We describe a novel gene fusion, EWSR1-CREM, identified in 3 cases of clear cell carcinoma (CCC) using anchored multiplex polymerase chain reaction, a next-generation sequencing-based technique. CCC is a low-grade salivary tumor recently characterized to have EWSR1-ATF1 fusions in the majority of cases. Three cases of malignant tumor presenting in the base of tongue, lung, and nasopharynx were studied. All cases shared a clear cell morphology with hyalinized stroma, presence of mucin and p63 positivity and were initially diagnosed as mucoepidermoid carcinoma but were negative for evidence of any of the expected gene fusions. Anchored multiplex polymerase chain reaction demonstrated a EWSR1-CREM fusion in all 3 cases to confirm a diagnosis of CCC. This finding is biologically justified as CREM and ATF1 both belong to the CREB family of transcription factors. EWSR1-CREM fusions have not been previously reported in CCC and have only rarely been reported in other tumors. We show that the ability to discover novel gene variants with next-generation sequencing-based assays has clinical utility in the pathologic classification of fusion gene-associated tumors.
- MeSH
- adenokarcinom z jasných buněk genetika patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- modulátor elementu responzivního pro cyklický AMP genetika MeSH
- nádory jazyka genetika patologie MeSH
- nádory nosohltanu genetika MeSH
- nádory plic genetika patologie MeSH
- onkogenní fúze MeSH
- protein EWS vázající RNA genetika MeSH
- senioři MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
UNLABELLED: In an attempt to explore infectious agents associated with nasopharyngeal carcinomas (NPCs), we employed our high-throughput RNA sequencing (RNA-seq) analysis pipeline, RNA CoMPASS, to investigate the presence of ectopic organisms within a number of NPC cell lines commonly used by NPC and Epstein-Barr virus (EBV) researchers. Sequencing data sets from both CNE1 and HONE1 were found to contain reads for human papillomavirus 18 (HPV-18). Subsequent real-time reverse transcription-PCR (RT-PCR) analysis on a panel of NPC cell lines identified HPV-18 in CNE1 and HONE1 as well as three additional NPC cell lines (CNE2, AdAH, and NPC-KT). Further analysis of the chromosomal integration arrangement of HPV-18 in NPCs revealed patterns identical to those observed in HeLa cells. Clustering based on human single nucleotide variation (SNV) analysis of two separate HeLa cell lines and several NPC cell lines demonstrated two distinct clusters with CNE1, as well as HONE1 clustering with the two HeLa cell lines. In addition, duplex-PCR-based genotyping showed that CNE1, CNE2, and HONE1 do not have a HeLa cell-specific L1 retrotransposon insertion, suggesting that these three HPV-18(+) NPC lines are likely products of a somatic hybridization with HeLa cells, which is also consistent with our RNA-seq-based gene level SNV analysis. Taking all of these findings together, we conclude that a widespread HeLa contamination may exist in many NPC cell lines, and authentication of these cell lines is recommended. Finally, we provide a proof of concept for the utility of an RNA-seq-based approach for cell authentication. IMPORTANCE: Nasopharyngeal carcinoma (NPC) cell lines are important model systems for analyzing the complex life cycle and pathogenesis of Epstein-Barr virus (EBV). Using an RNA-seq-based approach, we found HeLa cell contamination in several NPC cell lines that are commonly used in the EBV and related fields. Our data support the notion that contamination resulted from somatic hybridization with HeLa cells, likely occurring at the point of cell line establishment. Given the rarity of NPCs, the long history of NPC cell lines, and the lack of rigorous cell line authentication, it is likely that the actual prevalence and impact of HeLa cell contamination on the EBV field might be greater. We therefore recommend cell line authentication prior to performing experiments using NPC cell lines to avoid inaccurate conclusions. The novel RNA-seq-based cell authentication approach reported here can serve as a comprehensive method for validating cell lines.
- MeSH
- genom * MeSH
- HeLa buňky chemie MeSH
- kontaminace DNA MeSH
- lidé MeSH
- nádorové buněčné linie chemie MeSH
- nádory nosohltanu genetika MeSH
- polymerázová řetězová reakce MeSH
- sekvenční analýza RNA MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
- MeSH
- dítě MeSH
- dyspnoe etiologie MeSH
- lidé MeSH
- nádory nosohltanu diagnóza genetika klasifikace MeSH
- novorozenec MeSH
- polypy diagnóza klasifikace MeSH
- poruchy polykání MeSH
- teratom diagnóza MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
polo and CDC5 are two genes required for passage through mitosis in Drosophila melanogaster and Saccharomyces cerevisiae, respectively. Both genes encode structurally related protein kinases that have been implicated in regulating the function of the mitotic spindle. Here, we report the characterization of a human protein kinase that displays extensive sequence similarity to Drosophila polo and S. cerevisiae Cdc5; we refer to this kinase as Plk1 (for polo-like kinase 1). The largest open reading frame of the Plk1 cDNA encodes a protein of 68,254 daltons, and a protein of this size is detected by immunoblotting of HeLa cell extracts with monoclonal antibodies raised against the C-terminal part of Plk1 expressed in Escherichia coli. Northern blot analysis of RNA isolated from human cells and mouse tissues shows that a single Plk1 mRNA of 2.3 kb is highly expressed in tissues with a high mitotic index, consistent with a possible function of Plk1 in cell proliferation. The Plk1 gene maps to position p12 on chromosome 16, a locus for which no associations with neoplastic malignancies are known. The Plk1 protein levels and its distribution change during the cell cycle, in a manner consistent with a role of Plk1 in mitosis. Thus, like Drosophila polo and S. cerevisiae Cdc5, human Plk1 is likely to function in cell cycle progression.
- MeSH
- buněčný cyklus MeSH
- DNA vazebné proteiny genetika MeSH
- Drosophila melanogaster genetika MeSH
- fungální proteiny genetika MeSH
- genová knihovna MeSH
- geny MeSH
- HeLa buňky MeSH
- karcinom genetika MeSH
- klonování DNA MeSH
- lidé MeSH
- lidské chromozomy, pár 16 * MeSH
- mapování chromozomů MeSH
- mitóza * MeSH
- molekulární sekvence - údaje MeSH
- monoklonální protilátky imunologie MeSH
- nádory nosohltanu genetika MeSH
- otevřené čtecí rámce MeSH
- protein-serin-threoninkinasy genetika MeSH
- proteinkinasy genetika imunologie izolace a purifikace fyziologie MeSH
- proteiny buněčného cyklu * MeSH
- proteiny Drosophily * MeSH
- proteiny vázající RNA MeSH
- protoonkogenní proteiny MeSH
- regulace genové exprese u nádorů MeSH
- Saccharomyces cerevisiae - proteiny MeSH
- Saccharomyces cerevisiae genetika MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční seřazení MeSH
- subcelulární frakce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- MeSH
- genetické techniky MeSH
- HLA-B antigeny MeSH
- lidé MeSH
- nádory nosohltanu genetika MeSH
- Check Tag
- lidé MeSH
