Patients
The study included 92 patients with squamous cell carcinoma of the thoracic esophagus. All patients underwent R0 esophagectomy in the Department of Thoracic Surgery of the Fourth Hospital affiliated to the Hebei Medical University and Hebei General Hospital between Jun 2011 and Dec 2015. Primary cancer tissues and paired adjacent noncancerous tissues(≥ 2 cm from the edge of carcinoma) were snap-frozen in liquid nitrogen immediately after resection and then stored at -80 °C for RNA extraction. No patient received chemotherapy、radiotherapy、immunotherapy and traditional Chinese medicine prior to specimen collection. The patients were enrolled into our research according to the following criteria: 1)adult patients with pathologically confirmed esophageal squamous cell carcinoma;2)All of the studied patients underwent radical esophagectomy;3)No treatment strategy was performed before surgery:4) Age of 18 to 75 years. The exclusion criteria were as follows:1)The patients merged with other cancers;2)Cervical esophageal squamous cell carcinoma;3)Patients with severe hypertension、severe pulmonary function injury、massive myocardial infarction、cardiac function ≥ level 2(NYHA)、psychiatric history and severe diabetes complications;4)Patients participated in other clinical trial within the first 4 weeks.
Among the 92 patients with esophageal squamous cell carcinoma,54 patients were male, and 38 patients were female. The range of the patients’ ages at the time of diagnosis was 30–75 years (median, 56 years).The postoperative pathological stages were determined according to the UICC TNM staging system. 35 patients at early stage (TNM stages I), 57 patients at advanced stage (TNM stages II-III);52 patients included lymph-node metastasis,40 patients didn’t have lymph-node metastasis. No residual cancer cells were detected under the upper and lower cutting edge or the lateral margin. The esophageal cancer tissues and paired adjacent noncancerous tissues were confirmed via a pathological examination. This study was approved by the Medical Ethics Committee of the Fourth Hospital affiliated to the Hebei Medical University, and the consent in written form was obtained from all patients.
Surgical procedure
The scope of operation in three-field lymph node dissection(3FLND) included neck, chest, and abdomen. The specific surgical procedures were as follows:
The chest surgery: 1) The patients were intubated with single-lumen endotracheal tubes and were set in the lateral position. We commonly set a 12 mm trocar at the 9th intercostal space at the posterior axillary line for thoracoscope, a 12 mm trocar at the 7th intercostal space at posterior axillary line, two 5 mm trocars at 4th and 7th intercostal spaces at the anterior axillary line (insufflation of carbon dioxide 6–10 mmHg). 2)Mediastinal pleura was opened along the right vagus nerve and posterior margin of the upper esophagus,identify the root of the right recurrent laryngeal nerve(RLN), expose and protect the right RLN, dissect the right RLN and thoracic paraesophageal lymph nodes(LNs),completely mobilize the upper esophagus. 3) The azygos vein arch was mobilized and resected. 4) We freed the middle and lower segment of the esophagus. The lymph nodes along with the paraesophageal、hilar and esophageal hiatus were dissected. 5)Expose the left RLN, dissect the LNs of the left RLN, dissect the LNs under the carina and completely stop bleeding.The esophagus was dissected from the diaphragm to the apex of the chest, along with the mediastinal and hilar lymph nodes dissection, including the bilateral RLNs and subcarinal LNs.
The abdominal and cervical surgery: 1)Place the patients in the supine position and separate legs, expose the small bend in the stomach; cut off the left gastric artery,and lymph nodes along the left gastric artery、hepatic artery and the celiac axis were dissected. 2)The stomach was mobilized with reserve of the right gastroepiploic artery, meanwhile, dissect the perigastric LNs, separate the esophageal hiatus and mobilize the abdominal esophagus. 3)Make an 5 cm incision in the anterior border of the sternocleidomastoid muscle of the left cervix, separate the sternocleidomastoid, dissect the LNs in the left lower cervical esophagus [included left cervical paraesophageal (101) and supraclavicular (104)] and cut off the cervical esophagus at the level of thoracic inlet. 4)Make a 5 cm subxiphoid vertical incision in the centre of the abdomen, remove the stomach and esophagus with lesion, a tubular stomach was reconstructed using linear cutting stapler, lift the tubular stomach to the left neck by way of the esophageal hiatus and esophageal bed, a circular stapler was inserted to anastomose the greater curvature side of the tubular stomach and the cervical esophageal. Then, a linear cutting stapler was used to close the proximal end of the tubular stomach. 5) Make an 5 cm incision in the anterior border of the sternocleidomastoid muscle of the right cervix and dissect the LNs in the right lower cervical esophagus [included right cervical paraesophageal (101) and supraclavicular (104)].
RNA Extraction and qRT-PCR
Total RNA was extracted from specimens of esophageal cancer tissues and paired adjacent noncancerous tissues using Trizol reagent (Invitrogen,Carlsbad, CA).RNA was reversely transcribed into cDNAs using the SuperScript first-strand synthesis system (Invitrogen, Carlsbad, CA, USA).To evaluate SPRY4-IT1 expression levels, we used quantitative real time polymerase chain reaction (qRT-PCR) with 2 × TaqMan Premix Ex Taq (Takara, Japan), using an ABI7900 system (Applied Biosystems, CA,USA). Dissociation curve analysis was used to evaluate the PCR products. Glyceraldehyde 3-phosphate dehydrogenase(GAPDH) was used as an internal control. The comparative 2-ΔΔCt method was used for relative quantification and statistical analysis. The primers of GAPDH and SPRY4-IT1 were synthesized by Sangon Biotech (Shanghai, China). SPRY4-IT1expressionlevels were determined using 5′-AGCCACATAAATTCAGCAGA-3′ as a forward primer and 5′-CGATGTAGTAGGATTCCTTTCA3′ as a reverse primer sequence. Results were normalized to GAPDH using forward, 5′-GTCAACGGATTTGGTCTGTATT-3′and reverse 5′-AGTCTTCTGGGTGGCAGTGAT-3′primers.
Postoperative follow-up and the diagnosis of lymph-node recurrence
All patients underwent follow-up examinations every 3–6 months after surgery. These examinations included Doppler ultrasound, chest and abdominal enhanced CT examination, and, if necessary, PET-CT and endoscopy. Recurrence was defined as apparent recurrence on imaging studies during follow-up. The diagnosis of cervical lymph-node recurrence was mainly made based on the results of a physical examination, Doppler ultrasound, and fine-needle aspiration cytology. The diagnosis of mediastinal lymph-node recurrence was mainly based on CT findings. The diagnosis of abdominal lymph-node recurrence was mainly based on Doppler ultrasound and CT findings.
Statistical analysis
All statistical analyses were performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). The X2 test was used to analyze the relationship between SPRY4-IT1 expression and clinicopathological characteristics. The correlation between lymph-node metastasis、recurrence and SPRY4-IT1 expression was analyzed by X2 test or nonparametric test (Mann–Whitney U test). The Kaplan-Meier method was used to calculate the survival curves. Univariate analysis and multivariate analysis were performed using the Cox proportional hazard regression model. A p value less than 0.05 was considered statistically significant. All of the experiments were repeated three times.