Patient Selection
A total of 143 patients with ESCC supported by pathology who received radiotherapy at our institute from August 2018 to August 2020 were included in this study. Enrollment and exclusion criteria are as follows:
Enrollment criteria:
1) Initial diagnosis and treatment were received at our hospital and all the courses of radiotherapy plans were completed; 2) ESCC was diagnosed with pathological evidence before treatment, and classified by the 8th edition AJCC/UICC classification17; 3) There was no history of other previous malignancies; 4) Patients did not experience pleural effusion during radiotherapy; 5) Peripheral blood samples were collected as planned. Blood routine, blood biochemical and imaging examinations were regularly conducted. The case data were complete with good compliance; 6) No anti-angiogenic drugs were used during treatment; 7) The KPS (Karnofsky) score was greater than 70 points.
Exclusion criteria: 1) Previous history of other malignant tumors; 2) Previous radiotherapy and chemotherapy or tumor-related surgical treatment; 3) Receiving palliative or supportive treatment; 4) Except for tumors, patients with abnormal vascular proliferation diseases such as asthma, retinopathy, liver disease, peripheral vascular diseases etc.; 5) Patients with infection not associated with radiotherapy or other inflammatory diseases, such as pneumonia, ulcerative colitis, connective tissue diseases, rheumatism, acute infections, etc.; 6) Drugs that may affect peripheral blood biomarkers such as recombinant human granulocyte stimulating factor, thrombopoietin etc. used within 1 week before blood cell samples collection; 7) Patients lost to follow-up.
VEGF Dectection
4ml of peripheral blood from all enrolled patients was collected at five time points including 1 day before radiotherapy, during radiotherapy (15f), the end of radiotherapy, 1 month after radiotherapy, and 3 months after radiotherapy. Peripheral blood samples were placed in the anticoagulant EDTA tube for 30 minutes, centrifuged at 3500 r/min for 10 minutes, and stored in a refrigerator at minus 80 °C.
VEGF detection kits were provided by Beijing Jianping Jinxing Biotechnology Co., Ltd. (product registration number: Jingxi Zhuzhun 20152400398) and stored at 2-8 °C. Samples were tested by enzyme-linked immunosorbent assay (ELISA method). The brief operation steps are: 1) Preparation of lotion; 2) Dilution of calibrator; 3) Adding sample and incubating wash plate; 4) Adding enzyme; 5) Washing plate; 6) Color development; 7) Termination; 8) Calibration and measurement (450nm wavelength of microplate reader, reference wavelength 630nm); 9) Calculating and recording VEGF value according to the calibration curve and the OD (Absorbance) value measured by the microplate reader.
Data Collection and Definition
The age, gender, differentiation, imaging and other clinical data of all patients as well as the expression values of related inflammation indicators were found and recorded through the Website V1.1 of The Hospital (Nanjing Yijiantong Information Technology Co., LTD.) and subsequent follow-up. The relevant inflammation biomarkers were defined as follows:
GPS: the value was 1 for elevated C reactive protein combined with low albumin; only one abnormal phenomenon got 1. Both normal indicators were assigned with 0.
CAR: the ratio of C reactive protein to albumin.
CLR: the ratio of C reactive protein to lymphocyte.
Treatment Details
The radiotherapy was delivered with 6 MV photon beams for IMRT (Intensity Modulated Radiotherapy) in Eclipse treatment planning system (Varian Medical Systems, AAA 11.0). All patients underwent a spiral CT (Siemens Medical Systems, Iselin, NJ) scan before the treatment in the supine position. GTV (Gross Tumor Volume) obtained the primary lesion and positive regional lymph nodes. CTV (Clinical Tumor Volume) was expanded by a 0.5 cm radial margin around GTV, and was extended by 3.0cm to 5.0cm in the proximal and distal direction. PTV (Planning Tumor Volume) provided a 0.3 to 0.5 cm margin around CTV. The vital organs, including the spinal cord, heart, and bilateral lungs, were mapped and the optimal treatment plan was determined by experienced clinicians and physiotherapists according to the dose-volume histogram (DVH) and isodose curve. The prescription dose of PTV was 60 to 64Gy, 1.8 to 2.2 Gy/day, 5 days/week. Considering the patient’s age, basic state and other conditions, appropriate individualized treatment was conducted. The target area and dose can be reduced if necessary. The chemotherapy regimen uses the "TP" regimen, namely: liposomal paclitaxel 45 to 60 mg/m2, on the first day; cisplatin 20 to 25 mg/m2, from the first to third day, ivgtt (intravenously guttae), 21 days a cycle. Two cycles of chemotherapy were started simultaneously on the first day of radiotherapy until the end of radiotherapy.
Clinical Response Assessment
Response evaluation criteria in solid tumors (RECIST 1.1) was adopted18. The clinical response assessment was evaluated 2 to 3 weeks after the end of radiotherapy. According to the results of barium meal, CT (Computed Tomography) or MRI (Magnetic Resonance Imaging) examination before and after treatment, the clinical response was divided into complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD). CR showed that all lesions disappeared. PR indicated that the total maximum diameter of target lesions was reduced by 30%, PD revealed that the total maximum diameter of target lesions increased by 20% or new lesions appeared. SD showed that the reduction of target lesions did not reach PR or the increase failed reaching PD.
Follow-up
The adverse events were evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.0). According to CT or MRI examination at the end of radiotherapy, measure the short diameter (SD) of residual lymph nodes after radiotherapy. Telephone follow-up, outpatient follow-up etc. were used for follow-up. Follow-up were arranged a month after radiotherapy, then every 3 months in the first year, and every 6 months from the second year until the end of the follow-up or the end of the study. The time of recurrence, metastasis, or death of the patient was recorded. The deadline for follow-up was August 31, 2020. The PFS (progression-free survival) and OS (overall survival) were calculated.
Statistical Analysis
Descriptive statistics of patient baseline clinicopathological characteristics are expressed in medians and 95% confidence intervals (CIs). Survival analysis uses a log-rank test. The ROC (Receiver operating) curve was used to compare the area under the curve (AUC) at different time nodes. For each variable, the optimum cutoff value corresponding to the time node with the maximum AUC value was calculated using X tile 3.6.1 (Yale University, New Haven, CT, USA). Univariate and multivariate logistic regression model analysis was implemented to analyze the correlation between the risk factors and clinical response. The risk factors for PFS were calculated by univariate and multivariate cox regression model. To compare the accuracy of clinical response prediction and PFS between combined markers and independent markers, the ROC curve was applied. SPSS 24.0 (SPSS Inc., Chicago, IL, USA) was used to analyze the data. All tests were two-sided tests, P<0.05 was considered statistically significant.
Based on the Cox regression model analysis, a nomogram prediction model was developed by using the R×64 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria). It was further evaluated through calculating the value of C-index (Concordance index), ploting ROC curve, calibrating curve and and conducting DCA (Decision Curve Analysis). The installation packages involved are: Hmisc, survival, rms, pROC, lattice, Formula, ggplot2 and rmda.