RON, ROR1 and SUSD2 expression in tissues of endometrial carcinoma patients. Clinicopathological and prognostic implications

Endometrial carcinoma is now considered a common female gynecologic cancer with increasing in- cidence, with 13–25% of patients being still liable to recurrence and metasta- sis, which needs further studies to de- tect novel targets and new therapies. The aim of the study was evaluate tissue expression of RON, ROR1 and SUSD2 in endometrial carcinoma and atypical endometrial hyperplasia using immunohistochemistry and correlate their expression with clinical, patho- logical and prognostic parameters of patients.

We included samples from 100 patients with en- dometrial carcinoma. Sections from paraffin blocks were stained with RON, ROR1 and SUSD2 using immuno- histochemistry. Correlations between marker expression, clinicopathological features and prognostic samples were evaluated.

Upregulation of RON and ROR1 and downregulation of SUSD2 expression were found in endome- trial carcinoma more than atypical endometrial hyperplasia (p < 0.001). High RON and ROR1 expression levels were significantly associated with high grade (p < 0.001), presence of lymph node metastases (p = 0.003), distant metastases (p = 0.009), advanced In- ternational Federation of Gynecology and Obstetrics stage (p = 0.002), poor response to therapy (p = 0.046), and lower recurrence-free survival (RFS) rate (p = 0.002), progression-free sur- vival (PFS) rate (p = 0.008), distant metastasis-free survival (DMFS) rate (p = 0.019) and overall survival rate (p < 0.001). Low SUSD2 expression was significantly associated with older pa- tient age (p = 0.002), large tumor size (p = 0.003), high grade (p = 0.005), presence of adnexal invasion (p = 0.023), presence of lympho-vascular invasion (p = 0.021), extent of myometrial in- vasion (p = 0.002), lower RFS rate (p = 0.008), lower PFS rate (p = 0.023), and lower DMFS rate (p < 0.001).

Upregulation of RON and ROR1 and downregulation of SUSD2 lead to promotion of endometrial can- cer cell proliferation, migration, epi- thelial-mesenchymal transition, and invasion. Key words: endometrial carcinoma, RON, ROR1 and SUSD2, prognosis. Contemp Oncol (Pozn) 2022; 26 (2): 109–122 DOI: https://doi.org/10.5114/wo.2022.118245 Original paper RON, ROR1 and SUSD2 expression in tissues of endometrial carcinoma patients. Clinicopathological and prognostic implications Abeer M. Abdelbary1, Randa Mohamed Kaf1, Mohamed Elbakry Lashin2, Ahmed Z. Alattar3, Dalia Hamouda Elsayed3, Ahmed F. Amin4, Loay M. Gertallah5, Ahmed Mohamed Yehia6 1Pathology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt 2Gynecology and Obstetrics Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt 3Clinical Oncology and Nuclear Medicine Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt 4Medical OncologyDepartment, Faculty of Medicine, Zagazig University, Zagazig, Egypt 5Anesthesia and Intensive care Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt 6General Surgery Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Endometrial carcinoma is now considered a common female gynecologic can- cer with increasing incidence in developed countries. It usually has good prog- nosis but 13–25% of patients are still liable to recurrence and metastasis [1, 2], which needs further studies for detecting novel targets and new therapies. Cancer invasion and metastasis is responsible for poor patients’ progno- sis [3], and epithelial-mesenchymal transition (EMT) has vital roles in such processes [4]. Epithelial-mesenchymal transition is the biological process by which epi- thelial cells lose their normal criteria and acquire mesenchymal properties, thus enabling them to invade and metastasize [5], promoting cancer pro- gression and spread [6]. The tyrosine kinase receptor which is known as recepteur d’origine nantais (RON), discovered in 1993, has been found to be overexpressed in many can- cers [7, 8]. RON overexpression was associated with metastasis and poor prog- nosis [9]. Epithelial-mesenchymal transition, which explained invasion, metas- tases and progression of many cancers, was found to be mediated by several mediators as RON, ROR1 and SUSD2 (sushi domain containing 2) [10–12]. RON aberrant expression induces EMT in tumor cells, thus allowing mi- gration, invasion and metastases [10]. So, targeting RON could be a promis- ing cancer targeted therapy. Although the role of RON in cancer progression has been studied in many cancers, its prognostic roles in endometrial adenocarcinoma have not been sufficiently clarified. The recently detected Wnt receptor, ROR1 which is a tyrosine kinase-like orphan receptor, plays critical roles in embryogenesis. Aberrant ROR1 expres- sion has been found in many cancers [11, 13]. ROR1 has various oncogenic roles in cancers [14]. It plays an oncogenic role in many tumors by increasing tumor prolifera- tion, stimulating stemness [15], and activating EMT [12]. SUSD2 was primarily detected in mouse as a tumor-reversing gene. Human SUSD2 is located on chromosome 22 and encodes an 822-mino acid type I membrane protein containing Sushi domains, which play import- ant roles in adhesions between cells and cell-matrix adhesion [16]. Recent- 110 contemporary oncology ly, dysregulated expression of SUSD2 was found in many cancers and associated with cancer progression, pointing to its oncosuppressive roles in cancers. The aim of this study was to evaluate tissue expres- sion of RON, ROR1 and SUSD2 in endometrial carcinoma and atypical endometrial hyperplasia using immunohis- tochemistry and correlate their expression with clinical, pathological and prognostic parameters of patients.

