Establishment of PDX-derived salivary adenoid cystic carcinoma cell lines using organoid culture method.

To generate a reliable preclinical model system exhibiting the molecular features of salivary adenoid cystic carcinoma (ACC) whose biology is still unclear due to the paucity of stable cell cultures. To develop new in vitro and in vivo models of ACC, the techniques of organoid culture and patient-derived tumor xenograft (PDX), which have attracted attention in other malignancies in recent years, were applied. Tumor specimens from surgically resected salivary ACC were proceeded for the preparation of PDX and organoid culture. The orthotopic transplantation of patient-derived or PDX-derived organoids was demonstrated into submandibular glands of NSG mice and those histology was evaluated. PDX-derived organoid cells were evaluated for the presence of MYB-mediated fusion genes and proceeded for in vitro drug sensitivity assay. Human ACC-derived organoids were successfully generated in three-dimensional culture and confirmed the ability of these cells to form tumors by orthotopic injection. Short-term organoid cell cultures from two individual ACC PDX tumors were also established that maintain the characteristic MYBL1 translocation and histological features of the original parent and PDX tumors. Finally, the establishment of drug sensitivity tests on these short-term cultured cells was confirmed using three different agents. This is the first to report an approach for the generation of human ACC-derived organoids as in vitro and in vivo cancer models, providing insights into understanding of the ACC biology and creating personalized therapy design for patients with ACC.

FLCN alteration drives metabolic reprogramming towards nucleotide synthesis and cyst formation in salivary gland.

FLCN is a tumor suppressor gene which controls energy homeostasis through regulation of a variety of metabolic pathways including mitochondrial oxidative metabolism and autophagy. Birt-Hogg-Dubé (BHD) syndrome which is driven by germline alteration of the FLCN gene, predisposes patients to develop kidney cancer, cutaneous fibrofolliculomas, pulmonary cysts and less frequently, salivary gland tumors. Here, we report metabolic roles for FLCN in the salivary gland as well as their clinical relevance. Screening of salivary glands of BHD patients using ultrasonography demonstrated increased cyst formation in the salivary gland. Salivary gland tumors that developed in BHD patients exhibited an upregulated mTOR-S6R pathway as well as increased GPNMB expression, which are characteristics of FLCN-deficient cells. Salivary gland-targeted Flcn knockout mice developed cytoplasmic clear cell formation in ductal cells with increased mitochondrial biogenesis, upregulated mTOR-S6K pathway, upregulated TFE3-GPNMB axis and upregulated lipid metabolism. Proteomic and metabolite analysis using LC/MS and GC/MS revealed that Flcn inactivation in salivary gland triggers metabolic reprogramming towards the pentose phosphate pathway which consequently upregulates nucleotide synthesis and redox regulation, further supporting that Flcn controls metabolic homeostasis in salivary gland. These data uncover important roles for FLCN in salivary gland; metabolic reprogramming under FLCN deficiency might increase nucleotide production which may feed FLCN-deficient salivary gland cells to trigger tumor initiation and progression, providing mechanistic insight into salivary gland tumorigenesis as well as a foundation for development of novel therapeutics for salivary gland tumors.

Three Cases of Acute Calcific Retropharyngeal Tendinitis.

Acute calcific retropharyngeal tendinitis is an inflammation of the longus coli muscle characterized by the acute onset of neck pain, swallowing pain, and limitations of neck movement. Although symptoms subside spontaneously within one to two weeks, many cases are treated with antibiotics because clinical outcomes are similar to a severe infection of the retropharyngeal area such as a retropharyngeal abscess. We report herein on 3 cases of acute calcific retropharyngeal tendinitis. The first and second cases were hospitalized, had many examinations and were diagnosed retrospectively. The third patient was treated as an outpatient after a CT scan. Typical CT imaging shows prevertebral soft-tissue swelling without ring enhancement, and amorphous calcification just anterior to the atlanto-axial joint, allowing us early diagnosis.

Fibrinogen-to-lymphocyte Ratio Predicts the Outcomes of Hypopharyngeal Squamous Cell Carcinoma Treated With Definitive Radiotherapy.

BACKGROUND/AIM: Previous studies have identified several inflammatory biomarkers that are useful as prognostic biomarkers for various cancer types. However, the fibrinogen-to-lymphocyte ratio (FLR) has not been addressed in head and neck squamous cell carcinoma. Here, we aimed to examine the value of pretreatment FLR as a prognostic marker in patients who received definitive radiotherapy for hypopharyngeal squamous cell carcinoma (HpSCC). PATIENTS AND METHODS: This retrospective study included 95 patients treated with definitive radiotherapy for HpSCC between 2013 and 2020. The prognostic factors for progression-free (PFS) and overall (OS) survival were identified. RESULTS: The optimal cut-off value of pretreatment FLR for discriminating PFS was 2.46. Based on this value, 57 and 38 patients were classified into groups with high and low FLR, respectively. A high FLR was significantly associated with advanced local disease and overall stage, and with the development of synchronous second primary cancer compared with a low FLR. The high FLR group had significantly lower PFS and OS rates than the low FLR group. Multivariate analysis showed that having a high pretreatment FLR was an independent prognostic factor for poorer PFS and OS [PFS: hazard ratio (HR)=2.14, 95% confidence interval (CI)=1.09-4.19, p=0.026; OS: HR=2.86, 95% CI=1.14-7.20, p=0.024]. CONCLUSION: The FLR has a clinical effect on PFS and OS in patients with HpSCC, suggesting that it has potential application as a prognostic factor for patients with HpSCC.

