Minor Spliceosomal 65K/RNPC3 Interacts with ANKRD11 and Mediates HDAC3-Regulated Histone Deacetylation and Transcription.

RNA splicing is crucial in the multilayer regulatory networks for gene expression, making functional interactions with DNA- and other RNA-processing machineries in the nucleus. However, these established couplings are all major spliceosome-related; whether the minor spliceosome is involved remains unclear. Here, through affinity purification using Drosophila lysates, an interaction is identified between the minor spliceosomal 65K/RNPC3 and ANKRD11, a cofactor of histone deacetylase 3 (HDAC3). Using a CRISPR/Cas9 system, Deletion strains are constructed and found that both Dm65KΔ/Δ and Dmankrd11Δ/Δ mutants have reduced histone deacetylation at Lys9 of histone H3 (H3K9) and Lys5 of histone H4 (H4K5) in their heads, exhibiting various neural-related defects. The 65K-ANKRD11 interaction is also conserved in human cells, and the HsANKRD11 middle-uncharacterized domain mediates Hs65K association with HDAC3. Cleavage under targets and tagmentation (CUT&Tag) assays revealed that HsANKRD11 is a bridging factor, which facilitates the synergistic common chromatin-binding of HDAC3 and Hs65K. Knockdown (KD) of HsANKRD11 simultaneously decreased their common binding, resulting in reduced deacetylation of nearby H3K9. Ultimately, this study demonstrates that expression changes of many genes caused by HsANKRD11-KD are due to the decreased common chromatin-binding of HDAC3 and Hs65K and subsequently reduced deacetylation of H3K9, illustrating a novel and conserved coupling mechanism that links the histone deacetylation with minor spliceosome for the regulation of gene expression.

Prognostic Value of Lymph Node Necrosis at Different N Stages in Patients with Nasopharyngeal Carcinoma.

Background: Lymph node necrosis (LNN), including retropharyngeal nodal necrosis and cervical nodal necrosis, which is related to radiotherapy/ chemotherapy resistance, is a common phenomenon in nasopharyngeal carcinoma (NPC). This study was to assess the prognostic value of LNN at different N stages in NPC patients. Materials and Methods: In total, 1,665 newly diagnosed NPC patients at stage TxN1-3M0 from two centers were enrolled. Univariate and multivariate models were constructed to assess the association between LNN and long-term survival outcomes. The propensity score matching method was performed to balance treatment groups for baseline characteristics. Results: Of the 1,665, 540 patients (540/1665, 32.4%) were diagnosed with LNN, of which 54.1% (292/540) patients were at stage N1, 31.3% (169/540) at stage N2, and 14.6% (79/540) at stage N3. Univariate and multivariate analyses indicated LNN as an independent predictor for progression­free survival (PFS), overall survival (OS), distant metastasis-free survival (DMFS), and locoregional relapse-free survival (LRRFS) in stage N1-3 patients (all P<0.001). When patients were analyzed according to stage, similar findings were observed for N1 patients (all P<0.001); for N2 patients, LNN independently predicted PFS (P=0.003), OS (P=0.011), and DMFS (P=0.004), and for stage N3, LNN only independently predicted LRRFS (P=0.019). 123 pairs of patients who received induction chemotherapy plus concurrent chemoradiotherapy or only concurrent chemoradiotherapy were matched, adding induction chemotherapy improved 5-year OS, PFS and LRFFS, but the results were not statistically significant. Conclusions: In NPC patients, LNN could independently predict poor prognosis at all N1-3 stages and at each N stage (N1 to N3). The value of adding induction chemotherapy to concurrent chemoradiotherapy in patients with LNN still requires further prospective studies.

Expression Profiles and Prognostic Value of Multiple Inhibitory Checkpoints in Head and Neck Lymphoepithelioma-Like Carcinoma.

