Lower lymphocyte percentage and higher platelet count as poor prognostic factors for patients with epidermal growth factor receptor–mutated lung adenocarcinoma receiving tyrosine kinase inhibitors as first-line treatment

Background This study evaluated the effect of clinical factors on the treatment outcomes of patients with lung adenocarcinoma with active epidermal growth factor receptor (EGFR) mutations who received tyrosine kinase inhibitors (TKIs) as first-line treatment. Methods Patients with stage IIIb or IV lung adenocarcinoma with mutated EGFR were enrolled retrospectively between March 2010 and December 2017. The effects of various clinical features and hematologic markers on progression-free survival (PFS) and overall survival (OS) were analyzed. A total of 190 patients were enrolled in this study. In univariate analysis, the male sex, smoking history, EGFR mutation with L858R, and presentation with malignant pleural effusion at initial diagnosis were significantly associated with shorter PFS or OS. Among hematologic markers, lower lymphocyte percentage and higher platelet count were associated with significantly poor PFS and OS. Stepwise multivariate Cox regression analysis showed that smoking history, EGFR mutation with L858R, and lower lymphocyte percentage were independent poor prognostic factors for PFS and OS. Presentation with malignant pleural effusion and higher platelet count was an independent poor prognostic factor for OS only. Patients having

4 count had poorer prognoses compared with other patients. Additional studies are warranted to elucidate the underlying mechanisms.

Background
By the World Health Organization, lung cancers are the leading causes of cancer mortality and disability-adjusted life years [1]. Treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) as the first-line therapy for EGFR-mutant lung adenocarcinoma improved the response rates, progression-free survival (PFS) and overall survival (OS) significantly [2]. Although the response rate of EGFR TKIs as first-line treatment is high, there are still some patients with a dismal prognosis. Several biomarkers by previous reports, including CEACAM Evidence by previous studies had been showed changes in blood components and blood cells, including a white blood count and platelet, in cancer patients associated with the disease severity and survival [4][5][6][7][8]. By the previous study, lymphocytes played critical roles in promoting systemic immune responses against tumors, and lymphocytopenia is associated with poor outcomes in many malignancies [9][10][11].
High expression of CD8+ T lymphocytes, which predicts a favorable prognosis in lung adenocarcinoma was reported [12]. Platelet also played another important role in cancer prognosis. Thrombocytosis has been found which is associated with poorer cancer prognosis. Shorter OS rates observed for patients with many cancers, included ovarian cancer [4], lung cancer [5], and breast cancer [6] which was related to thrombocytosis at the time of diagnosis, and poor prognoses of patients 5 with colorectal cancer [7] and renal cancer [8] before surgical therapy are related to high platelet counts. Sylman et al. reported that platelet count is also a predictor of metastasis and venous thromboembolism in patients with cancer [13].
On the other hand, systemic inflammation also plays a role in cancer prognosis [14].
Inflammatory mediators are involved in cancer progression with apoptosis, angiogenesis, and DNA damage [15]. Numerous studies have shown the relationship between inflammation and cancer prognosis [16,17]. The markers included the neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR). Higher NLR or PLR has been reported to predict shorter progression-free survival (PFS) and OS in many solid cancers [18][19][20][21].
Awareness of newer prognostic factors might provide a potential direction for further improvement in treatment for EGFR-mutated non-small-cell lung cancer (NSCLC), especially adenocarcinoma, but no study has focused on hematologic and inflammatory markers in EGFR-mutant lung adenocarcinoma. In this study, we evaluated the effects of hematologic and inflammatory factors on the treatment outcomes of patients with advanced or metastatic lung adenocarcinoma with active EGFR mutations. All patients received TKIs as the first-line treatment.

