Prognostic significance of CDKN2A (p16) promoter methylation and loss of expression in 902 colorectal cancers: Cohort study and literature review.

A cyclin-dependent kinase inhibitor CDKN2A (p16/Ink4a) is a tumor suppressor and upregulated in cellular senescence. CDKN2A promoter methylation and gene silencing are associated with the CpG island methylator phenotype (CIMP) in colon cancer. However, prognostic significance of CDKN2A methylation or loss of CDKN2A (p16) expression independent of CIMP status remains uncertain. Using a database of 902 colorectal cancers in 2 independent cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study), we quantified CDKN2A promoter methylation and detected hypermethylation in 269 tumors (30%). By immunohistochemistry, we detected loss of CDKN2A (p16) expression in 25% (200/804) of tumors. We analyzed for LINE-1 hypomethylation and hypermethylation at 7 CIMP-specific CpG islands (CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1); microsatellite instability (MSI); KRAS, BRAF and PIK3CA mutations; and expression of TP53 (p53), CTNNB1 (ß-catenin), CDKN1A (p21), CDKN1B (p27), CCND1 (cyclin D1), FASN (fatty acid synthase) and PTGS2 (cyclooxygenase-2). CDKN2A promoter methylation and loss of CDKN2A (p16) were associated with shorter overall survival in univariate Cox regression analysis [hazard ratio (HR): 1.36, 95% CI: 1.10-1.66, p = 0.0036 for CDKN2A methylation; HR: 1.30, 95% CI: 1.03-1.63, p = 0.026 for CDKN2A (p16) loss] but not in multivariate analysis that adjusted for clinical and tumor variables, including CIMP, MSI and LINE-1 methylation. Neither CDKN2A promoter methylation nor loss of CDKN2A (p16) was associated with colorectal cancer-specific mortality in uni- or multivariate analysis. Despite its well-established role in carcinogenesis, CDKN2A (p16) promoter methylation or loss of expression in colorectal cancer is not independently associated with patient prognosis.

Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis.

Assays to measure DNA methylation, which are important in epigenetic research and clinical diagnostics, typically rely on conversion of unmethylated cytosine to uracil by sodium bisulfite. However, no study has comprehensively evaluated the precision and performance characteristics of sodium bisulfite conversion and subsequent quantitative methylation assay. We developed quantitative real-time polymerase chain reaction (MethyLight) to measure percentage of methylated reference (PMR, ie, degree of methylation) for the MGMT, MLH1, and CDKN2A (p16) promoters. To measure the precision of bisulfite conversion, we bisulfite-treated seven different aliquots of DNA from each of four paraffin-embedded colon cancer samples. To assess run-to-run variation, we repeated MethyLight five times. Bisulfite-to-bisulfite coefficient of variation (CV) of PMR ranged from 0.10 to 0.38 (mean, 0.21), and run-to-run CV of PMR ranged from 0.046 to 0.60 (mean, 0.31). Interclass correlation coefficients were 0.74 to 0.84 for the three loci, indicating good reproducibility. DNA mixing study with methylated and unmethylated DNA showed good linearity of the assay. Of 272 colorectal cancers evaluated, most showed PMR either <1 or >10, and promoter methylation (PMR >4) was tightly associated with loss of respective protein expression (P < 10(-16)). In conclusion, sodium bisulfite conversion and quantitative MethyLight assays have good precision and linearity and can be effectively used for high-throughput DNA methylation analysis on paraffin-embedded tissue.

Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component.

