To describe the frequency of MLH1 promoter methylation in colorectal cancer (CRC); to explore the associations between MLH1 promoter methylation and clinicopathological and molecular factors using a systematic review and meta-analysis.
A literature search of the PubMed and Embase databases was conducted to identify relevant articles published up to September 7, 2012 that described the frequency of MLH1 promoter methylation or its associations with clinicopathological and molecular factors in CRC. The pooled frequency, odds ratio (OR) and 95% confidence intervals (95% CI) were calculated.
The pooled frequency of MLH1 promoter methylation in unselected CRC was 20.3% (95% CI: 16.8–24.1%). They were 18.7% (95% CI: 14.7–23.6%) and 16.4% (95% CI: 11.9–22.0%) in sporadic and Lynch syndrome (LS) CRC, respectively. Significant associations were observed between MLH1 promoter methylation and gender (pooled OR = 1.641, 95% CI: 1.215–2.215; P = 0.001), tumor location (pooled OR = 3.804, 95% CI: 2.715–5.329; P<0.001), tumor differentiation (pooled OR = 2.131, 95% CI: 1.464–3.102; P<0.001), MSI (OR: 27.096, 95% CI: 13.717–53.526; P<0.001). Significant associations were also observed between MLH1 promoter methylation and MLH1 protein expression, BRAF mutation (OR = 14.919 (95% CI: 6.427–34.631; P<0.001) and 9.419 (95% CI: 2.613–33.953; P = 0.001), respectively).
The frequency of MLH1 promoter methylation in unselected CRC was 20.3%. They were 18.7% in sporadic CRC and 16.4% in LS CRC, respectively. MLH1 promoter methylation may be significantly associated with gender, tumor location, tumor differentiation, MSI, MLH1 protein expression, and BRAF mutation.
Citation: Li X, Yao X, Wang Y, Hu F, Wang F, Jiang L, et al. (2013) MLH1 Promoter Methylation Frequency in Colorectal Cancer Patients and Related Clinicopathological and Molecular Features. PLoS ONE 8(3): e59064. https://doi.org/10.1371/journal.pone.0059064
Editor: Anthony WI. Lo, The Chinese University of Hong Kong, Hong Kong
Received: February 7, 2012; Accepted: February 12, 2013; Published: March 29, 2013
Copyright: © 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by grants from National Natural Science Foundation of China for Priority Areas (NSFC 30972538), Education Bureau of Heilongjiang Province (11531100), and the Graduate Foundation, supported by the Scientific Research of Heilongjiang Province (YJSCX2009-224HLJ) and Harbin Medical University (HCXB2009009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Colorectal cancer (CRC) is one of the most common malignancies, representing the third most common cancer in men and the second in women worldwide . One of the genetic pathways in the development of CRC is the microsatellite instability (MSI) .
Microsatellites are repeated DNA sequences which occur approximately every 50–100 Kb base pairs throughout the human genome , . Multiple studies have indicated that about 90% of the Lynch Syndrome (LS) ,  and 10% to 15% of sporadic CRC can be detected of MSI , . MSI in LS and sporadic CRC occurs through two different mechanisms. In LS, MSI is mainly caused by germline mutation of mismatch repair genes . MSI in sporadic CRC is commonly due to methylation induced silencing of the MLH1 gene .
DNA methylation refers to the presence of a methyl group on a cytosine residue . DNA methylation of tumor suppressor genes leading to transcriptional inactivation has been identified as an important mechanism in human carcinogenesis , . MLH1 gene, as a number of suppressor genes, is prone to be silenced by promoter methylation in CRC , , .
Since the first report of MLH1 promoter methylation in sporadic colon tumors , the prevalence of MLH1 promoter methylation have been widely studied not only in sporadic but also in LS CRC. However, the results are inconsistent. The frequency of MLH1 promoter methylation in sporadic CRC varied from 0.0%  to 66.9% . It varied from 0.0%  to 21.4%  in LS CRC.
