Michael J. Crowley

A Panel of Four miRNAs Accurately Differentiates Malignant from Benign Indeterminate Thyroid Lesions on Fine Needle Aspiration

Purpose: Indeterminate thyroid lesions on fine needle aspiration (FNA) harbor malignancy in about 25% of cases. Hemi- or total thyroidectomy has, therefore, been routinely advocated for definitive diagnosis. In this study, we analyzed miRNA expression in indeterminate FNA samples and determined its prognostic effects on final pathologic diagnosis. Experimental Design: A predictive model was derived using 29 ex vivo indeterminate thyroid lesions on FNA to differentiate malignant from benign tumors at a tertiary referral center and validated on an independent set of 72 prospectively collected in vivo FNA samples. Expression levels of miR-222, miR-328, miR-197, miR-21, miR-181a, and miR-146b were determined using reverse transcriptase PCR. A statistical model was developed using the support vector machine (SVM) approach. Results: A SVM model with four miRNAs (miR-222, miR-328, miR-197, and miR-21) was initially estimated to have 86% predictive accuracy using cross-validation. When applied to the 72 independent in vivo validation samples, performance was actually better than predicted with a sensitivity of 100% and specificity of 86%, for a predictive accuracy of 90% in differentiating malignant from benign indeterminate lesions. When Hurthle cell lesions were excluded, overall accuracy improved to 97% with 100% sensitivity and 95% specificity. Conclusions: This study shows that that the expression of miR-222, miR-328, miR-197, and miR-21 combined in a predictive model is accurate at differentiating malignant from benign indeterminate thyroid lesions on FNA. These findings suggest that FNA miRNA analysis could be a useful adjunct in the management algorithm of patients with thyroid nodules. Clin Cancer Res; 18(7); 2032–8. ©2012 AACR.

Preoperative BRAF(V600E) mutation screening is unlikely to alter initial surgical treatment of patients with indeterminate thyroid nodules

Preoperative B‐type Raf kinase Val600Glu mutation, or BRAF(V600E), analysis has been proposed as a tool to guide initial surgery for indeterminate thyroid nodules. This study sought to determine if cytologic markers of malignancy are associated with the BRAF(V600E) mutation and if preoperative BRAF(V600E) testing would alter the initial management of patients with indeterminate nodules.

Intercellular Adhesion Molecule-1 (ICAM-1) is Upregulated in Aggressive Papillary Thyroid Carcinoma

BackgroundIntercellular adhesion molecule-1 (ICAM-1) is implicated in carcinogenesis. In this study we examined the expression of ICAM-1 in papillary thyroid cancer (PTC). We hypothesized that ICAM-1 correlates with indicators of tumor aggressiveness in PTC.MethodsThirty-five primary and metastatic PTCs, five follicular adenomas, five Hashimoto thyroiditis, five nodular hyperplasia, and eight normal thyroid tissue samples were analyzed for ICAM-1 gene expression using quantitative reverse-transcription polymerase chain reaction (RT-PCR). ICAM-1 gene expression was analyzed at protein level by immunohistochemistry (IHC) using a semiquantitative score. Gene expression and intensity levels were correlated with markers of tumor aggressiveness including BRAF V600E mutation, tumor size, extrathyroidal extension (ETE), angiolymphatic invasion, and lymph node metastasis.ResultsICAM-1 gene expression was higher in PTC (pxa0=xa00.01) and lymph node metastases (pxa0=xa00.03) when compared with benign tumors and Hashimoto’s. Furthermore, PTCs exhibiting BRAF V600E mutation (pxa0=xa00.01), ETE (pxa0<xa00.01), and lymph node metastasis (pxa0=xa00.02) were associated with higher ICAM-1 levels. Gene expression correlated with protein levels on IHC. Additionally, poorly differentiated thyroid carcinoma had a higher ICAM-1 intensity score compared with well-differentiated carcinoma (pxa0=xa00.03).ConclusionsICAM-1 expression is upregulated in papillary thyroid carcinoma. Furthermore, ICAM-1 upregulation correlated with aggressive tumor features such as BRAF V600E mutation, ETE, and lymph node metastasis, suggesting that ICAM-1 plays a role in thyroid cancer progression.

Vascular Platform to Define Hematopoietic Stem Cell Factors and Enhance Regenerative Hematopoiesis

Summary Hematopoietic stem cells (HSCs) inhabit distinct microenvironments within the adult bone marrow (BM), which govern the delicate balance between HSC quiescence, self-renewal, and differentiation. Previous reports have proposed that HSCs localize to the vascular niche, comprised of endothelium and tightly associated perivascular cells. Herein, we examine the capacity of BM endothelial cells (BMECs) to support ex vivo and in vivo hematopoiesis. We demonstrate that AKT1-activated BMECs (BMEC-Akt1) have a unique transcription factor/cytokine profile that supports functional HSCs in lieu of complex serum and cytokine supplementation. Additionally, transplantation of BMEC-Akt1 cells enhanced regenerative hematopoiesis following myeloablative irradiation. These data demonstrate that BMEC-Akt1 cultures can be used as a platform for the discovery of pro-HSC factors and justify the utility of BMECs as a cellular therapy. This technical advance may lead to the development of therapies designed to decrease pancytopenias associated with myeloablative regimens used to treat a wide array of disease states.

Risk stratification of indeterminate thyroid fine-needle aspiration biopsy specimens based on mutation analysis

BACKGROUNDnMutation analysis is potentially a powerful tool to enhance the diagnostic accuracy of thyroid fine-needle aspiration (FNA) biopsy specimens. However, some clinicians may rely on a negative mutation panel to exclude malignancy. We aimed to determine the malignancy rate in indeterminate lesions with negative mutation analysis.nnnMETHODSnA literature review established a mutation analysis model using the prevalence of BRAF, RET, RAS, and PAX8/peroxisome proliferator-activated receptor-γ mutations in indeterminate lesions. This model was applied retrospectively to a study cohort of 466 consecutive indeterminate lesions that underwent hemi- or total thyroidectomy for definitive diagnosis, to evaluate its accuracy for identifying malignancy.nnnRESULTSnOf 466 indeterminate lesions in the study, 30% (139) were malignant. These included 66 cases of papillary thyroid cancer, 45 cases of follicular variant of papillary thyroid cancer, 18 cases of follicular thyroid cancer, and 10 others. The risk of malignancy was 42% when cytologic atypia was present vs 17% without. The mutation analysis model would correctly identify only 48 of 139 (34%) of malignant indeterminate lesions. Therefore, when mutation analysis is negative, the overall risk of malignancy would be 23%. When atypia is present, the risk of malignancy would be 31% vs 13% in lesions without.nnnCONCLUSIONnIndeterminate lesions with a negative mutation analysis still carry a significant risk of malignancy, especially in the presence of atypia, requiring surgery for definitive diagnosis.

Thyroid stimulating hormone increases iodine uptake by thyroid cancer cells during BRAF silencing

BACKGROUNDnThe BRAF(V600E) mutation is present in 62% of radioactive iodine-resistant thyroid tumors and is associated with downregulation of the sodium-iodide symporter (NIS) and thyroid stimulating hormone receptor (TSHr). We sought to evaluate the combined effect of BRAF inhibition and TSH supplementation on (131)I uptake of BRAF(V600E)-mutant human thyroid cancer cells.nnnMATERIALS AND METHODSnWRO cells (a BRAF(V600E)-mutant follicular-derived papillary thyroid carcinoma cell line) were transfected with small interfering RNA targeting BRAF for 72 h in a physiological TSH environment. NIS and TSHr expression were then evaluated at three levels: gene expression, protein levels, and (131)I uptake. These three main outcomes were then reassessed in TSH-depleted media and media supplemented with supratherapeutic concentrations of TSH.nnnRESULTSnNIS gene expression increased 5.5-fold 36 h after transfection (P = 0.01), and TSHr gene expression increased 2.8-fold at 24 h (P = 0.02). NIS and TSHr protein levels were similarly increased 48 and 24 h after transfection, respectively. Seventy-two hours after BRAF inhibition, (131)I uptake was unchanged in TSH-depleted media, increased by 7.5-fold (Pxa0< 0.01) in physiological TSH media, and increased by 9.1-fold (P < 0.01) in supratherapeutic TSH media.nnnCONCLUSIONSnThe combined strategy of BRAF inhibition and TSH supplementation results in greater (131)I uptake than when either technique is used alone. This represents a simple and feasible approach that may improve outcomes in patients with radioactive iodine-resistant thyroid carcinomas for which current treatment algorithms are ineffective.

Dicer expression and microRNA dysregulation associate with aggressive features in thyroid cancer

BACKGROUNDnAltered miRNA expression and down-regulation of Dicer has been shown in various cancers. We investigated Dicer expression and global miRNA environment in correlation with malignant features of thyroid tumors.nnnMETHODSnDicer gene expression was assessed for 22 normal thyroids, 16 follicular adenomas, 28 papillary thyroid cancers (PTCs), 10 tall-cell variants of PTC, 11 follicular variants of PTC, as well as the four thyroid cell lines BCPAP, TPC1, KTC1, and TAD2 via quantitative polymerase chain reaction. BRAF((V600E)) mutation screening was completed for 31 neoplasms. Next-generation sequencing was performed on a subset of PTC and normal thyroid. Protein levels were assessed via Western blotting and immunohistochemistry.nnnRESULTSnDicer mRNA was down-regulated in malignant thyroid samples and cell lines compared with normal tissues, benign neoplasms, and the fetal cell line TAD2. Decreased Dicer gene expression in malignant tissues was correlated greatly with aggressive features: extrathyroidal extension, angiolymphatic invasion, multifocality, lymph node and distant metastasis, recurrence, and BRAF((V600E)) mutation. Conversely, increased levels of Dicer protein were observed in malignant tissues and cell lines. Sequencing yielded 19 differentially expressed miRNAs. Eight samples had a nonsignificant a global down-regulation in malignant tissues.nnnCONCLUSIONnDysregulation of Dicer and possibly altered expression of miRNAs are associated with aggressive features in thyroid cancers. These findings suggest that disruption in normal miRNA processing involving Dicer may play a role in thyroid cancer progression.

Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis.

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens.

Silencing of UCHL1 by CpG promoter hyper-methylation is associated with metastatic gastroenteropancreatic well-differentiated neuroendocrine (carcinoid) tumors.

BackgroundWell-differentiated gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare tumors with varying metastatic potential. The underlying molecular basis for metastasis by GEP-NETs remains undefined.MethodsQuantitative PCR and immunohistochemistry (IHC) staining for ubiquitin carboxyl-terminal esterase L1 (UCHL1) gene and protein expression was performed on a group of localized and metastatic well-differentiated GEP-NET samples acquired from a prospectively maintained tissue bank. The ability of extent of UCHL1 IHC staining to differentiate localized and metastatic tumors was compared with Ki-67 index.ResultsAmong 46 total samples, UCHL1 expression at both the gene and protein level was significantly greater among localized GEP-NETs compared with metastatic tumors and metastases (pxa0<xa00.001). Hypermethylation of the UCHL1 promoter was commonly observed among metastatic primary tumors and metastases (those with the lowest UCHL1 expression) but not among localized tumors (pxa0<xa00.001). Poor staining (<50xa0%) for UCHL1 was observed in 27xa0% of localized tumors compared with 87xa0% of metastatic tumors (pxa0=xa00.001). The presence of <50xa0% staining for UCHL1 was 88xa0% sensitive and 73xa0% specific for identifying metastatic disease. In contrast, there was no association between Ki-67 index and metastatic disease. In multivariable analysis, only UCHL1 staining <50xa0% [odds ratio (OR) 24.5, pxa0=xa00.035] and vascular invasion (OR 38.4, pxa0=xa00.03) were independent risk factors for metastatic disease at the time of initial surgery.ConclusionsLoss of UCHL1 expression by CpG promoter hypermethylation is associated with metastatic GEP-NETs. Extent of UCHL1 staining should be explored as a potentially clinically useful adjunct to Ki-67 index in evaluating GEP-NETs for aggressive features.

Prostate-Specific Membrane Antigen Is a Potential Antiangiogenic Target in Adrenocortical Carcinoma.

CONTEXTnAdrenocortical carcinoma (ACC) is a rare tumor type with a poor prognosis and few therapeutic options.nnnOBJECTIVEnAssess prostate-specific membrane antigen (PSMA) expression as a potential novel therapeutic target for ACC.nnnDESIGNnExpression of PSMA was evaluated in benign and malignant adrenal tumors and 1 patient with metastatic ACC.nnnSETTINGnThis study took place at a tertiary referral center.nnnPATIENTSnFifty adrenal samples were evaluated, including 16 normal adrenal glands, 16 adrenocortical adenomas, 15 primary ACC, and 3 ACC metastases.nnnMAIN OUTCOME MEASURESnDemographics, PSMA expression levels via real-time quantitative polymerase chain reaction and immunohistochemistry and whole-body positron emission tomography-computed tomography standardized uptake values for 1 patient.nnnRESULTSnqPCR demonstrated an elevated level of PSMA in ACC relative to all benign tissues (P < .05). Immunohistochemistry localized PSMA expression to the neovasculature of ACC and confirmed overexpression of PSMA in ACC relative to benign tissues both in intensity and percentage of vessels stained (78% of ACC, 0% of normal adrenal, and 3.27% of adenoma-associated neovasculature; P < .001). Those with more than 25% PSMA-positive vessels were 33 times more likely to be malignant than benign (odds ratio, P < .001). Whole-body positron emission tomography-computed tomography imaging showed targeting of anti-PSMA Zr89-J591 to 5/5 of the patients multiple lung masses with an average measurement of 3.49 ± 1.86 cm and a standardized uptake value of 1.4 ± 0.65 relative to blood pool at 0.8 standardized uptake value.nnnCONCLUSIONSnPSMA is significantly overexpressed in ACC neovasculature when compared with normal and benign adrenal tumors. PSMA expression can be used to image ACC metastases in vivo and may be considered as a potential diagnostic and therapeutic target in ACC.