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ObjectivesThis study concerns a new technique that aims to achieve precise interstitial brachytherapy of pelvic recurrent tumors under transvaginal ultrasound (US) guidance, enhance the conformity index of the brachytherapy (BT), and improve the curative effect of radiotherapy for gynecological oncology patients with pelvic relapse.MethodsA real-time transvaginal US-guided interstitial implant device was developed to assist in implant BT. Prior to implant brachytherapy, the size and location of the tumor in the pelvis and the interrelationship with adjacent organs were first assessed with intracavitary ultrasound. The transvaginal US-guided interstitial implant device was then placed on the endoluminal ultrasound probe, the probe was oriented intravaginally to determine a safe needle path, the implant needle was placed into the needle passage of the device, and the implant needle was inserted into the tumor tissue in the direction guided by the ultrasound puncture guide line. After the implant needle was placed in place, the cover of the transvaginal US-guided interstitial implant device was opened perpendicular to the ultrasound probe, and the needle was separated from the ultrasound probe smoothly, and then the cover was re-covered for subsequent implantation.ResultsIn this study, 56 patients who underwent real-time transvaginal ultrasound-guided implantation for gynecologic oncology were enrolled, and insertion of 736 implant needles was completed. Among them, 13 patients had recurrent pelvic tumors after cervical cancer surgery and 6 patients had recurrent pelvic tumors after endometrial cancer surgery. Thirty-two patients who underwent radical radiation therapy for cervical cancer did not have adequate regression of parametrial invaded tissue after completion of standard EBRT treatment; and 5 patients had recurrent tumors in the radiation field after previous standard course of pelvic radiotherapy. The accuracy of the implant therapy was improved. The radiotherapy dose for recurrent pelvic masses was successfully increased, and the cumulative dose of external irradiation combined with BT was augmented to 80–100 Gy. The use of a new device for transvaginal implant for recurrent masses located in the lateral wall of the pelvic cavity was successful.ConclusionThis intravascular US-guided interstitial implant device can realize interstitial implant with the shortest path under transvaginal US guidance. With convenient operation, high precision, and good security, the device not only improves the accuracy of implant therapy, but it also reduces the risks of anesthesia and organ injury, so it is suitable for widespread promotion and use.

Abstract Background DNMT3A mutations are frequently discovered in acute myeloid leukemia (AML), associated with poor outcome. Recently, a relapse case report of AML extramedullary disease has showed that AML cells harboring DNMT3A variation were detected in the cerebral spinal fluid. However, whether a causal relationship exists between DNMT3A mutation (D3Amut) and extramedullary infiltration (EMI) is unclear. Methods We took advantage of DNMT3A (R882C) mutation-carrying AML cell strain, that is, OCI-AML3, assessing its migration ability in vitro and in vivo. By RNA interfering technology and a xenograft mouse model, we evaluated the effect of DNMT3A mutation on cell mobility and explored the possible mechanism. Results OCI-AML3 displayed extraordinary migration ability in vitro and infiltrated into meninges of NOD/SCID mice after intravenous transfusion. We found that this leukemic migration or infiltration capacity was significantly compromised by the knockdown of DNMT3A mutant. Notably, TWIST1, a critical inducer of epithelial–mesenchymal transition, which underlies the metastasis of carcinomas, was highly expressed in association with R882 mutations. Abrogation of TWIST1 in DNMT3A mutated cells considerably weakened their mobility or infiltration. Conclusions Our results demonstrate that D3Amut in OCI-AML3 strain enhances leukemic aggressiveness by promoting EMI process, which is partially through upregulating TWIST1.

Abstract Currently, there is no strong evidence of the well-established biomarkers for immune checkpoint inhibitors (ICIs) in nasopharyngeal carcinoma (NPC). Here, we aimed to reveal the heterogeneity of tumour microenvironment (TME) through virtual microdissection of gene expression profiles. An immune-enriched subtype was identified in 38% (43/113) of patients, which was characterized by significant enrichment of immune cells or immune responses. The remaining patients were therefore classified as a non-Immune Subtype (non-IS), which exhibited highly proliferative features. Then we identified a tumour immune evasion state within the immune-enriched subtype (18/43, 42%), in which high expression of exclusion- and dysfunction-related signatures was observed. These subgroups were designated the Evaded and Active Immune Subtype (E-IS and A-IS), respectively. We further demonstrated that A-IS predicted favourable survival and improved ICI response as compared to E-IS and non-IS. In summary, this study introduces the novel immune subtypes and demonstrates their feasibility in tailoring immunotherapeutic strategies.

