Preoperative plasma level of endoglin as a predictor for disease outcomes after radical cystectomy for nonmetastatic urothelial carcinoma of the bladder

Abstract Elevated preoperative plasma level of endoglin has been associated with worse oncologic outcomes in various malignancies. The present large‐scale study aimed to determine the predictive and prognostic values of preoperative endoglin with regard to clinicopathologic and survival outcomes in patients treated with radical cystectomy (RC) for nonmetastatic urothelial carcinoma of the bladder (UCB). We prospectively collected preoperative blood samples from 1036 consecutive patients treated with RC for UCB. Logistic and Cox regression analyses were undertaken to assess the correlation of endoglin levels with pathologic and survival outcomes, respectively. The AUC and C‐index were used to assess the discrimination. Patients with adverse pathologic features had significantly higher median preoperative endoglin plasma levels than their counterparts. Higher preoperative endoglin level was independently associated with an increased risk for lymph node metastasis, ≥pT3 disease, and nonorgan confined disease (NOCD; all p < 0.001). Plasma endoglin level was also independently associated with cancer‐specific and overall survival in both pre‐ and postoperative models (all p < 0.05), as well as with recurrence‐free survival (RFS) in the preoperative model (p < 0.001). The addition of endoglin to the preoperative standard model improved its discrimination for prediction of lymph node metastasis, ≥pT3 disease, NOCD, and RFS (differential increases in C‐indices: 10%, 5%, 5.8%, and 4%, respectively). Preoperative plasma endoglin is associated with features of biologically and clinically aggressive UCB as well as survival outcomes. Therefore, it seems to hold the potential of identifying UCB patients who may benefit from intensified therapy in addition to RC such as extended lymphadenectomy or/and preoperative systemic therapy.


| INTRODUCTION
Due to the high intertumoral heterogeneity of urothelial carcinoma, a significant percentage of patients treated with radical cystectomy (RC) for urothelial carcinoma of the bladder (UCB) still experience disease progression. [1][2][3] Accurate identification of patients who could benefit from intensified perioperative systemic therapy remains an unmet clinical need. 4 Current prognostic models are mostly based on clinicopathologic features. [5][6][7][8][9] Nevertheless, preoperative patient selection for individualized treatment and follow-up scheduling remains challenging as we lack clinically reliable biomarkers for outcome prediction 6,[9][10][11][12][13][14][15][16][17][18] To accurately predict biologically and clinically aggressive disease as well as poor survival in UCB patients, novel biomarkers need to improve the current outcome prediction by a prognostically and clinically significant margin. 19 Angiogenesis has been proposed as a critical event in the initiation and progression of solid malignancies. 20 Endoglin is highly expressed by human vascular endothelial cells and has been reported as a marker of angiogenesis. 21 Elevated preoperative plasma levels of endoglin have been associated with worse oncologic outcomes in various malignancies. [22][23][24] Among urological malignancies, higher blood levels of endoglin have been found to be associated with higher preoperative serum prostate-specific antigen, adverse pathologic features, as well as biochemical progression in prostate cancer patients. [25][26][27] The association of circulating levels of endoglin with bladder cancer remains, however, uninvestigated.
We hypothesized that elevated preoperative endoglin plasma levels would be associated with features of biologically and clinically aggressive UCB as well as poor survival outcomes. To test this hypothesis, we studied the predictive and prognostic values of blood levels of endoglin in a large consecutive cohort of patients with nonmetastatic UCB treated with RC and pelvic lymphadenectomy.

| Data source and patient cohort
All procedures described in the present study were undertaken with the approval and oversight of the Institutional Review Board for the Protection of Human Subjects (IRB: 1011011386, 069826900). This study is a retrospective analysis of a prospectively collected consecutive cohort of patients who were treated with RC for nonmetastatic UCB at two medical centers. Sample collection took place between 2003 and 2015. The exclusion criteria were the following: patients with any concomitant secondary malignancies, including upper urinary tract carcinoma, as well as patients with missing data.

