Molecular Subtypes of Colorectal Cancer and Their Clinicopathologic Features, With an Emphasis on the Serrated Neoplasia Pathway

Molecular Subtypes of Colorectal Cancer and Their Clinicopathologic Features, With an Emphasis on the Serrated Neoplasia Pathway Jeong Mo Bae, MD; Jung Ho Kim, MD, PhD; Gyeong Hoon Kang, MD, PhD Context. Colorectal cancer is a heterogeneous disease entity with 3 molecular carcinogenesis pathways and 2 morphologic multistep pathways. Right-sided colon cancers and left-sided colon and rectal cancers exhibit differences in their incidence rates according to geographic region, age, and sex. A linear tendency toward increasing frequencies of microsatellite instability high or CpG island methylator phenotype high cancers in subsites along the bowel from the rectum to the cecum or the ascending colon accounts for the differences in tumor phenotypes associated with these subsites. The molecular subtypes of colorectal cancers exhibit different responses to adjuvant therapy, which might be responsible for differences in subtype-specific survival. Objectives. To review the clinicopathologic and molecular features of the molecular subtypes of colorectal cancer generated by combined CpG island methylator phenotype and microsatellite statuses, to integrate these features with the most recent findings in the context of the prognostic implications of molecular subtypes, and to emphasize the necessity of developing molecular markers that enable the identification of adenocarcinomas involving the serrated neoplasia pathway. Data Sources. Based on the authors own experimental data and a review of the pertinent literature. Conclusions. Because colorectal cancers arise from 2 different morphologic multistep carcinogenesis pathways with varying contributions from 3 different molecular carcinogenesis pathways, colorectal cancer is a heterogeneous and complex disease. Thus, molecular subtyping of colorectal cancers is an important approach to characterizing their heterogeneity with respect to not only prognosis and therapeutic response but also biology and natural history. (Arch Pathol Lab Med. 2016;140:406 412; doi: 10.5858/ arpa.2015-0310-ra) Colorectal cancer (CRC) is the third most common cancer cancers (cancer arising proximal to the splenic flexure) is and the third leading cause of cancer-related death in lower than the proportion of right-sided cancers in areas the United States, regardless of sex. The incidence of CRC with higher incidences of CRC. 5 Within the same geographic has decreased in both men and women in the United States since the mid 1980s. 1 3 In South Korea, there was a rapid increase in the incidence of CRC between 1999 and 2012, and CRC became the second and third most common cancer in males and females, respectively (www.cancer.go. kr; accessed May 1, 2015). The incidence of CRC varies markedly worldwide, with the highest incidence in Oceania area, the incidence of CRC and the proportion of right-sided cancers show age- and sex-related differences. 3,6 Men and older people are at higher risk of CRC than women and younger people, respectively. 7 Women and older people have a higher percentage of right-sided cancers than men and younger people, respectively. The proportion of women with CRC increases as the cancer location becomes more and Europe and the lowest incidence in Africa and South- right-sided, and the proportion of right-sided cancers Central Asia (globocan.iarc.fr; accessed May 1, 2015). increases with age, a characteristic observed in women Recently, high rates of CRC have been reported in newly more than in men. 3,8 These sex- and age-related differences developed countries in which the risk was once low, in the incidence of CRC and the proportion of right-sided including South Korea, Slovakia, and Hungary. 4 In areas cancers are found in both high- and low-incidence with lower incidences of CRC, the proportion of right-sided areas. 3,6,9,10 Furthermore, within the same geographic area, race and ethnicity influence both the incidence of CRC and the proportion of right-sided cancers. 