UBC Faculty Research and Publications

TERT promoter mutation in adult granulosa cell tumor of the ovary Cochrane, Dawn R.; Xia, Zhouchunyang; Aubert, Geraldine; Färkkilä, Anniina E.M.; Horlings, Hugo M.; Yanagida, Satoshi; Yang, Winnie; Lim, Jamie L.P.; Wang, Yi Kan; Bashashati, Ali; Keul, Jacqueline; Wong, Adele; Norris, Kevin; Brucker, Sara Y.; Taran, Florin-Andrei; Krämer, Bernhard; Staebler, Annette; Oliva, Esther; Shah, Sohrab P.; Kommoss, Stefan; Kommoss, Friedrich; Gilks, C. Blake; Baird, Duncan M.; Huntsman, David G.; Pilsworth, Jessica 2018

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 1TITLE: TERT promoter mutation in adult granulosa cell tumor of the ovary RUNNING TITLE: TERT promoter mutation in AGCT  AUTHORS: Jessica A. Pilsworth1,2, Dawn R. Cochrane2, Zhouchunyang Xia2,3, Geraldine Aubert4, Anniina E. M. Färkkilä5,6, Hugo M. Horlings2,3, Satoshi Yanagida7, Winnie Yang2 , Jamie L.P. Lim2, Yi Kan Wang2, Ali Bashashati2, Jacqueline Keul8, Adele Wong9, Kevin Norris10, Sara Y. Brucker8, Florin-Andrei Taran8, Bernhard Krämer8, Annette Staebler12, Esther Oliva9, Sohrab P. Shah2,11, Stefan Kommoss8, Friedrich Kommoss13, C. Blake Gilks3, Duncan M. Baird10, David G. Huntsman2,3  AFFILITIATIONS: 1Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada 2Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada 3Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada 4Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada 5Children’s Hospital and Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland  6Harvard Medical School, Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA, USA  27Department of Obstetrics and Gynecology, The Jikei University School of Medicine, Tokyo, Japan 8Tübingen University Hospital, Department of Women’s Health, Tübingen, Germany 9Department of Pathology, Massachusetts General Hospital, Boston, MA, USA 10Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom 11Department of Computer Science, University of British Columbia, Vancouver, BC, Canada 12Tübingen University Hospital, Institute of Pathology, Tübingen, Germany 13Institute of Pathology, Campus Bodensee, Friedrichshafen, Germany  Corresponding author: Dr. David G. Huntsman, BC Cancer Research Centre 4th Floor, 675 West 10th Avenue, Vancouver, BC, V5Z 1L3, Canada Email: dhuntsma@bccancer.bc.ca  Tel: +1-604-675-8205             3Abstract The telomerase reverse transcriptase (TERT) gene is highly expressed in stem cells and silenced upon differentiation. Cancer cells can attain immortality by activating TERT to maintain telomere length and telomerase activity, which is a crucial step of tumorigenesis. Two somatic mutations in the TERT promoter (C228T; C250T) have been identified as gain-of-function mutations that promote transcriptional activation of TERT in multiple cancers, such as melanoma and glioblastoma. A recent study investigating TERT promoter mutations in ovarian carcinomas found C228T and C250T mutations in 15.9% of clear cell carcinomas. However, it is unknown whether these mutations are frequent in other ovarian cancer subtypes, in particular, sex cord-stromal tumors including adult granulosa cell tumors (AGCTs). We performed whole genome sequencing on ten AGCTs with matched normal blood and identified a TERT C228T promoter mutation in 50% of tumors. We found that AGCT with mutated TERT promoter have increased expression of TERT mRNA compared to those with wild-type TERT promoter. AGCT with TERT C228T mutation exhibited significantly longer telomeres compared to AGCT with TERT wild-type promoter. Extension cohort analysis using allelic discrimination revealed the TERT C228T mutation in 51 of 229 primary AGCTs (22%), 24 of 58 recurrent AGCTs (41%), and 1 of 22 other sex cord-stromal tumors (5%). There was a significant difference in overall survival between patients with TERT C228T promoter mutation in the primary tumors and those without it (p = 0.00253, log rank test). In seven AGCTs, we found the TERT C228T mutation present in recurrent tumors and absent in the corresponding primary tumor. Our data suggests that TERT C228T mutations may have an important role in progression of AGCT.    4 Telomeres are conserved, repetitive (TTAGGG) DNA-protein complexes that are added to the ends of chromosomes by the enzyme telomerase to prevent DNA damage and maintain replicative potential1. Telomere attrition during DNA replication induces genomic instability that can result in tumorigenesis2. Telomerase consists of a catalytic protein subunit known as telomerase reverse transcriptase (TERT) and a functional RNA called telomerase RNA component (TERC)3. TERT is highly expressed in stem cells and is silenced upon differentiation in somatic cells4. Most cancer cells attain proliferative immortality by upregulating the TERT gene to maintain telomere length and telomerase activity5. The known mechanisms of telomerase activation include mutations in the TERT promoter, TERT gene amplification, CpG methylation at the TERT promoter, changes in alternative splicing of TERT pre-mRNA and upregulation of transcriptional activators6. Approximately 90% of cancers express TERT, while the remaining 10-15% of cancers maintain their telomere length through a telomerase-independent method called alternative lengthening of telomeres7.  TERT promoter mutations were first reported in familial melanoma and subsequently in sporadic melanoma8,9. There are two hot-spot TERT promoter mutations, C228T and C250T, each generates an identical 11 base pair sequence containing a consensus binding motif for ETS transcription factors, and functions as either a transcriptional activator or repressor to regulate telomerase expression8,9. These two mutations are implicated in the activation of telomerase in other malignances such as central nervous system tumors, hepatocellular carcinomas, bladder cancers and thyroid cancers10,11. A recent study on TERT promoter mutations in gynecological malignancies, including ovarian and uterine carcinomas, reported TERT hot-spot mutations in 15.9% of ovarian clear cell carcinomas12. However, it is unknown whether TERT promoter mutations are frequent in sex cord-stromal tumors, including adult granulosa cell tumors  5(AGCTs). In this study, we evaluated the biological and clinical significance of TERT promoter mutations, specifically C228T, in total of 251 primary ovarian sex cord-stromal tumors.   Materials and Methods Patient cohort description AGCTs  (n = 303) were collected from Helsinki University Hospital (Helsinki, Finland; n = 142), Tübingen University Hospital (Tübingen, Germany; n =49), Massachusetts General Hospital (Boston, USA; n = 41), Netherlands Cancer Institute (Amsterdam, Netherlands; n = 30), OvCaRe Gynecological Tumor Bank, (Vancouver, Canada; n = 20), the Jikei University School of Medicine (Tokyo, Japan; n = 14) and Referral Center for Gynecopathology (Mannheim, Germany; n = 7). The other sex cord-stromal tumors were collected from Vancouver General Hospital (Vancouver, Canada; n = 22). The respective institutional research ethics board approved the waiver for patient consent. The BC Cancer Agency and the University of British Columbia Research Ethics Boards approved the overall project methods. All cases were reviewed by at least one of the following expert pathologists (C.B.G., F.K., H.M.H., A.N.K., R.B., H.L-C., B.T.C., L.H.) to confirm the diagnosis of the sex cord-stromal tumor. A FOXL2 C402G allelic discrimination assay was used to validate AGCT diagnosis. Of the 229 primary AGCTs, 223 harbored a heterozygous FOXL2 C402G mutation and 6 harbored a homozygous, and were considered molecular defined AGCT (MD-AGCT) as previously described13. For the AGCT sequencing cohort, tumors were reviewed from frozen material, by at least two expert gynecological pathologists (H.M.H., A.N.K., H.L.