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Address correspondence to: Dr. Jingjing Deng, Department of Respiratory Medicine, The First Hospital of Jiaxing, Affiliated Hospital of Jiaxing University, No. 1882 South Zhonghuan Road, Jiaxing, Zhejiang, 314000, PR China.
Serum GDF15 concentration is of much clinical significance for lung cancer.
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It has great diagnostic value as a tumor marker for lung cancer patients.
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It could predict the efficacy of chemotherapy in lung cancer patients.
Abstract
Purpose
There is a need for efficient, convenient, and inexpensive methods to accurately diagnose the clinical stage of lung cancer and evaluate the efficacy of chemotherapy in patients with lung cancer. Although growth/differentiation factor 15 (GDF)-15 has great potential as a tumor marker, supporting clinical evidence is still lacking. In this study, we aimed to analyze the relationship between serum GDF15 concentration and the clinical characteristics of patients with lung cancer, and to assess the value of GDF15 in the diagnosis and curative effect of chemotherapy.
Methods
The study comprised 160 participants in total, of whom 88 had lung cancer, 31 had pneumonia, and 41 were control subjects. Among the 88 patients with lung cancer, 64 were willing to participate in follow-up chemotherapy-related studies and meet the inclusion criteria. The serum GDF15 concentration in 288 samples (31 cases, pneumonia group samples; 41 cases, control samples; 88 cases, lung cancer group samples; 64 cases, after 1 chemotherapy cycle; and 64 cases, after 2 chemotherapy cycles) with advanced lung cancer were detected by ELISA. The possible correlations between serum GDF15 level and sex, age, height, weight, body mass index, smoking history, diabetes status, and laboratory findings (hemoglobin, prealbumin, and lactate dehydrogenase) were analyzed using parametric and nonparametric tests. Thereafter, the sensitivity of GDF15 in diagnosing lung cancer was calculated. The serum levels of GDF15, carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and cytokeratin 19 fragment (CYFRA) 21-1 were determined in 64 patients with lung cancer, before and after chemotherapy reception. For the evaluation of the efficacy of chemotherapy, receiver operating characteristic curves were plotted.
Findings
Serum GDF15 concentration at baseline was significantly higher in the lung cancer group than were those in the pneumonia and control groups (both, P < 0.001). An increased expression of serum GDF15 was significantly correlated with diabetes, anemia, and clinical stage (tumor size, nodal involvement, and presence/absence of metastasis). After 2 cycles of chemotherapy among the 64 patients who received it, serum GDF15 concentrations were significantly different from baseline in those who had progressive disease (P = 0.003), stable disease (P < 0.001), or partial response (P = 0.039). The AUC of GDF15 was greater than those of CEA, NSE, and CYFRA 21-1 (0.851 vs 0.630, 0.720, and 0.654, respectively).
Implications
GDF15 is complementary to CEA, NSE, and CYFRA 21-1 in diagnosing lung cancer and, when used in combination, it could be of great diagnostic value and may facilitate correct predictions of the efficacy of chemotherapy. Therefore, serum GDF15 concentration is valuable in lung cancer diagnosis and in the evaluation of the efficacy of chemotherapy.
Despite recent advances in its diagnosis and treatment, the 5-year overall survival for lung cancer remains poor, and most cases are found with distal metastases.
Currently, the treatment options include chemotherapy, radiotherapy, targeted therapy, and immunotherapy; however, the prognosis remains poor, and no effective treatment is available.
In order to improve the diagnosis and prognosis of malignant lung cancer, it is urgent to clarify the underlying molecular mechanisms and to identify new reliable biomarkers and therapeutic targets.
Growth/differentiation factor (GDF)-15, also known as the macrophage inhibitory factor 1, belongs to the transforming growth factor (TGF)-β superfamily.
The GDF15 gene is located on chromosome 19p13.11 and consists of 2 exons separated by an intron sequence of approximately 1800 bp that codes for a 34-kDa protein.
