The role of the Dentist and, in particular, the role of the Oral Medicine specialist as a front-line sentinel in the interception of oral cancer has been consolidated since long time. However, over the last decade there have not been any recent developed diagnostic strategies that can facilitate neither a more precise general framework nor a definition of the degree of local and systemic involvement, as well as the type of behavior. Head and neck cancer (HNC), is the sixth most common type of cancer in the world and accounts for approximately 550,000 cases per year [1]. HNC encompasses a wide range of malignancies arising from the head and neck region, including the oral district, the oropharynx, and larynx. More than 90% of HNCs are squamous cell carcinomas (SCCs), which frequently develop from the mucous surfaces of the mouth [2]. When diagnosed at an advanced stage, treatment of OSCC involves particularly invasive procedures since, in most cases, the disease is fatal due to the high rate of early metastasis to regional cervical lymph nodes; treating these patients is a challenge for clinicians [3]. Early-stage OSCC, however, has a favorable prognosis and requires less aggressive treatment [4]. The 5-year overall survival rate for OSCC is less than 50% and has improved only modestly over the past 2 decades despite the dramatic improvement in treatment modalities [5]. Although tumor-node-metastasis (TNM) based on neoplastic staging is routinely used to predict tumor behavior and, consequently, to inform about the choice of treatment strategies of OSCC, patients with the same TNM stages may have non-overlapping clinical evolutions and a significantly different survival time [6]. These inconsistencies require the development of new and more indepth prognostic techniques and tools that can be used in the management of OSCC. For these reasons, the search for biomarkers which could be used for diagnosis and prognosis represents a growing hope to provide doctors with new tools for the treatment of the disease. MATERIALS AND METHODS The population to which the study is addressed is made up of patients with carcinoma of the oral cavity whose diagnosis was identified at the Oral Medicine clinic of the SC of Odontostomatology of the Circolo Hospital from 2008 to 2013 for a total of 9 patients, divided as follow: 3 non-metastatic oral squamous cell carcinomas nmOSCC, 3 metastatic oral squamous cell carcinomas mOSCC and 3 verrucous carcinomas OVC. The choice of the time period is dictated by the need to verify the survival rates, absence of disease or relapse after 5 years. At the Pathology Anatomy Service of Circolo Hospital, we recovered the stained slides in hematoxylin-eosin, and other immunohistochemical investigations carried out previously. Each sample included were in sufficient quantity to obtain the following useful indications for the retrospective study. The analysis on the expression profile of 84 lncRNA was conducted on histological samples from patients, which are stored in the Pathological Anatomy Service of the Circolo Hospital, using RT2 lncRNA PCR Array Kit for Human Cancer Pathway, Qiagen. The paraffin samples were processed using a mechanical sampling phase, thanks to an extraction template, which allowed the collection of both cancerous tissue and considered healthy tissue, which has been used as control, being at least 15mm away from the neoplastic tissue. The RNA was subjected to the retro-transcriptional phase and converted to complementary DNA (cDNA) to increase its stability using the RT 2 First Strand kit for the synthesis of Qiagen cDNA. Subsequent real-time PCR molecular biology investigations were then carried out using specific array assays, which were customized in Syber Green for the definition of the expression profile of 84 lncRNAs. These lncRNAs are known to be differentially expressed in numerous neoplasms of various anatomical districts, compared to normal tissue, using the RT 2 lncRNA PCR kit together with the RT2 SYBR Green Mastermix for qPCR, in association with a Quant Studio 3 Real Time digital instrument, Thermofisher Scientific. During the comparison between control oral mucosa and neoplastic tissue, the following data have been studied and quantified: the statistically significant levels of modification in the expression profile of the lncRNA transcripts detectable in neoplastic tissues, compared to control ones, or in healthy mucosa. It has also been defined the presence of a “alteration of the expression profile of a lncRNA based on the expression related to the transcript normalized with the expression of a standard reference gene called housekeeping, coamplified together with the target of interest, in particular β-actin”. RESULTS In the preliminary results, 9 carcinomas were identified, and three sub-categories were characterized: Verrucous carcinoma, Metastatic Squamous cell carcinoma