-C., and C.B.G.), and C.B.G. approved the final selected cohort. All AGCTs sequenced were primary tumor samples. Each respective  6institution for Helsinki, Boston, Tübingen, Amsterdam, Mannheim, and Tokyo cases collected clinical follow-up data.   Whole genome sequencing analysis of AGCT  A total of ten fresh-frozen AGCT tissues with >80% tumor cellularity (based on frozen hematoxylin and eosin slide review) were selected for cryosectioning and DNA extraction. Patient tumor and normal blood samples were collected at diagnosis during standard-of-care debulking surgery. DNA was extracted from matched normal (peripheral blood buffy coat) and tumor (frozen tissues) samples using QIAmp Blood and Tissue DNA (Qiagen, Mississauga, ON) and quantified using the Qubit Fluorometer with the dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific, Canada). Whole genome sequencing (WGS) was performed using Illumina HiSeq 2500 v4 chemistry (Illumina Inc., Hayward, CA) with the PCR-free protocol to eliminate PCR-induced bias and to improve coverage across the genome. WGS analysis was performed to identify somatic alterations in each case, including single nucleotide variants, small indels, copy number alterations and structural variants. For a full description of library construction and sequencing methods, please refer to Wang Y. K. et al., Nature Genetics, 201714.  Direct sequencing of TERT promoter in AGCT  TERT promoter (including positions 228 and 250) was PCR amplified from DNA extracted from all ten AGCTs and the recurrent AGCT-derived KGN cell line using the following primers: 5’-CAGCGCTGCCTGAAACTC-3’ (forward) and 5’-GTCCTGCCCCTTCACCTT-3’ (reverse). After denaturation at 94oC for 2 minutes, DNA was amplified over 35 cycles (94oC 30 seconds, 62oC 30 seconds, 68oC 30 seconds), followed by a final extension at 68oC for 5 minutes using an  7MJ Research (Ramsey, MN) Tetrad. PCR products were bi-directionally sequenced using an ABI BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).  Screening TERT C228T promoter mutation in extension cohort Allelic discrimination for TERT C228T promoter mutation in 229 primary AGCTs, 58 recurrent AGCTs and 22 other sex cord-stromal tumors was determined using a Custom TaqMan SNP Genotyping assay (Applied Biosystems, Foster City, CA). The sequences of the primers were as follows: 5’-CTGCCCCTTCACCTTCCA-3’ (forward) and 5’-GGGCCGCGGAAAGGAA-3’ (reverse). The wild-type specific probe (5’-VIC dye-CCCAGCCCCCTCCGG-NFQ (non-fluorescent quencher)) and mutant specific probe (5’-FAM dye-CCAGCCCCTTCCGG-NFQ) were included in the TaqMan Genotyping Master Mix (2x) and reactions were performed in the QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA) using 20 ng of DNA. After denaturation at 95oC for 10 minutes, DNA was amplified over 50 cycles (95oC 15 seconds, 60oC 90 seconds).  TERT mRNA expression quantification in AGCT  RNA was extracted using the miRNAeasy tissue kit (Qiagen, Mississauga, ON) and quantified using the NanoDrop ND1000 Fluorospectrometer (ThermoFisher Scientific, Canada). cDNA synthesis was performed using the SuperScript IV First-Strand Synthesis System on total RNA with 50 μM random hexamers (Invitrogen, Carlsbad, CA). TERT mRNA transcript expression was determined from the cDNA by real-time PCR in the QuantStudio 6 Flex Real-Time PCR System using the TERT TaqMan probe spanning exons 3 and 4 (Hs00972650_m1) with TaqMan  8Fast Advanced Master Mix (2x). The 18S (Hs03928990_g1) housekeeping gene was used for normalization (Applied Biosystems, Foster City, CA). The relative levels were calculated using the ΔΔCT method15. Telomere length evaluation in AGCT  Telomere length was determined using XpYp Single Telomere Length Analysis (STELA) as previously described16,17. Briefly, extracted DNA from all ten AGCTs was quantified by Hoechst 33258 fluorometry (Bio-Rad, Hercules, CA, USA) before dilution to 10 ng/μl in 10 mmol/l Tris-HCl, pH 7.5. A total of 10 ng of DNA was further diluted to 250 pg/μl in a volume of 40 μl containing 1 μmol/l Telorette2 linker and 1 mmol/l