Pre-operative Serum Hepatocyte Growth Factor and Inflammatory Prognostic Score as Prognostic Markers in Oral Squamous Cell Carcinoma

Summary

This study aimed to explore the potential association between preoperative serum hepatocyte growth factor (HGF) and the preliminary exploratory inflammatory prognostic score (IBPS) with postoperative overall survival (OS) and disease-free survival (DFS) in patients with oral squamous cell carcinoma.

Abstract

Oral squamous cell carcinoma (OSCC) is an aggressive malignancy that influences patient prognosis and quality of life. Identifying appropriate prognostic indicators may help improve clinical decision-making. This study aimed to evaluate the association of preoperative serum hepatocyte growth factor (HGF) and inflammatory prognostic score (IBPS) with postoperative pathological outcomes in patients with OSCC. A total of 158 subjects were enrolled in this retrospective study (2020–2022), including 79 cases in the A group (healthy control group) and 79 cases in the B group (OSCC group). Serum HGF and inflammatory markers were measured, and IBPS was calculated. Comparative analyses were performed between the A and B groups, and prognostic analyses were conducted within the B-group cohort. Logistic regression was used to examine associated factors, while survival and receiver operating characteristic (ROC) analyses were performed to assess prognostic performance. Baseline characteristics were comparable between the two groups (P > 0.05). HGF levels and IBPS were significantly higher in the B group than in the A group (P < 0.05). In the B group cohort, elevated HGF and IBPS were associated with poorer prognosis (HGF OR = 2.010, 95% CI: 1.046–3.863; IBPS OR = 2.603, 95% CI: 1.316–5.148, P < 0.05) and adverse postoperative pathological outcomes. The combination of HGF and IBPS showed improved discriminative capacity for prognostic prediction (AUC = 0.776, P < 0.001). Preoperative HGF and IBPS are abnormal in OSCC patients and are associated with postoperative pathological outcomes after 20-month follow-up. These observations may support further investigations into preoperative risk stratification and clinical monitoring for patients with OSCC.

Introduction

Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck, originating from the epithelial cells of the oral mucosa. OSCC contributes to a substantial disease burden worldwide, posing a threat to human health. According to relevant statistics, the number of newly diagnosed OSCC cases continues to grow each year, and its incidence shows certain geographical and population differences¹. From a pathological perspective, OSCC is a multifactorial, multistep, and complex process. Numerous studies have shown that long-term poor lifestyle habits, such as smoking, alcohol abuse, and human papillomavirus infection, are high-risk factors for the induction of OSCC. These factors can lead to mutations in the genes of oral mucosal epithelial cells, disrupting the normal regulation of cell proliferation, differentiation, and apoptosis, and eventually leading to the development of cancer cells. In the process of tumor evolution, cancer cells will continuously invade the surrounding tissues and organs, and at the same time, distant metastasis occurs through lymphatic vessels and blood vessels, which seriously affects the prognosis of patients^2,3.

In terms of clinical features, early symptoms of OSCC patients tend to be more insidious and may only manifest as local ulcers, hard nodules, or lumps in the oral mucosa, which are easily overlooked by patients⁴. As the disease progresses, patients may develop symptoms such as pain, bleeding, dysphagia, and speech disorders, which seriously affect their quality of life. When the disease progresses to an advanced stage, the tumor may invade important structures such as the jawbone and neck lymph nodes, leading to serious consequences such as facial deformities and enlarged lymph nodes in the neck⁵. Surgical resection is currently one of the mainstays of treatment for OSCC. However, despite the ability of surgery to remove tumor tissue visible to the naked eye, a significant proportion of patients still experience recurrence and metastasis after surgery. Studies have shown that the 5-year survival rate of OSCC patients after surgery is only about 60%, and more than half of them will recur within 2 years after the first surgical treatment⁶. Tumor recurrence and metastasis not only increase the difficulty of subsequent treatment but also greatly reduce patients’ chances of survival. In addition, surgery brings a series of physiological and psychological traumas to patients, such as changes in facial appearance and impaired chewing and swallowing functions, which seriously affect their postoperative quality of life⁷. Therefore, accurate and predictive assessment of postoperative pathological outcomes in OSCC patients is crucial for developing individualized treatment plans and improving patients’ survival and quality of life.

