19Background: Changes in DNA methylation over the course of life may provide 20 an indicator of risk for cancer. We explored longitudinal changes in CpG 21 methylation from blood leukocytes, and likelihood of a future cancer diagnosis. 22Methods: Peripheral blood samples were obtained at baseline and at follow-up 23 visit from 20 participants in the Health, Aging and Body Composition prospective 24 cohort study. Genome-wide CpG methylation was assayed using the Illumina 25 Infinium Human MethylationEPIC (HM850K) microarray. Results: Global 26 patterns in DNA methylation from CpG-based analyses showed extensive 27 changes in cell composition over time in participants who developed cancer. By 28 visit year 6, the proportion of CD8+ T-cells decreased (p-value = 0.02), while 29 granulocytes cell levels increased (p-value = 0.04) among participants diagnosed 30 with cancer compared to those who remained cancer-free (cancer-free vs. 31 cancer-present: 0.03 ± 0.02 vs. 0.003 ± 0.005 for CD8+ T-cells; 0.52 ± 0.14 vs. 32 0.66 ± 0.09 for granulocytes). Epigenome-wide analysis identified three CpGs 33 with suggestive p-values ≤ 10 -5 for differential methylation between cancer-free 34 and cancer-present groups, including a CpG located in MTA3, a gene linked with 35 metastasis. At a lenient statistical threshold (p-value ≤ 3 x 10 -5 ), the top 10 36 cancer-associated CpGs included a site near RPTOR that is involved in the 37 mTOR pathway, and the candidate tumor suppressor genes REC8, KCNQ1, and 38 ZSWIM5. However, only the CpG in RPTOR (cg08129331) was replicated in an 39 independent data set. Analysis of within-individual change from baseline to Year 40 6 found significant correlations between the rates of change in methylation in 41 RPTOR, REC8 and ZSWIM5, and time to cancer diagnosis. Conclusion: The 42 results show that changes in cellular composition explains much of the cross-43 sectional and longitudinal variation in CpG methylation. Additionally, differential 44 methylation and longitudinal dynamics at specific CpGs could provide powerful 45 indicators of cancer development and/or progression. In particular, we highlight 46CpG methylation in the RPTOR gene as a potential biomarker of cancer that 47 awaits further validation. 48 DNA methylation plays a central role in cell differentiation and in defining cellular 60 phenotypes. Differences in DNA methylation have been associated with a 61 growing list of morbidities, ranging from metabolic disorders and age-related 62 decline in health, to developmental and neuropsychiatric conditions. The 63 standard approach in an epigenome-wide association study (EWAS), which 64 attempts to link DNA methylation to disease, involves collection of a single 65 biospecimen from each participant (typically peripheral blood or saliva) and 66 performing cross-sectional analyses to compare methylation patterns in cases 67 against matched healthy controls [1, 2]. While differences in CpG methylation 68 between cases and controls may be directly related to disease, these case-69 control diff...

Background Changes in DNA methylation over the course of life may provide an indicator of risk for cancer. We explored longitudinal changes in CpG methylation from blood leukocytes, and likelihood of future cancer diagnosis. Methods Peripheral blood samples were obtained at baseline and at follow-up visit from 20 participants in the Health, Aging and Body Composition prospective cohort study. Genome-wide CpG methylation was assayed using the Illumina Infinium Human MethylationEPIC (HM850K) microarray. Results Global patterns in DNA methylation from CpG-based analyses showed extensive changes in cell composition over time in participants who developed cancer. By visit year 6, the proportion of CD8+ T-cells decreased ( p -value = 0.02), while granulocytes cell levels increased ( p -value = 0.04) among participants diagnosed with cancer compared to those who remained cancer-free (cancer-free vs. cancer-present: 0.03 ± 0.02 vs. 0.003 ± 0.005 for CD8+ T-cells; 0.52 ± 0.14 vs. 0.66 ± 0.09 for granulocytes). Epigenome-wide analysis identified three CpGs with suggestive p -values ≤10 − 5 for differential methylation between cancer-free and cancer-present groups, including a CpG located in MTA3, a gene linked with metastasis. At a lenient statistical threshold (p-value ≤3 × 10 − 5 ), the top 10 cancer-associated CpGs included a site near RPTOR that is involved in the mTOR pathway, and the candidate tumor suppressor genes REC8, KCNQ1 , and ZSWIM5 . However, only the CpG in RPTOR (cg08129331) was replicated in an independent data set. Analysis of within-individual change from baseline to Year 6 found significant correlations between the rates of change in methylation in RPTOR , REC8 and ZSWIM5 , and time to cancer diagnosis. Conclusion The results show that changes in cellular composition explains much of the cross-sectional and longitudinal variation in CpG methylation. Additionally, differential methylation and longitudinal dynamics at specific CpGs could provide powerful indicators of cancer development and/or progression. In particular, we highlight CpG methylation in the RPTOR gene as a potential biomarker of cancer that awaits further validation. Electronic supplementary material The online version of this article (10.1186/s40364-019-0161-3) contains supplementary material, which is available to authorized users.