ROR1 and ROR2 are receptor tyrosine kinases with altered expression in a range of cancers. Silencing ROR1 or ROR2 in different tumour types has been shown to inhibit proliferation and decrease metastatic potential. The aim of this study was to investigate the role of ROR1 and ROR2 in endometrial cancer via immunohistochemistry (IHC) in a large endometrial cancer patient cohort (n = 499) and through in vitro analysis in endometrial cancer cell lines. Correlation was assessed between ROR1/2 expression and clinicopathological parameters. Kaplan Meier curves were produced for 5-year progression free survival (PFS) and overall survival (OS) with low/moderate versus high ROR1/2 intensity. Cox multivariate regression was applied to analyse the effect of selected covariates on the PFS and OS. The effect of ROR1 and/or ROR2 modulation on cell proliferation, adhesion, migration and invasion was analysed in two endometrial cancer cell lines (KLE and MFE-296). We observed a significant decrease in OS and PFS in patients with high ROR1 expression. ROR1 silencing and ROR2 overexpression significantly inhibited proliferation of KLE endometrial cancer cells and decreased migration. This study supports the oncogenic role of ROR1 in endometrial cancer, and warrants investigation of future application of ROR1-targeting therapies in endometrial cancer patients. Endometrial cancer (EC) is the most prevalent gynaecological cancer and the sixth most common malignancy worldwide 1. Incidence has increased significantly over the last decade, particularly in developed countries 2. This escalating worldwide burden and poor survival outcomes from advanced stage and aggressive subtypes warrants further research into novel targets and new therapies. The pathogenesis for EC is multifactorial, with risk factors including genetic variants 3 , high BMI 4,5 , high number of cumulative menstrual cycles 6,7 , and infertility 8. In 1983, Bokhman 9 proposed the classic dualistic model which divided EC into estrogen driven endometrioid subtype (Type I) and the more aggressive nonendometrioid subtype (Type II). Based on the histopathological features, EC is also commonly classified into endometrioid adenocarcinoma, serous carcinoma, mucinous carcinoma, clear cell carcinoma mixed carcinoma etc. 10. There are certain overlaps between the two classification systems: Type I is generally endometrioid subtype and Type II is mostly serous. These traditional classification systems based on endocrine or histopathological features failed to take into account the heterogeneity of EC and were limited due to technical difficulties and controversies in histopathological assessment 11,12. In 2013, the Cancer Genome Atlas (TCGA) defined four genomic subgroups: Polymerase epsilon (POLE)-mutant tumours (ultrahypermutated), MSI (hypermutated), copy-number low (endometrioid) and copy-number high tumours (serous-like) through integration of multiomics data 13. Although this system is not yet in widespread clinical use, the identification of molecular targets correlate to dis...

BackgroundMethylation patterns in circulating cell-free DNA are potential biomarkers for cancer and other pathologies. Currently, bisulfite treatment underpins most DNA methylation analysis methods, however, it is known to fragment DNA. Circulating DNA is already short, and further fragmentation during bisulfite treatment is of concern, as it would potentially reduce the sensitivity of downstream assays.MethodsWe used high molecular weight genomic DNA to compare fragmentation and recovery following bisulfite treatment with 2 commercially available kits (Qiagen). The bisulfite treated DNA was visualised on an agarose gel and quantified by qPCR. We also bisulfite treated, visualised and quantitated circulating DNA from plasma.ResultsThere was no difference in DNA fragmentation between the two kits tested, however, the Epitect Fast kit gave better recovery than the standard Epitect kit, with the same conversion efficiency. We also found that bisulfite treated circulating DNA migrates as distinct bands on agarose gels, suggesting that, in contrast to genomic DNA, it remains largely intact following treatment. Bisulfite treatment of 129 and 234 base PCR products confirmed that this was due to the short length of the circulating DNA fragments. Compared to double stranded DNA, bisulfite treated single stranded DNA gives a very weak signal on gel electrophoresis.ConclusionsDNA fragmentation during bisulfite treatment does not contribute to loss of sensitivity in methylation analysis of circulating DNA. The absence of DNA fragments below approximately 170 bases from agarose gel images of purified circulating DNA raises the possibility that these fragments are single stranded following the DNA extraction step.

Background Endometrial cancer is one of the fastest rising cancers in women, and despite overall high survival, new therapies are required for women with aggressive subtypes of the disease. ROR1 and ROR2 are Wnt receptor tyrosine kinases whose expression is altered in a range of cancers. This study aimed to investigate the role of ROR1/2 in endometrial cancer (EC). Method Immunohistochemistry (IHC) of ROR1 and ROR2 was performed on an EC patient cohort (Australia-wide population-based Australian EC Study, ANECS, n=499). The association between ROR1/2 staining intensity (0-3) and clinicopathological parameters was analyzed, as well as association with 5-year progression free survival (PFS) and overall survival (OS). Cox multivariate regression was also applied to analyze the impact of selected covariates (age, BMI, FIGO stage, grade and subtypes) on the PFS and OS. The functional effect of ROR1 and ROR2 modulation was further investigated in two EC cell lines–KLE (high ROR1, low ROR2) and MFE-296 (low ROR1, high ROR2). For KLE, ROR1 silencing and ROR2 overexpression were performed. In contrast, ROR2 silencing and ROR1 overexpression were performed in MFE-296. ROR1 or ROR2 silencing was achieved via transfection with ROR1 or ROR2 siRNA. ROR1 pCMV3 plasmid or ROR2 pFLAG plasmid were transfected for ROR1 or ROR2 overexpression. All the conditions were compared to the negative control which was prepared by transfecting both non-targeting siRNA and pCMV3-NH plasmid. Effects on proliferation, adhesion, migration and invasion were measured. Result In the patient cohort, ROR1 expression level was significantly associated with tumor grade (p=0.004) and moderately associated with FIGO stage (p=0.057). A significant decrease in OS and PFS was observed in patients with high ROR1 expression (p=0.045 and 0.003 respectively). This correlation was not lost when filtering against multiple parameters including age, BMI, FIGO stage and tumor grade in COX regression (p=0.049). No significant correlation was observed for ROR2 expression with OS or PFS, though high ROR2 showed a trend towards better PFS. ROR1 downregulation or ROR2 upregulation in KLE cells decreased cell proliferation after 72 hours. The combination of the two further reduced cell proliferation significantly after 48h and 72h (p=0.04 and 0.004 respectively). Proliferation was not significantly altered in MFE-296, although ROR1 overexpression showed a trend for increasing cell proliferation after 72h. ROR1 silencing in KLE decreased the migration ability moderately (p=0.059) and its combination with ROR2 overexpression further reduced migration significantly (p=0.038). No significant change in invasion or adhesion was observed. Conclusion This study confirms the oncogenic role ROR1 plays in EC, which warrants the future application of ROR1-targeting therapies in EC patients. The role of ROR2 is more complex in EC and may be context dependent. Citation Format: Dongli Liu, Kate Gunther, Benjamin Daniels, Tracy O'Mara, Katrina Tang, Australian National Endometrial Cancer Study Group, Caroline Ford. ROR1: A novel target in endometrial cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2942.

No abstract