In this study of 117 well-characterized NSCLC subjects, we assessed the gene methylation status for 27 genes in tumor tissue blocks with a number of patient and tumor characteristics using quantitative MethyLight assays. Methylation at any level in at least one gene was present in 91% of tumor samples, and a high level of methylation (PMR≥4%) in at least one gene was present in 79% of tumor samples. Conversely, we previously showed that only one of the genes (APC) is methylated at high levels in more than 10% of matched noncancerous tissue from NSCLC patients and that only 5 of the other 26 genes in the current study (RARB, CCND2, CDH1, CDKN2A, and OPCML) were ever methylated (in 2-8% of patients) at high levels in noncancerous tissues (19
). In the current study we found that methylation of a number of genes was significantly associated with histologic type; adenocarcinoma was associated with increased methylation of APC, CCND2, KCNH5, and RUNX, and squamous cell carcinoma was associated with increased methylation of CDKN2A. There were trends towards larger tumors being associated with increased methylation frequency. Finally, females with lung cancer were significantly more likely than males to have methylation of a number of genes, including KCNH5, KCNH8, and CDH13.
Our findings of substantial differences in gene methylation patterns depending on histologic typing of NSCLCs have been reported to some degree in the literature. Similar to what we observed in the present study, a number of others (13
) have noted that APC is more frequently methylated in adenocarcinomas compared to squamous cell carcinomas, although this has not always been noted (38
). Similarly, methylation of RUNX was detected with higher frequency in adenocarcinomas compared to squamous cell carcinomas in previous studies (16
). Conversely, methylation of CDKN2A (also known as p16INK4a
) has previously been reported to be more common in squamous cell carcinomas compared to adenocarcinomas in most (13
) but not all (21
) prior studies. However, we believe that our findings that methylation of CCND2 is frequent in adenocarcinomas (46%) but not squamous cell carcinomas (3%) and that methylation of KCNH5 and KCNH8 is less frequent in adenocarcinomas (26% and 12%, respectively) but is specific to this class of tumors, are new to the literature. Aberrant methylation of cyclin D2 (CCND2) has been previously noted as potentially contributing to the pathogenesis of NSCLC (41
). However, in that report, potential differences in methylation of CCND2 between squamous and adenocaricomas in NSCLC tumors were not evaluated. Methylation of APC and CCND2, while common in cancer tissues, is also detected frequently in matched non-cancerous lung tissue (19
), suggesting that methylation of these two genes may not be a cancer-specific change, but may be the result of environmental exposure, perhaps due to smoking.
We noted a number of important differences in hypermethylation frequency in females compared to males, even after taking into account histologic type. Females with adenocarcinoma were significantly more likely to have methylated CDH13, KCNH5, KCNH8, and RARB compared to males. Interestingly, these differences were not observed in squamous cell carcinomas, although KCNH5 and KCNH8 were not hypermethylated in any squamous cell cancers, regardless of gender. Conversely, males with squamous cell carcinomas were more likely to have hypermethylation of APC, BVES, and CDKN2A (p16INK4a
). These substantial differences have not been consistently noted in the literature (16
), perhaps because few studies have stratified by histologic type when assessing hypermethylation differences by gender. Since females are more likely to have adenocarcinomas compared to males (43
), in other studies, differences in hypermethylation frequency due to gender may have been obscured by differences due to histologic type. These substantial differences in hypermethylation by gender suggest the possibility of differential pathways and/or risk factors for NSCLC between genders. Hypermethylation of RARB was similarly common (31%) in males and females with squamous cell carcinoma, but was substantially more common in females with adenocarcinama (54%) or large cell carcinoma (57%) compared to males (11% and 23%, respectively).
Gender differences have historically been noted and debated in the literature with respect to susceptibility and risk factors for NSCLC, especially with regards to the effect of cigarette smoking as well as survival and effectiveness of treatment. The relevance of these variations remains unclear (6
). Epidemiologic studies have generally noted that women with NSCLC are more likely to have developed adenocarcinoma, tend to be younger than men, and are more likely never to have smoked (4
). Additionally, for a given stage and treatment regimen, women may have better outcomes than men (7
). Hormonal factors may account for differences between men and women with regards to lung cancer (50
) although how hormones relate to gene methylation patterns in NSCLC remains unclear. In one study, estrogen receptor (ER) alpha promoter methylation was increased in males with NSCLC compared to females and was associated with poorer prognosis (52
). Another proposed explanation is that there is decreased DNA-repair capacity in women compared to men (53
), although we did not find evidence that methylation of DNA repair associated genes was differential in males compared to females in the five DNA-repair genes we studied (FHIT, MLH1, MGMT, FANCF, and GSTP1).
