br Chemokine receptors are G protein coupled recep tors and
Chemokine receptors are G-protein–coupled recep-tors and are differentially expressed in diverse cell types, including tumor cells. Chemotaxis mediated by chemokine receptors, such as chemokine (C-C motif) receptor-7 (CCR7) and chemokine (C-X-C motif) receptor-4 (CXCR4), plays a key role in the metastasis of oral SCC.4,5 Other roles of chemokine receptors in tumor growth, such as angiogenesis and angiostasis, also have been uncovered.6,7 Chemokine (C-C motif) receptor-4 (CCR4) is a CCX-type receptor expressed on various immune cells, especially on T-helper type 2 cells.8 It also has a biological function in the immunopathogenesis of hematologic tumors.9 Antibody therapeutics targeting the chemokine recep-tor CCR4 have been developed for clinical use, including for hematologic tumors and other dis-eases.10,11 To date, relatively little attention has been paid to the role of CCR4 in promoting the metastasis of some solid tumors.12-16 Tsujikawa et al17 reported that the CCR4 and C-C motif chemokine ligand-22 axis was involved in lymph node metastasis of head and neck SCC. It is interesting that the authors’ group recently reported that CCR4-positive staining could be observed in gastric cancer tissue of patients without lymphatic metastasis, indicating a novel invasive behavior of CCR4.18 The authors postulated that, even without lymphatic metastasis, patients with can-cer carrying certain chemokine receptors, such as CCR4, might be more likely to develop metastatic or recurrent disease. However, there were no data avail-able on CCR4 480-49-9 and its clinical value in patients with pN0 oral tongue cancer.
The purpose of this study was to investigate the expression and clinical relevance of CCR4 in pN0 oral tongue cancer. The authors hypothesized that CCR4 expression might be involved in tumor progression and act as a novel prognostic biomarker in pN0 oral tongue cancer. The specific aims of the study were to
1) detect CCR4 expression in nonmetastatic primary oral tongue cancer tissues, 2) analyze the association be-tween CCR4 expression and clinicopathologic factors 427
in surgically resected pN0 oral tongue cancer, and 3) evaluate the potential use of CCR4 as a biomarker for prognosis in pN0 oral tongue cancer.
Materials and Methods
To address the research purpose, the authors de-signed and implemented a retrospective cohort study. The study population was composed of all patients who underwent resection of oral tongue cancer in the Qilu Hospital of the Shandong University (Jinan, China) from 2005 through 2009. The pathologic stage of oral tongue cancer was evaluated according to the Pathology and Genetics of Head and Neck Tumors of the World Health Organization TNM classification at diagnosis based on the postoperative pathologic exam-ination of specimens.19 For controls, normal oral tis-sue samples were obtained from patients undergoing surgery for nonneoplastic and noninflammatory disor-ders, such as sleep apnea syndrome. Before sample collection, consent was obtained from all patients and approval was obtained from the ethics committee of the Qilu Hospital.
Patients were included in the study sample if they had no other primary cancers and had not received adjuvant therapy before surgery. Patients were excluded from the sample if they had lymph node and distant metastases.
The primary predictor variable in this study was CCR4 expression. Demographic and clinicopathologic features, including gender, age, histologic differentia-tion, tumor stage, and pathologic characteristics, were retrospectively collected and analyzed as second-ary predictor variables. Duration of follow-up was calculated from the date of operation to death or last follow-up, and follow-up was completed in December 2014. Disease-free survival (DFS) was calculated from the first day of diagnosis to the date of first recurrence. Overall survival (OS) was defined as the period from diagnosis to death.
IMMUNOHISTOCHEMICAL DATA COLLECTION
Immunohistochemistry (IHC) was performed on 4-mm-thick routinely processed paraffin sections mounted on glass slides. CCR4 expression was de-tected using a rabbit polyclonal anti-CCR4 antibody (dilution, 1:400; LS-A351; LifeSpan BioSciences, Seat-tle, WA) and a biotinylated second antibody according to a horseradish peroxidase and 3,30diaminobenzidine IHC kit (SP-9001; Zhongshan Golden Bridge Biotech-nology Co, Ltd, Beijing, China). Negative controls were obtained by displacing primary antibodies with
phosphate buffered saline. IHC results of patients with strong (+++), moderate (++), weak (+), and negative ( ) immunoreactivity for CCR4 were analyzed as pre-viously reported by the authors’ group.18,20