Annals of Oncology Advance Access published April 8, 2014 1
Prognostic Value of CpG Island Methylator Phenotype among Colorectal Cancer Patients: A Systematic Review and Meta-Analysis
Y. Y. Juo1, F. Johnston2, D. Zhang3, H. H. Juo4, H. Wang1, E. P. Pappou1, T. Yu3, H. Easwaran5, S. Baylin5,6, M. Engeland7, N. Ahuja1,5,8 Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, USA
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Department of Surgery, Medical College of Wisconsin, Milwaukee, USA
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Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, USA
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Department of Internal Medicine, Danbury Hospital, Danbury, USA
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Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, USA
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Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, USA
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Department of Pathology, Maastricht University, Maastrich, The Netherlands
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Department of Urology, Johns Hopkins University School of Medicine, Baltimore, USA
Address all correspondence to: Nita Ahuja, MD FACS, Vice Chair of Academic Affairs, SurgeryAssociate Professor of Surgery, Oncology and Urology, Chief, Section of Mixed Tumors-Surgical Oncology, Department of Surgery, Blalock 685, 600 N. Wolfe Street, Baltimore, MD 21287, USA,
Phone: (410) 502-6135, Email: [email protected] Key Message: "This is the first comprehensive qualitative and quantitative synthesis of the currently available literature regarding CIMP’s prognostic value among CRC patients. Our results showed significantly worse DFS and OS among CRC patients with CIMP compared to those without CIMP, and a potential survival benefit in CRC patients with CIMP who received adjuvant chemotherapy in comparison with those who received surgery alone. However, future research into the most appropriate operational definition of CIMP and the mechanism by which CIMP influenced disease prognosis would be of vital importance in helping us put past study results into proper perspective."
© The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
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ABSTRACT Background: Divergent findings regarding the prognostic value of CpG Island Methylator Phenotype (CIMP) in Colorectal Cancer (CRC) patients exist in current literature. We aim to review data from published studies in order to examine the association between CIMP and CRC prognosis. Materials and Methods: A comprehensive search for studies reporting Disease-Free Survival (DFS), Overall Survival (OS), or cancer-specific mortality of CRC patients stratified by CIMP is
hazard ratios (HR) as summary statistics. Results: 33 studies reporting survival in 10,635 patients are included for review. 19 studies provide data suitable for meta-analysis. The definition of CIMP regarding gene panel, marker threshold, and laboratory method varies across studies. Pooled analysis shows that CIMP is significantly associated with shorter DFS (pooled HR estimate 1.45; 95% CI 1.07~1.97, Q=3.95, I2=0%) and OS (pooled HR estimate 1.43; 95% CI 1.18~1.73, Q=4.03, I2=0%) among CRC patients irrespective of Microsatellite Instability (MSI) status. Subgroup analysis of Microsatellite Stable (MSS) CRC patients also shows significant association between shorter OS (pooled HR estimate 1.37; 95% CI 1.12~1.68, Q=4.45, I2=33%) and CIMP. Seven studies have explored CIMP’s value as a predictive factor on stage II and III CRC patient’s DFS after receiving adjuvant 5-FU therapy: of these, four studies showed that adjuvant chemotherapy conferred a DFS benefit among CIMP(+) patients, one concluded to the contrary, and two found no significant correlation. Insufficient data was present for statistical synthesis of CIMP’s predictive value among CRC patients receiving adjuvant 5-FU therapy.
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performed. Study findings are summarized descriptively and quantitatively, using adjusted
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Conclusion: CIMP is independently associated with significantly worse prognosis in CRC patients. However, CIMP’s value as a predictive factor in assessing whether adjuvant 5-FU therapy will confer additional survival benefit to CRC patients remained to be determined through future prospective randomized studies.
Keywords: CIMP, Prognosis, Colorectal Cancer, adjuvant chemotherapy, epigenetics, tumor Downloaded from http://annonc.oxfordjournals.org/ by guest on September 30, 2014
marker
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BACKGROUND CpG islands are genomic regions that contain a high frequency of cytosine and guanine nucleotides, connected with a phosphodiester bond. They are typically found in or near promoter regions of the genome, where transcription is initiated, and are present in as high as 40-50% of the human genes. Aberrant methylation of cytosine nucleotides within these CpG islands can lead to aberrant silencing of normal tumor-suppressor function and cancer formation.1, 2 A distinct molecular subtype of cancer, characterized by high degrees of methylation, has
unique molecular features, epidemiology, precursor lesions, and they represent approximately 15% of sporadic cases of colorectal cancer. However, the role CIMP plays in the pathogenesis of CRC is still poorly understood. It has been shown that hypermethylation secondary to CIMP leads to Microsatellite Instability (MSI) through the methylation of the MLH1 promoter and the consequent silencing of the MLH1 mismatch repair gene. Almost 70-80% of MSI CRCs can be attributed to CIMP and associated MLH1 methylation.4 CIMP, however, can exist with and without MSI. Studies have shown CIMP to be an independent negative prognostic factor in disparate subgroups of CRC patients5-7, although its influence is modified by other genetic factors including MSI8, 9 and KRAS/BRAF status10, 11. It has been hypothesized that CIMP-high colorectal cancer patients might have a better response to 5-fluorouracil (5-FU) chemotherapy due to observations that suppression of gamma-glutamyl hydrolase (GGH)12, one of the putative effects of CpG island methylation, is associated with an increase in intracellular folate level13, which magnifies the effect of 5-FU chemotherapy.14 Several clinical studies have come to
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been termed CpG Island Methylator Phenotype, or CIMP.3 CIMP cancers are characterized by
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conflicting conclusions regarding the predictive value of CIMP15, 16. Precise estimates of the predictive value of CIMP can allow for refinement in clinical management, especially among stage II CRC patients, where the value of 5-FU chemotherapy is still controversial17. The goal of this systematic review is to summarize published studies using standardized techniques to gain a better understanding of the prognostic significance of CIMP in CRC patients with respect to their Disease-Free Survival (DFS) and Overall Survival (OS).
SEARCH STRATEGY AND STUDY ELIGIBILITY Published studies were eligible if CRC patient survival was analyzed after stratification by CIMP status of primary tumor tissue. CIMP status was defined by the respective study authors with no restriction to laboratory method, gene panel, or marker threshold value. Primary outcome of the study was Disease Free Survival (DFS), defined as the length of time since initial cancer treatment during which no evidence of cancer recurrence or death was found. Secondary outcomes was Overall Survival (OS), defined as the length of time since initial cancer treatment that patients were still alive. Cohort studies of either a prospective or a retrospective nature were included. Case reports and reviews were excluded. There was no restriction with respect to patient age, gender, ethnicity, comorbidity, tumor type, disease stage, length of follow-up, or treatment received. (Study eligibility summarized in Table 1)
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METHODS
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STUDY IDENTIFICATION We followed MOOSE (Meta-analysis of Observational studies in Epidemiology) guidelines18 to identify eligible studies. Combination of text and exploded controlled vocabulary terms were used to search PUBMED, EMBASE, SCOPUS, and Web of Science electronic databases until June 6th 2013, relating to the following two concepts: (1) CIMP and (2) colorectal cancer. Citations in relevant reviews and studies were hand-searched to capture additional
conference abstracts and letters were included to capture grey literature. Each study was independently assessed for inclusion by at least two reviewers. Discrepancies within the reviewing pair were resolved via discussion. The flow chart of study identification was presented according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines in Figure 1.19 STATISTICAL METHODS Study characteristics including study country, patient characteristics, and CIMP assessment methods were summarized in a consistent manner for easy examination. Patient number and percentages among different gender and age groups were calculated using available data when they were not directly reported. Assignment of CIMP status into constitutive groups (CIMP-positive or CIMP-negative) or CIMP-high, CIMP-low, and CIMP-zero was performed according to the grouping in each study. The term CIMP was used synonymously with CIMP-positive and CIMP-high, depending on whether the study authors have di- or trichotomized CIMP status. During our discussion, CIMP-low and CIMP-zero were treated equivalently with CIMP-negative, but we performed
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potentially relevant studies. In addition to full publications, original studies in the form of
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separate statistical analyses for studies that dichotomized and studies that trichotomized CIMP status.Clinical and methodological sources of heterogeneity across studies were evaluated to assess the appropriateness of statistical synthesis. Factors taken into consideration included variation in patient disease stage, treatment received, and MSI status. Statistical heterogeneity was assessed by Breslow-Day test while Q and I2 statistics were presented as measures of inconsistency of CIMP prognostic values across studies attributable to heterogeneity.20 The Q statistic was a weighted sum of squares following a chi-squared distribution with k-1 (k=number
deviation from total homogeneity. In our study, we employed a p-value of 0.1 and an I2 statistic of less than 50% as indicating acceptable statistical heterogeneity. In order to summarize the association between CIMP status and survival across studies, a weighted average of the individual adjusted log Hazard Ratios (HR) was used, with the weights inversely proportional to the variance of the log hazard ratio of each study. Adjusted hazard ratios were sought among results of multivariate survival analysis using Cox proportional hazards regression model and the adjusted variables documented. When missing data regarding adjusted hazard ratio or variance was encountered, estimates were indirectly imputed from available numerical data where possible using confidence intervals, p-values, and event numbers by methods described by Parmar et al21. Recurrence-Free Survival (RFS) was interpreted as synonymous with DFS. Random-effects model was used in all numerical synthesis. Forest plots were presented with studies plotted in order of decreasing variance of the log HR. Horizontal lines represented 95% CIs. Each box represented the HR point estimate and its area was proportional to the weight of the study. The diamond on the bottom denoted the overall summary estimate, with CIs given by the width of the diamond. The vertical line signified the reference
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of included studies) degrees of freedom. The p-value calculated thereof gave an estimate of the
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line with HR=1.0. Sensitivity analyses were performed to assess the impact of including conference abstracts, imputed estimates, and studies with more than one risk of bias marked as high- or unclear risk. All numerical synthesis was performed using Review Manager 5.2.
RESULTS ELIGIBLE STUDIES
“colorectal cancer” as keywords for searching (Table 1). (Flow chart of study identification is summarized in Figure 1) The main reasons for exclusion of studies during the initial title/abstract screening were failure to examine disease prognosis, incorrect histology, or the studies were not original studies. 93 studies remained for full-text inspection after title/abstract screening (Figure1). Of these, 39 full-text studies were excluded because the primary outcomes of interest (OS and DFS) were not reported, 13 studies were excluded because no prognostic measure with regard to CIMP was reported, 6 studies were excluded due to overlapping patient population with included studies, and 2 studies were excluded because the study tissue was not primary tumor tissue. A total of 33 studies were included in the systematic review. (Study characteristics of these studies were summarized in Table 2.) Of these, only 19 studies were eligible for pooling hazard estimates in the meta-analysis. The reasons for exclusion were failure to present numerical hazard ratio estimates, failure to include CIMP in the multivariate analysis, and failure to present survival data with explicit numerical values.
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We initially identified 2,414 studies for potential inclusion using only “CIMP” and
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STUDY CHARACTERISTICS The 33 studies analyzed 10,635 patients for CIMP status and its relationship to disease prognosis. The mean number of patients for each study was 322 (range 33-990). Most studies included both colon and rectal cancer cases. 5 studies6, 7, 11, 22, 23 included colon cancers only and 3 studies24-26 rectal cancers only. In most studies, a mixture of different disease stages was included, however, eight studies6, 15, 22, 27-31 only examined stage II or III cancers and one study32
chemotherapy were included, while four other studies23, 29, 32, 35 included only Microsatellite Stable (MSS) tumor tissues for analysis and 1 study36 included only Microsatellite Instable (MSI) tumor tissues for analysis. The risk of bias of each included study is summarized in Table 3. While retrospective convenience sampling was employed for almost all studies, some studies stated their inclusion and exclusion criteria more explicitly than others and these studies were rated as low risk in selection bias. Almost all studies (31 out of 33 studies), including conference abstracts, have explicitly stated their method of assessment of CIMP and genetic mutations such as KRAS or BRAF. Median follow-up length, range, and loss-to-follow-up rate were satisfactorily reported in about half of the studies. Known or commonly discussed confounders in the relationship between CIMP and survival such as age, disease status, MSI status, KRAS/BRAF mutation were adjusted for in two-thirds of the studies, and in these studies the risk of confounding was rated as low risk. HETEROGENEITY IN CIMP DEFINITION Only studies that used primary tumor tissues were included in this review. 21 of the 33 included studies classified CIMP status in a dichotomized fashion (CIMP-positive vs. negative)
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only examined stage IV cancers. In three studies15, 27, 33, 34, only patients who received
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while CIMP was classified in a trichotomized fashion (CIMP-high/low/zero) in the remainder of the studies. Significant variation existed between the studies regarding the gene panel, marker threshold, and laboratory method used for defining CIMP. The Classic panel (MINT1, MINT2, MINT31, CKKN2A(p16), hMLH1) and the 5-gene panel devised by Weisenberger37 in 2006 (CACNA1G, IGF2, NEUROG1, RUNX3, SOCS1) were the two most commonly employed gene
number of genes assessed in each study ranged from 3 to 13 genes, with a median of 5 genes. Marker threshold for defining CIMP status was often chosen arbitrarily or based on previous literature; 2 studies7, 38 chose their threshold based on association with known clinicopathological features of CIMP-positive tumors. One study11 specifically compared the effect of using different CIMP panels and concluded that this variation could result in significantly different associations with survival outcomes. Sixteen7, 9, 10, 15, 22-26, 28, 29, 35, 39-41 studies used Methylation Specific PCR (MSP) as the laboratory method for methylation analysis, nine8, 11, 27, 32, 34, 38, 42 used MethyLight, five6, 16, 36, 43-45 used bisulfite pyrosequencing, one5 used COBRA, and in three studies, laboratory method was not specified. In general, no specific gene panel or laboratory method was observed to produce a consistently higher or lower CIMP prevalence. RELATIONSHIP BETWEEN CIMP AND PROGNOSIS DISEASE-FREE SURVIVAL The median prevalence of CIMP-positive or CIMP-high status amongst included studies was 18.2%, ranging from 4.6% to 46.5%. Overall median follow-up length was 45.3 months, ranging from 2.8 to 100.8 months.
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panels, although 17 of the 33 included studies opted to use a different gene panel(Table 2). The
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Eleven studies examined the relationship between CIMP status and Disease-Free Survival (DFS) amongst CRC patients. The majority6, 16, 27, 33, 34, 39, 42, 43 of studies (8 out of 11) found no significant relationship between CIMP and DFS, while 2 studies25, 44 found CIMP to be associated with unfavorable DFS. In addition, one6 found that CIMP, despite being an insignificant predictor for DFS in their overall study cohort, was associated with an unfavorable DFS among tumors of proximal colon. In contrast, Koo et al.31 found a combination of CIMP(+)/KRAS-wild type to be associated with favorable DFS compared with other
Stable (MSS), Zanutto et al.29 found that CIMP positivity was associated with a lower recurrence rate. In our pooled analysis, CIMP was associated with an unfavorable DFS after adjusting for other relevant confounders in each contributing study. Insufficient data was present among studies that categorized CIMP status in a trichotomized fashion for pooled analysis. Six studies that categorized CIMP status in a dichotomized fashion provided satisfactory adjusted HR estimates suitable for numerical synthesis. This accounted for a total of 1,454 patients. Their overall summary hazard ratio was 1.45 (95% CI 1.07-1.97), with a low heterogeneity (Q=3.95, I2=0%), indicating a worse DFS for CIMP-positive patients. From the forest plot (Figure 2), the confidence intervals of adjusted hazard ratios in five out of six studies was insignificant, but the majority of them had the same trend. Sensitivity analysis was performed to assess the effect of including imputed HR estimates by leaving out 2 studies with imputed HRs 25, 27. The exclusion did not change our conclusion (pooled HR=1.22, 95% CI, 0.83 to 1.79; Q=1.24, I2=0%). In a separate analysis, a subgroup of studies16, 25, 39 with
1 high risk of bias item were excluded.
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genetic/epigenetic profiles. Examining a subgroup of CRC patients who were Microsatellite
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This also did not change our conclusion although the summary estimate did not attain statistical significance (pooled HR=1.46, 95% CI, 0.79 to 2.67; Q=1.10, I2=0%). A subgroup of studies (7 of 33) also explored CIMP’s value as a predictive factor on the effect of adjuvant 5-FU therapy on CRC patients. Seven studies examined the relationship between CIMP status and DFS among patients who received 5-FU-based adjuvant chemotherapy after surgical resection of the primary tumor. (Table 4 shows the individual study details).
independent of MSI and p53 status. Min et al.34 found that stage II and III CRC patients with CIMP-high represented a cohort most likely to receive benefits from 5-FU therapy. Rijnsoever et al.15, in a study restricted to stage III CRC cases, also showed that CIMP(+) status was associated with favorable response to 5-FU therapy. In addition, Rijnsoever et al. found that CIMP, despite being associated with worse prognosis among patients treated by surgery alone, was associated with a trend for better DFS among patients receiving both surgery and 5-FU therapy.15 This finding was confirmed by Donada et al.22 in their study of stage II colon cancer cases. On the other hand, Jover et al.16 found that among stage II and III CRC cases, CIMP(+) cases represented a cohort that was unresponsive to 5-FU therapy while CIMP(-) cases received significant survival benefit from 5-FU therapy. Han et al.27 found both the predictive value of CIMP and the interaction between CIMP and adjuvant chemotherapy to be insignificant in their study of stage II and III CRC cases receiving FOLFOX treatment. Kim et al.33 found CIMP to be an insignificant predictor of DFS in their study of metastatic CRC cases treated with FOLFIRI. Insufficient (CIMP
adjuvant chemotherapy) interaction term estimates were available for
pooling so no statistical summary was attempted. Overall, four out of seven studies agreed that adjuvant chemotherapy conferred a DFS benefit among CIMP(+) stage II and III CRC patients;
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Elsaleh et al.30 showed that CIMP(+) cases received survival benefit from 5-FU therapy
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of the remaining three studies, one concluded to the contrary and two found CIMP to be an insignificant predictive factor. OVERALL SURVIVAL Nineteen studies provided data regarding the effect of CIMP status on Overall Survival (OS) among CRC patients of different disease stages and genetic profiles. Of these, 13 studies9-11, 23, 24, 26, 28, 35, 38-40, 42, 46
found that CIMP was associated with an unfavorable OS while no study found CIMP to be
associated with a favorable OS. Our pooled analysis showed that CIMP(+) status was associated with an unfavorable OS. A subset of 11 studies provided adjusted hazard ratio estimates with no restriction to MSI status suitable for pooling, accounting for a total of 3,559 patients. Of these, 7 studies have classified CIMP in a dichotomized fashion. Their overall summary hazard ratio estimate was 1.43 (95% CI: 1.18-1.73). No significant statistical heterogeneity was present (Q=4.03, I2=0%). Of the other four studies that have classified CIMP in a trichotomized fashion, both CIMP-high and CIMPlow were associated with a significantly worse OS in comparison with CIMP-zero (Summary HR 1.53 with 95% CI 1.11~2.12 and 1.33 with 95% CI 1.11~1.61, respectively). From the forest plot (Figure 3) we could infer that despite a lack of significance in many studies, they shared trends in the same direction which led to a significant overall summary HR. To assess the potential effect of including conference abstracts, a sensitivity analysis was performed by excluding studies that was not a full publication.47. The exclusion made no difference to our conclusion (pooled HR=1.59, 95% CI, 1.16 to 2.19; Q=2.22, I2=0%). Sensitivity analysis was also performed to assess the effect of including indirectly estimated HRs by leaving out three
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concluded that CIMP had no significant effect on OS. Six studies5, 32, 36, 44,
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studies7, 25, 45 with imputed log HR estimates. The exclusion made no difference to our conclusion (pooled HR=1.69, 95% CI, 1.24 to 2.31; Q=3.99, I2=0%). In order to assess the impact of including studies of variable risk of bias, a subgroup analysis excluding studies7, 9, 11, 25, 42
with
1 high risk of bias item showed essentially the same conclusions (pooled HR=1.83,
95% CI, 1.23 to 2.73; Q=3.49, I2=14%). Nine studies examined the relationship between CIMP and OS among Microsatellite
shorter OS in 6 studies7, 9, 10, 32, 36, 38, while 3studies11, 23, 35 found no significant association between CIMP and OS. Restricting the meta-analysis to the four studies with suitable summary HR estimates, accounting for a total of 1,112 MSS cases, produced a summary HR estimate of 1.37 (95% CI 1.12~1.68) with acceptable statistical heterogeneity (Q=4.45, I2=33%), suggesting that CIMP was associated with a significantly shorter OS amongst MSS tumors also (Figure 4). Four studies examined the relationship between CIMP and OS among Microsatellite Instable (MSI) tumors. Rhee et al.36 found CIMP(+) tumors to have shorter OS while Zlobec et al.45, Ward et al.9, and Barault et al.7 all found no significant association between CIMP and OS. Insufficient numerical estimates were available for pooling.
DISCUSSION Among patients included in our review, CIMP-positive accounted for almost one fifth (18.2%) of all sporadic colorectal cancers. Understanding the implication of its presence on patient prognosis would be crucial in making management decisions for CRC patients.
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Stable (MSS) tumors. In this cohort, CIMP was reported to be associated with a significantly
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One of the major confounding factors in a systematic review on topics relating to CIMP was the lack of a standardized operational definition of CIMP. No consensus existed regarding the most appropriate gene panel, marker threshold, or laboratory method for the assessment of CIMP.48 Several studies performed sensitivity analysis by employing different gene panels or marker thresholds and concluded that this could result in different conclusions regarding the prognostic value of CIMP.11, 32, 38, 44 Despite the methodological heterogeneity, our review showed no gene panel, laboratory method, or preservation method to be consistently associated
with the numerical synthesis. The results showed that CRC patients with CIMP were likely to experience both shorter DFS and OS after adjusting for their age, gender, disease stage and the treatment modalities they received. A number of other genetic profiles have also been cited as being significant effect modifiers in the relationship between CIMP and CRC patient prognosis, such as MSI status, mutation status of BRAF, KRAS, or anti-p53 autoantibody. While most studies adjusted for established confounders such as age, gender, and tumor stage, the genetic markers such as KRAS/BRAF and MSI were not consistently assessed and adjusted in all studies. This was most likely due to conflicting results in previous literature regarding the prognostic value of these markers49-51 and non-significant finding during univariate analysis in the respective studies. In our review, we were able to perform subgroup analysis for MSS CRC patients and we found that the survival disadvantage associated with CIMP persisted in this subgroup. There was not enough data among the included studies to allow data pooling for exploration of whether the same disadvantage held true among the MSI patients or other genetic subgroups. Of the included studies, Barault et al.7, Dahlin et al.8 and Ward et al.9 all found MSI status to be a significant
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with a better or worse prognosis in patients with CIMP, thus a decision was made to carry on
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effect modifier in the relationship between CIMP and survival; Kakar et al.35, 40 and Samowitz et al.23 found BRAF mutation to be a predictive factor for poor prognosis among MSS cases independent of CIMP status; in contrast, Lee et al.10 found KRAS/BRAF mutation to be an effect modifier in the relationship between CIMP and poor prognosis. Zlobec et al.45 argued for a combination of BRAF/CIMP as the most appropriate marker combination for classifying distinct prognostic groups. The dilemma of wishing neither to leave out potential confounders from the regression model nor overfit the model with mutually correlated markers could not be solved
BRAF, and KRAS play in it, had been elucidated. Besides the prognostic implication of CIMP among CRC patients, an even more important question was, should CIMP status influence the decision in giving adjuvant chemotherapy to CRC patients? According to National Comprehensive Cancer Network (NCCN) treatment guideline for colon cancer Version 3.201352, neither the use of adjuvant chemotherapy outside of clinical trials for stage II (T2N0M0) colon cancer patients nor the use of multi-gene assay in the decision-making process was recommended. However, if we were able to identify a subgroup of stage II tumors that were more susceptible to 5-FU, we could delay disease recurrence with adjuvant chemotherapy in this subgroup. Three out of four studies in our review showed that patients with CIMP experienced significantly longer DFS with the addition of adjuvant chemotherapy compared to receiving surgery alone. On the other hand, four out of five studies in our review showed that patients without CIMP had equivalent outcomes whether they received adjuvant chemotherapy or not (Table 4). According to Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK)53, the recommended summary statistic for evaluating the interaction of treatment and a binary (or categorical) marker like CIMP was the
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unless the pathway between CIMP and survival or treatment response, along with the roles MSI,
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interaction term. Of the seven studies in our review, interaction term was available in only one study16, thus we were unable to perform analysis beyond descriptive summary across studies for evaluation of the impact of CIMP on treatment response. CIMP’s value as a predictive factor in assessing whether adjuvant 5-FU therapy will confer additional survival benefit to CRC patients remained to be determined through future prospective randomized studies. The main limitations of our review were the clinical and methodological heterogeneity
patients with inflammatory bowel diseases, the distribution of cancer stage, treatments received, and genetic profiles varied between studies. In terms of the methodological heterogeneity, besides an inconsistency in CIMP definition, the length and completeness of follow-up, and the confounders assessed and adjusted for were also different from study to study. In this review, we have elected to use DFS and RFS synonymously, in order to include the maximal number of relevant clinical studies, acknowledging that we risk the bias that could be introduced through the small percentage of patients with second primary cancers. In summary, this is the first comprehensive qualitative and quantitative synthesis of the currently available literature regarding CIMP’s prognostic value among CRC patients. Our results showed significantly worse DFS and OS among CRC patients with CIMP compared to those without CIMP, and a potential survival benefit in CRC patients with CIMP who received adjuvant chemotherapy in comparison with those who received surgery alone. However, future research into the most appropriate operational definition of CIMP and the mechanism by which CIMP influenced disease prognosis would be of vital importance in helping us put past study results into proper perspective.