Tris-HCl, pH 7.5. Multiple polymerase chain reactions (PCRs; typically 6 reactions per sample) were carried out for each test DNA in 10-μl volume with 250 pg of DNA, 0.5 μmol/l of the telomere-adjacent and Teltail primers, 75 mmol/l Tris-HCl, pH 8.8, 20 mmol/l (NH4)2SO4, 0.01% Tween-20, 1.5 mmol/l MgCl2, and 0.5 units of a 10:1 mixture of Taq (ABGene, Thermo Scientific) and Pwo polymerase (Roche Molecular Biochemicals, Burgess Hill, West Sussex, UK). The reactions were cycled with an MJ PTC-225 thermocycler (MJ Research, Bio-Rad). The DNA fragments were resolved by 0.5% Tris acetate EDTA agarose gel electrophoresis, and detected by Southern blot hybridization with random-primed α-33P–labelled (Perkin Elmer, Waltham, Massachusetts, USA) TTAGGG repeat probe together with probes to detect the 1-kb (Stratagene, Agilent Technologies, Santa Clara, CA, USA) and 2.5-kb (Bio-Rad) molecular weight markers. The hybridized fragments were detected by phosphorimaging with a Typhoon FLA 9500 phosphorimager (GE Healthcare). The molecular weights of the DNA fragments were calculated using the Phoretix 1D quantifier (Nonlinear Dynamics, Newcastle upon Tyne, UK).   9Statistical analysis  Two-tailed Fisher exact tests were performed to compare the frequencies of TERT C228T mutation in AGCT and other sex cord-stromal tumors, and in primary versus recurrent AGCT. The frequency of C228T mutation in AGCTs collected from different institutions was evaluated using a two-tailed Fisher’s exact test. The TERT mRNA expression levels and telomere lengths of TERT mutant and TERT wild-type tumors were compared using a Mann-Whitney U test to determine whether TERT C228T promoter mutation was associated with increased TERT mRNA expression and longer telomeres, respectively. Overall survival, disease-specific survival and disease-free survival of AGCTs with and without TERT C228T promoter mutation were compared using the Kaplan-Meier method, followed by the log rank test to determine significance.   Results   TERT C228T promoter mutation is frequent in AGCT  Whole genome sequencing (WGS) analysis was performed on ten AGCTs and identified a TERT C228T mutation in 50% of tumors (Figure 1a). We previously performed WGS analysis on ovarian surface epithelial, including clear cell (CCOC), endometrioid (ENOC), and high-grade serous (HGSC) carcinomas. The frequency of TERT promoter mutations is summarized in Table 1. We found TERT promoter mutations in 2 of 35 CCOC (5.7%), 1 of 29 ENOC (3.4%) and 0 of 59 HGSC (0%)14. We did not identify any TERT C250T promoter mutations in AGCTs, thus our allelic discrimination analysis was performed for TERT C228T mutation in the extension cohort. The frequency of TERT C228T promoter mutation in the extension cohort of AGCTs and other  10sex cord-stromal tumors is summarized in Table 2. The C228T change was detected in 52 of 251 primary tumor samples (21%), with the majority occurring in AGCT. The mutation was present in 51 of 229 primary AGCT (22%) and 24 of 58 recurrent AGCT (41%), while only in 1 of 22 other sex cord-stromal tumors (5%). The mutation frequency between primary AGCT and other sex cord-stromal tumors was reaching significance (p = 0.05448, two-tailed Fisher’s exact test). The C228T mutation was also present in the KGN cell line, which was derived from a patient with recurrent AGCT. There was no significance difference in TERT C228T frequency among AGCTs collected from different countries (p =0.5843, two-tailed Fisher’s exact test, Figure 1b). TERT C228T promoter mutation is associated with elevated mRNA expression in AGCT  Among the ten AGCTs, we found that the five cases with a TERT C228T mutation showed a 10.8-fold increase of TERT mRNA expression as compared to the five tumors without TERT promoter mutations. The majority of AGCT harboring a TERT C228T mutation expressed higher levels of TERT mRNA than those with a wild-type promoter (Figure 1c). However, the difference was not significant (p  = 0.1043, Mann-Whitney U test). Interestingly, we found one wild-type TERT AGCT expressed a high level of TERT mRNA due to a copy number gain (n = 3). When this wild-type TERT AGCT was removed from the group, TERT promoter mutants expressed significantly higher levels of TERT mRNA compared to those with TERT wild-type promoter (p = 0.01587, Mann-Whitney U test). We excluded this wild-type TERT AGCT with the TERT copy number gain (VOA1405b) from subsequent analysis.  