The propeptide of macrophage inhibitory cytokine (MIC-1), a TGF-beta superfamily member, acts as a quality control determinant for correctly folded MIC-1.
GDF15 is involved in myocyte remodeling, cardiovascular disease, dysfunctional erythropoiesis, and general weight loss. Recently, GDF15 was reported to be overexpressed in various cancers and was associated with the proliferation, metastasis, and prognosis of lung cancer
Whether GDF15 is a potentially new biomarker and can be used in tumor diagnosis and prognosis for lung cancer has become a trending topic for research. Therefore, this study analyzed the role of serum GDF15 levels in the diagnosis of lung cancer and reports the efficacy of chemotherapy to provide a clinical reference.
Patients and Methods
This study protocol was approved by the institutional ethics committee, and all subjects gave informed consent.
Clinical Cases Data
A total of 160 participants were included in the study, of whom 88 had lung cancer and 31 had pneumonia. These individuals were hospitalized between December 2019 and August 2020 and met the inclusion criteria. The remaining 41 participants were healthy subjects concurrently selected from the physical examination center of The First Hospital of Jiaxing (Jiaxing, China). The participants were classified into 1 of 3 groups: the lung cancer group (n = 88), pneumonia group (n = 31), and control group (n = 41). The clinical staging of patients with lung cancer was strictly classified according to the 2017 TNM staging standard of the Eighth Edition Cancer Staging Manual of the International Lung Cancer Research Association.
The lung cancer group comprised 40 cases of adenocarcinoma, 24 cases of squamous cell carcinoma (SCC), and 24 cases of small-cell lung cancer (SCLC). Of these, 26 were in Stage III and 62 were in Stage IV. The inclusion criterion for the lung cancer group was a diagnosis of lung cancer, confirmed using lymph node biopsy, bronchoscopy, percutaneous lung puncture biopsy, thoracoscopic pleural biopsy, or other pathology examinations. The inclusion criteria for the pneumonia group included: (1) computed tomography of the chest showing pulmonary inflammatory changes without other space-occupying lesions; (2) available clinical data from the first day after admission; (3) no treatment at the time of blood collection; and (4) computed tomography of the chest at discharge showing relatively reduced or absent inflammation compared to that at admission. The exclusion criteria for both groups were the presence of: (1) a concurrent malignant tumor; (2) a blood system disease; and/or (3) concurrent hypertension and coronary heart disease.
Specimen Collection
On admission or during early morning physical examinations, 2 mL of excess venous blood was collected from the participants under fasting conditions. The specimen was stored in a separation gel–filled, negative-pressure vacuum tube that did not hinder normal sample testing and avoided increasing the patient's pain and physical damage. After spontaneous coagulation, each specimen was centrifuged at 3000 rpm for 15 min. The supernatant and aliquot were collected in a 1.5-mL Eppendorf tube and stored in a refrigerator at −20 °C. At least 2 portions of supernatant were collected from 3 groups of subjects to avoid repeated freeze–thaw cycles. Thereafter, serum GDF15 levels were measured using a human GDF15 serum detection kit produced by R&D Systems (Minneapolis, Minnesota).
The following clinical data were collected from all participants: sex; age; height; weight; body mass index (BMI [kg/m2] = Weight/Height2); history of smoking; diabetes status; and laboratory findings of hemoglobin, prealbumin, and lactate dehydrogenase (LDH). Laboratory data regarding carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and serum cytokeratin 19 fragment (CYFRA) 21-1 were additionally collected from patients with lung cancer. In a subset of patients with type 2 diabetes mellitus, their diagnosis met the American Diabetes Association diagnostic criteria (2018).
All laboratory tests were conducted by the laboratory department of the hospital.
Anemia was defined as a hemoglobin level of <120 or <110 g/L in men and women, respectively. Prealbumin of 180 mg/L was the lower limit of normal (cutoff value); upper limits were: LDH, 245 U/L; CEA, 5.0 ng/mL; NSE, 17.0 ng/mL; and CYFRA 21-1, 3.3 ng/mL.
