DNA methylation markers for blood-based detection of small cell lung cancer in mouse models

Lung cancer is the top cancer killer in the United States. This cancer starts in the cells lining the bronchi or other parts of the lung such as bronchioles or alveoli. It can be classified into two major groups: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Although SCLC is less common than NSCLC it is the most aggressive type of lung cancer. To this day, there are no effective serum biomarkers for detection, prediction of response or monitoring this disease. Such markers could be very valuable because currently most SCLC patients are diagnosed after their cancer has metastasized, and there is no way apart from imaging to test for response or recurrence. ❧ It has been amply demonstrated that epigenetic modifications, such as alterations in cytosine-5 DNA methylation, occur frequently in all types of cancer. DNA methylation can be detected in the serum of cancer patients using a sensitive assay called MethyLight. Our lab has developed blood-based DNA methylation markers for NSCLC that have also been shown to be present in the blood of small cell lung cancer patients. We were interested to determine whether these markers are also present in tumors derived from a mouse model of SCLC. These mice are important tools to develop new therapies, and identify molecular markers that could be used to determine whether the mice are responding to treatment or showing recurrent disease would be very useful. ❧ We extracted the DNA from normal and tumor tissue from mice with SCLC and disease-free control mice. Prior to DNA extraction, tissues were either microdissected from paraffin blocks or were directly taken from a frozen specimen. We performed MethyLight analysis on these DNAs and compared the methylation levels between normal lung and tumors. In order to setup the MethyLight assay for mouse DNA, we required a (positive control) fully in vitro methylated DNA sample to serve as reference in the MethyLight assay. We generated large amounts (3-5μg) of in vitro methylated DNA for two different strains of mice. This DNA is an important resource for the lab since it can be used as a positive control MethyLight assays for years to come, and eliminates the biases associated with using different batches of reference DNA. ❧ MethyLight analysis of normal/tumor DNA from the SCLC mouse model showed increased methylation in some of the tumors when the microdissected material was used, but little or no methylation when the frozen samples were examined. These results suggest that microdissection might be important in DNA methylation analysis. Because modest levels of methylation were detected but no marker showed high sensitivity, we suggest that a genome-wide analysis of DNA methylation patterns in these mice would be useful. It might uncover DNA methylation abnormalities that are specific for mice with SCLC.