11,12 In the United Accepted for publication September 28, 2015. States, the incidence is lower in Asian American individuals From the Department of Pathology, Seoul National University College of Medicine, Seoul, Korea. than in the white population and other races. African The authors have no relevant financial interest in the products or American individuals have the highest incidence of rightsided companies described in this article. and left-sided colon cancer, compared with the white Presented at the 14th Spring Seminar of the Korean Pathologists population and other races, and the reverse is true for rectal Association of North America (KOPANA); March 19 21, 2015; cancer. 13 In Southern Asia, the Chinese population has a Boston, Massachusetts. Reprints: Gyeong Hoon Kang, MD, PhD, Department of Pathology, significantly higher incidence of CRC than the Indian and Seoul National University College of Medicine, 110-799, Seoul, Malay populations. 5 In addition to differences in the sex and Korea (email: ghkang@snu.ac.kr). age of onset between right-sided and left-sided colon or 406 Arch Pathol Lab Med Vol 140, May 2016 Molecular Subtypes of Colorectal Cancer Bae et al

rectal cancers, the gross or histologic appearance of cancers can vary. The gross appearance of right-sided cancers is often exophytic, whereas that of the left-sided colon or rectal cancers is usually endophytic. Histologic variants, including medullary carcinoma, serrated adenocarcinoma, and cribriform comedo-type adenocarcinoma, are more common in the right-sided colon than in the left-sided colon or rectum. What causes such subsite-related differences in terms of age, sex, race, geography, or morphologic findings? The reasons for these variations may be related not only to genetic or behavioral factors, including diet and lifestyle, but also to differences in the proportions of molecular carcinogenesis pathways that are active in rightsided colon, left-sided colon, and rectal cancers. In this review, we examined (1) the association between molecular carcinogenesis pathways and sex, race, or age of onset; (2) the clinicopathologic and molecular features of CRCs according to molecular subtypes; (3) the prognostic implications of molecular subtypes; and (4) adenocarcinomas arising from the serrated neoplasia pathway and their phenotypic features. THREE MOLECULAR CARCINOGENESIS PATHWAYS AND 2 MORPHOLOGIC MULTISTEP PATHWAYS Colorectal cancer is a heterogeneous disease entity in terms of both molecular carcinogenesis and morphologic multistep pathways. Three molecular carcinogenesis pathways have been identified: (1) chromosomal instability (CIN), (2) microsatellite instability (MSI), and (3) CpG island methylator phenotype (CIMP) or epigenetic instability pathways. The 2 morphologic multistep pathways are the classical pathway (the so-called adenoma-carcinoma sequence) and the serrated neoplasia pathway. The CIN pathway is characterized by alterations in the number and structure of chromosomes and accompanying genetic mutations of proto-oncogenes and tumor suppressor genes. The MSI pathway features alterations in the number of nucleotide repeats located in the exons and subsequent frameshift mutations in tumor suppressor genes or tumor-related genes. The epigenetic instability (or CIMP) pathway is characterized by widespread hypermethylation of numerous promoter CpG island loci and consequent inactivation of tumor suppressor genes or tumor-related genes. The classical pathway (so-called adenoma-carcinoma sequence) begins with premalignant lesions comprising conventional adenomas, including tubular or tubulovillous adenomas, whereas the serrated neoplasia pathway begins with hyperplastic polyps or sessile or traditional serrated adenomas. These 2 morphologic pathways are driven by different molecular pathways: the classical pathway is driven by either CIN or MSI, whereas the serrated neoplasia pathway has epigenetic instability as its initial driving force as an optional secondary force. Although Lynch syndrome CRCs and sporadic MSI-high (MSI-H) CRCs both have a high level of MSI, their premalignant lesions are different because they develop through different morphologic multistep pathways: Lynch syndrome tumors follow the classical pathway and manifest their premalignant lesions as tubular or tubulovillous adenoma, 14,15 whereas the premalignant lesions of sporadic MSI-H CRCs are sessile serrated adenomas that arise through the serrated neoplasia pathway and undergo further hypermethylation-associated inactivation of MLH1 and subsequent acquisition of high level MSI. 16 TheCancerGenomeAtlas study results demonstrate that the CIN pathways are mutually exclusive. 