Lymph node density as an independent prognostic factor in node-positive patients with tonsillar cancer

ORIGINAL ARTICLE Lymph node density as an independent prognostic factor in node-positive patients with tonsillar cancer Jun-Ook Park, MD, PhD, 1 Young-Hoon Joo, MD, PhD, 2 Kwang-Jae Cho, MD, PhD, 2 Min-Sik Kim, MD, PhD 2 * 1 Department of Otolaryngology Head and Neck Surgery, Inje University College of Medicine, Haeundae Paik Hospital, Busan, South Korea, 2 Department of Otolaryngology Head and Neck Surgery, Catholic University College of Medicine, Seoul St. Mary s Hospital, Seoul, South Korea. Accepted 13 April 2015 Published online 15 June 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.24074 ABSTRACT: Background. The purpose of this study was to evaluate the utility of lymph node density for predicting the survival of patients with tonsillar cancer and positive neck nodes. Methods. We reviewed data for 64 patients diagnosed with tonsillar cancer with lymph node metastasis in a retrospective single center audit. Results. Lymph node density (0.05), extracapsular spread (ECS), number of ECS (3), vascular invasion, perineural invasion, and N classification (N1/N2a vs N2b/N2c) were significant predictors of 5-year diseasefree survival (DFS). Lymph node density (0.05), human papillomavirus (HPV) infection, and N classification (N1/N2a vs N2b/N2c) were significant predictors of 5-year overall survival (OS). In the multivariate analysis, categorical lymph node density remained a significant predictor of DFS (p 5.025*; hazard ratio [HR] 5 6.063) and no parameter was significantly predictive of OS. Conclusion. Lymph node density might be useful as an independent predictor of survival in patients with tonsillar cancer and positive lymph nodes. VC 2015 Wiley Periodicals, Inc. Head Neck 38: E705 E711, 2016 KEY WORDS: squamous cell carcinoma, head and neck, survival, neck dissection, lymph node density, tonsillar neoplasm INTRODUCTION The presence of clinically involved lymph nodes is commonly accepted as a major determinant of survival outcome in patients with head and neck cancer. 1,2 This is also accepted in cancer in the tonsillar fossa of the oropharynx; lymph node metastases are recognized as an adverse prognostic factor. 3 The American Joint Committee on Cancer guidelines on stage simply regards all patients with lymph node metastasis as stage III or IV. However, the prognosis is frequently not the same, but heterogeneous, in patients with the same stage, so that makes it difficult to predict prognostic outcome from staging alone. In some previous studies, nodal classification was shown not to reflect prognostic outcome effectively. 4,5 Some modern studies using multivariate analyses reported that, among patients with positive neck metastases, lymph node classification does not necessarily predict prognosis, especially after adjuvant radiotherapy. 6 8 Several authors have recently asserted that the relative proportion of positive nodes among dissected nodes, not the absolute number of positive nodes, can more effectively reflect outcome for patients with head and neck cancer. Lymph node density (the number of positive lymph nodes/total number of excised lymph nodes) has *Corresponding author: M.-S. Kim, Department of Otolaryngology Head and Neck Surgery, Catholic University College of Medicine, St. Mary s Hospital, 505 Banpodong Seochogu, The Catholic University of Korea, Seoul, Korea 137-040. E-mail: entkms@catholic.ac.kr emerged as an alternative staging system for predicting survival after surgery for squamous cell carcinoma of the oral cavity. 9 12 The extent of neck dissection, surgical technique, and the level of histopathological scrutiny determine the degree to which the regional lymph nodes are examined for neck metastasis and, hence, the probability of identifying metastasis in lymph nodes at risk. 13,14 The following factors are expected to affect lymph node density: (1) the number of positive lymph nodes (determined by the true characteristics of the tumor); (2) the number of dissected nodes (determined by the surgical extent of dissection and the surgical technique of the surgeon); and (3) the number of nodes analyzed pathologically (determined by the skill and experience of the pathologist). 