The immunomodulatory impact of naturally derived neem leaf glycoprotein on the initiation progression model of 4NQO induced murine oral carcinogenesis: a preclinical study.

Introduction: Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated. Methods: 4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status. Results: Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8+, IFNγ+, granzyme B+, CD11c+ cells in comparison to 4NQO treated mice with a decrease in Ki67+ and CD4+FoxP3+ cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP. Discussion: Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8+T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.

A unique case of epithelioid sarcoma involving the mandibular alveolar mucosa.

Epithelioid sarcoma (ES) is an infrequent, malignant, mesenchymal, soft-tissue neoplasm of uncertain histogenesis characterized by epithelioid cytomorphology. The lack of SMARCB1/INI1 (Integrase interactor 1) expression typifies this pathology. It usually presents as a painless, indolent, slowly enlarging lesion or rarely as an aggressive growth. Epithelioid sarcoma affects mostly pediatric patients or young adults. It recurs quite frequently and possesses a high degree of metastatic potential. Two predominant histomorphologic patterns include classic/conventional type and proximal types. Epithelioid sarcoma immunohistochemically shows vimentin, cytokeratin, epithelial membrane antigen, CD34 positivity, and negative staining with S100 and desmin. Management is generally multimodal, including wide surgical resection, radiotherapy, and chemotherapy. To date, to our knowledge, only 11 instances of head and neck ES have been reported in the literature. Our case deals with the diagnosis of intraoral ES in a 55-year-old female patient, probably the first one to involve the mandibular mucosa, based on relevant clinical-radiologic-pathologic-immunohistochemical findings.

Single-cell transcriptomic analysis of gingivo-buccal oral cancer reveals two dominant cellular programs.

Oral squamous cell carcinoma of the gingivo-buccal region (OSCC-GB) is the most common cancer among men in India, and is associated with poor prognosis and frequent recurrence. Cellular heterogeneity in OSCC-GB was investigated by single-cell RNA sequencing of tumors derived from the oral cavity of 12 OSCC-GB patients, 3 of whom had concomitant presence of a precancerous lesion (oral submucous fibrosis [OSMF]). Unique malignant cell types, features, and phenotypic shifts in the stromal cell population were identified in oral tumors with associated submucous fibrosis. Expression levels of FOS, ATP1A, and DUSP1 provided robust discrimination between tumors with or without the concomitant presence of OSMF. Malignant cell populations shared between tumors with and without OSMF were enriched with the expression of partial epithelial-mesenchymal transition (pEMT) or fetal cell type signatures indicative of two dominant cellular programs in OSCC-GB-pEMT and fetal cellular reprogramming. Malignant cells exhibiting fetal cellular and pEMT programs were enriched with the expression of immune-related pathway genes known to be involved in antitumor immune response. In the tumor microenvironment, higher infiltration of immune cells than the stromal cells was observed. The T cell population was large in tumors and diverse subtypes of T cells with varying levels of infiltration were found. We also detected double-negative PLCG2+ T cells and cells with intermediate M1-M2 macrophage polarization. Our findings shed light on unique aspects of cellular heterogeneity and cell states in OSCC-GB.

Corpus alienum (foreign body) embedded in the oral cavity of children: An agony of parents and diagnostic dilemma among clinicians.

Aspiration or ingestion of foreign bodies by children is a common problem globally. Corpus alienum or foreign bodies, embedded in the palate or other areas of the oral cavity, are unusual findings that can occasionally be muddled with other oral lesions. Studies reveal that the majority of cases occur in children, wherein 50% of the children lack a proper history. Since infants or very young children fail to provide proper history and are extremely scared of repeated oral examination clinical diagnosis is all the more difficult. The risks of respiratory obstruction, mucosal tear, nasopharyngeal inflammation, and gastrointestinal bleeding make these non-invasive foreign bodies potentially fatal.

Integrative analysis of genomic and transcriptomic data of normal, tumour, and co-occurring leukoplakia tissue triads drawn from patients with gingivobuccal oral cancer identifies signatures of tumour initiation and progression.