17 Whereas the CIMP pathway overlaps with the MSI pathway because of the presence of sporadic MSI-H CRCs, which are also usually CIMP-high (CIMP-H), the CIMP pathway does not appear to be in an exclusive relationship with the CIN pathway. CIMP-H/ CRCs show some copy number variations across the genome, although the degree of CIN is less pronounced than that of CIMP-negative or -low (CIMP- 0,L)/ CRCs. 18 This finding suggests that the CIMP pathway itself may not be sufficient for the malignant transformation of serrated polyps and requires collaboration with either the CIN or MSI pathway to promote successful malignant transformation. GEOGRAPHY-, AGE-, AND SEX-DEPENDENT DIFFERENCES IN THE PROPORTIONS OF MOLECULAR CARCINOGENESIS PATHWAYS The frequency of MSI in CRCs differs between Western and Eastern populations. MSI-H CRCs comprise approximately 15% of all CRCs in the United States but less than 10% of all CRCs in South Korea or Taiwan. 19 26 Because the prevalence of Lynch syndrome is approximately 3% and is similar in Eastern and Western patients with CRC, 27 30 sporadic MSI-H CRCs account for the difference in the proportion of MSI-H CRCs in all CRCs between populations in South Korea and the United States. Most sporadic MSI-H CRCs develop from sessile serrated adenomas through CIMP-associated inactivation of MLH1. The reason why the proportion of sporadic MSI-H CRCs is lower in South Korea is because the proportion of CIMP-H CRCs is lower in all CRCs in South Korea than in all CRCs in the United States. 31,32 The proportion of CIMP-H CRCs defined by at least 5 of 8 methylated CIMP panel markers is lowerinsouthkoreathanintheunitedstates(6% 7% versus 17 18%, respectively). 21,31,33,34 Accordingly, the mutation rate of BRAF is lower in CRCs in the Korean populationthanincrcsintheamericanpopulation(4% 5% versus 13% 15%, respectively). 26,31,35 38 Most Chinese studies have reported a relatively low frequency of BRAF mutation in CRCs (,7%). 39 Within the same geographic area, white individuals have a higher rate of CIMP-H in CRCs than do African American or Asian American individuals (14% 18% versus 8% 11% versus 7%, respectively). 35,40 Thus, the lower rate of MSI-H in African American than in white individuals reported in a population-based study (14% versus 7%, respectively) might be attributed to the lower rate of CIMP-H in African American than in white persons. 40,41 An association between MSI-H CRCs and female sex has been recognized but the association is valid only when MSI- H CRCs are sporadic. In Lynch syndrome, CRC risk is higher in male mutation carriers than in female mutation carriers. 42 In other words, an association between MSI-H CRCs and female sex is provided by CIMP-H because MSI- H CRCs are generated by CIMP-associated inactivation of MLH1, and CIMP-H is closely associated with female sex. The relationship between female sex and CIMP-H is persistent regardless of geographic location, race, or age. 40,43 45 Although CIMP-H is associated with increased patient age, 43,46,47 the proportion of CIMP-H is higher in females than in males regardless of age. 45 Arch Pathol Lab Med Vol 140, May 2016 Molecular Subtypes of Colorectal Cancer Bae et al 407

Table 1. Clinicopathologic and Molecular Features of 4 Molecular Subtypes of Colorectal Cancers Based on Combinatory Statuses of CIMP and Microsatellite Instability (MSI) a CIMP-0/ Non MSI-H, (n ¼ 464, 63%) MOLECULAR SUBTYPES BASED ON COMBINED CIMP AND MSI STATUSES Jass 15 classified CRCs into 5 molecular subtypes primarily by underlying types of genetic instability and the presence of DNA methylation: (1) CIMP-H/MSI-H, (2) CIMP-H/non MSI-H, (3) CIMP-L/, (4) CIMP-0/, and (5) CIMP-0/MSI-H. Jass subtypes 1 and 2 and subtype 4 reflect CRCs arising from the serrated neoplasia pathway and the classical pathway, respectively. Jass subtype 5 was suggested to be indicative of possible Lynch syndrome. However, with regard to Jass subtype 3, it is unclear whether CRCs arise from the classical pathway or the serrated neoplasia pathway. Furthermore, there are no consensus diagnostic criteria for CIMP-L. Conceptually, the term CpG island methylator phenotype L (CIMP-L) was coined to describe a group of tumors that show intermediate amounts of aberrant DNA methylation. 