Development of a high dimensional imaging mass cytometry panel to investigate spatial organization of tissue microenvironment in formalin-fixed archival clinical tissues.

To decipher the interactions between various components of the tumor microenvironment (TME) and tumor cells in a preserved spatial context, a multiparametric approach is essential. In this pursuit, imaging mass cytometry (IMC) emerges as a valuable tool, capable of concurrently analyzing up to 40 parameters at subcellular resolution. In this study, a set of antibodies was selected to spatially resolve multiple cell types and TME elements, including a comprehensive panel targeted at dissecting the heterogeneity of cancer-associated fibroblasts (CAF), a pivotal TME component. This antibody panel was standardized and optimized using formalin-fixed paraffin-embedded tissue (FFPE) samples from different organs/lesions known to express the markers of interest. The final composition of the antibody panel was determined based on the performance of conjugated antibodies in both immunohistochemistry (IHC) and IMC. Tissue images were segmented employing the Steinbock framework. Unsupervised clustering of single-cell data was carried out using a bioinformatics pipeline developed in R program. This paper provides a detailed description of the staining procedure and analysis workflow. Subsequently, the panel underwent validation on clinical FFPE samples from head and neck squamous cell carcinoma (HNSCC). The panel and bioinformatics pipeline established here proved to be robust in characterizing different TME components of HNSCC while maintaining a high degree of spatial detail. The platform we describe shows promise for understanding the clinical implications of TMA heterogeneity in large patient cohorts with FFPE tissues available in diagnostic biobanks worldwide.

TP53 mutation and human papilloma virus status as independent prognostic factors in a Norwegian cohort of vulva squamous cell carcinoma.

INTRODUCTION: Vulva squamous cell carcinoma (VSCC) develops through two separate molecular pathways-one involving high-risk human papilloma virus infection (HPV-associated), and the other without HPV infection (HPV-independent) often involving TP53 mutation. HPV-associated VSCC generally has a better progression-free survival than HPV-independent VSCC. The aim of this study was to determine TP53 mutation status using immunohistochemistry, compare different methods of HPV detection and correlate both with survival in a retrospective cohort of 123 patients with VSCC. MATERIAL AND METHODS: Immunohistochemistry for p53, Ki67 and p16INK4A (a surrogate marker for HPV infection) was performed on formalin-fixed paraffin-embedded tissues from a cohort of surgically treated VSCC patients to identify molecular subtypes of VSCC. Presence of HPV infection was detected by HPV DNA PCR and HPV mRNA in situ hybridization (ISH). The Pearson chi-square test and multivariable Cox regression model were used to investigate the association of different parameters with progression-free survival and disease-specific survival (DSS), and Kaplan-Meier curves were used to show the association of different parameters with survival. RESULTS: The results of p53 and p16INK4A immunohistochemistry confirmed three VSCC subtypes associated with different prognosis. The TP53 mutation status was identified as an independent prognostic factor of worse progression-free survival (p = 0.024) after adjustment for FIGO stage. p16INK4A immunohistochemistry, mRNA ISH, and DNA PCR had excellent concordance in terms of HPV detection. According to the multivariable Cox regression model, the presence of hrHPV mRNA correlated significantly with increased progression-free survival (p = 0.040) and DSS (p = 0.045), after adjustment for other confounders. CONCLUSIONS: p53 and p16INK4A immunohistochemistry stratify VSCC cohort into three subtypes with TP53mutated patients having the worst prognosis. The detection of hrHPV mRNA by ISH was an independent predictor of increased survival. Thus, the combined detection of p53 and HPV mRNA might improve risk stratification in VSCC.

Xeno-free generation of human induced pluripotent stem cells from donor-matched fibroblasts isolated from dermal and oral tissues.

