Exosomal double-stranded DNA as a biomarker for the diagnosis and preoperative assessment of pheochromocytoma and paraganglioma

In order to evaluate the DNA carried by exosomes, we isolated exosomes from PC12 cell culture medium, the serum of mice implanted with mutated xenografts, and the serum of PCC or PGL patients. The characteristics of exosomes were assessed (Fig. 1A-C). For further characterization, we isolated the exosomal DNA from PC12 supernatants and digested it with dsDNase and RNase. We observed that the majority of DNA in PC12 exosomes (Exo-DNA (I)) was digested by dsDNase rather than RNase (Fig. 1D a). The same pattern was observed for exosomal DNA isolated from the serum of xenograft-implanted mice (Exo-DNA (II)) and the serum of PCC or PGL patients (Exo-DNA (III)) (Fig. 1D b and c). Taken together, these results suggest that dsDNA is the predominant form of DNA in exosomes of tumor cell supernatants and the sera of mice with xenografts and patients with PCC and PGL. Our novel finding is consistent with the presence of dsDNA in the exosomes of pancreatic cancer and prostate cancer cells, and may differ from those in astrocytes and glioblastoma cells because exosomal DNA packaging methods may differ among cancer types [9]. All the methods and materials used during this study are included in Additional file 1.

RET, HIF2A, VHL, and SDHB are most frequently mutated genes in PCC and PGL and is usually monitoring for somatic mutations in sporadic PCC and PGL. To further explore somatic mutation testing in patients without germline mutations for PCC and PCL, we constructed plasmids expressing mutated human RET (c.1902C > G, c.1901G > A, c.1900 T > C, and c.1894G > A), HIF2A (c.1615G > T, c.1595A > G, and c.1591C > T), VHL (c.562C > G and c.293A > G), and SDHB (c.281G > A), and used them to transfect PC12 cells. We found that the plasmid-encoded mutations were detected in both the transfected PC12 cells and in the exosomes from the supernatants (Fig. 2A a-d). To evaluate the feasibility of screening DNA from circulating exosomes for susceptibility gene mutations, we separately established stably transfected PC12 cell lines with the 10 mutations described above. We implanted the transfected PC12 cells subcutaneously in the flanks of nude mice, and harvested their sera when the tumors reached the maximum size after 30 days, to ensure isolation of sufficient circulating exosomes for analysis. We analyzed exosomal DNA from the serum for mutations by Sanger sequencing (Additional file 2: Table S1).

Based on our observations in tumor cells and the animal model, we hypothesize that human serum exosomes may contain information regarding the mutation of RET, VHL, HIF2A, and SDHB in their parental cells. We analyzed samples from 12 PCC or PGL patients whose somatic tumor mutations had been identified by genetic diagnosis (Additional file 3: Table S2). We found that RET (Fig. 2B a), HIF2A (Fig. 2B b), VHL (Fig. 2B c), and SDHB mutations (Fig. 2B d) in serum exosomal DNA were definitively consistent with the somatic tumor mutations in patients with PCC or PGL. Collectively, these results provide evidence that serum exosomal dsDNA may serve as a primary somatic mutation diagnostic biomarker for PCC or PGL preoperative assessment.

To further determine if the dsDNA from circulating exosomes reflects the mutational status of their parental tumor cells, we compared the exosomal DNA from PC12 supernatants with its genomic DNA. The results of whole-genome sequencing demonstrated that exosomal DNA covered 97.7% of the single nucleotide polymorphisms (SNPs) of the parent PC12 cells (Fig. 3a and b and Additional file 4: Table S3). In order to confirm these results, we compared the exosomal DNA and paired tumor tissues from 3 PCC or PGL patients. Importantly, our results revealed that the entire genome is covered by the exosomal DNA in an unbiased manner (Fig. 3c). We examined the 12 driver susceptibility gene mutations in exosomal DNA, and found that the concordance rates of mutations in the exosomal and tumor tissue DNA were as high as 97.6–100% (Fig. 3d and Additional file 5: Table S4-S7). Taken together, our results demonstrate a high degree of consistency between serum exosomal DNA and paired tumor genomic DNA in patients with PCC or PGL.

Thus, our analysis revealed that the bulk of serum-derived exosomes of patients with PCC or PGL contained dsDNA that spanned all chromosomes and resembled nuclear genomic DNA (Fig. 3c). The presence of dsDNA in exosomes allows detection of somatic mutations in susceptibility genes before surgery in patients with PCC or PGL.