Chemotherapy Regimens and Efficacy Evaluation
The chemotherapy regimens in the lung cancer group were selected according to pathologic type. The duration of all chemotherapy cycles was 21 days. According to the National Comprehensive Cancer Network guideline (2018 Edition),
the following first-line chemotherapy regimens were recommended: for SCC, gemcitabine + platinum; for adenocarcinoma, pemetrix + platinum; and for SCLC, etoposide + platinum. Efficacy was evaluated after 2 cycles. After follow-up in 64 patients with lung cancer across 2 consecutive cycles of chemotherapy, the patients were evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) proposed in 2009.
The outcomes were classified as complete response, partial response, stable disease, and progressive disease.
Statistical Analysis
SPSS software version 26.0 (SPSS Inc, Cary, North Carolina) was used for statistical analysis. Measurement data that conformed to normal distribution are expressed as means (SD). The single-sample Kolmogorov–Smirnov test was used to analyze the distribution of serum GDF15 concentrations in each group, and the results showed that the data from the lung cancer, pneumonia, and control groups met normal distribution. The t test was used to compare the serum GDF15 concentration in a subset of patients with multiple metastases with that in a subset with pleural, bone, and brain metastases. An F test of 1-way ANOVA was used to compare pleural + bone + brain metastasis with multiple metastasis. The serum GDF15 concentrations in patients with Stage IV lung cancer with different metastatic sites were retested. Measurement data that did not conform to normal distribution are expressed as medians (interquartile range). Enumeration data are expressed as percentiles and were evaluated using the χ2 test.
A receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of GDF15, CEA, NSE, and CYFRA 21-1. The ROC curves were drawn using the serum GDF15 concentration in the lung cancer and pneumonia groups. The point with the highest sensitivity and specificity (1199.05 pg/mL) was taken as the diagnostic threshold value. To evaluate the value of the chemotherapy efficacy, the Mann–Whitney U test was used for comparing 2 groups; the Kruskal–Wallis H test was used for 3 or more groups. A P of <0.05 was considered indicative of a statistically significant difference. All P values are the results of 2-sided testing, and 2-sided CIs were determined with 95% confidence.
Results
There were no significant differences in sex, age, or BMI among the 3 groups (Table I). The mean (SD) serum GDF15 concentrations in the control, pneumonia, and lung cancer groups were 564.36 (166.88),1050.41 (203.88), and 1395.44 (237.91) pg/mL, respectively. The level of GDF15 in the lung cancer group was significantly higher than those in the pneumonia and control groups; the differences were statistically significant (both, P < 0.001; Table II).
Table IComparison of general data among the groups.
In the correlation analysis of serum GDF15 concentration and clinical baseline data in patients with lung cancer, the serum GDF15 concentration was not significantly correlated with sex, age, smoking history, BMI, or prealbumin or LDH level, and no statistically significant intergroup differences were found. Interestingly, the serum GDF15 levels in the subgroups with diabetes and anemia differed significantly from those without those respective conditions (P < 0.001 and P = 0.022, respectively; Table III).
Table IIISerum GDF-15 concentrations, by baseline characteristic, in patients with lung cancer (n = 88).
Serum GDF15 concentrations in patients with different pathologic types of lung cancer are shown in Table IV. On comparison, the concentrations did not significantly differ between the 3 pathologic types of lung cancer (P = 0.838). Serum GDF15 was also analyzed by clinical TNM stage. The results showed that serum GDF15 concentration was related to the T stage (P = 0.030) and to clinical stage (P = 0.041). However, the difference between N-stage groups was not statistically significant (P = 0.896). On comparing the serum GDF15 level in the subgroup with multiple metastases with that in the subgroup with pleural, bone, and brain metastases, the P values were 0.220, 0.588, and 0.578, respectively. The serum GDF15 concentrations did not significantly differ between the subgroup with pleural + bone + brain metastases and that with multiple metastases (P = 0.148). However, there were statistically significant differences in serum GDF15 concentrations between subgroups all with different distal metastases (P = 0.037<0.05).
