Quantitative Measurement of L1 Human Papillomavirus Type 16 Methylation for the Prediction of Preinvasive and Invasive Cervical Disease.

Background: Methylation of the human papillomavirus (HPV) DNA has been proposed as a novel biomarker. Here, we correlated the mean methylation level of 12 CpG sites within the L1 gene, to the histological grade of cervical precancer and cancer. We assessed whether HPV L1 gene methylation can predict the presence of high-grade disease at histology in women testing positive for HPV16 genotype. Methods: Pyrosequencing was used for DNA methylation quantification and 145 women were recruited. Results: We found that the L1 HPV16 mean methylation (±SD) significantly increased with disease severity (cervical intraepithelial neoplasia [CIN] 3, 17.9% [±7.2] vs CIN2, 11.6% [±6.5], P < .001 or vs CIN1, 9.0% [±3.5], P < .001). Mean methylation was a good predictor of CIN3+ cases; the area under the curve was higher for sites 5611 in the prediction of CIN2+ and higher for position 7145 for CIN3+. The evaluation of different methylation thresholds for the prediction of CIN3+ showed that the optimal balance of sensitivity and specificity (75.7% and 77.5%, respectively) and positive and negative predictive values (74.7% and 78.5%, respectively) was achieved for a methylation of 14.0% with overall accuracy of 76.7%. Conclusions: Elevated methylation level is associated with increased disease severity and has good ability to discriminate HPV16-positive women that have high-grade disease or worse.

Personalised management of women with cervical abnormalities using a clinical decision support scoring system.

OBJECTIVES: To develop a clinical decision support scoring system (DSSS) based on artificial neural networks (ANN) for personalised management of women with cervical abnormalities. METHODS: We recruited women with cervical abnormalities and healthy controls that attended for opportunistic screening between 2006 and 2014 in 3 University Hospitals. We prospectively collected detailed patient characteristics, the colposcopic impression and performed a series of biomarkers using a liquid-based cytology sample. These included HPV DNA typing, E6&E7 mRNA by NASBA or flow cytometry and p16INK4a immunostaining. We used ANNs to combine the cytology and biomarker results and develop a clinical DSSS with the aim to improve the diagnostic accuracy of tests and quantify the individual's risk for different histological diagnoses. We used histology as the gold standard. RESULTS: We analysed data from 2267 women that had complete or partial dataset of clinical and molecular data during their initial or followup visits (N=3565). Accuracy parameters (sensitivity, specificity, positive and negative predictive values) were assessed for the cytological result and/or HPV status and for the DSSS. The ANN predicted with higher accuracy the chances of high-grade (CIN2+), low grade (HPV/CIN1) and normal histology than cytology with or without HPV test. The sensitivity for prediction of CIN2 or worse was 93.0%, specificity 99.2% with high positive (93.3%) and negative (99.2%) predictive values. CONCLUSIONS: The DSSS based on an ANN of multilayer perceptron (MLP) type, can predict with the highest accuracy the histological diagnosis in women with abnormalities at cytology when compared with the use of tests alone. A user-friendly software based on this technology could be used to guide clinician decision making towards a more personalised care.

Artificial Neural Networks as Decision Support Tools in Cytopathology: Past, Present, and Future.

