Establishment and Characterization of an Epstein-Barr Virus-positive Cell Line from a Non-keratinizing Differentiated Primary Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC), a cancer that is etiologically associated with the Epstein-Barr virus (EBV), is endemic in Southern China and Southeast Asia. The scarcity of representative NPC cell lines owing to the frequent loss of EBV episomes following prolonged propagation and compromised authenticity of previous models underscores the critical need for new EBV-positive NPC models. Herein, we describe the establishment of a new EBV-positive NPC cell line, designated NPC268 from a primary non-keratinizing, differentiated NPC tissue. NPC268 can undergo productive lytic reactivation of EBV and is highly tumorigenic in immunodeficient mice. Whole-genome sequencing revealed close similarities with the tissue of origin, including large chromosomal rearrangements, while whole-genome bisulfite sequencing and RNA sequencing demonstrated a hypomethylated genome and enrichment in immune-related pathways, respectively. Drug screening of NPC268 together with six other NPC cell lines using 339 compounds, representing the largest high-throughput drug testing in NPC, revealed biomarkers associated with specific drug classes. NPC268 represents the first and only available EBV-positive non-keratinizing differentiated NPC model, and extensive genomic, methylomic, transcriptomic, and drug response data should facilitate research in EBV and NPC, where current models are limited. SIGNIFICANCE: NPC268 is the first and only EBV-positive cell line derived from a primary non-keratinizing, differentiated nasopharyngeal carcinoma, an understudied but important subtype in Southeast Asian countries. This model adds to the limited number of authentic EBV-positive lines globally that will facilitate mechanistic studies and drug development for NPC.

Calibration improves the agreement in grading oral epithelial dysplasia-Findings from a National Workshop in Malaysia.

INTRODUCTION: A major pitfall of many of the established oral epithelial dysplasia (OED) grading criteria is their lack of reproducibility and accuracy to predict malignant transformation. The main objective of this study was to determine whether calibration of practicing oral pathologists on OED grading could improve the reproducibility of the WHO 2017 and the binary OED grading systems. METHODS: A nationwide online exercise was carried out to determine the influence of calibration on the reproducibility of the WHO 2017 and the binary OED grading systems. RESULTS: A significant improvement was observed in the inter-observer agreement for the WHO 2017 OED grading system (K 0.196 vs. 0.448; Kw 0.357 vs. 0.562) after the calibration exercise. The significant difference (p = 0.027) in the level of agreement between those with five or more years and less than 5 years of experience was no more observed (p = 0.426) after the calibration exercise. The percent agreement for binary grading was significantly higher (91.8%) for buccal mucosal lesions as compared to lesions on the tongue after the calibration exercise. CONCLUSION: This study validates the significance of calibration in improving the reproducibility of OED grading. The nationwide exercise resulted in a statistically significant improvement in the inter-observer agreement for the WHO 2017 OED grading system among a large number of oral pathologists. It is highly recommended that similar exercises should be organized periodically by professional bodies responsible for continuing education among oral pathologists to improve the reliability of OED grading for optimal treatment of oral potentially malignant disorders.

Economic Evaluation of Oral Cancer Screening Programs: Review of Outcomes and Study Designs.

A lack of guidance on economic evaluations for oral cancer screening programs forms a challenge for policymakers and researchers to fill the knowledge gap on their cost-effectiveness. This systematic review thus aims to compare the outcomes and design of such evaluations. A search for economic evaluations of oral cancer screening was performed on Medline, CINAHL, Cochrane, PubMed, health technology assessment databases, and EBSCO Open Dissertations. The quality of studies was appraised using QHES and the Philips Checklist. Data abstraction was based on reported outcomes and study design characteristics. Of the 362 studies identified, 28 were evaluated for eligibility. The final six studies reviewed consisted of modeling approaches (n = 4), a randomized controlled trial (n = 1), and a retrospective observational study (n = 1). Screening initiatives were mostly shown to be cost-effective compared to non-screening. However, inter-study comparisons remained ambiguous due to large variations. The observational and randomized controlled trials provided considerably accurate evidence of implementation costs and outcomes. Modeling approaches, conversely, appeared more feasible for the estimation of long-term consequences and the exploration of strategy options. The current evidence of the cost-effectiveness of oral cancer screening remains heterogeneous and inadequate to support its institutionalization. Nevertheless, evaluations incorporating modeling methods may provide a practical and robust solution.

