Impact of Neutrophil-Lymphocyte Ratio on Long-term Outcomes of Patients with Coronary Chronic Total Occlusion After Percutaneous Coronary Intervention

Background: The impact of preprocedural neutrophil-lymphocyte ratio (NLR) level on subsequent adverse cardiovascular events and health status in coronary chronic total occlusion (CTO) patients after stent implantation is unclear. The present study aims to evaluate the impact of NLR level on long-term clinical outcomes and health status of CTO patients after percutaneous coronary intervention (PCI) in the drug eluting stent era. Methods: A total of 311 CTO patients who underwent successful PCI with new-generation drug-eluting stent and with follow-up were enrolled. Patients were classied into 3 groups according to the tertiles of NLR level at baseline. The primary endpoint (major adverse cardiac events, MACE) was a composite of cardiac death, target-vessel myocardial infarction (MI), and ischemia-driven target-vessel revascularization (TVR) on follow-up. Multivariable COX regression analysis was performed to estimate the relationships between the parameters and MACE. Results: Compared with the low and intermediate tertile groups, preprocedural platelet-lymphocyte ratio (PLR) level and age were signicantly higher in the high NLR tertile group. After a median follow-up of 32 (interquartile range: 20 to 44) months, MACE was observed in 10 patients (9.7%) in the low tertile, in 21 (20.2%) in the intermediate tertile, and in 31 (29.8%) in the high tertile group (P = 0.001). Kaplan-Meier analysis demonstrated a signicantly higher incidence of MACE, mainly driven by TVR, in patients with high tertile than the low and intermediate tertile groups. Multivariable COX regression analysis showed NLR and occluded length as independent predictors of MACE. No statistically differences were observed regarding the ve domains of Seattle Angina Questionnaire (SAQ) scores among the 3 groups (all P > 0.05). Conclusions: In conclusion, in patients who underwent PCI for CTO lesions, elevated NLR level was independently associated with MACE (driven by TVR) on follow-up.


Background
Chronic total occlusion (CTO) in coronary arteries is considered as the nal stage of coronary atherosclerosis, and these complex lesions are identi ed in approximately 15-30% of all coronary angiography [1,2]. Percutaneous coronary intervention (PCI) for CTO lesions is one of the major technical challenges in contemporary interventional cardiology. With the remarkable developments in interventional techniques in the last few years, the procedural success rate and safety of PCI in patients with CTO has improved [3][4][5]. Furthermore, successful PCI for CTO has been shown to reduce the risk of cardiac death, post-operative myocardial infarction (MI), and improves long-term survival compared with failed CTO PCI [6][7][8]. However, even after successful revascularization, the long term clinical outcomes of CTO patients were still unsatisfactory, and it has been identi ed as an independent predictor of the need for targetvessel revascularization (TVR) and subsequent adverse cardiac outcomes after CTO PCI [9,10]. Thus, attention has shifted to underlie the pathophysiology of atherosclerosis, and the in uence of early biomarkers on CTO patient's prognosis is worthy of exploration.
In ammation is a central factor for the initiation and progression of coronary atherosclerosis [11,12].
More recently, it has been demonstrated that NLR level is associated with the prevalence and severity of CAD, and the elevated NLR predicts the risk of adverse cardiac events in acute coronary syndrome patients [14,15]. Additionally, higher baseline NLR level was risk factor for the development of in-stent restenosis in CTO patients after PCI [16].
To date, few reports have evaluated the impact of preprocedural NLR level on major adverse cardiac events (MACE) at long-term follow-up in CTO patients after stent implantation, especially in Chinese individuals. Similarly, the long-term health status quanti ed by the Seattle Angina Questionnaire (SAQ) of this patient subgroup are poorly characterized. The aim of this study was to address these important questions.

