The serrated neoplasia pathway of colorectal tumors: Identification of MUC5AC hypomethylation as an early marker of polyps with malignant potential

The serrated neoplasia pathway accounts for 20–30% of colorectal cancers (CRC), which are characterized by extensive methylation (CpG island methylation phenotype, CIMP), frequent BRAF mutation and high microsatellite instability (MSI). We recently identified MUC5AC mucin gene hypomethylation as a specific marker of MSI CRC. The early identification of preneoplastic lesions among serrated polyps is currently challenging. Here, we performed a detailed pathological and molecular analysis of a large series of colorectal serrated polyps and evaluated the usefulness of mucin genes MUC2 and MUC5AC to differentiate serrated polyps and to identify lesions with malignant potential. A series of 330 colorectal polyps including 218 serrated polyps [42 goblet cell‐rich hyperplastic polyps (GCHP), 68 microvesicular hyperplastic polyps (MVHP), 100 sessile serrated adenoma (SSA) and eight traditional serrated adenoma (TSA)] and 112 conventional adenomas was analyzed for BRAF/KRAS mutations, MSI, CIMP, MLH1 and MGMT methylation, and MUC2 and MUC5AC expression and methylation. We show that MUC5AC hypomethylation is an early event in the serrated neoplasia pathway, and specifically detects MVHP and SSA, arguing for a filiation between MVHP, SSA and CIMP‐H/MSI CRC, whereas GCHP and TSA arise from a distinct pathway. Moreover, MUC5AC hypomethylation specifically identified serrated lesions with BRAF mutation, CIMP‐H or MSI, suggesting that it may be useful to identify serrated neoplasia pathway‐related precursor lesions. Our data suggest that MVHP should be recognized among HP and require particular attention.     

Molecular heterogeneity of colorectal cancer: Implications for cancer control

Colorectal cancer (CRC) is a multi-pathway disease. A molecular approach to the classification of CRC utilises: (1) the type of genetic instability, specifically microsatellite instability (MSI) versus stable (MSS), and (2) the presence of DNA methylation or the CpG island methylator phenotype (CIMP). The MSS/CIMP-neg subset evolves through the classical adenoma-carcinoma sequence while the MSI/CIMP-pos and MSS/CIMP-pos subsets evolve through the recently recognised 'serrated pathway'. This review will show that the existence of two or more independent pathways to CRC is relevant to cancer prevention. In particular, new strategies for detecting and managing sessile serrated polyps will need to be developed and evaluated.     

Genetic and epigenetic aberrations occurring in colorectal tumors associated with serrated pathway

To clarify molecular alterations in serrated pathway of colorectal cancer (CRC), we performed epigenetic and genetic analyses in sessile serrated adenoma/polyps (SSA/P), traditional serrated adenomas (TSAs) and high‐methylation CRC. The methylation levels of six Group‐1 and 14 Group‐2 markers, established in our previous studies, were analyzed quantitatively using pyrosequencing. Subsequently, we performed targeted exon sequencing analyses of 126 candidate driver genes and examined molecular alterations that are associated with cancer development. SSA/P showed high methylation levels of both Group‐1 and Group‐2 markers, frequent BRAF mutation and occurrence in proximal colon, which were features of high‐methylation CRC. But TSA showed low‐methylation levels of Group‐1 markers, less frequent BRAF mutation and occurrence at distal colon. SSA/P, but not TSA, is thus considered to be precursor of high‐methylation CRC. High‐methylation CRC had even higher methylation levels of some genes, e.g., MLH1, than SSA/P, and significant frequency of somatic mutations in nonsynonymous mutations (p < 0.0001) and insertion/deletions (p = 0.002). MLH1‐methylated SSA/P showed lower methylation level of MLH1 compared with high‐methylation CRC, and rarely accompanied silencing of MLH1 expression. The mutation frequencies were not different between MLH1‐methylated and MLH1‐unmethylated SSA/P, suggesting that MLH1 methylation might be insufficient in SSA/P to acquire a hypermutation phenotype. Mutations of mismatch repair genes, e.g., MSH3 and MSH6, and genes in PI3K, WNT, TGF‐β and BMP signaling (but not in TP53 signaling) were significantly involved in high‐methylation CRC compared with adenoma, suggesting importance of abrogation of these genes in serrated pathway.     

