Thomas Liggett

Differential methylation of cell‐free circulating DNA among patients with pancreatic cancer versus chronic pancreatitis

Although patients with chronic pancreatitis (CP) have an increased risk of pancreatic cancer (PanCa), the timely detection of PanCa often is difficult, because the symptoms of CP and PanCa are very similar. Moreover, secondary inflammation may be identified in PanCa, further complicating diagnosis. To improve the survival of patients with PanCa, a reliable test to differentiate CP from PanCa is needed. In this article, the authors describe a methylation profile of cell‐free plasma DNA that distinguished CP from PanCa with >90% accuracy.

Methylation patterns of cell-free plasma DNA in relapsing-remitting multiple sclerosis

BACKGROUND There is growing interest for identification of new targets for biomarker development in multiple sclerosis (MS). The goal of this study was to compare the concentration and the methylation patterns of cell-free plasma DNA (cfpDNA) in patients with relapsing-remitting multiple sclerosis (RRMS) and healthy individuals. METHODS Three 30-patient cohorts were examined: patients with RRMS, in either remission or exacerbation, and healthy individuals as controls. Concentration of cfpDNA was determined using a standard fluorometric assay. Patterns of methylation in 56 gene promoters were determined by a microarray-based assay (MethDet-56). The data were analyzed to identify statistically relevant differences among the study groups. RESULTS The concentration of cfpDNA in patients with RRMS was four to eight-fold higher compared to healthy controls. Significant differences in cfpDNA methylation patterns were detected in all three comparisons: RRMS patients in remission versus healthy controls were recognized with 79.2% sensitivity and 92.9% specificity; RRMS patients in exacerbation versus healthy controls were recognized with 75.9% sensitivity and 91.5% specificity; and RRMS patients in exacerbation versus those in remission were recognized with 70.8% sensitivity and 71.2% specificity. CONCLUSION Based on our findings, we conclude that patients with RRMS display unique disease- and state-specific changes of cfpDNA. Our findings are of clinical significance as they could be used in the development of potentially new biomarkers for MS. This is the first report in our knowledge describing such changes of cfpDNA in patients with MS.

Distinctive DNA methylation patterns of cell-free plasma DNA in women with malignant ovarian tumors

OBJECTIVE Epithelial ovarian carcinoma (OvCa) is rarely detected early, and it is also difficult to determine whether an adnexal mass is benign or malignant. Previously, we noted differences in methylation patterns of cell-free plasma DNA (cfpDNA) in women without disease compared to patients with OvCa. In this work, we investigated whether methylation patterns of cfpDNA can differentiate between benign and malignant tumors. METHODS Methylation patterns in cfpDNA were determined in three cohorts (30 samples each) using a microarray-based assay (MethDet 56). Principal component analysis, supervised clustering, linear discrimination analysis, and 25 rounds of 5-fold cross-validation were used to determine informative genes and assess the sensitivity and specificity of differentiating between OvCa vs. healthy control (HC), benign ovarian disease (mostly serous cystadenoma, BOD) vs. HC, and OvCa vs. BOD samples. RESULTS Differential methylation of three promoters (RASSF1A, CALCA, and EP300) differentiated between OvCa vs. HC with a sensitivity of 90.0% and a specificity of 86.7%. Three different promoters (BRCA1, CALCA, and CDKN1C) were informative for differentiating between BOD vs. HC, with a sensitivity of 90.0% and a specificity of 76.7%. Finally, two promoters (RASSF1A and PGR-PROX) were informative for differentiating between OvCa vs. BOD, with a sensitivity of 80.0% and a specificity of 73.3%. CONCLUSIONS This proof-of-principle data show that differential methylation of promoters in cfpDNA may be a useful biomarker to differentiate between certain benign and malignant ovarian tumors.

DNA methylation patterns in blood of patients with colorectal cancer and adenomatous colorectal polyps

Colorectal cancer (CRC) screening rates are currently suboptimal. Blood‐based screening could improve rates of earlier detection for CRC and adenomatous colorectal polyps. In this study, we evaluated the feasibility of plasma‐based detection of early CRC and adenomatous polyps using array‐mediated analysis methylation profiling of 56 genes implicated in carcinogenesis. Methylation of 56 genes in patients with Stages I and II CRC (N = 30) and those with adenomatous polyps (N = 30) were compared with individuals who underwent colonoscopy and were found to have neither adenomatous changes nor CRC. Composite biomarkers were developed for adenomatous polyps and CRC, and their sensitivity and specificity was estimated using five‐fold cross validation. Six promoters (CYCD2, HIC1, PAX 5, RASSF1A, RB1 and SRBC) were selected for the biomarker, which differentiated CRC patients and controls with 84% sensitivity and 68% specificity. Three promoters (HIC1, MDG1 and RASSF1A) were selected for the biomarker, which differentiated patients with adenomatous polyps and controls with sensitivity of 55% and specificity of 65%. Methylation profiling of plasma DNA can detect early CRC with significant accuracy and shows promise as a methodology to develop biomarkers for CRC screening.

Methylation patterns in cell-free plasma DNA reflect removal of the primary tumor and drug treatment of breast cancer patients.

Abnormal DNA methylation is a feature of most types of cancer, which is reflected in cell‐free circulating DNA in plasma. It is, however, unknown whether surgical removal of the tumor and subsequent therapy induces changes in plasma DNA methylation, which can be used to monitor treatment. In this pilot study, methylation in cell‐free plasma DNA of 20 breast cancer patients was determined by the previously developed MethDet‐56 technique. Samples at three time points were analyzed—before surgery (baseline), after surgery (to evaluate the effects of resection) and after surgery on tamoxifen therapy (to determine the effects of treatment). Methylation patterns of healthy controls were used as a reference for all comparisons. Seven promoters were differentially methylated (p < 0.05) in at least one comparison; three changed after surgery; another one changed after beginning of tamoxifen treatment; and four were differentially methylated in baseline versus combined treatment samples. Increased methylation of PR PROX, MDGI, PAX 5 and RARβ2 at baseline (presurgery) diminished toward the healthy controls with the lowest methylation in the combined treatment group. Surgery alone decreased methylation in PAX 5 and RARβ2, whereas tamoxifen treatment changed methylation only in the B promoter of ESR1. Methylation patterns in cell‐free plasma DNA change after surgery and tamoxifen treatment, most significantly—after combined treatment. The baseline (presurgery) patterns become similar to those of healthy controls, suggesting that methylation patterns in cell‐free plasma DNA may be used to monitor treatment.

Methylation of death-associated protein kinase is associated with cetuximab and erlotinib resistance

Anti-EGFR therapy is among the most promising molecular targeted therapies against cancer developed in the past decade. However, drug resistance eventually arises in most, if not all, treated patients. Emerging evidence has linked epigenetic changes, such as DNA methylation at CpG islands, to the development of resistance to multiple anticancer drugs. In addition, genes that are differentially methylated have increasingly been appreciated as a source of clinically relevant biomarker candidates. To identify genes that are specifically methylated during the evolution of resistance to anti-EGFR therapeutic agents, we performed a methylation-specific array containing a panel of 56 genes that are commonly known to be regulated through promoter methylation in two parental non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) cell lines and their resistant derivatives to either erlotinib or cetuximab. We found that death-associated protein kinase (DAPK) was hypermethylated in drug-resistant derivatives generated from both parental cell lines. Restoration of DAPK into the resistant NSCLC cells by stable transfection re-sensitized the cells to both erlotinib and cetuximab. Conversely, siRNA-mediated knockdown of DAPK induced resistance in the parental sensitive cells. These results demonstrate that DAPK plays important roles in both cetuximab and erlotinib resistance, and that gene silencing through promoter methylation is one of the key mechanisms of developed resistance to anti-EGFR therapeutic agents. In conclusion, DAPK could be a novel target to overcome resistance to anti-EGFR agents to improve the therapeutic benefit, and further evaluation of DAPK methylation as a potential biomarker of drug response is needed.

