WHAT IS ALREADY KNOWN ON THIS TOPIC

Recent studies have highlighted the effectiveness of epidermal growth factor receptor (EGFR)-targeted drugs in nasopharyngeal carcinomas (NPC), yet drug resistance has emerged as a challenge. SSTR2, highly expressed in NPC, is considered a potential tumour marker and therapeutic target.

WHAT THIS STUDY ADDS

This scientific article explores the correlation between SSTR2 and EGFR expression in NPC at the protein level and investigates their prognostic significance in response to different treatments. Through a retrospective analysis of clinical data and tissue samples, a significant correlation between SSTR2 and EGFR expression was observed, suggesting a potential functional relationship.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYIntroduction

Nasopharyngeal carcinoma (NPC) is an epithelial cancer that occurs in the mucosa lining of the nasopharynx. This cancer is caused by a combination of factors, with the most significant being infection with Epstein-Barr virus (EBV). Persistently elevated EBV antibody levels are considered a major risk factor for NPC.1–3 NPC is a regional cancer, with high incidence rates in southern China and Southeast Asia.2 4 The disease is classified into two types based on histological analysis: non-keratinising squamous cell carcinoma and keratinising squamous cell carcinoma. The majority of NPC cases in China are non-keratinising squamous cell carcinoma and characterised by high levels of EBV antibodies.2 5

Somatostatin (SST) is a potent neuroendocrine hormone that is widely expressed in the human body and plays a crucial role in regulating cell proliferation. In addition to SST receptor 2 (SSTR2), SST has four membrane surface receptors collectively known as SSTRs.6 7 It is widely accepted that SSTRs exert an antiproliferative effect on cells and trigger downstream signalling that promotes apoptosis and inhibits tumour growth factors.8–10 SSTR2 is a G-protein-coupled receptor (GPCR) with diverse biological functions in humans. The activation of the SSTR2 pathway is known to cause cell cycle arrest or apoptosis in low-grade neuroendocrine tumours. However, the opposite occurs in high-grade neuroendocrine tumours and small-cell lung cancers, where SSTR2 is upregulated, leading to tumour growth.1 11 12

Epidermal growth factor receptor (EGFR) belongs to the receptor tyrosine kinases family and overexpression of EGFR has been linked to poor prognosis and cancer progression. The EGFR plays a crucial role in maintaining homeostasis in epithelial tissues during normal physiological conditions. However, mutations or overexpression of EGFR occurred frequently in pathological conditions, leading to the development of tumours including head and neck squamous cell carcinomas. EGFR activates various signalling pathways that transmit signals from the cell surface to the nucleus, promoting cellular survival, proliferation and differentiation.13 14 Anti-human EGFR monoclonal antibodies have been developed as a treatment option for cancers such as NPC. The combination of chemoradiotherapy (CR) and EGFR-targeted therapy has been shown to significantly improve survival rates. LMP1, a proteins product of EBV, governs proliferative signalling pathways, including those associated with EGFR and NF-KB. In NPC, NF-KB signalling has been demonstrated to regulate SSTR2 expression, and additionally, EGFR-mediated MAPK/ERK signalling has also been shown to regulate SSTR2.1 5 8 15–17 However, the potential relationship between EGFR and SSTR2 has not been thoroughly investigated in NPC. Our study is the first to shed light on the intricate relationship between SSTR2 and EGFR in NPC and provides new insights into the potential benefits of EGFR targeted therapy for patients with high SSTR2 expression. In addition, SSTR2 has potential as a new biomarker for poor prognosis in NPC patients. These findings could potentially offer a theoretical foundation for investigating signalling pathways and advancing the development of future targeted therapeutic agents.

Materials and methodsBioinformatics analysis

It was obtained mRNA-seq data from 113 patients with NPC (GSE102349) from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). We normalised and analysed this data to investigate the correlation between EGFR and SSTR2.

Tissue specimens and clinical data

This study included 491 NPC samples and 50 non-cancerous nasopharyngeal epithelium samples. All samples were collected between 2016 and 2021 at The Second Xiangya Hospital in Changsha, China. Among the 491 NPC cases, 65 were clinically early (stages I and II) and 426 were clinically advanced (stages III and IV). A totall of 433 cases had lymph node metastases, and 37 cases had distant metastases. All participants had received a biopsy tissue prior to CR, and complete medical records as well as follow-up records were available. The pathological diagnosis of all specimens was made according to the latest WHO classification for head and neck tumours, and the patients were categorised according to the eighth edition of the UICC（ Union Internationale Contre le Cancer ）/AJCC（ American Joint Committee on Cancer）.

