Prognostic model on overall survival in elderly nasopharyngeal carcinoma patients: a recursive partitioning analysis identifying pre-treatment risk stratification.

BACKGROUND: We aimed to evaluate the optimal management for elderly patients with nasopharyngeal carcinoma (NPC) with intensity-modulated radiotherapy (IMRT). METHODS: A total of 283 elderly patients with NPC diagnosed from 2015 to 2019 were enrolled in the study. Overall survival (OS) was the primary endpoint. Univariate and multivariate Cox regression analyses were preformed to identify potential prognostic factors. The recursive partitioning analysis (RPA) was used for risk stratification. Kaplan-Meier survival curves were applied to evaluate the survival endpoints, and log-rank test was utilized to assess differences between groups. The prognostic index (PI) was constructed to further predict patients' prognosis displayed by nomogram model. The area under the receiver operating characteristic (ROC) curves (AUC) and the calibration curves were applied to assess the effectiveness of the model. RESULTS: Based on RPA-based risk stratification, we demonstrated that elderly NPC patients who were treated with IC followed by RT had similar OS as those with induction chemotherapy (IC) combined with concurrent chemoradiotherapy (CCRT) in the middle- (stage I-III and pre-treatment EBV > 1840 copies/ml) and high-risk groups (stage IVA). IMRT alone may be the optimal treatment option for the low-risk group (stage I-III with pre-treatment EBV ≤ 1840 copies/ml). We established an integrated PI which was indicted with stronger prognostic power than each of the factors alone for elderly NPC patients (The AUC of PI was 0.75, 0.80, and 0.82 for 1-, 3-, 5-year prediction of OS, respectively). CONCLUSION: We present a robust model for clinical stratification which could guide individual therapy for elderly NPC patients.

Impact of Radiotherapy Combined With Chemotherapy on Long-Term Outcomes of Patients With Recurrent Nasopharyngeal Carcinoma.

Background: It remains controversial whether the application of chemotherapy has an impact on recurrent nasopharyngeal carcinoma (rNPC) patients with salvage radiotherapy. Here, we aimed to evaluate treatment outcomes of rNPC patients and derive a prognostic model to assess the benefit of chemotherapy in patients with re-radiotherapy. Methods: This study was conducted as a retrospective study. In total, 340 rNPC patients treated with salvage intensity-modulated radiotherapy (IMRT) or radiochemotherapy (RCT) from October 2006 to September 2019 were included in this study. Overall survival (OS) was the primary outcome. Kaplan-Meier method was employed to detect the prognostic difference with Log-rank tests. The Cox regression analysis was performed to explore the potential prognostic factors while the multivariate Cox analysis was used to identify candidate variables for the prognostic model of OS. Results: The 5-year actuarial rates of OS, progression-free survival, loco-regional progression-free survival, and distant metastases-free survival did not show significant difference between the IMRT and RCT groups (P > .05). Age at recurrence and rT category were found to be the independent prognostic factors for OS. We found that rNPC patients suffered poor OS in the high-risk group (patients with higher age at recurrence and advanced rT category) (high-risk vs low-risk, HR = 1.87, 95% CI: 1.36-2.57, P < .001). Salvage RT alone may be superior to RCT for patients in the low-risk group (RCT group vs RT group, HR = 1.89, 95% CI: 1.11-3.20, P = .038). Conclusion: Salvage RT combined with chemotherapy cannot improve survival outcomes for rNPC. More novel clinical trials should be explored to develop individualized strategies to improve survival and minimize toxicities.

Profiling of Tumor Cell-Delivered Exosome by Surface Enhanced Raman Spectroscopy-Based Biosensor for Evaluation of Nasopharyngeal Cancer Radioresistance.

Although the advancement of radiotherapy significantly improves the survival of nasopharyngeal cancer (NPC), radioresistance associated with recurrence and poor outcomes still remains a daunting challenge in the clinical scenario. Currently, effective biomarkers and convenient detection methods for predicting radioresistance have not been well established. Here, the surface-enhanced Raman spectroscopy combined with proteomics is used to firstly profile the characteristic spectral patterns of exosomes secreted from self-established NPC radioresistance cells, and reveals specific variations of proteins expression during radioresistance formation, including collagen alpha-2 (I) chain (COL1A2) that is associated with a favorable prognosis in NPC and is negatively associated with DNA repair scores and DNA repair-related genes via bioinformatic analysis. Furthermore, deep learning model-based diagnostic model is generated to accurately identify the exosomes from radioresistance group. This work demonstrates the promising potential of exosomes as a novel biomarker for predicting the radioresistance and develops a rapid and sensitive liquid biopsy method that will provide a personalized and precise strategy for clinical NPC treatment.

