Genome-wide methylation profiling reveals differentially methylated genes in blood DNA of small-cell lung cancer patients

the epige-netic biomarkers under investigation. We here report a study that identified aberrantly methylated genes between current and former smokers among SCLC patients, revealing a set of candidate biomarkers in peripheral blood DNA for better stratifying patients with high risk.


Dear Editor,
Small-cell lung cancer (SCLC), one of the most-deadly malignant tumors, accounts for ∼15% of all lung cancers. 1,2 Although highly responsive to the standard chemoradiotherapy, the recurrence rate of SCLC is nearly unity, with median survival time ranging from 2-6 months. Almost all SCLC patients are inoperable, leading to the difficulty in obtaining adequate biopsy tissue for research. 3 Consequently, there is an urgent need to find noninvasive biomarkers to predict the risk and provide early diagnosis of SCLC. We aimed to identify potential epigenomic markers for SCLC development in blood DNA and reveal differentially methylated genes through a multi-omics approach, from genome-wide DNA methylation profiling, followed by pathway enrichment analyses and target gene validation utilizing Gene Expression Omnibus (GEO) and Oncomine microarray databases, to TCGA Pan-Cancer cohorts. It is known that virtually all patients with SCLC are victims of tobacco smoking, 4 but it is unknown whether stopping smoking before SCLC diagnosis would influence the epigenetic biomarkers under investigation. We here report a study that identified aberrantly methylated genes between current and former smokers among SCLC patients, revealing a set of candidate biomarkers in peripheral blood DNA for better stratifying patients with high risk.
Specifically, we analyzed 47 paired SCLC cases and controls with a pathological diagnosis of primary tumor at the Mayo Clinic (USA), under an Institution Review Board (IRB) approved protocol (225-99). Enrolment, diagnosis, and data collection processes are provided in the online supplementary material. By a matching design, no difference in age, sex or pack-year was found between cases and controls (Table S1, see online supplementary material). A total of 27 patients (57.4%) had limited-stage and 20 (42.6%) had extensive-stage SCLC. All subjects smoked cigarettes; >50% of cases (59.6%) and controls (53.2%) being current smokers at the time of diagnosis, with mean pack-years of 44.6 and 44.3, respectively.

Differentially methylated CpG sites in SCLC.
The magnitude of methylation of 25 550 CpG were analyzed between cases and controls ( Fig. 1A), resulting in 46 differential CpG sites between cases and controls (37 hyper-and 9 hypo-methylated). The distribution of differentially methylated CpGs showed that >60% were located outside of CpG islands (CGIs) [ Fig. S1 (see online supplementary material), 16 inside and 30 outside of CGIs]. For the 16 In-CGIs, the overall methylation status was significantly higher in cases than in controls (β = 0.58 ± 0.19 vs 0.53 ± 0.18, P < 0.001). For the 30 Out-CGIs, the overall methylation status remained higher in cases than in controls (β = 0.52 ± 0.18 vs 0.50 ± 0.17, P < 0.001). The Manhattan plot indicates that the 46 SCLC CpGs are spread across all autosomes (Fig. S2, see online supplementary material).

Differentially methylated CpGs stratified by smoking status.
To further classify the differentially methylated CpGs stratified by smoking status, we conducted four comparisons ( Fig. 1B): all subjects (G1), controls (G2), former smokers (G3), and current smokers (G4). As a result, 154 CpGs showed significant differences in all subjects, 141 CpGs showed significant differences in the controls, 550 CpGs showed significant differences in former smokers, and 93 CpGs showed significant differences in current smoker. All 46 CpGs in the overall analysis were identified as significantly differentially methylated CpGs in the subgroup analysis. As shown in Fig. 1B, the distribution of the 46 SCLC CpGs among the 4 comparisons was: 6 CpGs (4 hyper-and 2 hypo-) were found in all subjects (G1), 9 CpGs (8 hyper-and 1 hypo-) in former smokers (G3), 16 CpGs (12 hyper-and 4 hypo-) in current smokers (G4), 14 CpGs (13 hyper-and 1 hypo-) in both former and current smokers (G3 and G4), and 1 CpG with hypermethylation was identified in all groups (G1-G4).
Pathway analysis of genes with differentially methylated CpGs. These 46 differential CpGs were mapped to 43 genes (Table S2, see online supplementary material). A total of 16 genes with aberrantly methylated CpGs indicated a high risk for SCLC in current smokers, while 9 aberrantly methylated genes indicated a  high risk in former smokers. Fourteen methylated genes were determined to increase the risk of SCLC in both former and current smokers. Six methylated genes being identified only in the SCLC group suggested that they were hazardous to SCLC regardless of smoking status. One methylated gene was observed in all four comparison groups, indicating that it is a risk factor for smokers independent of SCLC. KEGG pathway analysis (Fig. S3, see online supplementary material) showed that the 43 genes were involved in apoptosis, inflammatory bowel disease, and hypoxia-inducible factor (HIF)-1 signaling. The 16 differentially expressed genes related to SCLC in current smokers were related to inflammatory bowel disease, adipocytokine signaling, and hematopoietic cell lineage. The 9 genes related to SCLC in former smokers were mainly enriched in drug metabolism. The 14 genes associated with SCLC in both former and current smokers were mainly enriched in transcriptional misregulation in cancer and apoptosis.

Validation of genes indicated by differentially methylated CpGs using GEO datasets.
To evaluate the reproducibility, we analyzed the methylation profile in 28 SCLC tissue samples and 13 noncancerous lung tissues from GSE50412. Of the significantly differentially methylated CpGs found in our study, only one aberrant methylation (cg04992673) of the HEPACAM2 gene showed a significant difference between SCLC and adjacent normal tissues in GSE50412. To further examine the biological relevance, we examined the gene expression using GSE43346. Eight differentially methylated genes showed differential expression (FDR < 0.01) in lung cancer: (up: SOD3, CBX7, RORC, ABHD14A, NDUFV1, LGALS, and PLD4; down: MPHOSPH9). In the subgroup analyses, SOD3, CBX7, and RORC genes were associated with susceptibility of SCLC in current smokers, while the ABHD14A gene was associated with susceptibility in former smokers; MPHOSPH9 and NDUFV1 genes were strongly associated with SCLC risk in both current and former smokers; and LGALS and PLD4 genes showed a high risk to the development of SCLC but independent of smoking status.
Oncomine analysis. To further confirm these findings, we identified 5 genes with significant dysregulation in lung cancer based on Oncomine microarray databases. The MPHOSPH9 gene showed significant upregulation in SCLC. 5,6 The PLD4, 7 ABHD14A, 7 NDUFV1, 7-9 and RORC 8 genes showed significant upregulation in non-SCLC rather than SCLC.
In summary, our findings indicate that methylation and genome profiles are significantly different between SCLC patients who continue smoking and those who have quitted before diagnosis, suggesting that the blood-based DNA methylated CpGs could be a potential marker for the early detection of SCLC, with differing candidates based on patients' smoking status.A potential mechanism of genes discovered in our study is the relevance to immune response in SCLC; particularly, some of the enriched Gene Ontology (GO) categories point to inflammation and even hematopoietic lineages. Therefore, we speculate that the changes in DNA methylation may correlate to, and/or be a result of changes in the immune component. Delineation of this mechanistic link will be the focus of future studies to investigate whether the differential methylation and methylation-indicated genes before vs. after smoking cessation constitute feasibly testable markers for early diagnosis of SCLC.

Supplementary data
Supplementary data is available at PCMEDI Journal online.