@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Medicine, Faculty of"@en, "Non UBC"@en, "Obstetrics and Gynaecology, Department of"@en, "Pathology and Laboratory Medicine, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:identifierCitation "Z. Xia, et al., LINE-1 retrotransposon-mediated DNA transductions in endometriosis associated ovarian cancers, Gynecologic Oncology (2017)"@en ; dcterms:creator "Xia, Zhouchunyang"@en, "Cochrane, Dawn Renee"@en, "Anglesio, Michael"@en, "Wang, Yi Kan"@en, "Nazeran, Tayyebeh"@en, "Tessier-Cloutier, Basile"@en, "McConechy, Melissa K."@en, "Senz, Janine"@en, "Lum, Amy"@en, "Bashashati, Ali"@en, "Shah, Sohrab P."@en, "Huntsman, David G."@en ; dcterms:issued "2018-10-08T00:00:00"@en, "2017-10-09"@en ; dcterms:description """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."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/63453?expand=metadata"@en ; skos:note "1 TITLE: LINE-1 retrotransposon-mediated DNA transductions in endometriosis associated 1 ovarian cancers 2 AUTHORS: 3 Zhouchunyang Xia1, Dawn R Cochrane2, Michael S Anglesio1,3, Yi Kan Wang2, Tayyebeh 4 Nazeran1, Basile Tessier-Cloutier1, Melissa K McConechy4, Janine Senz2, Amy Lum2, Ali 5 Bashashati2, Sohrab P Shah1,2, David G Huntsman*1,2. 6 AFFILIATIONS: 7 1. Department of Pathology and Laboratory Medicine, University of British Columbia, 8 Vancouver, BC, Canada 9 2. Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada 10 3. Department of Gynecology and Obstetrics, University of British Columbia, Vancouver, BC, 11 Canada 12 4. Department of Human Genetics, McGill University, Research Institute of the McGill 13 University Health Centre, Montreal, Quebec, Canada 14 15 *Corresponding author: 16 David Huntsman 17 BC Cancer Research Centre 18 675 West 10th Avenue 19 Vancouver, BC V5Z 1L3 20 1-604-675-8205 (phone) 21 1-604-675-8218 (fax) 22 dhuntsma@bccancer.bc.ca 23 2 Abstract: 24 Objective: Endometrioid (ENOC) and clear cell ovarian carcinoma (CCOC) share a common 25 precursor lesion, endometriosis, hence the designation endometriosis associated ovarian cancers 26 (EAOC). Long interspersed nuclear element 1 (LINE-1 or L1), is a family of mobile genetic 27 elements activated in many cancers capable of moving neighboring DNA through 3’ 28 transductions. Here we investigated the involvement of specific L1-mediated transductions in 29 EAOCs. 30 Methods: Through whole genome sequencing, we identified active L1-mediated transductions 31 originating within the TTC28 gene in 34% (10/29) of ENOC and 31% (11/35) of CCOC cases. 32 We used PCR and capillary sequencing to assess the presence of specific TTC28-L1 33 transductions in formalin-fixed paraffin-embedded (FFPE) blocks from six different anatomical 34 sites (five tumors and one normal control) for four ENOC and three CCOC cases, and compared 35 the results to the presence of single nucleotide variations (SNVs)/frame shift (fs) mutations 36 detected using multiplex PCR and next generation sequencing. 37 Results: TTC28-L1 mediated transductions were identified in at least three tumor samplings in 38 all cases, and were present in all five tumor samplings in 5/7 (71%) cases. In these cases, KRAS, 39 PIK3CA, CTNNB1, ARID1A, and PTEN mutations were found across all tumor sites whilst other 40 selected SNV/fs mutations of unknown significance were present at varying allelic frequencies. 41 Conclusion: The TTC28-L1 transductions along with classical driver mutations were near 42 ubiquitous across the tumors, suggesting that L1 activation likely occurred early in the 43 development of EAOCs. TTC28-L1 transductions could potentially be used to determine clonal 44 relationships and to track ovarian cancer progression. 45 3 1. Introduction: 46 Ovarian cancer is the most lethal gynecological cancer, and it is the 5th leading cause of 47 cancer related deaths in women in the United States [1]. Endometrioid ovarian carcinoma 48 (ENOC) and clear cell ovarian carcinoma (CCOC) each account for approximately 10% of all 49 ovarian cancer cases [2]. Both cancers are low grade subtypes that are often diagnosed at stages I 50 and II. When diagnosed at stages III/IV, ENOC has a more favorable prognosis compared to 51 CCOC, as CCOC tends to have a much lower response rate to chemotherapy [2, 3]. A recent 52 study of the genomic profile of ovarian cancers stratified ENOC into three subgroups: 53 ultramutator (high mutation load), micro-satellite instable (MSI), and micro-satellite stable 54 (MSS), and CCOC cases into two subgroups: APOBEC (localized hypermutation) and mutation 55 signatures similar to one previously associated with age, though not age-related in CCOC [4]. 56 Despite having different pathology and genomic profiles, both ENOC and CCOC are 57 associated with endometriosis, hence the designation endometriosis associated ovarian cancer 58 (EAOC). Endometriosis is characterized by ectopic growth of endometrial epithelium and 59 stroma. Women with endometriosis have a 2- to 3-fold increased risk of developing ENOC and 60 CCOC [5]. Common genes with mutations implicated to drive tumorigenesis include ARID1A 61 (30%), PTEN (20%), and CTNN1B (38-50%) in ENOC, and ARID1A (46-57%) and PIK3CA 62 (33-46%) in CCOC. Cancer driving mutations in ARID1A, PTEN, and PIK3CA have also been 63 found in endometriotic lesions both adjacent and distant to the tumors, indicating a clonal 64 relationship between these diseases [6-9]. In addition, canonical cancer mutations have been 65 found in deep infiltrating endometriosis that is not associated with cancer [7]. Given that driver 66 mutations in EAOCs are only found in a subset of patients, and certain changes can occur in 67 4 endometriosis without cancer, additional types of biomarkers are needed to distinguish women 68 with endometriosis at risk of developing cancer. 69 In our whole genome sequencing of 29 ENOC and 35 CCOC cases, we observed frequent 70 rearrangement events originating from an active LINE-1 (L1) retrotransposon located in the 71 TTC28 gene on chromosome 22q12. Long interspersed nuclear elements 1 (LINE-1 or L1), are 72 repetitive mobile DNA elements that accounts for 17% of the human genome [10-13]. L1s 73 encode an endonuclease and a reverse transcriptase that allows them to \"copy-and-paste\" their 74 own sequences into random genomic locations via RNA intermediates [10, 12]. They are also 75 capable of taking unique downstream DNA fragments with them during this genetic propagation 76 in a process called 3’ transduction [10]. While most L1s are not functional due to structural 77 rearrangements, there are approximately 80-100 full-length L1s that are potentially active [10]. 78 L1 activation is important in the germ cells during embryogenesis [14], but epigenetic silencing 79 typically occurs in adult [12]. In the cancer genomes, however, L1s become activated as part of a 80 general degradation of genomic and epigenetic integrity [12]. 81 The effects of L1 retrotransposon activation in cancer development and progression has 82 been studied in various cancers, including head and neck, prostate, ovarian, colorectal, gastric, 83 esophageal, pancreatic, and breast cancer [10, 11, 13, 15-21]. While most studies surveyed the 84 landscape of somatic L1 retrotranspositions by detecting the presence of novel inserted L1 85 sequences in tumors, Tubio and colleagues studied the effect of 3’ transductions events mediated 86 by a set of active L1s in cancers [10]. This specific TTC28-L1-mediated transduction from 87 chromosome 22q12 is one of the most frequently active L1s found in colon, head and neck, and 88 lung cancer samples and is the only active L1 element in 93% of breast cancer samples [10]. 89 Specific L1-mediated transductions have been less extensively studied in EAOCs [17, 21]. 90 5 Here we used PCR and capillary sequencing to validate the presence of TTC28-L1-91 mediated transductions in different tumor samplings of ENOC and CCOC, and we compared the 92 results to targeted re-sequencing of selected single nucleotide variations (SNVs)/frame shift (fs) 93 mutations in the same tissues to infer biological timing of L1 transductions. Our data showed that 94 these transduction events appeared with canonical driver mutations for majority of the cases, and 95 suggests that they likely occurred early in tumorigenesis. We hope to use TTC28-L1 mediated 96 DNA transductions to gain insights into the tumor development of EAOCs. 97 6 2. Methods: 98 2.1. Whole genome sequencing (WGS) 99 The cohort and methods have been described previously by Wang et al.[4]. Briefly, tumor 100 (frozen tissue) and matched normal (buffy coat) DNA libraries were constructed for 29 ENOC 101 and 35 CCOC cases, and sequenced using Illumina HiSeq 2500 V4 chemistry. Analysis were 102 performed using bioinformatic methods described in Wang et al. supplemental materials [4]. 103 104 2.2. Case selection 105 Four ENOC and three CCOC cases with the highest numbers of TTC28-L1-mediated 106 transductions detected by WGS were selected from our previous study [4]. All materials were 107 provided by the OVCARE gynecological tissue bank. H&E slides for all available formalin-fixed 108 paraffin-embedded (FFPE) blocks were reviewed by expert pathologists (T.N. and B.T-C.). Five 109 FFPE tumor tissue blocks with the highest cellularity (>90%) and one representative FFPE 110 normal tissue block were selected for each of the seven cases. Overall project processes were 111 approved by the BC Cancer Agency or the University of British Columbia Research Ethics 112 Board (REB #H08-01411 and #H09-02153). 113 114 2.3. DNA extraction from FFPE blocks 115 FFPE tissue blocks were cut 10um thick and applied on charged glass slides, where relevant 116 areas, identified by a pathologist (T.N.), were macrodissected. DNA was extracted from FFPE 117 tissues using the QIAamp DNA FFPE Tissue Kit (Qiagen) as per manufacturers’ protocols. All 118 DNA was quantified using the broad range DNA assay Qubit fluorometer (Life Technologies). 