This work was supported by Centro Grandi Strumenti Universit&#224; dell&#39;Insubria, Fondazione Comunitaria del Varesotto, Fondazione del Monte di Lombardia, and Fondazione Anna Villa e Felice Rusconi.

All the studies were performed according the Declaration of Helsinki (1975) with written informed consent and approved by the Ethical Committee of the University of Insubria in Varese.
1. Collection of the Samples
<ol>
	<li>Collect and divide all the human Formalin-Fixed Paraffin-Embedded (FFPE) samples into different subgroups according the WHO classification of Head and Neck Tumors14. 
	NOTE: Here we used the following samples: Normal sinonasal...

<ol>
	<li>Boncinelli, E., Simeone, A., Acampora, D., Gulisano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeobox+genes+in+the+developing+central+nervous+system.">Homeobox genes in the developing central nervous system.</a> Ann genet. 36 (1), 30-37 (1993).</li><li>Omodei, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+Brain+Transcription+Factor+OTX1+Occurs+in+a+Subset+of+Normal+Germinal-Center+B+Cells+and+in+Aggressive+Non-Hodgkin+Lymphoma.">Expression of the Brain Transcription Factor OTX1 Occurs in a Subset of Normal Germinal-Center B Cells and in Aggressive Non-Hodgkin Lymphoma.</a> American J. Pathol. 175, 2609-2617 (2009).</li><li>Levantini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unsuspected+role+of+the+brain+morphogenetic+gene+Otx1+in+hematopoiesis.">Unsuspected role of the brain morphogenetic gene Otx1 in hematopoiesis.</a> Proc. Natl. Acad. Sci USA. 100 (18), 10299-10303 (2003).</li><li>Larsen, K. B., Lutterodt, M. C., Mollgard, K., Moller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+homeobox+OTX2+and+OTX1+in+the+early+developing+human+brain.">Expression of the homeobox OTX2 and OTX1 in the early developing human brain.</a> J. Histochem. Cytochem. 58, 669-678 (2010).</li><li>Abate-Shen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deregulated+homeobox+gene+expression+in+cancer:+cause+or+consequence?.">Deregulated homeobox gene expression in cancer: cause or consequence?.</a> Nat. Rev. Cancer. 2, 777-785 (2002).</li><li>de Haas, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OTX1+and+OTX2+expression+correlates+with+the+clinicopathologic+classification+of+medulloblastomas.">OTX1 and OTX2 expression correlates with the clinicopathologic classification of medulloblastomas.</a> J. Neuropathol. Exp. Neurol. 65, 176-186 (2006).</li><li>Terrinoni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OTX1+expression+in+breast+cancer+is+regulated+by+p53.">OTX1 expression in breast cancer is regulated by p53.</a> Oncogene. 30 (27), 3096-3103 (2001).</li><li>Yu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OTX1+promotes+colorectal+cancer+progression+through+epithelial-mesenchymal+transition.">OTX1 promotes colorectal cancer progression through epithelial-mesenchymal transition.</a> Biochem. Biophys Res Commun. 44 (1), 1-5 (2014).</li><li>Glubrecht, D. D., Kim, J. H., Russel, L., Bamforth, J. S., Godbout, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+CRX+and+OTX2+expression+in+human+retina+and+retinoblastoma.">Differential CRX and OTX2 expression in human retina and retinoblastoma.</a> J. Neurochem. 111 (1), 250-263 (2009).</li><li>Coletta, R. D., Jedlicka, P., Gutierrez-Hartamn, A., Ford, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+the+cell+cycle+in+mammary+gland+development+and+tumorigenesis.">Transcriptional control of the cell cycle in mammary gland development and tumorigenesis.</a> J. mammary gland Biol. Neoplasia. 9, 39-53 (2004).</li><li>Cordes, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+phenotypic+analysis+of+poorly+differentiated+sinonasal+neoplasms:+an+integrated+approach+for+early+diagnosis+and+classification.">Molecular and phenotypic analysis of poorly differentiated sinonasal neoplasms: an integrated approach for early diagnosis and classification.</a> Hum Pathol. 40, 283-292 (2009).</li><li>Stelow, E. B., Bishop, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+from+the+4th+Edition+of+the+World+Health+Organization+Classification+of+Head+and+Neck+Tumours:+Tumors+of+the+Nasal+Cavity,+Paranasal+Sinuses+and+Skull+Base.">Update from the 4th Edition of the World Health Organization Classification of Head and Neck Tumours: Tumors of the Nasal Cavity, Paranasal Sinuses and Skull Base.</a> Head Neck Pathol. 11 (1), 3-15 (2017).</li><li>Llorente, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinonasal+carcinoma:+clinical,+pathological,+genetic+and+therapeutic+advances.">Sinonasal carcinoma: clinical, pathological, genetic and therapeutic advances.</a> Nat. Rev. Clin. Oncol. 11 (8), 460-472 (2014).</li><li>Sidransky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World health organization classification of tumors. Pathology and genetics of head and neck tumors. 9, (2005).</li><li>Radulesku, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+surgery+for+sinonasal+tumors:+The+transcribriform+approach.">Endoscopic surgery for sinonasal tumors: The transcribriform approach.</a> J Stomatol Oral Maxillofac Surg. 118 (4), 248-250 (2017).</li><li>Muller, P. A. J., Vousden, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+mutations+in+cancer.">p53 mutations in cancer.</a> Nature cell biology. 15 (1), 2-8 (2013).</li><li>Holmila, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profile+of+TP53+gene+mutations+in+sinonasal+cancer.">Profile of TP53 gene mutations in sinonasal cancer.</a> Mutat Res. 686 (1-2), 9-14 (2010).</li></ol>