Mutated HRAS Activates YAP1-AXL Signaling to Drive Metastasis of Head and Neck Cancer.

The survival rate for patients with head and neck cancer (HNC) diagnosed with cervical lymph node (cLN) or distant metastasis is low. Genomic alterations in the HRAS oncogene are associated with advanced tumor stage and metastasis in HNC. Elucidation of the molecular mechanisms by which mutated HRAS (HRASmut) facilitates HNC metastasis could lead to improved treatment options for patients. Here, we examined metastasis driven by mutant HRAS in vitro and in vivo using HRASmut human HNC cell lines, patient-derived xenografts, and a novel HRASmut syngeneic model. Genetic and pharmacological manipulations indicated that HRASmut was sufficient to drive invasion in vitro and metastasis in vivo. Targeted proteomic analysis showed that HRASmut promoted AXL expression via suppressing the Hippo pathway and stabilizing YAP1 activity. Pharmacological blockade of HRAS signaling with the farnesyltransferase inhibitor tipifarnib activated the Hippo pathway and reduced the nuclear export of YAP1, thus suppressing YAP1-mediated AXL expression and metastasis. AXL was required for HRASmut cells to migrate and invade in vitro and to form regional cLN and lung metastases in vivo. In addition, AXL-depleted HRASmut tumors displayed reduced lymphatic and vascular angiogenesis in the primary tumor. Tipifarnib treatment also regulated AXL expression and attenuated VEGFA and VEGFC expression, thus regulating tumor-induced vascular formation and metastasis. Our results indicate that YAP1 and AXL are crucial factors for HRASmut-induced metastasis and that tipifarnib treatment can limit the metastasis of HNC tumors with HRAS mutations by enhancing YAP1 cytoplasmic sequestration and downregulating AXL expression. SIGNIFICANCE: Mutant HRAS drives metastasis of head and neck cancer by switching off the Hippo pathway to activate the YAP1-AXL axis and to stimulate lymphovascular angiogenesis.

Image collection and annotation platforms to establish a multi-source database of oral lesions.

OBJECTIVE: To describe the development of a platform for image collection and annotation that resulted in a multi-sourced international image dataset of oral lesions to facilitate the development of automated lesion classification algorithms. MATERIALS AND METHODS: We developed a web-interface, hosted on a web server to collect oral lesions images from international partners. Further, we developed a customised annotation tool, also a web-interface for systematic annotation of images to build a rich clinically labelled dataset. We evaluated the sensitivities comparing referral decisions through the annotation process with the clinical diagnosis of the lesions. RESULTS: The image repository hosts 2474 images of oral lesions consisting of oral cancer, oral potentially malignant disorders and other oral lesions that were collected through MeMoSA® UPLOAD. Eight-hundred images were annotated by seven oral medicine specialists on MeMoSA® ANNOTATE, to mark the lesion and to collect clinical labels. The sensitivity in referral decision for all lesions that required a referral for cancer management/surveillance was moderate to high depending on the type of lesion (64.3%-100%). CONCLUSION: This is the first description of a database with clinically labelled oral lesions. This database could accelerate the improvement of AI algorithms that can promote the early detection of high-risk oral lesions.

High referral accuracy for oral cancers and oral potentially malignant disorders using telemedicine.

