Abstract LB-374: A novel method for the detection of circulating NK cells in archived blood reveals a decrease in NK cells in head and neck squamous cell carcinoma
Authors: Accomando William P. , Wiencke John K. , Houseman E. Andres , Butler Rondi , Zheng Shichun , Nelson Heather H. , Kelsey Karl T. 
Published: August 2012

Abstract
Patients with head and neck squamous cell carcinoma (HNSCC) frequently exhibit abnormal immune cell levels and activities, leading many to believe that progression of the disease is linked to evasion of immune surveillance. A more complete understanding of immune cell alterations could be obtained from analysis of archival samples, but this type of study is not possible using current immunodiagnostic methods. Combining information on cell lineage specific differentially methylated regions (DMRs) with methylation specific quantitative PCR (MS-qPCR) enables highly sensitive and accurate counts of individual cell types in archival blood and tissues. DNA methylation and mRNA expression in magnetic microbead (MACS) isolated, flow cytometry (FACS) validated normal human peripheral blood leukocytes were assessed using the Infinium HumanMethylation27 and Whole-Genome DASL HT (Illumina) microarrays. Cell-specific DMRs were identified by comparative analysis of DNA methylation and mRNA expression patterns between different cell lineages, and validated by MS-qPCR and pyrosequencing. Analysis of the microarray data revealed CpG loci significantly demethylated in NK cells compared to all other leukocyte lineages (q &lt; 0.1) within genes exhibiting significantly increased mRNA expression in NK cells (q &lt; 0.1). Pyrosequencing and MS-qPCR assays confirmed a NK cell specific demethylated region. The MS-qPCR assay was calibrated using known quantities of NK cell DNA to facilitate quantification of these cells in biological samples, and was then applied to archival peripheral blood DNA samples from 122 control donors and 122 HNSCC patients. Demethylation of the candidate region was significantly lower in HNSCC patient bloods compared with control bloods (p &lt; 0.001), but was not significantly associated with age, gender, HPV16 (E6 and/or E7) serology, cigarette smoking, alcohol consumption, BMI, or disease treatment. Measurements from control samples were used to establish normal demethylation tertiles. Proportion of total HNSCC cases decreased significantly with increasing tertile (p &lt; 0.001). Multivariate logistic regression controlling for age, gender, cigarette smoking, alcohol consumption, BMI, and HPV16 serology confirmed increased HNSCC risk for individuals in the lowest tertile (OR = 5.6, 95% CI: 2.0, 17.4) and second lowest tertile (OR = 4.9, 95% CI: 1.8, 16.1). The advent of DNA methylation based immunodiagnostic methods will make assessment of immune cell profiles in archived samples that have not undergone rigorous special handling requirements possible. Using a novel NK cell specific demethylated region to detect and quantify NK cells in the blood, we have detected a HNSCC associated decrease in circulating NK cells that is independent of disease treatments, known exposures and other putative risk factors.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-374. doi:1538-7445.AM2012-LB-374