119 120 7 2.4. PCR validation of TTC28-L1-mediated transduction events 121 To validate all L1 transduction events, primers were designed using Primer3 to generate 170-122 250bp amplicons, which spanned the transduction junction. Primers were validated on WGA 123 (whole genome amplified) DNA extracted from frozen tumor tissues (positive control) and buffy 124 coats (negative control) for each of the cases. Primers were tested at an annealing temperature of 125 60C, using PCR SuperMix High Fidelity mastermix (ThermoFisher). Successful PCR was 126 indicated by a single band at 200-300bp using gel electrophoresis. For failed PCRs (no band, a 127 smear, or multiple bands), a gradient PCR from 55C to 64C was performed. If the PCRs were 128 still unsuccessful, Platinum® Taq DNA Polymerase High Fidelity (ThermoFisher) were used in 129 substitution. Successful PCRs were validated via sequencing using ABI 3130XL automated 130 capillary sequencing as per manufacture protocol. Validated primers were then used on the 131 FFPE-extracted DNA samples with 50ng input. 132 133 2.5. Microfluidic PCR validation of selected SNV/Frameshift mutations 134 From the whole genome sequencing results, six likely pathogenic missense and/or frameshift 135 mutations were selected for each case for validations on the FFPE tissue blocks. Primer sets were 136 designed using Primer 3 to amplify the specific gene regions. Forward primers were tagged with 137 CS1 (5’-ACACTGACGACATGGTTCTACA-3’) and reverse primers with CS2 (5’-138 TACGGTAGCAGAGACTTGGTCT-3’) sequencing tags. PCR products (150-200bp) were 139 amplified using the Fluidigm 48X48 Access Arrays, as per manufacturers’ protocol, with input 140 of 50ng FFPE derived DNA. DNA barcodes (10bp) with Illumina cluster-generating adapters 141 were added to the libraries post-Fluidigm harvest, and cleaned-up using Agencourt AMpure XP 142 beads (Beckman Coulter). Barcoded PCR products were then quantified using the high 143 8 sensitivity DNA assay Qubit fluorometer (Life Technologies) and pooled to one total library by 144 normalizing to equal amounts of PCR product. In total, 48 samples were pooled and denatured 145 according to Illumina standard protocols, and sequenced using a MiSeq 300 cycle V2 kit on the 146 Illumina MiSeq for ultra-deep validations. Uni-directional barcode sequencing was performed. 147 Analysis was performed using the bam and VCF files generated through Illumina MiSeq 148 reporter. 149 9 3. Results: 150 3.1. TTC28-L1 mediated transductions are frequent events in clear cell and endometrioid 151 ovarian cancer. 152 Whole genome shotgun sequencing of 29 ENOC and 35 CCOC cases to a median 153 coverage of 51x and 37x for the tumour and matched normal DNA respectively was performed 154 in a previous study [4]. Here, we studied this same data set generated via a structural variant 155 caller (deStruct [22]), and found that the only recurrent rearrangement events originated from the 156 TTC28 gene at the chromosome 22q12 locus (Figure 1). Previously thought to be a fragile site 157 for translocation events, we now know that this is a L1-retrotransposon mediated 3’ transduction 158 event, as indicated by the clustering of breakpoints within 1 kb downstream of an active L1. This 159 retrotransposon-mediated transduction event was observed in 34% (10/29) of ENOC, and 31% 160 (11/35) of CCOC cases. None of the transductions targeted exons. There was no association 161 between TTC28-L1 mediated transduction and common mutations or landscape features (data 162 not shown). Because L1 elements are highly repetitive throughout the genome, there is difficulty 163 aligning the sequences. Consequently, there could be additional L1 retrotransposition events that 164 were undetected. 165 166 3.2. TTC28-L1 mediated transductions are early events in ENOC and CCOC oncogenesis 167 We validated these TTC28-L1 transductions on high quality gnomic DNA extracted from 168 patient frozen tumors and buffy coats via PCR, with primers designed to flank the sequences 169 around the insertion breakpoint, such that only samples with the transductions amplified. 170 Amplified PCR products are sequence on the Sanger to confirm the presence of breakpoints with 171 DNA sequences from both sides. While the TTC28-L1 transductions that failed at the PCR or 172 10 Sanger validation stages were excluded, each case had at least one validated transduction event 173 (for a list of validated TTC28-L1 transductions, see Supplemental Table S1). As expected, 174 validated TTC28-L1 transduction events were found only in the tumor and not the buffy coat 175 samples. 176 To infer the cellular timing of these TTC28-L1-mediated transductions, we took archival 177 FFPE tumor samplings from five different sites for each of the seven patients. We hypothesized 178 that the same transduction events detected at multiple tumor sites should indicate early 179 activation. For each case, we selected six somatic SNVs/fs mutations detected via WGS and with 180 highest frequencies and predicted impact, and performed targeted re-sequencing in the tumor 181 samplings to further characterize the timing of the TTC28-L1 mediated transductions relative to 182 WGS detected mutations (for a detailed list of SNVs/fs, see Supplemental Table S2). 183 We detected TTC28-L1 insertions in all FFPE tumor sites in the majority of the cases (5/7 184 cases, ENOC 1-4 and CCOC1) (Figure 2). In these cases, SNVs and fs mutations were detected 185 to varying allelic frequencies for each tumor block, reflecting both inter- and intra-tumour 186 heterogeneity (Figure 2 and Supplemental Table S2). For each case, SNVs/fs mutations that were 187 found in every tumor block sampled tend to be canonical cancer driver mutations, such as 188 mutations in ARID1A, PIK3CA, KRAS, PTEN, and CTNN1B (Figure 2 and Supplemental Table 189 S2). These results suggest that TTC28-L1 was likely activated early during tumorigenesis, and 190 was mediating DNA transductions along with SNVs/fs mutations in the earlier tumor clones. In 191 one of the five cases (ENOC 4) the TTC28-L1 transduction was also detected in the 192 histologically normal cervical tissue block used as a normal FFPE control, while the insertion 193 event was not found in the same patient’s buffy coat DNA. A second sampling from this tissue 194 11 block yielded the same result, which may indicate the presence of histologically normal cells 195 clonally related to the tumor, and suggest that L1 activation could occur even earlier. 196 In a single CCOC case (CCOC 2), we observed what appears to be two distinct clonal 197 populations within the same ovarian tumor, where three of the five tumor blocks surveyed had 198 highly similar SNVs/fs allelic frequencies in addition to presence of the TTC28-L1 transduction 199 event, while the other two tumor blocks contained neither SNVs/fs nor the TTC28-L1 200 transduction events. 201 Four cases in our cohort (ENOC 1, ENOC 2, ENOC4, and CCOC3) had synchronous 202 tumors in the ovary and the uterus. In the three endometrioid ovarian carcinoma cases (ENOC 1, 203 ENOC2, and ENOC 4), we observed the TTC28-L1 transduction event as well as several shared 204 SNV mutations in both the ovarian and uterine tumors. The observation of the same TTC28-L1 205 transductions in tumors at anatomically different locations further suggests that L1 activation 206 occurred early in these diseases. In the clear cell ovarian carcinoma case (CCOC 3), SNVs/fs 207 mutations and the TTC28-L1 event were found in all the samplings of the ovarian tumor but were 208 not seen in uterine tumor. 209 12 4. Discussion: 210 Herein we described the presence of DNA transductions mediated by a specific and 211 active retrotransposon, TTC28-L1, in the endometriosis associated ovarian carcinoma subtypes: 212 ENOC and CCOC. Our data showed that TTC28-L1 mediated transductions was detectable 213 across all tumor sites in the majority of the cases. The presence of TTC28-L1 transduction was 214 sometimes but not always accompanied by SNV/frameshift mutations at various allelic 215 frequencies. It is highly unlikely that the same retrotransposition event arose independently at 216 every tumor site, which indicates that TTC28-L1 transductions were not subclonal events, but 217 more likely events that occurred early in tumorigenesis. In addition, the presence of TTC28-L1 218 transductions correlated with high allelic frequencies of driver mutations implicated in the 219 development of these cancers, including mutations in ARID1A, CTNNB1, PIK3CA, and PTEN 220 [8], supporting our hypothesis that TTC28-L1 retrotransposons are activated early in EAOC 221 development. 222 Targeted resequencing was previously used by our group to describe a clonal relationship 223 between the endometrial and ovarian cancers in three of the four synchronous endometrioid and 224 ovarian tumors, ENOC 2, ENOC 4 and CCOC 3 [23]. We also found somatic mutations and 225 TTC28-L1 events shared between the uterine and ovarian tumors in ENOC 2 and ENOC 4, 226 however, we did not detect any of our selected mutations nor the TTC28-L1 event in the uterine 227 tumor from CCOC 3. CCOC 3 was unusual in several ways: different pathologies observed in 228 the ovary (clear cell) and uterus (endometrioid), and there was only one single shared somatic 229 event (a different event from the ones we repeated) as reported by Anglesio et al [23]. It is 230 possible this somatic event had occurred before either the seeding of endometriosis or the 231 activation of the TTC28-L1 retrotransposon. 232 13 It is interesting to note that 3’ transductions accounts for approximately one quarter of all 233 somatic L1 retrotranspositions, and the majority of L1 retrotranspositions result in solo L1 234 insertions, either as a full L1 or truncated at the 5’ end [10]. This meant that there are likely more 235 L1 insertions undetected in our cohort. Past studies on somatic L1 retrotranspositions in various 236 cancer types reported much more solo L1 insertions than 3’ transduction events; however, the 237 lack of L1 3’ transductions detected could be due to bias in L1 sequencing and analysis methods 238 [16, 18-20]. Nonetheless, we have found TTC28-L1 transduction events at a high frequency in 239 our cohort, which could be a unique feature of EAOCs. 