Molecular classification of papillary thyroid carcinoma: distinct BRAF, RAS, and RET/PTC mutation-specific gene expression profiles discovered by DNA microarray analysis.

Thyroid cancer poses a significant clinical challenge, and our understanding of its pathogenesis is incomplete. To gain insight into the pathogenesis of papillary thyroid carcinoma, transcriptional profiles of four normal thyroids and 51 papillary carcinomas (PCs) were generated using DNA microarrays. The tumors were genotyped for their common activating mutations: BRAF V600E point mutation, RET/PTC1 and 3 rearrangement and point mutations of KRAS, HRAS and NRAS. Principal component analysis based on the entire expression data set separated the PCs into three groups that were found to reflect tumor morphology and mutational status. By combining expression profiles with mutational status, we defined distinct expression profiles for the BRAF, RET/PTC and RAS mutation groups. Using small numbers of genes, a simple classifier was able to classify correctly the mutational status of all 40 tumors with known mutations. One tumor without a detectable mutation was predicted by the classifier to have a RET/PTC rearrangement and was shown to contain one by fluorescence in situ hybridization analysis. Among the mutation-specific expression signatures were genes whose differential expression was a direct consequence of the mutation, as well as genes involved in a variety of biological processes including immune response and signal transduction. Expression of one mutation-specific differentially expressed gene, TPO, was validated at the protein level using immunohistochemistry and tissue arrays containing an independent set of tumors. The results demonstrate that mutational status is the primary determinant of gene expression variation within these tumors, a finding that may have clinical and diagnostic significance and predicts success for therapies designed to prevent the consequences of these mutations.

Induction and regulation of Fas-mediated apoptosis in human thyroid epithelial cells.

Fas-mediated apoptosis has been proposed to play an important role in the pathogenesis of Hashimoto's thyroiditis. Normal thyroid cells are resistant to Fas-mediated apoptosis in vitro but can be sensitized by the unique combination of interferon-gamma and IL-1beta cytokines. We sought to examine the mechanism of this sensitization and apoptosis signaling in primary human thyroid cells. Without the addition of cytokines, agonist anti-Fas antibody treatment of the thyroid cells resulted in the cleavage of proximal caspases, but this did not lead to the activation of caspase 7 and caspase 3. Apoptosis associated with the cleavage of caspases 7, 3, and Bid, and the activation of mitochondria in response to anti-Fas antibody occurred only after cytokine pretreatment. Cell surface expression of Fas, the cytoplasmic concentrations of procaspases 7, 8, and 10, and the proapoptotic molecule Bid were markedly enhanced by the presence of the cytokines. In contrast, P44/p42 MAPK (Erk) appeared to provide protection from Fas-mediated apoptosis because an MAPK kinase inhibitor (U0126) sensitized thyroid cells to anti-Fas antibody. In conclusion, Fas signaling is blocked in normal thyroid cells at a point after the activation of proximal caspases. Interferon-gamma/IL-1beta pretreatment sensitizes human thyroid cells to Fas-mediated apoptosis in a complex manner that overcomes this blockade through increased expression of cell surface Fas receptor, increases in proapoptotic molecules that result in mitochondrial activation, and late caspase cleavage. This process involves Bcl-2 family proteins and appears to be compatible with type II apoptosis regulation.

Interleukin-1beta and tumor necrosis factor (TNF)-alpha sensitize human thyroid epithelial cells to TNF-related apoptosis-inducing ligand-induced apoptosis through increases in procaspase-7 and bid, and the down-regulation of p44/42 mitogen-activated protein kinase activity.

Primary thyroid cells are resistant to TNF-related apoptosis-inducing ligand (TRAIL). Previously we showed that the combination of IL-1beta and TNFalpha facilitated TRAIL-mediated apoptosis in these cells and enhanced cell surface expression of TRAIL receptors. The aim of this study was to further characterize the mechanism by which these cytokines sensitized primary thyroid cells to TRAIL-mediated apoptosis. IL-1beta and TNFalpha increased the concentrations of procaspase-7 and Bid. In contrast, the p44/42 MAPK (Erk) pathway was active in thyroid cells and this activity was significantly decreased after exposure to IL-1beta/TNFalpha. A MAPK kinase inhibitor (U0126) could enhance the cytokine-induced sensitization of thyroid cells to TRAIL, reinforcing the inhibitory role of Erk on TRAIL signaling. In conclusion, IL-1beta/TNFalpha treatment sensitizes human thyroid cells to TRAIL-mediated apoptosis through increased surface expression of TRAIL receptors, increased expression of procaspase-7 and Bid, and the inhibition of p44/42 MAPK (Erk) pathway.

