LINE-1 retrotranspositions in epithelial ovarian cancer : can we use DNA “parasites” for good purpose?

High grade serous ovarian cancer (HGSC), endometrioid ovarian cancer (ENOC) and clear cell ovarian cancer (CCOC) are the three most common subtypes of epithelial ovarian cancers (EOC). While HGSC arise from serous tubal intraepithelial carcinomas (STIC) lesions in the fallopian tube, ENOC and CCOC share a common precursor lesion, endometriosis (ectopic growth of uterine lining). Effective biomarkers of early cancer development and recurrence are lacking. We performed whole genome sequencing (WGS) and observed highly recurrent retrotransposition events originating from an active LINE-1 retrotransposon (L1) in the TTC28 gene in one-third of our ENCO and CCOC cohort. L1s are mobile genetic elements that encodes their own protein machineries to “copy-and-paste” their sequences into random genomic loci. A process called 3’ transduction occur when L1s insert the unique downstream DNA sequences along with their own sequences. All these processes may fuel genomic instability, as such L1s are epigenetically silenced in normal tissues, but are found to be re-activated in cancers and cancer precursor lesions. We hypothesize that L1s activate early in EOC tumorigenesis and that TTC28-L1 3’ transductions could be used as markers of tumor development and progression. Using conventional and multiplex PCR on formalin-fixed paraffin-embedded (FFPE) tumor tissues, we found that TTC28-L1 3’ transductions occurred early and preceded many somatic mutations. We developed a probe-based target capture sequencing method that could identify novel TTC28-L1 3’ transductions in frozen tumor and FFPE tissues, and potentially in circulating tumor DNA. Using immunohistochemistry (IHC), we observed high L1 protein expressions in HGSC and its precursor lesions. Our results suggest that TTC28-L1 events occur early in EOC development and L1 protein expressions may reflect pre-malignant transformations. The use of L1 protein IHC and our target capture assay could be explored as a potential method to track such development.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

LINE-1 retrotransposon-mediated DNA transductions in endometriosis associated 1 ovarian cancers

Objective: Endometrioid (ENOC) and clear cell ovarian carcinoma (CCOC) share a common precursor lesion, endometriosis, hence the designation endometriosis associated ovarian cancers (EAOC). Long interspersed nuclear element 1 (LINE-1 or L1), is a family of mobile genetic elements activated in many cancers capable of moving neighboring DNA through 3’ transductions. Here we investigated the involvement of specific L1-mediated transductions in EAOCs. Methods: Through whole genome sequencing, we identified active L1-mediated transductions originating within the TTC28 gene in 34% (10/29) of ENOC and 31% (11/35) of CCOC cases. We used PCR and capillary sequencing to assess the presence of specific TTC28-L1 transductions in formalin-fixed paraffin-embedded (FFPE) blocks from six different anatomical sites (five tumors and one normal control) for four ENOC and three CCOC cases, and compared the results to the presence of single nucleotide variations (SNVs)/frame shift (fs) mutations detected using multiplex PCR and next generation sequencing. Results: TTC28-L1 mediated transductions were identified in at least three tumor samplings in all cases, and were present in all five tumor samplings in 5/7 (71%) cases. In these cases, KRAS, PIK3CA, CTNNB1, ARID1A, and PTEN mutations were found across all tumor sites whilst other selected SNV/fs mutations of unknown significance were present at varying allelic frequencies. Conclusion: The TTC28-L1 transductions along with classical driver mutations were near ubiquitous across the tumors, suggesting that L1 activation likely occurred early in the development of EAOCs. TTC28-L1 transductions could potentially be used to determine clonal relationships and to track ovarian cancer progression.Medicine, Faculty ofNon UBCObstetrics and Gynaecology, Department ofPathology and Laboratory Medicine, Department ofReviewedFacultyResearche

TERT promoter mutation in AGCT

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 cordstromal 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. 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 potential. Telomere attrition during DNA replication induces genomic instability that can result in tumorigenesis. Telomerase consists of a catalytic protein subunit known as telomerase reverse transcriptase (TERT) and a functional RNA called telomerase RNA component (TERC). TERT is highly expressed in stem cells and is silenced upon differentiation in somatic cells. Most cancer cells attain proliferative immortality by upregulating the TERT gene to maintain telomere length and telomerase activity. 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 activators. 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 telomeres. TERT promoter mutations were first reported in familial melanoma and subsequently in sporadic melanoma. 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 expression. 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 cancers. 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 carcinomas. However, it is unknown whether TERT promoter mutations are frequent in sex cord-stromal tumors, including adult granulosa cell tumors (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.Medicine, Faculty ofScience, Faculty ofNon UBCComputer Science, Department ofMedical Genetics, Department ofPathology and Laboratory Medicine, Department ofUnreviewedFacult