Differential clinicopathological features in microsatellite instability-positive colorectal cancers depending on CIMP status

Microsatellite instability-positive (MSI+) colorectal cancers (CRCs) are divided into CpG island methylator phenotype-positive (CIMP+) and CpG island methylator phenotype-negative (CIMP−) tumors. The repertoire of inactivated genes in CIMP+/MSI+ CRCs overlaps with but is likely to differ from that of CIMP−/MSI+ CRCs. Because epigenotypic differences are likely to be manifested as phenotypic differences, CIMP+/MSI+ CRCs are expected to differ from CIMP−/MSI+ CRCs in some clinicopathological features. This study aimed to characterize both common and different features between the two subtypes. A total of 72 MSI+ CRCs were analyzed for their methylation status in eight CIMP panel markers using MethyLight assay. CIMP+/MSI+ and CIMP−/MSI+ CRCs were compared regarding clinicopathologic features and mutation in KRAS/BRAF. An independent set of MSI+ CRCs (n = 97) was analyzed for their relationship of CIMP+ status with clinical outcome. Eighteen cases (25%) were CIMP+, and this CIMP+ subtype was highly correlated with older age (P < 0.001). Polypoid gross appearance without ulceration was observed only in CIMP−/MSI+ CRCs (18.5%, P = 0.057). CIMP+/MSI+ CRCs were closely associated with poor differentiation, medullary appearance, signet ring cell appearance, and acinar-form appearance, whereas the CIMP−/MSI+ subtype was closely associated with intraglandular eosinophilic mucin and stratified nuclei (all P values <0.05). Patients with CIMP+/MSI+ CRCs showed worse overall survival than patients with CIMP−/MSI+ CRCs. Our results demonstrate heterogeneity in the clinicopathological features of MSI+ CRCs depending on CIMP status. The observation that CIMP+ and CIMP− subtypes showed different clinical behaviors may provide a clue for establishing subtype-specific therapeutic strategies for these two subtypes.     

The impact of CpG island methylator phenotype and microsatellite instability on tumour budding in colorectal cancer

Zlobec I, Bihl M P, Foerster A, Rufle A & Lugli A
(2012) Histopathology  61, 777–787 The impact of CpG island methylator phenotype and microsatellite instability on tumour budding in colorectal cancer Aims: In colorectal cancer, tumour budding, a process likened to epithelial mesenchymal transition, is an adverse prognostic factor which is rarely found in tumours with high‐level microsatellite instability (MSI‐H). Cases with MSI‐H or high‐level CpG island methylator phenotype (CIMP‐H) have similar histomorphological features, yet seemingly opposite prognosis. We hypothesized that tumour budding is related to CIMP, thus partially explaining this prognostic difference. Methods and results: MSI, KRAS, BRAF, CIMP and 0⁶‐methylguanine‐DNA methyltransferase (MGMT) were investigated in tissues from 127 colorectal cancer patients. Tumour budding was scored using pan‐cytokeratin‐stained whole tissue sections within the densest area of buds (×40). Tumour budding was not associated with KRAS, BRAF, MGMT or CIMP, but was correlated inversely with MSI‐H (P = 0.0049). Multivariate survival time analysis revealed that tumour budding was independent of all five molecular features and was predicted by MSI status [odds ratio (OR): 4.29, 95% confidence interval (CI) 1.5–12.1; P = 0.006)], but not CIMP (OR: 0.81, 95% CI 0.3–2.5; P = 0.714). Conclusions: These findings underline that MSI, rather than CIMP, plays a role in conferring a tumour budding phenotype. Budding retains its unfavourable prognostic effect independently of these five molecular features. Continued efforts to standardize the assessment of tumour budding are necessary to integrate this feature into daily diagnostic routine.     

