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Supplementary Figure 2 (a) The four potential binding sites (position 931,1497,1522,1635) on the 3'UTR were predicted by the PITA analysis. Bases labeled blue were the mutated nucleotides of potential binding sites. (b) Downregulated RND3 expression in BART2 expressing CNE2 cells was revealed by western blot. (c) RIP assay was performed with CNE2 cells with Vector or BART2 expression. The RT-PCR revealed that RND3 3'UTR was enriched on the RISC complex in BART2 expressing cells, but not in cells carrying vector. Input: cell lysate before immunoprecipitation. NC: precipitates pulled down by isotype control mouse IgG. Ago2: precipitates pulled down by anti-Ago2 antibody. Ago2 is core component of RISC. (d) Quality control of RIP assay. Western Blot confirmed that Ago2 was successfully and specifically pulled down by anti-Ago2 antibody. Light and heavy chains of primary antibody were also detected. (e) RND3 expression was inhibited in lung metastatic lesions formed by BART2 expressing cells. (f) Knock down efficiency of RND3 by shRNA was determined by western blot. (Left) Migration and invasion assays were performed to evaluate cell motility after knock down RND3. Migrated or invaded cells were counted and statistically compared in the bar chart. (Middle and Right) (g) Successful reconstitution of RND3 was confirmed by western blot. (Left) Cell migration and invasion was evaluated by transwell assay. Migrated or invaded cells were counted and statistically compared in the bar chart. (Middle and Right) (h) HONE-1 EBVï¼^+）and HONE-1 cells were transfected with Control mimic/siRNA targeting BART2-5p or infected by lentivirus to establish stable cell lines with Vector/RND3 expression. Migration assays were performed 8h after transient transfection or once Blasticidin selection finished. Migrated cells were counted under microscope for following statistical analysis. (i) HONE-1 EBVï¼^+）and HONE-1 cells were transfected with Control mimic/siRNA targeting BART2-5p or infected by lentivirus to establish stable cell lines with Vector/RND3 expression. Cell invasiveness was evaluated 8h after transient transfection or once Blasticidin selection finished. Invaded cells were counted under microscope for subsequent statistical analysis.

Supplementary tables include: Table.S1 Primers Table.S2 Clinical data of NPC patients Table.S3 Antibody list Table.S4 Multivariate analysis of Progression Free Time in nasopharyngeal carcinoma patients Table.S5 Association of expression level of Bart 2-5p with patients' clinical features in nasopharyngeal carcinoma Table.S6 Summary of metastatic status in spleen injection model

Nasopharyngeal carcinoma is an Epstein–Barr virus (EBV)-related malignancy. Recently, we found that the EBV-encoded miRNA BART2-5p was increased in the serum of patients with preclinical nasopharyngeal carcinoma and that the copy number positively correlated with disease progression. In this study, we established its role in nasopharyngeal carcinoma progression and explored underlying mechanisms and clinical significance. BART2-5p was an independent unfavorable prognostic factor for progression-free survival and its circulating abundance positively associated with distant metastasis. Ectopic expression of BART2-5p promoted migration and invasion of EBV-negative nasopharyngeal carcinoma cells, whereas genetic downregulation of BART2-5p in EBV-positive nasopharyngeal carcinoma cells decreased aggressiveness. Mechanistically, BART2-5p targeted RND3, a negative regulator of Rho signaling. Downregulation of RND3 phenocopied the effect of BART2-5p and reconstitution of RND3 rescued the phenotype. By suppressing RND3, BART2-5p activated Rho signaling to enhance cell motility. These findings suggest a novel role for EBV miRNA BART2-5p in promoting nasopharyngeal carcinoma metastasis and its potential value as a prognostic indicator or therapeutic target.Significance:This study shows that EBV-encoded BART2-5p miRNA suppresses expression of the RND3 Rho family GTPase, consequently promoting ROCK signaling, cell motility, and metastatic behavior of NPC cells.

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Supplementary Figure from Exploratory Study of NPC-0501 Trial: Optimal Cisplatin Dose of Concurrent and Induction/Adjuvant Chemotherapy for Locoregionally Advanced Nasopharyngeal Carcinoma

Purpose: The current recommendation for patients with locoregionally advanced nasopharyngeal carcinoma (NPC) is cisplatin-based induction chemotherapy (IC) or adjuvant chemotherapy (AC) plus concurrent chemoradiotherapy (CRT). However, data on the optimal platinum doses for each phase of combined regimens are lacking. Experimental Design: 742 patients with NPC in the NPC-0501 trial treated with CRT plus IC/AC and irradiated with intensity-modulated radiotherapy (IMRT) were analyzed. The optimal platinum dose to achieve the best overall survival (OS) in the concurrent and induction/adjuvant phases was studied. Results: Evaluation of the whole series shows the optimal platinum dose was 160 mg/m2 in the concurrent and 260 mg/m2 in the induction/adjuvant phase. Repeating the analyses on 591 patients treated with cisplatin throughout (no replacement by carboplatin) confirmed the same results. The cohort with optimal platinum doses in both phases had better OS than the cohort suboptimal in both phases (stage III: 90% vs. 75%; stage IVA–B: 80% vs. 56%, at 5-year). Multivariable analyses confirmed optimal platinum doses in both phases versus suboptimal dose in each phase are significant independent factors for OS, with HR of 0.61 [95% confidence interval (CI), 0.41–0.91] and 0.67 (95% CI, 0.48–0.94), respectively. Treatment sequence was statistically insignificant after adjusting for platinum doses. Conclusions: Both concurrent and IC/AC are needed for locoregionally advanced NPC, even for patients irradiated by IMRT; the concurrent platinum dosage could be set at ≥160 mg/m2 when coupled with adequate induction/adjuvant dosage at ≥260 mg/m2 (or at least ≥240 mg/m2). To achieve these optimal dosages, IC-CRT at conventional fractionation is favored.

Background Tuberculosis (TB) reactivation has been increasingly identified following immune checkpoint inhibitor (ICI) therapy for cancer patients. However there has been no report on TB reactivation in the gastrointestinal tract. In the report, we describe a patient who developed TB ileitis after pembrolizumab for her metastatic nasopharyngeal carcinoma (NPC). Rechallenge with pembrolizumab after its temporary interruption together with anti-TB therapy produced continuous tumor response but without further TB reactivation. Case presentation A 29-year-old lady with metastatic NPC involving the cervical nodes, lungs and bones started pembrolizumab after failure to multiple lines of chemotherapy. She complained of sudden onset of abdominal pain, vomiting and bloody diarrhea with mucus 21 months after pembrolizumab. Colonoscopy revealed terminal ileitis with multiple caseating granulomas with Langerhan cells. Serum interferon gamma release assay was strongly positive. She was treated with anti-TB medication and was later rechallenged with pembrolizumab for her progressive lung metastases without further TB relapse while her lung metastases were brought under control again. Conclusion To date, this is the first gastrointestinal TB reactivation after ICI therapy for cancer. Guidelines to screen for TB before initiation of ICIs in endemic areas should be established.