Background: Inhibitory checkpoints are promising antitumor targets and predictive biomarkers in a variety of cancers. We aimed to identify the expression levels and prognostic value of multiple inhibitory checkpoints supported by preclinical and clinical evidence in head and neck lymphoepithelioma-like carcinoma (HNLELC). Methods: The expression of seven inhibitory checkpoints were evaluated in the tumor nest (TN) and tumor stroma (TS) of 102 HNLELC specimens using immunohistochemistry and digital pathology, and an inhibitory checkpoint-based signature (ICS) was subsequently constructed using the LASSO Cox regression model. Results: PD-L1, B7H3, and IDO-1 were mostly expressed in the TN, with median H-score of TN vs TS: 63.6 vs 14.6; 8.1 vs 1.0; 61.5 vs 34.7 (all P < 0.001), whereas PD-1, TIM-3, LAG-3, and VISTA were mainly observed in the TS, with median H-score of TN vs TS: 0.2 vs 12.4, 3.4 vs 7.1, 6.2 vs 11.9, 16.4 vs 47.2 (all P < 0.001), respectively. The most common simultaneously expressed combinations consisted of PD-L1 + B7H3 + IDO-1 + TIM-3 + LAG-3 + VISTA and B7H3 + IDO-1 + TIM-3 + LAG-3 in the TN (both occurring in 8.8% of patients) and PD-L1 + B7H3 + IDO-1 in the TS (4.9%). In addition, high-ICS patients had shorter 5-year disease-free (40.6% vs 81.7%; P < 0.001), regional recurrence-free (63.5% vs 88.2%; P = 0.003), and overall survival (73.5% vs 92.9%; P = 0.006) than low-ICS patients. Multivariate analysis revealed that ICS represented an independent predictor, which could significantly complement the predictive performance of TNM stage for 3-year (AUC 0.724 vs 0.619, P = 0.014), 5-year (AUC 0.727 vs 0.640, P = 0.056), and 10-year disease-free survival (AUC 0.815 vs 0.709, P = 0.023). Conclusions: The expression of inhibitory checkpoints and ICS classifier may increase the prognostic value of the TNM staging system and guide the rational design of personalized inhibitory checkpoint blockade therapy in HNLELC.

Comprehensive Identification and Alternative Splicing of Microexons in Drosophila.

Interrupted exons in the pre-mRNA transcripts are ligated together through RNA splicing, which plays a critical role in the regulation of gene expression. Exons with a length ≤ 30 nt are defined as microexons that are unique in identification. However, microexons, especially those shorter than 8 nt, have not been well studied in many organisms due to difficulties in mapping short segments from sequencing reads. Here, we analyzed mRNA-seq data from a variety of Drosophila samples with a newly developed bioinformatic tool, ce-TopHat. In addition to the Flybase annotated, 465 new microexons were identified. Differentially alternatively spliced (AS) microexons were investigated between the Drosophila tissues (head, body, and gonad) and genders. Most of the AS microexons were found in the head and two AS microexons were identified in the sex-determination pathway gene fruitless.

Prognostic influence of prevertebral space involvement in nasopharyngeal carcinoma: A retrospective study.

PURPOSE: To evaluate how prevertebral space involvement (PSI) and degree of tumor extension within the space affects prognosis in nasopharyngeal carcinoma (NPC). PARTICIPANTS AND METHODS: Data of patients with newly-diagnosed nonmetastatic NPC (n = 757) were retrospectively analyzed. Patients were separated into groups according to presence or absence of PSI and degree of tumor spread. Overall survival (OS), failure-free survival (FFS), local relapse-free survival (LRFS), and distant metastasis-free survival (DMFS) were compared between the groups. RESULTS: Prevalence of PSI, simple prevertebral muscle involvement (PMI), and behind prevertebral muscle involvement (BPMI) were 44.9% (340/757), 22.5% (170/757), and 22.5% (170/757), respectively. OS, FFS, LRFS, and DMFS for patients with and without PSI were 64% vs. 84.8%, 68% vs. 85.6%, 85.8% vs. 94.4%, and 78.5% vs. 92.8%, respectively (all P < 0.001). PSI was an independent predictor of OS, FFS, LRFS, and DMFS. OS, FFS, and DMFS for patients with simple PMI and with BPMI were 72.7% vs. 54.8% (P = 0.002), 75.8% vs. 59.8% (P = 0.003), and 85.5% vs. 71.2% (P = 0.002), respectively. Degree of PSI extension was related to OS, FFS, and DMFS. OS, FFS, LRFS, and DMFS were significantly poorer in patients with PSI in T2-3 stage than in patients without PSI in T3 stage (P < 0.05), but comparable to those in patients with T4 stage (P > 0.05). CONCLUSIONS: PSI predicts poor prognosis in NPC. Survival is poorer in patients with BPMI than in those with simple PMI. NPC with PSI should be classified as T4 stage.

Influence of tumor necrosis on treatment sensitivity and long-term survival in nasopharyngeal carcinoma.

