CpG islands are DNA segments of at least 0.5 kb in size,

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1 Four Molecular Subtypes of Colorectal Cancer and Their Precursor Lesions Gyeong Hoon Kang, MD N Context. In addition to chromosomal instability and microsatellite instability (MSI), a third pathway, epigenetic instability, has been implicated in progression to colorectal carcinogenesis. CpG island methylator phenotype (CIMP) refers to a subset of colorectal cancers (CRCs) that occur through the epigenetic instability pathway and that are characterized by widespread hypermethylation of promoter CpG island loci, resulting in the inactivation of several tumor suppressor genes or tumor-related genes. Colorectal cancers can be classified into 4 molecular subtypes according to their CIMP and MSI statuses: CIMP+/MSI+, CIMP+/MSI2, CIMP2/MSI+, and CIMP2/MSI2. There are differences between Western (United States and European Union) and Eastern (Korea and China) populations in the number of CRCs that are MSI+, and in the number of MSI+ CRCs that are CIMP+. Objective. To review the clinicopathologic and molecular features of the 4 molecular subtypes of CRCs and their precursor lesions, and to emphasize geographic differences in CRCs between Eastern and Western populations. Data Sources. This article is based on the author s own experimental data and a literature review of relevant articles indexed in PubMed (US National Library of Medicine). Conclusion. The 4 molecular subtypes of CRC that are defined by their CIMP and MSI statuses are characterized by their own distinct clinicopathologic and molecular features and precursor lesions. In particular, the clinicopathologic features of MSI+ CRCs differ depending on the CIMP status. Further understanding of the heterogeneity in CRC molecular pathways may help to explain the diverse morphologic features of CRCs. (Arch Pathol Lab Med. 2011;135: ) CpG islands are DNA segments of at least 0.5 kb in size, with a high G:C ratio and CpG content, and are often located in the promoters or 59-exon sequences of genes. 1 Approximately 70% of human genes contain CpG islands in their promoter or 59-exon sequences. 2 More than 90% of CpG islands within promoters have generally been thought to be unmethylated in normal cells, except for those on the inactive X chromosome and those associated with imprinted genes, which are methylated. 3 Hypermethylation of CpG islands within promoters can occur in association with the development of cancer or aging, although the cause is unclear. Hypermethylation of CpG islands leads to the recruitment of methyl-dna binding proteins, and subsequently histone deacetylases. Deacetylation and methylation of the histone tails transforms the chromatin structure of the promoter into a closed structure, leaving it inaccessible to transcription factors and resulting in gene inactivation. 4,5 Hypermethylation of Accepted for publication December 8, From the Department of Pathology and Cancer Research Institute, Brain Korea 2nd Stage, Seoul National University College of Medicine, Seoul, Korea. The author has no relevant financial interest in the products or companies described in this article. Presented at the 9th Spring Seminar of the Korean Pathologists Association of North America; March 18 20, 2010; Washington, DC; in conjunction with the 99th Annual Meeting of the United States and Canadian Academy of Pathology. Reprints: Gyeong Hoon Kang, MD, Department of Pathology, Seoul National University College of Medicine, 28 Yongon-dong, Chongnogu, Seoul , South Korea ( ghkang@snu.ac.kr). promoter CpG islands is thought to act as an alternative mechanism to genetic mutation for the inactivation of tumor suppressor genes in human cancers. Virtually all types of human cancer display hypermethylation of promoter CpG islands, although the prevalence of such hypermethylation varies among tissue types. 