Minimum absolute lymphocyte count during radiotherapy as a new prognostic factor for nasopharyngeal cancer

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1 ORIGINAL ARTICLE Minimum absolute lymphocyte count during radiotherapy as a new prognostic factor for nasopharyngeal cancer Oyeon Cho, MD, Young-Taek Oh, MD, PhD,* Mison Chun, MD, PhD, O-Kyu Noh, MD, PhD, Jae-Sung Hoe, MD, Hwanik Kim, MD Department of Radiation Oncology, Ajou University School of Medicine, Suwon, Republic of Korea. Accepted 31 May 2015 Published online 2 September 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI /hed ABSTRACT: Background. The purpose of this study was to investigate whether the minimum absolute lymphocyte count (ALC) during radiotherapy (RT) could predict clinical outcome in patients with nasopharyngeal cancer (NPC). Methods. We analyzed 70 patients with NPC including 63 patients treated with chemoradiotherapy and used multivariate analysis to determine whether minimum ALC affected clinical outcome. Results. Patients were grouped by minimum ALC, with a cutoff of 245 cells/ll. Five-year disease-specific survival (DSS) and progression-free survival (PFS) for patients with minimum ALC 245 versus minimum ALC <245 were 88.1% versus 60.8% (p 5.004) and 71.2% versus 35.2% (p 5.004). All 10 patients with minimum ALC <120 experienced disease progression. Four of 6 patients (67%) with ALC <120 who died experienced disease progression within 6 months. Conclusion. Minimum ALC may predict poor 5-year DSS and should be evaluated by prospective studies. VC 2015 Wiley Periodicals, Inc. Head Neck 38: E1061 E1067, 2016 KEY WORDS: lymphocyte, nasopharyngeal cancer, prognosis, radiotherapy, minimum value INTRODUCTION Nasopharyngeal cancer (NPC) is an uncommon malignancy in Korea, and most cases are diagnosed at an advanced stage. Patients with advanced disease more commonly experience local progression or distant metastasis after concurrent chemoradiotherapy (CRT), ultimately leading to poor outcomes, compared with patients with localized disease. However, there were several reported cases of different outcomes, suggesting that various imaging studies cannot entirely explain the prognosis of patients and highlighting the need for identification of other prognostic factors. 1 For this reason, various methods to supplement TNM classifications have been suggested over the past 10 years. Methods using the Epstein Barr virus or the epidermal or hepatocyte growth factor receptor have been somewhat successful, but are often costly and time consuming. 2 4 There were the following biomarkers relevant to lymphocyte as the immune response, which is one of the hallmarks of cancer. 5 Studies have shown that pretreatment lymphopenia can predict overall survival in several advanced cancers and colorectal cancer. 6,7 Pretreatment neutrophil lymphocyte ratio (NLR), which is an inflammatory marker and reflects lymphopenia, is related to lung, liver, colorectal, esophageal, and pancreatic cancer, including NPC Some studies have noted that lymphopenia is relevant to *Corresponding author: Y.-T. Oh, Department of Radiation Oncology, Ajou University School of Medicine, 206 World cup-ro, Yeongtong-gu, Suwon , Korea. ohyoung@ajou.ac.kr treatment outcomes in patients with lung, head and neck, and pancreatic cancer 2 months after initiation of concurrent CRT Despite many studies about lymphopenia as a biomarker, there is rarely a study for the association between lymphocyte during concurrent CRT and clinical outcome. Yovino et al 18 suggested that radiation might suppress the immune response by inducing apoptosis of circulating lymphocytes. Past studies found that B, peripheral blood, T, and natural killer lymphocytes decreased to low levels regardless of cisplatin, and lymphopenia was related to poor response to radiation in patients undergoing pelvic radiotherapy (RT). 19,20 From these studies, we can assume that total lymphocytes will decline during RT, with or without cisplatin, in patients with NPC, and the degree of decline may vary between patients. Under these assumptions, we analyzed whether the minimum absolute lymphocyte count (ALC) during RT predicted clinical outcomes. MATERIALS AND METHODS This study was reviewed and approved by the Institutional Review Board of the Ajou University Hospital. We selected 70 patients who were newly diagnosed with NPC and treated at Ajou University Hospital between July 2001 and September Pediatric patients or patients who died during treatment, failed to complete treatment, or had distant metastasis at diagnosis were excluded. All diagnoses were based on imaging evaluations, such as positron emission tomography-ct or MRI, and confirmed histologically by biopsy. HEAD & NECK DOI /HED APRIL 2016 E1061