OBJECTIVE: This study aims to analyze the role of artificial neural networks (ANNs) in cytopathology. More specifically, it aims to highlight the importance of employing ANNs in existing and future applications and in identifying unexplored or poorly explored research topics. STUDY DESIGN: A systematic search was conducted in scientific databases for articles related to cytopathology and ANNs with respect to anatomical places of the human body where cytopathology is performed. For each anatomic system/organ, the major outcomes described in the scientific literature are presented and the most important aspects are highlighted. RESULTS: The vast majority of ANN applications are related to cervical cytopathology, specifically for the ANN-based, semiautomated commercial diagnostic system PAPNET. For cervical cytopathology, there is a plethora of studies relevant to the diagnostic accuracy; in addition, there are also efforts evaluating cost-effectiveness and applications on primary, secondary, or hybrid screening. For the rest of the anatomical sites, such as the gastrointestinal system, thyroid gland, urinary tract, and breast, there are significantly less efforts relevant to the application of ANNs. Additionally, there are still anatomical systems for which ANNs have never been applied on their cytological material. CONCLUSIONS: Cytopathology is an ideal discipline to apply ANNs. In general, diagnosis is performed by experts via the light microscope. However, this approach introduces subjectivity, because this is not a universal and objective measurement process. This has resulted in the existence of a gray zone between normal and pathological cases. From the analysis of related articles, it is obvious that there is a need to perform more thorough analyses, using extensive number of cases and particularly for the nonexplored organs. Efforts to apply such systems within the laboratory test environment are required for their future uptake.

Dysphagia Caused by Chronic Laryngeal Edema.

A rare case of a young female with chronic diffuse laryngeal edema causing severe swallowing difficulty is presented. The patient was previously treated with antibiotics and steroids with no improvement. Diagnosis was made with biopsy of the epiglottis under local anesthesia in the office.

Rhodotorula mucilaginosa associacted meningitis: A subacute entity with high mortality. Case report and review.

A fatal case of meningitis due to Rhodotorula mucilaginosa in a 28 year-old HIV-negative male with a history of Hodgkin lymphoma who underwent salvage chemotherapy is presented. Reviewing the literature we identified 13 cases with central nervous system infection due Rhodotorula spp. The disease usually occurs in HIV negative immunosupressed middle-aged males. It takes the form of subacute or chronic meningitis accompanied by fever with an overall mortality of 46.2% despite antifungal therapy.

A preliminary study of the potential of tree classifiers in triage of high-grade squamous intraepithelial lesions.

OBJECTIVE: To investigate the potential value of tree classifiers for the triage of high-grade squamous intraepithelial lesions. STUDY DESIGN: The dataset comprised 808 histologically confirmed cases having a complete range of the cytologic sample assessments--liquid-based cytology, reflex human papillomavirus (HPV) DNA test, E6/E7 HPV mRNA test, and p16 immunocytochemical examinations. Data include 488 histologically negative (cervical intraepithelial neoplasia [CIN] 1 and below) or clinically negative cases and 320 with histologic diagnosis of CIN 2 or worse. Cytologic diagnosis was made according to the criteria of the Bethesda System. Cases were classified in two groups according to histology: those with CIN 2 or worse and those with CIN 1 and below. Fifty percent were randomly selected as a training set and the remaining were as a test set. RESULTS: Application of tree classifier on the test set gave correct classification of 66.9% for CIN 2 and above cases and 97.3% for CIN 1 and below, producing overall accuracy of 91.5%, outperforming cytologic diagnosis alone. CONCLUSION: Application of tree classifiers, based on standard cytologic diagnosis and expression of studied biomarkers, produces improved classification results for cervical precancerous lesions and cancer diagnosis and

Human papillomavirus (HPV) detection using in situ hybridization in histologic samples: correlations with cytologic changes and polymerase chain reaction HPV detection.

Although in situ hybridization (ISH) and polymerase chain reaction (PCR) have extensively been used on cytology specimens, there have been limited reports of the usefulness of these techniques in relation to confirmed histologic findings. In this study, we used PCR and ISH to detect human papillomavirus (HPV) in cytologic and histologic specimens, respectively. By using positive and negative likelihood ratios, we attempted to identify any predictive role of ISH testing alone or in combination with PCR for the development of high-grade histologic lesions (cervical intraepithelial neoplasia [CIN] 2+). In our study, ISH was a useful method for detection of HPV, even in a large fraction of samples with normal cytologic or biopsy findings. We suggest that when used together and evaluated in conjunction with histologic sections, ISH is a useful tool for ancillary molecular testing of HPV infection in cervical lesions, especially in CIN 2+ histological lesions where its analytic sensitivities and specificities were as good as those of PCR testing.