9 Oropharyngeal cancer is a common head and neck cancer and, in particular, tonsillar cancer has increased recently. More than half of the patients with tonsillar cancer present with typically lymphatic metastasis (stage III or IV) and the prognosis is commonly predicted using the T classification (tumor size) and N classification (lymph node status). 15,16 To our knowledge, no report in the English-language literature has assessed the prognostic significance of lymph node density in predicting the risks of disease recurrence and survival in patients with oropharyngeal cancer, especially in patients with tonsillar cancer separately. We designed this study to assess the value of lymph node density in predicting survival after surgery for patients with tonsillar cancer and positive neck nodes. HEAD & NECK DOI 10.1002/HED APRIL 2016 E705

PARK ET AL. PATIENTS AND METHODS Patients We retrospectively reviewed the clinical and pathological data of 64 patients who were diagnosed with tonsillar squamous cell carcinoma with lymph node metastasis and who underwent surgery at our institute between 1997 and 2011. The Institutional Review Board approved this retrospective review of medical records and the use of archived tumor specimens. In total, 64 patients who met the following criteria were included: (1) surgically resectable squamous cell carcinoma of tonsil with lymph node metastasis without distant metastasis initially (T1 3, N1 2c, and M0); (2) no previous treatment for the same diagnosis (cancer of the head and neck) before hospitalization; and (3) patients who underwent lymph node dissection with surgery to the primary lesion before chemoradiation therapy. Patients who did not undergo neck dissection or who underwent dissection after definitive chemoradiation were excluded. Patients whose follow-up periods were <12 months were also excluded. Treatment modality Prophylactic selective neck dissection was performed in clinically negative neck sites, and a modified radical neck dissection was performed in clinically positive neck sites. Adjuvant treatment was considered when the cancer-free margin was insufficient (<5 mm), or when extracapsular invasion was found, or if multiple nodal metastases were found. Most patients were followed every 6 months for the first 2 years, and then every 12 months. The followup assessment tool was CT or MRI, and liver/bone scans or positron emission tomography/ct. The patients were staged according to the 2002 American Joint Committee on Cancer (AJCC) staging system. Histopathological review Resected tissue was fixed in 10% formaldehyde and embedded in paraffin wax. Tissues were evaluated by a pathologist who specialized in head and neck pathology. The lymph node density was calculated from the total number of positive nodes divided by the total nodal yield in each patient regardless of whether the patients had undergone unilateral or bilateral neck dissections. In situ hybridization for human papillomavirus In situ hybridization for human papillomavirus (HPV) was performed with an automated Benchmark system (Ventana Medical Systems, Tucson, AZ) using the INFORM HPV III Family 16 Probe (a cocktail of HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 66; Ventana Medical Systems) in accord with the manufacturer s protocol. The nuclear hybridization signal was assessed by a pathologist who specialized in head and neck pathology. TABLE 1. Demographic and clinical data of patients (n 5 64). Variables No. of patients % Mean age, y 55.95 6 8.447 (31 79) 64 100 Sex Male 50 78.1 Female 14 21.9 T classification T1 13 20.3 T2 42 65.6 T3 9 14.1 N classification N1 11 17.2 N2a 4 6.2 N2b 41 64.1 N2c 8 12.5 Treatment modality Surgery only 8 12.5 Surgery 1 radiation 14 21.9 Surgery 1 radiation 1 chemotherapy 42 65.6 Site of neck dissection Unilateral 15 23.4 Bilateral 49 76.6 Type of neck dissection Radical or modified radical 68 53.1 Selective 44 34.3 Follow-up, mo Mean 45.14 6 36.512 100 Statistical analyses All analyses were performed using the SPSS software (ver. 16.0; SPSS, Chicago, IL). A <.05 was deemed to indicate statistical significance. Five-year overall survival (OS), disease-free survival (DFS), locoregional control, and distant metastasis rates were calculated using the Kaplan Meier method; differences in survival rate were assessed using the log-rank test. A univariate association between individual clinical features and survival was determined with the log-rank test. A multivariate analysis using the Cox proportional hazards regression model was performed to compare the factors with prognostic potential, as indicated by the univariate analyses. For better interpretation of continuous variables, the continuous variables were categorized; age, lymph node density, and number of instances of extracapsular spread (ECS) were categorized using cutoff values based on the 5-year DFS using the Kaplan Meier method, logrank tests, and receiver operating characteristic curves, as used in a previous study in oral cavity cancer. 