Reference Samples to Compare Next-Generation Sequencing Test Performance for Oncology Therapeutics and Diagnostics.

OBJECTIVES: Diversity of laboratory-developed tests (LDTs) using next-generation sequencing (NGS) raises concerns about their accuracy for selection of targeted therapies. A working group developed a pilot study of traceable reference samples to measure NGS LDT performance among a cohort of clinical laboratories. METHODS: Human cell lines were engineered via CRISPR/Cas9 and prepared as formalin-fixed, paraffin-embedded cell pellets ("wet" samples) to assess the entire NGS test cycle. In silico mutagenized NGS sequence files ("dry" samples) were used to assess the bioinformatics component of the NGS test cycle. Single and multinucleotide variants (n = 36) of KRAS and NRAS were tested at 5% or 15% variant allele fraction to determine eligibility for therapy with the EGFR inhibitor panitumumab in the setting of metastatic colorectal cancer. RESULTS: Twenty-one (21/21) laboratories tested wet samples; 19 of 21 analyzed dry samples. Of the laboratories that tested both the wet and dry samples, 7 (37%) of 19 laboratories correctly reported all variants, 3 (16%) of 19 had fewer than five errors, and 9 (47%) of 19 had five or more errors. Most errors were false negatives. CONCLUSIONS: Genetically engineered cell lines and mutagenized sequence files are complementary reference samples for evaluating NGS test performance among clinical laboratories using LDTs. Variable accuracy in detection of genetic variants among some LDTs may identify different patient populations for targeted therapy.

Clinical validation of the next-generation sequencing-based Extended RAS Panel assay using metastatic colorectal cancer patient samples from the phase 3 PRIME study.

PURPOSE: To validate a next-generation sequencing (NGS)-based companion diagnostic using the MiSeqDx® sequencing instrument to simultaneously detect 56 RAS mutations in DNA extracted from formalin-fixed paraffin-embedded metastatic colorectal cancer (mCRC) tumor samples from the PRIME study. The test's ability to identify patients with mCRC likely to benefit from panitumumab treatment was assessed. METHODS: Samples from PRIME, which compared first-line panitumumab + FOLFOX4 with FOLFOX4, were processed according to predefined criteria using a multiplex assay that included input DNA qualification, library preparation, sequencing, and the bioinformatics reporting pipeline. NGS mutational analysis of KRAS and NRAS exons 2, 3, and 4 was performed and compared with Sanger sequencing. RESULTS: In 441 samples, positive percent agreement of the Extended RAS Panel with Sanger sequencing was 98.7% and negative percent agreement was 97.6%. For clinical validation (n = 528), progression-free survival (PFS) and overall survival (OS) were compared between patients with RAS mutations (RAS Positive) and those without (RAS Negative). Panitumumab + FOLFOX4 improved PFS in RAS Negative patients (P = 0.02). Quantitative interaction testing indicated the treatment effect (measured by the hazard ratio of panitumumab + FOLFOX4 versus FOLFOX4) differed for RAS Negative versus RAS Positive for PFS (P = 0.0038) and OS (P = 0.0323). CONCLUSIONS: NGS allows for broad, rapid, highly specific analyses of genomic regions. These results support use of the Extended RAS Panel as a companion diagnostic for selecting patients for panitumumab, and utilization is consistent with recent clinical guidelines regarding mCRC RAS testing. Overall, approximately 13% more patients were detected with the Extended RAS Panel versus KRAS exon 2 alone. CLINICAL TRIAL REGISTRY IDENTIFIER: NCT00364013 (ClinicalTrials.gov).

Final Analysis of Outcomes and RAS/BRAF Status in a Randomized Phase 3 Study of Panitumumab and Best Supportive Care in Chemorefractory Wild Type KRAS Metastatic Colorectal Cancer.