AGCT with TERT C228T promoter mutation tend to have longer telomere lengths  11To evaluate the relationship between TERT C228T mutation and telomere length, we assessed the XpYp telomere length in ten AGCTs (Figure 1d). Some of the tumors exhibited more heterogeneous telomere length distributions, for example VOA985c, VOA1173b, VOA1405b, and VOA1744b (Supplementary Figure 1a-b). Other tumours exhibited more homogenous telomere length distributions along with longer telomeres (Supplementary Figure 1a-b). TERT C228T promoter mutant AGCTs had significantly longer telomeres compared to those with wild-type TERT (p = 0.0303, Mann-Whitney U test). Three of four tumors  (VOA1617a; VOA3082b; VOA4047b) with a TERT mutation yield homogenous telomere length profiles that are typical of clonal growth as seen in the HT1080 Clone 2 control cell line and is most likely mediated by telomerase (Supplementary Figure 1b). The TERT promoter wild-type cases (VOA985c; VOA1405b) that express TERT mRNA exhibit a wide distribution of telomere lengths and is similar to what has been previously described in alternative lengthening of telomeres (ALT) cell lines, which use a recombination telomere maintenance process independent of telomerase (Supplementary Figure 1b).  Two of the remaining three AGCTs with no TERT mRNA expression yield heterogeneous telomere lengths and resemble ALT tumors. However, we interrogated the WGS data and did not find any mutations in ATRX or DAXX, the two commonly mutated genes described in ALT tumors18,19.  TERT C228T promoter mutation is present in recurrent AGCT but absent in corresponding primary tumors Despite the numerous studies on telomerase and TERT expression in multiple cancers, it is poorly defined at which stage telomerase is activated in tumorigenesis. Recent reports identified TERT promoter mutations in early stages of melanoma20, precursor lesions of hepatocellular  12carcinomas21, and benign urothelial lesions22, suggesting that TERT promoter mutation is an early genetic event in oncogenesis. To determine if TERT C228T promoter mutation is an early mutation event in AGCT, we compared the frequency of C228T mutation in primary (22%) and recurrent (41%) tumors and found the difference to be significant (p = 0.003334, two-tailed Fisher’s exact test, Figure 2a). We found seven AGCTs with no TERT C228T mutation in the primary tumor but with the mutation in either the first or second recurrent tumor (Figure 2b). The remaining five AGCTs with primary/recurrent pairs were TERT promoter wild-type.    AGCT patients with TERT C228T promoter mutation tend to have worse survival   In 186 primary AGCT patients with available follow-up data, there was a significant difference in overall survival between patients with TERT C228T promoter mutation and those with TERT wild-type promoter (p = 0.00253, log rank test, Figure 3a). When tested for differences in disease-specific survival, the p-value was reaching significance (p = 0.0754, log rank test, Figure 3b). For disease-free survival, there was no significant difference (p = 0.705,log rank test, Figure 3c).   