Recent progress in surface-enhanced Raman spectroscopy-based biosensors for the detection of extracellular vesicles.

Extracellular vesicles (EVs) are a type of mediator that enables intercellular communication. Moreover, EVs carry critical molecular information from parental cells, making them ideal biomarkers for clinical screening and diagnosis. Currently, several sensing technologies have been established to sensitively detect EVs. Among them, surface-enhanced Raman spectroscopy (SERS) has become a powerful analytical tool with high sensitivity and low detection limits. In this review, we first cover the biological characteristics of EVs and the principle of SERS amplification. Then, we describe the recent progress in SERS technology applied to detect EVs, including direct label-free methods and indirect labeling strategies, in which substrate fabrication and nanoprobe assembly were emphasized. Furthermore, SERS technology could also be used to characterize or monitor the behavior of programmable EVs. Finally, we discuss the prospects and issues to be addressed for the development of SERS technology for EV analysis.

Highly Efficient Blood Protein Analysis Using Membrane Purification Technique and Super-Hydrophobic SERS Platform for Precise Screening and Staging of Nasopharyngeal Carcinoma.

Early screening and precise staging are crucial for reducing mortality in patients with nasopharyngeal carcinoma (NPC). This study aimed to assess the performance of blood protein surface-enhanced Raman scattering (SERS) spectroscopy, combined with deep learning, for the precise detection of NPC. A highly efficient protein SERS analysis, based on a membrane purification technique and super-hydrophobic platform, was developed and applied to blood samples from 1164 subjects, including 225 healthy volunteers, 120 stage I, 249 stage II, 291 stage III, and 279 stage IV NPC patients. The proteins were rapidly purified from only 10 µL of blood plasma using the membrane purification technique. Then, the super-hydrophobic platform was prepared to pre-concentrate tiny amounts of proteins by forming a uniform deposition to provide repeatable SERS spectra. A total of 1164 high-quality protein SERS spectra were rapidly collected using a self-developed macro-Raman system. A convolutional neural network-based deep-learning algorithm was used to classify the spectra. An accuracy of 100% was achieved for distinguishing between the healthy and NPC groups, and accuracies of 96%, 96%, 100%, and 100% were found for the differential classification among the four NPC stages. This study demonstrated the great promise of SERS- and deep-learning-based blood protein testing for rapid, non-invasive, and precise screening and staging of NPC.

A Novel Necroptosis-Associated IncRNAs Signature for Prognosis of Head and Neck Squamous Cell Carcinoma.

Purpose: The prognosis of head and neck squamous cell carcinoma (HNSCC) is poor. Necroptosis is a novel programmed form of necrotic cell death. The prognostic value of necroptosis-associated lncRNAs expression in HNSCC has not been explored. Methods: We downloaded mRNA expression data of HNSCC patients from TCGA databases. Prognostic lncRNAs were identified by univariate Cox regression. LASSO was used to establish a model with necroptosis-related lncRNAs. Kaplan-Meier analysis and ROC were applied to verify the model. Finally, functional studies including gene set enrichment analyses, immune microenvironment analysis, and anti-tumor compound IC50 prediction were performed. Results: We identified 1,117 necroptosis-related lncRNAs. The Cox regression showed 55 lncRNAs were associated with patient survival (p < 0.05). The risk model of 24- lncRNAs signature categorized patients into high and low risk groups. The patients in the low-risk group survived longer than the high-risk group (p < 0.001). Validation assays including ROC curve, nomogram and correction curves confirmed the prediction capability of the 24-lncRNA risk mode. Functional studies showed the two patient groups had distinct immunity conditions and IC50. Conclusion: The 24-lncRNA model has potential to guide treatment of HNSCC. Future clinical studies are needed to verify the model.

