Prognostic signicance of platelet-lymphocyte ratio in extrapulmonary neuroendocrine carcinoma

Background Extrapulmonary neuroendocrine carcinoma (EP-NEC) is an aggressive type of cancer with poor prognosis. Although several biological and histological markers are prognostic factors in NEC, the correlation between prognosis and systemic inammation markers, such as the neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR), is unclear. This study evaluated the association between NLR or PLR and median overall survival (OS) or progression-free survival (PFS) in EP-NEC. response; stable


Background
Neuroendocrine carcinoma (NEC) is an aggressive type of cancer that can occur in various organs despite its rare incidence. More than 80% of NEC patients have metastasis at diagnosis and poor survival prognosis [1]. When determined according to the stage at diagnosis in one study, the median survival of localized NEC patients was 20.7 months, but that of patients with distant disease was 5.8 months.
Except for NECs of the small intestine (18.8 months) and anal canal (11.1 months), most NECs had a median survival of less than 6 months [2].
Several studies have reported that high NLR and high PLR are related to poor prognosis in various solid tumors, including colorectal cancer, gastric cancer, non-small-cell lung cancer, and pancreatic cancer [3][4][5][6][7][8][9][10]. In small-cell lung cancer, which is histologically similar to extrapulmonary NEC (EP-NEC), the high NLR group showed poor overall survival (OS) and progression-free survival (PFS) [11]. Thus, we can assume that the prognosis of EP-NEC would be associated with NLR and PLR. However, to date, it is unclear whether NLR or PLR is associated with the prognosis of EP-NEC patients who receive systemic chemotherapy. Therefore, we evaluated the prognostic signi cance of NLR and PLR in patients with EP-NEC.
Materials And Methods

Study subjects and data collection
This study included patients diagnosed with unresectable or metastatic EP-NEC according to the 2017 WHO classi cation from August 2007 to March 2019 at Chonnam National University Hwasun Hospital. We retrospectively reviewed the clinical information and laboratory and radiologic follow-up data from the hospital's electronic medical records. All patients were older than 20 years and received systemic chemotherapies. The peripheral blood samples were tested at diagnosis or before the rst chemotherapy.
NLR was de ned as the ratio of the absolute neutrophil count divided by the absolute lymphocyte count, and PLR was de ned as the ratio of the absolute platelet count divided by the absolute lymphocyte count. PFS was de ned as the time from initiation of chemotherapy to disease progression or death from any cause before disease progression. The OS was calculated as the time from diagnosis to death from any cause.

Statistical analysis
Continuous variables are presented as medians and ranges. The categorical variables are presented as the numbers of patients and percentages. The cut-off value for lactate dehydrogenase (LDH) was 1.5 times the upper limit of normal (ULN), and those for NLR and PLR were 3 [12] and 160 [11], respectively, as determined by reference to previous studies. The low-and high-NLR groups and the low-and high-PLR groups were compared using the Mann-Whitney U test (age and laboratory data), chi-square test (disease control after chemotherapy and second-line chemotherapy data), Fisher's exact test (sex, primary origin, and nal stage before chemotherapy data), or linear by linear association (ECOG performance status, and best response to initial chemotherapy data). Survival analyses and comparisons were performed using the Kaplan-Meier method and log-rank test, respectively. Multivariate analysis of survival was performed according to LDH and PLR data using the Cox proportional hazards model, and hazard ratios and 95% con dence intervals (CI) were calculated. SPSS 21.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. A P value of < 0.05 was considered statistically signi cant.

Ethics statement
The protocol was approved by the Institutional Review Board at Chonnam National University Hwasun Hospital (CNUHH-2020-019). The trial was conducted in accordance with the Declaration of Helsinki 1964.

Patient characteristics
In total, 38 patients with unresectable metastatic EP-NEC were analyzed in this study. The median age of the patients was 61.5 years (range 41-74), and there were 29 men (76.3%) and 9 women (23.7%). The primary origins of EP-NEC were the gastrointestinal tract (n = 15, 39.5%), hepatobiliary system (n = 10, 26.3%), and pancreas (n = 4, 10.5%), among others (n = 9, 23.7%). Surgical resection of the primary site at any stage was performed in 14 patients (36.8%), and of these, 8 patients underwent curative resection for stage II or III disease but experienced subsequent recurrence. The other 6 patients underwent palliative surgery, or undiscovered metastasis was detected in these patients after operation.

