Epigenomic landscape study reveals molecular subtypes and EBV-associated regulatory epigenome reprogramming in nasopharyngeal carcinoma.

BACKGROUND: Epstein-Barr virus (EBV) latent infection is associated with genome-wide epigenomic changes in several malignancies, but its role in epigenetic dysregulation remains unclear in nasopharyngeal carcinoma (NPC). METHODS: To investigate EBV-associated epigenetic dysregulation, we performed a multi-omics study by integrating whole-genome bisulfite sequencing (WGBS), assay for transposase-accessible chromatin using sequencing (ATAC-Seq), whole-exome sequencing (WES), and single-cell RNA sequencing (scRNA-Seq) data. FINDINGS: In addition to the known global DNA hypermethylated subtype, we discovered a novel subtype with global hypomethylation in EBV + NPC. The consistent EBV-specific differentially methylated regions (EBV-DMRs) in the human genome were identified from both subtypes and associated with loss of CTCF binding (P < 2.2e-16). Importantly, CTCF is a master chromatin regulator and CTCF protein was reduced in 45% of NPC cases, especially in those with advanced NPC (Stage IV vs. others: 62% vs. 38%, P = 0.034). This result links EBV with chromatin changes. The ATAC-Seq data suggest regulatory epigenome reprogramming through chromatin accessibility changes in EBV + NPC with altered CTCF binding and the switch of transcription factor binding from differentiation-associated KLF/SP family to the innate and adaptive immunity-related NF-ĸB and IRF families. Detailed chromatin accessibility analysis identified a potential EBV target gene CD74, which mediated EBV-specific cell-cell communications in the tumor microenvironment (TME) and was strongly correlated with T cell exhaustion (r2 = 0.55). INTERPRETATION: Our study reveals the unexpected epigenetic heterogeneity, providing insights into NPC pathogenesis and highlighting the involvement of host factors in virus-associated epigenetic changes. EBV infection is associated with epigenome reprogramming and may promote immune evasion. FUNDING: This study was funded by the Hong Kong Research Grants Council grant (AoE/M-06/08) to MLL, General Research Fund (17103218 and 17102619) and seed funding for basic research (201611159158) to WD, and General Research Fund (17119618) to HC.

Outcome of neoadjuvant chemoradiation in MRI staged locally advanced rectal cancer: Retrospective analysis of 123 Chinese patients.

BACKGROUND: For advanced rectal cancer with involved or threatened mesorectal fascia (MRF), current standard is pre-operative long course chemoradiotherapy (PLCRT) with either capecitabine or 5-fluorouracil (5-FU). However, few Chinese data on its clinical outcome are available, especially for those with pelvic MRI staging. METHODS: Between Jan-2009 and Oct-2014, 123 consecutive patients with biopsy proven adenocarcinoma of rectum, all with pelvic MRI staging, selected for PLCRT after multi-disciplinary team discussion were recruited. Their clinical records were retrospectively reviewed. RESULTS: Median follow-up was 1392 days (range: 48-2886) MRI defined poor risk factors as follows: MRF threatened or involved ≤1 mm 61.8% (n = 76), cT4 13.8% (n = 17), cN2 26.8% (n = 33) and low-lying tumor (≤5 cm from anal verge) 24.4% (n = 30). Five year OS and DFS were 63.9% and 68.3% respectively. Among 112 patients who received TME, 108 (96.4%) had microscopic clear resection (R0). Twelve and 32 individuals had pathological complete response and ypT0-2N0, respectively. Five local recurrences (4.5%) were detected. The incidence of grade 3 or above acute and late radiotherapy toxicity was 8.1% and 12.2% respectively. After multivariate adjustment, positive circumferential resection margin (CRM) status on pathology report was found to be significant factor for worse OS and DFS. CONCLUSION: The clinical outcomes of PLCRT in our institution are comparable with those in western literature. Our MRI staging lends support to the validity of data. CRM status is the most significant prognostic factor in OS and DFS, after multivariate adjustment.

The Role of Radiotherapy in Early-stage Primary Diffuse Large B-Cell Lymphoma of the Waldeyer Ring: A Retrospective Cohort Study.

