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Preoperative Risk Classification Using Neutrophil-to-Lymphocyte Ratio and Albumin for Upper Tract Urothelial Carcinoma Treated with Radical Nephroureterectomy

Purpose To improve the preoperative prediction of the outcomes of patients diagnosed with upper tract urothelial carcinoma (UTUC) treated with radical nephroureterectomy (RNU), we explored various preoperative laboratory factors and established a prognostic risk stratification method. Patients and Methods We retrospectively reviewed 232 UTUC patients who underwent RNU from September 2010 to October 2019 and analyzed their comprehensive clinicopathologic data and preoperative blood-based biomarkers. Kaplan–Meier analysis, receiver-operating characteristic (ROC) curves analysis and Cox regression analysis were performed to assess the relationship between these factors and the prognosis. Results The median follow-up and age were 24 months and 68.5 years, respectively. Preoperative elevated neutrophil-to-lymphocyte ratio (NLR > 3.44) and decreased albumin (ALB < 39.8 g/L) were negatively correlated with progression-free survival (PFS), cancer-specific survival (CSS) and overall survival (OS) in both univariate and multivariate analyses. Patients were sorted into three groups based on their NLR and ALB: the low-risk group (neither elevated NLR nor decreased ALB), intermediate-risk group (either elevated NLR or decreased ALB) and high-risk group (elevated NLR and decreased ALB). Their 5-year PFS rates were 77.8%, 52.6% and 32.3%; their 5-year CSS rates were 97.7%, 71.4% and 32.9%; and their 5-year OS rates were 92.7%, 70.4% and 29.2%, respectively (all P < 0.0001). ROC curves analysis showed that NLR plus ALB had a more accurate prognostic value (P < 0.05). Conclusion Preoperative risk classification using NLR and ALB was identified as an independent prognostic factor for patients with UTUC. The combination of NLR and ALB may help to determine the most appropriate treatment options before RNU.


Introduction
Upper tract urothelial carcinoma (UTUC), including ureter carcinoma and renal pelvicalyceal, is infrequent and only accounts for 5-10% of urothelial carcinomas. 1 Regardless of the actual site of the tumor, radical nephroureterectomy (RNU) is the first choice for the treatment of UTUC. 2 Despite the accuracy of diagnosis and the surgical resection of UTUC having been improved in the past few years, the survival outcomes of UTUC patients have not significantly improved. 3 Thus, recognizing the prognostic factors of UTUC after RNU to guide timely treatment is important. The current prognostic factors are mostly postoperative data, and preoperative prognostic factors have hardly been assessed. 4 Although pathological parameters have a strong predictive accuracy for the clinical outcome, this information cannot be obtained before surgery. Recently, a negative correlation between systemic inflammation and tumor prognosis has been reported, which can help guide the use of neoadjuvant therapy. 5 Patients routinely undergo the measurement of blood-based markers before surgery, and there is ample evidence that serum biomarkers reflect systemic inflammation, such as albumin (ALB), globulin (GLB), albumin-globulin ratio (AGR), neutrophil-to-lymphocyte ratio (NLR), platelet count (PLT), etc., which can all be used as prognostic indicators for a variety of cancers. [6][7][8][9][10] However, the preoperative blood-based markers that have prognostic value for UTUC have not yet been fully studied.
Herein, we aimed to assess the value of these bloodbased biomarkers in forecasting adverse pathological conditions and patient outcomes. Our goal was to establish a prognostic classification model that could be used before surgery.

Patients and Methods Patients
We retrospectively analyzed the data of 332 UTUC patients undergoing RNU from September 2010 to October 2019. Patients with missing data, the presence of other carcinomas, or those who had received neoadjuvant chemotherapy (NAC) or radiotherapy before surgery were excluded (n = 100). Therefore, a total of 232 patients were analyzed retrospectively. Lymphadenectomy was implemented if a suspicious lymph node status was revealed by preoperative imaging reports or positive lymph nodes were found during the operation.