OBJECTIVE: To evaluate the accuracy of MeMoSA®, a mobile phone application to review images of oral lesions in identifying oral cancers and oral potentially malignant disorders requiring referral. SUBJECTS AND METHODS: A prospective study of 355 participants, including 280 with oral lesions/variants was conducted. Adults aged ≥18 treated at tertiary referral centres were included. Images of the oral cavity were taken using MeMoSA®. The identification of the presence of lesion/variant and referral decision made using MeMoSA® were compared to clinical oral examination, using kappa statistics for intra-rater agreement. Sensitivity, specificity, concordance and F1 score were computed. Images were reviewed by an off-site specialist and inter-rater agreement was evaluated. Images from sequential clinical visits were compared to evaluate observable changes in the lesions. RESULTS: Kappa values comparing MeMoSA® with clinical oral examination in detecting a lesion and referral decision was 0.604 and 0.892, respectively. Sensitivity and specificity for referral decision were 94.0% and 95.5%. Concordance and F1 score were 94.9% and 93.3%, respectively. Inter-rater agreement for a referral decision was 0.825. Progression or regression of lesions were systematically documented using MeMoSA®. CONCLUSION: Referral decisions made through MeMoSA® is highly comparable to clinical examination demonstrating it is a reliable telemedicine tool to facilitate the identification of high-risk lesions for early management.

Time-to-Treatment of Oral Cancer and Potentially Malignant Oral Disorders: Findings in Malaysian Public Healthcare.

This study aims to evaluate the time-to-treatment of oral cancer and potentially malignant oral disorders (PMOD) in a Malaysian public healthcare setting while exploring its contributing factors. It consists of (1) a cross-sectional patient survey to quantify time to seek care and barriers faced, and (2) a retrospective medical record abstraction to determine treatment and management intervals. Time intervals were aggregated and analyzed by their primary contributor­patient, professional, or healthcare system. The average total time-to-treatment of the 104 patients investigated was 167 days (SD = 158). This was predominantly contributed by the patient interval of 120 days (SD = 152). In total, 67.0% of patients delayed their visit to primary healthcare centers because they assumed the lesions were not dangerous or of concern. Additionally, there was a significant difference between patients 'facing' and 'not facing' difficulties to seek care, at 157 vs. 103 days (p = 0.028). System and professional delays were comparably shorter, at 33 days (SD = 20) and 10 days (SD = 15) respectively. Both demonstrated a significant difference between oral cancer and PMOD, at 43 vs. 29 days (p < 0.001) and 5 vs. 17 days (p < 0.001). The findings reiterate the need to reform current initiatives to better promote early lesion recognition by patients and implement strategies for the elimination of their access barriers.

Transcriptional analysis highlights three distinct immune profiles of high-risk oral epithelial dysplasia.

Oral potentially malignant disorders (OPMD) are precursors of oral squamous cell carcinoma (OSCC), and the presence of oral epithelial dysplasia (OED) in OPMD confers an increased risk of malignant transformation. Emerging evidence has indicated a role for the immune system in OPMD disease progression; however, the underlying immune mechanisms remain elusive. In this study, we used immune signatures established from cancer to delineate the immune profiles of moderate and severe OED, which are considered high-risk OPMD. We demonstrated that moderate and severe OEDs exhibit high lymphocyte infiltration and upregulation of genes involved in both immune surveillance (major histocompatibility complex-I, T cells, B cells and cytolytic activity) and immune suppression (immune checkpoints, T regulatory cells, and tumor-associated macrophages). Notably, we identified three distinct subtypes of moderate and severe OED: immune cytotoxic, non-cytotoxic and non-immune reactive. Active immune surveillance is present in the immune cytotoxic subtype, whereas the non-cytotoxic subtype lacks CD8 immune cytotoxic response. The non-immune reactive subtype showed upregulation of genes involved in the stromal microenvironment and cell cycle. The lack of T cell infiltration and activation in the non-immune reactive subtype is due to the dysregulation of CTNNB1, PTEN and JAK2. This work suggests that moderate and severe OED that harbor the non-cytotoxic or non-immune reactive subtype are likely to progress to cancer. Overall, we showed that distinct immune responses are present in high-risk OPMD, and revealed targetable pathways that could lead to potential new approaches for non-surgical management of OED.

