Regulatory T-cells activated in metastatic draining lymph nodes possibly suppress cancer immunity in cancer tissues of head and neck squamous cell cancer.

Regulatory T cells (Tregs) play an important role in creating an immunosuppressive microenvironment in cancer tissues. However, the mechanisms by which Tregs are activated and suppress cancer immunity remain unclear. To elucidate these mechanisms, we performed a T cell receptor (TCR) repertoire analysis of Tregs and conventional T cells in peripheral blood, draining lymph nodes (DLNs), and cancer tissues of patients with head and neck squamous cell cancer (HNSCC). We found that the TCR repertoire was skewed in cancer tissue and metastatic DLNs (M-DLNs) compared with non-metastatic DLNs, and TCR repertoire similarities in Tregs and CD8+ T cells between M-DLNs and cancer tissue were high compared with those at other sites. These results suggest that Tregs and CD8+ T cells are activated in M-DLNs and cancer tissues by cancer antigens, such as neoantigens, and shared antigens and Tregs suppress CD8+ T cell function in a cancer antigen-specific manner in M-DLNs and cancer tissue. Moreover, M-DLNs might be a source of Tregs and CD8+ T cells recruited into the cancer tissue. Therefore, targeting Tregs in M-DLNs in an antigen-specific manner is expected to be a novel immunotherapeutic strategy for HNSCCs.

Immune Status of Cervical Lymph Nodes in Head and Neck Cancer-A Surgical Oncology Perspective.

Neck dissection for cervical lymph node metastasis is an established procedure for head and neck cancer (HNC). However, with the advent of immunotherapy, head and neck surgical oncologists need to rethink removing all lymph nodes, including those with immune function. We investigated the anti-cancer immune response of the cervical lymph nodes in four patients with human papillomavirus type 16 (HPV16)-positive head and neck squamous cell carcinoma. Using lymphocytes extracted from local, metastatic, and non-metastatic lymph nodes and peripheral blood from these patients, we performed an intracellular flow cytometric cytokine assay using anti-IFNγ and anti-TNF-α monoclonal antibodies to detect HPV16 E6- and E7-specific T cells. HPV status and p16 immunostaining were determined by in situ detection using the HPV RNAscope method and immunohistochemistry. In one case, E6-specific and E7-specific CD8+ T cells were detected in proximal metastatic nodes and distal non-metastatic nodes. This finding suggests that non-metastatic nodes should be preserved for their immune function during neck dissection and that the immune function of non-metastatic lymph nodes is important when administering immunotherapy. In this context, head and neck surgical oncologists treating HNC should consider the place of immunotherapy and neck dissection in the treatment of HNC.

Artesunate and cisplatin synergistically inhibit HNSCC cell growth and promote apoptosis with artesunate­induced decreases in Rb and phosphorylated Rb levels.

In the treatment of head and neck cancer, cisplatin is often used as a therapeutic agent; however, its efficacy is limited and it can cause renal dysfunction as an adverse effect. For this reason, the use of cisplatin is limited in elderly patients with reduced renal function. Recently, artemisinin, which was developed as an antimalarial drug, was found to have antitumor effects and is effective in combination with other anticancer drugs. In the present study, the antitumor effects of artemisinin and its derivatives as well as their combination with cisplatin and iron on head and neck squamous cell carcinoma cell lines, were investigated. Cell viability was determined by a cell viability assay, the cell cycle was analyzed by flow cytometry, cell death was assessed with annexin V and propidium iodide staining, and western blotting was used to analyze retinoblastoma protein (Rb), phosphorylated (p­)Rb, and other cell cycle­associated molecules. A total of four artemisinin compounds were examined and it was found that artesunate and dihydroartemisinin had a significant inhibitory effect on growth. It was also identified that the combination of artesunate, cisplatin, and iron inhibited cell proliferation and caused S/G2­M cell cycle arrest. In addition, western blotting of Rb, a molecule involved in the cell cycle, showed that artesunate induced the loss of not only Rb but also p­Rb. These results suggested that artesunate is a useful drug in combination with cisplatin.

Establishment of Mucoepidermoid Carcinoma Cell Lines from Surgical and Recurrence Biopsy Specimens.

Patients with advanced/recurrent mucoepidermoid carcinoma (MEC) have a poor prognosis. This study aimed to establish and characterize human mucoepidermoid carcinoma cell lines from the initial surgical specimen and biopsy specimen upon recurrence from the same patient to provide a resource for MEC research. MEC specimens from the initial surgical procedure and biopsy upon recurrence were used to establish cell lines. The established cell lines were cytogenetically characterized using multi-color fluorescence in situ hybridization and detection, and the sequence of the CRTC1-MAML2 chimeric gene was determined. Furthermore, the susceptibility of head and neck mucoepidermoid carcinoma to standard treatment drugs such as cisplatin, 5-fluorouracil, and cetuximab was investigated. We successfully established unique MEC cell lines, AMU-MEC1, from an initial surgical specimen and AMU-MEC1-R1 and AMU-MEC1-R2 from the recurrent biopsy specimen in the same patient. These cell lines exhibited epithelial morphology and developed in vitro-like cobblestones. They shared eight chromosomal abnormalities, including der(19)ins(19;11)(p13;?), which resulted in a chimeric CRTC1-MAML2 gene, indicating the same origin of the cell lines. The susceptibility of all cell lines to cisplatin and 5-fluorouracil was low. Interestingly, EGFR dependency for cell growth decreased in AMU-MEC-R1 and AMU-MEC-R2 but was retained in AMU-MEC1. These cytogenetic and biochemical findings suggest that the established cell lines can be used to investigate the disease progression mechanisms and develop novel therapeutics for MEC.

Pathological and Virological Studies of p16-Positive Oropharyngeal Carcinoma with a Good Response to Neoadjuvant Chemotherapy.

Human papillomavirus (HPV)-related, p16-positive oropharyngeal carcinoma is considered to be sensitive to anticancer drugs, and the standard treatment is therefore chemoradiotherapy, rather than surgery, especially for aggressive disease. However, with this higher sensitivity, chemotherapy alone may achieve a pathological complete response (CR), making radiation therapy unnecessary. A 46-year-old man with p16-positive squamous cell carcinoma (SCC) of the lateral oropharynx (palatine tonsil) underwent neoadjuvant chemotherapy. This achieved clinically significant tumor shrinkage and therefore surgery was performed for subsequent definitive treatment. Clinical and CT findings indicated a good effect of neoadjuvant chemotherapy on the tumor. A biopsy prior to chemotherapy revealed SCC, which demonstrated p16 immunoreactivity and positive signals for high-risk HPV by RNA in situ hybridization. The post-chemotherapy surgical specimen showed pathological CR and no p16 positive cells nor positive signals for high-risk HPV those were detected in the pre-chemotherapy specimen. There are some reports of chemotherapy alone achieving pathological CR in cases of p16-positive oropharyngeal carcinoma, but none have included high-risk HPV RNA findings. This is the first report of the disappearance of cancer cells as well as p16 staining and a positive signal for high-risk HPV. Achieving pathological CR confirmed by immunohistochemistry and high-risk HPV RNA in situ hybridization in a solid tumor with chemotherapy alone suggests that chemotherapy may have both an antitumor effect and an antiviral effect. Forgoing subsequent radiotherapy and undergoing surgery might be unnecessary and follow-up instead might be sufficient in such cases. Into the future, in an optimal tailored treatment approach, the option of neoadjuvant chemotherapy should be considered for management of p16-positive oropharyngeal carcinoma. Other options such as tumor immunotherapy are also expected to be effective.