240 While somatic L1 insertions are common in certain tumor types, there is little evidence 241 that they are oncogenic. Only two studies showed that somatic L1 retrotranspositions (solo L1 242 insertions) directly initiated oncogenesis in colorectal cancer via a somatic L1 insertion in an 243 exon of the tumor suppressor gene APC [24, 25]. The majority of somatic L1 insertions target 244 heterochromatic regions with no obvious functional impact [10, 12]. We also observe that our L1 245 transductions targeted non-coding regions within out cohort. 246 Most research in the field focuses on using L1 activations as a marker of tumor 247 development, by assessing the global hypomethylation of L1 loci and the expression of L1 248 mRNA and proteins throughout stages of cancer development and metastasis. The general 249 conclusion is that somatic L1 retrotranspositions are passenger events that occur after a 250 permissive environment has been established during cancer development, such as dysregulated 251 epigenetic control [10, 12, 17]. Evidence of L1 activation as an early event include a recent study 252 that found stepwise decrease of methylation across different L1 loci have been observed between 253 normal endometrium, contiguous endometriosis (endometriosis adjacent to tumor), and 254 ENOCs/CCOCs [17]. In gastrointestinal (GI) cancers, shared somatic L1 retrotranspositions 255 14 (solo insertions) have been found between precancerous lesions and colon tumors [20], as well as 256 between Barrett’s esophagus (precursor lesion of esophageal cancer) and concomitant 257 esophageal tumors [19], suggesting early activation of L1 during GI tumor development. This is 258 likely the scenario in our cohorts, where the TTC28-L1 was activated early during ENOC/CCOC 259 tumorigenesis, but after a retrotransposition-permissive environment was established. 260 The exact relationship between TTC28-L1 activation and malignant transformation in 261 these two ovarian cancer subtypes is challenging to elucidate. Further studies will be needed to 262 assay endometriosis tissues that are adjacent to tumors, distant from tumors, as well as 263 endometriosis without cancer involvement. Such studies could determine whether TTC28-L1 264 transduction is a useful marker for cancer risk of endometriosis. 265 EAOCs suffer from a lack of effective early detection tools. TTC28-L1 and other L1s can 266 potentially be used as biomarkers to identify early malignant transformations, especially in the 267 subset of CCOC/ENOC cases where common coding mutations are not present. In addition, a 268 better understanding of L1 activation and transduction patterns over the course of ENOC and 269 CCOC progression may help elucidate the underlying mechanisms of ovarian carcinogenesis and 270 progression. 271 272 15 Conflict of Interest Statement 273 Dr. David Huntsman reports that he is a founder and CMO of Contextual Genomics, a cancer 274 testing company; however, Contextual does not test for the features described in this manuscript. 275 Otherwise the authors have no conflict of interest. 276 16 Acknowledgement 277 The authors would like to thank the members of the Huntsman group and the OVCARE team for 278 all their technical supports. This research would not have been made possible without the 279 gracious donation of tissues from the women with ovarian cancer. This study was supported by 280 grants from the Canadian Cancer Society (Impact Grant # 701603), BC Cancer Foundation, and 281 the VGH & UBC Hospitals Foundations. 282 17 References 283 [1] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA: a cancer journal for clinicians. 284 2016;66:7-30. 285 [2] Gilks CB, Prat J. Ovarian carcinoma pathology and genetics: recent advances. Human 286 pathology. 2009;40:1213-23. 287 [3] Mackay HJ, Brady MF, Oza AM, Reuss A, Pujade-Lauraine E, Swart AM, et al. Prognostic 288 Relevance of Uncommon Ovarian Histology in Women With Stage III/IV Epithelial Ovarian 289 Cancer. International Journal of Gynecological Cancer. 2010;20:945-52. 290 [4] Wang YK, Bashashati A, Anglesio MS, Cochrane DR, Grewal DS, Ha G, et al. Genomic 291 consequences of aberrant DNA repair mechanisms stratify ovarian cancer histotypes. Nature 292 genetics. 2017;advance online publication. 293 [5] Pearce CL, Templeman C, Rossing MA, Lee A, Near AM, Webb PM, et al. 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Blue lines indicate ENOC 353 cases and red lines indicate CCOC cases. 354 22 Figure 2: a). TTC28-L1 retrotransposition presence and allelic frequencies for five selected 355 SNV/fs mutations at six anatomical sites for each of the seven ENOC and CCOC cases. 356 Three shades of grey portray differences in allelic frequencies: dark grey indicates high 357 frequency (>50%) at the point mutation; lighter grey indicates a frequency between 20% and 358 50%; lightest grey indicates low frequency (5%-20%). Red box indicates the presence of 359 TTC28-L1 retrotransposition, and white box indicates no variance or retrotransposition detected. 360 Numbers indicate the allelic frequencies in percentage. A.F., allelic frequencies; Ov, ovary; Cer, 361 cervix; Ut, uterus; R, right; L, left; N, normal; ND, not detected. b). Circos plot displays 362 retrotransposition events originating from TTC28 (Chr.22q12) for the seven selected cases. 363 All retrotransposition targets were in non-coding spaces. 364 23 Table S1. A list of the breakpoint positions (hg19 coordinates) of each validated transduction. 365 24 Table S2. A list of selected SNV/fs mutations for each case, accompanied by the allele count at 366 each FFPE block. 367 1 TITLE: LINE-1 retrotransposon-mediated DNA transductions in endometriosis associated 1 ovarian cancers 2 AUTHORS: 3 Zhouchunyang Xia1, Dawn R Cochrane2, Michael S Anglesio1,3, Yi Kan Wang2, Tayyebeh 4 Nazeran1, Basile Tessier-Cloutier1, Melissa K McConechy4, Janine Senz2, Amy Lum2, Ali 5 Bashashati2, Sohrab P Shah1,2, David G Huntsman*1,2. 6 AFFILIATIONS: 7 1. Department of Pathology and Laboratory Medicine, University of British Columbia, 8 Vancouver, BC, Canada 9 2. Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada 10 3. Department of Gynecology and Obstetrics, University of British Columbia, Vancouver, BC, 11 Canada 12 4. Department of Human Genetics, McGill University, Research Institute of the McGill 13 University Health Centre, Montreal, Quebec, Canada 14 15 *Corresponding author: 16 David Huntsman 17 BC Cancer Research Centre 18 675 West 10th Avenue 19 Vancouver, BC V5Z 1L3 20 1-604-675-8205 (phone) 21 1-604-675-8218 (fax) 22 dhuntsma@bccancer.bc.ca 23 2 Abstract: 24 Objective: Endometrioid (ENOC) and clear cell ovarian carcinoma (CCOC) share a common 25 precursor lesion, endometriosis, hence the designation endometriosis associated ovarian cancers 26 (EAOC). Long interspersed nuclear element 1 (LINE-1 or L1), is a family of mobile genetic 27 elements activated in many cancers capable of moving neighboring DNA through 3’ 28 transductions. Here we investigated the involvement of specific L1-mediated transductions in 29 EAOCs. 30 Methods: Through whole genome sequencing, we identified active L1-mediated transductions 31 originating within the TTC28 gene in 34% (10/29) of ENOC and 31% (11/35) of CCOC cases. 32 We used PCR and capillary sequencing to assess the presence of specific TTC28-L1 33 transductions in formalin-fixed paraffin-embedded (FFPE) blocks from six different anatomical 34 sites (five tumors and one normal control) for four ENOC and three CCOC cases, and compared 35 the results to the presence of single nucleotide variations (SNVs)/frame shift (fs) mutations 36 detected using multiplex PCR and next generation sequencing. 37 Results: TTC28-L1 mediated transductions were identified in at least three tumor samplings in 38 all cases, and were present in all five tumor samplings in 5/7 (71%) cases. In these cases, KRAS, 39 PIK3CA, CTNNB1, ARID1A, and PTEN mutations were found across all tumor sites whilst other 40 selected SNV/fs mutations of unknown significance were present at varying allelic frequencies. 41 Conclusion: The TTC28-L1 transductions along with classical driver mutations were near 42 ubiquitous across the tumors, suggesting that L1 activation likely occurred early in the 43 development of EAOCs. TTC28-L1 transductions could potentially be used to determine clonal 44 relationships and to track ovarian cancer progression. 45 3 1. Introduction: 46 Ovarian cancer is the most lethal gynecological cancer, and it is the 5th leading cause of 47 cancer related deaths in women in the United States [1]. Endometrioid ovarian carcinoma 48 (ENOC) and clear cell ovarian carcinoma (CCOC) each account for approximately 10% of all 49 ovarian cancer cases [2]. Both cancers are low grade subtypes that are often diagnosed at stages I 50 and II. When diagnosed at stages III/IV, ENOC has a more favorable prognosis compared to 51 CCOC, as CCOC tends to have a much lower response rate to chemotherapy [2, 3]. A recent 52 study of the genomic profile of ovarian cancers stratified ENOC into three subgroups: 53 ultramutator (high mutation load), micro-satellite instable (MSI), and micro-satellite stable 54 (MSS), and CCOC cases into two subgroups: APOBEC (localized hypermutation) and mutation 55 signatures similar to one previously associated with age, though not age-related in CCOC [4]. 56 Despite having different pathology and genomic profiles, both ENOC and CCOC are 57 associated with endometriosis, hence the designation endometriosis associated ovarian cancer 58 (EAOC). Endometriosis is characterized by ectopic growth of endometrial epithelium and 59 stroma. Women with endometriosis have a 2- to 3-fold increased risk of developing ENOC and 60 CCOC [5]. Common genes with mutations implicated to drive tumorigenesis include ARID1A 61 (30%), PTEN (20%), and CTNN1B (38-50%) in ENOC, and ARID1A (46-57%) and PIK3CA 62 (33-46%) in CCOC. Cancer driving mutations in ARID1A, PTEN, and PIK3CA have also been 63 found in endometriotic lesions both adjacent and distant to the tumors, indicating a clonal 64 relationship between these diseases [6-9]. In addition, canonical cancer mutations have been 65 found in deep infiltrating endometriosis that is not associated with cancer [7]. Given that driver 66 mutations in EAOCs are only found in a subset of patients, and certain changes can occur in 67 4 endometriosis without cancer, additional types of biomarkers are needed to distinguish women 68 with endometriosis at risk of developing cancer. 