Household Catastrophic Health Expenditure from Oral Potentially Malignant Disorders and Oral Cancer in Public Healthcare of Malaysia.

OBJECTIVE: Oral cancer causes a significant disease burden and financial distress, especially among disadvantaged groups. While Malaysia has achieved universal health coverage via its highly subsidized public healthcare, patient and family expenditure for treatment of oral potentially malignant disorders (OPMD) and oral cancer remains a concern in the equitability of care. This study thus aims to estimate household out-of-pocket (OOP) expenditures and the extent of catastrophic healthcare expenditure (CHE) while identifying its predictors. METHODS: This three-part study consists of a cross-sectional survey to collect sociodemographic and health utilization data of patients, a retrospective medical record abstraction to identify resources consumed, and cost modeling to simulate expenditures in two tertiary public hospitals. Loss of productivity was calculated based on absenteeism related to disease management in the hospital. OOP payments for transport, care in public healthcare facilities, and other healthcare expenditures were tallied. A CHE was defined as OOP spendings of more than 10% from total annual household income. Multivariable logistic regression was further applied to identify the association between sociodemographic factors and the incidence of CHE. RESULTS: A total of 52 patients with OPMD and 52 with oral cancer were surveyed and medical records were abstracted. A Kruskal-Wallis test showed a statistically significant difference in OOP share over household income between OPMD, early- and late-stage cancer, χ2(2)=51.05, p<0.001, with the mean percentage of 9%, 22%, and 65% respectively. This study found that the prevalence of CHE in the first year of diagnosis was 86.5% for oral cancer and 19.2% for OPMD. Indian ethnicity (OR=6.24, p=0.046) and monthly income group 'less than USD 2,722' (OR=14.32, p=0.023) were shown as significant predictors for CHE. CONCLUSIONS: Our study demonstrated the provision of subsidies may not be adequate to shield the more vulnerable group from CHE when they are diagnosed with OPMD and oral cancer.

Are There Betel Quid Mixtures Less Harmful than Others? A Scoping Review of the Association between Different Betel Quid Ingredients and the Risk of Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) is a potentially malignant condition of the oral cavity characterized by progressive fibrosis of the submucosal tissues. OSF is typically associated with the use of betel quid (BQ), a chewing package made of natural products (e.g., areca nut, betel leaves), with or without smokeless tobacco. BQ ingredients contain pro-carcinogenic bioactive compounds, but also potentially protective biomolecules, and we have shown recently that the chemical properties of different BQ recipes vary, which may explain the unequal prevalence of OSF and oral cancer in BQ users in different geographical regions. Hence, this scoping review was aimed at evaluating the existing literature regarding different BQ compounds and their association with OSF. The repository of the National Library of Medicine (MEDLINE/PubMed), medRxiv databases, Google scholar, Baidu scholar, CNKI, and EBSCO were used to search for publications that investigated the association between BQ chewing and OSF up to November 2021. The search terminology was constructed using the keywords "betel quid" and "oral submucous fibrosis", and their associated terms, with the use of Boolean operators. The search was conducted under Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines, together with clear inclusion and exclusion criteria. The review showed that the risk of developing OSF varied between different BQ recipes, and that chewing BQ mixtures containing betel inflorescence (BI) significantly increased the risk of OSF, as did the addition of tobacco. Conversely, the use of betel leaf in the mixture was likely to be protective, which may be due to the presence of polyphenols. Although further research is needed to determine the effect of individual BQ ingredients in the development of OSF, our pilot results provide the scope and rationale for informing future chemopreventive strategies for OSF and oral cancer in BQ chewers.

Rational Combinations of Targeted Therapy and Immune Checkpoint Inhibitors in Head and Neck Cancers.