PURPOSE: To assess the impact of tumor necrosis on treatment sensitivity and long-term survival in patients with nasopharyngeal carcinoma (NPC) treated using intensity-modulated radiation therapy (IMRT). PARTICIPANTS AND METHODS: In total, 757 patients with non-metastatic, histologically confirmed NPC were retrospectively examined. All patients were treated using IMRT; 93.7% patients with stage T3-T4/N1-N3 disease also received cisplatin-based chemotherapy. RESULTS: The incidence rates of tumor necrosis in primary tumor, retropharyngeal lymph nodes, neck lymph nodes, and total tumor were 2%, 17.7%, 21.5%, and 31.4%. Overall, 40.8% patients with necrosis of the total tumor achieved complete response (CR) and 54.7% patients without tumor necrosis achieved CR at the end of treatment (χ2 = 12.728, P < 0.001). The estimated 7-year overall survival (OS), failure-free survival (FFS), distant metastasis-free survival (DMFS), and loco-regional relapse-free survival (LRRFS) for patients with tumor necrosis and without tumor necrosis of the total tumor were 68.5% vs. 88.4%, 70.5% vs. 88.1%, 77.6% vs. 90.6%, and 85.9% vs. 91.3%, respectively (all P < 0.001). Multivariate analyses indicated that necrosis of the total tumor was an independent predictor of OS, FFS, DMFS, and LRRFS. The impact of lymph node necrosis on long-term survival was similar to that of necrosis of the total tumor. ROC curves verified that inclusion of lymph node necrosis improved the predictive value of the current N classification criteria (P = 0.006). CONCLUSIONS: Tumor necrosis served as a predictor of treatment sensitivity and poor prognosis for patients with NPC. Lymph node necrosis significantly improved the prognostic value of the current N classification criteria for NPC.

Combining tumor response and personalized risk assessment: Potential for adaptation of concurrent chemotherapy in locoregionally advanced nasopharyngeal carcinoma in the intensity-modulated radiotherapy era.

BACKGROUND AND PURPOSE: In the intensity-modulated radiotherapy (IMRT) era, the role of concurrent chemoradiotherapy (CCRT) after induction chemotherapy (IC) in locoregionally advanced nasopharyngeal carcinoma (LANPC) is undetermined, while concerns exist about CCRT-associated excessive toxicity. We aimed to combine tumor response and risk assessment to guide decisions about concurrent chemotherapy. MATERIALS AND METHODS: From April 2009 to December 2015, 744 LANPC patients treated with CCRT/IMRT after IC were included. Matching techniques were performed for treatment effect evaluation. Tumor response to IC was used for patient stratification. A nomogram was built based on multivariable Cox regression analysis to predict overall survival (OS). RESULTS: After IC, 508 patients (68.3%) had favorable tumor response (complete or partial response), among whom IC + CCRT achieved significantly superior 5-year disease-free survival and OS than IC + IMRT (82.2% vs. 72.5%, P = 0.025; 89.2% vs. 79.9%, P = 0.025). However, no significant difference was found in patients with unfavorable response (both P > 0.05). For favorable responders, a nomogram was built integrating age, smoking, T category, N category, pretreatment Epstein-Barr virus DNA and treatment modality. The concordance index was 0.713 and calibration was good. The nomogram determined three risk groups with distinct OS. High-risk patients benefited from CCRT after IC regarding disease-free survival, OS and distant metastasis-free survival, whereas low- and intermediate-risk patients did not. CONCLUSIONS: For LANPC patients with unfavorable response to IC, subsequent CCRT seems inadequate, rendering intensification necessary. For favorable responders with low risk, IC + IMRT represents a reasonable de-intensification approach, although confirmation by prospective data is needed.

PBA2, a novel compound, enhances radiosensitivity in various carcinoma cells by activating the p53 pathway in vitro and in vivo.