69 In our whole genome sequencing of 29 ENOC and 35 CCOC cases, we observed frequent 70 rearrangement events originating from an active LINE-1 (L1) retrotransposon located in the 71 TTC28 gene on chromosome 22q12. Long interspersed nuclear elements 1 (LINE-1 or L1), are 72 repetitive mobile DNA elements that accounts for 17% of the human genome [10-13]. L1s 73 encode an endonuclease and a reverse transcriptase that allows them to \"copy-and-paste\" their 74 own sequences into random genomic locations via RNA intermediates [10, 12]. They are also 75 capable of taking unique downstream DNA fragments with them during this genetic propagation 76 in a process called 3’ transduction [10]. While most L1s are not functional due to structural 77 rearrangements, there are approximately 80-100 full-length L1s that are potentially active [10]. 78 L1 activation is important in the germ cells during embryogenesis [14], but epigenetic silencing 79 typically occurs in adult [12]. In the cancer genomes, however, L1s become activated as part of a 80 general degradation of genomic and epigenetic integrity [12]. 81 The effects of L1 retrotransposon activation in cancer development and progression has 82 been studied in various cancers, including head and neck, prostate, ovarian, colorectal, gastric, 83 esophageal, pancreatic, and breast cancer [10, 11, 13, 15-21]. While most studies surveyed the 84 landscape of somatic L1 retrotranspositions by detecting the presence of novel inserted L1 85 sequences in tumors, Tubio and colleagues studied the effect of 3’ transductions events mediated 86 by a set of active L1s in cancers [10]. This specific TTC28-L1-mediated transduction from 87 chromosome 22q12 is one of the most frequently active L1s found in colon, head and neck, and 88 lung cancer samples and is the only active L1 element in 93% of breast cancer samples [10]. 89 Specific L1-mediated transductions have been less extensively studied in EAOCs [17, 21]. 90 5 Here we used PCR and capillary sequencing to validate the presence of TTC28-L1-91 mediated transductions in different tumor samplings of ENOC and CCOC, and we compared the 92 results to targeted re-sequencing of selected single nucleotide variations (SNVs)/frame shift (fs) 93 mutations in the same tissues to infer biological timing of L1 transductions. Our data showed that 94 these transduction events appeared with canonical driver mutations for majority of the cases, and 95 suggests that they likely occurred early in tumorigenesis. We hope to use TTC28-L1 mediated 96 DNA transductions to gain insights into the tumor development of EAOCs. 97 6 2. Methods: 98 2.1. Whole genome sequencing (WGS) 99 The cohort and methods have been described previously by Wang et al.[4]. Briefly, tumor 100 (frozen tissue) and matched normal (buffy coat) DNA libraries were constructed for 29 ENOC 101 and 35 CCOC cases, and sequenced using Illumina HiSeq 2500 V4 chemistry. Analysis were 102 performed using bioinformatic methods described in Wang et al. supplemental materials [4]. 103 104 2.2. Case selection 105 Four ENOC and three CCOC cases with the highest numbers of TTC28-L1-mediated 106 transductions detected by WGS were selected from our previous study [4]. All materials were 107 provided by the OVCARE gynecological tissue bank. H&E slides for all available formalin-fixed 108 paraffin-embedded (FFPE) blocks were reviewed by expert pathologists (T.N. and B.T-C.). Five 109 FFPE tumor tissue blocks with the highest cellularity (>90%) and one representative FFPE 110 normal tissue block were selected for each of the seven cases. Overall project processes were 111 approved by the BC Cancer Agency or the University of British Columbia Research Ethics 112 Board (REB #H08-01411 and #H09-02153). 113 114 2.3. DNA extraction from FFPE blocks 115 FFPE tissue blocks were cut 10um thick and applied on charged glass slides, where relevant 116 areas, identified by a pathologist (T.N.), were macrodissected. DNA was extracted from FFPE 117 tissues using the QIAamp DNA FFPE Tissue Kit (Qiagen) as per manufacturers’ protocols. All 118 DNA was quantified using the broad range DNA assay Qubit fluorometer (Life Technologies). 119 120 7 2.4. PCR validation of TTC28-L1-mediated transduction events 121 To validate all L1 transduction events, primers were designed using Primer3 to generate 170-122 250bp amplicons, which spanned the transduction junction. Primers were validated on WGA 123 (whole genome amplified) DNA extracted from frozen tumor tissues (positive control) and buffy 124 coats (negative control) for each of the cases. Primers were tested at an annealing temperature of 125 60C, using PCR SuperMix High Fidelity mastermix (ThermoFisher). Successful PCR was 126 indicated by a single band at 200-300bp using gel electrophoresis. For failed PCRs (no band, a 127 smear, or multiple bands), a gradient PCR from 55C to 64C was performed. If the PCRs were 128 still unsuccessful, Platinum® Taq DNA Polymerase High Fidelity (ThermoFisher) were used in 129 substitution. Successful PCRs were validated via sequencing using ABI 3130XL automated 130 capillary sequencing as per manufacture protocol. Validated primers were then used on the 131 FFPE-extracted DNA samples with 50ng input. 132 133 2.5. Microfluidic PCR validation of selected SNV/Frameshift mutations 134 From the whole genome sequencing results, six likely pathogenic missense and/or frameshift 135 mutations were selected for each case for validations on the FFPE tissue blocks. Primer sets were 136 designed using Primer 3 to amplify the specific gene regions. Forward primers were tagged with 137 CS1 (5’-ACACTGACGACATGGTTCTACA-3’) and reverse primers with CS2 (5’-138 TACGGTAGCAGAGACTTGGTCT-3’) sequencing tags. PCR products (150-200bp) were 139 amplified using the Fluidigm 48X48 Access Arrays, as per manufacturers’ protocol, with input 140 of 50ng FFPE derived DNA. DNA barcodes (10bp) with Illumina cluster-generating adapters 141 were added to the libraries post-Fluidigm harvest, and cleaned-up using Agencourt AMpure XP 142 beads (Beckman Coulter). Barcoded PCR products were then quantified using the high 143 8 sensitivity DNA assay Qubit fluorometer (Life Technologies) and pooled to one total library by 144 normalizing to equal amounts of PCR product. In total, 48 samples were pooled and denatured 145 according to Illumina standard protocols, and sequenced using a MiSeq 300 cycle V2 kit on the 146 Illumina MiSeq for ultra-deep validations. Uni-directional barcode sequencing was performed. 147 Analysis was performed using the bam and VCF files generated through Illumina MiSeq 148 reporter. 149 9 3. Results: 150 3.1. TTC28-L1 mediated transductions are frequent events in clear cell and endometrioid 151 ovarian cancer. 152 Whole genome shotgun sequencing of 29 ENOC and 35 CCOC cases to a median 153 coverage of 51x and 37x for the tumour and matched normal DNA respectively was performed 154 in a previous study [4]. Here, we studied this same data set generated via a structural variant 155 caller (deStruct [22]), and found that the only recurrent rearrangement events originated from the 156 TTC28 gene at the chromosome 22q12 locus (Figure 1). Previously thought to be a fragile site 157 for translocation events, we now know that this is a L1-retrotransposon mediated 3’ transduction 158 event, as indicated by the clustering of breakpoints within 1 kb downstream of an active L1. This 159 retrotransposon-mediated transduction event was observed in 34% (10/29) of ENOC, and 31% 160 (11/35) of CCOC cases. None of the transductions targeted exons. There was no association 161 between TTC28-L1 mediated transduction and common mutations or landscape features (data 162 not shown). Because L1 elements are highly repetitive throughout the genome, there is difficulty 163 aligning the sequences. Consequently, there could be additional L1 retrotransposition events that 164 were undetected. 165 166 3.2. TTC28-L1 mediated transductions are early events in ENOC and CCOC oncogenesis 167 We validated these TTC28-L1 transductions on high quality gnomic DNA extracted from 168 patient frozen tumors and buffy coats via PCR, with primers designed to flank the sequences 169 around the insertion breakpoint, such that only samples with the transductions amplified. 170 Amplified PCR products are sequence on the Sanger to confirm the presence of breakpoints with 171 DNA sequences from both sides. While the TTC28-L1 transductions that failed at the PCR or 172 10 Sanger validation stages were excluded, each case had at least one validated transduction event 173 (for a list of validated TTC28-L1 transductions, see Supplemental Table S1). As expected, 174 validated TTC28-L1 transduction events were found only in the tumor and not the buffy coat 175 samples. 176 To infer the cellular timing of these TTC28-L1-mediated transductions, we took archival 177 FFPE tumor samplings from five different sites for each of the seven patients. We hypothesized 178 that the same transduction events detected at multiple tumor sites should indicate early 179 activation. For each case, we selected six somatic SNVs/fs mutations detected via WGS and with 180 highest frequencies and predicted impact, and performed targeted re-sequencing in the tumor 181 samplings to further characterize the timing of the TTC28-L1 mediated transductions relative to 182 WGS detected mutations (for a detailed list of SNVs/fs, see Supplemental Table S2). 