Risk Factor Analysis of Intravesical Recurrence After Laparoscopic Nephroureterectomy for Upper Tract Urothelial Carcinoma

Background: One of the major concerns of patients with upper tract urothelial carcinoma (UTUC) treated with nephroureterectomy is intravesical recurrence (IVR). The purpose of the present study was to investigate the predictive risk factors for IVR after laparoscopic nephroureterectomy (LNU) for UTUC. Methods: Clinicopathological and surgical information were collected from the medical records of 73 patients treated with LNU for non-metastatic UTUC, without a history of or concomitant bladder cancer. The association between IVR after LNU and clinicopathological and surgery-related factors, including preoperative urine cytology and pneumoperitoneum time, was analyzed using Cox proportional hazards regression models and the Kaplan–Meier method with log-rank test. Results: During the median follow-up time of 39.1 months, 18 (24.7%) patients had subsequent IVR after LNU. The 3- and 5-year IVR-free survival rates were 76.5% and 74.3%, respectively. In the multivariate Cox regression analysis, positive preoperative urine cytology (hazard ratio [HR]: 3.55; 95% condence interval [CI]: 1.326–11.327; p=0.011) and prolonged pneumoperitoneum time of ≥ 210 min (HR: 3.40; 95% CI: 1.271–10.692; p=0.014) were independent prognostic factors for IVR-free survival. In patients with positive urine cytology, the Kaplan–Meier method with log-rank test revealed that the 3-year and 5-years IVR free survival rates were 46.3% and 39.7%, respectively, in patients with a prolonged pneumoperitoneum time of ≥ 210 min, which was signicantly lower than that in their counterparts (76% and 76%, respectively, p=0.041). Conclusions: when bladder tumor antigen, NMP22 nuclear mitotic apparatus protein 22; LVI, lymphovascular invasion; INF, inltrative growth; ASC, adjuvant systemic chemotherapy. results. Comparison between patients with pneumoperitoneum time of <210 min and ≥ 210 min (A) The 3-year and 5-year IVR-free survival rates of patients with negative urine cytology with pneumoperitoneum time of <210 min were 91.6% and 91.6%, respectively, and that of patients with pneumoperitoneum time of ≥ 210 min were 86.7 and 86.7%, respectively. (B) The 3-year and 5-year IVR-free survival rates of patients with positive urine cytology with pneumoperitoneum time of <210 min were 76% and 76%, respectively, and that of patients with pneumoperitoneum time of ≥ 210 min were 46.3% and 39.7%, respectively.

The purpose of the present study was to investigate the association between IVR after LNU for UTUC and clinicopathological and surgical factors, including preoperative urine cytology, urinary bladder tumor antigen (BTA), urinary nuclear mitotic apparatus protein 22 (NMP22), and pneumoperitoneum time.

Patient selection
We retrospectively identi ed 102 patients treated with LNU for non-metastatic UTUC at Nippon Medical School Hospital between 2012 and 2020. UTUC was diagnosed using computed tomography (CT), magnetic resonance imaging (MRI), and urine cytology. A diagnostic ureteroscopic biopsy was performed when required. All patients underwent preoperative cystoscopy. Of the 102 patients, 29 patients with a history of bladder cancer or concomitant bladder cancer were excluded from our study. Finally, 73 patients were included in the study.

Clinicopathological data
From the medical records, we collected clinicopathological and surgical information of the patients, including age, sex, laterality and location of the main tumor, presence or absence of hydronephrosis, preoperative urine cytology, preoperative urinary BTA level, preoperative urinary NMP22 level, necessity of diagnostic ureteroscopic biopsy, pneumoperitoneum time, total operating time, multifocality of the tumor, Organization classi cation [14].
Surgical procedure While performing LNU, laparoscopic procedures were performed using the retroperitoneal approach in the kidney position, with 8 mmHg CO 2 gas pressure in all cases. The CO 2 gas pressure was increased temporally when necessary. The maximum pressure of the CO 2 gas was 12 mmHg. In the laparoscopic procedure, we clamped the ureter after ligation of the renal arteries. A small iliac incision (Gibson incision) or lower abdominal midline incision was made to retrieve the kidney and ureter and to perform resection of the bladder cuff. In our institution, we have performed LNU in patients with non-metastatic localized or locally advanced UTUC (cTa-3N0M0). Therefore, lymphadenectomy was not performed in the present study.

