Maja Šutić1, Jurica Baranašić1, Lana Kovač Bilić2, Mario Bilić2, Antonija Jakovčević3, Luka Brčić4, Sven Seiwerth3, Marko Jakopović5, Miroslav Samaržija5, Ulrich Zechner6, Jelena Knežević7,8. 1. Division of Molecular Medicine, Laboratory for Advanced Genomics, Ruđer Bošković Institute, Zagreb, Croatia. 2. Department of Otorhinolaryngology, Head and Neck Surgery, Clinical Hospital Centre Zagreb, School of Medicine, University of Zagreb, Zagreb, Croatia. 3. Department of Pathology, School of Medicine, University of Zagreb, Zagreb, Croatia. 4. Diagnostic and Research Institute of Pathology, Medical University of Graz, Graz, Austria. 5. Department for Respiratory Diseases, Clinic for Respiratory Diseases Jordanovac, University of Zagreb, School of Medicine, University Hospital Centre Zagreb, Zagreb, Croatia. 6. Institute for Human Genetics, Johannes Gutenberg-University Mainz, Mainz, Germany. 7. Division of Molecular Medicine, Laboratory for Advanced Genomics, Ruđer Bošković Institute, Zagreb, Croatia. jknezev@irb.hr. 8. Faculty for Dental Medicine and Health, University of Osijek, Osijek, Croatia. jknezev@irb.hr.
Abstract
BACKGROUND: Patients with head and neck squamous cell carcinoma (HNSCC) can develop lung squamous cell carcinoma (LuSCC), which could be the second primary tumor or HNSCC metastasis. Morphologically it is difficult to distinguish metastatic HNSCC from a second primary tumor which presents a significant diagnostic challenge. Differentiation of those two malignancies is important because the recommended treatments for metastatic HNSCC and primary LuSCC differ significantly. We investigated if the quantification of the promotor methylation status in HNSCC and LuSCC differs. METHODS: Primary HNSCC (N = 36) and LuSCC (N = 17) were included in this study. Methylation status in the ASC/TMS1/PYCARD (apoptosis-associated speck-like protein containing a caspase recruitment domain; 8 CpG sites) and MyD88 (Myeloid differentiation primary response protein 88; 10 CpG sites) promoters was analyzed. Bisulfite converted DNA, isolated from tumor tissue was quantified using pyrosequencing. Results of pyrosequencing analysis were expressed as a percentage for each tested CpG site. Receiver-operating characteristic (ROC) curve analysis was used for the evaluation of the diagnostic properties of selected biomarkers. RESULTS: CpG sites located in the promoters of ASC/TMS1/PYCARD_CpG8 (- 65 upstream) and MyD88_CpG4 (- 278 upstream) are significantly hypermethylated in the HNSCC when compared with LuSCC (p ≤ 0.0001). By performing ROC curve analysis we showed that corresponding areas under the curve (AUC) were 85-95%, indicating that selected CpG sites are useful for a distinction between primary LuSCC and primary HNSCC. CONCLUSIONS: Results of the present study indicate that there is a significant difference in the methylation status of tested genes between primary HNSCC and LuSCC. However, to prove this approach as a useful tool for distinguishing second primary LuSCC from HNSCC metastasis, it would be necessary to include a larger number of samples, and most importantly, metastatic samples.
BACKGROUND:Patients with head and neck squamous cell carcinoma (HNSCC) can develop lung squamous cell carcinoma (LuSCC), which could be the second primary tumor or HNSCC metastasis. Morphologically it is difficult to distinguish metastatic HNSCC from a second primary tumor which presents a significant diagnostic challenge. Differentiation of those two malignancies is important because the recommended treatments for metastatic HNSCC and primary LuSCC differ significantly. We investigated if the quantification of the promotor methylation status in HNSCC and LuSCC differs. METHODS: Primary HNSCC (N = 36) and LuSCC (N = 17) were included in this study. Methylation status in the ASC/TMS1/PYCARD (apoptosis-associated speck-like protein containing a caspase recruitment domain; 8 CpG sites) and MyD88 (Myeloid differentiation primary response protein 88; 10 CpG sites) promoters was analyzed. Bisulfite converted DNA, isolated from tumor tissue was quantified using pyrosequencing. Results of pyrosequencing analysis were expressed as a percentage for each tested CpG site. Receiver-operating characteristic (ROC) curve analysis was used for the evaluation of the diagnostic properties of selected biomarkers. RESULTS: CpG sites located in the promoters of ASC/TMS1/PYCARD_CpG8 (- 65 upstream) and MyD88_CpG4 (- 278 upstream) are significantly hypermethylated in the HNSCC when compared with LuSCC (p ≤ 0.0001). By performing ROC curve analysis we showed that corresponding areas under the curve (AUC) were 85-95%, indicating that selected CpG sites are useful for a distinction between primary LuSCC and primary HNSCC. CONCLUSIONS: Results of the present study indicate that there is a significant difference in the methylation status of tested genes between primary HNSCC and LuSCC. However, to prove this approach as a useful tool for distinguishing second primary LuSCC from HNSCC metastasis, it would be necessary to include a larger number of samples, and most importantly, metastatic samples.
Entities:
Keywords:
CpG; Diagnostic biomarker; HNSCC; Lung; Methylation; Second primary tumor
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