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Updated: Aug 28, 2025
Serum and Plasma Copy Number Detection Using Real-time PCR
Published on: December 15, 2017
Analysis of BRCA2 Copy Number Loss and Genomic Instability in Circulating Tumor Cells from Patients with Metastatic
Ethan S Barnett1, Nikolaus Schultz2, Konrad H Stopsack1
1Genitourinary Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Background:
BRCA2 alterations predict for a response to poly-ADP-ribose polymerase inhibition in metastatic castration-resistant prostate cancer (mCRPC). However, detection is hindered by insufficient tumor tissue and low sensitivity of cell-free DNA for detecting copy number loss.
Objective:
To evaluate the BRCA2 loss detection using single-cell, shallow whole-genome sequencing (sWGS) of circulating tumor cells (CTCs) in patients with mCRPC.
Design, Setting, And Participants:
We analyzed CTC samples collected concurrently with tumor biopsies intended for clinical sequencing in patients with progressing mCRPC.
Outcome Measurements And Statistical Analysis:
Differences in proportions were evaluated using the chi-square test. Correlations between assays were analyzed in linear regression models. Associations between alterations and genomic instability were assessed on the single-cell level using mixed-effect negative binomial models.
Results And Limitations:
We identified 138 patients with concurrent CTC and biopsy samples. CTC sWGS generated copy number profiles in a similar proportion of patients to biopsy samples (83% vs 78%, p = 0.23), but was more effective than bone biopsies (79% vs 50%; p = 0.009). CTC sWGS detected BRCA2 loss in more patients than tissue at the ≥1 (42% vs 16%; p < 0.001) and ≥2 (27% vs 16%; p = 0.028) CTC thresholds. The overall prevalence of BRCA2 loss was not increased in CTCs using sample-level composite z scores (p = 0.4), but was significantly increased compared with a lower-than-expected prevalence in bone samples (21% vs 3%, p = 0.014). Positive/negative predictive values for CTC BRCA2 loss were 89%/96% using the ≥1 CTC threshold and 67%/92% using the composite z score. CTC BRCA2 loss was associated with higher genomic instability in univariate (1.4-fold large-scale transition difference, 95% confidence interval [CI]: 1.2-1.6; p < 0.001) and multivariable analysis (1.4-fold difference, 95% CI: 1.2-1.6; p < 0.001).
Conclusions:
Copy number profiles can reliably be generated using CTC sWGS, which detected a majority of tissue-confirmed BRCA2 loss and "CTC-only" losses. BRCA2 losses were supported by increases in genomic instability.
Patient Summary:
Current testing strategies have limitations in their ability to detect BRCA2 loss, a relatively common alteration in prostate cancer that is used to identify patients who may benefit from targeted therapy. In this paper, we evaluated whether we could detect BRCA2 loss in individual tumor cells isolated from patient blood samples and found this method to be suitable for further analysis.
Insights
Detecting BRCA2 loss in metastatic castration-resistant prostate cancer (mCRPC) is crucial for targeted therapy. Single-cell sequencing of circulating tumor cells (CTCs) offers a promising method for detecting BRCA2 loss, outperforming traditional tissue biopsies.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- BRCA2 alterations are key biomarkers for poly-ADP-ribose polymerase (PARP) inhibitor response in metastatic castration-resistant prostate cancer (mCRPC).
- Current methods for detecting BRCA2 loss face challenges due to insufficient tumor tissue and low sensitivity of cell-free DNA assays for copy number loss detection.
Purpose of the Study:
- To evaluate the efficacy of single-cell, shallow whole-genome sequencing (sWGS) of circulating tumor cells (CTCs) for detecting BRCA2 loss in patients with mCRPC.
- To compare the performance of CTC sWGS with traditional tumor biopsy methods for BRCA2 alteration detection.
Main Methods:
- Analysis of concurrent CTC and tumor biopsy samples from 138 patients with progressing mCRPC.
- Generation of copy number profiles using CTC sWGS and comparison with biopsy data.
- Statistical evaluation of BRCA2 loss detection rates, predictive values, and association with genomic instability.
Main Results:
- CTC sWGS successfully generated copy number profiles in a high proportion of patients, comparable to biopsy samples and superior to bone biopsies.
- CTC sWGS detected BRCA2 loss in significantly more patients than tissue biopsies, with high positive and negative predictive values.
- BRCA2 loss in CTCs was strongly associated with increased genomic instability.
Conclusions:
- CTC sWGS is a reliable method for generating copy number profiles and detecting BRCA2 loss, including "CTC-only" losses not identified in tissue.
- BRCA2 loss detection via CTC sWGS is supported by increased genomic instability, offering a viable alternative for patients with limited tissue samples.