BRAF and KRAS are important members of RAS/RAF/MAPK signaling pathway, which regulates cell growth, proliferation, differentiation, and apoptosis in malignant and nonmalignant cells . BRAF mutation has been shown to be associated with MLH1 promoter methylation , . Whereas, MLH1 promoter methylation was few detected in KRAS mutant CRC . The associations between MLH1 promoter methylation and BRAF and KRAS mutation in CRC have been widely studied with inconsistent results , , , . The associations between MLH1 promoter methylation and other clinicopathological and molecular characteristics of CRC such as tumor location, tumor staging, tumor differentiation, family history, MSI, and MLH1 protein expression were also widely studied. However, the results are inconsistent. Therefore, we conducted a systematic review and meta-analysis to accurately estimate the frequency of MLH1 promoter methylation in LS and sporadic CRC, and the associations between MLH1 promoter methylation and clinicopathological/molecular characteristics of CRC.
Search Strategy and Selection Criteria
We conducted a systematic literature search using PubMed and Embase from January 1, 1997 to September 7, 2012 to identify all the relevant English-language articles. The following keywords were used: “methylation” and “MLH1” and “promoter” and “colorectal cancer” and/or “carcinoma” or “tumor” or “neoplasm”. We also hand-searched the reference lists of the retrieved articles and reviews for additional articles.
The inclusion/exclusion criteria were as follows: (1) papers on MLH1 promoter methylation in unselected CRC were included. In contrast, papers that selected subgroups were excluded (such as selected based on age, tumor staging and ulcerative colitis-associated CRC); (2) sporadic CRC and/or LS related CRC remained as specific selected groups, often stratified by MSI status and/or MLH1 expression loss; (3) data regarding the DNA methylation of tumor tissue of CRC were included in the pooled analysis, whereas data regarding the DNA methylation of normal colonic mucosa , , , serum , , and peripheral blood leukocyte , ,  of CRC were excluded; (4) studies that investigated multiple CRCs were excluded , , ; (5) case reports were excluded; (6) repetitive reports were unified by using the latest or the largest edition; (7) paper with insufficient or duplicated data were excluded.
Two authors (X. and X.P) independently conducted literature searches to identify all possible papers that met the inclusion criteria. Disagreements were settled by consensus or a third review (Y.B.N) for adjudication. The following information were extracted from every eligible study: authors, publication year, continent, country, patient source, sample size, methylation detecting method, positive frequency, gender, family history, tumor location (proximal and distal), tumor staging, and promoter regions.
Classification of Family History
Patients had no family history of cancer regardless of the onset age were categorized as sporadic CRC. LS was diagnosed if a patient with family history met either Amsterdam criteria (I or II) ,  or Bethesda criteria (original or revised) ,  or confirmed with germline mutation in a DNA mismatch repair gene , . The unselected CRC tumors were defined as patients from nature population or hospital-based. The unselected CRC tumors included sporadic and LS CRC, which were defined as total CRC.
Tumor Staging and Differentiation
Tumor staging was categorized as I, II, III and IV stages based on the TNM classification (The Union for International Cancer Control [UICC]) . Stage I: Cancer has begun to spread, but is still in the inner lining. Stage II: Cancer has spread to other organs near the colon or rectum. It has not reached lymph nodes. Stage III: Cancer has spread to lymph nodes, but has not been carried to distant parts of the body. Stage IV: Cancer has been carried through the lymph system to distant parts of the body. Differentiation was graded on a scale of poor, moderate or well differentiation.
Promoter regions tested were noted as the A, B, C and D regions proposed by Deng et al. , where primer sequences were given. Promoter regions were checked against the sequence −1000 to −1, relative to the start codon of MLH1.
MSI is typically assessed by analyzing five microsatellite markers (BAT25, BAT26, D2S123, D5S346, and D17S250) suggested by the National Cancer Institute . One study expanded this panel to ten markers, which made the diagnosis of MSI CRC easier . In this meta-analysis, three categories of MSI status were defined according to the following criteria: two or more loci out of five loci with instability (or ≥30–40% of loci if a larger panel of markers was used) was defined as MSI-H; one locus with instability (or <30–40% of loci in larger panels) was defined as lower-level microsatellite instability (MSI-L); and no loci with instability (or no apparent instability in larger panels) was defined as microsatellite stable (MSS). For papers without detailed information about MSI-H and MSI-L, only two levels of microsatellite instability status could be categorized: MSI-positive (MSI) and MSI-negative (MSS). BRAF and KRAS status were classified into mutant and wild type. MLH1 protein expression status was defined as positive or negative.
