Intraductal carcinoma (IC) is the new World Health Organization designation for tumors previously called "low-grade cribriform cystadenocarcinoma" and "low-grade salivary duct carcinoma." The relationship of IC to salivary duct carcinoma is controversial, but they now are considered to be distinct entities. IC is a rare low-grade malignant salivary gland neoplasm with features similar to mammary atypical ductal hyperplasia or ductal carcinoma in situ, that shows diffuse S100 protein and mammaglobin positivity and is only partially defined genetically. (Mammary analogue) secretory carcinoma harboring ETV6-NTRK3, and in rare cases ETV6-RET fusion, shares histomorphologic and immunophenotypical features with IC. Recently, RET rearrangements and NCOA4-RET have been described in IC, suggesting a partial genetic overlap with mammary analogue secretory carcinoma. Here, we genetically characterize the largest cohort of IC to date to further explore this relationship. Seventeen cases of IC were analyzed by next-generation sequencing using the FusionPlex Solid Tumor kit (ArcherDX). Identified fusions were confirmed using fluorescence in situ hybridization break apart and, in some cases, fusion probes, and a reverse transcription polymerase chain reaction designed specifically to the detected breakpoints. All analyzed cases were known to be negative for ETV6 rearrangement by fluorescence in situ hybridization and for ETV6-NTRK3 fusion by reverse transcription polymerase chain reaction. Next-generation sequencing analysis detected a NCOA4-RET fusion transcript joining exon 7 or 8 of NCOA4 gene and exon 12 of RET gene in 6 cases of intercalated duct type IC; and a novel TRIM27-RET fusion transcript between exons 3 and 12 in 2 cases of salivary gland tumors displaying histologic and immunohistochemical features typical of apocrine IC. A total of 47% of IC harbored a fusion involving RET. In conclusion, we have confirmed the presence of NCOA4-RET as the dominant fusion in intercalated duct type IC. A novel finding in our study has been a discovery of a subset of IC patients with apocrine variant IC harboring a novel TRIM27-RET.

• MeSH
• DNA vazebné proteiny genetika   MeSH
• dospělí MeSH
• fenotyp MeSH
• fúze genů * MeSH
• genetická predispozice k nemoci MeSH
• hybridizace in situ fluorescenční MeSH
• imunohistochemie MeSH
• intraduktální neinfiltrující karcinom chemie   genetika   patologie   MeSH
• jaderné proteiny genetika   MeSH
• koaktivátory jaderných receptorů genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové biomarkery analýza   genetika   MeSH
• nádory slinných žláz chemie   genetika   patologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protoonkogenní proteiny c-ret genetika   MeSH
• registrace MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• stanovení celkové genové exprese přístrojové vybavení   MeSH
• transkriptom MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm). RESULTS: For evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted. CONCLUSIONS: We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step.

• MeSH
• lidé MeSH
• messenger RNA analýza   krev   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí přístrojové vybavení   metody   MeSH
• senzitivita a specificita MeSH
• stanovení celkové genové exprese přístrojové vybavení   metody   MeSH
• zdraví dobrovolníci pro lékařské studie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• diagnóza počítačová MeSH
• lékařská počítačová informatika MeSH
• mikročipová analýza statistika a číselné údaje   MeSH
• nádory diagnóza   epidemiologie   terapie   MeSH
• registrace MeSH
• rozhodování pomocí počítače * MeSH
• stanovení celkové genové exprese metody   přístrojové vybavení   MeSH
• Publikační typ
• práce podpořená grantem MeSH