BACKGROUND: Induced pluripotent stem cells (iPS) can be generated from various somatic cells and can subsequently be differentiated to multiple cell types of the body. This makes them highly promising for cellular therapy in regenerative medicine. However, to facilitate their clinical use and to ensure safety, iPS culturing protocols must be compliant with good manufacturing practice guidelines and devoid of xenogenic products. Therefore, we aimed to compare the efficiency of using humanized culture conditions, specifically human platelet lysate to fetal bovine serum, for iPS generation from different sources, and to evaluate their stemness. METHODS: iPS were generated via a platelet lysate or fetal bovine serum-based culturing protocol from matched dermal, buccal and gingival human fibroblasts, isolated from healthy donors (n = 2) after informed consent, via episomal plasmid transfection. Pluripotency, genotype and phenotype of iPS, generated by both protocols, were then assessed by various methods. RESULTS: More attempts were generally required to successfully reprogram xeno-free fibroblasts to iPS, as compared to xenogenic cultured fibroblasts. Furthermore, oral fibroblasts generally required more attempts for successful iPS generation as opposed to dermal fibroblasts. Morphologically, all iPS generated from fibroblasts formed tight colonies surrounded by a reflective "whitish" outer rim, typical for iPS. They also expressed pluripotency markers at both gene (SOX2, OCT4, NANOG) and protein level (SOX2, OCT4). Upon stimulation, all iPS showed ability to differentiate into the three primary germ layers via expression of lineage-specific markers for mesoderm (MESP1, OSR1, HOPX), endoderm (GATA4) and ectoderm (PAX6, RAX). Genome analysis revealed several amplifications and deletions within the chromosomes of each iPS type. CONCLUSIONS: The xeno-free protocol had a lower reprogramming efficiency compared to the standard xenogenic protocol. The oral fibroblasts generally proved to be more difficult to reprogram than dermal fibroblasts. Xeno-free dermal, buccal and gingival fibroblasts can successfully generate iPS with a comparable genotype/phenotype to their xenogenic counterparts.

The TP53 Codon 72 Arginine Polymorphism Is Found with Increased TP53 Somatic Mutations in HPV(-) and in an Increased Percentage among HPV(+) Norwegian HNSCC Patients.

BACKGROUND: Somatic TP53 mutations are frequent in head and neck squamous cell carcinoma (HNSCC) and are important pathogenic factors. OBJECTIVE: To study TP53 mutations relative to the presence of human papillomavirus (HPV) in tumors in HNSCC patients. METHODS: Using a custom-made next-generation sequencing (NGS) panel on formalin-fixed, paraffin-embedded tumor tissue, we analyzed somatic TP53 mutations and the TP53 single-nucleotide polymorphism (SNP) codon 72 (P72R; rs1042522) (proline → arginine) from 104 patients with HNSCC. RESULTS: Only 2 of 44 patients with HPV-positive (HPV(+)) HNSCC had a TP53 somatic mutation, as opposed to 42/60 HPV-negative (HPV(-)) HNSCC patients (p < 0.001). Forty-five different TP53 somatic mutations were detected. Furthermore, in HPV(-) patients, we determined an 80% prevalence of somatic TP53 mutations in the TP53 R72 polymorphism cohort versus 40% in the TP53 P72 cohort (p = 0.001). A higher percentage of patients with oral cavity SCC had TP53 mutations than HPV(-) oropharyngeal (OP) SCC patients (p = 0.012). Furthermore, 39/44 HPV(+) tumor patients harbored the TP53 R72 polymorphism in contrast to 42/60 patients in the HPV(-) group (p = 0.024). CONCLUSIONS: Our observations show that TP53 R72 polymorphism is associated with a tumor being HPV(+). We also report a higher percentage of somatic TP53 mutations with R72 than P72 in HPV(-) HNSCC patients.

Embryonic Stem Cells Can Generate Oral Epithelia under Matrix Instruction.

