Shoko Kure

PIK3CA Mutation Is Associated With Poor Prognosis Among Patients With Curatively Resected Colon Cancer

PURPOSE PIK3CA mutation and subsequent activation of the AKT pathway play an important role in colorectal carcinogenesis. However, little is known about the prognostic role of PIK3CA mutation in colon cancer. PATIENTS AND METHODS Using 450 resectable colon cancers (stage I to III) in two independent prospective cohorts, we detected PIK3CA mutation in 82 tumors (18%) by pyrosequencing. Cox proportional hazards models were used to calculate hazard ratios (HRs) of colon cancer-specific and overall mortalities, adjusted for patient characteristics and tumoral molecular features, including the CpG island methylation phenotype, microsatellite instability (MSI), LINE-1 hypomethylation, and p53, CIMP, KRAS and BRAF mutation. RESULTS Compared with patients with PIK3CA wild-type tumors, those with PIK3CA-mutated tumors experienced an increase in colon cancer-specific mortality according to univariate analysis (HR = 1.64; 95% CI, 0.95 to 2.86), which persisted after adjusting for other known or potential risk factors for cancer recurrence (including MSI; multivariate HR = 2.23; 95% CI, 1.21 to 4.11). The effect of PIK3CA mutation on cancer survival seemed to differ according to KRAS mutational status. Among patients with KRAS wild-type tumors, the presence of PIK3CA mutation was associated with a significant increase in colon cancer-specific mortality (HR = 3.80; 95% CI, 1.56 to 9.27). In contrast, PIK3CA mutation conferred no significant effect on mortality among patients with KRAS-mutated tumors (HR = 1.25; 95% CI, 0.52 to 2.96). CONCLUSION Among patients who undergo a curative resection of colon cancer, PIK3CA mutation is associated with shorter cancer-specific survival. The adverse effect of PIK3CA mutation may be potentially limited to patients with KRAS wild-type tumors.

Comprehensive Biostatistical Analysis of CpG Island Methylator Phenotype in Colorectal Cancer Using a Large Population-Based Sample

Background The CpG island methylator phenotype (CIMP) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. Recent investigations have selected 5 promoters (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1) as surrogate markers for CIMP-high. However, no study has comprehensively evaluated an expanded set of methylation markers (including these 5 markers) using a large number of tumors, or deciphered the complex clinical and molecular associations with CIMP-high determined by the validated marker panel. Metholodology/Principal Findings DNA methylation at 16 CpG islands [the above 5 plus CDKN2A (p16), CHFR, CRABP1, HIC1, IGFBP3, MGMT, MINT1, MINT31, MLH1, p14 (CDKN2A/ARF) and WRN] was quantified in 904 colorectal cancers by real-time PCR (MethyLight). In unsupervised hierarchical clustering analysis, the 5 markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1), CDKN2A, CRABP1, MINT31, MLH1, p14 and WRN were generally clustered with each other and with MSI and BRAF mutation. KRAS mutation was not clustered with any methylation marker, suggesting its association with a random methylation pattern in CIMP-low tumors. Utilizing the validated CIMP marker panel (including the 5 markers), multivariate logistic regression demonstrated that CIMP-high was independently associated with older age, proximal location, poor differentiation, MSI-high, BRAF mutation, and inversely with LINE-1 hypomethylation and β-catenin (CTNNB1) activation. Mucinous feature, signet ring cells, and p53-negativity were associated with CIMP-high in only univariate analysis. In stratified analyses, the relations of CIMP-high with poor differentiation, KRAS mutation and LINE-1 hypomethylation significantly differed according to MSI status. Conclusions Our study provides valuable data for standardization of the use of CIMP-high-specific methylation markers. CIMP-high is independently associated with clinical and key molecular features in colorectal cancer. Our data also suggest that KRAS mutation is related with a random CpG island methylation pattern which may lead to CIMP-low tumors.

