Association of BRAFV600E Mutation and MicroRNA Expression with Central Lymph Node Metastases in Papillary Thyroid Cancer: A Prospective Study from Four Endocrine Surgery Centers

Background: Studies have demonstrated an association of the BRAFV600E mutation and microRNA (miR) expression with aggressive clinicopathologic features in papillary thyroid cancer (PTC). Analysis of BRAFV600E mutations with miR expression data may improve perioperative decision making for patients with PTC, specifically in identifying patients harboring central lymph node metastases (CLNM). Methods: Between January 2012 and June 2013, 237 consecutive patients underwent total thyroidectomy and prophylactic central lymph node dissection (CLND) at four endocrine surgery centers. All tumors were tested for the presence of the BRAFV600E mutation and miR-21, miR-146b-3p, miR-146b-5p, miR-204, miR-221, miR-222, and miR-375 expression. Bivariate and multivariable analyses were performed to examine associations between molecular markers and aggressive clinicopathologic features of PTC. Results: Multivariable logistic regression analysis of all clinicopathologic features found miR-146b-3p and miR-146b-5p to be independent predictors of CLNM, while the presence of BRAFV600E almost reached significance. Multivariable logistic regression analysis limited to only predictors available preoperatively (molecular markers, age, sex, and tumor size) found miR-146b-3p, miR-146b-5p, miR-222, and BRAFV600E mutation to predict CLNM independently. While BRAFV600E was found to be associated with CLNM (48% mutated in node-positive cases vs. 28% mutated in node-negative cases), its positive and negative predictive values (48% and 72%, respectively) limit its clinical utility as a stand-alone marker. In the subgroup analysis focusing on only classical variant of PTC cases (CVPTC), undergoing prophylactic lymph node dissection, multivariable logistic regression analysis found only miR-146b-5p and miR-222 to be independent predictors of CLNM, while BRAFV600E was not significantly associated with CLNM. Conclusion: In the patients undergoing prophylactic CLNDs, miR-146b-3p, miR-146b-5p, and miR-222 were found to be predictive of CLNM preoperatively. However, there was significant overlap in expression of these miRs in the two outcome groups. The BRAFV600E mutation, while being a marker of CLNM when considering only preoperative variables among all histological subtypes, is likely not a useful stand-alone marker clinically because the difference between node-positive and node-negative cases was small. Furthermore, it lost significance when examining only CVPTC. Overall, our results speak to the concept and interpretation of statistical significance versus actual applicability of molecular markers, raising questions about their clinical usefulness as individual prognostic markers.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140269/1/thy.2015.0378.pd

Characteristics of contralateral carcinomas in patients with differentiated thyroid cancer larger than 1 cm

Purpose: Traditionally, total thyroidectomy has been advocated for patients with tumors larger than 1 cm. However, according to the ATA and NCCN guidelines (2015, USA), patients with tumors up to 4 cm are now eligible for lobectomy. A rationale for adhering to total thyroidectomy might be the presence of contralateral carcinomas. The purpose of this study was to describe the characteristics of contralateral carcinomas in patients with differentiated thyroid cancer (DTC) larger than 1 cm. Methods: A retrospective study was performed including patients from 17 centers in 5 countries. Adults diagnosed with DTC stage T1b-T3 N0-1a M0 who all underwent a total thyroidectomy were included. The primary endpoint was the presence of a contralateral carcinoma. Results: A total of 1

Characteristics of contralateral carcinomas in patients with differentiated thyroid cancer larger than 1 cm

textabstractPurpose: Traditionally, total thyroidectomy has been advocated for patients with tumors larger than 1 cm. However, according to the ATA and NCCN guidelines (2015, USA), patients with tumors up to 4 cm are now eligible for lobectomy. A rationale for adhering to total thyroidectomy might be the presence of contralateral carcinomas. The purpose of this study was to describe the characteristics of contralateral carcinomas in patients with differentiated thyroid cancer (DTC) larger than 1 cm. Methods: A retrospective study was performed including patients from 17 centers in 5 countries. Adults diagnosed with DTC stage T1b-T3 N0-1a M0 who all underwent a total thyroidectomy were included. The primary endpoint was the presence of a contralateral carcinoma. Results: A total of 1313 patients were included, of whom 426 (32 %) had a contralateral carcinoma. The contralateral carcinomas consisted of 288 (67 %) papillary thyroid carcinomas (PTC), 124 (30 %) follicular variant of a papillary thyroid carcinoma (FvPTC), 5 (1 %) follicular thyroid carcinomas (FTC), and 3 (1 %) Hürthle cell carcinomas (HTC). Ipsilateral multifocality was strongly associated with the presence of contralateral carcinomas (OR 2.62). Of all contralateral carcinomas, 82 % were ≤10 mm and of those 99 % were PTC or FvPTC. Even if the primary tumor was a FTC or HTC, the contralateral carcinoma was (Fv)PTC in 92 % of cases. Conclusions: This international multicenter study performed on patients with DTC larger than 1 cm shows that contralateral carcinomas occur in one third of patients and, independently of primary tumor subtype, predominantly consist of microPTC

Silencing Stmn1 Expression Decreases Aggressive Phenotype In Adrenocortical Carcinoma

