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Intra-tumor heterogeneity of MLH1 promoter methylation revealed by deep single molecule bisulfite sequencing.

Varley KE, Mutch DG, Edmonston TB, Goodfellow PJ, Mitra RD - Nucleic Acids Res. (2009)

Bottom Line: A single tumor may contain cells with different somatic mutations.By characterizing this genetic heterogeneity within tumors, advances have been made in the prognosis, treatment and understanding of tumorigenesis.We have characterized epigenetic heterogeneity within individual tumors using next-generation sequencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Center for Genome Sciences, Washington University School of Medicine, St Louis, Missouri, USA.

ABSTRACT
A single tumor may contain cells with different somatic mutations. By characterizing this genetic heterogeneity within tumors, advances have been made in the prognosis, treatment and understanding of tumorigenesis. In contrast, the extent of epigenetic intra-tumor heterogeneity and how it influences tumor biology is under-explored. We have characterized epigenetic heterogeneity within individual tumors using next-generation sequencing. We used deep single molecule bisulfite sequencing and sample-specific DNA barcodes to determine the spectrum of MLH1 promoter methylation across an average of 1000 molecules in each of 33 individual samples in parallel, including endometrial cancer, matched blood and normal endometrium. This first glimpse, deep into each tumor, revealed unexpectedly heterogeneous patterns of methylation at the MLH1 promoter within a subset of endometrial tumors. This high-resolution analysis allowed us to measure the clonality of methylation in individual tumors and gain insight into the accumulation of aberrant promoter methylation on both alleles during tumorigenesis.

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Representation of the different types of methylation observed in endometrial cancer specimens. The numerical patient identifier is followed by the bisulfite sequencing results for the distal and proximal MLH1 promoter in the tumor. (A) Methylated molecules from a tumor with dense homogenous promoter methylation. (B) Homogenous tumor with no promoter methylation. (C) and (E), Methylated molecules from tumors with distinct heterogeneous patterns of unmethylated cytosines in distal promoter. (D) and (F), Methylated molecules from tumors with distinct heterogeneous patterns of unmethylated cytosines in proximal promoter. Completely unmethylated molecules from the tumors in A, C, D, E and F were not included to allow for better resolution of the methylation patterns. Each column represents a CG dinucleotide. Each row represents a single molecule. The color of the boxes represents the methylation state of each cytosine. Red, methylated; Black, unmethylated.
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Figure 3: Representation of the different types of methylation observed in endometrial cancer specimens. The numerical patient identifier is followed by the bisulfite sequencing results for the distal and proximal MLH1 promoter in the tumor. (A) Methylated molecules from a tumor with dense homogenous promoter methylation. (B) Homogenous tumor with no promoter methylation. (C) and (E), Methylated molecules from tumors with distinct heterogeneous patterns of unmethylated cytosines in distal promoter. (D) and (F), Methylated molecules from tumors with distinct heterogeneous patterns of unmethylated cytosines in proximal promoter. Completely unmethylated molecules from the tumors in A, C, D, E and F were not included to allow for better resolution of the methylation patterns. Each column represents a CG dinucleotide. Each row represents a single molecule. The color of the boxes represents the methylation state of each cytosine. Red, methylated; Black, unmethylated.

Mentions: In four of eight of the MLH1 methylated tumors analyzed, we observed the expected dense homogeneous methylation (Table 1). For example, the tumor from patient #1569 is densely methylated at all CpGs in both the distal and proximal regions of the MLH1 promoter (Figure 3A). In tumors 1569, 1669, 1727 and 1789, dense MLH1 promoter methylation appears to have been an early event propagated throughout the tumor during the subsequent clonal expansion.Figure 3.


Intra-tumor heterogeneity of MLH1 promoter methylation revealed by deep single molecule bisulfite sequencing.

Varley KE, Mutch DG, Edmonston TB, Goodfellow PJ, Mitra RD - Nucleic Acids Res. (2009)

Representation of the different types of methylation observed in endometrial cancer specimens. The numerical patient identifier is followed by the bisulfite sequencing results for the distal and proximal MLH1 promoter in the tumor. (A) Methylated molecules from a tumor with dense homogenous promoter methylation. (B) Homogenous tumor with no promoter methylation. (C) and (E), Methylated molecules from tumors with distinct heterogeneous patterns of unmethylated cytosines in distal promoter. (D) and (F), Methylated molecules from tumors with distinct heterogeneous patterns of unmethylated cytosines in proximal promoter. Completely unmethylated molecules from the tumors in A, C, D, E and F were not included to allow for better resolution of the methylation patterns. Each column represents a CG dinucleotide. Each row represents a single molecule. The color of the boxes represents the methylation state of each cytosine. Red, methylated; Black, unmethylated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724279&req=5

Figure 3: Representation of the different types of methylation observed in endometrial cancer specimens. The numerical patient identifier is followed by the bisulfite sequencing results for the distal and proximal MLH1 promoter in the tumor. (A) Methylated molecules from a tumor with dense homogenous promoter methylation. (B) Homogenous tumor with no promoter methylation. (C) and (E), Methylated molecules from tumors with distinct heterogeneous patterns of unmethylated cytosines in distal promoter. (D) and (F), Methylated molecules from tumors with distinct heterogeneous patterns of unmethylated cytosines in proximal promoter. Completely unmethylated molecules from the tumors in A, C, D, E and F were not included to allow for better resolution of the methylation patterns. Each column represents a CG dinucleotide. Each row represents a single molecule. The color of the boxes represents the methylation state of each cytosine. Red, methylated; Black, unmethylated.
Mentions: In four of eight of the MLH1 methylated tumors analyzed, we observed the expected dense homogeneous methylation (Table 1). For example, the tumor from patient #1569 is densely methylated at all CpGs in both the distal and proximal regions of the MLH1 promoter (Figure 3A). In tumors 1569, 1669, 1727 and 1789, dense MLH1 promoter methylation appears to have been an early event propagated throughout the tumor during the subsequent clonal expansion.Figure 3.

Bottom Line: A single tumor may contain cells with different somatic mutations.By characterizing this genetic heterogeneity within tumors, advances have been made in the prognosis, treatment and understanding of tumorigenesis.We have characterized epigenetic heterogeneity within individual tumors using next-generation sequencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Center for Genome Sciences, Washington University School of Medicine, St Louis, Missouri, USA.

ABSTRACT
A single tumor may contain cells with different somatic mutations. By characterizing this genetic heterogeneity within tumors, advances have been made in the prognosis, treatment and understanding of tumorigenesis. In contrast, the extent of epigenetic intra-tumor heterogeneity and how it influences tumor biology is under-explored. We have characterized epigenetic heterogeneity within individual tumors using next-generation sequencing. We used deep single molecule bisulfite sequencing and sample-specific DNA barcodes to determine the spectrum of MLH1 promoter methylation across an average of 1000 molecules in each of 33 individual samples in parallel, including endometrial cancer, matched blood and normal endometrium. This first glimpse, deep into each tumor, revealed unexpectedly heterogeneous patterns of methylation at the MLH1 promoter within a subset of endometrial tumors. This high-resolution analysis allowed us to measure the clonality of methylation in individual tumors and gain insight into the accumulation of aberrant promoter methylation on both alleles during tumorigenesis.

Show MeSH
Related in: MedlinePlus