INTRODUCTION: Tumor rat sarcoma gene (RAS) status is a negative predictive biomarker for anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer (mCRC). We analyzed outcomes according to RAS and v-Raf murine sarcoma viral oncogene homolog B (BRAF) mutational status, and evaluated early tumor shrinkage (ETS) and depth of response (DpR) for patients with wild type RAS. PATIENTS AND METHODS: Patients with confirmed metastatic colon or rectum adenocarcinoma, wild type Kristen rat sarcoma gene tumor exon 2 status, clinical/radiologic disease progression or toxicity during irinotecan or oxaliplatin treatment, and no previous anti-EGFR therapy were randomized 1:1 to receive best supportive care (BSC) with or without panitumumab (6.0 mg/kg, intravenously, on day 1 of each 14-day cycle) in this open-label, multicenter, phase III study (20100007). RAS and BRAF mutation status were determined using Sanger sequencing. ETS was evaluated as maximum percentage change from baseline to week 8; DpR was calculated as the percentage change for tumor shrinkage at nadir versus baseline. RESULTS: Overall, 270 patients had RAS wild type mCRC (panitumumab with BSC, n = 142; BSC, n = 128). For patients with wild type RAS tumors, median overall survival (OS; hazard ratio [HR], 0.72; P = .015) and progression-free survival (PFS; HR, 0.45; P < .0001) were improved with panitumumab with BSC versus BSC. Similar improvements were seen for patients with wild type RAS, and wild type BRAF tumors (OS: HR, 0.75; P = .04; PFS: HR, 0.45; P < .0001). Median DpR was 16.9% for the evaluable panitumumab with BSC wild type RAS population. Overall, 69.5% experienced any type of tumor shrinkage at week 8; 38.2% experienced ≥ 20% shrinkage. Similar improvements in OS and PFS were seen with stratification according to ETS. CONCLUSION: This analysis showed that panitumumab improved outcomes in wild type RAS mCRC and indicated that ETS and DpR could be used as additional efficacy markers.

Diagnostic Quality Assurance Pilot: A Model to Demonstrate Comparative Laboratory Test Performance with an Oncology Companion Diagnostic Assay.

This Editorial highlights a model demonstrating laboratory test performance with a companion diagnostic assay.

A phase 3 trial evaluating panitumumab plus best supportive care vs best supportive care in chemorefractory wild-type KRAS or RAS metastatic colorectal cancer.

BACKGROUND: We assessed the treatment effect of panitumumab plus best supportive care (BSC) vs BSC on overall survival (OS) in patients with chemorefractory wild-type KRAS exon 2 metastatic colorectal cancer (mCRC) and report the first prospective extended RAS analysis in a phase 3 trial. METHODS: Patients with wild-type KRAS exon 2 mCRC were randomised 1 : 1 to panitumumab (6 mg kg-1 Q2W) plus BSC or BSC. On-study crossover was prohibited. RAS mutation status was determined by central laboratory testing. The primary endpoint was OS in wild-type KRAS exon 2 mCRC; OS in wild-type RAS mCRC (KRAS and NRAS exons 2, 3, and 4) was a secondary endpoint. RESULTS: Three hundred seventy seven patients with wild-type KRAS exon 2 mCRC were randomised. Median OS was 10.0 months with panitumumab plus BSC vs 7.4 months with BSC (HR=0.73; 95% CI=0.57-0.93; P=0.0096). RAS ascertainment was 86%. In wild-type RAS mCRC, median OS for panitumumab plus BSC was 10.0 vs 6.9 months for BSC (HR=0.70; 95% CI=0.53-0.93; P=0.0135). Patients with RAS mutations did not benefit from panitumumab (OS HR=0.99; 95% CI=0.49-2.00). No new safety signals were observed. CONCLUSIONS: Panitumumab significantly improved OS in wild-type KRAS exon 2 mCRC. The effect was more pronounced in wild-type RAS mCRC, validating previous retrospective analyses.

The State of the Art in Colorectal Cancer Molecular Biomarker Testing.

The number of molecular biomarkers to inform treatment decisions in patients with metastatic colorectal cancer (mCRC) continues to expand and with it the methodologies that can be employed to evaluate these biomarkers. Beyond standard diagnostic and prognostic biomarkers, such as those used for Lynch syndrome, mutations in KRAS exon 2 are well established as predictive for lack of response to the antiepidermal growth factor receptor therapies panitumumab and cetuximab. Recent studies have extended these findings by demonstrating that mutations in KRAS exons 3 and 4 and in NRAS exons 2, 3, and 4 (with all KRAS and NRAS mutations collectively referred to as RAS) are also predictive for treatment outcomes among patients with mCRC receiving panitumumab and cetuximab in combination with chemotherapy or as monotherapy. Consequently, evaluation of these additional loci has been incorporated into current clinical guidelines, and pathologists will need to develop testing procedures and algorithms to reliably and rapidly evaluate RAS status. With the increased number of mutations that must be examined to evaluate the status of RAS and other emerging biomarkers, next-generation sequencing technologies are likely to become increasingly important in mCRC testing. This review describes new considerations for pathologists that have arisen as a consequence of the incorporation of additional biomarker testing into clinical practice for mCRC.

