Overexpression of LAPTM4B-35 is a negative prognostic factor in head and neck squamous cell carcinoma

Overexpression of LAPTM4B-35 (lysosomal-associated transmembrane protein 4β-35) is associated with a poor prognosis in numerous malignant tumours. Expression patterns and effects of LAPTM4B-35 on head and neck squamous cell carcinomas (HNSCC) are unknown. The aim of this study was to investigate the prognostic relevance of LAPTM4B-35 in HNSCC. Tissue microarrays were constructed with primary tumours and associated lymph node metastases isolated from 127 patients. The expression of LAPTM4B-35 was investigated by immunohistochemistry and the results were correlated with survival data. LAPTM4B-35 in the primary tumour was highly expressed in 47.2% of the patients (60/127). LAPTM4B-35 expression was significantly associated with tumour stage. Moreover, overexpression of LAPTM4B-35 correlated with a significantly worse disease-free survival (10.23 years vs. not reached) and a higher recurrence rate (40.7% vs. 25%). High expression of LAPTM4B-35 in lymph node metastasis was found in 29.2% of cases. In 19.4% of cases, high LAPTM4B-35 expression was observed in both the primary tumour and corresponding lymph node metastases. In conclusion, our data indicates that overexpression of LAPTM4B-35 is associated with poor prognosis and may therefore serve as a new prognostic marker in HNSCC.


Correlation between LAPTM4B-35 expression and clinicopathological characteristics.
LAPTM4B-35 expression subgroups were then compared to the clinicopathological features of the patient cohort (Table 1). A chi-square test revealed that LAPTM4B-35 expression is significantly linked to UICC stage (p = 0.017). Regarding the T and N classifications, the statistical association was even more significant (p < 0.001). Furthermore, patients with high LAPTM4B-35 expression showed an increased rate of recurrence. After total follow-up, 40.7% of the patients with high LAPTM4B-35 expression had a recurrence compared to 25% in the LAPTM4B-35 low group. No significant differences in OS between high and low LAPTM4B-35 groups was observed, although more patients in the LAPTM4B-35 high group had died (52.5% compared to 41.2%). Moreover, univariate and multivariate analyses were performed to assess if LAPTM4B-35 expression is an independent marker for recurrence and survival ( Table 2). Hazard ratios in relation to LAPTM4B-35 expression, tumour localization, HPV infection, sex, age and classification (T and N) were calculated. Univariate analysis showed that high LAPTM4B-35 expression was associated with a significantly increased risk of recurrence (p = 0.041). In the multivariate analysis, however, LAPTM4B-35 expression association was not significant (p = 0.082).

Discussion
Treatment of head and neck squamous cell carcinoma is currently a therapeutic challenge. Therapeutic options available are often accompanied by significant side effects that lead to a reduction in quality of life 28 . The identification of new molecular biomarkers that predict patient response and outcome could help to distinguish the intensity of therapy applied in different patient subsets.
Expression of the LAPTM4B-35 oncogene has been shown to be a negative prognostic factor in a variety of malignant tumours 29 . This study is the first to investigate the HNSCC expression of LAPTM4B-35 in primary In 29.2% of the corresponding lymph node metastases, LAPTM4B-35 was expressed at a high intensity. This is in contrast to a study on axillary lymph nodes of breast cancer patients where high LAPTM4B-35 expression was found in 19 of 20 (95%) lymph node metastases 30 .   [31][32][33] . Correlation with clinicopathologic characteristics revealed that LAPTM4B-35 overexpression is associated with a significantly poorer DFS and a higher recurrence rate. These data are consistent with previously reported results for other types of cancer. In colorectal carcinoma, patients with high LAPTM4B-35 expression had a worse OS and DFS 17 .In non-small-cell lung cancer, LAPTM4B-35 overexpression was associated with significantly worse 5-year OS and progression free survival 34 . Moreover, high LAPTM4B-35 expression positively correlated with worse OS and progression free survival in breast cancer 13 . Similar results were found in glioblastoma, gastric cancer, prostate cancer, hepatocellular carcinoma and endometrial carcinoma 14,16,18,[35][36][37] . However, these studies did not correlate LAPTM4B-35 expression in lymph node metastases with patient survival data. This study shows that high nodal expression of LAPTM4B-35 in HNSCC is associated with a statistically significant worse DFS.
Head and neck tumours occur in distinct anatomical regions. In the oropharynx, a subset of carcinomas is related to HPV infection associated with a better prognosis 38,39 . We therefore correlated expression of LAPTM4B-35 with survival data of each anatomical region and HPV status in oropharyngeal carcinoma. The analysis showed that patients with HPV positive oropharyngeal cancer and a high LAPTM4B-35 expression had a significantly worse DFS. In cervical carcinomas, which in most cases are associated with HPV infection, high expression of LAPTM4B-35 was also related to poor OS and DFS 16 . A study with the cervical cancer cell line HeLa demonstrated that RNAi-mediated knockdown of LAPTM4B-35 inhibited proliferation, invasion and angiogenesis in vitro. Western blot analysis revealed that LAPTM4B-35 downregulation decreased the levels of vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, MMP-9, cyclin-dependent kinase 12 and hypoxia-inducible factor 1-α expression 40 .
In conclusion, this study examines for the first time the expression of LAPTM4B-35 in HNSCC tumour samples and in associated lymph node metastases at the protein level. Since high LAPTM4B-35 expression is associated with advanced tumour stage and significantly worse DFS as well as a higher recurrence rate, LAPTM4B-35 could serve as a negative prognostic marker in patients with HNSCC.