Immunotherapy, especially the immune checkpoint inhibitors (ICIs) such as the pembrolizumab and nivolumab have contributed to significant improvements in treatment outcomes and survival of head and neck cancer (HNC) patients. Still, only a subset of patients benefits from ICIs and hence the race is on to identify combination therapies that could improve response rates. Increasingly, genetic alterations that occur within cancer cells have been shown to modulate the tumor microenvironment resulting in immune evasion, and these have led to the emergence of trials that rationalize a combination of targeted therapy with immunotherapy. In this review, we aim to provide an overview of the biological rationale and current strategies of combining targeted therapy with the approved ICIs in HNC. We summarize the ongoing combinatorial clinical trials and discuss emerging immunomodulatory targets. We also discuss the challenges and gaps that have yet to be addressed, as well as future perspectives in combining these different drug classes.

Barriers to early detection and management of oral cancer in the Asia Pacific region.

OBJECTIVE: Oral cancer is amenable to early detection but remains a prominent cause of mortality in the Asia Pacific region. This study aimed to identify barriers to early detection and management of oral cancer in the Asia Pacific region. METHODS: A mixed-methods approach was employed triangulating findings from a survey and focus groups. The survey was conducted among seven representative members of the Asia Pacific Oral Cancer Network (APOCNET) across six countries. Focus groups were conducted to gain deeper insights into the findings of the survey. RESULTS: The identified barriers were a lack of national cancer control strategies and cancer registries and the limited availability of trained health care professionals. Overcoming these challenges in the Asia Pacific region where resources are scarce will require collaborative partnerships in data collection and novel approaches for continuous professional training including eLearning. Further, to overcome the lack of trained health care professionals, innovative approaches to the management of oral potentially malignant lesions and oral cancer including telemedicine were suggested. CONCLUSION: The findings of this study should be taken into account when charting national cancer control plans for oral cancer and will form the basis for future collaborative studies in evaluating effective measures to improve oral cancer detection and management in low- and middle-income countries.

Uncovering drug repurposing candidates for head and neck cancers: insights from systematic pharmacogenomics data analysis.

Effective treatment options for head and neck squamous cell carcinoma (HNSCC) are currently lacking. We exploited the drug response and genomic data of the 28 HNSCC cell lines, screened with 4,518 compounds, from the PRISM repurposing dataset to uncover repurposing drug candidates for HNSCC. A total of 886 active compounds, comprising of 418 targeted cancer, 404 non-oncology, and 64 chemotherapy compounds were identified for HNSCC. Top classes of mechanism of action amongst targeted cancer compounds included PI3K/AKT/MTOR, EGFR, and HDAC inhibitors. We have shortlisted 36 compounds with enriched killing activities for repurposing in HNSCC. The integrative analysis confirmed that the average expression of EGFR ligands (AREG, EREG, HBEGF, TGFA, and EPGN) is associated with osimertinib sensitivity. Novel putative biomarkers of response including those involved in immune signalling and cell cycle were found to be associated with sensitivity and resistance to MEK inhibitors respectively. We have also developed an RShiny webpage facilitating interactive visualization to fuel further hypothesis generation for drug repurposing in HNSCC. Our study provides a rich reference database of HNSCC drug sensitivity profiles, affording an opportunity to explore potential biomarkers of response in prioritized drug candidates. Our approach could also reveal insights for drug repurposing in other cancers.

DNA Vaccines Targeting Novel Cancer-Associated Antigens Frequently Expressed in Head and Neck Cancer Enhance the Efficacy of Checkpoint Inhibitor.

