Abstract B10: FOXL2 402C&gt;G mutation can be identified in the circulating tumor DNA of patients with adult-type granulosa cell tumor.
Authors: Färkkilä Anniina , McConechy Melissa K. , Yang Winnie , Talhouk Aline , Ng Ying , Morin Ryan , Riska Annika , McAlpine Jessica N. , Unkila-Kallio Leila , Anttonen Mikko , Huntsman David G. 
Published: May 2016

Abstract
Background: Adult granulosa cell tumors of the ovary are molecularly unique as 94% of tumours are defined by the pathognomonic FOXL2 402C&gt;G C134W mutation. More accurate methods are needed for the diagnosis and follow-up of AGCT patients. This study was designed to evaluate circulating tumor DNA (ctDNA) FOXL2 mutation as a diagnostic and monitoring tool in AGCT patients.
Methods: An optimized ddPCR assay was performed on free ctDNA extracted from 120 serial plasma samples (1-2mL) collected prospectively from 35 AGCT patients during 8/2007-4/2014 in Helsinki, Finland and in Vancouver, Canada. Clinical follow-up information was retrieved from hospital records. The median follow-up time was 6.3 (range 1.1 – 33.9) years. The statistical analyses were performed with JMP Pro 10 software.
Results: Of the 35 AGCT patients, 33 (31 Finland, 2 Vancouver) patients had measurable disease at the time of sample collection, and 12 (36%) harbored a detectable plasma ctDNA FOXL2 mutation. The FOXL2 mutation was identified in the ctDNA of 6/17 (36.3%) patients with primary, and 6/31 (19.4%) of patients with recurrent disease, respectively. Median tumor size was significantly larger in the mutation positive cases (13.5cm) compared to the mutation negatives (7.5cm; p=0.003), and the mutation was present in 9/16 (56%) of samples from patients with tumors over 10cm in diameter. Similarly, ctDNA FOXL2 mutation frequency positively correlated to tumor size (Spearman's Rho=0.32, p=0.003). The sensitivity of the ddPCR assay to detect AGCT disease presence was 25%, while the specificity was 90% (ROC-AUC 0.58). In patients with primary tumors, the ctDNA FOXL2 positivity did not correlate with tumor stage, and none of these patients has presented with a tumor recurrence during follow-up. This is putatively due to the relatively short follow-up time of (3.6 years, range 1.1-6.3 years). Given that the median time to relapse has been reported to be 7-8 years, no definitive conclusions can be drawn on the effect of ctDNA FOXL2 on the risk of AGCT recurrence.
We identified the presence of low-level ctDNA FOXL2 mutations in four patient's plasma that did not have clinical disease present. From patient ID 208, the first sample was drawn 7 months post primary tumor operation and already then the FOXL2 mutation was detected at 1.4% frequency. Subsequently, the FOXL2 ctDNA mutation was detected at two time points during follow-up, before the elevation of serum markers and clinical relapse with multiple metastases. From patient ID 221, we identified the ctDNA FOXL2 mutation in a plasma sample taken upon primary surgery. However, the FOXL2 mutation was identified in plasma samples taken at 10 and 34 months post primary surgery. This patient was last monitored with no clinical signs of relapse 49 months post surgery. In two patients (ID 213, 216), the FOXL2 mutation was detected at primary diagnosis, then once (at 24 months for ID 213, and at 38 months for ID 216) post primary surgery, and no subsequent relapse had been detected for these two patients during follow-up. In total, we identified three patients with the ctDNA FOXL2 mutation present in the plasma with no evidence of subsequent recurrence. Since the FOXL2 mutation is specific to AGCT, the only source of ctDNA FOXL2 mutation is likely to be from occult AGCT cells, and further monitoring of these patients over time will reveal if they will develop a clinical AGCT recurrence.
Conclusions: As a proof of concept, we established that specific molecular diagnosis and detection of primary and recurrent AGCT can be achieved non-invasively with a “liquid biopsy” and ctDNA FOXL2 mutation testing. Further studies are needed to increase the sensitivity and clinical applicability of ctDNA FOXL2 mutation testing in AGCT patients.
Citation Format: Anniina Färkkilä, Melissa K. McConechy, Winnie Yang, Aline Talhouk, Ying Ng, Ryan Morin, Annika Riska, Jessica N. McAlpine, Leila Unkila-Kallio, Mikko Anttonen, David G. Huntsman. FOXL2 402C&gt;G mutation can be identified in the circulating tumor DNA of patients with adult-type granulosa cell tumor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B10.