Materials and Methods
Patients. In total, 127 patients with freshly diagnosed HNSCC were included in the study. The study cohort was assembled by our research group as described previously 41 . All patients were treated with surgery and adjuvant radiotherapy at the Medical University of Vienna in the period between 2002 and 2012. Exclusion criteria were a second primary carcinoma, previous irradiation in the head and neck area and external treatment. Prior to treatment, all patients were presented to the institutional multidisciplinary tumour board. In some cases (i.e. extracapsular spread, perineural invasion), chemotherapy was administered additionally after surgery. This study was approved by the ethics committee of the Medical University of Vienna (EK 1311/2018). All subjects participating in this study gave written informed consent. All tests were performed in accordance with the Declaration of Helsinki and the guidelines for good scientific practice of the Medical University of Vienna.
Tissue microarray construction and immunohistochemistry. Formalin-fixed paraffin-embedded HNSCC specimens obtained from surgically resected specimens were selected. TMA were then constructed using a Galileo TMA CK Series-HTS Tissue computer assisted Microarray Platform (Integrated Systems Engineering Srl, Milan, Italy) as described previously 42 . Samples measured 2 mm in diameter and 4-6 mm in length. Haematoxylin and eosin (H&E) staining was performed to verify histology. Immunohistochemical staining was performed using the Lab Vision Ultra V Block kit (Thermo Fisher Scientific, Waltham, MA, USA) and the Lab Vision Ultravision LP detection system (Thermo Fisher Scientific) according to the manufacturer's protocol 41 .  www.nature.com/scientificreports www.nature.com/scientificreports/ Colon sections were stained as positive controls 17 to determine the appropriate antibody dilution and antigen retrieval buffer. Citrate buffer (pH 6.0) was used for antigen retrieval in the microwave and the anti-LAPTM4B-35 antibody (Abcam, Cambridge, United Kingdom) was diluted 1:200. Slides were incubated for one hour at room temperature. For negative controls, primary antibody was replaced by rabbit immunoglobulin G isotype control (Abcam). Samples were analysed using an Olympus BH-2 microscope (Olympus, Tokyo, Japan).
Based on the intensity of the cytoplasmic staining of neoplastic cells, all samples were assigned to one of four categories (negative, weak, moderate or strong). Analysis was performed by an experienced pathologist (F.O.). The percentage of stained neoplastic cells was then recorded and biopsies were divided into LAPTM4B-35 positive (>10% of cells staining positive) or LAPTM4B-35 negative (<10% of cells staining positive) groups. For statistical analysis, we combined patients with negative and weak expression of LAPTM4B-35 in the group of "LAPTM4B-35 low" patients and patients with moderate and strong expression in the group of "LAPTM4B-35 high" patients.
Statistical analysis. Descriptive statistics were used to report clinical data. To compare categorical data between two groups, the Fisher exact test was used. The chi square test was applied when evaluating three or more groups. OS and DFS were calculated using the Kaplan-Meier method. The log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests were used to compare statistical differences between the established patient groups. Hazard ratios with 95% confidence intervals (CI) were calculated to reflect a significance level of 0.05. Univariate logistic regression was used to assess the effects of demographic (age, sex) and tumour-specific (LAPTM4B-35 expression, HPV status, UICC stage, T & N classification, localization of the primary lesion) characteristics on OS and DFS. Variables with p < 0.2 in the univariate analyses were included in the multivariate logistic regression analysis. Multivariate analyses were performed using the Cox regression model to identify independent variables. P-values < 0.05 were determined as statistically significant. For data analysis and visualization, SPSS software (Version 21.0; SPSS Inc., Chicago, IL, USA) and GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA) were used.