Non-metastatic Oral Squamous Cell Carcinoma Research Articles

Abstract 3983: MicroRNAs and other small RNA molecules expressed in metastatic and non-metastatic oral squamous cell carcinoma

Abstract Small noncoding RNAs are regulatory molecules that play important roles in several aspects of cellular biology. They vary from 18 to 30 nucleotides in length and often act through the inactivation of complementary sequences. A variety of small RNA classes have been identified to date and the list is constantly growing, owing to the advent of new sequencing technologies. Among these molecules, microRNAs are the most extensively studied. They are known to play important roles in human diseases, including cancer. More recently, genome-wide analyses have showed that changes in the expression levels of other small noncoding RNAs are also associated with cancer, with correlations between RNA abundance and disease status. Oral squamous cell carcinoma, associated with chronic tobacco and alcohol consumption, is among the leading cancers in the world. The presence of cervical lymph node metastases is currently its strongest prognostic factor. The possibility to evaluate its metastatic potential is relevant to the clinical and molecular oncologist due to frequent asymptomatic development of such cancer in its early stages. In this study small RNA libraries from 30 oral squamous cell carcinoma samples were sequenced for the identification and quantification of known small RNAs. Samples were divided in two groups: those presenting lymph node metastasis at the time of diagnosis and those that did not present this characteristic. Additionally, plasma of 30 patients was accessed for the presence of microRNAs identified as differentially expressed between metastatic and non-metastatic tumor samples as a means to identify circulating molecules that could be considered potential biomarkers for lymph node metastasis. Global expression patterns of small RNA molecules were not associated with cervical metastases. MiR-21, miR-203 and miR-205 were highly expressed throughout samples, in agreement with their role in epithelial cell biology, but disagreeing with studies correlating these molecules with cancer invasion. Nineteen microRNAs, but no other small RNA class, varied consistently between metastatic and non-metastatic cancer samples, some of which were also detected at corresponding levels in plasma. MiR-31 and miR-130b, known to inhibit several steps in the metastatic process, were over-expressed in non-metastatic samples and the expression of miR-130b was confirmed in plasma of patients presenting non-metastatic oral squamous cell carcinoma. MiR-181 and miR-296 were associated with metastasis but were not detected in plasma. In conclusion, we identified microRNAs linked to the metastatic status in a group of patients diagnosed with oral squamous cell carcinoma, both in tissue and plasma samples. We also demonstrate that other small RNA classes are expressed and should, therefore, be involved and contribute to the metastatic phenotype of this disease. Citation Format: Patricia Severino, Liliane Santana Oliveira, Flavia Maziero Andreghetto, Natalia Torres, Otavio Curioni, Patricia Maluf Cury, Tatiana Natasha Toporcov, Alexandre Rossi Paschoal, Alan Mitchell Durham. MicroRNAs and other small RNA molecules expressed in metastatic and non-metastatic oral squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3983. doi:10.1158/1538-7445.AM2015-3983

Small RNAs in metastatic and non-metastatic oral squamous cell carcinoma.

BackgroundSmall non-coding regulatory RNAs control cellular functions at the transcriptional and post-transcriptional levels. Oral squamous cell carcinoma is among the leading cancers in the world and the presence of cervical lymph node metastases is currently its strongest prognostic factor. In this work we aimed at finding small RNAs expressed in oral squamous cell carcinoma that could be associated with the presence of lymph node metastasis.MethodsSmall RNA libraries from metastatic and non-metastatic oral squamous cell carcinomas were sequenced for the identification and quantification of known small RNAs. Selected markers were validated in plasma samples. Additionally, we used in silico analysis to investigate possible new molecules, not previously described, involved in the metastatic process.ResultsGlobal expression patterns were not associated with cervical metastases. MiR-21, miR-203 and miR-205 were highly expressed throughout samples, in agreement with their role in epithelial cell biology, but disagreeing with studies correlating these molecules with cancer invasion. Eighteen microRNAs, but no other small RNA class, varied consistently between metastatic and non-metastatic samples. Nine of these microRNAs had been previously detected in human plasma, eight of which presented consistent results between tissue and plasma samples. MiR-31 and miR-130b, known to inhibit several steps in the metastatic process, were over-expressed in non-metastatic samples and the expression of miR-130b was confirmed in plasma of patients showing no metastasis. MiR-181 and miR-296 were detected in metastatic tumors and the expression of miR-296 was confirmed in plasma of patients presenting metastasis. A novel microRNA-like molecule was also associated with non-metastatic samples, potentially targeting cell-signaling mechanisms.ConclusionsWe corroborate literature data on the role of small RNAs in cancer metastasis and suggest the detection of microRNAs as a tool that may assist in the evaluation of oral squamous cell carcinoma metastatic potential.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0102-4) contains supplementary material, which is available to authorized users.

