Identification of Mutations in Cell-Free Circulating Tumor DNA in Adrenocortical Carcinoma: A Case Series
Sara G. Creemers,1* Esther Korpershoek,2* Peggy N. Atmodimedjo,2 Winand N. M. Dinjens,2 Peter M. van Koetsveld,1 Richard A. Feelders,1 and Leo J. Hofland1
1Division of Endocrinology, Department of Internal Medicine, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam 3000 CA, The Netherlands; and 2 Department of Pathology, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam 3000 CA, The Netherlands
Context: The disease course of adrenocortical carcinoma (ACC) patients is heterogeneous. A marker for prognosis and treatment response would facilitate choices for diagnosis and therapy. In other cancer types, circulating cell-free tumor DNA predicted tumor dynamics.
Case Descriptions: The present pilot study included six patients. Next-generation sequencing (NGS) showed mutations in three ACC cases. From these patients, blood was drawn before (1 to 2 weeks) and after surgery and cell-free circulating DNA (cfDNA) was isolated. Tumor-specific mutations were found in the cfDNA of one of the three patients, with metastasized ACC at diagnosis. NGS of the tumor showed an NRAS mutation (c.182A>G:p.Q61R) in 78%, a TP53 mutation (c.856G>A:p.E286K) in 60%, and a TERT gene mutation (1295250C>T) in 28% of the reads. The preoperative cfDNA showed the same mutations at a frequency of 64%, 32%, and 2%, respectively. The postoperative cfDNA showed the same mutations but at lower frequencies (52%, 16%, and 3%, respectively). The patient was postoperatively treated with mitotane and chemotherapy. No mutations were detected in the corresponding leukocyte DNA or in the cfDNA from the two other patients.
Conclusions: To the best of our knowledge, we report for the first time mutations occurring at high levels in cfDNA collected before and after surgery from one of three patients, after previous identification in the tumor. However, in the cfDNA from two patients with known mutations, we were unable to reliably detect mutations in the cfDNA. Our results indicate that mutation detection in cfDNA can vary among ACC patients, and other approaches might be required to detect the tumor response and monitor progressive disease. (J Clin Endocrinol Metab 102: 3611-3615, 2017)
A drenocortical carcinoma (ACC) is a rare disease with heterogeneous treatment responses and prognosis (1). Several prognostic and predictive factors have been proposed, but all have had limited value (2). Many efforts in oncology have focused on noninvasive methods to monitor the disease course. The discovery of circulating cell-free DNA (cfDNA) in the blood and the increased concentrations found in cancer patients compared with healthy individuals raised attention for its application in monitoring tumor dynamics (3). The amount of cell-free circulating DNA derived from the tumor (ctDNA) largely
depends on the tumor type, disease stage, and therapeutic response (4, 5). ctDNA is derived from the primary tu- mor, metastases and micrometastases, and/or apoptotic circulating tumor cells (6). To identify ctDNA, tumor- specific aberrations can serve as personalized biomarkers. In ACCs, extensive efforts have led to the identification of several genes involved in ACC pathogenesis (2, 7). In the present case report, we aimed to detect ctDNA in the plasma of patients with ACC by identifying specific mutations present in both the primary tumor and the cfDNA using next-generation sequencing (NGS).
Received 19 January 2017. Accepted 27 June 2017.
First Published Online 30 June 2017
*These authors contributed equally to this study. Abbreviations: ACC, adrenocortical carcinoma; cfDNA, cell-free circulating DNA; CT, computed tomography; ctDNA, circulating tumor DNA; M-EDP, mitotane plus etoposide, doxorubicin, and cisplatin; MRI, magnetic resonance imaging; NGS, next-generation sequencing.
