Takanori Hama

Prognostic significance of epidermal growth factor receptor phosphorylation and mutation in head and neck squamous cell carcinoma.

The molecular status of the epidermal growth factor receptor (EGFR) has not been as well studied in head and neck squamous cell carcinoma (HNSCC) as in lung cancer. We examined the frequencies of EGFR mutations as well as the expression/phosphorylation status of the EGFR protein in HNSCC patients. Moreover, we tried to elucidate associations between EGFR molecular status and patient characteristics and disease-free survival. In this prospective cohort study, clinical data and samples were obtained from 82 consecutive patients who had not been treated with EGFR molecular targeting therapy. Full-length EGFR was sequenced, and expression and phosphorylation of the EGFR protein were measured by Western blotting. Four novel mutations (E709K, V765G, Ins770G, and G1022S) and one mutation well-known in lung cancer (L858R) were identified in six HNSCC samples (7%), but we could not find any mutations in the extracellular domain of EGFR, such as EGFRvIII, in this study. E709K and Ins770G as well as L858R appear to be functional mutations based on the use of Ba/F3 cells. In terms of patient characteristics, the number of metastatic lymph nodes and node stage were associated with phosphorylation of EGFR. No patients with EGFR mutations relapsed during the study period. Excluding mutated cases, patients whose tumor samples showed phosphorylated EGFR relapsed significantly earlier than those without phosphorylated EGFR. This finding was still significant after adjusting for mutation and overexpression of EGFR protein using the Cox proportional hazard model. In conclusion, phosphorylated EGFR without mutations may be a marker of poor prognosis in patients with HNSCC.

Mucosal eosinophilia and recurrence of nasal polyps - new classification of chronic rhinosinusitis.

BACKGROUND Eosinophils and nasal polyps are believed to affect the surgical outcome of chronic rhinosinusitis (CRS). CRS is classified based on the presence of nasal polyps in western countries. The majority of patients with CRS with nasal polyps (CRS with NP) are characterized by predominantly eosinophilic inflammation. However, Asian patients with CRS with NP show characteristics indicative of neutrophilic inflammation. Therefore, are eosinophils or nasal polyps more important for the classification of CRS? METHODS A prospective cohort study conducted from April 2007 to March 2008 classified patients with CRS based on the presence of nasal polyps and mucosal eosinophilia. The recurrence rate of nasal polyps was compared between the groups. Recurrence rate was analysed as a time-dependent variable by the Kaplan-Meier method. RESULTS Eosinophilic inflammation was found in 59.6% of patients with CRS with NP. Patients with mucosal eosinophilia had higher polyp recurrence rate than patients without mucosal eosinophilia, whereas patients with nasal polyps did not have higher polyp recurrence rate than patients without nasal polyps. CONCLUSIONS Presence of mucosal eosinophilia is a more important factor than nasal polyps for classifying CRS in terms of the surgical outcome.

Copy number amplification of the PIK3CA gene is associated with poor prognosis in non-lymph node metastatic head and neck squamous cell carcinoma.

BackgroundDeregulation of the EGFR signaling pathway is one of the most frequently observed genetic abnormalities that drives cancer development. Although mutations in the downstream components of the EGFR signaling pathway, including KRAS, BRAF and PIK3CA, have been reported in numerous cancers, extensive mutation and copy number analysis of these genes in clinical samples has not been performed for head and neck squamous cell carcinoma (HNSCC).MethodsWe examined the mutations and copy number alterations of KRAS, BRAF and PIK3CA in 115 clinical specimens of HNSCC obtained from surgically treated patients.We used DNA sequencing to detect mutations and the copy number changes were evaluated by qPCR and array comparative genomic hybridization (CGH) analysis.ResultsWe examined the mutations and copy number alterations of KRAS, BRAF and PIK3CA in 115 clinical specimens of HNSCC obtained from surgically treated patients. We identified 3 mutations (2.6%) in K-RAS and 3 mutations (2.6%) in PIK3CA. Copy number amplification was found in 37 cases (32.2%) for PIK3CA, 10 cases (8.7%) for K-RAS and 2 cases (1.7%) for BRAF. Kaplan-Meier survival analysis revealed that copy-number amplification of PIK3CA was markedly associated with cancer relapse in patients without lymph node metastasis. (Log-rank test, p = 0.026)ConclusionsCopy number amplification of the PIK3CA gene is associated with poor prognosis in HNSCC patients without lymph node metastasis. The PIK3CA copy number status will serve as a marker of poor prognosis in patients with HNSCC.

