Circulating tumor DNA: FOXL2 402

CirculaIng tumor DNA: FOXL2 402C-‐>G mutaIon can be idenIﬁed in plasma from adult granulosa cell tumor paIents with recurrent disease Melissa McConechy1,2★, Anniina Farkkila3, Winnie Yang1, Noora Andersson3, Ying Ng1, Leila Unkila-‐Kallio3, Jessica N McAlpine4, Blake C Gilks2, Mikko AnBonen3, and David Huntsman1,2 1BriIsh Columbia Cancer Agency, 2Dept of Pathology and Laboratory Medicine, University of BriIsh Columbia, 3 Dept of Obstetrics and Gynecology, University of Helsinki, 4Dept of Obstetrics and Gyneacology, University of BriIsh Columbia ABSTRACT KGN DILUTION SERIES RESULTS Background: Adult granulosa cell tumors (aGCT) represent 2-‐5% of all ovarian cancers. Most paIents 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 paIents die of aGCT. These women require prolonged surveillance for serum tumor markers such as, inhibin B, and AMH. In recent years, the detecIon of somaIc mutaIons in free circulaIng tumor DNA (ctDNA) in the plasma of cancer paIents has been invesIgated 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 paIents that are at risk to recur, which would involve tesIng for the presence of the FOXL2 402C-‐>G mutaIon. This mutaIon is known to be present in 95% of aGCTs; FOXL2 mutaIon tesIng is currently being used to aid in tumor diﬀerenIal diagnosis. PreampliﬁcaIon of 0.5ng KGN DNA Input Reliably Detects 5% FOXL2 MutaIon Frequency Hypothesis: FOXL2 mutaIons in free ctDNA can be detected in the plasma of aGCT paIents and may aid in diagnosis and monitoring of paIents. Methods: Plasma samples (1-‐2mL) were extracted for ctDNA using the Qiagen circulaIng 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 diluIon. Preampliﬁed ctDNA products were subjected to droplet formaIon and counted on the Raindance RaindropTM digital PCR instruments to detect wild type or mutant alleles. All analysis was performed using the Raindrop Analyst V2 soiware. ★ [email protected] PreampliﬁcaIon of 1ng KGN DNA Input Reliably Detects 0.5% FOXL2 MutaIon Frequency PreampliﬁcaIon of 10ng KGN DNA Input Reliably Detects 0.05% FOXL2 MutaIon Frequency PreampliﬁcaIon of 5ng KGN DNA Input Reliably Detects 0.5-‐0.05% FOXL2 MutaIon Frequency Results: Of two aGCT cases tested, both showed detectable plasma ctDNA FOXL2 mutaIons using Raindance RaindropTM digital PCR. For case 1, plasma from two subsequent Ime points were tested. The FOXL2 mutaIon was not detected in the ﬁrst Ime point, 12 years from diagnosis, at which Ime the paIent had relapsed disease and underwent chemotherapy. Surgery for extensive tumor burden was performed at the second Ime point, 6 months later, and a 16% allelic frequency of the FOXL2 mutaIon 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 mutaIon 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 mutaIon posiIve and later re-‐diagnosed as a relapsed aGCT. Conclusions: The analysis of two cases of plasma suggests that the pathognomonic aGCT associated FOXL2 mutaIon is present in recurrent disease. We also show that the presence of a ctDNA FOXL2 mutaIon conﬁrms the diﬀerenIal 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 prospecIve cohort with primary or recurrent aGCT will be analyzed to test the sensiIvity of FOXL2 ctDNA to predict tumor