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.

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.

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.

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.

Induction chemotherapy plus concurrent chemoradiotherapy versus concurrent chemoradiotherapy alone in locoregionally advanced nasopharyngeal carcinoma: a phase 3, multicentre, randomised controlled trial.

BACKGROUND: The value of adding cisplatin, fluorouracil, and docetaxel (TPF) induction chemotherapy to concurrent chemoradiotherapy in locoregionally advanced nasopharyngeal carcinoma is unclear. We aimed to compare TPF induction chemotherapy plus concurrent chemoradiotherapy with concurrent chemoradiotherapy alone in a suitably powered trial. METHODS: We did an open-label, phase 3, multicentre, randomised controlled trial at ten institutions in China. Patients with previously untreated, stage III-IVB (except T3-4N0) nasopharyngeal carcinoma, aged 18-59 years without severe comorbidities were enrolled. Eligible patients were randomly assigned (1:1) to receive induction chemotherapy plus concurrent chemoradiotherapy or concurrent chemoradiotherapy alone (three cycles of 100 mg/m2 cisplatin every 3 weeks, concurrently with intensity-modulated radiotherapy). Induction chemotherapy was three cycles of intravenous docetaxel (60 mg/m2 on day 1), intravenous cisplatin (60 mg/m2 on day 1), and continuous intravenous fluorouracil (600 mg/m2 per day from day 1 to day 5) every 3 weeks before concurrent chemoradiotherapy. Randomisation was by a computer-generated random number code with a block size of four, stratified by treatment centre and disease stage (III or IV). Treatment allocation was not masked. The primary endpoint was failure-free survival calculated from randomisation to locoregional failure, distant failure, or death from any cause; required sample size was 476 patients (238 per group). We did efficacy analyses in our intention-to-treat population. The follow-up is ongoing; in this report, we present the 3-year survival results and acute toxic effects. This trial is registered with ClinicalTrials.gov, number NCT01245959. FINDINGS: Between March 1, 2011, and Aug 22, 2013, 241 patients were assigned to induction chemotherapy plus concurrent chemoradiotherapy and 239 to concurrent chemoradiotherapy alone. After a median follow-up of 45 months (IQR 38-49), 3-year failure-free survival was 80% (95% CI 75-85) in the induction chemotherapy plus concurrent chemoradiotherapy group and 72% (66-78) in the concurrent chemoradiotherapy alone group (hazard ratio 0·68, 95% CI 0·48-0·97; p=0·034). The most common grade 3 or 4 adverse events during treatment in the 239 patients in the induction chemotherapy plus concurrent chemoradiotherapy group versus the 238 patients in concurrent chemoradiotherapy alone group were neutropenia (101 [42%] vs 17 [7%]), leucopenia (98 [41%] vs 41 [17%]), and stomatitis (98 [41%] vs 84 [35%]). INTERPRETATION: Addition of TPF induction chemotherapy to concurrent chemoradiotherapy significantly improved failure-free survival in locoregionally advanced nasopharyngeal carcinoma with acceptable toxicity. Long-term follow-up is required to determine long-term efficacy and toxicities. FUNDING: Shenzhen Main Luck Pharmaceuticals Inc, Sun Yat-sen University Clinical Research 5010 Program (2007037), National Science and Technology Pillar Program during the Twelfth Five-year Plan Period (2014BAI09B10), Health & Medical Collaborative Innovation Project of Guangzhou City (201400000001), Planned Science and Technology Project of Guangdong Province (2013B020400004), and The National Key Research and Development Program of China (2016YFC0902000).

Is maximum primary tumor diameter still a prognostic factor in patients with nasopharyngeal carcinoma treated using intensity-modulated radiotherapy?