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across the included studies. While most studies excluded hereditary colorectal cancers and
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DISCLOSURE The authors have declared no conflicts of interest. FUNDING This paper was supported by grants from National Cancer Institute K23 CA127141 (NA), the Downloaded from http://annonc.oxfordjournals.org/ by guest on September 30, 2014
American College of Surgeons/ Society of University Surgeons (NA).
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18. Berlin JA MS, Stroup DF, Olin I, Williamson GD, Rennie D, Moher D, Becker BJ, Sipe TA, Thacker SB. Meta‐analysis of observational studies in epidemiology: a proposal for reporting. Meta‐analysis Of OIbservational Studies in Epidemiology (MOOSE) group. JAMA 2000;283. 19. Moher D, Liberati A, Tetzlaff J, Altman DG. Preferred reporting items for systematic reviews and meta‐analyses: the PRISMA statement. Int J Surg;8:336‐41. 20. Higgins JP, Thompson SG. Quantifying heterogeneity in a meta‐analysis. Stat Med 2002;21:1539‐ 58. 21. Mahesh K. B. Parmar VT, Lesley Stewart. Extracting Summary Statistics to Perform Meta‐Analysis of the Published Literature for Survival endpoints. Statistics in Medicine 1998;17:2815‐34. 22. Donada M, Bonin S, Barbazza R, Pettirosso D, Stanta G. Management of stage II colon cancer ‐ the use of molecular biomarkers for adjuvant therapy decision. BMC Gastroenterol;13:36. 23. Samowitz WS, Sweeney C, Herrick J, et al. Poor survival associated with the BRAF V600E mutation in microsatellite‐stable colon cancers. Cancer Res 2005;65:6063‐9. 24. I.A. Al‐Sohaily CJH, N. Shin, J. Wu, J. Leong. Prognostic Value of Methylation Markers in Rectal Cancer. Australian Gastroenterology Week2011 2011. 25. Jo P, Jung K, Grade M, et al. CpG island methylator phenotype infers a poor disease‐free survival in locally advanced rectal cancer. Surgery;151:564‐70. 26. Samowitz WS, Curtin K, Wolff RK, Tripp SR, Caan BJ, Slattery ML. Microsatellite instability and survival in rectal cancer. Cancer Causes Control 2009;20:1763‐8. 27. Han SW, Lee HJ, Bae JM, et al. Methylation and microsatellite status and recurrence following adjuvant FOLFOX in colorectal cancer. Int J Cancer;132:2209‐16. 28. van Rijnsoever M, Grieu F, Elsaleh H, Joseph D, Iacopetta B. Characterisation of colorectal cancers showing hypermethylation at multiple CpG islands. Gut 2002;51:797‐802. 29. Zanutto S, Pizzamiglio S, Lampis A, et al. Methylation status in patients with early stage colon cancer: a new prognostic marker? Int J Cancer;130:488‐9. 30. H. E. Gender, Tumor Phenotype along with DNA Methylation Predict Survival Benefit from 5‐ fluorouracil in Colorectal Cancer. A Biological Basis for Chemosensitivity? Cancer Journal 2003;9:496. 31. D. H. Koo YSH, K. Kim, J. Lee, H. Chang, Y. Kang, C. S. Yu, K. C. Kim, M. Kim, S. J. Jang, T. W. Kim. CpG Island Methylator Phenotype and KRAS Mutation Status as Prognostic Markers in Patients with Resected Colorectal Cancer. Journal of Clinical Oncology 2011;29 (suppl; abstr 3595). 32. Ogino S, Meyerhardt JA, Kawasaki T, et al. CpG island methylation, response to combination chemotherapy, and patient survival in advanced microsatellite stable colorectal carcinoma. Virchows Arch 2007;450:529‐37. 33. S. H. Kim KHP, S. J. Shin, K. Y. Lee, T. I. Kim, N. K. Kim, S. Y. Rhal, J. K. Roh, J. B. Ahn. Association Between CpG Island Methylator Phenotype (CIMP) And Treatment Resopnse of FOLFIRI with Cetuximab in Patients with Metastatic Colorectal Cancer (MCRC). Annals of Oncology 2012;23:xi41‐xi2. 34. Min BH, Bae JM, Lee EJ, et al. The CpG island methylator phenotype may confer a survival benefit in patients with stage II or III colorectal carcinomas receiving fluoropyrimidine‐based adjuvant chemotherapy. BMC Cancer;11:344. 35. Kakar S, Deng G, Sahai V, et al. Clinicopathologic characteristics, CpG island methylator phenotype, and BRAF mutations in microsatellite‐stable colorectal cancers without chromosomal instability. Arch Pathol Lab Med 2008;132:958‐64. 36. Rhee YY, Kim MJ, Bae JM, et al. Clinical outcomes of patients with microsatellite‐unstable colorectal carcinomas depend on L1 methylation level. Ann Surg Oncol;19:3441‐8. 37. Weisenberger DJ SK, Campan M, Young J, Long TI, Faasse MA, Kang GH, Widschwendter M, Weener D,. Buchanan D, Koh H, Simms L, Barker M, Leggett B, Levine J, Kim M, French AJ, Thibodeau SN, Jass J, Haile R, Laird PW. CpG Island Methylator Phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet 2006;38:787‐93.
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38. Kim JH, Shin SH, Kwon HJ, Cho NY, Kang GH. Prognostic implications of CpG island hypermethylator phenotype in colorectal cancers. Virchows Arch 2009;455:485‐94. 39. Ju HX, An B, Okamoto Y, et al. Distinct profiles of epigenetic evolution between colorectal cancers with and without metastasis. Am J Pathol;178:1835‐46. 40. Kakar S, Deng G, Smyrk TC, Cun L, Sahai V, Kim YS. Loss of heterozygosity, aberrant methylation, BRAF mutation and KRAS mutation in colorectal signet ring cell carcinoma. Mod Pathol;25:1040‐7. 41. Simons CC, Hughes LA, Smits KM, et al. A novel classification of colorectal tumors based on microsatellite instability, the CpG island methylator phenotype and chromosomal instability: implications for prognosis. Ann Oncol. 42. Sanchez JA, Krumroy L, Plummer S, et al. Genetic and epigenetic classifications define clinical phenotypes and determine patient outcomes in colorectal cancer. Br J Surg 2009;96:1196‐204. 43. Kalady MF, Sanchez JA, Manilich E, Hammel J, Casey G, Church JM. Divergent oncogenic changes influence survival differences between colon and rectal adenocarcinomas. Dis Colon Rectum 2009;52:1039‐45. 44. Kim JC, Choi JS, Roh SA, Cho DH, Kim TW, Kim YS. Promoter methylation of specific genes is associated with the phenotype and progression of colorectal adenocarcinomas. Ann Surg Oncol;17:1767‐76. 45. Zlobec I, Bihl MP, Foerster A, Rufle A, Terracciano L, Lugli A. Stratification and Prognostic Relevance of Jass's Molecular Classification of Colorectal Cancer. Front Oncol;2:7. 46. Jo PJ, K.; Grade, M.; Conradi, L. C.; Wolff, H. A.; Ruschoff, J.; Schneider‐Stock, R.; Gaedcke, J.; Becker, H.; Ghadimi, B. M. CpG island methylator phenotype infers a poor prognosis in locally advanced rectal cancer. Langenbeck's Archives of Surgery;396:905. 47. Nam Jun Kim KJK, Byung‐Hoon Min, Kyoung‐Mee Kim, Jin Tong Kim, Dong Kyung Chang, Jae J. Kim, Jong Chui Rhee, Young‐Ho Kim. Sa1579 The Role of CpG Island Methylator Phenotype on Survival Outcome in Colon Cancer. Gastrointestinal Endoscopy 2011;73:AB213‐AB4. 48. Hughes LA, Khalid‐de Bakker CA, Smits KM, et al. The CpG island methylator phenotype in colorectal cancer: progress and problems. Biochim Biophys Acta;1825:77‐85. 49. Des Guetz G, Schischmanoff O, Nicolas P, Perret GY, Morere JF, Uzzan B. Does microsatellite instability predict the efficacy of adjuvant chemotherapy in colorectal cancer? A systematic review with meta‐analysis. Eur J Cancer 2009;45:1890‐6. 50. Cushman‐Vokoun AM, Stover DG, Zhao Z, Koehler EA, Berlin JD, Vnencak‐Jones CL. Clinical Utility of KRAS and BRAF Mutations in a Cohort of Patients With Colorectal Neoplasms Submitted for Microsatellite Instability Testing. Clin Colorectal Cancer. 51. Suppiah A, Alabi A, Madden L, Hartley JE, Monson JR, Greenman J. Anti‐p53 autoantibody in colorectal cancer: prognostic significance in long‐term follow‐up. Int J Colorectal Dis 2008;23:595‐600. 52. Benson AB, 3rd, Bekaii‐Saab T, Chan E, et al. Localized Colon Cancer, Version 3.2013. J Natl Compr Canc Netw;11:519‐28. 53. Douglas G. Altman LMM, Willi Sauerbreu, Sheila E. Taube. Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK): Explanation and Elaboration. PLoS Medicine 2012;9.
Figure 1: Study identification flowchart Legend: Using standardized protocol for a comprehensive search through 4 electronic databases, a total of 33 studies were included in this review for qualitative or quantitative analysis.
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Figure 2: Forest plots of hazard ratios (HRs) of Disease Free Survival (DFS) in studies of CRC patients associated with CIMP with no restriction to MSI status. Legend: CIMP is associated with a worse Disease Free Survival among CRC patients after adjusting for other prognostic factors.
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Figure 3: Forest plots of hazard ratios (HRs) of Overall Survival (OS) in studies of CRC patients associated with CIMP with no restriction to MSI status. Legend: CIMP is associated with a worse Overall Survival among CRC patients after adjusting for other prognostic factors.
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Figure 5: Forest plots of hazard ratios (HRs) of Overall Survival (OS) in studies of Microsatellite Stable (MSS) CRC patients associated with CIMP. Legend: CIMP is associated with a worse Overall Survival after adjusting for other prognostic factors among a subgroup of CRC patients with Microsatellite Stability (MSS).