burden. These studies will be imperaIve to determine the uIlizaIon of the plasma FOXL2 ctDNA mutaIon in rouIne diagnosis. BACKGROUND Count Threshold Use FOXL2 tesIng of primary tumor for primary diagnosis and diﬀerenIal diagnosis Count Threshold Count Threshold Count Threshold 95% of tumors harbor a heterozygous FOXL2 mutaIon 402C -‐>G (C134W) 5% •  To construct a sensiIve assay that can be used to detect the FOXL2 mutaIon (402C -‐>G, C134W) using Raindance RaindropTM digital PCR Dilute KGN DNA in a diluIon series with normal DNA of 0.005%, 0.05%, 0.5%, 5%, 25% and 50% FOXL2 mutaIon input With varying DNA input of 0.5, 1, 5 and 10ng. No PreampliﬁcaIon QuanIfy ctDNA (Qubit), determine ctDNA input between 2-‐10ng PreampliﬁcaIon (10 cycles FOXL2 Taqman probe) Raindance RaindropTM Digital PCR Preamp 0.05% FAM – MUTANT SPECIFIC FAM -‐ MUTANT FAM -‐ MUTANT Case 4 – Primary diagnosis 0.19% (40/20982) Mutant FOXL2 FAM -‐ MUTANT VIC – WILDTYPE VIC – WILDTYPE FAM -‐ MUTANT Case 6 – Recurrent disease 6.7% (1808/27075) Mutant FOXL2 Case 5 – Recurrent disease 3.4% (1255/34072) Mutant FOXL2 FAM -‐ MUTANT FAM -‐ MUTANT CONCLUSIONS AND FUTURE DIRECTIONS •  The pathognomonic aGCT associated FOXL2 mutaIon can be detected in aGCT plasma ctDNA in primary and recurrent disease. •  PreampliﬁcaIon of a low concentraIon of fragmented ctDNA can provide a means to detect low-‐level mutaIons in a high background of normal DNA. VIC – WILDTYPE SPECIFIC aGCT KGN cell line Heterozygous FOXL2 mutaIon VIC – WILDTYPE M
WT Case 3 – 7 months post primary diagnosis 1.4% (75/5463) Mutant FOXL2 Case 2 – Recurrent disease 6% (510/8445) Mutant FOXL2 VIC – WILDTYPE 2 Extract 1.0 – 2.0mL plasma ctDNA (fragmented DNA) VIC – WILDTYPE METHODS 1 0.05% FAM – MUTANT SPECIFIC VIC – WILDTYPE Case 1 – Recurrent disease 15.6% (1948/12293) Mutant FOXL2 Extract and shear DNA (100-‐300bp) No Preamp Preamp aGCT PATIENT ctDNA DIGITAL PCR •  To determine if the FOXL2 mutaIon can be detected in plasma circulaIng tumor DNA (ctDNA) using the digital PCR assay, and if this assay can be used for diagnosis, and disease monitoring to detect recurrence. M
WT 0.5% FAM – MUTANT SPECIFIC FAM – MUTANT SPECIFIC AIMS No Preamp Preamp VIC – WILDTYPE SPECIFIC Adult Granulosa Cell Tumor (H&E)1 – A rare ovarian cancer No Preamp VIC – WILDTYPE SPECIFIC Preamp VIC – WILDTYPE SPECIFIC No Preamp VIC – WILDTYPE SPECIFIC Use FOXL2 tesIng of plasma ctDNA to primary diagnosis and disease monitoring •  Raindance RaindropTM digital PCR using a FOXL2 Taqman assay can reliably detect low level mutaIons in a low input concentraIon of DNA. •  A large case series of plasma from aGCT paIents over their clinical course is currently being screened for FOXL2 ctDNA mutaIons. •  This study will be used to determine if plasma ctDNA FOXL2 mutaIon tesIng can be used for the clinical diagnosis and monitoring of disease recurrence in aGCT paIents. FAM – MUTANT SPECIFIC Digital PCR using FOXL2 Taqman primer/probe mix To detect a wild-‐type VIC labeled allele, and a mutant FAM labeled allele References 1. Shah SP, Kobel M, Senz J, et al. MutaIon of FOXL2 in granulosa-‐cell tumors of the ovary. The New England journal of medicine 2009; 360: 2719-‐2729.

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