Clinical and Pathologic Data
Experienced pathologists examined all surgical specimens in accordance with standard procedures. All serum biomarker levels were measured 1 day after the patients were hospitalized. Preoperative elevated NLR was defined as > 3.44, reduced ALB was defined as < 39.8 g/L, elevated GLB was defined as > 28.7 g/L, reduced AGR was defined as < 1.39 and an increased PLT was defined as > 254 � 10 9 /L. We determined the cutoff values of these serum biomarkers by using ROC curves analysis and evaluated the following clinical and pathological data to identify the relevant factors for prognostication: age at diagnosis of UTUC, sex, pathologic tumor stage (assessed by the TNM classification), 11 tumor grade (evaluated by the WHO International Society of Urological Pathology consensus classification), pathologic lymph node status (pN0, pNx or pN+), tumor size (we determined a threshold of 3.5 cm using ROC curves), tumor location (renal pelvicalyceal or ureter or both), tumor multifocality (two or more tumors were found), lymphovascular invasion (LVI) (the presence of malignant cells in the endothelial cell line), surgical margin status (the presence of malignant cells at the edge of the surgical specimen) and tumor histology (papillary tumor).

Follow-Up
Patients were usually examined at least once every 3-4 months in the first year, every 6 months in the second year, and at least once a year thereafter. The follow-up examinations included a cystoscopic examination, urine cytology, physical examination, blood tests, computed tomography (CT) or ultrasound and chest radiography. If there were clinical indications, a selective bone scan was performed. Disease progression was defined as a local recurrence, lymph node metastasis and/or distant metastasis that was not detected before the operation. The progression-free survival (PFS) was defined as the date of nephroureterectomy to the date of first progression on imaging examination, the cancer-specific survival (CSS) was defined as the date of nephroureterectomy to the date of cancer-specific mortality, and the overall survival (OS) was the date of followed-up death caused by any reason.

Statistics
All continuous variables are shown as median values and ranges. Pearson's chi-square test and Fisher's exact test were used to compare the distribution of categorical variables. Differences in variables with a continuous distribution among the three groups were tested using one-way ANOVA. Univariate and multivariate Cox regression analyses were implemented to assess the relationships among the parameters and PFS, CSS, and OS. Blood-based biomarkers and clinical/ pathological features that reached significance P < 0.05 in univariate Cox regression analysis were included in the multivariate Cox regression analysis. Kaplan-Meier analysis was used to determine the survival probability, and the Log rank test was used to compare the differences among the groups. ROC curve analysis was used to compare the prognostic values of NLR alone, ALB alone and NLR plus ALB for predicting survival outcomes. IBM SPSS Statistics version 26.0 (IBM, Armonk, New York, USA) was used to perform all statistical analyses, except for the Kaplan-Meier analysis and the comparisons of the ROC curves, which were performed using MedCalc version 19.1 (https://www.med calc.org). First, we analyzed the prognostic value of the reported predictive clinicopathological parameters for PFS, CSS and OS (Table 2). Univariate Cox regression analysis showed that NLR, ALB, LVI, pN-stage (N+) and positive surgical margin were associated with a shorter PFS, CSS, and OS (all P < 0.05). Although globulin (GLB), albumin-globulin ratio (AGR), platelet count (PLT), pT-stage (> T2), present papillary and tumor grade were related to a shorter CSS and OS, they were not associated with a shorter PFS. Blood-based biomarkers and clinical/pathological features that reached significance P < 0.05 for PFS, CSS, and OS by univariate Cox regression analysis were included in the multivariate Cox regression analysis. Multivariate Cox analysis revealed that elevated preoperative NLR, decreased ALB, and positive surgical margins were independent clinical risk predictors for shorter PFS, CSS and OS (all P < 0.05) (Table 3).

