The research associating methylation changes with the development of cancer have identified these epigenetic modifications as early events thought to often precede the appearance of tumor. Furthermore, investigations into whether therapeutic intervention of these epigenetic changes can alter the course of disease are being undertaken. A corollary to these observations is an interest in utilizing methylation changes as biomarkers to aid in the detection and monitoring of cancer. Though current screening methods for breast cancer have made significant strides in the detection of early stage breast cancer, the combination of mammography and MRI have a combined specificity of 77% and a sensitivity of 94% with increased cost.67
Whether the incorporation of MRI into the screening of high-risk women improves survival remains to be determined. The quest for more accurate detection of breast cancer in women has led to the analysis of nipple duct fluid as a means of direct assessment. However, the process has been hampered by difficulties in sampling of nipple duct fluid as well as the lack of predictive capability by cytologic analysis.68
In one series, the methylation status of 11 tumor suppressor genes from ductal epithelial cells predicted sporadic and hereditary cancer with an accuracy approaching 90%.69
Importantly, it appears that methylation changes are apparent in the ductal epithelial cells obtained from nipple duct fluid regardless of the location of the primary breast tumor.
In patients with non-small cell lung cancer (NSCLC), the heterogeneity in clinical behavior amongst patients with early stage disease has prompted the investigation of methods to improve prognostication. In a nested case-control study of patients with stage I NSCLC, promoter methylation of the cyclin-dependent kinase inhibitor 2A gene p16
and the H-cadherin gene CDH13
, was associated with an odds ratio of recurrent cancer of 25 when primary tumor and resected lymph nodes were evaluated by methylation specific PCR.63
This association was independent of age, race, sex, tumor histology, stage or smoking history. While efforts to predict the risk of recurrence in patients with an existing diagnosis of NSCLC will hopefully result in the ability to determine which patients need adjuvant therapy, research into methods to screen patients at risk for NSCLC may have even more far-reaching effects. In the first prospective study of its kind, Belinksy, et al. studied the methylation pattern of 14 genes in exfoliated cells from sputum.70
Study participants were recruited beginning in 1993 as part of the University of Colorado Cancer Center Sputum Screening Cohort Study and had a history of at least 30 pack-years of smoking with no prior diagnosis of cancer. Participants were followed periodically and sputum samples obtained routinely. The methylation patterns of sputum cells from patients who developed NSCLC were compared with those patients who did not. Of the 14 genes studied, 6 were identified as being associated with a greater than 50% likelihood of developing cancer. When 3 or more of the 6 genes were methylated, a 6.5 fold increased risk of cancer was noted. The sensitivity and specificity of this association was 64%. In a similar study, the sputum samples obtained from patients with and without squamous cell lung cancer participating in a lung cancer surveillance study were analyzed.71
The presence of methylation of the p16
and O6-methylguanine-DNA methyltransferase
(MGMT) promoters were determined by PCR. Aberrant methylation was detected in 100% of the patients with squamous cell carcinoma and was identified in samples from up to 3 years prior to diagnosis. The finding of p16 and MGMT methylation in high risk patients without cancer approximated their lifetime risk of lung cancer. At the time the study was published, two of these patients did in fact develop lung cancer. The search for a screening approach in patients at risk for NSCLC continues, however, results from prospective studies such as this certainly provide a basis for future investigations. The determination of additional methylated genes can perhaps increase the accuracy of sputum analysis and may provide a determination of which patients would benefit from frequent radiologic assessment.70
Hepatocellular carcinoma (HCC) poses challenges in early detection both in the U.S. and globally, and improvements in screening in high risk patients could have a far-reaching impact in this often incurable disease. Using blood samples from a community screening program in Taiwan, the DNA of patients with and without a diagnosis of HCC was extracted and methylation specific PCR was performed to detect the pre-diagnosis methylation status of three genes, p15, p16
and ras association domain family 1a (RASSF1A).72
Amongst the 50 cases of HCC, RASSF1A
was the most frequently methylated at a rate of 70%. Six HCC patients did not demonstrate methylation of any of the three genes. When combined with other risk factors such as hepatitis B surface antigen status, anti-hepatitis C antibody status, smoking and alcohol use, the hypermethylation biomarkers had a predictive accuracy of 89%, a sensitivity of 84% and a specificity of 94%. Methylation changes were detected in the samples up to 9 years prior to the diagnosis of HCC. The current method of screening patients at risk for HCC includes alpha-fetoprotein testing in combination with ultrasound. The effectiveness of such an approach remains controversial and the establishment of a molecular signature predicting for the development of HCC could prove useful, particularly in the setting of chemopreventive techniques. The ease of blood sampling may also increase compliance and potentially be more cost-effective and sensitive than routine radiologic screening.