In the present study we included samples from 100 pa- tients with endometrial carcinoma. Cases were primarily collected by endometrial biopsy in the Gynecology and Obstetrics Department diagnosed in the Pathology Department. Cases underwent the ope- rations of total abdominal hysterectomy and bilateral salpingo-oophorectomy with or without lymphadenectomy in the Gynecology and Obstetrics Department and General Surgery Department, Faculty of Medicine, Zagazig Univer- sity Hospitals. All surgically operated samples were sent to the Pathology Department for final diagnosis, grading and staging. Patients were sent to the Medical Oncology Department and to Clinical Oncology and Nuclear Medicine for further management and follow-up for survival and recurrence. We included 100 tissue blocks containing samples from patients with endometrial carcinoma and adjacent tissues of atypical endometrial hyperplasia. Exclusion criteria Cases with insufficient material in paraffin blocks and cases diagnosed with other endometrial malignancies such as endometrial stromal sarcoma and leiomyosarco- ma were excluded. Immunohistochemistry For immunohistochemistry sections were incubated with primary anti-RON (1 : 500; ab52927; Abcam, Cam- bridge, Cambridgeshire, UK), anti-ROR1 (1 : 50, #564464, BD Biosciences, USA) and anti-SUSD2 antibodies (Rabbit polyclonal, Sigma) using the Leica Bond RX system (Leica Microsystems, USA) at a dilution of 1 : 100 at 4°C overnight. Evaluation of RON, ROR1 and SUSD2 expression in stained tissues The stained sections were evaluated by assessment of staining intensity and staining percentage. The stain- ing intensity was divided into 0 (no stain), 1 (weak stain), 2 (moderate stain), and 3 (strong stain). The staining distribution percentages were 0 (no stain- ing ), 1 (1–25%), 2 (25–50%), and 3 (50–100%). Multiplying scores of intensity and percentage yields final stain scores from 0 to 12. We considered a score of 4 as a cut point below which scores are considered low and above which scores are considered high to facilitate statistical analysis. Statistical analysis All statistics were performed using SPSS Statistics 22.0 for Windows (IBM Corp.). Continuous variables were expressed as the mean ±SD and median (range), and the categorical variables were expressed as a number (percentage). Percent- ages of categorical variables were compared using Pearson’s c 2 test or Fisher’s exact test as appropriate. Recurrence free-survival (RFS) was calculated as the time from surgery to date of recurrence or the most re- cent follow-up contact that the patient was known to be recurrence free. Progression-free survival (PFS) was calcu- lated as the time from start of treatment to the date of disease progression or the most recent follow-up contact that the patient was known to be progression free. Over - all survival (OS) was calculated as the time from diagno- sis to death or the most recent follow-up contact (cen- sored). Stratification of RFS, PFS, distant metastasis-free survival (DMFS), and OS rates were estimated using a Ka- plan-Meier plot, and compared using the log-rank test. All tests were two sided. A p-value < 0.05 was considered significant. Table 1. Association between RON, ROR1 and SUSD2 expression and histopathology Factor Endometrial carcinoma (N = 100) Atypical endometrial hyperplasia (N = 100) p-value* n % n % Age ≤ 55 years 76 76 76 76 55 years 24 24 24 24 RON Low 46 46 90 90 < 0.001 High 54 54 10 10 ROR1 Low 24 42 80 80 0.001 High 58 58 20 20 SUSD2 Low 26 26 20 20 0.005 High 74 74 80 80 * chi-square test 111RON, ROR1 and SUSD2 expression in tissues of endometrial carcinoma patients. Clinicopathological and prognostic implications

