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.

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.

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.

Clinico-pathological study of malignant odontogenic tumours from a national referral centre.

BACKGROUND: Malignant odontogenic tumours are extremely rare tumours occurring within the jaws. Our study was performed to determine the demographic and clinico-pathological features of malignant odontogenic tumours amongst a multi-ethnic Asian population. METHODS: This was a retrospective cross-sectional study of malignant odontogenic tumours diagnosed at the Institute for Medical Research, Malaysia, from 2009 to 2019. All cases were independently reviewed and reclassified following the criteria set out in the latest edition of the World Health Organization 2017 reference text. Demographic and clinico-pathological data were recorded for each case. RESULTS: Twenty-four cases of malignant odontogenic tumours were identified. The patients' age ranged from 16 to 79 years with the mean age at diagnosis being 50.8 years (SD = 16.18). There was a male predominance (66.7%) in this cohort of patients. The ethnic distribution appeared to reflect the Malaysian population with most cases seen amongst the Malay ethnic group (66.7%). Ameloblastic carcinoma was the most frequently diagnosed malignant odontogenic tumour (45.8%) and was also predominantly seen in males (90.9%). All patients with clear cell odontogenic carcinoma were females. There was no obvious sex predilection in primary odontogenic carcinoma not otherwise specified (NOS). The mandible (79.2%) was more frequently involved compared to the maxilla. CONCLUSIONS: Diagnosis and management of malignant odontogenic tumours are challenging due to the rarity of these tumours. Our study has elucidated the clinico-pathological features of malignant odontogenic tumours seen in a multi-ethnic Asian population.

A retrospective analysis of oral and maxillofacial biopsied specimens in Malaysian newborns and infants.

BACKGROUND: Globally, research on oral and maxillofacial lesions among newborns and infants remains limited. AIM: To describe demographic patterns, histopathological findings, and locations of oral and maxillofacial lesions in newborns (birth-1 month) and infants (>1 month-2 years) reported over 51 years. DESIGN: A retrospective cross-sectional study on histopathological records of newborns and infants was conducted. Patients' demographic characteristics (age, gender, and race), histopathological diagnosis, and lesion's location were gathered. Pearson's chi-square or Fisher's exact test was performed to determine associations between demographic characteristics and different categories of lesions. RESULTS: Out of 66,546 specimens received, 0.44% (290 specimens) were from patients aged 2 years and younger (27 newborns and 263 infants). The most common category was inflammatory/reactive (44.2%), followed by tumour/tumour-like (42.0%), cystic/pseudocystic (6.6%), and miscellaneous lesions (5.5%). Mucous extravasation cysts (23.4%) and Langerhans cell histiocytosis (7.2%) were the most common histopathological diagnoses. Tumour/tumour-like lesions were significant in newborns (P = .021), and majority were congenital epulis (40.7%). Inflammatory/reactive lesions were significantly higher in male (P = .025) and infants (P = <.001) with predominant incidences of mucous extravasation cysts (24.7%). There was a more than one ten probability of malignant lesions (11.4%). CONCLUSION: Routine clinical head and neck examinations in newborns and infants are the key to early detection of lesions.

In vitro evaluation of dual-antigenic PV1 peptide vaccine in head and neck cancer patients.