183 We detected TTC28-L1 insertions in all FFPE tumor sites in the majority of the cases (5/7 184 cases, ENOC 1-4 and CCOC1) (Figure 2). In these cases, SNVs and fs mutations were detected 185 to varying allelic frequencies for each tumor block, reflecting both inter- and intra-tumour 186 heterogeneity (Figure 2 and Supplemental Table S2). For each case, SNVs/fs mutations that were 187 found in every tumor block sampled tend to be canonical cancer driver mutations, such as 188 mutations in ARID1A, PIK3CA, KRAS, PTEN, and CTNN1B (Figure 2 and Supplemental Table 189 S2). These results suggest that TTC28-L1 was likely activated early during tumorigenesis, and 190 was mediating DNA transductions along with SNVs/fs mutations in the earlier tumor clones. In 191 one of the five cases (ENOC 4) the TTC28-L1 transduction was also detected in the 192 histologically normal cervical tissue block used as a normal FFPE control, while the insertion 193 event was not found in the same patient’s buffy coat DNA. A second sampling from this tissue 194 11 block yielded the same result, which may indicate the presence of histologically normal cells 195 clonally related to the tumor, and suggest that L1 activation could occur even earlier. 196 In a single CCOC case (CCOC 2), we observed what appears to be two distinct clonal 197 populations within the same ovarian tumor, where three of the five tumor blocks surveyed had 198 highly similar SNVs/fs allelic frequencies in addition to presence of the TTC28-L1 transduction 199 event, while the other two tumor blocks contained neither SNVs/fs nor the TTC28-L1 200 transduction events. 201 Four cases in our cohort (ENOC 1, ENOC 2, ENOC4, and CCOC3) had synchronous 202 tumors in the ovary and the uterus. In the three endometrioid ovarian carcinoma cases (ENOC 1, 203 ENOC2, and ENOC 4), we observed the TTC28-L1 transduction event as well as several shared 204 SNV mutations in both the ovarian and uterine tumors. The observation of the same TTC28-L1 205 transductions in tumors at anatomically different locations further suggests that L1 activation 206 occurred early in these diseases. In the clear cell ovarian carcinoma case (CCOC 3), SNVs/fs 207 mutations and the TTC28-L1 event were found in all the samplings of the ovarian tumor but were 208 not seen in uterine tumor. 209 12 4. Discussion: 210 Herein we described the presence of DNA transductions mediated by a specific and 211 active retrotransposon, TTC28-L1, in the endometriosis associated ovarian carcinoma subtypes: 212 ENOC and CCOC. Our data showed that TTC28-L1 mediated transductions was detectable 213 across all tumor sites in the majority of the cases. The presence of TTC28-L1 transduction was 214 sometimes but not always accompanied by SNV/frameshift mutations at various allelic 215 frequencies. It is highly unlikely that the same retrotransposition event arose independently at 216 every tumor site, which indicates that TTC28-L1 transductions were not subclonal events, but 217 more likely events that occurred early in tumorigenesis. In addition, the presence of TTC28-L1 218 transductions correlated with high allelic frequencies of driver mutations implicated in the 219 development of these cancers, including mutations in ARID1A, CTNNB1, PIK3CA, and PTEN 220 [8], supporting our hypothesis that TTC28-L1 retrotransposons are activated early in EAOC 221 development. 222 Targeted resequencing was previously used by our group to describe a clonal relationship 223 between the endometrial and ovarian cancers in three of the four synchronous endometrioid and 224 ovarian tumors, ENOC 2, ENOC 4 and CCOC 3 [23]. We also found somatic mutations and 225 TTC28-L1 events shared between the uterine and ovarian tumors in ENOC 2 and ENOC 4, 226 however, we did not detect any of our selected mutations nor the TTC28-L1 event in the uterine 227 tumor from CCOC 3. CCOC 3 was unusual in several ways: different pathologies observed in 228 the ovary (clear cell) and uterus (endometrioid), and there was only one single shared somatic 229 event (a different event from the ones we repeated) as reported by Anglesio et al [23]. It is 230 possible this somatic event had occurred before either the seeding of endometriosis or the 231 activation of the TTC28-L1 retrotransposon. 232 13 It is interesting to note that 3’ transductions accounts for approximately one quarter of all 233 somatic L1 retrotranspositions, and the majority of L1 retrotranspositions result in solo L1 234 insertions, either as a full L1 or truncated at the 5’ end [10]. This meant that there are likely more 235 L1 insertions undetected in our cohort. Past studies on somatic L1 retrotranspositions in various 236 cancer types reported much more solo L1 insertions than 3’ transduction events; however, the 237 lack of L1 3’ transductions detected could be due to bias in L1 sequencing and analysis methods 238 [16, 18-20]. Nonetheless, we have found TTC28-L1 transduction events at a high frequency in 239 our cohort, which could be a unique feature of EAOCs. 240 While somatic L1 insertions are common in certain tumor types, there is little evidence 241 that they are oncogenic. Only two studies showed that somatic L1 retrotranspositions (solo L1 242 insertions) directly initiated oncogenesis in colorectal cancer via a somatic L1 insertion in an 243 exon of the tumor suppressor gene APC [24, 25]. The majority of somatic L1 insertions target 244 heterochromatic regions with no obvious functional impact [10, 12]. We also observe that our L1 245 transductions targeted non-coding regions within out cohort. 246 Most research in the field focuses on using L1 activations as a marker of tumor 247 development, by assessing the global hypomethylation of L1 loci and the expression of L1 248 mRNA and proteins throughout stages of cancer development and metastasis. The general 249 conclusion is that somatic L1 retrotranspositions are passenger events that occur after a 250 permissive environment has been established during cancer development, such as dysregulated 251 epigenetic control [10, 12, 17]. Evidence of L1 activation as an early event include a recent study 252 that found stepwise decrease of methylation across different L1 loci have been observed between 253 normal endometrium, contiguous endometriosis (endometriosis adjacent to tumor), and 254 ENOCs/CCOCs [17]. In gastrointestinal (GI) cancers, shared somatic L1 retrotranspositions 255 14 (solo insertions) have been found between precancerous lesions and colon tumors [20], as well as 256 between Barrett’s esophagus (precursor lesion of esophageal cancer) and concomitant 257 esophageal tumors [19], suggesting early activation of L1 during GI tumor development. This is 258 likely the scenario in our cohorts, where the TTC28-L1 was activated early during ENOC/CCOC 259 tumorigenesis, but after a retrotransposition-permissive environment was established. 260 The exact relationship between TTC28-L1 activation and malignant transformation in 261 these two ovarian cancer subtypes is challenging to elucidate. Further studies will be needed to 262 assay endometriosis tissues that are adjacent to tumors, distant from tumors, as well as 263 endometriosis without cancer involvement. Such studies could determine whether TTC28-L1 264 transduction is a useful marker for cancer risk of endometriosis. 265 EAOCs suffer from a lack of effective early detection tools. TTC28-L1 and other L1s can 266 potentially be used as biomarkers to identify early malignant transformations, especially in the 267 subset of CCOC/ENOC cases where common coding mutations are not present. In addition, a 268 better understanding of L1 activation and transduction patterns over the course of ENOC and 269 CCOC progression may help elucidate the underlying mechanisms of ovarian carcinogenesis and 270 progression. 271 272 15 Conflict of Interest Statement 273 Dr. David Huntsman reports that he is a founder and CMO of Contextual Genomics, a cancer 274 testing company; however, Contextual does not test for the features described in this manuscript. 275 Otherwise the authors have no conflict of interest. 276 16 Acknowledgement 277 The authors would like to thank the members of the Huntsman group and the OVCARE team for 278 all their technical supports. This research would not have been made possible without the 279 gracious donation of tissues from the women with ovarian cancer. 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Blue lines indicate ENOC 353 cases and red lines indicate CCOC cases. 354 22 Figure 2: a). TTC28-L1 retrotransposition presence and allelic frequencies for five selected 355 SNV/fs mutations at six anatomical sites for each of the seven ENOC and CCOC cases. 356 Three shades of grey portray differences in allelic frequencies: dark grey indicates high 357 frequency (>50%) at the point mutation; lighter grey indicates a frequency between 20% and 358 50%; lightest grey indicates low frequency (5%-20%). Red box indicates the presence of 359 TTC28-L1 retrotransposition, and white box indicates no variance or retrotransposition detected. 360 Numbers indicate the allelic frequencies in percentage. A.F., allelic frequencies; Ov, ovary; Cer, 361 cervix; Ut, uterus; R, right; L, left; N, normal; ND, not detected. b). Circos plot displays 362 retrotransposition events originating from TTC28 (Chr.22q12) for the seven selected cases. 363 All retrotransposition targets were in non-coding spaces. 364 23 Table S1. A list of the breakpoint positions (hg19 coordinates) of each validated transduction. 365 24 Table S2. A list of selected SNV/fs mutations for each case, accompanied by the allele count at 366 each FFPE block. 367 Sample Origin breakpoint Target breakpoint ENOC 1 chr22:29066036 chr5:145279611 ENOC 1 chr22:29065556 chr6:97785114 ENOC 2 chr22:29065619 chr11:32392341 ENOC 3 chr22:29065997 chr18:66643446 ENOC 4 chr22:29065742 chr18:27803952 CCOC 1 chr22:29065852 chr4:163516454 CCOC 2 chr22:29065604 chr18:37426280 CCOC 3 chr22:29065433 chr18:38206983 1 TITLE: LINE-1 retrotransposon-mediated DNA transductions in endometriosis associated 1 ovarian cancers 2 AUTHORS: 3 Zhouchunyang Xia1, Dawn R Cochrane2, Michael S Anglesio1,3, Yi Kan Wang2, Tayyebeh 4 Nazeran1, Basile Tessier-Cloutier1, Melissa K McConechy4, Janine Senz2, Amy Lum2, Ali 5 Bashashati2, Sohrab P Shah1,2, David G Huntsman*1,2. 6 AFFILIATIONS: 7 1. Department of Pathology and Laboratory Medicine, University of British Columbia, 8 Vancouver, BC, Canada 9 2. Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada 10 3. Department of Gynecology and Obstetrics, University of British Columbia, Vancouver, BC, 11 Canada 12 4. Department of Human Genetics, McGill University, Research Institute of the McGill 13 University Health Centre, Montreal, Quebec, Canada 14 15 *Corresponding author: 16 David Huntsman 17 BC Cancer Research Centre 18 675 West 10th Avenue 19 Vancouver, BC V5Z 1L3 20 1-604-675-8205 (phone) 21 1-604-675-8218 (fax) 22 dhuntsma@bccancer.bc.ca 23 2 Abstract: 24 Objective: Endometrioid (ENOC) and clear cell ovarian carcinoma (CCOC) share a common 25 precursor lesion, endometriosis, hence the designation endometriosis associated ovarian cancers 26 (EAOC). Long interspersed nuclear element 1 (LINE-1 or L1), is a family of mobile genetic 27 elements activated in many cancers capable of moving neighboring DNA through 3’ 28 transductions. Here we investigated the involvement of specific L1-mediated transductions in 29 EAOCs. 30 Methods: Through whole genome sequencing, we identified active L1-mediated transductions 31 originating within the TTC28 gene in 34% (10/29) of ENOC and 31% (11/35) of CCOC cases. 32 We used PCR and capillary sequencing to assess the presence of specific TTC28-L1 33 transductions in formalin-fixed paraffin-embedded (FFPE) blocks from six different anatomical 34 sites (five tumors and one normal control) for four ENOC and three CCOC cases, and compared 35 the results to the presence of single nucleotide variations (SNVs)/frame shift (fs) mutations 36 detected using multiplex PCR and next generation sequencing. 37 Results: TTC28-L1 mediated transductions were identified in at least three tumor samplings in 38 all cases, and were present in all five tumor samplings in 5/7 (71%) cases. In these cases, KRAS, 39 PIK3CA, CTNNB1, ARID1A, and PTEN mutations were found across all tumor sites whilst other 40 selected SNV/fs mutations of unknown significance were present at varying allelic frequencies. 41 Conclusion: The TTC28-L1 transductions along with classical driver mutations were near 42 ubiquitous across the tumors, suggesting that L1 activation likely occurred early in the 43 development of EAOCs. TTC28-L1 transductions could potentially be used to determine clonal 44 relationships and to track ovarian cancer progression. 45 3 1. Introduction: 46 Ovarian cancer is the most lethal gynecological cancer, and it is the 5th leading cause of 47 cancer related deaths in women in the United States [1]. Endometrioid ovarian carcinoma 48 (ENOC) and clear cell ovarian carcinoma (CCOC) each account for approximately 10% of all 49 ovarian cancer cases [2]. Both cancers are low grade subtypes that are often diagnosed at stages I 50 and II. When diagnosed at stages III/IV, ENOC has a more favorable prognosis compared to 51 CCOC, as CCOC tends to have a much lower response rate to chemotherapy [2, 3]. A recent 52 study of the genomic profile of ovarian cancers stratified ENOC into three subgroups: 53 ultramutator (high mutation load), micro-satellite instable (MSI), and micro-satellite stable 54 (MSS), and CCOC cases into two subgroups: APOBEC (localized hypermutation) and mutation 55 signatures similar to one previously associated with age, though not age-related in CCOC [4]. 56 Despite having different pathology and genomic profiles, both ENOC and CCOC are 57 associated with endometriosis, hence the designation endometriosis associated ovarian cancer 58 (EAOC). Endometriosis is characterized by ectopic growth of endometrial epithelium and 59 stroma. Women with endometriosis have a 2- to 3-fold increased risk of developing ENOC and 60 CCOC [5]. Common genes with mutations implicated to drive tumorigenesis include ARID1A 61 (30%), PTEN (20%), and CTNN1B (38-50%) in ENOC, and ARID1A (46-57%) and PIK3CA 62 (33-46%) in CCOC. Cancer driving mutations in ARID1A, PTEN, and PIK3CA have also been 63 found in endometriotic lesions both adjacent and distant to the tumors, indicating a clonal 64 relationship between these diseases [6-9]. In addition, canonical cancer mutations have been 65 found in deep infiltrating endometriosis that is not associated with cancer [7]. Given that driver 66 mutations in EAOCs are only found in a subset of patients, and certain changes can occur in 67 4 endometriosis without cancer, additional types of biomarkers are needed to distinguish women 68 with endometriosis at risk of developing cancer. 69 In our whole genome sequencing of 29 ENOC and 35 CCOC cases, we observed frequent 70 rearrangement events originating from an active LINE-1 (L1) retrotransposon located in the 71 TTC28 gene on chromosome 22q12. Long interspersed nuclear elements 1 (LINE-1 or L1), are 72 repetitive mobile DNA elements that accounts for 17% of the human genome [10-13]. L1s 73 encode an endonuclease and a reverse transcriptase that allows them to \"copy-and-paste\" their 74 own sequences into random genomic locations via RNA intermediates [10, 12]. They are also 75 capable of taking unique downstream DNA fragments with them during this genetic propagation 76 in a process called 3’ transduction [10]. While most L1s are not functional due to structural 77 rearrangements, there are approximately 80-100 full-length L1s that are potentially active [10]. 78 L1 activation is important in the germ cells during embryogenesis [14], but epigenetic silencing 79 typically occurs in adult [12]. In the cancer genomes, however, L1s become activated as part of a 80 general degradation of genomic and epigenetic integrity [12]. 81 The effects of L1 retrotransposon activation in cancer development and progression has 82 been studied in various cancers, including head and neck, prostate, ovarian, colorectal, gastric, 83 esophageal, pancreatic, and breast cancer [10, 11, 13, 15-21]. While most studies surveyed the 84 landscape of somatic L1 retrotranspositions by detecting the presence of novel inserted L1 85 sequences in tumors, Tubio and colleagues studied the effect of 3’ transductions events mediated 86 by a set of active L1s in cancers [10]. This specific TTC28-L1-mediated transduction from 87 chromosome 22q12 is one of the most frequently active L1s found in colon, head and neck, and 88 lung cancer samples and is the only active L1 element in 93% of breast cancer samples [10]. 89 Specific L1-mediated transductions have been less extensively studied in EAOCs [17, 21]. 90 5 Here we used PCR and capillary sequencing to validate the presence of TTC28-L1-91 mediated transductions in different tumor samplings of ENOC and CCOC, and we compared the 92 results to targeted re-sequencing of selected single nucleotide variations (SNVs)/frame shift (fs) 93 mutations in the same tissues to infer biological timing of L1 transductions. Our data showed that 94 these transduction events appeared with canonical driver mutations for majority of the cases, and 95 suggests that they likely occurred early in tumorigenesis. We hope to use TTC28-L1 mediated 96 DNA transductions to gain insights into the tumor development of EAOCs. 97 6 2. Methods: 98 2.1. Whole genome sequencing (WGS) 99 The cohort and methods have been described previously by Wang et al.[4]. Briefly, tumor 100 (frozen tissue) and matched normal (buffy coat) DNA libraries were constructed for 29 ENOC 101 and 35 CCOC cases, and sequenced using Illumina HiSeq 2500 V4 chemistry. Analysis were 102 performed using bioinformatic methods described in Wang et al. supplemental materials [4]. 103 104 2.2. Case selection 105 Four ENOC and three CCOC cases with the highest numbers of TTC28-L1-mediated 106 transductions detected by WGS were selected from our previous study [4]. All materials were 107 provided by the OVCARE gynecological tissue bank. H&E slides for all available formalin-fixed 108 paraffin-embedded (FFPE) blocks were reviewed by expert pathologists (T.N. and B.T-C.). Five 109 FFPE tumor tissue blocks with the highest cellularity (>90%) and one representative FFPE 110 normal tissue block were selected for each of the seven cases. Overall project processes were 111 approved by the BC Cancer Agency or the University of British Columbia Research Ethics 112 Board (REB #H08-01411 and #H09-02153). 113 114 2.3. DNA extraction from FFPE blocks 115 FFPE tissue blocks were cut 10um thick and applied on charged glass slides, where relevant 116 areas, identified by a pathologist (T.N.), were macrodissected. DNA was extracted from FFPE 117 tissues using the QIAamp DNA FFPE Tissue Kit (Qiagen) as per manufacturers’ protocols. All 118 DNA was quantified using the broad range DNA assay Qubit fluorometer (Life Technologies). 119 120 7 2.4. PCR validation of TTC28-L1-mediated transduction events 121 To validate all L1 transduction events, primers were designed using Primer3 to generate 170-122 250bp amplicons, which spanned the transduction junction. Primers were validated on WGA 123 (whole genome amplified) DNA extracted from frozen tumor tissues (positive control) and buffy 124 coats (negative control) for each of the cases. Primers were tested at an annealing temperature of 125 60C, using PCR SuperMix High Fidelity mastermix (ThermoFisher). Successful PCR was 126 indicated by a single band at 200-300bp using gel electrophoresis. For failed PCRs (no band, a 127 smear, or multiple bands), a gradient PCR from 55C to 64C was performed. If the PCRs were 128 still unsuccessful, Platinum® Taq DNA Polymerase High Fidelity (ThermoFisher) were used in 129 substitution. Successful PCRs were validated via sequencing using ABI 3130XL automated 130 capillary sequencing as per manufacture protocol. Validated primers were then used on the 131 FFPE-extracted DNA samples with 50ng input. 