2 CHO ET AL. TABLE 1. Patient characteristics. Characteristics No. of patients (%) or median (range) Evaluable patients Sex, female vs male 21 (30) vs 49 (70) 70 Median age, y at diagnosis (range) 53 (18 73) 70 Histology (WHO I vs II or III) 17 (24) vs 52 (76) 69 Smoking, yes vs no 48 (70) vs 21 (30) 69 ECOG, 0 vs (28) vs 50 (72) 69 T classification, 1 2 vs (60) vs 28 (40) 70 N classification, 0 1 vs (46) vs 38 (54) 70 Overall stage, 1 2 vs (29) vs 50 (71) 70 Induction chemotherapy, yes vs no 22 (31) vs 48 (69) 70 Concurrent CRT, yes vs no 63 (90) vs 7 (10) 70 NLR (range) 2.13 ( ) 63 Minimum ALC, cells/ll (range) 224 (58 581) 62 Abbreviations: WHO, World Health Organization; ECOG, Eastern Cooperative Oncology Group; CRT, chemoradiotherapy; NLR, neutrophil lymphocyte ratio; Minimum ALC, minimum absolute lymphocyte count during radiotherapy. All patients received RT alone or concurrent CRT with or without 1 or 2 cycles of cisplatin and 5-fluorouracilbased induction chemotherapy. Cisplatin 100 mg/m 2 was administered on days 1, 22, and 43 during RT. The complete blood count was assessed before RT and induction chemotherapy, as well as once a week during RT. In some cases, additional tests were performed because of the condition of the patient or previous complete blood count results. The median RT dose was 70 Gy (range, Gy) administered to the primary tumor volume and 60 Gy (range, Gy) to the involved regions of cervical lymph nodes, and 44 Gy (range, Gy) to the initial tumor and cervical lymph nodes. No patients were treated with adjuvant chemotherapy. Patients were followed up every 3 months for the first 3 years and every 6 months thereafter. The median follow-up period was 49 months (range, months), and the endpoints assessed were disease-specific survival (DSS), progression-free survival (PFS), locoregional PFS, and distant metastasis-free survival. Locoregional progression was diagnosed based on clinical symptoms, fiberoptic endoscopy, biopsy, and imaging evaluations, such as enhanced CT, MRI, and positron emission tomography-ct. Distant metastases were diagnosed based on clinical symptoms, physical examinations, and imaging tests. Patients were treated with proper management, such as reirradiation, chemotherapy, or surgery. Minimum absolute lymphocyte count We defined minimum ALC as the minimum ALC during RT with or without cisplatin. Radiation time (days), ALCs, and trend lines of all patients were described. Time zero meant the day when RT was started and ALCs of zero were pretreatment ALCs. The distribution between minimum ALCs of all patients and the days when minimum ALCs were measured was presented. Pretreatment lymphocytes A group of ALCs before initial chemotherapy was compared before concurrent CRT in 22 patients who undertook induction chemotherapy. The NLR was defined as the absolute neutrophil count divided by the ALC before initiation of RT. Statistical analysis All statistical analyses were performed using SPSS statistical analysis software, version 17.0 (SPSS, Chicago, IL). The t test was used to compare continuous variables. Cutoff values for NLR or minimum ALC demonstrated the maximum sensitivity and specificity for survival based on receiver operating characteristic curves. The chisquare test was used to compare the categorical date between 2 patient groups. We conducted the univariate and multivariate analysis using the Kaplan Meier method and the Cox proportional hazards model. This process was carried out in each group of advanced-stage patients (stages 3 and 4). RESULTS Patients The patients characteristics are shown in Table 1. There were 21 women and 49 men with a median age of 53 years (range, years). Regarding the World Health Organization (WHO) classification, 17, 1, and 51 