doi: 10.1210/jc.2017-00174
Methods
Blood was collected in EDTA tubes 1 to 2 weeks before surgery and 5 to 6 months after surgery. For patient 3, blood was also drawn at 14 months and 2 years postoperatively. The blood samples were processed directly after collection by centrifugation for 10 minutes at 1349g at 4℃. The supernatant was carefully removed and saved in 1-mL aliquots at -80℃ until analysis. The DNA from 1 to 2 mL of plasma, dependent on availability, was isolated using the QIAamp Circulating Nucleic Acid Kit and the vacuum-based QIAvac 24 Plus system (Qiagen). Leukocyte preparation and DNA isolation were performed using the DNA Isolation Kit for Mammalian Blood (Roche). Genomic DNA from the primary tumor was isolated from a formalin-fixed paraffin-embedded tissue sample, after selecting an area of high tumor content microscop- ically (>80%). DNA isolation was performed by overnight in- cubation at 56°C in 180 L Tris-EDTA-buffer (pH 7.5) containing 5% Chelex and 20 p.L proteinase K (20 mg/mL), followed by 8 minutes at 100℃. The DNA yield was measured using Qubit Fluorometric Quantitation (Thermo Fisher Scientific). NGS was performed sequencing a custom-made multigene panel using the Ampliseq website (available at: www.ampliseq.com) related to major cancer pathways, such as the WNT signaling, MAPK, and PI3K/AKT pathways, and has been previously reported (8). Ac- cordingly, the panel covered mutational hotspot areas of APC (exons 12 to 14), AXIN1 (exons 1 to 6), AXIN2 (exon 7), CTNNB1 (exon 3), BRAF (exons 11 and 15), KRAS (exons 2 to 4), NRAS (exons 2 to 4), HRAS (exons 2 to 4), EGFR (exons 18 to 21), PI3KCA (exons 9 and 20), AKT1 (exon 2), AKT2 (exon 2), AKT3 (exon 2), PTEN (exons 3 to 5 and 7), ALK (exons 23 to 25), ERBB2 (exons 19 and 20), PRKAR1a (exons 4 to 8), TP53 (exons 2 to 11), and the promoter region of TERT (UCSC:CRCh37/hg19 chromosome 5, nucleotides 1295228-250). NGS was performed as previously described (9). The detection threshold of a mutation was set at 1%. The patients provided informed consent, and the study was conducted under guidelines approved by the Erasmus Medical Center medical ethics committee.
NGS of the Primary Tumor and Leukocytes
In the present study, six patients with ACC were included. Of these six patients, mutations were found in the primary
tumor of three. In these patients, mutation analyses of germline DNA isolated from leukocytes revealed no mu- tations. cfDNA was isolated from plasma drawn before and after surgery and analyzed for the same mutations using NGS as described in the previous section.
Case Report
Patient 1
A 57-year-old male patient was referred to the Eras- mus Medical Center because of an adrenal mass. The computed tomography (CT) scan showed a 15-cm left adrenal mass (Fig. 1), with lung, peritoneal, omental, and right adrenal metastases. One day after admission, blood was drawn and processed as described. Open debulking adrenalectomy was performed 10 days later, with re- moval of the omental metastases. The histopathology report described a tumor with a Weiss score of 6, in- cluding necrosis, nuclear atypia, mitotic count >5/50 HPF, atypical mitoses, infiltration, and diffuse architec- ture. Immunohistochemically, calretinin, inhibin, and melan-A were focally positive, suggestive of preexisting adrenal tissue and supporting an adrenocortical origin of the tumor. The Ki67 index was 50%. The histopath- ological characteristics of the tumor in the greater omentum and cytomorphology of a fine needle aspirate from a lymph node revealed features similar to those observed in the primary tumor. Six days postoperatively, mitotane was started (1500 mg/d), with the aim of in- creasing the dosage according to tolerability. A dosage of 6000 mg/d was reached; however, the maximum plasma level in the first 3 months was only 2.4 mg/L. A plasma level of ≥14 mg/L is considered therapeutic (10). Three weeks later, an abdominal CT scan showed disease progression. The patient developed ascites, which was drained, and also cutaneous metastases, a rare presentation of disseminated
(a)
(b)
Preoperative
Postoperative
100-
NRAS: c.182A>G:p.Q61R
80
Frequency (%)
- TP53: c.856G>A:p.E286K
60
---- TERT: 1295250C>T
40
B 114.4mm
20
0-
Primary tumor
preoperative cfDNA
postoperative cfDNA
A: 147,1mm
ACC (11). Mitotane was subsequently combined with etoposide, doxorubicin, and cisplatin (M-EDP) accord- ing to the FIRM-ACT (First International Randomized Trial in Locally Advanced and Metastatic Adrenocortical Carcinoma Treatment) protocol, as palliative treatment. The cutaneous metastases decreased in size with che- motherapy. The mitotane levels during this period varied from 3.2 to 3.9 mg/L. Stable disease, determined by ra- diography, was achieved after two courses of chemo- therapy. However, 4 months after initiation, M-EDP was discontinued because of disease progression. Specifically, the amount of lesions in the mesentery and pelvis had increased, and subcutaneous, intramuscular, and retro- peritoneal lesions were still present with a mixed re- sponse. Four weeks later, blood was drawn again. At that point, signs suspicious for leptomeningeal metastases were seen on magnetic resonance imaging (MRI). The patient died 2 months later.