132 133 2.5. Microfluidic PCR validation of selected SNV/Frameshift mutations 134 From the whole genome sequencing results, six likely pathogenic missense and/or frameshift 135 mutations were selected for each case for validations on the FFPE tissue blocks. Primer sets were 136 designed using Primer 3 to amplify the specific gene regions. Forward primers were tagged with 137 CS1 (5’-ACACTGACGACATGGTTCTACA-3’) and reverse primers with CS2 (5’-138 TACGGTAGCAGAGACTTGGTCT-3’) sequencing tags. PCR products (150-200bp) were 139 amplified using the Fluidigm 48X48 Access Arrays, as per manufacturers’ protocol, with input 140 of 50ng FFPE derived DNA. DNA barcodes (10bp) with Illumina cluster-generating adapters 141 were added to the libraries post-Fluidigm harvest, and cleaned-up using Agencourt AMpure XP 142 beads (Beckman Coulter). Barcoded PCR products were then quantified using the high 143 8 sensitivity DNA assay Qubit fluorometer (Life Technologies) and pooled to one total library by 144 normalizing to equal amounts of PCR product. In total, 48 samples were pooled and denatured 145 according to Illumina standard protocols, and sequenced using a MiSeq 300 cycle V2 kit on the 146 Illumina MiSeq for ultra-deep validations. Uni-directional barcode sequencing was performed. 147 Analysis was performed using the bam and VCF files generated through Illumina MiSeq 148 reporter. 149 9 3. Results: 150 3.1. TTC28-L1 mediated transductions are frequent events in clear cell and endometrioid 151 ovarian cancer. 152 Whole genome shotgun sequencing of 29 ENOC and 35 CCOC cases to a median 153 coverage of 51x and 37x for the tumour and matched normal DNA respectively was performed 154 in a previous study [4]. Here, we studied this same data set generated via a structural variant 155 caller (deStruct [22]), and found that the only recurrent rearrangement events originated from the 156 TTC28 gene at the chromosome 22q12 locus (Figure 1). Previously thought to be a fragile site 157 for translocation events, we now know that this is a L1-retrotransposon mediated 3’ transduction 158 event, as indicated by the clustering of breakpoints within 1 kb downstream of an active L1. This 159 retrotransposon-mediated transduction event was observed in 34% (10/29) of ENOC, and 31% 160 (11/35) of CCOC cases. None of the transductions targeted exons. There was no association 161 between TTC28-L1 mediated transduction and common mutations or landscape features (data 162 not shown). Because L1 elements are highly repetitive throughout the genome, there is difficulty 163 aligning the sequences. Consequently, there could be additional L1 retrotransposition events that 164 were undetected. 165 166 3.2. TTC28-L1 mediated transductions are early events in ENOC and CCOC oncogenesis 167 We validated these TTC28-L1 transductions on high quality gnomic DNA extracted from 168 patient frozen tumors and buffy coats via PCR, with primers designed to flank the sequences 169 around the insertion breakpoint, such that only samples with the transductions amplified. 170 Amplified PCR products are sequence on the Sanger to confirm the presence of breakpoints with 171 DNA sequences from both sides. While the TTC28-L1 transductions that failed at the PCR or 172 10 Sanger validation stages were excluded, each case had at least one validated transduction event 173 (for a list of validated TTC28-L1 transductions, see Supplemental Table S1). As expected, 174 validated TTC28-L1 transduction events were found only in the tumor and not the buffy coat 175 samples. 176 To infer the cellular timing of these TTC28-L1-mediated transductions, we took archival 177 FFPE tumor samplings from five different sites for each of the seven patients. We hypothesized 178 that the same transduction events detected at multiple tumor sites should indicate early 179 activation. For each case, we selected six somatic SNVs/fs mutations detected via WGS and with 180 highest frequencies and predicted impact, and performed targeted re-sequencing in the tumor 181 samplings to further characterize the timing of the TTC28-L1 mediated transductions relative to 182 WGS detected mutations (for a detailed list of SNVs/fs, see Supplemental Table S2). 183 We detected TTC28-L1 insertions in all FFPE tumor sites in the majority of the cases (5/7 184 cases, ENOC 1-4 and CCOC1) (Figure 2). In these cases, SNVs and fs mutations were detected 185 to varying allelic frequencies for each tumor block, reflecting both inter- and intra-tumour 186 heterogeneity (Figure 2 and Supplemental Table S2). For each case, SNVs/fs mutations that were 187 found in every tumor block sampled tend to be canonical cancer driver mutations, such as 188 mutations in ARID1A, PIK3CA, KRAS, PTEN, and CTNN1B (Figure 2 and Supplemental Table 189 S2). These results suggest that TTC28-L1 was likely activated early during tumorigenesis, and 190 was mediating DNA transductions along with SNVs/fs mutations in the earlier tumor clones. In 191 one of the five cases (ENOC 4) the TTC28-L1 transduction was also detected in the 192 histologically normal cervical tissue block used as a normal FFPE control, while the insertion 193 event was not found in the same patient’s buffy coat DNA. A second sampling from this tissue 194 11 block yielded the same result, which may indicate the presence of histologically normal cells 195 clonally related to the tumor, and suggest that L1 activation could occur even earlier. 196 In a single CCOC case (CCOC 2), we observed what appears to be two distinct clonal 197 populations within the same ovarian tumor, where three of the five tumor blocks surveyed had 198 highly similar SNVs/fs allelic frequencies in addition to presence of the TTC28-L1 transduction 199 event, while the other two tumor blocks contained neither SNVs/fs nor the TTC28-L1 200 transduction events. 201 Four cases in our cohort (ENOC 1, ENOC 2, ENOC4, and CCOC3) had synchronous 202 tumors in the ovary and the uterus. In the three endometrioid ovarian carcinoma cases (ENOC 1, 203 ENOC2, and ENOC 4), we observed the TTC28-L1 transduction event as well as several shared 204 SNV mutations in both the ovarian and uterine tumors. The observation of the same TTC28-L1 205 transductions in tumors at anatomically different locations further suggests that L1 activation 206 occurred early in these diseases. In the clear cell ovarian carcinoma case (CCOC 3), SNVs/fs 207 mutations and the TTC28-L1 event were found in all the samplings of the ovarian tumor but were 208 not seen in uterine tumor. 209 12 4. Discussion: 210 Herein we described the presence of DNA transductions mediated by a specific and 211 active retrotransposon, TTC28-L1, in the endometriosis associated ovarian carcinoma subtypes: 212 ENOC and CCOC. Our data showed that TTC28-L1 mediated transductions was detectable 213 across all tumor sites in the majority of the cases. The presence of TTC28-L1 transduction was 214 sometimes but not always accompanied by SNV/frameshift mutations at various allelic 215 frequencies. It is highly unlikely that the same retrotransposition event arose independently at 216 every tumor site, which indicates that TTC28-L1 transductions were not subclonal events, but 217 more likely events that occurred early in tumorigenesis. In addition, the presence of TTC28-L1 218 transductions correlated with high allelic frequencies of driver mutations implicated in the 219 development of these cancers, including mutations in ARID1A, CTNNB1, PIK3CA, and PTEN 220 [8], supporting our hypothesis that TTC28-L1 retrotransposons are activated early in EAOC 221 development. 222 Targeted resequencing was previously used by our group to describe a clonal relationship 223 between the endometrial and ovarian cancers in three of the four synchronous endometrioid and 224 ovarian tumors, ENOC 2, ENOC 4 and CCOC 3 [23]. We also found somatic mutations and 225 TTC28-L1 events shared between the uterine and ovarian tumors in ENOC 2 and ENOC 4, 226 however, we did not detect any of our selected mutations nor the TTC28-L1 event in the uterine 227 tumor from CCOC 3. CCOC 3 was unusual in several ways: different pathologies observed in 228 the ovary (clear cell) and uterus (endometrioid), and there was only one single shared somatic 229 event (a different event from the ones we repeated) as reported by Anglesio et al [23]. It is 230 possible this somatic event had occurred before either the seeding of endometriosis or the 231 activation of the TTC28-L1 retrotransposon. 232 13 It is interesting to note that 3’ transductions accounts for approximately one quarter of all 233 somatic L1 retrotranspositions, and the majority of L1 retrotranspositions result in solo L1 234 insertions, either as a full L1 or truncated at the 5’ end [10]. This meant that there are likely more 235 L1 insertions undetected in our cohort. Past studies on somatic L1 retrotranspositions in various 236 cancer types reported much more solo L1 insertions than 3’ transduction events; however, the 237 lack of L1 3’ transductions detected could be due to bias in L1 sequencing and analysis methods 238 [16, 18-20]. Nonetheless, we have found TTC28-L1 transduction events at a high frequency in 239 our cohort, which could be a unique feature of EAOCs. 240 While somatic L1 insertions are common in certain tumor types, there is little evidence 241 that they are oncogenic. Only two studies showed that somatic L1 retrotranspositions (solo L1 242 insertions) directly initiated oncogenesis in colorectal cancer via a somatic L1 insertion in an 243 exon of the tumor suppressor gene APC [24, 25]. The majority of somatic L1 insertions target 244 heterochromatic regions with no obvious functional impact [10, 12]. We also observe that our L1 245 transductions targeted non-coding regions within out cohort. 246 Most research in the field focuses on using L1 activations as a marker of tumor 247 development, by assessing the global hypomethylation of L1 loci and the expression of L1 248 mRNA and proteins throughout stages of cancer development and metastasis. The general 249 conclusion is that somatic L1 retrotranspositions are passenger events that occur after a 250 permissive environment has been established during cancer development, such as dysregulated 251 epigenetic control [10, 12, 17]. Evidence of L1 activation as an early event include a recent study 252 that found stepwise decrease of methylation across different L1 loci have been observed between 253 normal endometrium, contiguous endometriosis (endometriosis adjacent to tumor), and 254 ENOCs/CCOCs [17]. In gastrointestinal (GI) cancers, shared somatic L1 retrotranspositions 255 14 (solo insertions) have been found between precancerous lesions and colon tumors [20], as well as 256 between Barrett’s esophagus (precursor lesion of esophageal cancer) and concomitant 257 esophageal tumors [19], suggesting early activation of L1 during GI tumor development. This is 258 likely the scenario in our cohorts, where the TTC28-L1 was activated early during ENOC/CCOC 259 tumorigenesis, but after a retrotransposition-permissive environment was established. 