patients were diagnosed with type I disease (ie, squamous cell carcinoma), type II disease (ie, nonkeratinizing differentiated carcinoma), and type III disease (ie, undifferentiated carcinoma), respectively. Regarding smoking status, 70% were current or past smokers. Among all patients, 28% had Eastern Cooperative Oncology Group- Performance Status (ECOG-PS) zero. Data for histology, smoking, and ECOG-PS had 1 missing value each. Patients were classified on the basis of the 2010 American Joint Committee on Cancer criteria. There were 23 patients (33%), 19 patients (27%), 15 patients (21%), and 13 patients (19%) with T classification 1 to 4, respectively; meanwhile, there were 9 patients (13%), 23 patients (33%), 23 patients (33%), and 15 patients (21%) with N classification 0 to 3, respectively. Furthermore, 3 patients (4.29%), 17 patients (24.29%), 22 patients (31.43%), and 28 patients (40%) had stage I to IV disease, respectively. Regarding treatment, 31% patients received induction chemotherapy, and 10% were treated with RT alone. The median NLR and minimum ALC E1062 HEAD & NECK DOI /HED APRIL 2016

3 LYMPHOPENIA DURING RT IN NASOPHARYNGEAL CANCER FIGURE 1. (A) The available absolute lymphocyte counts (ALCs) per radiation days and trend line of them about each of the 62 patients, including 61 with concurrent chemoradiotherapy and 1 with radiotherapy were described. (B) The minimum ALCs per the days when minimum ALC measured were plotted. Min ALC, minimum absolute lymphocyte count during radiotherapy. were 2.13 (range, ; n 5 63) and 224 cells/ll (range, cells/ll; n 5 62), respectively. Minimum absolute lymphocyte count The trend lines from 62 patients treated with concurrent CRT (n 5 61) or RT alone (n 5 1) were similar to convex quadratic function (Figure 1A). ALCs of each patient undergoing RT had minimum value, minimum ALC, and had a tendency to increase after passing it. Most of the minimum ALCs were between 20 and 50 days after starting RT, as shown in Figure 1B. The median number of available ALCs to decide minimum ALC was 7 (range, 2 25), and the median number of available ALCs for the last half of radiation was 4 (range, 1 13). Pretreatment lymphocytes There was no significant difference in the mean ALC before induction chemotherapy and concurrent CRT ( vs cells/ll; p 5.25). Survival analysis In this study, 36 of the 70 patients experienced disease progression. There were 26 patients with locoregional disease progression, 13 patients with distant metastases, and 3 patients with both. Residual tumor progression occurred in 10 of the patients with locoregional progression; the other 16 patients had disease recurrence. The 13 patients with distant metastases had disease spread to the following sites: lung (7 patients), bone (5 patients), liver (2 patients), skin (1 patient), and axillary metastases (1 patient). The 5-year DSS for all patients was 67.8%. Patients with localized disease (stages 1 and 2) had an 84.2% 5-year DSS rate, whereas patients with advanced stage disease (stages 3 and 4) had a 60.5% 5-year DSS rate (p 5.009). The 5-year PFS rate for all patients was 49.7%. For patients with localized and advanced-stage disease, the 5-year PFS rates were 67.4% and 43.3%, respectively (p 5.027). The 5-year locoregional PFS rate for all patients was 63.8%. For patients with localized and advanced-stage disease, the 5-year locoregional PFS rates were 78.8% and 57.8%, respectively (p 5.115). The 5-year distant metastasis-free survival rate for all patients was 78%. For patients with localized and advanced stage disease, the 5-year distant metastasis-free survival rates were 87.4% and 74.9%, respectively (p 5.092). A minimum ALC of 245 cells/ll achieved a sensitivity of 0.75 and specificity of for survival prediction. The characteristics of patients with respect to the cutoff of minimum ALC of 245 cells/ll is shown in Table 2. Female patients tended to be included in the group with a minimum ALC of <245 cells/ll than minimum ALC 245 cells/ll (p 5.058). There were significantly more stage 3 or 4 patients with a minimum ALC <245 cells/ ll than minimum ALC 245 cells/ll (85% vs 61%; p 5.041). No other characteristics differed significantly with respect to the cutoff. An NLR of 2.9 achieved a HEAD & NECK DOI /HED APRIL 2016 E1063

