Decreased NK Cells in Patients with Head and Neck Cancer Determined in Archival DNA.
ABSTRACT PURPOSE: 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.EXPERIMENTAL DESIGN: 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. Clin Cancer Res; 1-8. ©2012 AACR.
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ABSTRACT: Vaccine-based cancer immunotherapy has generated highly variable clinical results due to differing methods of vaccine preparation and variation in patient populations among other lesser factors. Moreover, these clinical responses do not necessarily correspond with the induction of tumor-specific cytotoxic lymphocytes. Here, we review the participation of natural killer (NK) cells as alternative immune components that could cooperate in successful vaccination treatment. NK cells have been described as helper cells in dendritic cell-based cancer vaccines, but the role in other kinds of vaccination strategies (whole cells, peptide, or DNA-based vaccines) is poorly understood. In this article, we address the following issues regarding the role of NK cells in cancer vaccines: NK cell anti-tumor action sites, and the loci of NK cell interaction with other immune cells; descriptions of new data on the memory characteristics of NK cells described in infectious diseases; and finally phenotypical and functional changes after vaccination measured by immunomonitoring in preclinical and clinical settings.Frontiers in Immunology 01/2015; 6:13.
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ABSTRACT: Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.Epigenetics: official journal of the DNA Methylation Society 10/2014; 9(10):1360-5. · 5.11 Impact Factor
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ABSTRACT: Background:Although many DNA methylation (DNAm) alterations observed in peripheral whole blood/leukocytes and serum have been considered as potential diagnostic markers for cancer, their origin and their specificity for cancer (e.g., vs inflammatory diseases) remain unclear.Methods:From publicly available datasets, we identified changes in the methylation of blood-borne DNA for multiple cancers and inflammatory diseases. We compared the identified changes with DNAm difference between myeloid and lymphoid cells extracted from two datasets.Results:At least 94.7% of the differentially methylated DNA loci (DM loci) observed in peripheral whole blood/leukocytes and serum of cancer patients overlapped with DM loci that distinguish between myeloid and lymphoid cells and >99.9% of the overlapped DM loci had consistent alteration states (hyper- or hypomethylation) in cancer samples compared to normal controls with those in myeloid cells compared to lymphoid cells (binomial test, P-value <2.2 × 10(-16)). Similar results were observed for DM loci in peripheral whole blood/leukocytes in patients with rheumatoid arthritis or inflammatory bowel diseases. The direct comparison between DM loci observed in the peripheral whole blood/leukocytes of patients with inflammatory diseases and DM loci observed in the peripheral whole blood of patients with cancer showed that DM loci detected from cancer and inflammatory diseases also had significantly consistent alteration states (binomial test, P-value <2.2 × 10(-16)).Conclusions:DNAm changes observed in the peripheral whole blood/leukocytes and serum of cancer patients and in the peripheral whole blood/leukocytes of inflammatory disease patients are predominantly determined by the increase of myeloid cells and the decrease of lymphoid cells under the disease conditions, in the sense that their alteration states in disease samples compared to normal controls mainly reflect the DNAm difference between myeloid and lymphoid cells. These analyses highlight the importance of comparing cancer and inflammatory disease directly for the identification of cancer-specific diagnostic biomarkers.British Journal of Cancer advance online publication 24 June 2014; doi:10.1038/bjc.2014.347 www.bjcancer.com.British Journal of Cancer 06/2014; 111(3). · 4.82 Impact Factor