We aimed to investigate whether molecular clues from the extracellular matrix (ECM) can induce oral epithelial differentiation of pluripotent stem cells. Mouse embryonic stem cells (ESC) of the feeder-independent cell line E14 were used as a model for pluripotent stem cells. They were first grown in 2D on various matrices in media containing vitamin C and without leukemia inhibitory factor (LIF). Matrices investigated were gelatin, laminin, and extracellular matrices (ECM) synthesized by primary normal oral fibroblasts and keratinocytes in culture. Differentiation into epithelial lineages was assessed by light microscopy, immunocytochemistry, and flow cytometry for cytokeratins and stem cell markers. ESC grown in 2D on various matrices were afterwards grown in 3D organotypic cultures with or without oral fibroblasts in the collagen matrix and examined histologically and by immunohistochemistry for epithelial (keratin pairs 1/10 and 4/13 to distinguish epidermal from oral epithelia and keratins 8,18,19 to phenotype simple epithelia) and mesenchymal (vimentin) phenotypes. ECM synthesized by either oral fibroblasts or keratinocytes was able to induce, in 2D cultures, the expression of cytokeratins of the stratified epithelial phenotype. When grown in 3D, all ESC developed into two morphologically distinct cell populations on collagen gels: (i) epithelial-like cells organized in islands with occasional cyst- or duct-like structures and (ii) spindle-shaped cells suggestive of mesenchymal differentiation. The 3D culture on oral fibroblast-populated collagen matrices was necessary for further differentiation into oral epithelia. Only ESC initially grown on 2D keratinocyte or fibroblast-synthesized matrices reached full epithelial maturation. In conclusion, ESC can generate oral epithelia under matrix instruction.

Degenerative bony changes in the temporal component of the temporomandibular joint - review of the literature.

Temporomandibular joint (TMJ) changes are quite frequent in adults, but not all changes are degenerative. A high prevalence of bone alterations in the TMJs was reported by different research groups. Disturbed remodeling of bony articulating structures occurs because of overloading masticatory forces or because the mechanical loading in the area out-weighs the adaptive capacity of the TMJ structures. Although most of the degenerative TMJ alterations are identified at the level of the condylar process, a complete evaluation of the degenerative modifications encountered in the temporal TMJ region should not be forgotten as they are important for a comprehensive assessment and further management of the clinical situation. Several research groups have described osseous remodeling in the temporal component of the TMJ. Evidence is scarce for degenerative modifications at the level of the articular eminence and thickening of the roof of the glenoid fossa has been associated with osteoarthritis.

Epithelial PD-L1 expression at tumor front predicts overall survival in a cohort of oral squamous cell carcinomas from Sudan.

BACKGROUND: We recently described the tumor immune microenvironment (TIME) in oral squamous cell carcinomas (OSCC) from Sudan by assessing the core of the lesions. However, the invasive tumor front (ITF) is the most active part of OSCC lesions; thus, TIME should also be characterized at the ITF in this patient cohort. OBJECTIVES: We aimed to evaluate patterns of immune cell infiltration at the ITF in a cohort of OSCC patients from Sudan previously investigated at the tumor center and their association with clinicopathological parameters. METHODS: This study was performed on a prospective cohort of 22 OSCC patients attending Khartoum Dental Teaching Hospital with a median follow-up of 48 months. Inflammatory infiltrate densities of CD4-, CD8-, FoxP3-, CD20-, CD66b-, M1 (CD80/CD68)-, M2 (CD163/CD68)-, and PD-L1-positive cells were assessed at the ITF by immunohistochemistry, followed by digital quantitative analysis at the stromal and epithelial compartments separately. Histopathological parameters such as the worst pattern of invasion, differentiation, and tumor budding (TB) were also assessed. Correlations between clinicopathological parameters and survival analysis were investigated using SPSS. RESULTS: All inflammatory cell subsets investigated were found to be higher in the stromal compartment as compared to the epithelial one, except for the PD-L1+ subset. Stromal infiltration with the CD8+ cell subset was associated with low TB. Kaplan-Meier analyses identified higher epithelial and stromal CD4+ cell subsets. The presence of PD-L1 was found to be associated with unfavorable overall survival. Further, Cox's regression analysis using an age- and tumor-stage-adjusted model identified epithelial PD-L1 expression at the ITF as the only independent prognosticator. CONCLUSIONS: Epithelial PD-L1 expression at the ITF was found to be an independent prognostic biomarker for OSCC in a cohort of Sudanese patients.

Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium.

Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast-derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D-organotypic (3D-OT) cultures were constructed. Various growth factors were added at a range of 0.1-100 ng/ml. 3D-OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D-OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage-colony stimulating factor (GM-CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM-CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and ß1-integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL-positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM-CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast-derived diffusible factor(s).

MicroRNA-138 Abates Fibroblast Motility With Effect on Invasion of Adjacent Cancer Cells.

Background: Recent studies have shown aberrant expression of micro-RNAs in cancer-associated fibroblasts (CAFs). This study aimed to investigate miR-138 dysregulation in CAFs in oral squamous cell carcinoma (OSCC) and its effects on their phenotype and invasion of adjacent OSCC cells. Methods: Expression of miR-138 was first investigated in OSCC lesions (n = 53) and OSCC-derived CAFs (n = 15). MiR-138 mimics and inhibitors were used to functionally investigate the role of miR-138 on CAF phenotype and the resulting change in their ability to support OSCC invasion. Results: Expression of miR-138 showed marked heterogeneity in both OSCC tissues and cultured fibroblasts. Ectopic miR-138 expression reduced fibroblasts' motility and collagen contraction ability and suppressed invasion of suprajacent OSCC cells, while its inhibition resulted in the opposite outcome. Transcript and protein examination after modulation of miR-138 expression showed changes in CAF phenotype-specific molecules, focal adhesion kinase axis, and TGFß1 signaling pathway. Conclusions: Despite its heterogeneous expression, miR-138 in OSCC-derived CAFs exhibits a tumor-suppressive function.

Characterization of immune cell infiltrate in tumor stroma and epithelial compartments in oral squamous cell carcinomas of Sudanese patients.

BACKGROUND: Tumor immune infiltrate has been explored in oral squamous cell carcinoma (OSCC), but studies on simultaneous characterization of multiple immune cell subtypes separately in stromal and intraepithelial tumor compartments are limited. OBJECTIVES: We aimed to investigate the immune cell infiltrate in OSCC by using immunohistochemistry (IHC) for a panel of inflammatory cells in stromal and epithelial tumor compartments for a better characterization of the tumors. METHODS: Thirty-six OSCC lesions and nine normal oral mucosa (NOM) samples from patients attending Khartoum Dental Teaching Hospital, Sudan were investigated for presence of tumor infiltrating lymphocytes, tumor-associated macrophages, tumor-associated neutrophils, and PD-L1 positive cells in the inflammatory infiltrate by single and double IHC. Digital quantitative analysis (Aperio Technologies Inc.) was performed separately for stromal and epithelial compartments. RESULTS: OSCC cases displayed a higher inflammatory infiltrate in the associated stroma, but not in the epithelial compartment when compared to NOM. The immunosuppressive type of inflammatory infiltrate, that is, T regulatory cells (FoxP3+ cells) was identified to be significantly higher in the epithelial compartment of tumors with advanced clinical state. An immunoscore developed by combining intraepithelial FoxP3+ and CD4+ cells was found significantly higher in lesions from elderly patients, localized at toombak dipping-related sites, poorly differentiated OSCCs, or with loco-regional lymph node spreading. CONCLUSIONS: Despite heavy immune cell infiltration in tumor-associated stroma, the majority of OSCCs in this cohort displayed a low intraepithelial immune infiltration. An immunoscore based on combined CD4 and FoxP3 intraepithelial expression may serve as an indicator of advanced tumor progression and should be further investigated for its use as potential prognostic biomarker in OSCC.

Knowledge, opinions, and practices related to oral cancer prevention and oral mucosal examination among dentists in Moldova, Belarus and Armenia: a multi-country cross-sectional study.

