Plasma DNA as a "liquid biopsy" incompletely complements tumor biopsy for identification of mutations in a case series of four patients with oligometastatic breast cancer

Abstract

Purpose: Circulating tumor DNA in plasma may present a minimally invasive opportunity to identify tumor-derived mutations to inform selection of targeted therapies for individual patients, particularly in cases of oligometastatic disease where biopsy of multiple tumors is impractical. To assess the utility of plasma DNA as a "liquid biopsy" for precision oncology, we tested whether sequencing of plasma DNA is a reliable surrogate for sequencing of tumor DNA to identify targetable genetic alterations.

Methods: Blood and biopsies of 1-3 tumors were obtained from 4 evaluable patients with advanced breast cancer. One patient provided samples from an additional 7 tumors post-mortem. DNA extracted from plasma, tumor tissues, and buffy coat of blood were used for probe-directed capture of all exons in 149 cancer-related genes and massively parallel sequencing. Somatic mutations in DNA from plasma and tumors were identified by comparison to buffy coat DNA.

Results: Sequencing of plasma DNA identified 27.94 ± 11.81% (mean ± SD) of mutations detected in a tumor(s) from the same patient; such mutations tended to be present at high allelic frequency. The majority of mutations found in plasma DNA were not found in tumor samples. Mutations were also found in plasma that matched clinically undetectable tumors found post-mortem.

Conclusions: The incomplete overlap of genetic alteration profiles of plasma and tumors warrants caution in the sole reliance of plasma DNA to identify therapeutically targetable alterations in patients and indicates that analysis of plasma DNA complements, but does not replace, tumor DNA profiling.

Trial registration: Subjects were prospectively enrolled in trial NCT01836640 (registered April 22, 2013).

Keywords: Advanced breast cancer; Cell-free DNA; Circulating tumor DNA; DNA sequencing.

Conflict of interest statement

Conflict of Interest: The authors declare that they have no conflict of interest.

Figures

Fig. 1-. Sequencing of plasma DNA identifies tumor-derived mutations.

DNA from plasma, tumors, and buffy coat (or normal liver sample) from 4 patients with metastatic breast cancer was used for sequencing of all exons in 149 genes. Plasma and tumor DNA sequences were compared to buffy coat or normal liver to identify somatic mutations in each patient. Mutations present in both plasma (≥5% AF) and at least one tumor specimen (≥10% AF) are indicated in heatmaps. Samples are labeled along y-axes with number of mutations found in plasma out of number of mutations called in parentheses. Mutations are indicated along x-axes. Locations of known tumors in each patient at the time of biopsy are indicated with boxes: green indicates a sequenced lesion; black indicates a non-sequenced lesion.

Fig. 2-. Plasma DNA sequencing identifies proportions of stem and non-stem mutations.

A) Proportions of stem mutations (found in all tumors) versus non-stem mutations (found in at least one but not all tumors) are shown for each patient. B/C) Proportions of non-stem (private/clade) (B) and stem (C) mutations identified in plasma DNA are shown.

Fig. 3-. Plasma DNA mutations with high allelic frequency tend to be detected in tumors.

Each vertical bar indicates one mutation. Plasma AFs of mutations detected in plasma at ≥5% AF are noted for each patient along y-axes. Red bars indicate variants present in both plasma and ≥1 tumor (≥10% AF). Black bars indicate variants present in plasma only. Numbers of tumors evaluated from each patient are noted.

Fig. 4-. Tumor DNA mutations at high allelic frequency are often detected in plasma DNA.

Each vertical bar indicates one mutation. Tumor AFs of mutations detected in a tumor(s) at ≥10% AF are noted for each tumor along y-axes. Red bars indicate variants present in both a tumor and plasma (≥5% AF). Black bars indicate variants present only in a tumor(s).

Fig. 5-. Plasma DNA reflects clinically occult and evident tumors in Patient #9A.

A) DNA from plasma acquired 3 months before death, buffy coat, and samples from 7 tumors acquired post-mortem from Patient #9 was used for DNA sequencing as in Fig. 1. Tumor sites sampled for DNA sequencing are indicated by green boxes. Additional tumor sites noted at autopsy (but not sampled) are indicated by black boxes. B) Mutant AFs were plotted according to organ site of tumor. For organ sites with multiple tumors, data were pooled. Data were analyzed by linear regression. C-D) Mutant AFs in plasma were compared to tumor size (mean from each organ site) determined from CT scans (C) and relative blood perfusion (mL/min) of tumor-containing organs (D) using Spearman correlation and linear regression analyses.