260 The exact relationship between TTC28-L1 activation and malignant transformation in 261 these two ovarian cancer subtypes is challenging to elucidate. Further studies will be needed to 262 assay endometriosis tissues that are adjacent to tumors, distant from tumors, as well as 263 endometriosis without cancer involvement. Such studies could determine whether TTC28-L1 264 transduction is a useful marker for cancer risk of endometriosis. 265 EAOCs suffer from a lack of effective early detection tools. TTC28-L1 and other L1s can 266 potentially be used as biomarkers to identify early malignant transformations, especially in the 267 subset of CCOC/ENOC cases where common coding mutations are not present. In addition, a 268 better understanding of L1 activation and transduction patterns over the course of ENOC and 269 CCOC progression may help elucidate the underlying mechanisms of ovarian carcinogenesis and 270 progression. 271 272 15 Conflict of Interest Statement 273 Dr. David Huntsman reports that he is a founder and CMO of Contextual Genomics, a cancer 274 testing company; however, Contextual does not test for the features described in this manuscript. 275 Otherwise the authors have no conflict of interest. 276 16 Acknowledgement 277 The authors would like to thank the members of the Huntsman group and the OVCARE team for 278 all their technical supports. This research would not have been made possible without the 279 gracious donation of tissues from the women with ovarian cancer. 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Blue lines indicate ENOC 353 cases and red lines indicate CCOC cases. 354 22 Figure 2: a). TTC28-L1 retrotransposition presence and allelic frequencies for five selected 355 SNV/fs mutations at six anatomical sites for each of the seven ENOC and CCOC cases. 356 Three shades of grey portray differences in allelic frequencies: dark grey indicates high 357 frequency (>50%) at the point mutation; lighter grey indicates a frequency between 20% and 358 50%; lightest grey indicates low frequency (5%-20%). Red box indicates the presence of 359 TTC28-L1 retrotransposition, and white box indicates no variance or retrotransposition detected. 360 Numbers indicate the allelic frequencies in percentage. A.F., allelic frequencies; Ov, ovary; Cer, 361 cervix; Ut, uterus; R, right; L, left; N, normal; ND, not detected. b). Circos plot displays 362 retrotransposition events originating from TTC28 (Chr.22q12) for the seven selected cases. 363 All retrotransposition targets were in non-coding spaces. 364 23 Table S1. A list of the breakpoint positions (hg19 coordinates) of each validated transduction. 365 24 Table S2. A list of selected SNV/fs mutations for each case, accompanied by the allele count at 366 each FFPE block. 367 Sample Origin breakpoint Target breakpoint ENOC 1 chr22:29066036 chr5:145279611 ENOC 1 chr22:29065556 chr6:97785114 ENOC 2 chr22:29065619 chr11:32392341 ENOC 3 chr22:29065997 chr18:66643446 ENOC 4 chr22:29065742 chr18:27803952 CCOC 1 chr22:29065852 chr4:163516454 CCOC 2 chr22:29065604 chr18:37426280 CCOC 3 chr22:29065433 chr18:38206983 Sample ID Gene Mutation Δ Position (hg19 coordinates) WGS A.F. Absolute read counts/allelic frequencies (A.F.) at different FFPE Sites Corresponding Location ENOC 1 B4 B6 D13 D9 D10 D2 (normal) B4 & B6 - left ovary ARID1A Q563* C>T 1:27057979 24% (2905/13155)22% (5469/12022)45% (1549/7082)22% (2524/5724)44% (4262/9672)44% (5/6164)0 D13 - left conu CSMD1 G38C G>T 8:4495054 50% (1228/10250)12% (4/9816)0 (8/8623)0 (2/7363)0 (9/7590)0 (2/5570)0 D9 & D10 - right conu CTNNA2 V91M G>A 2:80874884 43% (9857/10912)91% (3898/11899)33% (56/9919)1% (2651/5648)47% (5028/9785)52% (13/12768)0 D2 - posterior endocervix KRAS G12V G>T 12:25398284 36% (18363/40480)45% (12166/42854)28% (15075/41425)36% (23308/33202)70% (15132/36324)42% (544/33116)2% PIK3CA R88Q G>A 3:178916876 45% (57/13159)0 (7623/13760)55% (1841/11711)16% (6333/11353)56% (1470/11476)13% (253/14308)2% PIK3CA P449L C>T 3:178928068 39% (3438/8193)42% (1998/5683)35% (2741/7442)37% (0/107)0 (2077/4445)47% (2/4858)0 retrotransposition event validated 2 2 2 2 2 0 ENOC 2 synchronous case A1 A12 D9 D17 D2 D1 (normal) A1 & A12 - right ovary CTNNB1 S37F C>T 3:41266113 88% (10344/12018)86% (10267/13151)79% (6457/13571)48% (4198/10023)42% (4670/9841)48% (9/15117)0 D2 - cervix FAM208B G42S G>A 10:5762911 24% (3809/12485)30% (2173/8717)25% (305/11837)3% (22/11896)0 (20/9792)0 (25/12487)0 D9 & D17 - uterine NEB V4792M G>A 2:152410491 27% (27/13934)0 (30/15638)0 (18/13856)0 (11/13167)0 (20/9792)0 (18/14432)0 D1 - cervix PIGK S155N G>A 1:77632427 43% (3448/16699)21% (6528/14544)45% (1774/13418)13% (8591/13356)64% (1511/10653)14% (26/17126)0 PIK3CA E545K G>A 3:178936091 89% (14755/16811)88% (16008/16250)99% (11222/14536)77% (12104/14315)85% (11526/11799)98% (10/13491)0 TP53 E180* G>T 17:7578392 85% (6271/7925)79% (10198/11471)90% (7893/12607)63% (8687/11259)77% (7788/9295)84% (7/10620)0 retrotransposition event validated 1 1 1 1 1 0 ENOC 3 B18 B19 B20 B21 B22 B3 (normal) B18 & B19 - right ovarian DHX29 T673I C>T 5:54577291 66% (2058/31301)7% (27/24252)0 (7452/26508)28% (22/22006)0 (52/30106)0 (20/38479)0 B20 & B21 - right ovarian KMT2B C1435Y G>A 19:36218600 64% (7/8109)0 (5/8206)0 (3605/9461)38% (7/12114)0 (5/9342)0 (4/12048)0 B22 - ovarian nodule KMT2B T172fs 19:36210763-65 0 0 0 0 40% 0 B3 - anterior cervix NRAS Q61K C>A 1:115256530 93% (13863/16075)86% (5928/12947)46% (6828/8833)77% (9058/13939)65% (3821/13639)28% (9/18974)0 PIK3CA N345K T>A 3:178921553 54% (25452/55847)46% (16749/33593)50% (16267/47411)34% (4978/57014)9% (16046/42836)37% (21/39092)0 TTN S2146P T>C 2:179640155 36% (106/28470)0 (1680/18834)9% (9203/19948)46% (71/25915)0 (47/29284)0 (48/32216)0 retrotransposition event validated 1 1 1 1 1 0 ENOC 4 synchronous case A4 A5 A16 B22 B23 B6 (normal) A16 - left ovary CYTH2 A168V C>T 19:48977230 42% (8096/21371)38% (9226/15795)58% (8215/20459)40% (11/20489)0 (20/20011)0 (19/14995)0 A4 & A5 - left ovary DNAH9 R4205L G>T 17:11840793 25% (4950/14244)35% (5099/11610)42% (5424/14478)35% (5/14215)0 (5/13734)0 (1/10888)0 B22 & B23 - posterior endo. DOCK4 R972C C>T 7:111462434 27% (5670/15583)36% (1230/11298)11% (7275/14390)51% (5008/12557)40% (3911/10118)39% (18/11875)0 B6 - posterior cervix KRAS G12D G>A 12:25398284 67% (29394/63687)46% (13101/39951)33% (23646/52200)46% (22390/49973)46% (19738/46662)42% (46/41232)0 PIK3CA G118D G>A 3:178917478 81% (28792/30622)94% (17588/20579)85% (15779/26143)60% (14128/25231)56% (11763/16139)73% (14/24376)0 PTEN L265fs 10:89717768-70 98% 98% 99% 97% 98% 0 retrotransposition event validated 1 1 1 1 1 1 CCOC 1 B6 B7 B8 B12 B13 K2 (normal) B6-B13 - right ovary ARID1A Q548fs 1:27057934-36 0 0 0 0 34% 0 K2 - posterior cervix FAM98A E60Q G>C 2:33820580 33% (5498/24455)22% (2318/31724)7% (5038/36362)14% (7714/42400)18% (11105/32383)34% (7/41218)0 FAT3 S2855L C>T 11:92534629 56% (10/15186)0 (8/21868)0 (12/22579)0 (11/24887)0 (11/19376)0 (11/26646)0 GNA11 S242L C>T 19:3119041 30% (2689/5256)51% (1150/6722)17% (361/3782)10% (2173/7767)28% (1549/6887)23% (2/46)0 KRAS G12V G>T 12:25398284 45% (2733/32212)8% (2667/38939)7% (4336/49961)9% (11721/53627)22% (15245/39076)41% (32/56865)0 PCDHB6 E630K G>A 5:140531726 34% (2295/5180)44% (1734/8095)21% (2189/5485)40% (3213/12690)25% (2839/9173)31% 0 retrotransposition event validated 1 1 1 1 1 CCOC 2 A1 A4 A6 A11 A14 B2 (normal) A1-A14 - left ovary ELEFN1 P501S C>T 7:1785733 35% 0 0 0 0 0 0 B2 - posterior cervix HTR1E R220W C>T 6:87725710 38% (12493/33485)37% (7994/30042)27% (12858/29355)44% (475/34788)1% (49/48183)0 (39/23275)0 LONP1 R534C C>T 19:5699123 57% (5285/9263)57% (8294/12935)64% (7608/12891)59% (18/19351)0 (492/27196)2% (16/11148)0 PDAP1 D82N G>A 7:98998017 27% (8912/23305)38% (6100/22180)28% (8953/22371)40% (37/27474)0 (369/35843)1% (27/19598)0 PTGS2 R424S C>G 1:186644514 38% (13323/32092)42% (9449/33357)28% (11055/30301)36% (4/37242)0 (12/51601)0 (3/29148)0 SCN5A E555K G>A 3:38645430 48% (6917/17222)40% (5001/15456)32% (7009/16730)42% (10/20177)0 (802/27014)3% (8/13159)0 retrotransposition event validated 1 1 1 0 0 0 CCOC 3 synchronous case B3 B5 B6 B8 C14 C4 (normal) B3-B8 - right ovary ABCD3 G617V G>T 1:94980706 39% (14353/35382)41% (19581/41177)48% (12235/28525)43% (14983/31805)47% (31/20868)0 (28/27810)0 C14 - posterior endometrium ARID1A E2047* G>T 1:27106528 75% (11704/21408)55% (13002/24671)54% (9951/17632)56% (13189/18894)70% (5/12369)0 (7/15859)0 C4 - anterior cervix CPT1B R162W C>T 22:51015042 21% (4218/10878)39% (5193/12641)41% (2361/8053)29% (3829/9136)42% (14/6978)0 (6/8613)0 ITGB8 C634Y G>A 7:20444464 53% (4078/22334)18% (6299/27745)23% (5653/19408)29% (6269/20245)31% (224/15917)1% (24/17082)0 MTOR A2386V C>T 1:11174877 27% (9039/28130)32% (11319/32397)35% (8395/22873)37% (10277/27581)46% (15/18263)0 (22/21418)0 PTEN L108F C>T 10:89692838 30% (8002/23401)34% (9811/27560)35% (7143/19019)38% (7205/21088)34% (12/14125)0 (11/18464)0 retrotransposition event validated 1 1 1 1 0 0 "@en ; edm:hasType "Article"@en, "Postprint"@en ; edm:isShownAt "10.14288/1.0357358"@en ; dcterms:language "eng"@en ; ns0:peerReviewStatus "Reviewed"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "Elsevier"@en ; ns0:publisherDOI "10.1016/j.ygyno.2017.09.032"@en ; dcterms:rights "Attribution-NonCommercial-NoDerivatives 4.0 International"@* ; ns0:rightsURI "http://creativecommons.org/licenses/by-nc-nd/4.0/"@* ; ns0:scholarLevel "Faculty"@en, "Researcher"@en ; dcterms:title "LINE-1 retrotransposon-mediated DNA transductions in endometriosis associated 1 ovarian cancers"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/63453"@en .