Serum and Plasma Copy Number Detection Using Real-time PCR

Summary

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

Abstract

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.

We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

Introduction

Circulating cell free DNA (cfDNA) in blood has been demonstrated to be an optimal source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance^1,2. Many studies have shown a good concordance between DNA alterations (mutations, copy number variations, epigenetic modifications) in tissues and those found in corresponding plasma samples¹, confirming that circulating tumor DNA (ctDNA) is informative for primary and metastatic tumor tissue alterations³. The possibility of studying ctDNA thus allows for the reconstruction of g....

Protocol

The protocol consists of the isolation of DNA from serum or plasma samples to perform real-time PCR for copy number analysis. DNA extraction, DNA quantity control (spectrophotometer) and real-time PCR for specific targets were performed. In Figure 1, a summary of the procedures and time line are reported.

The protocol follows the guidelines of IRST Human Research Ethics Committee.

1. Serum Collection and Processing

1. Collect approximately 5 mL whole blood in a serum tube without anticoagulant to obtain serum. Maintain blood tubes at 4 °C until processing.
2. Cen....

Results

Total cfDNA concentration was quantifiable by spectrophotometry for all samples analyzed, showing a median of 6.12 ng/µL (range: 2.00 - 23.71 ng/µL) for serum samples and a median of 3.21 ng/µL (range: 2.31 - 8.49 ng/µL) for plasma samples. We analyzed a total of 115 samples by real-time PCR experiments.

Test sensitivity was assessed using mixed serum DNA from patients with high or low gain for AR, and DNA from healthy .......

Discussion

AR CN analysis in serum and plasma sample represents a new, non-invasive approach for the stratification of castration resistant prostate cancer (CRPC) patients. It has been recently demonstrated that the AR CN is able to predict outcomes in CRPC patients treated with abiraterone and enzalutamide, before and after chemotherapy^7,8,9,10.

The main adva.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD Vacutainer Serum Tubes	Becton Dickinson	367814	whole blood tube for serum
BD Vacutainer EDTA Tubes	Becton Dickinson	366643	whole blood tube for plasma
Ethanol absolute	VWR		the user could use also other companies
TaqMan Copy Number assay	Thermo Fisher Scientific	4400291	Pre-designed and validated assays with FAM-dye. We used the followig assay: AR_assay1: hs04107225 adn AR_assay2: hs04511283
TaqMan Copy Number assay (modified)	Thermo Fisher Scientific	4467084	Pre-designed assay modified with VIC-dye: AGO1: Hs02320401_cn
TaqMan Copy Number Reference Assay, human, RNase P	Thermo Fisher Scientific	4403326
TaqMan Universal PCR Master Mix	Thermo Fisher Scientific	4326708	Master mix for Real Time PCR
QIAamp DNA Mini Kit (50)	Qiagen	51304	DNA extraction kit
MicroAmp 96-Well Plates	Thermo Fisher Scientific	N8010560	plates for realt time PCR
NanoDrop 1000 Spectrophotometer	Thermo Fisher Scientific	-	the user could use also other spectrophotometric methods to quantify DNA
7500 Fast Real-Time PCR System	Applied Biosystem	-	the user could use also other real time instrument
CopyCaller Software	Applied Biosystem	-	Software for copy number analysis