Head and neck cancer bears substantial morbidity, mortality and treatment costs in the United States, which worsen with increasing stage at diagnosis. Thus there is a need for non-invasive tests that facilitate the detection of both early disease and recurrence, as well as those that improve the ability to predict risk of second primary cancers. Here, we describe a novel DNA methylation profile of 6 CpG loci in peripheral blood and assess its potential utility as a diagnostic biomarker of HNSCC.
We have demonstrated the ability of this profile to distinguish HNSCC cases from cancer-free control subjects with a high-degree of accuracy (AUC = 0.73), which further improves when age, gender, smoking, alcohol consumption and HPV16 serostatus are taken into account (AUC = 0.85). The profile also appears to have tumor specificity, as demonstrated by its inability to discriminate bladder cancer cases from controls. Moreover, we found no difference in stage at diagnosis of cases across classes, indicating that our methylation profile works with both early and advanced-stage disease. Although the sensitivity and specificity are not yet sufficient for clinical applications, it may be possible to improve diagnostic accuracy in the future by combining this profile with other biomarkers or diagnostic criteria. Furthermore, our results pave the way for research and development of DNA methylation biomarkers of HNSCC derived from blood. In the future, similar approaches may also allow for utility in predicting response to therapy, disease recurrence and risk of second primary cancer.
Different white blood cell types have different patterns of DNA methylation, and it is therefore conceivable that our observations are the result of significant, systematic changes in large cell populations in the hematopoietic system, such as might be observed in a specific immune response to the tumor.34
In fact, some, but not all, of the CpG loci that we identified are located within genes with either established or purported involvement in immune differentiation or function (FGD4, IL27
If our results are attributable to changes in cell populations it is likely that these changes will be systematically observed in most HNSCCs and that DNA methylation at the loci we identified is very specific for a particular subset of white blood cells. To our knowledge, none of the loci we discovered have been specifically described in the literature as differentially methylated in any subset of white blood cells. Our data suggest a need for further investigation of these loci and their role in any HNSCC immune response. If our findings are the result of an altered peripheral blood profile, we believe it to be important for them to be evaluated for an association with disease outcome, recurrence and second primary cancers.
Alternatively, our findings may be indicative of systemic, developmentally acquired alterations similar in their mechanism of occurrence to constitutional epimutations. There are likely to be DNA methylation events that occur in utero perhaps due to maternal exposures or stochastic errors in the setting of methylation marks. It is possible that certain of these may predispose individuals to HNSCC.38,39
While it is known that some epimutations are high-penetrance and have a very dramatic phenotype,39,40
it is also common for them to exhibit mosaicism and have a phenotype that is more complex. Going forward, the most important test of this hypothesis could come from prospective studies that can assess whether these profiles are present at times that predate disease diagnosis.
Finally, an additional possibility that could contribute to our findings might be an unaccounted cancer-associated exposure that systemically alters DNA methylation directly or alters a mechanism responsible for production of DNA methylation. However, we statistically controlled for the major HNSCC risk factors (smoking, alcohol consumption and HPV16) in our analyses, making it unlikely that any such effect stemming from these exposures is confounding our results. Barring these exposures, we are unaware of any such factor but cannot rule out the possibility that it might exist.
We decided to further explore any similarities in observed methylation patterns of HPV16 seropositive cases and HNSCC by looking at possible overlap between methylation-associated pathways and transcription factor binding sites (TFBS) associated with HNSCC and HPV16 infection. We did not identify any overlapping pathways between HNSCC and HPV16, although there were common functional groupings, but the significance of this, if any, is uncertain. Interestingly, all 6 of our CpG loci were located within 1 kb of a putative TFBS. There was overlap of the POU2F1 transcription factor-binding site, overrepresented in both HNSCC and HPV16 seropositive cases. POU2F1 is expressed in all cell types and plays a role in immunoglobulin production,41
regulation of genes encoding histone H2B42
and small nuclear RNA (snRNA) activation.42
It is involved in both inflammation and epigenetic regulation, in-line with our proposed explanations for alterations in blood methylation; conversely, it may be that the observed commonality of POU2F1 is simply due to chance.
We have identified a novel blood-based methylation profile that is indicative of HNSCC with a high degree of accuracy. Although not yet adequate for use in clinical settings, this profile demonstrates the potential of blood DNA methylation for development of non-invasive applications for detection of head and neck cancer. Future studies should be aimed at continued exploration of blood DNA methylation biomarkers using prospective studies and denser genome-wide methylation arrays with greater CpG coverage that might allow for continued discovery and improvement of blood methylation panels, alone or in conjunction other types of markers, for enhanced sensitivity and specificity. Translational efforts aimed at identification of practical DNA methylation biomarkers, such as these, may eventually result in improvement in the detection and treatment of HNSCC, helping to alleviate the overall public health burden of this disease.