Open AccessDetection of mutational patterns in cell‐free DNA of colorectal cancer by custom amplicon sequencing
Author(s) -
Herrmann Simon,
Zhan Tianzuo,
Betge Johannes,
Rauscher Benedikt,
Belle Sebastian,
Gutting Tobias,
Schulte Nadine,
Jesenofsky Ralf,
Härtel Nicolai,
Gaiser Timo,
Hofheinz RalfDieter,
Ebert Matthias P.,
Boutros Michael
Publication year - 2019
Publication title -
molecular oncology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.332
H-Index - 88
eISSN - 1878-0261
pISSN - 1574-7891
DOI - 10.1002/1878-0261.12539
Subject(s) - amplicon , multiplex , dna sequencing , biology , colorectal cancer , cold pcr , dna , mutation , cell free fetal dna , computational biology , genetics , cancer , point mutation , polymerase chain reaction , gene , pregnancy , fetus , prenatal diagnosis
Monitoring the mutational patterns of solid tumors during cancer therapy is a major challenge in oncology. Analysis of mutations in cell‐free (cf) DNA offers a noninvasive approach to detect mutations that may be prognostic for disease survival or predictive for primary or secondary drug resistance. A main challenge for the application of cf DNA as a diagnostic tool is the diverse mutational landscape of cancer. Here, we developed a flexible end‐to‐end experimental and bioinformatic workflow to analyze mutations in cf DNA using custom amplicon sequencing. Our approach relies on open‐software tools to select primers suitable for multiplex PCR using minimal cf DNA as input. In addition, we developed a robust linear model to identify specific genetic alterations from sequencing data of cf DNA . We used our workflow to design a custom amplicon panel suitable for detection of hotspot mutations relevant for colorectal cancer and analyzed mutations in serial cf DNA samples from a pilot cohort of 34 patients with advanced colorectal cancer. Using our method, we could detect recurrent and patient‐specific mutational patterns in the majority of patients. Furthermore, we show that dynamic changes of mutant allele frequencies in cf DNA correlate well with disease progression. Finally, we demonstrate that sequencing of cf DNA can reveal mechanisms of resistance to anti‐Epidermal Growth Factor Receptor( EGFR ) antibody treatment. Thus, our approach offers a simple and highly customizable method to explore genetic alterations in cf DNA .