Upregulation of PNCK Promotes Metastasis and Angiogenesis via Activating NF-κB/VEGF Pathway in Nasopharyngeal Carcinoma.

Background: Distant metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC). Thus, the identification of the molecular mechanisms and the development of novel therapeutic strategies are important. Previous studies suggest that PNCK promotes tumor growth by suppressing PI3K/AKT/mTOR signaling in NPC. However, the underlying regulatory mechanism of PNCK for NPC invasion and metastasis remains unclear. Methods: The PNCK expression level was evaluated in nonmetastatic and metastatic NPC specimens by mRNA sequencing and immunohistochemistry. In vitro migration and invasion and in vivo nude mouse metastasis model and zebrafish model were used to evaluate the effects of PNCK ectopic expression on the metastatic ability of NPC cells. Gene set enrichment and western blot analyses were used to investigate the PNCK downstream signaling pathway. Results: Human metastatic NPC samples showed elevated PNCK expression at both mRNA and protein levels. Upregulated PNCK promoted in vitro NPC cell migration, invasion, and the formation of lung metastases; the vascular-labeled fluorescence signal increased in the in vivo zebrafish model. Mechanistically, pathway analysis showed that the upregulation of PNCK may promote cell metastasis by activating the NF-κB/VEGF signaling pathway. Conclusions: These findings revealed the specific critical role of PNCK in promoting NPC metastasis and angiogenesis, which suggested that PNCK may have implications as a potential therapeutic target for individualized NPC treatment.

Identification of a ferroptosis-associated gene signature and the related therapeutic targets in head and neck squamous carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) has a poor prognosis due to its high rates of recurrence and metastasis. Herein, we designed and validated an individualized ferroptosis-associated gene signature (FGS) and further probed the potential survival mechanisms along with therapeutic targets for HNSCC. METHODS: The FGS risk score was constructed using stepwise regression analysis and validated in the GSE41613 cohort. Characterization of the tumor microenvironment (TME) in patients with HNSCC, involving immune cells and immunomodulatory genes, was performed to investigate the survival mechanisms and therapeutic targets associated with FGS. To validate the role of FGS in TME, multiplex fluorescent immunohistochemistry (mfIHC) was performed on tissue sections from 55 patients with oral squamous carcinoma. RESULTS: The risk score obtained from FGS showed good predictive power as an independent predictor of overall survival. From the tumor immune dysfunction and exclusion (TIDE) prediction, it was found that patients at low risk may benefit from immunotherapy. Furthermore, FGS was significantly associated with CD276, which was highly expressed in fibroblasts that enriched in angiogenesis and epithelial-mesenchymal transition pathways at a single-cell resolution, suggesting CD276 may play a critical mediator of the immunosuppressive microenvironment. Lastly, we identified ATG5 as a critical gene in FGS. And the immune-bioinformatics analysis combined with experimental validation showed a negative correlation between ATG5 expression and CD8 + T cells. CONCLUSION: The FGS model provides a novel and effective method to predict the prognosis of patients with HNSCC and their survival can be prolonged through TME-related therapeutic targets.

Optical biosensor based on SERS with signal calibration function for quantitative detection of carcinoembryonic antigen.

Monitoring the levels of cancer biomarkers is essential for cancer diagnosis and evaluation. In this study, a novel sandwich type sensing platform based on surface-enhanced Raman scattering (SERS) technology was developed for the detection of carcinoembryonic antigen (CEA), with a limit of detection (LOD) of 0.258 ng/mL. In order to achieve sensitive detection of CEA in complex samples, gold nanoparticle monolayer modified with CEA antibodies and with aptamer-functionalized probes was fabricated to target CEA. Two gold layers were integrated into the SERS platform, which greatly enhanced the signal of the probe by generating tremendous "hot spots". Meanwhile, the intensity ratio of Raman probes and the second-order peak of the silicon wafer was used to achieve dynamic calibration of the Raman probe signal. Excitingly, this sensing platform was capable of distinguishing cancer patients from healthy individuals via CEA concentrations in blood samples with the accuracy of 100%. This sandwich structure SERS sensing platform presented promising potential to be an alternative tool for clinical biomarker detection in the field of cancer diagnosis.

Characterization of METTL7B to Evaluate TME and Predict Prognosis by Integrative Analysis of Multi-Omics Data in Glioma.

