The major barrier to effective cancer therapy is the presence of genetic and phenotypic heterogeneity within cancer cell populations that provides a reservoir of therapeutically resistant cells. As the degree of heterogeneity present within tumours will be proportional to tumour burden, the development of rapid, robust, accurate and sensitive biomarkers for cancer progression that could detect clinically occult disease before substantial heterogeneity develops would provide a major therapeutic benefit. Here, we explore the application of chromatin conformation capture technology to generate a diagnostic epigenetic barcode for melanoma. The results indicate that binary states from chromatin conformations at 15 loci within five genes can be used to provide rapid, non-invasive multivariate test for the presence of melanoma using as little as 200 μl of patient blood.
The POC meter was user-friendly and performed well across a wide range of conditions. The meter was adequate for detection of pregnancy toxemia in sheep via whole blood BHB concentration. Results should be interpreted with caution when the POC meter is used to measure blood glucose concentrations.
Diffuse Large B-cell Lymphoma (DLBCL) is a common and aggressive type of Non-Hodgkin's Lymphoma. Gene expression profiling long ago identified two distinct subtypes: Activated B-Cell (ABC) and Germinal Center B-Cell (GCB), reflecting the biology associated with the cell of origin of the lymphoma (Alizadeh et al, Nature 2000). These subtypes are histologically non distinguishable, yet are associated with distinct outcomes. Emerging clinical data also suggests that they are predictive of outcome for novel therapeutics, including a BTK inhibitor, a proteasome inhibitor and an immune modulatory agent. Reliable diagnosis of the DLBLC subtype may therefore become a requirement for choosing the best treatment for DLBCL patients. Since gene regulation is known to be associated with changes in chromatin structures, we decided to investigate the chromatin conformation around key regulators of ABC/ GCB biology using a Chromatin Conformation Capture Assay (3C). For this assay, chromatin loops are cross-linked; the DNA is digested and re-ligated, joining topologically proximal ends that may be very distant along the length of the chromosome into distinct novel fusion products. After reversing the cross-links, the presence of these novel DNA fusions can be detected by PCR, resulting in a binary read-out. Composites of these PCR products form a signature that may be characteristic of disease, disease state or subtype. The EpiSwitchTM technology has been optimized to identify these epigenetic patterns not only in tissue, but also from blood borne tumor DNA. To investigate characteristic changes in chromatin conformation in ABC- and GCB- DLBCL cell lines, we focused on key genes known to be differentially expressed in the two sub-types, including Bcl-6, IRF-4 and LMO-2 (Wright et al. PNAS 2003,). We mapped several interactions indicating that these loci are subject to epigenetic regulation. We also applied the assay to blood from DLBCL patients and from healthy volunteers to exclude background signals from normal B-cell maturation. The assay was able to detect the same signatures as seen in cell lines. These results demonstrate the potential of this technology to detect DLBCL subtype specific chromatin conformation associated biomarkers in patient blood. Citation Format: Ron McCord, Megan Field, Philip Jordan, Ewan Hunter, Alexandre Akoulitchev, Kirsten E. Mundt. Chromatin signatures of DLBCL subtypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 462. doi:10.1158/1538-7445.AM2014-462
6063 Background: NPC is highly curable in early stages but 70% of NPC patients are diagnosed with advanced disease due to lack of effective screening. Genetic and epigenetic alterations involved in the pathogenesis of NPC are known. The higher order chromosomal structures reflecting aberrant transcriptional states of these genes can be measured via techniques such as chromosome conformation capture. Detection of these changes in peripheral blood may provide an accurate test for the early cancer detection. Methods: Blood samples have been collected from 84 patients with histologically confirmed NPC and 100 matched controls. Samples from 45 NPC patients and 68 controls have been analyzed. Fourteen genes known to be dysregulated in NPC were investigated. Potential higher order juxtaposition sites in the candidate genes were predicted using pattern recognition software. PCR primer sets were designed around the chosen sites to screen potential markers. Twenty-two markers showing predictability between NPC and control samples were analysed for optimal reproducibility using alternative primer sets. The optimal sets of markers were then tested amongst the complete set of samples. The dataset was processed by re-sampling using the synthetic minority oversampling technique. The overall sample was split into two groups (66% training set and 34% test set) in the classification. Results: Sixteen markers from 7 candidate genes were found to be optimal in differentiating between NPC and control samples in the first 103 samples. Using the multilayer perceptron (MLP) classification, the following results were obtained: Sensitivity 88.9%, 95% CI (79.2% - 98.6%); Specificity 72.7%, 95% CI (58.9% - 86.5%); PPV 72.7%, 95% CI (58.9% - 86.5%); NPV 88.9%, 95% CI (79.2% - 98.6%). The accuracy of the test was similar in detection of stage I and II NPC versus that of stage III or IV NPC. Conclusions: Using a PCR-based method to detect alterations in the cancer epigenome, the feasibility of developing a blood test of potential utility in early diagnosis of NPC was demonstrated. Analysis of larger numbers of patient samples and optimization of markers are ongoing. The performance characteristics of the test in the total population of 184 samples will be presented.
