Detection of Cell-Free Circulating Tumor DNA in Adrenocortical Cancer

Presentation Number: MON 363
Date of Presentation: April 3rd, 2017

Simon Garinet*1, Mario Neou2, Franck Letourneur3, Eric Pasmant4, Michel Vidaud4, Juliette Nectoux4, Rossella Libe5, Anne Jouinot6, Jerome Yves Bertherat7 and Guillaume Assie5
1Reference Center for Rare Adrenal Diseases, Assistance Publique Hopitaux de Paris, Hospital Cochin, INSERM U 1016, CNRS 8104, Institut Cochin, Paris Descartes University, Paris, France, 2Institut Cochin, INSERM U1016, CNRS UMR8104, Paris Descartes University, Paris, France, 3Genomic Platform, Insititut Cochin, INSERM U1016, CNRS UMR 8104, Institut Cochin Université Paris Descartes, Paris, 4Dpt of genetics, Cochin Hospital, Assistance Publique Hôpitaux de Paris, Paris, France, 5INSERM U 1016, CNRS 8104, Institut Cochin, Paris Descartes University, Paris, France, 6INSERM U1016, CNRS UMR 8104, Institut Cochin Université Paris Descartes, Paris, 7INSERM U 1016, Cochin Institute, Paris Descartes University, Paris, France



Cancers may release tumor DNA in blood flow, referred to as cell-free circulating tumor DNA (ctDNA). The ctDNA can be detected by searching in plasma somatic mutations previously identified in the tumor. Using ctDNA, it is possible to ascertain the presence of cancer in a blood sample (liquid biopsy).

Somatic mutations of ACC have recently been identified by exome sequencing. Twenty genes are recurrently mutated, found in 2/3 of ACC –those with the worse prognosis.

Objective: To demonstrate the presence of ctDNA in patients with ACC.

Patients and methods:

Twelve ACC patients were included, all presenting either the primary and/or metastases by the time blood was drawn. Plasma samples were collected using cell-free DNA BCT tubes (Streck).

For each patient, somatic mutations were searched by targeted NGS, using a specific panel including the 20 ACC genes (Ampliseq and PGM, Thermofisher) from frozen tumor and leukocyte DNA.

Plasma DNA was extracted using QIAamp DNA mini blood Kit (Qiagen), and quantified by Qubit (Life Technologies).

ctDNA was genotyped using two methods: either targeted NGS on an amplicon with high depth (AmpliSeq), or digital droplet PCR with a specific design for each mutation (QX100, BioRad).


A somatic mutation was found in 8/12 patients. Mutations were found in TP53 (n=5), CTNNB1 and NF1 (N=3), TERT (N=2), ATRX, RPL22, MED12 and MEN1 (N=1).

Median plasma DNA was quantified at 2.1 ng/µl [range 1-50].

In one patient, a TP53 substitution (c.C76T), with an allelic ratio of 71% in tumor, was studied. The specific amplicon carrying this mutation was amplified from plasma, and sequenced deeply by NGS (150k reads). Mutation was found in 16% of reads in plasma.

In a second patient, a CTNNB1 (coding for beta-catenin) mutation (c.C134T), with an allelic ratio of 92% in tumor, was studied. This mutation corresponds to the p.Ser45Phe hotspot mutation. The specific amplicon carrying this mutation was also sequenced by NGS (111k). Mutation was found in 13% of reads in plasma.

This c.C134T CTNNB1 mutation was also analyzed by digital droplet PCR, using a specific PCR assay. Mutation was found in 251/1649 (15.2%) droplets.

Analysis of ctDNA from the 6 remaining patients with a somatic mutation is ongoing.


The ctDNA is detectable in ACC. Sensitivity and quantitative value of ctDNA remain to be established, to properly determine the place of this technique for personal monitoring of ACC patients.


Nothing to Disclose: SG, MN, FL, EP, MV, JN, RL, AJ, JYB, GA