This study shows for the first time that, based on mRNA levels, the HB genes OTX1 and OTX2 are expressed in normal sinonasal mucosa and submucosal glands, inflammatory polyps, sinonasal Schneiderian papillomas, and in the different epithelial and neuroectodermal neoplasms, including squamous carcinomas, non-intestinal type sinonasal adenocarcinomas, salivary gland-type tumors, neuroendocrine neoplasms, and ONs.
Modifications and Troubleshooting:
To avoid RNA degradatio...

OTX HB genes are the vertebrate homologue of the Drosophila orthodenticle genes (otd) and they encode for transcription factors which are normally expressed during embryonic morphogenesis, but they can also be expressed in the adult organism with different functions. During embryonic development they control the specification of cell identity, cell differentiation, and the positioning of the body axis&#185;. The OTX family includes OTX1 and OTX2 genes which display different functions. OTX1 is involved in brain and sensory organ development. In the adult organism, it is expressed in sensory organs and is transcribed at low levels in the anterior lobe of the pituitary gland2; it also plays a role in hematopoiesis, being expressed in hematopoietic pluripotent and progenitor cells3. OTX2 is involved in the development of the rostral head and its translated protein acts as a morphogen because it generates a gradient through which other genes are activated or repressed in a spatio-temporal manner, thus contributing to cell proliferation and differentiation. In the adult organism, OTX2 is found exclusively in the choroid plexus and pineal gland4.
Mutations in OTX genes are often related to the appearance of human congenital, somatic, or metabolic defects. Gain or loss mutations in OTX genes could promote tumorigenesis if they are not able to properly control cellular growth and/or differentiation5. In leukemias and lymphomas as well as in many solid tumors (e.g., medulloblastomas6, aggressive non-Hodgkin lymphomas2, breast carcinomas7, colorectal cancers8, and retinoblastoma9), the deregulated expression of OTX HB genes is well documented10. In addition, OTX2 mutations have been demonstrated in cases of anophthalmia and microphtalmia11 due to the crucial role for this gene in the control of eye development.
In the context of solid neoplasms, the discovery of molecular and phenotypic markers is an important challenge for the diagnosis, classification, and treatment of several types of tumor11, including those that originate in the nasal cavity and paranasal sinuses. In fact, despite that these areas occupy only a modest anatomical space, mucosal epithelium, glands, soft tissues, bone, cartilage or neural/neuroectodermal, and hematolymphoid cells can be often the site for the origin of complex and histologically different groups of tumors. Different types of neoplasms involving the sinonasal tract present a variety of features that overcome what is usually seen in the upper aerodigestive tract or even throughout most parts of the body12.Sinonasal malignancies are rare and present an annual incidence of 1:100,000 inhabitants worldwide, and so this prevents studies regarding the pathways involved in the tumorigenesis and the testing of alternative treatment strategies.Despite this, the advances in imaging techniques, surgical approaches, and radiotherapy have improved the clinical management of sinonasal cancer.Moreover, the development of cell lines as well as animal models and cancer genetic profiling currently constitute the basis for the future targeted anticancer therapies13. To date, there are no reports regarding OTX1 and/or OTX2 expression in neoplasms of the nasal cavity, paranasal sinuses, and nasopharynx. Since we have previously observed that OTX1 and OTX2 are involved in breast cancer7, we wondered if these genes could be present not only in the normal nasal mucosa but also in tumors of the nasal cavity. To reach this goal we obtained from the Department of Pathology of the &#34;Ospedale di Circolo&#34; in Varese samples of normal mucosa, and nasal and sinonasal adenocarcinomas collected from 1985 to 2012 and classified according to the World Health Organization (WHO) classification of Head and Neck Tumors. We choose to analyze them through real-time PCR and immunohistochemistry analyses and we evaluated OTX1 and OTX2 expression to determine if they can be considered molecular markers for these types of tumors.

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			<td height="42" style="height:42px;width:216px;">3,3&#39;-diaminobenzidine tetrahydrocloride (DAB)</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5905</td>
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			<td height="21" style="height:21px;width:216px;">Harris Hematoxylin</td>
			<td style="width:140px;">Bioptica</td>
			<td style="width:119px;">0506004/L</td>
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			<td height="21" style="height:21px;width:216px;">Pertek</td>
			<td style="width:140px;">Kaltek SRL</td>
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identification of otx1 and otx2 as two possible molecular markers for sinonasal carcinomas and olfactory neuroblastomas

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations occurring in these genes are often related to tumorigenesis in human. No data are available today regarding the possible correlation between OTX genes and tumors of the nasal cavity. The aim of this work is to understand if OTX1 and OTX2 can be considered as molecular markers in the development of nasal tumors. We selected nasal and sinonasal adenocarcinomas to investigate the expression of OTX1 and OTX2 genes through immunohistochemical and real-time PCR analyses.Both OTX1 and OTX2 were absent in all the samples of sinonasal Intestinal-Type Adenocarcinomas (ITACs). OTX1 mRNA was identified only in Non-Intestinal Type Adenocarcinomas (NITACs) while OTX2 mRNA was expressed only in Olfactory Neuroblastomas (ONs). We have demonstrated that the differential gene expression for both OTX1 and OTX2 genes might be a useful molecular marker to distinguish the different types of sinonasal tumors.

In the normal mucosa we observed strong and homogenous nuclear reactivity for OTX genes both in the ciliated pseudostratified respiratory-type epithelium and in the submucosal glandular cells (Figure 1A). We found nuclear expression for OTX1 in all NITACs samples (Figure 1B), while little or absent immunoreactivity was highlighted in ITACs (Figure 1C). Intense immunoreactivity was present in all ONs ...

Watch this Scientific Journal Video about Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas at JoVE.com

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas

Homeobox genes are regulatory genes often associated with tumors in the adult organisms. We investigated their comparative expression by immunohistochemical and real-time PCR analysis, in normal and inflammatory nasal mucosae and in sinonasal neoplasms in order to use them as possible diagnostic and therapeutic targets.

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations ...

