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SOX17 Promoter Methylation in Circulating Tumor Cells and Matched Cell-Free DNA Isolated from Plasma of Patients with Breast Cancer M. Chimonidou, A. Strati, N. Malamos, V. Georgoulias, and E.S. Lianidou January 2013 www.clinchem.org/content/59/1/270.full © Copyright 2013 by the American Association for Clinical Chemistry © Copyright 2009 by the American Association for Clinical Chemistry Introduction Circulating Tumor Cells (CTCs) •CTCs play a critical role in the metastatic spread of carcinomas and their detection is associated with prognosis in many human cancers, while their enumeration has been cleared by the FDA for follow up of breast, colon, and prostate cancer patients with verified metastasis. •CTCs represent a promising new diagnostic tool, especially for advanced-stage cancer patients where they can be used as a “liquid biopsy,” allowing physicians to follow cancer changes over time and tailor treatment accordingly. •However, it is quite clear now that simple enumeration of CTCs is not enough. •CTC molecular characterization is very important since it can play a crucial role in understanding the biology of metastasis and in selecting patients for targeted therapy. © Copyright 2009 by the American Association for Clinical Chemistry Introduction Cell free DNA (cfDNA) •cfDNA circulates in plasma of patients with cancer at increased concentrations. •Many teams have focused on the development of assays that allow the specific detection of small amounts of tumor specific cfDNA in the peripheral blood of patients with cancer. •The detection of tumor specific DNA alterations such as mutations and methylation in cfDNA provides a less invasive, more easily accessible source of DNA for genetic analysis than tumor biopsies. •Several studies have described methylation of tumor suppressor genes in serum or plasma samples and in the corresponding primary breast tumors, while DNA methylation was not detected in plasma or serum of healthy donors. © Copyright 2009 by the American Association for Clinical Chemistry Introduction SOX17 •Is a member of the SOX (Sry-related high mobility group box) family of transcription factors. •Plays a critical role in the regulation of development and stem/precursor cell function partly through repression of the canonical Wnt/beta-catenin signaling pathway. •Global analysis of CpG island hypermethylation and gene expression in cancer cell lines revealed that SOX17 gene silencing is associated with DNA hypermethylation of a CpG island located in the promoter region •The SOX17 promoter is methylated in CTCs isolated from peripheral blood of patients with breast cancer. © Copyright 2009 by the American Association for Clinical Chemistry Question: Is there a direct connection between the presence of CTCs and cell free DNA in patients with operable breast cancer where the primary tumor has already been resected? To address this question, the authors chose to use the same marker and the same methodology in matched clinical samples. The authors evaluated whether SOX17 promoter methylation in CTCs was associated with the methylation pattern of this gene in matched cfDNA isolated from plasma of patients with breast cancer. © Copyright 2009 by the American Association for Clinical Chemistry Early breast cancer (N=55) Figure 1. Schematic diagram of the workflow of the study. Advanced breast cancer (verified metastasis) (N=59) Healthy Individuals (N=43) 21 ml peripheral blood in EDTA 20 mL: Isolation of PBMC’s by density gradient centrifugation (ficol) 1 mL: plasma separation Positive selection of CTC by immunomagnetic separation (EpCAM) Isolation of cfDNA from plasma (200μL) CTC fraction Isolation of total RNA (Trizol) Isolation of mRNA (oligo-dTbeads) cDNA synthesis RTq-PCR for CK-19 Isolation of genomic DNA (Trizol) SB Conversion of genomic DNA Real time MSP for SOX17 © Copyright 2009 by the American Association for Clinical Chemistry 20 mL Peripheral blood Methodology PBMCs Ficoll gradient Cell count CTCs isolation CellFreeDNA isolation Plasma DNA extraction From CTCs Positive selection (EpCAM) Apply magnet Figure 2. Outline of the extraction of cell