Multicenter evaluation of an automated, multiplex, RNA-based molecular assay for detection of ALK, ROS1, RET fusions and MET exon 14 skipping in NSCLC.

The current study assessed the performance of the fully automated RT-PCR-based Idylla™ GeneFusion Assay, which simultaneously covers the advanced non-small cell lung carcinoma (aNSCLC) actionable ALK, ROS1, RET, and MET exon 14 rearrangements, in a routine clinical setting involving 12 European clinical centers. The Idylla™ GeneFusion Assay detects fusions using fusion-specific as well as expression imbalance detection, the latter enabling detection of uncommon fusions not covered by fusion-specific assays. In total, 326 archival aNSCLC formalin-fixed paraffin-embedded (FFPE) samples were included of which 44% were resected specimen, 46% tissue biopsies, and 9% cytological specimen. With a total of 179 biomarker-positive cases (i.e., 85 ALK, 33 ROS1, 20 RET fusions and 41 MET exon 14 skipping), this is one of the largest fusion-positive datasets ever tested. The results of the Idylla™ GeneFusion Assay were compared with earlier results of routine reference technologies including fluorescence in situ hybridization, immunohistochemistry, reverse-transcription polymerase chain reaction, and next-generation sequencing, establishing a high sensitivity/specificity of 96.1%/99.6% for ALK, 96.7%/99.0% for ROS1, 100%/99.3% for RET fusion, and 92.5%/99.6% for MET exon 14 skipping, and a low failure rate (0.9%). The Idylla™ GeneFusion Assay was found to be a reliable, sensitive, and specific tool for routine detection of ALK, ROS1, RET fusions and MET exon 14 skipping. Given its short turnaround time of about 3 h, it is a time-efficient upfront screening tool in FFPE samples, supporting rapid clinical decision making. Moreover, expression-imbalance-based detection of potentially novel fusions may be easily verified with other routine technologies without delaying treatment initiation.

Identification and characterization of a novel homozygous deletion in the alpha-N-acetylglucosaminidase gene in a patient with Sanfilippo type B syndrome (mucopolysaccharidosis IIIB).

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disease that is caused by a deficiency of the lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). Over 100 different mutations in the NAGLU gene have been identified in Sanfilippo syndrome type B patients; however, no large deletions have been reported. Here we present the first case of a large homozygous intragenic NAGLU gene deletion identified in an affected child of consanguineous parents. Long range and multiplex PCR methods were used to characterize this deletion which encompasses exons 3 and 4 and is 1146 base pairs long. We propose that Alu element-mediated unequal homologous recombination between an Alu-Y in intron 2 and an Alu-Sx in intron 4 is the likely mechanism for this deletion, thereby contributing further insight into the molecular etiology of this disorder and providing additional evidence of its allelic heterogeneity.

Improved detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide and interleukin-2 stimulation: A Belgian multicentric study.

We performed a multicentric study to assess the impact of two different culture procedures on the detection of chromosomal abnormalities in 217 consecutive unselected cases with chronic lymphocytic leukemia (CLL) referred for routine analysis either at the time of diagnosis (n = 172) or during disease evolution (n = 45). Parallel cultures of peripheral blood or bone marrow were set up with the addition of either the conventional B-cell mitogen 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or a combination of CpG oligonucleotide (CpG) and interleukin-2 (IL-2). Cytogenetic analyses were performed on both cultures. Clonal abnormalities were identified in 116 cases (53%). In 78 cases (36%), the aberrant clone was detected in both cultures. Among these, the percentages of aberrant metaphases were similar in both conditions in 17 cases, higher in the CpG/IL-2 culture in 43 cases, and higher in the TPA culture in 18 cases. Clonal aberrations were detected in only one culture, either in CpG/IL-2 or TPA in 33 (15%) and 5 (2%) cases, respectively. Taken together, abnormal karyotypes were observed in 51% with CpG/IL-2 and 38% with TPA (P < 0.0001). Application of FISH (n = 201) allowed the detection of abnormalities not visible by conventional cytogenetic analysis in 80 cases: del(13q) (n = 71), del(11q) (n = 5), +12 (n = 2), del(14q) (n = 1), and del(17p) (n = 1). In conclusion, our results confirm that CpG/IL-2 stimulation increases the detection rate of chromosomal abnormalities in CLL compared with TPA and that further improvement can be obtained by FISH. However, neither conventional cytogenetics nor FISH detected all aberrations, demonstrating the complementary nature of these techniques.

