Methylation of MGMT Is Associated with Poor Prognosis in Patients with Stage III Duodenal Adenocarcinoma
Methylation of MGMT Is Associated with Poor Prognosis in Patients with Stage III Duodenal Adenocarcinoma
Tao Fu 0 1 2
Anup Sharmab 0 2
Fei Xie 0 1 2
Yanliang Liu 0 1 2
Kai Li 0 1 2
Weiwei Wan 0 1 2
Stephen B. Baylin 0 2
Christopher L. Wolfgang 0 2
Nita Ahuja 0 2
Dajun Deng, Beijing Cancer Hospital, CHINA
0 Competing Interests: Stephen B. Baylin has commercial grant funding and serves on the advisory board for MDx Health Inc. and BioNumerik Pharmaceuticals Inc. Nita Ahuja has grant funding
1 Department of Gastrointestinal Surgery II, Key Laboratory of Hubei Province for Digestive System Disease, Renmin Hospital, Wuhan University , Wuhan , China , 2 Department of Surgery, The Johns Hopkins University School of Medicine , Baltimore , Maryland, United States of America, 3 Department of Oncology, The Johns Hopkins University School of Medicine , Baltimore , Maryland, United States of America, 4 Department of Urology, The Johns Hopkins University School of Medicine , Baltimore, Maryland , United States of America
2 Institutes of Health grants CA140599 and CA127141, National Natural Science Foundation of China grants 81000898 and 81472289, and sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars , State Education Ministry of China
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
stage III DA.
O6-methylguanine-DNA methyltransferase (MGMT) methylation status has not been
extensively investigated in duodenal adenocarcinoma (DA). The aim of this study was to evaluate
the MGMT methylation status and examine its possible prognostic value in patients with
Demographics, tumor characteristics and survival were available for 64 patients with stage
III DA. MGMT methylation was detected by using MethyLight. A Cox proportional hazard
model was built to predict survival, adjusted for clinicopathological characteristics and
tumor molecular features, including the CpG island methylator phenotype (CIMP),
microsatellite instability (MSI), and KRAS mutations.
MGMT methylation was detected in 17 of 64 (26.6%) patients, and was not correlated with
sex, age, tumor differentiation, CIMP, MSI, or KRAS mutations. MGMT methylation was the
only one factor associated with both overall survival (OS) and disease-free survival (DFS)
on both univariate and multivariate analyses. In patients treated with surgery alone,
MGMTmethylated group had worse OS and DFS when compared with MGMT-unmethylated
group. However, in patients treated with chemotherapy/radiotherapy, outcomes became
comparable between the two groups.
Our results demonstrate MGMT methylation is a reliable and independent prognostic factor in DAs. Methylation of MGMT is associated with poor prognosis in patients with stage III DAs.
Primary adenocarcinoma of the duodenum (duodenal adenocarcinoma, DA) was initially
described by Hamburger in 1746, comprising less than 1% of all malignant neoplasms of the
gastrointestinal tract [
]. Because of its rarity, there is an insufficiency of well-designed
studies to guide management. In general, DAs have more favorable outcomes compared to other
periampullary malignancies and excision is considered the backbone of treatment for patients
with localized tumors or limited metastatic disease when feasible. Data regarding the effect of
adjuvant chemotherapy/radiotherapy are limited, with no faithful evidence of significant
benefit in survival in patients with DAs. A Cochrane review in 2007 failed to find suitable trials
eligible for meta-analysis to determine the role of adjuvant chemotherapy in the treatment of
adenocarcinoma of the small intestine [
]. Although adjuvant therapy is regularly used in this
disease, more studies are needed to evaluate the effectiveness of adjuvant therapy in the
management of DAs.