Several B-cell subsets with distinct functions and polarized cytokine profiles that extend beyond antibody production have been reported in different cancers. Here we have demonstrated that proliferating B cells were predominantly found in the peritumoral region of esophageal squamous cell carcinoma (ESCC). These B cells were enriched in tumor nests with high expression of high-mobility group box 1 (HMGB1). High densities of peritumoral proliferating B cells and concomitantly high intratumoral HMGB1 expression showed improved prognostic significance, surpassing prognostic stratification of ESCC patients based on HMGB1 positivity alone. This striking association led us to set up models to test whether cancer-derived HMGB1 could shape tumor microenvironment via modulation on B cells. Overexpression of HMGB1 in ESCC cell lines (KYSE510 and EC18) enhanced proliferation and migration of B cells. Transcriptomic analysis showed that migratory B cells exhibited high enrichment of proangiogenic genes. VEGF expression in proliferating B cells was induced upon co-culture of HMGB1-overexpressing tumor cells and B cells. Secretome array profiling of conditioned media (CM) from the co-culture revealed rich expression of proangiogenic proteins. Consequently, incubation of human umbilical vein endothelial cells with CM promoted angiogenesis in tube formation and migration assays. HMGB1 inhibitor, glycyrrhizin, abolishes all the observed proangiogenic phenotypes. Finally, co-injection of B cells and CM with HMGB1-overexpressing tumor cells, but not with glycyrrhizin, significantly enhanced tumor growth associated with increased microvascular density in ESCC xenograft mice model. Our results indicate that cancer-derived HMGB1 elevates angiogenesis in ESCC by shifting the balance toward proangiogenic signals in proliferating B cells. Supplementary Information The online version contains supplementary material available at 10.1007/s10456-021-09819-0.

BACKGROUND: This study investigated the feasibility of using a computer-assisted method to evaluate and differentiate the carotid plaque characteristics in radiation-induced and non-radiation-induced carotid atherosclerosis. METHODS: This study included 107 post-radiotherapy (post-RT) nasopharyngeal carcinoma (NPC) patients and 110 subjects with cardiovascular risk factors (CVRFs). Each participant had a carotid ultrasound examination, and carotid plaques and carotid intima-media thickness (CIMT) were evaluated with grey scale ultrasound. The carotid plaque characteristics were evaluated for grey-scale median (GSM) and detailed plaque texture analysis (DPTA) using specific computer software. In DPTA, five different intra-plaque components were colour-coded according to different grey scale ranges. A multivariate linear regression model was used to evaluate the correlation of risk factors and carotid plaque characteristics. RESULTS: Post-RT NPC patients have significantly higher CIMT (748±15.1 µm, P=0.001), more patients had a plaque formation (80.4%, P

Objective: The transcription factor forkhead box protein O1 (FOXO1) is critical for regulating cytokine and chemokine secretion. However, its function in the tumor microenvironment (TME) remains largely unexplored. In this study, we characterized the prognostic value of FOXO1 and the interaction between tumor-derived FOXO1 and M2 macrophages in esophageal squamous cell carcinoma (ESCC). Methods: FOXO1 expression and macrophage infiltration in clinical samples and mouse models were quantified using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry staining. Western blotting, qRT-PCR, and enzyme-linked immunosorbent assay were used to evaluate chemokine ligand 20 (CCL20) and colony stimulating factor 1 (CSF-1) expression in FOXO1(+) and FOXO1(-) tumor cells. Macrophage phenotypes were determined using qRT-PCR, flow cytometry, and RNA sequencing. Transcriptional activity was measured using chromatin immunoprecipitation (ChIP)-qPCR. Tumor viability was investigated using XTT proliferation and foci formation assays. Results: FOXO1 upregulation in tumor tissues was found to drive the polarization of M0 macrophages and infiltration of M2 macrophages into the TME, resulting in worse prognosis in ESCC patients. CSF-1, a vital factor inducing M0-to-M2 polarization, was upregulated via a FOXO1-mediated mechanism. RNA sequencing results corroborated that the FOXO1-induced macrophages exhibited similar molecular signatures to the IL4-stimulated M2 macrophages. The transwell assays showed that FOXO1 promoted the migration of M2 macrophages via CCL20 secretion, which could be inhibited using an anti-CCL20 antibody. FOXO1(+) tumor-induced M2 macrophages promoted tumor proliferation via the FAK-PI3K-AKT pathway and the PI3K inhibitor could effectively impede the oncogenical process. Conclusions: FOXO1 facilitated M0-to-M2 polarization and the recruitment of M2 macrophages in the TME via the transcriptional modulation of CCL20 and CSF-1. Our data deciphered the FOXO1-dependent mechanism in M2 macrophage infiltration in the TME of ESCC, which has implications for the development of novel prognostic and therapeutic targets to optimize the current treatment against ESCC.

Objective This study compared the efficacy of PF-based and CROSS-based neoadjuvant chemoradiotherapy for ESCC. Background PF-based regimen has been a standard regimen for ESCC, but it has been replaced by the CROSS regimen in the past few years, despite no prospective head-to-head comparative study has been performed. Methods This is a single center retrospective study. Records of all ESCC patients who have received neoadjuvant PF with 40 Gy radiotherapy in 20 daily fractions (PFRT Group) or CROSS with 41.4 Gy radiotherapy in 23 daily fractions (CROSS Group) during the period 2002 to 2019 were retrieved. Propensity score matching (1:1) was performed to minimize baseline differences. The primary and secondary endpoints were overall survival and clinicopathological response. Subgroup analysis ("CROSS Eligibility") was performed based on tumor length, cT-stage, cM-stage, age, and performance status. Results One hundred (out of 109) patients (CROSS group) and propensity score matched 100 (out of 210) patients (PFRT group) were included. Esophagectomy rates in CROSS and PFRT group were 69% and 76%, respectively (P = 0.268). R0 resection rates were 85.5% and 81.6% (P = 0.525) and the pathological complete remission rates were 24.6% and 35.5% (P = 0.154). By intention-to-treat, the median survival was 16.7 and 32.7 months (P = 0.083). For "CROSS Eligible subgroup," the median survival of the CROSS and PFRT group was 21.6 versus 44.9 months (P = 0.093). Conclusions There is no statistically difference in survival or clinicopathological outcome between both groups, but the trend favors PFRT. Prospective head-to-head comparison and novel strategies to improve the outcomes in resectable ESCC are warranted.

Preoperative chemoradiation (CROSS regimen) has been widely adopted worldwide. The survival advantage imparted is especially impressive for oesophageal squamous cell carcinoma (OSCC). This study aimed at investigating the efficacy of the CROSS regimen in real-world scenario. This is a retrospective study of all patients with OSCC intended for preoperative treatment using the CROSS regimen during 2012–2017. Patients were divided into two groups: those within the selection criteria in the CROSS trial and those beyond criteria, namely age > 75 years old, tumour length > 8 cm or clinical M1 stage of lymph node involvement (AJCC 6th edition). Clinical outcome and survival data were compared. Eighty-eight patients were included. There were 46 patients in the “CROSS eligible” group and 42 in the “CROSS ineligible” group. By intention-to-treat, the estimated median survival was 24.2 months vs. 12.7 months, respectively (p = 0.047). The results were much inferior compared to that published in the original CROSS trial. Univariable and multivariable analyses showed tumour length and resectability as independent variables affecting survival. In a real-world scenario, the clinical outcome remains suboptimal and the excellent results in the trial setting were not reproducible in this Asian cohort. Patient selection is one key element accountable for the difference. The efficacy of the CROSS regimen may not be adequate for patients with more advanced disease. The optimal multimodal therapy for this group of patients remains undefined.