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Table 1: Study eligibility criteria table Study Eligibility Criteria CIMP Definition Widespread CpG island methylation as defined by each study (no restriction regarding laboratory method, gene panel, or marker threshold value) Outcome Measure Disease-Free Survival, Overall Survival, or Cancer-Specific Mortality Study Design Prospective or retrospective cohort studies Anatomical Site Colon, Rectal, or Colorectal Study tissue Surgically resected primary tumor tissue Stage Any Therapy Any Length of Follow-up Any Downloaded from http://annonc.oxfordjournals.org/ by guest on September 30, 2014
Table 2: Summary of Included Study Characteristics Study Patient population characteristics Publication type
Country
Korea
Sample size (# patients) ,N 161
Ahn et al. 201110
Full Publication
III
Barault et al. 200811
Full Publication
France
582
Dahlin et al. 201012
Full publication
Sweden
Donada et al. 201328
Full Publication
Italy
Han et al. 201333
Full Publication
Korea
Jo et al. 201152
Full publication
Germany
Male Gend er, N (%) 93 (57.8 %)
Median Age, y/o (range) Mean 60.9 (31-84)
I-IV
333 (57.2 %)
604
I-IV
315 (52.2 %)
65y/ o: N=156/ 6675y/o: N=184/ >75y/o: N=242 69.9 (58-79)
120
II
57 (47.5 %)
322
150
Stage
II-III
NR
Inclusion Criteria
Specimen preservation
Gene panel
Lab method
CIMP classification
Marker Threshold
CIMP prevalence, N (%)
Sporadic colon CA
Cryopreservation
Bisulfite pyrosequen cing
CIMP -/+
CIMP+ 3/9 (plus additional genetic criteria)
29 (18.0%)
DFS
Sporadic colon CA without IBD
Cryopreservation
MINT1, MINT2, MINT31, hMLH1, p16, p14, SFRP1, SFRP2, WNT5A Classic panel
MSP
CIMP 0/low/high
CIMP high: 4-5/5 CIMP low: 1-3/5
CIMP high: 97 (16.7%) CIMP low: 199 (34.2%) CIMP 0: 286 (49.1%)
OS
Sporadic colorecta l CA
FFPE
MethyLight
CIMP 0/low/high
CIMP high: 6-8/8 CIMP low: 1-5/8
CIMP high: 74 (12.3%) CIMP low: 215 (35.6%) CIMP 0: 301 (49.8%)
Cancerspecific mortalit y
Mean 67.6
Colon CA
FFPE
MSP
CIMP 0/low/high
CIMP high:
DFS, OS
192 (59.6 %)
61.0 (30-78)
NR
CIMP 0/low/high
CIMP high:
107 (71% )
Mean 62.4
Sporadic colorecta l CA cases with complete resection and complete FOLFO X adjuvant treatment among stage II&III cases Locally advanced rectal CA cases
CIMP high: 22 (18.3%) CIMP low: 36 (30.0%) CIMP 0: 62 (51.7%) CIMP high: 25 (7.8%) CIMP low: 125 (38.8%) CIMP 0: 172 (53.4%)
CIMP -/+
CIMP+:
15 (10%)
DFS, OS
FFPE
CDKN2A, MLH1, CACNA1G, NEUROG1, RUNX3, SOCS1, IGF2, CRABP1 Weisenberger
CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, CDKN2A(p16 ), RUNX3, SOCS1
MethyLight
Weisenberger
MSP
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Study
Outcomes
CIMP assessment
3/5 CIMP low: 1-2/5
5/8 CIMP low: 1-4/8
3/5
DFS
Jover et al. 201120
Full publication
Spain
Ju et al. 201161
Full publication
Japan
Kakar et al. 200862
Full Publication
USA
Kakar et al. 201263
Full Publication
USA
Kalady et al. 200949
Full publication
USA
Kim et al. 200944
Full publication
Korea
Kim et al. 201050
Full publication
Korea
302
78
69
33
357
320
285
I-IV
I-IV
I-IV
I-IV
I-IV
I-IV
I-IV
185 (61.3 %)
Mean 70.3
51 (65.4 %) 39 (56.5 %)
Mean 63.9 =60y/o : 50
24 (72.7 %)
60y/o: 13
193 (54.1 %) 187 (58.4 %)
Mean 66.9
168 (58.9 %)
Mean 58.0
Lee et al.200814
Full Publication
Korea
81 (60.4 %)
Min et al. 201140
Full publication
Korea
245
I-IV
138 (56.3 %)
Ogino et al. 200638
Full publication
USA
31
IV
22 (71.0 %)
Mean 60.9
=60 y/o: 77 Mean 65.0 (33-83)
57.0 (31-81)
Sporadic colorecta l CA Sporadic colorecta l CA
FFPE
Cryopreservation
CACNAG1, SOCS1, NEUROG1, RUNX3, MLH1 Classic panel
Bisulfite pyrosequen cing
CIMP -/+
MSP
CIMP -/+
CIMP+:
89 (29.5%)
DFS
19 (24.4%)
DFS
16 (23.2%)
OS
16 (48.5%)
OS
78 (21.8%)
DFS
37 (11.6%)
OS
102 (35.8%) or 51 (17.9%)
DFS
42 (31.3%)
OS
3/5
CIMP+: 2/5
FFPE
hMLH1, p16, HIC1, RASSF2, ID4, MINT1, MINT31 hMLH1, p16, HIC1, RASSF2, ID4, MINT1, MINT31
MSP
Weisenberger
MethyLight
CIMP -/+
CIMP+: 3/7
Signet ring cell colorecta l carcinom a Sporadic colorecta l CA Sporadic colorecta l CA cases without neoadjuv ant therapy Sporadic colorecta l CA cases without neoadjuv ant therapy Sporadic colorecta l CA
FFPE
Sporadic colorecta l CA cases without neoadjuv ant therapy Advance d MSS colorecta
FFPE
Weisenberger
MethyLight
CIMP 0/low/high
CIMP high: >2/5 CIMP low: 1-2/5
CIMP high 34 (13.9%)
DFS
FFPE
CACNA1G, CDKN2A, CRABP1,
MethyLight
CIMP 0/low/high
CIMP high: >8/13 CIMP low:
CIMP high: 3 (10.0%) CIMP low:
OS
Cryopreservation
MSP
CIMP -/+
CIMP+: 3/7
CIMP -/+
CIMP+: 3/5
FFPE
NR
FFPE
CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, SOCS1 MLH1, MINT1, MINT2, MINT31, p16, p14, CACNA1G
MethyLight
Classic panel
MSP
CIMP -/+
CIMP+: 5/8
Bisulfite Pyrosequen cing
CIMP -/+
CIMP+: 2/7 or 3/7
CIMP -/+
CIMP+: 2/5
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134
I-IV
without preoperat ive chemoth erapy Sporadic colorecta l CA
l CA cases without metastas es
IGF2, MLH1, NEUROG1, RUNX3, SOCS1, MINT1, MINT31, IGFBP3, MGMT, WRN CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, SOCS1 CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, SOCS1 P16, MDR1, MINT2
Ogino et al. 200915
Full publication
USA
649
I-IV
283 (44% )
Mean 66.5
Primary colon CA cases
FFPE
Rhee et al. 201242
Full publication
Korea
207
I-IV
126 (60.9 %)
55y/o: 107
microsat ellite unstable colorecta l cancers
FFPE
Rijnsoeve r et al. 200234
Full publication
Australia
138 (50.2 %)
Colorect al CA
FFPE
Rijnsoeve r et al. 2003 19 Samowitz et al. 200529
Full publication
Australia
144 =71y/o Mean 60.9
Colorect al CA
83% FFPE; 17% cryopreservation
P16, MINT2, MDR1
MSP
Full Publication
USA
Classic panel
MSP
Full publication
USA
Sanchez et al. 200948
Full Publication
USA
Shen et al. 20079
Full publication
USA
Primary sporadic colon CA cases between 30~79 y/o without IBD Primary sporadic rectal CA cases between 30-79 y/o without IBD Colorect al CA cases without IBD Colorect al CA cases
FFPE
Samowitz et al. 200932
5/8 CIMP low: 1-5/8
CIMP high: 126 (19.4%) CIMP low: 252 (38.8%) CIMP 0: 271 (41.8%)
OS, cancerspecific mortalit y
MethyLight
CIMP 0/low/high
CIMP high:
NR
OS
CIMP -/+
CIMP+:
128 (46.5%)
OS
67 (32.5%)
DFS
246 (27.8%)
OS
103 (13.5%)
OS
83 (21.2%)
DFS, OS
28 (15.4%)
OS
95 (18.7%)
Cancerspecific
MSP
5/8 CIMP low: 1-4/8
2/3 CIMP -/+
CIMP+: 2/3
MSP
MethyLight
CIMP low/high
CIMP high:
CIMP low/high
CIMP high:
CIMP -/high
CIMP high:
2/5
2/5
3/5
FFPE
FFPE
MINT1, MINT2, MINT31, hMLH1, p14, p16 Weisenberger
MSP, COBRA
CIMP -/+
MSP
CIMP -/+
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275
1-8/13
CIMP+: 2/6
CIMP+:
%)
cases
Ward et al. 200313
Full publication
Australia
605
I-IV
323 (53.4 %)
Mean 68.3 (29-99)
Zlobec et al. 201151
Full publication
Switzerla nd
337
NR
156 (46.3 %)
Mean 69.9 (42-95)
Zanutto et al. 201135
Letter to the editor
Italy
42
II
NR
AlSohaily et al. 201130 Elsaleh et al. 200336 Kim et al. 201153
Conference Abstract
Australia
281
NR
NR
Conference Abstract Conference Abstract
USA
891
III
NR
NR
Korea
151
NR
NR
NR
Kim et al. 201239
Conference abstract
Korea
49
NR
NR
NR
Koo et al. 201137
Conference Abstract
Korea
191
III
NR
NR
3/5
mortalit y OS
Sporadic colorecta l CA cases without IBD Sporadic colorecta l CA
Cryopreservation
P16, MINT1, MINT2, MINT12, MINT31
MSP
CIMP -/+
CIMP+: >3/5
NR
FFPE
CRABP1, MLH1, p16INK4a, CACNA1G, NEUROG1
Bisulfite pyrosequen cing
CIMP 0/low/high
CIMP high:
66.1 (30-83)
MSS colorecta l CA
Cryopreservation
Classic panel
MSP
Methylation low / intermediate/ high
Methylation high: >3/5 Intermediat e 3/5
DFS
NR
Rectal CA
FFPE
Weisenberger
MSP
CIMP 0/low/high
CIMP high
CIMP high: 24 (7.1%) CIMP low: 145 (43.0%) CIMP 0: 168 (49.9%) High: 13 (31.0%) Intermediate : 15 (35.7%) Low: 14 (33.3%) 13 (4.6%)
Colorect al CA Colorect al CA
NR
NR
NR
NR
NR
NR
DFS
NR
Weisenberger
NR
CIMP -/+
CIMP+:
27 (17.9%)
OS
Colorect al CA cases with metastas es treated by 5-FU, leucovori n, irinoteca n, and cetuxima b Colorect al CA
FFPE
P16, p14, MINT1, MINT2, MINT31, hMLH1
Bisulfite Pyrosequen cing
CIMP -/+
NR
14 (28.6%)
DFS, OS
NR
Weisenberger
NR
CIMP-/+
CIMP+:
63 (33.0%)
DFS
4/5 CIMP low: 1-3/5
Cancerspecific mortalit y
OS
3/5
3/5
NR: Not reported; MSP: Methylation-Specific Polymerase Chain Reaction Classic panel: MINT1, MINT2, MINT31, CKKN2A(p16), hMLH1 Weisenberger: CACNA1G, IGF2, NEUROG1, RUNX3, SOCS
3/5
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Table 3: Risk of Bias of Included Studies
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Red circles represent studies with high risk of bias; green circles represent studies with low risk of bias; yellow circles represent studies that did not provide sufficient information for assessing risk of bias.