Cross-Immunoreactivity between Bacterial Aquaporin-Z and Human Aquaporin-4: Potential Relevance to Neuromyelitis Optica

Neuromyelitis optica (NMO) is a chronic inflammatory disease of the CNS that is mediated, in part, by a self-reactive Ab against the astrocyte aquaporin-4 protein. In the current study, we examined the possibility and the biological significance of cross-immunoreactivity between bacterial aquaporin-Z and human aquaporin-4 proteins. Sequence-alignment analysis of these proteins revealed several regions of significant structural homology. Some of the homologous regions were also found to overlap with important immune and disease-relevant epitopes. Cross-immunoreactivity between aquaporin-Z and aquaporin-4 was investigated and ascertained in multiple immune-based assays using sera from patients with neuromyelitis optica, immune mouse serum, and Abs raised against aquaporin-Z. The biological significance of this phenomenon was established in series of experiments demonstrating that induction of an immune response against aquaporin-Z or its homologous regions can also trigger an autoimmune reaction against aquaporin-4 and inflammation of the CNS. Our study indicates that the autoimmune response against aquaporin-4 in neuromyelitis optica may be triggered by infection-induced cross-immunoreactivity and presents a new perspective on the pathogenesis of this disease.

IRF-1 signaling in central nervous system glial cells regulates inflammatory demyelination

The present study provides evidence that interferon regulatory factor 1 (IRF-1) signaling in glial cells is involved in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Using a bone marrow chimera model of EAE, we demonstrated that CNS IRF-1 regulates inflammatory demyelination and disease severity independently of the peripheral immune cells. In addition, we identified Caspase 1, a pro-inflammatory and pro-apoptotic molecule, as an important transcriptional target of IRF-1. The findings of our study indicate that IRF-1 signaling in glial cells serves as a final common pathway of inflammatory demyelination and may have important clinical implications in MS.

Overexpression of the Dominant-Negative Form of Interferon Regulatory Factor 1 in Oligodendrocytes Protects against Experimental Autoimmune Encephalomyelitis

Interferon regulatory factor 1 (IRF-1) is a transcription factor that has been implicated in the pathogenesis of the human autoimmune demyelinating disease multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). The goal of the present study was to directly examine the role of IRF-1 in oligodendrocyte injury and inflammatory demyelination. For the purpose of this study, we generated a transgenic mouse line (CNP/dnIRF-1) that overexpresses the dominant-negative form of IRF-1 (dnIRF1) specifically in oligodendrocytes. CNP/dnIRF-1 mice exhibited no phenotypic abnormalities but displayed suppressed IRF-1 signaling in oligodendrocytes. The major finding of our study was that the CNP/dnIRF-1 mice, compared with the wild-type mice, were protected against EAE, a phenomenon associated with significant reduction of inflammatory demyelination and with oligodendrocyte and axonal preservation. The observed protection was related to suppressed IRF-1 signaling and impaired expression of immune and proapoptotic genes in oligodendrocytes. No significant differences in the peripheral immune responses between the wild-type and the CNP/dnIRF-1 mice were identified throughout the experiments. This study indicates that IRF-1 plays a critical role in the pathogenesis of EAE by mediating oligodendrocyte response to inflammation and injury. It also suggests that oligodendrocytes are actively involved in the neuroimmune network, and that exploring oligodendrocyte-related pathogenic mechanisms, in addition to the conventional immune-based ones, may have important therapeutic implications in MS.

Central nervous system expression of interferon regulatory factor 1 regulates experimental autoimmune encephalomyelitis.

Interferon regulatory factor 1 (IRF-1) is a transcription factor that has been implicated in the disease mechanisms of experimental autoimmune encephalomyelitis (EAE). In this study, we examined the role of central nervous system (CNS) expression of IRF-1 in the natural course of EAE. In an effort to dissect the CNS effects from the peripheral immune effects of IRF-1, we generated bone marrow chimera mice that differentially expressed IRF-1 in the CNS and in the immune system. We found that mice lacking IRF-1 in the CNS developed significantly milder clinical symptoms and shorter disease duration compared to those with wild-type background. Based on these results, we concluded that the CNS expression of IRF-1 regulates the disease process in EAE. Our findings are relevant to the inflammatory mechanisms involved in multiple sclerosis and may provide a basis for development of novel therapeutic targets of the disease.