To our knowledge, hypermethylation of KCNH5 and KCNH8, two voltage-gated, ether a go-go (Eag 1) family potassium channels, has not been previously studied with regards to human cancers, although KCNH2 has been reported to be downregulated in breast cancer (54
). The role of potassium channels in cancer has gained increasing interest in recent years as a possible target for tumor therapy (55
). In the present study, hypermethylation of KCNH8 was observed in tumors from six (25%) of 24 females with adenocarcinoma, but was not observed in any males with adenocarcinoma (n=27) or tumors from squamous cell carcinomas from either gender (n=39). Interestingly, hypermethylation of KCNH8 was present in five (71%) of 7 female cases but only one (8%) of 13 male cases of large cell carcinoma. With regards to hypermethylation of KCNH5, we observed similar but less dramatic differences. Importantly, we previously reported that hypermethylation of KCNH5 and KCNH8 is specific to cancerous tissue, as none of 49 matched noncancerous lung tissues from patients with NSCLC were hypermethylated (19
Our study, and its comparison to other's work, has a number of important limitations. Detection of methylation of the various genes is dependent on the primers and probes used to indicate specific sequence regions, and differences in gene-specific detection rates and as well as associations with clinical features of the tumors may be due to variation in the methodologies used across studies. In the current study, we used quantitative MethyLight, with a cut-off of 4% PMR, to determine associations with methylation. This method is considered sensitive and specific for gene-specific methylation (30
). Study findings were similar when a cut-off of 0% PMR was utilized. However, a number of methods, some quantitative and others less sensitive and/or specific qualitative procedures, have been utilized in previous reports, so direct comparisons in study findings across studies may not be entirely possible. In the literature, methylation of FHIT, GSTP1, and MGMT frequently occurs in NSCLC (21
) but in the current study, methylation of these genes was rarely detected. The gene-specific discrepancies we observed are likely due to a number of factors, such as assay-specific differences including target sites of CpG island loci, primers or probes, assay conditions including annealing temperatures and the conditions of sodium bisulfaite conversion, differences in tumor characteristics such as type of sample, tumor histology, and level of tumor heterogeneity, and finally, differences in patient populations with regards to age and gender. Primary tumor blocks were unavailable for many of the subjects in the original imaging study, potentially limiting the generalizability of our findings, and sample sizes within histologic subtypes and gender were limited. Further, we were unable to assess the effect of smoking status on methylation in those with NSCLC, as only 5 subjects (4%) were never smokers. Our findings may not be generalizable to populations in which a larger proportion of NSCLC is attributed to cigarette smoking. The distribution of histologic types in the current study was somewhat dissimilar to cases of NSCLC in the United States in general, as squamous and large cell carcinomas were somewhat overrepresented (43
). However, the large proportion of non-adenocarcinoma cases and relative heterogeneity of our study tissue samples with respect to histologic subtypes allowed us to identify important differences between adenocarcinomas and squamous cell NSCLCs, comparisons which have rarely been assessed in other studies due to the homogenous nature of their tumor samples. Finally, it is unclear how strongly methylation status is correlated with expression status for the examined genes. Gene expression analyses were not conducted in the current study. However, prior studies using MethyLight have shown that promoter methylation (PMR>4%) of CDKNsA, MLH1, and MGMT was strongly associated with respective loss of protein expression assessed by immunohistochemistry in colorectal cancers (32
). In a more recent study in diffuse large B-cell lymphoma, there was good correlation between the presence of MGMT protein expression and unmethylated status using MethyLight (34
) and MethyLight performed better than qualitative methylation-specific PCR (MSP). However, in another study, methylation of DAPK (using MSP) was not related with gene silencing in lung cancer cell lines and a number of CpG dinucleotides were often methylated in expression-negative tissues (59
). Clearly there may be mechanisms other than promoter hypermethylation contributing to down-regulation of gene expression.
In summary, in the present study of 117 patients with NSCLC, we found that methylation of a number of genes was strongly associated with histologic type; adenocarcinoma was associated with increased methylation of APC, CCND2, and RUNX, squamous cell carcinoma was associated with increased methylation of CDKN2A (p16INK4a), and large cell carcinoma was associated with increased methylation of KCNH8. Among patients with adenocarcinoma, females were significantly more likely to have methylated CDH13, KCNH5, KCNH8, and RARB compared to males. Conversely, males with squamous cell carcinomas were more likely to have hypermethylation of APC, BVES, and CDKN2A (p16INK4a).
The dissimilar pattern of gene hypermethylation in adenocarcinomas compared to other forms of NSCLC implies different pathogenesis of cancer by histologic type. Further, our findings of differential gene hypermethylation frequencies among those with adenocarcinoma or squamous cell cancers in females compared to males suggests that further investigation is warranted in order to more fully understand the potential disparate pathways and risk factors, especially with regard to smoking and hormones, as well as potential differential treatment modalities for NSCLC associated with histology and gender.