The serrated pathway to colorectal carcinoma: current concepts and challenges

Approximately 30% of colorectal carcinomas develop via a serrated neoplasia pathway, named for the pattern of crypts in the precursor polyps. Molecular abnormalities consistently involve methylation of CpG islands [CpG island methylator phenotype (CIMP)] of low degree (CIMP‐L) or high degree (CIMP‐H), and activating mutations of the mitogen‐activated protein kinase pathway components BRAF or KRAS. Microsatellite instability (MSI) of a high level (MSI‐H) is often present, allowing for a molecular classification of serrated pathway carcinoma as: (i) BRAF mutant/CIMP‐H with either a) MSI‐H or b) microsatellite stable (MSS); and (ii) KRAS mutant/CIMP‐L/MSS. Precursor polyps include sessile serrated adenoma (SSA), characterized by proximal location, crypt architectural disturbance, and BRAF mutation. Microvesicular hyperplasic polyp (MVHP) probably precedes the development of SSA, and borderline lesions between MVHP and SSA occur. Cytological dysplasia in SSA portends advanced genetic abnormality and a high risk of progression to carcinoma. The traditional serrated adenoma has a predilection for the left colon, tubulovillous architecture, eosinophilic cytoplasm, and frequent KRAS mutation. Serrated morphology carcinoma is a new World Health Organization subtype with well‐differentiated, mucinous or trabecular patterns. It has frequent KRAS or BRAF mutations and a poor prognosis. This review provides an insight into the histology and molecular mechanisms driving these serrated pathway lesions.     

Molecular characterization of “sessile serrated” adenoma to carcinoma transition in six early colorectal cancers

Colorectal cancer (CRC) is a heterogeneous group of diseases both from the morphological and molecular point of view. The sessile serrated adenoma/polyp (SSA/P) has been proposed as the precursor lesion of CRCs characterized by CpG island methylator phenotype (CIMP), DNA mismatch repair (MMR) system deficiency, and BRAF gene mutations. However, no study so far investigated the molecular landscape of “sessile serrated” adenoma to carcinoma transition in early CRCs. Six formalin-fixed paraffin-embedded CRCs developed within SSA/P were profiled for the immunohistochemical expression of MMR proteins (MLH1, MSH2, MSH6, PMS2, and Ep-CAM), p16, and β-catenin. DNA was extracted from the two components of each sample, after microdissection, and characterized for CIMP status and by applying a custom hotspot multigene mutational profiling of 164 hotspot regions of eleven CRC-associated genes (AKT1, APC, BRAF, CTNNB1, KIT, KRAS, NRAS, PDGFRA, PIK3CA, PTEN, and TP53). Five out of the six CRCs shared the same molecular profile (i.e. CIMP positive, MSI status, and BRAF mutation) with their SSA/P components. One out of five CRCs was also APC mutated, whereas another one showed an additional TP53 mutation. The remaining case was CIMP negative and MMR proficient in both the components, harbored a BRAF mutation in the SSA/P counterpart, whereas the CRC one was APC and TP53 mutated and showed p16 and β-catenin dysregulation. This study provides the molecular evidence that SSA/P, even without cytological dysplasia, is a precursor lesion of CRC and that conventional CRC might arise from mixed polyp.     

Molecular subtyping of gastric cancer combining genetic and epigenetic anomalies provides distinct clinicopathological features and prognostic impacts

Both genetic and epigenetic abnormalities play important roles in gastric cancer (GC) development. We investigated whether the molecular subtypes of gastric cancer by combining genetic and epigenetic anomalies define its clinicopathological features and prognosis. The CpG island methylator phenotype (CIMP), MLH1 methylation, TP53, and KRAS mutation statuses were characterized in 214 GCs in relation to their clinicopathological features and prognosis. The molecular subtypes based on CIMP and TP53 hot spot mutation status (R175, G245, R248, R273, and R282) best predicted prognosis of GC. These subtypes contained 120 CIMP‐positive (CIMP+) TP53 hot spot mutation‐negative (TP53 hot spot–) cases, 81 CIMP‐negative (CIMP–) TP53 hot spot– cases, 8 CIMP+TP53 hot spot mutation‐positive (TP53 hot spot+) cases, and 5 CIMP– TP53 hot spot+ cases. The CIMP–TP53 hot spot+ group presented the worst overall survival (OS) and progression‐free survival (PFS), followed by the CIMP+TP53 hot spot+, CIMP–TP53 hot spot– and CIMP+TP53 hot spot– groups (both P < 0.0001). These subtypes also correlated well with several aggressive clinicopathological features in that order. The molecular subtypes were independent factors for predicting overall survival (hazard ratio = 1.66, 95% CI = 1.07–2.57, P = 0.006). The molecular subtypes combining the CIMP and TP53 hot spot mutation status provide distinct clinicopathological features and prognostic impacts in GC.     

