Gingivo-buccal Squamous Cell Carcinoma Research Articles

A Transcriptomic Analysis to Identify Prevalent lncRNAs in Gingivobuccal Oral Cancer

Objective: Gingivobuccal cancer is a subtype of oral cancer, prevalent in developing countries where the use of tobacco and Areca (betel) nut is rampant. This study aims to search through the publically available NCBI data to find comparisons between the normal and cancer-affected transcripts of RNA, which are molecules that copied the genetic information from DNA and that will be “read” as instructions to produce other molecules in the body. Using bioinformatic tools, we analyzed the variations in the transcripts and examined how they were correlated with other molecular processes in the body. We specifically investigated the role of “long noncoding RNAs (lncRNAs)” because it is speculated that they play an active role in the causality of cancer. Our analysis found a correlation of CTA-384D8.35 lncRNA with gingivobuccal cancer after analyzing the Hazard Ratio using the Cox Proportional-Hazard Model and is expected to pave the way for future studies. Methods: In this study, transcriptomic analysis was performed on a publicly available gingivobuccal cancer dataset obtained from NCBI. We developed a novel method for identification of annotated lncRNAs, which helped us select and correlate the lncRNA transcripts with the gingivobuccal cancer disease. Furthermore, the results were validated using the GEPIA2 tool for integrated analysis of co-lncRNA and GEPIA databases. Findings: From 24 RNA-Seq samples (12 normal samples and 12 tumor samples), a total of 16,065 potential lncRNA transcripts were recognized. From these 16,065 lncRNA transcripts, 1201 lncRNA transcripts were further filtered out based on a threshold adjusted p value of <0.01. We were able to for the first time identify these 1201 lncRNA transcripts, which were not annotated for gingivobuccal squamous cell carcinoma previously. Integrated analysis of co-lncRNA and GEPIA databases revealed that a total of 11 lncRNAs were upregulated, whereas 29 lncRNAs were downregulated. Furthermore, CTA-384D8.35 showed a significant correlation with disease-free survival time in HNSC. CTA-384D8.35 which is downregulated in gingivobuccal cancer has been correlated with tumor stage, metastasis, cancer status, clinical stage, and grade. Novelty: In this report we are for the first time correlating the CTA-384D8.35 lncRNA with the gingivobuccal cancer disease with the help of statistical and survival analysis. Furthermore,1201 lncRNA transcripts, which have previously not been annotated for gingivobuccal squamous cell carcinoma, were identified in this study based on a threshold adjusted p value of <0.01. Finally we have also introduced a novel method of identifying annotated lncRNAs in a disease as well as improved upon the NGS data analysis pipeline by determining the strandness of the raw RNASeq data. Differential expression of lncRNAs could possibly play a significant role in carcinogenesis. Therefore the identification of lncRNAs gains priority, which are speculated to play a role in the mechanism and formation of various forms of cancer. For identification and analysis of lncRNAs, a comparison of their expression in normal and affected tissues is likely to confirm their involvement in the disease or the lack thereof. The introduction of a novel methodology along with the identification of CTA-384D8.35 lncRNA paves the way for further analysis pertaining to lncRNAs as prognostic or diagnostic biomarkers. Since CTA-384D8.35 lncRNA has previously been correlated with a number of cancer hallmarks, it gains significant traction to be considered as an important aspect in the biomarker identification pertaining to gingivobuccal cancer. This study has reported for the first time the lncRNAs that are prevalent in gingivobuccal squamous cell carcinoma. Keywords: Gingivobuccal cancer; lncRNA; oral cancer; epigenetics; NGS data analysis

Aspirin Inhibition of Group VI Phospholipase A2 Induces Synthetic Lethality in AAM Pathway Down-Regulated Gingivobuccal Squamous Carcinoma.

Background: To elucidate the role of iPLA2/PLA2G6 in gingivobuccal squamous cell carcinoma (GB-SCC) and to ascertain the synthetic lethality-based chemoprevention role of aspirin in arachidonic acid metabolism (AAM) pathway down-regulated GB-SCC. Methods: The in vitro efficacy of aspirin on GB-SCC cells (ITOC-03 and ITOC-04) was assessed by cell proliferation, colony formation, apoptosis, cell migration, cell cycle assay and RNA-seq, while inhibition of PLA2G6 and AAM pathway components was affirmed by qPCR, Western blot and immunofluorescence staining. The in vivo effect of aspirin was evaluated using NOD-SCID mice xenografts and immunohistochemical analysis. Results: We found that aspirin, which has been reported to act through the COX pathway, is inhibiting PLA2G6, and thereby the COX and LOX components of the AAM pathway. The findings were validated using PLA2G6 siRNA and immunohistochemical marker panel. Moreover, a pronounced effect in ITOC-04 cells and xenografts implied aspirin-induced synthetic lethality in the AAM pathway down-regulated GB-SCC. Conclusions: This study reveals that aspirin induces the anti-tumor effect by a previously unrecognized mechanism of PLA2G6 inhibition. In addition, the effect of aspirin is influenced by the baseline AAM pathway status and could guide precision prevention clinical trials of AAM pathway inhibitors.