The pooled frequency of MLH1 promoter methylation and 95% confidence intervals (95% CI) were estimated. The frequency of MLH1 promoter methylation was compared in different tumor characteristics. Heterogeneity among studies was evaluated with Cochran’s Q test  and the I2 statistic , . When heterogeneity was not an issue (I2 values <50%), a fixed effect model was used to calculate parameters. If there was substantial heterogeneity (I2 values ≥50%), a random-effects model was used to pool data and attempt to identify potential sources of heterogeneity based on subgroup analyses. The pooled OR was estimated for the association between MLH1 promoter methylation and clinicopathological, molecular features. P values tailed less than 0.05 were considered statistically significant.
Publication bias was evaluated with funnel plot, Begg’s rank correlation , and Egger’s regression . If publication bias existed, the trim and fill method was used to adjust the pooled frequency, pooled OR and 95% CI . Data were calculated with Comprehensive Meta-Analysis V2.
752 relevant articles were identified for initial review according to the inclusion and exclusion criteria. After screening, information for 10528 individuals from 96 studies was reviewed and included in the meta-analyses. Figure 1 showed the detailed selection process of articles.
Frequency of MLH1 Promoter Methylation in Total CRC Tumors
Overall, there were 19 studies with 5584 patients demonstrating MLH1 promoter methylation in total CRC tumors. The total frequency of MLH1 promoter methylation was 20.3% (95% CI: 16.8–24.1%) (Table 1, Figure 2). There was significant heterogeneity among the studies (I2 = 87.896%).
The frequency of MLH1 promoter methylation was 18.7% (95% CI: 14.7–23.6%) in 3583 sporadic CRC of 29 studies and 16.4% (95% CI: 11.9–22.0%) in 243 LS reported in eight studies (Table 1). The frequency of MLH1 promoter methylation in sporadic MSI-H and LS CRC MSI-H were 73.6% (95% CI: 67.3–79.0%) and 15.3% (95% CI: 8.8–25.4%), respectively (Table 2). For MSI-H CRC, significant association between MLH1 promoter methylation and family history was observed (pooled OR = 20.828, 95% CI: 4.056–106.950; P<0.001; I2 = 55.363%; Table 3), when pooled data on four studies , , , .
Gender information was available for 6 of the 19 studies with a total of 555 female and 699 male patients , , , , , . The MLH1 promoter methylation in female and male were 20.8% (95% CI: 15.6–27.2%) and 11.8% (95% CI: 6.9–16.5%) in total CRC group (pooled OR = 1.641, 95% CI = 1.215–2.215; P = 0.001; I2 = 33.819%) (Table 1 and Table 3).
MLH1 promoter methylation was observed in 29.6% (95% CI: 20.4–40.8%) of the 474 proximal tumors and 6.5% (95% CI: 3.0–13.4%) of the 698 distal tumors in six studies (Table 1). Significant association was observed between MLH1 promoter methylation and tumor location (pooled OR = 3.804, 95% CI: 2.715–5.329; P<0.001; I2 = 46.541%) (Table 3). These data were based on six studies covering a total of 1172 patients (Table 1).
The pooled prevalence of MLH1 promoter methylation and pooled OR for the association between MLH1promoter methylation and the UICC stage were estimated in four studies , , ,  (Table 1 and Table 3). The pooled prevalence of MLH1 promoter methylation in stages I & II and in stages III & IV were 22.4% (95% CI: 15.6–31.0%) and 25.5% (95% CI: 9.3–53.5%), respectively (Table 1). The association between MLH1 promoter methylation and tumor staging was not significant (pooled OR = 1.044, 95% CI: 0.441–2.471; P = 0.922; I2 = 42.854%) (Table 3).
