The value of preoperative neutrophil‐to‐lymphocyte ratio, platelet‐to‐lymphocyte ratio, and red blood cell distribution width in predicting positive surgical margin after laparoscopic radical prostatectomy

Abstract Background Prostate cancer (PCa) is one of the most common malignant tumors in men, and laparoscopic radical prostatectomy (LRP) is commonly used to treat localized and advanced PCa. Positive surgical margin (PSM) is one of the most frequent problems faced by surgeons. Aims This study aimed to explore the value of the neutrophil‐to‐lymphocyte ratio (NLR), platelet‐to‐lymphocyte ratio (PLR), and red blood cell distribution width (RDW) in predicting PSM after LRP. Methods and Results Three hundred and twenty patients with PCa were admitted and underwent LRP in Beijing Chaoyang Hospital from January 2017 to June 2023. Patients were randomly divided into a training set (225 cases) and a validation set (95 cases) in a 7:3 ratio. NLR, PLR, and RDW were significantly higher in the PSM group than in the negative surgical margins (NSM) group. In addition, the NLR, PLR, and RDW values correlated with clinical T stage, Gleason score, and seminal vesicle invasion in the PSM group. In training set, ROC curve analysis revealed that the optimal cutoff values of NLR, PLR, and RDW for predicting postoperative PSM in PCa were 2.31, 115.40, and 12.85%, respectively. Multivariate Logistic regression analysis showed NLR and RDW were the clinical independent predictors. The area under the curve (AUC, 0.770, 95% CI 0.709–0.831) for postoperative PSM was the highest when a combination of the three parameters was used, with sensitivity and specificity of 62.5% and 85.2%, respectively. In validation set, the AUC values for NLR, PLR, RDW and the three markers combined were 0.708, 0.675, 0.723, and 0.780, respectively. Correlation analysis showed that in the PSM group, NLR was positively correlated with PLR and RDW, and PLR was positively correlated with RDW. By contrast, in the NSM group, a positive association was only found between NLR and PLR. Conclusions Higher preoperative NLR, PLR, and RDW values were associated with postoperative PSM. Additionally, the three markers combined may be useful to predict PSM.

curve (AUC, 0.770, 95% CI 0.709-0.831)for postoperative PSM was the highest when a combination of the three parameters was used, with sensitivity and specificity of 62.5% and 85.2%, respectively.In validation set, the AUC values for NLR, PLR, RDW and the three markers combined were 0.708, 0.675, 0.723, and 0.780, respectively.Correlation analysis showed that in the PSM group, NLR was positively correlated with PLR and RDW, and PLR was positively correlated with RDW.By contrast, in the NSM group, a positive association was only found between NLR and PLR.

| INTRODUCTION
Prostate cancer (PCa) is one of the most common malignant tumors of the urogenital system in middle-aged and elderly men, posing a serious risk to psychological and physical well-being. 1 For patients with localized and advanced PCa, radical prostatectomy (RP)-either laparoscopic (LRP) or robot-assisted (RARP)-is one of the most established therapies, yielding a longer life expectancy. 2Positive surgical margin (PSM) is one of the most frequent problems faced by surgeons.
PSM depends on various factors, including prostate anatomy, tumor features (size, stage, and localization), and the surgical technique used. 3PSM is considered an adverse pathological feature that may lead to biochemical recurrence (BCR), metastasis, and cancer-specific death, exposing patients to the risk of further treatments, such as adjuvant or salvage radiotherapy with or without androgen blockade. 4,5Zhang et al. demonstrated that PSM is significantly associated with an increased risk of BCR and may serve as an independent prognostic factor in PCa patients. 6Based on the worse oncologic outcomes associated with PSM, preoperative judgment of the surgical margin is important to reduce PSM rates.[9] Cancer is widely thought to be caused by chronic inflammation. 10veral inflammatory markers, such as neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR), comprehensively reflect inflammation and immune status in patients with various cancers. 11,12e impact of NLR, PLR and other blood-derived metrics has been extensively studied across urological malignancies from the prognostic point of view. 13,14Another parameter, red blood cell distribution width (RDW), reflects the heterogeneity of peripheral blood erythrocyte volume and is primarily used to diagnose various clinical anemias.
RDW is also linked to long-term inflammation and poor health, while abnormal inflammation and nutritional status may be risk factors for carcinoma development. 15The higher the tumor stage, the more local or systemic inflammatory responses are promoted, leading to increased RDW values. 16Albayrak et al. showed that RDW was increased in PCa patients and was significantly associated with disease progression. 17 date, the predictive value of preoperative NLR, PLR, and RDW levels for postoperative PSM in PCa patients has not been assessed.
To address this research gap, in this study, we collected clinical and pathological characteristics as well as preoperative NLR, PLR, and RDW values for PCa patients who underwent LRP.We investigated the capacity of these parameters to predict PSM, with the aim of providing information that could be of potential benefit to urological surgeons carrying out this procedure in the future.

| Patients
We conducted a retrospective study on PCa patients who underwent The study was conducted in accordance with the Declaration of Helsinki and was approved by the ethics committee of Beijing Chaoyang Hospital, China.

| Data collection
Baseline characteristics and clinicopathological data were obtained from the medical records database.Clinical data mainly included age, weight, preoperative maximum serum prostate-specific antigen (PSA), prostate volume (PV), and clinical stage.Body mass index (BMI) values were classified by applying the World Health Organization criteria.PV was obtained by preoperative ultrasound or magnetic resonance imaging (MRI).f/t PSA was calculated as the ratio of free PSA to total PSA, and PSA density (PSAD) was determined by calculating the ratio of PSA to prostate volume.Clinical stage was derived based on the evaluation of the patients' clinical data, using the 8th edition of the American Joint Committee on Cancer staging system.Preoperative Gleason score (GS) and biopsy positive core (BPC) ratio were derived from preoperative biopsy pathology.Using the D'Amico classification, low-risk PCa was defined as PSA <10.0 ng/mL, GS <7, and clinical stage T1c to T2a at initial 10-core biopsy.Intermediate-risk PCa was defined as PSA ≥10 ng/mL and <20 ng/mL, GS of 7, and clinical stage T2b.High-risk PCa patients were PSA >20.0 ng/mL, GS >7, and clinical stage ≥ T2c.
The involvement of surgical margin positivity, seminal vesicle invasion, capsule invasion, and lymph nodes was determined by postoperative pathology.PSM was confirmed if, on evaluation of postoperative pathology by at least two pathologists, tumor cells were visible microscopically at the surgical margins of tumors.Venous blood (2 mL) was collected from patients within a week before surgery (in the morning) and placed in EDTA anticoagulation tubes and drying tubes.The samples were sent to the laboratory department where a hematology analyzer was used to obtain absolute neutrophil counts, absolute lymphocyte counts, blood platelet counts, and RDW (directly).NLR and PLR values were obtained according to the following formulae: NLR = absolute neutrophil count/lymphocyte count; PLR = blood platelet count/lymphocyte count.