Aberrant apoptosis in thyroid epithelial cells from goiter nodules.

The specific pathogenesis of nodular goiter and the role of apoptosis in goitrogenesis are not known. We sought to examine the regulation of the TNF-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL)-induced apoptosis pathways in primary thyroid cells from 17 patients with nodular goiter, using 10 normal thyroids as controls. Both goitrous and normal thyroid cells were resistant to recombinant human TRAIL and an agonist anti-Fas antibody under basal conditions. However, all normal thyrocytes could be sensitized by TNFalpha/IL-1beta or interferon gamma/IL-1beta to undergo apoptosis in response to TRAIL or FasL, respectively. In contrast, the majority of goiter-derived cells remained resistant to TRAIL (12 of 17 samples) or FasL (9 of 17 samples) after cytokine pretreatment; 14 of 17 goiter nodules were resistant to at least one death ligand. Goiter size was inversely correlated with the sensitivity to TRAIL-mediated apoptosis. The resistance of goiter cells to TRAIL did not appear to be due to transcriptional regulation or cell surface expression of death and decoy receptors. However, increased proteasome activity was found in a subset of goiter cells resistant to both death ligands, and proteasome inhibitors could sensitize these goiter cells to TRAIL-mediated apoptosis. In conclusion, goiter-derived thyroid cells are resistant to TRAIL and/or Fas-induced apoptosis in vitro, and this may represent a new aspect of aberrant growth regulation in goiter nodules. The increased proteasome activity associated with this resistance suggests that the proteasome may be an important regulator of apoptosis in nodular goiter.

The surgical management of MEN-1 pancreatoduodenal neuroendocrine disease.

BACKGROUND: The management of multiple endocrine neoplasia, type 1 (MEN-1) pancreatoduodenal neuroendocrine neoplasms (NENs) is controversial. An aggressive surgical approach is intended to control the functional syndromes and malignant potential for nodal or distant metastasis. METHODS: The results of treating 39 patients with MEN-1 pancreatoduodenal NENs over a 35-year period are available from chart reviews and patient interviews. This study focuses on pattern of disease, disease recurrence, and long-term functional outcomes. RESULTS: Between 1967 and 2003, 39 patients ages 19 to 58 years (mean age, 37) had abdominal operations for their pancreatoduodenal NENs: 26 with Zollinger-Ellison syndrome, 4 with hypoglycemia, 3 with both Zollinger-Ellison syndrome and hypoglycemia, and 6 with nonfunctional neoplasms. Fifteen of these 39 patients had malignant disease on initial abdominal operation; 24 of 39 patients have not required abdominal reoperation, 17 of whom have available follow-up data. Of these 17 patients, 11 have biochemical evidence of disease recurrence (increased serum concentrations of gastrin, insulin, or pancreatic polypeptide), while 6 have no biochemical evidence of recurrence. A total of 30 abdominal reoperations were performed in 15 patients; 14 of 15 patients undergoing 1 or more reoperations developed evident malignant disease by their most recent operation. Nine of 13 reoperative patients with follow-up data have evidence of disease recurrence. Functional outcomes available in 20 patients showed that 10 patients require insulin and that 6 require oral hypoglycemic medications. Ninety percent have no abdominal pain or nausea/vomiting, while 4 are unable to return to work secondary to this disease. CONCLUSIONS: Treatment of MEN-1 pancreatoduodenal NENs is met with frequent recurrence and some treatment-related morbidity and mortality. Most patients (22 of 39) eventually demonstrated malignant growth, but, with this strategy, few died of this disease.

Pitfalls in the surgical treatment of insulinoma.

BACKGROUND: In the 75 years since an insulinoma was first described, the challenge for the surgeon has been one of localization. The combination of endoscopic ultrasound (EUS) or intraoperative ultrasound and operative palpation has led to nearly 100% success rate at primary operation in experienced institutions. However, 13% of patients at referral centers undergo reexploration, which has an increased morbidity. With more successful localization modalities, the operative failures have become more challenging than ever. METHODS: During the past 35 years, we have treated 118 cases of sporadic insulinoma. Technical advances in EUS have led to preoperative identification of more than 90% of insulinomas. Despite this success we have become increasingly aware of the limitations of EUS in the surgical treatment of insulinoma. We present the pitfalls of 6 recent cases. RESULTS: The limitations of EUS include the assessment of malignancy, the identification of pedunculated or adjacent lesions, the evaluation of distal pancreatic lesions, and the differentiation of larger homogeneous tumors from surrounding parenchyma. CONCLUSIONS: The surgical challenge in the treatment of insulinomas has transformed from one of localization to one of recognizing the limitations of our current localization modalities. By sharing our experience with recent difficult cases, we guide the surgeon to greater success and the patient to decreased morbidity.

An alternative analysis of intraoperative parathyroid hormone data may improve the ability to detect multiglandular disease.

HYPOTHESIS: A nomogram based on regression analysis of intraoperative parathyroid hormone level decay discriminates single gland disease from multiglandular (MG) disease more accurately than the currently used 50% rule. DESIGN: Retrospective case series. SETTING: Academic health center. PATIENTS: Two hundred thirty-five patients (222 patients with single gland disease and 13 patients with MG disease) who underwent parathyroidectomy. INTERVENTIONS: Intraoperative parathyroid hormone level analysis at baseline, time 1 (about 5 minutes), and time 2 (about 10 minutes) after excision of the first gland. MAIN OUTCOME MEASURES: The mean slope was calculated at time 1 and time 2 and analyzed using one-way analysis of variance and the Fisher least significance difference post hoc tests using data normalized to baseline intraoperative parathyroid hormone levels to compare patients with single gland disease with patients with MG disease. A regression-based nomogram was created to analyze individual kinetic decay data. RESULTS: The mean (SEM) single gland disease slope was significantly steeper than the MG disease slope at both time 1 (-0.91 [0.02] vs -0.66 [0.05]; P<.01) and time 2 (-0.77 [0.01] vs -0.56 [0.05]; P<.01). When the standard threshold rule of a 50% decrease from baseline was used, only 23% of the patients with MG disease were correctly predicted by intraoperative parathyroid hormone values (77% false-positive result rate) at time 1. However, the nomogram correctly predicted 54% of the patients with MG disease at time 1 (46% false-positive result rate). At time 2, the standard threshold 50%-rule method correctly predicted 38% of the patients with MG disease (62% false-positive result rate), while the nomogram still correctly classified 54% of the patients with MG disease (46% false-positive result rate). CONCLUSIONS: A regression-based nomogram incrementally improves prediction of MG disease compared with the standard 50%-rule method and accounts for variability in the exact timing of samples. Slope analysis suggests that the earliest time point best isolates the kinetics of the excised gland. The nomogram will need to be validated prospectively.

Completion pancreatectomy and duodenectomy for recurrent MEN-1 pancreaticoduodenal endocrine neoplasms.

BACKGROUND: The purpose of this study is to describe outcomes of MEN-1 patients with recurrence requiring completion pancreatectomy and duodenectomy after initial treatment of pancreatic endocrine neoplasms (PENs) and hypergastrinemia with distal pancreatectomy, enucleation of pancreatic head PENs, and duodenotomy. METHODS: After undergoing this initial operation, 8 of 49 patients (16%) have required completion pancreatectomy and duodenectomy for recurrent PENs and hypergastrinemia. Retrospective review was performed. RESULTS: Median age was 39 years (27-51) at completion pancreatectomy compared to 31 years (20-40) at initial operation. Pathology revealed multiple PENs in 100%, duodenal neoplasms in 63%, and metastatic lymph nodes in 75%. There was no operative mortality and 88% of patients are currently alive. Preoperative gastrin levels were 934 +/- 847 pg/mL while postoperative levels are 93 +/- 79 pg/mL (normal 25-111 pg/mL). Mean Hemoglobin A1C levels are 8.3 +/- 3.3% (normal 3.8%-6.4%). Mean follow-up is 44 +/- 25 months. CONCLUSION: This initial operation may provide tumor control and prevent metastases but recurrent PENs are multifocal and progressive. Completion pancreatectomy and duodenectomy is arduous but outcomes are acceptable. Considering the radical nature of this treatment, individual consideration should be given to MEN-1 patients amenable to initial alternative pancreatic resections that preserve pancreatic mass and allow future pancreas-preserving reoperations.