O6-methylguanine-DNA methyltransferase (MGMT) is a ubiquitously expressed DNA
repair protein, and it removes methyl and chloroethyl groups from the O6 position of guanine
in a damage reversal reaction. In the absence of MGMT, O6-methylguanine in the DNA
generates point mutations and DNA double-strand breaks via cellular replication and DNA
mismatch repair that trigger cell death by apoptosis [
]. Methylation of the CpG islands located in
the promoter region of MGMT is primarily responsible for the inactivation of MGMT in
several tumor types [
]. Inactivation of MGMT can lead to it subsequently being unable to protect
tumors from cytotoxic damage induced by alkylating chemotherapeutics, i.e. methylating and
chloroethylating agents, and thus predicts benefit from these chemotheraptic agents. MGMT
methylation may also play a prognostic role in various cancers. To our knowledge, there is only
one previous study that has described MGMT methylation in DAs in a small number of
patients and there was no assessment of MGMT methylation frequency or prognostic
Microsatellite instability (MSI), developing from defects in other mismatch repair genes
MLH1, MSH2, MSH6, and PMS2, are seen in 18–35% of small bowel adenocarcinomas
including DAs [
]. MSI along with KRAS mutations represent the most common molecular
defects in DA [
7, 11, 12
]. MSI is associated with prognosis in patients with colorectal cancer.
Its prognostic value in DAs is worth investigation. MGMT methylation seems to favor
mutations in cancer-related genes (e.g. TP53 and KRAS). Kim et al. previously showed the
association between MGMT methylation and KRAS G-to-A transition in a group of patients with
carcinomas of the extrahepatic bile ducts, ampulla of Vater, and duodenum [
]. Due to the
small number of duodenal carcinomas in the previous study, this correlation still needs
The aims of this study were to assess the methylation status of MGMT gene in the largest
series of stage III DAs reported to date and to establish whether or not methylation of MGMT
might have prognostic or predictive value in patients with stage III DA.
Material and Methods
This retrospective cohort study included patients with pathologically confirmed DA who had a
surgical resection. Patients were identified from the Johns Hopkins Hospital Oncology Clinical
Information System from January 1997 to December 2009 and 155 duodenal adenocarcinomas
patients who underwent surgical resection at our institution were identified. Patients who
underwent preoperative chemotherapy/radiotherapy, lacked follow-up information or had
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missing archival primary tumors or corresponding matched normal samples were excluded.
Formalin-fixed, paraffin-embedded (FFPE) tissue blocks of primary tumors and corresponding
matched normal samples were collected from 107 patients. Tissue sections from the blocks
were then reviewed by an expert gastrointestinal pathologist. After excluding ampullary tumors
and low tumor cellularity sections, the remaining 64 stage III cases formed the final study
cohort (Table 1). Ascertainment of survival was performed by using the Johns Hopkins
electronic health records, the Cancer Registry and mortality was confirmed also within the Social
Security Death Index. The Johns Hopkins Hospital Institutional Review Board approved this
Analyses of KRAS mutations, and microsatellite instability
Genomic DNA was extracted from FFPE tissues. Polymerase chain reaction (PCR) and
sequencing targeted for KRAS codons 12 and 13 were performed [
MSI status was determined using D2S123, D5S346, D17S250, BAT25, and BAT26 [
Microsatellite sizes were compared with those of normal adjacent tissue, and tumors with 2 or
more of the markers exhibiting instability were classified as MSI-high. Tumors with only one
marker exhibiting instability or no markers with instability were classified as MSI-low or
microsatellite stable (MSS), respectively.
Bisulfite modification and methylation analysis
Purified DNA (2 μg) was bisulfite treated and purified using the EZ DNA methylation kit
(Zymo Research, Orange, CA) according to the manufacturer's instructions.
