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Updated: Apr 9, 2026
Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids
Published on: June 8, 2019
Sequential Analysis of cfDNA Reveals Clonal Evolution in Patients with Neuroblastoma Receiving ALK-Targeted Therapy
Charles Bobin1,2, Yasmine Iddir1,2, Charlotte Butterworth1,2
1SiRIC RTOP (Recherche Translationelle en Oncologie Pédiatrique), Translational Research Department, Institut Curie Research Center, PSL Research University, Institut Curie, Paris, France.
Purpose:
The study of cell-free DNA (cfDNA) enables sequential analysis of tumor cell-specific genetic alterations in patients with neuroblastoma.
Experimental Design:
Eighteen patients with relapsing neuroblastoma having received lorlatinib, a third-generation ALK inhibitor, were identified (SACHA national registry and/or in the institution). cfDNA was analyzed at relapse for nine patients and sequentially for five patients (blood/bone marrow plasma) by performing whole-genome sequencing library construction followed by ALK-targeted ddPCR of the hotspot mutations [F1174L, R1275Q, and I1170N; variant allele fraction (VAF) detection limit 0.1%] and whole-exome sequencing (WES) to evaluate disease burden and clonal evolution, following comparison with tumor/germline WES.
Results:
Overall response rate to lorlatinib was 33% (CI, 13%-59%), with response observed in 6/10 cases without versus 0/8 cases with MYCN amplification (MNA). ALK VAFs correlated with the overall clinical disease status, with a VAF < 0.1% in clinical remission, versus higher VAFs (>30%) at progression. Importantly, sequential ALK ddPCR detected relapse earlier than clinical imaging. cfDNA WES revealed new SNVs, not seen in the primary tumor, in all instances of disease progression after lorlatinib treatment, indicating clonal evolution, including alterations in genes linked to tumor aggressivity (TP53) or novel targets (EGFR). Gene pathway analysis revealed an enrichment for genes targeting cell differentiation in emerging clones, and cell adhesion in persistent clones. Evidence of clonal hematopoiesis could be observed in follow-up samples.
Conclusions:
We demonstrate the clinical utility of combining ALK cfDNA ddPCR for disease monitoring and cfDNA WES for the study of clonal evolution and resistance mechanisms in patients with neuroblastoma receiving ALK-targeted therapy.
Insights
Monitoring neuroblastoma with cell-free DNA (cfDNA) using ALK ddPCR and whole-exome sequencing (WES) detects relapse early and reveals resistance mechanisms. This approach aids in understanding tumor evolution during targeted therapy.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Neuroblastoma is a pediatric cancer where sequential analysis of tumor genetics is crucial.
- Cell-free DNA (cfDNA) offers a non-invasive method to track genetic alterations in tumors.
Purpose of the Study:
- To assess the utility of cfDNA analysis for monitoring neuroblastoma patients treated with lorlatinib.
- To investigate tumor clonal evolution and resistance mechanisms using cfDNA.
Main Methods:
- Whole-genome sequencing (WGS) library construction and ALK-targeted digital droplet PCR (ddPCR) for hotspot mutations.
- Whole-exome sequencing (WES) of cfDNA to evaluate disease burden and clonal evolution.
- Comparison of cfDNA analysis with tumor and germline WES.
Main Results:
- Lorlatinib showed a 33% overall response rate, with higher efficacy in patients without MYCN amplification.
- ALK variant allele fractions (VAFs) in cfDNA correlated with clinical disease status, detecting relapse earlier than imaging.
- cfDNA WES identified new somatic mutations and clonal evolution, including alterations in TP53 and EGFR, during treatment resistance.
Conclusions:
- Combining cfDNA ddPCR and WES is clinically useful for monitoring neuroblastoma patients on ALK-targeted therapy.
- This approach facilitates the study of clonal evolution and resistance mechanisms.
- cfDNA analysis provides valuable insights into treatment response and disease progression.
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