The role of radiologic studies in the evaluation and management of primary hyperaldosteronism.

BACKGROUND: Surgical treatment of primary hyperaldosteronism (PHA) requires demonstration of unilateral adrenal hypersecretion. Optimal methods for interpretation of imaging and invasive testing are still in development. METHODS: A retrospective review from 1996-2007 of 106 patients with PHA was undertaken. Patient demographics, biochemical studies, radiologic imaging, operative reports, and pathology were reviewed and comparisons made. Optimal ratios for adrenal vein sampling were tested with regard to sensitivity and specificity. Preoperative and postoperative medication requirements and blood pressures were compared among different treatment groups. RESULTS: Seventy-eight patients (62 surgically treated) met criteria for inclusion. Median arterial blood pressure at diagnosis was 150/86 mm Hg while taking 3 antihypertensive medications. 69.2% required potassium supplementation. Median aldosterone:renin ratio was 107.0. Forty-two AVS procedures changed the management of 15 patients (35.7%) when compared to CT results. AVS accuracy was 96.6 vs 88.9% for NP-59 scintigraphy. Operative patients remained on fewer antihypertensive medications (1 vs 3), and mean systolic pressure was lower (130 vs 146 mm Hg) compared with medically managed patients. CONCLUSION: When used together, pre-ACTH aldosterone ratios, normalized A/C:A/C ratios, ratios to define contralateral suppression, and post-ACTH stimulated values allowed for capture of episodically secreting tumors and subtle unilateral or bilateral hyperaldosteronism.

EUS detection of pancreatic endocrine tumors in asymptomatic patients with type 1 multiple endocrine neoplasia.

BACKGROUND: EUS is highly sensitive, specific, and cost-effective for localization of pancreatic neuroendocrine tumors. EUS screening of asymptomatic patients with multiple endocrine neoplasia type 1 has not been described. METHODS: EUS was used to evaluate all patients with known or suspected pancreatic neuroendocrine tumors. Asymptomatic patients with either a confirmed genetic or clinical diagnosis of multiple endocrine neoplasia type 1 were evaluated with EUS. The results were correlated with surgical and histopathologic findings. RESULTS: A total of 65 patients with multiple endocrine neoplasia type 1 underwent 132 EUS procedures over an 8-year period, including 13 asymptomatic patients. Five of the 13 asymptomatic patients had normal serum gastrin levels, and 8 had levels less than 4 times the upper limit of normal. EUS demonstrated pancreatic neuroendocrine tumors in 11 of these patients, ranging in size from 0.4 to 4 cm (mean 10 mm). Ten of the 11 patients with tumors at EUS underwent surgical exploration, and 23 of 28 (82%) tumors removed surgically had been identified by EUS. At a mean follow-up of 44 months, no liver or lymph node metastases were demonstrated in any patient. CONCLUSIONS: In this first cohort study of asymptomatic patients with multiple endocrine neoplasia type 1 undergoing screening EUS, pancreatic neuroendocrine tumors were identified before the development of significant biochemical test abnormalities. Aggressive early surgical treatment may improve the prognosis for these patients.

Regression of type II gastric carcinoids in multiple endocrine neoplasia type 1 patients with Zollinger-Ellison syndrome after surgical excision of all gastrinomas.

