Detection of circulating tumor DNA in early- and late-stage human malignancies

Abstract

The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction-based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.

Conflict of interest statement

Competing interests: K.W.K. and B.V. are consultants for Inostics. L.A.D. is a consultant for Amgen and Anaeropharma. L.A.D. and V.E.V. are co-founders and on the board of directors of Personal Genome Diagnostics. K.W.K., B.V., L.A.D., N.P., and V.E.V. all own Personal Genome Diagnostics stock, which is subject to certain restrictions under University policy. L.D.W. works as a paid consultant for Personal Genome Diagnostics. Johns Hopkins University has several patents related to the work presented in this paper. The terms of all these arrangements are managed by the Johns Hopkins University in accordance with its conflict-of-interest policies. A.B. is a shareholder and advisory board member of Horizon Discovery, and an advisory board member or Biocartis. L.A.F. is an advisory board member of Genentech-Roche. C.M.S. is a scientific advisor to Asuragen Inc. and a consultant to Redpath Inc.

Figures

Fig. 1

Potential applications of ctDNA.

Fig. 2. ctDNA in advanced malignancies

(A) Fraction of patients with detectable ctDNA. (B) Quantification of mutant fragments. Error bars represent the 95% bootstrapped confidence interval of the mean (tumor types with <4 samples were excluded from this figure).

Fig. 3. ctDNA in localized and nonlocalized malignancies

(A) Fraction of patients with detectable ctDNA in localized (stages I to III) and metastatic (stage IV) colorectal, gastroesophageal, pancreatic, and breast cancers. (B) Fraction of patients with detectable ctDNA. (C) Quantification of mutant fragments in cancer cases categorized by stage. Error bars represent SEM.

Fig. 4

Scatter plot correlating point mutations with rearrangements in the same plasma specimens.

Fig. 5. The relationship between ctDNA concentration (mutant fragments per milliliter) and 2-year survival

The association between survival and ctDNA concentration was assessed, holding known prognostic factors (age, ECOG PS, and CEA) constant. The 2-year survival was estimated on the basis of a multivariable Cox regression model, in which ctDNA concentration level was transformed with a natural spline function.

Fig. 6

Heat map of acquired resistance mutations to EGFR blockade in ctDNA from patients with metastatic CRC.