Comparison of Residual Dimethyl Sulfoxide (DMSO) and Ethylene Glycol (EG) Concentration in Bovine Ovarian Tissue During Warming Steps Between Slow Freezing and Vitrification.

BACKGROUND: DMSO and EG have been used as cryoprotectants for human ovarian tissue cryopreservation, but residual cryoprotectants concentration and safety have rarely been reported. OBJECTIVE: We aimed to compare residual cryoprotectants (DMSO, EG) concentration in bovine ovarian tissue during warming steps between one kind of common slow freezing method and two kinds of vitrification methods, which are usually used for cryopreservation of human ovarian tissue in Japan. MATERIALS AND METHODS: In this study, we used five bovine ovaries with an average age of 24.2 months divided into three kinds of cryopreservation methods. All ovarian cortices cut to 1 mm thickness were cryopreserved in slow freezing and two kinds of vitrification methods. Residual cryoprotectants before, during and after warming of cryopreserved ovarian cortices were measured using GC-MS and compared. RESULTS: Concentrations of residual cryoprotectants in the ovarian tissue just before transplantation into the body after warming were high after both vitrification methods but almost zero with the slow freezing method. CONCLUSION: We are concerned about the residual cryoprotectants in ovarian tissue, and continue to study the safety of cryopreservation methods to the woman after reimplantation and her baby.

A transportation network for human ovarian tissue is indispensable to success for fertility preservation.

PURPOSE: The purpose of this study was to examine the efficacy of an ovarian tissue transportation network for fertility preservation (FP) for cancer patients in Japan. METHODS: PubMed was searched for papers on transportation of human ovarian tissue for FP. We analyzed population, area, number of cancer patients for ovarian tissue cryopreservation (OTC), quality control/assessment and safety, cost of a cryopreservation center for the building for 30 years, and medical fees of cancer patients (operation, cryopreservation, and storage of ovarian tissue). RESULTS: More than twenty babies have been born in Denmark and Germany through a transportation system. Up to 400 new patients a year need OTC. The fees for removal, cryopreservation, and storage for 5 years, and transplantation of ovarian tissue are around €5,000, €4,000, and €5,000, respectively. It costs more than €5 million to establish and maintain one cryopreservation center for 30 years. If we have a few cryopreservation centers in Japan, we can cryopreserve 400 patients' ovarian tissue per year by safer slow freezing and maintain quality control/assessment. We need to lighten the patients' burden for easy to use FP by a government subsidy and medical insurance coverage. CONCLUSIONS: This model has been termed the Danish model ("the woman stays - the tissue moves"). This is truly patient-centered medicine. We can have maximum effects with the minimum burden. A transportation network like those of Denmark and Germany is the best strategy for FP in Japan. It may be the best system for cancer patients, medical staff, and the Ministry of Health, Labor, and Welfare.

Prognostic significance of PD-L1 expression on circulating tumor cells in patients with head and neck squamous cell carcinoma.

BACKGROUND: Successful application of programmed death 1 (PD1) checkpoint inhibitors in the clinic may ultimately benefit from appropriate patient selection based upon predictive biomarkers. Molecular characterization of circulating tumor cells (CTC) is crucial for the investigation of molecular-targeted therapies while predictive biomarkers for response to PD1 checkpoint inhibitors are lacking. We sought to assess whether overexpression of PD-L1 in CTCs could be detected at baseline and at different timepoints during treatment in a prospective cohort of head and neck squamous cell carcinoma (HNSCC) patients and used to predict clinical outcome after treatment with curative intent. PATIENTS AND METHODS: We developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA expression in EpCAM(+) CTCs. In a prospective cohort of 113 locally advanced HNSCC patients treated with curative intent we evaluated PD-L1 expression in the EpCAM(+) CTC fraction at baseline, after 2 cycles of induction chemotherapy (week 6) and at the end of concurrent chemoradiotherapy (week 15). RESULTS: PD-L1 overexpression was found in 24/94 (25.5%) patients at baseline, 8/34 (23.5%) after induction chemotherapy and 12/54 (22.2%) patients at the end of treatment. Patients with CTCs overexpressing PD-L1 at end of treatment had shorter progression-free survival (P = 0.001) and overall survival (P < 0.001). Multivariate analysis revealed that PD-L1 overexpression at end of treatment was independent prognostic factor for progression-free survival and overall survival. The absence of PD-L1 overexpression at the end of treatment was strongly associated with complete response with an odds ratio = 16.00 (95% CI = 2.76-92.72, P = 0.002). CONCLUSIONS: We demonstrate that detection of CTCs overexpressing PD-L1 is feasible and may provide important prognostic information in HNSCC. Our results suggest that adjuvant PD1 inhibitors deserve evaluation in HNSCC patients in whom PD-L1(+) CTCs are detected at the end of curative treatment.