Validation of a real-time PCR-based qualitative assay for the detection of methylated SEPT9 DNA in human plasma.

BACKGROUND: Epi proColon® is a new blood-based colorectal cancer (CRC) screening test designed to determine the methylation status of a promoter region of the SEPT9 (septin 9) gene in cell-free DNA isolated from plasma. We describe the analytical and clinical performance of the test. METHODS: Analytical performance at 4 testing laboratories included determination of limit of detection, precision, and reproducibility of the SEPT9 test. Clinical performance was evaluated in a prospective study by use of samples (n = 1544) from subjects enrolled in the PRESEPT clinical trial. Results were analyzed by comparison with colonoscopy, the reference standard. RESULTS: The limit of detection for methylated SEPT9 DNA was 7.8 pg/mL (95% CI 6-11 pg/mL) corresponding to <2 genome copies of methylated SEPT9 per milliliter of plasma. In the prospective clinical trial, sensitivity for all stages of CRC was 68% (95% CI 53%-80%) and for stage I-III CRC, 64% (48%-77%). Adjusted specificity, on the basis of negative colonoscopy findings, was 80.0% (78%-82%). SIGNIFICANCE: The Epi proColon test is a simple, real-time PCR-based assay for the detection of methylated SEPT9 DNA in blood that may provide a noninvasive CRC screening alternative for people noncompliant with current CRC screening guidelines.

Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer.

BACKGROUND: As screening methods for colorectal cancer (CRC) are limited by uptake and adherence, further options are sought. A blood test might increase both, but none has yet been tested in a screening setting. OBJECTIVE: We prospectively assessed the accuracy of circulating methylated SEPT9 DNA (mSEPT9) for detecting CRC in a screening population. DESIGN: Asymptomatic individuals ≥50 years old scheduled for screening colonoscopy at 32 US and German clinics voluntarily gave blood plasma samples before colon preparation. Using a commercially available assay, three independent blinded laboratories assayed plasma DNA of all CRC cases and a stratified random sample of other subjects in duplicate real time PCRs. The primary outcomes measures were standardised for overall sensitivity and specificity estimates. RESULTS: 7941 men (45%) and women (55%), mean age 60 years, enrolled. Results from 53 CRC cases and from 1457 subjects without CRC yielded a standardised sensitivity of 48.2% (95% CI 32.4% to 63.6%; crude rate 50.9%); for CRC stages I-IV, values were 35.0%, 63.0%, 46.0% and 77.4%, respectively. Specificity was 91.5% (95% CI 89.7% to 93.1%; crude rate 91.4%). Sensitivity for advanced adenomas was low (11.2%). CONCLUSIONS: Our study using the blood based mSEPT9 test showed that CRC signal in blood can be detected in asymptomatic average risk individuals undergoing screening. However, the utility of the test for population screening for CRC will require improved sensitivity for detection of early cancers and advanced adenomas. CLINICAL TRIAL REGISTRATION NUMBER: NCT00855348.

TFAP2E-DKK4 and chemoresistance in colorectal cancer.

BACKGROUND: Chemotherapy for advanced colorectal cancer leads to improved survival; however, predictors of response to systemic treatment are not available. Genomic and epigenetic alterations of the gene encoding transcription factor AP-2 epsilon (TFAP2E) are common in human cancers. The gene encoding dickkopf homolog 4 protein (DKK4) is a potential downstream target of TFAP2E and has been implicated in chemotherapy resistance. We aimed to further evaluate the role of TFAP2E and DKK4 as predictors of the response of colorectal cancer to chemotherapy. METHODS: We analyzed the expression, methylation, and function of TFAP2E in colorectal-cancer cell lines in vitro and in patients with colorectal cancer. We examined an initial cohort of 74 patients, followed by four cohorts of patients (total, 220) undergoing chemotherapy or chemoradiation. RESULTS: TFAP2E was hypermethylated in 38 of 74 patients (51%) in the initial cohort. Hypermethylation was associated with decreased expression of TFAP2E in primary and metastatic colorectal-cancer specimens and cell lines. Colorectal-cancer cell lines overexpressing DKK4 showed increased chemoresistance to fluorouracil but not irinotecan or oxaliplatin. In the four other patient cohorts, TFAP2E hypermethylation was significantly associated with nonresponse to chemotherapy (P<0.001). Conversely, the probability of response among patients with hypomethylation was approximately six times that in the entire population (overall estimated risk ratio, 5.74; 95% confidence interval, 3.36 to 9.79). Epigenetic alterations of TFAP2E were independent of mutations in key regulatory cancer genes, microsatellite instability, and other genes that affect fluorouracil metabolism. CONCLUSIONS: TFAP2E hypermethylation is associated with clinical nonresponsiveness to chemotherapy in colorectal cancer. Functional assays confirm that TFAP2E-dependent resistance is mediated through DKK4. In patients who have colorectal cancer with TFAP2E hypermethylation, targeting of DKK4 may be an option to overcome TFAP2E-mediated drug resistance. (Funded by Deutsche Forschungsgemeinschaft and others.).