and Non-Metastatic Squamous cell carcinoma. Higher expression levels of some LncRNA's, such as HOTAIR, were detected in tumor tissue, when we compared the samples of noncancerous matched tissue (CTR) with neoplastic tissue (K). It is possible to highlight a significant considerably alteration of the expression profile of 5 lncRNA in the neoplastic tissue associated with the mOSCC sample, and in particular LINC00312, NAMA, PRNCR1, TUSC7, XIST X compared to control tissues. As regards the nmOSCC group, an alteration of the expression profile of: GNAS-AS1, HOTAIR, HOTTIP, LINC01233, LSINCT5, PCAT1, PRNCR1, SPRY4-IT1, TERC was found. Regarding the last OVC group, the following have been identified as regulated: AIRN, GACAT1, HIF1A-AS1. The use of multivariate analysis in comparing the pattern of expression among groups: mOSCC, nmOSCC, OVC, associated with the use of both one and two components, found a good differentiation among the three groups in terms of expression. The two-component analysis made it possible to infer a ranking of distancing and maximization of variance, obtaining a good separation of the groups, with 45% of the total variance being described by the two main components. CONCLUSION We have turned our attention to the differential study of the expression profile of some possible tumor biomarkers between different subtypes of oral malignant neoplasms, associated with different behaviours, compared to healthy control tissues, to allow possible correlations on a diagnostic and possibly clinical level. The processing of the data emerging from the molecular biology analysis with RT PCR, in multivariate analysis with two-component variance maximization, demonstrated a wide variety of profiles between the samples under examination (mOSCC, nmOSCC, OVC) and - more generally - each group is characterized by the dysregulation of several lncRNAs. In the group of OSCC associated with metastases, the lncRNAs LINC00312, PRNCR1, XIST X, could represent, given their characteristic oncogenic action found in numerous other neoplasms, possible early indicators of an evolution in a metastatic sense, with possible implications in terms of staging. As regards the group of OSCCs not associated with metastases, lncRNA: HOTAIR, PCAT1, SPRY4-IT1, were found as possible biomarkers. HOTAIR and PCAT1 have emerged as important oncogenes remarkably expressed in several squamous tumoral cells of the head and neck district (HNSCC). They could also be configured as prognostic indicators at the oral level, even in cases not necessarily associated with metastatic spread, as demonstrated in our study. SPRY4-IT1 lncRNA, overexpressed in the group of oral cancer not associated with metastatic spread, has been studied in the gastroenterological field for its onco- suppressive action against gastric cancer. Reduced expression of this lncRNA, in fact, has been associated with larger tumoral size, advanced disease stage, greater depth of invasion and lymphatic metastases. Ultimately, in the OVC group, a reduced expression of GACAT1 was measured, which could significantly explain the reduced proliferation, invasion, and cell migration, all of them being characteristics of this histotype. The preventive evaluation of LncRNA’s expression, and a better understanding of their role, could give a more thorough perspective on the future treatment options for this cancer type. 1. Global Burden of Disease Cancer Collaboration. Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disabilityadjusted life-years for 32 cancer groups, 1990 to 2015: a systematic analysis for the global burden of disease study. JAMA Oncol. 2017;3(4):524–548 2. Vigneswaran N, Williams MD. 2014. Epidemiologic trends in head and neck cancer and aids in diagnosis. Oral Maxillofac Surg Clin North Am. 26(2):123–141. 3. Abu-Ghanem S, Yehuda M, Carmel NN, Leshno M, Abergel A, Gutfeld O, Fliss DM. Elective Neck Dissection vs Observation in Early-Stage Squamous Cell Carcinoma of the Oral Tongue With No Clinically Apparent Lymph Node Metastasis in the Neck: A Systematic Review and Meta-analysis. JAMA Otolaryngol Head Neck Surg. 2016; 142:857–865. 4. Hema KN, Smitha T, Sheethal HS, Mirnalini SA. 2017. Epigenetics in oral squamous cell carcinoma. J Oral Maxillofac Pathol. 21(2):252–259. 5. Le Campion ACOV, Ribeiro CMB, Luiz RR, da Silva Júnior FF, Barros HCS, Dos Santos KCB, Ferreira SJ, Gonçalves LS, Ferreira SMS. 2017. Low survival rates of oral and oropharyngeal squamous cell carcinoma. Int J Dent. 2017:5815493. 6. Bitu CC, Destro MF, Carrera M, da Silva SD, Graner E, Kowalski LP, Soares FA, Coletta RD. 2012. Hoxa1 is overexpressed in oral squamous cell carcinomas and its expression is correlated with poor prognosis. BMC Cancer. 12:146

Analisi del profilo di espressione di lncRNA nel carcinoma a cellule squamose del cavo orale / Fabio Croveri - : . , 2020. ((33. ciclo, Anno Accademico 2019/2020.