Association of Fusobacterium nucleatum with clinical and molecular features in colorectal serrated pathway

Human gut microbiota is being increasingly recognized as a player in colorectal cancers (CRCs). Evidence suggests that Fusobacterium nucleatum (F. nucleatum) may contribute to disease progression and is associated with CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) in CRCs; however, to date, there are no reports about the relationship between F. nucleatum and molecular features in the early stage of colorectal tumorigenesis. Therefore, we investigated the presence of F. nucleatum in premalignant colorectal lesions. In total, 465 premalignant lesions (343 serrated lesions and 122 non‐serrated adenomas) and 511 CRCs were studied. We determined the presence of F. nucleatum and analyzed its association with molecular features including CIMP, MSI and microRNA‐31 status. F. nucleatum was detected in 24% of hyperplastic polyps, 35% of sessile serrated adenomas (SSAs), 30% of traditional serrated adenomas (TSAs) and 33% of non‐serrated adenomas. F. nucleatum was more frequently detected in CIMP‐high premalignant lesions than in CIMP‐low/zero lesions (p = 0.0023). In SSAs, F. nucleatum positivity increased gradually from sigmoid colon to cecum (p = 0.042). F. nucleatum positivity was significantly higher in CRCs (56%) than in premalignant lesions of any histological type (p < 0.0001). In conclusion, F. nucleatum was identified in premalignant colorectal lesions regardless of histopathology but was more frequently associated with CIMP‐high lesions. Moreover, F. nucleatum positivity increased according to histological grade, suggesting that it may contribute to the progression of colorectal neoplasia. Our data also indicate that F. nucleatum positivity in SSAs may support the “colorectal continuum” concept.     

Expression of the MAP kinase phosphatase DUSP4 is associated with microsatellite instability in colorectal cancer (CRC) and causes increased cell proliferation

DUSP4 (MKP‐2), a member of the mitogen‐activated protein kinase phosphatase (MKP) family and potential tumor suppressor, negatively regulates the MAPKs (mitogen‐activated protein kinases) ERK, p38 and JNK. MAPKs play a crucial role in cancer development and progression. Previously, using microarray analyses we found a conspicuously frequent overexpression of DUSP4 in colorectal cancer (CRC) with high frequent microsatellite instability (MSI‐H) compared to microsatellite stable (MSS) CRC. Here we studied DUSP4 expression on mRNA level in 38 CRC (19 MSI‐H and 19 MSS) compared to matched normal tissue as well as in CRC cell lines by RT‐qPCR. DUSP4 was overexpressed in all 19 MSI‐H tumors and in 14 MSS tumors. Median expression levels in MSI‐H tumors were significantly higher than in MSS‐tumors (p < 0.001). Consistently, MSI‐H CRC cell lines showed 6.8‐fold higher DUSP4 mRNA levels than MSS cell lines. DUSP4 expression was not regulated by promoter methylation since no methylation was found by quantitative methylation analysis of DUSP4 promoter in CRC cell lines neither in tumor samples. Furthermore, no DUSP4 mutation was found on genomic DNA level in four CRC cell lines. DUSP4 overexpression in CRC cell lines through DUSP4 transfection caused upregulated expression of MAPK targets CDC25A, CCND1, EGR1, FOS, MYC and CDKN1A in HCT116 as well as downregulation of mismatch repair gene MSH2 in SW480. Furthermore, DUSP4 overexpression led to increased proliferation in CRC cell lines. Our findings suggest that DUSP4 acts as an important regulator of cell growth within the MAPK pathway and causes enhanced cell growth in MSI‐H CRC.     

Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas

BACKGROUND: Microsatellite instability (MSI) is seen in 10%-15% of sporadic colorectal cancers mostly in the right colon, but the precursors of cancers with MSI remain unknown. We examined whether sporadic cancers with MSI arise from pre-existing benign proliferative lesions (such as hyperplastic polyps or serrated adenomas [together denoted as "serrated polyps"]). METHODS: The frequency of benign epithelial lesions (serrated polyps and conventional adenomas) was determined by histologic review of resection specimens from individuals (n = 29) with sporadic colorectal cancer with MSI and from a matched control group (n = 29) with cancer showing microsatellite stability (MSS). MSI status, expression of mismatch repair enzyme (product of the human mut-L homologue 1 [hMLH1] gene), and hMLH1 gene promoter methylation in the benign lesions were determined. Data were analyzed by the chi-square test, by Wilcoxon's rank-sum test, and by conditional logistic regression as appropriate, and a two-sided probability less than.05 was considered to be statistically significant. RESULTS: Individuals with cancers showing MSI were more likely to harbor at least one serrated polyp than individuals with cancers showing MSS (odds ratio = 4.0; 95% confidence interval = 1.1 to 14.2; P =.03), but the frequency of conventional adenomas was the same in both groups (P =.52, Mann-Whitney test). Loss of hMLH1 protein expression was seen in lesions from 10 of 13 patients with MSI, but no loss was seen in lesions from four patients with MSS (P =.02, Fisher's exact test). Loss of hMLH1 protein expression was associated with MSI in assessable lesions. The hMLH1 promoter was methylated in all assessable serrated polyps from patients with cancers showing MSI but in none of the lesions from patients with MSS cancers. CONCLUSIONS: Some right-sided hyperplastic polyps may give rise to sporadic colorectal carcinomas with MSI. Methylation of the hMLH1 gene promoter within neoplastic cell subpopulations may be a critical step in the progression to carcinoma. The frequency with which benign lesions progress to cancer with MSI is unknown.     

结直肠锯齿状病变h-MLH1基因甲基化状态和蛋白表达意义研究

目的：通过研究结直肠锯齿状病变组织中h-MLH1基因启动子区CpG岛甲基化及蛋白表达,探讨h-MI．H1启动子甲基化状态与蛋白表达相关性及在锯齿状癌变通路中的作用,并分析hMLH1基因在不同年龄层段甲基化程度。方法：收集北京军区总医院病理科2007-0101-201212-31诊断为锯齿状病变标本225例,其中包括96例增生性息肉（hyperplasticpolyp,HP）、61例广基（无蒂）锯齿状腺瘤/息肉（sessileserratedadenoma／polyp,SSA／P）和68例传统锯齿状腺瘤（traditionalserratedadenoma,TSA）;同时收集同院同期的54例管状腺瘤（tubularadenoma,TA）、69例结直肠癌（colorectaIcancer,CRC）和42例正常结直肠黏膜组织作为对照。应用Taqman探针qPCR（MethyLight）方法检测各组织中h-MLH1基因CpG岛甲基化状态,同时采用免疫组织化学方法检测其蛋白表达水平,其中随机抽取锯齿状病变116例（HP52例、SSA／P41例、TSA23例）、TA20例、CRC24例和正常结直肠黏膜组织24例,并将其甲基化状态与相应的蛋白表达水平及临床病理学资料进行统计学分析。结果：正常、TA、HP、SSA／P、TSA和CRC组织中,MLH1基因启动子CpG岛甲基化阳性表达率分别为9．24％（4／42）、40．74％（22／54）、22．92％（22／96）、45．90％（28／61）、61．76％（42／68）和52．17％（36／69）。HP组与正常、SSA／P、TSA及CRC组之间比较,正常组与SSA／P及TSA组之间比较,差异均有统计学意义,P〈0．05;其余各组之间比较,差异均无统计学意义,P〉0．05。正常、TA、HP、SSA／P、TSA和CRC组织中,h-MLH1蛋白阳性表达率分别为100％（24／24）、80．00％（16／20）、98．08％（51／52）、75．61％（31／41）、69．57％（16／23）和70．83％（17／24）。正常组与HP、SSA／P、TSA、TA、CRC组之间比较,差异均有统计学意义,P％0．05;其余各组之间比较,差异均无统计学意义,P〉0．05。TA、SSA／P和TSA组中,hMLH1基因甲基化与其蛋白表达差异均有统计学意义（P〈o．05）,且呈负相关,相关系数．y分别为-0．553、-0．497、和-0．473。TA、HP、SSA／P和TSA组中,h-MLH1基因甲基化与年龄差异均有统计学意义（P〈0．05）,且呈正相关,相关系数7分别为0．475、0．289、0．450和0．575。结论：结直肠锯齿状病变组织中h-MLH1基因甲基化可能导致其蛋白表达下调,与锯齿状病变的发生和发展密切相关,同时,h-MLH1基因甲基化又可能与年龄相关,随年龄增长,甲基化程度越高。     

A distinct DNA methylation profile associated with microsatellite and chromosomal stable sporadic colorectal cancers