Despite widespread use of clinicopathologic staging, conventional parameters often fail to adequately stratify postoperative risk in individual patients. Single inflammatory biomarkers or ratios such as NLR, PLR, and LMR are widely used, but lack integrated prognostic information, and their performance remains inconsistent across cohorts. Similarly, serum biomarkers used in isolation often provide limited predictive accuracy⁸. To date, few studies have combined serum HGF with a comprehensive inflammatory score to improve preoperative risk stratification in OSCC. This represents an important knowledge gap that the present study aims to address⁹.

Hepatocyte growth factor (HGF) is a glycoprotein consisting of 728 amino acid residues, and its molecular structure contains α- and β-chains that are linked by disulfide bonds to form a heterodimer¹⁰. As a multifunctional cytokine, HGF is mainly secreted by mesenchymal stromal cells under physiological conditions and activates multiple signalling pathways, such as PI3K/AKT, by binding to c-Met receptors on the surface of target cells, thereby playing an important role in cell proliferation, migration, invasion, and angiogenesis¹¹. In recent years, more and more studies have focused on the mechanism of HGF’s role in tumorigenesis and development. In OSCC, preoperative serum HGF levels are closely associated with patient prognosis. Relevant studies have shown that preoperative serum HGF levels in OSCC patients are markedly higher than those in the healthy population, and their levels gradually increase with tumor progression¹². It was found that cancer patients with elevated serum HGF levels also had a markedly increased risk of postoperative recurrence and metastasis. This may be due to the fact that HGF promotes the proliferation, migration, and invasive ability of cancer cells, as well as inducing tumor angiogenesis, which provides the necessary nutrients and oxygen for tumor growth and metastasis^13,14. Therefore, preoperative serum HGF levels are expected to serve as an important indicator of postoperative pathological outcomes in OSCC patients.

Inflammation plays an important role in tumorigenesis, progression, and metastasis. The inflammation prognostic score (IBPS) is a comprehensive assessment of the body’s inflammatory status, which predicts the prognosis of tumor patients by integrating multiple inflammation-related factors or cell counts¹⁵. Currently, the specific scoring components of IBPS have not been fully standardized, and common inflammatory indicators include neutrophil, lymphocyte, platelet, and monocyte counts and their derived ratios, such as neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and lymphocyte-monocyte ratio (LMR)¹⁶. Studies have shown that there is a large infiltration of inflammatory cells in the tumor microenvironment of OSCC patients, and these inflammatory cells secrete cytokines and chemokines that promote the proliferation, migration, and invasion of tumor cells. At the same time, inflammation can also induce tumor angiogenesis, providing the necessary conditions for tumor growth. By analyzing peripheral blood inflammation indicators in OSCC patients, it was found that patients with higher IBPS also had a higher risk of postoperative recurrence and metastasis, and markedly shorter overall survival. Inflammation-related indicators such as NLR, PLR, and LMR, for example, have been shown to be closely associated with the prognosis of OSCC patients. High NLR and PLR levels usually suggest that patients have a poorer prognosis, whereas high LMR levels are associated with a better prognosis¹⁷. Therefore, IBPS can serve as a potential indicator for assessing postoperative pathological outcomes in OSCC patients, providing an important reference for clinical treatment decisions.

Compared with traditional clinicopathologic staging, single inflammatory ratios, or isolated serum biomarkers, the combined panel of HGF and IBPS offers several potential advantages: it is minimally invasive, cost-effective, routinely available from preoperative blood tests, and can be obtained before surgical intervention. Unlike imaging or histopathologic staging, this combined strategy allows early quantitative risk stratification rather than subjective or postoperative assessment alone^18,19. Notably, systemic infections, chronic inflammatory diseases, and certain medications may alter peripheral inflammatory markers and thus potentially influence IBPS values²⁰. This model aims to stratify preoperative risk for OSCC patients undergoing surgical treatment, but caution should be exercised when extrapolating these findings to other populations or clinical environments.