OBJECTIVES: The role of radiotherapy (RT) in improving survival of patients with diffuse large B-cell lymphoma (DLBCL) of the Waldeyer ring (WR) remains controversial. Therefore, this retrospective cohort study aimed to determine the role of RT in the treatment of DLBCL of the WR as well as the effects of associated covariates. MATERIALS AND METHODS: Patients (n=35) with stage I to II DLBCL of the WR who underwent treatment at our center between 1994 and 2010 were retrospectively investigated. All patients had histologic diagnosis and staging workup completed. Overall survival (OS), event-free survival (EFS), and disease-free survival (DFS) were analyzed. Variables with a P-value of <0.1 were subjected to multivariate Cox proportional hazards model analyses. RESULTS: The median OS was 8.1 years. The 5-year OS, DFS, and EFS rates were 59.4%, 53.8%, and 70.7%, respectively; the corresponding 10-year rates were 34.9%, 29.5%, and 51.0%, respectively. The 5-year OS rate was significantly higher in the RT group than in the non-RT group (65.4% vs. 36.4%, respectively; P=0.008). On multivariate analysis, RT was associated with improved OS (hazard ratio=0.15; 95% confidence interval [CI], 0.04-0.50; P=0.002) and EFS (hazard ratio=0.29; 95% CI, 0.095-0.86; P=0.026). An Eastern Cooperative Oncology Group performance status >1 and age above 60 years were also found to negatively influence OS and EFS. CONCLUSIONS: RT was associated with improved OS and EFS in stage I to II DLBCL of the WR. Future prospective studies are required to confirm these findings.

Throwing the dart blind-folded: comparison of computed tomography versus magnetic resonance imaging-guided brachytherapy for cervical cancer with regard to dose received by the 'actual' targets and organs at risk.

PURPOSE: Computed tomography (CT) is inferior to magnetic resonance imaging (MRI) in cervical tumor delineation, but similar in identification of organs at risk (OAR). The trend to over-estimate high-risk and low-risk clinical target volume (HRCTV, IRCTV) on CT can lead to under-estimation of dose received by 90% (D90) of the 'actual' CTV. This study aims to evaluate whether CT-guided planning delivers adequate dose to the 'actual' targets while spares the OAR similarly. MATERIAL AND METHODS: MRI-guided high-dose-rate image-guided brachytherapy (IGBT) was performed in 11 patients. The pre-brachytherapy CTs were retrospectively contoured to generate CT-guided plans. MRI-based contours (HRCTVmri, IRCTVmri, bladdermri, rectummri, and sigmoidmri) were fused to CT plans for dosimetric comparison with MRI-guided plans. Paired 2-tailed t-test and Wilcoxon signed-rank test were used to analyze data. RESULTS: 63.6% of CT plans achieved the HRCTVmriD90 constraint (≥ 7.2 Gy in one fraction), compared with 90.9% for MRI plans. > 90% of both modalities achieved the OAR's constraints (EMBRACE). The percentage of CT and MRI plans that achieved the aims (EMBRACE II) for bladder, rectum, and sigmoid were 36.4% vs. 81.8%, 63.6% vs. 63.6%, and 72.7% vs. 72.7%, respectively. There were no statistically significant differences in HRCTVmriD90, IRCTVmriD90, or dose received by the most exposed 2 cm3 (D2cc) of OARmri between the modalities. Excluding the CT plans not achieving HRCTVmriD90 constraint, there were significant increase in bladdermriD2cc, rectummriD2cc, and sigmoidmriD2cc, compared with MRI plans (0.9 Gy/Fr, 95% CI 0.2-1.5, p = 0.018; 0.9 Gy/Fr, 95% CI 0.3-1.4, p = 0.009; 0.5 Gy/Fr, 95% CI 0.2-0.9, p = 0.027, respectively). CONCLUSIONS: MRI-based IGBT remains the gold standard. CT planning may compromise HRCTVmriD90 or increase OARmriD2cc, which could decrease local control or increase treatment toxicity.

Aspirin and risk of multiple myeloma in adults: A systematic review and meta-analysis.

Multiple myeloma is a relatively uncommon plasma cell malignancy. Preclinical and clinical studies have suggested that aspirin might modify the risk of multiple myeloma. We performed a systematic review and meta-analysis of studies to examine the association between regular aspirin use and risk of multiple myeloma. Five observational studies including 332,660 adults were evaluated. The pooled estimate had a hazard ratio of 0.90 (95% confidence interval =0.58-1.39; P=0.638). Odds ratios from the two case-control studies were similar. The findings demonstrated that there was no significant association between aspirin use and the risk of multiple myeloma.

Metastasis-suppressing NID2, an epigenetically-silenced gene, in the pathogenesis of nasopharyngeal carcinoma and esophageal squamous cell carcinoma.

Nidogen-2 (NID2) is a key component of the basement membrane that stabilizes the extracellular matrix (ECM) network. The aim of the study is to analyze the functional roles of NID2 in the pathogenesis of nasopharyngeal carcinoma (NPC) and esophageal squamous cell carcinoma (ESCC). We performed genome-wide methylation profiling of NPC and ESCC and validated our findings using the methylation-sensitive high-resolution melting (MS-HRM) assay. Results showed that promoter methylation of NID2 was significantly higher in NPC and ESCC samples than in their adjacent non-cancer counterparts. Consistently, down-regulation of NID2 was observed in the clinical samples and cell lines of both NPC and ESCC. Re-expression of NID2 suppresses clonogenic survival and migration abilities of transduced NPC and ESCC cells. We showed that NID2 significantly inhibits liver metastasis. Mechanistic studies of signaling pathways also confirm that NID2 suppresses the EGFR/Akt and integrin/FAK/PLCγ metastasis-related pathways. This study provides novel insights into the crucial tumor metastasis suppression roles of NID2 in cancers.

IKBB tumor suppressive role in nasopharyngeal carcinoma via NF-κB-mediated signalling.

Tumor suppressor genes (TSGs) play a prominent role in cancer and are important in the development of nasopharyngeal carcinoma (NPC), which is endemic in Southern China as well as Southeast Asia. Apart from TSGs, aberrant signalling pathways are also commonly associated with tumor progression. Unsurprisingly, the NF-κB pathway is frequently associated with angiogenesis and promoting tumor growth and development. Functional complementation studies using microcell-mediated chromosome transfer helped to identify IKBB as a putative TSG in NPC. IKBB, an inhibitor of NF-κB, has recently been shown to be inversely associated with tumor growth and metastasis via inactivation of the NF-κB pathway, but its suppressive role is still only poorly understood. This study takes the lead in revealing the suppressive role of IKBB in NPC. IKBB is silenced in the majority of NPC tumor tissues in all stages. Its suppressive role is substantiated by perturbation in tumor formation, cell migration and angiogenesis. Interestingly, IKBB not only affects the 'seed', but also influences the 'soil' by downregulating the transcriptional level of proangiogenic factors Rantes, Upar, IL6, and IL8. For the first time, our data establish the importance of a novel tumor suppressive IKBB gene in abrogating angiogenesis in NPC via the NF-κB signalling pathway, which is likely mediated by crosstalk with the Akt/Gsk3ß signalling pathway.

NF-κB p65 Subunit Is Modulated by Latent Transforming Growth Factor-ß Binding Protein 2 (LTBP2) in Nasopharyngeal Carcinoma HONE1 and HK1 Cells.

NF-κB is a well-characterized transcription factor, widely known as a key player in tumor-derived inflammation and cancer development. Herein, we present the functional and molecular relevance of the canonical NF-κB p65 subunit in nasopharyngeal carcinoma (NPC). Loss- and gain-of-function approaches were utilized to reveal the functional characteristics of p65 in propagating tumor growth, tumor-associated angiogenesis, and epithelial-to-mesenchymal transition in NPC cells. Extracellular inflammatory stimuli are critical factors that trigger the NF-κB p65 signaling; hence, we investigated the components of the tumor microenvironment that might potentially influence the p65 signaling pathway. This led to the identification of an extracellular matrix (ECM) protein that was previously reported as a candidate tumor suppressor in NPC. Our studies on the Latent Transforming Growth Factor-ß Binding Protein 2 (LTBP2) protein provides substantial evidence that it can modulate the p65 transcriptional activity. Re-expression of LTBP2 elicits tumor suppressive effects that parallel the inactivation of p65 in NPC cells. LTBP2 was able to reduce phosphorylation of p65 at Serine 536, inhibit nuclear localization of active phosphorylated p65, and impair the p65 DNA-binding ability. This results in a consequential down-regulation of p65-related gene expression. Therefore, the data suggest that the overall up-regulation of p65 expression and the loss of this candidate ECM tumor suppressor are milestone events contributing to NPC development.