Adjuvant therapy and follow-up
Adjuvant intravesical therapy is not administered at our institution. Four courses of ASC, such as the gemcitabine/cisplatin regimen or gemcitabine/carboplatin regimen, were administered to select pT2-4 patients. Of these patients, those with an estimated glomerular ltration rate (eGFR) of <30 ml/min/1.73 m 2 received ASC with the gemcitabine/carboplatin regimen, and the other patients received ASC with the gemcitabine/cisplatin regimen. After LNU, all patients were generally followed-up using blood tests, urine analysis, urine cytology, cystoscopy, and CT scan every three months for two years, and every six months thereafter. We de ned IVR as a pathologically diagnosed bladder cancer after LNU.

Endpoint of the present study
The primary endpoint of the present study was to investigate the association between IVR after LNU for UTUC and clinicopathological and surgical factors, including preoperative factors of urine cytology, urinary BTA, urinary NMP22, and pneumoperitoneum time.

Statistical analysis
Statistical analyses were performed using JMP® 13 (SAS Institute Inc., Cary, NC, USA). The value of statistical signi cance was set at P<0.05. To determine independent factors predicting IVR after LNU, univariate and multivariate analyses were performed using the Cox proportional hazards regression model. Survival curves were constructed using the Kaplan-Meier method, and differences between the groups were evaluated using the log-rank test. The cut-off value of pneumoperitoneum time of LNU was 210 minutes, which was de ned as the maximum pneumoperitoneum time in the technical certi cation test of laparoscopic radical nephrectomy and LNU by the Japanese Society of Endourology [15].

Results
A total of 73 patients (56 men (76.7%), 17 women (23.3%); mean age: 72.9 years; range: 49-89 years) underwent LNU for UTUC in the present study (Table 1)  year IVR-free survival rates were 76.5% and 74.3%, respectively (Fig. 1). The histological type of bladder cancer in 18 patients was urothelial carcinoma. In 50% of these bladder cancers, the grade was lower than that of the initial UTUC diagnosis (Fig. 2). In the other 50% of bladder cancer cases, the grade was the same grade as the initial UTUC. None of the bladder cancers had a higher grade than the initial UTUC diagnosis.   year IVR-free survival rates were 86.7% and 86.7%, respectively, in patients with prolonged pneumoperitoneum time of ≥ 210 min, which were not signi cantly different from those with pneumoperitoneum time of < 210 min (91.6.% and 91.6%, respectively, p = 0.579) (Fig. 3a). In patients with positive urine cytology, the Kaplan-Meier method with log-rank test revealed that the 3-year and 5year IVR-free survival rates were 46.3.% and 39.7%, respectively, among patients with prolonged pneumoperitoneum time of ≥ 210 min, which were signi cantly lower than those with pneumoperitoneum time of < 210 min (76.0% and 76.0%, respectively, p = 0.041) (Fig. 3b). We also categorized the patients according to the duration of pneumoperitoneum and IVR rates (Fig. 4). In patients with negative urine cytology, IVR rates were 7.7% for pneumoperitoneum time of < 210 min, 25% for that of 210-270 min, and 14.3% for pneumoperitoneum time of > 270 min (Fig. 4A). In patients with positive urine cytology, IVR rates were 20% for pneumoperitoneum time of < 210 min, 55.6% for that of 210-270 min, and 62.5% for pneumoperitoneum time of > 270 min (Fig. 4B).