Table IVSerum GDF-15 concentrations, by pathologic characteristic, in patients with lung cancer.
From these results, it was concluded that GDF15 could be used as a diagnostic marker for lung cancer; therefore, the significance of serum GDF15 concentration in determining the efficacy of chemotherapy was evaluated. The Youden index was 0.492 at the diagnostic threshold value, and the AUC was 0.851, with a 95% CI between 0.776 and 0.926. The sensitivity and specificity of lung cancer diagnosis were 78.2% and 71.0%, respectively. According to the ROC curve, the sensitivity of GDF15 in the diagnosis of lung cancer was higher than that of the commonly used serum markers, namely CEA, NSE, and CYFRA 21-1, which had AUCs of 0.630, 0.720, and 0.654, respectively (Fig.).
FigureReceiver operating characteristic (ROC) curves of Sensitivity and specificity of serum markers. CEA = carcinoembryonic antigen; CYFRA = cytokeratin 19 fragment; GDF = growth/differentiation factor; NSE = neuron-specific enolase.
Serum GDF15 concentration showed good diagnostic sensitivity at different TNM stages in patients with lung cancer, and its distribution was superior to that of CEA, NSE, and CYFRA 21-1. The sensitivity of lung cancer diagnosis using serum GDF15 levels exclusively was higher than that of the other 3 serum markers combined. However, the sensitivity of Stage IV diagnosis using GDF15 levels was slightly lower than that by using a combination of serum markers. However, the sensitivity of the diagnosis by combining the 4 markers (GDF15, CEA, NSE, CYFRA 21-1) was much higher than that of each marker independently (Table V). As mentioned earlier, there were no statistically significant differences between pathologic categories; therefore, no further comparisons were made.
Table VDiagnostic sensitivity of serum markers, by clinical stage of lung cancer. Data are given as number (%) of patients.
Serum Marker
Stage III (n = 26)
Stage IV (n = 62)
All Patients (n = 88)
1. GDF-15
19 (73.1)
50 (80.6)
69 (78.4)
2. CEA
5 (19.2)
26 (41.9)
31 (35.2)
3. NSE
7 (26.9)
34 (54.8)
41 (46.6)
4. CYFRA 21-1
8 (30.8)
24 (38.7)
32 (36.4)
2–4 Combined
15 (57.7)
52 (83.9)
67 (76.1)
1–4 Combined
22 (84.6)
62 (100)
84 (95.4)
Parallel test, multi-indicator combination means that if any indicator was diagnosed as positive, the result was positive.
As shown in Table VI, the serum GDF15 concentrations were statistically different between the groups with adenocarcinoma, SCC, and SCLC before chemotherapy (P < 0.05). After 1 cycle of chemotherapy, this difference became statistically nonsignificant. The serum GDF15 concentrations measured in the groups with adenocarcinoma and SCLC after 2 cycles of chemotherapy significantly differed from those measured in them before chemotherapy (P < 0.001 and P = 0.004, respectively), but this difference was not seen in patients with SCC (P = 0.060). There were no statistically significant differences in serum CEA, NSE, or CYFRA 21-1 from before to after chemotherapy. According to the evaluation of imaging findings using the RECIST standard, 64 patients with lung cancer showed different chemotherapy effects. Partial response, stable disease, and progressive disease were seen in 11, 33, and 20 patients, respectively, who did not show a complete response (Table VII). Among the different curative effects, the serum GDF15 concentrations before and after chemotherapy in patients with progressive disease, stable disease, and partial response showed a statistically significant difference but only after 2 cycles of chemotherapy (progressive disease, P = 0.003; stable disease, P < 0.001; and partial response, P = 0.039). Serum CEA, NSE, and CYFRA 21-1 concentrations, however, did not significantly differ before to after chemotherapy.
Table VIConcentrations of serum markers, by pathologic type, before and after chemotherapy.
Tumor markers (TMs) are proteins in the blood, body fluids, or tissues of tumors and are widely used in tumor detection because their expression concentration is higher in tumors than that in normal tissues.