A 5-gene signature was used to assess the CpG island methylator phenotype (CIMP) status
of the primary tumor tissue: CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1 [
Methylation of these five genes and MGMT was quantified by MethyLight, a methylation-specific,
probe-based, real-time PCR technique [
12, 15, 16
]. Alu was used as a normalization control
reaction. All CIMP probes utilized a 5’ FAM fluorophore, a 3’ IBFQ quencher, and an internal
ZEN quencher (Integrated DNA Technologies, Coraville, IA). DNA methylation was reported
as the percent of methylated reference (PMR) = 100 × ((methylated
reaction/Alu)sample/(methylated reaction/Alu)M.SssI-reference) [
]. We classified each marker as methylated when PMR
4. The PMR cut-off levels were set at plus two standard deviations of the average methylation
levels observed in normal duodenal mucosa controls. Samples were considered CIMP+ if at
least 3 out of the five studied genes were methylated [
Differences in categorical variables between study groups were analyzed using χ2 test or
Fisher’s exact test. The primary end point for the study was disease-free survival (DFS), defined as
the time from surgery to death or recurrence of disease, whichever occurred first. Overall
survival (OS) was the secondary end point. Patients without evidence of death or recurrence were
censored at last follow-up. Survival was estimated by using the Kaplan-Meier method and
logrank statistics computed to test for differences between survival curves for various prognostic
factors. Univariate and multivariate Cox proportional hazard regression models included
MGMT methylation, sex, age, tumor differentiation, R0 resection, chemoradiation, CIMP, MSI
status, and KRAS mutations. Results of Cox regression are reported as hazard ratio (HR) with
corresponding 95% confidence intervals (CI). All hypotheses tests were two-sided, and results
were considered statistically significant for P values < 0.05. All calculations were performed
using SPSS 16.0 software (SPSS Inc, Chicago, IL).
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aMGMT-U versus MGMT-M, χ2 test unless indicated otherwise
bFisher’s exact test.
Abbreviations: CIMP, CpG island methylator phenotype; MSS, microsatellite stable; MSI, microsatellite instability; U, unmethylated; M, methylated.
Clinicopathologic characteristics and association with MGMT
methylation or MSI status
DNA extraction, MGMT methylation testing by MethyLight, and MSI status testing were
successful in all 64 patients. Seventeen patients (26.6%) out of the 64 patients tested were
MGMTmethylated (MGMT-M, Table 1). Fifteen patients (23.4%) displayed MSI-high; 9 patients
(14.1%) were MSI-low and 40 patients (62.5%) were MSS. Because extensive data indicate that
tumors with MSI-low are biologically similar to those exhibiting MSS, both tumors were
grouped together and henceforth are referred to as MSS in this study. Among the 17 (26.6%)
patients demonstrating the CIMP positive (CIMP+), 7 (41.2%) were MGMT-M as well
(Table 1). No correlation between CIMP and MGMT methylation status was observed
(P = 0.111, Table 1).
Median age at diagnosis of DAs was 64.5 years (64.2 ± 14.3; mean ± SD).
MGMT-unmethylated (MGMT-U) and MGMT-M subgroups showed no differences by gender, age, tumor
differentiation, CIMP, MSI and KRAS mutation status or the receipt of chemotherapy/
radiotherapy between the two groups (Table 1).
MGMT methylation status as a prognostic marker
The mean (SD) follow-up was 42.9 (28.5) months. There were 36 deaths, 24 recurrences, and
42 progressions at the end of follow-up. The median OS was 41.2 months (95% CI, 25.2 to 57.2
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Fig 1. Kaplan-Meier survival estimates between patients with stage III duodenal adenocarcinomas with MGMT methylated and those with MGMT
unmethylated. (A) overall survival, (B) disease-free survival.
months), and the median DFS was 18.8 months (95% CI, 5.6 to 32.1 months). In Kaplan-Meier
analysis of all patients, MGMT-M was associated with worse OS (log-rank P = 0.001; Fig 1A)
and DFS (log-rank P = 0.012; Fig 1B). The median OS was 51.9 months (95% CI, 22.5 to 81.3
months) vs. 14.5 months (95% CI, 9.7 to 19.3 months), and the median DFS was 29.2 months
(95% CI, 0 to 59.7 months) vs. 12.0 months (95% CI, 7.0 to 17.0 months) for patients with
MGMT-U tumor vs. MGMT-M tumor, respectively. In univariate models, MGMT-M was
associated with worse OS (HR, 3.01; 95% CI, 1.53 to 5.91; P = 0.001) and DFS (HR, 2.21; 95% CI,
1.17 to 4.17; P = 0.014). This remained statistically significant in multivariate models for OS
(HR, 4.25; 95% CI, 2.00 to 9.05; P = 0.000) and for DFS (HR, 2.80; 95% CI, 1.43 to 5.48;
P = 0.003; Table 2).