Table 4: Summary of study conclusions regarding interaction of CIMP and adjuvant chemotherapy Study
Study Cohort
Chemotherapy regimen
DFS among patients receiving surgery & adjuvant CT NR
Elsaleh et al. 200336
891 stage III CRC patients
5-FU-based chemotherapy
Min et al. 201140
124 stage II and III CRC patients
5-FU or capecitabine, leucovorin (Mayo regimen) 5-FU, leucovorin (Mayo regimen)
CIMP-H ~ CIMP-L/0
CIMP-H ~ CIMPL/0 (p=0.073).
Rijnsoever et al. 200319
206 stage III CRC patients
CIMP+ < CIMP(p=0.05)
CIMP+ > CIMP-
Donada et al. 201328
120 stage II colon cancer
5-FU, leucovorin
CIMP-H ~ CIMP-L/0 (p=0.6)
CIMP-H > CIMPL/0 (p=0.05)
Jover et al. 201120
196 stage II and III CRC patients,
5-FU-based chemotherapy
CIMP+ < CIMP(p=0.03)
CIMP+ ~ CIMP(p=0.1)
Han et al. 201233
322 stage II and III CRC who received surgery + CT 49 metastatic CRC cases who received CT
FOLFOX (5-FU, oxaliplatin, folinic acid)
NR
CIMP-H ~ CIMPL/0 (p=0.31)
FOLFIRI (5-FU, leucovorin, iriontecan) and cetuximab
NR
CIMP+ ~ CIMP-
Kim et al. 201239
NR: Not reported; CT: Chemotherapy; 5-FU: 5-fluorouracil CIMP-H=CIMP-high; CIMP-L=CIMP-low; CIMP-0=CIMP-zero
DFS among CIMP (+) patients
DFS among CIMP (-) patients
Surgery+CT > surgery alone (p=0.03) Surgery+CT > surgery alone (p=0.022)
Surgery+CT ~ surgery alone
Surgery+CT > surgery alone (p=0.002) NR
Surgery+CT ~ surgery alone
Surgery+CT ~ surgery alone (p=0.6) NR
Surgery+CT ~ surgery alone (p=0.6) Surgery+CT ~ surgery alone for CIMP-0, CIMPlow patients (p=0.7, p=0.9) surgery+CT > surgery alone (p=0.0001) NR
NR
NR
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DFS among patients receiving surgery alone NR
Prognostic Value of CpG Island Methylator Phenotype among Colorectal Cancer Patients: A Systematic Review and Meta-Analysis
Y. Y. Juo1, F. Johnston2, D. Zhang3, H. H. Juo4, H. Wang1, E. P. Pappou1, T. Yu3, H. Easwaran5, S. Baylin5,6, M. Engeland7, N. Ahuja1,5,8 Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, USA
2
Department of Surgery, Medical College of Wisconsin, Milwaukee, USA
3
Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, USA
4
Department of Internal Medicine, Danbury Hospital, Danbury, USA
5
Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, USA
6
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, USA
7
Department of Pathology, Maastricht University, Maastrich, The Netherlands
8
Department of Urology, Johns Hopkins University School of Medicine, Baltimore, USA
Address all correspondence to: Nita Ahuja, MD FACS, Vice Chair of Academic Affairs, SurgeryAssociate Professor of Surgery, Oncology and Urology, Chief, Section of Mixed Tumors-Surgical Oncology, Department of Surgery, Blalock 685, 600 N. Wolfe Street, Baltimore, MD 21287, USA,
Phone: (410) 502-6135, Email: [email protected] Key Message: "This is the first comprehensive qualitative and quantitative synthesis of the currently available literature regarding CIMP’s prognostic value among CRC patients. Our results showed significantly worse DFS and OS among CRC patients with CIMP compared to those without CIMP, and a potential survival benefit in CRC patients with CIMP who received adjuvant chemotherapy in comparison with those who received surgery alone. However, future research into the most appropriate operational definition of CIMP and the mechanism by which CIMP influenced disease prognosis would be of vital importance in helping us put past study results into proper perspective."
© The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
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1
2
ABSTRACT Background: Divergent findings regarding the prognostic value of CpG Island Methylator Phenotype (CIMP) in Colorectal Cancer (CRC) patients exist in current literature. We aim to review data from published studies in order to examine the association between CIMP and CRC prognosis. Materials and Methods: A comprehensive search for studies reporting Disease-Free Survival (DFS), Overall Survival (OS), or cancer-specific mortality of CRC patients stratified by CIMP is
hazard ratios (HR) as summary statistics. Results: 33 studies reporting survival in 10,635 patients are included for review. 19 studies provide data suitable for meta-analysis. The definition of CIMP regarding gene panel, marker threshold, and laboratory method varies across studies. Pooled analysis shows that CIMP is significantly associated with shorter DFS (pooled HR estimate 1.45; 95% CI 1.07~1.97, Q=3.95, I2=0%) and OS (pooled HR estimate 1.43; 95% CI 1.18~1.73, Q=4.03, I2=0%) among CRC patients irrespective of Microsatellite Instability (MSI) status. Subgroup analysis of Microsatellite Stable (MSS) CRC patients also shows significant association between shorter OS (pooled HR estimate 1.37; 95% CI 1.12~1.68, Q=4.45, I2=33%) and CIMP. Seven studies have explored CIMP’s value as a predictive factor on stage II and III CRC patient’s DFS after receiving adjuvant 5-FU therapy: of these, four studies showed that adjuvant chemotherapy conferred a DFS benefit among CIMP(+) patients, one concluded to the contrary, and two found no significant correlation. Insufficient data was present for statistical synthesis of CIMP’s predictive value among CRC patients receiving adjuvant 5-FU therapy.
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performed. Study findings are summarized descriptively and quantitatively, using adjusted
3
Conclusion: CIMP is independently associated with significantly worse prognosis in CRC patients. However, CIMP’s value as a predictive factor in assessing whether adjuvant 5-FU therapy will confer additional survival benefit to CRC patients remained to be determined through future prospective randomized studies.
Keywords: CIMP, Prognosis, Colorectal Cancer, adjuvant chemotherapy, epigenetics, tumor Downloaded from http://annonc.oxfordjournals.org/ by guest on September 30, 2014
marker
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BACKGROUND CpG islands are genomic regions that contain a high frequency of cytosine and guanine nucleotides, connected with a phosphodiester bond. They are typically found in or near promoter regions of the genome, where transcription is initiated, and are present in as high as 40-50% of the human genes. Aberrant methylation of cytosine nucleotides within these CpG islands can lead to aberrant silencing of normal tumor-suppressor function and cancer formation.1, 2 A distinct molecular subtype of cancer, characterized by high degrees of methylation, has
unique molecular features, epidemiology, precursor lesions, and they represent approximately 15% of sporadic cases of colorectal cancer. However, the role CIMP plays in the pathogenesis of CRC is still poorly understood. It has been shown that hypermethylation secondary to CIMP leads to Microsatellite Instability (MSI) through the methylation of the MLH1 promoter and the consequent silencing of the MLH1 mismatch repair gene. Almost 70-80% of MSI CRCs can be attributed to CIMP and associated MLH1 methylation.4 CIMP, however, can exist with and without MSI. Studies have shown CIMP to be an independent negative prognostic factor in disparate subgroups of CRC patients5-7, although its influence is modified by other genetic factors including MSI8, 9 and KRAS/BRAF status10, 11. It has been hypothesized that CIMP-high colorectal cancer patients might have a better response to 5-fluorouracil (5-FU) chemotherapy due to observations that suppression of gamma-glutamyl hydrolase (GGH)12, one of the putative effects of CpG island methylation, is associated with an increase in intracellular folate level13, which magnifies the effect of 5-FU chemotherapy.14 Several clinical studies have come to
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been termed CpG Island Methylator Phenotype, or CIMP.3 CIMP cancers are characterized by
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conflicting conclusions regarding the predictive value of CIMP15, 16. Precise estimates of the predictive value of CIMP can allow for refinement in clinical management, especially among stage II CRC patients, where the value of 5-FU chemotherapy is still controversial17. The goal of this systematic review is to summarize published studies using standardized techniques to gain a better understanding of the prognostic significance of CIMP in CRC patients with respect to their Disease-Free Survival (DFS) and Overall Survival (OS).
SEARCH STRATEGY AND STUDY ELIGIBILITY Published studies were eligible if CRC patient survival was analyzed after stratification by CIMP status of primary tumor tissue. CIMP status was defined by the respective study authors with no restriction to laboratory method, gene panel, or marker threshold value. Primary outcome of the study was Disease Free Survival (DFS), defined as the length of time since initial cancer treatment during which no evidence of cancer recurrence or death was found. Secondary outcomes was Overall Survival (OS), defined as the length of time since initial cancer treatment that patients were still alive. Cohort studies of either a prospective or a retrospective nature were included. Case reports and reviews were excluded. There was no restriction with respect to patient age, gender, ethnicity, comorbidity, tumor type, disease stage, length of follow-up, or treatment received. (Study eligibility summarized in Table 1)
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METHODS
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STUDY IDENTIFICATION We followed MOOSE (Meta-analysis of Observational studies in Epidemiology) guidelines18 to identify eligible studies. Combination of text and exploded controlled vocabulary terms were used to search PUBMED, EMBASE, SCOPUS, and Web of Science electronic databases until June 6th 2013, relating to the following two concepts: (1) CIMP and (2) colorectal cancer. Citations in relevant reviews and studies were hand-searched to capture additional
conference abstracts and letters were included to capture grey literature. Each study was independently assessed for inclusion by at least two reviewers. Discrepancies within the reviewing pair were resolved via discussion. The flow chart of study identification was presented according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines in Figure 1.19 STATISTICAL METHODS Study characteristics including study country, patient characteristics, and CIMP assessment methods were summarized in a consistent manner for easy examination. Patient number and percentages among different gender and age groups were calculated using available data when they were not directly reported. Assignment of CIMP status into constitutive groups (CIMP-positive or CIMP-negative) or CIMP-high, CIMP-low, and CIMP-zero was performed according to the grouping in each study. The term CIMP was used synonymously with CIMP-positive and CIMP-high, depending on whether the study authors have di- or trichotomized CIMP status. During our discussion, CIMP-low and CIMP-zero were treated equivalently with CIMP-negative, but we performed
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potentially relevant studies. In addition to full publications, original studies in the form of
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separate statistical analyses for studies that dichotomized and studies that trichotomized CIMP status.Clinical and methodological sources of heterogeneity across studies were evaluated to assess the appropriateness of statistical synthesis. Factors taken into consideration included variation in patient disease stage, treatment received, and MSI status. Statistical heterogeneity was assessed by Breslow-Day test while Q and I2 statistics were presented as measures of inconsistency of CIMP prognostic values across studies attributable to heterogeneity.20 The Q statistic was a weighted sum of squares following a chi-squared distribution with k-1 (k=number
deviation from total homogeneity. In our study, we employed a p-value of 0.1 and an I2 statistic of less than 50% as indicating acceptable statistical heterogeneity. In order to summarize the association between CIMP status and survival across studies, a weighted average of the individual adjusted log Hazard Ratios (HR) was used, with the weights inversely proportional to the variance of the log hazard ratio of each study. Adjusted hazard ratios were sought among results of multivariate survival analysis using Cox proportional hazards regression model and the adjusted variables documented. When missing data regarding adjusted hazard ratio or variance was encountered, estimates were indirectly imputed from available numerical data where possible using confidence intervals, p-values, and event numbers by methods described by Parmar et al21. Recurrence-Free Survival (RFS) was interpreted as synonymous with DFS. Random-effects model was used in all numerical synthesis. Forest plots were presented with studies plotted in order of decreasing variance of the log HR. Horizontal lines represented 95% CIs. Each box represented the HR point estimate and its area was proportional to the weight of the study. The diamond on the bottom denoted the overall summary estimate, with CIs given by the width of the diamond. The vertical line signified the reference
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of included studies) degrees of freedom. The p-value calculated thereof gave an estimate of the
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line with HR=1.0. Sensitivity analyses were performed to assess the impact of including conference abstracts, imputed estimates, and studies with more than one risk of bias marked as high- or unclear risk. All numerical synthesis was performed using Review Manager 5.2.
