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ORIGINAL ARTICLE Nodal metastasis and survival in oral cancer: Association with protein expression of SLPI, not with LCN2, TACSTD2, or THBS2 Rob Noorlag, MD,1 * Petra van der Groep, PhD,2,3 Frank K. J. Leusink, MD,1 Sander R. van Hooff, MSc,4 Micha€el H. Frank, DDS, MD,1 Stefan M. Willems, MD, PhD,2 Robert J. J. van Es, DDS, MD, PhD1 1 Department of Oral and Maxillofacial Surgery, University Medical Center Utrecht, Utrecht, The Netherlands, 2Department of Internal Medicine and Dermatology, University Medical Center Utrecht, Utrecht, The Netherlands, 3Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands, 4Department of Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands. Accepted 21 April 2014 Published online 19 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.23716 ABSTRACT: Background. Gene expression profiling revealed a strong signature predicting lymph node metastases in oral squamous cell carcinoma (OSCC). Four of the most predictive genes are secretory leukocyte protease inhibitor (SLPI), lipocalin-2 (LCN2), thrombospondin-2 (THBS2), and tumor-associated calcium signal transducer 2 (TACSTD2). This study correlates their protein expression with lymph node metastases, overall survival (OS), and disease-specific survival (DSS). Methods. Two hundred twelve patients with OSCC were included for protein expression analysis by immunohistochemistry. Results. SLPI expression correlates with lymph node metastases in the whole cohort, not in a subgroup of cT1 to 2N0. SLPI expression corre- INTRODUCTION Head and neck cancer is the sixth most common malignancy worldwide, of which one-third consists of oral squamous cell carcinoma (OSCC). Its incidence in The Netherlands, being 6.2 per 100 000 in 2010, is rising annually. Despite improvements in therapy, the 5-year survival rate has not changed over the past decades and remains approximately 50%.1–3 The prognosis depends on numerous clinical and pathological factors, of which cervical lymph node metastases is a major determinant.4 To perform appropriate treatment, it is therefore pivotal to determine the nodal status of the neck. However, in 30% to 40% of the patients, even optimal imaging is unable to detect nodal disease.5 To improve the negative predictive value for metastasis detection in OSCC, new diagnostic tools, such as molecular diagnosis and tumor profiling, have been developed.5 Roepman et al6 showed that microarray gene expression profiling could be used to predict lymph node metastases for OSCC. *Corresponding author: R. Noorlag, Department of Oral and Maxillofacial Surgery, University Medical Center Utrecht, G05.222, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: [email protected] Contract grant sponsor: Stefan M. Willems was funded by the Dutch Cancer Society (clinical fellowship: 2011-4964). Additional Supporting Information may be found in the online version of this article. 1130 HEAD & NECK—DOI 10.1002/HED AUGUST 2015 lates with OS (hazard ratio [HR] 5 0.61) and DSS (HR 5 0.47) in multivariate analysis. LCN2, THBS2, and TACSTD2 show no correlation with lymph node metastases, OS, or DSS. Conclusion. Although SLPI expression correlates with lymph node metastases, it has no additional value in determining lymph node metastases in early oral cancer. However, it is an independent predictor for both OS C 2014 and DSS and therefore a relevant prognostic biomarker in OSCC. V Wiley Periodicals, Inc. Head Neck 37: 1130–1136, 2015 KEY WORDS: oral cancer, secretory leukocyte protease inhibitor (SLPI), immunohistochemistry, lymph node metastases, survival Recently, this gene expression signature has been validated in a multicenter study and focused on the prediction of lymph node metastases in early oral cancer.7 Four of the strongest predictive genes in this signature encode for the proteins lipocalin-2 (LCN2), thrombospondin-2 (THBS2), tumor-associated calcium signal transducer 2 (TACSTD2), and secretory leukocyte protease inhibitor (SLPI). LCN2 participates in carcinogenesis by favoring iron uptake from the extracellular space within the tumor cell, a fundamental process for maintaining neoplastic cell multiplication. Although increased LCN2 plasma levels in patients with OSCC were found, no correlation was established with regional or distant metastases.8 THBS2 suppresses angiogenesis by inhibiting endothelial cell migration, inducing endothelial cell apoptosis and preventing the interaction of growth factors with the cell surface receptors of the endothelial cell.9 In supraglottic cancer, THBS2 gene expression seems inversely correlated with nodal metastases.10 TACSDT2, also known as TROP-2, belongs to a unique family of transmembrane glycoproteins that has a regulatory role in cell-cell adhesion and has a key controlling role in human cancer growth. Tumor development is quantitatively driven by TACSTD2 expression levels in many tumors.11 Fong et al12 correlated increased TACSTD2 expression in OSCC with decreased overall survival (OS), but found no correlation with nodal metastases. SLPI TABLE 1. Baseline characteristics. Variables Sex Female Male Age at diagnosis, y Median (range) Smoking Never Ceased >1 y Active smoker or ceased <1 y Missing Alcohol Never Occasionally 1–4 drinks/d 5 drinks/d Missing Clinical N classification cN0 cN1–3 Clinical T classification cT1 cT2 cT3 cT4 Pathological N classification pN0 pN1–3 Pathological T classification pT1 pT2 pT3 pT4 Infiltration depth <4.0 mm 4.0 mm Differentiation grade Good/moderate Poor/undifferentiated Vasoinvasion No Yes Missing Bone invasion No Yes Perineural growth No Yes Missing Invasive pattern Cohesive Noncohesive Missing Extracapsular spread No Yes No nodal metastasis High risk HPV status Negative Positive Abbreviation: HPV, human papillomavirus No. of patients (%) 84 (40) 128 (60) 61 (26–87) 43 (20) 34 (16) 133 (63) 2 (1) 46 (22) 49 (23) 71 (33) 44 (21) 2 (1) 146 (69) 66 (31) 44 (21) 79 (37) 19 (9) 70 (33) 97 (46) 115 (54) 44 (21) 73 (34) 22 (10) 73 (34) 19 (9) 193 (91) 173 (82) 39 (18) 39 (18) 169 (80) 4 (2) 152 (72) 60 (28) 122 (57) 80 (38) 10 (5) 44 (21) 167 (79) 1 (<1) 59 (28) 56 (26) 97 (46) 210 (99) 2 (1) EXPRESSION, NODAL METASTASIS, AND SURVIVAL IN ORAL CANCER SLPI, also known as antileukoproteinase, is a protease inhibitor of neutrophil elastase, cathepsin G, chymotrypsin, and trypsin,13,14 enzymes with extracellular matrix degradative properties, and associated with cancer development, invasiveness, and progression.15,16 SLPI expression has recently been associated with carcinogenesis and metastasis in various types of cancer, although its role remains controversial. In gastric and prostate cancer, increased SLPI expression is associated with invasiveness, metastases, and a worse survival.17–19 This is in contrast with the reports of SLPI expression in ovarian cancer, in which SLPI expression is associated with decreased tumor growth and fewer nodal metastases.20 In head and neck cancer, SLPI mRNA and protein levels seem to be increased compared to normal tissue.21 Reports correlating SLPI expression with lymph node metastases are contradicting.22,23 The purpose of this study was to present the correlation of the aforementioned protein expressions with lymph node metastases, OS, and disease specific survival (DSS), and evaluate their potential role as biomarkers for treatment decision and predictors of survival in OSCC. MATERIALS AND METHODS Patient selection Patients with histologically confirmed OSCC, whose primary treatment was by surgery between 1996 and 2005 in our institute were included in this study. Patients who had had a synchronous primary tumor or a previous malignancy in the head and neck region were excluded. Two hundred twelve patients