The study of methylation in FL to date has been predominantly restricted to analysis of small patient cohorts or focused on individual candidate tumour suppressor genes, using locus-specific non-quantitative methods. In our study we use array technology to quantitatively assess methylation profiles in a large, well-characterised cohort of newly diagnosed FL patients and sequential biopsies from patients who underwent transformation to DLBCL.
This approach generated a methylation profile which demonstrated extensive methylation changes between FL and benign lymph node allowing separation of tumour samples from non-tumour samples with a single exception (). This discriminatory ability was based primarily on tumour-specific gains of methylation within CpG islands. Haematopoietic control samples demonstrated remarkably consistent methylation profiles across a wide range of ages and pathological diagnoses. While many CpG loci were noted to have similar methylation levels across FL samples, the pattern of methylation at a number of loci was heterogeneous within the u-FL group. From our comparison with SNP profiles and analysis of purified B and T cell populations, we believe that the degree of change in methylation relative to the control group predominantly reflects the relative proportions of neoplastic B cells and non-neoplastic infiltrating immune cells contained within the biopsies rather than heterogeneity of methylation within tumour cells.
This potential confounding effect of non-neoplastic tissue on quantitative methylation analysis is often overlooked as the majority of methylation studies use whole tumour samples. This emphasises the importance of purified tumour populations in future attempts to define the epigenome of various cancers, which will undoubtedly gain impetus as technology to allow high-resolution large-scale methylation mapping becomes available.
Despite this, our study was able to discriminate all but 1 of 164 tumours from non-tumour tissue, indicating that changes in methylation in FL are sufficient to distinguish from benign samples regardless of any potential effect of tumour content in the vast majority of cases. This has important practical applications as it suggests the potential for methylation-based discriminators in cancer e.g. as a less invasive method of confirming disease recurrence (e.g. by fine needle aspiration rather than core needle or open biopsies) or as an adjunct to diagnosis when material is limited. Moreover, the recent findings by Killian et al. (34
) showing excellent reproducibility between matched frozen tissue and formalin-fixed, paraffin embedded tissue from individuals with FL confirms the potential clinical application of this methylation technology.
While the majority of loci examined showed similar methylation levels in both benign and malignant samples, the discriminative ability of the array was predominantly based on relative hypermethylation at selected loci in tumour samples relative to controls (; ; Supplementary Table S4
). Hypermethylated and hypomethylated groups were significantly associated with CpG location, within and outside of CpG islands, respectively. This finding supports the basic methylation theory that CpGs located within CpG islands in non-tumour tissue are unmethylated while CpGs outside CpG islands are methylated with the inverse pattern occuring in tumour tissue (1
The significant overrepresentation in the hypermethylated gene set of genes targeted for repression by Polycomb group proteins is in keeping with recent studies in carcinomas (35
). It has been proposed that methylation of these genes are early events which “lock in stem cell phenotypes” and lead to abnormal clonal expansion (35
). Indeed, similar findings have recently been reported in mature aggressive B cell lymphomas by Martin-Subero and colleagues who discussed the potential implications of this finding for our understanding of lymphomagenesis (39
). Recent observations suggesting that methylation of polycomb target genes is mediated by inflammation (40
) and that transcriptional activity of polycomb target genes may be regulated by enzymes expressed by macrophages (41
) are also intriguing, particularly given the role of the microenvironment in determining prognosis in FL (20
). Sequential analysis of biopsy material from 10 paired samples also suggests that changes in methylation are acquired early in the process of lymphomagenesis and conserved with treatment and transformation.
The hypermethylated CpG loci identified in this study include both previously reported and novel methylated genes with a wide range of functions. While numerous studies have identified methylated genes in a range of human tumours, it is unclear if these changes are causal events in tumourigenesis or are merely a consequence of tumour development (42
) - what have been referred to as ‘epipolymorphisms’ (43
). In this series it seems likely that many of the 199 CpG loci hypermethylated in u-FL will represent part of widespread epigenetic change brought about through instructive mechanisms rather than individual random methylation events leading to gene silencing (44
), particularly given the relatively low inverse correlation between gene expression and methylation. Previous studies have shown that many genes methylated in lymphoma are expressed at low level in benign haematopoietic tissue (34
). There are a number of other potential reasons for this low correlation. There may be a “threshold effect” in regulating gene expression whereby gene expression may not be unduly altered until a certain level of methylation has arisen(47
). In addition, the effect of the microenvironment on gene expression is unknown while other factors involved in methylation-associated gene silencing such as histone modifications could also contribute to these results.
In summary, this study confirms frequent aberrant tumour-specific methylation in FL in support of ongoing clinical trials of demethylating agents in FL. The similarities in the methylation profiles observed in sequential FL and t-FL biopsies and the overrepresentation within the hypermethylated gene set of genes that are targets for epigenetic repression by PRC2 in stem cells suggests that the widespread methylation may represent an early event in lymphomagenesis. It is likely that future studies will focus on elucidating the links between Polycomb group proteins and widespread tumour-specific methylation with a view to gaining insight into the timing and significance of these changes in lymphomagenesis. Methylation profiles provide a robust discriminator between benign and malignant tissue, irrespective of the confounding effect of non-neoplastic tissue, which prevented conclusive correlation with clinical outcome in this study. This is an important caveat in analysing studies attributing prognostic relevance to methylation of individual genes and future work will optimally require purified tumour populations using quantitative methylation studies to identify the true impact of methylation on tumour biology and outcome.