Comprehensive Genetic Characterization of Human Thyroid Cancer Cell Lines: A Validated Panel for Preclinical Studies

Abstract

Purpose: Thyroid cancer cell lines are valuable models but have been neglected in pancancer genomic studies. Moreover, their misidentification has been a significant problem. We aim to provide a validated dataset for thyroid cancer researchers. Experimental Design: We performed next-generation sequencing (NGS) and analyzed the transcriptome of 60 authenticated thyroid cell lines and compared our findings with the known genomic defects in human thyroid cancers. Results: Unsupervised transcriptomic analysis showed that 94% of thyroid cell lines clustered distinctly from other lineages. Thyroid cancer cell line mutations recapitulate those found in primary tumors (e.g., BRAF, RAS, or gene fusions). Mutations in the TERT promoter (83%) and TP53 (71%) were highly prevalent. There were frequent alterations in PTEN, PIK3CA, and of members of the SWI/SNF chromatin remodel-ing complex, mismatch repair, cell-cycle checkpoint, and histone methyl-and acetyltransferase functional groups. Copy number alterations (CNA) were more prevalent in cell lines derived from advanced versus differentiated cancers, as reported in primary tumors, although the precise CNAs were only partially recapitulated. Transcriptomic analysis showed that all cell lines were profoundly dedifferentiated, regardless of their derivation, making them good models for advanced disease. However, they maintained the BRAF V600E versus RASdependent consequences on MAPK transcriptional output, which correlated with differential sensitivity to MEK inhibitors. Paired primary tumor-cell line samples showed high concordance of mutations. Complete loss of p53 function in TP53 heterozygous tumors was the most prominent event selected during in vitro immortalization. Conclusions: This cell line resource will help inform future preclinical studies exploring tumor-specific dependencies.

Establishment and Characterization of Four Novel Thyroid Cancer Cell Lines and PDX Models Expressing the RET/PTC1 Rearrangement, BRAFV600E, or RASQ61R as Drivers

“…2B). These results are consistent with our Affymetrix gene expression data, showing expression of RET in the CUTC48 cells, as well as next-generation sequencing (25). We next performed Western blot analysis to evaluate RET/PTC1 protein expression in the CUTC48 cells, using the TPC1 cells as a positive control for RET/PTC1 expression, the HTh74 cells as a negative control, and the lung cancer cell lines Calu6, DMS-53, and H889, to monitor full length RET expression.…”

“…Interestingly, the CUTC60 cell line was resistant to BRAF (dabrafenib and vemurafenib IC 50 > 10 mmol/L) and MEK1/2 (selumetinib, PD-0325901, and trametinib IC 50 s > 9 mmol/L) inhibitors despite the presence of BRAF V600E mutation ( Supplementary Table S1). We have recently reported that the TCGA-derived BRAFV600E-RAS score (BRS) is a better predictor of MAPK pathway dependence than BRAF mutational status (6,25). Consistent with this, the CUTC5 cells have a lower BRS than the CUTC60 cells (CUTC5 BRS ¼ À0.341 vs. CUTC60 BRS ¼ 0.089), indicating the CUTC60 cells are less reliant on the MAPK pathway and, thus, resistant to MEK1/2 or BRAF inhibition.…”

“…The STR electropherograms were analyzed and alleles called using GeneMapper version 4.0 software (Thermo Fisher Scientific #4440915) and the results were confirmed by visual inspection. Matches to the resulting genotypes were searched for by comparing them individually to a collection of 6,678 STR genotypes consisting of in-house data and data from the consolidation of STR genotypes from DSMZ, ATCC, RIKEN, and JCRB tissue culture repositories (available at the DSMZ website https://www.dsmz.de/services/services-human-and-ani mal-cell-lines/online-str-analysis.html; downloaded on July 10, 2017), and the published STR data (17)(18)(19)(20)(21)(22)(23)(24)(25). Degree of matches were calculated by a modification of the Tanabe and colleagues formula (26): This formula was applied to allelic data from shared STR loci.…”

“…Mutational analysis was performed using the MSK-IMPACT targeted sequencing of 341 cancer genes (CUTC5, CUTC48), or a new panel covering 69 additional genes (410 genes total; CUTC60, CUTC61), as described previously (25,30).…”

Global computational alignment of tumor and cell line transcriptional profiles

“…Indeed, of the twelve skin samples in this cluster that were annotated by Tsoi et al, all three of the skin cell lines and seven of the nine skin tumors were annotated as being of an 'undifferentiated' subtype 41 . The majority (11/12) of the thyroid cell lines, which have been observed to be more dedifferentiated than thyroid tumors 34,45,46 , also belonged to this cluster. To further assess how distinct these cell line models are from their lineage-matched counterparts that co-clustered with tumors, we also looked at a set of lineagespecific transcription factors.…”