Radiotherapy is the main method used to treat human carcinoma; however, certain types of carcinomas are radiation-insensitive. The present study aimed to explore whether a novel compound, PBA2, could enhance the radiosensitivity of various carcinoma cells in vitro and in vivo, and investigate its underlying mechanism. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the cytotoxicity of PBA2. Colony formation assays were used to observe the radiosensitivity effect of PBA2 in vitro. Cell cycle distributions and cell apoptosis were estimated using flow cytometry. Comet assays and Immunofluorescence assays were used to analyze DNA damage. The intracellular RNA was extracted and analyzed by sequencing. Western blotting was used to determine protein levels. A stable cell line with TP53 (encoding p53) knockdown was constructed by cell transfection. A mouse xenograft model was used to assess the radiosensitivity effect of PBA2 in vivo. We found that PBA2 at a low concentration (0.1 µM) enhanced radiosensitivity in various carcinoma cells, including CNE1, MG63, KB, HEP2, GLC82, and SMMC7221, in vitro. Combined with PBA2, radiation induced significant cell apoptosis in CNE1 and MG63 cells, accompanied by increased DNA damage, but did not affect cell cycle arrest. Mechanistically, PBA2 promoted p53 expression significantly; however, when p53 was mutated, functionally impaired, or knocked down, PBA2 could not enhance the radiosensitivity of these cells. Additionally, the combination of PBA2 and radiation reduced the tumor volume and tumor weight in CNE1 xenograft models significantly, without obvious toxicities. Our results demonstrated that PBA2 enhanced the radiosensitivity of various carcinoma cells in vitro and in vivo. The underlying mechanism might involve increasing DNA damage and cell apoptosis via activating the p53 pathway.

Development and external validation of a nomogram for predicting the overall survival of patients with stage II nasopharyngeal carcinoma after curative treatment.

Objective: To facilitate decision-making support for individual patients, development and external validation of a nomogram was undertaken to reveal prognostic factors and predict the value of concurrent chemoradiotherapy (CCRT) compared with radiotherapy (RT) for stage-II nasopharyngeal carcinoma (NPC) patients. Methods: Clinical data of 419 and 309 patients with American Joint Committee on Cancer (2017) stage-II NPC in two institutions in China were collected retrospectively. Overall survival (OS) and progression-free survival were compared using Kaplan-Meier estimates. Cox regression analysis was used to identify the prognostic factors for building the nomogram. Predictive accuracy and discriminative ability were measured using the Concordance Index. Results: Finally, there were 24 and 20 deaths in the development and validation group, respectively. Patients with stage T2N1, N1 stage, involvement of retropharyngeal and unilateral cervical lymph nodes, and who had RT alone had worse OS (P=0.019, 0.035, 0.003 and 0.010, respectively; log-rank test) than patients with stage T1N1 and T2N0, N0 stage, involvement of retropharyngeal or unilateral cervical lymph nodes, and CCRT, respectively. After multivariate analysis of the training set, age, neutrophil-to-lymphocyte ratio, therapy type, and pretreatment plasma concentration of Epstein-Barr virus DNA were independent prognostic factors of OS. A nomogram was established externally by involving all the factors stated above. The Concordance Index for the established nomogram to predict the OS of the training set was 0.793 (95% CI 0.689-0.897), and 0.803 (95% CI 0.696-0.910) in the validation set. Conclusion: These data suggest that the nomogram was validated externally, could predict long-term outcome accurately, and enable accurate stratification of risk groups for stage-II NPC. Our model facilitated individualized care of NPC patients.

The development and external validation of simplified T category classification for nasopharyngeal carcinoma to improve the prognostic value in the intensity-modulated radiotherapy era.

BACKGROUND: Intensity-modulated radiotherapy (IMRT) provides excellent local control in nasopharyngeal carcinoma (NPC). We investigated whether simplifying 8th American Joint Committee on Cancer staging system T categories improves prognostic value. METHODS: We used 2191 NPC patients as a training set and 414 patients separately as an independent, external validation cohort. RESULTS: In the training set, local relapse-free survival (LRFS), disease-free survival (DFS), and overall survival (OS) were not significantly different between the 8th edition T2/T3 (P = 0.610, 0.380 and 0.353, respectively). Merging T2 and T3 to proposed T2 (proT2) provided significant differences in LRFS, DFS, and OS between proposed T categories. Proposed T categories had similar c-indices for LRFS, DFS, and OS (vs the 8th edition), which was validated in the external cohorts. Moreover, for DFS, the adjusted HRs of the proT2N0 (3.8), proT1N1 (3.8), and proT2N1 (6.0) subsets were similar; the adjusted HRs of the proT3N0 (7.0), proT3N1 (11.4), proT1N2 (11.0), proT2N2 (11.6), and proT3N2 (13.3) subsets were similar; the adjusted HRs of the proT1N3 (17.8), proT2N3 (15.3), and proT3N3 (26.4) subsets were similar; the results of the adjusted HRs for OS had the same rule. Defining proT1N0 as stage I; proT1N1/proT2N0-1 as stage II; proT3N0-2/proT1-2N2 as stage III; and proT1-3N3 as stage IVa generated orderly, significant differences in DFS and OS between stages in the training set and external validation cohort. CONCLUSIONS: In the IMRT era, three T categories are more reasonable (merging T2/T3 into T2) and proT3N0-2 (the 8th edition T4N0-2) should be down-staged to stage III.

