Sentinel Node Biopsy for the Head and Neck Using Contrast-Enhanced Ultrasonography Combined with Indocyanine Green Fluorescence in Animal Models: A Feasibility Study.

BACKGROUND: Sentinel node navigation surgery is gaining popularity in oral cancer. We assessed application of sentinel lymph node navigation surgery to pharyngeal and laryngeal cancers by evaluating the combination of contrast-enhanced ultrasonography and indocyanine green fluorescence in animal models. METHODS: This was a prospective, nonrandomized, experimental study in rabbit and swine animal models. A mixture of indocyanine green and Sonazoid was used as the tracer. The tracer mixture was injected into the tongue, larynx, or pharynx. The sentinel lymph nodes were identified transcutaneously by infra-red camera and contrast-enhanced ultrasonography. Detection time and extraction time of the sentinel lymph nodes were measured. The safety of the tracer mixture in terms of mucosal reaction was evaluated macroscopically and microscopically. RESULTS: Sentinel lymph nodes were detected transcutaneously by contrast-enhanced ultrasonography alone. The number of sentinel lymph nodes detected was one or two. Despite observation of contrast enhancement of Sonazoid for at least 90 minutes, the number of sentinel lymph nodes detected did not change. The average extraction time of sentinel lymph nodes was 4.8 minutes. Indocyanine green fluorescence offered visual information during lymph node biopsy. The safety of the tracer was confirmed by absence of laryngeal edema both macro and microscopically. CONCLUSIONS: The combination method of indocyanine green fluorescence and contrast-enhanced ultrasonography for detecting sentinel lymph nodes during surgery for head and neck cancer seems promising, especially for pharyngeal and laryngeal cancer. Further clinical studies to confirm this are warranted.

Validity of sentinel lymph node biopsy by ICG fluorescence for early head and neck cancer.

AIM: This study was designed to assess the validity of sentinel lymph node (SLN) biopsy using either the combination of indocyanine green (ICG) fluorescence and radioisotope (RI) or ICG-alone in SLN mapping for early head and neck cancer patients. PATIENTS AND METHODS: Nineteen patients received SLN biopsy with the following method. Thirteen patients received SLN biopsy with only RI, 2 patients with only ICG and 4 patients with the combination of ICG and RI. Detection time for each method of SLN biopsy was measured to evaluate the validity of SLN with the combination of ICG and RI. RESULTS: A total of 41 SLNs were identified by RI or ICG. All SLNs identified by ICG could be localized intraoperatively. The number of SLNs identified by the combination of ICG and RI was greater than that of SLNs identified by RI-alone. One of the patients who underwent SLN biopsy by RI-alone was diagnosed with a metastatic lymph node one year later, then underwent neck dissection. Mean detection time for SLN biopsy with ICG or with the combination of ICG and RI tended to be shorter than that of RI-alone. CONCLUSION: SLN biopsy with the combination of ICG and RI enabled us to identify SLNs more easily and rapidly than by using RI alone.

Feasibility study of alternate-day S-1 as adjuvant chemotherapy for head and neck cancer.

AIM: This study analyzed the safety and feasibility of alternate-day S-1, a mixture of tegafur, dehydroxypyrimidine and potassium oxonate, as adjuvant chemotherapy for head and neck cancers. PATIENTS AND METHODS: Patients with head and neck squamous cell carcinoma (HNSCC) who underwent primary treatment received alternate-day S-1 (80 mg/day for 1 year). The primary end-point was treatment completion rate. The secondary end-point was adverse events. Three-year overall survival (OS) and disease-free survival (DFS) were calculated using the Kaplan-Meier method. RESULTS: One-year completion rate was 65.6%. Out of 26 patients, 19.0% had grade III adverse events. All adverse reactions were tolerable and reversible. Three-year OS and DFS were 74.8% and 57.3%, respectively. CONCLUSION: S-1 therapy is an effective adjuvant treatment for head and neck cancer patients with relatively mild side-effects and does not adversely affect quality of life. A phase I/II study is warranted to investigate the appropriate dose for an alternate-day S-1 regimen.

Phase I study of nedaplatin prior to S-1 in patients with locally advanced head and neck squamous cell carcinoma.

BACKGROUND: We previously reported on the regimen of S-1 plus nedaplatin (NDP), with S-1 was administered orally for 14 days and NDP intravenously on day 8. The maximum tolerated dose (MTD) of NDP was determined to be 90 mg/m². The main toxicities were neutropenia and thrombocytopenia. This result was tolerated, but we believe there is a more effective and tolerable regimen. Thus, we investigated the S-1 regimen administered orally for 14 days, and NDP intravenously on day 1 in patients with locally advanced head and neck squamous cell carcinoma. PATIENTS AND METHODS: Oral administration of S-1 (days 1-14) and intravenous NDP (day 1) were tested for patients with advance head and neck cancer in a phase I setting. The dose of S-1 was fixed and the dose of NDP was escalated from 70 mg/m², with an increase of 10 mg/m² per step, to find the MTD. RESULTS: A total of 15 patients were registered. The MTD of NDP was determined to be 100 mg/m². The main toxicities were neutropenia and thrombocytopenia. The response rate (RR) was 57.1%. CONCLUSIONS: The recommended dose of NDP for a phase II study was determined to be 100 mg/m². We concluded that our regimen was well tolerated and that the RR was acceptable.

