Sequencing Analysis of Tumor DNA: Is It All in the Plasma?

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Britta Weigelt, PhD

Jorge S. Reis-Filho, MD, PhD, FRCPath

ctDNA is an undeniably appealing source of tumor material and may prove instrumental for the realization of the potentials of precision medicine.

—Britta Weigelt, PhD, and Jorge S. Reis-Filho, MD, PhD, FRCPath

Massively parallel sequencing analyses have demonstrated that most of the common malignancies display relatively complex repertoires of somatic genetic alterations, that the number of highly recurrent mutations is limited, and that a large number of genes is mutated in a small minority of tumors from a given cancer type.1 In addition, there is evidence to demonstrate that a substantial proportion of tumors are composed of multiple clones that in addition to the mutations present in all cells (the founder genetic events), have mutations restricted to some populations of cancer cells.2

This intratumor genetic heterogeneity has also been documented by differences in the mutations found in anatomically distinct biopsies of a cancer3 or between biopsies of the primary tumor and of its metastatic lesions.4 Importantly, some of the mutations present in minor populations of tumor cells within a cancer have been shown to mediate resistance to specific therapies.

Blood-Borne Biomarkers

Although the presence of circulating DNA in plasma has long been known, circulating cell-free plasma DNA (cfDNA) is detected in normal individuals, and its measure is of limited utility in oncology. With the development of highly sensitive sequencing technologies, there has been renewed interest in this blood-borne biomarker, as it may constitute a minimally invasive source of tumor material that can be employed for sequencing analyses (ie, “liquid biopsies”). Circulating tumor DNA (ctDNA) in plasma comprises a fraction of cfDNA, and is believed to be shed in the bloodstream by cancer cells through apoptosis, necroptosis, or secretion (eg, exosomes) not only from the primary tumor, but also metastatic lesions and minimal residual disease.5-7

Sequencing analysis of ctDNA has been envisaged as a means to overcome the challenges posed by intratumor and interlesion genetic heterogeneity. Evidence suggests that massively parallel sequencing analysis of ctDNA from patients with advanced disease allows for the identification of the entire constellation of somatic mutations found in cancer cells, either from the primary tumor or from metastatic lesions.4,6-8

Analyzing Circulating Tumor DNA to Monitor Disease

Although ctDNA levels have been shown to be detectable in the majority of patients with advanced disease—in particular, those with bladder cancer, colorectal cancer, gastroesophageal cancer, pancreatic cancer, breast cancer, and melanoma6—only a small minority of patients with gliomas have been reported to have detectable levels of ctDNA in plasma.6 Hence, ctDNA detection may only be useful for patients with specific tumor types.

Given that ctDNA levels correlate with tumor burden, its detection in patients with early-stage cancers has proven to be more challenging.9 By the same token, as variations in ­ctDNA levels are associated with variations in tumor burden, studies investigating whether ctDNA levels could be employed for disease monitoring have revealed that this approach may offer a substantial lead time over traditional methods for the detection of metastatic disease.4,6-8

Numerous challenges remain for the translation of the immense potential of this approach into benefit for cancer patients. Current technologies that allow for the identification of the entire repertoire of mutations in a cancer have a sensitivity of approximately 1%. As the amount of ctDNA in cfDNA varies (and is often low in patients with low tumor burden), de novo discovery of mutations by plasma DNA analysis is not trivial.5

The sequencing techniques currently available for the detection of known or “hotspot” mutations, however, offer incredibly high levels of sensitivity. In fact, there is evidence to suggest that these techniques may be useful for disease monitoring in cases where the primary tumor has been previously subjected to sequencing or for patients with tumor types characterized by the presence of highly recurrent mutations, as well as for the identification of emerging mutations already known to result in therapy resistance.

ctDNA Analysis in Clinical Decision-Making

ctDNA is an undeniably appealing source of tumor material and may prove instrumental for the realization of the potentials of precision medicine. Whether sequencing of ctDNA will render tumor biopsies redundant remains to be determined, particularly because of the strong association between ctDNA yield and disease burden and the technologic and bioinformatic developments required for de novo discovery of mutations based solely on ctDNA analysis.

Importantly, however, for the expeditious implementation of ctDNA analysis as a clinical tool, it will be essential to learn from experience with the translation of circulating tumor cell enumeration5 and molecular characterization10 into biomarkers and from previous high-throughput genetic analyses of tumors for clinical decision-making.

Disclosure: Drs. Weigelt and Reis-Filho reported no potential conflicts of interest.


1. Kandoth C, McLellan MD, Vandin F, et al: Mutational landscape and significance across 12 major cancer types. Nature 502:333-339, 2013.

2. Burrell RA, McGranahan N, Bartek J, et al: The causes and consequences of genetic heterogeneity in cancer evolution. Nature 501:338-345, 2013.

3. Gerlinger M, Rowan AJ, Horswell S, et al: Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 366:883-892, 2012.

4. De Mattos-Arruda L, Weigelt B, Cortes J, et al: Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: A proof-of-principle. Ann Oncol 25:1729-1735, 2014.

5. Bidard FC, Weigelt B, Reis-Filho JS: Going with the flow: From circulating tumor cells to DNA. Sci Transl Med 5:207ps14, 2013.

6. Bettegowda C, Sausen M, Leary RJ, et al: Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 6:224ra24, 2014.

7. Dawson SJ, Tsui DW, Murtaza M, et al: Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med 368:1199-1209, 2013.

8. Murtaza M, Dawson SJ, Tsui DW, et al: Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497:108-112, 2013.

9. Beaver JA, Jelovac D, Balukrishna S, et al: Detection of cancer DNA in plasma of patients with early-stage breast cancer. Clin Cancer Res 20:2643-2650, 2014.

10. Krebs MG, Metcalf RL, Carter L, et al: Molecular analysis of circulating tumour cells-biology and biomarkers. Nat Rev Clin Oncol 11:129-144, 2014.

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