6 8 In addition to its potential role in gene inactivation in human cancers, CpG island hypermethylation is now gaining attention as a molecular marker for tumor detection and prediction of clinical outcomes in cancer patients. THREE MOLECULAR PATHWAYS OF COLORECTAL CARCINOGENESIS Since the initial demonstration by Issa et al 9 that inactivation of the estrogen receptor gene in colorectal cancer (CRC) is associated with promoter CpG island hypermethylation, more than 100 genes have been shown to be inactivated by promoter CpG island hypermethylation in CRCs. 10,11 Recent articles 10,12 have demonstrated that the number of genes inactivated by promoter CpG island hypermethylation in CRC is greater than the number of genes that have undergone direct mutation. Accumulating evidence indicates that CRC has substantial genetic heterogeneity, and that CRC actually comprises several different genetic diseases all affecting the same organ. 13 Three mechanisms that increase the diversity of gene expression have been identified in CRC: microsatellite instability (MSI), chromosomal instability (CIN), and CpG island methylator phenotype (CIMP). Microsatellite instability occurs in 6% to 15% of CRCs and is caused by 698 Arch Pathol Lab Med Vol 135, June 2011 Molecular Subtypes of Colorectal Cancer Kang

2 inactivation of DNA mismatch repair genes Chromosomal instability is present in more than 50% of CRCs and is characterized by aneuploidy and frequent loss of heterozygosity, which facilitates the sequential inactivation of APC, DCC/SMAD4, and TP Up to 20% of CRCs demonstrate CIMP, which refers to the widespread hypermethylation of CpG island loci. 21,22 The original studies on CIMP in CRC identified associations with MSI, increased KRAS mutations, decreased TP53 mutations, proximal colon location, and increased age. 23,24 However, inconsistent results have been obtained concerning the association between CIMP and female preponderance or KRAS mutation, depending on the type of CIMP marker panel that was used to define CIMP FOUR MOLECULAR SUBTYPES OF COLORECTAL CANCER Colorectal cancers can be classified by global genomic/ epigenomic aberrations, including CIN, MSI, and CIMP. Classification based on CIN has many problems because the methods/markers and criteria currently used for analysis of CIN are not uniform. Because MSI and CIMP are relatively well defined when compared with CIN, CRCs are usually classified into 4 molecular subtypes on the basis of both CIMP and MSI statuses: CIMP+/MSI2 (5% 10%), CIMP2/MSI2 (75% 80%), CIMP+/MSI+ (10%), and CIMP2/MSI+ (5%). The frequency of each subtype among CRCs (shown in parentheses) is an approximate value obtained from CRCs in an American population, as obtained by Ogino and Goel. 22 We have obtained a largely similar distribution in our study of CRCs in a Korean population. The distributions were as follows: CIMP+/MSI2 (8%), CIMP2/MSI2 (79%), CIMP+/MSI+ (5%), and CIMP2/MSI+ (8%). 29 The ratio of the CIMP+/MSI+ subtype to the CIMP2/MSI+ subtype differs substantially between Western (2:1 ratio) and Korean populations (ratio ranging from 1:1.6 to 1:2.3). 22,30 This discrepancy does not appear to be related to differences in CIMP markers or the methodologies used in the 2 studies, as we (the Korean studies) used 3 different CIMP marker panels (Issa s classic 5-marker panel, 31 Laird s new 5-marker panel, 32 and Ogino s updated 8-marker panel 33 ) and methylation-specific polymerase chain reaction and MethyLight technology. 28,34 PROGNOSTIC IMPLICATIONS OF CIMP Of the 4 molecular subtypes, CIMP+/MSI2 subtype is associated with the worst clinical outcome, a finding that is similar in both Eastern and Western populations. 28,35 37 In our previous study on MSI2 tumors, 28 those that were both CIMP+ and mutated at KRAS/BRAF were associated with the worst clinical outcome, and there were no differences in the clinical outcomes for tumors that were either CIMP+ or mutated at KRAS/BRAF, or neither. This result indicates that the poor clinical outcome observed in patients with CIMP+/MSI2 tumors is related to the presence of coexisting mutations in KRAS or BRAF. Ward et al 35 and Hawkins et al 38 previously demonstrated that increasing levels of tumor methylation were significantly associated with decreased survival for patients with MSI2 tumors, and MSI2 tumors with methylation at 4 or more of the examined 5 CIMP markers were associated with decreased survival, compared with tumors with methylation at 1 to 3 markers or with no methylation. However, our previous study, 34 in which we determined CIMP status by using 2 different 5-marker panels and methylation-specific polymerase chain reaction, demonstrated a different pattern of survival in relation to methylation. MSI2 tumors with methylation at 1 to 3 markers tended to correlate with a worse clinical outcome than MSI2 tumors with no methylation, but a better clinical outcome than MSI2 tumors with methylation at 4 to 5 markers. Highly methylated, MSI2 CRCs were associated with an early rapid drop in postoperative survival, with prolonged survival in the remainder. This biphasic survival pattern raises the possibility that there may be 2 distinct subgroups within the highly methylated, MSI2 CRCs. Exploration of the molecular alterations revealed that the worse-outcome subgroup had a high frequency of KRAS/ BRAF mutations, whereas the better-outcome subgroup did not have KRAS/BRAF mutations. 