Background A current recommendation for the treatment of patients with locoregionally advanced nasopharyngeal carcinoma (NPC) is conventional fractionated radiotherapy (RT) with concurrent cisplatin followed by adjuvant cisplatin and 5-fluorouracil (PF). This randomized NPC-0501 trial evaluated the therapeutic effect of changing to an induction-concurrent sequence or accelerated-fractionation sequence, and/or replacing 5-fluorouracil with capecitabine (X). Methods Patients with American Joint Committee on Cancer/International Union Against Cancer stage III to stage IVB NPC initially were randomly allocated to 1 of 6 treatment arms (6-arm full-randomization cohort). The protocol was amended in 2009 to permit centers to opt out of randomization regarding fractionation (3-arm chemotherapy cohort). Results A total of 803 patients were accrued (1 of whom was nonevaluable) from 2006 to 2012. Based on the overall comparisons, neither changing the chemotherapy sequence nor accelerated fractionation improved treatment outcome. However, secondary analyses demonstrated that when adjusted for RT parameters and other significant factors, the induction-concurrent sequence, especially the induction-PX regimen, achieved significant improvements in progression-free survival (PFS) and overall survival. Efficacy varied among different RT groups: although no impact was observed in the accelerated-fractionation group and the 3-arm chemotherapy cohort, a comparison of the induction-concurrent versus concurrent-adjuvant sequence in the conventional-fractionation group demonstrated a significant benefit in PFS (78% vs 62% at 5 years; P = .015) and a marginal benefit in overall survival (84% vs 72%; P = .042) after adjusting for multiple comparisons. Comparison of the induction-PX versus the adjuvant-PF regimen demonstrated better PFS (78% vs 62%; P = .027) without an increase in overall late toxicity. Conclusions For patients irradiated using conventional fractionation, changing the chemotherapy sequence from a concurrent-adjuvant to an induction-concurrent sequence, particularly using induction cisplatin and capecitabine, potentially could improve efficacy without an adverse impact on late toxicity. However, further validation is needed for confirmation of these findings.

Nasopharyngeal carcinoma is an Epstein–Barr virus (EBV)-related malignancy. Recently, we found that the EBV-encoded miRNA BART2-5p was increased in the serum of patients with preclinical nasopharyngeal carcinoma and that the copy number positively correlated with disease progression. In this study, we established its role in nasopharyngeal carcinoma progression and explored underlying mechanisms and clinical significance. BART2-5p was an independent unfavorable prognostic factor for progression-free survival and its circulating abundance positively associated with distant metastasis. Ectopic expression of BART2-5p promoted migration and invasion of EBV-negative nasopharyngeal carcinoma cells, whereas genetic downregulation of BART2-5p in EBV-positive nasopharyngeal carcinoma cells decreased aggressiveness. Mechanistically, BART2-5p targeted RND3, a negative regulator of Rho signaling. Downregulation of RND3 phenocopied the effect of BART2-5p and reconstitution of RND3 rescued the phenotype. By suppressing RND3, BART2-5p activated Rho signaling to enhance cell motility. These findings suggest a novel role for EBV miRNA BART2-5p in promoting nasopharyngeal carcinoma metastasis and its potential value as a prognostic indicator or therapeutic target. Significance: This study shows that EBV-encoded BART2-5p miRNA suppresses expression of the RND3 Rho family GTPase, consequently promoting ROCK signaling, cell motility, and metastatic behavior of NPC cells.

Purpose: We hypothesized that radiation-induced lymphopenia could be predicted by the effective dose to the circulating immune cells (EDIC) in advanced esophageal squamous cell carcinoma treated with trimodality therapy according to the Dutch ChemoRadiotherapy for Oesophageal cancer followed by Surgery Study (CROSS) trial regimen. To test this hypothesis, we examined the effect of EDIC on the degree of lymphocyte drop (lymphocyte nadir). Methods and Materials: Patients with advanced nonmetastatic esophageal squamous cell carcinoma treated in a single tertiary cancer center from 2012 to 2018 were eligible for this study. All patients had to have a radiation therapy plan available for EDIC computation and received neoadjuvant chemoradiation according to the Dutch CROSS trial regimen before radical esophagectomy. The EDIC was calculated as a function of integral doses to the lung, heart, and total body with a verified mathematical model. The association between EDIC and lymphocyte nadir was studied, and the relationships of overall survival (OS) with lymphocyte nadir and EDIC were assessed using multivariable Cox regression model. Results: This analysis included 92 eligible consecutive patients (77 men and 15 women). The mean EDIC was 2.8 Gy (range, 0.6-4.4). EDIC was significantly correlated with lymphocyte nadir (Spearman coefficient = –0.505; P < .01), and lymphocyte nadir was a significant independent factor for shorter OS (hazard ratio = 0.63; P < .001). Lymphocyte nadir was also the most significant factor in determining OS among other clinical parameters. Exploratory analysis showed significant OS differences between EDIC groups (4 Gy). The 2–year OS rates were 66.7%, 42.7%, and 16.7% for EDIC 4 Gy, respectively. Conclusions: There was a significant correlation between radiation dose to circulating immune cells and lymphocyte nadir, which in turn affected OS in patients with advanced nonmetastatic esophageal squamous cell carcinoma treated by trimodality therapy.

The evolution of the tumor microenvironment (TME) is a cancer-dependent and dynamic process. The TME is often a complex ecosystem with immunosuppressive and tumor-promoting functions. Conventional chemotherapy and radiotherapy, primarily focus on inducing tumor apoptosis and hijacking tumor growth, whereas the tumor-protective microenvironment cannot be altered or destructed. Thus, tumor cells can quickly escape from extraneous attack and develop therapeutic resistance, eventually leading to treatment failure. As an Epstein Barr virus (EBV)-associated malignancy, nasopharyngeal carcinoma (NPC) is frequently infiltrated with varied stromal cells, making its microenvironment a highly heterogeneous and suppressive harbor protecting tumor cells from drug penetration, immune attack, and facilitating tumor development. In the last decade, targeted therapy and immunotherapy have emerged as promising options to treat advanced, metastatic, recurrent, and resistant NPC, but lack of understanding of the TME had hindered the therapeutic development and optimization. Single-cell sequencing of NPC-infiltrating cells has recently deciphered stromal composition and functional dynamics in the TME and non-malignant counterpart. In this review, we aim to depict the stromal landscape of NPC in detail based on recent advances, and propose various microenvironment-based approaches for precision therapy.

Emerging efficacy and safety data have led to the authorization of COVID-19 vaccines worldwide, but most trials excluded patients with active malignancies. This study evaluates the intended acceptance of COVID-19 vaccination in cancer patients in Hong Kong. Methods: 660 adult cancer patients received a survey, in paper or electronic format, between 31 January 2021 and 15 February 2021. The survey included patient’s clinical characteristics, perceptions of COVID-19 and vaccination, vaccine knowledge, cancer health literacy, and Hospital Anxiety and Depression scale (HADS). The primary outcome was the intended acceptance of COVID-19 vaccine in cancer patients. Multivariable analysis was performed to identify factors associated with intended acceptance. Results: The intended acceptance of COVID-19 vaccination was 17.9%. A total of 487 (73.8%) believed that vaccination could prevent them from infection. Over 70% worried about vaccine negative effects on cancer and its side effects. Factors associated with intended acceptance included higher level of “belief in vaccine on preventing them from getting COVID-19”, less worry about long-term side effects of vaccine, lower level of cancer health literacy, and normal HADS (Depression scale). Conclusions: To improve vaccine acceptance rate, public education campaigns specific to cancer patients to gain their trust in efficacy and relieve their worries are needed.