Aberrant DNA methylation, microsatellite instability (MSI) and chromosomal instability (CIN) are well-characterised molecular features of sporadic colorectal cancers (CRCs). In addition to CpG island methylator phenotype (CIMP) associated with MSI, an intermediate methylation subgroup is also a feature of non-MSI cancers. A large proportion of CRCs have no evidence of either MSI or CIN, here called Microsatellite and Chromosomal Stable (MACS), and require their methylation profile to be established. The clinical and molecular features of 170 sporadic CRC patients were investigated and stratified into MSI, CIN and MACS groups. MACS were most often found in the left colon and had a significantly lower BRAF mutation frequency (p < 0.001) compared with MSI. MACS had better survival [hazard ratio (HR) = 0.244, p = 0.017] compared with CIN, but were similar to MSI. The methylation status of 1,505 CpG loci from cancer-related genes was analysed in a subset of CRCs (n = 44 normal-tumour pairs) and compared with CIN, MSI and MACS status. Using two-way hierarchical clustering, three subgroups were identified, which associated with CIN, MSI and MACS status. Using significance analysis of microarray, 16 CpG loci demonstrating methylation changes associated with MACS were identified. A combination of six loci identified MACS with 81% sensitivity and 93% specificity. This result now requires independent validation. Hypomethylation of a CpG locus within the sonic hedgehog (SHH) promoter correlated with increased gene expression and was associated significantly with MACS cancers. In conclusion, we propose that MACS have distinct clinicopathological features and can be distinguished from other CRCs by a specific set of methylation loci.     

MicroRNA‐31 expression in relation to BRAF mutation,CpG island methylation and colorectal continuum in serrated lesions

The CpG island methylator phenotype (CIMP) is a distinct form of epigenomic instability. Many CIMP‐high colorectal cancers (CRCs) with BRAF mutation are considered to arise from serrated pathway. We recently reported that microRNA‐31 (miR‐31) is associated with BRAF mutation in colorectal tumors. Emerging new approaches have revealed gradual changes in BRAF mutation and CIMP‐high throughout the colorectum in CRCs. Here, we attempted to identify a possible association between miR‐31 and epigenetic features in serrated pathway, and hypothesized that miR‐31 supports the “colorectal continuum” concept. We evaluated miR‐31 expression, BRAF mutation and epigenetic features including CIMP status in 381 serrated lesions and 222 non‐serrated adenomas and examined associations between them and tumor location (rectum; sigmoid, descending, transverse and ascending colon and cecum). A significant association was observed between high miR‐31 expression and CIMP‐high status in serrated lesions with BRAF mutation (p = 0.0001). In contrast, miR‐31 was slightly but insignificantly associated with CIMP status in the cases with wild‐type BRAF. miR‐31 expression in sessile serrated adenomas (SSAs) with cytological dysplasia was higher than that in SSAs, whereas, no significant difference was observed between traditional serrated adenomas (TSAs) and TSAs with high‐grade dysplasia. The frequency of miR‐31, BRAF mutation CIMP‐high and MLH1 methylation increased gradually from the rectum to cecum in serrated lesions. In conclusion, miR‐31 expression was associated with CIMP‐high status in serrated lesions with BRAF mutation. Our data also suggested that miR‐31 plays an important role in SSA evolution and may be a molecule supporting the colorectal continuum.     

Distinct CpG island methylation profiles and BRAF mutation status in serrated and adenomatous colorectal polyps

A subset of colorectal cancers with CpG island methylator phenotype-high (CIMP-H) is frequently associated with MSI and BRAF V600E mutation. Since limited data are available on different histological types of colorectal polyps, we compared the pattern and the frequency of promoter methylation, CIMP-H, MSI, KRAS and BRAF V600E mutations and the relationship among these molecular parameters and the clinicopathologic characteristics in 110 serrated polyps (48 hyperplastic polyps, 32 sessile serrated adenomas and 30 serrated adenomas) and 32 tubular adenomas using 7 commonly used tumor-associated gene loci. No significant difference in the frequency of overall methylation frequency (86% vs. 100%) and CIMP-H (39% vs. 28%) between serrated polyps and tubular adenomas was observed, but proximally located serrated polyps showed more frequent methylation at 5 of 7 loci examined, and were more likely to be CIMP-H (62% vs. 22%). MGMT methylation was more common in tubular adenomas while MLH1 and HIC1 were more frequently methylated in serrated polyps. BRAF mutation was frequently present in all types of serrated polyps (80%), but was absent in tubular adenomas and was not associated with CIMP or MSI status. These results show comparable frequencies of promoter methylation of tumor-associated genes and CIMP-H, but distinct differences in gene-specific or colonic site-specific methylation profiles occur in serrated polyps and tubular adenomas. BRAF mutation occurs independently of CIMP and MSI in all types of serrated polyps and may serve as a marker of serrated pathway of colorectal carcinogenesis.     

