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An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage

Nature Medicine volume 20, pages 548–554 (2014)Cite this article

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Abstract

Circulating tumor DNA (ctDNA) is a promising biomarker for noninvasive assessment of cancer burden, but existing ctDNA detection methods have insufficient sensitivity or patient coverage for broad clinical applicability. Here we introduce cancer personalized profiling by deep sequencing (CAPP-Seq), an economical and ultrasensitive method for quantifying ctDNA. We implemented CAPP-Seq for non–small-cell lung cancer (NSCLC) with a design covering multiple classes of somatic alterations that identified mutations in >95% of tumors. We detected ctDNA in 100% of patients with stage II–IV NSCLC and in 50% of patients with stage I, with 96% specificity for mutant allele fractions down to ∼0.02%. Levels of ctDNA were highly correlated with tumor volume and distinguished between residual disease and treatment-related imaging changes, and measurement of ctDNA levels allowed for earlier response assessment than radiographic approaches. Finally, we evaluated biopsy-free tumor screening and genotyping with CAPP-Seq. We envision that CAPP-Seq could be routinely applied clinically to detect and monitor diverse malignancies, thus facilitating personalized cancer therapy.

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Figure 1: Development of CAPP-Seq.
Figure 2: Analytical performance.
Figure 3: Sensitivity and specificity analysis.
Figure 4: Noninvasive detection and monitoring of ctDNA.

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Acknowledgements

We thank S. Quake and members of his lab for suggestions and N. Neff for technical assistance. This work was supported by the US Department of Defense (M.D., A.A.A., A.M.N.), the US National Institutes of Health Director's New Innovator Award Program (M.D.; 1-DP2-CA186569), the Ludwig Institute for Cancer Research (M.D., A.A.A.), the Radiological Society of North America (S.V.B.; #RR1221), an Association of American Cancer Institutes Translational Cancer Research Fellowship (S.V.B.) and a grant from both the Siebel Stem Cell Institute and the Thomas and Stacey Siebel Foundation (A.M.N.). A.A.A. and M.D. are supported by Doris Duke Clinical Scientist Development Awards.

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Author notes

  1. Aaron M Newman and Scott V Bratman: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California, USA

    Aaron M Newman, Scott V Bratman, Chih Long Liu, Ash A Alizadeh & Maximilian Diehn

  2. Division of Oncology, Department of Medicine, Stanford Cancer Institute, Stanford University, Stanford, California, USA

    Aaron M Newman, Chih Long Liu, Joel W Neal, Heather A Wakelee & Ash A Alizadeh

  3. Department of Radiation Oncology, Stanford University, Stanford, California, USA

    Scott V Bratman, Jacqueline To, Jacob F Wynne, Neville C W Eclov, Leslie A Modlin, Billy W Loo Jr & Maximilian Diehn

  4. Division of Thoracic Surgery, Department of Cardiothoracic Surgery, Stanford School of Medicine, Stanford University, Stanford, California, USA

    Robert E Merritt & Joseph B Shrager

  5. Division of Hematology, Department of Medicine, Stanford Cancer Institute, Stanford University, Stanford, California, USA

    Ash A Alizadeh

  6. Stanford Cancer Institute, Stanford University, Stanford, California, USA

    Maximilian Diehn

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Contributions

A.M.N., S.V.B., A.A.A. and M.D. developed the concept, designed the experiments, analyzed the data and wrote the manuscript. S.V.B. performed the molecular biology experiments, and A.M.N. performed the bioinformatics analyses. C.L.L. helped develop analytical pipeline software. S.V.B., J.T., J.F.W., N.C.W.E., L.A.M., J.W.N., H.A.W., R.E.M., J.B.S., B.W.L. Jr. and M.D. provided patient specimens. A.A.A. and M.D. contributed equally as senior authors. All authors commented on the manuscript at all stages.

Corresponding authors

Correspondence to Ash A Alizadeh or Maximilian Diehn.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Methods (PDF 2806 kb)

Supplementary Table 1

NSCLC selector design and coordinates (XLSX 115 kb)

Supplementary Table 2

Quality control metrics of NGS libraries (XLSX 69 kb)

Supplementary Table 3

Clinical history of patients in this study and somatic variants discovered by CAPP-Seq (XLSX 31 kb)

Supplementary Table 4

Detection of somatic variants in plasma DNA from patients with NSCLC (XLSX 78 kb)

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Newman, A., Bratman, S., To, J. et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med 20, 548–554 (2014). https://doi.org/10.1038/nm.3519

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