A thickened, white patch - leukoplakia - in the oral cavity is usually benign, but sometimes (in ~9% of individuals) it progresses to malignant tumour. Because the genomic basis of this progression is poorly understood, we undertook this study and collected samples of four tissues - leukoplakia, tumour, adjacent normal, and blood - from each of 28 patients suffering from gingivobuccal oral cancer. We performed multiomics analysis of the 112 collected tissues (four tissues per patient from 28 patients) and integrated information on progressive changes in the mutational and transcriptional profiles of each patient to create this genomic narrative. Additionally, we generated and analysed whole-exome sequence data from leukoplakia tissues collected from 11 individuals not suffering from oral cancer. Nonsynonymous somatic mutations in the CASP8 gene were identified as the likely events to initiate malignant transformation, since these were frequently shared between tumour and co-occurring leukoplakia. CASP8 alterations were also shown to enhance expressions of genes that favour lateral spread of mutant cells. During malignant transformation, additional pathogenic mutations are acquired in key genes (TP53, NOTCH1, HRAS) (41% of patients); chromosomal-instability (arm-level deletions of 19p and q, focal-deletion of DNA-repair pathway genes and NOTCH1, amplification of EGFR) (77%), and increased APOBEC-activity (23%) are also observed. These additional alterations were present singly (18% of patients) or in combination (68%). Some of these alterations likely impact immune-dynamics of the evolving transformed tissue; progression to malignancy is associated with immune suppression through infiltration of regulatory T-cells (56%), depletion of cytotoxic T-cells (68%), and antigen-presenting dendritic cells (72%), with a concomitant increase in inflammation (92%). Patients can be grouped into three clusters by the estimated time to development of cancer from precancer by acquiring additional mutations (range: 4-10 years). Our findings provide deep molecular insights into the evolutionary processes and trajectories of oral cancer initiation and progression. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

"Loss of polarity of basal cells" - The term revisited in the light of genomics.

The concept of polarity in development, homeostasis and pathological alteration of tissues has emerged as an interesting aspect of the concerned biology. The epithelial cells exhibit apicobasal polarity which is maintained by Crumbs complex located at apical region of tight junction, 'PAR' complex at sub-apical region of tight junction and Scribble complex at adherens junction. Any functional perturbation of these proteins cause alteration of normal epithelial physiology en-route to epithelial pathology. In this maiden scientific exercise, we have tried to explore the association of expression of cell polarity proteins in OPMD and OSCC. Here, we have chosen DLG1 as a representative of polarity protein. RNA was isolated from the tissue samples. Then cDNA was prepared by RTPCR technique. qPCR was performed on cDNA samples. Expression data was analysed on the basis of Ct values. Paired t-test was performed with normalized Ct values of disease and normal tissue to determine whether there was any significant difference in expression between them. The statistical tests were done using SPSS software. Results of this study reflected increase in DLG1 expression in high grade dysplasia. There was no significant alteration in expression of DLG1 in WDSCC where there is formation of cluster of neoplastic cells ultimately producing epithelial islands and keratin pearls. But, in case of MDSCC, when the same neoplastic cells keep on invading with minimal keratin pearl formation, they again gain the mesenchymal character in full potential. And this phenomenon supports the upregulation of DLG1 in MDSCC in our study.

Genes and pathways monotonically dysregulated during progression from normal through leukoplakia to gingivo-buccal oral cancer.

Oral squamous cell carcinoma of the gingivo-buccal region (OSCC-GB) accounts for the highest cancer morbidity and mortality among men in India. It has been observed that about one-third of individuals with oral leukoplakia, a dysplastic precancerous lesion in the oral cavity, progress to oral cancer. We aimed to identify systematic transcriptomic changes as a normal tissue in the oral cavity progresses to frank OSCC-GB. Seventy-two OSCC-GB patients, from multiple hospitals, were recruited, and transcriptome analysis of tumor and adjacent normal tissue (of all patients) and adjacent leukoplakia tissue (of a subset of 25 unselected patients with concomitant leukoplakia) was performed. We have identified many differences in the transcriptomic profiles between OSCC-GB and squamous cell carcinoma of the head and neck regions. Compared to the normal/precancerous tissue, significant enrichment of ECM-receptor interaction, PI3K-Akt signaling, cytokine-cytokine receptor interaction, focal adhesion, and cell cycle pathways were observed in OSCC-GB. Using gene set enrichment analysis, we identified a profound role of interferon receptor signaling in tumor growth by activating immune evasion mechanisms. The role of tumor-infiltrating immune cells further supported the growth and immunosuppressive mechanism of tumor tissues. Some immune evasion genes-CD274, CD80, and IDO1-were found to be activated even in the precancerous tissue. Taken together, our findings provide a clear insight into the sequential genetic dysregulation associated with progression to oral cancer. This insight provides a window to the development of predictive biomarkers and therapeutic targets for gingivo-buccal oral cancer.

Study of Caspase 8 mutation in oral cancer and adjacent precancer tissues and implication in progression.

It is hypothesized that same driver gene mutations should be present in both oral leukoplakia and cancer tissues. So, we attempted to find out mutations at one of the driver genes, CASP8, in cancer and adjacent leukoplakia tissues. Patients (n = 27), affected by both of cancer and adjacent leukoplakia, were recruited for the study. Blood and tissue DNA samples were used to identify somatic mutations at CASP8 by next generation sequencing method. In total, 56% (15 out of 27) cancer and 30% (8 out of 27) leukoplakia tissues had CASP8 somatic mutations. In 8 patients, both cancer and adjacent leukoplakia tissues, located within 2-5 cm of tumor sites, had identical somatic mutations. But, in 7 patients, cancer samples had somatic mutations but none of the leukoplakia tissues, located beyond 5cm of tumor sites, had somatic mutations. Mutated allele frequencies at CASP8 were found to be more in cancer compared to adjacent leukoplakia tissues. This study provides mutational evidence that oral cancer might have progressed from previously grown leukoplakia lesion. Leukoplakia tissues, located beyond 5cm of cancer sites, were free from mutation. The study implies that CASP8 mutation could be one of the signatures for some of the leukoplakia to progress to oral cancer.

First case report of tongue squamous cell carcinoma in a neurofibromatosis type 1 patient and review of pathogenesis of carcinoma in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1), also known as von Recklinghausen's disease, is a type of genodermatoses having an autosomal dominant inheritance pattern and is recently considered as a RASopathy. Such patients are very much prone to develop mesenchymal tumors. However, carcinomas are quite rare in NF1 patients. This case study is the first case of oral squamous cell carcinoma (SCC) in tongue of an NF1 patient. A 35-year-old male reported to the Department of Oral Pathology in a tertiary care center with a chief complain of a painful ulcer on tongue for last 1 month. For confirmation of diagnosis of NF1, the "Diagnostic Criteria for Neurofibromatosis Type 1" was followed. Biopsied specimen of the tongue lesion was examined under microscope and histopathological features were suggestive of infiltrating SCC. Immunohistochemistry with Pan CK and beta-catenin was positive. RASopathy, WNT-beta-catenin pathway alteration, heat shock factor 1 production, and miRNA activity are investigated to explain the pathogenesis of malignancies in NF1 patients. In this first case of tongue SCC, we have found out the altered WNT-beta-catenin pathway.

A novel mutation at ANTXR1 in an Indian patient with growth retardation-alopecia-pseudoanodontia-optic atrophy syndrome.

OBJECTIVE: Growth retardation-alopecia-pseudoanodontia-optic atrophy (GAPO) syndrome (Online Mendelian Inheritance in Man [OMIM] ID 230740) is one of the rarest autosomal recessive syndromes. It is characterized by many phenotypes, including wide anterior fontanel, frontal bossing of the face, depressed nasal bridge, along with the 4 classic phenotypes contained in the name of the syndrome. Recent reports identified nonsense, missense, and splicing mutations at different exons of ANTXR1 responsible for GAPO syndrome in patients from different ethnic populations. Here, we are reporting a mutation at ANTXR1 in an Indian patient with GAPO syndrome. STUDY DESIGN: We describe an inherited mutation at ANTXR1 in a 6-year-old Indian boy with GAPO syndrome. RESULTS: Genomic DNA from the patient with the GAPO syndrome and his family members were screened for previously reported mutations at ANTXR1 by sequencing. Novel homozygous and heterozygous mutations in exon-3 of ANTXR1 (c.265 G > A, p.Gly89 Arg) were identified in the patient and in other members of the family, respectively. However, no mutated allele was identified in genomic DNA from unrelated healthy individuals. Bioinformatic analysis by different tools predicted the deleterious, damaging, or aberrant splicing effect of mutation on the protein. CONCLUSIONS: Functional inefficiency of ANTXR1 as a result of mutation might have led to GAPO syndrome.

Genome-wide mitochondrial DNA sequence variations and lower expression of OXPHOS genes predict mitochondrial dysfunction in oral cancer tissue.

Several studies reported that mtDNA mutations may play important roles in carcinogenesis although the mechanism is not clear yet. Most of the studies compared mtDNA sequences in a tumor with those in normal tissues from different individuals ignoring inter-individual variations. In this study, 271 SNPs, 7 novel SNPs (or SNVs), and 15 somatic mutations were detected in mtDNA of 8 oral cancer tissues with respect to reference (rCRS) and adjacent normal tissues, respectively, using Ion PGM next generation sequencing method. Most of the sequence variations (76 SNPs and 1 somatic) are present in D-loop region followed by CyB (36 SNPs), ATP6 (24 SNPs), ND5 (17 SNPs and 5 somatic), ND4 (18 coding and 2 somatic) and other non-coding and coding DNA sequences. A total of 53 and 8 non-synonymous SNPs and somatic mutations, respectively, were detected in tumor tissues and some of these variations may have deleterious effects on the protein function as predicted by bioinformatic analysis. Moreover, significantly low mtDNA contents and expression of several mitochondrial genes in tumor compared to adjacent normal tissues may have also affected mitochondrial functions. Taken together, this study suggests that mtDNA mutations as well as low expression of mtDNA coded genes may play important roles in tumor growth. Although the sample size is low, an important aspect of the study is the use of adjacent control tissues to find out somatic mutations and a change in the expression of mitochondrial genes, to rule out inter-individual and inter-tissue variations which are important issues in the study of mitochondrial genomics.

