Published OnlineFirst October 1, 2019; DOI: 10.1158/0008-5472.CAN-19-1019 Cancer Genome and Epigenome Research Histone-Related Genes Are Hypermethylated in Lung Cancer and Hypermethylated HIST1H4F Could Serve as a Pan-Cancer Biomarker Shihua Dong1,Wei Li1, Lin Wang2, Jie Hu3,Yuanlin Song3, Baolong Zhang1, Xiaoguang Ren1, Shimeng Ji3, Jin Li1, Peng Xu1, Ying Liang1, Gang Chen4, Jia-Tao Lou2, and Wenqiang Yu1 Abstract Lung cancer is the leading cause of cancer-related deaths lated in all 17 tumor types from TCGA datasets (n ¼ 7,344), worldwide. Cytologic examination is the current "gold stan- which was further validated in nine different types of cancer dard" for lung cancer diagnosis, however, this has low sensi- (n ¼ 243). These results demonstrate that HIST1H4F can tivity. Here, we identified a typical methylation signature of function as a universal-cancer-only methylation (UCOM) histone genes in lung cancer by whole-genome DNA methyl- marker, which may aid in understanding general tumorigen- ation analysis, which was validated by The Cancer Genome esis and improve screening for early cancer diagnosis. Atlas (TCGA) lung cancer cohort (n ¼ 907) and was further confirmed in 265 bronchoalveolar lavage fluid samples with Significance: These findings identify a new biomarker for specificity and sensitivity of 96.7% and 87.0%, respectively. cancer detection and show that hypermethylation of histone- More importantly, HIST1H4F was universally hypermethy- related genes seems to persist across cancers. Introduction to its low specificity, LDCT is far from satisfactory as a screening tool for clinical application, similar to other currently used cancer Lung cancer is one of the most common malignant tumors and biomarkers, such as carcinoembryonic antigen (CEA), neuron- the leading cause of cancer-related deaths worldwide (1, 2). Early specific enolase, CYFRA 21-1, etc. Therefore, effective biomarkers detection and surgery offer the best chance for survival, with the for early detection, diagnosis, prognosis, and monitoring of lung 5-year survival rate as high as 80% (3). However, most patients cancer are urgently needed (7). with lung cancer have been diagnosed with inoperable advanced Epigenetic and genetic abnormalities are hallmarks of stage with metastasis, and patients must undergo chemotherapy, lung cancer (8–10). Abnormal DNA methylation is the most radiotherapy, immunotherapy, or targeted therapy. The 5-year common epigenetic variation in the process of lung cancer. survival rate of patients in the advanced stage is below 10% (4, 5). Compared with DNA mutations, DNA methylation occurs Over the past decade, low-dose CT (LDCT) is the most commonly much earlier and is more stable in the early diagnosis of used screening method for lung cancer, which has been shown to tumors, and aberrant DNA methylation pattern can be used improve early detection and reduce mortality (6). However, due for predicting the liver cancer metastasis to lung (11). Although many DNA methylation biomarkers have been reported, they 1 are still under the exploration process and rarely used in clinical Shanghai Public Health Clinical Center and Department of General Surgery, fi Huashan Hospital, Cancer Metastasis Institute and Laboratory of RNA Epige- applications. Sensitivity and speci city of current methylation fi netics, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan markers are insuf cient with high false positives and false University, Shanghai, China. 2Department of Laboratory Medicine, Shanghai negatives risk (12, 13). Therefore, applying methylation mar- Chest Hospital, Shanghai Jiao Tong University, Shanghai, China. 3Department of kers to clinical applications is challenging, and searching for Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai, China. new biomarkers for the early detection of cancer is urgently 4 Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, needed (14). China. Histones are major essential components of chromatin and Note: Supplementary data for this article are available at Cancer Research conserved in eukaryotic cells (15). There are five major types of Online (http://cancerres.aacrjournals.org/). histones: H1, H2A, H2B, H3, and H4. Histones H2A, H2B, H3, S. Dong, W. Li, L. Wang, J. Hu, and Y. Song contributed equally to this article. and H4 are known as the core histones, whereas histone H1 is Corresponding Authors: Wenqiang Yu, Fudan University, 130 Dong'an Road, known as the linker histone (16). Histones are divided into West 13# Building, Room 419, Shanghai 200032, China. Phone: 8621-5423-7978; canonical replication-dependent histones that are expressed dur- Fax: 8621-5423-7339; E-mail: [email protected]; and Jia-Tao Lou, ing the S-phase