Methylation is but one of the many facets of what we now see
as causes of Cancer. We depict a short summary below.
We examine the work of Wojno et al which has received recent
interest. They examine the impacts of methylation upon 3 genes and see their
presence as prognostic of potential aggressive prostate cancer. Specifically
they conclude:
The diagnosis of prostate cancer is dependent on
histologic confirmation in biopsy core tissues. The biopsy procedure is
invasive, puts the patient at risk for complications, and is subject to
significant sampling errors.
An epigenetic test that uses methylation-specific
polymerase chain reaction to determine the epigenetic status of the prostate
cancer–associated genes GSTP1, APC, and RASSF1 has been clinically validated
and is used in clinical practice to increase the negative predictive value in
men with no history of prostate cancer compared with standard histopathology.
Such information can help to avoid unnecessary repeat biopsies.
The repeat biopsy rate may provide preliminary clinical
utility evidence in relation to this assay’s potential impact on the number of
unnecessary repeat prostate biopsies performed in US urology practices.
DNA methylation normally can result in the silencing of
genes by interrupting the normal process of promoters. CpG islands are often hypermethylated
and thus the gene which may regulate cell proliferation is silenced. This may
result in uncontrolled cell growth. For example genes controlling MYC are not
produced and MYC may then result in excess cell cycle proliferation.
Methylation is hypermethylated in the regions of intergenic regions and in
repetitive elements and this hypermethylation silences these regions and
facilitates normal cell DNA transcription of the gene. Disruption of DNA,
namely hypomethylation, in the intergenic and repetitive regions may result in
possible loss of imprinting. This hypomethylation is also related to the
production of lncRNAs which may in turn interfere with normal gene
transcription.
Decitabine is a DNMT inhibitor. Namely, it inhibits the DNA
methyltrasferases that facilitate methylation (such as DNMT3 which are de novo
and DNMT1 which is maintenance). Decitabine thus has then tendency on the
specific hematologic cell lines in MDS to remove methylations which have caused
the aberrant cell line proliferations and allow for the return of homeostasis.
MDS is a quasi-malignant condition originating in the bone marrow which may in
many cases result in Acute Myelogenous Leukemia. With the use of decitabine or
a similar DNMTI azacitidine, demethylation of these rapidly reproducing cells
may be achieved and possible a normal state of homeostasis achieved.
The use of pharmaceuticals that alter the methylation
patterns of DNA can have lasting effects because those patterns may last
through subsequent mitotic changes. On the one hand that may be beneficial as
is the case with MDS but such broad demethylation may also alter other segments
of the DNA altering essential control elements and pathways. In cell
development there are two sensitive periods; germ cell development and early
embryonic development. It is during these periods that methylation is cleared
and reset and that a drug-like a DNMTI would pose a serious risk to the proper
resetting of the marks and could result in substantial DNA expression damage.
In summary we will examine the three gene methylation
proposition with this test. We summarize this below:
Let us examine what the study presents in a bit more detail.
Basically it does the following:
1. It examines three gene products; GSTP1, APC, and
RASSF1
2. It determines if the genes are methylated so that the
gene products are suppressed.
3. If that is the case after a first biopsy which is deemed
normal, then a second biopsy is mandated due to the high incidence of a
positive result on the second biopsy for PCa.
Specifically from the paper by Partin et al on the same
topic the authors’ state:
The DOCUMENT multicenter trial in the United States
validated the performance of an epigenetic test as an independent predictor of
prostate cancer risk to guide decision making for repeat biopsy. Confirming an
increased negative predictive value could help avoid unnecessary repeat
biopsies. We evaluated the archived, cancer negative prostate biopsy core
tissue samples of 350 subjects from a total of 5 urological centers in the
United States. All subjects underwent repeat biopsy within 24 months with a
negative (controls) or positive (cases) histopathological result. Centralized
blinded pathology evaluation of the 2 biopsy series was performed in all
available subjects from each site.
Biopsies were epigenetically profiled for GSTP1, APC and
RASSF1 relative to the ACTB reference gene using quantitative methylation
specific polymerase chain reaction. Predetermined analytical marker cutoffs
were used to determine assay performance. Multivariate logistic regression was
used to evaluate all risk factors.
The epigenetic assay resulted in a negative predictive
value of 88% (95% CI 85-91). In multivariate models correcting for age,
prostate specific antigen, digital rectal examination, first biopsy
histopathological characteristics and race the test proved to be the most
significant independent predictor of patient outcome.
The DOCUMENT study validated that the epigenetic assay
was a significant, independent predictor of prostate cancer detection in a
repeat biopsy collected an average of 13 months after an initial negative
result. Due to its 88% negative predictive value adding this epigenetic assay
to other known risk factors may help decrease unnecessary repeat prostate
biopsies.
Recall that the negative predictive value or NPV is
defined as:
Thus an NPV of 88% for the sample size used implies that it
is fairly high in predicting True Negatives a priori but may still miss a
percentage. There of course is the issue of the pathologist missing the PCa as
well. This could be done by a sampling deficiency or confusion on a reading. It
is not clear if for example a HGPIN is read.
Thus focusing on methylated genes, specifically just 3 of
them, GSTP1, APC and RASSF1, they were able in a small sample to ascertain that
there would be no need of a second biopsy if they were found to be unmethylated
in the first.
Recall the effects of methylation as we show below:
From an article in Medical Express[1] as well as from an article
in Eureka[2] as well as from an article
in Science Codex[3] we
have the following summary:
More than one million prostate biopsies are performed
each year in the U.S. alone, including many repeat biopsies for fear of cancer
missed. Therefore there is a need to develop diagnostic tests that will help
avoid unnecessary repeat biopsies. Two independent trials have now validated
the performance of an epigenetic test that could provide physicians with a
better tool to help eliminate unnecessary repeat prostate biopsies, report
investigators in The Journal of Urology.
In the previously reported independent MATLOC
(Methylation Analysis To Locate Occult Cancer) trial, a multiplex epigenetic
assay (ConfirmMDx for Prostate Cancer) profiling the APC, GSTP1 and RASSF1
genes demonstrated a negative predictive value of 90%. GSTP1 methylation is a
specific biomarker for (prostate) cancer and this gene is methylated in up to
90% of prostate cancer cases. Additionally, APC and RASSF1 are important field
effect markers and increase the diagnostic sensitivity of the assay.
A second multicenter study, DOCUMENT (Detection Of Cancer
Using Methylated Events in Negative Tissue), has validated the performance of
the epigenetic assay used in the MATLOC trial as an independent predictor of
prostate cancer risk to guide decision making for repeat biopsy. In the
DOCUMENT study patients with a negative biopsy were evaluated to identify those
at low risk for harboring cancer missed, through biopsy sampling error, who
could forego an unnecessary repeat biopsy. The validation study resulted in a
negative predictive value of 88%.
"This epigenetic assay is a significant, independent
predictor and has been shown to be the most valuable diagnostic aid of all
evaluated risk factors in two independent trials," comments Alan W.
Partin, MD, PhD, of the James Buchanan Brady Urological Institute, The Johns
Hopkins University School of Medicine, Baltimore, Maryland. "Negative
findings of this assay could be used to reduce concern over unsampled cancer
and effectively avoid unnecessary repeat biopsies."
A total of 350 patients were enrolled in the DOCUMENT
trial from five geographically dispersed medical centers: Cleveland Clinic,
Eastern Virginia Medical School, Lahey Hospital & Medical Center, Johns
Hopkins University, and University of California Los Angeles. Patients were
grouped into those with two consecutive negative biopsies (controls) and those
with a negative biopsy followed by a positive biopsy within 24 months. The
initial archived, negative for cancer, prostate biopsy core tissue samples were
evaluated. All of the men underwent a repeat biopsy on average one year after
the initial biopsy.
Only biopsies with a minimum of eight cores per biopsy,
collected no earlier than 2007, were included in the study, while initial
biopsies with atypical cells suspicious for cancer, i.e. atypical small acinar
proliferation by the sites' pathologists, were excluded, since this would have
triggered a repeat biopsy based upon histopathology alone.
After correcting for age, prostate specific antigen
(PSA), digital rectal exam, histopathological characteristics of the first
biopsy, and race, this epigenetic test proved to be the most significant,
independent, and strongest predictor of patient outcome with an odds ratio of 2.69
as well as the most valuable diagnostic aid of all evaluated risk factors. The
slightly decreased sensitivity of the DOCUMENT trial compared to the MATLOC
trial is most likely associated with a higher PSA screening prevalence in the
DOCUMENT cohort.