Molecular differences in the microsatellite stable phenotype between left‐sided and right‐sided colorectal cancer

Differences in the pathogenesis of microsatellite stable (MSS) sporadic colorectal cancers (CRCs) between left‐sided CRC (LC) and right‐sided CRC (RC) have not been clarified. To identify pathogenesis‐related genomic differences between MSS CRCs within the two locations, we performed a comprehensive molecular analysis using crypt isolation with samples from 92 sporadic CRCs. Microsatellite instability (MSI; high and low/negative) and DNA methylation status (low methylation epigenome; intermediate methylation epigenome [IME] or high methylation epigenome [HME]) were determined using polymerase chain reaction (PCR) microsatellite analysis and PCR‐bisulfite pyrosequencing, respectively. Additionally, mutations in the TP53, KRAS, BRAF and PIK3CA genes were examined using PCR‐bisulfite pyrosequencing (for KRAS and BRAF mutations) or PCR‐single conformation polymorphism (for TP53 and PIK3CA mutations), followed by sequencing of aberrant bands. Finally, a genome‐wide study using a copy number alteration (CNA)‐targeted single nucleotide polymorphism array was performed. Ninety‐two CRCs were classified into 71 MSS and 21 MSI phenotypes. We examined 71 CRCs with the MSS phenotype (LC, 56; RC, 15). Mutations in KRAS were associated with RC with the MSS phenotype, whereas mutations in TP53 were more frequently found in LC with the MSS phenotype. There were significant differences in the frequencies of KRAS and TP53 mutations in the IME between LC and RC with the MSS phenotype. Although CNA gains were associated with LC with the MSS phenotype, CNA losses were not major alterations associated with the MSS phenotype. These findings suggested that the molecular pathogenesis of the MSS phenotype in LC was different from that in RC.     

Evidence of a preferred molecular pathway in patients with synchronous colorectal cancer

BACKGROUND: A small proportion of patients with colorectal carcinoma (CRC) have synchronous tumors at the time of diagnosis. A subset of sporadic CRCs display microsatellite instability (MSI) that is associated with MLH1 silencing due to promoter methylation. In the current study, the authors investigated the proportion of tumors with MSI in patients with synchronous colorectal carcinoma (SCRC) and the concordance in MSI status among tumors in a given individual. In addition, the authors examined MLH1 and MSH2 expression and MLH1 promoter methylation in SCRCs. METHODS: The current study included 77 patients, with a combined total of 170 invasive SCRCs, who were identified from a database of 2884 patients with CRC. Instability was determined by polymerase chain reaction (PCR) amplification using a set of five markers. Tumors that were unstable at two or more markers were considered unstable (MSI); otherwise, they were considered microsatellite stable (MSS). Expression of MLH1 and MSH2 was determined by immunohistochemistry. Methylation of the MLH1 gene promoter was determined by a methylation-specific PCR assay. Statistical comparisons were made using the chi-square test or the Student t test. RESULTS: Of the 77 patients in the study, 21 (27%) had a family history of hereditary nonpolyposis colon carcinoma-related malignancy, but none fulfilled the Amsterdam II criteria. Fifty-four of 170 tumors (32%) were found to be MSI. Patients with MSI tumors were older and more frequently female. All but 1 MSI tumor lacked expression of MLH1 (n = 44) or MSH2 (n = 8), or both (n = 1). All MLH1-negative tumors, compared with only 3 MLH1-positive tumors, were methylated at the MLH1 promoter. Most patients (n = 67; 87%) had either all MSS tumors (n = 48; 62%) or all MSI tumors (n = 19; 25%); 10 patients (13%) had both MSS and MSI tumors. The observed MSI/MSS distribution was significantly different from the distribution expected based on an assumption of independence (P < 0.0001). CONCLUSIONS: There is a strong concordance in MSI/MSS status among tumors in the same individual. This finding suggests that the tumors in patients with SCRC develop along a preferred molecular pathway.     