Based on the above background, the aim of this study was to investigate the prognostic value of preoperative serum HGF levels and IBPS on the postoperative pathological outcomes of OSCC patients. We hypothesized that elevated preoperative serum HGF levels and abnormal IBPS are associated with poor postoperative pathological outcomes in OSCC patients, and that by detecting patients’ preoperative serum HGF levels and calculating IBPS, more accurate prognostic prediction information can be provided to clinicians, which can guide the development of individualized treatment plans and improve patients’ survival rates and quality of life. The positive significance of this study lies in the following: on the one hand, if it can be confirmed that preoperative serum HGF levels and IBPS have good prognostic value for the postoperative pathological outcomes of OSCC patients, it will provide clinicians with new prognostic assessment indexes, which will help to assess the patients’ conditions more comprehensively and accurately, and provide a strong basis for the development of personalized treatment plans. On the other hand, by identifying high-risk patients at an early stage, clinicians can take more active treatment measures, such as strengthening postoperative follow-up and advancing adjuvant therapy, thereby reducing the risk of recurrence and metastasis and improving patient survival. In addition, this study may provide new ideas and directions for in-depth research on the pathogenesis and therapeutic targets of OSCC, with important theoretical and clinical significance.

Protocol

The study was performed in compliance with the Declaration of Helsinki and hospital ethical guidelines and was endorsed by Wuxi Second People’s Hospital, Jiangnan University Medical Center, and the ethical committees (Ethical No. 2024-Y-231). All human biological samples (venous blood and serum aliquots) were handled in strict compliance with biosafety regulations (GB 19489-2008). All operations were performed in a biosafety cabinet to avoid sample contamination and personnel exposure. Disposal of used biological samples, contaminated pipette tips, and microcentrifuge tubes was carried out in accordance with medical waste management standards: samples and contaminated materials were autoclaved at 121 ˚C for 30 min, then handed over to qualified medical waste disposal institutions for centralized treatment to ensure biosafety.

Research design
This was a single-centre retrospective cohort study to investigate the prognostic value of preoperative serum HGF levels and IBPS on the postoperative pathological outcomes of OSCC patients. This study design follows the REMARK (Tumor Marker Prognostic Study Report Recommendations) guidelines. The population attending the Dental Department of Wuxi Second People’s Hospital, Jiangnan University Medical Center, from January 2020 to December 2022 was selected as the study population. A total of 170 cases of information were collected, 165 cases were included after exclusion, 7 cases were lost during the follow-up period, and finally 79 cases in A group (healthy control group, healthy people who were admitted to the hospital for physical examination during the same period) and 79 cases in B group (OSCC group, patients with OSCC) were included. The flow chart of this study is illustrated in Figure 1. Retrieved eligible study subjects through the hospital electronic medical record system and the database of the physical examination center, and collected detailed clinicopathological data of the A group (healthy control group) and the B group (OSCC group). Performed a postoperative follow-up of B group patients during the study to record recurrence, metastasis, and survival. Included only a healthy control group to identify differential expression of inflammatory markers between OSCC patients and healthy individuals, providing a rational basis for setting the cutoff value for the self-constructed IBPS. Evaluated the prognostic value of IBPS and verified it within the OSCC patient cohort based on their postoperative pathological outcomes. This study design ensured the accuracy of the data and the scientific validity of the study through strict grouping criteria, standardized laboratory testing process and systematic follow-up mechanism, providing a rigorous research framework for analyzing the prognostic value of HGF levels and IBPS scores in the postoperative pathological outcomes of OSCC, which is expected to provide a scientific basis for the development of personalized prognostic assessment protocols and therapeutic strategies in the clinic.

Inclusion and exclusion criteria
Inclusion criteria: (1) Patients diagnosed with OSCC²¹; (2) Age 40–65 years old; (3) With complete clinical information; (4) Received a comprehensive sequential treatment plan based on surgery and supplemented by selective radiotherapy; (5) Patients with good compliance and willingness to cooperate with the treatment plan developed by the study; (6) The patients’ overall mental status is good, their bodies are basically healthy, and they can truthfully express their complaints about their symptoms and answer relevant questions from healthcare professionals; (7) The patients and their families were informed and agreed, and signed an informed consent form.