Glioma is the most common and aggressive type of primary brain malignant tumor with limited treatment approaches. Methyltransferase-like 7B (METTL7B) is associated with the pathogenesis of several diseases but is rarely studied in glioma. In this study, 1,493 glioma samples (data from our cohort, TCGA, and CGGA) expressing METTL7B were used to explore its prognostic value and mechanism in the immune microenvironment. Results showed that high expression of METTL7B is associated with poor prognosis and abundant immunosuppressive cells. Further, functional enrichment showed that METTL7B is involved in the negative regulation of immunity and carcinogenic signaling pathways. Moreover, a METTL7B-related prognostic signature constructed based on multi-omics showed a good prediction of the overall survival (OS) time of glioma patients. In conclusion, METTL7B is a potential prognostic biomarker. In addition, the prognostic prediction model constructed in this study can be used in clinical setups for the development of novel effective therapeutic strategies for glioma patients and improving overall survival.

A Novel Prognostic Signature Based on Metabolism-Related Genes to Predict Survival and Guide Personalized Treatment for Head and Neck Squamous Carcinoma.

Metabolic reprogramming contributes to patient prognosis. Here, we aimed to reveal the comprehensive landscape in metabolism of head and neck squamous carcinoma (HNSCC), and establish a novel metabolism-related prognostic model to explore the clinical potential and predictive value on therapeutic response. We screened 4752 metabolism-related genes (MRGs) and then identified differentially expressed MRGs in HNSCC. A novel 10-MRGs risk model for prognosis was established by the univariate Cox regression analysis and the least absolute shrinkage and selection operator (Lasso) regression analysis, and then verified in both internal and external validation cohort. Kaplan-Meier analysis was employed to explore its prognostic power on the response of conventional therapy. The immune cell infiltration was also evaluated and we used tumor immune dysfunction and exclusion (TIDE) algorithm to estimate potential response of immunotherapy in different risk groups. Nomogram model was constructed to further predict patients' prognoses. We found the MRGs-related prognostic model showed good prediction performance. Survival analysis indicated that patients suffered obviously poorer survival outcomes in high-risk group (p < 0.001). The metabolism-related signature was further confirmed to be the independent prognostic value of HNSCC (HR = 6.387, 95% CI = 3.281-12.432, p < 0.001), the efficacy of predictive model was also verified by internal and external validation cohorts. We observed that HNSCC patients would benefit from the application of chemotherapy in the low-risk group (p = 0.029). Immunotherapy may be effective for HNSCC patients with high risk score (p < 0.01). Furthermore, we established a predictive nomogram model for clinical application with high performance. Our study constructed and validated a promising 10-MRGs signature for monitoring outcome, which may provide potential indicators for metabolic therapy and therapeutic response prediction in HNSCC.

Characterization of Hypoxia Signature to Evaluate the Tumor Immune Microenvironment and Predict Prognosis in Glioma Groups.

Glioma groups, including lower-grade glioma (LGG) and glioblastoma multiforme (GBM), are the most common primary brain tumor. Malignant gliomas, especially glioblastomas, are associated with a dismal prognosis. Hypoxia is a driver of the malignant phenotype in glioma groups; it triggers a cascade of immunosuppressive processes and malignant cellular responses (tumor progression, anti-apoptosis, and resistance to chemoradiotherapy), which result in disease progression and poor prognosis. However, approaches to determine the extent of hypoxia in the tumor microenvironment are still unclear. Here, we downloaded 575 LGG patients and 354 GBM patients from Chinese Glioma Genome Atlas (GGGA), and 530 LGG patients and 167 GBM patients from The Cancer Genome Atlas (TCGA) with RNA sequence and clinicopathological data. We developed a hypoxia risk model to reflect the immune microenvironment in glioma and predict prognosis. High hypoxia risk score was associated with poor prognosis and indicated an immunosuppressive microenvironment. Hypoxia signature significantly correlated with clinical and molecular features and could serve as an independent prognostic factor for glioma patients. Moreover, Gene Set Enrichment Analysis showed that gene sets associated with the high-risk group were involved in carcinogenesis and immunosuppression signaling. In conclusion, we developed and validated a hypoxia risk model, which served as an independent prognostic indicator and reflected overall immune response intensity in the glioma microenvironment.