The abnormal increase or decrease of TMs can indirectly reflect the existence and growth of a tumor; this knowledge is helpful in the detection of metastasis and to determine the diagnosis, treatment, and prognosis of a patient.
At the initial diagnosis, 57% of the patients in this study who had lung cancer showed distal metastasis, which was determined through invasive biopsy.
Therefore, the assessment of TMs, as a noninvasive examination, is highly favored in clinical practice.
GDF15 is a secreted protein. The first synthesis of its monomer occurs in the form of a precursor protein within the cell. It comprises 308 amino acids, of which 29 are signal peptides, and 167 constitute the prematuration sequence. Once the precursor protein matures through enzyme digestion, 112 amino acids remain that are connected through disulfide bonds into bioactive homologous dimers that secrete extracellularly.
The propeptide of macrophage inhibitory cytokine (MIC-1), a TGF-beta superfamily member, acts as a quality control determinant for correctly folded MIC-1.
In the extracellular matrix, GDF15 is secreted in an inactive manner. After activation by TGF-kinase, GDF15 forms a heteropolymer with corresponding serine/threonine kinase receptors to activate a mothers against decapentaplegic homolog (SMAD) protein, which transduces signals to the nucleus to regulate the expression of target genes. GDF15 is a powerful stress protein.
Under stress or pathologic conditions, the expression of GDF15 increases in myocardial cells, lung tissues, and kidney tissues when ischemia and hypoxia occur.
GDF15 has been recognized as a novel molecular marker and its elevation is an independent risk factor for cardiovascular diseases such as coronary atherosclerosis.
Macrophage inhibitory cytokine-1 is increased in individuals before type 2 diabetes diagnosis but is not an independent predictor of type 2 diabetes: the Whitehall II study.
Increased serum concentrations of macrophage inhibitory cytokine-1 in patients with obesity and type 2 diabetes mellitus: the influence of very low calorie diet.
However, opinions differ as to whether the expression of GDF15 is increased or decreased in various tumors. Most studies have shown that the expression of GDF15 is increased in various tumors, and some results show that it is highly expressed in early and late tumors, but no obvious abnormalities are observed in the process of tumor progression. Our findings indicate that the serum GDF15 concentration in patients with lung cancer was related to the tumor size and distal metastasis, but not to the lymph node size and metastasis.
GDF15 ranges from 150 to 1150 pg/mL in the blood of normal adults and may be further increased in the elderly.
Macrophage inhibitory cytokine 1 biomarker serum immunoassay in combination with PSA is a more specific diagnostic tool for detection of prostate cancer.
measured serum GDF15 in 70 patients with prostate cancer, and the serum GDF15 concentration was increased, with a sensitivity and specificity of 78.6% and 89.3%, respectively. In terms of lung cancer, a previous study showed that the threshold value of GDF15 was 1000 pg/mL in 152 patients with Stage I and II non-small cell lung cancer, and the sensitivity and specificity were 70.4% and 99.0%, respectively, thus showing high diagnostic and prognostic monitoring value.
In our study, a serum GDF15 concentration of 1199.05 pg/mL was the threshold value for the diagnosis of lung cancer, and the sensitivity and specificity were 78.2% and 71.0%, respectively, which were superior to the sensitivity and specificity of CEA, NSE, and CYFRA 21-1. It is well-established that a biomarker used to predict clinical chemotherapeutic response has great significance in helping with treatment choice for patients with lung cancer.
Currently, there is no effective method to predict the clinical response of patients with lung cancer to chemotherapy. Meanwhile, the clinical evaluation of chemotherapeutic effect is based on RECIST. Although the evaluation of the curative effect is more accurate with RECIST than without, this method is expensive and time consuming and may result in radiation damage. However, evaluating the chemotherapeutic effect using tumor biomarkers is inexpensive and convenient.
Phase II trial evaluating the efficacy of sorafenib (BAY 43-9006) and correlating early fluorodeoxyglucose positron emission tomography-CT response to outcome in patients with recurrent and/or metastatic head and neck cancer.