Table 1 shows that upregulation of RON and ROR1 and down regulation of SUSD2 expression were found in endo- metrial carcinoma more than atypical endometrial hyper - plasia (p < 0.001) (Fig. 1). For RON expression in endometrial carcinoma and the association with clinicopathological parameters see Table 2 and Figure 2. High RON expression was significantly associated with large tumor size, high grade, presence of cervical stromal invasion, presence of adnexal invasion, presence of lym- pho-vascular invasion (p < 0.001), extent of myometrial invasion (p = 0.006), parametrial invasion (p = 0.008), serosal invasion (p = 0.005), positive peritoneal cytology (p = 0.004), presence of lymph node metastases (p = 0.003), distant metastases (p = 0.009), and advanced Internatio- nal Federation of Gynecology and Obstetrics (FIGO) stage (p = 0.002). No significant association was found between RON ex- pression and age of patients (Table 3). For RON expression in endometrial carcinoma and the association with prognostic and follow-up parameters see Tables 4 and 5 and Figures 5 and 6. High RON expression was significantly associated with poor response to therapy (p = 0.046), higher incidence of disease progression (p = 0.018) and higher incidence of tumor recurrence (p = 0.004). High RON expression was significantly associated with lower RFS rate (p = 0.002), PFS rate (p = 0.008), DMFS rate (p = 0.019) and OS rate (p < 0.001). For ROR1 expression in endometrial carcinoma and as- sociation with clinicopathological parameters see Table 2 and Figure 3. High ROR1 expression was significantly associat- ed with older age of the patient (p = 0.031), large tu- mor size (p = 0.003), high grade (p = 0.007), presence of cervical stromal invasion, presence of adnexal invasion, extent of myometrial invasion, presence of lymph node metastases, positive peritoneal cytology, advanced FIGO stage (p < 0.001), presence of lympho-vascular invasion (p = 0.003), parametrial invasion (p = 0.004), serosal in- vasion (p = 0.004) and presence of distant metastases (p = 0.029). For ROR1 expression in endometrial carcinoma and the association with prognostic and follow-up parameters see Tables 4 and 5 and Figures 5 and 6. High RON expression was significantly associated with higher incidence of disease progression (p = 0.044) and unfavorable survival (p < 0.001). No significant association was found between ROR1 expression and poor response to therapy or tumor recur - rence. High ROR1 expression was significantly associated with lower RFS rate (p = 0.032), PFS rate (p = 0.013), DMFS rate (p = 0.019) and OS rate (p < 0.001). For SUSD2 expression in endometrial carcinoma and the association with clinicopathological parameters see Table 2 and Figure 4. Low SUSD2 expression was significantly associat- ed with older patient age (p = 0.002), large tumor size (p = 0.003), high grade (p = 0.005), presence of adnexal invasion (p = 0.023), presence of lympho-vascular invasion (p = 0.021), extent of myometrial invasion (p = 0.002), se- rosal invasion (p = 0.005), presence of lymph nodes me- tastases (p = 0.003), distant metastases (p = 0.023), and advanced FIGO stage (p = 0.011). A B C Fig. 1. Low RON expression in endometrial hyperplasia (A), low ROR1 expression in endometrial hyperplasia (B), high SUSD2 expression in endometrial hyperplasia (C) 112 contemporary oncology No significant association was found between SUSD2 expression and cervical stromal invasion, positive perito- neal cytology or parametrial invasion. Characteristics EC patients (N = 100) n % Age ≤ 55 years 76 76 > 55 years 24 24 Size 4 cm 66 66 Grade Grade I 34 34 Grade II 36 36 Grade III 30 30 LVSI Absent 60 60 Present 40 40 Extent of myometrial invasion 50% 66 66 Cervical stromal invasion Absent 44 44 Present 56 56 Parametrial extension Absent 84 84 Present 16 16 Serosal invasion Absent 64 64 Present 36 36 Adnexal invasion Absent 64 64 Present 36 36 Peritoneal cytology Negative 60 60 Positive 40 40 Lymph node Negative 60 60 Positive 40 40 Distant metastasis Negative 80 80 Positive 20 20 FIGO stage Stage I 44 44 Stage II 20 20 Stage III 16 16 Stage IV 20 20 Table 2. Clinicopathological and follow-up parameters, RON, ROR1 