4 CHO ET AL. TABLE 2. Comparison between patients with a minimum absolute lymphocyte count <245 and 245 cells/ll. Minimum ALC <245 cells/ll Minimum ALC 245 cells/ll Characteristics (n 5 34) No. of patients (%) (n 5 28) No. of patients (%) p value (chi-square test) Female/male 14 (41)/20 (59) 5 (18)/23 (82).058 Age <50/50 y 15 (44)/19 (56) 11 (39)/17 (41).798 WHO I/II or III 7 (21)/26 (79) 10 (36)/18 (64).258 Smoking, yes/no 22 (67)/11 (33) 19 (68)/9 (32) ECOG 0/1 2 9 (27)/24 (73) 9 (32)/19 (68).781 T classification 1 2/ (50)/17 (50) 8 (28)/20 (72).120 N classification 0 1/ (62)/21 (38) 14 (50)/14 (50).443 Induction CTX, yes/no 12 (35) /22 (65) 9 (32)/19 (68) Concurrent CRT, yes/no 33 (97)/1 (3) 28 (100)/0 (0) Stage 1, 2/3, 4 5 (15)/29 (85) 11 (39)/17 (61).041 NLR <2.9/ (58)/14 (42) 18 (69)/8 (31).423 Abbreviations: minimum ALC, minimum absolute lymphocyte count during radiotherapy; WHO, World Health Organization; ECOG, Eastern Cooperative Oncology Group; CTX, chemotherapy; CRT, chemoradiotherapy; NLR, neutrophil lymphocyte ratio. Histology, smoking, and ECOG had 1 missing value each in the minimum ALC <245 cells/ll (n 5 61). NLR had 1 and 2 missing values in the minimum ALC <245 and 245 cells/ll groups, respectively (n 5 59). The p values <.05 are presented as a bold text. sensitivity of 0.5 and specificity of for survival prediction. The results of univariate and multivariate analyses are shown in Table 3. WHO type I disease, T classifications of 3 or 4, and minimum ALC <245 cells/ll were significant predictors of DSS among all patients in univariate analysis. In multivariate analysis, WHO type I disease and minimum ALC <245 cells/ll were significant predictors of DSS (hazard ratio [HR] ; 95% confidence interval [CI] ; p 5.001; HR ; 95% CI ; p 5.003, respectively). The patients with WHO type I disease had a significantly lower 5-year DSS rate than patients with WHO type II/III disease (55.2% vs 73.3%; p 5.038). The 5-year DSS rate differed significantly between patients with a minimum ALC <245 and 245 cells/ll (60.8% vs 88.1%; p 5.004). T classification 3 or 4, NLR 2.9, and minimum ALC <245 cells/ll were significant predictors of PFS in univariate analysis. Multivariate analysis showed that T classification 3 or 4 and minimum ALC <245 cells/ll were significant predictors of PFS (HR ; 95% CI ; p 5.012; HR ; 95% CI ; p 5.018, respectively). There was a significant difference in 5-year PFS between patients with T classification 3 or 4 versus 1 or 2 (30% vs 62.8%; p 5.005). The 5-year PFS rate was significantly lower in patients with a minimum ALC <245 than 245 cells/ll (35.2% vs 71.2%, respectively; p 5.004). The 5-year locoregional PFS rate differed significantly with respect to sex, histology, T classification, NLR, and minimum ALC in univariate analysis. However, in multivariate analysis, only T classification 3 or 4 and minimum ALC <245 cells/ll were significant predictors of 5-year locoregional PFS (HR ; 95% CI ; p 5.007; HR ; 95% CI ; p 5.076, respectively). The patients with a T classification of 3 or 4 had significantly lower 5-year locoregional PFS rate than those with a T classification of 1 or 2 (39.8% vs 79.7%, respectively; p 5.003). The 5-year locoregional PFS rate was significantly lower in patients with a minimum ALC <245 than 245 cells/ll (51% vs 80.2%, respectively; p 5.021). Age, smoking, performance status, N classification, induction chemotherapy, and RT alone were not associated with any endpoint. No variables were significantly associated with distant metastasis-free survival. Among advanced-stage patients, WHO type I disease and RT alone were predictors of DSS in multivariate analysis. The 5-year DSS was significantly lower in patients with WHO type I disease than type II or III disease (37.9% vs 69.8%, respectively; p 5.008). There was a significant difference in 5-year DSS between patients who received concurrent CRT and RT alone (63.9% vs 25%; p 5.01). There was no significant difference in the 5-year DSS between patients with a minimum ALC <245 and 245 cells/ll (57.7% vs 79.1%, respectively; p 5.088). There were significant differences in PFS with respect to minimum ALC. The 5-year PFS rate was significantly lower in patients with a minimum ALC <245 than 