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. 2019 Nov;31(6):836-843.
doi: 10.1177/1040638719880245. Epub 2019 Oct 4.

Quantification of plasma cell-free DNA levels in dogs with various tumors

Michihito Tagawa  1   2 , Genya Shimbo  1   2 , Hisashi Inokuma  1   2 , Kazuro Miyahara  1   2
Affiliations

Quantification of plasma cell-free DNA levels in dogs with various tumors

Michihito Tagawa et al. J Vet Diagn Invest. 2019 Nov.

Abstract

Circulating cell-free DNA (cfDNA) is extracellular DNA released into the bloodstream by apoptotic or necrotic tumor cells, with cfDNA determination proposed as a noninvasive, sensitive marker for the diagnosis of human cancer. We evaluated cfDNA quantification as a diagnostic and prognostic tool in dogs with various tumors. We quantified plasma cfDNA concentration by absolute real-time PCR of long interspersed nuclear elements in 50 dogs with malignant tumors, 13 dogs with benign tumors or nodules, and 11 healthy controls. Six patients with malignant tumors were followed-up, and plasma cfDNA was quantified throughout disease progression. We found that plasma cfDNA concentrations were significantly elevated in dogs with malignant tumors compared with dogs with benign nodules or healthy controls. The DNA integrity index (the ratio between long and short cfDNA fragments) was significantly lower in dogs with malignant tumors compared to healthy controls. Significantly higher cfDNA levels and a lower DNA integrity index were observed in dogs with lymphoma or leukemia, hemangiosarcoma, and distant metastasis; cfDNA levels correlated well with clinical stage and tended to increase during or before periods of disease progression, suggesting potential efficacy of cfDNA for the detection of distant metastasis and to monitor the clinical stage of neoplasia.

Keywords: cell-free DNA; dogs; liquid biopsy; tumor biomarker.

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Conflict of interest statement

Declaration of conflicting interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
A. Box-plot of plasma cell-free DNA (cfDNA) concentrations in dogs with malignant tumors, benign diseases, and healthy controls. B. cfDNA integrity index. Each box indicates the 25th and 75th percentiles. The horizontal line inside the box indicates the median, and the whiskers indicate the extreme measured values.
Figure 2.
Figure 2.
A. Box-plot of plasma cell-free DNA (cfDNA) concentrations in dogs with lymphoma or leukemia (LSA), hemangiosarcoma (HS), and other malignant tumors. B. cfDNA integrity index. Each box indicates the 25th and 75th percentiles. The horizontal line inside the box indicates the median, and the whiskers indicate the extreme measured values.
Figure 3.
Figure 3.
A. Box-plot of plasma cell-free DNA (cfDNA) concentrations in malignant-tumor dogs with distant metastasis (DM) or without DM. B. cfDNA integrity index. Each box indicates the 25th and 75th percentiles. The horizontal line inside the box indicates the median, and the whiskers indicate the extreme measured values.
Figure 4.
Figure 4.
Receiver operating characteristic and area under the curve (AUC) of plasma cell-free DNA (cfDNA) concentrations (A) and cfDNA-integrity index (B) to distinguish patients with distant metastasis from other patients and controls. The AUC for cfDNA concentrations and the cfDNA integrity index was 0.833 (95% CI: 0.714–0.952; p = 0.002) and 0.752 (95% CI: 0.611–0.893; p < 0.001), respectively.
Figure 5.
Figure 5.
Comparison of cell-free DNA (cfDNA) and cfDNA integrity index according to treatment response: cfDNA levels (solid line) and the cfDNA integrity index (dotted line) at various time points in 6 patients. The vertical axis on the left represents cfDNA concentration, and the vertical axis on the right represents the cfDNA integrity index. The horizontal axis labels represent the times of plasma-sampling day and tumor status. A. Patient 1, transitional carcinoma of the bladder treated with surgery and cyclooxygenase-2 inhibitor; B. Patient 2, undifferentiated sarcoma treated with surgery; C. Patient 3, splenic hemangiosarcoma treated with surgery and chemotherapy; D. Patient 4, lymphoma treated with chemotherapy; E. Patient 5, lymphoma treated with chemotherapy; F. Patient 6, leukemia treated with chemotherapy. CR = complete response; PD = progressive disease; PR = partial response.
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