title: Evaluation of exome sequencing to estimate tumor burden in plasma.
abstract: Accurate estimation of systemic tumor load from the blood of cancer patients has enormous potential. One avenue is to measure the presence of cell-free circulating tumor DNA in plasma. Various approaches have been investigated, predominantly covering hotspot mutations or customized, patient-specific assays. Therefore, we investigated the utility of using exome sequencing to monitor circulating tumor DNA levels through the detection of single nucleotide variants in plasma. Two technologies, claiming to offer efficient library preparation from nanogram levels of DNA, were evaluated. This allowed us to estimate the proportion of starting molecules measurable by sequence capture (<5%). As cell-free DNA is highly fragmented, we designed and provide software for efficient identification of PCR duplicates in single-end libraries with a varying size distribution. On average, this improved sequence coverage by 38% in comparison to standard tools. By exploiting the redundant information in PCR-duplicates the background noise was reduced to ∼1/35,000. By applying our optimized analysis pipeline to a simulation analysis, we determined the current sensitivity limit to ∼1/2400, starting with 30 ng of cell-free DNA. Subsequently, circulating tumor DNA levels were assessed in seven breast- and one prostate cancer patient. One patient carried detectable levels of circulating tumor DNA, as verified by break-point specific PCR. These results demonstrate exome sequencing on cell-free DNA to be a powerful tool for disease monitoring of metastatic cancers. To enable a broad implementation in the diagnostic settings, the efficiency limitations of sequence capture and the inherent noise levels of the Illumina sequencing technology must be further improved.
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Klevebring
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Mårten
authors.initials: M
authors.lastname: Neiman
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Simon
authors.initials: S
authors.lastname: Sundling
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Louise
authors.initials: L
authors.lastname: Eriksson
authors.affiliation: Department of Pathology and Cytology, Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Eva
authors.initials: E
authors.lastname: Darai Ramqvist
authors.affiliation: Department of Clinical Science and Education, Södersjukhuset, Stockholm, Sweden.
authors.forename: Fuat
authors.initials: F
authors.lastname: Celebioglu
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Kamila
authors.initials: K
authors.lastname: Czene
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Hall
authors.affiliation: Department of Pathology and Cytology, Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Lars
authors.initials: L
authors.lastname: Egevad
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Grönberg
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Lindberg
mesh_terms: D001943:Breast Neoplasms / Q000097:blood / Q000473:pathology*; D004273:DNA, Neoplasm / Q000097:blood*; D059472:Exome; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000097:blood / Q000473:pathology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D047368:Tumor Burden
chemical_list: D004273:DNA, Neoplasm