A quest for miRNA bio-marker: a track back approach from gingivo buccal cancer to two different types of precancers.

Deregulation of miRNA expression may contribute to tumorigenesis and other patho-physiology associated with cancer. Using TLDA, expression of 762 miRNAs was checked in 18 pairs of gingivo buccal cancer-adjacent control tissues. Expression of significantly deregulated miRNAs was further validated in cancer and examined in two types of precancer (leukoplakia and lichen planus) tissues by primer-specific TaqMan assays. Biological implications of these miRNAs were assessed bioinformatically. Expression of hsa-miR-1293, hsa-miR-31, hsa-miR-31* and hsa-miR-7 were significantly up-regulated and those of hsa-miR-206, hsa-miR-204 and hsa-miR-133a were significantly down-regulated in all cancer samples. Expression of only hsa-miR-31 was significantly up-regulated in leukoplakia but none in lichen planus samples. Analysis of expression heterogeneity divided 18 cancer samples into clusters of 13 and 5 samples and revealed that expression of 30 miRNAs (including the above-mentioned 7 miRNAs), was significantly deregulated in the cluster of 13 samples. From database mining and pathway analysis it was observed that these miRNAs can significantly target many of the genes present in different cancer related pathways such as "proteoglycans in cancer", PI3K-AKT etc. which play important roles in expression of different molecular features of cancer. Expression of hsa-miR-31 was significantly up-regulated in both cancer and leukoplakia tissues and, thus, may be one of the molecular markers of leukoplakia which may progress to gingivo-buccal cancer.

Association between risk of oral precancer and genetic variations in microRNA and related processing genes.

BACKGROUND: MicroRNAs have been implicated in cancer but studies on their role in precancer, such as leukoplakia, are limited. Sequence variations at eight miRNA and four miRNA processing genes were studied in 452 healthy controls and 299 leukoplakia patients to estimate risk of disease. RESULTS: Genotyping by TaqMan assay followed by statistical analyses showed that variant genotypes at Gemin3 and mir-34b reduced risk of disease [OR = 0.5(0.3-0.9) and OR = 0.7(0.5-0.9) respectively] in overall patients as well as in smokers [OR = 0.58(0.3-1) and OR = 0.68(0.5-0.9) respectively]. Among chewers, only mir29a significantly increased risk of disease [OR = 1.8(1-3)]. Gene-environment interactions using MDR-pt program revealed that mir29a, mir34b, mir423 and Xpo5 modulated risk of disease (p < 0.002) which may be related to change in expression of these genes as observed by Real-Time PCR assays. But association between polymorphisms and gene expressions was not found in our sample set as well as in larger datasets from open access platforms like Genevar and 1000 Genome database. CONCLUSION: Variations in microRNAs and their processing genes modulated risk of precancer but further in-depth study is needed to understand mechanism of disease process.

Genetic variations at microRNA and processing genes and risk of oral cancer.

Genetic variations at microRNA and microRNA processing genes are known to confer risk of cancer in different populations. Here, we studied variations at eight microRNA (miRNA) and four miRNA processing genes in 452 controls and 451 oral cancer patients by TaqMan genotyping assays. Variant allele-containing genotypes at mir-196a2 and variant allele homozygous genotype at Ran increased the risk of cancer significantly [adjusted odds ratio (OR) (95% confidence interval (CI)) = 1.3 (1-1.7) and 2.3 (1.1-4.6), respectively]. Conversely, variant allele-containing genotypes at mir-34b and variant allele homozygous genotype at Gemin3 reduced the risk of cancer significantly [adjusted OR (95% CI) = 0.7 (0.5-0.9) and 0.6 (0.4-1), respectively]. Cumulative risk was also increased by three times with increase in the number of risk alleles at these four loci. In tobacco stratified analysis, variant allele homozygous genotypes at mir-29a and Ran increased [adjusted OR (95% CI) = 1.5 (1-2.3) and 3 (1.1-8.4) respectively], while variant allele-containing genotypes at mir-34b decreased [adjusted OR (95% CI) = 0.6 (0.4-0.9)] the risk of cancer significantly. Thus, genetic variation at miRNA and processing genes altered the risk of oral cancer in this population thereby corroborating studies in other populations. However, it is necessary to validate this result in different Indian sub populations with larger sample sizes and examine the effect of these variations in tumour tissues to explain the mechanism of risk alteration.