Enterochromaffin-like (ECL) tumors are documented in patients with hypergastrinemia secondary to chronic atrophic gastritis or with Zollinger-Ellison syndrome and multiple endocrine neoplasia type 1 (ZES-MEN-1). In patients with ECL tumors and atrophic gastritis, normogastrinemia after antrectomy has resulted in resolution, regression, or stabilization of ECL tumors. The natural history of ECL tumors associated with ZES-MEN-1 following normalization of gastrin levels after gastrinoma resection has not been previously reported. The purpose of this study was to determine the course of ECL tumors in patients with ZES-MEN-1 following normalization of serum gastrin levels after gastrinoma resection. Two patients with ZES-MEN-1 had biopsy-proven ECL tumors on endoscopic evaluation. They then underwent surgical exploration that included distal pancreatectomy, enucleation of pancreatic head tumors, duodenotomy with excision of submucosal tumors, and peripancreatic lymphadenectomy. Gastric ECL tumors larger than 1.0 cm were locally excised. Patients underwent long-term follow-up with biochemical and endoscopic surveillance. Normogastrinemia was achieved and sustained following gastrinoma resection in two patients with ZES-MEN-1. Periodic endoscopic surveillance over a 6-year period showed complete resolution of the ECL tumors. The development of ECL tumors associated with ZES-MEN-1 is multifactorial. Studies identified a genetic influence on tumor growth with loss of heterozygosity at the MEN-1 gene locus in ECL tumors. The resolution of ECL tumors in ZES-MEN-1 patients who are normogastrinemic indicates that an elevated gastrin level is a primary initiator for development of these tumors. Therefore both genetic defects and hypergastrinemia are causative agents. Normalization of serum gastrin levels is critical for the prevention of aggressive forms of ECL tumors.

Palliative thyroidectomy for malignant lymphoma of the thyroid.

BACKGROUND: Current treatment of malignant lymphoma of the thyroid consists of chemotherapy and external beam radiation. The diagnosis can routinely be made by fine-needle aspiration, obviating the need for surgery. However, a significant number of patients present with symptoms of obstruction, necessitating thyroidectomy for palliation. METHODS: To determine the outcomes of patients with malignant thyroid lymphoma after palliative thyroidectomy, we reviewed our experience. Between 1980 and 2001, 27 patients with thyroid lymphoma and symptoms or signs of airway and/or esophageal obstruction were evaluated at 1 of 3 academic institutions. RESULTS: The mean age of the patients was 66 +/- 3 years, and the majority was female. Patients presented with symptoms of dyspnea/stridor (30%), dysphagia/pain (30%), or impending airway obstruction (40%). All underwent palliative surgery. In addition to surgery, 10 patients had combined chemo- and radiotherapy, 10 had radiotherapy alone, and 4 had only chemotherapy. Symptom-free survival after palliative surgery was determined by Kaplan-Meier analysis. The mean actuarial symptom-free survival of patients with symptomatic, malignant thyroid lymphoma was 10 years (95% confidence interval, 7.67 to 12.33 years). CONCLUSIONS: Patients with malignant lymphoma of the thyroid can present with obstructive symptoms requiring palliative intervention. In this group of patients, thyroidectomy can be associated with good long-term palliation and low morbidity.

Distinct transcriptional profiles of adrenocortical tumors uncovered by DNA microarray analysis.

Comprehensive expression profiling of tumors using DNA microarrays has been used recently for molecular classification and biomarker discovery, as well as a tool to identify and investigate genes involved in tumorigenesis. Application of this approach to a cohort of benign and malignant adrenocortical tissues would be potentially informative in all of these aspects. In this study, we generated transcriptional profiles of 11 adrenocortical carcinomas (ACCs), 4 adrenocortical adenomas (ACAs), 3 normal adrenal cortices (NCs), and 1 macronodular hyperplasia (MNH) using Affymetrix HG_U95Av2 oligonucleotide arrays representing approximately 10,500 unique genes. The expression data set was used for unsupervised hierarchical cluster analysis as well as principal component analysis to visually represent the expression data. An analysis of variance on the three classes (NC, ACA plus MNH, and ACC) revealed 91 genes that displayed at least threefold differential expression between the ACC cohort and both the NC and ACA cohorts at a significance level of P < 0.01. Included in these 91 genes were those known to be up-regulated in adrenocortical tumors, such as insulin-like growth factor (IGF2), as well as novel differentially expressed genes such as osteopontin (SPP) and serine threonine kinase 15 (STK15). Increased expression of IGF2 was identified in 10 of 11 ACCs (90.9%) and was verified by quantitative reverse transcriptase-polymerase chain reaction. Select proliferation-related genes (TOP2A and Ki-67) were validated at the protein level using immunohistochemistry and adrenocortical tissue microarrays. Our results demonstrated significant and consistent gene expression changes in ACCs compared to benign adrenocortical lesions. Moreover, we identified several genes that represent potential diagnostic markers and may play a role in the pathogenesis of ACC.