Cascaded learning vector quantizer neural networks for the discrimination of thyroid lesions.

OBJECTIVE: To investigate capability of combination of learning vector quantizer (LVQ) neural networks (NNs) in discrimination of benign from malignant thyroid lesions. STUDY DESIGN: The study included 335 liquid-based cytology, fine needle aspiration (FNA), Papanicolaou-stained specimens. All cases were compared to the histologic diagnosis. Features describing size, shape, and texture of -100 nuclei per case were extracted from cytologic images using a custom image analysis system. These features were used to classify each nucleus by LVQ type NNs. The nucleus classification results were used to classify individual lesions with a second LVQ NN. Cases were distributed by histologic diagnosis. Data from -50% from each category were used for training LVQ classifiers. Remaining data were used to test classifier performance. The system was used to discriminate to individual cellular level and individual patient level between benign and malignant nuclei. RESULTS: Application of the proposed algorithm combining two LVQ NNs allows discrimination between benign and malignant cell nuclei and lesions. CONCLUSION: Results indicate that use of NNs, combined with image morphometry, can provide information on thyroid lesion malignancy potential. The system could improve FNA diagnostic accuracy of the thyroid gland, especially in follicular neoplasms suspicious for malignancy and in Hürthle cell tumors.

Post-operative Aspergillus mediastinitis in a man who was immunocompetent: a case report.

INTRODUCTION: Aspergillus spp. infections mainly affect patients who are immunocompromised, and are extremely rare in immunocompetent individuals. CASE PRESENTATION: Aspergillus post-operative mediastinitis is considered to be a devastating infection, usually affecting patients undergoing cardiothoracic surgery with specific predisposing factors. We describe the case of an immunocompetent 68-year-old Caucasian man with severe chronic thromboembolic pulmonary hypertension, who underwent pulmonary thromboendarterectomy and developed post-operative mediastinitis due to Aspergillus flavus. The environmental control did not reveal the source of A. flavus infection and, despite combined antifungal therapy, our patient died as a result of septic shock and multiple organ failure. CONCLUSION: Aspergillus mediastinitis mainly affects patients after cardiosurgery operations with predisposing factors, and it is unusual in patients who are immunocompetent. The identification of the Aspergillus spp. source is often difficult, and there are no guidelines for the administration of pre-emptive therapy in this population of at-risk patients.

Hybrid capture vs. PCR screening of cervical human papilloma virus infections. Cytological and histological associations in 1270 women.

BACKGROUND: We evaluated two molecular methods of HPV detection and their correlation with cytological and histological diagnosis in a large sample of Greek women. METHODS: All women with liquid-based cytology performed at a University Hospital between 2000 and 2003 were included. The Hybrid Capture 2 (HC2) kit and in house Polymerase Chain Reaction (PCR) were used for HPV DNA detection. Cervical biopsy was performed for women with ASCUS+ cytology, HPV detection, or abnormal colposcopy. Positive (PLR) and negative (NLR) likelihood ratios were calculated for cytology and HPV molecular testing for the prediction of CIN2 and greater histology. RESULTS: Of the 1270 women evaluated 241 (18.5%) had abnormal cytology. Cytology diagnosed high-grade squamous intraepithelial lesion (HSIL) or invasive carcinoma in 21(1.7%) cases whereas 26 (2%) women had CIN2+ or greater histology. PCR detected HPV in 397/1270 (31.3%) and HC2 in 260/1270 (20.4%) samples. Both molecular tests exhibited high reproducibility (Cohen's kappa value 0.691, 95% CI: 0.664 - 0.718). Positive likelihood ratios (PLR) of 9.4, 3.8 and 3.4 and negative likelihood ratios of 0.13, 0.21, and 0 were noted for >or= LSIL, any positive HC2 or any positive PCR-HPV testing, for predicting CIN2+ histology, respectively. All CIN 3+ lesions harbored high risk oncogenic HPV type infections. CONCLUSIONS: HPV infection was found in a large proportion of this population and was associated with CIN 2/3 lesions and infiltrating carcinomas. Thin prep testing and HPV detection by HC2 or PCR performed very well with regards to identifying high grade lesions in an environment with experienced examiners.