were selected on the availability of both representative formaldehyde-fixed, paraffin-embedded tissue blocks and frozen tissue samples of the primary tumor. A dedicated head and neck pathologist examined all hematoxylin-eosin stained slides with special attention to the following pathological characteristics: type of tumor, differentiation grade, infiltration depth, invasive pattern, perineural growth, vasoinvasive growth, extracapsular spread, and bone invasion. A tissue microarray was made of the paraffinembedded tissue. For each tumor block, 3 central tissue cylinders and 3 tissue cylinders at the tumor front with a diameter of 0.6 mm were punched out, avoiding areas of necrosis, and arrayed in a recipient paraffin block. Normal epithelium from the floor of the mouth, gingiva, and tonsil was incorporated in each block to ensure similarity of staining between the different blocks, as described earlier.24 From each patient, clinical characteristics, clinical TNM classification (based on palpation, ultrasoundguided fine-needle aspiration, MRI or CT, and classified in a multidisciplinary panel), pathological TNM classification, and cause of death were retrieved from the medical records, as listed in Table 1. Gene expression For a subgroup of 83 tumors, normalized gene expression data were available from an earlier study for which methods has been described in detail earlier.7 In short, frozen tumor samples were sectioned, aliquoted in Trizol HEAD & NECK—DOI 10.1002/HED AUGUST 2015 1131 NOORLAG ET AL. (Life Technologies, Frederick, MD), and sent to Agendia Laboratories (Amsterdam, The Netherlands) for expression profile analysis. Tumor areas with a percentage of at least 50% were assessed on hematoxylin-eosin stained sections and taken in parallel; RNA was isolation and amplification. Tumor sample RNA was labeled as Cy3, and reference RNA was labeled Cy5. As a reference, the Universal Human Reference RNA (Agilent Technologies, Santa Clara, CA) was used. Samples were hybridized on full-genome Agilent arrays. Raw fluorescence intensities were quantified using Agilent Feature Extraction software and imported into R/Bioconductor (http://www.bioconductor.org/) for normalization (loess normalization using the LIMMA package) and additional analysis. Human papillomavirus type 16 analysis Human papillomavirus type 16 (HPV-16) active tumors were determined by p16 immunohistochemistry (IHC) followed by GP 51/61 polymerase chain reaction in positive p16 staining, a reliable algorithm for detection of HPV-16 in paraffin-embedded head and neck cancer specimens, as described by Smeets et al.25 Immunohistochemistry IHC was performed on 4-mm thick paraffin sections. The tissue sections were deparaffinized with xylene and rehydrated. Endogenous peroxidase activity was blocked for 15 minutes in a 0.3% hydrogen peroxide phosphatecitrate buffer. Then, tissue sections were washed in water and subsequently subjected to antigen retrieval by boiling the slides in ethylenediaminetetraacetic acid buffer, pH 9.0 (SLPI) or citrate buffer, and pH 6.0 (TACSTD2, LCN2, and THBS2) for 20 minutes. Sections were cooled down within the buffers for 30 minutes. After washing with phosphate-buffered saline (PBS) for 5 minutes, tissue slides were incubated with the primary antibody SLPI (clone 31; HyCult biotechnology, Uden, The Netherlands; dilution 1:50), primary antibody TACSTD2 (AF650, R&D Systems, Oxon, England; dilution 1:50), primary antibody LCN2 (MAB1757; R&D Systems, Oxon, England; dilution 1:50), or primary antibody THBS2 (sc12313, Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:50) for 60 minutes. After washing with PBS (3 times), incubation with poly-HRP Goat anti-Mouse/Rabbit/Rat (Brightvision, Immunologic, Duiven, The Netherlands; ready to use) for 30 minutes was followed by washing with PBS (3 times). Slides were then developed with diaminobenzidine