of the cell cycle and replication-independent Department of Laboratory Medicine, Shanghai Chest Hospital, 241 West Huaihai histone variants, which are expressed during each phase of the Road, Shanghai 200030, China. Phone: 86212-22000-01503; Fax: 8621-6280- cell cycle. Genes encoding canonical histones are intron-less and 8279; E-mail: [email protected] lack a polyA tail at the 30 end, having instead a stem-loop – Cancer Res 2019;79:6101 12 structure, and canonical histone genes also tend to be clustered doi: 10.1158/0008-5472.CAN-19-1019 in the genome. Genes encoding histone variants are usually not Ó2019 American Association for Cancer Research. clustered and have introns and polyA tails (17, 18). In the human www.aacrjournals.org 6101 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst October 1, 2019; DOI: 10.1158/0008-5472.CAN-19-1019 Dong et al. genome, histone genes mainly form histone cluster 1 (Chr6p21) The Cancer Genome Atlas DNA methylation data analysis and histone cluster 2 (Chr1q21; ref. 19). Other histone genes are The Illumina 450K methylation array level three data from The distributed randomly in the human genome. Although histone Cancer Genome Atlas (TCGA) database were downloaded from modifications have been extensively studied in chromatin regu- the UCSC Xena browser (https://xenabrowser.net/). For each lation, epigenetic variation in the family of histone genes them- histone gene, only probes within the gene-body region (listed selves is rarely considered. It has been shown that histone gene in Supplementary Table S2) were selected to calculate an average cluster 1 is occupied by abnormally higher order chromatin methylation value. Probes with "NA" values were excluded. The organization in breast cancer (20). However, DNA methylation absolute methylation values were calculated from the b values of alteration in histone genes' loci has not yet been systematically 450K methylation array [methylation value ¼ (b value þ 0.5) Â investigated, especially in cancer development. 100%]. For each gene, the final methylation value was calculated Here, through genome-wide DNA methylation analysis by the average of all CpG sites selected. The samples used from with an unusual strategy, we found that many histone gene TCGA database and the methylation levels of HIST1H4F are listed loci are abnormally hypermethylated in lung cancer, which in Supplementary Table S3. piqued our interest for further investigation. We demonstrate that methylation of histone genes can be used as a biomarker Clinical samples for early detection in bronchoalveolar lavage fluid (BALF) We collected 243 primary tissue samples and 265 BALF samples. Furthermore, histonegenelociarenotonlyabnor- samples from Shanghai Chest Hospital and Zhongshan Hos- mally hypermethylated in lung cancer but also specifically pital of Fudan University. Primary tissue samples included 25 methylated in various tumors. In particular, the HIST1H4F lung cancer and 25 paired para-cancer control samples, 12 gene is abnormally hypermethylated in 17 types of cancer, colorectal cancer and 12 paired para-cancer control samples, which could act as a potential universal-cancer-only methyla- 10 esophagus cancer and 12 paired para-cancer control sam- tion (UCOM) marker. We speculate that the methylation of ples, 20 liver cancer and 23 para-cancer control samples, nine HIST1H4F will be of great significance for early diagnosis, pancreatic cancer and nine paired para-cancer control samples, especially during the screening process of cancer in clinical 10 cervical cancer and 10 control samples, 10 gastric cancer and applications. 10 para-cancer control samples, 14 breast cancer and 14 paired para-cancer control samples, and 10 head and neck cancer and 10 paired para-cancer control samples. Clinical characters of Materials and Methods thesesamplesaresummarizedinSupplementaryTableS4. Whole genome bisulfite sequencing data analysis BALF samples contained a benign lung disease (BLD) control Whole genome bisulfite sequencing (WGBS) datasets were group and lung cancer group. BLD control group contained 59 downloaded from the Encode database (https://www.encodepro samples, including pneumonia, emphysema, tuberculosis, etc. ject.org/) and the SRA database (https://www.ncbi.nlm.nih.gov/ The lung cancer experimental group included 92 lung squa- sra); the serial numbers are summarized in Supplementary mous cell carcinoma (LUSC) samples, 70 lung adenocarcinoma Table S1. DNA methylation levels were calculated using BSMAP samples, and 44 small-cell lung carcinoma (SCLC) samples. software (21) as described previously (11), where hg19 human BALFsampleswererandomlyassignedtoatrainingsetanda genome assembly and University of California,