Carboplatin and paclitaxel in metastatic or recurrent cervical cancer.

OBJECTIVES: The purpose of this study was to evaluate the activity and toxicity of carboplatin and paclitaxel combination in advanced or recurrent carcinoma of the cervix. METHODS: Fifty-one eligible patients with measurable advanced or recurrent cervical carcinoma were treated with carboplatin (area under the curve, 5) and paclitaxel 175 mg/m every 3 weeks for 6 to 9 cycles or until disease progression or unacceptable toxicity. RESULTS: Eight complete (16%) and 19 partial responses (37%) occurred, for an overall response rate (RR) of 53% (95% confidence interval [CI], 39%-67%). The median progression-free survival was 6 months (95% CI, 5.4-6.5 months), and the median overall survival was 13 months (95% CI, 11.4-14.5 months). The RR was higher in patients with disease outside a previously irradiated site compared with those with disease in a previously irradiated field (68% vs 30%) (P = 0.011). Patients previously treated with chemoradiation had an RR of 28%, whereas in those previously treated with radiotherapy alone, the RR was 68% (P = 0.023). There was no statistically significant difference between histology and response to therapy. Patients with performance status of 0 or 1 had a higher RR than those with worse performance status. Toxicity was generally mild except for myelotoxicity. Neutropenia grade 3/4 was recorded in 44% of patients, and 6% experienced febrile neutropenia. Twenty-two percent of patients experienced anemia grade 3-4, whereas 14% had thrombocytopenia grade 3-4. Three patients (6%) developed grade 3 sensory neuropathy. CONCLUSION: The combination of carboplatin and paclitaxel seems to have activity in advanced or recurrent cervical carcinoma with an acceptable toxicity profile.

Combination chemotherapy with carboplatin, paclitaxel and pegylated liposomal doxorubicin for advanced or recurrent carcinosarcoma of the uterus: clinical experience of a single institution.

OBJECTIVES: The purpose of this study was to evaluate the activity and toxicity of carboplatin, paclitaxel and pegylated liposomal doxorubicin combination in advanced or recurrent of the uterine carcinosarcoma. METHODS: Twenty-nine eligible patients with measurable disease were treated with carboplatin [area under the curve (AUC) 5], paclitaxel 175 mg/m(2) and pegylated liposomal doxorubicin 25 mg/m(2) every 3 weeks for 6-8 cycles. RESULTS: There were 10 complete responses (CRs) (34%) and 8 partial responses (PRs) (28%) for an overall response rate (RR) of 62% (95% confidence interval [CI], 43-81%). The median progression-free survival (PFS) was 8.2 months (95% CI, 4.1-12.2 months) and the median overall survival (OS) was 16.4 months (95% CI, 14.7-18.0 months). There was no statistically significant difference between histology and response to therapy. Patients with PS of 0 or 1 had a higher RR than those with worst PS. Toxicity was generally mild except for myelotoxicity. Neutropenia grade 3/4 was recorded in 52% of patients and 10% experienced febrile neutropenia. Anemia grade 3 or 4 developed in 27% of patients and thrombocytopenia grade 3 or 4 in 31% of patients. Three patients (10%) developed grade 3 sensory neuropathy and only 2 patients (8%) grade 3 palmar-plantar erythrodysesthesias. No treatment-related deaths were recorded in our series. CONCLUSION: The combination of carboplatin, paclitaxel and pegylated liposomal doxorubicin appears to have activity in advanced, persistent or recurrent endometrial carcinosarcoma with an acceptable toxicity profile.

Antihistamine effects on experimental middle ear inflammatory model.