BACKGROUND: Intensity-modulated radiation therapy (IMRT) has represented a technical milestone that has facilitated the clinical implementation. The purpose of this study was to evaluate the prognostic value of maximum primary tumor diameter (MPTD) in patients with nasopharyngeal carcinoma (NPC) treated using IMRT. METHODS: Five-hundred and sixty-six patients with non-metastatic, histologically-confirmed NPC were retrospectively reviewed. MPTD was measured using magnetic resonance imaging (MRI). All patients were treated using IMRT; 87.5% (456/521) of patients with Stage T3-T4/N1-N3 disease also received cisplatin-based chemotherapy. Receiver operating characteristic (ROC) curves were used to identify the optimal MPTD cut-off point and examine the prognostic value of combining MPTD with the current T classification criteria. RESULTS: Median follow-up for all patients was 36 months (range, 1-52 months). The 3-year overall survival (OS), failure-free survival (FFS), distant metastasis-free survival (DMFS), and local relapse-free survival (LRFS) rates for patients with a MPTD ≤41 vs. >41 mm were 96.1% vs. 85.4%, 93.7% vs. 74.7%, 96.1% vs. 79.7%, and 98.1% vs. 92.9%, respectively (all P < 0.05). In multivariate analysis, MPTD was an independent prognostic factor for OS, FFS, DMFS and LRFS in all patients (all P < 0.05). Among stage T3-T4 patients, the 3-year OS, FFS, DMFS, and LRFS rates for patients with a MPTD ≤41 vs. >41 mm were 96.9% vs. 84.5%, 95.4% vs. 73.5%, 96.1% vs. 79.2%, and 99.3% vs. 92.6%, respectively (all P < 0.05). In multivariate analysis, MPTD was also an independent prognostic factor for OS, FFS and DMFS in stage T3-T4 patients (all P < 0.05), and the difference in LRFS was almost statistically significant (P = 0.05). ROC curves verified that inclusion of MPTD improved the predictive value of the current T classification criteria (P < 0.001). CONCLUSIONS: MPTD was an independent prognostic factor in patients with NPC treated using IMRT, and significantly improved the prognostic value of the current T classification criteria for NPC.

Primary tumor regression speed after radiotherapy and its prognostic significance in nasopharyngeal carcinoma: a retrospective study.

BACKGROUND: To observe the primary tumor (PT) regression speed after radiotherapy (RT) in nasopharyngeal carcinoma (NPC) and evaluate its prognostic significance. METHODS: One hundred and eighty-eight consecutive newly diagnosed NPC patients were reviewed retrospectively. All patients underwent magnetic resonance imaging and fiberscope examination of the nasopharynx before RT, during RT when the accumulated dose was 46-50 Gy, at the end of RT, and 3-4 months after RT. RESULTS: Of 188 patients, 40.4% had complete response of PT (CRPT), 44.7% had partial response of PT (PRPT), and 14.9% had stable disease of PT (SDPT) at the end of RT. The 5-year overall survival (OS) rates for patients with CRPT, PRPT, and SDPT at the end of RT were 84.0%, 70.7%, and 44.3%, respectively (P < 0.001, hazard ratio [HR] = 2.177, 95% confidence interval [CI] = 1.480-3.202). The 5-year failure-free survival (FFS) and distant metastasis-free survival (DMFS) rates also differed significantly (87.8% vs. 74.3% vs. 52.7%, P = 0.001, HR = 2.148, 95% CI, 1.384-3.333; 91.7% vs. 84.7% vs. 66.1%, P = 0.004, HR = 2.252, 95% CI = 1.296-3.912). The 5-year local relapse-free survival (LRFS) rates were not significantly different (95.8% vs. 86.0% vs. 81.8%, P = 0.137, HR = 1.975, 95% CI, 0.976-3.995). By multivariate analyses, the PT regression speed at the end of RT was the only independent prognostic factor of OS, FFS, and DMFS (P < 0.001, P = 0.001, and P = 0.004, respectively). The 5-year FFS rates for patients with CRPT during RT and CRPT only at the end of RT were 80.2% and 97.1%, respectively (P = 0.033). For patients with persistent PT at the end of RT, the 5-year LRFS rates of patients without and with boost irradiation were 87.1% and 84.6%, respectively (P = 0.812). CONCLUSIONS: PT regression speed at the end of RT was an independent prognostic factor of OS, FFS, and DMFS in NPC patients. Immediate strengthening treatment may be provided to patients with poor tumor regression at the end of RT.

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.

Progress report of a randomized trial comparing long-term survival and late toxicity of concurrent chemoradiotherapy with adjuvant chemotherapy versus radiotherapy alone in patients with stage III to IVB nasopharyngeal carcinoma from endemic regions of China.