Purpose: Reirradiation for locally recurrent nasopharyngeal carcinoma (NPC) is challenging because prior radiation dose delivered in the first course is often close to the tolerance limit of surrounding normal structures. A delicate balance between achieving local salvage and minimizing treatment toxicities is needed. However, high-level evidence is lacking because available reports are mostly retrospective studies on small series of patients. Pragmatic consensus guidelines, based on an extensive literature search and the pooling of opinions by leading specialists, will provide a useful reference to assist decision-making for these difficult decisions. Methods and Materials: A thorough review of available literature on recurrent NPC was conducted. A set of questions and preliminary draft guideline was circulated to a panel of international specialists with extensive experience in this field for voting on controversial areas and comments. A refined second proposal, based on a summary of the initial voting and different opinions expressed, was recirculated to the whole panel for review and reconsideration. The current guideline was based on majority voting after repeated iteration for final agreement. Results: The initial round of questions showed variations in clinical practice even among the specialists, reflecting the lack of high-quality supporting data and the difficulties in formulating clinical decisions. Through exchange of comments and iterative revisions, recommendations with high-to-moderate agreement were formulated on general treatment strategies and details of reirradiation (including patient selection, targets contouring, dose prescription, and constraints). Conclusion: This paper provides useful reference on radical salvage treatment strategies for recurrent NPC and optimization of reirradiation through review of published evidence and consensus building. However, the final decision by the attending clinician must include full consideration of an individual patient's condition, understanding of the delicate balance between risk and benefits, and acceptance of risk of complications. (C) 2021 Elsevier Inc. All rights reserved.

Introduction Older patients experience a higher risk of treatment-related toxicity (TRT). The G8 screening tool was developed to separate cancer older patients fit to receive standard treatment from those who are frail and experiencing functional decline due to reduced organ function and multiple comorbidities. The Cancer and Aging Research Group chemotherapy toxicity tool (CARG-tt) questionnaire was developed to predict chemotherapy toxicity in geriatric patients. This prospective observational study evaluated the performance of G8 and CARG-tt in predicting severe TRT in older Chinese cancer patients. Methods Chinese patients aged ≥65 with a diagnosis of solid malignancy and scheduled to receive anti-cancer treatment (chemotherapy or targeted therapy) were enrolled from March 2016 to July 2017 at the Department of Clinical Oncology at Queen Mary Hospital in Hong Kong. All patients completed the G8 and CARG-tt screening and pre-treatment assessments before starting treatment. Patients were monitored for any severe TRT, which was defined by grades 3–5 using the National Cancer Institute's Common Terminology Criteria for Adverse Events v4.03, treatment discontinuation, or unexpected hospitalization from starting to 30 days after treatment. Results A total of 259 patients (male: 154, 59.5%; median age: 73.4, age range: 65–93) were enrolled in the study. Two hundred and ten (81.1%) patients received chemotherapy while the rest (n = 49, 18.9%) received targeted therapy. Overall, 146 patients (56.8%) experienced severe TRT. The mean G8 score was 12.4 (SD: 2.8). The G8 score had a significant association with unexpected admission (cutoff: 14, 41.3% vs. 26.5%, p = 0.03) but not significant in other types of TRTs. The mean CARG-tt score was 7.67 (SD: 3.7); it was not associated with severe TRTs. Conclusions The G8 and CARG-tt demonstrated a weak prediction of severe TRT in older Chinese cancer patients. Future studies need to develop predictive tools for TRT in patients receiving novel antineoplastic therapies, with a focus on subgroup analysis for different populations.

Background Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) in endemic regions may have undetectable plasma EBV DNA. Methods We prospectively recruited 518 patients with non-metastatic NPC and measured their pre-treatment plasma EBV DNA. The stage distribution and prognosis between pre-treatment plasma EBV DNA-negative (0–20 copies/ml) and EBV DNA-positive (>20 copies/ml) patients following radical treatment were compared. Results Seventy-eight patients (15.1%) were plasma EBV DNA-negative, and 62 in this subset (12.0%) had 0 copy/ml. Only 23/78 (29.5%) plasma EBV DNA-negative patients with advanced NPC (stage III-IVA) had strong EBV encoded RNA (EBER) positivity (score 3) in their tumours compared to 342/440 (77.7%) EBV DNA-positive patients of the same stages (p p p Conclusions Patients with low-volume NPC may not be identified by plasma/serum tumour markers and caution should be taken in its utility as a screening tool for NPC even in endemic regions. Clinical trial registration Clinicaltrials.gov Identifier: NCT02476669.

Managing head and neck cancers is an excellent example of the importance of teamwork, with head and neck surgeons, clinical oncologists, radiologists, pathologists and other allied health professionals specialised in this disease site working together. The reliable imaging and dedicated pretreatment work-up entailing the comprehensive anatomical description of tumour involvement by the radiologists, the expertise of surgeons in performing en-bloc gross tumour resection, the uneventful speedy postoperative rehabilitation and recovery by the speech therapists and nutritionists, as well as the dedicated treatment planning of clinical oncologists in delivering precise preoperative or postoperative (chemo)radiotherapy to maximise the therapeutic potentials are the pillars of treatment success. A multidisciplinary tumour board involving all of these key players is essential to provide the highest level of recommendation based on evidence-based medicine and to bring patients new hopes and the best chance of cure. This review illustrates the seamless collaborative teamwork within a well-established multidisciplinary tumour board in managing one of the most intractable cancers in the East, taking enlightenment and inspiration from the West.

Deletion of Chromosome 3p is one of the most frequently detected genetic alterations in nasopharyngeal carcinoma (NPC). We reported the role of a novel 3p26.3 tumor suppressor gene (TSG) CHL1 in NPC. Down-regulation of CHL1 was detected in 4/6 of NPC cell lines and 71/95 (74.7%) in clinical tissues. Ectopic expressions of CHL1 in NPC cells significantly inhibit colony formation and cell motility in functional study. By up-regulating epithelial markers and down-regulating mesenchymal markers CHL1 could induce mesenchymal-epithelial transition (MET), a key step in preventing tumor invasion and metastasis. CHL1 could also cause the inactivation of RhoA/Rac1/Cdc42 signaling pathway and inhibit the formation of stress fiber, lamellipodia, and filopodia. CHL1 could co-localize with adhesion molecule Integrin-β1, the expression of CHL1 was positively correlated with Integrin-β1 and another known tumor suppressor gene (TSG) Merlin. Down-regulation of Integrin-β1 or Merlin was significantly correlated with the poor survival rate of NPC patients. Further mechanistic studies showed that CHL1 could directly interact with integrin-β1 and link to Merlin, leading to the inactivation of integrin β1-AKT pathway. In conclusion, CHL1 is a vital tumor suppressor in the carcinogenesis of NPC.

Summary Background & aims Little is known about the risk of nasopharyngeal carcinoma (NPC) in relation to vitamin D exposure. The aim of this study was to examine the associations of NPC risk with serum level of 25-hydroxyvitamin D (25OHD) and genetic predicted 25OHD, and potential effect modification by several putative risk factors of NPC. Methods Our multicenter case–control study in Hong Kong recruited 815 NPC cases and 1502 frequency-matched (by sex and age) hospital controls from five major regional hospitals, and recruited 299 healthy subjects from blood donation centers (2014–2017). Circulating level of 25-hydroxyvitamin D (25OHD) and genetic predicted 25OHD (rs12785878, rs11234027, rs12794714, rs4588 and rs6013897) were measured by validated enzyme immunoassay and the iPLEX assay on the MassARRAY System, respectively. Data were also collected on demographics, lifestyle factors, ultraviolet radiation exposure, and potential confounders using a computer-assisted, self-administered questionnaire with satisfactory test-retest reliability. Unconditional logistic regression models were used to estimate ORs and 95% CIs. Results Despite no significant association of NPC risk with circulating 25OHD and genetic predicted 25OHD, there was evidence for an inverse association in participants with normal body mass index (between 18.5 and 27.5) across categories of 25OHD (Ptrend = 0.003), and a positive association in those with low socioeconomic status across categories based on the genetic score (Ptrend = 0.005). In addition, risk of NPC diagnosed at an early stage was higher for genetically lower 25OHD level (adjusted OR = 3.09, 95% CI = 1.04–9.21, Ptrend = 0.022). Conclusions Findings of this first comprehensive study to investigate the positive association of NPC risk with vitamin D deficiency need to be confirmed and be best interpreted with results of further similar studies. Our findings may inform possible etiological mechanisms of the associations with several putative risk/protective factors of NPC.

