We recently constructed epigenetic profiles of HNSCC, reporting that DNA methylation events are common and associated with etiologically important exposures 
. Aberrant DNA methylation events have been hypothesized to accumulate initially in a stochastic fashion and, through positive selection, result in clones that have a growth advantage that leads to the genesis of a rapidly-dividing tumor. Here we expand upon these data and include an analysis of chromosomal integrity in these same tumors. Using genomic-level measurements, we observed a highly significant association between copy number and DNA methylation profiles, definitively showing that these modes of gene regulation are linked in HNSCC. These observations supplement recent evidence from Sadikovic et al.
that copy number alterations are generally correlated with both methylation and gene expression levels in osteosarcomas 
. At the same time, while specific targeting of genes through both mechanisms occurs in a deterministic manner within subgroups of patients, when we tested for regionally matching local (gene level) epigenetic and copy number events we only observed that global, rather than local, alterations were correlated. This indicates that coordinated two-hit gene inactivation (LOH followed by epigenetic silencing) is not the dominant character of somatic alteration over the genome. As the GoldenGate methylation array investigates nearly 800 cancer-involving genes and is enriched for tumor suppressor-associated loci, we were uniquely positioned to investigate just this question. Recent evidence supports our conclusion, as gene regulation by CNA and DNA methylation measured at 691 loci in meningiomas appears to be somewhat mutually exclusive 
. In addition, our combined analysis of the promoter regions of previously reported genes with allele loss or hypermethylation demonstrates that this situation is rare (see Table S2
), however a much larger investigation with higher resolution is needed to determine if these alterations occur systematically.
One possible explanation for the association between global profiles of DNA methylation and copy number is that amplification or loss of genetic material may result in a bias of measured methylation for CpGs within that region, potentially contributing to the inferred methylation profile (e.g. in our RPMM approach). Indeed, previous microarray-based methods to determine methylation status have been hindered by copy number changes that bias the measured relative methylation values at CpG loci 
. However, our recent work utilizing bead-arrays has shown that CNA produces little bias in absolute methylation data generated on the GoldenGate methylation panel, except in the case of homozygous deletion 
. We and others have previously demonstrated the validity of Illumina GoldenGate methylation array results with other high- and low-throughput technologies 
Integrative analysis revealed that several tumors with similar methylation profiles had large regions of chromosomal abnormality, particularly in chromosomes 8 and 3, consistent with the possible formation of isochromosomes i(8q) and i(3q) in aneuploid cells. These cytogenetic abnormalities commonly appear in HNSCC, possibly a result of chromosomal missegregation events during mitosis 
. We also observed that Methylation Class 3 tumor data reflect gross allelic amplification of 8q, which extends through the centromere and partially into 8p, possibly indicating a distinct mechanism of formation for this anomaly. Among tumors with an amplified 8q arm, several methylated CpG loci were observed in this region relative to tumors without this gross chromosomal alteration. Two mechanisms can be posited to explain this result. Firstly, epigenetic dysregulation may occur early in the genesis of these head and neck tumors and aberrant methylation marks are faithfully replicated despite the amplification event, which is consistent with previous reports implicating epigenetic modification as an early event in the progression of this disease [reviewed in 38]
. In fact, there is evidence that aberrant methylation in certain chromosomal regions, especially located near centromeres, predisposes the surrounding area to genetic alteration, including fragile breakpoint sites 
. On the other hand, it is possible that this differential methylation occurs following the chromosomal aberration, possibly in response to the genetic event and selective pressures. However, we are unable to distinguish between these possibilities in our data, highlighting the need for mechanistic studies.
Gain of 8q has been reported as a relatively common event in HNSCC, particularly at 8q24 
, which houses the MYC
oncogene, and 8q22, thought to be targeting LRP12 
. Similarly, in one-third of our cases we observed amplification of this entire arm, while putative tumor suppressor genes, such as SOX17
, within this amplified chromosomal arm are methylated. These findings are suggestive of a context wherein genetic modification (possibly a result of genomic instability) is responsible for perpetuating inappropriate oncogene expression with concomitant epigenetic silencing of local tumor suppressors ().
Molecular alterations in chromosome 3 have been previously reported as the most prevalent and potentially most important in HNSCC 
. Consistent with these findings, we observed extensive copy number and methylation alterations in this chromosome. For example, the gastric cancer-associated tumor-suppressor HRASLS
in the amplified q-arms were more highly methylated than those tumors which possessed normal 3q. In addition, the proto-oncogenic MST1R
loci, associated with poor prognosis through potentiation of cell scattering and invasion in breast cancer 
, were unmethylated in most tumors irrespective of chromosome 3p loss. However, we observed a number of genes that did not follow the expected directions of methylation within copy number variable loci, indicating that they may be hitchhikers or simply regulated by other genetic or epigenetic means. Overall, these structural modifications in chromosomes 3 and 8 are consistent with the literature and are thought to develop early during the genesis of disease 
Although 13 of the tumors examined (Methylation Classes 1, 3, and 6, ) demonstrated a preponderance of CNA, we observed a notable lack of CNA among the remaining tumors. We hypothesize that this may be due to these samples having higher levels of aberrant epigenetic or non-copy number altering genetic events such as mutation or chromosomal rearrangements. It is also possible that clinical stage could account for the observed levels of abnormal copy number, as this has been reported in other cancers 
. Other possible confounders include HPV16 status and tumor site, although our data do not indicate associations between any of these covariates and CNA. Larger future studies are required to investigate the nature of these notable differences with statistical rigor.
There was also an apparent relationship between global hypomethylation, represented by the extent of LINE-1 DNA sequence methylation, and the increased levels of allelic imbalance among HNSCC cases. This finding is consistent with the literature 
and with the hypothesis that global hypomethylation of transposable elements culminates in genomic instability. While it is apparent that the various modes of alteration are related, the timing of these events is less clear. Our data underscore the need for additional investigations into the chronology of multifaceted somatic alterations leading to the onset and progression of this deadly disease.
In our analysis to define the local relationships between copy number and methylation, we observed only one gene (where promoter-associated CpG alterations were averaged), HOXA11
, with a marginally significant correlation between methylation and copy number alteration, although a number of individual CpG loci reached significance, including sites within potentially oncogenic GRB10, IHH, and HOXA11
. While the strong positive correlation at these sites could indicate selection pressure for dual mechanism inactivation was occurring to promote neoplasm formation, there was little evidence of this pressure acting over the entire set of measured genes. Our finding that different genes are preferentially targeted through different mechanisms in HNSCC could reflect dramatic differences in the timing of these events (e.g. one type of somatic changes predominating early in clonal evolution with the other becoming dominant later in clonal evolution). Alternatively, it is possible that other simultaneous genetic events obviated the need for epigenetic modifications (e.g. copy number-activating mutations common in other cancers 
) or that sequence context (e.g. proximity to fragile sites or the CpG content of promoter regions) may interact with carcinogen exposure to select the order and the type (epigenetic or genetic) of alteration that inactivates genes. At the same, our stratified analysis of the two main biological methylation subgroups revealed that the events leading to abnormal copy number and CpG methylation are fundamentally different in each group, suggestive of an overall collateral relationship.
In sum, epigenetic profiles in HNSCC are significantly associated with the extent of CNA, but this global relationship is not widely reflected at the local level. Furthermore, the molecular targets of each are dissimilar. The precise mechanisms responsible for gene inactivation are obscure but in the framework of carcinogenic progression within Knudson's two-hit model, our data indicate that local, coordinate DNA methylation and copy number alteration do not dominate the profile of changes in primary HNSCC.