for 10 minutes, counterstained with hematoxylin, followed by dehydration, and mounted. Evaluation of immunohistochemical staining A core was considered inadequate/lost when the core contained <5% tumor tissue or when >95% of the core contained no tissue. Patients were only included in the study when one or more tumor cores were available. When two or more cores were available from one patient, the mean (SLPI, THBS2, or LCN2) or maximum (TACSTD2) score was calculated for that patient. The expression of SLPI and THBS2 in the primary tumor was evaluated by scoring the percentage of cytoplasm staining. The percentage of cytoplasm stained was 1132 HEAD & NECK—DOI 10.1002/HED AUGUST 2015 classified as 0 (<5%), 1 (5% to 30%), 2 (31% to 75%), or 3 (>75%), see Figure 1.22,26 Expression of TACSTD2 was evaluated by scoring the staining intensity of the cell membrane as 0 5 no, 1 5 weak, 2 5 moderate, or 3 5 strong staining. For LCN2 expression, both the intensity (0 5 no, 1 5 weak, 2 5 moderate, or 3 5 strong) and percentage of cytoplasm staining was scored, multiplying the intensity score with the percentage of staining classified as 1 (25%), 2 (26% to 50%), 3 (51% to 75%), or 4 (>75%) was used as a final score for LCN2 expression. Scores 3 were interpreted as negative, and scores >3 as positive.27 A dedicated head and neck pathologist (S.W.) and a researcher (R.N.), both blinded to the clinical characteristics of the patients, evaluated the protein expressions independently. Consensus was reached regarding discordant findings. Statistical analysis An interrater reliability analysis using the Spearman (for continuous data) and Kappa (for categorical data) statistic was performed to determine consistency of IHC scoring among raters. The Mann–Whitney test was used to determine differences in gene expression between lymph node-positive and lymph node-negative tumors. Receiver operating characteristic (ROC) curve analysis was used to determine cutoff points for the correlation of gene and protein expression and nodal metastases. Correlations between gene expression or protein expression and lymph node metastases were assessed by the chi-square test. OS was defined as the length of the time interval from surgery to death from any cause. DSS was defined as the time interval from surgery to either death because of or as a recurrence of the disease. ROC curve analysis was used to determine cutoff points for protein expression and survival. The OS and the DSS curves were constructed using the Kaplan–Meier method and the log-rank test was used to test for significance. Prognostic value was examined by univariate and multivariate analyses using the Cox proportional hazards regression model. Characteristics with a p < .10 in univariate analysis and potential confounders were included, and the model was created with backward logistic regression. All p values were based on 2-tailed statistical analysis and p < .05 was considered statistically significant. Statistical analysis was performed using the SPSS 20.0 statistical package (SPSS, Chicago, IL). RESULTS Human papillomavirus type 16 analysis Of our 212 tumor samples, 36 showed p16 overexpression on IHC. Of this group, only 2 samples (0.9%) proved to be true HPV-16 positive with polymerase chain reaction, see Table 1. Immunohistochemistry: descriptive analysis A total of 1080 cores (85%) stained with SLPI antibody, 1119 cores (88%) stained with THBS2 antibody, 1077 cores (85%) stained with LCN2 antibody, and 1077 cores (84%) stained with TACSTD2 antibody were available for analysis. There was at least 1 core of each tumor SLPI EXPRESSION, NODAL METASTASIS, AND SURVIVAL IN ORAL CANCER FIGURE 1. Scoring system for secretory leukocyte protease inhibitor (SLPI), lipocalin-2 (LCN2), tumor-associated calcium signal transducer 2 (TACSTD2), and thrombospondin-2 (THBS2). suitable for each staining