Six studies , , , , ,  addressed the frequency of MLH1 promoter methylation in total CRC according to tumor differentiation. The frequency of MLH1 promoter methylation was 31.0% (95% CI, 24.6–38.1%) in 182 poor-differentiated CRC and 17.6% (95% CI, 11.9–25.3%) in 769 moderate or well-differentiated CRC, respectively (Table 1). MLH1 promoter methylation in poor-differentiated CRC was significantly higher than in moderate or well-differentiated CRC (pooled OR = 2.131, 95% CI, 1.464–3.102; P<0.001; I2 = 0.000%) (Table 3).
For total CRC, MLH1 promoter methylation was detected in 62.6% (95% CI: 54.0–70.4%) of the 968 MSI-H CRC in 12 studies, 12.2% (95% CI: 3.0–38.2%) of the 344 MSI-L CRC in four studies, 55.8% (95% CI: 45.2–65.8%) of the 1325 MSI CRC in 16 studies, and 5.2% (95% CI: 2.2–11.6%) of the 1791 MSS CRC in 10 studies (P<0.001), respectively (Table 2). Significant differences were found between MSI vs. MSS, MSI-H vs. MSS, and MSI-H vs. MSI-L (P<0.001, P<0.001, and P<0.001, respectively). Whereas, no difference was observed between MSI-L and MSS (P = 0.380). For sporadic CRC, the pooled prevalence of MLH1 promoter methylation was 73.6% (95% CI: 67.3–79.0%) in MSI-H CRC, 67.3% (95% CI: 47.1–82.7%) in MSI CRC, and 17.5% (95% CI: 10.0–29.0%) in MSS CRC (P<0.001; Table 2). In addition, for the 10 studies , , , , , , , , ,  that provided both MSI and MSS status in total CRC, the pooled OR for the association between MLH1 promoter methylation and MSI status (MSI vs. MSS) was 27.096 (95% CI: 13.717–53.526; P<0.001; I2 = 59.001%; Table 3, Figure S1).
MLH1 Protein Expression
In tumors with a loss of MLH1 protein expression, MLH1 promoter methylation was detected in 66.5% (95% CI: 44.4–83.2%) of the 106 total CRC, 80.8% (95% CI: 75.3–85.3%) of the 247 MSI-H CRC, 69.8% (95% CI: 45.5–86.5%) of the 156 sporadic CRC, and 37.8% (95% CI: 25.3–52.1%) of the 169 LS tumors (P<0.001). Significant differences were observed when comparing LS tumors vs. total CRC, LS tumors vs. sporadic CRC, and LS tumors vs. MSI-H CRC tumors (P = 0.032, P<0.001, and P = 0.026, respectively). For tumors with MLH1 protein expression, MLH1 promoter methylation was detected in 11.9% (95% CI: 1.5–53.8%) of the 67 total CRC and in 9.8% (95% CI: 3.4–25.2%) of the 308 sporadic CRC (P = 0.862; Table 2). Six studies , , , , ,  provided expression status as a loss of MLH1 protein expression in 308 cases and MLH1 protein expression in 75 cases of sporadic CRC. The pooled analysis showed significantly association between MLH1 promoter methylation and MLH1 protein expression (OR = 14.919, 95% CI: 6.427–34.631%; P<0.001; I2 = 35.469%) (Table 3).
The pooled prevalence of MLH1 promoter methylation in 138 BRAF-mutated and 764 BRAF wild type CRC was 53.2% (95% CI: 27.7–77.2%) and 13.7% (95% CI: 5.1–32.0%) in three studies (pooled OR = 9.419; 95% CI: 2.613–33.953; P = 0.001; I2 = 67.030%) (Table 2 and Table 3). For the three studies that provided both MSI-H and BRAF mutation status in CRC, the pooled OR for the association between BRAF mutation status and MLH1 promoter methylation was 37.615 in MSI-H CRC (95% CI: 10.011–141.311; P<0.001; I2 = 0.000%) (Table 3).
