Quantification of human papillomavirus cell-free DNA from low-volume blood plasma samples by digital PCR
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Quantification of human papillomavirus cell-free DNA from low-volume blood plasma samples by digital PCR. / Rosing, Fabian; Meier, Matthias; Schroeder, Lea; Laban, Simon; Hoffmann, Thomas; Kaufmann, Andreas; Siefer, Oliver; Wuerdemann, Nora; Klußmann, Jens Peter; Rieckmann, Thorsten; Alt, Yvonne; Faden, Daniel L; Waterboer, Tim; Höfler, Daniela.
In: MICROBIOL SPECTR, Vol. 12, No. 7, 02.07.2024, p. e0002424.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Quantification of human papillomavirus cell-free DNA from low-volume blood plasma samples by digital PCR
AU - Rosing, Fabian
AU - Meier, Matthias
AU - Schroeder, Lea
AU - Laban, Simon
AU - Hoffmann, Thomas
AU - Kaufmann, Andreas
AU - Siefer, Oliver
AU - Wuerdemann, Nora
AU - Klußmann, Jens Peter
AU - Rieckmann, Thorsten
AU - Alt, Yvonne
AU - Faden, Daniel L
AU - Waterboer, Tim
AU - Höfler, Daniela
PY - 2024/7/2
Y1 - 2024/7/2
N2 - The incidence rate of human papillomavirus-driven oropharyngeal cancer (HPV-OPC) is increasing in countries with high human development index. HPV cell-free DNA (cfDNA) isolated from 3 to 4 mL blood plasma has been successfully used for therapy surveillance. A highly discussed application of HPV-cfDNA is early detection of HPV-OPC. This requires sensitive and specific cfDNA detection as cfDNA levels can be very low. To study the predictive power of pre-diagnostic HPV-cfDNA, archived samples from epidemiological cohorts with limited plasma volume are an important source. To establish a cfDNA detection workflow for low plasma volumes, we compared cfDNA purification methods [MagNA Pure 96 (MP96) and QIAamp ccfDNA/RNA] and digital PCR systems (Biorad QX200 and QIAGEN QIAcuity One). Final assay validation included 65 low-volume plasma samples from oropharyngeal cancer (OPC) patients with defined HPV status stored for 2-9 years. MP96 yielded a 28% higher cfDNA isolation efficiency in comparison to QIAamp. Both digital PCR systems showed comparable analytical sensitivity (6-17 copies for HPV16 and HPV33), but QIAcuity detected both types in the same assay. In the validation set, the assay had 80% sensitivity (n = 28/35) for HPV16 and HPV33 and a specificity of 97% (n = 29/30). In samples with ≥750 µL plasma, the sensitivity was 85% (n = 17/20), while in samples with <750 µL plasma, it was 73% (n = 11/15). Despite the expected drop in sensitivity with decreased plasma volume, the assay is sensitive and highly specific even in low-volume samples and thus suited for studies exploring HPV-cfDNA as an early HPV-OPC detection marker in low-volume archival material.IMPORTANCEHPV-OPC has a favorable prognosis compared to HPV-negative OPC. However, the majority of tumors is diagnosed after regional spread, thus making intensive treatment necessary. This can cause lasting morbidity with a large impact on quality of life. One potential method to decrease treatment-related morbidity is early detection of the cancer. HPV cfDNA has been successfully used for therapy surveillance and has also been detected in pre-diagnostic samples of HPV-OPC patients. These pre-diagnostic samples are only commonly available from biobanks, which usually only have small volumes of blood plasma available. Hence, we have developed a workflow optimized for small-volume archival samples. With this method, a high sensitivity can be achieved despite sample limitations, making it suitable to conduct further large-scale biobank studies of HPV-cfDNA as a prognostic biomarker for HPV-OPC.
AB - The incidence rate of human papillomavirus-driven oropharyngeal cancer (HPV-OPC) is increasing in countries with high human development index. HPV cell-free DNA (cfDNA) isolated from 3 to 4 mL blood plasma has been successfully used for therapy surveillance. A highly discussed application of HPV-cfDNA is early detection of HPV-OPC. This requires sensitive and specific cfDNA detection as cfDNA levels can be very low. To study the predictive power of pre-diagnostic HPV-cfDNA, archived samples from epidemiological cohorts with limited plasma volume are an important source. To establish a cfDNA detection workflow for low plasma volumes, we compared cfDNA purification methods [MagNA Pure 96 (MP96) and QIAamp ccfDNA/RNA] and digital PCR systems (Biorad QX200 and QIAGEN QIAcuity One). Final assay validation included 65 low-volume plasma samples from oropharyngeal cancer (OPC) patients with defined HPV status stored for 2-9 years. MP96 yielded a 28% higher cfDNA isolation efficiency in comparison to QIAamp. Both digital PCR systems showed comparable analytical sensitivity (6-17 copies for HPV16 and HPV33), but QIAcuity detected both types in the same assay. In the validation set, the assay had 80% sensitivity (n = 28/35) for HPV16 and HPV33 and a specificity of 97% (n = 29/30). In samples with ≥750 µL plasma, the sensitivity was 85% (n = 17/20), while in samples with <750 µL plasma, it was 73% (n = 11/15). Despite the expected drop in sensitivity with decreased plasma volume, the assay is sensitive and highly specific even in low-volume samples and thus suited for studies exploring HPV-cfDNA as an early HPV-OPC detection marker in low-volume archival material.IMPORTANCEHPV-OPC has a favorable prognosis compared to HPV-negative OPC. However, the majority of tumors is diagnosed after regional spread, thus making intensive treatment necessary. This can cause lasting morbidity with a large impact on quality of life. One potential method to decrease treatment-related morbidity is early detection of the cancer. HPV cfDNA has been successfully used for therapy surveillance and has also been detected in pre-diagnostic samples of HPV-OPC patients. These pre-diagnostic samples are only commonly available from biobanks, which usually only have small volumes of blood plasma available. Hence, we have developed a workflow optimized for small-volume archival samples. With this method, a high sensitivity can be achieved despite sample limitations, making it suitable to conduct further large-scale biobank studies of HPV-cfDNA as a prognostic biomarker for HPV-OPC.
KW - Humans
KW - DNA, Viral/blood
KW - Papillomavirus Infections/diagnosis
KW - Cell-Free Nucleic Acids/blood
KW - Polymerase Chain Reaction/methods
KW - Oropharyngeal Neoplasms/virology
KW - Papillomaviridae/genetics
KW - Female
KW - Sensitivity and Specificity
KW - Male
KW - Middle Aged
KW - Aged
KW - Human papillomavirus 16/genetics
KW - Human Papillomavirus Viruses
U2 - 10.1128/spectrum.00024-24
DO - 10.1128/spectrum.00024-24
M3 - SCORING: Journal article
C2 - 38829114
VL - 12
SP - e0002424
JO - MICROBIOL SPECTR
JF - MICROBIOL SPECTR
SN - 2165-0497
IS - 7
ER -