Stiff substrates increase YAP-signaling-mediated matrix metalloproteinase-7 expression.

Abnormally stiff substrates have been shown to trigger cancer progression. However, the detailed molecular mechanisms underlying this trigger are not clear. In this study, we cultured T84 human colorectal cancer cells on plastic dishes to create a stiff substrate or on collagen-I gel to create a soft substrate. The stiff substrate enhanced the expression of matrix metalloproteinase-7 (MMP-7), an indicator of poor prognosis. In addition, we used polyacrylamide gels (2, 67 and 126 kPa) so that the MMP-7 expression on the 126-kPa gel was higher compared with that on the 2-kPa gel. Next, we investigated whether yes-associated protein (YAP) affected the MMP-7 expression. YAP knockdown decreased MMP-7 expression. Treatment with inhibitors of epidermal growth factor receptor (EGFR) and myosin regulatory light chain (MRLC) and integrin-α2 or integrin-ß1 knockdown downregulated MMP-7 expression. Finally, we demonstrated that YAP, EGFR, integrin-α2ß1 and MRLC produced a positive feedback loop that enhanced MMP-7 expression. These findings suggest that stiff substrates enhanced colorectal cancer cell viability by upregulating MMP-7 expression through a positive feedback loop.

RAS/PI3K crosstalk and cetuximab resistance in head and neck squamous cell carcinoma.

PURPOSE: Cetuximab, an antibody directed against the EGF receptor, is an effective clinical therapy for patients with head and neck squamous cell cancer (HNSCC). Despite great clinical promise, intrinsic or acquired cetuximab resistance hinders successful treatment outcomes but little is known about the underlying mechanism. EXPERIMENTAL DESIGN: To study the role of oncogenic HRAS in cetuximab resistance in HNSCC, the frequency of oncogenic HRAS mutations was determined in a cohort of 180 genomic DNAs from head and neck cancer specimens. We also used a combination of cetuximab-resistant cell lines and a transgenic mouse model of RAS-driven oral cancer to identify an oncogenic RAS-specific gene expression signature that promotes cetuximab resistance. RESULTS: Here, we show that activation of RAS signaling leads to persistent extracellular signal-regulated kinase 1/2 signaling and consequently to cetuximab resistance. HRAS depletion in cells containing oncogenic HRAS or PIK3CA restored cetuximab sensitivity. In our study, the gene expression signature of c-MYC, BCL-2, BCL-XL, and cyclin D1 upon activation of MAPK signaling was not altered by cetuximab treatment, suggesting that this signature may have a pivotal role in cetuximab resistance of RAS-activated HNSCC. Finally, a subset of patients with head and neck cancer with oncogenic HRAS mutations was found to exhibit de novo resistance to cetuximab-based therapy. CONCLUSIONS: Collectively, these findings identify a distinct cetuximab resistance mechanism. Oncogenic HRAS in HNSCC promotes activation of ERK signaling, which in turn mediates cetuximab resistance through a specific gene expression signature. Clin Cancer Res; 20(11); 2933-46. ©2014 AACR.

Molecular profile of head and neck squamous cell carcinomas bearing p16 high phenotype.