The pooled frequency of MLH1 promoter methylation was 14.0% (95% CI: 10.2–19.0%) in 353 KRAS-mutated and 21.8% (95% CI: 13.2–33.8%) in 570 wild-type CRC, in three studies , ,  (pooled OR = 0.476; 95% CI: 0.322–0.703; P<0.001; I2 = 49.293%) (Table 2 and Table 3). Moreover, a statistically significant association was observed between MLH1 promoter methylation and KRAS mutation in MSI CRC (OR = 0.340; 95% CI: 0.167–0.693; P = 0.003; I2 = 0.000%) (Table 3).
For the frequency of MLH1 promoter methylation in MSI CRC and MSI-H CRC, the funnel plot seemed asymmetry (Figure S2 A and B). Funnel plot for the association between MLH1 promoter methylation and tumor location (proximal vs. distal) also seemed asymmetry (Figure S3). Begg’s rank correlation and Egger’s regression methods further supported the significant publication bias. With the trim and fill method, the adjusted frequency of MLH1 promoter methylation decreased from 55.8% to 36.7% in MSI CRC and from 62.6% to 53.5% in MSI-H CRC. The pooled OR for the association between MLH1 promoter methylation and tumor location decreased from 3.804 (95% CI: 2.715–5.329) to 3.172 (95% CI: 2.323–4.331).
Our meta-analysis suggested that the frequency of MLH1 promoter methylation in total CRC was 20.3%. They were 18.7% in sporadic CRC and 16.4% in LS CRC, respectively; significant associations were observed between MLH1 promoter methylation and gender, tumor location, tumor differentiation, MSI, MLH1 protein expression, and BRAF mutation.
The pooled MLH1 promoter methylation frequencies were 16.4% and 20.5% in 4 population-based studies and 16 hospital-based studies (One study  included 1061 population-based and 172 hospital-based CRC). In total CRC, the frequency of the MLH1 promoter methylation between hospital-based and population-based studies was not significantly different (P = 0.279) (Table 1).
A, B, C and D regions in the MLH1 promoter were commonly tested for methylation. However, only one study tested the MLH1 promoter methylation in “A” region , three studies tested the MLH1 promoter methylation in “C” region , , , other 15 studies did not provide the specific A, B, C or D regions in total CRC. The MLH1 promoter methylation frequency in “A” region (66.9%) was significantly higher than in “C” region in CRC (26.4%; P = 0.001) (Table S1). It may be due to the variation of methylation status in different regions of the MLH1 promoter.
The MLH1 promoter methylation was detected in total of 12 studies with 968 MSI-H CRC with a frequency of 62.6%. After the adjustment by trim and fill method, the pooled frequency decreased to 53.5%. The pooled MLH1 promoter methylation in 249 sporadic MSI-H CRC was 73.6%, significantly higher than in 95 LS MSI-H CRC (15.3%). The following may explain our results: in sporadic CRC, MSI-H was mainly caused by MLH1 promoter methylation , ; whereas, in LS CRC, MSI-H was mainly caused by MMR inactivation because of germline mutation .
In sporadic CRC, our meta-analysis indicated that the MLH1 promoter methylation frequency in 308 CRC with MLH1 protein expression (9.8%), which was lower than in 156 CRC without MLH1 protein expression (69.8%, P<0.001). In CRC with loss of MLH1 protein expression, the MLH1 promoter methylation was significantly higher in sporadic CRC (69.8%) than in LS CRC (37.8%, P = 0.026). In sporadic MSI-H CRC with loss of MLH1 protein, the MLH1 promoter methylation frequency was 86.3%. MLH1 promoter methylation could explain more fraction of MLH1 gene silencing in sporadic CRC than that in LS CRC. In this systematic review and meta-analysis, we can see that the highest frequency of MLH1 promoter methylation was in MSI-H CRC with loss of MLH1 protein, the following in sporadic CRC without MLH1 protein expression, and the lowest in the sporadic CRC with MLH1 protein expression.