Concurrent chemoradiotherapy with/without induction chemotherapy in locoregionally advanced nasopharyngeal carcinoma: Long-term results of phase 3 randomized controlled trial.

To report long-term results of a randomized controlled trial that compared cisplatin/fluorouracil/docetaxel (TPF) induction chemotherapy (IC) plus concurrent chemoradiotherapy (CCRT) with CCRT alone in locoregionally advanced nasopharyngeal carcinoma (NPC). Patients with stage III-IVB (except T3-4 N0) NPC were randomly assigned to receive IC plus CCRT (n = 241) or CCRT alone (n = 239). IC included three cycles of docetaxel (60 mg/m2 d1), cisplatin (60 mg/m2 d1), and fluorouracil (600 mg/m2 /d civ d1-5) every 3 weeks. Patients from both groups received intensity-modulated radiotherapy concurrently with three cycles of 100 mg/m2 cisplatin every 3 weeks. After a median follow-up of 71.5 months, the IC plus CCRT group showed significantly better 5-year failure-free survival (FFS, 77.4% vs. 66.4%, p = 0.019), overall survival (OS, 85.6% vs. 77.7%, p = 0.042), distant failure-free survival (88% vs. 79.8%, p = 0.030), and locoregional failure-free survival (90.7% vs. 83.8%, p = 0.044) compared to the CCRT alone group. Post hoc subgroup analyses revealed that beneficial effects on FFS were primarily observed in patients with N1, stage IVA, pretreatment lactate dehydrogenase ≥170 U/l, or pretreatment plasma Epstein-Barr virus DNA ≥6000 copies/mL. Two nomograms were further developed to predict the potential FFS and OS benefit of TPF IC. The incidence of grade 3 or 4 late toxicities was 8.8% (21/239) in the IC plus CCRT group and 9.2% (22/238) in the CCRT alone group. Long-term follow-up confirmed that TPF IC plus CCRT significantly improved survival in locoregionally advanced NPC with no marked increase in late toxicities and could be an option of treatment for these patients.

Prognostic value of gross tumor regression and plasma Epstein Barr Virus DNA levels at the end of intensity-modulated radiation therapy in patients with nasopharyngeal carcinoma.

PURPOSE: To assess gross tumor regression and plasma Epstein-Barr virus (EBV)-DNA levels at the end of intensity-modulated radiation therapy (IMRT) and its prognostic impact on patients with nasopharyngeal carcinoma (NPC). PARTICIPANTS AND METHODS: In total, 397 patients with non-metastatic, histologically confirmed NPC were retrospectively examined. All patients underwent magnetic resonance imaging of the nasopharynx and neck, and plasma EBV DNA assays before treatment and at the end of IMRT. RESULTS: The estimated 5-year loco-regional, local and regional relapse-free survival rates for patients with complete response (CR) and non-CR of the total tumor, primary tumor and metastatic lymph nodes at the end of IMRT were 94.9% vs. 85.8%, 96.6% vs. 87.3%, and 98.7% vs. 89.8%, respectively (P < 0.05). The estimated 5-year loco-regional relapse-free survival (LRRFS) rates for patients with persistent tumor with and without boost irradiation were 95.3% vs. 83%, respectively (P = 0.034). The estimated 5-year overall survival (OS), failure-free survival (FFS) and distant metastasis-free survival (DMFS) rates for patients with negative and positive plasma EBV DNA at the end of IMRT were 83.1% vs. 50.3%, 81.5% vs. 49.3%, and 87.6% vs. 61.5%, respectively (P < 0.001). Multivariate analyses indicated that regression of the total tumor and boost irradiation was an independent predictor of LRRFS, and plasma EBV DNA levels were independent predictors of OS, FFS and DMFS. CONCLUSIONS: Gross tumor regression and plasma EBV DNA levels at the end of IMRT served as predictors of poor prognosis for patients with NPC. The patients with persistent tumor and/or positive plasma EBV DNA might require timely strengthening treatment.