245 cells/ll (26.9% vs 76.5%; p 5.004). T classification 3 or 4 and minimum ALC <245 cells/ll were significant predictors of the locoregional PFS rate in multivariate analysis. Patients with a T classification of 1 or 2 had a significantly higher 5-year locoregional PFS rate than patients with a T classification of 3 or 4 (81.8% vs 39.8%; p 5.022). The 5-year locoregional PFS rate was significantly lower in patients with a minimum ALC <245 than 245 cells/ll (42.3% vs 82.4%; p 5.014). No variables were significantly associated with distant metastasis-free survival. There were 34 patients with a minimum ALC <245 cells/ll. Of the 24 patients with 120 minimum ALC <245cells/lL, 12 (50%) experienced disease progression; 3 of them with progressive disease (25%) progressed within 6 months of the initial diagnosis. All 10 patients (100%) with a minimum ALC <120 cells/ll experienced disease progression (locoregional progression in 9 patients and distant metastasis in 1 patient), and 6 of the 10 patients (60%) progressed within 6 months. The 5-year PFS rate was significantly lower in patients with a minimum ALC <120 cells/ll than 120 minimum ALC <245 cells/ll (0% vs 51.1%, respectively; p <.001, E1064 HEAD & NECK DOI /HED APRIL 2016

5 LYMPHOPENIA DURING RT IN NASOPHARYNGEAL CANCER TABLE 3. Univariate and multivariate analyses of patients with total and advanced-stage nasopharyngeal cancer. DSS PFS Locoregional PFS All HR (95% CI) p value HR (95% CI) p value HR (95% CI) p value Univariate analysis Female 2.25 ( ) ( ) ( WHO I 2.32 ( ) ( ) ( ).025 T classification 3, ( ) ( ) ( ).005 NLR < ( ) ( ) ( ).039 Minimum ALC < ( ) ( ) ( ).027 Multivariate analysis WHO I 4.75 ( ).001 T classification 2.63 ( ) ( ).007 ALC < ( ) ( ) ( ).076 Stages 3, 4 Multivariate analysis WHO I 4.98 ( ).002 T classification 3.07 ( ).031 RT alone 7.85 ( ).001 Minimum ALC < ( ) ( ).026 Abbreviations: DSS, disease-specific survival; PFS, progression-free survival; HR, hazard ratio; 95% CI, 95% confidence interval; WHO, World Health Organization; NLR, neutrophil to lymphocyte ratio; Minimum ALC, minimum absolute lymphocyte count during radiotherapy; RT, radiotherapy. The p values <.05 are presented as bold texts. A measure of Min ALC was cells/ll. Table 4). Of the 12 patients with a 120 minimum ALC <245 cells/ll, 9 with disease progression (75%) died of NPC; 2 of them (22.2%) were among those who had disease progression within 6 months. Meanwhile, among the 10 patients with a minimum ALC <120 cells/ll, 6 with disease progression (60%) died; 4 of them (67%) were among those with disease progression within 6 months. The 10 patients with a minimum ALC <120 cells/ll had a 5-year DSS rate of 29.2% compared to 73.3% in patients with a 120 minimum ALC <245 cells/ll (p 5.089). DISCUSSION TABLE 4. Survival rates of 34 patients with a minimum absolute lymphocyte count <245 cells/ll with respect to a cutoff of minimum absolute lymphocyte count <120 cells/ll. Survival rate Minimum ALC <120 cells/ll (n 5 10) 120 Minimum ALC <245 cells/ll (n 5 24) p value 5-y DSS 29.2% 73.3% y PFS 0% 51.1% <.001 Abbreviations: Minimum ALC, minimum absolute lymphocyte count during radiotherapy; DSS, disease-specific survival; PFS, progression-free survival. The scatter plot from Figure 1 shows that the change of ALCs by radiation was consistent with graphs from previous studies ,19 Studies of pelvic irradiation informed us that all types of lymphocytes decreased to low level regardless of cisplatin administration. 19,21 A review article about lymphopenia of high-grade glioma suggested that lymphopenia might be caused by apoptosis of lymphocytes passing radiation field. 18 Therefore, the association between RT and reduced lymphocytes might be established without consideration of cisplatin administration or specific lymphocyte subtypes if a high enough dose was administered to the target wide enough to cover large vessels like RT of NPC. We assumed that the 2 factors relevant to the degree of lymphocyte reduction were individual ability to produce lymphocytes and the extent of the tumor. Under these assumptions, severe lymphopenia occurs because of a reduction in productivity or because of increased lymphocyte infiltration from peripheral blood into peritumoral tissues as the cancer progresses. On the supposition that the tumor infiltrating ability of lymphocytes was similar, this could be true because the increase of tumor infiltrating lymphocytes was relevant to NPC progression. 