Frequency of NFKBIA deletions is low in glioblastomas and skewed in glioblastoma neurospheres.

The NF-kB family of transcription factors is up-regulated in inflammation and different cancers. Recent data described heterozygous deletions of the NF-kB Inhibitor alpha gene (NFKBIA) in about 20% of glioblastomas (GBM): deletions were mutually exclusive with epidermal growth factor receptor (EGFR) amplification, a frequent event in GBM. We assessed the status of NFKBIA and EGFR in 69 primary GBMs and in corresponding neurospheres (NS). NFKBIA deletion was investigated by the copy number variation assay (CNV); EGFR amplification by CNV ratio with HGF; expression of EGFR and EGFRvIII by quantitative PCR or ReverseTranscriptase PCR. Heterozygous deletions of NFKBIA were present in 3 of 69 primary GBMs and, surprisingly, in 30 of 69 NS. EGFR amplification was detected in 36 GBMs: in corresponding NS, amplification was lost in 13 cases and reduced in 23 (10 vs 47 folds in NS vs primary tumors; p < 0.001). The CNV assay was validated investigating HPRT1 on chromosome X in females and males. Results of array-CGH performed on 3 primary GBMs and 1 NS line were compatible with the CNV assay. NS cells with NFKBIA deletion had increased nuclear activity of p65 (RelA) and increased expression of the NF-kB target IL-6. In absence of EGF in the medium, EGFR amplification was more conserved and NFKBIA deletion less frequent point to a low frequency of NFKBIA deletions in GBM and suggest that EGF in the culture medium of NS may affect frequency not only of EGFR amplifications but also of NFKBIA deletions.

126 novel mutations in Italian patients with neurofibromatosis type 1.

Genetic analysis of Neurofibromatosis type 1 (NF1) may facilitate the identification of patients in early phases of the disease. Here, we present an overview of our diagnostic research spanning the last 11 years, with a focus on the description of 225 NF1 mutations, 126 of which are novel, found in a series of 607 patients (513 unrelated) in Italy. Between 2003 and 2013, 443 unrelated patients were profiled by denaturing high pressure liquid chromatography (DHPLC) analysis of 60 amplicons derived from genomic NF1 DNA and subsequent sequencing of heterozygotic PCR products. In addition, a subset of patients was studied by multiplex ligation-dependent probe amplification (MLPA) to identify any duplications, large deletions or microdeletions present at the locus. Over the last year, 70 unrelated patients were investigated by MLPA and sequencing of 22 amplicons spanning the entire NF1 cDNA. Mutations were found in 70% of the 293 patients studied by DHPLC, thereby fulfilling the NIH criterion for the clinical diagnosis of NF1 (detection rate: 70%); furthermore, 87% of the patients studied by RNA sequencing were genetically characterized. Mutations were also found in 36 of the 159 patients not fulfilling the NIH clinical criteria. We confirmed a higher incidence of intellectual disability in patients harboring microdeletion type 1 and observed a correlation between a mild phenotype and the small deletion c.2970_2972delAAT or the missense alteration in amino acid residue 1809 (p.Arg1809Cys). These data support the use of RNA-based methods for genetic analysis and provide novel information for improving the management of symptoms in oligosymptomatic patients.

Gene signatures distinguish stage-specific prostate cancer stem cells isolated from transgenic adenocarcinoma of the mouse prostate lesions and predict the malignancy of human tumors.

The relevant social and economic impact of prostate adenocarcinoma, one of the leading causes of death in men, urges critical improvements in knowledge of the pathogenesis and cure of this disease. These can also be achieved by implementing in vitro and in vivo preclinical models by taking advantage of prostate cancer stem cells (PCSCs). The best-characterized mouse model of prostate cancer is the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. TRAMP mice develop a progressive lesion called prostatic intraepithelial neoplasia that evolves into adenocarcinoma (AD) between 24 and 30 weeks of age. ADs often metastasize to lymph nodes, lung, bones, and kidneys. Eventually, approximately 5% of the mice develop an androgen-independent neuroendocrine adenocarcinoma. Here we report the establishment of long-term self-renewing PCSC lines from the different stages of TRAMP progression by application of the neurosphere assay. Stage-specific prostate cell lines were endowed with the critical features expected from malignant bona fide cancer stem cells, namely, self-renewal, multipotency, and tumorigenicity. Notably, transcriptome analysis of stage-specific PCSCs resulted in the generation of well-defined, meaningful gene signatures, which identify distinct stages of human tumor progression. As such, TRAMP-derived PCSCs represent a novel and valuable preclinical model for elucidating the pathogenetic mechanisms leading to prostate adenocarcinoma and for the identification of molecular mediators to be pursued as therapeutic targets.