Performance of epigenetic markers SEPT9 and ALX4 in plasma for detection of colorectal precancerous lesions.

BACKGROUND: Screening for colorectal cancer (CRC) has shown to reduce cancer-related mortality, however, acceptance and compliance to current programmes are poor. Developing new, more acceptable non-invasive tests for the detection of cancerous and precancerous colorectal lesions would not only allow preselection of individuals for colonoscopy, but may also prevent cancer by removal of precancerous lesions. METHODS: Plasma from 128 individuals (cohort I - exploratory study: 73 cases / 55 controls) was used to test the performance of a single marker, SEPT9, using a real-time quantitative PCR assay. To validate performance of SEPT9, plasma of 76 individuals (cohort II - validation study: 54 cases / 22 controls) was assessed. Additionally, improvement of predictive capability considering SEPT9 and additionally ALX4 methylation was investigated within these patients. RESULTS: In both cohorts combined, methylation of SEPT9 was observed in 9% of controls (3/33), 29% of patients with colorectal precancerous lesions (27/94) and 73% of colorectal cancer patients (24/33). The presence of both SEPT9 and ALX4 markers was analysed in cohort II and was observed in 5% of controls (1/22) and 37% of patients with polyps (18/49). Interestingly, also 3/5 (60%) patients with colorectal cancer were tested positive by the two marker panel in plasma. CONCLUSIONS: While these data confirm the detection rate of SEPT9 as a biomarker for colorectal cancer, they also show that methylated DNA from advanced precancerous colorectal lesions can be detected using a panel of two DNA methylation markers, ALX4 and SEPT9. If confirmed in larger studies these data indicate that screening for colorectal precancerous lesions with a blood-based test may be as feasible as screening for invasive cancer.

Circulating methylated SEPT9 DNA in plasma is a biomarker for colorectal cancer.

BACKGROUND: The presence of aberrantly methylated SEPT9 DNA in plasma is highly correlated with the occurrence of colorectal cancer. We report the development of a new SEPT9 biomarker assay and its validation in case-control studies. The development of such a minimally invasive blood-based test may help to reduce the current gap in screening coverage. METHODS: A new SEPT9 DNA methylation assay was developed for plasma. The assay comprised plasma DNA extraction, bisulfite conversion of DNA, purification of bisulfite-converted DNA, quantification of converted DNA by real-time PCR, and measurement of SEPT9 methylation by real-time PCR. Performance of the SEPT9 assay was established in a study of 97 cases with verified colorectal cancer and 172 healthy controls as verified by colonoscopy. Performance based on predetermined algorithms was validated in an independent blinded study with 90 cases and 155 controls. RESULTS: The SEPT9 assay workflow yielded 1.9 microg/L (CI 1.3-3.0) circulating plasma DNA following bisulfite conversion, a recovery of 45%-50% of genomic DNA, similar to yields in previous studies. The SEPT9 assay successfully identified 72% of cancers at a specificity of 93% in the training study and 68% of cancers at a specificity of 89% in the testing study. CONCLUSIONS: Circulating methylated SEPT9 DNA, as measured in the new (m)SEPT9 assay, is a valuable biomarker for minimally invasive detection of colorectal cancer. The new assay is amenable to automation and standardized use in the clinical laboratory.

Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA methylation assay.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC. METHODOLOGY/PRINCIPAL FINDINGS: Using a PCR assay for analysis of Septin 9 (SEPT9) hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls) and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls). 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48%) and 7/102 controls (7%). In the test study 73/126 CRCs (58%) and 18/183 control samples (10%) were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected) while maintaining 90% specificity (19/183 for controls). Positive rates for plasmas from the other cancers (11/96) and non-cancerous conditions (41/315) were low. The rate of polyp detection (>1 cm) was approximately 20%. CONCLUSIONS/SIGNIFICANCE: Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.

DNA methylation biomarkers for blood-based colorectal cancer screening.

BACKGROUND: Sensitive, specific blood-based tests are difficult to develop unless steps are taken to maximize performance characteristics at every stage of marker discovery and development. We describe a sieving strategy for identifying high-performing marker assays that detect colorectal cancer (CRC)-specific methylated DNA in plasma. METHODS: We first used restriction enzyme-based discovery methods to identify marker candidates with obviously different methylation patterns in CRC tissue and nonpathologic tissue. We then used a selection process incorporating microarrays and/or real-time PCR analysis of tissue samples to further test marker candidates for maximum methylation in CRC tissue and minimum amplification in tissues from both healthy individuals and patients with other diseases. Real-time assays of 3 selected markers were validated with plasma samples from 133 CRC patients and 179 healthy control individuals in the same age range. RESULTS: Restriction enzyme-based testing identified 56 candidate markers. This group was reduced to 6 with microarray and real-time PCR testing. Three markers, TMEFF2, NGFR, and SEPT9, were tested with plasma samples. TMEFF2 methylation was detected in 65% [95% confidence interval, 56%-73%] of plasma samples from CRC patients and not detected in 69% (62%-76%) of the controls. The corresponding results for NGFR were 51% (42%-60%) and 84% (77%-89%); for SEPT9, the values were 69% (60%-77%) and 86% (80%-91%). CONCLUSIONS: The stringent criteria applied at all steps of the selection and validation process enabled successful identification and ranking of blood-based marker candidates.

Comparative DNA methylation analysis in normal and tumour tissues and in cancer cell lines using differential methylation hybridisation.

Immortalized human cancer cell lines are widely used as tools and model systems in cancer research but their authenticity with regard to primary tissues remains a matter of debate. We have used differential methylation hybridisation to obtain comparative methylation profiles from normal and tumour tissues of lung and colon, and permanent cancer cell lines originally derived from these tissues. Average methylation differences only larger than 25% between sample groups were considered for the profiles and with this criterion approximately 1000 probesets, around 2% of the sites represented on the array, indicated differential methylation between normal lung and primary lung cancer tissue, and approximately 700 probesets between normal colon and primary colon cancer tissue. Both hyper- and hypomethylation was found to differentiate normal tissue from cancer tissue. The profiles obtained from these tissue comparisons were found to correspond largely to those from the corresponding cancer cell lines, indicating that the cell lines represent the methylation pattern of the primary tissue rather well. Moreover, the cancer specific profiles were found to be very similar for the two tumour types studied. Tissue specific differential methylation between lung and colon tissues, in contrast, was found to be preserved to a larger extent only in the malignant tissue, but was not preserved well in the cancer cell lines studied. Overall, our data therefore provide further evidence that permanent cell lines are good model systems for cancer specific methylation patterns, but deviate with regard to tissue-specific methylation.

Identification and validation of colorectal neoplasia-specific methylation markers for accurate classification of disease.

Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis.

Aristaless-like homeobox-4 gene methylation is a potential marker for colorectal adenocarcinomas.

BACKGROUND & AIMS: The identification of novel genetic and epigenetic markers indicative of changes in the pathogenesis of colon cancer, along with easier-to-use, more sensitive assay methods, may improve the detection, treatment, and overall prognosis of this malignancy. METHODS: Using methylation-specific arbitrarily primed polymerase chain reaction, a fragment of the Aristaless-like homeobox-4 (ALX4) gene that was highly methylated in colon adenomas and cancer was identified. Methylation of ALX4 was analyzed in colorectal adenomas and cancers, in the liver metastases of patients with colorectal cancer, and in 61 other neoplasias, including gastric, esophageal, and hepatocellular cancer and cholangiocarcinoma. ALX4 methylation was also analyzed in the serum of 30 patients with colon cancer. RESULTS: ALX4 gene methylation was confirmed in colon adenomas (11/13) and more frequently present in primary colorectal cancers (30/47) compared with the normal colon mucosa (0/21) (P < .0001). In addition, ALX4 methylation was frequently observed in adenocarcinomas of the esophagus (12/14), stomach (11/15), and bile ducts (4/5) compared with all other cancers (P < .001). ALX4 gene methylation was also more frequently found in sera of patients with colon cancer compared with noncancer controls (P < .0001). Using a cutoff of 41.4 pg/mL, sensitivity and specificity were 83.3% and 70%, respectively. CONCLUSIONS: Apart from colon adenomas and primary and metastatic colorectal cancers, ALX4 is frequently methylated in adenocarcinomas of the gastrointestinal tract. ALX4 gene methylation in sera of patients with cancer may thus serve as a methylation-specific test for colon and other gastrointestinal cancers.