Immunohistochemistry and scores

Immunohistochemistry (IHC) staining was conducted on paraffin-embedded of the NPC and non-cancerous nasopharyngeal epithelium tissues sections (4 µm). The tissues were first dewaxed and hydrated, followed by heating with EDTA repair solution for 7 min. Endogenous peroxidase was blocked using H2O2 (3%) for 30 min. Incubation with primary antibodies for SSTR2 (1:200; Rabbit monoclonal antibody; EP149; MXB) and EGFR (ready-to-use antibody; Rabbit monoclonal antibody; EP38Y; MXB) followed, and then secondary antibody incubation. The visualisation signal was obtained with 3, 3’-diaminobenzidine tetrachloride. We assessed staining only in epithelial cells of benign or malignant tissue and not lymphoid tissue. The staining revealed positive expression of SSTR2 and EGFR on the cytoplasm and cell membrane. The evaluation of protein expression was based on staining intensity and extent, which were semiquantitatively assessed under light microscopy by scorers who were blinded to the case data.18–20 Staining intensity was graded as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong), while staining extent was graded as 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%) and 4 (76%–100%). Scores were calculated by multiplying the staining intensity and extent. A score ≤2 for SSTR2 was considered low expression, while a score >2 was considered high expression. Similarly, an EGFR score of >2 was considered high expression based on data collected for NPC and previous studies.21–24

Statistical analysis

The statistical analyses and graphing in this study were performed using a variety of methods, including χ2 tests, Spearman correlations, univariate Cox regression and multivariate Cox regression analysis, all of which were performed using SPSS Statistics V.26 (SPSS) for macOS. Kaplan-Meier analysis, Bar graphs and Violin plots were conducted with GraphPad Prism V.9.4.1 (GraphPad, La Jolla, California, USA) for macOS. The progression-free survival (PFS) is defined as the period from the date of diagnosis until the patient dies or the disease progresses further. The overall survival (OS) is defined as the period from the date of diagnosis to the date of death or the last known date alive. The p values were analysed as two-sided statistics, and a p<0.05 was considered statistically significant.

ResultsPatients with NPC and those with non-cancerous nasopharyngeal epithelium exhibit differential expression levels of SSTR2 and EGFR

Initially, we performed bioinformatics analysis using the GEO database, which revealed a positive correlation between SSTR2 and EGFR in NPC (R=0.412, moderate positive association) (figure 1A). Subsequently, we investigated the expression levels of SSTR2 and EGFR proteins using IHC. The study demonstrated that patients diagnosed with NPC exhibited a significantly higher expression rate of SSTR2 (45.0%, 221 out of 491) and EGFR (71.7%, 352 out of 491) in high expression compared with the control group, which exhibited a SSTR2 high expression rate of only 6.0% (3 out of 50) and an EGFR high expression rate of 48.0% (24 out of 50) (figure 1B). Furthermore, the distribution of SSTR2 and EGFR scores between the NPC and control groups were represented using violin plots, which showed significant differences between the two groups (p<0.001) (figure 1C,D). These findings suggest that high expression levels of SSTR2 and EGFR are significantly associated with NPC rather than non-cancerous nasopharyngeal epithelium (p<0.05). Here, we present a partial IHC image of SSTR2 and EGFR protein expression in NPC and non-cancerous nasopharyngeal epithelium (figure 2).

Figure 1

Bioinformatics analysis and patients with NPC and non-NPC expressing different levels of SSTR2 and EGFR. (A) Scatter plot demonstrating a positive correlation between EGFR and SSTR2 at the mRNA level in the GEO database (R=0.412). (B) Bar graphs showing the expression levels of SSTR2 and EGFR in patients with NPC (n=491) and non-NPC (n=50) control group, as determined by immunohistochemistry. (C) Violin plots depicting the distribution of SSTR2 and EGFR scores between the NPC and control groups. The NPC group had a significantly higher proportion of patients with high SSTR2 and EGFR expression. EGFR, epidermal growth factor receptor; NPC, nasopharyngeal carcinomas. （*p<0.05,** p<0.01）

Figure 2

Representative IHC images of SSTR2 and EGFR protein expression in NPC and non-cancerous nasopharyngeal epithelium. (A, E) weak expression of SSTR2 and EGFR in non-cancer nasopharyngeal epithelium, while (B, F) weak expression of SSTR2 and EGFR in NPC. (C, G) Moderate expression of SSTR2 and EGFR in NPC, and (D, H) strong expression of SSTR2 and EGFR in NPC. The magnification of the light microscope used for these images is ×100x. EGFR, epidermal growth factor receptor; IHC, immunohistochemistry; NPC, nasopharyngeal carcinomas.