9 12 The following cutoff values were used: age (30 70 years), lymph node density (0.01 1), and number of ECS (1 8). In the presence of multiple cutoff values for a single variable, the Cox regression method (forward regression) was used to identify the cutoff with the highest statistical significance with respect to 5-year DFS. The primary endpoint was DFS, calculated as the length of time from diagnosis until first documented recurrence or death. The secondary endpoint was OS, calculated as the length of time from diagnosis until death or last follow-up. The following cutoffs achieved the highest statistical significance: lymph node density (<0.05 vs 0.05), age (<65 vs 65 years), and number of ECS (<3 vs3 nodes). RESULTS The study population consisted of 64 patients with primary tonsillar cancer with lymphatic metastasis treated by E706 HEAD & NECK DOI 10.1002/HED APRIL 2016

LYMPH NODE DENSITY IN PATIENTS WITH TONSILLAR CANCER TABLE 2. Univariate analysis of parameters for predicting prognosis in patients with N(1) tonsillar cancer (n 5 64). Parameter No. of patients 5-y locoregional control, % 5-y distant metastasis, % 5-y DFS, % 5-y OS, % Sex.981.159.926.253 Male 50 81.9 77.1 55.1 63.1 Female 14 80.8 70.7 61.9 85.7 Age, y.513.931.343.259 65 13 84.6 83.3 44.0 60.4 <65 51 84.0 72.8 56.8 66.9 Smoking status.977.916.619.824 Smoker 39 82.8 74.3 58.5 66.3 Nonsmoker 25 77.8 68.6 45.6 63.0 T classification.633.524.807.653 T1/T2 55 83.4 74.5 56.9 66.2 T3 9 71.4 58.3 41.7 64.8 N classification.06.595.035.041 N1/N2a 15 100 80.0 80.0 86.2 N2b/N2c 49 75.8 69.7 47.7 60.5 Bilateral metastasis.118.526.059.240 Yes 8 77.6 81.8 25.0 46.9 No 56 100 70.2 59.8 68.8 Lymph node density.013.010 <.0001.001 0.05 42 71.6 60.5 39.0 53.0 <0.05 22 100 95.2 90.9 95.5 Presence of ECS.083.032.020.108 Yes 37 72.1 59.6 43.5 60.7 No 27 92.6 88.5 70.5 73.5 No. of ECS.224.001.025.547 3 17 68.8 37.6 26.9 57.5 <3 47 85.9 86.1 65.5 68.8 HPV.550.301.125.021 Positive 23 85.0 81.8 69.6 85.4 Negative 41 79.8 67.5 47.7 56.2 Histological grade.088.881.080.118 Well/moderate 59 84.1 72.5 57.9 68.5 Poor 5 53.3 75 20.0 40.0 Margin status.322.543.254.982 Close margin (<1 mm) 18 73.3 63.5 43.7 66.2 Negative 46 84.6 75.5 58.9 65.8 Lymphatic invasion.104.461.717.838 Yes 52 77.3 71.1 53.9 65.4 No 12 100 79.5 57.1 66.8 Vascular invasion.021.113.035.129 Yes 7 47.6 40.0 14.3 57.1 No 57 86.0 76.2 58.2 67.0 Perineural invasion.480.002.005.152 Yes 5 80.0 26.7 20.0 40.0 No 59 82.3 75.8 57.6 68.0 Muscle invasion.782.616.544.895 Yes 37 78.5 74.8 52.0 64.8 No 12 77.1 77.9 56.2 62.7 Unknown 15 Treatment modality.047.038.061.068 Surgery only 8 100 100 56.2 56.2 Surgery 1 RT 14 100 92.9 84.4 90.9 Surgery 1 RT 1 chemotherapy 42 71.5 60.6 44.8 59.8 Abbreviations: ECS, extracapsular spread; HPV, human papillomavirus; RT, radiotherapy. The figures in bold indicate statistical significance. surgical ablation plus neck dissection between January 1997 and December 2011. Mean follow-up was 45 6 36 months. Details of the demographic and clinical data for the 64 patients are summarized in Table 1. In the total study population, 112 neck dissections were performed in 64 patients (49 bilateral procedures). The types of neck dissection were 68 (53.1%) levels I to V and 44 (34.3%) levels II to IV. The median nodal yield HEAD & NECK DOI 10.1002/HED APRIL 2016 E707