Impaired up-regulation of polo-like kinase 2 in B-cell chronic lymphocytic leukaemia lymphocytes resistant to fludarabine and 2-chlorodeoxyadenosine: a potential marker of defective damage response.

The functional evaluation of ataxia telangiectasia mutated (ATM) and p53 was recently developed in B-cell chronic lymphocytic leukaemia (B-CLL), a disease in which the response to DNA damage is frequently altered. We identified a novel biomarker of chemosensitivity based on the induction of DNA damage by the purine nucleoside analogues (PNA) fludarabine and 2-chlorodeoxyadenosine (CdA). Using genome-wide expression profiling, it was observed that, in chemosensitive samples, PNA predominantly increased the expression of p53-dependent genes, among which PLK2 was the most highly activated at early time points. Conversely, in chemoresistant samples, p53-dependent and PLK2 responses were abolished. Using a quantitative real time polymerase chain reaction, we confirmed that PNA dose- and time-dependently increased PLK2 expression in chemosensitive but not chemoresistant B-CLL samples. Analysis of a larger cohort of B-CLL patients showed that cytotoxicity induced by PNA correlated well with PLK2 mRNA induction. Interestingly, we observed that failure to up-regulate PLK2 following PNA and chemoresistance were not strictly correlated with structural alterations in the TP53 gene. In conclusion, we propose that testing PLK2 activation after a 24-h incubation with PNA could be used to investigate the functional integrity of DNA damage-response pathways in B-CLL cells, and predict clinical sensitivity to these drugs.

The nuclear factor kappaB-activator gene PLEKHG5 is mutated in a form of autosomal recessive lower motor neuron disease with childhood onset.

Lower motor neuron diseases (LMNDs) include a large spectrum of clinically and genetically heterogeneous disorders. Studying a large inbred African family, we recently described a novel autosomal recessive LMND variant characterized by childhood onset, generalized muscle involvement, and severe outcome, and we mapped the disease gene to a 3.9-cM interval on chromosome 1p36. We identified a homozygous missense mutation (c.1940 T-->C [p.647 Phe-->Ser]) of the Pleckstrin homology domain-containing, family G member 5 gene, PLEKHG5. In transiently transfected HEK293 and MCF10A cell lines, we found that wild-type PLEKHG5 activated the nuclear factor kappa B (NF kappa B) signaling pathway and that both the stability and the intracellular location of mutant PLEKHG5 protein were altered, severely impairing the NF kappa B transduction pathway. Moreover, aggregates were observed in transiently transfected NSC34 murine motor neurons overexpressing the mutant PLEKHG5 protein. Both loss of PLEKHG5 function and aggregate formation may contribute to neurotoxicity in this novel form of LMND.

Activity and safety of combined rituximab with chlorambucil in patients with mantle cell lymphoma.

We evaluated the combination of rituximab with chlorambucil in patients with mantle cell lymphoma (MCL) not eligible for aggressive therapy. Fourteen patients (male/female: 9/5) were included (two newly diagnosed, 12 relapsed/refractory). The toxicities were neutropenia, thrombopenia and infection. Nine (64%) patients responded; five (36%) achieved complete remission and four (29%) achieved partial remission. The median progression-free survival for responders was 26 months (95% CI, 4-48). Marrow polymerase chain reaction negativity was attained in seven responders. These results suggest that this schedule may have notable antitumour activity in patients with MCL, including patients in relapse after autologous stem cell transplantation.