Methods
Study populations 493 patients with CTO lesions were con rmed by coronary angiography in the Cardiology Department of the First A liated Hospital of Xi'an JiaoTong University (Xi'an, Shaanxi, China) undergoing PCI between June 2013 and October 2017. The inclusion criteria for the present study were: 1) only 1 CTO lesion per patient detected on diagnostic coronary angiography; 2) symptomatic angina and/or functional ischemia. Exclusion criteria were: 1) patients with signs of in ammation; 2) history of cardiogenic shock or cardiopulmonary resuscitation; 3) a self-identi ed history of stroke, renal failure or severe arrhythmia; 4) also had a malignant tumor; 5)previous coronary artery bypass graft surgery. Of these 493 patients, 182 were excluded because of unsuccessful PCI operation (n = 117), missing laboratory values (n = 27) or clinical follow-up data (n = 38), resulting in a study population of 311 patients who underwent successful PCI and follow-up. They were classi ed into 3 groups according to tertiles of NLR level. The periodic follow-up for adverse events were carried out via telephone contacts or outpatient visit every year between 2014 and 2018. The study was approved by both Research and Ethics Committees of the First A liated Hospital of Xi'an JiaoTong University.

De nitions
A "CTO lesion" was de ned as 100% luminal obstruction with an estimated duration of occlusion of at least 3 months with no antegrade ow through the lesion (TIMI 0 ow) [1,4]. The estimated duration of occlusion was based on the rst onset of angina, previous history of myocardial infarction (MI) in the target vascular region or proven by prior angiographic results. Technical success was de ned as residual stenosis of less than 30% with antegrade ow TIMI 3. Procedural success was de ned as technical success plus no in-hospital adverse cardiac effects, including MI, all-cause mortality and recurrence of cardiac symptoms requiring repeat target vessel PCI [4,10]. The clinical endpoint of MACE on follow-up were de ned as a composite of target-vessel MI, cardiac death, and ischemia-driven TVR in the treated CTO vessel [6,10]. All deaths were considered to have been from cardiac causes unless a explicit noncardiac cause could be documented. According to the fourth universal de nition of MI criteria [17], MI was de ned as an increase in creatine kinase-MB fraction or troponin-T/troponin-I to > 3 times the upper limit of reference value with ischemic symptoms or electrocardiographic ndings indicative of ischemia.

Seattle Angina Questionnaire
A total of 287 patient' views on their health status have been quanti ed by the SAQ Scores at the last follow-up. As shown in Table 6, no statistically differences were observed regarding physical limitation, angina frequency, angina stability, treatment satisfaction and quality of life among the 3 groups (all P > 0.05). Pearson correlation analysis indicated that there were no signi cant relationships between NLR level and SAQ scores in the ve domains (Data not shown).

Measurements Of Related Parameters
We collected several demographics, clinical and analytical parameters. Age, gender, height and weight were recorded. Patients either with persistent blood pressure > 140/90 mmHg or those currently taking antihypertensive drugs were considered hypertensive. Several biochemical parameters, including serum uric acid, lipid pro les, plasma homocysteine, hs-CRP and estimated glomerular ltration rate (eGFR) were measured. Total white blood cells, neutrophil, lymphocyte, and platelet counts, RDW, MPV were calculated using an automated blood cell counter (Model XE2100; Sysmex, Kobe, Japan). The NLR was computed using the absolute neutrophil count divided by the lymphocyte count, the PLR was calculated by dividing the platelet count by the absolute lymphocyte count [16].

Statistical analysis
Data are presented as means ± standard deviation or median and interquartile range for continuous variables and percentages for categorical variables. Continuous variables that had a normal distribution were evaluated using analysis of variance (ANOVA), whereas the Kruskal-Wallis test was used for nonnormally distributed data. Categorical variables and frequencies were compared with the chi-squared test.
Pearson correlation analysis was used to analyze the relationship between NLR and SAQ scores. The clinical outcomes occurring over time for MACE were described by Kaplan-Meier survival curves and compared by the log-rank test. Univariable COX regression analysis was used to explore the association between the variables and MACE, hypertension, diabetes mellitus and variables with P < 0.10 were included in the multivariable COX regression analysis. Strengths of associations were determined by estimating the hazard ratios (HRs) and their 95% con dence intervals (95% CIs). The predictive values of NLR in MACE was tested using receiver operating characteristic (ROC) analysis. In all cases, P < 0.05 was considered signi cant. All statistical analysis was performed retrospectively with SPSS 25.0 (SPSS, Inc., Chicago, IL) and R 3.1.0 (R Foundation for Statistical Computing).