EGFR and SSTR2 proteins expression and clinicopathological features

First, we classified and analysed NPC patients based on various factors, including age, sex, clinical stage and TNM stage. The resulting table (online supplemental table 1) revealed that NPC is more prevalent in older age groups and male. Furthermore, our study cohort included a larger proportion of patients with advanced clinical stages and lymph node metastases, while distant metastases were relatively rare. Subsequently, we investigated the correlation between SSTR2 and/or EGFR protein expression and clinicopathological features, such as gender, age, TNM stage, treatment strategy and disease progression. Online supplemental table 2 shows that patients with advanced T stage, lymph node metastases and distant metastases tended to exhibit higher expression levels of SSTR2 and/or EGFR than those with early T stage and no metastases, although this was not statistically significant (p>0.05). Furthermore, high expression levels of SSTR2 and EGFR were significantly associated with worse outcomes and a higher risk of progression (p<0.05).

Prognosis and correlation of SSTR2 and EGFR

The influence of various variables on PFS and OS was assessed using Cox univariate analysis, and the corresponding tables were created (table 1 and online supplemental table 3). Our findings revealed that high EGFR expression, advanced T stage, lymph node metastasis, distant metastasis and general CR were associated with a poor prognosis for both PFS and OS. However, high SSTR2 expression was identified as a poor prognostic factor only for PFS, while older age was found to be a poor prognostic factor solely for OS. Moreover, multivariate Cox analysis (table 1 and online supplemental table 3) was conducted to determine independent prognostic factors, which revealed that both M stage and treatment strategy were significant factors for both PFS and OS. However, SSTR2 and EGFR were independent prognostic factors only for PFS. Furthermore, K-M analysis was performed on a cohort of 491 patients (figure 3), which indicated that patients with high expression of EGFR, high coexpression of EGFR and SSTR2, and EGFR/SSTR2 anyone high expression had a poorer prognosis for both PFS and OS and high expression of SSTR2 had a poorer prognosis for PFS (p<0.05). Based on these studies, overexpression of SSTR2 and EGFR is detrimental to patients with NPC. SSTR2 has the potential to serve as a new biomarker for poor prognosis in patients with NPC. Additionally, we found a positive correlation between EGFR and SSTR2 at the mRNA level using the GEO database (R=0.412) as described previously. We also demonstrated that SSTR2 correlates with EGFR at the protein level via IHC (R=0.296, weak positive association) (data not shown).

Figure 3

Kaplan-Meier analysis of a cohort of 491 patients with NPC, evaluating the effect of different levels of SSTR2 and EGFR on PFS and OS. EGFR/SSTR2 anyone high expression includes EGFR (high) and SSTR2 (high), EGFR (high) and SSTR2 (low), and EGFR (low) and SSTR2 (high). EGFR, epidermal growth factor receptor; NPC, nasopharyngeal carcinomas; OS, overall survival; PFS, progression-free survival.

Table 1

Univariate and multivariate Cox regression analysis for PFS in NPC patients

EGFR-targeted therapy affected the prognosis associated with SSTR2 expression

Our study cohort consisted of 288 patients who were treated and reviewed at our institution, providing us with comprehensive and timely clinical information. Among the patients, 147 received only CR, while 141 received CR along with EGFR targeted therapy. We evaluated the clinical prognosis of these patients. First, the study found that patients receiving general CR with high expression of SSTR2, high expression of EGFR and high coexpression of both had a poorer prognosis in both PFS and OS (p<0.05). Although the prognosis did not reach a statistical difference between the EGFR/SSTR2 anyone high expression and others (low coexpression of EGFR and SSTR2) in PFS and OS, low coexpression had a better prognosis (p>0.05) (figure 4). Subsequently, we examined the prognosis of NPC patients treated with general CR combined with targeted therapy and found no significant differences in EGFR expression levels (p>0.05), indicating that EGFR targeted drugs improved the poor prognosis arising from high EGFR expression. Surprisingly, there was also no statistical difference in SSTR2 expression levels, between EGFR/SSTR2 anyone high expression and others (low coexpression of EGFR and SSTR2) in PFS and OS, and between high coexpression of EGFR and SSTR2 and other factors in OS (online supplemental figure 1). This finding led us to speculate whether EGFR targeted therapy could also improve the poor prognosis of NPC patients associated with high SSTR2 expression. To investigate further, we compared the prognosis of both treatment therapies and found that NPC patients with high expression of SSTR2, high expression of EGFR, high coexpression of EGFR and SSTR2, and EGFR/SSTR2 anyone high expression all had a better prognosis with CR combined with targeted therapy, with a significant statistical difference (p<0.05) (figure 5). Our findings suggest that CR combined with EGFR targeted therapy is more effective for NPC with high SSTR2 expression and high EGFR expression. EGFR targeted therapy significantly improves the poor prognosis of NPC patients with high expression of SSTR2 and EGFR.