Additional Cervical Lymph Node Biopsy is Not a Significant Prognostic Factor for Nasopharyngeal Carcinoma in the Intensity-Modulated Radiation Therapy Era: A Propensity Score-matched Analysis from an Epidemic Area.

Introduction: This study aimed to evaluate the prognostic value of cervical lymph node biopsy and whether different biopsy methods would lead different outcomes in NPC in the intensity-modulated radiotherapy (IMRT) era. Material and Methods: 1492 patients with biopsy-proven, non-metastatic NPC, and treated by IMRT with or without chemotherapy were retrospectively reviewed. Cervical lymph node biopsy was performed in 183 (12.3%) patients: 61(4.1%) by needle puncture and 118(7.9%) by excision biopsy. Propensity-score matching was used to match patients in both arms at an equal ratio. Overall survival (OS), distant metastasis-free survival (DMFS), locoregional relapse-free survival (LRFS), and nodal relapse-free survival (NRFS) were assessed using the Kaplan-Meier method and compared using the log-rank test. Independent prognostic factors were identified using the Cox proportional hazards model. Results: In the original cohort of 1492 patients, patients receiving cervical lymph node biopsy had comparable survival (OS: P = 0.736, DMFS: P = 0.749, LRFS: P = 0.538, NRFS: P = 0.093,) with patients receiving isolated napharynx biopsy. The results for the propensity-match cohort of 316 patients were similar. Interestingly, compared with the control group and needle puncture biopsy group, a slightly lower nodal recurrence rate was observed in the excision biopsy group (P = 0.082 and P = 0.072, respectively). Adjusting for the known prognostic factors in multivariate analysis, cervical biopsy did not cause a higher risk of death, distant metastasis, or nodal relapse. Conclusions: Pretreatment cervical lymph node biopsy is not associated with impaired survival in NPC, suggesting the resist of the biopsy and more aggressive treatment after the biopsy may be unnecessary.

Loss of MED12 Induces Tumor Dormancy in Human Epithelial Ovarian Cancer via Downregulation of EGFR.

A high rate of disease relapse makes epithelial ovarian cancer (EOC) the leading cause of death among all gynecologic malignancies. These relapses are often due to tumor dormancy. Here we identify the RNA polymerase II transcriptional mediator subunit 12 (MED12) as an important molecular regulator of tumor dormancy. MED12 knockout (KO) induced dormancy of EOC cells in vitro and in vivo, and microarray analysis showed that MED12 KO decreased expression of EGFR. Restoration of EGFR expression in MED12 KO cells restored proliferation. Additionally, MED12 bound to the promoter of EGFR, and correlation studies showed that MED12 expression positively correlated with EGFR expression in EOC patient samples. Clinical data demonstrated that chemotherapy-resistant patients expressed lower levels of MED12 compared with responsive patients. Overall, our data show that MED12 plays an important role in regulating dormancy of EOC through regulation of EGFR.Significance: MED12 is identified as a novel, important regulator of tumor dormancy in human ovarian cancer. Cancer Res; 78(13); 3532-43. ©2018 AACR.

Comparison of the seventh and eighth editions of the UICC/AJCC staging system for nasopharyngeal carcinoma: analysis of 1317 patients treated with intensity-modulated radiotherapy at two centers.

BACKGROUND: In the intensity-modulated radiotherapy (IMRT) era, great improvement has been made in survival of nasopharyngeal carcinoma (NPC). The 7th edition of the International Union against Cancer/American Joint Committee on Cancer (UICC/AJCC) staging system seems "outdated " as it mainly based on the study in 2D/3D era, and thus the 8th edition has made some amendments according to recent studies. We aimed to compare and evaluate these two editions of staging system for NPC in patients treated with intensity-modulated radiotherapy. METHODS: A total of 1317 patients with biopsy-proven, non-metastatic NPC treated with IMRT between 2009 and 2014 at two institutions were retrospectively assessed. All patients were assessed by magnetic resonance imaging and restaged according to the 7th and 8th editions. Prognostic factors for local relapse-free survival (LRFS), distant metastasis-free survival (DMFS), disease-free survival (DFS) and overall survival (OS) were assessed and compared using the Kaplan-Meier method and log-rank test. The Cox proportional hazards model was also used to calculate the hazard ratio (HR). RESULTS: In both 7th and 8th edition, insignificant difference could be observed between T2 and T3 disease, T2 and T4 disease (all P > 0.05) for LRFS, while the difference of LRFS between T3 and T4 disease was significant in the previous edition (P = 0.001) but insignificant (P = 0.279) after revision. For OS, highly similar survival curve could be seen between T2 and T3 disease in both edition (all P > 0.1). DMFS and OS were not significantly different between N3a and N1-3b categories of the 7th edition (all P > 0.05). In contrast, obvious segregation was observed between N3 and the other N categories after the revision and combination in the 8th edition (all P < 0.05). DFS and OS were not significantly different between stage IVA and IVB of the 7th edition (P = 0.057 and P = 0.365, respectively); therefore, combining these stages in the 8th edition was reasonable. CONCLUSION: The overall stages and N categories of the 8th edition of the UICC/AJCC staging system provide better segregation of survival outcomes than the 7th edition. The 8th edition is also more clinically applicable as it has reduced ambiguity and revised out-of-date definitions. However, the T categories need further optimizing as the 8th edition failed to solve the problem of similar survival between adjacent T-classification, which has been exited since 7th edition.