| Statistical analysis
Statistical analysis was performed using SPSS 26.0 (IBM Corp., Armonk, NY) and GraphPad Prism 9.5 (GraphPad Software, San Diego, CA).The independent t-test and χ 2 test were used to compare between-group differences.Multivariate Logistic regression analysis was used to determine the clinical independent predictors.Area under the curve (AUC), used as a summary measure of the receiver operating characteristics (ROC) curve, represents discrimination ability.AUC is expressed on a scale of 0.5 to 1 (the larger the AUC value, the better the classification effect).Sensitivity and specificity were defined using ROC curves, and differences in AUC values were analyzed using GraphPad Prism 9.5.Correlations among NLR, PLR, and RDW were analyzed using Spearman's correlation: r >0 indicates a positive correlation between two variables, while r < 0 indicates a negative correlation.0 < r < 1 indicates a certain degree of linear correlation between the two variables, as follows: r < 0.4 is a low linear correlation, 0.4 ≤ r < 0.7 is a significant correlation, and 0.7 ≤ r < 1 is a high linear correlation.Results were reported as numbers (n) and percentages (%), means and standard deviations, or AUC with 95% confidence intervals (CI), as appropriate, and were considered statistically significant at a p-value <.05 in two-tailed tests.1, there is no significant difference between training and validation sets.In Table 2, we performed univariate analysis of risk factors for surgical margins between NSM and PSM groups in training set.No significant differences in age, BMI, prostate volume, preoperative maximum PSA, f/t PSA, or PSAD were noted between the two groups (P > .05).The proportions of patients with low, intermediate, and high D'Amico risk classification in the NSM group were 23.5%, 35.8%, and 40.7%, respectively, while they were 11.1%, 30.6%, and 58.3% in the PSM group; these differences were significant (P = .014).Among the perioperative pathology variables, in the PSM group, BPC ratio (0.51 ± 0.28 vs. 0.41 ± 0.25, P = .008),biopsy GS (≤6, 7, ≥ 8: 16.7%, 34.7%, 48.6% vs. 29.6%,38.2%, 32.2%, P = .023),and clinical T stage (T2, T3 $ 4: 65.4%, 34.6% vs. 54.9%,45.1%, P = .009)were significantly higher than in the NSM group.As shown in Table 2, in terms of postoperative characteristics, there were significant differences in pathological GS (≤6, 7, ≥ 8: 14.8%, 58.0%, 27.2% vs. 7.6%, 50.7%, 41.7%, P = .047),seminal vesicle invasion (16.0% vs. 30.6%,P = .016),and pelvic lymph node involvement (3.7% vs. 10.4%,P = .048)between patients in the two groups.However, there was no significant difference in prostate capsule invasion (18.5% vs. 17.4%,P = .827).In addition, we performed the same analysis on validation set, and the results were similar (Supplementary Table S1).

| Correlation between NLR, PLR, and RDW values in the PSM and NSM groups
Correlation analysis showed that in the PSM group, NLR was positively correlated with PLR (r = 0.494, P < .001)and RDW (r = 0.315, P < .001),and RDW was also positively correlated with PLR (r = 0.271, P = .001)(Figure 2A-C).Comparison of the correlation coefficients showed that the correlation between NLR and PLR was stronger than the correlation between RDW and either NLR or PLR.
We also assessed the relationship between NLR, PLR, and RDW in the NSM group.The results showed that there was no significant correlation between RDW and either NLR (r = À0.122,P > .05)or PLR  (r = À0.121,P > .05),although we did find a significant correlation between NLR and PLR (r = 0.542, P < .001)(Figure 2D-F).

| DISCUSSION
RP is the standard first-line treatment modality for patients with localized PCa, particularly in intermediate and high-risk patients.In our study, the incidence of PSM was 64%, which is considerably higher than the previously reported figures of 10% to 35% among patients who have undergone RP. 4,18 This higher incidence may be related to the higher proportion of intermediate and high-risk patients in our study (264/320, 82.50%).0][21][22] Consistent with these findings, the results of the current study revealed that the PSM and NSM groups showed significant differences in BPC ratio, clinical T stage, D'Amico classification, GS, seminal vesicle invasion, and pelvic lymph node positivity.However, there were no significant differences in capsule invasion between the two groups, which could possibly be attributed to sample size limitations, as well as individual differences.
In our study, NLR, PLR, and RDW were significantly higher in the PSM group than in the NSM group and NLR and RDW were the independent predictors for PSM.Systemic inflammation is linked to tumor development and patient outcomes and is thought to be a feature of tumorigenesis and progression. 23,24Inflammation is also associated with obesity and metabolic diseases. 25The majority of patients in this study were overweight (217/320, 67.8%), which indicates these patients may have been in a state of chronic inflammation.
Inflammation can promote the migration of neutrophils to peritumor tissues, which then release reactive oxygen species, causing oxidative damage to DNA cells, and secrete large amounts of vascular endothelial growth factors.7][28] Tumor cells also produce cancer-associated inflammatory factors that promote neutrophil expansion. 29Platelet abnormalities and malignancy have been linked in both basic and clinical studies, while reactive thrombocytosis is common in solid tumors. 30,31Platelet activation promotes tumor angiogenesis, extracellular matrix degradation, and the release of adhesion molecules and growth factors and boosts tumor growth and metastasis. 32,33In addition, lymphocytes play an important role in tumor cell destruction and apoptosis and can activate anti-tumor immune factors directly or indirectly, thus inhibiting tumor metastasis and recurrence.Mantovani demonstrated that large numbers of neutrophils suppress the activation and antitumor activity of lymphocytes and natural killer cells. 34Furthermore, dependent on patient age and nutritional status, along with tumor progression, the immune system can become suppressed or weakened, resulting in reduced lymphocyte numbers.In summary, the inflammatory response is characterized by increased neutrophil levels and decreased lymphocyte levels.Thus, NLR and PLR reflect the level of systemic inflammation and the balance of the immune response, serving as potential biomarkers to characterize individual tumors in terms of angiogenesis, progression, and metastasis.In addition, RDW (which can be determined directly from blood tests) reflects the heterogeneity of red cell volume.Inflammatory cytokines have been shown to inhibit the stimulatory effects of erythropoietin on bone marrow erythrocyte stem cells, including the induction of cell maturation and suppression of apoptosis.Thus, inflammation causes more immature red blood cells to be released into the peripheral blood circulation, increasing the heterogeneity of peripheral red blood cells and leading to higher RDW values. 35tients with advanced cancers, including PCa, are also often in a state of malnutrition, which may result in deficiencies in iron, vitamin B12, and folic acid, and varying degrees of anemia, as well as increased RDW. 16bgroup analysis within the PSM group showed that increased NLR, PLR, and RDW were associated with clinical T stage, biopsy and pathological GS, capsule infiltration, and seminal vesicle invasion.The reason for these findings may be that patients with advanced carcinoma have faster disease progression and exhibit stronger inflammatory responses within the tumor tissue, resulting in abnormally high NLR, PLR, and RDW values.Our results are consistent with those of Rulando et al., 36 who showed that NLR was associated with higher GS (r = 0.572, P = .001).Gokce et al. also reported that high NLR was associated with higher GS, higher progression rates, and poorer prognosis. 37For PLR, Neofytou et al. showed that elevated PLR was related to poor tumor stage, pathological T stage, and degree of differentiation. 38Meanwhile, Huang et al. demonstrated that RDW was an independent risk factor for clinically significant PCa, 39 and Wang et al. found positive associations between tumor stage/grade and RDW, showing the utility of this parameter for predicting advanced carcinoma. 40 this study, ROC curve analysis showed that the optimal cutoff values of NLR, PLR, and RDW for predicting PSM after PCa were 2.31, 115.40, and 12.85%, respectively.Together, the three biomarkers yielded a greater AUC value, suggesting that it may be preferable to combine these indicators to improve the prediction of postoperative PSM.The result that the AUC of the three markers combined was 0.780 was validated by another group of patients, which was similar with training set.In the PSM group, NLR was positively correlated with PLR and RDW, and PLR was also positively correlated with RDW.In the NSM group, the only positive association was between NLR and PLR.It has been suggested that the higher the tumor risk, the stronger the interaction between NLR, PLR, and RDW and the higher the incidence of PSM  detecting surgical margin and pelvic lymph nodes, allowing a more accurate local staging and a prolonged biochemical RFS. 51The assessment of shaved prostate margins using fluorescence confocal microscopy (FCM) holds great potential as a valuable tool for secondary resection of spared neurovascular bundles in the case of PSM, aiming to maximize the preservation of functional tissue while pursuing oncological safety. 52The integrative approach including preoperative evaluation of clinicopathological features, intraoperative guidance of surgical margin, and postoperative follow-up can benefit patients with PCa during the process of treatment.
Although our study is the first to explore the use of inflammatory parameters to predict postoperative PSM, several limitations should be acknowledged.First, this was a retrospective and single-center study that involved a relatively small sample size.Hence, large-scale prospective studies are needed to confirm the results and exclude biases from unknown confounders.Additionally, the LRPs were not conducted by the same urologist, which may have contributed to bias and the high incidence of PSM.Moreover, NLR, PLR, and RDW could be influenced by other conditions, such as acute coronary syndromes, valvular heart diseases, and renal conditions, as well as liver diseases, inflammatory diseases, and some medications. 53However, we did not take these factors into account in this study.Finally, this study only The correlation between NLR, PLR, and RDW levels of PSM (A, B, C) and NSM (D, E, F) groups.s.
LRP at Beijing Chaoyang Hospital from January 2017 to June 2023.The inclusion criteria were as follows: (1) PCa was diagnosed by ultrasound-guided prostate biopsy and postoperative pathological results; (2) peripheral blood had been collected before LRP, with corresponding results provided; (3) patients had not received preoperative neoadjuvant endocrine therapy, radiotherapy, or chemotherapy; (4) the patients' clinicopathological data were complete, including previous hospitalization medical records, laboratory examination reports, and pathological diagnosis reports.The exclusion criteria were as follows: (1) patients suffering from other malignant tumors, acute and chronic infections, or autoimmune system diseases; (2) patients with severe heart, liver, or kidney diseases; (3) patients taking immunosuppressive drugs or suffering from hematologic diseases that might affect the results of routine blood tests.After these exclusions, 320 patients were enrolled in the study.Patients were randomly divided into a training set (225 cases) and a validation set (95 cases) in a 7∶3 ratio.Then, the patients were divided into PSM and negative surgical margin (NSM) groups according to post-operation pathology.