Background: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a poor prognosis and few therapeutic options. Stathmin1 (STMN1) is a cytosolic protein involved in microtubule dynamics, which has been implicated in carcinogenesis and more aggressive behavior in multiple epithelial malignancies. Here we aimed to evaluate the expression of STMN1 in ACC and to elucidate how this may contribute to its malignant phenotype. Methods: STMN1 was identified as a highly differentially expressed gene in human samples of adrenocortical carcinoma submitted for high throughput RNA-sequencing compared to benign adrenal tumors. Expression was confirmed by qRT-PCR, Western blot, and immunohistochemical (IHC) staining of a tissue microarray (TMA) from two independent cohorts. The biologic relevance of this target was investigated in the NCI-H295R ACC cell line by lentivirus-mediated silencing of STMN1. In vitro phenotypic changes were assessed for cell viability, cell-cycle alterations, invasion, migration, and anchorage-independent growth. Results: Differential gene expression demonstrated over an eight-fold increase in STMN1 mRNA in malignant compared to benign adrenal tissue (p<2.4E-07). IHC showed significantly higher expression of STMN1 protein in ACC compared to normal and benign tissues (p<0.05, p<0.01, respectively). We achieved more than 70% reduction of STMN1 expression in NCI-H295R by lentivirus-mediated short hairpin RNA (shRNA) delivery. STMN1 knockdown resulted in decreased cell viability (p<0.01), cell-cycle arrest at G0/G1 (p<0.01), and increased apoptosis (p<0.01) in stressed, serum-starved conditions compared to scramble shRNA controls. STMN1 knockdown also decreased migration (p<0.05), invasion (p<0.05), and anchorage-independent growth (p<0.01) compared to controls. Conclusion: STMN1 is overexpressed in human ACC samples and knockdown of this target in vitro resulted in a less aggressive phenotype of ACC, particularly under serum-starved conditions. Further study is needed to investigate the feasibility of interfering with STMN1 as a potential therapeutic target

Ntr1 activates the Prp43 helicase to trigger release of lariat-intron from the spliceosome

DEAD/H-box NTPases remodel the spliceosome at multiple steps during the pre-mRNA splicing cycle. The RNA-dependent NTPase Prp43 catalyzes dissociation of excised lariat-intron from the spliceosome, but it is unclear how Prp43 couples the energy of ATP hydrolysis to intron release. Here, we report that activation of Prp43’s inherently feeble helicase activity by the splicing factor Ntr1 is required for lariat-intron release. Lethal Prp43 mutants T384A and T384V, which are active for ATP hydrolysis and fail to dissociate lariat-intron from spliceosomes, are refractory to stimulation of RNA unwinding by Ntr1. An N-terminal 120-amino-acid segment of Ntr1 suffices for binding to Prp43 and for stimulating its helicase activity. We identify missense mutations in Prp43 and Ntr1 that disrupt protein–protein interaction and impair Ntr1 enhancement of Prp43 RNA unwinding. Our results demonstrate for the first time that regulating the motor activity of a DEAH-box protein by an accessory factor is critical for mRNA splicing

Characterization of the Schizosaccharomyces pombe Spt5-Spt4 complex

The Spt5-Spt4 complex regulates early transcription elongation by RNA polymerase II and has an imputed role in pre-mRNA processing via its physical association with mRNA capping enzymes. Here we characterize the Schizosaccharomyces pombe core Spt5-Spt4 complex as a heterodimer and map a trypsin-resistant Spt4-binding domain within the Spt5 subunit. A genetic analysis of Spt4 in S. pombe revealed it to be inessential for growth at 25°C–30°C but critical at 37°C. These results echo the conditional spt4Δ growth phenotype in budding yeast, where we find that Saccharomyces cerevisiae and S. pombe Spt4 are functionally interchangeable. Complementation of S. cerevisiae spt4Δ and a two-hybrid assay for Spt4-Spt5 interaction provided a readout of the effects of 33 missense and truncation mutations on S. pombe Spt4 function in vivo, which were interpreted in light of the recent crystal structure of S. cerevisiae Spt4 fused to a fragment of Spt5. Our results highlight the importance of the Spt4 Zn2+-binding residues—Cys12, Cys15, Cys29, and Asp32—and of Ser57, a conserved constituent of the Spt4-Spt5 interface. The 990-amino acid S. pombe Spt5 protein has an exceptionally regular carboxyl-terminal domain (CTD) composed of 18 nonapeptide repeats. We find that as few as three nonamer repeats sufficed for S. pombe growth, but only when Spt4 was present. Synthetic lethality of the spt51-835 spt4Δ double mutant at 34°C suggests that interaction of Spt4 with the central domain of Spt5 overlaps functionally with the Spt5 CTD

Functional interactions between Prp8, Prp18, Slu7, and U5 snRNA during the second step of pre-mRNA splicing

After the second transesterification step of pre-mRNA splicing, the Prp22 helicase catalyzes release of spliced mRNA by disrupting contacts in the spliceosome that likely involve Prp8. Mutations at Arg1753 in Prp8, which suppress helicase-defective prp22 mutants, elicit temperature-sensitive growth phenotypes, indicating that interactions in the spliceosome involving Prp8-R1753 might be broken prematurely at 37°C. Here we report that mutations in loop I of the U5 snRNA or in Prp18 can suppress the temperature-sensitive prp8-R1753 mutants. The same gain-of-function PRP18 alleles can also alleviate the growth phenotypes of multiple slu7-ts mutants, indicating a functional link between Prp8 and the second step splicing factors Prp18 and Slu7. These findings, together with the demonstration that changes at Arg1753 in Prp8 impair step 2 of pre-mRNA splicing in vitro, are consistent with a model in which (1) Arg1753 plays a role in stabilizing U5/exon interactions prior to exon joining and (2) these contacts persist until they are broken by the helicase Prp22