ATRX immunohistochemistry can help refine 'not elsewhere classified' categorisation for grade II/III gliomas.

Purpose: The 2016 WHO tumour classification highlights the role of IDH1/2 gene mutation and 1p/19q co-deletion in classifying grade II/III gliomas. A recent cIMPACT-NOW update proposes the use of the term 'Not Elsewhere Classified' (NEC) for IDH-mutant, non co-deleted tumours. Here we show how the incorporation of ATRX immunohistochemistry can be used to better delineate the NEC group. Methods: Clinical data was collected for 112 patients (59% male) treated at our unit. Mutations in IDH1/2 genes were detected by pyrosequencing or immunohistochemistry, 1p/19q co-deletion was assessed with fluorescence in situ hybridisation and ATRX status was determined using immunohistochemical techniques. Tumours were grouped on the basis of molecular markers and outcomes compared. Results: The mean age of diagnosis was 42.6 years (20-73 years). There were 88 oligodendrogliomas (II = 47, III = 41), 18 diffuse astrocytomas (II = 9, III = 9) and 6 oligoastrocytomas (II = 4, III = 2). The majority of gliomas (87.5%) had mutations in IDH1/2. 1p/19q co-deletion was significantly associated with oligodendroglial morphology (p = < 0.001) and was mutually exclusive with ATRX mutation. Classification on the basis of molecular information showed a significant different in survival between the groups. Conclusions: ATRX immunohistochemisty is a useful adjunct which can be used with IDH mutation status, 1p/19q co-deletion and histological findings to further define tumour groups. More work is needed to understand the molecular profiles and prognostic implications for non co-deletion, ATRX preserved cases.

Molecular epidemiology of Streptococcus zooepidemicus infection in naturally occurring equine respiratory disease.

The objective of the study was to characterise the molecular epidemiology of Streptococcus zooepidemicus infection among isolates collected sequentially from recently weaned, pasture maintained Welsh mountain ponies with naturally occurring respiratory disease. Weekly nasopharyngeal and tracheal lavage samplings over a 10-week period were conducted in 29 ponies. Two PCR typing methods based on characterisation of the M-protein hypervariable (HV) region and the 16S-23S rRNA gene intergenic spacer were then applied to isolates of S. zooepidemicus recovered from nasopharyngeal swab and tracheal wash samples. S. zooepidemicus infection was highly prevalent during the study, being isolated from 94% of tracheal washes and 88% of nasopharyngeal swabs. Among 39 different S. zooepidemicus types isolated, more were isolated from the trachea (n=33) than the nasopharynx (n=27). There was evidence from temporal patterns of infection for clonal succession over time by the more prevalent S. zooepidemicus types. Novel S. zooepidemicus types were identified, including previously untyped HV regions and intra-strain multiples of both the HV region and intergenic spacer types.

Association of the lymphotoxin-alpha gene Thr26Asn polymorphism with severity of coronary atherosclerosis.

A recent large-scale, genome-wide association study of single nucleotide polymorphisms showed a strong association between susceptibility to myocardial infarction and the Thr26Asn polymorphism in the lymphotoxin-alpha (LTA) gene. In the present study, we investigated whether the LTA Thr26Asn polymorphism was associated with the extent of coronary atherosclerosis in a large cohort (n=1082) of well-documented coronary artery disease patients. Thr26Asn genotypes showed a significant different distribution in male patients, when stratified according to the number of diseased coronary arteries, with an odds ratio of 1.98 (95% CI 1.22-3.22) for multiple-vessel disease in patients of the Asn/Asn genotype, compared with patients of the Thr/Thr or Thr/Asn genotype (P=0.006). Thus, further to the recent finding that LTA gene variation is associated with susceptibility to coronary heart disease, the present study provides evidence of an association between LTA genotype and the extent of coronary atherosclerosis.

Measurement of tumour reactive antibody and antibody conjugate by competition, quantitated by flow cytofluorimetry.

Binding of unlabelled monoclonal antibody preparations has been assessed by competition at saturation with fluorochrome labelled homologous antibody for binding to antigen bearing target cells. The extent of competition was measured by quantitative flow cytofluorimetry, and simple mathematical procedures have been developed to allow the interpretation of competition data in terms of antibody binding activity. In the system studied, non-specific (non-competitive) fluorescence was minimal, but an iterative method to calculate its contribution to the measured signal is given. This approach has the advantage that the antibody preparation to be tested does not need to be labelled or modified; this is particularly important when evaluating the binding activity of therapeutic antibody conjugates. Comparison with a well characterized standard antibody preparation provides a rapid, sensitive and accurate quality control procedure. This test is also simple to perform, requiring only the mixing of labelled and unlabelled antibodies with target cells, a single incubation, followed by analysis without washing of the target cells.

Kinetics of divalent monoclonal antibody binding to tumour cell surface antigens using flow cytometry: standardization and mathematical analysis.

Flow cytofluorimetric methods have been used to quantitate the interaction between a divalent monoclonal antibody and a tumour cell surface antigen. After standardization using fluorescein and 125I-labelled antibodies, kinetics of association and dissociation were measured, and antibody bound at equilibrium quantitated. A mathematical model was developed in conjunction with these experimental results which allowed the calculation of rates for monovalent association and monovalent and divalent dissociation, and a description of the contribution of each to the level of bound antibody at different antibody concns.