As research into the treatment of various cancers has progressed over the last few decades, the notion of customizing therapy to a particular tumor’s characteristics has gained increased interest. No example better illustrates this concept than the use of temozolomide in the treatment of glioma. The use of this alkylator in combination with radiation therapy became standard of care in the adjuvant treatment of glioblastoma with the demonstration of a clinically meaningful survival advantage of 2.5 months in the group that received temozolomide with radiation therapy as opposed to those who received radiation alone.73
Given that MGMT
repairs the damage caused by such alkyating agents as temozolomide, an investigation into whether MGMT
methylation affected response to the drug was initiated.74
promoter methylation status was evaluated in the tumors of patients enrolled in the adjuvant trial comparing radiation therapy with and without temozolomide described above. MGMT
promoter methylation was detected in 45% of the assessable cases and was associated with a more favorable prognosis regardless of the therapy administered. Among those patients who received temozolomide with radiation therapy, the presence of MGMT
promoter methylation was associated with a survival advantage with a median survival of 21.7 months compared to 15.3 months in the radiation alone cohort (p = 0.007). An association has also been described between MGMT
promoter methylation and the incidence of pseudoprogression on MRI (defined as the radiologic appearance of tumor progression due to increased contrast enhancement). This radiographic change is thought to be caused by blood-brain barrier disruption from radiation therapy resulting in capillary disruption, fluid transudation and finally brain edema. In a series of patients with glioblastoma treated with radiation therapy and temozolomide, MRI pseudoprogression was noted in 91% of patients with MGMT
promoter methylation and in 41% of those with unmethylated promoter (p = 0.0002).75
Overall survival was significantly longer in patients with MGMT
promoter methylation and in those patients who developed radiologic pseudoprogression (p = 0.001 and p = 0.045, respectively). Thus, methylation status may potentially be used to offer treatment to those patients with glioma for whom it has the most chance of efficacy while also providing a means to ensure that treatment is not prematurely discontinued.
One disease in which genetic alterations have had a significant impact on use of noninvasive screening methods is colorectal cancer. With the publishing of the most recent joint guidelines of the screening and surveillance for the early detection of cancer and adenomatous polyps by the American Cancer Society, US Multi-Society Task Force on Colorectal Cancer and the American College of Radiology, the first time incorporation of a fecal DNA test has the potential to significantly impact providers’ approach to colorectal cancer screening.76
Though the test does not include a search for methylation changes, such DNA alterations in fecal DNA are found in patients with colorectal cancer and could impact the further development of screening tests in the future. In a series studying the fecal DNA of patients with colorectal cancer and adenomas as well as samples from normal individuals revealed a higher rate of methylation of the target genes studied in the patients with cancer.77
In fact, of the three genes studied, over 96% of patients with cancer and 81% of patients with precancerous lesions had at least one methylated gene. In the normal individuals, only 1 patient of the 24 studied had a methylated gene. This analysis, which was performed using methylation-specific PCR had a sensitivity of 93.7% and a specificity of 77.1%. The presence of free-circulating tumor DNA in patients with colorectal cancer has provided an even easier option for testing in patients and a foundation on which to build further genetic testing.78,79
in circulating DNA of patients with colorectal cancer has been associated with advanced stage and has been found in patients with recurrent disease.80,81
Methylation changes in the serum yielding a diagnosis of colorectal cancer could further build on existing testing and provide an even less invasive means of diagnosis than fecal DNA testing. Epigenetic changes in the serum of patients already diagnosed with colorectal cancer provide a potential means of prognostication before and during therapy.
The disease most notably associated with hypermethylation due to the use of demethylating agents in its treatment is the myelodysplastic syndromes (MDS). In an assessment of methylation patterns of several key genes including p15INK4B
, E-cadherin (CDH1
), hypermethylated in cancer 1
), and estrogen receptor (ER
), hypermethylation was associated with leukemic transformation and poor prognosis in low-risk MDS.82
In particular, patients with early MDS who demonstrated hypermethylation of at least one gene had a significantly shorter survival (20 months vs. 102 months, p=0.002). Analysis of the methylation patterns of p15INK4B
in elderly patients with high risk MDS or acute myeloid leukemia (AML) following MDS who underwent standard induction therapy revealed absence of complete responses (CR) in those patients with hypermethylation of all three genes (p = 0.03).83
CR was achieved in over 50% of the patients with no hypermethylation which is the same or marginally better than CR rates in other studies of similar patients.884,85
This was the first study to demonstrate the effect of methylation on the response to treatment amongst patients with high risk MDS and AML arising from MDS. Whether incorporation of agents that modulate hypermethylation can improve outcomes in these patients remains to be determined.
The epigenetic changes well-established in MDS and AML arising from MDS have also been investigated in other types of AML. While the attainment of a complete response with standard induction therapy is feasible in most patients, the relapse rate is greater than 50%. Prognostication of individuals at greater risk of relapse could prove useful in the effort to develop risk-adapted therapy. In an evaluation of over 180 patients with and without AML, methylation of ERα
occurred specifically in patients with AML and in none of the controls.86
Increased methylation levels in one or both genes in patients in complete remission were associated with decreased relapse free survival. Importantly, all patients who demonstrated hypermethylation of ERα
relapsed whereas 17% of patients with methylation of neither gene had recurrence. The most obvious utility of studying methylation changes in cancer cells is the creation of novel agents; however, a very important potential outcome of this increasing knowledge is also in the arena of diagnostics. While the ultimate goal is the eradication of cancer through the development of preventive tactics, the fact remains that a significant proportion of the population will likely be affected by cancer and the development of biomarkers to use as diagnostic and monitoring tools can be an important part of our goals for the very near future.