Background: The majority of ovarian cancers (OvCa) are exquisitely sensitive to primary cytoreductive surgery and platinum-based agents, yet up to 80% of late-stage disease will relapse and develop deadly resistance to subsequent therapies. Identifying molecular mediators driving this acquired resistance is essential to improve the tragic 12- to 18-month prognosis for patients with recurrent disease. We hypothesize that relapsed ovarian cancers—which are largely uncharacterized—are molecularly distinct from primary disease and acquire druggable vulnerabilities throughout their life histories. To test this hypothesis, we undertook a transcriptome-wide analysis of 19 longitudinally collected patient-matched pairs of chemotherapy-naïve and recurrent cancers.
Materials and Methods: Ilumina TruSeq Total RNA-sequencing was performed on 19 flash-frozen patient-matched pairs of primary and recurrent OvCa. Time to recurrence was up to 64 months with a median of 25 months—a shared variant analysis confirmed all paired samples were patient-matched. Adapter-trimmed RNA-sequencing reads were quantified with k-mer based lightweight-alignment (Salmon v0.8.2) and transcript-abundance estimates were collapsed to gene-level with tximport. Differentially expressed genes were determined with DESeq2 using a paired model to account for patient-matched samples. To identify gains and losses in clinically actionable genes (DGIdb 2.0), pair-specific, outlier fold-change thresholds were defined as Q1/Q3 -/+ [1.5 X IQR], using each pairs’ expression fold-change values (recurrence vs. primary) as the distribution. These discrete, longitudinal transcriptional remodeling events (LTREs) in relapsed OvCa were then assessed for recurrence across all cases. Given that OvCa is thought to be driven largely by genomic structural variation, fusion RNAs were then called with FusionCatcher v0.99.7b. Identified fusions were filtered for cancer specificity by discarding fusions detected in normal tissue (Human Protein Atlas and BodyMap). The same fusion analysis was performed on CCLE OvCa cell line RNA-seq and selected fusions were validated with RT-PCR and Sanger sequencing.
Results: A suite of genes were consistently upregulated in OvCa recurrences, the most significant being NTRK2 (adjusted p-value < 0.001)—a targetable tyrosine kinase. LTREs were also common with the most shared LTRE gains in recurrences being INHBA (44%) and IGF1 (39%). 18 of 19 (95%) recurrent cancers acquired cancer-specific fusion RNAs that were undetectable in the primary lesion. An in-frame, recurrence-acquired fusion between TOP2A, a target of doxorubicin and known chemoresistance mediator, and STAU1 was confirmed with RT-PCR. Lastly, we discovered recurrent (2 of 19 cases), in-frame CCDC6-ANK3 fusions that persisted throughout therapy in both the primary and relapsed lesions, each with distinct breakpoints. A CCDC6-ANK3 fusion was also validated in the chemoresistant OVCAR3 cell line. 15 of 19 cases (79%) harbored additional preserved fusions, albeit none were shared between cases.
Conclusions: Collectively, these results define multimodal transcriptomic mechanisms of ovarian cancer evolution in late disease. Considering that some acquisitions are highly recurrent and readily druggable (NTRK2, IHBA, IGF1), further preclinical studies are demanded and currently ongoing. Lastly, we establish acquired fusions involving known chemoresistance modulators and preserved fusion transcripts—which are maintained throughout therapy—as common somatic events in OvCa. Because fusion breakpoints are cancer specific, they may serve as promising patient-specific nucleotide targets and biomarkers.
Citation Format: Nolan Priedigkeit, Sarah Taylor, Shannon Grabosch, Jahnik Kurukulasuriya, Peter C. Lucas, Silvia Liu, Ester Elishaev, Amit Lugade, Kevin Eng, Anda Vlad, George C. Tseng, Kunle Odunsi, Robert P. Edwards, Adrian V. Lee. Recurrent transcriptional remodeling events and acquired fusion RNAs in relapsed ovarian cancers. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B55.