1. Activation of themTORPathway in Primary Medullary Thyroid Carcinoma and Lymph Node Metastases

 

ÀúÀÚ: Anna Tamburrino, Alfredo A. Molinolo, Paolo Salerno, Rebecca D. Chernock, Mark Raffeld, Liqiang Xi, J. Silvio Gutkind, Jeffrey F. Moley, Samuel A. Wells, Jr., and Massimo Santoro

Àú³Î: Clin Cancer Res 2012;18:3532-3540

»ç¿ëÇÑ Á¦Ç°: Dynabeads Streptavidin MyOne C1 (Cat# 65001, 65002)

 

Abstract

 

Purpose: Understanding the molecular pathogenesis of medullary thyroid carcinoma (MTC) is prerequisite to the design of targeted therapies for patients with advanced disease.

Experimental Design: We studied by immunohistochemistry the phosphorylation status of proteins of the RAS/MEK/ERK and PI3K/AKT/mTOR pathways in 53 MTC tissues (18 hereditary, 35 sporadic), including 51 primary MTCs and 2 cases with only lymph node metastases (LNM). We also studied 21 autologous LNMs, matched to 21 primary MTCs. Staining was graded on a 0 to 4 scale (S score) based on the percentage of positive cells.Wealso studied the functional relevance of themTORpathway by measuring cell viability, motility, and tumorigenicity upon mTOR chemical blockade.

Results: Phosphorylation of ribosomal protein S6 (pS6), a downstream target of mTOR, was evident (S   1) in 49 (96%) of 51 primary MTC samples. This was associated with activation of AKT (phospho-Ser473, S > 1) in 79% of cases studied. Activation of pS6 was also observed (S   1) in 7 (70%) of 10 hereditary C-cell hyperplasia specimens, possibly representing an early stage of C-cell transformation. It is noteworthy that 22 (96%) of 23LNMshad a high pS6 positivity (S 3), which was increased compared with autologous matched primary MTCs (P ¼ 0.024). Chemical mTOR blockade blunted viability (P < 0.01), motility (P < 0.01), and tumorigenicity (P < 0.01) of human MTC cells.

Conclusion: The AKT/mTOR pathway is activated in MTC, particularly, in LNMs. This pathway sustains malignant features of MTC cell models. These findings suggest that targeting mTOR might be efficacious in patients with advanced MTC.

 

 

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RET and RAS genotyping

Cores of 0.6 mm in diameter were sampled from FFPE blocks. Fibrotic or amyloid-rich samples with less than 50% of tumor material were excluded from the analysis. DNA extraction was carried out from deparaffinized FFPE specimens using the DNeasy Blood and Tissue Kit (Qiagen). Targeted analysis of RET mutations in exons 10, 11, and 13 to 16 was done by pyrosequencing on a PyroMark Q24 instrument (Qiagen). Primers for the RET gene pyrosequencing reactions were designed in the Laboratory of Pathology, Center for Cancer Research, NCI, NIH and are available upon request. Germline RET mutations were present in all of the 18 familial MTC samples and in one CCH sample. The presence of RET mutations in the remaining 7 CCH only samples was documented previously at the Washington University School of Medicine (data not shown). Targeted analysis of RAS mutation was conducted in 28 samples, including 11 hereditary and 17 sporadic patients with MTC (7 RET mutant cases and 10 RET wild-type cases). RAS mutations were searched for the 3 RAS genes (H-, N-, and KRAS) by exome sequencing. Briefly, 3 mg of genomic DNA from each sample were sheared in 150 to 250 bp fragments using the CovarisTM S220 sonicator (Covaris Inc.). The ends of the DNA fragments were repaired using T4 and Klenow DNA polymerases (NEB) and an A-tail was added by using Klenow enzyme (NEB). Nucleotide adapters of about 100 bp (NEXTflex—BioScientific) specific for each sample were added at the ends of DNA fragments in areaction buffer containing T4DNAligase (Enzymatics Inc.). DNA fragments ranging from 250 to 400 bp were purified using the Caliper LabChip XT fractionation system (Caliper) according to manufacturer¡¯s instructions. The DNA fragments were amplified by PCR using NEXTflex primers according to manufacturer¡¯s instructions. PCR products were purified by using magnetic beads (Agencourt AMPure XP; Beckman) according to manufacturer¡¯s instructions. Finally, the quality of the DNA libraries was assayed by using the 2100 Bioanalyzer Agilent. Equal amounts of each DNA library were mixed in a hybridization buffer containing biotinylated RNAs complementary to the exons of RAS genes (SureSelect—target enrichment system, Agilent) according to manufacturer¡¯s instructions, and the hybridization reaction was run for 48 hours. Streptavidin magnetic beads (Dynabeads Myone streptavidin C1; Invitrogen) were used for DNA/RNA capturing. The captured DNA was amplified by PCR using NEXTflex primers and quantified by real-time PCR. Finally, DNA fragments were sequenced in an Illumina sequencer (Illumina GA II) according to manufacture¡¯s protocol. The results of the sequences were analyzed with the IGV 2.0 software.

 

 

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