Allergy is thought to be one of the etiologic factors in otitis media. The purpose of this study was to determine the histopathologic effects of H1 histamine receptor antagonists in an experimental histamine-induced middle ear inflammation model. In group A (20 rabbits), histamine challenge followed a 3-day intramuscular pretreatment with a single dose of 0.1 ml hydroxyzine hydrochloride (50 mg/ml) per day. In group B (20 rabbits), histamine challenge followed a 3-day pretreatment with a single dose 1.2 mg desloratadine per day orally. In group C (20 rabbits), histamine challenge followed a 3-day pretreatment with a single dose 1.2 mg levocetirizine per day orally. On the fourth day after baseline otomicroscopy 0.5 ml histamine 20 mg/ml was injected transtympanically on the right. Middle ear mucosa was removed and the following parameters were assessed: edema, vascular dilatation and congestion, inflammation, acute inflammatory component, presence of eosinophils, activity of the inflammation and fibrosis. A semi-quantitative grading system of 0-3 was used for grading of all parameters and statistical analysis performed by using the Mann-Whitney non-parametric test. Group A mucosae showed lower grades of all the parameters evaluated compared with those of group B. Histopathology of the mucosae of group C showed lower grade of inflammation compared with group B with significant statistical difference for the seven parameters tested. Our data validate the use of antihistamines in the treatment of refractory OME of allergic origin. Oldest H1 antagonists can be replaced by the newest agents who counteract successfully histamine effects, without any interactions or adverse effects from central nervous system.

Common femoral artery injury secondary to tennis ball strike.

Tennis is a popular, safe sport that exposes amateur or professional players to minimal risk of injury. Most of this risk is related to musculoskeletal injuries, and vascular trauma is extremely rare. We present a case of a 68-year-old man in whom intermittent claudication developed after his right groin was struck by a tennis ball. The patient sought medical evaluation 2 months after the accident, and a subtotal occlusion of his right common femoral artery was disclosed by angiography and treated by endarterectomy.

A simple digital image processing system to aid in melanoma diagnosis in an everyday melanocytic skin lesion unit: a preliminary report.

BACKGROUND: For early melanoma diagnosis, experienced dermatologists have an accuracy of 64-80% using clinical diagnostic criteria, usually the ABCD rule, while automated melanoma diagnosis systems are still considered to be experimental and serve as adjuncts to the naked-eye expert prediction. In an attempt to aid in early melanoma diagnosis, we developed an image processing program with the aim to discriminate melanoma from melanocytic nevi, establishing a mathematical model to come up with a melanoma probability. METHODS: Digital images of 132 melanocytic skin lesions (23 melanomas and 109 melanocytic nevi) were studied in features of geometry, color, and color texture. A total of 43 variables were studied for all lesions, e.g., geometry, color texture, sharpness of border, and color variables. Univariate logistic regression analysis followed by "-2 log likelihood" test and Spearman's rank correlation coefficient were used to eliminate inappropriate variables, as the presence of multi-collinearity among variables could cause severe problems in any stepwise variable selection method. Initially, "-2 log likelihood" and nonparametric Spearman's rho picked five variables to be included in a multivariate model of prediction. The five-variable model was then reduced to three variables and the performance of each model was tested. The "jackknife" method was performed in order to validate the model with the three variables and its accuracy was weighed vs. the five-variable model by receiver-operating characteristics (ROC) curve plotting. It was concluded that the reduced model did not compromise discriminatory power. RESULTS: Not all variables contributed much to the model, therefore they were progressively eliminated and the model was finally reduced to three covariates of significance. A predictive equation was calculated, incorporating parameters of geometry, color, and color texture as independent covariates for the prediction of melanoma. The proposed model provides melanoma probability with a 60.9% sensitivity and 95.4% specificity of prediction, an overall accuracy of 89.4% (probability level 0.5), and 8% false-negative results. CONCLUSIONS: Through a digital image processing system and the development of a mathematical model of prediction, discrimination between melanomas and melanocytic nevi seems feasible with a high rate of accuracy using multivariate logistic regression analysis. The proposed model is an alternative method to aid in early melanoma diagnosis. Expensive and sophisticated equipment is not required and it can be easily implemented in a reasonably priced portable programmable computer, in order to predict previously undiagnosed skin melanoma before histopathology results confirm diagnosis.