34 SUPERIORITY OF AN 8-MARKER PANEL IN METHYLIGHT-BASED DIAGNOSIS OF CIMP Three different marker panels are available for Methy- Light-based diagnosis of CIMP, 2 of which contain 5 markers, 31,32 and 1 of which contains 8 markers. 33 It is not yet clear which panel is optimal for CIMP diagnosis. We screened these 3 marker panels against 196 CRC cases and determined CIMP status by using two 5-marker panels and the 8-marker panel. For the 5-marker panels, CRC cases were considered CIMP+ if at least 3 of the markers were methylated; for the 8-marker panel, cutoff values of 5 and 6 markers were each tested for CIMP. Differently defined CIMP+ CRCs were compared by their associations with previously known clinicopathologic features of CIMP+ CRCs. Overall, the 8-marker panel with a cutoff value of 5 outperformed both of the 5-marker panels and the 8-marker panel with a cutoff value of 6, in all comparisons except for an association with old age. These results suggest that the 8-marker panel, with a cutoff value of 5, is best for CIMP diagnosis. 29 The combination of MSI status with CIMP status defined by the 8-marker panel classifies CRCs into 4 molecular subtypes, all of which have their own distinct clinicopathologic and molecular features (Table). DISCREPANCY IN THE PROPORTIONS OF CIMP IN MSI+ CRCS BETWEEN CRCS IN WESTERN AND EASTERN POPULATIONS The prevalence of MSI+ in CRCs in Western populations is approximately 15%, but tends to be lower in Korean populations (7% in 2800 consecutive cases of surgically resected CRC; G.H.K., unpublished data). Notably, the proportion of CIMP+ among MSI+ CRCs is different between the 2 populations. For CRCs in Western populations, approximately two-thirds of sporadic MSI+ CRCs are CIMP+, a result that is consistently obtained regardless of which CIMP panel markers and methylation analysis technologies are used. 15,22,30 In contrast, approximately one-third of MSI+ CRCs are CIMP+ in CRCs in Korean populations, which is a consistent finding regardless of the type of CIMP marker panel or methylation analysis used. 28,34 Environmental or ethnic differences, for example, diet, between Korean and Western populations may account for this discrepancy. Because the prevalence of hereditary nonpolyposis colon cancer is approximately 3% and similar in Eastern (Chinese and Japanese) and Western (European and Arch Pathol Lab Med Vol 135, June 2011 Molecular Subtypes of Colorectal Cancer Kang 699

3 Clinicopathologic and Molecular Features of 4 Molecular Subtypes of Colorectal Cancer Based on Microsatellite Instabilty (MSI) and CpG Island Methylator Phenotype (CIMP) Status a CIMP2/MSI2 (79%) CIMP2/MSI+ (8%) CIMP+/MSI2 (8%) CIMP+/MSI+ (5%) P Value Age, y Female, % Right colon, % ,.001 Differentiation (PD), % ,.001 Higher stage, % ,.001 Metastasis, % LN metastasis, % ,.001 BRAF mutation, % ,.001 KRAS mutation, % Abbreviations: LN, lymph node; PD, poorly differentiated. a A total of 197 cases of colorectal cancer were analyzed. American) patients with CRC, it is plausible that sporadic MSI+ CRCs which are believed to be due to CIMP-related silencing of the mismatch repair gene MLH1 account for the large difference in the proportion of MSI+ CRCs between CRCs in Western and Eastern populations. BRAF MUTATION IS CLOSELY ASSOCIATED WITH CIMP BUT NOT WITH MSI Although some controversy exists regarding the association of CIMP with KRAS mutations, 32,43 mutations in BRAF have been consistently associated with CIMP. Figure 1. Comparison of clinicopathologic and molecular features of CpG island methylator phenotype negative/microsatellite instability positive (CIMP2/MSI+) colorectal cancers (CRCs) (n 5 54) and CIMP+/MSI+ CRCs (n 5 18). The MSI+ CRCs (n 5 72) were retrieved from a