Study Population and Baseline Characteristics
A total of 311 patients with CTO (mean age: 67.6 ± 9.9 years; 82.3% men) were divided into tertiles according to baseline NLR level. The tertiles of NLR level were as follows: low tertile, ≤ 2.0; intermediate tertile, 2.1 to 3.2; and high tertile, ≥ 3.2. Baseline clinical characteristics and preoperative laboratory results of all subjects are summarized in Table 1 and Table 2. Compared with patients in the low and intermediate tertile groups, preprocedural PLR level and age were signi cantly higher in the high tertile group. However, patients with high tertile of NLR level had a lower left ventricular ejection fraction than other patients at admission. There were no statistically signi cant differences in the oral administration of cardiovascular drugs, white blood cell count, lipid pro les, eGFR and hs-CRP level among the 3 groups (all P > 0.05). Table 3 presents the angiographic and procedural characteristics of all CTO patients who underwent successful PCI and follow-up. No differences were observed regarding number of diseased vessels, the target CTO vessel, collateral ow, occluded length and the severity of calci cation. All patients in the present study received a second generation drug-eluting stents, there were no signi cant differences in stent parameters among these groups. Data are presented as n (%), mean ± standard deviation.

Discussion
The study was designed to evaluate the impact of NLR level on long-term clinical outcomes de ned as MACE and health status of CTO patients after PCI in the drug eluting stent era. The ndings of the study were as follows: 1) patients in the high tertile group (NLR > 3.2) had a higher rate of MACE than those in the low tertile and the intermediate tertile group at long period of follow-up; 2) elevated preprocedural NLR level was independently associated with MACE, mainly driven by TVR, on follow-up; 3) There were no differences in the long-term health status quanti ed by SAQ scores of CTO patients regardless of the NLR level.
Prior to the advancement of technique and devices for CTO PCI, CTO lesion is still a challenging subset for successful CTO revascularization. PCI for CTO patients was associated with lower procedural success rates and higher risk of operative complications than those with less complex lesions [19]. With the improvement of technique and equipment for CTO revascularization, such as the retrograde approach, CrossBoss catheter and dissection and re-entry techniques, success rates of CTO PCI have remarkably increased [3,5,20]. Moreover, new anti-ischemic drugs, drug-eluting stents, potent antiplatelet agents, and high-dose statins have contributed to reduce the progression of cardiovascular disease and atherosclerosis. However, CTO and in-stent restenosis have been shown to be a worsening clinical condition [9,10,21]. The successful treatment of CTO lesion was a strong predictor of subsequent adverse events, including cardiac death, ischemia or symptoms-driven TVR, and CTO PCI was not associated with a reduction in long-term MACE [10,21]. The pathophysiological mechanism of such recurrent events after PCI for CTO is largely unknown. Therefore, attention has shifted to underlie the pathophysiology of atherosclerosis, and there is a need to identify new markers to accurately predict the prognosis of CTO patients and allow better risk strati cation.
Indeed, in ammation plays an indispensable role in the pathogenesis of atherosclerosis, and an elevation in in ammatory biomarkers represents the activation of coronary artery damage [14]. Mounting evidence suggests that in ammation, including pre-existing systemic in ammation, local vascular in ammation secondary to PCI, plays a central role in promoting neointimal growth and in-stent restenosis [16,22].
After stenting for CTO lesions, platelet and brin are deposited around the stents. An acute in ammation response secondary to mechanical injury in icted by revascularization leads to endothelial rupture and dysfunction, plaque rupture and platelet activation [16]. And this initial acute in ammation is followed by granulation tissue reactions, including migration and proliferation of smooth muscle cells, neovascularization and chronic in ammatory cell in ltration occurs within a few weeks, which causes neointimal hyperplasia, atherosclerosis and in-stent restenosis [16,22,23]. Because in ammation plays an important role in the atherosclerosis process, there is increasing interest in various in ammatory biomarkers. In ammation has been associated with many diseases, such as acute myocardial infarction, chronic heart failure, metabolic disorders, cancer and other cardiovascular disease [12,24,25]. Currently, many in ammatory biomarkers have been used to investigate the relationship between in ammatory response and CAD, including white blood cell count, hs-CRP, PLR, NLR, MPV and interleukin-1 [12][13][14]16]. Furthermore, unlike several comorbidities and cardiovascular risk factors used by the Framingham Coronary Heart Disease Risk Score and the Atherosclerotic Cardiovascular Disease Risk Algorithm [26], some researchers have studied potential novel risk scores that also include in ammatory markers, such as the PLR and NLR values, which have also provided useful information for predicting cardiovascular events in patients with CAD [16,27,28].