Altered mRNA expression levels of the major components of sphingolipid metabolism, ceramide synthases and their clinical implication in colorectal cancer.
Macrophage inhibitory factor 1 acts as a potential biomarker in patients with esophageal squamous cell carcinoma and is a target for antibody-based therapy.
GDF15 predict platinum response during first-line chemotherapy and can act as a complementary diagnostic serum biomarker with CA125 in epithelial ovarian cancer.
The findings from the present study support the clinical value of GDF15 in evaluating the efficacy of chemotherapy for lung cancer. Serum GDF15 levels showed statistically significant differences before chemotherapy and after 2 cycles of chemotherapy. In this study, there is no reference for the use of GDF15 in the evaluation of the efficacy of chemotherapy for lung cancer, and the included population comprises patients with advanced lung cancer (Stage III and IV) but excludes patients with early lung cancer (Stage I and II). Therefore, the study findings may not be applicable to patients with Stage I and II lung cancer. At the same time, due to the study design, the sample size was small and may not represent the overall situation of patients with lung cancer.
Conclusions
Serum GDF15 concentration was significantly correlated with diabetes, anemia, and TNM stage. Used complementarily to CEA, NSE, and CYFRA 21-1, it may be useful in the diagnosis of lung cancer, and, when measured concurrently, can be of certain significance for chemotherapy patients and may be used to predict the efficacy of chemotherapy. However, the application of the findings from this study to other similar patients needs to be further explored. A study with a small sample size such as that in the present study can inform and warrant future clinical research with larger samples, and further consideration of serum GDF15 concentration before and after surgery among patients in the early stages of lung cancer and before and after gene-targeting drug delivery among patients receiving advanced lung cancer chemotherapy. Finally, evaluating the differences in overall survival and progression-free survival times through follow-up research and analysis can provide the basis for individualized treatment of lung cancer.
Author Contributions
J.J.D., M.Z., and H.L.Z. were responsible for the design of the study and interpretation of the data. C.L., H.X.H., Y.F., Z.X.F., and X.D.L. examined the archives and identified the databases included in the study, and critically revised the manuscript for important intellectual content. All of the authors contributed to the writing of the manuscript and read and approved the final manuscript.
Disclosures
The authors have indicated that they have no conflicts of interest with regard to the content of this article.
Acknowledgments
The present study was supported by Key Discipline of Jiaxing Respiratory Medicine Construction Project grant 2019-zc-04, Science Technology Project of Jiaxing grants 2020AD30044 and 2019AD32126, funds from the Jiaxing Key Laboratory of Precision Treatment for Lung Cancer, and The Early Diagnosis and Comprehensive Treatment of Lung Cancer Innovation Team Building Project.
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Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
The propeptide of macrophage inhibitory cytokine (MIC-1), a TGF-beta superfamily member, acts as a quality control determinant for correctly folded MIC-1.
Macrophage inhibitory cytokine-1 is increased in individuals before type 2 diabetes diagnosis but is not an independent predictor of type 2 diabetes: the Whitehall II study.
Increased serum concentrations of macrophage inhibitory cytokine-1 in patients with obesity and type 2 diabetes mellitus: the influence of very low calorie diet.
Macrophage inhibitory cytokine 1 biomarker serum immunoassay in combination with PSA is a more specific diagnostic tool for detection of prostate cancer.
Phase II trial evaluating the efficacy of sorafenib (BAY 43-9006) and correlating early fluorodeoxyglucose positron emission tomography-CT response to outcome in patients with recurrent and/or metastatic head and neck cancer.
Altered mRNA expression levels of the major components of sphingolipid metabolism, ceramide synthases and their clinical implication in colorectal cancer.
Macrophage inhibitory factor 1 acts as a potential biomarker in patients with esophageal squamous cell carcinoma and is a target for antibody-based therapy.
GDF15 predict platinum response during first-line chemotherapy and can act as a complementary diagnostic serum biomarker with CA125 in epithelial ovarian cancer.
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