Prevalence of Human Papillomavirus in Oropharyngeal Cancer: A Multicenter Study in Japan

Background: The incidence rates of oropharyngeal squamous cell carcinoma (OPSCC) have risen steadily in the USA and in northern Europe. These increases are thought to be a consequence of persistent infection with high-risk human papillomavirus (HPV) in OPSCC patients. HPV is an emerging etiologic factor in OPSCC. In Japan, the incidence of OPSCC has significantly increased over the last three decades. However, the population of HPV-positive OPSCC patients is currently unknown. We examined the nationwide trends with regard to HPV incidence in OPSCC patients at 21 specific sites, and examined the relationship between the presence of HPV and survival in OPSCC patients in Japan. Methods: Tumor samples were obtained from patients with OPSCC prior to treatment, and HPV infection was investigated by polymerase chain reaction (PCR). Hybrid Capture 2 (HC2) was also adopted for swab examination on the surface of fresh tumors. Results: HPV was detected by PCR in 79 (50.3%) out of 157 OPSCC patients. The clinical features of HPV-positive OPSCC were low differentiation, a tendency to involve the lateral wall, and high nodal staging. The sensitivity and specificity of HC2 were 93.7 and 96.2%, respectively, indicating its utility as a screening test. HPV-positive patients had significantly better overall survival and disease-free survival than HPV-negative patients.

Risk factors for complications of endoscopic sinus surgery for chronic rhinosinusitis.

Background Patients undergoing endoscopic sinus surgery (ESS) are at risk of complications because of the close proximity of the sinuses to the orbit and anterior skull base. The aim of this study was to evaluate the complications of ESS and to identify patient characteristics that were risk factors for the complications. Methods We conducted a prospective study of 706 patients who underwent ESS for chronic rhinosinusitis. Patients completed preoperative examinations that included computed tomography, endoscopic observation for nasal polyps, and tests for comorbidities including asthma and vascular disease. Perioperative complications were evaluated based on information provided by the surgeons. Multivariate analysis was performed to identify patient characteristics that were risk factors for complications. Results Overall, perioperative complications occurred in 41 patients (5.8%). A major complication, cerebrospinal fluid leakage, occurred in one patient (0.1%). Minor complications occurred in 40 patients (5.7%), with the most common being intraoperative hemorrhage (n = 18). Multivariate analysis indicated that presence of asthma and the total polyp score correlated significantly with the occurrence of complications. Conclusion The risk factors for perioperative complications were asthma and the polyp score. We conclude that the surgeon should confirm whether the patient has lower airway disease, especially asthma, before operating. The surgeon should also determine the grade of nasal polyps.

Middle ear mucosal regeneration with three-dimensionally tissue-engineered autologous middle ear cell sheets in rabbit model

The likelihood of recurrent retraction and adhesion of newly formed tympanic membrane is high when middle ear mucosa is extensively lost during cholesteatoma and adhesive otitis media surgery. If rapid postoperative regeneration of the mucosa on the exposed bone surface can be achieved, prevention of recurrent eardrum adhesion and cholesteatoma formation, for which there has been no definitive treatment, can be expected. Suture‐less transplantation of tissue‐engineered mucosal cell sheets was examined immediately after the operation of otitis media surgery in order to quickly regenerate middle ear mucosa lost during surgery in a rabbit model. Transplantable middle ear mucosal cell sheets with a three‐dimensional tissue architecture very similar to native middle ear mucosa were fabricated from middle ear mucosal tissue fragments obtained in an autologous manner from middle ear bulla on temperature‐responsive culture surfaces. Immediately after the mucosa was resected from middle ear bone bulla inner cavity, mucosal cell sheets were grafted at the resected site. Both bone hyperplasia and granulation tissue formation were inhibited and early mucosal regeneration was observed in the cell sheet‐grafted group, compared with the control group in which only mucosal removal was carried out and the bone surface exposed. This result indicates that tissue engineered mucosal cell sheets would be useful to minimize complications after the surgical operation on otitis media and future clinical application is expected. Copyright

Distinct effects of alcohol consumption and smoking on genetic alterations in head and neck carcinoma.

Background Tobacco and alcohol consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Recently, whole-exome sequencing clarified that smoking increased TP53 and other mutations in HNSCC; however, the effects of alcohol consumption on these genetic alterations remain unknown. We explored the association between alcohol consumption and somatic copy-number alterations (SCNAs) across the whole genome in human papillomavirus (HPV)-negative HNSCCs, and compared with the effects of smoking on genetic alterations. Methods SCNA and TP53 mutations in tumor samples were examined by high-resolution comparative genomic hybridization microarray 180K and by direct sequencing, respectively, and statistically analyzed for associations with alcohol consumption and smoking during the 20 years preceding diagnosis of HNSCC. Probes with a corrected p-value (=q-value) less than 0.05 and fold change greater than 1.2 or less than -1.2 were considered statistically significant. Results A total of 248 patients with HNSCC were enrolled. In the HPV-negative patients (n=221), heavy alcohol consumption was significantly associated with SCNAs of oncogenes/oncosuppressors that were previously reported to occur frequently in HNSCCs: CDKN2A (q=0.005), FHIT (q=0.005), 11q13 region including CCND1, FADD and CTTN (q=0.005), ERBB2 (HER2) (q=0.009), 3q25-qter including CCNL1, TP63, DCUN1D1 and PIK3CA (q=0.014), and CSMD1 (q=0.019). But, TP53 mutations were not affected. In contrast, smoking was associated with increased risk of TP53 mutations, but did not induce any significant SCNAs of oncogenes/oncosuppressors. Conclusion These results suggest that both alcohol consumption and smoking had distinct effects on genetic alterations in HNSCCs. Heavy alcohol consumption may trigger previously known and unknown SCNAs, but may not induce TP53 mutation. In contrast, smoking may induce TP53 mutation, but may not trigger any SCNAs.