Analisi del profilo di espressione di lncRNA nel carcinoma a cellule squamose del cavo orale

CROVERI FABIO
2020

Abstract

The role of the Dentist and, in particular, the role of the Oral Medicine specialist as a front-line sentinel in the interception of oral cancer has been consolidated since long time. However, over the last decade there have not been any recent developed diagnostic strategies that can facilitate neither a more precise general framework nor a definition of the degree of local and systemic involvement, as well as the type of behavior. Head and neck cancer (HNC), is the sixth most common type of cancer in the world and accounts for approximately 550,000 cases per year [1]. HNC encompasses a wide range of malignancies arising from the head and neck region, including the oral district, the oropharynx, and larynx. More than 90% of HNCs are squamous cell carcinomas (SCCs), which frequently develop from the mucous surfaces of the mouth [2]. When diagnosed at an advanced stage, treatment of OSCC involves particularly invasive procedures since, in most cases, the disease is fatal due to the high rate of early metastasis to regional cervical lymph nodes; treating these patients is a challenge for clinicians [3]. Early-stage OSCC, however, has a favorable prognosis and requires less aggressive treatment [4]. The 5-year overall survival rate for OSCC is less than 50% and has improved only modestly over the past 2 decades despite the dramatic improvement in treatment modalities [5]. Although tumor-node-metastasis (TNM) based on neoplastic staging is routinely used to predict tumor behavior and, consequently, to inform about the choice of treatment strategies of OSCC, patients with the same TNM stages may have non-overlapping clinical evolutions and a significantly different survival time [6]. These inconsistencies require the development of new and more indepth prognostic techniques and tools that can be used in the management of OSCC. For these reasons, the search for biomarkers which could be used for diagnosis and prognosis represents a growing hope to provide doctors with new tools for the treatment of the disease. MATERIALS AND METHODS The population to which the study is addressed is made up of patients with carcinoma of the oral cavity whose diagnosis was identified at the Oral Medicine clinic of the SC of Odontostomatology of the Circolo Hospital from 2008 to 2013 for a total of 9 patients, divided as follow: 3 non-metastatic oral squamous cell carcinomas nmOSCC, 3 metastatic oral squamous cell carcinomas mOSCC and 3 verrucous carcinomas OVC. The choice of the time period is dictated by the need to verify the survival rates, absence of disease or relapse after 5 years. At the Pathology Anatomy Service of Circolo Hospital, we recovered the stained slides in hematoxylin-eosin, and other immunohistochemical investigations carried out previously. Each sample included were in sufficient quantity to obtain the following useful indications for the retrospective study. The analysis on the expression profile of 84 lncRNA was conducted on histological samples from patients, which are stored in the Pathological Anatomy Service of the Circolo Hospital, using RT2 lncRNA PCR Array Kit for Human Cancer Pathway, Qiagen. The paraffin samples were processed using a mechanical sampling phase, thanks to an extraction template, which allowed the collection of both cancerous tissue and considered healthy tissue, which has been used as control, being at least 15mm away from the neoplastic tissue. The RNA was subjected to the retro-transcriptional phase and converted to complementary DNA (cDNA) to increase its stability using the RT 2 First Strand kit for the synthesis of Qiagen cDNA. Subsequent real-time PCR molecular biology investigations were then carried out using specific array assays, which were customized in Syber Green for the definition of the expression profile of 84 lncRNAs. These lncRNAs are known to be differentially expressed in numerous neoplasms of various anatomical districts, compared to normal tissue, using the RT 2 lncRNA PCR kit together with the RT2 SYBR Green Mastermix for qPCR, in association with a Quant Studio 3 Real Time digital instrument, Thermofisher Scientific. During the comparison between control oral mucosa and neoplastic tissue, the following data have been studied and quantified: the statistically significant levels of modification in the expression profile of the lncRNA transcripts detectable in neoplastic tissues, compared to control ones, or in healthy mucosa. It has also been defined the presence of a “alteration of the expression profile of a lncRNA based on the expression related to the transcript normalized with the expression of a standard reference gene called housekeeping, coamplified together with the target of interest, in particular β-actin”. RESULTS In the preliminary results, 9 carcinomas were identified, and three sub-categories were characterized: Verrucous carcinoma, Metastatic Squamous