| Biomarker measurements
Plasma samples were collected after a preoperative overnight fast on the morning of the day of surgery. Specimen collection and measurement have been described in detail elsewhere. 28 Briefly, blood was collected into Vacutainer CPT 8-ml tubes containing 0.1 ml of molar sodium citrate (Becton Dickinson) and centrifuged at room temperature for 20 min at 1500g. The top layer corresponding to plasma was decanted using sterile transfer pipettes. The plasma was immediately frozen and stored at −80°C in polypropylene cryopreservation vials (NalgeNunc). For quantitative measurements of endoglin level, we used commercially available quantitative immunoassays (R&D Systems). Every sample was run in duplicate, and the mean was calculated for data analyses. The coefficient of variation was less than 10%.

| Pathological review
All surgical specimens were processed according to standard pathological procedures. Genitourinary pathologists assigned tumor grades according to the 1973 WHO grading system. Pathological stage was reassigned according to the 2002 American Joint Committee on Cancer TNM staging system. The presence of concomitant carcinoma in situ (CIS) was defined as the presence of CIS in conjunction with another tumor other than CIS. 29 Pelvic lymph nodes were examined grossly, and all lymphoid tissue was submitted for histological examination. Positive soft tissue surgical margin was defined as the presence of tumor at inked areas of soft tissue on the RC specimen. 30 Urethral or ureteral margins were not considered soft tissue surgical margins. Lymphovascular invasion was defined as the unequivocal presence of tumor cells within an endothelium-lined space without underlying muscular walls. 31 Any nonorgan confined disease (NOCD) was defined as both ≥pT3 disease and lymph node metastasis.

| Follow-up
Clinical and radiological follow-up was performed in accordance with institutional protocols and current guidelines. Routine follow-up usually included physical examination, radiological imaging (CT of the thorax and abdomen), and urine cytology every 3 months for 2 years.
Between the second and the fifth year, follow-up was performed every 6 months. Afterward, in most cases, an annual follow-up was performed. Tumor recurrence was defined as the occurrence of locoregional recurrence or distant metastasis on radiological imaging.
Recurrence-free survival (RFS) time was calculated from the date of RC to tumor recurrence or last follow-up. Cause of death was abstracted from medical charts and/or from death certificates. 32 Overall survival (OS) time was calculated from the date of RC to death or last follow-up. Cancer-specific survival (CSS) time was calculated from the date of RC to death from disease or last follow-up. Binominal logistic regression analysis was performed using preoperative available variables to evaluate the association of preoperative plasma level of endoglin with lymph node metastasis, ≥pT3 disease, or any NOCD. The risk of events was expressed as odds ratios (ORs) and 95% confidence intervals (95% CIs). The area under the curve (AUC) of receiver operating characteristics (ROC) curves was calculated to determine the predictive accuracy of multiple logistic regression models. DeLong's test was used to assess the additional predictive value of preoperative endoglin after adding it to a reference model by comparing the AUCs of the models.

| Statistical analysis
Association between preoperative endoglin with RFS, CSS, and OS was assessed in univariable and multivariable Cox regression models. The risk of survival was expressed as hazard ratios (HRs) and 95% CI. Kaplan-Meier survival curves were used to depict the association between endoglin level and survival. The log-rank test was used to determine the statistical difference between the endoglin (<3.142 and ≥3.142) groups with respect to recurrence or death. Two LAUKHTINA ET AL.
separate Cox regression models that featured either preoperative clinical variables or postoperative histopathological variables were created. Clinical and pathological tumor grade was excluded as a variable for all predictive models as virtually all RC patients had highgrade UCB. The discriminative ability of the models before and after the inclusion of endoglin was tested and compared using Harrel's concordance indices (C-index) to assess the additional prognostic value of endoglin. The additional clinical net-benefit of endoglin was evaluated using DCA. 33 All reported p values were two-sided, and statistical significance was set at 0.05. All statistical analyses were performed using R Version 4.0.4.

