Smallridge, Robert C.

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 remodeling 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 BRAFV600E versus RAS-dependent 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.

Anaplastic thyroid carcinoma (ATC) is almost universally fatal. Elevated keratin-8 (KRT8) protein expression is an established diagnostic cancer biomarker in several epithelial cancers (but not ATC). Several keratins, including KRT8, have been suggested to have a role in cell biology beyond that of structural cytoskeletal proteins. Here, we provide evidence that KRT8 plays a direct role in the growth of ATCs. Genomic and transcriptomic analysis of >5000 patients demonstrates that KRT8 mutation and copy number amplification are frequently evident in epithelial-derived cancers. Carcinomas arising from diverse tissues exhibit KRT8 mRNA and protein overexpression when compared to normal tissue levels. Similarly, in a panel of patient-derived ATC cell lines and patient tumors, KRT8 expression shows a similar pattern. sh-RNA-mediated KRT8 knockdown in these cell lines increases apoptosis, whereas forced overexpression of KRT8 confers resistance to apoptosis under peroxide-induced cell stress conditions. We further show that KRT8 protein binds to annexin A2, a protein known to mediate apoptosis as well as the redox pathway.

We previously reported an extremely rare case of follicular dendritic cell sarcoma (FDCS) presented as a thyroid mass. Given the rarity of this disease, there are no personalized and molecularly targeted treatment options due to the lack of knowledge in the genomic makeup of the tumor. A 44-year-old white woman was diagnosed with an extranodal FDCS in thyroid. The patient underwent a total thyroidectomy, central compartment dissection, parathyroid re-implantation, and adjuvant radiation therapy. Tumor DNA sequencing of 236 genes by FoundationOne panel found truncating mutations in PTEN and missense mutations in RET and TP53. However, patient-matched germline DNA was not sequenced which is critical for identification of true somatic mutations. Furthermore, the FoundationOne panel doesn't measure genomic rearrangements which have been shown to be abundant in sarcomas and are associated with sarcoma tumorigenesis and progression. In the current study, we carried out comprehensive genomic sequencing of the tumor, adjacent normal tissues, and patient-matched blood, in an effort to understand the genomic makeup of this rare extranodal FDCS and to identify potential therapeutic targets. Eighty-one somatic point mutations were identified in tumor but not in adjacent normal tissues or blood. A clonal truncating mutation in the CLTCL1 gene, which stabilizes the mitotic spindle, was likely a driver mutation of tumorigenesis and could explain the extensive copy number aberrations (CNAs) and genomic rearrangements in the tumor including a chr15/chr17 local chromothripsis resulted in 6 expressed fusion genes. The fusion gene HDGFRP3→SHC4 led to a 200-fold increase in the expression of oncogene SHC4 which is a potential target of the commercial drug Dasatinib. Missense mutations in ATM and splice-site mutation in VEGFR1 were also detected in addition to the TP53 missense mutation reported by FoundationOne.

6087 Background: NTRK1-3 fusions, identified in pediatric radiation-associated thyroid cancers, are low frequency oncogenic drivers that can trigger constitutive activation of TrK1-3 tyrosine kinase domains with responsively heightened downstream signaling through RAS-MAPK, PI3K-AKT, and/or PLCγ-PKC. NTRK point mutations are rare and of largely uninvestigated oncogenic potential, but some appear oncogenic. Methods: Advanced thyroid cancer (TC) patients (pts) requiring systemic therapy at Mayo Clinic sites underwent tumor genetic interrogation using Foundation One analyses with the goal of informing salvage therapeutic strategies. In this IRB-approved study, data from pts with TCs bearing NTRK point mutations were retrospectively analyzed, as this occurrence was unexpected but potentially of prognostic and/or therapeutic relevance. Results: Five of 55 pts (9%) with advanced TCs subject to Foundation One tumor interrogation had NTRK1-3 point mutations (allele frequency, AF, 15-49%); none had fusions. All 5 tumors had microsatellite stability; 4/5 had low tumor mutation burden (2-5 mutations/megabase). NTRK mutations in 3 anaplastic TC (ATC) pts (15% prevalence) were: NTRK3 I749M (AF 15%; overall survival, OS, 15 mos), NTRK1 R744H (AF not available; OS 9.5 mos), and NTRK1 R583H (AF 20.1%; OS > 4.9 mos); NTRK2 S167Y (AF 49%; OS > 51 mos) or NTRK1 R6W (AF 42%; OS > 60 mos) were found in one papillary (PTC) and one Hürthle cell (HCC) TC pt respectively (summary table below). Conclusions: NTRK point mutations, but not fusions, were unexpectedly identified in 9% of assessed advanced TCs - but remarkably in 15% of ATCs. Mutated NTRKs, along with oncogenic p53 and TERT, may contribute to TC progression, especially in ATC, consequent to downstream activation of MAPK and PI3K-AKT pathways. We posit that NTRK point mutations may have therapeutic implications for treatment of refractory disease. [Table: see text]

Background: Anaplastic thyroid carcinoma (ATC) accounts for only 3% of thyroid cancers, yet strikingly, it accounts for almost 40% of thyroid cancer deaths. Currently, no effective therapies exist. In an effort to identify ATC-specific therapeutic targets, we analyzed global gene expression data from multiple studies to identify ATC-specific dysregulated genes. Methods: The National Center for Biotechnology Information Gene Expression Omnibus database was searched for high-throughput gene expression microarray studies from human ATC tissue along with normal thyroid and/or papillary thyroid cancer (PTC) tissue. Gene expression levels in ATC were compared with normal thyroid or PTC using seven separate comparisons, and an ATC-specific gene set common in all seven comparisons was identified. We investigated these genes for their biological functions and pathways. Results: There were three studies meeting inclusion criteria, (including 32 ATC patients, 69 PTC, and 75 normal). There were 259 upregulated genes and 286 downregulated genes in ATC with at least two-fold change in all seven comparisons. Using a five-fold filter, 36 genes were upregulated in ATC, while 40 genes were downregulated. Of the 10 top globally upregulated genes in ATC, 4/10 ( MMP1 , ANLN , CEP55 , and TFPI2 ) are known to play a role in ATC progression; however, 6/10 genes ( TMEM158 , CXCL5 , E2F7 , DLGAP5 , MME , and ASPM ) had not been specifically implicated in ATC. Similarly, 3/10 ( SFTA3 , LMO3 , and C2orf40 ) of the most globally downregulated genes were novel in this context, while 7/10 genes ( SLC26A7 , TG , TSHR , DUOX2 , CDH1 , PDE8B , and FOXE1 ) have been previously identified in ATC. We experimentally validated a significant correlation for seven transcription factors ( KLF16 , SP3 , ETV6 , FOXC1 , SP1 , EGFR1 , and MAFK ) with the ATC-specific genes using microarray analysis of ATC cell lines. Ontology clustering of globally altered genes revealed that “mitotic cell cycle” is highly enriched in the globally upregulated gene set (44% of top upregulated genes, p -value <10 −30 ). Conclusions: By focusing on globally altered genes, we have identified a set of consistently altered biological processes and pathways in ATC. Our data are consistent with an important role for M-phase cell cycle genes in ATC, and may provide direction for future studies to identify novel therapeutic targets for this disease.