Discussion  In this study, we found that AGCTs have the highest frequency of TERT C228T promoter mutation among tumors that have been studied in the ovary including surface epithelial carcinoma and sex cord-stromal tumors. AGCTs account for approximately 90% of sex cord-stromal tumors and are characterized by their slow, indolent growth23. They are thought to arise  13from proliferating granulosa cells of the pre-ovulatory stage, as the share several morphological and hormonal features, including estrogen and inhibin synthesis24. A single missense mutation in FOXL2 (c.402C>G; pC134W) is pathognomonic of AGCT and can aid in resolving diagnosis of AGCT for histologically challenging cases25,26. FOXL2 is a member of the forkhead box family of transcription factors and plays a fundamental role in ovarian folliculogenesis27. The majority of AGCTs are diagnosed at Stage I and therefore cured with surgery. However, late tumor recurrence is common and over 80% of patients with advanced-stage or recurrent tumors succumb to their disease.28 As the FOXL2 C402G mutation is present in essentially all AGCTs, it is likely that additional mutations drives tumor progression25. Our study is the first to use WGS to provide a comprehensive catalogue of coding and non-coding genetic events in AGCT, the most common among all sex cord-stromal tumors of the ovary. We compared the genomes of ten AGCT with their matched normal genomes with the objective to identify recurrent mutations that will aid in understanding and improving prognosis. The high frequency of TERT C228T promoter mutation in recurrent AGCT compared to primary AGCT highlights a potential mechanism for clonal expansion and recurrence.  Several studies have shown the two hot-spot TERT promoter mutations (C228T; C250T) generate a binding motif for ETS transcription factors and increase transcriptional activity of the TERT promoter11. Similarly, we observed higher TERT mRNA expression in the AGCTs harboring a TERT C228T promoter mutation and TERT mutant tumors had significantly longer telomeres compared to TERT wild-type tumors. One AGCT with TERT wild-type promoter with an identified copy number gain expressed high levels of TERT. This result highlights the importance of determining both the genotype and gene expression to identify copy number gains or epigenetic regulation. During ovarian follicle development, telomerase activity is tightly  14regulated in a temporal and spatial pattern, where TERT expression is dependent on cell type and folliculogenesis stage29. Studies have reported that TERT expression correlates with the length of telomeres in primary granulosa cells and that small pre-ovulatory follicles have the highest telomerase activity with the longest telomeres30. As the granulosa cells undergo differentiation into luteinized granulosa cells and the follicle develops into the corpus luteum, there is a large decline in telomerase activity31,32. This suggests that telomerase plays an essential role in granulosa cell apoptosis and follicular atresia31,33. Thus, persistent telomerase expression in granulosa cells would result in continued survival beyond the necessary cell divisions and could lead to the development of AGCT. Our findings are consistent with a revised model recently proposed by Chiba et al., whereby cells harboring activating TERT promoter mutations extend their proliferative capacity34. Our results highlight the importance of telomerase activity in tumor development, where TERT mRNA expression is seen in seven of ten cases, five of which harbor the TERT C228T promoter mutation. Allelic discrimination analysis of primary and recurrent AGCTs indicates that TERT C228T promoter mutation are already present in some primary tissues, but may be late events which occur during AGCT progression. Studies have reported TERT promoter mutations as an early genetic event in malignant transformation of multiple cancers20-22. In contrast, we found that TERT C228T mutations were present in a significantly higher proportion of recurrent compared to primary tumors. We observed TERT mutations in recurrent tumors where no mutations were detected in the corresponding primary tumor (Figure 2b), suggesting that TERT promoter mutations were acquired during their progression. However, it may also be possible that TERT C228T mutation was present