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01.12.2018 | Letter to the Editor | Ausgabe 1/2018 Open Access

Molecular Cancer 1/2018

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

Zeitschrift:
Molecular Cancer > Ausgabe 1/2018
Autoren:
Liang Wang, Ying Li, Xin Guan, Jingyuan Zhao, Liming Shen, Jing Liu
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12943-018-0876-z) contains supplementary material, which is available to authorized users.
Abbreviations
CTC
Circulating tumor cell
ctDNA
Circulating tumor DNA
dsDNA
Double-stranded DNA
NGS
Next-generation sequencing
PCC
Pheochromocytomas
PGL
Paragangliomas
SNP
Single nucleotide polymorphism

Main text

Pheochromocytomas (PCCs) and paragangliomas (PGLs), the most heritable endocrine tumors, demonstrate major genetic driver events including germline and somatic mutations [ 1, 2]. Specific genotype–phenotype correlations have been established between susceptibility gene mutations and their clinical presentations [ 1, 3, 4]. Thus, assessment of these susceptibility genes for mutations is recommended for the early diagnosis and preoperative assessment of all PCCs and PGLs [ 5, 6].
Germline mutation testing for clinical diagnosis has become well established in recent decades [ 7]. Since more than one-third of PCC and PGL patients harbor only somatic mutations, monitoring of somatic mutations is recommended even for patients who are negative for germline mutations [ 1, 8]. However, it is difficult to detect somatic mutations prior to surgery, which limits genetic testing applications in practice.
Exosomes are an effective biomarker source independent of tissues and contain RNA, DNA, and proteins for noninvasive diagnosis [ 9]. However, the presence of DNA in exosomes is usually dependent on cell type, and the ability of the exosomal DNA to reflect mutational status of the parental tumor cells in PCC or PGL patients is largely unknown. Thus, in this study, we focused on exosomal DNA from PCC or PGL exosomes, and its potential as samples for screening somatic mutations in the parental tumor cells. This could serve as a promising noninvasive biomarker for clinical genetic diagnosis and preoperative assessment of PCC and PGL.

Results and discussion

PCC and PGL exosomes contain double-stranded genomic DNA

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.

PCC and PGL exosomes contain dsDNA with RET, VHL, HIF2A, and SDHB mutations

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.

Serum Exosomes from PCC and PGL patients contain genomic dsDNA that covers all chromosomes

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.

Conclusions

This is the first study to reveal that PCC and PGL exosomes contain dsDNA that can reflect the mutation status of susceptibility genes and cover nearly all chromosomes. The definitive evidence of exosomal dsDNA presence suggests its use as a noninvasive genetic marker in one of the most effective somatic mutation screens for the genetic diagnosis and preoperative assessment of PCCs and PGLs.

Funding

This work was supported by the National Nature Science Foundation of China (Grant No. 31600614,81471308) and the Research Foundation for Talented Doctors of Liaoning Province (Grant No. 201601232).

Availability of data and materials

The datasets used and analyzed in the current study are available from the corresponding author in response to reasonable requests.

Ethics approval and consent to participate

The collection and use of the PCC/PGL tissue samples was reviewed and approved by the Institutional Ethics Committee of the First Affiliated Hospital of Dalian Medical University (China).

Consent for publication

Informed consents were obtained from the participants (or their parent or legal guardian) for the use of participant-derived material in the study and for the publication of the results.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://​creativecommons.​org/​licenses/​by/​4.​0/​), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://​creativecommons.​org/​publicdomain/​zero/​1.​0/​) applies to the data made available in this article, unless otherwise stated.
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