WRN Promoter CpG Island Hypermethylation Does Not Predict More Favorable Outcomes for Patients with Metastatic Colorectal Cancer Treated with Irinotecan-Based Therapy.

PURPOSE: WRN promoter CpG island hypermethylation in colorectal cancer has been reported to increase sensitivity to irinotecan-based therapies. We aimed to characterize methylation of the WRN promoter, determine the effect of WRN promoter hypermethylation upon expression, and validate a previous report that WRN promoter hypermethylation predicts improved outcomes for patients with metastatic colorectal cancer (mCRC) treated with irinotecan-based therapy. EXPERIMENTAL DESIGN: WRN methylation status was assessed using methylation-specific PCR and bisulfite sequencing assays. WRN expression was determined using qRT-PCR and Western blotting. WRN methylation status was correlated with overall survival (OS) and progression-free survival (PFS) in 183 patients with mCRC. Among these patients, 90 received capecitabine monotherapy as first-line therapy, and 93 received capecitabine plus irinotecan (CAPIRI) therapy as part of the CAIRO phase III clinical trial. RESULTS: WRN mRNA and WRN protein expression levels were low in colorectal cancer cell lines and in primary colorectal cancer and were largely independent of WRN methylation status. Patients with methylated WRN colorectal cancer had a shorter OS compared with patients who had unmethylated WRN colorectal cancer (HR = 1.6; 95% confidence interval [CI], 1.2-2.2; P = 0.003). Patients with unmethylated WRN showed a significantly longer PFS when treated with CAPIRI compared with capecitabine alone (HR = 0.48; 95% CI, 0.32-0.70; P = 0.0001). In contrast, patients did not benefit from adding irinotecan to capecitabine when WRN was methylated (HR = 1.1; 95% CI, 0.69-1.77; P = 0.7). CONCLUSIONS: WRN expression is largely independent of WRN promoter hypermethylation in colorectal cancer. Moreover, we could not validate the previous finding that WRN promoter hypermethylation predicts improved clinical outcomes of mCRC treated with irinotecan-based therapy and found instead the opposite result. Clin Cancer Res; 22(18); 4612-22. ©2016 AACR.

Biomarkers for detection and prognosis of breast cancer identified by a functional hypermethylome screen.

Breast cancer (BC) is a disease with diverse tumor heterogeneity, which challenges conventional approaches to develop biomarkers for early detection and prognosis. To identify effective biomarkers, we performed a genome-wide screen for functional methylation changes in BC, i.e., genes silenced by promoter hypermethylation, using a functionally proven gene expression approach. A subset of candidate hypermethylated genes were validated in primary BCs and tested as markers for detection and prognosis prediction of BC. We identified 33 cancer specific methylated genes and, among these, two categories of genes: (1) highly frequent methylated genes that detect early stages of BC. Within that category, we have identified the combination of NDRG2 and HOXD1 as the most sensitive (94%) and specific (90%) gene combination for detection of BC; (2) genes that show stage dependent methylation frequency pattern, which are candidates to help delineate BC prognostic signatures. For this category, we found that methylation of CDO1, CKM, CRIP1, KL and TAC1 correlated with clinical prognostic variables and was a significant prognosticator for poor overall survival in BC patients. CKM [Hazard ratio (HR) = 2.68] and TAC1 (HR = 7.73) were the strongest single markers and the combination of both (TAC1 and CKM) was associated with poor overall survival independent of age and stage in our training (HR = 1.92) and validation cohort (HR = 2.87). Our study demonstrates an efficient method to utilize functional methylation changes in BC for the development of effective biomarkers for detection and prognosis prediction of BC.

Protein inhibitor of activated STAT3 expression in lung cancer.

