QuantiGene Plex Represents a Promising Diagnostic Tool for Cell-of-Origin Subtyping of Diffuse Large B-Cell Lymphoma.

Emerging therapies targeting the molecularly distinct GCB and non-GCB/ABC subtypes of diffuse large B-cell lymphoma (DLBCL) have created the need to develop an accurate subtyping assay for routine use. We investigated the potential of QuantiGene Plex (QGP)-branched DNA signal amplification assay-for DLBCL subtyping. We performed in silico analysis of public DLBCL datasets to develop and validate a naïve Bayes classifier, and migrated the resulting 21-gene classifier to QGP and real-time quantitative PCR (qPCR) assays. Forty DLBCL formalin-fixed, paraffin-embedded tumors of known subtype (20 per subtype by gene expression profiling of paired fresh-frozen tissues) were reclassified, and results for QGP (on 38/40 for 21/21 targets) and qPCR (on 40/40 samples for 19/21 targets) compared for recapitulation of microarray data and classification accuracy. The 21-gene bayesian classifier achieved mean area under the curve values >0.9 on independent validation. QGP showed a higher correlation with microarray data (mean R(2) = 0.66 ± 0.05 versus 0.34 ± 0.07; P < 0.0001) and classification accuracy (92.1% versus 78.9%). The proportion of validated targets was also higher for QGP (85.7% versus 47.4%). The QGP protocol was rapid and simple to perform, at a cost similar to qPCR. These promising preliminary results strongly support ongoing work to develop a QGP companion diagnostic assay for DLBCL subtyping.

PLK1 and YY1 interaction in follicular lymphoma is associated with unfavourable outcome.

AIMS: Ying Yang 1 (YY1) is a transcription factor involved in both proliferation and apoptosis. It is prognostic in follicular lymphoma (FL), increased protein levels being associated with favourable outcome. PLK1 is a critical regulator of mitosis, playing a role in spindle formation and in regulation of the G2/M cell cycle checkpoint. PLK1 phosphorylates YY1 at the G2/M checkpoint with activation of YY1 and resultant progression from G2 into mitosis. METHODS: This study aims to investigate possible molecular coexpression and interaction of YY1 with PLK1 in FL using Duolink II in situ proximity ligation assay (PLA) in 51 FL samples in a tissue microarray. RESULTS: Positive PLA signals were present at variable frequency and Kaplan-Meier analysis showed association of signal frequency above the median with unfavourable outcome (p=0.0270). PLA signals were localised to the nuclear edge, with only one signal per cell, suggesting PLK1 and YY1 coexpression at the centrosome. In a minority of cells, two very close PLA signals were present in a single cell, and occasionally, there was a strong ring of semi-confluent fluorescent PLA signals round the nucleus of non-dividing cells, while rarely events were observed in the cytoplasm surrounding dividing cells. CONCLUSIONS: The results confirm association of YY1 and PLK1 with outcome in FL and suggest coexpression at the centrosome. Given the reported interaction of YY1 with PLK1 at the centriole and promotion of cell division at the G2/M checkpoint, the results would concord with the known association of higher proliferation with poor outcome in FL.

Analysis of optineurin in frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) can occur jointly with amyotrophic lateral sclerosis (ALS), and these 2 conditions share a genetic risk factor on chromosome 9. It has been reported that mutations in optineurin (OPTN) can cause ALS. Therefore, we sequenced OPTN in 371 FTLD cases but no mutations were detected, suggesting changes in OPTN do not cause FTLD.

Frontotemporal lobar degeneration genome wide association study replication confirms a risk locus shared with amyotrophic lateral sclerosis.

Frontotemporal lobar degeneration (FTLD) is a common cause of dementia especially in patients under the age of 65. FTLD has a high incidence of heritability with as many as 40% of patients reporting a family history of disease. Recently, the first genome wide association study was performed using only FTLD patients with a pathologically confirmed TDP-43 pathology. Genome wide significance was detected for a single gene (TMEM106B) on chromosome 7, though several other loci on chromosomes 1, 8, 9, 10 and 11 reached nominal significance. Here we have undertaken an attempt to replicate the association of these loci in FTLD cohorts of British origin. We failed to detect any association of TMEM106B in the Manchester or London cohort either when analyzed individually or when combined. Genotyping of the Manchester cohort failed to replicate any of the loci on chromosome 1, 8 and 10 but did detect association of the single SNP (rs2015747) on chromosome 11. Association was also observed in the London cohort but in the opposite direction. Combining the 2 datasets yielded no association. Analysis of the chromosome 9 locus, revealed strong association in the London FTLD cohort and the Manchester FTLD+ALS cases. These data confirm that FTLD and amyotrophic lateral sclerosis (ALS) share a common genetic risk factor on chromosome 9p.

RAS, FLT3, and C-KIT mutations in immunophenotyped canine leukemias.

OBJECTIVE: To determine the frequency of FLT3, C-KIT, and RAS mutations in canine leukemia patients. MATERIALS AND METHODS: Ethylenediamine tetra-acetic acid blood samples were recruited from dogs with suspected leukemia, categorized by quantitative and cytological evaluation and immunophenotyping. Flow cytometry was carried out using antibodies against CD3; CD3e; CD4; CD5; CD8; CD11a, b, c, and d; CD14; CD21; CD34; CD45 and 45RA; CD79a; CD90 (THY-1); major histocompatibility complex II; myeloperoxidase; MAC387; and neutrophil-specific antibody. Genomic DNA was extracted from whole blood and analyzed for mutations in N, H, and K-RAS, FLT3, and C-KIT genes by polymerase chain reaction and sequencing. RESULTS: Fifty-seven (77.0%) of 74 samples submitted from dogs with suspected leukemia had cytologically and immunophenotypically confirmed leukemia. There were 36 (63.2%) acute leukemias, 16 (28.1%) chronic, 3 (5.3%) prolymphocytic, 1 natural killer cell, and 1 chronic leukemia undergoing blast transformation. N-RAS mis-sense mutations were identified in 14 (25%) dogs with acute myeloid (AML) or lymphoid (ALL) leukemia, and also in one dog in the leukemic phase of lymphoma. Mutations in K-RAS were found in two dogs with AML. There were no H-RAS mutations. FLT3 internal tandem duplications were identified in three dogs with ALL, and a mis-sense mutation was found in one dog with ALL. C-KIT mutations were identified in three dogs with AML. Sixty-one percent of dogs with acute leukemia harbored mutations in N/K-RAS, FLT3, or C-KIT. CONCLUSION: RAS, FLT3, and C-KIT mutations, analogous to those found in human leukemia, occur commonly in acute canine leukemia.