The research project focuses on the analysis of tumor heterogeneity in patients with carcinoma. This evaluation is performed by analyzing tumor tissue samples obtained following tissue biopsy and liquid biopsy. The detection of Circulating Tumor Cells (CTC) and Circulating Tumor DNA (ctDNA) in patients' blood may represent a minimally invasive strategy for the diagnosis, staging and prognosis of various cancers. In a work previously published in 2018, we have demonstrated the existence of a release of CTCs from CRC lung metastases in the pulmonary artery and the existence of a correlation with CTCs isolated from peripheral blood with negative prognostic markers already known in metastatic CRC. Following these preliminary results, we had the opportunity to evaluate epigenetic alterations in CRCs. These epigenetic alterations play a key role in the progression of CRC and can be traced in the serum and plasma of cancer patients. One of the most frequent epigenetic alterations is DNA methylation, which leads to the silencing of gene expression, and which, in tumors, is frequently linked to the regulation of different functions, including cell proliferation, apoptosis, DNA repair. The hyper or hyporegulation of glycosyl transferases is well documented in various types of cancer and plays a central role in the regulation of cell growth, invasiveness, metastasis, and few studies so far have documented its epigenetic alterations. We proceeded with the analysis of the methylation levels of beta-1,4-galactosyltransferase 1 (B4GALT1) in CRC samples. This glycoprotein is a type II membrane glycoprotein that transfers UDP galactose to different acceptors, such as N-acetylglucosamine (GlcNAc). From previous studies, it has been shown that B4GALT1 gene expression is hyporegulated via hypermethylation in primary CRC tissues with a specificity of 91.7% and a sensitivity of 54%, and appears to be a hallmark of the invasive phenotype, being less frequent in the earliest lesions. Methylation levels of B4GALT1 have also been investigated in blood, both in ctDNA and in CTC, and confirmed the data in tissue of primary and metastatic CRC tumor, even when compared with normal mucosa of the same patient. The second part of the project is based on the concept of tumor heterogeneity in primitive tissue, namely the evaluation of tumor heterogeneity in primitive tissue samples preserved in formalin (FFPE). In order to evaluate the heterogeneity at the single cell level through the use of DEPArray and Ampli1, case histories and samples of different carcinomas have been obtained from different external structures. The DNA extracted from the tissue, deriving from FFPE samples collected in different years from 2015 to 2020, have been analyzed using the "QC kit" to determine the quality and integrity of the DNA. DNA integrity is in fact one of the fundamental factors and parameters for this type of analysis. The number of samples that reach the "QC score" suitable for single cell analysis will be investigated to evaluate the clinical application of this type of analysis.

Analysis of tumor eterogeneity in blood and tissue samples / Francesco Picardo - : . , 2021 Jun 21. ((33. ciclo

Analysis of tumor eterogeneity in blood and tissue samples

2021-06-21

Abstract

The research project focuses on the analysis of tumor heterogeneity in patients with carcinoma. This evaluation is performed by analyzing tumor tissue samples obtained following tissue biopsy and liquid biopsy. The detection of Circulating Tumor Cells (CTC) and Circulating Tumor DNA (ctDNA) in patients' blood may represent a minimally invasive strategy for the diagnosis, staging and prognosis of various cancers. In a work previously published in 2018, we have demonstrated the existence of a release of CTCs from CRC lung metastases in the pulmonary artery and the existence of a correlation with CTCs isolated from peripheral blood with negative prognostic markers already known in metastatic CRC. Following these preliminary results, we had the opportunity to evaluate epigenetic alterations in CRCs. These epigenetic alterations play a key role in the progression of CRC and can be traced in the serum and plasma of cancer patients. One of the most frequent epigenetic alterations is DNA methylation, which leads to the silencing of gene expression, and which, in tumors, is frequently linked to the regulation of different functions, including cell proliferation, apoptosis, DNA repair. The hyper or hyporegulation of glycosyl transferases is well documented in various types of cancer and plays a central role in the regulation of cell growth, invasiveness, metastasis, and few studies so far have documented its epigenetic alterations. We proceeded with the analysis of the methylation levels of beta-1,4-galactosyltransferase 1 (B4GALT1) in CRC samples. This glycoprotein is a type II membrane glycoprotein that transfers UDP galactose to different acceptors, such as N-acetylglucosamine (GlcNAc). From previous studies, it has been shown that B4GALT1 gene expression is hyporegulated via hypermethylation in primary CRC tissues with a specificity of 91.7% and a sensitivity of 54%, and appears to be a hallmark of the invasive phenotype, being less frequent in the earliest lesions. Methylation levels of B4GALT1 have also been investigated in blood, both in ctDNA and in CTC, and confirmed the data in tissue of primary and metastatic CRC tumor, even when compared with normal mucosa of the same patient. The second part of the project is based on the concept of tumor heterogeneity in primitive tissue, namely the evaluation of tumor heterogeneity in primitive tissue samples preserved in formalin (FFPE). In order to evaluate the heterogeneity at the single cell level through the use of DEPArray and Ampli1, case histories and samples of different carcinomas have been obtained from different external structures. The DNA extracted from the tissue, deriving from FFPE samples collected in different years from 2015 to 2020, have been analyzed using the "QC kit" to determine the quality and integrity of the DNA. DNA integrity is in fact one of the fundamental factors and parameters for this type of analysis. The number of samples that reach the "QC score" suitable for single cell analysis will be investigated to evaluate the clinical application of this type of analysis.
Liquid Biopsy; Heterogeneity
Analysis of tumor eterogeneity in blood and tissue samples / Francesco Picardo - : . , 2021 Jun 21. ((33. ciclo
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12610/68695
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