free DNA and CTCs. © Copyright 2009 by the American Association for Clinical Chemistry Methodology Methylation Specific PCR (MSP) MSP is a bisulfite conversion based PCR technique for the study of DNA CpG methylation. Sodium bisulfite converts all unmethylated, but not methylated, cytosines to uracil. © Copyright 2009 by the American Association for Clinical Chemistry How were the methylation specific primers and probes designed? © Copyright 2009 by the American Association for Clinical Chemistry Methodology Primer design for MSP For maximal discrimination between methylated and unmethylated alleles, primers should contain at least one CpG site at the most 3’-end. GATUUTGATTGC Other than CpG sites at the most 3’ end, more CpG sites in primer sequences are preferred. Both MSP primers and probe should contain T bases derived from modified unmethylated C regions so as to discriminate and amplify the converted from unconverted DNA. © Copyright 2009 by the American Association for Clinical Chemistry Specificity of the SOX17 MSP assay Bisulfite treatment of genomic DNA MSP using methylated specific primers and probe Real time Methylation Specific PCR 100%methylated control 50% methylated 1%methylated 0%methyaled © Copyright 2009 by the American Association for Clinical Chemistry Results- characteristic graphs Realtime-MSP for SOX17 using DNA isolated from CTCs fraction from: a) healthy donors b) operable breast cancer c) verified metastasis Realtime-MSP for SOX17 using DNA isolated from cfDNA from: a) healthy donors b) operable breast cancer c) 2009 verified metastasis © Copyright by the American Association for Clinical Chemistry RESULTS Heat map of SOX17 promoter methylation in matched samples: CTCs Fraction and cfDNA of patients with: (a) operable breast cancer (n=55), (b) verified metastasis (n=59) In parallel, the expression of the epithelial marker CK-19 is shown in CTCs fraction Early breast cancer (n=55) CTCs SOX17 CELL FREE DNA CK19 Verified metastasisis (n=59) CTCs SOX17 CELL FREE DNA CK19 Methylated SOX17 Unmethylated samples © Copyright 2009 by the American Association for Clinical Chemistry Operable breast cancer cell free DNA (N=55) meth SOX17 unmeth SOX17 Total meth SOX17 11 8 19 unmeth SOX17 8 28 36 Total 19 36 55 CTC fraction Table 1. Outline of the extraction of cell free DNA and CTCs. Agreement 39 of 55 (70.9%), P*=0.008 Verified Metastasis cell free DNA (n=59) meth SOX17 unmeth SOX17 Total meth SOX17 13 14 27 unmeth SOX17 11 21 32 Total 24 35 59 CTC fraction Agreement2009 34 ofby 59 the (57.6%), P*=0.283 © Copyright American Association for Clinical Chemistry Question Why is SOX17 promoter methylation in CTCs and in matched cfDNA highly correlated in primary breast cancer in contrast to verified metastasis? © Copyright 2009 by the American Association for Clinical Chemistry Discussion • SOX17 promoter methylation in CTCs and in matched cfDNA is highly correlated in early breast cancer but not in metastasis. • This finding leads toward a direct connection between the presence of CTCs and cfDNA in patients with operable breast cancer after surgical removal of the primary tumor. • In the group of patients with verified metastasis no such connection was observed, even if in many cases there was a concordance between SOX17 methylation in CTCs and cfDNA. • This could possibly be due to the fact that in this case the metastasis is already present and cfDNA can be also released from apoptotic cells escaping from the metastatic site. © Copyright 2009 by the American Association for Clinical Chemistry Conclusions • SOX17 promoter methylation in CTCs and in matched cfDNA is highly correlated in early breast cancer but not in metastasis. • This finding leads toward a direct connection between the presence of CTCs and cfDNA in operable breast cancer patients after surgical removal of the primary tumor. • Since this study was prospective, the clinical importance of this finding must be evaluated further, when the clinical outcome of these early breast cancer patients is known. © Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at www.clinchem.org Follow us © Copyright 2009 by the American Association for Clinical Chemistry