DNA methylation (DNAm) has been linked to changes in chromatin structure, gene expression and disease. The DNAm level can be affected by genetic variation; although, how this differs by CpG ...dinucleotide density and genic location of the DNAm site is not well understood. Moreover, the effect of disease causing variants on the DNAm level in a tissue relevant to disease has yet to be fully elucidated. To this end, we investigated the phenotypic profiles, genetic effects and regional genomic heritability for 196080 DNAm sites in healthy colorectum tissue from 132 unrelated Colombian individuals. DNAm sites in regions of low-CpG density were more variable, on average more methylated and were more likely to be significantly heritable when compared with DNAm sites in regions of high-CpG density. DNAm sites located in intergenic regions had a higher mean DNAm level and were more likely to be heritable when compared with DNAm sites in the transcription start site (TSS) of a gene expressed in colon tissue. Within CpG-dense regions, the propensity of the DNAm level to be heritable was lower in the TSS of genes expressed in colon tissue than in the TSS of genes not expressed in colon tissue. In addition, regional genetic variation was associated with variation in local DNAm level no more frequently for DNAm sites within colorectal cancer risk regions than it was for DNAm sites outside such regions. Overall, DNAm sites located in different genomic contexts exhibited distinguishable profiles and may have a different biological function.
Circulating tumor cell (CTC) enumeration is important clinically for identifying prognostic and predictive factors in patients with solid cancers. The CellSearch device (Veridex) is an immunomagnetic ...CTC selection and enumeration system used in clinical practice. The ImageStream (Amnis) combines the strengths of flow cytometry and fluorescent microscopy in a single platform and has potential application for CTC counting. The performance in CTC enumeration was compared between the ImageStream and CellSearch systems.
Various numbers of PANC‐1 tumor cells were spiked into 7.5 mL of peripheral blood from a healthy donor. Before cell analysis by the ImageStream, tumor cell enrichment was performed by immunomagnetic selection with anti‐EpPCAM. Anti‐CD45 and anti‐CK markers were used to discriminate between tumor cells and leukocytes. The ratios of tumor cells recovered from each dilution were calculated for both methods. The Wilcoxon rank test was applied to compare the results of the two methods and the reference value.
The results of the two tested methods differed significantly from the reference value, but did not differ between them. Nevertheless, lower level of trueness and precision was observed in ImageStream when fewer numbers of CTCs were analyzed.
Our results suggest that ImageStream platform for CTC enumeration has a potential value for the early diagnosis of disseminated disease, but needs an improvement of precision for the enumeration of low number of CTC.
The standard genetic test for Lynch syndrome (LS) frequently reveals an absence of pathogenic mutations in DNA mismatch repair genes known to be associated with LS. It was recently shown that germ ...line deletions in the last exons of EPCAM are involved in the etiology of LS. The aim of this study was to evaluate the prevalence of EPCAM deletions in a Spanish population and the clinical implications of deletion. Probands from 501 families suspected of having LS were enrolled in the study. Twenty-five cases with MSH2 loss were identified: 10 had mutations of MSH2 , five had mutations of MSH6 , and 10 did not show MSH2/MSH6 mutations. These 25 cases were analyzed for EPCAM deletions using multiplex ligation-dependent probe amplification, and deletions were mapped using long-range PCR analysis. One subject with no MSH2/MSH6 mutations had a large deletion in the EPCAM locus that extended for 8.7 kb and included exons 8 and 9. The tumor exhibited MSH2 promoter hypermethylation. EPCAM deletion analysis followed by MSH2 methylation testing of the tumor is a fast low-cost procedure that can be used to identify mutations that cause LS. We propose that this procedure be incorporated into clinical genetic analysis strategies and present a decision-support flow diagram for the diagnosis of LS.
Colorectal carcinoma is a common cause of cancer. Adjuvant treatments include: 5-fluorouracil administered together with folinic acid, or more recently, oral fluoropyrimidines such as capecitabine, ...in combination with oxaliplatin or irinotecan. Metastatic colorectal cancer patients can benefit from other additional treatments such as cetuximab or bevacizumab.
Using cell culture techniques, we isolated clonal populations from primary cultures of ascitic effusion derived from a colon cancer patient and after several passages an established cell line, HGUE-C-1, was obtained. Genetic analysis of HGUE-C-1 cells was performed by PCR of selected exons and sequencing. Cell proliferation studies were performed by MTT assays and cell cycle analyses were performed by flow cytometry. Retinoblastoma activity was measured by luciferase assays and proteins levels and activity were analysed by Western blot or immunohistochemistry.
We have established a new cell line from ascitic efussion of a colon cancer patient who did not respond to 5-fluorouracil or irinotecan. HGUE-C-1 cells did not show microsatellite instability and did not harbour mutations in KRAS, BRAF, PI3KCA or TP53. However, these cells showed loss of heterozygosity affecting Adenomatous Polyposis Coli and nuclear staining of β-catenin protein. The HGUE-C-1 cell line was sensitive to erlotinib, gefitinib, NVP-BEZ235, rapamycin and trichostatin, among other drugs, but partially resistant to heat shock protein inhibitors and highly resistant to AZD-6244 and oxaliplatin, even though the patient from which this cell line was derived had not been exposed to these drugs. Molecular characterization of this cell line revealed low expression levels and activity of Retinoblastoma protein and elevated basal levels of Erk1/2 activity and p70S6K expression and activity, which may be related to chemoresistance mechanisms.
HGUE-C-1 represents a novel and peculiar colon carcinoma model to study chemoresistance to chemotherapeutic agents and to novel anti-neoplasic drugs that interrupt signalling pathways such as the APC/βcatenin, Ras/Raf/Mek/Erk, PI3K/mTOR/p70S6K pathways as well as histone regulation mechanisms.
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