Effect of Cold Atmospheric Plasma Jet and Gamma Radiation Treatments on Gingivobuccal Squamous Cell Carcinoma and Breast Adenocarcinoma Cells

Effect of Cold Atmospheric Plasma Jet and Gamma Radiation Treatments on Gingivobuccal Squamous Cell Carcinoma and Breast Adenocarcinoma Cells

Utility of island bipaddle pectoralis major myocutaneous flap in reconstruction of full thickness lower gingivo-buccal sulcus defects

&lt;p class="abstract"&gt;&lt;strong&gt;Background:&lt;/strong&gt; Aims of the study was to evaluate the usefulness of island bipaddle pectoralis major myocutaneous flap in the reconstruction of through and through lateral gingivobuccal sulcus defects.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Methods:&lt;/strong&gt; This was a retrospective study carried out from 2013 - 2019 in the department of otorhinolaryngology and head and surgery at Sri Devaraj Urs Medical College and Hospital. After obtaining institutional ethical committee clearance. Sixty-one patients diagnosed with stage IV a lateral Gingivobuccal squamous cell carcinoma with skin involvement and reconstructed using island bipaddle pectoralis major myocutaneous flap were included in the study. &lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Results:&lt;/strong&gt; Majority of our patients were females. Nearly 50% of our patients had skin involvement secondary to Level-I nodal metastasis. Nine patients in our study developed complications. Among them seven patients had minor complications and in two patients the flap failed.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; In resource constraint places where microvascular reconstruction facilities are not available and where replacing bone is not mandatory, island bipaddle pectoralis major myocutaneous flap can be used effectively as single stage reconstruction option for full thickness lower gingivo-buccal sulcus defects.&lt;/p&gt;

Raman Spectroscopy: An Exploratory Study to Identify Post-Radiation Cell Survival.

Resistance to radiotherapy has been an impediment in the treatment of cancer, and the inability to detect it at an early stage further exacerbates the prognosis. We have assessed the feasibility of Raman spectroscopy as a rapid assay for predicting radiosensitivity of cancer cells in comparison to the conventional biological assays. Cell lines derived from breast adenocarcinoma (MCF7), gingivobuccal squamous cell carcinoma (ITOC-03), and human embryonic kidney (HEK293) were subjected to varying doses of ionizing radiation. Cell viability of irradiated cells was assessed at different time points using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Raman spectroscopy, and colony-forming capability was evaluated by clonogenic assay. Radiosensitivity observed using MTT assay was limited by the finding of similar cell viability in all the three cell lines 24 h post-irradiation. However, cell survival assessed using clonogenic assay and principal component linear discriminant analysis (PC-LDA) classification of Raman spectra showed correlating patterns. Irradiated cells showed loss of nucleic acid features and enhancement of 750 cm-1 peak probably attributing to resonance Raman band of cytochromes in all three cell lines. PC-LDA analysis affirmed MCF7 to be a radioresistant cell line as compared to ITOC-03 and HEK293 to be the most radiosensitive cell line. Raman spectroscopy is shown to be a rapid and alternative assay for identification of radiosensitivity as compared to the gold standard clonogenic assay.

Establishment and genomic characterization of gingivobuccal carcinoma cell lines with smokeless tobacco associated genetic alterations and oncogenic PIK3CA mutation

Smokeless tobacco associated Gingivobuccal squamous cell carcinoma (GB-SCC) is a major public health problem but available oral cancer cell lines are mostly from smoking associated tongue SCC raising the need for pertinent GB-SCC cell line models. As part of the International Cancer Genome Consortium (ICGC) Project, 4 novel cell lines, namely, Indian Tata Memorial Centre Oral Cancer (ITOC) −01 to −04 were established and characterized with conventional methods, karyotyping, ultrastructure, in vivo tumourigenicity, Whole exome sequencing (WES) and RNA sequencing. These hyperploid cell lines form xenografts in mice and show metabolically active and necrotic areas on fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging. WES of ITOC cell lines recapitulate the genomic tumor profile of ICGC GB-SCC database. We further identified smokeless tobacco associated genetic alterations (PCLO, FAT3 and SYNE2) and oncogenic PIK3CA mutation in GB-SCC cell lines. Transcriptome profiling identified deregulation of pathways commonly altered in cancer and down-regulation of arachidonic acid metabolism pathway, implying its possible role in GB-SCC. Clinical application of high throughput sequencing data depends on relevant cell line models to validate potential targets. Extensively characterized, these oral SCC cell lines are particularly suited for mechanistic studies and pre-clinical drug development for smokeless tobacco associated oral cancer.

Analysis of the whole transcriptome from gingivo-buccal squamous cell carcinoma reveals deregulated immune landscape and suggests targets for immunotherapy.

BackgroundGingivo-buccal squamous cell carcinoma (GBSCC) is one of the most common oral cavity cancers in India with less than 50% patients surviving past 5 years. Here, we report a whole transcriptome profile on a batch of GBSCC tumours with diverse tobacco usage habits. The study provides an entire landscape of altered expression with an emphasis on searching for targets with therapeutic potential.MethodsWhole transcriptomes of 12 GBSCC tumours and adjacent normal tissues were sequenced and analysed to explore differential expression of genes. Expression changes were further compared with those in TCGA head and neck cohort (n = 263) data base and validated in an independent set of 10GBSCC samples.ResultsDifferentially expressed genes (n = 2176) were used to cluster the patients based on their tobacco habits, resulting in 3 subgroups. Immune response was observed to be significantly aberrant, along with cell adhesion and lipid metabolism processes. Different modes of immune evasion were seen across 12 tumours with up-regulation or consistent expression of CD47, unlike other immune evasion genes such as PDL1, FUT4, CTLA4 and BTLA which were downregulated in a few samples. Variation in infiltrating immune cell signatures across tumours also indicates heterogeneity in immune evasion strategies. A few actionable genes such as ITGA4, TGFB1 and PTGS1/COX1 were over expressed in most samples.ConclusionThis study found expression deregulation of key immune evasion genes, such as CD47 and PDL1, and reasserts their potential as effective immunotherapeutic targets for GBSCC, which requires further clinical studies. Present findings reiterate the idea of using transcriptome profiling to guide precision therapeutic strategies.

Abstract 1499: Role of mutations and expression change of mitochondrial function related nuclear genes in oral gingivobuccal squamous cell carcinoma

Abstract Background: Mitochondria have long been suspected to have contribution in progression and sustenance of cancer. But mitochondria related nuclear genes have not been well-studied in oral cancer. The aim of this study is deciphering the impact of somatic mutations and expression deregulation of mitochondria related nuclear genes in oral gingivobuccal squamous cell carcinoma (GBSCC). Methods: Nuclear-encoded genes which are functionally involved with mitochondria were enlisted from Mitocarta 2.0 and IMPI databases. Whole exome sequencing was performed with 12 paired cancer-normal GBSCC samples and somatic mutations in mitochondria related nuclear genes were extracted from the data. Expression deregulation of mitochondria related nuclear genes were quantified from whole transcriptome data of 12 paired cancer-normal GBSCC samples. Expression was re-validated in another set of 12 cancer-normal paired samples. 5 cancer-normal paired samples with whole transcriptome data were also used for reduced representation bisulfite sequencing (RRBS). Probable impact of non-synonymous somatic mutations were predicted using SIFT and POLYPHEN2 tools. Genes with possible damaging mutations and expression deregulation were taken for literature search and KEGG pathway analysis to understand their contribution in the function of mitochondria in GBSCC. Result: Total 1561 mitochondria related nuclear genes were identified from two databases and included in the study. A total of 977 somatic mutations were identified in 583 such genes in 12 oral GBSCC samples. These mutations were distributed in exonic (346 mutations), intronic (288 mutations), splicing (4 mutations), UTR3’ (270 mutations) and UTR5’ (69 mutations) regions. Ten recurrently mutated genes (AK3, AK4, ATP10D, CASP8, ERBB4, MRPL10, NT5DC3, RYR3, SMURF1and TP53) were identified in at least 25% (3/12) of samples. Significant expression deregulation in 147 mitochondrial related nuclear genes was found from whole transcriptome data. Differential methylation was found in some of these 147 genes in 5 oral GBSCC samples. Thirty-seven genes were selected from 147 genes for expression re-validation by qPCR and expressions of 32 genes were re-validated in another set of 12 GBSCC samples. Somatically mutated and expression deregulated genes were different except two genes, ACADM and NNT, which were mutated and deregulated. Literature survey and KEGG pathway analysis revealed that most of the mutated or deregulated genes were functionally involved with biological pathways like mitochondrial metabolism, oxidative phosphorylation, apoptosis and molecular transport. It indicates possible disruption of these pathways in oral GBSCC. Conclusion: Mitochondrial function related nuclear genes may have significant impact on oral GBSCC in maintenance and progression, but needs to be validated in larger set of samples. Citation Format: Esita Chattopadhyay, Richa Singh, Roshni Roy, Bidyut Roy. Role of mutations and expression change of mitochondrial function related nuclear genes in oral gingivobuccal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1499. doi:10.1158/1538-7445.AM2017-1499