NGS of the primary tumor showed an NRAS mutation (c.182A>G:p.Q61R) in 78% of the reads and a TP53 mutation (c.856G>A:p.E286K) in 60% of the reads. A mutation in the promoter region of the TERT gene (1295250C>T) was reported at a frequency of 28%. Isolation of cfDNA from the preoperative plasma yielded 123.6 ng/ml plasma. In contrast, the DNA yield from the postoperative sample was 1.99 ng/mL. NGS of the pre- operative cfDNA showed the same NRAS mutation as found in the primary tumor in 64% of the reads, the TP53 mutation in 32%, and the TERT mutation in 2% of the
reads. cfDNA from the plasma collected postoperatively showed the same three mutations in 52%, 16%, and 3% of the reads, respectively (Table 1). No mutations were found in DNA isolated from the leukocytes from the same blood samples.
Patient 2
A 5-cm right-sided cortisol-producing ACC was di- agnosed in a 61-year-old female patient, with no signs of lymph node or distant metastases as determined by CT, fludeoxyglucose positron emission tomography/CT, and MRI. Blood was drawn 2 weeks before the first surgery. After open adrenalectomy, the histopathology report described a tumor with a Weiss score of 5. The tumor showed immunoreactivity against synaptophysin, kera- tin, and melan-A, pointing toward a primary ACC. Be- cause of positive microscopic margins, mitotane was initiated at 6000 mg/d 1 month after surgery. At that point, a CT scan revealed no lung metastases or local recurrence. Because of toxicity, the mitotane levels did not reach 14 mg/mL in the 5 months after surgery. At 5 months postoperatively, CT and MRI scans revealed local recurrence, with a lesion in the liver suspicious for metastasis. Blood was drawn at that time.
NGS of the primary tumor showed a CTNNB1 mu- tation (c.100G>A: p.G34R) in 34% of the reads. NGS of the preoperative (yield, 6.50 ng/ml) and postoperative (yield, 8.93 ng/ml) cfDNA showed no mutations (Table 1).
| Patient and Sample | Yield (ng/ml Plasma) | Input DNA (ng) | Mutation 1 | %ª | Coverageb | Mutation 2 | %ª | Coverageb | Mutation 3 | %ª | Coverageb | Mean Coverage |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | ||||||||||||
| Primary tumor | 10.0 | NRAS: c. 182A> | 78 | 1337 | TP53: c.856G> | 60 | 694 | TERT: | 28 | 122 | 1299 | |
| sample | G:p.Q61R | A:p.E286K | 1295250C>T | |||||||||
| cfDNA sample | ||||||||||||
| Preoperative | 123.6 | 6.18 | 64 | 4580 | 32 | 2566 | 2 | 134 | 716 | |||
| 6 mo | 1.99 | 0.48 | 52 | 2408 | 16 | 1127 | 3 | 128 | 125 | |||
| postoperative | ||||||||||||
| 2 | ||||||||||||
| Primary tumor | 10.0 | CTNNB1: c.100G> | 34 | 1641 | NA | NA | NA | NA | NA | NA | 1074 | |
| sample | A: p.G34R | |||||||||||
| cfDNA sample | ||||||||||||
| Preoperative | 6.50 | 1.56 | ND | 2104 | 2350 | |||||||
| 5 mo | 8.93 | 2.10 | ND | 1422 | 1532 | |||||||
| postoperative | ||||||||||||
| 3 | ||||||||||||
| Primary tumor | 10.0 | TP53: c.542G> | 27 | 1191 | NA | NA | NA | NA | NA | NA | 1074 | |
| sample | A:p.R181H | |||||||||||
| cfDNA sample | ||||||||||||
| Preoperative | 15.88 | 3.81 | ND | 626 | 605 | |||||||
| 6 mo | 44.38 | 10.0 | ND | 2239 | 1961 | |||||||
| postoperative | ||||||||||||
| 14 mo | 13.00 | 3.00 | ND | 379 | 854 | |||||||
| postoperative | ||||||||||||
| 2 y postoperative | 19.75 | 4.74 | ND | 3997 | 109 |
Abbreviations: NA, not applicable; ND, not detectable (or below detection limit).
aFrequency of reads with mutations determined using NGS.
bTotal amount of reads per nucleotide position of the mutation.