Prognostic Significance of Vitamin D Receptor Polymorphisms in Head and Neck Squamous Cell Carcinoma

Background In patients with advanced non-small-cell lung cancer, vitamin D receptor (VDR) polymorphisms and haplotypes are reported to be associated with survival. We hypothesized that a similar association would be observed in patients with head and neck squamous-cell carcinoma (HNSCC). Methods In a post-hoc analysis of our previous prospective cohort study, VDR polymorphisms including Cdx2 G/A (rs11568820), FokI C/T (rs10735810), BsmI A/G (rs1544410), ApaI G/T (rs7976091), and TaqI T/C (rs731236) were genotyped by sequencing in 204 consecutive patients with HNSCC who underwent tumor resection. Progression-free survival was compared between VDR polymorphisms using Kaplan-Meier survival curves with log-rank tests and Cox proportional hazard models adjusting for age, gender, smoking status, primary tumor sites, postoperative stages, existence of residual tumor, and postoperative treatment with chemotherapy or radiotherapy. Results During a median follow-up of 1,047 days, tumor progression and death occurred in 76 (37.3%) and 27 (13.2%) patients, respectively. The FokI T/T genotype was associated with poor progression-free survival: median survival for T/T was 265 days compared with 1,127 days for C/C or C/T (log-rank test: P = 0.0004; adjusted hazard ratio, 3.03; 95% confidence interval, 1.62 to 5.67; P = 0.001). In contrast, the other polymorphisms (Cdx2, BsmI, ApaI, TaqI) showed no significant association with progression-free survival. The A-T-G (Cdx2-FokI-ApaI) haplotype demonstrated a significant association with a higher progression rate (P = 0.02). Conclusion These results suggest that VDR polymorphisms and haplotypes may be associated with prognosis in patients with HNSCC, although the sample size is not large enough to draw definitive conclusions.

Functional mutation analysis of EGFR family genes and corresponding lymph node metastases in head and neck squamous cell carcinoma

Tumors with certain mutations in the epidermal growth factor receptor (EGFR) family genes dramatically respond to EGFR inhibitors. Therefore, these mutations are important factors that influence disease progression and patient survival. We previously studied the mutation status of EGFR in patients with head and neck squamous cell carcinoma (HNSCC). However, the mutation status of lymph node metastases and the frequency of mutations in EGFR family genes have not been extensively studied. In this study, we sequenced the catalytic domains of the three other members of the EGFR family, HER2, HER3, and HER4 in 92 clinical samples of HNSCC. We identified a HER2 mutation (K716E) in one sample but no mutations were found in HER3 or HER4. Next to investigate the relationship between EGFR mutations and tumor metastasis, we compared the DNA sequences of the EGFR gene between the primary tumor and the lymph node metastasis in 31 clinical samples. Only one of the patients with an EGFR mutation in the primary HNSCC carried the same mutation (L858R) in the lymph node metastasis. Finally, we explored the tumorigenic potential of the EGFR mutations that we had previously identified and their sensitivity to two different EGFR tyrosine kinase inhibitors (CL-387785, OSI-420). Ba/F3 cells transformed with mutant EGFR genes were sensitive to treatment with lower concentrations of CL-387785 than of OSI-420. These results contribute to our understanding of the genetic basis of drug sensitivity and will help design drugs that specifically target different subtypes of HNSCC.

Autologous human nasal epithelial cell sheet using temperature‐responsive culture insert for transplantation after middle ear surgery

Postoperative mucosal regeneration of the middle ear cavity and the mastoid cavity is of great importance after middle ear surgery. However, the epithelialization of the mucosa in the middle ear is retarded because chronic inflammation without epithelialization aggravates gas exchange and clinical function. These environmental conditions in the middle ear lead to postoperative retraction and adhesion of the newly‐formed tympanic membrane. Therefore, if the mucosa on the exposed middle ear bone surface can be rapidly regenerated after surgery, the surgical treatments for cholesteatoma and adhesive middle ear disease can potentially be improved. In this study, we successfully generated a cell sheet designed for the postoperative treatment of cholesteatoma. We used nasal cells to create an artificial middle ear mucosal cell sheet with a three‐dimensional (3D) configuration similar to that of the middle ear mucosa. The sheets consisted of multi‐layered mucosal epithelia and lower connective tissue and were similar to normal middle ear mucosa. This result indicates that tissue‐engineered mucosal cell sheets would be useful to minimize complications after surgical operations in the middle ear and future clinical applications are expected. Copyright