Abstract 2425: Delving into molecular phenomena of methylation, polyadenylation and mutation suggests their importance in regulating gene expression in GBSCC

Abstract Background: Gingivo-buccal squamous cell carcinoma (GBSCC) is one of the most prevalent types of oral cancers in India. Although whole exome studies in oral cancer have reported somatic mutational driver genes, studies on methylation, mutations and alternative polyadenylation (APA) to identify differentially expressed transcripts and exons, respectively, are rare. The aim of our study is to investigate possible causes of expression deregulation due to methylation, APA or mutations. Methods: Whole transcriptome data was generated from 12 tumor-normal paired GBSCC tissues and analyzed for differential gene expression, differential exon usage and predicting APA site. Whole exome sequencing was performed with same sample set and the data was analyzed to identify somatic mutations. Reduced representation of bisulphate sequencing (RRBS) was also done for some of these samples to inquire about the methylation status of deregulated genes. Results: A total of 465 genes were found to have differential exon usage in tumor leading to expression of different isoforms and change in overall gene expression. About 194 genes were predicted to utilize alternate polyadenylation site in tumors. Amongst these, 11 genes including ERBB3, CDC25B and LTBP4 were shown to have differential exon usage in tumor with altered gene expression hinting upon the influence of polyadenylation in expression change. From the exome sequencing study, 180 SNVs and 11 indel somatic mutations per sample were observed. Apart from other non-silent exonic mutations at TP53, CASP8, DIS3 and DLG1, splice site mutations were also found in 37 and 4 genes due to point and indel mutations respectively. Genes such as TPM3, NBN, CXCL12 along with others showed differential exon usage and were found to harbor somatic mutations and APA site. This suggests for coupled regulation by APA and mutation in altering expression of those genes. Apart from this, a subset of deregulated genes (n=54) were observed to have differential methylation in tumors. Expression change of TAP1, SLN, ELN etc. corroborated with their methylation status suggesting the influence of methylation in regulating expression of these genes. Conclusion: Differential exon usage suggested different transcript formation leading to overall change in gene expression in tumor. Upon excavating into the regulatory causes, a subset of genes showed change in methylation pattern while few others showed change in APA site with/without somatic mutation effect. These molecular phenomena explain how different mechanisms are involved in expression deregulation of genes observed in GBSCC which would help in further functional research for cure of the disease. Citation Format: Richa Singh, Esita Chattopadhyay, Roshni Roy, Bidyut Roy. Delving into molecular phenomena of methylation, polyadenylation and mutation suggests their importance in regulating gene expression in GBSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2425. doi:10.1158/1538-7445.AM2017-2425

Quadriplegia secondary to cervical spondylotic myelopathy—A rare complication of head and neck surgery

Free tissue reconstruction after ablation of head and neck malignancy often requires extensive cervical manipulation, which may exacerbate preexisting cervical spondylosis and result in progression to cervical myelopathy. We present a rare case of postoperative quadriplegia caused by cervical spondylotic myelopathy after head and neck reconstruction. A 63-year-old man without a history of cervical spondylosis underwent resection of a gingivo-buccal squamous cell carcinoma with immediate reconstruction with free fibula osteocutaneous flap. On postoperative day 4, the patient was found to have quadriplegia. MRI demonstrated severe cervical myelopathy. Decompressive laminectomy was performed. The patient underwent an extensive rehabilitation program but only realized moderate improvement. Cervical spondylotic myelopathy is a rare but disastrous complication of head and neck surgery. We hypothesize that it is potentially avoidable with heightened awareness of this disease entity, preoperative identification of patients at risk, and prophylactic interventions

Tumour thickness and relationship to locoregional failurein cancer of the buccal mucosa

AimsThe TNM system is the main parameter in treatment planning and the prediction of survival in oral cancer. Here, we investigate the role of tumour thickness as a predictor of locoregional failure and survival in node-negative patients.MethodsWe studied 176 node-negative and early stage gingivo-buccal squamous cell carcinoma patients retrospectively. Clinico-pathological factors investigated for tumour failure prediction were: T stage; tumour differentiation; tumour thickness; and treatment. Tumour thickness was measured using an ocular micrometer.ResultsLocoregional tumour failure was found in 34% of cases (60 of 176). Tumour differentiation was found not to be statistically significant in predicting tumour failure. The covariates predicting tumour failure were T stage (P<0.05); type of surgical treatment (P<0.05); and tumour thickness (P<0.001). The mean tumour thickness found was 4 mm. Tumours 4 mm in thickness behaved similarly in all three stages and those >4 mm had a higher rate of lymph-node metastasis. No distant metastasis was found in this series.ConclusionTumour thickness was found to be an accurate predictor of locoregional failure in early stage cancer of the buccal mucosa.