PARK ET AL. FIGURE 1. Kaplan Meier curves for the prognostic outcomes in all patients according to lymph node density (LND; n 5 64). (A) Locoregional control, (B) distant metastasis, (C) disease-free survival, and (D) overall survival. per neck dissection was 23 (range, 10 89). In total, 3283 lymph nodes were dissected in 112 neck sites in 64 patients and 368 (11.2%) lymph nodes were positive. In each neck dissection specimen, there were 17 to 118 (mean 6 SD, 51.30 6 22.372) lymph nodes, and 1 to 41 (5.75 6 7.272) of them were positive. The mean lymph node density was 0.1323 6 0.16357 (range, 0.01 0.83). Ten patients developed locoregional recurrence; of these, 9 (90%) died with a mean survival of 10.7 months (SD 6 16.84 months) from the time of recurrence and mean survival of 27.6 months (SD 6 11.63 months) from the time of initial diagnosis. On univariate analysis, lymph node density (0.05), vascular invasion, and treatment modality were significant predictors of 5-year locoregional recurrence. On Cox regression analysis, after adjusting for lymph node density, vascular invasion, and treatment modality in a multivariable analysis, there was no significant parameter in predicting locoregional recurrence. Distant metastases were seen in 13 patients, at a mean follow-up of 37.1 months (SD 6 33.05 months) from initial treatment. Of them, 8 (61.5%) died with a mean survival of 17.8 months (SD 6 21.75 months) from the time of recurrence and a mean survival of 46.9 months (SD 634.02 months) from the time of initial diagnosis. On univariate analysis, lymph node density (0.05), ECS, number of ECS (3), perineural invasion, and treatment modality were significant predictors of distant metastasis. On Cox regression analysis, after adjusting for lymph node density (0.05), ECS, number of ECS (3), perineural invasion, and treatment modality in the multivariate analysis, no parameter was significantly predictive of distant metastasis. When total recurrence (combined locoregional and distant recurrence) was analyzed, it differed significantly according to the lymph node density on multivariate analysis (<0.05 vs 0.05; p 5.001*). The median DFS was 65 months (mean 6 SD, 95.3 6 11.1 months) and the median OS was 101 months (mean 6 SD, 107.1 6 11.7 months). The 5-year DFS rate was 54.8% and 5-year OS rate was 66%. Lymph node density (0.05), ECS, number of ECS (3), vascular invasion, perineural invasion, and N classification (N1/ N2a vs N2b/N2c) were significant predictors of 5-year DFS. Lymph node density (0.05), HPV, and N classification (N1/N2a vs N2b/N2c) were significant predictors of 5-year OS (Table 2). Kaplan Meier curves for the prognostic outcomes in all patients according to lymph node density are summarized in Figure 1. E708 HEAD & NECK DOI 10.1002/HED APRIL 2016

LYMPH NODE DENSITY IN PATIENTS WITH TONSILLAR CANCER FIGURE 2. Kaplan Meier curves for the survival outcomes in N2 classification patients substratified to lymph node density (LND) <0.05 and 0.05 (n 5 53). (A) Disease-free survival and (B) overall survival in N2 classification patients. (C) Disease-free survival and (D) OS in N2b classification patients. Significant prognostic variables identified in the univariate analyses were further evaluated in the Cox regression multivariate model. After adjusting for the other retained variables, categorical lymph node density remained a significant predictor of DFS (p 5.025*; hazard ratio [HR] 5 6.063; 95% confidence interval 5 1.250 29.408). No parameter was significantly predictive of OS (Table 3). Of 64 patients, 53 were classified as N2 (N2a: 4; N2b: 41; and N2c: 8). The 5-year DFS rate was 49.6% and the 5-year OS rate was 61.5% in patients with N2 classification disease. Survival was reevaluated using the clinical N classification system with specific consideration of lymph node density. N2 classification patients were subclassified into 2 groups according to the lymph node density (<0.05 vs 0.05). The higher lymph node density (0.05) group consisted of 41 patients and the lower lymph node density (<0.05) group had 12 patients. The 5-year DFS rate was 91.7% and 5-year OS rate was 91.7% in lymph node density <0.05 and N2 classification patients. The 5-year DFS rate was 40% and the 5-year OS rate was 54.3% in lymph node density 0.05 and N2 classification patients (Figure 2A and 2B). We also analyzed N2b classification patients (with ipsilateral multiple positive nodes) separately and they were subclassified into 2 groups according to the lymph node density (<0.05 vs 0.05). The higher lymph node density (0.05) group consisted of 30 patients and the lower lymph node density (<0.05) group had 11 patients. The 5-year DFS rate was 90.9% and the 5-year OS rate was 90.9% in lymph node density <0.05 