in the primary tumors at a very low frequency that was undetectable. This discordance of TERT mutation in paired primary and recurrent AGCT  15suggests that these tumors exhibit intratumoral genetic heterogeneity. Future studies using targeted deep sequencing at the TERT promoter would elucidate whether TERT mutations are present at low frequencies in primary tumors. It is notable that we found the TERT C228T promoter mutation in the recurrent AGCT-derived cell line KGN. Similarly, addition of TERT expression was required for immortalization of the SVOG cell line that was derived from primary granulosa cells, suggesting the need for telomerase activity in AGCT. Previous studies have associated TERT promoter mutations with a worse prognosis in melanoma35, glioblastoma, and bladder cancer patients36. In contrast, previous analysis of disease-specific survival in clear cell ovarian carcinoma comparing mutated TERT promoter and wild-type TERT promoter was not significant (p = 0.9592, log rank test)12. Although in our analysis there was a trend of worse disease-specific survival for TERT C228T mutant primary AGCTs, it was not significant (p = 0.0754, log rank test). Additional studies investigating all TERT promoter mutations, including the less common ones C250T, C242T and C243T37, would be required to accurately compare the prognosis of TERT promoter mutant and wild-type AGCT patients. Overall, we found that TERT C228T promoter mutation was most common in AGCTs among the ovarian carcinomas and sex cord-stromal tumors. Our data confirms the activation of telomerase in AGCTs via TERT C228T promoter mutation, although alternative telomerase activation methods in AGCTs may exist. Our results suggest that TERT activation may play a role in AGCT recurrence. As such, telomere biology may be important for the progression of AGCTs.     16Acknowledgements: We wish to thank all the women who generously donated the samples used in this study. This work is supported by the Terry Fox Research Institute Program Project grant #1021, the British Columbia (BC) Cancer Foundation, the Vancouver General Hospital (VGH)–University of British Columbia (UBC) Hospital Foundation (to the OvCaRe ovarian cancer research team, Vancouver) and Cancer Research UK (C17199/A18246). JAP is supported by the UBC Four-Year Doctoral Fellowship. We are grateful for the clinicians from each institution for patient recruitment. We would like to thank the pathologists, Drs. C. Blake Gilks, Friedrich Kommoss, Ralf Buzow, Hugo M. Horlings, Anthony N. Karnezis, Hector Li Chang, Basile Tessier-Cloutier, and Lien Hoang, for reviewing slides. We would also like to thank our technicians, Amy Lum and Janine Senz, for their advice.   Conflict of interest: DGH and SPS are founders and shareholders of Contextual  Genomics Inc. GA holds part time employment with Repeat Diagnostics Inc. DMB declares no conflict of interests other than co-authorship of a patent application based on STELA. The other authors declare no conflicts of interest.  Statement of Authors Contributions: Conception and design: JAP, DRC, DGH   Provision of study materials or patients: AF, HMH, SY, EO, SK, FK, DGH Collection and assembly of data: JAP, DRC, ZX, GA, DMB, AF, HMH, SY, SK Data analysis and interpretation: JAP, DRC, YW, GA, DMB Manuscript writing: JAP, DRC, GA, DGH Final approval of manuscript: All authors  17References: 1 Akincilar, S. C., Unal, B. & Tergaonkar, V. Reactivation of telomerase in cancer. Cell Mol Life Sci 2016;73:659-1670. 2 Jafri, M. A., Ansari, S. A., Alqahtani, M. H. et al. Roles of telomeres and telomerase in cancer, and advances in telomerase-targeted therapies. Genome Med 2016;8:69. 3 Zhang, Q., Kim, N. K. & Feigon, J. Architecture of human telomerase RNA. Proc Natl Acad Sci U S A 2016;108:20325-20332. 4 Chiba, K., Johnson, J. Z., Vogan, J. M. et al. Cancer-associated TERT promoter mutations abrogate telomerase silencing. Elife 2015;4:7918. 