Recently, NLR has been considered as a potential marker of in ammation in cardiovascular diseases. Neutrophils secrete a variety of in ammatory mediators, which can cause endothelial injury and vascular wall degeneration. Lymphocytes have an anti-atherosclerotic role and regulate the in ammatory response. Consequently, a high NLR represents two immune pathways, making it more predictive than the method used to evaluate either parameter individually [23,29]. Several studies have shown that the white blood cell count and its subtypes, neutrophils and lymphocytes, were associated with the prevalence and severity of CAD and its prognosis [14,15,28,30]. Meta-analysis shows that NLR was an independent predictor of hospitalization, long-term cardiovascular events and cardiac mortality in patients with STsegment elevation MI after PCI [31]. Arbel Y et al. reported that in patients with ST-segment elevation MI, higher NLR was independently associated with lower left ventricular ejection fraction and higher all-cause mortality up to 5 years [30]. Therefore, NLR appears to be an effective marker of systemic in ammation and was considered to be a predictor for worse clinical outcomes under different conditions, including acute coronary syndrome, ST-segment elevation MI and in-stent restenosis.
However, few reports have evaluated the impact of preprocedural NLR level on subsequent adverse cardiovascular events at long-term follow-up in CTO patients after stent implantation. Elevated NLR level was reportedly associated with the impaired development of coronary collaterals in CTO patients [32]. Li C et al. observed that preprocedural NLR level was a powerful risk factor for early in-stent restenosis in patients who underwent successful PCI for CTO lesions [16]. To our knowledge, only one study showed that NLR △ , de ned as the change of NLR level between on admission and post-PCI, was independently associated with higher risks of in-stent restenosis and MACE in 160 CTO patients at 1-year follow-up [33]. However, the NLR △ level may be involved in vascular mechanical injury secondary to PCI. After a median follow-up of 32 months, we found that in CTO patients who underwent successful PCI, the incidence of MACE in high NLR tertile group was signi cantly higher than those in the other groups, and elevated preprocedural NLR level was independent predictor for long-term MACE in patients with CTO lesions.  [37]. In our study, we rst found that there were no signi cant differences regarding physical limitation, angina frequency, angina stability, treatment satisfaction and quality of life among the 3 groups, and the preprocedural NLR level was not associated with SAQ scores in the ve domains. Therefore, the preprocedural NLR level had no effect on the long-term health status quanti ed by the SAQ scores of CTO patients after PCI.
Our study has several limitations. First of all, it shares all the limitations of observational, single center studies. Second, although there were no statistically signi cant differences in most potential confounding factors among the 3 groups, we were unable to correct for unmeasured potential variables. Third, this was a retrospective study unpowered to identify all changes to patients' medical treatment strategies during follow-up. Finally, the sample size was limited, so this study was considerably underpowered, and our results might not be generalizable to other studies.

Conclusions
In conclusion, the present study suggests that NLR value appears additive to conventional cardiovascular risk factors and commonly used biomarkers in predicting MACE after PCI for CTO. A collaborative, largersample and multicenter study is needed to investigate the potential predictive value of baseline and postoperative in ammatory biomarker levels for long-term outcomes in patients with CTO lesions after PCI. Kaplan-Meier curves for MACE according to tertiles of NLR level Kaplan-Meier curves describing the risk of MACE according to tertiles of baseline NLR level. MACE: major adverse cardiac events; NLR, neutrophillymphocyte ratio.