Development and validation of a gene expression-based signature to predict distant metastasis in locoregionally advanced nasopharyngeal carcinoma: a retrospective, multicentre, cohort study.

BACKGROUND: Gene expression patterns can be used as prognostic biomarkers in various types of cancers. We aimed to identify a gene expression pattern for individual distant metastatic risk assessment in patients with locoregionally advanced nasopharyngeal carcinoma. METHODS: In this multicentre, retrospective, cohort analysis, we included 937 patients with locoregionally advanced nasopharyngeal carcinoma from three Chinese hospitals: the Sun Yat-sen University Cancer Center (Guangzhou, China), the Affiliated Hospital of Guilin Medical University (Guilin, China), and the First People's Hospital of Foshan (Foshan, China). Using microarray analysis, we profiled mRNA gene expression between 24 paired locoregionally advanced nasopharyngeal carcinoma tumours from patients at Sun Yat-sen University Cancer Center with or without distant metastasis after radical treatment. Differentially expressed genes were examined using digital expression profiling in a training cohort (Guangzhou training cohort; n=410) to build a gene classifier using a penalised regression model. We validated the prognostic accuracy of this gene classifier in an internal validation cohort (Guangzhou internal validation cohort, n=204) and two external independent cohorts (Guilin cohort, n=165; Foshan cohort, n=158). The primary endpoint was distant metastasis-free survival. Secondary endpoints were disease-free survival and overall survival. FINDINGS: We identified 137 differentially expressed genes between metastatic and non-metastatic locoregionally advanced nasopharyngeal carcinoma tissues. A distant metastasis gene signature for locoregionally advanced nasopharyngeal carcinoma (DMGN) that consisted of 13 genes was generated to classify patients into high-risk and low-risk groups in the training cohort. Patients with high-risk scores in the training cohort had shorter distant metastasis-free survival (hazard ratio [HR] 4·93, 95% CI 2·99-8·16; p<0·0001), disease-free survival (HR 3·51, 2·43-5·07; p<0·0001), and overall survival (HR 3·22, 2·18-4·76; p<0·0001) than patients with low-risk scores. The prognostic accuracy of DMGN was validated in the internal and external cohorts. Furthermore, among patients with low-risk scores in the combined training and internal cohorts, concurrent chemotherapy improved distant metastasis-free survival compared with those patients who did not receive concurrent chemotherapy (HR 0·40, 95% CI 0·19-0·83; p=0·011), whereas patients with high-risk scores did not benefit from concurrent chemotherapy (HR 1·03, 0·71-1·50; p=0·876). This was also validated in the two external cohorts combined. We developed a nomogram based on the DMGN and other variables that predicted an individual's risk of distant metastasis, which was strengthened by adding Epstein-Barr virus DNA status. INTERPRETATION: The DMGN is a reliable prognostic tool for distant metastasis in patients with locoregionally advanced nasopharyngeal carcinoma and might be able to predict which patients benefit from concurrent chemotherapy. It has the potential to guide treatment decisions for patients at different risk of distant metastasis. FUNDING: The National Natural Science Foundation of China, the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period, the Natural Science Foundation of Guang Dong Province, the National Key Research and Development Program of China, the Innovation Team Development Plan of the Ministry of Education, the Health & Medical Collaborative Innovation Project of Guangzhou City, China, and the Program of Introducing Talents of Discipline to Universities.

Survival and Toxicities of IMRT Based on the RTOG Protocols in Patients with Nasopharyngeal Carcinoma from the Endemic Regions of China.

Background: This study evaluated the survival outcomes and toxicities of intensity-modulated radiation therapy (IMRT) based on the RTOG 0225/0615 RT protocols in patients with nasopharyngeal carcinoma (NPC) from a region of China where this tumor type is endemic. Methods: A total of 455 patients with non-metastatic, histologically-confirmed NPC were retrospectively reviewed. All patients were treated by IMRT using the RTOG 0225/0615 RT protocols; 91.1% (288/316) of patients with stage III-IVb NPC received concurrent chemotherapy +/- induction chemotherapy or adjuvant chemotherapy. Results: Estimated four-year overall survival (OS), failure free survival (FFS), local relapse free survival (LRFS), regional relapse free survival (RRFS) and distant metastasis free survival (DMFS) were 83.8%, 80.5%, 94.3%, 96.7% and 85.8%, respectively. T and N category were significant prognostic factors for OS, FFS, RRFS and DMFS; and T category, for LRFS. In-field failure was the major loco-regional failure pattern. During RT, 206 (45.3%) patients experienced acute grade 3-4 toxicities. The most common acute toxicity was mucositis; 124 (27.2%) patients experienced grade 3-4 mucositis; 46 (10.1%) experienced serious late toxicities. The most common late toxicity was MRI-detected radiation-induced temporal lobe necrosis (6.8%). Conclusions: The RTOG IMRT protocols are feasible for patients with NPC from the endemic regions of China.

Prognostic value of total tumor volume in patients with nasopharyngeal carcinoma treated with intensity-modulated radiotherapy.

BACKGROUND: Few studies have evaluated the prognostic value of total tumor volume (TTV), which reflects both the primary tumor volume and nodal tumor volume, in NPC. Furthermore, the relationship between TTV and survival remains unknown. The purpose of this study was to evaluate the prognostic value of TTV in patients with NPC treated with intensity-modulated radiation therapy (IMRT). METHODS: TTV was retrospectively assessed in 455 patients with newly diagnosed, non-metastatic NPC. All patients were treated using IMRT; 91.1% (288/316) of patients with stage III-IVb also received cisplatin-based chemotherapy. Receiver operating characteristic (ROC) curves were used to identify the optimal TTV cut-off point and examine the prognostic value of combined TTV with current clinical stage. RESULTS: Mean TTV was 11.1 cm3 (range, 0.3-27.9 cm3) in stage I, 22.5 cm3 (1.3-92.4 cm3) in stage II, 40.6 cm3 in stage III (3.2-129.2 cm3), and 77.5 cm3 in stage IVa-b (7.1-284.1 cm3). For all patients, the 4-year estimated FFS, OS, DMFS, and LRRFS rates for patients with a TTV ≤ 28 vs. > 28 cm3 were 93 vs. 71.4% (P < 0.001), 95.1 vs. 75.4% (P < 0.001), 94.5 vs. 79.4% (P < 0.001), and 96.2 vs. 88% (P = 0.001). TTV was an independent prognostic factor for FFS, OS, DMFS and LRRFS in all patients. In stage III-IVb, 4-year estimated FFS, OS, DMFS, and LRRFS for a TTV ≤28 vs. >28 cm3 were 88.9 vs. 70.5% (P = 0.001), 96.2 vs. 72.7% (P < 0.001), 91.2 vs. 78.3% (P = 0.008), and 93.8 vs. 87.6% (P = 0.063). TTV was an independent prognostic factor for FFS, OS and DMFS in stage III-IVb. Receiver operating characteristic (ROC) curve analysis curves revealed adding TTV to clinical stage had superior prognostic value for treatment failure compared to clinical stage alone (P = 0.016). CONCLUSIONS: TTV is an important prognosticator for treatment outcome and significantly improves the prognostic value of the current staging system for patients with NPC treated with IMRT.

Application of single-cell technology in cancer research.

In this review, we have outlined the application of single-cell technology in cancer research. Single-cell technology has made encouraging progress in recent years and now provides the means to detect rare cancer cells such as circulating tumor cells and cancer stem cells. We reveal how this technology has advanced the analysis of intratumor heterogeneity and tumor epigenetics, and guided individualized treatment strategies. The future prospects now are to bring single-cell technology into the clinical arena. We believe that the clinical application of single-cell technology will be beneficial in cancer diagnostics and treatment, and ultimately improve survival in cancer patients.