Results
Second, we performed Kaplan-Meier analysis to assess the risk classification of preoperative NLR and ALB. Patients with an elevated preoperative NLR had significantly worse PFS, CSS and OS (all P < 0.0001) ( Figure 1). The patients with an ALB ≥ 39.8 g/L had significantly better PFS, CSS and OS (all P < 0.001) than those with an ALB < 39.8 g/L ( Figure 2). ROC curve analysis showed that both NLR and ALB had diagnostic value for PFS, CSS, and OS (all P < 0.05) (Figures 3 and 4). These results indicated that NLR and ALB are useful predictors of PFS, CSS, and OS in UTUC patients after RNU.

Discussion
In the present study, we have demonstrated that elevated preoperative NLR and decreased ALB are independent prognostic factors for patients with UTUC and can be used as predictors of patient outcomes before surgery. We also proved that the risk stratification  based on preoperative NLR and ALB can accurately predict patient outcomes. As far as we know, this is the first proposal to use the combined preoperative NLR and ALB to classify risk groups among UTUC patients.
Currently, the main restriction of treatment plans for UTUC patients is the difficulty in achieving accurate staging before surgery. The tissue obtained by preoperative endoscopic biopsies can be used to diagnose and assess the tumor    Due to limited studies on UTUC, the NAC for UTUC is derived from that used to treat bladder cancer. 13 Compared to immediate cystectomy in muscle-invasive bladder cancer, platinum-based neoadjuvant therapies have shown significant survival benefits. 14 Because RNU will cause nephron loss, patients may have difficulty with cisplatin-based adjuvant chemotherapy after surgery. Therefore, NAC may be more suitable for high-risk patients with UTUC than adjuvant chemotherapy. Several retrospective studies have evaluated the use of NAC to improve the survival of patients with UTUC. 15,16 Ongoing Phase II and Phase III clinical trials of NAC and adjuvant therapy applied to patients with UTUC will provide sounder guidance.
Meanwhile, it is important to determine the most suitable patients for NAC to increase the pathological downgrading rate and to improve survival outcomes. 17  If the patient's tumor stage decreases after receiving NAC, the treatment plan may be re-evaluated according to the tumor stage. Therefore, it is important to identify risk stratification factors for UTUC patients before surgery. Several prognostic models have been reported, including the use of clinical and pathological variables to predict survival outcomes after RNU, but none have been validated externally. 18 The limitation of these models is that they mainly include postoperative variables, and therefore cannot be used for preoperative risk stratification. It has been reported that some preoperative biomarkers, including molecular markers assessed by immunohistochemistry on preoperative tumor biopsy tissue, can increase the accuracy of prognostication of UTUC. 19 However, these molecular markers require special tests to be performed and they require a certain amount of intact tissue for evaluation.
There is increasing evidence that preoperative bloodbased biomarkers can serve as prognostic indicators for UTUC. 20,21 The European Association of Urology guidelines updated in 2017 has suggested preoperative NLR as a prognostic factor for CSS in UTUC. 3 Patients with UTUC routinely undergo laboratory tests, including blood tests and biochemical tests, before surgery. Therefore, using these laboratory parameters in a prognostic model could provide potentially prognostic information without increasing costs. In this research, we retrospectively analyzed data from 232 UTUC patients who underwent RNU to explore the prognostic significance of preoperative serum biomarkers: NLR, ALB, AGR and PLT. Although univariate Cox regression analysis showed that GLB, AGR and PLT were associated with CSS and OS, NLR and ALB were the most significant and were also related to PFS. Moreover, according to the results of the Kaplan-Meier and ROC curve  analyses, preoperative NLR and ALB are significant predictors of PFS, CSS, and OS in UTUC patients after RNU. Thus, we developed a preoperative risk classification model for patients with UTUC using preoperative NLR and ALB.
Kaplan-Meier analysis showed that the high-risk group had significantly worse PFS, CSS and OS than the other groups. The ROC curve analysis also showed that NLR plus ALB had a greater diagnostic value than NLR or ALB alone regarding CSS and OS. Considering the differences in 5-year survival rates among the different groups, the treatment options for patients in these different risk groups should be individualized. For the low-risk group, since the patients have a 5-year survival rate greater than 90%, adjuvant chemotherapy may not be needed to avoid overtreatment. For the high-risk group, these patients may have higher staging and poor survival outcomes, so they may benefit more from NAC. 17 For the intermediate-risk group, the treatment options should be considered comprehensively. Therefore, this preoperative risk classification model using NLR and ALB will be a valuable tool for predicting survival outcomes and for guiding treatment options in UTUC patients.