It is important to note the following:
1. The genes selected have been studied for over two decades
and especially as regards to their hypermethylation status.
2. The test is an early prognostic test which when combined
with prostate biopsy data, especially a benign reading on the prostate biopsy.
3. The test has reasonable statistics given its small sample
size but as we shall see there is substantial variability in these tests.
Methylation is but one of the very many changes we see in
cancers. Whether they are cause or effect is yet to be determined. We know, for
example, that methylation is causal in MDS, myelo-dysplasia syndrome, a
precursor often of ALL. However, the cause of this methylation is still
problematic. Drugs like DNMT inhibitors, azacitidine and decitabine are
methylation inhibitors that seem to work for a while in this disorder. However
what they do to other cells is uncertain.
In this study which we examine, there is a multiplicity of
questions.
1. What causes the methylation?
There is still a lack of clear process as to how methylation
occurs. As we will note shortly there are hypothesis that it is a result from
inflammatory states and others that there may be secondary effects of
autoimmune conditions. The elements of the process are understood to some
degree but no true full causal chain is established.
2. Why are these gene sites methylated?
Why specific CpG islands at specific genes are methylated is
still unknown. Are these initial locations or are the related to other as yet
to be determined sites. Are certain CpG site more susceptible? Is there some
histone issue wherein the histones are demethylated opening the DNA and thus
allowing CpG methylation?
3. Why not try the DNMT inhibitors if these methylations are
causal for PCa?
DNMT inhibitors are used for MDS with some short term
success. Can they be used here as a step towards reversal? The problem is that
we do not know what DNMTI sequellae are. The unintended consequences could be
significant. Can we develop cell specific DNMTIs or are they even too potent?
4. Are many cancers caused by methylation or demethylation?
If so which ones and why?
We have examined several different cancers and there is a
large putative collection of genes, translocations, miRNAs, SNPs, methylations,
and the like. Almost weekly and perhaps soon daily we will be seeing alleged
markers for every element of a cancer development. Two decades ago we saw just
gene issues. Today we have a problem of ascertaining the chicken or the egg.
This is both a challenge and an opportunity. The problem, however, is that each
time one of these has been developed, we see a Press release frenzy as it be a
sine qua non.
5. Are the observations made by the researchers causal or
coincidental?
This is always a critical question. Is this specific
methylation of genes the cause of ultimate PCa or the coincidental effect of
some other factor?
6. Are the methylation observations drivers or therapeutic
approaches?
This question is a follow on to our question regarding
DNMTIs.
7. Should we be examining more cancers from an epigenetic
point of view rather than a genetic mutation perspective?
We have examined a few cancers for methylation as drivers
for malignancy and metastatic behavior.
8. If as we have seen anecdotally, patients determined to
have widespread HGPIN on a first 24 core biopsy and then none on a second 24
core biopsy, is this possibly a demethylation result, a stem cell result, or
some other factor.
We have examined HGPIN in previous analyses. Although not
considered a true PCa it has been considered as a natural pre-cursor. Namely
many assume that HGPIN will always turn into PCa. However we have anecdotally
seen patients where the HGPIN actually regresses, to the level of being
unidentifiable at on a high density core biopsy. Thus we have asked if the
first biopsy actually precipitated the remission, if so how and why, or was
there some other factor. Unfortunately there is inadequate data for this study.
9. What of the stem cell factor in PCa?
PCa stem cells are always a concern. We have discussed them
at length and basically research continues into these cells in PCa. Yet it does
beg the question; what cells need be methylated and are certain cells more
likely than others? Furthermore we can ask; how do we segregate prostate cells
so as to ascertain the severity of methylation?
10. PTEN mutations have been proposed as a major causal
effect of PCa. How does this relate to epigenetic factors?
We have shown that PTEN is controlled in the process of p53
and in turn the MDM2 and its RAS precursor control. Thus the focus on RAS
derivatives is consistent with the PTEN argument.
11. Are epigenetic factors the results of inflammatory
states? Namely in a patient with for example Type 2 Diabetes and elevated
inflammatory status, does this become an initiating factor for methylation?