BACKGROUND: We sought to determine biomarker expression differences in head and neck squamous cell cancers (HNSCCs) based on p16/human papillomavirus (HPV) classification. In addition, our aim was to explore how expression of biomarkers is modulated after E6/E7 repression in HPV16⁺ oropharyngeal cancer cells. METHODS: HPV16⁺ and HPV⁻ HNSCC cells were infected with retroviruses expressing short hairpin RNA targeting HPV16 E6/E7. Components of the epidermal growth factor receptor (EGFR) pathway before and after E6/E7 gene silencing were analyzed by immunoblotting and qRT-PCR. Protein expression of 13 biomarkers was analyzed using AQUA on a tissue microarray (TMA). The HPV16 status was determined using HPV16 in situ hybridization (ISH). RESULTS: In HPV16⁺ cells, E6/E7 silencing was associated with PTEN upregulation and reduction of phosphorylated EGFR. Tumors were classified into four categories based on the HPV and p16 status. HPV⁺/p16⁺ tumors expressed significantly higher levels of E-cadherin (P = 0.003), PTEN (P = 0.004), lower levels of PI3Kp110 and ß-catenin (P = 0.07). There was a significant difference in overall survival (OS, P = 0.016) among the four subsets. The median OS was 24.83 months for p16⁻/HPV⁻ patients, 11.63 for p16⁻/HPV⁺ patients and was not reached for p16⁺/HPV⁻ and p16⁺/HPV⁺ groups. CONCLUSIONS: Aberrant EGFR signaling contributes to malignant conversion of HPV16⁺ HNSCC cells. These results validate ß-catenin as a distinct biomarker in HPV⁺/p16⁺ HNSCC. Wnt signaling inhibitors merit exploration in HPV⁺/p16⁺ HNSCC.

Bioassay technique using seed shrimps for comparative studies regarding the aquatic acute lethality of biodegradable lubricants.

To evaluate the environmental load resulting from the spillage of biodegradable lubricants in aquatic systems, a comparative acute lethality test wherein an oil-water interfacial area could be examined was considered. In this study, oleic acid was employed as a model biodegradable lubricant. Measurements of the pH value and dissolved oxygen (DO) level of water during the exposure tests indicate that water degradation depends on the oil-water interfacial area, exposure duration, and water temperature. Furthermore, 72 h acute lethality tests were performed using two types of freshwater ostracods (seed shrimps) as test organisms: the large species Stenocypris hislopi and the small species Cypretta seurati. The longevity of the small species, which was physically more active, was strongly affected by water pollution. During the exposure test, the DO in water was significantly consumed by the degradation of the lubricant floating on it. Water exposed to a lubricant containing copper (Cu) demonstrated strong toxicity even after the recovery of the pH value and DO level by aging. The decrease in the DO level of water and increase in the concentration of metal compounds are dominant factors responsible for the mortality of aquatic organisms.

Xenografting of testicular tissue from an infant human donor results in accelerated testicular maturation.

BACKGROUND: Grafting of testicular tissue into immunodeficient mice has been used to differentiate the neonatal testes from different animal species up to the level of complete spermatogenesis; however, this approach has not been successful for human testicular tissue. The aim of this study was to evaluate the capacity for differentiation of infant human testicular tissue grafts. METHODS AND RESULTS: Testicular tissue from a 3-month-old patient with testicular cancer was grafted into immunodeficient nude mice. At the time of grafting, A spermatogonia were the only germ cells present in the testicular tissue. B spermatogonia and first spermatocytes were observed at 7 months and 1 year after grafting, respectively. Positive immunostaining with antibodies against BOULE and CDC25A suggested that spermatocytes in the graft were not arrested but in meiosis. Furthermore, ultrastructural and immunohistochemical analyses showed that the onset of both Sertoli cell maturation and partial differentiation of Leydig cells preceded the appearance of spermatocytes. Differentiation of testicular cells was accelerated compared with in vivo development. CONCLUSIONS: Spermatogenesis in the xenograft of infant human testicular tissues proceeded successfully from the stage of spermatogonial stem cells until pachytene spermatocyte formation. The differentiation of Sertoli cells and Leydig cells was reproduced in a manner similar to that in normal testicular development. Grafting of infant human testicular tissue may be a powerful tool to examine the early period of human spermatogenesis and may pave the way for fertility preservation among infant patients.

p52-Independent nuclear translocation of RelB promotes LPS-induced attachment.