so no tumors were excluded from analysis. The level of interrater concordance was high, with a Spearman’s rank correlation of 0.975 (p < .001) for continuous data and a Kappa of 0.874 (95% confidence interval, 0.806–0.942; p < .001) for categorical data, scatter plot in Supplemenary Figure 1, online only. The IHC results are given in Table 2. LCN2 (p < .001), TACSTD2 (p 5 .002), and THBS2 (p 5 .001), see Figure 2. Optimal cutoff points determined with ROC curve analysis (Supplemenary Figure 2 and Supplementary Table 1, online only) revealed that gene expression is a significant predictor of lymph node metastasis for all 4 genes. SLPI, LCN2, and TACSTD2 mRNA are downregulated and THBS2 mRNA is upregulated in lymph node-positive patients, see Table 3. Gene expression and lymph node metastases Analysis of 83 OSCCs show a statistically significant differential gene expression between lymph node-positive and lymph node-negative patients for SLPI (p 5 .001), Immunohistochemistry and lymph node metastases In the whole cohort, SLPI expression is significantly correlated with lymph node metastases (p 5 .010), using HEAD & NECK—DOI 10.1002/HED AUGUST 2015 1133 NOORLAG ET AL. TABLE 2. Immunohistochemical descriptive results. Variables SLPI THBS2 TABLE 3. Gene expressions correlated with lymph node metastases. LCN2 TACSTD2 No. of cores (%) Tumor 1080 1119 1077 (85) (88) (85) No tumor 50 (4) 86 (7) 68 (5) No core 142 67 (5) 127 (10) (11) No. of cores per tumor (212 tumors) 6 110 107 109 5 50 60 53 4 26 20 23 3 16 18 16 2 8 6 7 1 2 1 4 Score per tumor (212 tumors) Score 5% 103 24 3 126 6% to 30% 90 100 >3 86 31% to 75% 19 87 >75% 0 1 Lymph node metastases 1074 (84) Gene expression 73 (6) 125 (10) SLPI M 0.130 M >0.130 LCN2 M -0.360 M >-0.360 TACSTD2 M 0.027 109 51 24 16 9 3 Score 0 11 21 31 22 63 73 54 No. of patients No (%) 57 26 18 (32) 19 (73) 39 (68) 7 (27) <.001 43 40 11 (26) 26 (65) 32 (74) 14 (35) <.001 47 14 (30) 33 (70) 36 23 (64) 13 (36) 34 49 7 (79) 30 (61) 27 (21) 19 (39) Yes (%) p value .002 M >0.027 THBS2 M >0.100 M 0.100 <.001 Abbreviations: SLPI, secretory leukocyte protease inhibitor; THBS2, thrombospondin-2; LCN2, lipocalin-2; TACSTD2, tumor-associated calcium signal transducer 2; M, log2 (sample/reference pool). Abbreviations: SLPI, secretory leukocyte protease inhibitor; THBS2, thrombospondin-2; LCN2, lipocalin-2; TACSTD2, tumor-associated calcium signal transducer 2. 30% as the cutoff point, with a negative predictive value of 74%. However, in a subgroup of early cancers, which were clinically lymph node-negative (cT1–T2N0), significance disappeared. Analysis of protein expression of LCN2, TACSTD2, and THBS2 revealed no significant correlation with lymph node metastases in the whole cohort or in a subgroup of cT1-T2N0 tumors (Table 4). For ROC curves, see the Supplementary Figure 3 and Supplementary Table 2, online only. Immunohistochemistry and survival Kaplan–Meier curves show a significant difference between SLPI expression for both OS and DSS. The 5year OS and DSS of patients with 5% or more staining is, respectively, 62% and 76%, in contrast with the patients with <5% staining with a 5-year OS and DSS of, respec- tively, 41% and 53%, see Figure 3. For ROC curves, see Supplementary Figure 4 and Supplementary Table 3, online only. LCN2, TACSTD2, and THBS2 expression showed no correlation with OS or DSS (data not shown). Cox proportional hazard regression model revealed SLPI protein expression as an independent predictor for both OS and DSS. Other independent predictors of OS are age, clinical N classification, the pathological characteristics of vasoinvasion, noncohesive invasive pattern, and bone invasion, see Table 5. For DSS, clinical N classification, and extracapsular spread are other independent predictors, see Table 6. DISCUSSION Biomarkers with diagnostic and prognostic value for determining lymph node metastases and predicting survival in OSCC are crucial for determining treatment TABLE 4. Biomarkers correlated with lymph node metastases. Whole cohort (212 tumors) Biomarker expression FIGURE 2. Gene expression and nodal status. Mann–Whitney test, **p < .01, ***p < .001. SLPI, secretory leukocyte protease inhibitor; THBS2, thrombospondin-2; LCN2, lipocalin-2; TACSTD2, tumor-associated calcium signal transducer 2; pN1, pathologic lymph node positive; pN0, pathologic lymph node negative. 