International Phase IV Field Study for the Reliability and Validity of the European Organisation for Research and Treatment of Cancer Thyroid Cancer Module EORTC QLQ-THY34.

Purpose: The aim of this study was to validate the new European Organisation for Research and Treatment of Cancer Quality of Life Thyroid Cancer Module (EORTC QLQ-THY34). Methods: We enrolled 437 thyroid cancer patients from 17 countries. One group (n = 303), undergoing treatment or best supportive care, completed the questionnaires at three time points (before therapy [t1], 6 weeks later [t2], and 6 months after t2 [t3]). A second group (survivors ≥2 years after diagnosis, n = 134) completed it at a random baseline time point and a second time 1 week later. We determined internal consistency (using Cronbach's alpha), the scale structure (with confirmatory factor analysis), and discriminant validity (using known-group comparisons). Group 1 data were used to assess responsiveness and group 2 data to determine test-retest reliability using intra-class correlations (ICC). Results: All 34 items fulfilled the criteria to be kept in the questionnaire. Cronbach's alpha was >0.70 in 8 of the 9 multi-item scales. All standardized factor loadings exceeded 0.40, confirming the proposed scale structure. The ICC was >0.70 in all scales expressing good test-retest reliability. Differences in scale scores between patients with different histology were >5 points in all scales. In all but one of the pre-specified scales (Dry Mouth), changes over time were ≥|4| points between at least two time points. Conclusion: The EORTC QLQ-THY34 with its 9 multi-item and 8 single-item scales is a reliable and valid tool to measure quality of life in thyroid cancer patients and can be used in future trials and studies.

NGS based identification of mutational hotspots for targeted therapy in anaplastic thyroid carcinoma.

CONTEXT: Anaplastic thyroid carcinoma (ATC) represents one of the most aggressive carcinomas with no consistent survival benefit when treated with conventional radiochemotherapy. Approaches targeting "oncogene addiction" of ATC are increasingly explored and first promising results have been reported in single case studies. OBJECTIVE: To determine the prevalence of mutations in known thyroid oncogenes and signalling pathways amendable to targeted therapy in a large cohort of ATC. RESULTS: In 118 ATC (57 male/ 61 female) a total of 165 mutations were found. Genes involved in the MAPK/ERK and PI3K pathway (BRAF 11.0%, HRAS 4.2%, KRAS 7.6%, NRAS 7.6%, PI3KCA 11.8%) were altered in 33%. Targetable receptor tyrosine kinases were mutated in 11%. The most frequently altered genes were TERT in 86/118 (73%) and p53 in 65/118 (55%) cases. No mutations were found analysing ALK, KIT, MET and mTOR. MATERIALS AND METHODS: Next generation sequencing (NGS) was performed in FFPE samples from 118 ATC using MiSeq (Illumina) and CLC Cancer Research Workbench (CLCbio; Qiagen) for mutation analysis in: ALK, BRAF, CDKN2A, EGFR, ERBB2, HRAS, KIT, KRAS, MET, mTOR, NRAS, PDGFRA, PI3KCA, p53, RB1, RET and TSC2. Sanger sequencing was used to detect TERT promotor mutations. CONCLUSIONS: To our knowledge this is the largest study analysing mutations for targeted therapy of ATC. We found that 33% of ATC harbour mutations in pathways amendable to targeted therapy. Molecular screening in ATC is suggested for targeted therapies since current conventional treatment for ATC proved mainly futile.

The long N-terminus of the human monocarboxylate transporter 8 is a target of ubiquitin-dependent proteasomal degradation which regulates protein expression and oligomerization capacity.

Monocarboxylate transporter 8 (MCT8) equilibrates thyroid hormones between the extra- and the intracellular sides. MCT8 exists either with a short or a long N-terminus, but potential functional differences between both variants are yet not known. We, therefore, generated MCT8 constructs which are different in N-terminal length: MCT8(1-613), MCT8(25-613), MCT8(49-613) and MCT8(75-613). The M75G substitution prevents translation of MCT8(75-613) and ensures expression of full-length MCT8 protein. The K56G substitution was made to prevent ubiquitinylation. Cell-surface expression, localization and proteasomal degradation were investigated using C-terminally GFP-tagged MCT8 constructs (HEK293 and MDCK1 cells) and oligomerization capacity was determined using N-terminally HA- and C-terminally FLAG-tagged MCT8 constructs (COS7 cells). MCT8(1-613)-GFP showed a lower protein expression than the shorter MCT8(75-613)-GFP protein. The proteasome inhibitor lactacystin increased MCT8(1-613)-GFP protein amount, suggesting proteasomal degradation of MCT8 with the long N-terminus. Ubiquitin conjugation of MCT8(1-613)-GFP was found by immuno-precipitation. A diminished ubiquitin conjugation caused by K56G substitution resulted in increased MCT8(1-613)-GFP protein expression. Sandwich ELISA was performed to investigate if the bands at higher molecular weight observed in Western blot analysis are due to MCT8 oligomerization, which was indeed shown. Our data imply a role of the long N-terminus of MCT8 as target of ubiquitin-dependent proteasomal degradation affecting MCT8 amount and subsequently oligomerization capacity.

The impact of CLAUDIN-1 on follicular thyroid carcinoma aggressiveness.

CLAUDIN-1 belongs to the family of transmembrane tight junction proteins tightening the paracellular cleft of epithelial cells. In human malignancies, CLAUDIN-1 is often dysregulated and located in subcellular compartments, particularly in the nucleus where it may influence cellular behaviour. Here, we studied CLAUDIN-1 in relation to the biological characteristics of follicular thyroid carcinoma (FTC). CLAUDIN-1 immuno-staining showed loss of membrane expression and increased nuclear CLAUDIN-1 localization in FTC metastases. CLAUDIN-1 function was further investigated in two different follicular thyroid carcinoma cell lines: FTC-133 isolated from a regional lymph node metastasis and FTC-238 derived from a lung metastasis. In both cell lines CLAUDIN-1 expression was demonstrated in the cell nuclei with a significantly higher protein expression in FTC-238 compared to FTC-133 cells. Interestingly, in vitro scratch assay revealed enriched nuclear CLAUDIN-1 expression near the scratch. Furthermore, the increase of the pathogenic character of FTC-133 cells by RASV12 transfection was associated with elevated CLAUDIN-1 expression and enhanced cell migration, invasion and proliferation. Likewise over-expression of nuclear CLAUDIN-1 in FTC-133 cells resulted in increased cell migration and invasion. Conversely, CLAUDIN-1 downregulation in FTC-238 cells by siRNA resulted in decreased cell migration and invasion and was accompanied by reduced phosphoPKC expression. Moreover, activation and inhibition of PKC resulted in CLAUDIN-1 up- and downregulation in FTC cells respectively. These data suggest an impact of CLAUDIN-1 on follicular thyroid carcinoma aggressiveness, which could potentially be influenced by PKC activity.