48 CIMP-L is defined when the number of methylated CIMP panel markers exceeds 0 but does not reach the cutoff value for CIMP-H. In the 8-marker panel, CIMP-L tumors have methylation at 1 to 4 or 5 markers, whereas in the 5-marker panel, CIMP-L has methylation at 1 to 2 or 3 markers. 45,48 Because conventional adenomas or CRCs contiguous with conventional adenomas often harbor methylation of 1 or more of the 8-panel markers or of 1 or 2 of the 5-panel markers, 49 CIMP-L/ CRCs are thought to arise from the classical pathway as well as the serrated neoplasia pathway. 15 Thus, the clinicopathologic and molecular features overlap substantially between subtypes 3 and 4. 15 CLINICOPATHOLOGIC OR MOLECULAR FEATURES OF 4 MOLECULAR SUBTYPES OF CRCS In our previous study, 31 we analyzed the CIMP statuses of surgically resected CRCs (n ¼ 734). Of the 5 molecular subtypes according to Jass classification, the CIMP-L/ Non MSI-H, (n ¼ 174, 24%) CIMP-L,0/ MSI-H, (n ¼ 46, 6%) CIMP-H/ Non MSI-H, (n ¼ 31, 4%) CIMP-H/ MSI-H, (n ¼ 19, 3%) P Value b Age, average, y 61.4 61.1 52.5 61.2 68.8,.001 Female 179 (39) 66 (38) 20 (44) 16 (52) 8 (42).63 Right-sided colon location 99 (21) 44 (25) 24 (52) 19 (61) 14 (74),.001 Stage III or IV 241 (52) 89 (51) 16 (35) 25 (81) 12 (63).002 Distant metastasis 81 (18) 25 (14) 3 (7) 11 (36) 2 (11).01 Nodal metastasis 222 (48) 86 (49) 14 (30) 25 (81) 12 (63),.001 Poor differentiation 6 (1) 8 (5) 3 (7) 8 (26) 4 (21),.001 Dirty necrosis c 431 (93) 155 (89) 34 (74) 28 (90) 11 (58),.001 Crohn-like lymphoid reaction c 238 (51) 81 (47) 36 (78) 14 (45) 15 (79),.001 Luminal serration c 10 (2) 12 (7) 7 (15) 6 (19) 7 (37),.001 Mucin production c 85 (18) 53 (31) 29 (63) 12 (39) 15 (79),.001 Medullary appearance c 0 0 1 (2) 1 (3) 3 (16),.001 BRAF mutation (n ¼ 730) 19 (4) 13 (8) 1 (2) 5 (17) 3 (16).005 KRAS mutation (n ¼ 696) 107 (24) 53 (32) 10 (22) 8 (30) 4 (22).37 TP53 loss or overexpression d (n ¼ 719) 370 (82) 138 (81) 23 (51) 26 (84) 6 (32),.001 KRT20 loss e (n ¼ 727) 17 (4) 7 (4) 3 (7) 1 (3) 7 (37),.001 CDX2 loss e (n ¼ 727) 7 (2) 11 (6) 2 (5) 8 (28) 11 (58),.001 Abbreviations: ANOVA, analysis of variance; CIMP, CpG island methylator phenotype. a A total of 734 cases of colorectal cancers were analyzed. b An ANOVA test was conducted to compare the mean value among groups and a v 2 test was performed to compare the other parameters. c If the analyzed feature was present in more than 5% of the entire tumor area, the feature was considered to be present. d By immunohistochemistry, loss of nuclear expression or overexpression of TP53 in all tumor cells of a tissue microarray core. e By immunohistochemistry, KRT20 loss and CDX2 loss were considered to be present if loss of expression was present in more than 90% of tumor cells in a tissue microarray core. CIMP-0/ subtype was the most common, comprising 63% (n ¼ 464) of the CRCs, whereas the CIMP-H/MSI-H subtype was the least common, comprising 3% (n ¼ 19) of the CRCs (Table 1). The age of onset was the highest for the CIMP-H/MSI-H subtype and the lowest for the CIMP-0,L/MSI-H subtype. The 5 molecular subtypes showed distinct clinicopathologic and molecular features. Compared with the CIMP-0/ subtype, which is the most common subtype, the CIMP-H/ subtype was associated with a more common right-sided colon location, poor differentiation, luminal serration, nodal metastasis, distant metastasis, and BRAF mutation. Compared with the other 3 molecular subtypes, the CIMP-0,L/ MSI-H subtype was associated with the youngest age of onset, less frequent nodal metastasis, less frequent distant metastasis, and near absence of BRAF mutation. Regardless of CIMP status, the 2 MSI-H subtypes showed a more frequent right-sided colon location, less frequent dirty necrosis, more frequent Crohn-like lymphoid reaction, more frequent mucinous histology, and less frequent KRAS mutation than the CIMP-0,L/ subtype. Despite these shared clinicopathologic and molecular features, the CIMP-H/MSI-H and CIMP-0,L/MSI-H subtypes differ in several clinicopathologic features, including age of onset, tumor multiplicity in the large bowel, cancer stage, and frequency of BRAF mutation. These differences were corroborated in a large-scale study of MSI-H CRCs. 50 Although extensive exonal microsatellite alterations and increased point mutations caused by mismatch repair defects bestow very considerable overlap on the altered gene expression signatures between the 2 MSI-H subtypes, the serrated neoplasia pathway associated gene signature can be found in the CIMP-H/MSI-H subtype. 51 Because serrated polyps, including hyperplastic polyps, sessile serrated adenomas, and traditional serrated adenomas, are associated with aberrant gastric-type differentiation, the 408 Arch Pathol Lab Med Vol 140, May 2016 Molecular Subtypes of Colorectal Cancer Bae et al