BACKGROUND: The objective of this study was to evaluate the long-term survival and late toxicities of concurrent-adjuvant chemotherapy in patients with stage III through IVB nasopharyngeal carcinoma (NPC) from endemic regions of China. METHODS: Patients with stage III to IVB NPC were assigned randomly to receive radiotherapy (RT) alone (the RT group) or RT plus concurrent adjuvant chemotherapy (the CRT group). CRT patients received concurrent cisplatin (40 mg/m2) weekly during RT followed by cisplatin (80 mg/m2) and fluorouracil (800 mg/m(2) daily for 5 days) every 4 weeks for 3 cycles. The primary endpoint was overall survival. RESULTS: In total, 316 patients underwent randomization, with 158 to each group. At a median follow-up of 70 months, the 5-year overall survival rate was 72% for the CRT group and 62% for the RT group (hazard ratio, 0.69; 95% confidence interval, 0.48-0.99; P = .043). Failure-free survival was significantly higher in the CRT group (P = .020). Most late toxicities were similar (33% vs. 26%; P = .089), except for cranial neuropathy (P = .042), peripheral neuropathy (P = .041), and ear damage (P = .048), which were significantly increased in the CRT group. CONCLUSIONS: The addition of concurrent adjuvant chemotherapy to RT provides survival benefits to patients with stage III through IVB NPC in endemic regions of China, and it does not increase most late toxicities apart from cranial neuropathy, peripheral neuropathy, and ear damage.

Prognostic significance of maximum primary tumor diameter in nasopharyngeal carcinoma.

BACKGROUND: To evaluate the prognostic value of maximum primary tumor diameter (MPTD) in nasopharyngeal carcinoma (NPC). METHODS: Three hundred and thirty-three consecutive, newly-diagnosed NPC patients were retrospectively reviewed. Kaplan-Meier analysis and the log-rank test were used to estimate overall survival (OS), failure-free survival (FFS), distant metastasis-free survival (DMFS) and local relapse-free survival (LRFS). Cox proportional hazards regression analysis was used to assess the prognostic value of MPTD. RESULTS: Median follow-up was 66 months (range, 2-82 months). Median MPTD in stage T1, T2, T3 and T4 was 27.9, 37.5, 45.0 and 61.3 mm, respectively. The proportion of T1 patients with a MPTD ≤ 30 mm was 62.3%; 72% and 62.9% of T2 and T3 patients had a MPTD > 30-50 mm, and 83.5% of T4 patients had a MPTD > 50 mm. For patients with a MPTD ≤ 30 mm, > 30-50 mm and > 50 mm, the 5-year OS, FFS, DMFS and LRFS rates were 85.2%, 74.2% and 56.3% (P < 0.001); 87%, 80.7% and 62.8% (P < 0.001); 88.7%, 86.4% and 72.5% (P = 0.003); and 98.2%, 93.2% and 86.3% (P = 0.012), respectively. In multivariate analysis, MPTD was a prognostic factor for OS, FFS and DMFS, and the only independent prognostic factor for LRFS. For T3-T4 patients with a MPTD ≤ 50 mm and > 50 mm, the 5-year OS, FFS and DMFS rates were 70.4% vs. 58.4% (P = 0.010), 77.5% vs. 65.2% (P = 0.013) and 83.6% vs. 73.6% (P = 0.047), respectively. In patients with a MPTD ≤ 30 mm, 5-year LRFS in T1, T2, T3 and T4 was 100%, 100%, 88.9% and 100% (P = 0.172). CONCLUSIONS: Our data suggest that MPTD is an independent prognostic factor in NPC, and incorporation of MPTD might lead to a further refinement of T staging.

Concurrent chemoradiotherapy plus adjuvant chemotherapy versus concurrent chemoradiotherapy alone in patients with locoregionally advanced nasopharyngeal carcinoma: a phase 3 multicentre randomised controlled trial.

