New insights into intranuclear inclusions in thyroid carcinoma: Association with autophagy and with BRAFV600E mutation


Autoři: Suzan Schwertheim aff001;  Sarah Theurer aff001;  Holger Jastrow aff002;  Thomas Herold aff001;  Saskia Ting aff001;  Daniela Westerwick aff001;  Stefanie Bertram aff001;  Christoph M. Schaefer aff001;  Julia Kälsch aff001;  Hideo A. Baba aff001;  Kurt W. Schmid aff001
Působiště autorů: Institute of Pathology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany aff001;  Institute of Anatomy and Electron Microscopy Unit of Imaging Center Essen, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany aff002;  Department of Gastroenterology and Hepatology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226199

Souhrn

Background

Intranuclear inclusions (NI) in normal and neoplastic tissues have been known for years, representing one of the diagnostic criteria for papillary thyroid carcinoma (PTC). BRAF activation is involved among others in autophagy. NI in hepatocellular carcinoma contain autophagy-associated proteins. Our aim was to clarify if NI in thyroid carcinoma (TC) have a biological function.

Methods

NI in 107 paraffin-embedded specimens of TC including all major subtypes were analyzed. We considered an inclusion as positive if it was delimited by a lamin AC (nuclear membrane marker) stained intact membrane and completely closed. Transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF) and 3D reconstruction were performed to investigate content and shape of NI; BRAFV600E mutation was analyzed by next generation sequencing.

Results

In 29% of the TCs at least one lamin AC positive intranuclear inclusion was detected; most frequently (76%) in PTCs. TEM analyses revealed degenerated organelles and heterolysosomes within such NI; 3D reconstruction of IF stained nuclei confirmed complete closure by the nuclear membrane without any contact to the cytoplasm. NI were positively stained for the autophagy-associated proteins LC3B, ubiquitin, cathepsin D, p62/sequestosome1 and cathepsin B in 14–29% of the cases. Double-IF revealed co-localization of LC3B & ubiquitin, p62 & ubiquitin and LC3B & p62 in the same NI. BRAFV600E mutation, exclusively detected in PTCs, was significantly associated with the number of NI/PTC (p = 0.042) and with immunoreactivity for autophagy-associated proteins in the NI (p≤0.035). BRAF-IHC revealed that some of these BRAF-positive thyrocytes contained mutant BRAF in their NI co-localized with autophagy-associated proteins.

Conclusions

NI are completely delimited by nuclear membrane in TC. The presence of autophagy-associated proteins within the NI together with degenerated organelles and lysosomal proteases suggests their involvement in autophagy and proteolysis. Whether and how BRAFV600E protein is degraded in NI needs further investigation.

Klíčová slova:

Cytoplasm – Cytoplasmic staining – Immunostaining – Lamins – Membrane staining – Nuclear membrane – Thyroid carcinomas – Cytoplasmic inclusions


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2019 Číslo 12