Clinicopathological features of CpG island methylator phenotype-positive colorectal cancer and its adverse prognosis in relation to KRAS/BRAF mutation

CpG island methylator phenotype (CIMP) is a recently described subset of colorectal cancers (CRC) with widespread methylation of multiple promoter CpG islands. But the prognostic implication of CIMP in CRC has not been clarified. Thus, the aim of the present study was to differentiate the unique characteristics of CIMP from those of microsatellite instability (MSI)-high CRC, especially with regard to prognosis. CIMP, MSI, and mutations of KRAS codons 12 and 13 and of BRAF codon 600 were evaluated in 134 sporadic CRC. Patient survival and other clinicopathological variables were correlated with CIMP or genetic changes. High CIMP, high MSI, and mutations in KRAS or BRAF were detected in 31.3%, 14.2%, 33.6%, and 4.5% of overall CRC, respectively. High CIMP was closely associated with MSI and BRAF mutation but not with KRAS mutation. CIMP-high, microsatellite-stable (MSS) CRC were significantly associated with proximal location and nodal metastasis and had close but non-significant associations with liver metastasis. A worse clinical outcome was found for CIMP-high, MSS CRC with KRAS/BRAF mutation but not for those lacking KRAS/BRAF mutation. The findings support the contention that CIMP-high CRC have distinct clinicopathological and epidemiological features and suggest that the alleged poor clinical outcome of CIMP-high CRC patients is closely associated with the presence of KRAS/BRAF mutation.     

Molecular subtypes of glioma identified by genome‐wide methylation profiling

Recent studies have indicated a prognostic role for genome‐wide methylation in gliomas: Tumors that show an overall increase in DNA methylation at CpG sites (CIMP+; CpG island methylator phenotype) have a more favorable prognosis than CIMP? gliomas. Here, we have determined whether methylation profiling can identify more and clinically relevant molecular subtypes of glioma by performing genome‐wide methylation profiling on 138 glial brain tumors of all histological diagnosis. Hopach (Hierarchical ordered partitioning and collapsing hybrid) clustering using the 1,000 most variable CpGs identified three distinct glioma subtypes (C+_1p19q, C+_wt, and C?) and one adult brain subtype. All “C+_1p19q” and “C+_wt” tumors were CIMP+ whereas most (50/54) “C?” tumors were CIMP?. The C? subtype gliomas contained many glioblastomas and all pilocytic astrocytomas. 1p19q LOH was frequent in the C+_1p19q subtype. Other genetic changes (IDH1 mutation and EGFR amplification) and gene‐expression based molecular subtypes also segregated in distinct methylation subtypes, demonstrating that these subtypes are also genetically distinct. Each subtype was associated with its own prognosis: median survival for C?, C+_1p19q, and C+_wt tumors was 1.18, 5.00, and 2.62 years, respectively. The prognostic value of these methylation subtypes was validated on an external dataset from the TCGA. Analysis of recurrences of 14 primary tumors samples indicates that shifts between some C+_wt and C+_1p/19q tumors can occur between the primary and recurrent tumor, but CIMP status remained stable. Our data demonstrate that methylation profiling identifies at least three prognostically relevant subtypes of glioma that can aid diagnosis and potentially guide treatment for patients. © 2013 Wiley Periodicals, Inc.     