Intraoperative molecular assessment for lymph node metastasis in head and neck squamous cell carcinoma using one-step nucleic acid amplification (OSNA) assay.

BACKGROUND: Conventional intraoperative pathological examination for Sentinel node navigation surgery (SNNS) has been controversial. We evaluated the efficacy of one-step nucleic acid amplification (OSNA) assay for intraoperative diagnosis of cervical lymph node (CLN) metastasis compared with histopathological examination in patients with head and neck squamous cell carcinoma (HNSCC). METHODS: A total of 175 CLNs dissected from 56 patients with HNSCC who underwent surgery at Aichi Cancer Center, Kyorin University, Gunma University or Fukushima Medical University, between April 2008 and December 2011 were enrolled. CLN samples were sectioned into four equal pieces, with two of each used for OSNA assay and other histopathological examinations. The diagnostic value of OSNA assay in HNSCC patients in predicting the results of histopathological diagnosis was evaluated using the area under the receiver operating characteristic (AUROC) curve. RESULTS: OSNA assay showed acceptable efficacy in the detection of pathological CLN metastasis (AUROC 0.918, 95 % confidence interval [CI] 0.852-0.984). Regarding the CK19mRNA cutoff value, the optimum cutoff point in HNSCC patients was 131 copies/µl (sensitivity: 82.4, 95 % CI 65.5-93.2; specificity: 99.3, 95 % CI 96.1-100.0; positive likelihood ratio 116.1; negative likelihood ratio 0.2]. CONCLUSIONS: We demonstrated that OSNA assay is useful in intraoperative diagnosis for CLN metastasis in patients with HNSCC. OSNA assay could be applied for SNNS in HNSCC patients.

Expression and function of glycogen synthase kinase-3 in human hair follicles.

Beta-catenin is involved in the hair follicle morphogenesis and stem cell differentiation, and inhibition of glycogen synthase kinase-3 (GSK-3) increases beta-catenin concentration in the cytoplasm. To examine the effects of GSK-3 inhibition on the hair follicle epithelium, we first examined the expression of GSK-3 in plucked human hair follicles by RT-PCR and found GSK-3 expression in hair follicles. Western blotting with a GSK-3beta-specific antibody, Y174, also demonstrated GSK-3beta expression in the follicles. Moreover, GSK-3beta immunostaining with Y174 showed that GSK-3beta colocalized with hair follicle bulge markers. Contrary to GSK-3beta, GSK-3 alpha was widely expressed throughout the follicles when immunostained with a specific antibody, EP793Y. We then investigated the influence of GSK-3 inhibition. A GSK-3 inhibitor, BIO, promoted the growth of human outer root sheath cells, which could be cultured for up to four passages. The BIO-treated cells exhibited smaller and more undifferentiated morphology than control cells. Moreover, in organ culture of plucked human hair, outer root sheath cells in the middle of a hair follicle proliferated when cultured with BIO. These results indicate that GSK-3beta is expressed in hair bulge stem cells and BIO promotes the growth of ORS cells, possibly by regulating the GSK-3 signaling pathway.

Inhibition of glycogen synthase kinase-3 enhances the expression of alkaline phosphatase and insulin-like growth factor-1 in human primary dermal papilla cell culture and maintains mouse hair bulbs in organ culture.

Dermal papilla (DP) at the hair follicle base is important for hair growth. Recent studies demonstrated that mouse vibrissa DP cells can be cultured in the presence of fibroblast growth factor-2 (FGF-2), but lose expression of versican and their follicle-inducing activity during the culture, and that activation of the Wnt signal, which is inhibited by glycogen synthase kinase-3 (GSK-3), in the DP cells promotes hair growth activity. We therefore investigated the influence of a GSK-3 inhibitor, (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO), on the growth of human DP cells and mouse vibrissa follicles in culture. We first demonstrated that, similarly to mouse DP cells, human DP cells were able to be cultured up to 15 passages in the presence of FGF-2, and lost the expression of alkaline phosphatase (ALP). When human DP cells later than ten passages were treated with BIO, the expression of ALP as well as insulin-like growth factor-1 (IGF-1), another DP marker, was significantly elevated. Nuclear and perinuclear translocation of beta-catenin was also observed. We then cultured mouse vibrissa follicles. In the presence of BIO, the follicles could be maintained for at least 3 days without detectable regression of the hair bulbs. The morphology and ALP expression were well preserved. BIO successfully retrieved the expression of DP marker molecules, such as ALP and IGF-1 in cultured human DP cells, and maintained mouse hair bulbs. Thus, treatment with BIO may be useful to prepare DP cells with hair follicle-inducing activity.