Background: Cells present as minimal residual disease (MRD) following frontline therapy drive chemoresistant tumour relapse, thus their detection and targeting is a major priority in cancer therapy. Neuroblastoma (NB) is an aggressive neural crest-derived malignancy of infants and young children. A hallmark of NB is its clinical heterogeneity and the majority of infants with NB have a unique biology that results in spontaneous remission through differentiation in the absence of therapy, despite disease that may be disseminated to the liver, skin, and bone marrow at diagnosis. These infants (with stage 4S disease) have survival rates that exceed 95%. Children with high-risk NB, however, largely characterized by amplification of the MYCN oncogene, often have a disease that is therapy resistant. Although modest improvements in outcome have been achieved as a result of therapy intensification, 5-year event free survival in high-risk patients remains approximately 40-50%. Typically, high-risk neuroblastoma recurs after near complete remission is achieved and the development of effective strategies directed at MRD remains an unmet clinical need. Detection of MRD in neuroblastoma is usually performed using real-time quantitative (RQ)-PCR of neuroblastoma-specific transcripts. Tumor-selective mRNA markers levels are highly dependent on gene expression, which can vary between patients (by a factor of up 1000) and change during treatment. Furthermore, the applicability of a PCR target for MRD detection is also determined by its background expression in hematologic cells. Chromatin conformation patterns integrate the spatial arrangement of the chromatin and build a signature for the physiological status of the cell representing early changes in the genetic and epigenetic regulation. These signatures form the basis for a new technology, Episwitch™, which can identify epigenetic patterns linked with cancer progression and aggressiveness. Results: Based on a screen of human NB cell lines containing amplification of MYCN or expression of high levels of MYCN protein versus NB cell lines lacking expression of MYCN, we have identified a panel of four NB-associated genetic markers: HDM2, PHOX2B, TERT and TH. HDM2 and TERT are direct transcriptional targets of MYCN. This epigenetic signature is currently being validated in primary NB samples. An in vivo proof of concept study utilizing the equivalent mouse epigenetic signature is being performed in a relapse model of MYCN-driven NB. Conclusion: In this study we have developed and tested a novel blood test based on Episwitch™ technology that discriminates MYCN-associated NB. Our data suggest this non-invasive test can be used to monitor remission/relapse status quantitatively with high sensitivity in high-risk MYCN-associated neuroblastoma, thus informing treatment decisions. Citation Format: Kevin Petrie, Megan Field, Zai Ahmad, Mehrnoush Dezfouli, Karen Barker, Magdalena Jeznach, Laura Glass, Albert Hallsworth, Yordan Sbirkov, Howard Womersley, Arthur Zelent, Philip Jordan, Alexandre Akoulitchev, Louis Chesler. A novel epigenetic blood test to monitor minimal residual disease in high-risk neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-87. doi:10.1158/1538-7445.AM2013-LB-87
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