Development of a highly sensitive and specific method for detection of circulating tumor cells harboring somatic mutations in non-small-cell lung cancer patients.
BACKGROUND
Oncogenic mutations are powerful predictive biomarkers for molecularly targeted cancer therapies. For mutation detection patients have to undergo invasive tumor biopsies. Alternatively, archival samples are used which may no longer reflect the actual tumor status. Circulating tumor cells (CTC) could serve as an alternative platform to detect somatic mutations in cancer patients. We sought to develop a sensitive and specific assay to detect mutations in the EGFR gene in CTC from lung cancer patients.


METHODS
We developed a novel assay based on real-time polymerase chain reaction (PCR) and melting curve analysis to detect activating EGFR mutations in blood cell fractions enriched in CTC. Non-small-cell lung cancer (NSCLC) was chosen as disease model with reportedly very low CTC counts. The assay was prospectively validated in samples from patients with EGFR-mutant and EGFR-wild type NSCLC treated within a randomized clinical trial. Sequential analyses were conducted to monitor CTC signals during therapy and correlate mutation detection in CTC with treatment outcome.


RESULTS
Assay sensitivity was optimized to enable detection of a single EGFR-mutant CTC/mL peripheral blood. CTC were detected in pretreatment blood samples from all 8 EGFR-mutant lung cancer patients studied. Loss of EGFR-mutant CTC signals correlated with treatment response, and its reoccurrence preceded relapse.


CONCLUSIONS
Despite low abundance of CTC in NSCLC oncogenic mutations can be reproducibly detected by applying an unbiased CTC enrichment strategy and highly sensitive PCR and melting curve analysis. This strategy may enable non-invasive, specific biomarker diagnostics and monitoring in patients undergoing targeted cancer therapies.
Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany.
Frank
F
Breitenbuecher
Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany.
Sandra
S
Hoffarth
Institute of Pathology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany.
Karl
K
Worm
Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany.
Diana
D
Cortes-Incio
Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany ; Division of Thoracic Oncology, Ruhrlandklinik, West German Lung Center, University Duisburg-Essen, Essen, Germany.
Thomas C
TC
Gauler
Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany.
Jens
J
Köhler
Institute of Pathology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany ; German Cancer Research Center, Heidelberg, Germany ; German Cancer Consortium (DKTK), Heidelberg, Germany.
Thomas
T
Herold
Institute of Pathology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany.
Kurt Werner
KW
Schmid
Division of Interventional Pneumology, Ruhrlandklinik, West German Lung Center, University Duisburg-Essen, Essen, Germany.
Lutz
L
Freitag
Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany.
Stefan
S
Kasper
Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany ; Division of Thoracic Oncology, Ruhrlandklinik, West German Lung Center, University Duisburg-Essen, Essen, Germany ; German Cancer Consortium (DKTK), Heidelberg, Germany.
Martin
M
Schuler
D000328:Adult; D000368:Aged; D000970:Antineoplastic Agents / Q000627:therapeutic use; D014408:Biomarkers, Tumor / Q000235:genetics*; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009364:Neoplasm Recurrence, Local / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology / Q000517:prevention & control; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology; D009691:Nucleic Acid Denaturation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D059010:Single-Cell Analysis
D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; C512478:EGFR protein, human; D066246:ErbB Receptors