E-cadherin, β-catenin, and α2β1 and α3β1 integrin expression in primary oral squamous cell carcinoma and its regional metastasis.

To investigate E-cadherin, β-catenin, and α2β1 and α3β1 integrins in 40 samples of non-metastatic and metastatic oral squamous cell carcinoma (OSCC) with positive cervical lymph nodes (LN). Immunohistochemistry was used to evaluate expression in the lesion center (LC) and invasive tumor front (ITF) of non-metastatic (n=18) and metastatic (n=22) OSCC and in the LN on the metastatic neoplastic cells (MNC; n=22). In metastatic OSCC, E-cadherin and β-catenin presented significantly lower cytoplasmic membrane expression in the ITF and MNC when compared to the LC and lower cytoplasmic expression in MNC when compared to the LC and ITF (p<0.05). Integrins α2β1 and α3β1 showed high cytoplasmic expression in the LC, ITF and MNC (p>0.05). A positive correlation was observed between E-cadherin cytoplasmic expression and α2β1 (p=0.860) and α3β1 (p=0.975) expression. When comparing the primary sites of metastatic and non-metastatic disease, β-catenin presented lower cytoplasmic membrane (p=0.013) expression in metastatic OSCC. E-cadherin presented low expression and the integrins high expression in both groups. Abnormal expression of β-catenin and E-cadherin associated with high expression of α2β1 and α3β1 integrins contribute to LN metastasis in OSCC.

441 The role of méthylation in metastasis of oral squamous cell carcinoma: understanding the OSCC methylome

Background: Oral Squamous Cell Carcinoma (OSCC) is characterized by an increasing incidence and a 60% 5-year survival. The most important prognostic factor for OSCC is the presence of lymph node (LN) metastases but the detection of LN metastases in the clinic by palpation and imaging is inaccurate resulting in under and overtreatment of patients. To improve LN diagnosis new detection methods are needed. DNA methylation studies can be used to identify novel biomarkers, as epigenetics have been established as an important regulator of metastatic potential. The aim of this study is to identify new DNA methylation markers that predict LN metastasis in OSCC. Materials and Methods: Genome-wide methylation patterns of metastatic (n = 6) and non-metastatic OSCC (n = 6) were assessed using MethylCap- Seq. Subsequently, analysis was performed on the most differentially methylated loci to identify pathways and genomic loci associated with LN metastasis. Additionally, the methylation data were combined with expression data of 222 OSCC patients acquired by an expression microarray with a 696 gene panel associated with N-status in OSCC. Finally, we validated these findings on the OSCC samples (n = 174) in The Cancer Genome Atlas (TCGA). Results: We found that genes on chromosome 7 are the most hypermethylated in metastatic OSCC in comparison to non-metastatic OSCC, more specifically a four million bp loci around the EGFR gene. In total 26 genes were found to be differentially methylated by MethylCap- Seq as well as differentially expressed by microarray. In the OSCC TCGA validation cohort, all 26 genes are significantly differentially expressed between metastatic and non-metastatic OSCC. For these 26 differentially expressed genes 88 probes were found in the TCGA Infinium 450k methylation data that are both significantly differentially methylated between the two groups and correlated with mRNA levels. Finally, six of these probes overlapped with the genomic regions annotated by MethylCap-Seq, representing five genes. Conclusion: We identified chromosomal loci, pathways and gene promoters associated with LN metastasis in OSCC patients. For 10 genes the differential methylation, expression and correlation between methylation and expression was validated on an independent OSCC cohort from the TCGA database. Increased understanding of the metastatic OSCC methylome will contribute to the identification of DNA methylation markers, pathways and potential therapeutic targets in OSCC primary tumors that will improve chances of providing the proper lymph node treatment and may result in improvement of survival and quality of life.

