title: Decreased NK cells in patients with head and neck cancer determined in archival DNA.
abstract: OBJECTIVE
  Natural killer (NK) cells are a key element of the innate immune system implicated in human cancer. To examine NK cell levels in archived bloods from a study of human head and neck squamous cell carcinoma (HNSCC), a new DNA-based quantification method was developed.
  
  
  METHODS
  NK cell-specific DNA methylation was identified by analyzing DNA methylation and mRNA array data from purified blood leukocyte subtypes (NK, T, B, monocytes, granulocytes), and confirmed via pyrosequencing and quantitative methylation specific PCR (qMSP). NK cell levels in archived whole blood DNA from 122 HNSCC patients and 122 controls were assessed by qMSP.
  
  
  RESULTS
  Pyrosequencing and qMSP confirmed that a demethylated DNA region in NKp46 distinguishes NK cells from other leukocytes, and serves as a quantitative NK cell marker. Demethylation of NKp46 was significantly lower in HNSCC patient bloods compared with controls (P < 0.001). Individuals in the lowest NK tertile had over 5-fold risk of being a HNSCC case, controlling for age, gender, HPV16 status, cigarette smoking, alcohol consumption, and BMI (OR = 5.6, 95% CI, 2.0 to 17.4). Cases did not show differences in NKp46 demethylation based on tumor site or stage.
  
  
  CONCLUSIONS
  The results of this study indicate a significant depression in NK cells in HNSCC patients that is unrelated to exposures associated with the disease. DNA methylation biomarkers of NK cells represent an alternative to conventional flow cytometry that can be applied in a wide variety of clinical and epidemiologic settings including archival blood specimens.
authors.affiliation: Department of Pathology and Laboratory Medicine, Department of Epidemiology, Brown University, Providence, Rhode Island 02912, USA.
authors.forename: William P
authors.initials: WP
authors.lastname: Accomando
authors.forename: John K
authors.initials: JK
authors.lastname: Wiencke
authors.forename: E Andres
authors.initials: EA
authors.lastname: Houseman
authors.forename: Rondi A
authors.initials: RA
authors.lastname: Butler
authors.forename: Shichun
authors.initials: S
authors.lastname: Zheng
authors.forename: Heather H
authors.initials: HH
authors.lastname: Nelson
authors.forename: Karl T
authors.initials: KT
authors.lastname: Kelsey
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000378:metabolism; D002138:Calibration; D002294:Carcinoma, Squamous Cell / Q000276:immunology* / Q000473:pathology; D016022:Case-Control Studies; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D019175:DNA Methylation; D005260:Female; D006258:Head and Neck Neoplasms / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D006801:Humans; D007694:Killer Cells, Natural / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D055627:Natural Cytotoxicity Triggering Receptor 1 / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D015415:Biomarkers; C112260:NCR1 protein, human; D055627:Natural Cytotoxicity Triggering Receptor 1; D012333:RNA, Messenger; D004247:DNA