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Updated: Aug 19, 2025
Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids
Published on: June 8, 2019
Circulating versus cellular tumor DNA for the detection of BTK resistant CLL clones
Arne Trummer1, Wiebke Schier1, Jürgen Krauter1
1Department of Hematology and Oncology, Städtisches Klinikum Braunschweig, Braunschweig, Germany.
Abstract:
Resistance mutations can be detected in 75% of CLL patients progressing under BTK inhibitor therapy. Using semiquantitative wild-type-blocking (WTB) RT-PCR for BTK and Sanger sequencing for PLCG2 mutations, we compared detection sensitivity of cellular versus circulating tumor DNA (ctDNA) in 20 sample pairs of 13 consecutive patients. With an assay sensitivity of 0.06%, 7 patients had a BTK-C481S and one a PLCG2-G667E mutation. Cellular DNA was positive in 10 but ctDNA only in 6 samples, giving false-negative results in samples with low mutational burden. In summary, WTB-PCR is cost-effective and routinely applicable but misses low frequency mutations when using ctDNA.
Insights
Detecting resistance mutations in chronic lymphocytic leukemia (CLL) is crucial. Wild-type blocking PCR on cellular DNA is more sensitive than circulating tumor DNA (ctDNA) for detecting low-frequency mutations.
Area of Science:
- Hematology
- Oncology
- Molecular Biology
Background:
- Bruton's tyrosine kinase (BTK) inhibitors are effective treatments for chronic lymphocytic leukemia (CLL).
- Resistance mutations, particularly in BTK and PLCG2, can emerge during BTK inhibitor therapy, leading to disease progression.
- Accurate and sensitive detection of these resistance mutations is critical for patient management.
Purpose of the Study:
- To compare the detection sensitivity of cellular DNA versus circulating tumor DNA (ctDNA) for resistance mutations in CLL patients undergoing BTK inhibitor therapy.
- To evaluate the utility of semiquantitative wild-type blocking (WTB) RT-PCR for BTK and Sanger sequencing for PLCG2 mutations.
Main Methods:
- Analysis of 20 sample pairs (cellular DNA and ctDNA) from 13 consecutive CLL patients progressing on BTK inhibitor therapy.
- Utilized semiquantitative wild-type blocking (WTB) RT-PCR for detecting BTK mutations.
- Employed Sanger sequencing for identifying PLCG2 mutations.
Main Results:
- Resistance mutations were detected in 75% of CLL patients progressing under BTK inhibitor therapy.
- The assay demonstrated a sensitivity of 0.06%.
- Cellular DNA detected mutations in 10 samples, whereas ctDNA detected mutations in only 6 samples, indicating false negatives in samples with low mutational burden.
Conclusions:
- WTB-PCR is a cost-effective and routinely applicable method for detecting resistance mutations in CLL.
- Cellular DNA analysis offers higher sensitivity compared to ctDNA for detecting low-frequency resistance mutations.
- ctDNA analysis may miss low-frequency mutations, potentially impacting treatment decisions in CLL patients.