Abstract 2091: Metastatic oral cancer cell adhesion to vascular and lymphatic endothelial cells is sialyl Lewis A/X-dependent

Abstract Background: The vast majority of mortalities from cancer, including oral cancer, are due to metastatic disease. Previous studies have shown that sialylated cell surface glycoconjugates mediate the extravasation of circulating tumor cells from several different tumor types. The aim of this study was to investigate whether metastatic oral squamous cell carcinoma (OSCC) cells overexpress and employ sialyl Lewis A/X (sLeA/X) to adhere to E-selectin on endothelial cells. Materials and methods: Twenty-one matched-pairs of primary and nodal metastatic OSCC tissue sections were evaluated immunohistochemically using anti-sLeA (clone KM231) and anti-sLeX (clone KM93) monoclonal antibodies (mAbs). q-PCR was performed to measure relative transcript expression of a panel of glycogens potentially involved in sLeA/X synthesis in OSCC cells. sLeA/X cell surface expression was assessed by flow cytometry and fluorescence immunocytochemistry. sLeA/X involvement in OSCC cell adhesion behavior was evaluated using static and flow adhesion assays employing recombinant E-selectin (rE-selectin) and TNF-α-stimulated human dermal microvascular endothelial cells (HuDMEC) or human lymphatic endothelial cells (HLEC), respectively. Several approaches to prevent OSCC cell binding to rE-selectin or HuDMEC were tested including the use of anti-sLeA/X mAbs, neuraminidase, fucosidase, IELLQAR®, chondroitin sulfate, swainsonine and FUT3 siRNA. Results: Immunohistochemistry analysis showed sLeA/X to be mainly expressed by well- to moderately-differentiated OSCC. sLeA/X expression on primary OSCC correlates with lymph node metastasis but not tumor differentiation status. The metastatic OSCC cells, TR146, displayed higher sLeA/X expression than the non-metastatic OSCC cells, SCC4 and Cal27, and this was at least partially related to up-regulated gene expression of fucosyltransferase III. Knocking down FUT3 expression in TR146 cells reduced sLeX but not sLeA levels. TR146 cells adhered to rE-selectin and TNF-α-stimulated HuDMEC/HLEC in significantly greater numbers than SCC4 cells. Attachment of TR146 cells to rE-selectin was significantly reduced following incubation with anti-sLeA/X mAbs, neuraminidase and FUT3 siRNA. Treatment of TR146 cells with neuraminidase also caused a significant decrease in cell adhesion to HuDMEC under hydrodynamic flow conditions. Conclusions: Elevated levels of sLeX in metastatic OSCC cells results from increased FUT3 expression. Metastatic OSCC cell binding to HuDMEC/HLEC is sLeA/X-mediated and may be a target for anti-metastasis therapy. Citation Format: Ahmed Alkishi, Syed Ali Khurram, Martin Thornhill, Craig Murdoch. Metastatic oral cancer cell adhesion to vascular and lymphatic endothelial cells is sialyl Lewis A/X-dependent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2091. doi:10.1158/1538-7445.AM2014-2091

High nuclear SOX2 expression is associated with radiotherapy response in small (T1/T2) oral squamous cell carcinoma.

Expression of the stem cell transcription factor SOX2 is often used to imply stemness and poor prognosis in cancer. However, its role in oral squamous cell carcinoma (OSCC) is not fully elucidated. Tumour tissues from 62 patients with primary, node negative and non-metastatic OSCCs were used to evaluate SOX2 expression by immunohistochemistry. The results were correlated to clinicopathology, treatment and disease recurrences. The majority of the OSCCs (88%) expressed SOX2. Patients with higher nuclear SOX2 staining intensity in the invasive front compared to the adjacent normal epithelium, had a remarkable longer disease-free period if they received adjuvant post-operative radiotherapy (P = 0.001). This was in particular evident for highly differentiated OSCCs, as none of the high SOX2-expressing tumours reoccurred in contrast to all low SOX2-expressing OSCCs. High nuclear SOX2 expression in the invasive front was associated with dramatic longer disease-free period than low SOX2-expressing carcinomas after post-operative radiotherapy in small OSCCs. The result suggested that high nuclear SOX2 expression at the invasive front may predict radiosensitivity.

Assessment of Tissue Eosinophilia as a Prognosticator in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma—An Image Analysis Study

Association of tissue eosinophilia with oral squamous cell carcinoma has shown variable results ranging from favourable to unfavourable or even having no influence on prognosis. Also, very few studies have been done to know the role of eosinophils in premalignancy. So the present study investigated role of eosinophilic infiltration in oral precancer and cancer and its possible use as a prognosticator. 60 histopathologically proven cases (20 cases each of metastatic and nonmetastatic oral squamous cell carcinoma and oral leukoplakia with dysplasia of various grades) were included. Congo red is used as a special stain for eosinophils. Each specimen slide was viewed under high power in 10 consecutive microscopic fields for counting of eosinophils. As a result, a significant increase in eosinophil count was found in oral carcinomas compared to dysplasia. Nonmetastatic cases showed higher counts than metastatic carcinomas. So, it is concluded that eosinophilia is a favourable histopathological prognostic factor in oral cancer. Moreover, higher eosinophil counts in carcinoma group compared to dysplasia group proved that they might have a role in stromal invasion thus suggesting that quantitative assessment of tissue eosinophilia should become a part of the routine histopathological diagnosis for oral precancer and OSCC.