Figure 4

Kaplan-Meier analysis of a cohort of 147 patients with NPC who underwent CR, evaluating the effect of different levels of SSTR2 and EGFR on PFS and OS. EGFR/SSTR2 anyone high expression includes EGFR (high) and SSTR2 (high), EGFR (high) and SSTR2 (low), and EGFR (low) and SSTR2 (high). CR, chemoradiotherapy; EGFR, epidermal growth factor receptor; NPC, nasopharyngeal carcinomas; OS, overall survival; PFS, progression-free survival.

Figure 5

Kaplan-Meier analysis of different treatment options for NPC, evaluating the effect of different levels of SSTR2 and EGFR on PFS and OS. EGFR/SSTR2 anyone high expression includes EGFR (high) and SSTR2 (high), EGFR (high) and SSTR2 (low), and EGFR (low) and SSTR2 (high). EGFR, epidermal growth factor receptor; NPC, nasopharyngeal carcinomas; OS, overall survival; PFS, progression-free survival.

Discussion

The SSTR2 receptor plays a critical role in regulating cell proliferation and acts as a growth suppressor in various biological processes. Several studies have shown that SSTR2 can inhibit tumour growth in low-grade neuroendocrine tumours and prostate cancer. However, high levels of SSTR2 expression have been linked to promoting tumour growth in high-grade neuroendocrine tumours and small cell lung cancer. The study of EGFR proteins has gained significant attention in recent years due to their involvement in activating various signalling pathways and regulating cell proliferation and survival.15 25–28 The findings of our study have significant implications for the identification of SSTR2 and EGFR as biomarkers associated with poor prognosis provides opportunities for targeted therapies. Targeting SSTR2 could be a potential strategy to improve the survival outcomes of NPC patients, particularly those with high SSTR2 expression. However, our results also suggest that the use of EGFR targeted therapy in combination with chemotherapy could be a more effective approach for patients with high SSTR2 and EGFR expression levels.

The surprising result of EGFR targeted therapy suppressing the effects of high SSTR2 expression warrants further investigation. It is possible that EGFR targeted therapy indirectly affects SSTR2 expression or that it inhibits signalling pathways that promote tumour growth in SSTR2 overexpressing cells. These findings could pave the way for developing novel treatment strategies that target both EGFR and SSTR2 in NPC patients.

In conclusion, our study provides valuable insights into the role of SSTR2 and EGFR in the prognosis of NPC patients. Our results suggest that targeting both biomarkers could be a promising strategy to improve the survival outcomes of NPC patients. Further studies are needed to validate our findings and to explore the mechanisms underlying the interaction between EGFR and SSTR2 in NPC.

Compared with previous research on the role of SSTR2 in NPC, our study stands out with a larger sample size of 491 patients. Overall, our study is the first to shed light on the intricate relationship between SSTR2 and EGFR in NPC and provides new insights into the potential benefits of EGFR targeted therapy for patients with high SSTR2 expression. Future studies could further investigate the molecular mechanisms underlying this relationship and explore potential alternative therapies for patients with high SSTR2 expression. Additionally, efforts should be made to address the toxicity concerns associated with EGFR targeted therapy and optimise treatment strategies to improve patient outcomes.15

Abstract translationThis web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.Data availability statement

No data are available.

Ethics statementsPatient consent for publication

Not applicable.

Ethics approval

This study involves human participants and the Ethical Review Committee of Xiangya Second Hospital of Central South University approved the study protocols (Scientific and Research Ethics Committee, No. K022). Participants gave informed consent to participate in the study before taking part.