The NF-kappaB signaling pathways have a critical role in the development and progression of various cancers. In this study, we demonstrated that the small cell lung cancer cell line (SCLC) H69 expressed a unique NF-kappaB profile as compared to other cancer cell lines. The p105/p50, p100/p52, c-Rel, and RelB protein and mRNA transcripts were absent in H69 cells but these cells expressed RelA/p65. The activation of H69 cells by lipopolysaccharide (LPS) resulted in the induction of RelB and p100 expression. The treatment also induced the nuclear translocation of RelB without the processing of p100 to p52. Furthermore, LPS-induced beta1 integrin expression and cellular attachment through an NF-kappaB-dependent mechanism. Blocking RelB expression prevented the increase in the expression of beta1 integrin and the attachment of H69. Taken together, the results suggest that RelB was responsible for the LPS-mediated attachment and may play an important role in the progression of some cancers.

Density fluctuations in the presence of spinodal instabilities.

Density fluctuations resulting from spinodal decomposition in a nonequilibrium first-order chiral phase transition are explored. We show that such instabilities generate divergent fluctuations of conserved charges along the isothermal spinodal lines appearing in the coexistence region. Thus, divergent density fluctuations could be a signal not only for the critical end point but also for the first-order phase transition expected in strongly interacting matter. We also compute the mean-field critical exponent at the spinodal lines. Our analysis is performed in the mean-field approximation to the Nambu-Jona-Lasinio model formulated at finite temperature and density. However, our main conclusions are expected to be generic and model independent.

Identifying and reducing errors with surgical simulation.

The major determinant of a patient's safety and outcome is the skill and judgment of the surgeon. While knowledge base and decision processing are evaluated during residency, technical skills-which are at the core of the profession-are not evaluated. Innovative state of the art simulation devices that train both surgical tasks and skills, without risk to patients, should allow for the detection and analysis of errors and "near misses". Studies have validated the use of a sophisticated endoscopic sinus surgery simulator (ES3) for training residents on a procedural basis. Assessments are proceeding as to whether the integration of a comprehensive ES3 training programme into the residency curriculum will have long term effects on surgical performance and patient outcomes. Using various otolaryngology residencies, subjects are exposed to mentored training on the ES3 as well as to minimally invasive trainers such as the MIST-VR. Technical errors are identified and quantified on the simulator and intraoperatively. Through a web based database, individual performance can be compared against a national standard. An upgraded version of the ES3 will be developed which will support patient specific anatomical models. This advance will allow study of the effects of simulated rehearsal of patient specific procedures (mission rehearsal) on patient outcomes and surgical errors during the actual procedure. The information gained from these studies will help usher in the next generation of surgical simulators that are anticipated to have significant impact on patient safety.

Prognostic significance of p16 protein levels in oropharyngeal squamous cell cancer.

PURPOSE: Functional inactivation of p16 is an early and frequent event in head and neck squamous cell cancers. In this study, we sought to determine whether p16 expression is of prognostic importance in oropharyngeal squamous cell carcinoma. EXPERIMENTAL DESIGN: p16 protein expression was evaluated by immunohistochemistry in a tissue microarray composed of 123 oropharyngeal squamous cell cancers with a mean patient follow-up time of 33 months. RESULTS: p16 overexpression was associated with more advanced Tumor-Node-Metastasis stage and higher histologic grade. Despite this association with unfavorable features, p16 overexpression was associated with decreased 5-year local recurrence rates (11 versus 53%) and increased 5-year disease-free survival (62 versus 19%) and overall survival (60 versus 21%). In multivariate analysis, p16 expression status remained an independent prognostic factor for local recurrence, disease-free survival, and overall survival. CONCLUSIONS: In patients with oropharyngeal squamous cell carcinoma, overexpression of p16 as determined by immunohistochemistry is associated with significantly improved prognosis and lower local recurrence rates.

Cisplatin, fluorouracil, and leucovorin induction chemotherapy followed by concurrent cisplatin chemoradiotherapy for organ preservation and cure in patients with advanced head and neck cancer: long-term follow-up.