Immunohistochemical expression of endothelin protein in oral squamous cell carcinoma

Endothelin-1 (ET-1) plays a significant role in cancer biology and is considered as a potential target for molecular therapy. Alteration of ET-1 was detected in oral squamous cell carcinoma (OSCC). To evaluate the immunohistochemical expression and distribution of ET-1 in OSCC and to correlate its expression with the degree of histopathologic differentiation as well as the regional metastatic status of OSCC. The immunohistochemical expression and distribution of ET-1 was evaluated in total number of 30 cases of OSCC. The specimens were retrieved as formalin-fixed, paraffin-embedded blocks from the archival files. Detection of ET-1 expression in different grades of metastatic and non-metastatic OSCC was quantified using an image analyzer computer system. Statistical software package SPSS v. 19. ET-1 immunoreactivity was observed in all studied specimens. On comparing optical density values for ET-1 expression in different grades of OSCC, poorly differentiated OSCC showed a significantly greater expression than moderately differentiated OSCC, which was also significantly greater than well-differentiated OSCC ( P = 0.001). Moreover, ET-1 expression was higher in metastatic OSCC than that in non-metastatic OSCC ( P = 0.018). Increased ET-1 expression could enhance the aggressive behavior of poorly differentiated OSCC, especially metastasis. Accordingly, ET-1 could be a therapeutic target in OSCC.

Stromal myofibroblasts in nonmetastatic and metastatic oral squamous cell carcinoma: An immunohistochemical study

Background and Aims:Myofibroblasts are one of the important components of the tumor microenvironment which could possibly play an important role in tumor progression. The purpose of this study was to compare the presence of α-smooth muscle actin (α-SMA) and CD34 positive fibroblasts in nonmetastatic and metastatic oral squamous cell carcinoma and to evaluate their role in tumor metastasis.Materials and Methods:Ten cases each of histologically proven metastatic and nonmetastatic oral squamous cell carcinoma formed the study group. The tissue sections were stained immunohistochemically for α-SMA and CD34. The stromal spindle cells positive for these markers in the study groups were counted and compared.Results:α-SMA positive cases were more in the metastatic group and CD34 positive cases were found to be more in the nonmetastatic tumors.Conclusions:Though difference in the staining pattern was statistically nonsignificant, the inverse relationship between α-SMA and CD34 positive cells is indicative of dynamic nature and the influence of tumor stroma in tumor progression and metastasis.

Comparison of external beam radiation therapy and radioactive implants in the management of nonmetastatic oral squamous cell cancer: an analysis of the surveillance epidemiology and end results database

Radiotherapy in oral squamous cell carcinoma (OSCC) could be either through external beam (EBRT) or brachytherapy (BT). In third-world countries, access to novel treatment like BT is limited. This study aimed to compare survival in patients with OSCC treated with EBRT with BT recipients. We examined cases of nonmetastatic OSCC between January 1, 1987, and December 31, 2007, in the surveillance epidemiology and end results (SEER) registry. A Cox proportional hazards model was used to determine the adjusted relationship between radiation type and survival. There was no significant difference in survival (adjusted) between patients who were treated with BT and those treated with EBRT (P = .330). However, combining EBRT + BT was associated with significantly better survival compared with EBRT alone (hazard ratios [HR] = 0.61, P < .001) or BT alone (HR = 0.70, P = .024). The effect of BT on survival was not significantly different from EBRT.

Abstract 4983: Integrative analysis of DNA copy number and gene expression identifies G3BP1 and HIRA as potential therapeutic targets against metastatic oral squamous cell carcinoma