Annexin A10 expression correlates with serrated pathway features in colorectal carcinoma with microsatellite instability

Annexin A10 (ANXA10) has recently been identified as a marker of sessile serrated adenomas/polyps of the colorectum. Although the serrated neoplasia pathway is thought to be involved in the majority of microsatellite instability‐high (MSI‐H) sporadic colorectal carcinomas (CRCs), the clinicopathological implications of ANXA10 expression in CRC are unknown. Here, we evaluated ANXA10 expression status in 168 MSI‐H CRCs by immunohistochemistry. Among 168 MSI‐H CRCs, nuclear staining for ANXA10 in tumor cells revealed 28 cases (17%) with ANXA10‐positive (ANXA10+) tumors. Most of the ANXA10+ tumors were located in the proximal colon (96%, p < 0.001). The ANXA10+ phenotype in MSI‐H CRC was significantly associated with female gender (68%, p = 0.016), CpG island methylator phenotype‐high (CIMP‐H) (68%, p < 0.001), MLH1 promoter hypermethylation (61%, p < 0.001), loss of MLH1 expression (82%, p = 0.019), and wild‐type KRAS status (96%, p = 0.023). Survival analysis revealed no prognostic significance of ANXA10 expression in MSI‐H CRC. In conclusion, ANXA10+ MSI‐H colon carcinomas are characterized by serrated pathway features, including proximal location, female predominance, and high frequencies of CIMP‐H status and MLH1 methylation.     

Methylation‐induced loss of miR‐484 in microsatellite‐unstable colorectal cancer promotes both viability and IL‐8 production via CD137L

Colorectal cancer (CRC) exhibiting MSI (microsatellite instability) represents a well‐defined subtype characterized by a deficient mismatch repair pathway and typical clinico‐pathological features. Our objective was to identify the entire miRNome and its molecular pathological roles in MSI CRCs. We profiled miRNA expression in MSI CRCs and compared it with MSS counterparts. Microarray and qRT‐PCR analysis identified eight miRNAs that could distinguish the MSI status of CRCs. MiR‐484 was the most significantly decreased miRNA in MSI CRCs, primarily mediated by the CpG island methylator phenotype. MiR‐484 functions as a tumour suppressor to inhibit MSI CRC cell viability in vitro and in vivo. Moreover, miR‐484 repressed CD137L expression and thereby attenuated IL‐8 production by MSI CRC cells. Our results contribute to a better understanding of the roles of dysregulated miRNAs in the distinct phenotypic features of MSI CRCs and indicate an option for early diagnosis and gene therapy for these patients. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

CpG island hypermethylator phenotype in gastric carcinoma and its clinicopathological features

Gastric carcinoma (GC) is one of the human cancers in which promoter CpG island hypermethylation is frequently found. CpG island methylator phenotype (CIMP) refers to a subset of GCs which harbor concordant methylation of multiple promoter CpG island loci. However, little is known regarding clinicopathological features of CIMP-positive (CIMP-high) GC. Our study aimed to characterize clinicopathological features of CIMP-high GC. We analyzed 196 cases of GCs for their methylation status in 16 cancer-specific CpG island loci using MethyLight assay and arbitrarily defined CIMP-high GC as those with methylation at 13 or more CpG island loci. With exclusion of microsatellite instability-positive GC and EBV-positive GC from the analysis, CIMP-high GC (n = 10, 6.7%) demonstrated tendency toward higher cancer stage, infiltrative growth type, poor differentiation, and diffuse or mixed type of Lauren classification. CIMP-high GC showed significantly shortened survival compared with that of CIMP-negative GC. When CIMP-negative GC (methylation at 12 or less) was divided into CIMP-intermediate and CIMP-low (methylation at one or none), CIMP-low exhibited better clinical outcome than CIMP-intermediate. Hypermethylation at 14 CpG island loci or more was closely associated with poor clinical outcome and found to be an independent prognostic factor. Our findings that CIMP-high GCs were featured with characteristic clinicopathological parameters, including poor prognosis are distinct from previous studies. More extensive, large-scaled study is necessary to validate the findings of the present study.     