Hypermethylation of the TPEF/HPP1 gene in primary and metastatic colorectal cancers.

The role of promoter methylation in the process of cancer cell metastasis has, however, not yet been studied. Recently, methylation of the TPEF (transmembrane protein containing epidermal growth factor and follistatin domain) gene was reported in human colon, gastric, and bladder cancer cells. Using the Methylight assay, TPEF/HPP1 gene methylation was assessed in primary colorectal cancers (n = 47), matched normal colon mucosa, as well as in the liver metastasis of 24 patients with colorectal cancer, and compared to the methylation status of the TIMP-3, APC, DAPK, caveolin-2, and p16 genes. TPEF was frequently methylated in primary colorectal cancers (36 of 47) compared to the normal colon mucosa (1 of 21) (P < .0001), and TPEF mRNA expression in colon cancer cell lines was restored after treatment with 5-aza-2'-deoxycytidine. The p16 and APC genes were also frequently methylated in primary colorectal cancers (P < .02) compared to the normal colon mucosa. Interestingly, promoter methylation was significantly more frequent in proximal, nonrectal cancers (P < .05). Furthermore, a high degree of methylation of the TPEF gene was also observed in liver metastasis (19 of 24). In summary, we observed frequent TPEF methylation in primary colorectal cancers and liver metastases, indicating that epigenetic alterations are not only present in the early phases of carcinogenesis, but are also common in metastatic lesions. The high frequency of TPEF methylation in this series of colorectal cancers underscores the importance of epigenetic changes as targets for the development of molecular tests for cancer diagnosis.

DNA methylation markers in patients with gastrointestinal cancers. Current understanding, potential applications for disease management and development of diagnostic tools.

DNA methylation, the modification of a cytosine nucleotide immediately preceding a guanine base in a stretch of DNA, is rapidly gaining strength in the diagnostic field as a powerful tool to be utilized for the discrimination of neoplastic tissue from its healthy counterpart. This epigenetic modification occurs often in the promoter region of genes and is associated with transcriptional silencing of tumor suppressors or other genes important for normal cellular function. These changes have been found to occur at very early stages in the progression of healthy to malignant phenotype in many cancer types. We are taking a targeted approach to finding methylation-based markers that can be used not only for the early detection of cancer but also for determining risk, monitoring patient response to therapy and even determining the degree of aggressiveness of a tumor. In this paper, we review the progress in our understanding of methylation in gastrointestinal tumors, the potential clinical applications of methylation-based markers and our process for the discovery and validation of highly specific and sensitive markers for the use in these applications.

Expressed CpG island sequence tag microarray for dual screening of DNA hypermethylation and gene silencing in cancer cells.

We present a novel concept by using expressed CpG island sequence tags (ECISTs)for dual analysis of DNA methylation and gene expression in cancer cells. ECISTs are present in the genome and are DNA fragments expected to be located in the promoter and first exon region of genes. Their GC-rich segments can be used for screening hypermethylated CpG sites in cancer cells, and their exon-containing portions can be used for measuring levels of the corresponding transcripts. A total of 1162 loci met the criteria of ECISTs from an initial screening of 7776 CpG island tags. This ECIST panel was used to analyze the breast cancer cell line MDA-MB-231, which was treated with a demethylating agent. Microarray profile analysis identified 30 methylation-silenced genes, the expression of which could be directly reactivated by demethylation. An additional group of 96 up-regulated genes was also identified but appeared to be downstream from this epigenetic cascade. Thus, this study shows that the ECIST microarray can be used to differentiate the primary and secondary causes of demethylation and provides an effective tool to elucidate the mechanisms of aberrant DNA methylation in cancer.