BACKGROUND: The effect of the addition of adjuvant chemotherapy to concurrent chemoradiotherapy in locoregionally advanced nasopharyngeal carcinoma is unclear. We aimed to assess the contribution of adjuvant chemotherapy to concurrent chemoradiotherapy versus concurrent chemoradiotherapy alone. METHODS: We did an open-label phase 3 multicentre randomised controlled trial at seven institutions in China. Randomisation was by a computer-generated random number code. Patients were stratified by treatment centre and randomly assigned in blocks of four. Treatment allocation was not masked. We randomly assigned patients with non-metastatic stage III or IV (except T3-4N0) nasopharyngeal carcinoma to receive concurrent chemoradiotherapy plus adjuvant chemotherapy or concurrent chemoradiotherapy alone. Patients in both groups received 40 mg/m(2) cisplatin weekly up to 7 weeks, concurrently with radiotherapy. Radiotherapy was given as 2·0-2·27 Gy per fraction with five daily fractions per week for 6-7 weeks to a total dose of 66 Gy or greater to the primary tumour and 60-66 Gy to the involved neck area. The concurrent chemoradiotherapy plus adjuvant chemotherapy group subsequently received 80 mg/m(2) adjuvant cisplatin and 800 mg/m(2) per day fluorouracil for 120 h every 4 weeks for three cycles. Our primary endpoint was failure-free survival. We did efficacy analyses in our intention-to-treat population. Our trial is ongoing; in this report we present the 2 year survival results and acute toxic effects. This trial is registered with ClinicalTrials.gov, number NCT00677118. FINDINGS: 251 patients were assigned to the concurrent chemoradiotherapy plus adjuvant chemotherapy group and 257 to the concurrent chemoradiotherapy alone group. After a median follow-up of 37·8 months (range 1·3-61·0), the estimated 2 year failure-free survival rate was 86% (95% CI 81-90) in the concurrent chemoradiotherapy plus adjuvant chemotherapy group and 84% (78-88) in concurrent chemoradiotherapy only group (hazard ratio 0·74, 95% CI 0·49-1·10; p=0·13). Stomatitis was the most commonly reported grade 3 or 4 adverse event during both radiotherapy (76 of 249 patients in the concurrent chemoradiotherapy plus adjuvant chemotherapy group and 82 of 254 in the concurrent chemoradiotherapy alone group) and adjuvant chemotherapy (43 [21%] of 205 patients treated with adjuvant chemotherapy). INTERPRETATION: Adjuvant cisplatin and fluorouracil chemotherapy did not significantly improve failure-free survival after concurrent chemoradiotherapy in locoregionally advanced nasopharyngeal carcinoma. Longer follow-up is needed to fully assess survival and late toxic effects, but such regimens should not, at present, be used outside well-designed clinical trials. FUNDING: Sun Yat-sen University Clinical Research 5010 Programme (No 2007037), Science Foundation of Key Hospital Clinical Programme of Ministry of Health PR China (No 2010-178), and Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2010).

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.

The N staging system in nasopharyngeal carcinoma with radiation therapy oncology group guidelines for lymph node levels based on magnetic resonance imaging.

PURPOSE: To evaluate the prognostic value of variables including nodal size, level, laterality, extranodal neoplastic spread (ENS), and necrosis in patients with nasopharyngeal carcinoma (NPC) and further explore the feasibility of an N-staging system using Radiation Therapy Oncology Group (RTOG) guidelines for lymph node levels based on magnetic resonance imaging (MRI). EXPERIMENTAL DESIGN: The MRI scans of 924 patients with histologically diagnosed nondisseminated NPC were reviewed retrospectively. The distribution of the tumors was mapped using RTOG guidelines and laterality. The multiplicity of each tumor was calculated, as well as the size and status of ENS and the necrosis of individual nodes. RESULTS: Nodal level, cervical lymph node laterality, and ENS were independent prognostic factors for disease failure and distant failure in multivariate analyses. There was no significant difference in the hazard ratios (HR) for distant failure between level II and retropharyngeal, level Ib, level V, or level III involvement, whereas patients with level IV and supraclavicular fossa involvement had a significant increase in HRs. The subsets that made up a given N stage group had similar HRs for distant failure. Both the HRs for disease failure and distant failure by the proposed N staging system between one stage and the next were statistically significant (P < 0.05). The survival curves of disease-free survival and distant metastasis-free survival for all subclassifications of N stage showed significant difference from the adjacent stage (P < 0.05). The overall distribution pattern of the proposed N staging was more equitable than that of the 6th American Joint Committee on Cancer N staging. CONCLUSIONS: Nodal variables including level, cervical lymph node laterality, and ENS are independent prognostic factors for NPC. The proposed N staging system of NPC using RTOG guidelines based on MRI is highly predictive and may provide a more objective method for staging NPCs.

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.