Cyclin D1 is frequently overexpressed in microsatellite unstable colorectal cancer, independent of CpG island methylator phenotype

Aims: Cyclin D1 and cyclin‐dependent kinases are commonly activated in colorectal cancer. Microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) are important molecular classifiers in colorectal cancer. The aim was to clarify the relationship between cyclin D1, MSI and CIMP. Methods and results: Among 865 colorectal cancers with MSI and CIMP data, 246 tumours (28.4%) showed cyclin D1 overexpression by immunohistochemistry. DNA methylation in p14 and eight CIMP‐specific promoters (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) was quantified by real‐time polymerase chain reaction (MethyLight). Both MSI‐high and CIMP‐high were associated with cyclin D1 overexpression (P < 0.0001). After tumours were stratified by MSI and CIMP status, the relationship between MSI‐high and cyclin D1 persisted (P ≤ 0.02), whereas the relationship between CIMP‐high and cyclin D1 did not. Cyclin D1 overexpression was correlated with BRAF mutation (P = 0.0001), p27 loss (P = 0.0007) and p16 loss (P = 0.02), and inversely with p53 expression (P = 0.0002) and p21 loss (P < 0.0001). After stratification by MSI status, the inverse relationship between cyclin D1 and p21 loss still persisted (P < 0.008). Conclusions: Cyclin D1 activation is associated with MSI and inversely with p21 loss in colorectal cancers. Cyclin D1 may play an important role in the development of MSI‐high tumours, independent of CIMP status.     

WRN promoter methylation possibly connects mucinous differentiation, microsatellite instability and CpG island methylator phenotype in colorectal cancer.

Werner syndrome is a premature aging syndrome characterized by early onset of cancer and abnormal cellular metabolism of glycosaminoglycan. The WRN helicase plays an important role in the maintenance of telomere function. WRN promoter methylation and gene silencing are common in colorectal cancer with the CpG island methylator phenotype (CIMP), which is associated with microsatellite instability (MSI) and mucinous tumors. However, no study has examined the relationship between mucinous differentiation, WRN methylation, CIMP and MSI in colorectal cancer. Utilizing 903 population-based colorectal cancers and real-time PCR (MethyLight), we quantified DNA methylation in WRN and eight other promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) known to be specific for CIMP. Supporting WRN as a good CIMP marker, WRN methylation was correlated well with CIMP-high diagnosis (> or =6/8 methylated promoters), demonstrating 89% sensitivity and 81% specificity. WRN methylation was associated with the presence of any mucinous component and > or =50% mucinous component (P<0.0001). Because both MSI and CIMP were associated with mucinous tumors and WRN methylation, we stratified tumors into 9 MSI/CIMP subtypes, to examine whether the relationship between WRN methylation and mucin still persisted. In each MSI/CIMP subtype, tumors with mucinous component were persistently more common in WRN-methylated tumors than WRN-unmethylated tumors (P=0.004). No relations of WRN methylation with other variables (age, sex, tumor location, poor differentiation, signet ring cells, lymphocytic reactions, KRAS, BRAF, p53, p21 or 18q loss of heterozygosity) persisted after tumors were stratified by CIMP status. In conclusion, WRN methylation is associated with mucinous differentiation independent of CIMP and MSI status. Our data suggest a possible role of WRN methylation in mucinous differentiation, and may provide explanation to the enigmatic association between mucin and MSI/CIMP.     