Study on the chemodrug-induced effect in nasopharyngeal carcinoma cells using laser tweezer Raman spectroscopy.

To explore the effect in nasopharyngeal carcinoma (NPC) cells after treatment with chemodrugs, Raman profiles were characterized by laser tweezer Raman spectroscopy. Two NPC cell lines (CNE2 and C666-1) were treated with gemcitabine, cisplatin, and paclitaxel, respectively. The high-quality Raman spectra of cells without or with treatments were recorded at the single-cell level with label-free laser tweezers Raman spectroscopy (LTRS) and analyzed for the differences of alterations of Raman profiles. Tentative assignments of Raman peaks indicated that the cellular specific biomolecular changes associated with drug treatment include changes in protein structure (e.g. 1655 cm-1), changes in DNA/RNA content and structure (e.g. 830 cm-1), destruction of DNA/RNA base pairs (e.g. 785 cm-1), and reduction in lipids (e.g. 970 cm-1). Besides, both principal components analysis (PCA) combined with linear discriminant analysis (LDA) and the classification and regression trees (CRT) algorithms were employed to further analyze and classify the spectral data between control group and treated group, with the best discriminant accuracy of 96.7% and 90.0% for CNE2 and C666-1 group treated with paclitaxel, respectively. This exploratory work demonstrated that LTRS technology combined with multivariate statistical analysis has promising potential to be a novel analytical strategy at the single-cell level for the evaluation of NPC-related chemotherapeutic drugs.

Radiation-induced small extracellular vesicles as "carriages" promote tumor antigen release and trigger antitumor immunity.

Rationale: Accumulating evidence supports the importance of radiation therapy in the induction of antitumor immunity. Small extracellular vesicles (sEVs) play essential roles in tumor antigen loading and delivery. However, the role of sEVs in radiation-induced antitumor immunity remains unclear. It is therefore important to determine the role and regulatory mechanisms of sEVs in radiation-induced immunity. Methods: Tumor cells were irradiated (8 Gy), and sEVs were purified via ultracentrifugation. Primary tumor and experimental lung metastasis models were established in mice to evaluate antitumor immunity triggered by immunization with sEVs. Proteomic and bioinformatic analyses were performed to identify altered cargos in sEVs induced by radiation. Peptides derived from up-regulated proteins in sEVs were designed and synthesized as vaccines according to major histocompatibility complex (MHC) I binding and immunogenicity. Results: Here, we demonstrated that sEVs derived from irradiated tumor cells could trigger antitumor immunity against primary tumor and experimental lung metastasis by enhancing CD8+ and CD4+ T cell infiltration. Radiation may also enrich sEVs with tumor antigens and heat-shock proteins. Furthermore, CUB domain-containing protein 1 (CDCP1) derived from radiation-induced sEVs was identified as a novel tumor-associated antigen and developed as a peptide vaccine that may generate antitumor immune responses. Conclusions: Our results demonstrate that the use of sEVs secreted by irradiated tumor cells constitutes an efficient approach for tumor antigen delivery and presentation and highlight the role of sEVs in radiation-triggered antitumor immunity.

Raman profile alterations of irradiated human nasopharyngeal cancer cells detected with laser tweezer Raman spectroscopy.

Radiotherapy has been widely used for nasopharyngeal carcinoma (NPC) treatment, which causes DNA damage and alterations of macromolecules of cancer cells. However, the Raman profile alterations of irradiated NPC cells remain unclear. In the present study, we used laser tweezers Raman spectroscopy (LTRS) to monitor internal structural changes and chemical modifications in NPC cells after exposure at a clinical dose (2.3 Gy) to X-ray irradiation (IR) at a single-cell level. Two types of NPC cell lines, CNE2 (EBV-negative cell line) and C666-1 (EBV-positive cell line), were used. The Raman spectra of cells before and after radiation treatment were recorded by LTRS. The analysis of spectral differences indicated that the IR caused Raman profile alterations of intracellular proteins, DNA base and lipids. Moreover, by using the multivariate statistical analysis including principal component analysis (PCA) and linear discriminant analysis (LDA) algorithm, an accuracy of 90.0% for classification between CNE2 cells before and after IR could be achieved, which was 10% better than that of C666-1 cells. The results demonstrated that CNE2 cells were more sensitive to IR in comparison to C666-1 cells, providing useful information for creating a treatment strategy in clinical practice. This exploratory study suggested that LTRS combined with multivariate statistical analysis would be a novel and effective tool for evaluating the radiotherapeutic effect on tumor cells, and for detection of the corresponding alterations at the molecular level.