Treatment patterns
All patients received an etoposide plus cisplatin regimen as rst-line chemotherapy, and the median number of chemotherapy cycles was 6 (range 2-13). Second-line chemotherapies were administered to 22 patients. After rst-line chemotherapy, the numbers of patients who showed complete remission (CR), partial remission (PR), stable disease (SD), or progressive disease (PD) as the best response were 2, 17, 12, and 7, respectively (Table 1).
Univariate analyses for PFS and OS were performed. Ki-67 and AST are well-known prognostic factors in NEC, but these factors were not associated with prognosis in this study. NLR had a higher hazard ratio (HR) than several other factors, but the difference was not signi cant. However, the high-PLR group showed a signi cant association with decreased PFS (HR 2.1, 95% CI 1.0-4.0, P = 0.036) and OS (HR 2.6, 95% CI 1.3-5.3, P = 0.009). Moreover, patients with high LDH levels (more than 1.5 times the ULN) showed signi cantly poorer PFS (HR 2.6, 95% CI 1.2-5.7, P = 0.015) and OS (HR 2.8, 95% CI 1.2-6.3, P = 0.015) than those with low LDH levels. In multivariate analysis, high PLR and LDH were independently associated with poor prognosis (Table 3).
Previously, several studies have validated the prognostic factors in NENs. Although the survival times of NEN patients vary according to the stage, grade, and origin [2], only a few studies have distinguished between NETs and NECs. Therefore, this study evaluated patients with unresectable or metastatic EP-NEC who received platinum-based chemotherapy.
Some authors have reported that LDH and AST are independent factors representing poor prognosis in NEC [14][15][16]. Similarly, LDH was one of the impactful prognostic factors identi ed in this study. The characteristics of NECs, such as high glucose consumption, high lactate production, a hypoxic tumor environment because of poor vascularization, and high proliferation, could increase LDH levels [15,17,18].
However, AST was not a signi cant prognostic factor in this study. A previous study suggested a cut-off value of AST as 2 times the ULN [15], but there was only one patient with more than 2 times the ULN in this study. Therefore, to determine the prognostic signi cance of AST, a large number of patients with high AST levels should be included.
The Ki-67 protein is a cellular marker for cell proliferation [19], and the NORDIC NEC study by Sorbye et al. showed that NEC patients with Ki-67 < 55% had signi cantly longer survival than their counterparts with Ki-67 ≥ 55% [14]. However, Ki-67 was not a signi cant prognostic factor in this study. The NORDIC NEC study included patients with grade 3 NET, which was regarded as NEC according to the previous WHO classi cation, but we excluded these patients according to the recent classi cation. This discrepancy in classi cation could be responsible for the difference in results.
The relationship between the systemic in ammatory response and the outcome of cancer has been a focus of research in recent years. In particular, NLR and PLR have been established as prognostic factors in various solid tumors for predicting treatment outcomes. Recently, Salman et al. reported that these markers were associated with PFS in NETs as well [1]. Tumor-associated neutrophils (TANs) have been investigated for their protumorigenic, anti-apoptotic, and angiogenic roles and for promoting tumor progression, tumor invasion, and metastasis [20][21][22]. NLR is assumed to re ect an increase in TANs, and thus, NLR is considered an easily veri able surrogate marker for TANs [11]. Although this study could not prove the signi cance of NLR in survival analysis, there was a remarkable difference in survival between patients with high NLR and those with low NLR (Fig. 1A and 1D). However, it is necessary to investigate the effect of NLR in a large cohort study.
In contrast, the high-PLR group had signi cantly shorter PFS and OS than the low-PLR group ( Fig. 1B and  1E). This can be explained by the role of platelets in the in ammatory response. Platelet activation is stimulated by chemokines and proin ammatory lipids and is connected with neutrophil recruitment. Therefore, PLR is a well-known in ammatory marker [23,24]. Moreover, platelets play a remarkable role in tumor proliferation and distant metastasis by shielding tumor cells from immune responses and facilitating cancer growth and dissemination [25][26][27]. Therefore, PLR is an effective and independent prognostic factor in EP-NEC.
In addition to increased neutrophil and platelet counts, high NLR and PLR represent decreased lymphocyte counts. Lymphocytes play a major role in the cytotoxic cell death of tumor cells and the inhibition of tumor cell proliferation and migration [28,29]. Thus, decreased lymphocyte counts might be associated with tumor progression and poor prognosis. This is an additional reason that explains the association of NLR and PLR with cancer prognosis.
PLR at diagnosis is easily measurable via peripheral blood samples, and it may be one of most signi cant and earliest prognostic markers for NEC. It may be helpful to estimate patient prognosis and to determine therapeutic strategies for clinicians.
There were several limitations to this study. First, because the sample size was relatively small, a large prospective cohort study is necessary to generalize these results. Second, although all patients had the same pathologic diagnosis of NEC, the origins of the NECs were quite heterogeneous. Nevertheless, to our knowledge, this is the rst study to show that PLR is a signi cant prognostic factor for PFS and OS in EP-NEC. Further prospective studies optimizing the cut-off values are needed to con rm our results.

Conclusion
In addition to high LDH, high PLR was also signi cantly associated with decreased survival of EP-NEC patients who received platinum-based chemotherapy. Therefore, PLR may be an independent and easily measurable prognostic factor in EP-NEC at the beginning of diagnosis.