PURPOSE: The poor functional outcome in patients with advanced head and neck squamous cell carcinoma (HNSCC) with surgery and radiation has led to alternative approaches to advanced disease. We conducted a phase II study of induction chemotherapy followed by concurrent chemoradiotherapy for organ preservation in patients with advanced resectable and unresectable (nasopharyngeal) tumors. PATIENTS AND METHODS: Forty-two patients with stage III to IV resectable HNSCC and nasopharyngeal tumors received induction chemotherapy with two courses of cisplatin (20 mg/m2/d continuous infusion [CI]), fluorouracil (800 mg/m2/d CI), and leucovorin (500 mg/m2/d CI; PFL) for 4 days followed by concurrent therapy with cisplatin (100 mg/m2/d on days 1 and 22) and approximately 70 Gy of external-beam radiotherapy. RESULTS: Response to induction chemotherapy included partial response rate of 52% and complete response rate of 24%. The most common grade 3 or 4 toxicity was neutropenia (59%). After cisplatin chemoradiotherapy the complete response rate was 67%. Toxicities of cisplatin chemoradiotherapy consisted of grade 3 or 4 mucositis (79%) and neutropenia (51%). At a median follow-up of 71.5 months, 43% of the patients are still alive and disease-free. The 5-year progression-free survival (PFS) rate was 60%, and the 2- and 5-year overall survival (OS) rates were 67% and 52%, respectively. Three patients died of second primaries. Late complications of treatment included xerostomia and hoarseness. One patient had persistent dysphagia and required laser epiglotectomy 108 months after treatment. CONCLUSION: Induction chemotherapy with PFL followed by concurrent cisplatin chemoradiotherapy is well tolerated and results in a good likelihood of organ preservation and excellent PFS and OS.

Comparison between tube compensation and pressure support ventilation techniques on respiratory mechanics.

In the intubated patient, the presence of an endotracheal tube increases the work of breathing during spontaneous breathing. The tube compensation technique was developed as a new ventilator mode that can compensate for that additional the work of breathing. We investigated the respiratory parameters during the pressure support ventilation 0, 5, 10 cmH2O and tube compensation 100% modes of the Puritan Bennett 840 ventilator in ten postoperative patients who had undergone radical surgery for oesophageal cancer. Measurements were performed just before extubation. The tidal volume, respiratory rate and other respiratory parameters were measured with a Ventrak respiratory monitor, and the duty ratio, mean inspiratory flow, and rapid shallow breathing index were calculated. In particular, we performed a comparison between pressure support ventilation 5 cmH2O and tube compensation 100%, because pressure support ventilation 5 cmH2O is the usual ventilating mode before the extubation in our intensive care unit. The tidal volume of pressure support ventilation 10 cmH2O was significantly larger and the respiratory rate was significantly lower than the other three modes. There was no significant difference in the minute volume, tidal volume, and respiratory rate between pressure support ventilation 5 cmH2O and tube compensation 100%. The duty ratio of pressure support ventilation 10 cmH2O was significantly smaller than the other three modes. There was no significant difference in the duty ratio and rapid shallow breathing index between pressure support ventilation 5 cmH2O and tube compensation 100%. It was concluded that the assist levels of pressure support ventilation 5 cmH2O and tube compensation 100% were almost equal for clinical purposes.

A case of paroxysmal kinesigenic dyskinesia with spastic paraparesis.

We report the first case of paroxysmal kinesigenic dyskinesia (PKD) with spastic paraparesis. A 17-year-old male began to show a dystonic posture in both his upper limbs when walking at age 12 years. Neurological examination revealed bilateral talipes cavus, spasticity in all extremities with general hyperreflexia and pathological reflexes. On starting to walk, he showed a dystonic posture in bilateral maniphalanx, wrists, elbows, and toes. Magnetic resonance imaging (MRI) revealed high T2-weighted signal intensity in bilateral pyramidal tract. Although the combination of pyramidal and the basal ganglia disorders is very rare, the present case suggests an inter-relation of the pyramidal and the basal ganglia systems.