Maintenance chemotherapy using S-1 following definitive chemoradiotherapy in patients with N3 nasopharyngeal carcinoma.

BACKGROUND: Patients with N3 stage nasopharyngeal carcinoma (NPC) are at high risk for treatment failure. This study aims to assess the efficacy of maintenance chemotherapy (MC) using S-1 (MC-S1), a novel oral fluoropyrimidine agent, following definitive chemoradiotherapy (CRT) using intensity-modulated radiotherapy (IMRT) in patients with N3 nasopharyngeal carcinoma (N3-NPC). METHODS: A retrospective review was conducted for all N3-NPC treated with CRT with MC (CRT-MC) or without MC (CRT-non-MC) during 2014-2016. Toxicities with MC were recorded. Overall survival (OS), locoregional failure-free survival (LFFS) and distant metastasis free survival (DMFS) were compared between CRT-MC vs. CRT-non-MC cohorts. RESULTS: A total of 130 N3 patients were identified, of whom 21 (16.2%) were treated with CRT-MC, and 109 (83.8%) with CRT-non-MC. Patient characteristics did not significantly differ between the CRT-MC and CRT-non-MC groups, with the exception of the number of cycles of neoadjuvant chemotherapy. Following IMRT 69 patients achieved a complete response (CR) (CRT-MC: 10; CRT-non-MC: 59), 61 had a partial response (PR) (11 vs. 50), and none maintained stable disease (SD) or developed progression of disease (PD). After a median follow-up of 41 months for surviving patients, a significant differences in OS (76.3% vs. 95.2%, p = 0.046) and DMFS (70.3% vs. 90.5%, p = 0.043) but not LFFS (84.9% vs. 100%, p = 0.091) at 3 years were observed between the CRT-non-MC and CRT-MC groups. Skin hyperpigmentation, leucopenia, fatigue, neutropenia, anorexia and nausea were the common but not severe (grade 1-2) toxicities of MC. CONCLUSIONS: Using MC-S1 in N3-NPC patients following IMRT achieved superior survival to the CRT-non-MC patients. The toxicities of MC-S1 were mild and tolerable. Further clinical trials are required to evaluate the efficacy of MC-S1 in N3-NPC patients.

Early Diffusion-Weighted Imaging and Proton Magnetic Resonance Spectroscopy Features of Liver Transplanted Tumors Treated with Radiation in Rabbits: Correlation with Histopathology.

In this study, we sought to determine how diffusion-weighted imaging (DWI) and proton magnetic resonance spectroscopy (1H-MRS) features are associated with histopathological results, and explored the cellular mechanisms of DWI and 1H-MRS in early radiosensitivity of transplanted liver tumors. VX2 tumors were implanted into the hind leg muscles of 60 New Zealand White Rabbits. All rabbits were randomly divided into ten subgroups according to treatment: irradiated or nonirradiated and according to different times postirradiation. Magnetic resonance scanning was then performed one day before irradiation and on days 1, 3, 5 and 7 postirradiation. Differences in tumor volume, apparent diffusion coefficient (ADC) value, choline/creatine ratio and lipid/creatine ratio, and their associations with histopathological findings, were assessed. Tumor volumes in the irradiated groups were smaller than control values, while ADC values increased gradually with time postirradiation; choline/creatine ratios were reduced while lipid/creatine ratios were larger compared to control values. Bax protein levels after irradiation increased with time. Interestingly, the ADC value and Bax-positive grade showed the same increasing trend (r = 0.900, P < 0.001). Additionally, choline/creatine and lipid/creatine ratios were respectively significantly associated with Bax-positive grade. Furthermore, significant associations of tumor volume with ADC value, choline/creatine ratio and lipid/creatine ratio were observed. These findings demonstrated that ADC value, choline/creatine ratio and lipid/creatine ratio, indicators of early radiosensitivity, are related to cell apoptosis.