A
total of 320 PCa patients who had undergone LRP were enrolled in this study.In training set, patients were divided into two groups based on surgical margin status obtained from pathological results: negative surgical margin (NSM, 81/225, 36%) and PSM (144/225, 64%).The number of NSM and PSM were 35 (36.8%) and 60 (63.2%) in validation set.Comparisons of the clinical and pathological data are shown in Table

T A B L E 1
Clinical and pathological characteristics of patients received LRP between training and validation sets.

F
I G U R E 1 ROC curve of preoperative NLR, PLR, and RDW levels predicting postoperative PSM of PCa patients in training (A) and validation (B) sets.outcomes.The guidance of augmented reality (AR) has mainly been used as a navigation system intraoperatively, and studies show a potential use of AR for more accurately identifying tumor margins and accuracy of detection of capsular involvement. 50Multiple preclinical and clinical studies have shown the usefulness of indocyanine green fluorescence in identifying and guiding treatment for PCa such as Univariate analysis of risk factors for surgical margins between NSM and PSM groups in training set.
Multivariate Logistic regression analysis of independent risk factors affecting surgical margins in training set.
T A B L E 4 Relationship between NLR, PLR, RDW levels and clinicopathological characteristics in PSM group of training set.
Value of preoperative NLR, PLR, and RDW levels predicting postoperative PSM in training and validation set.Abbreviations: AUC, area under the curve; CI, confidence interval; NLR, neutrophil to lymphoctye ratio; PLR, platelte to lymphocyte ratio; PSM, positive surgical margins; RDW, red blood cell distribution width.
491][42][43][44]alues for NLR, PLR, and RDW can be obtained from routine blood tests in daily clinical practice, which is convenient, reproducible, and low cost.Numerous studies have confirmed that NLR, PLR, and RDW have better predictive value for patient prognosis in different cancers.[41][42][43][44]Forexample,Guetal.demonstrated that elevated NLR was associated with poor overall survival (OS) and progression-free survival (PFS)/ recurrence-free survival (RFS) in 16 266 patients with PCa.45Similarly, a meta-analysis conducted by Li et al. reported that in urological cancers (except for bladder cancer), elevated PLR was negatively related48Conversely, although RDW was found to be significantly related to tumor tissue size, stage, and necrosis in the study of Lee et al., no association was found with prognosis.49Basedon our results, intraoperative guidance including fluorescence and artificial intelligence can be used as an additional tool for patients with high NLR, PLR, or RDW before operation to improve T A B L E 5 *Means the P-value<.05 is considered statistically significant.