consecutive series of 989 patients who underwent curative surgery for CRCs in Seoul National University Hospital, Seoul, Korea, from 2004 to a, The average age of patients with CIMP+/MSI+ CRC was significantly higher than that of patients with CIMP2/MSI+ CRC (Student t test, P,.001). b, The number of tumorinfiltrating CD8 + lymphocytes per high-power field (HPF) was significantly higher in CIMP+/ MSI+ CRCs than in CIMP2/MSI+ CRCs (Student t test, P 5.05). c, A comparison of parameters, such as proximal colon location, polypoid gross morphology, and advanced cancer stage, with marginal differences indicated by an asterisk (*) (P value between.05 and.09) and significant differences indicated by a double asterisk (**) (P,.05) (2-sided Fisher exact test). MLH1 and MSH2, DNA mismatch repair genes. 700 Arch Pathol Lab Med Vol 135, June 2011 Molecular Subtypes of Colorectal Cancer Kang

4 mutations in MSI+ sporadic CRCs. However, recent studies 33,50 provide conflicting evidence on the association between BRAF mutations and MSI. Microsatellite instability status does not correlate with the frequency of BRAF mutations, although the BRAF mutation is positively correlated with CIMP, independent of MSI status. Figure 2. Survival analysis (Kaplan-Meier log-rank test) was performed for 72 microsatellite instability positive (MSI+) colorectal cancers (CRCs). Solid line indicates CpG island methylator phenotype negative (CIMP2)/MSI+ CRCs and dotted line indicates CIMP+/MSI+ CRCs. Although the prevalence rate of CIMP+ CRCs in Eastern populations is only marginally lower than in Western populations, 14,29,32,44 the BRAF mutation rate is lower in CRCs from Korean than Western populations (4.4% versus 9% 17%). 45 A review of the literature 14,25,45 47 indicates that the rates of BRAF mutations are lower in CRCs from Asian populations than US or European populations (4.2% 5.1% versus 9.5% 17%, respectively). In our previous study, % (2 of 19) of MSI+ sporadic CRCs had BRAF mutations, although other studies 48,49 have reported substantially higher occurrences of BRAF CIMP STATUS RELATED DIFFERENCES IN THE CLINICOPATHOLOGIC FEATURES OF MSI+ CRCS MSI+ CRCs have distinct clinicopathologic features compared with MSI2 CRCs: proximal location, marked mucin production, lower tumor stage, larger tumor size, and reduced frequency of nodal or liver metastasis. However, MSI+ tumors are heterogeneous and can be categorized as CIMP+/MSI+ and CIMP2/MSI+. CIMP+/ MSI+ tumors undergo gene inactivation by CIMP, in addition to those inactivated by MSI, which are shared by both subsets of MSI+ CRCs. 51,52 We speculated that the difference in the repertoire of inactivated genes between CIMP+/MSI+ and CIMP2/MSI+ CRCs would be reflected in phenotypic differences of the tumors. To test this hypothesis, we compared the clinicopathologic and molecular features of CIMP+/MSI+ and CIMP /MSI+ tumors and found that features such as old age, poor differentiation, tumor-infiltrating lymphocytes, and the BRAF mutation were significantly more frequent in CIMP+/MSI+ tumors than in CIMP2/MSI+ tumors. Proximal location and advanced tumor stages were also more frequent in CIMP+/MSI+ than in CIMP /MSI+ tumors; however, these features only just reached statistical significance, indicating that they will have to be confirmed in future studies (Figure 1). A polypoid tumor without ulceration (Borrmann type I) was only found in CIMP2/MSI+ tumors. Of note, the molecular difference between these 2 CRC subtypes was associated with critical differences in clinical outcome, since the survival rate after surgery for patients with CIMP+/MSI+ CRC was significantly lower than for patients with CIMP2/MSI+ CRC (Figure 2) (manuscript in preparation). Figure 3. a, Colorectal carcinomas with contiguous adenoma on the margin of the carcinoma (presumed carcinoma ex adenoma). Thirty-nine cases of carcinoma were associated with contiguous conventional adenoma and 3 cases of carcinoma were accompanied by serrated adenoma. b, The methylation status of 8 CpG island methylator phenotype panel markers were analyzed in adenoma and carcinoma components. Arch Pathol Lab Med Vol 135, June 2011 Molecular Subtypes of Colorectal Cancer Kang 701