Distribution of 5 molecular subtypes along bowel subsites. Abbreviations: CIMP-H, CpG island methylator phenotype-high; CIMP-L, CpG island methylator phenotype-low; CIMP-0, CpG island methylator phenotypenegative; MSI-H, high level of microsatellite instability. CIMP-H/MSI-H subtype is characterized by a gain of gastric differentiation (ANXA10, TFF2, and MUC5AC expression) and loss of intestinal expression (CDX2 and KRT20 loss). 51,52 Furthermore, histologic features that differ between the 2 MSI-H subtypes include sheeting appearance, eosinophilic cytoplasm, acinar appearance, signet ring cell formation, and stratified nuclei. The first 4 of these features are more frequent in the CIMP-H/MSI-H subtype, whereas the last feature is more frequent in the CIMP-0,L/MSI-H subtype. 50 BOWEL SUBSITE DISTRIBUTION OF MOLECULAR SUBTYPES OF CRCS Right-sided and left-sided colon cancers and rectal cancers differ in various clinicopathologic and molecular features. These differential clinicopathologic and molecular features do not change abruptly at the borders between the right-sided colon and the left-sided colon or between the left-sided colon and the rectum, but change gradually along the large bowel. 34 Yamauchi et al 34,53 examined the clinicopathologic and molecular features of CRCs at 9 bowel subsites from the rectum to the cecum and found that the frequencies of female sex, poor differentiation, mucinous histology, CIMP-H, MSI-H, and BRAF mutation increased linearly along the bowel from the rectum to the ascending colon. Our previous study 31 also found a linear trend toward increasing female occurrence, advanced T category, N category, overall stage, poor differentiation, absence of luminal necrosis, luminal serration, mucinous histology, Crohn-like lymphoid reaction, CIMP-H, -H from the rectum to the cecum. These findings support the concept of the large bowel as a continuum rather than as 2 or 3 large-bowel subsections in terms of molecular and clinicopathologic features. The Figure illustrates the proportions of the 5 molecular subtypes in each bowel subsite from the cecum to the rectum. 31 For the cecum or ascending colon, the CIMP-0/ subtype comprises approximately 50% of tumors, whereas this molecular subtype constitutes approximately 70% of the tumors in the sigmoid and rectosigmoid colon and the rectum, which may account for the observed differences in clinicopathologic features between right-sided cancers and left-sided or rectal cancers. PROGNTIC IMPLICATIONS OF MOLECULAR SUBTYPES OF CRCS The association between molecular subtypes and survival of colon cancers or CRCs was first explored by Ward et al 54 who showed that, of the 4 molecular subtypes generated by combinations of CIMP (CIMP-H versus CIMP-L,0) (MSI-H versus ) statuses, the CIMP-H/non MSI-H subtype was associated with the shortest overall survival and disease-free survival time (Table 2). Ward et al 54 analyzed the CIMP panel markers CDKN2A (p16), MINT1, MINT2, MINT12, and MINT31 for their methylation statuses by using bisulfite or methylation-specific polymerase chain reaction in study subjects who included adjuvanttreated or untreated patients with stage I to IV CRC. Our previous studies, which analyzed stage I to IV CRC cases with or without adjuvant treatment, also found that the CIMP-H/ subtype was associated with the shortest overall survival time. 43,55 In contrast, in a study by Ogino et al, 56 the CIMP-0,L/ subtype showed the worst cancer-specific survival in the cohort of study patients with stage I to IV colon cancer. A recent study 57 assessed the association between molecular subtypes and survival of patients with stage III colon cancer who were randomly assigned to treatment with adjuvant fluorouracil and leucovorin (FU/LV) alone or with irinotecan (IFL) (a phase III intergroup trial [C89803]) and found that the CIMP-H/ subtype was associated with the worst overall survival or disease-free survival in patients treated with adjuvant FU/LV but not in patients treated with adjuvant IFL. 57 In adjuvant IFL treated patients, no survival difference was identified among the 4 molecular subtypes. This finding suggests that differences in the response to adjuvant therapies contribute to subtype-specific survival Arch Pathol Lab Med Vol 140, May 2016 Molecular Subtypes of Colorectal Cancer Bae et al 409