Adjuvant treatment with fluorouracil-based chemotherapy/radiotherapy was administered in
47 patients, while 17 patients were treated with surgery alone. There was no significant
improvement in OS for patients treated with adjuvant therapy when compared with patients
who were not treated (HR, 1.13; 95% CI, 0.51 to 2.51; P = 0.759). When comparing DFS, there
was no difference based on adjuvant treatment (HR, 0.85; 95% CI, 0.41 to 1.73; P = 0.648;
In patients treated with surgery alone (n = 17), MGMT-M was associated with worse OS
(HR, 7.88; 95% CI, 1.83 to 34.00; P = 0.006) and DFS (HR, 5.33; 95% CI, 1.40 to 20.30;
P = 0.014) on univariate analysis. This remained statistically significant in multivariate models
for OS (HR, 7.49; 95% CI, 1.04 to 53.84; P = 0.045) and OS (HR, 4.11; 95% CI, 1.03 to 16.40;
P = 0.046). However, no association was observed between MGMT methylation status and
both OS (HR, 1.85; 95% CI, 0.84 to 4.11; P = 0.130) and DFS (HR, 1.56; 95% CI, 0.74 to 3.30;
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Abbreviations: OS, overall survival; DFS, disease-free survival; HR, hazard ratio; CI, confidence interval; M, methylated; U, unmethylated; CIMP, CpG island
methylator phenotype; MSS, microsatellite stable; MSI, microsatellite instability. A backward elimination with threshold of P = 0.300 was used to select
variables in the final models
P = 0.243; Table 3) in patients treated with chemotherapy/radiotherapy. In Kaplan–Meier
analysis, there were also significant differences in survival time distributions between patients with
MGMT-M and those with MGMT-U in the group treated with surgery alone (log-rank
P = 0.001 for OS, Fig 2A; log-rank = 0.006 for DFS, Fig 2B). The median OS was not reached
vs. 9.4 months (95% CI, 0 to 25.7 months), and the median DFS was not reached vs. 9.4 months
(95% CI, 0 to 25.7 months) for patients with MGMT-U tumor vs. MGMT-M tumor,
respectively. No significant differences were found between patients with MGMT-M tumor and those
with MGMT-U tumor in the group treated with chemotherapy/radiotherapy (log-rank
P = 0.123 for OS, Fig 3A; log-rank = 0.239 for DFS, Fig 3B).
The present study was designed to better understand the contribution of methylation of
MGMT for patients with stage III DAs and to determine its effect in response to
fluorouracil6 / 13
based adjuvant chemotherapy/radiotherapy in a cohort of patients. Our results indicate that,
MGMT methylation is a reliable and independent prognostic factor in DAs. MGMT
methylation is associated with poor prognosis in patients with stage III DAs. It seems that
fluorouracilbased chemotherapy/radiotherapy does not improve outcomes in patients with stage III DAs.
However, in the subsets of DAs with MGMT methylation fluorouracil-based chemotherapy/
radiotherapy may confer a survival benefit.
MGMT methylation has been associated with various cancers. Specifically, MGMT
methylation was seen in 39–53% of CRCs [
], 11% of gastric cancer , 30–38% of lung cancer [
], 34–72% of esophageal cancer , 34% of soft tissue sarcomas [
], 58% of breast cancer
Fig 2. Kaplan-Meier survival estimates between patients with stage III duodenal adenocarcinomas with MGMT methylated and those with MGMT
unmethylated in group treated with surgery alone. (A) overall survival, (B) disease-free survival.
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Fig 3. Kaplan-Meier survival estimates between patients with stage III duodenal adenocarcinomas with MGMT methylated and those with MGMT
unmethylated in group treated with fluorouracil-based chemotherapy/radiotherapy. (A) overall survival, (B) disease-free survival.
], and 30–70% of glioblastoma [
]. In this study, we analyzed a large cohort of patients
with stage III DAs and showed that MGMT methylation existed in 26.6% (17/64) of the tumors.
It was reported that inactivation of MGMT by promoter methylation was theoretically
associated with the presence of KRAS G>A transitions in CRC [
]. Their data suggested that
epigenetic silencing of MGMT by methylation was strongly associated with, and preceded, G>A
mutations in KRAS in colorectal tumorigenesis. Some studies proved this possible association
in CRCs [
], however, we did not find this link between these two events in DAs
(P = 0.226; data not shown). This can be secondary to various causes including methodology
issues (type of methylation assay, small sample size, intratumor heterogeneity) and most
importantly, alternative molecular mechanisms that cause DAs. The concurrence of these
epigenetic and genetic lesions in different tumors suggests a more complex relationship between
these events. For example, MGMT methylation is common , but KRAS mutations are
relatively rare in glioblastoma [
]. Nagy et al. also showed that no conclusions could be drawn
with regard to mutation type and methylation in endometrial cancers [
]. In a study of 62
gastric cancer tissue samples, KRAS mutations were detected in only one (1.6%) sample and
MGMT methylation was detected in 13 (21%) samples, and no connection was shown between
KRAS mutations and MGMT methylation [
]. Similar results were shown in a study of 62 soft
tissue sarcomas with MGMT methylation 33.9% (21/62) and KRAS mutations 3.7% (2/62)
]. In a large cohort study with 1123 CRC, a strong association with MGMT methylation was
found with KRAS mutations both in univariate analysis (OR 2.3, 95% CI 1.7–3.0, P < 0.0001)
and multivariate analysis (OR 1.9, 95% CI 1.5–2.6, P < 0.0001). But on classification of the
KRAS mutant cancers by mutation type, no association was found between MGMT
methylation and G>A mutations compared with non-G>A mutations, and in fact frequency of
MGMT-M and MGMT-U tumors was approximately equal for each mutation category [
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In previous studies, the significance of the correlation between MGMT methylation and
prognosis of patients was controversial [
21, 25, 34–36
]. In present study, the impact of MGMT
methylation on patient survival was assessed by univariate and multivariate analyses. Cox
proportional hazard models indicated that methylation of MGMT was strongly associated with
poor survival in DAs patients.
Despite the absence of prospective randomized data clarifying the role of adjuvant therapy
in DAs, the use of adjuvant therapy has increased. Data from the National Cancer Database
shows a spread use of adjuvant chemoradiation in small bowel cancers (including 49.1%-58.8%
DAs) from 8.1% in 1985 to 22.2% in 2005 (P < 0.0001) [
]. In all likelihood, this trend reflects
the poor outcome of high-risk dissected DAs, the known efficacy of systemic chemoradiation
in the metastatic setting and the significant survival benefit of adjuvant therapy in patients
Several studies have individually examined the results of adjuvant therapy after resection of
DA. In 1980, Alwmark et al. suggested that chemoradiation might improve the survival of
patients with DA [
]. Since then, advances in chemotherapy and radiotherapy have developed,
but chemoradiation has commonly been reserved for palliation of DAs. Our institution has
previously published a pilot study on 14 patients with node-positive DA who underwent
pancreaticoduodenectomy followed by adjuvant fluorouracil-based chemoradiation [
study suggested that adjuvant chemoradiation contributed improved local control compared
with historical controls treated with surgery alone (93% vs. 67%), but did not lengthen overall
survival (5 year, 44% vs. 43%). However, in this follow up study from our institution of a larger
cohort of patients we were unable to reproduce this positive effect of chemoradiation for either
local control or OS [
]. Another retrospective study of 103 patients with DA (including 46
stage III DAs) from Massachusetts General Hospital compared patients who underwent
resection alone with those who received resection and adjuvant and/or neoadjuvant chemotherapy/
chemoradiation and found no marked improvement in OS, or time to recurrence [
]. A similar
study of 32 patients with DA from Duke University Medical Center also failed to show a
beneficial effect of adjuvant chemoradiation both in terms of OS (44% vs. 57%), disease-free survival
(44% vs. 54%) or local control (49% vs. 70%) [
]. In an analysis of 1,611 cases on long-term
outcome after resection of DA by utilizing the Surveillance, Epidemiology, and End Results
(SEER) database, a large population-based cancer registry showed that the use of radiation was
associated with improvements in survival on univariate analysis, but this effect disappeared
after controlling for other variable [
In this study, we showed that patients treated with adjuvant therapy had similar prognosis
to those treated with surgery alone. In patients treated with surgery alone, patients with
MGMT-M tumor had worse OS and DFS compared with those with MGMT-U tumor.