The tumor microenvironment (TME) of nasopharyngeal carcinoma (NPC) harbors a heterogeneous and dynamic stromal population. A comprehensive understanding of this tumor-specific ecosystem is necessary to enhance cancer diagnosis, therapeutics, and prognosis. However, recent advances based on bulk RNA sequencing remain insufficient to construct an in-depth landscape of infiltrating stromal cells in NPC. Here we apply single-cell RNA sequencing to 66,627 cells from 14 patients, integrated with clonotype identification on T and B cells. We identify and characterize five major stromal clusters and 36 distinct subpopulations based on genetic profiling. By comparing with the infiltrating cells in the non-malignant microenvironment, we report highly representative features in the TME, including phenotypic abundance, genetic alternations, immune dynamics, clonal expansion, developmental trajectory, and molecular interactions that profoundly influence patient prognosis and therapeutic outcome. The key findings are further independently validated in two single-cell RNA sequencing cohorts and two bulk RNA-sequencing cohorts. In the present study, we reveal the correlation between NPC-specific characteristics and progression-free survival. Together, these data facilitate the understanding of the stromal landscape and immune dynamics in NPC patients and provides deeper insights into the development of prognostic biomarkers and therapeutic targets in the TME., The tumor microenvironment can influence patient survival response to therapy. Here, the authors used single-cell sequencing to investigate the microenvironment of nasopharyngeal cancer and identify tumor-specific signatures in five stromal clusters of cells that may influence patient survival.

Background The role of dietary fiber intake on risk of nasopharyngeal carcinoma (NPC) remains unclear. We examined the associations of dietary fiber intake on the risk of NPC adjusting for a comprehensive list of potential confounders. Methods Using data from a multicenter case-control study, we included 815 histologically confirmed NPC incident cases and 1502 controls in Hong Kong, China recruited in 2014–2017. Odds ratios (ORs) of NPC (cases vs controls) for dietary fiber intake from different sources at different life periods (age 13–18, age 19–30, and 10 years before recruitment) were evaluated using unconditional logistic regression, adjusting for sex, age, socioeconomic status, smoking and drinking status, occupational hazards, family history of cancer, salted fish, and total energy intake in Model 1, Epstein-Barr virus viral capsid antigen serological status in Model 2, and duration of sun exposure and circulating 25-hydroxyvitamin D in Model 3. Results Higher intake of total dietary fiber 10 years before recruitment was significantly associated with decreased NPC risk, with demonstrable dose-response relationship (P-values for trend = 0.001, 0.020 and 0.024 in Models 1–3, respectively). The adjusted ORs (95% CI) in the highest versus the lowest quartile were 0.51 (0.38–0.69) in Model 1, 0.48 (0.33–0.69) in Model 2, and 0.48 (0.33–0.70) in Model 3. However, the association was less clear after adjustment of other potential confounders (e.g. EBV) in the two younger periods (age of 13–18 and 19–30 years). Risks of NPC were significantly lower for dietary fiber intake from fresh vegetables and fruits and soybean products over all three periods, with dose-response relationships observed in all Models (P-values for trend for age 13–18, age 19–30 and 10 years before recruitment were, respectively, 0.002, 0.009 and 0.001 for Model1; 0.020, 0.031 and 0.003 for Model 2; and 0.022, 0.037 and 0.004 for Model 3). No clear association of NPC risk with dietary fiber intake from preserved vegetables, fruits and condiments was observed. Conclusion Our study has shown the protective role of dietary fiber from fresh food items in NPC risk, but no association for total dietary fiber intake was observed, probably because total intake also included intake of preserved food. Further studies with detailed dietary information and in prospective settings are needed to confirm this finding, and to explore the possible underlying biological mechanisms.

Purpose We compared, in this pooled analysis, the differences in efficacy and safety between three induction chemotherapy regimens including gemcitabine plus cisplatin (GP), cisplatin plus fluorouracil (PF) and cisplatin plus capecitabine (PX) in patients recruited into our two prospective studies for previously untreated locoregionally advanced nasopharyngeal carcinoma (NPC). Methods GP, PF or PX followed by radical concurrent chemoradiotherapy was given to patients with previously untreated locoregionally advanced (stage III to IVA) NPC prospectively recruited into our two prospective studies. The study endpoints included progression-free survival (PFS) and overall survival (OS), locoregional recurrence-free survival (LRFS), distant metastasis-free survival (DMFS), cancer-specific survival (CSS), and major acute and late treatment-related toxicities (grade ≥ 3). Results From 2006 to 2016, 278 patients were enrolled (84, 94 and 100 patients in GP, PF and PX group respectively). After a median follow-up of 80 months, the 3-year PFS, OS, LRFS, DMFS and CSS of the whole population were 78.7%, 88.1%, 84.9%, 80.9% and 89.8%, respectively. There were no significant differences in prespecified survival endpoints among GP, PF and PX in both stage III and stage IVA patients. GP had lower incidences of severe (grade ≥ 3) anemia and diarrhea in stage III patients, as well as severe anemia, dehydration, renal impairment and vomiting in stage IVA patients. The incidences of grade ≥ 3 late toxicities were similar among these 3 induction regimens. Conclusion GP had similar efficacy and potentially fewer treatment-related complications compared with PF and PX as induction chemotherapy for previously untreated locoregionally advanced NPC.

Background We investigated the relationship of Epstein-Barr virus viral capsid antigen (EBV VCA-IgA) serostatus with ambient and personal ultraviolet radiation (UVR) and vitamin D exposure. Methods Using data from a multicenter case-control study, we included 1026 controls subjects in 2014–2017 in Hong Kong, China. Odds ratios (ORs) and 95% confidence intervals (CIs) of the association between UVR exposure and EBV VCA-IgA (seropositivity vs seronegativity) were calculated using unconditional logistic regression models adjusted for potential confounders. Results We observed a large increase in seropositivity of EBV VCA-IgA in association with duration of sunlight exposures at both 10 years before recruitment and age 19–30 years (adjusted OR = 3.59, 95% CI = 1.46–8.77; and adjusted OR = 2.44, 95% CI = 1.04–5.73 for ≥8 vs Conclusions Our results suggest that personal UVR exposure may be associated with higher risk of EBV reactivation, but we did not find clear evidence of vitamin D exposure (observational or genetic), a molecular mediator of UVR exposure. Further prospective studies in other populations are needed to confirm this finding and to explore the underlying biological mechanisms. Information on photosensitizing agents, and serological markers of EBV, and biomarkers related to systemic immunity and inflammation should be collected and are also highly relevant in future studies.