and N2b classification patients. The 5-year DFS rate was 42.4% and the 5- year OS rate was 55.5% in lymph node density 0.05 and N2b classification patients (Figure 2C and 2D). DISCUSSION The prognosis of oropharyngeal cancer is commonly predicted using the T classification (tumor size) and N classification (absolute number and size of positive lymph nodes). However, the results of this study showed that the T classification and N classification was not a significant predictor of disease-free or OS on multivariate analysis in patients with tonsillar cancer with lymph node metastasis. Only lymph node density remained as a significant HEAD & NECK DOI 10.1002/HED APRIL 2016 E709

PARK ET AL. independent prognostic predictor. There may be several possible reasons why the T classification was not a valuable prognostic indicator. We included only advanced stage patients with lymph node metastasis, so the size of the primary tumor may not greatly affect the prognosis. Another possible reason is that, given that most of the patients (85.9%) in our study had the same T1/T2 classification, comparing T classification might be meaningless in this population. The N classification in the current AJCC (TNM) staging system gives substantial weight to the absolute number, size, and site of positive lymph nodes. 17 However, the N classification is greatly dependent on the adequacy of the neck dissection and, thus, the adequacy of the nodal yield. The total nodal yield of a neck dissection is affected by the surgical extent and by the skill of the surgeon. It can also be affected by the technique of analysis (eg, serial sectioning, immunohistochemistry) used by the pathologist. However, there is still no literature defining parameters for adequate nodal yield. Thus, considering these biases, lymph node density may be a superior indicator of real nodal burden than the N classification (the absolute number of positive lymph nodes). 12,18,19 We did not analyze patients who underwent different types of neck dissection (selective neck dissection vs modified radical neck dissection) separately; we analyzed them together, using the same cutoff value of lymph node density. A previous report by Liao et al 10 on oral squamous cell carcinoma (OSCC) showed that the median value and optimal cutoff value of lymph node density between selective neck dissection (I III) and modified radical neck dissection were approximately the same, even though there was a 2-fold difference in the mean number of positive nodes. In our study, a mean of 51.3 lymph nodes (range, 17 118) were dissected, which is higher than in previous studies. Of these, a mean 5.75 lymph nodes (range, 1 41) were positive and the mean lymph node density was 0.13 6 0.16, which is also higher than in previous studies on OSCC. In previous articles about patients with OSCC, the mean number of dissected lymph nodes was 34.3 (range, 6 154) and the mean number of positive nodes was 3.4 (range, 1 130), so the mean value of lymph node density was 0.09. 9 12,19 In our neck dissection specimen, there were 17 118 lymph nodes (mean 6 SD, 51.30 6 22.372) and only 4 patients had a nodal yield <20, all of whom showed lymph node density 0.05. Nodal yield, which is determined by the surgeon s experience and by the pathologist s techniques, was sufficient in our study, so higher values of mean lymph node density indicate higher nodal burden in tonsillar cancer, a characteristic of oropharyngeal carcinoma. In this study, in the survival analysis of 64 patients, it was not obvious that the presence of HPV conferred a clear-cut benefit for prognosis. This contrasts several studies in which HPV-positive tumors have an improved prognosis versus patients with HPV-negative tumors. 