HPV-independent head and neck squamous cell carcinoma (HNSCC) is a common cancer globally. The overall response rate to anti-PD1 checkpoint inhibitors (CPIs) in HNSCC is ~16%. One major factor influencing the effectiveness of CPI is the level of tumor infiltrating T cells (TILs). Converting TILlow tumors to TILhigh tumors is thus critical to improve clinical outcome. Here we describe a novel DNA vaccines to facilitate the T-cell infiltration and control tumor growth. We evaluated the expression of target antigens and their respective immunogenicity in HNSCC patients. The efficacy of DNA vaccines targeting two novel antigens were evaluated with or without CPI using a syngeneic model. Most HNSCC patients (43/44) co-expressed MAGED4B and FJX1 and their respective tetramer-specific T cells were in the range of 0.06-0.12%. In a preclinical model, antigen-specific T cells were induced by DNA vaccines and increased T cell infiltration into the tumor, but not MDSC or regulatory T cells. The vaccines inhibited tumor growth and improved the outcome alone and upon combination with anti-PD1 and resulted in tumor clearance in approximately 75% of mice. Pre-existence of MAGED4B and FJX1-reactive T cells in HNSCC patients suggests that these widely expressed antigens are highly immunogenic and could be further expanded by vaccination. The DNA vaccines targeting these antigens induced robust T cell responses and with the anti-PD1 antibody conferring excellent tumor control. This opens up an opportunity for combination immunotherapy that might benefit a wider population of HNSCC patients in an antigen-specific manner.

Provider cost of treating oral potentially malignant disorders and oral cancer in Malaysian public hospitals.

Oral cancer has been recognized as a significant challenge to healthcare. In Malaysia, numerous patients frequently present with later stages of cancers to the highly subsidized public healthcare facilities. Such a trend contributes to a substantial social and economic burden. This study aims to determine the cost of treating oral potentially malignant disorders (OPMD) and oral cancer from a public healthcare provider's perspective. Medical records from two tertiary public hospitals were systematically abstracted to identify events and resources consumed retrospectively from August 2019 to January 2020. The cost accrued was used to estimate annual initial and maintenance costs via two different methods- inverse probability weighting (IPW) and unweighted average. A total of 86 OPMD and 148 oral cancer cases were included. The initial phase mean unadjusted cost was USD 2,861 (SD = 2,548) in OPMD and USD 38,762 (SD = 12,770) for the treatment of cancer. Further annual estimate of initial phase cost based on IPW method for OPMD, early and late-stage cancer was USD 3,561 (SD = 4,154), USD 32,530 (SD = 12,658) and USD 44,304 (SD = 16,240) respectively. Overall cost of late-stage cancer was significantly higher than early-stage by USD 11,740; 95% CI [6,853 to 16,695]; p< 0.001. Higher surgical care and personnel cost predominantly contributed to the larger expenditure. In contrast, no significant difference was identified between both cancer stages in the maintenance phase, USD 700; 95% CI [-1,142 to 2,541]; p = 0.457. A crude comparison of IPW estimate with unweighted average displayed a significant difference in the initial phase, with the latter being continuously higher across all groups. IPW method was shown to be able to use data more efficiently by adjusting cost according to survival and follow-up. While cost is not a primary consideration in treatment recommendations, our analysis demonstrates the potential economic benefit of investing in preventive medicine and early detection.

Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway.

New therapeutic targets for oral squamous cell carcinoma (OSCC) are urgently needed. We conducted genome-wide CRISPR-Cas9 screens in 21 OSCC cell lines, primarily derived from Asians, to identify genetic vulnerabilities that can be explored as therapeutic targets. We identify known and novel fitness genes and demonstrate that many previously identified OSCC-related cancer genes are non-essential and could have limited therapeutic value, while other fitness genes warrant further investigation for their potential as therapeutic targets. We validate a distinctive dependency on YAP1 and WWTR1 of the Hippo pathway, where the lost-of-fitness effect of one paralog can be compensated only in a subset of lines. We also discover that OSCCs with WWTR1 dependency signature are significantly associated with biomarkers of favorable response toward immunotherapy. In summary, we have delineated the genetic vulnerabilities of OSCC, enabling the prioritization of therapeutic targets for further exploration, including the targeting of YAP1 and WWTR1.

