Exploration of the association between HIF3α mRNA and lncRNA MALAT1 in laryngeal squamous cell carcinoma by correlation analysis.

Laryngeal squamous cell carcinoma (LSCC) is a significant global health burden, for which there has been limited evidence of improved survival rates. Although the roles of hypoxia-inducible factor (HIF)1α and HIF2α have been well documented in hypoxia, the involvement of HIF3α, particularly in LSCC, has been inadequately explored. The present study aimed to investigate the correlation between HIFα subunits and the hypoxia-related long noncoding RNAs (lncRNAs) MALAT1 and HOTAIR in 63 patients diagnosed with LSCC. Total RNA was extracted from fresh-frozen laryngeal tumor and adjacent normal tissues, and was subjected to reverse transcription-quantitative PCR for target detection. Statistical analyses were conducted using SPSS software, with significance set at P<0.05. The present study is the first, to the best of our knowledge, to report a positive moderate monotonic correlation (rs=0.347) and moderately strong positive linear correlation (r=0.630) between HIF3α mRNA and lncRNA MALAT1 in LSCC. Regression analysis revealed a direct association between 39.6% of both variables. Additionally, a positive correlation was observed between lncRNAs MALAT1 and HOTAIR (rs=0.353); HIF2α mRNA and lncRNA MALAT1 (rs=0.431); HIF3α mRNA and lncRNA HOTAIR (rs=0.279); and HIF3α mRNA and HIF2α mRNA (rs=0.285). Notably, a significant negative correlation (rs=-0.341) was detected between HIF3α mRNA and HIF1α mRNA, potentially indicative of the HIF switch or negative regulation. In addition, the present study investigated the association between HIFα subunits and the clinicopathological characteristics of patients. The results revealed a notable association between HIF1α transcript levels and the location of LSCC; specifically, subglottic tumors exhibited elevated HIF1α levels compared with glottic and supraglottic LSCC. Furthermore, a significant association was identified between HIF3α transcript levels and patient age (P=0.032) and positive family history (P=0.047). In conclusion, the present findings suggested a pivotal role for HIF3α in LSCC development, potentially involving direct regulation of lncRNA MALAT1. However, further research is warranted to elucidate its precise mechanisms.

Peritumor Mucosa in Advanced Laryngeal Carcinoma Exhibits an Aberrant Proangiogenic Signature Distinctive from the Expression Pattern in Adjacent Tumor Tissue.

The field cancerization theory is an important paradigm in head and neck carcinoma as its oncological repercussions affect treatment outcomes in diverse ways. The aim of this study is to assess the possible interconnection between peritumor mucosa and the process of tumor neoangiogenesis. Sixty patients with advanced laryngeal carcinoma were enrolled in this study. The majority of patients express a canonical HIF-upregulated proangiogenic signature with almost complete predominancy of HIF-1α overexpression and normal expression levels of the HIF-2α isoform. Remarkably, more than 60% of the whole cohort also exhibited an HIF-upregulated proangiogenic signature in the peritumoral benign mucosa. Additionally, the latter subgroup had a distinctly shifted phenotype towards HIF-2α upregulation compared to the one in tumor tissue, i.e., a tendency towards an HIF switch is observed in contrast to the dominated by HIF-1α tumor phenotype. ETS-1 displays stable and identical significant overexpression in both the proangiogenic phenotypes present in tumor and peritumoral mucosa. In the current study, we report for the first time the existence of an abnormal proangiogenic expression profile present in the peritumoral mucosa in advanced laryngeal carcinoma when compared to paired distant laryngeal mucosa. Moreover, we describe a specific phenotype of this proangiogenic signature that is significantly different from the one present in tumor tissue as we delineate both phenotypes, quantitively and qualitatively. This finding is cancer heterogeneity, per se, which extends beyond the "classical" borders of the malignancy, and it is proof of a strong interconnection between field cancerization and one of the classical hallmarks of cancer-the process of tumor neoangiogenesis.

Global microRNA expression profile in laryngeal carcinoma unveils new prognostic biomarkers and novel insights into field cancerization.