A clinical study of optical biopsy of the uterine cervix using a multispectral imaging system.

OBJECTIVE: To present the clinical application of the multispectral imaging colposcopic system (MIS colposcopy). METHODS: MIS colposcopy was performed on 123 enrolled women. After a 3% acetic acid application, sequential images were captured, analyzed, and stored automatically. Directed biopsies were taken from distinct marked acetic acid-responsive tissue areas indicated on the monitor, while a real-time assessment of the curves of intensity of the backscattered light (IBSL) vs. time was performed. Blind biopsies were taken from non-acetowhitening areas. Histological findings were correlated with MIS colposcopy results and compared with conventional colposcopy and Pap test results. RESULTS: Acetic acid-tissue interaction resulted in temporal and spatial alterations to the light scattering properties of the abnormal tissue that was analyzed. The shape of IBSL curve and the "relaxation time" (the time it takes for IBSL to decay to 1/e of its peak value) changed in accordance with the underlying lesion. More severe CIN lesions lead to higher maximum IBSL; longer durations of acetowhitening lead to increasingly delayed exponential decay of IBSL curve. To compare with histological examination, MIS colposcopy had a 1.7% false-diagnostic rate, while PAP test and conventional colposcopy had 24.4% and 22% false-diagnostic rates, respectively. A triple exponential function created a "pseudocolor" image that comprised the grade map of the lesion, and this is frequently representative of the duration/degree of the induced alterations. CONCLUSION: Improved diagnostic information can be gained by recording the optical information in a narrow spectral range with high spatial resolution. MIS colposcopy can be used in the diagnosis of uterine cervix pathological conditions and in the differentiation between CIN lesions.

Effect of single dose irradiation on human glioblastoma spheroids in vitro.

Investigation of the predictive value of a radiosurgery-relevant treatment of glioblastoma spheroids. Organotypic multicellular spheroids were cultured and irradiated (20 Gy). Morphology, apoptosis and immunohistochemical expression of p53, p21, MIB-1, TGF-beta and VEGF were examined 4 h, 24 h, 7 days, and 14 days following treatment. Cell proliferation decreased, while apoptosis was increased. No morphological damage was observed. p53 expression was significantly increased after 4 h. TGF-beta and VEGF expression were only slightly altered. Particularly early changes in proliferation and apoptosis can be observed in spheroids. Individual response differences suggest spheroids of human gliomas to be useful for monitoring radiosurgery effects.

Lump sensation in the throat caused by tumors in the preepiglottic space.

Lump sensation in the throat is a common symptom, which accounts for 4% of first patient visits to Otorhinolaryngologic clinics. The etiology includes abnormalities of the thyroid gland, cysts, tumors and chronic infections of the pharynx, larynx, esophagus and tongue base, gastroesophageal reflux and anxiety disorders. In this article we describe two rare cases of lump sensation caused by masses in the preepiglottic space and we stress the importance of thorough investigation to exclude any possible non functional causes.

Hidradenitis suppurativa associated with Crohn's disease and spondyloarthropathy: response to anti-TNF therapy.

An association of hidradenitis suppurativa with Crohn's disease is supported by previous repent. We here report a patient with hidradenitis suppurativa who subsequently developed peripheral arthritis, sacroiliitis, and Crohn's disease. A significant attenuation of bowel, cutaneous, and joint symptoms was achieved after treatment with monoclonal antibody against tumor necrosis factor (TNF). The pathogenetic aspects according to the literature and response to the various therapeutic measures applied are also presented.