Clinical significance of programmed cell death‐ligand 1 expression and the immune microenvironment at the invasive front of colorectal cancers with high microsatellite instability

Immunotherapy is reportedly effective in colorectal cancers (CRCs) with high microsatellite instability (MSI‐H); however, the specific cell types that respond to immune checkpoint therapy are unclear. Herein, we aimed to examine the expression of programmed cell death‐ligand 1 (PD‐L1) and related proteins in MSI‐H and microsatellite‐stable (MSS) CRCs to investigate the immune microenvironment at the tumor's invasive front. The MSI status was retrospectively assessed in 499 patients undergoing surgical resection of primary CRC; of these, 48 were classified as MSI‐H. Propensity score matching was performed, and tissues from 36 and 37 patients with MSI‐H and MSS CRCs, respectively, were immunohistochemically evaluated for PD‐L1, PD‐1, CD8 and CD68. PD‐L1 expression was evaluated separately for tumor cells (PD‐L1 [T]) and tumor‐infiltrating myeloid cells in the stroma (PD‐L1 [I]). PD‐L1 (T) was positive in only 5.4% and 36.1% of MSS and MSI‐H CRCs, while PD‐L1 (I) was positive in 27% and 72.2% of these CRCs, respectively. The PD‐L1 (T) and PD‐L1 (I) expression levels in MSI‐H CRCs significantly correlated with poor differentiation, lymphatic invasion and vascular invasion (p < 0.05), and with early‐stage adenocarcinoma and high budding grade (p < 0.05), respectively. Significantly more PD‐L1 (I), CD8‐positive cells and CD68‐positive macrophages were present at the invasive front than in the central tumor in MSI‐H CRCs (p < 0.005). PD‐L1 was expressed on both tumor cells and CD68/CD163‐positive (M2) macrophages at the invasive front of MSI‐H CRCs. In conclusion, PD‐L1‐positive tumor cells and M2‐type tumor‐associated macrophages may contribute to tumor invasion and immune escape at the invasive front.     

Left-Sided microsatellite unstable colorectal cancers show less frequent methylation of hMLH1 and CpG island methylator phenotype than right-sided ones

CONDENSED ABSTRACT: We compared the methylation status of several loci between right-sided and left-sided colorectal cancers (CRCs). To the best of our knowledge, this is the first to report that left-sided CRCs show significantly less frequent methylation at several loci and CpG island methylator phenotype (CIMP) than right-sided ones. BACKGROUND AND OBJECTIVES: MSI CRC occurs in 10-20% of unselected series of patients with CRC. Somatic hMLH1 promoter methylation is reported to cause MSI in sporadic cases. Many researchers report that MSI CRCs are more frequently located in the right-side colon than MSS CRCs. Though the number is very small, some MSI CRCs are located in the left-side colorectum. We focused on the existence of left-sided MSI CRCs and investigated whether they arise through hMLH1 methylation as they do in right-sided ones. METHODS: Thirty-eight sporadic MSI CRCs were included in the study. The methylation status of the promoter of hMLH1, p16, MINT1, 2 and 31 were examined and the proportions of methylated samples for each locus were compared. RESULTS: The left-sided group showed significantly less frequent methylation in hMLH1, p16, MINT1, 2 and 31. The frequency of CIMP+ samples in the left-sided group was significantly lower than the right-sided group. CONCLUSIONS: Left-sided MSI CRCs show significantly less frequent methylation of hMLH1. They also showed significantly less frequent occurrence of CIMP+ than right-sided ones. It is possible that left-sided MSI CRCs differ from the right-sided ones in the way of acquiring MSI.     

Microsatellite instability,MLH1 promoter methylation,and BRAF mutation analysis in sporadic colorectal cancers of different ethnic groups in Israel

BACKGROUND:

The molecular mechanisms that underlie colorectal cancer (CRC) include microsatellite instability (MSI), chromosomal instability, and the CpG island methylator phenotype. There is evidence to suggest that CRC incidence varies among different ethnic populations worldwide. The authors of this report hypothesized that environmental factors and lifestyle differences among various ethnic groups may differentially influence the epigenetic regulation of tumor suppressor genes in CRC.