MSI status is associated with distinct clinicopathological features in BRAF mutation colorectal cancer: A systematic review and meta-analysis

BackgroundMicrosatellite stable (MSS) BRAF p.V600E mutation colorectal cancer (BRAF-CRC) has a poor prognosis, whereas microsatellite instability (MSI) in BRAF-CRC is associated with a favorable prognosis. Although usually considered a single clinical entity, the MSI BRAF-CRC subtype shows some distinct characteristics in comparison with the MSS BRAF-CRC subtype.MethodsWe conducted a meta-analysis to investigate the influence of clinicopathological features on MSI status in BRAF-CRC. We searched publications up to March 2019 from PubMed, Embase, and the Cochrane Library. The effect of MSI status on outcome parameters was assessed using odds ratios (ORs) with 95% confidence intervals (CIs) and fixed- or random-effects models according to the heterogeneity.ResultsAfter reviewing 2839 reports, 16 eligible studies including 1381 patients with BRAF-CRC met the criteria. The MSI BRAF-CRC subtype was associated with older age, female sex (OR = 1.70; 95% CI = 1.35–2.14; P < 0.00001), proximal tumor location (OR = 5.10; 95% CI = 3.70–7.03; P < 0.00001), early TNM stage (OR = 5.28; 95% CI = 3.93–7.09; P < 0.00001), and poor differentiation (OR = 2.29; 95% CI = 1.60–3.28; P < 0.00001).ConclusionsMSI was significantly correlated with distinct favorable clinicopathological characteristics in BRAF-CRC. These results suggest that MSI status should be considered as a stratification factor for better management of the BRAF-CRC.     

Nuclear maspin expression correlates with the CpG island methylator phenotype and tumor aggressiveness in colorectal cancer

It has been suggested that nuclear expression of maspin (mammary serine protease inhibitor; also known as SERPINB5) in colorectal cancer (CRC) is associated with proximal colonic tumor location, mucinous and poorly differentiated histology, microsatellite instability-high (MSI-H), and poor prognosis. Based on these findings, there may be a potential association between nuclear maspin expression and the CpG island methylator phenotype (CIMP) in CRC, but no study has elucidated this issue. Here, we evaluated maspin protein expression status by immunohistochemistry in 216 MSI-H CRCs. CIMP status was also determined by methylation-specific quantitative PCR method (MethyLight) using eight CIMP markers (MLH1, NEUROG1, CRABP1, CACNA1G, CDKN2A (p16), IGF2, SOCS1, and RUNX3) in 216 MSI-H CRCs. Associations between maspin expression status and various pathological, molecular, and survival data were statistically analyzed. Among the 216 MSI-H CRCs, 111 (51%) cases presented nuclear maspin-positive tumors. Nuclear maspin-positive MSI-H CRCs were significantly associated with proximal tumor location (P = 0.003), tumor budding (P < 0.001), lymphovascular invasion (P = 0.001), perineural invasion (P = 0.008), absence of peritumoral lymphoid reaction (P = 0.045), lymph node metastasis (P = 0.003), distant metastasis (P = 0.005), advanced AJCC/UICC stage (stage III/IV) (P = 0.001), and CIMP-high (CIMP-H) status (P < 0.001). Patients with nuclear maspin-positive tumors showed worse disease-free survival than patients with nuclear maspin-negative tumors (log-rank P = 0.025). In conclusion, nuclear maspin expression is molecularly associated with CIMP-H rather than MSI-H, and clinicopathologically correlates with tumor aggressiveness in CRC.     

Serrated polyps of the large intestine

Serrated polyps of the large intestine comprise a family of lesions bearing some histological similarities, including an overall serrated configuration caused at least in part by inhibition of apoptosis by mutations in one of two genes. Over the past decade, it has become apparent that these lesions can be subdivided by histological criteria into lesions with differing degrees of relationship to the development of carcinoma, including sporadic microsatellite instable (MSI) carcinomas and probably carcinomas demonstrating the CpG island methylator phenotype (CIMP), which includes both MSI and microsatellite stable tumors. These differing histological subtypes can in part predict some of the molecular features of these lesions, and the combination of histological and molecular features is beginning to give us better insight into the potential natural history and therefore management of these lesions. This review will present the histological classification of these lesions, relate that histological classification to molecular aspects of the lesions, and present recommendations for management.     