20 22 The results of our previous study on oropharyngeal cancer also showed that patients positive for high-risk HPV had improved survival compared to patients who were negative for high-risk HPV. The limitation of this study was the small sample size, which might have led to the lack TABLE 3. Multivariate analysis of 5-year locoregional control, distant metastasis, disease-free survival, and overall survival rates in patients with N(1) tonsillar cancer (n 5 64). Outcomes HR 95% CI Locoregional control Lymph node density 0.05.964 Vascular invasion.262 Treatment modality.950 Distant metastasis Lymph node density 0.05.212 Presence of ECS.813 No. of ECS 3.236 Perineural invasion.107 Treatment modality.116 DFS 6.063 1.250 29.408 Lymph node density 0.05.025 Presence of ECS.204 No. of ECS 3.674 Vascular invasion.268 Perineural invasion.124 N classification.496 OS 8.103 0.921 71.255 Lymph node density 0.05.059 Positive for HPV.112 N classification.496 Abbreviations: HR, hazard ratio; CI, confidence interval; ECS, extracapsular spread; DFS, disease-free survival; OS, overall survival; HPV, human papillomavirus. The figures in bold indicate statistical significance. of statistically significant results. As we strictly excluded patients who did not meet the inclusion criteria and included only previously untreated squamous cell carcinoma of the tonsil (the epicenter of the cancer is regarded as the tonsil, not another site in the oropharynx) with lymph node metastasis without distant metastasis initially (T1 3, N1 2c, and M0), the number of patients was relatively small. A study with a larger sample size might indicate a significant difference in prognosis according to HPV status. In our study, lymph node density adversely affected disease-specific survival but not OS on multivariate analysis. However, lymph node density was not found to affect locoregional or distant metastasis in the multivariate analysis (Table 3). Given the small sample size, there does not seem to be a difference in locoregional or distant metastasis according to the lymph node density (<0.05 vs 0.05), as it was analyzed separately. When total recurrence (combined locoregional and distant recurrence) was analyzed, it differed significantly according to the lymph node density. This could account for the difference in survival according to lymph node density (ie, lymph node density adversely affected disease control and locoregional/distant control). In our previous study, the presence or absence of extratonsillar invasion to pharyngeal constrictor muscles seemed to be a reliable predictor of lymphatic metastasis and prognostic outcomes in early tonsillar cancer. 23 We supposed that the fibrous barrier surrounding the muscle may work as a barrier to cancer invasion; once the tumor penetrates the barrier, it seems easier for the cancer to then spread into the parapharyngeal space and the cervical lymphatic networks. In this study, only patients with E710 HEAD & NECK DOI 10.1002/HED APRIL 2016

LYMPH NODE DENSITY IN PATIENTS WITH TONSILLAR CANCER advanced-stage tonsillar cancer (T1 3, N1 2c) were included and there was no prognostic difference according to pharyngeal constrictor muscle invasion among the patients with tonsillar cancer with lymph node metastasis. A few data about lymph node density in patients with OSCC are currently available. 9 12,19 However, to our knowledge, this is the first reported study in which lymph node density was analyzed in patients with homogeneous tonsillar cancer with positive lymph nodes. The main limitation of this study was that its retrospective, singlecenter nature limits the generalizability of the results, and larger multicenter studies are needed. CONCLUSION Many patients with tonsillar cancer present with typically lymphatic metastasis initially. The nodal classification, according to the AJCC staging system, seems not to reflect prognostic outcome effectively in these patients. Considering several biases that may influence nodal classification, lymph node density seems to be a superior indicator, reflecting true nodal burden. Lymph node density may be a useful independent predictor of prognosis in patients with tonsillar cancer and positive lymph nodes. REFERENCES 1. Kramer S, Marcial VA, Pajak TF, MacLean CJ, Davis LW. Prognostic factors for loco/regional control and metastasis and the impact on survival. Int J Radiat Oncol Biol Phys 1986;12:573 578. 2. Schuller DE, McGuirt WF, McCabe BF, Young D. The prognostic significance of metastatic cervical lymph nodes. Laryngoscope 1980;90:557 570. 3. Wang X, Xie FY, Han F, Hu WH, Li JS, Xu HM. Tonsillar carcinoma: analyses of the therapy and prognostic factors [in Chinese]. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2009;44:848 852. 4. Freeman DE, Mendenhall WM, Parsons JT, Million RR. Does neck stage influence local control in squamous cell carcinomas of the head and neck? 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