Table 2. Summary of Studies Exploring the Prognostic Value of Molecular Subtypes of Colorectal Cancers (CRCs) Based on CIMP and Microsatellite Instability (MSI) Statuses in Colorectal Cancers Source, y Patients Adjuvant Molecular Markers Ward et al, 54 2003 612 stage I IV CRCs Surgery only or FUbased regimen or radiotherapy Lee et al, 55 2008 134 stage I IV CRCs Surgery only or FUbased regimen Kim et al, 44 2009 318 stage I IV CRCs Surgery only or FUbased regimen Ogino et al, 56 2009 649 stage I IV colon Not informed CIMP (8 markers) cancers Sanchez et al, 68 2009 391 stage I IV CRCs Not informed Dahlin et al, 69 2010 574 stage I IV CRCs Surgery only or FUbased CIMP (8 markers) regimen or radiotherapy Han et al, 58 2013 Shiovitz et al, 57 2014 Phipps et al, 35 2015 Sinicrope et al, 59 2015 322 stage II III CRCs 615 stage III colon cancers 2080 stage I IV CRCs 2720 stage III colon cancers FOLFOX FU/LV or IFL Surgery only or variable treatment FOLFOX or FOLFOX þ cetuximab differences. In a study of patients with stage III or high-risk stage II CRC who were treated with adjuvant FU/LV plus oxaliplatin (FOLFOX), the 4 molecular subtypes did not show significant differences in disease-free survival. 58 In lieu of the CIMP-H/ subtype, concurrent methylation of 2 CIMP panel markers, CDKN2A (p16) and NEUROG1, was associated with recurrence after adjuvant FOLFOX treatment in stage II/III CRCs. In a recent population-based study that evaluated differences in survival across CRC subtypes defined by CIMP, MSI, BRAF mutation, and KRAS mutation status at stages I to IV, Phipps et al 35 suggested that the CIMP-H/ subtype with BRAF mutation had the highest disease-specific mortality. However, this study was not stratified according to the difference in adjuvant therapy regimens. Additional studies are needed to test whether the CIMP-H/ subtype with BRAF mutation has the highest diseasespecific mortality in adjuvant FOLFOX treated patients with stage II or III CRC. In a recent study of a large number of patients with stage III colon cancer who were treated with adjuvant FOLFOX or adjuvant FOLFOX plus cetuximab, Sinicrope et al 59 reported that colon cancers with KRAS mutations or BRAF mutations were associated with poorer disease-free survival than colon cancers with wild-type KRAS and BRAF. Additionally, MSI- H colon cancers did not differ from colon cancers with wild-type KRAS and BRAF in terms of diseasefree survival. Overall, the findings suggest that the CIMP-H/ subtype is associated with the shortest survival time in adjuvant FU treated patients with stage III CRC but not in adjuvant IFL or FOLFOX treated patients with stage CIMP (8 markers), KRAS, BRAF, MSI KRAS, BRAF, MSI No. of Subtypes; Subtype With the Worst Outcome non-msi-h 4 subtypes; CIMP- 0,L/ 6 subtypes; CIMP-H/ CDKN2A (p16) and NEUROG1 methylation in FU/ LV setting 5 subtypes; CIMP-H/ /BRAF mutation 5 subtypes; non MSI-H and mutations of either KRAS or BRAF Survival and III CRC. In adjuvant FOLFOX treated patients with stage III CRC, CRCs with mutations in either KRAS or BRAF appear to have the worst outcomes. SERRATED PATHWAY ADENOCARCINOMAS The associations of reduced survival with (1) the CIMP-H/ subtype in patients with CRC who are treated with FU-based adjuvant therapy and (2) CRCs with BRAF or KRAS mutations in patients with CRC who are treated with adjuvant FOLFOX suggest that CRCs arising from the serrated neoplasia pathway may be more aggressive or more resistant to adjuvant therapy than those arising from the classical adenoma-carcinoma pathway. Serrated pathway adenocarcinomas refer to CRCs resulting from the serrated neoplasia pathway. Serrated adenocarcinoma is a newly introduced disease entity that defines a subset of colorectal adenocarcinomas characterized by epithelial serrations, eosinophilic or clear cytoplasm, abundant cytoplasm, vesicular nuclei, distinct nucleoli, absence of necrosis, extracellular mucin production, and cell balls and papillary fronds in mucinous areas. 