Peptide vaccines derived from tumour-associated antigens have been used as an immunotherapeutic approach to induce specific cytotoxic immune response against tumour. We previously identified that MAGED4B and FJX1 proteins are overexpressed in HNSCC patients; and further demonstrated that two HLA-A2-restricted 9-11 amino acid peptides derived from these proteins were able to induce anti-tumour immune responses in vitro independently using PBMCs isolated from these patients. In this study, we evaluated the immunogenicity and efficacy of a dual-antigenic peptide vaccine (PV1), comprised of MAGED4B and FJX1 peptides in HNSCC patients. We first demonstrated that 94.8% of HNSCC patients expressed MAGED4B and/or FJX1 by immunohistochemistry, suggesting that PV1 could benefit the majority of HNSCC patients. The presence of pre-existing MAGED4B and FJX1-specific T-cells was detected using a HLA-A2 dimer assay and efficacy of PV1 to induce T-cell to secrete cytotoxic cytokine was evaluated using ELISPOT assay. Pre-existing PV1-specific T-cells were detected in all patients. Notably, we demonstrated that patients' T-cells were able to secrete cytotoxic cytokines upon exposure to target cells expressing the respective antigen post PV1 stimulation. Furthermore, patients with high expression of MAGED4B and FJX1 in their tumours were more responsive to PV1 stimulation, demonstrating the specificity of the PV1 peptide vaccine. Additionally, we also demonstrated the expression of MAGED4B and FJX1 in breast, lung, colon, prostate and rectal cancer suggesting the potential use of PV1 in these cancers. In summary, PV1 could be a good vaccine candidate for the treatment of HNSCC patients and other cancers expressing these antigens.

Trigeminal neuralgia: a retrospective multicentre study of 320 Asian patients.

OBJECTIVES: This study was performed to obtain the clinicodemographic data regarding patients with trigeminal neuralgia (TN) treated at oral-maxillofacial medicine clinics, as there is a paucity of such information in the Asian setting. STUDY DESIGN: Retrospective multicenter study involving clinicodemographic information of 320 patients with TN diagnosed between 2001 and 2012 at eight regional oral-maxillofacial medicine clinics and followed up for at least 6 months. Statistical tests were performed to assess the associations among the clinicodemographic factors. RESULTS: TN was mostly diagnosed during the seventh and sixth decades of life, with a median of 58.2 years (interquartile range = 13.0). Females were more commonly affected (61.6%). TN affected the right side more frequently, and the mandibular branch was most commonly involved (58.5%). Carbamazepine was the first-line drug of choice (87.5%). CONCLUSIONS: Asian patients with TN exhibited features similar to those in Caucasian patients except for the increased affliction of the mandibular division.

Genetically-defined novel oral squamous cell carcinoma cell lines for the development of molecular therapies.

Emerging biological and translational insights from large sequencing efforts underscore the need for genetically-relevant cell lines to study the relationships between genomic alterations of tumors, and therapeutic dependencies. Here, we report a detailed characterization of a novel panel of clinically annotated oral squamous cell carcinoma (OSCC) cell lines, derived from patients with diverse ethnicity and risk habits. Molecular analysis by RNAseq and copy number alterations (CNA) identified that the cell lines harbour CNA that have been previously reported in OSCC, for example focal amplications in 3q, 7p, 8q, 11q, 20q and deletions in 3p, 5q, 8p, 18q. Similarly, our analysis identified the same cohort of frequently mutated genes previously reported in OSCC including TP53, CDKN2A, EPHA2, FAT1, NOTCH1, CASP8 and PIK3CA. Notably, we identified mutations (MLL4, USP9X, ARID2) in cell lines derived from betel quid users that may be associated with this specific risk factor. Gene expression profiles of the ORL lines also aligned with those reported for OSCC. By focusing on those gene expression signatures that are predictive of chemotherapeutic response, we observed that the ORL lines broadly clustered into three groups (cell cycle, xenobiotic metabolism, others). The ORL lines noted to be enriched in cell cycle genes responded preferentially to the CDK1 inhibitor RO3306, by MTT cell viability assay. Overall, our in-depth characterization of clinically annotated ORL lines provides new insight into the molecular alterations synonymous with OSCC, which can facilitate in the identification of biomarkers that can be used to guide diagnosis, prognosis, and treatment of OSCC.

Co-Expression of TWIST1 and ZEB2 in Oral Squamous Cell Carcinoma Is Associated with Poor Survival.

