Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA

Amanda Frydendahl; Mads Heilskov Rasmussen; Sarah Østrup Jensen; Tenna Vesterman Henriksen; Christina Demuth; Mathilde Diekema; Henrik Jørn Ditzel; Sara Witting Christensen Wen; Jakob Skou Pedersen; Lars Dyrskjøt; Claus Lindbjerg Andersen

doi:10.3390/ijms25084252

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA

Amanda Frydendahl, Mads Heilskov Rasmussen^*, Sarah Østrup Jensen, Tenna Vesterman Henriksen, Christina Demuth, Mathilde Diekema, Henrik Jørn Ditzel, Sara Witting Christensen Wen, Jakob Skou Pedersen, Lars Dyrskjøt, Claus Lindbjerg Andersen^*

^*Corresponding author af dette arbejde

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

5 Citationer (Scopus)

Abstract

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.

Originalsprog	Engelsk
Artikelnummer	4252
Tidsskrift	International Journal of Molecular Sciences
Vol/bind	25
Nummer	8
ISSN	1661-6596
DOI	https://doi.org/10.3390/ijms25084252
Status	Udgivet - apr. 2024

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10.3390/ijms25084252Licens: CC BY

Citationsformater

Frydendahl, A., Rasmussen, M. H., Jensen, S. Ø., Henriksen, T. V., Demuth, C., Diekema, M., Ditzel, H. J., Wen, S. W. C., Pedersen, J. S., Dyrskjøt, L., & Andersen, C. L. (2024). Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA. International Journal of Molecular Sciences , 25(8), Artikel 4252. https://doi.org/10.3390/ijms25084252

@article{851e94a0171c46219aa9d072537a1dcd,

title = "Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA",

abstract = "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.",

keywords = "cell-free tumor DNA, cfDNA, clonal hematopoiesis, colorectal cancer, ctDNA, DNA mutation, liquid biopsy, minimal residual disease, next-generation sequencing, UMI-mediated error suppression, Circulating Tumor DNA/genetics, Humans, Middle Aged, Male, High-Throughput Nucleotide Sequencing/methods, Sensitivity and Specificity, Aged, 80 and over, Female, Adult, Biomarkers, Tumor/blood, Gene Frequency, Colorectal Neoplasms/genetics, Cell-Free Nucleic Acids/genetics, Aged, Mutation",

author = "Amanda Frydendahl and Rasmussen, {Mads Heilskov} and Jensen, {Sarah {\O}strup} and Henriksen, {Tenna Vesterman} and Christina Demuth and Mathilde Diekema and Ditzel, {Henrik J{\o}rn} and Wen, {Sara Witting Christensen} and Pedersen, {Jakob Skou} and Lars Dyrskj{\o}t and Andersen, {Claus Lindbjerg}",

note = "Publisher Copyright: {\textcopyright} 2024 by the authors.",

year = "2024",

month = apr,

doi = "10.3390/ijms25084252",

language = "English",

volume = "25",

journal = "International Journal of Molecular Sciences ",

issn = "1661-6596",

publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",

number = "8",

}

Frydendahl, A , Rasmussen, MH , Jensen, SØ , Henriksen, TV , Demuth, C , Diekema, M, Ditzel, HJ, Wen, SWC, Pedersen, JS , Dyrskjøt, L & Andersen, CL 2024, 'Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA', International Journal of Molecular Sciences , bind 25, nr. 8, 4252. https://doi.org/10.3390/ijms25084252

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA. / Frydendahl, Amanda ; Rasmussen, Mads Heilskov ; Jensen, Sarah Østrup et al.
I: International Journal of Molecular Sciences , Bind 25, Nr. 8, 4252, 04.2024.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

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

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 -

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA

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