and SUSD2 expression and outcome of 100 patients with endometrial carcinoma Characteristics EC patients (N = 100) n % RON Low 44 44 High 56 56 ROR1 Low 84 42 High 58 58 SUSD2 Low 26 26 High 74 74 Treatment Surgery 50 50 Concurrent chemoradiation 30 30 Chemotherapy 20 20 Response to CRT (n = 50) OAR 28 56 NR 22 44 Follow-up duration (months) Mean ±SD 36.50 ±15.77 Median (range) 38.50 (12–60) Recurrence (n = 78) Absent 66 84.6 Present 12 15.4 Progression (n = 56) Absent 30 53.6 Present 26 46.4 Metastasis (n = 40) Absent 80 80 Present 20 20 Mortality (N = 100) Alive 64 64 Died 36 36 CRT – cardiac resynchronization therapy, EC – endometrial cancer, FIGO – Inter- national Federation of Gynecology and Obstetrics, LVSI – lymph-vascular space invasion, NR – non-response, OAR – organs at risk For SUSD2 expression in endometrial carcinoma and association with prognostic and follow-up parameters see Tables 4 and 5 and Figures 5 and 6. Low SUSD2 expression was significantly associated with poor response to therapy (p = 0.005), higher inci- dence of disease progression (p = 0.021) and higher inci- dence of tumor recurrence (p = 0.023). Low SUSD2 expression was significantly associated with lower RFS rate (p = 0.008), PFS rate (p = 0.023), and DMFS rate (p < 0.001).

In the present study we assessed levels of RON at the protein level using immunohistochemistry in endometrial 113RON, ROR1 and SUSD2 expression in tissues of endometrial carcinoma patients. Clinicopathological and prognostic implications carcinoma and found that RON was overexpressed in tis- sues and it was associated with poor clinicopathological parameters and unfavorable outcomes. These results were similar to results obtained by Qin et al. [9] and Zhuang et al. [17]. Qin et al. [9] found that high RON expression mark- edly increased the proliferation, migration, and invasion of malignant cells, while its knockdown had the opposite effect; additionally, targeted therapy against RON, with the RON inhibitor BMS777607, antagonizes the effects of RON overexpression. All these results collectively showed the role of RON in increasing proliferation and progression of endometrial adenocarcinoma. Similar roles of RON were found in different types of cancers [18, 19]. The roles of RON in cancer invasion, spread and pro- gression are mostly due to activation of EMT , the process that was incriminated in cancer progression. RON activation was shown to induce spindle morpholo- gy in tumor cells, thus facilitating invasion and spread [20]. Moreover, RON overexpression was found to affect many EMT biomarkers such as E-cadherin, which is a marker of intercellular adhesions in epithelial cells, and vimentin [21]. Moreover, RON expression allows endome- trial carcinoma progression and promotes deep invasion into the myometrium [22]. RON also controls vimentin expression in endometri- al carcinoma tissues; previous studies showed that in- creased vimentin expression promotes the migration and invasion of tumor cells [23]. So, RON played roles in facilitating cancer invasion and progression through induction of EMT of endometrial ad- enocarcinoma. Additionally, RON could control EMT through several pathways, such as RAS-MAPK and PI-3K-Akt pathways [24], and SMAD and JAK pathways [25, 26]. Based on our results and results of previous studies, RON could regulate cell proliferation and EMT , which allow invasion and metastases in endometrial cancer (EC) cells, which promote the development of EC. For further confirmation of the role of RON in progres- sion of endometrial carcinoma we assessed levels of 2 oth- er incriminated markers of EMT in endometrial carcinoma: ROR1 andSUSD2. We found that ROR1 was overexpressed in endometrial carcinoma tissues and its expression was positively associ- ated with progression and unfavorable patients’ outcome. Fig. 2. RON expression in endometrial carcinoma (EC). High RON expression in serous EC high grade stage III 400× (A), high