br mucosa irradiation and dosimetric parameters are not
mucosa irradiation and dosimetric parameters are not enough to predict this event . Therefore, it is necessary to find specific biomarkers to customize the RT dose administration and predict esophageal toxicity, leading to a personalized therapy.
Different molecular events have been described as potentially responsible for radiation-induced normal tissue damage and inter-individual genetic variations appear to be promising biomarkers to discriminate patients with high risks of treatment-related toxicities in different types of cancer [11–14]. Genes within oxidative stress and IMP1088 signaling networks have being widely studied for their influence to RT response [15,16]. Among them, the Transforming growth factor beta-1 (TGFB-1) plays important roles in inflammation and cell proliferation, which appear to be linked to radiation-induced fibrosis progression . TGFB1 is activated as a feedback anti-inflammatory signal fac-ing the release of cytokines, chemokines and growth factors during the early immune inflammatory response to radiation . Previ-ous studies attempted correlations of different candidate single nucleotide polymorphisms (SNPs) along TGFB1 sequence and increased risk of RIET with the potential of being prognostic and/ or predictive biomarkers for clinical practice [19–21]. Additionally, SNPs along Heat shock protein beta-1 (HSPB1) sequence, a chaper-one responsible for remediating damage to proteins in response to high levels of stress, inhibition of apoptosis, regulation of cell development, and cell differentiation, have been associated with cellular radiosensitivity in LC patients [22–24].
To explore new planning and treatment technology in RT, SNPs along TGFB1 and HSPB1 might be valuable genetic biomarkers for the identification of patients’ individual RIET susceptibility. Conse-quently, we have performed a multicenter prospective study in 247 LC patients for a better understanding of contributions of dif-ferent SNPs to predict esophageal radio-sensitivity in LC patients.
Materials and methods
The study was approved by the Ethics Committee for clinical research and complies with the tenets of the declaration of Hel-sinki and the Institutional Review Board of the participating cen-ters. Written informed consent for molecular genetic studies was obtained from all participants.
Subjects for this prospective analysis were recruited at the radi-ation oncology department of 3 different institutions between Jan-uary 2012 and December 2016. Patient selection criteria were as follows: Patients 18 years old, newly diagnosed stage I-IV patients (undergoing thoracic RT with radical or palliative intent) with small cell, or non-small cell lung cancer were included. Patients presenting lung cancer recurrence were also included if no previous radiation therapy was administered in the first treat-ment course. The LC cohort consisted of 247 patients, 213 men (86%) and 34 (14%) women (Table 1).
Patient immobilization and treatment planning were performed with the patient in the supine position. A vacuum sealed cradle for immobilization was made when necessary. All patients were scanned (contrast enhanced computed tomography [CT] scan in 0.5-cm thickness) from the atlas (C1) level to the second lumbar vertebra (L2) level, approximately, to include the whole neck and lungs. In brief, the gross tumor volume (GTV-primary and GTV-node) consisted of the lesion diagnosed by biopsy and therefore
visible in the subsequent CT scan. The regions of tumor visible by endoscopy but not seen on CT images were also included in the GTV-primary. The GTV was expanded by 6–8 mm around the pri-mary tumor and selected lymph node region to obtain a clinical target volume (CTV) which was further expanded laterally and ver-tically by 10 mm to obtain a planning target volume (PTV). The esophagus was contoured beginning at the level of cricoid cartilage on every CT image, until and including the gastroesophageal junction.
Patient evaluation and follow-up
During the course of radiotherapy, patients were seen at least weekly and more often if they needed clinical evaluation and dis-ease management. They were evaluated at approximately 1– 3 months after completion of therapy and then every 3 months. The follow-up evaluations consisted of a history and physical examination. Computerized axial tomography scans were obtained at intervals of 3–6 months. Double-contrast esophagography was performed if clinically indicated.
Genotyping method and SNP selection
Genomic DNA was isolated from peripheral blood sample using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). DNA concentrations and purity were determined using the NanoDrop 2000 UV–Vis spectrophotometer (Nano Drop Technologies, Wilm-ington, DE, USA).