An amendment to this paper has been published and can be accessed via the original article.

DNA methylation is an important epigenetic change in carcinogenesis. However, the function and mechanism of DNA methylation dysregulation in nasopharyngeal carcinoma (NPC) is still largely unclear. Our previous genome-wide microarray data showed that NFAT1 is one of the most hypermethylated transcription factor genes in NPC tissues. Here, we found that NFAT1 hypermethylation contributes to its down-regulation in NPC. NFAT1 overexpression inhibited cell migration, invasion, and epithelial-mesenchymal transition in vitro and tumor metastasis in vivo. We further established that the tumor suppressor effect of NFAT1 is mediated by its inactivation of ITGA6 transcription. Our findings suggest the significance of activating NFAT1/ITGA6 signaling in aggressive NPC, defining a novel critical signaling mechanism that drives NPC invasion and metastasis and providing a novel target for future personalized therapy.

Abstract Background Increasing evidence support an important role for DNA methylation in nasopharyngeal carcinoma (NPC). Here, we explored the role of circadian clock gene Aryl Hydrocarbon Receptor Nuclear Translocator-Like (ARNTL) methylation in NPC. Methods We employed bisulfite pyrosequencing to determine the epigenetic change of ARNTL in NPC cell lines and tissues. ARNTL mRNA and protein expression in cell lines and tissues were detected by real-time PCR and western blotting. Then, we constructed cell lines overexpressing ARNTL and knocked down ARNTL to explore its function and effect on chemotherapy sensitivity of NPC cell lines to cisplatin in vitro and vivo. Finally, we investigated the potential molecular mechanism of ARNTL by gene set enrichment analysis (GSEA), dual Luciferase reporter assay and chromatin immunoprecipitation assay. Results ARNTL was hypermethylated, and its mRNA and protein were significantly down-regulated in NPC cell lines and tissues. When treated by 5-aza-2′-deoxycytidine, mRNA expression was up-regulated. Overexpression of ARNTL could suppress NPC cells proliferation in vitro and vivo while silencing of ARNTL using shRNA achieved opposite results. GSEA assay found that ARNTL was associated with cell cycle and ectopic ARNTL overexpression could induce G2-M phase arrest. Then, we identified and validated cyclin-dependent kinase 5 (CDK5) as the targeting gene of ARNTL by dual Luciferase reporter assay and chromatin immunoprecipitation assay. When transiently infected ARNTL-overexpression cells with PENTER-vector or PENTER-CDK5 plasmids, the later could reverse the suppressive effects of ARNTL on NPC cell proliferation. Moreover, ARNTL significantly enhanced sensitivity to cisplatin in NPC cells. Conclusions ARNTL suppresses NPC cell proliferation and enhances sensitivity to cisplatin by targeting CDK5. ARNTL may represent a novel therapeutic target for NPC.

Background Tumor-infiltrating lymphocytes have been reported as prognostic markers in tumors. We aimed to assess the prognostic value of total T cell (CD3+) density, cytotoxic T cell (CD8+) density and memory T cell (CD45RO+) density in patients with nasopharyngeal carcinoma (NPC).Methods The expression of CD3, CD8 and CD45RO was detected by immunohistochemistry in the training (n=221) and validation cohorts (n=115). The densities of these three markers were quantified by digital pathology both in the tumor and stroma. Then, we developed the immune score based on the density of these three markers and further analyzed its prognostic value.Results The high density of CD3+, CD8+ and CD45RO+ T cells both in the tumor and/or stroma were significantly associated with the decrease in mortality in the training cohort, respectively. High immune score predicted a prolonged overall survival (OS) (HR 0.34, 95% CI 0.18 to 0.64, p=0.001, disease-free survival (DFS) (HR 0.44, 95% CI 0.25 to 0.78, p=0.005) and distant metastasis-free survival (DMFS) (HR 0.43, 95% CI 0.21 to 0.87, p=0.018) in NPC patients. The findings were confirmed in the validation cohort. Multivariate analysis revealed that immune score remained an independent prognostic indicator for OS, DFS and DMFS. In addition, we established a nomogram with the integration of all independent variables to predict individual risk of death.Conclusions We established an immune score model, which provides a reliable estimate of the risk of death, disease progress and distant metastasis in NPC patients.