22 We estimate that both of these variables can result in inferior treatment efficacy in patients. In addition, the ALC during treatment may cover up interference related to increases in lymphocytes because of usual viral infection or decreases in lymphocytes because of cardiovascular disease. 23 ALC measurements taken during treatment can be used to minimize the possible disruptive factors in lymphocyte counts in the pretreatment NLR. Campian et al, 15 Balmanoukiana et al, 16 and Campian et al 17 studied the association between treatment-related lymphopenia and prognosis in several types of cancer. They showed radiation-induced lymphopenia, which did not take back to initial ALC for nearly 1 year and neoadjuvant chemotherapy was not relevant to reduction of ALCs in patients with stage III nonsmall cell lung cancer. 15 Induction chemotherapy did not affect reduction of ALCs in our data, although we could not evaluate radiation-induced lymphopenia because of deficiency of raw data. Their lymphopenia point was 2 months, and the interruption-like infection was possible at that point when RT was finished. In addition, the immune response at 2 months did not reflect the tumor extent within the RT period; therefore, the degree of lymphopenia during treatment, rather than pretreatment or posttreatment, may reflect both the immune response and tumor HEAD & NECK DOI /HED APRIL 2016 E1065

6 CHO ET AL. FIGURE 2. (A) Five-year disease-specific survival rate and (B) 5-year progression-free survival rate according to minimum absolute lymphocyte count (ALC) level for all patients. Min ALC; minimum absolute lymphocyte count during radiotherapy. extent, with fewer compounding factors. The degree of lymphopenia during treatment can therefore be used to comprehensively evaluate the status of patients treated with RT. Minimum ALC was the only variable consistently associated with DSS, PFS, and locoregional PFS (Table 3 and Figure 2). No independent variable was significantly associated with distant metastasis-free survival, because there were so few cases of distant metastasis. Patients with a minimum ALC <245 cells/ll had poor DSS, PFS, and locoregional PFS in univariate analysis, and poor DSS and PFS in multivariate analysis. The strength of the associations of minimum ALC with locoregional PFS and PFS in univariate analysis (p and p 5.007, respectively) and multivariate analysis (p and p 5.018, respectively) indicates the contribution of distant metastasis. In patients with advanced-stage disease, minimum ALC was associated with PFS and locoregional PFS in univariate and multivariate analyses (Table 3). The 5- year DSS rate of patients with a minimum ALC <245 cells/ll tended to be inferior, but the difference was not statistically significant (p 5.088); this is likely because the number of patients with disease progression was small, making it difficult to ascertain its relationship with DSS. The strength of the associations of minimum ALC with locoregional PFS and PFS in multivariate analysis (p and p 5.008, respectively) indicates the contribution of distant metastasis. In the multivariate analysis of the total patients, T classification was a significant predictor of PFS and locoregional PFS, with p values and HRs superior to those of minimum ALC. We can estimate the contribution of locoregional PFS to PFS on the basis of the fact that the p value for PFS (p 5.012) is greater than that for locoregional PFS (p 5.007). In patients with advanced-stage disease, T classification was a significant predictor only for locoregional PFS in multivariate analysis; these results suggest T classification is most relevant to the 5-year locoregional PFS. Regarding histology, WHO type I disease was significantly associated with worse outcomes compared with WHO type II or III disease. Histology was associated with DSS and locoregional PFS in univariate and DSS in multivariate analysis among both the total and advanced-stage disease. These results are consistent with studies reporting that squamous cell carcinomas have inferior outcomes compared with nonkeratinizing undifferentiated carcinomas. 