RESULTS ELIGIBLE STUDIES
“colorectal cancer” as keywords for searching (Table 1). (Flow chart of study identification is summarized in Figure 1) The main reasons for exclusion of studies during the initial title/abstract screening were failure to examine disease prognosis, incorrect histology, or the studies were not original studies. 93 studies remained for full-text inspection after title/abstract screening (Figure1). Of these, 39 full-text studies were excluded because the primary outcomes of interest (OS and DFS) were not reported, 13 studies were excluded because no prognostic measure with regard to CIMP was reported, 6 studies were excluded due to overlapping patient population with included studies, and 2 studies were excluded because the study tissue was not primary tumor tissue. A total of 33 studies were included in the systematic review. (Study characteristics of these studies were summarized in Table 2.) Of these, only 19 studies were eligible for pooling hazard estimates in the meta-analysis. The reasons for exclusion were failure to present numerical hazard ratio estimates, failure to include CIMP in the multivariate analysis, and failure to present survival data with explicit numerical values.
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We initially identified 2,414 studies for potential inclusion using only “CIMP” and
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STUDY CHARACTERISTICS The 33 studies analyzed 10,635 patients for CIMP status and its relationship to disease prognosis. The mean number of patients for each study was 322 (range 33-990). Most studies included both colon and rectal cancer cases. 5 studies6, 7, 11, 22, 23 included colon cancers only and 3 studies24-26 rectal cancers only. In most studies, a mixture of different disease stages was included, however, eight studies6, 15, 22, 27-31 only examined stage II or III cancers and one study32
chemotherapy were included, while four other studies23, 29, 32, 35 included only Microsatellite Stable (MSS) tumor tissues for analysis and 1 study36 included only Microsatellite Instable (MSI) tumor tissues for analysis. The risk of bias of each included study is summarized in Table 3. While retrospective convenience sampling was employed for almost all studies, some studies stated their inclusion and exclusion criteria more explicitly than others and these studies were rated as low risk in selection bias. Almost all studies (31 out of 33 studies), including conference abstracts, have explicitly stated their method of assessment of CIMP and genetic mutations such as KRAS or BRAF. Median follow-up length, range, and loss-to-follow-up rate were satisfactorily reported in about half of the studies. Known or commonly discussed confounders in the relationship between CIMP and survival such as age, disease status, MSI status, KRAS/BRAF mutation were adjusted for in two-thirds of the studies, and in these studies the risk of confounding was rated as low risk. HETEROGENEITY IN CIMP DEFINITION Only studies that used primary tumor tissues were included in this review. 21 of the 33 included studies classified CIMP status in a dichotomized fashion (CIMP-positive vs. negative)
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only examined stage IV cancers. In three studies15, 27, 33, 34, only patients who received
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while CIMP was classified in a trichotomized fashion (CIMP-high/low/zero) in the remainder of the studies. Significant variation existed between the studies regarding the gene panel, marker threshold, and laboratory method used for defining CIMP. The Classic panel (MINT1, MINT2, MINT31, CKKN2A(p16), hMLH1) and the 5-gene panel devised by Weisenberger37 in 2006 (CACNA1G, IGF2, NEUROG1, RUNX3, SOCS1) were the two most commonly employed gene
number of genes assessed in each study ranged from 3 to 13 genes, with a median of 5 genes. Marker threshold for defining CIMP status was often chosen arbitrarily or based on previous literature; 2 studies7, 38 chose their threshold based on association with known clinicopathological features of CIMP-positive tumors. One study11 specifically compared the effect of using different CIMP panels and concluded that this variation could result in significantly different associations with survival outcomes. Sixteen7, 9, 10, 15, 22-26, 28, 29, 35, 39-41 studies used Methylation Specific PCR (MSP) as the laboratory method for methylation analysis, nine8, 11, 27, 32, 34, 38, 42 used MethyLight, five6, 16, 36, 43-45 used bisulfite pyrosequencing, one5 used COBRA, and in three studies, laboratory method was not specified. In general, no specific gene panel or laboratory method was observed to produce a consistently higher or lower CIMP prevalence. RELATIONSHIP BETWEEN CIMP AND PROGNOSIS DISEASE-FREE SURVIVAL The median prevalence of CIMP-positive or CIMP-high status amongst included studies was 18.2%, ranging from 4.6% to 46.5%. Overall median follow-up length was 45.3 months, ranging from 2.8 to 100.8 months.
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panels, although 17 of the 33 included studies opted to use a different gene panel(Table 2). The
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Eleven studies examined the relationship between CIMP status and Disease-Free Survival (DFS) amongst CRC patients. The majority6, 16, 27, 33, 34, 39, 42, 43 of studies (8 out of 11) found no significant relationship between CIMP and DFS, while 2 studies25, 44 found CIMP to be associated with unfavorable DFS. In addition, one6 found that CIMP, despite being an insignificant predictor for DFS in their overall study cohort, was associated with an unfavorable DFS among tumors of proximal colon. In contrast, Koo et al.31 found a combination of CIMP(+)/KRAS-wild type to be associated with favorable DFS compared with other
Stable (MSS), Zanutto et al.29 found that CIMP positivity was associated with a lower recurrence rate. In our pooled analysis, CIMP was associated with an unfavorable DFS after adjusting for other relevant confounders in each contributing study. Insufficient data was present among studies that categorized CIMP status in a trichotomized fashion for pooled analysis. Six studies that categorized CIMP status in a dichotomized fashion provided satisfactory adjusted HR estimates suitable for numerical synthesis. This accounted for a total of 1,454 patients. Their overall summary hazard ratio was 1.45 (95% CI 1.07-1.97), with a low heterogeneity (Q=3.95, I2=0%), indicating a worse DFS for CIMP-positive patients. From the forest plot (Figure 2), the confidence intervals of adjusted hazard ratios in five out of six studies was insignificant, but the majority of them had the same trend. Sensitivity analysis was performed to assess the effect of including imputed HR estimates by leaving out 2 studies with imputed HRs 25, 27. The exclusion did not change our conclusion (pooled HR=1.22, 95% CI, 0.83 to 1.79; Q=1.24, I2=0%). In a separate analysis, a subgroup of studies16, 25, 39 with
1 high risk of bias item were excluded.
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genetic/epigenetic profiles. Examining a subgroup of CRC patients who were Microsatellite
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This also did not change our conclusion although the summary estimate did not attain statistical significance (pooled HR=1.46, 95% CI, 0.79 to 2.67; Q=1.10, I2=0%). A subgroup of studies (7 of 33) also explored CIMP’s value as a predictive factor on the effect of adjuvant 5-FU therapy on CRC patients. Seven studies examined the relationship between CIMP status and DFS among patients who received 5-FU-based adjuvant chemotherapy after surgical resection of the primary tumor. (Table 4 shows the individual study details).
independent of MSI and p53 status. Min et al.34 found that stage II and III CRC patients with CIMP-high represented a cohort most likely to receive benefits from 5-FU therapy. Rijnsoever et al.15, in a study restricted to stage III CRC cases, also showed that CIMP(+) status was associated with favorable response to 5-FU therapy. In addition, Rijnsoever et al. found that CIMP, despite being associated with worse prognosis among patients treated by surgery alone, was associated with a trend for better DFS among patients receiving both surgery and 5-FU therapy.15 This finding was confirmed by Donada et al.22 in their study of stage II colon cancer cases. On the other hand, Jover et al.16 found that among stage II and III CRC cases, CIMP(+) cases represented a cohort that was unresponsive to 5-FU therapy while CIMP(-) cases received significant survival benefit from 5-FU therapy. Han et al.27 found both the predictive value of CIMP and the interaction between CIMP and adjuvant chemotherapy to be insignificant in their study of stage II and III CRC cases receiving FOLFOX treatment. Kim et al.33 found CIMP to be an insignificant predictor of DFS in their study of metastatic CRC cases treated with FOLFIRI. Insufficient (CIMP
adjuvant chemotherapy) interaction term estimates were available for
pooling so no statistical summary was attempted. Overall, four out of seven studies agreed that adjuvant chemotherapy conferred a DFS benefit among CIMP(+) stage II and III CRC patients;
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Elsaleh et al.30 showed that CIMP(+) cases received survival benefit from 5-FU therapy
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of the remaining three studies, one concluded to the contrary and two found CIMP to be an insignificant predictive factor. OVERALL SURVIVAL Nineteen studies provided data regarding the effect of CIMP status on Overall Survival (OS) among CRC patients of different disease stages and genetic profiles. Of these, 13 studies9-11, 23, 24, 26, 28, 35, 38-40, 42, 46
found that CIMP was associated with an unfavorable OS while no study found CIMP to be
associated with a favorable OS. Our pooled analysis showed that CIMP(+) status was associated with an unfavorable OS. A subset of 11 studies provided adjusted hazard ratio estimates with no restriction to MSI status suitable for pooling, accounting for a total of 3,559 patients. Of these, 7 studies have classified CIMP in a dichotomized fashion. Their overall summary hazard ratio estimate was 1.43 (95% CI: 1.18-1.73). No significant statistical heterogeneity was present (Q=4.03, I2=0%). Of the other four studies that have classified CIMP in a trichotomized fashion, both CIMP-high and CIMPlow were associated with a significantly worse OS in comparison with CIMP-zero (Summary HR 1.53 with 95% CI 1.11~2.12 and 1.33 with 95% CI 1.11~1.61, respectively). From the forest plot (Figure 3) we could infer that despite a lack of significance in many studies, they shared trends in the same direction which led to a significant overall summary HR. To assess the potential effect of including conference abstracts, a sensitivity analysis was performed by excluding studies that was not a full publication.47. The exclusion made no difference to our conclusion (pooled HR=1.59, 95% CI, 1.16 to 2.19; Q=2.22, I2=0%). Sensitivity analysis was also performed to assess the effect of including indirectly estimated HRs by leaving out three
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45, 47
concluded that CIMP had no significant effect on OS. Six studies5, 32, 36, 44,
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studies7, 25, 45 with imputed log HR estimates. The exclusion made no difference to our conclusion (pooled HR=1.69, 95% CI, 1.24 to 2.31; Q=3.99, I2=0%). In order to assess the impact of including studies of variable risk of bias, a subgroup analysis excluding studies7, 9, 11, 25, 42
with
1 high risk of bias item showed essentially the same conclusions (pooled HR=1.83,
95% CI, 1.23 to 2.73; Q=3.49, I2=14%). Nine studies examined the relationship between CIMP and OS among Microsatellite
shorter OS in 6 studies7, 9, 10, 32, 36, 38, while 3studies11, 23, 35 found no significant association between CIMP and OS. Restricting the meta-analysis to the four studies with suitable summary HR estimates, accounting for a total of 1,112 MSS cases, produced a summary HR estimate of 1.37 (95% CI 1.12~1.68) with acceptable statistical heterogeneity (Q=4.45, I2=33%), suggesting that CIMP was associated with a significantly shorter OS amongst MSS tumors also (Figure 4). Four studies examined the relationship between CIMP and OS among Microsatellite Instable (MSI) tumors. Rhee et al.36 found CIMP(+) tumors to have shorter OS while Zlobec et al.45, Ward et al.9, and Barault et al.7 all found no significant association between CIMP and OS. Insufficient numerical estimates were available for pooling.
DISCUSSION Among patients included in our review, CIMP-positive accounted for almost one fifth (18.2%) of all sporadic colorectal cancers. Understanding the implication of its presence on patient prognosis would be crucial in making management decisions for CRC patients.
