Lauren and I were intrigued by the role
of epigenetics in cancerous growth and chose to further investigate this correlation in our Cancer Project, so imagine my excitement after an epigenetic cancer
study was introduced in my general psychology class!
This study, conducted by Bellvitge Biomedical Research Institute; University of Coimbra Centre
for Neurosciences and Cell Biology; Kings College London Department of Twin
Research and Genetic Epidemiology; University of Iceland Molecular and Cell
Biology Research Laboratory; and University of Barcelona School of Medicine,
looked at 15 monozygotic (MZ) twin pairs discordant for breast cancer, with
further examination into the different degrees of methylation affecting gene
rich segments within the cancerous cells. In other words, despite the genetic
equivalence within each twin pair, an extraneous variable, presumably
differences in epigenetics, resulted in one twin exhibiting breast cancer while
the other was cancer free. By comparing isolated DNA from blood and cell
samples obtained from the cancer positive twin with that of the cancer negative twin,
this study isolated 403 differentially methylated CpG sites, or
Cytosine-Guanine DNA regions, many of which contain specific genes referred to
as “novel breast cancer genes,” genes highly correlated with breast cancer incidence.
After each blood and tumor/normal
tissue sample was macroscopically examined, DNA regions were isolated from the whole
blood and triple negative breast tumors of the cancer positive twins and normal
breast tissue of the cancer negative twins (n=15). The DNA samples were then
randomly distributed into a 96-well plate to eliminate experimenter bias, after
which, an EZ DNA methylation kit was
used to determine methylation degrees of specific regions per sample. DNA
methylation levels were normalized, then presented in decimal form ranging from
0 to 1 and referred to as the Beta-value, Beta-values falling outside the
significance value (p > 0.01) were thereby eliminated from the sample. Following
Beta-value attribution, a Wilcoxon rank
sum paired test was performed to identify consistently differentially methylated
sites between each cancer positive and cancer negative twin pair with a p value
< 0.05. This test identified the statistically significant 403
differentially methylated sites, demonstrating data much in line with the
hypothesis that epigenetic regulation plays a role in cancer development.
Epigenetic regulation of genes and
corresponding CpG sites accounts for activation and suppression of
functionality and transcription via alterations in DNA methylation, offering
more insight into controllable influencers of cancer. Thus, much research has
focused more recently on methylation patterns in these novel breast cancer
genes, as apposed to predetermined genetic susceptibility, with the aim of
determining potential epigenetic indicators of Breast Cancer. This study aimed
to narrow down the original 403 differentially methylated CpG sites to a
handful of consistent regions known as bcDMPs, or “differentially methylated
CpG positions in breast cancer.” Regions identified as bcDMPs, hold the promise
of serving as potential epigenetic indicators of breast cancer, in both early
and late states of the disease, and eventually helping to better tailor the
understanding of effective treatment and pathology.
So, following the first round of
results, the study conducted a genome-wide DNA methylation technique to analyze
whole blood samples from an additional sample of monozygotic twin pairs (n=21),
again discordant for breast cancer. However, this time a new variable was
introduced: point of diagnosis, 16 twin pairs incorporated a cancer twin that
had already been diagnosed with breast cancer, the other 5 incorporated a twin
suspected of breast cancer but not yet formally diagnosed. Using locus-specific pyrosequencing for gene
alteration, 14 CpG sites were isolated as potential bcDMPs.
One of these 14 CpG sites, Docking Protein 7 (DOK7) a known receptor tyrosine kinase activator, presented statistically significant methylation variation between the cancer positive and cancer negative twin after completing the Wilcoxon signed rank test. The cancer twin exhibited hypermethylation of the DOK7 region, a finding consistent across all the twin pairs, with exactly four additional CpG sites (directly upstream of DOK7) also presenting clear patterns of significant hypermethylation. The DOK7 regions and same additional four CpG sites were isolated in six healthy breast samples; interestingly enough, these cancer-free cells displayed clear hypomethylation, that is, decreased levels of methylation. This whole 5-site loci, DOK7 plus the additional 4 CpG sites, was thus confirmed to be a bcDMP.
One of these 14 CpG sites, Docking Protein 7 (DOK7) a known receptor tyrosine kinase activator, presented statistically significant methylation variation between the cancer positive and cancer negative twin after completing the Wilcoxon signed rank test. The cancer twin exhibited hypermethylation of the DOK7 region, a finding consistent across all the twin pairs, with exactly four additional CpG sites (directly upstream of DOK7) also presenting clear patterns of significant hypermethylation. The DOK7 regions and same additional four CpG sites were isolated in six healthy breast samples; interestingly enough, these cancer-free cells displayed clear hypomethylation, that is, decreased levels of methylation. This whole 5-site loci, DOK7 plus the additional 4 CpG sites, was thus confirmed to be a bcDMP.
But
what about the yielded differences in pre versus post diagnosis cancer twins? In
fact, three of the five suspected cancer twins displayed blood and tissue
samples with a significant gain of DOK7 methylation, and these samples were
taken prior to actual diagnosis, with a mean of 4.7 years before concrete
diagnosis. In other words, DOK7 displayed significant enough methylation to be
detected as a breast cancer indicator in patients about 4.7 years before their
cancer was symptomatic and fully diagnosable. While this represents a very
small sample size, such visible DOK7 methylation prior to actual diagnosis of
breast cancer presents a very promising avenue of early detection.
This
is why this study, though not fully perfect, is incredibly exciting. This study
has first identified 403 differentially methylated regions within important DNA
regions, highlighting a significant epigenetic difference in cancer positive
and cancer negative twins, independent of genetic influencers. Furthermore, these
403 regions were narrowed down to 14 possible bcDMPs, and finally 1 confirmed
bcDNP, DOK7. DOK7 represents one of many possible bcDMPs that not only
demonstrates significant methylation differences between tumor samples and
normal tissues post diagnosis, but also unlocks the possibility for
pre-symptomatic detection, epigenetic monitoring of disease development, and
eventually epigenetic treatment of cancer!
Source Citation
Heyn, Holger, et al. "DNA methylation profiling in breast cancer discordant identical twins identifies
DOK7 as novel epigenetic biomarker." Carcinogenesis (2012): 102-08.