However, in patients undergoing adjuvant fluorouracil-based chemotherapy/radiotherapy,
outcomes became comparable between patients with MGMT-M tumor and those with MGMT-U
tumor. This might be, to some extent, due to differential responses to
chemotherapy/radiotherapy between these two subtypes of tumor. Nevertheless, this phenomenon deserves further
investigation. The finding is potentially of great significance, as the addition of adjuvant
chemotherapy/radiotherapy in DAs is currently a matter of great debate.
Alkylating agent temozolomide is now the chemotherapeutic agent most regularly used in
patients with newly diagnosed glioblastoma. It is well established that MGMT methylation is a
promising predictor of prolonged prognosis in patients with glioblastoma receiving
]. In a pivotal randomized trial investigating the value of temozolomide added to
radiotherapy in patients with glioblastoma, median survival in patients with methylated
MGMT promoter increased from 15.3 months (95% CI 13.0–20.9) with radiotherapy alone to
21.7 months (17.4–30.4) with radiotherapy and temozolomide (hazard ratio [HR] 0.51, 95% CI
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0.31–0.84). However, patients with unmethylated MGMT promoter in the tumor showed only
a marginal benefit from addition of temozolomide, with a median survival of 12.7 months
(95% CI 11.6–14.4) compared with 11.8 months (9.7–14.1) for patients treated with
radiotherapy alone (HR 0.69, 95% CI 0.47–1.02) . However, the value of MGMT methylation as a
prognostic or predictive marker for patients treated with other specific regimens of anticancer
agents remains a matter of debate to date. A previous study has shown that CRC patients who
received oral fluorouracil-based adjuvant chemotherapy had a low recurrence rate when the
tumor revealed methylation in its MGMT promoter [
]. Their in vitro study also proved an
enhancement of fluorouracil anti-tumor effect for CRC and other malignancies with MGMT
methylation by controlling the levels of MGMT in tumor [
]. It was hypothesized that tumor
cells with methylation of MGMT are likely to remain in G2/M checkpoint, resulting in
increased sensitivity to chemoradiation [
Our results show that MGMT methylation is an important prognostic factor in stage III
DAs. Our data also suggest a possible role for fluorouracil-based chemotherapy/radiotherapy
in management of stage III DAs patients with MGMT methylation and MGMT-M may also
then have a predictive role. Further studies in larger samples will help validate these.
S1 File. SPSS file for statistical analysis.
We would like to thank Kathy Bender and Joann Murphy for administrative support. We
would also like to thank Sharon Metzger-Gaud, Theresa Sanlorenzo-Caswell, and the Johns
Hopkins Cancer Registry for assistance with the primary cancer databases.
Conceptualization: TF NA.
Data curation: TF NA.
Formal analysis: TF.
Funding acquisition: TF NA.
Methodology: TF NA.
Project administration: TF NA.
Resources: AS CLW NA.
Supervision: TF SBB CLW NA.
Validation: TF SBB CLW NA.
Visualization: TF NA.
Writing – original draft: TF.
Investigation: TF AS FX YL KL WW CLW NA.
Writing – review & editing: TF AS FX YL KL WW SBB CLW NA.
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