Novel application of structural equation modeling to correlation structure analysis of CpG island methylation in colorectal cancer

The CpG island methylator phenotype (CIMP-high, CIMP1) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. Recent evidence suggests the presence of KRAS mutation-associated CIMP subtype (CIMP-low, CIMP2). We used cluster analysis, principal component analysis (PCA), and structural equation modeling (SEM), a novel strategy, to decipher the correlation structure of CpG island hypermethylation. Using a database of 861 colon and rectal cancers, DNA methylation at 16 CpG islands [CACNA1G, CDKN2A (p16/ink4a), CHFR, CRABP1, HIC1, IGF2, IGFBP3, MGMT, MINT-1, MINT-31, MLH1, NEUROG1, p14 (CDKN2A/arf), RUNX3, SOCS1, and WRN] was quantified by real-time PCR. Tumors were categorized into three groups: Group 1 with wild-type KRAS/BRAF (N = 440); Group 2 with mutant KRAS and wild-type BRAF (N = 308); and Group 3 with wild-type KRAS and mutant BRAF (N = 107). Tumors with mutant KRAS/BRAF (N = 6) were excluded. In unsupervised hierarchical clustering analysis, all but six markers (CACNA1G, IGF2, RUNX3, MGMT, MINT-1, and SOCS1) were differentially clustered with CIMP-high and CIMP-low according to KRAS and BRAF status. In SEM, the correlation structures between CIMP, locus-specific CpG island methylation, and MSI differed according to KRAS and BRAF status, which was consistent with PCA results. In conclusion, KRAS and BRAF mutations appear to differentially influence correlation structure of CpG island methylation. Our novel data suggest two distinct perturbations, resulting in differential locus-specific propensity of CpG methylation.     

Distinct patterns of promoter CpG island methylation of breast cancer subtypes are associated with stem cell phenotypes

Although DNA methylation profiles in breast cancer have been connected to breast cancer molecular subtype, there have been no studies of the association of DNA methylation with stem cell phenotype. This study was designed to evaluate the promoter CpG island methylation of 15 genes in relation to breast cancer subtype, and to investigate whether the patterns of CpG island methylation in each subtype are associated with their cancer stem cell phenotype represented by CD44+/CD24- and ALDH1 expression. We performed MethyLight analysis of the methylation status of 15 promoter CpG island loci involved in breast cancer progression (APC, DLEC1, GRIN2B, GSTP1, HOXA1, HOXA10, IGF2, MT1G, RARB, RASSF1A, RUNX3, SCGB3A1, SFRP1, SFRP4, and TMEFF2) and determined cancer stem cell phenotype by CD44/CD24 and ALDH1 immunohistochemistry in 36 luminal A, 33 luminal B, 30 luminal-HER2, 40 HER2 enriched, and 40 basal-like subtypes of breast cancer. The number of CpG island loci methylated differed significantly between subtypes, and was highest in the luminal-HER2 subtype and lowest in the basal-like subtype. Methylation frequencies and levels in 12 of the 15 genes differed significantly between subtypes, and the basal-like subtype had significantly lower methylation frequencies and levels in nine of the genes than the other subtypes. CD44+/CD24- and ALDH1+ putative stem cell populations were most enriched in the basal-like subtype. Methylation of promoter CpG islands was significantly lower in CD44+/CD24-cell (+) tumors than in CD44+/CD24-cell (-) tumors, even within the basal-like subtype. ALDH1 (+) tumors were also less methylated than ALDH1 (-) tumors. Our findings showed that promoter CpG island methylation was different in relation to breast cancer subtype and stem cell phenotype of tumor, suggesting that breast cancers have distinct patterns of CpG island methylation according to molecular subtypes and these are associated with different stem cell phenotypes of the tumor.     