Abstract The presence of lymph node metastasis is associated with a 50% reduction of 5-year survival in patients with oral squamous cell carcinoma (OSCC). We set out to combine DNA copy number aberrations (CNAs) with gene expression profiles, to identify CNAs-associated genes dysregulated in metastatic OSCC, and determine whether these genes can be targeted to selectively kill metastatic OSCC tumor cells. Toward this end, we interrogated DNA and RNA of the same OSCC cell populations laser micro-dissected from non-metastatic primary tumors (n=17) and metastatic lymph nodes (n=20) using Affymetrix 250K Nsp single-nucleotide polymorphism (SNP) arrays and U133 Plus 2.0 arrays, respectively. With a false discovery rate (FDR) &amp;lt; 5%, 1988 transcripts were found to be differentially expressed between primary and metastatic OSCC. Out of these, 114 transcripts showed significant correlation between their DNA copy number alternations (estimated using SNPs within 250 kb upstream and downstream of the transcript) and gene expression (FDR &amp;lt; 0.01). Among these CNA-correlated transcripts, 95 had significantly different DNA copy numbers between metastatic and non-metastatic OSCC (FDR &amp;lt;0.01 by Wilcoxon rank test). These 95 transcripts (representing 87 genes) mainly clustered around three genomic locations: 3p25.3-22.1, 9p24.1- 22.3 and 18q21.1-22.3. Among these 87 genes, we selected 28 of the 58 genes that were over-amplified and over-expressed in metastatic OSCC and conducted a high-throughput siRNA-mediated gene knockdown screen in five cell lines derived from primary and lymph node metastatic OSCC. The expression-knockdown of 18 genes showed 30% or more growth suppression in at least one cell line as compared to mock controls in which the cells were treated with transfection reagent only without the siRNA (P &amp;lt; 0.01). In particular, knocking-down G3BP1 and HIRA selectively suppressed the growth of all OSCC cell lines derived from lymph node metastases when compared to non-metastatic lines. Further investigation is warranted to confirm these findings and to examine the biological role of G3BP1 and HIRA in OSCC metastasis and their potential as therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4983. doi:10.1158/1538-7445.AM2011-4983

O106. Profiling of metastatic and non-metastatic oral squamous cell carcinoma using array comparative genomic hybridization

O106. Profiling of metastatic and non-metastatic oral squamous cell carcinoma using array comparative genomic hybridization

Bone morphogenetic protein-2/4 and bone morphogenetic protein receptor type IA expression in metastatic and nonmetastatic oral squamous cell carcinoma

Purpose The study aimed to analyze the expression of bone morphogenetic protein-2/4 (BMP-2/4) and its receptor BMPR-IA (BMP receptor type IA) in metastatic and nonmetastatic oral squamous cell carcinoma (OSCC) and its implications for disease prognosis. Materials and methods The experimental group included 16 cases of OSCC without metastasis and 7 cases of OSCC with metastasis. The presence or absence of nodal metastasis was used as a parameter for the evaluation of disease prognosis. Ten cases of oral fibroepithelial hyperplasia were selected as the control group. The expression of BMP-2/4 and BMPR-IA was analyzed by immunohistochemistry. Results In the experimental group with metastasis, strong expression of BMP-2/4 was observed in most cases (71.4%), whereas BMPR-IA exhibited weak expression (85.7%). In the experimental group without metastasis, there was strong expression of BMP-2/4 (62.5%) and BMPR-IA (100%). A significant association was observed between the prognosis of OSCC and the intensity of BMP-2/4 staining ( P = .002). Weak immunoreactivity to BMP-2/4 and BMPR-IA was observed in all control specimens. Conclusions The results suggest that strong expression of BMP-2/4, associated with low expression of BMPR-IA, observed in metastatic OSCC has a prognostic value, with the loss of responsiveness to BMPs through the loss of expression of their receptors being indicative of the development of metastasis.

Quantification of Serum Proteins of Metastatic Oral Cancer Patients Using LC-MS/MS and iTRAQ Labeling

Metastasis is a critical event in oral squamous cell carcinoma (OSCC) progression. In this study, we have performed quantitative analysis of serum proteins from non-metastatic (lymph-node metastasis free) and metastatic OSCC patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with iTRAQ labeling (isobaric tagging for relative and absolute quantitation). To eliminate highly abundant proteins, the serum samples were initially separated by SDS-PAGE and only low abundant protein bands were excised for subsequent in-gel tryptic digestion. The resulting peptides were then extracted from each sample gels and labeled with iTRAQ reagent 114 (control), 116 (non-metastatic) and 117 (metastatic), respectively. Afterwards, the labeled samples were combined and subjected to LC-MS/MS analysis using linear ion trap (LIT) MS with pulsed Q collision induced dissociation (PQD). A total of 64 proteins were identified and quantified by this approach. Our study showed that iTRAQ labeling and LIT-MS with PQD is a valuable approach to quantification of serum proteins. We also demonstrated the presence of differentially expressed serum proteins between non-metastatic and metastatic OSCCs that may be further validated as biomarkers for metastatic OSCC. However, in order to comprehensively quantify low abundant serum proteins, a more efficient approach is needed to deplete highly abundant proteins prior to quantitative serum proteome analysis of OSCC.