Introduction: The prognostic role of plasma Epstein–Barr virus (EBV) DNA clearance when intensity-modulated radiotherapy (IMRT) and the 8th edition of American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) TNM Staging Classification are fully implemented remains undeciphered. We investigated if its half-life clearance during radical treatment for non-metastatic nasopharyngeal carcinoma (NPC) was an early prognosticator.Patients and methods: Patients with previously untreated non-metastatic NPC were prospectively treated with radical IMRT and concurrent chemotherapy +/– induction/adjuvant chemotherapy from 2014 to 2018. Their plasma EBV DNA was measured immediately before treatment followed by weekly schedules until 0 copy/ml in two consecutive measurements. Cox regression models were employed to identify prognostic factors.Results: Forty-five patients were prospectively recruited and analyzed. After a median follow-up of 30.3 months, 2 (4.5%), 1 (2.3%), and 6 (13.6%) patients experienced local, regional, and distant relapses, respectively. The median half-life clearance of plasma EBV DNA was 7.92 days. Those with half-life clearance of >15 days had a worse 3-years progression-free survival (PFS) (79.5 vs. 25.0%, p = 0.005), distant metastasis-free survival (DMFS) (85.0 vs. 31.3%, p = 0.009), and overall survival (OS) (91.3 vs. 75.0%, p = 0.024) when compared to those with a shorter half-life. Multivariable analyses demonstrated that only half-life (>15 days) was prognostic of DMFS [HR (95% CI): 4.91 (1.31; 18.39), p = 0.01] and OS [HR (95% CI): 5.24 (1.06; 26.05)] while half-life (>15 days) [HR (95% CI): 5.14 (1.28; 22.73), p = 0.02] and sum of pretreatment gross tumor volumes of the primary nasopharyngeal tumor and the radiologically positive neck nodes (GTV_P+N) [HR (95% CI): 1.01 (1.00; 1.03), p = 0.02] were prognostic of PFS.Conclusion: The half-life clearance of plasma EBV DNA was prognostic in non-metastatic NPC staged and treated in the contemporary era. Earlier biomarker surveillance during treatment should be considered.Clinical Trial Registration: This study has been registered with ClinicalTrials.gov (Identifier: NCT03830996).

Despite pronounced associations of major histocompatibility complex (MHC) regions with nasopharyngeal carcinoma (NPC), causal variants underlying NPC pathogenesis remain elusive. Our large-scale comprehensive MHC region deep sequencing study of 5689 Hong Kong Chinese identifies eight independent NPC-associated signals and provides mechanistic insight for disrupted transcription factor binding, altering target gene transcription. Two novel protective variants, rs2517664 (Trs2517664 = 4.6%, P = 6.38 × 10−21) and rs117495548 (Grs117495548 = 3.0%, P = 4.53 × 10−13), map near TRIM31 and TRIM39/TRIM39-RPP21; multiple independent protective signals map near HLA-B including a previously unreported variant, rs2523589 (P = 1.77 × 10−36). The rare HLA-B*07:05 allele (OR, Here the authors report a major histocompatibility complex (MHC) association analysis for nasopharyngeal carcinoma in Chinese individuals from Hong Kong, finding 8 independent associated loci associated with lower risk for developing nasopharyngeal carcinoma. Two non-human leukocyte antigen (HLA) genes are E3 ubiquitin ligases, TRIM31 and TRIM39, having a role in the innate immune response and implicating the importance of host Epstein-Barr virus interactions in this cancer.

(1) Background: Early predictive markers to track treatment responses are needed for advanced esophageal squamous cell carcinoma (ESCC) patients. We examined the prognostication and risk stratification role of liquid biopsy serial monitoring for this deadly cancer. (2) Methods: Circulating tumor cells (CTCs) and plasma cell-free DNA (cfDNA) were isolated from 60 ESCC patients treated by chemotherapy (CT) at five serial timepoints: baseline (CTC1/cfDNA1), CT pre-cycle III (CTC2/cfDNA2), CT post-cycle IV, end of CT and relapse. (3) Results: In 45/57 ESCC patients with evaluable CTC counts at CT pre-cycle III, positive CTC2 (&ge, 3 CTCs) is independently associated with response at interim reassessment and progression-free survival (PFS) in multivariate analysis. In 42/57 ESCC patients with changes of CTC1/CTC2 and cfDNA1/cfDNA2, patients categorized into four risk groups based on the number of favorable and unfavorable changes of CTC1/CTC2 and cfDNA1/cfDNA2, were independently associated with overall survival (OS) by multivariate analysis. (4) Conclusions: CTC counts at pre-cycle III are independently associated with response at interim reassessment and PFS. Combined changes of CTC counts and cfDNA levels from baseline to pre-cycle III are independently associated with OS. Longitudinal liquid biopsy serial monitoring provides complementary information for prediction and prognosis for CT responses in advanced ESCC.

Purpose: The treatment of nasopharyngeal carcinoma requires high radiation doses. The balance of the risks of local recurrence owing to inadequate tumor coverage versus the potential damage to the adjacent organs at risk (OARs) is of critical importance. With advancements in technology, high target conformality is possible. Nonetheless, to achieve the best possible dose distribution, optimal setting of dose targets and dose prioritization for tumor volumes and various OARs is fundamental. Radiation doses should always be guided by the As Low As Reasonably Practicable principle. There are marked variations in practice. This study aimed to develop a guideline to serve as a global practical reference.Methods and Materials: A literature search on dose tolerances and normal-tissue complications after treatment for nasopharyngeal carcinoma was conducted. In addition, published guidelines and protocols on dose prioritization and constraints were reviewed. A text document and preliminary set of variants was circulated to a panel of international experts with publications or extensive experience in the field. An anonymized voting process was conducted to rank the proposed variants. A summary of the initial voting and different opinions expressed by members were then recirculated to the whole panel for review and reconsideration. Based on the comments of the panel, a refined second proposal was recirculated to the same panel. The current guideline was based on majority voting after repeated iteration for final agreement.Results: Variation in opinion among international experts was repeatedly iterated to develop a guideline describing appropriate dose prioritization and constraints. The percentage of final agreement on the recommended parameters and alternative views is shown. The rationale for the recommendations and the limitations of current evidence are discussed.Conclusions: Through this comprehensive review of available evidence and interactive exchange of vast experience by international experts, a guideline was developed to provide a practical reference for setting dose prioritization and acceptance criteria for tumor volumes and OARs. The final decision on the treatment prescription should be based on the individual clinical situation and the patient's acceptance of optimal balance of risk. (C) 2019 Elsevier Inc. All rights reserved.

Purpose: Target delineation in nasopharyngeal carcinoma (NPC) often proves challenging because of the notoriously narrow therapeutic margin. High doses are needed to achieve optimal levels of tumour control, and dosimetric inadequacy remains one of the most important independent factors affecting treatment outcome. Method: A review of the available literature addressing the natural behaviour of NPC and correlation between clinical and pathological aspects of the disease was conducted. Existing international guidelines as well as published protocols specified by clinical trials on contouring of clinical target volumes (CTV) were compared. This information was then summarized into a preliminary draft guideline which was then circulated to international experts in the field for exchange of opinions and subsequent voting on areas with the greatest controversies. Results: Common areas of uncertainty and variation in practices among experts experienced in radiation therapy for NPC were elucidated. Iterative revisions were made based on extensive discussion and final voting on controversial areas by the expert panel, to formulate the recommendations on contouring of CTV based on optimal geometric expansion and anatomical editing for those structures with substantial risk of microscopic infiltration. Conclusion: Through this comprehensive review of available evidence and best practices at major institutions, as well as interactive exchange of vast experience by international experts, this set of consensus guidelines has been developed to provide a practical reference for appropriate contouring to ensure optimal target coverage. However, the final decision on the treatment volumes should be based on full consideration of individual patients' factors and facilities of an individual centre (including the quality of imaging methods and the precision of treatment delivery). (C) 2017 Elsevier B.V. All rights reserved.