P4HA1 Promotes Cell Migration and Colonization in Hypopharyngeal Squamous Cell Carcinoma.

BACKGROUND/AIM: This study aimed to identify key molecules associated with the survival of patients with hypopharyngeal squamous cell carcinoma (HpSCC) by combining in silico and in vitro analyses. MATERIALS AND METHODS: Differentially expressed genes (DEGs) were screened using the Gene Expression Omnibus database. For DEGs, we performed functional enrichment and protein-protein interaction network analyses to identify potential biological functions and hub genes. Functional analysis of HpSCC cell lines verified the critical roles of the hub genes. RESULTS: DEGs were associated with the extracellular matrix. Among the hub genes, high expression of prolyl 4-hydroxylase subunit alpha 1 (P4HA1) was significantly associated with shorter survival. In addition, P4HA1 knockdown inhibited cell migration and colonization. Suppression of cell proliferation was demonstrated using P4HA1-selective inhibitors. CONCLUSION: P4HA1 may be a useful therapeutic target for the treatment of HpSCC.

Combination of Performance Status and Lymphocyte-monocyte Ratio as a Novel Prognostic Marker for Patients With Recurrent/Metastatic Squamous Cell Carcinoma of the Head and Neck.

Background/Aim: We previously presented the real-world treatment outcomes of the EXTREME regimen as a first-line therapy for recurrent/metastatic squamous cell carcinoma of the head and neck (R/M SCCHN). This study aimed to evaluate the prognostic significance of pretreatment inflammatory biomarkers in patients with R/M-SCCHN treated with the EXTREME regimen as first-line therapy as a supplementary study of our previous retrospective cohort study. Patients and Methods: The treatment outcomes of 100 patients with R/M-SCCHN treated with the EXTREME regimen as first-line therapy were compared according to patient characteristics and pretreatment inflammatory biomarkers using a Cox proportional hazards regression model. Survival was evaluated using the Kaplan-Meier method. Results: In multivariate analysis, a lymphocyte-to-monocyte ratio (LMR) of <1.944 and Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 1 were independent risk factors for poor overall and progression-free survival. Furthermore, we found that the PS-LMR score based on the ECOG PS and LMR could stratify patients to extract the poor prognostic characteristics of R/M-SCCHN patients treated with the EXTREME regimen as first-line therapy. Conclusion: Further evaluation is warranted to study the reliability and applicability of this novel scoring system in predicting the prognosis of R/M-SCCHN patients in the future.

FosL1 Regulates Regional Metastasis of Head and Neck Squamous Cell Carcinoma by Promoting Cell Migration, Invasion, and Proliferation.

BACKGROUND/AIM: We evaluated the impact of FosL1, a member of the activated protein-1 family, on the pathways leading to regional metastasis of head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: We examined the influence of small interfering RNA (siRNA) and short heparin RNA (shRNA) mediated knockdown of FosL1 on cell migration, invasion, and proliferation in vitro as well as on regional metastasis in vivo. The prognostic significance of FosL1 was also analyzed using the Kaplan- Meier plotter using data from an HNSCC patient database. RESULTS: Down-regulation of FosL1 inhibited cell migration, invasion, and proliferation in vitro, decreased the incidence of regional metastases, and prolonged the survival of mice in vivo. We also determined that HNSCC patients with higher expression levels of FosL1 had a significantly shorter survival time than those with low expression of FosL1. CONCLUSION: FosL1 plays a crucial role in promoting cell migration, invasion, and proliferation in HNSCC.

Fibrinogen and Neutrophil-to-lymphocyte Ratio Predicts Survival in Patients with Advanced Hypopharyngeal Squamous Cell Carcinoma.

BACKGROUND/AIM: The purpose of this study is to investigate the prognostic significance of the pretreatment F-NLR score, which is based on fibrinogen (F) and neutrophil-to-lymphocyte ratio (NLR) in patients with advanced hypopharyngeal carcinoma (HPC). MATERIALS AND METHODS: A total of 111 advanced HPC patients treated with radiotherapy, chemoradiotherapy, or bioradiotherapy were classified into three groups: F-NLR score of 2 (fibrinogen ≥341 mg/dl and NLR≥3.59), score of 1 (fibrinogen ≥341 mg/dl or NLR≥3.59), and score of 0 (fibrinogen <341 mg/dl and NLR<3.59). RESULTS: F-NLR score of 2 was an independent prognostic factor for overall (OS) and progression-free survival (PFS) in patients with advanced HPC in the multivariate analysis. Both OS and PFS were significantly lower in patients with an F-NLR score of 2 than in those with an F-NLR score of 0. CONCLUSION: F-NLR score was useful to stratify patients to extract poor prognostic characteristics in patients with advanced HPC.