Oral squamous cell carcinoma (OSCC) is an aggressive disease accounting for more than 260,000 cancer cases diagnosed and 128,000 deaths worldwide. A large majority of cancer deaths result from cancers that have metastasized beyond the primary tumor. The relationship between genetic changes and clinical outcome can reflect the biological events that promote cancer's aggressive behavior, and these can serve as molecular markers for improved patient management and survival. To this end, epithelial-mesenchymal transition (EMT) is a major process that promotes tumor invasion and metastasis, making EMT-related proteins attractive diagnostic biomarkers and therapeutic targets. In this study, we used immunohistochemistry to study the expression of a panel of transcription factors (TWIST1, SNAI1/2, ZEB1 and ZEB2) and other genes intimately related to EMT (CDH1 and LAMC2) at the invasive tumor front of OSCC tissues. The association between the expression of these proteins and clinico-pathological parameters were examined with Pearson Chi-square and correlation with survival was analyzed using Kaplan Meier analysis. Our results demonstrate that there was a significant differential expression of CDH1, LAMC2, SNAI1/2 and TWIST1 between OSCC and normal oral mucosa (NOM). Specifically, CDH1 loss was significantly associated with Broder's grading, while diffused LAMC2 was similarly associated with non-cohesive pattern of invasion. Notably, co-expression of TWIST1 and ZEB2 in OSCC was significantly associated with poorer overall survival, particularly in patients without detectable lymph node metastasis. This study demonstrates that EMT-related proteins are differentially expressed in OSCC and that the co-expression of TWIST1 and ZEB2 could be of clinical value in identifying patients with poor survival for appropriate patient management.

Four-protein signature accurately predicts lymph node metastasis and survival in oral squamous cell carcinoma.

The presence of lymph node (LN) metastasis significantly affects the survival of patients with oral squamous cell carcinoma (OSCC). Successful detection and removal of positive LNs are crucial in the treatment of this disease. Current evaluation methods still have their limitations in detecting the presence of tumor cells in the LNs, where up to a third of clinically diagnosed metastasis-negative (N0) patients actually have metastasis-positive LNs in the neck. We developed a molecular signature in the primary tumor that could predict LN metastasis in OSCC. A total of 211 cores from 55 individuals were included in the study. Eleven proteins were evaluated using immunohistochemical analysis in a tissue microarray. Of the 11 biomarkers evaluated using receiver operating curve analysis, epidermal growth factor receptor (EGFR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (HER-2/neu), laminin, gamma 2 (LAMC2), and ras homolog family member C (RHOC) were found to be significantly associated with the presence of LN metastasis. Unsupervised hierarchical clustering-demonstrated expression patterns of these 4 proteins could be used to differentiate specimens that have positive LN metastasis from those that are negative for LN metastasis. Collectively, EGFR, HER-2/neu, LAMC2, and RHOC have a specificity of 87.5% and a sensitivity of 70%, with a prognostic accuracy of 83.4% for LN metastasis. We also demonstrated that the LN signature could independently predict disease-specific survival (P = .036). The 4-protein LN signature validated in an independent set of samples strongly suggests that it could reliably distinguish patients with LN metastasis from those who were metastasis-free and therefore could be a prognostic tool for the management of patients with OSCC.

Ameloblastoma of the jaws: a retrospective analysis of 340 cases in a Malaysian population.