2629 Background: The complex cell interactions within the tumor microenvironment (TME) have become a crucial point in cancer research. Yet, the cell interactions might not only depend on the frequency of immune cells, but also on the inter-individual distances as cells might interact via soluble factors and/or cell-cell contact. Accordingly, the mapping of TME has recently gained importance. The aim of this study is to investigate the alternations between galectin-9 (G9) and its natural immunosuppressive receptor, T cell immunoglobulin and mucin domain 3 (Tim3) in nasopharyngeal cancer (NPC). Methods: Using multiplexed quantitative immunofluorescence, we measured the levels of G9 and Tim3 in 95 NPC patients cancerous and 8 normal specimens in tissue microarray format. Cell densities and cell-to-cell distances were quantified. The interaction between G9-expressing tumor cell lines and T cells were also studied. Results: G9-expressing tumor cells were detected in all NPC cases and were significantly higher than normal tissue. Elevated G9 was associated with shorter overall survival (OS: 89% vs 70.5% at 7 years, p: 0.019). Incremental percentages of Tim3+ cells were shown in top 10% cases strongly positive for G9-expressing tumor cells. The number of Tim3+ cells was calculated at 15µm intervals from the nearest G9-expressing tumor cells, of which a significant difference of Tim3+ cells was observed at the 0-15µm distance from G9-expressing cell in cancerous compared to normal tissues. Epithelial short distances were associated with a unfavourable prognosis. Observed short distance were hypothesized to represent Tim3+ cells actively interacting with G9-expressing tumor cells. Accordingly, In vitro cocultured of G9-ovexpressing NPC cell lines induced Tim3 expression on T cells which suppressed the T-cell mediate cytotoxicity on tumor cells. Conclusions: Our findings indicate a specific preexisting profile of Tim3+ and G9-expressing tumor cells and demonstrated that Tim3+ cells were mainly found intratumorally within 15µm of a NPC cell. The relevance of Tim3+ and G9+ distances reflect a potential marker of their functional interaction. Our results could have important implications for clinical therapeutic strategies. Since high G9 expression have poorer OS, they would deserve a different therapeutic strategy.[Table: see text]

Objective. To determine if the perfusion parameters by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) of regional nodal metastasis are helpful in characterizing nodal status and to understand the relationship with those of primary tumor of nasopharyngeal carcinoma (NPC). Materials and Methods. Newly diagnosed patients imaged between August 2010 and January 2014 and who were found to have enlarged retropharyngeal/cervical lymph nodes suggestive of nodal disease were recruited. DCE-MRI was performed. Three quantitative parameters, Ktrans, ve, and kep, were calculated for the largest node in each patient. Kruskal-Wallis test was used to evaluate the difference in the parameters of the selected nodes of different N stages. Spearman’s correlation was used to evaluate the relationship between the DCE-MRI parameters in nodes and in primary tumors. Results. Twenty-six patients (7 females; 25~67 years old) were enrolled. Ktrans was significantly different among the patients of N stages (N1, n=3; N2, n=17; N3, n=6), P=0.015. Median values (range) for N1, N2, and N3 were 0.24 min−1 (0.17~0.26 min−1), 0.29 min−1 (0.17~0.46 min−1), and 0.46 min−1 (0.29~0.70 min−1), respectively. There was no significant correlation between the parameters in nodes and primary tumors. Conclusion. DCE-MRI may play a distinct role in characterizing the metastatic cervical lymph nodes of NPC.

Craniopharyngioma is a rare low-grade neoplasm in children. Tumor progression occurs frequently, and survivors are at risk of long-term disease and treatment-related morbidities. We reviewed the population-based experience of managing pediatric craniopharyngioma in Hong Kong. The Hong Kong Pediatric Hematology/Oncology Study Group database was interrogated for patients with craniopharyngioma younger than 18 years between 1999 and 2018. Patient demographics, clinical characteristics, outcomes, and long-term morbidities were summarized. Twenty-eight patients with craniopharyngioma were included (approximate incidence of 1.1 per 1,000,000 individuals). The treatment approaches were heterogeneous and included surgery only, surgery with adjuvant radiation, and surgery with intracystic interferon. With a median follow-up of 6.1 years, 12 (43%) patients experienced disease progression, and 3 patients died of progression, postoperative complication, and gastrointestinal bleeding. The 5-year progression-free survival (PFS) and overall survival (OS) rates were 56.8% (± 10.0%) and 92.0% (± 5.4%), respectively. The 10-year PFS and OS rates were 37.3% (± 11.4) and 92.0% (± 5.4%), respectively. Patients receiving treatment in a high-volume center had significantly better outcomes than did those treated at other centers (PFS, p = 0.007; OS, p = 0.029). Period of diagnosis, sex, age at diagnosis, greatest tumor dimension, extent of resection, and radiotherapy use did not significantly affect patient survival. Long-term visual impairment (60%) and endocrinopathies (92%) were common. Prognosis of pediatric craniopharyngioma in Hong Kong compares unfavorably with published reports. Centralization and standardization of treatment may prove valuable in mitigating patient outcomes.

Background Nasopharyngeal carcinoma (NPC) is an important cancer in Hong Kong. We aim to utilise liquid biopsies for serial monitoring of disseminated NPC in patients to compare with PET-CT imaging in detection of minimal residual disease. Method Prospective serial monitoring of liquid biopsies was performed for 21 metastatic patients. Circulating tumour cell (CTC) enrichment and characterisation was performed using a sized-based microfluidics CTC chip, enumerating by immunofluorescence staining, and using target-capture sequencing to determine blood mutation load. PET-CT scans were used to monitor NPC patients throughout their treatment according to EORTC guidelines. Results The longitudinal molecular analysis of CTCs by enumeration or NGS mutational profiling findings provide supplementary information to the plasma EBV assay for disease progression for good responders. Strikingly, post-treatment CTC findings detected positive findings in 75% (6/8) of metastatic NPC patients showing complete response by imaging, thereby demonstrating more sensitive CTC detection of minimal residual disease. Positive baseline, post-treatment CTC, and longitudinal change of CTCs significantly associated with poorer progression-free survival by the Kaplan–Meier analysis. Conclusions We show the potential usefulness of application of serial analysis in metastatic NPC of liquid biopsy CTCs, as a novel more sensitive biomarker for minimal residual disease, when compared with imaging.

Gastric cancers are highly prevalent in both the East and the West, although they differ in aetiology and prognostic outcome. Management of gastric cancer from screening to definitive treatment varies substantially between Eastern and Western countries and regions, owing to numerous factors, including government incentives to carry out population-wide screening programmes to detect early disease, differences in clinical and biological tumour behaviours and responsiveness to treatment, patient accessibility to effective treatment, etc. This review highlights and contrasts the differences in tumour aetiology and histology, as well as the management approaches between the East and the West, which gives important insights and inspirations on future international multicentre research collaboration to combat this dreadful malignancy.