RON expression in endometroid EC high grade stage III 400× (B), low RON expression in endometroid EC low grade stage II 400× (C), negative RON expression in endometroid EC low grade stage I 400× (D) A B C D 114 contemporary oncology Table 3. Association between RON, ROR1 and SUSD2 expression and clinicopathological features in endometrial cancer patients (N = 100) Characteristics All (N = 100) RON p-value ROR1 p-value SUSD2 p-value Low (n = 46) High (n = 54) Low (n = 42) High (n = 58) Low (n = 26) High (n = 74) n % n % n % n % n % n % n % Age ≤ 55 years 76 76 38 50 38 50 0.128* 38 50 38 50 0.031* 10 13.2 66 86.8 0.002* > 55 years 24 24 6 25 18 75 4 16.7 20 83.3 16 66.7 8 33.3 Size < 4 cm 34 34 30 88.2 4 11.8 4 cm 66 66 14 21.2 52 78.8 12 18.2 54 81.8 26 39.4 40 60.6 Grade Grade I 34 34 30 88.2 4 11.8 < 0.001** 30 88.2 2 11.8 0.007** 0 0 34 100 0.005** Grade II 36 36 12 33.3 24 66.7 12 33.3 12 66.7 10 27.8 26 72.2 Grade III 30 30 2 6.7 28 93.3 0 0 15 100 16 53.3 28 46.7 LVSI Absent 60 60 38 63.3 22 36.7 0.005* 38 63.3 22 36.7 0.003* 10 16.7 50 83.3 0.021* Present 40 40 6 15 34 85 4 10 36 90 16 40 24 60 Extent of myometrial invasion < 50% 34 34 30 88.2 4 11.8 0.006* 30 88.2 4 11.8 50% 66 66 14 21.2 52 78.8 12 18.2 54 81.8 26 39.4 40 60.6 Cervical stromal invasion Absent 44 44 34 77.3 10 22.7 < 0.001* 34 77.3 10 22.7 < 0.001* 8 18.2 36 81.8 0.264* Present 56 56 10 17.9 46 82.1 8 14.3 48 85.7 18 32.1 38 67.9 Parametrial extension Absent 84 84 48 59.4 36 40.6 0.008* 48 59.4 36 40.6 0.004* 22 18.8 62 81.2 0.180* Present 16 16 6 16.7 10 83.3 4 11.1 12 88.9 4 38.9 12 61.1 Serosal invasion Absent 64 64 38 59.4 26 40.6 0.005* 38 59.4 26 40.6 0.002* 12 18.8 52 81.2 0.018* Present 36 36 6 16.7 30 83.3 4 11.1 32 88.9 14 38.9 22 61.1 Adnexal invasion Absent 64 64 42 65.6 22 34.4 < 0.001* 42 65.6 22 34.4 < 0.001* 10 15.6 54 84.4 0.023* Present 36 36 2 5.6 34 94.4 0 0 36 100 16 44.4 20 55.6 Peritoneal cytology Negative 60 60 38 63.3 22 36.7 0.004* 38 63.3 22 36.7 < 0.001* 10 16.7 50 83.3 0.065* Positive 40 40 6 15 34 85 4 10 36 90 16 40 24 60 115RON, ROR1 and SUSD2 expression in tissues of endometrial carcinoma patients. Clinicopathological and prognostic implications Characteristics All (N = 100) RON p-value ROR1 p-value SUSD2 p-value Low (n = 46) High (n = 54) Low (n = 42) High (n = 58) Low (n = 26) High (n = 74) n % n % n % n % n % n % n % Lymph node Negative 60 60 38 63.3 22 36.7 0.003* 38 63.3 22 36.7 < 0.001* 10 16.7 50 83.3 0.003* Positive 40 40 6 15 34 85 4 10 36 90 16 40 24 60 Distant metastasis Negative 80 80 42 52.2 38 47.5 0.009* 42 52.5 38 47.5 0.029* 16 20 64 80 0.023* Positive 20 20 2 10 18 90 0 0 20 100 10 50 10 50 FIGO stage Stage I 44 44 34 77.3 10 22.7 0.002** 34 77.3 10 22.7 < 0.001** 6 13.6 38 86.4 0.011** Stage II 20 20 4 20 16 80 4 20 16 80 4 20 16 80 Stage III 16 16 4 25 12 75 4 25 12 75 6 37.5 10 62.5 Stage IV 20 20 4 10 18 90 2 10 18 90 10 50 10 50 FIGO – International Federation of Gynecology and Obstetrics, LVSI – lymph-vascular space invasion * chi-square test, ** chi-square test for trend Table 3. Cont. Table 4. Association between RON, ROR1 and SUSD2 expression and patients’ outcome in endometrial cancer patients (N = 100) Outcome All RON p-value* ROR1 p-value* SUSD2 p-value* Low High Low High Low High No. % No. % No. % n % n % n % n % Response to CRT (N = 50) (N = 10) (N = 40) (N = 8) (N = 42) (N = 26) (N = 24) OAR 28 56 10 100 18 45 0.046 8 100 20 47 0.105 18 69.2 10 41.7 0.005 NR 22 44 0 0 22 55 0 0 22 52. 