PURPOSE: Ameloblastoma of the human jaw is an uncommon but clinically significant odontogenic epithelial neoplasm. The aim was to analyze the clinicopathologic characteristics of ameloblastoma in a Malaysian population. MATERIALS AND METHODS: This is a retrospective study (1993 through 2008) of consecutive ameloblastoma cases accessioned in 2 main oral pathology diagnostic centers: the Unit of Stomatology, Institute for Medical Research and the Department of Oral Pathology, Oral Medicine, and Periodontology, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia. Data on patient demographics, tumor location, symptomology, duration, radiographic appearance, preoperative diagnosis, clinicopathologic subtypes, treatment, and recurrence were analyzed. RESULTS: Three hundred forty cases of ameloblastoma were reviewed. These were from 197 male patients (57.9%) and 143 female patients (42.1%), with a male-to-female ratio of 1.4:1. A wide age range (7 to 85 years), mean onset age of 30.3 ± 16.3 years, and peak incidence in the second decade of life were recorded. Most were mandibular tumors (n = 311/340, 91.5%). These consisted of 95 (28%) unicystic ameloblastomas, 221 (65%) solid/multicystic ameloblastomas, 22 (6.4%) desmoplastic ameloblastoma, and 2 (0.6%) peripheral ameloblastomas. Unicystic ameloblastoma (41.1%) and solid/multicystic ameloblastoma (52.0%) mostly affected Malays patients, whereas desmoplastic ameloblastoma (59.1%) was prevalent in Chinese patients. Unicystic ameloblastoma (56.8%) and solid/multicystic ameloblastoma (47.1%) occurred predominantly in the body and posterior mandible, whereas desmoplastic ameloblastoma (36.4%) preferentially involved the anterior jaw segment. Most tumors presented as multilocular radiolucencies (36.8%). Enucleation (n = 42/92, 45.7%) was the treatment of choice. About 18 cases (13.3%) presented with recurrence. CONCLUSIONS: Because ameloblastoma subsets differ in their biologic behavior, the present data are significant as baseline references for clinicians and pathologists.

An updated clinical and epidemiological profile of the adenomatoid odontogenic tumour: a collaborative retrospective study.

BACKGROUND: Adenomatoid odontogenic tumour (AOT) is a benign odontogenic jaw lesion. The aim of this study was to update the biological profile of AOT. MATERIAL AND METHODS: Cases published in the literature and cases in files of co-authors were included. RESULTS: 550 new cases were retrieved, and of a total of 1082 cases analysed, 87.2% were found in the second and third decades. The M:F ratio was 1:1.9. 70.8% were of the follicular variant (extrafollicular: 26.9%, peripheral: 2.3%). 64.3% occurred in the maxilla. 60% of follicular AOTs were associated with unerupted canines. Nineteen cases of AOT (2.8%, M:F ratio was 1:1.4) were associated with embedded third molars. Twenty-two peripheral AOTs (2.3%, M:F ratio was 1:5.3) were recorded. The relative frequency (RF) of AOT ranged between 0.6% and 38.5%, revealing a considerably wider AOT/RF range than hitherto reported (2.2-7.1%). CONCLUSIONS: This updated review based on the largest number of AOT cases ever presented, confirms the distinctive, although not pathognomonic clinicopathological profile of the AOT, its worldwide occurrence, and its consistently benign behaviour.

HPV infection and the alterations of the pRB pathway in oral carcinogenesis.

Inactivation of the retinoblastoma (pRB) pathway is a common event in oral squamous cell carcinoma particularly through the aberrant expression of the components within this pathway. This study examines the alterations of molecules within the pRB pathway by looking at the presence of homozygous deletions in p16(INK4A) and the expression patterns of pRB, cyclin D1 and CDK4, as well as the presence of human papillomavirus (HPV) in our samples. In our study, 5/20 samples demonstrated deletions of p16(INK4A) exon 1alpha. pRB overexpression was found in 20/20 samples, the expression was mainly observed in all layers of the epithelia, particularly in the basal layer where cells are actively dividing and aberrant pRB expression was found in 12/20 samples. Cyclin D1 and CDK4 overexpression was detected in 6/20 and 2/20 samples respectively in comparison to hyperplasias where both proteins were either not expressed or expressed at minimal levels (<10%). Strikingly, HPV was found to be present in all of our samples, suggesting that HPV plays a significant role in driving oral carcinogenesis. Notably, 17/20 of our samples showed more than one alteration in the pRB pathway, however, we did not find any significant relationship between the presence of HPV, homozygous deletion of p16(INK4A) and overexpression of pRB, cyclin D1 and CDK4. Collectively, this data demonstrates that alterations in the pRB pathway are a common event and involve the aberration of more than one molecule within the pathway. Furthermore, the involvement of HPV in all our samples suggests that HPV infection may play an important role in oral carcinogenesis.

Establishment and characterization of Asian oral cancer cell lines as in vitro models to study a disease prevalent in Asia.

We have established 3 cell lines ORL-48, -115 and -136 from surgically resected specimens obtained from untreated primary human oral squamous cell carcinomas of the oral cavity. The in vitro growth characteristics, epithelial origin, in vitro anchorage independency, human papilloma-virus (HPV) infection, microsatellite instability status, karyotype and the status of various cell cycle regulators and gatekeepers of these cell lines were investigated. All 3 cell lines grew as monolayers with doubling times ranging between 26.4 and 40.8 h and were immortal. Karyotyping confirmed that these cell lines were of human origin with multiple random losses and gains of entire chromosomes and regions of chromosomes. Immunohistochemistry staining of cytokeratins confirmed the epithelial origin of these cell lines, and the low degree of anchorage independency expressed by these cell lines suggests non-transformed phenotypes. Genetic analysis identified mutations in the p53 gene in all cell lines and hypermethylation of p16INK4a in ORL-48 and -136. Analysis of MDM2 and EGFR expression indicated MDM2 overexpression in ORL-48 and EGFR overexpression in ORL-136 in comparison to the protein levels in normal oral keratinocytes. Analysis of the BAT-26 polyadenine repeat sequence and MLH-1 and MSH-2 repair enzymes demonstrated that all 3 cell lines were microsatellite stable. The role of HPV in driving carcinogenesis in these tumours was negated by the absence of HPV. Finally, analysis of the tissues from which these cell lines were derived indicated that the cell lines were genetically representative of the tumours, and, therefore, are useful tools in the understanding of the molecular changes associated with oral cancers.

Gingival myofibroma in children: report of 4 cases with immunohistochemical findings.

Oral myofibroma is a rare tumour which usually occurs in children and has been reported in the mandible, tongue, buccal mucosa with only a few cases reported from the gingiva. It appears alarming clinically due to its fast growth which may mimic a malignancy. However, it is completely benign and is usually treated by complete surgical excision with excellent prognosis. Clinically, myofibroma presents as a single swelling when it occurs on the gingiva, and more common lesions such as fibrous epulis, pyogenic granuloma and peripheral odontogenic fibroma, myofibroma are usually considered in the differential diagnosis. We present 4 additional cases of gingival myofibroma in children. Their ages ranged from 7 to 14 years. Three were girls and 1 patient was a boy. All presented with solitary gingival growths, ranging from 3 weeks to 2 months in duration, and raised the clinical diagnoses of peripheral giant cell granuloma, pyogenic granuloma and fibrous epulis. Histopathology of incisional biopsies revealed proliferation of streaming and whorled fascicles of spindle cells around slit-like vascular spaces. The spindle cells were cytologically bland and were immunopositive for vimentin and smooth muscle actin, but were negative for desmin and S-100 protein. All were treated by surgical excision.

Alterations of the p14ARF-p53-MDM2 pathway in oral squamous cell carcinoma: MDM2 overexpression is a common event.

The majority of global incidences of oral cancer occur in Asia, and the aetiology of oral cancer is different in Asia as it is in the West. However, whereas there is a growing understanding of the molecular mechanisms of oral cancer progression in the West, there is little progress in this understanding in Asia. In particular, the role of the p53 pathway in modulating cancer progression in Asian oral cancer remains unclear. In this study, we micro-dissected and analysed 20 well-differentiated oral squamous cell carcinoma specimens for alterations in the p53 pathway. We found that 6/20 samples contained mutations in the p53 gene which occurred in three hotspots, at codon 203, 218 and 296. Furthermore, 6/20 samples had a homozygous deletion of p14ARF, but notably p14ARF deletion and p53 mutation events were often independent and mutually exclusive. Strikingly, MDM2 was upregulated in 20/20 samples, but not in 3/3 normal tissue specimens. Taken together, these data suggest that inactivation of the p53 pathway is a frequent event in oral squamous cell carcinoma, which occurs by an aberration in one of a number of players in the p53 pathway.