A Droplet PCR-Based Next Generation Sequencing Assay to Track Plasma DNA Mutation Dynamics in Estrogen Receptor Positive Metastatic Breast Cancer

Summary

Here we describe a protocol that uses droplet polymerase chain reaction for target enrichment followed by next generation sequencing of blood plasma circulating tumor DNA. This technique was used to characterize mutations in the genes ESR1 (all coding regions), TP53 (all coding regions), PIK3CA (hotspots), PIK3R1 (hotspots) and POLE (exonuclease domain).

Abstract

The estrogen receptor gene (ESR1) is expressed in approximately two-thirds of breast cancers (BCs) and predicts sensitivity to endocrine therapy. Mutations in ESR1 have recently been associated with endocrine therapy resistance in patients with estrogen receptor positive metastatic breast cancer (ER+ MBC). Thus, monitoring the status of ESR1 mutations may facilitate personalized therapy decisions for ER+ MBC patients. Additionally, mutations in PIK3CA and TP53 are also prevalent in ER+ MBC and may influence therapeutic responses. Recent studies demonstrate mutational heterogeneity in metastatic breast cancer (MBC), highlighting a need to monitor for the emergence of new mutations over time. The analysis of blood plasma circulating tumor DNA (ctDNA) by next generation sequencing (NGS) has emerged as an attractive approach to address the mutation heterogeneity and evolution of MBC over time. However, the high costs and intensive bioinformatics required for plasma ctDNA NGS analysis limit its utility in clinical studies that require longitudinal monitoring. We have recently developed and validated an assay for plasma ctDNA mutation profiling that utilizes droplet PCR-based multiplexed target enrichment followed by NGS, which we have termed dPCR-Seq. Here we describe the protocol for dPCR-Seq, illustrating its relative simplicity in library preparation and bioinformatics analysis to detect ESR1 (all coding regions), TP53 (all coding regions), PIK3CA (hotspots), PIK3R1 (hotspots), and POLE (exonuclease domain) mutations in breast cancer patients. We have validated a subset of the ESR1 mutations identified by dPCR-Seq using allele-specific digital PCR (dPCR) assays, demonstrating exceptional concordance in the measurement of mutant allele frequency (MAF) in clinical plasma ctDNA specimens. We anticipate that dPCR-Seq may have practical utility in future studies that investigate longitudinal monitoring of plasma ctDNA mutations as potential biomarkers of therapeutic response in ER+ MBC patients.

Introduction

Breast cancer is the most common cancer diagnosed in women worldwide¹. Endocrine therapy, which inhibits estrogen receptor signaling with tamoxifen, fulvestrant, or aromatase inhibitors (AIs), is a mainstay of treatment for ER+ MBC^2,3,4,5. However, most metastatic breast cancer patients will develop resistance to endocrine therapy. Ligand-binding domain mutations in ESR1 have been identified as a key mechanism for acquired endocrine therapy resistance^6,7

Protocol

The collection and analysis of blood samples follows the ethics guidelines of the University of North Carolina at Chapel Hill. Informed written consent was obtained from each patient, and our study was approved by the Institutional Review Board of University of North Carolina at Chapel Hill.

1. Blood collection from breast cancer patients

1. Collect 10 mL of blood from breast cancer patients in a glass blood collection tube. Please refer to Kumar et al. for patient characteristics

Discussion

Digital PCR has become an important tool in translational research that is used widely to track hotspot mutations in cancer patients. Genome-wide and targeted NGS of liquid and solid biopsy samples has also successfully been used to identify mutations in breast cancer patients³⁶. We have developed a five gene panel to track mutations in ESR1, PIK3CA, PIK3R1, POLE, and TP53 mutations in plasma ctDNA isolated from metastatic breast cancer patients. Altern.......

Materials

Name	Company	Catalog Number	Comments
10 μL Aerosol Barrier tips	VWR	10017-062	Can be replaced with other equivalent product
10 mL Serological Pipettes	VWR	13-675-20	Can be replaced with other equivalent product
10-100 μL 8-channel pipette	Eppendorf	3125000036	Can be replaced with other equivalent product
1250 μL Aerosol Barrier tips	VWR	10017-092	Can be replaced with other equivalent product
2 μL Aerosol Barrier tips	VWR	10010-364	Can be replaced with other equivalent product
20 μL Aerosol Barrier tips	VWR	10017-064	Can be replaced with other equivalent product
200 μL Aerosol Barrier tips	VWR	10017-068	Can be replaced with other equivalent product
20-200 ul 8-channel pipette	Denville Scientific	463230067	Can be replaced with other equivalent product
2100 Bioanalyzer	Agilent	G2939BA
5 mL Serological Pipettes	VWR	13-675-22	Can be replaced with other equivalent product
8-strip 0.2 mL PCR tubes & caps	Axygen	PCR-0208-CP-C
Absolute Ethanol	Sigma	E7023-500ML
Agilent DNA 1000 Kit	Agilent	5067-1504
Betaine Anhydrous	Sigma	B2629-100G
Bio-Rad’s C1000 Touch Thermal Cycler with 96–Deep Well Reaction Module	Bio-Rad	1851197
Cell-Free DNA BCT tubes, RUO	Streck	218962
Centrifuge 5810R	Eppendorf	22625101	Suitable for 15 ml conical tubes; Can be replaced with other equivalent instrument
DMSO	Sigma	D8418-100ML
dNTP Solution Mix	New England Biolabs	N0447L
DynaMag-96 magnetic plate	Life Technologies	12331D
Falcon 15ml Conical Centrifuge Tubes	Corning	352096	Can be replaced with other equivalent product
Falcon 50mL Conical Centrifuge Tubes	Corning	352098	Can be replaced with other equivalent product
Heating block (for 1.7 ml microcentrifuge tubes )	Denville Scientific	I0540
Microcentrifuge	Eppendorf	5424	Suitable for 1.5-2.0 ml tubes; Can be replaced with other equivalent instrument
MiSeq Reagent Kit v3	Illumina	MS-102-3003
MiSeq Sequencer	Illumina	SY-410-1003
Nuclease Free Water	Integrated DNA Technologies	11-05-01-04	Can be replaced with other equivalent product
P10 pipettes	Denville Scientific	355022105	Can be replaced with other equivalent product
P1000 pipettes	Denville Scientific	455060205	Can be replaced with other equivalent product
P2 pipettes	Denville Scientific	455010336	Can be replaced with other equivalent product
P20 pipettes	Denville Scientific	355032002	Can be replaced with other equivalent product
P200 pipettes	Denville Scientific	45505009	Can be replaced with other equivalent product
Platinum Taq DNA Polymerase High Fidelity	Life Technologies	11304-029
Portable Pipet-Aid XP Pipette Controller	Drummond Scientific	4-000-101
QIAamp Circulating Nucleic Acid Kit	Qiagen	55114
Qubit fluorimeter	Thermo Fisher Scientific	Q33226
Qubit Assay Tubes	Thermo Fisher Scientific	Q32856
Qubit dsDNA HS Assay Kit	Thermo Fisher Scientific	Q32851
RainDrop System Source Instrument	Raindance	20-04401
SPRIselect Reagent Kit	Beckman Coulter	B23318
TaqMan Genotyping Master Mix	Life Technologies	4371355
ThunderBolts Cancer Panel Consumables Pack	Raindance	20-07205
Tris Base	Thermo Fisher Scientific	BP152-500
Vortex mixer	Denville Scientific	Vortexer 59A	Can be replaced with other equivalent product