Signet ring cell carcinoma and mucinous carcinoma are distinct subtypes of colorectal adenocarcinoma. The morphologic and molecular spectra of colorectal carcinomas with various signet ring cell components and colorectal carcinomas with various mucinous components, compared to non-mucinous adenocarcinomas, have not been examined. The study groups consisted of 39 carcinomas with various signet ring cell components ('the signet group'), 167 carcinomas with various mucinous components ('the mucinous group'), and 457 nonmucinous adenocarcinoma. We visually estimated the amounts of signet ring cell and mucinous components in tumors, and subclassified the signet and mucinous groups according to the amount of each component (< or = 19, 20-49, and > or = 50%). We sequenced BRAF and KRAS, analyzed for microsatellite instability (MSI) and 18q loss of heterozygosity (LOH), and performed immunohistochemistry for TP53, cyclooxygenase-2 (COX2), MLH1, O-6-methylguanine DNA methyltransferase (MGMT), p16 (CDKN2A), and fatty acid synthase (FASN). Signet ring cell carcinoma (> or = 50% signet ring cell tumors) and < or = 49% signet ring cell tumors showed similar molecular features. Except for MSI and MGMT, > or = 50% mucinous tumors and < or = 49% mucinous tumors also showed similar molecular features. BRAF mutations, MSI, and MLH1 loss were more frequent in both the signet and mucinous groups than nonmucinous carcinoma. More frequent KRAS mutations and less frequent p16 loss and TP53 positivity were observed in the mucinous group than nonmucinous carcinoma. 18q LOH and COX2 overexpression were less common in the signet group than nonmucinous carcinoma. FASN levels were highest in the mucinous group, followed by nonmucinous carcinoma, and lowest in the signet group. In conclusion, a minor (< or = 49%) signet ring cell or mucinous component in colorectal carcinoma suggests molecular features similar to > or = 50% signet ring cell or mucinous carcinoma, respectively. Signet ring cell carcinoma and mucinous carcinoma are related subtypes of colorectal adenocarcinoma, but have molecular features distinct from each other.

Molecular alterations in tumors and response to combination chemotherapy with gefitinib for advanced colorectal cancer.

PURPOSE: Recently, activating mutations of the epidermal growth factor receptor (EGFR) gene were discovered in non-small cell lung cancers sensitive to gefitinib (ZD1839, an EGFR tyrosine kinase inhibitor) but not in gefitinib-resistant cancers. Abnormalities of EGFR and related pathways may have an effect on responsiveness of advanced colorectal cancer to combination chemotherapy with gefitinib. EXPERIMENTAL DESIGN: We examined patients with previously untreated metastatic colorectal cancer, who were enrolled into two phase I/II trials of combination chemotherapy (irinotecan, leucovorin, and 5-fluorouracil) and daily oral gefitinib. We obtained paraffin tissue blocks of primary tumors from 31 patients, sequenced the EGFR, KRAS, and BRAF genes, and did immunohistochemistry for EGFR, phosphorylated AKT1, p53, p21, and p27. RESULTS: Twelve (39%) of the 31 patients experienced a partial objective response to the therapy. A novel EGFR mutation in exon 18 (c.2170G>A, p.Gly724Ser) was identified in only one patient who did not experience an objective tumor response. EGFR immunohistochemistry was not predictive of responsiveness. In contrast, loss of p21 was associated with a higher response rate to therapy (P = 0.05). Moreover, the response rate among patients whose tumors maintained p21 expression and possessed a mutation in p53 was only 9% (1 of 11, P = 0.005). Overexpression of phosphorylated AKT1 also seemed to predict a trend towards resistance to the therapy. CONCLUSIONS: p21 expression in colorectal cancer, especially in combination with p53 mutation, is a predictor of resistance to the combination chemotherapy with gefitinib. Activating EGFR mutations are rare in colorectal cancer and do not seem to confer sensitivity to gefitinib and chemotherapy.

Sensitive sequencing method for KRAS mutation detection by Pyrosequencing.

Both benign and malignant tumors represent heterogenous tissue containing tumor cells and non-neoplastic mesenchymal and inflammatory cells. To detect a minority of mutant KRAS alleles among abundant wild-type alleles, we developed a sensitive DNA sequencing assay using Pyrosequencing, ie, nucleotide extension sequencing with an allele quantification capability. We designed our Pyrosequencing assay for use with whole-genome-amplified DNA from paraffin-embedded tissue. Assessing various mixtures of DNA from mutant KRAS cell lines and DNA from a wild-type KRAS cell line, we found that mutation detection rates for Pyrosequencing were superior to dideoxy sequencing. In addition, Pyrosequencing proved superior to dideoxy sequencing in the detection of KRAS mutations from DNA mixtures of paraffin-embedded colon cancer and normal tissue as well as from paraffin-embedded pancreatic cancers. Quantification of mutant alleles by Pyrosequencing was precise and useful for assay validation, monitoring, and quality assurance. Our Pyrosequencing method is simple, robust, and sensitive, with a detection limit of approximately 5% mutant alleles. It is particularly useful for tumors containing abundant non-neoplastic cells. In addition, the applicability of this assay for DNA amplified by whole-genome amplification technique provides an expanded source of DNA for large-scale studies.