The overall goal of this experiment is to identify useful molecular markers for sinonasal carcinomas and olfactory neuroblastomas in a simple and efficient way. This method can help answer key question in the field of olfactory neuroblastoma and sinonasal carcinoma, such as the identification of molecular markers to discriminate tumor subtype. The main advantage of this technique is that we can obtain result in less than six hour, or within a day for the hospital report.Demonstrating the procedure of RNA extraction and real-time PCR will be Dr.Giorgia Millefanti, a PhD student from my lab. And demonstrating the procedure of immunohistochemistry will be Giovanni Micheloni, also a PhD student from my lab. To begin the procedure, collect and divide all the human FFPE samples into different sub-groups, according to the WHO classification of head and neck tumors.Heat the samples slides in an oven at 60 degrees Celsius for 30 minutes. Next, rehydrate the three micrometer thick FFPE sections by briefly washing them in xylene for 10 minutes. Then, wash the slides in 100%95%85%and 75%alcohol serially for five minutes each wash.Rinse the slides for five minutes in distilled water. Subsequently, block the endogenous activity by placing the slides in 3%aqueous hydrogen peroxide for 12 minutes. Afterward, perform antigen-retrieval by treating the slides with 10 millimolar citrate buffer, and microwaving for 10 minutes.After 10 minutes, wash the sections in TBS buffer. Then, add 0.2%of Triton X, and incubate them overnight at four degrees Celsius, with Goat Anti-Human OTX2 Antibody, diluted at a 1 to 100 ratio. The next day, add biotinylated rabbit anti-goat secondary antibody, diluted at 1 to 200, and incubate the sections for one hour at room temperature.Then, treat the samples with the ABC peroxidase complex. Afterward, develop the immunoreaction using diaminobenzene tetrahydrochloride, and counter stain the nuclei with Harris hematoxylin. Then, dehydrate the sections in a crescent alcohol scale, and clarify them in a clearing substance of turpine origin.Next, mount the sections on a slide with mounting medium. After RNA extraction and reverse transcription, perform quantitative real time PCR analysis with probe-based technology and a thermal cycler. Prepare the PCR reaction mix using 12.5 microliters of probe-based master mix, 1.25 microliters of each OTX1, OTX2, and ACTB probes, 50 nanograms of CDNA, and nuclease-free water up to 25 microliters of total volume.Perform all the reactions in triplicate using ACTB gene as the endogenous control to normalize gene expression levels. Next, centrifuge the plate at 1109 times gravity for three minutes. Then store the plate protected from light at four degrees Celsius, until the experiment.To perform real time PCR, place the samples in the machine. Then, set the thermal cycler profile with an initial hot start cycle at 50 degrees Celsius for two minutes, and 95 degrees Celsius for 10 minutes, followed by 40 cycles at 95 degrees Celsius for 15 seconds and a final cycle at 60 degrees Celsius for one minute. For gene expression level analysis, normalize the gene expression levels through the comparative cycle threshold method using ACTB gene as the endogenous control.Evaluate the gene expression levels using the comparative cycle threshold method, and plot the results. Then, perform statistical analysis using student's t-test, considering statistically significant results with p<0.05. In the normal mucosa, strong and homogenous nuclear reactivity for OTX genes both in ciliated pseudostratified respiratory type epithelium and in the submucosal glandular cells were observed.Nuclear expression for OTX1 was found in all non-intestinal type adenocarcinoma samples, while little or absent immunoreactivity was highlighted in intestinal type adenocarcinomas. Intense immunoreactivity was present in all olfactory neuroblastomas. Among all poorly differentiated neuro-endocrine carcinomas, OTX expression varied in intensity and percentage of positive cells.While attempting this procedure, it is important to remember to perform every single step of the RNA extraction in sterile conditions using also sterile equipment. In parallel with this procedure, other methods like x-ray analysis, endoscopic exam of nasal cavity, CT scan, magnetic resonance imaging, and biopsy, can be performed in order to confirm the results obtained from our technique. After its development, this technique paved the way for researchers in the field of oncology to explore this disease in cell models.After watching this video, you should have a good understanding of how to identify molecular markers to discriminate tumor subtypes using the immunohistochemistry and real time PCR. Don't forget that working with these reagents, instrumentation, and samples can be extremely hazardous and precautions such as DPI should always be taken when performing these procedure.

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Cancer Research

5.7K vistas.  University of Insubria. El objetivo general de este experimento es identificar marcadores moleculares útiles para los carcinomas sinonasales y los neuroblastomas olfativos de una manera simple y eficiente. Este método puede ayudar a responder a preguntas clave en el campo del neuroblastoma olfativo y el carcinoma sinonasal, como la identificación de marcadores moleculares para discriminar el subtipo tumoral. La principal ventaja de esta técnica es que podemos obtener resultado en menos de seis horas, o dentro de...