| Association of preoperative plasma endoglin level with clinicopathologic features
A total of 1036 patients were included in the analysis. The median age of the entire cohort was 67 years (IQR: 60-73). Patient characteristics are shown in Table 1. Median plasma levels of endoglin were significantly higher among patients with adverse pathologic features such as lymphovascular invasion (p < 0.001), lymph node metastasis (p < 0.001), contaminant CIS (p < 0.01), and advanced pathologic tumor stage (p < 0.001).
On multivariable logistic regression modeling, elevated preoperative plasma levels of endoglin were significantly associated with an increased risk of lymph node metastasis, ≥pT3 disease, and any NOCD (all p < 0.001) ( Table 2). ROC curve analyses showed that the addition of preoperative plasma levels of endoglin to a reference model comprising age, sex, and clinical tumor stage improved the discriminatory ability for the prediction of lymph node metastasis (10%, p < 0.001), ≥pT3 disease (5%, p < 0.001), and any NOCD (5.8%, p < 0.001).
On DCA for prediction of lymph node metastasis, the addition of preoperative endoglin plasma levels to the preoperative standard model resulted in the improved clinical net-benefit between a threshold probability of 30%-60% ( Figure 1A

| Association of survival outcomes within a preoperative model
Median follow-up of patients alive was 37 months (IQR: 14.5-108.5).
In a multivariable Cox regression model that included established available preoperative variables (age, sex, and clinical tumor stage), a higher preoperative plasma level of endoglin was associated with worse RFS, CSS, and OS (all p < 0.001) ( Table 3). The addition of preoperative plasma levels of endoglin slightly improved the Cindices of the same model for prediction of early RFS (4%), CSS   F I G U R E 1 Decision curve analyses (DCA) for the evaluation of the clinical net-benefit using the log models for the prediction of (A) lymph node metastasis, (B) ≥ pT3 disease, and (C) any nonorgan confined disease margin was minimal. This is in agreement with previous studies reporting preoperative plasma endoglin to improve the accuracy for the prediction of pelvic lymph node metastasis in patients treated with radical prostatectomy for clinically localized prostate cancer. 25,27 In contrast, Gomceli et al. failed to find an additive predictive value to endoglin levels in patients with gastric or colorectal carcinoma. 34,35 Conversely, in accordance with our findings, Daly et al. found endoglin levels to increase in the transition from node-positive disease to disseminated disease in patients with lung adenocarcinoma. 24 Similarly, several independent research groups reported an association of plasma endoglin with distant metastasis in patients with both colorectal and breast cancers. 22,36 Findings from these studies suggested that plasma endoglin levels are associated with the metastatic process and may be useful in the identification of early metastases.
However, it seems that the prediction probability of endoglin highly Nevertheless, conventional multivariable analyses and the change in C-index or AUC that quantify the ability of the model to discriminate between patients with and those without the outcome of interest are not sufficient to demonstrate that a biomarker provides a clinical benefit. 19 To explore the net benefit of adding of endoglin to the standard models, we performed DCA, a method that combines simplicity with efficient computations. 19 According to DCA, It is also important to consider that combining preoperative plasma endoglin with other blood-based biomarkers is more likely to capture a higher predictive value than any single biomarkers. 9,19,37,38 Additionally, its combination with tissue expression of endoglin, as a marker of angiogenesis, in a specimen from transurethral resection of

| CONCLUSION
Preoperative plasma endoglin holds potential in identifying UCB patients who may benefit from intensified therapy in addition to RC due to its association with features of the biologically and clinically aggressive disease as well as poor survival outcomes. In particular, with respect to the prediction of lymph node metastasis, preoperative endoglin offers a high discriminatory power, which warrants inclusion into future predictive models. Nico С. Grossmann is supported by the Zurich Cancer League.