Exclusion criteria: (1) Patients with combined malignant tumors other than OSCC; (2) Patients with a combination of severe systemic diseases or immune system diseases; (3) Patients with combined hemorrhagic coagulation dysfunction, or severe cardiac, hepatic or renal functional defects, severe cardiovascular disease or other more serious diseases; (4) Combined chronic infectious diseases, combined with other sexually transmitted diseases (e.g. syphilis, gonorrhea, etc.); (5) Combined brain, heart, liver and kidney function abnormalities; (6) Patients who have been involved in clinical drug trials or clinical studies; (7) Requesting to stop treatment or automatic discharge for personal reasons; (8) Other conditions that, in the opinion of the study physician, should not be included; (9) Other circumstances affecting the indicators of follow-up observation.

Grouping method
Divided the study subjects into two groups: A group: healthy control group (n = 79), B group: OSCC group (n = 79).

Indicator collection
Collected the basic information about patients in both groups.

Sample collection
Collected fasting venous blood samples (5 mL) from all participants early in the morning before surgery and maintained at room temperature (25 ± 2 ˚C) during transportation. Processed the blood samples within 30 min (1,800 s) of collection. Transferred the supernatant serum into 0.5 mL aliquots using sterile polypropylene microcentrifuge tubes after centrifugation at 3,000 × g for 15 min at room temperature (25 ± 2 ˚C). Immediately placed all aliquots in a -80 ˚C ultra-low temperature freezer for long-term storage until analysis, and avoid repeated freeze-thaw cycles (no more than 1 freeze-thaw cycle was allowed if necessary).

Serum HGF level
Determined the serum HGF concentrations using a commercial enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions²². Briefly, thawed serum samples at 4 ˚C for 30 min to avoid protein denaturation, then dilute at a 1:1 ratio with the kit-provided dilution buffer. Added standards, controls, and diluted serum samples (100 µL per well) to the ELISA plate and incubated at 37 ˚C for 60 min. After washing the plate 5 times with the provided washing buffer, added the primary antibody conjugate and incubated at 37 ˚C for another 30 min. Following a second round of washing, the chromogenic substrate was added, and the reaction was incubated at 37 ˚C in the dark for 15 min, and the reaction was terminated with the stop solution. Measured the absorbance at 450 nm using an ELISA reader within 10 min after termination. Implemented standard quality control procedures throughout the assay, including the use of kit-provided calibrators and controls. The intra-assay coefficient of variation (CV) was < 5% and the inter-assay CV was < 8%, confirming reliable analytical performance.

IBPS calculation
Analyzed the serum samples using the XN3000 automatic haematology analyser and the accompanying reagents²³. Thawed the serum samples at 4 ˚C for 20 min and equilibrated to room temperature (25 ± 2 ˚C) for 10 min before testing. Routine blood tests were performed at 25 ± 2 ˚C to obtain the counts of leukocytes, neutrophils, lymphocytes, and platelets, and then the NLR (Neutrophil count/lymphocyte count) and PLR (Platelet count/lymphocyte count) were calculated. Regular calibration of testing instruments was performed at 25 ˚C, with intra-batch CV < 3% and inter-batch CV < 5% for blood routine indicators. Used an immunoturbidimetric assay to measure the serum CRP content in the automatic biochemical analyzer. The serum samples were thawed at 4 ˚C for 20 min and equilibrated to 37 ˚C for 5 min before detection. Performed the assay at 37 ˚C with an incubation time of 10 min, and monitored the reaction continuously at 546 nm. The instrument was operated in strict accordance with the instruction manual to complete the test. CRP detection with intra-batch CV < 4% and inter-batch CV < 6%. Scoring was performed according to our homemade IBPS scale. The IBPS scoring system refers to the construction method of previous inflammation prognosis scores, and adjusts the weights of indicators based on the pre-experimental results of this study. NLR value ≤3.0 was scored as 0 points, 3.0–5.0 was scored as 1 point, >5.0 was scored as 2 points. PLR values ≤150 were scored as 0 points, 150–300 were scored as 1 point, and >300 were scored as 2 points. CRP content ≤50 mg/L was 0 points, 50–100 mg/L was 1 point, and >100 mg/L was 2 points. Total IBPS score = CRP score + NLR score + PLR score. Stratification criteria: ≤ 4 points for medium-low risk, 5–6 points for high risk. The cutoff values and scoring criteria for NLR, PLR, and CRP were determined based on previously published inflammatory prognostic scores combined with exploratory data analysis in the present study population, rather than using statistical optimization such as the Youden index or receiver operating characteristic curve analysis.