8 30.8 14 58.3 Recurrence (N = 78) (N = 44) (N = 34) (N = 42) (N = 36) (N = 18) (N = 60) Absent 66 84.6 44 100 22 64.7 0.004 40 95.2 26 72.2 0.077 12 66.7 54 90 0.023 Present 12 15.4 0 0 12 35.3 2 4.8 10 27.8 6 33.3 6 10 Progression (N = 56) (N = 12) (N = 44) (N = 10) (N = 46) (N = 26) (N = 30) Absent 30 53.6 12 100 18 40.9 0.018 10 100 20 43.5 0.044 20 76.9 5 33.3 0.021 Present 26 46.4 0 0 26 59.1 0 0 26 56.5 6 23.1 10 66.7 Metastasis (N = 80) (N = 42) (N = 38) (N = 42) (N = 38) (N = 16) (N = 64) Absent 64 80 38 90.5 26 68.4 0.120 38 90.5 26 68.4 0.120 6 37.5 58 90.6 0.004 Present 16 20 4 9.5 12 31.6 4 9.5 12 31.6 10 62.5 6 9.4 Mortality (N = 100) (N = 44) (N = 56) (N = 42) (N = 62) (N = 26) (N = 74) Alive 64 64 40 90.9 24 42.9 < 0.001 38 90.5 26 44.8 0.001 14 53.8 50 67.6 0.504 Died 36 36 4 9.1 32 57.1 4 9.5 36 55.2 12 46.2 24 32.4 CRT – cardiac resynchronization therapy, NR – non-response, OAR – organs at risk * chi-square test 116 contemporary oncologyTable 5. Association between RON, ROR1 and SUSD2 expression and survival rates in endometrial cancer patients (N = 100) Outcome All RON p-value* ROR1 p-value* SUSD2 p-value* Low High Low High Low High RFS (N = 78) (n = 44) (n = 34) (n = 42) (n = 36) (n = 18) (n = 40) Mean (months) (95% CI) 54.31 (50–58.62) 45.90 (41.29–50.51) 32.76 (24.74–40.78) 0.002 59 (57.14–60.85) 44.65 (37.44–51.85) 0.032 29.30 (24.13–34.47) 57.02 (53.53–60.52) 0.008 1 year RFS (%) 97.4 100 94.1 100 94.4 100 96.7 2 year RFS (%) 89.6 100 76.5 100 77 63.5 96.7 3 year RFS (%) 89.6 100 76.5 100 77 – 96.7 4 year RFS (%) 84.9 100 65.5 100 66 – 91.6 5 year RFS (%) 79.2 – – 90 – – 85.5 PFS (N = 56) (n = 12) (n = 44) (n = 10) (n = 46) (n = 26) (n = 30) Mean (months) (95% CI) 32.11 (23.96–40.26) 37.67 (25.29–50.04) 17.54 (12.42–22.66) 0.008 41.20 (30.08–52.31) 17.65 (12.77–22.53) 0.013 31.23 (27.35–35.10) 23.40 (12.53–34.26) 0.023 1 year PFS (%) 64.3 100 54.5 100 56.5 92.3 40 2 year PFS (%) 64.3 100 54.5 100 56.5 92.3 40 3 year PFS (%) 64.3 100 21.8 100 22.6 55.4 32 4 year PFS (%) 45 – 21.8 – 22.6 – 32 5 year PFS(%) – – – – – – – DMFS (N = 80) (n = 42) (n = 38) (n = 42) (n = 38) (n = 16) (n = 64) Mean (months) (95% CI) 52.26 (47.38–57.14) 57.38 (53.91–60.84) 41.61 (33.63–49.59) 0.019 57.38 (53.91–60.84) 41.61 (33.63–49.59) 0.019 30.55 (24.80–36.29) 55.89 (51.41–60.37) < 0.001 1 year DMFS (%) 97.4 100 94.1 100 94.1 87.5 100 2 year DMFS (%) 92.1 100 82.4 100 82.4 87.5 93.3 3 year DMFS (%) 77.4 90.5 74.9 90.5 58.2 – 89.9 4 year DMFS (%) 77.4 90.5 58.2 90.5 58.2 – 89.9 5 year DMFS (%) 77.4 90.5 – 90.5 – – 89.9 OS (N = 100) (n = 44) (n = 56) (n = 42) (n = 58) (n = 26) (n = 74) Mean (months) (95% CI) 46.13 (40.82–51.45) 57.61 (54.48–60.75) 34.18 (27.40–40.96) < 0.001 57.61 (54.48–60.75) 34.63 (27.94–41.32) < 0.001 32.86 (28.61–37.11) 47.41 (41.16–53.67) 0.070 1 year OS (%) 88 100 78.6 100 79.3 92.3 86.5 2 year OS (%) 79.3 100 61.2 100 62.8 92.3 75.5 3 year OS (%) 66.9 90.5 44.5 90.5 45.6 18.5 72.7 4 year OS (%) 64.4 90.5 39 90.5 39.9 – 72.7 5 year OS (%) 56.8 90.5 – 90.5 – – 64.1 DMFS – distant metastasis-free survival, OS – overall survival, PFS – progression-free survival, RFS – recurrence-free survival * log rank test 117RON, ROR1 and SUSD2 expression in tissues of endometrial carcinoma patients. Clinicopathological and prognostic implications A B C D Fig. 3. ROR1 expression in endometrial carcinoma (EC). High ROR1 expression in serous EC high grade stage III 400× (A), high ROR1 expres- sion in endometroid EC high grade stage III 400× (B), low ROR1 expression in endometroid EC low grade stage II 400× (C), negative ROR1 expression in endometroid EC low grade stage I 400× (D) Similar results were reported by Liu et al. [14] and Henry et al. [27], who confirmed the role of ROR1 as a novel prog- nostic marker and therapeutic target in EC. We observed that patients with high expression of ROR1 had lower OS and PFS rates in comparison with patients with low ROR1 expression levels. We found an association between ROR1 expression and higher grade and advanced stage of endometrial carcino- ma, which was similar to the results of Liu et al. [14] and those of Zhang et al. [28] in ovarian and pancreatic cancer. ROR1 was found to play a role in cancer progression through activation of EMT through activation of Wnt sig- naling [29, 30], which was proved to be associated with metastasis in ovarian cancer [31]. Previous studies showed that ROR1 played an import- ant