Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive sarcomas with variable patient survival and few known prognostically relevant genomic biomarkers. To identify survival-associated genomic biomarkers, we performed high-resolution array-based comparative genomic hybridization (aCGH) on a large set of MPNSTs.Candidate gene alterations identified by aCGH in 38 MPNSTs were validated at the DNA, RNA, and protein levels on these same tumors and an independent set of 87 MPNST specimens.aCGH revealed highly complex copy number alterations, including both previously reported and completely novel loci. Four regions of copy number gain were associated with poor patient survival. Candidate genes in these regions include SOX5 (12p12.1), NOL1 and MLF2 (12p13.31), FOXM1 and FKBP1 (12p13.33), and CDK4 and TSPAN31 (12q14.1). Alterations of these candidate genes and several others of interest (ERBB2, MYC and TP53) were confirmed by at least 1 complementary methodology, including DNA and mRNA quantitative real-time PCR, mRNA expression profiling, and tissue microarray-based fluorescence in situ hybridization and immunohistochemistry. Multivariate analysis showed that CDK4 gain/amplification and increased FOXM1 protein expression were the most significant independent predictors for poor survival in MPNST patients (P < 0.05).Our study provides new and independently confirmed candidate genes that could serve as genomic biomarkers for overall survival in MPNST patients.
A recurrent somatic mutation frequently found in cytogenetically normal acute myeloid leukemia (AML) is internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene (FLT3). This mutation is generally detected in the clinical laboratory by PCR and electrophoresis-based product sizing. As the number of clinically relevant somatic mutations in AML increases, it becomes increasingly attractive to incorporate FLT3 ITD testing into multiplex assays for many somatic mutations simultaneously, using next-generation sequencing (NGS). However, the performance of most NGS analysis tools for identifying medium-size insertions such as FLT3 ITD mutations is largely unknown. We used a multigene, targeted NGS assay to obtain deep sequence coverage (>1000-fold) of FLT3 and 26 other genes from 22 FLT3 ITD-positive and 29 ITD-negative specimens to examine the performance of several commonly used NGS analysis tools for identifying FLT3 ITD mutations. ITD mutations were present in hybridization-capture sequencing data, and Pindel was the only tool out of the seven tested that reliably detected these insertions. Pindel had 100% sensitivity (95% CI = 83% to 100%) and 100% specificity (95% CI = 88% to 100%) in our samples; Pindel provided accurate ITD insertion sizes and was able to detect ITD alleles present at estimated frequencies as low as 1%. These data demonstrate that FLT3 ITDs can be reliably detected in panel-based, next-generation sequencing assays.
Abstract Long non-coding RNAs (lncRNA) act as transcriptional regulators, scaffolds, and signaling modulators, but their role in normal or malignant lymphocytes is unknown. We identified lncRNAs with altered expression in >100 human Non-Hodgkin Lymphoma (NHL) and normal B cell samples. One of these lncRNA genes is upstream of PLCG2 and parallels its expression, but the transcript is localized in the cytoplasm, making transcriptional regulation of PLCG2 unlikely. As expected, knock-out (KO), knock-down (KD), or over-expression of lncRNA-PLCG2 did not affect PLCG2 levels. PLCG2 is a B-cell specific phospholipase C enzyme that stimulates Ca2+ signaling after BCR activation. We assessed Ca2+ signaling via IgM stimulation of the BCR in multiple lncRNA-PLCG2 KO/KD NHL cell lines and observed defects in Ca2+ efflux from the ER and influx through Orai channels. To identify lncRNA-PLCG2 interacting proteins, we performed RNA pull-down experiments using sense (S) and anti-sense (AS, control) lncRNA-PLCG2 as bait incubated with B cell lysates. Mass spectrometry of eluted proteins showed several proteins significantly enriched in S versus AS, including PLD1, DICER1 and DHX9; we confirmed these by Western blot. We next performed RNA-binding protein Immunoprecipitation (RIP) using antibodies for PLD1, DHX9 and DICER1, which showed 2–10-fold enrichment of lncRNA-PLCG2 compared to IgG isotype control. Ongoing studies are mapping lncRNA-PLCG2-protein interactions and evaluating the impact of lncRNA-PLCG2 on PLD1 activity. Taken together, these results suggest that lncRNA-PLCG2 modulates BCR-mediated Ca2+ signaling via interaction with BCR downstream signaling proteins, a previously unrecognized mechanism that may promote lymphomagenesis.