Tratamento clínco de perfuração esofágica após tratamento endoscópico de divertículo de Zenker: relatro de caso / Clinical treatment of esophageal perforation after endoscopic treatment for Zenker

O divertículo de Zenker é uma alteração da anatomia esofágica que acomete adultos de idade avançada, caracterizadao pela presença de um divertículo posterior, proximal ao músculo cricofaríngeo. De forma precoce, apresenta-se como disfagia transitória e, como o evoluir da doença, como sensação de massa no pescoço e regurgitação. seu tratamento pode ser realizado por endoscopia flexível ou por correção cirúrgica através de miotomia cricofaríngea com diverticulectomia. O objetivo deste trabalhoo é relatar uma possível complicação do tratamento do divertículo de Zenker por endoscopia flexível. Descreve-se o caso de uma paciente feminina, 83 anos, portadora de divertículo de Zenker com início da sintomatologia havia seis meses, submetida a diverticulotomia endoscópica e que algumas horas após o procedimento evoluiu dor e efisema subcutâneo bilateral em região cervical. Esofagografia com contrate iodado evidenciou extravasamento em região cervical. Foi então, instituido manejo conservador, com alta hospitalar assintomática no sexto dia pós-procedimento. Portanto, apesar de considera modalidade de tratamento segura, a diverticulotomia endoscópica não é isenta de riscos

Cancer of the pharynx and larynx.

Carcinoma of the head and neck is among the most debilitating forms of cancer. Survival rates for these tumors vary and depend on the presence of early symptoms, anatomic accessibility, and lymphatic supply. Despite advances in therapy and novel surgical approaches, early diagnosis remains the best predictor of survival. This article reviews the diagnosis, staging criteria, and treatment strategies for nasopharyngeal carcinoma, hypopharyngeal carcinoma, and laryngeal carcinoma in an effort to heighten the clinical and endoscopic recognition of these lesions.

Temporal and spatial differences of PSA-NCAM expression between young-adult and aged rats in normal and ischemic brains.

Highly polysialylated neural cell adhesion molecule (PSA-NCAM) is transiently expressed specifically in newly generated cells, and is important for migration and neurite outgrowth. To investigate the effect of aging on the migration of neural stem cell (NSC) after brain ischemia, the spatiotemporal expressions of immunoreactive PSA-NCAM were examined at 4 h or 1, 3 or 7 days after 90 min of middle cerebral artery occlusion (MCAO) in the young-adult or aged rats. In the sham control brain, PSA-NCAM staining was slightly observed both in dorsal and ventral parts of subventricular zone (SVZ) in the aged brain, but only in the dorsal part of SVZ in the young brain. After transient MCAO, immunoreactivity for PSA-NCAM increased in the number and the intensity in SVZ ipsilateral to MCAO in the young-adult brains and became the peak at 1 day, while that was at 3 days in the aged brains. These findings suggest that PSA-NCAM was located in different spatial distribution in normal condition between young and old rats. PSA-NCAM was induced after ischemia, and the temporal expression was also different after transient MCAO between young and older rats.

Role of hydrophilic interaction in binding of hydroxylated 3-deoxy C(19) steroids to the active site of aromatase.

As part of our investigation into the structure-activity relationship of a novel class of aromatase inhibitors, C(19) steroids having no oxygen function at C-3, we tested aromatase inhibition activity of polar diol compounds 4,19-dihydroxyandrost-5-en-17-ones (25 and 27) and 6,19-dihydroxyandrost-4-en-17-ones (36 and 37). 4alpha,19-Diol 25 was synthesized from tert-butyldimethylsilyoxyandrost-4-ene steroid (9) through its OsO(4) oxidation, giving the 4alpha,5alpha-dihydroxy derivative 12, as a key reaction. Acetylation of 5beta,6alpha-dihydroxy-19-acetate 30 and its 5alpha,6beta-analogue 31 followed by dehydration with SOCl(2) and alkaline hydroxysis gave 6alpha,19-diol 36 and its 6beta-isomer 37, respectively. The stereochemistry of a hydroxy group at C-4 of compound 25 and that at C-6 of compounds 36 and 37 were determined on the basis of (1)H NMR spectroscopy in each case. 4beta,19-Diol 27, previously synthesized, was identified as an extremely powerful competitive inhibitor of aromatase (K(i) = 3.4 nM). In contrast, its 4alpha,19-dihydroxy isomer 25 and other series of diol compounds, 6,19-dihydroxy-4-en-17-one steroids, were moderate to poor competitive inhibitors (K(i) = 110-800 nM). Through this series of analyses, it was concluded that hydrophilic interaction of a 4beta,19-diol function with the active site of aromatase plays a critical role in the tight binding of 3-deoxy-5-ene steroids.