The frequency of MLH1 promoter methylation in BRAF mutated total CRC was 53.2%, significantly higher than in BRAF wild type total CRC 13.7% (P = 0.001). In contrast, the MLH1 promoter methylation frequency in KRAS mutated total CRC (14.0%) was significantly lower than in KRAS wild type total CRC (21.8%) (P<0.001). The largest population-based study  observed similar results, the frequencies of MLH1 promoter methylation were 46.8% (51/109) in BRAF mutated CRC and 17.4% (97/559) BRAF wild type CRC (P<0.001); whereas, they were 15.5% (40/258) in KRAS mutated CRC and 26.2% (112/428) KRAS wild type CRC (P = 0.001). In MSI CRC, similar results were also observed. The MLH1 promoter methylation frequency in BRAF mutated MSI-H CRC (94.5%) was significantly higher than in BRAF wild MSI-H CRC (28.2%) (P<0.001). Whereas, the MLH1 promoter methylation frequency in KRAS mutated MSI CRC (25.9%) was significantly lower than in KRAS wild MSI CRC (50.4%) (P = 0.003). The following may explain the results: colon tumors progress by distinct genes of the RAS/RAF/MAP kinase pathway, depending on the genetic/epigenetic event underlying MMR deficiency (mutation and loss induced by MLH1 promoter methylation). MSI-H tumors with MMR gene mutations (hereditary and sporadic forms) my preferentially target KRAS, whereas, MSI-H tumors with MLH1 promoter methylation may preferentially target the BRAF gene . In addition, the methylation of MGMT was associated with KRAS mutant CRC but not of BRAF mutant CRC could also support the results of our meta-analysis .
Our study suggested that the frequency of MLH1 promoter methylation was higher in female, proximal tumor location, and poor differentiation. Study reported that MSI CRC had a very distinct clinicopathological phenotype, which was commonly mucinous, poorly differentiated, presenting at earlier Dukes’ stage, and in the proximal side of the colon . MSI CRC was also commonly female and older at diagnosis. Moreover, in sporadic CRC, MSI was mainly caused by MLH1 promoter methylation. Therefore, MSI CRC and MLH1 promoter methylation CRC may have similar clinicopathological phenotype. However, the underlying mechanisms need to be investigated.
Heterogeneity persisted in our meta-analysis. The followings may explain the sources of heterogeneity. Firstly, MLH1 promoter methylation was tested in different promoter regions. One study tested in “A” region; three studies tested in “C” region; other 15 studies did not supply specific regions tested. Additionally, various ages of the study subjects may also explain the heterogeneity. Genes of individual are progressively methylated with aging due to chromosomal instability . However, only three of 19 studies provided the information of age of study subjects , , .
Although this meta-analysis provides some robust results, limitations also existed like all the meta-analysis. Firstly, dietary factors, smoking and drinking alcohol may affect the MLH1 promoter methylation. Lack of these original data of the studies reviewed limited our further evaluation of their effect on MLH1 promoter methylation , , . Secondly, lacking of the original data limited our further evaluation of the interactions between the clinicopathological and molecular variables in CRC. Thirdly, the prevalence of MLH1 promoter methylation in CRC may increase with aging. However, the majority of studies did not provide this information, which limited our further evaluate their effect on MLH1 promoter methylation.
In summary, this systematic review and meta-analysis yield some conclusions: the MLH1 methylation in total CRC was 20.3%; they were 18.7% in sporadic CRC and 16.4% in LS CRC, respectively; MLH1 promoter methylation may be significantly associated with gender, tumor location, tumor differentiation, MSI, MLH1 protein expression, and BRAF mutation.
Forest figure for association of MLHI promoter methylation and MSI status in CRC tumors (MSI vs. MSS).
Funnel plot of the log (event rate) versus its standard error, for the frequency of MLH1 promoter methylation in CRC tumors: (A) MSI and (B) MSI-H.
Funnel plot of the log odds ratio versus its standard error, for the frequency of MLH1 promoter methylation in CRC tumors: Proximal vs. Distal.
Pooled frequency of MLH1 promoter methylation in colorectal cancer patients with other subgroup analysis.
Revised the paper: YZ. Conceived and designed the experiments: YZ. Performed the experiments: XL XY YW. Analyzed the data: FW LJ YL. Contributed reagents/materials/analysis tools: GS DW. Wrote the paper: XL FH YZ.
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