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Stable (MSS) tumors. In this cohort, CIMP was reported to be associated with a significantly
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One of the major confounding factors in a systematic review on topics relating to CIMP was the lack of a standardized operational definition of CIMP. No consensus existed regarding the most appropriate gene panel, marker threshold, or laboratory method for the assessment of CIMP.48 Several studies performed sensitivity analysis by employing different gene panels or marker thresholds and concluded that this could result in different conclusions regarding the prognostic value of CIMP.11, 32, 38, 44 Despite the methodological heterogeneity, our review showed no gene panel, laboratory method, or preservation method to be consistently associated
with the numerical synthesis. The results showed that CRC patients with CIMP were likely to experience both shorter DFS and OS after adjusting for their age, gender, disease stage and the treatment modalities they received. A number of other genetic profiles have also been cited as being significant effect modifiers in the relationship between CIMP and CRC patient prognosis, such as MSI status, mutation status of BRAF, KRAS, or anti-p53 autoantibody. While most studies adjusted for established confounders such as age, gender, and tumor stage, the genetic markers such as KRAS/BRAF and MSI were not consistently assessed and adjusted in all studies. This was most likely due to conflicting results in previous literature regarding the prognostic value of these markers49-51 and non-significant finding during univariate analysis in the respective studies. In our review, we were able to perform subgroup analysis for MSS CRC patients and we found that the survival disadvantage associated with CIMP persisted in this subgroup. There was not enough data among the included studies to allow data pooling for exploration of whether the same disadvantage held true among the MSI patients or other genetic subgroups. Of the included studies, Barault et al.7, Dahlin et al.8 and Ward et al.9 all found MSI status to be a significant
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with a better or worse prognosis in patients with CIMP, thus a decision was made to carry on
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effect modifier in the relationship between CIMP and survival; Kakar et al.35, 40 and Samowitz et al.23 found BRAF mutation to be a predictive factor for poor prognosis among MSS cases independent of CIMP status; in contrast, Lee et al.10 found KRAS/BRAF mutation to be an effect modifier in the relationship between CIMP and poor prognosis. Zlobec et al.45 argued for a combination of BRAF/CIMP as the most appropriate marker combination for classifying distinct prognostic groups. The dilemma of wishing neither to leave out potential confounders from the regression model nor overfit the model with mutually correlated markers could not be solved
BRAF, and KRAS play in it, had been elucidated. Besides the prognostic implication of CIMP among CRC patients, an even more important question was, should CIMP status influence the decision in giving adjuvant chemotherapy to CRC patients? According to National Comprehensive Cancer Network (NCCN) treatment guideline for colon cancer Version 3.201352, neither the use of adjuvant chemotherapy outside of clinical trials for stage II (T2N0M0) colon cancer patients nor the use of multi-gene assay in the decision-making process was recommended. However, if we were able to identify a subgroup of stage II tumors that were more susceptible to 5-FU, we could delay disease recurrence with adjuvant chemotherapy in this subgroup. Three out of four studies in our review showed that patients with CIMP experienced significantly longer DFS with the addition of adjuvant chemotherapy compared to receiving surgery alone. On the other hand, four out of five studies in our review showed that patients without CIMP had equivalent outcomes whether they received adjuvant chemotherapy or not (Table 4). According to Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK)53, the recommended summary statistic for evaluating the interaction of treatment and a binary (or categorical) marker like CIMP was the
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unless the pathway between CIMP and survival or treatment response, along with the roles MSI,
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interaction term. Of the seven studies in our review, interaction term was available in only one study16, thus we were unable to perform analysis beyond descriptive summary across studies for evaluation of the impact of CIMP on treatment response. CIMP’s value as a predictive factor in assessing whether adjuvant 5-FU therapy will confer additional survival benefit to CRC patients remained to be determined through future prospective randomized studies. The main limitations of our review were the clinical and methodological heterogeneity
patients with inflammatory bowel diseases, the distribution of cancer stage, treatments received, and genetic profiles varied between studies. In terms of the methodological heterogeneity, besides an inconsistency in CIMP definition, the length and completeness of follow-up, and the confounders assessed and adjusted for were also different from study to study. In this review, we have elected to use DFS and RFS synonymously, in order to include the maximal number of relevant clinical studies, acknowledging that we risk the bias that could be introduced through the small percentage of patients with second primary cancers. In summary, this is the first comprehensive qualitative and quantitative synthesis of the currently available literature regarding CIMP’s prognostic value among CRC patients. Our results showed significantly worse DFS and OS among CRC patients with CIMP compared to those without CIMP, and a potential survival benefit in CRC patients with CIMP who received adjuvant chemotherapy in comparison with those who received surgery alone. However, future research into the most appropriate operational definition of CIMP and the mechanism by which CIMP influenced disease prognosis would be of vital importance in helping us put past study results into proper perspective.
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across the included studies. While most studies excluded hereditary colorectal cancers and
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DISCLOSURE The authors have declared no conflicts of interest. FUNDING This paper was supported by grants from National Cancer Institute K23 CA127141 (NA), the Downloaded from http://annonc.oxfordjournals.org/ by guest on September 30, 2014
American College of Surgeons/ Society of University Surgeons (NA).
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38. Kim JH, Shin SH, Kwon HJ, Cho NY, Kang GH. Prognostic implications of CpG island hypermethylator phenotype in colorectal cancers. Virchows Arch 2009;455:485‐94. 39. Ju HX, An B, Okamoto Y, et al. Distinct profiles of epigenetic evolution between colorectal cancers with and without metastasis. Am J Pathol;178:1835‐46. 40. Kakar S, Deng G, Smyrk TC, Cun L, Sahai V, Kim YS. Loss of heterozygosity, aberrant methylation, BRAF mutation and KRAS mutation in colorectal signet ring cell carcinoma. Mod Pathol;25:1040‐7. 41. Simons CC, Hughes LA, Smits KM, et al. A novel classification of colorectal tumors based on microsatellite instability, the CpG island methylator phenotype and chromosomal instability: implications for prognosis. Ann Oncol. 42. Sanchez JA, Krumroy L, Plummer S, et al. Genetic and epigenetic classifications define clinical phenotypes and determine patient outcomes in colorectal cancer. Br J Surg 2009;96:1196‐204. 43. Kalady MF, Sanchez JA, Manilich E, Hammel J, Casey G, Church JM. Divergent oncogenic changes influence survival differences between colon and rectal adenocarcinomas. Dis Colon Rectum 2009;52:1039‐45. 44. Kim JC, Choi JS, Roh SA, Cho DH, Kim TW, Kim YS. Promoter methylation of specific genes is associated with the phenotype and progression of colorectal adenocarcinomas. Ann Surg Oncol;17:1767‐76. 45. Zlobec I, Bihl MP, Foerster A, Rufle A, Terracciano L, Lugli A. Stratification and Prognostic Relevance of Jass's Molecular Classification of Colorectal Cancer. Front Oncol;2:7. 46. Jo PJ, K.; Grade, M.; Conradi, L. C.; Wolff, H. A.; Ruschoff, J.; Schneider‐Stock, R.; Gaedcke, J.; Becker, H.; Ghadimi, B. M. CpG island methylator phenotype infers a poor prognosis in locally advanced rectal cancer. Langenbeck's Archives of Surgery;396:905. 47. Nam Jun Kim KJK, Byung‐Hoon Min, Kyoung‐Mee Kim, Jin Tong Kim, Dong Kyung Chang, Jae J. Kim, Jong Chui Rhee, Young‐Ho Kim. Sa1579 The Role of CpG Island Methylator Phenotype on Survival Outcome in Colon Cancer. Gastrointestinal Endoscopy 2011;73:AB213‐AB4. 48. Hughes LA, Khalid‐de Bakker CA, Smits KM, et al. The CpG island methylator phenotype in colorectal cancer: progress and problems. Biochim Biophys Acta;1825:77‐85. 49. Des Guetz G, Schischmanoff O, Nicolas P, Perret GY, Morere JF, Uzzan B. Does microsatellite instability predict the efficacy of adjuvant chemotherapy in colorectal cancer? A systematic review with meta‐analysis. Eur J Cancer 2009;45:1890‐6. 50. Cushman‐Vokoun AM, Stover DG, Zhao Z, Koehler EA, Berlin JD, Vnencak‐Jones CL. Clinical Utility of KRAS and BRAF Mutations in a Cohort of Patients With Colorectal Neoplasms Submitted for Microsatellite Instability Testing. Clin Colorectal Cancer. 51. Suppiah A, Alabi A, Madden L, Hartley JE, Monson JR, Greenman J. Anti‐p53 autoantibody in colorectal cancer: prognostic significance in long‐term follow‐up. Int J Colorectal Dis 2008;23:595‐600. 52. Benson AB, 3rd, Bekaii‐Saab T, Chan E, et al. Localized Colon Cancer, Version 3.2013. J Natl Compr Canc Netw;11:519‐28. 53. Douglas G. Altman LMM, Willi Sauerbreu, Sheila E. Taube. Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK): Explanation and Elaboration. PLoS Medicine 2012;9.
Figure 1: Study identification flowchart Legend: Using standardized protocol for a comprehensive search through 4 electronic databases, a total of 33 studies were included in this review for qualitative or quantitative analysis.
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Figure 2: Forest plots of hazard ratios (HRs) of Disease Free Survival (DFS) in studies of CRC patients associated with CIMP with no restriction to MSI status. Legend: CIMP is associated with a worse Disease Free Survival among CRC patients after adjusting for other prognostic factors.
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Figure 3: Forest plots of hazard ratios (HRs) of Overall Survival (OS) in studies of CRC patients associated with CIMP with no restriction to MSI status. Legend: CIMP is associated with a worse Overall Survival among CRC patients after adjusting for other prognostic factors.
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Figure 5: Forest plots of hazard ratios (HRs) of Overall Survival (OS) in studies of Microsatellite Stable (MSS) CRC patients associated with CIMP. Legend: CIMP is associated with a worse Overall Survival after adjusting for other prognostic factors among a subgroup of CRC patients with Microsatellite Stability (MSS).
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Table 1: Study eligibility criteria table Study Eligibility Criteria CIMP Definition Widespread CpG island methylation as defined by each study (no restriction regarding laboratory method, gene panel, or marker threshold value) Outcome Measure Disease-Free Survival, Overall Survival, or Cancer-Specific Mortality Study Design Prospective or retrospective cohort studies Anatomical Site Colon, Rectal, or Colorectal Study tissue Surgically resected primary tumor tissue Stage Any Therapy Any Length of Follow-up Any Downloaded from http://annonc.oxfordjournals.org/ by guest on September 30, 2014
Table 2: Summary of Included Study Characteristics Study Patient population characteristics Publication type
Country
Korea
Sample size (# patients) ,N 161
Ahn et al. 201110
Full Publication
III
Barault et al. 200811
Full Publication
France
582
Dahlin et al. 201012
Full publication
Sweden
Donada et al. 201328
Full Publication
Italy
Han et al. 201333
Full Publication
Korea
Jo et al. 201152
Full publication
Germany
Male Gend er, N (%) 93 (57.8 %)
Median Age, y/o (range) Mean 60.9 (31-84)
I-IV
333 (57.2 %)
604
I-IV
315 (52.2 %)
65y/ o: N=156/ 6675y/o: N=184/ >75y/o: N=242 69.9 (58-79)
120
II
57 (47.5 %)
322
150
Stage
II-III
NR
Inclusion Criteria
Specimen preservation
Gene panel
Lab method
CIMP classification
Marker Threshold
CIMP prevalence, N (%)
Sporadic colon CA
Cryopreservation
Bisulfite pyrosequen cing
CIMP -/+
CIMP+ 3/9 (plus additional genetic criteria)
29 (18.0%)
DFS
Sporadic colon CA without IBD
Cryopreservation
MINT1, MINT2, MINT31, hMLH1, p16, p14, SFRP1, SFRP2, WNT5A Classic panel
MSP
CIMP 0/low/high
CIMP high: 4-5/5 CIMP low: 1-3/5
CIMP high: 97 (16.7%) CIMP low: 199 (34.2%) CIMP 0: 286 (49.1%)
OS
Sporadic colorecta l CA
FFPE
MethyLight
CIMP 0/low/high
CIMP high: 6-8/8 CIMP low: 1-5/8
CIMP high: 74 (12.3%) CIMP low: 215 (35.6%) CIMP 0: 301 (49.8%)
Cancerspecific mortalit y
Mean 67.6
Colon CA
FFPE
MSP
CIMP 0/low/high
CIMP high:
DFS, OS
192 (59.6 %)
61.0 (30-78)
NR
CIMP 0/low/high
CIMP high:
107 (71% )
Mean 62.4
Sporadic colorecta l CA cases with complete resection and complete FOLFO X adjuvant treatment among stage II&III cases Locally advanced rectal CA cases
CIMP high: 22 (18.3%) CIMP low: 36 (30.0%) CIMP 0: 62 (51.7%) CIMP high: 25 (7.8%) CIMP low: 125 (38.8%) CIMP 0: 172 (53.4%)
CIMP -/+
CIMP+:
15 (10%)
DFS, OS
FFPE
CDKN2A, MLH1, CACNA1G, NEUROG1, RUNX3, SOCS1, IGF2, CRABP1 Weisenberger
CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, CDKN2A(p16 ), RUNX3, SOCS1
MethyLight
Weisenberger
MSP
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Study
Outcomes
CIMP assessment
3/5 CIMP low: 1-2/5
5/8 CIMP low: 1-4/8
3/5
DFS
Jover et al. 201120
Full publication
Spain
Ju et al. 201161
Full publication
Japan
Kakar et al. 200862
Full Publication
USA
Kakar et al. 201263
Full Publication
USA
Kalady et al. 200949
Full publication
USA
Kim et al. 200944
Full publication
Korea
Kim et al. 201050
Full publication
Korea
302
78
69
33
357
320
285
I-IV
I-IV
I-IV
I-IV
I-IV
I-IV
I-IV
185 (61.3 %)
Mean 70.3
51 (65.4 %) 39 (56.5 %)
Mean 63.9 =60y/o : 50
24 (72.7 %)
60y/o: 13
193 (54.1 %) 187 (58.4 %)
Mean 66.9
168 (58.9 %)
Mean 58.0
Lee et al.200814
Full Publication
Korea
81 (60.4 %)
Min et al. 201140
Full publication
Korea
245
I-IV
138 (56.3 %)
Ogino et al. 200638
Full publication
USA
31
IV
22 (71.0 %)
Mean 60.9
=60 y/o: 77 Mean 65.0 (33-83)
57.0 (31-81)
Sporadic colorecta l CA Sporadic colorecta l CA
FFPE
Cryopreservation
CACNAG1, SOCS1, NEUROG1, RUNX3, MLH1 Classic panel
Bisulfite pyrosequen cing
CIMP -/+
MSP
CIMP -/+
CIMP+:
89 (29.5%)
DFS
19 (24.4%)
DFS
16 (23.2%)
OS
16 (48.5%)
OS
78 (21.8%)
DFS
37 (11.6%)
OS
102 (35.8%) or 51 (17.9%)
DFS
42 (31.3%)
OS
3/5
CIMP+: 2/5
FFPE
hMLH1, p16, HIC1, RASSF2, ID4, MINT1, MINT31 hMLH1, p16, HIC1, RASSF2, ID4, MINT1, MINT31
MSP
Weisenberger
MethyLight
CIMP -/+
CIMP+: 3/7
Signet ring cell colorecta l carcinom a Sporadic colorecta l CA Sporadic colorecta l CA cases without neoadjuv ant therapy Sporadic colorecta l CA cases without neoadjuv ant therapy Sporadic colorecta l CA
FFPE
Sporadic colorecta l CA cases without neoadjuv ant therapy Advance d MSS colorecta
FFPE
Weisenberger
MethyLight
CIMP 0/low/high
CIMP high: >2/5 CIMP low: 1-2/5
CIMP high 34 (13.9%)
DFS
FFPE
CACNA1G, CDKN2A, CRABP1,
MethyLight
CIMP 0/low/high
CIMP high: >8/13 CIMP low:
CIMP high: 3 (10.0%) CIMP low:
OS
Cryopreservation
MSP
CIMP -/+
CIMP+: 3/7
CIMP -/+
CIMP+: 3/5
FFPE
NR
FFPE
CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, SOCS1 MLH1, MINT1, MINT2, MINT31, p16, p14, CACNA1G
MethyLight
Classic panel
MSP
CIMP -/+
CIMP+: 5/8
Bisulfite Pyrosequen cing
CIMP -/+
CIMP+: 2/7 or 3/7
CIMP -/+
CIMP+: 2/5
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134
I-IV
without preoperat ive chemoth erapy Sporadic colorecta l CA
l CA cases without metastas es
IGF2, MLH1, NEUROG1, RUNX3, SOCS1, MINT1, MINT31, IGFBP3, MGMT, WRN CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, SOCS1 CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, SOCS1 P16, MDR1, MINT2
Ogino et al. 200915
Full publication
USA
649
I-IV
283 (44% )
Mean 66.5
Primary colon CA cases
FFPE
Rhee et al. 201242
Full publication
Korea
207
I-IV
126 (60.9 %)
55y/o: 107
microsat ellite unstable colorecta l cancers
FFPE
Rijnsoeve r et al. 200234
Full publication
Australia
138 (50.2 %)
Colorect al CA
FFPE
Rijnsoeve r et al. 2003 19 Samowitz et al. 200529
Full publication
Australia
144 =71y/o Mean 60.9
Colorect al CA
83% FFPE; 17% cryopreservation
P16, MINT2, MDR1
MSP
Full Publication
USA
Classic panel
MSP
Full publication
USA
Sanchez et al. 200948
Full Publication
USA
Shen et al. 20079
Full publication
USA
Primary sporadic colon CA cases between 30~79 y/o without IBD Primary sporadic rectal CA cases between 30-79 y/o without IBD Colorect al CA cases without IBD Colorect al CA cases
FFPE
Samowitz et al. 200932
5/8 CIMP low: 1-5/8
CIMP high: 126 (19.4%) CIMP low: 252 (38.8%) CIMP 0: 271 (41.8%)
OS, cancerspecific mortalit y
MethyLight
CIMP 0/low/high
CIMP high:
NR
OS
CIMP -/+
CIMP+:
128 (46.5%)
OS
67 (32.5%)
DFS
246 (27.8%)
OS
103 (13.5%)
OS
83 (21.2%)
DFS, OS
28 (15.4%)
OS
95 (18.7%)
Cancerspecific
MSP
5/8 CIMP low: 1-4/8
2/3 CIMP -/+
CIMP+: 2/3
MSP
MethyLight
CIMP low/high
CIMP high:
CIMP low/high
CIMP high:
CIMP -/high
CIMP high:
2/5
2/5
3/5
FFPE
FFPE
MINT1, MINT2, MINT31, hMLH1, p14, p16 Weisenberger
MSP, COBRA
CIMP -/+
MSP
CIMP -/+
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275
1-8/13
CIMP+: 2/6
CIMP+:
%)
cases
Ward et al. 200313
Full publication
Australia
605
I-IV
323 (53.4 %)
Mean 68.3 (29-99)
Zlobec et al. 201151
Full publication
Switzerla nd
337
NR
156 (46.3 %)
Mean 69.9 (42-95)
Zanutto et al. 201135
Letter to the editor
Italy
42
II
NR
AlSohaily et al. 201130 Elsaleh et al. 200336 Kim et al. 201153
Conference Abstract
Australia
281
NR
NR
Conference Abstract Conference Abstract
USA
891
III
NR
NR
Korea
151
NR
NR
NR
Kim et al. 201239
Conference abstract
Korea
49
NR
NR
NR
Koo et al. 201137
Conference Abstract
Korea
191
III
NR
NR
3/5
mortalit y OS
Sporadic colorecta l CA cases without IBD Sporadic colorecta l CA
Cryopreservation
P16, MINT1, MINT2, MINT12, MINT31
MSP
CIMP -/+
CIMP+: >3/5
NR
FFPE
CRABP1, MLH1, p16INK4a, CACNA1G, NEUROG1
Bisulfite pyrosequen cing
CIMP 0/low/high
CIMP high:
66.1 (30-83)
MSS colorecta l CA
Cryopreservation
Classic panel
MSP
Methylation low / intermediate/ high
Methylation high: >3/5 Intermediat e 3/5
DFS
NR
Rectal CA
FFPE
Weisenberger
MSP
CIMP 0/low/high
CIMP high
CIMP high: 24 (7.1%) CIMP low: 145 (43.0%) CIMP 0: 168 (49.9%) High: 13 (31.0%) Intermediate : 15 (35.7%) Low: 14 (33.3%) 13 (4.6%)
Colorect al CA Colorect al CA
NR
NR
NR
NR
NR
NR
DFS
NR
Weisenberger
NR
CIMP -/+
CIMP+:
27 (17.9%)
OS
Colorect al CA cases with metastas es treated by 5-FU, leucovori n, irinoteca n, and cetuxima b Colorect al CA
FFPE
P16, p14, MINT1, MINT2, MINT31, hMLH1
Bisulfite Pyrosequen cing
CIMP -/+
NR
14 (28.6%)
DFS, OS
NR
Weisenberger
NR
CIMP-/+
CIMP+:
63 (33.0%)
DFS
4/5 CIMP low: 1-3/5
Cancerspecific mortalit y
OS
3/5
3/5
NR: Not reported; MSP: Methylation-Specific Polymerase Chain Reaction Classic panel: MINT1, MINT2, MINT31, CKKN2A(p16), hMLH1 Weisenberger: CACNA1G, IGF2, NEUROG1, RUNX3, SOCS
3/5
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Table 3: Risk of Bias of Included Studies
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Red circles represent studies with high risk of bias; green circles represent studies with low risk of bias; yellow circles represent studies that did not provide sufficient information for assessing risk of bias.
Table 4: Summary of study conclusions regarding interaction of CIMP and adjuvant chemotherapy Study
Study Cohort
Chemotherapy regimen
DFS among patients receiving surgery & adjuvant CT NR
Elsaleh et al. 200336
891 stage III CRC patients
5-FU-based chemotherapy
Min et al. 201140
124 stage II and III CRC patients
5-FU or capecitabine, leucovorin (Mayo regimen) 5-FU, leucovorin (Mayo regimen)
CIMP-H ~ CIMP-L/0
CIMP-H ~ CIMPL/0 (p=0.073).
Rijnsoever et al. 200319
206 stage III CRC patients
CIMP+ < CIMP(p=0.05)
CIMP+ > CIMP-
Donada et al. 201328
120 stage II colon cancer
5-FU, leucovorin
CIMP-H ~ CIMP-L/0 (p=0.6)
CIMP-H > CIMPL/0 (p=0.05)
Jover et al. 201120
196 stage II and III CRC patients,
5-FU-based chemotherapy
CIMP+ < CIMP(p=0.03)
CIMP+ ~ CIMP(p=0.1)
Han et al. 201233
322 stage II and III CRC who received surgery + CT 49 metastatic CRC cases who received CT
FOLFOX (5-FU, oxaliplatin, folinic acid)
NR
CIMP-H ~ CIMPL/0 (p=0.31)
FOLFIRI (5-FU, leucovorin, iriontecan) and cetuximab
NR
CIMP+ ~ CIMP-
Kim et al. 201239
NR: Not reported; CT: Chemotherapy; 5-FU: 5-fluorouracil CIMP-H=CIMP-high; CIMP-L=CIMP-low; CIMP-0=CIMP-zero
DFS among CIMP (+) patients
DFS among CIMP (-) patients
Surgery+CT > surgery alone (p=0.03) Surgery+CT > surgery alone (p=0.022)
Surgery+CT ~ surgery alone
Surgery+CT > surgery alone (p=0.002) NR
Surgery+CT ~ surgery alone
Surgery+CT ~ surgery alone (p=0.6) NR
Surgery+CT ~ surgery alone (p=0.6) Surgery+CT ~ surgery alone for CIMP-0, CIMPlow patients (p=0.7, p=0.9) surgery+CT > surgery alone (p=0.0001) NR
NR
NR
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DFS among patients receiving surgery alone NR