Discussion
One of the greatest concerns of UTUC patients treated with NU remains to be the occurrence of IVR. Previous studies have reported that IVR after NU occurs with an incidence of approximately 22-47% [1][2][3][4]. In the present study, the rate of IVR incidence was 24.7%, which was consistent with previous studies.
In this study, we demonstrated that positive urine cytology  [20]. Therefore, the CO 2 gas pressure during the laparoscopic procedure of LNU differs between institutions. In recent studies on LNU, laparoscopic procedures were performed with 10-14 mmHg CO 2 gas pressure [8,16,17]. It was suggested that a low CO 2 gas pressure of 8 mmHg in the present study might have in uenced the low IVR rates.
In the present study, 210 min, which is close to the mean and median pneumoperitoneum time, was used as the cut-off value for pneumoperitoneum time. In patients with negative urine cytology, IVR-free survival did not differ between patients with prolonged pneumoperitoneum time (≥ 210 min) and those with pneumoperitoneum time of < 210 min (Fig. 3A) (p = 0.579). On the other hand, in patients with positive urine cytology, IVR-free survival was signi cantly lower in patients with prolonged pneumoperitoneum time (≥ 210 min) than in those with pneumoperitoneum time of < 210 min (p = 0.041) (Fig. 3B). In addition, in positive urine cytology, the rate of IVR incidence tended to increase as the operation time was prolonged (Fig. 4B). The present study is the rst to demonstrate that in patients with positive urine cytology, prolonged pneumoperitoneum time increases the frequency of IVR after LNU for UTUC. Further studies comparing different CO 2 gas pressures are required to investigate the impact of CO 2 gas pressure on IVR after LNU.
Recent molecular genetic studies have suggested that intraluminal seeding is one of the main mechanisms of IVR after NU [18][19][20]. It was also reported that continuous intravesical irrigation with distilled water or physiological saline solution during LNU decreased the rate of IVR incidence [21]. They concluded that continuous intravesical irrigation might eliminate cancer cells oating in the bladder during surgery before they become engrafted on the mucous membrane of the bladder. This result suggests that IVR after NU occurs due to intraluminal seeding. Recent studies demonstrated that prolonged pneumoperitoneum time and diagnostic ureteroscopic biopsy are independent factors of IVR after NU [5,8]. Based on these results of past studies, long-term pneumoperitoneum pressure to the tumor and direct destruction of the tumor by diagnostic ureteroscopic biopsy might contribute to intraluminal seeding. In the present study, in patients with preoperative positive urine cytology, prolonged pneumoperitoneum time was a risk factor for IVR after LNU. UTUC with positive urine cytology is a type of cancer that releases cancer cells into the urine, and long-term pneumoperitoneum pressure during LNU might promote the release of cancer cells into the urine. In the present study, the grade of bladder cancer with IVR was not higher than that of initial UTUC (Fig. 1). It has also been suggested that IVR tumors are caused by intraluminal seeding from UTUC.
The BTA test detects the human complement factor H-related protein secreted in the urine. While the NMP22 test detects the protein level of the nuclear mitotic apparatus. Positive urinary BTA and NMP22 have been reported as predictors of the presence of bladder cancer and UTUC, along with positive urine cytology [22][23][24][25]. In the present study, the risk factor for IVR was not positive urinary BTA or urinary NMP22, but positive urine cytology. Urinary BTA and NMP22 are considered unsuitable for predicting IVR after LNU because the values of urinary BTA and NMP22 generally have a positive correlation with tumor volume; however, urinary BTA and NMP22 do not directly detect cancer cells.
Recently, two prospective randomized trials have demonstrated that a single early intravesical chemotherapy cycle using mitomycin C or pirarubicin after NU decreased the risk of IVR [26,27]. However, the type of patients that will bene t from this treatment remains unclear. From our results, we strongly recommend that patients with positive urine cytology with pneumoperitoneum time of ≥ 210 min should receive a single early intravesical chemotherapy after LNU with 8 mmHg CO 2 gas pressure.
The present study has several limitations. UTUC is a relatively uncommon condition. We excluded patients with a history of bladder cancer or concomitant bladder cancer, because the purpose of the present study was to investigate the risk factors for IVR after LNU for UTUC. In addition, this study was conducted in a single institution; therefore, the cohort in this study was small. Since the study was a retrospective analysis, there might be a selection bias for the surgeons. In this study, 13 surgeons performed the LNU procedure. However, three experienced surgeons who had performed more than 100 laparoscopic surgeries performed or supervised all of the LNU procedures. In addition, the rate of IVR incidence in our study was lower than that reported in previous studies. Based on these facts, we believe that the participation of inexperienced surgeons in LNU had little impact on the IVR rate in the present study. To reduce these limitations, prospective studies with larger cohorts from several institutions are required.

Conclusions
Positive urine cytology was the strongest factor for IVR after LNU with 8 mmHg CO 2 gas pressure. In patients with positive urine cytology, a prolonged pneumoperitoneum time of ≥ 210 min was a signi cant factor for IVR after LNU with 8 mmHg CO 2 gas pressure. For patients with positive urine cytology, it is necessary to devise a strategy to shorten the pneumoperitoneum time. In addition, when the pneumoperitoneum time is prolonged (≥ 210 min) in LNU with 8 mmHg CO 2 gas pressure for patients with positive urine cytology, strict follow-up after LNU is highly recommended. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Competing interests Figure 2 The percentage of change in grade of tumor of IVR compared to grade of initial UTUC. In 50% of IVR tumors, the grade was lower than that of the initial UTUC, and in the other 50% of IVR tumors, the grade was the same as the initial UTUC. None of the IVR tumors had a higher grade than the UTCU.  Recurrence rate by pneumoperitoneum time (A) IVR rate of patients with preoperative negative urine cytology (B) IVR rate of patients with preoperative positive urine cytology