Evolving DNA methylation and gene expression markers of B-cell chronic lymphocytic leukemia are present in pre-diagnostic blood samples more than 10 years prior to diagnosis

Abstract

Background: B-cell chronic lymphocytic leukemia (CLL) is a common type of adult leukemia. It often follows an indolent course and is preceded by monoclonal B-cell lymphocytosis, an asymptomatic condition, however it is not known what causes subjects with this condition to progress to CLL. Hence the discovery of prediagnostic markers has the potential to improve the identification of subjects likely to develop CLL and may also provide insights into the pathogenesis of the disease of potential clinical relevance.

Results: We employed peripheral blood buffy coats of 347 apparently healthy subjects, of whom 28 were diagnosed with CLL 2.0-15.7 years after enrollment, to derive for the first time genome-wide DNA methylation, as well as gene and miRNA expression, profiles associated with the risk of future disease. After adjustment for white blood cell composition, we identified 722 differentially methylated CpG sites and 15 differentially expressed genes (Bonferroni-corrected p < 0.05) as well as 2 miRNAs (FDR < 0.05) which were associated with the risk of future CLL. The majority of these signals have also been observed in clinical CLL, suggesting the presence in prediagnostic blood of CLL-like cells. Future CLL cases who, at enrollment, had a relatively low B-cell fraction (<10%), and were therefore less likely to have been suffering from undiagnosed CLL or a precursor condition, showed profiles involving smaller numbers of the same differential signals with intensities, after adjusting for B-cell content, generally smaller than those observed in the full set of cases. A similar picture was obtained when the differential profiles of cases with time-to-diagnosis above the overall median period of 7.4 years were compared with those with shorted time-to-disease. Differentially methylated genes of major functional significance include numerous genes that encode for transcription factors, especially members of the homeobox family, while differentially expressed genes include, among others, multiple genes related to WNT signaling as well as the miRNAs miR-150-5p and miR-155-5p.

Conclusions: Our findings demonstrate the presence in prediagnostic blood of future CLL patients, more than 10 years before diagnosis, of CLL-like cells which evolve as preclinical disease progresses, and point to early molecular alterations with a pathogenetic potential.

Keywords: Biomarkers of risk; Epigenomics; Molecular epidemiology; Prospective cohort; Transcriptomics; miRNA.

Fig. 1

Flowchart of data analyses

Fig. 2

Top: PCA based on 1308 CpG sites significant (FDR < 0.05) in CLL cases and with minimal variation between WBC sub-populations. Bottom: similar analysis with 1308 CpG sites randomly selected from among those with FDR > 0.8 in CLL cases and with minimal variation between WBC sub-populations. The signal intensities employed were denoised for various parameters, including B-cell content (see Methods). The numbers in the Figures on the right indicate the fractional B-cell content of the samples. The Figures on the left show all subjects while those on the right show only the CLL case subjects

Fig. 3

Comparison of denoised case-control methylation differences (Δβ) (left) and expression differences (foldchange ratio) (right), obtained from the comparison of all cases with controls with <10% B-cells (vertical axes) or with all controls (horizontal axes). The light lines show slope = 1

Fig. 4

Normalized least squares means (LSM) of DM and DE signal values in the controls and the two TtD groups. Top left: 238 DM signals Bonferroni-significant in the short TtD subgroup; bottom left: 291 DE signals FDR-significant in the short TtD subgroup. Top right: top 20 DM signals observed in all cases; bottom right: Bonferroni -significant DE signals observed in all cases. In the Figures on the right are named the genes associated with the 3 DM or DE signals with the largest changes in each direction (only 2 DM genes show increased methylation with decreasing TtD)

Fig. 5

Interaction network of the DM + DE hubs (STRING)