role in the EMT process, which has a critical role in con- trolling cancer metastases [32, 33]. Regarding the association between ROR1 expression and survival rates, it was found that ROR1 overexpression was related to unfavorable survival rates and it could be considered a promising novel therapeutic target for recent management of EC. The recently detected ROR1 inhibitor cirmtuzumab is a monoclonal antibody that has proven to be effective in inhibiting ROR1 signaling in ovarian cancer and chronic lymphocytic leukemia (CLL) [34, 35]. Another ROR1-targeting therapy which is called ROR1 chimeric antigen receptor T cell therapy, as described by Berger et al. [36], was tried as targeted therapy in CLL and triple negative breast carcinoma. These new ROR1 inhibitors and targeted therapies could be beneficial to EC patients, particularly those with high expression of ROR1. Our study confirms the prognostic role of ROR1 in en- dometrial carcinoma progression and points to the future benefits of ROR1-targeting therapies in EC patients. We observed a positive association between RON and ROR1 expression in tissues of endometrial carcinoma; both biomarkers performed their work through activation of EMT and both are associated with cancer progression and unfavorable outcomes. We assessed expression of another marker, SUSD2, which has variable prognostic roles in different cancer types. Several studies have assessed associations between SUSD2 expression and prognosis of many cancers, e.g. breast, colon [16, 37], but its role in endometrial carcinoma has not been clarified yet. 118 contemporary oncology Our study demonstrated that tissue protein expression of SUSD2 was downregulated in endometrial carcinoma tissues, its low expression was related to unfavorable prognostic and clinicopathological parameters and its ex- pression was inversely associated with RON and ROR1 ex- pression in tissues of endometrial carcinoma. Similar results were obtained in hepatocellular carcino- ma tissues in the study of Liu et al. [38], which showed that SUSD2 expression was reduced in malignant tissues more than adjacent non-neoplastic tissues, suggesting that SUSD2 down-regulation plays important roles in car - cinogenesis and cancer progression. A possible explanation of our results is that SUSD2 downregulation led to an increased rate of cell prolifera- tion and reduced apoptosis; thus, SUSD2 had a tumor sup- pressor role and inhibited tumor growth [38]. Additionally we observed that high levels of SUSD2 markedly reduced invasiveness and migration ability of endometrial carcinoma cells, and we found that it was in- versely associated with the EMT markers ROR1 and RON1. Moreover, we found that high levels of SUSD2 expres- sion in the tumor were associated with improved patient survival, which points to the tumor suppressor role of SUSD2. Similar results were obtained in serous ovarian carcinoma by Sheets et al. [39, 40], who reported that high SUSD2 expression could inhibit ovarian carcinoma metas- tasis. Different results from ours were obtained in some stud- ies on different cancer types. Zhang et al. [41] reported that SUSD2 expression is up- regulated in EC cells, associated with unfavorable progno- sis, and its downregulation improves patients’ prognosis. Additionally they concluded that SUSD2 could be consid- ered a chemotherapeutic target in EC. Their explanation for their results was that SUSD2 participates in apoptosis and cell senescence in EC cells and thus affects invasive- ness and spread. Also the results of Xu et al. [42] showed that high ex- pression of SUSD2 increased ovarian cancer metastasis. Those contrasting results could be explained by differ - ences in methods of tissue expression, subcellular local- ization of SUSD2 and different metastasis mechanisms [43, 44]. Also the results of Watson et al. [16] showed that high SUSD2 expression in breast cancer enhanced cancer cell invasion and metastases.