Laryngeal carcinoma is still a worldwide burden that has shown no significant improvement during the last few decades regarding definitive treatment strategies. The lack of suitable biomarkers for personalized treatment protocols and delineating field cancerization prevents further progress in clinical outcomes. In the light of this perspective, MicroRNAs could be promising biomarkers both in terms of diagnostic and prognostic value. The aim of this prospective study is to find strong prognostic microRNA biomarkers for advanced laryngeal carcinoma and molecular signatures of field cancerization. Sixty patients were enrolled and four samples were collected from each patient: tumor surface and depth, peritumor normal mucosa, and control distant laryngeal mucosa. Initially, a global microRNA profile was conducted in twelve patients from the whole cohort and subsequently, we validated a selected group of 12 microRNAs with RT-qPCR. The follow-up period was 24 months (SD ± 13 months). Microarray expression profile revealed 59 dysregulated microRNAs. The validated expression levels of miR-93-5p (χ2(2) = 4.68, log-rank p = 0.03), miR-144-3p (χ2(2) = 4.53, log-rank p = 0.03) and miR-210-3p (χ2(2) = 4.53, log-rank p = 0.03) in tumor samples exhibited strong association with recurrence-free survival as higher expression levels of these genes predict worse outcome. Tumor suppressor genes miR-144-3p (mean rank 1.58 vs 2.14 vs 2.29, p = 0.000) and miR-145-5p (mean rank 1.57 vs 2.15 vs 2.28, p = 0.000) were significantly dysregulated in peritumor mucosa with a pattern of expression consistent with paired tumor samples thus revealing a signature of field cancerization in laryngeal carcinoma. Additionally, miR-1260b, miR-21-3p, miR-31-3p and miR-31-5p were strongly associated with tumor grade. Our study reports the first global microRNA profile specifically in advanced laryngeal carcinoma that includes survival analysis and investigates the molecular signature of field cancerization. We report two strong biomarkers of field cancerization and three predictors for recurrence in advance stage laryngeal cancer.

MiRNA expression profiling in adenocarcinoma and squamous cell lung carcinoma reveals both common and specific deregulated microRNAs.

The current study investigated the expression signatures of miRNAs in lung adenocarcinoma (LUAD) and squamous cell lung carcinoma (LUSC). miRNA profiling was performed using microarray in 12 LUAD and 12 LUSC samples and adjacent normal tissues. In LUAD, 107 miRNAs were significantly deregulated, whereas 235 miRNAs were deregulated in LUSC. Twenty-six miRNAs were common between the 2 cancer subtypes and 8 were prioritized for validation, in addition to 6 subtype-specific miRNAs. The RT-qPCR validation samples included 50 LUAD, 50 LUSC, and adjacent normal tissues. Eight miRNAs were validated in LUAD: 3 upregulated - miR-7-5p, miR-375-5p, miR-6785-3p, and 5 downregulated - miR-101-3p, miR-139-5p, miR-140-3p, miR-144-3p, miR-195-5p. Ten miRNAs were validated in the LUSC group: 3 upregulated - miR-7-5p, miR-21-3p, miR-650, and 7 downregulated - miR-95-5p, miR-140-3p, miR-144-3p, miR-195-5p, miR-375, miR-744-3p, and miR-4689-3p. Reactome pathway analysis revealed that the target genes of the deregulated miRNAs in LUAD were significantly enriched in cell cycle, membrane trafficking, gene expression processes, and EGFR signaling, while in LUSC, they were enriched in the immune system, transcriptional regulation by TP53, and FGFR signaling. This study identified distinct miRNA profiles in LUSC and LUAD, which are common and specific miRNAs that could be further investigated as biomarkers for diagnosis and prognosis.

Novel insights into laryngeal squamous cell carcinoma from association study of aberrantly expressed miRNAs, lncRNAs and clinical features in Bulgarian patients.

PURPOSE: Laryngeal cancer is one of most common and aggressive head and neck cancers with poor prognosis and great necessity for improvement of treatment modalities. MicroRNAs (miRs) are among the most investigated molecules recently due to their potential as diagnostic and prognostic biomarkers in cancer. The purpose of our study was to explore the association of certain clinicopathological features with the expression levels of some known cancer associated non-coding (nc) RNAs: miR-21 and miR-31 in both of their isoforms, miR-145-5p, miR-55-5p, miR-196a-5p, miR-210-3p, miR-221-3p, miR-222-3p, miR-424-5p, lncRNA MALAT1 and lncRNA HOTAIR. METHODS: Expression levels of the chosen markers were investigated in laryngeal squamous cell carcinoma (LSCC) and normal samples in 82 Bulgarian patients via RT-qPCR, and the results were analyzed with SPSS v23.0 statistical software. RESULTS: All of the explored ncRNAs were significantly deregulated in LSCC samples, suggesting their involvement in laryngeal carcinogenesis. New significant association were found between the expression levels of miR-21-5p, miR-222-3p, HOTAIR and family history. Moreover, miR-424-5p showed potential as marker for subglottic LSCC location, and "passenger" miR-31-3p was significantly upregulated in well and moderately differentiated LSCC. CONCLUSION: Our results enrich the knowledge about ncRNA involvement in LSCC tumorigenesis. Further studies are needed to evaluate the clinical utility of the differently expressed ncRNAs as potential diagnostic and prognostic biomarkers in LSCC.