Abstract Background Increasing evidence recognizes that DNA methylation abnormalities play critical roles in cancer development. Our previous genome-wide methylation profile showed that tumor necrosis factor-alpha-induced protein 8 like 3 (TIPE3) was hypermethylated in nasopharyngeal carcinoma (NPC). However, the relationship between TIPE3 methylation and its mRNA expression, as well as its biological roles in NPC are unknown. Methods Bisulfite pyrosequencing and quantitative RT-PCR were performed to quantify the TIPE3 methylation and expression levels. Kaplan-Meier curves and Cox regression analysis were used to estimate the correlation between TIPE3 methylation levels and survival in two patient cohorts collected from two hospitals (n = 441). The MTT, colony formation, Transwell migration and invasion assays, and xenograft tumor growth and lung metastatic colonization models were used to identify the functions of TIPE3 on NPC cells. Results We found that TIPE3 CpG island (CGI) was hypermethylated and its mRNA levels were downregulated in many cancers, including NPC. TIPE3 downregulation was associated with its CGI hypermethylation. Furthermore, NPC patients with high TIPE3 CGI methylation levels had poorer clinical outcomes than those with low methylation levels. The TIPE3 CGI methylation level was an independent prognostic factor. Moreover, restoring TIPE3 expression significantly inhibited NPC cell proliferation, migration and invasion in vitro, and suppressed tumor growth and lung metastatic colonization in vivo, while silencing TIPE3 acted in an opposite way. Conclusions TIPE3 downregulation correlates with its CGI hypermethylation in several solid cancers. TIPE3 acts as a tumor suppressor in NPC, providing a further insight into NPC progression and representing a potential prognostic biomarker for NPC.

Abstract Background Epigenetic abnormalities play important roles in nasopharyngeal cancer (NPC), however, the epigenetic changes associated with abnormal cell proliferation remain unclear. Methods We detected epigenetic change of ZNF671 in NPC tissues and cell lines by bisulfite pyrosequencing. We evaluated zinc finger protein 671 (ZNF671) expression in NPC cell lines and clinical tissues using real-time PCR and western blotting. Then, we established NPC cell lines that stably overexpressed ZNF671 and knocked down ZNF671 expression to explore its function in NPC in vitro and in vivo. Additionally, we investigated the potential mechanism of ZNF671 by identifying the mitotic spindle and G2/M checkpoint pathways pathway downstream genes using gene set enrichment analysis, flow cytometry and western blotting. Results ZNF671 was hypermethylated in NPC tissues and cell lines. The mRNA and protein expression of ZNF671 was down-regulated in NPC tissues and cell lines and the mRNA expression could be upregulated after the demethylation agent 5-aza-2′-deoxycytidine treatment. Overexpression of ZNF671 suppressed NPC cell proliferation and colony formation in vitro; silencing ZNF671 using a siRNA had the opposite effects. Additionally, overexpression of ZNF671 reduced the tumorigenicity of NPC cells in xenograft model in vivo. The mechanism study determined that overexpressing ZNF671 induced S phase arrest in NPC cells by upregulating p21 and downregulating cyclin D1 and c-myc. Conclusions Epigenetic mediated zinc finger protein 671 downregulation promotes cell proliferation and enhances tumorigenicity by inhibiting cell cycle arrest in NPC, which may represent a novel potential therapeutic target.

The B7 family and tumor necrosis factor receptor (TNFR) superfamily play a vital role in the T-cell co-stimulatory and co-inhibitory pathways, regulating T-cell activation, tolerance, and exhaustion; therapeutic modulation of these pathways is translated into effective new cancer treatments. Better understanding of the immune molecular landscapes of the B7 and TNFR families would guide head and neck immuno-oncology clinical research. We performed comprehensive molecular profiling of 10 B7 and 6 TNFR family members in head and neck cancer. Over 20% of patients had B7 and TNFR gene alterations. B7 gene amplifications were relatively more common (3–11%) than TNFR gene amplifications (0–5%). Analysis of 496 sequenced samples revealed that all genes were upregulated: B7 and TNFR mRNA were upregulated in 158 cases (> 30%) and 83 cases (∼15%), respectively. B7-H1 (PD-L1) mRNA upregulation was the most common (∼10%). Promoter methylation analysis indicated an epigenetic basis for B7 and TNFR gene regulation (especially B7-H1, which was relatively strongly correlated with promoter methylation). B7-H1 expression was significantly associated with worse overall survival, and its expression was increased in cases with gene amplifications. Human papillomavirus (HPV) status correlated significantly with B7-H1 alterations at genetic level. Almost half (47.1%) of HPV-negative patients had deep or shallow B7-H1 deletion; >90% of HPV-positive patients had diploid, copy number gain, or amplification of B7-H1. This is the first study elucidating the immune molecular landscapes of the B7 and TNFR families in head and neck cancer, providing a potential novel rationale for clinical investigations.