Blood-based RAS mutation testing: concordance with tissue-based RAS testing and mutational changes on progression.

Aim: To determine the concordance between plasma and tissue RAS mutation status in metastatic colorectal cancer patients to gauge whether blood-based testing is a viable alternative. We also evaluated the change in mutation status on progression. Materials/methods: RAS testing was performed on plasma from patients commencing first-line therapy (OncoBEAM™ RAS CEIVD kit). Results were then compared with formalin-fixed paraffin embedded tumor samples. Results: The overall percentage agreement (concordance) was 86.0% (86/100), which demonstrates that blood-based testing is an alternative to tissue-based testing. Reproducibility was 100% between three laboratories and 20% showed changes in their RAS mutational status on progression. Conclusion: These results show good concordance between tissue and plasma samples and suggest the need for longitudinal plasma testing during treatment to guide management decisions.

Development of a diagnostic microarray assay to assess the risk of recurrence of prostate cancer based on PITX2 DNA methylation.

Prostate cancer is among the most common cancers. Although it has a relatively good prognosis, 15 to 30% of men with prostate cancer experience a relapse after radical prostatectomy. Identifying patients with an aggressive tumor will therefore help to improve prostate cancer management. DNA methylation of PITX2 has been established in several studies as a prognostic biomarker for breast and prostate cancer. These case control studies were conducted using research assay components; to facilitate its use in a diagnostic setting, the PITX2 biomarker was transferred to a validated diagnostic platform, the Affymetrix GeneChip System. A customized microarray (Epichip PITX2) was designed using features in high redundancy to ensure a robust determination of the methylation state of the PITX2 promoter. The developed method allowed for accurate assessment of prognosis in prostate cancer patients who underwent radical prostatectomy. Determination of PITX2 methylation in formalin-fixed and paraffin-embedded tissue samples from a cohort of 157 prostatectomy patients resulted in an excellent level of concordance of the clinical classification, as well as the measured output between the research assay and the Epichip PITX2. These analytical performance results describe the Epichip PITX2 as a robust and reliable diagnostic tool for assessing the methylation status of PITX2, enabling an improved outcome prediction in cancer patients following radical prostatectomy.

Multicenter study using paraffin-embedded tumor tissue testing PITX2 DNA methylation as a marker for outcome prediction in tamoxifen-treated, node-negative breast cancer patients.

PURPOSE: We recently reported DNA methylation of the paired-like homeodomain transcription factor 2 (PITX2) gene to be strongly correlated with increased risk of recurrence in node-negative, hormone receptor-positive, tamoxifen-treated breast cancer patients using fresh frozen specimens. Aims of the present study were to establish determination of PITX2 methylation for routine analysis in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue and to test PITX2 DNA methylation as a biomarker for outcome prediction in an independent patient cohort. PATIENTS AND METHODS: Real-time polymerase chain reaction (PCR) technology was validated for FFPE tissue by comparing methylation measurements in FFPE specimens with those in fresh frozen specimens from the same tumor. The impact of PITX2 methylation on time to distant metastasis was then evaluated in FFPE specimens from hormone receptor-positive, node-negative breast cancer patients (n = 399, adjuvant tamoxifen monotherapy). RESULTS: Reproducibility of the PCR assay in replicate measurements (r(s) > or = 0.95; n = 150) and concordant measurements between fresh frozen and FFPE tissues (r(s) = 0.81; n = 89) were demonstrated. In a multivariate model, PITX2 methylation added significant information (hazard ratio = 2.35; 95% CI, 1.20 to 4.60) to established prognostic factors (tumor size, grade, and age). CONCLUSION: PITX2 methylation can be reliably assessed by real-time PCR technology in FFPE tissue. Together with our earlier studies, we have accumulated substantial evidence that PITX2 methylation analysis holds promise as a practical assay for routine clinical use to predict outcome in node-negative, tamoxifen-treated breast cancer, which might allow, based on future validation studies, the identification of low-risk patients who may be treated by tamoxifen alone.