Abstract 5610: Circulating tumor DNA: FOXL2 402C-G mutation can be identified in plasma from adult granulosa cell tumor patients with recurrent disease
Authors: McConechy Melissa K. , Farkkila Anniina , Yang Winnie , Andersson Noora , Ng Ying , Unkila-Kallio Leila , McAlpine Jessica N. , Gilks Blake , Anttonen Mikko , Huntsman David G. 
Published: May 2015

Abstract
Adult granulosa cell tumors (aGCT) represent 2-5% of all ovarian cancers. Most patients present with stage 1 disease and have a good prognosis, however up to 30% of aGCTs will recur (median=7 years), and 50% of these patients die of aGCT. These women require prolonged surveillance for serum tumor markers. In recent years, the detection of somatic mutations in free circulating tumor DNA (ctDNA) in plasma has been investigated as a non-invasive method to diagnose and monitor different cancers. The analysis of plasma DNA, which is a mixture of ctDNA and normal DNA, can be extended to aGCT patients that are at risk to recur, which would involve testing for the presence of the FOXL2 402C-&gt;G mutation. This mutation is known to be present in 95% of aGCTs; FOXL2 mutation testing is currently being used to aid in tumor differential diagnosis.
FOXL2 mutations in free ctDNA can be detected in the plasma of aGCT patients and may aid in diagnosis and monitoring of patients.
Plasma samples (1-2mL) were extracted for ctDNA using the Qiagen circulating nucleic acid kit. The ctDNA was preamplified for 10 cycles using the C134W FOXL2 Taqman primer/probe, ABI genotyping mix, followed by a 1:5 dilution. Preamplified ctDNA products were subjected to droplet formation and counted on the Raindance Raindrop digital PCR instruments to detect wild type or mutant alleles. All analysis was performed using the Raindrop Analyst V2 software.
Of two aGCT cases tested, both showed detectable plasma ctDNA FOXL2 mutations using Raindance Raindrop digital PCR. For case 1, plasma from two subsequent time points were tested. The FOXL2 mutation was not detected in the first time point, 12 years from diagnosis, at which time the patient had relapsed disease and underwent chemotherapy. Surgery for extensive tumor burden was performed at the second time point, 6 months later, and a 16% allelic frequency of the FOXL2 mutation was detected in the plasma DNA. For case 2, two plasma samples were tested, the first from the primary surgery, the second at relapse two years later. The FOXL2 ctDNA mutation was present at 6% in the recurrent plasma sample, but not at primary diagnosis. The recurrence was originally diagnosed as a high-grade serous ovarian carcinoma, however the tumor was FOXL2 mutation positive and later re-diagnosed as a relapsed aGCT.
The analysis of two cases of aGCT plasma suggests that the pathognomonic aGCT associated FOXL2 mutation is present in recurrent disease. We also show that the presence of a ctDNA FOXL2 mutation confirms the differential diagnosis of recurrent disease. This is the first proof of principle that plasma DNA may be a non-invasive method to detect recurrent aGCT. Plasma samples from a large prospective cohort with primary or recurrent aGCT will be analyzed to test the sensitive of FOXL2 ctDNA to predict tumor burden. These studies will be imperative to determine the utilization of the plasma FOXL2 ctDNA mutation in routine diagnosis.
Citation Format: Melissa K. McConechy, Anniina Farkkila, Winnie Yang, Noora Andersson, Ying Ng, Leila Unkila-Kallio, Jessica N. McAlpine, Blake Gilks, Mikko Anttonen, David G. Huntsman. Circulating tumor DNA: FOXL2 402C-G mutation can be identified in plasma from adult granulosa cell tumor patients with recurrent disease. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5610. doi:10.1158/1538-7445.AM2014-5610