Genetic imbalances detected by multiplex ligation-dependent probe amplification in a cohort of patients with oral squamous cell carcinoma—the first step towards clinical personalized medicine

Oral tumors are a growing health problem worldwide; thus, it is mandatory to establish genetic markers in order to improve diagnosis and early detection of tumors, control relapses and, ultimately, delineate individualized therapies. This study was the first to evaluate and discuss the clinical applicability of a multiplex ligation-dependent probe amplification (MLPA) probe panel directed to head and neck cancer. Thirty primary oral squamous cell tumors were analyzed using the P428 MLPA probe panel. We detected genetic imbalances in 26 patients and observed a consistent pattern of distribution of genetic alterations in terms of losses and gains for some chromosomes, particularly for chromosomes 3, 8, and 11. Regarding the latter, some specific genes were highlighted due to frequent losses of genetic material—RARB, FHIT, CSMD1, GATA4, and MTUS1—and others due to gains—MCCC1, MYC, WISP1, PTK2, CCND1, FGF4, FADD, and CTTN. We also verified that the gains of MYC and WISP1 genes seem to suggest higher propensity of tumors localized in the floor of the mouth. This study proved the value of this MLPA probe panel for a first-tier analysis of oral tumors. The probemix was developed to include target regions that have been already shown to be of diagnostic/prognostic relevance for oral tumors. Furthermore, this study emphasized several of those specific genetic targets, suggesting its importance to oral tumor development, to predict patients’ outcomes, and also to guide the development of novel molecular therapies.     

Permeation of Light Gases through Hexagonal Ice

Gas separation using porous solids have attracted great attention due to their energetic applications. There is an enormous economic and environmental interest in the development of improved technologies for relevant processes, such as H₂ production, CO₂ separation or O₂ and N₂ purification from air. New materials are needed for achieving major improvements. Crystalline materials, displaying unidirectional and single-sized pores, preferentially with low pore tortuosity and high pore density, are promising candidates for membrane synthesis. Herein, we study hexagonal ice crystals as an example of this class of materials. By slowly growing ice crystals inside capillary tubes we were able to measure the permeation of several gas species through ice crystals and investigate its relation with both the size of the guest molecules and temperature of the crystal.     

Validation of quantitative analysis of intravascular ultrasound images

This study investigated the accuracy and reproducibility of a computer-aided method for quantification of intravascular ultrasound. The computer analysis system was developed on an IBM compatible PC/AT equipped with a framegrabber. The quantitative assessment of lumen area, lesion area and percent area obstruction was performed by tracing the boundaries of the free lumen and original lumen.Accuracy of the analysis system was tested in a phantom study. Echographic measurements of lumen and lesion area derived from 16 arterial specimens were compared with data obtained by histology. The differences in lesion area measurements between histology and ultrasound were minimal (mean ± SD: –0.27±1.79 mm², p>0.05). Lumen area measurements from histology were significantly smaller than those with ultrasound due to mechanical deformation of histologic specimens (–5.38±5.09 mm², p<0.05). For comparison with angiography, 18 ultrasound cross-sections were obtainedin vivo from 8 healthy peripheral arteries. Luminal areas obtained by angiography were similar to those by ultrasound (–0.52±5.15 mm², p>0.05). Finally, intra- and interobserver variability of our quantitative method was evaluated in measurements of 100in vivo ultrasound images. The results showed that variations in lumen area measurements were low (5%) whereas variations in lesion area and percent area obstruction were relatively high (13%, 10%, respectively).Results of this study indicate that our quantitative method provides accurate and reproducible measurements of lumen and lesion area. Thus, intravascular ultrasound can be used for clinical investigation, including assessment of vascular stenosis and evaluation of therapeutic intervention.Dr. C.D. Mario, Div. of Cardiology, Vicenza, Italy, is the recipient of the E.S.C. Research Fellowship 1991.     

Oesophageal echocardiography

Summary The diagnostic value of oesophageal echocardiography is most striking in patients in whom precordial studies are of inadequate quality or fail to establish a definitive diagnosis. Oesophageal studies have excellent image quality, can be completed within 10 minutes without complications and, in most instances, enables the clinical question to be answered. In 50 patients referred for suspected thoracic aorta pathology, oesophageal echocardiography correctly excluded or diagnosed the type of aortic dissection, aortic aneurysm or the site of coarctation. Of 35 patients referred with suspected infective endocarditis, oesophageal echocardiography revealed complications in 18 patients, including vegetation, mycotic aneurysm, abscess or chordal rupture. Oesophageal echocardiography is extremely helpful to visualize intracardiac mass lesions. In 27 patients with a history of systemic or pulmonary embolism, the technique confirmed the presence, size and position of a mass lesion in 11 patients. Oesophageal color Doppler flow imaging further expands the diagnostic capabilities, particularly in patients with mitral valve prosthesis. Our experience indicates that oesophageal echocardiography significantly extends the diagnostic potential of echocardiography. Detailed knowledge of cardiothoracic anatomy and its pathologic sequelae is, however, a prerequisite for the efficient and safe application of this method.     