Mean coverage per sample or mean depth.
Patient 3
A 76-year-old male patient was referred to the Erasmus Medical Center because of a right adrenal mass of 14 cm. A CT scan revealed a 14-cm right adrenal mass with prom- inent pretracheal and aortocaval lymph nodules but no signs of metastases. Open adrenalectomy was performed. The histopathology report described a tumor with a Weiss score of 5. At 3 weeks postoperatively, treatment with mitotane was started; mitotane levels of 8.4 mg/L were reached within 2 weeks. However, the patient had to withdraw from mitotane 2 months after the start of treatment because of toxicity. At 8 months postoperatively, a CT scan showed no signs of local recurrence or residual disease; however, it did show an enlarged ileocecal lymph node, for which colo- noscopy was performed, and the findings ruled out colon carcinoma. Two weeks later, blood was drawn. Sub- sequently, 6 months later, a CT scan showed increased nodules in the ileocecal and paraduodenal region on the right side, suspicious for local recurrence. Thus, blood was drawn again. Two months later, hemicolectomy and lymph node dissection were performed. The histopathology report confirmed locoregional recurrence of the primary ACC. Two months after surgery, mitotane was started but again was not tolerated by the patient. At the fourth blood sample drawn, a CT scan showed lesions in the right perirenal region suspicious for locoregional recurrence.
A mutation in the TP53 gene (c.542G>A:p.R181H) was found in 27% of the reads in the primary tumor. At all measurement points (preoperatively and post- operatively), the TP53 mutation was not found in the cfDNA (Table 1).
Discussion
To the best of our knowledge, we have shown for the first time the possibility of identifying ctDNA in patients with ACC. However, from the three patients in whom we identified gene mutations in the primary tumor, we were able to identify the mutations in the cfDNA of only one patient, suggesting this minimally invasive approach will only be suitable for monitoring disease progression in a subgroup of patients with ACC.
The cfDNA from both blood samples of patient 1 appeared to include high percentages of ctDNA, as in- dicated by the relatively high mutation frequencies. As expected, the mutation frequencies were lower in the cfDNA than in the primary tumor DNA, because only ctDNA includes mutations and the fraction of ctDNA will be influenced by tumor heterogeneity and the cfDNA amount released by apoptotic cells from healthy or inflamed tissues unrelated to the tumor. Several reports have already shown that the fraction of circulating DNA derived from the tumor varies greatly, from 0.01% to
90% (5). The three mutations found in patient 1 were previously reported to be associated with ACC patho- genesis (12). However, the frequencies of the TERT mutation (2% and 3%) and the coverage of this position in the cfDNA from patient 1 were low. No quantification of the background noise can be performed as yet for cfDNA with this panel; therefore, we could not consider this mutation as “real” in the cfDNA. The absence of mutations in the leukocytes served as a negative control.
