A representative series of mesotheliomas and their matched peripheral blood-derived DNA as referents (n=23) from the total study population were subjected to 500K SNP mapping arrays to assess copy number (CN) alteration (Supplementary Table 1
). A summary of the copy number alterations identified is provided in Supplementary Table 2
. Consistent with previous reports (36
), we observed prevalent allele loss at 1p36 (35%), 1p21.3 (30%), 4q22 (30%), 4q31–32 (35%), 3p21.3 (44%), 6q25 (39%), 9p21 (39%), and 22q (44%). In addition, we observed prevalent gains at1q23 (35%), 5p (22%), 7p (22%), and 8q24 (22%). Across tumor samples, the overall prevalence of CN losses and CN gains varied widely, with a mean prevalence of CN loss events of 8% (Standard Deviation (SD) = 9%), and a mean prevalence of CN gains of 5% (SD = 6%, Supplementary Table 3
). Tests for association between extent of CN alteration and age, gender, histology, and asbestos exposure burden were not statistically significant.
Using prior data generated from interrogating DNA methylation in 158 mesotheliomas (24
) we applied recursively partitioned mixture modeling (RPMM) to infer methylation profile classes wherein the 23 tumors profiled for CN alterations were included. This approach built classes of samples based on profiles of methylation with data from all measured autosomal loci using a mixture of beta distributions to recursively split the tumors into parsimoniously differentiated classes (39
). Since methylation profile classes from the established model of all 158 mesotheliomas represent a more stable classification, we did not model the subset of 23 tumors with CN data separately. Instead, original class membership data were used in our analyses and among the seven RPMM methylation classes, the 23 tumors with CN data had a distribution of class membership that included all but methylation class five. Tumors in methylation class two were previously shown to be from patients with significantly lower asbestos burden than other classes, and relative to these class two cases, classes four and seven had significantly poorer prognosis (24
directly compares copy number alterations and DNA methylation state in side-by-side, chromosome-specific plots of these alterations arranged by methylation class membership. Remaining chromosomes are illustrated in Supplementary Figure 1
. Qualitatively, when focusing on single loci or specific genes, there was no obvious or consistent pattern of altered methylation when CN alterations were present. However, we next quantitatively evaluated whether – consistent with Knudson’s two hit paradigm of gene inactivation – coordinate dysregulation of CN state and methylation was present. No loci were found to have a significant correlation (Q
< 0.05) between CN and methylation state across the examined tumors (Supplementary Table 4
Figure 1 Side by side plots of chromosome-specific mesothelioma copy number alteration and DNA methylation profiles ordered by RPMM methylation class. Left panel illustrates copy number alteration, SNP loci are matched to the nearest CpG locus and oriented coordinately (more ...)
By averaging CN and methylation state within gene-specific regions we expanded this approach to a gene-level query. Only ITGB1
had significantly correlated (Q
< 0.05) CN and methylation among 663 examined genes (Supplementary Table 4
). However, shows that these significant correlations were not due to coordinate allele loss and hypermethylation. Other genes with highly correlated CN and methylation were also plotted. CDNKN2A
had widespread methylation of one of its target CpGs, though this site is normally methylated in non-tumor pleura (). Only two genes – TGFB2
– had any visually detectable coordinate methylation and CN loss and in each case only one tumor had both alterations (). Though not among the genes in Supplementary Table 4
is commonly inactivated in mesothelioma and in our samples 7 tumors (30%) had allele loss, 2 (22%) were hypermethylated (), and consistent with results above, only one tumor had coordinate methylation and CN loss.
Figure 2 Gene-specific integrated copy number and methylation plots indicate a lack of coordinate copy number loss and hypermethylation events in mesotheliomas. Each ordinate tick mark represents a tumor sample, equivalently ordered across individual plots. In (more ...)
To assess whether global, rather than discrete trends for methylation and/or CN alteration might be observed, we plotted the magnitude and direction of CN and methylation alterations (log2 ratios versus a ratio of 0 for unaltered CN and tumor versus non-tumor methylation at each gene) to genes by their associated P-value (). The volcano plot of CN alterations was skewed to the left, indicating genome-wide trends for gene-level CN losses (15,790 genes). Similarly, the volcano plot of methylation alterations was also skewed to the left, indicating a genome-wide trend for loss of methylation relative to non-tumor pleural samples (663 genes). In order to determine whether CN and methylation alterations differ based on methylation profile, we split samples by the first partition of RPMM methylation classes. More specifically, the unsupervised nature of the RPMM model allows stratification of samples by branches of its associated dendrogram, since the model recursively partitions the methylation data, class membership can be retraced to the original parent methylation classes, the left branch and right branch class. Here, the “left branch” samples are those belonging to methylation classes 1 or 2 (n=10) the daughter classes of the initial left branch class while the “right branch” samples are those belonging to methylation classes 3 – 7 (n=13), the daughter classes of the initial right branch RPMM class. The volcano plots for CN alterations were strikingly different between methylation class branches: tumors in the left branch classes of the RPMM had far more loci with significantly altered CN compared to tumors in the right branch of RPMM (). Tumors in the left branch had a wider range of methylation alteration compared to tumors in the right branch, indicating that the trend toward an overall increase or decrease in the degree of methylation was associated with allele copy number loss. These results prompted further investigation of the global association between CN alterations and methylation state.
Figure 3 Log significance plots for mean copy number and methylation alterations indicate global copy number losses, global hypomethyaltion, and differential copy number alteration by methyaltion class. Each region was compared to its expected normal value (normal (more ...)
The evidence for a global relationship between CN alterations and methylation alterations could suggest that CN alterations of master epigenetic regulatory genes influence overall tumor methylation. Maintenance methyltransferase DNMT1
had CN loss in 7 tumors (30%, Supplementary Figure 1
, Chromosome 19) and de novo
had no CN alterations (Supplementary Figure 1
, Chromosome 20). Plotting the average CpG methylation for tumors with DNMT1
allele loss versus tumors without loss, we observed a significant trend for increased methylation among tumors with no allele loss at DNMT1
compared to tumors with allele loss (, P
= 0.05). Further, DNMT1
allele loss was associated with significantly reduced patient survival in a Cox proportional hazards model controlling for age, gender and tumor histology (HR, 5.07; 95% C.I. 1.23 – 20.9, Supplementary Table 5
). In similar investigations of alterations of critical DNA double strand break repair genes (e.g. ATM
) prevalent CN alterations or methylation silencing events were not observed.
Figure 4 Tumors without DNMT1 allele loss have increased methylation relative to tumors with DNMT1 allele loss. Mean average β methylation values for tumors with allele loss at DNMT1 are plotted versus mean average β methylation for tumors without (more ...)
In an effort to better visualize and test the global trends of association between methylation and CN alterations we plotted the CN alteration profiles of tumors based on RPMM methylation class membership (). This plot illustrates the significant difference in the extent of CN alterations among methylation classes (Permutation test P < 0.02). Tumors in methylation classes two, six, and seven exhibit a greater extent of CN alteration than tumors in methylation classes one, three or four (). The means and standard errors of class-specific percents of SNPs with CN alterations are also illustrated in .
Figure 5 Plot of mesothelioma copy number alteration profiles based on class membership from recursively partitioned mixture model (RPMM) of DNA methylation data indicates a global correlation between methylation class and extent of copy number alteration. The (more ...)