5 Kim NW, P. M., Prowse K.R., Harley C.B. et al. 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Molecular pathogenesis of granulosa cell tumors of the ovary. Endocr Rev 2012;33:109-144. 29 Liu, J. P. & Li, H. Telomerase in the ovary. Reproduction 2010;140:215-222. 30 Russo, V., Berardinelli, P., Martelli, A. et al. Expression of Telomerase Reverse Transcriptase Subunit (TERT) and Telomere Sizing in Pig Ovarian Follicles. Journal of Histochemistry & Cytochemistry 2006;54:443-455.  2031 Yamagata, Y., Nakamura, Y., Umayahara, K. et al. Changes in Telomerase Activity in Experimentally Induced Atretic Follicles of Immature Rats. Endocrine Journal 2002;49:589-595. 32 Kossowska-Tomaszczuk, K., De Geyter, C., De Geyter, M. et al. The multipotency of luteinizing granulosa cells collected from mature ovarian follicles. Stem Cells 2009;27:210-219. 33 Lavranos, T. C., Mathis Jm Fau - Latham, S. E., Latham Se Fau - Kalionis, B. et al. Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles. Biol Reprod 1999;2:358-366 34 Chiba, K., Lorbeer, F. K., Shain, A. H. et al. Mutations in the promoter of the telomerase gene TERT contribute to tumorigenesis by a two-step mechanism. Science 2017;6358:1416-1420. 35 Nagore, E., Heidenreich, B., Rachakonda, S. et al. TERT promoter mutations in melanoma survival. Int J Cancer 2016;139:75-84. 36 Killela, P. J., Reitman, Z. J., Jiao, Y. et al. TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal. Proceedings of the National Academy of Sciences of the United States of America 2013;110:6021-6026. 37 Scott, G. A., Laughlin, T. S. & Rothberg, P. G. Mutations of the TERT promoter are common in basal cell carcinoma and squamous cell carcinoma. Mod Pathol 2014;27:516-523.   21Figure Legends Figure 1. (a) TERT promoter mutation in adult granulosa cell tumors (AGCTs). Shown is the schematic diagram of the TERT promoter region, representative chromatograms of the two hot-spot mutations (C228T; C250T). (b) Mutation status of TERT C228T promoter in AGCT separated by the country in which they were collected from: red, TERT C228T mutated promoter; blue, TERT wild-type promoter (c) AGCTs with mutated TERT promoter have increased expression of TERT mRNA; black horizontal bar, mean (d) Comparison of XpYp STELA telomere length mean between AGCTs with and without TERT promoter mutation; black horizontal bar, group mean  Figure 2. (a) Mutation status of TERT C228T promoter in primary and recurrent AGCTs: red, TERT C228T mutated promoter; blue, TERT wild-type promoter. (b) An example of acquired TERT C228T promoter mutations in seven AGCTs with primary and recurrent tumour pairs: red, TERT C228T mutated promoter; blue, TERT wild-type promoter  Figure 3. Kaplan-Meier survival curves of AGCTs patients (a) Overall survival in patients with TERT C228T promoter mutation is worse than patients with wild-type TERT promoter (p = 0.00253, log rank test) (b) Disease-specific survival in patients with TERT C228T promoter mutation is similar to patients with wild-type TERT promoter (p = 0.0754, log rank test) (c) Disease-free survival in patients with TERT C228T promoter mutation is similar to patients with wild-type TERT promoter (p = 0.705, log rank test)   Table 1. Prevalence of TERT C228T and C250T promoter mutations in ovarian cancer C228T C250T Mutated/Total % Ovarian cancer         Adult granulosa cell tumors 5 0 5/10 50      Clear cell ovarian cancer 1 1 2/35 5.7      Endometrioid ovarian cancer 1 0 1/29 3.4      High-grade serous ovarian cancer 0 0 0/59 0  Table 2. Prevalence of TERT C228T promoter mutation in ovarian sex cord-stromal tumors  Mutated/Total % Ovarian sex cord-stromal tumor      Primary Adult Granulosa Cell Tumor 51/229 22      Recurrent Adult Granulosa Cell Tumor 24/58 41      Adult Granulosa Cell Tumor KGN Cell Line 1/1 100      Sertoli-Leydig Cell Tumour 1/5 20      Fibroma/Thecoma 0/14 0      Gynandroblastoma  0/1 0      Unclassified 0/2 0   


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