INTRODUCTION: Moldova, Belarus, and Armenia are post-Soviet countries with a high rate of heavy smokers and a relatively high age-standardized incidence of oral cancer. However, to our knowledge, there is lack of available information on dentists' knowledge on prevention of oral cancer in the countries in question. Accordingly, this study aimed to assess the knowledge, opinions, and practices related to oral cancer prevention and oral mucosal examination among dentists in Moldova, Belarus, and Armenia. METHODS: This was a multi-country, cross-sectional study based on a self-administered questionnaire. A structured questionnaire was distributed to 3534 dentists (797 in Chisinau, Moldova, 1349 in Minsk, Belarus, and 1388 in Yerevan, Armenia). Dentists' knowledge about risk factors for oral cancer development and its clinical picture, current practices and opinions with regard to oral mucosal screening and oral cancer prevention, and their consistency to perform oral mucosal examination were assessed. A knowledge score ranging from 0 to 14 points was generated based on each dentist's answer to the questionnaire. RESULTS: A total of 1316 dentists responded, achieving an overall response rate of 37.2% (34.5% in Moldova; 52.3% in Belarus; 24.2% in Armenia). Most dentists in the three countries correctly identified tobacco (83.8-98.2%) and prior oral cancer lesions (84.0-96.3%) as risk factors for oral cancer. Most dentists correctly identified leukoplakia as a lesion with malignant potential (68.7% in Moldova; 88.5% in Belarus; 69.9% in Armenia), while erythroplakia was identified by much fewer in all three countries. Less than 52% of dentists identified the tongue, rim of tongue, and floor of mouth as the most common sites for oral cancer. The mean knowledge score for all countries combined was 7.5 ± 2.7. The most commonly reported barriers to perform oral mucosal examination were lack of training, knowledge, and experience. CONCLUSIONS: This study highlights the need for improved oral cancer-related education and training on oral mucosal examination for dentists in Moldova, Belarus, and Armenia. Such skills are essential to enhance oral cancer prevention and to improve the prognostic outcome by early detection.

Ectopic Bone Tissue Engineering in Mice Using Human Gingiva or Bone Marrow-Derived Stromal/Progenitor Cells in Scaffold-Hydrogel Constructs.

Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation and bone regeneration capacity of mesenchymal stromal cells (MSC). Gingiva-derived progenitor cells (GPC) represent a less invasive alternative to bone marrow MSC (BMSC) for clinical applications. The aim of this study was to test the in vivo bone forming potential of human GPC and BMSC cultured as 3D spheroids or dissociated cells (2D). 2D and 3D cells encapsulated in constructs of human platelet lysate hydrogels (HPLG) and 3D-printed poly (L-lactide-co-trimethylene carbonate) scaffolds (HPLG-PLATMC) were implanted subcutaneously in nude mice; cell-free HPLG-PLATMC constructs served as a control. Mineralization was assessed using micro-computed tomography (µCT), histology, scanning electron microscopy (SEM) and in situ hybridization (ISH). After 4-8 weeks, µCT revealed greater mineralization in 3D-BMSC vs. 2D-BMSC and 3D-GPC (p < 0.05), and a similar trend in 2D-GPC vs. 2D-BMSC (p > 0.05). After 8 weeks, greater mineralization was observed in cell-free constructs vs. all 2D- and 3D-cell groups (p < 0.05). Histology and SEM revealed an irregular but similar mineralization pattern in all groups. ISH revealed similar numbers of 2D and 3D BMSC/GPC within and/or surrounding the mineralized areas. In summary, spheroid culture promoted ectopic mineralization in constructs of BMSC, while constructs of dissociated GPC and BMSC performed similarly. The combination of HPLG and PLATMC represents a promising scaffold for bone tissue engineering applications.

Profiling and Functional Analysis of microRNA Deregulation in Cancer-Associated Fibroblasts in Oral Squamous Cell Carcinoma Depicts an Anti-Invasive Role of microRNA-204 via Regulation of Their Motility.

Background: Knowledge on the role of miR changes in tumor stroma for cancer progression is limited. This study aimed to investigate the role of miR dysregulation in cancer-associated fibroblasts (CAFs) in oral squamous cell carcinoma (OSCC). Methodology: CAF and normal oral fibroblasts (NOFs) were isolated from biopsies of OSCC patients and healthy individuals after informed consent and grown in 3D collagen gels. Total RNA was extracted. Global miR expression was profiled using Illumina version 2 panels. The functional impact of altered miR-204 expression in fibroblasts on their phenotype and molecular profile was investigated using mimics and inhibitors of miR-204. Further, the impact of miR-204 expression in fibroblasts on invasion of adjacent OSCC cells was assessed in 3D-organotypic co-cultures. Results: Unsupervised hierarchical clustering for global miR expression resulted in separate clusters for CAF and NOF. SAM analysis identified differential expression of twelve miRs between CAF and NOF. Modulation of miR-204 expression did not affect fibroblast cell proliferation, but resulted in changes in the motility phenotype, expression of various motility-related molecules, and invasion of the adjacent OSCC cells. 3' UTR miR target reporter assay showed ITGA11 to be a direct target of miR-204. Conclusions: This study identifies differentially expressed miRs in stromal fibroblasts of OSCC lesions compared with normal oral mucosa and it reveals that one of the significantly downregulated miRs in CAF, miR-204, has a tumor-suppressive function through inhibition of fibroblast migration by modulating the expression of several different molecules in addition to directly targeting ITGA11.

Predicting intention of Norwegian dental health-care workers to use nanomaterials: An application of the augmented theory of planned behavior.

Due to the rapid development of nanotechnology and its integration into dentistry, there is a need for information on the factors influencing the decision of dental health-care workers to use nanomaterials. Based on a national survey among Norwegian dentists and dental hygienists, this study applied the theory of planned behavior (TPB), augmented with past behavior and perceived risk, to predict the intention to use dental nanomaterials in the future and to assess whether an augmented TPB model operates equivalently across professional groups. Structural equation modelling was used to assess whether the hypothesized model fits the data. Of 1792 eligible participants, 851 responded to an electronic survey. Attitudes and perceived behavioral control had the strongest effect on intention, followed by past behavior and subjective norms. Risk perceptions had an indirect effect on intention. Multigroup comparison confirmed invariance of the model across professional groups. This study supports the validity of the augmented TPB model to explain the intention of Norwegian dentists and dental hygienists to use nanomaterials. The strongest influence on intention is given by the attitudes toward nanomaterials and perceived confidence in their use. The findings of the study have implications for management of the use of nanomaterials in dentistry by policy makers.

EGFR Regulates the Hippo pathway by promoting the tyrosine phosphorylation of MOB1.

The Hippo pathway is frequently dysregulated in cancer, leading to the unrestrained activity of its downstream targets, YAP/TAZ, and aberrant tumor growth. However, the precise mechanisms leading to YAP/TAZ activation in most cancers is still poorly understood. Analysis of large tissue collections revealed YAP activation in most head and neck squamous cell carcinoma (HNSCC), but only 29.8% of HNSCC cases present genetic alterations in the FAT1 tumor suppressor gene that may underlie persistent YAP signaling. EGFR is overexpressed in HNSCC and many other cancers, but whether EGFR controls YAP activation is still poorly understood. Here, we discover that EGFR activates YAP/TAZ in HNSCC cells, but independently of its typical signaling targets, including PI3K. Mechanistically, we find that EGFR promotes the phosphorylation of MOB1, a core Hippo pathway component, and the inactivation of LATS1/2 independently of MST1/2. Transcriptomic analysis reveals that erlotinib, a clinical EGFR inhibitor, inactivates YAP/TAZ. Remarkably, loss of LATS1/2, resulting in aberrant YAP/TAZ activity, confers erlotinib resistance on HNSCC and lung cancer cells. Our findings suggest that EGFR-YAP/TAZ signaling plays a growth-promoting role in cancers harboring EGFR alterations, and that inhibition of YAP/TAZ in combination with EGFR might be beneficial to prevent treatment resistance and cancer recurrence.

Targeted Next-Generation Sequencing of Cancer-Related Genes in a Norwegian Patient Cohort With Head and Neck Squamous Cell Carcinoma Reveals Novel Actionable Mutations and Correlations With Pathological Parameters.

BACKGROUND: Targeted next-generation sequencing (NGS) is increasingly applied in clinical oncology to advance personalized treatment. Despite success in many other tumour types, use of targeted NGS panels for assisting diagnosis and treatment of head and neck squamous cell carcinomas (HNSCC) is still limited. AIM: The focus of this study was to establish a robust NGS panel targeting most frequent cancer mutations in long-term preserved formalin-fixed paraffin-embedded (FFPE) tissue samples of HNSCC from routine diagnostics. MATERIALS AND METHODS: Tumour DNA obtained from archival FFPE tissue blocks of HNSCC patients treated at Haukeland University Hospital between 2003-2016 (n=111) was subjected to mutational analysis using a custom made AmpliSeq Library PLUS panel targeting 31 genes (Illumina). Associations between mutational burden and clinical and pathological parameters were investigated. Mutation and corresponding clinicopathological data from HNSCC were extracted for selected genes from the Cancer Genome Atlas (TCGA) and used for Chi-square and Kaplan-Meier analysis. RESULTS: The threshold for sufficient number of reads was attained in 104 (93.7%) cases. Although the specific number of PCR amplified reads detected decreased, the number of NGS-annotated mutations did not significantly change with increased tissue preservation time. In HPV-negative carcinomas, mutations were detected mainly in TP53 (73.3%), FAT1 (26.7%) and FLG (16.7%) whereas in HPV-positive, the common mutations were in FLG (24.3%) FAT1 (17%) and FGFR3 (14.6%) genes. Other less common pathogenic mutations, including well reported SNPs were reproducibly identified. Presence of at least one cancer-specific mutations was found to be positively associated with an extensive desmoplastic stroma (p=0.019), and an aggressive type of invasive front (p=0.035), and negatively associated with the degree of differentiation (p=0.041). Analysis of TCGA data corroborated the association between cancer-specific mutations and tumour differentiation and survival analysis showed that tumours with at least one mutation had shorter disease-free and overall survival (p=0.005). CONCLUSIONS: A custom made targeted NGS panel could reliably detect several specific mutations in archival samples of HNSCCs preserved up to 17 years. Using this method novel associations between mutational burden and clinical and pathological parameters were detected and actionable mutations in HPV-positive HNSCC were discovered.

Analysis of Salivary Mycobiome in a Cohort of Oral Squamous Cell Carcinoma Patients From Sudan Identifies Higher Salivary Carriage of Malassezia as an Independent and Favorable Predictor of Overall Survival.

Background: Microbial dysbiosis and microbiome-induced inflammation have emerged as important factors in oral squamous cell carcinoma (OSCC) tumorigenesis during the last two decades. However, the "rare biosphere" of the oral microbiome, including fungi, has been sparsely investigated. This study aimed to characterize the salivary mycobiome in a prospective Sudanese cohort of OSCC patients and to explore patterns of diversities associated with overall survival (OS). Materials and Methods: Unstimulated saliva samples (n = 72) were collected from patients diagnosed with OSCC (n = 59) and from non-OSCC control volunteers (n = 13). DNA was extracted using a combined enzymatic-mechanical extraction protocol. The salivary mycobiome was assessed using a next-generation sequencing (NGS)-based methodology by amplifying the ITS2 region. The impact of the abundance of different fungal genera on the survival of OSCC patients was analyzed using Kaplan-Meier and Cox regression survival analyses (SPPS). Results: Sixteen genera were identified exclusively in the saliva of OSCC patients. Candida, Malassezia, Saccharomyces, Aspergillus, and Cyberlindnera were the most relatively abundant fungal genera in both groups and showed higher abundance in OSCC patients. Kaplan-Meier survival analysis showed higher salivary carriage of the Candida genus significantly associated with poor OS of OSCC patients (Breslow test: p = 0.043). In contrast, the higher salivary carriage of Malassezia showed a significant association with favorable OS in OSCC patients (Breslow test: p = 0.039). The Cox proportional hazards multiple regression model was applied to adjust the salivary carriage of both Candida and Malassezia according to age (p = 0.029) and identified the genus Malassezia as an independent predictor of OS (hazard ratio = 0.383, 95% CI = 0.16-0.93, p = 0.03). Conclusion: The fungal compositional patterns in saliva from OSCC patients were different from those of individuals without OSCC. The fungal genus Malassezia was identified as a putative prognostic biomarker and therapeutic target for OSCC.

Combined loss of expression of involucrin and cytokeratin 13 is associated with poor prognosis in squamous cell carcinoma of mobile tongue.

BACKGROUND: This study aimed to evaluate the prognostic significance of expression levels of involucrin (IVL), cytokeratin (CK)-10 and -13 at different intratumor sites (tumor center and invading area) of oral tongue squamous cell carcinoma (OTSCC). METHODS: IVL, CK13 and CK10 expression levels were examined in a multicenter cohort of 146 OTSCCs using immunohistochemistry. External mRNA datasets were used for expression analysis and/or to validate survival associations. RESULTS: External transcriptomic datasets showed downregulation of IVL and KRT13 in oral malignancies including OTSCC as compared to normal controls. The combined loss of IVL and CK13 expression at the invading core but not at the center core was significantly associated with poor differentiation and reduced 5-year overall survival. Multivariate Cox analysis confirmed the loss of CK13 and IVL expression to be an independent prognostic factor. Transcriptomic dataset corroborated immunohistochemistry results. CONCLUSIONS: Combined expression levlels of IVL and CK13 might be useful as prognostic biomarkers in OTSCC.

Feasibility of a Portable Electronic Nose for Detection of Oral Squamous Cell Carcinoma in Sudan.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is increasing at an alarming rate particularly in low-income countries. This urges for research into noninvasive, user-friendly diagnostic tools that can be used in limited-resource settings. This study aims to test and validate the feasibility of e-nose technology for detecting OSCC in the limited-resource settings of the Sudanese population. METHODS: Two e-nose devices (Aeonose™, eNose Company, Zutphen, The Netherlands) were used to collect breath samples from OSCC (n = 49) and control (n = 35) patients. Patients were divided into a training group for building an artificial neural network (ANN) model and a blinded control group for model validation. The Statistical Package for the Social Sciences (SPSS) software was used for the analysis of baseline characteristics and regression. Aethena proprietary software was used for data analysis using artificial neural networks based on patterns of volatile organic compounds. RESULTS: A diagnostic accuracy of 81% was observed, with 88% sensitivity and 71% specificity. CONCLUSIONS: This study demonstrates that e-nose is an efficient tool for OSCC detection in limited-resource settings, where it offers a valuable cost-effective strategy to tackle the burden posed by OSCC.

Combined In Situ Hybridization and Immunohistochemistry on Archival Tissues Reveals Stromal microRNA-204 as Prognostic Biomarker for Oral Squamous Cell Carcinoma.

Micro-RNAs (miRs) are emerging as important players in carcinogenesis. Their stromal expression has been less investigated in part due to lack of methods to accurately differentiate between tumor compartments. This study aimed to establish a robust method for dual visualization of miR and protein (pan-cytokeratin) by combining chromogen-based in situ hybridization (ISH) and immunohistochemistry (IHC), and to apply it to investigate stromal expression of miR204 as a putative prognostic biomarker in oral squamous cell carcinoma (OSCC). Four different combinations of methods were tested and ImageJ and Aperio ImageScope were used to quantify miR expression. All four dual ISH-IHC methods tested were comparable to single ISH in terms of positive pixel area percentage or integrated optical density of miRs staining. Based on technical simplicity, one of the methods was chosen for further investigation of miR204 on a cohort of human papilloma virus (HPV)-negative primary OSCC (n = 169). MiR204 stromal expression at tumor front predicted recurrence-free survival (p = 0.032) and overall survival (p = 0.036). Multivariate Cox regression further confirmed it as an independent prognostic biomarker in OSCC. This study provides a methodological platform for integrative biomarker studies based on simultaneous detection and quantification of miRs and/or protein and reveals stromal miR204 as a prognostic biomarker in OSCC.