1134 HEAD & NECK—DOI 10.1002/HED AUGUST 2015 SLPI 30% > 30% LCN2 Score 3 Score > 3 TACSTD2 0 to 11 21 to 31 THBS2 5% > 5% pN0 (%) pN1 (%) cT1–2N0 (101 tumors) p value pN0 (%) pN1 (%) p value 83 (43) 110 (57) .01 55 (63) 14 (74) 5 (26) 9 (69) 33 (37) 4 (31) NS 51 (41) 75 (59) 46 (54) 40 (46) NS 33 (60) 31 (67) 22 (40) 15 (33) NS 35 (41) 50 (59) 62 (49) 65 (51) NS 31 (65) 33 (62) 17 (35) 20 (38) NS 13 (52) 12 (48) 84 (45) 103 (55) NS 7 (78) 57 (62) 2 (22) 35 (38) NS Abbreviations: pN0, pathologic lymph node-negative; pN1, pathologic lymph node-positive; SLPI, secretory leukocyte protease inhibitor; NS, not significant; THBS2, thrombospondin-2; LCN2, lipocalin-2; TACSTD2, tumor-associated calcium signal transducer 2. SLPI EXPRESSION, NODAL METASTASIS, AND SURVIVAL IN ORAL CANCER FIGURE 3. Secretory leukocyte protease inhibitor (SLPI) expression and survival. Log-rank test, overall survival (OS) p 5 .002, disease-specific survival (DSS) p < .001. planning and possible targets for personalized treatment in the future. Gene expression profiling revealed LCN2, THBS2, TACSTD2, and SLPI as genes with a strong statistically significant differential gene expression between pN1 and pN0 patients with early oral cancer.7 Although the precise function of these genes is yet not fully understood, an explanation might be their joint role in matrix remodeling.28 In our cohort of OSCC, LCN2, THBS2, and TACSTD2 showed no correlation with lymph node metastases or survival on protein expression level despite significant differences in staining between tumors on IHC. There are several reasons for the poor correlations between mRNA and protein expression levels. First, there is the undervalued role of complicated and varied posttranscriptional and translational mechanisms, which are not yet sufficiently defined. Second, proteins differ substantially in degradation and in vivo half-lives.29,30 Finally, both protein and mRNA experiments contain a significant amount of errors and noise that limits our ability to get a clear picture.30 A combination of one or more of these factors may explain these poor correlations, which is in line with several studies that report discrepancies between mRNA and protein correlations with prognostically relevant outcomes.31–33 Therefore, mRNA levels cannot be used as surrogates for corresponding protein levels without validation. To our knowledge, this is the first study that shows the correlation between SLPI expression by IHC and lymph TABLE 5. Cox regression analysis of overall survival. Variables HR (95% CI) p value 0.56 (0.39–0.81) .002 0.61 (0.41–0.89) 1.04 (1.02–1.06) 3.81 (2.58–5.65) 1.59 (1.02–2.46) 2.69 (1.53–4.73) 1.75 (1.17–2.63) .010 <.001 <.001 .040 .001 .007 node metastases, OS, and DSS in a large cohort of patients with OSCC. Despite a significant correlation between SLPI protein expression and lymph node metastases in the whole cohort, SLPI expression has no additional diagnostic value as a predictor for lymph node metastases in a subgroup of early cancers, which are clinically lymph nodenegative in this cohort of OSCC. Previous studies report different results correlating SLPI expression with lymph node metastases in head and neck squamous cell carcinoma. Westin et al23 found no significant correlation, whereas Cordes et al22 found a strong correlation between lower SLPI protein expression and an increased risk of lymph