5 Figure 4. Premalignant lesions of 4 molecular subtypes. Abbreviations: CIMP, CpG island methylator phenotype; CIN, chromosomal instability; CRC, colorectal cancer; HNPCC, hereditary nonpolyposis colon cancer; MSI, microsatellite instability. PRECURSOR LESIONS OF 4 MOLECULAR SUBTYPES Almost all CRCs are thought to develop from preexisting adenomas, and several lines of evidence support this adenoma-to-carcinoma sequence. 53,54 However, CIMP+ carcinomas do not arise from conventional adenomas, such as tubular adenoma, tubulo-villous adenoma, and villous adenoma, but instead develop from sessile or traditional serrated adenomas When we analyzed the methylation status of 8 CIMP markers in CRCs with contiguous, conventional adenoma on the margin of the carcinoma lesion, no difference in the number of methylated genes was noted between the adenomatous and carcinomatous lesions, and the number of methylated genes was low (Figure 3). This result indicates that there is no further increase in CpG island hypermethylation during malignant transformation, and that CIMP+ carcinomas do not arise from conventional adenomas. In our surgical data files, we found 3 cases of CRCs with a contiguous serrated adenoma, which were also analyzed for their methylation status in 8 CIMP markers. The serrated adenoma components and the carcinoma components of all 3 cases were positive for CIMP,withnodifferencesinthenumbersofmethylated markers between the different lesion types (Figure 3). Several researchers have reported a high frequency of BRAF mutations, and a low frequency of KRAS mutations, in serrated polyps (sessile serrated adenoma, traditional serrated adenoma, and hyperplastic polyp), and a low frequency of BRAF mutations and a high frequency of KRAS mutations in conventional adenomas. 59,60 This observation provides further evidence to support the hypothesis that serrated polyps are precursor lesions of CIMP+ CRCs, which have a high frequency of BRAF mutations and a low frequency of KRAS mutations. The precursor lesions of the 4 molecular subtypes are summarized in Figure 4. CIMP+/MSI+ CRCs arise from sessile serrated adenomas admixed with dysplasia, and MSI is thought to be a late event in the multistep progression to carcinogenesis in this molecular subtype. 15,55,56,61 In contrast, MSI is an initiating event and driving force for the progression of the lesion in the CIMP2/MSI+ subtype (hereditary nonpolyposis colon cancer). 15,39 For CIMP2/MSI+ CRCs, the precursor lesion is not serrated polyps but conventional adenomas. 39,62,63 CIMP+/MSI2 tumors arise from either sessile serrated adenomas or traditional serrated adenomas. 57,58,64 In conclusion, CRC is a heterogeneous disease with various pathologic features and clinical behaviors, which are associated with abnormalities in 3 different major molecular pathways: CIN, MSI, and CIMP. The 4 molecular subtypes of CRCs, as defined by combinations of their CIMP and MSI statuses, differ with respect to demographics, clinical features, prognosis, response to therapy, repertories of activated or inactivated genes, and histomorphologic features that can be readily recognized by surgical pathologists. The fact that each molecular subtype of CRCs has its own precursor lesion indicates that the molecular pathways are determined at an early evolutional stage and are already fully established within precursor lesions. The role of surgical pathologists now extends beyond furnishing an accurate tissue diagnosis to providing prognostic information and additional findings relevant to patient management. This approach to the molecular classification of CRCs should accelerate understanding of causation, have an impact on clinical management, and facilitate the development of new ways to prevent and treat CRCs. This study was supported by a grant from the Seoul National University Hospital Research Fund ( ), a grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea ( ), and a Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology ( ). References 1. Takai D, Jones PA. Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci U S A. 2002;99(6): Saxonov S, Berg P, Brutlag DL. A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc Natl Acad Sci U S A. 2006;103(5): Eckhardt F, Lewin J, Cortese R, et al. DNA methylation profiling of human chromosomes 6, 20 and 22. Nat Genet. 2006;38(12): Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet. 2002;3(6): Laird PW. Cancer epigenetics. 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