Combinatorial detection of autoreactive CD8<Superscript>+</Superscript> T cells with HLA-A2 multimers: a multi-centre study by the Immunology of Diabetes Society T Cell Workshop

Aims/hypothesis

Validated biomarkers are needed to monitor the effects of immune intervention in individuals with type 1 diabetes. Despite their importance, few options exist for monitoring antigen-specific T cells. Previous reports described a combinatorial approach that enables the simultaneous detection and quantification of multiple islet-specific CD8⁺ T cell populations. Here, we set out to evaluate the performance of a combinatorial HLA-A2 multimer assay in a multi-centre setting.

Methods

The combinatorial HLA-A2 multimer assay was applied in five participating centres using centralised reagents and blinded replicate samples. In preliminary experiments, samples from healthy donors were analysed using recall antigen multimers. In subsequent experiments, samples from healthy donors and individuals with type 1 diabetes were analysed using beta cell antigen and recall antigen multimers.

Results

The combinatorial assay was successfully implemented in each participating centre, with CVs between replicate samples that indicated good reproducibility for viral epitopes (mean %CV = 33.8). For beta cell epitopes, the assay was very effective in a single-centre setting (mean %CV = 18.4), but showed sixfold greater variability across multi-centre replicates (mean %CV = 119). In general, beta cell antigen-specific CD8⁺ T cells were detected more commonly in individuals with type 1 diabetes than in healthy donors. Furthermore, CD8⁺ T cells recognising HLA-A2-restricted insulin and glutamate decarboxylase epitopes were found to occur at higher frequencies in individuals with type 1 diabetes than in healthy donors.

Conclusions/interpretation

Our results suggest that, although combinatorial multimer assays are challenging, they can be implemented in multiple laboratories, providing relevant T cell frequency measurements. Assay reproducibility was notably higher in the single-centre setting, suggesting that biomarker analysis of clinical trial samples would be most successful when assays are performed in a single laboratory. Technical improvements, including further standardisation of cytometry platforms, will likely be necessary to reduce assay variability in the multi-centre setting.

     

Rotavirus serotypes and electropherotypes identified among hospitalised children in São Luís, Maranhão, Brazil

During June 1997-June 1999 rotavirus infection was screened in infants aged up to 2 years and hospitalised with acute diarrhoea in S?o Luís, Northeastern Brazil. Altogether, 128 stool samples were collected from diarrhoeic patients and additional 122 faecal specimens from age and temporal matched inpatients without diarrhoea were obtained; rotavirus positivity rates for these groups were 32.0% (41/128) and 9.8% (12/122), respectively (p < 0.001). Both electropherotyping and serotyping could be performed in 42 (79.2%) of the 53 rotavirus-positive stool samples. Long and short electropherotypes were detected at similar rates--38.1% and 40.5% of specimens, respectively. Overall, a G serotype could be assigned for 35 (83.3%) of specimens, the majority of them (66.7%) bearing G1-serotype specificity. Taking both electropherotypes and serotypes together, G1 rotavirus strains displaying long and short RNA patterns accounted for 30.9% and 19.0% of tested specimens, respectively; all G2 strains had short electropherotype. Rotavirus gastroenteritis was detected year-round and, in 1998, the incidence rates tended to be higher during the second semester than in the first semester: 45.2% and 26.1% (p = 0.13), respectively. Rotavirus infections peaked at the second semester of life with frequencies of 30.1% and 13.5% for diarrhoeic children and controls, respectively. While the six rotavirus strains bearing G2-type specificity were circulating throughout the whole study period, G1 serotypes (n = 27) emerged as from June 1998 onwards, 20 (74.1%) of which clustering in 1998. These data underscore the importance of rotaviruses in the aetiology of severe infantile gastroenteritis in Northeastern Brazil and sustain the concept that a future vaccine should confer protection against more than one serotype.     

Valor Prognóstico de Níveis Elevados de Troponina I Isolados em Pacientes sem Síndrome Coronariana Aguda Admitidos no Serviço de Emergência

Background:Although non-ischemic troponin elevation is frequently seen in patients admitted to the emergency department (ED), consensus regarding its management is lacking.Objectives:This study aimed to characterize patients admitted to the ED with non-ischemic troponin elevation and to identify potential mortality predictors in this population.Methods:This retrospective observational study included ED patients with a positive troponin test result between June and July of 2015. Patients with a clinical diagnosis of acute coronary syndrome (ACS) were excluded. Data on patient demographics and clinical and laboratory variables were extracted from medical records. Follow-up data were obtained for 16 months or until death occurred. The statistical significance level was 5%.Results:Troponin elevation without ACS was found in 153 ED patients. The median (IQR) patient age was 78 (19) years, 80 (52.3%) were female and 59(38.6%) died during follow-up. The median (IQR) follow-up period was 477(316) days. Survivors were significantly younger 76 (24) vs. 84 (13) years; p=0.004) and featured a higher proportion of isolated troponin elevation (without creatine kinase or myoglobin elevation) in two consecutive evaluations: 48 (53.9%) vs. 8 (17.4%), p<0.001. Survivors also presented a lower rate of antiplatelet treatment and same-day hospitalization. In the multivariate logistic regression with adjustment for significant variables in the univariate analysis, isolated troponin elevation in two consecutive evaluations showed a hazard ratio= 0.43 (95%CI 0.17–0.96, p=0.039); hospitalization, previous antiplatelet treatment and age remained independently associated with mortality.Conclusions:Isolated troponin elevation in two consecutive measurements was a strong predictor of survival in ED patients with troponin elevation but without ACS.