In this study, the results raised the question of how preoperative elevated NLR and decreased ALB are related to the survival outcomes of UTUC after RNU. There is increasing evidence that chronic inflammation may be associated with the occurrence and progression of malignant tumors through multiple inflammatory signaling pathways. 22 However, the best biomarkers have not yet been found. The NLR has been considered as an accurate, simple, cheap, standardized and widely available index of systemic inflammation, and it is associated with a worse prognosis of several types of cancers, including UTUC. 23 An elevated NLR may imply an increase in tumorassociated neutrophils and/or a decrease in lymphocytes, which reflects the imbalance of tumor immunity and inflammation. 24 Studies have shown that tumorassociated neutrophils have a pivotal role in the tumor microenvironment, promoting extracellular matrix modification and further activation of epithelial-mesenchymal transformation. 25,26 Moreover, lymphopenia has been reported as an independent predictor of poor survival in several carcinomas, and the increase of tumor-infiltrating lymphocytes is related to cytotoxic treatment and an improved prognosis of cancer patients. 27 ALB is mainly manufactured by the liver and is not only considered as a nutritional marker, is but also used to assess the inflammatory status of patients. 28 Low levels of albumin, also called hypoalbuminemia, might due to the increases of catabolism, inflammatory responses and chronic malnutrition. 29 A deficiency of nutritional status is often correlated with impaired immune responses, including a decrease in cell-mediated immunity and reduced complement system components. Therefore, a few studies have suggested that low serum albumin independently predicts poor oncology outcomes for UTUC after RNU. 30 It is reasonable to believe that systemic inflammation has a pivotal role in the survival outcomes of UTUC patients after RNU. Although there is increasing evidence of an association between systemic inflammation and poor oncological outcomes in patients with various malignancies, the underlying mechanisms of these phenomena have not been fully elucidated. One possible explanation is that the tumor inflammatory microenvironment may be critical for cancer development and progression. The inflammatory tumor microenvironment enables tumor cells to avoid immune responses, facilitates the production of cytokines, and promotes tumor development and metastasis. Inflammation is also thought to be a pivotal event in the early development of cancer. 31 Both NLR and ALB are good indicators of inflammation, and the combination of NLR and ALB can more comprehensively reflect the inflammatory status of UTUC patients, which helps us to judge the prognosis of patients more accurately.
The present study also had several limitations. First, it is a retrospective study with a limited number of cases from a single center, which may cause various biases. Second, the operating techniques (open vs laparoscopic surgery vs robotic assisted laparoscopic surgery, lymphadenectomy or not) differed among the patients. Third, other inflammatory factors were not calculated, including the levels of CRP, fibrinogen, and inflammatory cytokines.

Conclusions
Elevated preoperative NLR and decreased ALB were identified as independent prognostic factors of oncological outcomes in UTUC patients who underwent RNU. The addition of ALB improved the prognostic significance of NLR alone. Thus, we recommend a sample risk classification model based on NLR and ALB, which may be routinely used in clinical practice to provide an objective preoperative prognostic assessment. Our research requires more validation, such as additional data from multicenter and prospective studies.

Data Sharing Statement
The datasets analyzed during the current study are not publicly available as the Ethics Committee of Nanjing Drum Tower Hospital does not have permission to provide the data of individual participants, but they are available from the corresponding author on reasonable request.

Ethics Approval and Consent to Participate
The present study was approved by the Ethics Committee of Nanjing Drum Tower Hospital in accordance with the Declaration of Helsinki. As the present study collected only retrospective clinical data and offered no risk to the participants, written informed consent was not required.