node metastases (p < .001). However, there are some drawbacks in comparing these studies. First, they did not analyze whether SLPI had additional value as a predictor for lymph node metastases. Second, most cancers in the Cordes study were located in the larynx, oropharynx, and hypopharynx (87.6%). This might explain the difference with our findings in oral cancer, as also for other genes, such as EGFR, pAkt, and PTEN, it is known that its expressions vary between oral and oropharyngeal carcinomas.34 Although Won et al34 suggested initially that the difference in HPV-related pathogenesis of the tumors could be the reason for different protein expression in head and neck subsites, a later study by Hoffmann et al35 identified SLPI expression to be an HPV-independent predictor for lymph node metastases in head and neck cancer. In addition, their cohort contained mainly laryngeal and oropharyngeal carcinomas. Another possibility for the TABLE 6. Cox regression analysis of disease-specific survival. SLPI expression Multivariate model SLPI expression Age cN classification Vasoinvasion Noncohesive invasive pattern Bone invasion Variables Abbreviations: HR, hazard ratio; CI, confidence interval; SLPI, secretory leukocyte protease inhibitor. SLPI expression Multivariate model SLPI expression cN classification Extracapsular spread HR (95% CI) p value 0.43 (0.27–0.67) <.001 0.47 (0.29–0.75) 2.14 (1.38–4.19) 1.93 (1.10–3.38) .002 .002 .022 Abbreviations: HR, hazard ratio; CI, confidence interval; SLPI, secretory leukocyte protease inhibitor. HEAD & NECK—DOI 10.1002/HED AUGUST 2015 1135 NOORLAG ET AL. discrepancy could be the amount of tumors with moderate/strong immunoreactivity, which, in our cohort, was 9.0% compared to 31.4% in the Cordes cohort.22 As a result, the group of tumors with moderate/strong immunoreactivity in our study could be too small to have additional value as a predictor for lymph node metastases. We identified SLPI as an independent predictor for OS and DSS in OSCC. Patients with low SLPI protein expression had a worse OS and DSS compared with patients with any SLPI expression, see Supplementary Figures S3 and S4, online only. Earlier studies suggested a role for SLPI expression as a prognostic biomarker in head and neck cancer. Westin et al23 correlated stronger SLPI expression with well-differentiated tumors in a group of 26 head and neck cancers and suggested its use as a prognostic tool, although they found no significant relationship with lymph node metastases and did not correlate its expression with survival. Alkemade et al36 found the same significant correlation between SLPI expression and tumor differentiation in skin cancer. In addition, Wen et al37 demonstrated inverse correlations of SLPI expression with multiple tumor invasion parameters, which suggests a protective role of SLPI against OSCC invasion. They also suggested SLPI as a potential biomarker in evaluating prognosis and treatment of the clinically lymph node-negative neck, although they did not correlate SLPI expression with lymph node metastases or survival. In conclusion, this is, to our knowledge, the first study that links SLPI expression with both lymph node metastases and survival in a large cohort of patients with OSCC. Although SLPI expression is correlated with lymph node metastases in the whole cohort, it has no additional value in determining lymph node metastases in early cancers that are clinically lymph node-negative. On the other hand, SLPI seems to be an independent predictor for both OS and DSS. Therefore, SLPI IHC might be relevant as a prognostic biomarker for patients with OSCC. However, its molecular role in progression and metastasis of different head and neck cancer subsites needs further investigation. REFERENCES 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. 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