Severe ileitis associated with capecitabine: Two case reports and review of the literature.

Capecitabine is a commonly used anticancer drug, which has been associated with adverse events, including skin and gastrointestinal symptoms, such as vomiting and diarrhea. We herein present treated two rare cases of capecitabine-associated ileitis. In one of the patients, ileitis occurred during combination chemotherapy for metastatic colon cancer, despite previous good tolerance to this drug; the other patient developed ileitis following adjuvant single-agent treatment. The first case is unlike previously reported cases, in which patients had no past exposure to capecitabine. Ileitis may be severe but reversible with early diagnosis and proper supportive treatment, and patients may resume chemotherapy following capecitabine discontinuation.

Treatment results of 1070 patients with nasopharyngeal carcinoma: an analysis of survival and failure patterns.

BACKGROUND: The aim of this analysis was to evaluate the outcomes of patients with nasopharyngeal carcinoma (NPC) treated primarily by external beam irradiation (ERT) and to explore for possible ways to improve the treatment results. METHODS: One thousand seventy patients with nonmetastatic NPC treated from 1990 to 1998 were retrospectively analyzed. The distribution according to the Union Internationale Contre le Cancer (UICC) (1997 edition) staging system at initial diagnosis was as follows: stage I, n = 113; stage IIA, n = 38; stage IIB, n = 360; stage III, n = 306; stage IVA, n = 136; stage IVB, n = 117; T1, n = 284; T2a, n = 88; T2b, n = 398; T3, n = 149; T4, n = 151; N0, n = 321; N1, n = 393; N2, n = 238; N3a, n = 29; N3b, n = 89. Two hundred eight patients were given neoadjuvant chemotherapy. Ninety-seven patients were diagnosed with locally persistent disease and were salvaged with high dose rate intracavitary brachytherapy. Multivariate analysis was performed with the Cox regression proportional hazards model. RESULTS: The 5-year actuarial local failure-free survival, regional failure-free survival, distant metastasis-free survival, progression-free survival, cancer-specific survival, and overall survival rates were 80.9%, 93.3%, 77.2%, 62.7%, 71.4%, and 66.5%, respectively. Isolated distant metastasis occurred in 191 patients (18%). The distributions were as follow: stage I, 2.1% (two of 95); stage IIA, 5.7% (two of 35); stage IIB, 14.9% (45 of 302); stage III, 26.4% (62 of 235); stage IVA, 40% (40 of 100); stage IVB, 47.1% (40 of 85). Results of the multivariate analysis of various clinical endpoints were discussed. By studying these failure patterns, it is hoped that we could refine future treatments according to the failure patterns of patients with different risks of locoregional and distant failure. CONCLUSIONS: The 18% incidence of isolated distant metastasis is too high to be ignored. Maximizing the local control and minimizing the risk of distant metastasis and late complications should be the key objectives in designing future clinical trials.

Survival outcome of patients with nasopharyngeal carcinoma with first local failure: a study by the Hong Kong Nasopharyngeal Carcinoma Study Group.

