serum-and-plasma-copy-number-detection-using-real-time-pcr

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS

Patients with non-muscle invasive bladder cancer (NMIBC) generally have a high risk of relapsing locally after primary tumor resection. The search for new predictive markers of local recurrence thus represents an important goal for the management of this disease. We studied the copy number variations (CNVs) of 24 oncogenes (MDM4, MYCN, ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2, TOP2A, AURKA, AR and BRAF) using multiplex ligation probe amplification technique to verify their role as predictive markers of recurrence. Formalin-fixed paraffin-embedded tissue samples from 43 patients who underwent transurethral resection of the bladder (TURB) were used; 23 patients had relapsed and 20 were disease-free after 5 years. Amplification frequencies were analyzed for all genes and MDM4 was the only gene that showed significantly higher amplification in non recurrent patients than in recurrent ones (0.65 vs. 0.3; Fisher's test p=0.023). Recurrence-free survival analysis confirmed the predictive role of MDM4 (log-rank test p=0.041). Our preliminary results indicate a putative role for the MDM4 gene in predicting local recurrence of bladder cancer. Confirmation of this hypothesis is needed in a larger cohort of NMIBC patients.

Copy number analysis of 24 oncogenes: MDM4 identified as a putative marker for low recurrence risk in non muscle invasive bladder cancer.

The detection of tumor-specific markers in urine has paved the way for new early noninvasive diagnostic approaches for prostate cancer. We evaluated the DNA integrity in urine supernatant to verify its capacity to discriminate between prostate cancer and benign diseases of the urogenital tract.

Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients.

In the present study, we aimed to evaluate the association of circulating AR copy number (CN) and outcome in a cohort of patients with advanced castration-resistant prostate cancer (CRPC) treated with enzalutamide after docetaxel. Fifty-nine CRPC patients were evaluated. AR CN was analyzed with real-time and digital PCR in the serum collected at starting of treatment. Progressive disease was defined on the basis of Prostate Cancer Working Group 2 criteria. AR CN gain was found in 21 of 59 (36%) patients. Median baseline PSA, alkaline phosphatase and lactate dehydrogenase levels were higher in the AR CN gained group (p = 0.007, p = 0.003, p = 0.0009, respectively). Median PFS of patients with AR CN gain was 2.4 (95%CI: 1.9-3.2) vs. 4.0 months (95%CI: 3.0-6.5) of those with no gain (p = 0.0004). Median OS of patients with AR CN gain was 6.1 (95%CI: 3.4-8.6) vs. 14.1 months (95%CI: 8.2-20.5) of those with no gain (p = 0.0003). At multivariate analysis, PSA decline ≥ 50% and AR CN showed a significant association with PFS (p = 0.008 and p = 0.002, respectively) and OS (p = 0.009 and p = 0.001, respectively). These findings indicate that the detection of circulating AR CN gain is a promising non-invasive biomarker for outcome prediction to enzalutamide treatment in CRPC patients.

Circulating AR copy number and outcome to enzalutamide in docetaxel-treated metastatic castration-resistant prostate cancer.

Epigenetic silencing mediated by CpG island methylation is a common feature of many cancers. Characterizing aberrant DNA methylation changes associated with prostate carcinogenesis could potentially identify a tumour-specific methylation pattern, facilitating the early diagnosis of prostate cancer. The objective of the study was to assess the methylation status of 40 tumour suppressor genes in prostate cancer and healthy prostatic tissues.

Methylation pattern analysis in prostate cancer tissue: identification of biomarkers using an MS-MLPA approach.

GSTP1 belongs to the GSTs family, a group of enzymes involved in detoxification of exogenous substances and it also plays an important role in cell cycle regulation. Its dysregulation correlates with a large variety of tumors, in particular with prostate cancer. We investigated GSTP1 methylation status with methylation specific PCR (MS-PCR) in prostate cancer (PCa) and in benign tissue of 56 prostatectomies. We also performed immunohistochemistry (IHC) so as to correlate gene methylation with gene silencing. GSTP1 appears methylated in PCa and not in healthy tissue; IHC confirmed that methylation leads to protein underexpression (p < 0.001). GSTP1 is highly expressed in basal cell layer and luminal cells in benign glands while in prostatic intraepithelial neoplasia (PIN) it stains only basal cell layer, whereas PCa glands are completely negative. We demonstrated that methylation leads to underexpression of GSTP1. The progressive loss of GSTP1 expression from healthy glands to PIN and to PCa glands underlines its involvement in early carcinogenesis.

GSTP1 Methylation and Protein Expression in Prostate Cancer: Diagnostic Implications.

The increasing knowledge of the molecular pathogenesis of cancer and the rapid development of new molecular techniques are promoting the study of early molecular alterations involved in cancer development in body fluids. Specific genetic and epigenetic alterations could be found in plasma, serum, and urine cell-free DNA (cfDNA) and could potentially be used as diagnostic biomarkers for several types of cancers. This review focuses on the role of cfDNA in diagnosis: a PubMed search was performed by selecting papers according to journal impact factor and robustness of statistical analysis. A comprehensive evaluation of "liquid biopsy", including cfDNA analysis, will be one of the critical challenges to better understand the early mechanisms of cancer development.

Article	Year
Copy number analysis of 24 oncogenes: MDM4 identified as a putative marker for low recurrence risk in non muscle invasive bladder cancer. International journal of molecular sciences\| PubMed ID: 25026175	2014
Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients. Disease markers\| PubMed ID: 26412928	2015
Circulating AR copy number and outcome to enzalutamide in docetaxel-treated metastatic castration-resistant prostate cancer. Oncotarget\| PubMed ID: 27191887	2016
Methylation pattern analysis in prostate cancer tissue: identification of biomarkers using an MS-MLPA approach. Journal of translational medicine\| PubMed ID: 27576364	2016
GSTP1 Methylation and Protein Expression in Prostate Cancer: Diagnostic Implications. Disease markers\| PubMed ID: 27594734	2016
Cell-free DNA as a diagnostic marker for cancer: current insights. OncoTargets and therapy\| PubMed ID: 27822059	2016

Giorgia Gurioli

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