A Prospective Cohort Study Shows Unique Epigenetic, Genetic, and Prognostic Features of Synchronous Colorectal Cancers

BACKGROUND & AIMS Synchronous colorectal neoplasias (2 or more primary carcinomas identified in the same patient) are caused by common genetic and environmental factors and can be used to study the field effect. Synchronous colon cancers have not been compared with control solitary cancers in a prospective study. METHODS We analyzed data collected from 47 patients with synchronous colorectal cancers and 2021 solitary colorectal cancers (controls) in 2 prospective cohort studies. Tumors samples were analyzed for methylation in LINE-1 and 16 CpG islands (CACNA1G, CDKN2A [p16], CRABP1, IGF2, MLH1, NEUROG1, RUNX3, SOCS1, CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14 [ARF], and WRN); microsatellite instability (MSI); the CpG island methylator phenotype (CIMP); 18q loss of heterozygosity; KRAS, BRAF, and PIK3CA mutations; and expression of beta-catenin, p53, p21, p27, cyclin D1, fatty acid synthase, and cyclooxygenase-2. RESULTS Compared with patients with solitary colorectal cancer, synchronous colorectal cancer patients had reduced overall survival time (log-rank, P = .0048; hazard ratio [HR], 1.71; 95% confidence interval [CI]: 1.17-2.50; P = .0053; multivariate HR, 1.47; 95% CI: 1.00-2.17; P = .049). Compared with solitary tumors, synchronous tumors more frequently contained BRAF mutations (P = .0041), CIMP-high (P = .013), and MSI-high (P = .037). Methylation levels of LINE-1 (Spearman r = 0.82; P = .0072) and CpG island methylation (P < .0001) correlated between synchronous cancer pairs from the same individuals. CONCLUSIONS Synchronous colorectal cancers had more frequent mutations in BRAF, were more frequently CIMP- and MSI-high, and had a worse prognosis than solitary colorectal cancers. Similar epigenomic and epigenetic events were frequently observed within a synchronous cancer pair, suggesting the presence of a field defect.

Colorectal Cancer Expression of Peroxisome Proliferator-Activated Receptor γ (PPARG, PPARgamma) Is Associated With Good Prognosis

BACKGROUND & AIMS The peroxisome proliferator-activated receptor gamma (PPARG, PPARgamma) is a nuclear receptor that regulates expression of mediators of lipid metabolism and the inflammatory response. There is controversy over the pro-oncogenic or antioncogenic effects of PPARG, and little is known about its prognostic significance in colon cancer. METHODS Among 470 patients with colorectal cancer (stages I-IV) identified in 2 independent prospective cohorts, PPARG expression was detected in 102 tumors (22%) by immunohistochemistry. Cox proportional hazards models were used to compute hazard ratios (HRs) of colorectal cancer-specific and overall mortalities, adjusted for patient characteristics and molecular features including cyclooxygenase 2, fatty acid synthase, KRAS, BRAF, PIK3CA, p53, p21, beta-catenin, LINE-1 hypomethylation, microsatellite instability (MSI), and the CpG island methylation phenotype (CIMP). RESULTS Compared with patients with PPARG-negative tumors, patients with PPARG-positive tumors had significantly lower overall mortality, determined by Kaplan-Meier analysis (P=.0047), univariate Cox regression (HR, 0.55; 95% confidence interval [CI], 0.37-0.84; P=.0053), and multivariate analysis (adjusted HR, 0.43; 95% CI, 0.27-0.69; P=.0004). Patients with PPARG-positive tumors experienced lower colorectal cancer-specific mortality (adjusted HR, 0.44; 95% CI, 0.25-0.79; P=.0054). The relationship between PPARG and lower mortality did not appear to be significantly modified by MSI, CIMP, LINE-1, or the other clinical and molecular variables examined (all P(interaction)>.05). CONCLUSIONS Tumor expression of PPARG is independently associated with longer survival of patients. PPARG expression appears to mark an indolent subset of colorectal cancers.

DNMT3B Expression Might Contribute to CpG Island Methylator Phenotype in Colorectal Cancer

Purpose: DNA methyltransferase-3B (DNMT3B) plays an important role in de novo CpG island methylation. Dnmt3b can induce colon tumor in mice with methylation in specific CpG islands. We hypothesized that cellular DNMT3B level might influence the occurrence of widespread CpG island methylation (i.e., the CpG island methylator phenotype, CIMP) in colon cancer. Experimental Design: Utilizing 765 colorectal cancers in two cohort studies, we detected DNMT3B expression in 116 (15%) tumors by immunohistochemistry. We assessed microsatellite instability, quantified DNA methylation in repetitive long interspersed nucleotide element-1 (LINE-1) by Pyrosequencing, eight CIMP-specific promoters [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and eight other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, and WRN) by real-time PCR (MethyLight). Results: Tumoral DNMT3B overexpression was significantly associated with CIMP-high [≥6/8 methylated CIMP-specific promoters; odds ratio (OR), 3.34; 95% confidence interval, 2.11-5.29; P < 0.0001]. The relations between DNMT3B and methylation in 16 individual CpG islands varied substantially (OR, 0.80-2.96), suggesting variable locus-to-locus specificities of DNMT3B activity. DNMT3B expression was not significantly related with LINE-1 hypomethylation. In multivariate logistic regression, the significant relation between DNMT3B and CIMP-high persisted (OR, 2.39; 95% confidence interval, 1.11-5.14; P = 0.026) after adjusting for clinical and other molecular features, including p53, β-catenin, LINE-1, microsatellite instability, KRAS, PIK3CA, and BRAF. DNMT3B expression was unrelated with patient outcome, survival, or prognosis. Conclusions: Tumoral DNMT3B overexpression is associated with CIMP-high in colorectal cancer. Our data support a possible role of DNMT3B in nonrandom de novo CpG island methylation leading to colorectal cancer.

SIRT1 histone deacetylase expression is associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer

The class III histone deacetylase SIRT1 (sir2) is important in epigenetic gene silencing. Inhibition of SIRT1 reactivates silenced genes, suggesting a possible therapeutic approach of targeted reversal of aberrantly silenced genes. In addition, SIRT1 may be involved in the well-known link between obesity, cellular energy balance and cancer. However, a comprehensive study of SIRT1 using human cancer tissue with clinical outcome data is currently lacking, and its prognostic significance is uncertain. Using the database of 485 colorectal cancers in two independent prospective cohort studies, we detected SIRT1 overexpression in 180 (37%) tumors by immunohistochemistry. We examined its relationship to the CpG island methylator phenotype (CIMP), related molecular events, clinical features including body mass index, and patient survival. We quantified DNA methylation in eight CIMP-specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1) and eight other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, and WRN) by MethyLight. SIRT1 overexpression was associated with CIMP-high (≥6 of 8 methylated CIMP-specific promoters, P=0.002) and microsatellite instability (MSI)-high phenotype (P<0.0001). In both univariate and multivariate analyses, SIRT1 overexpression was significantly associated with the CIMP-high MSI-high phenotype (multivariate odds ratio, 3.20; 95% confidence interval, 1.35–7.59; P=0.008). In addition, mucinous component (P=0.01), high tumor grade (P=0.02), and fatty acid synthase overexpression (P=0.04) were significantly associated with SIRT positivity in multivariate analysis. SIRT1 was not significantly related with age, sex, tumor location, stage, signet ring cells, cyclooxygenase-2 (COX-2), LINE-1 hypomethylation, KRAS, BRAF, BMI, PIK3CA, HDAC, p53, β-catenin, COX-2, or patient prognosis. In conclusion, SIRT1 expression is associated with CIMP-high MSI-high colon cancer, suggesting involvement of SIRT1 in gene silencing in this unique tumor subtype.

A Cohort Study of Cyclin D1 Expression and Prognosis in 602 Colon Cancer Cases

Purpose: Cyclin D1 and cyclin-dependent kinases (CDK) are commonly activated in colorectal cancer. The activity of cyclin D1 can be blocked by CDK inhibitors, including p27 (CDKN1B) and p21 (CDKN1A, which is induced by p53). However, prognostic significance of tumoral cyclin D1 remains uncertain, and no previous study has considered potential confounding effect of p53, p21, p27, and related molecular events [microsatellite instability (MSI), CpG island methylator phenotype, and LINE-1 hypomethylation]. Experimental Design: Among 602 colon cancer patients (stage I-IV) in two prospective cohort studies, cyclin D1 overexpression was detected in 330 (55%) tumors by immunohistochemistry. Cox proportional hazard models computed hazard ratios (HR) of colon cancer–specific and overall mortalities, adjusted for patient characteristics and tumoral molecular features, including p53, p21, p27, cyclooxygenase-2, fatty acid synthase, LINE-1 methylation, CpG island methylator phenotype, MSI, BMI, KRAS, and BRAF. Results: Cyclin D1 overexpression was associated with a low cancer-specific mortality in Kaplan-Meier analysis (P = 0.006), and in both univariate Cox regression [unadjusted HR, 0.64; 95% confidence interval (CI), 0.47-0.88; P = 0.0063] and multivariate analyses (adjusted HR, 0.57; 95% CI, 0.39-0.84; P = 0.0048). Similar findings were observed for an overall mortality (adjusted HR, 0.74; 95% CI, 0.57-0.98; P = 0.036). Notably, the effect of cyclin D1 on survival might differ by MSI status (Pinteraction = 0.008). Compared with tumors that were both cyclin D1–negative and MSI-low/microsatellite stable, the presence of either cyclin D1 or MSI-high or both seemed to confer better clinical outcome (adjusted HR point estimates, 0.10-0.65). Conclusions: Cyclin D1 overexpression is associated with longer survival in colon cancer.

A cohort study of STMN1 expression in colorectal cancer: body mass index and prognosis.

OBJECTIVES:STMN1 (stathmin or oncoprotein-18) destabilizes microtubules and reorganizes cytoskeleton, and functions in cell cycle progression and cell migration. STMN1 activity is influenced by p53, p27, and the PI3K/AKT pathway. However, its prognostic significance in colon cancer is uncertain.METHODS:Utilizing 546 colorectal cancers (stage I–IV) from two independent prospective cohort studies (the Nurses’ Health Study and Health Professionals Follow-up Study), STMN1 expression was detected in 297 (54%) tumors by immunohistochemistry. Cox proportional hazard models computed hazard ratios (HRs) of mortality, adjusted for clinical and tumoral features, including microsatellite instability (MSI), CpG island methylation phenotype (CIMP), LINE-1 hypomethylation, KRAS, BRAF, PIK3CA, p53, p21, p27, cyclin D1, β-catenin, fatty acid synthase, FASN, and COX-2.RESULTS:Five-year colorectal cancer–specific survival was 78% in STMN1-positive patients and 76% in STMN1-negative patients (log-rank P=0.30). STMN1-positivity was not significantly associated with cancer-specific survival in univariate analysis with HR of 0.82 (95% confidence interval (CI), 0.59–1.14), which became significant in multivariate analysis (adjusted HR=0.60; 95% CI, 0.41–0.87; P=0.0078). Notably, the prognostic effect of obesity (body mass index, BMI≥30 kg/m2) significantly differed by STMN1 (Pinteraction=0.005). Obesity was associated with high cancer-specific mortality among STMN1-positive patients (adjusted HR=2.36; 95% CI, 1.18–4.69), whereas obesity was not associated with high mortality among STMN1-negative patients (adjusted HR=0.51; 95% CI, 0.24–1.07).CONCLUSIONS:STMN1 overexpression in colorectal cancer is independently associated with improved survival. The adverse prognostic effect of obesity was limited to patients with STMN1-positive tumors. Our findings suggest the presence of a tumor (STMN1)–host (BMI) interaction that potentially determines clinical outcome.

Vitamin D receptor expression is associated with PIK3CA and KRAS mutations in colorectal cancer.