Comprehensive analysis of CpG island methylator phenotype (CIMP)-high, -low, and -negative colorectal cancers based on protein marker expression and molecular features

CpG island methylator phenotype (CIMP) is being investigated for its role in the molecular and prognostic classification of colorectal cancer patients but is also emerging as a factor with the potential to influence clinical decision-making. We report a comprehensive analysis of clinico-pathological and molecular features (KRAS, BRAF and microsatellite instability, MSI) as well as of selected tumour- and host-related protein markers characterizing CIMP-high (CIMP-H), -low, and -negative colorectal cancers. Immunohistochemical analysis for 48 protein markers and molecular analysis of CIMP (CIMP-H: ≥ 4/5 methylated genes), MSI (MSI-H: ≥ 2 instable genes), KRAS, and BRAF were performed on 337 colorectal cancers. Simple and multiple regression analysis and receiver operating characteristic (ROC) curve analysis were performed. CIMP-H was found in 24 cases (7.1%) and linked (p < 0.0001) to more proximal tumour location, BRAF mutation, MSI-H, MGMT methylation (p = 0.022), advanced pT classification (p = 0.03), mucinous histology (p = 0.069), and less frequent KRAS mutation (p = 0.067) compared to CIMP-low or -negative cases. Of the 48 protein markers, decreased levels of RKIP (p = 0.0056), EphB2 (p = 0.0045), CK20 (p = 0.002), and Cdx2 (p < 0.0001) and increased numbers of CD8+ intra-epithelial lymphocytes (p < 0.0001) were related to CIMP-H, independently of MSI status. In addition to the expected clinico-pathological and molecular associations, CIMP-H colorectal cancers are characterized by a loss of protein markers associated with differentiation, and metastasis suppression, and have increased CD8+ T-lymphocytes regardless of MSI status. In particular, Cdx2 loss seems to strongly predict CIMP-H in both microsatellite-stable (MSS) and MSI-H colorectal cancers. Cdx2 is proposed as a surrogate marker for CIMP-H.     

Correlation of CpG island methylator phenotype with poor prognosis in hepatocellular carcinoma

CpG island methylator phenotype (CIMP), in which multiple genes are concurrently methylated, is an important mechanism in hepatocellular carcinoma development. We determined a hypermethylation profile in hepatocellular carcinoma (HCC).We examined the promoter methylation status of 10 genes in 60 cases of hepatocellular carcinoma (HCC), 60 cases of paired non-tumor tissues, and 6 cases of normal tissues by methylation-specific PCR.The average methylated gene numbers were significantly different between HCC and nontumor tissues (p < 0.001). We found metastasis, γ-glutamyl transpeptidase (GGT) and tumor node metastasis (TNM) stage were significantly different among patients with different CIMP status. Patients with high frequency CIMP tumors had significantly worse survival than patients with intermediate frequency or no CIMP tumors (p < 0.01 and p < 0.05, respectively).Our results suggested that CIMP could serve as a molecular marker of late stage and poorly prognostic HCC development.     

Molecular analysis of isolated tumor glands from endometrial endometrioid adenocarcinomas

We studied the extensive molecular alterations of endometrial endometrioid adenocarcinoma (EEA) using a crypt isolation method. We analyzed copy number variation (CNV) using a single nucleotide polymorphism (SNP) array, genetic mutations (KRAS, BRAF, p53, PIK3CA), DNA methylation and microsatellite instability (MSI) status. In addition, loss of PTEN protein expression was examined. Increased chromosome copy numbers of 1q21.2–44 (22%) and 10q11.21–23.31 (28%) were seen relatively frequently in EEA, and copy‐neutral loss of heterozygosity (LOH) was also observed in 10q22.1–26.3 (22%). The CNV patterns of EEA were classified into four groups through hierarchical cluster analysis. Cluster 1 had many CNVs of 10q, and cluster 2 was characterized by MSI status. In cluster 3, increased CNVs of 1q were often seen. In cluster 4, p53 mutations were detected. KRAS and PIK3CA mutations and reduced PTEN protein expression were common to all groups. On the other hand, CpG island methylator phenotype (CIMP) was rare in all groups. The data indicated an association with chromosomal gain of 1q and 10q or 10q copy‐neutral LOH in some cases. We suggest that EEA consists of four groups that are characterized with molecular alterations.