BACKGROUND: The purpose of this article is to report the overall survival (OS) outcome of patients with nasopharyngeal carcinoma (NPC) with local failure who received salvage treatment and to identify prognostic factors for OS. METHODS: Between January 1996 and December 2000, 2915 patients received primary radiotherapy (RT) with or without chemotherapy for nonmetastatic NPC. At a median follow-up of 3.1 years, 319 patients had developed local failure as the first failure, with or without synchronous regional/distant failure. OS was calculated from the start of primary RT. Univariate and multivariate analyses were performed to identify prognostic factors for OS in patients with isolated local failure. RESULTS: The T classification distribution of the local failure (rT classification) was as follows: 68 (21%) rT1 to T2a, 92 (29%) rT2b, 82 (26%) rT3, and 77 (24%) rT4. The rT classification was the same as the initial T classification in 82% of patients. Two hundred seventy-five patients (86%) had isolated local failure, and 232 (84%) of them did not have any distant metastasis or regional failure develop during follow-up. Salvage treatment was given to 200 patients (73%) with isolated local failure. One hundred fifty-nine patients (80%) received reirradiation (108 external beam RT [EBRT], 44 brachytherapy, and seven EBRT plus brachytherapy), 22 patients (11%) underwent nasopharyngectomy with or without postoperative RT, and 19 patients (9%) were treated with chemotherapy alone. Four patients died of RT complications, and one died of chemotherapy toxicity in the absence of active NPC. The 3-year actuarial OS for patients with isolated local failure was 74%. On multivariate analysis, advanced initial T classification (hazard ratio [HR], 1.44; p = .0006) and the use of salvage treatment (HR, 0.54; p = .0038) were independent prognostic factors. For the subgroups of patients who had the same recurrent and initial T classification, salvage treatment was associated with improved OS only in the subgroup with T1 to T2 local failure (n = 127; p = 0.0446), but not in the subgroups with T3 (n = 48) or T4 (n = 54) disease. CONCLUSIONS: Most patients with first local failure have localized disease. Salvage treatment is feasible in most of the patients with clinically isolated local failure. Patients who had early initial T classification have a more favorable prognosis. Subgroup analysis suggests that salvage treatment only prolongs survival in patients with T1 to T2 recurrent disease.

High dose rate brachytherapy for early stage oral tongue cancer.

BACKGROUND: High dose rate (HDR) interstitial brachytherapy of the oral tongue is a new treatment modality. Our study evaluates the outcomes of patients with early stage oral tongue cancer as treated by HDR interstitial implant. METHODS: We reviewed the records of 19 patients who were seen between 1994 and 2000 with carcinoma of the oral tongue and whose primary tumors were treated solely with interstitial implant using HDR remote afterloading technique. Ten patients had T1 N0 disease, and the remaining 9 had T2 N0 disease. Elective neck treatment was withheld for 12 patients. The remaining seven patients had ipsilateral elective neck dissection. The male-female ratio was 1:0.9, and the median age was 60 years (range, 32-81 years). The median follow-up time was 43 months (range, 6-78 months). The afterloading catheters were positioned by the submandibular approach with the assistance of a template set. Fifteen patients had single planar implants, and the remaining four had double planar implants. The median number of catheters inserted was 5 (range, 4-9). The median dose given was 55 Gy in 10 fractions over 6 days. The minimal interfraction interval was 7 hours for the first 7 patients and was extended to 8 hours for the other 12. Mandibular shields were inserted before treatment. RESULTS: The mucositis lasted for 6 to 20 weeks (median, 9 weeks). One patient had local failure, and the 4-year local failure-free survival rate was 94.7%. Three of the 12 patients without elective neck treatment had ipsilateral regional failure develop. They were salvaged by neck node dissection and regionally remained in control. One patient with multiple nodal metastases and extracapsular spread had biopsy-proven liver metastases and died 6 months after implant. One of the seven patients who were treated with elective neck dissection had multiple nodal metastases and extracapsular spread. She was treated with postoperative radiotherapy to the neck. She died 30 months after implant with evidence of regional and distant failure. One patient treated with double planar implant had grade II necrosis of the soft tissue and bone develop. The necrosis resolved with conservative treatment. Another four patients had small area of soft tissue deficit of the tongue attributed to aggressive debulking or biopsy before brachytherapy. CONCLUSIONS: Our experience in treating early stage tongue cancer with HDR remote afterloading technique is encouraging, because it gives a local control rate of 94.7% at 4 years with acceptable morbidity. Further studies are eagerly awaited to delineate the optimum schedule for this new treatment modality.