Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from cancer patients.
BACKGROUND
When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA.


METHODS
Our panel ('SiRe') covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice.


RESULTS
SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression.


CONCLUSIONS
SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.
Department of Public Health, University of Naples Federico II, Via S. Pansini 5, Naples 80131, Italy.
Umberto
U
Malapelle
Laboratory of Oncology/Pangaea Biotech, Quiron Dexeus University Hospital, Barcelona, Spain.
Clara
C
Mayo de-Las-Casas
Department of Oncology, Azienda Ospedaliera di Rilievo Nazionale 'Vincenzo Monaldi', Naples, Italy.
Danilo
D
Rocco
Laboratory of Oncology/Pangaea Biotech, Quiron Dexeus University Hospital, Barcelona, Spain.
Monica
M
Garzon
Department of Public Health, University of Naples Federico II, Via S. Pansini 5, Naples 80131, Italy.
Pasquale
P
Pisapia
Laboratory of Oncology/Pangaea Biotech, Quiron Dexeus University Hospital, Barcelona, Spain.
Nuria
N
Jordana-Ariza
Department of Public Health, University of Naples Federico II, Via S. Pansini 5, Naples 80131, Italy.
Maria
M
Russo
Department of Public Health, University of Naples Federico II, Via S. Pansini 5, Naples 80131, Italy.
Roberta
R
Sgariglia
Department of Public Health, University of Naples Federico II, Via S. Pansini 5, Naples 80131, Italy.
Caterina
C
De Luca
Department of Public Health, University of Naples Federico II, Via S. Pansini 5, Naples 80131, Italy.
Francesco
F
Pepe
Instituto Oncológico Rosel (IOR), Quiron Dexeus University Hospital, Barcelona, Spain.
Alejandro
A
Martinez-Bueno
Instituto Oncológico Rosel (IOR), Quiron Dexeus University Hospital, Barcelona, Spain.
Daniela
D
Morales-Espinosa
Instituto Oncológico Rosel (IOR), Quiron Dexeus University Hospital, Barcelona, Spain.
María
M
González-Cao
Instituto Oncológico Rosel (IOR), Quiron Dexeus University Hospital, Barcelona, Spain.
Niki
N
Karachaliou
Instituto Oncológico Rosel (IOR), Quiron Dexeus University Hospital, Barcelona, Spain.
Santiago
S
Viteri Ramirez
Department of Public Health, University of Naples Federico II, Via S. Pansini 5, Naples 80131, Italy.
Claudio
C
Bellevicine
Laboratory of Oncology/Pangaea Biotech, Quiron Dexeus University Hospital, Barcelona, Spain.
Miguel Angel
MA
Molina-Vila
Instituto Oncológico Rosel (IOR), Quiron Dexeus University Hospital, Barcelona, Spain.
Rafael
R
Rosell
Department of Public Health, University of Naples Federico II, Via S. Pansini 5, Naples 80131, Italy.
Giancarlo
G
Troncone
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics*; D002289:Carcinoma, Non-Small-Cell Lung / Q000097:blood / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D015179:Colorectal Neoplasms / Q000097:blood / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics*; D005260:Female; D005500:Follow-Up Studies; D005770:Gastrointestinal Neoplasms / Q000097:blood / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D046152:Gastrointestinal Stromal Tumors / Q000097:blood / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D008297:Male; D008545:Melanoma / Q000097:blood / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D009367:Neoplasm Staging; D011379:Prognosis; D011446:Prospective Studies; D012372:ROC Curve
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm