Decreased NK Cells in Patients with Head and Neck Cancer Determined in Archival DNA

Department of Neurological Surgery, University of California San Francisco, San Francisco, California
Clinical Cancer Research (Impact Factor: 8.72). 09/2012; 18(22). DOI: 10.1158/1078-0432.CCR-12-1008
Source: PubMed


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.

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.

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.

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    • "It has been shown that NK cells are abundantly present in the tissue of preneoplastic cervical lesions but much less cells have been detected in cervical squamous cell carcinomas [15] [16]. In patients with HNSCC, decreased numbers of NK cells in the peripheral blood have been detected independently of the etiology of the tumor [17] [18]. Additionally, it has been shown that a lower number of NK cells in the blood of patients with HNSCC predict their poor outcome [19]. "
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    ABSTRACT: Background: Human papillomaviruses (HPVs) have been proved as one of the etiological factors of oropharyngeal squamous cell carcinoma (OPSCC). Patients with tumors of viral etiology have a lower recurrence rate and better prognosis. OPSCC is linked to an alteration in the immune system. Only a limited number of studies have correlated both the immunological parameters and HPV status with patient prognosis. The aim of this study was to determine whether HPV infection and the immunological status influence patient prognosis individually or in concurrence. Material and methods: Sixty patients with oral and oropharyngeal carcinomas were enrolled. They were divided into HPV-positive and HPV-negative groups based on the expression of HPV 16 E6 mRNA. Basic lymphocyte subpopulations were determined in the peripheral blood by means of flow cytometry. Results: Significantly better disease-specific survival (DSS) was observed in patients with HPV-positive tumors. Nodal status, tumor grade, recurrence, and CD8+/Tregs ratio were identified as factors influencing DSS. A higher level of Tregs and a lower ratio of CD8/Tregs influenced overall survival (OS) independently of HPV status and age. Patients with HPV-positive tumors and high levels of Tregs survived significantly better than patients from the other groups. Conclusion: Better survival is associated with HPV positivity and elevated Tregs levels. Our data suggest that HPV infection and Tregs do not influence patient prognosis in concurrence.
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    • "Of course, it is now recognized that the expression of these cellular surface protein markers is controlled epigenetically. In fact, normal leukocyte lineage-specific differentiation is directed by differences in gene expression associated with distinct patterns of DNA methylation, with differentially methylated regions (DMRs) delineating distinct leukocyte subtypes [3-6,11,12,26]. "
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    ABSTRACT: Cell lineage-specific DNA methylation patterns distinguish normal human leukocyte subsets and can be used to detect and quantify these subsets in peripheral blood. We have developed an approach that uses DNA methylation to simultaneously quantify multiple leukocyte subsets, enabling the investigation of immune modulations in virtually any blood sample including archived samples previously precluded from such analysis. Here we assess the performance characteristics and validity of this approach. Using Illumina Infinium HumanMethylation27 and VeraCode GoldenGate Methylation Assay microarrays, we measure DNA methylation in leukocyte subsets purified from human whole blood and identify cell lineage-specific DNA methylation signatures that distinguish human T-cells, B-cells, NK cells, monocytes, eosinophils, basophils and neutrophils. We then employ a bioinformatics-based approach to quantify these cell types in complex mixtures, including whole blood, using DNA methylation at as few as 20 CpG loci. A reconstruction experiment confirms that the approach could accurately measure the composition of mixtures of human blood leukocyte subsets. Applying the DNA methylation-based approach to quantify the cellular components of human whole blood, we verify its accuracy by direct comparison to gold standard immune quantification methods that utilize physical, optical and proteomic characteristics of the cells. We also demonstrate that the approach is not affected by storage of blood samples, even under conditions prohibiting the use of gold standard methods. Cell mixture distributions within peripheral blood can be assessed accurately and reliably using DNA methylation. Thus, precise immune cell differential estimates can be reconstructed using only DNA rather than whole cells.
    Genome biology 03/2014; 15(3):R50. DOI:10.1186/gb-2014-15-3-r50 · 10.81 Impact Factor
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    • "cell activity contributes to an increase in incidence of urethaneinduced lung lesions in the C57BL/6 mouse strain, the strain used here. Furthermore, an association was also demonstrated between suppression of NK cell activities and a higher risk of developing head and neck cancers in humans, as well as with a higher incidence of 3-methylcholanthrene-induced sarcomas in mice (Accomando et al., 2012; O'Sullivan et al., 2012). "
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    ABSTRACT: Abstract Pteridium aquilinum (bracken fern), one of the most important toxic plants in the world, contains the toxic norsequiterpene ptaquiloside that induces cancers in humans and farm animals. Previous studies in the laboratory demonstrated immunotoxic effects produced by ptaquiloside, which are characterized by suppression of natural killer (NK) cell activity (i.e. cytotoxicity and interferon [IFN]-γ production). However, it is unknown whether these immunosuppressive effects could contribute to carcinogenesis in situ in general because of the important function of NK cells in innate killing of tumor cells. This study assessed the impact of P. aquilinum-induced immunosuppression on urethane-induced lung cancer in C57BL/6 mice. Adult mice were treated with an extract of P. aquilinum (30 g/kg/day) by gavage once daily for 14 days, followed by gavage (5 days/week) during an 11-week period that was accompanied by treatment with urethane (1 g/kg) via once-weekly intraperitoneal injection; 20 weeks after the end of the treatment period, all lungs were evaluated. The results indicated there was a significant increase in lung nodule number as well as in multiplicity of lesions in mice treated with both P. aquilinum and urethane (PU group) compared to values in mice treated only with the urethane (U group). In addition, histologic evaluation revealed a 76% increase in the rate of lung adenomas and a 41% increase in rate of bronchiolization of alveoli in the mice from the PU group compared to levels seen in mice within the U group. Taken together, the results here show for the first time that immunosuppressive effects of P. aquilinum could increase the risk of cancer formation in exposed hosts.
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