24 NLR was significantly associated with PFS and locoregional PFS in univariate analysis but not in multivariate analysis. In patients with advanced-stage disease, high NLR was not associated with any clinical outcome. NLR had fewer associations with survival rates than minimum ALC. Therefore, additional patients are required to verify the associations between NLR and clinical outcomes. We suspect the lack of associations is due to interference by inflammatory factors other than NPC. Women had inferior DSS and locoregional PFS compared with men in univariate analysis (p and p 5.015, respectively); this is likely because 20 of 43 of male patients (46.5%) had a minimum ALC <245 cells/ll compared to 14 of 19 of female patients (73.7%; Table 2). Although concurrent CRT is generally associated with better outcomes than RT alone, 25 it is unwise to compare the concurrent CRT and RT alone groups in the present study, because the RT alone group was too small (n 5 7). Cisplatinbased and 5-fluorouracil based induction chemotherapy was not significantly associated with any endpoints, consistent with previous studies. 26,27 Age, smoking status, performance status, and N classification were also not associated with any endpoints. Nevertheless, a larger study is required to verify these findings. E1066 HEAD & NECK DOI /HED APRIL 2016

7 LYMPHOPENIA DURING RT IN NASOPHARYNGEAL CANCER How can minimum ALC aid the treatment of nasopharyngeal carcinoma? The standard treatment for patients with advanced NPC is concurrent CRT favoring the intensity-modulated RT technique, but the benefit of adjuvant chemotherapy remains unclear. 25,28,29 Patients with a minimum ALC <245 cells/ll; including total patients and those with stage 3 or 4 disease; may be a high-risk group for adjuvant chemotherapy because they had poor PFS in the present study (Table 3 and Figure 2). The 10 patients, including 9 who underwent locoregional progression with a minimum ALC <120 cells/ll, exhibited rapid progression and early death (Table 4); this group included 4 patients who progressed within 6 months. Therefore, this group might require dose escalation with accelerated fractionation of gross tumors using intensity-modulated RT when minimum ALC falls below 120 cells/ll during concurrent CRT or adjuvant chemotherapy. This study had 2 major limitations. First, the constancy of minimum ALC is uncertain, because the ALC of the 62 patients was not measured regularly. Some patients received additional testing because of fever, anemia, leukocytosis, leukopenia, thrombocytopenia, etc. Others refused blood tests, or ALC could not be determined because the differential white blood cell count was not performed. This might result in a lack of sensitivity and specificity for survival prediction. Second, the survival analysis of this study was limited by its retrospective design and small population, with data collected over a long period of time. Despite these limitations, this is the first study of lymphopenia during RT in patients with NPC. Furthermore, the results suggest lymphopenia can be used for survival prediction. Therefore, these results warrant a large-scale meta-analysis or prospective studies in the near future. In conclusion, minimum ALC predicts poor DSS, PFS, and locoregional PFS in patients with NPC. This might help determine high-risk groups for adjuvant chemotherapy or radiation dose escalation. The rapid progression of cancer followed by death in patients with a minimum ALC <120 cells/ll might require aggressive treatment. Finally, the minimum ALC may predict survival outcome and aid treatment decisions for patients with NPC. Therefore, minimum ALC should be evaluated by welldesigned prospective studies. Acknowledgments The authors thank all the staff related to the treatments and all the patients suffering from NPC. This work was supported by 2012 grant from the Department of Medical Sciences at The Graduate School of the Ajou University. REFERENCES 1. Wang HY, Sun BY, Zhu ZH, et al. Eight-signature classifier for prediction of nasopharyngeal [corrected] carcinoma survival. J Clin Oncol 2011;29: Leung SF, Zee B, Ma BB, et al. 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