We conclude that upregulation of RON and ROR1 in ad- dition to downregulation of SUSD2 might be considered A B C D Fig. 4. SUSD2 expression in endometrial carcinoma (EC). High SUSD2 expression in endometroid EC low grade stage I 400× (A), high SUSD2 expression in endometroid EC low grade stage II 400× (B), low SUSD2 expression in serous EC high grade stage III 200× (C), low SUSD2 expression in serous EC high grade stage III 400 × (D) 119RON, ROR1 and SUSD2 expression in tissues of endometrial carcinoma patients. Clinicopathological and prognostic implications A C B D Cum survival Cum survival Cum survival Cum survival 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 Fig. 5. Five-year disease-free survival (DFS) rate of included patients with endometrial carcinoma (A), DFS rate in association with RON ex- pression (B), DFS rate in association with RON and ROR1 expression (C), DFS rate in association with SUSD2 expression (D) DFS – disease-free survival Survival function Survival function Survival function Survival function 0 10 20 30 40 50 60 DFS [months] Survival function Censored 0 10 20 30 40 50 60 DFS [months] ROR1 Low High Low-censored High-censored 0 10 20 30 40 50 60 DFS [months] RON Low High Low-censored High-censored 0 10 20 30 40 50 60 DFS [months] SUSD2 Low High Low-censored High-censored 120 contemporary oncology A C B D Cum survival Cum survival Cum survival Cum survival 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 Fig. 6. Five-year overall survival (OS) rate of included patients with endometrial carcinoma (A), OS rate in association with RON expression (B), OS rate in association with RON and ROR1 expression (C), OS rate in association with SUSD2 expression (D) OS – overall survival Survival function Survival function Survival function Survival function 0 10 20 30 40 50 60 OS (months) Survival function Censored 0 10 20 30 40 50 60 OS (months) ROR1 Low High Low-censored High-censored 0 10 20 30 40 50 60 OS (months) RON Low High Low-censored High-censored 0 10 20 30 40 50 60 OS (months) SUSD2 Low High Low-censored High-censored 121RON, ROR1 and SUSD2 expression in tissues of endometrial carcinoma patients. Clinicopathological and prognostic implications a promoting factor for EC cells’ proliferation, migration, EMT , and invasion. Recommendations We recommend performing further studies on gene expression of studied markers, aiming at their appraisal as novel therapeutic targets for treatment of endometri- al adenocarcinoma. Moreover, we recommend assessing markers’ ex- pression in different variants of endometrial carcinoma in the recent molecular classification of endometrial cancer, to detect their roles. Pre-operative detection of glandular cells (GC) in cer- vical smear analysis might be a predictor for endometri- al cancer local recurrence [45]. Relative telomere length (RTL) in cell-free DNA (cfDNA) for EC, and cfDNA RTL analysis might be a potential diagnostic tool for early EC detection, progression, staging, and grading. However, further studies are needed to confirm these

focusing on high-risk patients who might benefit from this tool, as TL shortening is not specific for EC [46]. We recommend investigating correlations between studied markers’ expression, pre-operative detection of GC in cervical-smear analysis and cfDNA RTL analysis for early detection of endometrial cancer recurrence. The authors declare no conflict of interest.

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