Protein Inhibitor of Activated Signal Transducer and Activators of Transcription 3 (PIAS3) is an endogenous inhibitor of STAT3 transcriptional activity. We have previously demonstrated the concentration-dependent negative regulatory effect of PIAS3 on STAT3 signaling and its capacity to decrease lung cancer proliferation and synergize with epidermal growth factor inhibition. We now investigate PIAS3 expression in both non-small cell lung cancer (NSCLC) cell lines and human resected NSCLC specimens. We also investigated the mechanism by which some lung cancers have significantly decreased PIAS3 expression. Expression of PIAS3 is variable in lung cancer cells lines with 2 of 3 squamous cell carcinoma (SCC) cell lines having no or little PIAS3 protein expression. Similarly, the majority of human SCCs of the lung lack PIAS3 expression by immunohistochemistry; this despite the finding that SCCs have significantly higher levels of PIAS3 mRNA compared to adenocarcinomas. High PIAS3 expression generally correlates with decreased phosphorylated STAT3 in both SCC cell lines and human specimens compatible with the negative regulatory effect of this protein on STAT3 signaling. To investigate this variable expression of PIAS3 we first performed sequencing of the PIAS3 gene that demonstrated single nucleotide polymorphisms but no mutations. Exposure of lung cancer cells to 5-azacytidine and trichostatin A results in a significant increase in PIAS3 mRNA and protein expression. However, methylation-specific PCR demonstrates a lack of CpG island methylation in the promoter region of PIAS3. Exposure of cells to an agent blocking proteosomal degradation results in a significant increase in PIAS3. Our data thus shows that SCC of the lung commonly lacks PIAS3 protein expression and that post-translational modifications may explain this finding in some cases. PIAS3 is a potential therapeutic molecule to target STAT3 pathway in lung cancer.

Identification and validation of the methylated TWIST1 and NID2 genes through real-time methylation-specific polymerase chain reaction assays for the noninvasive detection of primary bladder cancer in urine samples.

BACKGROUND: Accumulating evidence suggests that DNA methylation markers could serve as sensitive and specific cancer biomarkers. OBJECTIVE: To determine whether a panel of methylated genes would have the potential to identify primary bladder cancer (BCa) in voided urine samples. DESIGN, SETTING, AND PARTICIPANTS: A pharmacologic unmasking reexpression analysis in BCa cell lines was initially undertaken to unveil candidate methylated genes, which were then evaluated in methylation-specific polymerase chain reaction (MSP) assays performed on DNA extracted from noncancerous and cancerous bladder tissues. The most frequently methylated genes in cancerous tissues, with 100% specificity, were retained for subsequent MSP analysis in DNA extracted from urine samples to build and validate a panel of potential methylated gene markers. Urine samples were prospectively collected at three urologic centres from patients with histologically proven BCa and processed for use in real-time MSP and cytologic analysis. Patients with nonmalignant urologic disorders were included as controls. MEASUREMENTS: A urine sample was classified as valid when > or = 10 copies of the gene encoding ß-actin were measured in the urine sediment genomic DNA. Sensitivity, specificity, and predictive values of the MSP and cytology tests were assessed and compared. RESULTS AND LIMITATIONS: MSP assays performed on 466 of the 496 (94%) valid urine samples identified two genes, TWIST1 and NID2, that were frequently methylated in urine samples collected from BCa patients, including those with early-stage and low-grade disease. The sensitivity of this two-gene panel (90%) was significantly better than that of cytology (48%), with comparable specificity (93% and 96%, respectively). The positive predictive value and negative predictive value of the two-gene panel was 86% and 95%, respectively. CONCLUSIONS: Detection of the methylated TWIST1 and NID2 genes in urine sediments using MSP provides a highly (> or = 90%) sensitive and specific, noninvasive approach for detecting primary BCa. TRIAL REGISTRATION: BlCa-001 study - EudraCt 2006-003303-40.

Validation of real-time methylation-specific PCR to determine O6-methylguanine-DNA methyltransferase gene promoter methylation in glioma.

Epigenetic silencing of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy, such as with temozolomide, in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test, suitable for high-throughput analysis of small amounts of formalin-fixed, paraffin-embedded tumor tissue. A direct, real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the beta-actin gene, provides a quantitative test result. We analyzed 134 clinical glioma samples, comparing the new test with the previously validated nested gel-based MSP assay, which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results, supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohen's kappa = 0.80; 95% confidence interval, 0.82-0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gel-based MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy.