Jeffrey A. Knauf

Deoxyribonucleic Acid Profiling Analysis of 40 Human Thyroid Cancer Cell Lines Reveals Cross-Contamination Resulting in Cell Line Redundancy and Misidentification

CONTEXT Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated. OBJECTIVE We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis. RESULTS Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines. CONCLUSIONS Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.

Mutational profile of advanced primary and metastatic radioactive iodine-refractory thyroid cancers reveals distinct pathogenetic roles for BRAF, PIK3CA, and AKT1.

Patients with poorly differentiated thyroid cancers (PDTC), anaplastic thyroid cancers (ATC), and radioactive iodine-refractory (RAIR) differentiated thyroid cancers have a high mortality, particularly if positive on [(18)F]fluorodeoxyglucose (FDG)-positron emission tomography (PET). To obtain comprehensive genetic information on advanced thyroid cancers, we designed an assay panel for mass spectrometry genotyping encompassing the most significant oncogenes in this disease: 111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1, and other related genes were surveyed in 31 cell lines, 52 primary tumors (34 PDTC and 18 ATC), and 55 RAIR, FDG-PET-positive recurrences and metastases (nodal and distant) from 42 patients. RAS mutations were more prevalent than BRAF (44 versus 12%; P = 0.002) in primary PDTC, whereas BRAF was more common than RAS (39 versus 13%; P = 0.04) in PET-positive metastatic PDTC. BRAF mutations were highly prevalent in ATC (44%) and in metastatic tumors from RAIR PTC patients (95%). Among patients with multiple metastases, 9 of 10 showed between-sample concordance for BRAF or RAS mutations. By contrast, 5 of 6 patients were discordant for mutations of PIK3CA or AKT1. AKT1_G49A was found in 9 specimens, exclusively in metastases. This is the first documentation of AKT1 mutation in thyroid cancer. Thus, RAIR, FDG-PET-positive metastases are enriched for BRAF mutations. If BRAF is mutated in the primary, it is likely that the metastases will harbor the defect. By contrast, absence of PIK3CA/AKT1 mutations in one specimen may not reflect the status at other sites because these mutations arise during progression, an important consideration for therapies directed at phosphoinositide 3-kinase effectors.

Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer

Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1-8 of the AKAP9 gene and exons 9-18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.

Targeted Expression of BRAFV600E in Thyroid Cells of Transgenic Mice Results in Papillary Thyroid Cancers that Undergo Dedifferentiation

The BRAFT1799A mutation is the most common genetic alteration in papillary thyroid carcinomas (PTC). It is also found in a subset of papillary microcarcinomas, consistent with a role in tumor initiation. PTCs with BRAFT1799A are often invasive and present at a more advanced stage. BRAFT1799A is found with high prevalence in tall-cell variant PTCs and in poorly differentiated and undifferentiated carcinomas arising from PTCs. To explore the role of BRAFV600E in thyroid cancer pathogenesis, we targeted its expression to thyroid cells of transgenic FVB/N mice with a bovine thyroglobulin promoter. Two Tg-BRAFV600E lines (Tg-BRAF2 and Tg-BRAF3) were propagated for detailed analysis. Tg-BRAF2 and Tg-BRAF3 mice had increased thyroid-stimulating hormone levels (>7- and approximately 2-fold, respectively). This likely resulted from decreased expression of thyroid peroxidase, sodium iodine symporter, and thyroglobulin. All lines seemed to successfully compensate for thyroid dysfunction, as serum thyroxine/triiodothyronine and somatic growth were normal. Thyroid glands of transgenic mice were markedly enlarged by 5 weeks of age. In Tg-BRAF2 mice, PTCs were present at 12 and 22 weeks in 14 of 15 and 13 of 14 animals, respectively, with 83% exhibiting tall-cell features, 83% areas of invasion, and 48% foci of poorly differentiated carcinoma. Tg-BRAF3 mice also developed PTCs, albeit with lower prevalence (3 of 12 and 4 of 9 at 12 and 22 weeks, respectively). Tg-BRAF2 mice had a 30% decrease in survival at 5 months. In summary, thyroid-specific expression of BRAFV600E induces goiter and invasive PTC, which transitions to poorly differentiated carcinomas. This closely recapitulates the phenotype of BRAF-positive PTCs in humans and supports a key role for this oncogene in its pathogenesis.

Molecular genotyping of papillary thyroid carcinoma follicular variant according to its histological subtypes (encapsulated vs infiltrative) reveals distinct BRAF and RAS mutation patterns

The follicular variant of papillary thyroid carcinoma usually presents as an encapsulated tumor and less commonly as a partially/non-encapsulated infiltrative neoplasm. The encapsulated form rarely metastasizes to lymph node, whereas infiltrative tumor often harbors nodal metastases. The molecular profile of the follicular variant was shown to be close to the follicular adenoma/carcinoma group of tumors with a high RAS and very low BRAF mutation rates. A comprehensive survey of oncogenic mutations in the follicular variant of papillary thyroid carcinoma according to its encapsulated and infiltrative forms has not been performed. Paraffin tissue from 28 patients with encapsulated and 19 with infiltrative follicular variant were subjected to mass spectrometry genotyping encompassing the most significant oncogenes in thyroid carcinomas: 111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1 and other related genes. There was no difference in age, gender, tumor size and angioinvasion between encapsulated or infiltrative tumors. Infiltrative carcinomas had a much higher frequency of extrathyroid extension, positive margins and nodal metastases than encapsulated tumors (P<0.05). The BRAF 1799T>A mutation was found in 5 of 19 (26%) of the infiltrative tumor and in none of the encapsulated carcinomas (P=0.007). In contrast, RAS mutations were observed in 10 of 28 (36%) of the encapsulated group (5 NRAS_Q61R, 3 HRAS_Q61, 1 HRAS_G13C and 1 KRAS_Q61R) and in only 2 of 19 (10%) of infiltrative tumors (P=0.09). One encapsulated carcinoma showed a PAX8/PPARγ rearrangement, whereas two infiltrative tumors harbored RET/PTC fusions. Encapsulated follicular variant of papillary thyroid carcinomas have a molecular profile very close to follicular adenomas/carcinomas (high rate of RAS and absence of BRAF mutations). Infiltrative follicular variant has an opposite molecular profile closer to classical papillary thyroid carcinoma than to follicular adenoma/carcinoma (BRAF>RAS mutations). The molecular profile of encapsulated and infiltrative follicular variant parallels their biological behavior (ie, metastatic nodal and invasive patterns).

Relief of Feedback Inhibition of HER3 Transcription by RAF and MEK Inhibitors Attenuates Their Antitumor Effects in BRAF-Mutant Thyroid Carcinomas

The RAF inhibitor vemurafenib (PLX4032) increases survival in patients with BRAF-mutant metastatic melanoma, but has limited efficacy in patients with colorectal cancers. Thyroid cancer cells are also comparatively refractory to RAF inhibitors. In contrast to melanomas, inhibition of mitogen-activated protein kinase (MAPK) signaling by PLX4032 is transient in thyroid and colorectal cancer cells. The rebound in extracellular signal-regulated kinase (ERK) in thyroid cells is accompanied by increased HER3 signaling caused by induction of ERBB3 (HER3) transcription through decreased promoter occupancy by the transcriptional repressors C-terminal binding protein 1 and 2 and by autocrine secretion of neuregulin-1 (NRG1). The HER kinase inhibitor lapatinib prevents MAPK rebound and sensitizes BRAF-mutant thyroid cancer cells to RAF or MAP-ERK kinase inhibitors. This provides a rationale for combining ERK pathway antagonists with inhibitors of feedback-reactivated HER signaling in this disease. The determinants of primary resistance to MAPK inhibitors vary between cancer types, due to preferential upregulation of specific receptor tyrosine kinases, and the abundance of their respective ligands.

Increased density of tumor associated macrophages is associated with decreased survival in advanced thyroid cancer

Thyroid cancers are infiltrated with tumor-associated macrophages (TAMs), yet their role in cancer progression is not known. The objectives of this study were to characterize the density of TAMs in well-differentiated (WDTC), poorly differentiated (PDTC), and anaplastic thyroid cancers (ATC) and to correlate TAM density with clinicopathologic parameters. Immunohistochemistry was performed on tissue microarray sections from WDTC (n=33), PDTC (n=37), and ATC (n=20) using macrophage-specific markers. Electronic medical records were used to gather clinical and pathologic data. Follow-up information of PDTC patients was available for 0-12 years. In total, 9 out of 33 WDTC (27%), 20 out of 37 PDTC (54%), and 19 out of 20 ATC (95%) had an increased density of CD68(+) TAMs (> or = 10 per 0.28 mm(2); WDTC versus PDTC, P=0.03; WDTC versus ATC, P<0.0001; PDTC versus ATC, P<0.002). Increased TAMs in PDTC was associated with capsular invasion (P=0.034), extrathyroidal extension (P=0.009), and decreased cancer-related survival (P=0.009) compared with PDTC with a low density of TAMs. In conclusion, the density of TAMs is increased in advanced thyroid cancers. The presence of a high density of TAMs in PDTC correlates with invasion and decreased cancer-related survival. These results suggest that TAMs may facilitate tumor progression. As novel therapies directed against thyroid tumor cell-specific targets are being tested, the potential role of TAMs as potential modulators of the thyroid cancer behavior will need to be considered.

Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers

BACKGROUND Poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) are rare and frequently lethal tumors that so far have not been subjected to comprehensive genetic characterization. METHODS We performed next-generation sequencing of 341 cancer genes from 117 patient-derived PDTCs and ATCs and analyzed the transcriptome of a representative subset of 37 tumors. Results were analyzed in the context of The Cancer Genome Atlas study (TCGA study) of papillary thyroid cancers (PTC). RESULTS Compared to PDTCs, ATCs had a greater mutation burden, including a higher frequency of mutations in TP53, TERT promoter, PI3K/AKT/mTOR pathway effectors, SWI/SNF subunits, and histone methyltransferases. BRAF and RAS were the predominant drivers and dictated distinct tropism for nodal versus distant metastases in PDTC. RAS and BRAF sharply distinguished between PDTCs defined by the Turin (PDTC-Turin) versus MSKCC (PDTC-MSK) criteria, respectively. Mutations of EIF1AX, a component of the translational preinitiation complex, were markedly enriched in PDTCs and ATCs and had a striking pattern of co-occurrence with RAS mutations. While TERT promoter mutations were rare and subclonal in PTCs, they were clonal and highly prevalent in advanced cancers. Application of the TCGA-derived BRAF-RAS score (a measure of MAPK transcriptional output) revealed a preserved relationship with BRAF/RAS mutation in PDTCs, whereas ATCs were BRAF-like irrespective of driver mutation. CONCLUSIONS These data support a model of tumorigenesis whereby PDTCs and ATCs arise from well-differentiated tumors through the accumulation of key additional genetic abnormalities, many of which have prognostic and possible therapeutic relevance. The widespread genomic disruptions in ATC compared with PDTC underscore their greater virulence and higher mortality. FUNDING This work was supported in part by NIH grants CA50706, CA72597, P50-CA72012, P30-CA008748, and 5T32-CA160001; the Lefkovsky Family Foundation; the Society of Memorial Sloan Kettering; the Byrne fund; and Cycle for Survival.

Conditional BRAFV600E expression induces DNA synthesis, apoptosis, dedifferentiation, and chromosomal instability in thyroid PCCL3 cells.

The activating mutation BRAF(T1796A) is the most prevalent genetic alteration in papillary thyroid carcinomas (PTC). It is associated with advanced PTCs, suggesting that this oncoprotein confers thyroid cancers with more aggressive properties. BRAF(T1796A) is also observed in thyroid micropapillary carcinomas and may thus be an early event in tumor development. To explore its biological consequences, we established doxycycline-inducible BRAF(V600E)-expressing clonal lines derived from well-differentiated rat thyroid PCCL3 cells. Expression of BRAF(V600E) did not induce growth in the absence of thyrotropin despite increasing DNA synthesis, which is likely explained because of a concomitant increase in apoptosis. Thyrotropin-dependent cell growth and DNA synthesis were reduced by BRAF(V600E) because of decreased thyrotropin responsiveness associated with inhibition of thyrotropin receptor gene expression. These results are similar to those obtained following conditional expression of RET/PTC. However, in contrast to RET/PTC, BRAF activation did not impair key activation steps distal to the thyrotropin receptor, such as forskolin-induced adenylyl cyclase activity or cyclic AMP-induced DNA synthesis. We reported previously that acute RET/PTC expression in PCCL3 cells did not induce genomic instability. By contrast, induction of BRAF(V600E) expression increased the frequency of micronuclei by both clastogenic and aneugenic events. These data indicate that BRAF(V600E) expression confers thyroid cells with little growth advantage because of concomitant activation of DNA synthesis and apoptosis. However, in contrast to RET/PTC, BRAF(V600E) may facilitate the acquisition of secondary genetic events through induction of genomic instability, which may account for its aggressive properties.

Small-molecule MAPK inhibitors restore radioiodine incorporation in mouse thyroid cancers with conditional BRAF activation

Advanced human thyroid cancers, particularly those that are refractory to treatment with radioiodine (RAI), have a high prevalence of BRAF (v-raf murine sarcoma viral oncogene homolog B1) mutations. However, the degree to which these cancers are dependent on BRAF expression is still unclear. To address this question, we generated mice expressing one of the most commonly detected BRAF mutations in human papillary thyroid carcinomas (BRAF(V600E)) in thyroid follicular cells in a doxycycline-inducible (dox-inducible) manner. Upon dox induction of BRAF(V600E), the mice developed highly penetrant and poorly differentiated thyroid tumors. Discontinuation of dox extinguished BRAF(V600E) expression and reestablished thyroid follicular architecture and normal thyroid histology. Switching on BRAF(V600E) rapidly induced hypothyroidism and virtually abolished thyroid-specific gene expression and RAI incorporation, all of which were restored to near basal levels upon discontinuation of dox. Treatment of mice with these cancers with small molecule inhibitors of either MEK or mutant BRAF reduced their proliferative index and partially restored thyroid-specific gene expression. Strikingly, treatment with the MAPK pathway inhibitors rendered the tumor cells susceptible to a therapeutic dose of RAI. Our data show that thyroid tumors carrying BRAF(V600E) mutations are exquisitely dependent on the oncoprotein for viability and that genetic or pharmacological inhibition of its expression or activity is associated with tumor regression and restoration of RAI uptake in vivo in mice. These findings have potentially significant clinical ramifications.