Dedifferentiation of Human Primary Thyrocytes into Multilineage Progenitor Cells without Gene Introduction

While identification and isolation of adult stem cells have potentially important implications, recent reports regarding dedifferentiation/reprogramming from differentiated cells have provided another clue to gain insight into source of tissue stem/progenitor cells. In this study, we developed a novel culture system to obtain dedifferentiated progenitor cells from normal human thyroid tissues. After enzymatic digestion, primary thyrocytes, expressing thyroglobulin, vimentin and cytokeratin-18, were cultured in a serum-free medium called SAGM. Although the vast majority of cells died, a small proportion (∼0.5%) survived and proliferated. During initial cell expansion, thyroglobulin/cytokeratin-18 expression was gradually declined in the proliferating cells. Moreover, sorted cells expressing thyroid peroxidase gave rise to proliferating clones in SAGM. These data suggest that those cells are derived from thyroid follicular cells or at least thyroid-committed cells. The SAGM-grown cells did not express any thyroid-specific genes. However, after four-week incubation with FBS and TSH, cytokeratin-18, thyroglobulin, TSH receptor, PAX8 and TTF1 expressions re-emerged. Moreover, surprisingly, the cells were capable of differentiating into neuronal or adipogenic lineage depending on differentiating conditions. In summary, we have developed a novel system to generate multilineage progenitor cells from normal human thyroid tissues. This seems to be achieved by dedifferentiation of thyroid follicular cells. The presently described culture system may be useful for regenerative medicine, but the primary importance will be as a tool to elucidate the mechanisms of thyroid diseases

Multi-center real-world comparison of the fully automated Idylla (TM) microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer

Microsatellite instability (MSI) is present in 15-20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla (TM) MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla (TM) testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla (TM) results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla (TM) MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had >= 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla (TM) MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla (TM) MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla (TM) MSI Assay and routine tests.Peer reviewe

12. Jahrestagung der Sektion Molekulare Diagnostik der DGKL am 6. und 7. Juni 2013 in der Evangelischen Akademie Tutzing / Report on the 12th Annual Meeting of the Section of Molecular Diagnostics of the DGKL on 6th/7th June 2013 in Tutzing.

Die diesjährige 12. Jahrestagung der Sektion Molekulare Diagnostik der Deutschen Vereinten Gesellschaft für Klinische Chemie und Laboratoriumsmedizin (DGKL) stand unter dem Leitthema „Molekulare Signaturen“ und fand vom 06.–07.06.2013 in Tutzing statt. Molekulare Signaturen ergeben sich aus der Bewertung mehrerer gleichzeitig bestimmter Biomarker mit dem Ziel einer verbesserten Prävention und gezielteren Therapie von Erkrankungen. Mit diesem zentralen Aspekt der personalisierten Medizin befassten sich die vier Arbeitsgruppen der Sektion Molekulare Diagnostik aus ihren jeweiligen Blickwinkeln: Der Fokus der einführenden Sitzung der Arbeitsgruppe Genomics aus der Sichtweise der Pathologie und Laboratoriumsmedizin lag auf onkologischen Fragestellungen. In der darauf folgenden Sitzung widmet sich die Arbeitsgruppe Biobanken der Erhebung molekularer Signaturen aus archivierten Geweben und Körperflüssigkeiten. Die Arbeitsgruppe Bioinformatik ging der Frage nach, wie viele Biomarker man konkret für eine aussagekräftige Signatur benötigt. In der abschließenden Sitzung der Arbeitsgruppe Proteomics/Metabolomics wurden diese Erkenntnisse auf der Metabolitenebene am Beispiel endokrinologischer und maligner Erkrankungen in die Praxis übersetzt. Im Rahmen der Jahrestagung fand eine Übergabe des Vorsitzes von Michael Neumeier (Heidelberg-Mannheim), der die Sektion seit ihrer Gründung geleitet hatte, an Daniel Teupser (München) statt. An dieser Stelle möchten die Autoren Herrn Professor Neumeier ihren tiefempfundenen Dank für den Aufbau und die sehr erfolgreiche Leitung der Sektion in den zurückliegenden Jahren aussprechen. Ihm ist es gelungen, die Molekulare Diagnostik als Zukunftsgebiet weit über die Grenzen der Fachgesellschaft hinaus bekannt zu machen. Dies wird auch aus dem fachübergreifenden Rednerspektrum der diesjährigen Tagung evident

Telomerase activity and telomerase subunits gene expression patterns in neuroblastoma: A molecular and immunohistochemical study establishing prognostic tools for fresh-frozen and paraffin-embedded tissues

Purpose: We have recently demonstrated that telomerase activity (TA) is an independent prognostic factor in neuroblastomas. in the present study, the prognostic impact of TA and gene expression of the three major telomerase subunits is evaluated by molecular and immunohistochemical techniques in fresh-frozen and paraffin-embedded tissues. Patients and Methods: One hundred thirty-three neuroblastomas of all stages were analyzed for TA, The TA levels of 75 neuroblastoma cases were correlated with gene expression of telomerase subunits hTRT, human telomerase RNA (hTR), and telomerase protein 1 (TP1) by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), using an innovative approach on the LightCycler instrument (Roche Diagnostics, Mannheim, Germany). For selected cases, the applicability of RT-PCR and immunohistochemistry for hTRT expression analysis was investigated in paraffin-embedded tissues. TA and subunit expression patterns were correlated with traditional prognostic indicators and disease outcome. Results: TA was present in a total of 39 (29.3%) of 133 neuroblastomas and in 31 (29.8%) of 104 initial neuroblastomas without cytotoxic pretreatment, TA was significantly correlated with both event-free and overall survival (P <.0001), Furthermore, we found a significant correlation between expression levels of TA and hTRT (P <.0001) as well as hTR (P <.001), Multivariate analysis revealed only TA and tumor stage but not serum lactate dehydrogenase, MYCN amplification, or age at diagnosis as independent prognostic factors. Conclusion: The significant correlation with clinical outcome strongly recommends that analysis of TA be incorporated into the clinical investigation of each individual neuroblastoma at the time of diagnosis. Because the mere presence or absence of TA without further quantification is sufficient basis for predicting disease outcome, the telomeric repeat amplification protocol assay could be complemented with but not replaced by analysis of hTRT or hTR expression. J Clin Oncol 18:2582-2592, (C) 2000 by American Society of Clinical Oncology