FOXL2 402C>G Mutation Can Be Identified in the Circulating Tumor DNA of Patients with Adult-Type Granulosa Cell Tumor  Anniina Färkkilä, Melissa K. McConechy, Winnie Yang, Aline Talhouk, Ying Ng, Amy Lum, Ryan D. Morin, Kevin Bushell, Annika Riska, Jessica N. McAlpine, C. Blake Gilks, Leila Unkila-Kallio, Mikko Anttonen, David G. Huntsman  The Journal of Molecular Diagnostics  Volume 19, Issue 1, Pages 126-136 (January 2017) DOI: 10.1016/j.jmoldx.2016.08.005 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Optimization of a FOXL2 digital droplet PCR (ddPCR) assay. Panels A–D show three figures each; the graphs in the left panels shows the digital droplet counts based on the percentage of mutant FOXL2 input. The black line depicts the approximate threshold of counts considered above background. The right panels show the ddPCR output graphs of the digital counts from the lowest threshold detected in each panel. The middle graphs show no preamplification (No Preamp), and the rightmost graphs are with Preamp. The wild-type droplets are on the y axis, and the mutant droplets are on the x axis. A: 0.5 ng of total DNA input, with the lowest detection at 5% FOXL2 mutant DNA with Preamp. In the rightmost graph, mutant droplets are clearly visible, but they are not present in the middle graph with No Preamp. B: One nanogram of total DNA input, with the lowest detection at 0.5% FOXL2 mutant DNA with Preamp. C: Five nanograms of total DNA input, with the lowest detection at 0.05% FOXL2 mutant DNA with Preamp. D: Ten nanograms of total DNA input, with the lowest detection at 0.05% FOXL2 mutant DNA with Preamp. Data are expressed as means ± SD. MUT, mutation; NEG, negative; WT, wild type. The Journal of Molecular Diagnostics 2017 19, 126-136DOI: (10.1016/j.jmoldx.2016.08.005) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Flowchart for the digital droplet PCR (ddPCR) assay to detect the FOXL2 circulating tumor DNA (ctDNA) mutations in adult granulosa cell tumor patient plasma. The flowchart documents the steps from acquisition of patient plasma samples to extraction of ctDNA, quantification, preamplification, ddPCR using the RainDrop Source and Sense instruments, and final droplet analysis. Additional details can be found in Materials and Methods. MUT, mutation; NEG, negative; qPCR, real-time quantitative PCR; WT, wild type. The Journal of Molecular Diagnostics 2017 19, 126-136DOI: (10.1016/j.jmoldx.2016.08.005) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 The circulating tumor DNA (ctDNA) FOXL2 402C>G mutation correlates to tumor size. The median tumor diameter in correlation to plasma ctDNA FOXL2 402C>G mutation positivity (Mann-Whitney U-test). Data are presented in quantile box plots. Long whiskers represent the range; short whiskers are the 5th and 95th percentiles. The lowest 25th quantile is below the box, 50th quantile is the the lower portion of the box (median), and 75th quantile is the upper box. Dashed lines represents the means. ∗P < 0.05. The Journal of Molecular Diagnostics 2017 19, 126-136DOI: (10.1016/j.jmoldx.2016.08.005) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Representative graphs of FOXL2 mutation detection in individual adult granulosa cell tumor (AGCT) patients. A: A 59-year-old patient (Patient 232) underwent surgery for a stage IA AGCT, and the ctDNA FOXL2 mutation was detected at primary surgery at 0.2% frequency, with simultaneous elevation of the serum markers. The FOXL2 mutation has not been detected thereafter, and the patient has not relapsed during follow-up of 36 months. B: A 60-year-old patient (Patient 216) underwent surgery for a primary 17-cm stage IA AGCT, and the mutation was detected at primary surgery at 3.02% frequency and after 25 months of follow-up at 0.63%, with no subsequent relapse. C: A 55-year-old patient (Patient 213) underwent surgery for a primary 14-cm stage IA AGCT, and the FOXL2 mutation was detected at primary surgery at 0.31% frequency. In a plasma sample obtained at the last clinical control visit 39 months after the primary surgery, the FOXL2 mutation is detected at 1.03% frequency; however, no clinical relapse was detected. D: A 60-year-old patient (Patient 221) underwent emergency laparotomy for intra-abdominal bleeding. A spontaneously ruptured 20-cm stage IC AGCT presenting with tumor torsion and acute hemoperitoneum was diagnosed. The ctDNA FOXL2 mutation was detected at 0.4% frequency in the sample taken at surgery. Complete cytoreduction was achieved, and the patient received four cycles of adjuvant chemotherapy [bleomycin-etoposide-carboplatin (BEC)]. However, the FOXL2 mutation was identified in the plasma samples taken 10 months (6.2%) and 32 months (0.4%) after primary surgery. This patient was last monitored with no clinical relapse 42 months after surgery. E: A 61-year-old patient (Patient 208) underwent laparoscopy for a 6-cm AGCT. The tumor ruptured intraoperatively, and she received no adjuvant chemotherapy. In the first plasma sample collected at a symptomless control visit 7 months after surgery, the FOXL2 mutation was present at 1.4% frequency. Subsequently, the FOXL2 ctDNA mutation was detected at 0.1% at 23 months, and again at 1.3% at 55 months after her primary surgery. Then 6 months later, clinical relapse with multiple subcutaneous and intraperitoneal metastases was detected. At the time of the operation, the FOXL2 mutation frequency was 0.15%; however, the number of mutated alleles detected (2 mutants/1359 normal alleles) was below the threshold to be counted positive. After complete cytoreductive surgery and four cycles of chemotherapy with BEC, the patient was disease free and the FOXL2 levels, as well as the serum marker levels, were normalized. However, 3 months later, serum marker levels started rising, and diffuse peritoneal carcinomatosis was detected by CT, and chemotherapy with BEC was reintroduced. F: A 31-year-old patient (Patient 51) had undergone surgery for a stage IC AGCT 21 years before the first plasma sample in the graph. She had undergone multiple operations for tumor recurrences 9, 14, and 17 years after the initial diagnosis. At 21 years after diagnosis, another tumor recurrence was detected in the abdominal cavity; at this point, the first plasma sample was tested to be ctDNA FOXL2 mutation negative. However, the second plasma sample was collected with a high tumor load at surgery and revealed a high level of FOXL2 mutation (15.8%). The surgery was incomplete, and chemotherapy (carboplatin and taxol) was initiated; however, the FOXL2 mutation was detected at 4.4% frequency 4 months after surgery, indicating residual disease. This patient died of her recurrent AGCT disease 10 months later. AMH, anti-Müllerian hormone; Ch, chemotherapy; freq, frequency; Op, operation; Prim, primary; S, serum. The Journal of Molecular Diagnostics 2017 19, 126-136DOI: (10.1016/j.jmoldx.2016.08.005) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions