The Role of Pretreatment Serum Neutrophil-to-Lymphocyte Ratio in Hypopharyngeal Cancer Treated with Definitive Chemoradiotherapy: A Pilot Study

Serum neutrophil-to-lymphocytes ratio (NLR) is a potential predictive and prognostic marker in head and neck cancers. This study aimed to determine the role of pretreatment serum NLR in patients with hypopharyngeal cancer (HPC) treated with definitive chemoradiotherapy. We retrospectively investigated the correlation between clinicopathological parameters and NLR status and analysed its impact on therapeutic response and survival. A total of 120 patients treated at a single institution between 2009 and 2015 were included. The median follow-up time was 24.1 months. High NLR (NLR ≥ 4) was associated with advanced T classification (p = 0.01*) and advanced stage (p = 0.02*) based on chi-square test. We also found that high pretreatment NLR was correlated with poor treatment response (HR = 2.42, 95% CI: 1.08–5.44, p = 0.03*). Pretreatment NLR was also an independent prognostic factor for progression-free survival (HR = 1.71, 95% CI: 1.01–2.90, p = 0.046*) and overall survival (HR = 1.99, 95% CI: 1.21–3.28, p = 0.01*) while correcting for known prognostic factors. Overall, these findings support that NLR is a potential biomarker for host response to tumour aggressiveness, therapeutic response to chemoradiotherapy and survival in HPC patients. This study is limited by its retrospective nature and further validation is warranted.


Treatment and outcomes.
Of the 120 total patients, 117 completed definitive radiotherapy and 3 developed major complications and terminated treatment early. Fifty-three patients received induction chemotherapy followed by radiotherapy or concurrent chemoradiotherapy, while 67 patients underwent concurrent chemoradiotherapy. As multiple prospective studies have not shown a survival difference between concurrent and induction chemotherapy 18 , we analysed all 120 patients together. The median total dose of the cohort was 7020 cGy (range, 700 to 7920 cGy), with a median overall radiotherapy treatment time of 54 days (range, 10 to 94 days). Of the 120 patients, 63 (52.5%) achieved complete response after definitive radiotherapy. The median follow-up time was 24.1 months (range, 3.1 to 111.3 months). The median OS was 31.3 months and the median PFS was 22.5 months.
Prognostic significance of baseline NLR and other parameters. Analysis of the odds ratio of NLR on treatment response showed that high pretreatment NLR (NLR ≥ 4) was correlated with poor response to definitive chemoradiotherapy (HR = 2.42, 95% CI: 1.08 to 5.44, p = 0.03*). In the survival analysis, anaemia was associated with poor overall survival (OS); other known prognostic factors, such as performance status, T classification and overall stage, were associated with both progression-free survival (PFS) and OS. In the univariate analysis, prolonged radiotherapy time and radiotherapy response predicted inferior PFS and OS. NLR was also significantly associated with PFS (Table 3, Fig. 2) and OS (Table 4, Fig. 2). All significant factors in the univariate analysis were then incorporated into the multivariate analysis, except for T classification as it was not independent from overall stage. In the multivariate analysis, pretreatment NLR and treatment response were shown to be independent prognostic factors of PFS (NLR ≥ 4 vs. NLR < 4, HR = 1.71, 95% CI: 1.01 to 2.90, p = 0.046*) and overall survival (NLR ≥ 4 vs. NLR < 4, HR = 1.99, 95% CI: 1.21 to 3.28, p = 0.01*).

Discussion
This study reports three major findings. First, we found that high pretreatment NLR was correlated with advanced T classification and advanced stage. Second, high pretreatment NLR was associated with poor response to definitive chemoradiotherapy. Third, high pretreatment NLR was significantly correlated with worse PFS and OS for HPC patients receiving definitive chemoradiotherapy. To the best of our knowledge, this is the first study of the significance of pretreatment NLR in HPC patients treated with definitive chemoradiotherapy. NLR can provide insight into the immune-related host response of the tumour microenvironment in various cancers. Elevated NLR levels may result from increased neutrophils and/or decreased lymphocytes. Neutrophils are known to secrete matrix metalloproteinase, vascular endothelial growth factor, fibroblast growth factor, platelet-derived growth factor, TNF-alpha, IL-1 and IL-6 19 . These secreted cytokines and molecules create a microenvironment for extracellular matrix remodelling, endothelial cell migration and tumour cell dissociation  20 . Neutrophils also inhibit the cytolytic activity of active T lymphocytes and natural killer cells 21 . Collectively, high NLR represents an unfavourable tumour microenvironment, which subsequently contributes to tumour growth. In this study of HPC patients, high NLR was significantly associated with advanced T classification and advanced stage. The elevated neutrophil activity and decreased lymphocytic activity may have contributed to accelerated growth of tumour cells. Thus, this is the first study suggesting that NLR is a biomarker of unfavourable tumour microenvironment in HPC patients. NLR has been reported as a biomarker for predicting therapeutic response in many cancers, regardless of chemotherapy or target therapy [22][23][24] . In patients with oral cavity squamous cell carcinoma and oesophageal cancer, therapeutic response to radiotherapy-based treatment was better predicted by adding NLR levels 25,26 . Consistent with such previous work, our study revealed that high pretreatment NLR was correlated with poor response to definitive chemoradiotherapy in HPC patients. The result of the current study provides more confidence to physicians in suggesting alternative treatments for HPC patients with high pre-treatment NLR, since poor response is anticipated.
Elevated NLR is associated with poor treatment response as well as with poor survival outcomes. Chua 16 . A recent meta-analysis that included 40559 patients revealed a strong prognostic influence of NLR 28 . In line with these previous findings, we also found that elevated NLR was correlated with poor PFS and OS in HPC patients. We acknowledge that our study is subject to several limitations. First, due to its retrospective nature, selection and survival biases are unavoidable. Thus, our findings warrant further confirmation in a prospective study. In addition, the study population was treated at a single institution and the population was almost exclusively Taiwanese; extrapolation of these results to other Asian or Caucasian populations is necessary and should be done with care.
In conclusion, our study revealed that high pretreatment NLR was associated with host response to tumour aggressiveness. Further, high pretreatment NLR was correlated with poor therapeutic response and was an independent predictor of inferior PFS and OS in HPC patients treated with definitive chemoradiotherapy.

Data collection and definitions of parameters.
Previously reported prognostic factors were retrieved from medical records, including age, sex, performance status, body mass index (BMI), comorbidity, smoking history, TNM stage and tumour differentiation. Performance status was documented according to the ECOG system. BMI less than 18.5 was regarded as underweight 29,30 . Comorbidity was scored using the ACE-27 index 31 . The ACE-27 index groups comorbidity conditions as follows: cardiovascular, gastrointestinal, respiratory, renal, neurological, psychiatric, endocrine, immunological, rheumatologic, malignancy prior to index malignancy or concomitant malignancy with index malignancy, substance abuse and obesity. Overall cogent comorbidity was classified by the severity of organ decompensation using four categories: none, mild, moderate, or severe.
Smoking history was recorded as packs per year; more than 15 packs/year was regarded as a heavy smoker 32 . Primary tumour classification (T), nodal classification (N) and overall stage were re-defined based on the 7 th edition of the American Joint Committee on Cancer (AJCC) staging manual 33 . We also recorded pretreatment blood count data collected within two weeks prior to any treatments. Pretreatment haemoglobin, white blood cell count, absolute neutrophil count and lymphocyte count were extracted. We calculated the neutrophil-to-lymphocyte ratio (NLR) by dividing the absolute neutrophil count by the absolute lymphocyte count. An NLR value of 4 or greater was considered high 34,35 . Definitive radiotherapy. All patients were treated with intensity-modulated radiotherapy using 6 or 10 MV linear accelerators (Clinac iX, Varian Medical Systems, Palo Alto, USA). Briefly, a total radiation dose of 7000-7200 cGy (200 cGy/fraction, 5 days per week) was administered to the gross hypopharyngeal tumour and enlarged neck lymph nodes, while 5000 cGy (200 Gy/fraction, 5 days per week) prophylactic irradiation was given to the bilateral neck lymph node regions. All patients were treated by three senior radiation oncologists with over ten years of experience at the same institution according to institutional guidelines. With the same technique and treatment plan of definitive radiotherapy, there were three treatment combination: induction chemotherapy followed by concurrent chemoradiotherapy, concurrent chemoradiotherapy and induction chemotherapy followed by radiotherapy alone. The choice of treatment combination was based on the physician's judgement.

Definition of endpoints and statistical considerations.
The study endpoints were treatment response, progression-free survival (PFS) and overall survival (OS). Treatment response evaluation was performed six weeks after completion of radiotherapy utilizing the head and neck CT scan. If complete radiological response was achieved, it was recorded as complete response; if it was hard to determine complete radiological response, biopsy was performed for confirmation. If the biopsy was negative for malignancy, response was then documented as complete response (CR); if not, response was documented as less than CR. OS was defined from the date of diagnosis until death from any cause, or censored at last follow-up. PFS was defined from the date of diagnosis to the date of any recurrence or death from any cause, or censored at last follow-up. All variables were grouped as categorical data according to clinically meaningful cut-off values. The Chi-square tests (or Fisher's exact probability when appropriate) were used for analysing the relationship between NLR and clinicopathological parameters. Odds ratio estimation was used to calculate the relationship between pretreatment NLR and treatment response. For univariate analysis, we plotted survival curves with the Kaplan-Meier method and compared differences between groups using the log-rank test. We evaluated potential prognostic factors with univariate and multivariate regression analysis with respect to PFS and OS; factors with p < 0.05 in univariate analysis were selected for inclusion in multivariate analysis. Multivariate analysis was performed using the Cox proportional hazards model. A two-tailed p value < 0.05 was considered statistically significant. Statistical analysis was conducted with MedCalc Statistical Software version 15.2.2 (MedCalc Software bvba, Ostend, Belgium). All data generated or analysed during this study are provided with this published article.