Patients and data collection
Patients with diagnosis as stage IIIb or IV lung adenocarcinoma in Buddhist Tzu Chi General Hospital, Hualien, Taiwan, from March 1, 2010, to December 31, 2017, and harbored mutated EGFR were enrolled in this retrospective study. According to the World Health Organization pathology classification, lung adenocarcinoma was confirmed pathologically. All patients received serial imaging studies at the initial 6 diagnosis for staging, including computed tomography (CT), whole-body bone scan, positron emission tomography scan (PET), and brain imaging. Tumor staging was recorded by the seventh American Joint Committee on Cancer staging system. All the patients had an EGFR mutation examination of the tumor specimen, and the results showed active EGFR mutations in exons 18, 19, 20, or 21 in all patients. And then they received EGFR TKIs as first-line therapy, including afatinib, erlotinib, or gefitinib. The choice of EGFR TKI was by the discretion of their attending physicians.
Patients were excluded if they had previously undergone palliative chemotherapy.
Baseline clinical characteristics were determined through a retrospective chart review, including age at diagnosis, sex, staging, smoking status, mutation type, and TKIs used. Smoking status was categorized to ever smoker or never smoker.
Malignant pleural effusion was diagnosed by either pleural effusion cytology or a pleural biopsy. Complete blood counts, including total leukocyte counts with a different count, hemoglobin, and platelet count, were also recorded. NLR and PLR were also calculated from the data of complete blood counts.
Mutational analysis of EGFR gene was done as described in a previous study [22].
Briefly, formalin-fixed, paraffin-embedded tissues of histologically were used for confirming NSCLC. An EGFR RGQ Kit (Qiagen, Hilden, Germany) was used for the analysis of mutations in EGFR, which utilizes amplification refractory mutationspecific polymerase chain reaction and Scorpion technologies for detection and direct sequencing.
PFS was recorded as the duration between the start of TKI treatment and the date of progression. And OS was defined as the duration from the start of TKI treatment to the time of all-cause death. All enrolled patients were followed up until death or the end of December 2018. This retrospective study was approved by the Institutional 7 Review Board of Buddhist Tzu Chi General Hospital (IRB108-48-B). Informed written consent was waived because the study was a retrospective data analysis.

Statistical analyses
Data were analyzed by MedCalc (Mariakerke, Belgium) with median and hazard ratios (HRs) and their 95% confidence intervals (CIs). PFS and OS were analyzed using Kaplan-Meier curves and the log-rank test. Univariate and multivariate analyses were executed using Cox proportional-hazards regression. All variables were included for multivariate analysis to assess the effect of each variable after univariate analyses. Stepwise variable selection was used to develop a reduced multivariate model, including variables with P < 0.1 and removing variables with P > 0.2. Two-sided with the level of statistical significance set at P < 0.05 was used in all results.  In the multivariate analysis with a stepwise model, only higher lymphocyte percentage was significantly associated with better prognosis in PFS (HR = 1.50, 95% CI: 1.08-2.09, P = 0.0159). Regarding OS, higher lymphocyte percentage (HR = 2.10, 95% CI: 1.39-3.17, P = 0.0004) was also associated with a better prognosis, but higher platelet count (HR = 0.63, 95% CI: 0.43-0.93, P = 0.0211) was associated with a significantly poorer prognosis.  In our study, patients with a lymphocyte percentage of >20.8% had a better prognosis than did other patients. Higher lymphocyte percentage and lower lymphocyte percentage were associated with median OS periods of 40.63 and 18.07 months, respectively, and these were significantly different between univariate and multivariate analyses.

Discussion
High platelet count has been reported with a poor prognosis in various cancers [11,36]. The mechanism may be related to thymidine phosphorylase, which is a plateletderived endothelial cell growth factor with potent angiogenic activity. An increase in thymidine phosphorylase levels may be associated with a poor prognosis in various

Conclusions
In our study of patients with stage IIIb or IV lung adenocarcinoma with EGFR mutations who received TKIs as the first-line treatment, a lower lymphocyte percentage and higher platelet counts were significantly associated with shorter PFS and OS. Stepwise multivariate Cox regression analysis also showed that lower lymphocyte percentage was an independent poor prognostic factor for both PFS and 13 OS, but a higher platelet count was only for OS. Further research is necessary to confirm the possible mechanism of poor prognoses.

Consent for publication
Informed written consent was waived by the Institutional Review Board of Buddhist Tzu Chi General Hospital because the study was a retrospective data analysis.
14 Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests    Kaplan-Meier curves of progression-free survival and overall survival Kaplan-Meier curves of