Many types of cancer now have 'targeted treatments', which specifically home in on genes cancer cells rely on for survival. But there are very few of these treatments available for the most common type of mouth cancer, oral squamous cell carcinoma, which around 350,000 people are diagnosed with each year. Designing targeted treatments relies on detailed knowledge of the genetic makeup of the cancer cells. But, little is known about which genes drive oral squamous cell carcinoma, especially among patients living in Asia, which is where over half of yearly cases are diagnosed. One way to resolve this is to use gene editing technology to find the genes that the cancer cells need to survive. Now, Chai et al. have used a gene editing tool known as CRISPR to examine 21 cell lines from patients diagnosed with oral squamous cell carcinoma. Most of these lines were from Asian patients, some of whom had a history of chewing betel quid which increases the risk of mouth cancer. By individually inactivating genes in these cell lines one by one, Chai et al. were able to identify 918 genes linked to the survival of the cancer cells. Some of these genes have already been associated with the spread of other types of cancer, whereas others are completely unique to oral squamous cell carcinoma. The screen also discovered that some cell lines could not survive without genes involved in a signalling pathway called Hippo, which is known to contribute to the progression of many other types of cancer. Uncovering the genes associated with oral squamous cell carcinoma opens the way for the development of new targeted treatments. Targeted therapies already exist for some of the genes identified in this study, and it may be possible to repurpose them as a treatment for this widespread mouth cancer. But, given that different cell lines relied on different genes to survive, the next step will be to identify which genes to inactivate in each patient.

An Overview on Betel Quid and Areca Nut Practice and Control in Selected Asian and South East Asian Countries.

Background: Areca nut (AN) and betel quid (BQ) chewing are ancient practices followed by an extensive proportion of the world's population. These practices are endemic in larger parts of South and Southeast Asia and selected Western Pacific countries. The prevalence of these habits varies across regions, age, gender, cultural practice, and socioeconomic status groups. Considerable variations exist between countries with respect to prevention/intervention programs, and policy guidelines of BQ usage. Objectives: (1) To provide an overview of the BQ chewing prevalence, practices, preventive interventions and policies in selected Asian and Western Pacific countries. (2) To explore the different terminologies associated with BQ use. Method: A narrative review of the current literature related to BQ, AN, and oral cancer was conducted by searching PUBMED, CINAHL, and GOOGLE databases. Results: The literature review revealed that the prevalence of BQ was found to be highest in Papua New Guinea, followed by Bangladesh, India, Pakistan, Myanmar and Sri Lanka. While, Cambodia, Malaysia, Indonesia and Taiwan had comparatively lower prevalence. Smokeless tobacco, BQ with tobacco, BQ without tobacco, AN were some of the terminologies used for BQ in various studies. Conclusions: The prevalence, and the interventional policies related to BQ and AN chewing habits varies widely among the selected countries. With the increasing awareness and association of BQ with oral cancer, there is a need to have better awareness, prevention and interventional strategies in place. We also found considerable variation in the use of terminologies associated with BQ.

Label-Free, High-Throughput Assay of Human Dendritic Cells from Whole-Blood Samples with Microfluidic Inertial Separation Suitable for Resource-Limited Manufacturing.

Microfluidics technology has not impacted the delivery and accessibility of point-of-care health services, like diagnosing infectious disease, monitoring health or delivering interventions. Most microfluidics prototypes in academic research are not easy to scale-up with industrial-scale fabrication techniques and cannot be operated without complex manipulations of supporting equipment and additives, such as labels or reagents. We propose a label- and reagent-free inertial spiral microfluidic device to separate red blood, white blood and dendritic cells from blood fluid, for applications in health monitoring and immunotherapy. We demonstrate that using larger channel widths, in the range of 200 to 600 µm, allows separation of cells into multiple focused streams, according to different size ranges, and we utilize a novel technique to collect the closely separated focused cell streams, without constricting the channel. Our contribution is a method to adapt spiral inertial microfluidic designs to separate more than two cell types in the same device, which is robust against clogging, simple to operate and suitable for fabrication and deployment in resource-limited populations. When tested on actual human blood cells, 77% of dendritic cells were separated and 80% of cells remained viable after our assay.