocyte Antigen-DRB1 Alleles with Nasopharyngeal Carcinoma and Its Clinical Significance in Xinjiang Uyghur Autonomous Region of China.

BACKGROUND: Genetic susceptibility is one of the major etiological factors for nasopharyngeal carcinoma (NPC). Among the genetic predisposing factors, human leukocyte antigen (HLA) genes have been reported to be associated with NPC. This study aimed to investigate the associations of HLA-DRB1 alleles with NPC and the clinical significance of HLA-DRB1 alleles in NPC. METHODS: From January 2009 to December 2013, 140 NPC patients (118 Han patients and 22 Uyghur patients) and 158 healthy controls (81 Han individuals and 77 Uyghur individuals) from Xinjiang Province were genotyped for HLA-DRB1 using the polymerase chain reaction-sequence specific primer technique. Chi-square analysis was used when comparing allele frequencies between groups. The clinical outcomes were evaluated by Kaplan-Meier method and Cox regression model. RESULTS: Compared with healthy controls, the allele frequency of HLA-DRB1*0701 was increased in the Uyghur patients (P = 0.008) but not in the Han patients (P = 0.869). HLA-DRB1*0101 allele was presented with higher frequency in clinical Stage I + II group compared with clinical Stage III + IV group in the Han patients (P = 0.015) but not in the Uyghur patients (P = 1.000). Higher frequency of HLA-DRB1*1501 allele was observed in patients aged <45 years compared with those in patients aged ≥45 years (P = 0.002). Neither HLA-DRB1*0701 nor HLA-DRB1*0101 had a statistically significant association with 3-year survival. CONCLUSIONS: This study found HLA-DRB1*0701 in Uyghur population was associated with an increased risk of developing NPC. In Han population, we found HLA-DRB1*0101 was associated with protection from disease progression, and HLA-DRB1*1501 was associated with early age of onset. HLA-DRB1 could not be identified as a prognostic indicator for NPC in either Han or Uyghur patients.

HLA-A*02-B*46 haplotype: an adverse prognostic factor in Han patients with nasopharyngeal carcinoma.

Epidemiological studies have shown that human leukocyte antigen (HLA) allelic polymorphisms are closely correlated to susceptibility to nasopharyngeal carcinoma (NPC), and in a previous study, we showed that HLA-B*46 and HLA-A*02-B*46 haplotypes were strongly associated with NPC susceptibility. In this retrospective study, we investigated the phenotype of the HLA-A and HLA-B alleles and haplotypes and correlated these data to the clinical and pathological parameters of NPC to understand the role of HLA alleles and haplotypes in NPC prognosis. The cohort comprised 117 NPC patients from a Han population in Xinjiang. The local recurrence-free survival (LRFS), distant metastasis- free survival (DMFS), disease-free survival (DFS), and overall survival (OS) were analyzed. The 5-year DMFS of the HLA-A*02-B*46 haplotype carriers and non-carriers was 66.4% and 90.3%, respectively. In addition, age was found to be a prognostic factor for LRFS, DFS, and OS (P=0.032, 0.040, and 0.013, respectively). We found that the HLA-A*02-B*46 haplotype might be a prognostic marker in addition to the traditional TNM staging in patients with NPC.

Small hairpin RNA-mediated Krüppel-like factor 8 gene knockdown inhibits invasion of nasopharyngeal carcinoma.

The present study aimed to characterize the expression of Krüppel-like factor 8 (KLF8) in nasopahryngeal carcinoma (NPC) cell lines and determine its effect on tumor development and invasion following KLF8 gene knockdown by small hairpin RNA (shRNA). KLF8 expression in four NPC cell lines was examined by quantitative polymerase chain reaction (qPCR) and western blotting. KLF8 was knocked down in the SUNE1-5-8F/Sh-KLF8 cell line using shRNA, and the resulting stable cell line SUNE1-5-8F-sh-KLF8 was transplanted into nude mice in order to observe tumor formation and invasion. The results obtained from qPCR and western blotting revealed that, of the four NPC cell lines, KLF8 expression was lowest in the CNE-1 cells and highest in the SUNE1-5-8F cells. The tumor xenograft mouse models revealed that SUNE1-5-8F/Sh-KLF8 cells had a reduced ability for tumor formation and invasion compared with the control group. These results demonstrated for the first time that KLF8 modulates the formation and invasive ability of nasopharyngeal carcinoma.