METHODS:

In the current study, microdissection and DNA extraction were performed on 128 samples of CRC from Israeli patients (85 Jews and 43 Arabs). MSI analysis, mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2) protein expression levels, and MLH1 promoter methylation were investigated by combined bisulfite restriction analysis. The v‐raf murine sarcoma viral oncogene homolog B1 (BRAF) valine‐to‐glutamic acid mutation at residue 600 was investigated by direct DNA sequencing.

RESULTS:

High MSI (MSI‐H), MLH1 methylation, and BRAF mutations were observed in 11.6%, 9.4%, and 23.5% of Jews, respectively, and in 16.2%, 17.6%, and 20.9% of Arabs, respectively (P value nonsignificant). MLH1 promoter methylation was observed in 22.6% of microsatellite‐stable (MSS) tumors and in 53.8% of MSI‐H tumors (P < .015). Extensive methylation (covering both 5′ and 3′ promoter regions) was present in all MSI‐H tumors with loss of MLH1 expression. BRAF mutation was observed in 15.6% and 46.1% of MSS tumors and MSI‐H tumors, respectively (P < .007). BRAF mutation was observed in 66%, 22.2%, and 14.7% of patients who had tumors with extensive MLH1 promoter methylation, methylation of the 5′ region alone, or without methylation, respectively (P < .006).

CONCLUSIONS:

There was no difference in molecular signatures examined between Jewish and Arab patients with CRC in Israel. Extensive promoter methylation was associated with MLH1 inactivation, MSI, and BRAF mutation. Cancer 2009. © 2009 American Cancer Society.     

Very low prevalence of germline MSH6 mutations in hereditary non-polyposis colorectal cancer suspected patients with colorectal cancer without microsatellite instability

Hereditary non-polyposis colorectal cancer (HNPCC) is caused by mutations in one of the mismatch repair genes MLH1, MSH2, MSH6, or PMS2 and results in high-level microsatellite instability (MSI-high) in tumours of HNPCC patients. The MSI test is considered reliable for indicating mutations in MLH1 and MSH2, but is questioned for MSH6. Germline mutation analysis was performed in 19 patients with an MSI-high tumour and absence of MSH2 and/or MSH6 protein as determined by immunohistochemistry (IHC), without an MLH1 or MSH2 mutation, and in 76 out of 295 patients suspected of HNPCC, with a non-MSI-high colorectal cancer (CRC). All 295 non-MSI-high CRCs were analysed for presence of MSH6 protein by IHC. In 10 patients with an MSI-high tumour without MSH2 and/or MSH6 expression, a pathogenic MSH6 mutation was detected, whereas no pathogenic MSH6 mutation was detected in 76 patients with a non-MSI-high CRC and normal MSH6 protein expression. In none of the 295 CRCs loss of MSH6 protein expression was detected. The prevalence of a germline MSH6 mutation is very low in HNPCC suspected patients with non-MSI-high CRC. Microsatellite instability analysis in CRCs is highly sensitive to select patients for MSH6 germline mutation analysis.     

p53 mutation is common in microsatellite stable, BRAF mutant colorectal cancers

The majority of "serrated pathway" colorectal cancers have mutation of the BRAF oncogene and display the CpG island methylator phenotype (CIMP). Half these cancers have microsatellite instability (MSI) and an excellent prognosis. In the absence of MSI (microsatellite stable, MSS), BRAF mutation has been associated with a particularly poor prognosis. "Traditional pathway" cancers are BRAF wild type. Mutation of p53 is common and this correlates with advanced stage. We therefore hypothesized that p53 mutation would be common in MSS/BRAF mutant colorectal cancer. One thousand and eighty-one colorectal cancers were screened for BRAF mutation to identify two BRAF mutant study groups (MSI: n = 77; MSS: n = 69) and a BRAF wild type control group (n = 101). These were screened for p53 mutation by high resolution melt analysis and classified for CIMP and MGMT methylation by quantitative methylation specific PCR. Molecular data were compared to patient age, gender, tumor location and stage. p53 was mutated significantly more frequently in MSS/BRAF mutant (28/69, 40.6%) compared to MSI/BRAF mutant cancers (13/77, 16.9%), but this mutation rate did not differ from MSS/BRAF wild type cancers (47/101, 46.5%)(p < 0.0001). CIMP was less common in MSS/BRAF mutant (26/47, 55.3%) compared to MSI/BRAF mutant cancers (41/54, 75.9%), but was more common than in MSS/BRAF wild type cancers (3/85, 3.5%) (p < 0.0001). MSS/BRAF mutant cancers were more commonly proximal (38/54, 70.3%), but were similar to MSS/BRAF wild type cancers in terms of patient age, gender distribution and stage at presentation. MSS/BRAF mutant cancers share molecular and clinical features of both the serrated and traditional pathways of colorectal tumorigenesis.     

