TY - JOUR
T1 - Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA
AU - Frydendahl, Amanda
AU - Rasmussen, Mads Heilskov
AU - Jensen, Sarah Østrup
AU - Henriksen, Tenna Vesterman
AU - Demuth, Christina
AU - Diekema, Mathilde
AU - Ditzel, Henrik Jørn
AU - Wen, Sara Witting Christensen
AU - Pedersen, Jakob Skou
AU - Dyrskjøt, Lars
AU - Andersen, Claus Lindbjerg
N1 - Publisher Copyright:
© 2024 by the authors.
PY - 2024/4
Y1 - 2024/4
N2 - Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.
AB - Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.
KW - cell-free tumor DNA
KW - cfDNA
KW - clonal hematopoiesis
KW - colorectal cancer
KW - ctDNA
KW - DNA mutation
KW - liquid biopsy
KW - minimal residual disease
KW - next-generation sequencing
KW - UMI-mediated error suppression
KW - Circulating Tumor DNA/genetics
KW - Humans
KW - Middle Aged
KW - Male
KW - High-Throughput Nucleotide Sequencing/methods
KW - Sensitivity and Specificity
KW - Aged, 80 and over
KW - Female
KW - Adult
KW - Biomarkers, Tumor/blood
KW - Gene Frequency
KW - Colorectal Neoplasms/genetics
KW - Cell-Free Nucleic Acids/genetics
KW - Aged
KW - Mutation
U2 - 10.3390/ijms25084252
DO - 10.3390/ijms25084252
M3 - Journal article
C2 - 38673836
AN - SCOPUS:85190954755
SN - 1661-6596
VL - 25
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 8
M1 - 4252
ER -