60,61 Given that 20% to 30% of CRCs are attributed to the serrated neoplasia pathway 32,61 and that 7% to 12% of CRCs are serrated adenocarcinomas, 62 64 approximately one-third to one-half of serrated pathway adenocarcinomas are serrated adenocarcinomas. However, there has been no study examining the CIMP status of serrated adenocarcinomas. Thus, little is known regarding the proportions of CIMP-L and CIMP-H in serrated adenocarcinomas and regarding the proportion of serrated adenocarcinomas in CIMP-H CRCs. In other CSS and CSS and CSS and Abbreviations: CIMP, CpG island methylator phenotype; CIMP-H, CIMP-high; CIMP-0,L, CIMP-negative or -low; CSS, cancer-specific survival;, disease-free survival; FOLFOX, FU plus LV plus oxaliplatin; FU, fluorouracil; IFL, FU plus LV plus irinotecan; LV, leucovorin; MSI-H, MSI-high;, overall survival. 410 Arch Pathol Lab Med Vol 140, May 2016 Molecular Subtypes of Colorectal Cancer Bae et al

words, whether all CIMP-H CRCs show histologic features of serrated adenocarcinomas remains to be determined. Because conventional adenomas can harbor aberrant hypermethylation of CIMP panel markers, 49 CIMP-L CRCs can arise from either the serrated neoplasia pathway or the classical pathway. At present, methylation assays of CIMP panel markers cannot differentiate between CIMP-L serrated pathway adenocarcinomas and CIMP-L CRCs arising from the classical pathway. There is a need to develop auxiliary markers to identify serrated pathway adenocarcinomas. Immunohistochemical markers associated with the serrated neoplasia pathway include ANXA10, CLDN18, CTSE, MUC5AC, MUC6, TFF2, and VSIG2, which are overexpressed in hyperplastic polyps, sessile serrated adenoma/polyps, and traditional serrated adenomas, compared with conventional adenomas. 51,52,65 67 Little information is available regarding the expression statuses of these markers in CIMP-L CRCs and the differential clinicopathologic features of CIMP-L CRCs associated with the expression of these markers. In summary, although the factors triggering the CIMP pathway are unknown, the CIMP pathway is associated with the right-sided colon, female sex, older age, and white patients. Familiarity with the differential clinicopathologic features of CRCs associated with molecular carcinogenesis pathways is a requisite for a better understanding of the mechanisms that lead CRCs to exhibit bowel subsite associated differences with regard to age of onset, sex, race, and geographic area. Different responses of the molecular subtypes of CRCs to adjuvant therapy underlie the survival differences between sexes or between right-sided colon cancers and left-sided colon or rectal cancers. Clinical trials of adjuvant chemotherapies or targeted therapies should be conducted taking into account the molecular subtypes of CRCs. It is recognized that a considerable portion of serrated pathway adenocarcinomas do not exhibit any of CIMP-H, serrated adenocarcinoma histology, or direct contiguity with serrated polyps but instead show CIMP-L. Because CIMP-L CRCs can arise from both the classical pathway and the serrated neoplasia pathway, it is necessary to develop molecular or immunohistochemical markers to identify CIMP-L serrated pathway adenocarcinomas. With the help of these biomarkers, a better understanding of the clinicopathologic and molecular features of CIMP-L serrated pathway adenocarcinomas will be reached. This study was supported by a grant from the Basic Science Research Program through the National Research Foundation (NRF) funded by the Korean Ministry of Education (2013R1A1A2059080), an NRF grant funded by the Korean Ministry of Science, ICT and Future Planning (2011-0030049), a grant (2009-0093820) from the Priority Research Centers Program of the NRF, and a grant from the Korea Health Technology R&D Project of the Korea Health Industry Development Institute funded by the Korean Ministry of Health and Welfare (HI14C1277). References 1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64(1):9 29. 2. Cheng L, Eng C, Nieman LZ, Kapadia AS, Du XL. Trends in colorectal cancer incidence by anatomic site and disease stage in the United States from 1976 to 2005. Am J Clin Oncol. 2011;34(6):573 580. 3. Siegel R, Desantis C, Jemal A. Colorectal cancer statistics, 2014. CA Cancer J Clin. 2014;64(2):104 117. 4. Center MM, Jemal A, Smith RA, Ward E. 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