Era of universal testing of microsatellite instability in colorectal cancer

Colorectal cancer (CRC) incidence and mortality are constantly decreasing, but CRC still remains the third most prevalent cancer and the third most common cause of cancer death in both males and females in the United States. Recent rapid declines in CRC incidence rates have largely been attributed to increases in screening that can detect and remove precancerous polyps, and the decrease in death rates for CRC largely reflects improvements in early detection, treatment and the understanding of molecular/genetic basis of CRC. One of the important molecular/genetic findings is the presence of microsatellite instability (MSI) in CRCs. Many studies have shown the importance of MSI testing in diagnosing Lynch syndrome and predicting prognosis and response to chemotherapeutic agents in CRCs. Increased emphasis has been placed on the importance of MSI testing for all newly diagnosed individuals with CRCs. Both immunohistochemical staining (IHC) and polymerase chain reaction (PCR)-based MSI testing show high sensitivity and specificity in detecting MSI. The current clinical guidelines and histopathology features are indicative of, but not reliable in diagnosing Lynch syndrome and CRCs with MSI. Currently, there are evidences that universal testing for MSI starting with either IHC or PCR-based MSI testing is cost effective, sensitive, specific and is getting widely accepted.     

散发性结直肠癌的分子机制研究进展

结直肠癌是最常见的恶性肿瘤之一,其病因和发病机制十分复杂。大部分肠癌都是非遗传性的“散发性肠癌”,散发性肠癌是在环境因素影响下遗传和表观遗传学相继改变并累积,最终导致体细胞突变而发生的复杂的异质性疾病。其中最主要的三类基因变化是:染色体的不稳定性(CIN)、微卫星的不稳定性(MSI)和CpG岛甲基化表型(CIMP),表观遗传学变化是DNA的甲基化、组蛋白修饰、MicroRNA的改变。有些患者可能会有二至三种不同变化途径的同时呈现。更加透彻得理解肠癌发生发展背后分子生物学途径的变化将会有助于改善我们对肠癌的预防、监测、诊断和治疗策略。本文围绕结直肠癌发病的分子生物学机制的最新研究进展做一综述。     

Biomarkers for the identification of precursor polyps of colorectal serrated adenocarcinomas

Background

In contrast to conventional colorectal carcinomas (CCs), which develop through a so-called chromosome instability or suppressor phenotype pathway, the sequence of events leading from precursor polyps/adenomas to serrated adenocarcinomas (SACs), which are more aggressive and exhibit a poorer survival than CCs, is as yet not clearly defined. Here, we aimed at detecting protein and DNA biomarkers for SAC in a series of primary colorectal polyps.

Methods

In total 303 colorectal polyps were included: 121 serrated polyps (33 hyperplastic polyps, 37 sessile serrated adenomas (SSA), 51 traditional serrated adenomas (TSA)), 143 conventional polyps (72 tubular polyps, 34 tubulovillous polyps, 37 villious adenomas), and 39 bi-phenotypic serrated-conventional polyps. The protein biomarkers tested were deduced from previously published SAC and CC expression profiling studies. A representative subset of 106 polyps was selected for DNA biomarker analyses, i.e., proto-oncogene mutation and microsatellite instability (MSI) status. In order to confer proper weight to each biomarker, a multivariate logistic regression model was employed.

Results

We found that serrated and conventional polyps differed in most of the SAC biomarkers tested. Of these biomarkers, FSCN1 showed the largest difference in expression (p = 0.0001). Despite sharing a serrated morphology, we found that SSAs and TSAs differed considerably with respect to anatomical location, expression of EPHB2 and PTCH1, presence of the V600E BRAF mutation and MSI status. Logistic regression analysis revealed that SSA was the polyp type that shared most biomarkers with SAC.

Conclusion

Based on the shared presence of protein and molecular biomarkers, especially FSCN1 expression, SSA may serve as a precursor lesion of SAC. Biomarker assessment may help in discerning colorectal carcinogenic routes with distinct prognostic implications.