Background: Health-related quality of life (HRQoL) is important for differentiated thyroid cancer survivors, but data for Asian survivors is lacking. This study aimed to have an overview of, and identify any disease-or treatment-related factors associated with, HRQoL in Asian differentiated thyroid cancer survivors. Patients and Methods: Thyroid cancer survivors were recruited from the thyroid clinics at Queen Mary Hospital, Hong Kong from February 2016 to December 2016. All adult differentiated thyroid cancer patients with stable disease more than or equal to 1 year received a survey on HRQoL using the European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) and Thyroid cancer specific quality of life (THYCA-QoL) questionnaire. Clinical information was collected retrospectively from the computerized clinical management system. To identify factors associated with poor HRQoL, univariable and stepwise multivariable regression analysis were performed. Results: A total of 613 survivors completed the questionnaires (response rate: 82.1%; female: 80.1%; median survivorship: 7.4 years (range: 1.0-48.2 years)). The QLQ-C30 summary score mean was 84.4 (standard deviation (SD): 12.7) while the THYCA-QoL summary score mean was 39.9 (SD: 9.7). The 2 highest symptom subscales were fatigue (mean: 26.4, SD: 20.6) and insomnia (mean: 26.2, SD: 27.6). Factors associated with worse HRQoL included serum thyrotropin (TSH) greater than 1.0 mIU/L, unemployment, and concomitant psychiatric disorders. Concomitant psychiatric illness (n = 40/613, 6.5%) also showed significant association with most of the symptom and functional subscales. Conclusions: Fatigue and insomnia were the 2 most common symptoms experienced by our differentiated thyroid cancer survivors. Long-term survivorship care with monitoring serum TSH level, supporting return-to-work and screening for concomitant psychiatric disorders should be offered.

e18519 Background: A better understanding of peripheral cellular phenotypes in association with checkpoint inhibitors (CPI) responsiveness in recurrent/metastatic NPC could profoundly impact our knowledge of NPC immunopathology. Methods: Blood were collected from 11 patients with recurrent/metastatic NPC who received CPI (pembrolizumab) after failing second-line chemotherapy. Response to pembrolizumab was assessed by imaging and EBV DNA. Patients who achieved CR/PR were considered responsive and those with SD/PD were considered non-responsive. Four patients in the responsive group also had baseline blood before pembrolizumab. PBMC were freshly isolated and stored until analysis. For surface staining, cells were rested overnight at 37°C before resuspended in BD Brilliant stain with 2.5ng/µl Fc block for 15mins. Cells were then co-stained: 7-AAD, CD19-BV510, CD3-Alexa Fluo 700, CD4-BV510, CD279(PD-1)-BB515, CD197(CCR7)-BV421, CD45RO-APC, CD45RA-Pe-Cy5, CD8-APC-H7, CD27-PE, CD95-PE-Cy7, incubated on ice for 30mins. Samples were washed and acquired on NovoCyte Quanteon. Immune phenotypes were correlated with clinical response. Results: We found that: 1) %CD3 was upregulated in responsive group; 2) CD4/CD8 ratio did not directly stratify drug responsiveness; 3) frequency of PD1-expressing CD8+ T cells was significantly reduced in responsive group; 4) lower frequency of CCR7+PD1+CD8+ T cells in responsive group, suggesting that these may be highly differentiated and have the ability to move into peripheral sites in response to inflammatory chemokines; 5) frequency of naïve and TEMRA CD8+ T cells, but not Tscm was upregulated in responsive group; 6) finally, an interesting finding of sustained CD19+ subsets was observed in non-responsive group. Conclusions: Our results suggest that peripheral blood analysis may provide valuable insights into NPC patients’ responses to PD-1-targeted therapies. [Table: see text]

Whether certain variants of Epstein-Barr virus (EBV) are linked to the pathogenesis of nasopharyngeal carcinoma (NPC), which shows a marked geographic restriction, remains an unresolved issue. We performed a case-control study comparing genomic sequences of EBV isolated from saliva samples of 142 population carriers with those from primary tumour biopsies derived from 62 patients with NPC of Hong Kong. Cluster analysis discovered five EBV subgroups 1A-C and 2A-B amongst the population carriers in contrast to the predominance of 1A and -B in the majority of NPC. Genome-wide association study (GWAS) identified a panel of NPC-associated single nucleotide polymorphisms (SNPs) and indels in the EBER locus. The most significant polymorphism, which can be found in 96.8% NPC cases and 40.1% population carriers of Hong Kong, is a four-base-deletion polymorphism downstream of EBER2 (EBER-del) from coordinates 7188-7191 (p = 1.91 × 10-7 ). In addition, the predicted secondary structure of EBER2 is altered with likely functional consequence in nearly all NPC cases. Using the SNPs and indels associated with NPC, genetic risk score is assigned for each EBV variant. EBV variants with high genetic risk score are found to be much more prevalent in Hong Kong Chinese than individuals of other geographic regions and in NPC than other EBV-associated cancers. We conclude that high risk EBV variants with polymorphisms in the EBER locus, designated as HKNPC-EBERvar, are strongly associated with NPC. Further investigation of the biological function and potential clinical application of these newly identified polymorphisms in NPC and other EBV-associated cancers is warranted.

Background We aim at subdividing M1 stage to better predict survival of metastatic nasopharyngeal carcinoma (NPC) patients whose outcomes could vary greatly. Methods Patients with previously untreated metastatic NPC (training cohort) were recruited prospectively from 2007 to 2018 and were re-staged based on 8th edition of American Joint Committee on Cancer system. All patients had baseline plasma EBV DNA at diagnosis of metastasis. Characteristics of metastases (site, number and size of metastatic lesions) were confirmed by MRI and PET-CT. We used recursive partitioning analysis (RPA) incorporating baseline plasma EBV DNA and/or metastatic characteristics with internal validations to subdivide M1 stage. The two models were externally validated using an independent data set of 67 NPC patients who were non-metastatic at diagnosis but later developed distant metastases after radical treatment (validation cohort). Performance of survival prediction between the two models was compared with paired t-test under 1000 bootstrapping samples. Results The training cohort of 69 patients had a median follow-up of 40.8 months and 3-year overall survival (OS) of 36%. Model 1 incorporating pre-treatment plasma EBV DNA subdivided M1 stage into two groups: M1a (EBV DNA ≤2500 copies/ml; OS 74%) and M1b (EBV DNA >2500 copies/ml; OS 17%) (P 2500 copies/ml) (HR 4.7 (95% CI 1.9-11.5); P=.001) and metastatic site (coexisting liver and bone metastasis) (HR 2.2 (1.0-4.7); P=.046) were prognostic of OS. Model 1 demonstrated better model fit in predicting OS (Model 1: mean AIC 246.9 (95% CI 187.8-303.6) vs Model 2: mean AIC 257.7 (200.6-313.2); P Conclusions A novel RPA-based M1 stage set incorporating baseline plasma EBV DNA had a significantly better survival prediction, providing important values on prognosis and treatment decision making. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

The eighth edition of the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) stage classification (TNM) for nasopharyngeal carcinoma (NPC) was launched. It remains unknown if incorporation of nonanatomic factors into the stage classification would better predict survival. We prospectively recruited 518 patients with nonmetastatic NPC treated with radical intensity-modulated radiation therapy ± chemotherapy based on the eighth edition TNM. Recursive partitioning analysis (RPA) incorporating pretreatment plasma Epstein-Barr virus (EBV) DNA derived new stage groups. Multivariable analyses to calculate adjusted hazard ratios (AHRs) derived another set of stage groups. Five-year progression-free survival (PFS), overall survival (OS) and cancer-specific survival (CSS) were: Stage I (PFS 100%, OS 90%, CSS 100%), II (PFS 88%, OS 84%, CSS 95%), III (PFS 84%, OS 84%, CSS 90%) and IVA (PFS 71%, OS 75%, CSS 80%) (p < 0.001, p = 0.066 and p = 0.002, respectively). RPA derived four new stages: RPA-I (T1-T4 N0-N2 & EBV DNA