Hartnett and Egan have recently written:
Recently, epigenetic alterations, in particular
alterations in DNA methylation, have been observed during inflammation and
inflammation-associated carcinogenesis. The mediators of this, the significance
of these changes in DNA methylation and the effect this has on gene expression
and the malignant transformation of the epithelial cells during IBD and CAC are
discussed in this review. The recent advances in technologies to study
genomewide DNA methylation and the therapeutic potential of understanding these
molecular mechanisms are also highlighted.
Dayeh et al focus on these types of methylation induced by
Type 2 Diabetes in a recent paper as well.
Also it is worth examining the summary by Kundu and Surh on
inflammation and cancer. They state:
DNA methylation, the covalent addition of a methyl group
to the 5-position of cytosine base in the DNA, represents a critical epigenetic
control of gene expression. The perturbation of methylation patterns as either
aberrant loss of cytosine methylation in transforming genes or inappropriate
gain of cytosine methylation in tumor suppressor genes has been involved in
various human malignancies.
The most predominant aberrant DNA methylation is
hypermethylation that typically occurs at the CpG islands located in the
promoter regions of tumor suppressor genes. Promoter hypermethylation of tumor
suppressor genes, such as p16, von-Hippel Lindau (VHL), adenomatous polyposis
coli (APC), breast cancer susceptibility gene (BRCA1), retinoblastoma (Rb),
E-cadherin (CDH1), p73, p53, and p57, results in transcriptional silencing . By
analyzing the methylation status of 11 candidate cancer-related genes in
cutaneous squamous cell carcinomas, Murao et al. have demonstrated that the
promoter hypermethylation of CDH1, p16, Rb1 and p14 results in the loss of
respective protein production.
Therefore, the epigenetic silencing of tumor suppressor
genes by promoter CpG island hypermethylation perturbs cell cycle control,
apoptosis, DNA repair and cell adhesion, and is recognized as an important
mechanism in the tumorigenesis. However, global hypomethylation of certain
genes, e.g., insulin-like growth factor-2 (IGF-2), can also result in genomic
instability, accelerating malignant transformation.
As Donkena et al state:
“Oxidative stress” is the state of a cell, which is
characterized by excess production of reactive oxygen species (ROS) and/or a
reduction in antioxidant defenses responsible for metabolism. ROS are formed as
a natural byproduct of the normal metabolism of oxygen. Under normal
circumstances, the cell is able to maintain an adequate homeostasis between the
formation of ROS and its removal through enzymatic pathways or via
antioxidants. If, however, this balance is disturbed, then oxidative stress
occurs.
This generates an imbalance of production/removal of ROS,
which is either directly or indirectly involved in initiation, promotion, and
progression phases of carcinogenesis. Oxygen radicals may cause damage to DNA
and chromosomes, induce epigenetic alterations, interact with oncogenes or
tumor suppressor genes, and impart changes in immunological mechanisms.
The extent of ROS induced oxidative damage can be
exacerbated by a decreased efficiency of antioxidant defense mechanisms. ….
Hypermethylation of a combination of genes including APC, RASSF1A, PTGS2,
PDLIM4, and MDR1 could distinguish cancer from benign prostate tissues with
sensitivities of 97.3%–100% and specificities of 92%– 100%. The increase in
methylation of these genes with cancer progression indicates that they could be
used for biomarkers for both diagnosis and risk assessment
Furthermore, we showed significant differences in the
frequency of methylation at individual CpG sites of PITX2, PDLIM4, KCNMA1,
GSTP1, FLNC, EFS, and ECRG4 in recurrent and nonrecurrent subtypes of prostate tumors.
Indeed, hypermethylation of a CpG island in PITX2 portended an increased risk
of prostate cancer recurrence and was a predictor of distant disease recurrence
in tamoxifen-treated, node-negative breast cancer patients.
12. HGPIN has always been an issue of concern. In some cases
it appears to revert to fully benign states and in others it progresses to PCa.
The question is: How useful would such a test be in HGPIN conditions?
The concern always is the HGPIN cells are confined to the
glandular portions of luminal and basal cells. Is it necessary to get HGPIN
cells to test or can the test be performed on other cells? Specifically how
large a sample is necessary to get adequate sensitivity and specificity?
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