Highly sensitive noninvasive early lung cancer detection using DNA methylation topology in sputum-derived epithelial cells.

Objective: Sputum is a source of exfoliated respiratory epithelial cells transformed early in lung carcinogenesis. Malignant cells are hypomethylated and contain less genomic 5-methylcytosine (5mC). Validating a test that recognizes and quantifies aberrantly hypomethylated cells in sputum, we assessed its potential as a screening tool for detecting early-stage non-small cell lung cancer. Methods: Cells extracted from sputum were immunofluorescence labeled with an anti-5-methylcytosine antibody and counterstained with 4',6-diamidino-2-phenylindole (DAPI) delineating global nuclear DNA (gDNA). Via confocal scanning and 3-dimensional image analysis, fluorescence 5mC and DAPI signals were measured in segmented cell nuclei, and a 5mC/DAPI co-distribution map was generated for each imaged cell. Cells were classified as hypomethylated based on 5mC load and 5mC/DAPI co-distribution. The proportion of hypomethylated epithelial cells in the sputum determines whether a patient has lung cancer. Results: A total of 88 subjects were enrolled: 12 healthy subjects; 34 high-risk subjects with benign chronic lung disorders (10 with chronic obstructive pulmonary disease, 24 with idiopathic pulmonary fibrosis), and 43 subjects with non-small cell lung cancer (27 with stage I-II and 16 with stage III-IV). The test identified early-stage non-small cell lung cancer and distinguished it from the high-risk group with 95.8% (95% confidence interval, 78.9-99.9) sensitivity and 41.2% (95% confidence interval, 24.6-59.3) specificity applying only 5mC, 95.8% (95% confidence interval, 78.9-99.9) sensitivity and 26.5% (95% confidence interval, 12.9-44.4) specificity using solely 5mC/DAPI index, and 100% (95% confidence interval, 98.7-100) sensitivity and 26.1% (95% confidence interval, 26.2-27.8) specificity with the combined parameters. Conclusions: We tested and validated a novel, noninvasive, highly sensitive screening test for non-small cell lung cancer. With the use of sputum, our test may impact lung cancer screening, evaluation of pulmonary nodules, and cancer surveillance algorithms.

DNA methylation topology differentiates between normal and malignant in cell models, resected human tissues, and exfoliated sputum cells of lung epithelium.

Background: Global DNA hypomethylation is a prominent feature of cancer cells including lung cancer, that has not been widely explored towards cancer diagnosis. In this study we assess the comparative distribution of global DNA methylation in normal cells versus cancer cells in various specimen models. Methods: We used in situ immunofluorescence labeling of overall 5-methylcytosine (5mC) and covisualization of global DNA (gDNA) by 4',6-diamidino-2-phenylindole (DAPI), confocal microscopy and 3D image analysis to derive 5mC/DAPI colocalization patterns in human cell lines (BEAS-2B, A549, H157) and upper respiratory epithelial cells derived from various sources (i.e., sputum from healthy and cancer patients, and resected tissues from normal parenchyma and lung tumors). Results: By introducing 5mC/DAPI colocalization index as a metric we could distinguish between normal epithelial cells and aberrantly hypomethylated cancer cells. Cultured lung cancer cells (H157 and A549) had significantly lower indices compared to normal cells (BEAS-2B). Furthermore, we were able to identify such extensively hypomethylated low-index cells in tumor tissues and the matching sputum from cancer patients. In contrast, the indices of cells derived from sputum of healthy individuals had more similarity to epithelial cells of normal parenchyma and the phenotypically normal BEAS-2B cells. Conclusions: The results suggest that 5mC topology using high-resolution image cytometry shows potential for identifying hypomethylated cancerous cells in human tissues and amongst normal cells in matching sputum, which may render a valuable surrogate for biopsied tissues. This promising feature deserves further validation in more comprehensive studies.

Insulin Receptor-Expressing T Cells Appear in Individuals at Risk for Type 1 Diabetes and Can Move into the Pancreas in C57BL/6 Transgenic Mice.

Insulin receptor (IR) expression on the T cell surface can indicate an activated state; however, the IR is also chemotactic, enabling T cells with high IR expression to physically move toward insulin. In humans with type 1 diabetes (T1D) and the NOD mouse model, a T cell-mediated autoimmune destruction of insulin-producing pancreatic ß cells occurs. In previous work, when purified IR+ and IR- T cells were sorted from diabetic NOD mice and transferred into irradiated nondiabetic NOD mice, only those that received IR+ T cells developed insulitis and diabetes. In this study, peripheral blood samples from individuals with T1D (new onset to 14 y of duration), relatives at high-risk for T1D, defined by positivity for islet autoantibodies, and healthy controls were examined for frequency of IR+ T cells. High-risk individuals had significantly higher numbers of IR+ T cells as compared with those with T1D (p < 0.01) and controls (p < 0.001); however, the percentage of IR+ T cells in circulation did not differ significantly between T1D and control subjects. With the hypothesis that IR+ T cells traffic to the pancreas in T1D, we developed a (to our knowledge) novel mouse model exhibiting a FLAG-tagged mouse IR on T cells on the C57BL/6 background, which is not susceptible to developing T1D. Interestingly, these C57BL/6-CD3FLAGmIR/mfm mice showed evidence of increased IR+ T cell trafficking into the islets compared with C57BL/6 controls (p < 0.001). This transgenic animal model provides a (to our knowledge) novel platform for investigating the influence of IR expression on T cell trafficking and the development of insulitis.

Insulin receptor based lymphocyte trafficking in the progression of type 1 diabetes.

The insulin receptor (IR) is a transmembrane receptor which recognizes and binds the hormone insulin. We describe two models that were devised to explore the role of IR over-expression on T-lymphocytes and their chemotactic motility in the progression of type 1 diabetes. FVB/NJ-CD3-3×FLAG-mIR/MFM mice were generated to selectively over-express 3×FLAG tagged murine IR in T-lymphocytes via an engineered CD3 enhancer and promoter construct. Insertion of the 3×FLAG-mIR transgene into FVB/NJ mice, a known non-autoimmune prone strain, lead to a minor population of detectable 3×FLAG-mIR tagged T-lymphocytes in peripheral blood and the presence of a few lymphocytes in the pancreas of the Tg+/- compared to age matched Tg-/- control mice. In order to induce stronger murine IR over-expression then what was observed with the CD3 enhancer promoter construct, a second system utilizing the strong CAG viral promoter was generated. This system induces cell specific IR over-expression upon Cre-Lox recombination to afford functional 3×FLAG tagged murine IR with an internal eGFP reporter. The pPNTlox2-3×FLAG-mIR plasmid was constructed and validated in HEK-Cre-RFP cells to ensure selective Cre recombinase based 3×FLAG-mIR expression, receptor ligand affinity towards insulin, and functional initiation of signal transduction upon insulin stimulation.