The lower cfDNA yield and the lower mutation fre- quencies in the postoperative plasma of patient 1 could potentially be explained by the lower tumor load, because the primary tumor and most metastases were surgically removed. The patient had also undergone treatment with M-EDP before the postoperative blood sample was taken, which could have influenced ctDNA release. The mutation frequencies in the cfDNA from patients 2 and 3 were very low (<0.05%) and should therefore be interpreted as noise. For patients with very limited ctDNA in the plasma, it might be useful and required to use more sensitive techniques to detect tumor DNA. Although it will be difficult to interpret very low (<1%) mutation frequencies in cfDNA, the clinical relevance to date is also not known. Potential other explanations for the absence of tumor- specific mutation detection in cfDNA include the absence of circulating tumor cells and intermittent or no leakage of DNA by the tumor. The differences in relative mutation frequencies detected in the tumor tissue and blood samples between and within patients are indicative of intra- or intertumor molecular heterogeneity or might result from regional differences in ctDNA stability. That ctDNA was not detected in the same patient at different disease stages (local vs metastasized disease) might indicate that ctDNA release, stability, or clearance is also, to an extent, tumor or patient related. The absence of mutations in the plasma sample from patients whose tumor was found to have a mutation has been described previously in other types of cancer (13). cfDNA is also known to be fragmented. In other types of cancer, ctDNA released by apoptosis is thought to be approximately 166 bp. In contrast, necrosis releases larger fragments of about 10,000 bp (14, 15). DNA fragmentation analysis at 100 to 12,000 bp revealed fragments in part of our cfDNA samples, which might indicate that our cfDNA samples also harbored larger DNA fragments (>12,000 bp) or that more sensitive methods are necessary to detect DNA fragments at lower DNA concentrations (Supplemental Table 1).
Our approach considers the mutations detected by NGS in the primary tumor for identification of these tumor-specific mutations in the cfDNA. Consequently, the limitations of this approach include the required presence of ACC-associated mutations in the primary tumor and the minimal necessary DNA yield for NGS. To
enable discrimination between true mutations at low frequency and background noise due to sequencing artifacts and, thus, improve the sensitivity of NGS, mo- lecular barcodes could be used. Hence, mutations could be identified in reads originating from different mole- cules, which would allow for more reliable detection of mutations. This should be investigated in more detail.
Isolation and quantification of ctDNA in ACC has several potential clinical applications. However, on the basis of this case series, it might only be applicable for a subgroup of patients with ACC, potentially those with large tumors. Research could focus on the value of ctDNA as biomarker for diagnosis, tumor dynamics, treatment response, or prognosis. Finally, it would be interesting to identify the mechanisms underlying the response to sys- temic therapies, because the unresponsive clones of ctDNA will potentially remain in the circulation during treatment.
In conclusion, to the best of our knowledge, this is the first study reporting mutations in the cfDNA in a patient with ACC. In addition, our results showed variability in the fraction of ctDNA in the cfDNA between patients. Our results provide a basis for further research using innovative NGS approaches to enable minimally invasive techniques for monitoring progressive disease and detecting a tumor response in patients with ACC.
Acknowledgments
Financial Support: This study was financially supported by the Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 259735 (ENS@T-Cancer).
Correspondence and Reprint Requests: Leo J. Hofland, PhD, Division of Endocrinology, Department of Internal Medicine, Erasmus Medical Center, P.O. Box 2040, Rotterdam 3000 CA, The Netherlands. E-mail: l.hofland@erasmusmc.nl.
Disclosure Summary: The authors have nothing to disclose.
References
1. Fassnacht M, Libé R, Kroiss M, Allolio B. Adrenocortical carci- noma: a clinician’s update. Nat Rev Endocrinol. 2011;7(6): 323-335.
2. Creemers SG, Hofland LJ, Korpershoek E, Franssen GJ, van Kemenade FJ, de Herder WW, Feelders RA. Future directions in the diagnosis and medical treatment of adrenocortical carcinoma. Endocr Relat Cancer. 2016;23(1):R43-R69.
3. Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. 1977;37(3):646-650.
4. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, Bartlett BR, Wang H, Luber B, Alani RM, Antonarakis ES, Azad NS, Bardelli A, Brem H, Cameron JL, Lee CC, Fecher LA, Gallia GL, Gibbs P, Le D, Giuntoli RL, Goggins M, Hogarty MD, Holdhoff M, Hong SM, Jiao Y, Juhl HH, Kim JJ, Siravegna G, Laheru DA, Lauricella C, Lim M, Lipson EJ, Marie SK, Netto GJ, Oliner KS,
Olivi A, Olsson L, Riggins GJ, Sartore-Bianchi A, Schmidt K, Shih M, Oba-Shinjo SM, Siena S, Theodorescu D, Tie J, Harkins TT, Veronese S, Wang TL, Weingart JD, Wolfgang CL, Wood LD, Xing D, Hruban RH, Wu J, Allen PJ, Schmidt CM, Choti MA, Velculescu VE, Kinzler KW, Vogelstein B, Papadopoulos N, Diaz LA Jr. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014;6(224):224ra24.
5. Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, Thornton K, Agrawal N, Sokoll L, Szabo SA, Kinzler KW, Vogelstein B, Diaz LA Jr. Circulating mutant DNA to assess tumor dynamics. Nat Med. 2008;14(9):985-990.
6. Alix-Panabières C, Schwarzenbach H, Pantel K. Circulating tumor cells and circulating tumor DNA. Annu Rev Med. 2012;63: 199-215.
7. Assié G, Letouzé E, Fassnacht M, Jouinot A, Luscap W, Barreau O, Omeiri H, Rodriguez S, Perlemoine K, René-Corail F, Elarouci N, Sbiera S, Kroiss M, Allolio B, Waldmann J, Quinkler M, Mannelli M, Mantero F, Papathomas T, De Krijger R, Tabarin A, Kerlan V, Baudin E, Tissier F, Dousset B, Groussin L, Amar L, Clauser E, Bertagna X, Ragazzon B, Beuschlein F, Libé R, de Reyniès A, Bertherat J. Integrated genomic characterization of adrenocortical carcinoma. Nat Genet. 2014;46(6):607-612.
8. Papathomas TG, Duregon E, Korpershoek E, Restuccia DF, van Marion R, Cappellesso R, Sturm N, Rossi G, Coli A, Zucchini N, Stoop H, Oosterhuis W, Ventura L, Volante M, Fassina A, Dinjens WN, Papotti M, de Krijger RR. Sarcomatoid adrenocortical car- cinoma: a comprehensive pathological, immunohistochemical, and targeted next-generation sequencing analysis. Hum Pathol. 2016; 58:113-122.
9. Papathomas TG, Oudijk L, Zwarthoff EC, Post E, Duijkers FA, van Noesel MM, Hofland LJ, Pollard PJ, Maher ER, Restuccia DF, Feelders RA, Franssen GJ, Timmers HJ, Sleijfer S, de Herder WW, de Krijger RR, Dinjens WN, Korpershoek E. Telomerase reverse transcriptase promoter mutations in tumors originating from the adrenal gland and extra-adrenal paraganglia. Endocr Relat Cancer. 2014;21(4):653-661.
10. Terzolo M, Baudin AE, Ardito A, Kroiss M, Leboulleux S, Daffara F, Perotti P, Feelders RA, deVries JH, Zaggia B, De Francia S, Volante M, Haak HR, Allolio B, Al Ghuzlan A, Fassnacht M, Berruti A. Mitotane levels predict the outcome of patients with adrenocortical carcinoma treated adjuvantly following radical re- section. Eur J Endocrinol. 2013;169(3):263-270.
11. Assié G, Antoni G, Tissier F, Caillou B, Abiven G, Gicquel C, Leboulleux S, Travagli JP, Dromain C, Bertagna X, Bertherat J, Schlumberger M, Baudin E. Prognostic parameters of metastatic adrenocortical carcinoma. J Clin Endocrinol Metab. 2007;92(1): 148-154.
12. Forbes SA, Beare D, Gunasekaran P, Leung K, Bindal N, Bout- selakis H, Ding M, Bamford S, Cole C, Ward S, Kok CY, Jia M, De T, Teague JW, Stratton MR, McDermott U, Campbell PJ. COSMIC: exploring the world’s knowledge of somatic mutations in human cancer. Nucleic Acids Res. 2015;43(Database issue): D805-D811.
13. Kim ST, Lee WS, Lanman RB, Mortimer S, Zill OA, Kim KM, Jang KT, Kim SH, Park SH, Park JO, Park YS, Lim HY, Eltoukhy H, Kang WK, Lee WY, Kim HC, Park K, Lee J, Talasaz A. Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients. Oncotarget. 2015;6(37):40360-40369.
14. Mouliere F, Rosenfeld N. Circulating tumor-derived DNA is shorter than somatic DNA in plasma. Proc Natl Acad Sci USA. 2015;112(11):3178-3179.
15. Mouliere F, Robert B, Arnau Peyrotte E, Del Rio M, Ychou M, Molina F, Gongora C, Thierry AR. High fragmentation charac- terizes tumour-derived circulating DNA. PLoS One. 2011;6(9): e23418.