cell carcinoma and Non-Metastatic Squamous cell carcinoma. Higher expression levels of some LncRNA's, such as HOTAIR, were detected in tumor tissue, when we compared the samples of noncancerous matched tissue (CTR) with neoplastic tissue (K). It is possible to highlight a significant considerably alteration of the expression profile of 5 lncRNA in the neoplastic tissue associated with the mOSCC sample, and in particular LINC00312, NAMA, PRNCR1, TUSC7, XIST X compared to control tissues. As regards the nmOSCC group, an alteration of the expression profile of: GNAS-AS1, HOTAIR, HOTTIP, LINC01233, LSINCT5, PCAT1, PRNCR1, SPRY4-IT1, TERC was found. Regarding the last OVC group, the following have been identified as regulated: AIRN, GACAT1, HIF1A-AS1. The use of multivariate analysis in comparing the pattern of expression among groups: mOSCC, nmOSCC, OVC, associated with the use of both one and two components, found a good differentiation among the three groups in terms of expression. The two-component analysis made it possible to infer a ranking of distancing and maximization of variance, obtaining a good separation of the groups, with 45% of the total variance being described by the two main components. CONCLUSION We have turned our attention to the differential study of the expression profile of some possible tumor biomarkers between different subtypes of oral malignant neoplasms, associated with different behaviours, compared to healthy control tissues, to allow possible correlations on a diagnostic and possibly clinical level. The processing of the data emerging from the molecular biology analysis with RT PCR, in multivariate analysis with two-component variance maximization, demonstrated a wide variety of profiles between the samples under examination (mOSCC, nmOSCC, OVC) and - more generally - each group is characterized by the dysregulation of several lncRNAs. In the group of OSCC associated with metastases, the lncRNAs LINC00312, PRNCR1, XIST X, could represent, given their characteristic oncogenic action found in numerous other neoplasms, possible early indicators of an evolution in a metastatic sense, with possible implications in terms of staging. As regards the group of OSCCs not associated with metastases, lncRNA: HOTAIR, PCAT1, SPRY4-IT1, were found as possible biomarkers. HOTAIR and PCAT1 have emerged as important oncogenes remarkably expressed in several squamous tumoral cells of the head and neck district (HNSCC). They could also be configured as prognostic indicators at the oral level, even in cases not necessarily associated with metastatic spread, as demonstrated in our study. SPRY4-IT1 lncRNA, overexpressed in the group of oral cancer not associated with metastatic spread, has been studied in the gastroenterological field for its onco- suppressive action against gastric cancer. Reduced expression of this lncRNA, in fact, has been associated with larger tumoral size, advanced disease stage, greater depth of invasion and lymphatic metastases. Ultimately, in the OVC group, a reduced expression of GACAT1 was measured, which could significantly explain the reduced proliferation, invasion, and cell migration, all of them being characteristics of this histotype. The preventive evaluation of LncRNA’s expression, and a better understanding of their role, could give a more thorough perspective on the future treatment options for this cancer type. 1. Global Burden of Disease Cancer Collaboration. Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disabilityadjusted life-years for 32 cancer groups, 1990 to 2015: a systematic analysis for the global burden of disease study. JAMA Oncol. 2017;3(4):524–548 2. Vigneswaran N, Williams MD. 2014. Epidemiologic trends in head and neck cancer and aids in diagnosis. Oral Maxillofac Surg Clin North Am. 26(2):123–141. 3. Abu-Ghanem S, Yehuda M, Carmel NN, Leshno M, Abergel A, Gutfeld O, Fliss DM. Elective Neck Dissection vs Observation in Early-Stage Squamous Cell Carcinoma of the Oral Tongue With No Clinically Apparent Lymph Node Metastasis in the Neck: A Systematic Review and Meta-analysis. JAMA Otolaryngol Head Neck Surg. 2016; 142:857–865. 4. Hema KN, Smitha T, Sheethal HS, Mirnalini SA. 2017. Epigenetics in oral squamous cell carcinoma. J Oral Maxillofac Pathol. 21(2):252–259. 5. Le Campion ACOV, Ribeiro CMB, Luiz RR, da Silva Júnior FF, Barros HCS, Dos Santos KCB, Ferreira SJ, Gonçalves LS, Ferreira SMS. 2017. Low survival rates of oral and oropharyngeal squamous cell carcinoma. Int J Dent. 2017:5815493. 6. Bitu CC, Destro MF, Carrera M, da Silva SD, Graner E, Kowalski LP, Soares FA, Coletta RD. 2012. Hoxa1 is overexpressed in oral squamous cell carcinomas and its expression is correlated with poor prognosis. BMC Cancer. 12:146
LncRNA, oral squamous cell carcinoma, OSCC
Analisi del profilo di espressione di lncRNA nel carcinoma a cellule squamose del cavo orale / Fabio Croveri - : . , 2020. ((33. ciclo, Anno Accademico 2019/2020.
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