An Optimized Workflow to Evaluate Estrogen Receptor Gene Mutations in Small Amounts of Cell-Free DNA.
Author(s): Vitale SR, Sieuwerts AM, Beije N, Kraan J, Angus L, Mostert B, Reijm EA, Van NM, van Marion R, Dirix LY, Hamberg P, de Jongh FE, Jager A, Foekens JA, Vigneri P, Sleijfer S, Jansen MPHM, Martens JWM
Publication: J Mol Diagn, 2019, Vol. 21, Page 123-137
PubMed ID: 30296589 PubMed Review Paper? No
Purpose of Paper
This paper optimized a digital PCR (dPCR) assay for the detection of mutations in plasma collected from metastatic breast cancer patients by comparing mutation detection and variant allele frequency obtained using single-locus versus multiplex pre-amplification and different volumes of plasma and cell-free DNA (cfDNA) as input. Mutational profiles from dPCR analysis and next-generation sequencing were compared as were those from longitudinal specimens.
Conclusion of Paper
Preamplification with the multiplex dPCR assay identified the same mutations with a VAF >1% as a locus-specific preamplification approach. The allele frequency of the spiked-in D538G ESR1 mutation in the dPCR assay was comparable among the different volumes of plasma used as input (200 µL to 4 mL plasma) regardless of whether the sample underwent preamplification or not. However, when the amount of cfDNA used for dPCR-based preamplification was less than 2000 ng the VAF tended to be slightly lower. Increasing the input volume to 2 µL, as opposed to 0.5 µL, of DNA permitted the detection of a third mutation in one of the 26 specimens; increasing the input volume further did result in the detection of additional mutations. NGS failed to detect mutations in 8 patients and only detected 4 of the 5 ESR1 mutations that were detected by dPCR. However, NGS detected an additional mutation that was not included in the dPCR preamplification step. ESR1 mutations were detected at a higher frequency in patients who had progressed on endocrine therapy than in patients who were receiving endocrine therapy for the first time (34.1% versus 3.9%, P=0.0001). The authors identified several mutations that demonstrated a longitudinal change in allele frequency that coincided with disease progression and treatment.
Studies
-
Study Purpose
This study optimized a dPCR assay for the detection of mutations in plasma collected from metastatic breast cancer patients by comparing mutation detection and variant allele frequency obtained using single-locus versus multiplex pre-amplification, and different volumes of plasma and cfDNA as input. Mutational profiles from dPCR analysis and next-generation sequencing were compared as were those from longitudinal specimens. Blood for this study had been previously collected from 156 patients diagnosed with metastatic breast cancer (95 patients had received endocrine therapy and 43 patients had received chemotherapy) and ten healthy blood donors. Blood was collected in Vacutainer EDTA tubes. Additionally, 2- 5 longitudinal specimens were collected from 17 patients. Blood was transported at ambient temperature, and plasma and circulating tumor cells (CTC) were separated within 24 h of venipuncture. Plasma was separated by centrifugation at 1711 g for 10 min and stored at -80°C. Before thawing, 5 mmol/L dithiothreitol was added to plasma specimens. Plasma was centrifuged a second time at 12,000 g for 10 min at 4°C before cfDNA isolation from 200 µL plasma using the QIAamp Circulating Nucleic Acid Kit with 20 µL elution buffer applied to the column three times. cfDNA was quantified using the Qubit dsDNA HS Assay Kit and stored at -30°C for < 1 week or at -80°C. cfDNA was preamplified using a TaqMan PreAmp Kit and either a single locus-specific (ESR1) approach or a multiple loci (ESR1, BRAF, KRAS, PIK3CA, and TP53) approach. Samples were then amplified using hotspot mutation-specific assays and the QuantStudio 3D Digital PCR System. A total of 152 different mutations were sequenced in specimens from 17 breast cancer patients and ten healthy volunteers using the Ion Torrent Oncomine Breast cfDNA Assay on an Ion Torrent S5XL NGS system a range of DNA inputs (<1 to 10.41 ng DNA). A comparison of different plasma volumes was conducted by extracting DNA from plasma spiked with D538G ESR1.
Summary of Findings:
Preamplification with the multiplex dPCR assay identified the same mutations with a VAF >1% as a locus-specific preamplification approach. Further, the VAF was highly reproducible when a single pooled cfDNA extract was subjected to multiple independent locus-specific and multiplex preamplification steps. The allele frequency of the spiked-in D538G ESR1 mutation in the dPCR assay was comparable among the different volumes of plasma used as input (200 µL to 4 mL plasma) regardless of whether the sample underwent preamplification or not. However, when the amount of cfDNA used for dPCR-based preamplification was less than 2000 ng the VAF tended to be slightly lower. Increasing the input volume to 2 µL, as opposed to 0.5 µL, of DNA permitted the detection of a third mutation in one of the 26 specimens; increasing the input volume further did result in the detection of additional mutations. The limit of detection was set for each mutation at three times that of the background noise and ranged from 0.52-1.02% depending on the mutation. Among the 17 patients that had a DNA input amount <5 ng, NGS failed to detect mutations in 8 but 22 variants were detected in the remaining 9 patients. Importantly, 4 of the 5 ESR1 mutations detected in seven patients with metastatic breast cancer by dPCR were also detected by NGS. The mutation missed by NGS had a VAF of 1.9% and the authors state the loss of detection may be attributable to the lower input in the NGS assay. An additional mutation not included in the dPCR preamplification was also detected by NGS at very low VAF (0.48%). ESR1 mutations were detected at a higher frequency in patients who had progressed on endocrine therapy than in patients who were receiving endocrine therapy for the first time (34.1% versus 3.9%, P=0.0001). The authors identified several mutations that demonstrated a longitudinal change in allele frequency that coincided with disease progression and treatment.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Digital PCR DNA Fluorometry DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Digital PCR Specific Template/input amount 641 ng-309,706 ng
0.5 µL
2 µL
8 µL
10 µL
Digital PCR Specific Template modification Preamplified
Not preamplified
Digital PCR Specific Technology platform Ion Torrent Oncomine Breast cfDNA Assay
dPCR assays
Biospecimen Aliquots and Components Aliquot size/volume 500 µL
1 mL
2 mL
4 mL
Digital PCR Specific Priming method Multiplex
Single-locus
Biospecimen Acquisition Time of biospecimen collection Longitudinal sampling