The present study advances our understanding of immune factors in HNSCC in two ways: firstly we developed a novel and sensitive biomarker of NK cells that is highly useful in archival blood specimens, and second we assessed this new NK biomarker in relationship to risk factors and clinical prognostic factors in HNSCC. Applying this new method to archived blood samples from a case-control study of HNSCC revealed a decreased NK cell fraction in the peripheral blood of HNSCC patients compared to cancer free controls. Past studies have indicated that NK cell suppression is a feature of HNSCC (15
), but none have conclusively shown a disease-associated decrease in circulating NK cells relative to other leukocytes in the peripheral blood. One study did find decreased levels of the more regulatory CD56bright
NK cell subset in the peripheral blood of patients with HNSCC compared to controls (14
). It is likely that the HNSCC-associated circulating NK cell decrease we have observed in this study is primarily the result of a diminished CD56dim
NK cell compartment in the peripheral blood of patients with HNSCC, since the vast majority of NK cells circulating in the periphery belong to the CD56dim
NK cell subset. In addition, our NKp46
qMSP assay seems to be more specific to the detection of CD56dim
NK cells than CD56bright
NK cells based on measurements of MACS isolated human leukocytes. This may, in part, be an artifact of antibody-based method of cell isolation. The populations of CD56dim
NK cells obtained by MACS may not be entirely distinct, but exist along a spectrum of NK cells with varying cytotoxic activities. Recent evidence suggests that CD56bright
NK cells can give rise to CD56dim
NK cells through a differentiation process that results in decreased proliferative potential and increased cytolytic activity (33
). It is possible that, as CD56bright
NK cells proceed down a path of differentiation to become CD56dim
NK cells, regions of cytotoxic genes undergo demethylation to prime them for expression in the effector cells. Thus, some of the NK cells that were classified as the more regulatory, CD56bright
subset using antibody-based methods could be further along the spectrum towards CD56dim
than others, and therefore exhibit NKp46
demethylation. If there is a complex spectrum of NK cell subtypes that cannot be fully distinguished by traditional protein based methods of cell isolation and quantification, then careful examination of DNA methylation profiles could reveal hitherto unknown cell lineages.
Previous work has shown that NK cell activity has prognostic significance (34
). Our strategy for developing a DMR that would identify NK cells was aimed at assessing NK cell numbers, and not to specifically inform us about the functional activities of NK cells that are present. However, future work could also define NK cell DMRs associated with activation. Comparing patterns of DNA methylation in isolated NK cells exhibiting different types of cytotoxic activation using the same methodology employed in this work might reveal important biomarkers. NK cell activity-associated DMR assays could then be run in tandem with the NKp46 demethylation assay to assess both NK cell numbers and activities.
Other studies have indicated that cigarette smoking can attenuate the cytotoxic activity of NK cells. However, this is not accompanied by a difference in the number of NK cells in the blood or lung tissue of smokers compared to non-smokers (27
). Consistent with these findings, our data confirmed that peripheral blood NK cell levels are not associated with cigarette smoking. In our data, peripheral blood NK cell levels were not associated with age, gender, HPV16 (E6 and/or E7) serology, alcohol consumption or BMI. We investigated primarily men with lean BMIs (< 30), limiting our power to detect differences in NK cell levels associated with gender and BMI. In addition, since blood samples were collected as close to the diagnosis date as possible, it was not possible to use these data to analyze the longer-term effects of chemotherapy or radiation treatment on circulating NK cell levels. However, this study design allowed us to rule out oncologic treatment as the underlying cause of the observed decrease in NK cells. There were also no differences in peripheral blood NK cell levels between HNSCC patients with oral, laryngeal and pharyngeal tumors. Thus, we have detected a HNSCC associated decrease in circulating NK cells that is independent of tumor site, known exposures and other putative risk factors and prognostic elements.
These data were not sufficient to determine whether the observed alterations in the balance of peripheral blood leukocytes associated with HNSCC occurred before or after onset of the disease. It is possible that the lower numbers of circulating NK cells in patients with HNSCC is an underlying cause of the disease that is independent from other risk factors. Individuals with lower levels of NK cells could be predisposed to developing cancers, such as HNSCC, due to reduced anti-cancer immune surveillance, since NK cells play a prominent role in the recognition and elimination of incipient cancer cells. The underlying cause of the disease may also be systemic inflammation, which causes the peripheral blood to be flooded with granulocytes hereby causing an apparent decrease in the proportion of NK cells in a blood sample. Alternatively, the lower numbers of circulating NK cells in the peripheral blood of patients with HNSCC could be a result of the disease itself. Tumors manipulate their microenvironment by secreting chemokines that induce suppression and apoptosis of cytotoxic lymphocytes (38
), particularly the CD56dim
NK cell subset (39
). The induction of NK cell death by secreted chemical signals emanating from immunoedited tumor cells may occur to such an extent that systemic alterations in immune cell levels can be detected in the peripheral blood. Likewise, the tumor may stimulate systemic inflammation by secreting proinflammatory cytokines. This could potentially increase the amount of granulocytes in the blood, causing an apparent decrease in the proportion of NK cells.
Prospective studies would provide the best evidence to determine if individuals with lower NK cell numbers are pre-disposed to developing cancer, and whether or not NK cells levels are altered by presence of head and neck cancer. This type of study is more feasible than even before, since our novel DNA methylation-based approach can assess NK cell levels in almost any blood sample, regardless of freshness or special handling regimes. Prospective studies of HNSCC could also shed light on the relationship between circulating NK cell levels and disease outcome. It is possible that extreme changes in the balance of circulating blood cells could compromise the body’s ability to monitor for malignant cells outside the tumor microenvironment, providing favorable conditions for local invasion and metastasis. In addition, immune status may be associated with effectiveness of any treatment, as a proper balance of immune cells could contribute to recovery.