Vitamin D is associated with decreased risks of various cancers, including colon cancer. The vitamin D receptor (VDR) is a transcription factor, which plays an important role in cellular differentiation and inhibition of proliferation. A link between VDR and the RAS–mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K)–AKT pathway has been suggested. However, the prognostic role of VDR expression or its relationship with PIK3CA or KRAS mutation remains uncertain. Among 619 colorectal cancers in two prospective cohort studies, 233 (38%) tumors showed VDR overexpression by immunohistochemistry. We analyzed for PIK3CA and KRAS mutations and LINE-1 methylation by Pyrosequencing, microsatellite instability (MSI), and DNA methylation (epigenetic changes) in eight CpG island methylator phenotype (CIMP)–specific promoters [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by MethyLight (real-time PCR). VDR overexpression was significantly associated with KRAS mutation (odds ratio, 1.55; 95% confidence interval, 1.11-2.16) and PIK3CA mutation (odds ratio, 2.17; 95% confidence interval, 1.36-3.47), both of which persisted in multivariate logistic regression analysis. VDR was not independently associated with body mass index, family history of colorectal cancer, tumor location (colon versus rectum), stage, tumor grade, signet ring cells, CIMP, MSI, LINE-1 hypomethylation, BRAF, p53, p21, β-catenin, or cyclooxygenase-2. VDR expression was not significantly related with patient survival, prognosis, or clinical outcome. In conclusion, VDR overexpression in colorectal cancer is independently associated with PIK3CA and KRAS mutations. Our data support potential interactions between the VDR, RAS–MAPK and PI3K–AKT pathways, and possible influence by KRAS or PIK3CA mutation on therapy or chemoprevention targeting VDR. (Cancer Epidemiol Biomarkers Prev 2009;18(10):2765–72)

1033 Genetic and Epigenetic Features in Synchronous Colorectal Cancer Cases Versus Solitary Cancers, Utilizing 1243 Colorectal Cancer Patients From Two Large Prospective Cohort Studies

Background & Aims: Synchronous colorectal cancers refer to two or more primary colorectal carcinomas detected in a single individual at the time of the first diagnosis of colorectal cancer. Synchronous neoplasias, which arise in a background of common etiologic (genetic or environmental) factors, can provide a unique model to examine molecular aberrations. Previous studies have reported several molecular features in synchronous colorectal cancers. However, in all of these studies, control solitary cases were retrospectively selected, thereby subject to potential selection bias. In this study, we analyzed data collected from 32 patients with synchronous colorectal cancers and 1211 solitary cancers (controls) in 2 well-characterized, large prospective cohort studies. Methods: Tumor tissue specimens were analyzed for methylation in LINE-1 and 16 CpG islands (CACNA1G, CDKN2A [p16], CRABP1, IGF2, MLH1, NEUROG1, RUNX3, SOCS1, CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14 [CDKN2A/ARF], and WRN); microsatellite instability (MSI) status; CpG island methylator phenotype (CIMP) status; KRAS, BRAF, and PIK3CA mutations; and expressions of DNA methyltransferase-3B (DNMT3B) and JC virus T-antigen (JCVT). Results: BRAF mutations were significantly more common in synchronous cancers (30%=9/30; p=0.0080) than in solitary cancers (13%=158/1198). Likewise, synchronous cancers were more commonly CIMP-high (p=0.047) and MSI-high (p=0.030), compared to solitary controls, whereas no association was found between tumor synchronicity and other molecular markers. Considering the confounding effect by age, we constructed a logistic regression model including age, BRAF mutation, CIMP-high, MSI-high and synchronicity status as an outcome variable. As a result, the association between BRAF mutation (p=0.010), CIMP-high (p=0.038), MSIhigh (p=0.033) and tumor synchronicity persisted, while age was no longer associated with tumor synchronicity. Among the 32 synchronous cancer cases, 12 synchronous cases could provide cancer tissues from both of two cancers in synchronous pairs. We found that methylation levels of LINE-1 (Spearman r=0.76; p=0.0062) and levels of CpG island methylation (p<0.0001) correlated between synchronous pairs from the same individuals. Conclusions: Compared with solitary cancers, synchronous colorectal cancers more commonly show BRAF mutations, CIMP-high, and MSI-high. In addition, synchronous colorectal cancer pairs exhibit a significant correlation of LINE-1 methylation levels and concordance in CpG island methylation. Our study may be an important one step forward to elucidate clinical and molecular characteristics of synchronous colorectal cancers.