Rapid nodal staging of head and neck cancer surgical specimens with flow cytometric analysis.

BACKGROUND: Detection of metastatic spread of head and neck cancer to cervical lymph nodes is essential for optimal design of therapy. Undetected metastases lead to mortality, which can be prevented by better detection methods. METHODS: We analysed 41 lymph nodes from 19 patients with oral squamous cell carcinoma (OSCC). Each lymph node was divided in two, one half processed for histopathology and the other half dissociated into single-cell suspension, stained for the carcinoma cell markers cytokeratin 5/8 (CK5/8), epithelial cell adhesion molecule (EpCAM) and epithelial mucin (MUC-1), and analysed with flow cytometry. Flow cytometry data were compared with histopathology performed on serial sections and immunohistochemistry. Six cervical lymph nodes from cancer-free patients were used to establish baseline levels in flow cytometry. RESULTS: Flow cytometry analysis (fluorescence-activated cell sorting; FACS) detected all six metastases confirmed by histopathology as well as the histologically negative nodes. Importantly, among nine sentinel lymph nodes, FACS analysis detected <1% malignant cells in four cases, not found in histopathology. Results from flow cytometry analysis can be obtained within 3 h of the time of biopsy. CONCLUSIONS: We show that flow cytometric analysis of nodal tissue is sensitive and reliable in identifying metastases of OSCC. Flow cytometry is inexpensive and fast, providing a possibility of perioperative diagnostics and immediate treatment planning.

Nitric Oxide Is Locally Produced in the Human Middle Ear and Is Reduced by Acquired Cholesteatoma.

OBJECTIVE: To find out if nitric oxide (NO) can be locally produced in the middle ear and if chronic otitis media (COM) and acquired cholesteatoma affect the production. DESIGN: Case-control study. SETTING: Two tertiary-referral hospitals. PATIENTS: Gaseous NO from 11 patients with unilateral perforations or grommet openings was measured with chemiluminescence. Middle ear mucosa from 48 patients with COM and 26 patients with cholesteatoma was investigated. MAIN OUTCOME MEASURES: Detection of NO. Expression of nitric oxide synthase (NOS) mRNA, in mucosa from COM, cholesteatoma and healthy controls, quantified using polymerase chain reaction. RESULTS: The gaseous NO from ears with a unilateral tympanic membrane perforation or a grommet was higher (9 ±â€Š3 ppb, n = 11) than among the controls (4 ±â€Š1 ppb, n = 11, p = 0.04). Lower levels of eNOS (2.64 ±â€Š0.86 mol/100,000 mol ACTB) were detected in the pooled samples from the COM group (n = 48), compared with the control group (140.48 ±â€Š92 mol/100,000 mol ACTB, n = 45, p = 0.010). In the cholesteatoma group (n = 26), a lower expression of nNOS (5.78 × 10-6 ±â€Š1.13 × 10-6 ΔCt) was found in comparison with the controls (1.23 × 10-4 ±â€Š3.18 × 10-5 ΔCt, n = 15, p = 0.011). CONCLUSIONS: NO is likely a natural and permanent part of the gas mixture in the human middle ear. The presence of NOS enzymes in the middle ear mucosa indicates an ongoing NO production and the reduction of NOS in ears with cholesteatoma, and pooled samples from ears with COM, suggest a role for locally produced NO in middle ear disease.

JAK/STAT Dysregulation With SOCS1 Overexpression in Acquired Cholesteatoma-Adjacent Mucosa.

IMPORTANCE: Surgery remains the gold standard in cholesteatoma treatment. However, the rate of recurrence is significant and the development of new nonsurgical treatment alternatives is warranted. One of the possible molecular pathways to target is the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. OBJECTIVE: To investigate the JAK/STAT pathway in the middle ear mucosa in patients with acquired cholesteatoma compared with middle ear mucosa from healthy controls. DESIGN: Case-control study. SETTING: Linköping University Hospital, Sweden, and Karolinska Institutet, Stockholm, Sweden. Sampling period: February 2011 to December 2016. PARTICIPANTS: Middle ear mucosa from 26 patients with acquired cholesteatoma undergoing tympanoplasty and mastoidectomy, and 27 healthy controls undergoing translabyrinthine surgery for vestibular schwannoma or cochlear implantation was investigated. MAIN OUTCOMES/MEASURES: The expression of Interleukin-7 receptor alpha, JAK1, JAK2, JAK3, STAT5A, STAT5B, and suppressor of cytokine signaling-1 (SOCS1) were quantified using quantitative polymerase chain reaction. In addition, expression level of cyclin D2, transforming growth factor beta 1, thymic stromal lymphopoietin, CD3, and CD19 was evaluated. RESULTS: In cholesteatoma-adjacent mucosa, SOCS1 was significantly upregulated (p= 0.0003) compared with healthy controls, whereas STAT5B was significantly downregulated (p = 0.0006). The expression of JAK1, JAK2, JAK3, and STAT5A did not differ significantly between groups. CONCLUSIONS AND RELEVANCE: To the best of our knowledge, this is the first article reporting dysregulation of the JAK/STAT pathway in cholesteatoma-adjacent mucosa. The main finding is that important players of the aforementioned pathway are significantly altered, namely SOCS1 is upregulated and STAT5B is downregulated compared with healthy controls.

Extensive qPCR analysis reveals altered gene expression in middle ear mucosa from cholesteatoma patients.

The middle ear is a small and hard to reach compartment, limiting the amount of tissue that can be extracted and the possibilities for studying the molecular mechanisms behind diseases like cholesteatoma. In this paper 14 reference gene candidates were evaluated in the middle ear mucosa of cholesteatoma patients and two different control tissues. ACTB and GAPDH were shown to be the optimal genes for the normalisation of target gene expression when investigating middle ear mucosa in multiplex qPCR analysis. Validation of reference genes using c-MYC expression confirmed the suitability of ACTB and GAPDH as reference genes and showed an upregulation of c-MYC in middle ear mucosa during cholesteatoma. The occurrence of participants of the innate immunity, TLR2 and TLR4, were analysed in order to compare healthy middle ear mucosa to cholesteatoma. Analysis of TLR2 and TLR4 showed variable results depending on control tissue used, highlighting the importance of selecting relevant control tissue when investigating causes for disease. It is our belief that a consensus regarding reference genes and control tissue will contribute to the comparability and reproducibility of studies within the field.