Quantification of plasma DNA as a prognostic indicator in canine lymphoid neoplasia

Abstract

Dogs have a similar incidence of spontaneous cancers as people, and a noninvasive test to monitor disease status in dogs would be of great value. Humans with cancer often have increased levels of cell-free circulating DNA in their plasma, which has shown promise for diagnosis, prognosis and detection of residual disease. We hypothesized that dogs with cancer have increased circulating DNA compared with healthy dogs or dogs with non-neoplastic diseases. Plasma DNA was measured in 40 healthy dogs, 20 dogs with non-neoplastic diseases and 80 dogs with cancer. The reference interval for plasma DNA in healthy dogs was 1-15 ng mL(-1). Dogs with lymphoma and lymphoid leukaemia had significantly higher concentrations (range: 0-91 ng mL(-1), P < 0.0001). Antigen receptor rearrangement assays suggest that plasma DNA had the same clonality as the primary lymphoid tumours. Dogs with lymphoid neoplasia and plasma DNA >25 ng mL(-1) had shorter remission times than those with < 25 ng mL(-1) (P = 0.0116). In contrast to humans, where increased plasma DNA is seen in many diseases, dogs with nonlymphoid malignancies and non-neoplastic diseases had plasma DNA concentrations similar to healthy dogs. This study shows that a portion of dogs with lymphoid neoplasia have increased tumour-derived plasma DNA, which serves as a negative prognostic indicator.

Investigation of cell-free DNA in canine plasma and its relation to disease

“…In the present paper, cfDNA was analyzed without DNA extraction, thus, eliminating the loss of small DNA fragments in the extraction process. Using the technique described in this manuscript, the lowest concentration of cfDNA was 439 ug/L, whilst the range of concentrations reported by Schaefer et al (2007) was 1-91 ug/L, which was similar to the experimental data reported by Uzuelli et al (2009), and in both studies, DNA extraction was performed with QIAmp DNA blood mini Kits, prior to DNA quantification. Standard DNA extraction protocols are extremely insensitive at conserving small DNA fragments (<100 bp), resulting in up to eight-fold variations in the human literature in cfDNA concentrations when DNA extraction was performed using standard extraction techniques (Jung et al 2010).…”

“…The concentration of cfDNA was increased in dogs with both neoplastic and non-neoplastic diseases. This contrasts with the findings of Schaefer et al (2007), who reported that the cfDNA concentration was only elevated in dogs with lymphoma (Schaefer et al 2007). It is most likely that the contrast is due to the difference in sample handling between the two studies.…”

“…Due to the large variety of breeds analyzed in this study, the presence of any significant breed variation in cfDNA concentration was unable to be determined. The previous study on canine cfDNA by Schaefer et al found no impact of breed on cfDNA concentration, although that study also had too few of any particular breed for a firm conclusion (Schaefer et al 2007). Thus, a breed effect cannot be excluded at this stage and requires further investigation.…”

“…In the study by Schaefer et al (2007), a column filtration based DNA extraction protocol was used, whereas, no filtration step was used in the present study. Although column filtration greatly improves DNA purification, it removes a significant quantity of DNA in the process (Goldstein et al 2009;Xue et al 2009;Jung et al 2010); thus, it is possible that an increase in cfDNA concentration in non-neoplastic patients was not seen by Schaefer et al (2007) because of the extraction technique. In the present paper, cfDNA was analyzed without DNA extraction, thus, eliminating the loss of small DNA fragments in the extraction process.…”

“…The tumor-specific DNA was undetectable following surgical excision of the tumor in four of the five dogs, but persisted in one, which was later attributed to a pulmonary metastasis. Finally, cfDNA concentrations were measured in 40 clinically normal dogs, in 20 dogs with non-neoplastic diseases and in 80 dogs with cancer (Schaefer et al 2007). Dogs with lymphoma had a significantly higher cfDNA than clinically normal dogs.…”