Follow-up visits
In this study, a standardized 20-month follow-up was conducted to assess postoperative outcomes in patients. The follow-up start time was the day of the surgical operation for the B group (OSCC group) patients, and the end time was August 31st, 2024. A combination of outpatient follow-up and telephone follow-up was adopted. Performed the physical examination and imaging examination at each follow-up to confirm the presence of tumor recurrence or metastasis. The primary prognostic indicators were overall survival (OS) and disease-free survival (DFS). OS was defined as the time from the day of surgery to death due to any cause. DFS was defined as the time from the day of surgery to tumor recurrence, progression, or death from tumor-related causes.

Sample size calculation
This study used G*Power 3.1.9.7 for sample size estimation to clarify the prognostic value of HGF levels and IBPS scores for postoperative pathological outcomes in OSCC patients. Set a test level of α of 0.05 (bilateral), test efficacy of 85%, and a predicted inflammatory factor AUC of 0.75, as calculated by the software, and 75 cases in each group were needed to meet the statistical requirements of ROC curve analysis and multifactorial regression. Considering the uncertainties of potential dropout and missing data, the sample size of this study was finally set at 79 cases per group to ensure that the study had sufficient statistical efficacy to draw reliable conclusions.

Statistical analysis
Used SPSS 28.0 statistical software for data analysis. GraphPad Prism 9.0 is used to draw survival curves, while Lucidchart is used to draw flowcharts. Tested the data in the study for normal distribution first. Compare baseline characteristics between the A group and the B group using an independent samples t-test for measurement data and a χ² test for count data. Described baseline characteristics as count [n (%)] and measurement data (expressed as x ± s). Indicators including HGF, NLR, PLR, and CRP were expressed as x ± s, and intergroup comparisons of these indicators were also performed using an independent samples t-test. Binary logistic regression analysis was used to investigate the association between HGF levels, IBPS scores, and postoperative pathological outcomes (dependent variable, 1 = adverse, 0 = favorable), with positive regression coefficients indicating a positive correlation between indicators and adverse outcomes. The Kaplan-Meier method was used to plot the OS and DFS curves of the A group and the B group, and the log-rank test was applied to compare the statistical differences in survival rates between groups. The prognostic value of the above metrics for OSCC patients was analyzed using the receiver operating characteristic (ROC) curve and area under the curve (AUC) to determine the optimal cutoff value, sensitivity, and specificity. The count data were presented as n (%), and the comparison between the A group and the B group was analyzed using the χ² test. All statistical tests were two-sided, and P < 0.05 indicated a statistically significant difference.

Results

Diagnostic analyses (OSCC group vs. Healthy control group)
Basic information
This study included 79 patients diagnosed with OSCC at Wuxi Second People’s Hospital, Jiangnan University Medical Center, between January 2020 and December 2022, and 79 healthy people who were admitted for physical examination were also selected as controls. The aim was to analyse the prognostic value of HGF levels and IBPS scores on the postoperative pathological outcomes of OSCC patients. The results s...

Discussion

OSCC is the most common type of oral cancer. Its pathological characteristics are mainly defined by squamous differentiation of tumor cells. Based on similarities in cell morphology and structure with normal squamous cells, OSCC can be classified into highly differentiated, moderately differentiated, and poorly differentiated subtypes. Cancer cells in highly differentiated OSCC are similar to normal squamous cells in morphology and show a relatively regular structure, whereas poorly differentiated tumors exhibit abnormal...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automatic haematology analyser	Sysmex Corporation	XN3000	RRID: SCR_019145 / Routine blood test (leukocytes, neutrophils, lymphocytes, platelets count)
ELISA kit	Shanghai Qincheng Biotechnology Co. Ltd	QC-HGF-Hu	RRID: AB_2898765 / Serum HGF concentration measurement
GraphPad Prism	GraphPad Software, Inc.	9	RRID: SCR_002798 / Survival curve plotting
G*Power	Heinrich-Heine-Universität Düsseldorf	3.1.9.7	RRID: SCR_013756 / Sample size estimation
Immunoturbidimetric assay reagent for CRP	Shanghai Kewa Bioengineering Co., Ltd	BH031M	RRID: AB_2898766 / Serum CRP content measurement
SPSS	IBM Corporation	28	RRID: SCR_002865 / Data statistical analysis