Relative quantitative expression of hypoxia-inducible factor-1α, -2α and -3α, and vascular endothelial growth factor A in laryngeal carcinoma.

The aim of the present study was to determine the relative quantitative expression of hypoxia-inducible factor (HIF)-1α, -2α and -3α, and VEGF-A in laryngeal carcinoma. A total of 63 patients with carcinoma of the larynx were enrolled in the study. Total RNA was isolated from fresh, frozen normal and tumor tissues of each patient, and quantitative polymerase chain reaction was performed. HIF-1α was upregulated in the majority of patients (44 patients; 69.84%). By contrast, only 7 (11.11%) patients from the whole group displayed HIF-2α overexpression, while the HIF-3α isoform was silenced in the majority of patients (48 patients, 76.19%). A small group of 5 (7.94%) patients exhibited significant overexpression of the HIF-3α isoform. VEGF-A expression was significantly higher (P<0.05) in patients with upregulated HIF-1α (2.72±1.41 RQ) compared with patients without upregulated HIF-1α (1.86±1.46 RQ). There was a moderate positive correlation between mRNA expression levels of HIF-1α and VEGF-A (rs=0.392; P<0.005). To the best of our knowledge, this study is first to report quantitative data with regard to the expression of all three HIF isoforms in malignant neoplasms. The findings suggest the existence of specific phenotypes of HIF expression in laryngeal carcinoma, where the HIF switch is absent.

Mutational Status of CDKN2A and TP53 Genes in Laryngeal Squamous Cell Carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is the second most common tumour of the head and neck. It is characterized by frequent aberrations in two cell-cycle regulators--CDKN2A and TP53. However, LSCC has been often studied as a part of the group of head and neck cancers and not as an individual entity. In the current study we aimed to examine mutation status of CDKN2A and TP53 genes in 108 LSCC patients. DNA was extracted from fresh-frozen tumour tissues; exons 1-3 of CDKN2A and exons 5-8 of TP53 were screened for mutations by direct sequencing. Genetic aberrations in CDKN2A were found in 16 (14.2%) and those in TP53--in 56/108 (51.9%) tumours. Seven mutations (two insertions, three deletions, one missense and one silent) detected in CDKN2A were not described previously. Also, we found seven novel deletions and a novel indel in TP53. No significant associations with clinical features were found. However, TP53 mutations were predominantly observed in smokers with advanced stage tumours. Screening for genetic aberrations in a defined group of LSCC contributes to the knowledge about laryngeal carcinogenesis. Further investigations are required to confirm the observed trends in associations with clinical features.

IDH1/IDH2 but not TP53 mutations predict prognosis in Bulgarian glioblastoma patients.

Mutations in genes encoding isocitrate dehydrogenase isoforms 1 (IDH1) and 2 (IDH2) have been associated with good prognosis for patients with brain neoplasias and have been commonly found together with mutated TP53 gene. To determine the prevalence of IDH1, IDH2, and TP53 mutations and their impact on overall survival 106 glioblastoma patients were analysed. IDH1 mutations were detected in 13 and IDH2 mutation in one patient. Two homozygous samples with R132H mutation in IDH1 gene and a novel aberration K129R in IDH2 gene were found. Sixty-four percent of IDH1/IDH2 mutated tumours harboured also a mutation in TP53 gene. Genetic aberrations in TP53 were present in 37 patients. Statistical analysis of the impact of the studied factors on the overall survival showed that the mutations in IDH1/IDH2, but not the ones in TP53, were associated with longer survival. Also, the impact of age on prognosis was confirmed. This is the first comprehensive study on glioblastomas in Bulgaria. Our results suggest that IDH1/IDH2 but not TP53 mutations together with other prognostic factors such as age might be applied in clinical practice for prediction of outcome in patients with glioblastomas.