Increased DNA Integrity in Colorectal Cancer

Background: Blood-based DNA integrity, defined as the relation of long to small fragments of cell-free circulating DNA, is known to be increased in various types of cancers. Since different DNA fragments and formulae are used by different researchers, conflicting results on the relevance of this marker for cancer diagnosis have been reported. Patients and Methods: Sera from 24 patients with colorectal cancer, 11 patients with benign gastrointestinal diseases, and 24 healthy individuals were investigated. After DNA isolation, ALU repeats with 115 bp and 247 bp length were measured using quantitative polymerase chain reaction and resulting DNA integrities were calculated by the two formulae of Umetani et al. (DNA Int 1) and Wang et al. (DNA Int 2) Results: DNA integrity by both formulae correlated strongly with each other. DNA integrity was significantly higher in patients with colorectal cancer when compared with healthy controls (p=0.005 and p=0.006, respectively), while there was no significant difference from those with benign colorectal diseases. In receiver operating characteristic curve analysis, areas under the curve of 0.74 and 0.73 and sensitivities of 71% at 75% specificity (DNA Int 1 and 2, respectively) were achieved for the discrimination between patients with colorectal cancer and healthy controls. Conclusion: DNA integrity is significantly increased in patients with colorectal cancer and may be useful in prospective trials

Relevance of Histone Marks H3K9me3 and H4K20me3 in Cancer

Background: Circulating nucleosomes are valuable biomarkers for therapy monitoring and estimation of prognosis in cancer disease. While epigenetic and genetic modifications of DNA have been reported in blood of cancer patients, little is known about modifications of histones on circulating nucleosomes. Patients and Methods: Sera of 45 cancer patients (21 colorectal, 4 pancreatic, 15 breast, 5 lung cancer), 12 patients with benign gastrointestinal and inflammatory diseases, and 28 healthy individuals were investigated. Histone modifications were detected by chromatin-immunoprecipitation (ChIP) using antibodies for triple hi stone methylations at sites wH3K9me3 and H4K20me3 and subsequent real-time polymerase chain reaction using primers for the centromeric satellites SAT2. Additionally, the amount of circulating nucleosomes, as well as of carcino-embryonic antigen (CEA) and cancer antigen (CA) 19-9 were measured. Results: Levels of SAT2 on H3K9me3 (median 0.507 ng/ml) and on H4K20me3 (0.292 ng/ml) were elevated in sera of patients with breast cancer when compared with healthy controls (0.049 and 0.035 ng/ml), but were lower in patients with colorectal cancer (0.039 and 0.027 ng/ml). Both histone marks were correlated with each other but did not correlate with CEA or CA 19-9 in cancer patients. When H3K9me3 and H4K20me3 were normalized to nucleosome content in sera, ratios were significantly higher in all types of cancer as well as in colorectal and breast subtypes when compared with healthy controls. Best discrimination was achieved by normalized H4K20me3 reaching areas under the curves (AUC) of 79.1%, 90.4% and 81.2% in receiver operating characteristic (ROC) curves of these three comparisons. Conclusion: SAT2 levels on H3K9me3 and H4K20me3 are up-regulated in breast cancer and down-regulated in colorectal cancer. Normalization to total nucleosome content enables better discrimination between cancer and control groups

Characterization of H3K9me3- and H4K20me3-associated circulating nucleosomal DNA by high-throughput sequencing in colorectal cancer

Modified histone tails in nucleosomes circulating in the blood bear the potential as cancer biomarkers. Recently, using chromatin immunopecipitation (ChIP)-related quantitative PCR, we described reduced plasma levels of the two pericentric heterochromatin-specific histone methylation marks H3K9me3 and H4K20me3 in patients with colorectal cancer (CRC). Here, by utilizing ChIP-related high-throughput sequencing, we further characterized these modifications in circulation. Plasma DNA from nucleosomes immunoprecipitated by H3K9me3- and H4K20me3-specific antibodies from patients with CRC (N = 15) and healthy subjects (N = 15) was subjected to the Roche 454 FLX sequencing, and the generated array of ChIP-enriched sequences were compared to the human reference genome. The total number of nucleosomes, of sequence reads and of diverse DNA repetitive elements were statistically compared between the study groups. Total nucleosome amount was not different in both groups. Concerning both histone modifications, lower numbers of sequence reads were detected in CRC patients as compared with healthy controls (medians in H3K9me3: 32 vs. 61; p < 0.01; in H4K20me3: 54 vs. 88; p < 0.01). Size of fragments was not different in both groups. Most abundant sequences were repetitive LINE and SINE elements while simple repeats, LTR, DNA, SAT, and low complexity elements were less frequent. Best discrimination between both groups was achieved by total number of H3K9me3 reads (AUC 0.90) and H3K9me3 LINE elements L1 (AUC 0.93) und L2 (AUC 0.91). The present results confirm earlier findings of lower H3K9me3 levels in CRC and show LINE elements to be the most frequent and best discriminative markers on modified histones